ADVANCES IN INSECT PHYSIOLOGY VOL 4 APL, Volume 4 (v. 4)

Advances in

INSECT PHYSIOLOGY

VOLUME 4

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Advances in

Insect Physiology Edited by

J. W. L. BEAMENT, J. E. TREHERNE and V. B. WIGGLESWORTH Department of Zoology, The University, Cambridge, England

VOLUME 4

1967

ACADEMIC PRESS London and New York

ACADEMIC PRESS INC. (LONDON) LTD BERKELEY SQUARE HOUSE BERKELEY SQUARE LONDON, W.1

US.Edition published by ACADEMIC PRESS INC.

11 1 FIFTH AVENUE 10003

NEW YORK, NEW YORK

Copyright 0 1967 By Academic Press Inc. (London) Ltd

All Rights Reserved NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS

Library of Congress Catalog Card Number: 63-14039

Printed in Great Britain by William Clowes and Sons, Limited, London and Beccles

Contributors to Volume 4 D. J. AIDLEY, Department of Zoology, University of Oxford, England (p. 1)

E. BURSELL,Department of Biological Sciences, University of Rhodesia and Nyasaland, Salisbury, Rhodesia @. 33) L. I. GILBERT, Department of Biological Sciences, Northwestern University, Evanston, Illinois, U.S.A. (p. 69) A. C . NEVILLE, Department of Zoology, University of Oxford, England (p. 213) G. R. WYATT, Department of Biology, Yale University, New Haven, Connecticut, U.S.A. (p. 287)

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Contents CONTRIBUTORSTO VOLUME4

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V

EXCITATION OF INSECT

SKELETAL. D. J. AIDLEY

I. Introduction

.

11. The Resting Potential

MUSCLES

.

A. The effect of potassium ions . B. The effect of chloride ions . C. The effect of sodium ions . D. The effect of divalent cations . E. The effects of carbon dioxide F. Temperature dependence . 111. Neuromuscular Transmission . A. The innervation of insect muscle . B. Excitatory responses . C. The quanta1 release of transmitter substance . D. Inhibitory responses . , IV. The Electrical Excitability of the Muscle Fibre Membrane A. The electrical properties of electrically excitable responses B. The ionic basis of electrically excitable responses . C. Spontaneous activity . . V. The Excitation-contraction Coupling Process . A. Depolarization . B. The importance of calcium ions . C. The action of carbon dioxide . D, “Fast” and “slow” muscles . References .

.

1 2 2 4 5 6 6 6 7 7 8 15 17 20 20 21 23 23 23 24 26 26 27

THEEXCRETION OF NITROGEN IN INSECTS E. BURSELL

I. Introduction . 11. The Formation of Nitrogenous End Products A. The uricolytic pathway . . B. The uricotelic pathway . C. The formation of urea . D. The formation of ammonia . E. Amino acids . F. Miscellaneous materials . III. The Excretion of Nitrogenous End Products A. Collembola . B. Orthoptera . C. Odonata .

vii

.

33 34 36

40

.

41 42 43 44 44 45 46

47

viii

CONTENTS

D. Dermaptera

E. Hemiptera . F. Coleoptera G. Neuroptera . H. Hymenoptera I. Diptera . J. Lepidoptera 1V. Conclusions . References .

.

48 48 50 51 51 52 54 56 61

.

. .

LIPIDMETABOLISM AND FUNCTION IN INSECTS LAWRENCE I. GILBERT

I. Introduction . A. General . B. Definition and classification . 11. Lipid Content . A. Expression of data . B. Alterations during metamorphosis . C. Nature of insect lipids . III. Lipid Utilization . A. Digestion and absorption . B. Lipid release and transport . C. Extra-digestive lipases . D. Fatty acid catabolism . IV. Lipid Biosynthesis . A. General mechanism of fatty acid synthesis B. Fatty acid biosynthesis in insects . C. Phospholipid and triglyceride . D. Fatty acids in nutrition . E. Substrate interconversion . V. Hydrocarbons and Waxes . A. Cuticle B. Extra-cuticular . VI. Isoprenoid Compounds . A. Nutritional studies . B. Isoprenoid biosynthesis . C. Isoprenoid content . D. Sterol modification . E. Function F. Insect hormones . W.Conclusions . References . Addenda .

70 70 71 71 71 81 89 97 97 102 110 116 127 127 130 134 145 147 152 152 155 157 157 161 168 170 175 176 186 187 208

.

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.

CHITIN ORIENTATION IN CUTICLE AND

ITS

CONTROL

A. C. NEVILLE

I. Introduction

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II. Orientation and the Mechanicai Properties. of &tic& molecules

.

213

Macro:

217

CONTENTS

ix

. A. Parallel orientation . B. Crossed fibrillar orientation . C. Lamellar structure . D. Functional aspects . 1V. Orientation Control . A. Circadian organization . B. Metabolic oscillators and “switches” C. “Dermal” light sense . D. Implantation experiments . E. Nervous control . F. Discussion . V. Orientation Mechanisms . A. Primary orientation . B. Secondary orientation . C. Protein orientation . D. Hypothesis . M.Conclusion . References .

220 220 220 223 229 233 233 246 254 257 260 260 262 263 265 270 272 279 280

III. Types of Chitin Architecture

THEBIOCHEMISTRY OF SUGARS AND POLYSACCHARIDES IN INSECTS G. R. WYATT

1. Introduction . II. The Occurrence of Sugars in Insects . A. Glucose and reducing substances . B. Trehalose . C. Sugar content of insect hsmolymph . D. Sugar content of whole insects and insect tissues . 111. Intestinal Absorption and the Physiology of Hemolymph Sugar Levels . A. Absorption from the gut . B. Regulation of blood sugar . IV. Biosynthesis and Utilization of Sugars . A. Glucose . B. Use of monosaccharides other than glucose . C. Biosynthesis of trehalose . D. Cleavage and use of trehalose . E. Physiological roles of trehalose and trehalase . F. Dormancy and the properties of trehalose . .* . V. Glycogen . A. Glycogen in insects . B. Accumulation and conversion during growth and metamorphosis C. Glycogen in insect flight D. Metabolism of glycogen VI. Hormonal Effects on Carbohydrate Metabolism .

.

1.

. .

287 289 289 289 29 1 295 297 297 299 301 301 302 304 309 317 324 325 325 327 329 329 336

CONTENTS

X

W.Glycoproteins and Chitin

.

A. Glycoproteins in insects B. Metabolism of chitin . Vm. Glycerol and Sorbitol . . References

AUTHORINDEX .

SUBJECT INDEX

.

.

.

. .

340 340 341 345 347

.

361

. .

. 375

The Excitation of Insect Skeletal Muscles D. J. AIDLEY Department of Zoology, University of Oxford, England I. Introduction

.

11. The Resting Potential

1

.

A.

The effect of potassium ions B. The effect of chloride ions . C. The effect of sodium ions . D. The effect of divalent cations . E. The effects of carbon dioxide . F. Temperature dependence . UI. Neuromuscular Transmission . A. The innervation of insect muscle . B. Excitatory respomes . C. The quanta1 release of transmitter substance . D. Inhibitory responses . IV. The Electrical Excitability of the Muscle Fibre Membrane . A. The electrical properties of electrically excitable responses B. The ionic basis of electrically excited responses . C. Spontaneous activity . . V. The Excitation-contraction Coupling Process A. Depolarization . B. The importance of calcium ions . C. The action of carbon dioxide . D. “Fast” and “slow ” muscles References .

. . .

. . . . . . .

. . . . . . .

.

2 2 4

s

6 6 6 7 7 8 1s 17 20 20 21 23 23 23

. 2 4 26 . 26 . 27

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I. INTRODUCTION

In the resting condition, muscle cell membranes ‘are electrically polarized so that the inside of the cell is a few tens of millivolts negative to the outside. Stimulation of the motor nerves supplying the muscle results in a reduction of this membrane potential, which is followed by contraction of the muscle. This article will attempt to view our knowledge of this chain of events in insect muscles mainly in the light of the much greater understanding of vertebrate muscles. I

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D . J . AIDLEY

11. THERESTINGPOTENTIAL A . THE EFFECT OF POTASSIUM IONS

According to the theory developed by Boyle and Conway (1941) to explain the ionic distribution between the cell and the extracellular fluid in frog sartorius muscle, the distribution of potassium and chloride ions follows a Donnan equilibrium set up by the presence of indiffusible anions inside the fibre, the system being maintained in osmotic equilibrium by the presence of an effectively indiffusible cation (sodium) in the extracellular fluid. The resting potential at equilibrium (E) is then given by the equations

where R is the gas constant, T the absolute temperature, F is Faraday’s constant, and the subscripts and ,refer to the intracellular and extracellular media, respectively. These equations also define the potassium equilibrium potential, EK,and the chloride equilibrium potential, Eel. Changes in [Kl0 or [CI], result in movements of KCl across the membranes so as to restore the equilibrium condition given by eqs. (1) and (2). Since movement of a given quantity of KCI results in much greater relative changes in [CI], than in [K],, changes in [Kl0 cause much larger changes in membrane potential at equilibrium than do changes in [Cl],, although large transient changes in membrane potential may be produced by changes in [Cl], (Hodgkin and Horowicz, 1959). One method of testing the applicability of eq. (1) is to measure [K], and [K], and see if the membrane potential calculated from the equation agrees with that actually found. This was done by Wood (1963) for leg muscle fibres of Locusta, Periplaneta and Carausius after equilibration for four to six hours in salines of ionic composition approximating to that of the haemolymph, with [K], between 10 and 18 m ~ In. each case the actual resting potential was close to that predicted from eq. (1). A more usual method of testing the applicability of eq. (1) is by measuring membrane potentials at different values of [K],. It is necessary either to measure [K], at each value of [Kl0 (Conway, 1957), or to ensure that there is no change in [K], during the course of the experiment by maintaining the [K],[Cl], product constant or by eliminating chloride from the system by using extracellular solutions

THE EXCITATION O F INSECT SKELETAL MUSCLES

3

containing sulphate as the major anion (Adrian, 1956; Hodgkin and Horowicz, 1959). If [K]*is constant, eq. (1) predicts that the membrane potential should be related to [K], by means of a straight line with a slope (at 18") of 58 mV per unit change in log,,[K],. Hoyle (1953) studied the effects of substitution of potassium chloride for sodium chloride in the external saline solution on the membrane potential of fibres in the metathoracic retractor unguis of Schistocercu (Fig. 1). At concentrations greater than 10 mM, the relation between

Potassium concentration (mu)

RQ.1. The relation between potassium ion concentration and membrane potential in the metathoracic retracto: unguis muscle of Schisrocerca. Chloride ion concentration constant. (From Hoyle, 1953.)

membrane potential and log,,[K], was linear, with a slope of about 50 mV. Similar results have been obtained by Hagiwara and Watanabe (1954) from locust flight muscle (slope 25 mV) and Wood (1957) on the prothoracic flexor tibialis of Curuusius (slope 36 mv). In each case the slope of the straight line section of the curve was less than the 58 mV predicted by eq. (1). However, the method used in these experiments involves a change in the [K],[CI], product so" that the system will initially be displaced from equilibrium (so that EE is not equal to Ec3

4

D . J . AIDLEY

and can only reach a new equilibrium position by a change in [K],; both these effects will tend to reduce the slope of the line. When the external potassium ion concentration is low, the membrane potential is less than that predicted from eq. (I), so that the graph of membrane potential against log,,[K], tends to level out in this region (Fig. 1). This phenomenon is common to a number of types of cell, and can be ascribed to a constant small permeability of the membrane to sodium ions and a decrease in permeability to potassiuni ions when [K], is low (Hodgkin, 1951). The membrane potential can then be described by the equation

(Hodgkin and Katz, 1949), where PK,PNaand PClare permeability coefficients. There seems to be little doubt that this equation could be successfully applied to the results obtained from the experiments so far mentioned. There are, however, observations on some insects which are very difficult to reconcile with the potassium electrode hypothesis for the resting potential. Belton (1958, 1960) reported that the membrane potentials of muscle fibres of various Lepidoptera were unaffected by changes in [K],. Belton and Grundfest (1962) obtained similar results from Tenebrio larvae; here the membrane potential was not affected by changes in [K],up to about 100 m, but at higher concentrations there was a logarithmic relation between membrane potential and [Kl0with a slope of 58 mV. They also measured the potassium concentration of the muscle fibres (72 m) and the haemolymph (40 m),and pointed out that the value of EKcalculated from these figures (- 17 mv) is much less than the actual membrane potential (- 50 to - 60 mV in muscles bathed in haemolymph). B . T H E EFFECT O F C H L O R I D E IONS

The relationship between external chloride ion concentration and the membrane potential has been investigated by Wood (1965), in the leg muscle fibres ofbcusta and Periplaneta. In both insects there was an increase of a few millivoltsas [CI], was raised from 0 to 150mM. This implies that the membrane is permeable to some extent to chloride ions. At first sight, the fact that the membrane potential was not related to [Cl], by a straight line with a slope of 58 mV per unit change in logl0[C1], might be explained by movement of KCl out of the cell when [Cl], was less than about 100 mM. However, Wood found that [Cl], was almost independent

THE EXCITATION OF INSECT SKELETAL MUSCLES

5

of [Cl], in this range. He suggested that the results implied that the muscle

cell membrane is much less permeable to chloride than to potassium ions, with the possibility that there is some active transport of chloride across the membrane. Thus the insect muscle cell membrane would seem to be, in these respects, similar to the squid giant axon (Keynes, 1963) rather than the frog muscle fibre membrane, where the chloride permeability is high (Hodgkin and Horowicz, 1959). The importance of such a relatively low chloride permeability might be that it allows the membrane potential to be changed by increase of PCl during inhibition (see Section 111, D, below). However, Wood’s results cannot be taken as implying that the chloride conductance of the membrane is negligible. If this were so, it would be difficult to explain the departure of the slope of the relation between membrane potential and log,,[K], from the 58 mV predicted by eq. (1). A critical investigation of this problem would include measurements of the transient changes in membrane potential caused by changes in [Kl0 and [Cl],, such as were made by Hodgkin and Horowicz (1959) on frog muscle fibres. Usherwood and Grundfest (1965) noticed that transient depolarizations of 7 to 15 mV occurred when a chloride-free solution was substituted for the normal Ringer solution perfusing Romalea muscle fibres ;the reverse procedure caused transient hyperpolarizations (Usherwood, personal communication). C . THE EFFECT OF S O D I U M IONS

Wood (1961, 1963) showed that a decrease in external sodium ion concentrationresulted in a fall in the resting potential of muscle fibres of Lmusta, Periplaneta and Carausius. The tonicity of the low-sodium salines was maintained by substitution of sucrose or choline chloride for sodium chloride. In crustacean muscle fibres, Fatt and Katz (1953) observed a similar decrease in the resting potential when sucrose was substituted for sodium, but only a very small decrease when sodium was replaced by choline. In the case of sucrose substitution it is evident that the associated decrease in chloride concentration (and, probably, consequent movements of KC1) would be expected to lower the membrane potential, although it is not clear whether these effects would be sufficient to account for the magnitude of the observed changes. Obviously this explanation cannot be applied to the fall in membrane potential which occurs in insect muscles when choline chloride is used as a substitute for sodium chloride. Hence it seems (Wood, 1961) that there is some direct influence of sodium ions on the permeability of the resting muscle fibre membrane.

6

D. J. AIDLEY

There is at the time of writing no evidence for the existence of an active sodium extrusion mechanism in insect muscle fibres, but it would be most surprising if such a mechanism did not exist. D . T H E EFFECT O F D I V A L E N T C A T I O N S

It is well known that, in vertebrate muscles, decrease in the calcium ion concentration of the Ringer's solution causes a fall in resting potential, and that this fall can be prevented by the presence of other divalent cations (Jenden and Reger, 1963). This phenomenon has apparently not been looked for in insect muscles, but Wood (1957) showed that an increase in external calcium ion concentration caused an increase in the resting potential of Carausius muscle fibres ; increase in magnesium ion concentration did not affect the resting potential. High concentrations of barium ions cause a fall in the resting potentials of Romalea muscle fibres (Werman, McCann and Grundfest, 1961). E . T H E EFFECTS O F C A R B O N D I O X I D E

In Schistocerca spiracular muscle, introduction of carbon dioxide into the tracheal system causes a slight depolarization of the muscle fibre membranes, associated with a decrease in the membrane resistance (Hoyle, 1960). The ionic basis of this depolarization is not known, but an increase in sodium permeability seems to be the obvious candidate. Similar slight depolarization by carbon dioxide occurs in the flight muscles of the beetles Pissodes and Tenebrio, but very large depolarizations are produced in the flight muscles of the wasp Vespula and the fly Sarcophaga (McCann and Boettiger, 1961). In the pupal spiracular muscles of the silkmoths Hyalophora and Telea, application of carbon dioxide causes a hyperpolarization of the fibre membrane (van der Kloot, 1963). F . TEMPERATURE DEPENDENCE

Kerkut and Ridge (1 96 1) compared the immediate effects of a change in temperature from 15 to 25" on the resting potentials of muscle fibres of Carcinus, Rana and Periplaneta. The Qlo values over this range were 1.064 & 0.004(s.E.)for the crab, 1.060 2 0.004 for the frog, and 1.296 f 0.046 for the cockroach. These values are all significantly different from the value of 1.035 expected if the system obeys eq. (I), and the value for the cockroach is significantly higher than those for the frog and the crab. Kerkut and Ridge concluded that the resting potential is directly dependent upon metabolic processes. Since the measurements of membrane

THE EXCITATION O F I N S E C T S K E L E T A L MUSCLES

7

potential were made after about 1 min. at the new temperature, it is improbable that this effect is dependent upon changes in ionic concentrations in the cell. If there is a sodium extrusion mechanism which is not electrically neutral (for example, if there is no 1 :1 exchange with potassium ions) the resting potential will be dependent to some extent on the activity of the pump, being more negative when the pump is active (as it presumably is at higher temperatures within the physiological range). However, this is not the only possible interpretation of these results. As we have seen, eq. (3) gives a better description of the resting potential than does eq. (l), particularly at external potassium ion concentrations less than about 10 mM; in the salines used by Kerkut and Ridge the potassium ion concentration was approximately 5 mM. Now the Qlo of membrane potential given by eq. (3) will only be 1.035 for the range 15 to 25” if the Qlos of the permeability coefficients PK,P,, and Pcl are identical. In particular, if the Qlo of PNais less than those of PK and Pel, increase in temperature will result in a hyperpolarization greater than that predicted by eq. (1). It should be possible to distinguish between these two alternatives by measuring the effect of temperature changes on the membrane potential of fibres poisoned with some suitable metabolic inhibitor. 111. NEUROMUSCULAR TRANSMISSION A. THE I N N E R V A T I O N O F I N S E C T MUSCLE

This subject has been extensively discussed in a recent review by Hoyle (1965), and will therefore be but briefly examined here. Insect muscle fibres possess multiterminal innervation, there being numerous motor nerve endings on each fibre which are spaced at intervals of 30 to 80 p apart along the length of the fibre. Polyneuronal innervation also occurs, so that many muscle fibres are innervated by more than one motor axon. In these cases stimulation of the different motor axons causes different responses in the muscle fibre. The motor axons can be divided into two broad categories: excitor axons, which produce depolarization of the muscle fibre membrane leading to contraction of the fibre, and inhibitor axons, which produce hyperpolarization and can counteract, to some extent, the action of the excitor axons. In many cases fibres are innervated by more than one excitor axon, and stimulation of these axons produces electrical and mechanical responses of different magnitude; axons producing larger responses are then known as “fast” axons, and those producing,smallerresponses are known as ‘“slow” axons (Pringle, 1939; Hoyle, 1955a, b). Some muscles (such as the extensor tibiae of

8

D . J . AIDLEY

locusts) are innervated by only one set of axons, while others (such as the flexor tibiae of locusts) are innervated by a number of axons of each type. The latter may thus be said to possess a number of motor units, although it is obvious that the definition of a motor unit in a system in which there may be overlapping fields of motor innervation, between axons producing different types of response, is largely a matter of opinion. These points may be illustrated by reference to two muscles of the locust Schistocerca gregaria, in which the pattern of motor innervation has been closely examined. The metathoracic extensor tibiae (the jump ing muscle) is innervated by three motor axons (Hoyle, 1955a, b), a “fast” excitor, a “slow” excitor and an inhibitor. About 80% of the muscle fibres are innervated by the “fast” exciter, 20 to 30% are innervated by the “slow” excitor, and about 10% (all of them also innervated by the ‘‘slow’’ excitor) are innervated by the inhibitor (Usherwood and Grundfest, 1965). Thus some fibres are innervated by all three axons, some are innervated by the “ slow” excitor and either the inhibitor or the “fast” excitor, and the majority are innervated by the “fast” excitor only. In the meso- and metathoracic dorsal longitudinal flight muscles of Schistocerca, there are five motor units each separately supplied by one axon, the response to stimulation being of the “fast” type (Neville, 1963). It is probable that there is no “slow” or inhibitor innervation of these muscles. B. EXCITATORY RESPONSES

1. The general nature of the excitatory responses Stimulation of “slow” type excitatory axons causes a depolarization of the muscle fibre membrane, the postsynaptic potential (also known as the junction potential or end plate potential), which rises fairly rapidly to a peak and then falls more slowly (Fig. 2). The size of the response varies between different preparations and between different fibres of the same preparation; Hoyle (1957) gives a range of 2 to 50 mV for the size of the “slow” postsynaptic potential in the mesothoracic extensor tibiae of Schistocerca. Stimulation of “fast” type excitatory axons causes a larger electrical response (Fig. 3), which frequently overshoots the zero level of membrane potential so that the inside of the fibre becomes briefly positive to the outside. In many cases the “fast” response consists of two components, a postsynaptic potential and an electrically excited response

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9

(sometimes called the “active membrane response”) caused by the depolarization constituting the postsynaptic potential (see Section IV below). This electrically excited component may be absent in some preparations, such as the metathoracic spiracular muscle of Schistocerca (Hoyle, 1959). There does not seem to be any fundamental difference between “slow” and “fast” responses. “ Slow” responses may summateto give a depolarization large enough to elicit an electrically excited response (Cerf et al., 1959),and “fast” responses lose their regenerativecomponents and look

FIG.2. The electrical response to stimulation of the “fast” axon in the mesothoracic extensor tibiae of Schistocercu. Intracellular records from three different fibres. The upper trace shows zero potential initially, then twitch tension.

just like “slow” responses when the muscle is subjected to low temperatures (del Castillo et al., 1953) or neuromuscular blocking agents (Hill and Usherwood, 1961; Hoyle, 1955~). 2. The ionic basis of excitatory postsynaptic potentials In their classic study of the postsynaptic potential in frog sartorius muscle, Fatt and Katz (1951) suggested that the chemical transmitter substance (acetylcholine in this case) released from the motor nerve ending causes a brief increase in the ionic permeability of the postsynaptic membrane, so that the resting potential is effectively shortcircuited at the motor end plate during the action of the transmitter

10

D. J . A I D L E Y

substance. In accordance with this hypothesis, it was found that the size of the postsynaptic potential increased proportionately when the membrane potential was increased by passing current through a second microelectrode inserted in the (curarized) muscle fibre. A similar effect

. . c-w-!=Qc

I 100 rnsec

FIG.3. Theelectrical response to stimulation of the “slow” axon in the mesothoracic extensor tibiae of Schistocerca. Intracellular records from four different fibres (a-d). Upper trace shows zero potential and stimulus monitor. Note reflex responses in c and summation in the right-hand record from ‘0.

was found by del Castillo et al. (1953) for the “fast” responses of locust leg muscles, the postsynaptic potential component of the electrical response being just over half the value of the membrane potential over the range - 50 to - 150 mV. Further confirmation of this view was provided by Cerf et al. (1959), who showed that depolarizations large enough to make the inside of the fibre positive to the outside resulted in a

THE EXCITATION OF INSECT SKELETAL MUSCLES

11

reversal of the sign of the “slow” postsynaptic potential. Similarly, depolarization of the muscle fibre membrane by increasing the external potassium ion concentration causes a decrease in the size of the postsynaptic potential (Hoyle, 1955~;Wood, 1957). In frog muscles, voltage clamp studies have shown that sodium and potassium are the ions mainly involved in the increase in permeability during the postsynaptic potential (Takeuchi and Takeuchi, 1960). There is as yet no information of this kind for the excitatory postsynaptic potentials of insect muscles.

3. The blocking effect of tryptamine and similar compounds Hill and Usherwood (1961) showed that perfusion of the metathoracic flexor and extensor tibiae muscles of Schistocerca with solutions containing 1 to 10 DIM tryptamine diminished both the electrical and

FIG.4. The effect of tryptamine on the “fast” response of the metathoracicflexor tibiae of Schistocerca. The upper trace shows tension and, initially, zero potential. Time signal 500 c/s. Trace 1 shows the normal response. Traces 2-4 show responses at 5 sec. intervals after the application of 10 mM tryptamine. Traces 6-10 show responses at 5 sec. intervals after removal of tryptamine. (From Hill and Usherwood, 1961.)

mechanical responses to “fast” axon stimulation (Fig. 4). Similar results were obtained with 5-hydroxytryptamine, lysergic acid diethylamide, bromolysergic acid diethylamide, 5,6-dimethoxytryptamine and 3-(pyrrolidino-methyl)-thionapthene. These substances did not affect the conduction of action potentials in the crural nerve trunk, and neither tryptamine nor 5-hydroxytryptamineaffected the muscle fibre membrane resistance or the electrically excited responses of the membrane to depolarization via an internal electrode. It was concluded that these substances act as neuromuscular blocking agents, possibly by competing with the transmitter substance for receptor sites 04 the postsynaptic membrane.

