No title

Advances in

Insect Physiology

Volume 15

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Advances in Insect Physiology edited 4 y

M. J. BERRIDGE J. E. TREHERNE and V. B. WIGGLESWORTH Department of Zoology, The University Cambridge, England

Volume 15

1980

ACADEMIC PRESS A Subsidlaw of Harcourt Brace Jovanovlch, Publishers

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British Lihrury Cutuloguing in Puhlicution Dutu Advances in insect physiology Vol. IS 1 . Insects - Phy\iology I . Brrridgc. Michael John 11. Treherne, John Edwin I l l . Wigglcsworth. Sir Vincent SV5.7'01 QL4VS 63-1.1039 ISBN 0-1 2-024215-X ISSN 0065-2806

P R I N T E D IN G R E A T B R I T A I N B Y W & J M A C K A Y LIMITED. C H A T H A M

Contri but0 Peter D. Evans

Agricultural Re.yearch Council, Unit of Invertebrate Chemistry and Physiology, Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, U K A. R. Gilby

Division of Entomology, C.S.I.R.O., P.O. Box 1700, Canberra City, A C T 2601, Australia Nancy J. Lane

Agricultural Research Council, Unit of Invertebrate Chemistry and Physiology, Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, U K Stuart E. Reynolds

School of Biological Sciences, University of Bath, Claverton Down, Bath BA2 7AY, U K David B. Sattelle

Agricultural Research Council, Unit of Invertebrate Chemistry and Physiology, Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, U K Helen leB. Skaer

Agricultural Re.rearch Council, Unit of Invertebrate Chemistry and Physiology, Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, U K

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Contents Contributors

V

Transpiration, Temperature and Lipids in Insect Cuticle A. R . GILBY

1

Intercellular Junctions In Insect Tissues NANCY J. LANE and HELEN leB. SKAER

35

Acetylcholine Receptors of Insects DAVID B. SATELLE

215

Biogenic Amines in the Insect Nervous System PETER D. EVANS

317

Integration of Behaviour and Physiology in Ecdysis STUART E. REYNOLDS

475

Subject Index

597

Cumulative List of Authors

621

Cumulative List of Chapter Titles

623

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Transpiration, Temperature a n d Lipids in Insect Cuticle A. R. Gilby Drvrsion of Entomology,

C S l R 0 , Canberra, Australra

1 Introduction 1 2 Biophysics of cuticular transpiration 3 2.1 Mass transfer of water 3 2.2 Energy budget during evaporation 6 3 Water loss and temperature 9 3.1 Water loss measurements 9 3.2 Critical temperature - dynamic experiments 12 3.3 Energy budget analysis 16 4 Transpiration and cuticular lipids 20 4.1 Insect integument as a limiting membrane 20 4.2 Cuticular lipids and water loss 21 4.3 Chemical composition of cuticular lipids 22 4.4 Hypotheses o n lipid functioning 24 5 Conclusion 29 Acknowledgement 30 References 30

1 Introduction

The water relations of arthropods, and particularly of insects, have attracted the interest of scientists of many disciplines during half acentury. The ability of terrestrial insects to conserve water has been emphasized as one vital basis for the unique success of insects in colonizing a wide range of environments, despite their relatively small size. The penalty of a large ratio of area to volume has somehow been overcome. Given the large interface with the environment, it is inevitable that the insect cuticle should be the focus of attention in attempts to suggest mechanisms by which this has occurred. Early preoccupation with the loss of water from insects has given way to the realization that other factors such as the rapid absorption of water 1

2

A. R . GlLBY

vapour are important in vivo. Recent work has shown that, while some of the structures involved are integumental derivatives, the site of water uptake is not the external integument itself. This has been reviewed recently by Wharton and Richards (1978) and mechanisms are discussed by NobelNesbitt (1977). Furthermore, in the living animal the layer of epidermal cells beneath the cuticle may well be important in controlling the passage of water (Berridge, 1970). Treherne and Willmer (1975) have produced evidence that integumentary water loss from a cockroach may be influenced hormonally. Also, the beetle Cryptoglossa virrucosa was shown by Hadley (1979) to produce in direct response to exposure to low humidity a surface mesh of wax filaments which he associated with lower cuticular transpiration rates. Much of current emphasis in the subject is on aspects such as those characteristic of processesin vivo.However they will not be dealt with here. In a previous article in this series, Beament (1964) gave an extensive exposition of his work over 20 years on active transport and passive movement of water in insects. This represents essentially the current position in the evolution of his ideas and his hypotheses involving detailed models of the functioning of lipids in insect cuticle and the control of cuticular water relations. It follows an earlier comprehensive account (Beament, 1961). Subsequent publications by Beament have not radically changed the viewpoints advanced (Beament 1965, 1967, 1976). An extremely useful and discerning contribution which covers the whole subject is the recent book on water balance in land arthropods written by Edney (1977). Another valuable review is that by Ebeling (1974). These recent publications eliminate the need for any general review and provide an exhaustive reference list. The present article is fairly restricted in its scope. Much of published experimental data on the outward passage of water through insect cuticle is useful in comparative physiology and ecology. However, inferences from the influence of temperature on the permeability of cuticle to water have formed one of the main bases on which have been erected hypotheses on physico-chemical mechanisms involved in water retention. The literature on this topic is in a state of confusion. Much of the experimental data is either inappropriate or too limited for biophysical analysis and the analytical framework for the design and interpretation of experiments is generally unsound, mainly through the neglect of important independent variables. Many authors over-interpret their results and speculate on possible but unsupported mechanisms. 'The effect of these possibly wrong conclusions flowing on into text-books and general articles amounts to proof by repeated affirmation. This paper offers a personal view of current knowledge (or the lack of it) on the interactions of transpiration and temperature in insects and the role of epicuticular lipids. Because of the paucity of soundly based data, it amounts to a critique of the current situation with no possibility of drawing

TRANSPIRATION, TEMPERATURE A N D LIPIDS

3

definitive conclusions on the validity of current or alternative hypotheses except that they lack evidence.

2

Biophysics of cuticular transpiration

Before proceeding to a consideration of experimental data on the effects of temperature on water loss, it is necessary t o set down briefly some biophysical principles. Some of the mathematical expressions depend for their validity on assumptions concerning the mechanism of water loss and the properties of cuticular membranes. Others, however, depend only upon the application of the laws of physics to insects without such assumptions. Some of the parameters have not been experimentally determined but limiting values can reasonably be assigned. Restriction of consideration to passive diffusion and dead insects enables metabolic terms to be ignored.

2.1

MASS TRANSFER O F WATER

2.1.1 Membrane permeability At any point within a membrane, the molecular diffusion of a component, j, in a direction x is described by Fick’s law F.(x)

=

dc . -D.-J dx

where F,(x) is the net flux density of component j at x , g m-’ s-’ D j is the diffusion coefficient of component j, mz s-l and dc,/dx is the concentration gradient of component j, g m-4. Since the actual concentration gradient within the membrane is not known, it is usual to assume homogeneity in the membrane and to replace -dcj/dx by Acj/Ax, where Ac, is the difference in the concentrations of component j in the phases separated by the membrane and Ax is the thickness of the membrane, so that Acj F ~ ( x )= D . JAX It is often preferred to express the flux density by

Acj Rj

Fj(X) = -

where R j is the resistance to diffusion of component j, s m-’.

(3)

A. R . GILBY

4

2.1.2 Permeability of cuticle to water - evaporative resistances When water passes through insect cuticle into an unsaturated atmosphere, the exact place within the integument where the change from liquid to vapour occurs is not known. The dermal and cuticular structure of insects is complex and varied and will not be dealt with here. Between the haemolymph and the cuticle there are several layers, such as the basement membrane and the epidermal cells which contain aqueous components. The implications of such layers acting as the barriers to the passage of water have been proposed by Berridge (1970) and discussed by Edney (1977). An alternative route for the loss of water is provided by the tracheae which pierce the integument through all its component layers. Without at this stage taking into account further subdivisions of the various structures, the major components which can offer resistance to the passage of water are represented in Fig. 1(a), which is not drawn to any scale. The various possible resistances to the flux of water vapour can be incorporated into a resistance network outlined in Fig. l( b ) as an electrical circuit analogue. If water passes from the haemolymph to the surface as vapour, it diffuses through the dermal layers, resistance id, and the cuticle, resistance rc. These two resistances are in series so that their combined integumental resistance, R,, is their sum Rs = rd + rc

(4)

An alternative parallel pathway is transpiration through the tracheae, resistancer,, so that the combined internal resistance to diffusion of water due to the insect, R , , is

In series with the internal resistances is the stagnant layer of air, resistancer,, at the cuticle surface. The total resistance to the diffusion of water, R,, is thus R,

=

Ri

+ ro

(7)

Depending on the relative magnitudes of the various resistances, these expressions can be simplified. Thus if rd can be neglected either because

TRANSPIRATION, TEMPERATURE A N D LIPIDS (0

5

1 Atmosphere

Stagnant oir layer

fa

fC

................ ............... ................

........................................... ..........................................

rd

Trachea

(b)

R, ,integurnental resistance

haernolymph fd

\

fa

fC

V

in atmosphere

,

R,,, total resistance for water vapour Fig. 1 (a) Schematic representation of structures and resistances possibly involved in the flow of water vapour from haemolymph to atmosphere. (b) Resistances involved in transpiration arranged in electrical circuit analogue

liquid water is able to penetrate into the cuticle, e.g. through the pore canals, or because re >> r d , and also if r , >>re, e.g. because spiracles form a small fraction of the area of cuticle or the spiracular valves are efficiently closed, then the total resistance, R,, becomes equal to rc + To. Equation (3) can be applied to the mass transport of water and rewritten

A . R . GILBY

6

E

-

P S

-

P G

(9)

Rw

where E is the water loss rate, g m-2 s-' p, is the water vapour density at the liquid water surface within the insect, g m-3. pa is the ambient water vapour density in the atmosphere, g m-3. and R , is the vapour diffusion resistance of eqn (8), s m-'. The liquid vapour density, ps, may be taken as the saturated vapour density of water at cuticle temperature because the change in vapour density accompanying even large changes in osmotic pressure is small. The solutes in haemolymph have little effect. Because vapour pressure is related to vapour density by the perfect gas law, the driving force for diffusion, P , - P, can be replaced by the saturation deficit of the air if due account is taken of any temperature differences from the cuticle and suitable adjustment is made to the dimensions of R,. The concentration term in Fick's law can be expressed in yet other ways but the utmost vigilance must be exercised in any subsequent derivations to avoid involuntary assumptions concerning the variables concealed in different parameters. Thus, if the chemical potential gradient is considered as the driving force, it follows by the reasoning explained in Noble (1974), pp. 312-313, that the diffusion resistance, R,, thenobtained depends inversely on the water vapour concentration. This is undesirable. Expressions equivalent to eqn (9) are widespread in descriptions of transpiration in biology. As defined in eqn (9), the diffusion resistance R , is &/p,,,RT where p, is the mobility of water in the limiting membrane. 2.2

ENERGY B U D G E T D U R I N G EVAPORATION

Evaporation of water from an insect results in the loss of latent heat of vaporization of water from the animal. The environment interacts with the insect at its surface through the flow of energy. Energy, usually as some form of sensible or radiant heat, arriving at the insect will tend to increase its temperature unless the energy is consumed in metabolic or physiological processes. Because the energy input to an insect less the energy loss must equal the heat stored in the insect, an energy budget equation can be set up. This is based on the principle of the conservation of energy. An energy budget equation contains terms which depend on the physical characteristics of the insect and its environment, but it does not rely on assumptions concerning the architecture of anatomicalstructures or details of physiological mechanisms. The energy balance equation for flux densities into an insect is H + R +(l-a)R, + M - LE-G-q

=

0

(10)

TRANSPIRATION, TEMPERATURE A N D LIPIDS

7

where H is the sensible heat flux from the air to the surface, W m-’ R is the net long wave flux to the surface, W m-* R, is the short wave irradiance to the surface, W m-2 a is the short wave reflectivity of the surface M is the metabolic heat per unit area of surface, W m-’ L E is the latent heat flux due to evaporation of water, W m-2 G is the net heat flux by conduction, W m-2 and q is the rate of heat storage in the insect, W m-2. The terms in eqn (10) will be considered briefly as they apply to experimental conditions for the determination of water loss from insects. 2.2.1 Sensible heatflux The sensible heat flux arises from the exchange of heat between the insect and the surrounding air. It is proportional to the temperature difference between the insect and the air and inversely proportional to the resistance to heat transfer. The sensible heat flux is given by

H

= h(T, - T,)

where h is the heat transfer coefficient, W m-’ K-’ and T, and T, are the temperatures of ambient air and of cuticle respectively. The heat transfer coefficient, h , is equal to pcp/rH,the ratio of the volumetric heat capacity of air pcp, to the resistance to heat transfer r,. An increase in the movement of air will cause a decrease in r,, i.e. an increase in h and in the sensible heat flux. If the cuticle is at a lower temperature than the air, such an increase in h will tend to raise the cuticle temperature.

2.2.2 Radiant energy Jtitxes The second and third terms in eqn (10) relate to the exchange of radiant energy. Any insect is always exposed to streams of thermal radiation from surrounding objects and itself contributes to the general radiation flux. The major source of short wave radiation is the sun either directly or by reflection or scattering. In laboratory experiments, R , is likely to be negligible unless the design of apparatus is particularly unfortunate, e.g. by allowing sunlight or other strong radiation to penetrate the specimen chamber. If R, were not negligible, its strongly directional character would impose problems in calculating its effects. For objects near room temperature, the radiation emitted is of long wavelength. An analysis of the net radiation flux is greatly simplified if the insect is surrounded by a radiating surface at a uniform temperature equal to

A. R . G l L B Y

8

the air temperature. This is usually so in quantitative measurementsof water loss which otherwise would have little meaning. Application of the Stephen-Boltzmann law then gives R

= E

m(Ta4 - TC4)

(12)

where E is the emissivity of the cuticle u is the Stephan-Boltzmann constant, W ni-’ K-4 and Toand T, are the temperatures of ambient air and of cuticle respectively. For most natural biological surfaces E is close to unity.

2.2.3 Latent heatflux The evaporation of water from the insect results in latent heat loss as water is removed as vapour from the insect by diffusion and air movement. The energy consumed by transpiration is L E where L is the latent heat of evaporation of water, Jg-’, and E is the rate of water loss, g m-’ s-’. 2.2.4

Metabolic heat, heat lost by conduction and storage

By restricting analysis to dead insects, metabolic heat is eliminated. For an insect supported on a poor conductor with a small temperature difference and with a small area of contact, the heat lost by conduction will be negligible. Under steady-state conditions there is no storage of heat. When these conditions apply, M , G and q in eqn (10) each equal zero.

2.2.5

SimpliJied energy budget equation

Substitution of eqns (11) and (12) in eqn (lo), with the assumptions just outlined, results in the energy balance equation h(T, -- T,)

+ m(Ta4

-

T):

=

LE

(13)

A consequence of eqn (13) is that, in any situation where an insect is enclosed in a chamber of unifrom temperature under subdued illumination, so long as water is evaporating under steady-state conditions then the cuticle temperature will be lower than the air temperature. The cuticle temperature in turn determines the water vapour density or pressure within the cuticle and hence the driving force for transpiration [eqn (9)]. The resistances to evaporation included in R , may also change with temperature and with air speed. The heat transfer coefficient, h , of eqn (13) also depends on air speed. The influence of ambient conditions on rate of water loss and on cuticle temperature is thus very complex.

TRANSPIRATION, TEMPERATURE A N D LIPIDS

3

9

Water loss and temperature

Although there is a large body of experimental information on water loss from insects, none of it has been obtained under rigorously controlled conditions where all the data for a complete physical analysis are known. The earliest measurements by Gunn (1933) on the effect of temperature on transpiration were made on live insects. He interpreted an observed increase in the rate of water loss from Bfatta orientalis at about 30°C as due to the onset of active movement of air into and out of tracheae by a muscular pumping action. Such physiological and metabolic reactions, important also in water uptake, complicate attempts to detect biophysical mechanisms affecting transpiration. Although dead insects can exhibit higher transpiration rates than living insects, a relatively small initial rate of respiratory transpiration can assume a markedly increased contribution to the rate of water loss of live insect:, as the temperature is increased (Ahearn, 1970). Fick's Law [eqn (3)J has been applied to calculate the cuticular resistance to water loss in living insects, e.g. by Vannier (1974), but in no investigation on the influence of temperature on transpiration from living insect5 has all the necessary parameters which would enable an analysis, such as both air temperature and cuticle temperature, been reported. Consideration is thus restricted mostly to data from freshly killed insects.

3.1

W A T E R LOSS M E A S U R E M E N T S

The history of the development of experimentation and interpretation of the effects of temperature on transpiration stems from the first systematic investigations by Ramsay (1935a, b). He was concerned with the physics of methods of measuring the evaporation of water and studied the effects of temperature, humidity and wind speed upon the rate of evaporative water loss from living or dead specimens of the cockroach, Periplaneta urnericanu. A wind tunnel was used for experiments up to 35°C. Unfortunately a different apparatus, consisting of a jar with desiccant in a thermostated water bath, was used for experiments between 35-50°C so that the wind speed, induced by a slowly rotating fan, was different and not known. Cuticle temperatures were not measured. From his measurements of the weight loss due to transpiration from freshly killed cockroaches with blocked spiracles, Ramsay plotted the rate of evaporation at a constant saturation deficit against air temperature. Rather than connect the points with a smooth curve, he drew two intersecting straight lines and concluded there was, at about 30°C, a sudden increase in the rate of evaporation of water through the body surface. The interpretation given was equivalent to attributing the

10

A . R. G l L B Y

sudden increase in the raie of water loss to an abrupt decrease in the cuticular resistance to water loss at the particular temperature. Subsequently, Wigglesworth (1945) extended observations to several other species of insect. Groups of dead insects with spiracles occluded were weighed before and after t:xposure to dry, unstirred air over phosphorus pentoxide in flasks immersed in a water bath. Plots of rate of water loss, in mg cm-2 h-’ with no consideration of saturation deficit, against temperature, mostly at 10°C intervals, gave curves increasing with temperature, the increase being steeper at higher temperatures, The curve for each species was interpreted as exhibiting a “critical temperature” at which the rate of water loss was considered to increase abruptly. The critical temperatures differed between species and between developmental stages of the same species. Similar results were obtained by Lees (1 947) with several species of ticks. Holdgate and Seal (1956), using freshly killed pupae of Tenebrio mofitor and nymphs of Rhodnius prolixus and Holdgate (1956), using several species of aquatic insects, investigated the effect of temperature on rate of water loss into dry air by methods similar to those of Wigglesworth (1945). The measurements on the terrestrial insects were conducted in still air and those on the aquatic insects in air that was stirred. All their experimental data fitted smooth and continuous curves. They rejected the hypothesis of a mathematical discontinuity in the previously published insect water loss curves and postulated the apparently sudden rise in the rate of water loss to be an artefact due to the choice of scales for the axes of what were basically exponential curves. With considerable justification, Holdgate and Seal questioned t h e validity of the critical temperature concept. Mead-Briggs (1956) reached similar conclusions from his experiments on several species of insects and emphasized the inconclusive nature of all results to that time. The situation did not remain static for long before the approach to the subject was revolutionized by Beament. First with Peripfaneta americana (Beament, 1958), and later with many other species of insect (Beament, 1959), Beament refined hitherto very crude experimental techniques and published results which appeared to re-establish convincingly the existence of critical temperatures in the water loss-temperature relations of insects. Bearnent used thermistors and thermocouples to measure air temperature and cuticle temperature and emphasis was placed on the necessity for extreme care in handling insects to avoid cuticle damage. Manipulations such as the blocking of spiracles were therefore precluded. The rate of water loss was determined from the weight change of single insects suspended on a beam balance incorporated in the thermostatted apparatus. Attention was paid to the circulation of air and to ensuring that the evaporation occurred into dry air. Air temperature, cuticle-air temperature difference and weight

TRANSPIRATION, TEMPERATURE A N D LIPIDS

11

loss could be measured without interference with the specimens. Results for permeability were expressed as mg/animal/mm Hg/h which is consistent with eqn (9) and allows for the saturation deficit of the air as a measure of the driving force for evaporation of water. When plotted against cuticle temperature, Beament's results indicate an extremely sharp, virtually a first order, increase in permeability to water at a critical temperature characteristic of each individual specimen of eleven species of terrestrial insect. Two exceptions were Tipula sp., which is a soil insect with damaged cuticle, and Schistocerca nymphs which both gave smoothly increasing curves. The pronounced discontinuities evident in the curves relating cuticle permeability to water and the cuticle temperature published by Beament have influenced the interpretation of results by most authors publishing on insect water loss in the last 20 years. However, in spite of repeated emphasis, e.g. Beament (1961), on the need for experimental rigour if results are to be interpreted in terms of the permeability of cuticle to water, the majority of investigators have continued to ignore parameters which must be measured, such as cuticle temperature, or have inadequately controlled experimental conditions. The bibliography by Edney (1977) should be consulted for specific references. The simultaneous measurement of water loss and cuticle temperature is experimentally very difficult. Oloffs and Scudder (1965) quoted both air and cuticle temperatures in their work on water loss from Cenocorixa expletu but their measurements were at 5 "C increments in air temperature. This is too coarse for their interpretation of the results in terms of critical temperature to be convincing. In a recent publication, Toolson (1978) advocated the use of the chemical potential gradient as a measure of the driving force for the diffusion of water vapour in transpiration from arthropods. Using results on water loss from a scorpion, he claimed that this procedure resulted in a smooth curve relating the conductivity coefficient, L,, to air temperature as an exponential function, whereas the conductivity coefficient, D,, calculated by the use of saturation deficit as the driving force, exhibited a stepped curve. Similar smoothing effects were claimed when other published data for insects were analysed using the chemical potential equations. However, in the example given on water loss from a scorpion, Toolson fitted an exponential curve to the experimental points for L, but drew a stepped line through the actual points for D,.The subjective impression of a smooth curve in the one case but not in the other is due to the transformations carried out on the data and not to any more fundamental cause. In the equations derived from the use of chemical potentials, the driving force involves the logarithm of the vapour pressure of water. This introduces difficulties in treating transpiration into dry air because the driving force then formally becomes infinite. As mentioned already in Section 2.1.2, the diffusion resistance and hence the

A. R . G I L B Y

12

conductivity coefficient depend on the water concentration as well as the mobility of water in the membrane if they are derived from chemical potentials. In seeking an indicator for water mobility in cuticle, it is preferable to use the diffusion resistance calculated from eqn (9) where R , depends on the water mobility and the temperature but not on water concentration as well. The use of chemical potential, which is not linearly related to concentration, also introduces logical difficulties in the application of the mathematics. Toolson does not take account of the effects of evaporative cooling on cuticle temperature. It will be shown later that errors in published measurements of cuticle temperature are a likely cause of spurious discontinuities in permeability - temperature curves. 3.2

CRITICAL TEMPERATURE - DYNAMIC EXPERIMENTS

An alternative approach to the common method of making measurements on an insect sequentially at a stepped series of fixed temperatures is to record changes as the temperature is varied continuously. Such “dynamic” experiments can result in a significant lag in cuticle temperature in addition to temperature differences due to latent heat of vapourization of water. Choice of a suitable rate of temperature change can minimize any lag and information relevant to the existence of critical temperatures can be gained. 3.2.1 Dynamic experiments on water loss Edney and McFarlane (1974) have studied the effect of temperature on transpiration in two cockroaches, Arenivugu investigatu and PeripEaneta americanu. Although there are unexplained inconsistencies between temperatures quoted in their Table 1 and Tables 2 and 3, they claimed it was possible to estimate approximate cuticle temperatures up to 50°C. With insects suspended from an electrobalance, the weight of each specimen was recorded continuously as transpiration occurred into circulating dry air while the temperature was steadily raised. They analysed their results in two ways. The recorded curve of weight against time was inspected for any abrupt change, and, the rate of water loss was determined graphically from the weight curves at points corresponding to any selected cuticle temperature. From these measurements and with due allowance for saturation deficit, permeability-cuticle temperature curves were constructed. Although the results showed that the permeability of the cuticles increased faster at higher temperatures, both types of curve were smooth and they provided no evidence for a critical temperature. I have carried out similar experiments on P . americana (Gilby, unpublished). Nymphs were from a stock culture maintained in the laboratory.

T

Before use in experiments, individuals were taken from the culture and, with access to food and water, kept isolated in glass jars for at least 5 days to allow recovery from any accidental damage to the cuticle. One day before experiment, food and water were withdrawn. Insects were killed by short exposure to HCN gas about 1h before transfer to the apparatus. The alternative use of H2Sdid not affect results. At all times the avoidance of mechanical damage to cuticle was regarded as of the utmost importance. The apparatus used was similar in principle to that illustrated by Edney and McFarlane (1974) but the air was vented and not recycled. The insect rested on a small copper loop on a terylene thread connected to the electrobalance and reaching into a glass tube immersed in a thermostatted water bath. A stream of dry air was passed through 10 m of copper tube immersed in the water bath to control the air temperature and then passed over the specimen at 100 cmlmin, with the insect head-to-wind. The output of the electrobalance was recorded as the temperature was raised from 20°C to 45°C contindously at a rate which was not linear but averaged a little over 20"C/h. The curves, equivalent to weight-temperature curves, increased smoothly in a fashion similar to those published by Edney and McFarlane (1 974). Alternatively, the recorder output was fed to a Cahn Time Derivative Computer, which enabled the rate of water loss to be recorded continuously. Such a curve, as illustrated in Fig. 2 , does not provide evidence for a step-like break at a critical temperature. However, it should be emphasized that no measurement was made of cuticle temperature, nor was allowance made for the saturation deficit of the air or any alternative indicator of the driving force for the diffusion of water.

3.2.2

Dynamic experiments on cuticle temperature

Examination of Figure 4, and particularly Figure 5, in Beament's (1958) paper where allowance is made for the saturation deficit of the air, reveals that it is the use of experimental values for T,, the cuticle temperature, rather than To, the air temperature, in the presentation of results which produces the abrupt step in the rate of water loss-temperature curve of P. americana. A similar situation applies for Rhodnius prolixus nymphs in Figure 6 of Beament (1959), but To is not shown for other species. Correct measurements of T, are therefore crucial to establishing whether the critical temperature interpretation is valid. Experimentally this is very difficult, much more so than the measurement of water loss. An investigation has been undertaken (Gilby, unpublished) of the cuticle temperature of P . americana nymphs under a regime of steadily increasing air temperature. The apparatus was based on that described earlier (Section 3.2.1). Dry air was passed through 10 m of copper pipe in a water bath and then over the specimen, supported head-to-wind on its legs on a fine nylon

A. R. GILBY

I

45'

z

35°C

1

05

10 Time I h 1

I 5

Fig. 2 Recorder trace of rate of change of weight of a P. americana nymph, weight 44.5 mg, in continuously rising ambient air temperature (indicated). Air flow 100 cm/min

net in a horizontal glass tube in the bath, before being vented. Insects were killed with HCN as described above and placed in position after having been isolated for several days to minimize cuticle damage. Towas measured with a copper-constantan thermocouple made from 6.1 X cm diameter wire placed in the air stream in front of the insect. Identical fine thermocouples were located on the insect, one placed under the posterior edge of the pronotum and another similarly placed on the abdomen under the second tergum. This was done with the utmost care. It was necessary to place the thermocouple wires laterally across the insect to avoid any spearing action and to keep the maximum length of wire in contact with the cuticle to minimize conduction errors. Merely placing the tip in contact with the cuticle gave variable results. The design of the apparatus was such that these preparative procedures were performed in the open before the glass tube was sealed in place and lowered into the water bath. The thermocouples had a common cold junction immersed in ice-water. The output from each

15

TRANSPIRATION. TEMPERATURE A N D LIPIDS

0

1 Trme ( h '

2

Fig. 3 Recorder traces of air temperature (T,) and cuticle temperature, measured at pronotum (T, pronotum) and second tergum (T, tergum), of P . americana nymph, weight 1055 mg, in continously rising ambient air temperature. Air flow 100 cmimin

thermocouple was taken to a multichannel potentiometric recorder and temperatures could be read to 0.1"C. The response of cuticle temperature of a large P . americana nymph to a steady change in air temperature from 25°C to 45°C during slightly more than 2 h is shown in Fig. 3. At this rate of heating there was only a minor lag in cuticle temperature. When the air reached its upper steady temperature of 45"C, the cuticle was within 0.25 "C of its equilibrium temperature, which it reached about 3 minutes later. A slower change in body temperature of P . americana was reported by Coenen-Stass and Kloft (1977) but they did not detect any lowering of body temperature at less than 32°C. With the size of specimen which gave the results in Fig. 3, the cuticle temperatures measured at the pronotum and the tergum were scarcely distinguishable. With small nymphs, differences of up to 1"C were sometimes evident but temperature measurements on small insects were relatively unreliable due to difficulty in placement of thermocouples and other physical causes. The cuticle

A . R . GILBY

16

temperature increased in a smooth curve (Fig. 3). Any sudden increase in transpiration rate should have been reflected in a diminished cuticle temperature because of the higher rate of energy consumption. This was not supported by experiment. In Fig. 4, the differences between air temperature (To) and

Air ternperature,T,,

(“C )

Fig. 4 Differences between air temperature and cuticle temperature of insects at different from ambient air temperatures. 0 ,from data of Beament (1958) for P . antericana nymphs; 0, data of Oloffs and Scudder (1965) for C’. e.rplern adults; A,from Fig. 3 for P. crmericarru nymphs

cuticle temperature (T, tergum) derived from Fig. 3 are plotted against air temperature. After the initial brief lag, a smoothly increasing curve results. This contrasts with the temperature differences shown in Fig. 4 measured from the results of Beament (1958) for large P. americana nymphs. Again, the curve in Fig. 4 derived from the results of experiments with C. expleta (Oloffs and Scudder, 1965) showed a smooth increase in temperature difference with increasing air temperature. 3.3

ENERGY B U D G E T ANALYSIS

3.3.1 Latent heat flux densities The quantitative comparison of results from different publications is almost impossible because of the great variety in the species of insect used and the lack of important information in many papers. For example, several investigators have worked with P. americana nymphs but Beament (1958) does not specify the size of his specimens, information which is needed to calculate flux densities. However a comparison was sought by assuming the reasonable value of 8 cm2for the surface area of a “large nymph” as used in his work. Calculations were made of the latent heat flux from three sets of data on the rate of water loss and these are shown in Fig. 5 plotted against air temperature. The results of Beament and of Gilby were each from single

17

TRANSPIRATION, TEMPERATURE A N D LIPIDS

b

10

?

20

A

0 .

30

40

50

Air temperature (TI

Fig. 5 Latent heat flux calculated for transpiration from P. americana nymphs at different ambient air temperatures. 0 ,from data of Beament 1958. Weight of nymph assumed 1 g; 0, from data of Edney and McFarlane (1974); A, from data of Gilby (unpublished). Weight of nymph 115 mg

insects but of disparate size, while those of Edney and McFarlane were an average from ten insects. It is evident from Fig. 5 that there is good agreement between the investigators in the measurements of water loss from P. arnericana into dry air at different temperatures. 3.3.2 Energy budget equation On the basis of the energy budget equation derived earlier, h(T, - T,) + (+(TU4 -T ):

=L E

(13)

it would be possible to calculate the cuticle temperature of any insect if the latent heat flux density, L E , (calculated from the rate of water loss) and the sensible heat transfer coefficient, h, were known for any given air temperature. The value of h , which depends very strongly on environmental conditions such as air movement, is not known experimentally for any insect, let alone under conditions where transpiration has been measured. However, it is possible for certain limiting assumptions to be made which enable existing results on temperature depression to be tested against an extreme estimate. Since the value of a,the Stephan-Boltzmann constant, is known and L E can be calculated as the product of the flux density of water from the insect

A. R . GILBY

18

surface and the latent heat of vaporization of water, the major problem is to provide an estimate of h. Calculations of h for still air conditions gave the results in Table 1. The method used is based on principles described by T A B L E 1 Cuticle temperature depression a n d transpiration. Experimental values a n d values calculated from a n energy budget equation Insect species an d Ref.

27.0 28.3 33.2 37.0 45.7

Beament (1958)

P. americana Edn ey an d Mcfarlane

(1 974)

P . arnericana Gilby (unpublished)

C. expleta

Oloffs an d Scudder (1 965)

Beament (1 959)

LE’ (w m-2) (a)

P. arnericana

R . prolixus

To

2.8 3.3 11.0 30.1 61.8

(b) 2.4 2.9 9.5 26.0 53.4

AT Experimental

2.4 2.4 3.7 7.3 9.4

AT2 Calculated

(a) 0.26 0.31 0.93 2.32 4.34

(b) 0.23 0.27 0.82 2.06 3.84

10 20 30 40 50

1.3 2.0 8.3 30.1 76.8

0.3 0.9 1.4 2.2 6.2

0.14 0.20 0.72 2.26 5.14

30 35 45

4.2 8.3 38.9

0.08 0.20 0.75

0.12 0.24 1.11

20 25 30 35 40 45

23.0 32.7 48.0 73.5 121.5 183.9

0.75 1.09 1.49 2.19 3.93 5.99

1.17 1.60 2.24 3.27 5.13 7.41

6.8 14.3 22.1 33.3 44.2

0.8 2.0 1.8 3.2 4.0

58.6 61.2 63.0 65.2 67.8

(c) 0.50 1.00 1.50 2.17 2.80

(4

0.44 1.88 1.31 1.91 2.46

’

Surface areas used in calculation of LE for P. americana were derived from the insect weight by the empirical relation S = 8.8 w’’~ (,.S cm’, w g) established experimentally.

Results of Edney and McFarlane were averaged from their Table 2 Diameters of equivalent cylinders used in solutions of energy budget equation for P. americana were derived from the insect weight by the empirical relation w = 1. 5 9 d 3(w g,d cm) established experimentally ( a ) Insect assumed to weigh 0.8 g i.e. d = 0.80 ( b ) Insect assumed to weigh 1.0 g, i.e. d = 0.86 ( c ) Equivalent cylinder assumed d = 0.7 approximating fed nymph ( d ) Equivalent cylinder assumed d = 0.4 approximating unfed nymph

TRANSPIRATION, TEMPERATURE A N D LIPIDS

19

Gates (1962). In heat transfer by convection, the physics of heat engineering establishes three dimensionless variables, the Grashof number, the Prandtl number and the Nusselt number, which relate basic properties of the air, such as heat conductivity and viscosity, to characteristic dimensions of the object. Using the expression relating the Nusselt number, Nu, to the other two for a cylinder under free convection and the further expression h =Nuk/pc,d, where k, p andlc, are the thermal conductivity, the density and the specific heat of air respectively and d is the diameter of the cylinder, an algebraic expression was established for h for inclusion in eqn (13). For the diameter of the cylinder equivalent to an insect, half the sum of the width and depth was used. An empirical relationship between this and insect weight was experimentally determined for P . americana. Likely dimensions derived from the literature were used for the other insects. The only unknown terms remaining in eqn (13) were then T, and T,. At any air temperature for which the latent heat flux was known, eqn (1 3) could then be solved iteratively for T,. This was done until the error in T, was 0.01 "Cto give the calculated values for AT, the temperature depression in the cuticle, in Table 1. In the calculation of AT from the energy budget equation, the expression used for the sensible heat transfer coefficient, h , was derived for conditions of free convection, i.e. still air. O n the other hand, the experiments which provided the values of the latent heat flux, L E , were all performed in moving air. The inconsistency was deliberately introduced to bias the calculation to yield maximum values for AT. Under the experimental conditions of forced convection, h would be larger than in free convection so that the calculated values of AT represent an upper limit. Comparison of calculated values of A T with experimental values set out in Table 1indicates that the energy budget analysis works well for the results of Oloffs and Scudder for C. expleta and the results of Gilby for P . americana. In both cases, experimental values of AT are less than the calculated values. In the other investigations with P. americana, the data of Edney and McFarlane is equivocal in that the experimental values are generally larger than those calculated. However, examination of their results on individual insects, rather than averages, reveals big variations which are not explained. Furthermore, the temperatures quoted in their Table 2, the source of data for the present calculations, conflict with their Table 1 and their Figure 2. The values of AT found by Beament are all much larger than the maximum values predicted. Some variations in the latter are possible because of unspecified parameters, e.g. size of insect which affects both L E and AT. However the calculations are not sensitive to the size of insect in the 0.8-1 g range as illustrated by the two examples in Table 1. A similar situation exists for R . prolixus where the experimental values of AT exceed the theoretical

A. R. GILBY

20

maxima calculated and the latter are not sensitive to the dimensions of the model. The evidence suggests that several published measurements of cuticle temperature during transpiration are unsound and therefore they are not a valid basis from which to draw conclusions about the existence of critical temperatures deduced from transpiration curves. This is particularly so because too large values of A T compress the temperature scale and expand the permeability scale simultaneously. It is not clear why cuticle temperatures as measured are lower than they should be. The most likely physical errors would produce the opposite effect. However, if the thermocouple probe damaged the cuticle, any damp spot resulting could give rise to exaggerated local cooling. Some evidence for such an effect was noted in the experiments on cuticle temperature measurements described earlier. If thermocouples were aligned towards the head rather than across the cockroach abdomen abnormally low cuticle temperatures were occasionally indicated. In some preliminary measurements of cuticle temperatures of P. americana an infrared thermometer, a non-invasive method of measurement, appeared preferable to thermocouples. However, its use imposed limits to the minimum size of insect which could be studied.

4

4.1

Transpiration and cuticular lipids I N S E C T I N T E G U M E N T AS A L I M I T I N G M E M B R A N E

In Section 2.1.2 and Fig. 1, the various possible resistances to the passive diffusion of water from inside an insect to the atmosphere were enumerated and incorporated into a resistance network in series and parallel. It is not easy to decide the relative contributions of the various resistances in eqn (8) to the total resistance, R,. On available information it is impossible to do so rigorously. Consideration of the energy budget of an insect shows the problem to be complex. Transpiration rate and cuticle temperature are functions of the air temperature, relative humidity, wind speed, net radiation and insect dimensions, as well as the internal diffusion resistances of the integument. The inherent complexity of the system has not been generally recognized. For example, some authors have sought to deduce the contribution of the stagnant air layer to the total diffusion resistance from the effect of air speed on transpiration rate without consideration of the energy flow and its effect on cuticle temperature. Even under circumstances where ro is negligible compared with other diffusive resistances, the alteration to h , the sensible heat transfer coefficient in eqn (13), caused by change in air speed will produce a change in cuticle temperature. This can be quite considerable. For

TRANSPIRATION, TEMPERATURE A N D LIPIDS

21

example (Gilby, unpublished), a thermocouple under the tergum of a 1.1 g P . arnericana nymph in the apparatus described earlier registered a cuticle temperature of 38.2"C at an air temperature of 40°C and an air flow of 100 cm/min. When the air speed was increased to 24 mlmin, the cuticle temperature rose to a steady 39.3"C. On resumption of the original air speed the cuticle temperature was again 38.2 "C. Neglect of such effects invalidates any deductions concerning diffusive resistances. However there are a number of indications that the resistance of the integument, R,, is a major component of the total resistance, R,. A thorough and extensive body of work on transpiration from plants, reviewed by Noble (1974), indicates that the leaf cuticular resistance is large compared with the resistance of the stagnant air layer. As pointed out by Edney (1977), a similar situation probably applies even more markedly to insects which are generally much less permeable to water than are plants. Also many of the experiments on water loss from insects have been carried out on insects with the spiractular openings mechanically blocked. Details may be consulted in the review by Edney (1977). Most comparisons of water loss from dead insects with spiracles blocked or unblocked indicate that water loss from the tracheae is of minor importance and sometimes undetectable (Loveridge, 1968). Microscopic examination of cockroaches killed with HCN showed that the spiracular valves normally remain closed. A further indication that the integument is the limiting membrane comes from the results of Beament (1945) on the evaporation of water through isolated cuticles and exuviae of Rhodnius into still air. The permeability to water was of the same order as that from dead insects (Wigglesworth, 1945). As will be discussed later, superficial damage to a cuticle, e.g. by abrasion or washing with water, can cause a big increase in its permeability to water. It is probable that the major barrier to loss of water from dead insects lies in the integument. 4.2

C U T I C U L A R L I P I D S A N D WATER LOSS

The postulate that the epicuticle and its lipids have an important function in transpiration from insects stems from very early work, e.g. Ramsey (1935b). The justification rests on experiments with whole insects and with model systems. Hadley (1978) has found correlations of water loss with epicuticular hydrocarbon composition in several species of tenebrionid beetles. The classical work involving killed or living whole insects is that of Wigglesworth (1945). H e measured the rates of water loss from insects treated superficially in various ways. The application of inert dusts caused a big increase in the rate of water loss. The situation has since been complicated by a controversy over whether mechanical abrasion is necessary to produce the effect (Ebeling, 1974) and the suggestion by Richards (private

A. R . GILBY

22

communication; Wharton & Richards, 1978) that in live insects the epiderma1 cells become involved. However, Wigglesworth associated the altered transpiration with damage he observed to an epicuticular wax. Also, he found that extraction of Rhodnius nymphs with chloroform or detergents increased the rate of water loss by a factor of up to twenty five. In some other insects exposure to chloroform vapour was sufficient to produce similar effects.Although solvents, too, remove more than superficial lipids and may affect other components (e.g. extraction with chloroform makes the cuticle rigid), the cuticular lipids are strongly implicated. These conclusions were supported by companion experiments on model systems by Beament (1945). Lipids were extracted from exuviae of several insects and, when spread on suitable supporting substrates, like tanned gelatin or butterfly wings, formed membranes which behaved in a manner consistent with Wigglesworth’s results on insects when treated with dusts or solvents. Furthermore, the changes in transpiration through the artificially made lipid-membranes with temperature resembled those of intact insects. It should be remembered, however, that this work was before the later refinements in techniques now considered essential. Following earlier observations by Ramsay (1 935b) on the preservation of droplets of water on cockroach cuticles, Beament (1958) reported experiments on the changes in size of droplets of water coated with wax extracted from P. americana exuviae and supported on a silicone treated metal plate in air at different temperatures. His results indicated, even without corrections for surface temperature, an abrupt increase in the rate of water loss from a drop at about 33°C. Whether this can be interpreted correctly as support for the concept of a transition temperature based on lipids in intact cuticle is extremely doubtful. For example, it is possible that at a particular temperature, the lipid on a water drop might spread to the hydrophobic plate due to a decrease in viscosity as noted by Ramsay (1935b) for films of cockroach grease. Further, the conditions of energy flow to the surface of the drop on a conductive plate are so different from those for an insect in air that it would be surprising if they showed similar transpiration - temperature responses. Indeed, on a polystyrene plate (a poor conductor of heat) smooth curves were obtained (Beament, 1958). It has been shown above that experimental support for sharp temperature effects in cuticles is lacking. 4.3

CHEMICAL COMPOSITION OF CUTICULAR LIPIDS

Much of the work on transpiration from insects was done at a time when there was almost no knowledge on the chemical composition of cuticular lipids. From the early 1960s, modern chromatographic and spectroscopic methods of separation and analysis, were applied to the identification of

TRANSPIRATION, TEMPERATURE A N D LIPIDS

23

cuticular lipids (Baker el al., 1960, 1963; Gilby and Cox, 1963). There is now a considerable amount of information available on the composition of cuticular lipids from many species of insect. Summaries of the data have been given by Hackman (1974) and by Jackson and Blomquist (1976). Considerable variation in composition is found in the cuticular lipids of different species. However, of the chemical classes of lipid, hydrocarbons and free fatty acids are probably present in the cuticles of all insects. Hydrocarbons predominate overwhelmingly in many species and, for example, often comprise more than 50%, and sometimes over 90%, of the cuticular lipids of cockroaches and grasshoppers. The hydrocarbons are usually n-alkanes, together with methyl branched alkanes or either mono- or di-unsaturated alkenes, and range in chain length from C20-C50.Nelson (1 979) has recently reviewed the surface hydrocarbons, particularly branched compounds of arthropods. Triglycerides and steroids are commonly reported as minor constituents of cuticular lipids and, more rarely, traces of phospholipids. Sometimes, but not always, these compounds might be suspected to be contaminants from the body. Wax esters of long chain acids and alcohols have been found in the cuticles of only a few species, sometimes in considerable proportion, e.g. a recent report of about 65% in a beetle (Baker, 1978). Aliphatic aldehydes have also been detected. Free fatty acids, which appear to be ubiquitous, are commonly of chain length CI4-C2,,, saturated and unsaturated but rarely branched. There are very few reports of alcohols in cuticular lipids. Primary alcohols would be of particular interest because of their known capacity to reduce the evaporation of water. Apart from two reports as a minor constituent, the only substantial occurrence is in the larvae of Samia cynthia ricini (Bowers & Thompson, 1965) where n-triacontanol is the major component. However, this is present as a powder coating the larva and is not laid down on the epicuticle as a discrete layer. Minor amounts of secondary alcohols occur in the cuticular lipids of some grasshoppers (Jackson and Blomquist, 1976). The major part (over 50%)of an insect cuticular lipid is therefore typically made up of non-polar materials which are the hydrocarbons, either saturated or unsaturated. If unsaturated hydrocarbons predominate, as in P. americana (Beatty and Gilby, 1969), the lipid is likely to be a fluid grease. The saturated long chain hydrocarbons are solids. Oxygenated long chain compounds of varying degrees of polarity make up a complex mixture of substances each usually a minor fraction. Phospholipids and sterols, the common polar lipids of the membranes of animal cells and tissues, are minor constituents or absent. It is possible that some, at least, of the oxygenated compounds may derive from chemical degradation of hydrocarbons.

A. R . GILBY

24

4.4

HYPOTHESES O N LIPID FUNCTIONING

Various physicochemical interpretations have been advanced which seek to explain transpiration from insects in terms of specific arrangements, either molecular or crystalline, of cuticular lipids. Most of the hypotheses were made in ignorance of the chemical nature of the lipids and some were made on chemical assumptions now known to be false. An important role is commonly assigned to amphipathic molecules with their dual polar - nonpolar nature. Modern chemical analyses show these to differ so greatly between species that any hope for an explanation common to all insects might well be futile. Furthermore, there is a lack of agreement on the morphological structure of the epicuticle (Edney, 1977; Neville, 1975) which may be due partly to differences between species. It is basic to any understanding of how lipids might function to know whether or not the epicuticular filaments observed by electron microscopy in some insects are wax canals of the kind suggested by Locke (1961), but the nature of which was disputed by Filshie (1970a), and to know what is their relation to the pore canals. The hard, thin, resistant wax layer of the epicuticle of Rhodnius, which is very different in morphology from the mobile grease covering Periplaneta, was stated to comprise a complex of lipids with other materials (Wigglesworth, 1975). Locke (1965) interpreted electron micrographs of Calpodes cuticle as showing a lipid monolayer in the epicuticle, but it is doubtful whether the techniques of the time were capable of detecting such a structure. Further uncertainties regarding the molecular organization of the lipids in insect cuticle arise from the fact that the vast volume of biophysical data which exists on the structure and properties of lipid films is almost entirely for systems based on the lipids of cellular membranes. These are chemically very different from cuticular lipids. Moreover, analogies with films of phospholipids and sterols are entirely speculative and may be quite misleading. It is therefore difficult to apply any definitive tests to existing hypotheses concerning insect cuticular lipids. 4.4.1

The monolayer hypothesis

The monolayer hypothesis, which was first considered tentatively and inconclusively by Ramsay (1935b), owed its principal development to Beament (1958, 1961, 1964). The hypothesis attributed the major influence on the permeability of cuticle to water to an oriented monolayer of lipid molecules underlying a thicker layer of wax randomly arranged on the epicuticle. Much of the argument was based on work with cockroaches, specifically P. americana. From his experiments on cockroaches with much of the lipid

TRANSPIRATION, TEMPERATURE A N D LIPIDS

25

removed by a water spray, Beament claimed that the monolayer is five times as impermeable to water as is the randomly arranged grease. Most attention was given to explanations of the so-called critical temperature phenomenon in transpiration. These explanations involved hypothetical transitions in the organization of the monomolecular film either by disruption of the monolayer at the critical temperature so that its organization was destroyed or by a change in phase within the monolayer to a less condensed state of higher permeability (Beament, 1961). Subsequently however, Beament (1964) adopted a very explicit model with amphipathic molecules of lipid in fixed positions on the epicuticle with their hydrocarbon chains oriented at an angle of 24.5" to the vertical and locked together by van der Waals forces so that the zig-zag shape of the methylene groups of the paraffin chains interfit. H e further proposed that, at a critical temperature, thermal energy would overcome the attractive forces so that the hydrocarbon chains assume a mean vertical position with increased interchain space for migration of water molecules through the monolayer. The proposal requires that the polar groups of lipid molecules in the monolayer occupy fixed positions on the surface. This would place extraordinarily specific requirements on the correspondence of the geometry of such sorption sites with the molecular architecture of the lipids to accommodate the model. Aside from arguments concerning any changes which might occur at a particular temperature, it is unlikely that lipids such as are found in insect cuticle would form monolayers substantially impermeable to water. There are compounds such as fatty acids present in the waxes which could, and no doubt do, form an oriented monolayer on the epicuticle surface. However, as pointed out by Gilby and Cox (1963), fatty acid monolayers are effective to control water evaporation only if artificially held in a state of high compression on a film balance and their permeabilities are sensitive to the presence of impurities. The presence of non-polar hydrocarbons characteristic of insect cuticular lipids would disrupt the cohesive forces between the hydrocarbon chains of molecules in a monolayer. Unsaturation and branched chains especially cause expansion in monolayers. Moreover, a monolayer beneath a wax layer is at an interface where the outer phase is an oil rather than air. Such a system is characterised by the formation of expanded monolayers. Experiments on the surface properties of films of cockroach lipid support these expectations (Lockey, 1976). The force-area curve obtained for a film of cuticular lipid from P. arnericana on water showed that a tightly packed monolayer was not formed. The film was readily collapsed and only weakly adherent to the water surface. A more direct assessment of the possible contribution of a monolayer to the restriction of water loss can be made from comparisons with published data on insect systems. Where the rate of water loss per unit area is known, it

A. R. GILBY

26

is possible by the use of eqn (9), with suitable attention to units, to calculate the evaporation resistance (s cm-l) of the system. This was done to give the results in Table 2. Because the measurements on which the calculations are based have been made under different conditions, a close quantitative TABLE 2

Resistances to evaporation of water from insect cuticle and lipid films System

Temperature ("C)

Evaporation resistance (s cm-')

Ref. for permeability data

P. americana nymph'

27

199

Beament (1958)2

Pieris wing coated with 1 p m film of cockroach grease

25

193

Beament (1945)

Water drop coated with film of cockroach grease

30.6

28

Beament (1958)

Condensed monomolecular film of stearic acid at maximum compression

25

2

Barnes and La Mer (1 962)

Weight of nymph assumed to be 1 g for flux density calculation Similar values obtained from results of Edney and McFarlane (1974) or Gilby (unpublished)

comparision is not justified. For example, the evaporative resistance of the stagnant air layer may differ but it is basic to the whole subject that this should be small compared to the resistance of the membrane, as discussed earlier. The permeability data used were those at the lowest temperature available and below any suggested critical temperature. The results in Table 2 show that the resistances to evaporation of water from a cockroach and a 1 p m membrane of cockroach grease are each some 6-7 times larger than the resistance of a film of cockroach grease on a water drop, which is in turn an order of magnitude greater than the resistance of a compressed monolayer of stearic acid. The resistance of the cuticle is equivalent to some 100 condensed monolayers of stearic acid in series. It is conceivable, however, that the net area of cuticle through which water evaporates might be only 1% of the total area. If that were so, the water flux density would be correspondingly greater and the value of the resistance to evaporation from eqn (9) correspondingly less. Nevertheless, this would create new discrepancies between the resistances to evaporation of the insect and those of both the wing coated with lipid and the water drop coated with lipid, whilst the resistance of the film on the water drop would remain equivalent to many condensed monolayers. Consequently it seems that the monolayer hypothesis is scarcely tenable.

TRANSPIRATION, TEMPERATURE AND LIPIDS

27

4.4.2 Crystal structure and other hypotheses Some other hypotheses on the role of lipids in transpiration from insects postulate the existence of crystalline forms in epicuticular lipids. Unfortunately there is very little evidence, so that the suggestions remain highly speculative. Included in this category is the supposition of Gilby and Cox (1963) that cuticular lipids might form a system analogous to the relatively thick duplex films of paraffins and polar lipids, possibly with polymerized structures in the interface, which can reduce water evaporation efficiently. Also, Locke (1965, 1974) attributes a key role to the occurrence of lipidwater liquid crystals in the epicuticular canals to explain changes in transpiration with temperature. Changes in crystal structure might explain changes in permeability to water. The artificial lipid systems from which analogies are drawn with insect waxes differ in chemical composition from cuticular lipids. Direct evidence for any of the postulated structures is lacking. A similar situation exists for the suggestion by Davis (1974) that phase transitions known to occur with phospholipids and sterols may occur in cuticular waxes of ticks. An early hypothesis (Beament, 1945) attributed abrupt changes in cuticle permeability with temperature to changes of crystal form in the lipids from an orthorhombic to a close-packed hexagonal system. Hurst (1950) developed this suggestion and carried out electron diffraction studies on the puparium of Calliphora erythrocephala and on artificial membranes in collodion. If he had used mixtures, his choice of n-fatty acids, unsaturated acids and n-paraffins for the artificial membranes would have proved to be remarkably appropriate as a model for insect waxes but unfortunately he applied the lipids singly and the larval cuticle which becomes the puparium of cyclorapphous flies is probably not typical in that it lacks a discrete lipid layer (Filshie, 1970b). Hurst found that the electron diffraction patterns both of the collodion-lipid films and of the puparium indicated a threedimensional arrangement of orthorhombic microcrystals which were less oriented ior paraffin films. After heating in an intense electron beam, the fatty acid films and the puparium showed a reduction in crystal orientation and a change to a hexagonal packing. It was suggested that such changes in crystalline form were correlated with changes in permeability to water. Where alignment of crystals occurred, it was believed to be due to the orienting effect of a monolayer underlying the wax. Measurements of dielectric constants were made in supporting observations by Chefurka and Pepper (1955) on beeswax, a system which is chemically a poor model. On the other hand, observations by Holdgate and Seal (1956) on wax from the pupa of Tenebrio molitor gave no indication by electron diffraction of any crystalline transition before meltiiig.

28

A. R . GlLBY

Since these attempts to detect microstructure in sheets of insect cuticular lipids, there has been an increase of interest in the study of thermotropic transitions in lipids and biological membrane systems in general. The literature is now very large. The lipids in many membranes are believed to have a liquid crystalline structure (Larsson and Lundstrom, 1976). Various physical methods have been used to detect transitions in lipids, model membrane preparations and biomembranes. They include infrared and raman spectroscopy, dilatometry, X-ray diffraction, calorimetry, nuclear magnetic resonance spectrometry, electron spin resonance spectrometry and fluorescent labelling (Andersen, 1978). Unfortunately, the data available on natural and artificial systems involve phospholipids almost exclusively (Chapman, 1975). The chemical compositions of insect waxes are so different from those of cell membranes that it is not justified to assume they are analogous. Electron paramagnetic resonance (EPR) spectroscopy has been used to study the lipids on the cuticle of a scorpion, Centruroides sculpturatus, by Toolsonet al. (1979). A spin label was applied to the cuticle of living animals which were sacrificed and E P R measurements made between 20-70°C on dissected cuticle. The main contribution to the E P R spectrum was probably in the surface epicuticular lipids. The results indicated a change in the mobility of the spin-labelled molecules at about 35°C but this was not associated with a sudden breakdown of water retention. Conversely, at higher temperatures where the rate of water loss was increasing rapidly, no changes were detected by EPR. Exploratory investigations on some insect cuticular lipids have been carried out using differential thermal analysis (Gilby, unpublished) with a Mettler TA2000. Endothermic processes occurred over the whole range of biological temperatures in waxes extracted from the exuviae of four species. In lipids from P. americana and Periplaneta brunnea, continuous broad peaks were observed from below 10°C to about 40°C. With the lipids from Lucilia cuprina puparia and Austracris guttulosa the peaks extended to over 50°C. Lipid mixtures such as are obtained from natural sources characteristically show broad transitions (Melchior and Steim, 1976). Calorimetry is a sensitive technique to reveal changes in the physical structures of lipids and ordered-disordered transitions (Spink and Wadso, 1976) but does not enable interpretation at a molecular level unless supplemented by other methods. The experiments showed that changes occurred in the extracted cuticular waxes with temperature but there was no sharp transition at any particular temperature. Attempts to detect similar transitions in samples of cuticle cut from cast skins of P. americana failed, possibly because of lack of sensitivity due to the minute quantities of lipid involved. If it should eventuate that mesomorphic structures in cuticular lipids do influence the permea-

TRANSPIRATION, TEMPERATURE A N D LIPIDS

29

bility of insect cuticle to water and if the liquid crystals are similar to those in phospholipid membranes, the interpretation of the effects of temperature on transpiration may be further complicated by the effects of changing water concentration in the epicuticle. Small amounts of water affect the melting behaviour of phospholipids (Chapman and Wallach, 1968).

5 Conclusion

The foregoing discussion reveals a distressing lack of well-established information or theory. The relationship of an insect to the physical properties of its environment is a very complex problem. Cuticle temperature and transpiration rate depend on such variables as air temperature and relative humidity, wind speed, the net amount of radiation, the geometry of insect and the diffusion resistances to the passage of water. This is a multidimensional system in which a change in any of the independent variables can alter the influence of the others. Thus, changes in air temperature at one wind speed may affect cuticle temperature and transpiration in quite a different manner from the same changes at a different wind speed because, in addition to any changes in cuticle permeability, there are also changes in the energy fluxes. For a rigorous analysis of experimental results, it is necessary to know physical properties of the insect, such as absorptivities and emissivities to the various streams of radiation and the effective geometry of the structures transferring energy and matter, in addition to environmental parameters. T o analyse the dynamic approach to a steady state after a change in external conditions would also require information on heat conductivity and specific heat of the insect. A complete treatment involving analyses of energy exchange and diffusive resistances has not been made for any insect. Most experimental results in the literature are not useful for assessing and interpreting the effects of temperature on the permeability of cuticle to water because essential parameters have been ignored, usually at least by the failure to measure cuticle temperature. This is very difficult to measure experimentally, especially with thermocouples which themselves interfere with the measurement. The establishment of sharp discontinuities in water permeability-temperature curves at a critical temperature depends entirely on measurements of temperatures in the cuticle. Where the original data are available, an energy budget analysis indicates that the measured values of the cuticle temperature in this work are impossibly low by several degrees. It is therefore not possible to conclude that these critical temperatures are real. The evidence points rather to a smooth increase in permeability with temperature. If there are no abrupt changes in cuticle permeability at a particular

TT

temperature, the incentive to invoke specific physico-chemical phenomena such as changes in crystallinity in epicuticular lipids or the organization of lipid monolayers is greatly diminished. Experimental evidence for such postulated effects in insects is very nebulous. At present there is insufficient known on the distribution of epicuticular lipids, to support detailed hypotheses on molecular mechanisms. Generalizations may be inappropriate because of interspecific differences which exist in morphology and in the chemical composition of lipids. Until soundly based experiments on insects define the phenomena to be explained and until hypotheses can be tested by experiments on model systems of appropriate composition, speculation will remain unproductive. Insect physiologists have not appreciated the biophysical complexities of the problems and even tend to a simplistic view of the integument. A similar confusion existed in plant eco-physiology until about ten years ago. The analytical framework for the study of plant biophysics is now well established and it is to be hoped that a similar prospect is in store for the study of transpiration in relation to cuticle structure and environmental conditions for insects. Acknowledgement

I am indepted to D r A. R . G. Lang, Division of Environmental Mechanics, C.S.I.R.O.,.for his advice on energy budget analysis.

References Ahearn, G. A. (1970).The control of water loss in desert tenebrionid beetles./. Exp. Biol. 53, 573-595 Andersen, H. C. (1978). Probes of membrane structure. Ann. Rev. Biochem. 47,

359-383 Baker, J. E. (1978). Cuticular lipids of larvae of Attagenus negatoma. Insect Biochem. 8,287-292 Baker, G. L., Pepper, J. H., Johnson, L. H. and Hastings, E. (1960).Estimation of the composition of the cuticular wax of the mormon cricket, Anabrus simplex Hald. J. Insect Physiol. 5 , 47-60 Baker, G.L.,Vroman, H. E. and Padmore, J. (1963). Hydrocarbons of the American cockroach. Biochem. Biophys, Res. Commun. 13, 360-365 Barnes, G.T. and La Mer, V. K. (1962).The evaporation resistances of monolayers of long-chain acids and alcohols and their mixtures. In “Retardation of Evaporation by Monolayers” (Ed. V. K. La Mer) pp. 9-33.Academic Press, New York and London Beament, J. W.L. (1945).Thecuticularlipoidsofinsects.J.Exp. Biol. 21,115-131 Beament, J. W.L. (1958).The effect of temperature on the waterproofing mechanism of an insect. J. Exp. B i d . 35, 494-519 Beament, J. W. L. (1959). The waterproofing mechanism of arthropods. 1. The

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effect of temperature on cuticle permeability in terrestrial insects and ticks. J . Exp. Biol. 36, 391-422 Beament, J. W. L. (1961). The water relations of the insect cuticle. Biol. Rev. 36, 281 -320 Beament, J. W. L. (1964). Active transport and passive movement of water in insects. A d v . Insect Physiol. 2 , 67-129 Beament, J. W. L. (1965). The active transport of water: evidence, models and mechanisms. Symp. SOC. Exp. Biol. 19, 273-298 Beament, J. W. L. (1967). Lipid layers and membrane models. In “Insects and Physiology” (Eds J. W. L. Beament and J. E. Treherne) pp. 303-313. Oliver and Boyd, London Beament, J. W. L. (1976). ‘The ecology of cuticle. In “The Insect Integument” (Ed. H. R. Hepburn) pp. 359-374. Elsevier, Amsterdam Beatty, I. M. and Gilby, A. R. (1969). The major hydrocarbon of a cockroach cuticular wax. Naturwissrnschafren 56, 373-374 Berridge, M. J. (1970). Osmoregulation in terrestrial arthropods. In “Chemical Zoology” (Eds M. Florkin and B. T. Schier) pp. 287-319. Academic Press, New York and London Bowers, W. S. and Thompson, M. J. (1965). Identification of the major constituents of the crystalline powder covering the larval cuticle of Samia Cynthia ricini (Jones). J . Insect Physiol. 11, 1003-1011 Chapman, D. (1975). Phase transitions and fluidity characteristics of lipids and cell membranes. Quart. Rev. Biophys. 8, 185-235 Chapman, D. and Wallach, D. F. H. (1968). Recent physical studies of phospholipids and natural membranes. In “Biological Membranes” (Ed. D. Chapman) pp. 125-202. Academic Press, New York and London Chefurka, W. and Pepper, J. H. (1955). On the physical nature of the transition region of insect waxes. Can. Entomol. 87, 163-1 7 1 Coenen-Stass, D. and Kloft, W. J. (1977). Auswirkungen der Verdunstungskiihlung und der Stoffwechselwarme auf die Korpertemperatur der Schnabenarten Periplaneta americana und Blaberus trapezoideus. J . Insect Physiol. 23, 1397-1406 Davis, M. T. B. (1974). Critical temperature and changes in cuticular lipids in the rabbit tick, Haemaphysalid leporispalustris. J . Insect Physiol. 20, 1007-1 100 Ebeling, W. (1974). Permeability of insect cuticle. In “Physiology of Insecta” (Ed. M. Rockstein) Vol. VI, pp. 271-343. Academic Press, New York and London Edney, E. B. (1977). “Water Balance in Land Arthropods” (Zoophysiology and Ecology, Vol. 9) Springer-Verlag, Berlin, Heidelberg and New York Edney, E. B. and McFarlane, J. (1974). The effect of temperature on transpiration in the desert cockroach, Arenivaga investigata, and in Periplaneta americana. Physiol. Zool. 47, 1-12 Filshie, B. K. (1970a). The resistance of epicuticular components of an insect to extraction with lipid solvents. Tissue & Cell 2 , 181-190 Filshie, B. K. (1970b). The fine structure and deposition of the larval cuticle of the sheep blowfly (Lucilia cuprina). Tissue & Cell 2 , 479-489 Gates, D. M. (1962). “Energy Exchange in the Biosphere” pp. 94-142. Harper and Row, New York Gilby, A. R. and Cox, M. E. (1963). The cuticular lipids of the cockroach, Periplaneta americana L. J. Insect Physiol. 9, 671-681 G u m , D. L. (1933). The temperature and humidity relationsof the cockroach BIatta orientalis. 1. Desiccation. J . Exp. Biol. 10, 274-285

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Hackman, R. H. (1974). Chemistry of the insect cuticle. In “Physiology of Insecta” (Ed. M. Rockstein) Vol. VI, pp. 216-270. Academic Press, New York and London Hadley, N. F. (1978). Cuticular permeability of desert tenebrionid beetles: correlation with epicuticular hydrocarbon composition. Insect Biochem. 8, 17-22 Hadley, N. F. (1979). Wax secretion and color phases of the desert tenebrionid beetle Cryptoglossa virrucosa (Le Conte). Science 203, 367-369 Holdgate, M. W. (1 956). Transpiration through the cuticles of some aquatic insects. J. Exp. Biol. 33, 107-118 Holdgate, M. W. and Seal, M. (1956). The epicuticular wax layers of the pupa of Tenebrio molitor L. J. Exp. Biol. 33, 82-106 Hurst, H. (1950). An electron diffraction study of the crystalline structure of the lipids in the pupal exuviae of Calliphora erythrocephala. J . Exp. Biol. 27, 238-252 Jackson, L. L. and Blomquist, G. J. (1976). In “Chemistry and Biochemistry of Natural Waxes” (Ed. P. E. Kolattukudy) pp. 201-238. Elsevier, Amsterdam, Oxford and New York Larsson, K. and Lundstrom, I. (1976). Liquid crystalline phases in biological model systems. A d v . Chem. Ser. 152, 43-70 Lees, A. D. (1947). Transpiration and the structure of the epicuticle in ticks. J . Exp. Biol. 23, 397-410 Locke, M. (1941). Pore canals and related structures in insect cuticle. J . Biophys. Biochem. Cytol. 10, 5 89-6 18 Locke, M. (1965). Permeability of insect cuticle to water and lipids. Science 147, 295-298 Locke, M. (1974). The structure and formation of the integument of insects. In “Physiology of Insecta” (Ed. M. Rockstein). Vol VI, pp. 124-213. Academic Press, New York and London Lockey, K . H. (1976). Cuticular hydrocarbons of Locusta, Schistocerca and Periplaneta, and their role in waterproofing. Insect Biochem. 6 , 457-472 Loveridge, J. P. (1968). The control of water loss in Locusta migratoria migratorioides R & F. 1. Cuticular water loss. J . Exp. Biol. 49, 1-13 Mead-Briggs, A. R. (1956). The effect of temperature upon the permeability to water of arthropod cuticles. J . Exp. Biol. 33, 737-749 Melchior, D. L. and Steim, J. M. (1976). Thermotropic transitions in biomembranes. Ann. Rev. Biophys. Bioeng. 5 , 205-238 Nelson, D. R. (1979). Long-chain methyl-branched hydrocarbons: occurrence, biosynthesis and function. A h . Insect Physiol. 13, 1-33 Neville, A. C. (1975). “Biology of Arthropod Cuticle”. Springer-Verlag, Berlin, Heidelberg and New York Noble, P. S. (1974). “Introduction to Biophysical Plant Physiology”. W. H. Freeman, San Francisco Noble-Nesbitt, J. (1977). Active Transport of Water Vapour. In “Transport of Ions and Water in Animals” (Eds B. L. Gupta, R. B. Moreton, J. L. Oschman and B. J. Wail) Vol. 5 , pp. 571-597. Academic Press, New York and London Oloffs, P. C. and Scudder, G. G. E. (1965). The transition phenomenon in relation to the penetration of water through the cuticle of an insect, Cenocorixa expleta (Hungerford). Can. J. Zool. 44, 621-662 Ramsay, J. A. (1935a). Methods of measuring the evaporation of water from animals. J . Exp. Biol. 12, 355-372

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Ramsay, J . A . (1935b). The evaporation of water from the cockroach. J . Exp. Biol. 12,373-383 Spink, C. and Wadso, I. (1'176). Calorimetry as an analytical tool. Method. Biochem. Anal. 23, 1-159 Toolson, E. C. (1978). Diffusion of water through the arthropod cuticle: thermodynamic consideration of the transition phenomenon. J . Thermal Biol. 3, 69-73 Toolson, E. C. White, T. R.. and Glaunsinger, W. S. (1979). Electron paramagnetic resonance spectroscopy of spin-labelled cuticle of Centruroides sculpturatus (Scorpiones: Buthidae): correlation with thermal effects on cuticular permeability. J . Insect Physiol. 25, 271-275 Treherne, J . E. and Willnier, P. G. (1975). Hormonal control of integumentary water loss: evidence for a novel neuroendocrine system in an insect (Periplaneta americana). J . Exp. Biol 63, 143-159 Vannier, G. (1974). Calcul d e la resistance cuticulaire B la diffusion de vapour d'eau chez un insecta Collembola. C.R. Acnd. Sc. Paris Ser. D. 278, 625-628 Wharton, G. W. and Richards, A. G. (1978). Water vapor exchange kinetics in insects and acarines. Ann. Rev. Entomol. 23, 309-328 Wigglesworth, V. B. (1945). Transpiration through the cuticle of insects. J . Exp. Biol. 21, 97-114 Wigglesworth, V. B. (1975). Incorporation of lipid into the epicuticle of Rhodnius (Hemiptera). J . Cell Sci. 19, 459-485

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Intercellular Junctions in Insect Tissues Nancy J. Lane and Helen leB. Skaer A. R. C. Unit of Invertebrate Chemistry and Physiology, Department of Zoology, Cambndge, UK

1 Introduction 36 1.1 Techniques 37 2 Septate junctions 43 2.1 Introduction 43 2.2 Pleated septate junction - structural features 44 2.3 Smooth septate junction - structural features 54 2.4 Occurrence in insects 62 2.5 Distribution in other invertebrates 65 2.6 Occurrence in vertebrates 67 2.7 Functional significance 69 2.8 Formation of septate junctions 73 3 Desmosomes 75 3.1 Introduction '75 3.2 Structural features 76 3.3 Occurrence in insects 79 3.4 Distribution in other invertebrates 8 0 3.5 Functional significance 83 3.6 Development 84 4 Gap junctions 85 4.1 Introduction 85 4.2 Structural features 87 4.3 Model derived from structural evidence 93 4.4 Distribution of gap junctions 94 4.5 Structural differences between arthropod and vertebrate gap junctions 98 4.6 Functions of gap junctions 100 4.7 Dynamics of gap junctional formation and disassembly 109 4.8 Co-existence with other junctional types 118 5 Tight junctions 120 5.1 Historical introduction: d o tight junctions exist in insect tissues? 5.2 Structural features 126 5.3 Model derived from structural evidence 131 35

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6

7

8 9

5.4 Distribution and localization in arthropod tissues 132 5.5 Co-existence with other junctional types 138 5.6 Homo- and heterocellular tight junctions 138 5.7 Comparison with vertebrate tight junctions 138 5.8 Functional significance 141 5.9 Developmental stages in tight junction formation 146 5.10 Tight junction degradation 149 5.1 1 Phylogenetic and evolutionary position 150 Specialized junctions of glia 151 6.1 Introduction 151 6.2 Axo-glial junctions 152 6.3 Tracheo-glial junctions 157 Scalariform junctions 157 7.1 Introduction 157 7.2 Thin-section appearance 159 7.3 Preparations treated with tracer 162 7.4 Freeze-fracture replicas 162 7.5 Model derived from structural evidence 166 7.6 Distribution in insect tissues 168 7.7 Physiological significance 170 7.8 Junctional development 172 Reticular septate junctions 172 8.1 Rectal papillae of dipteran insects 172 8.2 Peripheral retina of dipteran insects 177 Concluding remarks 180 9.1 Development of junctions 180 9.2 Functional considerations 181 9.3 Correlation of insect physiology with junctional structure Acknowledgements 183 References 184

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GLOSSARY O F TERMS

smooth septate junction = continuous junction zonula adhaerens = belt desmosome fascia adhaerens = discontinuous belt desmosome macula adhaerens = spot desmosome gap junction = nexus = macula comrnunicans = close junction tight junction = zonula occludens = occluding junction terminal bar = zonula adhaerens plus zonula occludens The material illustrated in the plates derives from adult specimens unless otherwise stated

1 Introduction

Over recent years the number of studies on intercellular junctions in invertebrate tissues has enormously increased. Moreover our understanding of the ultrastructure and functioning of these cell-to-cell associations has

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improved due to a variety of technical improvements and ingenious experimental designs. Reviews of insect junctional complexes were last published in 1973 (Satir and Fong, 1973; Satir and Gilula, 1973) although an abbreviated account has appeared more recently (Lane, 1978)*. However, a number of developments have occurred since then. These include more detailed analyses of the regulation and physiology of cell coupling by gap junctions, more precise distinctions between septate and continuous (smooth septate) junctions and the accumulation of evidence that establishes the existence of tight junctions in arthropod tissues. More information on scalariform junctions has also been forthcoming and other membrane modifications, possibly junctional, have been reported (axo-glial junctions and reticular septate junctions). Studies on developing systems have been carried out which elucidate details of junctional formation. Moreover, a fuller understanding of the functional significance of certain intercellular junctions has also been achieved. 1.1

TECHNIQUES

Studies on intercellular junctions generally incorporate one or more of the following four techniques: ( i ) En bloc uranyl acetate (UA) stained thin sections of fixed tissues which show the arrangement of the unit membranes of the adjacent cells with respect to one another and make possible a clear indication of the size of the intercellular cleft; (ii) lanthanum impregnation of the material which stains the extracellular space between the two apposed cells and reveals the details of the true membrane surface as well as any cross-linking structures within the space, and (iii) freeze-cleaving that displays the features of the fractured faces of the cell membranes consisting of E face and P facet, which possess different components characteristic in each case of the junction under consideration. (iv) Some idea of the permeability of the junctional structures may be gained from in vivo incubation of the tissue in physiological saline containing tracers. Each of these techniques will now be considered in slightly more detail, because the first three, at least, are essential in order to categorize junctions correctly. * Since this review was submitted, a review concerned with septate and scalariform junctions in arthropods has appeared, Noirot-Timothbe and Noirot, 1979 t The terminology of Branton et al. (1975) has been used in this review whereby the fracture face overlying the extracellular space is designated by EF, and that adhering to the cytoplasm, PF (see Fig. 1)

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NANCY J. LANE AND HELEN leB. SKAER CYTOPLASM

-/

/-

E FACE

-1

Fig. 1 Diagram to illustrate the way the membranes of two apposed insect cells appear after freeze-fracturing and after lanthanum-incubation. Freeze-fracturing reveals intramembranous substructure, in that the membranes cleave along the mid-line producing an outer membrane half or E face (as shown in the top membrane), and an inner membrane half or P face (as shown in the bottom membrane). Only one of the two fracture faces is revealed in any given area of replica but the plane of cleavage may pass from the PF (or EF) of one cell to the EF (or PF) of the adjacent cell. Since the fracture plane then passes through the intercellular space, some estimate of the size of the cleft as well as the degree of complementarity of intramembranous structures on the two faces can he made. Lanthanum, which infiltrates the space (*) between membranes, will reveal features of this extracellular space only but illuminates very well any structure that is present there, for example, septa or columns. A combination of these two techniques will reveal most features of junctional modifications

1.1.1 Revealing intercellular dimensions b y en bloc U A staining Staining en bloc with uranyl acetate (Farquhar and Palade, 1963) clarifies the membrane image and so allows a more precise assessment of the intercellular space in accurately cut transverse sections. This has been of particular importance in distinguishing gap junctions (2-3 nm intercellular space) from tight junctions (intercellular space occluded) (see Sections 4.2.1 and 5.2.1). 1.1.2

Revealing intercellular structures b y “negative” staining

In order to reveal the fine structural details of the intercellular space and the junctional modifications therein, staining of the extracellular cleft matrix with heavy metals is frequently employed, so that the components of the junctions will be, in effect, “negatively” stained. The junctional elements remain unstained and electron-lucent, but their features, arrangement and distribution are highlighted by the electron density of the stained matrix background in which they lie.

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This technique was originally employed using colloidal lanthanum nitrate as the negative stain (Revel and Karnovsky, 1967; Goodenough and Revel, 1970). However, any molecule which will bind to the often highly-charged matrix is suitable, and substances such as tannic acid (van Deurs, 1975) and ruthenium red (Luft, 1971), have also been employed. In addition, when material is treated with fixatives including calcium, subsequent treatment with uranyl acetate in the presence of calcium leads to images comparable to lanthanum-treated ones, as the uranium fills the extracellular space, delineating the structures that lie therein. Whether the uranium replaces calcium or binds to a mordant formed by the calcium in the extracellular matrix, is not yet clear, but this method may be referred to as “uranium calcium en bloc staining” (Wallet al., 1975), in contrast to the more conventional “uranyl acetate en bloc staining” (Farquhar and Palade, 1965) for enhancing membrane structure as referred to earlier. Depending on the plane of section, the stained intercellular cleft will reveal the non-opaque intercellular structures either along the longitudinal axis or transverse axis, while tangential sections elucidate the threedimensional structure or interrelationships of these junctional components. Such methods often clarify the detailed features of the junction under consideration because exclusion of tracer by a structure usually gives a clearer impression of its characteristics than positive staining, which cannot distinguish structure from background as easily or effectively. This method, when applied to a junctional region in conjunction with freeze-fracturing, enables the investigator to work out both intercellular and intramembranous structures and at the same time to determine in what ways such structures may be coincident and hence how they might be physically interrelated. From such evidence, three-dimensional models of junctions can be constructed. Without applying both negative-staining by tracers and freeze-fracturing to any given junction, it is not possible to discover all the intricacies of its structure. 1.1.3 Revealing intramembranous structures by freeze-fracture Freeze-fracturing (or freeze-cleaving) is a technique which enables the investigator to see an en face view of membranes in a single unique fracture plane, and hence to observe the relative size and position of any intramembranous components that may be present. Earlier investigations demonstrated that the plane of cleavage passes through the interior of membranes, not along the surface (Branton, 1966; Pinto da Silva and Branton, 1970; Tillack and Marchesi, 1970). Such a procedure exposes two fracture faces which were originally referred to as A and B but are now called the P and E face respectively (Branton et al., 1975). The P face refers

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to the cytoplasmic half, and the E face to the extracellular half, of the membrane (Fig. 1). The true external surface of membranes can only be revealed by etching, or sublimation of the surrounding ice. When a membrane is fractured, two complementary surfaces are revealed (Fig. 2) so that for every particle seen on, for example, the P face, a

Fig. 2 Freeze-fracture replica from an insect transporting epithelium (Culliphoru salivary gland) to demonstrate the characteristic appearance of the two fracture faces of the cell membranes. The P face (PF) possesses many intramembranous particles (IMPs) which vary in size and have an irregular distribution. The E face (EF) has fewer IMPs which lie scattered through the lipid layer; this interpretation assumes that the membrane structure conforms to the fluid mosaic model formulated by Singer and Nicholson (1972). x 81 300

corresponding pit or depression is to be found on the E face. These pits are often less obvious than the intramembranous particles (IMP), for the angle of shadowing may be too shallow to reveal their presence or they may in other ways become obscured. The smooth areas of the membrane faces are assumed to be the central hydrophobic part of the lipid bilayer, while the IMPs are generally considered to be intercalated protein of some kind (Branton and Deamer, 1972; Pinto da Silvaet al., 1971), possibly enzymatic, structural or antigenic. Recent evidence, however, indicates that lipid by itself can form IMPs and that such particles exhibit complementarity; these may represent inverted micelles of phospholipid (Verkleij et al., 1979). It

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seems clear, therefore, that care must be taken in the interpretation of the nature of any IMPs. Frequently, the P face of membranes possesses a n abundance of IMPs while the E face is less well endowed; this occurs because the fracture goes along the path of least resistance and particles remain attached t o the PF because they a r e thought to be more firmly bonded t o that face in t h e frozen state (Bullivant, 1974). T h e existence of particles within the plane of the membrane is consistent with the fluid mosaic model of biological membrane structure (Singer and Nicolson, 1972). When intercellular junctional membranes a r e cleaved, it must b e remembered that when o n e sees a P face and E face together, the P face will belong to the cell underneath, and the E face to the cell overlying it, so that the space between these particular two faces represents the intercellular cleft (Figs 1 and 2). This enables o n e t o see any reduction o r expansion in this extracellular space. Hence, when o n e structure o n t h e P F is seen, the comparable structure in the EF is said to be complementary, but it is not in fact in the other half of the same membrane. Complementary replicas a r e required t o see both halves of the same membrane. However, with junctions, the structures within the membranes on either side of the extracellular space are usually symmetrical a n d so the term complementary is still used in these circumstances, and will be throughout this review. For the uninitiated, it is worth noting that with freeze-fracturing, the observer is looking at a replicu of the fractured material. T h e tissue is removed by chemical agents following shadowing and carbon-backing the fractured surface, and the thin metallic replica can then be viewed by transmission electron microscopy. T h e micrographs of freeze-fracture replicas are by convention mounted with the metallic shadow coming from the bottom o r side, a n d all the freeze-fracture illustrations used in this review are mounted in this way. 1.1.4 Permeability studies by in vivo incubation with tracers The presence and distribution of certain kinds of junctions can be determined by the demonstration of a restriction to free permeability. Ions and molecules of various sizes can h e employed as in vivo tracers in fine structural studies t o reveal the sites of any such diffusion barriers. Ionic lanthanum chloride is often used (Machen, et al., 1972; Lane, 1972; Lane et al., 1975a, 1977a) because it is relatively small a n d the hydrated lanthanum ion is 0.92 nm in diameter (Robinson a n d Stokes, 1970); however, since it carries a high positive charge there is a tendency t o associate with structures that are negatively-charged a n d care must therefore be taken in interpreting results. In studies on permeability, however, it is important t o carry out these physiological in vivo incubationsprior to fixation, since fixatives may modify the fine structure of diffusion barriers. I n vivo ionic lanthanum incubations

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prior to fixation may, therefore, yield different information from the study of tissue fixed in the presence of colloidal lanthanum. Incubation in vivo with solutions of exogenous enzymatic proteins such as microperoxidase (M. W. 1900) (Feder, 1970) and horseradish peroxidase (HRP) (M.W. 40000) (Brightman and Reese, 1969) has shown these substances to be useful tracer molecules but, being macromolecular, their usefulness in determining the accessibility of very small openings or spaces is limited. Caution must also be exercised with respect to dosage and enzymatic activity since these tracers can produce permeability changes at high concentration (see Lewis and Knight, 1977). The movement of ions and molecules through extracellular channels gives an indication of the extent to which they can freely diffuse before junctions interfere with their entry; however, when barriers are demonstrated, they restrict the movement of that particular tracer, and perhaps not necessarily of other molecules of a smaller size or different chemical structure. In this respect, HRP and microperoxidase can be used to give an indication as to whether or not a diffusion barrier exists, and hence whether junctional complexes are likely to be present, forming its morphological basis. A smaller molecular weight molecule such as lanthanum, however, is likely to give more information about the subtleties of the junctional distribution, revealing at greater resolution the finer structural details.

Throughout this review, representative citations are given for the various points raised. In some especially crucial cases, all the known supportive reports will be listed, but ordinarily a range of examples will be presented in order to give the reader an idea of the scope of the papers that touch upon that particular issue. The reasons for this of course are obvious, since in many instances there have been innumerable passing references made to junctions without any further details as to their structure or function. There is one aspect of junctional elaboration that we do not intend to consider in this article. These are the specialized structures developed where three junction-bearing membranes become connected, coined tricellular junctions. Distinctive junctional structures are found in these areas and have not always been recognized as relating to tricellular associations but have been described as novel elaborations in the established structures of various junctional types, These misinterpretations as well as a detailed consideration of arthropod tricellular junctions of the septate and scalariform types has been included in a recent review (Noirot-TimothCe and Noirot, 1979).

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2.1

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Septate junctions INTRODUCTION

Septate junctions, so called because of their ladder-like appearance in transverse section, were first described as septate desmosomes in Hydra by Wood (1959) although Fernhndez-Moran (1958) had previously shown their presence in the insect eye but identified them as “ultratracheoles”. Subsequently junctions having closely similar, though not necessarily identical, characteristics have been described very widely in many invertebrate phyla (for example, Overton, 1963; Wiener et af., 1964; Locke, 1965; Gouranton, 1967; Messier and Sandborn, 1967; Noirot and NoirotTimothCe, 1967; Bullivant and Loewenstein, 1968; Danilova et af., 1969; Gilula et a f . , 1970; Leik and Kelley, 1970; Hand and Gobal, 1972; Flower 1972; Noirot-TimothCe and Noirot 1973; Satir and Gilula, 1973; Flower and Filshie, 1975; Dallai, 1975, 1976; Filshie and Flower, 1977; Wood, 1977; Noirot-TimothCe et al., 1978; Lane and Harrison, 1978; Skaer et af., 1979). Reports of septate junctions in vertebrate tissues (for example, Bargmann and Linder, 1964; Laatsch and Cowan, 1966; Peters, 1966; Hirano and Danbitzer, 1967; 1969; Barros and Franklin, 1968; Lasansky, 1969,1971; Gobel, 1971; Friend and Gilula, 1972; SoteloandLlinBs, 1972; Enders, 1973; Livingston et a f . , 1973; Schwartz, 1973; Aetorfer and Hedinger, 1975; Connell, 1978; Nistal et af., 1978a,b) bear only superficial resemblances to the invertebrate junctions and may in most cases be considered as separate, unrelated junctions (for further discussion see Section 2.6) Berridge and Oschman (1969) and Danilova et af. (1969) independently suggested that septate junctions in invertebrate species could be subdivided into more than one type on the basis of their morphology. Danilova et af. (1969) specified two types, the true “septate desmosome”, found in Hydra, and the “comb desmosome” found in insects. Furthermore they speculated that some other modifications of the septate junction might be found and indeed shortly afterwards Leik and Kelly (1970) carried this subdivision a step further by suggesting a phylogenetic progression from junctions with parallel shelves of septa (hydroids and flatworms) to pleated shelves (leeches and bivalves) to hexagonal or honeycomb arrangements (insect tissues). Flower and Filshie (1975) subsequently suggested that a junction, relatively newly characterized in insects and previously described as a “continuous” junction (Noirot and Noirot-TimothCe, 1967, 1972; Dallai, 1970; Oschman and Wall, 1972; Satir and Fong, 1972; Reinhardt and Hecker, 1973; Noirot-TimothCe and Noirot, 1974), should in fact be recognized as a further variant of the septate type. This junction they called the smooth

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septate junction to contrast with the previously established honeycomb septate junction (which they termed “pleated” septate junction). The distinction between these two types depends on clearly distinct morphological characteristics rather than phylogenetic occurrence. Green (1 978), in a brief report, enumerated eight morphological types of septate junction which he clamed to be phylogenetically distinct. Green’s published evidence is solely from tissues infiltrated with lanthanum but corroborates previous findings of other workers (e.g. Baskin, 1976). Further evidence (in press) from thin-sections and freeze-fracture (Green el al., 1979a; Green and Flower, 1980) should, when published, shed light on this proliferation of septate junctional types. Until recently, only two types of true septate junction have been described in insect tissues, the original “comb desmosome” or pleated septate junction, as it is now normally called, and the continuous or smooth septate junction. (For a discussion of other variants of septate-like junctions found in insect tissues see Sections 7 and 8). 2.2

P L E A T E D S E P T A T E JLINCTION - S T R U C T U R A L FEATURES

2.2.1 Thin-section appearance In thin section (Figs 3 and 6, insert) pleated septate junctions can be distinguished by the regular intercellular spacing of the membranes which are fairly rigidly separated by 15 nm. The intercellular space is punctuated by septa of uniform thickness (normally 8-9 nm, although Caveney and Podgorski (1975) reported septa only 2-3 nm wide in Tenebrio epidermis) which in some areas may show a periodic separation (the precise spacing Fig. 3 Pleated septate junction from the tracheole investment of the eye of the locust, Schistocerca gregaria showing the regular interseptal distance and prominent septa typical of this junction in thin-section. The circular insert shows an enlargement allowing resolution of the septal substructure (arrow) and a darker spot traversing the membrane at the point of contact with the septum (arrowheads). Insert (lower left-hand corner) shows a septate-like junction that is found between axons in the central nervo,ussystem of the housefly, Musca domestica; this displays certain differences from the conventional pleated septate junction in that the intercellular space is narrower, the septa are of different dimensions, and the junction is limited in distribution, presumably to a macular area between the two apposingaxon surfaces X 158 300; circular insert x 239 100; inset x 142 700 Fig. 4 Thin-section passing obliquely through the pleated septate junctions of the epidermis of Rhodnius prolixus. The honeycomb appearance deriving from the juxtaposition of the pleated septa is clear (see text for further explanation of this phehomenon). The arrow indicates the one region of transversely-sectioned septate junction that is present, straddled by the areas of oblique-section, cutting through the intercellular septal sheets. X 50 600 Fig. 5 Pleated septate junction from the testis of the cockroach, Periplaneta americana. The preparation was fixed in the presence of lanthanum hydroxide which has permeated the intercellular space, revealing the septa in negative contrast. Note the variation in thickness of the septa as the angle of section relative to the orientation of the septum alters. x 164 300

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NANCY J. LANE AND HELEN leB. SKAER

46

appears to vary with species but is in the region of 16-20 nm; NoirotTimothee and Noirot, 1973, reported it to be as low as 9 nm) or which may show a considerable variation in the interseptal distance. Indeed the degree of order in the spacing of the septa can vary considerably. Some of this variation is due to the angle of sectioning relative to the regular undulations of the septa (as explained in the original description, see Danilova et al., 1969, Figure 10; see also Flower and Filshie, 1975, Figure l ) , but even bearing this in mind, the transverse sections of some septate junctions present a much more ordered appearance than others ( c j Figs 3 and 6 insert with insert to Fig. 7). The septate junctions that occur between insect nerve cells (Fig. 3 square insert) appear to be rather different from the normal epithelial ones. They seem likely to be macular rather than zonular, and their structure, for example that of septa, appears to be distinctive (see also Smith, 1967). The septa themselves appear at higher power to have some kind of subunit structure (Fig. 3, circular insert) such as has been suggested previously by Noirot-TimothC and Noirot (1973). They concluded that each septum had two pairs of side arms, projecting at right angles to the septum and on either side of it. This model is consistent with the image arrowed in Fig. 3 (circular insert). The enlarged area in the circular insert also illustrates that the membrane itself may be transversed by an electron dense spot at the point of contact between septum and membrane. Such images have also been seen by Reinhardt and Hecker (1973) after staining with periodic acid-TCH silver proteinate (Thiery, 1967). Fortuitous sections passing tangentially through the junctional membrane sometimes reveal details of the septa in en face view (Fig. 4). It was sections such as these that allowed the original distinction to be made between the insect septate junctions and those of Hydra (Danilova et al., 1969). The septa present an overall honeycomb arrangement but close inspection reveals that individual septa follow a sinusoidal pattern and that it is the alignment of adjacent septa 180" out of phase with one another that produces the overall hexagonal appearance (Fig. 36, insert A) (see Gilula et d., 1970 for optical diffraction of the junction in en face view).

2.2.2

Lanthanum infiltration

After infiltration with lanthanum salts the intercellular space appears electron dense and solid structures in the intercellular cleft appear electron Fig. 6 Freeze-fracture preparation from the testis of Periplanetu americanu to show the rows of particles on the P face (PF) with corresponding pits on the E face (EF). The rows of particles and grooves are fairly regularly spaced in some areas and the junction shows a relatively high degree of order. The insert shows the thin section appearance of a similarly ordered junction from tracheole cells in the tissues of the eye of the locust. X 94 100; insert X 127 000

INTERCELLULAR JUNCTIONS I N INSECT TISSUES

47

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SKAER

lucent. The intercellul:ir s p i c e in the ;ire;i\ ot the junction. in transversc section. is \ l i s h t l y larger ( c t r . I 7 n m ) than in convcnticnally \t;iinecl preparations d u e t o optical f u \ i o n o f the electrov-dense intercellular pace with the osmiophilic outer nienihrane leiitlets on either aide o f it. I he w p t a show up a s electron-lucent b a r s often nar-I-owcr- (4-5 n m ; Satir a n d (iilula. 1973) than i n nci n - in ti It r;i t ecl \ peci ni e n \ . In t;ingenti;il section\ t n c septa are \trikinglj reve;iled ;I\ clear undulating rihtmns set in ; i n electron-de:iw lake ot \tain (Fig. 5 ) . 'l'hc ril,lx)n\ :'un together in more 01- les\ pi-:ilIel t>;ind\ with occ;ision;il iii-cas w h e r e the tight n e \ 4 o f pa c I\ ing 1 ()o\c n \ ;I n d in d iv id 11;I I \c p t ;I ;I cc()ni p I is ti w h (1I- I \ o I- I o o ps alone ( w e . f o r example. illu\ti-ations in N o i r o t - ' l ' ~ n ~ o t h i .acn d Ncirot, 1953). .[his paper ( N o i r o t - I in1othi.e and N o i l - o t . 1073) ;i!so reve;il\ that there i s con\idcral,le \ a r i ; i t r o n in the thickne\\ o f t h e w p t a a n d t h ; i t an individual septum inah var! in thicknes4 a l o n g i t \ Icngth ( f r o n i 3- 1 1 rim. see their Plate IV: \ec al\o Figure 5). T h i \ i\ p i - o t ~ ~ due i l ~ to ~ ~the prcciw angle of wetion w i t h re\pcct to the \epta thc~ni\cl\.c~\. (C'a\cney a n d Podgo:-\ki. 1075) h i i t might. ;I\ Noir-~,t-.l'iriic)thi.ei t n d Noirot \ugge\t. indic;ite ;I v ; i r i i i t i o i i in the width o f i h c \eptum :icro\\ the intcrcellular \piice. .l'he regularity o f the sinii\oid;il u n d u l a t i o n \ of the wpta a r e elearl! w e n after ianthanum infiltrat i o n (Fig. 5 ) : the periodicit! o f the w ; i ~ eha\ twcn reported b y various author4 ; I \ 2 0 - 7 2 n n i ( ( i i l u l a c'i t i / . , 1 9 7 0 ; Noirot-~l'iniottiiie a n d K o i r o t . 197.3: StacLhelin. lO7-4: Noii-ot-.l'iriiottii.c('I t i / , , 1978). 1n ce rt ;Ii n pre p;i i-:it io n \. \c c t io 114 ( ) f I ;I 11t h ;I 11 I I ni -in fi I t ir:it cd ni ;i t e ria I \how sidearms ar-i\ing f r o n i the ci-c\t of tach w a c~(Noirot-~!im~)tlii.e a n d Noirot. 1073; (ireen 1978): i i i \ome c;ise\ the \ide a r m s f r o n i :!dJ:iccnt wpta ,join t o form pattern\ o f complete I:c\agon\ ((ireen. 1978; c/'. ( i i I ~ i l ; i ('I t i / . , 1970). Even without \.i\il,lc \idcar-m\. ;idlacent i-itition\ 180" o u t o f phase with one another give ri\c to the appearance iiescrihed ;I\ honeyconit, b y Loeke (1975) a n d I ) : i n i l o \ ; i ( ' I t r / . ( 1 % 0 ) (Fig, 3 a n d inw1-t A in Fig. 3 6 ) . This honelcomt-,appcai-ancc ill lie enha:iccti if the adjacen! w p t a . r u n n i n g 1x0" out o f ph;i\e. ;II-C clow together and the p;itter:i M i l l he tiirthci- I-cinforccd where the :implit uclc of the \cptiil \v:i\ e i \ regular ;ind o f con\idei-:il~lc:.izc. The rihhons riiay h o \ \ c \ c r aI\o irun together in ph;iw (Figure IV o f N o i r o t Tiniothiie and Noirot. 107.3) 01-cornplctel! o u t o f register. 'l'hii\. in t h i \ I-espect ; i l l the model\ t h ; i t ha\.c heen pi-opo\ed ( 1 3 i i l l i \ , a n t a i ~ d1,oeweristcin. 1968; (iilula ci t i / , , 1970;Yoi:-ot-.l'imotiiiic and Noirot. 197.;; Staehelin. 1073; C'a\cncy a n d Podgoi-\ki. 1075) ( a n d t l i i i t pi-opo\cci i n thi\ revie\\. Figure 10). reprewnt ideali/ed gencr;ili/;ition\ o f ;I \truetiire e\hihiting considcral>lc \ iiri:ition.

INTERCELLULAR JUNCTIONS IN INSECT TISSUES

49

2.2.3 Freeze-fracture Freeze-fracture of septate junctions was first carried out on Mollusc tissues by Flower (1970, 1971); Gilula et al. (1970) and Gilula and Satir (1971). The replicas revealed characteristic modifications of the membranes associated with the septate junctions, ranks of more or less parallel rows of particles were found on the PF, corresponding rows of depressions or pits occurring on the E F (as in Figs 6 and 36). Their results have subsequently been confirmed and extended t o the septate junctions of insects (e.g. Flower, 1971; Satir and Fong, 1972; Noirot-Timothee and Noirot, 1973; Satir and Gilula, 1973; Dallai et al., 1977; Noirot-TimothCe et al., 1978). The particles are normally 8.5 nm in diameter (Noirot-TimothCe et al. (1978) give a range of 7-10 nm) and show a somewhat variable separation (although Gilula et al., 1970, examining mollusc tissues, claim a regular centre-tocentre spacing of 21 nm confirmed by optical diffraction). A recent report by Noirot-Timothkeetal., (1978), in which a number of species are considered, gives variations as large as 9.5-20 nm. The variation in spacing between adjacent rows of particles and the degree to which they run parallel to one another is also variable (see Figs 7 and 8); Flower (1970, 1971) g'ives a minimum figure of between 13 and 1 7 nm, and Noirot-TimothCe et al. (1978) a separation of 16 to 20 nm, when the rows are running parallel. Few junctional particles are found on the E F of the junction (Figs 6 and 36) although complementarity of the P F particle rows and the E F depressions can be seen where the fracture passes through the intercellular space and the two membranes of the junction are revealed in adjacent areas (Figs 6 and 36). Chemical fixation has a marked effect on the fracturing characteristics of smooth septate junctions in some tissues (see Section 2.3.3) but this has not been reported for pleated septate junctions. In the latter, the particles fracture into the P F and the depressions appear on the EF whether the tissue has been fixed prior to glycerination and freezing (e.g. Gilulaetal., 1970) or not (e.g. Flower, 1970). The degree of order displayed by the bands of particle rows shows considerable variation both within a single junction but also and to a greater extent between the junctions of different tissues or from different regions of the same tissue. This is illustrated in Figs 6, 7 and 9, which are freeze-fracture preparations from the testis of the locust and the cockroach. Thin sections reveal similar variations in septa1 organization (as shown in Fig. 3 and insert to Fig. 6, in comparison with the insert to Fig. 7). In the testis, septate junctions between cyst cells that envelope the groups of developing germ cells are very loosely organized, with rows of particles that are widely separated and show no indication of lining up in parallel (Fig. 7). In the peripheral layers of cells ensheathing each follicle (the follicle cell layers),

50

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.

0

.

INTERCELLULAR JUNCTIONS I N INSECT TISSUES

51

the septate junctions display considerably more complex arrays of particle rows (Figs 6 and 9). Junctions showing an intermediate organization are found where the cyst cell layers merge into the follicle cell layers. A septate junction exhibiting a very loose organization and no E F elaborations in freeze-fracture replicas has recently been described in the rectal sheath cells of termites and cockroaches (Noirot et ul., 1979). Smooth septate junctions are known to show topographical variations in the degree of septa1 and freeze-fracture particle (ridge) organization (Flower and Filshie, 1975; Graf, 1978a; Skaer et ul., 1979; Noirot-TimothCe and Noirot, 1979; Juperthie-Jupeau, 1979). As yet this has not been described for pleated septate junctions although variations such as those illustrated in Figs 7-9 might well be topographically related. There do, however, appear to be genuine tissue differences. Only highly organized junctions are encountered, in our experience, in salivary gland, certain regions of the gut, follicle cell layers of the testis and between epidermal cells. Loosely organized junctions (or those with an intermediate organization) on the other hand, are frequently found in the ensheathing cells within the testis, between the perineurial cells of the central nervous system and in the eye (Skaer, 1979a). 2.2.4

Structural model

Various models have been put forward since septate junctions were first described in insect tissues by Locke (1961). Locke (1965) proposed a possible pattern of orientation of the lipid component of the membrane within the junctions, a facet that does not seem to have been explored further. The other models proposed (Wood, 1959; Bullivant and Loewenstein, 1968; Gilula et al., 1970; Flower 1971; Satir and Gilula, 1973; Noirot-Timothte and Noirot, 1973; Staehelin, 1974; Caveney and Podgorski, 1975) have been concerned with the arrangement of the septa within the intercellular space and the relationship between the septa and the rows of particles seen in freeze-fracture. Common to all models are the septa, either pleated or in honeycomb arrays, traversing the intercellular cleft Fig. 7 Freeze-fracture preparation of the testis of Schistocerca gregaria showing the PF of the loosely organized pleated septate junction, characteristic of the cyst cell layer. Insert shows the thin-section appearance of this type of loose septate junction; the septa are infrequent, irregular and may not lie perpendicular to the junctional membranes. X 47 900; insert X 121 400 Fig. 8 Pleated septate junction from the rectum of Periplaneta americana. The PF particle rows form a slightly more coherent pattern than was illustrated in Fig. 7, joining to run in parallel rows over some distance. However large areas of non-junctional membrane lie between many of the particle rows. x 66 800 Fig. 9 PF of the pleated septate junction found in the inner follicle cell layer of the testis of the locust Schbtocerca gregaria. The particles line up to form broad bands of parallel rows. This junction of intermediate organization should be compared with the more highly ordered type illustrated in Fig. 6. x 60 400

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NANCY J. LANE A N D HELEN leB. SKAER

perpendicular to the cell membranes on either side. Two areas of controversy remain, firstly the degree to which the septa are inserted into the cell membranes of the junction, prophetically illustrated by Wood (1959, Figure l ) , and secondly the precise structure of the septa themselves. Authors vary in the degree to which they suppose that the septa and the cell membranes are interconnected. Wood (1959), Locke (1965) Gouranton (1967) and Flower (1971) suggested that the septa were not separated from the cell membranes at all but represented out-pushings from the adjacent cell membranes. Gouranton (1967) and Flower (1971) proposed that the septa are made up of two halves, one from each junctional membrane, which meet in the centre of the intercellular cleft. Gouranton (1967) supported this with micrographs of impressive clarity. Flower (1971) furthermore suggested that a dilation occurs in the septum whenever an intramembrane particle was present. If these dilations were offset, the septal undulations could be explained. Bullivant and Loewenstein (1968) and Gilulaet al. (1970), in papers favouring the septate junction as the structure underlying a direct route of intercellular communication, also proposed that the septa were continuous with the membranes. The route of communication proposed in each case was slightly different: Bullivant and Loewenstein (1968) suggested that the septa were arranged to enclose hexagons of high permeability membrane, sealed by the continuity of septa with membrane. O n the other hand. Gilula et al. (1970) on the basis of “negatively stained” oblique sections and freeze-fracturing, arranged the septa in their model as undulating ribbons of regular periodicity and with adjacent septa 180”out of phase, so creating the honeycomb pattern. This latter model forms the basis of subsequent ones (e.g. Staehelin, 1974; Flower and Filshie, 1975). However, Gilula and co-workers (1970) proposed that the septa insert into the membranes at alternate vertices of the pleats, the intramembrane structures responsible for this being the particles revealed by freeze-fracture. The continuity of particle-septum-particle they argued was the route of intercellular communication. Since septate junctions have been superceded by gap junctions as the structures underlying communication (reviewed by Bennett, 1978 and see Section 4.6.3), authors have exercised considerably more caution in assuming that the septa insert into the membrane (as in NoirotTimothCe and Noirot, 1973; Flower and Filshie, 1975; Caveney and Podgorski, 1975). Recently, in a study of several different insect tissues, Noirot-TimothCe et af. (1978) have shown that there is poor correlation between the periodicity of the septal undulations and the separation of the freeze-fracture particles within the rows. From this they conclude that there can be “no structural continuity throughout the thickness of the septate junction”. Their evidence does not show this but does indicate that insertion of the septa at regular intervals along the undulations (as proposed by Gilula

INTERCELLULAR JUNCTIONS I N INSECT TISSUES

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etal., 1970; Satir and Gilula, 1973; Satir and Fong, 1973; Staehelin, 1974) is unlikely. Noirot-TimothCe et al. (1978), as well as evidence presented here, show that there is a clear correlation between the topography of particle rows and septal bands and moreover that both fuse to give double structures and end abruptly. It therefore seems plausible to admit some relationship between the two structures (Fig. lo), although the exact nature of their association must await further elucidation. /-CYTOPLASM-/

'-'

FACE -'LANTHANUM IN INTERCELLULAR

Fig. 10 Model of the pleated septate junction to illustrate the way in which the images of the junction obtained by the different methods discussed may be united into a three-dimensional structure. For purposes of simplicity, controversial aspects such as the substructure of the septa and the precise relationship between them and the intramembrane particle rows have not been illustrated although we have assumed that the septa are attached to the membranes at points coincidental with the rows of particles seen in freeze-fracture. The ladder-like appearance is produced by transverse sections through the junction, as in Fig. 3 and insert in Fig. 6 . The E and P face correspond to freeze-fracture images as in Figs 6-9 and the lanthanum-stained intercellular space corresponds to the undulating ribbons seen in Fig. 5

The early models of insect septate junctions assumed the septa to be solid (Bullivant and Loewenstein, 1968; Danilovaet al., 1969; Gilulaetal., 1970) but, on the basis of heterogeneity observed in the intercellular space following lanthanum infiltration, various modifications have been suggested. Indeed the septa may be made up of morphologically distinguishable subunits as shown in Fig. 3 (circular insert). Staehelin (1974) suggested, on the basis of evidence published by Noirot-TimothCe and Noirot (1973), that the septa may consist of thin bands of material, limited to the central regions of the intercellular space, and slung between rows of thin pegs, whose insertion into the membrane is marked by the particles seen in freeze-fracture preparations (see Figure 59 in Staehelin, 1974). An open septal substructure would explain the permeability of the junction to tracers such as lanthanum salts but the evidence for anything other than solid septa is not yet convincing and the junctional permeability to tracers can be explained in terms of

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the frequent breaks in septal continuity (see for example Noirot-Timothke et al., 1978). Caveney and Podgorski (1975) have extended this idea in proposing structures at right angles to the septa. They suggested that the intercellular space is bridged by narrow septal ribbons (3 nm wide) but that these septal ribbons are connected by two platforms, about 1.5 nm in thickness, which lie in the central region of the intercellular space and are coplanar with the plasma membranes (see Figure 1 5 in Caveney and Podgorski, 1975). The chemical nature of the septa has been investigated in a number of systems. Wood, 1959, Weineret al., 1964, Locke, 1965, Gouranton, 1967, Gilula et al., 1970 and Flower, 1971 suggested that they are an extension of the cell membrane. Noirot-TimothCe and Noirot (1973) on the basis of cytochemical studies suggest that they contain glycoprotein but probably lack lipid. Caveney and Podgorski (1975), consider that both the septa and the interseptal platform, which they suggest link adjacent septa, are formed by elaborations of the glycocalyx. However in the smooth septate junctions of the mosquito Aedes, the septa appear to lack polysaccharides (Reinhardt and Hecker, 1973) unlike those of the pleated septate junction (NoirotTimothCe and Noirot, 1973). Recent observations (Humbert, 1979) on the smooth septate junctions of a collembolan species, demonstrate the glycoprotein nature of the intercellular elaborations. The intercellular material has not been specifically characterized. Gilula et al. (1970) found that the intercellular matrix of a Mollusc pleated junction stained with ruthenium red, indicating the presence of acid mucopolysaccharide. Dallai (1970) demonstrated the presence of glycoproteins in the interseptal space of the smooth septate junction. Djaczenko and CalendaCimmino (1974) report that the “extracellular substance” of septate junctions from the epidermis of a variety of annelid species can be identified cytochemically as predominantly polysaccharide in nature. In view of the possible significance of this extracellular matrix (see Section 2.7) further studies concerning its nature and variation in junctions from different tissues would be of interest.

2.3 2.3.1

SMOOTH SEPTATE J U N C T I O N - STRUCTURAL FEATURES

Thin-section appearance

Continuous or smooth septate junctions are characterized in thin sections both by the regularity of the intercellular separation (14-17 nm wide) and, in general by the uniform electron opacity of the intercellular space (Fig. 11). However in some instances septa can be made out, either indistinctly (e.g. Noirot-Timothke and Noirot, 1967; Hudspeth and Revel, 1971; Dal-

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Fig. 11 Thin-section appearance of the smooth septate junction from the apical part of the lateral border from the midgut of the housefly, Muscu dornesticu. The regularity of the intercellular space and the featureless electron opaque intercellular material are clear. At the apical extremity of the junction, just below the microvilli (MV), the junction is revealed cut tangentially (arrow) and the presence of fairly straight septa can be resolved. X 65400 Fig. 12 Sub-apical area of the smooth septate junction from Muscu domesticu midgut. This thin section includes an area exhibiting no clear septa and also a finger-like interdigitation cut transversely to reveal a ring of junction indicating clearly the septa1 nature of the intercellular structures. X 106400 Fig. 13 A preparation infiltrated with lanthanum hydroxide duringfixation from the midgut of the horseshoe crab, Limufus pofyphemus. The septa of the smooth septate junction are highlighted in negative contrast (as at arrows). x 77 400

lai, 1975; Reinhardt, 1975; Graf, 1978) or more clearly (see Fig. 12) (Flower and Filshie 1975; Skaer et al., 1979). Where septa are distinct it is difficult, on the basis of thin-sections alone, to distinguish smooth septate junctions from pleated septate junctions. Ancillary techniques such as infiltration of the extracellular spaces with stain (lanthanum salts or ruthenium red), special staining methods (e.g. the uranium calcium method described by Walletal., 1975) or freeze-fracture are necessary to classify the junction with certainty. As a result, various reports in the literature describe septate junctions that may well, on more comprehensive examination, prove to be smooth septate junctions (for example Messier and Sandborn, 1967; Bullivant and Loewenstein, 1968; Smithet al., 1969b; Hudspeth and Revel, 1971; de Priester, 1971; Herman and Preus, 1972; Johnson et aE., 1973;

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Reinhardt and Hecker, 1973; Reinhardt, 1974; Houk and Beck, 1975; Mills et al., 1976; Hecker, 1977). 2.3.2

Lanthanum infiltration

Transverse sections of the junction, in tissue infiltrated with lanthanum salts, reveal non uniformity in the slightly enlarged intercellular space. Frequent clear bars which exclude the stain traverse the intercellular cleft (Figs 13 and 14). Oblique sections show that these striations or bars are due to septa which run, without the regular undulations familiar in insect pleated septate junctions (see Section 2.2.2), over considerable tracts of the cell surface (Figs 14-18). The septa are fairly uniform in diameter, 4-6 nm wide, (Satir and Gilula, 1973; Flower and Filshie, 1975; Dallai, 1975) or slightly larger, 7-8 nm, (Skaer et al., 1979). The exact width will vary with nonperpendicular alignment to the plane of the section (see for example Fig. 16). Variation in width is also produced by lateral fusion of adjacent septa (thick arrow in Fig. 17). As with pleated septate junctions (Section 2.2.2), in Figs 14-19 All the preparations illustrated in this plate have been infiltrated with lanthanum hydroxide duringfixation and are concerned with the fine structure of smooth septate junctions Fig. 14 Preparation of Limulus polyphemus midgut where the plane of section has cut some parts of the smooth septate junction transversely and some obliquely. In oblique section the intercellular structures are more clearly delineated; relatively straight septa are separated by rows of electron-lucent particles, interpreted as sections through colums. Insert: at higher magnification the septa can themselves be seen to have a particulate substructure. x 140 000; insert x 295 000 Fig. 15 Part of the smooth septate junction from the Malpighian tubule OfRhodniusprolixus. The clear septa are separated by variable distances; some lie closely adjacent to each other forming doublets (thick arrows) and, where they are more widely separated, electron-lucent spots or colums in cross-section are evident. The thin arrows indicate blind-ending septa. X 259 700 Fig. 16 Apical region of the lateral border of the midgut of Musca domestica. The septa are closely packed with only occasional electron-lucent spots between adjacent septa. The junction can also be seen in transverse section, the intercellular space filled with lanthanum and giving only a suggestion of intercellular structure. The arrow indicates a blind-ended septum. X 163 500 Fig. 17 Sub-apical region of the lateral border of Musca domestica midgut. The septa are, for the most part, separated by one or more rows of electron-lucent spots, or transversely-sectioned columns. Septamay, however, fuse (thick arrow) andmay also terminate abruptly(white arrow). X 195 600 Fig. 18 Smooth septate junction from the midgut of Limulus polyphemus. The septa are separated in this region by double rows of electron-lucent particles arranged into a regular Maltese cross pattern. A septa1 termination is arrowed. In some cases septa run side by side in double rows (thick arrow). x 180200 Fig. 19 A slightly oblique section passing through the sub-apical region of the midgut of Musca domestica. The section is more nearly transverse at the top o f the figure where continuous thin clear lines can be made out (arrowheads). As the Dlane of the section deviates from the strictly transverse, the electron-lucent pegs or columns appear as spots or particles (arrow). x 237 800

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favourable sections, a subunit structure of the septa may be made out (insert in Fig. 14; Lane and Harrison 1978; Skaeret al., 1979). The septa appear to be made up of adjacent subunits which seem to be arranged in groups of 2 , 4 or 6. Adjacent septa may lie close together with only a thin line of stain separating them (2-3 nm interseptal distance) (Figs 15-18) or they may be separated by larger and more variable distances and, in this case, electron lucent spots (2-4 nm in diameter) are normally found in the interseptal spaces (Figs 14-18), although Dallai (1976) reports that in the Malpighian tubules of Periplanetu these transparent pegs are not encountered. In most other tissues studied, however, and in particular the midgut, they seem always to be present, albeit exhibiting variability in distribution and numbers. Where they do occur these spots may line up to form single (Figs 1 6 and 17) or double (Fig. 18) rows between adjacent septa. More commonly, however, the septa are more widely separated with variable numbers of electron lucent spots in the interseptal space (Figs 14 and 15). These spots are thought to represent columns that span the intercellular space as can be seen from sections in which they are cut obliquely (Fig. 19, arrow heads; see also Figure 5, Flower and Filshie, 1975, in which the columns are shown cut obliquely both perpendicular to the septa and in parallel with and between them). The septa run for considerable lengths over the cell surface but are not continuous structures; blind endings are fairly frequently encountered in oblique sections (arrows in Figs 15-18). 2.3.3 Freeze-fracture Freeze-fracture replicas of the smooth septate junction are typified by series of ridges on one membrane with complementary rows of grooves on the other (Figs 20 and 21). The location of the ridges is dependent in some tissues on the treatment prior to freezing. In fixed tissues, the ridges appear on the PF with grooves on the EF; the ridges are approximately 10 nm in width and appear to be made up of short rods (Fig. 20). The EF grooves are overlaid by a variable number of particles (see Flower and Filshie, 1975). In certain tissues a different pattern emerges if the material is frozen unfixed. The ridges, more clearly moniliform in nature (particles about 10 nm in Fig. 20 Freeze-fracture preparation of the fixed midgut of Musca domestica. The apical extremity of the border is identified by the terminal microvilli (MV) and the smooth septate junction can be followed down towards the basal part of the cells. Its ordered appearance loosens as it extends basally. The ridges on the P face (PF) are complemented by E face (EF) grooves. Insert: in this preparation the gut was not fixed before processing for freeze-fracture. In this case, in contrast with the fixed tissue, the more clearly moniliform ridges cleave with the EF and the complementary grooves are found on the PF. Arrows indicate ridge termination. x 22 500; Insert x 65 700

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diameter), are found on the EF, with grooves virtually free of overlying particles on the P F (insert in Fig. 20, and Fig. 21). The influence of fixation has been reported in midgut by several authors (Flower and Filshie, 1975; Dallai et al., 1977; Lane and Harrison, 1978; Skaer et al., 1979) and also in Malpighian tubule (Dallai. 1976; Skaer et al., 1979). However, particularly in the latter tissue, the fracturing characteristics in fixed material are very variable so that in some cases the PF ridges predominate and in other the EF grooves are so decorated with particles as to appear almost ridge-like (cf Figures 11 and 1 2 in Dallai, 1976). In our experience (Skaeret al., 1979), Malpighian tubules show little change in fracturing pattern with fixation. The particle rows are always found on the EF with clear grooves on the PF. There is considerable variation in the packing of adjacent ridges, correlating with findings of diverse patterns of septa1 spacing in oblique sections of lanthanum-infiltrated tissue. Further correlations are found in that ridges or rows of particles may fuse laterally to give double structures (arrows in Fig. 22) and they terminate abruptly (arrows in insert to Fig. 20). Freeze-fracture replicas allow analysis of topographical variations in the organization of the junction (see Fig. 20) in a way that is rarely possible with the relatively restricted regions revealed in oblique sections (see Figs 1 6 and 17 and also Giusti, 1976). In this way it has been possible to show that in the midgut of Musca and Rhodnius the junction is tightly organized apically and loosens as the junction extends basally (Fig. 20) (Skaeret al., 1979; Figure 22 in Lane, 1978a). Similar variations are found in lanthanum-infiltrated specimens in those regions whose position in the apical-basal axis of the border can be analysed. This topographical variation combined with parallel findings in freeze-fracture replicas and lanthanum-infiltrated preparations in terms of double rows (Figs 1 5 and 22) and terminating rows (Figs 15-1 8 and insert in Fig. 20), leads to the suggestion that there is a correlation between the freeze-fracture ridges or rows of particles and the intercellular septa (discussed further in Section 2.2.4). However no authors have reported freeze-fracture correlates of the electron-lucent columns of lanthanuminfiltrated preparations of smooth septate junctions.

Fig. 21 Freeze-fracturepreparationof the lateral border of the midgut of an unfixed specimen of the moth, Manduca sexta, bearing smooth septate junctions. The particle rows on the EF are complemented by clearly marked grooves on the PF. The highly convoluted nature of the lateral border can be deduced from the complex fracturing pattern of membranes. X 65 600 Fig. 22 Junction from the proventriculusof Rhodnius prolixus, prepared for freeze-fracture without prior fixation. The moniliform EF ridges sometimes run very close together to give a double structure (arrows) (compare with Figs 15 and 18). x 68 800

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62

2.3.4 Model derived from evidence Few models have been put forward to explain the structure of smooth septate junctions. Flower and Filshie (1975) proposed that the previously named continuous junctions were in fact a form of septate junction, and their model reflects this view. Figure 23 is largely derived from this model; /-CYTOPLASM

-. /

/-E

FACE-I

-

P FACE-\

Fig. 23 Model of a smooth septate junction to illustrate a three-dimensional structure built up from the images obtained by various preparative techniques. The freeze-fracture cleaving pattern illustrated is for unfixed tissues. An idealised situation is shown in which the septa are regularly spaced and separated by a single row of intercellular columns (stippling). Although the evidence is not unequivocal, the septa are shown as inserting into the membrane via the particles therein. The columns seem not to insert into any obvious intramembranous structure

the septa are unpleated and the points of septa1 contact with the membranes are marked by the freeze-fracture ridges or particle rows. However, Flower and Filshie (1975) are non-committal about the degree of correspondence between the intercellular septa and the intramembrane ridges (this point is discussed more fully in Section 2.2.4). The chief difference between the pleated and smooth septate junctions are the intercellular colums, which appear to have no freeze-fracture correlate and so are thought not to insert into the junctional membranes. 2.4

OCCURRENCE I N IXSECTS

The septate junction in insects is associated primarily with epithelial tissues, although not exclusively (as suggested with one exception by Caveney and Podgorski, 1975). In fact septate junctions are found in almost all insect tissues including the epidermis (e.g. Locke, 1965; Hagopian, 1970; Noirot and Noirot-Timothee, 1973; Noirot and Quennedy, 1974; Caveney and Podgorski, 1975; Lawrence and Green, 1975), epidermal glands (e.g. Stuart and Satir, 1968; Crossley and Waterhouse, 1969; Noirot and Quennedy, 1974), imaginal disc (e.g. Poodry and Schneiderman, 1970; van Ruiten and

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Sprey, 1974), partsof the gut (e.g. Satir and Fong, 1972,1973; Bode, 1977; Credland, 1978; Lane, 1978a,c, 1979; Noirot-Timothte et af., 1978; Skaer and Lane, 1979; Noirot et af., 1979), salivary gland (e.g. Bullivant and Loewenstein, 1968; Kendall, 1969; Burger and Uhrik, 1972; Gaudecker, 1972; Lane et af., 1972; Leslie and Robertson, 1973; Lauverjat, 1973; Skaer et af., 1975), Malpighian tubule (e.g. Bullivant and Loewenstein, 1968; Berridge and Oschman, 1969; Taylor, 1971; Noirot and NoirotTimothte, 1973; Walletaf., 1975; Dallai, 1976; Peacock and Anstee, 1977; Skaer et a f . , 1979), the gonads and associated structures (e.g. testis, Danilovaetaf., 1969; Szollozi and Marcaillou, 1977; Skaer and Jones, 1979; seminal vesicle, Cantacuzene, 1972; accessory collateral gland, Flower, 1972; sperrnotheca, Gupta and Smith, 1969; transiently in ovary, Mahowald, 1972; Lane, unpublished observations on locust and cockroach ovary), the nervous system (reviewed by Lane, 1974); between perineurial cells (e.g. Maddrell and Treherne, 1967; Schiirmann and Wecksler, 1969; Skaer and Lane, 1974; Lane et a f . ,1977a), glial cells (Osborne, 1975; Ribi, 1977), axons and glia (Smith, 1967) and nerve cells (Smith, 1967; Boschek, 1971; Corbikre-Tichant and Bermond, 1972; Sohal and Sharma, 1973), sense organs, for example the eye (e.g. Eley and Shelton, 1976; Shaw 1978; Chiet a f . ,1979; Ribi, 1979; Lane and Skaer, unpublished), antenna (Zachruk et al., 1971), and rnechanoreceptor (Chi and Carlson, 1976), myocardial tissue (Sanger and McCann, 1968), possibly between encapsulating haemocytes (e.g. Grimstone et af., 1967) and between cells of the tracheolar system (e.g. Gupta and Berridge, 1966; Smith, 1968; Laneetaf., 1977a). In view of their apparent ubiquity, perhaps of greater interest is their absence in a few instances. Skeletal muscle does not appear to have septate junctions, although they do occur between myocardial cells (Sanger and McCann, 1968; McCann, 1970). Septate junctions do not appear to have been found in fat body (Clarke, 1973). Moreover, while pleated septate junctions are found in the perineurium in the majority of insect species examined (reviewed by Lane, 1974); they are absent from this tissue in the moth, Manduca sexta (Lane, et al., 1977a; Lane and Swales, 1 9 7 9 ~ ) . Septate junctions have also been observed in in vitro conditions (Poodry and Schneiderman, 1970; Epstein and Gilula, 1977). These latter authors have identified septate and gap junctions between homologous cells in culture. Cells derived from a Homopteran line showed both junctions whereas those from a Lepidopteran line displayed only gap junctions. Pleated septate junctions are found between heterologous cells (Stuart and Satir, 1968; Kloetzel and Laufer, 1969; Satir and Gilula, 1970; Rose, 1971; Noirot and Quennedy, 1974; Flower and Filshie, 1975; NoirotTimothee et al., 1979) as well as more commonly between homologo~lscells. They may also be formed between processes of the same cell and this is

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particularly well illustrated by the mestracheon system (Smith, 1968; Poodry and Schneiderman, 1970) but occur in other cells as well (Maddrell and Treherne, 1967; Stuart and Satir, 1968; Osborne, 1972; Lane 1974; for further references see Woirot-TimothCe and Noirot, 1979). Smooth septate junctions have been reported only from Malpighian tubules (Dallai, 1976; Lacombe, 1976; Meyran, 1977; Skaer et al., 1979; Green et al., 1979b) and midgut (Noirot and Noirot-TimothCe, 1967,1972; Dallai, 1970, 1975; Andries, 1972; Oschman and Wall, 1972; Satir and Fong, 1972; Reinhardt and Hecker, 1973; Foldi, 1973; Noirot-Timothee and Noirot, 1974; Flower and Filshie, 1975; Ito et al., 1975; Burgos and Gutierriez, 1976; Lacombe, 1976; Houk, 1977; Graf, 1978; Lane, 1978a; Humbert, 1979; Skaer et al., 1979) although junctions closely resembling them, particularly in freeze-fracture appearance, have been found in the eye (Carlson and Chi, 1979). Houk and Beck (1975) claim to have found smooth septate junctions between perineurial cells, although their published micrographs are not at sufficiently high magnification to verify their assertion. Initially it was thought that smooth septate junctions were a substitute for pleated septate junctions (Noirot and Noirot-TimothCe, 1972; Satir and Gilula, 1973); thus pleated septate junctions were found in the posterior regions of the gut (Satir and Fong, 1973; Noirot and Noirot-Timothie, 1977; Noirot-TimothCe et al., 1978), while smooth septate junctions were found in the midgut (Noirot and Noirot-Timothee, 1967). More recently, however, smooth and pleated septate junctions have been found side by side on the lateral borders of Malpighian tubule (Dallai, 1976; Lacombe, 1976; Meyran, 1977; Skaer et al., 1979) although as early as 1969, Filshie is quoted as having found two types of septa in Malpighian tubule (Leik and Kelly, 1970). Pleated septate junctions have also been found in the midgut (Gouranton, 1967; Ito et al., 1975; Skaer, unpublished observations), in some cases coexisting with smooth septate junctions (Itoet al., 1975). Foldi (1973) also reports the coexistence of smooth and pleated septate junctions in the "filter chamber" of an Homopteran midgut. However the junctional identification in this case is based solely on thin-section evidence. Freezefracture and/or lanthanum infiltration is necessary t o confirm this finding. Smooth septate junctions, like pleated septate junctions, are found between heterologous as well as homologous cells (Flower and Filshie, 1975). However, to our knowledge, there do not appear to have been reports yet of smooth septate junctions forming between processes of the same cell.

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DISTRIBUTION IN OTHER INVERTEBRATES

Various types of septate junction have been described throughout almost all the invertebrate phyla (see Section 2.1). Pleated septate junctions have however been reported only in the chaetognathes (Duvert et a f . , 1978); platyhelminths (Storch and Welsch, 1977); nemertines (Vernet e f al., 1979); annelids (Coggeshall, 1966; Lasansky and Fuortes, 1969; White and Walther, 1969; Storch and Welsch, 1970, 1972; Boilly-Marer, 1972; Baskin, 1976; Bilbaut, 1980); molluscs (Gilula er al., 1970; Satir and Gilula 1970; Flower 1971 ; Newel1 and Skelding, 1973; Ryder and Bowen, 1977) and other classes of the arthropods in addition to the insects (Crustacea: Lang 1977; Shivers and Chauvin, 1977; Arachnida: Foelix et al., 1975; Lane, unpublished observations; Myriapoda: Juberthie-Jupeau, 1975). Although most authors agree that the septate junctions described in platyhelminths, annelids, molluscs and arthropods are pleated, various distinctions have been made. Baskin (1976) claims that the pleated septate junctions of polychaete epidermis shows unusual characteristics, reminiscent both of the smooth and the Hydra-type of septate junction. The lack of septa in some specimens, more characteristic of smooth septate junctions, might be explained by section angle. In some specimens Baskin found clear septa. An independent examination of the epidermis of another polychaete (Ficopofamus enigmatica) by thin-section, lanthanum staining and freezefracture, clearly reveals pleated junctions (Skaer, 1979b). The Hydra-like elaborations of the intercellular architecture are also described by Green (1978), who claims that these are common to all pleated junctions. Another freeze-fracture study of the epidermal septate junctions of two different annelid species (Welsch and Buchheim, 1977) emphasizes the fundamental similarities between annelid and arthropod septate junctions, while supporting Baskin’s (1976) view that annelid septate junctions may also exhibit distinctive characteristics of their own. Giusti (1976) finds pleated septate junctions in annetids and molluscs but claims that in the molluscs the junctions are characterized by tubular structures scattered randomly between and occasionally within the septa. These tubular structures have also been reported in a cephalopod mollusc by Boucaud-Camou (1978). White and Walther (1969) report similar structures in the photoreceptors of the leech. Pleated septate junctions have been found in the Onychophora with similar tubular structures, which however are not randomly arranged but assembled in clusters (Dallai et al., 1977). The significance of these tubular structures is not clear although Giusti (1976) suggests that they might be isolated gap junctional particles. Such structures are absent in the arthropod classes which show little deviation from the insect pleated septate junction.

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Smooth septate junctions have, for the most part, been found only in arthropods. Apart from insect species, they have been found in a myriapod (Scutigerella midgut; Juberthie-Jupeau, 1979); crustacea (crayfish hepatopancreas; Gilula, 1972b, 1974; Satir and Gilula 1973; Gammnrus hepatopancreas; Schultz, 1975; Orchestia posterior caeca; Graf, 197th; the midgut of a copepod; Amaud, Brunet and Mazza, 1978; possibly also in the hepatic caecum of Daphnia; Hudspeth and Revel, 1971) and in a slightly unusual form in a chelicerate (midgut and hepatopancreas of Limulus polyphemus; Lane and Harrison 1978). They have also been found in ticks (Binnington, personal communication). Mills et al. (1976) report a septate junction in the midgut of the crayfish Cambarus which exhibits “a very regular array of transverse septa”. Without lanthanum-infiltration or freeze-fracture studies, it cannot be ascertained whether this is a pleated or smooth septate junction . Satir and Gilula (1973) claim to have seen smooth septate junctions in a nematode and in Peripatus (Onychophora); this accords with previous observations in that Lavallard (1967) reported a structure thought to be a smooth septate junction in Peripatus, although Dallai et al. (1977) query this finding. However these authors have demonstrated a smooth septate-like junction in the midgut of Peripatopsis moseleyi (Dallai and Giusti, 1978). These junctions display the typical smooth septate junction appearance on lanthanum-infiltration but freeze-fracture reveals an unusual intramembranous organization; the undulating EF ridges are decorated on their surface with rows of isolated 9 nm particles. Greven (1976) has described a zonuia continua in the midgut of a tardigrade and Mme Auber-Thomay (personal communication in Noirot and Noirot-Timothee, 1967) has found smooth septate junctions in a nematode intestine. Whether further smooth septate junctions will be found in the midguts of other invertebrate species remains to be seen but it seems highly likely given the ubiquitly observed thus far. Green (1978) and Green et al. (1979a) describe anastomosing septate junctions in both echinoderms and hemichordates. Green (1978) considers that these junctions form an evolutionary link between the invertebrate septate junction and the vertebrate tight junction. The lowest vertebrate phylum, the tunicates, display true tight junctions in which the intercellular space is occluded and freeze-fracture reveals an anastomosing network of P face ridges (Cloney, 1972; Lorber and Rayns, 1972). The evolutionary link between junctions displaying an enlarged extracellular space (invertebrate septate junction ca. 14-17 nm) and those with an occluded intercellular space (vertebrate tight junctions) is not clear, $spite their superficial similarity after lanthanum infiltration or freeze-fracture. This similarity of appearance arises from quite separate structures; in the first case the lanthanum-excluding septa and the corresponding rows of particles within

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the membrane and in the second, the obliteration of the intercellular space by the fusion of ridge-like structures in the junctional membrane. 2.6

OCCURRENCE I N VERTEBRATES

A variety of “septate-like” junctions have been described in vertebrate tissues. As yet they have been identified only in certain tissues, the nervous system (Laatsch and Cowan, 1966; Peters 1966; Hirano and Dembitzer, 1967, 1969; Gobel 1971; Sotelo and Llinhs, 1972; Livingstoner af., 1973; Cabellero et af., 1978), the retina (Lasansky, 1969, 1971; Schwartz, 1973) vascular endothelium (Simionescu et af ., 1976), endocrine tissues (Bargmann and Lindner, 1964; Friend and Gilula, 1972; Enders, 1973), testis (Aetorfer and Hedinger, 1975; Connell, 1974, 1975, 1976, 1978) and transiently in the ovary (Albertini and Anderson, 1974). They have also been described in pathological tissues (Nistal er af., 1978a; Tani et af.,1971) and moreover have been shown to arise artifactually (Bulger and Trump, 1968; Nistal et af., 1978b). Where their existence is established, the septatelike junctions vary both in their structure and in the extent of their resemblance to invertebrate septate junctions. In the adrenal cortex, Friend and Gilula (1972) described a junction with septa formed by 10-15 nm extracellular particles, often circular in profile, spanning the 21-30 nm intercellular space. Freeze-fracture preparations revealed no specialized intramembrane organization associated with these structures. Other descriptions of septate junctions in endocrine tissues (Bargmann and Lindner, 1964; Enders, 1973) have not included evidence from freeze-fracture. In the retina of turtles (Lasansky, 1969, 1971), the adjacent membranes of the septate-like junctions are separated by a uniform 16 nm space, similar to that of the invertebrate junctions. The separation of the septa ( C U . 19 nm) and the observation in oblique sections that they form a regular network is also reminiscent of the invertebrate junctions. Schwartz (1973) has described a similar junction in the retina of the rat. However without freeze-fracture evidence, parallels with the invertebrate septate junction cannot be made. As with other septate-like junctions, the degree to which those in the nervous system parallel the invertebrate septate junction is probably slight. Gobel (1971) and Sotelo and Llintis (1972) have described junctions between axons in the vertebrate cerebellar cortex where 5-8 nm thick septa bridge the 11-18 nm intercellular space at a repeating distance of 16-22 nm. In oblique sections, these septa appear as undulating ribbons sometimes giving a honeycomb appearance of hexagonally arranged “cells” 1 7 nm in diameter (Sotelo and Llinhs, 1932). These features superficially resemble

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the invertebrate pleated septate junctions, but without freeze-fracture, the existence of a true cell junction, displaying membrane specialization in freeze-fracture replicas, as opposed to a cell contact with no intramembrane specialization (Friend and Gilula, 1972b) cannot be established. Glial-axonal junctions (Laatsch and Cowan, 1966; Peters, 1966; Hirano and Dembitzer 1967,1969; Livingston et al., 1973; Schnapp and Mugnaini, 1975,1976; Schnappet al., 1976) display septa, 10-15 nm wide, and with a repeat distance of 25-30 nm. However, here the intercellular space is only 2-3 nm. Lanthanum infiltration (Hirano and Dembitzer, 1969) reveals straight septa in this intercellular space. Freeze-fracture preparations (Livingstonetal., 1973; Schnapp and Mugnaini, 1975; Schnappetal., 1976) reveal regular convolutions of the glial and axonal membranes, which take the form of complementary ridges and grooves in the glial and axonal P and E faces (ridges on the PF corresponding with grooves on the EF). These regular undulations are separated by 25-30 nm and follow a helical path with respect to the long axis of the axon with its glial wrapping. Both the axon and glial membranes display 8-13 nm particles, associated with the P F ridges in the axon and with either the PF ridges or EF grooves in the glial cells. Schnapp and Mugnaini (1975) and Schnapp et al. (1976) present a model of this axo-glial junction in which straight septa, spanning the narrow intercellular space, insert into the membranes at the point of minimal intercellular distance, where the undulations bring the membranes closest together. This explains the identical spacing of the septa in thin-sections, with that of the ridges and grooves in freeze-fracture. This junction has clear analogies both with invertebrate septate junctions and also with gap junctions. Suggestions as to the functional significance of these junctions reflects this. Livingston et al. (1973) suggest that the particles of the junction could traverse the narrow intercellular cleft and, as in gap junctions, allow the passage of ions between the axonal and glial cytoplasm. Schnapp et al. (1976) propose that a more likely function is adhesive, following the analogy of the junction with a nut and bolt assembly (Petersetal., 1970). Intercellular occlusion is not considered, since these junctions occur in close juxtaposition with tight junctions (Schnapp and Mugnaini, 1975, 1976). Connell (1974, 1975, 1976, 1978) describes a type of septate junction between the Sertoli cells of mammalian testis and claims, on the basis of thin sections, lanthanum staining and freeze-fracture evidence, that these junctions resemble closely the invertebrate septate junction (Connell, 1978). The resemblance is closer to the smooth rather than the pleated type. Her lanthanum infiltrated preparations do resemble the double septa described by Green (1978) from echinoderm epithelium and by Duvert et al. (1978) from the intestine of a chaetognathe (Sagitta). Although there are similarities between the Sertoli cell septate junctions and those in inverte-

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brate tissues, there are also differences. The uniformity of the intercellular space, a very characteristic and almost diagnostic feature of the invertebrate septate junction, is not clear in the testis (see Connell, 1978, Figures 2 and 3) and the intercellular space, rather than being enlarged (14-17 nm), is slightly reduced (ca. 9 nm). In these two last examples of septate-like junctions (the glial-axonal complexes of the central nervous system and the Sertoli cell junctions of the testis), the junctions are in close co-existence with tight junctions. Moreover, it is suggested that they perform a physiological role distinct from the tight junctions (Schnapp et al., 1976; Connell, 1978), a feature to be borne in mind when considering claims of functional equivalence of invertebrate septate junctions and vertebrate tight junctions (see Section 2.7). 2.7

FUNCTIONAL SIGNIFICANCE

The functional significance of septate junctions is still not entirely clear. The role of intercellular communication assigned to them (Loewenstein and Kanno, 1964; Wieneret al., 1964; Bullivant and Loewenstein, 1968; Gilula etal., 1970; Satir and Gilula, 1970; Rose, 1971) is now generally considered to be fulfilled not by septate junctions (Hagopian, 1970; Poodry and Schneiderman, 1970; Flower 1971; Hudspeth and Revel, 1971;Burger and Uhrik, 1972; Caveney and Podgorski, 1975; Snigirevskaya et al., 1977; Noirot-TimothCeetal., 1978) but by gap junctions (see Section 4.6.1). It is, however, difficult to exclude this possibility with certainty as septate junctions tend to occur in association with gap junctions (see Section 4.8). Intercellular adhesion is generally agreed to be one of the functions of septate junctions (see for example Wood, 1959, 1977; Poodry and Schneiderman, 1970; Berridge and Oschman 1972; Noirot-Timothte and Noirot, 1974, 1979; Baskin, 1976; Noirot-TimothCe et al., 1978; Lane, 1979c) and is a corollary of the structural models proposed for the insertion of the septa into the junctional membrane (see Section 2.2.4.) Although there is little direct experimental evidence for the adhesive function of septate junctions, Filshie and Flower (1977) report a parallel loss of structural stability in Hydra with the disappearance of the septate junctions after glycerol treatment. Correlating with this finding, extensive septate junctions are commonly found in those tissues subject to rapid dilations as for example, the gut and the epidermis in fluid feeders. It has also been suggested that septate junctions function as a transepithelial permeability barrier (Wood, 1959, 1977; Dan, 1960; Berridge and Oschman, 1969; Newel1 and Skelding, 1973; Lord and di Bona, 1976; Filshie and Flower, 1977; Szollosi and Marcaillou, 1977; Noirot-TimothCe et al., 1978; Graf, 1978a; Noirot-TimothCe, and Noirot, 1979; Greenet a/.,

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1979a). Evidence conflicting with this supposition has been presented by several authors (Treherne et al., 1973; Ryder and Bowen, 1977; Lane 1978c, 1979a, c) who have shown penetration of septate junctions by tracers. Authors vary in the methods they employ to test the penetrability of junctions by tracers but in a number of cases, tracer molecules are added to the fixative solution and conclusions drawn about the permeability of the intercellular space by the depth to which the tracer penetrates the junction in moribund‘tissue (e.g. Hudspeth and Revel 1971; Hand and Gobel, 1972; Newel1 and Skelding, 1973; Gilula, 1974; Baskin, 1976; Filshie and Flower, 1977; Graf, 1978a). Physiological conclusions based on the movements of tracers are always difficult to make since the properties of the tracer may interfere with the physiology of the system and at best can indicate only the permeability of the tissue to the particular tracer molecule under consideration. However the value of such studies is considerably enhanced if the tracer molecule is applied in conditions as near to the in vivo state as possible. Such conditions were first defined for the use of ionic lanthanum by Machen et al. (1972) and have subsequently been used in the study of invertebrate tissues (e.g. Lane, 1972; Lane and Treherne, 1972; Treherneet al., 1973; Leslie, 1975; McLaughlin, 1974b; Ryder and Bowen 1977; Szollosi and Marcaillou, 1977; Lane and Swales, 1978a, b, 1979a, c; Lane, 1978c, 1979a, c; Lane and Harrison, 1979b). Of thesein vivo incubations all except Szollosi and Marcaillou have demonstrated complete penetration of the septate junctions by the applied tracer. Szollosi and Marcaillou (1977) show septate junctions in the basal stain-excluding compartment of the locust testicular follicle. In this tissue, however, both septate (Figs 6 , 7 and 9) and simple tight junction-like ridges are found (Fig. 47 and Skaer and Jones, 1979 and Szollosi, personal communication). Thus until further analysis of junctional position and tracer penetration is completed, the identity of the sealing junction cannot be made with certainty. Lord and di Bona (1976) demonstrated that blisters are produced between the septa of planarian epidermal junctions when subjected to osmotic stress (the normal external < 6 mosmol fluid was enriched with NaCl, KCl, urea, mannitol or urea to levels in excess of 300 mosmol). They drew an analogy with similar blistering behaviour previously demonstrated in vertebrate epithelial tight junctions and so concluded that both junctions can be functionally classified as “limiting junctions” constituting “ratelimiting barriers to the passive, paracellular flow of water and small molecules”. The significance of blister-production by elevating the osmolarity of the external environment to completely unphysiological levels is questionable. A similar criticism could be levelled at a parallel study on the midgut of crayfish (Mills et al., 1976). The existence of a potential difference across an epithelium has been

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taken to indicate that the intercellular junctions seal the paracellular pathway (e.g. insect salivary gland, Loewenstein and Kanno, 1964; and see Noirot-TimothCe and Noirot, 1979). In tissues where polarized pumps have been shown to be active such as insect midgut (Harvey et al., 1968), Malpighian tubule (Maddrell, 1971) and salivary gland (Berridge et al., 1976a), the maintenance of a transepithelial potential need not depend on sealed intercellular spaces but is a function of the relationship between the electrical resistance of the epithelium and the rate of ion transport. The resistance of the epithelium moreover, will be a function not only of intercellular leakiness bur also the permeability of the cell membranes and will therefore also depend on the relative area presented by each of these surfaces. This latter ratio is variable but in the case of Malpighian tubule, the relative permeabilities in different species can be related to the geometry of the cells which determines the area of basal (or apical) cell surface, relative to that of the lateral borders (Maddrell, 1979a). Lane and Swales (1978) have shown the absence of septate junctions in the nervous system of a moth, Manduca sexta, where a physiological bloodbrain barrier has been demonstrated (Pichon et al., 1972; Lane, 1972). Furthermore, the upper Malpighian tubule of Rhodnius in which smooth septate junctions are found (Skaer et al., 1979) apparently offers little restriction to the transepithelial movement of a range of organic solutes which include such substances as sucrose and inulin, that could scarcely d o other than cross the epithelium by the intercellular route (Maddrell and Gardiner, 1974). However, as calculated by Filshie and Flower (1977), the presence of complex arrangements of septa, even if they are relatively short with frequent blind endings, dramatically reduces the cross-sectional area available for intercellular permeation and increases the transepithelial path length. Thus the rate of passive flow through the junction is bound to be reduced and, if the molecule is large enough, its entry might be blocked (as with ferritin (Zylstra, 1972; Ryder and Bowen, 1977)). Moreover, the interseptal space may contain electron-dense material, especially in the smooth septate junction (Noirot and Noirot-TimothCe, 1967,1979; Dallai 1970). In the midgut of several insects this material has been shown to be glycoprotein in nature (Dallai, 1970). Some specificity in the otherwise unselective retardation of passive flow through the junction, imposed by the architecture of the septa, might therefore be conferred by the chemical nature and charge characteristics of the intercellular matrix trapped between the septa. Some evidence to support this comes from a study by Gupta et al. (unpublished data, 1972) in which the septate junctions of Calliphora Malpighian tubule were shown to be permeable to the anion, sulphate, but not to a heavy metal cation, barium. A similar suggestion relating to the septate junctions of

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Hydra has been made by Staehelin (1974) and the idea of the intercellular space and cement as an ion-exchange resin, molecular sieve, gel filter and chromatographic column is considered by Oschman (1978). The situation in Hydra and other cnidaria, in which a structurally distinctive type of septate junction has been described, is rather different from the insects. In this phylum, tight junctions have not been described. Moreover, several investigators have concluded, on a variety of grounds, that the septate junctions act as permeability barriers (e.g. Wood, 1959,1977; Leik and Kelly, 1970; Hand and Gobel, 1972; Staehelin, 1974; Flower and Filshie, 1977; King and Spencer, 1979). Although some of the criticisms cited above can be levelled at permeability studies carried out on these tissues, it may be that these “Hydra”-type septate junctions restrict transepithelial permeability. Finally Dallai (1 976) has suggested that septate junctions (both pleated and smooth) might confer greater rigidity on the intercellular membranes, preventing the occlusion of the intercellular space and so facilitating the rapid flow of water through the intercellular cleft. In view of the considerations mentioned above concerning the effect of the septa on path lengths and cross-sectional area of the intercellular space, as well as the finding that the septa normally run perpendicular to, rather than parallel with, the direction of fluid flow, this suggestion seems rather unlikely. Moreover in a tissue where bulk transport of fluids take place, a specialised junction (the reticular septate junction) has been described, which, it is suggested, is involved in ensuring a ready paracellular flow (Lane, 1979c, and see Section 8.1.6). Throughout this discussion it has been assumed that the roles of smooth and pleated septate junctions are identical. It was originally thought that they represented analogous structures and that smooth septate junctions might be associated with regenerating tissues or those of endodermal origin, whereas pleated septate junctions were associated with non-regenerating tissues or those of ectodermal origin (Noirot and Noirot-TimothCe, 1967). The results described by Dallai (1967), Lacombe (1 976), Meyran (1 977) and Skaer et af. (1979) conflict with this interpretation since smooth septate junctions occur in Malpighian tubules which are thought to be ectodermal in origin and in which constant cell turnover does not occur. The embryological origin of Malpighian tubules is, however, open to discussion (see Wall ef al., 1975). Both Wall et af. (1975) and Dallai (personal communication) consider it possible that some parts of the Malpighian tubule may be of endodermal origin. This however still leaves unexplained the occurrence of smooth and pleated septate junctions side by side in Malpighian tubules, which argues against the two types of junction being analogous structures.