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D. J . A I D L E Y

Hill and Usherwood also found that tryptamine increases the synaptic delay, and that this effect appears to be more long-lasting than the blocking effect on the size of the post-synaptic potential. In the mesothoracic extensor tibiae of Schistocerca tryptamine also blocks transmission from the “slow” axon (D. J. Aidley, unpublished). 4. The nature of the excitatory transmitter substance A number of studies have been made on the effects of acetylcholine on

insect muscles. Harlow (1958) found that acetylcholine had no effect on resting locust leg muscles, and Wood (1958) obtained similar results for the prothoracic flexor tibiae of Carausius. These authors also reported that d-tubocurarine and adrenaline did not affect the responses to nervous stimulation. On the other hand, it seems that neurally evoked contractions can be increased in size in the presence of acetylcholine. This effect has been described by Harlow (1958) and Hill (1963) in locusts, and by Kerkut et al., (1965a) in the cockroach. The nature of the mechanisms behind this increase in contraction size is obscure; there is an urgent need for microelectrode studies on the form of the electrical responses to stimulation under these conditions. Similar increase in the contraction size is brought about by L-glutamic acid and, to a lesser extent, D-glutamic and L-aspartic acids (Kerkut et al., 1965a). The presence of glutamate in perfusates of the femoral muscles of Periplaneta has recently been demonstrated by Kerkut et al. (1965b). They found that the amount of glutamate produced was proportional to the number of stimuli to the motor nerves, and that the muscles contracted when perfused with low concentrations of glutamate, the contraction increasing in size as the glutamate concentration was increased. Similar results were obtained for crab and snail muscles. Usherwood and Grundfest (1965) state that glutamate causes a depolarization of insect muscle fibres. It is evident from these results that a number of substances have excitatory effects on insect muscles, although it is impossible at present to say which of them, if any, is the naturally occurring excitatory transmitter substance. Glutamate, or a glutamate complex, appears to have the strongest claims in this respect. It is possible that the “slow” and “fast” responses to nervous stimulation are brought about by different transmitter substances, but it seems more likely that “fast” responses are brought about by release of more transmitter substance per impulse than are “slow” responses. Qualitatively, the effects of blocking agents appear to be the same on

T H E E X C I T A T I O N OF I N S E C T S K E L E T A L MUSCLES

13

both systems. This opinion would have to be revised if it were shown that the reversal potentials for the two types of postsynaptic potential were different. 5. Calcium-magnesium antagonism Increase of the magnesium ion concentration of the external solution causes a diminution in the size of the “fast” postsynaptic potential in Locusta and Periplaneta (Hoyle, 1955~).This effect is antagonized by a corresponding increase in calcium ion concentration. “ Slow ” postsynaptic potentials are similarly affected @. J. Aidley, unpublished). These results are similar to those obtained by del Castillo and Engbaek (1954) from frog sartorius muscles. They concluded that the depressant action of magnesium occurs presynaptically, part of the evidence for this being that the very small postsynaptic potentials produced in high magnesium ion concentrationsshow quanta1fluctuations in size, thought to correspond with “packets” of acetylcholine released from the motor nerve ending (del Castillo and Katz, 1954). Similar fluctuations in postsynaptic potential size were observed by Usherwood (1963a) in Schistocerca leg muscles in solutions containing high magnesium ion concentrations, and hence it would seem probable that the site of this calcium-magnesium antagonism is presynaptic in locust muscle. The blood of some herbivorous insects may contain relatively large concentrations of magnesium ions (Duchilteau et al., 1953); in Carausius, for example, investigated by Wood (1957), the average magnesium concentration was 53 mM, whereas that for calcium was only 7.5 m. This situation is of some interest, for concentrations of this order are sufficient to block completely neuromuscular transmission in locusts and frogs. Wood found that the size of the “fast” postsynaptic potential decreased as the calcium ion concentration was reduced below its value in the blood, as in locusts and frogs. However, the relation which he found between the size of the total electrical response to stimulation of the “fast” axon (postsynaptic potential plus electricallyexcited response) and the magnesium ion concentration is unique among those preparations which have been investigated. The electrical response increased from a value of about 21 mV in the absence of magnesium ions to a maximum of about 43 mV at a concentration of approximately 75 mM, thereafter falling with increasing magnesium ion concentration to reach zero at about 200 m. This fall in the electrical response at very high magnesium concentrations could not be antagonized by calcium ions, and may imply, as Wood suggested, that a high”magnesiumion concentration reduces the sensitivity of the postsynaptic membrane to the

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D . J. AIDLEY

transmitter substance, as appears to be the case in the frog sartorius muscle (del Castillo and Engbaek, 1954). It is not clear whether the increase in the size of the postsynaptic potential with increasing magnesium ion concentration over the range 0 to 75 mM is a presynaptic or a postsynaptic effect. 6. The action of carbon dioxide Postsynaptic potentials of Schistocerca metathoracic spiracular muscle (Hoyle, 1960) and the flight muscles of certain beetles and the bug Nazzara (McCann and Boettiger, 1961)are reduced in the presence of high concentrations of carbon dioxide. It is not clear whether the carbon dioxide acts presynaptically or postsynaptically in these cases.

7 . Time-dependentproperties of the postsynaptic potentials When the interval between motor nerve impulses is short enough, postsynaptic potentials readily summate so as to give a depolarization greater than that following a single impulse. In some cases, repetitive stimulation of the “slow” axon results in a “plateau” of depolarization whose degree is proportional to the stimulation frequency (Hoyle, 1955b; Wood, 1958). When the postsynaptic potential is accompanied by an electrically excited response (as in typical “fast” potentials) the situation is complicated by the presence of refractoriness in the electrically excited component, but it is sometimes possible to discern summation of the postsynaptic potential components when the stimulus interval is short enough (Wood, 1958). Some degree of facilitation is frequently present when the postsynaptic potentials are very small, but this phenomenon does not seem to be as well developed in insects as it is in crustacea. By estimating the quanta1 content of postsynaptic potentials in crustacean muscle fibres, Dude1 and Kuffler (1961) were able to show that facilitation is a presynaptic phenomenon, being caused by an increase in the amount of transmitter substance released from the motor nerve ending. It is probable that a similar mechanism occurs in insect muscles. Becht et al. (1960)claimed that, in some of the coxal muscles of the cockroach, the postsynaptic potential component of the second of two responses to “fast” axon stimulation showed the effects of refractoriness in that it was smaller than usual for about 15 msec. after the first response. The interpretation of this result is complicated by the presence of the electrically excited component associated with the first response, but it may well be that a process of depression (the reverse of facilitation) occurred.

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C. THE Q U A N T A L RELEASE OF TRANSMITTER SUBSTANCE

1. Spontaneous miniature postsynaptic potentials If a microelectrode is inserted into a frog sartorius muscle fibre in the region of the end plate, small “miniature postsynaptic potentials” can be seen (Fatt and Katz, 1952). These are similar in their pharmacology to neurally evoked postsynaptic potentials, and are probably caused by the spontaneous release of acetylcholine in “quantal” units derived from the vesicles which occur in the presynaptic nerve ending (Katz, 1962). Usherwood (1961, 1963a) has observed similar spontaneousminiature potentials in the muscle fibres of Schistocerca, Blaberus and Periplanetu (Fig. 5). The amplitude and time course of these miniature potentials were more variable than in the frog sartorius, an effect which is almost certainly due to the multiterminal innervation of insect muscles, so that potentials originate at different distances from the recording site, with consequent differences in the degree of attenuation of the potentials. In accordance with this view, Usherwood showed that the frequency distribution of the amplitudes of the potentials was much more skew in fibres which were long in relation to their length constant than in short ones. Analysis of the frequency distribution of the time intervals between successive potentials in a long series showed that the discharge is a random process. The average frequency of occurrence of the spontaneous miniature potentials could be increased by raising the external calcium ion concentration, and decreased by raising the external magnesium ion concentration. Concentrations of magnesium greater than 10 m~ also reduced the amplitude of the potentials; it is thought that this effect is by means of a reduction in the sensitivity of the postsynaptic membrane to the transmitter substance. Increase in external potassium ion concentration caused an increase in the average frequency of the potentials, probably by depolarizing the motor nerve endings. Application of salines made hypertonic with sucrose caused a transient increase in the frequency of the potentials. In all these respects the miniature potentials are like those of vertebrate muscles, except that, as Usherwood points out, the insect system seems to be rather less sensitive to magnesium ions. Since there seems to be much evidence for the statement that changes in frequency indicate presynaptic actions whereas changes in amplitude indicate postsynaptic actions (Katz, i962), an examination of the effects of calcium and magnesium ion concentration on the

16

D. J . AIDLEY

frequency of discharge of miniature potentials in Carausius might give some indication of the site of action of these ions in this case. Curare, acetylcholine and prostigmine had no effect on the miniature potentials, but 5hydroxytryptamine reduced their amplitude, resulting in complete block at high concentrations. Thus the pharmacology of

FIG. 5. Spontaneous miniature potentials from six different muscle fibres of Bluberus, recorded on a free-running trace. Calibrations 1 mV and 400 c/s. (From Usherwood, 1961.)

the miniature potentials appears to be similar to that of the neurally evoked postsynaptic potentials. After denervation of locust muscles, “ giant’’ miniature potentials up to 10 mV in height are observed at about the same time that impulse transmission from the nerve to the muscle fails (Usherwood, 1963b).

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It seems likely that these are connected with a disorganization of the motor nerve endings.

2. The quantal nature of the postsynaptic potential The presynaptic nerve endings at chemically transmitting synapses contain numbers of small vesicles. These are thought to contain “packets ” of transmitter substance which are released spontaneously in small numbers (producing miniature postsynaptic potentials) and in large numbers in the depolarization of the presynaptic nerve membrane. According to this interpretation, the postsynaptic potential is composed of a large number of quantal units, each resulting from the action of one of these packets on the postsynaptic membrane. The evidence for this is mainly derived from the observations of the sizes of the reduced postsynaptic potentials seen in frog muscles after treatment with Ringer solutions containing high magnesium ion concentrations. These potentials fluctuate in size by amounts equal to those of single miniature potentials. Similar “quantal ” fluctuations have been observed by Ushenvood (1963a,b) in postsynaptic potentials of locust muscle fibres after treatment with high magnesium ion concentrations or following denervation of the muscle. Presynaptic vesicles occur at insect neuromuscular junctions (Edwards et al., 1958). From these results, it seems that the intimate nature of the transmission process at insect neuromuscular junctions is similar to that at other chemically transmitting synapses. D . INHIBITORY RESPONSES

1. Peripheral inhibition in insects

Hoyle (1955b) showed that the locust metathoracic extensor tibiae muscle is innervated by three axons, one of which produces a small hyperpolarization of the muscle fibre membrane on stimulation. He was, however, unable to show that stimulation of this axon produced any inhibitory effect and suggested that its function was to enhance the amplitude of the “fast” electrical response so as to produce a larger contraction. As a consequence, the existence of peripheral inhibition in insects was until recently considered doubtful. Ripley and Ewer (1951) and Becht (1959) showed that the contraction of certain muscles could be reduced by increasing the intensity of the stimulus applied to the nerve trunk supplying the muscle, although in the absence of electrical records from the muscle, explanations other than inhibition are possible (see Hoyle, 1955b). More recently, Ikeda and

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D. J . AIDLEY

Boettiger (1965a,b) have demonstrated the presence of hyperpolarizing responses in the dorsoventral flight muscle of the bumble bee Bombus and in the basalar muscles of the rhinoceros beetle Oryctes, where it was shown that these hyperpolarizing potentials could cause attenuation of the excitatory postsynaptic potentials. The most complete study of this phenomenon is that performed by Usherwood and Grundfest (1964,1965) on the metathoracic extensor tibiae muscles of Schistocerca and Romalea, in which they were able to show both electrical and mechanical attenuation of the responses to excitatory stimuli; the rest of this section deals with their results. 2. Inhibitory postsynaptic potentials Stimulation of the inhibitory axon results in hyperpolarizing potentials with an amplitude of 1 to 25 mV (Fig. 6). These potentials summate on repetitive stimulation. In fibres in which the potentials are small in size, the potentials may show facilitation; in other cases this is not so.

IOOmsec

FIG.6. Intracellular records of the interaction of inhibitory and "slow" excitatory postsynaptic potentials in Schistocercu. The excitatory response was elicited at varying times after the inhibitory response. (From Usherwood and Grundfest, 1965.)

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By applying brief current pulses across the membrane via an intracellular electrode during the inhibitory postsynaptic potential, it was shown that the membrane conductance rises sharply at the beginning of the rising phase of the potential and then falls less rapidly. The duration of the inhibitory potentials is variable (40 to 300 msec.), but always longer than the excitatory potentials (15 to 150 msec.) in any one fibre. 3. Pharmacology of inhibition y-Aminobutyric acid (GABA) reduces the membrane resistance, causes hyperpolarization, and diminishes the excitatory postsynaptic potentials of fibres which are innervated by the inhibitor axon. Hence it apparently produces the same effects on the muscle fibre membrane as the inhibitory transmitter substance. Picrotoxin reverses these effects, and also diminishes the size of the inhibitory postsynaptic potentials. In these respects the pharmacology of inhibition in insects is identical to that of the crustacea (van der Kloot, 1960). 4. The ionic basis of inhibitory postsynaptic potentials

As mentioned earlier, the inhibitory potential is associated with an increase in membrane conductance. If the muscle membrane is hyperpolarized slightly, the inhibitory potential decreases in size, and further hyperpolarization results in the inhibitory potential being a depolarization. The level of membrane potential at which this changeover occurs (the reversal potential) is usually about - 70 mV (range - 55 to - 75 mV) and is thus near the value of the equilibrium potentials for potassium and chloride ions. In experiments in which all of the chloride in the saline solution was replaced by propionate, so altering the equilibrium potential for chloride ions, the inhibitory postsynaptic potentials reversed in sign. The reversal potential for the changes in membrane potential produced by GABA could be estimated by passing square current pulses through the membrane and finding at what potential the current-voltage curves obtained in the presence and absence of GABA crossed. This potential was -68 mV in potassium-free solutions and -67 mV in solutions with a potassium ion concentration of 30 mM, indicating that the reversal potential for the action of GABA was not the potassium equilibrium potential. It was concluded from these experiments that the action of the inhibitory transmitter substance, and of GABA (which may be the same thing), was to increase the permeability of the

20

D. J. AIDLEY

postsynaptic membrane to chloride ions. Here, again, the insect inhibitory system is similar to that of crustacea (Boistel and Fatt, 1958).

5. Interaction of inhibitory and excitatory responses Fig. 6 shows the effects of an inhibitory postsynaptic potential on a subsequent “slow” excitatory postsynaptic potential. Marked attenuation of the latter occurs if it arises during the initial phase of the inhibitory potential. Similar results have been obtained by Ikeda and Boetigger (1965b). This rather precise dependence of the interaction of the excitatory and inhibitory potentials on the timing of the two potentials may explain Hoyle’s failure to observe the phenomenon (Usherwood and Grundfest, 1964). The tetanic tension produced by stimulation of the slow axon in Romalea could be reduced by stimulation of the inhibitor axon, the attenuation increasing with increasing frequency of inhibitory stimulation. Complete inhibition of the mechanical response to stirhulation of the “slow” excitor axon at 12 impulses/sec. was obtained by stimulation of the inhibitor at 200/sec. In Schistocerca, such complete inhibition was never obtained, since not all of the muscle fibres supplied with the “slow” excitor axon are also innervated by the inhibitor.

Iv. THE ELECTRICAL EXCITABILITY OF THE MUSCLE FIBRE MEMBRANE A . T H E E L E C T R I C A L P R O P E R T I E S OF E L E C T R I C A L L Y E X C I T E D RESPONSES

When depolarizing current pulses are passed through the muscle fibre membrane by means of an intracellular electrode, the voltage recorded by a second electrode shows a further depolarization beyond that attributable to the resting resistance of the membrane jdel Castillo et a/., 1953; Cerf et a/., 1959). This electrically excited response differs from the propagated action potentials of nerve axons and vertebrate “twitch” muscle fibres in being a graded phenomenon (i.e. the size of the response is dependent upon the size of the stimulus). As a consequence, it is not propagated without decrement along the length of the fibre, but dies away within a few millimetres (Cerf et al., 1959). A similar phenomenon is seen in the responses to stimulation of the “fast” excitor axon in many preparations (del Castillo et al., 1953). In this case the stimulus for the production of the electrically excited response is the postsynaptic potential, and the point at which the

T H E E X C I T A T I O N OF I N S E C T S K E L E T A L MUSCLES

21

electrically excited response arises from the postsynaptic potential is given by the point of inflection on the rising phase of the response (Fig. 2). Here, again, the electrically excited component is a graded response, being proportional in size to the depolarization produced by the postsynaptic potential, as is evident in Fig. 4, where the size of the postsynaptic potential component is reduced in size by means of tryptamine. These electrically excited responses differ from the postsynaptic potentials in that they show refractoriness, so that there is a brief period after a response during which no second response can be elicited (the absolute refractory period) followed by a longer period during which the second response is reduced in size (the relative refractory period). Refractoriness occurs whether the electrically excited response is elicited by direct depolarization or by nervous stimulation (Cerf et al., 1959). The time course of recovery from refractoriness will probably be dependent on the size of the initial response, but this question remains to be investigated. If depolarizing pulses of long duration are passed through the membrane, the initial electrically excitable response is followed by a damped oscillation of the membrane potential (Cerf et al., 1959). B. T H E I O N I C B A S I S O F E L E C T R I C A L L Y E X C I T A B L E RESPONSES

The electrically excitable responses of insect muscle fibres are similar in type to the subthreshold “local responses” (Hodgkin, 1938) of nerve fibres. The well established theory of the ionic basis of the propagated action potential in squid giant axons (Hodgkin and Huxley, 1952) provides a convincing explanation of the mechanism of these responses. In a normal nerve action potential, depolarization causes an increase in the sodium conductance of the membrane which itself produces further depolarization, so that the membrane potential moves rapidly towards the sodium equilibrium potential. The return to the normal resting potential is brought about by two delayed consequences of depolarization: an increase in potassium conductance and a decrease in sodium conductance (the sodium inactivation process). Local responses arise because the increase in sodium conductance brought about by the subthreshold stimulus is too small and proceeds too slowly to counteract the effects of increase in potassium conductance. It seems very reasonable to suggest that the mechanism of the electrically excitable responses of insect muscle fibres is of the same type as the local responses of nerve axons (Hoyle, 1962). It is probable that the Hodgkin-Huxley equations would predict such responses if adjusted by

22

D . J . AIDLEY

suitable changes in the rate constants of the sodium and potassium activation processes. More specifically, it has been suggested that the increase in potassium conductanceis more rapid in the graded responses of insect muscles (Werman et ul., 1961). The presence of a sodium inactivation process operative at the normal resting potential seems to be implied by some results of Cerf et ul. (1959), in which the electrically excitable response arose at a much more negative membrane potential after the fibre had been hyperpolarized for a short time. While the general principles of this argument seem to be fairly sound, it is not so clear that the ion involved in the specific, regenerative increase in permeability (i.e. the ion responsible for the rising phase of the electrically excitable response) is sodium, or sodium alone. Wood (1963) has investigated the effects of sodium ion concentration in the size of the electrical responses to “fast” axon stimulation in the leg muscles of Locustu, Periplunetu and Curuusius, at the same time measuring the internal sodium concentrations of muscles subjected to similar treatments, so that estimates of the sodium equilibrium potential could be made. In Locustu, the peak of the response occurred at an average membrane potential of - 19 mV in a solution containing no sodium ions, rising to + 5 mV in a solution with a sodium ion concentration of 150 m ~The . change was rather less for PeripZunetu (the corresponding figures were - 15 and + 2 mv), and much less for Caruusius (-3 and +4.5 mv). The values obtained at nominally zero sodium ion concentrations are interesting in that they are more positive than the sodium equilibrium potential, even if the actual sodium ion concentration were as much as 5 m~ (which is most unlikely). Interpretation of these results is complicated by the fact that the quantity measured was total electrical responses to nervous stimulation, which would include (and in zero sodium salines may entirely consist of) a large postsynaptic potential component. Since sodium is the major cation in the haemolymph of Locustu and PeripZunetu and since the membrane potential at the peak of the response is moderately sensitive to the sodium ion concentration, it is probable that sodium is the major ion involved in the rising phase of the electrically excitable response, although participation of other cations, such as calcium, is not excluded. In Curuusius, however, where the haemolymph sodium concentration is only 15 m~ (giving a sodium equilibrium potential of about +4.6 mV; Wood, 1963) and the potential at the peak of the response is only slightly affected by the sodium ion concentration, it seems rather unlikely that sodium is the only, or indeed the major ion involved. In view of the dependence of the amplitude of the electrical response to stimulation

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on the magnesium ion concentration, and the very high magnesium ion concentration in the haemolymph, it seems probable that magnesium carries the action current in this case (Wood, 1958). The observations of Treherne (1965) on nervous conduction in the abdominal nerve cord of Carausius are of interest in this connection; he found that nervous conduction ceased in the absence of either sodium or magnesium ions. If the conclusion that magnesium ions carry a major part of the action current in Carausius is correct, then there is a very high probability that an active transport system concerned with maintaining the magnesium ion concentration gradient across the cell membrane exists. C . SPONTANEOUS ACTIVITY

If locust muscle fibres are perfused with calcium- and magnesium-free saline solutions, there follows a brief period during which the membrane is hyperexcitable, producing repetitive spike-like depolarizations (D. J. Aidley, unpublished). Similar effects have been seen in frog skeletal muscle (Bulbring et al., 1956). Van der Moot (1963) has reported some interesting observations on the spiracular muscles of the pupae of the silkmoths Hyalophora and Telea. These undergo spontaneous activity which consists of slow depolarizations of the membrane (pacemaker potentials), each followed by a spike-like component. The pacemaker potentials were accompanied by an increase in membrane conductance. If the membrane potential was set to various levels by passing current through an intracellular electrode, the pacemaker potentials always reached a plateau at the same value, - 29 mV. This suggests that the potentials are produced by an increase in the membrane conductance for some ion which has an equilibrium potential at - 29 mV, but what this ion is is far from clear. If the membrane is depolarized beyond - 29 mV, the potentials reverse in sign; this is rather extraordinary, since it implies that the cyclic nature of the phenomenon is based on changes in membrane permeability which are not determined by changes in membrane potential. V. THE EXCITATION-CONTRACTION COUPLING PROCESS A . DEPOLARIZATION

The evidence that depolarization of the cell membrane is necessary for the contraction of the skeletal muscles of vertebrates (Kuffler, 1946; Sten-Knudsen, 1960) and crustacea (Orkand, 1962) is considerable. The same situation appears to exist in insect skeletal muscles. 2-kA.I.P.

4

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D. J. A I D L E Y

If the amplitude of the electrical response to nervous stimulation is reduced by means of neuromuscular blocking agents, the twitch tension is also reduced, as is shown in Fig. 4. Perfusion of a muscle with salines containing high potassium ion concentrations causes depolarization and contraction. In the metathoracic spiracular muscle of locusts (Hoyle, 1961), the threshold for the development of this contracture occurs at a potassium ion concentration of about 30 m~ (membrane potential - 34 mv) ; contracture tension increases with increasing potassium ion concentration to reach the maximum level at about 70 m~ (membrane potential - 18 mv). A similar result has been obtained from the mesothoracic extensor tibiae of Schistocerca (Aidley, 1965a). A more subtle influence of membrane potential on tension development is seen in frog “ twitch ” fibres, where the potassium contracture lasts only for a few seconds (Hodgkin and Horowicz, 1960). If the membrane is then repolarized by perfusion with a solution containing a lower potassium ion concentration, a restorative (“priming”) process occurs, so that the fibre is again able to contract on depolarization. The extent and rapidity of this “priming” process increases with increasing (more negative) membrane potentials. Potassium contractures of a similar short duration occur in the metathoracic extensor tibiae (Hoyle, 1961) and flexor tibiae (D. J. Aidley, unpublished) of Schistocerca, and it is not unlikely that a “priming” process similar to that described by Hodgkin and Horowicz could be demonstrated in these and some other insect muscles. B. THE I M P O R T A N C E O F C A L C I U M IONS

If the mesothoracic extensor tibiae of Schistocerca is perfused with a calcium-free solution containing the chelating agent ethylenediaminetetra-acetate (EDTA), it will no longer contract when depolarized by an isotonic solution of potassium chloride (Aidley, 1963, 1965a). In this condition, contraction can be elicited by addition of calcium chloride to the potassium chloride solution bathing the muscle (Fig. 7). The effect of solutions containing various concentrations of calcium ions (in the absence of EDTA) is slower, but it can be shown that the maximum contracture tension is related to the external calcium ion concentration, with a threshold at about 0.03 m~ and a saturation value in the region 2 to 4 mM. Thus it seems that insect muscles are similar to vertebrate muscles (Frank, 1960; Niedergerke, 1956; Edman and Schild, 1962) in requiring calcium ions for contraction. In vertebrate muscles, the presence of calcium ions in the sarcoplasm appears to be necessary for the interaction of actin, myosin and adeno-

-

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25

sine triphosphate so as to produce contraction, and the calcium ion concentration is probably controlled by a “relaxing factor” system composed of vesicular elements derived from the endoplasmic reticulum (see, for example, Needham, 1960, and Weber et al., 1964). A relaxing factor whose action is antagonized by calcium ions has been isolated from locust skeletal muscles by Tsukamoto and his colleagues (quoted in Maruyama, 1965). The contraction of glycerinated fibres from the

A -

C

-

7 ]log

5 min

FIG.7. The effects of treatment with EDTA and subsequent addition of calcium chloride on the potassium contracture of the mesothoracic extensor tibiae of Schistocerca. The horizontal line under each record shows the duration of perfusion with KCl solution. Before each perfusion with KCl solution the muscle was treated as follows: (A) perfusion with a standard Ringer solution (4 mM CaCI2); (B) perfusion for 40 min. with a Ca-free Ringer containing 4 m~ EDTA; and, (C) perfusion for a further 60 min. with standard Ringer. The plateau of a tetanus produced by stimulation of the motor nerves is shown at the beginning of record (A). (From Aidley, 1965.)

fight muscles of the giant water bugs Lethoceros and Hydrocirius in the presence of adenosine triphosphate is dependent upon the presence of calcium ions (Jewell et al., 1964). This contractile action of calcium ions appears to be rather specific, in that strontium and a number of other divalent cations do not form adequate substitutes for calcium in the promotion of potassium contracture (Aidley, 1965b). Application of solutions containing strontium ions to calcium-depleted muscles depolarized by potassium chloride solution results in submaximal, phasic contractures;but successive$responses to depolarization decline in the continued absence of calcium

26

D . J . AIDLEY

ions, suggesting that the small contractions mediated by strontium ions are brought about by a displacement of calcium ions from some intracellular store. Similar results have been obtained by Frank (1962) on frog toe muscles. C . THE ACTION OF CARBON DIOXIDE

Hoyle (1961) has shown that application of carbon dioxide to the metathoracic spiracular muscle of Schistocerca causes relaxation when the muscle is in potassium contracture, although the membrane potential of the depolarized fibres was unaffected. This result implies that carbon dioxide has some direct effect on the coupling process in this muscle in addition to its effects on the resting membrane and the size of the postsynaptic potential. Contracture tension of the spiracular muscle is maintained for many hours in isotonic solutions of potassium chloride or sulphate, but falls to zero in about half an hour in isotonic potassium bicarbonate (Hoyle, 196 1). The mechanisms of the relaxations occurring in the presence of bicarbonate or carbon dioxide are not known, although it is conceivable that bicarbonate acts by reducing the calcium ion concentration. Further evidence for a direct action of carbon dioxide on the coupling process of the locust spiracular muscle has been adduced by Hoyle from experiments in which he measured the effects of a brief “pulse” of carbon dioxide on the size of the postsynaptic potentials of individual fibres and the twitch tension of the whole muscle (Hoyle, 1960). It was found that a “hysteresis” effect occurred, in which the size of the postsynaptic potential fell and rose more rapidly than the twitch tension. These experiments cannot be considered conclusive, however, since the whole cycle of depression and recovery lasted only a few seconds, a time which is probably comparable with the diffusioii delay which must occur between the carbon dioxide affecting fibres in different parts of the muscle. D.

“

FAST

”

AND

“

SLOW

”

MUSCLES

Skeletal muscle fibres of frogs can be divided into two clearcut types, known as “twitch” (or “fast”) and “slow” fibres. Some muscles, such as the sartorius, are composed entirely of “fast” fibres, others, such as the iliofibularis and rectus abdominis, contain a high proportion of “slow” fibres (Kuffler and Vaughan Williams, 1953). “Slow” fibres differ from “fast” fibres in that they are multiterminally innervated, electrically inexcitable, contract at much slower velocities and do not

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relax after a few seconds when immersed in solutions containing high potassium ion concentrations. These distinctions cannot be usefully applied to insect muscles, which in many cases can be classified as either “fast” or “slow” according to the criteria used. For example, if we compare the metathoracic and mesothoracic extensor tibiae muscles of locusts, we find that the fibres of both muscles are electrically excitable (as in “fast” muscles), that they are both multiterminally innervated (as in “slow” muscles), and that the potassium contracture is brief (as in “fast” muscles) in the metathoracic muscle whereas it is maintained (as in “slow” muscles) in the mesothoracic muscle (Hoyle, 1961; Aidley, 1963). Becht and Dresden (1956) suggested that the coxal muscles of the cockroach could be divided into two types (termed “fast” and “slow”) on the basis of their mechanical responses to stimulation. However, later investigations using more sophisticated techniques failed to uphold this view (Becht et al., 1960; Usherwood, 1962). It is clear that there are differences in the mechanical properties of various insect muscles, but a clearcut division into two types is not at present possible. I am very grateful to Dr. P. N. R. Usherwood and Dr. A. C. Neville for their comments on an early draft of this article. REFERENCES

Adrian, R.H. (1956). The effect of internal and external potassium concentration on the membrane potential of frog muscle. J. Physiol. 133, 631-658. Aidley, D. J. (1963). Influence of calcium ions on potassium contracture in an insect leg muscle. Nature, Lond. 198,591-592. Aidley, D.J. (1965a). The effect of calcium ions on potassium contracture in a locust leg muscle. J. Physiol. 177, 94-102. Aidley, D.J. (1965b). The effects of strontium and other divalent cations on potassium contracture in a locust leg muscle. J. Physiol. 177, 103-111. Becht, G. (1959). Studies on insect muscles. Bijdr. Dierk. 29, 5-40. Becht, G. and Dresden, D. (1956). Physiology of the locomotory muscles in the cockroach. Nature, Lond. 177,836-837. Becht, G., Hoyle, G. and Usherwood, P. N. R. (1960). Neuromuscular transmission in the coxal muscles of the cockroach. J. Insect Physiol. 4, 191-201. Belton, P. (1958). Membrane potentials recorded from moth muscle fibres. J. Physiol. 142,20P. Belton, P. (1960). Effect of ions on potential in lepidopteran muscle fibres. Biol. Bull. mar. biol. Lab., Woods Hole 119, 289. Belton, P.and Grundfest, H. (1962). Potassium activation and K spikes in muscle fibres of the mealworm larva (Tenebrio molitor). Am. J. Physiol. 203, 588594.