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FORMATION O F SEPTATE JUNCTIONS

Gilula (1972a, 1973) has studied the formation of septate junctions in sea urchin embryos. The assembly of the junctions can be followed from the detection of the presumptive junctional site at 9-12 hours to its maturation at 72 hours. In thin-sections, the development can be followed by the appearance of electron-dense septa spanning the intercellular cleft. In parallel with this, the 8.5 -9 nm particles, revealed by freeze-fracture (EFcf. PFin insects), arise in the presumptive region and gradually become aligned. Initiaily they line up into simple rows and subsequently into swathes of parallel rows, possibly in association with a nucleation furrow, and finally the multiple rows form a continuous belt around the cells with the P F displaying furrows complementary with the particle rows. The development of pleated septate junctions in insects has been followed during the embryonic and larval development of Calliphora (Lane and Swales, 1978a). The junctions in thin-section show incomplete formation of the septa in early stages of development. As the junctions mature, the septa develop and join the junctional membranes to give the regular appearance in the fully-formed larva. In freeze-fracture, the PF particles are disorganized in their arrangement in the early larva. They gradually line up, initially into short, abruptly terminating rows and subsequently into the extensive meandering rows, typical of the mature junction between Calliphora perineural cells. To date, the development of smooth septate junctions has not been investigated, though it has been mentioned that in certain insect species the formation of the junction must occur during embryonic life since the mature junction is present at each instar (Rhodnius midgut; Lane 1978a). However during the remodelling of the gut of Manduca sexta during pupation, the smooth septate junction reforms and developmental stages can be found (Fig. 24, and Lane 1979e). Short, moniliform ridges are found on the E F in unfixed tissue, often collected together into disorganised stacks (insert, Fig. 24), while meandering P F grooves or E F ridges can frequently be seen, not aligned, and often terminating abruptly (arrows, Fig. 24). Even in mature tissues, such as the gut, where cell replacement occurs, the reformation of cell junctions is inevitably involved. One might therefore expect to find stages in this cycle of breakdown and re-establishment of smooth septate junctions. Lane (1978a, 1979b) considers this possibility and shows short, isolated PF ridges (Lane, 1979b, Figure 28) in the proventriculus of Rhodnius which could represent a developmental stage in the formation of the continuous junctions that are found in this tissue. In this context, of interest also are the macular continuousjunctions of Graf (1978a) in Orchestia midgut. These macular junctions are situated below and

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contrast with the belt of septa representing the apical zonular junction. It is possible that they could be developmental stages, although Graf does not comment on this possibility. Houk (1977) however suggests that the smooth septate junctions of the flea (Culex tarsalis) are able to dissociate and reform after the junction has reached a mature state. He cites evidence (Milesetal., 1973) to indicate that the passage of virus particles from the gut lumen to the haemocoel occurs sufficiently rapidly to preclude the transcellular route. Furthermore, the morphology of the flea gut alters very greatly during ingestion of the blood meal, the columnar epithelium becoming squamous. Houk suggests that this alteration may entail breakdown and reassociation of junctions. Howard (1962), also working with flea, showed that after bloodmeal ingestion, polystyrene spheres of 5 p m diameter could penetrate the intercellular cleft to within a few microns of the basal lamina. The spheres are too large to pass through the septate junctions and so, discounting a transcellular route, this observation would imply the temporary dissolution of the junction.

3 Desmosomes 3.1

INTRODUCTION

The majority of work published on desmosomes is concerned exclusively with vertebrate material. Thus our knowledge and interpretation of desmosome-like structures found in insect tissues is set against the background of a wealth of information on the various desmosomal types characteristic of vertebrate tissues. Some of the information about these vertebrate junctions will be summarized first so that similarities and differences of the insect junctions can be assessed. Desmosomes in vertebrates can be distinguished on morphological grounds into two distinct types; the macula adhaerens or spot desmosome and zonula (and fascia) adhaerens or belt desmosome (Farquhar and Palade, 1963). The junctions are characterized not only by their geometry on the cell surface but also by their appearance in thin-section and by the degree of elaboration they display in the intramembrane planes revealed by freezefracture. Hemi-desmosomes have an appearance identical with half a macula adhaerens and so will be described with them. Fig. 24 Freeze-fracture preparation of midgut from a fixed Manduca sexta pupa. The smooth septate junction, in the process of reforming between the cells of the potential adult midgut, displays a very loosely ordered appearance with frequent terminations, both of the PF ridges and EF grooves (arrows). This is particularly clearly seen in the insert of unfixed tissue, which shows an array of very short EF ridges, apparently becoming aligned. x 73 700; insert x 65 900

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3.2 3.2.1

STRUCTURAL FE.ATURES

Thin-section appearance

(a) Vertebrates The spot desmosome is typified by its shape (oval, 0.2-0.5 p m in length) and by an enlarged intercellular space (25-35 nm) which contains material often formed into a central dense stratum lying parallel to the cell membranes, and whose subjacent cytoplasm is elaborated into dense fibrillar plaques. The junctional intercellular space is continuous with the non-junctional intercellular space but is distinguished from it by the electron-dense stratum which in some cases is a double structure, 4 nm separating the two dense lines, and which appears to be connected to the two junctional membranes by a complex array of connecting 4 nm filaments. These, in some cases, (Rayns et al., 1969) show a square array (7-8 nm centre-to-centre spacing). This latter observation was made after infiltration with lanthanum hydroxide to which the stratum and interconnecting filaments are impermeable. The intercellular material has been shown to stain with ruthenium red (Kelly, 1966; Luft, 1971) and amixture of chromic acid and phosphotungstic acid (Rambourg, 1969) and this, combined with its susceptibility to trypsin treatment (Overton, 1968; Berry and Friend, 1969), suggests that it contains glycoprotein. The cytoplasmic plaques have a filamentous appearance in thin-section and are separated from the membrane surface by a slightly less electrondense zone, The plaques are the sites of attachment of the numerous tonofilaments, 10 nm in diameter. Kelly (1966) concluded, on the basis of studying stereo-pairs, that the tonofilaments, rather than terminating in the plaques, loop through them and terminate either in the tonofilament bundle in the cytoplasm or, if there are further desmosomes arranged in series, the filaments may course through the cytoplasm linking one plaque with the next (Fawcett, 1958; Lentz and Trinkaus, 1971). It has recently been suggested that secondary filaments, derived from the 10 nm tonofilaments, traverse the membrane and anchor on particles making up the intercellular midline (Leloup et al., 1979). In vertebrates, a second type of desmosome has also been described, the zonula adhaerens. In shape, it can either be belt-like (zonula)or have a more interrupted topography Cfascia) but beyond this they are distinguished from maculae adherentes on the basis of several structural features. In thinsection, the separation of the membrane is smaller (15-20 nm), no central intercellular stratum is seen, although the intercellular space stains to reveal fine filamentous material. The cytoplasmic plaque is closely applied to the membrane but itself is more loosely organized and the filaments associated with it have a diameter of 7 nm as compared with 10 nm for the spot desmosomes. These filaments appear to have some actin-like properties

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since they bind heavy meromyosin (Ishikawa et al., 1969; Tilney and Mooseker, 1971). ( b ) Arthropods The foregoing structural features have been summarized by Campbell and Campbell (1971), McNutt and Weinstein (1973), Overton (1974), Staehelin (1974) and Staehelin and Hull (1978) who deal largely with vertebrate material. The desmosomes of arthropods are not as readily classified, for example, some authors claim that spot desmosomes are very infrequent (Satir and Gilula, 1973) or absent (Noirot-Timothte and Noirot, 1979) whereas their presence is asserted in a number of individual reports. Moreover, insect desmosomes exhibit some distinct differences from their vertebrate counterparts. The intercellular separation is variable as is the organization of the intercellular material. The most striking difference in thin-section is the association of the cytoplasmic plaques with microtubules rather than with tonofilaments (Fig. 25). This is especially well established for the much studied sites of muscle attachment where both hemidesmosomes and more extensive desmosomes (sometimes termed fascia) are found (Fig. 25) (Auber, 1963; Shafiq, 1963; Lai-Fook, 1967; Beaulaton, 1968; Caveney, 1969; Hagopian, 1970). However the association with microtubules is also found in other tissues (Satir and Stuart, 1965; Stuart and Satir, 1968; Moulins, 1968; Smith, 1968; Ashhurst, 1970; Friedman, 1971; Corbitre-Tichant, 1971) although not universally (Gupta and Rerridge, 1966; Stuart and Satir, 1968; Reinhardt and Hecker, 1973; Reger, 1974; Noirot and Noirot-Timothte, 1976). Where they occur, the microtubules appear to run parallel with, rather than insert into, the cytoplasmic plaque and a role of skeletal stiffening of the junctional membrane has been attributed to them (Ashhurst, 1970). In the majority of cases the blanket term “desmosome” appears to be used in describing these junctions in insects. Where a distinction is made between different types it seems to be solely on the basis of junctional shape. Thus the junctions involved in muscle attachment in cockroach epidermis are termed zonulae adhaerentes (Hagopian, 1970), the apical desmosome region of the columnar cells of the sternal gland is called the terminal bar (Stuart and Satir, 1968), the myoepidermal connections of Calpodes and Rhodnius are designated fasciae adhaerentes (intermediate junctions) (Lai-Fook, 1971), whereas the spot desmosomes found between cells are called maculae adhaerentes on the basis of their distinctive, restricted shape rather than a detailed analysis of their structural characteristics. Both zonular and macular desmosomes are described by Noirot and Noirot-Timothte (1976) in the rectum of various cockroach species where according to the authors, these junctions are comparable with their vertebrate equivalents though structurally simpler. Cytochemical investigations of insect desmosomes appear to be limited to

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Fig. 25 Numerous hemi-desmosomes at the site of insertion of skeletal muscle in the locust Schistocerca gregaria. The hemi-desmosomes occur in pairs, occupying opposite sides of the dilated extracellular spaces, which are filled with an electron-opaque material. Note also the cytoplasmic plaques of electron-dense fibrillar material and the high density of microtubules. Insert: Macular desmosome joining glial cells in Calliphora nervous system. The elaboration of the extracellular material into striations, the relative uniformity of the intercellular space, cytoplasmic fibrillar thickening and microtubules lying close to, but not intimately associated with the junction are all demonstrated. x 60 100; insert X 90 000

the study of Reindardt and Hecker (1973), who found that maculae adhaerentes and hemidesmosomes of the mosquito midgut did not stain with periodic acid-TCH-silver-proteinate (specific for polysaccharide) but that the cytoplasmic mat stained strongly with phosphotungstic acid (specific for basic amino acid-rich protein)

3.2.2

Freeze-fracture appearance

Studies on vertebrate tissues have shown that spot desmosomes (maculae adhaerentes) are associated with intramembranous particle aggregates.

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These particles fracture onto both the EF and the P F although the P F is normally more richly endowed than the EF. This however can alter with fixation or developmental stage (see Staehelin, 1974). The particles are of an irregular, normally elongate shape, about 12 to 20 nm in diameter. The intercellular stratum is evident as an etch-resistant line 5 to 7 nm in width, which in some preparations can be seen to be made up of small particles (e.g. Leloup et al., 1979). In contrast, the zonulae and fasciae adhaerentes reveal very little in the way of intramembrane elaborations. Freeze-fracture shows no differentiation of the membranes in the area of these junctions. Friend and Gilula (1972b), Satir and Gilula (1973) and Satir and Fong (1973) point out that structures such as these, having a clear intercellular and cytoplasmic differentiation but no intramembranous specializations should be called intercellular contacts and not junctions. O n this basis, the majority of insect desmosome-like junctions should be designated contacts and not junctions. There are very few examples of freeze-fracture preparations of insect tissues that show any intramembrane elaborations and these are of spot desmosomes (e.g. Skaer and Lane, 1974; Baerwald, 1975; Chi et al., 1979). In the rare instances where freezefracture images are obtained, the membranes show symmetrical fracturing characteristics, plaques of small irregularly organized particles being found both on the E F and PF. More commonly, in tissues where spot desmosomelike junctions are very common, no freeze-fracture evidence for their presence can be found (Lane and Swales, 1978a, b). Freeze-fracture preparations of tissues, in which fasciar or zonular junctions have been described, also show no specialized intramembrane features (e.g. Graf, 1978a; cf. Figures l A , PIId; Chi et al., 1979, Figure 13).

3.3

OCCURRENCE I N INSECTS

Desmosomes are found almost universally in insect tissues (see Smith, 1968) and only selected examples of their occurrence will be given in this review. Zonular junctions are found at the extremities of lateral borders in epithelia (Locke, 1965; Stuart and Satir, 1968; Oschman and Berridge, 1970; Noirot and Noirot-Timothie, 1976; Lane, 1979c), although Flower and Filshie (1975) claim that they are found only in ectodermal epithelia where cuticle is present external to the cellular layer, and hence are absent from Lepidopteran midgut. Further Noirot et al. (1979) have noted the absence of an apical belt desmosome in the rectal sheath cells of a termite and cockroach, where a cuticular lining is found. Spot desmosomes are found in more varied positions along intercellular borders and may be found in very

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large numbers in certain tissues such as muscle where desmosomes are involved in skeletal attachment via myoepidermal junctions (Bouligand, 1962; Auber, 1963; Shafiq, 1963; Lai-Fook, 1967; Caveney, 1969; Hagopian, 1970; Kuo, McCully and Haggis, 1971) and in an analogous “connective-epidermo-cuticular” connection found in the hypopharyngeal cavity of Blabera craniifer (Moulins, 1968). Desmosomes may also be found in the nervous system both between glial cells (Fig. 25, insert and Schurmann and Wechsler 1969; Laneetal., 1977a; Ribi, 1977; Lane and Swales, 1978a, b, 1979a) and also in the perineurium (Skaer and Lane, 1974, Lane et al., 1977a; Lane and Swales, 1978a). Structures termed interfibrillar junctions and resembling desmosomes are also found associated with the intercalated disc of insect myocardium (Sanger and McCann, 1968; McCann, 1970). The intercellular space containing “basement membranelike material” varies from 20-55 nm in width and the junctional membranes display cytoplasmic plaques of increased electron density into which the muscle thin filaments insert. Hemi-desmosomes, although found in regions of intercellular dilation on the lateral borders of epithelia, are found in their greatest numbers in positions where the cell membrane abuts onto connective tissue (basal lamina) (Fig. 25). Desmosomes appear to form between homologous cells, heterologous cells and between different regions of the membrane derived from the same cell, when an autodesmosorne is formed (Smith, 1968; Ishizaki, 1973; and for examples of all three types see Noirot and Noirot-Timothte, 1976).

3.4

DISTRIBlJTlON I N OTHER INVERTEBRATES

3.4.1 Arthropod tissues Desmosomes are also found widely in the other classes of arthropods. A slightly unusual type of desmosome, exhibiting marked asymmetry and not associated with microtubules has been found in the myoepidermal attachments in an Acarid mite (Kuo et al., 1971). The attachment of copepod muscles is via similar desmosomes, which however are associated with fine tubules designated “tonofilaments” by the original author (Bouligand, 1962) but which Lai-Fook (1 967) suggests are microtubules. Desmosomes in crustacean muscle are discussed by Komuro (1970) and Anderson and Smith (1 971), and muscle attachment in the venomous spider Latrodectus mactans is also associated with desmosomes between cells packed with microtubules (Smith et al., 1969a). Graf (1978a) has shown the presence of an apical “zone of adhesion” in the

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midgut of an amphipod Orchestia carimana. In thin-section the junction shows a regular, but reduced intercellular space of 11-12 nm, with electron-dense cytoplasmic plaques and intercellular material that is sometimes organized into broad septa and which does not stain with lanthanum. In freeze-fracture preparations, very little alteration of the intramembrane regions are found although there is a slight diminution in the population of intramembrane particles. Contrasting with this situation, a recent investigation using freezefracture has revealed the most highly organized intramembrane specializations so far reported as being associated with hemidesmosomes (Smith et al., 1979, working with crayfish muscle). Each hemidesmosome consists of a “stripe” of 7-8 nm particles on the P F with complementary EF grooves. “Stripes” up to 3 p m in length are found lying parallel at regular intervals (80 -90 nm centre-to-centre spacing). This elaboration resembles those reported in molluscs by Prescott and Brightman (1976), who suggested that they might represent desmosomes. However, Franzini-Armstrong (1979) describing similar freeze-fracture images from the striated muscle of a spider, does not regard them as junctional structures. Shivers and his co-workers have applied freeze-fracture techniques t o a variety of crustacean tissues. They have described in some detail the structure of both mature and forming hemi-desmosomes (Shivers and Brightman, 1977; Shivers, 1977). However, their images are much more closely comparable to the arthropod gap junctions (see Section 4.2). The striking similarities to gap junctions are in the fracturing characteristics ( E F particles), the enlarged size of these particles, the linear particle configurations and loose plaques associated with developing junctions (see Section 4.7.1) and the involvement of two membranes, which would be inconsistent with a designation of hemidesmosome for these junctions (see Shivers, 1977, Figures 5 and 6; Shivers and Brightman, 1977, Figures 3, 6-8, and 18). A similar interpretation is made in the antenna1 gland where junctions are described, which in both thin-section (upper circles Figures 10 and 11 in Shivers and Chauvin, 1977) and freeze-fracture (EF particles of enlarged size Figure 9, PF pits with occasional particles Figures 13 and 14 in Shivers and Chauvin, 1977) appear more similar to gap junctions than maculae adhaerentes.

3.4.2 Non-arthropod tissues Desmosomes and desmosome-like structures occur so widely in the invertebrates that they are reported at least in passing in almost any ultrastructural study of a particular tissue. As a result a comprehensive survey of their

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occurrence is beyond the scope of this review and an indication of the variety of organisms in which they are found is all that will be covered. Structures not altogether unlike desmosomes have been reported between prokaryotic and eukaryotic cells (Wagner and Barnett, 1974) in that intercellular filaments apparently derived from both cells traverse a regular and enlarged (30-40 nm) “intercellular space”. Hemidesmosomes and desmosomes have been found in flagellates (autodesmosome) (e.g. Brooker, 1970) and rotifers (e.g. Koehler and Hayes, 1969) and miniature autodesmosomes have been reported in trypanosomes (e.g. Vickerman, 1969; Smith et a f . , 1974). This latter study includes evidence from freezefracture but suggests that there is no intramembrane specialization associated directly with the desmosomes. In coelenterates, the situation appears confused as to whether desmosomes comparable in structure with those of insects exist (e.g. Roosen-Runge and Szollosi, 1965). Areas have been found where the intercellular space is very regular and the membranes are closely associated with cytoplasmic vesicles and these have been suggested as possible sites of adherence. However specialized cells termed desmocytes are also found in Cnidaria (Mackie, 1962; Chapman, 1969; Knight, 1970; Bouillon and Levi, 1971;Marcum and Diehl, 1978) and these cells are richly endowed with filaments ( 7nm in Cordyfophora;Marcum and Diehl, 1978) and, by their attachment to the perisarc and mesoglia, are thought to act as skeletal structures (for further details see Chapman, 1969). Wood (1 977), however, claims the occurrence of intermediate junctions beween myoid processes in Hydra; these junctions resemble fasciae adhaerentes and like them reveal little intramembrane specialization in freeze-fracture replicas. Cells from echinoderm tissues may be linked by desmosomes (e.g. Holland, 1971; Pentreath and Cobb, 1972) and desmosome-like junctions are found in planarians (e.g. Oaks, 1978) and chaetognathes (Duvert, 1977; Duvert et a / , 1978). Desmosomes of the macula adhaerens type as well as terminal bars have been described in the Pogonophora (Gupta et a f . ,1966; Gupta and Little, 1970). Zonulue adhaerentes are reported in the epidermis of annelids (e.g. Baskin, 1976; Storch and Welsch, 1970, 1972; Knapp and Mill, 1971; Boilly-Marer, 1972; Michel, 1972; Bilbaut, 1980; Skaer, 1979b) and both desmosomes (homo- and heterocellular) and hemidesmosomes have been found in the nervous system of earthworms (Hama, 1959; Coggeshall and Fawcett, 1964; Coggeshall, 1965; Giinther, 1976), the leech (Zimmerman, 1967) and ragworms (Baskin, 1971); these junctions are also found in many other annelid tissues. Similarly in molluscs, desmosomes are commonly found (e.g. Guptaetal., 1969; Lane and Treherne, 1972; Newel1 and Skelding, 1973; Kataoka, 1976; Ryder and Bowen, 1977).

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FUNCTIONAL SIGNIFICANCE

The adhesive role of desmosomes, both macular, fasciar, zonular, and hemi-, appears t o be generally agreed. This adhesion is apparently achieved by the attachment of fibres projecting from the cell surface either to an extracellular matrix (as in the case of hemidesmosomes) or to similar fibres of another cell. In this latter case, the fibrillar attachments may be mediated by other intercellular structures, seen in thin-section as the stratum or midline (see Leloup et al., 1979 for a detailed model), Underlining the adhesive significance of desmosomes, they are found in insects in those tissues or in those positions in tissues subject to mechanical stress. Thus they are found at the attachment sites of insect muscles and in tissues subject to volume changes such as the epidermis (especially of fluid-sucking bugs, e.g. Rhodnius prolixus), the midgut of certain blood-sucking insects (where the intercellular spaces decrease in size during tissue distention after a bloodmeal (Reinhardt and Hecker, 1973)), salivary gland, Malpighian tubules and rectal tissues. They are also found in the ensheathing structures of the nervous system but especially noteworthy is their abundance in the glia of Manduca sexta where the nervous system, by virtue of its attachment to the musculature of the body wall, is in a state of constant agitation (Lane, 1972; Lane et al., 1977a). Desmosomes are also found where the relative position of neighbouring cells is physiologically critical; thus, for example, they are found in the rhabdomeres of the eye (e.g. Eley and Shelton, 1976; Shaw, 1978; Schinz, 1978; Nickel and Scheck, 1978). In vertebrate desmosomes, cytoplasmic tonofilaments are found in association with the electron dense plaques and, in the case of zonulae and fasciae adherentes, these filaments appear to be actin-like in composition. Moreover filaments may run through the cytoplasm of some cells forming an interconnected system, locating on the cell membranes at the sites of the desmosomes (see Staehelin and Hull, 1978 for a diagrammatic representation). This type of system would allow the transmission of tension through the cell. Where the filaments are contractile (7 nm), they may also be involved in controlling cell shape. On the other hand, the non-contractile filaments (1 0 nm) appear to form a structural framework for the cytoplasm and may also be involved in the passive distribution of shearing forces within the tissue. Whether such a network exists in insect cells is not known but clearly few desmosomes are associated with systems of tonofilaments as complex as in vertebrates and, where microtubules are present, the intimacy of their relationship with the junction is not clear.

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3.6

DEVELOPMENT

Little detailed work seems; to have been done on the formation of desmosomes in invertebrates, in contrast to the situation in vertebrate tissues. Considerations of the morphology of formation, timing in development of the appearance (and disappearance) of desmosomes, the stability of the forming and mature junctions and the involvement of the two cells in the process and symmetry of development are discussed and summarised by Campbell and Campbell ( 1971), McNutt and Weinstein (1973), Staehelin (1974) and Overton (1974). Within the invertebrates, Shivers and Brightman (1977) have followed the development of hemidesmosomes in the regenerating nerve root sheath of crayfish by means of freeze-fracture techniques. They show the alignment and clustering of 12 to 13 nm EF particles as the junctions develop. However there are doubts as to the classification of these junctions (see Section 3.4.1 and Lane, 1978a), and the evidence indicates that these clustering particles could be forming gap junctions (see Section 4.7.1) which are known to be present in the sheath of the crayfish nerve cord (Lane and Abbott, 1975). Lane and Swales (1978a) describe the formation of gliallperineurial and glial/glial maculae adhaerentes in the development of Calliphora larva. The development of cytoplasmic plaques appears to precede the intercellular elaborations. The intercellular separation appears regular and the electron density of the plaques is striking at a stage when the intercellular material is still amorphous. This material becomes at first faintly striated and then, as the junction elongates to its mature dimensions, the intercellular substance becomes clearly fibrillar (Fig. 25, insert). An even earlier stage in the development of desmosomes has been described in the pupal nervous system of Manduca sexta (Lane and Swales, 1979c). Here the membranes become aligned, showing a very regular intercellular space. However, at this stage, there are no cytoplasmic electrondense plaques nor is the intercellular material structured in any way. It is of interest to note that the precise alignment of membranes is achieved in the absence of observable cytoplasmic o r intercellular superstructures of filaments or microtubules. Although desmosomes are found in many mature tissues, they may be formed only transiently at some stages in development, and may be lost as soon as the junctions of the mature tissue are established (Poodry and Schneiderman, 1970; Reinhardt et al., 1976). A similar situation has been described in vertebrate tissues (see Overton, 1974) where, however, the transient desmosomes are found to differ in structure when compared with their mature counterparts, being simpler, smaller and with no fibrous attachments.

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Gap junctions I N T R O D u CT I O N

Gap junctions were originally described as such (Revel and Karnovsky, 1967) because the cell membranes in the regions in question were separated by a narrow gap of only 2 to 4 nm. They had been earlier referred to as a “nexus” (Robertson, 19F3); Dewey and Barr, 1964), and more recently have been termed macula communicans (Simionescu et al., 1975), because they appear to be the sites of cell-to-cell communication. These sites of communication are in the form cf special low resistance pathways between cells, through which the direct exchange of substances can occur; that which is transferred may be ions and/or metabolites and the cells are then said to be ionically or metabolically coupled. The importance of arthropods as experimental material in the elucidation of the function of gap junctions was evident from the beginning in that the first ionically coupled cells to be described were neurones of the crayfish (Furshpan and Potter, 1957). Subsequent studies on such coupling between excitable cells were made in crustacea (for e.g. Asada et al., 1967; Payton, Bennett and Pappas, 1969; Peracchia, 1973a, b, 1974) and other excitable tissues (see Bennett, 1977, 1978; Sotelo, 1977) where intracellular microelectrode techniques demonstrated low resistance pathways between the cells. Transfer of tracers indicated the degree of their permeability and fine structural studies elucidated their characteristic thin-section and freezefracture appearance. These specialized electrical synapses are now referred to as electrotonic, low-resistance or gap junctions. Low-resistance pathways have also been found to occur between nonexcitable cells in epithelia and other tissues, as well as as between cultured cells. Again, arthropod material has featured significantly since the salivary glands of dipteran flies (Loewenstein and Kanno, 1964; Loewenstein, 1976, 1977; Loewensteinet al., 1978b) have been used as an elegant test system to study such parameters as the molecular weight range of substances capable of being transferred between coupled cells and the effects of cations on junctional permeability. Cultured insect cell lines have also been employed to study uncoupling and the factors which affect this phenomenon (Gilula and Epstein, 1976). Gap junctions have also been implicated in metabolic coupling or cooperation, by which is implied the cell-to-cell transfer of metabolites (SubakSharpe et al., 1969). This phenomenon has been little studied in arthropod systems and has tended to be most frequently analysed in cultured mammalian cells where it has been shown that metabolically coupled cells may also be ionically coupled (Gilula et al., 1972). Exchange of regulatory molecules between coupled cells in the coordination of development has

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also been suggested in a variety of different systems, but in insects, thus far, gap junctional communication has been found to play no clear-cut role in differentiation, for example, of cuticular pattern (Warner and Lawrence, 1973; Lawrence and Green, 1975). There is suggestive evidence in the insect nervous system, however, where interglial gap junctions are present throughout almost the entire larval and pupal development (Lane and Swales, 1978a, b, 1979a, c), to indicate that exchange of regulatory substances may take place throughout larval and pupal growth. The actual disappearance of gap junctions has often been thought to be the developmental cue for cell determination (see Section 4.7.2) and here again relatively few arthropod systems have been studied, save, for example, grasshopper embryonic CNS (Goodman and Spitzer, 1979) and the locust eye (Eley and Shelton, 1976) where it is possible that molecules exchanged between cells, prior to the uncoupling or junctional disappearance, may provide the signals to trigger off the next stage of development. In the arthropods, as well as many other invertebrates, nonexcitable tissues are found to be coupled by gap junctions, which often co-exist with septate junctions (for example, Hudspeth and Revel, 1971;Gilula and Satir, 1971; Rose, 1971; Hand and Gobel, 1972; Skaer and Lane, 1974; Caveney and Podgorski, 1975; Lane et al., 1977a; King and Spencer, 1979). This originally led to difficulties in determining which junction was the actual site of cell-to-cell communication (see Section 4.6.3). The septate junction was the more obvious structure, indeed the only one seen in early investigations, and hence seemed the likely candidate (Wiener et al., 1964; Loewenstein and Kanno, 1964; Gilula et al., 1970; Loewenstein, 1973). However, the fact that in crayfish septate axons (Asada et af., 1967) and also in many vertebrate tissues, cells known to be coupled, apparently possessed only one type of junctional specialization, the gap junction (for example, Revelet al., 1971; Pinto da Silva and Gilula, 1972; Gilulaetal., 1972; Pinto da Silva and Martinez-Palomo, 1975; Larsen, 1975), helped clarify the situation. The gap junction has now emerged as the single intercellular structure which is organized in such a way as to permit coupling by allowing interchange of ions and small molecules between cells via the channels present in their component particles. X-ray diffraction studies have enabled the molecular architecture to be analyzed so that models can be produced (Caspar et al., 1977; Makowski et al., 1977). Although the mere presence of gap junctions cannot alone be taken as proof that molecules are being exchanged between the cells thus coupled, there is evidence from a variety of sources to verify the fact that different substances can by physically interchanged from cell to cell via the gap junctions. For example, low resistance connections have been found to be associated with the capacity for cell-to-cell transfer of fluorescent dyes such

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as Procion yellow or fluorescein (Kanno and Lowenstein, 1964; Payton et al., 1969), labelled nucleotides (Subak-Sharpe et al., 1969) and labelled amino acids and small peptides (Simpson et al., 1977). (For tables giving details of examples of coupled cells and permeant molecules, see Bennett, 1978; Loewenstein, 1976; Loewenstein et al., 1978b). In recent years several reports have appeared which review the structural features found in gap junctions in vertebrates (Bennett, 1977,1978; Gilula 1977, 1978; Larsen, 1977a; Revel, 1978) but the structural features in the invertebrates may show some deviation from these (Flower, 1977; Lane, 1978a). A description of those found in arthropods follows.

4.2 4.2.1

STRUCTURAL FEATURES

Thin-section appearance

In thin-sections, gap junctions are characterized by a reduction of the usual intercellular cleft between the membranes of apposing cells down to 2 to 4 nm (Fig. 26). Because of this they have also often been referred to as regions of “close” membrane apposition or close junctions in insect tissues (Hagopian, 1970; Burger and Uhrik, 1972; Chi and Carlson, 1976b). There is often a cross-striated appearance across this gap (Figs 26 and 27) which is no doubt due to the component junctional particles which bridge the cleft between the cells. This very narrow gap is usually only visible when en bloc uranyl acetate staining is employed (Farquhar and Palade, 1965); without this additional membrane enhancement the adjacent membranes of such junctions seem to be directly apposed o r fused. Such membrane apposition was mistakenly referred to as being a tight or occluding junction by earlier investigators of insect material (for example, Locke, 1965; Osborne, 1966; Berridge and Gupta, 1967; Grimstone et al., 1967; Maddrell and Treherne, 1967; Smith, 1968; Stuart and Satir, 1968; Oschman and Wall, 1969; Smith et al., 1969b; Schurmann and Wechsler, 1969; Lane and Treherne, 1969, 1970; Treherne e f a/., 1970; Zacharuk et al., 1971; Leslie and Robertson, 1973; McLaughlin, 1974a; Peacock and Anstee, 1977b) because they had not en bloc stained the tissues and the intercellular space could not be resolved. The appearance of a gap junction is often referred to as being septilaminar (heptalaminar or 7-layered), where the true tight junction (and indeed the less highly resolved image produced without en bloc staining) is a pentalaminar (or 5-layered) structure. Gap junctions being extensive regions of close membrane apposition are distinguishable from tight junctions which usually take the form of tiny punctate appositions. Recognizable septilaminar gap junctions were first seen in insect tissue in epithelial cells (Hagopian, 1970). In this context, however, it is of interest that as early as

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Fig. 26 Thin-section of a lengthy gap junction, from the perineurium of the cockroach, Periplanera americana,en bloc stained with uranyl acetate. Note the close apposition of the two unit membranes separated by a reduced intercellular cleft of 2-4 nm, with the occasional appearance of cross-striations. x 181 500 Fig. 27 Thin-section of a lanthanum-impregnated gap junction from the rectal pad cells of a cockroach. The normal intercellular clefts (C) can be seen to narrow where the gap junction begins; as the section starts to cut tangentially across the gap junction, through the plane of the membrane, the gap junctional sub-units can be resolved as non-opaque particles in negative contrast against the lanthanum-stained background. In this preparation the sub-unit packing is fairly regular and the central channel can be seen in some of the particles. The central pore is more clear-cut in the insert, which is from the primitive arthropod, Limulus, after lanthanum staining. x 146 400: insert x 289 400

1965, in examining the so-called zonulae occludentes of the caterpillar Calpodes, Locke commented that “the intercellular material in the tight junctions is frequently discontinuous with a repeat pattern like the junctions in synaptic disc membrane complexes”. Since he refers here to Robertson’s (1963) work which first described the nexus or gap junction, it is clear that what Locke was actually seeing, although he did not appreciate the fact, were gap junctions! Equally Trujillo-Cenoz (1965) showed a “synaptic contact” in fly eye which is undoubtedly a gap junction and similarly,

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Landholt and Ris, in 1966, studying soma-somata interneuronal junctions in the wood ant, described electrical junctions, which were “characterized by a diminished width of the (synaptic) cleft as well as by a typical crossdirectional ‘subunit pattern’ in the junctional membranes”. 4.2.2

Lanthunum staining

After treatment with lanthanum, either colloidal (Revel and Karnovsky, 1967) or ionic (Machen et al., 1972; Lane et al., 1977a) the gap junctional cleft becomes heavily stained. This intercellular space associated with gap junctions can also be negatively stained with tannic acid, ruthenium red and phosphotungstate or uranyl salts (van Deurs, 1975; Zampighi and Robertson, 1973). The extracellular space becomes, in these circumstances, indistinguishable from the dense outer leaflets of the membranes of the two adjacent coupled cells, producing a pentalaminar appearance with a gap of about 7.5 to 8.5 nm (Fig. 27) instead of the usual ca. 3 nm. Again, cross striations may be seen (Fig. 27), while, in tangential sections, when the junction is viewed en face, a lattice is apparent, with the component subunit particles revealed as electron lucent spots or particles lying in an electrondense network-like background (Fig. 27). The en face images demonstrate the relatively loose packing of the particles in the arthropod macular gap junctions. These non electron-dense spots are presumed to be the points where the intra-membranous junctional particles of the two apposed and coupled cells lie opposite each other, with particle subunits in contact, and the junctional channels of the two opposed particles in register; the central channels frequently take up the lanthanum and appear electron-opaque (insert, Fig. 27). This feature provides evidence for the presence of a central pore through which the exchange of ions and small molecules is thought to occur. 4.2.3 Freeze-fructured uppeurar1c.e Evidence for gap junctions by freeze-cleaving in insects was first put forward by Flower in 1972 and the differences they exhibited from those of vertebrates were mainly in terms of their cleaving characteristics; the junctional particles showed preferential fracturing onto the E face (then termed the B face) so that they were termed B-type or inverted gap junctions. The particles were about 13 nm in diameter, and left complementary pits in the P face (then termed A face) (Fig. 28). The reduced intercellular cleft is obvious when the fracturing plane cleaves across the junction (insert to Fig. 28) and most subsequent reports reveal that the tendency for the particles to fracture onto the E F (although some may remain on the PF- see Fig. 29 and

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Section 4.4.1) and to measure approximately 13 nm (the range is about 12 to 14 nm) is fairly universal in arthropod tissues. Although a range of smaller subunit sizes has been reported in lanthanum-stained insect gap junctions, for example 6.5 to 9 nm (Caveney and Podgorski, 1975) or larger, it must be remembered that the shadowing that occurs when replicas are made produces an increase in the actual size due to the heavy metal deposition (see discussion in Skaer and Lane, 1974). Particle separation is irregular, or, if uniform, is usually about 12 nm (Flower, 1977) and occasionally fused aggregates of two of more junctional particles occur (double arrows in Fig. 28). However, here the possibility of shadowing artefacts should be borne in mind. The junctions appear as irregular macular aggregates of intramembranous particles; each junction may vary from about 30 nm to several pm in diameter and may contain only a few particles or many 100s of particles (Fig. 30). In some cases, linear E F arrays of 13 nm gap junctional particles are observed in mature tissues (Fig. 31) (see Section 4.5, point 4). Central channels may be observed in the individual gap junctional particles after shadowing (as in Fig. 33, insert). The size of these pores in insects has been calculated on the basis of the molecular weights of molecules permeating these ,junctionalchannels (Simpsonetal ., 1977) and ranges from about 1 to 2 nm Comparable pores have been seen in replicas of such arthropod tissues as the moth (Lane and Swales, 1979a), collembola (Dallai, 1975),Lirnulus (Johnsonet al., 1973; Lane, 1978b) and the crayfish (Peracchia, 1973b). Although some earlier investigators claimed that the channels seen in replicas are artifacts due to decoration effects or plastic deformation (Plattner et al., 1975), the more recent X-ray diffraction studies tend to corroborate their existence, at least in mammalian tissues (Casper et al., 1977; Makowski et al., 1977). In some cases, the resolution may be sufficiently good to discern the subunits which compose each particle and surround the central pore; originally, in crayfish tissue, there appeared to be 6 of these hexamers (Peracchia, 1973b) but recent studies suggest that there may in some cases only be 4 tetramers (Peracchia and Peracchia, 1978). Structural diversity may occur in the gap junctions within a single organism, even within the same tissue. Thus in the caecal epithelium of the crustacean Orchestia two types occur that differ in size, spacing and polarity of their intermembranous particles (Graf, 1978b). Also, in crayfish giant Fig. 28 Freeze-fracture replica showing typical circular arthropod gap junctions with E face (EF) particle aggregates and P face (PF) pits. Some particles lie so close to one another as to appear fused (double arrows). Note the presence of PF particles, occasionally aligned into linear ridges (PF arrow) with complementary EF grooves (EF arrow). There is a reduction in the intercellularcleft at the gap junctional regions (* and in insert). x 44 100;insert x 79 200 Fig. 29 Replica of gap junctions with E face (EF) particles and complementary PF pits with a number of particles which fracture onto the P face. x 45 400

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axon gap junctions, the component globules are organized into two different arrangements, both hexagonally packed but with different unit “cells” (Peracchia, 1973a, b); these, however, have been shown to be due to changes in junctional membrane permeability, from high to low coupling resistance (Peracchia and Dulhunty, 1976) (for a further discussion of this, see Section 4.6.3b). Morphologically distinct kinds of gap junctions with regard to particle distribution, size and spacing in freeze-fracture replicas have been found to occur between different cell types in the same organism as well. For example, in locust heart muscle, linear gap junctions occur (Fig. 31) while other locust tissues possess typical macular plaques. In Planaria, three such types exist; these are thought to relate to the specialized functions of the different tissues in which they are found, so that, perhaps, they have differing permeabilities, one shuttling food reserves, another ionic currents, and a third, exchanging metabolic or development signals (Quick and Johnson, 1977). Although such structural differences between the gap junctions in insect tissues are not common, subtle distinctions in function need not necessarily be reflected in obvious ultrastructural differences, since a small change in channel diameter, for example, would undoubtedly not be recognizable (Pappas et al., 1971; Bennett, 1978). Whether tissues are glycerinated before or after fixation and whether they are fixed or unfixed, may affect the fracturing properties of the gap junctional particles (Flower, 1977). For example, the anastomosing particulate network appearance of unfixed B-type gap junctions is artifactual and induced by glycerol (Flower, 1977). 4.3

MODEL DERIVED FROM STRUCTURAL EVIDENCE

After a thorough analysis of the information gleaned about gap junctions from thin-sections, lanthanum-impregnated material and freeze-fractured replicas, it is possible to construct a model to explain their three-dimensional structure insofar as the evidence permits (Fig. 32). Although a number of reconstructions have been proposed for vertebrate gap junctions (for example, McNutt and Weinstein, 1973; Staehelin, 1974; Pappas, 1975; Makowskietal., 1977; Bennett, 1977; Staehelin and Hull, 1978) there seem to have been only very simplified versions put forward for those of arthropods (Pappas et al., 1971; Satir and Gilula, 1973; Peracchia, 1973b; Fig. 30 Freeze-fracture replica from the moth, Manduca sexfa, showing the EF particle aggregatestypical of insect gap junctions. Note the variability in size and number of component particles in these gap junctions, their relatively loose packing, and the frequency with which they are to be found. x 79 600 Fig. 31 Freeze-cleave replica from the dorsal vessel or heart of the locust, Schistocerca gregaria. These 13 nm EF particles have the features characteristicof gap junctions but occur in a linear instead of a macular configuration. x 74 000

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Loewenstein, 1976). The one presented here (Fig. 32) attempts to incorporate information from all possible perspectives; it draws attention to the EF fracturing position of the junctional particles and the complementary pits produced in the P face as well as indicating the appearance in both /-

CYTOPLASM-/

Fig. 32 Model of an arthropod gap junction showing the junctional particles which lie in apposition so as to straddle the extracellular space with the central channels aligned. In replicas the junctional particles fracture onto the E face and the central channel can be resolved; complementary pits are to be found on the P face. Lanthanum infiltrates the intercellular space as well as the pores within the junctional particles, Although depicted as being regularly packed, the particles are in fact usually irregularly arranged. Each particle is subdivided into sub-units which may be 4 or 6 in number (not shown on diagram). These particles are seenen face in tangential sections as non-opaque spots against an electron-dense background

lanthanum-infiltrated sections and en bloc uranyl acetate-stained thinsections. The lanthanum sits in the extracellular space around the junctional particles and also frequently, presumably artefactually (see Bennett et al., 1972) enters the central channel that links opposing particles; this gives rise to the striations seen in transverse sections and to the lattice or network effect around the unstained particles in en face views. In Fig. 32 the particles that comprise the junction are depicted as single globules. The X-ray diffraction evidence thus far available, in support of 6 hexameric sub-units making up each particle, is limited to vertebrates (Casparet al., 1977; Makowski, et al., 1977). Freeze-fracture studies on crustacean septate and other gap junctions suggests hexamers (Peracchia, 1973b) or tetramers (Peracchia and Peracchia, 1978) so there is no general concensus yet as to the number of sub-units per particle in arthropod systems. 4.4

DISTRIBUTION OF GAP JUNCTIONS

4.4.1 Homocellular, heterocellular and autocellular junctions in arthropod tissues Gap junctions are commonly homocellular and link cells of the same morphological type, which then can presumably be synchronized physiologically.

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In thin-sections, these homocellular gap junctions have been found between a wide variety of insect cell types and include salivary gland cells (Oschman and Berridge, 1970; Rose, 1971; Burger and Uhrik, 1972; Leslie and Robertson, 1973; Berridge et a[., 1976b), cells of various parts of the gut including midgut, proctodeum and rectum (Berridge and Gupta, 1967; Reger, 1970;NoirotandNoirot-TimothCe, 1971a,b, 1976,1977; Wall and Oschman, 1973; Noirot-TimothCe and Noirot, 1974b; Ito et al., 1975; Dallai 1975; Lane, 1978a, 1979a, c; Noirot-TimothCe et al., 1979; Skaer et al., 1979), haemocyte capsules (Baerwald, 1975), cyst cells of the testis (Szollosi and Marcaillou, 1977; Jones, 1978; Skaer and Jones, 1979), follicular epithelial cells of the ovary (Huebner and Anderson, 1972; Mahowald, 1972; Woodruff, 1979), glial cells of the nervous system (Lane and Treherne, 1972a, 1973; Skaer and Lane, 1974; McLaughlin, 1974a, b; Lane et al., 1977a; Ne’eman and Spira, 1977a; Lane and Swales, 1978a, b, 1979a, b; Lane 1978a) or nerve cells (Osborne, 1966; Landholt and Ris, 1966; Morris and Steel, 1977), sub-cuticular surface epidermal cells (Hagopian, 1970; Caveney and Podgorski, 1975; Lawrence and Green, 1975),cellsoftheeye(EleyandShelton,1976; Ribi, 1978; NickelandSheck, 1978; Shaw, 1979; Carlson and Chi, 1979; Lane, 1979d), fat body cells (Skaer and Lane, unpublished observations), cardiac muscle cells (Fig. 3 l), glandular epithelial cells (Stuart and Satir, 1968), the undifferentiated cellsof haemocytopoietic organs (Monpeyssin and Beaulaton, 1978), imaginal disc cells (Poodry and Schneiderman, 1970) and cells of Malpighian tubules (Wall et al., 1975; Dallai, 1975; Skaer 1979; Green et al., 1979). After Flower’s (1972) initial freeze-cleave study on insect tissue, freezefracture reports of gap junctions in arthropods have been numerous and include, not only tissues from insects (for example, Noirot-TimothCe and Noirot, 1974; Skaer and Lane, 1974; Flower and Filshie, 1975; Skaer ef al., 1975; Baerwald, 1975; Lane et al., 1977a; Flower, 1977; Ne’emen and Spira, 1977a. b; Lane and Swales, 1978a, b, 1979a, b) but also tissues from other arthropods as well, such as from the xiphosauran, Lirnulus (Gilula, 1973; Johnson et al., 1973; Lane and Harrison, 1978; Harrison and Lane, 1980), from ticks (Binnington and Lane, 1980) and from crustacea (Gilula, 1972b; Peracchia, 1973a, b, 1974; Perrachia and Dulhunty, 1976; Graf, 1978b). Shivers and co-workers report on desmosomes and hemi-desmosomes in crayfish tissue (Shivers and Chauvin, 1977; Shivers and Brightman, 1977) that bear all the freeze-fracture attributes of the gap junctions which they probably actually are (see Section 3.4.1). For the most part, these arthropod gap junctions have EF particles. However, there are exceptions. The perineurial/glial gap junctions of crayfish, seen in thin-sections (Lane and Abbott, 1975; Lane et al., 1977b),

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are reported, by freeze-fracture, either to possess conventional EF particles and P F pits (Brightman et al., 1975) or to exhibit particles that are irregularly segregated on both P and E faces (Perracchia and Dulhunty, 1976). In other crustacean tissues, such as caecal epithelium, the gap junctions are found to have mainly P F particles (Graf, 1978b). Gap junctions are sometimes heterocellular; that is, they may link two different cell types within the same organism which are then able to exchange substances and so are functionally coupled. These usually are indistinguishable structurally from homocellular gap junctions (Johnson et al., 1973). For example, the ooplasm of oocytes may be coupled to the encompassing follicle cells in moth ovary (Woodruff, 1979), the principal cells of the insect rectum may be associated by gap junctions to basal cells (Noirot and Noirot-Timothte, 1976, 1977), perineurial cells are linked to underlying glial cells by communicating junctions in the central nervous system of a variety of insects (Lane, 1978a; Lane and Swales, 1979a), neuroblast cells are electrically coupled to epithelial cells in grasshopper embryos (Goodman and Spitzer, 1979) the epithelial cells of the midgut of Limulus are frequently coupled to reserve cells (Johnson et al., 1973), glial and visual cells in Limulus eye are associated by “quintuple-layered junctions” (Lasansky, 1967), and epithelial and underlying interstitial cells are coupled by gap junctions in the midgut of certain fresh water crustacea and terrestrial Arachnids (Reger, 1970). There are a number of examples of heterocellular gap junctions in vertebrate tissues, and the details of these associations are described by Larsen (1977a). It has been established, by co-culturing insect and mammalian cells lines, that low-resistance heterocellular gap junctions cannot be extensively established between two such different cell types (Epstein and Gilula, 1975, 1977). This led these authors to suggest that different gap junctional phenotypes must exist, as is suggested by their differing fracturing characteristics and particle sizes. Although a low incidence of coupling was found between heterologous cell lines derived from different arthropod orders, insect cell lines from the same order will couple extensively (Epstein and Gilula, 1977), suggesting an insect interorder specificity. In this vein, it is interesting that rat granulosa cells may transmit hormonal stimuli through permeable gap junctions formed in culture with mouse myocardial cells: these stimuli were shown to be passed by means of a second messenger, thought to be cyclic AMP (Lawrence et al., 1978) and clearly here functional heterocellular gap junctions have formed between cells from two different genera. No communication specificity appears to exist, therefore, in this instance. Very few examples of such communication-specificity have been as yet documented of which one is the insect interorder specificity (Epstein and Gilula, 1977).

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The functional significance of autocellular gap junctions, referred to as “reflexive” gap junctions (Herr, 1976), which occur between two projections of the same cell (for examples in vertebrates see Larsen, 1977a) is still obscure. Perhaps these have to do with the exchange of ions or trophic molecules which are important locally but which have difficulty in diffusing any great distance. Autocellular gap junctions have been reported between salivary gland cells in the adult blowfly (Oschman, 1976, in Larsen, 1977a), and also occur in tracheal cells where the mestracheon exhibits gap junctions (Lane and Skaer, unpublished observations). They often appear between attenuated cytoplasmic processes of cells in insect tissues [for example, glial cells (Lane et al., 1977a) where diffusion could well be a problem to be overcome. 4.4.2

Other invertebrate tissues

Gap junctions are fairly ubiquitous and are also found in a wide range of other invertebrate groups. In the molluscs (Flower, 1971; Gilula and Satir, 1971), chaetognaths (Duvert et al., 1978) and tunicates (Lorber and Rayns, 1977) they are vertebrate-like in that their component particles fracture onto the P face leaving E F pits, and the particle separation is usually 9-10 nm. Some particles also cleave onto the PF in Hydra (Hand and Gobel, 1972; Filshie and Flower, 1977; Wood, 1977), and in some, but not all, planarian worms (Quick and Johnson, 1977; Flower, 1977). In annelid worms, the situation is complex, but it appears that if the tissue is fixed the particles are mainly PF (Bilbaut, 1979), although some fracture onto the EF (Skaer, 1979b), while in unfixed material, they are primarily EF particles (Flower, 1977). In centipedes, the junctional particles fracture onto the EF in fixed preparations (Juperthie-Jupeau, 1979). A number of particles in the gap junctions of coelenterates adhere to the E face (Wood, 1977; Filshie and Flower, 1977) and here strong intercellular adhesive forces exist too, since fragments of cytoplasm often cover the junctions. This phenomenon can also be observed in some insect tissues (for example see Fig. 36, insert B) and annelid tissues (Skaer, 1979b). There is a good deal of variation in the shape of gap junctions in invertebrates; round or oval macular plaques occur in hydra, planaria, ragworms, centipedes and some arthropod tissues, but other arthropod tissues may have more irregular outlines (Flower, 1977). The centre-to-centre spacings of the invertebrate gap junctional particles is variable because they are rarely very tightly packed (Figs 28 and 30). In those cases where figures have been given the centre-to-centre separation for insects measures from 1 0 to 12 nm (Flower, 1972; Satir and Gilula, 1973; Baerwald, 1975; Dallai, 1975; Caveney and Podgorski, 1975) and

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holds also for crustacea (Hudspeth and Revel, 1971). In planaria and ragworms it appears to be about 12 nm too (Flower, 1977), while in Hydra and other coelenterates it is 9.5-11 nm (Hand and Gobel, 1972; King and Spencer, 1979) and in molluscs it is 10 nm (Flower, 1977). A larger repeat of 18-20 nm has been reported in crayfish nerve fibres (Peracchia, 1973b) but this may reduce to a smaller repeat when the cells are uncoupled (Peracchia and Dulhunty, 1976). 4.5

STRUCTURAL DIFFERENCES BETWEEN ARTHROPOD A N D VERTEBRATE GAP J U N C T I O N S

In thin-sections, the gap junctions of arthropods (for example, Hagopian, 1970; Reger, 1970; Noirot andNoirot-Timothie, 1971b; Laneetal., 1977a) appear virtually identical to those found in vertebrate cells although certain reports indicate that the intercellular gap is slightly larger (3-4 nm) than that of vertebrates(Paytoneial., 1969; Hudspeth and Revel, 1971; Rose, 1971). There is also the exception of the so-called “septate” gap or electrotonic junctions that couple the giant axons of crayfish (Peracchia, 1973a) when fixed in glutaraldehyde-H202solutions; the space between two axonal membranes may be as much as 4-5 nm wide (Zampighi et al., 1978). Electrotonic junctions in vertebrates have been divided into asymmetrical and symmetrical gap junctions, the former exhibiting greater electron opacity in the dendritic rather than the axonal side (Bennet et al., 1967) but this has not been observed in the insect electrotonic junctions thus far studied (Ribi, 1978). The only clear-cut morphological evidence of asymmetry is the presence of 80 nm vesicles in the cytoplasm near the presynaptic membrane of the rectifying electrotonic synapse in crayfish giant axons (Hanna et al., 1978). In freeze-fracture preparations, the insect junctions also have the same basic structure as vertebrate gap junctions, in that they are both composed of macular arrays of intramembranous particles (see McNutt and Weinstein, 1973; Staehelin, 1974; Gilula, 1974,1977; Bennett, 1977) and in that there is a reduction in the intercellular space when fracture faces cleave across the membranes composing a gap junction. Vertebrate gap junctions are, like those of insects, fairly ubiquitous, in their distribution between cells of different tissue types; they appear to be present in most kinds of tissue except for skeletal muscle, red blood cells and spermatozoa (Revel, 1978) and are absent in many differentiating neurons (Gilula, 1978). However, there are some freeze-fracture differences between aphropod and vertebrate gap junctions, in spite of their basic similarity. 1 Arthropod gap junctional particles in general tend to adhere to the E face, leaving complementary P face pits; the opposite fracture face possesses

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the junctional particles in vertebrates. There are, however, some recent reports suggesting that under different circumstances, with respect to fixing or cryoprotection, some of these particles in arthropod tissues may adhere to the PF (Flower, 1977; Wood, 1977; Graf, 1978b), but these are very much in the minority. 2 Arthropod gap junctional particles measured in freeze-fracture replicas are approximately 13 nm in diameter (Flower, 1972; Satir and Fong, 1973; Satir and Gilula, 1973; Flower, 1977; Gilula, 1977; Lane, 1978a) which is much larger than the 6 nm (Revel, 1978) to 7-8 nm (Gilula, 1978) particles that make up vertebrate gap junctions. However, since those of arthropods are larger particles, with different fracturing characteristics, they are readily distinguishable from the tight junctional particles which are smaller, 6-10 nm, and fracture onto the P face. This convenient distinction is not found in vertebrates where PF particles of “ambiguous identity” may occur (Larsen, 1977a). In studying such phenomena as the mode of formation of gap junctions, in vertebrate tissues, where tight and gap junctions co-exist on the same membrane face, no distinction can be drawn between the particles destined to form one of the two junctional types (Revel, 1978) until they have assumed the distribution which is characteristic of their mature state. This is not so in arthropods which is a distinct advantage in following the changes in particle distribution that occur during junction formation (Lane and Swales, 1978a, 1979a, b; Lane, 1978a, b). 3 The gap junctions of arthropods are composed of intramembranous particles which tend to lie in loose formations (Figs 28 and 30) either as disordered arrays or with a 1 2 nm spacing (Flower, 1977), not in the closely-packed hexagonal arrays with a 9-1 0 nm spacing that are characteristic of vertebrate macula communicans (see for example, Gilula, 1974). Differences in the degree of order with which the particles are arrayed may relate to whether or not they are coupled (Peracchia and Dulhunty, 1976; Perrachia, 1978). In this context it is of interest that two types of hexagonal arrays have been reported in vertebrates, one with the usual 9-10 nm spacing (Staehelin, 1974) and the other with a 19-20 nm spacing (Staehelin, 1972). Factors affecting coupling will be considered in greater detail in the later section on the functional significance of gap junctions (Section 4.6.3). Recent studies on mammalian gap junctions with a rapid-freezing technique involving liquid helium-cooled copper block surfaces, has produced freeze-fracture replicas with randomly-distributed junctional particles, rather than hexagonally-packed ones (Raviola et af., 1978). Using this system, the junctions in tissues suffering from anoxia contract into closely packed arrays, suggesting that they are uncoupled and in a high resistance state. This indicates that the hexagonally-packed gap junctional particle arrays normally seen in vertebrate tissue have been uncoupled by the

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preparative procedures. In coupled cells, low resistance junctions should therefore appear as randomly distributed particles, or connexons, with little mutual affinity (Raviolaet a [ . , 1978). This work supports the hypothesis put forward by Peracchia that states that closely-packed particles represent the uncoupled state. It is interesting to note that insect gap junctions often appear in a relatively loosely packed state; this may mean that conventional fixing does not abolish electrical coupling between insect cells (cf. vertebrate and crayfish cells, Bennett et al., 1972) or that their packing arrangements do not accurately reflect their state of coupling. 4 The size of the gap junctional aggregates in arthropods is highly variable; the plaques in insect tissue may range widely both in diameter and in the number of component particles (Fig. 30), from 5 to 10, up to many 100s. Such small plaques are less frequent in vertebrate tissues, although they have been reported in such situations as embryonic tissues (Argue110 and Martinez Palomo, 1975). Ordinarily, the insect junctional areas are not particularly extensive but, like the vertebrates, larger areas of cell surface may occasionally be junctional, especially in the case of developing junctions (Fig. 34). Although the particles in arthropod tissues are usually in the form of macular arrays, they may sometimes be ring-shaped (Lane, 1978b) or in the case of insect cardiac muscle, linear configurations may occur (Fig. 31). Interestingly, linear arrays have also been reported in cardiac tissue of vertebrates (Larsen, 1977a; Mazet, 1977; Kensler et al., 1977; Shibata and Yamamoto, 1979) as well as in certain other tissues (Raviola and Gilula, 1973; Pricam et al., 1974; Fujisawa et al., 1976; Simionescu et al., 1976). This is highly unusual and more typically such irregular patterns only occur during development; there are also certain distinctions in the mode of formation of gap junctions in arthropods in comparison with those of vertebrate cells which will be considered later (see Section on Developing gap junctions 4.7.1).

4.6

FUNCTIONS O F G A P IUNCTIONS

4.6.1 Adhesion Since gap junctions serve to maintain a very close cell-to-cell association between the cells that are coupled, they inadvertently must serve in an adhesive capacity to some extent. This has been particularly stressed in certain situations, (for example, Lorber and Rayns, 1977; Pannese et al., 1977; Wood, 1977) and in insect tissues, cytoplasm above gap junctions frequently adheres to these as though held by strong adhesive forces (Fig. 36, insert B). In particular, insect interglial gap junctions have been thought

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to be a specific device for cell-to-cell adhesion (Ribi, 1978), while in dipteran midgut, polysaccharides found in the intercellular 2 to 3 nm gap, have been interpreted to function as a “cement” substance for cellular adhesion (Reinhardt and Hecker, 1973). In further support of this, gap junctional complexes are found to be very resistant to disruption by mechanical or physical means, or by treatments such as divalent cation removal or proteolysis (Gilula, 1974; 1977). When cells are separated by these means, one of the dissociated cells retains the intact gap junctional complex (Berry and Friend, 1969). When unfixed coelenterate tissue is exposed to 25% glycerol, commonly used as a cryoprotectant, the septate junctions were lost after 5 min, whereas the gap junctions did not separate, even though considerable distortion of the tissues often took place (Filshie and Flower, 1977); this was taken to suggest that gap junctions may have a “localized bonding function”. Only treatment with hypertonic sucrose effectively splits the gap junctional complex (Goodenough and Gilula, 1974) but such a procedure has not thus far been applied to insect junctions. 4.6.2 Sieve-area effects In some instances, gap junctions have been considered to have a rather different function, in forming an intercellular sieve area for small molecules, that is, acting as a specialized barrier for controlled entry between cells (Forssmannetal., 1975). For example, such regulation of the speed of entry of molecules seems likely to be one function of gap junctions in the crustacean CNS. In the crayfish, unlike the insects, there is no ultimate blood-brain barrier to the entry of substances, but the rate of penetration through the perineurium is significantly slower than in “open” systems (Abbott et al., 1977). This partial restriction seems likely to be due to gap junctions which are found in the perineurium of the CNS (Lane and Abbott, 1975) but which are relatively infrequent or absent in the peripheral nerves which are quite patent to tracers (Lane et al., 1977b). 4.6.3

Cell-to-cell communication and low-resistance path ways

( a ) Historical introduction: gap junctions versus septate junctions as communication channels. Although the cytoplasm of cells has a low specific resistance, that of the plasma membrane is high. In many cases, however, this resistance can be overcome and adjacent cells are found to be electrically coupled, with low resistance pathways between them so that they behave like a synctium. The morphological basis for this coupling appears to be gap junctions. In recent years, in attempts to understand how these are regulated, many experiments have been carried out on the uncoupling of

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adjacent cells by reducing the permeability of the gap junctions. The most extensively studied systems are both arthropod in nature: the epithelial cells of dipteran salivary glands and the crayfish septate giant axons. These have been studied both electrically with intracellular microelectrodes and by exogenous tracer exchange; substances that uncouple the cells electrically are also found to inhibit the intercellular flow of tracers. However, as will be discussed later, the systems are more complex than this suggests and modulation in the degree of uncoupling is possible. In dipteran salivary glands, much of the early work on coupling was carried out by Loewenstein and his co-workers (Loewenstein and Kanno, 1964; Lowenstein, 1973, 1977). It is now clear that the cells of Chironomous or Drosophila salivary glands have both septate and gap junctions. However, it seemed originally that the former were providing the pathway for intercellular exchange (Bullivant and Lowenstein, 1968; Gilula et al., 1970) since the latter did not occupy a sufficiently large fraction of the lateral cell surface to be responsible for the degree of intercellular coupling observed (Rose, 1971). Doubt, however, began to be expressed as to the role of the septate junction in communication in the developing insect in 1970 (Poodry and Schneiderman). It was then found that when iso-osmotic fixatives were used, which d o not lead to the damaging cell shrinkage that otherwise would occur with a non-isosmotic fixing solution, very extensive regions of gap junctions were observed to co-exist with the septate junctions in dipteran salivary glands (Burger and Uhrik, 1972). In these salivary glands, although electrical coupling is easily disrupted, septate junctions are stable structures, and are unmodified by uncoupling (Bullivant and Loewenstein, 1968; Rose, 1971). Gap junctions are sensitive to osmotic disruption (Burger and Uhrik, 1972) and this suggested that the gap junctions were possible candidates for the sites of intercellular coupling. Poodry and Schneiderman (1970) eliminated the septate junction as the prime communication channel in Drosophila imaginal discs because they occur most frequently afier cell determination and pattern formation. Other evidence with microelectrode measurements and fluorescent labelling indicates that no low resistance pathways are present in the septate junctions of gregarines (Sniginevskaya et al., 1977). On structural grounds, as well as on the basis of morphometric analysis, it was found that in the epidermal cells of the larval beetle Tenebrio, the gap junctional membrane alone can account for the high electrotonic coupling recorded in this epidermal sheet (Caveney and Podgorski, 1975); analysis of the septate junction cast doubt on the possibility that it could serve as the communicating channel between these beetle cells. Although some investigators have not yet rejected the possiblity that the septate junctions may play some role in the intercellular coupling phenome-

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non (Gilula, 1978), the weight of current evidence may now be seen to support the thesis that the gap junctions represent the communication pathways; the more recent freeze-fracture and lanthanum studies, the evidence from the isolation and biochemical characterization of vertebrate gap junctions (Goodenough and Stoeckenius, 1972; Goodenough, 1974), together with the X-ray diffraction data (Casper et al., 1977; Makowski et al., 1977), all indicate that their structure is uniquely organized so as to permit transport of ions and small molecules through the central channel found in each of their component particles (see Fig. 32). Conversely, the conclusions as to the function of the septate junctions are still indefinite (see Section 2.7). The other arthropod system that has been studied in depth is that of the crayfish septate giant axons. After the original demonstration (Furshpan and Potter, 1957) of electrical transmission in certain synapses of the crayfish nerve cord, other studies established that procion yellow (Payton et al., 1969) and mixtures of fluorescein and microperoxidase (Reese et al., 1971), when injected intra-axonally could move into adjacent axons. Peracchia (1973a, b) demonstrated the existence of two kinds of particle packing within the gap junctions of crayfish giant axon synapsis, the channels of which are thought to be the sites of exchange of these molecules. The gap junctions of arthropod eyes have also been implicated in low resistance pathways, and, for example, are found coupling photoreceptor axons in the optic ganglion of the fly (Chi and Carlson, 1976; Ribi, 1978; Carlson and Chi, 1979); these are found at sites where electrical transmission is observed physiologically (Shaw, 1979). The junctions are symmetrical, and it has been suggested that they are sites of electrotonic synapses so that those retinular cells which share the same visual field are linked together through these low resistance pathways (Ribi, 1978). The functional significance of this coupling of the retinular cells in attaining optimal visual acuity seems clear-cut, since signal enhancement can occur through frequency-selective signal averaging via the gap junctions that connect photoreceptor axons (Shaw, 1979). Electrotonic coupling between adjacent photoreceptor cells in the lateral eye of Lirnulus (Lasansky, 1967) may have a similar physiological basis. There is also some evidence from other invertebrates in support of ionic coupling between excitable epithelial cells having its basis in recognizable gap junctions. In annelid worms (Bilbaut, 1979), the gap junctions in the bioluminescent scales have been analysed by both electro-physiological and freeze-fracture techniques and correlations are drawn between the communicating structures and low-resistance pathways. Moreover, electrophysiological evidence for electrical coupling in Hydru has been reported (Hufnagel and Kass-Simmon, 1976). This is taken to support the contention

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that the gap junctions are the structural basis of cell-to-cell communication, since gap junctions occur between cell processes extending into the mesoglica, and no other junctional type is present at these particular points, although homocellular septate junctions occur between the ectodermal cells and also between endodermal cells (Hufnagel and Kass-Simon, 1976). However, in the vast majority of cases, the presence of gap junctions in non-excitable tissues has been reported without any accompanying experimental evidence to show that molecules, possibly regulatory, are exchanged between the cells thus coupled. Any specific biological function of such communication is, therefore, as yet unclear (Gilula and Epstein, 1976). It is assumed that transport from cell-to-cell does occur, because in cultured non-excitable cells, a cell type with defective gap junctions has been shown to be communication defective (Gilula et al., 1972) but what the nature of the molecules exchanged may be is still obscure. ( 6 ) Regulation of gap junction permeability: effects of Ca++,hormones and transmission of hormonal stimuli Much of our information about the effects of calcium on gap junctional permeability stems from the work of Loewenstein and his colleagues. Originally, removal of calcium from coupled dipteran salivary glands led to loss of cell coupling (Nakaset al., 1966), while elevated levels of intracellular calcium were also found to induce uncoupling. It now seems that the permeability of the gap junctional membrane channels depends on the cytoplasmic Ca++concentration (Rose and Loewenstein, 1975a). This dependence has been shown by experiments on Chironomus salivary glands in which Ca++is injected into a cell, or the cytoplasmic Ca++concentration is elevated by Ca++ionophores or metabolic inhibitors, while the free cytoplasmic Ca++concentration is monitored in the gap junctional region by the luminescent protein aequorin. This protein is a specific Ca++indicator (Rose and Loewenstein, 1975b; Loewenstein, 1977) since its light emission is approximately proportional to Ca++concentration. At normal concentrations of Ca++,of about 1 0 - 7o~r less, the channels are permeable to a wide range of molecular sizes up to peptides of about 1200 to 1900 daltons (Simpson, Rose and Loewenstein, 1977) or to tracer molecules of up to M.W. 1000 (Caveney and Podgorski, 1975). However, it appears that within the Ca++range from less than ~ O - ’ M and up to 5 x ~O-’M (above which permeability €or all molecular species falls dramatically), there is a graded control by Ca++of junctional permeability (Rose et al., 1977). Although one cannot discount the possibility that a non-selective reduction in channel number takes place, this phenomenon may be due to a binding of Ca++to the junctional membrane in a way that induces a graded change either in the channel’s fixed charge or in its molecular configuration so p to reduce its effective size or induce channel misalignment (Loewenstein, 1977).

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Support for the effects of Ca++on the junctions include evidence that exists for calcium deposits in those regions of the plasmalemma where the junctional arrays occur (Oschman and Wall, 1972; Larsen, 1975). It is also claimed that ionic lanthanum can be used as a marker of calcium-binding sites (Weihe et al., 1977) and certainly it stains the outer membrane leaflets of gap junctions, as well as the central channel (Fig. 27). In crayfish septate axons, as in insect salivary gland, varying the intercellular calcium concentration also led to the observation that uncoupling occurred with elevated or depressed Ca++levels (Asada and Bennett, 1971). It has subsequently been demonstrated in freeze-fractured preparations of these crayfish giant axons that an increase in the degree of close packing of the component particles of these gap junctions occurs with the elevation of Ca++ (Peracchia and Dulhunty, 1976). This is paralleled by changes in permeability leading to electrical uncoupling; these changes are thought to be due to a conformational change in certain components of the gap junctional particles (Peracchia and Dulhunty, 1976) that effectively close the junctional channels. A decrease in junctional thickness and possibly also particle size occur as well. Recent studies on crayfish gap junctions by Zampighi (personal communication) reveal that, in vivo , ionic lanthanum binds to the intraparticle pore simulating the site of Ca++ binding. This supports the contention that perhaps Ca++acts to change the configuration of the gap junctional particle sub-units during graded permeability by binding directly to them. In this same system (Peracchia and Dulhunty, 1976; Zamphighi et al., 1978; Hanna et al., 1978) vesicles are present in the cytoplasm adjacent to these junctional regions in the presynaptic, but not postsynaptic giant fibre. The function of such vesicles, typical of conventional synapses rather than electrotonic junctions, is not clear, although it has been suggested (Potter, in Peracchia and Dulhunty, 1976) that they may be Ca++-sequestering organelles which would presumably release Ca++in response to appropriate stimuli. Support for the effects of Ca++on uncoupling arthropod cells is also t o be found from experiments with cultured cells. An insect cell line has been found to be rapidly affected by treatment with an ionophore which increases intracellular calcium; within minutes the cell population begins to uncouple, and by the end of one hour's exposure the cells are all rounded and disassociated (Gilula and Epstein, 1976). Such results are compatible with the observations on dipteran salivary gland cells and crustacean giant axons where rises id cytoplasmic calcium lead to uncoupling. Lowering the intracellular pH, independent of Ca++,has also been found to uncouple embryonic cells of vertebrates, such as Xenopus (Turin and Warner, 1977); this may mean that the acidity of the solutions rather than elevated <:a++may be affecting gap junctions in both isolated preparations

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(Peracchia, 1978) and in intact cells (Peracchia, 1977). Recent experiments show that, in vertebrate cells, exposure of gap junctions to lowered pH alone does lead to changes in their packing, producing crystalline arrays, so that both Ca++ and H+ may independently cause cell uncoupling by acting directly on the junctional molecules (Peracchia and Peracchia, 1978). Certain studies on uncoupling in insect salivary glands support this, in that it has been shown that both acidic and alkaline pH can lead to uncoupling and, in addition, agents that influence cell membrane components such as EGTA, lipase and trypsin also produce uncoupling at high concentrations (Suzuki et af.,1978). These agents also were considered to decrease gland stiffness and so are thought to influence the structural components of the cell membranes. Although it is not stated, some of the uncoupling here must be due to conformational changes in the junctional channels induced by the various exogenous agents. In contrast, other recent evidence, using the Chironomus salivary gland preparation, indicates that there is not a good correlation between uncoupling or channel closure and changes in intracellular pH (Rose and Rick, 1978); these authors contend that H+ may therefore not play an important role in the regulation of cell-to-cell channel permeability, at least not at pHs above 6.5 in insect cells. Hence, at the moment, no unequivocal statement can be made regarding the effects of pH on the conformational state of the gap junctional channels. With regard to the biological relevance of the effects of calcium and perhaps of pH, as well as Ca++levels being able to uncouple a cell community from a damaged cell, the graded control of gap junctional permeability by Ca++provides a means of selective transmission of intercellular molecular signals, offering advantages for the physiological regulation of cell-to-cell communication (Rose, Simpson and Loewenstein, 1977). An example of changes in what can move through the gap junctional channel occurs during insect development. In larval Tenebrio epidermal cells, lowered intercellular resistivity prior to metamorphosis represents an enhanced coupling between cells already linked by low resistance pathways (Caveney, 1976,1978). This could readily be interpreted as increasing the pore size of the already existing channels although it could also be due to the formation of new junctional channels. In either case, this transient elevation of ionic flux is presumed to be a means of tightly coordinating synthetic activities in the epidermis at metamorphosis and is thought to represent hormonal regulation of intercellular communication between the epidermal cells. Insect epidermal cells have been long known to be associated with one another by homocellular gap junctions (Hagopian, 1970), and in larval Tenebrio, gap junctions have been found to make up to 20 p’ercent of the junctional membrane (Caveney and Podgorski, 1975). There are several other reports on insect tissues that also implicate gap junctional involvement in the

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exchange of developmentally-significant molecules (Stuart and Satir, 1968; Poodry and Schneiderman, 1970). Support for this suggestion of hormonal regulation of coupling comes from the observation that the normal changes in coupling that occur in Tenebrio epidermal cells during metamorphosis can be mimicked in vitm with 0-ecdysone (Caveney, 1978). Electrical coupling is increased initially, suggesting exchange of signals to trigger cell proliferation and the generation of new epidermal cell spatial patterns; these changes are reversed by elevation of intracellular cyclic AMP or Ca++(Caveney, 1978). As discussed earlier, heightened Ca++concentration uncouples gap junctions in insect salivary gland (Rose and Loewenstein, 1975; Popowich and Caveney, 1976), crustacean giant axons (Peracchia and Dulhunty, 1976), and cultured insect leafhopper cells (Gilula and Epstein, 1976). Moreover, the effect of cyclic AMP on uncoupling is probably also due to increased cytoplasmic Ca++(Caveney, 1978). It seems clear that cyclic AMP passes through gap junctional channels (Lawrence et al., 1978) but that Ca++, which binds to the junctional molecules, does not (Rose and Loewenstein, 1976). It may therefore be that developmental events that are stimulated by hormones or Ca++changes,may be coordinated by cyclic nucleotides which diffuse via gap junctions between the cells of the affected tissue (Caveney, 1978). Interestingly, although the segmental boundaries in the epidermis of the milkweed bug Oncopeltus are compartment borders, marked by a change of cell shape and sometimes pigmentation difference, they too are linked by gap junctions (Lawrence and Green, 1975). This compartment border therefore allows for intersegmental exchange of small ions; these border junctions may however carry current rather than small information-bearing molecules such as cyclic AMP (Warner and Lawrence, 1973; Caveney, 1976) since the segmental boundary is known to delimit functional developmental compartments (Lawrence, 1971). Hence, although low resistance pathways may be required to establish pattern in developing epidermal fields, the boundaries are not defined by their absence (Lawrence and Green, 1975). In vertebrate tissues, gap junctions may be intimately associated with the production of cyclic AMP in that the junctions themselves may be aggregates of macromolecular complexes made up of adenylate cyclase and peptide hormone receptors (Albertini et al., 1975; Larson, 1977b). These complexes are thought to aggregate at points of cell contact to form gap junctions. Support for this contention came from the observation that the internalization of gap junctions is stimulated by proteinaceous hormone activity (Larsen, 1977b). It seems that hormones that act via adenylate cyclase may also stimulate gap junctional turnover which is thought, by some

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investigators, to occur by interiorization followed by degradation of the annular gap junctional vesicles so formed (for a detailed account of the intricacies of the various hormone effects, see Larson 1977b). There is no supportive evidence that this occurs in insect cells, particularly since annular gap junctions, signifying internalization, have not thus far been reported in arthropod tissues (Lane, 1978a) (see Section 4.7.2). (c) Structural modification by hormonal stimulation Not only does it seem that gap junctions serve to transmit hormonal stimuli but the assembly or break-down of the gap junctions themselves is controlled by hormones. In insects, ecdysone initiates pupal development by terminating diapause (Chippendale, 1977), or, in non-diapausing lepidopteran or dipteran pupae, by initiating the changes of tissue organization characteristic of pupal differentiation; in the CNS this appears to involve, in glial cells, the stimulation of gap junction disaggregation (Lane and Swales, 1978b, 1979b, c). Hence, although in other systems hormones may act to stimulate junction assembly (Brownet al., 1979), in the insect pupae, they induce junctional breakdown. Freeze-fracture studies of pupal CNS show particle migration and gap junctional disassembly in parallel with the break-up of the glial gap junctions as the glial cells migrate and become reoriented during the nerve cord shortening and ganglia fusion that occurs between larval and adult life (Lane and Swales, 1978b, 1979b, c). However, in other systems, such as insect eye, the disappearance of gap junctions, perhaps hormonally-stimulated, may be the event that heralds cell determination (Eley and Shelton, 1976); presumably in this situation the regulatory substances have already been exchanged so that the cells’ fates are destined and gap junctions are no longer required. There is no evidence in the eye as to whether or not these gap junctions disappear by particle disaggregation since no freeze-fracture studies were made. Ecdysterone injection into the primitive arthropod Limufus leads to changes in the shape of gap junctions and the density of their particle packing in the cells of the midgut. Three days after treatment the experimental animals had more loosely-packed, irregular or elongated junctions, similar to those seen in premoult Limufus tissues (Johnson et al., 1974b) and in forming junctions in other arthropod systems (Lane, 1978a). This may represent, as the authors thought, an increase in junction formation, or the images seen could be junctions undergoing disaggregation prior to the cellular transformations of the moult. Hormones have also been implicated in the enhancement of gap junctional coupling in insect epidermis which could be interpreted as due to an increase in the number of junctions. As mentioned earlier, ecdysterone controls the cell-to-cell resistance in Tenebrio epidermal cells and stimulates a resistance drop just before the mitoses indicative of metamorphic epidermal growth (Caveney, 1976).

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Recent evidence implicates gap junctions in hormone action in a variety of vertebrate tissues as well (Merk et al., 1973; Decker and Friend, 1974; Albertini et al., 1975; Larsen, 1977b; Lawrence et al., 1978; Brown et al., 1979). Amplification of gap junctions in vertebrate tissues can often be correlated with altered biochemical conditions that result from the administration of exogenous hormones. For example, thyroxine-induced differentiation of ependymoglial cells in frog embryo (Decker, 1976a) leads to increased gap junctional formation, while ACTH treatment of adrenocortical tumour cells (Decker, 1976b), vitamin A-induced metaplasia in cultured chick epidermis (Elias and Friend, 1976), gonadotropin application to rat ovary (Burghardt and Anderson, 1979) and stimulation of insulin secretion (Medaet al., 1979) all lead to the growth and development of gap junctions too. Ablation of the pituitary, moreover, may result in reductions in the size and number of gap junctions in ovarian tissue (Burghardt and Anderson, 1979). Gap junctional contacts, although reduced, are maintained in ovarian interstitial cells, along with the appropriate hormonal receptors, and this clearly may facilitate the return to a more highly differentiated and more coupled state, in response to elevated levels of circulating hormone (Burghardt and Anderson, 1979). These observations, combined with analyses of the results of hormonal stimulation, have led to the suggestion that the effect of hormones is to modulate the junctional surface area. This could well be the case for insect tissues too, for example, membrane modifications are observed when gap junctional particle dispersal occurs during junctional breakdown in early moth pupae (Lane and Swales, 1979b). The fact that a direct correlation has been demonstrated between the mean area of gap junctional contacts calculated from freeze-cleaved replicas, and electrophysiological measurements of mean junctional conductance (Sheridan et al., 1978), suggests that these decreased or increased numbers of gap junctional plaques due to hormone stimulation are reflected in decreased or increased flow of physiologically-important ions or small molecules via the particle channels of the communicating junctions.

4.7

DYNAMICS OF GAP JUNCTIONAL FORMATION A N D DISASSEMBLY

4.7.1 Gap junction formation; origin, determination, development and turnover Since an examination of a whole range of arthropod tissues shows that the cells within them are associated by gap junctions, they are clearly very ubiquitous structures. As described earlier, they are also present in very large numbers, often lie very close together and may vary tremendously in their diameter (see Fig. 30). It is not yet possible, however, to determine

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whether these different junctions have subtly diverse functions, such as allowing different kinds, or sizes, of molecules through their pores. The question of both the origin and the determination of position of these junctions also arises. Although we now have some information about their mode of formation and the role of hormones in stimulating their development, we still are ignorant of the forces determining their relative positions and sizes. In insects, mature rnacular gap junctions are present in adult tissues (Lane, et af., 1977a) and also in all larval stages (see Lane and Swales, 1978a, 1979a); hence the larval junctions must form during embryonic development (Lane and Swales, 1978a, 1979a; Lane etaf., 1979), although some junctions develop during the growth and multiplication of cells that occurs in larval life (Lane and Swales, 1979a). In holometabolous insects, pupation produces a second round of gap junction formation in late pupae, after the breakdown of the particle plaques by disaggregation in at least some tissues in early pupae (Lane and Swales, 1978b, 1979b), as already mentioned. Finally, gap junction formation can also occur in adult arthropods in tissues that undergo turnover; here, the cells that are replacing old ones must establish contact with the other cells (as, for example, in the midgut (Lane, 1978b)). In each of these situations, details of the mode of gap junctional formation can be followed. As indicated above, what is not yet clear, nor even hinted at, is how the determination of size, frequency and distribution of gap junctions occurs. The two cells to become coupled must recognize one another so that the matching junctional particles in the apposed membranes may come to lie in intimate association, with their intraparticle channels aligned (as in Fig. 32). It is impossible to gauge whether or not the features of gap junctions, such as their component number of particles and their distribution, over the cell membranes, have a random element, somewhat dependent upon the developmental pattern of the coupled cells, or whether they are arranged in a fixed pattern, identical in every organism. The latter is a situation in which, it seems, the substructure of the cell membranes would have to be so rigidly controlled that they would probably lack the flexibility required of them to adapt to any new signal. The established fluid nature of the plasmalemma (Singer and Nicholson, 1972) argues against this latter possibility, but some reasonable degree of regulation seems likely, such as the relative number, or approximate positioning of the junctions. The total amount of gap junctional membrane on the lateral border of epidermal cells in newly moulted larval beetles is ca. 26% (Caveney and Podgorski, 1975). This average area ratio of gap junctionljunctional membrane declines at the intermoult to an average of 20%.Since the frequency at which areas of gap junctions are sectioned is constant in both newly moulted

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and intermoult individuals, and the area fraction varies, then the size of individual gap junctions must fluctuate during the moult cycle (Caveney and Podgorski, 1975) from a mean diameter of 0.24 p m in intermoult to 0.33 pm in newly moulted tissues. It can be calculated that the newly moulted cell must have twice as many gap junctions as the intermoult cell, each junction with a greater surface area (Caveney and Podgorski, 1975). Here the relative number of junctions does seem to be regulated. However, whether the precise distribution of plaques is always the same, from cell to cell, and how the numbers are altered from newly moulted to intermoult, is not yet clear. In vertebrate tissues, the gap junctional particles have been shown by biochemical assay to be protein in nature (Goodenough, 1974), but no assays have yet been made on arthropod tissues. However, it seems probable that the component gap junctional particles in insects will be protein too. These have to be inserted into the plasma membrane at some point in development and became incorporated between the lipid molecules. Gap junctions are able to form relatively rapidly after cells come into contact with each other, between a few minutes up to an hour (Johnson e l al., 1974a); hence it is likely that the particle precursors for gap junction formation are readily available in the cells or at the cell surface. Although in certain cultured cell preparations, coupling between dissociated cells does not require protein synthesis or ATP (Epstein et al., 1977), other studies (e.g. Decker, 1976; Griepp and Bernfield, 1978; Revel, 1978) show that junction formation may be an active metabolic event, blocked by such compounds as cycloheximide and involving proteolytic activity. The analysis of gap junction formation has not been very extensively carried out in invertebrates, and has been studied mainly in insects in the glial cells of the nervous system of the moth Manduca (Lane and Swales, 1979a, b, c) and of the blowfly Calliphora (Lane and Swales, 1978a, b); tissues from late embryos, early hatchlings, various larval instars, pupae and adults, have all been studied for stages in junctional formation, as has adult material (Lane, 1978a, b). Gap junction formation is heralded first by free 13 nm E F particles (Fig. 34) already possessing a central pore (Fig. 33, insert) which is presumably closed; these are to be found in embryos, early hatchlings, mid-pupae and adult tissue undergoing turnover when cells are inserting between previously established cells. In all cases, these free 13 nm E F particles can be seen becoming arranged in clusters, or in linear aggregates (Fig. 34) in later stages (or within the same tissue at the same stage, since not all the junctions are developing synchronously). Ultimately the particles coalesce into loose (Fig. 3 9 , then more tightly packed maculae (Figs 28 and 30). Although these final mature junctions are never as closely packed as those of the vertebrates,

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with their hexagonally-packed arrays, they are clearly macular EF structures with complementary PF pits (Figs 28 and 29) not random associations of IMPS. The significance of gap junctions forming in the CNS at late embryo/early hatchling stages may be that they are required to permit exchange of regulatory molecules during growth and development of the larval insect (Lane and Swales, 1978a, 1979a). The gap junctions that form during late pupal stages may be required by the adult CNS for synchronized cellular interaction (Lane and Swales, 1978b, 1 9 7 9 ~ ) . In adult tissues undergoing turnover, the cells involved are only those which are actually in the process of becoming coupled to a newly inserted cell (as in Fig. 34). The others remain coupled to the other cells with which they are already associated (as in Fig. 30). The only other mention of gap junctional formation in insect tissues has been in studies of developing salivary gland (Berridge et al., 1976b) and Malpighian tubules (Wall, Oschman and Schmidt, 1975) where, in thin-sections, short lengths of gap junctions become transformed to longer ones as the systems differentiate. During insect gap junction formation, there is no smooth formation plaque area as in vertebrates (Johnson, Hammer, Sheridan and Revel, 1974; Decker, 1976a) and no obvious larger precursor particles such as occur in vertebrates (Revel, 1974; Decker and Friend, 1974; Johnsonet al., 1974a; Gros et al., 1977; Gilula, 1977; Yee and Revel, 1978; Revel et al., 1978) have been recognized. This would tend not to support Revel’s (1974) contention that the larger particles may represent the intramembranous locus of a determinant for cell recognition, playing a role in enabling cells to contact each other during development. However, the general pattern of assembly is similar to that in vertebrates (Yee 1972; Revel et al., 1973; Fig. 33 Changes in gap junctional structure during early pupal disaggregation of the E F macular particle arrays. The junctional breakdown, here shown in the pupal stages of the moth, Manduca sexta, appears to commence by the particles within a macular aggregate being “pulled out” in one direction (see large arrows). This seems to be associated with a change in membrane structure in that the E face shows a lengthy depression along the side of the gap junction with which the particles are associated. Pupal stages studied five days later show that the junctions are completely dispersed, with separate particles scattered over the fracture face. The Insert shows that the EF gap junctional particles contain a central channel (arrows) even when disassociated prior to undergoing reaggregation during cell rearrangement. x 36 000; insert X 160200 Fig. 34 During arthropod gap junctional turnover, when two cells are becoming newly coupled, EF particles, 13 nm in diameter, are to be found lying free on the fracture face, or are beginning to coalesce with clusters or linear arrays which then lead into aggregates. These then will become condensed into more closely packed macular arrays (as in Fig. 35). The P face (PF) shows the complementary pits. X 55 S O 0 Fig. 35 Freeze-fracture replica of gap junctions from the insect nervous system in the process of formation. The 13 nm EF particles are becoming clustered into arrays of different sizes. Linear processes leading into the clusters are found and the plaques are much more loosely packed than in the adult (see Figs 28 and 30). x 41 600

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Johnson et al., 1974a; Revel, 1974; Benedetti et al., 1974; Decker and Friend, 1974; Albertini and Anderson, 1974a; Decker, 1976a; Mazet, 1977; Gros et al., 1977; Ne’eman et al., 1977; Revel et al., 1978; Yee and Revel, 1978) in that individual particles become aligned into linear arrays which ultimately coalesce into macular plaques that gradually may become enlarged. This dynamic interpretation of the static freeze-fracture micrographs is dependent upon acceptance of the fluid mosaic nature of the plasmalemma (Singer and Nicholson, 1972) which permits movement of protein particles translaterally in the lipid layers. Recently, more detailed analyses of the gap junctional formation process have been made in vertebrates. During this formation process, it is suggested (Revel et al., 1978) that each gap junctional protein, or monomer, has to become associated with 5 others to become a hexameric unit particle, called a connexon; the protein composing the particles is called connexin (Goodenough, 1974). The subsequent stages in formation often occur on a smooth formation plaque area (Decker, 1976a) although this need not be present (Ne’eman et al., 1977; Revel, 1978; Yancey et al., 1979). It is thought that the six subunits first form a proconnexon (10-1 1 nm in apparent diameter), after which a proteolytic cleavage of these precursors leads to a conformational change in the protein so that the particles appear smaller (6-9 nm); this also establishes end-to-end connexon association between adjacent cells and concomitantly the junctional channels are formed between the two neighbouring membranes (Revel et d., 1978; Revel, 1978). This speculative sequence of events is based on enzymatic and inhibitor studies which tend not to support the alternative possibility that protein inhibitors interfere with gap junction formation by blocking production of an enzyme required to degrade the extracellular material that would otherwise not allow for cell-to-cell contact prior to junction formation. Comparable studies have not yet been made with insect tissues so that it is not yet clear what, if any, event, parallels the hypothetical transformation of the large precursor granules into smaller particles during vertebrate gap junctional development. The apparent absence of any significantly larger particles in arthropods may mean either that a comparable proteolytic event does not occur or that any such change in particle configuration in arthropods is so subtle as to be beyond the resolution of current techniques. If Revel’s theory is correct, one would not expect to see large precursor particles in insect tissues during gap junction formation in late pupal stages, because the IMPS are already in the membrane and, insofar as one can judge, are not altered by the disassembly or aggregation stages; hence no conformational changes would be required. In addition to the distinctions concerning formation plaques and precursor granules mentioned earlier, other differences between arthropods and

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vertebrates include the fact that the final size of arthropod gap junctions is much smaller and, as already discussed, the packing of their component particles is much looser. Moreover, component particles of the arthropods fracture onto the EF, which is otherwise relatively particle-free. This makes it possible to distinguish particles involved in forming gap junctions from those involved in forming tight junctions, since the latter fracture onto the PF. This convenient attribute, not shared by the vertebrates which possess both PF gap junctional maculae and PF tight junctional ridges, makes studies of developing junctions in arthropods more readily interpretable than those of vertebrates (Lane, 1978a; Lane and Swales, 1979b). It should be noted that formation plaques are not always found in vertebrate systems however (Ne’eman et al., 1977). For example, during the reappearance of hepatic gap junctions in regenerating liver they are rarely seen (Yancey et al., 1979). In insects, their absence may be related to the fact that the junctional particles fracture onto the E face, which, as noted, is relatively particle-free in any case. An IMP-free plaque area would not be as obvious there as it would be in a particle-enriched P face where the forming junctions occur in the vertebrates. Using conductance measurements, studies on gap junction formation have been made in vitro when a pair of cells are brought into contact. After a short period of latency, a junctional conductance develops, rising to a final steady value; this can be interpreted as the progressive increase in the number of open channels during the development of a permeable gap junction (Ito et al., 1974). This explanation is consistent with the findings described earlier whereby the number of particles in a forming gap junction increases with time (for e.g. Johnson et al., 1974a). Moreover, it has now been shown that the opening of nascent gap junctional channels in isolated embryonic cells at least, is indicated by stable quanta1 increments in cell-tocell conductance (Loewenstein et at., 1978a). Correlations of this sort have not been made for intact systems thus far, so that this aspect of junctional formation remains supposition for the great majority of tissues, including those of insects. It does seem reasonable, however, to predict that this should be a general phenomenon. 4.7.2

Gap junction uncoupling, breakdown and disaggregation

Although there are a variety of reports on arthropod and other cells, particularly when in culture (Gilula and Epstein, 1976), becoming uncoupled after treatment with one of a variety of agents, for example, change of H+ or Ca++concentration, trypsin addition, etc. (Peracchia and Dulhunty, 1976; Peracchia, 1977; Simpson et al., 1977; Peracchia and Peracchia, 1978), it seems that this uncoupling of arthropod cells is simply due to a

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closure of the central pore. It is unlikely that there is any necessity for the particles forming the junctions to move about or to become disaggregated from their macular arrangements. In other cases, for example in the ovary of vertebrates, developmental changes may lead not just to uncoupling, but to loss and degeneration of the gap junctions. The cells become uncoupled, and the junctional membrane appears to be pinched off; the resulting vesicles seem then to be disposed of by “internalization” or “interiorization”. This removal of gap junctions is thought to be by an endocytotic process and has been observed variously in vertebrates during ovarian follicle differentiation (Albertini and Anderson, 1974a, b; Albertinietal., 1975; Anderson, 1977), degeneration of the decidua (Amsterdam et al., 1976), in granulosa cells (Larsen and Hai-Nan, 1978), embryonic chick otocyst (Ginzberg and Gilula, 1979) and in a variety of other situations where the profiles are circular and referred to as “annular” gap junctions (see references in Larsen and HaiNan, 1978). Although these may be, to a large extent, sections through interdigitating cell processes that carry normal gap junctions (Fawcett, 1978), some of them display hydrolytic activity which suggests that the internalization leads to their ultimate degradation (Larsen and Hai-Nan, 1978). However, annular gap junctions are not always encountered in tissues in which gap junctions are known to be disappearing (Yancey et al., 1979) and there is sometimes little evidence for endocytosis either (Yancey et al., 1979). In insects, the breakdown of gap junctions in developing systems during metamorphosis appears to differ from the vertebrate system. When cell distribution is modified during pupal changes, for example, as the glial cells around axons become detached from the axons while both cell types migrate to take up their adult positions, then it appears that the gap junctions established as macular assemblies in larval life, disappear. No indication of internalization occurs, however, but instead disaggregation or dispersal of the gap junctional particles takes place (Fig. 33) (Lane and Swales, 1978b, 1979b; Lane, 1978a). This disaggregation of particle plaques takes the form of the once fairly tightly packed 13 nm E F particle arrays becoming loosened and separated out into first strands of particles and then individual particles (Lane and Swales, 1978b, 1979b). This appears to start gradually (Fig. 33) resulting in many individual particles that are no longer associated in clusters (see Figures 5 and 6 in Lane and Swales, 1978b). Near the beginning of pupal metamorphosis, initiation of the junctional breakdown is indicated when part of the gap junctional assembly begins to be “pulled out”, in that linear particle strands appear at the edge of the macular plaques (Fig. 33). The changes observed strongly suggest than an alteration in the membrane structure, possibly its fluidity, has occurred. Since pupation itself is hormon-

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ally triggered, it may be a hormone effect, either directly or indirectly, which is acting on the plasma membrane, modifying its properties and allowing the streaming out of the junctional particles (Lane and Swales, 1979b). It is interesting that our suggestion of intramembranous particle dispersal during gap junctional breakdown in early insect pupae in vivo (Lane and Swales, 1978b, 1979b) allows for re-utilization of the same particles for subsequent reformation of macular gap junctions in late pupae. The plausibility of this is supported not only by statistical analyses (Lane and Swales, 1978b) and theoretical considerations (Metz et al., 1977; Revel, 1978), but also by a comparable suggestion which has now been made for tight junctional breakdown. These junctions become dispersed into particles in a cell line of human colon adenocarcinoma (Polak-Charcon and Ben-Shaul, 1979) where the same junctional particles are thought to be preserved for re-use; in this way, immediate junction reformation can occur without the necessity for protein synthesis if cells become reassociated. In support of this contention, protein synthesis, as mentioned earlier, is not always required for gap junction formation (Cox et al., 1976; Epstein et al., 1977). The formation of gap junctions during the process of pupal growth may be a developmental cue, triggering off differentiation since signals can now be exchanged. However, it is also true that the reduction in or the loss of gap junctions together with the loss of ionic communication may also be important in differentiation both in insects (Eley and Shelton, 1976; Goodman and Spitzer, 1979) and in vertebrates (Dixon and Cronly-Dillon, 1972; Lopresti et al., 1974; Keeter et at., 1975; Blackshaw and Warner, 1976; Fugisawaet al., 1976; Kelly and Fallon, 1976; Meyeretal., 1977; Panneseet al., 1977; Gilula et al., 1978; Ginzberg and Gilula, 1979). In grasshopper embryos, for example, extensive electrical coupling exists not only between the neuroblast cells but also between neuroblasts and morphologically distinct epithelial cells; the neuroblasts are also dye coupled to one another in that injected Lucifer yellow spreads rapidly from cell to cell (Goodman and Spitzer, 1979). These cells become uncoupled as differentiation proceeds, although the cells become dye uncoupled while they are still electrically coupled, suggesting a progressive restriction of the molecular size of substances that can pass from cell to cell via the gap junctional channels. It seems that the substances exchanged between the coupled cells are unlikely to be involved in early cell differentiation, since in many cases this has already begun, linked cells forming, for example, morphologically distinct axon arborizations. However, the role of the electrical uncoupling, with respect to further differentiation, is not yet clear, although it occurs at the same time as the advent of excitability (Goodman and Spitzer, 1979). Nor is it known in what way these embryonic gap junctions disappear (by particle dispersal or by internalization, see above).

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4.8

N A N C Y J. LANE A N D HELEN leB. SKAER CO-EXISTENCE WITH OTHER J U N C T I O N A L TYPES

In certain situations where septate junctions exist, isolated rather than clustered “gap junctional” particles are found lying between the septa1 sheets; these have been described as “tubular” structures (9 nm in diameter) in molluscan epithelium (Giusti, 1976) because they possess a 2-2.5 nm wide central channel into which lanthanum penetrates. They are rather similar to the tubular structures (15 -1 8 nm in diameter) described in insect Malpighian tubules (Fain-Maurel and Cassier, 1972) as part of the scalariform junctions. Another example of isolated 12 nm “isodiametric” particles intercalated into pleated septate junctions occurs in leech photoreceptor cells (White and Walther, 1969); these also have a central pore and are thought to represent gap junctional elements. However, correlated thin-section studies are required to establish the intercellular cleft size, and its structural features, before accurate identification is possible. Macular gap junctions also frequently co-exist along the same lateral cell border with other junctions such as septate junctions (Fig. 36) so that it was originally difficult to distinguish the specific function of each junctional type. Gap and septate junctions tend to lie in intimate association with one another, the macular EF particle aggregates of the former can be seen in freeze fracture replicas, lying intercalated between the rows of EF pits or grooves of the latter (Fig. 36). These have been shown to co-exist in thin-sections, lanthanum-stained sections (Fig, 36, insert A) or freezefracture replicas (Fig. 36, insert B) of arthropod tissues. For example, they have been shown to co-exist in such tissues as scorpion sensory organs (Foelix et al., 1975), Calliphora salivary gland (Rose, 1971; Berridge el al., 1976b; Skaer et al., 1975), the salivary glands of Chironomus (Berger and Uhrik, 1972), wing hypodermis (Seligman et al., 1975), Malpighian tubules of the cockroach (Wall et al., 1975), Schwann or glial cell mesaxons of a variety of insects (Lane, 1974; Osborne, 1975), glandular epithelia (Flower, 1972), epidermal cells (Hagopian, 1970; Lawrence and Green, 1975; Caveney and Podgorski, 1 9 7 9 , cells of the oesophagus and rectum (Wall and Oschman, 1973,1975; Lane, 1978a, 1979a, c), cells of the anal sac (Noirot Fig. 36 Freeze-fracture replica of gap junctions intercalated between septate junctions from a tracheole in the moth M U ~ ~ K sexta. C U The plaques of EF particles are nestled into the undulating tracts of EF pits. Note the variable number of particles comprising each of the gap junctions. The arrow indicates the point where the fracture plane cleaves from P F up into the EF. Such co-existence of junctions can also be seen in lanthanum-impregnated thin-sections (insert A) where the characteristic, non-opaque septa of the septate (SJ) and hollow particles of the gap (GJ) junctions lie against an opaque background of tracer. Strong adhesive forces hold membranes together at these junctions so that in tissues such as the perineurium (insert B), where attenuated processes bearing gap and septate junctions lie over one another, they tend to adhere so that the PF of one junction (septate junction arrowed in insert B) is found overlying the EF of the other (gap junction in Insert B). x 88 200; insert A, x 83 100; insert B, X 52 200

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and Noirot-TimothCe, 1971; Noirot-TimothCe and Noirot, 1973), and other insect gland cells (Noirot and Quennedy, 1974). Gap junctions and continuous or smooth septate junctions also occur together (Fig. 37) in a variety of tissues, for example, in Musca Malpighian tubules and midgut (Skaer et al., 1979), Rhodnius midgut (Lane, unpublished observations), crayfish hepatopancreas (Gilula, 1972), amphipod hepatopancreas (Schultz, 1976), crustacean caecal epithelium (Graf, 1978a), Collembolu midgut (Dallai, 1975), mosquito midgut (Houk, 1977), rat flea midgut (It0 et al., 1975), midgut and hepatopancreas of Limulus (Lane and Harrison, 1978) and various other epithelia (Noirot-Timothke and Noirot, 1974). In lanthanum-infiltrated specimens the reduced cleft of the gap junctions is often very striking (Fig. 37, insert) as it sits in the middle of the much wider clefts of the smooth septate junctions. Nearly always the continuous junctions are near the luminal surface while the gap junctions lie below them (Fig, 37) near the base of the cells. In some earlier papers, continuous junctions were thought to be normal septate junctions, and their co-existence with gap junctions was reported in this belief, for example, in Limulus midgut (Johnsonetal., 1973) and mothmidgut (Smithetal., 1969). Gap junctions can also be found to co-exist with a number of other junctional types, such as desmosomes (Figs 38,39 and 40) or tight junctions (Figs 38 and 39): the latter are those found, for example, in the CNS. Desmosomes either macular or zonular are very commonly found to co-exist with all other kinds of cell junction. The co-existence of more than one kind of junction on the same lateral border, suggests that each junctional type has its own specific function, and monitoring their association may help to elucidate the precise role of each junctional modification (see Section 9).

5 Tight junctions 5.1

H I S T O R I C A L I N T R O D U C T I O N : DO T I G H T J U N C T I O N S E X I S T I N INSECT TISSUES?

Tight junctions, or zonulae occludentes have in general been considered not Fig. 37 Freeze-fracture replica of smooth septate junctions (CJ) co-existing on the same membrane face with macular gap junctions (GJ), from the midgut of the housefly M u m dornestica. The tissue is fixed and the majority of the intramembranous smooth septate junctional particles therefore remain associated with the P face (PF) although some still adhere t o the EF. The particles tend to be fused, as is typical, into short ridgeson the PF. The Insert is a lanthanum-infiltrated thin-section from the midgut of the Xiphosuran horse-shoe crab Lirnulus,and exhibits the conventional smooth septate junctional appearance of only a few very thin non-opaque structures (arrows) visible against the density of the lanthanum. The area of reduction in the intercellular cleft width is a gap junction (GJ) which lies in the midst of the smooth septate junctional region. x 51 100; insert X 116 500

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U3V)lS ‘gal N313H a N V 3 N V l ‘ f A 3 N V N

zz 1

Fig. 38 Thin-section from the CNS of the moth Manduca sexta showing interglial junctions of several sorts co-existing between the same two membrane surfaces. A macular desmosome (D) occurs, followed by a punctate tight junction (TJ) and then a gap junction (GJ). X 173000 Fig. 39 Thin-section from an adult specimen of the blowfly, Calliphora erythrocephala showing gap junctions (GJ), a desmosome (D) with associated microtubules, and tight junctions (TJ) co-existing between the glial cells which ensheath the nerve cells. x 80400 Fig. 40 Co-existence of a rnaculadesmosome (D) with a gap junction (GJ) between two adjacent cells in Calliphora. X 92 000 Fig. 41 Thin-section through the perineurial cell layer ensheathing the CNS in an adult moth, Manduca sexta, where the lateral borders interdigitate extensively. The gap junctions (GJ) reveal their component particles (small arrows) when cut tangentially. Several punctate tight junctional membrane appositions are evident (larger arrows). x 185 900 Fig. 42 Perineurial cell sheath in the CNS of an early Manduca hatchling; this thin-section reveals numerous punctuate appositions (arrows) between the cell membranes along the undulating lateral border. Such tight junctions are particularly numerous in the attenuated perineurium of newly hatched first instar stages. The insert shows a higher magnification of an insect tight junction with the occluded intercellular cleft characteristic of these structures. X 85 300; insert x 239 300 Figs 43,44,45 These micrographs are all freeze-fracture preparations from the insect nervous system, demonstrating the P face (PF) ridges and E face (EF) grooves that occur towards the base of the perineurium in the CNS. These examples are from the cockroach (Figs 43 and 44) and the moth, Manduca sexta (Fig. 4 5 ) . They illustrate that th tight junctional ridges may be discontinuous, and overlapping, and that they may lie in para112 as 2 to 3 closely apposed fibrils (at arrows in Fig. 44) or in parallel as separate structures (Fig. 45). Fig. 43: x 60 500, Fig. 44: x 67 900, Fig. 45: x 65 100

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to occur in invertebrates (Satir and Fong, 1973; Satir and Gilula, 1973; Noirot and Quennedy, 1974; Filshie and Flower, 1977; Noirot-Timothee et al., 1978) mainly because it has been considered that septate junctions have a sealing role (Filshie and Flower, 1977; Noirot-Timothee, et al., 1978; Green, 1978) and hence represent the invertebrate equivalent of the vertebrate tight junction. Furthermore no highly complex anastomosing network of fibrils, as occurs in vertebrate tissues, have been found in freeze-fracture replicas of invertebrate tissues. The issue is further complicated by early reports (references in Section 4.2.1) in which those junctions originally termed “tight” when observed in thin-sections of invertebrate tissues, turned out with the advent of en bloc U A staining (see Section 1.1.1) actually to be gap junctions. However, since a good deal of evidence has recently been accumulating which indicates that septate junctions do not always play an entirely occluding role (see discussion in Section 2.7) they cannot be held to be the morphological basis of all invertebrate barrier systems. The gradual recognition that tight junctions might actually exist in insects, began when examinations of insect tissues revealed that tracers fail to penetrate certain systems, including the insect central nervous system (Figs 4 8 , 4 9 and 52) (Lane and Treherne, 1970,1971,1972a; Lane, 1972) where a blood-brain barrier can also be demonstrated electrophysiologically (Treherne e f a / . , 1970; Treherne and Pichon, 1972; Pichon et al., 1972, the blood-germ cell barrier of the insect testis (Fig. 51) (Szollosi and Marcaillou, 1977; Jones, 1978; Skaer and Jones, 1979), the blood-rectal barrier situated in the basal portion of the lateral cleft in the insect rectal pads (Fig. 50) (Lane 1978a, 1979a, 1979c) and the insect blood-eye barrier (Shaw, 1977, 1978). Subsequent investigations have revealed that punctate tight junctional contacts can be found in thin-sections of these regions (Figs 41 and 42) (Lane et al., 1977a; Lane, 1978a; Lane and Swales, 1979a), while freeze-fracture studies of these same areas exhibit simple linear systems of P F ridges and EF grooves (Figs 43-47) (Lane et al., 1977a; Lane and Swales, 1978a, b, 1979a; Lane, 1978a, 1979a, c; Skaer and Jones, 1979; Skaer 1979a). The previous lack of recognition of these structures may well be related to the fact that they do not occupy an obvious or extensive site in tissues, unlike vertebrate tight junctions (Claude and Goodenough, 1973).

Figs 46, 47 Freeze-fracture replicas from tissues that possess “barrier” systems, the locust blood-eye barrier (Fig. 46), the locust blood-testis barrier (Fig. 47) and the cockroach bloodrectal cleft barrier (Fig. 47, insert). Note the presence of P face ridges (arrows), which may be very lengthy (Fig. 46) or discontinuous (Fig. 47), as well as EF grooves (G in Fig. 46 and Fig. 47, insert). The ridges are composed of fused 8-10 nrn intramembranousparticles (Fig. 47) and lie parallel to each other. Fig. 46: x 39 100, Fig. 47: x 115 100, insert, Fig. 47: x 47 500

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5.2

STRUCTURAL FEATURES

5.2.1 Thin-section appearance

In thin-sections, the tight junctions of insects are not often immediately obvious. Firstly, the punctate nature of both vertebrate and insect tight junctions makes their identification in thin-section critically dependent on section plane and angle. In addition, in vertebrates the belt of junctionbearing membrane is readily located and identified as it is restricted to the terminal bar (Farquhar and Palade, 1965); such is not the case in insects. Insect tight junctions are found as a belt around the system they seal off, but they occur as more widely separated punctate appositions and are more basally located within a highly interdigitating lateral cell border (Fig. 54). When observed, they show ii single punctate point of fusion between the two plasma membranes thus associated (Figs 41 and 42) and at this point of fusion, their total width, like vertebrate tight junctions, is reduced to less than that of the two component cell membranes. En bloc staining is required to be able to see these points of fusion, but they are quite distinct from the lengthy gap junctions which are separated by a gap of 2-3 nm (as in Fig. 41) and which have so often been confused with tight junctions (see Section 4.2.1). In considering the structure and distribution of tight junctions it must be remembered that before the advent of en bloc staining the 2-3 nm gap between the membranes of gap junctions was not visible. Hence many reports on insect tissues published before (or even after!) the era of en bloc staining began, refer to gap junctions as being tight or occluding junctions (see references in Section 4.2.l), because the membranes appeared to be sealed together along the length of the gap junction. It is clear that care must be taken in interpreting reports on tight junctions that do not incorporate en bloc staining in their protocol. In contrast to the relatively straight junctional membrane appositions of gap junctions (as in Fig. 2 6 ) , tight junctional membranes show undulations as the membranes bend inwards to fuse and then out again, in a wave-like appearance (Figs 41 and 42) which may be repeated if more than one tight junctional punctate apposition is present. 5.2.2. Tracers and negative-stained appearance

When tissues possessing tight junctions are incubated in tracer-containing solutions, the tracers are unable to move past the barrier represented by the junctional occlusions which are found as a complete band around the lateral cell borders. This restriction to the entry of exogenous molecules is an important criterion that needs to be satisfied if an occlusion of the intercellu-

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lar space is thought t o be present. There is no doubt that incubation in physiological saline to which tracers have been added, prior to fixation, gives more information (see Figs 48-52) than simply adding tracers to the fixative (see Section 1.1.4). This must be borne in mind during the interpretation of data purporting to demonstrate physiological barriers. Tracers commonly used include horseradish peroxidase (HRP), microperoxidase (MP), ferritin and ionic lanthanum. The validity of using ionic lanthanum as a physiological tracer in carrying out permeability studies on insects has been demonstrated, since in the insect nervous system it neither increases the Na’ nor K+ permeability of the perineurial barrier, nor does it affect nerve conduction (Thomas and Leslie, 1976). Hence substances such as lanthanum would not ordinarily be expected to move past a tight junction and into the cleft beyond to reveal the details of the extracellular architecture as tracers so conveniently do for the other junctions; this could only happen if the junctions were disrupted (see Goodenough and Revel, 1970) or in the process of formation. Since it is in any case rare to see the tight junctions in insect tissues, it is not surprising that in thin-sections, after lanthanum impregnation, it is also rare to see lanthanum that has penetrated beyond an incomplete, discontinuous or disrupted, yet still recognizable, tight junction. This occurs during embryonic formation (Lane, unpublished observations) and it is assumed that it takes place because the junctions are not yet complete. One other way of viewing the details of the cleft between tight junctions is by staining the cell membrane very heavily with en bloc uranyl acetate or with uranium calcium en bloc. The external membrane leaflet may stain everywhere except where the fused ridges run and there, non-opaque strands appear as white fibrils or ribbons against a dense background (comparable to the stained intercellular space in Fig. 53). This phenomenon has been strikingly observed in the tight junctions in the perineurial layer of spider CNS (Lane and Chandler, 1980). A comparable situation has been observed in the tissues of vertebrates (Goodenough and Revel, 1970; Neaves, 1973), where lanthanum was found to penetrate the tight junctions after their disruption by various non-physiological treatments. In en face views of junctions so treated, the tracer revealed electron-lucent ribbons which demonstrated blind-ends; these discontinuities in the ribbons had presumably been created by trauma. Comparable experiments which have thus far been made on insect material, however, have not led to tracers getting past the tight junctions (Treherne et a f . , 1973). Other attempts to disrupt the tight junctions in the perineurium of the insect CNS have involved mechanical techniques such as the surgical removal of the neural lamella and perineurium (Treherne and Maddrell, 1967; Treherne t’r a f . , 1970; Pichon et a f . , 1972) or the stretching of the

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central nervous connectives (Treherne et al., 1970). The results of these experiments was to produce extensive perineurial damage rather than slight leakage around the tight junctions and the glial cytoplasm was, as a result, penetrated by the tracer molecules employed (Lane and Treherne, 1969, 1970). Junctional disruptions of some sort must have occurred since the tracers, under such conditions, can move into the intercellular system (Lane and Treherne, 1972a), but no clear-cut en face views of tight junctional fibrils were obtained. 5.2.3

Freeze-fractured replicas

In freeze-cleaved replicas, tight junctions, which are best characterized thus far in the CNS of insects, consist of P F ridges arranged as linear arrays lying parallel to the outer surface of the tissue, and at right angles to the direction of diffusion of substances into the system (Figs 43-45). These linear P F ridges are usually single, but sometimes may be double or triple with interweaving ridges (Fig. 44); they are often discontinuous (see Figs. 43-45) but frequently have “back up” ridges lying in parallel to them, rather like the vertebrate testis (Gilula et al., 1976; Nagano and Suzuki, 1976a, b; Connell, 1978). The EF grooves appear to be complementary to the ridges, but fractures across the intercellular space at the point of membrane apposition are extremely rare. These PF ridges are composed of fused particles that measure 8 to 10 nm in diameter and which are aligned together Figs 48,49,50, 51 All these micrographs are thin-sections taken from tissues that have been incubated in physiological saline to which ionic lanthanum was added Fig. 48 Cross-section through the ventralnervecord ofthecockroach toreveal theperineurium (PN) ensheathing the nervous tissue. This area is from an interganglionic connective so that the glial cells (G) surround the axons (A) rather than nerve cell bodies. Note that the lanthanum does not penetrate beyond the perineurial/glial clefts (arrows). The acellular neural lamella (NL) that surrounds the nerve cord contains neurosecretory-bearing axons (NS) and small axons (*). It is clear that the lanthanum permeates freely through to their surfaces since they possess no perineurium and only a thin glial covering, if any, which does not display occluding junctions. This contrasts with the basal perineurial glial layer which exhibits tight junctions. x 10 500 Fig. 49 Transverse section through the ventral nerve cord of the moth, Manduca sexta, which possesses no septate junctions in the perineurium (PN) so that the observed restriction (at arrows) to the inward movement of the tracer must be due to the demonstrated presence of tight junctions, NL, neural lamella; G , glial cells; A, axons. x 8100 Fig. 50 Cross-section through the rectal pads of the cockroach, feriplaneta, showing the basal region that lies next to the haemolymph and basement membrane (BM). When incubated with lanthanum from the haemolymph (basal) side, the tracer moves into the clefts and then is abruptly stopped (arrow). This is the region where tight junctions are found in thin-sections and in replicas. x I4 100 Fig. 51 Transverse section through the peripheral layers of the cockroach testis. Lanthanum is found to penetrate some way through the ensheathing cell layers but is stopped (arrows) before the central compartment (C) in which the developing germ cells are found. x 7700

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Fig. 52 Thin-section through the perineurium (PN) forming the blood-brain barrier in the CNS of the cockroach, Penplaneiu, after incubation in 10 mM ionic lanthanum in physiological saline for 1.5 h. The tracer has traversed the neural lamella (NL) and entered the perineurial clefts but it has not penetrated beyond the occluding perineurial junctions (at arrows). Insert shows at higher magnification the dense lanthanum stopping (arrow) at a punctuate tight junction. X 34 900; insert x 85 100

in rows (see Figs 43 and 44). Depending on the shadow, the points of fusion may be or may not be noticeable so that the ridges may look moniliform or bead-like or may look more like solid rods. In tight junctions, it appears that the adjacent cell membranes contain rows of particles tightly packed into ridges, and it is these ridges that make contact o r perhaps fuse to form the observed punctate appositions (Figs 41 and 42), so that the intercellular space at that point is obliterated. The lines of attachment form "sealing" strands that will prevent molecules from running across them, thereby forming an occluSion or barrier to the free passage of substances from the external to the internal compartment thus formed.

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5.3

M O D E L DERIVED FROM STRUCTURAL EVIDENCE

The model presented here for insect tight junctions (Fig. 53) indicates the simple nature of the P F ridges or fibrils that together with the complementary EF grooves, comprise the occluding junctions. It can be seen how in thin-sections (as in Figs 41 and 42) the punctate appositions represent the points where the ridges are apposed, so that thin-sections of extensive gap junctional membrane apposition, in the past sometimes mistakenly referred to as “tight” junctions, could not correspond to these ridge structures seen after freeze-cleaving. The lack of complexity and consequent relative infrequency of the ridges observed in freeze-fracture replicas together with the concomitant rare fracture plane through ridges and grooves, make it impossible to determine whether the fractured ridge structure of insects is a single or double fibril as has been debated for vertebrate tight junctions (Staehelin, 1973; Wade and Karnovosky, 1974; Bullivant, 1978); in Fig. 53 it is -CYTOPLASM

I-F

FACF

-

/-%

-LANTHANUM IN INTERCELLULAR SPACE-

-

P FACE

-

Fig. 53 Model of an insect tight junction showing the appearance of the two fractured faces, with the P face exhibiting the junctional ridges and the E face the grooves. The intercellular space is shown as it would appear with lanthanum leaking past a ridge that had been disrupted, or that was in the process of forming. Once completed, the tracer would not move past the junctional structure, as indicated in the left hand side of the model where the punctate apposition between adjacent membranes is illustrated. Although this is drawn here as a double fibril structure, there is insufficient evidence as yet to differentiate between this and the single fibril model

indicated as double and not “offset” but there is no definitive evidence for this. Similarly, although in the diagram we indicate lanthanum migrating around the discontinuous ridges and past others, as might occur after disrupting the ridge system in a way comparable with that done in vertebrates (see Goodenough and Revel, 1970; Neaves, 1973), such images are difficult to identify unequivocally. In hypertonic urea-treated insect central nervous system, unlike vertebrates (see Ussing, 1968), the blood-brain barrier does not break down insofar as ionic tracer uptake is concerned (Treherne et al., 1973). Although the barrier to the entry of potassium and the loss of sodium was impaired under these conditions, this damage was thought to be due to a selective change in permeability of the perineurial membrane or of the tight

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junctions to these ions, since there was no indication of lanthanum leaking around the modified junctions into the underlying glial-axonal clefts. In developing CNS, with tight junctions cut in thin-sections, images that are suggestions of lanthanum leaking around a forming ridge element have been observed (Lane, unpublished observations); these have been seen both in normal cross section where the intercellular space narrows, and in en face views, revealing non-opaque strands against a dense background as would be expected in such “negatively-stained” preparations (compare with Goodenough and Revel, 1970; Neaves, 1973). 5.4

DISTRIBUTION A N D 120CALIZATION IN ARTHROPOD TISSUES

Tight junctions of the sort described above appear to be restricted to the insects. Some preliminary freeze-fracture studies carried out in our laboratory, however, suggest that tight junctions may also exist in some of the terrestrial arachnids, for example, spiders (Lane and Chandler 1980). There have been no reports of tight junctions in other groups except for the crustacea, and here they have been observed only as macular structures in thin-sections (Lane and Abbott, 1975) or in the regenerating nervous system of the crayfish in freeze-fracture (Shivers 1977). In the latter tissue, they took the form of discontinuous linear arrays of 11 nm EF particles arrayed in intimate spatial association with plaques of large EF particles termed desmosomes. Another interpretation of these results is that in regenerating tissues, gap junctions would be undergoing reformation and that probably these linear array of particles are gap junctions in the process of formation; the particles are large for tight junctions and fracture on the wrong face of the membrane to be typical ofzonufae occludentes. Moreover, there have been many reports that insect desmosomes lack any insertion sites into the membrane that are decipherable in freeze-fracture replicas (see discussion in 3.2.2), so that the macular particle arrays near the linear ones were probably also gap junctions already formed. This would appear to be substantiated from a close inspection of other replicas from comparable crustacean tissue (see Figure 6 in Shivers and Brightman, 1977). The insect tissues in which tight junctions have been found now include the central nervous system, compound eye, the testis, and rectal pads. In several cases, supportive electrophysiological evidence is available and in all these systems there has been shown to be (i) an intercellular barrier to the entry of exogenous tracer molecules, (ii) punctate tight junction-like membrane appositions which have been seen in thin-sections, and (iii) simple PF ridges and complementary EF grooves which have been found in freezefracture replicas.

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5.4.1 Insect central nervous system

( a ) Perineurial cells In the central nervous system (CNS) in insects, a blood-brain barrier has been shown to exist electrophysiologically (Treherne et al., 1970); Treherne and Pichon, 1972; Pichon et al., 1972). Incubation in physiological saline with tracers such as horseradish peroxidase, microperoxidase or ionic lanthanum reveals that the exogenous tracers can penetrate no further than between the clefts of the outer cellular layer (Figs 4 8 , 4 9 and 52) (Lane and Treherne, 1970,1971, 1972a; Lane, 1972, 1974, 1978a; MacLaughlin, 1974b; Lane et al., 1977a; Lane and Swales, 1978a, b, 1979a); this is a layer of modified glial cells called the perineurium and so these cells are thought to be the morphological basis of the insect blood-brain barrier. It must be remembered that the insect central nervous system is avascular so that substances can only reach the axonal surfaces by diffusion through the sheaths that surround them. The axons are often surrounded by glia, and bundles of them are ensheathed by the perineurial cells and acellular neural lamella; in certain special cases, such as the stick insect Carausius, a fat-body sheath lying beyond the neural lamella also surrounds the entire ventral nerve cord. This sheath does not, however, inhibit the inward flow of ions and molecules (Lane and Treherne, 1971). Tracers readily penetrate the neural lamella (Lane and Treherne, 1970), and have ready access to many small peripheral nerves (Lane and Treherne, 1972a) or neurosecretory axons and terminals (Lane, Leslie and Swales, 1975) that lie in the neural lamella (Fig. 48); these small nerves either lack a glial investment or have no interglial occluding junctions. The perineurial layer, however, which possesses tight junctions, is able to exclude tracers such as horseradish peroxidase (Lane and Treherne, 1970), microperoxidase (Lane and Treherne, 1972a), or lanthanum (Lane and Treherne, 1972a; Lane, 1972) (Figs 48 and 49). This barrier effect has been shown in the nervous system of a variety of insects including the cockroach and locust (Lane and Treherne, 1970, 1972a), the stick insect Carausius (Leslie, 1975), the moth Manduca sexta (Pichon et al., 1972; Lane, 1972; McLaughlin, 1974b; Lane et al., 1977a; Lane and Swales, 1979a, c), the blow-fly Calliphora (Lane and Swales, 1978a, b), the blood-sucking bugRhodniusprolixus (Laneetal., 1975a) and the European corn borer, Ostrinia (Houk and Beck, 1975). When the perineurial cell layer is removed or disrupted, tracers can move into the nervous system both between cells into the clefts and also into the ground cytoplasm of the disrupted cells (Lane and Treherne, 1969,1970,1972a) as already mentioned. The perineurial cells are associated on their undulating and interdigating lateral borders by a variety of junctional types including the occasional desmosome, septate junctions, gap junctions and tight

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junctions. In earlier communications the extensive gap junctions were incorrectly referred to as “tight” junctions (Maddrell and Treherne, 1967) because no en bloc staining was employed (see Section 5.1). The septate junctions appear to be absent entirely in the larval and adult moth, Manduca sexta (Lane et a!., 1977a; Lane and Swales, 1979a, c) yet tracers are prevented from proceeding beyond the perineurium (Fig. 49). Although they have been reported to be present in pupal CNS (McLaughlin, 1974a) we have never found them to be present in the perineurium of Manduca pupa (Lane and Swales, 1979c), in spite of prolonged searching. Septate junctions are of course to be found in the tracheolar and tracheal cells that penetrate the moth CNS, and these are especially numerous in later larval and early pupal systems. In any case, even when septate junctions are present in other insect genera, tracers move through them and through the gap junctions (Treherne et a f . , 1973) in the perineurium, so neither of these junctional types seems capable of forming the basis of a barrier. Hence, the tight junctions seem to be the likely candidates, for in fortuitously-cut lanthanum-treated tissues, the tracer can be seen to stop at what appear to be the tight junctional elements (arrows in Fig. 52). In addition, freeze-fracture replicas from these areas reveal that the basal perineurial layer and the peripheral glial layer, against which the former is closely applied and associated by both gap and tight junctions (Lane and Swales, 1979a), possess a series of PF ridges and E F grooves (Lane et a f . , 1977a; Lane and Swales, 1978a, b, 1979a; Lane, 1978a) which appear to be complementary. These ridges are composed of 8-10 nm particles and are non-anastomosing linear arrays, usually single (Fig. 43) but sometimes double or triple ridges (Fig. 44). ( b ) Gliul cells The glial cells that surround the axons or nerve cell bodies in the insect CNS are often associated by gap junctions (see discussion in Section 4.4.1) (Lane and Treherne, 1973; Lane et a f . , 1977a; Lane and Swales, 1978a, b, 1979a) and in some cases by tight junctions, probably not zonular; these latter appear as punctate membrane appositions (Figs 38 and 39) (Laneet al., 1977a; Lane and Swales, 1979a) or as linear PF particle ridgesor EF grooves (Fig. 59) (Laneetal., 1977a; Lane and Swales, 1978a, b). In some cases it is difficult to tell if these are glial-glial or axo-glial junctions, since axo-glial junctions can also be found in thin sections (Fig. 58) (Lane and Treherne, 1973; Lane and Swales, 1978a, b), while axonal ridges are commonly found in insect nerve cells (Fig. 57) (Laneetaf ., 1975b, 1977a; Wood eta!., 1977; Lane and Swales, 1978b, 1979b; Lane, 1978a, 1979b) (see Section 6.2.1 ). A somewhat idiosyncratic suggestion has been made that glial cells in the insect CNS may be of importance in any “enzymatic” blood-brain barrier (Houk and Beck, 1975) by providing “an additional regulatory function”, perhaps by being involved in degradative processes,

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but there has been no indication that there is any junctional involvement in this hypothetical glial barrier. Some tight junctions have been reported between glial cells in the peripheral nerves in thin-sectioned preparations (Osborne, 1975) but given the nature of the membrane appositions these appear more likely to be gap junctions. Larger peripheral nerves exhibit a barrier to tracer entry (Lane and Treherne, 1973; Laneetaf., 1975a) and hence possess modified glial, or perineurial, cells with occluding junctions; very small peripheral nerves may lack a perineurial envestment (Fig. 48) (Lane and Treherne, 1972a, 1973) and their glial cells d o not prevent tracer entry. Neurosecretory terminals which may lack much ensheathing glia, are accessible to substances in the haemolymph such as tracers (Fig. 48) (Lane er al., 1975a). For further discussion see Section 6.2. 5.4.2 Insect compound eye In the insect eye, there have been reports of tight junctions in thin-sections (Eley and Shelton, 1976) although these are not totally convincing. More impressive are the electrophysiological studies of Shaw (1977) which indicate the presence of a blood-eye barrier in the locust compound eye, for which there must be some structural basis. Studies on tracer penetration from the surface of the eye show a zone of restriction in the area of receptor axons and pigmented glial cells (“Zone band 2”) and narrowing clefts occur between glia and axons here (Shaw, 1978). However, the precise morphological basis of this barrier remains obscure. Recently, freeze-fracture evidence on insect eyes from several laboratories shows that simple tight junction-like PF ridges and E F grooves occur in various parts of the eye of the worker honey bee (Nickel and Scheck, 1978), the house fly (Carlson and Chi, 1979; Lane, 1979d) and the locust (Fig. 46) (Skaer, 1979a). When tracers are applied to the eye of the locust not at the surface, but by introduction into the blood system (Shaw, 1978), they then stop in a slightly different site, that is, between the cells that line the haemolymph channels; these cells are comparable to those that form the perineurium of the insect CNS. These haemolymph-containing extracellular channels run through the locust eye (Shaw 1978) and they are lined by a basement membrane that resembles the neural lamella. This then represents an “outside-in” version of the normal ventral nerve cord arrangement (see Lane, 1974). It appears that the perineurial cells and overlying neural lamella that ensheath the CNS and run up to the eye regions have folded in at these points so as to produce pockets of haernocoele at intervals within the eye (Northrop and Gugnion, 1970; Shaw, 1977). The observed “perineurial” occlusion of tracers applied to the eye via the blood coincides exactly with the situation in the ventral

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nerve cord (Lane, 1974,1978a) where the tight junctions in the perineurium appear to prevent tracers entering, thereby forming the morphological basis of the insect blood-brain barrier. In the locust, PF ridges of fused 10 nm particles are found lying in the zones where Shaw observes the blood-eye barrier to occur (Skaer, 1979a) (Fig. 46); these may be very extensive linear arrays which lie in parallel with other similar ones. Although it is still not clear whether some of the ridges may be axo-glial, axo-axonal or between pigmented glial cells, they bear similarities to the perineurium in that a series of parallel ridges and complementary grooves may be found, lying parallel to each other as if forming a multiple impedance, comparable to the arrangement in the CNS. Whether it is they rather than the septate junctions that impede the observed entry of tracers cannot yet be established but the ridges appear to occur in the appropriate topographical zones to do so. Moreover, recently, more extensive, vertebrate-like tight junctions have also been found in dipteran eyes (Lane, unpublished data). In the eye of the housefly, Musca, “close” junctions were first reported (Chi and Carlson) in 1976, and turned out to be gap junctions (Ribi, 1978), while quite distinct axo-axonal or glial-axonal ridge/groove systems have also been observed more recently in freeze-fracture replicas (Carlson and Chi, 1979;Chietal., 1979; Lane, 1979d) (Figs 71 to 73).Theseridgesarecomposed of lOnm particles fused into linear arrays as seen at higher powers (Fig. 72, insert), but in many cases the intercellular clefts seem unreduced (Fig. 73), suggesting that they cannot represent typical tight junctions (see Section 8.2). 5.4.3 Insect testis A barrier to the entry of exogenously-applied tracer substances has been found in the testisof the locust (Szollosi and Marcailliou, 1977; Jones, 1978; Marcaillou et al., 1978; Skaer and Jones, 1979); this occurs at the inner margin of the ensheathing cell layer that surrounds the developing germ cells of both the locust and cockroach (Fig. 51). The barrier appears to have its foundation in tight junctions (Skaer and Jones, 1979) since moniliform PF ridges which may be lengthy o r discontinuous and unanastomosing, are to be found in the inner ensheathing cell layers (Fig. 47) which corresponds to the sites of exclusion of extracellular tracers (Fig. 51). The blood/germ cell barrier in male insects may exist because, as in vertebrates (Fawcett, 1978), the germ cells require a specialized environment for meiosis and subsequent spermatid maturation and development; there is some evidence for this in that both the testicular fluids and the fluid in the spermatheca are enriched with K’ at the expense of other cations (see Jones, 1978). The barrier in the testis does not develop until the 2nd instar in the heterometabolous locust

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and there is some evidence that there is a barrier-stimulating factor to induce its formation (Jones, 1978). Freeze-fracture studies underway on the development of the tight junctions found in the adult (Skaer and Jones, 1979), should indicate whether the stages in development are comparable with those observed elsewhere. 5.4.4

Tracheae and tracheoles

The tracheal system in insects exhibits autocellular junctions in that each tracheal element has a mestracheon whereby the helically-folded tracheal cuticular intima is continuous with the cell surface (Smith, 1968). This mestracheon is crossed by septate junctions (Fig. 63), desmosomes, gap junctions and sometimes punctate membrane appositions. In freezefractured replicas, the septate junctions, exhibiting characteristic PF particle rows, sometimes have P F ridges lying in association with them within the membrane (Fig. 41). Heterocellular junctions may also exist in that the outer membranes of tracheae or tracheoles are often associated with glial cells by what may be fasciar tight junction-like membrane modifications (Lane et al., 1977a; Lane, 1979b). These appear either in thin-sections as sites of focal membrane apposition (Fig. 62) or as moniliform PF ridges in the surface of the outer tracheal membrane (Fig. 60). How these interact with the glial cells, with which they are presumed to be associated to form a partial barrier, is not clear, but they may be important if leakage occurs from the haemolymph when tracheoles penetrate the CNS (Lane et al., 1977a); for further discussion see Section 6.3.

5.4.5 Orthopterun rectal pads In the rectum of the cockroach, Periplaneta americana, the rectal pads possess tight junctional punctate appositions near the basal surface by the subepithelial sinus (Lane, 1978a, 1979a, c). Incubation with exogenous tracers shows that ionic lanthanum application from the basal surface results in it being taken up only a short way into the intercellular cleft (Fig. 50). These occlusion sites could serve to prevent back-flow of substances into the tissue from the haemolymph, which, if it occurred, would interfere with the unidirectional flow of solutes from the lumen of the gut, via the various clefts and sinuses (Fig. 65) to the circulating haemolymph. Freeze-fracture studies of this area reveals basal PF ridges and EF grooves (Fig. 47, insert) which appear to represent the tight junctional elements. Again, as in other insect tissues, these are non-anastomosing, discontinuous structures, which may overlap and which lie in a parallel series, parallel also to the basal surface (Lane, 1979a).

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In other insects such as Bfaberus (Wall and Oschman, 1973), a functionally analogous structure exists, a multilayered sheath which lies on the basal surface of the rectal pads, between the sites of the tracheolar sinuses; this is thought to form a permeability barrier in that the cells comprising the sheath cells are said to be “tightly” fused. However, the extensive and even areas of membrane appositions do not look unlike the gap junctions previously confused with tight junctions in other systems (see Section 4.2.1). 5.5

CO-EXISTENCE WITH OTHER JUNCTIONAL TYPES

In the various types of insect tissue in which tight junctions occur they have been found to co-exist with a variety of other intercellular associations, in particular, in the CNS, eye, testis, tracheae and rectal pads, with gap junctions (Figs 38 and 39); they also are often found in intimate spatial relationship with septate junctions (Fig. 61) and also with scalariform junctions (Fig. 65) in rectal pad tissue (see Section 7.2). 5.6

HOMO- A N D HETEROCELLULAR TIGHT JUNCTIONS

Tight junctions in insects are normally homocellular. An example of an autocellular arrangement might be in the insect mestracheon (Fig. 61). However, heterocellular tight junction-like appositions have been observed in thin-sections between axons and glia (Fig. 58) as well as between tracheoles and glial cells (Fig. 62); the freeze-fracture correlates of these have also been seen in the form of intramembranous PF ridges in glial and tracheal membranes (Fig. 60), as well as E F grooves (Fig. 59). Moreover, perineurial-glial tight junctions have also been seen in thin-sections (see Figure 6 in Lane and Swales, 1979a) and very probably some of the ridgegroove systems seen at the base of the perineurium in freeze-fracture replicas are perineurial-glial associations as well.

5.7

COMPARISON WITH VERTEBRATE TIGHT JUNCTIONS

In vertebrate tissues the terminal bar of a variety of epithelia possesszonulue occludentes or tight junctions arranged as a band around the apical cell margins (Farquhar and Palade, 1965). These are restricted to this relatively non-interdigitated luminal area of intercellular contact (see Fig. 54B) and it can be seen how these junctions differ in distribution, position and complexity from their insect equivalents (Fig. 54A). The junctions in vertebrates possess P F ridges or fibrils and complementary VF furrows in freeze-fracture (Chalcroft and Bullivant, 1970) which correspond to the focal contacts observed in thin-sections (Staehelin, 1974). The ridges and grooves are

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often interwoven into an anastomosis or reticulum and this may be very extensive and deeply cut, or less complex and more shallow, depending on the tissue examined (Claude and Goodenough, 1973). They are, on the whole, much more complex than those of insects. Models of vertebrate tight junctions have been drawn up to show how the fibrils form the sealing element of the junction by fusing across and occluding the intercellular space. Examination of the heights of the ridges led Wade and Karnovsky (1974) to suggest that a single set of fibrils span both membranes while Bullivant (1978) has proposed an “offset” double fibril hypothesis; insufficient evidence is available from insect tissues to enable any conclusions to be drawn as to whether a single or double set are present. Although the tight junctions of insect cells are comparable in structure with those of vertebrates in that they are composed ofca. 10 nm PFparticles fused into ridges with what appear to be complementary grooves, they are much less frequent and less extensive than the latter and never seem to form as elaborate an anastomosing reticulum. They never occur at the luminal surface nor at a relatively straight border and always seem to be in the midst of complicated cellular interdigitations lying at the basal surface of the epithelial layer with which they are associated (see Fig. 54A for the diagram summarizing these points). Here they seem to be in a special formation, lying parallel to one another and to the surface and occasionally overlapping. Perhaps as a result of this, the tight junctional ridges of insects seem never to be as deeply sunk into grooves in the membrane as those of vertebrates but are always rather shallow. They can always be distinguished from the particles comprising gap junctions, with which they are frequently associated., because of their size and fracturing characteristics; the same cannot he said of vertebrate tight and gap junctions, which may be indistinguishable (see Section 5.8.1). Insect tight junctions are more akin to the discontinuous linear tight junctional arrays observed in parts of the vertebrate CNS (Brightman, Shivers and Prescott, 1975; Dermietzel, 1975), and between cells of the renal proximal tubule (Roesinger et al., 1978), ovarian follicle (Toshimori and Yasuzumi, 1979) or endothelium, (Yee and Revel, 1975; Staehelin, 1975; Simionescu et al., 1975, 1976), than they are to the complex, anastomosing tight junctions described earlier. Simple, non-anastomosing parallel particle rows also form the morphological basis of the tight junctions in testis (Neaves, 1973; Nagano and Suzuki, 1976a, b; Gilula et al., 1976; Connell, 1978) while parallel ridges represent the tight junctions at the borders of myelin in Schwann cells of vertebrate CNS (Mugnaini and Schnapp, 1974; Schnapp and Mugnaini, 1975, 1976). Again these are structurally rather more like those of insects, and they too have been shown to form barriers.

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A variety of experimental regimes have been applied to tight junctions, in an attempt to elucidate some of their features. For example, the permeability of tight junctions in vertebrates can be altered by the passage of current across the tissue; depolarization increases permeability and the increased flow of solutes may give rise to junctional blistering (Di Bona, 1972; Erlij and Martinez-Palorno, 1978). Comparable effects may be expected to occur in insect tissues, but for the most part, the appropriate experiments have not been done. Chemical agents such as urea, can also modify junctional permeability and this has been seen in insects (Treherne et al., 1973). In addition, when the central nervous system of insects is incubated in external hyperosomotic solutions, the blood-brain barrier is disrupted, the junctions appear to “open” and lanthanum gains access to the intercellular spaces between axons and glia throughout the system (Leslie, 1975). This effect is very like that observed in vertebrates after treatment with hypertonic urea or other substances (Rapaport et af., 1971) when the tight junctions were opened or made leaky in some way. Such results suggest that tight junctions play a role in regulating epithelial function, with “leaky” tight junctions being more sensitive to such effects as, for example, changes in osmotic concentrations (Diamond, 1978; Erlij and MartinCz-Palomo, 1978). There is an ever-increasing literature on modifications of vertebrate tight junction permeability such as described above by altering the tonicity of the bathing medium, by passing current across the tissue, by the addition of chemical agents or by changing physiological conditions; the effects of these have been recently summarized and reviewed by Erlij and MartinCz-Palomo (1978) where further details may be obtained.

5.8

FUNCTIONAL SIGNIFICANCE

5.8.1 Adhesion There can be little doubt that in the act of creating an effective occlusion of the intercellular space, the tight junction is also maintaining the physical Fig. 54 Diagram to represent the chief differences between the distribution and structure of insect tight junctions (A) and those of the vertebrates (B). The insect lateral borders are highly interdigitated and the basal tight junctions have a simple, linear intramembranous structure (as indicated in rectangle on right). The vertebrate tight junctions are near the apical border, and the adjacent cell membranes at this terminal bar region have a relatively straight, noninterdigitating apposition. In freeze-fracture, as shown in rectangle on the right, the vertebrate tight junctions are more reticular and anastomosing than those of insects. It would appear that although such complex structures are required to form the basis of an effective barrier in vertebrate cells, in the insects, on the other hand, the complex interwoven cell surfaces heighten the effectiveness of the much simpler tight junctions so that they too can be occluding. GJ, gap junctions; SJ, septate junctions; TJ, tight junctions; D, desmosomes

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integrity of any tissue system; this particular function has been emphasized in certain vertebrate tissues (Pannese, 1968; Simionescu et al., 1975; Yee and Revel, 1975; Shinowara et al., 1977). However, a role in adhesion would appear ordinarily to be secondary to their primary significance in producing seals or barriers, particularly since desmosomes and, in insect tissues, septate junctions can maintain physical associations very effectively. 5.8.2 Barrier functions and regulation of permeability As mentioned earlier, the sealing strands of tight junctions provide a permeability barrier. The tightness of the seal is indicated by the transepithelial resistance to the movement of ions over it; if there is a high electrical resistance, a steep concentration gradient may be formed across the epithelium and the two compartments may be maintained as distinct from one another. It has been suggested that there is a positive correlation in vertebrates between the number of sealing strands and higher transepithelial resistance or greater impermeability (Claude and Goodenough, 1973; Claude, 1978). For example, small intestine and urinary bladder which are very “tight”, have a complex reticulum of tight junctional ridges and grooves; proximal kidney tubule or gall bladder, which are leakier, have far fewer strands and a less complex network. Insect tight junctions do not exhibit such a range of complexity in structure. If the electrical resistance is changed by bathing in hypertonic solutions, the tight junctions of epithelia in vertebrates become altered and blisters form (Di Bona, 1972); this suggests that the tight junctions are accessible to water and probably limit, rather than prevent, t h e paracellular passage of water and ions. Hence tight junctions should more properly be termed “limiting junctions” (Di Bona, 1972). Such studies, together with the results of Erlij and MartinCz-Palomo (1 978), MBllgSrd et al. (1976) and MartinezPalomo and Erlil (1975), suggest that the differences in transepithelial resistance of tissues cannot be explained solely by the number of tight junctional strands and that some differences in the chemistry of the junctional molecules (Claude, 1978) may be the determining factor of junctional permeability. This bears on the functioning of the tight junctions in the perineurial cells of the insect CNS where a finite leak to K+ has been found (Pichonetal., 1971) and where addition of urea enhances t h e leak of K+ and Na+ whilst still limiting the entry of tracers which are of a higher molecular weight (Treherne et al., 1973). Modifications in perineurial membrane permeability or in the tight junctions themselves were thought to be responsible for these effects so that such experimental evidence could irnplicatc thc ’sy tight junctions in roles such as that of regulating epithelial pe~~ieabiliiy differential sensitivity to different substances.