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Boistel, J. and Fatt, P. (1958). Membrane permeability change during transmitter action in crustacean muscle. J. Physiol. 144, 176-191. Boyle, P. J. and Conway, E. J. (1941). Potassium accumulation in muscle and associated changes. J. Physiol. 100, 1-63. Bulbring, E.,Holman, M. and Lullman, H. (1956). Effects of calcium deficiency on striated muscle of the frog. J. Physiol. 133, 101-1 17. Cerf, J. A., Grundfest, H., Hoyle, G. and McCann, F. V. (1959). The mechanism of dual responsiveness in muscle fibres of the grasshopper Romalea microptera. J . gen. Physiol. 43, 377-395. Conway, E. J. (1957). Nature and significance of concentration relations of potassium and sodium ions in skeletal muscle. Physiol. Rev. 37, 84-132. del Castillo, J. and Engbaek, L. (1954). The nature of the neuromuscular block produced by magnesium. J. Physiol. 124, 370-384. del Castillo, J. Hoyle, G. and Machne, X. (1953). Neuromuscular transmission in a locust. J. Physiol. 121, 539-547. del Castillo, J. and Katz, B. (1954). Quanta1 components of the end-plate potential. J. Physiol. 124, 560-573. Duchgteau, G., Florkin, M. and Leclercq, J. (1953). Concentrations des bases fixes et des types de compositions de la base totale de l’hemolymph des insectes. Arch. int. Physiol. 61, 518-549. Dudel, J. and Kuffler, S. W. (1961). Mechanism of facilitation at the crayfish neuromuscular junction. J. Physiol. 155,530-542. Edman, K. A. P. and Schild, H 0. (1962). The need for calcium in the contractile responses induced by ecetylcholine and potassium in the rat uterus. J. Physiol. 161,4244ll. Edwards, G. A., Ruska, H. and de Harven, E. (1958). Neuromuscular junctions in flight and tymbal muscles of the Cicada. J. biophys. biochem. Cytol. 4,251-255. Fatt, P. and Katz, B. (1951). An analysis of the end-plate potential recorded with an intracellular electrode. J. Physiol. 115,320-370. Fatt, P. and Katz, B. (1952). Spontaneous subthreshold activity at motor nerve endings. J. Physiol. 117, 109-128. Fatt, P. and Katz, B. (1953). The electrical properties of crustacean muscle fibres. J. Physiol. 120, 171-204. Frank, G. B. (1960). Effects of changes in extracellular calcium concentration on the potassium-induced contracture of frog’s skeletal muscle. J. Physiol. 151, 518-538. Frank, G. B. (1962). Utilization of bound calcium in the action of caffeine and certain multivalent cations on skeletal muscle. J. Physiol. 163,254-268. Hagiwara, S. and Watanabe, A. (1954). Action potential of insect muscle examined with intracellular electrode. Jap. J. Physiol. 4, 65-78. Harlow, P. A. (1958). The action of drugs on the nervous system of the locust (Locusta migratoria). Ann. appl. Biol. 46,55-73. Hill, R.B. (1963). The effects of acetylcholine on twitches in the locust leg. Comp. Biochem. Physiol. 10, 203-208. Hill, R. B. and Usherwood, P. N. R. (1961). The action of 5-hydroxytryptamine and related compounds on neuromuscular transmission in the locust, Schistocerca gregaria. J. Physiol. 153,393-401. Hodgkin, A. L. (1938). The subthreshold potentials in a crustacean nerve fibre. Proc. R. SOC.B 126,87-121.

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Hodgkin, A. L. (1951). The ionic basis of electrical activity in nerve and muscle. Biol. Rev. 26, 339-409. Hodgkin, A. L. and Horowicz, P. (1959). The influence of potassium and chloride ions on the membrane potential of single muscle fibres. J. Physiol. 148, 127-160. Hodgkin, A. L. and Horowicz, P. (1960). Potassium contractures in single muscle fibres. J. Physiol. 153,386-403. Hodgkin, A. L. and Huxley, A. F. (1952). A quantitative description of conduction and excitation in nerve. J. Physiol. 117, 500-544. Hodgkin, A. L. and Katz, B. (1949). The effect of sodium ions on the electrical activity of the giant axon of the squid. J. Physiol. 108,37-77. Hoyle, G.(1953). Potassium ions and insect nerve muscle. J. exp. Biol. 30,121-135. Hoyle, G . (1955a). The anatomy and innervation of locust skeletal muscle. Proc. R. SOC.B 143,281-292. Hoyle, G. (1955b). Neuromuscular mechanisms of a locust skeletal muscle. Proc. R. SOC.B 143,343-367. Hoyle, G. (195%). The effects of some common cations on neuromuscular transmission in insects. J. Physiol. 127, 90-103. Hoyle, G. (1957). Nervous control of insect muscles. In “Recent Advances in Invertebrate PhysiQlogy ” (B. T. Scheer, ed.), pp. 73-98. University of Oregon Publications. Hoyle, G. (1 959). The neuromuscular mechanism of an insect spiracular muscle. J. Insect Physiol. 3, 378-394. Hoyle, G.(1960). The action of carbon dioxide gas on an insect spiracular muscle. J. Insect Physiol. 4, 63-79. Hoyle, G.(1961). Functional contracture in a spiracular muscle. J. Insect Physiol. 7, 305-314. Hoyle, G . (1962). Comparative physiology of conduction in nerve and muscle. Am. Zoologist 2, 5-25. Hoyle, G. (1965). Neural control of ske!etal muscle. In “The Physiology of Insects" (M. Rockstein, ed.) vol. 2, pp. 407-449. Academic Press, New York and London. Ikeda, K. and Boettiger, E. G. (1965a). Studies on the flight mechanism of insects. 11. The innervation and electrical activity of the fibrillar muscles of the bumble bee, Bombus. J. Insect Physiol. 11, 779-789. Ikeda, K. and Boettiger, E. G. (1965b). Studies on the flight mechanism of insects. 111. The innervation and electrical activity of the basalar fibrillar flight muscle of the beetle, Oryctes rhinoceros. J. Insect Physiol. 11, 791-802. Jenden, D. J. and Reger, J. F. (1963). The role of resting potential changes in the contractile failure of frog sartorius muscles during calcium deprivation. J. Physiol. 169,889-901. Jewell, B. R., Pringle, J. W. S. and Riiegg, J. C. (1964). Oscillatory contraction of insect fibrillar muscle after glycerol extraction. J. Physiol. 173,6-8P. Katz, B. (1962). The transmission of impulses from nerve to muscle, and the subcellular unit of synaptic action. Proc. R. SOC.B 155, 455-479. Kerkut, G. A. and Ridge, R. M. A. P. (1961). The effect of temperature changes on the resting potential of crab, insect and frog muscle. Comp. Biochern. Physiol. 3, 64-70.

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Kerkut, G. A., Leake, L. D., Shapira, A., Cowan, S. and Walker, R. J. (1965b). The presence of glutamate in nerve-muscle perfusates of Helix, Carcinus, and Periplaneta. Comp. Biochem. Physiol. 15,485-502. Kerkut, G. A., Shapira, A. and Walker, R. J. (1965a). The effect of acetylcholine, glutamic acid and GABA on the contractions of the perfused cockroach leg. Comp. Biochem. Physiol. 16, 37-48. Keynes, R. D. (1963). Chloride in the squid giant axon. J. Physiol. 127, 90-103. Kuffler, S. W. (1946). The relation of electrical potential changes to contracture in skeletal muscle. J. Neurophysiol. 9, 367-377. Kuffler, S. W. and Vaughan Williams, E. M. (1953). Properties of the “slow” skeletal muscle fibres of the frog. J. Physiol. 121, 318-340. Maruyama, K. (1956). The biochemistry of the contractile elements of insect muscle. In “The Physiology of Tnsecta” (M. Rockstein ed.) Vol 2, pp. 451-483. Academic Press, New York and London. McCann, F. V. and Boettiger, E. G. (1961). Studies on the flight mechanism of insects. I. The electrophysiology of fibrillar flight muscle. J. gen. Physiol. 45, 1 25-1 42. Needham, D. M. (1960). Biochemistry of muscular action. In “The structure and function of muscle” (G. H. Bourne, ed.) Vol. 2, pp. 55-104. Academic Press, London and New York. Neville, A. C. (1963). Motor unit distribution of the dorsal longitudinal flight muscles in locusts. J. exp. Biol. 40, 123-136. Niedergerke, R. (1956). The potassium chloride contracture of the heart and its modification by calcium. J. Physiol. 134, 584-599. Orkand, R. K. (1962). The relation between membrane potential and contraction in single crayfish muscle fibres. J. Physiol. 161, 143-159. Pringle, J. W. S. (1939). The motor mechanism of the insect leg. J. exp. Biol. 16,220-23 1. Ripley, S. H. and Ewer, D. W. (1951). Peripheral inhibition in the locust. Ncture, Lond. 167, 1066. Sten-Knudsen, 0. (1960). Is muscle contraction initiated by internal current flow? J. Physiol. 151, 363-384. Takeuchi, A. and Takeuchi, N. (1960). On the permeability of the end-plate membrane during the action of transmitter. J. Physiol. 154, 52-67. Treherne, J. E. (1965). Some preliminary observations on the effects of cations on conduction processes in the abdominal nerve cord of the stick insect, Carausius morosus. J. exp. Biol. 42, 1-6. Usherwood, P. N. R. (1961). Spontaneous miniature potentials from insect muscle fibres. Nature, Lond. 191, 814-815. Usherwood, P. N. R. (1962). The nature of “slow” and “fast “ contractions in the coxal muscles of the cockroach. J. Insect Physiol. 8, 31-52. Usherwood, P. N. R. (1963a). Spontaneous miniature potentials from insect muscle fibres. J. Physiol. 169, 149-160. Usherwood, P. N. R. (1963b). Response of insect muscle to denervation. 11. Changes in neuromuscular transmission. J. Insect Physiol. 9, 81 1-125. Usherwood, P. N. R. and Grundfest, H. (1964). Inhibitory postsynaptic potentials in grasshopper muscle. Science, N. Y. 143, 817-818 Usherwood, P. N. R. and Grundfest, H. (1965). Peripheral inhibition in skeletal muscle of insects. J. Neurophysiol. 28, 497-518.

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van der Kloot, W. G. (1960). Picrotoxin and the inhibitory system of crayfish muscle. In “Inhibition in the nervous system and y-aminobutyric acid” (E. Roberts, ed.) pp. 409-412. Pergamon Press, New York and Oxford. van der Kloot, W. G. (1963). The electrophysiology and the nervous control of the spiracular muscle of pupae of giant silkmoths. Comp. Biochem. Physiol. 9,317-333.

Weber, A., Herz, R. and Reiss, I. (1964). The regulation of myofibrillar activity by calcium. Proc. R. SOC.B 160, 489-499. Werman, R., McCann, F. V. and Grundfest, H. (1961). Graded and all-or-none electrogenesis in arthropod muscle. I. The effects of alkali-earth cations on the neuromuscular system of Romalea microptera. J. gen. Physiol. 44, 979-995.

Wood, D. W. (1957). The effect of ions upon neuromuscular transmission in a herbivorous insect. J. Physiol. 138, 119-139. Wood, D. W. (1958). The electrical and mechanical responses of the prothoracic flexor tibialis muscle of the stick insect. J. exp. Biol. 35, 850-861. Wood, D. W. (1961). The effect of sodium ions on the resting and action potentials of locust and cockroach muscle fibres. Comp. Biochem. Physiol. 4, 42-46.

Wood, D. W. (1963). The sodium and potassium composition of some insect skeletal muscle fibres in relation to their membrane potentials. Comp. Biochem. Physiol. 9, 151-159. Wood, D. W. (1965). The relationship between chloride ions and resting potential in skeletal muscle fibres of the locust and the cockroach. Comp. Biochem. Physiol. 15, 303-312.

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The Excretion of Nitrogen in Insects E. BURSELL Department of Biological Sciences, University College of Rhodesia and Nyasaland, Salisbury, Rhodesia I. Introduction . 11. The Formation of Nitrogenous End Products A. The uricolytic pathway . B. The uricotelic pathway. . C. The formation of urea D. The formation of ammonia . E. Amino acids . F. Miscellaneous materials . LU. The Excretion of Nitrogenous End Products A. Collembola . B. Orthoptera . C. Odonata . D. Dermaptera . E. Hemiptera F. Coleoptera G. Neuroptera . H. Hymenoptera . I. Diptera . J. Lepidoptera . N. Conclusions . References .

.

. .

33 34 36

.

41 42 43

. 4 0

. .

. 4 4 .

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

4

4 45 46 47 48 48 50 51 51 52 54 56 61

I. INTRODUCTION The occurrence of uric acid in the excreta of insects was established before the turn of the century and its presence has been confirmed subsequently by a large number of investigators (see reviews in Wigglesworth, 1950; Prosser, 1952; Roeder, 1953; Chauvin, 1956). Earlier findings were generalized in statements such as those of Needham (1935): “. . . in the insect, excretion of uric acid as the main end product of nitrogen metabolism is widespread, if not universal”, and of Florkin (1945): “I1 est bien Ctabli que chez les insectes adultes, l’acide urique reprksente le terme pridominant du catabolisme protidique”. On this basis the insect was assessed as being predominantly uricotelic in a general classification of excretory metabolism (Needham, 1950). Uricotelism was seen as an adaptation to a terrestrial mode of life; the 33

34

E. B U R S E L L

excretion of toxic end products, like ammonia, or of soluble end products, like urea, was seen as being militated against by the shortage of water. This view of the insects as a class still prevails (e.g. reviews by Craig, 1960; Stobbart and Shaw, 1964), subject to certain exceptions. In recent years, however, data have accumulated which suggest that nitrogenous end products other than uric acid may figure largely in the excreta of a variety of insects; the exceptions are becoming more numerous, and the validity of the earlier generalization appears to be threatened. In the following account, an attempt has been made to summarize the available information, and, in this way, to provide some basis for a reassessment of the situation. 11. THE FORMATION OF NITROGENOUS END P R O D U C T S The metabolic interrelationships of the main nitrogenous end products in insects, and the sources from which they derive, are summarized in Fig. 1. Two essentially opposing metabolic pathways appear to be involved.

(a) One has its origin in the nucleic acids, and may be termed the uricolytic pathway. It involves deamination and oxidation of the purine raw material (adenine and guanine) followed by the breakdown of the ring system to progressively simpler nitrogenous waste products. The last steps are thought to play little, if any, part in the excretory metabolism of insects, and they have been indicated by dotted lines. (b) The other has its origin in the proteins and their constituent amino acids, and may be termed the uricotelic pathway. It involves the synthesis of purines from amino acid nitrogen. In some insects the uricotelic pathway appears to be inoperative, and amino acid nitrogen is excreted as ammonia. In many others, urea appears as a minor constituent of the excreta, being apparently derived from protein nitrogen. As a third possibility, in certain insects specific amino acids are excreted as such; these alternative pathways are indicated in the diagram. Values for the nitrogen content and the solubility of nitrogenous end products have been included in Fig. 1. Ammonia is highly soluble, but a most efficient vehicle for the removal of waste nitrogen, with a nitrogen content of more than 80%; urea contains substantially less nitrogen and is of the same order of solubility as ammonia. The purines and their near derivatives contain between 30 and 40% nitrogen, but are

35

THE EXCRETION O F NITROGEN I N INSECTS

very much less soluble than ammonia and urea, while two of the amino acids listed have a nitrogen content not much lower than the purines, but are relatively soluble. Further reference to these differences and

I Nucleic Acid I

260

33

6

Allantoin

35

60

Allantoic Acid

32

slightly soluble

46

119,300

82

89,000

32 27 6

15,000 soluble

Uric Acid

’’ Arginine Histidine Cystine

Solubility mg/lOO ml

37

Xanthine

I Protein HAmino Acids I

% Nitrogen

Ammonia

.

FIG. 1. Nitrogenous end products and their metabolic interrelationships. (Data from “Handbook of Chemistry and Physics” ed. Hodgman, C. D., Chemical Rubber Publishing Co., Cleveland, Ohio.)

their implications in relation to nitrogenous excretion will be deferred until the excretory metabolism of different insects has been reviewed. The pyrimidines constitute another nitrogenous component of nucleic acid, but little information is available concerning their metabolism in insects. Recent studies (Bruno and Cochran, 1965) suggest

11

36

E. B U R S E L L

that it may conform in part to the vertebrate pattern, but further work will need to be done before their possible role as nitrogenous end products can be assessed. A . THE URICOLYTIC P A T H W A Y

The starting material for this reaction chain is nucleic acid, arising in part as a product of digestion, and in part from processes of tissue

Adenine

Hypoxanthine

1

(xanthine dehyhogenase)

Xanthine

Guanine

1

(xanthine dehydrogenase)

I

Uric Acid

maintenance and repair. The primary breakdown products are the nucleosides adenosine and guanosine, and these may be deaminated directly under the influence of the corresponding enzymes, adenosine deaminase and guanosine deaminase. In the presence of purine nucleosidases, the ribose sugar is then split off to liberate the purine bases.

T H E EXCRETION OF N I T R O G E N I N INSECTS

37

Enzymes responsible for such actions have been demonstrated in the cockroach (Cochran and Bruno, 1963; Cordero and Ludwig, 1963), and adenosine deaminase activity has been established in extracts of the larval fat body of blowflies (Desai and Kilby, 1958a) and in homogenates of Drosophila (Wagner and Mitchell, 1948). On the other hand, Duchlteau et al. (1940) were unable to show the presence of adenosine or guanosine deaminase in certain insects, and it is possible that in such species deamination occurs after hydrolysis of the nucleosides, as shown opposite. Adenase and guanase, the enzymes responsible for deamination of the free purine bases, have been shown to occur in a number of different insects (Duchfiteau et al., 1940; Desai and Kilby, 1958a; Prota, 1961). In extracts from the fat body of the silkworm the activity of guanase is much greater than that of adenase (Hayashi, 1961), and this appears to be so in other insects as well (Anderson and Patton, 1955). The deaminated purines, xanthine and hypoxanthine, are oxidized to uric acid under the influence of a single enzyme, xanthine oxidase, or xanthine dehydrogenase as it should more properly be called in view of the investigations of Irzykiewicz (1955); this author has shown that, unlike its mammalian counterpart, the insect enzyme cannot use molecular oxygen, but can reduce methylene blue. It is possible that nicotinamide-adenine dinucleotide (NAD) may act as the hydrogen acceptor for this reaction under normal conditions. Early workers failed to find evidence of the oxidation of xanthine in insects (Truszkowski and Chajkinowna, 1935), but xanthine dehydrogenase activity has subsequently been demonstrated in a variety of species, and the enzyme appears to be widely distributed throughout the class (Duchlteau et al., 1940; Florkin and Duchlteau, 1941; Anderson and Patton, 1954; Desai and Kilby, 1958b; Lisa and Ludwig, 1959; Ursprung and Hadorn, 1961; Hayashi, 1962; Cordero and Ludwig, 1963; Keller and Glassman, 1963; Parzen and Fox, 1964). In fact, there are indications that there may be more than one molecular form of the enzyme in Drosophila (Keller et al., 1963; Smith et al., 1963). The uric acid formed under the influence of xanthine dehydrogenase may be subjected to a series of degradative reactions in the presence of uricolytic enzymes as summarized overleaf. The first step is the oxidation of uric acid to allantoin under the iduence of the enzyme uricase. This enzyme has long been known to be active in insects (Leifert, 1935; Truskowski and Chajkinowna, 1935;Brown, 1938a; ROCCO, 1938),and more recent work has confirmed

38

E. BURSELL

its wide distribution within the class (Razet, 1952,1953, 1954,1956,1957, 1961;Lisa and Ludwig, 1959; Corder0 and Ludwig, 1963; Nelson, 1964), though it has been reported absent in some species (Florkin and DuchC teau, 1943; Razet, 1961). In Popillia japonica uricase is present in extracts of the embryo and of larval stages, but absent from the prepupa and pupa (Ross, 1959); a similar loss of uricolytic activity during pupal development has been reported for the blowfly Lucilia (Brown, 1938a).

I I

Allantoic acid O=C

(uricase)

(allantoinme)

YH2 COOH I \,cH. H

N\H2

c=o

N’

H

1

(allanroicme)

COOH

I

CHO

/NH2

-t-2O=C,

Glyoxylic acid

NH, Urea

I

I

(weme)

2 C 0 2 + 4NHs

T H E E X C R E T I O N OF N I T R O G E N I N I N S E C T S

39

The hydrolysis of allantoin to allantoic acid, which constitutesa further breakdown of the purine ring system, is catalysed by the enzyme allantoinase. Active extracts of this enzyme have also been obtained from a variety of insects (Rocco, 1936 and 1938; Manunta, 1948 and 1949; Razet, 1952-1957), but it appears to be somewhat less widespread than uricase (Razet, 1961). The next step in the sequence of uricolytic breakdown is the hydrolysis of allantoic acid under the influence of allantoicase, leading to the formation of urea and glyoxylic acid. This enzyme appears to have a very restricted distribution. It has been reported for larval stages of Bombyx (Manunta, 1948), but Razet (1961) failed to confirm its presence in this species. Feebly active preparations have been made from insects belonging to a number of different orders, but only from two of the many species tested by Razet (1961) have highly active extracts been obtained, namely from Xenylla wekhii (Collembola) and Achetu domesticu (Orthoptera). Since urea is absent from the excreta, or present in very small amounts, even in those insects which possess an active allantoicase, it seems safe to conclude that uricolytic breakdown beyond the stage of allantoicacid is of little significancein the excretorymetabolism of insects. The last in the chain of uricolytic enzymes is urease, which catalyses the hydrolysis of urea to ammonia and carbon dioxide. The presence of this enzyme has not been unequivocally established in insects. It has been reported to occur in the meat-eating larvae of certain Diptera (Tomita and Kumon, 1936; Baker, 1939; Robinson and Baker, 1939; Robinson and Wilson, 1939), and in a small number of other insects (Razet, 1961), but the methods used for its detection have not been above reproach, and the evidenceshould be accepted with caution. Lennox (1941) failed to find trace of its existence in a careful study of the enzymes responsible for ammonia production in larvae of the blowfly Luciliu cuprina. Even if the presence of urease were accepted for the species concerned, it is doubtful if it could be considered as a uricolytic enzyme, since earlier members of the chain are missing (Razet, 1961). A considerable amount of work has been done on the localization of enzymes involved in purine metabolism. The activity of xanthine dehydrogenase is high in the fat body of Peripluneta (Anderson and Patton, 1954 and 1955), and in larvae of Drosophila the fat body is more active than the Malpighian tubules (Ursprung and Hadorn, 1961). In Tenebrio the enzyme appears to be more concentrated in extracts from the mid gut and fat body than from other tissues (Irzykiewicz, 1955). Uricase has also been shown to occur in the fat body ofinsects (Leifert, 1935; Lisa and Ludwig, 1959; Pierre, 1964), but in the species studied by Razet

40

E. BURSELL

(1961) highest activities were usually found in extracts of the Malpighian tubules, with substantially lower concentrations occurring in mid gut and fat body extracts; and in Musca domestica, too, uricase activity is concentrated in the Malpighian tubules (Nelson, 1964). In insects which possess an active allantoinase, the enzyme distribution appears to be similar to that of uricase, with highest activity in the Malpighian tubules, followed by fat body and mid gut (Razet, 1961). B . T H E URICOTELIC P A T H W A Y

While the breakdown of uric acid in insects has been the subject of extensive investigations during the last decade, its synthesis has received relatively little attention. It has long been realized that the large amounts of uric acid which occur in the excreta of insects cannot derive solely from nucleic acid sources; the bulk of it must originate from protein nitrogen, but the synthetic pathways involved have not yet been fully elucidated. An early suggestion was made that arginine and histidine might be involved as intermediaries in uric acid synthesis (see Baldwin, 1948; Hoskins and Craig, 1935),but the existence of such a pathway has not been confirmed. The possibility of identifying precursors of uric acid in insects has been investigated both in vivo (Brighenti and Colla, 1940) and in vitro (Leifert, 1935;Anderson and Patton, 1955; Desai and Kilby, 1958b), and various non-purine substances including urea, malonate, ammonium salts, monoethyloxaloacetate and 4-amino-5-imidazole carboxamide riboside have been reported to stimulate the production of uric acid in certain cases. A more direct approach to the problem was made by McEnroe and Forgash (1957 and 1958) who investigated the fate of radioactive formate in Periplaneta umericana.Their results suggest that the pathway of uric acid synthesis may be the same as that in birds (Buchanan, 1951), where it involves a building up of the purine ring system on a basis provided by ribose sugar phosphate, by successive reaction with glutamine, glycine, formate, glutamate, carbon dioxide, aspartate and formate, as shown schematically opposite (from Baldwin, 1963). This view receives some support from the work of Heller and Jezewska (1959) who show that uric acid synthesis in Antherea may be stimulated by the simultaneous addition of precursors like formate, ribose-5phosphate, glutamate and aspartate, in the presence of added adenosine triphosphate (ATP). ATP is required at nearly every step of purine synthesis, so that the process is an expensive one in terms of metabolic energy. But upon completion of the ring system, ribose-5’-phosphate is split off to

THE EXCRETION O F NITROGEN I N INSECTS

41

liberate hypoxanthine, and this is subsequently oxidized first to xanthine and then to uric acid. In view of the demonstration that a dehydrogenase system is involved (Irzykiewicz, 1955), it may be that a substantial proportion of energy is recaptured at this stage. The recent work of Ito and Mukaiyama (1964) provides confirmation of the role of hypoxanthine and/or xanthine as intermediaries in the metabolism of protein nitrogen. These authors show that an increase in the protein intake of silkworms causes an increase in the activity of .*

(carbon dioxide).; * a .

(formare)

8

O= -pyrophosphate

xanthine oxidase. On the basis of these results, and of the earlier findings of Hayashi (1961) that adenase activity of silkworms extracts is low compared with the guanase activity, they suggest that a pathway involving guanine and xanthine may be of importance in protein catabolism, but this does not seem to follow. It should be emphasized that evidence for the existence of a reaction sequence in insects similar to the one which has been established for birds is far from complete. Only a few of the steps have been unequivocally confirmed, and some of the results obtained (e.g. the stimulating effect of malonate and urea) cannot easily be interpreted on this basis. The possibility cannot at present be excluded that different metabolic pathways may be operative in the synthesis of uric acid by insects. C . THE FORMATION O F UREA

Urea has been reported as a minor constituent of the haemolymph (see review by Buck, 1953), of the tissues (e.g. Leifert, 1935; Brown, 1938b) and of the excreta (e.g. Powning, 1953; Razet, 1961) of many insects. Since none of the species investigated in this respect possess an active allantoicase, it seems unlikely that the urea could represent a purine breakdown product, nor can it be considered to derive from the diet in all of the species concerned. The question of its metabolic derivation in insects must therefore be considered.

42

E. B U R S E L L

One possibility is that it might be produced from amino acid nitrogen through the mediation of the ornithine cycle (see Gilmour, 1961), which has been shown to be operative in other uricotelic groups like the birds and reptiles (Cohen and Brown, 1960). Arginine, citrulline and ornithine occur as intermediaries in this reaction sequence, and all of these amino acids have been detected in certain insects (Garcia et al., 1956a); and arginase, the enzyme responsible for the hydrolysis of arginine to form ornithine and urea, has been detected in several species (Kilby and Neville, 1957; Garcia et al., 1956b; 1957 and 1958; Szarkowska and Porembska, 1959). On the other hand, arginase appears to be absent from extracts of fat body of Calliphora (Desai and Kilby, 1958a), and in Celerio euphorbiae, the ornithine cycle has been shown to be incomplete, despite the presence of its three constituent amino acids (Porembska and Mochnacka, 1964). It is natural to associate the presence of arginase with the operation of an ornithine cycle, but this may well involve a misinterpretation, as has been pointed out by Kilby (1963); it is quite possible that the function of arginase in insects is not the production of urea, but the supply of ornithine “for some as yet unknown purpose”. Direct biochemical evidence in favour of the existence of the ornithine cycle is thus very inconclusive, and the results of relevant nutritional studies are hardly less equivocal. Hinton (1955) showed that Drosophila has a developmental requirement for arginine, and that arginine can be partially replaced by citrulline, but not at all by ornithine; similar results have been obtained by Davis (1962) with larvae of Oryzaephilus. The question of the synthesis of urea in insects must clearly be left open. D. THE FORMATION OF AMMONIA

Ammonia occurs as a minor excretory product in many insects, but it is only in aquatic forms (cf. Staddon, 1955) or in meat-eating fly larvae (Brown, 1938b) that it constitutes a substantial proportion of the nitrogenous waste. The manner of its formation has not yet been satisfactorily established. Where it occurs in small quantities it is possible that it may derive entirely from nucleic acid breakdown products, by deamination of purines under the influence of adenase, guanase or adenosine deaminase, all of which enzymes have been found in insects (see above). But in species which excrete large amounts of ammonia, the nitrogen must be assumed to derive from protein sources. Attempts to demonstrate deamination of amino acids by homogenates of dipteran larvae have not been successful, nor does a diet of

THE EXCRETION OF N I T R O G E N IN INSECTS

43

free amino acids lead to an increase in ammonia production by them. A protein diet, on the other hand, causes a substantial increase in ammonia output, and homogenates are capable of causing the liberation of ammonia from various peptone mixtures (Brown and Farber, 1936; Brown, 1938b). It would seem, therefore, that the ammonia produced by feeding larvae results from a deamination of proteins or higher breakdown products, but neither the mechanism nor the precise substrate have been identified. In other insects it is likely that ammonia arises in part by direct deamination of amino acids (Bheemeswar, 1959), since amino acid oxidases have been demonstrated in the fat body of a number of insects (Kilby and Neville, 1957; Desai and Kilby, 1958a; Auclair, 1959). Alternatively, amino acids may undergo transamination with a-ketoglutaric acid as the amino acceptor, and the glutamic acid formed may be deaminated under the influence of glutamic dehydrogenase : L-amino acid a-keto acid

x

NH3

a-ketoglutaric acid

.

glutamic acid

Y (tmnsaminase)

:

NAD .H NAD

Y (gluramic dehydrogenase)

Glutamic dehydrogenase activity has been demonstrated in the cockroach Periplunetu (McAllan and Chefurka, 1961b) and in extracts from the fat body of locusts (Kilby and Neville, 1957), and the presence of transaminases has been detected in a variety of insects (Kilby and Neville, 1957; McAllan and Chefurka, 1961a, b; Zandee et al., 1958; Murphy and Micks, 1964; Chen and Bachmann-Diem, 1964; Emmerich et ul., 1965). Clearly there are a number of ways in which ammonia may be formed from protein nitrogen in insects ; but what contribution the different reactions make to the total ammonia output of a given insect cannot be decided on the basis of available evidence. E. AMINO ACIDS

Small quantities of a variety of amino acids have been found in the excreta of different insects (e.g. Harrington, 1961; Mitlin et ul., 1964), but it is possible that their presence should in many cases be interpreted as a loss of amino acid rather than as an excretion of amino nitrogen. In plant-sucking Hemiptera (e.g. Mittler, 1958) the quantities involved

44

E. B U R S E L L

are substantial in relation to the total nitrogen output, but in these insects the amino acids should perhaps be regarded as in the nature of a faecal material. In a few insects, however, large quantities of particular amino acids are excreted (Powning, 1953; Bursell, 1964a), and here there can be little doubt that a disposal of metabolic waste is involved. F. MISCELLANEOUS MATERIALS

A number of other nitrogen-containing substances have been identified as components of insect excreta; for instance, creatine has been identified in the excreta of Luciliu (Brown, 1938a) and of Rhodnius (Wigglesworth, 1931), and haematin in that of the tsetse fly (Bursell, 1964b); but in view of the diet of these insects, it seems likely that the substances represent products of digestion rather than of nitrogenous metabolism. Pteridines have been identified in the excreta of the milkweed bug, Oncopeltus fusciutus (Bartel et ul., 1958) and has been shown to make up over 5% of excretory nitrogen (Hudson et ul., 1959); but little is known of the metabolism of these substances in insects, and they will not be further discussed.

111. THEEXCRETION OF NITROGENOUS END P R O D U C T S The data presented in Section I1 have shown that enzymes capable of catalyzing the formation of a variety of end products are widely distributed among insects. But the occurrence of an enzyme which catalyses a given reaction does not necessarily mean that the product of that reaction constitutes an important component of nitrogenous excretion. Many of the substances considered may have specific parts to play in the general metabolism of the insect, and this could account for the occurrence of the corresponding enzymes. It is only by an analysis of excretory material that the contribution of different metabolic pathways to the disposal of nitrogenous waste materials can be accurately evaluated. A great deal of information is available on this point, but unfortunately it is seldom complete. The aims of investigators in this field have differed greatly. Some have been concerned simply with demonstrating the presence or absence of a specific substance in excretory material; some have focused their attention on certain classes of substance, purine degradation products for example, to the neglect of others, and so on. It is only in very few cases that the excretory material of a given insect has been accurately partitioned among all the possible nitrogenous waste products. But even with the somewhat fragmentary information which is available, it is possible to get a

THE EXCRETION OF NITROGEN I N INSECTS

45

reasonable indication of the tremendous diversity of excretory patterns which exists within the class, and the picture that emerges is not at first sight easy to reconcile with the traditional view of insects as a uricotelic group of animals. In view of the large number of species which have been investigated during recent years, it will be convenient to give a separate account of each of the orders whose members have been studied, and the classification adopted by Imms (1958) will be used for this purpose. The proportion of nitrogenous waste in the faeces of insects varies enormously, depending to a large extent on feeding habits. In herbivorous species, for instance, the nitrogen load may be light and undigested plant material often makes up the bulk of faecal matter, so that uric acid may comprise no more than 0.1% of the dry weight (e.g. Razet, 1961); in blood-sucking insects, on the other hand, the nitrogen load is heavy, there is little indigestible material, and uric acid may account for as much as 60% of the dry weight of faeces (e.g. Bursell, 1964a). In order to facilitate comparison of values which may differ by several orders of magnitude, the quantity of nitrogen excreted in different nitrogenous end products will be expressed as a proportion of nitrogen in the predominant end product. For instance, an insect which excretes 100 mg of uric acid, 10 mg of urea and 2 mg of ammonia, which would be 33.3 mg of uric acid nitrogen, 4.6 mg of urea nitrogen and 1.6 mg of ammonia nitrogen, will be listed as: Uric acid Urea Ammonia 1-00 0.14 0.05 showing that for every 100 mg of uric acid nitrogen excreted, 14 mg of urea nitrogen and 5 mg of ammonia nitrogen are disposed of. It must be emphasized that values of this kind indicate only the proportionate composition among the substances assayed. In some cases it is possible that unidentified substances, or substances not quantitatively estimated, may exceed the listed products in importance. Furthermore, values quoted for different species in the tables which follow should be regarded as no more than generally indicative of the proportionate composition in different species. Variability within species is known to be great, and in many cases determinations have been based on single samples (Razet, 1961). A . COLLEMBOLA

In view of the occurrence in a member of this order of three of the uricolytic enzymes, uricase, allantoinase and allantoicase (Razet, 1961),

46

E. BURSELL

it is a pity that no data are available on the composition of excreta, so that the extent to which degradation of uric acid is a feature of nitrogenous excretion must remain in doubt. B . ORTHOPTERA

Some of the available information concerning the nitrogenous end products in members of this order has been summarized in Table I. A number of other species have been investigated (Razet, 1961), but in TABLE I Excretory products in the Orthoptera, Odonata and Dermaptera Uric Allantoic Amino acid Allantoin acid Urea Ammonia acids

Author

Order ORTHOPTERA Schistocerca gregaria 1.00

0.00

0.00

+

+

Locusta migratoria

1-00

0.00

0.10

-

-~

MeIanopIus bivittatus

1.00

-

-

0.07

053

Acheta dornesticus

1.00

0.01

0.01

0.00

-

Mantis religiosa Periplaneta americana BIatta orientalis Carausis morosus

1-00

0.01

0.00

-

-

1.00

0.00 0.64

0.00

-

__

-

0.64 0.69

1.00

-

-

- Razet, 1961

0.44

-

-

--

Razet, 1961

Order ODONATA Aeshna cyanea (larva)

0.08

-

-

Staddon, 1959

Order DERMAPTERA ForficuIa auricuIaria 1.00

-

1-00

_.

0.00

-

1.00

-

-{

Chauvin, 1941 Razet, 1961 Razet, 1961 Nation and Patton, 1961

0.20 Brown, 1937

Razet, 1961 Nation and Patton, 1961 - Razet, 1961

-{

Razet, 1961

- Razet, 1961

the interests of brevity a selection, illustrative of the range of variation within the order, has been made for this group, as for most of the others. Uric acid is clearly the predominant end product in many Orthoptera, with other components appearing in relatively small amounts. Blattu orientalis, however, excretes most of its nitrogen as allantoic acid, while the stick insect, Curuusis morosus, has a preponderance of allantoin in its excreta. Such differences do not seem to be correlated at all

THE EXCRETION OF NITROGEN I N INSECTS

47

closely with the presence of the corresponding uricolytic enzymes. Achetu has been shown to possess active uricase, allantoinase and allantoicase,yet it excretes virtually none of the correspondingproducts; Cuurausishas a highly active allantoinase (Poisson and Razet, 1952), yet allantoin predominates in its excreta. Nor is there any apparent correlation with feeding habit, since uric acid predominates in the excreta of herbivorous, and omnivorous as well as carnivorous species. In fact, consideration of just this one order illustrates the sort of diversity in respect of nitrogenous excretion which succeeding pages will show to characterize the class as a whole. The deposition of uric acid in cells of the fat body seems to be a feature of many orthopteran species, particularly among the Blattidae (e.g. Srivastava and Gupta, 1960), and uric acid may comprise as much as 10% of the total dry weight of such insects (Razet, 1961). Haydack (1953) has shown that the amount of uric acid stored in the fatty tissues of the American cockroach is greatly affected by the level of protein intake. On a high protein diet the fat body becomes enlarged, and is filled with white deposits of uric acid. If such insects are transferred to a low protein diet the uric acid deposits largely disappear. The readiness with which uric acid may be mobilized from fat body deposits in these, as in other, insects, has led to the suggestion that it may serve in part as a reserve of nitrogen for synthetic purposes (Ludwig, 1954; Ross, 1959), and there is some indication that endocrine control mechanisms may be involved (Bodenstein, 1953). Recent work by Roth and Dateo (1964 and 1965) has shown that in males of certain cockroaches the accessory sex glands play a special role in the storage of uric acid, and that as much as 5% of the live weight of males may be attributed to uric acid deposited in the utriculi majores of these glands. Most of this uric acid is poured over the spermathecae during copulation, and the sex glands may thus be said to serve as accessory excretory organs. C . ODONATA

Excretion in the aquatic larva of Aeshna cyuneu has been investigated by Staddon (1959). In starving animals most of the excretory nitrogen appeared as ammonia, with uric acid making up less than a tenth of the ammonia nitrogen (see Table I). When the larvae were fed on protein (heat-coagulated egg white) the output of ammonia increased greatly, while uric acid excretion remained constant. It would seem that ammonia is the main end product of protein catabolism, while uric

48

E . BURSELL

acid represents the end product of purine metabolism, and is therefore unaffected by protein intake: this is in accord with the absence of uricolytic enzymes in the species investigated by Razet (1961). D . DERMAPTERA

The excreta of Forjicula has been shown to contain a high proportion of uric acid (see Table I), and despite the presence of an active allantoinase, no allantoic acid could be demonstrated (Razet, 1961). E . HEMIPTERA

1. Sub-order Heteroptera The excretory material of a number of plant bugs has been investigated, and some of the results are summarized in Table 11. Allantoic acid has not been recorded for any member of this group, and this is in TABLE I1 Excretory products in the Heteroptera Uric Allantoic Amino acid Allantoin acid Urea Ammonia acids

____

Micrelytra fossularum Verlusia rhombea Palomena prasina Rhaphigaster griseus Dysdercus fasciatiis Rhodniusprolixus

0.65

0.02 1.00 1.00

0.00 0.00

1.00 1.00

1.00 0.87

1 .oo

0.00

~

0.00 0.00 0.00

0.00 ~~

-

0.26 0.03

Author -

.__-

-

Razet, 1961 Razet, 1961 Razet, 196:

- Razet, 1961 0.24 Berridge, in press trace Wigglesworth, 1931 Harrington, 1956 and 1961

accord with the apparent absence of the corresponding enzyme, allantoinase, in the large number of species examined by Razet (1961). Allantoin, on the other hand, occurs in all species, and is the predominant end product in several, which relates well to the widespread presence of a highly active uricase among members of this suborder (Poisson and Razet, 1953; Razet, 1961). Allantoin is the predominant end product in Dysdercus, but substantial quantities of amino nitrogen have been recovered from the excreta, and urea is also an important component (Berridge, 1966);

THE EXCRETION OF NITROGEN I N INSECTS

49

in view of the general absence of allantoinase and allantoicase which characterizes the group as a whole, the urea is unlikely to represent a product of uricolysis, and the possibility of faecal contamination can be excluded in this insect, since the alimentary canal is discontinuous. Urea has also been found in the excreta of Oncopeltus fasciatus, but in this species uric acid constitutes the predominant end product (Nation and Patton, 1961). The blood-sucking hemipteran RhochiusproZixus is a member of the suborder, and its excretory metabolism has been the subject of several studies (Wigglesworth, 1931; Harrington, 1956, 1961). Uric acid is the main end product, but urea accounts for 2.3% of the dry weight of excreta, and a number of amino acids have been detected together making up 0.2% of the total dry weight, with histamine and histidine predominant among them. In view of the fact that the food of this insect is composed almost entirely of amino acids, arising by hydrolysis of blood proteins, the appearance of such very small quantities of amino acid nitrogen in the excreta should perhaps be interpreted as a loss, rather than as an excretion, of amino acids. 2. Sub-order Homoptera Investigations of excretory metabolism in this group of insects has centred upon the occurrence of large amounts of amino acid in the honeydew of various aphids and coccids (see review by Auclair, 1963). These substances account for 13.2% of the dry weight of honeydew in Myzus circumflexus (Maltais and Auclair, 1952) and make up the bulk of nitrogenous material also in Brevicoryne brassicae (Lamb, 1959). The pattern of amino acids in honeydew is qualitatively similar to that of the plant juices ingested, though the concentration may be slightly lower due to absorption of nitrogenous material (Mittler, 1958 with Tuberolachnus salignus), or somewhat higher (Auclair, 1958 with Acyrthosiphon pisum) possibly due to a concentration of fluids in the insect associated with evaporative losses of water. But it would seem that the insects are ingesting nitrogenous substances in excess of their requirements, and that this excess is voided as a faecal material. If this is the correct interpretation then such amino acids have not been subjected to metabolic manipulation, and should hardly be considered as end products of nitrogenous metabolism. Gray and Fraenkel(l954) have reported the occurrence of uric acid in Pseudococcus citri on the basis of qualitative, colour reactions, but Mittler (1958) failed to find evidence of uric acid or of ammonia in Tuberolachnur.In Breuicornis neither uric acid nor urea, allantoin or

50

E. BURSBLL

allantoic acid could be detected (Lamb, 1959), but ammonia was found to constitute 0.5% of the total nitrogen in honeydew. In view of the rapid throughput of liquid in these insects it is not impossible that even such low concentrations of ammonia may represent a high proportion of truly excretory nitrogen. Uricase has been demonstrated in homogenates of Aphis brassicae, but other members of the group which have been investigated appear to be devoid of uricolytic enzymes (Razet, 1961). F. C O L E O P T E R A

Table I11 summarizes results of investigations of the excretory metabolism in beetles. Uric acid is the predominant end product in three of the species examined, and in these uricolytic activity has been shown to be absent or feebly developed (Razet, 1961). In Chrysobothris TABLE I11 Excretory products in the Coleoptera and Neuroptera Uric Allantoic Amino acid Allantoin acid Urea Ammonia acids Author Order COLEOPTERA 1.00

0*00

0.00

-

-

-

1.00 1.00

0.01 0.10

0.03 0.00

-

-

- Razet, 1961 Razet, 1961

0.83

1.00 -

0.00 -

-

0.72

1.00

0.57

- Razet, 1961 0.50 Pomhg, 1953

Order NEUROPTERA Uroleon nostras (larva) 1.00

0.03

0.00

-

-

Melolontha vulgaris Chaetocarabus intricatus Procrustes coriaceus Chrysobothris afinis Attagenuspiceus

Razet, 1961

-

- Razet, 1961

the quantity of allantoin recovered in excreta exceeds that of uric acid, which suggests the presence of an active uricase, but this has not been directly verified. In the carpet beetle, Attagenus, urea is the predominant end product identified, substantial quantities of ammonia are excreted, and the presence of the sulphur-containing amino acid, cystine, accounts for a considerable proportion of excretory nitrogen (Pawning, 1953); its occurrence is correlated with the high cystine content of keratin, which forms the major component of diet in this species, and it is possible that this amino acid should be considered to represent a faecal material rather than an excretory product. It must be noted

THE EXCRETION O F NITROGEN I N INSECTS

51

that the nitrogenous components listed for this insect make up only a proportion of the total nitrogen excreted, and some unidentified excretory products must be involved. The excreta of the mealworm beetle, Tenebrio molitor, contains mostly uric acid, but the presence of urea and allantoin has been established (Nation and Patton, 1961) although none of the uricolytic enzymes could be demonstrated in this species (Razet, 1961). Urea and ammonia have been found as constituents of the excreta of other members of the order (Delaunay, 1931) and ammonia has been identified in the excreta of Ephestia kuhniella (Payne, 1936). The excreta of the boll weevil, Anthonomus grandis has been examined for the presence of non-protein amino acids, and these were found to constitute 3.2% of the total nitrogen, with about a third attributable to free amino acids (Mitlin et al., 1964). Storage of uric acid in cells of the fat body has been reported for one of the species of beetle examined by Gupta and Sinha (1960), but storage excretion does not appear to be a general characteristic of the group, to judge by the generally low uric acid contents recorded by Razet (1961). G . NEUROPTERA

Data for the larva of Uroleon have been included in Table 111. Since the alimentary canal is discontinuous, excretory material could be collected free of faecal contamination. 33% of the dry weight was uric acid, and only a small quantity of allantoin was present; allantoic acid could not be detected. In the larva of Chrysopa carnea the deposition of uric acid in cells of the fat body has been reported (Spiegler, 1962). The presence of large quantities of ammonium bicarbonate in excretory fluids of the aquatic larva of Sialis lutaria was demonstrated by Shaw (1955). The excretory metabolism of this insect was further investigated by Staddon (1955) who showed that 86% of the nitrogen output of starving animals appeared in the form of ammonia. H . HYMENOPTERA

The excreta of certain herbivorous larvae belonging to this group has been investigated by Razet (1961), and some of his results are summarized in Table IV. Here again some species excrete predominantly uric acid, in others allantoin or allantoic acid may be important constituents, and in some they achieve dominance. The differences between

52 E. BURSELL species cannot be correlated with differencesin host plant, nor do they accord with the distribution of uricolytic enzymes. Uricase is poorly developed in both the genera listed; and Pteronidea sulicis, which has a highly active allantoinase, excretes a very small proportion of allantoic acid, while in P. ribesi allantoinase activity is slight, but allantoic acid forms a high proportion of the excretory material. TABLE IV Excretory products in the Hymenoptera and Diptera Uric Allantoic Amino acid Allantoin acid Urea Ammonia acids Author Order HYMENOPTERA Hemichroa alni

1.00

0.00

0.00

- Razet, 1961

1.00

0.03

0.06

- RBWt, 1961

0.78

1.00

0.90

- R m t , 1961

1.00 1-00 1.00

0.04

0.30 0.30

0.00 0.62

0.05

1.00

(larva) Pteronidea salicis

(larva) Pteronidea ribesi

(larva) Order DIPTERA Compsilura concinnata Tipulapaludosa Lucilia sericata Lucilia sericata Oarva) Lucilia sericata

(pupa) Bibio marci (larva) Aedes aegypti Anopheles guadrimaculatus Culex pipiens Glossina morsitans

I

0.45

1*0° 1.00

-

-

0-30

0.02

-

1.00

0.00 1.00

-

0.15

0-36

-

0.22

+

0.18

- Razet, 1961 - X m t , 1961

-

-{

Brown, 1936; 1938a and b

-

- Razet, 1961

0.11 Irrevere and Terzian, 1959 0.22 Bursell, 1964b

I . DIPTERA

A number of insects belonging to this order have been studied, and results are included in Table IV. Most of the adults examined are predominantly uricotelic, though in Tipula, where active uricolytic enzymes have been demonstrated (Razet, 1961), substantial amounts of allantoin and allantoic acid are present in the excreta. In the sheep ked, Melophugus ovinus, uric acid is the main end product, but quantities of xanthine and hypoxanthine may appear in the excreta of pregnant females (Nelson, 1958).

53 The excretory metabolism of several blood-sucking members has been the subject of detailed investigation. Uric acid is the dominant excretory product in mosquitoes, but urea and ammonia occur in fairly high concentration, and amino acids make up a substantial proportion of total nitrogen; there is little difference between the three species examined, and in Table IV results have been averaged. The effect of nutrition on the excretion of Aedes aegypti has been studied by Terzian and his colleagues (1957), who show that on a diet of sugar there is a decrease in the nitrogen output, and the proportion of uric acid in the excreta falls to about 4%. Following a blood meal the nitrogen output increases greatly, and the proportion of uric acid rises to SO%, thus confirming that uric acid is the end product of protein metabolism. In the tsetse fly also, the predominant excretory product is uric acid; urea occurs in trace amounts, and a substantial proportion of nitrogen is excreted in the form of histidine and arginine. These two amino acids, which together make up about 10% of the protein amino acids in human blood, are particularly rich in nitrogen (see Fig. 1). It would presumably be uneconomical to deaminate materials of this kind, since any benefit which the insect might derive from deamination products would be offset by metabolic losses involved in uricotelic detoxication of the nitrogen they contain. In view of this, a quantitative elimination of ingested arginine and histidine might be expected, and this is roughly in accord with observation (Bursell, 1964b). There is some evidence that all of the amino acids liberated during digestion are absorbed from the midgut, and that arginine and histidine are subsequently excreted, so that they cannot be regarded simply as faecal materials. Indeed, recent work has shown that arginine becomes rapidly labelled following injections of C14-glutamate(E. Bursell, unpublished), and it is possible that this substance may play a more active role in excretion than was originally envisaged. The occurrence of small quantities of ammonia has been reported in the excreta of adult Diptera by a number of workers (Brown, 1938b; Lennox, 1940; Hitchcock and Haub, 1941; Sedee, 1958), but it is only in the meat-eating larvae of species like Calliphora and Lucilia that this product becomes dominant (Brown, 1936; 1938a, b; Robinson, 1935; Robinson and Baker, 1939). Allantoin and uric acid are present in relatively small amounts in the larval excreta of Luciliu (see Table IV), and the proportion between them appears to depend in part on larval diet; with larvae fed on casein allantoin predominates, but on a meat diet relatively little allantoin is produced (Brown, 1938a). THE EXCRETION O F NITROGEN I N INSECTS

54

E. B U R S E L L

Allantoin is an important constituent of excreta in larvae of Bibio, and allantoic acid occurs as well, in accord with the presence of an active allantoinase in larval extracts (Razet, 1961). The excretory products which accumulate in the meconium during pupal development have been examined by Brown (1938a, b) in Lucilia. A considerable amount of ammonia was found, but uric acid constitutes the bulk of excretory nitrogen and no allantoin could be detected. The absence of allantoin is correlated with a loss of uricase activity at this stage of the life history; the enzyme disappears suddenly when larvae leave the meat, to reappear immediately after emergence from the pupa. The results of investigations of dipteran excretion provide a striking illustration of the lability which appears to characterize this aspect of metabolism, with the distribution of nitrogenous end products varying widely from stage to stage in the life history of a single species. J. LEPIDOPTERA

A great deal of information is available concerning the uricolytic metabolism of this group, thanks to the work of Razet (1961). A selection of results has been summarized in Table V, where the proportions of uric acid, allantoin and allantoic acid in the excreta of different stages in the life history of members of the order have been set out. Variability within the group is considerable; many of the adults are predominantly uricotelic, but allantoin and allantoic acid occur as minor components in several species, and in some allantoic acid is the predominant end product. In pupal stages uric acid is predominant in all the species listed, including those whose adults show a preponderance of allantoic acid; but the proportions of allantoic acid and allantoin are often appreciable, and in some species exceed those found in adult excreta (e.g. Aglais urticae). In larval stages the three earcretory products are more evenly distributed in the excreta of different species; where allantoic acid is the end product, uric acid occurs in substantial proportion, and vice versa. But there is little correlation between adult and larval excretory metabolism, for some of the species whose adults are predominantly uricotelic show a preponderance of allantoin in larval excreta, while some of the species whose adults have allantoic acid as the main end product have uricotelic larvae. In certain species it has been demonstrated that larval diet may have an important effect on excretory metabolism. For instance, with

55

THE EXCRETION O F NITROGEN I N INSECTS

larvae of Lasiocampa trifolii fed on leaves of Hordeum murinum the predominant excretory product is allantoic acid, while a diet of Betulus aZba produces a predominance of uric acid. Such effects cannot be attributed to the pre-existence of nitrogenous end products in the diet, since of all the host species examined only one, the cabbage Brassica TABLE V Excretory products in the Lepidoptera (Razet, 1961) Uric acid

1. ADULTS Mammestra brassicae Agritos comes Aglais urticae Trigonophora meticulosa Pieris brassicae Vanessa atalanta Conistra vaccinii 2. PUPAE M . brassicae A. comes A . urticae T. meticulosa P. brassicae V. atalanta C. vaccinii

3. LARVAE M . brassicae A. comes A. urticae T. meticulosa P. brassicae V. atalanta C. vaccinii

1.00 1.oo 1.oo 1.00 1.00 0.89 0.12

1.00 1.00 1.00 1.00

Allantoin -

0.00 0.01 0.04 0.14 -

-

0.29 -

1.oo 1.00 1.00

0-03 0.14 0.04

0.49 0.67 0.56 1-00 0.28 1.oo 1.00

0.11 0.02 0.18 0.02

0.16 0.26 0.06

Allantoic acid 0.00 0.01

0.06 0.05 0.01

1.00 1.00

0.03 0.01 0.43 0.19 0.05 0.29 0.04 1.00 1.00 1.00 0.12 1.00 0.20 0.05

oleracea, could be shown to contain small quantities of allantoin and allantoic acid. The occurrence of uricolytic products in the excreta of different members of the order is in general accord with the presence, in most species, of the corresponding uricolytic enzymes, but in point of detail there is little agreement. In some species where enzymatic activity is 34-A.I.P.