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One very effective barrier created by tight junctions is that at the bloodbrain interface; this has been much studied in vertebrates (Reese and Karnovsky, 1967; Brightman and Reese, 1969; Brightmanet al., 1975) and was the first kind of barrier to be characterized in insect tissues (see Treheme and Pichon, 1972). One of the features of the insect tight junctions in the CNS, as well as in other tissues, is their relative simplicity; being linear and non-anastomosing, they are clearly far less ordered in their arrangement than those of vertebrates. Can they then effectively form a “tight” barrier to the entry of ions and molecules? This problem has been considered in detail elsewhere (Lane et al., 1977a, pp. 192-194). In essence the assumption is that, given the complex geometry of the intercellular interdigitations (see Fig. 54A), a series of simple linear ridges that represent points of membrane fusion, aligned in parallel and parallel to the outer epithelial surface, would be sufficient to prevent entry of most molecules. The lack of tight junctions in invertebrate groups other than insects (and tunicates) may be related to the absence of systems requiring a tight seal or barrier (see also Section 9). The CNS can be shown to be open and lacking a barrier in other invertebrate groups such as annelids (Nicholls and Kuffler, 1964; Coggeshall and Fawcett, 1964; Skaer et al., 1978), molluscs (Pentreath and Cottrell, 1970; Sattelle and Lane, 1972, Lane and Treherne, 1972a; Sattelle and Howes, 1975; Browning, 1979), the Xiphosuran Limulits (Harrison and Lane, 1980) and acarines (Binnington and Lane, 1980). As would be expected, in none of these areas arezonulae occludentes to be found in the perineurial cells surrounding the CNS. In some gastropod molluscs such as Helix, no tight junctions are found between peripheral glial cells yet it is thought that peri-neuronal glia may act as a haemolymph-neuron barrier (Reinecke, 1976); it is not clear how this could operate in the absence of junctions, especially since in other gastropods such as Lirnnaea, the glial cells may not even form a complete investment around the nerve cells (Sattelle and Lane, 1972). However, without freeze-fracturing it cannot be determined whether tight junctions are truly absent so that this system obviously requires further investigation. In the crustacea, no definitive blood-brain barrier is present (Abbott, 1970,1972; Kristenssonetal., 1972; Lane, Swales and Abbott, 1977); there is a delay measured physiologically to the entry of ions and molecules, but no ultimate restriction (Abbott et al., 1977). The perineurium in this case contains many gap junctions and what appear to be some non-zonular tight junctions which no doubt lead to the slowing down of entry of substances (Lane and Abbott, 1975). In peripheral nerves, the perineurium is reduced to a single or incomplete cell layer with no intercellular junctions (Lane and Abbott, 1975) and exogenous tracers enter freely (Kristenssonet al., 1972; Lane rt al., 1977b). It is clear, therefore, that the entry of tracers can be

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correlated with the absence of an electrophysiological barrier and of occluding junctions so that it is valid to assume that when tracers are prevented from entry into any system, occluding junctions must be present to seal off the intercellular pathway. It seems, then, that in groups of animals lower than insects, the nervous system possesses a more primitive form of homeostasis that does not necessarily involve junctional specializations at the blood-brain interface. If some form of homeostasis i s required in such systems and no barrier has evolved, it seems likely that other mechanisms, such as ion-binding to a charged extracellular matrix, or glial-neuronal regulation, may function to produce short-term neural regulation (Abbott and Treherne, 1977; Abbott et al., 1977). In the case of systems other than the nervous system, there are reports of occlusions to the entry of exogenous tracers in such organisms as planarians (Lord and Di Bona, 1976) and coelenterates (Wood, 1977; Filshie and Flower, 1977) but in these cases only septate and gap junctions have been found to be present. It may be that in more primitive metazoans, particularly perhaps in diploblastic organisms, barrier functions are carried out by the septate junctions (see discussion in Section 2.7). These could then be retained in higher invertebrate metazoans for adhesive purposes and for some partial occluding roles, whilst the tight junctions, as in the vertebrates, have evolved to deal with more physiologically-critical barrier functions (for further discussion see Section 9). 5.8.3

Compartmentalization

The role of tight junctions near the lumen in the terminal bar in establishing and maintaining the polarity of cells is important in vertebrate cells (Staehelin and Hull, 1978); the junctional fusions provide a physical barrier to the migration of intramembranous particles from the outer surface of an epithelium to the inner. Differences in IMP populations between the apical region and the lateral and basal regions, have been established to substantiate this (De Camilli et al., 1974). In insect tissues, however, the tight junctions are basally situated. It may be suggested, therefore, that this compartmentalization is one of the roles of the septate junction, since they tend to occur closer to the luminal surface of the cells in insects. Alternatively, it is possible that this division of IMP into ( a ) luminal, and ( b ) lateral and basal regions by terminal bar tight junctions in vertebrate epithelia, is, in the insects, more correctly divided into ( a ) luminal and lateral, and ( b ) basal regions, by the tight junctions located on the lateral borders near the basal surface in insect cells. For example, in the perineurium, both luminal and lateral surfaces of the plasma membrane display very prominent and close-

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packed IMPS, in contrast with the other membrane faces seen in the CNS (Lane et al., 1977a). Such compartmentalization in the insect CNS might correlate with the observed high levels of ion transport across the outwardly directed perineurial membrane faces (Treherne and Schofield, 1978). 5.8.4 Functional implications of tight junctional ridge morphology It has been noted that irregular unlinked or loosely interconnected arrays of tight junctional ridges in vertebrate tissues, in contrast to more regularly interconnecting, richly anastomosing ridges, may be related to the physiology of the particular tissue under consideration (Hull and Staehelin, 1975; Staehelin and Hull, 1978). The former arrangement of tight junctional ridges occurs in tissues that have to expand or stretch under the tension of an accumulating secretory product or from some other strain. In insects, the cells of the rectum may be under tension with water and solute flow while the insect CNS may be subject to a good deal of stress; for example, the ventral nerve cord in some adults is in a constant state of agitation due to movement of the attached musculature (Lane, 1972). This could relate to the lack of cross-linking in insect tight junctions. It has previously been observed (Lane et al., 1977a) that the large number of interglial desmosomes in Calliphoru (Lane and Swales, 1978a, b) and Manduca (Lane, 1972), together with the dense mucopolysaccharide-like material that fills the extracellular dilations between gljal cells in the ventral nerve cord of such insects as the cockroach, locust and stick insect (Lane and Treherne, 1970; Ashhurst and Costin, 1971; Lane, 1974; Skaer and Lane, 1974), may all contribute to the maintenance of the structural integrity of the axoglial arrangements in the nervous system when subjected to external stress or motion. This suggests that it may be important to the insect to possess perineurial tight junctions which are less highly ordered and with few or no cross-links to allow for variability in cell shape. The tight junctions in the vertebrate blood-testis barrier which occur between Sertoli cells, also, like insects, have no anastomosing network of fibrils but only parallel particle arrays (Neaves, 1973; Nagano and Suzuki, 1976a, b; Gilulaet al., 1976; Connell, 1978). Equally, the tight junctions at the borders of myelinated axons in a range of vertebrates are also composed of only simple parallel ridges (Dermietzel, 1974a; Mugnaini and Schnapp, 1974; Schnapp and Mugnaini, 1975,1976). The significance of these structural differences in tight junctions in relation to their functional roles makes for intriguing speculation; for example, Suzuki and Nagano (1978) suggest that the tight junctional strand geometry in the testis may be related to passive shape changes in the cells due to pressure of luminal contents and muscular contraction.

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NANCY J. LANE AND HELEN leB. SKAER DEVELOPMENTAL STAGES I N TIGHT JUNCTION FORMATION

Very little has been done on the development of tight junctions in insects. What has been carried out has been done on the developing blood-brain barrier of late embryos/early hatchlings and early pupae of Calliphora (Lane and Swales, 1978a, b) and the moth, Manduca (Lane and Swales, 1979a). Here individual PF particles are seen first in perineurial cells of embryonic CNS. These become aligned into short rows (Fig. 55, insert), which themselves seem to become lined up (Fig. 56) into longer ridges which have row ends touching or overlapping (Lane and Swales, 1979a). A number of such ridges become aligned in parallel with each other and with the outer surface of the CNS (Fig. 55). By the time of hatching, these are usually fully functional junctional structures in that the insect has an effective bloodbrain barrier, which is capable of excluding tracers and acting to prevent the inward leakage of undesrrable large ions and molecules. Interestingly, the embryonic development of the blood-brain barrier seems to occur at different times in different insects. In the blowfly, Calliphora, the barrier forms just after hatching (Lane and Swales, 1978a), in the moth,Manduca, it arises during the last day of embryonic development before hatching (Lane and Swales, 1979a), while in the locust, Schisrocerca, it forms quite early on in embryonic development. In the stick insect, Carausius, the blood-brain barrier is also already established at the time of hatching (Leslie, 1973), a feature that seems particularly vital in this phytophagous species given the unusual ionic composition of its haemolymph which would be incompatible with the conventional nervous condition that occurs in this insect (Treherne and Maddrell, 1967). In these studies, although the development of tight junctional ridges and grooves can be followed, the penetration or occlusion of tracers serves as an immediate and rapid way of determining whether or not a blood-brain barrier has formed. In vertebrate tissues, comparable tracer studies enable one to determine exactly when the blood-brain barrier develops by observing, over a period of time, the sudden exclusion of a tracer, for example, horseradish peroxidase (HRP) from the nerve cell and neuropile areas Fig. 55 Freeze-fracture replica from the developing CNS in the late embryo of the moth, Manduca sexta. This area is the forming perineurium that abuts onto the neural lamella. The interdigitating perineurial cell processes can be seen to possess membrane P face ridges (at arrows) and E face grooves (C). The insert shows one of these forming ridges, at higher magnification, in order to demonstrate their bead-like nature. As can be seen in the micrograph, the 8-10 nm particles seem to be lining up into shorter segments (thick arrows) which then coalesce to fuse into long thin ridges (arrows). x 17 300; insert x 77 300 Fig. 56 Embryo of a moth showing the perineurial P face fromhhe CNS with tight junctions in the process of formation. Note that the short ridges of fused particles (arrows) become aligned, as in Fig. 55, insert, into lengthy ridges which lie parallel to the surface of the nervous system. x 33 700

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(Wakai and Hirokawa, 1978). Studies on tracer exclusion coupled with freeze-fracture show that there is a correlation between the presence of tight junctional ridges and this lack of exogenous tracer accessibility. For example, during tight junction development, as the ridges form, first HRP and then lanthanum, are excluded from entry (Tice et al., 1977), while after bile duct ligation, on the contrary, the tight junctional ridges degenerate by breaking down and as they do so, the system becomes leaky to HRP (Metzet al., 1977). In vertebrate tight junctions, the free junctional particles also become lined up into short ridge lengths; these fuse into beaded ridges and progressively interconnect and coalesce into the confluent smooth-ridged network or reticulum characteristic of mature vertebrate tight junctions. This sequence of events has been shown to occur in such developing tissues as foetal cells (Revel et a[., 1973; Decker and Friend, 1974; Ducibella et al., 1974; Montesano et al., 1975; Humbert et al., 1976; Luciano et al., 1976; Magnuson et al., 1977; Tice et al., 1977; Schneeberger et al., 1978) and cultured cells (Porvaznik and Johnson, 1974; Porvaznik et al., 1976; RonveauxDupal and Wanson, 1976; Elias and Friend, 1976; Dermietzel et al., 1977; Meyer et al., 1978). It has been shown that the junctions can become established within 30 minutes (Hudspeth, 1975) thereby demonstrating a capacity for rapid restoration of tissue integrity after cell loss or tissue disruption (Erlij and Lazaro, 1975). In considering the assembly of tight junctional ridges from free particles, the process is more plausible if one invokes the offset two fibril model of Bullivant (1978) rather than the single fibril model of Wade and Karnovsky (1974). With insects, however, we do not have enough information to be able to judge which is the appropriate model. In some cases, as for example in the vertebrate blood-testis barrier, the occluding junctions may be, as described earlier, simple parallel intramembranous particle rows; during their development a random discontinuous arrangement of short segments is found (Nagano and Suzuki, 1976a, b; Gilulaetal., 1976; Meyer, Posalaky and McGinley, 1977; Connell, 1978) to which pattern and final form the insect tight junctions are rather more comparable. Recent analyses of tight junctional development in foetal mesothelium and Sertoli cells (Suzuki and Nagano, 1978) suggest that the initial stage in the formation of tight junctions is the close apposition of the two cell membranes without particles; the junctional particles appear to become incorporated subsequently. However, in insect developing junctions, no precise correlation between close membrance apposition and particle alignment has yet been possible, so that the actual moment o f particle involvement cannot be ascertained.

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A problem facing investigators analysing the intramembranous changes in vertebrate developmental events is that the tight junctional and the gap junctional particles are both about the same diameter, and both usually fracture onto the P face (except for some rare cases such as in oligodendrocytes (Dermietzel etul., 1978) and Sertoli cells (Nagano and Suzuki, 1976a, b; Gilula et al, 1976) where the tight junctions are of the inverse type with EF particles). Since these two junctions are often found together in epithelial cells (Staehelin and Hull, 1978), this can lead to difficulties in interpreting which junctional particles belong to which junctional type (Revel and Brown, 1976); this problem never arises in insects as mentioned earlier, because although the tight junctional particles are PF, the gap junctional ones fracture onto the EF and are of a larger diameter.

5.10

T I G H T JIJNCTION DEGRADATION

In holometabolous insects, the nervous system frequently undergoes considerable changes in arrangements between larval and adult stages and the question arises as to how the blood-brain barrier and its component tight junctions are affected by these structural alterations. In Culliphoru pupae, the barrier is disrupted, fragments of tight junctional ridges are observed and tracers freely enter the extracellular spaces beneath the perineurium between axons and glia (Lane and Swales, 1978b); it appears that the junctional particles become dispersed as the ridges break down. In Manduca, although studies on the abdominal nerve cord indicate that the blood-brain barrier there continues to exclude tracers throughout pupation (McLaughlin, 1974a, b), when thoracic ganglia are examined the situation is less straightforward. Partial disruption of the barrier seems to occur (Lane and Swales, 1979c) in those places where the thoracic ganglia fuse with one another during pupal nerve cord shortening (see Pipa and Woolever, 1964). In diapausing pupae, the perineurial cells which form the basis of the blood-brain barrier are very similar to those of non-diapausing insects (Houk and Beck, 1975; Lane and Swales, 1979b, c) with intercellular junctions exhibiting no obvious differences from the late larval state (Lane and Swales, 1979a, b, c); they d o not display junctional breakdown as d o normal pupae (Lane and Swale, 1979b). In vertebrates, tight junctions undergoing degradation have interrupted or disarranged strands with ridges diminishing in number and increasing dissociated particles (Revel et al., 1973; Revel, 1974; Decker and Friend, 1974; Dermietzel et al., 1977; Metz et al., 1977; Metz et al., 1978; PolakCharcon and Ben-Shaul, 1979). It has been found that tight junctions are susceptible to mechanical shear (Metz et ul., 1978), to trypsinization (Dermietzel et al., 1977) and to changes in Ca++concentration (Brenna and

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Meldolesi, 1976; Meldolesi et al., 1978). In some cases, they disappear after a certain stage in development and are clearly only temporary structures (Pannese, 1968). Their mode of breakdown may either be that described above which enables the cell t o preserve the intramembranous particles for re-utilization or that involving internalization and exclusion of tight junctional elements by bleb formation (Polak-Charcon and Ben-Shaul, 1979); in the latter an endocytotic mode of digestion (Porvaznik, 1979) with degradation by lysosomes (Staehelin, 1973, 1974) seems probable. It is likely that events comparable to the first possibility occur when insect tight junctions disaggregate in early pupal life, since no striking evidence has been found to suggest internalization (Lane and Swales, 1978b, 1 9 7 9 ~ )However, . their relative infrequency and basal orientation renders their disassembly more difficult to follow than that of the luminal tight junctions of vertebrates. When vertebrate cell tight junctional breakdown occurs in vitro after trypsinization, the degradative mechanisms are not the same (Dermietzel et al., 1977) as those observed in vivo (Revelet al., 1973; Decker and Friend, 1974; Elias and Friend, 1976). The breakdown due to trypsin produces a diagonal orientation of subunit structures (Dermietzel et al., 1977) called “abortive” tight junctions (Yee and Revel, 1975). After an in vitro disassembly, the junctional reformation that occurs is said to be a “hemiconservative” process (Dermietzel et al., 1977); remnants serve as condensation points from which the remodelling carries on in a way comparable to that of later stages in normal junctional formation. In insects, no in vitro studies have yet been made on tight junctional development but the reformation of tight junctions in the CNS in late pupae after their breakdown in early pupal stages (Lane and Swales, 1978b) may reutilize junctional remnants still present in the perineurial membranes. In this sense, it too may be a “hemiconservative” process.

5.11

PHYLOGENETIC AND EVOLUTIONARY POSITION

The tight junctions seen in the tissues of tunicates (Lorber and Rayns, 1972; Cloney, 1972), which are positioned phylogenetically above the invertebrates as lower chordates, appear as punctate appositions in thin-sections and produce an intramembranous particle distribution that is reticular-like; this, it is claimed, i s similar to that of the smooth septate junction (Green, 1978) although in a more anastomosing form. The membranes fuse in thin-section, but are said to be “leaky” to lanthanum (Green, 1978). Since this study involved colloidal lanthanum added to the fixative, rather than incubation in ionic lanthanum in physiological saline, the term ‘‘leaky’’is not really meaningful. Green (1 978) considers that the invertebrate septate

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junctions are occluding, although no unequivocal evidence in support of this is cited, and suggests that they, rather than invertebrate tight junctions, may form an evolutionary link with the vertebrate tight junctions. This proposal suffers from a disregard of the fact that the septate junctions are characterized by a 1 5 to 20 nm intercellular cleft, while the vertebrate tight junctions have fused membranes with a complete absence of any intercellular space. The intramembranous particles in each are dissimilar and have quite different functions; in the septate junctions they are rows of separated particles that may possibly serve as insertion sites for the septa, while in tight junctions, the particles are fused together laterally into ridges which serve to fuse further onto directly opposite ridges from the adjacent cell. Moreover, the evidence in support of an occluding role for the septate junctions is slight compared with that indicating that they produce negligible or only limited obstruction to the movement of ions and molecules (see details in Section 2.7). Hence it seems likely that insect tight junctions, rather than septate junctions, either represent the evolutionary forerunners of the vertebrate tight junctions or form their functional equivalent in invertebrate tissues.

6

6.1

Specialized junctions of glia INTRODUCTION

Junctional specializations between arthropod neurones and the innermost glial layer than enfolds them, while akin to the junctional types described in this review, exhibit differences that seem to us to merit a short section devoted to their description and evaluation. Desmosomes do not appear to have been found linking these two cell types, apart from the description of small asymnietric junctions in a sensory terminal in Culliphora (van Riuten and Sprey, 1.974), and septate junctions (probably of the pleated type) have only rarely been described (Smith, 1967). In this section, we shall consider evidence for the existence of junctions similar to tight junctions as well as junctions bearing a resemblance both to tight and smooth septate junctions. A variety of particle arrays, revealed by freeze-fracture studies, may characterize the axo-glial membranes; these will be described and their status as cell junctions assessed. Glial cells may also form junctions with tracheal cells in those regions (e.g. the glial lacunar system) where the tracheal system penetrates the perineurial investment of the CNS.

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6.2 6.2.1

AXO-GLIAL JUNCTIONS

Tight junction-like appositions

Tight junction-like structures between axons and their glial covering are revealed in thin sections of the central nervous system as isolated punctuate appositions (Fig. 58) not unlike those of the perineurium (Figs 41, 42). However in freeze-fracture replicas (Figs 57 and 59), the P face ridges and E face grooves are much shorter and less coherently organized than in the perineurium, although they tend to lie roughly parallel to the longitudinal axis of the cell (Fig. 57). The ridges are most commonly associated with the P face of the axonal membrane (Fig. 57); the grooves occurring on the glial E face (Figs 57 and 59). The ridges appear to be made up of 10 nm particles which may fuse or remain clearly moniliform (Fig. 57). The degree of complementarity between the ridges and grooves is not clear since fractures crossing the intercellular space to reveal the two membrane faces in the region of a ridge/groove are rarely encountered. These axo-glial tight junction-like associations have been found in replicas between nerve cells and glia in the central nervous system of the cockroach, Periplaneta americana (Lane et al., 1975b, 1977a; Wood et al., 1977), the locust, Schistocerca gregaria (Lane et al., 1977a; Lane, 1979b), where the axonal ridges are found with an increased frequency in hatchlings and pupae compared with adults (Lane, 1979b), the moth, Manduca sexta (Lane et al., 1977a; Lane and Swales, 1979a; Lane, 1979b), the blowfly, Calliphora erythrocephala (Fig. 58 and Lane et al., 1977a), and the bug, Rhodnius prolixus (Lane, 1979b), where axonal ridges have also been encountered in the peripheral nervous system. Thin-sections reveal the occurrence of punctate appositions between neighbouring glial cells (Figs 3 8 , 3 9 and Figure 1 6 in Laneet al., 1977a) and it is possible that some of the freeze-fracture ridges and grooves represent glial-glial junctions rather than axo-glial associations. Short, bead-like P F ridges have been described in a variety of insect

Figs 57-59 This plate deals with axo-glial “tight” junctions, and presents some evidence for their existence in insects Fig. 57 Replica from the central nervous system of the insect Calliphora showing the discontinuous axonal ridges (at arrows) which are frequently encountered, as well as overlying glial grooves (G). x 50500 Fig. 58 Thin-section from the central nervous system of the blowfly Calliphora showing the regions where the glial (G) membrane and that of the axon (A) are in close apposition (arrows), in an apparent axo-glial junction. GGJ, glial gap junction. X 67 800 Fig. 59 Freeze-cleaved replica from a glial cell membrane in the CNS of Calliphora exhibiting the short E F grooves (arrows) complementary to the PF ridges seen in axon and glial membranes, and hence representing a possible basis for axo-glial junctions. X 113 800

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tissues including tracheole cells (see Sections 5.4.4 and 6.3 and Lane et al., 1977a; Lane, 1979b), proventriculus, midgut (see Figure 8 in Ito et al., 1975), rectum (Lane, 1979b), muscle cell membranes (Smith and Aldrich, 1971; Lane, 1979b) and fibroblast cells (Lane, 1979b). In arthropods other than the insects, similar truncated ridges on the P face have been found in the tissues of the horse-shoe crab Limulus (Lane, 1979b) and in the muscle of crustaceans (Smith et al., 1979). In the vertebrates, in addition to the septate-like axo-glial junctions described in Section 2.6, the nervous system displays P face ridges and E face grooves more clearly allied to vertebrate tight junctions while still being distinct from them. These may be axo-glial (e.g. Schnapp et al., 1976), between oligodendrocytes and the oligodendroglia (e.g. Tani et al., 1977; Dermietzel et al., 1978), or glial-glial (in myelin) (e.g. Schnapp and Mugnaini, 1975,1976). In these instances, the junctions are considered to have a barrier function (Dermietzel, 1975; Schnapp and Mugnaini, 1975, 1976) although their possible significance as mechanical stabilizing junctions is also suggested (Dermietzel, 1975; Dermietzel et al., 1978). A recent paper by Shivers (1979) on the CNS of the chameleon, Anolis carolinensis, presents evidence to support the mechanical rather than occluding role of mesaxon tight junctions. Freeze-fracture images of the junctions show discontinuous ridges and the tissue appears to be patent to horseradish peroxidase. However, the permeability of the blood-brain barrier had been experimentally increased by injection of D-glucose, a procedure that Shivers assumed did not affect the mesaxon tight junctions. Penetration of myelin by other tracers has been reported [in vivo lanthanum pellet implants (Hirano and Dembitzer, 1969), lanthanum in fix (Revel and Hamilton, 1969) and microperoxidase (Feder, 1971)] but these reports and that of Shivers (1969) are diametrically opposed to those of other investigators (Hirano et al., 1969; Feder, 1971; Hall and Williams, 1971; Reier, 1976; Reier et al., 1978) who show the exclusion of tracers from the myelin sheath. The functional significance of axo-glial tight junction-like associations in the insect nervous system is not clear. Their resemblance to simple ridges and grooves in non-nervous tissues argues against a specialized role in impulse generation or conduction. Moreover, while they could represent receptor sites, a number of the particle arrays that have been observed thus far in the excitable tissues of invertebrates (see Section 6.2.3) have also been thought to be receptors - it seems relatively unlikely that both particle arrays and linear ridges could be intramembranous receptor sites and the linearity of the ridges suggests that their role might involve directionality. It has been suggested that they could be involved in contact guidance of outgrowing neurones. This suggestion (Lane, 1979b) would explain their increased frequency in the early stages of development and at those stages in

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holometabolous insects when rearrangement of the nervous system is underway (Lane 1979b; Lane et al., 1980). 6.2.2 Smooth septate-like junctions Axo-glial associations have been reported in the compound eye of dipteran insects (Carlson and Chi, 1979); the picture of the cellular associations is not yet entirely clear, however, so that there may be some confusion between axo-axonic, axo-glial and interglial junctions. After freeze-fracture, the structures that are found have some of the morphological characteristics of the smooth septate junctions described in such tissues as insect midgut (see Section 2.3.3). They display a regular intercellular cleft of about 15-20 nm and in replicas feature ridges and grooves of which some are complementary. No negative-stained preparations are yet available so no unequivocal interpretation can be made. Somewhat similar structures have been found on the photoreceptor cells in Musca and Calliphora (Figs 71-73) but these may well not be axo-glial. They appear perhaps more similar to the reticular septate junctions of the rectal papillae (see Section 8.1). 6.2.3 Particle arrays Arrays of closely-packed particles are occasionally found in freeze-fracture replicas of insect nervous tissue. The particles, of about 10 nm in diameter, are found, packed with approximately rhomboidal symmetry, on the P F of the axon with a barely decipherable image of EF pits on the overlying glial membrane (Lane et a l . , 1977a; Lane, 1979b). Such particle clusters have been found in various parts of the insect nervous system [axons (references cited above and Ne’eman and Spira, 1977) post-synaptic membrane (Rheuben and Reese, 1976) photoreceptor cells (Gemne, 1969)] as well as in the nervous systems of crustacea (e.g. Perrachia, 1974), ticks (Binnington and Lane, 1580) and other invertebrate phyla (planarians, Quick and Johnson, 1977; annelids, Rosenbluth. 1972; Skaer, unpublished observations). However, these regular arrays of particles are not restricted to nervous tissues but are found in other excitable cells such as muscle cells, (Smith and Aldrich, 1971; Rosenbluth, 1974; Franzini-Armstrong, 1976, 1979; Prestcott and Brightman, 1976; Quick and Johnson, 1977; Eastwood, Franzini-Armstrong and Peracchia, 1977; Smith et a l . , 1979a). They have also been described in a variety of invertebrate epithelia (Hydra surface epithelium, Wood, 1974; mollusc gill epithelium, Porvaznik et a l . , 1979; insect salivary gland, Skaeret a f . ,1973; and rectal epithelium, Lane, 1979b). The occurrence of particle arrays in vertebrate tissues is also well

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documented. These assemblies may be associated with axons (Livingston et al., 1973; Dermietzel 1973,1974b; Landis and Reese, 1974; Schnappetal., 1976; Miller and Pinto da Silva, 1977; Elfvin and Forsman, 1978), muscle cells (Rash and Ellisman, 1974; Orcietal., 1974; McNutt, 1975; Smithetal., 1975), with other epithelia (Albertinietal., 1975; Kachadorian etal., 1975; Severs and Hicks, 1975; Dermietzel and Leibstein, 1978; Severs and Warren, 1978) or between cells grown in culture (Porvaznik et d., 1976). There are, however, distinctions between vertebrate and invertebrate particle arrays, in that in the former the particle size is smaller and the packing of particles is tighter than in the latter. Furthermore, the majority of reports on invertebrates are concerned with excitable tissues or with epithelia that are innervated (Skaer et al., 1975; Porvaznik et al., 1979) or that may be sensitive to hormones (Lane, 1979b), while in vertebrates, particle arrays have been reported from a very wide variety of tissues and even from cells in culture. The majority of the reports concerning these regular arrays of particles in both vertebrate and invertebrate tissues conclude that the membrane specializations are not associated with junctions. Where they occur on free surface membranes (e.g. Severs and Hicks, 1975; Skaer et al., 1975) they cannot form junctions and despite their superficial resemblance, in some instances, to gap junctions, the details of their freeze-fracture morphology (larger particle size, wider centre-to-centre spacing, absence of central depression in the particle) mitigates against their being gap junction-like structures. The functional significance of rhombic arrays of particles is not clear. It has been suggested that they may represent receptor sites in subsynaptic membranes (e.g. Orci et al., 1974; Rash and Ellisman, 1976; FranziniArmstrong, 1976; Rosenbluth, 1978) or in the membrane of an innervated epithelium (Porvaznik et al., 1979), they may affect the electrical properties of membranes (Quick and Johnson, 1977) or may permit ionic or metabolic exchange between cells (in a junctional specialization, Perracchia 1974; Binnington and Lane, 1980) or between cells and the environment (Landis and Reese, 1974; Franzini-Armstrong, 1979). They have also been implicated as sites of adhesion with extracellular or intracellular elements (Prestcott and Brightman, 1976; Smith et al., 1979a; Franzini-Armstrong, 1979). Although the arrays of particles thus far described bear strong resemblances one to another, their varied distribution with respect to tissues and phyla, minor 'differences in their arrangement, particle size, packing and fracturing characteristics may well underly significant differences in their composition and stereochemical properties and therefore in their functional significance. This is an area in which further analysis as well as technical and

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experimental developments will yield a rich harvest in terms of our understanding of these structures. 6.3

TRACHEO-GLIAL JUNCTIONS

The insect central nervous system is penetrated by tracheae and tracheoles in certain specialized regions, termed the glial lacunar system (Wigglesworth, 1960). In these regions, the tracheole cells are found abutting onto the glial cells and, in some instances, forming junctions with them. In freeze-fracture replicas, tight junction-like ridges may be found on the P face of the glial cell membranes or the tracheole cell membranes (Fig. 60), with grooves on the complementary E face. In thin-section, punctuate appositions may be found where the intercellular space between the two cells is occluded (arrowed in Fig. 62). In freeze-fracture replicas, PF ridges are found on the tracheal membranes in other tissues as well and occasionally a rather unusual association of pleated septate junctional P face particles and tight junction-like ridges occurs (Fig. 61). Pleated septate junctions are very commonly found in the mestracheal folds both in replicas and thin sections (Fig. 63 and Section 2.4) but the “enclosing” of the intramembranous septate junctional specialization with ridges running parallel to the course of the particle rows (as in Fig. 61) is unusual. The functional significance of these tight-like junctions between tracheole and glial cells is not yet known. Such associations have been found in the nervous systems of a variety of insects, both holo- and hemi-metabolous (see Figure 38 in Lane, 1979b) and it is not yet clear whether they represent non-junctional or junctional structures. They might be involved in the process of contact guidance as the cells seek their final location, in which case further study should reveal an increase in their frequency in the tissues of animals undergoing metamorphosis. It has, however, also been suggested that they may be involved in maintaining the spatial relationship between the two cell types (Lane, 1979b) or in creating a tracheo-glial barrier, which could act as a functional substitute for the perineurial blood-brain barrier, in those areas where it is breached by the invading tracheal supply (Lane et al., 1977a) (see Section 5.4.4).

7 Scalariform junctions

7.1

INTRODUCTION

Scalariform junctions are so-called because of their cross-striated appearance in thin-sections (Fain-Maurel and Cassier, 1972; Staehelin, 1974; Noirot and Noirot-TimothCe, 1976; Lane, 1979c; Noirot-TimothCe, Noirot,

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Smith and Cayer, 1979; Noirot-Timothte and Noirot, 1979). This is the result of the presence of rather faint filamentous intercellular crossstriations which are far less distinct (Figs 65 and 66, insert) than the septa of septate junctions. These junctional areas are characterized by a uniform spacing of about 1 0 to 20 nm between adjacent cell membranes, so that, like septate and smooth septate junctions, a regular and equidistant intercellular channel is established wherever these cell appositions exist. Scalariform junctions were first reported as such in thin-sections of the excretory system of the insect Petrobius (Fain-Maurel and Cassier, 1972) and are now known to exist primarily in rectal tissues but also in nephridial and some other secretory tissues (Noirot and Noirot-Timothte, 1976; Noirot-TimothCe et al., 1979); there is also some evidence implicating their presence in nervous tissues (Lane, 1968,1974; Lane and Treherne, 1970).

7.2 7.2.1

THIN-SECTION APPEARANCE

Cytological features

The two apposed plasma membranes in regions of scalariform junctions appear rigidly maintained with a constant intercellular space which appears inextensible (Fain-Maurel and Cassier, 1972; Noirot and Noirot-TimothCe, 1976; Lane, 1978a, 1979c; Noirot-TimothCe et al., 1979). They are always intimately associated spatially with mitochondria. The regular intercellular cleft has a range in width, depending on the species examined, so that it may be as little as 10 nm in Apis (Kiimmel and Zerbst-Boroffka, 1974), increasing to 15 nm in Calliphora (Gupta and Berridge, 1966; Oschman and Berridge, 1970) and the mosquito Aedes (Hopkins, 1967), or as much as 20 to 25 nm in the Thysanura (Noirot and Noirot-Timothee, 1971a; Fain-Maurel and Cassier, 1972; Bode, 1977) and the Dictyoptera (Oschman and Wall, 1969; Wall and Oschman, 1973; Noirot and Noirot-TimothCe, 1976; Lane 1978a, 1 9 7 9 ~ )The . junctional Figs 60-63 All these micrographs are of structures associated with the membranes of tracheae and glial cells Fig. 6 0 Freeze-cleave preparation of a tracheal cell from the mothManducasewtn showing the PF ridges found on the outer tracheal membrane. X 79 800 Fig. 61 Freeze-fracture replica of a trachea from Rhodnius prolixus showing conventional septate junctions (SJ) surrounded by PF ridge structures (arrows). X 85 300 Fig. 62 Thin-section through a tracheole cell (T) near its cuticle-lined cavity (C). A thin glial investment (G) accompanies the tracheole and is closely associated with it at one point (arrow) which may represent a glial-tracheal junction. x 78 700 Fig. 63 Thin-section through the mestracheon system typical of tracheal cells wherever they occur. An autocellular septate junction can be seen running from the cavity of the trachea ( C ) to the extracellular space (E). x 75 600.

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membranes are interconnected by thin-striations which appear variable both in their diameter and in their centre-to-centre spacing (Fig. 65 and insert in Fig. 66). It is thought that these may be either fibrous trabeculae or, less frequently, columnar processes; the range in diameter of these, respectively, is from 2 to 3 nm and from 4 to 5 nm (Noirot TimothCe et al., 1979). Other reports cite values of 6-10 nm (Lane, 1979c) or 15-18 nm (FainMaurel and Cassier, 1972). In some cases, their diameter is smaller near the middle of the extracellular space, and greater near the membrane (Lane, 197912;Noirot-Timothte and Noirot, 1979). The spacing of these intercellular columns is rather erratic (Noirot-TirnothCe et a1 ., 1979; Lane 1979c), but there may occasionally, in tangential sections, be a hexagonal pattern (Lane, 1979c) with a regularity in centre-to-centre spacing of 15 to 25 nm (FainMaurel and Cassier, 1972; Bode, 1977; Noirot-TimothCe et al., 1979). The columns may be aligned perpendicular to the membranes or may be set at an oblique angle (Noirot-TimothCe et al., 1979; Lane, 1 9 7 9 ~ ) .When the cross-striations appear to be columnar, they then seem to have a hollow centre (Fain-Maurel and Cassier, 1972; Bode, 1977; Lane, 1 9 7 9 ~ ) . 7.2.2 Dijrerent types The scalariform junctions which have been observed in different organs and in different insect species can be categorized with respect both to the proportion of the junctional zone that is intimately associated with mitochondria and to the relative precision of the positioning of the mitochondria (Noirot-TimothCe et al., 1979). These include two types: Type I, where the whole length of all the junctional zones are intimately associated with mitochondria which have a very regular position with respect to the junctional membranes (for example, the rectal pads of cockroaches (Oschman and Wall, 1969; Noirot and Noirot-TimothCe, 1976; Lane, 1978a, 1979c), (Fig. 65) and Type 11, where the whole length of the Fig. 64 Thin-section through the rectal papillae in the hind-gut of the blowfly, Cdliphoru. The lateral intercellular borders of the component cells are associated by gap junctions (GJ) and are thrown into stacked membrane arrays (S), that may exhibit cross-striations typical of scalariform junctions (large arrow) and which are intimately associated with mitochondria (M). The inter-stack regions may be dilated into lacunae (L) or may show the more normal dimensions of the intercellular clefts. These regions, however, exhibit specialised features termed by us the “reticular septate” (RSJ) junctions. x 52 700 Fig. 65 Thin-section through the rectal padsof the cockroach, Periptanetuumericanu, showing the undulating and interdigitating lateral borders of the component cells. These usually take the form of scalariform junctions (see Section 7.2) in that cross-striations can be seen in the intercellular spaces (arrows). Note the close association with the mitochondria (M), here scarcely visible in the unstained tissue. Gap junctions (GJ) occur at intervals and the intercellular cleft may also be thrown open into larger intercellular spaces (S). Towards the base of the tissue, tight junctions (TJ) occur. x 58 000

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junctional zone is not entirely associated with mitochondria, which, in turn, have a rather less precise positioning (for example, the rectal papillae of Calfiphora (Gupta and Berridge, 1966; Berridge and Gupta, 1967) (Fig. 64). Independent recognition of the basic similarity of these two types of cell association as typified by scalariform junctions in rectal pads and rectal papillae has also been reported elsewhere (Lane, 1 9 7 9 ~ ) . 7.3

PREPARATIONS T R E A T E D WITH TRACER

In lanthanum-impregnated preparations of both Type I and Type I1 scalariform junctions either the tracer fails to reveal any structure and masks the whole intercellular space, (Fain-Maurel and Cassier, 1972) or it suggests the presence of columns (Lane, 1 9 7 9 ~ )This . latter situation is indicated in longitudinal sections, where filaments are found lying as non-opaque striations against a background of high electron density due to the presence of tracer (Lane, 1 9 7 9 ~ )Moreover . in cross-section, the junctional areas contain non-opaque spheres lying in a densely stained intercellular substance which may be interpreted as being en face views of the columns, the sections having cut transversely across them; under these conditions, the central channel of the particles is also stained (Lane, 1979c) but the columns measure less than the figure quoted (15-18 nm) by Fain-Maurel and Cassier (1972) who also observed that they were hollow. However different groups of insects - here the Orthoptera in contrast to the Thysanura - may well have different dimensions with respect to the thickness of the intercellular striations, as they do with regard to the width of the cleft (see citations in Section 7.2.1). No septa appear to be present in oblique sections so that columns alone appear to maintain the uniform intercellular cleft. Neither pronase treatment nor acetone extraction affects the integrity of these columns; although other tests for polysaccharides are negative, ruthenium red stains a 5 nm surface layer lying round the outside of the columnar processes (Fain-Maurel and Cassier, 1972). 7.4

FREEZE-FRACTURE REPLICAS

Most of the studies on the scalariform junctions have not involved freezecleaving and so little has been published on their intramembranous features. Fig. 66 Freeze-fracture replica of the scalariform junctions that comprise the bulk of the lateral border of the rectal pad cells of the cockroach. Note that their P face (PF) bears many intramembranous particles while the E face (EF) displays only a few particles and some complementary pits. The mitochondria (M) lie at intervals but in intimate association with the scalariform junctions. GJ, gap junction. The insert shows how thin-sections demonstrate the presence of cross-striations (arrows) in the scalariform regions as they lie around the mitochondria (M). x 32 200; insert X 119 500

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However recent investigations on freeze-fracture replicas of cockroach rectal pads (Lane, 1978a, 1979c; Noirot-TimothCeet al., 1979) and on housefly or blow-fly rectal papillae (Flower, personal communication; Lane, 1979c) reveal that both the scalariform junctional areas of rectal pads (Fig. 66 and Lane, 1978a, 1979c; Noirot-TimothCe et al., 1979) and the comparable areas of stacked membranes in papillae (Fig. 67; Lane 1979c) exhibit large numbers of intramembranous particles (IMPs). These lie in the P face while complementary pits, less easily seen, and a smaller number of particles, appear in the E face. These PF particles measure about 4 to 12 nm in diameter, exhibit no obvious regular pattern or arrangement, and are only very rarely aligned to any extent. They never lie in undulating rows, as occurs in septate junctions (see Section 2.2.3), nor do they fuse together to form intramembranous ridges, as occurs in smooth septate junctions (see Section 2.3.3). Unlike the latter, fixing or lack of fixing seems to make no difference in the fracturing face to which the particles adhere; they consistently fracture onto the PF (Lane, 1 9 7 9 ~ ) . The PF particles have been reported to range, ordinarily, from 6-10 nm (Lane, 1979c) to 7 or 11 nm (Noirot-TimothCe et af., 1979) in diameter; sometimes however, two other populations of particles are described, larger ones(12-13 nm) and smaller ones (5-6 nm) so that there are three different sub-populations of IMPs (Noirot-TimothCe et al., 1979). Other reports categorize the particles as falling into two main populations, 8-12 nm and 4-8 nm in diameter, respectively (Lane, 1 9 7 9 ~ ) .Clearly, until more is known about the characterization of particles within membranes it will not be possible to judge whether there are 2, 3 or more categories of IMP here either in terms of chemical differences or in terms of physiological distinctions. In addition, it must be recognized that any division of IMPs into classes may have no real significance since the proportion of particles exposed on the fracture faces may vary with the viscosity of the membrane lipid, variations in which may lead to differential vertical displacement of the particles (Borochov and Shinitsky, 1976; Rivnay and Shinitsky, 1976). Fig. 67 Freeze-fracture replica from rectal papillae of Cuffiphorashowing the stacked membrane arrays ( S ) flanked by the “inter-stack” regions of lateral membrane. The stacks consist of piles of scalariform junctions, with numerous IMPs in the PF as shown here and pits on the E face (EF); the extracellular space in these areas is of a regular width. The inter-stack regions possess reticular septate junctions which feature ridges and particles on the P face (PF) with grooves in the E face (EF); the intercellular space in these junctional regions is variable (C). Insert shows a P face from the stacked region which is unusual in displaying P F ridges as well as the numerous IMPs typical of scalariform junctions; the ridges are part of the adjacent reticular septate junctional regions. x 49 500; insert x 24 500 Fig. 68 Area of reticular septate junctions in an inter-stack region of rectal papillae; the P face (PF) ridges and E face (EF) grooves may be complementary (at arrows). Between the linear ridges lie scattered intramembrane particles. x 80 000

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The P fracture face is characterized by an unusually high particle density (Figs 66 and 67) (Lane, 1978a, 1979c; Noirot-Timothkeetal., 1979), about 5500 particles per pm’, with the E face bearing relatively few particles numbering less than 200 per wm2 (Noirot-TimothCe et al., 1979). In some cases, the EF is said to be without complementary depressions from the PF particles (Noirot-Timothee et al., 1979) but in other instances there is an indication of complementary pits (Lane, 1 9 7 9 ~ )The . apparent lack of these could be due to the angle of shadowing in the depth of the pits; if the former is low and the pits are shallow, they will not be revealed at all in the replica. Moreover, when membrane particles do not penetrate deeply into the other half of the lipid bilayer, the complementary depressions or pits out of which they are pulled will not be visible because lipid collapse will lead to their disappearance (Bullivant, 1977); the term lipid collapse refers to the observation that after fracturing, the lipid halves of a membrane may become thinner. Although Noirot-Timothee and Noirot (1979) comment that the large number of IMPS, conspicuously higher in their view than the number of intercellular columns, disproves any precise correspondence between intercellular and intramembranous structures, they have failed to take into consideration the fact that it is impossible to estimate accurately the number of columns present given the variability observed and the possibility that perhaps only a certain percentage of the intramembranous particles are related to the intercellular columns. The differences in particle size could reflect a functional distinction and only one of the populations of particles, distinguishable by their larger or smaller size, may be insertion sites for the columns which traverse the intercellular space (Lane, 1 9 7 9 ~ )This . need not necessarily be so, but it is at least a possibility. 7.5

MODEL DERIVED FROM S T R U C T U R A L E V I D E N C E

On the basis of the thin-section tracer and freeze-fracture studies, correlations between the observed intercellular and intramembranous structures can be drawn up. A model which purports to classify the interrelationships derived from the results of these studies is shown in Fig. 69. It can be seen from analysis of the ruthenium red and lanthanum evidence that the structures that cross the extracellular space in scalariform junctions are columns or pegs, not continuous septa. The lack of alignment of intramembranous particles into rows in freeze-cleaved scalariform junctions supports this contention that columns, not septa, exist in the intercellular space; alignment of particles into rows or ridges typically occurs when septa exist (see Section 2.2.4). These columns in addition to any other function, apparently operate to maintain the uniform intercellular space found throughout these

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junctions. In this diagram we have not included the possibility that the columns are hollow although there is some evidence, as already presented, that this may be the case; Fain-Maurel and Cassier (1972) include this feature in a model based on their thin-sectioned material. Difficulties are encountered, however, when attempting to correlate these intercellular columns with the intramembranous structures seen with freeze-fracture. From studies of smooth septate junctions, although septa have an apparent intramembranous correlate with particle rows or ridges, columns appear to have no freeze-cleave counterpart (Flower and Filshie, 1975; Lane, 1978a). /--CYTOPLASM-/

/-

-E

FACE

-/

LANTHANUM IN INTER

Fig. 69 Model of insect scalariform junctions showing that although the intercellular space possesses columns which are stained in negative contrast by lanthanum, it is not yet clear whether or not they insert into the membrane via particles. The intramembranous PF particles appear to be more numerous than the columns and are of variable size so that some could be insertion sites for columns while others could be involved in the ion transport which is known to take place across these junctional membranes. The evidence that the columns are hollow seems equivocal, so a central pore has not been included in this diagram

Moreover, the lack of order observed in scalariform junctions makes it difficult to discern any correlation in pattern between the distribution of IMPS and the intercellular columns. The columns may represent strands of glycoprotein arising from the membrane surface, which merely span the intercellular space without any direct intramembranous insertion. If this is so, scalariform junctions would be more closely allied to desmosomes than to septate junctions, since the former also possess cross-fibrillae, often, in insects, with no obvious freeze-fracture indication of an intramembranous insertion (Satir and Fong, 1973; Lane and Swales, 1978a, b, 1979a). In this respect, they would both be cell “contacts” rather than cell junctions (Friend and Gilula, 1972b). Further, the majority of the numerous intramembranous particles in the scalariform junctions are more likely to have another function, that of ion transport, given that they are present in fluid transporting epithelia which are rich in ATPase (Berridge and Gupta, 1968). However one cannot ignore the possibility that a percentage of the IMPS may

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represent insertion sites of the columns and this we have incorporated into the model, although it can only be speculative in view of the limited data currently available. 7.6

D I S T R I B U T I O NI N INSECT T I S S U E S

7.6.1 Occurrence in transporting epithelial cells These junctions occur between lateral borders in a variety of insect secretory tissues but have not always been recognized as a junctional modification. However, it is now possible in the light of our present knowledge to reexamine published micrographs of many such tissues and find regions of the lateral intercellular channels which are very similar to those that are now called scalariform junctions (see references in Noirot-Timothke et al., 1979). Scalariform junctions are sometimes designated by a variety of other terms, including the mitochondrial-scalariform junction complex or MS complexes (Noirot and Noirot-Timothee, 1976; Noirot-Timothee et a1 ., 1979) and the plasmalemma mitochondria1 complex (Oschman and Wall, 1969). As these names imply, scalariform junctions are nearly always intimately associated with mitochondria which appear to act as an energy supply for the ATP-dependent activities which occur in these regions. The junctional clefts appear to be inextensible, and are considered to be involved in the flow of water and solutes from the lumen of tubular organs out through to the haemolymph. It is, therefore, not surprising to find that they appear to occur in transporting epithelia involved in ion and solute reabsorption such as, for example, the proctodeum (Noirot and Noirot-Timothee, 1971a; Strambi and Zylberberg, 1972), the rectal pads in cockroaches (Oschman and Wall, 1969; Wall and Oschman, 1973, 1975; Noirot and NoirotTimothee, 1976; Noirot-TimothCe et a/., 1979; Lane 1978a, 1979c), termites (Noirot and Noirot-Timothie, 1977), honey bees (Kiimmel and Zerbst-Boroffka, 1974) and tettigonids (Peacock and Anstee, 1977b) and the salivary glands of flies (Oschman and Berridge, 1970). In the rectal papillae of dipteran flies “stacks” of lateral border membrane occur in large numbers (Fig. 64) (Noirot and Noirot-Timothee, 1960, 1966; Gupta and Berridge, 1966; Berridge and Gupta, 1967; Hopkins, 1967; NoirotTimothee and Noirot, 1967; Wessing and Eichelberg, 1973; Lane 1979c) and it has been suggested (Lane, 1979c; Noirot-Timotheeet al., 1979) that the membrane clefts within the stacks are analogous to the scalariform junctions in that the thin-sectioned and freeze-fractured images of the two are very similar (compare PF and EF in Fig. 66, Type I, with those of the stacks, Type 11, S in Fig. 67). Scalariform junctions are only rarely associated

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with Malpighian tubules and nephridial tissues (Fain-Maurel and Cassier, 1972; Wall et aZ., 1975; Noirot-Timothte et aZ., 1979) but are present between the epithelial cells of the seminal vesicle of the locust, which exhibits a glandular activity, commencing at the time of the imaginal molt (Cantacuz;re, 1972). There have been brief reports of scalariform-like junctions in the insect nervous system, referred to as (septate) desmosomes or continuous junctions; these were found between glial cells or perineurial cells in the grasshopper (Lane, 1968), in the cockroach and the stick insect (Lane and Treherne, 1970; Lane, 1974). In some cases, they exhibited cross filaments in the intercellular space but always were characterized (Lane, 1974) by a uniform cleft of about 20 nm which was relatively short but frequently associated with mitochondra on either side (see Figure 5 in Lane, 1968). Recent experiments on the glial cells of developing CNS in the pupal moth Manduca (Lane and Swales, 1979b) and on the glial cells around the neurones of Limulus CNS (Harrison and Lane, 1980) reveal comparable areas with a regular intercellular cleft, in lanthanum-treated preparations. Intercellular cross-striations reminiscent of columns cut lengthwise occur; in oblique en face sections, non-opaque spots exist as could be characteristic of transverse sections through columns. These are always in close association with mitochondria and with gap junctions; also, like scalariform junctions proper, in freeze-fracture replicas they produce n o distinguishable freezefracture image other than intramembranous particles, that, in the perineurium, a tissue involved in ion transport, are present in very large numbers (Skaer and Lane, 1974). 7.6.2 Absence in non-arthropod systems When published micrographs of certain crustacean tissues are re-examined, Noirot-Timothte and Noirot (1979) state that these also possess scalariform junctions; such tissues include ion-transporting gill epithelia and salt secreting or glandular cells which tends to corroborate the suggestion that the junctions are implicated in transport phenomena. N o scalariform junctions as such have been described in vertebrate tissues. A somewhat similar structure has been reported between a procaryotic cell and a eucaryotic one derived from vertebrate tissue (Wagner and Barnett, 1974); here filamentous cross-striations form around the procaryotic bacterium where it nestles into the eucaryotic cell surface. This could, of course, also represent a desmosomal structure (see Section 3.4). Although a distinctive, sometimes cross-striated, cell contact has been observed in rat adrenal cortex (Friend and Gilula, 1972a) that bears some resemblance to the scalariform junctions, it is not a rigorously comparable structure. Other

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reports of similar junctions in the vertebrate literature refer to “septatelike” structures, so these have been considered under septate junctions (see Section 2.6.). 7.6.3

Autocellular, homocellular and heterocellular junctions

In most cases scalariform junctions appear to be homocellular (NoirotTimothCe et al., 1979) but in the rectal pads of some cockroaches they may also be heterocellular when they occur between principle and basal cells (Noirot and Noirot-TimothCe, 1976). Autocellular scalariform contacts undoubtedly occur too, especially in rectal tissues, but they are often difficult to discern from homocellular junctions because of the complexity of the lateral infoldings. It has been stressed (Noirot-TimothCe and Noirot, 1979) that scalariform junctions are autocellular when they occur along blind invaginations of the lateral face of the cell membrane; here only one end is open, and it faces the system of intercellular sinuses. It is, however, often difficult to follow these well enough to be absolutely convinced of such a distribution of open ends linking up with blind invaginations. 7.6.4 Co-existence with other junctions In many situations, for example rectal tissues (Noirot and Noirot-TimothCe, 1976; Lane, 1979c), the scalariform junctions co-exist along the same lateral border with gap junctions (Figs 64,65 and 66) as well as with septate junctions and desniosomes at luminal and basal surfaces (Noirot and Noirot-TimothCe, 1976; Lane, 1978c, 1 9 7 9 ~ ) In . glandular epithelia of seminal vesicles, septate and scalariform junctions also co-exist along with desmosomes on the lateral intercellular cleft (Cantacuzene, 1972). In rectal pads, they co-exist with tight junctions near the basal surface (Fig. 6 5 ) (Lane, 1978a, c, 1979a, c) where an occlusion to the backflow of fluid from the haemolymph is required (Noirot and Noirot-TimothCe, 1976; Lane, 1979a, c). The presence of such a number of different junctions between lateral cell borders suggests that each must have a specialized function since it is otherwise difficult to conceive why so many surface modifications should have evolved. 7.7

PHYSIOLOGICAL SIGNIFICANCE

The channels in which the scalariform junctions are found exhibit a rigid and uniform intercellular spacing, presumably maintained by the intercellular columns; this would appear to be important in reducing flow resistance within these regions and in preventing occlusion of the intercellular space

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(Fain-Maurel and Cassier, 1972). The transporting epithelia in which these junctions occur are involved in the reabsorption, against a concentration gradient, of water and ions which necessitates the pumping of ions across the scalariform junctional membrane into the channels. This creates an osmotic gradient to pull water across and so it is important that fluid flow through these areas should be unimpeded (Oschman and Wall, 1969, 1975; Berridge, 1970; Berridge and Oschman, 1972; Wall and Oschman, 1973,1975; Oschman et af., 1974; Noirot and Noirot-Timothte, 1976; Wall, 1977; Maddrell, 1978; Gupta and Hall, 1979). The scalariform junctions themselves are inextensible (Oschman and Wall, 1969; Noirot and NoirotTimothie, 1976), but there often are lacunae or sinuses at intervals between them which may become dilated with excess solute flow throughout the system; this is particularly true in the case of the stacks in rectal papillae which represent type I1 scalariform junctions. Although the inter-stack areas may become distended, the stacks remain organized in regular arrays even under conditions of massive fluid flow (Berridge and Gupta, 1967). The inextensible junctions may function in creating the high solute concentration within the junctional clefts by restricting water entry into these regions; this ensures a high osmolarity of the solution emergent from the junctions into the sinuses. Water would then flow into these sinuses due to the osmotic gradient thus created. The scalariform junctions are closely associated with mitochondria, presumably for the supply of ATP required in the active pumping across the membranes, and their membranes have been shown to possess ATPase (Berridge and Gupta, 1968); this enzyme activity would make available the energy necessary for transport stored in the ATP, as required. Moreover, electron microprobe studies now directly support the theory of ion pumping over the scalariform junctions as Na+ and K+ are present in higher concentrations in the scalariform junctional stacks of the rectal papillae than elsewhere in the cells (Gupta et al., 1977). The rectal papillae, by virtue of having “stacks”, possess more extensive arrays of type I1 scalariform junctions than rectal pads of simple rectal stratified epithelium. These differences in complexity seem related to habitat rather than the position they occupy on the evolutionary ladder (Noirot and Noirot-Timothte, 1977; Noirot-TimothCe and Noirot, 1979); the scalariform junctions of rectal pads are more highly developed where reabsorption of water against a concentration gradient is required in a dry environment. For example, in certain families of termites, the rectal pads are reduced and these are found to be living in a relatively moist environment, while more primitive termites living in dry conditions, exhibit maximal complexity (Noirot and Noirot-TimothCe, 1977). Hence, although some may act as insertion sites for the intercellular columns, most of the intramembranous particles (IMPS) found in the

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scalariform junctions, are doubtless involved in the ion pumping which takes place in these areas as described above. Increased numbers of IMPS have been implicated in ion transport or as sites of active pumping across membranes in a variety of arthropod tissues (for example Skaer et al., 1975; Lane, Skaer and Swales, 1977a; Lane, 1979c; Noirot-TimothCe and Noirot 1979). The role of the scalariform junctions in adhesion or as a permeability barrier seems unlikely, in the first instance, because many other adhering junctions are present, such as septate junctions and desmosomes, and in the second, because columns are unlikely to offer significant resistance to paracellular flow of solutes. 7.8

JUNCTIONAL DEVELOPMENT

No studies have been made on the development of scalariform junctions hitherto. Such analyses could not be carried out using freeze-fracture, for the particulate structures implicated do not form a structure that is sufficiently discrete so as to be recognizable, unlike the regular linear or macular arrays observed elsewhere in other junctions. The stages in the formation of non-discrete IMP arrays could therefore not be easily followed by trying to observe changes in intramembranous particle distribution with time. Since the distribution of the intercellular columns is very variable, a thin-section study of developing tissues would also be difficult to interpret.

8 Reticular septate junctions

8.1

RECTAL PAPILLAE OF DIPTERAN INSECTS

8.1.1 Junctional location and structure The cells of the insect rectum are often highly modified in order to be effective in the reabsorption of vital ions and water which are conserved by these tissues to maintain homeostasis (see Section 7.7). The structural modifications to the transporting epithelia observed in the rectal papillae of the Dipferu are more elaborate than those found in the rectal pads of cockroaches; they involve the formation of separate papillae which project into the rectal lumen. These consist of cortical cells, covered by the rectal cuticle and lying above the cone-shaped medullary region penetrated by tracheae. The lateral cell borders of the cortical cells in these tissues are so highly infolded that they form stacks (S in Fig. 64) (Gupta and Berridge, 1966; Berridge and Gupta, 1967; Hopkins, 1967; Wessing and Eichelberg, 1973; Bode, 1977; Lane, 1 9 7 9 ~ )These . stacked areas consist of scalariform

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junctions in the form of attenuated cytoplasmic folds with 15-20 nm intercellular clefts between them (S in Fig. 64) (see Section 7.2). Thus there is clearly a vast increment in the surface area of lateral membrane between adjacent cells that is available for ion transport. Between these stacks there is an intercellular cleft which links the various stacked regions; these interstack areas are the sites of the so-called reticular septate junctions. 8.1.2 Thin-section and lanthanum stained appearance In these rectal papillae, the scalariform junctions, as already indicated (Section 7.2), are represented by the multiple piles of stacked membrane arrays (Fig. 64) lying along the length of the lateral cell borders. The membranous stacks are associated with one another by lateral intercellular clefts and lacunae (L in Fig. 64) which exhibit some gap junctions (GJ in Fig. 64) and the reticular septate junctions. These display little fine-structural detail save for an occasional indistinct striation (RSJ in Fig. 64). After lanthanum-impregnation these striations appear to be columnar (insert in Fig. 70), being non-opaque fibrils against an electron-dense background (Lane, 1 9 7 9 ~ )The . width of the extracellular channels o r lacunae that form the reticular septate junctions and link fhe stacks is variable in comparison with the more consistently equidistant apposing membranes of the scalariform junctions (as in Fig. 64). 8.1.3

Freeze-fiacture appearance

These reticular septate junctions that occur in the variable inter-stack lacunae are characterized by a very striking freeze-fracture appearance (Figs 67,68 and 70) (Lane, 1979c, d; Flower, personal communication). This is in contrast to the scalariform junctions which form the stacks (S in Fig. 67) where, as in the rectal pad scalariform junctions (Fig. 66), the P-face is enriched with numerous intramembranous particles arranged, for the most part, with no apparent order. The inter-stack membranes exhibit irregular arrays of free or interwoven PF ridges (Fig. 67), composed of fused 8 to 1 0 nm particles (Fig. 68), and EF grooves (Fig. 70) which are sometimes, although not always, complementary (Fig. 68). The ridges fracture onto the PF whether fixed before cryoprotection or not (Lane 1 9 7 9 ~ )The . intercellular space is variable but frequently is between 15 to 20 nm as in septate junctions (Figs 64,67 and insert to Fig. 70). The reticular or semi-anastomosing appearance of these ridges and grooves has led to their being termed reticular septate junctions (Lane, 1979c, d) and they appear to pervade all of the inter-stack membrane areas that lie around each stacked membrane array (Fig, 67).

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175

Co-existence and occurrence in other organisms

These reticular septate junctions can be seen to co-exist with gap junctions along the lateral intercellular border (Figs 64, 70), with the scalariform junctions of the stacks (Fig. 67) and also with septate junctions (Fig. 70) which lie at either end of the lateral clefts at both luminal and basal surfaces. Little is yet known about these reticular septate junctions except that they occur in the rectal papillae of the blow-fly Calliphora (Lane, 1979c, d) and the house fly Musca (Flower, personal communication; Skaer, unpublished observations). They appear in these situations to be auto- or homocellular since they always occur along the lateral borders of the homologous cortical cells that comprise the rectal papillae. No model can yet be constructed relating to their fine structure. This is partly because in some cases their reticular P F ridge arrays are not complementary to the grooves found in the E face of the adjacent cell membrane (Lane, 1979c) and hence it is not clear that they are junctional in the true sense throughout the whole of this inter-stack region. There is also the problem of the variable width of the intercellular cleft which is not typical of a junctional structure. If intercellular columns only exist over part of the inter-stack area, then these could not insert into the membrane via the ridges, because the ridges seem to lie throughall of the inter-stack region. However, a number of small particles are present on the PF of the inter-stack membrane lying in random arrays between the ridges (Fig. 68); some of these could perhaps act as anchoring sites for the occasional intercellular column (Lane, 1979~). 8.1.5

Comparison with other junctions

Reticular septate junctions bear a certain similarity to the continuous or smooth septate junction whose particles are also often fused into ridges (see Section 2.3.3). But in contrast, in the former the ridges are often discontinuous, less well organized, not always complementary and d o not shift from one fracturing face to another depending on whether or not they are fixed. Morover, the extracellular space in smooth septate junctions is usually in the form of an equidistant 15 to 20 nm channel while that of these reticular septate junctions is variable. There can be little doubt that the reticular septate junctions are not a Fig. 70 Freeze-cleaved replica through the reticular septate junctions of Calliphoru rectal papillae where they abut onto the peripheral septate junctions ( S J ) . The E face (EF) shows the numerous linear grooves that occur, with intercalated clusters of gap junctional particles (GJ). The P face (PF) displays the characteristic interweaving reticular septate ridges. The insert shows lanthanum staining the extracellular space in the reticular septate area, indicating the presence of the occasional intercellular column (at arrows). x 40 000; insert x 149 500

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variation on a theme of the tight junction since the intercellular cleft is distinctly not reduced o r occluded. However, it seems that they could be considered a new “species” of the generic septate junctional type in that there is ( i ) a substantial and over some length, equidistant, intercellular channel; there are ( i i ) cross-striations, albeit occasional, traversing this channel, and (iii)rowsof aligned intramembranous particlesand pitsabound. A rather comparable structure has also recently been revealed in the optic cartridges of dipteran flies (Carlson and Chi, 1979; Lane, 1979d) where PF particles are fused into a network of often discontinuous ridges. This is considered later (see Section 8.2.1 -3). No developmental studies have yet been made on the reticular septate junctions since they have only so very recently been discovered. 8.1.6 Possible function Given the presence of the reticular septate junctions in rectal papillae, which possess stacked scalariform junctions, and their absence in rectal pads, which lack stacks, it seems reasonable to suppose that the reticular septates have a role in the maintenance of the stacks as integral structures. This could take the form of impeding any random translateral movement of the IMPs involved in ion transport, which are concentrated in the stack membranes where the main pumping activity occurs. It is, however, tempting to speculate that the reticular septate junctional ridges allow for the extensive dilatation that can occur in these inter-stack areas (Berridge and Gupta, 1967) when there is a considerable fluid flow. Perhaps they also act to regulate the speed and extent of the membrane distension; with increased hydrostatic pressure the ridges could produce tissue resistivity to prevent the channels opening too rapidly (Lane, 1 9 7 9 ~ )This . kind of regulation would ensure an adequate hydrostatic pressure as required to support solute flow. By spreading the pressure applied to these membranes by solute flow via the ridge system, an even pushing apart of the membranes to increase the extracellular space would be ensured; this would also allow for a more even flow of solutes (Lane, 1 9 7 9 ~ )Although . these remarks are highly speculative, it seems clear that some such special function must be attributed to the reticular septate junctions. since they co-exist with septate, gap and scalariform junctions, as well as desmosomes. Each junction is apt to have its own specific role since it is unlikely that two or more junctions with the same function should be present along the same lateral border. An explanation for the IMPs that lie between the PF ridges in the interstack regions of the reticular septate junctions (Figs 67 and 68) may be related to the finding that ATPase activity is present there as well as in the stacks (Berridge and Gupta, 1968). These IMPs may represent sites of ion

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pumping, in addition to those in the stacks. The possibility also exists that some of these may serve as insertion sites for the intercellular columns observed occasionally in thin-sections (insert in Fig. 70). 8.2

PERIPHERAL RETINA OF DIPTERAN INSECTS

8.2.1 Junctional location, structure and type In dipteran eyes, the retina possesses photoreceptor cells organized into retinulae; these have a complex morphology, displaying microvilli on their rhabdomeric facet and synaptic terminations at their proximal extremity and are associated with pigment and Semper cells (Carlson and Chi, 1979). The peripheral retina is an epithelial layer with regions of specialized contact between retinula cells; these regions in the housefly Musca sometimes show septate-like characteristics with an intercellular cleft of 1 2 nm (Carlson and Chi, 1979) together with tight junction-like features (PF ridges and EF grooves) so that it has been suggested that they may possess sealing properties (Carlson and Chi, 1979). A more detailed description of these particular structures (Chi et al., 1979) indicates that they may be a kind of smooth septate (continuous) junction and some pleated septate junctions also seem to be present. Thin-sections reveal that the extracellular cleft in the retinular cell junctions varies from 100-200 nm in width down to 10-12 nm (Chi et al., 1979), a discontinuity which is similar to that observed in the reticular spetate junctions of rectal papillae. Moreover, indistinct septa are sometimes observed in the regions where the intercellular space is 1 2 nm (Chi et al., 1979); these striations are not unlike those in the scalariform junctions or those found in the regular cleft interspaces in reticular septate junctions (Lane, 1 9 7 9 ~ )Often, . however, the grooves on the E face of the retinular cell junctions do not correspond to the particle ridges on the PF face (Chi et al., 1979; Lane, 1979d); in contrast with this, a brief abstract has appeared (Schinz, 1978) on the photoreceptor cells of Drosophila which possess PF ridges that have corresponding furrows which lie in a loose and irregular pattern. Since a gap of 1 5 nm is found between the neighbouring photoreceptor membranes, these cell associations in Drosophila have been termed continuous (smooth septate) junctions (Schinz, 1978). These may in fact be the same structures referred to as septate junctions (Carlson and Chi, 1979) or continuous junctions (Chi et al., 1979) in Musca. Comparable structures have recently been observed between retinular cells in the eyes of Calliphora as well as Musca (Lane, 1979d) (Figs 71-73). The features observed by Carlson and Chi (1979), Chi et al. (1979) and Schinz, (1978), as well as our findings in the photoreceptor cells of Musca and Calliphora, suggest that a reticular septate junction may exist in insect eye between the adjacent retinular cells.

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8.2.2 Freeze-fracture appearance In the fly Musca riornestica, or in Calliphora, striking arrays of P F ridges and EF grooves that are sometimes complementary have been found around the lateral edges of the retinular cells of the optic cartidges (Fig. 71; Lane, 1979d). These nearly always are to be found, very like the reticular septate junctions of the rectum, as network-like arrays which have not fused into a complete continuum. These tend to be aligned so that the ridges of fused particles lie parallel to the outer edge of the rhabdomere (as in Fig. 72). This is not always the case for in a few rare instances, the ridges are aligned at right angles to the outer edge of the retina (Fig. 71). These ridges are composed of 8-10 nm particles, fused into variable lengths of ridges (Fig. 72, insert) which may lie at odd angles to one another although with a definite tendency to lie parallel to each other and with the periphery. In some cases, t h e complementarity of ridges and grooves is very striking (Fig. 73, insert) while in others there seems to be no such clear-cut association (Fig. 73). The intercellular clefts appear to be considerable (Fig. 73) so that by this criterion the junction is more septate-like than tight-like. These junctions are presumably in the membranes of the retinular axon bundles, but may be at the glia-axonal interface, for marginal glial cells enclose the cartidge bases and escort axons leaving the lamina, making contact with many other glial cells (Carlson and Chi, 1979). Since Shaw (1977,1978) showed a retina-lamina barrier in locust eye, these membranle specializations could be important in the maintenance of a blood-eye barrier or in maintaining localized concentration gradients by restricting horizontal diffusion currents (Chi et al., 1979). However, the arrangement of these ridges is very different from that of the tight junctional ridges of the locust eye (Skaer, 1979) or that of the worker honey bee (Nickel and Scheck, 1978). Moreover, there are differences in the organization of the locust eye and the eye of the fly, and little evidence is forthcoming about the dipteran blood-retina barrier except for a report by Campos-Ortega (1974) who Figs 71 -73 All these micrographs are freeze-fracture replicas from the compound eye of dipteran flies such as Musca and Caliiphora Fig. 7 1 Rhabdomere region of the insect eye showing the typical microvilli-like structures (MV) and the P face ridges (PF) that lie in these photoreceptor cell membranes. In this preparation, the orientation of the ridges is atypical in lying parallel to the microvilli, since they usually lie at right angles to them (as in Figs 72 and 73). x 43 400 Fig. 72 Another area of retinular or rhabdomere cells from dipteran eye to show the way the PF ridges are interwoven. The insert indicates that they are formed by fused 8-10 nm particles. X 38 100; insert x 87 000 Fig. 73 Dipteran eye junctions with P face (PF) ridges and E face (EF) grooves. (*) indicates the intercellularcleft. Some, but not all, of these ridges and grooves are complementary. Insert shows two that are complementary (arrows). x 58 300; insert, x 70 800

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commented that the optic cartidges of Musca lie in almost “perfect isolation’’ by means of three different kinds of glial cells. 8.2.3 Possible function One possible role of the reticular septate junctions has already been considered, involving the regulation of the distention of the inter-stack cleft in rectal papillae during solute flow in this transporting epithelium. There is no such clear-cut transporting function for these junctions in the photoreceptor cells in the dipteran eye. However, it may be that the retinulae must be kept completely separated by a flexible and non-collapsible system which these junctions could provide. The advent of volume or positional changes in the glia and pigment cells which could lead to distention of the kind just described, is one possibility. Again, septate junctions are already found elsewhere in the insect eye, (Eley and Shelton, 1976; Nickel and Scheck, 1978; Skaer, 1979a) so the role of the reticular septate junctions is unlikely to be purely to maintzin physical integrity, although it has been suggested in Musca that they could have the cohesive qualities necessary to maintain the twist that the retinula displays along its longitudinal axis (Chietal., 1979). A role in the maintenance of local concentration gradients in the intercellular spaces between retinular cells has also been put forward (Chi et al., 1979). However, further investigations are required to clarify the precise physiological significance of these junctions.

9 Concluding remarks 9.1

DEVELOPMENT OF JUNCTIONS

Studies that have been made on developing junctions show that pleated septate, smooth septate and tight junctions, being linear structures, tend to differentiate in a stepwise assembly process, while gap junctions being macular, form by “streaming” of particles towards a focal point. Intercellular junctional development, as seen in thin-sections, tends to be concurrent with the ordered IMP appearance in freeze-cleaved replicas so it seems likely that both of the apposing cell membranes, associated with the forming junctions, develop simultaneously. Where the IMPS of the junction have some association with intercellular specializations, the formation of the junctional areas may involve the translateral movement of “IMPcomplexes” (the IMP with its associated extracellular components through the membrane; this phenomenon could be likened to the movement of icebergs through a sea of lipid (Satir and Fong, 1973)). This presupposes that the membrane is “fluid” (Erye and Edidiu, 1970; Pinto da Silva, 1972)

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as expressed in the fluid-mosaic model of membrane structure (Singer and Nicolson, 1972). Different kinds of junctions clearly have different component particles and “IMP-complexes”; certainly their size and fracturing features differ markedly. Yet more than one junction can exist on the same membrane surface. Hence, different kinds of IMP must move about during development without interfering with each other, establishing their final specific association, through, it must be supposed, some sort of recognition factor that stimulates them to aggregate together. Moreover in some cases, for example the septate junction, the IMPs are kept separated in the mature junction at a distance apart of about 20-30nm; some regulating device must be responsible for this characteristic spacing and it may have to do with the intercellular structure with which the IMPs may be associated. In the case of gap junctions, it is possible to imagine that some spectrin-like molecule could hold them in position together, but this molecule would then have to be broken down during the early pupal stages in insects, possibly stimulated by the hormones that trigger off pupation. Given our increasing understanding of the coupled and uncoupled state of gap junctions, it seems likely that while they are in the process of formation or when undergoing disaggregation, the conformational arrangement of the component gap junctional particles is such that the channels are effectively closed. Revel and his colleagues ( 1978) have postulated configurational changes in large precursor IMPs that give rise to mature, smaller, gap junctional particles in vertebrate tissue, but there is no evidence that precursor IMPs exist or that such a process could occur in insects. In the arthropods, there is the additional problem that the macular gap junctional plaques disperse into separate particles during metamorphosis rather than being disposed of by internalization and this cannot easily be explained as the reverse of Revel’s suggestion. Further studies are clearly required before the basis of these and related phenomena can be understood. 9.2

FUNCTIONAL CONSIDERATIONS

There can be no doubt that all junctional complexes have an effect upon the structural integrity of the system in that adjacent cells, being associated by some membranous modification, remain in contact with each other as long as that association persists. The question is whether or not there is some other function performed by any given junction in addition to that of holding cells together. There seems little question about the role of the desmosomes and hemi-desmosomes which seems to be predominantly that of maintaining spatial association. Other functional roles that junctions appear to perform in tissues are intercellular communication, the enhancement of intercellular flow, the

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restriction of intercellular movements and the sealing of the intercellular cleft. Although initially septate junctions were suggested as the structure underlying cell-to-cell communication, the weight of evidence now favours gap junctions for this role. Similarly, septate junctions were suggested as the structures underlying the maintenance of an open intercellular space to ensure rapid fluid flow but this role appears to be fulfilled by scalariform or scalariform-like junctions, at least in rectal tissues. The restriction, selectively, of solute flow through the intercellular cleft seems to represent a plausible role for the septate junctions, the basis of selectivity being the chemical nature of the intercellular and interseptal matrix rather than the barrier effect of the septa themselves. Our contention is that the sealing of the intercellular cleft is achieved by tight junctions, albeit simpler structures than the complex, anastomosing systems in vertebrates. This is not an uncontested view, since many authors favour septate junctions in this role but leave unexplained both the absence of such junctions in a tissue shown on electrophysiological grounds to possess a barrier to the diffusion of ions (the perineurium of Manduca sextu) and the presence of such junctions in epithelia that have been shown to be permeable to molecules of relatively high molecular weight (the transepithelial passage of inulin in Rhodnius Malpighian tubules). 9.3

CORRELATION O F INSECT PHYSIOLOGY WITH J U N C T I O N A L S T R U C T U R E

The insects display striking differences in their intercellular junctions compared with other groups and these are highlighted when compared with the other intensively studied group, the vertebrates. Insects have unusual gap junctions with larger freeze-fracture particles which fracture onto the EF rather than the PF [hence Flower's (1972) original term "inverted gap junction"], they are characterized by septate junctions of two distinct kinds, the pleated and smooth, they are unique in exhibiting scalariform and reticular septate junctions, and they alone of the invertebrate phyla (apart from the hemichordates) exhibit tight junction-like structures. The significance of many of these characteristic features is not yet fully understood but the key to our understanding of them must come from an appreciation of the distinctive physiology of the Znsecta. To take one instance, Maddrell(l978, 1979a, b) has considered the systems involved in absorption, secretion and excretion in the insect. Insects, like vertebrates, exhibit highly efficient mechanisms for homeostatic control but unlike them appear to be remarkably tolerant of changes in the fluid and chemical composition of their body fluids (Maddrell, 1979b). Research to date shows that in insects, unlike the vertebrates, the gut appears not to be selective in its uptake. Surprisingly, almost all the solutes

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appearing in the gut lumen readily pass through into the haemolymph (Treherne, 1967). Thus the composition of the haemolymph is subject to rapid and quite substantial changes both in volume (as for example in plant bugs or blood-suckers) and chemical composition (especially in phytophagous species). This alteration in the haemolymph would be deleterious in certain systems where constancy of the extracellular fluids is crucial to the normal functioning of the component cells (as for example in the nervous system and certain sensory extensions of the nervous system) or where a specialized and highly controlled extracellular medium is essential (as for example in the maturation of the gametes). The hypothesis put forward in this review article is that these systems are sealed off from the haemolymph by tight junctions analogous to, although of a simpler structure than, those described in vertebrate epithelia. Accordingly, simple tight junctions are found in the ensheathing structures of the central nervous system, in the eye and in the follicular layers of the testis. Thus the insect may seal off systems vulnerable to the fluctuations of haemolymph composition but homoestatic adapations are nonetheless present in the form of the excretory systems, the Malpighian tubules and rectal complexes. As in the gut, the Malpighian tubules are permeable to the lower molecular weight components of the haemolymph (blood cells and proteins are held back). Virtually all the solutes contained in the haemolymph, both beneficial and toxic, are able to pass into the primary excretory fluid in the lumen of the Malpighian tubules (Ramsay, 1958), albeit at a very slow rate in some species (for the significance of this see Maddrell (1979b)) and this despite the presence of smooth and/or pleated septate junctions. However as this fluid passes down the Malpighian tubules and the rectal complexes, reabsorption takes place, but only of those constituents for which there is an active mechanism of uptake. In this way any novel, potentially toxic compounds are expelled by purely passive mechanisms, the insect need in no way be “programmed” to deal with such molecules. The adaptive advantages of such a system are evident; in this way insects can make maximum use of the potentials of the environment since no specific uptake mechanism for individual molecules is necessary and yet they can protect themselves from the possible harmful effects of novel toxic compounds by a passive system for their excretion.

Acknowledgements

We are extremely grateful to Mr J. Barrie Harrison and Mr William M. Lee for their untiring assistance in producing the final photographic plates; we are also deeply indebted to Mr John Rodford who prepared the final

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diagrams and models used in this chapter. Our thanks are also extended to our colleagues in the A R C Unit of Invertebrate Chemistry and Physiology for their helpful concern during the preparation of this review but we are especially grateful to D r Simon H. P. Maddrell for many stimulating discussions on the physiological role of junctions in insect tissues. We also thank Mrs Margaret Clements and Mrs Vanessa Rule for their patience and care in preparing the final typescript. We would also like to acknowledge our collaborators with whom some of the micrographs used in this review were produced; the work on developing Manduca and Calliphora tissues was carried out in collaboration with Lesley S. Swales and thestudieson Limulus gut with J . Barrie Harrison(N.J.L.); that on Musca mid-gut and Malpighian tubule with William M. Lee and J. Barrie Harrison and on locust testis with Dr Therasa Jones (H.1eB.S.). The literature survey for this review was completed in July 1979. References Abbott, N. J. (1970). Absence of blood-brain barrier in a crustacean, Carcinus maenas L. Nature, Lond. 225, 291 -293 Abbott, N. J. (1972). Access of ferritin to the interstitial space of Carcinus brain from intracerebral blood vessels. Tissue & Cell 4, 99-104 Abbott, N. J. and Treherne, J. E. (1977). Homeostasis of the brain microenvironment: a comparative account. In “Transport of Ions and Water in Animals” (Eds B. L. Gupta, R. B. Moreton, J. L. Oschman and Wall B. J.) Ch. 19, pp. 481-510 Academic Press, London Abbott, N. J., Pichon, Y. and Lane, N. J. (1977). Primitive forms of potassium homeostasis: observations on crustacean central nervous system with implications for vertebrate brain. Exp. Eye. Res. Suppl. 25, 259-271 Albertini, D. F. and Anderson, E. (1 974a). The appearance and structure of intercellular connections during the ontogeny of the rabbit ovarian follicle with particular reference to gap junctions. J . Cell Biol. 63, 234-250 Albertini, D. F. and Anderson, E. (1974b). Structural modifications of lutein cell gap junctions during pregnancy in the rat and the mouse. Anat. Rec. 181, 171-194 Albertini, D. F., Fawcett, D. W.and Olds, P. J. (1975). Morphological variations in gap junctions of ovarian granulosa cells. Tissue & Cell 7 , 389-405 Altorfer, J. and Hedingel-, C. (1 975). Septate-like junctions between spermatogonia in human seminiferous epithelium. Experientia 31, 105-107 Amsterdam, A., Bratosin, S. and Lindner, H. R. (1976). Gap junctions between decidual cells in the rat uterus. In “Electron Microscopy 1976”. Proc. 6th European Congr. on Electron Microscopy Vol. 2. Biological Sciences (Ed. Y. BenShaul) pp. 372-373. Tal International, Jerusalem Anderson, E. (1977). Junctional complexes in the developingovarian follicle and the pre-implar.tation mammalian embryo with particular reference to gap junctions. Res. In Reprod. 9 , 2-3 Anderson, M. E. and Smith, D. S. (1 971). Electrophysiological and structural studies on the heart muscle of the lobster, Homarus americanus. Tissue & Cell 3,191 -205 Andries, J.-C. (1 972). Genese intraepithkliale des microvillosi tCs de 1’epithClium

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Congr. on Electron Miscroscopy” (Ed, J. M. Sturgess) Vol. 11, pp. 332. Imperial Press, Toronto, Canada Suzuki, K., Sangworasil, M. and Higashino, S. (1978). On correlation between gland stiffness and cell coupling in salivary gland of Chironomus plumosus Larva. Cell Struct. Funct. 3, 161--172 Szollosi, A. and Marcaillou, C. (1977). Electron microscope study of the blood-testis barrier in an insect: Locusta migratoria. J. Ult. Res. 59, 158-172 Tani, E., Ametani, T., Higashi, N. and Fujihara, E. (1971). Atypical cristae in mitochondria of human glioblastoma multiforme cells. J. Ult. Res. 36, 21 1-221 Tani, E., Itagaki, T. and Nakano, M. (1977). Tight junctions of oligodendrocytes. Cell Tiss. Res. 184, 139-142 Taylor, H. H. (1971). Water and solute transport by the Malpighian tubules of the stick insect, Carausius morosus. The normal structure of type 1cells. Z. Zellforsch. 118,333-368 ThiCry, J.-P. (1967). Mise en evidence des polysaccharides sur coupes fines en microscopie Clectronique. J. Microscopie 6, 987-101 8 Thomas, M. V. and Leslie, R. A. (1976). The physiological effects of ionic lanthanum on the insect blood-brain barrier. Experientia 32, 720-721 Tice, L. W., Carter, R. L. and Cahill, M. C. (1977). Tracer and freeze fracture observations on developing tight junctions in fetal rat thyroid. Tissue & Cell 9, 395-417 Tillack, T. W. and Marchesi, V. T. (1970). Demonstration of the outer surface of freeze etched red blood cell membranes. J. Cell Biol. 45, 649-653 Tilney, L. G. and Mooseker, M. (1971). Actin in the brush-border of epithelial cells of the chicken intestine. Proc. Nat. Acad. Sci. USA 68, 2611-261s Toshimori, K . and Yasuzumi, F. (1979). Tight junctions between ovarian follicle cells in teleost (Plecoglossus altivelis). J Ult. Res. 67, 73-78 Treherne, J. E. (1967). Gut absorption. Ann. Rev. En?. 12,43-58 Treherne, J. E. and Maddrell, S . H. P. (1967). Axonal function and ionic regulation in the central nervous system of a phytophagous insect (Carausius morosus). J. exp. Biol. 47, 235-247 Treherne, J. E. and Pichon, Y . (1972). The insect blood-brain barrier. In “Advances in Insect Physiology”. (Eds J. E. Treherne, M. J. Berridge and V. B. Wigglesworth) Vol. 9, pp. 257-313. Academic Press, London Treherne, J. E. and Schofield, P. K. (1978). A model for extracellular sodium regulation in the central nervous system of an insect (Periplaneta americana) J. exp. Biol. 77, 251-254 Treherne, J. E., Lane, N. J., Moreton, R. B. and Pichon, Y . (1970). A quantitative study of potassium movements in the central nervous system of Periplaneta americana. J. exp. Biol. 53, 109-136 Treherne, J. E., Schofield, P. K. and Lane, N. J. (1973). Experimental disruption of the blood-brain barrier system in an insect (Periplaneta americana)J. exp. Biol. 59, 711-723 Trujillo-Cen6z, 0. (1965). Some aspects of the structural organization of the arthropod eye. Cold Spring Harbour Symp. Quant. Biol. 30, 371-382 Turin, L. and Warner, A. (1977). Carbon dioxide reversibly abolishes ionic communication between cells of early amphibian embryo. Nature 270, 56-57 Ussing, H. H. (1968). T h e effect of urea on permeability and transport of frog skin. Excerpta Medica in?. Congress Series, No. 195, 138-148 Van Buren, J. M., Akert, K., Sandri, C. and Moor, H. (1977). Neuritic growth cone

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and ependymal gap junctions in the feline subfornical organ during early development. Cell Tiss. Res. 181, 27-36 Van Deurs, B. (1975). The use of a tannic acid-glutaraldehyde fixative to visualize gap and tight junctions. J. Ult. Res. 50, 185-192 Van Ruiten, Th. M. and Sprey, Th. E. (1974). The ultrastructure of the developing leg disk of Calliphora erythrocephala. Z . Zellforsch. 147, 373-400 Verkleij, A. J., Mombers, C., Lennissen-Bijrelt, J. and Ververgaert, P. H. J. Th. (1979). Lipid intramembranous particles. Nature 279, 162-163 Vernet, G., RuC, G. and Gontcharrof, M. (1979). Etude par les techniques d’imprCgnation au lanthane et de cryofracture des jonctions septCes de I’CpithClium de la paroi du corps de Lineus ruber (HCtCronemerte). J. Ult. Res. 67, 225-227 Vickerman, K. (1 969). On the surface coat and flagellar adhesion in trypanosomes J. Cell Sci. 5, 163-194 Wade, J. B. and Karnovsky, M. J. (1974). The structure of the zonula occludens. A single fibril model based on freeze-fracture. J . Cell Biol. 60, 168-180 Wagner, R. C. and Barnett, R. J. (1974). The fine structure of prokaryotic eukaryotic cell junctions. J. Ult. Res. 48, 404-413 Wakai, S. and Hirokawa, N. (1978). Development of the blood-brain barrier to horseradish peroxidase in the chick embryo. Cell Tiss. Res. 195, 195-203 Wall, B. J. (1977). Fluid transport in the cockroach rectum. In “Transport of Ions and Water in Animals” (Eds B. L. Gupta, R. B. Moreton, J. L. Oschman, and B. J. Wall) Ch. 23, pp. 599-612. Academic Press, London Wall, B. J. and Oschman, J. L. (1973). Structure and function of rectal pads in Blattella and Blaberus with respect to the mechanism of water uptake. J. Morph. 140,105-118 Wall, B. J. and Oschman, J. L. (1975). Structure and function of the rectum in insects. Forschritte der Zoologie 23, 193-222 Wall, B. J., Oschman, J. L. and Schmidt, B. A. (1975). Morphology and function of Malpighian tubules and associated structures in the cockroach, Periplaneta americana. J. Morph. 146, 265-306 Warner, A. E. and Lawrence, P. A. (1973). Electrical coupling across developmental boundaries in insect epidermis. Nature 245, 47-48 Weihe, E., Hartschuh, W., Metz, J. and Briihl, U. (1977). The use of ionic lanthanum as a diffusion tracer and as a marker of calcium binding sites. Cell Tiss. Res. 178, 285-302 Welsch, U. and Buchheim, W. (1977). Freeze fracture studies on an annelid septate junction. Cell Tiss. Res. 185, 527-534 Wessing, A. and Eichelberg, D. (1973). Electronenmikroskopische Untersuchungen zur Struktur und Funktion der Rectalpapillen von Drosophila melanogaster. Z . Zelljorsch 136, 415-432 White, R. H. and Walther, J. B. (1969). The leech photoreceptor cell: ultrastructure of clefts connecting the phaosome with extracellular space demonstrated by lanthanum deposition. Z . Zellforsch. 95, 102-108 Wiener, J., Spiro, D., and Loewenstein, W. R. (1964). Studies on an epithelial (gland) cell junction. 11. Surface structure. J. Cell Biol. 22, 587-598 Wigglesworth, V. B. (1960). The nutrition of the central nervous system in the cockroach Periplaneta americana L. J . exp. Biol. 37, 500-512 Wood, M. R., Pfenninger, K. H. and Cohen, M. J. (1977). Two types of presynaptic configurations in insect central synapses: an ultrastructural analysis. Bruin Res. 130, 25-45

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Wood, R. L. (1959). Intercellular attachment in the epithelium of Hydra as revealed by electron microscopy. J . biophys biochem. Cytol. 6 , 343-352 Wood, R. L. (1974). A closely packed array of membrane intercalated particles at the free surface of Hydra. J . Cell Biol. 62, 556-560. Wood, R. L. (1977). The cell junctions of Hydra as viewed by freeze-fracture replication. J . Ult. Res. 58, 299-315 Woodruff, R. I. (1979). Electrotonic junctions in Cecropia moth ovaries. Devel. Biol. 69, 281-295 Yancey, S. B., Easter, D. and Revel, J.-P. (1979). Cytological changes in gap junctions during liver regeneration. J . Ult. Res. 67, 229-242 Yee, A. G. (1972). G a p junctions between hepatocytes in regenerating liver. J. Cell Biol. 55, 294A Yee, A. G. and Revel, J.-P. (1975). Endothelial cell junctions. J . Cell Biol. 66, 200-204 Yee, A . G. and Revel, J.-P. (1978). Loss and reappearance of gap junctions in regenerating livers. J . Cell Biol. 78, 554-564 Zacharuk, K.Y. Ru-Siu, Yin. L. and Blue, S. (1971). Fine structure of the antenna and its sensory cone in larvae of Aedes aegypti (L.). J . Morph. 135, 273-298 Zampighi, G. and Robertson, J. D . (1973). Fine structure of the synaptic discs separated from the goldfish medulla oblongata. J. Cell Biol. 56, 92-105 Zampighi, G., Ramon, F. and Durin, W. (1978). Fine structure of the electrotonic synapses of the lateral giant axons in a crayfish (Procambarus clarkii). Tissue & Cell 10, 4 13-426 Zimmerman, P. (1967). Fluoreszenmikroskopishe Studien uber die Verteilung und Regeneration der Faserglia bei Lumbricus terrestris L. Z . Zellforsch. 81,190-220

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Acetylcholine Receptors of Insects David B. Sattelle A.R.C. Unrt of Invertebrate Chemistry and Physiofogy, Department of Zoology, Cambridge, UK

1 Introduction 215 2 Biochemical characterization of putative acetylcholine receptors 21 8 2.1 Experimental approaches 218 2.2 Binding of reversible ligands to high-speed extracts 220 2.3 ['ZSI]-a-bungarotoxinbinding to low-speed extracts 227 2.4 [3H]-quinuclidinyl benzilate binding to low-speed extracts 236 3 Autoradiographic localization of binding sites 240 3.1 Distribution 240 3.2 Pharmacology 242 4 Electrophysiological responses of neurones to cholinergic ligands 243 4.1 Experimental approaches 243 4.2 Multifibre preparations 248 4.3 Single neurones 253 4.4 Single identified neurones 260 5 Comparative pharmacology of CNS acetylcholine receptors 265 5.1 Insects 266 5.2 Invertebrates other than insects 271 5.3 Vertebrates 276 6 Genetic and developmental studies 279 6.1 Genetic approaches to receptor structure and function 279 6.2 Receptors in development 282 7 Receptor actions of toxins and insecticides 288 7.1 Receptor-active toxins 288 7.2 Cholinergic receptors as sites of insecticide action 289 8 Conclusions 293 References 295

1 Introduction

Chemical synaptic transmission is one mechanism by which many neurones communicate with each other and with muscle and gland cells. Neurotransmitter chemicals mediate this transfer of information between cells (Hall et af., 1975; Triggle and Triggle, 1976). At cholinergic synapses, in response to 215

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depolarization of the presynaptic nerve terminal, acetylcholine molecules are released into the synaptic cleft between pre- and postsynaptic cells (Katz, 1969). As a consequence of the interaction of acetylcholine molecules with specific receptor (recognition) sites on the postsynaptic cell, changes in the ionic permeability of the postsynaptic membrane are induced. These changes in ionic conductance may either excite (depolarize) or inhibit (hyperpolarize) the postsynaptic cell depending on the ionic species involved and whether or not the permeabilities are increased or decreased. The functional unit that accounts for this regulation of postsynaptic ion permeability - the acetylcholine receptor - therefore comprises at least two categories of sites ( a ) the recognition site which binds acetylcholine and related compounds and ( b ) the ionophore which is the site of ion translocation (Heidmann and Changeux, 1978). The concept of the neurotransmitter-receptor dates back to the early years of this century. Based on observations of the effects of curare on the nicotine stimulation of vertebrate skeletal muscle, Langley (1905) first postulated the existence of chemically specific recognition sites which when occupied either initiated or inhibited the response of the postsynaptic cell. Acetylcholine can interact with more than one specific recognition site as first shown by Dale (1914, 1937a, b) who demonstrated that two kinds of acetylcholine receptor could be distinguished using nicotine and muscarine, two pharmacological agents that can activate the receptors (agonists). Receptors of the target tissues of the parasympathetic nervous system are “muscarinic” whereas ganglionic receptors of the autonomic nervous system and at the neuromuscular junction are “nicotinic”. Pharmacological agents that block acetylcholine receptor-controlled responses (antagonists) can further characterize vertebrate cholinergic receptors. d-Tubocurarine is a more effective antagonist than atropine at nicotinic receptors, whereas the reverse is the case at muscarinic receptors (Koelle, 1975). In addition, it has been established that hexamethonium is a potent antagonist at ganglionic nicotinic receptors (Volle and Koelle, 1975) whereas decamethonium, an effective blocking agent at the neuromuscular junction, is only weakly antagonistic at ganglionic nicotinic receptors (Paton and Perry, 1953, Barlow, 1964). Our current understanding of the molecular pharmacology of acetylcholine receptors has been greatly enhanced by preparations that are amenable to both biochemical and electrophysiological analysis (Rang, 1975; Changeux, 1975). In particular, the use of receptor-specific ligands radiolabelled to high specific activity has enabled binding to receptor sites to be studied directly rather than simply being inferred from the pharmacological responses of tissues. Binding studies have proved to be of particular value in investigating the properties, distribution and function of central nervous

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system (CNS) acetylcholine receptors of vertebrates (Yamamura et al., 1978; Schmidt, e t a l . , 1979). There is an urgent need to combine biochemical and electrophysiological approaches to the study of cholinergic receptors of a variety of organisms, particularly in view of recent findings which indicate that the receptor classification outlined above may be inappropriate for many invertebrate acetylcholine receptors (Michelson, 1973;Michelson and Zeimal, 1973; Gerschenfeld, 1973; Ascher and Kehoe, 1976). Acetylcholine is considered to be an excitatory neurotransmitter at synapses in the insect central nervous system (CNS) and does not appear to be involved in insect neuromuscular transmission. Evidence for this has been summarized by several authors (Faeder et al., 1970; Pitman, 1971; Gerschenfeld, 1973; Callec, 1972, 1974; Pichon, 1974; Sattelle, 1977). Many of the essential components of a cholinergic system have been demonstrated in the CNS of several insect species. Acetylcholine, choline acetyltransferase (the acetylating enzyme responsible for synthesis) and acetylcholinesterase (the hydrolytic enzyme which terminates the synaptic actions of acetylcholine) are all present at high concentrations (see Colhoun, 1963; Pichon, 1974). The ability of insect central nervous tissue to synthesize acetylcholine in vitro has also been demonstrated (Tobias et al., 1946; Lewis, 1953; Smallman, 1956, 1975; Frontali, 1958; Prescott et al., 1977). Nevertheless the technical difficulties of proving that any putative neurotransmitter has a functional role at a particular central synapse are acute and the case usually rests on (a) its release following nerve stimulation and ( b ) its ability to qualitatively mimic the effects of presynaptic nerve stimulation. The release of acetylcholine from insect ganglia by presynaptic nerve stimulation has not so far been demonstrated, but pharmacological experiments have revealed a high sensitivity of certain insect central neurones to locallyapplied or bath-applied acetylcholine following the inhibition of endogenous acetylcholinesterase (Kerkut et al., 1969a, b; Shankland, et al., 1971; Callec, 1974; Sattelle et al., 1976; David, 1979). The actions of a range of cholinergic agonists (ligands which mimic the actions of acetylcholine) and antagonists (ligands which inhibit the actions of acetylcholine) provide supporting evidence for the existence of functional acetylcholine receptors at some central synapses (Pitman, 1971; Callec, 1974; Sattelle 1978). In recent years, a considerable body of new data has emerged on the properties and functions of insect acetylcholine receptors. The techniques of radiolabelled-ligand binding and single-cell electrophysiology have provided the bulk of this information. This review attempts to correlate, for the first time, these two approaches to the study of insect acetylcholine receptors. Apart from brief surveys covering particular aspects of this work (Eldefrawi et al., 1978; Jones et al., 1979; Dudai, 1979) no detailed review has appeared to date even though the first ligand-binding experiments were

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published a decade ago (Eldefrawi and O’Brien, 1970). The study of insect acetylcholine receptors is of comparative and evolutionary interest. Moreover, the insects provide experimental material ideally suited to genetic and developmental analysis of receptor function - aspects of receptor biology that cannot be tackled in higher organisms. Finally, a number of insecticidally-active, molecules are considered to act at acetylcholine receptors (Corbett 1974; Eldefrawi, 1976; O’Brien, 1978). An improved understanding of the mechanisms of action of such molecules will result from a detailed characterization of the properties of the various insect acetylcholine receptors.

2

2.1

Biochemical characterization of putative acetylcholine receptors EXPERIMENTAL APPROACHES

The recognition and binding of a neurotransmitter molecule by its receptor precedes the transduction events leading to a biological response. This initial step in neurotransmitter-receptor interactions can be studied by investigating the binding of radiolabelled ligands to membrane extracts prepared from a particular tissue. Many central nervous system and peripheral neurotransmitter receptors have now been characterized by radiolabelled-ligand binding techniques (cf. Yamamura et al., 1978). It is essential to establish however that the binding of a radiolabelled ligand t o a particular tissue or to subcellular fractions derived from that tissue is specific binding to the putative receptor rather than non-specific binding to other sites. To achieve this, the following criteria must be satisfied (cf: Birdsall and Hulme, 1976; Creese, 1979): ( a ) a component of specific binding should saturate with increasing concentrations of the radiolabelled ligand, indicating a finite number of sites; ( b )specific binding should increase linearly with increasing tissue concentrations; (c) pharmacologically effective concentrations of receptor-active ligands should displace the saturable component of binding, whereas pharmacologically effective concentrations of drugs with different receptor specificity should be ineffective; ( d ) the saturable component of binding should be localized to specific regions of tissues known from pharmacological experiments to contain the receptor. Ideally estimates of receptor occupancy as a function of ligand concentration derived from binding studies should agree with estimates obtained from the analysis of ligand-induced cellular responses. However, direct comparisions should be undertaken cautiously in view of possible departures from the expected correlation, which may result from the intervention of a complex sequence of transduction steps between the initial binding of the cholinergic ligand and the production of a measurable physiological

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response. Another criterion is sometimes listed. This is the isolation and purification of the specific binding component, followed by its reconstitution in a model membrane system which should then exhibit conductance changes with the ligand sensitivity and specificity of in vivo receptors on the cells from which the binding component originates (Triggle and Triggle, 1976; Eldefrawi, 1976). Although a degree of success has been obtained in reconstitution studies using purified vertebrate nicotinic cholinergic receptors (Michaelson and Raftery, 1974; Michaelson et al., 1976; Shamoo and Eldefrawi, 1975) and more recently using the purified subunit ionophore (Sobel e f al., 1977) this criterion remains difficult to fulfil. Three main approaches have been employed in binding studies on cholinergic receptors. First, reversibly acting agents such as cholinergic agonists can be used to determine the ability of putative receptor fractions to bind these ligands in amounts and with affinities paralleling those of the functional receptor in the intact cell (Eldefrawi et al., 1971a, b, c, 1972; Changeux et al., 1971). Reversible antagonists, in particular the potent muscarinic antagonist quinuclidinyl benzilate (Yamamura and Snyder, 1974a, b, c; Yamamuraet al., 1974a, b ; Snyderet al., 1975), have also been employed effectively to the same end. A variety of methods including equilibrium dialysis, centrifugation, filter-binding and gel-filtration have been used to measure the reversible binding of radiolabelled ligands to macromolecules (O’Brien et al., 1974; Synder and Bennett, 1976). Binding to the acetylcholine receptor is distinguished from binding to other proteins by its high affinity for specific ligands. The detection of similar concentrations of ligand-binding sites for a range of specific, reversible ligands, has been used as evidence of binding to an acetylcholine receptor (O’Brien et al., 1969; Eldefrawi et af., 1971c; Schleifer and Eldefrawi, 1974). Secondly, affinity label compounds such as 4-(N-maleimido)-5benzyltrimethylammonium iodide have been used to bind covalently to the disulphide group close to the active site of the nicotinic receptor, following reduction of this -S-S- group by dithiothreitol (Karlin et al., 1971, 1973; Karlin, 1974). Since non-specific interactions with sulphide groups might also be expected, the following criteria for a specific receptor action must be fulfilled: ( a ) specific binding of 4-(N-maleimido)-5benzyltrimethylammonium iodide should saturate at physiological concentrations; (b) the specific binding should be abolished by pretreatment with dithiobischoline which reoxidises the disulphide group; (c) specific binding should be inhibited by reversible competitive ligands. Finally,a number of highly specific antagonists, which form stable, largely irreversible, ligand-receptor complexes, have greatly facilitated the study of cholinergic receptors. These include a-bungarotoxin, a polypeptide purified from the venom of the Taiwan banded krait Bungarus rnulticinctus (Lee and

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Chang, 1966; Lee 1972), which was introduced by Changeux er al. (1970) and Miledi er a l . (1971) and is now a widely used chemical tool for the identification and characterization of nicotinic cholinergic receptors. Benzilylcholine mustard (Fewtrell and Rang, 1971, 1973) and propylbenzilylcholine mustard (Hiley et af., 1972; Burgen ef al., 1974a, b; Burgen and Hiley, 1975) have proved to be useful irreversible probes of muscarinic cholinergic receptors. Overall, neurotransmitter antagonists have proved the most useful ligands in receptor characterization studies as they normally exhibit a higher receptor affinity than agonists and hence a slower dissociation rate from their binding sites. The characteristics of binding are therefore easier to investigate. The receptor affinity of a particular ligand can be expressed in terms of its dissociation constant (KD) (see Cuatrecasas and Hollenberg, 1976). It may also be expressed as an inhibitor constant ( K i ) defined as:

Ki = 1 +- [LI KD where K , is the apparent dissociation constant determined from binding studies and [L] is the concentration of radiolabelled ligand under the assay conditions (see Levitzki et a f . , 1975). The concentration of test ligand that displaces 50% of the binding of the radiolabelled ligand (I5,,) is also commonly used when the relative affinities of several receptor-active compounds are compared. In the last ten years, several laboratories have performed radiolabelled-ligand binding studies in attempts to characterize insect acetylcholine receptors. This work is reviewed in the remainder of Section 2. 2.2

B I N D I N G O F R E V E R S I B L E L I G A N D S TO H I G H - S P E E D EXTRACTS

The first indications of binding of cholinergic ligands to a “receptor-like” component of insect extracts were reported by Eldefrawi and O’Brien (1970). These authors used equilibrium dialysis to characterize the binding of the reversible cholinergic agonist [3H]-muscarone to aqueous extracts of heads of houseflies (Musca dornestica). Muscarone was chosen as a suitable receptor probe for the following reasons: (a) it was effective at both nicotinic and muscarinic synapses; (b) it appeared to have fewer non-specific effects than antagonists such as d-tubocurarine (Eldefrawi et af.,1 9 7 1 ~ )and ; (c) it was known not to be hydrolyzed by acetylcholinesterase (O’Brien and Gilmour, 1969). The bulk of the binding was present in the supernatant fluid after centrifugation at 100 OOOxg for 60 min. For example, using a sample homogenate containing 200 mg headslml, the supernatant bound 924 pmole

ACETYLCHOLINE RECEPTORS OF I N S E C T S

22 1

muscarone per g original tissue, whereas the pellet bound only 125 pmole per g (Eldefrawi and O'Brien, 1970). The amount of muscarone bound to the high-speed supernatant was linearly related to the amount of tissue used to prepare the supernatant (in the range 50-200 mg heads/ml and for a concentration of 1 0 - 6 muscarone). ~ A binding constant of 2.4 X 1 0 - 6was ~ determined from a study of the extent of binding as a function of concentration (Fig. 1). A high level of reversible [3H]-muscaronebinding equivalent to

i

10

t

t 5t

0

4

8

Fig. 1 Linweaver-Burke plot of the binding of muscarone (B, in molelg of heads) to the supernatent fluid fraction (isolated from a suspension of heads of house-flies Musca dornestica at 100 OOOxg for 60 mm), as a function of the concentration of muscarone (S, molar concentration). Some points at low values of S are omitted because they are far off the scale. The plotted slope was computed from all points by a weighted regression method. From Eldefrawi and O'Brien (1970)

70 nmol/g head protein was reported. The extent and affinity of the binding of ['H]-muscarone to fly heads was comparable to that found for the binding of this reversible cholinergic ligand to electroplax of Torpedo (O'Brienet al., 1969). [3H]-muscarone binding was found to be inhibited by nicotinic ligands (nicotine, d-tubocurarine, gallamine) and muscarinic ligands (atropine and pilocarpine) at about the same concentrations. It was concluded therefore that ['HH]-muscarone was binding to an acetylcholine receptor of mixed affinity (Eldefrawi and O'Brien, 1970). Some of the earlier studies on housefly head cholinergic binding molecules utilized chloroform-methanol extraction in conjunction with the

DAVID 6.SATTELLE

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detection of binding by Sephadex LM-20 chromatography (Cattell and Donnellan, 1972; Donnellanetal., 1975). Although the proteolipid isolated by this means showed a similar subcellular distribution and pharmacology to the putative receptor present in aqueous extracts (Eldefrawi et al., 1971b; Donnellan et al., 1 9 7 9 , the use of organic solvents has been abandoned in more recent studies. Levinson and Keynes (1971) have demonstrated the possibility of artefacts when gel-filtration is used in conjunction with organic solvents. Furthermore, it has been shown by Barrantes et al. (1975) that there is no cross-reaction between the cholinergic proteolipid extracted by chloroform-methanol from electric organ membranes and rabbit antisera against detergent-extracted receptor from the same tissue. Because of these difficulties, the use of extraction by organic solvent has not been pursued. TABLE 1 Binding constants of cholinergic ligands and concentrations of binding sites in extracts of housefly (Musca) heads and fish (Torpedo) electroplax. From Eldefrawi et a f . (1971b)

Ligand

Muscarone Nicotine Decamethonium Dimethyl d-tubocurarine Atropine

Binding constants

Concentrations of binding sites

Musca Torpedo ( X 1o-6M)

Musca Torpedo (nmolelg fresh wt)

2.4 2 0 . 4 0.72k0.06 3.220.3 3.2 f 1.7 2.5 kO.5 2.32 1.1 0.19?0.02 0.4320.03 3.0k0.07 1.3 2 0.2 2.3 k 0.6 23.3 2 2.3 2.1 k 0 . 8 31.0 277.2" 2.220.6

1.0 k 0.03 1.3k0.2 2.120.04 1.3 k 0.3 4.6k13.7

'The high variance of atropine binding to Torpedo is due to the poor affinity, which leads to measuring small amounts of binding at high concentrations, and hence computing a small number by subtraction of two large values

Following the initial studies with [3H]-muscarone (Eldefrawi and O'Brien, 1970), radiolabelled nicotine, decamethonium, dimethyl d-tubocurarine and atropine were also shown to bind to the lOOOOOxg supernatant derived from an aqueous extract of housefly heads with binding constants in the micromolar range (Eldefrawiet al., 1971b). Table 1 shows that, with the exception of dimethyl d-tubocurarine, all ligands exhibited approximately the same number of binding sites (2.2-3.2 nmole/g tissue). The detection of an order of magnitude greater number of binding sites for dimethyl d-tubocurarine was interpreted as being due to non-specific binding. In a comparison of the binding of [3H]-nicotineand [3H]-muscaroneto the high-speed supernatant of housefly heads, Eldefrawi et al. (1970) pointed to the similar numbers of binding sites for both ligands. In the same study it was shown that the inhibitory effects of six cholinergic ligands were similar in both cases (Table 2). Further, the binding molecules for both

ACETYLCHOLINE RECEPTORS OF INSECTS

223

TABLE 2 Blockade of binding to housefly head extracts of t'H]-nicotine and . ['H]-muscarone (at 1.0 x 10%) by cholinergic ligands (at 1.0 X 1 0 - 4 ~ ) From Eldefrawi et al. (1970) 96 blockade

Ligand Decamethoniurn d-Tubocurarine 3-Pyridylmethyldirneth ylamine N-(3-pyridylmethyl)-morpholine Atropine Pilocarpine

Nicotine

Muscarone

75 55 66 23 80

76

84

84

54

92 8

72

radiolabelled ligands were sensitive to trypsin and chymotrypsin but not phospholipase C. The authors concluded that both nicotine and muscarone were binding to the same macromolecule(s) in the high speed (100 OOOxg) supernatant. Mansour et al. (1977) have purified by 25-fold the cholinergic binding molecules from the high-speed supernatant of housefly (Musca dornesfica) heads by gel-filtration on Sephadex G-200 (Fig. 2). The purified form of this putative receptor bound 1000 pmole/mg protein of [3H]-decamethonium with an apparent K D of 1.5 X 1 0 - 7 ~Acetylcholine . (in the presence of the

Elution Volume I m l )

Fig. 2 Gel filtration of the lOOOOOxg supernatent of housefly head extract on Sephadex G-200. Void volume is 400 ml and each fraction is 5 ml. From Mansourer al. (1977)

DAVID

224

B. SATTELLE

anticholinesterase diisopropyl fluorophosphate (DFP) at a concentration of 1.0 X 1 0 - 5 ~competitively ) blocked this binding with an inhibitor constant (Ki) of 1.0 x lO-’w. A variety of nicotinic and muscarinic ligands at 1.0 x ~O-’M competitively blocked the binding of 1.0 x ~O-’M [3H]decamethonium and [3H]-nicotine (Table 3). The potent nicotinic antagonists a-bungarotoxin and cobratoxin, however, did not inhibit binding when . non-cholinergic, putative neurotransmitter tested at 2.5 x 1 O W 6 ~ The serotonin was similarly ineffective. Further purification proved difficult. For TABLE 3 Effects of cholinergic drugs and neurotoxins on the binding of [’HInicotine (1.0 x ~O-’M) and [’HI-decamethonium (1.0 x ~ O - ’ M ) to purified cholinergic binding molecules from housefly head extracts (Sz)* measured by equilibrium dialysis. From Mansour et af. (1977) % blockade of binding“ [’HI-Decamethonium [’HI-Nicotine

Drug or Toxin (X ~~

10-7~ ____

Acetylcholine Diisopropyl fluorophosphate (DFP) Acetylcholine + DFP Carbamylcholine Succinylcholine Benzylcholine Nicotine Decamethonium d-Tubocurarine Atropine Pilocarpine Scopolamine Arecoline Isopropamide Hexocyclium methylsulphate Serotonin a-Bungarotoxin‘ Cobratoxin’

5.4 t 1.2’ 0 53.3 % 0.7 35.5 t 6.0 54.0 t 8.2 75.2 t 7.1 50.0 t 3.3

7 . 0 2 1.5’ 0 54.6 2 0.8 64.8 2 7.0

98.92 4.4 40.4 t- 7.2 80.3 t 10.4 83.5 ? 0.9 74.8% 5.9 52.9 t 3.1 51.5 % 7.7 90.2 +- 8.9 0 0 0

72.1 2 4 . 1 78.2 2 0.5

0 0

Blockade values are the means of three experiments ? standard deviations. Zero values are used when there is no signlficant change in binding (p<0.05) Since acetylcholinesterase is present in this S2 preparation, in the absence of an acetylcholinesterase inhibitor such as DFP (KDfor acetylcholine+ DFP is 1.0 X ~ O - ’ M ) all acetylcholine should be hydrolyzed and the low blockage observed is suggested to be due to the effects of choline All drugs are added to the dialysis bath with the radiolabelled ligand, but in the case of the neurotoxins they are added in addition to the Szpreparation at the final concentration stated, then after 60 min at 23°C dialysis is started. Final concentrations of toxins used are 2.5 x 1 0 - 6 ~ for a-bungarotoxin and 2.8 x 10-O~for the cobratoxin * The Sz supernatant fraction is produced by gel filtration on Sephadex G-200 of the S, (100 OOOxg, 1 h; 4°C) supernatant prepared from an homogenate of housefly heads. After gel filtration and a further 100 OOOxg spin (1 h; 4°C) the supernatant (6) is collected

ACETYLCHOLINE RECEPTORS OF I N S E C T S

225

example, affinitychromatography gave further purification but no increase in specific binding due to some loss of binding activity. Also, using semipreparative disc-gel electrophoresis, a loss of binding activity was noted (Mansour et al., 1977). The same authors detected two rapidly migrating protein bands (M.W. 60 000 daltons) and a third band (M.W. 94 000 daltons) by means of acrylamide gel-electrophoresis of cholinergic binding material purified by affinity chromatography. These bands accounted for all the binding activity. Clarke and Donnellan (1975) confirmed the subcellular localization and nature of the binding activity in a 1OOOOOxg supernatant fraction, also prepared from heads of Musca domestics. The same laboratory also showed that a-bungarotoxin, the largely irreversible nicotinic antagonist, did not appear to bind the high-speed supernatant fraction (Donnellan et al., 1975, 1977). Components that bind decamethonium, acetylcholine, nicotine and atropine were reported (Cattell and Donnellan, 1975; Donnellan et al., 1975). These authors showed that acetylcholine binds to two sites - a high affinity site (Ki = 1.5 X ~O-'M)and a site of lower affinity (Ki = 8.3 X 10-6M). The respective concentrations of these high and low affinity sites were 1.5 pmole/mg protein and 19.2 pmole/mg protein. Decamethonium also bound to two classes of sites ( K i = 5 . 2 x 1 0 - 7 ~ ;17.9 pmole/mg protein and Ki = 6.7 x 1 0 - 6 ~71.5 ; pmole/mg protein), whereas nicotine and atropine ) about the same each bound with similar affinity (Ki= 7.8 x 1 0 - 6 ~ to number of sites (42-47 pmole/mg protein). Jewess et al. (1975) purified a protein fraction from housefly heads using differential centrifugation and gel-filtration. The purified material bound, with high affinity, acetylcholine and the potent muscarinic ligands dexetimide and atropine, in addition to ligands such as nicotine and decamethonium which also bind strongly to nicotinic receptors of vertebrate muscle (cJ Colquohoun and Rang, 1976). For all the ligands tested there appeared to be a single site for binding to this purified material suggesting that the two site binding profile reported earlier for the impure extracts (Cattell and Donnellan, 1972; Donnellan et al., 1975) may have been the result of binding to other sites in addition to the receptor. Dissociation constants varied from 6.2 x ~O-'M (dexetimide) to 5.4 x 1OT6hf (pilocarpine) as shown in Table 4.The concentration of binding sites was in the range 381 nmole/mg protein (in the case of atropine) to 560 nmole/mg protein (for pilocarpine). In the same study, isoelectric focusing of the putative receptor fraction yielded an isoelectric point (PI) of 4.8 for the binding material, a value similar to that reported for the nicotinic receptor of the eel Electrophorus (Biesecker, 1973). A molecular weight of 360 000 daltons was determined for the fly head receptor using gel-permeation chromatography (Jewess er a1., 1975). Polyacrylamide-gel electrophoresis in the presence of

-

-

DAVID B. SATTELLE

226

TABLE 4 Binding of reversible ligands to a purified Sepharose 6B extract from heads of Musca domestica. The binding of tritiated ligands was measured by an ultrafiltration assay (Clarke and Donnellan, 1974) using a Sepharose 6B extract in the presence of the anticholinesterase paraoxon (1 .O X lo-'^). For each ligand the number of separate binding runs is indicated - each involving 8-10 ligand concentrations. From Jewess et al. (1975) Ligand Acetylcholine Decamethonium Nicotine Atropine (+) Dexetimide Pilocarpine

Dissociation constant Concentration of binding sites Number ( X lo-%) (nmole/g protein) of runs

* * *

5.027 1.253 0.476 f 0.09 4.445 0.484 5.223 2 0.621 0.062 0.009 5.425 f 0.719

* * *

453 59 575 37 508 +: 29 381 25 579* 9 560 +: 40

2 3 2 2 2 2

SDS revealed two populations of subunits (M.W. 83000 daltons and M.W. 90 000 daltons). Donnellan and Harris (1977) have recently reported that cross-linkage studies of these subunits can produce a protein of molecular weight similar to that of this putative receptor, suggesting an a& configuration of four subunits for the protein. The same authors also showed that the putative receptor, in its native form, and the subunits all stained as glycoproteins. Mannose, glucose and galactose appeared to be the major neutral sugars of the native receptor. Results in broad agreement with the findings of Donnellan and Harris (1977) were recently obtained by Tripathief at. (1979) who also detected a single type of [3H]-decamethonium binding to a soluble fraction of housefly head extract. In this case a molecular weight of 342 000 daltons was determined by sedimentation analysis, and subunits of molecular weights 64 000 and 90 000 were detected by gel electrophoresis. Of particular interest was the improved purification of this putative receptor by affinity chromatography using decamethonium to elute material from the column. The reversible binding of [3H]-nicotine to a 40 OOOxg supernatant prepared from homogenates of cockroach nerve cords (thoracic and abdominal regions of the cord) has also been described (Aziz and Eldefrawi, 1973). Dissociation constants of approximately one micromolar were reported and the number of binding sites was 30.6 pmole/mg protein in the case of Gromphodorhina porfenfosa and 37 ( k 5 ) pmole/mg protein in the case of Periplanefa americana (Table 5). It was noted however by Aziz and Eldefrawi (1973) that the nicotine-binding component in nerve cords of Gromphodorhina exhibited a low affinity for acetylcholine (Ki = 1.9 x 1 0 - 3 ~ ) . These same authors although not pursuing in detail the cockroach putative receptor did point out that the cockroach nerve cord preparation provides

ACETYLCHOLINE RECEPTORS OF I N S E C T S

227

TABLE 5 Dissociation constants and maximal binding of [3H]-nicotineto nerve cord extracts of cockroaches. From Aziz and Eldefrawi (1973) Dissociation constant Species Gromphodorhina portentosa Peripianeta americana

(X

10-6M)

1.5 f 0.6 1 . 1 k0.2

Concentration of binding sites (pmole/mg protein) 30.6 f 8 37 2 5

an ideal source of material for the study of insect acetylcholine receptors in that it is readily amenable to both biochemical and physiological studies. Thus using a range of reversibly acting cholinergic ligands a putative receptor of “mixed” pharmacological specificity, has been characterized in considerable detail in high-speed supernatant extracts prepared from heads of Musca domestica, and in less detail using nerve cord extracts of Periplaneta americana and Gromphodorhina portentosa. 2.3

[1251]-(y-BUNGAROTOXIN B I N D I N G TO L O W - S P E E D EXTRACTS

The polypeptide a-bungarotoxin (M.W. 7800 daltons - Lee, 1972) isolated from the venom of Bungarus multicinctus is a potent, essentially irreversible, antagonist of nicotinic acetylcholine receptors of mammalian skeletal muscle and fish electroplax (Lee and Chang, 1966; Miledi and Potter, 1971; Changeux et al., 1970). Radiolabelled a-bungarotoxin has been used to isolate peripheral nicotinic receptors and to characterize their pharmacological properties in tisse homogenates of vertebrate muscle and CNS (Cohen and Changeux, 1975; Rang, 1975; Changeux, 1975; Heidmann and Changeux, 1978; Schmidt et at., 1979). In 1975 Hall and Teng reported that [‘251]-a-bungarotoxinbound in a non-uniform way to frozen sections of Drosophila rnelanogaster. This paper was of considerable interest since the early work on putative insect acetylcholine receptors had been confined to an a-bungarotoxin-insensitive component (see Section 2.2). Within a short period, several laboratories began to characterize the a-bungarotoxin-binding component from Drosophila. A particulate form of the receptor was in all cases prepared from homogenized heads of Canton-S wild type strain by differential centrifugation. SchmidtNielsenet al. (1977) found that, after filtration to remove chitinous material and a low speed (2000xg) spin to remove pigment granules and other debris, the bulk of the toxin-binding activity remained in the 2000xg supernatant fraction. Following a 20 OOOxg centrifugation, almost all the toxin binding material was pelleted but only one third of the protein was pelleted. A second 20 OOOxg centrifugation removed additional protein without

DAVID 6.SATTELLE

228

significant loss of toxin-binding activity. The specific toxin-binding properties of this pellet were assessed by Schmidt-Nielsen et al. (1977). A somewhat similar extract was prepared by Rudloff (1978) who, following a low speed (1OOOxg) spin of the homogenate, centrifuged the supernatant at 20 OOOxg and then exposed the pellet to a hyposmotic shock. A final spin at 9500xg to remove soluble proteins resulted in a pellet which was used in subsequent binding studies. Dudai and collaborators have utilised a 500 xg supernatant prepared from head homogenates (Dudai 1977; Dudai and Amsterdam 1977; Dudai 1978). The similarity of the extraction procedures allows comparison of the data from the three laboratories. Using either a centrifugal assay (Schmidt-Nielsenetal., 1977; Rudloff, 1978; Dudai, 1977, 1978) or a filter-binding assay (Schmidt-Nielsen et al., 1977; Dudai 1977; Dudai and Amsterdam 1977; Dudai, 1978), the presence of a specific toxin-binding activity, that under the assay conditions was linearly proportional to extract concentration, has been demonstrated.

L

,'

T O T A L [ ~ ~ ~ ~ - ~ - B U N G A R O T O( XnI N~ )

Fig. 3 Saturability of ['Z51]-a-bungarotoxin binding to the 40 OOOxg pellet by differential centrifugation of extracts from whole flies (Drosophila melanogaster). Duplicate assay mixtures containing increasing concentrations of ['2SI]-a-bungarotoxin (specific activity 92 Ci-nmol) and a constant amount of extract equivalent to 10 mg flies (wet weight of starting material) were incubated for 1 h at 21 "C.Toxin binding was determined by a centrifugal assay. 0 -- - - - 0, total [1Z51]-a-bungarotoxinbound; A - - - - - A, background binding of [lZSI]-a-bungarotoxin to heat-inactivated extract; 0 0 net toxin binding calculated as total binding minus background binding. From Schmidt-Nielsen et al. (1977)

A requirement for specific binding is that binding should saturate with increasing concentrations of ['2'I]-a-bungarotoxin (see Section 2.1). This has been fulfilled (see for example Fig. 3) in the case of binding to Drosophila extracts by Schmidt-Nielsen et al. (1977) and by Dudai (1977, 1978). From the net concentration of toxin bound under saturating condi-

ACETYLCHOLINE RECEPTORS OF I N S E C T S

229

tions, the concentration of toxin binding sites in the extract can be calculated. For example, concentrations close to 1.0 x 1 0 - 9 ~a-bungarotoxin binding sites per gram of extract protein have been reported corresponding to 7-13 pmole binding sites per g of flies (Schmidt-Nielsenetal., 1977). One laboratory, having determined a figure of 6.8 x moles binding sites per single fly head, has used this figure to estimate that the surface of a single neurone may contain at least 5000 receptor sites (Rudloff, 1978). Inview of the uncertainties of the assumptions on which this calculation is based it seems premature to attempt such estimates. Saturability studies have also enabled estimates of the apparent K Dfrom the concentration of free toxin that produces half-maximal binding. Values ~ 1977),1.9 X 1 0 - 9(Dudai ~ 1978) and of 1.1 x 1 0 - 9(Schmidt-Nielsenetaf., ~ 1978) were reported for Drosophila. Studies of the 1.8 X 1 0 - 9 (Rudloff, binding as a function of time yielded an association rate constant of 2.4 x 10-5~-1s-1(Dudai, 1978) and a dissociation rate constant of 1.4 x 10-4s-' (Dudai, 1978). The K D calculated from these values was ~ 1978) which was probably a better estimate than the 0.6 x 1 0 - 9 (Dudai, apparent K , s estimated from binding isotherms (cf: Cuatrecasas and Hollenberg, 1976). Toxin binding to Drosophila extracts was inhibited by high salt (NaC1, CaC12) conditions (Dudai 1978). Calcium was more effective than sodium at an equivalent ionic strength. Similar inhibitory effects of inorganic cations were reported for the nicotinic receptors of Torpedo electroplax (Schmidt and Raftery, 1974) and in mammalian brain (Schmidt, 1977). Dudai (1980) has shown that the Hill coefficient for binding of [lZ5I]-abungarotoxin was 0.96 indicating no co-operativity. By contrast, the Hill coefficient for the binding of acetylcholine measured by displacement of toxin-binding was found to be only 0.5. This low value may be due either to negative co-operativity in the binding of acetylcholine or to heterogeneity of agonist binding sites. By preincubation of extracts with different concentrations of test ligands, the relative abilities of cholinergic ligands to inhibit the specific binding of [1Z51]-a-bungarotoxincan be tested. The pharmacological specificity of the a-bungarotoxin-binding component in Drosophila described by SchmidtNielsen et al. (1977) is illustrated in Fig. 4. From such binding curves it is possible to estimate the ligand concentration needed to inhibit by 50% the maximum specific binding to extracts (I5o).Table 6 summarizes 15,,values obtained for a range of cholinergic ligands by several laboratories. Nicotine, d-tubocurarine and acetylcholine were particularly strong inhibitors of a-bungarotoxin binding. Drugs such as D,L-muscarine, atropine and pilocarpine which were effective at submicromolar concentrations on the putative muscarinic receptor only weakly inhibited a-bungarotoxin binding

DAVID B. SATTELLE

230

a

Ligand Concentration ( M )

ACETYLCHOLINE RECEPTORS OF I N S E C T S

23 1

to Drosophila extracts. Decamethonium which proved to be a potent inhibitor of toxin binding to Torpedo electroplax (Cohenet al., 1974) and rat muscle (Colquhoun and Rang, 1976) was less effective in preventing binding to Drosophila extracts. Also, higher concentrations of carbamylcholine were required to block toxin-binding in Drosophila (Schmidt-Nielsen et al., 1977) when compared to Torpedo (Cohen et al., 1974). Thus, although some differences have emerged between putative nicotinic cholinergic receptors from various tissues it is clear that the ['251]-~-bungarotoxin binding component in Drosophila closely resembles a nicotinic receptor. The prospects of further characterization and purification of this putative nicotinic receptor of Drosophila were enhanced by the demonstration that extract treated with the detergent Triton X-100 also exhibited a saturable component of [1251]-~-bungarotoxin binding with a pharmacological specificity not significantly different from that observed in particulate extracts (Schmidt-Nielsen et al., 1977). Recent attempts at purification of the a-bungarotoxin binding component have resulted in enrichment for the putative receptor. This has not, however, been achieved without some difficulties. For example, Dudai (1978) showed that conditions yielding a total solubilization of the nicotinic receptor of vertebrate CNS (Lowy et al., 1976) were inefficient when applied to Drosophila. However starting with fly heads of Drosophila, the

Fig. 4 Inhibition by cholinergic ligands of ['251]-a-bungarotoxin binding to Drosophila extracts. Either the membrane-bound or the Triton X-100 treated extracts were preincubated with the ligands. [1251]-a-bungarotoxinwas added, and the amount of bound toxin was assayed. One hundred percent binding was defined as the [LZ51]-a-bungarotoxin bound in the absence of added ligand. Background binding to heat-inactivated extracts has been subtracted. The plotted ligand concentrations are the final concentrations after radioactive toxin addition. The inhibition by acetylcholine was measured in the presence of 1 O - h neostigmine to inhibit acetylcholinesterase. (a) A membrane-bound preparation (equivalent to 8.8 mg whole flies in starting material) was preincubated for 30 minutes with 1.2 times the final concentration of ligand in a total volume of 100 p1. 20 p1 of [1251]-a-bungarotoxinwas added, and the mixtures were incubated for another 30 minutes and assayed by the centrifugal binding assay. For nicotine, decamethonium, and atropine, the [LZSI]-a-bungarotoxin concentration (sp. act. 31 Ci/mmol) was 2.5 x 1 0 - 9 ~ .For neostigmine, acetylcholine, d-tubocurarine, and a-bungarotoxin, the ['2SI]-ol-bungarotoxinconcentration (sp. act. 90 Ci/mmol) was 1.5 X 10-9M.(b), (c), 100 p1 ofTriton X-100 treatedextract waspreincubated with 1.17 times the final concentration of ligand for 10 min. in a total volume of 300 pl. ['251]-a-bungarotoxin(sp. act. 119-148 Ci/mmol) was added to a final concentration of 0.57 x 1 0 - 9 and ~ the mixtures were H aBgt, incubated for 10 minutesand assayed by the DEAEfilter binding assay. H a-bungarotoxin; 0 . . . . . . . .O Nic, nicotine; + - - - - - + dTC, d-tubocurarine chloride; 0 0 ACh, acetylcholine chloride; A A Atr, atropine sulfate; A . . . . . . . .A Deca, decamethonium bromide; 0- - - - Neo, neostigmine bromide; X . . . . . . . . X Ese, eserine sulfate; H - - - - - H Carb, carbamylcholine chloride; . . . . . . . .+ Hexa, hexamethoniurn chloride; 0 0 Pilo, pilocarpine HCI; A - - - - - A Cho, choline chloride. From Schmidt-Nielsen et a f .(1977)

+

DAVID B . SATTELLE

232

TABLE 6 Pharmacological profiles of putative acetylcholine receptors determined by Drug concentration (M) Ligand (Particulate)’ a-Bungarotoxin Dihydro-8 -erythroidine Nicotine d-Tubocurarine Acetylcholine Gallamine Atropine Hexamethonium Carbamylcholine Pilocarpine Decamethonium Dexetimide D,L-Muscarine Mecamylamine Oxotremorine Phenyltrimtheylammonium Nereistoxin 2-Isothiocyanatoethyltrimethylammonium iodide Pempidine Hyoscamine Choline Eserine Neostigmine BW 284C51 Diethyllluorophosphate Trimethapan a + neostigmine (~O-’M); mine ( 1 0 - 5 ~ )

Drosophila melanogaster (Solubilized)’ (Purified)’ (Particulate)’

(Particulate)4

4.3 x 10-10

3.6x

5X10-’’

2.6X10-n 4 . 5 ~10-7 4.6~ 3.5x10-’ 5X 2.7~10-~ 6 . 5 ~ 1 0 - ~ 6 . 6 ~ 1 0 - ~ 3 x “7.9X 10-6 “2.8~ 3.0X b3x 10-6 1.6~ 7.4x10-’ 5.7 X lo-’ 1.8 x 10-’ 3.6 x 10-4 7X10-5 1.2x10-~ 2.3X 7.9X10-4 1.5 X 10-4 2 . 4 lo-‘ ~ 2.5~10-4 6 . 3 lo-’ ~ 6.0X10-’ 1 . 4 lo-’ ~ 6.8X

gx 2x10-7 ‘2X 10-5 5X10-’ 5X10-5

1x 1 0 - ~ 2x10-4 9x10-4 2x10-4 10-3 10-3 5X10-4 2x10-~

3.8 x 10-4 6.9x10-6 1.3 x

10-3

4.9~10-~ 5.4X10-6 1.4X10-6 4.5 x 5.4~

6X10-6

lo-’ 2x 10-5

1.3 X

+ eserine (5X 1 0 - 6 ~ ) ; + diethyllluorophosphate

(IO-’M);

+ neostig-

Insect data were obtained o n fly head material with the exception of (5) and (6) in which abdominal nerve cord extracts were used. I Schmidt-Nielsen et al. (1 977) Hall (1980); Gepner (1979) Rudloff (1978) Dudai (1978) Sattelle (1980) Gepner et al. (1978) Hams et al. (1979) Cattell et al. (1979) S h ah et al. (1974) lo Thomas et al. (1978) ’I Schmidt (1977) Colquohoun and Rang ( I 976)

’ ’ ’ ’

ACETYLCHOLINE RECEPTORS OF I N S E C T S

233

inhibition of ['251]-a-bungarotoxin binding to tissue extracts required for 50%)maximum effect (Is,,) Periplaneta Musca Aplysia Limulus americana domestica Rat brain Rat muscle californica polyphemus (Partic~late)~,' ( P a r t i c ~ l a t e )(~Particulate)9 .~ (Particulate)'" (Particulate)" (Particulate)" (Solubilized)" ~

<<10-7 2.7X10-6 2 . 4 10-7 ~ d8.3x10-6

4-9~10-~ 3-7x 1.8~10-~

7.2~10-~ 10-~ 2.1 x 10-5 1.7 x

2 x lo-' 2 ~ l O - ~ 3~10-~ 3x10-4

LOX

1-5 x 10-4 1.3 x 10-3

2x10-4

<< 10-7 l.oxlo-s 3.1 x 1.9xlO-' 3.0~10-~ 3.5X10-6 3 ~ 1 0 - ~ 1.6x10-' 9.0~10-~ 6 ~ 1 0 - ~ 9.0~10-~ 1.o x 10-3 5.0~10-~ 2xlO-' 7x 2X10-'

2.2XlO-' 3.7X10-7 4.7~10-~ 4 . 2 ~ l . 2 ~ l O - ~ 8.9X10-' 3 . 5 ~ 1 0 - ~ 6.3x10-' 2.1~10-~ 8.6~10-~

1.ox lo+ 3.O x 10-3 2.0~10-3

1.8x10-4 1.6X

3 . 7 10-5 ~ 4x10-' 2 x 10-4

purification scheme shown in Fig. 5 was employed by Gepner and Hall (Gepner, 1979; Hall, 1980). A membrane preparation (P4) was first prepared by differential centrifugation and the toxin-binding activity was solubilized in Triton X-100 and applied to an affinity column consisting of cobratoxin covalently linked to sepharose 4B. The adsorbed material was recovered from the column by elution with 0.2 M carbamylcholine and re-applied to a second cobratoxin-sephadex affinity column. The second column was washed with buffer containing high salt (0.5 M NaCl) and 0.1% sodium deoxycholate to remove contaminating material. Finally, the toxinbinding activity was eluted with 0.2 M carbamylcholine. In this way the a-bungarotoxin-binding component was purified 1200-fold from Drosophila heads.

D A V I D B. SATTELLE

234 PURIFICATION SCHEME

I HEAD O HOMOGENATE 1 P

Pooled 2000 x g supernatants (S1-3)

buffer 2 or buffer 2

2OOOx g pellet (PI )

deoxycholate

off inity column I1 Elute with carbamylcholine in deoxycholate buffer 2

component from Fig. 5 Scheme for purification (1200-fold) of [1251]-~-bungarotoxin-binding heads of Drosophila rnefanogasfer.From Gepner (1979)

Rudloff et al. (1980) found that treatment of a membrane fraction with sodium deoxycholate at pH 9.0 was an effective way to solubilize the Drosophila a-bungarotoxin-binding component. Affinity chromatography was also used by these authors to achieve about the same level of purification reported by Hall and collaborators (cf. Hall, 1980). The molecular weight of the purified “receptor-toxin” complex was estimated by gel-filtration chromatography to be 500 000 daltons (Gepner, 1979; Hall, 1980). As pointed out by Gepner (1979) this estimate is likely to be high as it does not take into account the contribution of bound detergent to the Stokes radius. By means of velocity sucrose-gradient analysis, a

ACETYLCHOLINE RECEPTORS OF INSECTS

235

sedimentation coefficient of 11.5 S was estimated which would be equivalent to a molecular weight of approximately 300 000 daltons for a soluble globular protein (cf. Martin and Ames, 1961). This is slightly larger than the value of 9-9.5 determined for crude and purified acetylcholine receptor of fish electroplax (de Robertis and Schacht, 1974). The specific activity of the purfied receptor, which is a glycoprotein, is in the range 0.6-1.1 pmoles of toxin binding sites per g protein (Gepner, 1979; Hall, 1980), an order of magnitude lower than that reported for the electroplax receptor (cf. Heidmann and Changeux, 1978) indicating that the Drosophila receptor is about 10% pure. Using association (7.8 X lO-'M-'s-') and dissociation (1.2 x 10-4s-') rate constants, the K D for the purified receptor-toxin complex was calculated to be 1.5 x ~O-"M (Gepner 1979). The pharmacological specificity of the purified ['Z51]-a-bungarotoxin-bindingcomponent was found to be comparable to that of the particulate material (Table 6). Toxin-binding components with similar properties to those described for Drosophila have been reported in other insects. For example, detergent extracts prepared from the brain of the moth Manduca sexta (Sanes et al., 1977) contained an a-bungarotoxin binding component with some of the properties expected of a nicotinic acetylcholine receptor. Recently, Harris, et al. (1979) reported the existence of a putative nicotinic receptor in extracts of housefly (Musca domestica) heads. d-Tubocurarine and nicotine inhibited toxin binding in this preparation much more effectively than both decamethonium and D,L-muscarine (see Table 6). Abdominal nerve cord extracts of the cockroach Periplaneta americana also contained an a-bungarotoxin-binding component with the specificity expected of a nicotinic acetylcholine receptor (Gepner et al., 1978; cf. Sattelle, 1980). This toxin binding activity was pelleted by centrifugation at 40 OOOxg for 30 minutes and saturation binding studies indicated that there were 112 pmoles of toxin binding sites per g of abdominal nerve cord (wet weight). Toxin bound with high affinity and no dissociation was detected during a 5-hour test period (Fig. 6). The most effective inhibitors of toxinbinding were d-tubocurarine, nicotine, and acetylcholine in the presence of neostigmine. Pilocarpine, carbamylcholine, atropine and decamethonium were all less effective than acetylcholine in inhibiting ['251]-a-bungarotoxin binding. The cockroach CNS preparation is of particular interest in that it readily enables direct comparison between ligand-binding studies and electrophysiological studies on identified neurones. An ['251]-a-bungarotoxinbinding component that is saturable and exhibits the pharmacological specificity expected of a nicotinic acetylcholine receptor has been demostrated, therefore, in low-speed extracts of heads from Drosophilu melanogaster, abdominal nerve cords from Periplaneta americana, heads from Musca domestica and brain tissue from Manduca sextu. This putative

DAVID 6 . SATTELLE

236

2

4

6

I0

8

Total I'zSl]-oBunqarotoxin

12

14

(nM)

I 100

P

cj

__

0

I

I

I

2

1%

Time ( h l

Fig. 6 ["51]-a-bungarotoxin binding to cockroach (Periplanera americana) abdominal nerve cord extracts. (a) Saturability of [1Z51]-a-bungarotoxinbinding to the particulate component. Toxin binding was determined by DEAE filter binding assay. In all cases net binding was determined by subtracting background binding to heat-inactivated extract. ( b ) "On" kineticsof binding of [1251]-a-bungarotoxinto cockroach abdominal nerve cord extracts. (c) "Off' kinetics of ['i51]-a-bungarotoxin binding. From Sattelle (1980)

acetylcholine receptor is located in particulate fractions as would be predicted for a membrane receptor.

2.4

[ 3 H ] - Q U I N U C L I D I N Y L B E N Z I L A T E B I N D I N G TO L O W - S P E E D EXTRACTS

In vertebrate brain and smooth muscle tissue several antagonists have been used to characterize a membrane component with the expected

ACETYLCHOLINE RECEPTORS OF INSECTS

237

properties of a muscarinic acetylcholine receptor. (cf. Birdsall and Hulme, 1976). [3H]-quinuclidinyl benzilate, a potent muscarinic antagonist, has proved a particularly useful receptor probe in studies on brain tissue (Yamamura and Snyder, 1974a, b, c; Yamamuraetal., 1974 a, b; Snyderet al., 1975) and isolated brain synaptosomes (Yamamura and Snyder, 1974a). Recently Dudai and Ben-Barak (1977) demonstrated that [3H]quinuclidinyl benzilate binds to a component of head homogenates of Drosophila melanogaster. Following separation from bodies by freezing and shaking, and homogenization in 0.32 M sucrose, a Drosophila head homogenate was centrifuged at 500xg for 10 min and the supernatant fraction was used for kinetic and pharmacological studies. Using a filter-binding assay, Dudai and Ben-Barak (1977) showed that the binding of [3H]-quinuclidinyl benzilate to the particulate component of the supernatant fraction was linearly proportional to the amount of head homogenate present. Background binding (-5% total binding) in the presence of 1.0 x 1 0 - 4 atropine ~ was routinely subtracted and a saturable component of specific binding was obtained with [3H]-quinuclidinyl benzilate concentrations above 5.0 x 1 0 - 9 ~ ( F i g7). . Half-saturationwasnotedat approximately2.0 x 1 0 - 9 ~ . From the amount of binding under saturating conditions, Haimetal. (1979) estimated the concentration of [3H]-quinuclidinyl benzilate binding sites to be 65 ? 0.6 pmole per g protein. The same laboratory in an earlier study estimated the concentration of ['251]-a-bungarotoxin binding sites in Drosophila head homogenates to be 400-800 pmole per g protein (Dudai and Amsterdam, 1977).

Fig. 7 The level of binding of [3H]-quinuclidinyl benzilate to Drosophila rnelanogaster head homogenates after incubation in the presence of various concentrations of ligand. Aliquots of the supernatant of 5OOxg centrifugation for 10 min were incubated with [3H]-quinuclidinyl benzilate for 60 min at 25°C in the presence or in the absence of 1 x 1 0 - s ~atropine. 0 0 ,specific binding i.e. total binding minus binding in the presence of 1 X 1 O - h atropine; A A, binding in the presence of 1 X IO-'M atropine. Inset: doublereciprocal plot of specific binding. From Haim et al. (1979)

DAVID B. SATTELLE

238

The time-dependence of [3H]-quinuclidinyl benzilate binding has been described (Dudai and Ben-Barak, 1977; Haim et a f . , 1979). Under the conditions of the assay, binding was half-maximal after about 5 min and maximal values were reached in about 40 min. An on-rate constant of 2 X 10-6rK1s-1was calculated for the formation of the [3H]-quinuclidinyl benzilate-receptor complex (Haim et al., 1979). The pharmacological properties of [3H]-quinuclidinyl benzilate binding sites in Drosophifa have been investigated (Dudai and Ben-Barak, 1977; Haim et al., 1979). Concentrations of cholinergic ligands that inhibited binding by 50% are given in Table 7 (data of Dudai and Ben-Barak, 1977). The muscarinic ligands dexetimide, scopolamine and atropine were most potent in protecting against [3H]-quinuclidinyl benzilate binding. D,L-Muscarine which was without effect on the binding of a-bungarotoxin to Drosophila putative nicotinic receptors at concentrations up to 1 X 1 0 - 3 (Dudai, ~ 1977) inhi. bited [3H]-quinuclidinyl benzilate binding with an Is0 of 6 x 1 0 - 5 ~AcetylTABLE 7 Pharmacological profiles of putative acetylcholine receptors determined by inhibition of [3H]-quinuclidinyl benzilate binding to tissue extracts ~

Ligand Quinuclidinyl benzilate Scopolamine Dextetimide Atropine d-Tubocurarine Acetylcholine Pilocarpine Oxotremorine D,L-Muscanne Carbamylcholine Diethylfluorophosphate Nicotine Gallamine Mecamylamine Decamethonium Hexamethonium Eserine a-Bungarotoxin

~

~

~

Estimated dissociation constant ( K d (MI

Drug concentration inhibiting binding by 50% (M)

Fly head' Rat brain' (Drosophila rnelanogaster)

Human frontal cortex 1x

1x 10-9 1x10-9 4~10-~ 5x10-6 5 x 10-6 8x

8~10-~ 2~ 10-5 >10-5

>10-3 >10-3

5x10-'u 4x10-"' 1x 10-9 4~10-~ I x 10-5 5 x 10-6 1x 10-6 2x10-5

8 X 10-10

2x10-9 1 x 10-9 1x 10-5 SxlO-'

>10-5 6~10-~ 6x lo-'

>1 0 - ~ >10-3 >10-6

' Haim et al. (1979) * Ben-Barak and Dudai (1979)

5~10-~ >loWastek and Yamamura (1978)

ACETYLCHOLINE RECEPTORS OF I N S E C T S

239

choline, in the presence of the anticholinesterase DFP (1 x 1 0 - 5 ~ )also , inhibited [3H]-quinuclidinyl benzilate binding (I5,)= 3 x 1O+M), whereas nicotine which has a high affinity for putative nicotinic receptors in Drosophila (Dudai, 1977; Schmidt-Nielsen, et al., 1977) was ineffective in protecting [3H]-quinuclidinyl benzilate binding sites at concentrations up to 1 x 1 0 - 3 ~(Dudai and Ben-Barak, 1977). The last named authors also showed that a-bungarotoxin at 1 x 1 0 - 5 had ~ no significant effect on [3H]quinuclidinyl benzilate binding. Differential centrifugation has been employed to determine the subcellular distribution of the [3H]-quinuclidinyl benzilate binding sites. Both in isotonic sucrose and Pow ionic strength buffer, the bulk of the activity sedimented between 500xg and 20000xg (Haim e t a l . , 1979). In the same study it was shown by discontinuous sucrose-gradient centrifugation that most of the [3H]-quinuclidinyl benzilate binding activity sedimented between 1.O M and 1.7 M sucrose whereas a large part of the acetylcholinesterase activity sedimented to lower densities. Thus, the sedimentation profile of Drosophila [3H]-quinuclidinyl benzilate binding sites closely corresponds to that for Drosophila ['251]-a-bungarotoxinbinding sites (Dudai, 1978). This indicates that in both cases the binding sites are membrane-located. Several treatments have been applied to Drosophila extracts in attempts to solubilize the muscarinic receptor sites (Table 8). T o date it has not proved possible to solubilize the [3H]-quinuclidinyl benzilate-binding component. TABLE 8 Effect of various treatments on [3H]-quinuclidinyl benzilate binding levels in Drosophila. Aliquots of Drosophila head homogenate were preincubated for 1 h at 25°C with the indicated agent in 0.025 M Tris-C1, p H 7.6. Binding of [3H]quinuclidinyl benzilate was measured. Each value represents mean f S.E.M.for 3-5 experiments. In the case of Triton treatment, ammonium sulfate, precipitation and D E A E adsorption assays were also tried but yielded similar results. From Haimet al. (1979) Treatment Buffer 1 M NaCl 2 M NaCl 0.6% Triton X-100 M dithiothreitol 2 x lo-' M CaClz M EDTA 0.5% (w/w protein) trypsin 1.6% (w/w protein) phospholipase C Boiling 3 min

[3H]- quinuclidinyl benzilate bound (fmol) 12.821.3 14.3f0.3 13.7f0.6 1.3*1.1 11.421.0 14.0f1.0 13.8k0.7 0.620.4 11.820.5 0.1

D A V I D B. SATTELLE

240

A specific [3H]-quinuclidinyl benzilate binding component has recently been reported in extracts of the terminal abdominal ganglion of the cricket Acheta domestica with a dissociation constant calculated from the Hill plot . and Edwards, 1980). In the same study the data of 5.1 x 1 0 - 9 ~(Meyer density of binding sites in this cricket ganglion was estimated to be 480 pmoles per g protein. Thus head extracts of Drosophila contain a [3H]-quinuclidinyl benzilate binding component with the expected properties of a muscarinic acetylcholine receptor. The pharmacological specificity of these binding sites closely resembles that reported for vertebrate muscarinic receptors. However, whereas mammalian brain has about an order of magnitude more muscarinic receptors than nicotinic receptors (Yamamura and Snyder 1974a; Salvaterra et al., 1975; Segal et al., 1978), in Drosophila head extracts the situation is reversed and nicotinic receptors are about an order of magnitude more abundant (Dudai and Ben-Barak, 1977; Haim, et al., 1979).