4

56

E. BURSELL

relatively high the corresponding end product is poorly represented in the excreta (e.g. allantoinase in adults of Aglais urticae), while species with relatively low enzymatic activity may show a preponderance of the reaction product in their excreta (e.g. Conistra vaccinii). The excretory metabolism of lepidopteran larvae at different stages of larval life has also been investigated by Razet (1961), and the results serve further to emphasize the lability of this function within the life history of the individual, for the ratio of allantoin to uric acid may fluctuate enormously from day to day. In most of the species studied there is a general tendency for uric acid to become increasingly important with larval age. A number of end products other than these three have been assayed in the excreta of different members of the order. In Bombyx mori, amino acids have been demonstrated in excretory material of the larva (Yoshitaka and Aruga, 1950); uric acid is the predominant end product in this species, and the quantity exsreted has been shown to increase on a diet rich in protein (It0 and Mukaiyama, 1964). In larvae, of Corcyra cephalonica uric acid is again predominant in the excreta, but small quantities of urea and of xanthine have been demonstrated (Srivastava, 1962) ; both xanthine and hypoxanthine have been identified in the excreta of the wax moth, Galleria melonella (Nation, 1963; Nation and Patton, 1961), with hypoxanthine making up as much as 10% of the total; ammonia has also been identified in the larval excreta of this species (Zielenska, 1952). In the clothes moth, TineoZa bisselliella, uric acid also predominates, but for every 100 mg of uric acid nitrogen excreted, 24 mg of ammonia nitrogen and 10 mg of urea nitrogen are disposed of. In addition, the excreta of this species contains a substantial amount of amino nitrogen in the form of cystine, which correlates with its diet of keratin (Waterhouse, 1952; Powning, 1953). I V . CONCLUSIONS The preceding pages have given some indication of the tremendous progress which has been made in the field under review during the last 15 years. A number of uricolytic enzymes have been investigated in tissue homogenates, their distribution among Werent tissues has been established and some of their properties have been described. A great deal has been discovered about xanthine oxidase, or xanthine dehydrogenase as it should more properly be called, and here studies have advanced to the stage of enzyme purification. But unfortunately there remain several gaps in our knowledge of nitrogenous metabolism

THE EXCRETION OF N I T R O G E N I N INSECTS

57

at the level of enzyme studies. The contribution made by different reactions (adenase and guanase deamination, oxidative deamination of amino acids, the glutamic dehydrogenase system) to the appearance of ammonia remains under dispute. The origin of urea is still uncertain, and, above all, the precise pathway of uric acid synthesis has not been unequivocally settled. The evidence concerning these aspects of nitrogenous metabolism is often contradictory, and the problems stand in urgent need of resolution. The application of radioactive tracer techniques, coupled wit% the use of insects whose nutrition, in terms of protein and nucleoprotein intake, can be rigidly controlled, would seem to offer prospects of rapid advance in this field. The role of urea in nitrogenous excretion has never been fully understood; it is present in the excreta of many insects, but usually forms a rather minor component. It cannot be regarded as a purine degradation product, since so far no insect has been shown to possess the enzymes required for the degradation of uric acid to this stage. The presence of arginase in many insects suggests that it may originate by hydrolysis of arginine, but evidence for the existence of the complete omithine cycle is weak. It seems possible that arginine may be the raw material for its production, but that its occurrence in insects should be seen as a reflection of the use of arginine as a phosphagen in this class of animals (Razet, 1964) ;the precise implications of this suggestion have, however, yet to be worked out. The data summarized in Section I1 of this review give some indication of the complexities involved in the excretory metabolism of insects. In the face of such complexities one may be forgiven for looking back with a degree of nostalgia to the early generalization-that insects as a class are uricotelic, and that the production of uric acid as the main end product of nitrogenous metabolism can be seen as a reflection of the shortage of water to which most members of the group are subject. This shortage of water would make toxic substances, like ammonia, and%oluble substances, like urea, unsuitable as waste products. It was possible to accommodate certain exceptions within the general framework of this generalization; for instance, the predominance of ammonia as an excretory product of aquatic larvae, of certain dipteran larvae and perhaps of certain plant-sucking bugs, could be interpreted on the basis of the ready availability of water associated with these modes of life. Similarly, the excretion of substantial proportions of specific amino acids could be seen as an adaptation to special diets; insects which ingest a lot of keratin excrete a lot of the sulphur-containing amino acid which this diet contains; blood-sucking insects, whose

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diet contains a high proportion of nitrogen-rich amino acids, may eliminate these unchanged, though why this should occur in the tsetse fly and to some extent in mosquitoes, but not in Rhodnius, is unclear. Up to this point it would appear permissible to retain the view of uricotelism as an insect characteristic. But what investigations of the last ten years have brought to light is the high proportion of purine derivatives other than uric acid that occur in the excreta of many insects. These include not only degradation products like allantoin and allantoic acid, but also some of the reduced derivatives like xanthine and hypoxanthine. Since these substances may occur in the excreta of insects in amounts far exceeding any that could be attributed to nucleic acid catabolism, they must derive largely from amino acid nitrogen. The situation, therefore, seems to be that amino acid nitrogen is detoxicated by incorporation in a purine ring system at the expense of considerable amounts of metabolic energy; and that the purine ring system is then broken down in a sequence of hydrolytic reactions which are unproductive of metabolic energy, and lead to the formation of reaction products that differ appreciably from uric acid neither in respect of solubility nor in respect of nitrogen content. Coupled with the apparent lack of correlation between the proportion of degradation products in the excreta of different insects and their mode of nutrition and way of life, this provides small basis for an interpretation in terms of water balance or of excretory efficiency. In seeking for an alternative basis of interpretation, perhaps the most sigmficant finding is the localization of enzyme activity in cells of the Malpighian tubules and of the midgut, both of which are regions which have been implicated in the elimination of excretory material. It seems possible that the degradation of the purine ring may in some way be associated with the transfer of material across a secretory epithelium (M. J. Berridge, in the press), perhaps by some form of facilitated diffusion. The ability to break the purine ring to the stage of allantoic acid appears to be widely, and maybe universally, distributed within the class. It is true that negative results of enzyme assays have been reported for a number of insects (Razet, 1961), but this may mean not that the enzymes are absent, but that they are present in concentrations too low to be detected by the methods used. The nature of the results obtained by Razet does not allow a great deal of reliance to be placed on negative results. In many cases the quantity of substrate broken down is inversely related to the amount of enzyme present, and Razet suggests the possibility that inhibitory factors may be involved, coming into play at high homogenate concentrations. Similar effects could be involved in

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extracts from species showing no detectable activity; indeed, the development of more refined methods led to the detection of uricase in species which had previously been thought devoid of activity (Razet, 1964). It would seem that investigations with dialysed enzyme preparations would be required for unequivocal demonstration of the absence of uricolytic activity. These considerations suggest that the development of uricotelism in insects has not been associated with a loss of uricolytic enzymes. The ability to synthesize these enzymes appears to have been preserved in the genotype, perhaps because certain of the reaction products have special parts to play in general metabolism. It is the degree to which this ability is reflected in the phenotype which is subject to immense variation from order to order within the class, from species to species within the orders, from stage to stage in the life history of a species, and from time to time within a given stage. The main problem which confronts the investigator today is to discover what is the biological significance of these variations, and with what aspect of species biology they are correlated. The demonstration that different nitrogenous end products may achieve dominance in the excretory metabolism of different insect species calls, however, for some reassessment of the generalization that terrestrial insects as a group are uricotelic. Either this statement must be qualified to take account of recent discoveries, or it must be abandoned in favour of a more complex statement of the situation. The second alternative is the one favoured by P. Razet (in the press) in his recent review of nitrogenous metabolism in insects. He proposes that insects should be assigned to one or other of a number of different categories depending on the nature of the predominant excretory end product. The terms uricotelic, ureotelic and ammonotelic would be retained to indicate a preponderance in the excreta of uric acid, urea and ammonia respectively, and two new categories would be added : allantoinotelic for insects which excrete mainly allantoin, and allantoicotelic for insects which excrete mainly allantoic acid. Apart from the rather cumbersome nature of these new epithets, a classification of this sort does not seem very helpful at the present stage. In the first place it bears a largely random relation to the generally accepted taxonomy of the class, and representatives of a given category may occur equally among primitive and advanced members. Secondly, the proposal to classify species on the basis of the predominance of a given end product would run into difficulty in the case of certain Diptera in which the larva is ammonotelic and the adult uricotelic; or

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certain Lepidoptera, which may be uricotelic in the larval stage and allantoicotelic as adults, or whose larvae may switch from one category to another depending gn age. What is described would here cease to be a species characteristic; the terms would become descriptive simply of a particular moment in the life history, and would thus be deprived of their generally accepted connotations. Even if it should be found desirable to institute a new system of classification of the kind proposed by Razet, the time to apply it to the orders of insect would not be now. For while the information available is reasonably extensive in respect of certain groups (e.g. the Lepidoptera), it is very meagre in respect of others (e.g. Collembola; Dermaptera), and these latter might have to be assigned to their place in classification on the basis of a single species. In view of the wide spectrum of end product predominance discovered in all the groups which have been thoroughly studied, it seems certain that any scheme proposed would be shortlived and subject to continual adjustment in the light of new information. For these reasons it seems to the present reviewer that some attempt should be made to retain the older generalization for the time being, if this can be done by suitable qualification. Granted the complexities reviewed in Section 11, it still remains true to say that uric acid is an important excretory product in nearly all terrestrial insecfs, and to this limited extent the statement that uricotelism is a characteristic of the class remains valid. It is not always the predominant excretory product, but where it is not, its place seems invariably to be taken by allantoin or allantoic acid, as far as present information goes. Since these substances differ from uric acid in neither of the properties which are chiefly relevant to the disposal of nitrogenous waste, namely nitrogen content and solubility, it would seem permissible to regard them all as belonging, to a single class of excretory substance, and to widen the definition of uricotelism accordingly-to regard an animal as uricotelic if it excretes uric acid or one of its primary degradation products, allantoin or allantoic acid, or some mixture of these three substances, as the predominant waste product. With such a qualification the view of insects as a uricotelic class, in line with thsir terrestrial mode of life, remains tenable, and the centre of interest in the field of nitrogenous metabolism then shifts from further cataloguing of the relative proportions of these end products in the excreta of insects to what seems at present a more rewarding problem, namely a study of the biological significance of such differences in the proportion of end products, possibly in relation to the mechanism of excretion. The most promising approach to this study, and through it to the general problem of the distribution of

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nitrogenous end products, would seem to lie with the species which show a pronounced shift in excretory metabolism during the course of their life history. Here there would be some possibility of relating observed changes in the proportion of different end products to the level and type of nutrition and to the availability of water, and in this way to gain some insight into the significance of such changes. The work of the last decade has adequately demonstrated the diversity and complexity of excretory metabolism in the insects as a whole. What seems to be required now are intensive studies of selected species in which the problem of excretory metabolism can be set squarely in the context of species biology.

ACKNOWLEDGEMENTS My sincere thanks are due to Dr. P. Razet for his kindness in presenting me with a copy of his thesis, and for allowing me to read and to refer to his review of nitrogenous metabolism before its publication.

REFERENCFB Anderson, A. D. and Patton, R. L. (1954). Determination of xanthine oxidase in insects with tetrazolium salts. Science, N.Y. 120, 956. Anderson, A. D. and Patton, R. L. (1955). In vitro studies of uric acid synthesis in insects. J. exp. 2001.128, 443-451. Auclair, J. L. (1958). Honeydew excretion in the pea aphid, Acyrthosiphon pisum (Harr.) (Homoptera: Aphididae). J. Insect Physiol. 2, 330-337. Auclair, J. L. (1959). Amino acid oxidase activity in the fat body and Malpighian tubules of some insects. J. Insect Physiol. 3, 57-62. Auclair, J. L. (1963). Aphid feeding and nutrition. A. Rev. Ent. 8, 439490. Baker, F. C. (1939) The preparation of a relatively stable urease concentrate of blow-fly larvae (Lucilia sericata Meig.). J. Parasit. 25, 280. Baldwin, E. (1948). “Dynamic aspects of biochemistry”, (1st Ed.) Cambridge University Press, London. Baldwin, E. (1963). “Dynamic aspects of biochemistry”, (4th Ed.) Cambridge University Press, London. Bartel, A. H., Hudson, B. W. and Craig, R. (1958). Pteridines in the milkweed bug, Oncopeltus fasciatus Dallas. 1. Identification and localizatien. J. Insect Physiol. 2, 348-354. Berridge, M. J. (1966). The physiology of excretion in the cotton stainer Dysdercus fasciatus Signoret (Hemiptera, Pyrrhocoridae). J. exp. Biol. in press. Bheemeswar, B. (1959). Some aspects of amino acid metabolism in insects. 4th Znt. Congr. Biochem. Vienna, 1958. XII. Biochenfistry of Insects, 78-89. Pergamon Press, Oxford.

62

E. B U R S E L L

Bodenstein, D. (1953). Studies on the humoral mechanisms in growth and metamorphosis of the cockroach Periplaneta americana. 111. Humoral effects on metabolism. J. exp. 2001. 124, 105-115. Brighenti, A. and Colla, A. (1940). Sul process di formazione dell’acido urico uli bachi da seta. Boll. SOC.ital. Biol. sper. 15, 197-198. Brown, A. W. A. (1936). The excretion of ammonia and uric acid during the larval life of certain muscoid flies. J. exp. Biol. 13, 131-139. Brown, A. W. A. (1937). Studies on the excreta of a grasshopper (Melanoplus bivittatus Say.). J. exp. Biol. 14, 87-94. Brown, A. W. A. (1938a). The nitrogen metabolism of an insect (Lucilia sericata Mg.) I. Uric acid, allantoin and uricase. Biochem. J. 32, 895-902. Brown, A. W. A. (193813). The nitrogen metabolism of an insect (Lucilia sericata Mg.) 11. Ammonia and other metabolites. Biochem. J. 32, 903-911. Brown, A. W. A. and Farber, L. (1936). On the deaminating enzyme of flesh-fly larvae. Biochem. J. 30, 1107-1 118. Bruno, C. F. and Cochran, D. (1965). Enzymes from insect tissues which catabolize pyrimidine compounds. Comp. Biochem. Physiol. 15, 113-124. Buchanan, J. M. (1951). Biosynthesis of the purines. J. cell. comp. Physiol. 38, 143-1 71. Buck, J. B. (1953). Physical properties and chemical composition of insect blood. In “Insect Physiology”, (K. D. Roeder, ed.). John Wiley and Sons, New York. Bursell, E. (1964a). Nitrogenous waste products in the tsetse fly. 12th Int. Congr. Ent. 797. Bursell, E. (1964b). Nitrogenous waste products of the tsetse fly, Glossina morsitans. J. Insect Physiol. 11, 993-1001. Chauvin, R. (1941). Physiologie du criquet pklerin (Schistocerca gregaria Forsk.). Annls SOC.ent. Fr. 9, 139-272. Chauvin, R. (1956). “Physiologie de 1’Insecte.” Inst. nat. Rech. Agronomique, Paris. Chen, P. S. and Bachmann-Diem, C. (1964). Studies on the transamination reactions in the larval fat body of Drosophila melanogaster. J. Insect Physiol. 10, 819-829. Cochran, D. C. and Bruno, C. F. (1963). A partial pathway for the synthesis of uric acid in the American cockroach. 16th Int. Congr. Zool. 2, 92. Cohen, P. P. and Brown, G. W. (1960). Ammonia metabolism and urea biosynthesis. In “Comparative Biochemistry”, (M. Florkin and H. S. Mason, eds.) vol. 2, pp. 161-244. Academic Press, New York and London. Cordero, S. M. and Ludwig, D. (1963). Purification and activities of purine enzymes from various tissues of the American cockroach Periplaneta americana L. (Orthoptera, Blattidae). J. N. Y. ent. SOC. 41, 66-73. Craig, R. (1960). The physiology of excretion. A. Rev. Ent. 5, 53-68. Davis, G. R. F. (1962). Quantitative L-arginine requirements of larvae of the saw-toothed grain beetle, Oryzaephilus surinamensis (L.) (Coleoptera, Silvanidae). J. Insect. Physiol. 8, 377-382. Delaunay, H. (1931). L’excrCtion azotCe des invertCbrCs. Biol. Rev. 6,265-301. Desai, R. M. and Kilby, B. A. (1958a). Some aspects of nitrogen metabolism in the fat body of the larva of Calliphora erythrocephala. Archs int. Physiol. Biochem. 66, 248-259.

THE EXCRETION O F NITROGEN I N INSECTS

63

Desai, R. M. and Kilby, B. A. (1958b). Experiments on uric acid synthesis by insect fat body. Archs int. Physiol. Biochem. 66, 282-286. Duchbteau, G., Florkin, M. and Frappez, G. (1940). Sur les ferments du catabolisme purique chez les insectes. C. r. SPanc. SOC.Biol. 133,436. Emmerich, H., Zahn, A. and Schmialek, P. (1965). Uber die Aktivitat einiger Dehydrogenasen und Transaminasen bei Tenebrio molitor unter den Einfluss von Farnesylmethylather. J. Insect Physiol. 11, 1161-1 168. Florkin, M. (1945). L’tvolution du mttabolisme des substances azotks chez les animaux. ActualitPs Biochim. No. 3, 66 pp. Florkin, M. and Duchbteau, G. (1941). Sur la distribution de la xanthine-oxidase. Bull. Acad. r. Belg. CI. Sci. 27, 114-178. Florkin, M. and Duchlteau, G. (1943). Les formes du systbme enzymatique de l’uricolyse et l’tvolution du catabolisme purique chez les animaux. Archs int. Physiol. Biochem. 53, 267. Garcia, I., Couerbe, J. and Roche, J. (1957). Sur le mttabolisme oxydatif de la 1-arginine chez les insectes. Activitts catalisique 1-amino-acide oxydasique. C. r. SPacne. SOC.Biol. 151, 1844-1847. Garcia, I., Couerbe, J. and Roche, J. (1958). Sur la prksence d’arginase chez les Sauterelles Schistocerca gregaria Forsk. et Eyprepocnemis plorans Charp. C. r. Skanc. SOC.Biol. 152, 1646-1649. Garcia, I., Roche, J. and Tixier, M. (1956a). Sur le mttabolisme de l’arginine chez les insectes. Bull. SOC.Chim. biol. 38, 1423-1433. Garcia, I., Roche, J. and Tixier, M. (1956b). Sur le mttabolisme hydrolytique de l’arginine chez les insectes et sa signification mttaboliques. C . r. Skanc. SOC. Biol. 150, 632-634. Gilmour, D. (1961). “Biochemistry of Insects”. Academic Press, New York and London. Gray, H. E. and Fraenkel, G. (1954). The carbohydrate components in honeydew. Physiol. 2061. 27, 56-65. Gupta, P. D. and Sinha, R. N. (1960). Excretion and its products in some storedgrain-infesting beetles. Ann. ent. SOC.Am. 53, 632-638. Harrington, J. S. (1956). Histamine and histidine in excreta of the blood-sucking bug, Rhodnius prolixus. Nature, Lond. 178, 268. Harrington, J. S. (1961). Studies of the amino acids of Rhodnius prolixus. 11. Analysis of the excretory material. Parasitology, 51, 3 19-326. Hayashi, Y.(1961). Studies on the xanthine oxidase system in the silkworm Bombyx mori L. I. Guanine-xanthine dehydrogenase system of the fat body of the silkworm larva. J. seric. Sci., Tokyo 30, 305-312. Hayashi, Y.(1962). On the properties of xanthine dehydrogenase of the silkworm Bombyx mori L. I. Isolation and components of the silkworm enzyme. J. seric. Sci., Tokyo 31, 25-31. Haydack, M. H. (1953). Influence of the protein level of the diet on the longevity of cockroaches. Ann. ent. SOC.Am. 46, 547-560. Heller, J. and Jezewska, M. M. (1959). The synthesis of uric acid in the Chinese Tussur moth (Antherea pernyi). Bull. Acad. pol. Sci. CI. ZZ Skr. Sci. biol. 7, 1-4. Hinton, T. (1955). Effect of arginine, ornithine and citrulline on the growth of Drosophila. Archs Biochem. Biophys. 62, 78. 3’

64

E. B U R S E L L

Hitchcock, F. A. and Haub, J. G. (1941). The interconversion of foodstuffs in the blow-fly (Phormia regina) during metamorphosis. Ann. ent. SOC.Am. 34, 17-25.

Hoskins, W. M. and Craig, R. (1935). Recent progress in insect physiology. Physiol. Rev. 15, 525-596. Hudson, B. W., Bartel, A. H. and Craig, R. (1959). Pteridines in the milkweed bug, Oncopheltus fasciatus (Dallas). 11. Quantitative determination of pteridine content of tissues during growth. J. Insect Physiol. 3, 63-73. Imms, A. D. (1948). “A general textbook of entomology”. Methuen, London. Irreverre, F. and Terzian, L. A. (1959). Nitrogen partition in excreta of three species of mosquitoes. Science, N. Y. 129, 1358-1359. Irzykiewicz, H. (1955). Xanthine oxidase of the clothes moth, Tineola bisselliella, and some other insects. Aust. J. biol. Sci., 8, 369-377. Ito, T. and Mukaiyama, F. (1964). Relationship between protein contents of diets and xanthine oxidase activity in the silkworm, Bombyx mori L. J. Insect Physiol. 10, 789-796. Keller, E. C. and Glassman, E. (1963). Xanthine dehydrogenase: differences in activity among Drosophila strains. Science, N. Y. 143, 40-41. Keller, E. C., Saverance, P. and Glassman, E. (1963). Paper electrophoresis of xanthine dehydrogenase from Drosophila. Nature, Lond. 198, 286-287. Kilby, B. A. (1963). The biochemistry of the insect fat body. Advan. Insect Physiol. 1, 112-174. Kilby, B. A. and Neville, E. (1957). Amino-acid metabolism in locust tissues. J. exp. Biol. 34,276-289. Lamb, K. P. (1959). Composition of the honeydew of the aphid Brevicoryne brussicae (L.)feeding on swedes (Brassica napobrassica DC). J. Insect Physiol. 3, 1-13. Leifert, H. (1935). Untersuchungen uber den Exkretstoffwechsel bei Eiern, Raupen und Puppen von Antherea pernyi. Zool. Jb. Abt. Allg. Zool. Physiol. 55, 131-190.

Lennox, F. G. (1 940). Distribution of ammonia in larvae of Lucilia cuprina Wied. Nature, Lond. 146, 268. Lennox, F. G. (1941). Studies of the physiology and toxicology of blowflies. 8. Rate of ammonia production by larvae of Lucilia cuprina and its distribution in this insect. 9. The enzymes responsible for ammonia production by larvae of Lucilia cuprina. Aust. Council Sci. Ind. Res. Pamphlet 109, 64 pp. Lisa, J. D. and Ludwig, D. (1959). Uricase, guanase and xanthine oxidase from the fat-body of the cockroach (Leucophaea madera). Ann. ent. SOC.Am. 52, 548-551.

Ludwig, D. (1954). Changes in the distribution of nitrogen in blood of the Japanese beetle, Popillia japonica Newman, during growth and reproduction. Physiol. Zoa”1. 27, 325-334. Maltais, J. B. and Auclair, J. L. (1952). Occurrence of amino acids in the honeydew of the crescent-marked lily aphid, Myzus circumflexus (Buck.). Can. J. ZOO^. 30, 191-193. Manunta, C. (1948). Sul metabolismo dell’azoto in Bombyx mori L. Afti Accad. naz. Lincei Rc. Sed. Solen. 4, 117-121. Manunta, C. (1949). Nitrogen metabolism in silkworms. Hereditus Suppl. 624.

T H E E X C R E T I O N OF N I T R O G E N I N I N S E C T S

65

McAllan, J. W. and Chefurka, W. (1961a). Some physiological aspects of glutamate-aspartate transamination in insects. Comp. Biochem. Physiol. 2, 290-299.

McAllan, J. W. and Chefurka, W. (1961b). Properties of transaminases and glutamic dehydrogenase in the cockroach Periplaneta americana. Comp. Biochem. Physiol. 3, 1-19.

McEnroe, W. D. and Forgash, A. J. (1957). The in viuo incorporation of C1*formate in the ureide groups of uric acid by Periplaneta americana (L.). Ann. ent. SOC.Am. 50,429-43 1.

McEnroe, W. D. and Forgash, A. J. (1958). Formate metabolism in the American cockroach, Periplaneta americana L. Ann. ent. SOC.Am. 51, 126-129. Mitlin, N., Vickers, D. H. and Hedin, P. A. (1964). End products of metabolism in the boll weevil, Anthonomus grandis Boheman: Non-protein amino acids in the faeces. J. Insect Physiol. 10,393-397. Mittler, T. E. (1958). Studies in the feeding and nutrition of Tuberolachnus salignus (Gmelin) (Homoptera, Aphidae) 11. The nitrogen and sugar composition of ingested phloem sap and excreted honeydew. J. exp. Biol. 35,7484. Murphy, M. R. V. and Micks, D. W. (1964). Transamination in Aedes aegypti. J. econ. Ent. 57, 12-14. Nation, J. L. (1963). Identification of xanthine in excreta of the greater wax moth, Galleria melonella (L.). J. Insect Physiol. 9, 195-200. Nation, J. L. and Patton, R. L. (1961). A study of nitrogen excretion in insects. J. Insect Physiol. 6, 299-308. Needham, J. (1 935). Problems of nitrogen catabolism in invertebrates. 11. Correlation between uricotelic metabolism and habitat in the phylum Mollusca. Biochem. J. 29, 238-251. Needham, J. (1950). “Biochemistry and morphogenesis.” Cambridge University Press, London. Nelson, W. A. (1958). Purine excretion by the sheep ked Melophagus ovinus L. Nature, Lond. 182, 115. Nelson, M. (1964). Some properties of uricase from the housefly, Musca domestica. Comp. Biochem. Physiol. 12,37-42. Parzen, S.D. and Fox, A. S. (1964). Purification of xanthine dehydrogenase from Drosophila melanogaster. Biochim. biophys. Acta 92,465. Payne, N. M. (1936). Excretory products of the mediterranean flour moth Ephestia kuhniella Zell. Anat. Rec. 67, 37. Pierre, L. L. (1964). Uricase activity of isolated symbionts and the aposymbiotic fat body of a cockroach. Nature, Lond. 201, 54-55. Poisson, R. and Razet, P. (1952). Recherches sur les urCides glyoxyliques et leur enzymes chez le phasme Carausius morosus Br. C. r. Sdanc. SOC.Biol. 234, 1804-1806.

Poisson, R. and Razet, P. (1953). Recherches sur la dkgradation de l’acide urique chez les Hemipttres aquatiques. C. r. SJanc. SOC.Biol. 237, 1362-1363. Porembska, Z. and Mochnacka, I. (1964). The ornithine cycle in Celerio euphorbiae. Acta Biochim. pol. 11, 109-117. Powning, R. F. (1953). Studies on the digestion of wool by insects. VIII. The significance of certain excretory products of the clothes moth, Tineola bisselliella and the carpet beetle Attagenus piceug Aust. J. biol. Sci, 6, 109-117.