3 Autoradiographic localization of binding sites

In addition to demonstrating a saturable component of binding with the pharmacological specificity expected of an acetylcholine receptor it is necessary, in order to satis@ the criteria for identification of a specific binding component as a receptor, to show that binding of the radiolabelled ligand is localized to synaptic areas in the central nervous system. Autoradiographic studies of the localization of radiolabelled-ligand binding sites are only available to date in the case of ['251]-a-bungarotoxin and these studies have to date been largely confined to Drosophila melanogaster, Manduca sexta and Periplaneta americana. 3.1

DISTRIBUTION

The distribution of [*Z"I]-a-bungarotoxinbinding in insect tissues was first studied by Hall and Teng (1975) who showed that its distribution in frozen, serial 10 p m sections of the fruit fly Drosophila melanogaster was not uniform. In a subsequent detailed investigation by the same laboratory, it was demonstrated that toxin-binding was confined to neural tissue in the head and thorax of Drosophila. Non-neural tissues exhibited only background levels of binding. Specific binding to neural tissue was reduced to background levels by preincubation of the sections with 1.O x ~O-'M unlabelled a-bungarotoxin (Hall and Teng, 1975; Schmidt-Nielsen et al., 1977). Rudloff (1978) was unable to introduce ['2SI]-a-bungarotoxin into intact

ACETYLCHOLINE RECEPTORS OF I N S E C T S

24 1

whole brain tissue and all studies to date of the distribution of toxin-binding have been carried out using frozen sections. The brain of a dipterous fly such as Drosophila bridges the head capsule between the laterally situated compound eyes and consists of: the median protocerebrum and its lateral optic lobes; the anteriorly located deuterocerebrum the neuropile of which contains many glomeruli (synaptic loci for neurones of the antennal nerve); and the tritocerebrum (cf. Miller, 1950). Each optic lobe consists of three readily distinguishable regions - the outer lamina ganglionaris, the medulla and the lobula (Cajal and Sanchez, 1915; Strausfeld, 1976). Toxin-binding to frozen sections of Drosophila heads was shown by three laboratories to be confined to neuropile areas of the brain (Schmidt-Nielsen et al., 1977; Dudai and Amsterdam, 1977; Rudloff, 1978). No binding was detected, for example, over axons in the antennal nerve. Binding in neuropilar (synaptic) regions was not however uniform (Fig. 8). Schmidt-Nielsen et at. (1977) reported that in the medulla of the optic lobes binding was layered in a manner resembling the distribution of neuronal branching found in Calliphora brain by Cajal and Sanchez (1915) and in Musca brain by Strausfeld (1976). By contrast, little or no binding was found over the lamina of the optic lobes. All three laboratories have concluded from autoradiographic studies that toxin binding is confined to synaptic areas of neuropile and is absent on nerve tracts and cell bodies in the CNS of Drosophila. That the pattern of binding appeared to reflect synaptic distribution was consistent with the view that a-bungarotoxin was binding to synaptic nicotinic receptors in Drosophila. Rudloff (1978) provided the first autoradiograms of frozen sections of larvae and pupae of Drosophila. A non-uniform distribution of toxin binding activity was reported in both cases and features of the adult pattern of binding were discernible in the three-day old pupae. Toxin-binding to frozen sections of the brain of Manduca sexta has been studied by Hildebrand and colleagues (1979). The organization of the antennal lobes of the brain of this insect enables a histological distinction to be made between synaptic and non-synaptic regions. Sensory axons from the ipsilateral antenna terminate in condensed areas of neuropile known as the glomeruli which contain the primary afferent synapses. Groups of cell bodies of second order antennal neurones are located peripherally. [12sI]a-bungarotoxin-binding sites were heavily concentrated over the glomeruli (see Fig. 28). Few binding sites were detected over non-synaptic regions such as the non-synaptic neuropile, the cell body regions and the antennal nerves. In recent studies on the localization of [ 1251]-a-bungarotoxinbinding in sections of the sixth abdominal ganglion of Periplaneta arnericana an intriguingpatternof bindingsitesemerged (Sattelleetal. ,1981a). Two distinct areas

DAVID B. SATTELLE

242

Fig. 8 Distribution of [1251]-a-bungarotoxinbinding in the head of Drosophila melanogaster. The small arrow points to the antenna1 nerve running from the antenna into the brain. ( a ) The section is viewed by transmitted light. ( b ) This is the same field shown in ( a ) but viewed by dark-field illumination. From Schmidt-Nielsen et al. (1977)

of densely concentrated toxin-binding sites were located in the central neuropile on either side of the midline of the ganglion. This is the region of the ganglion in which cercal afferent fibres make synaptic contacts with the extensive dendritic brances of the giant interneurones. Densely concentrated toxin-binding sites were also detected in the periphery of the ganglion, a non-synaptic region occupied by glial cells and neuronal cell bodies.

3.2

P H A R M A c o LO G K

Schmidt-Nielsen et al. (1977) showed that binding of [12SI]-cy-bungarotoxin to frozen sections of Drosophila brain was blocked by preincubation with

ACETYLCHOLINE RECEPTORS OF INSECTS

243

1 x ~O-’M unlabelled toxin and 1 x 1 0 - 4 d-tubocurarine. ~ Pre-incubation ~ reduced binding but did not completely eliminate it. with 1 x 1 0 - 4 atropine Rudloff (1978) also showed that 1 x ~O-’M unlabelled a-bungarotoxin prevented toxin binding to sections. Dudai and Amsterdam (1977) noted that ~ prevented binding. pretreatment with 1x l W 3 nicotine In pharmacological studies designed to test the specificity of toxin binding to sections of the brain of Manduca sexta, Hildebrand and colleagues (1979) showed that 1 x 1 0 - 3 acetylcholine ~ in the presence of 1 X 1 0 - 3 neostig~ mine blocked toxin binding. d-Tubocurarine (1 X 1 0 - 3 ~ )completely ) blocked toxin binding whereas quinuclidinyl benzilate (1 x 10 - 3 ~ only slightly reduced toxin binding. Thus autoradiographic studies of the [1251]a-bungarotoxin binding sites in Drosophila melanogaster, Manduca sexta and Periplaneta americana reveal a distribution and pharmacological specificity consistent with the notion that they are acetylcholine receptors.

4

4.1

Efectrophysiological responses of neurones to cholinergic ligands EXPERIMENTAL APPROACHES

In order to ascribe a functional role to any of the putative acetylcholine receptors characterized by radiolabelled ligand binding techniques it is necessary (1) to show a comparable pharmacological specificity in vivo to that demonstrated in the binding studies and (2) in cases where a radiolabelled receptor-specific antagonist is used for receptor characterization (see Sections 2.3 and 2.4), to demonstrate a synaptic blocking action of the receptor probe. A variety of electrophysiological recording techniques have been utilized to monitor the actions of cholinergic ligands on insect central neurones. A brief consideration of experimental methods is an essential preliminary to a survey of the results of pharmacological experiments of this kind. External hook-electrode recordings (see for example Roeder, etal., 1947; Twarog and Roeder, 1957; Yamasaki and Narahashi, 1958,1960; Shanklandet al., 1971) and suction electrode recordings (for example Kerkut etal., 1969b) have been used to monitor synaptic transmission in insect ganglia. In all cases a large number of neurones contribute to the observed recording. Using such techniques it is difficult to obtain more than an estimate of the threshold concentration of a ligand that will produce a response and the response cannot be attributed to specific neurones. However, using sucrosegap recordings (Fig. 9) of excitatory postsynaptic potentials (EPSPs) and average postsynaptic polarization derived from a population of neurones,

244

DAVID B. SATTELLE

dose-response curves can be constructed for the actions of Iigands on cercal-afferent, giant-interneurone synaptic transmission in the cockroach (Callec and Sattelle, 1973). Although quantitative comparisons of the synaptic actions of ligands can be achieved by this technique, the responses can be ascribed only to a group of neurones. Experiments on multifibre preparations in conjunction with bath-application of drugs can therefore lead only to rather general conclusions concerning the pharmacological properties of receptors in such tissues.

Evoked Monosynaptic

EPSP

I2mv

-

toms

Fig. 9 Schematic representation of the oil-gap and sucrose-gap recordingtechniques. Excitatory postsynaptic potentials (EPSPs) evoked by the application of depolarizing pulses to nerve 11 (nXI) and recorded by these two methods are illustrated. Mechanical stimulation (MS) can be applied to single mechanoreceptor hairs. Electrical stimulation (ES) of many cercal mechanoreceptor afferents can be achieved by hook electrodes applied to nerve 1 1 . From Callec et al. (1980)

Studies on single invertebrate neurones, and in particular identified cells, have proved fruitful in the study of a variety of neurotransmitter-receptors ( c f . Gerschenfeld, 1973; Ascher and Kehoe, 1976). Two complementary experimental approaches to the pharmacology of single insect neurones were developed in the late 1960s. Boistel, Callec, and collaborators pioneered the microdissection of axons of giant interneurones (Fig. 10) and the oil-gap, single-fibre recording technique (cf. Boistel, 1968; Callec, 1972) for monitoring synaptic phenomena in single giant interneurones of the sixth abdominal ganglion of the cockroach Periplaneta americana. A t about the same time, Kerkut and collaborators (cf. Pitman 1971) made a detailed microelectrode study of the cholinergic sensitivity of certain nerve cell bodies (somata) of cockroach ganglia. The particular advantage of micro-

ACETYLCHOLINE RECEPTORS OF INSECTS a

-

245

b

300pm

Fig. 10 Stages in the microdissection of the axon of giant interneurone 2 of the cockroach Peripfaneta americana. ( a ) Ventral view of the isolated sixth abdominal ganglion. (b) From one of the paired connectives linking the fifth and sixth abdominal ganglia the nerve sheath is split and the cut ends retracted towards the ganglia. (c) The axon is isolated from adjacent fibres using fine stainless-steel needles. (d) The axon is dissected to within about 150 p m of the ganglion so that in the experimental chamber it is located close to the barrier between the oil and saline compartments. From Callec (1972)

electrode recording is that the precise site of recording can be defined. The method is widely applicable and has been extensively employed for investigating soma membrane chemosensitivity. The non-electrolyte gap techniques (oil-gap and sucrose-gap) enable prolonged, stable recordings of synaptic events, and in the case of the oil-gap technique (Fig. 9) these can be ascribed to a single identifiable postsynaptic neurone. Also, pre- and postsynaptic drug actions can be simultaneously assessed (Callec et al., 1980). However, these non-electrolyte gap techniques are less widely applicable,

DAVID 6.SATTELLE

246

TABLE 9 Comparison of nonelectrolyte-gap and microelectrode techniques for recording synaptic transmissionin the terminal abdominal ganglion of the cockroach (Peripfaneta americana). From Callec et al. (1980)

Properties Unitary EPSPs Unitary IPSPs Evoked - EPSP Evoked - IPSP Postsynaptic action potential

Manni tol-gap (whole or part Microelectrode Oil-gap of the (in giant fibre) (single giant fibre) connective) 0.7-3.2 mV 0.5-2 mV 15-20 mV 5 mV Up to 75 mV

0.5-2 mV 0.04-0.32 mV 3-8 mV up to 4 mV up to 115 mV

0.2-0.5 mV 0.15 mV 2-5 mV up to 1 mV up to 35 mV (compound response) 1.5-3 mV (giant axon)

although they are particularly well suited to investigations of the cercalafferent, giant-interneurone synapses of the cockroach. Table 9 compares some of the synaptic phenomena that can be recorded by different electrophysiological techniques from the sixth abdominal ganglion of the cockroach Periplaneta americana. Although most pharmacological studies have employed bath-application of cholinergic ligands to the cell or tissue under investigation, more recently localized application of ligands to individual neurones has been achieved using microsyringe application (Callec and Boistel, 1967) and microiontophoresis (Kerkut et a/..,l968,1969a, b; Pitman and Kerkut, 1970; Callec and Boistel, 1971; Callec 1972; David and Pitman, 1979; David, 1979; Goodman and Spitzer, 1979a, b; Sattelle et al., 1980). By these means, the substance under investigation is delivered to the cell from a micropipette. In the first case ejection is achieved by the application of a pressure pulse and in the second case ejection is induced by the application to the micropipette of rectangular current pulses of appropriate polarity. Before discussing in detail the results of electrophysiological experiments some consideration will be given to the limitations and advantage of the methods of ligand application. When pharmacological agents are bath applied to a ganglionic preparation, although the final concentration in the bath is known, this may depart from the true concentration at the cell surface. Also, all cells sensitive to the ligand will respond. Furthermore, appreciable time is required for equilibrium of the agonist concentration at the cholinoceptive membrane. This is

ACETYLCHOLINE RECEPTORS OF I N S E C T S

247

undoubtedly the cause of considerable desensitization which precludes repeated applications of test compounds (Fig. 11). The limitations imposed by this method of application to insect ganglia have been discussed in detail (Sattelle el al., 1976). A further possible problem resulting from bath application stems from the observations on electroplax (Karlin 1967) and 30

-20

a

I

I

I

-

> E

v

C

210 m

-

-

N

m 0

I

Concentration

(M)

Fig. 11 Dose-response data for carbamylcholine-induced depolarization recorded from the sixth abdominal ganglion of the cockroach Periplaneta americana. In ( a ) results of experiments on 18 ganglia are summarized. The mean depolarization for a particular dose is determined. Vertical bars show twice the standard error. In ( b ) , the upper curve ( 0 )is constructed by plotting results obtained from 13 ganglia which were tested once or twice only; the lower curve (0)is derived from an experiment in which a single ganglion was challenged successively by increasing concentrations of carbamylcholine. Between each test pulse repolarization was observed in normal Ringer. From Sattelle et al. (1976)

muscle (Jenkinson and Terrar, 1973) tissue that prolonged exposure to acetylcholine can cause ionic changes inside the cell. Nevertheless, when care is taken to eliminate physical and metabolic barriers that may restrict access of the ligand to the neuronal surface, it remains a valuable method for comparative studies since under these conditions dose-response data can be accumulated with the ligand dose expressed in terms of molarity. Localized application of a ligand to the cell body region of an individual neurone can be achieved via micropipettes. In the same way, ligands can be delivered to localized regions within the neuropile although clearly in this

DAVID

248

B. SATTELLE

case it is not possible to confine the actions to a single cell. By varying the duration of the pressure pulse (in the case of pressure ejection) or by changing the iontophoretic current amplitude (in the case of microiontophoresis) the amound of ligand discharged can be varied and a doseresponse curve constructed (see Fig. 12 for an example). By means of localized, brief applications of ligands many of the problems of desensitization that limit bath-application experiments can be avoided. Although only charged ligands can be applied by microiontophoresis, this restriction does not apply to pressure ejection via micropipettes. In the remainder of this section consideration is given to the results of experiments in which acetylcholine and related compounds are applied to insect neurones.

4.2

MULTIFIBRE FREPARATIONS

4.2.1 Actions of acetylcholine The relative insensitivity of ganglionic synaptic transmission in insects to bath-applied acetylcholine was consistently reported in the earlier pharmacological investigations using extracellular recording electrodes. Transmission across the cercal-afferent, giant-interneurone synapses of the sixth abdominal ganglion of Periplaneta americana, first described by Pumphrey and Rawdon-Smith (1937), was unaffected by acetylcholine at concentra~ ganglia with the nerve sheath removed (Roeder tions below 1.0 x 1 0 - 3 for et al., 1947; Roeder, 1948; Tawrog and Roeder, 1956, 1957). The same authors showed that the anticholinesterase agent eserine (1.0 x 1 0 - 6 ~ ) ~ desheathed ganglia. reduced the threshold concentration to 1.0 x 1 0 - 4for Yamasaki and Narahashi (1958, 1960) showed that the threshold concentration for the same preparation but with the nerve sheath intact was ~ pretreatment with eserine reduced from 1.0 x 1 0 - ’ ~to 1.Ox 1 0 - 3following (1.0 x 10%). Also, application of acetylcholine by perfusion or by direct addition to the saline surrounding the metathoracic ganglion of Periplaneta increased the spike activity recorded from the fifth thoracic nerve (Kerkut et al., 1969b). The threshold level for this action of acetylcholine was esti. studies on the grasshopper Crampsockis mated to be about 5.5 X 1 0 - 5 ~In buergeri (Suga and Katsuki, 1961), 5.5 x 1 0 - * ~acetylcholine produced excitatory effects on the auditory synapses of the prothoracic ganglion. It has been suggested that the nerve sheath surrounding the CNS presents a diffusion barrier to the penetration of acetylcholine (Twarog and Roeder, 1956; O’Brien, 1957; O’Brien and Fisher, 1958). Nevertheless Treherne and Smith (1965a) noted that radiolabelled acetylcholine rapidly penetrated nerve cords of the cockroach Periplaneta americana. The most likely explanation of the insensitivity of ganglia to bath-applied acetylcholine, an

ACETYLCHOLINE RECEPTORS OF I N S E C T S

IONTOPHORETIC

CURRENT

249

( nA

I

Fig. 12 Dose-response curve obtained by iontophoretic application of acetylcholine to a neuronal cell body in the sixth abdominal ganglion of the cockroach Periplanefa americana. The relationship between the amount of acetylcholine applied to the nerve cell (iontophoretic current) and the depolarization of the cell. Retaining current = 60 nA. Acetylcholine-filled electrode had a resistance of 12 M a . From Kerkut et al. (1969b)

insensitivity that persists even following removal of the nerve sheath, has been advanced by Smith and Treherne (1965) and Treherne and Smith (1965a, b). These authors proposed that the high concentration of cholinesterase in the CNS is responsible for the hydrolysis of most of the applied acetylcholine so that the final concentration in the extracellular spaces adjacent to cholinoceptive membranes in the CNS is much lower than the applied concentration. For example, Treherne and Smith (1965a) calculated that when the abdominal nerve cord was bathed in 1.0 X ~ O - * Macetylcholine, the extracellular concentration was approximately 8.1 x 1O+M. This notion of a biochemical barrier to the penetration of bath-applied acetylcholine is supported by the report from several laboratories that carbamylcholine, a cholinergic agonist that is not subject to hydrolysis by cholinesterase is more potent than acetylcholine when bath applied to intact ganglia (see Fig. 13). The first study that attempted to examine the detailed cholinergic pharmacology of the cercal-afferent, giant-interneurone synapses, giving due

30

I

I

I

I

I

I

I

1

ACE T Y LCHOI-INE j ACh) 0 ACh + ESERItdE 0 ACh . CURAFE 0

20 -

ACh 4 ATROPINE

mv 10 -

0

'

I 0

NICOTINE

a CARBACHOL Q

PILOCARPINE

20 -

M

Fig. 13 Actions of acetylcholine and various cholinergic agonists (bath applied) on synaptic transmission between cercal afferent fibres and giant interneurones in the sixth abdominal ganglion of the cockroach Periplanefa amerzcana. Ganglionic (postsynaptic) depolarization (in mV) recorded by the sucrose-gap technique is plotted against ligand concentration (M). Vertical bars represent twice the standard error of the mean. From Sattelle (1978)

consideration to the t (me required for ligand diffusion in the ganglion and the enzymic hydrolysis of bath-applied choline esters by endogenous cholinesterase, was that of Shanklandet al. (1971). These authors reasoned that the response of the synapses to bath applied acetylcholine and cholinergic drugs would be subject to interference by (1) endogenous transmitter, and (2) the degrading actions of endogenous cholinesterase in cases where the drug was susceptible to hydrolysis by this enzyme. Pretreatment of the desheathed sixth abdominal ganglion with hemicholinium-3 (which competitively inhibits choline transport in membranes - Birks and MacIntosh, 1961 - thereby blocking acetylcholine synthesis), and either dichlorvos or paraoxon (which are potent cholinesterase inhibitors O'Brien, 1960), followed by washing, resulted in preparations highly sensitive to bath-applied acetylcholine. Three quarters of these pretreated ganglia responded to 1.0 x 1 0 - 6 ~acetylcholine and Shankland er al. (1971)

ACETYLCHOLINE RECEPTORS

OF I N S E C T S

251

estimated the average minimal effective concentration to be between 1.0 X 1 0 - 7and ~ 1.O x 1 0 - 6 ~In. subsequent studiesusing the sucrose-gap recording technique, dose-response data were obtained for the actions of acetylcholine on cercal-afferent, giant-interneurone synaptic transmission in the sixth abdominal ganglion of Periplaneta americana (Callec and Sattelle, 1973; Sattelle etal., 1976).It was shown, for example, that pretreatment of the ganglion with 1.O x 1O-% eserine considerably shifted the dose-response curve for the post-synaptic depolarizing action of acetylcholine so that the preparation was consistently sensitive to 1.0 x 1 0 - 6 acetylcholine ~ (Fig. 13).

4.2.2 Actions of cholinergic ligands A variety of cholinergic agonists have been applied to insect central nervous tissue and their effects monitored using external-electrode recordings from multifibre preparations. Twarog and Roeder (1975) demonstrated that muscarine and pilocarpine when bath applied to the sixth abdominal gang~ 1.0 X 1 0 - 4 ~ lion of Periplaneta at concentrations in the range 1.0 x 1 0 - 3 evoked asynchronous bursts of spikes in giant interneurones. By contrast nicotine and acetyl-P-methylcholine (see Table 10) excited the same postsynaptic giant fibres at concentrations as low as 1.0 X ~ O - ' M (Flattum and Sternberg, 1970a, b; Shankland, et al., 1971; Flattum and Shankland, 1971). Urocanylcholine induced bursts of large and small spikes followed by a synaptic block in the same preparation (Twarog and Roeder, 1957). The metathoracic ganglion-fifth nerve preaparation of Periplanefa was also shown to be most sensitive to nicotinic agonists, the order of effectiveness being nicotine>carbamylcholine>pilocarpine>acetylcholine(Kerkut et al., 1969). The auditory synapses of Gampsocleis were stimulated by the perfusion of butyrylcholine (5.5 x ~O-'M) over the prothoracic ganglion (Suga and Katsuki, 1961). Using sucrose-gap techniques the actions of a variety of cholinergic agonists and antagonists were studies at cercal-afferent, giant-interneurone synapses of Periplaneta (Sattelle et al., 1976; Sattelle, 1978; Sattelle et al., 1980). Nicotine was the most potent of the ligands used (Fig. 13) producing postsynaptic depolarization at concentrations of 1.O x 1 0 - * ~ and above. Acetylcholine (in the presence of 1.O x 1 0 - 6 .eserine) ~ at 1.O x and higher concentrations also produced a postsynaptic depolarizing action (see Section 4.2.1). Carbamylcholine and pilocarpine were three to four orders of magnitude less effective than nicotine (Fig. 13). Several cholinergic antagonists have been shown to block transmission at the synapses in the sixth abdominal ganglion of Periplaneta americana. d-Tubocurarine a nicotinic antagonist which in earlier pharmacological

DAVID B. SATTELLE

252

TABLE 10 Actions of cholinergic ligands on synaptic transmission in the insect central nervous system using multifibre preparations Ligand

Nicotine Acetylcholine d-Tubocurarine Hexamethonium Acetyl-fl-methylcholine Carbamylcholine Decamethonium Atropine Pilocarpine

Threshold concentration (M) for minimal detectable response at cercal-nerve giant fibre synapses in the sixth abdominal ganglion of the cockroach Periplaneta americana Hook-electrodel recordings

Sucrose-gap2 recordings

10-8

10-8

10-7-

10-6

10-7 - I O - ~

10-6 10-8 10-~ 10-7 -

10-6 10-7 10-5 -

1o

-

-~

-

10-5

Shanklandetal. (1971) Sattelle (1978); Sattelle (1980)

studies had been reported to have no effect at 1.0 x ~ O - * M(Roeder, 1948), was more recently shown to suppress transmission at cercal-afferent, giantinterneurone synapses at concentrations as low as 1.0 X ~O-’M.(see Table 10). This same antagonist inhibited the excitatory effects of acetylcholine in the auditory synapses of Gampsocleis (Suga and Katsuki, 1961). Antagonism of the actions of applied acetylcholine by d-tubocurarine was noted at cercal-afferent, giant-interneurone synapses in the cockroach in both hook-electrode recording tests (Shankland et al., 1971) and sucrose-gap experiments (Sattelle, 1978). At high concentrations d-tubocurarine blocked conduction in cockroach axons (Friedman and Carlson, 1970). Atropine, an antagonist at vertebrate muscarinic receptors was reported to inhibit transmission at cercal-afferent, giant-interneurone synapses ~ al., 1971) and the at a threshold concentration of 1.0 x 1 0 - 7(Shanklandet same authors demonstrated its ability to suppress the excitatory effects of acetylcholine on the same preparation. Sucrose-gap experiments have also shown that atropine can completely block transmission at cercal-afferent, giant-interneurone synapses but only at much higher concentrations (1 .O X 1 0 - 4 ~ than ) required for d-tubocurarine block. Using the same method it was also shown that atropine was less effective than d-tubocurarine in displacing the dose-response curve for acetylcholine (Fig. 13). Recent sucrose-gap studies demonstrated that benzoquinonium

ACETYLCHOLINE RECEPTORS OF I N S E C T S

2 53

(1.0 x ~O-’M) and hexamethonium (1.0 x 1 0 - 3 ~will ) block cercal-afferent, giant-interneurone synaptic transmission (cf Sattelle, 1980). Decamethonium also blocked synaptic transmission across the terminal abdominal ganglion of the cockroach (Twarog and Roeder, 1957). Shankland et al. (1971) estimated that the concentrations required to block were 1.0 x 1 0 - 6in ~ the ~ the case of decamethonium. case of hexamethonium and 1.0 x 1 0 - 3 in Twarog and Roeder (1957) reported that tetramethylammonium (1.O X 1 0 - 3 ~ )hydroxyphenyltrimethylammonium , (1 .O x 10-*h1) and benzoquinonium (1.0 x 1 0 - 2 ~produced ) synaptic block in this preparation. The reasons for the discrepancies between the effective concentrations of hexamethonium and benzoquinonium reported by different laboratories has yet to be resolved. Only in the case of the sucrose-gap experiments, however, have dose-response curves for a series of cholinergic antagonists been prepared following bath-application for a fixed period of time (Fig. 14). The rank order of effectiveness of antagonists revealed in this way was as follows: a-bungarotoxin > d-tubocurarine > benzoquinonium > hexamethonium and atropine.

Fig. 14 Actions of cholincrgic antagonists on synaptic transmission between cercal-afferents and giant interneurones. Antagonists were bath applied and their effects on transmission were monitored by the sucrose-gap recording technique. Abbreviations: a-Bgt, a-bungarotoxin; d-TC, d-tubocurarine; Benzo, benzoquinonium; Hexa, hexamethonium; Atr, atropine. From Sattelle. (1980)

4.3

SINGLE NEURONES

This section is confined to a consideration of pharmacological data obtained in studies on single neurones which were not subsequently identified anatomically using single-cell marking methods.

DAVID B. SATTELLE

254

4.3.1 Actions of acetylcholine Depolarization accompanied by a decrease in membrane resistance (Rm) has been observed to follow bath-application of acetylcholine (3.0 X 3.0 x ~ O - ’ M ) to the desheathed sixth abdominal ganglion of Periplaneta, using an oil-gap, single-fibre technique to record from single giant interneurones (Fig. 15). In an attempt both to circumvent the peripheral

OlO

mV

mV

Time (min)

Fig. 15 Actions of bath applied acetylcholine on a giant interneurone in a desheathed sixth abdominal ganglion of the cockroach Periplaneta americana recorded using the oil-gap technique. The cell depolarizes in response to 3.0 x 1 0 - 3acetylcholine ~ (ACh), ultimately giving rise to a volley of action potentials. Acetylcholine-induced depolarization is accompanied by a progressive reduction in membrane resistance (Rm) and the amplitude of the monosynaptic EPSP evoked by contralateral electrical stimulation of ipsilateral nerve 11. Repolarization and recovery of the EPSP and Rm follow re-exposure to normal saline. Em = membrane potential. From Callec (1972)

cholinesterase barrier and to apply acetylcholine close to the synapses, Callec used microiontophoresis of acetylcholine (Callec and Boistel, 1971; Callec, 1972). With an acetylcholine-filled electrode located in the dendritic field of an interneurone, the injection of acetylcholine induced a transitory depolarization of the giant interneurone (Fig. 16). The response obtained increased as the applied current, and hence the quantity of acetylcholine discharged, was increased. This acetylcholine-induced depolarization was accompanied by a decrease in the membrane resistance of the giant interneurone. During the acetylcholine potential, the EPSPs obtained by stimulation of cercal nerve 11 decreased in amplitude, with a corresponding decrease in Rm (Fig. 16). Changes in amplitude of both the EPSP and the acetylcholine-induced potential as a function of the polarizing current were also investigated (Fig. 17). With hyperpolarizing current the acetylcholine potential increased in amplitude, but when depolarizing currents were applied it decreased (Callec, 1974). These changes followed closely the changes in the EPSP. Both responses have similar reversal potentials and are therefore likely to be supported by the same ionic currents. Reversal potentials for the EPSP and the acetylcholine potential of around -35mV were

ACETYLCHOLINE RECEPTORS OF I N S E C T S

255

A mV 7-

6-

-z

.-

5 -

C

+

2

4 -

1

0 Q

3-

't!

-

AC h

500 m s

I

100

200

I

300

I

I

400

500

~~~

600

nA

B 1

Fig. 16 Effects on a giant interneurone of iontophoretic injection of acetylcholine in the region of neuropile in the sixth abdominal ganglion of Periplaneta containing part of the cell's dendritic tree. Oil-gap, single-fibre recordings are used to show: (A), the relation between the amplitude of the acetylcholine-induced potential response (see insert) and the injection current (nA); (B,), that during the application of acetylcholine the monosynaptic EPSP evoked by electrical stimulation of nerve 11, which was initially subthreshold, is now able t o generate an action potential; (BJ, the reduction in membrane resistance (Rm) during the acetylcholineinduced potential. From Callec (1972)

estimated by extrapolation (Callec, 1974). Evidence available to date therefore has established that acetylcholine has a depolarizing action which is accompanied by an increase in the conductance of the postsynaptic membrane. It also appears that the acetylcholine potential and the EPSP originate from the same ionic currents. The ionic basis of these synaptic currents remains to be explored.

D A V I D B. SATTELLE

256 mV 14, 0

0

12 ' 0

--

0 .

oo2\

ACh

-

I2OnA

300ms

O

Em.

Fig. 17 Relation between the amplitude of the monosynaptic EPSP (evoked by ipsilateral electrical stimulation of cercal nerve 11) and the amplitude of the iontophoretic acetylcholine response. Data were obtained from the same giant interneurone in the sixth abdominal ganglion of the cockroach (Periplunetu umericuna) at a variety of different membrane potentials Em. The amplitude of the response to iontophoretically applied acetylcholine was initially adjusted to be equal to the EPSP amplitude. From Callec (1972)

Excitatory postsynaptic potentials have also been recorded from insect neuronal somata (Hagiwara and Watanabe, 1956, Kerkut ef al., 1969a, b, Crossman ef al., 1971). The cell bodies of insect neurones are not in synaptic contact with presynaptic fibres. Interpreting the data obtained on nerve cell bodies therefore requires caution in that the effects of any given cholinergic ligand on the neuronal cell body membrane may differ from its actions on synaptic membranes. In 1967 Callec and Boistel impaled cell bodies located near the dorsal midline of the cockroach sixth abdominal ganglion. These authors showed that previously silent and autoactive cell bodies responded to the localized application of acetylcholine at concentrations above 5 x 1 0 - 6by ~ membrane depolarization and an associated increase in action potential activity. Later it was shown that iontophoretic ejection of acetylcholine from a microelectrode located close to a recording microelectrode inserted into a nerve cell body of the same region of the sixth abdominal ganglion (Kerkut et al., 1968; 1969a, b; Pitman and Kerkut, 1970) resulted in a transient

2 57

ACETYLCHOLINE RECEPTORS OF I N S E C T S

depolarization (Fig. 18). The acetylcholine response and the EPSP evoked by stimulating the right anterior connective both increased in amplitude when the membrane potential was increased. Based on these observations, Pitman and Kerkut (1970) estimated, by extrapolation of plots of the amplitude of transient responses versus membrane potential, reversal potentials of -45.3 t 3 . 1 mV for the EPSP and -40.3 -+ 1.6 mV for the acetylcholine depolarization, These findings suggested that the same ionic currents were involved in both responses. Kerkutetal. (1969b) also showed that acetylcholine-induced depolarization of the soma membrane was reduced in the absence of external sodium ions (Fig. 19), pointing to a role for this cation in the generation of the EPSP. The threshold concentration of acetylcholine required to produce a response was estimated by extrapolating the depolarization amplitude-iontophoretic dose curve (Kerkut et al., 1969a, b). A value of 1.31 x 1 0 - 1 3 was ~ obtained. This resembles the value obtained for snail neurones and is not far removed from the value of 1 0 - l ' ~ reported for the vertebrate neuromuscular junction (del Castillo and Katz, 1955). b

a

-

lJ

66

-761

--93 831

,

20s

-86] -96

\ \

c

i

41

-

030

O - k -

M.P. mV

-50

-70

-90

-110

M.P. mV

Fig. 18 The effects on (u) the EPSP driven through the right anterior connective and (b) the iontophoretic acetylcholine response of hyperpolarizing the membrane of a neuronal cell body of the cockroach sixth abdominal ganglion in 10 mV steps (c) shows a plot of the amplitude of the EPSP against the membrane potential of the cell. The EPSP reversal potential is -44 m V ( d ) shows a graph of the amplitude of the iontophoretic acetylcholine (ACh) response against the membrane potential of the cell. The acetylcholine reversal potential is -38 mV. From Pitman and Kerkut (1970)

2 58

DAVID B . SATTELl F

a

I 5OOpg

ACh.

b

10 min

Na+ FREE

C

/ I

,

"I

1 I

1

10 rnin WASH

Fig. 19 Effects of reducing the external sodium concentration on the response of a neuronal cell body to acetylcholine. Removal of external sodium reduced the effect of acetylcholine. From Kerkut et al. (1969b)

4.3.2 Actions of cholinergic ligands The application of nicotine to the desheathed sixth abdominal ganglion of PeripEaneta resulted in a rapid depolarization of a single giant interneurone at concentrations of 1.0 x 1 0 - 6 to ~ 4.0 x 1 O w 6 (Callec ~ 1972). In the same study it was noted that acetyl-8-methylcholine depolarized a single giant interneurone at concentrations of 5.0 x 1 0 - 3 ~to 3.0 x ~ O - ' M . d-Tubocurarine (1.0 x ~O-'M to 8.0 x 1 0 - 4 ~induced ) a progressive decline

A C E T Y L C H O L I N E RECEPTORS OF I N S E C T S

259

in the unitary and evoked EPSP at cercal-afferent, giant-interneurone synapses. The blocking action of d-tubocurarine was reversible and did not result in a significant change in either the membrane potential or the resistance of the postsynaptic cell. If acetylcholine is the neurotransmitter released by the excitatory cercal nerve terminals then it would be predicted that antagonists would have similar effects on the potentials caused by the natural transmitter and those induced by artificially injected acetylcholine. ) observed to progressively Bath applied d-tubocurarine (7.0 x 1 0 - 4 ~was block both the monosynaptic EPSP recorded from a giant interneurone in response to the stimulation of cercal nerve 11 and the acetylcholine potential resulting from the microapplication of acetylcholine into the neuropile at a depth of 210 p m below the ganglion surface (Fig. 20). Both potentials

RI

5

4 2 nin

6

53mn

7

I

9 2 rnn

Fig. 20 Actions of d-tubocurarine (d.TC) on a giant interneurone in a desheathed sixth abdominal ganglion of Periplanetu arnericunu. Using the oil-gap technique changes in the monosynaptic EPSP evoked by stimulation of ipsilateral nerve 11 and the depolarization resulting from iontophoretic application of acetylcholine into the dendritic tree region are followed in a single giant interneurone during the bath application of d-tubocurarine (7.0 x 1 0 - 4 ~ )The . two responses decline at approximately the same rates. Times in minutes after exposure to test solution are shown. Traces 1 and 8 were obtained with the preparation bathed in normal saline (Ri). Traces 2-7 show progressive blocking actions of d-tubocararine. From Callec (1972)

were largely reversible when the ganglion was rebathed in normal saline. The uniformity of action of d-tubocurarine on the EPSP and the acetylcholine potential indicated that the same postsynaptic receptors were involved in both cases. Atropine, a classic muscarinic antagonist, also blocked cercal-afferent, giant-interneurone synaptic transmission in Periplaneta (Callec and Boistel 1971c; Callec, 1972). Effective concentrations

DAVID B. SATTELLE

260

required for complete block (2.0 x - 1.0 x 1 0 - 3 ~were ) higher than those required for d-tubocurarine. The acetylcholine potential was also blocked by these rather high concentrations of atropine. A comparative study of the effects of various cholinergic ligands has been made on cell bodies of Peripfaneta central neurones. The following sequence of effectiveness was detected: nicotine > carbamylcholine > pilocarpine > acetylcholine (Kerkut et ~ l . 1969b). , In dissociated neuronal cell bodies isolated from the thoracic ganglia of adult locusts (Schistocerca gregaria), membrane depolarization resulted from bath application of acetylcholine at concentrations above 1.0 x 1 0 - 6 ~ (Holden et al., 1977). Iontophoretic application of acetylcholine also depolarized the isolated neurones and this response was antagonized by d-tubocurarine and atropine. Although there appear to be broad similarities in their sensitivity to cholinergic ligands between cell body (extrasynaptic) membranes and synaptic membranes, to date a direct comparison of the properties of synaptic and extrasynaptic receptors of a particular insect neurone has not been performed. 4.4

S I N G L E IDENTIFIEI) NEURONES

A limited number of studies have recently been initiated aimed at characterizing the pharmacological properties of single identified neurones in the insect central nervous system. Giant interneurones 2 and 3 of the sixth abdominal ganglion of Peripfaneta americana and the fast (Df) and slow (D,) coxal depressor motoneurones of the third thoracic ganglion of the same insect, have been investigated. 4.4.1

Giant interneurones

Using a novel, single-axon backfill technique, selective cobalt staining of individual giant interneurones in the sixth abdominal ganglion of the cockroachPeripfanetaamericanahas beenachieved (Harrowetaf., 1979; 1980a,b). Each of the three giant interneurones (GI 1-3) investigated has unique morphological features (Fig. 21). Cell body position, neurite shape, dendritic branching pattern, the presence or absence of an axon collateral and the characteristic position of the axon in sections of the fifth abdominal ganglion provide anatomical criteria for identifying giant interneurones (Harris and Smyth, 1971; Camhi, 1976; Harrow et al., 1980a). Since it is possible to select by dissection one of these three giant interneurones it is possible to complement neuroanatornical studies with pharmacological studies of synaptic transmission in selected, identified interneurones using the oil-gap, single-fibre recording technique. In this way it has been shown, for giant interneurone 3, that a-bungarotoxin (1 .O x ~O-'M)when bath applied to the

261

ACETYLCHOLINE RECEPTORS OF I N S E C T S

b

Y I

,

\

Fig. 21 Morphology of giant interneurones of the cockroach (Periplaneta americana, L.). ( a ) Camera lucida drawing of a section through the fifth abdominal ganglion showing the relative positions of giant axons. (b-d) Camera lucida representations of cobalt-backfilled, silverintensified, single, giant interneurones in the sixth abdominal ganglion: ( b ) GI 1; (c) GI 2; ( d ) GI 3. ( e ) Photograph of the sixth abdominal ganglion containing the cobalt-filled giant interneurone (GI 3) prior to intensification. Scale bar represents 100 pm.From Harrowetal. (1980a)

DAVID 6.SATTELLE

262

desheathed sixth abdominal ganglion, completely and irreversibly blocks unitary excitatory post-synaptic potentials (EPSPs) recorded in response to the deflection of a single cercal mechanoreceptor (Fig. 22). Cobaltbackfilling (Harrowetal., 1979) confirmed that giant interneurone 3 was the SALINE 0 rnin

W-Bgt

30 rnin

SALINE after 210 min wash

Fig. 22 ( a ) The unitary EPSP recorded from giant interneurone 3 (GI 3) in response to mechanical stimulation of a single cercal mechanoreceptor is irreversibly blocked by a-bungarotoxin (I x ~ O - * M ) . ( b ) The EPSP evoked by electrical stimulation of nerve 11 and recorded from GI 3 is irreversibly blocked by 1 X lo-% a-bungarotoxin. ( a ) from Harrower a l . (1979); ( b ) from Harrow efal. (1980b)

postsynaptic cell under investigation (Fig. 23). Using the same combination of techniques, it was shown for giant interneurone 2 that both the unitary EPSP and the EPSP evoked by electrical stimulation of cercal nerve 11were completely blocked by 1.O x 1 0 - * a-bungarotoxin ~ (Sattelleetal., 1981). This neurotoxin is a specific, irreversible antagonist of vertebrate peripheral cholinergic receptors and some vertebrate central cholinergic receptors (Heidmann and Changeux, 1978). The blocking action of a-bungarotoxin at cockroach synapses at which nicotinic agonists and antagonists are particularly active, and at concentrations close to the KDs(see Table 11) estimated for binding to various insect extracts, provides strong evidence for a functional role in synaptic transmission of the membrane component that binds a-bungarotoxin. 4.4.2

Fast coxal depressor motoneurone

The fast coxal depressor motoneurone of the third thoracic ganglion of the cockroach Periplaneta americana, numbered cell 28 by Cohen and Jacklett (1967), was designated D, by Pearson and lles (1970). Located on the ventral surface of the ganglion, the cell body of this motoneurone is 8 0 p m in diameter and can readily be located visually in the whole ganglion. This cell

-

ACETYLCHOLINE RECEPTORS OF I N S E C T S

263

innervates the coxal depressor muscles 177d, 177e, 178 and 179 through the fifth ganglionic nerve trunk (Carbonell, 1947, Pearson and Iles, 1970). Recently, David and Pitman (1979) studied the sensitivity of the cell body membrane to both bath-applied and iontophoretically-applied acetyl-

lo&m

Fig. 23 Camera-lucida drawing of a cobalt-stained, silver-intensified, giant interneurone of the sixth abdominal ganglion of Peripfaneta americana following an experiment in which all cercal afferent input to the cell via nerve 11 was blocked by 1.0 X 1 0 - ' ~a-bungarotoxin. The criteria of cell body position, neurite shape, major dendritic branching pattern and the position of the axon in sections of the fifth abdominal ganglion confirmed that the cell under test was GI 3. From Harrow et al. (1 979)

choline. Depolarization of the cell membrane followed the application of acetylcholine and repeated applications produced desensitization of the response. The sensitivity of D, cell bodies to acetylcholine (David, 1979) appeared to be similar to that of the dorsal unpaired median (DUM) cells of the metathoracic and sixth abdominal ganglia reported earlier (Kerkut er al., 1969a, b; Pitman and Kerkut, 1970). Carbamylcholine also depolarized the cell body membrane when locally applied to D, using iontophoretic techniques (David, 1979). Axotomy did not result in a change in sensitivity to carbomylcholine (David and Pitman, 1979). These authors showed that the anticholinesterases (physoconcentrations produced up stigmine and neostigmine) at 1.0 x 1 0 - 7 ~ to a 1000-fold potentiation of the normal acetylcholine response indicating the presence of large amounts of cholinesterase near the soma membrane. It was also concluded that the increased acetylcholine sensitivity of axotomized Df motoneurones may in part be attributable to a fall in the activity of acetylcholinesterase at the cell surface. Recent experiments on D r (Sattelle et al., 1980) demonstrated that a-bungarotoxin (5.0 x ~O-'M)irreversibly reduced the sensitivity of this cell to iontophoretically applied acetylcholine without any appreciable change in the membrane resistance and resting potential (Fig. 24). Thus it appears that in Periplaneta there are

DAVID

264

B. SATTELLE

16

-> E

v

r e 0 .c

m

N ._ I

m 0

a a,

n

C 1

10

102

I 103 1 o4 Charge ( n C )

Fig. 24 a-Bungarotoxin suppresses the response to acetylcholine of the cell body membrane of the right coxal depressor motoneurone (Df) of the metathoracic ganglion of Periplaneta americana. As shown in the upper traces a-bungarotoxin (5.0 x lo-%) substantially reduced the depolarizing response of the cell body membrane to iontophoretically applied acetylcholine. Changes in the dose-response curve are shown following a 2 h exposure to a-bungarotoxin. From Sattelle et al. (1980)

acetylcholine receptors on the soma membrane of Dfwhich in their sensitivity to a-bungarotoxin resemble the receptors mediating cercal-afferent, giantinternpiirnne cvnantir- trancmiccinn in t h e ciyth ahclnminal onnolinn

4.4.3

Trochanteral hairplate-to-rnotoneurone D,reflex

The pharmacological properties of the monosynaptic connection between the trochanteral hair plate afferents and motoneurone D, of the metathoracic ganglion of Periplaneta americana (Fourtner et al., 1978) were investigated by Carr and Fourtner (1978). Electrical stimulation of the

ACETYLCHOLINE RECEPTORS

OF I N S E C T S

265

hairplate produced a 1:1 reflex activation of D,. Changes in the D, reflexresponse to stimulation and changes in the level of activity in D, were monitored during perfusion of cholinergic ligands. A variety of antagonists including a-bungarotoxin, nicotine, hexamethonium and atropine blocked synaptic transmission at this central synapse. 4.4.4

Dorsal unpaired median (DUM) neurones

Goodman and Spitzer (1979a) have examined the sensitivity of the cell bodies of identified embryonic DUM neurones in the grasshopper Schistocerca nitens to bath application and iontophoresis of various cholinergic ligands. The iontophoretic application of acetylcholine resulted in a transitory depolarization of the soma membrane. For the acetylcholine response, a reversal potential of +20 mV was estimated by extrapolation. The acetylcholine-induced depolarizations were blocked following the substitution of sodium in the physiological saline by choline and were reduced by the bath application of the nicotinic antagonists d-tubocurarine (1.O X 1 0 - 4 ~ ) , ) decamethonium (1.0 x lo-’~).Nicotine hexamethonium (1.0 x 1 0 - 3 ~and was found to be an agonist, but a-bungarotoxin (1.0 x 1 0 - 6 ~failed ) to block the acetylcholine response. The muscarinic antagonists quinuclidinyl benzi) atropine ( 1 x lo-%) were also without effect. Thus, late (1.0 x 1 0 - 7 ~and although the cholinergic receptors on embryonic DUM cell somata of Schistocerca nitens appear to be nicotinic in nature, they differ from the receptors on D r motoneurone cell bodies of adult Periplaneta americana in their sinsitivity to a-bungarotoxin (see also Section 4.4.2). In recent studies on DUM cells of the metathoracic ganglion of adult Periplaneta, a relative insensitivity to a-bungarotoxin has been noted (Sattelle et af., 1980) similar to that reported for the embryonic DUM cells of Schistocerca (Goodman and Spitzer, 1979a). However, whereas the responses to acetylcholine of the embryonic DUM neurone cell bodies (Schistocerca) did not desensitize (Goodman and Spitzer, 1979a), desenstization was always noted in the case of the acetylcholine-induced responses of adult DUM neurone cell bodies (Periplaneta) (Kerkut et al., 1969a, b). Clearly a more detailed comparison of the acetylcholine sensitivity of embryonic and postembryonic DUM neurones of a single species would be desirable.

5 Comparative pharmacology of CNS acetylcholine receptors Having surveyed the results of biochemical, localization and electrophysiological studies, it is now possible to ask whether or not a physiological role can be ascribed to any of the three putative acetylcholine receptors

DAVID B. SATTELLE

266

characterized by radiolabelled ligand-binding techniques. Having attempted this, it will then be appropriate to compare the pharmacological properties of insect acetylcholine receptors with acetylcholine receptors of other invertebrates and vertebrates. 5.1

INSECTS

Three putative acetylcholine receptors have been reported in insect central nervous tissue (see Section 2). As a first step in considering a possible physiological role €or one or more of these receptors, we have compared in Table 11 the sedimentation properties of these putative receptors and the density of binding sites. Clearly the bulk of the [1251]-a-bungarotoxin binding sites and many of the [3H]-quinuclidinyl benzilate binding sites sediment in membrane or particulate fractions (pelleted at 20 00040 000 xg). Also, the difficulties noted in solubilizing these binding components indicate that they are tightly bound (intrinsic) to the membrane as would be expected for receptors. The density of binding sites defined by these two receptor probes is within the range reported for other tissues such as vertebrate CNS (Snyder and Bennett, 1976) and electroplax tissues (Changeux et al., 1970). The main difference is that whereas in vertebrate brain [3H]-quinuclidinyl benzilate (muscarinic) binding sites are more abundant by one or two orders of magnitude than the ['251]-abungarotoxin-binding sites (Yamamura et al., 1974; Segal et al., 1978), the reverse appears to be the case in the one insect preparation (Drosophila rnelanogaster) for which comparative data is available (Dudai, 1978; Haimet al., 1979). The extremely high density of binding sites defined by various reversible radiolabelled ligands in Musca heads (Eldefrawi et al., 1971; Jewess et al., 1975) and the solubilization of the cholinergic binding molecules by homogenization in water distinguishes this putative receptor from all other putative cholinergic receptors. Of the three putative acetylcholine receptors reported in insect central nervous tissues (see Section 2) only in the case of the abungarotoxin-sensitive receptor is there substantial evidence for a physiological role in synaptic transmission. Although the [ '2'I]-a-bungarotoxin-binding component has been most fully characterized in Drosophila rnelanogaster, the evidence that it is a constituent of a synaptic acetylcholine receptor stems largely from experiments on Periplanetu arnericana, the central nervous system of which has proved amenable to both radiolabelled ligand binding studies and single-cell electrophysiology. The evidence can be summarized as follows: (a) Extracts from abdominal nerve cords of Periplaneta contain a compowith many of the expected nent of specific [ 1251]-a-~bungarotoxin-binding

TABLE 11 Comparison of putative CNS acetylcholine receptors from different insects

z

a-Bungarotoxin binding parameters

rn

n Insect preparation Periplaneta americana (abdominal nerve cord extracts) Drosophila rnelanogasfer (whole fly extracts)

Receptor probe

ki k-, K D = k - , / k , pmoles (lo-'%) binding sites (lO5W1s-') (lo-%')

[~2511-~-

5.7

Irreversible binding

112

bungarotoxin

76% pelleted after 40000xg for 30 min

0 rn W

+

Gepner er al. (1978)

V)

Sattelle (1980)

51 z

[ q ~ - ~ 7.7 -

[w-

-

6.2

0.81

10

-

0.21

88

a-Bungarotoxin does not bind

6.5

a-Bungarotoxin does not bind

3000

decame ttronium (head extracts)

Reference

0 Y

bungarotoxin (head extracts treated with [1251]-aTriton X-100) bungarotoxin (head extracts) ['HIquinuclidinyl benzilate Musca domestica (head extracts)

Sedimentation properties

[12511-~-

bungarotoxin

89% pelleted after 40000xg for 30 min not done 48% pelleted after 20 000% for 20 min

In supernatant after 100 OOOxg for 60 min

0.3

2.07

6.8

-

Schmidt-Nielsen ef a l . (1977), Gepner (1979) Schmidt-Nielsen ef al. (1977) Haimetal. (1979)

Mansour et al. (1977). Eldefrawi et al. ( 1 971 b) Harriser al. (1979)

V)

rn

v)

268

D A V I D 6.SATTELLE

properties of an acetylcholine receptor. For example, toxin-binding is saturable and sediments in a membrane fraction. Nicotine and d-tubocurarine are highly effective at displacing a-bungarotoxin from its binding site, whereas atropine and pilocarpine are much less effective. ( b ) Nicotinic ligands are similarly more effective in modifying synaptic transmission between cercal afferent neurones and giant-interneurones in the sixth abdominal ganglion of Periplunetu. In fact, a close correspondence has been reported for the pharmacological specificity of the [1251]-abungarotoxin-binding component and the pharmacological specificity of cercal-afferent, giant-interneurone synapses. This is illustrated in Table 12 in which the ligand concentrations required for inhibition by 50% of the binding [1251]-a-bungarotoxinare compared to concentrations estimated to produce half-maximal physiological actions at cercal-afferent, giantinterneurone synapses. ( c ) The most effective blocking agent of all those tested to date for their ability to block cercal-afferent, giant-interneurone synapses has proved to be a-bungarotoxin, which completely blocks transmission at nanomolar concentrations. This is close to values for the K D estimated from toxinbinding studies on Drosophilu (Schmidt-Nielsen et ul., 1977; Gepner, 1979). No K Dvalues are available for Periplunetu since toxin bound essentially irreversibly but from saturability experiments it was noted that half~ (see Fig. 6). maximal binding was achieved at 1.1 x 1 0 - 9 a-bungarotoxin ( d ) Autoradiographic studies have shown specific binding of [1751]-abungarotoxin in the neuropile of the sixth abdominal ganglion, the region known to contain the cercal-afferent, giant-interneurone synapses. Thus there is strong evidence for a functional role in synaptic transmission at Peripfunetu cercal-afferent, giant-interneurone synapses of an acetylcholine receptor for which a-bungarotoxin is a specific irreversible probe. Recent microelectrode studies on the cell body membrane of the coxal depressor motoneurone (Df) of the metathoracic ganglion of Periplunetu have shown that the depolarizing response to iontophoretically applied acetylcholine can be largely suppressed by the bath application of 5.0 x ~ O - * M a-bungarotoxin (Sattelle et ul., 1980). This provides further evidence for the existence of a neuronal a-bungarotoxin-sensitive acetylcholine receptor. Dudai and collaborators (1980) have shown that cholinergic drugs administered to Drosophilu by injection could result in the death of the insect. When LD5{,values were compared, it was clear that nicotinic ligands were far more active than muscarinic drugs, but the results of such whole-animal responses to injected ligands are difficult to interpret in terms of possible receptor actions of the ligands. A physiological role for the other two putative acetylcholine receptors of insects has yet to be demonstrated. It seems unlikely that these putative

,

D 0 rn -I

s 0

I

-

TABLE 12 Comparative pharmacology of insect cholinergic receptors as determined electrophysiologically and by binding studies

z

Concentration (M)for 50%of maximum effect

0

m

P Ligand

Penpianera amncana '

Peripianera amencana '

Electrophysiology

["'I]-a-Bungarotoxin hindlng

~

~

0.8 x 0.3 X '1.3 x 5.0 x 15.0 x 6.6 X 5.4 x

lo-* lo-'' lo-"

lo-'

['i~I]-a-Bungarotox~n binding (15")

(150)

Nicotine d-Tubocurarine Acetylcholine Carbamylcholine Decamethonium Pilocarpine Atropine PrBCIvf'

Drosophila mclanogrrrter'

2.7 x lo-" 2.4 x 10-7 '8.3 x lo-" 1.0 x 10-4 1.7~10-~ 2.1 x 10-5 7.2 x 10-5

4.5 x lo-' '2.8 x lo-'' 1.2 x 10-4 2.5 x 7.9 x low 5.7 x lo-'

rn

Musca domesrrca'

Musca domesrxa'

Drosophila meianogasfer'

Labelled ligand

['HI-Decamethonium

[1H]-Qumuclidmyl

binding

binding

bemilate binding

( K Oor K,)

(15")

3.0 - 4.4 X 1.6 X lo-' -d5.0

X

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~

~

" + eserine ( 10-6M); + neOStigmine (10-'M); '+ neostigmine (lo-'#); + paraoxon (1W'M); ' + diethylfluorophosphate lo-'^); 'PrBCM = propylbenzilylcholinemustard 'Sattelle (1978; 1980) *Schmidt-Nielsenet al. (1977) 'Eldefrawi et al. (1971b), Aziz and Eldefrawi (1973); Jewess et al. (1975) Tattell et al. (1979) 'Dudai and Ben-Barak (1977); Haim et al. (1979)

(ED,,

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270

DAVID 6 . SATTELLE

receptors contribute substantially to acetylcholine-mediated transmission at cercal-afferent, giant-interneurone synapses since both the muscarinic receptor (characterized by [3H]-quinuclidinyl benzilate binding) and the mixed receptor (characterized by reversible ligand binding) are unaffected by micromolar a-bungarotoxin (Mansouretal., 1977; Haimet al., 1979). By contrast, cercal-afferent, giant interneurone synaptic transmission is completely blocked by nanomolar toxin concentrations (Harrow et al., 1980b). Nevertheless, some support for the existence of a-bungarotoxin-insensitive acetylcholine receptors on certain insect neurones has emerged from recent electrophysiological experiments. For example. DUM neurone cell bodies of the grasshopper (Schistocerca nitens) and cockroach (Periplaneta arnericana) metathoracic ganglia are sensitive to acetylcholine applied by microiontophoresis, but the resulting depolarization is not blocked by 10 - 6 ~ a-bungarotoxin (Goodman and Spitzer 1979a; Sattelleetal., 1980). Further studies are needed to define the detailed pharmacological specificity of these acetylcholine responses of insect neurones which are relatively insensitive to a-bungarotoxin. In this context, it is of interest to note that Greenspan et al. (cited in Dudai, 1980) have shown that Drosophila mosaics with acetylcholinesterase-less mutant tissue in the lamina lack a synaptic component of the electroretinogram - the “off” transient. Dudai (1980) obtained the same modification of the electroretinogram by feeding flies with the acetylcholinesterase inhibitor neostigmine. Since the lamina contains very few [L251]-a-bungarotoxinbinding sites, it may be that a-bungarotoxin-insensitive receptors mediate the “off” response recorded from the retina. Confirmation of the identity of putative insect acetylcholine receptors by immunological methods would be highly desirable and work in this area has recently been initiated (see Eldefrawi and Eldefrawi, 1980). Several antibodies form against the nicotinic acetylcholine receptor purified from Torpedo (Lindstrom et al., 1977; Eldefrawi, 1978; Gomez, et al., 1979) all of which react with the receptor antigen to precipitate it from solution. In double-diffusion tests Mansour et al. (1977) compared the electric-organ receptor with the housefly putative receptor purified from high-speed supernatants (see Section 2.2). No line appeared between the purified Torpedo receptor and the purified housefly cholinergic binding proteins (Fig. 25a). Sharp lines did appear between the well containing a 1:1mixture of antisera against the pure Torpedo and housefly proteins and the two wells containing their respective antigens (Fig. 25b). However, these lines did not connect smoothly indicating that the antigenic determinants were distinct. The same laboratory employed a radioimmune assay to test for crossreactivity between Torpedo acetylcholine receptor and the Triton-extracted [‘251]-a-bungarotoxin-binding component from houseflies (Eldefrawi and

ACETYLCHOLINE RECEPTORS OF I N S E C T S

27 1

Eldefrawi, 1980). No such cross-reaction was detachable. On the other hand, the sheep antiserum against Torpedo nicotinic receptor did precipitate the [1251]-a-bungarotoxinlabelled Torpedo receptor. These authors concluded that the toxin-binding proteins of Torpedo electroplax and housefly heads differ at least in respect of the antigenic site(s) against which these antibodies are produced.

Fig. 25 Double diffusion assay of ( a ) rabbit antisera for the isolated housefly proteins (bottom well) against the two isolated housefly proteins (top left well) and the high-speed (100 000 x g) supernatant extract S , (top right well) and ( b ) 1: 1 mixture of rabbit antisera for the purified housefly and Torpedo ACh receptor (bottom well) against the purified Torpedo ACh receptor (top left well) and the two isolated housefly proteins (top right well). From Mansour et al. (1977)

Thus biochemical and electrophysiological experiments have shown that several insect species (Drosophila melanogaster, Musca domestica and Periplaneta arnericana) possess more than one type of CNS putative acetylcholine receptor. Evidence has been presented for a physiological role in synaptic transmission of an acetylcholine receptor for which nicotine and acetylcholine are potent agonists and d-tubocurarine and a-bungarotoxin are potent antagonists.

5.2

INVERTEBRATES OTHER THAN INSECTS

In this section the properties of acetylcholine receptors of the central nervous tissues of invertebrate organisms other than insects are considered. Emphasis will be given where possible to those groups of organisms for which both biochemical and electrophysiological data are available. Few data are available for the Annelida although it has been established that acetylcholine (Kerkut etal., 1970; Kerkut and Walker, 1967); and nicotinic agonists (Woodruff et al., 1971) depolarize and excite the cell body membrane of Retzius cells in the central nervous system of the leech Hirudo

272

D A V I D 6.SATTELLE

medicinalis. The excitatory action of acetylcholine was blocked by benzoquinonium. Muscarinic agonists at low concentrations were able to produce inhibition but at higher concentrations their application resulted in excitation (Woodruff, et af., 1971). a-Bungarotoxin was ineffective in blocking the acetylcholine-induced depolarization of Retzius cells (Magazanik, 1976). Sargent et al. (1977) showed that whereas cell body membranes of pressure-sensitive (P) and nociceptive (N) sensory neurones of the CNS of Hirudo were depolarized by acetylcholine, the touch sensitive (T) cells exhibited a biphasic response (depolarization followed by hyperpolarization). Of the motoneurones investigated, the A E cells which control the formation of skin ridges were hyperpolarized by acetylcholine, but in the L neurones which control shortening of the animal a biphasic response was elicited. Nicotine and d-tubocurarine were highly effective ligands at the cholinergic receptors of N cells. Signhcant differences were noted between extrasynaptic (cell body) and synaptic receptors in respect of their sensitivity to d-tubocurarine - the extrasynaptic receptors being much more sensitive. No radiolabelled ligand binding studies have been performed to date on annelid central nervous tissues. The nerve cell soma membranes of various species of Mollusca have been the subject of the most detailed pharmacological analysis of CNS acetylcholine receptors in invertebrates (Ascher and Kehoe, 1976; Walker, 1980). Acetylcholine, applied iontophoretically to soma of identifiable Aplysia neurones or released by synaptic activation of cholinergic cells, can give a variety of responses which result from the activation of three different acetylcholine receptors each with a distinct pharmacological specificity (Kehoe, 1972 a, b, c; Kehoe et al., 1976; Shain et a f . , 1974; Yarowsky and Carpenter, 1978). For example, a receptor mediating an increased membrane permeability to sodium ions generated an EPSP that was sensitive to hexamethonium and d-tubocurarine, but relatively insensitive to a-bungarotoxin.This receptor is, therefore, comparable to the acetylcholine receptor giving rise to the rapid EPSP in vertebrate sympathetic ganglion cells ( c t Barlow, 1964: Volle and Koelle, 1975). In addition, a receptor mediating an increase in permeability to chloride ions generated a rapid inhibitory postsynaptic potential, that was sensitive to d-tubocurarine and a-bungarotoxin but insenstive to hexamethonium. This receptor therefore exhibits a pharmacological profile resembling that of the acetylcholine receptor of vertebrate skeletal muscle (cf. Barlow, 1964; Koelle, 1975). Finally a receptor mediating an increased permeability to potassium ions generated a slow IPSP. This inhibitory postsynaptic potential was insensitive to d-tubocurarine, atropine and a-bungarotoxin but was blocked by tetrethylammonium ions. There is no vertebrate or invertebrate counterpart to this receptor which is not readily incorporated into traditional schemes of

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273

receptor classification. Three similar acetylcholine-induced responses were reported in Helix neurones (Gerschenfeld and Tauc, 1961; Kerkut et al., 1973; Chadetal., 1979; Yavarietal., 1979) and the sodium-dependent and chloride-dependent responses were also observed in neurones of Limnaea, Planorbarius (Zeimal and Vulfius, 1967) and Navanax (Levitan and Tauc, 1972). A biphasic response to acetylcholine was recorded from neurones of the pedal ganglion of Planorbarius (Ger and Zeimal, 1977). The initial (depolarizing) phase was mimicked by nicotinic ligands, whereas the slower (hyperpolarizing) phase was mimicked by muscarinic agonists such as 2-methyl-4-trimethylammonium-1, 3 dioxolane (F-2268). Recently, Walker and colleagues (Walker and Kerkut, 1977; Chadetal., 1977; Yavari et al., 1979) obtained evidence for the existence of an excitatory muscarinic receptor on cell E4 of Helix aspersa. The effects of a-bungarotoxin on the three types of acetylcholine-induced responses in Aplysia neurones have been investigated. Shain et al. (1974) were able to block all three acetylcholine responses at toxin concentrations as low as 2.0 x ~O-'M. However, Kehoe et al. (1976) found a-bungarotoxin (from Bungarus multicinctus) completely ineffective on the sodium and potassium mediated responses but able to produce reversible blockade of the response resulting from an increase in chloride permeability when . al. (1976) also showed applied at high concentrations (1 x 1 0 - 5 ~ )Kehoeet that a-bungarotoxin from B. caeruleus was ineffective on all three cholinergic responses. They suggested that the results of Shain et al. (1974) might have been due to contamination of their toxin samples by acetylcholinesterase. Other a-toxins, the Dendroaspis toxins from the green mamba Dendroaspis aspis, were also only effective on the chloride dependent response (Szczepaniak, 1974). Thus it appears that the so-called long toxins (e.g. B. multicinctus and D. aspis) are effective on the chloridemediated response but the short toxins (e.g. B. caeruleus toxin) are ineffective. Differences have been observed between the chloride-mediated response of Aplysia neurones and vertebrate skeletal muscle acetylcholine receptors. For example, whereas only the toxin of B. multicinctus was effective on Aplysia neurones, both B. multicinctus and B. caeruleus toxins were effective on vertebrate neuromuscular receptors (Kehoe et al., 1976). Also, the a-bungarotoxin block reported for Aplysia neurones was achieved only at high (1.O x ~O-'M)concentrations and was reversible, whereas neuromuscular junction receptors of vertebrates were irreversibly antagonized by ~ and Potter, 1971). Using [1251]-aa-bungarotoxin at 8.0 x l W 9 (Miledi bungarotoxin, Shain et al. (1974) demonstrated the existence of a saturable component of binding with some of the expected properties of a nicotinic acetylcholine receptor (Table 6). It is premature to consider this binding

274

DAVID

B. SATTELLE

component as a constituent of the receptor mediating chloride permeability changes, in view of the lack of correlation between the physiological and biochemical findings. Also, a number of ligands formerly considered to act at the receptor recognition site (including d-tubocurarine) are now known to exert their actions a t the receptor ionophore (Ascher et al., 1978). A high concentration of a-bungarotoxin-binding sites was reported in squid optic ganglia (Kato and Tattrie, 1976) and a-bungarotoxin-sensitive receptors were found on the Schwann cells enveloping the squid giant axon (Villegas, 1975). Of the arthropods other than insects that have been investigated both ligand-binding studies and electrophysiological results are available but in only one case to date (the Xiphosuran, Limulus polyphemus) have these complementary approaches been applied to the same tissue. Walker and James (1 978) found that acetylcholine depolarized Limulus neurones and this response was blocked by hexamethonium. Although nicotine was the most effective agonist, d-tubocurarine was a weak antagonist. An ['251]-a-bungarotoxin-binding component with the properties of a nicotinic acetylcholine receptor was characterized in homogenates prepared from the CNS of Limulus polyphemus (Thomas et al., 1978). As shown in Table 6, nicotine was a more effective inhibitor of toxin-binding than carbamylcholine and of the antagonists tested d-tubocurarine was more effective than atropine. Another acetylcholine binding component was demonstrated in axons of Limuluspolyphemus with a low affinity for both acetylcholine and a-bungarotoxin (Jonesetal., 1973). The sensitivity of Crustacean neurones to acetylcholine has been described by several laboratories (see Wiersma etal., 1953; McLennan and York, 1966; Barker eta/., 1972). For example, Barkerelal. (1972) showed that the depolarizing response to acetylcholine, iontophoretically-applied to cell bodies in lobster (Homarus americanus) CNS was blocked by 7.0 x 10 - 4 ~ d-tubocurarine. Atropine and hexamethonium at similar concentrations were less effective. The same workers noted that when bath applied to the abdominal ganglion cell. M15, d-tubocurarine and atropine (7.0 x 1 0 - 4 ~ ) blocked the depolarization recorded from the soma membrane in response to electrical stimulation of the slowly-adapting muscle receptor neurone. Hexamethonium, mecamylamine, choline and succinylcholine were either less effective or induced depolarization of the soma membrane (Barker, et al., 1972). The amine containing neurones of the second roots of lobster thoracic ganglia were depolarized by iontophoretically applied acetylcholine (Konishi and Kravitz, 1978). Both the response to iontophoretically applied acetylcholine and synaptic potentials evoked by nerve stimulation were blocked by 7.0 x ~ O - ' M hexamethonium. Nicotine and d-tubocurarine were the most effective of the ligands tested for their ability to suppress

ACETYLCHOLINE RECEPTORS OF I N S E C T S

275

the synaptic response and a-bungarotoxin (1.0 x 10-6-1.0 x 1 0 - 5 ~were ) ineffective even after perfusion for 30 min. Neurones of the stomatogastric ganglion of the crab Cancer pagurus showed several responses to iontophoretically applied acetylcholine, the predominant one being a depolarizing response (Marder, 1977; Marder and Paupardin-Tritsch, 1978) which was mimicked by agonists of vertebrate nicotinic ganglionic acetylcholine receptors such as TMA and DMPP ( c t Volle and Koelle, 1975). These responses were blocked by antagonists of vertebrate nicotinic ganglionic transmission such as mecamylamine and hexamethonium. Decamethonium, a potent ligand at vertebrate skeletal muscle acetylcholine receptors, had no effect on the stomatogastric ganglion cell depolarizing response to acetylcholine. Evidence was also obtained for the existence on stomatogastric ganglion neurones of acetylcholine receptors comparable to the muscarinic receptors of vertebrates (Marder, 1977; Marder and Paupardin-Tritsch, 1978). Thus studies on a variety of Crustacean neurones have revealed more than one kind of response to acetylcholine, but common to several species is an acetylcholine receptor resembling somewhat the properties of the vertebrate ganglionic nicotinic receptor. Few radiolabelled ligand binding studies have been performed on tissues of Crustacea. However. an acetylcholine-binding component has been demonstrated in axons of the lobster (Homarus americanus) (Denburget al., 1972; Denburg, 1973; Denburg and O’Brien, 1973; Joneset al., 1977) but not in axons of crabs such as Cancer pagurus (Balerna et al., 1975), Maia squinado (Balerna et al., 1975) and Callinectes sapidus (Jonesetal., 1977). Nevertheless this component has a low affinity for acetylcholine and binds a-bungarotoxin with low affinity and reversibly. This binding component to which no function has yet been ascribed does not appear to be either an axonal or glial acetylcholine receptor (Jones et al., 1977). Thus work on insects and other invertebrates has revealed a diversity of neuronal acetylcholine receptor types. Although there is some evidence for the existence of acetylcholine receptors resembling vertebrate muscarinic receptors, most invertebrate cholinergic receptors are characterized by a high affinity for nicotine and nicotinic ligands. Several invertebrate species possess more than one kind of CNS acetylcholine receptor and identified molluscan and annelid neurones with more than one kind of cholinergic receptor have been described. Extrasynaptic (cell body) receptors and synaptic receptors of Aplysia neurones share the same pharmacological profile, whereas differences have been detected in the case of Hirudo neurones.

DAVID 6.SATTELLE

276

5.3

VERTEBRATES

Acetylcholine receptors in the central nervous systems of vertebrates have been investigated using both electrophysiological and radiolabelled ligand binding techniques. Several distinct responses to acetylcholine have been recorded from vertebrate neurones. Fast excitatory responses resulting from an increased cation permeability have been well documented in autonomic ganglion cells (Blackman et al., 1963; Dennis et al., 1971) Renshaw cells (Curtis and Ryall, 1966a, b) and a variety of other regions of the CNS (cf. Krnjevic 1974). These responses are nicotinic in nature but the most effective blocking agents are not d-tubocurarine and its derivatives, but hexamethonium and tetraethylammonium (cfi Volle and Koelle, 1975). In studies on a cholinergic pathway in the frog spinal cord, Miledi and Szcepaniak (1975) showed that, although four neurotoxins from Dendroaspis venom appeared to block neuronal acetylcholine receptors, abungarotoxin (8.0 x LO-'M) was ineffective. This pathway in amphibia corresponds to the motoneurone-Renshaw cell pathway of higher vertebrates. Hunt and Schmidt (1978) have shown that Renshaw-like cells in the anterior horn of the rat spinal cord possess binding sites for ['*51]- a-bungarotoxin. Recently a saturable component of [ '251]-cr-bungarotoxin binding with a nanomolar dissociation constant ( K , ) and a pharmacological specificity corresponding to that of a nicotinic acetylcholine receptor was reported in the following nervous tissues of vertebrates: mammalian brain (Moore and Loy, 1972; Salvaterra and Moore, 1973; Eterovic and Bennett, 1974; Moore and Brady, 1976; McQuarrieetal., 1976; Schmidt, 1977; McQuarrie et al., 1977); mammalian autonomic (sympathetic) ganglia (Fumagallietal., 1976; Greene, 1976); cultured sympathetic neurones (Patrick and Stallcup, 1977a; Carbonetto et al., 1978); mammalian adrenal medulla (Wilson and Kirschner, 1977); avian retina (Yazulla and Schmidt, 1977); avian autonomic (parasympathetic) ganglia (Chiappinelli and Giacobini, 1978); fish brain (Oswald and Freeman, 1979) and amphibian brain (Oswald and Freeman 1977). The receptor-like properties of these sites was established and a good correlation demonstrated between the distribution of binding sites and sites of known central cholinergic pathways (cf. Schmidt et al., 1979). Also, in autonomic (sympathetic) ganglion preparations, toxin-binding sites appeared to be confined to neurones rather than to non-neuronal cells (Greene et al., 1973; Fumagalli et al., 1976). Synaptic localization of toxin binding sites has been confirmed by electronmicroscopy (Daniels and Vogel, 1975; Lentz and Chesher, 1977; Hunt and Schmidt, 1978). However,

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attempts to block nicotinic receptors with a-bungarotoxin and related a-neurotoxins have met with variable results. Several laboratories have failed to block transmission in vertebrate sympathetic ganglia (Chou and Lee, 1969; Magazanik et al., 1974; Brown and Fumagalli, 1977). Also the responses to nicotinic ligands of cultured vertebrate sympathetic neurones were not blocked by a-bungarotoxin (Obata, 1974;Nurse and O’Lague, 1975; Giller et al., 1975; KOet al., 1976; Carbonetto et af., 1978). a-Bungarotoxin was also unable to block the nicotine-induced release of catecholamines in the adrenal medulla (Wilson and Kirschner, 1977). Patrick and Stallcup (1977b) working on clone PCR, a cell line from a rat pheochromocytoma showed that receptor activation (measured as carbamylcholine-induced sodium ion fluxes) was unaffected by a-bungarotoxin. The same authors also showed that antibodies raised against Torpedo acetylcholine receptors inhibited receptor function but did not precipitate the toxin-binding macromolecules. Thus it appears that a-bungarotoxin binds to a nicotiniclike binding site on sympathetic neurones but that occupation of this site does not inhibit receptor activation. The visceral (parasympathetic) ganglia of rabbit, guinea pig, and chick were unaffected by a-bungarotoxin and no binding of radiolabelled toxin was detected (Bursztajn and Gershon, 1977), whereas in chick ciliary (parasympathetic) ganglion specific toxin binding sites were detected (Chiappinelli and Giacobini, 1978) and some, but not all, batches bf a-bungarotoxin proved effective at blocking transmission though at rather ) (Chiappinelli and Zigmond, 1978). A high (1.O x 1 0 - 6 ~ concentrations recent investigation of the inhibition of neuronal acetylcholine sensitivity by a-toxins from B. multicinctus venom has been performed using ciliary ganglion neurones in cell culture (Ravdin and Berg, 1979). These authors have identified six separate a-toxins. The widely used a-bungarotoxin (aBgt 2.2) is one of these fractions. Although a high-affinity specific toxin-binding site was detected on these cells, this particular a-toxin did not affect the response of the cultured cells to iontophoretically applied acetylcholine at 1.O X 1O-’M (a concentration which should saturate the high-affinity binding site). Higher concentrations of a-bungarotoxin (1.O x 1 0 - 5 ~ ) produced a partial inhibition of acetylcholine sensitivity. Whether this indicates that the cultured neurones possess two classes of binding sites or that other a-toxins present as minor components in the aBgt 2.2 were responsible for the blockade remains to be determined. In contrast to the results on spinal cord neurones and autonomic ganglia, other laboratories have provided physiological evidence that abungarotoxin acts at a site that is a constituent of a functional acetylcholine receptor. For example, excitatory postsynaptic potentials recorded from neurones in toad tectum (Freeman, 1977) are irreversibly blocked by

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a-bungarotoxin at a concentration of 1.0 x ~O-’M.This tissue contains a specific toxin-binding component. a-Bungarotoxin also blocks acetylcholineinduced dopamine release from rat brain synaptosomes (de Belleroche and Bradford, 1978) and cholinergic transmission in mammalian retinal ganglion cells (Masland anti Ames, 1976). It is possible that the complex results that have emerged from studies on vertebrate neuronal nicotinic acetylcholine receptors reflect an underlying heterogeneity of nicotinic receptor types, in particular those sensitive to, and those insensitive to a-bungarotoxin. In this context it might be recalled that certain neuromuscular junction nicotinic receptors of vertebrates are also insensitive to a-bungarotoxin (Burden et al., 1975). Alternatively, it is possible that all the a-bungarotoxin binding sites are constituents of the receptor complex. The different actions of a-bungarotoxin could then be explained if the binding sites for small ligands (including acetylcholine) and a-bungarotoxin vary in the degree to which they overlap (cf. Schmidt et a1., 1979). The techniques of electrophysiology and radiolabelled ligand binding have also been applied to the study of muscarinic acetylcholine receptors in the CNS of vertebrates. A slow prolonged excitation in response to acetylcholine and muscarinic agonists that was blocked by atropine and hyoscine, was recorded from ganglion cells and from a variety of central neurones (cf. Krnjevic, 1974). This type of response proved to be the most common excitatory response of acetylcholine in the vertebrate CNS and was recorded from neurones that, in addition, exhibited nicotinic responses (Phillis, 1970). Inhibitory actions of acetylcholine were reported in many areas of the CNS, even in cells predominantly excited by acetylcholine (cf. Krnjevic, 1974). These responses appeared to be due solely to the activation of muscarinic receptors. In recent years, the binding of a variety of reversible tritiated muscarinic antagonists to vertebrate CNS tissues, and to subcellular fractions derived from CNS tissues has been investigated. These include atropine (Paton and Rang, 1965; Schleifer and Eldefrawi, 1974; Birdsall et al., 1975; Hulme et al., 1976) quinuclidinyl benzilate (Yamamura and Snyder, 1974a, b, c Yamamura et al., 1974a, b; Snyder et al., 1975) and propylbenzilylcholine (Birdsall et al., 1976; Hulme et al., 1976). The tritiated form of the irreversible ligand propylbenzilylcholine mustard has also been used as a muscarinic receptor probe (Hiley et al., 1972; Burgen et al., 1974b; Hiley and Burgen, 1974; Hiley and Bird, 1974; Birdsall and Hulme, 1976; Birdsall et al., 1978). By these methods, a specific, saturable binding component with the expected pharmacological properties of a muscarinic receptor has been characterized. Also, the distribution of [3H]-antagonist binding sites in the central and peripheral nervous system detected by autnradinpranhv rlncelv n n m l l e l s the dictrihlltinn eynepted frnm eler-

ACETYLCHOLINE RECEPTORS OF INSECTS

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trophysiological studies (Kuhar and Yamamura, 1975; Yamamura and Snyder, 1974b). The cerebral cortex is particularly rich in muscarinic binding sites (Table 7). Thus several general conclusions emerge from a comparative study of CNS acetylcholine receptors of insects, other invertebrates and vertebrates. For example, several different kinds of receptor can be recognized with reference to both pharmacological specificity and receptor mediated cellular responses. That several acetylcholine receptors appear to be present in vertebrate, insect and molluscan central nervous tissues indicates that this diversity may have been established early in animal evolution. It is also clear that the traditional classification based on experiments on vertebrates becomes an inadequate framework when both invertebrate and vertebrate receptors are considered. The existence of more than one type of acetylcholine receptor on a single neurone has been demonstrated for both vertebrate and invertebrate receptors, so the ability of a single neurotransmitter to exert different effects on a particular postsynaptic cell also appears to have been established early in evolution. Finally, the density of acetylcholine receptors is comparable in both vertebrate and invertebrate organisms, though it appears that whereas muscarinic receptors predominate over nicotinic receptors in the mammalian brain the reverse is true in the insect brain. Comparative studies of this kind may point to distinct functional roles for the various acetylcholine receptors.

6 Genetic and developmental studies

6.1

G E N E T I C A P P R O A C H E S TO RECEPTOR S T R U C T U R E A N D F U N C T I O N

The wealth of genetic information that has accumulated on the fruit fly Drosophila melanogaster provides scope for a detailed genetic analysis of receptor structure, function and role in behaviour and development. As a first step, it is necessary to isolate mutants with altered receptors that can be used for subsequent genetic analysis. Although the existence of a nicotineresistant mutant of Drosophila was briefly reported over ten years ago (Lindsley and Grell 1968), the most detailed study was recently performed by Hall and collaborators (1979). These authors exposed Canton-S wildtype Drosophila to 3 mM nicotine in the culture medium. This concentration of nicotine killed 96% of the Canton-S strain and was routinely used to screen for nicotine-resistant strains in wild-type populations. One of the eight resistant strains that the authors identified was designated H R (isolated from Hikone-R wild type stock) and 50% of this resistant strain survived when exposed to 3 m M nicotine. To identify resistant strains with modified receptors, Hall and co-workers

DAVID B . SATTELLE

280

subjected a solubilized receptor-[ 1251]-cy-bungarotoxincomplex to isoelectric focusing. As shown in Fig. 26 whereas the receptor-toxin complex in extracts from the Canton-S strain focused as a single peak with an isoelectric point of 6.60, the receptor-toxin complex from the nicotine-resistant H R strain focused as a single peak at pH 6.69. By running a mixture of extracts

N tc ot i ne - res i st ant H - R slroin

150-

C

%

7

@bacD, %No

1 100 E

M l x t u r e of

Canton-S and H-R

50.-

20

40

60

Fraction

Fig. 26 Isoelectric focusing of t h e acetylcholine receptor-a-bungarotoxin complex from wildtype Canton4 and nicotine-resistant H R strains of Drosophila. Extracts were prepared and run on isoelectric focusing gels: ( a ) Canton-S strain alone; (b) Nicotine-resistant H R strain alone; ( c ) Mixture of extracts of Canton-S and H R strains. From Hall ef al. (1978)

from the two strains on the same gel, Hall et al. (1979) demonstrated that this difference in isoelectric point was due to an alteration in receptor properties and not due to variations between gels. In this way, a hereditary alteration in acetylcholine receptor structure has been identified. Not all nicotine-resistant strains would be expected to affect the structure of the nicotinic acetylcholine receptor. Alterations in components involved in

ACETYLCHOLINE RECEPTORS OF INSECTS

28 1

nicotine metabolism permeability and membrane environment of the receptor could also conceivably produce nicotine resistance. Of the eight nicotine-resistant strains identified by Hall et al. (1979), three of the stocks (including the resistant HR strain) show shifts in isoelectric point relative to Canton-S. These authors concluded that the iso-electric focusing variants represented either mutations in structural genes coding for the polypeptide subunits of the receptor or mutations in genes coding for enzymes which modified the receptor complex. It has been noted that the mutations identified so far have resulted in changes in the receptor which do not appear to greatly modify its function, since normal locomotor behaviour is observed in the nicotine-resistant strains. Recently using reciprocal crosses to test for X-chromosome linkage the same laboratory has shown that the gene causing the PI shift detected by isoelectric focusing is X-linked (Hall, 1980). When this PI variant has been mapped, the location of a gene affecting receptor structure will be identified. The major nicotine-resistance factor also segregated with the X-chromosome. Genetic mapping experiments could determine whether or not these two phenotypes are the result of a change in the same gene. Hybrid females carrying genetic information for two different forms of the variant polypeptide always revealed material which migrated to an isoelectric point between that of the parental types. The HR locus therefore codes for a structural polypeptide in the a-bungarotoxin-binding complex. Also at least two copies of this polypeptide must be present in the receptor complex. As Hall (1980) has pointed out, mapping the isoelectric focusing point variants will enable the determination of both the number and gene location of loci affecting receptor structure and could also provide information on receptor subunit composition. The structural gene for acetylcholinesterase has been identified on the third chromosome of Drosophita by Hall and Kankel(l976). Dudai (1978) tested acetylcholinesterase-less mutants (both point mutations and deletions covering the locus) for their ['2'I]-a-bungarotoxin-binding activity. This author showed that flies which were heterozygous for mutations in that gene and contained only half the normal acetylcholinesterase activity exhibited normal toxin-binding activity. In addition, pharmacological studies established that a-bungarotoxin did not bind to the active site of acetylcholinesterase and subellular fractionation revealed that the toxin-binding and enzyme activities did not co-purify (Dudai 1978). Furthermore, the fact that deletions covering the acetylcholinesterase locus from both sides did not abolish receptor activity was a strong indication that the genes coding for the toxin-binding receptor of Drosophila were not situated adjacent to the gene for acetylcholinesterase (Dudai, 1978). In a parallel study from the same laboratory Haim et al. (1979) using similar genetic techniques demonstrated

DAVID B. SATTELLE

282

that the [3H]-quinuclidinyl benzilate binding component could be distinguished from acetylcholinesterase and genes coding for the muscarinic receptor were not located adjacent to the acetylcholinesterase gene. These findings for the muscarinic receptor (Table 13) are of interest in the light of earlier reports that muscarinic ligands bind to peripheral sites on acetylcholinesterase molecules (Kato et al., 1972). In their studies on the [3H]-decamethonium binding component in head extracts of Musca, Tripathi et al. (1979) have compared a wild type with a mutant strain that shows a remarkable insensitivity to poisoning by organophosphates and carbamates. The mutant showed a four fold greater affinity for [3H]-decamethonium binding but the total amount of binding was reduced by seven fold. Thus the initial genetic studies on insect acetylcholine receptors have proved fruitful and the prospects are optimistic for an increased understanding of receptor structure, function and role in behaviour and development.

6.2

RECEPTORS I N DEVELOPMENT

Insects provided experimental material well suited to the analysis of pre- and postembryonic neuronal development. Identifiable neurones are a particularly attractive source of material since it is possible to follow individual cells from birth to maturity. It is therefore possible to ask at what stage in development functional acetylcholine receptors first appear. Also, the ease with which experimental manipulations can be performed early in development enables analysis of neuronal development in the absence of normal synaptic input. The embryonic development of the dorsal unpaired median (DUM) neurones of the grasshopper (Schistocerca nitens) was recently investigated (Goodman et al., 1979; Goodman and Spitzer, 1979). On the seventh day of embryonic development neither the DUM neuroblast nor its progeny showed responses to bath application of acetylcholine. Beginning on day 8, the cell bodies of the older DUM neurones exhibited sensitivity to bath application and iontophoretic application of acetylcholine (Fig. 27). Simultaneously, processes of the older DUM neurones became sensitive to acetylcholine. It appears therefore that soon after the first appearance in the cell membrane of functional acetylcholine receptors, they are distributed over the whole surface of the cell (Goodman and Spitzer, 1979; 1980). The same authors showed that sensitivity to acetylcholine develops whilst the cells are still electrically coupled. The reversal potential (+ 20 mV) and the ionic dependence of the acetylcholine-induced depolarization of the cell body membrane appeared to be unchanged between days 8-1 8 of embryonic development.

D

0

rn

-I

< TABLE 13 [3H]-QNB binding in acetylcholinesterase-deficient mutants. Heads (400/ml) were homogenized in 0.32 M-sucrose and assayed for specific [3H]-QNB binding and acetylcholinesterase activity. Mutants were kept in heterozygous state (homozygous is lethal) over the chromosomal balancer MRS. As seen in the table, the balancer itself had no significant effect on esterase activity or QNB-binding. Values are percentages - mean one standard error with number of experiments in parentheses. From Haim et nl, (1979)

*

Acetylcholinesterase activity Strain

Description

c-s

Wild type

z

rn

['HIQNB binding

Absolute activity (%) Specific activity (%) Absolute activity (%) Specific activity (%)

-I

0 SJ

Heterozyygous for a point mutation in the acetylcholinesterase gene Heterozygous for a point mutation 1(3)m38/MRS in the acetylcholinesterase gene Df(3R)126d/MRS Heterozygous for a small deletion covering the acetylcholinesterase gene Df(3R)ry615/+ Heterozygous for a larger deletion covering the acetylcholinesterase gene + /MRS Control for the balancer chromosome employed in part of the above experiments

100 (22 ? 1 nmol/min/ 10 PI) (1 1)

100 (0.52 ? 0.02 pmol/ min/ mg protein) (11)

100 (2.7 2 0.2 fmol/ 10 p1) (6)

100 (62 2 2 fmol/mg protein) (6)

cn

: z

cn

1(3)m15/MRS

rn

52

*3 (6)

5 3 k 2 (8)

* 2 (8)

87 k 10 (2)

101 ? 11 (4)

97 2 7 (2)

80 2 9 (4)

55?2(6)

43

56 ? 2 ( 6 )

51?6(8)

loo? 12 (2)

105 "20 (4)

61 k 3 (3)

54? 5 (3)

86 5 2 (4)

9 6 ? 3 (3)

103 k 4 (7)

82?4(7)

92?7(7)

77?9(7)

0

-I

cn

N

m W

DAVID 8 . SATTELLE

284

1

4

10

rnvL 2s

b

13 D A Y I

ACh

GABA

Fig. 27 Chemosensitivity of identified DUMneurones in the grasshopper (Schistocera nitens). ( a ) Response to iontophoretic application of acetylcholine in a day 8 embryo. The diagram

shows the embryonic neuropile of a single segmental ganglion (T3), as viewed from the dorsal surface(anteri0rat top). The DUMneuroblast(MNB)isshown withitspacketofprogeny; those most anterior (oldest) send their axons anteriorly in a median bundle of processes which cross the posterior commissure (PC) and bifurcate near the anterior commissure (AC). The extent of branching of an individual neurone was determined by intracellular injection of Lucifer Yellow. The longitudinal connectives extend into the ventral nerve cords (VNC) and are seen on either side. The lateral neuropile extends (beyond the margin of the figure) into three fibre tracts which become peripheral nerves 3,4 and 5. The responses to applicationof acetylcholine at the points indicated by the arrows were recorded by an intracellular electrode in the cell body. Processes are sensitive to ACh over their whole length. Small vertical or lateral displacements of the iontophoretic electrode abolished the response. (b) Responses to sequential iontophoretic application of acetylcholine and y-aminobutyric acid from a double-barrelled micropipette, recorded with an intracellular electrode in a day 13 embryo. At a resting potential of -55 mV, acetylcholine depolarizes the cell to threshold, eliciting overshooting action potentials, while y-aminobutyric acid hyperpolarizes the cell. When the cell is hyperpolarized by injected current, the response to acetylcholine is larger, but remains subthreshold; the cell is then depolarized by y-aminobutyric acid. From Goodman and Spitzer (1979a)

ACETYLCHOLINE RECEPTORS OF I N S E C T S

285

In the same study the first spontaneous synaptic activity (origin of input unknown) was reported in DUM neurones at 15 days, one week after the onset of chemosensitivity. Although causal relations in the sequence of phenotypes in embryonic neurones have yet to be established, Goodman and Spitzer (1979) have pointed out that several events appear to be closely linked. For example, outgrowth of processes and sensitivity to acetylcholine occur at about the same time. A possible regulatory function of acetylcholine receptors in the maintenance of synaptic connections has been proposed based on experiments on amphibian retino-tectal synapses (Freeman 1977). Insect neurones which can be identified during development could provide a direct test of this hypothesis. The role of innervation in the normal development of excitable tissues has been most fully documented in the case of the regulation of the distribution of acetylcholine receptors in vertebrate skeletal muscles developing in vitro andin vivo (cf. Fambrough, 1979). It is established that in response to motor innervation clusters of acetylcholine receptors are formed in the subsynaptic membrane. In the first comparable study of the development of receptors during de novo synapse formation in the central nervous system, Hildebrand and colleagues (1979) have investigated the antennal lobes of the brain of the moth, Manduca sexta. The antennal lobes are well suited to such a study as they appear to have a high concentration of cholinergic synapses, a simple anatomical organization and their development is amenable to experimental manipulation (Sanes and Hildebrand, 1976a, Sanes and Hildebrand 1976b, Sanes et al., 1977, Hildebrand, 1980). The antennal lobes are the first synaptic stations for processing most of the antennal sensory inputs to the central nervous system (Strausfeld, 1976). Both the antennae and the antennal lobes of Manduca develop de novo during adult development, commencing at about the time of the metamorphic moult of the larva to the pupa (Sanes and Hildebrand 1976, Saneset al., 1977; Prescott, et al., 1977). Sensory neurones with cell bodies in the antennae send fibres into the lobes. Antennae and antennal nerves contain acetylcholine. The antennae synthesize and store [14C]acetylcholine whereas several other putative neurotransmitters do not accumulate. Also, the presence of choline acetyltransferase is further evidence that the sensory axons under investigation are cholinergic (Sanes and Hildebrand, 1976b). Levels of acetylcholine, choline acetyltransferase and acetylcholinesterase rise dramatically in the antennal lobes as these sensory (probably cholinergic) axons grow into the lobes through the antennal nerves (Sanes et al., 1977). An ['251]-cu-bungarotoxinbinding activity was shown to develop in the antennal lobes with a time-course different from that of the other cholinergic components, rising steadily throughout metamorphosis (Sanes et al., 1972). This toxin-binding activity was specific

286

DAVID B. SATTELLE

to nervous tissue and was blocked by a range of cholinergic ligands (Saneset a f . ,1977). The pharmacological properties of this toxin-binding component indicated that it closely resembled the nicotinic acetylcholine receptor characterized by [1251]-a-bungarotoxinbinding in several other insect species (see Section 2 . 3 ) . Antenna1 lobes were deprived of their normal antennal sensory inputs throughout adult development by deafferentation. (Hildebrand et a f., 1979). This was routinely achieved by removal of an antenna from one side of the head, the remaining antenna serving as a control. Following deafferentation, levels of acetylcholine, choline acetyltransferase and acetylcholinesterase were greatly reduced in the antennal lobe but the toxinbinding activity of the lobe was not significantly lowered (Sanesetaf., 1977). Furthermore, the deafferented lobe, although somewhat stunted and ectopic, displayed histological features similar to those of the normal antennal lobe (Hildebrand et a f . , 1979). These same authors showed that deafferentation did not lead to the appearance of toxin- binding sites over other regions of the neuropile or over the cell bodies of neurones in the antennal lobe (Fig. 28). To check that localized toxin-binding activity was not due to the influence of the unoperated side on the contralateral side of the brain bilaterally deafferented animals were investigated. Both deafferented lobes showed the same pattern of toxin-binding noted in the deafferented lobe of a unilaterally deantennated animal. In the complete absence of antennal sensory input to the brain, therefore, the developing antennal lobes nevertheless develop toxin-binding activity in the neuropile region normally destined to receive afferent synaptic inputs (Hildebrand et a f . , 1979). Finally, these authors were unable to detect any affects of deafferentation on the histology or toxin-binding activity of higher-order neuropile regions. Thus, antennal sensory neurones of Manduca appear to undergo normal development following deafferentation as judged by morphological, neurochemical and electrophysiological criteria. The deafferented antennal lobes produce a characteristically organized neuropile and elaborate acetylcholine receptors in the appropriate region of the neuropile. This finding for the innervation of central neurones is in contrast with the situation in vertebrate muscle ( c j Fambrough, 1979). These initial autoradiographic studies reveal a pattern of development in excitable cells that is distinct from that of vertebrate neuromuscular junctions. In the antennal system of the moth Manduca sexta, therefore, there is strong evidence that neurones complete much or all of their development autonomously, independent of their normal synaptic input.

ACETYLCHOLINE RECEPTORS OF INSECTS

Fig. 28

287

Effects of deaffererltation on [li5]-a-Bgt binding in the antennal lobes of Manduca on the left were taken with bright-field illumination and those on the right, with dark-field illumination. ( a ) Frontal section of antennal lobes from a unilaterally deantennated animal showing normal distribution of toxin-binding sites on the unoperated, control side and dense toxin binding confined to protoglomeruli (pgl) on the deafferented side. ( b ) Same section as that shown in ( a ) . Note the absence of binding over cell bodies (cb). (c) Nearly frontal section through the antennal lobes and subesophageal ganglion (SEG) of a bilaterally deantennated animal showing a-Bgt binding to the condensed protoglomeruli. (d) Same section as that shown in (c). ( e ) Horizontal section of a unilaterally deantennated animal showing a-Bgt binding to the mushroom bodies (mb) in the protocerebrum. The unoperated, control antennal lobe is marked with the AL, but the deafferented lobe is not visible in this plane of section. cf) Dark-field view of same section as that shown in ( e ) . Scale bar = 100 pm. From Hildebrand er a l . (1979)

sexta. Micrographs of toluidine blue stained sections

DAVID 6.SATTELLE

288

7 Receptor actions of toxins and insecticides 7.1 7.1.1

RECEPTOR-ACTIVE TOXINS

a-Neurotoxins

Several of the potent snake a-neurotoxins have been tested for receptor activity in insects using both electrophysiological and radiolabelled ligandbinding techniques. Irreversible blockade of cercal-afferent, giantinterneurone synaptic transmission in Periplaneta americana at nanomolar concentrations was noted (Harrow et a f . , 1979; 1980b). Suppression of the depolarization induced by iontophoretic application of acetylcholine to the cell body membrane of the Df motoneurone of Peripfaneta at 5.0 x 1 0 - 8 ~ was also demonstrated (Sattelle et al., 1980). By contrast, DUM neurone cell bodies from adults of Peripfaneta (Sattelleet al., 1980) and embryos of Schistocerca nitens (Goodman and Spitzer, 1979; 1980) were unaffected by (see also Section 4.4.2). In these experia-bungarotoxin at 1.0 X 1 0 - 6 ~ ments, the a-bungarotoxin used corresponded in all cases to the fraction aBgt 2.2 from the venom of Bungarus multicintus (nomenclature of Ravdin and Berg, 1979). Other a-toxins purified from the venom of Bungarus multicintus were ineffective on the acetylcholine-induced depolarization of embryonic DUM neurone cell bodies (Goodman and Spitzer, 1979a, b). These included: aBgt 3.1; aBgt 3.2; aBgt 3.3; aBgt 3.4 (Goodman and Spitzer, 1980). The a-neurotoxin from the Siamese cobra (Naja naja siamensis) was ineffective in blocking the response of DUM neurone cell bodies to iontophoretic application of acetylcholine (Goodman and Spitzer, 1980). An a-toxin from the green mamba (Dendroas viridis) which blocked a vertebrate central cholinergic synapse (Szcepaniak and Miledi, 1975) was also ineffective in blocking the acetylcholine response of DUM neurone cell bodies at 1.0 x ~O-’M (Goodman and Spitzer, 1980). The highly selective nature of the receptor blocking actions of the snake a-toxins could be further exploited in order to characterize in more detail the differences between neuronal acetylcholine receptors of different organisms. 7.1.2 Nereistoxin Nereistoxin (NTX) isolated from the marine annelid Lumbriconereis heteropoda is a potent insect neurotoxin (Nitta, 1934, Sakai, 1964; Narahashi, 1972). Of the variety of synthesized derivatives of NTX, those toxic to insects are the 4-alkylamino-1, 2-dithiolanes and the 2-dimethylamino-l , 3-propane dithiols (Sakai, 1966). Nereistoxin blocks cholinergic synaptic transmission but its mechanism of action is complex.

ACETYLCHOLINE RECEPTORS OF INSECTS

289

Deguchi et a f . (1971) showed that the toxin reduced the amount of acetylcholine released from presynaptic nerve terminals of neuromuscular junctions. The same authors found that NTX reduced the sensitivity of the postsynaptic nicotinic receptors to applied acetylcholine. In addition to exhibiting antagonistic actions at nicotinic synapses, NTX acted as an agonist at certain vertebrate muscarinic synapses. For example, the toxin both decreased mammalia heart rate and increased salivary gland secretion, both effects being antagonized by atropine (Nitta, 1941). The actions of NTX at both nicotinic and muscarinic receptors of mammals led to the suggestion (Eldefrawi, 1976) that this compound did not act at the ligand binding site of these acetylcholine receptors. Some confirmation of this notion comes from the actions of NTX on the insect central nervous system. Nereistoxin blocked cercal-afferent, giant-interneurone synapses in the sixth abdominal ganglion of Periplaneta (Sakai, 1967; Bettini e f al., 1973; Sattelle and Callec, 1977). Concentrations as low as 5 X ~ O - ' M NTX partly suppressed excitatory postsynaptic potentials in Periplaneta giant interneurones (Sattelle and Callec, 1977; Sattelle, 1977). Nevertheless, a discrepancy was noted between the ability of NTX to suppress transmission at synapses at which nicotinic cholinergic receptors appear to mediate transmission (see Fig. 29) and its ability to inhibit the binding of [1251]-abungarotoxin to Periplaneta extracts. The concentration of NTX that produced an inhibition of 50%of the toxin binding was 5 x 1 0 - 4 (Sattelleetal., ~ 1981b). One possible explanation is that NTX acts on the nicotinic receptor not at the ligand binding site but on the ionophore. The amphibian toxin histrionicotoxin is considered to act in this way at vertebrate neuromuscular junctions (Kato et al., 1975; Eldefrawi et a f . , 1977). However, NTX did not inhibit the binding of ['HI-perhydrohistrionicotoxin to Torpedo membranes rich in nicotinic receptors (Eldefrawi et a f . , 1980). Although its detailed receptor interactions are not resolved, NTX primarily acts as an antagonist at insect and vertebrate nicotinic receptors.

7.2

C H O L I N E R G I C RECEPTORS AS SITES OF INSECTICIDE ACTION

For a limited number of insecticidally active molecules acetylcholine receptors have been proposed as a possible site of action (see Narahashi, 1973; Corbett 1974; Eldefrawi, 1976). The present discussion will consider the potential of specific cholinergic receptors as target sites for insecticides in the light of recent advances in the characterization of insect acetylcholine receptors. Clearly, the demonstration of receptor activity of an insecticide or related compound does not necessarily indicate that the molecule acts primarily at this site. Also, metabolism, permeability barriers and indirect effects of the compound may conspire to produce only a weak correlation

DAVID B. SATTELLE

290

--tDrosophila (150-6.6 x 6 ' M ) --o-- Periplaneta

/>

( I ~ ~ -xI ~. ~6 5 ~ )

l

l

l

l

l

l

-4 10 1 Nereistoxin concentration (M)

18

8

Fig. 29 ( a ) Concentration dependence of inhibition by Nereistoxin of [1Z51]-~-bungarotoxin binding to extracts of both whole flies (Drosophila melnnogaster) and cockroach abdominal nerve cords (Periplaneta americana). Results are expressed as a percentage of toxin bound to extract in the absence of Nereistoxin. Each point is the average of three replicates and vertical bars denote one standard deviation. Concentrations for 50% inhibition of toxin binding are estimated to be 6.6 x lo-% (Drosophila; 0 )and 1.8 X 1 0 - 4 (Periplaneta; ~ 0). ( b )Effects of a range of nereistoxin concentrations on the amplitude of the evoked EPSP recorded from the cercal-nerve, giant-interneurone synapses in the sixth abdominal ganglion of the cockroach Periplaneta americana by means of a sucrose-gap recording technique. Clearly the inhibition by nereistoxin of an ['Z51]-a-bungarotoxin-sensitivereceptor does not fully account for its synaptic blocking action. Modified from Sattelle er al. (1981b)

between insect toxicity and receptor activity even in those cases where there are strong indications of a primary site of action on an acetylcholine receptor. Nevertheless, in approaching a rational design of environmentally more acceptable insecticides it is instructive to test the actions of both currently used and future potential insecticides at specific receptors. Nicotine, one of the earliest commercial insecticides (Corbett, 1974), is a potent cholinergic agonist when applied to insect neurones (see Section 3), and binds both to the nicotinic receptor and the mixed receptor characterized by radiolabelled ligand binding to insect extracts (see Section 2). For example, studies on the mixed receptor of Musca have shown that nicotine binding is inhibited by both optical isomers of nicotine and by toxic nicotinoids but not by non-toxic nicotinods (Eldefrawietal., 1970). The common

ACETYLCHOLINE RECEPTORS OF I N S E C T S

291

requirement for toxicity was 3-pyridylmethylamine with a basic amino nitrogen (Yamamoto et al., 1962, 1968; Kamimura et al., 1963). The correlation between toxicity measured as LDSousing houseflies and the blockade of binding of [3H]-muscarone to the mixed receptor was poor possibly as a result of metabolism and barriers to penetration. Furthermore, in studies on Periplanetu it was shown that the receptor actions of nicotine can largely account for its observed effects on cercal-afferent, giant-interneurone synaptic transmission in the sixth abdominal ganglion (Gepner et ul., 1978). So, there is strong evidence that nicotine acts at two cholinergic receptors in insects, one of which appears to have a functional role in synaptic transmission (see Section 2.3). As an insecticide, however, nicotine is largely of historical interest. Cartap (4-N,N-dimethylamino-1, 2-dithiolane) was the first synthetic insecticide based on the structure of a natural toxin and in vivo appears to be metabolized to Nereistoxin (NTX) (Sakai and Sato, 1971). Although most studies have applied NTX directly to insect nerve preparations (see Section 7.1.2), some studies have been performed with cartap. Similar results to those reported for NTX were obtained (Bettini et al., 1973). The bulk of the present generation of insecticides including the organophosphates and carbamates are anticholinesterase agents (cf. Corbett, 1974). They are considered to exert their primary action by retarding the hydrolysis of acetylcholine and thereby prolonging the actions of the neurotransmitter at cholinergic synapses. Nevertheless, in electrophysiological experiments on Electrophorus electroplax, evidence has accumulated that the organophosphate anticholinesterases DFP, paraoxon and phos) pholine can act as receptor antagonists at high (1-8 X 1 0 - 3 ~concentrations (Bartels and Nachmanson, 1969). Eldefrawietal. (1971d) obtained an inhibition of acetylcholine binding to electroplax tissue by similar concentrations ( 1 0 - 4 ~of) the anticholinesterases DFP, Guthoxon, Tetram and 2- (0, S-dimethylthiophosporylimino) 3-ethyl-5-methyl-l , 3-oxazolindine (known as R-16661). The carbamates neostigmine and pyridostigmine also inhibited the binding of acetylcholine to electroplax tissue (Eldefrawi et a1 ., 1972). Since this competitive effect was only noted at high concentrations, Eldefrawi (1976) has suggested that it may result from an electrostatic attraction between the anionic site of the receptor and the positively charged carbamates. It has also been shown that the anticholinesterase edrophonium has a high affinity for peripheral nicotinic receptors (Seifert and Eldefrawi, 1974) which is consistent with earlier electrophysiological evidence of its agonistic actions (Riker, 1953). Thus, in vertebrates, actions as cholinergic receptor agonists and antagonists have been reported for concentrations of anticholineresterases in excess of the concentrations required for enzyme inhibition.

292

DAVID 6.SATTELLE

A number of anticholinesterase insecticides inhibited [3H]-decamethonium binding to housefly brain (Eldefrawiet al., 1971d). These were in order of effectiveness Tetram > Guthoxon > R-16661> DFP. Cholinesterase inhibitors eserine and neostigmine also inhibited the binding of [ '251]-a-bungarotoxin to extracts of Drosophila. In these studies I,,, values of 1.0 x 1 0 - 5 ~were reported for eserine (Schmidt-Nielsen et al., 1977) and values of 2.0 x 1 O W s were ~ reported for neostigmine (Schmidt-Nielsen et a!., 1977). It may be, therefore, that certain anticholinesterase insecticides owe some of their toxicity to interactions with acetylcholine receptors (Eldefrawi et al., 1971d; Jones et al., 1979). Recent studies on isothiocyanates as potential insecticides by Baillie and collaborators (1975) have shown their considerable potency as choline acetyltransferase inhibitors. When applied to the isolated abdominal nerve cord of Periplaneta at concentrations higher than those required to inhibit the enzyme in vitro, postsynaptic actions consistent with those of a cholinergic agonist were recorded (Sattelle and Callec, 1977). A good correspondence was noted between the postsynaptic blocking actions of 2-isothiocyanato-ethyltrimethylammoniumiodide and its ability to inhibit [1251]-a-bungaratoxinto extracts of Periplaneta nerve cords (Fig. 30). Although this particular molecule of the series of isothiocyanates synthesized is not a likely candidate insecticide because of its charge and water solubility, it is possible that related compounds in the series may owe part of their toxic action to a cholinergic receptor action in addition to an inhibitory action on the enzyme choline acetyltransferase. In 1976, Bigg and Purvis reported that a range of muscarinic agonists showed acaricidal activity against both organophosphate resistant and susceptible strains of the tick Boophilus microplus. In the case of oxotremorine the compounds were and 1-(4-Dimethylaminobut-2-ynyl)pyrrolid-2-one, more effective on the susceptible strain than nicotine. Several muscarinic agonists were active against mites but were completely inactive against the following insects: Musca dornestica (adults), Aedes aegyptii (larvae), and Lucilia pericata (larvae). Oxotremorine at 9 x 10% inhibited 50% of the binding of [3H]-quinuclidinylbenzilate to Drosophila extracts (Haim et a1., 1979). It is of interest to note that a much greater abundance of nicotinic receptors compared to muscarinic receptors has been reported in extracts of insect CNS (Dudai and Ben Barak, 1977; Haimetal., 1979). The potency of muscarinic ligands on ticks and mites may point to fundamental biochemical differences between these two groups of arthropods. Thus some differences between acetylcholine receptors of insects and other organisms have emerged (Section 5 ) . Also, evidence presented above shows that acetylcholine receptor ligands show differential toxicity to differ-

A C E T Y L C H O L I N E RECEPTORS OF I N S E C T S

-

20 ms

I

lo-@

10.'

293

m

I

lo4

\ I 10.~

lsothlocyanate Concentration ( M I

Fig. 30 ( a ) Concentration dependence of inhibition by 2-isothiocyanatoethyltrimethylammonium iodide of [1251]-a-bungarotoxinbinding to extracts of both whole flies (Drosophila melarrogaster) and cockroach abdominal nerve cords (Periplaneta americana). Each point is the average of three replicates and vertical bars denote one standard deviation. Concentrations for 50% inhibition of toxin binding are estimated to be 6.9 x 1 0 - 6 ~ (Drosophila; 0) and 1.6 x 1 0 - 5(Periplunetu; ~ 0 ) .(b) Effects of various isothiocyanate concentrations on the amplitude of the evoked EPSP recorded from the cercal-nerve, giant interneurone synapses in the terminal abdominal ganglion of P. americana using a sucrose-gap recording technique. Concentration for 50% suppression of the EPSP is estimated to be 2.6 X 1 0 - 5 ~Inset . shows the EPSP recorded in normal saline (100%). From Gepner et al. (1978)

ent groups of arthropods. Such differences might conceivably be exploited in the future design of pesticides.

8 Conclusions

Insects have provided material well suited to the investigation of CNS acetylcholine receptors. The central nervous tissues of these organisms have proved amenable to both radiolabelled ligand-binding studies and singlecell electrophysiology of identifiable neurones. Binding studies have resulted in the characterization in vitro of three putative acetylcholine recognition sites each with a distinct pharmacological specificity. Of these

294

D A V I D B . SATTELLE

three putative acetylcholine receptors defined biochemically, the a-bungarotoxin-sensitive receptor has been most fully investigated. The ['251]-a-bungarotoxin-binding components characterized from Drosophila melanogaster (heads), Musca domestica (heads), Periplaneta americana (abdominal nerve cords) and Manduca sexta (brain) are indistinguishable in their pharmacological properties. A highly purified form of the solubilized receptor has been prepared from Drosophila melanogaster which maintains the pharmacological profile of the membrane-bound receptor. A physiological role has been established for this receptor at synapses between cercal mechanoreceptor afferent neurones and giant interneurones (GI 2 and GI 3) in the sixth abdominal ganglion of the cockroach Periplaneta americana. The cell body membrane of the coxal depressor motoneurone (Df) of Periplaneta americana also contains a-bungarotoxin-sensitive acetylcholine receptors. Pharmacological differences have emerged between this insect receptor and the nicotinic receptor of vertebrate muscle and electroplax tissue. The insect receptor is, for example, much less sensitive to decamethonium and carbamylcholine. Also the receptors mediating cercal-afferent, giantinterneurone synaptic transmission are much more sensitive to a-bungarotoxin and less sensitive to hexamethonium than the nicotinic receptors of vertebrate autonomic ganglia. Two other putative insect acetylcholine receptors both of which are insensitive to a-bungarotoxin have been investigated by radiolabelledligand binding methods. A putative receptor in Drosophila heads characterized by its high affinity for [3H]-quinuclidinyl benzilate closely resembles vertebrate muscarinic receptors in its pharmacological properties. A putative receptor exhibiting a pharmacological profile quite different from any acetylcholine receptor so far described has been characterized in heads of Musca dornestica. This putative receptor which has been highly purified is present at an unusually high density and is very readily solubilized when compared to most other membrane receptors studied. Caution is needed in interpreting these two binding components as true receptors until physiological evidence for a functional role is available. Nevertheless abungarotoxin-insensitive acetylcholine responses have been reported for the cell body membranes of dorsal unpaired median (DUM) neurones of Schistocera nitens and Periplaneta americana . Further studies are needed to determine the detailed pharmacological specificity of these toxin-insensitive acetylcholine responses. Insect material is particularly well suited for developmental and genetic approaches to receptor biology. Using ['251]-a-bungarotoxin as a receptor probe, work on Manduca sexta has provided the first analysis of the development of acetylcholine receptors duringde novo synapse formation in

ACETYLCHOLINE RECEPTORS OF I N S E C T S

295

the central nervous system. A pattern of development has emerged quite different from that of the vertebrate neuromuscular junction. Acetylcholine receptors on insect neurones appear to cluster and develop normally in the absence of presynaptic inputs. Also, studies on acetylcholine receptors of embryonic DUM neurones of Schistocera nitens have shown that the oldest progeny of the DUM neuroblast become sensitive to acetylcholine at day 8 of embryonic life. On this day, both the cell body and processes acquire sensitivity to acetylcholine indicating that functional receptors are distributed over the whole surface of the cell soon after they first appear. Using these embryonic neurones it may be possible to determine the distribution and properties of acetylcholine receptors on an identified neurone during synapse formation. Genetic studies offer another approach to the study of receptor biology for which insect material is supremely well suited. Hereditary changes in acetylcholine receptor structure (detected as isoelectric point (PI) variants) have been reported in nicotine-resistant strains of Drosophila melanogaster. The gene responsible for this structural change is on the X-chromosome. Gene mapping techniques could also provide information on receptor subunit composition. In addition temperature-sensitive mutants in which the receptor is active, or not, depending upon the ambient temperature should be useful for determining the role of receptors in behaviour and in development. Arising from genetic studies on Drosophila is the finding that the gene causing the PI shift and the gene controlling a major resistance factor are both located on the X-chromosome. These two phenotypes may result from a change in the same gene. If the mechanism of nicotine-resistance does involve a change in receptor structure this would be of considerable interest in view of the recent demonstrations that several insecticidally active molecules are receptor-active. The molecular basis of one type of insecticide resistance would then be directly accessible to experimental analysis.

References Abe, T., Alema, S. and Miledi, R. (1977). Isolation and characterization of presynaptically acting neurotoxins from the venom of Bungarus snakes. Eur. J . Biochem. 80, 1-12 Ascher, P. and Kehoe, J. S. (1976). Amine and amino-acid receptors in gastropod neurones. In “Handbook of Psychopharmacology” (Eds L. L. Iversen, S. D. Iversen and S. H. Snyder) 4, 265-309. Plenum Press, New York Ascher, P., Marty, A . and Neild, T. 0. (1978). The mode of action of antagonists of the excitatory responses to acetylcholine in Aplysia neurones. J. Physiol. 278, 207-235

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Sakai, M. (1966a). Studies on the insecticidal action of nereistoxin, 4N,N-dimethylamino-l,2-dithiolane. 11. Symptomatology. Bochu-Kagaku 31, 53-61 Sakai, M. (1966b). Studies on the insecticidal action of nereistoxin, 4N,N-dimethylamino-l,2-dithiolane. 111. Antagonism to acetylcholine in the contraction of rectus abdominis muscle of frog. Bochu-Kagaku 31, 61-67 Sakai, M. (1967). Studies on the insecticidal action of nereistoxin, 4N,N-dimethylamino-l,2-dithiolane. V. Blocking action of the cockroach ganglion. Bochu-Kagaku 32, 21-33 Sakai, M. (1970). Nereistoxin and its derivatives; their ganglionic blocking and insecticidal activity. In “Biochemical Toxicology of Insecticides” (Eds R. D. O’Brien and I. Yamamoto) pp. 33-40. Academic Press, New York Sakai, M. and Satn, Y. (1971). Metabolic conversion of the nereistoxin-related compounds into nereistoxin as a factor of their insecticidal action. In “Abstr. 2nd Int. Congr. Pestic. Chem”. Tel-Aviv Salvaterra, P. M. and Moore, W. J. (1973). Binding of [‘251]-a-bungarotoxin to particulate fractions of rat and guinea pig brain. Biochem. Biophys. Res. Comm. 55, 1311-1318 Salvaterra, P. M., Mahler, H. R. and Moore, W. J. (1975). Subcellular and regional distribution of *2SI-labeleda-Bungarotoxin-binding in rat brain and its relationship to acetylcholinesterase and choline acetyltransferase. J . Biol. Chem. 250, 6469-6475 Sanes, J. R. and Ilildebrand, J. G. (1975). Nerves in the antennae of pupal Manduca sexta Johanssen (Lepidoptera: Sphingidae). Wilhelm Roux’ Archiv. 178, 71-78 Sanes, J. R. and Hildebrand, J. G. (1976a). Structure and development of antennae in a moth, Manduca sexta. Devel. Biol. 51, 282-299 Sanes, J. R. and Hildebrand, J. G. (1976b). Acetylcholine and its metabolic enzymes in developing antennae of the moth Manduca sexta. Devel. Biol. 52, 105-120 Sanes, J. R., Hildebrand, J. G. and Prescott, D. J. (1976). Differentiation of insect sensory neurons in the absence of their normal synaptic targets. Devel. Biol. 52, 121-127 Sanes, J. R., Prescott, D. J . and Hildebrand, J. G. (1977). Cholinergic neurochemical development of normal and deafferented antenna1 lobes during metamorphosis of the moth Manduca sexta. Bruin Res. 119, 389-402 Sargent, P. B., Yau, K.-W. and Nicholls, J. G. (1977). Extrasynaptic receptorsoncell bodies of neurons in the central nervous system of the leech. J . Neurophysiol. 40, 446-452 Sattelle, D. B. (1977a). Cholinergic synaptic transmission in invertebrate central nervous systems. Trans. Biochem. SOC.5, 849-852 Sattelle, D. B. (1977b). A simple assay for the actions of toxic agents on synaptic transmission in the insect CNS. In “Crop Protection Agents: Their Biological Evaluation” (Ed. N. R. McFarlane) pp. 41 1-423. Academic Press, London and New York Sattelle, D. B. (19’78). The insect central nervous system as a site of action of neurotoxicants. 111 “Pesticide and Venom Neurotoxicity” (Eds D. L. Shankland, R. M. Hollingworth and T. Smyth Jr) pp. 7-26. Plenum Press, New York Sattelle, D. B. (1980). Cholinergic pharmacology of identified cells and pathways in the insect central nervous system. In “Neurotransmitters of Invertebrates” (Ed. K. S.-R6zsa). Hungarian Academy of Sciences

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Sattelle, D. B. and Callec, J. J. (1977a). Actions of isothiocyanates on the central nervous system of Periplaneta americana. Pestic. Sci. 8, 735-747 Sattelle, D. B. and Callec, J. J. (1977b). Actions of nereistoxin at an invertebrate central synapse. Proc. XXVIII Int. Congr. IUPS. Paris, p. 662 Sattelle, D. B., McClay, A. S., Dowson, R. J. and Callec, J. J. (1976). The pharmacology of an insect ganglion: actions of carbamylcholine and acetylcholine J . exp. Biol. 64, 13-23 Sattelle, D. B., David, J. A,, Harrow, I. D. and Hue, B. (1980). a-Bungarotoxin blocks acetylcholine responses in identified neurones of the cockroach Periplaneta americana (L.). In “Receptors for Neurotransmitters, Hormones and Pheromones in Insects” (Eds D. B. Sattelle, L. M. Hall and J. G. Hildebrand) pp. 125-139. Elsevier/North-Holland, Amsterdam Sattelle, D. B., Harrow, I. D., Hue, B., Gepner, J. I. and Hall, L. M. (1981a). a-Bungarotoxin blocks synaptic transmission between cercal afferent neurones and an identified interneurone of the cockroach Periplaneta americana (L.).J. exp. Biol. (in press) Sattelle, D. B., Harrow, I. D., Pelhate, M., Callec, J . J., Gepner, J . I. and Hall, L. M. (1981b). Nereistoxin: synaptic and axonal actions in the central nervous system of the cockroach Periplaneta americana (L.). J. exp. Biol. (in press) Schleifer, L. S. and Eldefrawi, M. E. (1974). Identification of the nicotinic and muscarinic acetylcholine receptors in subcellular fractions of mouse brain. Neuropharmacology 13, 53-63 Schlieper, P. and De Robertis, E. (1977). Lipid layers and liposomes in reconstitution experiments with cholinergic proteolipid from Torpedo electroplax. Biochem. Biophys. Res. Comm. 75, 886-894 Schmidt, J. (1977). Drug binding properties of an a-bungarotoxin-binding component from rat brain. Mol. Pharmacol. 13, 283-290 Schmidt, J. and Raftery, M. A. (1974). The cation sensitivity of the acetylcholine receptor from Torpedo californica. J . Neurochem. 23, 617-623 Schmidt, J., Hunt, S. and Polz-Tejera, G. (1979). Nicotinic receptors of the central and autonomic nervous system. I n “Neurotransmitters, Receptors and Drug Action” (Ed. W. B. Essman). Spectrum Publications, New York Schmidt-Nielsen, B. K., Gepner, J. I., Teng, N. N. H. and Hall, L. M. (1977). Characterization of an a-bungarotoxin binding component from Drosophila melanogaster. J. Neurochem. 29, 1013-1029 Segal, M., Dudai, Y. and Amsterdam, A. (1978). Distribution of an a-bungarotoxinbinding cholinergic nicotinic receptor in rat brain. Brain Res. 148, 105-119 Seifert, S. A. and Eldefrawi, M. E. (1974). Affinity of myasthenia drugs to acetylcholinesterase and acetylcholine receptor. Biochem. Med. 10, 258 Shain, W., Greene, L. A,, Carpenter, J. O . , Sytkowski, A. J. and Vogel, Z. (1974). Aplysia acetylcholine receptors: blockade by and binding of a-bungarotoxin. Brain Res. 72, 225-240 Shamoo, A. E. and Eldefrawi, M. E. (1975). Carbamylcholine and acetylcholinesensitive cation selective ionophore as part of the purified acetylcholine receptor. J . Membrane Biol. 25, 47-63 Shankland, D. L., Rose, J. A. and Donniger, C. (1971). The cholinergic nature of the cercal nerve-giant fibre synapse in the sixth abdominal ganglion of the American cockroach, Periplaneta americana (L.). J. Neurobiol. 2, 247-262 Smallman, B. N. (1956). Mechanism of acetylcholine synthesis in the blowfly. J . Physiol. Lond. 132, 343-357

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Smallman, B. N. (1975). Synthesis of acetylcholine in the blowfly (Calliphora erythrocephala). Pestic. Biochem. Physiol. 5, 170-183 Smith, D. S. and Treherne, J. E. (1965). The electron microscopic localization of cholinesterase activity in the central nervous system of an insect Periplaneta americana. J . Cell Biol. 26, 445 -465 Snyder, S. H. and Bennett, J. P. Jr (1976). Neurotransmitter receptors in the brain: biochemical identification. Ann. Rev. Physiol. 38, 153-175 Snyder,S. H.,Chang, K. J.,Kuhar,M.J. and Yamamura, H. I. (1975). Biochemicalidentificationof the mammalianmuscariniccho1inergicreceptor.Fedn.Proc. 34,191 5 -1 92 1 Sobel, A., Heidmann, T., Hoffler, J. and Changeux, J.-P. (1977). Distinct protein components from Torpedo marmorata membranes carry the acetylcholine receptor site and the binding site for local anaesthetics and histrionicotoxin. Proc. Natn. Acad. Sci. USA 75, 510-514 Strausfeld, N. J. (1976). “Atlas of an Insect Brain”. Springer-Verlag, Berlin Suga, N. and Katsuki, Y. (1961) Pharmacological studies on the auditory synapse in a grasshopper. J . exp. Biol. 38, 759-770 Szczepaniak, A. C. (1974). Effect of a-bungarotoxin and Dendroaspis neurotoxins on acetylcholine responses of snail neurones. J . Physiol. Lond. 241, 55-56P Thomas, W. E,., Brady, R. N. and Townsel, J. G . (1978). A characterization of a-bungarotoxin-binding in the brain of the horseshoe crab, Limulus polyphemus. Arch. Biochem. Biophys. 187,53-60 Tobias, J. M., Kollross, J. J. and Savit, J. (1946). Acetylcholine and related substances in the cockroach fly and crayfish and the effect of DDT. J . Cell Comp. Physiol. 28, 159-182 Treherne, J . E. and Smith, D. S. (1965a). The penetration of acetylcholine into the central nervous tissue of an insect Periplaneta americana. J . exp. Biol 43, 13-21 Treherne, J. E. and Smith, D. S. (1965b). The metabolism of acetylcholine in the intact central nervous system of an insect Periplaneta americana. J . exp. Biol. 43, 441-454 Triggle, D. J. and Triggle, C. R. (1976). “Chemical Pharmacology of the Synapse”. Academic Press, London Tripathi, R. K., Tripathi, H. L. and O’Brien (1979). Properties and affinity purification of the mixed-type putative acetylcholine receptor from wild and a mutant strain of house flies. Biochim. Biophys. Acta 586, 624-631 Twarog, B. M. and Roeder, K. D. (1956). Properties of the connective tissue sheath of the cockroach abdominal nerve cord. Bzol. Bull. 111, 278-286 Twarog, B. M. and Roeder, K. D. (1957). Pharmacological observations on the desheathed last abdominal ganglion of the cockroach. Ann. Ent. SOC. Amer. 50, 231 -236 Tyrer, N. M. and Bell, E. M. (1974). The intensification of cobalt-filled neuron profiles using a modification of the Timm’s silver sulphide method. Brain Res. 73, 151-154 Villegas, J. (1975) Characterization of acetylcholine receptors in the Schwann cell membrane of the squid nerve fibre. J . Physiol. Lond. 249, 679-689 Volle, R. L. and Koelle, G. B. (1975) Ganglionic stimulating and blocking agents. In “The Pharmacological Basis of Therapeutics” (Eds L. S. Goodman and A. Gilman) 5th edn, pp. 565-574. Macmillan, London Walker, R. J. and Hedges, A. (1968). The effect of cholinergic antagonists on the response to acetylcholine, acetyl-P-methylcholine and nicotine of neurones of Helix aspersn. Comp. Biochem. Physiol. 23, 979-989

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Walker, R. J. and James, V. A. (1978). The action of putative transmitters and related compounds on neurones in the abdominal ganglion of the horseshoe crab Limulus polyphemus Neuropharmac. 17, 765-769 Walker, R. J. and Kerkut, G. A. (1977). The actions of nicotinic and muscarinic cholinomimetics and a series of choline esters on two identified neurones in the brain of Helix aspersa. Comp. Biochem. Physiol. 56C, 179-187 Walker, R. J., Hedges, A. and Woodruff, G. N. (1968). The pharmacology of the neurones of Helix aspersa. Symp. Zool. SOC.lond. 22, 33-74 Walker, R. J., James, V. A., Roberts, C. J. and Kerkut, G. A. (1980). Neurotransmitter receptors in invertebrates. In “Receptors for Neurotransmitters, Hormones and Pheromones of Insects” (Eds D. B. Sattelle, L. M. Hall and J. G. Hildebrand) pp. 41 -52. Elsevier/North-Holland, Amsterdam Waser, P. G. (1960). The Cholinergic Receptor. 1. Pharm. Pharmac. 12, 577-594 Wastek, G. J. and Yamamura, H. I. (1978). Binding of [3H]-quinuclidinyl benzilate to human cerebral cortex. Mol. Pharmacol. 14, 768-780 Weber, M. and Changeux, J.-P. (1974). Binding of Naja nigricollis [3H]-a-toxin to membrane fragments from Electrophorus and Torpedo electric organs. Mol. Pharmacol. 10, 15-34 Wiersma, C. A. G., Furshpan, E. and Florey, E. (1953). Physiological and pharmacological observations on muscle receptor organs of the crayfish Cambarus clarkii Girard. J . Exp. Biol. 30, 136-150 Wilson, S. P. and Kirschner, N. (1977). The acetylcholine receptor of the adrenal medulla. J . Neurochem. 28, 687-695 Witzemann, V. and Raftery, M. (1978). Ligand-binding sites and subunit interactions of Torpedo californica acetylcholine receptor. Biochemistry 17,3598-3603 Woodruff, G. N., Walker, R. J. and Newton, L. C . (1971). The action of some muscarinic and nicotinic agonists on the Retzius cells of the leech. Gen. comp. Pharrnacol. 2, 106-1 17 Yamamoto, I., Kamirnura, H., Yamamoto, R., Sakai, S. and Goda, M. (1962). Studies on nicotinoids as an insecticide. I. Relation of structure to toxicity. Agr. Biol. Chem. 26, 709--716 Yamamoto, I., Soeda, Y., Kamimura, H. and Yamamoto, R. (1968). Studies on nicotinoids as an insecticide. VII. Cholinesterase inhibition by nicotinoids and pyridylalkylamines - its significance to mode of action. Agr. Biol. Chem. 32, 1341 Yamamura, H. I., and Snyder, S. H. (1974a). Muscarinic cholinergic binding in rat brain. Proc. Natn. Acad. Sci. USA 71, 1725-1729 Yamamura, H. I. and Snyder, S. H. (1974b). Muscarinic cholinergic receptor binding in the longitudinal muscle of the guinea pig ileum with [3H]-quinuclidinyl benzilate. Mol. Pharmacol. 10, 861 -867 Yamamura, H. I. and Snyder, S. H. ( 1 9 7 4 ~ )Postsynaptic . localization of muscarinic cholinergic receptor binding in the rat. Brain Res. 78, 320-326 Yamamura, H. I., Kuhar, M. J., Greenberg, D. and Snyder, S. H. (1974a). Muscarinic cholinergic receptor binding: regional distribution in monkey brain. Brain Res. 66,541 -546 Yamamura, H. I., Kuhar, M. J. and Snyder, S. H. (1974b). In vivo identification of muscarinic cholinergic receptor binding in rat brain. Brain Res. 80, 170-176 Yamamura, H. I., Enna, S. J. and Kuhar, M. J. (Eds) (1978). “Neurotransmitter Receptor Binding”. Raven Press, New York Yamasaki, T. and Narahashi, T. (1958). Synaptic transmission in the cockroach. Nature, Lond. 182, 1805-1806

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Yamasaki, T. and Narahashi, T. (1960). Synaptic transmission in the last abdominal ganglion of the cockroach. J. Insect. Physiol. 4, 1-13 Yarowsky, P. J. and Carpenter, D. 0. (1978). A comparison of similar ionic responses to y-Aminobutyric acid and Acetylcholine. J. Neurophysiol. 41, 531-541 Yavari, P., Walker, R. J. and Kerkut, G. A. (1979). The pA2 values of cholinergic antagonists on identified neurons of the snail Helix aspersa. Comp. Biochem. Physiol. 63C, 39-52 Yazulla, S. and Schmidt, J. (1977). Two types of receptors for a-bungarotoxin in the synaptic layers of pigeon retina. Brain Res. 138, 45-57 Zeimal, E. V. and Vulfius, E. A. (1968). The action of cholinomimetics and cholinolytics on Gastropod neurons. In “Neurobiology of Invertebrates” (Ed. J. Salanki) pp. 255-265. Acad. Kiado, Budapest

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Biogenic Arnines in the Insect Nervous System Peter D. Evans A. R. C. Unit of Invertebrate Chemistly and Physiology, Department of Zoology, Cambridge, UK

I 2

3 4

5

6

7

Introduction 318 The distribution of biogenic amines in the insect nervous system 320 2.1 Quantitative studies on biogenic amine distribution - an historical perspective 321 2.2 Cellular localization of biogenic amines 330 2.3 Subcellular location of biogenic amines 346 Metabolic studies on biogenic amines 349 3.1 Synthesis of biogenic arnines 350 3.2 Inactivation of biogenic amines 356 Octopamine and the dorsal midline neurones 365 4.1 Are the individual cells of the dorsal midline group uniquely identifiable? 367 4.2 The octopaminergic nature of D U M cells 373 4.3 DUMETi and the modulation of a myogenic rhythm 376 4.4 DUMETi and the potentiation of neuromuscular transmission 381 4.5 Studies on the terminal abdominal ganglion 387 4.6 Functions of D U M neurones and parallels with other systems modulated byamines 389 Biogenic amines and firefly light organs 394 5.1 Innervation of light organs 394 5.2 Pharmacology of light responses 397 5.3 Mode of action of neurotransmitter 400 5.4 Future studies on firefly light organs 402 Dopamine and insect salivary glands 402 6.1 Catecholamine distribution and innervation pattern 403 6.2 Effects of nerve stimulation and application of biogenic amines 406 6.3 Further studies on dopaminergic transmission in insect salivary glands 412 Biogenic amines and the insect heart 414 7.1 Innervation pattern 414 7.2 Biogenic amine distribution 417 7.3 Pharmacology of responses to biogenic amines 418 317

P E T E R D. EVANS

318

8 Biogenic amines in the control of gut muscle 420 8.1 Innervation of gut muscle 421 8.2 Pharmacological studies on gut muscle 422 9 Amines and neurohaemal organs 426 9.1 Corpora cardiaca 427

9.2 Median neurohaemal organs 429 9.3 Function of aniines in neurohaemal organs 433 10 Amine-stimulated adenylate cyclase activity 436 10.1 Studies on insect preparations 437 10.2 Comparisons with other invertebrate and vertebrate preparations 442 10.3 Cellular location of responses 444 10.4 Functional role in insects 444 11 Conclusions 445 Acknowledgements 449 References 449

1 Introduction

In the insect nervous system the presence of the catecholamines, dopamine and noradrenaline, and the indolalkylamine, 5-hydroxytryptamine (5-HT), has been known for some time and the presence of the phenolamine, octopamine, has recently been demonstrated (see Fig. 1 for structures). The

5 -HYDROXY TRYPTAMINE

H

w

ADRENALINE

Fig. 1 Structures of some biogenic amines

functional roles played by these amines in the insect nervous system have remained obscure, however, due to a lack of correlation between biochemical and histochemical data on the one hand, and physiological and pharmacological information from amine-containing neurones on the other. Several previous reviews have critically examined the evidence for the role of biogenic amines as potential neurotransmitters in the insect nervous system (Pitman, 1971; Murdock, 1971; Klemm, 1976). Relevant information is also collated in certain sections of other reviews (Davey, 1964;

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319

Kerkut, 1973; Gerschenfeld, 1973; Pichon, 1974; Axelrod and Saavedra, 1977; Hicks, 1977; Robertson and Juorio, 1976; Lafon-Cazal, 1978). The aim of the present review is to take a wider view of biogenic amines in the insect nervous system and to assess critically the evidence for their roles as neurotransmitters, neuromodulators and neurohormones. The term neurotransmitter will be used to define a chemical messenger that is released at a specialized synaptic structure and then diffuses across a narrow synaptic cleft to act on a specialized region of membrane on a post-synaptic cell. This cell may be another neurone, a muscle cell or a specialized gland cell. The specificity in this form of communication is dependent on the anatomical distribution of the synapses made by the pre-synaptic cell. The term neurohormone will be used to refer to chemical messengers released from the nervous system into the circulatory system of the insect, but will be modified to “local neurohormone” to describe the release of a messenger within a localized region of the nervous system or a particular end organ. In the case of neurohormones, both general and local, the chemical message is not necessarily confined to a single anatomically apposed post-synaptic cell, but can affect many post-synaptic cells, the specificity of the system relying on the distribution of cells with appropriate receptors. The currently favoured term “neuromodulator” will be used to designate the special case of a neurohormone that either changes the quality of the information being passed through a conventional synapse, or changes the spontaneous activil. of a receptive neurone or muscle cell. In the case of neurohormones, lo,d neurohormones, and neuromodulators, no specific synaptic structures are present and release often occurs from “blindly-ending neurosecretory terminals”. The above definitions obviously represent the extremes of a continuum, making it difficult to define absolutley the point where one category finishes and another starts. The terms, however, appear to convey useful distinctions. This review will attempt to answer the following questions. Where are biogenic amines located in the insect nervous system? How are they synthesized and inactivated? What physiological roles d o they perform and how do they bring about their effects? Particular emphasis will be placed on the correlation of biochemical, physiological, pharmacological and anatomical information from systems containing identified aminergic neurones. Attempts will be made to point out instances where data from one or more of the above approaches is either missing o r conflicts with that from another and experiments will be suggested to resolve these points. When appropriate, the function of biogenic amines in the insect nervous system will be compared with that in the vertebrate nervous system. In this context it is appropriate to summarize here what is known of the role of biogenic amines as chemical messengers in the vertebrate nervous system.

320

PETER

D. E V A N S

In vertebrates, biogenic amines serve as neurotransmitters, and neuromodulators, and also as true hormones, which are released into t h e circulatory system. Dopamine, noradrenaline and 5-HT act as neurotransmitters in the central nervous system (KrnjeviC, 1974) whilst recent evidence suggests that noradrenaline and 5-HT may also serve as central neuromodulators (Dismukes, 1977a). In addition, noradrenaline is released from the terminals of the peripheral sympathetic nervous system where it acts as a transmitter or local neurohormone (Smith, 1973). The third well known member of the catecholamine family, adrenaline, is a circulatory hormone released from the cells of the adrenal medulla and has widespread actions throughout the body (Douglas and Rubin, 1963; see also Berridge, 1975). Evidence is also accumulating for an adrenaline based neural pathway in the vertebrate brain, but the extent of this pathway appears small compared to the corresponding dopamine and noradrenaline pathways, and its physiology and function have yet to be investigated (Moore and Bloom, 1979). Recently attention has also been focussed on the possible role of the phenolamines, octopamine and tyramine, as neurotransmitters or neuromodulators in the vertebrate nervous system, but little positive evidence has been presented to date (see Boulton, 1976; Hicks, 1977; Evans, 1978a; Hicks and McLennan, 1978a, b). Finally, it is worthwhile to re-emphasize one of the major themes of the present review. In contrast to the vertebrate nervous system, the insect nervous system, along with those of many other invertebrates, often presents the researcher with the advantage of working with single, physiologically identified aminergic neurones. This advantage arises from the large size of many of the neuronal somata, which can be isolated for biochemical studies, and also from the relative ease with which the same individual neurone can be identified from one preparation to the next (e.g. for 5-HT see McAdoo, 1978; Qsborne, 1978; for dopamine see Berry and Pentreath, 1978; for octopamine see Evans, 1978b; for histamine see Weinreich, 1978). But, for the most part these attributes have not yet been fully exploited in studies of biogenic amines in the insect nervous system. It is hoped that this review will serve to focus attention on potentially rewarding areas of future research.

2

The distribution of biogenic amines in the insect nervous system

Studies on the distribution of biogenic amines in the insect nervous system have attempted to answer a number of questions of increasing complexity. Initially the questions asked were, which amines are present, in which ganglia are they contained, and how much of each is present? Subsequently,

BlOGENlC A M l N E S I N THE INSECT NERVOUS SYSTEM

321

questions were raised about which cells in the ganglia contained the amines and where they were localized at a subcellular level. More recently attention has centered on the functional roles fulfilled by the various amines present in identified aminergic neurones. In the present section, information on the cellular and subcellular distribution of biogenic amines in the insect nervous system will be reviewed from an historical viewpoint. Throughout the section attempts will be made to examine the extent to which information on the distribution of biogenic amines has contributed to an understanding of their different functional roles. 2.1

QUANTITATIVE STUDIES O N BIOGENIC AMINE DISTRIBUTION - A N HISTORICAL PERSPECTIVE

2.1.1 Fluorescence-based assays The use of fluorescence-based assays has provided evidence for the presence of dopamine, noradrenaline and 5-HT in the insect nervous system. Early studies on biogenic amines in insects were made on homogenates of whole insects using relatively insensitive fluorescent techniques, e.g. Wense (1939); Ostlund(1953); Dresseetal. (1960). These studiessuggested that a wide variety of insect species contained dopamine, noradrenaline and adrenaline, but conflicting results were obtained by Gregerman and Wald (1952) and von Euler (1961) who were unable to detect any adrenaline. The results of the above studies on whole insects tell us very little about the aminergic content of the insect nervous system for several reasons. First, dopamine and its derivatives are found in insect cuticle, where they are thought to be involved in the tanning process (Sekeris and Karlson, 1966; Andersen, 1979). Second, catecholamines occur in the venom glands of some Hymenoptera (Owen, 1971; Ishay et al., 1974) and third, insect haemocytes contain many enzymes capable of synthesising and metabolizing biogenic amines (see section 3 present review). One interesting result, that has emerged from studies on catecholamine levels in homogenates of whole insects, comes from the genetic selection experiments of Tunnicliff et al. (1969). These authors selected two behakioural strains of Drosophila for locomotor activity. They found that noradrenaline levels were highest in the active strain and lowest in the inactive, whilst dopamine levels were highest in the inactive strain m d lowest in the active strain. Unselected control strains had intermediate levels of both amines. These results are obviously open to all the criticisms outlined above and tell us nothing about changes in amine levels within the nervous system, but they suggest an interesting area for research on the role of catecholamines in the modulation of behaviour, especially if more precise techniques for the localization of catecholamines in the nervous system are employed.

PETER D. EVANS

322

The advent of the Falck-Hillarp histochemical technique for the localization of catecholamines and indolalkylamines (see Falck and Owman, 1965) led to the demonstration of the presence of catecholamines in the insect nervous system. Frontali and Norberg (1966) and Frontali (1968) were able to show the presence of a primary catecholamine (either dopamine or noradrenaline -it was not resolved which), but not adrenaline, in the central body and p-lobes of the corpora pedunculata (mushroom bodies) of the cockroach, Periplaneta americana (see Fig. 2 for details of anatomy). The GC

1

MC

LC

I

I

Fig. 2 Diagram of the brain (cerebral ganglia) of the adult cockroach, viewed from the front, to show position of regions of highly structured neuropile. The basic arrangement of a typical globuli cell is illustrated schematically within each corpus pedunculatum. The various regions of the corpus pedunculatum are labelled as follows: A, a-lobe; B, P-lobe; P, pedunculus; MC, medial calyx; LC, lateral calyx; GC, globuli cells. Other labelling: PROTO, protocerebrum; DEUTO, deutocerebrum; TRITO, tritocerebrum; PI, pars intercerebralis: PB, protocerebral bridge; CB, central body; OL, optic lobe; ASN, antennal sensory nerve; and GA, glomeruli within antennal sensory lobe. (Adapted from Weiss, 1974)

Falck-Hillarp technique is able to localize catecholamines to cell bodies and nerve processes but the fluorophore reaction is not quantitative (Lindvall et al., 1974). The amounts of dopamine and noradrenaline present in cockroach brain were measured using a trihydroxyindole based fluorescence assay (Frontali and Haggendal, 1969). The results from this study, together with those of other fluorescence based determinations of catecholamines in insect nervous tissue (Hiripi and S.-Rozsa, 1973; Klemm and Axelsson, 1973; Kusch, 1975) are summarized in Table 1 . In all cases much more dopamine was found than noradrenaline and no adrenaline could be detected in the majority of the studies. The weight of evidence is thus against the presence of adrenaline in the insect nervous system. Only two studies have presented evidence against this conclusion. In the first, adrenaline is reported to be present in the brain of

323

B l O G E N l C A M l N E S IN T H E I N S E C T NERVOUS S Y S T E M

TABLE 1 Biogenic amine content of insect nervous tissue expressed as nglg wet weight Species

Tissue

Periplaneta americana

Cerebral ganglion Suboesophageal ganglion Thoracic nerve cord Prothoracic ganglion Mesothoracic ganglion Metathoracic ganglion Abdominal ganglia 1-5 6th Abdominal ganglion

Schirrocerca Cerebral ganglion gregariu Optic lobes Brain minus optic lobes Whole C.N.S. Leg muscle Salivary gland Fat body Locusta migratoria

Cerebral ganglion Suboesophageal ganglion

Melamplus CNS sanguinipes Acheta domesticus

Octopamine

1 3450,425w 9 4 ~

Dopamine )2500c,

107@

Noradrenaline 5-HT

) 370c

21 50'

20504 1460' 1580' ll5od 27md

490' 350d 1804 130d 310d

} lolw

8706, 800' 66@ 7706

24306 39106 860 18206 9w <23. <18'

]lob,

70'*

790-1470'

33w

>

1310*

240k

2340*

5 OW

1430-2600'

Brain

Formica rufa Brain

9480"

4960-7920' 8090"

6470-9990'

~

Blaberus giganticus

Nerve cord

Anabolia nervosa

Whole head Whole thorax

Munduca sexta

Thoracic ganglion

30W

Photuris

Light organ containing segments

125"

versicolis

* Sum of adrenaline and noradrenaline n.d. = non-detectable ' Robertson and Steele (1974) Robertson (1976) ' Frontali and Haggendal (1969) Kusch (1975) ' Klemm and Axelsson (1973) Robertson (1 975) Robertson and Juorio (1976)

'

<20k 3520, 3260' 5100, 2420"

320, 750,

290' 350"

* Hiripi and S.-Rozsa (1973)

n.d.

' Muszynska-Pytel and Cymborowski (1978a)

Kostowski et al. (1975a) Welsh and Moorehead (1960) Klemm and Bjorkland (1971) Bjorkland et al. (1970) " Robertson and Catlson (1976) Kostowski etal. (1975b) 1

'

324

PETER D. E V A N S

the ant, Formica rufa, at levels (2800-5970 ng/g) equal to or slightly lower than those of noradrenaline (Kostowski et al., 1975a; Kostowski et al., 1975b). It should be noted, however, that the results of Kostowski et al. (1975a, b) for dopamine and noradrenaline (see Table 1)appear to be about an order of magnitude higher than most of the other results obtained for insect nervous tissue. It is not clear at present if these anomalous results are due to the non-specificity of the fluorimetric assay method used, that of Chang (1964), or alternately represent species differences between ants, cockroaches and locusts. In the second, Armati and Gilmour (1976) report the possible presence of adrenaline in the brain and ventral nerve cord of the Queensland fruit fly, Dacus tryoni, using the trihydroxyindole method of Angelakos and King (1967) which is reported to be specific for secondary catecholamines, such as adrenaline. Doubts, however, seem to exist over the specificity of the latter reaction for adrenaline since it did not work on sections of the adrenal medulla, a tissue known to contain large amounts of this catecholamine (Angelakos and King, 1967). Unfortunately, no further studies have been reported on either F. rufa or D . tryoni to allow us to determine the reproducibility of the above observations. It would be of considerable interest to see if the radioenzymatic assay methods for catecholamines, which have to date failed to demonstrate the presence of adrenaline in any insect nervous system (see below), would give positive results with nervous tissue from the above two species. 5-Hydroxytryptamine (5-HT, serotonin) has been shown to be present in the nervous system of the cockroach, P. americana, by the use of chromatography, UV absorption and spectrofluorometric techniques (Gersch et al., 1961; Colhoun, 1963, 1967). The amount of 5-HT present has also been quantified in nervous tissue from the locusts, Schistocerca gregaria (Klemm and Axelsson, 1973) and Locusta migratoria migratorioides (Hiripi and S.-Rozsa, 1973) and from the brain of the ant F. rufa (Kostowski et al., 1975a). In the cockroach, P. americana, Kusch (1975) demonstrated that large amounts of 5-HT could be measured in all the ganglia of the central nervous system, the highest levels being found in the sixth abdominal ganglion. Osborne and Neuhoff (1974a) also estimated the amount of 5-HT in P. americana nervous tissue by the use of a microdansylation prodecure but it should be noted that their values for “brains” were obtained by using metathoracic ganglia. There does however seem to be some species differences in the 5-HT content of insect nervous tissue since Klemm and Bjorklund (1971) could not detect any 5-HT in extracts of caddisfly (Anabolia nervosa) brains despite the fact that its presence had been demonstrated histochemically in the stomatogastric system of the same insect (Klemm, 1968). The available quantitative data on 5-HT distribution in insect nervous tissue is summarized in Table 1.

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325

It has been suggested on several occasions that the activity levels of insects can be correlated with diurnal rhythms in the 5-HT content of their brains. Hinks (1967) is often quoted as demonstrating changes in the 5-HT content of certain median protocerebral neurosecretory cells in the brain of noctuid moths. However, Hinks’ investigations only appear to demonstrate a “large concentration of tryptophan” (but could be tryptophan-containing proteins or peptides?) in these cells, not 5-HT itself, and the paper does not report anything about the diurnal activity pattern of these cells. Hinks does report that injections of 5-HT, but not of adrenaline, noradrenaline, tryptamine and tyramine, cause an increase in the duration and amplitude of night flight in these moths. Fowler et af. (1972) describe diurnal fluctuations in 5-HT levels of Drosophila meianogaster which appear to correlate with their locomotor patterns, but the estimations were done on whole insects and the extent of changes in the 5-HT content of the nervous system is unknown. The recent work of Muszynska-Pytel and Cymborowski (1978a) could find no correlation between circadian rhythms of locomotor activity in the cricket, Achetu domesticus, and 5-HT levels in brains or haemolymph. Nevertheless, in a companion paper Muszynska-Pytel and Cymborowski (1 978b) claim to provide histochemical evidence for diurnal fluctuations in 5-HT levels in the central body. The latter brain region is hypothesised to liberate 5-HT during the night to inhibit the inhibitory effect of neurosecretory cells in the pars intercerebralis, in turn resulting in an increase in locomotor activity. However, as the authors point out, their histochemical technique for 5-HT localization (that of Adams, 1957) is not specific for 5-HT and also reacts with tryptophan, 5-hydroxytryptophan and 5-hydroxyindole acetic acid. Thus at present the evidence for diurnal changes in 5-HT levels regulating locomotor activity in insects is very poor. However, in mammals and birds the pineal gland is thought to be involved in the regulation of rhythmic locomotor activity, possibly through its production of the hormone melatonin (see Kasal et al., 1979). Melatonin production is controlled by marked diurnal changes in the activity of the enzyme serotonin-N-acetyltransferase (Klein and Weller, 1970; Kasal et al., 1979) which is also the enzyme responsible for regulating the 5-HT content of the pineal gland (Klein et al., 1971). In mammals the above system appears to be controlled by noradrenaline released from a sympathetic input to the pineal gland from the superior cervical ganglion (Deguchi and Axelrod, 1972; Axelrod, 1974). Thus the diurnal content of 5-HT in insect brain needs to be measured using a specific radioenzymatic assay ( e g Saavedra et af ., 1973) and correlated with the activity levels of N-acetyltransferase, to see if the other biogenic amines present in the insect CNS, such as octopamine and noradrenaline, can exert a “sympathetic” control on this system.

PETER D. EVANS

326

2.1.2 Radioenzymatic assays Recent investigations on biogenic amines in insect nervous tissue have employed radioenzymatic assays of much greater sensitivity than the fluorescence based assays described above (Robertson and Steele, 1974; Robertson, 1976; Evans, 1978c; Dymond and Evans, 1979). The use of radioenzymatic assays besides giving more accurate estimates of the amounts of catecholamines present in the insect nervous system has also demonstrated the presence of large amounts of the monophenolic amine, octopamine, which does not form a fluorescent derivative with the FalckHillarp technique. Octopamine was demonstrated to be present in nervous tissue from the cockroach, P. americana, by Robertson and Steele (1974) using the radioenzymatic assay devised by Molinoff et al. (1969). Robertson (1976) later used this method to measure octopamine in a partial analysis of the nervous system of the locust, Schistocerca gregaria and also presented data on the distribution of dopamine and noradrenaline, assayed using the catechol-0-methyl-transferase method of Cuello et al. (1973). No adrenaline was detected by Robertson (1976). The above studies only reported biogenic amine measurements for a limited number of regions of the insect nervous system and they are compared in Table 1 with the values obtained from assays based on fluorescent techniques. More extensive measurements of biogenic amine levels (Evans, 1978c; Dymond and Evans, 1979) are discussed below (Section 2.1.3). 2.1.3

Concentration versus total amount per structure

In the studies summarized in Table 1 the results have been expressed on the basis of the amount of biogenic amine present per unit wet weight of nervous tissue. Whilst providing useful data on the amounts of each amine present in a given piece of insect nervous system, this method of expressing the data has certain drawbacks (Evans, 1 9 7 8 ~ ) . First, the wet weight of particular portions of the insect nervous system varies with the age of the adult animal in different ways (Sbrenna, 1971; Evans, 1 9 7 8 ~ )the ; amount of connective tissue in the ganglionic sheath, for instance, increases considerably with the age of the animal. The biogenic amine content, however, appears to remain unchanged (see Evans, 1978c, for octopamine values) so that many of the previously made comparisons on biogenic amine levels in insects are only of limited usefulness as the ages of the insects used and the weights of individual pieces of tissue were not presented. Second, comparisons of the biogenic amine content in nervous tissue from different species expressed on a unit wet weight basis (e.g. as in Robertson

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327

and Juorio, 1976) may also be misleading since the cell density may vary between different species (see Klemm, 1976 for some examples). This is particularly important from a neurobiological point of view where one is trying to estimate the number of amine-containing cells and their innervation patterns in a certain portion of the nervous system. It seems that differences in neuronal density may account for many of the differences in the quantitative distribution reported for biogenic amines between various species and not necessarily reflect any functional differences for different amines in different species. A more useful comparison can probably be made on the basis of the relative amounts of different biogenic amines present in a given sample of nervous tissue (Robertson and Juorio, 1976). At the present time the most useful way to express the biogenic amine content of portions of insect nervous tissue appears to be on the basis of the number of moles of the biogenic amine present per piece of tissue analysed taking a careful note of the age of the donor (e.g. for octopamine Evans, 1978c; and for dopamine and noradrenaline, Dymond and Evans, 1979). An understanding of the significance of the differing amounts of each amine present will require the identification of the aminergic neurones present and a description of their innervation patterns. The most extensive studieson the distribution of biogenic amines in insect nervous tissue have employed radioenzymatic assay techniques and have concentrated upon the nervous systems of the locust and the cockroach (Evans, 1978c; Dymond and Evans, 1979. See also Table 2). The above studies point out the similarity in the biogenic amine distribution between the nervous systems of the locust and the cockroach. In each case the amines are highly localized in the ventral ganglia and, especially, in the cerebral ganglia. In the ventral ganglia some of the octopamine is presumably associated with the medial group of dorsal unpaired cells, which in the locust (Evans and O’Shea, 1977, 1978) and the cockroach (Dymond and Evans, 1979) has been shown to contain some,octopaminergic cells. In both of these systems, the cells of the dorsal unpaired medial system have been shown to selectively stain with the dye Neutral red (Evans and O’Shea, 1977, 1978; Dymond and Evans, 1979). Neutral red specifically stains amine-containing cells in the nervous systems of the leech (Stuart et al., 1974) and the lobster (Wallace etal., 1974; Evansetal., 1976b). As no cell bodies are found in the interganglionic connectives of the locust and cockroach, the octopamine in these regions is presumably contained in the processes of cells with their somata in the ganglia. The functional role of the dorsal unpaired medial cells will be considered in a later section (see Section

4). In most of the ganglia of the cockroach ventral nerve cord there appear to be about 2 times as much octopamine as dopamine and about one tenth as

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328

TABLE 2 Biogenic arnine content of locust and cockroach nervous tissue as estimated by radioenzymatic assay Locust

Nervous tissue

Neurohaemal tissue

Cerebral ganglion Optic lobes (per pair) Retina Lamina Medulla Lobula Suboesophageal ganglion Corpora cardiaca (per pair) Corpora allata (per pair) Whole thoracic nerve cord Prothoracic ganglion Pro-mesothoracic connectives (per pair) Mesothoracic ganglion Mesothoracic NHO medial ' Meso-meta thoracic connectives (per pair) Metathoracic ganglion

Cockroach

Octo- Octo- Dopa- Noradrenpamine" pamine" mineb aline' Ratiob Ratiob pmole pmole prnole pmole DA/NA DA/OCT

11.58

14.68

26.81

5.53

4.84

1.83

17.16 0.33 0.34 6.10 2.03

7.24 0.62 0.35 1.90 0.75

4.72

5.60

3.29

0.33

9.97

0.59

0.47

1.20

0.19

0.11

1.73

0.16

0.15

0.03

0.02

n.d.

-

0.67

8.27

-

-

-

2.61

0.29

9.00

0.49

2.25

0.23

9.78

0.52

-

-

-

5.42

5.28

0.70

1.39

6.23

4.33

0.25 0.63

1.22

4.26

5.32

-

2.14

0.16

13.38

0.40

much noradrenaline as dopamine. The amount of 5-HT present varies from 2 times to 7 times the amount of dopamine in the different ganglia of the cockroach nerve cord. However, in the terminal abdominal ganglion there is 3-4 times as much octopamine and 9 times as much 5-HT as the amount of dopamine. In the cerebral ganglia of both the cockroach and locust there appears to be roughly the same amounts of octopamine and dopamine. The optic lobes of the cockroach have three times as much octopamine as dopamine (7.2 pmoles per pair for OCT and 2.4 pmoles per pair for dopamine) (Dymond and Evans, 1979), whilst those of the locust have six times as much (Robertson, 1976). The above data (Table 2) also suggest that relatively large amounts of octopamine are found in the isolated

BlOGENlC A M l N E S I N THE I N S E C T N E R V O U S S Y S T E M

Locust

Nervous tissue

Neurohaemal tissue Metathoracic medial NHO 1st' Metathoracic medial NHO 2nd' Metathoracic medial NHO 3rd' Metathoracic medial NHO 4th'

Whole abdominal nerve cord Abdominal ganglion' Abdominal connectives (per pair) Abdominal ganglion 1 2 3 4 5 Terminal abdominal gangliond

329 Cockroach

Octo- Octo- Dopa- Noradrenpamine" pamine" mineb alineb Ratiob Ratiob pmole pmole pmole pmole DA/NA DA/OCT 0.25

-

0.15

-

n.d.

-

n.d.

-

-

-

1.04 -

-

-

1.12 1.04 1.02 1.13 1.44

0.23 0.24 0.18 0.24 0.21

4.87 4.33 5.67 4.71 6.86

0.72 0.67 0.65 0.72 0.92

2.73

1.53

0.05

30.60

0.29

0.05

0.33

0.14

2.36

4.71

0.36 -

Abdominal medial NHO'

Cockroach: Periplaneta americana; Locust: Schistocerca americana gregaria . "Data reproduced from Evans, 1978c; Data reproduced from Dymond & Evans, 1979; Information on standard errors and number of observations can be obtained from original papers; ' Values obtained from locust abdominal ganglia 1-4 pooled and cockroach abdominal

-

ganglia 1-5 pooled. Values expressed per abdominal ganglion. Terminal abdominal ganglion is 5th and 6th abdominal ganglion of locust and cockroach respectively; Values per neurohaemal organ; n.d. none detectable - = not determined

medulla compared to the other neuropile regions of the optic lobe in both the locust and cockroach (see Section 2.2.4 for a detailed discussion of biogenic amines in the optic neuropile). The association of biogenic amines with the medial neurohaemal organs of insects is also indicated and will be discussed in a separate section (see Section 9). It can be seen in the present section that the most detailed studies on the distribution of biogenic amines have concentrated upon the nervous systems of the cockroach and the locust. As yet, very little information is available on the distribution of amines in other insect species. Studies on other insects could well reveal interesting anomalies worthy of further study. The above data, whilst providing us with valuable information on the distribution of

PETER D. E V A N S

330

biogenic amines in insect nervous tissue, makes very little contribution to our understanding of the functional roles played by the biogenic amines. It emphasizes the need to determine the cellular location of the amines and to work on single physiologically identified neurones Cvhenever possible.

2.2.

CELLULAR LOCALIZATION OF BIOGENIC AMINES

An understanding of the roles played by biogenic amines in the insect nervous system requires the identification of the cells containing the amines. The histo-fluorescence based technique of Falck-Hillarp has provided the bulk of information, to date, on the location of cell bodies and nerve fibres containing catecholamines and 5-HT in the insect nervous system. More recently, Neutral red staining has provided information about the cellular localization of other biogenic amines that d o not form fluorescent derivatives with the Falck-Hillarp method, e.g. octopamine. The application of radioenzymatic assays to groups of microdissected identified cells has also been recently used to study the functional role of octopamine in insect nervous tissue and potentially offers a most powerful technique for similar studies of other biogenic amine-containing neurones in insects. Studies on the histochemical localization of the biogenic amines dopamine, noradrenaline and 5-HT, using the Falck-Hillarp technique have concentrated on the insect “brain”, or cerebral ganglia, contained in the head. An excellent reveiw of these studies has been presented by Klemm (1 976) and the present article will only consider these results briefly, paying specific attention to more recent studies and reveiwing the information available on the roles of the biogenic amines in the various regions of the insect brain. The insect cerebral ganglia can be divided up into a number of clusters of cell bodies and specific neuropil regions such as the corpora pedunculate, central body complex, optic lobes, deutocerebrum and tritocerebrum (see Fig. 2). The location of biogenic amines will be considered in each of these regions in turn.

2.2.1 Location of aminergic cell bodies The first histochemical demonstration of catecholamine containing neurones in insect nervous tissue was made on the brain of the cockroach, Periplaneta americana by Frontali and Norberg (1966) and Frontali (1968). A schematic drawing of their findings showing the distribution of catecholamine-containing cell bodies in the brain of the cockroach is shown in Fig. 3A. For comparison a map of the catecholamine and indolalkylamine-containing cells of the locust brain is shown in Fig. 3B. Klemm (1976) has pointed out that cell bodies with catecholamine fluorescence

BlOGENlC AMlNES I N THE INSECT

NERVOUS SYSTEM

33 1

A GC

B

LO

c1

PI

Fig. 3 Schematic representation of monoamine-containing structures in the insect brain as revealed by histofluorescent techniques. A. The brain of cockroach, Peripluneta umericana. An anterio-posterior series of frontal sections, showing the components of the corpora pedunculata. The groups of cells which develop yellow-green (specific) fluorescence are marked in black (from Frontali, 1968). B. The brain of an adult locust, Schistocerca gregariu Forsk. Right side, frontal plane; left side, caudal plane. Stippled areas indicate where monoamine-containing fibres are found. The densely stippled areas have a dense supply of catecholamine fibres, whereas the moderately stippled areas have only a sparse supply. The widely stippled areas contain fibres with a fluorescence of indole type (LO, PB, and outer part of LA). The filled circles represent catecholamine-containing cell bodies, whereas the open circles represent cell bodies with indoleamine-induced fluorescence. C,, 3 yellow fluorescent cell bodies in the caudal part of the pars intercerebralis; C,, yellow fluorescent cell bodies in the optic lobe; C, calyx; D, deutocerebrum; LA, lamina; LO, lobula; M,medulla; all other labelling as in Fig. 2. (From Klernm and Axelsson, 1973)

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332

have been found in all regions of the cell body layer of the brains of all insect species examined to date. These cell bodies are arranged in groups or occur singly. They are frequently localized in the posterior regions of the pars intercerebralis (Frontali, 1968; Klemm, 1971; Klemm and Axelsson, 1973). Monoamine-containing cell bodies are also located among the neurosecretory cells of the anterior pars intercerebralis of several species including silver fish (Lepisima saccharina), house crickets (Acheta domesticus), locusts (Schistocerca gregaria and Locusta migratoria , Klemm and Falck, 1978) and nymphs and adults of dragonflies (Aeschna viridis and Ae. cyanae) (see Klemm, 1976 for additional references). The relationship between the various groups of biogenic amine-containing cell bodies and the amine-containing nerve fibres of the various neuropile regions to be discussed below has not yet been resolved. 2.2.2

The corpora pedunculata or mushroom bodies

( a ) Distribution ojbiogenic amines The corpora pedunculata or mushroom bodies are highly structured regions of neuropile found in the protocerebra of most insects (see Fig. 2). The details of their morphology vary from species to species but they typically consist of one or two calyces, attached to one or two pedunculi (or stalks) which bifurcate to give rise to one a-lobe and either one or two @-lobes.The majority of the fibres present in this structure are the axons of the globuli cells (Schiirmann, 1973) (equivalent to Kenyon cells, cf. Kenyon, 1896 and intrinsic cells, Pearson, 1971) which enter through t h e calyx and give rise to the intrinsic fibres of the pedunculi and the a and 0 lobes. The most highly developed insect mushroom bodies are found in the social Hymenoptera, such as female wasps and bumble bees where they can occupy up to 50% of the protocerebrum, whilst they are absent in the apterygote, Machilidae (see Bullock and Horridge, 1965, and Klemm, 1976 for more details of different species). The detailed distribution of monoamine-containing fibres in the mushroom bodies varies from species to species (see Klemm, 1976). In general, no monoamines have been detected histochemically in the globuli cell bodies or in the calyx itself. The globuli cell bodies even remain nonfluorescent after in vitro treatment with highly fluorogenic compounds such as a-methyl noradrenaline, 6-hydroxytryptamine and L-Dopa (Klemm, 1976). In the calyx, a monoamine-containing fibre system has only been shown to be present in some Hymenoptera, such as the hornet Vespa crabro and the honey bee Apis mellifera, where it may represent a migration of a visual integrative centre from the optic lobes to the mushroom bodies (Klemm, 1976). In other species, only occasional monoamine-containing fibres can be found at the margins o r penetrating the calyx, e.g. in locusts

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333

(Klemm, 1976), in the cricket Acheta domesticus (Schurmann and Klemm, 1973), in the noctuid moth Spodoptera and in the blowfly, Calliphora vomitoria (Klemm, 1976). In all these examples, however, the location of the cell bodies of these aminergic fibres to the calyces remains unknown. The distribution of monoamine-containing fibres in the pedunculus, as seen in histofluorescent studies, again varies from species to species (see Klemm, 1976). They are totally absent in the cockroach, Periplaneta americana (Frontali, 1968), the silverfish, Lepisma, and in various species of Trichoptera (see Klemm, 1976). In the locust, occasional longitudinally oriented fibres can be found (Klemm and Axelsson, 1973) whilst in the cricket, A . domesticus, the longitudinal zonation is very pronounced in the half of the pedunculus furthest from the calyx and there is a fluorescencefree cylinder in the middle of the pedunculus (Schurmann and Klemm, 1973). The pedunculi of bees, hornets and scorpionflies are filled completely with green fluorescent varicose fibres (Klemm, 1476). No monoaminecontaining fibres have been described to enter the pedunculus (Klemm, 1976). In the a and 8 lobes of the mushroom bodies, biogenic amine fluorescence has been detected In most species examined. An exception is in the silverfish L . saccharina where catecholamines appear to be located in bud-like regions on the pedunculus and a- and 8-lobes (Klemm, 1976). In some species a very pronounced banding pattern of zones of different fluorescent intensities is found in both lobes, e.g. in Periplaneta (Frontali, 1968). ( b ) Are theglobuli cells adrenergic? The location of the cell bodies giving rise to the catecholamine-containing fibres of the pedunculus and the a and 8 lobes seems to be a matter of debate. On the one hand it has been suggested that the globuli cells themselves are adrenergic (Schurmann and Klemm, 1973; Klemm, 1976) and give rise to the intrinsic adrenergic fibres in the mushroom body. In contrast, the intrinsic fibres have been suggested to be non-adrenergic, non-serotonergic and non-cholinergic, whilst the adrenergic fibres of the mushroom body are said to be of extrinsic origin, from cell bodies of unknown location (Frontali and Mancini, 1970; Frontali et al., 1971). If the globuli cells are adrenergic ,it is difficult to understand why they d o not fluoresce even when they are incubated with highly fluorogenic compounds (Klemm, 1976) and also why fluorescence is absent from the calyces and pedunculi of many species. Schurmann and Klemm (1973) argue that the cell bodies d o not fluoresce because they contain only a very low concentration of catecholamines, most of which are concentrated in the highly aggregated terminal regions of the cells in the fluorescent regions of the pedunculus and a and 8 lobes. The above argument would also suggest that the dendritic arborizations of

334

PETER D. EVANS

the globuli cells in the neuropile of the calyx (see Pearson, 1971; Strausfeld, 1976) would also lack sufficient amounts of catecholamines to produce a significant fluorescence. This would mean that the cells were capable of selectively directing adrenergic vesicles along their main axons, preventing them from being concentrated in their dendritic regions in the calyx. Such a theory would be of great interest since it seems to be contradictory to recent results on the accumulation and release of catecholamines from the dendrites of vertebrate catecholaminergic neurones (Bjorklund and Lindvall, 1975; Sladek and Parnavelas, 1975; see also Dismukes, 1977b). It will be of interest to see if the dendritic arborizations of invertebrate monopolar neurones also accumulate and release transmitter substances. Strausfeld (1976) points out that the zones of fluorescence in the pedunculi and lobes correspond to the zonation of spines found on the globuli cell fibres, whilst Schiirmann and Klemm (1973) point out that these fluorescent zones also correspond to the regions of the mushroom bodies containing the largest number of vesicles, as observed in the electron microscope. However, neither of these two pieces of information helps to resolve which cell type contains the catecholamines. It is not clear if the globuli cell spines are input or output regions for the cells and in either case they will, presumably, be associated with extrinsic fibres. Also the association of vesicle density with fluorescent intensity does not provide evidence for a pre- or postsynaptic location of the fluorogenic amines. Schiirmann and Klemm (1973) and Klemm (1976) also argue that because many of the adrenergic fibres in the lobes and pedunculi of locusts and crickets run longitudinally, they must be the intrinsic fibres from the globuli cells. However, Strausfeld (1976) figures several extrinsic neurones that enter the lobes and pedunculi, and then divide to give longitudinally oriented fibres. Evidence from electron microscopical studies on the mushroom bodies is somewhat equivocal. Although many terminal structures contain vesicles with similar characteristics to known adrenergic vesicles in other tissues (Schurmann and Klemm, 1973; Mancini and Frontali, 1970), positive evidence that they represent the terminals of the globuli cells, and for the actual identity of the transmitter they contain, is lacking. It is thus obvious that the evidence presented above is not sufficient to determine if the globuli cells are adrenergic or not. A possible resolution of the above, apparently conflicting evidence, would be provided if the globuli cells were octopaminergic (Dymond and Evans, 1979). The globuli cell somata of the cockroach, P . americana (Dymond and Evans, 1979) (Fig. 4), the locust, Schistocerca americana gregaria, and the crickets, Acheta domesticus and Gryllus campestris (Evans, unpublished), all stain selectively with the dye Neutral red, indicating their possible aminergic

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335

Fig. 4 Light micrographs of regions of cockroach (Periplaneta arnericana) cerebral ganglion as revealed by neutral red staining. (A) Whole cerebral ganglion viewed from the rear. Note the intense staining of the mushroom bodies (m). S, suboesophageal ganglion; C, circumoesophageal connective. Scale bar 500 Fm. (B) Isolated mushroom body from right half of ganglion showing double calyx viewed from above. Anterior is towards top of picture. (C) Isolated mushroom body from left half of ganglion viewed from the side; P, pedunculus; a, a-lobe; b, P-lobe. Anterior is towards the right hand side of the picture. (D) Cross-section through calyx region of mushroom body from right half of ganglion. Note the intense stainingof the globuli cell layer (g) on the surface of the calyces (c). Scale for B, C and D is 100pm. (From Dymond and Evans, 1979)

nature. Since they do not fluoresce with the Falck-Hillarp technique (see Klemm, 1976 and above), this suggests the possibility that they could contain a non-fluorogenic amine, such as octopamine. Microdissected clusters of globuli cell bodies and also the isolated calyx itself have recently been shown to contain considerable quantities of octopamine, in the cockroach (Dymond and Evans, 1979) and also in the locust (Evans, unpublished). In

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PETER D. EVANS

the cockroach, the calyx is doubled (see Fig. 2), but the inner and outer calyx contain equal amounts of octopamine (Evans, unpublished). Octopamine has also been demonstrated to be present in samples consisting of isolated calyces with globuli cell bodies still attached in the two species of cricket referred to above (Evans, unpublished). The presence of intrinsic octopamine-containing fibres would also be consistent with the electron microscopical evidence that intrinsic fibres contain large, dense cored or “neurosecretory” granules (Schiirmann and Klemm, 1973; Mancini and Frontali, 1970; cf, Hoyle et ul., 1974). The possibility that the globuli cells are octopaminergic is consistent with the suggestion of Frontali and Mancini (1970) and Frontalietal. (1971) that these cells were non-adrenergic, non-serotoninergic and non-cholinergic. The Neutral red staining procedure indicates that all the globuli cells might be aminergic in character. However, the biochemical radioenzymatic octopamine assays on isolated clusters of globuli cells do not indicate whether the octopamine is equally distributed in every cell, or is specifically confined to a sub-population. Globuli cells can be subdivided morphologically on the basis of their dendritic endings into “clawed” and “spiny” categories (Pearson, 1971; Strausfeld, 1976) but it is not clear at present if the morphological subdivision corresponds to any differences in transmitter content. (c) Function of mushroom bodies The functional role of aminergic fibres in the mushroom body remains a matter of sp6culation as does the role of the mushroom body itself. In the cockroach the mushroom bodies have been suggested to represent second order antennal sensory processing centres (Weiss, 1974). The majority of extrinsic fibres appear to come from the antennal lobe, entering the mushroom body through the calyx (Weiss, 1974; Strausfeld, 1976) where each large extrinsic fibre is contacted by many small globuli cell processes. The mushroom bodies consist of highly ordered regions of neuropile where the intrinsic fibres from the globuli cells proceed along the pedunculus to the a-and /3-lobes in parallel bundles which intertwine, but do not exchange fibres (Pearson, 1971). The bundles appear to be arranged in a regular relationship to their cell bodies (Pearson, 1971; Howse, 1974, 1975; Strausfeld, 1970, 1976). Along their length the intrinsic fibres synapse with each other and with extrinsic fibres (Mancini and Frontali, 1970). In the a-and /3-lobes each globuli cell fibre appears to contact many extrinsic efferent neurones. Recent electrophysiological studies on the mushroom bodies of the bee, Apis mellifera carnica, reveal that a high percentage of the units recorded give multimodal responses to light, scent, taste, and mechanical stimulation (of the antennae) (Suzuki et a f . , 1976; Erber and Menzel, 1977; Erber, 1978). Thus the mushroom bodies, of the bee at least, cannot be regarded

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exclusively as second order integration centres for olfactory input. They can rather be considered as association centres where one sensory modality can modulate the response characteristics of a particular neurone to an input from a second sensory modality (Erber and Menzel, 1977). At present, physiological evidence is lacking on the degree of multimodality of mushroom body units in other species. No direct anatomical connections with the optic lobe neuropiles have been described in cockroaches (Weiss, 1974), butterflies and moths (Pearson, 1971), locusts or house-flies (see Strausfeld, 1976) but indirect connections may exist in butterflies, moths and locusts. Thus anatomical studies alone are not sufficient to predict all the modes of sensory input to the mushroom bodies, and the findings from such studies need to be correlated with physiological recordings. It is possible that differences in the relative importance of various sensory modalities in different insect species could account for some of the morphological variations in mushroom body structure. Thus mushroom bodies may not play exactly the same integrative roles in all species. The roles played by the amines present in the various regions of the mushroom body are not clear at present. In view of the modulatory interactions of different sensory modalities, outlined above, it is tempting to speculate that some of the amines may be functioning as central neuromodulators as has been recently suggested for amines in the vertebrate central nervous system (Dismukes, 1977a) and insect peripheral nervous system (Evans and O’Shea, 1977,1978; O’Shea and Evans, 1979). Dopamine has, for instance, been shown to inhibit the spontaneous firing of units in the mushroom bodies of the ant (Steiner and Pieri, 1969). In the future it is hoped that combined biochemical, pharmacological and physiological studies on anatomically defined sets of neurones will reveal the exact neuromodulatory or neurotransmitter roles played by biogenic amines in the mushroom bodies.

2.2.3 The central body complex and protocerebral bridge The central complex of the insect brain consists of the central body, protocerebral bridge and ventral bodies. It is present in all insects (Strausfeld, 1976). Its highly complex neuronal architecture is elegantly described by Strausfeld (1976) for the housefly and by Williams (1975) for the locust, whilst Klemm (1976) provides a useful comparative survey of the literature on this structure. Histochemical analysis has shown that biogenic amines are present in the central body of all insects studied (Frontali and Norberg, 1966; Frontali, 1968; see Klemm, 1976 for other references). In general, the biogenic amine fluorescence was found to be arranged in a series of vertical stripes. In

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the caddisfly, dopamine was the predominant biogenic amine present (Klemm and Bjorklund, 1971) whilst in the locust pure dopamine fluorescence, as well as a mixed dopamine/noradrenaline fluorescence, could be detected (Klemm and Axelsson, 1973). Indolalkylamine - induced yellow fluorescent fibres are also present in the central body of the locust (Klemm, 1976). The protocerebral bridge does not exhibit any biogenic amine fluorescence in Lepisma, Trichoptera, Panorpa, Spodoptera and Apis but contains yellow (indole) fluorescence in dragonflies and locusts and catecholamine fluorescence in Calliphora (Klemm, 1976). A t the present time the location of the cell bodies providing the aminergic input to the central complex is unknown. The central complex receives anatomical projections from all the higher order sensory neuropiles of the insect brain. It is believed to play an integrative role in an arousal system regulating the responsiveness of the animal and possibly providing a gating mechanism for the selection of appropriate command neurones (Klemm, 1976; Strausfeld, 1976). The roles of the biogenic amines contained in the nerve fibres in this region of the brain, as possible neurotransmitters and neuromodulators, require further investigation. 2.2.4

Optic lobes

The compound eyes of insects are connected to the brain by three regions of integrative neuropile contained in the ganglia of the optic lobes, that is, in the lamina, the medulla and the lobula (Bullock and Horridge, 1965; see also Fig. 3B). The distribution of biogenic amines in these optic ganglia varies considerably from one insect species to the next, none being detectable, histochemically, in the optic lobes of the bee, Apis mellifera (Elofsson and Klemm, 1972; Klemm, 1976). Very little is known about the functions performed by any of the aniines that are present in insect optic ganglia or about the locations of the cell bodies that give rise to many of the histofluorescent aminergic fibres. The locust is one insect for which a considerable amount of physiological data is available on the processing of visual information in the optic lobes (e.g. Rowell and O’Shea, 1976a, b; O’Shea and Rowell, 1976; Rowelletal., 1977). The histochemical distribution of amine fluorescence (Elofsson and Klemm, 1972; Klemm and Axelsson, 1973; Klemm, 1976) and the biochemical distribution of octopamine (Evans, 1978c) are also known for locust optic lobes (Fig. 5). In the locust lamina, fibres demonstrating amine histofluorescence are arranged in an obvious cartridge-like pattern with a proximal (facing the brain) dopamine-containing region and a more distal (facing the retina)

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L.O.

0 la)

..

I

Fig. 5 Schematic representation of the distribution of monoamine-containing neuropile and cell bodies in the optic lobe of different insects as revealed by histofluorescent techniques. The species are ordered according to phylogenetic rank. Dots represent neuropile with yellow fluorophore (indoleamine), stripes represent moderate density of catecholamine-containing fibres, dark areas denote intense green fluorescent neuropile. (a),catecholamine-containing cell bodies; (0),5-HT-containing cell bodies. L.a.-lamina; L.0.-lobula; M.e.-medulla. (a) Silverfish (Lepisrno sacchurina L.), ( 6 ) dragonfly nymph (Aeschna viridis Evers), ( c ) desert locust (Schistocerca gregaria Forsk.), ( d ) house cricket (Achetu domesticus L.), ( e ) caddisfiy (Lirnneyhilidae), U, noctuid moth (Spodoptera littoralis, Bois), (g) fly (Calliphora vornitoria L.), ( h ) worker of the honey bee (Apis rnellifera L.). (From Klemm, 1976)

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PETER D. EVANS

region exhibiting only a yellow fluorescence corresponding to a tryptamine fluorophore. In the medulla, biogenic amine fluorescence is arranged in a longitudinal striated pattern of five separate bands (Fig. 6). The widest band lies some distance from the distal surface of the medulla and next to it is a thinner highly fluorescent layer. The other three thin bands are equally

Fig. 6 Frontal section of the medulla of an adult locust (Schistocerca gregaria) to show distribution of monomaine fluorescence. C2, 5-HT-containing cell bodies (see Fig. 3B). G.c., cell bodies of globuli type with a fluorescent catecholamine compound; Pr, processes of the cell bodies; d, distal, non-fluorescent part of the medulla; m, highly fluorescent middle layer of medulla. (From Klemm and Axelsson, 1973)

spaced in the proximal portion of the medulla. Microspectrofluorometric recordings reveal the presence of both dopamine and noradrenaline fluorophores, that of the latter being concentrated in the high-intensity fluorescent band in first instar locusts but appearing to be replaced by dopamine in adults (Klemm and Axelsson, 1973). 5-HT-containing cell bodies give rise to yellow fluorescent fibres which intermingle with the catecholamine-containing fibres of the two most distal strata. The arrangement of aminergic fibres in the lobula is less ordered than that in the medulla and consists of both catecholamine and 5-HT-containing fibres (Elofsson and Klemm, 1972). A loosely arranged bundle of catecholamine-containing fibres originates from the ventral side of the lobula and traverses the protocerebrum to the contralateral lobula. The most distal fibre system at the outer edge of the locust medulla does

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not normally fluoresce with the standard Falck-Hillarp technique but it can be induced to do so when incubated with highly fluorogenic amine substrates such as a-methyl-noradrenaline, 6-hydroxytryptamine or L-DOPA (Klemm, 1976). This suggests that some of the nerve processes in this region possess an uptake mechanism for the accumulation of these amines. Klemm (1976) takes this as evidence for the presence of fibres containing a nonfluorogenic amine such as octopamine. It should be noted, however, that the same technique did not induce fluorescence in the globuli cells of the mushroom body calyx which have recently been suggested to contain octopamine(DymondandEvans, 1979; seeSection2.2.26).It ispossible that the uptake processes are more highly localized in the terminal regions of octopaminergic neurones and that the usefulness of the above technique may be restricted to the localization of such terminal regions rather than cell bodies. The radioenzymatically determined distribution of octopamine in the optic lobes of the locust (Table 2) shows that it is also highly localized in the medulla (Evans, 1 9 7 8 ~ )Thus . the endogenous octopamine of the locust medulla may in part be localized in the outer stratum of the medulla. In view of the recently proposed modulatory role for octopamine at the locust neuromuscular junction (Evans and O’Shea, 1977; O’Shea and Evans, 1979) it is possible that the octopamine of the locust medulla is involved in a form of central modulation or arousal, such as dishabituation (Rowell and Horn, 1968) which may be located in this region of the locust visual system (O’Shea and Rowell, 1976). Due to the lack of detailed information on the distribution of different cell types in the locust optic lobe, it is not possible, at present, to relate the biogenic amine distribution, described above, to the fibre distribution of any particular cell type. Thus it is only possible to speculate, by analogy with other nervous systems, such as those of flies and vertebrates, on the functional roles played by biogenic amines in the optic lobes of the locust. In the medulla of the blowfly, Culliphoru, there are three longitudinal layers of green catecholamine fluorescence, a strongly fluorescent middle band and two weakly fluorescing distal bands (Elofsson and Klemm, 1972) (see Fig. 5). The detailed neuronal geometry of the optic lobe of the housefly, Musca dornestica has been described by Strausfeld (1976) using the Golgi impregnation technique. This reveals that the longitudinally oriented fibre systems in the medulla are derived from either extrinsic tangential fibres or from the layered arrangement of varicose and spiny amacrine cell processes. The association of catecholamines, such as dopamine, with amacrine cells in the locust medulla would be of much interest since Rowell et al. (1977) favour the hypothesis that a lateral inhibitory circuit, which gives protection against habituation, is probably mediated by amacrine neurones in the most proximal layers of the medulla.

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Rowel1 et al. (1977) also point out that there are many similarities and parallels between the mechanisms producing lateral inhibition in locusts and those in the vertebrate retina. In this context, it is of interest to note that a specific subpopulation of amacrine cells, containing dopamine, in the vertebrate (goldfish and rabbit) retina are thought to play a modulatory role regulating the lateral inhibitory effects mediated by horizontal cells and by other types of amacrine cell (Hedden and Dowling, 1978; Dowling and Ehinger, 1978). The possibility that some of the dopamine present in the locust optic lobes is also contained in amacrine type cells responsible for the modulation of lateral inhibition bears further investigation. However it must remain a speculation until a coordinated multidisciplinary investigation of the system is undertaken, combining electrophysiological recordings, the pharmacological application of dopamine and blocking agents, the histochemical and electron microscope localization of dopamine containing neurones (cf. Dowling and Ehinger, 1978) and detailed neuroanatomical studies. It is to be emphasised that such multidisciplinary studies need to be performed on the same insect species to understand the functional roles of biogenic amines in the optic lobes and also in other regions of the insect brain. 2.2.5

The deutocerebrurn

The deutocerebrum contains two regions of neuropile, a well developed antennal lobe that receives olfactory information from the antennae and a more diffuse area receiving mechanosensory information. The antennal lobe neuropile consists of a series of rounded glomeruli where the incoming sensory fibres branch profusely in the outer layers, and converge on a number of intrinsic neurones and on outgoing elements which travel to the calyces of the mushroom body along the antennoglomerular tract (Strausfeld, 1976). In Musca, Strausfeld (1976) defines at least four different classes of glomeruli based on the types of neuronal elements present. Some of the intrinsic neurones link only a limited number of glomeruli whilst others send projections to many. A similar diversity is also noted for the dendritic fields of the neuronal elements of the antennoglomerular tract. The glomeruli of the antennal lobe have been shown to contain catecholamines by fluorescent histochemistry in Periplaneta (Frontali, 1968; Rutschke and Thomas, 1975), Trichoptera (Klemm, 1968, 1971), Acheta, Panorpa, Spodoptera and Apis (Klemm, 1976). However, no fluorogenic amines could be detected in this region by the standard EalckHillarp method in various species of flies (Klemm, 1976) oi in the locusts, Locusta migratoria (Plotnikova and Govyrin, 1966) and Schistocerca gregaria (Klemm and Axelsson, 1973).

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In the cockroach, the distribution and intensity of fluorescence in the antennal lobes are not homogeneous. It is very intense at the edges of the glomeruli, where it is reported that the fluorophore is located in varicosities and is associated with a particular vesicle type (Ruschke and Thomas, 1975). The fluorophore has been identified as that of dopamine and its intensity, as well as the number and the size of the vesicles present, can be reduced by treatment with reserpine and a-methyl DOPA (Frontali, 1968; Rutschke and Thomas, 1975). The antennal glomeruli incorporate large quantities of 3H-dopamine as judged by autoradiographic techniques whilst a lateral population of deutocerebral neurones also readily accumulate 3H-5-HT (Rutschke et al., 1976). The latter observation correlates well with the distribution of yellow 5-HT histofluorescence (Rutschke and Thomas, 1975). Octopamine can also be shown to be present in the antennal lobes of the cockroach by radioenzymatic assay (Dymond and Evans, 1979) but as with the biogenic amines discussed above, its cellular location and functional role remain unknown. In the locust, Klemm and Schneider (1975) demonstrated that nerve fibres at the edges of the antennal glomeruli selectively take up 6-hydroxytryptamine indicating their serotoninergic nature. They presume that these fibres normally contain low levels of 5-HT due to the low number of vesicles observed in electron microscope sections and that this could account for their non-fluorescent nature under standard histochemical techniques. The cell bodies giving rise to these nerve fibres have not been located but the fibres have been observed to be restricted to the ipsilateral side and to pass between the different antennal glomeruli. Klemm (1976) speculates that these fibres may function at the glomerular surfaces, to alter the threshold of non-aminergic interneurones to the cholinergic input from the sensory cells of the antennae. The distribution of several prominent groups of monoamine containing cell bodies in the deutocerebral region are marked in Figs 3 A and B, but little is known about the distribution of their processes and the functional roles of these cells.

2.2.6 The tritocerebrum and stomatogastric system The tritocerebrum is the region of the insect brain that connects it with the suboesophageal ganglion and also with the frontal ganglion, the first ganglion of the stomatogastric (or vegatative) nervous system. The detailed neuroanatomy of the tritocerebrum has been studied in locusts by means of cobalt chloride backfilling of nerves (Aubele and Klemm, 1977) and in the housefly Musca dornestica by Golgi staining (Strausfeld, 1976). The tritocerebral neuropile is much more diffuse and less ordered than those of

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the protocerebrum and deutocerebrum described above. The main sensory input to the ti itocerebral neuropile in locusts appears to come from sensory cells in the mouth region and epipharyngeal wall, via the labral nerve (Aubele and Klemm, 1977). The neuropile is directly linked to many regions of the brain including the mushroom bodies and optic lobes. Some tritocerebral neurones project to the corpora cardiaca along nervus corporis cardiaci I while others are motoneurones directly innervating visceral muscle (Aubele and Klemm, 1977). In some species, such as the bee Apis mellifera and the housefly Musca dornestica the connectives between the brain and the suboesophageal ganglia are very short and filled with neuropile (Strausfeld, 1976) whereas in locusts and grasshoppers the connectives are much longer and consist purely of axon bundles. The majority of fibres in these bundles traverse the tritocerebrum without branching (Aubele and Klemm, 1977). The tritocerebra of all insects studied to date contain isolated fluorescent cell bodies exhibiting a green catecholamine fluorescence, as well as many catecholamine-containing nerve fibres (Plotnikova, 1968; Frontali, 1968; Klemm, 1968, 1971, 1976). In the cockroach, Periplaneta americana, a single pair of highly fluorescent cells were found (Frontali, 1968). In locusts, Klemm (1976) has provided evidence for the presence of varicose fibres and cell bodies exhibiting a yellow fluorescence (serotonergic?). Monoaminecontaining nerve fibres pass along all the tritocerebral pathways described above, but at presenf no information is available on their functions or on the functions of the biogenic amines present in the tritocerebrum itself. The suboesophageal ganglion of the cockroach contains one pair of large strongly fluorescent cells as well as a group of smaller less fluorescent cells and numerous catecholamine-containing nerve fibres (Frontali, 1968). In contrast, the same ganglion in caddisflies contains two pairs of fluorescent cells, one pair located dorso-medially and the second located caudoventrally (Klemm, 1968). The function of these catecholamine-containing cells is not yet clear but could possibly provide the dopaminergic innervation of some insect salivary glands (see Section 6). The stomatogastric nervous system of insects provides innervation to the insect foregut and salivary glands, and has been shown to contain biogenic amines (Klemm, 1968; 1971; Chanussotetal., 1969). In the frontal ganglion it has recently been reported that the processes of putative intrinsic aminergic neurones are closely apposed to the endings of neurosecretory fibres from the tritocerebrum (Ude et al., 1978) suggesting that they may exert a modulatory influence on the release of neurosecretory material. The relationship between the biogenic amines contained in the various ganglia of the stomatogastric nervous system and their effects on the functioning of

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insect visceral gut muscle will be discussed in detail in a later section (see Section 8). 2.2.7

Ventral nerve cord

Despite the large amount of information available, from spectrofluorometric and radioenzymatic assays, on the amounts of various biogenic amines present in the different ganglia of the insect ventral nerve cord (see Tables 1 and 2 above), very little is known about the cellular location of these amines. The great success at localizing catecholamine and 5-HT containing cell bodies in the brains (cerebral ganglia) of insects using the Falck-Hillarp technique has not been followed by an equal success in parallel studies on the ventral nerve cord. In the thoracic ganglia of locusts, a specific intense green fluorescence (dopamine?) has been found localized in nerve terminals in the regions of the medial and lateral nuclei of the unpaired nerves (Plotnikova and Govyrin, 1966; Plotnikova, 1968). These nuclei represent regions where the neurones that leave the ganglia via the median nerve arborize to produce dendritic fields. However, it is not clear which of the neuronal elements present in these nuclei contain the catecholamines and the location of their cell bodies is unknown. In one exceptional case, the location of dopamine-containing cell bodies has been reported in the thoracic and abdominal ganglia of Trichoptera (Bjorklundet al., 1970; Klemm, 1971). Two pairs of fluorescent cell bodies were present in each ganglion. One pair was located caudo-ventrally and the second pair dorso-medially to dorso-caudally. In general, the position of the cell bodies was very variable, especially in the abdominal ganglia, and in some cases a second pair of dorsal cell bodies was found. It is not clear at present if the above dopamine-containing neurones are homologous to some of the putative aminergic neurones revealed by Neutral red staining in the ventral ganglia of the locust and cockroach (Evans and O’Shea, 1977, 1978; Dymond and Evans, 1979). Some of the most prominently staining cells are those of the dorsal medial neurosecretory group. These cells do not fluoresce with the Falck-Hillarp technique for catecholamines and 5-HT in the locust (Hoyle and Barker, 1975; Evans, unpublished) or in the cockroach (Dymond and Evans, 1979). This suggests that they contain a nonfluorogenic amine, such as octopamine. The presence of octopamine in the cells of this group has been confirmed by radioenzymatic assays on single physiologically identified cells from the locust (Evans and O’Shea, 1977, 1978) and on groups of unidentified somata from the cockroach (Dymond and Evans, 1979). The octopaminergic nature and physiological roles of these cells will be discussed in detail below (see Section 4).

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Where is the noradrenaline?

The studies discussed in the previous sections have been highly successful in locating specific pathways containing dopamine and 5-HT, and more recently the non-fluorogenic monophenolamine, octopaminL. However, no specific noradrenergic cell bodies or fibre pathways have, as yet, been positively identified. In the locust, the noradrenaline fluorophore is only detectable in specific cell bodies or nerve fibres that also contain dopamine, such as in the mushroom bodies and central body complex (Klemm and Axelsson, 1973). These cells are thought to be different from the majority of catecholaminecontaining cells in the locust, which evince only a pure dopamine fluorophore. In the medulla of the locust (Klemm and Axelsson, 1973) the noradrenaline fluorophore is detectable in the most highly fluorescent second layer in the first instar but in adults dopamine is detectable in this layer. It is not clear if both dopamine and noradrenaline containing terminals occur in this region and that in the adult an increase in dopamine levels masks a relatively weak noradrenaline fluorescence, or if both amines are found in the same terminals. In caddisflies, noradrenaline could also be detected intraneuronally within the stratus caudalis and the optic medulla but the noradrenaline terminals appear to be intermingled with the dopamine terminals and no isolated noradrenaline structures could be found (Klemm and Bjorklund, 1971). In the latter study, only dopaminecontaining cell bodies could be located microspectrographically but it was pointed out that many cell bodies in the brain had such a low fluorescence intensity that it was not possible to analyse it. The absence of positive evidence for the presence of specific noradrenergic neurones in the insect must lead us to speculate whether or not the relatively small quantities of this amine detected in insect nervous tissue (see Tables 1 and 2) are due to the presence of a small class of, as yet unidentified, noradrenergic neurones or arise as the result of a metabolic artefact. Such an artefact could arise if small quantities of released dopamine were somehow taken up by octopaminergic neurones. The latter cells contain the enzyme tyramine-P-hydroxylase which can also convert dopamine to noradrenaline. The noradrenaline in this case would be classed as a false transmitter! It would seem that the answer to this problem will only be found by the assay of biogenic amines in microdissected single identified neurones. 2.3

SUBCELLULAR LOCATION OF BIOGENIC AMINES

The application of histofluorescent techniques to insect nervous tissue has confirmed the intraneuronal localization of biogenic amines, especially in

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terminal regions or varicosities (see Klemm, 1976). As yet, however, only circumstantial evidence exists for the localization of biogenic amines within vesicles and storage granules in insect nervous tissue. The rigorous isolation and purification of aminergic storage granules which has been performedion I vertebrate tissues (see Nelson and Molinoff, 1976) has not yet been attempted on insect tissues. At the electron microscope level a large variability in size and electron density has been reported for putative amine-containing vesicles in different vertebrate tissues but it seems generally agreed that noradrenaline is contained in at least two types of membrane-bound granules, small dense-cored vesicles (30-60 nm in diameter) and large dense-cored vesicles (60-150 nm) (see Nelson and Molinoff, 1976). The dense cores of the small vesicles are frequently only seen after permanganate fixation (Hokfelt, 1968). Klemm (1976) has pointed out that comparisons between animals of different phyla may be of little use, so it is of interest to exflmine the range of vesicle characteristics found in different putative aminergic neurones in a range of insects. Table3 contains such a comparision and also includes some comparative data from studies on other invertebrates. With the exception of the study of Lafon-Cazal and Arluison (1976) on the corpora cardiaca and hypocerebral ganglia of locusts, where catecholamines were suggestdd to be present in large (250 nm in diameter) clear vesicles, all the studies using aldehydelosmium fixation suggest that putative aminergic neurones,contain two populations of vesicles, small clear vesicles (30-60 nm in diameter) and large dense-cored vesicles (50-200 nm in diameter). In many cases the amine present has not been positively identified, so that the data must be regarded with some caution, but it is of interest to note the similarity in the vesicle populations between octopaminergic and catecholaminergic systems. Some comment is also appropriate about the results obtained in insect tissues with the permanganate fixation technique of Hokfelt (1968). This technique was originally introduced for the specific localization of noradrenaline-containing vesicles and was said, in vertebrates at least, not to show up dopamine-containing vesicles, although the latter could be dernonstrated by prsincubation of the tissue with a related amine, such as a-methyl-noradrenaline or 6-hydroxydopamine (Hokfelt, 1968). However, the uptake systems for such amines in insect nervous tissue are relatively unspecific (e.g. see Evans, 1978d) so caution is necessary in the interpretation of the results from such studies since related amines can be taken Up into a range of different aminergic neurones. In the salivary glands of the moth, Munducu sextu, where dopamine is thought to be the most likely neurotransmitter (Robertson, 1975), Robertson (1974) reported th’at small dense-cored vesicles (30-40 nm) were present after permanganate fixation,

TABLE 3 Electron microscopical characteristics of vesicles from putative aminergic neurones ~

:

Fixation Aldehyde-osmium Species

Tissue

Amine

Salivary gland Salivary gland

Dopamine Doparnine

DUMETi terminals

Octopamine

Adult light organ

Clear vesicles (nm)

Permanganate

Dense-cored Dense-cored (nm) ( 4

Reference

INSECTS

Nauphoera cinerea Manduca sexta Schistocerca gregaria Photuris pennsylvanica Photuris pennsylvanica Periplaneta americana Blabera craniifer and Schistocerca gregaria Blaberilr craniifer Schistocerca gregaria and Locus ta migratoria

44 30-40

92 50-100

33

99-165

Hoyle et al. (1974)

Octopamine(?)

20-40

60-120

Smith (1963)

Larval light organ

Octopamine(?)

40-65

90-135

Mushroom bodies

Catecholamine(?)

-

42-47

Oertel et al. (1975) Mancini and Frontali (1970)

lngluvial ganglion Stomatogastric system and lateral cardiac nerves

Catecholamine

65

Catecholamine

100-300

Corpora cardiaca Catecholamine and Hypocerebral ganglion Indolylamine

250

200

30-40

32

Maxwell (1978) Robertson (1974)

Chanussot et al. (1969) 100-125

G e r s c h e t a l . (1974)

Lafon-Cazal and Arluison (1976)

100 -0

rn

OTHER INVERTEBRATES

Homarus americanus Panulirus interruptus Spisula solida Quadrula pustulosa

Second thoracic roots Stomatogastric ganglion Ganglionic neuropil Pedal ganglion

Octopamine 5-HT Dopamine

200 50-100

Schaeffer et al. (1978) Friend (1 976)

Dopamine

40-100

Cottrell (1968)

Dopamine

75-100

Myers (1974)

Small pleiomorphic

-I rn

II 0 rn

<

b

2

v,

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apparently without the need to incubate in the presence of an exogenous amine. There seem to be two possible explanations for these conflicting results. Either noradrenaline is the true transmitter, not dopamine, or more likely there are differences in the results obtained with the permanganate fixation technique between insects and vertebrates. Further research is obviously needed to resolve this point. In the two other studies where this technique has been applied to insects (Gersch et al., 1974; Mancini and Frontali, 1970), preparations were incubated with a-methyl-noradrenaline at a concentration of 10pg/ml. Klemm (1976) points out that this is a high concentration (4.6 x 1 0 - 5 ~and ) could well lead to a non-specific uptake into a range of aminergic or even non-aminergic neurones. The results obtained from studies using the amine-depleting drug, reserpine, have also been taken as evidence for the localization of amines in storage vesicles in insects (Frontali 1968; Rutschke and Thomas, 1975; Robertson, 1976). This drug is thought to act by inhibiting the amine concentrating mechanism of storage granules (Carlsson et al., 1963; Guldberg and Broch, 1971). However, relatively high doses of reserpine (50-60 pg/g) have had to be used to demonstrate effects in insects. In the cockroach brain, a concentration of reserpine that completely abolished catecholamine histofluorescence had relatively little effect on the number, appearance or size distribution of the vesicle population (Mancini and Frontali, 1970). From this evidence it appears likely that biogenic amines are stored in several different size ranges of vesicles in insect nervous tissue, as has been found for vertebrates. Further research using a combination of biochemical and electron microscopical techniques will be required to provide definitive evidence on the size and shape of storage vesicles for the different amines present in the insect nervous system.

3

Metabolic studies on biogenic amines

Very few studies have examined the metabolic pathways involved in the synthesis and metabolism of biogenic amines in the insect nervous system. Most studies on the metabolism of biogenic amines in insects have concentrated on the role of these compounds and their derivatives in the tanning of cuticle (see Sekeris and Karlson, 1966; Murdock, 1971; Andersen, 1979). To date none of the synthetic or inactivating enzymes for biogenic amines have been isolated and purified from insect neural tissue, so that it has not been possible to compare their properties with those of the corresponding enzymes isolated from vertebrates. Most of our information on the metabolism of biogenic amines in insect nervous tissue has come from studies where intact pieces of tissue have been incubated with radioactive precursors and

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the products formed identified. Subsequently, possible metabolic pathways have been suggested, by analogy with those known to occur in vertebrates. The present section will deal with biogenic amine metabolism in the insect n e r v o h system. The available evidence for the synthesis of various biogenic amines will be considered, together with the evidence for possible inactivation mechanisms of amines released as chemical messengers. Evidence will be considered for both enzymatic inactivation mechanisms and for the presence of high-affinity concentrative uptake mechanisms.

3.1

S Y N T H E S I S O F BIOGENIC AMlNES

3.1.1 Catecholamines and phenolamines In the vertebrate nervous system it is well established that the catecholamines, dopamine and noradrenaline, are synthesized from tyrosine by a pathway involving the enzymes, tyrosine hydroxylase, DOPA decarboxylase and dopamine 6-hydroxylase (see Fig. 7 and Chapter 3 on catecholamines in Hallet a/., 1974). The latter two enzymes are also thought to be involved in the production of the phenolamines, tyramine and octopamine (Axelrod et al., 1976). All three enzymes have been isolated from vertebrate tissue and their properties characterized. The same pathway has also been demonstrated (except for the production of noradrenaline) in neural tissue from various invertebrate species such as the lobster, Homarus americanus (Barker et al., 1972; Wallace, 1976) and + no

+

+ TlRAMlNE

I H

c

f

1

HO

PNMT

NORADRENALINE

I

CH:N%H3

HO

SINEPHRINE

DBH

I

+

- OCTOPAMINE H3

1

OOPAMINE

DBH

Ho

+

PNMT

PlORENALlNE

Fig. 7 The pathway of catecholamine and phenolamine production from tyrosine. The reactions are catalyzed by the following enzymes. TH, tyrosine hydroxylase; D D , DOPA decarboxylase; DBH, dopamine 8-hydroxylase; PNMT; phenylethanolamine-N-methyltransferase. (From Evans, 1978a)

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the marine mollusc, Aplysia (see Saavedra, 1978). In the lobster, the enzyme tyramine-p-hydroxylase that converts tyramine to octopamine, has been isolated, and its properties characterized and compared to mammalian dopamine-p-hydroxylase (Wallace, 1976). In the insect nervous system, the evidence for the existence of the above metabolic pathway for the synthesis of biogenic amines is fragmentary. Tyrosine has been shown to be metabolised to the phenolamines tyramine and octopamine in nervous tissue from the locust (Hoyle and Barker, 1975; Evans, unpublished) and from the moth, Manduca sexta (Maxwell, Tait and Hildebrand, 1978). Octopamine has also been reported to be synthesized from tyrosine by nervous tissue of the cockroach Gromphadorhina portentom (Nelson, Drickamer, Maxwell and Hildebrand, personal communication, quoted in Evans, 197%) and from tyramine by the nervous system of another cockroach, Periplaneta americana (Robertson and Steele, 1974). The above results thus present circumstantial evidence for the presence of tyrosine decarboxylase and tyramine-P-hydroxylase activities in insect nervous tissue. The study of Vaughan and Neuhoff (1976), however, presents results which are difficult to reconcile with those from the above studies. In their study on locust nervous tissue these authors report no incorporation of radioactive label into octopamine using either tyrosine or tyramine as the precursor, despite the fact that they did find some incorporation of label into tyramine from tyrosine. The major metabolites formed from both precursors were N-acetyl-dopamine and N-acetyl-tyramine. In other studies N-acetylated derivatives may have been formed in such incubations but they were not specifically reported (e.g. Hoyle and Barker, 1975). Recent experiments indicate that in studies where locust (Schistocerca americana gregaria) metathoracic ganglia are incubated (3 hours) in the presence of , tyrosine (54 Ci/mmole and 10 pCi/ml), at high specific activity ~ - [ 35-3H] least 3% of the counts recovered from the tissue are found in N-acetylated derivatives of biogenic amines, whilst 2% are found in octopamine and 0.3% in tyramine (Evans, unpublished). Possible explanations for the apparent lack of label in octopamine in the experiments of Vaughan and Neuhoff (1976) are the very low levels of radioactively labelled L - [ U ~ ~ tyrosine C] used in the incubations, its low specific activity (405 mCi/mmole) and the absence of any energy source in the incubation medium (cf. Hoyle and Barker, 1975 and Evans, unpublished above, where 5 mM trehalose was present in the incubation medium). In view of these criticisms, the results and the pathways suggested by Vaughan and Neuhoff (1976) should be treated with caution, and obviously require repetition with more critical experiments. In some vertebrate tissues e.g. hog kidney, aromatic amino acids, such

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as tyrosine, phenylalanine, tryptophan, histidine as well as 5 hydroxytryptophan, can be decarboxylated by a single enzyme, which has been called aromatic amino acid decarboxylase (AAD) (Christenson et al., 1970). The presence of non-specific A A D activity has been reported in insect nervous tissue by several authors (Dewhurst et al., 1972; Murdock et al., 1973; Emsonet al., 1974). In the locust brain its activity is reported to be high enough to synthesize the total amount of dopamine present in twentytwo seconds (Murdock, 1971). As yet, this is the only enzyme in the catecholamine/phenolamine pathway in insect nervous systems for which direct activity measurements have been made. In vertebrate brain, experiments indicate that separate enzymes, other than AAD, decarboxylate 5-hydroxytryptophzn, and dihydroxyphenylalanine (DOPA) (Sims et al., 1973) but no equivalent evidence exists for insect nervous tissue. The rate-limiting step in the production of catecholamines in vertebrates is catalyzed by the enzyme tyrosine hydroxylase. This enzyme converts tyrosine to D O P A by the addition of a second hydroxyl group to the aromatic ring. No direct evidence is available on the activity of this enzyme in insect nervous tissue. Robertson and Juorio (1976) report, in passing, that the incorporation of label from [‘4C]-tyrosine in locust nervous tissue was ten-fold higher into tyramine than into dopamine but unfortunately no quantitative data were presented. Hoyle and Barker (1975) also reported “little, if any” incorporation of label from tyrosine into dopamine or noradrenaline in the same insect. The only study that provides quantitative evidence for the production of dopamine from tyrosine is that of Maxwell et al. (1978) on the moth, Manduca sexta. This study provides an interesting comparison of the abilities of various regions of the Manduca nervous system to synthesize putative neurotransmitters (see Fig. 8). Dopamine was shown to be synthesized from tyrosine in large amounts in thoracic and abdominal ganglia and in lower amounts in the optic lobes and “protocerebrum” (equivalent to protocerebrum plus tritocerebrum plus deutocerebrum minus antenna1 lobes in this study). In all the above studies direct evidence for the presence of tyrosine hydroxylase activity is lacking and even the results on Manduca do not rule out the possible production of dopamine from an alternative pathway via tyramine. The lack of incorporation of radioactive label from tyrosine into dopamine in locust nervous tissue is surprising in view of the large quantities of dopamine present (Robertson, 1976; and see Section 2 above). One possible explanation could be that the rate of turnover of the phenolamines (tyramine and octopamine) in insect nervous tissue is much higher than that of the catecholamines (dopamine and noradrenaline) so that a preferential incorporation of label is found in the phenolamines (Hoyle and Barker, 1975; Vaughan and Neuhoff, 1976). Even in Manduca, where some incor-

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;,

poration of label into dopamine from tyrosine was reported (Maxwell et al., 1978), the incorporation of label into tyramine was five to ten times higher in some regions of the brain. It is known in vertebrates that octopamine also turns over six times faster than noradrenaline (Molinoff and Axelrod, >ti1 tiA

Iil

0 . 0

0

Fig. 8 Relative specific synthesis of neurotransmitter candidates in CNS structures of the moth, Munduca sextu. Large, medium and small circles correspond to high, intermediate and low specific synthesis. Comparisons are made between CNS structures for a given neurotransmitter candidate AL, antenna1 lobe; OL, optic lobe; PR, “protocerebrum” see text; SEG, suboesophageal ganglion; TG, thoracic ganglion; AG, abdominal ganglion; ACh, acetylcholine; GABA, gamma-aminobutyric acid; DA, dopamine; TA, tyramine; OA, octopamine; SHT, 5-hydroxytryptamine; HA, histidine. (From Maxwell et al., 1978)

1972). A second possibility suggested by Vaughan and Neuhoff (1976) is that decarboxylation of tyrosine (to tyramine) may be the major metabolic pathway in insect neural tissue, rather than its 0-hydroxylation (to DOPA) as occurs in the vertebrate nervous system. The decarboxylation of tyrosine to tyramine and the subsequent p-hydroxylation to octopamine by insect haemolymph is known to be an important step in the production of the metabolites involved in the tanning processes (Whitehead, 1969; Lakeetal., 1970; Lake and Mills, 1975). Vaughan and Neuhoff (1976) further suggest that the conversion of tyramine to N-acetyldopamine supports the suggestion that tyramine and not tyrosine is the preferred substrate for 0hydroxylation in locust nervous tissue. This suggestion, however, is unfounded as they present no direct evidence for the conversion of tyramine to dopamine and it is possible that the N-acetylated dopamine is only produced from N-acetyltyramine (see Sekeris and Karlson, 1966) in extracellular connective tissue, rather than intracellularly in neurones. The above suggestion also seems unlikely in view of the fact that very little free dopamine is labelled even under conditions where a considerable amount of label accumulates in the tryamine pool (Hoyle and Barker, 1975; Vaughan and Neuhoff, 1976). A third possibility is that the incubation conditions

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used in the above studies are not suitable for the expression of tyrosine hydroxylase activity. This is the rate-limiting enzyme of catecholamine synthesis in vertebrates and its activity depends critically upon the availability of molecular oxygen (Nagatsu et a l . , 1964). In the insect studies mentioned above the neuronal tissues were incubated whilst detached from their tracheal systems, which normally supply oxygen to the tissues. In this connection it is interesting to note that Truman (1978) could obtain behavioural responses to eclosion hormone in the isolated abdominal nervous system of the silkworm, Hyafophora cecropia, only when air was supplied to the CNS through its normal route, i.e. through the tracheal system. Noradrenaline is present in insect nervous tissue in amounts about an order of magnitude below those of dopamine (see Section 2.1), but no synthesis of noradrenaline has been demonstrated in any insect neural tissue. This could obviously be related to the difficulties of demonstrating the synthesis of its presumed precursor dopamine in many insect nervous systems (e.g. cockroach and locust). Even in the nervous system of Manduca, where dopamine can be demonstrated to be synthesized from tyrosine, Maxwellet a l . (1978) failed to observe any synthesis of noradrenaline. They point out that if noradrenaline synthesis occurred at a rate which was two orders of magnitude lower than that of dopamine then they would have difficulty in detecting it reliably. 3.1.2 5-Hydroxytryptamine ( 5 - H T ) 5-HT is synthesized in insect nervous tissue from tryptophan by a ring hydroxylation by the enzyme tryptophan hydroxylase to produce 5-hydroxytryptophan (5-HTP) (Osborne and Neuhoff, 1974b) which is subsequently decarboxylated to 5-HT (Colhoun, 1963; Osborne and Neuhoff, 1974b) (see Fig. 9). It is not clear at present if a specific 5-HTP decarboxylase activity exists in insect nervous tissue distinct from the aromatic amino acid decarboxylase activity mentioned above. 3.1.3 Relation between synthetic ability and neurotransmitter function If a particular biogenic amine is to be accepted as a neurotransmitter or neuromodulator in a certain region of the insect nervous system then one of the criteria that must be satisfied, is that the region synthesizes the amine in question. However, it should be emphasised that metabolic studies on the production of radioactively-labelled biogenic amines from labelled precursors measure the amount of radioactivity that accumulates in a particular pool as the result of the interplay of synthetic and degradative processes under the particular incubation conditions used. Such results are not a