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E. B U R S E L L

Prosser, C. L. (ed.) (1952). ‘‘Comparative animal physiology”. Saunders, Philadelphia. Rota, C. D. (1961). Enzymes in the hemolymph of the mealworm Tenebrio molitor L. J. N . Y. ent. SOC.69, 59-67. Razet, P. (1952). Catabolisme des purines chez les Collembole Xenyllu welchii Folsom (Insecte, Aptdrygote). C . r. Sdanc. SOC.Biol. 234, 25662568. Razet, P. (1953). Recherches sur la localisation des enzymes uricolytiques chez les insectes. C. r. Sdanc. SOC.Biol. 236, 1304-1306. Razet, P. (1954). Sur l’dlimination d’acide allantoique par quelques insectes Ldpidopteres. C. r. Sdanc. SOC.Biol. 239, 905-907. Razet, P. (1956). Sur l’dlimination simultanke d‘acide urique et d‘acide allantoique chez les insectes. C . r. Sianc. SOC.Biol. 243, 185-187. Razet, P. (1957). L’uricolyse chez les insectes. Arch. Orig. Serv. Docum. C.N.R.S., 361. Razet, P. (1961). Recherches sur l’uricolyse chez les insectes. These Doct. Sc. Nat., Imprimerie Bretonne, Rennes. Razet, P. (1964). Le probleme de l’acide urique chez les arthropodes antennates. 12th Znt. Congr. Ent. 220-221. Razet, P. (in the press). Les elements terminaux du catabolisme azote chez les insectes. Annke biol. Robinson, W. (1935). Allantoin, a constituent of maggot excretions, stimulates healing of chronic discharging wounds. J. Parasit., 21, 354-358. Robinson, W. and Baker, F. C. (1939). The enzyme urease and the occurrence of ammonia in maggot-infected wounds. J. Parasit. 25, 149-155. Robinson, W. and Wilson, G. S. (1939). Changes in the concentration of urease during pupal development of the blowfly Phormia regina. J. Parasit. 25, 455459. Rocco, M. L. (1936). Prdsence de l’allantoinase chez les insectes. C. r. Sdanc. SOC.Biol. 202, 1947-1948. ROCCO,M. L. (1938). Le mdtabolisme des composds d’origine purique chez les insectes. C . r. Sdanc. SOC.Biol. 207, 1006-1008. Roeder, K. D. (ed.) (1953). “Insect Physiology”. John Wiley and Sons, New York. Ross, D. J. (1959). Changes in the activity of uricase and xanthine oxidase during the life cycle of the Japanese beetle, Popillia japonica Newm. Physiol. Z00“l. 32, 239-245. Roth, L. M. and Dateo, G. P. (1964). Uric acid in the reproductive system of males of the cockroach Blatalla germanica. Science, N . Y. 146, 782-784. Roth, L. M. and Dateo, G. P. (1965). Uric acid storage and excretion by accessory sex glands of male cockroaches. J. Insect Physiol. 11, 1023-1030. Sedee, J. W. (1958). Dietetic requirements and intermediary protein metabolism of an insect (Calliphora erythrocephala Meig.). Entomologia exp. appl. 1, 38-40. Shaw, J. (1955). Ionic regulation and water balance in the aquatic larva of Sialis lutaria. J. exp. Biol. 32, 353-382. Smith, K. D., Ursprung, H. and Wright, T. R. F. (1963). Xanthine dehydrogenase in Drosophila: Detection of isozymes. Science, N. Y. 142, 226-227. Spiegler, P. E. (1962). Uric acid and urate storage in the larva of Chrysopu carnea Stephens (Neuroptera, Chrysopidae). J. Insect Physiol. 8, 127-1 32.

THE EXCRETION O F NITROGEN I N INSECTS

67

Srivastava, P. N. (1962). Physiology of excretion in the larva of Corcyra cephaIonica Stainton (Lepidoptera, Pyralidae). J. Insect Physiol. 8, 223-232. Srivastava, P. N. and Gupta, P. D. (1960). Excretion of uric acid in Periplaneta americana L. J. Insect Physiol. 6, 163-167. Staddon, B. W. (1955). The excretion and storage of ammonia by aquatic larvae of Sialis lutaria (Neuroptera). J. exp. Biol. 32, 84-94. Staddon, B. W. (1959). Nitrogen excretion in nymphs of Aeshna cyanea (Mull.) (Odonata, Anisoptera). J. exp. Biol. 36, 566-574. Stobbart, R. H. and Shaw, J. (1964). Salt and water balance: excretion. In “Physiology of Insecta”, (M. Rockstein, ed.) Academic Press, New York and London. Szarkowska, L. and Porembska, Z. (1959). Arginase in Celerio euphorbiae. Acta biochim. pol. 6,273-276. Terzian, L. A., Irreverre, F. and Stahler, N. (1957). A study of nitrogen patterns in the excreta and body tissues of adult Aedes aegypti. J. Insect Physiol. 1, 221-228. Tomita, M. and Kumon, T. (1936). Zur Chemie der Fliegen-larven. HoppeSeyler’s Z. physiol. Chem. 238, 101--104. Truszkowski, R. and Chajkinowna, S. (1935). Nitrogen metabolism of certain invertebrates. Biochem. J. 29, 2361-2365. Ursprung, H. and Hadorn, R. (1961). Xanthinedehydrogenase in Organen von Drosophila melanogaster. Experientia, 17, 230-23 1. Wagner, R. P. and Mitchell, H. K. (1948). Enzymic assay for studying the nutrition of Drosophila melanogaster. Archs Biochem. 17, 87-96. Waterhouse, D. F. (1952). Studies on the digestion of wool by insects. IV. Absorption and elimination of metals by lepidopterous larvae, with special reference to the clothes moth, Tineola bisselliella (Humm.). Aust. J. scient. Res. (B), 5, 143-168. Wigglesworth, V. B. (1931). The physiology of excretion in a blood-sucking insect, Rhodnius prolixus (Hemiptera, Reduviidae). J . exp. Biol. 8, 41 1-451. Wigglesworth, V. B. (1950). “Principles of Insect Physiology.” Methuen, London. Yoshitaka, N. and Aruga, H. (1950). Studies on the amino acids in the silkworm. IV. On the free amino acids in the silkworm faeces. J. seric. Sci., Tokyo 19, 536. Zandee, D. L., Nijkamp, H. J., Roosheroe, L., De Waart, J., Sedee, P. D. J. W. and Vonk, H. J. (1958). Transaminations in invertebrates. Archs int. Physiol. Biochim. 66, 220-227. Zielenska, Z. M. (1952). Studies on the biochemistry of Galleria melonella. Nitrogen metabolism of the larva. Acta Biol. exp. Vars. 16, 171-186.

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Lipid Metabolism and Function in Insects LAWRENCE I. GILBERT Department of Biological Sciences, Northwestern University, Evanston, Illinois, U.S.A. I. Introduction . A. General . B. Definition and classification 11. Lipid Content . A. Expression of data . B. Alterations during metamorphosis . C. Nature of insect lipids . III. Lipid Utilization . A. Digestion and absorption . B. Lipid release and transport C. Extra-digestive lipases . D. Fatty acid catabolism . IV. Lipid Biosynthesis . A. General mechanism of fatty acid synthesis B. Fatty acid biosynthesis in insects C. Phospholipid and triglyceride . D. Fatty acids in nutrition . E. Substrate interconversion . V. Hydrocarbons and Waxes A. Cuticle B. Extra-cuticular . VI. Isoprenoid Compounds . A. Nutritional studies . B. Isoprenoid biosynthesis . C. Isoprenoid content . D. Sterol modification . E. Function . F. Insect hormones VII. Conclusion . References .

.

.

. . . . . . .

.

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

70 70 71 71 71 81 89 97 97 102 110 116 127 127 130 134 145 147 152 152 155 157 157 161 168 170 175 176 186 187

In the course of this paper, the reader will encounter the following abbreviations: ADP-adenosine diphosphate; ATP-adenosine triphosphate; CoA or COASHcoenzyme A; Co@-coenzyme Q ; CTP4ytidine triphosphate; DGLAiglyceride; FAD-flavine adenine dinucleotide; FFA-free or unestersed fatty acids ; MGLmonoglyceride; NAD-nicotinamide adenine dinucleotide; NADP-nicotinamide PTC-phosphatidylcholine; adenine dinucleotide phosphate; PL-phospholipid; FTE-phosphatidylethanolamine ;TGL-triglyceride. 69

70

LAWRENCE I . GILBERT

I. INTRODUCTION A . GENERAL

The awarding of the Nobel prize in medicine to Bloch and Lynen for their contributions to the study of lipid metabolism is a heartening occurrence for many biologists and biochemists. This act pointed out once again the importance of the study of lipids, their synthesis and catabolism. It was in fact in this research area that the first definitive data were obtained on the dynamics of any protoplasmic constituents when Schoenheimer and Rittenberg (1936) studied fatty acid turnover utilizing heavy water. One prime impetus to the study of lipid biochemistry in insects in recent years has been the finding that many, if not all of the insect growth hormones, pheromones and sex attractants are lipoidal (cf. Gilbert, 1964). An understanding of the biosynthetic pathway and means of catabolizing these humoral and air-borne messengers necessitates a vigorous experimental approach to the fields of insect lipid chemistry and biochemistry. As will be discussed subsequently, lipids are also of vital importance to many insects as substrates for embryogenesis, metamorphosis and flight. And several members of this large category of biochemical compounds are without peers as vital nutritional growth factors. The purpose of this review is not so much to tabulate all knowledge of insect lipid biochemistry as to point out the gaps in our knowledge, in an attempt to attract others to this increasingly important research area. When this paper was begun, the last tome on insect lipids was more than a decade out of date (Scoggin and Tauber, 1950). In recent years however, several reviews on aspects of insect lipid chemistry and biochemistry have appeared (Clayton, 1964; Fast, 1964; Gilby, 1965 and Tietz-Devir, 1963),while other reviews that consider the metabolism of lipids along with other biochemicals have also been published (Chefurka, 1965; Gilbert and Schneiderman, 1961a; Gilmour, 1961; Kilby, 1963). Numerous papers on the biochemistry of lipids in microorganisms, fowl and mammals have appeared in the recent past and will be alluded to when considered important to the present discussion. It is of interest to note that several journals dealing exclusively with lipid biochemistry have appeared in the last eight years (Journal of Lipid Research, Lipid section of Biochimica Biophysica Acta, Steroids, as well as Advances in Lipid Research) but one has difficulty finding more than a dozen publications considering insect material in the cumulative

L I P I D METABOLISM A N D F U N C T I O N I N I N S E C T S

71

editions of these journals. It may be that as many reviews have appeared on this subject in recent years as breakthroughs in this research area. We shall concentrate on developments within the past ten years but will discuss older literature when applicable to the historical development of the topic or when it buttresses the argument. However, not all of the older literature will be discussed. Some aspects of the topic will be treated in a cursory manner, some omitted and others dwelled upon at length. The hierarchy of discussion is in part due to the author’s own interests and it is possible that topics not treated in depth (e.g. nutrition) are of more importance than those treated extensively. A final point worthy of note is that few generalizations can be made regarding all members of this largest animal class. In most cases, sophisticated experiments have been conducted on only a few species of “domesticated” insects and one cannot extrapolate the results to all insects. The multitude of different ecological niches and behavioural characteristics are no doubt reflected in a great number of metabolic variations on perhaps more than one basic theme. Notwithstanding the above, the past five years have brought us a clearer understanding of at least some aspects of the lipid biochemistry of insects. Before considering these advances it is of importance to agree on a vocabulary. B. DEFINITION A N D CLASSIFICATION

There has as yet been no rigorous definition of the term “lipid” that has been accepted categorically by all workers in the field. Generally, all compounds that are insoluble or only sparingly soluble in water and are extractable with, or soluble in, organic solvents such as diethyl ether, chloroform, acetone, etc., are termed lipids. For our present purposes, “lipids” will be defined as all compounds falling into the classification outlined in Table I, which is adapted and modified from similar tables by numerous authors, but notably Deuel (1951) and Strickland (1963). 11. LIPIDCONTENT A . EXPRESSION OF D A T A

One of the fundamental questions concerning the role of lipids in the physiology of the insect concerns the quantity of lipid contained upon, and interior to, the rigid exoskeleton. The exact amount will of course vary with physiological state, but also appears to vary according to the method of extraction and whether the resqlts are expressed in terms of dry weight, wet weight or lean dry weight. At first glance it

72

LAWRENCE I . GILBERT

would appear most desirable to express the quantity of lipid in terms of dry weight of the insect or tissue since this value can be obtained easily by drying the tissue to constant weight at about 100” and subsequently extracting the lipid. Unfortunately this procedure usually TABLE I Classification of lipids

A. Simple Lipids 1. Glycerol esters of fatty acids (mainly long-chain fatty acids; commonly called “neutral fat ”; includes monoglycerides, diglycerides and triglycerides) 2. Esters of long chain monohydric alcohols (commonly called “waxes”) 3. Sterol esters (sterol plus fatty acid) 4. Glycerol ethers (containing palmityl, stearyl or oleyl alcohols) B. Compound Lipids (esters of fatty acids and alcohol containing one or more additional groups) 1. Phosphoglycerides (commonly called phospholipids; esters of phosphate and free a hydroxyl of an a, 8, diglyceride or monoglyceride) (a) phosphate-monoester (phosphatidic acid) (b) phosphate-diester (e.g. phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine) (c) phosphate-triester (complexes of phosphatidylethanolamine or phosphatidylserine) 2. Sphingolipids (sphingosine containing N-fatty acyl group) (a) sphingomyelin (containing phosphorus and choline) (b) cerebrosides (containing sugar; usually galactose) (c) sulfatides (cerebroside plus sulfate residue) (d) gangliosides (commonly called mucolipid; contains sialic acid) C. Derived Lipids (products of A. and B. still possessing lipid characteristics) 1. Fatty acids 2. Long-chain alcohols 3. Sterols 4. Terpenes 5. Hydrocarbons 6. Sphingosine D. Complex Lipids 1. Lipoprotein 2. Proteolipid (lipid plus peptide) 3. Phosphatide peptide (contains sphingosine, inositol, phosphorus and nitrogen)

leads to profound changes in the nature of the lipid (e.g. oxidation) and the lipid becomes virtually useless for further analysis. It is therefore not unusual to express the lipid content in terms of fresh or wet weight. This procedure can lead to large errors due to the fluctuations in the

LIPID METABO LISM A N D F U N C T I O N I N I N S E C T S

73

water content of the insect. From our experience, lean dry weight is a more constant value and is obtained by extracting the tissue with organic solvents (ethanol-ether or chloroform-methanol) and weighing the air-dried residue. The lipid can then be treated gently (e.g. evaporation under nitrogen) and used further for identification of its individual components. The lean dry weight then is the dry weight minus the weight of the lipid. For reviews of lipid extraction procedures see Haahti (1961) and Horning (1964). As Table I1 demonstrates, there is a wide variation in lipid content of insects of different orders and even within a single family. This is not difEcult to understand when one considers the large diversity of insects, TABLE I1 Lipid content of various insect species ~~

Insect COLEOPTERA Bruchidae Pachymerus bactris Callosobruchus chinensis Laria irresecta Buprestidae Eurythyrea marginata Carabidae Pterostichus vulgaris Pterostichus nigrita Pseudophonus pubescens Harpalus pubescens Harpalus griseus Cebrionidae Cebrio gigas Cerambycidae Ergates faber Rhagium inquisitor Plagionotus arcuatus Cerambyx scopolii Chrysomelidae Leptinotarsa decemlineata ¶¶ ¶¶

,,

¶¶ ¶¶ ¶¶

Galerucella luteola Colaspidema atrum

Stage

Per cent lipid

41 D 33-2W 23.6W 12.2w

57w 4.8W 28*4D 4.8W 6.0W 144W 13.0W 7.0W 14.4W 8.5W 4.2W 3.4w 13.4W 39.6D 7.0W 3.9w continued

74

LAWRENCE I . GILBERT

Insect Aulacophora fumolaris Cistelidae Heliotaurus menticornis Curculionidae Balaninus elephas Rhynchophoruspalmarum Anthonomus grandis

,,

Dermestidae Dermestes sp

,,

9,

Dytiscidae Dytiscus marginalis Lampyridae Luciola vitticollis Meloidae Mylabris pustulata Lytta vesicatoria Scarabaeidae Oryctes nasicornis Melolontha hippocastani Melolontha vulgaris Cetonia aurata Phyllophaga rugosa Geotrupes stercoralis Popillia japonica 99

9, 99

9,

9,

,,

Tenebrionidae Tenebrio molitor 9,

,,

9,

3,

,,

,?

LEPIDOPTER A Arctiidae Arctia caja Estigmene acraea Bombycidae Bombyx mori ,¶

99

,, 9,

Stage

Per cent lipid 22

w

6 W 28 W 22.3W 12.8W 35 D 47 D 21 D 6.2W 7.3w 4.8W 12.5D 13-5D 15.0W 6.1W 16.9W 1.9W 9.0 7.7 4.0W 3.2W 3.8W 4.0W 3.8W 17.4W 154W 14.9W 12.9W

4.1W 13.OD 12 w 343W 24.4D continued

75

LIPID METABOLISM AND FUNCTION IN INSECTS

Insect Bombyx mori 99

Carposinidae Carposina niponensis

,,

,,

9)

3,

9,

,,

Citheroniidae Citheronia regalis Eacles imperiaris

27.6W

Gelechiidae Pectinophora gossypiella

,, ,, ,, ,,

99

,, ,,

9,

Hesperidae Acentrocneme hesperiaris Lasiocampidae Malacosoma franconicrim Malacosoma americanum

,,

99

Lithosiidae Asura conferta Lymantridae Lymantria dispar Euproctis chrysorrhoea (Linn.) Noctuidae Alabama argillacea Heliothis armigera Prodenia ornithogalli Laphygma frugiperda Lycophotia margaritosa Euxoa segetum Agrotis segetum 99

¶3

9)

9)

27*1W 85.4D 35-7w 81.6D 17.4W 55W 6*0W

Cossidae Cossus ligniperda Danaidae Danaus plexippus

99

Per cent lipid 5.9w 9.1W

99

99

Stage

Laphygma exempta solitary

23 W 12 w 15.3W 44.8D 16.7W 50.5D 16-1W 33*4D 10

w

1.9w 82 D 24.7D 1.ow 1.5w 4-9w 24.4D 20.9D 18.1D 13.1D 0.9W 6.4W 23.1D 30.1D 13.5W 15W continued

76

LAWRENCE I . GILBERT

Insect Laphygma exempta gregarious Spodoptera abyssinia solitary Spodoptera abyssinia gregarious Spodoptera abyssinia solitary Spodoptera abyssinia gregarious Acronycta rumicis Colocasia coryli Daseochaeta at'pium Scoliopteryx libatrix PIusia gamma Notodontidae Pha lera bucephala Nymphalidae Vanessa urticae Olethreutidae Carpocapsa pomenella Papilionidae Papilio troilus Papilio turnus Papilio zolicaon Pieridae Pieris brassicae 99

>¶

Pieris napi 9)

Y9

Pieris rapae 9,

99

,,

,,

9,

99

9,

$9

,I

Y9

Psychidae Thyridopteryx sp. Pyralidae Galleria mellonella 99

>,

Myelobia smerintha Ephestia figulilella Loxostege similalis Loxostege sticticalis

Stage

Per cent lipid 2.7W 3.1W 4.9w 7.1 W 9.3w 25.5D 29.5D 12.5D 18.2W 334W 34.8W 10.3W 3.7w 44.2D 3-3w 4.6W 7.6W 10.8W 1.9w 6.0W 2.2w 3.6W 16.0d 7.7D 2.3W 12.1D 6.4W 26.1D 11.4W 29.7D 5-6W 9.1w 56-2D 21-0w 22 w 21-6W 18.8D 29.7D

continued

LIPID METABOLISM A N D FUNCTION I N INSECTS

Insect Loxostege sticticalis

,,

Y9

,,

9)

,,

9,

Pyrausta nubilalis 3,

YY

99

9,

Stage

77

Per cent lipid 5-9w 7.5w 30.2D 96W 3.2W 26.5W 9-7w 143W

Saturniidae Saturnia pyri Antheraea pernyi Y9

9,

Y9

,,

*Antheraea mylitta *Antheraea polyphemus *Antheraea roylei *Hyalophora cecropia *Hyalophora euryalus *Actias luna Adelocephala heiligbrodtii *Attacus atlas

*Attacus canningii *Samia Cynthia Automeris io

* Callosamia promethea * Calosaturnia mendocino *Rothschildiaforbesi

*Rothschildia orizaba

4.0W 1.ow 4*6W 14.8D 17.8D 4-OW 6.0W 21.6W 6-1W 38.3W 5.ow 216W 4-2W 22.8W 4.4w 17.6W 1*9W 2.8W 9-9w 41.3W 8.3W 40-OW 8.0W 36-7W 9.8W 18.2W 2.9W 13.4W 2.9W 22.8W 294W 3.7w 32.2W

Sphingidae Deilephila sp. Sphinx elpenor

35w 4.9w continued

78

LAWRENCE I . GILBERT

Insect

Stage

Sphinx ebenor Sphinx ligustri

,, 39

6-1W 7.8W 3.8W 6.1W 2*8W

9,

9,

Sphinx euphorbiae Thaumetopoeidae Thaumetopoea pityocampa

35w

HEMIPTERA Aphididae Aphis rumicis Aphis rosae Aphis fabae Hyalopterus pruni Coccidae Coccus cacti Eriosomatidae Pemphigus utricularias Jassidae Euttetix tenellus

7.5 w 6.5W 12 w 6.2W 8 W

20

9,

,,

9,

36.3D 11-6W 5-2W 6-2W 6-2W 32.7D

ORTHOPTERA Acrididae Acrida bicolor Anacridium aegyptium Orthocanthacris aegyptium Dociostaurus maroccanus Oxya japonica Locusta migratoria 9s

,,

Locusta migratoria solitaria

iAi ,, Locusta migratoria migratorioides ( A ) (A) Locusta migratoria solitaria (N) 9)

99

99

99

w

34.2D 11.5D

Lygaeidae Blissus leucopterus Oncopeltus fasciatus Notonectidae Notonecta glauca Pyrrhocoridae Pyrrhocoris apterus 99

Per cent lipid

,

9,

I

3.2W 3-8W 2.6W 3.3w 3 w 24W 2.8W 2.8W ll*ID 3-6W 14.0D 7.5D continued

79

L I P I D METABOLISM A N D F U N C T I O N I N INSECTS

Insect

Stage

Locusta migratoria migratorioides

(N) ?(A) $.(A) (A) (A) (A) (A) (A)

,>

99

Locusta pardalina solitary Locusta pardalina solitary Locusta pardalina gregarious 9,

,,

9,

Per cent lipid 10-7D 10.4W 147W 2.9W 12.8D 3.7w 14-6D 10 w 5.3D 9.3D 3.8W 3-2W 12 w 3.5w 25w

Schistocerca gregaria Schistocerca gregaria solitary (N) Schistocerca gregaria gregarious (N) Pezotettix giornae ?(A) Melanoplus atlanis (A) Melanoplus differentialis (E) 99 (A) Sphenarium purpurescens (A) Bacunculidae Dixippus morosus (A) Blattidae BIatta orientalis (A) ,* (A) BlattelIa germanica (N) ,, ?(A) d(A) (A> Periplaneta americana ?(A) $.(A) (N) ?(A) $.(A) Tettigoniidae Tettigonia uiridisima (A) 9,

2*4W 4.3w 3.2W 5.7w 4.8W 1-7W 17.1D 13.9D 14.5D 7.7w 8.9W 7-1W

9,

9

9,

9,

9,

73

3.8W

ODONATA Aeschnidae Aeschna sp. ISOPTERA (unspecified)

25w

(N)

6.1W

(L)

5-6W

(L) ?(A) $.(A)

30-OD 3.3w 34w

DIPTERA Anthomyiidae Pegomyia ulmaria Calliphoridae Calliphora erythrocephala

,,

')

continued

80

LAWRENCE I . GILBERT

Insect Calliphora erythrocephala Calliphora vomitoria Lucilia sericata

,.

9,

99

9,

,,

99

Phormia terraenovae Phormia regina 99

3,

9,

,,

Chironomidae Chironomus sp. Tanytarsus lewisi Culicidae Culex pipiens 3,

9,

Y9

99

Anopheles atroparuus Anopheles stephensi Anopheles gambiae

Glossinidae Glossina palpalis Glossina morsitans

Muscidae Musca domestica

Oestridae Gastrophilus intestinalis Syrphidae Eristalis tenax

Stage

Per cent lipid 12.7D 7.8D 19 D 31 D 27 D 20 D 12.2w 8.9W 6.8W 52W 8.3W 11.7D 27.9W 3-7w 32*7D 4.0W 5-9w 57w 8.4W 15-OD 9.8D 22.7D 21.9D 16.8D 352? 2.7W 7.1W 6-3W 4.2W 2.5W 4.9w 4.2W 2.6W 2*9W

5w 1.8W continued

LIPID METABOLISM A N D F U N C T I O N I N INSECTS

Insect

Stage

81

Per cent lipid

HYMENOPTERA Apidae Apis dorsata Apis melrifera workers

,, ,, ,, 9,

,9

,, ,9

,,

9,

,, ,, ,, ,, ,, ,, ,, ,,

7,

),

,, 77

,, 9,

Y,

,, 9,

,, drones 79

freshly hatched wax producing forager winter worker Queen worker Queen summer winter

Formicidae Camponotus vagus Cremastogaster scuiellaris Tenthredinidae Croesus septentrionalis Vespidae Vespa cincta

(N) (P) (A) (PI (A) (A) (A) (A) (A) (L) (L) (P) (P) (A) (A)

4.7w 4.1W 0.9w 6.1W 1.5W 9.4D 10.2D 95D 7.3D 3-7w 4.9w 3.7w 5-3w 13.OD 12*2D

(L)

(A)

2.4W 10.9W

(PP)

26.OD

(I-)

67W

(E)egg; (L)larva; (N)nymph; (PP)prepupa; (P)pupa; (A) adult; (W) wet weight; (D) dry weight; * abdomens only. Modified from review by Fast (1964); consult Fast (1964) for original references and other data.

their modes of living and varying habitats. In most insects the female contains more lipid than the male, as lipid is a most efficient substrate for egg development. However, the reverse may be true for many species and this is especially evident when we consider the Lepidoptera (Table 11). This must be borne in mind since most of the author's research has utilized the American silkmoth Hyalophora cecropia, and this insect w ill be used to exemplify many points in this review. B . ALTERATIONS D U R I N G METAMORPHOSIS

In the course of a study on the alterations in juvenile hormone content during the life cycle of H.cecropia, we noted a sexual dimorphism in lipid content in the adult stage, and conducted a number of experiments to determine its basis (Gilbert and Schneiderman, 1961b). By preparing lipid extracts of insects at various stages in the life cycle the observations in Table I11 were made. Lipid content per individual larva

00 h)

TABLE I11 Lipid content of H. cecropia from egg to adult emergence

Stage

Number of animals

unfertilized eggs embryos 1st instar larvae 2nd instar larvae 3rd instar larvae 4th instar larvae 4th instar larvae 5th instar larvae 5th instar larvae 5th instar larvae prepupae Diapausing pupae 3 Diapausing pupae 9 Chilled pupae d Chilled pupae d Chilled pupae 0 Chilled pupae 0 Developing adult 3

750 1000 200 20 17 6 6 1 4 3 3 20 20 20 2 20 2 4

Time sacrificed freshly laid 7 clays

Fresh weight (€9

Lipid Lipid Dry Percent Grams as % as % weight dry lipid: fresh dry (g) weight individual weight weight

3.80 4.30

0.00025 0.00019

4.94 4.30

0.00012 0.00109 0.00922 0.0204 0-0416 0-0460 0-1189 0.2538 0-3723 0.2890 0.3150 0.3970 0.27 18 0.2435 0.2135 0.3100

4-53 2.27 0-98 1.34 1-28 1.40 1.63 1.86 5-68 7-34 5.57 8.68 6.44 5.43 4-24 8.67

0.516 newly hatched 0.957 early 15.879 late 9.122 early 19.463 late 3.285 early 29.233 mid 40-976 late shortened body 19.6648 1 month old 78.8 113.1 1 month old 6O, 6 months 9 1-50 6O, 6 months 8.439 6O, 6 months 91.50 6O, 6 months 10.067 day 2 14.30

0.125 0-131 2.316 1~207 2-300 0.334 5.141 6-690 5.361

24.22 13-69 14.59 13-23 11.82 10.17 17.59 16-33 27.26

-

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2.209

26.19

2-485

24.69

-

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Comment

includes chorion includes chorion and yolk 18.72 whole animal 16.56 whole animal 6.77 whole animal 10.13 whole animal 10.85 whole animal 13.77 whole animal 9-25 whole animal 11.38 whole animal 20.84 whole animal - whole animal whole animal whole animal 24-59 whole animal whole animal 17.18 whole animal - abdomens only and pupal cuticle