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measure of the total stores of a transmitter (Maxwellet a f . , 1978). Thus if a particular biogenic amine does not appear to be synthesized by a certain region of the insect nervous system, this in itself cannot rule out a transmitter function for this amine. The incubation conditions might be inappropriate

Q-r <:::TRYPTOPHAN

I

TH

‘coo5-HYDROXYTRYPTOPHAN

5-HVDROXYTRYPTAMINE N H :H :

Fig. 9 Metabolic pathway for the synthesis of 5-hydroxytryptamine (5-HT) in insects. TH, tryptophan hydroxylase activity and D, 5-hydroxytryptophan decarboxylase activity

for its production, its rate of turnover under resting conditions could be very low or it could have a high rate of turnover but a very small pool size. In the locust nervous system, for example, it has proved difficult to label the dopamine pool from tyrosine even though large amounts of endogenous dopamine can be shown to be present. The above examples emphasise the need for a multidisciplinary approach to the problem with data on the synthesis of biogenic amines being correlated with information on their endogenous levels in various regions of the nervous system from the same insect species. Only then can the significance of such intriguing observations as the high levels of tyramine synthesis in certain regions of the brain of Manduca (Maxwell er ul., 1978) be determined. The results obtained from comparative metabolic studies of the relative synthetic abilities of different regions of the nervous system are also difficult to interpret on their own. In the nervous system of Manducu, for instance, several brain regions, as well as the thoracic and abdominal ganglia, all synthesize approximately equal amounts of octopamine from tyrosine when the results are expressed as ferntomoles/structure, but when expressed as nanomoles/g protein, the specific synthesis appears higher in the nerve cord ganglia than in the brain regions (Maxweller a f . , 1978). From this synthetic data alone, it is impossible to determine the number of octopamine synthesizing cells in each region. There could be more cells synthesizing

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octopamine in the nerve cord ganglia than in the brain regions, or alternatively, there could be equal numbers of such cells in all regions examined, but in the brain regions there are larger amounts of protein not connected with octopamine synthesis (see Section 2.1.3). The presence of high endogenous levels of octopamine and dopamine in insect nervous tissue suggests the presence of specific octopaminergic and dopaminergic neurones. The octopamine-containing cells would lack the enzyme tyrosine hydroxylase and be rich in tyramine-P-hydroxylase activity, whilst the dopamine-containing cells would be rich in tyrosine hydroxylase activity but low in dopamine P-hydroxylase activity. Similar reasoning lead to the discovery of specific octopamine-containing neurones in the nervous system of the lobster (Barkeret al., 1972; Wallaceet al., 1974). No noradrenaline can be detected in the nervous system of the lobster. In contrast, in the insect nervous system low quantities of noradrenaline are detectable (see Section 2.1). These could result from low levels of dopamine-P-hydroxylase activity in dopamine cells or from the uptake and metabolism of small quantities of dopamine, by octopamine cells. A third possibility is that a small population of as yet unidentified noradrenergic neurones exist in insect nervous tissue. These various alternatives have not yet been resolved but it is important to bear in mind, that if such noradrenergic neurones do exist, then they will contain all the enzymes necessary to synthesize octopamine from tyrosine. Thus synthetic criteria alone should not be used to identify octopaminergic neurones in insects (c5 Hoyle and Barker, 1975). 3.2

INACTIVATION OF BIOGENIC A M I N E S

In the vertebrate nervous system biogenic amines are inactivated at release sites by specific uptake mechanisms (Iversen, 1973). High-affinity uptake mechanisms are used to transfer released amines back into presynaptic terminals, presumably to help to conserve the transmitter, whilst lower affinity uptake mechanisms may be used to take up amines into nonneuronal tissues where they can be enzymatically inactivated (Iversen, 1967). The major amine metabolizing enzymes in vertebrates are monoamine oxidase (MAO) and catechol-0-methyl transferase (COMT) (Iversen, 1967; Sharman, 1973). M A 0 activity is located within mitochondria and works in series with the high-affinity uptake system of nerve terminals. COMT activity appears to be widely dispersed in non-neuronal tissue and will act in series with the lower-affinity uptake system (see Trendelenburg, 1979). In many insect species the biogenic amines involved in the cuticular tanning process an: synthesized from tyrosine by enzymes contained in the

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cells of the haeniolymph (Lakeet al., 1970; Koeppe and Mills, 1975). These amines may be protected from oxidation by conjugation with sulphate, phosphate or sugar groupings, and also bound to specific carrier proteins for translocation to the epidermis and cuticle (Bodynark et al., 1974; Koeppe and Gilbert, 1974). The interest in biogenic amines in insects as possible neurotransmitters, neuromodulators and neurohormones has had to face two problems. First, how is the nervous system protected from the high levels of circulating amines related to the tanning processes? It needs to be pointed out, however, that the actual free haemolymph concentrations of these biogenic amines, and their derivatives, during the tanning period is unknown, as is the extent to which they would interfere with the functioning of the nervous system. Second, how are the amines released as chemical messengers inactivated? A certain amount of confusion has arisen from the study of biogenic amine metabolism in several insect species, since it is not clear whether the pathways being studied are involved in the production of tanning intermediaries, in the detoxification of amines or perhaps serve a common mechanism for both functions. Also since the insect nervous system is surrounded by a non-neuronal sheath of specialized glial cells, it is not clear if the results from experiments on amine metabolism in isolated, intact nerve cords, represent solely the activity of a peripheral non-neuronal enzymic barrier or whether the activity also represents an inactivation mechanism for amines released as transmitters or modulators from neurones within the nervous system itself. The rest of this section will examine the evidence for the inactivation of biogenic amines in insects by concentrative uptake systems and also their metabolism by various enzymic pathways subsequent to their cellular uptake. The structures of some of the possible metabolic derivatives of biogenic amines are illustrated using octopamine as an example in Fig. 10. 3.2.1

Uptake systems

In the vertebrate nervous system the major inactivation mechanism for biogenic amines, such as noradrenaline, dopamine and 5-HT, is by reuptake intonerve terminals (Whitbyetal., 1961; Wolfeetal., 1962; Iversen, 1967). In the nerve cord of the cockroach, Periplaneta americana, Evans (1978d) has described a concentrative, high-affinity, sodium-dependent uptake mechanism for octopamine. This system has many parallels with the uptake of noradrenaline into isolated rat heart (Iversen, 1965b) and into rat brain (Puglsey and Lippman, 1976). In the cockroach CNS the uptake of DLoctopamine can be divided into three components, higher and lower affinity sodium-sensitive components (Kms 0.5 and 19.8 PM respectively) and a sodium-sensitive component (see Fig. 11). The uptake of

PETER D. E V A N S

358 +coo-

p-HYDROXVMANDELIC ACID H

N-ACETVLOCTOPAMINE

no OCTOPAMINE

OCTOPAMINE

0-SULPHATE

,

Fig. 10 Possible routes of metabolism of biogenic amines in insects, illustrated by reference to octopamine. 1, monoamine oxidase activity; 2, N-acetyltransferase activity; 3 , sulphate conjugation; 4, p-alanine cpnjugation

DL-noradrenaline into rat heart also shows a similar division into high and low affinity systems (Kms 0.67 and 252 FM respectively) (Iversen, 1963, P965a) which are presumed to represent neuronal and non-neuronal uptake systems respectively. However, it has recently been shown that the Km is much lower (between 3 and 12 p ~ for ) the "extra-neuronal 0-methylating system" in rat heart, in which the catecholamines are first taken up and then metabolized (see Trendelenburg, 1979). ( a ) Sodium-sensitive uptake The high-affinity uptake mechanisms, for octopamine into cockroach CNS and for noradrenaline into rat heart, show marked parallels, especially in their structure specificity and their drug sensitivity. The structure specificity of the receptors in both uptake systems has been investigated in competition experiments (Iversen, 1965b; Evans, 1978d). The effectiveness of competitors is reduced in both cases by the removal of a hydroxyl group from the dromatic ring, by the methylation of a ring hydroxyl group and by the substitution of a methyl group for one of the hydrogens of the terminal primary amino group. The major difference between the two uptaEe processes is that the uptake of octopamine into the cockroach CNS prefers phenolarnines and the uptake of noradrenaline into rat heart prefers catecholamines. Thus the most potent inhibitor of octopamine uptake in cockroach nerve cord will be a non-0-hydroxylated amine with an unsubstituted primary amine grouping on the a-carbon and a single hydroxyl on the aromatic ring, i.e. tyramine. So the insect nkrve cord, as well as using the high-affinity uptake systeni to inactivate released octopamine, could also use

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10

000

30

20 OCTOPAMINE

I B

359

p~

0

4C0 -

I

l

l

l

l

l

l

l

10

l

l 20

OCIOPAMINE

l

l

l

l

l 30

1tM

Fig. 11 A. The rate of uptake of octopamine into cockroach (Pertplanera arnericana) abdominal nerve cord is plotted against concentration of oL-octopamine in the bathing medium. The total uptake represents the uptake rate from normal saline (120 mM Na+) and the Na+insensitive uptake represents influx from a Na'-free medium where Na+ was replaced by Tris. The bars represent 2. S.E. and n = 6. B. Na+-sensitiveuptake components obtained by subtracting the Na+- insensitive component from the total uptake rate at each octopamine concentration. (From Evans, 1978d)

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it to accumulate tyramine, the immediate precursor of octopamine (Evans, 1978d). The tyramine taken up could then be rapidly metabolized to its 0-hydroxylated congenor, octopamine, which could then be readily stored in arninergic storage granules. The high-affinity sodium-sensitive uptake of octopamine into cockroach CNS is also effectively inhibited by tricyclic antidepressant drugs such as desimipramine, imipramine, chlorimipramine and protriptyline (Evans, 1978d). This is very similar to the drug sensitivity of noradrenaline uptake by isolated rat heart (Iversen, 1965b) and rat brain (Pugsley and Lippman, 1976), which suggests that the insect nervous system has the potential to provide a useful and rapid screening system for new drugs which may have potent effects on peripheral and central noradrenergic systems in vertebrates (Evans, 1978d). ( b ) Sodium-insensitive uptake The sodium-insensitive uptake of octopamine into cockroach nerve cord shows no saturation kinetics in the concentration range examined (0.07-100 FM) (Evans, 1978d). Competition experiments reveal that the receptors mediating this component of the octopamine uptake have a different structure-specificity profile for competitors and a different drug sensitivity from those mediating the sodiumdependent high-affinity system. The best blocking drug for the sodiumindependent uptake was the a-adrenergic blocking agent, phentolamine. The antipsychotic drugs, a-flupenthixol and fluphenazine, which are potent blockers of dopamine receptors in vertebrates (Iversen, 1975) were also effective blockers of this component of octopamine uptake into cockroach CNS. It is also of interest to note that tetracycline and its derivatives also significantly reduced this uptake component in the cockroach. They have been shown to inhibit the binding of catecholamines to the collagen and elastin of vertebrate connective tissue (Powis, 1973). This binding is insensitive to the ionic concentration of the bathing medium. The neural lamella surrounding the insect nerve cord consists of a connective tissue sheath rich in collagen fibrils (Ashhurst, 1968) which could represent the site of this component of the octopamine uptake by the cockroach CNS. Such a peripherally located barrier in the insect CNS would be ideally suited to protect it from the transient fluctuations in the amine content of the haemolymph that are thought to occur during the processes of cuticle tanning. 3.2.2 Monoamine oxidase and catechol- 0-methyltransferase The activities of the enzymes monoamine oxidase (MAO) and catechol0 -methyltransferase (COMT) from the brain of the fruitfly, Drosophilu, are very low, especially when compared either to the activity of N-acetyl-

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transferase from t h e same tissue or to the respective activities of the enzymes in vertebrate nervous tissue (Dewhurst et al., 1972) M A 0 activity is also reported to be low in homogenates of the brain of the bee, Apis mellifera (Evans and Fox, 1975). In studies on a wide range of insect tissues, including nervous tissue, a significant M A 0 activity has only been found in Malpighian tubules (Blaschko et al., 1961; Hayashi et al., 1977). According to a recent report, locust brain contains a significant M A 0 activity that is inhibited by the insecticides, chlordimeform and dieldrin (Gripois et al., 1977). However, the results from this study are somewhat in doubt since the authors measured M A 0 activity using the method of Wurtman and Axelrod (1963). Although reportedly specific for M A 0 activity this method cannot be used in insects to distinguish between oxidation of tryptamine by M A 0 and metabolism by N-acetyltransferase without a careful chromatographic separation of the reaction products (Evans and Fox, 1975). There is, however, an apparent contradiction between the results obtained by biochemical measurements of M A 0 activity in insect brain and those obtained by the use of histochemical methods. The former indicate little or no M A 0 activity to be present in insect nervous tissue, whilst the latter give a positive reaction indicating its presence (Richter and Rutschke, 1977; Houk and Beck, 1978). Houk and Beck (1978) examined the brain of the European corn borer, Ostrinia nubilalis, for M A 0 activity with a histochemical method using a relatively new tetrazolium salt [2-(2’benzothiozoly1)- 5- stryl- 3- (4’- phthalhydrazidyl) tetrazolium chloride (f3S PT)] and tryptamine as a substrate. They were able to detect a specific reaction product within the outer membrane complex of mitochondria within the perineurial type I cells. The deposition of the reaction product was sensitive to the M A 0 inhibitors, tranylcypromine and nialamide. Richter and Rutschke (1977) reported M A 0 activity specific for dopamine, in dopamine-containing regions of the cockroach brain using a light microscope based histochemical method. The activity was inhibited by the addition of the M A 0 inhibitor pargyline but could not be demonstrated using 5-HT as a substrate. It is clear that M A 0 activity plays an important role in the breakdown of biogenic amines in the nervous systems of vertebrates and also of molluscs, such as the octopus (Blaschko and Hope, 1957; Juorio and Killick, 1972) and gastropods (Osborne et al., 1975). However, no gastropod M A 0 activity could be demonstrated against 5-HT (Goldman and Schwartz, 1977). In the insect nervous system very little M A 0 activity appears to be present as judged by biochemical assays. Histochemical data however indicates that M A 0 activity appears to be concentrated in specific regions of the insect brain where it could play a local role in the metabolism of aminergic neurotransmitters after their cellular reuptake. It is apparent that there are

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differences between various insect species and that further work is needed to determine if the histochemical demonstrations of M A 0 activity represent a significant localised metabolizing system for biogenic amines which is of general importance in all insect nervous systems. 3.2.3 N-acetyltransferase The bulk of the available evidence in the literature suggests that the major enzymatic pathway for the metabolism of biogenic amines in insect nervous tissue is by N-acetylation. The enzyme N-acetyltransferase replaces a hydrogen on the free amino group with an acetyl grouping (see Fig. 10). In Drosophila nervous tissue, tyramine was the best substrate tested but the N-acetyltransferase also had a significant activity towards dopamine and 5-HT (Dewhurst ' e t al., 1972; Maranda and Hodgetts, 1977). Brain homogenates of the honeybee, Apis mellifera, also contain enzymes capable of N-acetylating tryptamine and 5-HT (Evans and Fox, 1975). In the locust, Locusta migratoria, N-acetyltransferase activity towards octopamine, dopamine and 5-HT was high in both the nervous system and Malpighian tubules. It was also relatively high in a number of other locust tissues, except for skeletal muscle where it was relatively low (Hayashi et al., 1977). The latter finding is in agreement with that of Moore, Maxwell and Hildebrand (1978) in the moth Manduca sexta where it was found that N-acetyltyramine was produced, from tyramine, in substantial amounts by nervous tissue, tracheae and connective tissue, but not by muscle. The existence of N-acetyltransferase activity towards a wide variety of biogenic amines has also been reported in rat brain (Yang and Neff, 1976a, b). There were no large differences in enzyme activity between the different regions of the rat brain. The activity of serotonin N-acetyltransferase in the pineal glands from rat brain has been shown to exhibit a circadian rhythm (Ellison et al., 1972) but the physiological role of this enzyme in the mammalian brain remains unknown at the present time (see also Section 2.1.1). In insects, N-acetylated derivatives of biogenic amines, notably N-acetyldopamine, are known to play important roles in the sclerotization of certain insect cuticles (Sekeris and Karlson, 1966; Andersen, 1979). This raises the important question of whether the N-acetyltransferase activity found in isolated nervous tissue actually represents an inactivation mechanism or is just part of a widespread enzyme system producing tanning intermediaries. The persistence of the widespread distribution of this enzyme activity in adult insects, after the tanning process has finished, argues against this latter hypothesis. Maranda and Hodgetts (1977) could not distinguish separate types of N-acetyltransferase activity for sclerotization and for the inactivation of neurogenic amines in their studies on Drosophila, but Evanset

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al. (1980) suggest that in the corn borer, Ostrinia nubilalis the neural enzyme may be different fromathat found in non-neural tissues. The widespread distribution of the enzyme would seem to ideally suit it for a role as a general inactivation mechanism for biogenic amines which are released into the haemolymph as circulating neurohormones and subsequently taken up into a wide range of tissues for metabolism. In this respect it has many parallels with the non-neuronal COMT metabolizing system of vertebrates. One puzzling question is the low activity levels of this enzyme found in skeletal muscle in locusts and moths, particularly in view of the neuromodulatory role proposed for octopamine at the locust neurornuscular junctions (Evans and O’Shea, 1977; O’Shea and Evans, 1979). Perhaps one of the other inactivation mechanisms, such as a concentrative high-affinity uptake system, is responsible for inactivating the octopamine in this situation. In view of the fact that the cellalar location of the N-acetyltransferase activity of insect nerve cord has not yet been reported, it remains to be seen if it has a role in the inactivation of biogenic amines released within the insect nervous system. 3.2.4

Sulphate conjugation

It has recently been shown that conjugation with sulphate is an important step in the metabolism of the biogenic amines octopamine, dopamine and 5-HT in the lobster nervous system (Kennedy, 1978). In the lobster, phenylethylamine, which lacks an hydroxyl group on the aromatic ring, did not form a conjugate. with sulphate, indicating that the sulphate group becomes attached to the aromatic hydroxyl groups in the other amines (see Fig. 10). Sulphate conjugation appears to be a general inactivation mechanism for biogenic amines in species ranging from arthropods to mammals (Eccleston and Ritchie, 1973; Meek and Neff, 1973; Kennedy, 1978). In the cockroach, Periplaneta americana , the concentration of dopamine 3-0-sulphate is high in the newly emerged insect but decreases sharply as the sclerotization of the cuticle proceeds (Bodnaryk and Brunet, 1974). This sulphate derivative is suggested to protect dopamine against oxidation in the haemolymph. N-Acetyl dopamine can also be sulphated, or phosphorylated, by cockroach haemolymph and these derivatives may represent the form in which the cuticular sclerotizing agent, N-acetyldopamine, is transported from the blood into the epidermis by a carrier protein (Bodnaryketal., 1974). Moore et al. (1978) presented preliminary evidence for the conversion of tyramine to tyramine-0-sulphate in the intact isolated nervous system of the moth, Manduca sexta, but Maxwell et al. (1980) reported that this product is probably a sugar conjugate of N-acetyltyramine. The enzyme responsible for this conversion was again widespread in different tissues of the insect, including tracheae, connective tissue and muscle. In the same way as for the

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N-acetyltransferase activity discussed above, it is not clear, at present, if sulphate conjugation in intact isolated insect nerve cords is related to a diffuse system for producing protected intermediates for use in tanning, to a general inactivation mechanism for released amines or to a multifunctional system. It does appear, however, that sulphate conjugation may be a general mechanism for monoamine metabolism in Arthropods.

3.2.5 /3-Alanine conjugation A second major pathway for the metabolism of octopamine, and possibly of amines in general, in the lobster nervous system is 0-alanine conjugation (see Fig. 10). In this system, /3-alanine is linked by an amide bond to the amino group of octopamine or to t h e amino group of the sulphated conjugate of octopamine (Kennedy, 1977). Moore et al. (1978) presented preliminary evidence for the production of an N-aminoacyl derivative of tyramine in the nerve cord of the moth Manducasexta and recently Maxwellet al. (1980) identified p-alanine as the amino acid conjugated with the amino group of tyramine in this derivative. The significanceof the production of the latterconjugate in the insect nerve cord is also unknown, but it is thought that in various insects the formation of dipeptides in the haemolymph forms a convenient store of aromatic amino acids such as tyrosine (see Bodnaryk, 1978). For instance, it has been suggested that the dipeptide, celerin (L-tyrosyl-o-acetyldopamine), from the blood of the Lepidopteran, Celerio euphorbiae, participates in the process of melanization and sclerotization during pupation (Sienkiewicz and Piechowska, 1973). Similarly, the dipeptides, /3-alanyl-L-tyrosine (sarcophagine) (Levenbook et al., 1969) and y-L-glutamyl-L-phenylalanine (Rodnaryk, 1970), accumulate in the haemolymph of the third instar larvae of Diptera, and disappear abruptly during puparium hardening. They are hydrolyzed before the incorporation of the aromatic amino acid, or one of its derivatives, into the cuticle. In view of the widespread tissue distribution of the /3-alanine conjugatiton process in Manduca, it is again impossible to determine if this system is involved in the production of a storage form of tanning intermediaries, represents a functional system for inactivating released amines or subserves both functions. 3.2.6

Consequences for the functional role of biogenic amines

At present we have no direct evidence that any of the above metabolic pathways or uptake systems fulfill physiologically important roles in the insect nervous system, or that different systems predominate in different species. It should be possible to tackle these questions by the use of specific inhibitors of the enzymatic pathways and of the different components of the

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uptake systems (Evans, 1978d) and to see how these inhibitors interfere with the functioning of defined aminergic pathways. It is unclear which of the cellular elements of the isolated nerve cord are responsible for the enzymic activities and uptake properties described above. Studies locating these cellular elements will undoubtedly give a clearer picture of the possible functional involvements of the enzymatic and uptake processes. It is obvious that effective metabolic pathways for inactivation, together with the binding to serum proteins (Kirksey et al., 1974; Koeppe and Gilbert, 1974) will have important consequences on the ability of biogenic amines to function as circulating hormones in insects. Many of the enzymic inactivation processes discussed above are widely distributed in different insect tissues, as well as, perhaps, being localized in glial cells which form a peripheral barrier around the nervous system. It is thus tempting to speculate that these enzymatic inactivation processes may be associated with low affinity non-neuronal uptake systems and fulfill a dual function. First they would keep the levels of circulating amines low during sclerotisation, thus preventing the desensitization of aminergic receptors, and second they would terminate the actions of biogenic amines released into the circulation as neurohormones. It is possible that high-affinity uptake systems might be responsible for the local inactivation of biogenic amines released as neurotransmitters or local neurohormones within the nervous system.

4

Octopamine and the dorsal midline neurones

The presence of a highly specialized median group of cells on the dorsal surface of insect thoracic and abdominal ganglia has been known for many years. The unusual anatomical characteristics of a specific subset of these cells was first reported in the locust, Locusfa migraforia, by Plotnikova (1969). The cells of this group appear to be unpaired in locust ganglia; that is they have bifurcating axons which project symmetrically into left and right peripheral nerve roots of the ganglia. This feature distinguishes them from most of the known locust motoneurones, that are bilaterally paired and have axons leaving the central ganglia in the nerve roots of one side only (but see below for spiracular motoneurones). For these reasons, the dorsal median cells of the locust metathoracic ganglion were later referred to as Dorsal Unpaired Median (or DUM) neurones (Hoyleet al., 1974). Cells of similar morphology were also described in the metathoracic ganglia of the crickets, Teleogryllus oceariicus and Acheta domesticus, (Bentley, 1973; Clark, 1976; Davis and Alanis, 1979) and of the hemipteran, Dysdercus fulvoniger (Davis, 1977) as well as in the thoracic and abdominal ganglia of the moth, Manduca sexfa (Casaday and Camhi, 1976; Taylor and Truman, 1974).

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These cells are also unusual physiologically, in that their somata are capable of generating large, overshooting action potentials of 60-1 1 0 mV in amplitude (Jego et a[., 1970; Crossman et al., 1971; Rowe and Will, 1971; Heitler and Goodman, 1978) This contrasts with the situation in the somata of the majority of insect motoneurones and interneurones, so far examined, where only attenuated action potentials (-3-6 mV) are recorded, resulting from the electrotonic propagation of potentials initiated at distant regions of active membrane. The large overshooting action potentials are characteristically of long duration, a feature which has been suggested to be typical of the action potentials of neurosecretory neurones (see Maddrell, 1974 for references). It needs to be emphasized that not all the cells in the dorsal midline of insect ganglia are unpaired (see below) and conform to the above anatomical pattern. Also this distinctive anatomical pattern is itself not restricted to cells in the dorsal midline. Many somata in the dorsal midline belong to cells of a variety of neurosecretory types (Delphin, 1965) some of which send their axons out of the median nerve (Chalaye, 1967; 1974a, b; Smalley, 1970; Lewis et al., 1973; Ali and Pipa, 1978). Other somata apparently belong to intra- and inter-ganglionic interneurones (Hoyle, 1978a; Goodman and Spitzer, 1978). Taylor and Truman (1974) describe a pair of dorsally located midline cells in the fourth abdominal ganglia of M . sextu larvae that each send out a posteriorly directed neurite which bifurcates to give a branch in both the left and right connectives. These branches run to the fifth abdominal ganglion where they pass to the periphery along the dorsal segmental nerves. These cells are not present in the three to five day old adult. Some midline cells that occur on the ventral surface of insect ganglia have identical branching patterns to the DUM cells described above. Examples of these cells are the presumed motoneurones in the abdominal ganglia of M . sextu (Taylor and Truman, 1974) and interneurones in the abdominal ganglia of the locust, Schistocercu gregaria (Seabrook, 1970). The paired spiracular motoneurones of insect thoracic ganglia also have ventrally located somata. Each neurone sends out a single axon into the median nerve, where it bifurcates to send processes along the left and right branches of the transverse nerve to innervate the appropriate spiracular muscles, on both the left and right sides of the animal (Zawarzin, 1924; Case, 1957; Miller, 1960; Burrows, 1975). In the pregenital abdominal ganglion of the locust, the transverse nerve arises directly from the posterior surface of the ganglion and a pair of posterior median cells each send a single neurite anteriorly which bifurcates to give branches to both the left and right transverse nerves (Seabrook, 1968). The following sections will examine the evidence for the identification of

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individual dorsal midline neurones and for the association of biogenic amines with the cells of this group. The functions of an identified octopaminergic neurone from this group will be described and considered as a possible model for the functioning of similar aminergic modulatory cells in the central and peripheral nervous system. 4.1

A R E THE I N D I V I D U A L CELLS O F THE D O R S A L M I D L I N E GROUP UNIQUELY

IDENTIFIABLE?

One difficulty frequently encountered in studies on nervous tissue, especially in vertebrates, is that of returning to a uniquely identifiable neurone in different individual preparations. In invertebrates, however, the smaller numbers of cells in the nervous system present the neurobiologist with a distinct advantage in this respect. Thus in the insect nervous system, for example, it has been possible to identify the cell bodies of many of the motoneurones innervating the muscles of the wings and legs (Hoyle and Burrows, 1973; Burrows and Hoyle, 1973, see also Hoyle’s review, 1975a). It is desirable that work on DUM cells also be carried out at a level of the identified neurone so that physiological, pharmacological and biochemical data from the same cell can be correlated. In the locust metathoracic ganglion one of the D U M cells has been shown to project only to the extensor tibiae muscle (ETi muscle) of the two hind legs, and has been called DUMETi (Hoyle et al., 1974). The bilateral distribution of the DUMETi axons provides a convenient criterion for its physiological identification, since the three motoneurones innervating this muscle are paired, one cell from each pair innervates the left, and the other the right extensor muscle (Pearson and Bergman, 1969; Hoyle and Burrows, 1973). Thus, to locate the soma of DUMETi, DUM somata can be penetrated randomly with an intracellular microelectrode until a neurone is found where the action potentials initiated in its soma by depolarizing current are transmitted 1 :1 to both the left and right extensor tibiae nerves (nerve 5bl, Pringle, 1939) (Evans and O’Shea, 1977, 1978). This distinguishes the DUMETi neurone soma from those of the other DUM cells since the action potentials in the identified cell can account for all the action potentials found simultaneously in the left and right extensor nerves. In repeated experiments in which many D U M cells were penetrated in a single locust metathoracic ganglion only one cell was found to meet the above criteria for the identification of DUMETi. The anatomy of this neurone can be seen from experiments where it is filled with dyes such as Procion yellow (Hoyle et al., 1974, see Fig. 12A) or with cobaltous ions (Heitler and Goodman, 1978, see Fig. 12B). In both cases the median neurite can be seen to pass ventrally and then anteriorly before making a T-junction from which

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A

B

Fig. 12 Anatomy of DUMETi neurone from the metathoracic ganglion of the locust. A. Schistocerca gregaria. Reconstruction based upon projections of serial sections of a specimen injected with Procion yellow and viewed with ultraviolet light (from Hoyle et al., 1974). B. Schistocerca nitens (i) Semi-diagrammatic drawing of whole-mount of metathoracic ganglion where anatomy of DUMETi has been revealed by cobaltous sulphide staining, and (ri) reconstruction from horizontal sections of the ganglion intensified by precipitation of silver in a modification of Timm's method. Calibration: (i) 0.6 m m (approx.), (ii) 200 pm. (From Heitler and Goodman, 1978)

branches pass into nerve 5 on each side. The smaller branches of the neurite passing ventrally and posteriorly in the Procion reconstruction (Hoyle et al., 1974) were not reported in the cobalt study (Heitler and Goodman, 1978). Extensive dendritic branching can be seen in the region of the bifurcation, especially in the cobalt fill. In both cases, a small tuft of branches arises just before the axon branches leave the ganglion. The axon diameter does not increase at this point but Heitler and Goodman (1978) note that these fine branches ramify amongst the large axons of motoneurones leaving the ganglion through nerves four and five. The position of the DUMETi soma in the locust metathoracic ganglion

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(Evans and O’Shea, 1978) varies a good deal more than do the positions of the identified motorneurones in the same ganglion (Burrows and Hoyle, 1973). The position of the D U M cells within the group also varies as can be seen from Neutral red-staining (Evans and O’Shea, 1978; see Fig. 13). This variability was also noticed in cockroach preparations by Crossman et a l . (1971) and in locust and grasshopper preparations by Hoyle (1978a). The Neutral red (Evans and O’Shea, 1977, 1978) and other staining procedures (Hoyleet al., 1974; Hoyle, 1978a) have revealed two major size categories of DUM cell somata. The large somata are 40-80 p m in diameter and the small somata are about 30 p m in diameter. The total number of DUM cells stained in the locust metathoracic ganglion is variable but Neutral red staining typically reveals 8-1 1 large cells and 14-21 small cells. The Neutral red staining technique, which is believed to be specific for amine-containing cells (Stuart et al., 1974), also reveals the presence of a variable number of intensely staining small cells (-10 p m in diameter) in the midline of the “abdominal” portions of the locust metathoracic ganglion (see Fig. 13, X). (The locust metathoracic ganglion is a fused ganglionic mass consisting of the true metathoracic ganglion and the ganglia of the first three abdominal segments). These very small cells could represent the neurosecretory cells that send processes into the median nerves (Delphin, 1965; Chalaye, 1967; see Section 9). Neutral red staining of the cockroach metathoracic ganglion reveals a cluster of eight dorsal midline cells of 40-60 p m in diameter (Dymond and Evans, 1979) (see Fig. 14) which is in agreement with the observations of Crossman et al. (1971). It is not clear at present if the variability in total DIJM cell numbers observed with the various staining techniques in the locust metathoracic ganglion represents a true variability in total numbers, is the result of the variable staining properties of the cells, or is due to the most dorsal layer of densely stained cells obscuring other stained cells lying underneath them. It is to be noted, however, that a variation in the position and total numbers of octopamine cells has also been observed in the peripheral nerve roots of the lobster (Wallace et al., 1974; Evans et al., 1976b). Thus the physiological identification of the DUMETi soma is required in each preparation because soma size, position in the ganglion and soma position in relation to the other DUM somata are all variable and cannot alone be used as criteria for identification. The variability in total number of cells also suggests some nonessential function for the cells of this group, such as the modulatory role described below. Recent studies on this dorsal group of cells in the thoracic ganglia of the locust have suggested that it may be possible to identify uniquely other DUM neurones. Hoyle (1978a) reports that in the locust Schistocerca gregaria and the grasshopper, Romalea microptera, a single DUM cell sends out branches in the wing nerve (nerve 1) to innervate the dorsal longitudinal

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D. EVANS

A

D

Fig. 13 Light micrographs of the dorsal surface of five metathoracic ganglia (A-E) from the locust (Schistocerca americana gregaria) showing the organization of the DUM neurone somata as revealed by neutral red staining (0.01 mg/ml in isotonic saline for 3 h). The soma of DUMETi is presumably present in each example but cannot be identified due to the variable organization of the group. X indicates position of very small (10-15 pm) neutral red staining cells in “abdominal portion” of the fused metathoracic ganglionic mass. Scale bar, 100 pm. (From Evans and O’Shea, 1978)

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c

Fig. 14 Light micrographs of dorsal surfaces of cockroach (Peripfaneta americana) thoracic (A-D) and abdominal ( E and F) ganglia showing the organization of the dorsal median cell group as revealed by neutral red staining. A, prothoracic ganglion; B, mesothoracic ganglion; C and D, two examples of metathoracic ganglia to show variable organization of cells; E, a typical abdominal ganglion; F, sixth abdominal ganglion showing several groups of cells indicating fused segmental origin of this ganglion. In each case the anterior edge of the ganglion is towards the top of the figure. Scale bars 100 pm. (From Dymond and Evans, 1979)

muscles. Hoyle christened this cell DUMDL and it appears to be the same neurone figured by Plotnikova (1969) in Locusta migratoria, by Bentley (1973) and Clark (1976) in the cricket, Teleogryllus oceanius and by Davis and Alanis (1979) in the cricket Acheta domesticus. A certain amount of doubt exists, however, about the uniqueness of this cell in locusts as Altman and Tyrer (1977) suggest that there are two dorsal medial neurones in each

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ganglion with axons in both the right and left nerve 1, though it is rare to fill both from one side. In view of this finding it appears that DUMDL cannot be regarded as a unique identified cell solely on the basis of the presence of its axons in nerve 1. It is possible that further work on this pair of neurones in locusts will show that they have different peripheral innervation patterns which could serve as a basis for their unique identification. It is also possible that locusts and crickets have different numbers of D UMDL cells, since Davis and Alanis (1979) claim that it is only possible to identify one DUMDL somata when nerve 1 is simultaneously filled with cobaltous ions from both the right and the left sides. Further investigation is obviously required to resolve this point. It is also becoming increasingly apparent that not all of the other dorsal median cells are unpaired and that many of them are interneurones (Hoyle er af., 1974; Hoyle, 1978a; M. O’Shea unpublished observations and C. S. Goodman, personal communication quoted in O’Shea and Evans, 1979). Hoyle (1978a) points out that the figure of a “general” DUM neuron obtained by Crossman et af. (1972) was not constructed from direct dye filling but from electrical excitation and recording. On the basis of his own dye injections he concludes that their drawing is wrong. Hoyle et af. (1974) suggest that, in the locust, four of the large DUM neurones are probably interneurones with one major meurite either in the left or right anterior connective of the metathoracic ganglion, and that the smaller DUM neurones are local intraganglionic interneurones. Hoyle (1978a) revised the estimate of the large DUM neurones to suggest that six are probably paired, and may be interneurones. Doubts exist however about the branching pattern and dendritic fields of many of the dye-filled neurones figured by Hoyle (1978a). In many of the attempts to cobalt fill single DUM neurones, multiple fills of neurones occurred (Hoyle 1978a) in which it is difficult to distinguish the branching pattern of individual neurones. In the fourth abdominal ganglion of the moth, Manduca sexta, both the dorsally and ventrally located cells that have a similar branching pattern to locust DUM cells, are all paired (Taylor and Truman, 1974). Goodman and Spitzer (1978), working on the thoracic ganglia of developing locust embryos, describe some D U M cells that are confined to the ganglion, and others that have bilaterally symmetrical peripheral axons. Other DUM cells send axons into one or other, or even both, connectives. It is thus questionable whether all DUM neurones are “unpaired” in the adult. The term unpaired is, however, appropriate if one considers the unique origin of many of the neurones from the single, unpaired median neuroblast (Goodman and Spitzer, 1978,1979). It is not clear at present, however, if all the median neurones that stain with Neutral red have a common origin. At this time, the usefulness of a change in generic name does not seem appro-

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priate but it may become necessary as more is discovered about individuals of this group of neurones (O’Shea and Evans, 1979). 4.2

T H E O C T O P A M I N E R G I CN A T U R E O F

DUM

CELLS

The selective staining of DUM cells by the dye Neutral red has been taken as evidence for their aminergic nature in the nervous systems of the locust (Evans and O’Shea, 1977, 1978) and the cockroach (Dymond and Evans, 1979), by analogy with the effects of Neutral red in other systems such as the leech (Stuart et al., 1974) and lobster (Wallace et a[., 1974; Evans et a[., 1976b). The first indications of the possible octopaminergic nature of insect DUM cells came from three pieces of evidence. First, tissue removed from the dorsal region of the locust metathoracic ganglion, which was presumed to include the somata of some DUM cells, synthesized octopamine when incubated with radioactive tyrosine (Hoyle and Barker, 1975). Second, the only known physiological effect of stimulating DUM cells, namely the slowing of an intrinsic rhythm of contraction and relaxation found in the extensor tibiae muscle of the locust hind leg, (see below) could be mimicked by applying octopamine to the muscle (Hoyle, 1975b). Third, the cells did not fluoresce with the Falck-Hillarp technique for the localization of catecholamines (Hoyle and Barker, 1975). The above evidence was taken by Hoyle (1975b) and subsequent reviewers (e.g. Robertson and Juorio, 1976) to indicate that the DUM neurone innervating the extensor tibiae muscle (DUMETi) and DUh4 neurones in general, were octopaminergic. This conclusion (although, as it turns out a correct one, see below) was premature at the time due to the inadequacies of the experimental evidence. In the biochemical studies (Hoyle and Barker, 1975) the specific ability of DUM cells to synthesize octopamine was inferred from analysis of heterogenous tissue containing unidentified cells. Furthermore, the only products of the control incubations on whole ganglia were tyramine and octopamine; dopamine and noradrenaline are also present in locust nervous tissue and should also have been sythesized (see Section 3.1.1). Their absence from the labelled products raises the possibility that the incubation conditions of Hoyle and Barker (1975) were not optimal for the synthesis of dopamine and noradrenaline and thus did not permit normal metabolism. The reported correspondence between the physiological effects of octopamine application and DUM cell stimulation on the intrinsic rhythm is also difficult to interpret. Many of the cells in the D UM group will cause a slowing of. the rhythm (Hoyle, 1974, 1975b). In these experiments DUMETi was not uniquely identified and was therefore not shown to affect the rhythm. In addition, the effects of octopamine on the rhythm were not shown to be specific; octopamine may have been the most physiologically

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active amine of the limited number tested by Hoyle (1975b), but subsequent studies indicate that other amines are either equal or more active than octopamine ,on the ,same preparation (Evans and O'Shea, 1978). The octopaminergic nature of DUMETi was positively demonstrated by Evans and O'Shea (1977; 1978). In these studies, physiologically identified DUMETi somhta were dissected from locust metathoracic ganglia and analysed for their octopamine content (see Table 4). Each soma was shown TABLE 4 Octopamine content of isolated cells A Identified DUMETi neurone from metathoracic ganglion, of the locust Schistocerca americana gregaria' DUMETi (experiment no.) 1 2 3 4 5

Mean t S.E. FETi B

No. of somata (10)

(8) (10) (10) (5) (47)

Octopamine/soma (pmol) 0.086 0.190 0.083 0.063 0.074 0.099 k 0.023 t none detectable

Unidentified dorsal median somata from the cockroach Periplaneta americanad (n) Octopamine/soma (pmol 2S.E.) 0.085 & 0.014 6th Abdominal ganglion (16)" 0.140 k 0.038 Metathoracic ganglion (6)b

5 to 30 somata Estimated in groups containing 7 to 10 somata Data from Evans and O'Shea (1 978) Data from'Dymond and Evans ( 1 979)

* Estimated in groups containing

to contain about 0.1 pmoles of endogenous octopamine. Octopamine was also shown to be present in samples of peripheral nerve which contained the DUMETi axon, with the octopamine concentration in the axon being four times that of the soma. This finding agrees well with the distribution of amines between the axon and soma in other adrenergic neurones, such as the d'istribution of norahenaline in the cell bodies and terminal regions of vertebrate sympathetic neurones (Norberg and Hamberger, 1964; Dahlstrom and Haggendal, 1966). No octopamine could be detected in tlfe soma of an identified motorneurone to the same muscle (Fast extensor tibiae motoneurone; FETi - Hoyle and Burrows, 1973) so that octopamine is at least 800 times more concentrated in the soma of DUMETi. Recent evidence ihdicates that DUM cells from the metathoracic and 6th abdominal ganglia of the cockroach, Periplaneta americana, also contain octopamine in about the same amounts per soma as that of DUMETi from

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the locust (Dymond and Evans, 1979; see Table 4); but as yet measurements have only been performed on phsyiologically unidentified cells in the cockroach. It is of interest to note that some paired dorsal medially located cells in the thoracic and abdominal ganglia of Trichoptera (caddisflies) have been shown microspectro-fluorometrically to contain dopamine (Klemm, 1971). The position of these cells varies, especially in the abdominal ganglia. The branching pattern of one of these dorsal cells from the second abdominal ganglion is shown in Fig. 15. The single media9 neurite bifurcates to give rise

B

Fig. 15 A dorso-medially located dopamine cell from the second abdominal ganglion of the caddisfly, Anabolia nervosa, as revealed by histofluorescence. A. Sagital section to show dorsal cell body (2)with a thick granular cytoplasm (Zf) in the branches and terminal varicosities (marked with arrows). The median neurite passes ventrally and then bifurcate;. B. Reconstruction of same cell as in A. (From Klemm, 1971)

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to two separate dendritic regions. It is not clear at present if these cells have any peripheral axons and if they are homologous to the DUM cells or other median Neutral red staining cells of locusts, crickets and cockroaches. The above evidence, taken together with the fact that all the known phsyiological effects of DUMETi stimulation can be mimicked by octopamine application (see below) provides convincing evidence for the octopaminergic nature of DUMETi in locusts. The various physiological roles of the DUMETi neurone will be discussed in the following sections. 4.3

DUMETi

A N D T H E MODULATION OF A MYOGENIC RHYTHM

One of the first physiological functions demonstrated for D U M cells in the locust was the slowing of a myogenic rhythm of contraction and relaxation found in the extensor tibiae muscle of the locust hindleg (Hoyle and O'Shea, 1974; Hoyle, 1974). Firing of the identified DUMETi neurone can also be shown to inhibit this rhythm, the degree of inhibition being related to the firing frequency (Evans and O'Shea, 1978; Hoyle, 1978b) (Fig. 16). This L RI

I

57 Ten\ ion

1

/I

1 :"ov 30

(RI)

b

Fig 16 The effect of stimulating DUMETi on the myogenic rhythm of the extensor tibiae muscle in hindleg of the locust (Schistvcercaamericanagregar~a).Spikes in DUMETi, identified from intracellular soma recordings (R,) are initiated at 1Hz by antidromic stimulation from the left extensor nerve (S,) Stimulation artefacts are shown on the middle trace Myogenic contractures of the right extensor muscle are monitored on the lower trace A single spike in DUMETi (arrowed) is capable of lengthening the interval between two myogenic contractures At 1 Hz the interval IS lengthened considerably and the amplitude reduced (From Evans and O'Shea, 1978)

effect can be mimicked by the application of low concentrations of octopamine (Hoyle, 1975b; Evans and O'Shea, 1978) (Fig. 17). The myogenic rhythm is confined to a bundle of tonic muscle fibres at the proximal end of the muscle, and it is possible to dissect out the bundle without inhibiting the rhythm (Burns and Usherwood, 1978). The myogenic bundle possesses at least two types of aminergic receptor, one which slows the rhythm and another which accelerates the rhythm, in addition to receptors for GABA and glutamate (Evans and O'Shea, 1978). Figure 18 sum-

377

BlOGENlC A M I N E S I N THE INSECT N E R V O U S SYSTEM A

-

C

-

+

C

C

~

~

~

-

4

-

-

~

~

-

k+c--bL_

i

Fig. 17 The response of the myogenic rhythm to octopamine applied alone and in the presence of a-and p-adrenergic blocking agents. The figure shows a continuous recording of the myogenic rhythm in a detached leg; it has been divided into three parts (A,B,C) for ~ in the convenience. The arrival on the myogenic bundle of a pulse of 1 0 - 6 DL-octopamine saline superfusate is marked (f). The end of each pulse and the return to saline is also indicated (J). Trace A shows the inhibiting effect of octopamine alone. Trace B shows a similar response to octopamine in the presence of 1 0 - 6 oL-propranolol ~ (p-adrenergic blocker). Trace C shows a slight accelerating effect of octopamine in the presence of 10-6M-phentolamine (a-adrenergic blocker), the slowing effect of octopamine having been blocked. (From Evans and O’Shea, 1978)

marizes the effects of the various transmitters on the myogenic bundle. Blocking experiments with picrotoxin indicate that the effects of octopamine are directly on the muscle rather than on the terminals of the inhibitory motoneurone. The time courses of the effects of the application of glutamate and GABA, and of the stimulation of the slow excitatory motoneurone and the common inhibitor are also much shorter than those induced by DUMETi stimulation and octopamine application (Evans and O’Shea, 1978). Thus the effects of octopamine are not mediated by an interaction with either the glutamate or GABA receptors. 4.3.1 Octopamine receptor-mediated slowing of the rhythm The aminergic receptor that mediates the slowing of the rhythm is maximally sensitive to monophenolic amines and exhibits several of the characteristics of the classical vertebrate a-adrenergic receptor (Evans and O’Shea, 1978) (see Fig. 17). This finding contrasts to that of Hoyle (1975b) who found that p-adrenergic blocking agents were more effective than a-adrenergicblocking

378

PETER soul cc

D. EVANS

1 SET1

1

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Bloc h I rig .Igcnl

E l k l ,111 rll\llllll

Fig. 18 Diagram summarizing effects of various transmitters and neuro-hormones on the myogenic rhythm of extensor-tibiae muscle of locust hindleg. The sources of the active agents and blocking drugs for the responses are indicated. Increases (1) and decreases (J) in amplitude (a) and frequency (f) of the rhythm are indicated. SETi, slow extensor-tibiae motoneurone; CI, common inhibitor; DUMETi, dorsal unpaired median cell to extensor-tibiae muscle; GABA, gamma-amino butyric acid; 5HT, 5-hydroxytryptamine. (From Evans and O'Shea, 1978)

agents in inhibiting the effects of amines on this rhythm. This discrepancy cannot be explained as Hoyle (1975b) did not specify the blocking agents used or their concentrations. However, a-adrenergic blocking agents are more effective than 0-adrenergic blocking agents at the octopamine receptors that mediate the activation of adenylate cyclases in lobster blood cells (Battelle and Kravitz, 1978), in cockroach brain (Harmer and Horn, 1977), and in firefly light organs (Nathanson, 1979). Similarly, two other defined octopamine receptors, those in crayfish heart (Florey and Rathmayer, 1978) and those mediating autoregulation of peripheral octopamine cells in lobster (Konishi and Kravitz, 1978), are preferentially blocked by a-adrenergic blocking agents. A recent detailed pharmacological study of the octopamine receptor on the locust myogenic bundle confirms the a-adrenergic-like character of the receptor. The most potent synthetic antagonists of octopamine were WB4101 and phentolamine, whilst the most potent synthetic agonists were effective in the following order: clonidine > tolazoline > tramazoline > methoxamine > phenylephrine (Evans, unpublished). The amine most potent in causing a slowing of the myogenic rhythm is DL-synephrine, but little or no endogenous synephrine could be detected in assays of isolated DUMETi somata (Evans and O'Shea, 1978). This sug-

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gests that the endogenous activator of this receptor would be octopamine, released from the terminals of the DUMETi neurone. Recent studies have shown that the slowing response is stereospecific for the naturally-occurring D(-)isomer of octopamine (Evans, unpublished). In the firefly light organ DL-synephrine is again a more potent agonist than DL-octopamine (Carlson, 1968) but the former is not an endogenous constituent of the light organ containing segments, whilst octopamine is present. (Robertson and Carlson, 1976). The structure specificity and drug sensitivity of the receptor mediating the slowing of the myogenic rhythm in the locust (Evans and O'Shea, 1978) is similar to that found for the receptors mediating the activation of octopamine-sensitive adenylate cyclases (Battelle and Kravitz, 1978; Harmer and Horn, 1977; Nathanson, 1979). This suggests that the prolonged effects of octopamine on the myogenic rhythm may also be mediated via the action of a specific octopamine-sensitive adenylate cyclase. 4.3.2

Receptor-mediated acceleration of the rhythm

The aminergic receptor that accelerates the myogenic rhythm has a lowaffinity for octopamine and a high-affinity for the indolalkylamine, 5-hydroxytryptamine (5-HT) (Evans and O'Shea, 1978). The function of this receptor which accelerates the rhythm is not clear. Since none of the neurones innervating the myogenic bundle appear significantly to activate this receptor, it is likely to be the target of a blood-borne factor. This factor may be an amine such as 5-HT; 5-HT at low concentrations is known to accelerate the beating of the insect heart (Miller, 1975a), and is thought to be a neurohormone in some insects (Berridge and Prince, 1972). The fact that exogenously applied 5-HT accelerates the rhythm, however, does not mean that 5-HT is the natural activator of the myogenic rhythm. It has been reported on several occasions that 5-HT is capable of activating receptors whose natural agonists are peptides (e.g. Maddrell et al., 1971; Pilcher, 1971). It has also been reported that the pentapeptide proctolin, and related peptides, are capable of inducing myogenic contractions in the extensor tibiae muscle of the locust, Locusta migratoria (Piek and Mantel, 1977; Piek et al., 1979). However, in another locust, Schistocerca gregaria, where low concentrations of proctolin (5 x 1 0 - I ' ~ )increase both the frequency and amplitude of the ongoing myogenic rhythm, superfusion of the preparation with bromo-LSD (at a concentration that completely blocked the responses to 5-HT) did not modify the response of the myogenic rhythm to proctolin (May et al., 1979). This suggests that proctolin and 5-HT are acting on separate receptors on the myogenic bundle, both of which mediate increases in the frequency and the amplitude of the rhythm.

380

PETER D. EVANS

It is, however, interesting to note that at high concentrations octopamine also activates the aminergic accelerating receptor (Evans and O’Shea, 1978). This can be particularly well demonstrated in preparations where the slowing response to octopamine has been blocked with phentolamine. It is thus possible that the aminergic accelerating receptor is involved in the acceleration of the rhythm sometimes observed on stimulating some unidentified DUM cells (Hoyle, 1978b). This effect could either be mediated through the release of octopamine from DUM cell endings or by the release of 5-HT (or perhaps a peptide) into the circulation from cells in the same region. The resolution of this point awaits the physiological identification of the cells responsible for the accelerating response described by Hoyle (1978b). 4.3.3 Function of the myogenic rhythm

It seems likely that the myogenic rhythm found in a part of the extensor tibiae muscle of the hind legs of locusts and grasshoppers, and described above, is identical to the rhythm first described in this muscle by Voskresenskaya (1959). This author described the appearance of “spontaneous rhythmic contractions” in this muscle in denervated leg preparations from the migratory locust, Locusta migratoria, and the Egyptian locust, Anacridium aegyptium. The rhythmic contractions were suggested to be of myogenic origin and could be induced in quiescent preparations by the application of acetylcholine. Similar spontaneous rhythmic contractions were also reported in the flexor tibiae muscle of L . migratoria three days after denervation. Hoyle (1978b) rediscovered the same phenomenon and he points out that the rhythm in extensor tibiae preparations from Locusta migratoria is seldom evident unless the leg has been denervated for some hours. H e goes on to state that “in all insects that show it, a few days of denervation, followed by excision of the leg, are sufficient to reveal it is an intrinsic feature, at least of the extensor tibiae muscle”. Hoyle (1978b) also observes that a “hidden” rhythm can be recovered by adding acetylcholine ( ~ O - ’ M ) and eserine to the saline. Hoyle (1974) has suggested that the rhythm may represent some form of intrinsic exercise rhythm for the muscle. A more plausible suggestion is that it functions to aid in the flow of blood along the long narrow hindleg (Usherwood, 1974). Evans and O’Shea (1978) have also suggested that in addition it might assist in the ventilation of the leg. Myogenic pumping structures are present at the base of many insect appendages, including the antennae and wings, where they are thought to be involved in haemolymph circulation (see Wigglesworth, 1965 and Jones, 1977 for references).

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The myogenic bundle preparation of the locust extensor tibiae muscle has provided a large amount of useful information on octopamine receptors and on the actions of an identified octopaminergic neurone. Nonetheless, it seems unlikely that the modulation of this myogenic rhythm is the sole function, or even a major physiologically important one, for DUMETi for the following three reasons. First, physiological, biochemical and anatomical evidence indicates that DUMETi projects to parts of the extensor tibiae muscle that do not exhibit any myogenic rhythm (Evans and O’Shea, 1978; Hoyle, 1 9 7 8 ~ )Second, . although D U M cells exist in other thoracic ganglia of the locust, the rhythm in the extensor muscle is reportedly confined to the metathoracic segment (Hoyle and O’Shea, 1974). Burns and Usherwood (1978), however, report that the extensor muscles of the pro- and mesothoracic legs gradually develop low levels of tension in the absence of any neural excitation and that small osciliations in base tension were observed during low frequency stimulation of the slow excitatory motoneurone to this muscle. They point out that in these muscles the tonic fibres do not seem to be arranged in separate bundles as they are, at least in part, in the metathoracic leg. This diffuse distribution of tonic fibres would make it difficult for these muscles to produce synchronized myogenic contractions. Third, myogenic rhythms are apparently absent from the leg muscles of the cockroach (Hoyle and O’Shea, 1974) even though DUM cells are present in their thoracic ganglia (Crossmanet al., 1971; Dymond and Evans, 1979; see Fig. 14). Thus the functions of DUMETi in other regions of the extensor muscle may be more indicative of the general functions shared by other DUM cells. 4.4

DUMETi

A N D T H E P O T E N T I A T I O NO F N E U R O M U S C U L A R T R A N S -

MISSION

The proposal that DUM cells may have some “regulatory function” on neuromuscular transmission in various locust muscles was first put forward by Plotnikova (1969). Recent evidence on the modulatory role of octopamine released from DUMETi terminals in the extensor tibiae muscle of the locust hind leg is consistent with such a proposal (Evans and O’Shea, 1977; O’Shea and Evans, 1977, 1979). Direct stimulation of DUMETi has very little effect on the muscle fibres of the locust extensor tibiae muscle, but has a very pronounced effect on neuromuscular transmission from the slow excitatory motorneurone (SETi) to this muscle (Fig. 19). (Evans and O’Shea, 1977; O’Shea and Evans, 1979). Stimulating DUMETi increases the strength of the SETi-induced tension twitches by about 30% and the potentiation is detectable for at least two minutes after the end of the DUMETi stimulation period. The SETi

PETER

382

D. EVANS

induced excitatory junctional potentials (EJPs) are also potentiated by DUMETi stimulation and the effect has the same time course as the effect on twitch tension. The effects on EJP size are generally smaller than those on twitch tension. This probably reflects the fact that EJPs are recorded from single end plates whereas the tension responses represent the summed

~

-

-

,NTRA

DUMETi EXTRA

I Z!”

1

0 1 mV

25 8

Fig. 19 The effect of stimulating DUMETi at 10 Hz (lower traces) on the tension (upper trace) generated in the extensor muscle of the tibiae of the locust hindleg by stimulating the slow extensor motoneurone (SETi) (10 Hz). The DUMETi intracellular (INTRA) and extracellular (EXTRA) potentials are shown. Note the slow potentiation and long-lasting effect of DUMETi on the SETi tension. (From Evans and O’Shea, 1977)

effectsover the whole muscle. Factors such as the distance of the recording electrode from the end plate will effect E J P size as well as differences in the responses of individual muscle fibres to SETi stimulation, which are known to vary in different parts of the muscle (Hoyle, 1 9 7 8 ~ ) . The effects of DUMETi stimulation can be mimicked by the application of low concentrations of octopamine to the muscle (Evans and O’Shea, 1977; O’Shea and Evans, 1979) (Fig. 20). The effects of octopamine application and DUMETi stimulation far outlast the period of stimulation. The magnitude of the octopamine effects are proportional to the length of exposure and are concentration dependent (O’Shea and Evans, 1979). They are also stereo-specific for the naturally occurring D(-)isomer of octopamine (Evans, unpublished). The effect on twitch tension is also highly specific for monophenolic biogenic amines, so that the best agonist of the response is again DL-synephrine (cf. myogenic rhythm slowing receptor) whereas dopamine, tyramine, N,N-dimethyloctopamine,noradrenaline, adrenaline, phenylethanolamine and 5-HT, all are without effect when applied for 30 s at 1 0 - 6 ~Higher . concentrations of 5-HT (10-3-10-2~)have been shown to block quickly the excitatory nerve-muscle transmission in this muscle, probably by non-specifically blocking the actions of the natural transmitter at post-synaptic sites (Hill and Usherwood, 1961). Besides affecting the magnitude of SETi-induced twitch tension, octopamine and synephrine affect the rate of relaxation of twitch tension

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SFl'i tension

I I0

v

111

30 s

Fig. 20 The potentiation by a pulse of 1 0 - ' ~DL-octopamine of the amplitude of tension and EJPs induced by the SETi motoneurone in the locust. The SETi motoneurone is stimulated at 1Hz by extracellular stimulation of nerve 3b of the metathoracic ganglion. Note that the slow rise in SETi-induced tension (upper trace) is accompanied by a smaller and more variable rise in the amplitude of SETi induced EJPs (lower trace). Both effects far outlast the presence of octopamine. (From O'Shea and Evans, 1979)

(O'Shea and Evans, 1979). This relaxation response is also observed for twitch tension induced by stimulation of the fast excitatory motoneurone (FETi) to this muscle, but in this case no potentiation of tension is found (see Fig. 21). It seems likely that the relaxation and potentiation effects produced by octopamine result from different sites of action. First, the two responses differ in their concentration thresholds, that for the relaxation effect being lower. Second, the magnitude of the responses differ, with the relaxation effect being bigger than that of potentiation. Third, the rate of development of the relaxation response is faster than that of the potentiation response. Fourth, the a-adrenergic blocking agent phentolamine appears to preferentially block the potentiating effect of octopamine. It has been suggested that the change in the rate of relaxation is a direct effect of the amines on the muscle whereas the potentiation of tension is, at least in part, a presynaptic effect (O'Shea and Evans, 1979). The presence of presynaptic octopamine receptors can be demonstrated on the terminals of the SETi neurone in experiments where octopamine has been shown to affect the frequency but not the amplitude of spontaneous miniature endplate potentials (mepps) (Fig. 2 2 ) , (O'Shea and Evans, 1979). This effect is also blocked by the a-adrenergic blocking agent, phentolamine and again contrasts with that produced by high concentrations (1 0 - 3 ~of ) 5-HT, which reduces the amplitude but has very little effect on the frequency of mepps (Usherwood, 1963). Thus although the presence of presynaptic

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384

SLTi

D. E V A N S

XTi

Fig. 21 Effect of lO-'u DL-octopamine and DL-synephrine on the time course and amplitude of twitches generated by stimulating SETi (A and B), and the effect of 10-6M-DL-octopammeon FETi-induced tension (C) in the locust. The oscilloscope time base is triggered by the rise in tension caused by stimulating either the SETi or FETi motoneurone. In A and B three sweeps are superimposed which represent twitches initiated prior to octopamine or synephrine application (l),about 20 s after application (2) and about 60 s after application (3). In C two twitches are shown, the first (1) initiated prior to octopamine application and the second (2) about 30 s after application. Note that in A and B the effect of octopamine and synephrine on the rate of relaxation occurs prior to the potentiating effect on amplitude of peak tension. Twitches induced by FETi are not increased in amplitude by octopamine but there is a marked effect on the rate of relaxation (C). (From O'Shea and Evans, 1979)

receptors for octopamine can be demonstrated, it is not yet proven that they alone are responsible for the effects of octopamine on SETi-induced twitch tension. Indeed, in a similar modulatory system in the marine mollusc, Apfysia, the 5-HT containing metacerebral cells act to increase the twitch tension of certain buccal muscles both by presynaptic effects on the motorneurones and by direct post-synaptic actions on the muscle (Weiss et a f . , 1975, 1978). A possible explanation for the potentiating effects of octopamine on SETi neuromuscular transmission could be that its action was secondary to a suppression of tonic release of transmitter (GABA) from the inhibitory motoneurone. Such a tonic release of GABA has been suggested to occur at the neuromuscular junction of the crab (Parnas et a f . , 1975). This explanation would be consistent with the observation that octopamine reduces the amplitude of inhibitory junctional potentials from the common inhibitory motoneurone to the extensor tibiae muscle of the locust (Evans and O'Shea, 1977). However, the potentiating effects of octopamine on SETi neuromuscular transmission are not blocked by the GABA blocking agent picrotoxin (O'Shea and Evans, 1979). Thus the effects of octopamine appear to be

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direct and not secondary consequences of reducing tonic, spontaneous release of GABA. The relaxation effects of octopamine on SETi-induced twitch tension (O’Shea and Evans, 1979) and of octopamine and DUM cell stimulation on basal tonus in the extensor tibiae muscle of the locust (Hoyle, 1975b; 1978b; O’Shea and Evans, 1979) suggest a ready explanation for the apparently contradictory results of Mayet al. (1979). The latter authors were unable to demonstrate any potentiation of SETi-induced tension by DUMETi stimulation or octopamine application in the isolated bundle of tonic muscle fibres from the locust extensor tibiae muscle. It is known that in this isolated bundle preparation that SETi-induced twitch contractions summate at very low frequencies (< 1HZ) (Burns and Usherwood, 1978). Thus, since Mayet al. (1979) used a stimulation regime for SETi of 10-15 HZ, for periods of 2-3 s every 15-20 s, they were looking at the effects of octopamine and DUMETi stimulation, not on individual SETi-induced twitch contractions, but rather on summated tetanic contractions of the muscle. It is thus likely that their observed antagonistic actions of octopamine and DUMETi stimulation on neurally evoked contractions are due to the relaxation effects described above reducing the summation responses of the muscle. In view of the marked effects of DUMETi on SETi neuromuscular transmission, and its apparent relative lack of effect on FETi neuromuscular transmission ,the reported distribution of DUMETi terminals on the extensor tibiae muscle (Hoyle 1978c) is rather curious. Hoyle ( 1 9 7 8 ~ )reports that DUMETi terminals are found only in association with muscle bundles that receive innervation from FETi, irrespective of whether they also receive input from SETi and the common inhibitor. DUMETi terminals are also reported to be absent from the proximal fan region of the muscle which contains the myogenic bundle responsible for generating the myogenic rhythm discussed above (Hoyle, 1 9 7 8 ~ )This . is again a curious finding in view of the potent effects of DUMETi stimulation on this rhythm (Evans and O’Shea, 1978). Indeed, Mayet al. (1979) working on the isolated tonic muscle bundle (myogenic bundle), which does not contain any fibres innervated by FETi, were able to present electrophysiological evidence for the presence of a DUMETi innervation. It is possible that the techniques used by Hoyle ( 1 9 7 8 ~ did ) not accurately reveal all the terminals of the DUMETi neurone, due to the sparseness of their distribution in the muscle. Alternatively, the distribution of DUMETi terminals might not be a true reflection of their site of action. This would be particularly true of a neurone that releases its neuroeffector as a local neurohormone, to affect the activity of many cells in the area of its terminal arborisations. Indeed, Hoyle et al. (1974) could detect no discrete DUMETi synapses in the extensor tibiae muscle and described the terminals of DUMETi as “blindly ending

PETER D. E V A N S

386 c

2

=

L

0

I

I

40

I

I

80

I

I

I

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I

l(10

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I

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Fig. 22 Effect of lO-’u-m-octopamine (black bar) on the spontaneous release of neurotransmitter from the terminals of the SETi motoneurone on the extensor tibiae muscle of the locust. Sample intracellular records from a muscle fibre which receives SETi input ale shown in the upper part of the figure (A,B,C). These recordings are representative of the condition before (A), during (B) and after (C) application of octopamine. A plot of frequency of mepps (mean frequency per second of five consecutive seconds) against time is shown in the lower part of the figure and arrows indicate from where the sample recordings were taken. Note the slow rise in mepp frequency and the long-lasting effect of octopamine. (From O’Shea and Evans, 1979)

neurosecretory terminals” (see Fig. 23). The parallels between the DUMETi system and other known neuromodulatory systems that release biogenic amines as local neurohormones will be considered later (see Section 4.6).

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Fig. 23 Nerve terminal of DUMETi containing dense-cored vesicles (from the locust, Schistocercugreguriu). This was located after tracing the DUMETi action potential into a final nerve twig. The twig supplied a bundle of fast muscle fibres innervated by the fast axon to the extensor tibiae (FETi) but neither the slow axon nor the common inhibitor. The fast axon branch to the muscle fibre branches to supply several terminals elsewhere on the fibre. The DUMETi axon continues into only a single strip terminal with varicosities, the whole of which contains dense-cored vesicles. Scale bar, 1 pm. (From Hoyle et al., 1974)

4.5

STUDIES ON THE TERMINAL ABDOMINAL GANGLION

The terminal abdominal ganglion in insects, such as cockroaches, crickets and locusts, is much larger than the other abdominal ganglia, and is produced by the fusion of several ganglia from the last abdominal segments. This is reflected in the presence of several groups of dorsal median neurones that stain selectively with the dye Neutral red in this ganglion. In the cockroach terminal abdominal ganglion (6th abdominal ganglion), Dymond and Evans (1979) reported the presence of three groups of from eighteen to twenty-five cells selectively stained with Neutral red, thus indicating their possible aminergic nature (see Fig. 14). The cells in these three groups did not exhibit any fluorescence characteristic of catecholamine-containing cells when subjected to the Falck-Hillarp histochemical technique. However, isolated unidentified cell bodies from these groups were shown to contain octopamine in about the same amounts as that found for isolated DUMETi somata in the locust (Dymond and Evans, 1979; see Table 4). The corresponding cells in the terminal abdominal ganglion of the cricket have also been reported to stain selectively with Neutral red (O’Shea and Murphey, 1978). It has been known for some time that the physiological properties (Jego et al., 1970; Crossman et al., 1971) and some morphological properties (Seabrook, 1968, 1970) of dorsal median neurones in the terminal abdominal ganglia of cockroaches and locusts are similar to those described for the corresponding cells in the metathoracic ganglia (Plotnikova, 1969; Crossrnan et al., 1971; Evans and O’Shea, 1977; Hoyle 1978a).

388

PETER D. E V A N S

The terminals of the intraganglionic neurosecretory interneurones described by Farley and Milburn (1969), in the sixth abdominal ganglion of the cockroach, have many similarities with octopamine containing nerve terminals in the locust (Hoyle et al., 1974) and the lobster (Evans et al., 1975, 1976a; Schaeffer e f al., 1978). The terminals of these cockroach neurosecretory cells were found to be closely associated with the cercalgiant fibre synapses, usually adjacent to the cercal sensory ending (Farley and Milburn, 1969) and in close association with the medial giant interneurone dendrite in the cricket (O’Shea and Murphey, 1978). Farley and Milburn (1 969) suggested that these neurosecretory terminals contained biogenic amines “such as catecholamines or indoles” on the basis of the size of their neurosecretory granules (100 nm in diameter). They proposed that these endings might “regulate in some manner the transmission at the cercal-giant fibre synapse”. Recently, Caste1 et al. (1976) reported similar neurosecretory terminals in association with the processes of giant fibres in the metathoracic ganglion of the cockroach. The above evidence suggests the possibility that these neurosecretory terminals might represent the neuropilar processes of the octopamine-containing dorsal median neurones in the cockroach sixth abdominal ganglion. Early pharmacological studies on the cercal afferent giant fibre synapse in the cockroach sixth abdominal ganglion revealed its sensitivity to the application of high concentrations ( 10-3-10-4~)of catecholamines such as adrenaline and noradrenaline (Twarog and Roeder, 1957). Hodgson and Wright (1963) reported that the system was insensitive to phenylephrine (neo-synephrine or rn-synephrine). They assumed that the system was preferentially activated by catecholamines, and that adrenaline itself was probably the neurologically active substance in insects. However, more recent studies have failed to substantiate the original identification of adrenaline (Ostlund, 1953) in insect nervous tissue (see Section 2.1). It has also been shown that the sixth abdominal ganglion of the cockroach contains a higher ratio of octopamine to dopamine than any other ganglion in the nerve cord (Dymond and Evans, 1979). Unfortunately, the early pharmacological studies mentioned above did not test octopamine or p-synephrine, which has been shown to be a potent agonist of several other insect octopamine receptors (Carlson, 1968a, b; Evans and O’Shea, 1978; O’Shea and Evans, 1979). It is possible that if they had, then the functional importance of octopamine in insect nervous tissue would have been realised ten years earlier. O’Shea and Murphey (1978) have recently presented preliminary evidence for the sensitizing action of octopamine on the response to sound of the medial giant interneurones in the cricket terminal abdominal ganglion. This effect was blocked by the a-adrenergic blocking agent phentolamine, as

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are other effects of octopamine in insects (Evans and O’Shea, 1978; O’Shea and Evans, 1979). It is thus tempting to speculate that exogenously applied octopamine mimicks the action of octopamine released from the terminals of the dorsal medial neurones in this ganglion. Direct evidence for this hypothesis will require studies on physiologically identified, individual dorsal median neurones in the terminal abdominal ganglion.

4.6

FUNCTIONS OF

DUM

N E U R O N E S A N D P A R A L L E L S W I T H OTHER

SYSTEMS MODULATED B Y AMINES

4.6.1

Vertebrate skeletal neuromuscular junctions

Biogenic amines can modulate transmission at the vertebrate neuromuscular junction (Orbeli, 1923; Bowman and Zamis, 1958; Kuba, 1970). The defatiguing effect of sympathetic nerve stimulation and of the application of adrenaline to vertebrate skeletal muscle has been called the “Orbeli phenomenon”. However, the natural release sites for the modulatory amines and the physiological significance of the experimental effects observed have yet to be established (Bowman and Nott, 1969). The effects could be mediated by circulating amines such as adrenaline, the levels of which increase markedly under stressful conditions. They could also be produced by the release of noradrenaline from the endings of sympathetic nerves in the skeletal muscles. At the present time the sympathetic fibres associated with vertebrate skeletal muscle are not thought to innervate the muscle fibres directly but rather to supply blood vessels within the muscle. Nonetheless, the innervation of the peripheral sympathetic nervous system is very diffuse and it is possible that some of the released noradrenaline “spills over” on to the muscle fibres. The actions of biogenic amines on neuromuscular transmission in vertebrates has many similarities with the actions of octopamine at the SETi neuromuscular junction of the locust (see Section 4.4). Presynaptic a-adrenergic receptors are involved in the potentiating effect of noradrenaline at the vertebrate skeletal neuromuscular junction (Bowman and Nott, 1969; Kuba, 1970) and similar a-aminergic presynaptic receptors mediate some of the actions of octopamine at the locust skeletal neuromuscular junction (Evans and O’Shea, 1977; O’Shea and Evans, 1979). Again, in both cases, the magnitude of twitch tension and E J P size are increased and the frequency, but not the amplitude, of spontaneous mepps is increased when the respective amines are applied (Kuba, 1970; O’Shea and Evans, 1979). In vertebrate fast twitch muscle, adrenaline increases the maximum twitch tension and the time to peak tension (Lewis and Webb, 1976) but the rate of development of tension is not significantly affected (Bowman and

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Nott, 1969). This is again similar to the effects of octopamine on twitch tension at the SETi neuromuscular junction of the locust (O’Shea and Evans, 1979). 4.6.2 Invertebrate skeletal neuromuscular junctions In contrast to the situation described above for vertebrate skeletal muscle, invertebrate skeletal muscle has been shown recently to receive a direct aminergic innervation, in addition to its motor innervation, in, for instance, the marine mollusc, Aplysia, (Weiss et al., 1975, 1978) and in the locust (Hoyle et al., 1974; Evans and O’Shea, 1977, 1978; O’Shea and Evans, 1979; see also above). In Aplysia, an identified aminergic modulatory neurone is involved in the control of feeding behaviour. The giant serotonin-containing metacerebral cells (MCC) produce a long-lasting modulatory action on the motoneurones of the buccal mass, associated with the food-arousal state of the animal (Weissetal., 1975, 1978). The MCC appear to exert a modulatory effect on the ongoing activity of certain buccal motoneurones both centrally and peripherally. In the central nervous system they act through conventional excitatory synapses onto the motoneurones but in the peripheral musculature of the buccal mass the MCC appear to have little direct effect on the muscle when stimulated alone. Rather, they modulate the effectiveness of neuromuscular transmission from the motoneurones. The MCC potentiation appears to be the result of a small transient increase in EJP size together with a more prolonged enhancement of excitation-contraction coupling, which may well be mediated via increased levels of cyclic AMP (Weiss et al., 1976,1979). The above MCC effects could also be mimicked by the application of serotonin. Heterosynaptic facilitation mediated by serotonin through an increase in cyclic AMP levels has also been demonstrated to occur in central nervous pathways in Aplysia (Brunelli et al., 1976; Kandel et al., 1976; Shimahara and Tauc, 1977). Recently a modulatory role has also been proposed for dopamine in neuromuscular transmission in the gill of Aplysia (Swann et al., 1978). In the lobster, octopamine has been shown to increase the tension generated in skeletal muscle by an excitatory motoneurone (Evans et al., 1975; Kravitz et al., 1976). It is released into the blood from a group of peripheral octopaminergic neurones (Wallace et al., 1974; Evanset al., 1975; Evanset al., 1976a, b). Lobster skeletal muscle, however, unlike that of the locust (Evans and O’Shea, 1977,1978) and ofAplysia (Weissetal., 1975,1978) is not known to receive any direct aminergic innervation. Thus any of the variety of amines that affect it must presumably reach their target sites through the blood (Dudel, 1965; Kravitz et al., 1976). Some of the active

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amines in lobster, such as 5-HT, are better at potentiating the muscle tension produced by the stimulation of an excitatory motoneurone than is octopamine (Kravitz et af., 1976). 5-HT however had no effect on SETi neuromuscular transmission in the locust at a concentration of 1 0 - 6 ~ , despite its pronounced effects on the myogenic bundle of the same muscle (Evans and O’Shea, 1978; O’Shea and Evans, 1979). Thus the nonspecificity of the potentiation effect at the lobster neuromuscular junction is in sharp contrast to the specificity for monophenolic amines exhibited by the locust preparation. 4.6.3

Neurosecretory innervation to skeletal muscle

A discrete neurosecretory innervation appears to be present in a variety of skeletal muscles from both vertebrates and invertebrates. It has been reported in certain muscles of molluscs (Barrantes, 1970), insects (Osborne et af., 1971) and crustaceans (Huddart and Bradbury, 1972). In general, it appears as a series of blindly ending neurosecretory terminals which do not appear to form discrete synapses with the muscle fibres. This description is very similar to that of the endings of DUMETi in the extensor tibiae muscle of the locust (Hoyle et al., 1974) (see Fig. 23) and may represent an analogous modulatory input in these other muscles. Similar neurosecretory terminals have been described in frog skeletal muscle (Osborneetal., 1971). They have been suggested to provide anatomical evidence for an adrenergic innervation which could be responsible for the modulatory actions of amines (see Section 4.6.1). However, Osborne et al. (1971) note that such neurosecretory axons frequently run alongside a blood capillary, so that it seems possible that they may represent a form of sympathetic control for blood vessels. It should also be pointed out that anatomical evidence for neurosecretory terminals does not provide any information on the chemical nature of the neurosecretory material itself. The size of the electron-dense granules observed by Osborne et al. (1971) was 100-170 nm in diameter, which falls in the area of overlap reported for granule size, between presumed aminergic and presumed peptidergic neurosecretion (see Knowles, 1967). Recent evidence has also shown that skeletal muscles of cats may receive a peptidergic innervation from insulin and gastrin containing neurones (Uvnas and Uvnas-Wallensten, 1978; Uvnas-Wallensten and Uvnas, 1978). These observations were made by demonstrating the release of insulin-like and gastrin-like immunoreactivities upon nerve stimulation in the perfused hind limb of the cat. Thus the whole question of the distribution of neurosecretory terminals in vertebrate skeletal muscle needs to be reinvestigated with a combination of anatomical, biochemical and physiological techniques. The exact distribution

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of the terminals in the muscle needs to be described, the identity of the neurosecretory product found and its physiological role determined.

4.6.4

Presynaptic receptors

The presence of presynaptic receptors for octopamine (O’Shea and Evans, 1979) and for acetylcholine (Fulton and Usherwood, 1978) at excitatory neuromuscular junctions in the locust, has many parallels with vertebrate peripheral and central synapses. It is becoming increasingly apparent that presynaptic a-adrenergic receptors are involved in the modulation of the release of a number of different neurotransmitters (Starke, 1977). It is becoming equally clear that presynaptic terminals in general may have specific receptors for many different hormones and neurotransmitters which modulate either the release or the synthesis of transmitter (Westfall, 1977). The significance of these complex modulatory systems is not clear at present since in many cases there is no physiological evidence to indicate that the synapses ever get exposed in vivo to many of the compounds they have been demonstrated to respond to in vitro.

4.6.5

Vertebrate central nervous system

The idea that amines may play roles as local modulatory hormones also receives support from studies in the vertebrate central nervous system (see Dismukes, 1977b; Moore and Bloom, 1979; Kupfermann, 1979). In the cerebral cortex, for instance, it has recently been reported that only about 5% of the terminals of serotonergic and noradrenergic inputs actually form discrete synapses. The rest appear to end as neurosecretory terminals which probably release their amines into a general area of the brain where they may simultaneously affect and coordinate the activities of large neuronal assemblies (Descarries, Beaudet and Watkins, 1975; Descarries, Watkins and Lapierre, 1977). This again makes a striking parallel with the peripheral innervation pattern of DUMETi in the locust. Physiological evidence for a modulatory role for biogenic amines in the vertebrate brain is also emerging from studies on the effects of microiontophoretic application of amines on the unitary activity of spontaneously firing neurones in the rat cerebral cortex (Hicks and McLennan, 1978a; Reader et al., 1979; Henwood et al., 1979) and in the dorsal horn of the spinal cord (Hicks and McLennan, 1978b). The predominant effect of the application of dopamine, noradrenaline or 5-HT was an inhibition of the spontaneous firing rate, in most cases, whilst octopamine produced both excitatory and inhibitory effects (Hicks and McLennan, 1978a, b). In the

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cerebellum it has been shown that noradrenaline, besides inhibiting the spontaneous firing rates of Purkinje cells, also serves to bias or modulate the responses produced by synaptic inputs such as those from the mossy fibres or those from the climbing fibres (Freedman et al., 1977). The effects of noradrenaline application can be mimicked by the activation of noradrenergic pathways from the locus coeruleus to the cerebellum (Hoffer et al., 1973). It thus appears that the modulatory function for biogenic amines is an important and general function which has been confirmed in both vertebrates and invertebrates and in both the central and peripheral nervous systems.

4.6.6 Function of D U M neurones The various long-lasting modulatory effects of biogenic amines have led to the idea that they may function in the long-term regulation of behavioural responsiveness, such as in learning, in the control of motivational state, in arousal or in sensitization (Weiss et al., 1975, 1978; Susswein et al., 1976; O’Shea and Evans, 1979; Kupfermann, 1979). This idea is supported by recent evidence showing that amines can function in the vertebrate CNS to selectively bias or modulate the effects of outputs produced by conventional neurotransmitters (Hokfelt and Fuxe, 1969; Hoffer et al., 1973; Freedman et al., 1977). The example of the action of the octopaminergic DUMETi neurone in the locust is probably the simplest way in which the intensity of behaviour can be increased; it is done without altering the motor output, but by placing a bias directly at the neuromuscular junction (O’Shea and Evans, 1979). It is thus tempting to speculate that one of the functions for DUM cells in insects could be a general “arousal” phenomenon which, by analogy with the vertebrate sympathetic nervous system, would be activated by the animal under conditions of stress. A similar function has been proposed for the peripheral octopaminergic neurones in the lobster nervous system (Konishi and Kravitz, 1978; Evans, 1978b). Many modes of sensory input are known to feed into the DUM cell system (O’Shea and Evans, 1977; Hoyle and Dagan, 1978) which would then be in a position to enhance the effectiveness of other sensory inputs and to potentiate specific muscular responses, whilst perhaps providing a resistance to muscular fatigue. At the moment, little is known about the central effects of DUMETi and other DUM neurones, but it has been speculated (O’Shea and Evans, 1979) that neuromuscular function can provide a simple model for central effects.

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5

Biogenic amines and firefly light organs

It has been known for many years that biogenic amines are able to stimulate light production in firefly light organs. Kastle and McDermott in 1910 noted that adrenaline, when injected into a firefly, acted as a “powerful stimulus, inducing a strong and steady glow” (see also Creighton, 1926). Since this time our detailed knowledge of the pharmacological responses of light organs, to a variety of biogenic amines, has been much expanded (see Carlson, 1969). Ultrastructural studies have, however, revealed differences in the innervation patterns of light organs from larval and adult fireflies, as well as a diversity of patterns among adults of different species. At present, although we have a reasonable amount of evidence for the chemical identity of the natural activator of the light organ and a detailed understanding of the chemistry of light production (McElroy and DeLuca, 1973), our knowledge of the mechanism by which the released transmitter triggers light production is still very poor. This section will outline the different innervation patterns reported for various firefly light organs, to provide a basis for a discussion of their pharmacological and physiological responses to biogenic amines. The possible modes of action of the neurotransmitter will be discussed and likely approaches for future studies on firefly light organs considered.

5.1

INNERVATION OF LIGHT ORGANS

The neural control of light production in fireflies has been demonstrated by the direct stimulation of the nerves to the light organs (see Buck 1948 and Hanson, 1962; Buck and Case, 1961; Carlson, 1972; Oertel and Case, 1976). It is generally assumed that differences in the location of the nerve terminals in the light organs, from larval and adult fireflies (see Fig. 24), are responsible for the differences in their pattern of light production. Larval fireflies produce a continuous slow glow, whereas adults are capable of producing very complex flashing patterns that are thought to represent species-specific mating signals (see Lloyd, 1971). Electron microscopical investigations of the ultrastructure of the light organ of the adult firefly (Photuris pennsylvanica) showed that fine nerve endings were present within the tracheal end organs (Kluss, 1958). The latter structures consist of a complex arrangement of epithelial cells (tracheal epithelia1 cell; tracheal end cell; and tracheolar cell) which surround the bifurcation point of the tracheal twig into separate tracheoles (see Fig. 24A). Smith (1963) further showed that the nerve terminals in this species ended as axonal swellings inserted between the tracheal end cell and the tracheolar cell. They did not innervate directly the cells of the light

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Fig. 24 Diagramatic representation of the innervation patterns of firefly light-organs. A. Adult light organ of American genera e.g. Phofuris and Phofinus, where tracheal end-cell completely surrounds tracheolar cell and nerve endings (adapted from Smith, 1963). B. Adult light organ of Asiatic genera, e.g. Pferopfyx,Pyrophanes and Luciofa (adapted from Peterson and Buck, 1968). C. Larval light organ of Photurts (adapted from Oertel, Linberg and Case, 1975). T, lantern trachea; TE, tracheal epithelial cell; tt, tracheal twig; TEC, tracheal end cell; NT, nerve terminals; TC, tracheolar cell; t, tracheoles; P, photocytes; n, nucleus of photocyte

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producing epithelium (the photocytes). The terminals contained two populations of membrane bound vesicles, one with small (20-40 nm) clear vesicles, and a second with larger (60-120 nm) dense cored “neurosecretory granules”. Although Smith (1963) could find no specialized regions of synaptic contact, Case and Linberg (quoted in preparation in Oertel et al., 1975) are reported to have found many synaptic specializations within the membranes of the nerve terminals where they were contiguous with the tracheal end organ. The innervation pattern of the light organs of certain Asiatic genera of firefly (Pteroptyx, Pyrophanes and Luciola) appears to differ slightly from that of the American genera (Photuris and Photinus) (Peterson and Buck, 1968) (see Fig. 24B) In the former, the tracheal end cell does not completely surround the nerve terminals and the tracheolar cell in the region of the bifurcation of the tracheal twig. This contrasts with the situation found in the American genera (see above) and means that in the Asiatic genera the nerve terminals not only make contact with the components of the tracheal end organ, but also lie next to the photocytes themselves. The nerve terminals, however, appear to be more closely apposed to the membranes of the tracheal end organ complex than to those of the photocytes, and no synaptic contacts are observed between the nerve terminals and the photocytes (Peterson and Buck, 1968). Complex tracheal end organs have been shown to be present in all firefly species capable of producing sharp flashes, and absent in species producing long-lasting glows (Buck, 1948). Tracheal end organs are also absent in the light organs of larval fireflies (Peterson, 1970; Oertel et al., 1975). Oertel et al. (1975) found a direct innervation of the photocytes in Photuris larvae (see Fig. 24C). The nerve terminals showed synaptic specializations with electron dense, bar-shaped structures, continuous with the nerve membrane and surrounded by clusters of small (40-65 nm) clear vesicles. As in the nerve endings in adults, large (90-135 nm) electron dense, dark-cored vesicles were also found. It appeared that larger numbers of light vesicles were found in naked (terminal?) regions of the axons. The larval light organs are innervated from the 8th (last) abdominal ganglion. The nerve entering the light organ contains two axons which branch profusely, giving numerous endings amongst the photocytes, which in the larvae are randomly arranged, as opposed to the regular array of columns and rosettes in the adult (Oertel et al., 1975). It is not known if both axons innervate all the photocytes in the larvae, or if different subpopulations exist. It is also not known if there are functional differences between the two axons. Thus in Photuris, the light organs of both adult and larvae are innervated by neurones that form discrete synaptic structures. Two populations of vesicles (small clear and large dense cored) are present in the nerve termi-

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nals in each case. The size distribution and staining characteristics of the two vesicle populations are consistent with the presence of an aminergic neurotransmitter (see Table 3). However, despite the direct innervation of the photocytes in the larval light organ, its light response develops at a rate about 100 times slower, and lasts 50 times longer, than that of the adult (Oertel et al., 1975). It thus seems that the presence of the tracheal end organ complex in the adult facilitates the rapid turning on and off of the light response.

5.2

PHARMACOLOGY OF L I G H T RESPONSES

Despite differences in the innervation patterns of the light organs of adult and larval fireflies the results of all the pharmacological studies performed to date seem to suggest a common aminergic response mechanism. Smalley (1965), working on the light organs of the adult firefly, Photuris pyrulis was able to confirm the original observations of Kastle and McDermott (1910) and of Creighton (1926) that injection of adrenaline stimulated light production. She extended the study to show that injections of noradrenaline and amphetamine could also induce light production. Pretreatment of the light organs with reserpine, a drug that was presumed to deplete the levels of endogneous adrenergic substances from the nerve terminals, did not block the actions of noradrenaline and adrenaline (Smalley, 1965) or tyramine (Borowitz and Kennedy, 1968) in causing light production. This suggested that these amines act directly upon post-synaptic receptors, rather than by releasing an endogenous amine from the nerve terminals. This action of tyramine was different from its well known actions of displacing noradrenaline from storage sites in vertebrate sympathetic systems and provided the first indications of a postsynaptic site of action for phenolamines in this system. Reserpine treatment, however, as well as long-term denervation of the light organs, blocked the effects of amphetamine, suggesting that the latter compound acts by releasing transmitters from nerve terminals. Amphetamine may also act postsynaptically since it reduced the effectiveness of exogenously applied tyramine (Borowitz and Kennedy, 1968). Smalley (1965) was also able to demonstrate that the actions of the amines in causing light production were mediated by a-adrenergic receptors. Electrical stimulation of the light organ induces a short-latency quick flash, due to direct stimulation of the photocytes and also a longer latency slow flash, which is due to neural activation and transmitter release. The slow flash was blocked, or delayed, by the a-adrenergic blocking agents yohimbine and dibenzyline (phenoxybenzamine). The p-adrenergic agonist, isoproterenol proved ineffective in eliciting light responses from the

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adult light organ (Borowitz and Kennedy, 1968). The above evidence suggested that normal neural excitation of the light organ involved an a-adrenergic mechanism, rather than a cholinergic mechanism as had been suggested by McElroy and Hastings (1955). Further investigations on an in vitro preparation derived from the light organs of the larval stagesof the firefly, Photuris, (Carlson 1968a, b) showed that the light organs were in fact maximally sensitive to the application of the phenolamines, synephrine and octopamine, rather than to the catecholamines. Synephrine was about six times more effective than an equivalent amount of octopamine. Carlson (1968a) was also able to show that the effects of reserpine and amphetamine upon the larval light organ were similar to those upon the adult organ. Surprisingly, he found that the response of the larval light organ to noradrenaline was not stereospecific (Carlson, 1968a). As yet the stereospecificity of the responses to synephrine and octopamine d o not seem to have been tested. O n the basis of potency in inducing luminescence, Carlson (1968b) reasoned that the transmitter was most probably a monophenolic amine, but warned that it was not possible to rule out the possibility that a catecholamine was the true transmitter, rendered less potent than the monophenolic amines by an inactivation mechanism. In the larval light organ the effects of neurally released transmitter were compared with those caused by the application of exogenously applied synephrine (Carlson, 1972). Their actions shared the following similarities. Both had direct actions on the photocytes, had relatively long latencies, stimulated ATP production, did not induce the production of long-lived intermediates, were unaffected by M A 0 inhibitors, were rapidly blocked by chlorpromazine and showed similar responses to dichloroisoproterenol. The one major difference Carlson (1972) found between the two modes of activation of the light organs was that the luminescence induced by transmitter release was extinguished much more rapidly than that caused by synephrine. He argued, however, that this difference could be related to the mode of application of the active compounds in the two cases. In the case of neural release, transmitter might be rapidly inactivated by a reuptake process localized in the nerve terminals, whereas exogenously applied synephrine would become more widely distributed in the light organ and might even have its actions on non-synaptic extrajunctional receptors. In a similar preparation where intracellular electrophysiological records were made from larval photocytes, whilst simultaneously monitoring light output and recording nerve activity extracellularly, Oertel and Case (1976) were able to compare the electrical effects induced by transmitter release with those resulting from the application of biogenic amines. They were able to demonstrate that neural activation of the light organ resulted in a small

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depolarization and conductance change (peak at one second) in the photocyte, which was followed by light production (peak at seven seconds). However, the application of the biogenic amines, noradrenaline, adrenaline or synephrine, in the concentration range to 1 0 - ’ ~ induced , light emission but no depolarization. The authors suggest that the depolarization might be an indirect consequence of neurotransmitter action. Oertel and Case (1976) suggest that the transmitter may activate a nucleotide cyclase, resulting in an increased level of cyclic nucleotide that then triggers light production and also indirectly, produces a change in membrane permeability. However, if this is the case, it could be argued that exogenous application of the aminergic neurotransmitter, or related compounds, should also activate the cyclase and then produce the same side effects on membrane permeability. This disturbing difference could well be explained by the idea presented by Carlson (1972) that exogenously applied amines are acting at extrajunctional receptors in the light organ, rather than mimicking the actions of the released transmitter at the junctions themselves. This suggestion would not, of course, preclude the possibility that the transmitter was a biogenic amine, but would mean that activation of the junctional and non-junctional receptors might have different side effects on membrane permeability. It is obvious that a major step in our understanding of the firefly system will be achieved by the positive identification of the transmitter released from the nerve terminals. Attempts to demonstrate the presence of the catecholamines, adrenaline and noradrenaline, in the nerve terminals in the light organs, by the use of potassium dichromatic fixation at acid and neutral pH, have not been successful (Oertel and Case 1976). Unfortunately, the use of fluorescent histochemical techniques is precluded by the high background fluorescence of the tissue (Oertel et a f . , 1975). Thus no positive evidence is available on the catecholamine content of firefly light organs. Radioenzymatic assays have, however, provided evidence for the presence of octopamine, but not of synephrine, in the last three light organ-containing segments of adult fireflies (Robertson and Carlson, 1976). This does not ensure that octopamine is the transmitter, since the last three abdominal segments contain neuronal ganglia, fat body, intestine and cuticle, as well as the light organs. The isolated light organs of the glow worm (Lampyris noctiluca) have, however, been shown to contain octopamine but not synephrine (Evans, unpublished). The above studies suggest that the actual transmitter of the firefly light organ may well be octopamine rather than synephrine, despite the fact that synephrine is the most potent of the exogenously applied amines in inducing light production. This observation is not unexpected since in other invertebrate systems synephrine is found to be a very potent agonist of octopamine

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receptors (Harmer and Horn, 1977; Battelle and Kravitz, 1978; Evans and O’Shea, 1978; O’Shea and Evans, 1979). Further evidence for the octopaminergic nature of transmission in the firefly light organ is provided by the demonstration of the presence of a specific phenolamine activated adenylate cyclase in homogenates of the light organ-containing segments (6 and 7) of the abdomen of adult Photuris (Nathanson, 1979). In the same study, Nathanson was able to demonstrate that in homogenates of isolated light ~ stimulated basal enzyme activity by approxiorgans, 10 - 5 octopamine mately 20-fold, with a maximal activation at 1 0 - 3 of ~ greater than 25-fold. In contrast, octopamine stimulation was less than three fold in the surrounding non-lantern tissue which included ganglia, fat body, intestine, reproductive organs and cuticle. Structure-activity relationships support the idea of the activation of a specific octopamine receptor in this preparation whilst additivity experiments suggest that all exogenously added catecholamines and phenolamines are acting on a single octopamine receptor. The indolalkylamine, 5-hydroxytryptamine, caused no enzyme stimulation which is consistent with its lack of effect on light production (Smalley, 1965). Alpha-adrenergic blocking agents were again more effective than @-blockingagents in preventing octopamine activation of adenylate cyclase in the homogenates of the firefly light organs (Nathanson, 1979). This is similar to their actions on light production in fireflies (Smalley, 1965; Oertel and Case, 1976), on the activation of other adenylate cyclases by octopamine (Harmer and Horn, 1977; Battelle and Kravitz, 1978) and also to their actions on other octopamine receptors in insects (Evans and O’Shea, 1978; O’Shea and Evans, 1979). Octopamine has also been found to be a potent stimulator of adenylate cyclase activity in broken-cell preparations from the light organs of larval fireflies (Photuris) (Nathanson and Hunnicutt, 1979).

5.3

M O D E O F ACTION O F NEUROTRANSMITTER

The effects of biogenic amines on firefly light organs could be mediated via their activation of an adenylate cyclase. This hypothesis is supported by the fact that the phosphodiesterase inhibitors theophylline and aminophylline can induce glowing in the larval light organ (Oertel and Case, 1976). The increased levels of cAMP could increase the amount of energy available for light production by activating a phosphorylase, thus causing an increased rate of breakdown of stored glycogen. A similar activation pathway has been described for the action of octopamine on glycogenolysis in insect nerve cord (Robertson and Steele, 1972,). Alternatively, the raised levels of cAMP could play some modulatory role in the photocyte light emission reactions. Another possibility, suggested by Carlson (1968b), is that light produc-

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tion is triggered by the pyrophosphate liberated during the production of CAMPfrom ATP when the amine stimulates adenylate cyclase. It is known from the work of McElroy and Hastings (1955) that the enzyme luciferase can be released from an inhibitory oxyluciferyl-adenylate complex by pyrophosphate. This would be very attractive to explain the functioning of the larval light organ where the photocytes are directly innervated. It would, however, be more difficult to conceive of such a mechanism working in the adult light organ, where the innervation of the tracheal end organ complex is presumed to play an important role in the rapid onset and extinction of the light response. This has lead to the suggestion that in the adult light organ, oxygen availability is the rate limiting factor in light production. Under hypoxic conditions, the introduction of an oxygen pulse can induce a “pseudoflash” in the light organ (Buck, 1948). Buck, however, decided that this was unlikely to be an in vivo control mechanism due to the difficulties involved in isolating the photocytes at the necessary lowered oxygen tension compared to the surrounding tissues. Smith (1963) also pointed out the lack of any specialised sphincter structure in the light organ that might control the oxygen supply, and concluded that the architecture of the light organ precluded this mode of control under normal conditions. Recently however, the finding of highly-specialized, reinforced tracheoles in the firefly light organ prompted Ghiradella (1977, 1978) to re-examine this theory. The latter author suggested that these tracheoles are designed to withstand stresses that would fold or collapse ordinary tracheoles. The structural reinforcement and the resemblance of the end cells to other cellular structures considered to be active in the transport of ions and fluid, has been taken as evidence that an osmotic mechanism may be involved in turning on and off the oxygen supply to the light organ (Ghiradella, 1977). This is an intriguing idea but at present definitive evidence for the physiological mechanisms involved is lacking. It would be interesting to know if the lack of specialized cuticular thickenings on the tracheal twig (see Fig. 24) means that this branch is more susceptible to collapse under appropriate osmotic conditions than other parts of the tracheal system. It seems likely that one function of the tracheal end cell (especially in the American genera of firefly, where it completely surrounds the origin of the tracheolar cell) could be to control the ionic environment in the immediate vicinity of the tracheolar cell and perhaps, set up an ionic potential gradient across its outer membrane. This gradient could then provide a potential energy source which could be used in the activation of the light organ when the neurally released transmitter caused a specific permeability change in the tracheolar cell membrane. The latter cell is anatomically well-designed to convey electrical disturbances deep into the photocyte mass and may thus act very much like the T-tubule system of striated muscIe.

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5.4

F U T U R E S T U D I E S O N FIREFLY L I G H T O R G A N S

It can be seen from the preceding discussion that we are still in the realms of speculation as regards the exact mechanism of coupling transmitter action to the better understood process of light production. The bulk of the evidence to date supports the case for the presence of an aminergic transmitter substance in the light organs, probably octopamine or a closely related compound. There are, however, a number of gaps in our knowledge that need to be filled before we can finally accept this theory. First, the octopamine present in the light organs must be demonstrated to be present in the nerve terminals. One approach, that would certainly help in this matter, would be the identification of the cell bodies of the neurones supplying the innervation to the light organs. The cell bodies could then be dissected out and analysed for their amine content and synthetic abilities. The identification of these cells and subsequent electrical activation would also enable investigations on the innervation pattern of the photocytes. It might then be possible to determine the significance of the two axons innervating each light organ (Oertel et al., 1975) and see if there are any differences between their effects. It would also be possible to perform transmitter release experiments and, perhaps, also provide definitive evidence on the presence, or absence, of catecholamines in the light organs and their innervating neurones. Second, the cellular location of the octopamine-sensitive adenylate cyclase needs to be determined. This information is essential to the understanding of how the transmitter activates the light response, especially in the adult light organs. The fact that the stimulation of adenylate cyclase activity by octopamine is ten-fold greater in firefly light organ homogenates than in any other broken cell preparation from excitable tissue, makes it ideal for the study of phenylethylamine receptors. Nathanson (1979) suggests that this greater activity of the enzyme may be related to the “redundancy of the lantern cellular organisation and associated innervation which, in some ways, seems analogous to the arrangement present in the electric organs of certain fishes which contain high concentrations of cholinergic receptors”.

6

Dopamine and insect salivary glands

A dopaminergic innervation appears to be present in the salivary glands of many insect species. Thus they represent very useful preparations for studying the pharmacology and physiology of dopaminergic transmission in insects, since the peripherally located neuroglandular interactions are much more accessible to study than the corresponding dopaminergic synaptic

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structures in the central nervous system. In this section, the innervation pattern of various insect salivary glands will be briefly outlined and the distribution of catecholamines considered. The effects of nerve stimulation will be compared to the pharmacological responses of the glands to the application of biogenic amines and the properties of the biogenic amine receptors discussed. This section will concentrate on the innervated salivary glands of insects and will only refer briefly to the non-innervated salivary gland of the blowfly, Calliphora for comparative purposes. A more detailed account of fluid secretion and its regulation by 5-hydroxytryptamine (5-HT) in the salivary gland of Calliphora is given by Berridge (1977). 6.1

CATECHOLAMINE DISTRIBUTION A N D INNERVATION PATTERN

Fluorescence histochemistry of insect salivary glands using the Falck-Hillarp technique has revealed the presence of neuronal varicosities containing catecholamines, but not 5-hydroxytryptamine (5-HT), in the locust, Schistocerca gregaria (Klemm, 1972), in the cockroach, Nauphoeta cinerea (Blander al., 1973) and in the moth, Manduca sexra (Robertson, 1975). In the locust, microspectrofluorometrical recordings indicate that the fluorescent fibres contain dopamine. The presence of dopamine, but not noradrenaline or adrenaline, has been confirmed by radioenzymatic assay in the salivary glands of Nauphoeta (DA 0.55 ng/gland; Fry et al., 1974) and Manduca (DA 0.34 pglg; Robertson, 1975). Similar amounts of dopamine (0.74 p g / g ) were found in the salivary glands of another arthropod, the ixodid tick, Boophilus microplus, but in this case a significant amount of noradrenaline (0.45 pg/g) was also detected (Megaw and Robertson, 1974). The innervation pattern of insect salivary glands varies from species to species. The paired racemose (acinar-type) glands of the cockroach and locust are dually innervated. They receive one input from the central nervous system via the paired salivary duct nerves of the suboesophageal ganglion, and a second input from branches of the stomatogastric nerve (e.g. in Periplanera americana, Whitehead, 1971; in N . cinerea, Bowser-Riley, 1978; and in S. gregaria, see Klemm , 1972). Branches of the nerves from both sources innervate the salivary gland reservoirs, ducts and the secretory cells themselves in the acini. Each acinus contains peripheral cells that transport water and ions, and central cells that secrete enzymes. Many of the acini towards the midline are innervated from both the left and right salivary duct nerves (Ginsborg and House, 1976). The salivary duct nerve contains two large axons (about 7 p m in diameter) and several smaller axons in P. americana (Whitehead, 1971), and in N . cinerea the nerve has a similar composition (House, 1977). Methylene blue staining reveals that the salivary gland duct nerve in N . cinerea branches

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profusely to form a plexus on the surface of the acinus where the axons exhibit swellings or varicosities at irregular intervals (Bowser-Riley, 1978). These varicosities are of a similar size to those observed in the acinar nerve plexus of cockroaches (Whitehead, 1971) and also cats (Garrett, 1966). They are also of similar size to those observed to run over the acinar surface in scanning electron microscope studies and to those observed by fluorescence histochemistry to contain catecholamines (Bowser-Riley, 1978). The branch of the stomatogastric nerve to the salivary glands contains about a dozen axons, each of less than 2 p m in diameter in P. americana (Whitehead, 1971). The distribution of the stomatogastric nerve branches is less extensive than that of the salivary duct nerve and in N . cinerea the former were not observed to contribute to an acinar plexus (Bowser-Riley, 1978). They did, however, connect with a complex network of multipolar neurones on the surfaces of the anterior regions of both salivary reservoirs. These cells have been suggested to be sensory neurones that monitor pressure changes in the salivary reservoirs (Bowser-Riley, 1978). Although a great deal of information is available on the branching patterns of the nerves to the salivary glands no information is available on the location of the cell bodies of the neurones that supply this innervation. Electron microscopical observations of the salivary duct nerve of N . cinerea reveal that the two largest axons contain dense-cored vesicles (Bland and House, 1971; House, 1977). Within the acini two types of axon have been described (Maxwell, 1978). The most widespread (Type A) had small (44 nm in diameter) agranular elliptical vesicles and large electron dense vesicles (92 nm in diameter). These axons were found in the basement membrane of the acinus, where they were still mostly wrapped in glial cells, and also deeper in the acinus where they ran between the peripheral and central cells. In many cases the distance between the axons and the surface of the acinar cells was not more than 20 nm and the axons were only infrequently surrounded by glial cells. The second type of axon (Type B) was less numerous than the first. It contained large dense cored vesicles (138 nm in diameter) similar to those found in other insect neurosecretory axons (Miller, 1975a). The type B axons were common on the surfaces of the acini and ducts, and also in nerve bundles traversing the acini, but none were observed deep in the core of the acini or seen to have any obvious releasing sites (Maxwell, 1978). Maxwell (1978) has suggested that the Type A axons represent the catecholaminergic innervation to the glands, based on the size distribution of the vesicles (see Section 2.3), the frequency of occurrence of the axons, and the fact that this axon type reacts with the permanganate fixation technique of Hokfelt (1968) to give electron dense precipitates (Maxwell unpublished, quoted in Maxwell, 1978). It is not clear at present if the Type B axons represent the input to the acinus from the stomatogastric

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nerve, which has been postulated to modify or augment salivary secretion rather than to initiate it (Bowser-Riley, 1978). Very little evidence has been presented for the existence of discrete synaptic junctions in insect salivary glands. Each acinus is traversed by numerous axons, some of which appear to loose their glial sheaths when they penetrate the acinus, whilst those that remain on the surface only rarely loose their glial sheath. Thus the proposed aminergic innervation to the cockroach salivary glands has many parallels with the sympathetic innervation to vertebrate salivary glands, in that true synaptic specializations are rare and transmitter may be released “en passant” from varicosities running both on the surface and also between the cells within the acinus (Garrett, 1966; Tandler and Ross, 1969). Thus transmitter may be released from the same neurone at several different sites and diffuse as a local neurohormone to affect the secretory activity of many target cells. The tubular salivary glands of some saturniid moths (Kafatos, 1968; Robertson, 1974) and of the blowfly, Calliphora (Oschman and Berridge, 1970) are much simpler in structure than the acinar-type of locusts and cockroaches. They consist of a single epithelial layer and contain only a single cell type in any given region of the gland. The salivary glands of the moth, Manduca sexta, receive only a single innervation from the frontal ganglion of the stomatogaaric nervous system via branches of the unpaired oesophageal nerve (Robertson, 1974). Only the proximal fluid secreting portion of the gland appears to be innervated and Robertson (1974) hypothesises that the cells of the more distal protein secretory regions, either produce salivary enzymes continuously, or are regulated by blood-borne factors. The nerve endings on the glands from the oesophageal nerve, contain both large dense-cored granules (50-100 nm in diameter), as well as small electron-lucent vesicles (30-40 nm in diameter). In permanganate fixed glands the small vesicles have electron-dense cores indicating their possible catecholaminergic nature (Robertson, 1974,1975) (but see Section 2.3). Axons without glial investment are found in intercellular areas throughout the tissue, where they approach to within 20-25 nm of the effector cell membranes. Possible release sites were only found infrequently and were characterized by accumulations of large dense-cored vesicles and electron-lucent vesicles. In contrast to the above, Oschman and Berridge (1970) could find no evidence for innervation of the salivary glands of the blowfly Calliphora. It is currently believed that in this species the secretory activity of the glands is controlled by 5-I-IT,released into the haemolymph and carried to the glands as a blood-borne hormone (Berridge, 1975, 1977; Berridge and Prince, 1972). The release sites of the 5-HT and the location of the neurones involved are not known at the present time.

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6.2

EFFECTS O F NERVE STIMULATION A N D APPLICATION O F BIOGENIC AMINES

Further evidence for dopaminergic transmission in the innervated insect salivary gland has been obtained by comparing the electrical and secretory responses of the glands to neural stimulation and to the application of biogenic amines. T o date these studies have been carried out exclusively on the isolated salivary glands of Nauphoeta cinerea. 6.2.1

Effects of electrical stimulation

Intracellular recordings froin acini during the electrical stimulation of the salivary duct nerves reveals a long latency (- 1 s) hyperpolarizing response. This is sometimes followed by a smaller and slower depolarization (Ginsborg and House, 1976). The hyperpolarizing phase of the response varies in amplitude not only with the number of stimuli applied (Fig. 25 lower trace), D A I O a~~

t

--

It

II

5x10

it

it

10 -'\I

'td

I

i t

If

-I I J_ \f

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It

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Fig. 25 Intracellular recording showing the effects of dopamine (DA) and nerve stimulation on the resting potential of a cell in the salivary gland of the cockroach, Nuuphoeta cinereu. The duration of application of dopamine is the interval between the arrows. Responses evoked by nerve stimulation are marked by asterisks and the number of stimuli delivered is given above each response. The maximum responses to nerve stimulation (lower trace) were obtained after the dopamine responses (upper trace). The resting potential of this cell was approximately - 35 mV. (From Bowser-Riley and House, 1976)

but also with the stimulus intensity. This latter observation has been interpreted as evidence for the possible multiple innervation of the acini by neurones that are recruited at different intensities of stimulation. However, Ginsborg and House (1976) warn, that since the cells of a given acinus are electrically coupled, the observation says nothing about the innervation

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pattern of individual cells. Direct evidence for multiple innervation of acini towards the midline is provided by the fact that they receive axons from branches of both the left and the right salivary duct nerves. It has been suggested that the more variable depolarizing response could result from the activation of receptors other than those mediating the hyperpolarization (Ginsborg and House, 1976). Under a number of experimental conditions, it is possible to reduce the amplitude of the hyperpolarizing component without having much effect on the amplitude and the time course of the subsequent depolarization (see Fig. 26). The relationship A

Fig. 26 Independence of depolarizing and hyperpolarizing phases of response to salivary duct nerve stimulation in the cockroach. A downward deflexion corresponds to hyperpolarization. The stimulus artefact at the beginning of each trace marks the duration of the period of stimulation. A, preparation taken from an animal pre-treated with reserpine. Responses shown to trains of 100 stimuli at 100 Hz (a) before and (b) after several exposures to dopamine. B, preparation had been exposed to a-flupenthixol. Responses to trains of (a) 50 and (b) 5 stimuli at 100 Hz. The peak value of the hyperpolarization in (a) was 25 mV, too large to be recorded on this channel. All the responses shown were evoked at about the same resting potential. (From Ginsborg and House, 1976)

between neuronal stimulus intensity and the electrical response of the gland suggests that several axons in the salivary duct nerve could be activated together. Thus, since the axons of the neurones that mediate the hyperpolarizing responses have not yet been identified, and the salivary duct nerve contains two large and several small axons, it is possible that the different components of the biphasic electrical response could be caused by different transmitters released from different neurones. Recent evidence, however, indicates that the iontophoretic application of dopamine t o the acini can mimic both the hyperpolarizing and depolarizing responses to electrical Stimulation (Blackman et al., 1979a). In some instances a depolarizing response was obtained with neural stimulation but could not be mimicked by iontophoretic application of dopamine to the same acinar cell, suggesting that perhaps the depolarizing receptors are less accessible to applied

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dopamine than the hyperpolarizing receptors. A differential sensitivity of the two receptor types to applied dopamine is also possible and the effects of the blocking agent a-flupenthixol would also be consistent with this suggestion (see Fig. 26). Electrical stimulation of the salivary duct nerve also leads to fluid secretion from the isolated salivary glands of N . cinerea (House and Smith, 1978). The submaximal secretory responses were frequency dependent. The exact temporal and causal relationships between the electrical responses of the acini and the production of saliva have yet to be established. The effects of direct stimulation of the second neural input to the salivary glands, that is the one from the oesophageal nerve, have not yet been reported. Ginsborg and House (1976) point out that the effects of salivary duct nerve stimulation were comparable to those obtained by field stimulation of the gland administered via a fine platinum wire placed close to the impaled cell and a large bath electrode. Thus they concluded that the oesophageal nerve did not make any conspicuous contribution to the initiation of salivary secretion. In Periplaneta americana, Whitehead (1 970) came to a similar conclusion since there was no apparent relationship between electrical activity in the nerve and salivary secretion. 6.2.2 Effects of biogenic amine application Pharmacological studies indicate that a number of biogenic amines, namely 5-HT, adrenaline, noradrenaline and dopamine, cause a hyperpolarization of the acinar cells (Bowser-Riley and House, 1976). Figure 25 (upper trace) shows a series of intracellularly recorded responses to dopamine application at different concentrations in the bathing medium. The amplitude of the response to dopamine is concentration dependent (see Fig. 27) and the maximum response obtained is the same as that for the maximal neurally evoked response. Dose-response curves (Fig. 27) indicate that dopamine is clearly the most potent agonist in producing hyperpolarization, whilst noradrenaline and adrenaline are almost equally potent and 5-HT is apparently only a partial agonist, since it failed to produce a maximum response equal to that of the neurally-evoked responses (Bowser-Riley and House, 1976). It has been suggested that the receptors mediating the responses to noradrenaline and adrenaline are different from those activated by dopamine. The average slope of the dose-response curve for adrenaline is steeper than that found for dopamine or noradrenaline (see Fig. 27) and this has been suggested to indicate that the receptor for adrenaline may be different to those for the other catecholamines (Bowser-Riley and House, 1976). More recent evidence on the additivity effects of biogenic amines in

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inducing the secretory response of cockroach salivary glands also indicates that the adrenaline and dopamine receptors are different (House and Smith, 1978). Similar experiments have also indicated that separate receptors for dopamine and noradrenaline can be distinguished. The possibility that the results of the additivity experiments described above could be explained by a

Agonisl conceiitralioii ( X I )

Fig. 27 Typical log dose-response curves of certain biogenic amines for the hyperpolarization response of acinar cells in the cockroach salivary gland. Each agonist response has been divided by the corresponding maximum evoked response for each cell. These maxima were 7 6 , 6 5 , 5 8 and 68 m V for dopamine (DA), noradrenaline (NA), adrenaline (Ad) and 5-hydroxytryptamine (5-HT) curves, respectively. (From Bowser-Riley and House, 1976)

reduction of dopamine binding or cellular uptake was thought to be unlikely by House and Smith (1978) on the basis that this effect should have been larger at lower dopamine concentrations, a finding not consistent with their observations. The receptors mediating the hyperpolarizing response to 5-HT are also thought to be distinct from those mediating the dopamine responses since, as mentioned above, 5-HT did not produce a maximal response equal to that of the neurally-evoked response. The 5-HT receptors could also be distinguished from the catecholamine receptors mediating both the electrical and secretory responses of the glands on the basis of differences in their competitive antagonism to the a-adrenergic blocking agent phentolamine (House and Smith, 1978; Bowser-Riley et al., 1978). The hyperpolarizing receptors activated by dopamine display a high degree of specificity for this amine. A structure-activity study has revealed that mono- or di-N-methylated derivatives were still active but that any substitution on the catechol ring hydroxyl groups dramatically reduced

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activity (Ginsborg et al., 1976b). N-acetyl dopamine, an important intermediate in the tanning processes of insect cuticle, was also without effect. The drug sensitivity of the dopamine receptors mediating the hyperpolarizing responses in the acinar cells of the cockroach salivary gland is very similar to that of specific dopamine receptors in the vertebrate brain (see Iversen, 1975). In both cases, the rigid structural analogue of dopamine, ADTN (2 amino, -6,7 dihydroxy -1,2,3,4, tetrahydronaphthalene) is a potent agonist and the antipsychotic drug, cis-a-fl upenthixol, is a potent antagonist (House and Ginsborg, 1976). The latter drug antagonizes both the hyperpolarizing responses to applied dopamine and to nerve stimulation in the cockroach salivary gland. Further evidence that the dopamine-induced hyperpolarizing responses of cockroach salivary glands were mediated by specific dopamine receptors, and not by a- or 13-adrenergic receptors, came from studies with a and 13 agonists and antagonists. The a-adrenergic agonists, amidephrine and methoxamine, and the B-adrenergic agonist, isoprenaline, were without effect at low concentrations, as was the 13-adrenergic blocking agent, propranolol, on the hyperpolarizing response induced by nerve stimulation (Ginsborg et al., 1976a). The classical a-adrenergic blocking agent phentolamine, however, turned out to be a reversible, competitive antagonist of the hyperpolarizing response induced by neural stimulation and dopamine application, as well as of the secretory responses elicited by both methods of stimulation (Ginsborg et al., 1976a; House and Smith, 1978; Bowser-Riley et al., 1978). The hyperpolarizing response induced by 5-HT is also antagonized by phentolamine but to a lesser extent than either that due to dopamine or to nerve stimulation. However, the affinity constant of phentolamine for the catecholamine receptors in the cockroach salivary gland is about 100-1000 times less than is usually found for classical a-adrenergic receptors in vertebrates (Bowser-Riley, et al., 1978; Furchgott, 1972). Thus at higher concentrations phentolamine blocks a range of other receptors in addition to the classical a-adrenergic receptor. It, for instance, antagonizes the dopamine-induced activation of adenylate cyclase in the rat superior cervical ganglion (Kebabian and Greengard, 1971) as well as the activitiesof octopamine, dopamine and 5-HT-stimulated adenylate cyclases in a variety of insect neuronal ganglia (Nathanson and Greengard, 1974; see also Table 6, Section 10). Phentolamine also antagonizes dopamine responses at a number of other receptors in molluscs and vertebrates (see Bowser-Riley et al., 1978, for references). The dopamine agonist apomorphine (see Iversen, 1975) also induces a dopamine-like response at high concentrations in the cockroach salivary gland (Ginsborg et al., 1976a). Methylsergide and ergometrine are potent antagonists of dopamine responses in several systems (see Bowser-Riley et

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41 1

al., 1978). They also block the hyperpolarizing responses both to dopamine and to nerve stimulation but the effect was again not specific as the action of 5-HT was also antagonized. In contrast, methylsergide and ergometrine did not block either the dopamine-induced or neurally-induced secretory responses of these glands. Bowser-Riley et al. (1978) point out that directly opposite effects of these drugs are known to occur at a number of different dopamine receptors in other preparations. However, this observation again raises the question of what relationship the hyperpolarizing response has to the secretory response, and whether or not they are mediated by the same dopamine receptors. This is obviously a very important question that requires further investigation. Octopamine was a relatively poor agonist of secretion in the cockroach salivary gland, and at best only produced a small hyperpolarization at a concentration of 1 0 - 5 ~Even . at concentrations of to 1 0 - 3 it ~ gave responses of less than 25% of the maximum neurally-evoked response (Bowser-Riley and House, 1976). However, when applied at 1 0 - 7 ~ , octopamine potentiated the neurally-evoked secretory potentials by about 30%, whilst at ~ O - ’ M the increase was followed by a pronounced decline during a prolonged exposure (Bowser-Riley and House, 1976). This effect suggests a parallel with the potentiating effect of octopamine at the neuromuscular junction of the locust, where octopamine increases the size of neurallyevoked excitatory junctional potentials (Evans and O’Shea, 1977; O’Shea and Evans, 1979; see Section 4.4). The frequency and size of “minature secretory potentials” in unstimulated salivary glands can be enhanced by adrenergic drugs such as tyramine and methoxamine (Ginsborgetal., 1976b) and bretylium (Silinsky, 1974) and also by some methylated derivatives of dopamine such as epinine (Ginsborget al., 1976a) (see Fig. 28). These results suggest the presence of presynaptic aminergic receptors on the terminals of the presumptive dopaminergic neurones in the cockroach salivary gland. These presynaptic receptors would presumably be involved in the regulation of the release and synthesis of transmitter (see Section 4.6.4). Acetylcholine has also been demonstrated to increase the submaximal responses produced by nerve stimulation, but does not increase the dopamine sensitivity of the cells. Furthermore, acetylcholine in the presence of physostigmine gives rise to random potentials, some of large size with a time course similar to that of evoked secretory potentials (BowserRiley and House, 1976). The above evidence has been tentatively suggested to indicate the presence of presynaptic acetylcholine receptors at the neuroglandular junctions (House, 1977). It is of interest to note that presynaptic acetylcholine receptors have also been described at the locust neuromuscular junction (Fulton and Usherwood, 1978). In view of the absence of any known cholinergicinput to these regions on both preparations,

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the significance of such receptors is unclear. It seems possible that they are not functionally important in these locations, but rather reflect the fact that the innervating neurones are sensitive to cholinergic sensory inputs on their dendrites in the central nervous system. 1 I'M a d r e n a l i n e

10 m V

1 min

Control

1 r n M rncrhoxamine

Fig. 28 Increase in miniature hyperpolarizations in cockroach salivary gland. A, effect of 1 p ~ tyramine, present in the bathing solution during the periods indicated by the horizontal bar. The onset is shown in ( a ) ;the individual miniature hyperpolarizations may be more clearly seen in ( b ) ,from the same experiment at higher speed and gain. B, effect of 1 p~ adrenaline; the large hyperpolarization is the response to a short train of stimuli to the salivary duct nerves. C , onset ( a ) and details (c) of the action of 1 mM methoxamine; b is a control record from the same acinus in the absence of methoxamine. The large hyperpolarizations in Aa, B and Ca are responses to short trains of stimuli to the salivary duct nerves. (From Ginsborg et al., 1976a)

6.3

FURTHER STUDIES ON DOPAMINERGIC TRANSMISSION I N INSECT SALIVARY GLANDS

The extensive studies on the salivary glands of the cockroach Nauphoefu cinerea, reviewed above, provide strong evidence for their dopaminergic innervation from the suboesophageal ganglion. There are still, however, several pieces of evidence lacking before dopamine can be positively identified as the transmitter at this neuroglandular junction. We need to know, for instance, the identity of the neurones in the suboesophageal

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ganglion responsible for this innervation and need to demonstrate that they contain endogenous dopamine and can release it from their terminals in the salivary gland. It will also be of interest to learn the function of the smaller neurones in the salivary duct nerve, and to discover which of the neuronal elements is responsible for the depolarizing potentials. The question of whether the receptors mediating the depoalirizing response are segregated in some way from those mediating the hyperpolarization is also intriguing. In addition it will be important to determine the relationship between the secretory and electrical responses of the salivary gland. The very long latencies of the hyperpolarizing response to nerve stimulation (- 1 s) can be reduced only to 380 ms by the iontophoretic application of dopamine to the salivary glands from a pipette placed as close as possible to the acinar surface (Blackman et ul., 1979a). However, Blackman et u f . (1979b) conclude that the long latency is not due to diffusion barriers to the applied dopamine, but rather to processes subsequent to receptor activation. A t the present time no evidence is available as to the nature of the mechanism of this slow response which could be the result of a sequence of chemical reactions (Ginsborg et af., 1979b). In view of the involvement of the enzyme adenylate cyclase in mediating the actions of dopamine in the vertebrate brain (see Iversen, 1975) and in mediating the actions of 5-HT in the non-innervated salivary glands of the blowfly, Cuffiphora(see Berridge, 1975, 1977), this enzyme system seems a worthwhile possibility for further investigation in the cockroach salivary glands. A further question that needs to be answered concerns the function of the enigmatic stomatogastric input to the cockroach salivary glands. It is interesting to note that in the salivary glands of the moth Munduca, the stomatogastric input is the only one present and also appears to be dopaminergic. It has been suggested in the cockroach that the stomatogastric input might serve some modulatory role (Bowser-Riley, 1978). It thus seems a possibility that this input provides a neuronal basis for the observed octopaminergic modulation of the secretory potentials. The significance of the presence of 5-HT receptors on the cockroach salivary glands, despite the apparent absence of this amine in the neurones innervating the gland, is unclear. One explanation would be that the cockroach glands may be activated by both neural and hormonal pathways, in contrast to those of the blowfly which are only activated by hormonal pathways. It would seem that the insect salivary gland is an excellent preparation for the study of the actions of biogenic amines in insects and will, in the future, answer many important questions of relevance to the functioning of biogenic amines in the insect central nervous system.

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7 Biogenic amines and the insect heart In 1926 Alexandrowicz summed up the knowledge on the neuronal control of the insect heart by stating that “the function of the nervous elements can be presented only in the form of a hypothesis, because the physiological data are meager and the morphological not sufficient”. A t the present time, some fifty years later, despite a considerable volume of literature on the subject, we are still in the realm of speculation concerning the role of the nervous system in the control of the insect heart. Insect heart muscle is probably the most well studied of all insect visceral systems but the great variability in the innervation pattern from species to species, the complex interactions between information from the central nervous system with that from peripheral ganglion cells, and the problems of differentiating between the effects of neurohormones and neurotransmitters, have all led to difficulties in the interpretation of the functions of the different neural elements present. In the present section some of the wide variety of innervation patterns of insect hearts will be briefly outlined to provide a background for a discussion of the localization of biogenic amines and the pharmacological effects of their application t o the insect heart.

7.1

I N N E R V A T I O NPATTERN

The insect heart consists of a dorsally situated muscular tube that is suspended in position by segmental sets of alary muscles. The exact anatomy varies from species to species (see Wigglesworth, 1965, and Jones, 1977). It is generally believed that the insect heartbeat is of myogenic origin, but is modulated by neural or hormonal mechanisms. In some species e.g. Anopheles larvae and pupae (Jones, 1954) no synaptic endings have been reported on the heart itself, even though various nerves run close to its surface, and it is assumed that all modulation must be mediated by hormones. Where a neuronal innervation is present, its extent varies considerably from one species to the next (see Wasserthal and Wasserthal, 1977; Jones 1977). It consists of a segmental input from the ventral nerve cord via branches of either the segmental nerves or of the transverse branch of the medial nerve. In some species, an additional input is present from lateral cardiac nerves which originate from the ganglia of the retrocerebral neurosecretory complex (stomatogastric nervous system). A great deal of confusion seems to have arisen about the innervation pattern of the heart in several insect classes, e.g. in Lepidoptera (see Wasserthal and Wasserthal, 1977), due to the difficulties involved in the interpretation of anatomical

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studies without adequate electrophysiological studies to back them up. The cardiac nervous system reaches its most complex form in primitive insect orders such as the Dictyoptera (cockroaches) (Miller and Usherwood, 1971). Here the heart is innervated by neurones from the central nervous system via segmental nerves, and also from peripheral lateral cardiac nerve cords running on either side of the dorsal vessel (see Fig. 29). In the cockroach, Periplanefa urnericana, Miller (1968a) reports the presence of the somata of two types of spontaneously active neurones in the lateral cardiac nerve cords. The first, the cardiac ganglion cells, are presumed to act as motorneurones, and the second, the neurosecretory cells, are found at the points where the segmental nerves join the lateral cardiac nerves. The total number of intrinsic cardiac neurones varies from one cockroach species to the next (e.g. at least 40 per lateral nerve in Blatta orientalis, Alexandrowicz, 1926; and 32 per lateral nerve in P . arnericuna, McIndoo, 1945) but at the present time the proportions of the different cell types is not known. The lateral cardiac nerve cords are made up of an outer region of mainly neurosecretory axons and an inner region of mostly non-neurosecretory axons (Johnson, 1966). In the cockroach, the frequency of the heart beat is not altered by the removal of the lateral cardiac nerve cords (Miller and Metcalf, 1968; Miller and Usherwood, 1971). Thus neurones from the ventral nerve cord and the intrinsic cardiac neurones in the lateral cardiac nerve cords, all act either directly or indirectly to regulate an endogenous myogenic rhythm of contractility in the cockroach heart muscle. The cardiac ganglion cells are spontaneously active but they can produce bursts of action potentials in response to mechanical deformation of the tissues (Miller, 1968a) and thus probably serve to help coordinate the simultaneous beating of the different myocardial chambers (Miller and Usherwood, 1971). The segmental nerves from the central nervous system contain three types of nerve fibre, two “neurosecretory” and one “ordinary” (Miller and Usherwood, 1971). One type of neurosecretory fibre contains large densecored granules and does not synapse with either the myocardium or the intrinsic cardiac neurones (Johnson, 1966). It probably releases its contents into the environment of the lateral cardiac nerve cord at various sites along this peripheral system and may also have effects on the pericardial cells on the walls of the myocardium (Johnson, 1966). A second type of neurosecretory fibre with small granules synapses directly with the heart muscle, each fibre innervating more than one heart chamber. In addition, the fibres may also synapse directly with the cardiac ganglion cells (Miller and Usherwood, 1971). The “ordinary” axons apparently also synapse directly with the cardiac ganglion cells, but do not innervate muscle fibres. Thus in the cockroach, the myogenic activity of the heart is regulated directly via a neurosecretory input from the central nervous system and also via inputs

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A MG

\

6AG

VNC

/

LCNC

B sv

DV-

Fig. 29 A. Diagramatic representation of the abdominal heart of the adult cockroach to show the connexion with the central nervous system (adapted from Alexandrowicz, 1926, and Miller and Usherwood, 1971). B. Enlargement of enclosed box from A to show details of cardiac nervous system (adapted from Miller, 1968). MG, metathoracic ganglion; VNC, ventral nerve cord: 6AG, sixth abdominal ganglion; SN,segmental nerve; DV, dorsal vessel or heart; SV, segmental vessel; LCNC, lateral cardiac nerve cord with intrinsic neurones; BW, body wall; OS, ostial valve; AL, alary muscles; NS, neurosecretory cells; CG, ganglion cell or motoneurone

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from the two types of intrinsic cardiac neurones. The intrinsic cells are in turn modulated by inputs from the central nervous system (Miller, 1968a; Miller and Usherwood, 1971). The existence of lateral cardiac nerves has also been confirmed for several other species of adult hemimetabolous insects, including dragonflies (Zawarzin, 191 l ) , and stick insects (Opoczyhska-Sembratowa, 1936), although the presence of intrinsic cardiac neurones was not reported in the former example (see Jones, 1977 for more references). In several species of Lepidoptera only segmental heart nerves have been confirmed, at the present time, but groups of peripheral neurosecretory cells are located in this nerve as it runs over the alary muscles (Hinks, 1975; Wasserthal and Wasserthal, 1977). In most insect species electrical stimulation of the segmental nerves leads to cardioacceleration (Miller 1968a), although evidence for an inhibitory innervation has been presented in Cafliphora (Normann, 1972).

7.2

BIOGENIC AMINE DISTRIBUTION

Direct evidence for the association of biogenic amines with the neural elements of the cardiac regulatory system is very limited. Hiripi and S.-Rozsa, (1973) demonstrated the presence of dopamine (2.42 pg/g) and 5-HT (2.69 pg/g) in the heart of the locust, Locusta migratoria migratorioides, using a spectrophotofluorimetric assay. In several preparations, electron microscopical evidence has been presented for the presence of axons containing dense-cored granules, of less than 150 nm in diameter, in association with the different structures of the insect heart (e.g. in Periplanefa americana, heart, Johnson, 1966; alary muscle, Adams et al., 1973; segmental vessel valve, Miller and Rees, 1973; in Cafliphora erythocephafa heart, Normann, 1972; in Sphinx figustri,axons of segmental nerve on alary muscles, Wasserthal and Wasserthal, 1977). The presence of dense cored granules of this type corresponds to the proposed “B type” aminergic innervation category of Knowles (1967) but there is no direct evidence in the above examples that the fibres contain biogenic amines. Some of the neurosecretory endings in the cardiac nerve cord of the cockroach, P. americana, have, however, been claimed on cytochemical evidence (Miller and Rees, 1973) to contain free amine groups associated with the electrondense granules. Miller and Rees (1973) showed that silver grains were associated with these granules using the silver cytochemical technique of Tramezzani, Chiocchio and Wassermann (1964). This technique is claimed to demonstrate the presence of free amine groups. It was not clear, however, which type of neurosecretory fibres reacted (i.e. either those intrinsic to the cardiac nerve cord or those from the ventral nerve cord) and furthermore,

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the specificity of the technique for biogenic amines seems doubtful (i.e. would it also react with small peptides?). More recently, Gersch et al. (1974) demonstrated that dopamine and 5-HT are present in the lateral cardiac nerve cords of the cockroach, Blaberus craniifer, by use of the Falck-Hillarp fluorescence technique. In the same study, the authors found that the permanganate fixation technique of Hokfelt (1968), for the demonstration of catecholamines, produced densecores in granules of 100-125 nm in diameter, whereas those of greater diameters i.e. 125-300 nm, remained electron-lucent. From this evidence, the larger granules were presumed to contain a peptidergic neurosecretory material. But, the source of the aminergic fibres is, as yet, unresolved. There appear to be many similarities between the association of biogenic amines with neurosecretory elements in the lateral cardiac nerve cord of insects and the pericardial organs of crustacea (Cooke and Goldstone, 1970; Evans et al., 1976a). Secretory material released from both systems is ideally suited to modulate the activity of the heart and also to be effectively dispersed around the whole body of the animal. 7.3

PHARMACOLOGY O F R E S P O N S E S TO BIOGENIC A M I N E S

A considerable literature exists on the effects of the application of biogenic amines to insect heart preparations and it has been extensively reviewed (Pitman, 1971; Jones, 1974, 1977). However, the results obtained in the majority of these studies are very difficult to interpret in terms of in vivo roles for biogenic amines as neurotransmitters, or neurohormones, in the control of the myogenic activity of insect heart. First, the effects observed generally vary with the concentration of the compound used and also the species being studied. Second, the complexity of the innervation patterns of the heart in many insect species, as outlined above, makes it difficult to determine the site of action of the applied amines. Third, many studies have applied biogenic amines, such as adrenaline, that are not thought to be present in the insect nervous system (see Jones, 1974). Fourth, in many cases the pharmacology of the responses has not been adequately studied, so that the nature of the receptors mediating the responses are unknown. Fifth, it is now becoming obvious that there are large differences in the pharmacological responsiveness of intact and isolated heart preparations e.g. in the blowfly, Calliphora (Normann, 1972) and in the locust, Locusta (Roussel, 1974) (see also Mordue and Goldsworthy, 1969). Sixth, the pharmacological responsiveness of the insect heart can apparently alter with the age of the insect. The heart beat of the cricket, Acheta domesticus, is accelerated by threshold concentrations of acetylcholine, 5-HT and dopamine in young adult animals, but in older adults (6-7 weeks) the same

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concentrations all produce inhibition (McFarlane, 1967). In a few instances, however, attempts have been made to determine the sites of action of the applied amines and some of these studies will be considered below. Studies on the effects of biogenic amines on the activity of the intrinsic neurones of the cockroach lateral cardiac nerve cord have lead to conflicting results. In Periplaneta americana, Miller (1968b) reported that dopamine stimulated the spontaneous activity of the intrinsic ganglion cells, but did not affect that of the neurosecretory cells. In contrast, in similar studies on another cockroach, Blaberus craniifer, Richter and Gersch (1974) reported that dopamine had no effect on ganglion cell activity, but rather that it selectively increased the activity of the neurosecretory cells in a dosedependent manner (between and 1 0 - 3 ~ and ) that the response was blocked by dihydroergotamine, also in a dose-dependent manner. Both the above studies are in agreement with the observation that aceylcholine activated the ganglion cells, but Richter and Gersch (1974) found that it also activated the neurosecretory cells, again in a dose-dependent manner (between and ~O-’M). Miller (1968b) reported no effect of acetylcholine on these cells in P . americana. Richter and Gersch (1974) also reported that the neurosecretory cells were activated by neurohormone D but not by adrenaline or noradrenaline. At the present time, it is difficult to explain the differences between these two studies. Both used extra-cellular recording techniques, where the two classes of cells were identified on the basis of spike height alone, so that a possibility of an error in the classification of the units recorded exists in both cases. Also Miller (1968b) used only a single concentration of dopamine ( 1 0 - 6 ~ )which , was applied to the preparation in a 50 p1 drop, and thus became substantially diluted. Richter and Gersch (1974) on the other hand, employed a much wider range of dopamine concentrations. It is also possible that the discrepancies between the two studies could be explained on the basis of species differences. The above observations that acetylcholine stimulates the activity of the intrinsic neurones of the lateral cardiac nerve cord is consistent with the later findings of Collins and Miller (1977) on the “semi-isolated heart preparation” of P . americana. This preparation consists of the heart and associated lateral cardiac nerve cords isolated from the ventral nerve cord and its segmental input to the heart. In the latter study, the stimulatory effects of acetylcholine, on the rate of the heart beat, were found to be abolished by the removal of the lateral cardiac nerve cords containing their two types of intrinsic neurones. This evidence is compatible with the idea that the ganglion cells of the lateral cardiac nerve cord receive a cholinergic sensory input from sense organs activated by the stretch of the heart tissue, and that they are thus involved in some form of feedback loop helping to ensure the coordinated contractions of the various heart chambers.

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Collins and Miller (1977) also used the same semi-isolated heart preparation of P. arnericunu in a pharmacological study of the actions of exogenously applied biogenic amines. They found that 5-HT increased the rate of heart beat and decreased its amplitude, but in contrast to their findings for acetylcholine, the effects of 5-HT were not lost on sectioning of the heart from the lateral cardiac nerve cords. This suggested that the actions of 5-HT were directly on the myocardium itself. This preparation showed a decreasing responsiveness to biogenic amines in the order 5-HT > synephrine > octopamine > tryptamine > dopamine > tyramine. The results of applying a competitive antagonist (501c, Wellcome) suggested that 5-HT and octopamine were acting at different sites on the myocardium. The results of Collins and Miller (1977) tell us very little about the in vivo role of biogenic amines in the control of the insect heart. It was not established if the applied amines were acting at synaptic or extrasynaptic sites, o r if they were acting at receptors specific for the biogenic amines. It is to be noted that biogenic amines such as 5-HT can activate receptors whose natural activators are thought to be peptide hormones e.g. in the Malpighian tubules of insects (Maddrell et al., 1971). The use of the “drop-on” assay technique by Collins and Miller (1977) also makes it impossible to determine the actual concentrations of the drugs being applied to the preparation. In addition it needs to be emphasized that differences in the potencies of various cardioaccelerators have been observed between experiments using semi-isolated and intact heart preparations, with the latter system generally being less responsive than the former (Mordue and Goldsworthy, 1969; Normann, 1972; Roussel, 1974; see also Miller, 1975a, b). In summary, it can be seen from the above discussion that biogenic amines are present in the lateral cardiac nerve cord and that the activity of some of the intrinsic neurones may be sensitive to dopamine. At the present time, we d o not know which of the cellular elements present in the cardiac nerve cords contains the amines, and where and under what circumstances they are released. The relationship between the modulating effects of biogenic amines and other cardioaccelerating agents, such as peptides, from corpora cardiaca and other sources (see Goldsworthy and Mordue, 1974; Miller, 1975a), in the control of the insect heart is also unknown. It would again seem advantageous to investigate this problem with an integrated, biochemical, pharmacological and physiological approach using physiologically identified neuronal elements. 8 Biogenic amines in the control of gut muscle

The involvement of biogenic amines as neurotransmitters or local neurohormones (or neuromodulators) in the control of t h e activity of insect gut

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42 1

muscle has provided a basis for much speculation (see Pitman, 1971; Miller, 1975a, b). However, very little information is available on the degree of its direct aminergic innervation. Most of the published data deals with the effects of exogenously applied amines on the spontaneous contractile activity of gut muscle. As with the studies on insect heart muscle discussed above, there are many problems in interpreting the data from such studies. In many cases it is not possible to determine if the amines are acting directly on the gut muscle, or perhaps indirectly, by interactions with its neuronal input. Also a lack of detailed pharmacological studies makes it difficult to determine if the applied amines are acting on specific aminergic receptors or merely acting non-specifically at other receptors, perhaps on those for peptide hormones. The bulk of the evidence obtained to date comes from studies on cockroaches and locusts, and it is not clear to what extent the results obtained on these species are of general applicability to other insect species. In this section, the evidence for a direct aminergic innervation to insect gut muscle will first be examined, followed by a consideration of the pharmacology of the responses of gut muscle to applied biogenic arnines. 8.1

INNERVATION OF GUT MUSCLE

The neuronal input to insect gut muscle appears to provide a coordinating influence which regulates the force and duration of the contractions of the various parts of the gut (Miller, 1975b). It also operates the muscular valves that separate the various regions and serves to override the intrinsic rhythmic myogenic contractions of the gut muscle. For instance, removal of the frontal ganglion of the locust restricts the passage of food along the oesophagus (Clarke and Anstee, 1971). The degree of spontaneous activity exhibited by denervated preparations varies from species to species (see Miller, 1975b), being rather low in the locust Schistocerca, on removal of the ventricular ganglion, (Clarke and Grenville, 1960) but much more pronounced in Diptera (Jones, 1960; Knight, 1962). Electrical stimulation of the nerves t o the gut indicates the presence of a polyneuronal excitatory innervation, which generates excitatory junctional potentials (EJPs). These facilitate at low frequencies of stimulation (Nagai and Brown, 1969; Brown and Nagai, 1969; Nagai, 1973). In cockroach proctodeal muscle fibres, EJPs above a certain threshold trigger the production of all or none action potentials, not graded electrical responses as in somatic muscle fibres (see Usherwood, 1974). The strength of the contractions produced by neural stimulation is proportional to the frequency of stimulation in the cockroach hindgut and reaches a maximum at 50 Hz (Brown, 1975). The insect foregut (the oesophagus and pharynx) is innervated from the

PETER

422

D. E V A N S

stomatogastric nervous system, which includes the frontal, hypocerebral and ingluvial or ventricular ganglia (Cook et a f . , 1969; Mohl, 1972; Klemm, 1972). Recently in the locust, Aubele and Klemm (1977) have described an additional direct innervation of foregut muscles by tritocerebral neurones via a pathway which passes through the frontal ganglion. The association of biogenic amines with neurones in the stomatogastric ganglia has been well documented by histofluorescent techniques (Chanussot et al., 1969; Chanussot, 1972; Klemm, 1972,1976; Lafon-Cazal and Arluison, 1976). In contrast, there is little evidence for the aminergic innervation of the gut muscles themselves. An exception is the work of Klemm (1972) which provides histofluorescent evidence that dopamine-containing nerve fibres are localized in the muscle layer of the posterior pharyngeal part of the oesophagus in the locust. In this study, no fluorescent cell bodies could be detected in the foregut and the fluorescent fibres were observed to originate from the ganglia (occipital and ventricular) of the stomatogastric nervous system. The hind gut of cockroaches is innervated bilaterally by the paired proctodeal nerves from the terminal abdominal ganglion (Cook and Holman, 1975; Miller, 1975b), but may also be innervated from peripheral nerve cells lying on the surface of the gut (Brown, 1975). No information appears to be available on whether the hindgut receives any direct aminergic innervation. The terminal abdominal ganglion has been shown to contain octopamine, dopamine and noradrenaline by radioenzymatic assays (Evans, 1978a; Dymond and Evans, 1979) and 5-HT using a fluorimetric assay (Kusch, 1975) but it is not known as yet which neurones contain these amines. Electron microscopical studies on rectal muscle fibres in the cockroach, Periplaneta americana, have demonstrated the presence of a presumed neurosecretomotor innervation, with nerve endings containing electrondense granules (100-200 nm in diameter) (Nagai and Brown, 1969; Nagai, 1973). Similar neurosecretory granules can be found in endings of the proctodeal nerve of another cockroach, Leucophaea maderae (Holman and Cook, 1972). This type of innervation has been suggested to be peptidergic (Brown, 1975) but as pointed out by Miller (1975a) it also falls into the B type aminergic category of Knowles (1967). Thus further experimentation is needed to identify the chemical nature of the active principle released from these neurosecretory endings. 8.2

PHARMACOLOGICALSTUDIES O N GUT MUSCLE

The responsiveness of insect gut muscle to biogenic amines appears to vary from species to species, and also from foregut to hindgut in the same species.

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Most studies have concentrated on their neuromodulatory or neurohormonal roles and have examined the ability of exogenously applied amines to initiate or to modulate the frequency and amplitude of the myogenic contractions of gut muscle. A few studies have attempted to compare the effects of neural stimulation with the effects of biogenic amine application. A summary of the effects of biogenic amines on the myogenic activity of gut muscle is given in Table 5 (see also Davey, 1964). 5-HT appears to have a general excitatory effect on all the preparations examined. In P. americana, the foregut was more sensitive than the hindgut (Brown, 1965) but in Locusra migratoria the converse was true (Freeman, 1966). Dopamine, noradrenaline and tyramine all appear to have inhibitory effects on myogenic contractions in the gut muscles of L. maderue (Holman and Cook, 1970; Cook and Holman, 1978). In contrast, on L. rnigratoria they are excitatory, except for the action of dopamine on the foregut, which was inhibitory (Freeman, 1966). As mentioned above, the effects of the application of biogenic amines on intact (innervated) gut preparations could be mediated indirectly via effects on the neuronal input. However, Brown (1965) showed that the effects of 5-HT on the foregut of P . americana were not affected by the removal of the ingluvial ganglion. Cooketal. (1969) also showed that the stimulatory effects of 5-HT, noradrenaline and acetylcholine on the foregut of Blaberus giganteus were independent of whether the preparation was denervated or left undissected. Octopamine and its N-methylated analogue, synephrine, which is a very potent agonist of many octopamine receptors in insects, do not yet appear to have been tested. They would be expected to have antagonistic actions to that of 5-HT in view of their potent effects on the myogenic rhythm of a bundle of muscle fibres in the locust hindleg (Evans and O’Shea, 1978). The identity of the excitatory neurotransmitter released upon electrical stimulation of the nerves to insect gut muscle has been the subject of much debate. It seems unlikely to be 5-HT, despite its pronounced excitatory effects described above, since bromolysergic acid diethylamide (bromoLSD) blocks the action of 5-HT on P. americana hindgut, but has no effect on contractions evoked by electrical stimulation of the proctodeal nerves (Brown, 1975). The two principal candidates, so far, are glutamate and the pentapeptide, proctolin (Arg-Tyr-Leu-Pro-Thr). The work of Brown and Starratt on the hindgut of P. americana (Brown, 1975, 1977; Brown and Starratt, 1975; Starratt and Brown, 1975) has provided evidence for proctolin as the excitatory transmitter. The application of proctolin to the hind gut mimics the effects of neural stimulation, both actions being antagonized by the presence of tyramine. Cook and Holman (1978), however, working on the gut muscle of another cockroach L. maderue, point out that tyramine suppresses the spontaneous contractions of both the foregut and the hindgut

P

TABLE 5 Effects of biogenic amines and other compounds on myogenic activity of insect gut muscle DA

5-HT Species

F

H

F

NA

H

F

H

ADR F H

TYR F H

PROC F

H

GLU F H

h)

P

References

Locusta migratoria Freeman (1966) Leucophaea maderae

+

+

-(?)

+

Holman and Cook (1970) Cook et al. (1975) Cook and Holman (1978)

(3x10-* (3x10-" MI

M)

+

Holman and Cook (1979) Kooistra (1950) Brown (1965)

Periplaneta americana

+ NO

+

Blaberus giganteus Galleria mellonella

+

Dytiscus marginalis

+

NO

+

Brown (197.5) Freeman (1966) Cook etal. (1969) Beard (1960) 71

rn -I

Ten Cate (1924)

Abbreviations: 5-HT, 5-hydroxytryptamine; DA, dopamine; NA, noradrenaline; ADR, adrenaline; TYR, tyramine; PROC, proctolin; GLU, glutamate; F, foregut; H, hindgut; +, excitatory; -, inhibitory; +/NO does not initiate contractions but stimulates ongoing myogenic activity. Concentrations listed refer to thresholds for responsiveness; NO, no effect drug in concentration range 0.5-.50fig/ml

rn

30

0

2

D

z v)

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42 5

in this cockroach. They found that tyramine could also block the actions of 5-HT and glutamate on their preparation. This contrasts with the finding of Brown (1975) on the hindgut of P . americana where tyramine was not found to block the actions of glutamate. It thus seems likely from the work of Cook and Holman (1978) that the actions of tyramine on gut muscle are direct actions on perhaps octopamine or dopamine receptors, rather than a true competitive action at proctolin receptors. On the myogenic bundle of the locust hindleg tyramine is an agonist of the octopamine receptors (Evans and O’Shea, 1978) which are distinct from those receptors mediating the actions of proctolin, the two sets of receptors having antagonistic effects (May et al., 1979). Cook and Holman (1978) suggest that some of the above discrepancies may be due to different sensitivity profiles of gut muscle in the different species of cockroach. They also point out that the hindgut contains much more proctolin and is much more sensitive to proctolin than the foregut in L . maderue (Cook and Holman, 1978; Holman and Cook, 1979). They claim that the evidence for proctolin as a true neurotransmitter is not yet convincing and that it could equally well be interpreted to indicate a neuromodulatory role for the peptide (Holman and Cook, 1979), the real neurotransmitter being perhaps the amino acid glutamate (Holman and Cook, 1970; Cook and Holman, 1975). However, in P . americana the glutamate induced contractions of the hindgut never approach the magnitude of the maximum contractions evoked by neural stimulation (Brown, 1975). This again contrasts to the situation found by Cook and Holman (1978) in L. maderue where glutamate and neurally evoked contractions had the same maxima. Further, Brown (1975) found that in P . americana the glutamate contractions were localized to muscles in the region of the rectal valve and appeared to be correlated with the presence of a peripheral nerve cell innervation on the surface of the muscle fibres. Perhaps differences in the degree of such peripheral networks could account for the difference in the glutamate sensitivity of the two species. Thus the identity of the excitatory neurotransmitter of insect gut musculature remains to be determined. It seems unlikely to be a biogenic amine, but the evidence available at present is insufficient to distinguish between the conflicting claims made for glutamate and proctolin. The presence of dopamine containing nerve fibres (Klemm, 1972) and the inhibitory actions of dopamine on locust foregut (Freeman, 1966) suggest that dopamine may act as an inhibitory neurotransmitter or neuromodulator in insect gut muscle. The role, if any, of 5-HT in the control of insect gut muscle is more difficult to evaluate at the present time. The lack of any demonstration of 5-HT containing nerve fibres in gut muscle suggests that it could serve as a circulatory neurohormone affecting gut muscle activity. However, a detailed

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PETER D. EVANS

study of 5-HT receptor pharmacology in insect gut muscle is required, to determine if it is acting via a specific 5-HT receptor, such as is found on the salivary glands of C d i p h o r a (Berridge, 1972) or on a peptide hormone receptor that is also responsive to 5-HT, such as is found on the Malpighian tubules of several insects (Maddrell, et a l . , 1971). The physiological identification of the neurones innervating the gut musculature, in both the stomatogastric nervous system and the terminal abdominal ganglion, would undoubtedly facilitate further studies on the identity of the neurotransmitters of gut muxle and on the role of biogenic amines in the control of its activity.

9 Amines and neurohaemal organs

The association of biogenic amines with neurohaemal organs has been noted in vertebrates and invertebrates, but the functional roles of these amines have yet to be resolved (Scharrer and Scharrer, 1944; Knowles, 1965; Dellmann, 1973; Miller, 1975a; Klemm, 1976; Weiner and Ganong, 1978). Studies on the chemical nature of the neurosecretory products present in insect neurohaemal organs have centred around two approaches, defining the histochemical staining properties of the cells and nerve fibres, and extracting physiologically active materials (Maddrell, 1974). Information obtained from studies on the histochemical staining properties of insect neurosecretory cells is difficult to correlate with the nature of their active physiological principles, since the bulk of the staining presumably reflects the nature of the carrier proteins they contain. Furthermore, it has been suggested that there is a variation in the staining properties of some cells with the age of the insect and its physiological state (for references see Miller, 1975a; Rowell, 1976). Rowell (1976) notes that the above variations, together with the different nomenclature adapted by different authors, has produced a semantic chaos in the classification of insect neurosecretory cells, and Maddrell(l974) questions whether such a classification scheme is possible at all. More recently, attempts have been made to correlate t h e size and appearance of neurosecretory granules found in the neurohaemal organs in electron microscope studies, with the presence of specific physiologically active products, but to date this approach has only met with a limited success. It has thus proved difficult to distinguish the terminals of the neurones containing the biogenic amines, from those containing other neurosecretory products in the neurohaemal organs. Studies on the extraction of physiologically active principles from insect neurohaemal organs have been suggested to provide evidence for the presence of aminergic secretory products. However, most of these studies only

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IN THE INSECT NERVOUS S Y S T E M

427

suggest similarities between the effects of exogenously applied amines and those of the extracts. In many cases, the amines proposed to be present in the extracts have not been positively identified and have not been shown to be released under normal physiological conditions. Thus care is needed in the interpretation o f the results of such studies. In the present section, evidence will be examined for the association of biogenic amines with two insect neurohaemal structures, the corpora cardiaca and the median neurohaemal organs. The function of the amines in these neurohaemal organs will then be discussed. Detailed reviews of neurosecretory systems in insects have been published recently (Goldsworthy and Mosdue, 1974; Maddrell, 1974; Rowell, 1976; see also Berlind, 1977). 9.1

CORPORA CARDIACA

The most intensively studied neurosecretory system present in insects is that of the pars intercerebralis - corpus cardiacum complex. This complex has been suggested to have many parallels with the hypothalamus-pituitary complex of the vertebrates, (Scharrer and Scharrer, 1944; Hanstrom, 1953) both in functional terms (e.g. the presence of diuretic and hyperglycaemic factors) and in the association of biogenic amines (Axelrod, 1974; Weiner and Ganong, 1978). Fluorescence histochemistry has demonstrated the presence of dopamine, and a yellow fluorescence characteristic of an indolalkylamine (as yet unidentified) in the corpora cardiaca of the locust, Schistocerca gregaria (Klemm, 1971; Lafon-Cazal and Arluison, 1976; Klemm and Falck, 1978) and of the cockroach, Blaberus craniifer (Gersch et al., 1974). In the cockroach Periplaneta americana, biochemical studies on extracts of corpora cardiaca indicate the presence of 5-HT (Gerschet al., 1961; Colhoun, 1963; Migliori-Natalizi et al., 1970). More recently, radioenzymatic assays have confirmed the presence of dopamine, and indicated the presence of small amounts of noradrenaline, in the corpora cardiaca of the cockroach (Dymond and Evans, 1979) and the presence of octopamine in the corpora cardiaca of both locusts and cockroaches (Evans, 1 9 7 8 ~ ) . The histofluorescence studies mentioned above indicate that the catecholamines and indolalkylamines are restricted to the storage (neurohaemal, extrinsic) lobe of the corpora cardiaca of locusts, which in this species is morphologically distinct from the glandular (intrinsic) lobe. In general, no formaldehyde-induced fluorescence could be detected in the glandular region of the locust corpora cardiaca except where the continuation of certain fluorescent fibres from the storage lobe extended into the peripheral part of the glandular lobe. Here they were observed to pass

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between the perikarya of the intrinsic endocrine cells (Klemm and Falck, 1978). In addition to the fluorescent granules and fibres in the extrinsic region of the corpora cardiaca, a specific green fluorescence, characteristic of catecholamines, was occasionally observed in cell bodies of probable glial origin (Klemm and Falck, 1978). It has previously been suggested that some glial cells can be stained by histochemical techniques for the demonstration of neurosecretory material in both vertebrate and invertebrate central nervous systems (Koritsanszky, 1967). The significance of this glial localization is not clear at present. Autoradiographical studies at the electron microscope level suggest that 3H-5-HTcan be taken up selectively into axon terminals containing densecored granules of 100 nm i n diameter, in the extrinsic lobe of the locust corpus cardiacum during in vitro incubations in media containing low concentrations of 5-HT (Lafon-Cazal and Arluison, 1976). These terminals were never observed to form any true synapses, suggesting that any 5-HT they contain would be released non-synaptically into the haemolymph as a neurohormone or perhaps locally as a neuromodulator to control the release of other peptide hormones. The use of higher concentrations resulted in a further, but non-specific, uptake into axons without granules, into axons with dense granules from 100-300 nm in diameter, and into axons with clear vesicles 250 nm in diameter. The results of uptake studies with 3Hnoradrenaline are more difficult to interpret as only relatively high concen~ 5 x 1 0 - 4 ~were ) used (Lafon-Cazalet al., 1973) and trations (5 x 1 0 - 5and the specificity of the uptake process was not determined. This is particularly important due to the presence of octopamine in locust corpora cardiaca (Evans, 1978c) and the presence of a high-affinity uptake mechanism for octopamine in insect nervous tissue (Evans, 1978d). It is also difficult to interpret in view of the recent observations that biogenic amine precursors can be taken up into peptide containing cells (see below). The heterogeneity of apparently morphologically similar axon terminals in the locust corpus cardiacum is further emphasized in the above study on 'H-5-HT uptake (Lafon-Cazal and Arluison, 1976) by the fact that axons containing dense granules of' 100 nm in diameter in the glandular lobe were not selectively labelled under any incubation conditions. Lafon-Cazal and Arluison (1976) took this observation to mean that probably the glandular cells are not controlled by a monoaminergic input. The glandular lobes of the corpus cardiacum in the locust Schistocerca gregaria synthesize adipokinetic hormone, which on release causes the eievation of the lipid levels of the haemolymph (Goldsworthy et al., 1972). Physiological evidence for the involvement of biogenic amines in the release of adipokinetic hormone in this locust has been presented by Samaranayaka (1976). This finding emphasises the great care needed in the interpretation of morpho-

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429

logical data in functional terms, without adequate data from physiological controls. The neurosecretory cells from the anterior region of the pars intercerebralis pass to the corpora cardiaca along the nervi-corporis cardiaca I (NCCI) which also carries a smaller bundle of nerve fibres originating in the tritocerebrum (Mason, 1973). The NCCI tract was devoid of aminergic fluorescence in its intercerebral regions except in occasional preparations where it contained small amounts of green and yellow fluorescent material accumulated as droplets in the distal part or as occasional fine greenfluorescent varicose fibres at its edges (Klemm and Falck, 1978). At the periphery of the brain, however, the NCCI become highly fluorescent before entering the extrinsic lobes of the corpora cardiaca. In this region the green catecholamine fluorescence present in fine varicose fibres dominated and tended to mask the yellow. The cells of the pars intercerebralis form part of the median neurosecretory system of insects which will be discussed below (see Section 9.2; see also Section 4). This region of the locust brain contains large numbers of neurosecretory cells that are chrome-haematoxylin positive and aldehyde-fuchsin positive (A cells) together with phloxinophilic cells (B cells) (Highnam, 1961). By a combination of fluorescent histochemistry and classical neurosecretory staining, Klemm and Falck (1978) were able to show, in the locust, that the monoamine containing neurones found in the pars intercerebralis were distinct from the A and B classes of peptide containing neurosecretory cells. They also found non-fluorescent cells that did not contain A or B type neurosecretory material intermingled with the above three types of cells. Klemm and Falck (1978) have suggested that monoamines are not present in the neurosecretory systems of all insects. None have been demonstrated in the neurosecretory system of Trichoptera or in that of the lepidopteran, Spodoptera littoralis. All these studies however were performed using histofluorescent techniques which will not show up the presence of phenolamines, such as octopamine, which is now known to be widely distributed in insect nervous tissue (Evans, 1 9 7 8 ~ ) . 9.2

MEDIAN NEUROHAEMALORGANS

The presence of segmentally arranged swellings on the medial nervous system of insects has been known for some considerable time (Lyonet, 1762). Because of their association with tracheae and spiracles, Newport (1 834) suggested that the system might be some sort of sympathetic nervous system concerned with the control of respiration. Alexandrowicz (1952) reported that the swellings in the transverse branches of the median nerves of the cockroach contained a dense network of fibres but no ganglion cells.

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He suggested that similar structures on the nerves of stomatopods might be “instrumental in the liberation of certain substances into the blood”. The more recent work of Raabe (1966) on stick insects, de BessC (1967) on cockroaches, and Chalaye (1967) on locusts provided evidence for the neurosecretory nature of these structures and suggested they serve to release neurosecretory material from the terminals of “C-cells” (cells which are azan positive but paraldehyde-fuchsin and chrome haematoxylinphloxin negative). Brady and Maddrell (1967) confirmed the neurosecretory nature of these organs in several insect species and noted that ultrastructural evidence suggested that the organs released substances from the medial nervous system into the blood. The association of biogenic amines with the neurohaemal organs of the median nervous system has recently been shown. Octopamine has been demonstrated by radioenzymatic assay to be present in the medial neurohaemal organs of cockroache,s and locusts (Evans, 1978c) whilst dopamine and small amounts of nordrenaline have been demonstrated in the abdominal median neurohaemal organs of the cockroach, Periplaneta americana (Dymond and Evans, 1979). In another cockroach, Gromphadorhina portentosa, the abdominal median neurohaemal organs have been demonstrated to synthesize octopamine (Nelson, Drickamer, Maxwell and Hildebrand, personal communication). Catecholamine histofluorescence has also been reported to be associated with the median and lateral nuclei of the unpaired nerves of the locust metathoracic ganglion (Plotnikova, 1968). However, it is not clear which fibres in the nuclei contain the catecholamines and the location of the cell bodies concerned was not reported. In the cockroach, Smalley (1970) reported the uptake of 3Hdopamine by the abdominal median neurohaemal organs and associated ganglia. The latter author also provided evidence for the innervation of these neurohaemal organs by the median midline cells of the abdominal ganglia. The significance of the labelling of these cells is unclear since the specificity of the uptake system was not examined, and as Smalley admits, it was not determined if the label in the midline median neurosecretory cells was still in dopamine or in one of its metabolites. The presence of biogenic amines in insect median neurohaemal organs seems established. However, the same organs also contain other physiologically active products that are reported to be involved in the regulation of the heart beat, diuresis, blood protein concentrations and the tanning of cuticle (bursicon) (see Raabeet al., 1974). Thus the identity of which nerve endings contain which secretion is not clear. As mentioned previously, Knowles (1967) has suggested the classification of neurosecretory nerve endings into A type (peptidergic - containing dense granules 100-300 nm in diameter) and B type (aminergic - containing dense granules not exceeding

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100 nm in diameter). However as can be seen from Table 3 biogenic amines have been suggested to be associated with a much broader range of densecored granules (from 40-200 nm in diameter) in the insect nervous system. The ultrastructure of the abdominal median neurohaemal organs of the cockroach, locust and stick insect have been examined by Brady and Maddrell (1967). They point out that in the locust, Schistocerca gregaria and in the stick insect, Carausius morosus, the main neurosecretory product consists of small electron transparent vesicles of rather irregular shape, 40-80 nm and 110-250 nm in diameter respectively. In addition, the organs of these species also contain a very small number of endings containing electron-dense granules (60-120 nm in diameter). In the cockroach, Periplaneta americana, the major neurosecretory product of the median neurohaemal organs consists of a heterogenous population of vesicles, some containing electron-dense and some electron-transparent material. The transparent vesicles are larger (80-150 nm in diameter) and more numerous than the smaller opaque granules (55-100 nm in diameter) (Brady and Maddrell, 1967). In the median neurohaemal organs of the stick insect and the locust, Brady and Maddrell(l967) suggested that the electron transparent material was comprised of “C type” neurosecretion (azan positive). A similar conclusion was reached by Chalaye (1967, 1974a, b) for another locust, Locusfa migratoria migratorioides, although in this case the ultrastructure of the neurosecretory material was different. In the abdominal median neurohaemal organs of this species, a single type of ending was found with ovoid dense-cored granules of 200-500 nm in diameter ( C , type neurosecretion). In contrast, the neurohaemal organs of the metathoracic ganglion contained three types of axon terminal, the first, with small densecored spherical granules (<150 nm in diameter) and the second, with large dense cores (<300 nm in diameter). The third and most numerous type of ending contained both clear and dense vesicles (200-250 nm in diameter) ( C , type neurosecretion). At the present time it is not clear which type of neurosecretory ending contains the biogenic amines in the median neurohaemal organs. A direct demonstration of the location of the cell bodies of the neurones giving rise to the median neurohaemal organs can be obtained by backfilling the median nerve at the neurohaemal organ, using cobaltous chloride and subsequently precipitating the cobalt with ammonium sulphide (Pitman et al., 1972). Using this technique Ali and Pipa (1978) were able to demonstrate that the abdominal median neurohaemal organs of the cockroach, P . americana were innervated by four groups of cells, two located anterior dorsally, one posterior dorsally and one posterior ventrally. All four groups of cells appeared to correspond in cell size and number to the groups of neurosecretory cells localized by staining with azocarmine and

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paraldehyde-fuchsin in the same study. The granules of the neurohaemal organ also stained with both azocarmine and paraldehyde-fuchsin in this study. This contrasts with the findings by Brady and Maddrell (1967), de BessC (1967) and Chalaye (1967) where the secretory material of the neurohaemal organs was azocarmine positive but paraldehyde-fuchsin negative. Ali and Pipa (1978) were unable to account for this discrepancy. Cobalt backfilling of the median nerves of the abdominal portion of the fused metathoracic ganglionic mass of the locust, Schisfocerca americana gregaria, from the level of the neurohaemal organs, reveals dorsal midline neurones (- 10 p m in diameter) (see Fig. 30), as well as several groups of

Fig. 30 Dorsal median neurones projecting into medial nerves from “abdominal” portions of fused metathoracic ganglionic mass of the locust, Schisfocerca americana gregaria, as demonstrated by backfilling of medial nerves with cobaltous ions from regions of neurohaemal organs. A, is from a backfill of the second median nerve, and B, from a backfill of the third median nerve. Dorsal cell bodies are indicated by the filled somata and ventral cell bodies by the open ones. Scale bars, 100 pm. (Evans unpublished)

median and lateral neurones on the ventral surface of the ganglion (Evans, unpublished). The dorsal median cells are similar in size and position to some of the presumed aminergic neurones revealed by Neutral red staining of the same preparation (Evans and O’Shea, 1978; see Fig. 13) and also to the azan-positive cells in the same ganglion of Locusta (Chalaye, 1967). In the locust and cockroach, some of the dorsal median neurones have recently been shown to be octopaminergic (Evans and O’Shea, 1977,1978; Dymond and Evans, 1979) (see also Section 4). It is thus possible that at least some of

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the C-type neurosecretory material found in the abdominal neurohaemal organs could result from the presence of the endings of octopaminergic neurones, since octopamine is known to be present in these organs along with dopamine and a small quantity of noradrenaline (Evans, 1978c; Dymond and Evans, 1979). At present it is not possible to positively identify any of the above vesicle types with specific biogenic amines or to give any direct evidence for the functions of the biogenic amines in the neurohaemal organs of the medial nervous system. 9.3

FUNCTION OF AMINES IN NEUROHAEMAL ORGANS

The studies described above establish the presence of biogenic amines in insect neurohaemal organs, but otherwise little is known of the functional roles of these amines and their cellular locations. Studies on the physiological effects of extracts of corpora cardiaca from various insect species have attempted to show parallels with the effects of amines but there have been no coordinated approaches which include the identification of a particular amine in an extract, show its release from the corpora cardiaca and determine its consequent effects. Corpora cardiaca extracts from different species have been claimed to exhibit physiological and pharmacological effects similar to those of several monoamines. Lafon-Cazal and Arluison (1976) list examples of such studies for the following amines: 5-HT (Davey, 1961; Brown, 1965; Freeman, 1966; Cook et af.,1969; Hart and Steele, 1969; Mordue and Goldsworthy, 1969); adrenaline (Barton-Browne et al., 1961); bufotenine (Maddrell et af., 1971); dopamine (Richter and Gersch, 1974) and octopamine (Robertson and Steele, 1972). In several of the above cases, however, it is known that although the amines mimic the effects of the corpus cardiacum extracts, they are not the active principles responsible for the observed physiological effects. The hyperglycaemic activity of the extracts, for instance, is thought to be due to a peptide factor (Brown, 1965; Migliori-Natalizi and Frontali, 1966) not to octopamine, despite the presence of the latter in the corpora on the effects of adrenaline e.g. Bartoncardiaca (Evans, 1 9 7 8 ~ )Studies . Browne et al. (1961), are difficult to interpret as no adrenaline has been detected in the insect nervous system (see Klemm, 1976; and Section 2.1). It is to be hoped that any future studies on physiologically active extracts will also include a detailed pharmacological study of the receptors mediating the responses. The association of biogenic amines with neurosecretory systems in vertebrates and other invertebrates, such as crustacea, is well known. The pericardial organs of the lobster, for instance, contain dopamine, 5-HT (Cooke and Goldstone, 1970) and octopamine (Evanset af., 1976a; Evans

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PETER D. E V A N S

er al., 1976b). Here again, the function of the endogenous amines is