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LIPID METABOLISM A N D FUNCTION I N INSECTS

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84

LAWRENCE I . GILBERT

was observed to increase with age and size as expected, but the rate of increase varied in the various instars when compared to the net increase in non-lipid constituents. The newly hatched larva contains an appreciable amount of lipid, but this percentage decreases to a more or less constant value in the fourth and fifth instars (1-3-1.9% fresh weight; 9-3 to 13.8% dry weight), although the lipid content increases as the larva grows. The prepupa loses water rapidly and lipid content increases, so that at pupation lipids constitute between 5 and 7% of the wet weight of the insect. At this time a sexual dimorphism in lipid content is evident; the male pupa contains 50% more lipid than the female per gram fresh weight. This dimorphism also exists in pupae chilled for 6 months at 6". During the pupal-adult transformation this dimorphism increases markedly until in the adult moth the tissues of males contain about 5 times as much lipid per gram fresh weight as the tissues of the female. During adult development, the female apparently utilizes far more lipid than the male (see also Domroese and Gilbert, 1964). A 5 g previously chilled male pupa contains about 360 mg of lipid while the newly emerged adult moth contains approximately the same amount. For the female, the picture is quite different. A similar 5 g female pupa contains about 230 mg of lipid. The correspondingnewly emerged adult with eggs contains about 185 mg. In short, during adult development females appear to use more lipid than males. Because the abdominal tissues of female moths have a greater water content than those of males, the sexual dimorphism in lipid content is not as impressive when calculated on a dry weight basis. The results of a typical series of extractions reveal that during the first week of adult life the abdomens of males contain from 51-79% lipid per gram dry weight while the abdomens of females contain from 19-25%. After the sixth day of adult life there appears to be a rapid utilization of lipid in the males indicating a significant increase in catabolism at this time. The same is true of female moths, with the most rapid utilization occurring after the tenth day of adult life: senile females may contain as little as 8% lipid per gram dry weight as contrasted with about 20% in young females. To determine the extent of the lipid metabolism associated with egg development during adult development, a series of female pupae were , castrated and allowed to develop. Two days after eclosion the moths were extracted and the lipid content determined. The abdomens of castrated females contained about twice as much lipid as normal eggless female abdomens but still less than as much as male abdomens (less

+

LIPID METABOLISM A N D FUNCTION I N INSECTS

85

than 3% on a fresh weight basis). Thus, although egg development involves some utilization of lipids, it cannot by itself account for the sexual difference in lipid content between males and females. It also appears that during adult development both normal and castrated females utilize far more lipid than males. Castration of males had no effect on their lipid content. When ovaries were implanted into male pupae, the adults which emerged contained large masses of eggs. Extraction revealed that these egg-filled male abdomens contained less lipid than normal abdomens but twice the amount of those of castrated or normal females. This result implies that egg development involves lipid catabolism but that certain non-ovarian tissues of the developing female utilize more lipid than corresponding male tissues. Parabiotic union of male and female pupae resulted in parabiotic moths. This technique resulted in the elevation of the lipid content of the female but a normal content in the male. What conclusions can we draw from this data? As was expected, the lipid content of individual larvae increased with size and age reflecting the synthesis of lipid from leaf material. Since no attempt was made to sex the larvae, nothing can be said regarding any sexual dimorphism in lipid content at this stage. The 50% decrease in lipid content per individual between egg and first instar larva indicates lipid utilization during embryogenesis. Although we shall return to this point later, this does confirm observations made on insects of other orders (Rothstein, 1952)and on B. mori (Niemierko et al., 1956), although the lipid content is much lower in H. cecropia eggs (as per cent fresh weight) than in the diapausing egg of Bombyx. This could be due to both the greater weight of the chorion of the Cecropia egg and to greater substrate storage in the diapause egg which supports a living embryo over winter. In the fifth instar larva when the water content reaches the extraordinarily high level of about 90% of the total weight, the animal begins to synthesize lipid at a high rate. The fifth instar larva has about five times as much total lipid as the early fifth and a lower water content. During spinning, when a large quantity of protein is used for constructing the cocoon, there is a further increase in total lipid and a loss of water. Whether this large increase in lipid during spinning reflects a release of lipids firmly bound to the proteins used in construction of the cocoon or whether the increase is a result of enhanced lipid synthesis has not been definitely answered. The fact that male pupae and adults contain more lipid than females seems peculiar to Lepidoptera. In most insects, the female usually contains more lipid (Table 11). Our results are consistent with the findings of

86

L A W R E N C E I . GILBERT

Niemierko et al., (1956; see also Vaney and Maignon, 1906; TimonDavid, 1930; Yamafuji, 1937) in Bombyx mori, and Demyanovsky and Zubova (1957) in Antheraea pernyi. Niemierko and his colleagues found that the lipid content of newly emerged male Bombyx was 48% per gram dry weight while that of the female was only 25% (17.5% in males and 6% in females on a fresh weight basis). The females used 50% of their stored lipid between spinning and adult emergence while the males utilized only 30%. In A . pernyi, Demyanovsky and Zubova found the sexual dimorphism in lipid content evident as early as the fifth instar larva and it continued until adult emergence. Our results (Gilbert and Schneiderman, 1961b) extend this dimorphism to a total of seven families of Lepidoptera. The above results indicate that lipid is used during the pupal-adult transformation of female Lepidoptera but that if any lipid is used for energy to underwrite the many syntheses occurring during construction of the male adult, other substrates are presumably being converted to lipid. Biochemical systems that accomplish such conversions have already been established (see Section IV). Why is there a lipid-sparing system operating during adult development of male Cecropia? The higher concentration of lipid in the male moth is most likely correlated with mating behaviour, since the male moth flies relatively great distances in search of the virgin female. As we shall see subsequently (Section 111) the male appears to utilize lipid as the primary substrate for flight. The monarch butterfly is a continental migrant and also utilizes lipid during flight. Prior to migration this specieslays down a store of fat as do migratory birds (Beall, 1948). Since its only food is nectar which does not contain lipid, it evidently converts carbohydrates to lipid during periods of repose and then uses it during flight. In migratory moths, it appears that there is no sexual dimorphism in lipid content (Williams, 1945) since both sexes migrate and presumably use equal amounts of lipid. The fact that the female Cecropia moth utilizes a significant portion of her pupal lipid during adult development and contains much less than the male, can be explained in part by her adult role. First, the female converts a large percentage of her endogenous substrate into the egg (yolk, cytoplasm, chorion, etc.). Secondly, the female does only limited flying after emergence and therefore needs little flight fuel. A by-product of the female’s lipid utilization during adult development is metabolic water, which is reflected in the greater water content of the female as compared to the male. During adult life, lipid appears to be the favored substrate of both

L I P I D METABOLISM A N D F U N C T I O N I N INSECTS

87

sexes. The utilization of lipid at this stage when the animal has left its desiccation-proofcocoon and heavy pupal cuticle, provides these moths with enough metabolic water to offset desiccation. It is possible that the male converts protein to lipid during the first week of adult life when the lipid content drops only slightly, since there is very little carbohydrate present at this time (Domroese and Gilbert, 1964). Perhaps these non-feeding adults ultimately die of lipid depletion. What is the basis of the sexual dimorphism in lipid content in these Lepidoptera? Our data reveal that egg development can explain in part

0.72

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FIG. 1. The respiratory quotient of male H. cecropia during adult development and adult life. Determinations were made on five animals, each designated by a different symbol. (From Domroese and Gilbert, 1964.)

the low lipid content of female moths. However, the surgical operations performed never reversed or even equalized the lipid differences between the sexes. Since this dimorphism exists in the pupal and even in the larval stages (Demyanovsky and Zubova, 1957) we believe it to be a genetic difference which may find its first phenotypic expression during embryonic life. The above experimental findings were based almost entirely on direct lipid extraction of animals at different stages of development. To explore this sexual dimorphism further, we (Domroese and Gilbert, 1964) determined the respiratory quotients (RQ) of both sexes of H . cecropia during pupal-adult development. The results of these determinations show a differencein pattern between males and females (Figs. 1,2). In the female, kA.1.P.

4

88

LAWRENCE I . GILBERT

the RQ decreases from higher values early in development to values between 0.75-0.80 shortly after the first week of development. The RQ remains at this characteristic level throughout the rest of development and in adult life. In terms of the role of lipid as an energy source for development, the RQ pattern suggests that after the first week of development lipid provides a greater portion of the energy expended in the female. In the male, the RQ is maintained for the most part at a level well above 0.80 until the seventeenth day of development, at which time it falls to levels indicative of greater lipid utilization. The RQ persists at this lower 1.00

8

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FIG.2. The respiratory quotient of female H. cecropia during adult development and adult life. Determinations were made on five animals, each designated by a different symbol. (From Domroese and Gilbert, 1964.)

value during the remainder of adult development and in adult life. This drop in RQ characteristically occurred simultaneously with the appearance of the large dark pigmented wing spot on the seventeenth day of development. This RQ pattern as well as the data previously cited indicates that the male uses more of the non-lipid substrates for energy during adult development with a conservation of the lipid stores for use during adult life. In Section 111, we will discuss in detail the role of lipid in the adult life of the male moth. Among the various deductions that can be made regarding the above/ one fact is quite clear. The amount of lipid varies considerably during the life of an insect as well as between various species. The difference in the quantity of lipid expressed as per cent fresh weight has been reported to

L I P I D METABOLISM A N D F U N C T I O N I N INSECTS

89

vary from 1% in some Lepidoptera to 50% in the beetle Puchyrnerus (Niemierko, 1959). Examples of the high lipid content of some insects can be seen in the older work of Maciuca (1935) who showed that in the fall, Pyrrhocoris upterus contains 32.7% lipid (dry weight) while Fulton and Chamberlain (1934) noted that the lipid content of E. tenellus may rise as high as 42.2% of the dry weight of the female. Sinoda and Kurata (1932) showed that ether extracts of Dermestes larvae account for as much as 47% of the dry weight of the insect. As Scoggin and Tauber (1950) point out, many factors influence the lipid content of insects including stage of development, nutrition, environmental temperature, sex, starvation, diapause, cold hardiness, whether migratory or not and finally the systematic position of the organism under study. Rather than pursuing the most laborious task of discussing the lipid content of diverse insect species, let us now turn to the qualitative nature of insect lipids. C . NATURE OF INSECT LIPIDS

1. Glycerides In general, almost all of the compounds listed in Table I have been identified in one insect or another. These will be discussed in some detail throughout the paper, but there is no doubt that the major lipid component in insects as in other animals, is triglyceride (TGL) : the glycerol esters of long chain fatty acids. We can think of triglycerides as a secondary energy source since they must be hydrolyzed before energy can be made available to the cell. This results when the individual fatty acids cleaved from the glycerol moiety undergo B-oxidation within the mitochondria (see Section 111). In general, these TGL form the greater part of the lipid content of the insect at all developmental stages (Fig. 3). In Muciosiphium burri, about 80% of the extractable lipid is TGL and less than 3% exists as unesterilied fatty acid (FFA) (Strong, 1963a, b). This predominance of neutral lipid has been demonstrated in many insects, both in total body extracts (cf. Fast, 1964) and isolated fat body (Chino and Gilbert, 1964, 1965a; see also reviews of Kilby, 1963 and Gilby, 1965). Large quantities of FFA have been reported in the older literature (cf. Scoggin and Tauber, 19Sl) but in many cases one can “ see” the esterified lipids hydrolyzing before one’s eyes while reading the methods sections of these papers (see, however, Albrecht, 1961). In a study of the lipid of adult Anthonornus grundis by silicic acid chromatography, Lambremont et ul. (1964) showed that newly emerged

90

LAWRENCE I . G I L B E R T

adult boll weevils had 2 4 % body lipid of which only 2% was triglyceride. After 2-3 weeks of feeding, non-diapausing adults contained 6 1 0 % lipid (40-60% of which is TGL), whereas diapausing adults had 18-25% body lipid containing 75-85% TGL. It thus appears that lipid serves as the energy source for this diapausing insect which must last over winter, a suggestion made earlier by Braze1 and Newsom (1959).

Tube no.

FIG.3. Column chromatography of lipids from male H. cecropia moths. The column support was Florisil and elution was according to Chino and Gilbert (1965a). Tubes 12 to 22 represent triglycerides. (From Domroese and Gilbert, unpublished observations.)

Lambremont and his colleagues also demonstrated that the major fatty acids in the boll weevil were oleic and palmitic acids, and that the diet of the adult was the determining factor controlling the type of fatty acids incorporated into TGL.

2. Fatty acids (a) General characteristics. Protoplasmicfatty acids usually vary from C2to C,,in chain length and are most commonly composed of an even number of carbons, with oleic acid being the most abundant in nature. Of the many unsaturated fatty acids, the majority have a double bond between Cgand Clo (Fig. 4). Isomers of any particular fatty acid differ from one another by the position of the double bond, cis-trans isomerism, or both. There is a large variety of fatty acid types including

LIPID METABOLISM A N D FUNCTION I N INSECTS

91

branched, unsaturated (up to six double bonds), hydroxy saturated or unsaturated, and cyclic. Saturated fatty acids having ten or less carbon atoms are liquid at room temperature and are thus easier to handle in experimental situations although more prone to auto-oxidation. All fatty acids with the exception of acetic acid have a density of less than one. The most common form for fatty acids in living things is as constituents of glycerides which are usually mixed (i.e. contain more than one variety of fatty acid per molecule). Although we speak of unesterified fatty acids as free fatty acids, in the cell or in the hemolymph they are usually in the form of the cationic salt (e.g. sodium palmitate) and can be referred to as palmitate or oleate rather than palmitic acid or oleic acid. 0

1I

CH3(CH2)7CHaCH2(CH&C--OH I. Stearic acid

0

It

CH~(CH&CHL-CN(CH~)~C-OH 11. Oleic acid (AS:l0) 0

CH3(CHa)rCHSHCHaCH=CH(CH& !! -OH 111. Linoleic acid (Aa:loJ2:13 )

i

CH3CH2CHSHCHaCH=CHCH2CH=CH(CH2)7 -OH IV. Linolenic acid (A9:10.12:13.15:16 1

FIG.4. The structure of some important C,, fatty acids.

(b) Fatty acid composition. The fatty acid composition of numerous insect species has been reviewed by Fast (1S64), and only the more recent work will be discussed here. The older literature suffers from a lack of sophisticated analytical tools (i.e. gas chromatography) and in many cases the unsaturated fatty acids were handled without respect, undoubtedly leading to oxidative deterioration in many cases. In a study of the lipids of homopterous insects, Strong (1963a) determined the fatty acid composition of 21 species of aphids and 6 species of leafhoppers. The percentage of the various fatty acids varied quite a bit between groups and even between species of a single group. In general, the aphids had a relatively large percentage of C14saturated fatty acid while the leafhoppers had a predominance of C18(especially

92

L A W R E N C E I . GILBERT

C,,,,) fatty acid. Strong concludes that “the fatty acid composition of an aphid appears to be a species characteristic not especially influenced by the host plant.” Honeydew secreted by Myzus persicae contains for the most part free sterols and fatty acids and appears to originate from the phloem sap (Strong, 1964). It seems from Strong’s work that the fatty acid composition of a species is rather specific and if this is true, it may be that gas chromatography is the “ultimate weapon” for the taxonomist. In examining the fatty acid content of the boll weevil, Lambremont and Blum (1963) identified 23 fatty acids varying in chain length from six to twenty carbons. Of these, 8 fatty acids (myristate, palmitate, palmitoleate, heptadecanoate, stearate, oleate, linoleate, linolenate) constitute 98% of the total, with 62% of the fatty acids containing at least one point of unsaturation. Palmitate makes up about 31% of the total while oleate constitutes about 30% of the total. Of the eight predominant fatty acids in the boll weevil, all but palmitoleate, heptadecanoate and stearate were identified in Drosophila melanogaster (Anders, 1960). It is possible that Anders’ paper chromatographic system was too insensitive for detection of the other three. Fawzi et al. (1961) have also shown that palmitate and oleate are the most common fatty acids in the TGL of Locusta migratoria. Almost all of the studies on fatty acids in insects utilized extraction of whole insects and generally palmitate and oleate predominate. The situation in the arachnids seems to be similar (Blum et al., 1963). Other more exotic fatty acids have been identified in insects and insect products, but are the exception rather than the rule. In bees for example, hydroxy fatty acids are common in queen bee larvae and royal jelly. Royal jelly is of course the nutrient fed to young larvae causing them to develop into queens. In 1957, Butenandt and Rembold identified 10-(OH)-decenoic acid in royal jelly. Weaver and Law (1960) studied the heterogeneity of fatty acids in this most interesting nutrient and showed the presence of: decandioic acid; monohydroxymonocarboxylic acids ; dihydroxymonocarboxylic acids. Weaver et al. (1964) reported that 9-13% of the dry weight of royal jelly is lipid of which 90% is FFA. Pain and her colleagues (1962) had demonstrated previously that royal jelly contains adipic, pimelic and suberic acids. Brown and his group (Brown and Felauer, 1961; Brown et al., 1961) showed the presence of 9-(OH)-dec-Zenoic acid, lO-(OH)-dec-2-enoic acid, 10-(OH)-decanoic acid, dec-Zendioic acid, p-hydroxybenzoic acid and sebacic acid. The presence of these acids is now known but their function is still a matter of speculation. They may have an anti-

LIPID METABOLISM A N D FUNCTION I N INSECTS

93

bacterial action (cf. Slepecky and Gilbert, 1962) or may have morphogenetic effects in inducing those larvae fed on royal jelly to develop into queens. Several of these fatty acids and their derivatives have been identified in the products of hydrolysis of queen bee larval lipid (Pain et al., 1962). Identified by the French group in larval lipids were myristate, palmitate, dec-Zenedioic acid, suberic acid and trans-10(OH)-dec-Zenoic acid. This latter acid accounted for about 20% of the total fatty acids present but its function is unknown. The wax of Apis mellifera was thought to be composed largely of fatty acids and alcohols containing the even numbered homologues of CZ4to C34fatty acids (Chibnall et al., 1934) but the use of gas chromatography has revealed the presence of some odd numbered fatty acids as well (Downing et al., 1961). The cuticular wax of B. mori is composed for the most part of fatty acid-alcohol esters with fatty acid chain lengths of from c16 to C,, (Amin, 1960; Bergmann, 1934; Shikata, 1960). The most complete survey by one individual of the fatty acid constitution of insects is that of Barlow (1964), who by gas chromatography analysed 30 species of insects (Table IV). Unfortunately, only one analysis was conducted on most of the species due to lack of material. Barlow’s findings indicate that the fatty composition is characteristic of the species. For the family aphidoidea (Homoptera) for instance, more than SO% of the fatty acid was saturated C14 whereas in most species outside of this group, this fatty acid only constituted about 15% (19 to 60%) of the total. All the Diptera had a high proportion of while most other species yielded no more than about 2.2% of this acid. That some insects can synthesize this c16:1is no longer in doubt since A . aflnis contained a high concentration of this fatty acid even when grown on a diet devoid of it. In general, Barlow’s work suggested that the stage of the insect had little effect on the fatty acid composition (see, however, the subsequent discussion of the work of Herodek and Farkas). [The new rash of papers on fatty acid composition of insects (and other living things) is due in great part to the classic efforts of Martin and Synge (1941) who first described the concept of gas chromatography in which gas is the transient phase rather than a liquid (as in paper or column chromatography).] A relationship appears to exist between the temperature to which a tissue is constantly exposed and the degree of unsaturation of the fatty acids present in the tissue. This appears to be true of all animals and has been demonstrated in insects. Newly emerged female Culex tarsalis for example possess a greater percentage of unsaturated fatty acids

TABLE IV Total fatty acid composition of some insect lipid

% fatty acid1 Stage

Insect Coleoptera Calosoma calidum (Fabricius) Harpalus caliginosus (Fabricius) Sitona scissifrons (Say) Leptinotarsa decemlineata (Say) Trirhabda virgata Lec. Pyropyga decipiens Harris Tetraopes tetraophthalmus (Forster) Neuroptera Corydalus cornictus (Linnaeus) Trichoptera Unidentified Homoptera-Aphididae Dactynotus ambrosiae (Thomas) S.L. Aphis pomi de Gier Acyrthosiphon pisum (Harris) Tuberolachnus salignus (Gmelin) Pemphigus populicaulis (Fitch) Homoptera-o ther Cicadidae unidentified Campylenchia latipes (Say) Stictocephala diceros (Say) Philaenus spumarius (Linnaeus)

12

14

16:O

16:l

18 13 10 11 9 11 8

5

2 3 1 1 1 2

adult adult adult Pupa adult adult adult

<1

larva

21

10

22

11

larva

1

1

46

10

nymphs and adults nymphs and adults nymphs and adults nymphs and adults nymphs and adults

1 1 9 4 3

9 9 4 3 3 5 5 7 8 5 3 2

3 4 3 4

adult adult adult adult

-

1 10 25 3

2 2 1 1 1 2 2 1

1 1 99+1 1 12 1

5

-

3 3 3 1 1

18:O 18:l 2 2 10 10 13 10 1 4

30 23

18:2 18:3

25 16 42 6

21 58 12 32 25 9 16

6

14

8

5

35

1

50

21

-

14 19 25

2

12

c1

8

7

-

-

5 3 2 1 1 3 5 4 4

4

25

12 16 4 11 63

-

-

-

-

-

14 11

1

2

1

4

15 48

66 23

?

M

Diptera Agria afinis (FallCn) Musca domestica (Linnaeus) Drosophila melanogaster Meigen Aedes aegypti (Linnaeus) Hylemya brassicae (BouchC) Lepidoptera Nymphalis antiopu (Linnaeus) Galleria mellonella (Linnaeus) ffyphantria cunea (Drury) Calophasiu lunula (Hufnagel) Hymenoptera Neodiprion sertifer (Geoffroy) Acunthomyops claoiger (Roger) Acanthomyops claoiger (Roger) Orthoptera Melanoplus sanguinipes (Fabricius)

19 28 22 20

Pupa Pupa Pupa Pupa Pupa

1 1 2 1 1

2 4 20 3 4

Pupa Pupa Pupa larva

_

6

<1

9

-

1 1

31 24 18

larva worker pupae sexual pupae

1 2 98

2 4 1

9 29 1

1

1

11

adult

-

_

15

29 21 21 19 60 <1 1 4 2

4 1

1 6

-

13 25 16 16

-

I 6

11 43 41 24

11 17 5

-

4 4

-

33 54

-

45 1

-

1

4

19

20

1

6

7

32 19 18 35 20

-

15

Lauric (12:0), myristic (14:0), palmitic (16:0), palmitoleic (16:1), stearic (18:0), oleic (18:1), linoleic (18:2), and linolenic (18:3) acids are expressed as a percentage of their total. (-): not observed. (from Barlow, 1964)

96

LAWRENCE I . GILBERT

when raised at low temperature (Hanvood and Takata, 1965). In addition, photoperiod appears to play some role in this phenomenon since a short day also causes a greater degree of unsaturation. Since these and perhaps other environmental conditions affect the consistency of the fatty acids in some insects, it is imperative that workers describe the conditions under which their experimental animals were raised. In some insects, the body lipid may contain up to 70% of polyenoic fatty acids with linoleic and linolenic acids the most commonly encountered (cf. Schmidt, 1964). Of these, some tetraenes and pentaenes are present. In Formica polyctena females for instance, fatty acids with two to five double bonds have been detected (Schmidt, 1963). In an analysis of the lipids of pre-pupating larvae of Phalera bucephala (Lepidoptera), Schmidt and Osman (1962) showed the lipid to comprise 32.7% of the dry weight, most of which was glycerides. Of the fatty acids present, palmitate, oleate and linolenate exceeded the concentration of stearate and linoleate. In addition, trace quantities of a tetraenic fatty acid were present. A tetraenic fatty acid was also identified in the total body lipid of adult Locusta migratoria by Fawzi et al. (1961). This insect’s lipid contains predominantly oleate, linoleate and palmitate plus lesser quantities of myristate, stearate, linolenate and arachidonate. In Musca domestica, there is a general trend toward synthesis of fatty acids during early larval growth and subsequent lipid utilization during metamorphosis (Pearincott, 1960). Alterations in individual fatty acid content do occur during metamorphosis but are usually masked when the entire FFA fraction is studied. Individual analysis of the fatty acid content during metamorphosis is rare. Herodek and Farkas (1960) conducted such analyses of unsaturated fatty acids during the embryonic and post-embryonic development of Bombyx mori. The percentage of total unsaturated fatty acids declined only slightly from embryo to last instar larva but fell from 80 to 70% in the prepupa. During adult development, the percentage of unsaturated fatty acids increased in the female where they may play a role in egg development, but fell in the male. On an individual fatty acid basis, the percentage of oleate, linoleate and linolenate remained rather constant during embryogenesis. Once the fist-instar larva hatched however, linoleate increased from about 10% of the total to more than 50% by the third instar, presumably at the expense of oleate and linolenate which decreased during this period. Subsequent to the third-instar, the concentration of linoleate decreased to reach a level of about 15% in prepupae after

L I P I D METABOLISM A N D F U N C T I O N I N INSECTS

97

which the concentration remained rather constant. Coincidentally with this decrease in percentage of linoleate, increases occurred in both linolenate and oleate. In all instances, the percentage of individual fatty acids was greater in the female imago than in the male. These results indicate that either there is an interconversion of linoleate to and from oleate and linolenate by hydrogenation and dehydrogenation, or a pronounced utilization of linoleate during the larval-pupal moult. Other than their value as a source of energy, little is known regarding the role of individual fatty acids in molting and metamorphosis. If the drastic alterations in fatty acid concentration discussed above can be extended to other species, it would appear that these unsaturated fatty acids may play a crucial role in the growth of the insect. With increasingly more sophisticated apparatus, future studies along this line are assured. According to Gilby and Cox (1963), the cockroach odor which is familiar to all of us who have worked with Periplaneta americana is due to an unidentified unsaturated fatty acid. Once we have a general idea of the fatty acid composition of insects there are several questions to be asked. How do these fatty acids come to be in the insect tissues and what is their purpose? Essentially then, we wish to know the biosynthetic scheme for fatty acids and their role in providing energy for the organism. Insects are famous (or infamous) for ingesting almost anything in the plant and animal kingdoms. When the food source is lipoidal or contains some lipid, degradation by digestive enzymes can lead ultimately to fatty acid. But how can an insect on a lipid-free diet build up a lipid store? To answer the last question we must entertain a discussion of molecular interconversion of carbohydrates and proteins to lipid.