unknown. It is possible that in both the insect and crustacean examples, that the amines serve as releasing factors for peptide hormones, as has been suggested to be the case in several vertebrate neurosecretory systems, including the anterior pituitary (see Weiner and Ganong, 1978). In the latter example, dopamine has been shown to act as a local neurohormone inhibiting the secretion of prolactin, whilst other amines such as adrenaline, noradrenaline and 5-HT are thought to act as neurotransmitters involved in the transfer of neural information to hypothalamic hormone-secreting neurones (Weiner and Canong, 1978). There is little direct evidence for such a suggestion in insects, but the recent work of Samaranayaka (1976) has established the presence of an aminergic link in the release of adipokinetic hormone from the intrinsic cells of the glandular lobes of the locust corpora cardiaca. These cells are innervated by a group of cells in the lateral area of the protocerebrum which send axons along NCCII into the glandular lobe (Rademakers, 1977). The terminals of these neurones contain dense-cored granules (100 nm in diameter) and make discrete synapses with the glandular cells. It is tempting to speculate that they represent the anatomical correlate for the aminergic control of the release of adipokinetic hormone. The presence of amines in the median neurohaemal organs of the locust, Schistocerca americana, could be well associated with the modulation of the myogenic activity of the ventral diaphragm in this species (Guthrie, 1962) as the neurohaemal organs are located immediately below it, and the amines they contain such as octopamine, are known to exert a profound influence on the myogenic activity of other insect muscles (e.g. Evans and O’Shea, 1978). The insect median nervous system has itself been suggested to represent the insect analogue of the vertebrate adrenergic sympathetic nervous system and to be involved in controlling the level of responsiveness of locust flight muscle (Ivanova, 1956; Voskresenskaya, 1959; Voskresenskaya and Svidersky, 1960) and cicada tympana1 muscles (Voskresenskaya and Svidersky, 1961). However, in many of these Russian studies, the effects of the median nervous system were obtained from ablation experiments. At that time it was not realized that the median nerves also carried the motorneurones to the spiracular muscles. Thus, it is not clear to what extent the observed effects were due to interference with the innervation of the respiratory muscles or due to the removal of the hypothesized sympathetic input. In view of the potent effects of adrenaline in increasing the responsiveness of some preparations which had had their median nervous system ablated (Voskresenskaya, 1959), it would seem worthwhile reinvestigating these claims bearing in mind that the likely sympathetic effector of the insect nervous system appears to be octopamine, rather than adrenaline. An alternative explanation for the localization of biogenic amines in

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insect neurohaemal organs might be that they are probably the most efficacious sites for the release of material into the circulatory system as neurohormones. Octopamine, for instance, has been suggested to be the mediator of the excitation-induced hypertrehalosemic (EXIT) response in the cockroach, Periplaneta arnericana (Downer, 1979a). This is an interesting suggestion but no evidence has been presented for the release of octopamine during the EXIT response. Also from the limited pharmacological study of the amines capable of mimicking the EXIT response, the specificity of the receptor involved appears to be different from those of all the octopamine receptors known to date (see Sections 4 and 5 ) , in that synephrine, the N-methylated analogue of octopamine was not a potent agonist of the hypertrehalosemic response. It thus seems possible that octopamine could be acting non-specifically as a receptor for a hyperglycaemic peptide, in a parallel fashion to the way 5-HT acts at other peptide receptors (Maddrell et a!., 1971). Further experimentation with specific antagonists and agonists is required to resolve these possibilities. Downer (1979a) suggested that the time course of the EXIT response made it unlikely to be mediated by the same hyperglycaemic peptide found in the corpus cardiacum, being released at other sites along the nerve cord, but could not rule out the existence of a second rapidly acting hyperglycaemic peptide released from these regions. Downer (1979a) tested a series of biogenic amines for their hyperglycaemic inducing activity by injecting them into the haemocoel of the cockroach. The responses he observed could have been due to the injected amines releasing other active compounds. Thus in a parallel study Downer (1979b) demonstrated that octopamine and dopamine could increase the production of trehalose from glycogen in isolated pieces of fat body. Tyramine on the other hand had no effect on the fat body and Downer (1979b) thus suggested that its effects in mimicking the EXIT response were probably indirect. Octopamine has also been shown to increase the activity of glycogen phosphorylase and to stimulate glycogenolysis in cockroach nerve cord (Robertson and Steele, 1972) and also to increase the rate of glucose oxidation in locust flight muscle (Candy, 1978). The above effects of octopamine are all consistent with its being able to act as the sympathetic circulatory neurohormone, possibly being released from the median neurohaemal organs, and serving to increase the availability of energy to the muscles under stressful conditions. It needs to be emphasized that, as yet, octopamine has not been demonstrated to be released from these organs in response to appropriate stimuli and in sufficient quantities to produce high enough circulating concentrations to bring about the observed physiological effects. The specifity of the receptors mediating these responses also requires further investigation.

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PETER D. E V A N S

An intriguing possibility is that some of the amines and peptide hormones present in insect neurohaemal organs may actually be localized in the same nerve terminals. It has been known for some time that some peripheral endocrine cells in vertebrates, notably in the gastrointestinal tract, appear to store and/or synthesize concomitantly a peptide hormone and a biogenic amine. These cells have been termed “APUD” cells (amine precursor uptake and decarboxylation) (Pearse, 1969). Dopamine and 5-HT seem to be natural constitutents of several endocrine cells which produce polypeptide hormones in the pineal gland (Owman, et af., 1973). Also the parafollicular cells of the thyroid, which store calcitonin, can take up 5-HT or catecholamines into the same granules as contain the hormone when supplied with appropriate precursors (Ericson, 1972). It has also been recently shown by immunofluorescent techniques that certain peripheral sympathetic noradrenergic neurones in the guinea pig and rat contain both the enzyme dopamine P-hydroxylase and somatostatin or a somatostatin-like peptide (Hokfelt et af., 1977). Somatostatin is a peptide known to inhibit the release of growth hormone from the anterior pituitary (see Weiner and Ganong, 1978). It has been suggested to act as a neurotransmitter or neuromodulator in other parts of the vertebrate CNS, but its function in the peripheral nervous system is unknown. Immunohistochemical evidence has also recently been provided for the presence of Substance P-like immunoreactivity in the somata of some 5-HT containing neurones in the rat central nervous system (Hokfelt et af ., 1978; Chan-Palay et af., 1978). It should be noted that the above neuronal immunolocalizations of somatostatin and Substance P were confined to neuronal somata and as yet no amine containing nerve terminals have been demonstrated to contain these peptides or to release them simultaneously with their endogenous amines. Thus Dale’s principle of one neurone releasing a single transmitter is not violated by the above observations. The physiological significance of biogenic amines in insect neurohaemal organs, and their possible association with peptide-containing neurones, await further experimentation. This is an exciting area for research into the functional roles of biogenic amines in insects and should add to our understanding of the relationship of amines and peptides in similar neurohaemal organs in other invertebrates and also in those of vertebrates.

10 Amine-stimulated adenylate cyclase activity

It is becoming increasingly apparent that many neurotransmitters mediate their actions on post-synaptic cells through the stimulation of the enzyme adenylate cyclase (E.C 4.6.1.1.) which results in an increase in the level of

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cyclic AMP in the cell (see reviews by Nathanson, 1976, 1977; Greengard, 1976; Kebabian, 1977). Beam and Greengard (1976) point out, however, that many criteria need to be fulfilled before it can be concluded that a post-synaptic response to a particular neurotransmitter is mediated directly by a change in the intracellular concentration of a cyclic nucleotide. It needs to be demonstrated that the neurotransmitter is capable of eliciting a change in the cyclic AMP levels and that this change occurs specifically in the post-synaptic cells. The change in cyclic AMP levels should also be elicited by synaptic stimulation. Also the post-synaptic potential change or cell response initiated by the transmitter should be mimicked by the application of exogenous cyclic AMP or one of its related derivatives. Finally, drugs which modify the post-synaptic membrane response should also alter the magnitude of the cyclic nucleotide response. All the above criteria have only been met in a very few cases, such as in the mammalian sympathetic ganglion (Greengard, 1976) and in the Purkinje cells of the vertebrate cerebellum (Bloom et al., 1975). In most cases, the only information available is that biogenic amines are able to change the levels of cyclic AMP in a tissue. Thus, for the vertebrate central nervous system, Iversen (1977) points out that the hypothesis that cyclic nucleotides mediate post-synaptic responses to catecholamines depends largely on argument by analogy with the role played by cyclic AMP in peripheral systems (Robison et al., 1971) and on circumstantial evidence. The same is true for the bulk of the information we have on the role of amine-stimulated adenylate cyclases in the insect nervous system. Here amine-stimulated adenylate cyclases have been shown to be present in whole ganglia, or in tissue homogenates, from several insect species. However, until this information can be correlated with physiological information from specific neuronal pathways, the other criteria outlined by Beam and Greengard (1976) cannot be approached, and the involvement of the amine-stimulated cyclases in neuronal transmission must remain hypothetical. In this section, studies on amine-sensitive adenylate cyclases in insect nervous tissue will be reviewed and their findings compared to those on similar preparations from vertebrates and other invertebrates. The cellular locations of the enzyme will be considered and its function in insect nervous tissue discussed. 10.1

S T U D I E S O N INSECT PREPARATIONS

10.1.1 Neuronal ganglia Studies on amine-stimulated adenylate cyclase activity in the ganglia of the insect nervous system have used either intact or homogenized preparations.

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In intact cockroach ganglia, the stimulation of adenylate cyclase activity caused by octopamine is equal to or greater than that produced by the catecholamines dopamine and noradrenaline (Nathanson, 1976). In intact ganglia, however, the exogenously applied amines could initially release other factors which then activate the adenylate cyclase, or the amines could in some way be interfering with the metabolism of ATP. Additionally, since the steady state levels of cyclic AMP are the net result of synthetic and degradative processes, we need to know if the effects are actually due to a stimulation of adenylate cyclase activity, or to an inhibition of phosphodiesterase activity (Nathanson, 1976). To overcome the above problems several authors have studied the activation of adenylate cyclase by biogenic amines in homogenates or washed particle preparations from insect neural tissue. Using such preparations the presence of both octopamine- and dopamine-sensitive adenylate cyclases have been described in cockroach (Periplaneta americana) thoracic ganglia (Nathanson and Greengard, 1973), in cockroach brain (Harmer and Horn, 1977) and in the brain of the bertha armyworm (moth) Mamestra configurata (Bodnaryk, 1979a, b). A specific 5-HT-sensitive adenylate cyclase has also been shown in cockroach thoracic ganglia (Nathanson and Greengard, 1973,1974) and in nerve cord preparations from the moth Manduca sexta (Taylor and Newburgh, 1978) but not in the “brain” preparations mentioned above. The octopamine- and dopamine-sensitive enzymes from the brain preparations more closely resemble each other with respect to the magnitude of the increases above basal activity caused by dopamine and octopamine than those of the corresponding enzymes from the thoracic ganglia (Bodnaryk, 1979a). There are however many similarities between the “brain” enzymes and the “thoracic ganglion” enzymes and the following description of their properties, unless otherwise specified, applies to all of the preparations examined. The octopamine-sensitive enzyme was stimulated by low concentrations of octopamine e.g. the thoracic enzyme of the cockroach responded to concentrations as low as 0.03 p~ and had a half maximal activation (K,) of about 1.5 p ~Its. maximal stimulation occurred at about 30 p ~ Relatively . high concentrations of octopamine had no effect on endogenous phosphodiesterase activity, indicating that the observed increases in cyclic AMP were due to a stimulation of adenylate cyclase activity rather than to an inhibition of phosphodiesterase activity. Noradrenaline was found to produce the same maximal effects as octopamine but at a much higher concentration. Dopamine produced a lower maximal stimulation of adenylate cyclase activity than did octopamine in all the above cases, but it had an equally low K,, e.g. 2 p ~in, the cockroach thoracic ganglion preparation (Fig. 3 1) (Nathanson and Greengard, 1973). The 5-HT-sensitive adenylate cyclase activity of the thoracic ganglion prepara-

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tion was again specifically activated by low concentrations of 5-HT ( K , 0.5 PM) in a dose-related manner, but the magnitude of its maximal stimulation was small compared to that of octopamine (Fig. 31). This contrasts with the situation in lobster skeletal muscle where the activation by 5-HT was much greater than that due to octopamine (Kravitz et al., 1976; Battelle and Kravitz, 1978).

1

A. Hornogenate

400

350

,)oo

t

1

6 .intact Tissue

I

1100

900

t 500 150

1

J

.x

300

t

100

0 001

0.t 1 10 100 Biagen$c Amins Cacenlralion ( p M 1

1000

5000

COnllDl

Thsophylline 1OmM

OCtOD8mlne

OcIODarnlne

250pM TheODhyllma

Fig. 31 Effect of octopamine on cyclic AMP accumulation in (A) homogenates and (B) intact tissue preparations of cockroach (PerQdunetu urnericuna) thoracic ganglia. (A) Effect of various concentrations of DL-octopamine, dopamine, and serotonin (5-HT) on adenylate cyclase activity in homogenates. The control activity, per milligram of protein, in the absence of added biogenic amine was 10.02 1.5 pmolesimin. (B) Effect of 250 ~ C L MDL-octopamine and lOmM theophylline, alone and in combination, on the accumulation of cyclic AMP in "intact hemiganglia". The control was 17.7 -r- 2.7 pmole per milligram of protein. The values shown in both (A) and (B) are the means and ranges for two to three replicate samples, each assayed in duplicate. (Re-drawn from Nathanson and Greengard, 1973)

A detailed structure-activity relationship for the stimulation of the octopamine-sensitive adenylate cyclase has been worked out in the cockroach brain preparation (Harmer and Horn, 1977). This study indicated that ( a ) the response was stereospecific for the naturally-occurring D( -) isomer of octopamine which was over 200 times as potent as the L( +) isomer, ( b ) the presence of a p-hydroxyl group was essential for potent activity, (c) the absence of a p-hydroxyl group reduced activity, ( d ) the presence of a rn-hydroxyl group drastically reduced potency, ( e ) the addition of a methoxy group to the rneta position also decreased potency; however, rn-methoxyphenyl-ethylamineswere more potent than the corresponding rn-hydroxyl derivatives, 01 a-methylation of octopamine resulted in about a

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sixty-fold decrease in potency, (9) N-methylation slightly reduced potency and substitution of the still larger isopropyl group reduced potency substantially. Thus, of the biogenic amines known to be present in the insect nervous system octopamine was the most effective agonist at activating this particular adenylate cyclase. Additivity experiments have been used to determine the number of different types of specific amine receptor present. Such experiments reveal that the activities of dopamine and octopamine (and also of 5-HT in the thoracic preparation) are totally additive, indicating the existence of specific receptors for these amines. In contrast, the activities of tyramine, noradrenaline, adrenaline, phenylethylamine and phenylethanolamine were not totally additive to those of octopamine and dopamine. This suggests the absence of separate receptor sites for these amines listed above. Thus the pronounced effects on adenylate cyclase activity observed by the addition of noradrenaline to the insect preparations is probably due to a partial activation of the receptor sites for octopamine, dopamine and possibly 5-HT (Nathanson, 1976). It also seems likely that the activation of adenylate cyclase by adrenaline and noradrenaline in the nerve cord of the Madagascar cockroach (Gromphadorhina portentosa) (Rojakovick and March, 1972) is produced by the partial activation of octopamine and dopamine receptors, and does not necessarily indicate a physiological role for adrenaline and noradrenaline in this system. This idea is further supported by evidence for only low levels of endogenous noradrenaline and the complete absence of adrenaline in insect nervous tissue (see Section 2.2). Studies with specific agonists and antagonists also provide evidence for the presence of specific octopamine, dopamine and 5-HT receptors activating adenylate cyclase in the appropriate sections of the insect nervous system. Table 6 summarizes the available data on the antagonists which inhibit the stimulation of adenylate cyclase activity by various biogenic amines. It can be seen that phentolamine preferentially blocks the effects of octopamine and noradrenaline, but nonetheless has a significant blocking action on the effects of dopamine and 5-HT. The dopamine-blocking agent haloperidol is most effective against dopamine stimulation, but also has a significant effect against 5-HT stimulation. In the cockroach thoracic ganglion preparation, it can be seen that LSD, BOL, and to a lesser extent cyproheptadine, are highly specific blocking agents for 5-HT stimulation. Interestingly, Harmer and Horn (1977) found that cyproheptadine was a very potent blocking agent of both dopamine and octopamine stimulation in the cockroach brain preparation where they could detect no 5-HT stimulation of adenylate cyclase. It would be of much interest to know the effects of LSD and BOL on the octopamine and dopamine stimulation of the cockroach brain preparation and to see if their use could unmask any hidden 5-HT

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sensitive adenylate cyclase activity. The antipsychotic drugs a-and p-flupenthixol were more effective at blocking the effects of dopamine than those of octopamine. In no case was it possible to differentiate between the effect of octopamine and noradrenaline by the use of blocking agents, again suggesting that there is no specific noradrenaline receptor. The lack of effect of propranolol on noradrenaline-stimulated activity supports the idea that a p-adrenergic receptor is absent in this system. Propranolol did however, produce a moderate blocking action on the stimulation due to 5-HT. TABLE 6 Inhibition by antagonists of the stimulation of insect ganglion adenylate cyclase activity by various biogenic amines Antagonist

OCT

Phentolamine ( p ~ )

0.5 0.4

Approximate K , NA DA

Propranolol ( p ~ ) >loo Haloperidol ( p ~ ) 10 BOL ( n M ) >10OOQ LSD (nM) >10000 Cyproheptadine ( p ~ ) 1.o 0.07 a-Flupenthixol ( p ~ ) 3.2 0-Flupenthixol ( p ~ ) 11.0

0.6 0.5 >loo 10 >10000 >10000 1 .o

2 5

>loo 0.2 100

>10000 1.o 0.06 0.02 0.35

5-HT

Ref.

3 6 5 0.5 5 5 0.25

Abbreviations: OCT, octopamine; N A , noradrenaline; D A , dopamine; 5-HT, 5-hydroxytryptamine; BOL, 2-bromo-d-lysergicacid diethylamide; LSD, d-lysergic acid diethylamide a Nathanson and Greengard (1974) ' Nathanson (1976) ' Harmer and Horn (1977)

Harmer and Horn (1977) note that there was very little structural similarity between the most effective antagonists of octopamine stimulation of adenylate cyclase observed in cockroach brain homogenates. The most potent drugs were cyproheptadine (a potent antagonist of histamine and 5-HT), phentolamine (an imidazoline with potent a-adrenoceptor blocking activity) and promethazine (a phenothiazine with histamine-blocking activity). Nathanson (1976) points out that in the cockroach thoracic ganglion preparation that curiously (in view of the potent action of the a-adrenergic antagonist phentolamine on octopamine stimulation) the a-adrenergic agonist, phenylephrine, is only a relatively weak activator of adenylate cyclase. He suggests that the octopamine receptor might have characteristics different from that of a classical a-adrenergic receptor. In view of the apparent diversity of a-adrenoreceptor types in vertebrates, the latter

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suggestion needs to be examined by making a much more detailed pharmacological investigation (Starke, 1977). 10.1.2 Firefly light organ A recent study on the amine activation of adenylate cyclase activity in the light organs of the firefly, Photuris (Nathanson, 1979; Nathanson and Hunnicutt, 1979), reveals that broken cell preparations from this tissue contain an enzyme specifically activated by octopamine. This enzyme has many similarities to the corresponding enzyme in cockroach ganglion homogenates discussed above. In the firefly light organ homogenates, however, other biogenic amines such as noradrenaline, tyramine and dopamine were less effective than octopamine and 5-HT had no effect at all on enzyme activity. Additivity experiments on this preparation indicated the presence of a single amine receptor that was preferentially activated by octopamine. This octopamine receptor was similar to those activating adenylate cyclase in cockroach ganglia and to those mediating the actions of octopamine in locust skeletal muscle (Evans and O’Shea, 1978; O’Shea and Evans, 1979). All were preferentially blocked by the a-adrenergic blocking agent phentolamine and were activated by synephrine. The degree of octopamine stimulation of adenylate cyclase activity in the light organ preparation was ten fold greater than that found in any other broken cell preparation of excitable tissue. The functional role of octopamine in the firefly light organ has been discussed in a previous section (see Section 5 ) . 10.2

COMPARISONS WITH OTHER INVERTEBRATE A N D VERTEBRATE

PREPARATIONS

The properties of the dopamine-sensitive adenylate cyclase in homogenates of cockroach brain are similar to those observed in rat corpus striatum homogenates (Miller et al., 1974). The dopamine agonists epinine and ADTN were comparable in potency to dopamine as stimulants of insect adenylate cyclase, and evoked similar maximal responses. At higher concentrations, LSD acted as an agonist of adenylate cyclase in the cockroach thoracic ganglion preparation, presumably via the 5-HT receptor. This is similar to its known concentration-dependent actions in physiological studies, and suggests that cockroach ganglia may provide a model system for some of the effects of LSD on humans (Nathanson and Greengard, 1974). The pharmacological properties of the 5-HT-sensitive adenylate cyclase are similar to those of 5-HT receptors in rat uterus (Cerletti and Doepfner, 1958) and A-receptors in molluscan ganglia (Gerschenfeld, 1971).

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In the vertebrate nervous systems, so far tested, there is little evidence for the presence of a specific octopamine-activated adenylate cyclase. Harmer and Horn (1977) found no significant stimulation of adenylate cyclase by 0.3 mM DL-octopamine in homogenates of different regions of the rat brain (including the hypothalamus, striatum, pons-medulla, cerebellum, cortex or cervical spinal cord). Octopamine caused almost no stimulation in the rat caudate nucleus, a tissue with a known dopamine receptor, or in the rabbit heart, which contains a p-adrenergic receptor (Nathanson, 1976). Nathanson (1976) reports that in tissues where a small degree of stimulation by octopamine is found (bovine pineal gland and rabbit liver) that this amine is much less potent than either adrenaline or noradrenaline. In the rat salivary gland, Nathanson (1976) reports that octopamine causes a small stimulation of adenylate cyclase at relatively low concentrations. This appears to be independent of that due to noradrenaline and may reside in a part of the salivary gland anatomically distinct from that containing a noradrenalinesensitive adenylate cyclase. The octopamine-sensitive adenylate cyclase of insect neural tissue has many similarities with those found in other invertebrate tissues. In Aplysia, for instance, in an intact cell preparation from a neuronal ganglion, octopamine increases cyclic AMP content which is reported to lead to a delayed phosphorylation of a specific particulate protein, probably found in the neuropile (Levitan and Barondes, 1974; Levitan et al., 1974). The phosphorylation induced by octopamine is again blocked by phentolamine, which also blocks the increase in cyclic AMP due to octopamine, and is mimicked by the direct application of dibutyryl cyclic AMP. The octopamine-sensitive adenylate cyclase found in several tissues of the lobster, including skeletal and heart muscle, and blood cells (Battelle and Kravitz, 1978), is also similar to that of insect nervous tissue in many ways. In both cases, the structure specificity of the octopamine receptors is similar, and the lobster receptors are also blocked by a-adrenergic antagonists, such as phentolamine and dibenamine (Battelle and Kravitz, 1978). In the vertebrate nervous system adrenergic receptors can be divided into two broad categories (p-adrenergic and a-adrenergic) on the basis of their interactions with agonists and antagonists. Further, p-adrenergic receptors are thought to mediate their actions through the activation of an adenylate cyclase, whilst a-adrenergic receptors mediate their actions by gating calcium. In invertebrates, the drugs that act as agonists and antagonists at vertebrate a-adrenergic receptors also appear to act at octopamine receptors. Paradoxically the invertebrate octopamine receptors seem in many cases to mediate their actions via the stimulation of an adenylate cyclase. Thus it may be that invertebrate octopamine receptors are only similar to vertebrate a-adrenergic receptors in the specificity of some elements of their

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binding sites and the two classes of receptors may not act in the same way. This suggests that in future studies on octopamine receptors it needs to be ensured that any effects observed on adenylate cyclase activity are direct and not secondary to changes in calcium levels. 10.3

CELLULAR LOCATION OF RESPONSES

The studies mentioned above on intact tissues, and also those on tissue homogenates, however, do not reveal the cellular location of the aminesensitive adenylate cyclases. This is a particularly difficult problem in such a heterogeneous tissue as the nervous system, which contains many different neuronal elements and glial cells. There is a certain amount of evidence that a glial fraction from the central nervous system of the moth, Manduca sexta, contains an adenylate cyclase responsive to 5-HT and to a lesser extent noradrenaline (Taylor et a1 ., 1976). Primary cultures of vertebrate glial cells also exhibit a @-adrenoceptorcoupled adenylate cyclase activity (Schimmer, 1971; Schubert et al., 1976). The functional role of the amine-stimulated adenylate cyclase of glial cells remains unknown in any preparation, but its involvement in regulating cerebral glycogenolysis has been suggested (Nahorski et al., 1975). It has also been suggested that a dopamine binding site on glial cells in the caudate nucleus of the bovine brain may be associated with an adenylate cyclase, and that this may be a site of action for certain neuroleptic drugs (Henn et al., 1977). It is thus very obvious that the contribution of glial cells to the amine-sensitive adenylate cyclase activity found in insect nervous tissue deserves much closer attention. 10.4

FUNCTIONAL ROLE I N INSECTS

At present we know little o f the physiological significance of amine-sensitive adenylate cyclases in insect nervous tissue. Robertson and Steele (1972) report that octopamine causes a stimulation of phosphorylase activity in cockroach nerve cords via an increase in cyclic AMP levels. In the absence of appropriate additivity experiments, however, it is not possible to say if octopamine is producing this effect through a specific receptor or is acting at a receptor site for a hyperglycaemic peptidergic hormone, perhaps from the corpus cardiacum. As described earlier there are now several examples of arnines being potent agonists of receptors for peptide hormones in insects. The best evidence for a physiological involvement of the adenylate cyclase system in insects is circumstantial. In the firefly light organ, there are many similarities between the properties of the octopamine receptors mediating the activation of adenylate cyclase activity (Nathanson, 1979) and the receptors responsible for light production (Carlson, 1968a, b, 1969; Oertel

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and Case, 1976). Also, in the extensor tibiae muscle of the locust hindleg, the octopamine receptors responsible for the slowing of the myogenic rhythm of contractility (Evans and O’Shea, 1978) and those mediating the potentiation of neuromuscular transmission (O’Shea and Evans, 1979) have properties similar to those capable of stimulating adenylate cyclase in several tissues (Harmer and Horn, 1977; Battelle and Kravitz, 1978). In addition, it has recently been shown that cyclic AMP may facilitate transmitter release at a neuromuscular junction in the cockroach (Wareham, 1978). It thus seems likely that a definitive demonstration of the involvement of cyclic nucleotides in aminergic neurotransmission in insects must await the results from studies on identified aminergic neurones where the remaining criteria outlined by Beam and Greengard (1976) can be directly tested. 11 Conclusions

Biogenic amines fulfill many different roles in the insect peripheral and central nervous systems and some of these are classified in Table 7. The TABLE 7 Functions of biogenic amines in the insect nervous system Location

Amine

Function

Peripheral “Sympathetic effects” Skeletal muscle Visceral muscle Stimulation of carbohydrate metabolism in skeletal muscle, fat body and nerve cord

Octopamine Dopamine 5-Hydroxytryptamine Octopamine Octopamine (?)

Neurornodulator Neurotransmitters/ Neuromodulators

Dopamine 5-H ydroxytryptamine Octopamine 5-Hydroxytryptamine (?)

Neurotransmitter Neurohormone Neurotransmitter Neurotransmitter

Neurohormone

“Epithelial effects” Salivary glands Light organs Epidermisplasticization of cuticle Epidermis-mosquito antennae

?

Neurotransmitter

Central Brain and ventral nerve cord

Octopamine, Dopamine 5-hydroxytryptamine Noradrenaline (?)

Neurotransmitters/ Neuromodulators?

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analogy between the actions of the vertebrate sympathetic nervous system and certain of the actions of biogenic amines in insects, has been drawn on many occasions. Within the category of sympathetic effects of biogenic amines in insects one can include the neurotransmitter/neuromodulatory roles of biogenic amines on the visceral muscle of the heart and the intestine, as well as the modulatory role of octopamine at the insect skeletal neuromuscular junction. The latter role, together with the proposed neurohormonal role for octopamine in the stimulation of carbohydrate metabolism in several tissues such as skeletal muscle, fat body and nerve cord, suggest that octopamine is ideally suited to function as part of an arousal system for the animal under stressful circumstances. Not only would the magnitude of the responses of individual muscles be increased, but the amount of energy available for their immediate use would also be increased. It remains to be seen if there is an increased release of biogenic amines into the haemolymph of insects under stressful conditions. Biogenic amines also appear to be involved in controlling the functions of various epithelial tissues in insects. Secretory epithelia such as that of the salivary glands are controlled either by a direct dopaminergic innervation or by the neurohormonal actions of 5-HT released into the circulatory system, from as yet undefined sites in the nervous system. The highly specialized epithelial structures of the firefly light organ also appear to be under aminergic control, possibly through an octopaminergic innervation, but in the light organ of the adult the actual amine-responsive cells have not been identfied. Recent studies indicate that insect epithelia in general may receive a direct aminergic innervation. In the blood sucking bug, Rhodnius, for instance, the plasticization of the abdominal cuticle following a blood meal is under neural control (Maddrell, 1966), and evidence has been provided that this effect is probably mediated directly by specific 5-HT receptors on the epidermal cells (Reynolds, 1974). Reynolds (1974) warns, however, that further studies are needed to demonstrate the presence of 5-HT in the nerves to the epidermis, as well as its release upon nerve stimulation, before we can accept 5-HT as the transmitter in this preparation. Another example of an aminergically innervated epithelium is found in the antennae of certain species of mosquito. In this preparation a direct aminergic innervation controlling hair erection has been proposed to act on the effector cells via a-adrenergic receptors (Nijhout, 1977). Here again further work is needed to establish firmly the nature of this proposed aminergic innervation. These examples do, however, raise the possibility that the activities of many other insect epithelia may be controlled by a direct aminergic innervation. The majority of the studies on the physiological roles of biogenic amines in the insect nervous system have concentrated on the peripheral nervous

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system, rather than the central nervous system, because of the greater accessibility of the former. Similarly, studies on vertebrates initially elucidated the roles of biogenic amines in the peripheral, sympathetic nervous system, rather than their central roles. However, the recent application of techniques such as sensitive radioenzymatic assays for biogenic amines and their synthetic enzymes, sensitive histofluorescent techniques for the location of amine containing cell bodies and nerve terminals, together with the results obtained from microiontophoretic application of drugs to identified populations of neurones, has provided much information on the distribution and function of aminergic pathways in the vertebrate brain (see Moore and Bloom, 1978, 1979). The biogenic amines present in the vertebrate brain appear to act either as neurotransmitters in specific pathways conveying information from one part of the brain to another (e.g. dopamine in the nigrostriatal pathway; see Moore and Bloom, 1978) or as neuromodulators, which are released from more diffuse aminergic networks to bias the activity and responsiveness of whole regions of the brain (e.g. the noradrenaline containing pathways from the locus coeruleus; see Moore and Bloom, 1979). The data available on the effects of biogenic amines on the insect central nervous system do not provide any direct evidence about the function of the amines in the control of the behaviour of the whole animal. In many studies, biogenic amines have been injected into whole insects (Hodgson and Wright, 1963; Kostowskietal., 1975b; see also Murdock, 1971) and it is not clear if the resulting behavioural change is due to the actions of the amines themselves, to their metabolites or to other active substances released by the amines. Similarly, experiments that attempt to assess the effects of biogenic amines on the spontaneous activity, or effectiveness of synaptic transmission in the insect CNS are also difficult to interpret (e.g. Twarog and Roeder, 1957; Hodgson and Wright, 1963; Kostowski et al., 1975b, see also Murdock, 1971 and Pichon, 1974). This is a result of a general lack of information on the specificity of the receptors mediating the observed responses and also of the difficulty in ascertaining whether the observed effects are primary or secondary. A third approach, the measurement of the levels of biogenic amines in insect brains after the induction of different “behavioural states”, such as inter- and intra-specific aggression in ants (Kostowski et al., 1975a), also suffers from the difficulty of ascertaining whether the effects are primary or secondary. Thus at present our information on the roles of biogenic amines in the insect CNS and their involvement in the control of insect behaviour is particularly limited. In the future, new and more specific approaches will be required to elucidate the roles of biogenic amines in the insect central nervous system. This review has emphasized the importance of two lines of approach towards

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the study of biogenic amines in insects, namely the use of identified neuronal systems and the use of integrated multidisciplinary studies. The first is essential for the comparison of the results obtained from individual animals of the same species. The second is essential if we are to understand the functional role of the biogenic amines; clearly information from physiological studies needs to be correlated with that from biochemical, pharmacological and anatomical studies on the same preparation. Previous sections in this review have pointed out the dangers of extrapolation on the basis of evidence obtained from one approach only. An example is the extrapolation of functional roles from anatomical evidence without adequate physiological controls. The biochemical analysis of identified cell bodies from the ganglia of the insect central nervous system isone obvious way to establish the distribution of biogenic amines, and has already proved particularly useful in the study of octopamine (Evans and O’Shea, 1977, 1978). However, if we are going to understand the functional roles of biogenic amines in the insect brain, positive techniques for the identification of the amine-containing terminals at the electron microscope level will be essential. Immunocytochemical techniques for the localization of synthetic enzymes, or even the biogenic amines themselves, should prove particularly useful in this respect. Information obtained from such studies could then be compared with physiological and pharmacological data obtained from the local microiontophoretic application of biogenic amines to specific regions of the insect CNS (see Steiner and Pieri, 1969 for iontophoretic application of dopamine to mushroom bodies of the ant). If biogenic amines in the insect CNS are involved in modulatory systems, as in the vertebrate CNS, then such systems would seem particularly amenable to studies on structural modifications induced by mutations. Such studies have provided much information on the functional roles of cells in the mouse CNS (see Caviness and Rakic, 1978). The insect CNS with its limited number of cells also provides a simple system where cell lineages can be followed (e.g. Goodman and Spitzer, 1978,1979) and the further application of this technique is likely to provide much interesting information on the development of identified aminergic neurones in insects (e.g. Goodman et af., 1979). It seems likely that at the level of the thoracic nerve cord studies on single identified neurones could be interpreted directly in behavioural terms, but when considering the more complex parallel distributed networks that occur in the insect brain (e.g. in the optic lobes, mushroom bodies and central body), it should be remembered that the emergent properties of the neuronal assemblies may be more directly correlated with brain function than are the responses of any individual neurone. This point has been recently stressed in studies on insect brain by Strausfeld (1970,1976) and in

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studies on vertebrate brain by Pellionisz and Llinas (1979). It would seem that the diffuse modulatory control systems currently being described for biogenic amines in the vertebrate CNS, would be ideally suited for the simultaneous control of the activities of whole assemblies of neurones. The existence of similar aminergic control systems must obviously be considered in attempts to understand the functional roles of biogenic amines in the highly structured neuropiles of the insect brain, such as occur in optic lobes and mushroom bodies. It is obvious that studies on the functional roles of biogenic amines in the insect nervous system are entering a new and exciting era. Such studies are of great economic importance and will undoubtedly uncover principles of biogenic amine functioning of general applicability to all nervous systems.

Acknowledgements

I would like to thank Dr M. V. S. Siegler for critically reading the manuscript and making many useful suggestions. 1 would also like to thank Mrs M. Clements, Mrs I. Prince and Mrs V. Rule for their skill and patience in the typing of the manuscript.

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Integration of Behaviour and Physiology in Ecdysis Stuart E. Reynolds School of B/O/Og/Ca/Saences, Un/vers/ty of Bath, UK

1 Introduction 476 2 The timing of ecdysis 476 2.1 Environmental stimuli 476 2.2 Endogenous factors: circadian rhythms 478 2.3 Developmental “readiness” 480 3 Ecdysial behaviour 482 3.1 Hatching behaviour 482 3.2 Exopterygote ecdyses 487 3.3 Adult eclosion in large moths 496 3.4 Adult eclosion in blowflies 499 3.5 Puparium formation in blowflies 502 3.6 Stereotypy and plasticity in ecdysis 503 3.7 Behavioural switching in ecdysis 514 4 The mechanics of ecdysis 519 4.1 Splitting the old cuticle 519 4.2 Escaping from the old cuticle 523 4.3 Inflating the new cuticle 525 5 Ecdysial physiology and its integration with behaviour 530 5.1 Initiation and termination of behaviour patterns 530 5.2 Modification of behaviour patterns before and after ecdysis 5.3 Cuticle plasticization 537 5.4 Cuticle hardening 541 5.5 Tracheal air-filling 546 5.6 Cuticle deposition 549 5.7 Changes in blood volume: post-ecdysial diuresis 553 5.8 Discharge of dermal glands 557 5.9 Changes in heart rate 558 5.20 Metabolism during ecdysis 560 5.11 Post-ecdysial cell death 561 5.12 Integrative mechanisms 567 6 Failures of ecdysis 569 6.1 Natural failures 569 6.2 Experimentally-induced failures 575 Acknowledgements 579 References 579 47 5

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1 Introduction

One of the most obvious features of the lives of arthropods is the way in which the processes of growth and development are punctuated by the periodic shedding of the cuticle. Only the lives of insects and allied groups are so completely dominated by the cyclic need to deposit a new cuticle and then to shed the old one. Much attention has been devoted to the endocrine physiology of this cyclic development, but rather less to the physiological events which accompany the actual event of ecdysis -the act of shedding the old cuticle. Often ecdysis is thought of as “merely” the necessary conclusion of the previous round of development. In fact, ecdysis is a complex feat of escapology for the insect, which is of course both aided and hampered in its efforts at this time by the soft, flexible condition of its new cuticle. It is the purpose of this review to bring together what is known of the physiology of ecdysis with what is known of the behaviour of ecdysing insects. and to try to show how one is integrated with the other. Hatching is of course akin to ecdysis, in that it involves the escape of the insect, clothed in an unhardened cuticle in most cases, from the confines of an outmoded body case which is no longer useful to it. Indeed, an essential part of the hatching process is, in many cases, the first true larval ecdysis, whereby the so-called embryonic cuticle is shed. This first larval ecdysis may, or may not, be coincident with escape from the eggshell proper, as we shall see below. Rather less is known of the behaviour and physiology of hatching insects than is the case for ecdysis, doubtless because the former are usually too small for experimental work. Also, of course, before hatching begins it is difficult to observe the behaviour of the animal unless the eggshell is transparent. Such observations on hatching insects as there are, have been included in this review as being relevant to the study of ecdysis. Insects are particularly vulnerable at this time, and the review is concluded by a brief consideration of some of the things which can go wrong during ecdysis. 2

The timing of ecdysis

2.1 2.1.1

ENVIRONMENTAL STIMULI

Hatching

Once larvae are “ready” to emerge from their eggs many stimuli can promote hatching behaviour. Bernays (1 972a) reports that in locusts a drop in temperature (but not a mild increase) and also a reduction in humidity are both stimuli for the initiation of hatching behaviour. Mechanical stimulation of the eggs is a hatching stimulus too. This was shown directly in an experi-

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ment, but it also probably accounts for the observation that whereas most of the eggs in a pod hatch within 2-3 h if left undisturbed in the substratum in which they were laid, when separated the eggs hatch over a much longer period. Presumably the movements of their newly hatched neighbours are a stimulus for the larvae to hatch. However, mechanical disturbance of the eggs is not a necessary pre-requisite for hatching to occur. Where the eggs are separated, each egg hatches when it is “ready”, though the influence of small, local environmental changes as possible hatching stimuli cannot be ruled out. Provine (1976a) also found that although mechanical stimulation was not necessary to initiate “hatching” behaviour in cockroach pharate 1st instar, which he had removed from their ootheca, it was very effective in advancing the time at which hatching occurred. In one experiment 49 out of 52 larvae shed their embryonic cuticles within 30 min of beginning gentle tactile stimulation (stroking), whereas only 3 of 53 unstimulated controls shed their cuticle in the same time. Provine suggests that this response acts to synchronize hatching of the larvae within the ootheca. Hatching stimuli have been described for many insect species and I can do no more than quote a few examples here. For instance, eggs of the human warble fly Dermatobia haminis which are laid on the body surface of mosquitoes and transported thus to their warm-blooded hosts, are stimulated to hatch by the warmth of their intended victim (Bates, 1943). On the other hand the principal stimulus to hatching in the horse bot fly, Gasterophilus intestinalis, which lays its eggs on the horse’s forelegs, seems to be the mechanical stimulation of being licked (Dinulescu, 1932). Aquatic larvae will only hatch in water, and wetting may be a hatching stimulus. However, other conditions are often required too. For instance,Aedes eggs hatch most readily when the pOz of the water is low (indicating bacterial growth); the longer the larvae remain, fully developed, within the egg, the less ready they are to hatch unless the p 0 2 is low (Judson et al., 1965). An interesting consequence of this dependence on low pOz has been described by Gillett el al. (1977). The first larvae of anAedes egg mass to hatch are able to delay the hatching of their fellows by grazing away the film of bacterial growth from the remaining unhatched eggs. This keeps PO, in their vicinity high and thus inhibits hatching, reducing competition for what bacterial food sources there may be. On the other hand, Jackson (1958) reports that larvae of the water beetle, Agahus, will only hatch when PO, is high. 2.1.2 Ecdysis Similarly, in later life, environmental variables may influence the time at which ecdysis occurs. Insects generally seek out a suitable spot for

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ecdysis and the onset of ecdysial behaviour may be delayed if such a site is unavailable (Hughes, 1980a). The restless locomotor behaviour involved in searching for a suitable site has been included by Carlson (1977a) in his “preparatory phase” of ecdysis in pharate adults of the cricket Teleogryllus oceanicus. Vincent (1971) describes similar behaviour in pharate adult Locusta, commenting that at this stage the locust is irritable and easily disturbed, whereas once the business of shedding the old cuticle is under way, the locust becomes oblivious to even quite intense stimulation. A t this stage of preparatory behaviour, Vincent was able to prevent ecdysis from occurring for up to about a day by tumbling the locusts in a shaker. Hughes (1980a) prevented locusts from ecdysing by removing all perch sites from their cage. On the othzr hand, the emergence of blowflies from their puparia can be stimulated by mechanical disturbance, (author’s observation) as can the emergence of adult Manduca sexta from the pupal cuticle, once eclosion hormone has been released (Reynolds et al., 1979). Although the time at which adult Drosophilapseudoobscura emerge from the puparium is broadly controlled by the operation of an internal circadian clock, so that eclosion occurs at about dawn (see below), it can be inferred from the shape of the histogram of frequency of emergence during the peak, that emergence is inhibited during the dark (Saunders, 1976). Similarly, although subject to the control of a circadian rhythm, the emergence of adult Manduca is evidently stimulated by a laboratory transition from light to dark (see emergence data given in Reynolds et al., 1979).

2.2

E N D O G E N O U S FACTORS: C I R C A D I A N RHYTHMS

In many insects there are die1 rhythms of hatching and of ecdysis. Saunders (1976; pp. 2 3 0 - 2 3 3 ) gives references to a number of examples. Hatching rhythms are noted to occur in 5 orders, and rhythms of ecdysis in 6 orders of both exopterygote and endopterygote insects. It is quite certain that this phenomenon is very widespread. In at least some of these cases, the rhythm appears to be endogenous, i.e. hatching or ecdysis results from a cue given by a circadian clock somewhere in the insect’s body. Presumably the clock is located in the central nervous system; the only unambiguous identification of the site of such a clock controlling ecdysis is that of Truman (1972a), where the clock timing the initiation of eclosion behaviour in pharate adult Antheraea pernyi was found to be in the protocerebral lobes of the brain. In the case of D . pseudoobscura such evidence as is available suggests that the clock controlling adult eclosion is somewhere in the head (Zimmerman and Ives, 1971), but that neither the eyes nor the ocelli are required for entrainment to environmental light cycles (Engelmann and Honeggar, 1966). This is also the case for the silkmoth eclosion clock, which evidently uses an

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extra-retinal photoreceptor for entrainment purposes (Truman, 1972a). The modus operandi of the circadian clock controlling adult eclosion in Drosophila pseudoobscura has been investigated in great detail, perhaps more so than any other physiological clock, by Pittendrigh and his coworkers. Rather little is known about the biochemistry and/or physiology of this, or any other clock at the cellular level, but much is known about its functional characteristics. All this has been reviewed recently (Brady, 1974; Saunders, 1974, 1976) and will not be the subject of further review here. However, it should be pointed out that the existence of a diel rhythm of hatching or ecdysis affords an experimental opportunity to investigate the physiological, biochemical and behavioural events which occur before the event, as well as those which occur during it and afterwards. This opportunity has been rather little exploited, probably because those common laboratory insects, Prriplaneta americana and Schistocerca gregaria, do not show very marked diel rhythms of ecdysis or of hatching. Truman has, however, made use of this experimental tool to investigate both the control of larval and adult ecdyses in large moths. In larvae of M. sexta, the timing of ecdysis shows a diel rhythm. The time at which ecdysis occurs, however, is different for each instar and is strongly temperature dependent. The results of neck- and thorax-ligation experiments suggest that the rhythm of shedding of the old cuticle is not due to the independent gating of the time of ecdysis itself, but is the result of the previous “gating” of the time at which prothoracicotropic hormone (PTTH; “brain hormone”) is released. The pulse of PTTH activates the prothoracic glands, which in turn secrete ecdysone to initiate apolysis and the deposition of the new cuticle. Ecdysis then follows when development of the cuticle is complete and the insect is “ready” (Truman, 1972b). It was observed that a recognizable, although weak, ecdysial behaviour pattern occurred at the normal time even in the absence of the head, so that evidently higher central nervous centres are not required to trigger ecdysis. In the case of the larval-pupal ecdysis of M . sexta, the observed diel rhythm is also due to the previous gating of the release of PTTH (Truman and Riddiford, 1974a). In this case, there are two releases of PTTH, the second pulse of PTTH (and consequently of ecdysone) being the one which actually initiates pupal development per se. The first pulse of PTTH (and thus of ecdysone) is responsible for the change in “commitment” of the epidermis which is only expressed later, when the second pulse is seen (Riddiford, 1976). As in the case of the larval ecdyses once development has been initiated by ecdysone, the timing of the subsequent ecdysis is fixed, although temperature dependent. It seems likely that in many cases the observed diel rhythms of insect ecdysis will prove to be due to the previous gating of the release of developmental

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hormones. Recently Wright (1977) has shown that although larval ecdyses in the butterfly, Heliconius melpomene, are synchronous and occur only during a relatively restricted period around dawn, it is not ecdysis itself which is gated, but the release of PTTH. This is a similar situation to the case in M . sexta, except that whereas in Manduca PTTH release is always at the same time, and as the time necessary for development increases with each instar, the actual time of ecdysis is delayed more and more, inHeliconius, the time of ecdysis is always the same, so that the gate for PTTH release must be shifted backward through the hours of the day. However, the adult ecdysis, or eclosion, of a number of endopterygote insects appears to be controlled differently. Skopik and Pittendrigh (1967) observed (contrary to the claims of Harker, 1965a, b) that a number of “marker” events during the development of pharate adult Drosophila pseudoobscura occurred randomly at any time of day, whereas emergence from the puparium alone appeared to be directly “gated” according to the instructions of a circadian clock. The way in which ecdysis is initiated by the clock remains unknown in Drosophila, but Truman and Riddiford (1970) have provided an explanation for a similar rhythm of adult eclosion which occurs in silkmoths. Here, eclosion is initiated by the release of a hormone from the corpora cardiaca (CC), the eclosion hormone. The timing of hormone release seems to be directed by a circadian clock located in the protocerebral lobes of the brain (Truman, 1972a). The way in which the eclosion hormone triggers eclosion behaviour will be discussed further in Section 5.1. So far, eclosion hormone is only known to occur in Saturniid and Sphingid moths, but it seems more than likely that a similar hormone triggers eclosion in other endopterygote insects showing an eclosion rhythm. However, it should be noted that the timing of adult eclosion in the saltmarsh mosquito, Aedes taeniorhynchus, is known not to be gated directly, but to occur in a die1 rhythm which is dependent on the previous pattern of pupation (Nayar, 1967). This does not mean to say, of course, than an eclosion hormone-like factor cannot be involved in the adult emergence of this insect. As we shall see (Section 5 ) , the eclosion hormone has functions other than just that of initiating eclosion behaviour. 2.3

DEVELOPMENTAL “READINESS”

The susceptibility of the timing of ecdysis to the influence of external cues from the environment, and to internal cues from circadian clocks, gives us cause to think about the concept of the larva within the egg, or the pharate insect within its old cuticle, being in some way “ready” to hatch or to ecdyse. In some cases, development within the egg may appear to have been completed, and yet the larva is not “ready” to hatch, e.g. Antheraea yamamai,

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Lymantria dispar, Malacosoma testacea, M . disstria, (Lepidoptera); Campsocleis buergeri, Melanoplus bivittatus, (Orthoptera); Tinarcha tenebricosa, T. violacea-nigra (Coleoptera), all of which diapause at this stage (Lees, 1955). On the other hand mosquito larvae (Aedes) are “ready” to hatch, even after several weeks of dry storage, and emerge from the egg within minutes of being immersed in water with a suitably low p 0 2 (Judson et al., 1965). The nature of this state of “readiness” is not clear but must involve changes in the nervous system which allow the expression of hatching or ecdysial behaviour. However, this is not just a matter of having the requisite programme of behaviour ready to hand in the nervous system’s repertoire: Provine (1977) observed that cockroach larvae showed typical “hatching behaviour”, albeit at a low level of intensity and frequencey, several days before hatching. At this stage they are clearly not “ready” to hatch, and even if the hatching efforts were stronger, they would have little effect on the still undigested eggshell. Truman (1 976) has similarly noted that “peeled” adult A . pernyi moths show spontaneous eclosion movements (and other types of typically adult behaviour) 2 and 3 days before the expected time of eclosion. Interestingly, both Provine (1977) and Truman (1976) found that such movements were reduced on the last day before hatching and eclosion respectively. At this time the eggshell, or the pupal cuticle, in each case would be considerably weakened and might well be prematurely split if such movements did occur. Truman suggested that in the case of the silkmoth some kind of central inhibition might be involved, supporting this claim by his observation that injections of picrotoxin (an antagonist of the inhibitory transmitter, GABA) caused the release of some of the “inhibited” adult behaviours. Perhaps this inhibition may have something to do with the state of “readiness”; thus, all that is needed to hatch o r to eclose is to remove the inhibition. Truman’s discovery that adult eclosion in Hyalophora cecropia and in other giant silkmoths, is triggered by the release into the blood of a hormone, the eclosion hormone, (Truman and Riddiford, 1970), does not unfortunately help very much in solving the problem of the nature of being “ready” to undergo ecdysis. The ability of injected silkmoths to respond to the eclosion hormone is almost always, but not invariably, restricted to those individuals which have completed moulting fluid resorption. In these species, resorption is not gated (i.e. takes place at any time of day or night) and insects which become “ready” to respond, must wait until the next gate for the release of eclosion hormone (early morning in H . cecropia; late afternoon in A . pernyi). In Manduca sexta, however, moulting fluid resorption is gated, as also is the ability to respond to injections of eclosion hormone, which develops at

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about the same time as moulting fluid resorption is being completed (Reynolds ef a f . , 1979). This is some hours before the eclosion hormone itself is due to be released. This might suggest that moulting fluid resorption and “readiness” to respond are in some way linked together, However, brainless individuals of both silkmoths (Truman and Riddiford, 1970) and M. sexfa (Truman, personal communication) are able to emerge despite the fact that no eclosion hormone is released. These animals also emerge “wet”, having failed to resorb their moulting fluid. Also some individuals of A . pernyi and H . cecropia will respond to exogenous eclosion hormone while still “wet”. In these cases, the resorption of moulting fluid is evidently accelerated by the hormone, so that the moths are “dry” by the time that they eclose (Truman, personal communication). The fact that both moulting fluid resorption and the acquisition of responsiveness to eclosion hormone are gated events in M. sexta, both occurring before the release of eclosion hormone (Reynolds et al., 1979), appears to mean that in Manduca, at least, there must be some synchronizing event which occurs late in adult development, but before eclosion hormone release. Such an event might be the release of another, as yet unknown, hormone. The existence of such gated events during the development of adult Drosophila melanogaster, enclosed within the pupal cuticle, has been postulated by Harker (1965a, b). This suggestion was criticized by Skopik and Pittendrigh (1967) and Pittendrigh and Skopik (1970), on the basis of experiments with both D . melanogaster and D . pseudoobscura, in which they failed to find any evidence for such “internal gates”. However, perhaps the problem should be re-examined. The experiments with Manduca indicate that such gates can exist, and may provide an experimental “handle” with which to investigate the nature of “readiness” to undergo ecdysis.

3 3.1

Ecdysial behaviour HATCHING BEHAVIOUR

There is a variety of hatching mechanisms which are briefly reviewed by Chapman (1969) and Wigglesworth (1972), both of which provide good introductions to the older literature. There are some good descriptions of hatching in Sikes and Wigglesworth (1931), but most of the older descriptions either lack detail, or were observed with some other purpose in mind, so that the classification of behaviour into separable, motor acts (rather than functional units) is difficult. Two very closely observed descriptions of prehatching and hatching behaviour have been given recently for the locust, Schisfocerca gregaria (Bernays, 1971, 1972a, b) and for the cockroach,

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Periplaneta arnericana (Provine, 1976a, 1977) and these will be reviewed here in some detail. Aspects of hatching mechanics are considered in Section 4. The term “hatching” will be used to describe breaking the egg-shell and escaping from it. “Eggshell” in this context includes the chorion, the vitelline membrane, and the serosal cuticle. Where an ootheca (communal egg case) is present, “hatch” will also do to describe escape from this also. Provine (1976a, 1977) also uses the term “eclosion” to denote escape from the embryonic cuticle. In cockroaches this takes place simultaneously with hatching. In locusts, it does not, the vermiform larvae only ecdysing after they are free of the substrate in which the egg-pod was buried. Bernays (1972b) calls this ecdysis the intermediate moult, or first true larval ecdysis. To add to these terminological difficulties, Provine describes the cockroach larvae within the egg as being “pharate first instar larvae”. When they are removed experimentally from the ootheca, still in their embryonic cuticle he calls them “first instar larvae” and after “eclosion” they became “second instar larvae”. Clearly this terminology of Provine’s cannot be correct. I will follow Bernays (1972a), where the first instar larva in the egg is described as being enclosed by both the eggshell and an embryonic cuticle. These two coverings may be shed together (cockroach) or separately (locust). But in both cases, the insect which emerges at the end is a first instar larva. 3.1.1 Prehatching and hatching behaviour Bernays (1972a) observed hatching movements of locusts in dechorionated eggs, and analyzed them cinematographically. Prehatching peristaltic movements, which occur quite sporadically, are distinguished from what are called “hatching efforts”. These appear suddenly, and occur repeatedly until the eggshell is burst. The movement consists of a simultaneous dorsalventral contraction of all abdominal segments. At 25”C, each “effort” is maintained for over a second. Unsuccessful hatching efforts are followed by a wave of contraction which moves forward from the abdomen to the thorax and head, taking about 3 seconds. The larva tends to move forward within the egg during a period of many unsuccessful efforts. After making a typical transverse slit in the eggshell, over the cervical ampullae (see below, Section 4.1), the vermiform larva forces its way out of the eggshell without further breaking of the shell. The frequency of peristaltic waves increases as soon as the shell is ruptured. The abdominal contraction is now seen to take the form of a peristaltic wave which passes forward extremely rapidly. Also evident is an arching of the first four segments of the abdomen, which is strongest when the abdomen is lengthening. It is not clear whether this is a different kind of movement from the earlier “hatching

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efforts” or whether the latter are just a modification of the former, being a result of the resistance offered to forward movement by the unbroken eggshell. Bernays’ observations on digging movements in vermiform larvae (see below) suggest that this may well be the case. As the larva escapes from the shell, the frequency of the movements increases, and the speed at which the waves move is greater. At 25”C, the frequency increases from 5 min-’ to 10-20 min-’. Once the larva is free from the confines of the shell, of course, changes in body shape during the cycles of movement become much more pronounced, since there are now no external constraints. Provine (1977) describes “eclosion” movements of cockroach larvae still within their embryonic cuticles but which had been removed from their eggshells and oothecae. The analysis was visual, using an event recorder, and thus lacks the detail of Bernays’ work. Hatching movements begin suddenly. They may be sustained until the larva escapes, or halt and then restart. When sustained, their frequency is about 10-12 min-’. Each movement is a peristaltic wave directed anteriad. When the wave reaches the thorax it is propagated as a segment-to-segment ventral flexure. Dorsal thoracic flexures and rotational movements of the abdomen are occasionally interspersed with these behaviours. Provine does not say whether the performance of such movements affected the timing of the subsequent peristaltic movement (i.e. whether the movement “replaced” a peristalsis, or whether it was an “extra”). Air-swallowing is concurrent with eclosion movements. The embryonic cuticle splits along the dorsal surface of the thorax (as is the case in later ecdyses in this species). The exuvia is then passed over the head and is moved posteriad along the body. During eclosion, the cockroach larvae show no response to tactile stimulation, even when this is quite violent (e.g. pinching the cerci with forceps). Once the larva is free of the exuvia, however, and eclosion movements cease, the larva reacts to such stimuli in typical fashion. Such mature patterns of behaviour as walking and escape reactions are never observed prior to this (see Section 3.7). The “eclosion” behaviour of cockroach larvae, described above, is strictly speaking the equivalent of the first larval ecdysis described for locusts by Bernays (1972b). Provine (1977) mentions escape from the eggshell and ootheca (his “hatching”) relatively briefly, commenting that eclosion (i.e. first larval ecdysis) and hatching normally occur simultaneously, and that they involve similar movements. All the larvae (12-16) hatch from the ootheca together, surging rhythmically headfirst out of the mouth of the ootheca. The embryonic cuticles are left behind in the mouth of the ootheca, which closes immediately after the larvae have hatched.

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3.1.2 Intermediate moult, or first larval ecdysis In the locust, unlike the cockroach, shedding of the embryonic cuticle is separated from “hatching”. However, the distinction may not represent such a major difference between the two species as appears at first sight. Bernays (1972b) says that the major stimulus to ecdysis in the vermiform larva of the locust is the lack of all-round contact. If enclosed, the larva will continue to dig far up to 8 h without attempting to ecdyse. On reaching a space where there is not an all-round contact, however, ecdysis will occur. If eggs are allowed to hatch on the surface, then shedding of the embryonic cuticle is concurrent with the later stages of hatching. The essential stimuli may be from sensilla in the neck and prothorax, since the larvae often begin to swallow air (an ecdysial as opposed to a hatching behaviour) when only the neck and prothorax are free of the serosal cuticle. The club-shaped hairs of the pharate first instar integument may be involved (Bernays, 1971). Thus the delay between hatching and the first larval ecdysis in locusts is probably associated with their habit of laying their eggs underground. The behaviour associated with the first larval ecdysis is as follows. A few minutes after reaching a suitable situation (i.e. above ground), the larva begins to swallow air. The rate of swallowing builds up rapidly to a peak just before ecdysis. A t 28°C o r 38”C, only 1-2 min elapse from the beginning of swallowing to the moment when the embryonic cuticle splits. After this, the rate of swallowing falls rapidly to zero (1-2 min). Once swallowing is underway, the insect will swallow water or oil instead of air, indicating that sensory feedback cannot be very important. The swallowed air distends the gut, and increases the larva’s volume by about 25%. Within 0.5 min of the onset of air-swallowing (at 20°C) peristaltic waves begin to pass anteriad along the body, each taking about 5 s to do so. As the wave passes the neck, swallowing is interrupted. As each wave begins, the tip of the abdomen is flexed ventrally. The wave itself consists of segmental dorso-ventral contractions, weakening in the thorax and petering out in the prothorax (though as noted above, the wave’s effects are seen anterior to this). In the abdomen, narrowing of the segments precedes shortening, and widening precedes lengthening, so that segments can move forward relative to the ensheathing embryonic cuticle. The effect of this is that the exuvia passes posteriad along the body, wrinkling up as it goes. The mechanics of this are considered further below, (Section 4.2). Once the cuticle has split, the locust’s body arches in a more pronounced way, until the head is pulled free of the exuvia. Now the larva straightens up and the antennae and mouthparts are pulled free. The legs assist in gaining their freedom by flexing, their movements being co-ordinated with the wave of contraction, which passes forward from the abdomen. Generally, the right

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and left legs in a particular segment do not move together, but rather one after the other, so that a side-to-side movement results. Leg movements are more pronounced once the head is free. 3.1.3 Digging behaviour of vermiforrn larvae

This is of interest in that it serves to link hatching and the first larval ecdysis. Digging is a part of normal ecdysial behaviour both in vermiform larvae of locusts, and also in newly emerged adults of some flies and moths (see below) and appears to utilize the basic peristaltic patterning of both hatching and ecdysial movements. Digging in vermiform larvae of locusts has been described by Bernays (1971) who has made a detailed cinematographic analysis. The basic pattern of digging movements is again the peristaltic wave, passing anteriad along the body. Cycles of contraction follow one another “almost without a pause”. The details of the movements are different to those used in hatching and in the first larval ecdysis. The contractions do not result in telescoping of the presumptive sclerites, but rather in a worm-like distortion of the whole body. Shortening of a segment therefore results in some wrinkling of the cuticle in the abdomen. Widening of the segment is simultaneous with its shortening, unlike the situation in the later stages of hatching and in the first larval ecdysis, where narrowing of the segment precedes shortening, and widening precedes lengthening, at least in the more posterior abdominal segments (Bernays 1972a, b). Five phases are distinguished in the digging movements: ( i ) abdomen shortens and widens; (ii) thorax shortens and widens; (iii) thorax relaxes, cervical ampullae are withdrawn, head tilted back; ( i v ) thorax continues to lengthen, ampullae protruded, abdomen shortens; (v) sustained dorsoventral contraction. No direct comparisons are drawn, but the frequency and speed of the movements seem to be somewhat faster than those seen in hatching and in the first larval ecdysis at similar temperatures. Digging brings the vermiform larva to the surface by a combination of negative geotaxis, and seeking out the easiest path (which in vivo is usually through the foam plug of the egg pod; Ewer, 1977). It is interesting that throughout digging, the larval tracheal system remains fluid-filled, so that exchange of respiratory gases must be considerably impaired (see Section 5.10). The digging response is lost very soon after the first larval ecdysis (see Section 3.7).

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487

EXOPTERYGOTE ECDYSES

As we have seen above, Bernays (1972b) has given a closely observed account of the specialized first larval ecdysis of the locust, Schistocerca gregaria. Another very detailed account of behaviour associated with ecdysis is given by Carlson and Bentley (1977) and Carlson (1977a, b), in this case from a specifically neurobiological point of view. The larval-adult ecdysis of the cricket, Teleogryllus oceanicus, was analysed using videotape, and observable behaviour was correlated with electromyographic recordings of motorneurone activity. Ecdysis is described as a “mechanically difficult task” and the entire behaviour, which takes about 4 h, is divided into 4 distinct phases: (i) a preparatory phase, which loosens and splits the exuvia and anchors it to the substrate. This involves restless locomotor and grooming activity; rhythmic leg movements which fix the tarsal claws to the substrate; abdominal contractions and air-swallowing; the assumption of a characteristic posture which facilitates emergence. (ii) An ecdysial phase, which extracts the insect from the old cuticle. Peristaltic abdominal waves propel the body forward and widen the ecdysial split; appendages are extracted by a complex sequence of muscle contractions, pulling them up and forward. (iii) an expansional phase inflates the new cuticle, protects appendages during cuticle sclerotisation, and subsequently folds the wings. This is a period of general immobility, except for the small set of motor patterns necessary for proper expansion and hardening of the cuticle. ( i v ) anexuvial phase, which involves eating the exuvia. It commences with a release from immobility which can be quite abrupt. Additional behaviours, which Carlson relates to proper hardening of the cuticle occur, such as alternate bending of the ovipositor valves in females. Further to this division of the total behaviour pattern into 4 phases, each phase is further subdivided into a number of “motor programmes” each of which is defined as a series of contractions of particular muscles in a particular pattern of co-ordination. Each motor programme subserves a particular mechanical task. Carlson (1977a) identifies a large number (48) of these motor programmes (Table 1). A typical motor programme (illustrated in Fig. 1)results in alternating contractions of bilaterally homologous muscles in the prothorax, which gradually draw the forelegs out of their old exoskeletal sheaths. The motor programmes are described as being recruited in a stereotyped sequence (Fig. 2) and terminating independently of one another, although their activities are co-ordinated while they occur together (Fig. 3).

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TABLE 1 Motor programmesof the ecdysial behaviour of the cricket, Teleogryllus oceanicus. (From Carlson, 1977a) The preparatory. .phase A1 Push-ups A2 Pumping A3 Anchoring A4 Lateral antennal abduction A5 Antennal beating A6 Antennal scissoring The ecdysial phase B l Abdominal peristalsis B2 Cercal scissoring B3 Alterations B4 Wingbud folding B5 Head tucking B6 Antennal folding B7 Proximal leg segment extrication B8 Mouthpart extrication B9 Labral extrication B10 Antennal loosening B11 Front distal leg segment extrication B 12 Proximal labial palp flexion B13 Antennal guiding (by maxillae) B14 Low frequency distal flexion of labial palps B15 Labial palp lowering B16 Head raising B17 Middle tarsal depression B18 Front leg tetanus B19 High frequency labial antennal clasping B20 Middle leg extension

The ecdysial phase (cont.) B21 Front tarsal depression B22 Hind distal leg segment extrication B23 Labial palp tetanus (antennal clasping) B24 Front leg tugging B25 Hind leg tetanus B26 Maxillary palp distal segment flexion B27 Middle leg antennal clasping (vertical) B28 Maxillary palp distal segment spreading B29 Lateral antennal abduction B30 Hind leg kicking The expansional phase C1 Cercal scissoring C2 Hind tarsal depression C3 Abdominal tetanus C4 Hind tibia1 extension C5 Front wing fluttering C6 Push-ups C7 Hind wing fluttering C8 Front wing compression The exuvial phase D1 Front wing spreading D 2 Eating of the exuvia D3 Bending of the ovipositor D4 Hind wing spreading

Transcending this organization into both phases and motor programmes is another structuring of the behaviour into “bouts” of activity (Carlson and Bentley, 1977) which are separated by quiescent “interbout” periods. Each of the phases of ecdysial behaviour is a period where such bouts increase in frequency. Within each phase, the frequency of bouts tends to increase, peak, and then decline, but the bout frequency may vary irregularly in the short term within a single phase (Fig. 4).Evidently what is being modulated is theprobability that a bout will occur, rather than a rigid structuring of the pattern of bouts. The intensity with which any particular motor programme

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A

B 1

C D

Fig. 1 A typical ecdysial motor programme: the foreleg extraction motor programme from the adult ecdysis of Teieogryilus ocranicus. Recordings of action potentials in the two motor units of prothoracic muscle 71d resulting in leg extraction. In each record the upper trace is from the left muscle, and the lower from the right. ( A ) The complete output of the muscle during the ecdysial phase. ( B ) Expansion of (A), showing that the programme is divided into rhythmically occurring periods of activity (bouts). All concurrently active motor programmes have the same bout rhythm. (C) Expansion of ( B ) showing that a single bout consists of rhythmical bursts of action potentials, resulting in alternating contractions of the homologous muscles. (D) Expansion of (C) showing that a burst consists of multiple action potentials in two motor units. Calibrations: (A) 100 s; ( B ) 5 s; (C) 0.5 s; (D) 50 ms. (From Carlson and Bentley, 1977, copyright 1977 by the American Association for the Advancement of Science)

is performed within a bout is low when that programme is first recruited, but then rises, and often decreases again toward the end of its currency. However, in some cases certain programmes may be terminated abruptly within a single bout (of successful appendage extraction, for instance), indicating that sensory feedback must be important in controlling its expression. The extremely fine level of analysis employed by Carlson (1977a, b), dividing the insect’s behaviour into individual movements of body parts is obviously essential in order to identify those elements which are relatively fixed and those which are not. But the detail tends to obscure their overall co-ordination. To show this whole-animal level of organization, Carlson (1977a) illustrates the timing of a number of motor programmes which

STUART E . REYNOLDS

490 Preparatory

Expansional

Exuvial

1

2

A

D

I

2

'

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I

8

-9 10

11 12-

13 141516 - 71

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3 Fig. 2 The sequence of participation of motor programmes in a typical ecdysis of Teleogryllus oceanicm. The entire 4 h period of ecdysis is illustrated by the upper part of the figure (time

calibration, 10 min). Major phases are separated by dotted vertical lines. The ecdysial phase is illustrated below on an expanded timescale (calibration, 1 min). Horizontal bars represent durations, times of recruitment and termination for the 48 motor programmes which can be observed externally. The programmes are identified by numbers as in Table I. (From Carlson, 1977a)

occur together within bouts occurring at three points in the ecdysial phase (Fig. 3). It can be seen that such organization may take the form of waves of activity passing from the cerci through the abdomen to the thoracic segments, with appropriate, but different, muscular activity occurring in each segment (Fig. 3A); of bilateral coincidence or alternation (Fig. 3B); or of relatively complex coordination in time and space to provide coordinated movements of various appendages (Fig. 3C). Hughes (1980a) finds that the organization of the ecdysial behaviour of adult locusts (Schistocerca) is very similar to that Teleogryllus, although there are differences in detail. H e recognizes six stages in locust ecdysis (Table 2); however stages 1 and 6 include the quiescent behaviour which

491

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4

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3 1 2-

B7

[T 3 - T 2 1

T1-

-

1 I

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813,

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Fig. 3 Coordination of simultaneously active motor programmes during the adult ecdysis of Teieogryffus oceanicus. Each figure represents the activity of individual motor programmes during a single bout. (A) An early bout in the ecdysial phase, which shows longitudinal co-ordination. The solid bars represent activity in muscles of the left side only. The motor programmes are ( B l ) Abdominal peristalsis; (B2) cercal scissoring; (B7) Proximal leg segment extrication. A peristaltic wave culminates in the first two cycles of leg contractions. Numerals (2-8) refer to abdominal segment contractions. Leg contractions are designated by thoracic segment numbers; T,, metathorax; T,, metathorax; T, prothorax. (8)A bout occurring at the height of leg extrication, immediately preceding the freeing of the labrum. (B10) Antenna1 loosening; (B9) labral extrication; (B8) mandibular extrication; (B7) proximal leg segment extrication. (C) A bout illustrating motor programmes active during the early stages of antennal clasping, when the legs (B l l ) ,maxillae (B13) and the labrum (B 15) cooperate in the task of guiding the antennae toward the midline. The relevant motor programmes are (B 11) distal leg segment extrication; (B12) labial antennal clasping; (B13) antennal guiding; (B15) labial palp lowering. (From Carlson, 1977a)

occupy the 24 h periods before and after ecdysis respectively, so that effectively we are mostly concerned with stages 2-5. Stage 2 is recognizable pre-ecdysial behaviour and occupies up to 30 min. Following the splitting of the old cuticle, the timing of the next 3 stages is quite invariant, indicating a high degree of stereotypy and a probable central origin of the behaviour patterns employed. Like Carlson (1977a), Hughes (1980b) recognizes the principle of the

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270 I

1

Fig. 4 The motor programme, bout and phase organization of ecdysis in Tefeogryffus oceanicus (A) Bout frequency during an entire ecdysis. Each of the 4 phases of ecdysis corresponds to a period of activity during which the bout frequency rises and falls (the subpeak within the preparatory phase is not a consistent feature). (B) Expansion ( A ) to show bout frequency during the most active part of ecdysis. Also shown are sequences of vertical bars which represent the intensity of expression of eight different sample motor programmes. The programmes are numbered as in Table I. Each line represents a single bout, and its height signifies the number of separate muscle contractions (i.e. motor acts) in the bout. It can be seen that each motor programme has its own activity peak, independently of the changes of bout frequency. (From Carlson and Bentley, 1977, copyright 1977 by the American Association for the Advancement of Science)

organization of behaviour into bouts. However, unlike that of the cricket, the locust’s ecdysis is only divided into bouts during a part of the behaviour. Recognizable bouts first appear late in the pre-ecdysial behaviour of stage 2, and stage 3 is clearly organized into bouts. The expansional motor programme of stages 5 and 6 (see below) is also divided into bout-like periods, but Hughes ( 1 9 8 0 ~prefers ) not to use the term “bout” in describing these as it is not certain that the central mechanism underlying their organization is the same as that of the bouts of stage 3. Hughes (1980b) also distinguishes 3 phases within each bout in stage 3 (see Fig. 5), emphasizing the progression of behaviour from posterior to anterior. Carlson (1977a) does not subdivide the bouts occurring in Teleogryllus, but it is clear from Fig. 3 that such a subdivision could be made, separating abdominal and thoracic motor programmes, which d o not occur

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TABLE 2 Stages in the imaginal ecdysis of Schistocercagregaria, at 25"C, according to Hughes (1980a) Stage 1 Larva ceases t o feed, becomes quiescent, selects site for emergence Stage 2 Larva hangs from chosen perch, slowly inflates gut Stage 3 Gut inflation proceeds rapidly; old cuticle is split; head, thorax, pro- and mesothoracic legs emerge Stage 4 Metathoracic legs emerge; adult climbs old cuticle, pulls tip of abdomen free, selects site for expanding new cuticle Stage 5 Expansional stage; new cuticle stretched to adult size and shape during initial period of hardening. Wings expanded and folded Stage 6 Adult quiescent as new cuticle hardens, gut deflated

Duration approx. 24 h U p to 30 rnin 15.3 + 1.9 min

11.9 + 3.0 rnin 44.3

+ 6.0 min

Approx. 24 h

concurrently. Hughes also makes clear the distinction between the motor programme, an abstraction which represents the underlying organization of the particular behaviour which is identified, and the motor act which is its actual expression. A motor act is a single cycle of a repetitive motor one bout 1 ~

-2

-

1

:-=

3

first phase

second phase

third Dhase

contraction runs appendages. anteriorly along abdomen ventilation of tracheal gut inflation

Fig. 5 Functional organization of a single bout of behaviour during stage 3 of the imaginal ecdysis of Schistocercagregaria. The bout is divided into three phases, numbered 1 to 3. (From Hughes, 1980b)

programme. Each bout contains a number of individual motor acts belonging to different motor programmes; a particular motor act may occur more than once during a bout. Hughes (1980a) identifies fewer motor programmes in locust ecdysis than does Carlson (1977a) for the cricket, but this probably does not reflect the

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relative complexity of locust and cricket ecdysial behaviours. The mechanical constraints of the insects’ exoskeletons are such that many of the programmes are common to both species. Abdominal peristalsis (which Hughes calls the “waves of contraction” motor programme) is prominent in stage 3 of locust ecdysis, as it is in the ecdysial phase of ecdysis in Teleogryllus, for instance. On the other hand, the abdominal motor programmes which contribute to the expansion of the new cuticle after the insect emerges from the exuvia are quite different. In Teleogryllus, as the cerci clear the old cuticle, an abdominal tetanus is released which is maintained throughout the first half of the expansional phase, whereas inSchistocerca, stages 5 and 6 of ecdysis are characterized by the “expansional motor programme” of the abdomen, in which long, maintained compressions, with all the spiracles closed, are each punctuated briefly by a number of fast, ventilatory strokes, and by a deep inspiration, prior to another maintained compression (see Fig. 6). The expansional motor programme was first described by Miller and

10 5 Fig. 6 The expansional motor programme of Schistocerca gregaria. Activity is recorded from muscle L 176, which contracts during the tonic compression, and during the expiratory phase of each ventilatory stroke. Thus, the silent periods in the record indicate inspiration. Part of the record is shown on an expanded time scale, as indicated. (From Hughes, 1978)

Mills (1976), and is explored in more detail by Hughes ( 1 9 8 0 ~ )There . is good evidence of its central origin (Section 3.6). Like the cricket’s abdominal tetanus, the expansional motor programme only appears when the old cuticle is cast off. This strongly implies a role for sensory input to the CNS in its initiation. Hughes’ (1980a) division of locust ecdysial behaviour into stages relies on the occurrence of external markers, rather than on the waxing and waning of bout frequency, as does Carlson’s (1977a) division of cricket ecdysis into phases. Nevertheless, bout frequency in the locust is modulated during the behavioral sequence in a similar way to that in the cricket. The period of bouts in stage 3 falls to a minimum about 10 min after the cuticle is first

495

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split (Fig. 7), and the period of individual cycles of the expansional motor programme falls and then rises again during its currency in stages 4 and 5, being abruptly reset at the beginning of stage 6, as wing folding is completed (see Fig. 8).

- 15-

m

.

D

.

2 !

.. .. .

b 10a 5-

0-

r

I 5

I

I

10 Time from start of stage 3 ( rnin )

0

15

Fig. 7 Changes in the period of bouts of activity during stage 3 of a normal ecdysis in adult Schistocerca gregaria. The bout period falls to minimum (i.e. bout frequency is greatest) about 7-8 min after the beginning of stage 3. Subsequently, bout frequency falls, and bouts are initiated more and more irregularly. (From Hughes, 1978)

r/!

..

60

r -20

I

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I

I

I

I

I

I

I

I

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0

10

20

30

40

50

60

70

80

Time from start of stage 5 ( m i n )

Fig. 8 Changes in the period of the expansional motor programme during stages 4,5 and 6 of a normal ecdysis in adult Schistocerca gregaria. The period is long and irregular during the late part of stage 4,falling to a minimum at the onset of stage 5 . During stage 5 , the period progressively increases, being abruptly reset at the onset of stage 6. The pattern of this change in period length is not affected by sensory manipulation (see text). (From Hughes, 1978)

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As in Tefeogryffus,the intensity of expression of the motor programmes also rises and falls. For example, Hughes (1980d) investigated the airswallowing motor programme of the locust in some detail. H e found that sporadic swallowing movements occur throughout stage 2, but the rate of swallowing is greatly increased at the onset of stage 3, largely due to an increase in the number of air-swallowing acts per bout. The rate of swallowing rises to a maximum and then decreases again during the first phase of air-swallowing, which is modulated by sensory feedback from the foregut (see Section 3.6); with the emergence of the eyes from the old cuticle of the head, a second, continual phase of air-swallowing is initiated, which is no longer sensitive to sensory feedback. This also shows a gradual increase and decrease in frequency, which is due to changes in the number of motor acts per bout, rather than changes in bout frequency. 3.3

A D U L T ECLOSION IN LARGE M O T H S

In Saturniid and Sphingid moths, adult eclosion is triggered by a hormone, the eclosion hormone (see Section 5.1). In Hyalophora cecropia, at least the basic features of the behaviour are known to be centrally programmed (see Section 3.6). The behaviour associated with escape from the pupal cuticle is described by Truman (1971a) and Truman and Sokolove (1972). The movements of the abdomen have received most attention, since this part of the insect may be isolated and caused to perform the normal behaviour pattern when injected with eclosion hormone, at least in H . cecropia. A detailed comparison of the hormonally induced behaviour in intact, isolated abdomens and of the pattern of nervous activity elicited by the hormone in completely isolated abdominal nervous systems in vitro, has been given by Truman (1978). The behaviour pattern consists of 3 distinct phases: (i) a hyperactive period (approx. 0.5 h) which involves abdominal rotations and twitches. The pattern of movements is quite variable from animal to animal, as is the frequency of movements. (ii) a quiescent phase (approx. 0.5 h) which is also somewhat variable. During this period, activity ranges from a rduced frequency of abdominal rotations and twitches to a complete cessation of movement. (iii) a second hyperactive phase in which movements consist primarily of strong, peristaltic waves directed anteriad. Although earlier publications described the entire sequence of behaviour as the “pre-eclosion behaviour” (e.g. Truman and Sokolove, 1972) more recently, the first (early) hyperactive period, together with the quiescent period, have been called the “pre-eclosion behaviour”, and the second (late) hyperactive period has been termed the “eclosion behaviour” (Truman,

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1978). This terminology is more in keeping with other authors’ descriptions of ecdysial behaviour (see above), and emphasizes the fact the pre-eclosion and eclosion behaviour patterns, although both triggered by the same hormone appear to have separate neural origins (Truman, 1978). The central origin of these behaviour patterns will be further discussed below (Section 3.6). In silkmoths other than H . cecropia, the behaviour associated with adult eclosion is not so highly stereotyped (Truman, 1971a). In both Antheraea pernyi and A . polyphernus the initiation of the pre-eclosion behaviour (terms as above) is ill-defined, and no quiescent phase intervenes between pre-eclosion and eclosion behaviour. Instead, the frequency of movements increases gradually to a maximum at the point when the pupal cuticle is split. Truman’s accounts of pre-eclosion and eclosion behaviour in silkmoths deal principally with abdominal movements. However, there are important thoracic movements too. These are “shrugging” movements of the wing bases which in eclosion behaviour are co-ordinated with the forward movement of the peristaltic wave into the thorax from the abdomen (we have seen this progression before in the accounts by Bernays, 1972b, and Carlson, 1977a, respectively). These thoracic movements can be quite violent, and probably play an important part in splitting the pupal cuticle. These movements are analagous to those described by Bernays (1972b) in the first instar locust. The pupal cuticle splits longitudinally along the dorsal thorax as far forward as the facial mask, which is pushed out of the way as the moth creeps forward by means of the peristaltic movements of its abdomen. The legs are hardly used at all in this movement. In the Sphingid, Manduca sexta, on the other hand, the situation is rather different. Here there is no regularly recognizable pre-eclosion behaviour, although occasional typical eclosion movements may occur singly when the moth is nearly ready to eclose, particularly if the insect is disturbed. When eclosion movements begin, usually no more than 3 or 4 cycles of movement are required to split the pupal cuticle, and a few more to complete the moth’s escape. Each cycle of movement consists of an intense (peristaltic) wave of contraction which passes forward along the abdomen rather rapidly. When the wave reaches the thorax, the wing bases shrug violently and the legs push strongly downward. These shrugging movements have been studied using electrophysiological techniques by Kammer and Kinnamon (1977). The motor pattern is characterized by alternating activity in the flight muscles and increasing numbers of muscle potentials per burst in successive cycles of wing movements. The pattern of activity is quite different from that of flight. Instead of splitting longitudinally along the dorsal thorax, as in the Saturniids, in M . sexfa the pupal cuticle is broken across the top of the proboscis and around an enlarged facial mask which includes the cuticle over the adult

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STUART E. REYNOLDS

legs and antennae. Once the cuticle is split, the legs push off this mask and begin to scrabble at the substrate. At the same time, the peristaltic movements of the abdomen continue, and by a combination of abdominal peristaltsis and walking forward, the moth leaves the exuvia behind. When the moth is above ground, peristaltic movements continue for a few cycles as the newly-emerged moth walks away from the cast pupal case. In Manduca, digging normally follows immediately on escape from the pupal cuticle, since under natural conditions the pupae are buried in the soil. Digging consists largely of alternating scrabbling movements with the forelegs, together with a dorsal arching of the abdomen, pushing backward with its tip. This movement of the abdomen is quite distinct from the peristaltic contractions seen during escape from the old cuticle. The silkmoths, on the other hand, are not buried, but their pupae are encased in silken cocoons. If. cecropia, like many other Saturniids, has an escape valve built into the structure of the cocoon through which it can creep (Van der Kloot and Williams, 1953).Ahtheraea spp., however, do not have such an easy option, and instead use an enzyme, cocoonase, to digest an opening through which an escape can be made. The enzyme is secreted a few days before eclosion as a crystalline solid on the surface of the galea, and is activated, subsequent to eclosion, by the secretion of an alkaline buffer from the labial glands (Kafatos and Williams, 1964). When their pupae have previously been removed from the cocoon, the emerging adults of A. pernyi creep out of the old pupal cuticle soon after it has been split, but A. polyphernus becomes quiescent after splitting the exuvia, and only makes its escape after a delay of 1-2 h (Truman, 1971a). During this time, the labial glands are secreting fluid, and the delay presumably allows the cocoonase to d o its work. In A . pernyi this delay is not “built in” to the eclosion behaviour programme, but when the emerging adult meets an obstacle to its escape from the pupal cuticle, as it does in the cocoon, then the moth ceases to struggle and waits for the cocoon to be digested. Manduca and other Sphingids do not produce a labial gland secretion after eclosion. A characteristic action at this time, however, is the complete extension of the proboscis, which has been stored in the pupal case in a folded condition. At this time, the proboscis is extended right to its tip and is then coiled up in the normal adult position under the head (Truman and Endo, 1974). Having escaped from the soil or the cocoon, two further behaviour patterns remain before the moth achieves its final form. The first is termed by Truman (1971a) “post-ecdysis activity”. This relates to the searching-out of a suitable place to spread the wings. This behaviour can hardly be said to be stereotyped at all, except in so far as it is oriented to a very specific goal.

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Truman comments that the behaviour is “most easily influenced by the environment” of the various phases of eclosion-related behaviour. The moth may walk any distance from a few steps to a few metres. It may be that other factors in addition to environmental cues may influence this behaviour, since two animals, newly eclosed, may walk very different distances under apparently identical conditions before being satisfied as to a wing-spreading site. The moth’s main requirements for such a spot are that it be a vertical surface (or nearly so) and readily gripped by the prothoracic legs. During post-ecdysis activity, the moths are irritable and easily disturbed. Once wing-spreading behaviour has begun, the moths are relatively oblivious to external stimulation. Manduca never begins wing-spreading until a suitable site has been attained. Of the silkmoths, however, at least A. pernyi will usually begin to attempt wing-spreading after a few minutes, however unsuitable its location (even when confined in a glass vial, for example; Truman 1971a). This may well be related to differences in the control of bursicon release between the two moths (see Section 5.7). Wing-spreading behaviour in M. sexta is described by Truman and Endo (1974). Its onset is readily identifiable by the rotation of the wings, hitherto held close to the body, dorso-laterally away from it. Simultaneously, the abdomen assumes a taut, dorsally-flexed position which presumably functions to generate a high blood pressure, which in turn is responsible for wing-inflation (insects with their abdomens removed fail to inflate their wings). Shortly after this, the wings begin to increase in size. By 15 min after the start of wing-spreading behaviour, the wings are fully expanded and are held straight behind the body, dorsal faces together in the “butterfly position”. The wings are held like this for about 45 min, when they are rapidly rotated about their bases to assume the normal “moth position”, in which the wings rest in a tent-like fashion over the abdomen. Truman and Endo (1974) found the length of this behaviour pattern to vary very little, indicating a high degree of stereotypy. The form of the observable behaviour was constant despite a number of experimental treatments, although its total duration did seem to be influenced by sensory feedback from the wings, being prolonged when the wings did not expand or harden properly.

3.4

ADULT ECLOSION I N BLOWFLIES

Cyclorrhaphous Diptera emerge as adults from the puparium, a specially shaped and hardened protective container fashioned from the discarded cuticle of the third instar larva, in which they pupate (see Section 3.5). I do

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not know of any studies of pre-eclosion behaviour in pharate adult blowflies, but the behaviour at eclosion, and subsequently, has been described by Fraenkel (1935a), Laing (1935) and Cottrell (1962c, e). The peristaltic movements which Cottrell (1962c) observed during digging in Calliphora and in Sarcophaga, are probably very similar to those which are responsible for eclosion. Certainly, the ptilinum, a special eversible sac on the head which is evidently a “hatching” structure, is instrumental in causing the puparium to crack along its line of weakness, a longitudinal line around the anterior end, which meets a circular line extending around the anterior margin of what was the 4th visible segment of the larvae cuticle (Laing, 1935). In digging, the ptilinum is expanded by the force of blood pressure as the abdomen contracts. The contraction passes in a wave along the body and results in the insect shifting forward. In digging, both the ptilinum and the backward-pointing spines of the thorax act as anchors to prevent the body slipping back during abdominal contraction (Cottrell, 1 9 6 2 ~ )In . eclosion, the ptilinum must also act to concentrate the pressure generated by eclosion movements on the weak part of the puparium (see Section 4.1). Once the puparium has been broken, the fly surges forward using the digging action just described, scrambling free when its legs leave the confines of the puparial shell. Peristalsis ceases as soon as the legs are free, but buried flies will continue to dig for as long as 60 h (Cottrell, 1962e). Once free of the substrate, the flies enter into a sequence of behaviour which Cottrell (1962e) divides into 3 phases (see Fig. 9): ( i ) running (with intermittent cleaning) (ii) cleaning (with intermittent air-pumping) (iii) continuous air-pumping; expansion of the wings. Phase (iii) may be subdivided into: (iiia) increasing rate of air-pumping; rhythmic muscular efforts (iiib) decreasing rate of air-pumping; wing-straightening movements of the hind legs performed for the first time. Clearly phases (i ) and (ii) correspond to Truman’s (1971) “post-ecdysis activity” in silkmoths, and phase (iii) to Truman’s “wing-spreading be haviour” . Cottrell describes the cleaning movements of phases ( i ) and (ii) as being quite stereotyped and obviously different to those which will be used in later adult life. Air-swallowing begins erratically at first, but soon begins to increase rapidly in frequency (see Fig. 9). The proboscis is now held extended continuously, and from now on it is very difficult to disturb the fly. The wings begin to expand at this time, and become fully extended when the rate of

phase

phase

phase

phase

i

ii

iiia

iiib

150

100 rate of swallowing ( min-’1

5c

C 0

10

20

30

40

50

60

70

min

Fig. 9 Events during the expansion of newly-emerged adult b l o d i e s (Cufliphoru) at 22°C. (Modified from Cottrell, 1962e)

swallowing is at its highest. In this phase (iiia), air-swallowing is accompanied by “muscular efforts”. These are similar to the abdominal contractures which earlier would have produced eversion of the ptilinum. Now, however, eversion is prevented by the action of muscles in the head. These “efforts7’occur rhythmically at about 3 per min in Calliphora and 4 per min in Sarcophuga (Cottrell, 1962e). Cottrell ( 1 9 6 2 ~ )measured the internal pressure excess in normal flies and in flies with denervated abdomens. Normally, there is a gradual rise in pressure during phase (iiia)up to about 60 mm Hg in Caftiphora,90 mm Hg in Sarcophaga, which then falls during phase (iiib). Superimposed on this gradual increase is a series of brief pressure pulses of up to 30 mm Hg in size. These pulses do not appear in phase (iiib) after muscular “efforts” have ceased, nor do they occur in flies with denervated abdomens. These denervated flies fail to expand their wings properly, probably because the abdominal “efforts” are important in forcing blood forward from the abdomen, making it available for the expansion of the appendages. The ingestion of air results not only in the expansion of the wings but also in a tremendous increase in size of the whole insect, which is quite different in appearance when it emerges, from its later, more familiar, final form. Evans (1935) noted that Lucilia increased in size by 117% (males) and 133% (females). Fraenkel(1935a) found an increase in volume of 128%for

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an unsexed group of C‘ulliphora. More will be said of this increase in size below (see Sections 4.3 and 5.3). The swallowing of air and the expansion of the body and wings also mark the beginning of the process of cuticular hardening and darkening (see Section 5.4). 3.5

P U P A R I U M FORMATION I N B L O W F L I E S

This is an event in the life of Cyclorrhaphous flies which is not strictly an ecdysis, nor even a modified form of ecdysis. However, it has considerable relevance to the problems of physiological and behavioural integration being considered here. Fraenkel and his associates have studied puparium formation in Calliphoru and other blowflies for more than 40 years, and the study of the endocrinology of puparium formation (Fraenkel 1935b) has turned out to be of paramount importance to the whole of insect physiology. However, although the classical picture of puparium formation has stressed the importance of the moulting hormone, ecdysone, in initiating the process, it has recently become clear that this is not the only endocrine factor involved in coordinating the process (Fraenkel, 1975). Once ecdysone has acted to begin puparium formation, several distinct morphological events take place (Zdarek and Fraenkel, 1972): ( i ) the anterior and larval segments are irreversibly retracted; (ii) the body contracts by muscular action to about +of its original length; (iii) simultaneously with ( i i ) a longitudinal shrinkage occurs in the cuticle giving it a smooth surface; (iv) the cuticle now becomes stabilized, in the sense that it can no longer be stretched, and it no longer collapses if it is pricked; (v) tanning starts almost as soon as the smooth barrel shape of the puparium (“White puparium stage”) is attained. The whole process of transformation, from anterior retraction to white puparium stage, takes about 30-40 min at 22°C in Sarcophaga, Calliphoru and Phormiu. Tanning follows about 5-10 min after that. The process is broadly similar, corresponding to the above outline, in the first two genera. In Phormiu, however, contraction to the overall puparium shape takes place first, followed by the secretion of a drop of saliva, which is smeared over the ventral surface of the body. This is the salivary glue which fixes the puparium to the substrate in Phormia, Drosophila and some other Dipteran genera (Fraenkel and Brookes, 1953). The body wall is still folded at this stage. Now anterior retraction and cuticular shrinkage take place (3040 min) and tanning follows this after 5-10 min. A number of experiments with a variety of drugs have suggested that the processes of anterior retraction, cuticular shrinkage and cuticular tanning

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are separately and independently controlled, probably by neurosecretory hormones. (Zdarek and Fraenkel, 1972). These control systems will be further discussed below (Sections 5.1 and 5.4).

3.6

STEREOTYPY A N D PLASTICITY IN ECDYSIS

Truman and Sokolove (1972) and Carlson and Bentley (1977) have drawn attention to the behaviour shown by ecdysing insects as being an example of a long and quite complex sequence of behaviour which is apparently to a large extent pre-patterned within the nervous system. Not only are individual actions within the overall pattern of ecdysial behaviour highly stereotyped [i.e. conforming to the definition of a “fixed action pattern” (FAP; Hinde, 1970, p. 19)] but both sets of authors stress that the linking together of these individual acts into a complex whole is subject to “overall co-ordinating and controlling elements” within the CNS (Carlson and Bentley, 1977). Many of the observations of ecdysial and hatching behaviour detailed in Sections 3.1-3.5, above, are consistent with such an interpretation. It should be remembered that attributing a particular behaviour to the operation of an underlying central pattern generator does not deny that sensory input may be important in shaping the details of the way in which that behaviour is actually expressed. Also, whether or not particular component acts are relatively fixed, there may be higher levels of control which dictate the probability of those acts being performed. The details of the performance of this overall behavioural programme may be subject to modification by sensory feedback, just as is the case at lower levels. As we shall see, the evidence available suggests that whereas the performance of individual component behaviours patterns (“motor programmes”) is quite often subject to sensory modification, it appears that the overall behavioural programme is relatively fixed. An exception to this is where “behavioural switches” operate to terminate one part of an overall programme, and initiate the next. This is discussed in Section 3.7. T o the extent that the overall behavioural programme which we are postulating to exist in the CNS will still be subject at various levels in its hierarchy of control to the influences of behavioural switches and sensory feedback, it may be misleading to liken its operation to the “playing out” of a “motor tape” (Hoyle, 1970). In a striking metaphor, Carlson and Bentley (1977) point out the complexities of neural organization which must be involved in the control of ecdysis, referring to the “neural orchestration of a complex behavioural performance”. In this particular piece, it is evident that the score leaves the individual players some degree of freedom in the execution of their parts. Truman’s work on adult eclosion in the silkmoth, H . cecropia, provides

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the best evidence that not only are the individual component acts prepatterned within the CNS, but also the overall sequence of behaviour and the succession of the elements which comprise it. The behaviour displayed in intact animals is extremely stereotyped, and can be triggered in its complete form in isolated abdomens by injections of eclosion hormone (Truman, 1971a). In addition, a long and complex sequence of behavioural output, which is very similar in its organization to the behaviour seen in vivo, can be triggered by the eclosion hormone when applied to the abdominal CNS, either deafferented within the body (Truman and Sokolove, 1972) or completely isolated, in vitro (Truman, 1978; see also Fig. 10). It is clear from such results that a great deal of both the fine structure and the overall programme of silkmoth eclosion behaviour must arise within the CNS, and may be “released” by an appropriate signal (i.e. the eclosion hormone). The first hyperactive phase of pre-eclosion behaviour (rotatary movements), and eclosion behaviour itself (peristaltic movements) are each composed of repeated stereotyped movements which may be regarded as individual FAPs. Truman (1978) has analysed the temporal patterning of the whole sequence of behaviour, both in vivo and in vitro, and comes to the conclusion that the pre-eclosion behaviour (itself composed of a hyperactive period and a quiet period) and the eclosion behaviour are not merely different phases of the same overall behavioural programme, but represent quite separate programmes sequentially activated within the CNS by the action of the eclosion hormone. The main arguments for making this distinction are: (i) the temporal patterning of each of the two programmes is internally consistent, but that of eclosion behaviour cannot be predicted from the preceding pre-eclosion behaviour; (ii) the two programmes may be separated under certain circumstances (viz. when threshold doses of eclosion hormone are used, or when the moth’s abdomen is allowed to “age” after it has been isolated from the rest of the body). The patterning of the individual component acts of both pre-eclosion and eclosion behaviour (which appear as “bursts” in Truman’s suction electrode recordings from cut peripheral nerves) is very similar to that which must occur in vivo. However, sensory feedback does have an important influence on the way in which these component acts are put together into the overall pattern. Although the manner in which individual movements follow one another in the intact moth is very similar to that seen in the isolated abdomen and isolated abdominal CNS, the frequency with which the movements occur is not. This is particularly so for the eclosion behaviour. In an intact moth, the peristaltic movements of the abdomen follow one another rapidly

505

Fig. 10 The effect of adding eclosion hormone to the isolated abdominal nervous system of pharate adult Hyalophora cecropia. Records show the integrated motor activity from the nerves supplying the longitudinal intersegmental muscles. Lefi: Schematic diagram of the chain of abdominal ganglia, showing positions of motorneurones. Numbers refer t o electrode placements. Top: Complete integrated record of the motor activity recorded through electrode 1, showing the active and quiet phases of the pre-eclosion behaviour, followed by the eclosion behaviour. Eclosion hormone was added 22 min before the first burst on the record. Letters refer to bursts displayed in expanded form, below. Bottom: High-speed integrated records showing the fine structure of individual motor bursts as recorded simultaneously through the four electrodes. (A and B) Rotational bursts; (C and D) eclosion (peristaltic) bursts. (From Truman and Riddiford, 1977)

and cease soon after the insect emerges from the pupal skin. In an isolated abdomen, the waves occur at a reduced frequency and go on for much longer (an hour or so, usually). In the isolated abdominal CNS, peristaltic bursts continue for hours (apparently indefinitely) but at a very much reduced frequency (Truman, 1978). Evidently, sensory cues (perhaps from the pupal cuticle) are important in maintaining the frequency of contractions, and also in terminating the behaviour. Termination may be an aspect of behavioural “switching” from one central pattern generator to another, in Truman’s

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(1971a) analysis, eclosion behaviour being followed by “post-eclosion activity”. Higher centres, perhaps in the head, are probably important in this switching of behaviour. This will be discussed in Section 3.7. A very interesting feature of Truman’s in vitro work on the abdominal CNS of H . cecropia, is that the total lenth of the complete behavioural programme was found to be quite variable from preparation to preparation, usually being much longer than the behaviour in v i v o , although not invariably so (Truman, 1978). ‘The lengths of the individual rotatory bursts were not related to the length of the pre-eclosion behaviour within which they occurred, although the inter-burst intervals were. This argues that the pattern within the bursts is determined separately from the overall pattern of bursts within the behavioural programme. Truman is thus led to propose a hierarchical scheme for the organization of the two sequential behaviour patterns (Fig. 11). The scheme remains a model, of course, but explains Truman’s observations reasonably well. Carlson and Bentley (1977) also propose a hierarchical scheme to account

Time after hormone addition ( m i d

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Eclosion bursts

Fig. 11 Truman’s model of the central control of pre-eclosion and eclosion behaviour in adult Hyalophora cecropia. The behaviours are organized separately; in each case a burst timer drives an independent motor pattern generator to produce bursts of patterned activity. It i s envisaged that each burst timer is activated by eclosion hormone independently, and the succession of pre-eclosion and eclosion behaviours is produced by the different latencies of the respective neural programmes. The quiet period is directed by the pre-eclosion burst timer, and may represent a period of active inhibition of subsidiary pattern generators. As indicated, the site of this inhibition is not known. (From Truman, 1978)

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for adult ecdysis in T. oceanicus. Basing their analysis on detailed, simultaneous visual and electromyographic observations (Carlson, 1977a, b), they suggest that not only are the basic behavioural units of ecdysis centrally patterned, but also at least the broad outlines of the whole behavioural programme. The principal experimental evidence for this conclusion is that if the old larval cuticle is peeled from the pharate adult cricket during the early part of the preparatory phase of ecdysis, then the insect nevertheless goes on to perform a pantomime ecdysis which is in many ways normal, despite the absence of the old cuticle. The usual preparatory motor programmes (Section 3.2) are recruited in the correct order, at the right time, and the preparatory phase is followed by the ecdysial phase in the normal way. Carlson does not say if the behavioural programme progresses any further than this. When the metathoracic legs, abdomen and cerci are “peeled” in the ecdysial phase, motor programmes which would normally be xtivated as these structures emerged from the old cuticle, are not triggered immediately, but appear after a delay, indicating that these component acts are not reflex responses to sensory information, but are recruited centrally. In this case, behaviour carries on at least as far as to include an expansional phase. However, in these and other experiments, the sequence of behaviour was evidently not completed normally. Continuing sensory feedback must be necessary for the proper performance of the overall pattern of behaviour. Carlson (1977b) says, for instance, that peeling the abdomen and cerci at any time during the whole ecdysial behaviour will release a general abdominal tetanus, identical to that occurring in the expansional phase. This behaviour would normally appear just at the moment when the cerci clear the exuvia and it seems clear that this particular motor programme is integrated into the overall sequence of behaviour only with the aid of specific sensory mechanisms. Thus if the cerci and abdomen are peeled while the thorax and head are left covered, then abdominal tetanus is released and components of the ecdysial phase are omitted from the sequence. But if the head and thorax are peeled in addition to the abdomen and cerci, then no . abdominal tetanus is observed, and after a delay, the component acts of the ecdysial phase are recruited in their usual sequence. Evidently, abdominal tetanus is a reflex response which can be overridden. Thus in the cricket, as in the silkmoth, some aspects of the overall organization of ecdysial behaviour are apparently centrally patterned, while others require specific sensory cues for normal performance. While recognizing the importance of sensory contributions to the organization of ecdysial behaviour, Carlson and Bentley (1977) choose to emphasize the role of central programming. They propose a structure of ecdysial behaviour which involves the following elements:

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(i) sequential recruitment of component motor programmes, (ii) a “bout rhythm generator” which co-ordinates the activities of concurrently active component motor programmes, (iii) an overall modulation of the bout rhythm, dividing the behaviour as a whole into 4 phases, each with its own characteristic component motor programmes. Since no deafferentiation studies were done, it is not possible to comment on the reality of the large number of central pattern generators (motor programmes) proposed in (i). T o this reviewer, at least, it seems that at least some of the activities described (Table l ) , particularly those involved with the details of extracting appendages from the exuvia, might well be goaldirected behaviour rather than the result of rigid central patterning. Their apparent stereotypy could well be due to the mechanical constraints of the arthropod exoskeleton. Certainly, the recruitment of “back-up motor programmes” when initial attempts fail is suggestive of this. Nevertheless, it seems more than likely that Carlson and Bentley are right in identifying many of the individual movements of ecdysis as being centrally patterned. Carlson and Bentley’s control of elements (ii) and (iii) are, at present, based largely on inference from observation rather than experiment. Nevertheless, it is striking that their model is very similar to that of Truman (1978), in that it proposes that the CNS elements which generate particular motor acts are under the control of other elements which time the occurrence of bouts of activity. Truman’s model does not include any element comparible to (iii) in Carlson and Bentley’s scheme. Instead, he proposes that the succession of the silkmoth pre-eclosion and eclosion behaviours is governed by the differing latencies of the two behavioural programmes following their simultaneous activation by eclosion hormone. However, Truman’s (1978) paper only deals with a relatively restricted part of the overall ecdysial behaviour programme, and the progress of the programme beyond the escape of the moth from the old cuticle might well be organised on different principles. Hughes’ (1980a-d) observations of locust ecdysis are mostly compatible with Carlson and Bentley’s model. But a number of experimental manipulations of the process afford some insight into the nature of the “bout rhythm generator” in locusts, and also into the way in which different stages of ecdysis succeed one another. During stage 3 of locust ecdysis, immediately after the escape from the exuvia, behaviour is organised into bursts, which increase and then decrease in frequency. Hughes (1980b) found that an insect which became stuck in the old cuticle early in stage 3 did not show an increase in the frequency of bouts as normally occurs (Fig. 7), instead the bout period remained at the initial level throughout the period in which stage 3 would usually be com-

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pleted, eventually rising progressively as bouts were initiated more and more irregularly. On the other hand in another locust which became stuck late in stage 3 (during the extraction of the hind legs), the normal modulation of bout frequency occurred. It is evident that sensory feedback must be important in controlling the modulation of bout frequency during stage 3. Hughes localized the source of this sensory feedback, by interfering with the shedding of various regions of the exuvia. When the old cuticle over the thorax and abdomen was shed, but that over the head remained in place, the modulation of bout period was normal. However, when only the abdominal cuticle was allowed to be shed, the bout period remained constant throughout the normal duration of stage 3. In addition, where emergence of only the head was prevented, stage 3 was succeeded by stage 4 (expansional behaviour) in the usual way, but where the thorax remained in the old cuticle, the insect was “stuck” in stage 3, and the expansional motor programme was never recruited. This indicates that both the modulation of bout frequency, and the termination of stage 3 behaviour, are dependent on sensory information from receptors in the thorax. In this case, it does not seem as if an intrinsic programming element of Carlson and Bentley’s type (iii) is required to explain Hughes’ observations of bout frequency during locust stage 3 ecdysial behaviour. The same is true for the modulation of air-swallowing during the early part of stage 3, when the frequency of air-swallowing movements rises, largely due to an increase in the number of swallowing acts per bout (Hughes, 1980d). The frequency of swallowing reaches a peak some seven or eight minutes after stage 3 begins, and then falls again (see Fig. 12). During that period, the frequency of air-swallowing is apparently sensitive to sensory feedback. If the gut is experimentally inflated during rapid air-swallowing, the number of swallowing acts per bout is reduced, or swallowing may even be temporarily abolished. If the gut is deflated, then air-swallowing increases in frequency. The sensory information concerned in these responses arises from stretch receptors in the foregut wall, and is conveyed to the swallowing pattern generator via the outer oesophageal nerves of the stomatogastric nervous system. If these are cut, the air-swallowing programme “free runs” at a high rate, no longer subject to sensory modulation. During the later part of stage 3, about 15 min after stage 3 begins, air-swallowingincreases in frequency again, now apparently no longer responsive to sensory feedback. This transition occurs abruptly at about the time that the eyes emerge from the old cuticle of the head. Swallowing acts are now no longer restricted to the first phase of each bout, as they were during the earlier, modulated phase of air-swallowing, and the number of acts per bout increases rapidly until the head is pulled free from the exuvia, when swallowing frequency again declines (Fig. 12). During this “continuous

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phase” of the air-swallowing motor programme, the frequency of motor acts is apparently unaffected by sensory feedback of any kind. This phase of the programme bears great similarity to the “free running” of the swallowing programme which is produced by cutting both the outer oesophageal nerves

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Fig. 12 Air swallowing during the adult ecdysis of Schistocerca gregaria. The rate of air swallowing (continuous line) and the volume of air in the gut (dotted line) are shown diagrammatically. The rate of air swallowing was monitored as the number of contractions of muscle 37 per 20s interval. Each contraction represents a single motor act. Volume of air is shown as ml g-l body weight. (Modified from Hughes, 1980d)

and the neck connectives. These operations free the programme from the influences of sensory feedback, and of the “bout rhythm generator” (see below) respectively. The cessation of air-swallowing at the end of stage 3 depends on the final removal of the old cuticle. Pulling the insect free from the exuvia prematurely brings air-swallowing to an abrupt halt. This does not occur when the neck connectives have previously been severed, indicating that the necessary nervous information must be conveyed to the swallowing pattern generator via the ventral nerve cord. However, air-swallowing can subsequently be reinitiated, even in stage 4, by experimentally deflating the gut. Thus, the frequency of air-swallowing is modulated during the early part of stage 3 by the influence of sensory feedback, but in the second part of stage 3, frequency is determined by a central programming element of Carlson and Bentley’s type (iii). However, the expansional motor programme of phases 5 and 6 appears to be relatively unaffected by sensory feedback at any time (Hughes 1 9 8 0 ~ ) . Mechanical disturbance may result in occasional periods of exceptional

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length, but the normal course of change in period length (see Fig. 8) is unaffected by preventing expansion of the wings, either by waxing them in place immediately on emergence, or by preventing the shedding of the head cuticle, which in turn prevents the initiation of wing-spreading behaviour (see Section 3.7). The time course of changes in the expansional motor programme’s period length is also very similar in ecdysing fifth instar locusts, which do not, of course, have wings to expand. Severance of the connectives in the neck does not affect the normal decrease and subsequent increase in period length during stage 5 . Thus, in this case, we may infer that the modulation of period length of the expansional motor programme during stage 5 is likely to be centrally programmed, by an element of Carlson and Bentley’s type (iii). At the beginning of stage 6, when the expanded wings are refolded, the period of the expansional motor programme is abruptly reset. This does not occur in fifth instar locusts, but it does occur at the usual time in adult locusts which have been prevented from inflating their wings, even though these insects do not apparently move their wings at this time. This might indicate that the resetting of period at the start of stage 6 is also centrally programmed, but Hughes ( 1 9 8 0 ~ cautiously ) points out that movements of the wing bases may still have occurred even in those insects whose wings were waxed in the emergence position. If this were the case, such movements could provide a cue for the resetting of the expansional motor programme’s period. The origin of the bout rhythm of stage 3, and of the expansional motor programme of stages 5 and 6, has been shown to be in the metathoracic ganglion. Miller and Mills (1976) were able to record a pattern of activity in spiracular motorneurones which is characteristic of the expansional motor programme, even after the connectives on both sides of the ganglion had been severed. Hughes (1980b, c) confirms this, and also shows that the organization into bouts of motor acts which occurs in other parts of the body during stage 3, is dependent on the rhythm generated by this ganglion. When the ventral nerve cord behind the metathoracic ganglion was cut, the activity of a part of the abdominal waves of contract.ion motor programme was uncoupled from the bout rhythm (abdominal sequents 1 and 2 are not affected by this operation, as they are innervated by the metathoracic ganglion). When the nerve cord was cut between the meso- and metathoracic ganglia, anterior motor programmes such as air-swallowing and appendage extraction were expressed irregularly and were uncoupled from the normal bout structure. Evidently during a considerable part of locust ecdysis, the organization of behaviour into bouts is dependent on the activity of some organizing centre in the metathoracic ganglion. Hughes (1980b) envisages the presence of

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coordinating interneurones which run from that ganglion to the rest of the CNS, and which are responsible for the timing of the motor acts within each bout, and for the occurrence of the bouts themselves. Truman and Endo (1974) have investigated the extent to which wingspreading behaviour in eclosion of Munduca is dependent on sensory feedback. Two components of that behaviour were recognised; wing-folding (a series of wing movements produced by thoracic muscles) and wing-inflation (expansion of the wings to their full size by means of tonic contractions of the abdominal muscles, producing an elevated blood pressure). The extreme stereotypy of these two components suggests a central origin. Both are performed essentially unchanged even when sensory feedback is experimentally modified. Thus if the wings are clipped, tonic abdominal contracture persists and the moth exsanguinates itself. If the abdomen is ligated and removed, then wing-folding behaviour still occurs (it goes on longer than usual; see Section 5.1), even though without the abdomen, the blood pressure cannot be elevated, and the wings do not inflate. In both cases, once the behaviour has been initiated, decapitation has no effect on the behaviour’s performance (although the head is required for initiation of wing spreading behaviour; see Section 3.7). It seems likely that these two aspects of wingspreading behaviour in moths are centrally patterned within the thoracic CNS. Not only are the motor programmes which produce the necessary movements centrally determined, but also the times at which they occur. However, since no deafferentation studies have been done, it is necessary to be cautious in this conclusion. In particular, an experiment by Moreau (1974) suggests that in Bombyx, wing inflation behaviour may require proper sensory feedback for its expression. Whereas newly-emerged adult moths show a considerably elevated blood pressure during the period after eclosion, when wing inflation is taking place, individuals which have been made apterous by removing their wing imaginal discs during larval life, show n o sign of any attempt to raise their blood pressure. Partially apterous insects (i.e. in which only some of the wing discs have been ablated) show essentially normal behaviour, except that the expansion of the remaining wings is achieved in a rather shorter time than usual. Also Moreau and Bounhiol(l967) have shown that it is necessary for Bombyx to swallow some of its moulting fluid before eclosion, if wing inflation is subsequently to occur normally. They showed that the presence of fluid in the crop is the crucial factor in this requirement. Evidently, the performance of wing-spreading behaviour in this species is subject to at least some sensory constraints. A number of incidental observations in the literature also suggest that some of the behaviours associated with ecdysis may be centrally patterned. For instance, Fraenkel (1935a) reported that the expansion of the newly-

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emerged adult blowfly could be prevented by pricking the insect’s gut with a pin. Nevertheless, pharyngeal swallowing movements and abdominal pumping still occurred. The same was true of flies in which the proboscis had been blocked (Cottrell, 1962c), although in these experiments, the air-swallowing movements were found to go on a little longer than they normally would have done (45-55 min instead of 40-45 min; Cottrell, 1962d). In flies with blocked probosces, abdominal muscular efforts were made as usual, ceasing at about the same stage in the air-swallowing cycle as they normally would have done in an unoperated fly. Further, the pressure pulses resulting from these efforts decreased in size toward the end of the time in which they occurred, even though in this case, there was no progressive build-up of blood pressure due to air-swallowing. Thus, the cessation of abdominal muscular efforts must be centrally programmed rather than due to sensory feedback from stretch receptors. Nevertheless, it is obvious that many other components of ecdysial behaviour must be strongly influenced by sensory input. For instance, neither adult blowflies (Fraenkel, 1935a), adult Manduca (Truman, 1973a), nor newly-hatched first instar Schistocerca (Bernays, 1972b) will proceed to the next stage of ecdysial behaviour until they have freed themselves by digging from the substrate in which they emerged. When they do escape, they go on to perform activities which are strongly influenced by their surroundings. The blowfly spends some 10 min running, and intermittently cleaning, and then a further 10 min in more intense cleaning activity before beginning the more stereotyped behaviour of air-swallowing (Cottrell, 1962e). Similarly, Manduca spends some minutes in seeking out a suitable site on which to hang, before beginning wing-spreading behaviour (Truman and Endo, 1974). This phase of behaviour in Manduca may be greatly prolonged by keeping the insect moving on a flat surface (Reynolds; unpublished). Hatching behaviour, like that of ecdysis, is subject to both central and peripheral control mechanisms. Provine (1976a) described hatching movements in cockroaches (his “eclosion movements”) as “a stereotyped pattern of behaviour”. Sensory information is obviously an important factor in initiating hatching (see Section 2.1) and also in terminating it. Provine showed this by peeling the embryonic cuticle from the pharate first instar larva (which brought hatching movements to an end) and by gluing a cast skin onto the larva, so that it could not be shed (in this case, hatching movements persisted for an extended period of time). This is very like the situation in the adult ecdyses of Teleogryllus and Schistocerca. However, ligation experiments gave results which suggested that individual hatching movements were centrally patterned (Provine, 1976b, 1977). Neither the head nor the thorax was necessary for the initiation of individual hatching

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movements, although the interval between movements varied more in the insects without both head and thorax than it did in those without the head alone. Variation was even less in intact insects. Also the amplitude of the movements was less in the ligated insects. The conclusion from this study seems to be that the individual hatching movements are probably centrally patterned, with only the details of their execution subject to modification by sensory feedback, but that sensory information is important in the initiation and termination of behaviour. The work of Bernays on hatching in locusts, (1971; 1972a, b) in which individual motor acts were more thoroughly analysed, suggests that sensory input may play a more important role in the shaping of particular movements than Provine’s work indicates. For instance the length of phase (v) of the digging movements of a vermiform larva, which consists of a sustained dorso-ventral contraction (see Section 3.1), is very dependent on the nature of the surrounding medium. When digging is easy, this movement can disappear altogether, but when the going is difficult, it is greatly prolonged. Bernays’ descriptions suggest, in fact, that a single, basic, central pattern underlies the movements of hatching, digging, and the subsequent first larval ecdysis, being modified in each phase by the different sensory contexts in which they occur. Thus we must conclude that the control of ecdysial and hatching behaviour includes contributions from both central pattern generators and elements which are dependent on sensory feedback. In such a complex behavioural performance this can scarcely be surprising, but it is interesting that the overall programming of the behaviour (at the level of the control of bout frequency, and recruitment of motor programmes) relies so much on central elements. Determining the nature of these higher level control elements should prove an interesting challenge to insect neurobiologists. In identifying the metathoracic ganglion as the location of a centre controlling bout organization and coordination in Schistocerca, Miller and Mills (1976) and Hughes (1980b, c) have pointed the way for further studies. 3.7 B E H A V I O U R A L S W I T C H I N G I N E C D Y S I S Hatching and ecdysis provide a number of examples of sudden changes in behaviour which I shall call “behavioural switching”. This implies that the insect “chooses” to follow one of two or more alternative behavioural pathways. Camhi (1977) has used the term to describe the transformation of tactile reflexes during righting behaviour in the cockroach Gromphadorhina portentosa. In this example, the behavioural switch operates on each occasion that the cockroach’s legs lose contact with the ground, or regain it, but in

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ecdysis, the behavioural switches which I shall identify appear to operate on a long-term basis, either irreversibly or at least until the next ecdysis. Bernays (1971) reports that in locusts, the digging response to being buried, which is characteristic of the vermiform larva, disappeared soon after the first larval ecdysis. Newly ecdysed first instar larvae would resume digging movements, even though their efforts were now ineffectual. Larvae which had experienced five or more minutes of freedom, however, did not dig. Thus, “the behavioural response to the environment is in some way altered during the first few minutes after ecdysis”. It would be interesting to know how long the newly ecdysed larvae which did resume digging continued in this activity, and whether the loss of the ability to dig is a gradual or an all-or-none change. The club shaped hairs on the head and thorax of first-stage larvae of Schistocerca may well be involved in this response (Bernays, 1972a; Bernays, Cook and Padgham, 1976). Newly-emerged adult blowflies must also dig their way free of the substrate, and here too, there is a rather rapid loss of the digging response (Cottrell, 1 9 6 2 ~ )After . full wing extension (at about the time that abdominal muscular efforts cease), reburial will no longer elicit digging movements. Cottrell tested the possibility that the loss of the digging movements might be due to tanning of the cuticle by injecting “active” blood (i.e. containing bursicon) into a digging fly and causing premature sclerotisation. The digging reaction continued indicating that the loss of digging ability is triggered by some aspect of the normal performance of expansion and wing-inflation behaviour, rather than its physiological correlates. The muscles which are used in digging in blowflies degenerate within 2 days after eclosion (Section 5.11); however, it hardly seems likely that the extreme rapidity of the behavioural switching seen here can be due to the inability of the muscles to contract. The eclosion behaviour of Manduca provides another example of behavioural switching. Unlike H . cecropia, where the eclosion hormone can trigger eclosion behaviour in isolated abdomens (Section 5.1), in Manduca, the presence of the head is necessary for the eclosion hormone to be able to initiate eclosion behaviour. Performance of the behaviour does not follow hormone release immediately but some time later ( 1-2 h; Reynolds et al., 1979). At any time after the hormone has been released, however, decapitation triggers immediate performance of eclosion behaviour, presumably by the release of some kind of inhibition. Thus it seems as though a correlate of eclosion hormone release in Manduca is the operation of some kind of behavioural “switch” in the head, which activates the neural machinery necessary for eclosion behaviour. The performance of this behaviour is prevented, however, by an inhibitory centre in the head. Whether this inhibitory centre is also subject to eclosion hormone action is not known.

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Not the least interesting point about this particular behavioural switch is that it evidently acts a t a distance. The activation of the switch is evidently in the head, but the neural elements which are switched are in the thoracic and abdominal CNS (Reynolds and Truman, unpublished). A similar sequence of events involving a behavioural switch follows the escape of the moth from the pupal case. If the insect is decapitated immediately on emergence, then neither wing folding nor wing inflation can occur. However, if decapitation is delayed until after proboscis coiling ( 5 s after eclosion), then not only can both of these behaviours occur normally, but they d o so immediately, regardless of circumstances (Truman and Endo, 1974). Again it seems as though some sort of “switch” in the head is required to register that eclosion has been completed satisfactorily, and to activate the neural circuitry in the thorax which is responsible for the organization of the wing-spreading behaviour. As before, the actual performance of the behaviour is normally inhibited by a centre in the head until suitable circumstances for wing-spreading have been found. Interestingly, Truman and Endo’s experiments showed that the two component behaviour patterns of wing-spreading are not “switched on” at exactly the same moment, since decapitation after eclosion, but before proboscis-coiling (i.e. 1-5 s) allowed wing-folding, but not wing-inflation to occur. Presumably, the “switch” which activates these behaviours is normally activated by sensory cues signalling the end of confinement. Moreau (1973) has observed the operation of a similar “switch” in the adult eclosion behaviour of Bombyx mori. Bilateral section of connectives between thorax and head did not prevent eclosion, but did prevent wing inflation. Unilateral connective section did not prevent wing inflation, so that the operation of the cephalic “switch” can evidently be signalled to the rest of the CNS via only one of the two connectives. In the Lepidopteran examples discussed, inflation behaviour is delayed for a variable period after emergence from the old cuticle, so that it has been possible to examine the working of the behavioural switch experimentally. In the Orthopterans Teleogryllus and Schistocerca, inflation behaviour follows immediately on escape from the exuvia; nevertheless there are indications that a behavioural switch may be at work here too. Carlson (1977a) found that abdominal tetanus (which generates elevated blood pressure for expansion) was released immediately the cerci cleared the exuvia, even if this was the result of “peeling” the abdomen prematurely. Only if the whole exuvia was peeled could the occurrence of premature abdominal tetanus be avoided. The transition from the ecdysial to the expansional phase of ecdysis must be dependent on a particular set of sensory cues, in addition to the operation of an internal programme. Hughes ( 1 9 8 0 ~ )also finds that the expansional motor programme of

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locust ecdysis is released by specific sensory cues, resulting in this case from the emergence of the thorax early in stage 3 , well before expansion behaviour begins. If removal of the thoracic cuticle is prevented, then stage 3 is prolonged and stage 4 never begins. Another “switch” which operates during stage 3 appears to utilise sensory cues from the emergence of the head from the old head cuticle as a trigger. If this is prevented, then the second, continual phase of air-swallowing never occurs, and wing inflation does not occur. Interestingly this operation also prevents hardening of the cuticle. Evidently the initiation of continual air-swallowing, wing-spreading and the release of bursicon may all be triggered by the same sensory cues, just as we have seen in Calfiphora and Manduca. An interesting aspect of the eclosion behaviour of moths is that eclosion and the behaviour patterns associated with it can only be triggered by the eclosion hormone once (Truman, unpublished). Evidently either the interaction of the hormone with the nervous system, as perhaps the performance of the behaviour itself, in some way prevents reinitiation of the behaviour by eclosion hormone. It is possible that either receptors for the hormone disappear, or that the mechanism whereby the behaviour is activated is altered; at the moment there is no evidence to help decide between these alternatives. Interestingly, it appears that under some circumstances individual components of eclosion behaviour may be exhibited without activation by eclosion hormone. For instance, components of eclosion behaviour were seen in 84% of developing pharate adult A . pernyi peeled on day 16 of adult development (3 days prior to the normal time of eclosion). These components of eclosion behaviour are not organized into their proper sequence at this time, however (Truman, 1976). Kammer and Kinnamon (1977) report being able to trigger “eclosion behaviour” in Manduca on several days both before and after eclosion, by restraining the insects. In our experience, however (Truman, Taghert and Reynolds, unpublished) the normal and complete sequence of eclosion behaviour cannot be triggered at any other time than at eclosion. Another aspect of ecdysial behaviour which strongly indicates the operattion of one or more central nervous “switches” is the changeover from pupal to adult behaviour patterns which occurs at eclosion in Lepidoptera. Prior to this event, the pharate adult moth behaves like a pupa, showing few of the behaviour patterns which characterize the adult. This is true even when the confining pupal cuticle is removed (Blest, 1960; Truman, 1971a, 1976). However, the entire repertoire of adult behaviour patterns is subsequently established within a span of a few hours at the time of eclosion. Apparently the eclosion hormone, in addition to triggering the performance of the behaviour involved in escaping from the pupal cuticle, also “turns on” the whole range of typically adult behaviour patterns which the moth henceforth

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shows, and “turns off” the pupal activities which have up to this time characterized its behaviour (Truman, 1971a, 1973c, 1976). Although one of the functions of the eclosion hormone is to initiate cell death (Section 5.1 l) , both in the peripheral musculature and in the CNS, these degenerative changes are much too slow to account for the rapid behavioural switching seen here. In fact, adult behaviours do not appear in the newly-eclosed adult moth completely without precedent. Blest (1960) showed in Automeris memusae that parts of the adult rocking display and flight behaviour could be elicited from peeled pharate adults; Kammer and Rheuben (1976) have shown that both the pre-flight warm-up (shivering) and flight motor patterns can be recorded from developing A . polyphemus and A . pernyi, still within their pupal cases; and Truman (1976) has observed that a number of adult behaviours (wing flapping, walking, leg extension, leg grasping, tarsi on substrate) may be seen in peeled adults of H . cecropia on the last day before eclosion, though at a low level in most cases. Peeled A . pernyi were much less active, but some moths still showed adult behaviours at a very low level. The response to injections of eclosion hormone in Truman’s (1976) study was dramatic. Whereas, before injection of the hormone, peeled A . pernyi showed no walking behaviour at all, and H . cecropia spent on average only 5% of their time walking, after treatment with eclosion hormone had elicited a pantomime eclosion, the insects spent 77% and 51% of their time, respectively, in locomotary behaviour. Interestingly, between the injection of the hormone and the onset of pre-eclosion behaviour, those moths which had previously been active now showed much reduced levels of activity (e.g. in H . cecropia, time spent wing-flapping was reduced from 26% of the time to only 5% of the time). The fact that these behaviours can occur (though at a low level) indicates that the neural elements from which the behaviour arises are already present in the developing adult some time before the behaviour is normally displayed. In the case of the flight motor patterns recorded by Kammer and Rheuben (1976), motor output is generated, even though the moth is still enclosed in the pupal cuticle and the behaviour cannot thus be of any “use”. Such observations are not confined to Lepidoptera. Motor output patterns typical of singing can be recorded from larval crickets, Teleogyrllus commodus, which do not yet have the necessary cuticular structures for sound production (Bentley and Hoy, 1970), and rhythmic firing of flight motorneurones develops during larval life both in T. commodus (Bentley and Hoy, 1970) and inSchistocercagregaria (Kutsch, 1971). Although these motor patterns are essentially complete in the last instar, they are not normally expressed. However, larval crickets can be induced to “sing” prematurely by lesions in certain, presumed inhibitory, regions of the brain

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(Huber, 1960; Bentley and Hoy, 1970). The conclusion from these experiments is that the central pattern generator for cricket singing is normally subject to central inhibition, originating in the brain, which suppresses its output until a suitable time (i.e. until after adult ecdysis). In the case of the pharate adult moths, discussed above, a similar scheme is supposed by Truman (1976) to apply. In Automeris, Blest (1960) found that display behaviour could no longer be elicited on the day before eclosion, and Truman (1976) found that in A . pernyi, the eclosion movements which are triggered by peeling on days 15-18 of adult development, disappeared entirely on the day on which eclosion occurs (day 19). Kammer and Kinnamon (1977) likewise report that in Manduca, eclosion movements can be triggered by peeling 8-12 h prior to eclosion, but were much less frequent 5-7 h before eclosion. Again the interpretation favoured is that the central organization appropriate to these behaviour patterns is already complete some time before eclosion, but is centrally inhibited. Truman (1976) supported this hypothesis by showing that injections of picrotoxin (an antagonist of the inhibitory neurotransmitter, y-aminobutyric acid, could release the performance of an adult behaviour pattern (walking) in A . pernyi, which normally never shows such behaviour on day 19. Although Truman points out the actions of injected picrotoxin, and of eclosion hormone, have little in common other than the fact that both result in the moth eventually showing walking behaviour, he nevertheless goes on to suggest that eclosion hormone might activate adult behaviour patterns by removing them from tonic central inhibition. This is a reasonable hypothesis, but it has yet to be suitably tested. Thus behavioural switching “on” of behaviour patterns at ecdysis may involve a release from central inhibition. The way in which other behaviour patterns are switched “off” is unknown.

4 The mechanics of ecdysis

4.1

S P L I T T I N G ‘THEO L D C U T I C L E

Patterns of ecdysial movement have been discussed above (Section 3). These contribute to the bursting of the old cuticle in the following ways.

4.1.1 General aj7plication of pressure This results from an increase in the volume of the pharate insect, as it swallows air. This is important for cockroaches (Eidmann, 1924); locusts (Duarte, 1939; Clarke, 1956; Bernays, 1972c; Allum, 1973; Hughes,

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1980d); adult dragonflies (de Bellesme, 1877; Brocher, 1919) and many other species. Aquatic larvae may swallow water; e.g. dragon flies (Shafer, 1923); Dyriscus (Brunck, 1923), and mosquitoes (Wigglesworth, 1938). Hinton (1946) has described how in those Endopterygote pupae which have a sealed mouth, and thus cannot swallow air directly, air may nevertheless be able to enter the space between the pupal and adult cuticles via pupal spiracles which are not connected to adult tracheae. This air can be swallowed by the pharate adult. On the other hand, many insects do not swallow air until after the old cuticle is split; e.g. adult blowflies (Cottrell, 1962e); adult Pieris (Cottrell, 1964). Before hatching, the pharate larva may swallow extraembryonic fluid in order to increase its bulk (Sikes and Wigglesworth, 1931; Bernays, 1972a), but this would not result in the exertion of any more pressure on the surrounding eggshell. It seems unlikely that air-swallowing can be very important until the chorion has already been pierced, although Sikes and Wiggleworth (1931) say that larvae of the louse Pofypfax may swallow air which diffuses through it, once the serosal cuticle has been cut by hatching spines on the head. Air-swallowing is important though, for the shedding of the embryonic cuticle, which is often coincident with the escape from the eggshell. Thus Cimex swallows air as it emerges from the egg, and is consequently able to undergo its 1st larval ecdysis before it has fully escaped from the chorion (Sikes & Wigglesworth (1931). O n the other hand, Schisrocerca hatches without swallowing air, and defers this until it undergoes its 1st larval ecdysis at the surface (Bernays, 1972b). The absorption of water from the environment may cause the egg contents to have a slight positive pressure prior to hatching (Bernays, 1972a; Lees, 1976) and doubtless this is of some slight assistance in hatching. But it cannot be a crucial factor in hatching in Schisrocerca, since eggs which had lost water, and whose shells were visibly flaccid, were nevertheless able to hatch normally (Bernays, 1972a). Sikes and Wigglesworth (1931) found no evidence that the growth of the larva within the shell results in an increased internal pressure as had been suggested by Heymons (1926). The extent of the internal pressure excess which resulted from preecdysial water-swallowing in dragonflies was measured by Shafer (1923) using a water-filled capillary tube. He estimated a value of about 65 mm of water at the peak. However, it is likely that this method would have given low values. Bernays (1972b) measured blood pressure during the 1st larval ecdysis of Schistocercu and found that the maximum values occurred just about the time of splitting the old cuticle, varying between 55 and 65 mm Hg. During the post-ecdysial air swallowing of adult dragonflies Shafer (1923) recorded a pressure of 75 mm H,O. In adult blowflies, the baseline

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pressure (i.e. that due to air-swallowing alone) reaches values of 60 to 95 mm Hg (Cottrell, 1962c) and in Bombyx mori and Pieris brassicae, it reaches 40 to 50 mm Hg and 20 to 30 mm Hg respectively (Moreau, 1974). The development of a high blood pressure need not be correlated with airor water-swallowing. S16ma (1976) found that pressure built up from 50 mm H,O to 300 mm H20 during the 3 h prior to pupal ecdysis in Dermestes vulpinus, increasing suddenly to a maximum of about 400 mm H,O about one min before the larval cuticle was ruptured. Neither change is due to air swallowing, but is due to muscular action. 4.1.2

Local application of pressure

This is probably of paramount importance in most cases. Even where there is a general increase in blood pressure due to air-swallowing, it is likely that this pressure is concentrated by muscular action so as to act at the ecdysial line(s) of weakness in the old cuticle. In ecdysing 1st instar locusts, the maximum internal pressure excess measured by Bernays (1972b) was about 60 mm Hg. Yet injections of saline solution causing artificial pressures of up to 170 mm Hg failed to split the embryonic cuticle. Even higher pressures caused the injected insects to burst at various points, but not in the middorsal thoracic position of the ecdysial line. The ecdysial line is a region of the integument where the exocuticle is thinner than usual. Underneath, the exocuticle may be thicker than it is elsewhere to compensate for this (Shelton, 1979), but of course prior to ecdysis the endocuticle is digested and resorbed, leaving a line of weakness. In many cases this is longitudinal and in the midline of the dorsal thorax, but this is by.no means universal. For instance soft bodied larvae have their ecdysial line in the hard cuticle of the head capsule. The puFarium of blowflies splits at the anterior end along both circumferential and longitudinal lateral lines. This leaves a substantial hole through which the adult can escape. In Manduca the adult emerges by pushing off the pupal cuticle overlying the head and ventral thorax using its legs. Subsidiary ecdysial lines may exist in the head capsule (e.g. Hinton, 1963). Eggshells also have hatching lines of weakness. Their positions are very variable. Even when they are present, they may not be functional (e.g. Hinton and Cole, 1965). Where the ecdysial line is in the standard, dorsal, longitudinal position, this would appear t o be good mechanical sense. An increase in internal pressure will exert its maximum effect so as to pull the cuticle away from the line of weakness (the circumferential tension in the walls of a cylindrical pressure vessel is twice that in the longitudinal direction). However, there are other constraints too. It is generally essential to ecdysial success that the

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exuvia be left whole, so that the location of the ecdysial split must allow the appendages to be drawn out of their exuvial sheaths at the same time as the insect emerges from the rest of the cuticle. This means that the dorsal thorax is in most cases the only possible choice. The position in which the ecdysial line develops appears to be determined by a morphogenetic gradient within the integument (Shelton, 1979). The movements which result in the concentration of pressure on the ecdysial line mostly appear to be derived from a general peristalsis. This may be more or less evident on observation (see Section 3 for descriptions). The same is true for hatching. These movements tend to produce a forward movement of the body relative to the old cuticle so that pressure is concentrated on anterior structures. Bernays (1972a) comments that in locusts hatching from the egg, continued hatching efforts cause the pharate larva to move forward in the egg. She considers that this forward movement must stretch the eggshell above the thorax, and that this stretching may well explain why the eggshell is normally split transversely above the thorax, instead of longitudinally as would be predicted if the split were due to pressure alone (when eggs were inflated artificially, they did indeed split in the long axis). There is no apparent line of weakness in the eggshell of Schistocerca. In this case, the application of pressure to the eggshell in this region is probably facilitated by special hatching structures, the cervical ampullae of the pharate 1st instar larvae, which underly the position of the split (Bernays, 1972a). These are also important in digging, when they act as an anchor. (Bernays, 1971). This emphasizes the similarity of the behavioural patterns underlying hatching and digging. On the other hand, Bernays (1972b) rejects the suggestion that the ampullae might be important in the 1st larval ecdysis. The forces exerted locally must be considerably greater than those due to the general pressure increase following air-swallowing. Obviously it is difficult to measure local tensions within the cuticle. Cottrell ( 1 9 6 2 ~ re) corded intrathoracic pressures in newly-emerged adult blowflies. In digging flies, pressure pulses could be as large as 100 mm Hg. Since Cottrell(l964) considers that the mechanism of escape from the puparium involves similar movements to those of digging, the magnitude of the pressures exerted during eclosion must be at least as great, perhaps greater, since the puparium’s wall will be less deformable than the adult integument. The puparium is split over the ptilinum; Cottrell ( 1 9 6 2 ~ found ) that the forces exerted by the ptilinum of digging flies enclosed in a capillary tube could vary between 9 and 23 g. These were calculated to correspond to pressures of 50 to 140 mm Hg, suggesting that the pressure within the ptilinum was probably about the same as that in the thorax. Bernays (1972a) performed an experiment to test the force required to burst a locust eggshell, and con-

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cluded that pressures in the order of 28 g cm-* (about 20 mm Hg) must be exerted by the hatching larva,

4.1.3 Special cutting structures In some cases, the eggshell is cut, rather than burst, by the hatching larva. A wide variety of special structures for the purpose (“egg bursters”) has been described (e.g. Sikes and Wigglesworth, 1931; Van Emden, 1946; Southwood, 1956). although it is by no means certain that these are always functional. They are generally on the head, and may be a part of the embryonic cuticle, or of the underlying 1st instar cuticle. They take the form of hooks, spines, etc., sometimes mounted on protrusible membranes. In some cases, the mouthparts may be used to break the eggshell. For instance, the hatching larva of the fly Lucilia sericata uses its mouth hooks partly as a piston, to break the chorion at its weak point, and then as a cutting tool to pierce the vitelline membrane (Sikes and Wigglesworth, 1931). Lepidopteran larvae eat their way out of their eggshells, and according to David and Gardiner (1962), newly hatched larvae of Pieris may eat the tops from neighbouring eggshells, saving the larvae inside the trouble of doing so themselves. Hinton and Cole (1965) report that larvae of the cabbage root fly, Erioischia brassicae, bite through their eggshells, despite the provision in the latter of hatching lines. Such mechanical aids to tearing the exuvia do not seem to be so much used in ecdysis, although they are usually employed where the pharate adult escapes from a cell or cocoon before rather than after eclosion. In species with decticous pupae, the pupal mandibles may be used by the pharate adult to cut through the cocoon. Where the pupa is adecticous, a “cocoon cutter” on the head is often used to pierce the walls of the cocoon (Hinton, 1946).

4.2

ESCAPING FROM THE OLD CUTICLE

Having split the exuvia, the next task is to escape from it. It seems clear that the peristaltic movements which are the main propulsive force for this, are derived from the same basic motor programme as those employed in the preceding phase, though their detailed form may be modified by the changing sensory feedback as the insect escapes (see Section 3). However, not all of the escape may rely on peristaltic movements. Shafer (1923) says that in larval dragonflies, the anterior motion of the body relative to the old cuticle is caused by the expansion of the abdomen, which lengthens as the insect swallows water. The extrication of some of the appendages may be to a large extent

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passive, following from the foward movement of the body due to peristalsis (e.g. extraction of the wings and antennae at the adult ecdysis in Schistocerca; Hughes, 1980), but generally, the mouthparts and the legs assist in gaining their own freedom by various retraction, adduction or abduction movements. Carlson (1977a) describes a number of motor programmes which are normally associated with leg extraction in Teleogryllus, and also some additional “back-up” patterns of behaviour which are only recruited when things go wrong. Hughes (1980a) also notes the existence of such “back-up” patterns of behaviour used only by Schistocerca when the extraction of the long, metathoracic jumping legs proves difficult. The forward movement of the body during escape from the old cuticle is probably facilitated by the existence of various devices which prevent backward slippage. For instance, Hinton (1946) attributes just such a function to the backward-pointing spines which are common on the abdominal cuticle of endopterygote pupae. They only occur in those species which escape from the pupation cell o r cocoon, as the pharate adult, still encased in the pupal cuticle. O n a more microscopic scale Bernays (1972b) has described areas of the cuticle of the 1st instar larvae of Schistocerca which are apparently specially modified to prevent forward movement of the overlying exuvia during the 1st larval ecdysis. In these areas, which are distributed in patches, laterally on each abdominal segment, and which are larger in the posterior segments, the cuticular plates produced by individual epidermal cells are each armed with a backward facing spine. The ventral surfaces of the paraprocts are also covered with similar plates, which bear larger spines than elsewhere. Sculpturing of the epicuticle is quite general in insects, most commonly consisting of a pattern of polygonal plates, each corresponding to the area of cuticle secreted by a single epidermal cell (Hinton, 1970). The spined plates described above are a modification t o the basic pattern in which spines are not present. Such a pattern is present on the embryonic cuticle of crickets, Achetu domesticus, and McFarlane (1 962) has suggested that the resulting irregularity of the cuticle surface might help to reduce adhesion between the embryonic cuticle and the chorion during hatching. Presumably a similar function would be served in ecdysis. Bernays (1972a) suggested that what is left at the time of hatching of the extra-embryonic fluid might function as a lubricant, easing the exit of the larva from the eggshell. Bernays (1972b) describes small, heavily sclerotised knobs, one at the base of each cercus, in 1st instar larvae of Schistocerca. They are covered with spines which are directed forward and ventrally. These structures, the brustia, are prehardened before hatching, and appear to be specially designed to assist in gathering up the embryonic cuticle during the 1st larval ecdysis. The old cuticle is gripped between the brustia and the 10th

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abdominal tergite. With each cycle of abdominal peristalsis, the embryonic cuticle is drawn back and collects in front of the cerci, which are held erect during ecdysis. During each cycle, when the special muscles connecting the 10th tergite with the base of each cercus relax, high blood pressure forces the brustia and the 10th segment apart, and allows the new fold of cuticle to be gathered up with those already in this region. With the start of the next peristaltic wave, the muscles contract, and the new fold is trapped. The muscles associated with the brustia degenerate following the 1st larval ecdysis, but the brustia themselves do not disappear in subsequent instars. Bernays (1972b) suggests that the fact that only the 1st larval ecdysis takes place on the ground may explain this. At an subsequent ecdyses, the insect hangs from a perch, and emerges to hang upside down by the tip of the abdomen from the old cuticle. The old abdominal cuticle is still gathered up by the brustia, but now gravity and the weight of the newly emerged locust, hanging supported by its brustia, are sufficient to hcid it in place (Hughes, 1980b). 4.3

INFLATING THE N E W CUTICLE

All the activities described above which effect the splitting of the old cuticle by exerting pressure on the body wall, will also at the same time tend to expand the new cuticle which lies folded up beneath the old. Indeed in most cases it is likely that the inflation of the new cuticle is already substantially accomplished by the time the insect has escaped from the exuvia. Bernays ( 1 9 7 2 ~ measured ) femur length during the 1st larval ecdysis of Schistocerca and found that more than 90% of the increase in length which takes place at this time occurs during the 5 minutes over which the escape took place. I have measured tibia1 lengths from photographs of ecdysing adult locusts, and could detect no increase in length once the leg had emerged from the 5th instar cuticle (unpublished). An interesting exception occurs immediately after the 1st larval ecdysis, or intermediate moult, of the bugs Cirnex and Rhodnius (Sikes & Wigglesworth, 1931). Here, immediately after shedding the embryonic cuticle, the larva swallows large quantities of air, and at the same time contracts dorso-ventral muscles, to assume its characteristic size and shape. However, some body parts, in particular the wings, do remain to be expanded. These may be unfolded to some extent during the process of pulling free of the old cuticle of the wing-buds, but remain substantially uninflated at the moment of emergence. In adult female crickets (Tefeogryllus), the ovipositor valves also remain to be inflated (Carlson, 1977a), and in adult dragonflies the abdomen lengthens considerably during the 2.5 h after the larval cuticle is shed (Brocher, 1919).

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The wings (and other structures) may be inflated immediately, as in locusts (Hughes, 1980a) or after a variable delay, as in blowflies (Fraenkel, 1935a) and in Manducu (Truman, 1973a). In these species, the delay is associated with the time taken to dig free of the substrate in which the pupae were buried. Not all endopterygotes wait until they escape from the cell or cocoon, however, before inflating their wings. Hinton (1946) points out that a number of Dipteran families, and all Hymenoptera, Strepsiptera and Coleoptera, inflate their wings within the cocoon or pupation cell, only emerging when their cuticle is sufficiently tanned. Obviously in these cases, the insect must take the precaution of constructing a cocoon or cell which is sufficiently large to allow this. In locusts, the wings are inflated by a special “expansional” motor programme (Hughes, 1980c) which serves to fill the tracheal system maximally with air, and then to compress both the closed tracheae and the air-filled gut, so that the resulting internal pressure excess is transmitted to the wings (and other structures) by the blood. Interestingly, this motor programme is first recruited during the preceding stage 4, when the locust is still hanging upside down from the tip of its abdomen, and during which the wings do not become inflated. This must mean that the increased blood pressure is not transmitted to the wings at this time, perhaps because the blood flow is occluded when the wings are folded. Certainly in locusts, as in other insects (e.g. Manduca; Truman and Endo, 1974), the wings are held in a characteristic position during their inflation, probably to ensure that blood pressure is adequately transmitted to the wing veins. During wing expansion in locusts, the abdominal movements of the expansional motor programme are accompanied by wing movements which serve to spread out the expanding wings (Hughes, 1980a). These movements may ensure the proper distribution of blood in the wings during the inflation process. In Manduca (Truman and Endo, 1974) the abdomen contracts tetanically to generate pressure, rather than cyclically as in the locust. Here, wing movements do not occur during inflation. It is not entirely clear from Carlson’s (1977a) description, whether wing movements occur during the expansion process in Teleogryllus, which also has an abdominal tetanus at this time; it would seem that such movements are reserved until after completion of expansion, just before wing-folding. However, movements of the ovipositor valves, which may help in their expansion, are described. Moreau and Lavenseau (1975) have described changes in the rate of beating both of the abdominal dorsal vessel (the heart) and of the accessory thoracic hearts at the bases of the wings, which accompany the expansion of the wings in Bombyx (see Section 5.9). The increased rate of beating of the dorsal vessel might be envisaged to assist in pumping blood into the wings,

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but since the accessory hearts at the wing bases pump bloodout of the wings, some other explanation must be found for their increased activity at this time. During expansion, the wings come to contain a considerable amount of blood, which must be removed when expansion is complete (Moreau, 1973). It is unlikely that the fluid “evaporates” (Kroon et a f . , 1952), and the accessory hearts may help in the blood‘s removal. Also their activity will help to ensure that blood in the wings actually circulates during inflation, rather than merely accumulating. This is important as the haemolymph carries tanning precursors and hormones which are required for physiological processes going on at this time in the wings. Specifically, eclosion hormone and bursicon both control wing cuticle extensibility (see Section 5.3); bursicon controls both cuticle tanning (Section 5.4) and the disaggregation of the epidermal cell processes which connect the upper and lower surfaces of the wings (Section 5.11). In the above account of the mechanics of wing inflation it was supposed that the only force causing cuticle unfolding is the application of pressure to the blood space within the wing veins. This would cause each vein to be blown up like a long cylindrical balloon. The behaviour of the wing during expansion appears to be in accord with this idea, being progressively inflated starting at the end closest to the articulation with the thorax and ending with the inflation of the wing tip. As all who have attended Christmas parties will know, long, thin balloons blow up in just this way. However, it should be pointed out that the situation may not be so simple as this. In particular, the pressure within the wing veins may not be uniform all over the wing. If the pressure in veins at the proximal end of the wing were greater than in those at the distal end, this would also lead to the balloon-like inflation observed. In addition, Glaser and Vincent (1979) have recently observed that if wings are removed from adult locusts in the act of ecdysing, then these wings will expand on their own, without the benefit of elevated blood pressure from abdominal contractions. I have observed a similar phenomenon in excised wings of M. sexta and A . pernyi after injection of the wings with bursicon. Significantly, in Glaser and Vincent’s experiments with Locustu, wings would probably also have been exposed to bursicon, since the hormone is released in this species as the head emerges from the old cuticle, (Hughes, 1978). In the case of Munduca, however, I have never seen excised wings become fully inflated, only partially so. A possible explanation of autoinflation may be that bursicon acts on the wing epidermis to cause tanning, which not only causes molecular crosslinking of cuticle proteins, but also results in cuticle dehydration and shrinkage. Estimates of cuticle shrinkage during tanning vary. In Sarcophaga

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larval, puparium formation results in a 36% decrease in surface area, with no change in thickness (Fraenkel and Rudall, 1940). In Schistocerca 1st instar hoppers, tanning resulted in a 25% decrease in thickness in femur cuticle (Bernays, 1 9 7 2 ~ ) .At any rate, it is clear that shrinkage does accompany tanning, and it could well be that this shrinkage could cause a decrease in the diameter of the wing veins, compressing the blood within them. In turn, t h e blood could cause straightening of cuticle folds and inflation of the wing; the extent to which this would proceed would depend on the extent of the cuticle shrinkage, and the extent to which local increases in blood pressure could be relieved by bleeding. Glaser and Vincent (1979) found that autoinflation of Locustu wings was inhibited by cyanide. This would also inhibit tanning, and thus cuticle shrinkage. More investigation is obviously needed. The relative contributions of cuticle unfolding and cuticle stretchingper se remain to be properly worked out. Bernays ( 1 9 7 2 ~considers ) that unfolding can account for all 01 the observed increase in length of the metathoracic femur of ecdysing 1st instar locusts. The ratio of unfolded: folded lengths decreases during ecdysis from 1.36 to 1.06during ecdysis, and falls very little further in the next few hours. I have measured changes in the linear dimensions of the wings of A . pernyi during adult eclosion (unpublished), and find that different parts of the wing change in their size during inflation to quite markedly different extents. In the proximal region of the costal edge of the fore wing, the ratio oi the inflated : uninflated distance between two prominent markers was 2 . 0 2 2 0.11 (n = 11). In the same region, microscopic examination of sections of fresh material showed that the unfolded : folded ratio for the length of wing vein cuticle was 2.40 ? 0.42 (n = lo), which suggests that the increase in size of the wing, in this region, can be accounted for in terms of unfolding alone. This is not to say the wing cuticle cannot stretch. The same region of the wing ofA. pernyi can show an increase of between 2.5 to 3.0 times its starting length when subjected to a modest maintained load (3.5 g). The maximum value of the strain achieved is the same whether the wing is plasticized or unplasticized (see Section 5.3). In the former case, then this limit to the wing's extensibility is achieved more quickly, and with a smaller imposed stress. There are cases where an increase in linear dimensions of the cuticle can be attributed to stretching per se, however. The most notable example is probably the huge increase in size of the abdominal cuticle of Rhodnius which occurs when the insect feeds (Bennet-Clark, 1962), but an example related to ecdysis is provided by the stretching of the body cuticle of newly-emerged adult blowflies (Cottrell, 1962d). Here the body cuticle is not appreciably folded, and the considerable increases in linear dimensions

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observed in life or during artificial inflations, (in the order of 30%; Cottrell, 1962c; Reynolds, 1976) must be due to stretching. In Rhodnius, the limit to which the cuticle can be stretched is set by the unfolding of the highly sculptured epicuticle, and not by the stretching of the underlying procuticle, which is not folded (Bennet-Clark, 1963). It was proposed that this might be a principle generally applicable to the increase in size which occurs at ecdysis. The ultimate size of an insect during any instar would be set by the size of the epicuticle laid down immediately after the preceding apolysis. Since it seems that cuticle stretching per se does not occur all over the body during ecdysis this would not always be the case, as proposed by Bennet-Clark, but the relationship might well hold in those cases where stretching did occur. A possible restraint on cuticle stretching neglected by this hypothesis, however, is the limit to extension which might be imposed by the chitin microfibres within the cuticle. The orientation of these must change when the cuticle is stretched more in one surface dimension than in another, as has been discussed by Neville (1967,1975, p. 298). If the initial arrangement of microfibres is helicoidal, in the plane of the cuticle surface, then a unidirectional strain will re-orient them so that there is a net orientation in the axis of the imposed stress. As this occurs, the extensibility of the cuticle is likely to decrease; very markedly so when the extent of reorientation of the microfibres approaches its logical limit of complete orientation in the stress axis (Wainwrightetal., 1976; p. 165). This could set a limit on the extent to which the cuticle could be stretchedin vivo. Evidence that substantial re-orientation of chitin does actually occur is available from X-ray diffraction (e.g. in blowfly puparium formation; data of Fraenkel and Rudall, 1947, reinterpreted by Neville, 1975, p. 299) and from electron microscopy (e.g. during the post-ecdysial terminalial inversion of Aedes aegypti; Chevone and Richards, 1977). Whether or not such secondary re-orientation is responsible in these cases for limiting the extent of cuticle stretching is unknown. Incidentally it is worthwhile pointing out here that the well known results of Kroon et a f . (1952), often quoted as showing that secondary re-orientation of chitin occurs along the length of wing-veins following inflation in the butterfly, Aglais urticae, in fact d o nothing of the kind. Since all the chitin microfibres in the cuticle lie in the plane of the surface, when viewed edge-on the walls of the wing-veins will appear to contain chitin microfibres oriented along their length, even when their orientation is in fact helicoidal within the cuticle. All that the “reorientation” seen in Kroon et a l . ’ ~X-ray diffraction pictures implies, is that the orientation of the veins has changed, as would be expected. Previously highly folded (i.e. “random”), the walls of the veins are now unfolded (i.e. “unidirectional”)! Hepburn and Levy (1975) found that samples of soft larval cuticles from a

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beetle (Agenius zebra) and a silkworm (Bombyx mori) showed a decrease in extensibility during successive cycles of tensile testing of mechanical properties. Additional data on femoral cuticle from newly ecdysed locusts (Locusra migratoria) was presented in Hepburn and Chandler (1976). Such a change during hysteresis testing is termed “work hardening”. It was proposed that this might be of significance during ecdysis. A soft body cuticle which could be made more rigid by work-hardening during and prior to ecdysis would provide greater purchase for the application of hydrostatic pressure against the old cuticle’s ecdysial line. However, in a subsequent report (Hepburn and Chandler, 1976b) the previous findings of work-hardening were refuted. Apparently the cuticle samples tested had lost water during the testing procedure. Glaser and Vincent (1979) report that locust wings become less extensible as they autoinflate, and reiterate the suggestion that the expanding cuticle might be subject to a form of strain-hardening. However, in this case it is possible that bursicon mediated tanning may be occurring during the inflation process (see above), and at present it seems likely that work-hardening does not play an important part in stabilizing the new cuticle as it expands.

5

5.1

Ecdysial physiology and its integration with behaviour I N I T I A T I O NA N D T E R M I N A T I O N O F B E H A V I O U R P A T T E R N S

5.1.1 Eclosion hormone As we saw in Section 2, provided the insect is “ready”, hatching or ecdysial behaviour may be initiated by environmental cues, or by endogenous factors, such as the operation of a circadian clock. In many cases it may well be that the behaviour is initiated directly by “command” elements within the CNS (see Kennedy (1976) for a discussion of command cells in invertebrate nervous systems), but at least in the case of the Saturniid moths, H . cecropia and A . pernyi (Truman and Riddiford, 1970), it is clear that ecdysial behaviour is triggered by the release of a neurosecretory hormone into the blood Ecdysial behaviour is probably largely programmed within the CNS, but once initiated, the details of its performance may be more or less influenced by sensory feedback according to the species, and which part of the behaviour is being considered (see Section 3.6 for a discussion of this). The exceptional stereotypy of the abdominal movements involved in the preeclosion and eclosion behaviour of H . cecropia (Truman, 1971a) and the confinement of eclosion to a narrow, temporal “gate” by a circadian clock in the brain (Truman and Riddiford, 1970) were key factors which led to the

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discovery of the role of the eclosion hormone in initiating eclosion be haviour . As well as in H . cecropia and A . pernyi, ecdysial behaviour is known to be released by eclosion hormone in the Sphingid, M . sexta (Truman, 1973a). Little is known about the possible endocrine initiation of ecdysis in other insects. Truman (1973b) tested for the presence of eclosion hormone in the brains and CC/CA complexes of a cockroach (Leucophaea maderue) and a Hemipteran (Pyrrhocoris apterae), but failed to find any activity using the A . pernyi assay. But of course, a hormone which triggered ecdysis in these insects might well have been inactive in a Lepidopteran bioassay. Carlson (1977a) and Miller and Mills (1976) have reported preliminary experiments which suggest that neuroendocrine factors might possibly be involved in triggering ecdysial behaviour in crickets and locusts, respectively. However, details of these experiments have not yet been given. In the dragonfly, Aeshna cyanea, destruction of the neurosecretory cells of the anterior protocerebrum blocks ecdysis in insects otherwise “ready” to proceed, and Charlet and Schaller (1976) have suggested that these cells may secrete an eclosion hormone-like factor which initiates ecdysis. Outside the insects, Maissat and Graf (1973) and Charmentier and Trilles (1976) have proposed that an “exuviation factor” might initiate ecdysis in Isopod Crustaceans. The release of this factor is apparently inhibited by ecdysone. A number of authors have reported that hatching or ecdysis is accompanied by the discharge of neurosecretory materials. For instance Khan and Fraser (1962) found that there was a decrease in stainable material in the CC at hatching in Periplaneta americana. Steel and Harmsen (1971) noted an apparent reduction in PAF positive material in median neurosecretory cells of the brain at, or just before, the time of ecdysis in Rhodnius. It was suggested that this material was discharged from the CC. Of course, this is not to say that the neurosecretory materials which are apparently liberated must be instrumental in triggering ecdysial behaviour patterns. As we shall see, there are many other physiological functions occurring at this time, and in many insects bursicon, the tanning hormone, is known to be released just at the moment of ecdysis. It is possible that additional, unknown hormones may also be released at this time. The evidence that silkmoth eclosion behaviour is initiated directly by eclosion hormone is as follows. H . cecropia and A. pernyi emerge from the pupal cuticle only at a particular time of day, during relatively narrow gates determined by a circadian clock (Truman and Riddiford, 1970). If the pharate adults are removed from their pupal cases (i.e. “peeled”) during the few hours before the normal eclosion gate, then they continue to show predominantly pupal behaviour. In the case of A . pernyi this is particularly striking, the peeled moth showing very little spontaneous motor activity at

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all (Truman, 1971a, 1976b). However, at the time of the normal eclosion gate, the moths go through a pantomime emergence, despite having been free of the pupal case for several hours. Truman (1971a) showed that the presence of the brain was necessary for a normal sequence of eclosion behaviour to occur. When the brain was removed surgically from pupae, or early in adult development, the resulting brainless adults did not show the normal eclosion behaviour. But if the connectives to the brain were cut and the brain reimplanted in the abdomen, then behaviour occurred normally, and at the right time of day. Thus, the brain must control eclosion behaviour hormonally. This was confirmed by injecting extracts of brains and CC/CA complexes into pharate adults a few hours before the normal eclosion gate. These produced premature eclosion, the pre-eclosion behaviour following within a few minutes of injection. The extracts must have contained a hormone, which Truman named the “eclosion hormone”. It was subsequently shown (Truman, 1973b) that this hormone is in fact present in the blood of eclosing adult moths, a further piece of evidence that eclosion hormone actually initiates ecdysial behaviour in vivo. That the hormone released by the brain serves only as a trigger, and does not influence the detailed form of the behaviour displayed, was shown by transplanting brains reciprocally between H . cecropia and A . pernyi. When A . pernyi received a brain from H . cecropia, then it eclosed during the H . cecropia gate, but nevertheless showed typical A. pernyi behaviour during eclosion. On the other hand, in H . cecropia, an A . pernyi brain triggered eclosion behaviour typical of the host, but during the donor’s eclosion gate (Truman, 1971b). This ingenious experiment not only showed that the eclosion behaviour must be patterned within the host’s CNS, other than the brain, but also showed that the circadian clock, which controls the eclosion hormone’s release, must be located in the brain. It should be noted that brainless silkmoths can still escape from their pupal cuticle despite the absence of eclosion hormone (Truman and Riddiford, 1974b), though rather few show typical pre-eclosion and eclosion behaviour (Truman, 1971a), and none go on to inflate thei.r wings (Truman and Riddiford, 1974b). This is not due to the absence of proper nervous connections between the brain and the rest of the CNS, since brainless moths with an implanted brain in the abdomen perform these activities normally, but must be due to the absence of eclosion hormone. Apparently, without the hormone, some parts of the behaviour associated with eclosion can appear spontaneously, but are not integrated into their proper context. The initial parts of the behavioural sequence, the pre-eclosion and eclosion behaviours (see Section 3.3 for descriptions), are patterned entirely within the abdominal CNS. Isolated abdomens of H . cecropia respond to

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injected eclosion hormone by going through the normal sequence of preeclosion and eclosion behaviour in an essentially normal fashion (Truman, 1971a). The behaviour is expressed more or less independently of sensory feedback, as was shown by Truman and Sokolove (1972) when they recorded spontaneousmotor activity from the deafferentedCNS ofH . cecropia. The application of eclosion hormone-containing extracts of CC/CA complexes resulted in the performance of the normal sequence of behaviour (see Section 3.6). “Behaviour” is here, of course, interpreted as the patterning of bursts of action potentials in cut motor nerves. In these experiments, the deafferented abdominal CNS was left in situ, but recent improvements in technique have allowed the H . cecropia abdominal CNS to be completely isolated, and eclosion behaviour elicited in vitro. (Truman, 1978). Since purified eclosion hormone still triggers normal pre-eclosion and eclosion behaviour under these conditions (Reynolds and Truman, 1980), it follows that there can now be no reasonable doubt that, at least in the abdominal CNS of H. cecropia, the eclosion hormone acts directly on the CNS to cause the initiation of ecdysial behaviour. The evidence for M . sexta is less complete, and it appears that the action of the eclosion hormone in initiating eclosion behaviour in this insect may not be quite the same as in H . cecropia. Eclosion hormone is present in the brain and CC of M . sexta (Truman, 1973b) just as it is in the Saturniids. The hormone is released into the blood during a brief period some hours before eclosion (Reynolds e t a l . , 1979), and its peak concentration is considerably in excess of that which is known to be able to elicit premature eclosion in injected animals (Reynolds, 1977), but this is dependent on the adult being “ready” to respond to the hormone. Responsiveness to eclosion hormone does not develop in M . sexta until about 4 h before the time at which the hormone would normally be released (Reynolds et al., 1979). Since the acquisition of responsiveness is a gated event, this implies that prior exposure to another, unidentified co-ordinating hormone may be necessary. This is quite different to the situation in the Saturniids, where responsiveness to eclosion hormone can develop at any time during the previous 24 h. M . sexta differs from H. cecropia in another respect. Whereas, in H . cecropia, essentially normal eclosion behaviour can be triggered in isolated abdomens, this is not the case in M . sexta. The presence of the head at the time of eclosion hormone injection is necessary for the exogenous hormone to induce eclosion behaviour, even though the behaviour can subsequently be performed in its absence. This is further discussed in Section 3.7. The mode of action of the eclosion hormone on the CNS is not well understood. The hormone is a peptide, of approximately 8500 D molecular weight (Reynolds and Truman, 1980). Whether the hormone has many or

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few target cells in the CNS is not known. Exposure of the isolated abdominal CNS o f H .cecropia to the eclosion hormone for only a few minutes can result in the subsequent performance of the complete sequence of pre-eclosion and eclosion hehaviours, so that the hormone must act as a trigger rather than a modulator of behaviour (Truman, 1978). There is evidence that this action of the eclosion hormone is mediated by an increase in the level of cyclic 3’,5’-guanosine monophosphate (cGMP) in the CNS (Truman et al., 1980). Injection of a number of cyclic nucleotides can mimic the effects on behaviour of the eclosion hormone, but cGMP is by far the most effective. When substantially purified preparations of eclosion hormone were used to trigger eclosion behaviour, the content of cGMP in the abdominal CNS was found to increase, attaining a peak value about 15 min after injection which was more than twice the resting level. By 30 min after hormone injection, cGMP content had fallen to the starting level. This is consistent with a trigger role for the hormone. The use of purified eclosion hormone in these experiments is important. In previous workon the mechanism of hormone action, Trumanetal. (1976) had used crude CC/CA extracts to trigger eclosion. These extracts were found to cause a massive increase in the CNS content of CAMP,and this was misinterpreted as evidence for a role for cAMP in the mediation of eclosion hormone action. Using purified hormone preparations, Trumanet al. (1980) did not observe any change in the level of cAMP in the CNS, while the much smaller increase in the level of cGMP remained. The original observation of an increase in cAMP in the CNS must have been due to the presence of some other hormonally active material in the CC/CA extracts, which has an effect on the CNS which is unconnected with changes in behaviour. 5.1.2 Bursicon In blowflies (Cottrell, 1962a; Fraenkel and Hsaio, 1962) and in M. sexta (Truman, 1973a) bursicon is not released until expansion behaviour begins. Its release is extremely rapid. In collecting blood samples from blowflies at the beginning of inflation behaviour, I have found that blood taken from flies which have just begun swallowing air, invariably shows bursicon activity (unpublished). In M . sexta, the blood titre of bursicon reaches a maximum in less than 10 min. once the moth comes to rest on a vertical surface (Reynolds et al., 1979). However, there is no suggestion here that the release of bursicon is responsible for the initiation of expansion behaviour; rather the hormone’s release may be regarded as being an early event in the expansional behaviour pattern. Fraenkel and Hsaio (1965) found that airpumping and bursicon release could be experimentally dissociated. When the connectives in the neck were severed the great majority of flies still

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initiated air-swallowing, although they did not release bursicon. A small number of flies released bursicon, but did not pump. In M . sexta, Truman and Endo (1974) found that abdomenless moths (which cannot release bursicon) nevertheless attempted to inflate their wings, indicating that the hormome cannot be responsible for the initiation of this behaviour. In adult cockroaches (Srivavista and Hopkins, 1977) and in 1st instar locusts (Padgham, 1976a) bursicon is released within a few minutes, at most, of the old cuticle being split. In neither case was bursicon activity observed in pre-ecdysial insects, so that bursicon is unlikely to have a role in initiating ecdysial behaviour here. On the other hand, although bursicon may not initiate behaviour, there is some evidence that it may be involved in terminating behaviour, although perhaps indirectly. Truman & Endo (1974) found that in abdomenless moths, the duration of wing-spreading behaviour was greater than normal. However, if extracts of the abdominal nerve cord (which contain bursicon) were injected into such moths, then the duration of the behaviour was reduced, although it was still significantly longer than usual. This effect could be due to a direct action of bursicon on the CNS, but it is perhaps more likely that the hormone is acting by causing the wing cuticle to tan, and that it is sensory information from receptors in the hardening cuticle which act to terminate wing-spreading behaviour. 5.1.3 ARF The “Anterior Retraction Factor” (ARF) of blowfly larvae is a neurosecretory hormone which initiates the irreversible withdrawal of the three most anterior segments of the larva (Fraenkel et al., 1972). This piece of behaviour is normally the first event in the complex sequence of changes which accompany the formation of the puparium (Zdarek and Fraenkel, 1972; see Section 3.5). Unfortunately very little is known about the way in which this hormone acts to initiate behaviour, or about the details of the behaviour itself. Unlike the puparial tanning factor (PTF) which initiates tanning in fly larvae, its action does not seem to involve any effects on transcription or translation (Seligman et al., 1977), although the release of ARF can apparently be prevented by inhibitors of translation (delayed release) and transcription (complete prevention of release). CAMP seems not tobe involvedin the behaviouralresponse to ARF(Fraenkeletal., 1977).

5.2

M O D I F I C A T I O N OF B E H A V I O U R PATTERNS B E F O R E A N D AFTER

ECDYSIS

The approach of ecdysis may often be recognized by a change in the insect’s behaviour. Locusts become “restless” and begin searching for a suitable

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spot for ecdysis. They also stop feeding, and their ability to make escape jumps is much impaired (Hughes, 1980a). Caterpillars stop feeding, and some may construct silken holdfasts. Final instar endopterygote larvae commonly leave their food and “wander”, subsequently constructing a cocoon, or burying themselves and making a pupation cell. Hoyle (1956) found that the tension generated by a single twitch in the extensor tibiae of the locust’s jumping leg was reduced at and around the time of ecdysis. This could account for the reduced jumping power of locusts at this time. It was argued that this reduced efficiency of synaptic transmission was caused by the markedly higher activity of K+ ions in the locust’s blood around the time of ecdysis. The concentrations of Na and K found in the haemolymph could cause a similarly reduced twitch size when an artificial saline with this composition was used to bathe the muscle. A similar reduction in the efficiency of synaptic transmission at the neuromuscular junction (n.m.j.) has been described by Kammer and Kinnamon (1979) for the flight muscles of M . sextu. In the pharate adult, up until about 20-30 h before eclosion, the performance of “practice” flight motor rhythms does not result in movements of the flight muscles. The impulses in the motor nerves give rise only to small psps which do not initiate muscle contractions. However, more prolonged bursts of motor impulses give rise to larger psps which do give rise to contractile activity. This explains how it is possible to stimulate immature insects to eclose prematurely (Kammer and Kinnamon, 1977; see Section 3.7), despite the low efficiency of synaptic transmission at the n.m.j. Of course in this case, the efficiency of synaptic transmission is not reduced only just before ecdysis as in the locust, but is maintained at this low level during adult development within the pupa. Kammer and Kinnamon do not speculate on possible causes, ionic or otherwise. However, other factors are also involved in the suppression of activity which would be undesirable in the pre-ecdysial insect. Haskell and Moorhouse (1963) discovered that a blood-borne substance, which was tentatively identified with ecdysone, could depress the level of spontaneous motorneurone activity in the isolated CNS of locusts. The target site of the factor is unknown. Since this factor was present in the blood of pre-ecdysial locusts (as ecdysone would be) it was argued that this would explain the reduced spontaneous activity of the insects at this time. Interestingly, this depression in motor output was accompanied by an increase in the level of interneuronal activity within the connectives, probably of an inhibitory nature. Central inhibition of motor activity in the period before ecdysis may be a general phenomenon. Truman (1976) has suggested that the expression of adult behaviour patterns in pharate adult A . pernyi is subject to inhibition in

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the last few days before eclosion, and Kammer and Kinnamon’s (1977) observations on Manduca also suggest that activity may be suppressed (see Section 3.7). Both Truman (1976) and Kammer and Kinnamon (1979) speculate that this period of reduced activity might be important in preventing “accidental” rupture of the pupal cuticle. It is interesting that during this period, injections of the non-protein amino acid, L-canaline (which acts as a CNS toxin in Manduca) can induce the performance of adult behaviours which result in the rupture of the thin pupal cuticle (Krammer et al., 1978). The induction of specific behaviour patterns which are appetitive to ecdysis is also not well understood. It seems extremely likely that the release of ecdysone which occurs in endopterygote larvae at the onset of the wandering stage (see Bollenbacher et al., 1975, for titres in Manduca, for instance), is responsible for the switchover from activities typical of the feeding stage to those characteristic of the post-feeding period. Until recently this has been a correlation, rather than an experimentally demonstrated causal link. Lounibos (1976) was able to induce precocious cocoonspinning in H . cecropia by infusing 20-hydroxyecdysone (p-ecdysone) in a single instance, although most of his experimental insects did not respond to the hormone. 0. S. Dominick (personal communication) has been more successful in inducing wandering behaviour in Manduca larvae using the same technique, by paying careful attention to the dose given and the time of infusion. 5.3

CUTICLE PLASTICIZATION

It has been shown in a number of cases, that where cuticular structures must undergo extreme changes in size during a short period of time, this expansion is facilitated by a temporary increase in the cuticle’s extensibility. This change in mechanical properties is called plasticization. Cuticle plasticization has been shown to occur at ecdysis in a number of insects. Adult blowflies emerge from the puparium with their body cuticle relatively inextensible. By the time that the fly begins to swallow air, and to expand the cuticle of its wings and body, the cuticle of the thorax is found to be more extensible than it was previously (Cottrell, 1 9 6 2 ~ )This . change is brought about by a circulating hormone, apparently identical with bursicon (Reynolds, 1976), rather than by the direct nervous route used in Rhodnius. In emerging adult Manduca, the cuticle of the wings undergoes two distinct, successive increases in its extensibility (Reynolds, 1977). The first occurs shortly before eclosion, and is caused by the eclosion hormone. The second occurs after emergence, at the time of wing spreading, and is controlled by the tanning hormone, bursicon. The wings of emerging adult A . pernyi can

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also be plasticized by bursicon (Reynolds, unpublished). Glaser and Vincent (1979) have shown that the wings of adult locusts become more extensible as the insect begins to inflate them. In this case, it is not known whether cuticle plasticization is under hormonal control, but since bursicon is released within a very short time of ecdysis beginning in locusts (Vincent, 1972; Padgham, 1976a), control by this hormone is quite possible. Reynolds (1974b) showed that the abdominal cuticle of Rhodnius underwent a brief plasticization just after ecdysis. Unlike the post-prandial plasticization, this was not under direct nervous control, but was apparently induced hormonally. The hormone concerned was not identified, but could well have been bursicon. Reynolds (1974b) was unable to find a function for the post-ecdysial plasticization of the abdominal cuticle in Rhodnius, and suggested that it might be an evolutionary relic. The account of behaviour during hatching in Climex and Rhodnius given by Sikes and Wigglesworth (1931) may provide a clue, though. Immediately after the newly hatched bug has shed the embryonic cuticle, it begins to swallow air and expand its body cuticle. By this means, together with muscular action, it changes its more or less cylindrical form into the characteristic flattened shape of later life. This post-ecdysial expansion doubtless involves cuticle stretching, and may well be facilitated by plasticization of the abdominal integument. Doubtless more examples could have been quoted if more mechanical testing had been done on the appropriate structures of ecdysing insects. It should be noted, by the way, that cuticle plasticization is not only useful in those cases where the bulk of the cuticle is actually stretchedperse, but also in those cases where it is merely unfolded during expansion (see Section 4, for this distinction). Even in unfolding, it is necessary for cuticular macromolecules to move past one another, and plasticizatim will facilitate this. The mechanism whereby cuticle mechanical properties are altered is unknown in any of the cases where plasticization occurs at ecdysis. However, the increase in cuticle extensibility which is seen in the abdominal cuticle of the blood-sucking bug Rhodnius when it feeds (Bennet-Clark, 1962), is better understood, and may provide a model for the ecdysial cases. As in the newly ecdysed insect, the abdominal cuticle of Rhodnius is untanned, so that the two cases may reasonably be compared. Rhodnius has the experimental advantage that cuticle plasticization can be induced pharmacologically at any time without waiting for ecdysis to occur (Reynolds, 1974a). The post-prandial increase in extensibility of the abdominal cuticle of Rhodnius has been shown to be under nervous control (NuAez, 1963; Maddrell, 1966). The effect of the natural plasticizing agent, presumably released from neurosecretory terminals supplying the epidermal cells directly (Maddrell, 1965), is mimicked by 5-hydroxytryptarnine and a num-

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ber of related chemicals (Reynolds, 1974a), but the nature of the agent acting in vivo is unknown. The changed mechanical properties of plasticized Rhodnius cuticle appear to result from a reduced number of secondary (i.e. non-covalent) interactions between cuticular macromolecules (Reynolds, 1975a). There is no evidence that the rupture of primary (covalent) bonds is involved; indeed, mechanical testing suggests that these are unimportant in determining extensibility anyway (Reynolds, 1975a; Hillerton, 1979). For this reason, and others (see below), the suggestion of Wigglesworth (1970, 1975) that plasticization might be a result of enzymic digestion of structural lipids in the cuticle is to be regarded as unlikely. There is no apparent change in the macromolecular composition of the abdominal cuticle on plasticization (Hillerton, 1978), although there is an increase in the cuticle's water content (from 26% to 3 1%) and a small change in the content of major cations (Reynolds, 1975b). The most likely explanation for the increased extensibility of plasticized Rhodnius cuticle, is that a change in the ionic environment within the cuticle disturbs the secondary interactions between macromolecules, so that the potential energy barrier to the movement of molecules past one another is reduced. Reynolds (1975b) examined a number of possible ways to account for the increased extensibility seen in vivo, using loops of abdominal cuticle in vitro as a model system. It was concluded that the mechanism which was most likely to operate in vivo was a change in the intracuticular pH. Some evidence that a suitable p H change does in fact occur was obtained by experiments using an indicator dye. The pH in the cuticle was found to be greater than 6.0 in the normal state, but was lowered to less than 6.0 in the plasticized condition. Better evidence than this would be more satisfying, but it is of course very difficult to measure pH within a hard extracellular matrix. However, the observed pH change was in the right range of values, and in the right direction to explainin vivo observations; the extensibility of the Rhodnius abdominal cuticle does indeed change markedly in the region of pH 6.0, increasing as the p H falls. How does pH affect extensibility? It is possible that specific bonds between cuticle components are pH-labile. A similar situation was noted by Harkness (1970) in collagen from the tails of young rats, where Schiff base linkages between collagen molecules are broken at low pH. However, it is at least as likely that no specific bonds are involved, but that as the p H is changed to values further away from the isoelectric points of cuticular proteins, the extent of secondary interaction between the matrix proteins is reduced, so that cuticle extensibility is increased. Reynolds (1975b) d'1scusses some of the ways in which this reduced secondary bonding might be caused. Hackman (1975) examined the proteins of Rhodnius abdominal

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cuticle by isoelectric focussing and found that the majority of the proteins had PI values of 7.1 to 8.6. This was considered to support the hypothesis of the pH control of cuticular extensibility. The nature of the secondary interactions in the Rhodnius cuticle which would be disrupted is not known. It was originally suggested that these might be between the matrix proteins (Reynolds, 1975b), but interactions between chitin and protein may be involved. Hackman and Goldberg (1978) have studied the binding of soluble cuticle proteins (from the soft bodied larvae of a fly and a beetle) with purified chitin (from a crab). It was found that the chitin bound quite large quantities of protein. The binding was reversible, but complete removal of the protein required solvents able to break hydrogen bonds. Protein binding was found to be dependent on the prevailing ionic conditions, being greatest at low ionic strength and at pH values close to the isoelectric point of the proteins concerned. These observations, applied to the case of the Rhodnius abdominal cuticle, suggest that the change in extensibility on plasticization might be due at least in part to the disruption of chitin-protein secondary bonds. Hackman and Goldberg (1978) comment that under normal conditions in the cuticle, it is extremely likely that chitin is “saturated” with protein. This would most particularly be the case in Rhodnius abdominal cuticle which has a low chitin content (11%; Hackman, 1975). The relative scarcity of chitin in the Rhodnius cuticle may explain why, even in the unplasticized state, the interaction of chitin microfibres with the protein matrix does not lead to more evident network properties, leaving the cuticle with a relatively low modulus of extensibility (Reynolds, 1975a). It is of some interest that the abdominal cuticle of adult female ticks (Boophilus microplus), which also undergoes massive stretching on feeding, has a gross amino acid composition which is almost identical to that of Rhodnius, despite their considerable taxonomic separation. O n this ground, Hackman (1975) has suggested that Boophilus abdominal cuticle might undergo a post-prandial plasticization similar to that of Rhodnius. However, no mechanical measurements are available. If the extensibility of the Rhodnius abdominal cuticle is indeed controlled by the intracuticular pH, then this system has a remarkable parallel in the plant kingdom. The rate of cell elongation in a variety of rapidly growing plant cells has been shown to be dependent on the extracellular pH, which is controlled by the cells under the direction of the plant hormone, indoleacetic acid. When the pH is reduced below 5.8 the primary cell walls of the growing cells become more extensible, and the cells are enabled to elongate. In this case, the effect is thought to depend on the activation by H+ ions of an enzyme (as yet unidentified) within the cell wall which can cleave loadbearing primary bonds (Rayle and Cleland, 1977).

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5.4

54 1

CUTICLE HARDENING

The means by which cuticle hardening and darkening (“tanning”) is achieved is still not well understood. Possible means of chemical crosslinking have been reviewed recently by Hackman (1974), Neville (1975), and Anderson (1979), so that it is unnecessary to discuss this here. It is evident that although much new information has been collected since Cottrell’s (1964) review, the overall view of the field is not much clearer. In any case, Vincent and Hillerton (1979) have recently suggested that the decreased extensibility shown by tanned cuticles may be due not so much to the formation of primary bonds between cuticular proteins, as has usually been supposed, but rather to the tanned cuticle’s decreased water content, and the consequent increase in secondary bonding between macromolecules. However, the purpose of the present work is to attempt to show how cuticle hardening is integrated with other ecdysial events, and a reasonably clear picture of how this is done has emerged. Simultaneous and independent reports by Cottrell(1962a, b) and Fraenkel and Hsaio (1962, 1963) showed that a hormone controlled cuticle tanning in newly emerged adult blowflies. This hormone was subsequently named “bursicon” (Fraenkel and Hsaio, 1965). Its presence in a wide variety of insects, in addition to blowflies, has since been shown (Table 3). In addition, a single report (Fingerman and Yamamoto, 1964) suggests that Crustaceans may make use of a similar factor, originating in the eyestalk. However, Fraenkel and Hsaio (1965) could not find any bursicon activity when they tested eyestalk extracts on blowflies. Bursicon is a neurosecretory hormone. Although it is widely distributed in the CNS, including the brain, its principal release site appears t o be from the ventral nerve cord. The exact location varies: in blowflies, the fused thoracic/abdominal ganglion is involved (Fraenkel and Hsaio, 1965) although release appears to be under the control of a centre in the head. Thus flies neck-ligatured immediately after eclosion are unable to release the hormone. In Periplaneta release is from the terminal abdominal ganglion (Mills et al., 1965); in Leucophaea (Srivavista and Hopkins, 1975), first instar larvae of Schistocerca (Padgham, 1976a), and Tenebrio (Grillot et al., 1976), the release site is in the thoracic nervous system; and in Locusta (Vincent, 1972) and Manduca (Truman, 1973a) release is from the abdominal CNS generally. Where the problem has been specifically investigated, it appears that release occurs from specialised neurohaemal sites, either the perivisceral organs (PVOs) (Truman, 1973a; Grillot et al., 1976), or from the surface of small segmental nerves (Padgham, 1976a), rather than from the ganglia themselves. The original discovery of bursicon in blowflies was made possible by the

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TABLE 3

Species in which bursicon has been shown to be present

Orthoptera

Periplaneta americana Leucophaea maderae Schistocerca gregaria Locusta migratoria Carausius morosus

a. b.

d.f. 8 d

Hemiptera

Oncopeltus fasciatus

Lepidoptera

Galleria mellonella Pieris brassicae Manduca sexta Antheraea pernyi

b

Tenebrio molitor

Diptera

Calliphora vicina (= C. erythrocephala) Lucilia spp. Phormia regina Sarcophaga spp. Aedes aegypti

Fraenkel and Hsaio (1962)

’Fraenkel and Hsaio (1965)

‘Mills et aI. (1965) dVincent (1972) ‘Srivavista and Hopkins (1975) ’Cottrell (1962b)

d

d

b

Coleoptera

C.

P

h

J b,

k

0 ,

b.f

f. (I,6 a,

b.f

Padgham (1 976a, b) Post and de Jongh (1 973) ‘Truman (1973a) Reynolds (unpublished) Delachambre (1971) ‘Seligman and Doy (1972,1973)

separation in time of eclosion and the subsequent release of the tanning hormone (Cottrell, 1962a; Fraenkel and Hsaio, 1962). This is also the case in Manduca (Truman, 1973a), and is occasioned by the fact that the newly emerged adults must in both cases dig free of the substrate before they can expand and harden their cuticle. In many other insects this is not the case, and bursicon is released early in ecdysis, as the old cuticle is split (e.g. Srivavista and Hopkins, 1975; Padgham, 1976a). In Tenebrio (Delachambre, 1971) and in Pieris (Post and de Jongh, 1973) bursicon has been detected in the blood considerably before ecdysis, and it has been suggested that pre-ecdysial tanning may be under bursicon’s control, as well as the post-ecdysial tanning with which the hormone is more usually associated. Puparium formation in blowfly larvae appears to be a special case. Here, as in the cases discussed above, cuticle tanning is controlled by a neurosecretory hormone. However, the hormone is not bursicon (Sivasubramanian e f al., 1974a). Initially designated “factor X,”(Fraenkel e f al., 1972), the hormone concerned is now known as “PTF” (“puparium tanning factor”; Sivasubramanian et al., 1974a). The hormone is found in the CNS

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and may be liberated from peripheral neurohaemal organs, although the exact site of release is unknown (Fraenkel et al., 1972). Its mode of action may be somewhat different from that of bursicon. The principal action in both cases seems to be to allow the access of tanning precursors, principally tyrosine, to the enzymes of the tanning process. Bursicon has been shown to increase the rate of tyrosine hydroxylation seen in whole adult flies (Seligmanetaf ., 1969), and also enhances the rate of tyrosine decarboxylation in cockroach haemocytes (Whitehead, 1969). Both tyrosine hydroxylase (Mills et af., 1968) and tyrosine decarboxylase (Whitehead, 1969) are found in Peripfaneta haemocytes, and not in the plasma. Mills and Whitehead (1970) proposed that these actions of bursicon were best explained by supposing that the hormone did not modulate enzyme activity directly, but rendered the cell membrane of the haemocyte more permeable to tyrosine. In support of this hypothesis it has been shown that bursicon can enhance the rate of tyrosine uptake into haemocytes of Periplaneta (Mills and Whitehead, 1970) and of Pieris (Post, 1972). In Mills and Whitehead’s (1970) model, it was supposed that Dopa decarboxylase was located only in the haemocytes, and that in order for the supposed tanning substrate, N-acetyldopamine, to be produced, it would be necessary for dopamine to be released into the plasma and taken up by the epidermal cells, where the necessary transacetylase was located. They showed that treatment with a diuretic hormone (D.H.) preparation (postulated to be identical with bursicon; see Section 5.7) could enhance the incorporation of dopamine into the integument, so that bursicon may increase cell permeability to dopamine as well as to tyrosine. In fact, it is likely that both epidermal cells and haemocytes are involved in the conversion of tyrosine to dopamine, as the necessary enzymes are present in both tissues (Hopkins and Wirtz, 1976). Once tanning substrates are formed, unless they are to be used immediately they must be stored in an unreactive form. A variety of conjugated forms have been identified in insects, including D-glucosides, phosphates and sulphates (reviewed by Neville, 1975). In order to become available for tanning, these conjugates must be cleaved by appropriate enzymes. Whitehead (1971) has shown that the activity of D-glucosidase in emerging adult blowflies is increased several-fold after emergence, and suggests that this increase is controlled by bursicon. In addition to the B-glucosides and other conjugates discussed above, tanning substrates are known to be complexed in the haemolymph with carrier proteins (Koeppe and Mills, 1972; Koeppe and Gilbert, 1974; Vacca and Fingerman, 1975a, b). It has been suggested that these carrier proteins are translocated across the epidermis and become incorporated into the

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cuticle intact (Koeppe and Gilbert, 1973), and that the transport of the protein-diphenol complexes is under the control of bursicon (Koeppe and Mills, 1972) or its Crustacean homologue (Vacca and Fingerman, 1975b). Rather less is known about the mode of action of PTF than is the case for bursicon. The metabolic pathways involved in tanning the puparium are thought to be the same as in the general case. Dopa decarboxylase is a key enzyme, and as in the case of ecdysis, its activity increases greatly just before puparium formation. Karlson and Sekeris (1966) have shown that its de novo synthesis is under the control of ecdysone. As the ecdysone titre rises, so does the activity of the enzyme (Shaaya and Sekeris, 1965). However, tanning does not begin until some time after DOPA decarboxylase activity begins to rise (Seligmanetal., 1977) so that this cannot be the point at which immediate control of tanning is exerted by PTF. This is supported by the observation that injections of various catecholamines, including DOPA, will accelerate tanning in blowfly larvae, even in the absence of PTF (Fraenkel et al., 1977). When a - M D H is used to block DOPA decarboxylase, then only decarboxylated compounds (Dopamine, adrenaline) are able to induce tanning; DOPA is unable to reverse the a-MDH block. Since tyrosine is unable to induce tanning under any circumstances, this places the site of PTF control either at tyrosine uptake, or at tyrosine hydroxylation, as was concluded for bursicon. Both bursicon and PTF appear to exert their effects via an increase in the level of 3', 5'-cyclic adenosine monophosphate (CAMP)in the cells of their target tissues. Von Knorre et al. (1972) and Vandenburg and Mills (1974) observed that cAMP could mimic bursicon activity in neck-ligated, adult Calliphora and in the ligated thorax of Periplaneta, respectively. Seligman and Doy (1972) showed that cAMP (and also its deaminated analogue cIMP) enhanced both cuticle tanning and DOPA synthesis in neck-ligated adult Lucilia , as does bursicon; also the phosphodiesterase inhibitor, theophylline, was capable of potentiating the effects of low doses of bursicon. Delachambre et al. (1979a) have shown that levels of cAMP in the epidermis of Tenebrio rise just at the time of bursicon secretion, and subsequently (Delachambre et al., 1979b) that exogenous bursicon induces a brief elevation of epidermal cAMP content. Fraenkel et al. (1977) have found that during puparium formation in blowfly larvae, cAMP and some of its analogues, are capable of accelerating tanning of the puparium, just as PTF does. The effects of low doses of PTF are potentiated by theophylline, as would be expected if cAMP synthesis were an essential step in the action of PTF. However, the resemblance between the actions of bursicon and PTF may end here. Bursicon and PTF are not the same. Their molecular characteristics are quite different (Sivasubramanian et al., 1974) although both are

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polypeptides. According to Seligman et al. (1977) haemolymph from newly-emerged adult flies, which is rich in bursicon, has no detectable PTF activity, whereas blood from orange puparia, rich in PTF, does not have any bursicon activity. In addition, it appears that PTF‘s action includes a transcription-related step not found in that of bursicon. Seligmanetul. (1 977) found that 5-bromo-2’-deoxyuridine (Brd Urd) and actinomycin D (inhibitors of RNA synthesis) as well as puromycin and cycloheximide (inhibitors of protein synthesis), completely blocked PTFinduced tanning in blowfly larvae. The block could be reversed by both dopamine and by DOPA, suggesting that the block took its effect at the level of tyrosine uptake, or tyrosine hydroxylation, already known to be the hormone’s site of action (Fraenkel et al., 1977). Interestingly, cAMP could not reverse the block caused by inhibitors of translation, although it did overcome the block caused by inhibition of transcription. A model was proposed to account for these results (Fig. 13), in which PTF exerts independent actions on cAMP synthesis, and on the transcription of genetic material in the nucleus. The effect on cAMP metabolism would be achieved by the interaction of PTF with a cell surface receptor linked to adenylate cyclase, but the effect on mRNA synthesis would be independent

+,

TARGET CELL

HEMOLYMPH

NSC

Tronscrlptionol and

PTF&>

Translational PiF inhibitors

Chromosome

m

5’

-

RNA

4

Activation

Synlhesis Metabolic

‘\-?oL \ a

Tyrosine

1-

-

-

+ c

DOPA

-

a-MDH

Tanning substrates

Fig. 13 Hypothetical interrelationships between ecdysone, PTF, and cAMP in the regulation of puparial tanning in the blowfly, Surcophugp bullutu. See text for explanation. (From Seligman et al., 1977)

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of CAMP. They postulate that a tanning-specific mRNA is transcribed, which is then translated into a tanning-specific protein. The effect of this protein would be dependent on elevated intracellular levels of CAMP.The way in which these two intracellular regulators would affect tyrosine metabolism is not known. A difficulty with this model is that cAMP can reverse the effects of inhibition of transcription, but not of translation. Seligmanet al. (1977) suppose that low levels of the tanning-specific mRNA and its translation product may exist in the absence of PTF, and that this would suffice to enable artificially high levels of cAMP to accelerate tanning. However, since transcription-blocked larvae never tan unless cAMP is added, PTF itself is evidently unable to reverse the block, where exogenous cAMP can. Why this should be so is not clear from the model. The doses of inhibitor used were rather high, and the authors point out that these levels were somewhat toxic to the larvae. The reversal of the effects of the inhibitors caused by DOPA and dopamine was only partially effective, so that some doubt remains as to whether the tanning caused by these agents was in fact of the normal kind. Nevertheless, it is clear that PTF action involves components which are not present in bursicon’s action in controlling cuticle tanning. Fogal and Fraenkel (1969) failed to find any effects of inhibitors of RNA and protein synthesis on bursicon-induced tanning in newly-emerged adult blowflies. This does not mean to say that bursicon has no effects on the genome. Trepte (1976) observed the appearance of puffs on the chromosomes of bristle cells, in the integument of emerging adult Sarcophaga, which he attributed to the action of bursicon, but this might not relate to the action of the hormone in promoting tanning. As we shall see, bursicon has other actions (notably on endocuticle deposition) which might be expected to involve effects on the genome (see Section 5.6). 5.5

TR A C H E A L A I R - F I L L I N G

The tracheal system of the insect within the egg is completely formed before any air appears within it. The nature of the fluid within the lumen is unknown, but it is removed rather rapidly at about the time of hatching, SO that the tracheae fill with air. This process may occur just before the eggshell is broken, as in Tenebrio, or just after, as in Cimex (Sikes and Wigglesworth, 1931). In both these cases, the fluid in the tracheae is withdrawn as soon as the spiracles are first allowed access to the air, but in other species, air-filling may occur without such access, both in aquatic (e.g. Chironomid larvae; Keilin, 1924) and in terrestrial forms (e.g. larvae of the moth Sitotroga; Sikes and Wigglesworth, 1931). In these cases, gas must be liberated from solution and surrounding tissue fluids, and indeed if dissolved gases are excluded, then filling does not occur (Sikes and Wigglesworth, 1931:

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actually it might be objected that in such cases, the failure of the tracheae to fill with air might equally be due to the effects of anoxia; Keister and Buck, 1949). Later in life, new tracheae are formed at each moult, and these are also fluid-filled up until the moment of ecdysis. Presumably the mechanism of fluid resorption at ecdysis is similar to that employed at the time of hatching. Throughout life, fluid can be secreted into or resorbed from the tracheoles, depending on the state of activity of the tissue which they supply. Wigglesworth (1930) suggested that the accumulation of t h e products of metabolism might lead to locally elevated osmotic pressure in active tissues, which in turn would lead to the osmotic withdrawal of fluid from the lumen of the tracheoles. Beament (1964) has criticized this idea, and concluded on the basis of calculations of the likely surface tension forces operating within tracheoles, that the removal of fluid from the lumen can only be accounted for by the active transport of water. In hatchingAedes (Wigglesworth, 1938) and ecdysing Sciara larvae (Keister and Buck, 1949), it has been observed that fluid absorption can occur from tracheal trunks which have no tracheolar endings, so that at ecdysis absorption must occur generally through the walls of the tracheae, as well as in tracheoles. Unlike tracheolar absorption, the general air-filling of the tracheal system can only occur at the time of hatching or ecdysis. Larvae of Aedes need access to the surface in order to fill their tracheal system with air. Wigglesworth (1938) found that air filling could be prevented by chloroform narcosis, and suggested that this provided evidence that the process must be due to some kind of active transport (as opposed to an osmotic effect), and also that the process was under nervous control. O n the other hand, decapitating ecdysing larvae did not prevent air-filling, SO that any such nervous control could not be dependent on the cerebral ganglia. There is an interesting difference between the tracheal air-filling which occurs in Aedes at hatching, and that occurring at later ecdyses. If hatching larvae are prevented from reaching the surface, they gradually lose the ability to empty their tracheae of fluid when access is eventually allowed, but nevertheless the capacity for air-filling does persist for quite some time (up to 3d). However, the situation is quite different at later ecdyses, where air-filling ability is lost within 0.5 h of ecdysis. Wigglesworth (1938) draws a parallel here between the control of air-filling in hatchingAedes, and the way in which cuticle expansion and tanning may be delayed in newly-emerged adult blowflies which are forced to keep on digging (Fraenkel, 1935a). If this analogy is correct, it suggests a role for a hormone (bursicon?) in bringing an end to the possibility of tracheal air-filling, perhaps by tanning the tracheal cuticle and making it impermeable to water. Vincent (1972) also proposed a role for bursicon in controlling the air-

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filling of the tracheae at ecdysis in Locusta. H e ligatured off the last abdominal 'segments of ecdysing locusts and found that the cuticle of this segment did not tan, and neither did the tracheae fill with air, regardless of whether the previous instar's tracheae were withdrawn. Vincent suggested that this was due to the lack of bursicon release in the ligatured segments, and that the failure of air-filling might be associated with the failure of the tracheal cuticle to tan. The mechanism whereby fluid is absorbed from the tracheac is still obscure. The principal experimental observations to be accounted for, are as follows: (i) gas filling of the trachea at hatching and/or ecdysis can be prevented by chloroform (Wigglesworth, 1938), cold (Keister and Buck, 1949), and by complete anoxia (i.e. < 0.3% 0,;Keister and Buck, 1949); (ii) gas filling cannot be prevented by 99.7% CO, (Keister and Buck, 1949); (iii) premature gas-filling can be induced, in Sciara larvae about to undergo ecdysis, by exposure to concentrations of 0, in the range 0.3-0.75% (Buck and Keister, 1955); (iv) in such pre-ecdysial larvae, exposure to 0, concentrations > 1% reverses the gas-filling previously brought about as in (iii). The process can be repeated several times (Buck and Keister, 1955); (v) this reversibility of gas-filling is maintained during pre-ecdysial behaviour, but declines during the shedding of the old cuticle. After ecdysis, air-filling cannot be reversed (Buck and Keister, 1955); ( v i ) O n the other hand, complete anoxia does not reverse air-filling previously induced as in (iii) as does exposure to ordinary air, but can only prevent air-filling from occurring in the first place. Buck and Keister (1955) attempted to reconcile these findings by a synthesis of previously-proposed hypothetical mechanisms. They supposed that tracheal air-filling is subject to an equilibrium between capillarity in the trachea (which tends to maintain them in a liquid-filled state), and two separate imbibing forces; one an active uptake mechanism, and the other a physical mechanism due to the existence of an osmotic pressure gradient between the tracheal fluid and the tissues. The premature air-filling induced by 0.3-0.75% 0, is explained by supposing that these low concentrations of 0, do not completely suppress metabolism, but rather lead to the accumulation of its products. Buck and Keister (1955) suggest that at normal levels of 0, this does not occur, and under complete anoxia, metabolism is suppressed, so that only under 0.3-0.75% 0, does an osmotic force develop which can empty the tracheae. On return to ordinary air, the local accumulation of metabolites can be dispersed and the osmotic withdrawing force is removed, so that fluid

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returns to the tracheae. Although Beament (1964) has criticizedthe osmotic hypothesis of tracheolar fluid withdrawal, on physical grounds (see above), it should be noted that the surface tension forces opposing fluid withdrawal from the much larger tracheal trunks would be considerably less, so that his objection need not apply to the general air-filling of the tracheal system. The air-filling induced in pre-ecdysial larvae by 0.3-0.75% O2 is, of course, entirely an experimental phenomenon. Although Buck and Keister (1955) point out that any theory of tracheal fluid absorption must be able to account for it, it should be realized that the osmotic forces postulated to be responsible for experimentally-induced air-filling may have little to d o with the mechanism of the reabsorption of tracheal fluid occurring naturally at ecdysis. Perhaps the most significant observation in Buck and Keister’s (1955) paper is that there is a change in the properties of the tracheae at the time of ecdysis, so that exposure to aerobic conditions no longer leads to the refilling with fluid of previously emptied tracheae. As suggested above, this change could well have something to do with the tanning of the tracheal cuticle. Wigglesworth (1953) has pointed out that the luminal surfacesof the tracheae became more hydrofuge at the time of ecdysis, perhaps due to wax secretion, perhaps due to cuticle tanning. This would have the dual effect of reducing the surface tension forces which must be overcome in order to empty the lumen of fluid, and also to render the walls of the trachea less permeable to water. This would explain why tracheal air-filling cannot be effected later, if for some reason it is delayed. Altogether, our knowledge of this subject must be considered to be in an unsatisfactory state. 5.6

CUTICLE DEPOSITION

Prior to ecdysis, new cuticle is deposited underneath the old under the influence of the two developmental hormones, ecdysone and juvenile hormone (Gilbert and King, 1973). However, cuticle deposition continues after ecdysis and is influenced by the physiological events of ecdysis itself. The cuticle deposited before ecdysis may be distinguished from that which is laid down afterwards. The former is (usually) tanned in the period immediately after ecdysis and is termed exocuticle. The latter (usually) remains untanned and is calledendocuricle. There are exceptions to this. For instance, the pre-ecdysial cuticle of the abdomen in larval Rhodnius remains untanned (Wigglesworth, 1933), whereas in locusts, cross-linking of the post-ecdysial cuticle goes on throughout adult (but not larval) life (Andersen, 1973; 1974). The supramolecular architecture of the pre-ecdysial and post-ecdysial cuticle differ in many cases. Generally, in the exocuticle, chitin microfibres are oriented in a continuous helicordal array, whereas in

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endocuticle, this organization is subject to the imposition of daily (or nondaily) growth layers, in which chitin is unidirectionally oriented (Neville, 1970). In some beetles, however, there are areas in which the deposition of cuticle with the typical organization of endocuticle begins before adult ecdysis. Generally these areas are those where extensive pre-ecdysial tanning has occurred (Zelazny and Neville, 1972). Fogal and Fraenkel(l970) have noted that a similar phenomenon occurs in adult blowflies. This suggests that the formation of typical endocuticle might be induced by the same mechanism which controls tanning, a point which is developed below. The deposition of the cuticle stops before ecdysis, and resumes again afterwards. The discontinuity between the pre- and post-ecdysial cuticle leads to the appearance in cross-sections of the cuticle of an “emergence line” (Neville, 1975, p. 8). This can be seen even in apparently homogenous cuticles (see Neville, 1963; Hillerton, 1978). At the moment of ecdysis, and up to 1 h afterward, in adult blowflies, the subcuticle (“deposition zone” of Neville, 1975) is absent according to Fogal and Fraenkel(l970) as would be expected if deposition of cuticle materials had stopped. Nothing is known about how this temporary halt in the synthetic activity of the epidermis is brought about. Presumably some kind of hormonal signal is involved, but it is not known how or when this would act. Cuticle deposition is resumed again after ecdysis. The first evidence that deposition of post-ecdysial cuticle might be under a hormonal control quite distinct from that of the two major developmental hormones was the observation by Locke er al. (1965) that intermoult synthesis of endocuticle in larvae of Culpodes was inhibited when they were neck-ligated, even though they subsequently pupated (i.e. deposited a pupal exocuticle) more or less normally. Apparently, some factor from the head was required even in the presence of ecdysone. The secretion of wax in the epidermis seems to be subject to a similar control system (Locke, 1965). Wielgus and Gilbert (1978) have recently shown that a similar requirement exists in Manducu. Deposition of endocuticle in vivo required the presence of the head, and experiments in vitro showed that co-culture of the integument with isolated brains could increase the amount of endocuticle deposited. The addition of fat body alone to the culture medium could not enhance cuticle deposition, but fat body and brains together were superior in this respect to brains alone. This was interpreted in terms of a model where a factor released by the brain could cause the fat body to synthesize and/or release components necessary for cuticle deposition. These would probably be proteins. The identity of the hormonal factor from the brain is unknown. Presumably it is released continuously once ecdysis has been completed. However, it seems that in some insects at least, endocuticle deposition is

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“switched on” at ecdysis by a hormonal trigger. Fogal and Fraenkel(l969) have shown that the tanning hormone, bursicon, is required for the resumption of cuticle synthesis after adult ecdysis in blowflies. Whereas, by 24 h after eclosion, normal flies have not only tanned their pre-ecdysial (exo-) cuticle, but also deposited an appreciable layer of post-ecdysial (endo-) cuticle, flies ligatured around the neck immediately after eclosion were found to have deposited no endocuticle at all by this time. Endocuticle was recognized by its staining properties with Mallory’s triple stain. Synthesis and deposition of endocuticle could be restored in the neck-ligated flies by injections of “active” blood, taken from control flies at the time of tanning. The hormonal activity in the blood was indistinguishable from bursicon on partial purification by gel filtration and polyacrylamide gel electrophoresis. The time of release of the factor which promoted cuticle deposition was identical to that of bursicon, as determined by neck-ligation experiments, and there is every reason to suppose that it is bursicon which is in fact responsible for the effects observed by Fogal and Fraenkel (1969). Unlike bursicon’s action in promoting cuticle tanning (see Section 5.4) the action of the hormone in triggering cuticle deposition is apparently dependent on new synthesis of both RNA and protein, as both actinomycin D and puromycin were found by Fogal and Fraenkel to be capable of inhibiting endocuticle formation, while cuticle tanning was unaffected in the same flies. On the other hand, tanning could be selectively inhibited by a a-MDH without affecting post-ecdysial cuticle deposition. Evidently these two actions of bursicon are independently mediated at the level of the epidermal cells. Experiments in which RNA synthesis was monitored autoradiographically, by following the incorporation of 3H-uridine,failed to show any effect of bursicon in stimulating the general level of RNA metabolism in either epidermal or fat body cells. However, protein synthesis (also measured autoradiographically by the incorporation of 3H-leucine) was clearly stimulated (by 5 to 6 times) in the epidermal cells, although hardly at all in fat body cells. Interestingly, the apparent incorporation of 3H-leucine into the epidermis was much higher where these cells were adjacent to fat body cells, whether or not they were stimulated by bursicon. This was interpreted as being evidence in support of the hypothesis that proteins synthesized in the fat body are released into the haemolymph and then translocated into the cuticle by the epidermal cells. Given that mixing of the haemolymph pool of protein is slow, one might expect that cells close to the fat body would take up more of the newly synthesized (and thus 3H-labelled) proteins than those epidermal cells which were not adjacent to fat body cells. Fogal and Fraenkel (1969) showed that bursicon promotes endocuticle formation at similar concentrations in the blood to the levels at which it

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causes cuticle tanning. Since bursicon does not persist at these levels in the blood for more than a few hours after eclosion in blowflies (Cottrell, 1962b; Fraenkel and Hsaio, 1965), bursicon must act as a trigger in promoting endocuticle deposition, not being required subsequent to the brief period in which it is present at high levels. However, it is possible that in blowflies bursicon does not act alone to promote cuticle growth after eclosion. Schlein (1972a, b) claims that an additional hormonal factor, produced and secreted by neurosecretory cells in the ocellar nerve, is required. He has proposed the name “auximon” for this new hormone, which is distinct from bursicon, and for which no other function is known (Schlein, 1972b). Experiments based on neck-ligatures (Schlein, 1972a) and surgical extirpation of the ocellar nerve (Schlein, 1972b) showed that this hormone is secreted into the blood of pharate adult blowflies from about 28 h before eclosion. If release of auximon is prevented by either ligature or surgery, then endocuticle deposition is absolutely prevented, even though a high proportion of the surviving ocellar nerveextirpated flies inflate and tan their (exo-) cuticle normally, indicating that bursicon must have been released. The appearance of daily growth bands along the edges of two thoracic apodemes was used as a criterion of endocuticle deposition. At first sight, the results of these experiments appear to conflict with those of Fogal and Fraenkel (1969). However, inspection of Schlein’s (1972a) data shows that prevention of bursicon release by neck-ligation immediately after eclosion does indeed completely prevent endocuticle deposition for up to 2 days after adult emergence. Schlein found that only after this was cuticle growth detectable in these flies. Although no comment is made on this by Schlein, neck-ligated flies kept for periods as long as this usually show a progressive “non-specific” tanning (Cottrell, 1962a), and when this occurs, this is associated with endocuticle formation (Fogal and Fraenkel, 1969). Consequently, it seems unwise to conclude, as does Schlein (1972b) that bursicon does not initiate endocuticle deposition. However, Schlein’s evidence does indicate that previous exposure to auximon is also necessary for post-ecdysial cuticle growth to occur, even when bursicon is released normally. It would seem that auximon must be released progressively over the period from about 28 h before eclosion, UP to emergence itself. Since post-ecdysial cuticle deposition in flies neckligated 28 h before eclosion can be restored by parabiosis with newlyeclosed flies, auximon must still be present in the blood at the time of emergence. To add to the complexity of this situation, in parabiosis experiments between flies ligatured at eclosion and adult flies of various ages (4 h-21 days), none of the ligated flies ever showed post-ecdysial cuticle growth,

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whereas control flies, neck-ligated at the same time, but not joined in parabiosis to another fly, all showed some endocuticle deposition by 3-7 days after eclosion. Schlein (1972a) interprets this in terms of the presence in older flies of an inhibitory factor, which prevents cuticle growth. This is puzzling, since this factor obviously does not inhibit post-ecdysial cuticle deposition in the older flies themselves. The identity of the inhibitory factor is unknown, and it should be noted that it might not be hormonal in nature. So far, Schlein’s results stand alone. His ligation experiments apparently were accompanied by heavy mortality, so that some doubt must attach to the results obtained from the small percentage of survivors. Confirmation of Schlein’s conclusions would be most welcome. 5.7

CHANGES IN BLOOD VOLUME: POST-ECDYSIAL D l U R E S l S

A number of authors have noted that insects have a high blood volume at the time of ecdysis, and reduce it thereafter. As pointed out by Cottrell(1962~), this high blood volume is associated with the use of a hydrostatic skeleton in many of the movements of ecdysis. Quantitative data on changes in blood volume are given for Schistocerca by Lee (1967); for Periplaneta by Wheeler (1963), Wharton et al. (1965), Mills and Whitehead (1970); for Achera by Woodringet al. (1977); for Calliphora by Cottrell(1962c); and for Pieris by Nicolson (1976a). According to Lee (1967), the increase in blood volume which occurs before ecdysis is largely at the expense of cellular and gut water, the total percentage dry weight of the whole animals remaining more or less constant. In Periplaneta, however, Mills and Whitehead (1970) found that the blood’s content of an antidiuretic hormone, capable of causing resorption of water from the rectum, increased by more than 5 times in the 3 days prior to ecdysis (Fig. 14). In larvae of Calpodes, Ryerse (1978) has shown that the Malpighian tubules are “switched off” prior to preparation by the increased titre of ecdysone at this time. After ecdysis, blood volume decreases very quickly in Periplaneta, declining by almost 30% in 7 h (Mills and Whitehead, 1970). InSchistocerca, however, the blood volume is maintained at a high level until 24 h after ecdysis, declining over the next day or two by about 35% in larval stages and by about 45% in the adult (Lee, 1967). The decrease in blood volume in locusts appears to be due to a net loss of water from the blood to the environment, since the percentage of cellular water remains steady and the proportion of dry weight is greatly increased. Some of the post-ecdysial water loss is undoubtedly due to the increased rate of transpiration at this time. This has been documented for Rhodnius, where the rate of water loss on the day following ecdysis is more than

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-i260 -g 240 3

220

8

180

d

16C

’200 0

0.4 $ 2

n

0

7248240 I h before

I

0

2 3 4 5 6 7

h a f t e r ecdysis

Fig. 14 Hormonal control of haemolymph volume before and after the last larval ecdysis in Periplaneta americana. ( A ) Changes in haemolymph volume, determined using 14C-carboxy inulin. (B) Antidiuretic and diuretic (0)activity in haemolymph, as determined by the ability of blood samples to cause volume changes in isolated rectal sacs. Also shown is the titre of bursicon in the blood (A).(Modified from Mills and Whitehead, 1970)

(e),

doubled (Wigglesworth and Gillett, 1936), and in Tenebrio, where the rate of water loss of newly emerged adults is on average some 6 times greater than was that of the pupa, and about 5 times greater than that of the young adults a day or so later (Wigglesworth, 1948). However, the very substantial water loss observed in many insects suggests that an active diuresis may be involved. This conclusion is supported by the results of Mills and Whitehead (1970), who found that the blood titre of a diuretic hormone (which causes a net transport of water into an isolated rectal sac, in vitro) was greatly increased during the first few hours after ecdysis (Fig. 14). As the titres of bursicon and of the diuretic hormone increased and decreased in parallel, it was suggested that the two hormones might in fact be identical. Both hormones have molecular weights in excess of 30 000 D (Mills and Nielsen, 1967; Goldbard et al., 1970), and a partially purified preparation of the diuretic hormone was able to cause increased uptake of tyrosine into cockroach haemocytes - a function which is consistent with bursicon’s function in promoting tanning (Mills and Whitehead, 1970: see Section 5.4). Following adult eclosion, the butterfly Pieris brassicae, also undergoes a drastic post-ecdysial diuresis, so that the newly emerged insects can be seen to void numerous drops of clear urine. The blood volume is reduced by 75%

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in a few hours following ecdysis (Nicolson, 1976a). Diuresis is under the control of a hormone, which can be shown to be present in the haemolymph of diuresing insects by assay on an in vitro Malpighian tubule preparation (Nicolson, 1976b). The hormone is present in substantial amounts only in the brain and CC/CA of the pharate adult insect, and it seems likely that the CC/CA complex is its normal site of release. The identity of the hormone is unknown. Following Mills and Whitehead’s (1970) work with Periplaneta, we might suppose that it was bursicon. Is this possible in Pieris? In another Lepidopteran, Manduca, bursicon is contained in, and released from, the abdominal nerve cord, rather than the CC/CA Complex (Truman, 1973b). However, there is no reason why bursicon should not be present in the CC/CA of Pieris; in fact, in another moth, the Saturniid, A . pernyi, bursicon is both contained in and is released from the CC/CA (Reynolds, unpublished). In Manduca, bursicon release is delayed until wing-spreading behaviour has begun (Truman, 1973b), whereas in Pieris, diuretic hormone is apparently released during ecdysis (Nicolson, 1976b). But in A . pernyi, bursicon is released during ecdysis (Reynolds, unpublished) as it is in a number of other insects. Diuretic hormone from Pieris CC/CA is apparently stable at room temperature for at least 20 h, and is unharmed by boiling for 2 min. These properties are quite compatible with what is known of the properties of blowfly bursicon: although this is not stable when present in active blood, the hormone is stable to boiling when obtained by homogenizing brain neurosecretary cells (Fraenkel et al., 1966). It might be supposed that the post-eclosion diuresis could not be due to bursicon, because this hormone is known to be present in the blood at other times in the insect’s life (Post and de Jongh, 1973). However, the responsiveness of the adult Malpighian tubules to CC/CA extracts, and also to CAMP appears to be limited to the period around ecdysis (Nicolson, 1976b), so that this need not be an obstacle to our supposition that diuretic hormone and bursicon might be identical in Pieris. Of course all this is only circumstantial evidence, and more work is needed on this point. Post-ecdysial blood volume reduction need not necessarily be effected via the orthodox route of the Malpighian tubules and hindgut. In adult silkmoths, a significant loss of water occurs through the secretory activity of the labial glands. The total amount of fluid secreted by the glands is dependent on how quickly the moth succeeds in escaping from confinement in its cocoon. Where escape is prevented, secretion may continue for up to 3 h, and as much as 0.2 ml of fluid may be secreted (Kafatos, 1968). Evidently, sensory stimuli regulate secretory activity in the labial glands. This control pathway may be disrupted by the anticholinesterase drug, physostigmine, which causes a profuse secretion by the glands (Kafatos, 1968). In the

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Sphingid, Manduca, secretion by the salivary glands is under nervous control (Robertson, 1974), but it is not known if this is the case in silkmoths. At any rate it is very likely that physostigmine acts on the CNS, rather than peripherally, to stimulate fluid secretion. The capacity of the labial glands to secrete fluid varies with the developmental age of the moth. Kafatos (1968) reports that in A . pernyi both the rate of secretion and the total volume secreted in response to injected physostigmine were “limited” in pharate adult moths which were still damp with moulting fluid. Similarly, in Manduca, salivation in response to physostigmine cannot be elicited until shortly before eclosion (Reynolds and Truman, unpublished). Following eclosion the ability of the glands to secrete declines quite rapidly in A.pernyi. Whereas immediately after eclosion up to 200 p1 could be secreted in response to a physostigmine injection, only 30-41 pl was produced at 10 h after emergence, and about 5 pl at 1 5 h (Kafatos, 1968). However, Edwards (1964) found that in H.cecropia some individuals retained the ability to secrete large amounts of fluid in response to injections of salt solution throughout adult life. The development in the labial glands of the capacity to secrete at just about the time of ecdysis has a parallel in some other fluid transporting epithelia. At the time of eclosion, the salivary glands of blowflies do not respond to 5-hydroxytrypramine, applied at a concentration (10 nM) which is later supramaximal. At about 2 h after emergence, the glands first show a secretory response, and by 3 h, all flies have glands which respond maximally (Berridgeetaf., 1976). It is possible that thegland’ssecretoryapparatusisnot properly functional at the time of eclosion. In particular, Berridge et al. (1976) observed that in electron micrographs of glands from eclosing flies, the development of the secretary canaliculi on the luminal surface of the cells appeared incomplete. However, it was noted that the lack of responsiveness to 5-HT at eclosion could also be due to a number of other factors, such as the absence of functional receptor-adenylate cyclase complexes or the non-availability of proper energy supplies. Malpighian tubules may also be subject to this kind of change in responsiveness a t the time of ecdysis. Nicolson (1976b) found that the response of tubules from adult Pieris to CC/CA extracts (i.e. diuretic hormone) was greatest just at the time of ecdysis, and declined quite quickly thereafter. Whereas, just before eclosion, the rate of secretion in vitro could be increased by about 8 times, by 1 day later, the basal rate of secretion could only be increased about 2 times, the response declining still further in the next 2 days. A similar pattern was evident in the tubules’ responses to CAMP. Unfortunately, the ability of Malpighian tubules to respond to diuretic hormone before eclosion was not tested, but the response to CAMP developed only in the 3 days prior to emergence, reaching a maximum just at

,

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the time of ecdysis. In Calliphora, the Malpighian tubules are relatively insensitive to the fly’s diuretic hormone at the time of adult eclosion. The stimulated rate of fluid transport more than doubles in the first 12 h after eclosion and remains high thereafter (Schwartz and Reynolds, unpublished). In none of these cases is the cause of the sudden onset of responsiveness to stimulation at ecdysis known. The precision of the timing involved suggests that some kind of hormonal trigger may be involved. 5.8

DISCHARGE OF DERMAL GLANDS

There exists a large variety of forms of dermal glands (reviewed by Noirot and Quennedy, 1974), not all of which are concerned with the physiological events of ecdysis. However, a class of dermal glands which do function during ecdysis is typified by the type B dermal glands of Rhodnius (Wigglesworth, 1933,1947; Lai-Fook, 1970) and the Verson’s glands of Lepidoptera (Verson, 1889; Barbier, 1968; Lai-Fook, 1972, 1973). These glands increase in size during the formation of the new cuticle, and discharge their accumulated contents rapidly at about the time of ecdysis. In Rhodnius, the glands subsequently regress and are renewed with the initiation of the next moulting cycle (Lai-Fook, 1972). The Verson’s glands of Calpodes, however, do not degenerate, but renew their secretory activity once ecdysis is completed. Lai-Fook (1973) comments that there is no time at which the gland in Culpodes is not undergoing some change in its activity during the moult/intermoult cycle. According to Lai-Fook (1973), the discharge of the Verson’s glands’ contents occurs just before the old cuticle is shed in Culpodes, but in Rhodnius and Tenebrio the glands are emptied just afterwards (Wigglesworth, 1933,1948). The function of the dermal gland secretions seems to be in the formation of the cement layer of the cuticle (Wigglesworth, 1947, 1948; Way, 1950; Pihan, 1967). Suggestions that the glands might have a role prior to ecdysis, in secreting the moulting fluid (Wigglesworth, 1933; Barbier, 1968) have been discounted on the grounds that the glands do not appear active at the right time (Wigglesworth, 1947; Lai-Fook, 1973). Little is known about the nature of the secretions of this kind of dermal gland. It is clear that more than one type of secretion is produced and it may be that the cuticle-lined saccule of the Verson’s gland may act as a mixing chamber, in which reactive components can come together prior to discharge (Lai-Fook, 1970,1973). In Rhodnius, the A- and B-type gland secretions may be mixed after discharge to the same effect (Wigglesworth, 1948). Almost nothing is known about how the discharge of the dermal glands is powered, nor about the way in which discharge is controlled. According to

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Lai-Fook (1973) the Verson’s glands of Culpodes are not supplied with either nerves or muscles. It might be that the glands discharge under the pressure of their own contents, or it might be that the mechanical activity of ecdysis is responsible for emptying the glands. Lai-Fook (1973) has observed that cellular processes, which fill the saccule of the Culpodes gland, are withdrawn to permit emptying of the glands, and that this might be an important event in the control of discharge. A suggestion (Lai-Fook, 1970) that cellular microtubules might reinforce the €3- glands of Rhodnius, and maintain the shape of the saccule so as to prevent premature discharge was not supported by the result of an experiment which attempted to disrupt these microtubules in the cold. Instead, Lai-Fook (1970) supports the suggestion of Wigglesworth (1947) that the removal of a plug in the gland’s duct might be important in controlling the gland’s discharge. 5.9

CHANGES I N HEART RATE

Moreau and Lavenseau (1975) measured the rate of beating of the heart and of the thoracic pulsatile organs at the bases of the wings, using implanted electrodes to record their activity. In Bornbyx rnori,prior to adult eclosion, the 3 organs beat independently with rather irregular rhythms, periods of activity being interspersed with periods of quiescence. At eclosion, the overall rate of beating is increased in all 3 organs, both by reducing and then eliminating the quiescent periods, and also by increasing the rate of beating during active periods. Unfortunately, details of changes in the rate of beating during the splitting of the exuvium, and escape from it, are not given (perhaps the placing of electrodes did not allow this), but changes in the rate of beating during the inflation of the wings are shown in Fig. 15. The rate of beating of all 3 organs increases considerably during the first two minutes of wing inflation and declines thereafter. At the peak, the rate of beating of all 3 organs is almost double the resting rate some 2 h later. Although the overall pattern of change in rate is similar for each organ, the detailed pattern of beating is independent in each. The period during which the rates of beating are elevated is quite brief (10-20 min). The increased rate of beating of the heart and the thoracic pulsatile organs is almost certainly nothing to do with the generation of an increased blood pressure. These pulsatile vessels are too flimsy to develop appreciable pressure excesses, and the elevation of blood pressure is achieved by the use of the abdominal intersegmental muscles. Blood pressure in the abdomen of Bombyx does not reach its maximum value until 10 min after the onset of inflation behaviour, and not until after 20 min in the wings (Moreau, 1974). By this time, the elevated rate of beating in the heart and the thoracic pulsatile organs has already declined substantially.

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However, the distribution of blood during inflation is probably important, and the heart and accessory hearts may influence this considerably. The heart may be involved in moving blood from the abdomen to the thorax during the early stages of inflation, and as discussed above (Section 4.3) the thoracic accessory hearts are probably important both in maintaining the flow of blood through the wing veins and in subsequently emptying them of accumulated blood.

r 150 beots per mi n

100 meso

50 heart

0

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Fig. 15 Rate of beating of the metatergal and mesotergal pulsatile organs, and of the heart during wing inflation in Bombyx mori. The beginning of wing expansion behaviour is designated t = 0. Rates of beating were monitored using implanted electrodes. (Modified from Moreau and Lavenseau, 1975)

It is not known how the increased rate of beating which accompanies wing inflation is controlled. The heart of Bombyx, like that of other insects (Miller, 1974) is probably myogenic, but subject to nervous control. In addition, a number of heart-accelerating factors from the CC have been described, at least some of which can be released into the general circulation (Kater, 1968). However, evidence that the rate of beating of the thoracic pulsatile organs in Bombyx is unlikely to be controlled by circulating factors during ecdysis was obtained by Moreau and Lavenseau (1975). Apterous insects, produced by previous ablation of wing imaginal discs, showed only a slight elevation of the rate of beating of the thoracic accessory hearts at the time when wing spreading ought to have occurred. Where partially apterous insects were produced (by ablating only some of the discs), only where the pulsatile organ was next to a normal wing was its rate of beating increased during inflation behaviour. This indicated that each organ can be separately controlled.

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In Bombyx which had undersized wings, as a result of partial regeneration of the ablated imaginal discs, the rate of beating in the thoracic organs was “proportional to the dimensions of the wings”. These observations suggest that the rate of beating of the accessory hearts may be determined either by local nervous influence, or by a direct effect of the circulatory load on the organ itself. 5.10

METABOLISM DCRING ECDYSIS

A number of authors have found that the act of ecdysis is accompanied by an increased level of metabolism, as reflected in the insect’s respiratory gas exchange. Taylor (1927) found that the pupal-adult ecdysis of a number of species of flies and moths was accompanied by a large increase in oxygen consumption, though in most cases he did not properly quantify it. Slhma (1960) saw a brief peak of oxygen uptake “just after” each ecdysis during the life of Pyrrhocoris apterus. He commented that since his results were expressed in terms of O2 consumption per unit body weight, some of this increase at ecdysis could be accounted for by the loss of weight at ecdysis when the insect shed its old cuticle. However, the increase was quite large (in the order of 30%) so that weight change would be unlikely to account for all of this. Slama speculated that the process of cuticle tanning might require extra oxygen uptake. Roussel (1963) also observed a sudden increase (about 30%) in oxygen uptake at ecdysis in Locusta. In this case, the elevated rate fell only gradually thereafter, during the course of the instar. Fourche (1 967) found a brief peak of oxygen uptake at puparium formation in Drosophila and a sharp rise again at adult emergence in both Drosophila and Bombyx. In the latter case, oxygen consumption began to increase from about 10 h before eclosion. Moreau and Gourdoux (1971) examined both oxygen uptake and carbon dioxide elimination in Pieris and Tenebrio. While Pieris showed a conventionally elevated rate of oxygen uptake at eclosion, Tenebrio actually showed a considerable decrease. Taylor (1927) noted that such a decrease sometimes (but not always) occurred in Diptera and Lepidoptera just before adult eclosion, and Houlihan and Newton (1979) observed such a decrease in the blowfly Calliphora. The decrease in oxygen uptake might be caused by obstruction of the tracheal airways during ecdysis. This cannot be due to fluid-filled tracheae (see Section 5.5) in Calliphora, since in this species fluid resorption takes place some 2-3 h before eclosion (Houlihan and Newton, 1979). At any rate, metabolism is not halted, and in Tenebrio, Moreau and Gourdoux (1971) found that the output of CO, was actually increased during the period of depressed oxygen consumption. This makes for a very high respiratory quotient, but under these conditions of disturbed gas exchange, it is doubtful if the RQ means

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very much. Pieris also showed a considerably augmented release of carbon dioxide during ecdysis. After the old cuticle has been shed, oxygen uptake remains high in Drosophila (Fourche, 1967) but this is probably not due to any ecdysisrelated metabolic activities; oxygen uptake declines very rapidly in Bombyx (Fourche 1967), Pieris and Tenebrio (Moreau and Gourdoux, 1971), to levels similar to those seen immediately before ecdysis. Thus wing expansion and cuticle tanning evidently do not require substantially more oxygen than “resting” levels of metabolism. The implication of the increased oxygen uptake and carbon dioxide output during emergence itself, is that ecdysis is a metabolically expensive process. Hughes ( 1 9 8 0 ~ observes ) that the expansional motor programme of adult ecdysis in Schistocerca is of necessity a compromise between its function in generating pressure (when spiracles are closed) and allowing respiration (when the spiracles are open). The proportion of time devoted to ventilatory strokes in each cycle of the programme is increased under artificially-induced respiratory stress (exposure to CO,), and also rises once wing expansion is completed. 5.11

POST-ECDYSIAL CELL DEATH

A number of structures which are useful only at ecdysis degenerate following its successful completion. This particularly the case for a number of muscles known to be specially concerned with ecdysis. Thus the ventral intersegmental muscles of the abdomen in Rhodnius undergo a cycle of development and involution at each moult, so that they are in peak condition just at the time of ecdysis (Wigglesworth, 1956). The structure of these muscles, which is in itself unexceptional, has been described by AuberThomay (1967), Toselli and Pepe (1965) and Warren and Porter (1969). It might be surmised that the appearance of ecdysone in the blood acts as a signal for the renewed growth and development of these muscles, but proof is lacking on this point. Anwyl and Finlayson (1973) have described neurosecretory endings in the nerves supplying these muscles and suggest that the axons have a dual motor/trophic function. If this is so, it may be that the involution of the muscles subsequent to ecdysis may be due to cessation of neurosecretory activity, and thus the lack of sufficient trophic factor(s). On the other hand, it might be that the neurosecretory products released at the time of ecdysial activity are actually the cause of involution. Anwyl and Finlayson (1974) recorded from these nerves and found spontaneous activity both near the time of ecdysis, when the muscles are functional, and during the intermoult, when they are degenerate. Some of the activity recorded on either occasion appeared to be peripherally generated, and conducted antidromically to the CNS, but the significance of this is not

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known. At any rate, since the level of spontaneous activity did not differ much according to the state of the muscles, we are left no wiser as to the nature of the control mechanism of the muscles’ cyclic regeneration and involution. This system of cyclic development has not been described for ecdysial musculature in any other insect, and may be an adaptation to Rhodnius’ life-style, which involves long periods of starvation, during which every resource must be utilized, punctuated by huge meals. Other insects, which develop more continuously, keep muscles which are involved mainly in ecdysis until it is certain that they will never be required again. Usually this means that the ecdysial musculature is maintained until the final ecdysis to the adult stage has been accomplished. Thus Locusta and other acridids have muscles in the thorax which degenerate after adult ecdysts (Thomas, 1954; Ewer, 1954; Wiesend, 1957), as is the case in crickets (Van Schreven, 1938). However, if the usefulness of a particular muscle does not continue throughout larval life, it may degenerate sooner than this. Bernays (1972b, d) describes accessory cercal muscles in newly-hatched first instar Schistocerca, which are not present in second instar larvae. These are apparently concerned with control of the brustia during the specialized first larval ecdysis (see Section 4.2) and are not subsequently required. Similar principles apply in the Endopterygotes where pupal muscles concerned with ecdysis degenerate after eclosion of the adult. Such muscles have been noted to occur in the head (Laing, 1935) and abdomen (Cottrell, 1962c) of blowflies, and in the abdomen of various Lepidoptera (Finlayson, 1956) and Coleoptera (Murray and Tiegs, 1935). Earlier phases of muscle degeneration occur immediately after puparium formation, and after head eversion in the early pupa, in Culliphora (Crossley, 1965). Similarly many larval muscles degenerate after the larval-pupal ecdysis in Saturniids and other Lepidopterans (Finlayson, 1956). Patterns of post-embryonic muscle degeneration are reviewed by Finlayson (1975). The term “caducous” has been proposed (Finlayson, 1956) to describe those muscles “destined to die” but the term has not achieved wide usage. Lockshin and Williams (1965a) called the rapid degeneration of abdominal intersegmental muscles after adult eclosion in silkmoths “programmed cell death”, which describes the phenomenon rather well. Degeneration following ecdysis is not restricted to the musculature, but also occurs in the nervous system. Following adult ecdysis in Manduca, of the 21 motorneurones which send their axons out from each abdominal ganglion via the dorsal nerve, some 13 degenerate within 3 days. Many interneurones degenerate too (Taylor and Truman, 1974). These neurones must have been concerned with typically pupal behaviour patterns, no longer appropriate to the free-living adult; probably, the motorneurones

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which degenerate are those which controlled the activity of the degenerating intersegmental muscles. In the giant silkmoth, H . cecropia, however, the histolysis of the intersegmental muscles is not accompanied by the degeneration of their corresponding motorneurones (Truman, personal communication). Post-ecdysial degeneration also occurs in other tissues. Once adult development is completed, the prothoracic glands are no longer required to secrete ecdysone, and thus may be sacrificed. But they do not degenerate until after adult ecdysis, when they do so rather quickly (reviewed by Herman, 1967). In newly-emerged adult blowflies, the upper and lower surfaces of the wings are held together by epidermal cell processes packed with strengthening microtubules. As the wings are expanded and hardened in their final shape, these processes break down, liberating large numbers of characteristic cell fragments into the haemolymph (Seligman and Doy, 1973; Seligman et al., 1975). Larval fat body cells, which persist during pupal and adult development in Drosophila, and in blowflies, break down only after the adult flies have emerged from their puparia (Wigglesworth, 1949; Whitten, 1968; Butterworth, 1972). The cellular events which lead to degeneration after ecdysis have been extensively studied in the case of the intersegmental muscles of adult silkmoths, and have been reviewed by Lockshin and Beaulaton (1974a). An early event (the primary event?) in the programmed cell death of the silkmoth intersegmental muscle is the synthesis of new RNA and protein. At this stage (0-2, h after eclosion) the muscles can still be protected from subsequent degeneration by cycloheximide and actinomycin D , inhibitors of translation and transcription respectively (Lockshin, 1969). By 2 h, the number of primary lysosomes has increased three-fold (Lockshin and Beaulaton, 1974b). This formation of new lysosomes can be inhibited by cycloheximide and actinomycin D (Lockshin and Beaulaton, 1 9 7 4 ~ )By . 7 h, there is evidence of considerable breakdown of the contractile machinery in electron micrographs of the muscles, but the fibres are still capable of contraction. They have normal resting membrane potentials, although the membrane’s input resistance is increased somewhat at this stage. The lysosomes seem not to be primarily involved with the destruction of the actomyosin complex, but rather with the breakdown of mitochondria and other cell organelles. Autophagic vacuoles containing mitochondria can be seen at 1 2 h after eclosion, but none containing myofilaments at this time. Lyosomal acid phosphatase does not appear in the cytoplasm (Lockshin and Beaulaton, 1974b) and an inhibitor of lysosomal proteases, pepstatin, fails to prevent loss of birefringence from the muscles (Lockshin, 1975a). Loss of contractility finally occurs at about 1 5 h after eclosion, and is correlated with a drastic fall in both the membrane’s resting potential and its

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input resistance (Lockshin, 1973). A marked loss of birefringence also occurs at this time, presumably reflecting major disorganization of the contractile machinery. By this time, however, the loss of muscle proteins must already have been in progress for a considerable period, since these can be identified in the blood by immunological means from as early as 3-5 h after ecdysis (Lockshin, 1975b). Morphological signs of nuclear dysfunction appear only relatively late in the process of degeneration, at about 15-20 h (Lockshin and Beaulaton, 1974b). The eventual fate of the doomed intersegmental muscles is determined long before ecdysis, at the outset of the previous round of development, being influenced by the balance of ecdysone and juvenile hormone (Lockshin and Williams, 1964). Changes in the muscles actually begin well before ecdysis, and the extent to which ecdysis itself is important in influencing the course of degeneration seems to vary considerably between different species. In Manduca, a Sphingid, there is a similar loss of abdominal intersegmental muscles after adult eclosion, but Lockshin et al. (1977) have shown that loss of muscle proteins begins as early as 4 days before eclosion. Some are lost at a faster rate than others, but the loss is greatly accelerated after eclosion. Interestingly, an ATPase of unknown function actually increases its relative activity by some 10 times in the 4 days before eclosion (Lockshin et al., 1977) and a particular isozyme of lactic dehydrogenase (LDH) also increases activity during this time, rising t o a peak at about 10 h after eclosion at a time when other respiratory enzymes are being lost at a greatly increased rate (Bidlack and Lockshin, 1976). This same isozyme does not appear in non-labile muscles, and it was suggested that its increased activity might indicate some kind of respiratory lesion in the degenerating muscles. Muscle loss in the Saturniid,A. polyphemus, is much more dependent on ecdysis itself. Here, there is a post-eclosion increase in the L D H isozyme can be blocked by cycloheximide, which also blocks breakdown. The nature of the signal acting at the time of ecdysis to promote muscle breakdown is not well understood. Originally, Lockshin and Williams (1965b) argued that the immediate cause of degeneration in the intersegmental muscles of adult silkmoths was the cessation of impulses in the muscles’ motor nerves. They found that in some cases electrical stimulation of the nerves could preserve the muscles in isolated abdomens. Injections of pilocarpine and eserine (which produce paralytic symptoms, associated with hyperactivity in the motor nerves) could also in some cases preserve the muscles (Lockshin and Williams, 196%). However, improvements in the experimental technique of isolating the abdomens revealed that there was a change in the properties of the abdomen at the time of eclosion, so that those isolated after ecdysis almost always showed normal loss of muscles, while in those isolated before eclosion, the

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muscles frequently did not degenerate (Lockshin, 1969). This change was supposed to be brought about by some centre in the head and thorax acting via the CNS, being correlated with the changes in neural activity accompanying ecdysis. As we now know (Truman and Riddiford, 1970), eclosion in silkmoths is brought about by the release from the CC of the eclosion hormone, and it is not surprising to find that this hormone is required for normal breakdown of the intersegmental muscles (Truman, 1970, 1 9 7 3 ~ )Whether . the hormone acts on the muscles via the CNS, as suggested by Lockshin’s work, or whether its action might be direct on the muscles themselves h a s not yet been satisfactorily resolved. Undoubtedly the CNS does exert some kind of trophic influence on the musculature, both during development, and in maintaining those already differentiated (reviewed by Finlayson, 1975). However, it is by no means certain that postecdysial degeneration is induced by the withdrawal of such a trophic factor, and argument about whether the trophic influence is (Lockshin, 1971) or is not (Runion and Pipa, 1970) identical with “classical” nervous activity may be quite irrelevant to the problem of muscle breakdown. The work of Lockshin and Williams (1965b, c), and Lockshin (197 1) indicating that silkmoth intersegmental musclescan be preserved after eclosion by abnormal nervous stimulation, does not prove that the action of eclosion hormone in initiating breakdown must necessarily be via the CNS. Finlayson (1975) suggested that the control of muscle degeneration may prove to be multifactional in nature, and recently L. M. Schwartz (personal communication) has shown that when abdomens of A . polyphernus are isolated before exposure to eclosion hormone has occurred, the entire abdominal CNS may be removed without provoking intersegmental muscle breakdown. However, when eclosion hormone is injected into such denervated abdomens, cell death follows rapidly, indicating that the hormone must act directly on the muscles in this case. By contrast, the breakdown of wing epidermal processes in blowflies is initiated not by the eclosion hormone, but by bursicon (Seligman and Doy, 1973). Cell breakdown can also be initiated experimentally by CAMP,and by its deaminated analogue, cIMP, indicating that an early event in the programmed cell death of this particular tissue may be the hormonally-directed synthesis of CAMPin target cells (Seligman and Doy, 1972). This is in accord with what is known of the mode of action of bursicon in promoting cuticle tanning (see Section 5.4). Nothing is known of the later biochemical events which lead to histolysis, but these must occur remarkably swiftly, since the number of cell fragments appearing in the haemolymph reaches a maximum only 75 min after ecdysis, and falls precipitately thereafter, so that by 3 h, practically none can be found (Seligman and Doy, 1973). The first sign of the degenerative process in the cells of the wing epidermis is the appearance of

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vacuoles, and the cells then fragment apparently without the aid of lysosomal action. or of phagocytic haemocytes (Seligman et al., 1975). The circulating cellular fragments are subsequently removed by phagocytic haemocytes, however. The hormonal and/or nervous signals which initiate post-ecdysial cell death in other tissues are much less well understood. Bodenstein (1953) and Wigglesworth (1955) both came to the conclusion that the degeneration of the prothoracic glands after the final ecdysis to adult required both a previous exposure to ecdysone, in the absence of juvenile hormone, and a subsequent exposure to an unknown factor which acted during adult ecdysis, or at about this time. If the glands did not experience this second event, then they did not degenerate. Wigglesworth (1955) was able to transplant prothoracic glands from pharate adult Rhodnius, up to a few hours before ecdysis, into fed adult insects, without provoking their breakdown. But if the donor insect was in the act of ecdysis when the glands were removed, then the glands would subsequently degenerate. Whatever the nature of the factor which triggers cell death, its source must be other than in the head, since decapitation of pharate adult Rhodnius on the day before ecdysis did not prevent prothoracic gland breakdown. Bodenstein’s (1953) results with Peripfaneta differ from those of Wigglesworth, in that it was possible in the case of Peripfaneta to prevent the degeneration of the prothoracic glands by transplanting corpora allata into the adult, so that the prothoracic gland cells were exposed immediately after ecdysis to juvenile hormone. Cassier and Fain-Maurel(l970) have found essentially the same antagonism in Locusta between juvenile hormone and the unknown factor acting at ecdysis. However, Wigglesworth (1955) was unable to postpone the degeneration of Rhodnius prothoracic glands by transplanting corpora allata into newly emerged adults, perhaps indicating that the histolysis-promoting factor in Rhodnius acts very quickly and irreversibly. The factor(s) which acts t o induce the lysis of larval fat-body cells in newly-ecdysed adult Diptera is not known. According to Butterworth (1972) the internal environment in young adult Drosophila is in some way “lytic” i.e. promotes breakdown. As the adult fly ages, this lytic influence decreases, so that the environment becomes progressively more “permissive”. Cell death probably involves lysosomes which accumulate in late larval life in the form of “protein granules” in the larval fat body cells. Despite a full complement of these granules, cells from 96 h old larvae degenerate only slowly when transplanted into the lytic environment of a young adult host (Butterworth, 1973). This indicates that some additional developmental change must occur during metamorphosis to render the cells susceptible to the action of the lytic factor in the adult blood. Presumably this degeneration-promoting factor is hormonal in nature, but its identity is

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unknown. Butterworth (1972) showed that the blood of young adult male upterous4 (up4) Drosophilu is less lytic than that of wild types, the larval fat-body cells dying 1.6 times faster in the latter. up4 cells are competent to degenerate at the normal rate when transplanted into a wild-type host, so that the genetic lesion must reside in the interval environment. up4 flies are female-sterile, but since Butterworth (1972) used only males as hosts, the failure of vitellogenesis cannot provide an explanation for the permissive nature of the up4 environment. It is now known that the failure of vitellogenesis in up4 is due to a deficiency of J H production in adult female flies (Postlethwaitetul 1976), and it is possible that a similarly lowered J H titre in male flies might account for the slower rate of fat body cell death. Day (1943) found that fat body cell death was greatly retarded in allatectomised blowflies (Luciliu),so that this is a reasonable hypothesis, but the possibility of other endocrinological malfunctions in up4 cannot be ruled out. Whatever the nature of the lytic factor, its action is irreversible. When fat boqy cells have been exposed to the lytic environment of a newly-emerged adult for 24 h, transplantation to an older host fails to effect any amelioration of the subsequent rate of cell death (Butterworth, 1972). 5.12

I N TE G R A T I v I: M E c H A N I s M s

During ecdysis a considerable number of distinct behavioural patterns and physiological changes occur simultaneously or in succession. It is important to the success of this potentially dangerous undertaking that each of these events should occur at the proper time in relation to the others. A considerable amount of information (reviewed above) has been gathered on the ways in which individual changes are controlled in particular species, but knowledge of how they are integrated is poor. Probably the best known case is that of the adult eclosion of the Saturniid silkmoth H . cecropia. A diagrammatic representation of the way in which ecdysial behaviour and physiology are integrated in this insect is shown in Fig. 16. Details of each of the individual control mechanisms have been given above. The principal features of the scheme are: (i) The timing of ecdysis is determined by a circadian clock in the brain, which directs the release of eclosion hormone. But the release of the hormone is dependent on the pharate insect having completed that part of its adult development which occurs within the pupal case. Thus the insect must “know” that it is “ready” (see Section 2.3). This state of “readiness” might correspond to the removal of a prohibition on the release of eclosion hormone, as directed by the circadian clock, or alternatively might be a specific direction to release the hormone at the first available gate. (ii) All the physiology and behaviour which is initiated before the insect

STUART E. REYNOLDS

568 COMPLETED

Is1 MAJOR PHASE

BEHAVIOUR

CONTEiT SENSORY

POST-ECLOSION ACTIVITY

d

w

:7”:

LABIAL GLAND SECRETION

2nd MAJOR PHASE

CUTICLE DEPOSITION

Fig. 16 Integration of behaviour and physiology during adult eclosion in large moths, based principally on H . cmropia. All the individual control mechanisms are discussed in the text. The principal features of the overall scheme are discussed in Section 5.12. A number of hypothetical control mechanisms which have not been demonstrated in large moths are grouped together and are marked with an asterisk (*)

escapes from the pupal cuticle is triggered by the eclosion hormone. The succession of the pre-eclosion and eclosion behaviours is assured by their different latencies. The group of physiological and behavioural events which are triggered by the eclosion hormone represent the first major phase of eclosion. (iii) Once the insect has escaped the pupal cuticle, its activity is determined by the sensory context in which it finds itself. The insect may walk, climb, display righting behaviour etc. Labial gland secretion is a physiological event which is likewise modulated at this time by sensory cues. This group of physiological and behavioural changes forms a “bridge” between the first major phase of eclosion, dominated by eclosion hormone, and the second major phase [see (iv) below]. But all these responses to the environment are dependent on previous exposure to the eclosion hormone, which may be said to “permit” the insect to behave appropriately. (iv) It is particularly important that the inflation and stiffening of the new

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cuticle of the wings take place rapidly, and also that these two irreversible processes are closely linked together. This is achieved by initiating a second major phase of eclosion, which is dominated by the release of bursicon. The release of the hormone is linked to the initiation of wing inflation behaviour. Here the hormone does not initiate behaviour, as is the case in the first major phase of eclosion; rather the release of the hormone may be considered to be a part of the behaviour pattern. (v) The initiation of wing spreading behaviour marks an irrevocable step taken to begin the second major phase of the eclosion process. The initiation of a number of physiological changes associated with ecdysis is delayed until the second phase in other insects, and bursicon has been shown to be the controlling agent for the initiation of post-ecdysial cuticle deposition, diuresis, and cell death in various cases. It is likely that bursicon may have similar functions in large moths, in addition to its role in controlling cuticle extensibility and initiating cuticle tanning, but this has not been specifically shown. (vi) Physiological and behavioural events at eclosion are triggered by hormones rather than continuously modulated by them. Speed is obviously the most important factor, and fine control of these processes is superfluous once they are initiated. This means that the exact titres in the blood of eclosion hormone and bursicon need not be closely controlled. Indeed, both hormones are massively released into the blood at levels far higher than necessary (Reynoldsetal., 1979). On the other hand, the timing of release is critical, and this is closely controlled. The principles on which the scheme of Fig. 16 operates may prove to be generally applicable, although the details of individual control mechanisms will probably not. The scheme is incomplete, in that a number of physiological processes which occur at eclosion have not been included, because their control is not well understood. Also the role of behavioural switches in controlling the succession of the various behaviour patterns has been left undefined. Behavioural switches are known to be important in eclosion in Manduca (see Section 3.7), but this may not be the case in H. cecropia.

6

6.1

Failures of ecdysis NATURAL FAILURES

Ecdysis is described by Carlson and Bentley (1977) as being “a mechanically difficult task” and we might expect that its performance would be accompanied by an appreciable failure rate. Very little published information seems t o be available on this point, though most of those who have kept

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insects in culture will agree that it is those insects which are already in poor condition which most often succumb during ecdysis. Failure might result from a number of causes. One kind of failure would be where the insect did not succeed even in splitting the old cuticle, remaining trapped in the exuvia. Later, and more obviously to the casual observer, failure might occur due to the insect’s inability to escape from the old cuticle, once this had been split. The insect might succeed in escaping the old cuticle but fail to expand wings or other appendages properly. Finally, tanning of the new cuticle might occur too soon, so that expansion o r escape was not yet complete, or too late or even not at all, leaving the insect vulnerable, weak and liable to desiccation. Both splitting the cuticle and the subsequent escape are activities which require sustained muscular activity and presumably considerable expenditure of energy (see Section 5.10). Richards (1957) and Dean and Hartley (1977) have interpreted poor hatching success at low temperatures in Oncopeltus fasciatus and Ephippiger cruciger respectively, as being due to the lack of a sufficient energy supply for the hatching process. However, there is no direct evidence on this point. Another cause of failure at these early stages is a low blood volume, perhaps caused by excessive water loss prior to ecdysis. Hatching failures in Schistocerca (Bernays, 1972a) and Tenebrio (Buxton, 1931) have been correlated with water loss, which presumably leads to a decreased blood volume, and Mellanby (1938) has emphasized the need for high blood volume in emerging adults of Lucilia. Loss of blood is probably the direct cause of failure in ecdysis when an insect is physically injured, perhaps by some sharp object in the environment, by a predator, or by the insect’s own efforts during ecdysis (e.g. in diflubenzuron-poisoned insects; see Section 7.2, below). Where the blood volume is low, the insect cannot exert pressure on the old cuticle properly, and thus fails to escape. In those insects where there is substantial pretanning of the body cuticle (e.g. adult Lepidoptera), escape from the old cuticle may be successful, but the subsequent inflation of the wings may be impaired. The maintenance of a high internal pressure excess during ecdysis by air-swallowing can lead to excessive bleeding in injured insects, and those damaged at this time are quite capable of more or less completely exsanguinating themselves, a fact sometimes taken advantage of when insect blood must be collected. In most cases, the behavioural programme for air-swallowing is relatively unaffected by sensory feedback and once the internal pressure begins to increase, the programme cannot be halted in response to injury. This renders the insect vulnerable, but since the tanning hormone, bursicon, has already been released by this time (Section 5.1.2), and hardening of the cuticle will follow quickly, there is no possibility of pausing

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571

to allow the wound to seal itself and prevent loss of blood. Evidently the selective advantage to be gained from rapid fixation of the new cuticle’s size and shape is greater than the selective disadvantage of vulnerability to injury. The inflexibility of the air-swallowing programme can lead to a curious kind of ecdysial failure. Bernays (1972) reports that at the 1st larval ecdysis, Schistocerca will swallow water or even mineral oil instead of air once swallowing is under way. How often such “mistakes” occur under natural conditions can only be the subject of speculation, but one might expect that such an error would have fatal consequences. Failure to shed the old cuticle properly may result from aberrant splitting of the exuvium. Blowfly puparia may sometimes split in the middle or at the rear, instead of at the usual anterior end, when they are laterally compressed. The pharate adult is then generally unable to escape from the puparial case. Similarly in large moths, if the cuticle over the tip of the abdomen breaks away from the rest of the pupal case, the insect is generally unable to shed the remainder of the exuvia. In Manduca, sometimes the facial mask of the pupal cuticle breaks so that although the legs are free, the head and proboscis are still enclosed. When this occurs, the insect is unable to remove it. This generally happens in those insects which are undersized and in poor condition, but accidents in handling healthy individuals can lead to the same result. Lepidopterous larvae occasionally fail to escape completely from the old cuticle, which remains bunched up around the posterior part of the body. As the larva continues to feed and increases in size during the instar, the exuvium fails to stretch to accommodate the caterpillar’s increased girth, and eventually becomes a ligature, preventing the passage of food along the gut. Death usually ensues. All these are casual observations. No published information on the causes of ecdysial failures, under natural, as opposed to laboratory conditions has come to my attention. The only work that I know which deals in a quantitative way with failures of ecdysis in the field, is that of Corbet (1957) on the dragonfly, Anax imperator, living on a pond in Berkshire, England. Corbet found that the incidence of “incomplete ecdysis” (i.e. where the exuvia was not shed) was “very low” when the numbers of adult insects emerging on any one night was low. However, when the number was high, then there was competition for ecdysis sites, and insects which emerged early were likely to be disturbed by those following later. Corbet concluded that individual incompetence was seldom the cause of ecdysial failure, and that overcrowding was the chief factor. On the modal night of the first peak of ecdysis, failure to shed the exuvia accounted for the loss of 2.4% of the day-group. But the average over the year was much less (see Table 4).

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E. REYNOLDS

Corbet does not comment on the causes of his “incomplete ecdyses”, and indeed such information would be very difficult to gather. TABLE 4 Minimum estimates of mortality during ecdysis in amatural population of Anax imperator (from Corbet 1957) % mortality

1 Incomplete ecdysis (i.e. failure to shed exuvium) 2 Incomplete expansion of wings 3 Predation by birds Total (Total number emerging)

1952

1953”

1.2

1.9

4.3 3.0

8.9 5.0 15.8 (2944)

8.5

(4368)

Corbet ascribes higher mortality during 1953 mainly to a period of exceptionally cold weather, with high winds, which coincided with the main peak of emergence

In the case of the adult silkmoth, having extricated itself from the old cuticle, the insect must still escape the confines of a cocoon. A bizarre example of ecdysial failure is given by Waldbauer and Sternberg (1976) for H . cecropia, of which they found a number of individuals which had been trapped in the cocoon, because the “escape valve” had been blocked by foreign objects. These were seeds which the authors speculated might have been inserted by birds. Newly-ecdysed insects digging through the substrate can sometimes be misled into following unproductive paths in their efforts to reach the surface. Although gravity is important to their orientation, newly hatched vermiform larvae of Schistocerca will follow the line of least resistance. Normally this is provided by the foam plug of the egg pod (Ewer, 1977), but in sand or soil life may not be so simple. Bernays (1971) shows an example of a larva which followed a circular path in the vertical plane of an experimental sand bed, making at least twelve revolutions during the period of observation. Since digging vermiform larvae of locusts, like newly emerged adult flies (Fraenkel, 1935a) and Sphingid moths (Truman, 1973b), can delay bursicon release for considerable periods whilst digging, such mistakes may not have very serious consequences. Much worse, however, is the error which many adult blowflies make when emerging from puparia packed closely together, as they often are in the laboratory. Here, it frequently occurs that the newly emerged fly creeps out o f its own puparial case headfirst straight into another empty one. This is invariably fatal since the new case cannot be split and the digging programme does not provide for reversing the direction of movement.

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573

Digging might well be thought to be a somewhat hazardous occupation for newly-emerged adult flies and moths, which have soft, unexpanded wings which are easily torn. The delay of cuticle plasticization in flies (which does not occur until air-swallowing begins; Cottrell, 1962d) might be explained partially in terms of the advantage to be gained in preventing the premature distortion and/or injury of the wings during digging. However, in Manduca, although wing cuticle plasticization at the time of wing spreading does still occur in response to bursicon, it is preceded by a plasticization of the cuticle which is due to eclosion hormone, released some time before ecdysis (Reynolds, 1977). This is puzzling as it might well be imagined that the wings would need to be less extensible at this time, rather than more so. An important component of failure during metamorphic ecdyses must be failure to expand the wings properly. As mentioned above, lack of blood can seriously impair this process, but perhaps the most important cause of failure here is simple, mechanical interference. Insects appear to take some care in seeking out a suitable site for this hazardous activity. Wing spreading does not generally begin until a vary variable, preceding phase of appetitive behaviour has taken place. Very little is known about the criteria the insect uses to “‘decide” on an appropriate spot. For large moths, such a place is generally a vertical, o r near-vertical, surface which can be clung to with a sufficiently firm grip, and which offers a suitably large air-space into which the inflating wings may be expanded. Locusts prefer a perch a t approximately 45 degrees to the horizontal (Hughes, 1980a), while dragonflies (Anax) prefer a vertical one (Corbet, 1957). If the insects are dislodged from these carefully chosen positions during the wing inflation process, disaster almost always ensues, and the wings will be deformed. In fact, the insects are usually very difficult to disturb during this time, and even quite gross stimuli (pinching etc.) fail to cause them to take evasive action (Hughes, 1980a). Nevertheless, in a crowded laboratory culture, interruptions not infrequently occur, so that deformed wings can usually be seen in such circumstances. Corbet (1957) observed in his study ofAnax that incomplete expansion of the wings was a major cause of mortality at emergence. As many as 16%of a day group could perish in this way. Disturbance by other emerging dragonflies was almost certainly the main cause, and thus failure was density-dependent.The average loss from this cause over the whole year was much less (see Table 4). Corbet also ascribed this kind of failure to low temperatures and to dislodging by wind. The silkmoth A. pernyi is apparently unable to delay the release of bursicon after it ecloses (author’s unpublished work) and consequently it must begin to inflate its wings as soon as possible before their cuticle begins to harden. Wing-spreading behaviour will take place in this species in the most unsuitable circumstances, as for instance, when the moths are allowed

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to eclose into glass vials (Truman, 1973b). Evidently this insect does not have to cope with delays between eclosion and subsequent wing inflation in its normal life obtside the laboratory. It may be that environmentaI factors can directly affect the success of wing inflation. Van den Heuvel (1963) noticed that in mosquitoes emerging at low temperatures, the wings became longer during inflation than they did at higher temperatures, presumably because the cuticle has longer in which to expand at the lower temperatures because the process of cuticle tanning, which puts a stop to further extension, is slowed down by the lower temperature. Relative humidity may be important also. Where this is too low, the expanding wing cuticle may harden prematurely. Pittendrigh (1958) has suggested that the rhythm of adult eclosion in Drosophila (which emerges principally at about dawn) may serve to synchronize emergence with periods of high environmental humidity and thus to limit water loss. Apparently in D . pseudoobscura eclosion is more successful at higher values of R.H. Nutritional factors may also affect the subsequent success of wing spreading. It is well-known to those who rear insects on artificial diets that a deficiency of linolenic acid can lead to poor success at the imaginal ecdysis, the major effect being on the inflation of the wings (see for example, Chippendale et al., 1964, 1965). The reason for this effect is obscure. As a result of the need to stay still during ecdysis, and also perhaps because of the soft, new cuticle of the emerging insect, predation is probably a considerable hazard for ecdysing insects. Doubtless this is one of the selective pressures which keeps the total time required for the process short, despite some of the dangers resulting from this (see above). Reiter and Jones (1976) have suggested also, that one of the functions of the light-entrainable circadian rhythm of eclosion shown by mosquitoes (and other insects) might be to “prevent eclosion at a time when the new adults are particularly vulnerable”. Again, there is very little information available on the extent of such predation. Corbet (1957) give estimates of the mortality of Anax due to predation. The major predators at his site were a pair of blackbirds, although newts took a small number of larvae on their way to the shore, and some newly-emerged adults which were dislodged into the water. Corbet suggests that the birds probably take a given number of insects each year, working close to their asymptotic capacity, so that mortality does not increase with population density. Mortality can be high. O n day 6 of the emergence season in 1953, a minimum of 43 emerging adults, out of a total of 193 (i.e. 22%) were taken by the birds. But over the year, the loss was less (see Table 4). Nagamine et al. (1975) give life-tables for the cicada, Mogannia iwasakii, which include details of deaths due to predation by ants, during and just after

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575

the final ecdysis. According to W. F. Humphreys (personal communication) Australian wolf spiders, Geolycosa godefroyi, which live in burrows, spin silk doors to their holes before ecdysing. When the door is damaged, predation by ants (which do not normally attack the spiders) ensues. An interesting case of predation in this context would be cannibalism within a group of insects all hatching together. Needless to say, insects take care to avoid this, and Eisenberg and Hurd (1977) have described the strategy of hatch timing adopted by the mantid Tenodera d i f o f a sinensis in order to minimize such wastage. Fungal infections can result in death at any time, but some insects fail to complete ecdysis as a result of such infections. This could be due to a number of causes, but Zacharuk (1973) suggests that the old cuticle may be attached to the new by fungal hyphae which penetrate them both. In those insects which manage to shed the exuvium normally after surface inoculation with fungal conidia, it is probable that ecdysis occurs before the secondary appressoria become attached to the new cuticle.

6.2

EXPERIMENTALLY-INDUCED FAILURES

The lethal effects of insect growth regulators (IGRs) are characteristically expressed some time after the contact of the target insect with the poison, rather than immediately, or at least soon after, as is the case with “conventional” insecticides. As it happens, these delayed lethal effects are most often seen when the insect subsequently undergoes ecdysis. The largest group of IGRs is that based on the juvenile hormones. The application of natural juvenile hormones, or of synthetic JH analogues, interferes with ecdysis. Slhma et al. (1974) comment that the “inability of insects to cast off the old cuticle during ecdysis represents the most common side effect associated with juvenoid action”. They distinguish three types of ecdysial failures: ( a ) the old cuticle remains fixed to the body at the tips of the legs, tip of the abdomen or the distal parts of the wings. Otherwise normal; ( 6 ) the old cuticle is digested and split, and the new cuticle is properly formed; ecdysial movements occur, but the exuvia is not shed. The insects quickly desiccate and die; ( c ) complete absence of ecdysial behaviour. The first two of these are probably due to the disturbed biometrical relations of the separate parts of the pharate integument. The larger the dose of juvenoid, the greater is the morphological change and the higher is the incidence of ecdysial failure. It is significant that Slhma et a f . (1974) found that for treated Pyrrhocoris, the highest incidence of ecdysial failure was found in those cases where the pharate insect had about 50% adultoid

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morphology, i.e. those individuals which deviated most from both old and new developmental stages. Slima et al. (1974) did not observe an increased incidence of ecdysial failure in early larval instars when these were treated with juvenoids, but in successfully ecdysed extra-larval instars there was subsequently a very high (65%) rate of failure at the next extra-larval ecdysis. The @)-type effect, in which ecdysis is not even attempted, apparently occurs mainly in pupal stages. It is not necessarily fatal. Some insects, like Dermestes vulpinus, have a tremendous ability to survive within the old cuticle and will, according to Slima et al. (1974) go on to produce multiple extra-pupal cuticles one within the other. A real ecdysis never occurs. Slfima et al. (1974) ascribe the (c)-type effect to the necessity in pupaladult ecdyses for a special ecdysial musculature. Perhaps the juvenoids prevent proper development of these muscles. In these cases, they say that only adultoids with less than about 30% morphological change are able to escape. There is some support for this idea from the results of Bhaskaran (1972). He distinguished two groups of affected insects when he treated developing adults of Sarcophaga bullara with an unidentified “JH”. The larger group all showed some degree of inhibition of differentiation. The main effects were on the formation of abdominal adult structures, probably due to the inhibitory effects of JH on the spreading and differentiation of the abdominal imaginal histoblasts, which are responsible for the formation of the adult cuticle. Since escape from the puparium is effected by abdominal muscle action (see Sections 4.1 and 4.2), interference with the development of abdominal structures must be expected to lead to a failure to escape from the puparium. However, Bhaskaran (1972) also identified a second type of affected insect, which appeared morphologically normal, but which nevertheless failed to emerge, indicating that “some process associated with emergence is inhibited”. Spielmann and Skaff (1967) also observed inhibition of adult ecdysis in JH-treated mosquitoes, and Madhaven (1973) in Drosophila. Madhaven suggested that an effect on the production or secretion of an eclosion hormone-like factor might be involved in the last case, although this must be regarded as somewhat speculative in the present absence of evidence for any such factor in Diptera. The effects of J H on ecdysis also extend to hatching. Street (1978) treated eggs of Pieris brassicae with J H analogues and found inhibition of embryonic development in many cases. But where development did proceed as far as hatching, deaths were only found to occur at the time of hatching or at subsequent larval ecdyses. Another class of IGR is that based on chemicals which inhibit the syn-

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thesis of chitin. Diflubenzuron (Dimilin; PH 60-40; T H 6040) is the prototype. This compound’s effects are also most characteristically expressed at the ecdysis subsequent to treatment, when failure occurs. This is due to a reduction in the strength of the cuticle (Hunter and Vincent, 1974) which is caused in turn by the cuticle’s reduced content of chitin (Post and Vincent, 1973; Hunter and Vincent, 1975; Sowa and Marks, 1975; Ker, 1977). The severity with which ecdysis is affected depends on dosage and length of exposure. The most severely affected locusts examined by Ker (1 977) were unable t o split the exuvium (which was normal). Less severely affected animals might manage to split the old cuticle, but would tear the new arthrodial membranes of the basal joints of their legs. Some locusts would shed the exuvia successfully, but would be unable to expand their wings because of tears in the cuticle. L. Clarke (personal communication) finds that untanned cuticles, such as arthrodial membranes, are particularly severely affected by diflubenzuron, since it seems that protein secretion cannot occur without the concommitant chitin secretion, and as a result cuticle deposition is inhibited more or less completely. This is like the situation in the peritrophic membranes of locusts (Clarke et al., 1977). Another experimental treatment which has profound effects on the success of ecdysis is X-irradiation. In Dipteran pupae, there are striking similarities between the effects produced by X-rays and those induced by J H treatment (Sivasubramanian et al., 1970a, b; Bhaskaran, 1972; Sasaki and Sakka, 1976). Both can result in an inhibition of differentiation to a varying extent depending on dosage and time of exposure. In both cases, the abdomen is particularly sensitive because the abdominal imaginal histoblasts differentiate later than the discs of the head and thorax. In both cases, there is a sharp decline in sensitivity after the eversion of the head when abdominal differentiation is well on its way. The failure of abdominal structures to develop after X-irradiation is likely to lead t o these insects being unable to eclose, because of the lack of the necessary musculature (Sivasubramanian et al., 1970a), just as has been suggested for JH-treated insects (see above). Also as in the case of JH-treatment, X-irradiation can lead to the production of pharate adults which appear to be morphologically normal, but which are unable to eclose (Sivasubramanian et al., 1970b). Partial irradiation studies have shown that the target mechanism for this inhibition of eclosion is located in the anterior one third of the body, i.e. the head or thorax. The developmental state of the abdomen may not be very important in determining whether eclosion can be attempted (Nair et al., 1967). Bhaskaran (1972) speculates that the similar inhibition of eclosion by J H might be due to its action on the same target in the head and/or thorax. Ely and Jungreis (1977a, b) studied the effects of X-rays on pupae and

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developing adults of M . sexta and came to similar conclusions. Here the target for X-irradiation could be localized more exactly, and it was found that although the abdomen was relatively insensitive, both thorax and head were each individually sensitive to X-rays (Ely and Jungreis, 1977b). The most sensitive period (7 days after the pupal ecdysis), coincides well with the time of maximum ecdysone titre in the blood; also radiosensitivity could be increased at other times by injecting ecdysone 24 h previously. Evidently the target mechanism is some developmental event which is initiated by ecdysone. EIy and Jungreis speculate that the target in the head might be the development of the eclosion hormone secreting system, and present some evidence that the content of eclosion hormone in irradiated insects may be reduced. But this cannot explain the existence of a target in the thorax, as Ely and Jungreis (1977b) point out. Obviously some other target must be present. This is not the thoracic musculature, so that neural circuitry in the CNS seems likely. At an earlier stage, X-irradiation can delay pupation in the Coleopteran, Tenebrio molitor (Jayaraman and Ducoff, 1970) in the Lepidopteran Ephestia kuhniella (Kuzin et al., 1968) and in the Dipteran Ceratitis capitata (Cals-Usciati, 1971a, 1972). This seems to be due to the inhibition of ecdysone secretion (Cals-Usciati, 1971a, 1972) rather than an effect on the events of ecdysis themselves. X-rays also affect puparium formation in flies, causing a delay in the time at which this occurs, when the larva is irradiated before the time of ecdysone release (Sivasubramanian, et al., 1974b). These authors confirmed previous suggestions (Villee 1946; Bourgin et al., 1956; Horikawa and Sugahara, 1960) that the delay could be caused by damage to the ring gland, which secretes ecdysone, but also discovered other components in the response to irradiation. In particular, irradiation of the hind part of the body alone could delay pupation, the delay being proportional to the area irradiated. This was interpreted in terms of damage to the mechanism whereby ecdysone (aecdysone) is converted to 20-hydroxyecdysone (p-ecdysone). Perhaps of more relevance here is the observation by Sivasubramanian et al. (1974b) that not only was pupation delayed when larvae were X-irradiated before ecdysone release, but (at doses above 2500 R) the shape of the resulting puparia was deformed, due to the inhibition of anterior segment retraction and cuticle shrinkage. Irradiation after ecdysone release did not cause these effects, so that the target mechanism must be completed by this time. The target was found to be located in the anterior one third of the larva, probably in the CNS. Sivasubramanian et al. (1974b) interpret these findings in terms of interference with a hypothetical CNS mechanism which controls the neuromuscular events of puparium formation, several hours later. This mechanism is distinct from the synthesis and secretion of

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the neurosecretory factors X, X, (Fraenkel et al., 1972) which control anterior retraction and cuticle tanning, because these factors were found to be manufactured and released normally in the irradiated larvae, which nevertheless produced mis-shapen puparia. But no further clues to the nature of this CNS mechanism were obtained. Acknowledgements

I would like to thank Oliver Dominick, Larry Schwartz and Paul Taghert for discussing some of the ideas presented here, and particularly Jim Truman for reading and commenting on the manuscript. Thanks are also due to Tim Hughes for lending me a copy of his Ph.D. thesis, and showing me the MSS of several papers. References Allum, R. C. (1973). Surgical interference with the anterior stomatogastric nervous system and its effect upon growth and moulting in Locusta migratoria migratorioides R. and F. Ph.D. thesis, University of Nottingham Anderson, S. 0. (1973). Comparison between the sclerotisation of adult and larval cuticle in Schistocerca greguriu. J. Insect Physiol. 19, 1603-1614 Anderson, S. 0.(1974). Cuticular sclerotisation in larval and adult locusts, Schistocerca gregaria. J . Insect Physiol. 20, 1537-1552 Anderson. S. 0. (1979). Biochemistry of insect cuticle. Ann. Rev. Ent. 24, 29-61 Anwyl, R. and Finlayson, L. H. (1973). The ultrastructure of neurons with both a motor and a neurosecretory function in the insect Rhodniusprolixus. 2.zellforsch. mikr. Anat. 146, 267-274 Anwyl, R. and Finlayson, L. H. (1974). Peripheral and centrally generated action potentials in neurons with both a motor and a neurosecretory function in the insect Rhodnius prolixus. J. comp. Physiol. 91, 135-145 Auber-Thomay, M. (1967). Modifications ultrastructurales au cours de la dCgCnCrescence et la croissance de fibres musculaires chez un insecte. J. microscopie 6,627-638, Barbier, R . (1968). Etude de la glande Verson chez Galleria mellonella (LCpidoptkre; Pyralidae) au cours du cycle cuticulaire: histologie, cytochimie et microscopie Clectronique. C. R. Acad. Sci. Paris 267, 522-525 Bates, M. (1943). Mosquitoes as vectors of Dermatobia in eastern Colombia. Ann. Ent. SOC.A m . 36, 21-24 Beament, J. W. L. (1964). The active transport and passive movement of water in insects. A d v . Insect Physiol. 2, 67-129 Bellesme, J. de (1877). Phenomknes qui accompagnent la metamorphose chez la Libellule dCprimCe. C . R . Acad. Sci. Paris 85, 448-450 Bennet-Clark, H. C., (1962). Active control of the mechanical properties of insect endocuticle. 8, 627-633. Bennet-Clark, H. C. (1963). The relation between epicuticular folding and the subsequent size of an insect. J. Insect Physiol. 9, 43-46

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Truman, J. W. and Endo, P. T. (1974). Physiology of insect ecdysis: neural and hormonal factors involved in wing-spreading behaviour of moths. J . exp. Biol. 61, 47-56 Truman, J. W., Fallon, A. M. and Wyatt, G. R. (1976). Hormonal release of programmed behavior in silkmoths: probable mediation by cyclic AMP. Science, Wash. 194, 1432-1434 Truman, J. W., Mumby, S. M. and Welch, S. K. (1980). Involvement of cyclic GMP in the release of stereotyped behaviour patterns in moths by a peptide hormone. 1. exp. Biol. 84, 201-212 Truman, J. W. and Riddiford, L. M. (1970). Neuroendocrine control of ecdysis in silkmoths. Science, Wash. 167, 1624-1626 Truman, J. W. and Riddiford, L. M. (1974a). Physiology of insect rhythms, 111. The temporal organization of the endocrine events underlying pupation of the tobacco hornworm. J. exp. Biol. 60, 371-382 Truman, J. W. and Riddiford, L. M. (1974b). Hormonal mechanisms underlying insect behaviour. Adv. Insect Physiol. 10, 297-367 Truman, J. W. and Riddiford, L. M. (1977). Invertebrate systems for the study of hormonal effects on behaviour. Vitam. Horrn. 35,283-352 Truman, J. W. and Sokolove, P. G. (1972). Silkmoth eclosion: hormonal triggering of a centrally programmed pattern of behavior. Science, Wash. 175, 14911493 Vacca, L. L. and Fingerman, M. (1975a). The mechanism of tanning in the fiddler crab, Uca pugilator, I. Tanning agents and protein carriers in the blood during ecdysis. Comp. Biochem. Physiol. 51B, 475-482 Vacca, L. L. and Fingerman, M. (1975b). The mechanism of tanning in the fidler crab, Uca pugilator, 11. The cyclic appearance of tanning agents and attached carrier proteins in the blood during the moulting cycle. Comp. Biochem. Physiol. 51B, 483-488 Vandenburg, R. D. and Mills, R. R. (1974). Hormonal control of tanning by the American cockroach: cyclic AMP as a probable intermediate.J . Insect Physiol. 20, 623-627 Van den Heuvel, M. J. (1963). The effect of rearing temperature on the wing length, thorax length, leg length and ovariole number in the adult mosquito,Aedes aegypti (L). Trans. R. Ent. Soc. Lond. 115, 197-216 Van der Kloot, W. G. and Williams, C. M. (1953). Cocoon construction by the cecropia silkworm, I. The role of the external environment. Behaviour 5,141 -1 56 Van Emden, F. 1. (1 946). Egg bursters in some more families of polyphagous beetles, and some general remarks on egg bursters. Proc. R. Ent. SOC.Lond. A 21,89-97 Verson, E. (1890). Hautdriisensystem bei Bombyciden. Zool. Anz. 30, 118-120 Villee, C. A. (1946). Some effects of x-rays on development in Drosophila. J. exp. ZOO/.101, 261 -280 Vincent, J. F. V. (1971). Effects of bursicon on cuticular properties in Locusta migratoria migratorioides. J. Insect Physiol. 17, 625 -636 Vincent, J. F. V. (1972). The dynamics of release and the possible identity of bursicon in Locusta migratoria rnigratorioides. J . Insect Physiol. 18, 757-780 Vincent, J. F. V. and Hillerton, J. E. (1979). The tanning of insect cuticle - a critical review and a revised mechanism. J . Insect Physiol. 25, 653-658 Von Knorre, D., Gersch, M. and Kusch, T. (1972). Zur Frage der Beeinflussung des “tanning” phanomens durch zyklisches-3‘, 5’-AMP Zool. Jb. (Physiol). 76, 434-440

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Wainwright, S. A., Biggs, W. D., Currey, J. D. and Gosline, J. M. (1976). “Mechanical Design in Organisms”. Halsted Press, New York Waldbauer, G. P. and Sternburg, J. G . (1976). Emergence of Hyalophora cecropia (Saturniidae) blocked by seeds in the cocoon valve. 1. Lep. SOC.30, 131-132 Warren, R. H. and Porter, K. R. (1969). A n electron microscope study of differentiation of the molting muscles of Rhodnius prolixus. A m . J . Anat. 124, 1-30 Way, M. J. (1950). The structure and development of the larval cuticle of Diataraxia oleracea (Lepidoptera). Quart. J . micr. Sci. 91, 145-182 Wharton, D. R. A . Wharton, M. L. and Lola, J. (1965). Blood volume and water content of the American cockroach, Periplaneta americana (L). Methods and the influence of age and starvation. J . lnsect Physiol. 11, 391-404 Wheeler, R. E. (1963). Studies on the total haemocyte count and haemolymph volume in Periplaneta americana (L) with special reference to the last moulting cycle. J . Insect Physiol. 9, 223-235 Whitehead, D. L. (1969). New evidence for the control mechanism of sclerotization in insects. Nature, Lorid. 224, 72 1-723 Whitehead, D. L. (1971). Some evidence for the likely mechanism of action of the hormone that initiates sclerotization. In “Insect Endocrines” (Eds V. J. A. Novak and K . Slama) pp. 157-166. Suppl. Acta ent. Bohemoslov. Academia, Prague Whitten, J. M. (1968). Metamorphic changes in insects. In “Metamorphosis: a Problem in Developmental Biology” (Eds W. Etkin and L. I. Gilbert) pp. 43-105. Appleton-Century-Crofts, New York Wigglesworth, V. B. (1930). A theory of tracheal respiration in insects. Proc. R . SOC. Lond. B 106,229-250 Wigglesworth, V. B. (1933). The physiology of the cuticle and of ecdysis in Rhodnius prolixus (Triatomidae, Hemiptera); with special reference to the function of the oenocytes and of the dermal glands. Quart. J . micr. Sci. 76, 270-318 Wigglesworth, V. B. (1938). The absorption of fluid from the tracheal system of mosquito larvae at hatching and moulting. J . exp. Biot. 15, 248-254 Wigglesworth, V. B. (1947). The epicuticle of an insect, Rhodnius prolixus (Hemiptera). Proc. R . SOC. Lond. B 134, 163-180 Wigglesworth, V. B. (1949). The utilization of reserve substances in Drosophila during flight. J . exp. Biol. 26, 150-163 Wigglesworth, V. B. (19.53).Surface forces in the tracheal systemof insects. Quart. J . micr. Sci. 94, 507-522 Wigglesworth, V. B. (1955). The breakdown of the thoracic gland in the adult insect Rhodnius prolixus. J . exp. Biol. 32, 485-491 Wigglesworth, V. B. (1956). Formation and involution of striated muscle fibres during the growth and moulting cycles of Rhodniusprolixus. Quart. J . micr. Sci. 97, 465-480 Wigglesworth, V. B. (1970). Structural lipids in the insect cuticle and the function of the oenocytes. Tissue & Cell 2, 155-1 79 Wigglesworth, V. B. (1972). “The Principles of Insect Physiology”, 7th edn. Methuen, London Wigglesworth, V. B. (1975). Distribution of lipid in the lamellate endocuticle of Rhodnius prolixus (Hemiptera). J . Cell. Sci. 19, 439-457 Wigglesworth, V. B., and Gillett, J. D. (1936). The loss of water during ecdysis in Rhodnius prolixus Stil. Proc. R . Ent. SOC.Lond. A 11, 104-107 Woodring, J. P., Clifford, C . W., Roe, R. M. and Mercier, R. R. (1975). Relation of

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blood composition to age in the larval female house cricket, Acheta domesticus. J. Insect Physiol. 23, 559-568 Wright, S. (1977). Photoperiod, body size and the neuroendocrine system: integrated factors controlling onset of the moulting cycle in Heliconius melpomene L. (Lepidoptera). Ph.D. thesis, University of Nottingham Zacharuk, R. Y. (1973). Penetration of the cuticular layers of Elaterid larvae (Coleoptera) by the fungus Metarrhizum anisopliae, and notes on a bacterial invasion. J. Invert. Pathol. 21, 101-106 Zdarek, J., and Fraenkel, G. (1972). The mechanism of puparium formation in flies. J. exp. Zool. 179, 315-324 Zelazny, B. and Neville, A. C. (1972). Endocuticle layer formation controlled by non-circadian clocks in beetles. J . Insect Physiol. 18, 1967-1979 Zimmerman, W. F., and Ives, D. (1971). Some photophysiological aspects of circadian rhythmicity in Drosophila. In “Biochronometry” (Ed. M. Menaker) pp. 381-391. Nat. Acad. Sci. Press, Washington

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Subject Index Acarid mites, desmosomes in, 80 Accessory collateral gland, septate junctions in, 63 Acetylcholine binding to Musca domestica head extracts, 225 effect on cell bodies of Periplaneta central neurones, 260 on electrophysiology of the sixth abdominal ganglion of Periplaneta, 251 on Periplaneta fast coxal depressor motoneurone, 263 on salivary gland stimulation by biogenic amines, 41 1 on single neurones, 254-258 in cholinergic system, 217 inhibition of a-bungarotoxin binding by, 229 presynaptic receptors at neuromuscular junctions, 392 receptors, 215-316 in development, 282-288 putative, biochemical characterisation, 218-240 Acetylcholinesterase in cholinergic system, 217 structural gene in Drosophila for, 281 Acetyl-P-methylcholine, effect on sixth abdominal ganglion of Periplaneta, 251,258 N-Acetyltransferase, biogenic amine inactivation in, 362-363 Acheta Deutocerebrum, biogenic amine cell localization in, 342 ecdysis, blood volume and, 553 Acheta domesticus biogenic amine, distribution, 323 cell localization, 332 corpora pedunculata, biogenic amine distribution in, 333 dorsal midline neurones, octopamine and, 365

DUMDL cells, 371 ecdysis, escape from cuticle, 524 globuli cells, 334 heart preparations, biogenic amine effect on, 418 5-HT distribution in, 325 terminal abdominal ganglion extracts, 3H-quinuclidinyl benzilate binding component, 240 ACTH, gap junction permeability and, 109 Actinomycin D, PTF induced tanning and, 545 Adenosine monophosphate 3',5'-cyclic, gap junction permeability and, 107 in cuticle hardening, 544 post ecdysial cell death and, 565 Adenylate cyclase activity, biogenic amines and, 436-445 function, 444-445 in fireflies, cellular location, 402 light organ, 442 Adhesion desmosomes and, 83 gap junctions and, 100-101 intercellular, septate junctions and, 69 tight junctions and, 141-142 Adrenal cortex, septate junction occurrence in, 67 Adrenal medulla, mammalian, acetylcholine receptors, 276 Adrenaline application to salivary glands, 408 function in corpora cardiaca, 433 in central nervous system, 320 in insect nervous system, 321 stimulation of Photuris pyralis light organs, 397 ADTN, adenylate cyclase activity and, 442 Aedes comb desmosome, 54

597

598

Aedes-(contd. ) hatching, developmental readiness, 480 timing, 477 scalariform junctions, thin section appearance, 159 trachael air filling in ecdysis, 547 Aedes aegypti ecdysis, bursicon and, 542 cuticle inflation, 529 Aedes taeniorhynchus, ecdysis, circadian rhythms and, 480 Aequorin, 104 Aeschna cyanae biogenic amine cell localization, 332 eclosion hormone in, 531 Aeschna viridis, biogenic amine cell localization, 332 Agabus, hatching, timing, 477 Agenius zebra, ecdysis, cuticle inflation, 530 Air filling, trachea, in ecdysis, 546-549 Air swallowing in adult eclosion. 500 in ecdysis, 509 failure in ecdysis and, 570 P-Alanine, biogenic amine conjugation by sulphates in, 363 Alcohols in insect cuticular lipids, 23 Aldehydes in insect cuticular lipids, 23 Amacrine cells, 342 Amidephrine, effect on salivary gland stimulation by biogenic amines, 410 Amino acids, cell lo cell transfer, 86-87 Aminophylline, firefly light organ stimulation by, 400 Ammonium, hydroxyphenyltrimethyl-, interneurone synaptic transmission and, 253 Ammonium, 2isothiocyanato-ethyltrimethyl-, iodide, receptor actions, 292 Ammonium, 4-

SUBJECT INDEX

Anabolia nervosa biogenic amine distribution, 323 dopamine cell, 375 5-HT distribution in, 324 Anacridium aegyptium, myogenic rhythm, function, 380 Anax imperator, failures in ecdysis, 571, 572 Annelid worms desmosomes in, 82 gap junction in, 97, 103 septate junction in, 65 Anolis carolinensis, axo-glial tight junction-like associations, 154 Anopheles, heart, innervation pattern, 414 Antenna, septate junctions in, 63 Anterior retraction factor, ecdysis and, 535 Antheraea spp., eclosion, 498 Antheraea pernyi ecdysis, bursicon and, 542 circadian rhythms, 478 eclosion, 497, 498 behavioural switching and, 518 eclosion hormone in, 530 Antheraea polyphemus eclosion, 497, 498 behavioural switching and, 518 Antheraea yarnamai, hatching, developmental readiness, 480 Apis deutocerebrum, biogenic amine cell localization in, 342 protocerebral bridge, biogenic amine cell localization, 338 scalariform junctions, thin section appearance, 159 Apis mellifera N -acetyltransferase in, biogenic amine inactivation and, 362 biogenic amine inactivation in, 360 corpora pedunculata, biogenic amine distribution in, 332 (N-maleimido)-5-benzyltrimethyl-, optic lobes, biogenic arnine cell localiodide, in binding studies of ization, 338 acetylcholine tritocerebrum, biogenic amine cell receptors, 219 localization in, 344 Amphetamine, stimulation of Photuris Apis mellifera carnica, mushroom pyralis light organs, 397 bodies, function, 336

S U B J E C T INDEX

599

Autonomic ganglia Aplysia avian, acetylcholine receptors, 276 adenylate cyclase activity, octopamine mammalian, acetylcholine receptors, and, 443 276 catecholamine synthesis in, 351 Autoradiographic localization, binding neuromuscular junctions, biogenic sites, 240 amines and, 390 Auximon, 552 neuromuscular transmission, 5-HT Axo-glial junctions and, 384 neurones, acetylcholine receptors, smooth septate-like junctions, 155 tight junction-like appositions, 272,273,275 152-155 Aplysia californica, putative acetylAxons, septate junctions in, 63 choline receptors, pharmacological profiles, 2,33 Barrier functions, tight junctions and, Apomorphine, effect on salivary gland 142- 144 stimulation by biogenic amines, Bees wax, dielectric constant, 27 410 Behaviour APUD cells, 436 in ecdysis, integration of physiology Arachnida, septate junction in, 65 and, 475-595 A ren i vaga in vrstigata , transpiration , physiology and, 530-569 temperature and, 12 Belt desmosome See Zonula adhaerens ARF See Anterior retraction factor Aromatic amino acid decarboxylases, Benzoquinonium circle-giant-interneurone synaptic 352 Arousal, DUM neurones and, 393 transmission and, 253 interneurone synaptic transmission Arthropods and, 253 desmosomes in, 80-81 Benzilylcholine mustard in cholinergic gap junctions, vertebrate and, 98 spot desmosomes, thin section receptor studies, 220 appearance, 77 aBgt 3.1, receptor actions, 288 aBgt 3.2, receptor actions, 288 tight junctions in, 132-138 aBgt 3.3, receptor actions, 288 Atropine aBgt 3.4, receptor actions, 288 binding to Musca dornestica head Binding sties, autoradiographic localextracts, 222, 225 ization, 240-243 effect on dorsal unpaired median Binding studies neurones, 265 central nervous system acetylcholine on Periplaneta motoneurone D,, receptors, 2 16 265 on sixth abdominal ganglion of radiolabelled-ligand, acetylcholine Periplaneta , 25 9 receptors, 21 8 inhibition of a-bungarotoxin binding Biogenic amines by, 229 application to insect heart preparanicotinic receptor antagonist, 216 tions, 418-420 synaptic transmission and, 252 to salivary glands, 408-412 Austracris guttulosa, cuticular lipids, difcellular localization, 330-346 ferential thermal analysis, 28 distribution in cardiac regulatory Autodesmosomes in flagellates, 82 system, 417-418 A utomeris ,eclosion, behavioural switchin insect nervous system, 320-349 ing and, 519 firefly light organs and, 394-402 Automeris rnemusae, eclosure, fluorescence-based assays of distribubehaviour switching and, 5 18 tion, 321-325

600

Biogenic amines-(contd.) functional role, 364-365 in neurohaemal organs, 433-436 heart and, 414-420 in control of gut muscles, 420-426 in nervous system, 317-473 inactivation, 356-365 metabolism, 349-365 neurohaemal organs and, 426-436 radioenzymatic assays, 326 salivary glands nerve stimulation and, 406-412 subcellular location, 346-349 synthesis, 350-356 Bivalves, septate junctions in, 43 Blaberus, tight junctions, 138 Blaberus craniifer cardiac nerve cords, biogenic amines in, 418 corpora cardiaca, biogenic amines in, 427 desmosomes in, occurrence, 80 heart, response to biogenic amines, 419 putative amiriergic neurones, vesicle characteristics, 348 Blaberus giganticus biogenic amine distribution, 323 gut muscle, biogenic amine effect on, 424 pharmacological studies, 423 Blatta orientalis heart, innervation pattern, 41 5 water loss, 9 Blood volume in ecdysis, 553-557 failure in ecdysis and, 570 BOL, adenylate cyclase activity and, 44 1 Bombyx, wing expansion in ecdysis, 512, 526 Bombyx mori ecdysis, behavioural switching in, 5 16 cuticle inflation, 530 heart rate in, 558 Boophilus microplus cuticle plasticization in ecdysis, 540 salivary glands, catecholamine in, 403 Brain amphibian, acetylcholine receptors,

S U B J E C T INDEX

eclosion behaviour and, 532 extracts, 3H-quinuclidinyl benzilate binding components, 237 fish, acetylcholine receptors, 276 mammalian, acetylcholine receptors, 276 Bretylium, effect on salivary gland stimulation by biogenic amines, 411 Bufotenine, function in corpora cardiaca, 433 Bumble bees, corpora pedunculata, biogenic amine distribution in, 332 a-Bungarotoxin binding to Musca domestica head extracts, 224, 225 effect on dorsal unpaired median neurones, 265 on Periplaneta fast coxal depressor motoneurone, 263 giant interneurone 3, 260 motoneurone D,, 265 1251-,binding in insect tissues, distribution, 240 binding to low speed extracts, 227-236 pharmacology, 242-243 in cholinergic receptor studies, 219 receptors, 294 Bursicon blood volume in ecdysis and, 555 cuticle plasticization and, 537 cuticle tanning and, 541 ecdysis and, 534-535,542 endocuticle formation and, 55 1 in tracheal air filling in ecdysis, 547-548 post ecdysial cell death, 565 tyrosine hydroxylation and, 543 Butterflies, mushroom bodies, function, 337 Butyrylcholine, effect on electrophysiology of the sixth abdominal ganglion of Periplaneta, 251 Caddisfly central body complex, biogenic amine localization in, 338 stomatogastric system, biogenic amine

SUB.JECT I N D E X

Calcium, gap junction permeability and, 104 Cailitrectes sapidus, neurones, acetylcholine receptors, 275 Calliph o ra adult eclosion, 500 axo-glial smooth septate-like junctions, 155 desniosome development in, 84 ecdysis, blood volume and, 553 mt:tabolism and, 560 gap junctions, 161 formation, 1 1 1 heart preparations, biogenic amine effect on, 418 medulla, biogenic amine localization in, 341 peripheral retina, reticular septate junctions, 177 reticular septate junctions, freezefracture appearance, 179 pleated septate junction development in, 73 post ecdysial cell death, 562 protocerebral bridge, biogenic amine cell localization, 338 puparium formation, 502 reticular septate junctions, 175 scalariform junctions, 162 freeze-fracture replica, 164 thin section appearance, 159 sensory terminal, asymmetric junctions, 151 septate junctions, function, 71 tight junctions in, 133 degradation, 1 4 9 development, 146 ridge morphology, 145 tubular salivary glands, 405 Calliphora erythrocephala axo-glial junction-like associations, 152 cuticular lipids, crystal structure, 27 heart, biogenic amine distribution in, 417 interglial junctions, thin section, 122 Calliphora vicina, ecdysis, bursicon and, 542 Calliphora vomitoria, corpora pedunculata, biogenic amine distribution in, 333

60 1

Calpodes cuticle deposition prior to ecdysis, 550 cuticular lipids, function, 24 myoepidermal connections, 77 Verson’s glands, 557 Zonulae occludentes, 88 Cambarus, septate junction in, 6 6 Campsocleis buergeri, hatching, developmental readiness, 480 Cancer pagurus, neurones, acetylcholine receptors, 275 Carausius tight junctions in, 133 development, 146 Carausius morosus ecdysis, bursicon and, 542 median neurohaemal organs, biogenic amines in, 431 Carbamylcholine effect on cell bodies of Periplaneta central neurones, 260 on electrophysiology of the sixth abdominal ganglion of Periplaneta, 25 1 o n Periplaneta fast coxal depressor motoneurone, 263 toxin binding inhibition by, 23 1 Cartap, receptor actions, 291 Catecholamines in nervous system, 318 in salivary glands, innervation pattern and, 403-405 synthesis, 350 Catechol-0-methyltransferase in biogenic amine inactivation, 360-362 Celerin, 364 Celerio euphorbiae celerin from, 363 Cell death, post-ecdysial, 561 -567 Cenocorixa expleta energy budget analysis, 1 9 water loss, measurement, 11 Centipedes, gap junction in, 97 Central body complexes, biogenic amines cell localization in, 337-338 Central nervous system acetylcholine receptors, binding studies, 216-217 comparative pharmacology, 265-279

602

S U B J E C T INDEX

Central nervous system-(contd.) cuticular lipids composition, 23 tight junctions in, 132 epidermis, Zonulae adhaerentes, 77 vertebrates, biogenic amines as horglobuli cell bodies, octopamine in, 335 mones and, 392 mushroom bodies, function, 337 Centruroides sculpturatus, cuticular nerve cords, extract, binding properlipids, electron paramagnetic resoties, 226 nance, 2 8 nervous tissue, biogenic amines disCeratitis capitata, ecdysis, X-irradiation tribution, 328 and, 578 octopamine distribution in, 327 Chaetognaths rectal pads, scalariform junctions, 168 desmosornes in, 82 testis, tight junctions, 136-137 gap junction in, 97 tight junctions in, 133 septate junction in, 65 water loss from, 2 Chemical potential gradient in insect Coelenterates water loss, 11 desmosomes in, 82 Chironomous gap junction in, 97 ' Comb desmosomes, 44 salivary glands, gap junctions, 102 gap junction permeability, calcium definition, 43 and, 104 freeze-fracture, 49-5 1 lanthanum infiltration, 46-48 p H and, 106 Chitin structural features, 44-54 cuticle plasticization in ecdysis and, structural model, 5 1-54 thin section appearance, 44-46 540 synthesis, insect growth regulators and, Communication cell to cell, gap junctions and, 85, 576-577 Choline acetyltransferase in cholinergic 101-109 intercellular, septate junctions and, 69 system, 217 Cholinergic ligands, electrophysiojunctions and, 181-182 logical responses of neurones to, Compartmentalization, tight junctions 243-265 and, 144-145 Cholinergic receptors as sites of insecti- Compound eye, tight junctions in, 132, cide action, 289-293 135-136 Chromatographic columns, septate Conduction, evaporation of water from junctions as, 72 insects and, 8 Ciliary ganglion, chick, acetylcholine Connexin, 114 receptors, 277 Connexon, 114 Cimex Continuous junction See Smooth septate junction cuticle inflation in ecdysis, 525 cuticle plasticization in ecdysis, 538 Continuous septate junctionsee Smooth tracheal air filling in ecdysis, 546 septate junction Circadian rhythms, ecdysis and, Cordylophora, desmosomes in, 82 478-480 Corpora cardiaca Close junction See Gap junction biogenic amines and, 427-429 Cnidaria, desrnosomes in, 82 function in, 433 Cobratoxin, binding to Musca domesCorpora pedunculata tics head extracts, 224 biogenic amine distribution in, Cockroach (See also Periplaneta 332-337 americana) function, 336-337 central nervous system extract, toxin Crayfish binding component, 235 gap junction, 91, 98

S U B J E C T INDEX

hemidesmosomes, development, 8 4 neurones, ionically coupled cells, 85 septate axons, gap junction permeability, calcium and, 105 septate giant axons, 103 septate junction in, 66 tight junctions in, 132 Critical temperature, insect transpiration and, monolayer hypothesis, 25 in insect water loss, 10 dynamic experiments, 12-16 Crustacea gap junctions, 85, 95 muscle, desmosomes in, 80 septate junction in, 65, 6 6 tissues in, 81 Cryptoglossa virrucosa, water loss, 2 Crystal structure in insect epicuticular lipids, 27 Culex tarsalis, septate junction formation in, 75 Curare, effect on nicotine stimulation of skeletal muscle, 216 Cuticle deposition prior to ecdysis, 549-553 escape from, in ecdysis, 523-525 hardening in ecdysis, 541-546 inflation in ecdysis, 525-530 lipids in, 1-33 permeability to water, temperature and, 2 vapourization and, 4 plasticization in ecdysis, 537-540 shedding, failures in ecdysis and, 571 splitting in ecdysis, 519-523 temperature, 1-33 measurement, 29 transpiration, 1-33 Cutting, cuticle, in ecdysis, 523 Cyproheptadine, adenylate cyclase activity and, 441 Cyclohexidine, PTF induced tanning and. 545 Dacus tryoni, adrenaline distribution in, 324 Daphnia, septate junction in, 6 6 Decamethonium binding to Musca dornestica head

603

extracts, 222, 224, 225 circle-giant-interneurone synaptic transmission and, 253 effect on dorsal unpaired median neurones, 265 ganglionic nicotinic receptor antagonist, 216 toxin binding inhibition by, 231 3H-Decamethonium binding component in Musca dornestica head extracts, 282 Dendroas viridis, a-toxin, 288 Dendroaspis, neurones, acetylcholine receptors, 273 Dermal glands discharge in ecdysis, 557-558 type B, Rhodnius, 557 Derrnatobia hominis, hatching, timing, 477 Desmosomes, 75-84 co-occurrence with gap junctions, 120 development, 84 functional significance, 8 3 in arthropods, 80-81 in glia, 1 51 Deutocerebrum, biogenic amine cell localization in, 342-343 Development gap junctions and, 85 junctions, 180-181 Dexetimide, binding t o Musca dornestica head extracts, 225 DFP, receptor actions, 291, 292 Dichlorvos, ganglionic synaptic transmission sensitivity t o acetylcholine and, 250 Dictyoptera heart, innervation pattern, 41 5 scalariform junctions, thin section appearance, 159 Differential thermal analysis, insect cuticular lipids, 28 Differentiation, gap junctional communication and, 8 6 Diflubenzuron, synthesis, insect growth regulators and, 576-577 Digging failures in ecdysis and, 572 in adult eclosion, 500 vermiform larvae, 486

604

Diptera peripheral retina, reticular septate junctions, 177- 180 rectal papillae, reticular septate junctions, 172-173 salivary glands, gap junctions, 85 Discontinuous belt desmosome See Fascia adhaerens 1,2-Dithiolane, 4-(N,N-dimethylamino) See Cartap Diuresis in ecdysis, 555 Dopamine adenylate cyclase activity and, 438 application to salivary glands, 408 conjugation with sulphates, 363 -containing cell bodies, in Trichoptera, 345 distribution in cockroach, 328 in Schistocerca gregaria, 326 function in corpora cardiaca, 433 in Anabolia nervosa, 375 in central nervous system, 320 in corpora cardiaca, 427 in insect heart, 417 in insect nervous system, 321 in Manduca sexta salivary glands, 347 in median neurohaemal organs, 430 in mushroom bodies of ants, 337 in nervous system, 31 8 inactivation, 357 salivary glands, 402-413 synthesis, 350 Dorsal midline neurones identifiability, 367-373 octopamine and, 365-393 Dorsal unpaired median neurones, 265 Drosophila acetylcholine receptors, 294 acetylcholinesterase, structural gene, 281 N-acetyltransferase in, biogenic amine inactivation and, 362 biogenic amine distribution, in, 321 biogenic amine inactivation in, 360 ecdysis, failures, juvenile hormones and, 576 metabolism and, 560 peripheral retina, reticular septate junctions, 177 post ecdysial cell death, 563

SUBJECT INDEX

puparium formation, 502 salivary glands, gap junctions, 102 Drosophila rnelanogaster acetylcholine receptor, genetics and, 279 'Z51-a-bungarotoxinbinding sites distribution in, 240 central nervous system, acetylcholine receptors, comparisons, 267 cholinergic receptors, comparative pharmacology, 269 heads, 'Z51-a-bungarotoxin-binding component from, purification, 234 extracts, 3H-quinuclidinyl benzilate binding components, 238 homogenates, 3H-quinuclidinyl benzilate binding components, 237 5-HT distribution in, 325 low speed extracts, '251-a-bungarotoxin binding to, 227 putative acetylcholine receptors, pharmacological profiles, 232 Drosophila pseudoobscura ecdysis, circadian rhythms and, 480 timing, environment, 478 DUM cells, octopaminergic nature, 373-376 DUM neurones functions, 389-393 identifiability, 369 DUMDL cells, 371, 372 DUMETi axons, 367 DUMETi cells myogenic rhythm and, 376-379 neuromuscular transmission potentiation and, 381-387 octopaminergic nature, 374 Dysdercus fulvoniger, dorsal midline neurones, octopamine and, 365 Dytiscus rnangrnalis, gut muscle biogenic amine effect on, 424 Earthworms, desmosomes in, 82 Ecdysis behaviour, 482-519 behaviour pattern before and after, 535-537 behavioural switching in, 514-519 exopteryogote, 487-496 failures, 569-579

SUBJECT INDEX

experimentally induced, 575 first larval, 485-486 integration of behaviour and physiology in, 475-595 mechanics, 519-530 physiology, behaviour and, 530-569 plasticity in, 503-514 stereotypy in, 503-514 timing, 476-482 environment and, 477-478 Ecdysone, gap junction permeability and, 108 P-Ecdysone E , gap junction permeability and, 107 Ecdysterone, gap junction permeability and, 108 Echinoderm desmosomes in, 82 septate junction in, 66 Eclosion hormone, 481, 530-534 adult eclosion and, 496 eclosion behaviour and, 517 Edrophonium, receptor actions, 291 Electrical stimulation, salivary glands, 406-408 Electron paramagnetic resonance, insect lipids, 28 Electrophorus, nicotinic receptor, 225 Electrophysiology , neurone response to cholinergic ligands, 243-265 Encapsulating haemocytes, septate junctions in, 63 Endocrine tissues, septate junction occurrence in, 67 Endocuticle, 549 Energy, failures in ecdysis and, 570 Energy budget for insect water loss, equation, 17-20 in evaporation of water from insects, 6-8 insect water loss, analysis, 16-20 simplified equation for insects water loss, 8 Energy flux, radiant, insect transpiration and, 7-8 Environment, ecdysis timing and, 476-478 Ephestia kuhniella, ecdysis, X-irradiation and, 578 Ephippiger cruciger, ecdysis, failures, 570

605

Epidermal cells, gap junction in, 95 Epidermal glands, septate junctions in, 62 Epidermis, septate junctions in, 62 Epinine adenylate cyclase activity and, 442 effect on salivary gland stirnulation by biogenic amines, 41 1 Epithelial tissues, septate junctions in, 62 Ergometrine, effect on salivary gland stimulation by biogenic amines, 410 Erioischia brassicae, ecdysis, cutting in, 523 Eserine electrophysiological responses of neurones to, 248 receptor actions, 292 Esters in insect cuticular lipids, 23 Evaporation from insects, energy budget during, 6-8 permeability of insect cuticle to water and, 4 Extraction, Musca domestica heads, 222 Eyes septate junctions in, 63, 64 Falck-Hillarp histochemical technique, 322 Fasciae adhaerentes, freeze-fracture appearance, 79 Fast coxal depressor motoneurone, 262-264 Fat body cells gap junction in, 95 post ecdysial lysis, 566 septate junctions in, 6 3 Fatty acids in insect cuticular lipids, 23 monolayer hypothesis, 25 Ferritin, tight junctions and, 127 Fick’s Law, 3 Ficopotamus enigmatica, septate junction in, 65 Fireflies See Photuris pennsylvanica Flagellates, desmosomes in, 82 Flatworms, septate junctions in, 43

606

Flea See Culex tarsatis Flies, salivary glands, scalariform junctions in, 168 Fluid mosaic model, biological membrane structure, 41 Fluorescein, cell to cell transfer, 86-87 a-Flupenthixol, adenylate cyclase activity and, 441 P-Flupenthixol, adenylate cyclase activity and, 441 Formation, gap junctions. 109-1 15 Formica rufa biogenic amine distribution, 323, 324 5-HT distribution in, 324 Freeze-cleaving, intercellular junction study and, 37, 3 9 Freeze fracturing See Freeze-cleaving Frogs, spinal cord, acetylcholine receptors, 276 Fungal infections, failure in ecdysis and, 574 Galleria mellonella ecdysis, bursicon and, 542 gut muscle biogenic amine effect on, 424 Gammarus, septate junction in, 6 6 G a p junctions, 85-120 annular, 116 arthropod vs vertebrates, 9 8 break down, 115-117 co-occurrence, with desrnosomes, 120 with septate junctions, 118 with smooth septate junctions, 120 disaggregation, 115-1 17 distribution, 94-98 formation, 109-1 15 freeze-cleaving, 89-93 functions, 100-109 lanthanum staining, 89 models, 93-94 permeability regulation and, 104 septate junctions in association with, 6 9 thin section appearance. 87-89 uncoupling, 115-117 Gasterophilus intestinalis, hatching, timing, 477 Gel filter, septate junctions as, 72 Genetics, acetylcholine receptors and, 279-282

SUBJECT INDEX

Glandular epithelial cells, gap junction in, 95 Glial cells septate junctions in, 6 3 specialized junctions, 151-157 tight junctions in, 134 Glial-axonal junctions, occurrence in vertebrates, 68 Globuli cells adrenergicity, 333-336 octopaminergic, 336 Glycoproteins in comb desmosomes, 5 4 in Musca domesrica head extracts, 226 in septate junctions, 71 Gonadotropin, gap junction permeability and, 109 Gonads, septate junctions in, 6 3 Grampsocleis buergeri, acetylcholine effect on auditory synapses of the prothoracic ganglion, 248 Grashof number, 1 9 Grasshoppers, cuticular lipids composition, 23 Gromphadorhina portentosa, adenylate cyclase activity, biogenic amines and, 440 binding sites, 226 ecdysis, behavioural switching in, 5 1 4 median neurohaemal organs, biogenic amines in, 430 octopamine biosynthesis in, 351 Gryllus campestris, globuli cells, 334 3',5'-Guanosine monophosphate, cyclic, eclosion hormone and, 534 Gut, septate junctions in, 63, 64 Gut muscles biogenic amines in control of, 42 1-426 innervation, 421 -422 pharmacological studies, 422-426 Guthoxon, receptor actions, 291, 292 Haemocyte capsules, gap junction in, 95 Haemocytopoietic organs, gap junction in, 95 Haloperidol, adenylate cyclase activity and, 441 Hardening, cuticle, in ecdysis, 541 -546

SUBJECT INDEX

Hatching behaviour, 482-486, 513 and physiology in, 476 prehatching and, 483-484 timing, 476-477 Heart, biogenic arnines and, 414-420 Heart rate in ecdysis, 558 Heat flux between insects and surrounding air, 7 Heat storage, evaporation of water from insects and, 8 Heliconius melpomene, ecdysis, circadian rhythms and, 480 Helix neurones, acetylcholine receptors, 27 3 tight junctions in, 143 Helix uspersa, neurones, acetylcholine receptors, 273 Hemicholinium-3, ganglionic synaptic transmission sensitivity to acetylcholine and, 250 Hemichordates, septate junction in, 66 Hernidesmosomes, 75 development, 8 4 mosquito midgut, 78 Hexamethonium circle-giant-interneurone synaptic transmission and, 253 effect on dorsal unpaired median neurones, 265 on Periplanetu motoneurone D,, 265 ganglionic nicotinic receptor antagonist, 216 Hirudo, neurones, acetylcholine receptors, 275 Hirudo medicinalis, central nervous system, acetylcholine receptors, 271 Homarus americanus acetylcholine receptors, 274 catecholamine synthesis in, 350 neurones, acetylcholine receptors, 275 putative aminergic neurones, vesicle characteristics, 348 Homoptera, septate junctions in, 63 Honey bee compound eye, tight junctions in, 135

607

scalariform junctions in, 168 Hormones gap junction permeability and, 104 water loss from cockroach and, 2 Horseradish peroxidase intercellular junction permeability studies and, 42 tight junctions and, 127 Houseflies central body complex, biogenic amine localization in, 337 compound eye, tight junctions in, 135 mushroom bodies, function, 337 5-HT See Tryptamine, 5-hydroxyHumidity, insect water loss and, 9 Hyalophora cecropia adult eclosion, 503 biogenic amine biosynthesis in, 354 ecdysis, integrative processes, 567 eclosion, 498 behaviour, 506 behavioural switching and, 518 developmental readiness, 481 eclosion hormone and, 496 eclosion hormone, 505, 530 post ecdysial cell death, 563 Hydra desmosomes in, 82 gap junction in, 97, 103 septate desmosomes in, 4 3 septate junctions, function, 72 Hydrocarbons in insect cuticular lipids, 23 Hydroids, septate junctions in, 4 3 Hymenoptera biogenic amine distribution in, 321 corpora pedunculata, biogenic amine distribution in, 332 Imaginal disc gap junction in, 95 septate junctions in, 6 2 Infrared thermometer in insect water loss measurements, 2 0 Innervation heart, 414-417 salivary glands, catecholamine distribution and, 403-405 Inosine monophosphate, cyclic, postecdysial cell death and, 565

608

Insect growth regulators, ecdysis failures and, 575 Insecticides, receptor actions of. 290-295 Integument, insect transpiration and, 20-21 Intercellular junctions insect tissues, 35-213 techniques, 37 Intramembranous particles, 40 Invertebrates desmosome development in, 84 septate junctions, 43, 65-67 skeletal neuromuscular junctions, biogenic amines and, 390 Ion exchange resin, septate junctions as, 72 Ion pumping reticular septate junctions, 176-1 77 scalariform junctions and, 171 Isoprenaline, effect on salivary gland stimulation by biogenic amines, 410 Isoproterenol, stimulation of Photuris pyralis light organs, 397 Isothiocyanates, receptor actions, 292 Junctions development, 180-1 8 1 functions, 181-182 Juvenile hormone, ecdysis failures and, 575

Lactrodectus mactans, desmosomes in, 80 Lampyris noctituca, octopamine in, 399 Lanthanum intercellular junction study and, 37 tight junctions and, 127 Lanthanum chloride in permeability studies of intercellular junctions in vivo, 4 1 -42 Lanthanum nitrate in intercellular junction study, 3 9 Latent heat flux evaporation of water from insects and, 8 in insect water loss, 16-17 Learning, DUM neurones and, 393 Leeches, septate junctions in, 43, 6 5 Lepidoptera, septate junctions in, 63

SUBJECT I N D E X

Lepisma corpora pedunculata, biogenic amine distribution in, 333 protocerebral bridge, biogenic amine cell localization, 338 Lepisima saccharina biogenic amine cell localization, 332 corpora pedunculata, biogenic amine distribution in, 333 Leucophaea, eclosion, bursicon in, 541 Leucophaea maderae ecdysis, bursicon and, 542 eclosion hormone in, 531 gut muscle, biogenic amine effect on, 424 innervation, 422 pharmacological studies, 423 Light organs firefly, adenylate cyclase activity in, 442 biogenic amines and, 394 future studies, 402 innovation, 394-397 Light responses, pharmacology, 397 Limnaea neurones, acetylcholine receptors, 273 tight junctions in, 143 Limulus axo-glial tight junction-like associations, 154 central nervous system, scalariform junctions, 169 eye, gap junction in, 96, 103 gap junction, 88, 95 permeability and, 108 Limulus polyphernus acetylcholine receptors, 274 putative acetylcholine receptors, pharmacological profiles, 233 septate junction in, 6 6 smooth septate junction, 57 thin section appearance, 55 Lipids insect, functions, 24 cuticles, 1-33 chemical composition, 22-23 transpiration and, 20-29 water loss and, 21-22

SUBJECT I N D E X

Lobster biogenic amine conjugation by sulphates in, 363 skeletal neuromuscular junction, octopamine and, 390 Locust (See also Schistocerca americana) biogenic amine synthesis, 351 central body complex, biogenic amine localization in, 337, 338 compound eye, tight junctions in, 135-136 ecdysis, metabolism and, 560 wing inflation, 528 globuli cell bodies, octopamine in, 335 heart preparations, biogenic amine effect on, 418 mushroom bodies, function, 337 nervous tissue, biogenic amines distribution, 328 optic lobes, biogenic amine cell localization, 338 post ecdysial cell death, 562 testis, tight junctions, 136-137 tight junctions in, 133 timing, environment, 478 tracheal air filling in ecdysis, bursicon and, 548 tritocerebrum, bingenic amine cell localization in, 343 Locusta migratoria N-acetyltransferase in, biogenic amine inactivation and, 362 biogenic amine, cell localization, 332 distribution, 323 deutocerebrum, biogenic amine cell localization in, 342 dorsal midline neurones, octopamine and, 365 DUMDL cells, 371 ecdysis, bursicon and, 542 cuticle inflation, 530 gut muscle, biogenic amine effect on, 424 pharmacological studies, 423 myogenic rhythm, function, 380 receptor mediated acceleration, 379 putative aminergic neurones, vesicle characteristics, 348

609

Locusta migraroria migrutorioides heart, biogenic amine distribution in, 417 5-HT distribution in, 324 median neurohaemal organs, biogenic amines in, 431 LSD, adenylate cyclase activity and, 441,442 Luciferase, firefly light organ stimulation by, 401 Lucilia ecdysis, bursicon and, 542 wing expansion, 501 Luciliu cuprina, cuticular lipids, differential thermal analysis, 28 Lucilia sericala, ecdysis, cutting in, 523 Luciola, light organs, innovation, 396 Lumhriconereis heteropoda, nereistoxin from, 288 Lymantria dispar, hatching, developmental readiness, 480 Machilidue, corpora pedunculata, biogenic amine distribution in, 332 Maculae adhaerentes, 75, 77 arthropods, thin section appearance, 77 development, 84 freeze-fracture appearance, 78-79 in vertebrates, thin section appearance, 76 intercellular, 77 mosquito midgut, 7 8 Macula commurticans See G a p junctions Main squinado, neurones, acetylcholine receptors, 275 Mulacosoma disstria, hatching, developmental readiness, 480 Malacosoma testacea, hatching, developmental readiness, 480 Malanoplus bivattatus, hatching, developmental readiness, 480 Malpighian tubules ecdysis, 556 embryological origin, 72 gap junction in, 95 formation, 113 scalariform junctions in, 1 6 9 septate junctions in, 63, 64

61 0

Mamestra condfigurata,adenylate cyclases in, biogenic amine effect on, 438 Manduca cuticle, deposition prior to ecdysis, 550 inflation in ecdysis, 526 dopamine biosynthesis, 354 ecdysis, behavioural switching in, 515 eclosion, bursicon in, 541 wing spreading in, 512 gap junction formation, 111 glial cells, scalariform junctions, 169 post ecdysial cell death, 562 tight junctions, degradation, 149 development, 146 ridge morphology, 145 timing, environment, 478 Manduca sexta acetylcholine receptors in antenna1 lobes of the brain, development and, 285 N-acetyltransferase in, biogenic amine inactivation and, 362 adenylate cyclases in biogenic amine effect on 438, 444 axo-glial junction-like associations, 152 biogenic amine, biosynthesis, neurotransmitter function and, 355 conjugation, by p-alanine in, 364 by sulphates in, 363 distribution, 323 synthesis, 351 brain, a-bungarotoxin binding component, 235 toxin binding distribution in, 241 desmosome development in, 8 4 dopamine biosynthesis in, 352 dorsal midline neurones, octopamine and, 3 6 5 , 3 6 6 D U M cells, 372 ecdysis, bursicon and, 542 circadian rhythms and, 479 eclosion, 497 eclosion hormone in, 481, 531 gap and septate junctions, 118 gap junctions, 93 development, 113 glia, desmosomes in, functional

SUBJECT INDEX

significance, 8 3 interglial junctions, thin section, 122 putative aminergic neurones, vesicle characteristics, 348 salivary glands, catecholamine in, 403 dopamine in, 347 septate junctions in, 6 3 development in, 73 in nervous system, 71 smooth septate junction, freezefracture, 60, 75 tight junctions, 123, 129, 133, 134 development, 146 timing, environment, 478 tracheal cell, membrane junctions, 159 tubular salivary glands, 405 Mass transfer, water, 3-6 Mechanoreceptors, septate junctions in, 6 3 Melanoplus sanguinipes, biogenic amine distribution, 323 Melatonin, production, 5-HT and, 325 Membranes, permeability, 3 Mestracheon system, septate junctions in, 64 Metabolic coupling, gap junctions and, 85 Metabolic heat, evaporation of water from insects and, 8 Metabolism in ecdysis, 560-561 Methoxamine, effect on salivary gland stimulation by biogenic amines, 410,411 Methylsergide, effect on salivary gland stimulation by biogenic amines, 410 Microperoxidase intercellular junction permeability studies and, 42 tight junctions and, 127 Microelectrode recording, 244 Midgut gap junction in, 95 septate junctions in, 64 Molecular sieve, septate junctions as, 72 Molluscs comb desmosomes, freeze-fracture, 49 desmosomes in, 82

SUBJECT I N D E X

gap junction in, 97 nerve cell soma membranes, acetylcholine receptors, 272 septate junction in, 65 Monoamine oxidase in biogenic amine inactivation, 360-362 Monolayer hypothesis, insect cuticular lipids, 24 Mosquitos, midgut, hemidesmosomes, 78 Moths, mushroom bodies, function, 337 Multifibre recordings of electrophysiological responses of neurones to cholinergic ligands, 248-253 Musca axo-glial smooth septate-like junctions, 155 compound eye, tight junction, 136 peripheral retina, reticular septate junctions, 177 reticular septate junctions, 175 Musca domestica acetylcholine receptors, 294 brain, a-bungarotoxin binding component, 235 central nervous system, acetylcholine receptors, comparisons, 267 cholinergic receptors, comparative pharmacology, 269 comb desmosome, thin section appearance, 44 deutocerebrum, biogenic amine cell localization, 342 head extracts, 'H-decamethonium binding component, 282 high-speed head extracts, binding of reversible ligands to, 220-227 high-speed supernatant extracts, purification, 223 medulla, biogenic amine localization in, 341 peripheral retina, reticular septate junctions, freeze-fracture appearance, 179 putative acetylcholine receptors, pharmacological profiles, 233 smooth septate junction, 5 7 and gap junctions, freeze-fracture, 120

61 1

freeze-fracture, 58, 60 thin section appearance, 55 tritocerebrum, biogenic amine cell localization in, 343 Muscarine acetylcholine receptors and, 21 6 effect on electrophysiology of the sixth abdominal ganglion of Periptuneta, 25 l D,L-Muscarine, inhibition of abungarotoxin binding by, 229 Muscarone, binding to high-speed extracts of Musca domestica, 220 Mushroom bodies See Corpora pedunculata Myocardial cells, septate junctions in, 63 Myocardial tissue, septate junctionsin, 63 Myocardium, intercalated disc, desmosomes in, 80 Myogenic rhythm DUMETi cells and, 376-379 function, 380-381 receptor mediated acceleration, 379-380 Myriapods, septate junction in, 65, 6 6 Nuju nuju siamensis, a-neurotoxins, 288 Nuuphoetu cinerea putative aminergic neurones, vesicle characteristics, 348 salivary duct nerve, 404 salivary glands, biogenic amines and, 406 catecholamine in, 403 suboesophageal ganglion, dopaminergic innervation, 412 Navanax, neurones, acetylcholine receptors, 273 Nematodes, septate junction in, 66 Nemertines, septate junction in, 65 Neostigmine electroretinogram and, 270 receptor actions, 291, 292 Nephridial tissues, scalariform junctions in, 169 Nereistoxin, receptor actions, 288-289 Nerve cells, gap junction in, 95 Nerve stimulation, salivary glands, biogenic amines and, 406-412

612

Nervous system biogenic amines in, 317-473 desmosomes in, 80 gap junction in, 95 scalariform junctions in, 169 septate junctions in, 63 occurrence in, 67 Neurohaemal organs biogenic amines and, 426-436 function, 433-436 median, biogenic amines and, 429-433 Neurohormones, definition, 319 Neuromodulators, definition, 319 Neuromuscular transmission, potentiation, DUMETi cells and, 381-387 Neuronal ganglia, adenylate cyclase activity, biogenic aniines and, 437-442 Neurones cultured sympathetic, acetylcholine receptors, 276 electrophysiological responses to cholinergic ligands, 243-265 single, electrophysiological responses to cholinergic ligancls, 253-260 identified, electrophysiological responses to cholinergic ligands, 260-265 invertebrate, pharmacology, 244 Neurosecretory innervation t o skeletal muscle, 391-392 a-Neurotoxins, receptor actions of, 288 Neurotransmitters definition, 319 function, biogenic amine synthetic ability and, 354-356 Nexus See G a p junction:i Nicotine acetylcholine receptors and, 21 6 binding to Musca domrstica head extracts, 222, 224, 225 effect on cell bodies of Periplaneta central neurones, 260 on dorsal unpaired median neurones, 265 on electrophysiology of the sixth abdominal ganglion of Periplaneta, 251

SUBJECT INDEX

on Periplaneta motoneurone D,, 265 o n sixth abdominal ganglion of Periplaneta, 258 inhibition of a-bungarotoxin binding by, 229 receptors, 294 actions, 290 Noradrenaline adenylate cyclase activity and, 438 application to salivary glands, 408 biosynthesis, 354 distribution in cockroach, 328 in Schistocerca gregaria, 326 high affinity uptake mechanism, 358 in central nervous system, 320 in corpora cardiaca, 427 in median neurohaemal organs, 430 in nervous system, 318, 321 in salivary glands, 403 inactivation, 357, 358 localization in insect nervous system, 346 stimulation of Photuris pyralis light organs, 397 subcellular location, 347 synthesis, 350 Nucleotide cyclase in firefly light organ stimulation, 399 Nucleotides, cell to cell transfer, 86-87 Nusselt number, 19 Nutrition, failure in ecdysis and, 574 Occluding junct. n See Tight junctions Octopamine adenylate cyclase activity and, 438, 439-440,443 /3-alanine conjugation, 364 biogenic arnine cell localization, 338-342 biosynthesis, 351 in Manduca sexta, 355 conjugation with sulphates, 363 distribution in locust, 328 in optic lobes, 338 dorsal midline neurones and, 365-393 effect o n salivary glands, 41 1 function, 446 in corpora cardiaca, 433

S U B J E C T INDEX

high affinity uptake mechanism, 358 in corpora cardiaca, 427 in firefly light organs, 399,402 in globuli cell bodies, 335 in median neurohaemal organs, 430 function, 434,435 in nervous system, 3 18 inactivation, 357 in Periplaneta americana, 359 in lobster skeletal neuromuscular junctions and, 390 localization in optic lobes, 341 myogenic rhythm and, 377 neuromuscular transmission and, 382 presynaptic receptors at neuro muscular junctions, 392 radioenzymatic assays, 326 sodium sensitive uptake, 360 stimulation of Photuris pyralis light organs, 398 synthesis in DUM cells, 373 in DUMETi cells, 374 Oil-gap technique, 245 Oncopeltus, gap junction permeability, 107 Oncopeltus fasciatus ecdysis, bursicon and, 542 failures, 570 Onychophora, septate junction in, 65 Orbeli phenomenon, 389 Orchestia gap junction, 91 septate junction in, 66 development in, 73 Orchestia carimana, midgut, desmosomes in, 81 Ostrinia, tight junctions in, 133 Ostrinia nubilalis, biogenic amine inactivation in, 360 Ovaries gap junction in, 95 septate junctions in, 63, 67 1,3-Oxazolidine, 2(0,s-dimethylthiophosporylimino) -3-ethyl-5-methyl-, receptor actions, 291, 292 Oxotremorine, receptor actions, 292 Oxygen, firefly light organ stimulation by, 401

613

Panorpa deutocerebrum, biogenic amine cell localization in, 342 protocerebral bridge, biogenic amine cell localization, 338 Panulirus interruptus, putative aminergic neurones, vesicle characteristics, 348 Paraoxon ganglionic synaptic transmission sensitivity to acetylcholine and, 250 receptor actions, 291 Particle arrays in insect nervous tissue, 155-157 Pathological tissue, septate junction occurrence in, 67 Peptides, cell to cell transfer, 86-87 Perineurial ceiis septate junctions in, 63, 64 tight junctions in, 133-134 Perineurium, septate junctions in, 63 Peripatopsis moseleyi, septate junction in, 66 Peripatus, septate junction in, 66 Periplaneta deutocerebrum, biogenic amine cell localization in, 342 ecdysis, blood volume and, 553 eclosion, bursicon in, 541 mobile grease covering, function, 24 smooth septate junction, 58 tight junctions, 129, 130 Periplaneta americana abdominal nerve cord extracts, a-bungarotoxin binding component, 235 1251-a-bungarotoxinbinding component, 236 adenylate cyclases in, biogenic amine effect on, 438 axo-glial junction-like associations, 152 binding sites, 226 biogenic amine, conjugation by sulphates in, 363 distribution, 322, 323 blood volume in ecdysis, 554 brain, catecholamine-containing cell bodies, 330 central nervous system, acetylcholine receptors, comparisons, 267

614

Periplaneta americana -(contd. ) cholinergic receptors, comparative pharmacology, 269 comb desmosome, 47 freeze-fracture, 51 thin section appearance, 44 corpora cardiaca, biogenic amines in, 427 corpora pedunculata, biogenic amine distribution in, 333 cuticle temperature, measurement, 13,15 cuticular lipids composition, 23 differential thermal analysis, 28 film from, force-area curve, 25 monolayer hypothesis, 24 dorsal median cell group, 371 DUM cells, octopamine in, 374 ecdysis, bursicon and, 542 circadian rhythms and, 479 eclosion hormone in, 531 energy budget analysis. 19 gap junction, 88 globuli cells, 334 gut muscle, biogenic amine effect on, 424 innervation, 422 pharmacological studies, 423 hatching behaviour, 483 heart, biogenic amine distribution in, 417 innervation pattern, 415 response to biogenic amines, 419 5-HT distribution in, 324 latent heat flux in water loss, 1 6 median neurohaemal organs, biogenic amines in, 430, 43 1 metathoracic ganglion, motoneuron D,, 264-265 nerve cords, acetylcholine penetration of, 248 neurohaemal organs, octopamine in, function, 435 a-neurotoxin receptor activity in, 288 octopamine, biosynthesis in, 351 distribution in, 326 inactivation, 357, 359 putative acetylcholine receptors, pharmacological profiles, 233 putative aminergic neurones, vesicle

SUBJECT INDEX

characteristics, 348 rectal pads, tight junctions, 137 salivary glands, catecholamine in, 403 scalariform junctions, 161 sixth abdominal ganglion, '251-abungarotoxin binding site distribution in, 241 individual giant neurones, electrophysiological response to cholinergic ligands, 260 synaptic phenomena single giant interneurones, 244 third thoracic ganglion, fast coxal depressor motoneurone, 262 transpiration, cuticular lipids and, 21 temperature and, 12 tritocerebrum, biogenic amine cell localization in, 344 water loss, measurement, 9, 10 water loss-temperature curve, 13 Periplaneta brunnea , cuticular lipids, differential thermal analysis, 28 Permeability insect cuticle, to water, vapourization and, 4 intercellular junctions, tracer studies in V ~ V O 41-42 , junctional structures, 37 membranes, 3 regulation, gap junction and, 1 0 4 tight junctions and, 142-144 tight junctions, 141 Permeability barrier septate junctions as, 72 transepithelial, septate junctions and, 69 Petrobius, scalariform junctions, 159 pH, cuticle plasticization in ecdysis and, 539 Phenolamines, synthesis, 350 Phentolamine adenylate cyclase activity and, 441 effect on salivary gland stimulation by biogenic amines, 410 L-Phenylalanine, y-L-glutamyl-, in Diptera third instar larvae, 364 Phormia, puparium formation, 502 Phormia regina, ecdysis, bursicon and, 542 Phospholine, receptor actions, 291

SUBJECT INDEX

Phospholipids in insect cuticular lipids, 23 Photinus, light organ innervation, 396 Phoruris, light organs, innervation, 396 Photuris pennsylvanica light organs, adenylate cyclase activity in, 442 biogenic amines and, 394 future studies, 402 putative aminergic neurones, vesicle characteristics, 348 Photuris pyralis, light organs, pharmacology, 391 Photuris versicolis, biogenic amine distribution, 323 Physiology ecdysis, behaviour and, 530-569 integration of behaviour and, 475-595 Pieris ecdysis, blood volume and, 553 cutting in, 523 metabolism and, 560 Pieris brassicae ecdysis, blood volume and, 554 bursicon and, 542 failures, juvenile hormones and, 576 Pilocarpine binding to Musca domestica head extracts, 225 effect on cell bodies of Periplaneta central neurones, 260 effect on electrophysiology of the sixth abdominal ganglion of Periplaneta, 25 l inhibition of a-bungarotoxin binding by, 229 Planaria, gap junction, 93, 97 Planarians desmosomes in, 82 gap junction in, 97 Planobarius, neurones, acetylcholine receptors, 273 Plants, transpiration, leaf cuticular resistance and, 21 Plasticity in ecdysis, 503-514 Plasticization, cuticle, in ecdysis, 537 -540 Plathyelminthes, septate junction in, 6 5

615

Pleated septate junction See Comb desrnosome Pogonophora, desmosomes in, 82 Polysaccharides in comb desmosomes, 54 Potential difference across epithelium, septate junctions and, 7 0 Prandtl number, 19 Predation, failure in ecdysis and, 574 Prehatching behaviour, 483-484 Pressure in splitting cuticle during ecdysis, 519-523 Presynaptic receptors for acetylcholine, 392 for octopamine, 392 Procion yellow, cell to cell transfer, 86-87 Proconnexon, 114 Proctodeum gap junction in, 95 scalariform junctions in, 168 Proctolin, myogenic rhythm in Schistocerca gregaria and, 379 Propranolol adenylate cyclase activity and, 441 effect on salivary gland stimulation by biogenic amines, 410 Propylbenzilylcholine mustard in cholinergic receptor studies, 220 Proteins, chitin binding in cuticle, 540 Prothoracic glands, post ecdysial degeneration, 566 Protocerebral bridge, biogenic amines cell localization in, 337-338 Pteroptyx, light organ, innervation, 396 PTF See Puparium tanning factor Puparium tanning factor, 542 in cuticle hardening, 544 Puromycin, PTF induced tanning and, 545 Pyridostigmine, receptor actions, 291 Pyrophanes, light organs, innovation, 396 Pyrrhocoris apterus ecdysis, metabolism and, 560 eclosion hormone in, 531 Pyrrolid-Zone, 1-(4-dimethylaminobut-2-ynyl)-, receptor actiotis, 292

616

Quadrula pustulosa, putative aminergic neurones, vesicle characteristics, 348 Quinuclidinyl benzilate effect on dorsal unpaired median neurones, 265 muscarinic antagonist, 210 'H-Quinuclidinyl benzilate, binding to low speed extracts, 237-240

SUBJECT INDEX

smooth septate junction, freezefracture, 60 transpiration, cuticular lipids and, 21 type B dermal glands, 557 water loss, cuticular lipids and, 22 Rhodnius prolixus axo-glial j unction-like associations, 152 comb desmosome, thin section appearance, 44 Rats desmosomes in, functional septate junction occurrence in, 67 significance, 8 3 brain, head extracts, 3H-q~~inuclidinyl energy budget analysis, 19 benzilate binding components, 238 smooth septate junction, 57 putative acetylcholine receptors, freeze-fracture, 60 pharmacological profiles, 233 tight junctions in, 133 muscle, putative acetylcholine receptracheal cell, membrane junctions, tors, pharmacological profiles, 233 159 Rectal pads water loss, measurement, 10 orthopteran, tight junctions in, water loss-temperature curve, 13 137-138 RNA, cuticle deposition and, 55 1 scalariform junctions in, 168 Rornalea rnicroptera, DUM cell tight junctions in, 132 identifiability, 369 Rectum gap junction in, 95 Rotifers, desmosomes in, 82 Relative humidity, failure in ecdysis Ruthenium red in intercellular junction and, 574 study, 3 9 Reticular septate junctions, 172-180 coexistence and occurrencca in other Salivary glands organisms, 175 dopamine and, 402-413 freeze-fracture appearance, 173 gap junction in, 95 function, 176-177 formation, 113 in peripheral retina, function, 180 scalariform junctions in, 168 lanthanum stained appearance, 173 septate junctions in, 6 3 thin section appearance, 173 Samia Cynthia ricini, cuticular lipids Retina, septate junction occurrence in, composition, 23 67 Surcophaga Rigidity, septate junctions and, 72 adult eclosion, 500 Rho d n ius ecdysis, bursicon and, 542 abdominal cuticle plasticization in, puparium formation, 502 446 Sarcophuga bullata, ecdysis, failures, cuticle, inflation in ecdysis, 525 juvenile hormones and, 576 plasticization in ecdysis, 538 Sarcophagine in Diptera third instar ecdysis, blood volume and, 553 larvae, 364 cuticle inflation in, 528 Saturniid moths, tubular salivary eclosion hormone in, 531 glands, 405 epicuticular lipids, function, 24 Scalariform junctions, 157-172 myoepidermal connections, 77 autocellular, 170 post-ecdysial cell death, 561 coexistence with other junctions, 170 septate junction, development in, 7 3 development, 172 in malpighian tubules, 71 distribution in insect tissues, 168-170

SUBJECT INDEX

freeze-fracture replicas, 162-1 6 6 heterocellular, 170 homocellular, 170 models, 166-168 physiological significance, 170-1 72 thin sections appearance, 159-162 tracers and, 162 Schistocerca adult ecdysis, 513 ecdysial behaviour. 490 ecdysis, behavioural switching in, 5 15, 516 blood volume and, 553 escape from cuticle, 5 2 4 failures, 570 digging and, 572 eclosion, bursicon in, 541 gut muscle, innervation, 421 tanning in ecdysis, 528 tight junctions, development, 146 water loss, measurement, 11 Schistocerca americana , median neurohaemal organs, function of biogenic amines in, 434 Schistocerca americana gregaria biogenic arnine biosynthesis in, 351 D U M neurones, 370 globuli cells, 334 median neurohaemal organs, biogenic amines in, 432 myogenic rhythm, DUMETi cells and, 376 Schistocerca gregaria axo-glial junction-like associations, 152 biogenic amine, cell localization, 332 distribution, 323, 340 comb desmosomes, freeze-fracture, 49,51 thin section appearance, 44 corpora cardiaca, biogenic amines in, 427,428 deutocerebrum, biogenic amine cell localization in, 342 dorsal midline neurones, octopamine and, 366 D U M cell identifiability, 369 DUMETi neurones, 368 ecdysis, 487 behaviour, 495

617

behavioural switching in, 5 18 bursicon and, 542 circadian rhythms and, 479 motor programme, 494 gap junction, 9 3 hatching behaviour, 482 hernidesmosomes, 78 5-HT distribution in, 324 imaginal ecdysis, 4 9 3 median neurohaemal organs, biogenic amines in, 431 myogenic rhythm, receptor mediated acceleration, 379 octopamine distribution in, 326 putative aminergic neurones, vesicle characteristics, 348 salivary glands, catecholamine in, 403 Schistocerca nitens dorsal unpaired median neurones, 265 DUMETi neurones, 368 neurones, dorsal unpaired median, development and, 282 a-neurotoxin receptor activity in, 288 Sciara, tracheal air filling in ecdysis, 547 Scorpion, water loss, measurement, 11 Sea urchins, septate junction formation, 73 Seminal vesicle, septate junctions in, 63 Sense organs, septate junctions in, 63 Sensible heat transfer coefficient, insect water loss, 17, 19 Septate desmosomes, definition, 4 3 Septate junction, 43-75 coexistence with gap junctions, 118 formation, 73-75 functional significance, 69-72 morphological types, 44 occurrence in insects, 62-64 Serotonin binding t o Musca domestica head extracts, 224 invertebrate skeletal neuromuscular junctions and, 390 Sertoli cells, septate junction in, 6 8 Seutigerella, septate junction in, 6 6 Sieve-area effects, gap junctions, 201 Singing, Teleogyrullus commodus, ecdysis and, 518

618

Sifotroga, tracheal air filling in ecdysis, 546 Skeletal muscle, septate junctions in, 63 Smooth septate junctions, 44 co-occurrence with gap junctions, 120 freeze-fracture, 58-62 lanthanum infiltration, 56-58 models, 62 structural features, 54-62 thin section appearance, 54-56 Somatostatin, function, 436 Sphinx ligusfri, heart, biogenic amine distribution in, 417 Spiders, tight junctions in, 132 Spisula solida, putative aminergic neurones, vesicle characteristics, 348 Spodoptera corpora pedunculata, biogenic amine distribution in, 333 deutocerebrum, biogenic amine cell localization in, 342 protocerebral bridge, biogenic amine cell localization, 338 Spodopteru Iittorulis, corpora cardiaca, biogenic amines in, 429 Spot desmosome See Maculae adhaerentes Staining negative, intercellular junctions and, 38-39 uranium calcium en bloc, intercellular junctions and, 39 Stereotypy in ecdysis, 503-514 Steroids in insect cuticular lipids, 23 Stomatogastric system, biogenic amine cell localization in, 343-345 Storage vesicles, biogenic amine localization in, 340 Sucrose-gap technique, 245 Sulphates, biogenic amine conjugation by, 363 Synaptic junctions in salivary glands, 405 DL-Synephrine myogenic rhythm and, 378 neuromuscular transmission and, 382 stimulation of Phofuris pyrulis light organs, 398

SUBJECT I N D E X

Tannic acid in intercellular junction study, 39 Tanning cuticle, in ecdysis, 541 in ecdysis, 528 Tardigrades, septate junction in, 66 Tectum fish, acetylcholine receptors, 277 toad, acetylcholine receptors, 277 Teleogry llus adult ecdysis, 513 cuticle inflation in ecdysis, 525 ecdysis, behavioural switching in, 516 escape from cuticle, 524 Teleogryllus cornmodus, ecdysis, singing and, 518 Teleogryllus oceanicus adult ecdysis, 507 dorsal midline neurones, octopamine and, 365 DUMDL cells, 371 ecdysis, 487 behaviour, 488 motor programmes, 490,491,492 timing, environment and, 478 Temperature in insect cuticles, 1-33 dynamic experiments, 13-16 measurement, 29 permeability to water and, 2 insect water loss and, 9-20 Tenebrio comb desmosome, thin section appearance, 44 ecdysis, blood volume and, 554 failures, 570 metabolism and, 560 epidermal cells, gap junction, 102 gap junction permeability, 108 calcium and, 106 tracheal air filling in ecdysis, 546 Tenebrio molitor ecdysis, bursicon and, 542 X-irradiation and, 578 water loss, measurement, 10 wax crystal structure, 27 Terminal abdominal ganglion, octopamine and, 387 Terminal bar See Zonulu udhaerens; Zonulu occludens

SUBJECT INDEX Termites, scalariform junctions in, 168 Testes gap junction in, 95 insect, tight junctions, 136-137 septate junctions in, 63, 68 occurrence in, 67 tight junctions in, 132 Tetram, receptor actions, 291, 292 Tetramethylammonium, interneurone synaptic transmission and, 253 Tettigonids, scalariform junctions in, 168 Theophylline, firefly light organ stimulation by, 400 Thyroxine, gap junction permeability and, 109 Thysanura, scalariform junctions, thin section appearance, 159 Ticks, septate junction in, 66 Tight junctions, 120-151 Calpodes, 88 coexistence with other junctional types, 138 degradation, 149-150 development and, 146-149 evolutionary position, 150-151 freeze-fractured replicas, 129-1 30 functional significance, 141-145 heterocellular, 138 homocellular, 138 in arthropods, distribution and localization, 132-138 models, 131 - 132 negative stained appearance, 126-1 29 phylogenesis, 150-1 5 1 ridge morphology, functional implications, 145 thin section appearance, 126 tracers and, 126-129 vertebrate, comparison with insect, 138-141 Tinarcha tenebricosa, hatching, developmental readiness, 480 Tinarcha violacea-nigra, hatching developmental readiness, 480 Tipula sp., water loss, measurement, 11 Tissues, insect, intercellular junctions, 35-213 Torpedo, nicotinic acetylcholine receptor from, 270

619

Toxins, receptor actions of, 288-293 Trachea air filling, in ecdysis, 546-549 tight junctions in, 137 Tracheo-glial junctions, 157 Tracheolar system septate junctions in, 63 tight junctions in, 137 Transpiration critical temperature, monolayer hypothesis, 25 cuticles, 1-33 biophysics, 3-8 lipids and, 20-29 Transporting epithelial cells, scalariform junction in, 168-169 n-Triacontanol in insect cuticular lipids, 23 Tricellular junctions, 42 Trichoptera corpora pedunculata, biogenic amine distribution in, 333 deutocerebrum, biogenic amine cell localization in, 342 protocerebral bridge, biogenic amine cell localization, 338 Triglycerides in insect cuticular lipids, 23 Tritocerebrum, biogenic amine cell localization in, 343-345 Trochanteral hairplate-to-motoneurone D, reflex, 264-265 Trypanosomes, desmosomes in, 82 Tryptamine, 5-hydroxyapplication to salivary glands, 408 biosynthesis, 354 conjugation with sulphates, 363 distribution in Periplanetu americuna, 3 24 function in corpora cardiaca, 433 in central nervous system, 320 in corpora cardiaca, 427 in heart, 417 in nervous system, 318, 321 inactivation, 357 myogenic rhythm and, 379 d-Tubocurarine effect on dorsal unpaired median neurones, 265 on electrophysiology of the sixth

620

d-Tubocurarine-(contd.) abdominal ganglion of Periplaneta, 251,258 inhibition of a-bungarotoxin binding by, 229 nicotinic receptor antagonist, 216 d-Tubocurarine, dimethyl-, binding to Musca dornestica head extracts, 222 Tunicates gap junction in, 97 septate junction in, 66 Turtles, septate junction occurrence in, 67 Tyramine effect on salivary gland stimulation by biogenic amines, 41 1 stimulation of Ph0turi.r pyralis light organs, 397 Tyrosine, hydroxylation, bursicon and, 543 Tyrosine hydroxylase, 352 Uranyl acetate, intercellular junctions and, 37,38 Uridine, 5-bromo-2’-deoxy-, PTF induced tanning and, 545 Urocanylcholine, effect on electrophysiology of the sixth abdominal ganglion of Periplaneta, 251 Vapourization See Evaporation Vascular endothelium, septate junction occurrence in, 67 Ventral nerve cord, biogenic amine cell localization in, 345 Vermiform larvae, digging behaviour, 486 Verson’s glands, 557 Vertebrates biogenic amines in, 320 central nervous system, acetylcholine receptors, 276-279 biogenic amines as hormones and, 392 desmosomes in, 75 gap junction, arthropods and, 98-100 formation, 114

SUBJECT INDEX

Maculae adhaerentes , freeze-fracture appearance, 78 -79 thin section appearance, 76 scalariform junctions, 169 septate junctions, 43 occurrence in, 67-69 skeletal neuromuscular junctions, biogenic amines and, 389-390 Vespa crabro, corpora pedunculata, biogenic amine distribution in, 332 Visceral ganglia, vertebrates, acetylcholine receptors, 277 Vitamin A, gap junction permeability and, 109 Wasps, corpora pedunculata, biogenic amine distribution in, 332 Water mass transfer, 3-6 permeability of insect cuticle to, vapourization and, 4 Water loss from insects, dynamic experiments, 12-13 measurements, 9-12 temperature and, 9-20 insect cuticular lipids and, 21-22 Waxes, insect epicuticular, water loss and, 22 Wind speed, insect water loss and, 9 Wing expansion failure in ecdysis and, 573 in adult eclosion, 501 in ecdysis, 526 in Manduca eclosion, 512 Xenopus, gap junction permeability, calcium and, 105 X-irradiation, ecdysis failure and, 577-579 Zonulae adhaerentes, 75 cockroach epidermis, 77 freeze-fracture appearance, 79 in vertebrates, thin section appearance, 76 Zonufa occludens See Tight junctions

Cumulative List of Authors Numbers in bold face indicate the volume numbers of the series Aidley, D. J., 4, 1 Andersen, Sven Olav, 2, 1 Ashini, E., 6, 1 Ashburner, Michael, 7, 1 Baccetti, Baccio, 9, 315 Barton Browne, L., 11, 1 Beament, J. W. L., 2, 67 Berridge, Michael J., 9, 1 Bodnaryk, Robert P., 13, 69 Boistel, J., 5, 1 Brady, John, 10, 1 Bridges, R. G., 9, 51 Burkhardt, Dietrich, 2, 131 Bursell, E., 4, 33 Burtt, E. T., 3, 1 Carlson, A. D., 6, 51 Catton, W. T., 3, 1 Chen, P. S., 3, 53 Calhoun, E. H., 1, 1 Cottrell, C. B., 2, 175 Crossley, A. Clive, 11, 117 Dadd, R. H., 1 , 4 7 Dagan, D., 8, 96 Davey, K. G., 2, 219 Edwards, John S., 6, 97 Eisenstein, E. M., 9, 111 Elsner, Norbert, 13, 229 Engelmann, Franz, 14, 49 Evans, Peter D., 15, 317 Gilbert, Lawrence I., 4, 69 Gilby, A. R., 15, 1 Goodman, Lesley, 7, 97

Harmsen, Rudolf, 6, 139 Harvey, W. R., 3, 133 Haskell, J. A., 3, 133 Heinrich, Bernd, 13, 133 Hinton, H. E., 5, 65 Hoyle, Graham, 7, 349 Jungreis, Arthur M. 14, 109 Kafatos, Fotis C., 12, 1 Kammer, Ann E., 13, 133 Kilby, B. A., 1, 111 Lane, Nancy J., 15, 35 Lawrence, Peter A., 7, 197 Lees, A. D., 3, 207 Linzen, Bernt, 10, 117 Machin, John, 14, 1 Maddrell, S. H. P., 8, 200 Michelsen, Axel, 10, 247 Miles, P. W., 9, 183 Miller, P. L., 3, 279 Morgan, E. David, 12,17 Narahashi, Toshio, 1, 175; 8, 1 Nelson, Dennis R., 13, 1 Neville, A. C., 4, 213 Njio, K. Djie, 14, 185 Nocke, Harold, 10, 247 Palka, John, 14, 251 Parnas, I., 8, 96 Pichon, Y., 9,257 Piek, Tom, 14, 185 Poole, Colin, F., 12, 17 Popov, Andrej V., 13, 229 Prince, William T., 9, 1

621

Pringle, J. W. S., 5, 163 Reynolds, Stuart, E., 15, 475 Riddiford, Lynn M., 10, 297 Rowell, C. H. F., 8, 146; 12, 63 Rudall, K. M., 1, 257 Sacktor, Bertram, 7,268 Sander, Klaus, 12, 125 Sattelle,DavidB., 15, 215 Shaw, J., 1,315 Skaer, Helen leB., 15, 35 Smith, D. S., 1, 401 Staddon, Brian W., 14, 351 Steele, J. E., 12, 239 Stobbart, R. H., 1, 315 Telfer, William H., 11, 223 Thomson, John A., 11, 32 1 Treherne, J . E., 1, 401; 9, 257 Truman, James W., 10, 297 Usherwood, P. N. R., 6, 205 Waldbauer, G. P., 5, 229 Weis-Fogh, Torkel, 2, 1 White, Richard H., 13,35 Wigglesworth, V. B., 2, 247 Wilson, Donald, M., 5, 289 Wyatt, G. R., 4, 287 Ziegler, Irmgard, 6, 139

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Cumulative List of Chapter Titles Numbers in bold face indicate the volume number of the series

Acetylcholine Receptors of Insects, 15, 215 Active Transport and Passive Movement of Water in Insects, 2, 67 Atmospheric Water Absorption in Arthropods, 14, 1 Amino Acid and Protein Metabolism in Insect Development, 3, 53 Biochemistry of Sugars and Polysaccharides in Insects, 4, 287 Biochemistry of the Insect Fat Body, 1, 111 Biogenic Amines in the Insect Nervous System, 15, 317 Biology of Pteridines in Insects, 6, 139 Biophysical Aspects of Sound Communication in Insects, 10, 247 Cells of the Insect Neurosecretory System: Constancy, Variability, and the Concept of the Unique Identifiable Neuron, 12, 63 Cellular Mechanisms Underlying Behaviour - Neuroethology, 7, 349 Chitin Orientation in Cuticle and its Control, 4, 213 Chitin/Protein Complexes of Insect Cuticles, 1, 257 Choline Metabolism in Insects, 9, 51 Colour Discrimination in Insects, 2, 131 Comparative Physiology of the Flight Motor, 5, 163 Consumption and Utilization of Food by Insects, 5, 229 Control of Polymorphism in Aphids, 3, 207 Control of Visceral Muscles in Insects, 2, 219 Cytophysiology of Insect Blood, 11, 117 Development and Physiology of Oocyte-Nurse Cell Syncytium, 11, 223 Effects of Insecticides in Excitable Tissues, 8, 1 Electrochemistry of Insect Muscle, 6, 205 Excitation of Insect Skeletal Muscles, 4, 1 Extraction and Determination of Ecdysones in Arthropods, 12, 17 Excretion of Nitrogen in Insects, 4, 33 Feeding Behaviour and Nutrition in Grasshoppers and Locusts, 1, 47 Frost Resistance in Insects, 6, 1 Function and Structure of Polytene Chromosomes During Insect Development, 7, 1 Functional Aspects of the Organization of the Insect Nervous System, 1, 401 Functional Organization of Giant Axons in the Central Nervous System of insects: New Aspects, 8, 96 Hormonal Control of Metabolism in Insects, 12, 239 Hormonal Mechanisms Underlying Insect Behaviour, 10, 297 Hormonal Regulation of Growth and Reproduction in Insects, 2, 247 Image Formation and Sensory Transmission in the Compound Eye, 3, 1 Insect Blood-Brain Barrier, 9, 257 Insect Ecdysis with Particular Emphasis on Cuticular Hardening and Darkening, 2, 175 Insect Flight Metabolism, 13, 133 623

624

C U M U L A T I V E LIST OF CHAPTER TITLES

Insect Sperm Cells, 9, 3 15 Insect Visual Pigments, 13, 35 Insect Vitellogenin: Identification, Biosynthesis, and Role in Vitellogenesis, 14,49 Intercellular Junctions in Insect Tissues, 15, 35 Integration of Behaviour and Physiology in Ecdysis, 15, 475 Learning and Memory in Isolated Insect Ganglia, 9, 111 Lipid Metabolism and Function in Insects, 4, 69 Long-Chain Methyl-Branched Hydrocarbons: Occurrence, Biosynthesis, and Function, 13, 1 Major Patterns of Gene Activity During Developments in Holometabolous Insects, 11, 321 Mechanisms of Insect Excretory Systems, 8, 200 Metabolic Control Mechanisms in Insects, 3, 133 Morphology and Electrochemistry of Insect Muscle Fibre Metabolism, 14, 185 Nervous Control of Insect Flight and Related Behaviour, 5 , 289 Neural Control of Firefly Luminescence, 6, 51 Neuroethology of Acoustic Communication, 13, 229 Osmotic and Ionic Regulation in Insects, 1, 315 Physiology of Insect Circadian Rhythms, 10, 1 Physiology of Moulting in Insects, 14, 109 Physiological Significance of Acetylcholine in Insects and Observations upon other Pharmacologically Active Substances, 1, 1 Polarity and Patterns in the Postembryonic Development of Insects, 7, 197 Postembryonic Development and Regeneration of the Insect Nervous System, 6,97 Properties of Insect Axons, 1, 175 Regulation of Breathing in Insects, 3, 279 Regulation of Intermediary Metabolism, with Special Reference to the Control Mechanisms in Insect Muscle, 7,268 Regulatory Mechanisms in Insect Feeding, 11, 1 Resilin. A Rubberlike Protein in Arthropod Cuticle, 2, 1 Role of Cyclic AMP and Calcium in Hormone Action, 9, 1 Saliva of Hemiptera, 9, 183 Scent Glands of Heteroptera, 14, 351 Sequential Cell Polymorphism: A Fundamental Concept in Developmental Biology, 12, 1 Specification of the Basic Body Pattern in Insect Embryogenesis, 12, 125 Spiracular Gills, 5, 65 Structure and Function of the Insect Dorsal Ocellus, 7, 97 Structure and Function of Insect Peptides, 13, 69 Theories of Pattern Formation in Insect Neural Development, 14, 251 Transpiration, Temperature and Lipids in Insect Cuticle, 15, 1 Tryptophan+Ommochrome Pathway in Insects, 10, 117 Variable Coloration of the Acridoid Grasshoppers, 8, 146