111. LIPIDUTILIZATION A . DIGESTION A N D ABSORPTION

Since the bulk of ingested lipid is usually in the form of TGL which may not be able to penetrate the gut wall, the function of the digestive enzymes is to split these glycerol esters into simpler molecules, namely, diglycerides (DGL), monoglycerides (MGL) and FFA. We will not discuss these digestive processes in detail nor the transport of lipid from the digestive tract into the hemolymph because little is known regarding the former in insects and what is known regarding both phenomena has been recently reviewed (House, 1965; Treherne, 1962). The main

98

LAWRENCE I . GILBERT

digestive enzyme acting upon lipids is a lipase that mediates the hydrolysis of TGL. Contrary to what we might assume, hydrolysis most likely does not go to completion in the gut. In mammals for instance, a controversy has embroiled the field for many years regarding the exact form in which lipid passes from the gut to the circulatory system. In a concise review of the work in his laboratory, Isselbacher (1965) discusses the metabolism and transport of lipid by the mammalian intestinal mucosa. The major hydrolytic products of ingested lipid in the intestinal lumen are FFA and MGL. These end products then enter the mucosal cell where they are utilized in the resynthesis of TGL. The FFA are acylated and react with a-glycerophosphate to yield phosphatidic acid derivatives (i.e., glycerides and phospholipids, see Section IV). All the enzymes necessary for the synthesis of TGL appear to be located in the microsomal fraction of the mucosal cells. It may be of interest to those working on hormone action that mucosal microsomes from adrenalectomized rats have less than half the capacity of normal mucosal microsomes to esterify palmitate, indicating regulation of lipid absorption by the corticosteroids. In an excellent biochemical and electron microscopic investigation, Ashworth and Johnson (1963) found that the fatty acid released by the action of digestive lipase is taken into the intestinal epithelial cell by an infolding of the cell membrane. It is only when the fatty acid is internalized and transported further into the cell that it is resynthesized into TGL. It is then the TGL which is released into the intestinal lymphatic system. Studies of this calibre have not been reported in insects, but the need for such research is surely great if we are to understand the complete cycle of lipid utilization in insects. Digestive lipases have been the objects of study in several insects (Baker and Paretsky, 1958; Duspiva, 1934; Gaeta and Zappanico, 1959; Schlottke, 1937; Yamafuji and Yonezawa, 1935), and they appear to have the same function as the pancreatic lipase of mammals (cf. Desnuelle and Savary, 1963). In mammals, the main products of this hydrolysis are unesterified fatty acids and 2-monoglycerides, and there appear to be two competing reactions for the MGL absorbed into the intestinal cell. On the one hand there is the tendency to re-esterify to the higher glycerides utilizing acyl thioesters and on the other hand the tendency for the final hydrolysis of the MGL into FFA and glycerol (Senior, 1964). The major goal in the absorption of lipid in the mammalian intestine is the conversion of ingested TGL to lymph TGL with ’ the least expenditure of energy and minimal alteration in the structure of the fatty acids. In insects we know that the presence of FFA aids in the absorption of lipid out of the alimentary canal (Eisner, 1955; cf.

LI PI D METABOLISM A N D F U N C T I O N I N INSECTS

99

Treherne, 1962) but have little idea of the chemical form in which these materials enter the hemolymph. The aim of lipid absorption in insects is to convert ingested TGL into an unknown form of hemolymph lipid and thence to fat body TGL with a minimum expenditure of energy. The mechanics and biochemistry of this conversion are virgin research areas. In fact, there have been few detailed studies on the lipid content and fatty acid identification in the hemolymph. Wyatt’s (1961) excellent review on the biochemistry of hemolymph scarcely mentions lipids. In the recent 41-page review on the composition of insect hemolymph (Florkin and Jeuniaux, 1964) lipids are completely omitted. Hopf (1940) found that the larval hemolymph of Phormia contains about 1% lipid while Levenbook (1950) has demonstrated the presence of 137 mg % of fatty acids and sterols in the hemolymph of Gastrophilus larvae. More recently, Tietz-Devir (1963) has demonstrated the presence of 28.8 p equivalents of esterified fatty acids and 0.1-0.2 p equivalents of FFA in the hemolymph of L. rnigratoria. Most of the work in this connection deals with lipid transport from the fat body and will be discussed in Section 111. However, in one investigation of the hemolymph of the adult cricket, A . domesticus (Nowosielski and Patton, 1965), it was shown that the lipid level varies between 1.5-2-7%. It is at a minimum in the larval instars, increases to a maximum in 5-10 day old adults and drops once again after the fifteenth day of adult life. The latter drop is more precipitous in females than males and the authors suggest that the hemolymph lipid may be used for egg maturation since sexual maturity is attained between days 5-10 of adult life. This is a reasonable assumption that should be tested by employing castrated females. However, this tells us little of lipid absorption or transport, as these changes may also be a result of lipid release from and not to the fat body. Figures 5 and 6 demonstrate the pH optima and time course of hydrolysis of TGL by a supernatant fraction separated from the midgut of P . americana. The time course approaches zero order kinetics for the first two hours. There have been several studies on the digestive lipase of this cockroach, among which are those of Wigglesworth (1928), Eisner (1955) and Treherne (1958). Our finding (Gilbert et al., 1965) that the lipase of the mid-gut displays dual pH optima (PH 5.0 and pH 7.2) is of some interest. Wigglesworth (1928) had shown that the gut lipase of this insect was maximally active at an alkaline pH while Eisner (1955) demonstrated it to be quite active even at pH 5.0 (the normal pH of the crop). In general, mammalian pancreatic lipase displays an alkaline pH optimum, although dual acid and alkaline pH

100

LAWRENCE I . GILBERT

optima have been reported for mammalian adipose tissue lipase (Lynn and Perryman, 1960). The greater activity of the mid-gut lipase at pH 5.0 is reasonable since this approaches the in vivo environment. The enhancement of activity by calcium ions compares well with the response of pancreatic lipase to this activator (Gilbert et al., 1965). The similarities between the mammalian and insect digestive lipase would lead us to assume at this point that the absorption products from gut to hemolymph are probably similar to those in the mammals.

2

4

5

6

7

8

9

1

0

PH

FIQ.5. Effect of pH on the hydrolysis of triolein by the digestive lipase of P.americana. Lipolytic activity is expressed in ctslmin in FFA released per mg protein per hour. (From Gilbert et al., 1965.)

At least one species of insect,Galleriamellonella, has a special problem regarding lipid ingestion. The waxmoth larvae invade bee combs and feed upon the wax. The method by which the larvae extract the essentials from the wax and digest them has been an object of experimentation by Niemierko and his colleagues for over a decade. Unfortunately the problem remains unsettled although progress has been made. Niemierko and Wlodawer (1950, 1952a, b) suggested that the rapid diminution of unsaponifiable material and the constant fatty acid level in the larval body during an initial period of starvation is due to a transformation of unsaponifiable matter to fatty acid in the gut. As fatty acid is meta-

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101

bolized, more unsaponifiable material is converted. The unsaponifiable substances containing hydrocarbons and alcohols may be transformed in part by the intestinal flora, but these workers believe that the larval enzymes play a predominant role. During the growth of GaZZeria larvae, glycerides are accumulated and the phospholipid (PL) concentration increases, but at a rate below that of the neutral lipid (Niemierko, 1952). About 47% of the total lipid in the larval intestine is PL (Wlodawer, 1956) although the honeycomb on which the larva feeds is devoid of PL. The PL concentration drops dramatically during starvation and Wlodawer postulates that the products of the hydrolytic 3000 r

L 180

0

60

120

Time (min)

FIG.6. Time course of hydrolysis of tnolein by P. arnericano digestive lipase. Lipolytic activity is expressed in ctslmin in FFA released per mg protein per hour. (From Gilbert et al., 1965.)

breakdown of wax during digestion penetrate the gut wall in a phosphorylated form. An enzyme in the gut wall is presumed to split the PL. Thus, PL most likely play a role in the absorption and transport of ingested lipid in this insect (Niemierko, 1959). This theory gains support from the recent work of Young (1964a, b). In addition, Young found that the Galleria larva has the capacity to convert the iso- or hydroxyacids of the honeycomb into more common (palmitic and oleic acids are predominant) saturated and unsaturated fatty acids. This wax utilization is a most intriguing problem but is easily obscured by the presence of symbiotic micro-organisms in the digestive tract which may be in part responsible for this phenomenon. Subsequent to feeding and absorption of lipid (in some unknown form) into the hemolymph, it is proposed that the fatty acids are then

102

LAWRENCE I . GILBERT

reconstituted into triglycerides in the fat body (Chino and Gilbert, 1964, 1965a). This is not merely supposition but is based on two types of experimental approach. First, TGL as mentioned previously are the major lipid store in the fat body and the hemolymph does not have the capacity to synthesize TGL (Chino and Gilbert, 1965a). Secondly, there have been many in vivo and in vitro experiments that demonstrate that labelled fatty acids injected into the hemolymph or incubated with isolated fat body are to a great extent recovered in the TGL fraction (see Section IV). These fat body triglycerides are then stored in the fat body until metabolic demands are made upon the animal (e.g. flight, molting) whereupon these storage lipids are released into the hemolymph and made available to the appropriate tissue. B . LIPID RELEASE A N D T R A N S P O R T

We have a great deal more information on the release of lipid from the fat body into the hemolymph than on how the ingested lipid finally reaches the storage stage. When in vivo measurements are taken of the lipid concentration in the hemolymph it is clearly difficult to discern between fatty acid influx into the fat body for storage as glycerides, and lipid release from the fat body during the mobilization phase. One approach is to separate the digestive tract-hemolymph system from the fat body-hemolymph system by in vitro studies. When working with an in vitro system it should always be borne in mind that extrapolation back to the whole organism may not necessarily reveal a linear relationship. The ingestion of lipids or conversion of other substrates into lipoidal form, the breakdown in the digestive tract, the reconstitution in the fat body would all be to no avail unless the insect had an efficient mechanism for transporting this potential ATP to a body site where it could be utilized. The most general energy consuming processes in insects are movement (especially flight), oogenesis, embryogenesis and molting (particularly at metamorphosis). How does lipid reach the maturing oocyte or embryo or the flight muscle? What happens once it arrives at the site of utilization? These are the questions that will be at least partially answered in this section. Before turning to the insect, let us look at the situation in the mammal so that parallels can be drawn if indeed they exist. At the pH of mammalian plasma, less than 1% of the FFA would be dissociated, most being bound to protein (Fredrickson and Gordon, 1958) and perhaps the same is true for insect hemolymph. Although each albumin molecule

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103

in mammalian blood bears two sites with a very high affinity for fatty acids, the mechanism of binding remains unknown. Fredrickson and Gordon (1958) suggest that the union is probably electrostatic between the carboxylate ion of the fatty acid and a positively charged group on the protein surface. We know that insect hemolymph contains some FFA as well as protein with an electrophoretic mobility approaching albumin, but are as yet completely ignorant of how and in what form FFA are transported. In mammals, the unique problem of how lipid with its very low solubility in aqueous solutions is transported, is solved by circulation in the form of high or low density lipoproteins or as chylomicrons. The chylomicrons are emulsified particles of lipid in the lymph or blood and are comprised of three zones; a central droplet composed mainly of TGL; a covering of surfactant material (e.g. phospholipid, cholesterol, traces of fatty acid) ; an outer atmosphere of loosely absorbed protein (Dole and Hamlin, 1962). In all of the transport forms in mammals, the major component is TGL although some cholesterol has been identified in all of the lipoprotein complexes. It may be that cholesterol plays a vehicular role in mammals. The physical structure of the lipid droplets in insect hemolymph remains an open question due to the lack of work on the problem. However, we are not completely ignorant of how lipid is transported in insects. An electrophoretic analysis of the hemolymph of P. americana nymphs (Siakotos, 1960a,b) resulted in the finding that three of the five plasma fractions are lipoproteins. Two of these fractions contained protein, phospholipid and carbohydrate while the third had a high neutral lipid and sterol content. Siakotos suggested that the presence of a /?-like lipoprotein may be important in the transport of lipid to and from the reserve tissues. This supposition was supported by the experiments of Tietz (1962) who showed that the locust fat body would synthesize glycerides when incubated in oitro with [14C]palmitate. When this labelled fat body was then incubated in fresh unlabelled medium, glycerides were released into the medium. The specific activity of the released glycerides was about 10 times greater than that present in the fat body. She suggested that in uioo, glycerides are likely mobilized from the fat body into the hemolymph where they are transported as lipoproteins. This suggestion has received extensive confirmation through our work (Chino and Gilbert, 1964, 1965a), and we have identified the form in which the lipid is transported as well. Our studies indicate that diglyceride-hemolymph protein conjugates are the means by which lipid is most likely transported in insects. Although most of the experiments were o n pupae and adults of 5-A.I.P.

4

104

LAWRENCE I . GILBERT

H . cecropia, critical experiments utilizing P . americana and Melanoplus differentialis yielded data indicating the same conclusion. Because of the import of these findings based on electrophoretic, thin layer and column chromatographic techniques as well as radioassay, the results and interpretation of this two-year study will be discussed. In addition, some information on the lipid composition of fat body and hemolymph will also be offered. Table V reveals that the lipid content of adult Cecropia fat body is almost three times greater than that of pupal fat body. In both cases, almost 98% of the glycerides are TGL with the remainder being TABLE V Content of lipid in fat body and hemolymph of H . cecropia Male diapausing pupa Fat body Hemolymph 500 mg 1000 mg (fresh weight) (fresh weight) Content Content

Total lipid 1 . Triglyceride 2. Diglyceride 3. Monoglyceride 4. Sterol Sum o f I , 2, 3, and 4

Male adult ~Fat body 500 mg (fresh weight) Content . _ _ ~

mg

%

mg

%

mg

%

70.0 66.5 0.9 < 0.5 0.8

100 95.0 1.3 0.7 1.1

3.9 0.7 2.6 < 0.2 0.3

100 17.9 66.6 5.1 7.7

233.0 224.7 5.4 < 0.5 1.3

100 96.4 2.3 0.2 0.6

68.7

98.1

3.8

97.3

231.9

99.5

From Chino and Gilbert, 1965a.

diglycerides (DGL) and monoglycerides (MGL). However, in the pupal hemolymph, about 75% of the neutral lipid is DGL, and because of this extraordinary finding we conducted several experiments to ensure that this fraction was indeed DGL (Chino and Gilbert, 1965a). When fat body is incubated with [14C]palmitate, or this fatty acid is injected into pupae, we obtain fat body with high specific activity in the glyceride fraction, the major portion being labelled TGL. This fat body is termed pre-labelled fat body in these experiments. Hemolymph that, had been incubated with this pre-labelled fat body was extracted and the lipid applied to a Florisil column. As shown in Fig. 7, almost all the radioactivity was recovered in the 25% ether fraction, the fraction containing DGL. Standard [14C]DGLwas then mixed with the labelled

_

L I P I D M E T A B O L I S M A N D F U N C T I O N I N INSECTS

105

hemolymph lipid and the whole applied to the Florisil column. Figure 7 demonstrates that peaks of the standard and the labelled hemolymph lipid, and of the mixture of the two, are all eluted at the same time. Though it is possible that sterol and DGL overlap in this chromatographic procedure, we demonstrated through a series of chemical techniques that the radioactive lipid in the 25% ether fraction is not

n

5000r

4000

c

1000

1

I

I!' TGL

-A-&A-A .

0

Herone+5%

4-&

10 ether b 1 5 % ether in hexane in hexone

20

30

Fraction number 2 5 % ether in h e x a n e - t f + 2 % 4 methanol in ether

4% acetic acid in ether

FIG.7. Chromatographic separation of I4Clabelled hemolymph lipid from H . cecropia. I4Clabelled hemolymph lipid. A-A 14C labelled standard diglyceride.0-0 Mixure of standard 14C labelled diglyceride and I T labelled hemolymph lipid. (From Chino and Gilbert, 1965a.)

-.-

cholesterol or any other sterol that forms a digitonide. Finally, thin layer chromatography before and after saponification of the labelled hemolymph lipid demonstrated conclusively that the major lipid in the hemolymph is indeed DGL. This has been since confirmed in L. migrutoriu by Dr. A. Tietz-Devir (personal communication). Figure 8 shows the time course of [I4C]DGLrelease from pre-labelled adult Cecropia fat body into pupal hemolymph. The release of DGL continued through the incubation period whereas the release of TGL

I06

LAWRENCE I . GILBERT

was negligible. Furthermore, this experiment demonstrates that the release of DGL is not a simple “leaking” since only a minute amount is released into phosphate saline. That the release of DGL is rather specific for insect hemolymph is seen in Table VI. This table also shows that [14C]palmitate initially taken up by the fat body is also released into the hemolymph as FFA. However, this release was not specific for insect hemolymph but was also enhanced by serum albumin. 50,OOC

JO.CO0 I

.E. ”7

”

+

1 ~

30,OOL‘

a,

m

m 0 ~

* *

v)

?

; 20,000

-

> -

w

U

u

10,000

0 Incubation lime (min)

FIG.8. Time course of I4C glyceride release from male adult H . cecropia fat body into the hemolymph in uifro. )-u Diglyceride released into hemolymph. -0 Diglyceride released into phosphate saline. A--A Triglyceride released into hemolymph. (From Chino and Gilbert, 1965a.)

The labelled TGL was also released into all incubation media suggesting that the labelled TGL recovered from the incubation media may have “leaked” from the fat body during incubation. In fact, when the pre-labelled fat body was incubated in undiluted hernolymph, TGL release was extremely low. To determine whether DGL release occurs in insects other than Cecropia, similar experiments were conducted with the fat body of the grasshopper and cockroach. We showed that [14C]DGL was rapidly released from the fat body of these insects and that Cecropia hemo-

107

L I P I D METABOLISM AND F U N C T I O N I N INSECTS

lymph stimulated this process as much as the hemolymph of the corresponding insect. Recent work regarding the synthesis of TGL from long chain fatty acids in mammals indicates that DGL is an intermediate TABLE VI Release of glycerides from the fat body of H. cecropia Expt. Incubation No. medium

Triglyceride in medium

% released

Diglyceride in medium cts/min 7; released

Free fatty acid in medium cts/min % released ~.

0.2 ml adult hemolymph 0.2 ml chilled 1 pupal hemolymph 0.3 rnl 5% serum albumin 1 ml phosphate saline 0.3 ml diapause pupal hemolymph 2 I ml diapause pupal hemolymph 1 ml phosphate saline 0.3 ml chilled pupal hemolymph 1 ml chilled pupal hemolymph 0.5 ml rat plasma 0.3 ml rat plasma 1 ml phosphate saline ~~~

~~

944

1.7

91.240

37.7

3,006

4.9

830

I .5

63,180

26.1

2,120

3.5

550

1

.o

2,463

1.7

9,250

15.1

60 I

1.1

2,617

1.8

1,208

2.0

734

0.8

100.070

28.2

I82

0.2

106,530

29.8

3,160

10.9

665

0.7

4,885

1.4

1,110

3.8

1,300

1.4

64.635

28.1

-

150 1,140 1,005

0.2 1.2

29.1 1.8 1.6

.~ .

1.1

66,620 4,120 3,580

1.1

3,225

1.4

995 ~

.-

~~~

~~

Pre-labelled fat body was incubated with incubation media listed below in the presence of 1-2 pmoles GSH at 25" for 60 min. Phosphate saline (pH 6.7) was added to I ml final volume. Expt. 1 :I70 mg pre-labelled fat body containing 54,900 cts/min of TGL, 242,400 cts/min of DGL, 61,200 cts/min of FFA. Expt. 2:180 mg pre-labelled fat body containing 94,100 cts/min of TGL, 357,200 cts/min of DGL, 29,200 cts/min of FFA. Expt. 3:150 mg pre-labelled fat body containing 89,500 ctslmin of TGL, 229,700 ctslmin of DGL, 19,100 cts/min of FFA. (From Chino and Gilbert, 1965a.)

108

LAWRENCE I. GILBERT

and that ATP is required (see Section IV). If the synthesis of TGL and DGL from labelled palmitate by Cecropia fat body proceeds by a similar mechanism, one would expect inhibition of glyceride synthesis by cytochrome inhibitors, uncouplers and other substances that interfere with oxidative phosphorylation. Our data revealed that inhibitors such as KCN, NaN, or 2,4-dinitrophenol prevent the normal incorporation of palmitate into glycerides by the fat body and result in an accumulation of FFA in the tissue. One may ask whether the release of DGL is an active process and may also be affected by these inhibitors. When pre-labelled fat body was incubated with pupal hemolymph in the presence of the above inhibitors, there was a marked inhibition of release of [14C]DGL. In contradistinction, FFA were released at an accelerated rate. Further work led to the conclusion that these inhibitors accelerate the release of FFA from the fat body into the hemolymph by interfering with the incorporation of FFA into neutral lipid. Since the hemolymph itself cannot synthesize glycerides from labelled palmitate, we can exclude this phenomenon as contributing to the DGL content of the hemolymph. Finally, we incubated pupal hemolymph and pre-labelled fat body, and subjected this hemolymph to paper electrophoresis. As illustrated in Fig. 9, four distinct protein bands were resolved, three of which were lipoproteins. Subsequent determination of the distribution of radioactivity of [14C]DGLon the strip clearly revealed that almost all of the DGL was concentrated and recovered in the lipoprotein fraction having a relative mobility of 0.13. From these experiments we can conclude that DGL is rapidly and specifically released from the insect’s fat body into the hemolymph and that very little or no TGL release occurs. This DGL release occurs in uiuo as well as in uitro (Chino and Gilbert, 1964). When [14C]palmitate is injected into a Cecropia pupa, this labelled fatty acid is first incorporated into glyceride by the fat body and subsequently released as DGL. Consequently, the specific radioactivity of the glycerides in the hemolymph is much greater than that in the fat body because TGL is the major component of the neutral lipid in the fat body. In other words, DGL with an extremely high specific radioactivity is continuously being released into the hemolymph, while the low specific activity TGL remains for the most part in the fat body. The demonstration of DGL release from the fat body of four species of insects (silkmoth, grasshopper, locust, cockroach) suggests that this may be a phenomenon common to many or all insects.

Distonce from o r i g i n ( c m )

FIG.9. Separation of H . cecropia hernolymph proteins and associated 14C labelled diglycerides by paper electrophoresis. Ordinate: cts/min of ["C] diglyceride found in 1 cm piece of electrophoretogram. LP is strip stained by Sudan black, lipoprotein. P is strip stained by brornophenol blve, protein. (From Chino and Gilbert, 1965a.)

110

LAWRENCE I . GILBERT

In addition the data suggest that the release of DGL is an endergonic process and requires ATP as is the case in glyceride synthesis. In contradistinction, the release of FFA seems to follow a concentration gradient and depends on the level of FFA in the fat body. This then is a passive process. Thus, we might expect that in an actively metabolizing tissue like adult Cecropia fat body, DGL release is high, while FFA release is high in tissues of lower metabolism such as the fat body of a diapausing pupa. How does this compare with the transport mechanism in mammals to which we have already alluded? In mammals, the main components in the transport of long chain fatty acids are most likely TGL and FFA. In fact, the major neutral lipid component in mammalian plasma is TGL with DGL and MGL present as only minor constituents. This differs from the insect case in that DGL replaces TGL in the insect. In mammals, the TGL found in the plasma is conjugated with plasma proteins as the low density lipoprotein, chylomicron, etc. It appears that in the hemolymph of the insect, DGL is carried by, and perhaps conjugated to, one of the hemolymph proteins. These findings indicate that DGL-protein complexes and FFA (perhaps also bound to protein) are the means by which insects transport lipid. That acetate can be oxidized by insect muscle is now taken for granted since it was demonstrated almost 20 years ago (Barron and Tahmisian, 1948). In 1955, Sacktor suggested that stored lipid is broken down to acetate in the fat body of Musca domestica and transported in that form to the flight muscle. The work discussed above demonstrates that this is not true for several insect forms although we have not yet completed our investigation of the Diptera. The physiological significance of the transport of DGL will be discussed in Sections 111 and IV. At this time, we can say that in the Cecropia moth for instance, the flight muscles utilize long chain fatty acids almost exclusively but store only little lipid. Thus lipid must be transported to this active tissue perhaps in the form of DGL. In addition, long chain fatty acids are necessary for normal oogenesis and must be transported from the fat body to the ovaries in a similar way. Furthermore, of importance is the fact that DGL are intermediates not only in TGL synthesis but also in phospholipid synthesis (Fig. 15; see Section IV). C . EXTRA-DIGESTIVE LIPASES

Prior to discussing the final utilization of the transported DGL as a source of energy, we should consider one other problem in the sequence

L I P I D METABOLISM A N D F U N C T I O N IN INSECTS

111

of events beginning with the ingestion of lipoidal material and ending with the oxidation of fatty acids. If lipid is stored as TGL within the fat body and subsequently released as DGL, it follows that at least one fatty acid moiety must be cleaved from each TGL molecule in the fat body before transport can occur. Until very recently, no true extradigestive lipase has been demonstrated in insects by the use of biochemical procedures. Histochemical work has demonstrated the presence in the fat body of an enzyme that will hydrolyze chemical such as a-naphthyl acetate (see for example Wigglesworth, 1958), but these histochemicals are certainly not the true substrate in the insect. There have been a large number of reports in the past by Prof. J. C. George and his group on the ability of various insect (and vertebrate) tissues to hydrolyze tributyrin (Bhakthan, 1964; George and Bhakthan, 1960a, b, 1961, 1963; George and Eapen, 1959a, b; George et a/., 1958, etc.). Although this work is of real interest, it does not elucidate the action of a lipase, contrary to what the authors state. Although their work demonstrated that honey bee flight muscle can oxidize butyrate, this C, fatty acid is more likely present in the insect as a result of the /3-oxidation of longer chain fatty acids (see Section 1II.D.) rather than of the hydrolysis of tributyrin. It is more likely that most of the work of the Indian group is conducted on esterases such as that described by Van Asparen (1959). She showed the presence of potent ali-esterase activity in various body regions of Musca domestica. The enzyme(s) was extremely efficient in hydrolyzing both triacetin and tributyrin. Of interest is the fact that Van Asperen found 22% of the esterase activity responsible for the hydrolysis of tributyrin to be localized in the head of the insect. Since the head contains little or no fat body, but is composed mainly of nervous tissue, the enzyme described is most likely not a true lipase. The ability of an enzyme to cleave butyrate from tributyrin may be far removed from its primary action. Our laboratory has been engaged in an attempt to characterize a true extra-digestive lipase in various stages of the life cycle of H . cecropia for several years. Almost every conventional technique has been employed including titrimetric determination, spectrophotometric assay and manometry. In each case no true lipase could be demonstrated, but potent esterase activity was found in several tissues. Table VII reveals that hemolymph, fat body and gut from several developmental stages show no lipase activity when assayed by manometFy. All tissues are capable of hydrolyzing triacetin, with gut exhibiting the greatest

112

L A W R E N C E 1. G I L B E R T

activity. In both the pupal and adult stages, the male fat body shows more active esterase than that of the female, and the adult tissues are far more potent in splitting the ester bond than the respective tissues of the pupa. In no case could we detect the release of FFA from the water-insoluble glycerides (olive oil). Since the flight muscle is the most metabolically active tissue in the moth we believed that it might be the source of the enzyme we were seeking. However, repeated attempts to demonstrate a true lipase in TABLE VII Esterase activity in tissues of If. cecropia Hydrolytic activity Stage

Enzyme source

(pl C 0 2 / m g protein

N/hr) Triacetin Chilled male pupa Chilled female pupa 20-Day-old male Developing adult 1-Day-old male adult 1-Day-old female adult

whole blood fat body homogenate gut homogenate whole blood fat body homogenate gut homogenate whole blood fat body homogenate fat body homogenate fat body homogenate

6.02 266.34 890.79 2.40 56.54 375.70 16.49 27 1.57 408.50 266.70

. _ _-

Olive oil

0 0 -~

0

0 0 0 0

Manometric assay. Reaction mixture contained 1 ml substrate; I ml enzyme source; 1 ml bicarbonate buffer. Triacetin added as 10% solution in buffer. Olive oil added as 10% emulsion in buffer prepared in Waring Blender with 1% polyvinyl alcohol. Run at 25", pH 7.4. (From Gilbert et a/., 1965.)

this tissue failed. The homogenates did split the ester bonds of tributyrin and ethyl acetate, but not those of higher fatty acid glycerides. The conditions of the assay were not limiting since the reactions that did occur were linear with time. The optimum pH for flight muscle esterase is about pH 7.4 and activity rapidly drops when assayed one pH unit below this value (Gilbert et al., 1965). T h w various tissues from Cecropia possess the ability to hydrolyze water-soluble esters, but showed no lipase activity. All our other data (see previous section) indicated that this insect, obviously utilizing products of glyceride hydrolysis, must possess a means of converting the storage lipids to a

L I P I D METABOLISM AND F U N C T I O N I N INSECTS

113

utilizable state. If the lipase present was only minimally active, perhaps the available assays were below the level of necessary sensitivity. We therefore developed a highly sensitive assay for lipase activity utilizing [14C]labelledglycerides (Chino and Gilbert, 1965b). Utilization of this sensitive assay provided proof for the existence of a true lipase that hydrolyzes triolein in the fat body of the Cecropia moth (Gilbert et al., 1965; Figs. 10 and 11). Table VIII indicates the activity of lipase from several tissues on the DGL and TGL. It is TABLE VIII Hydrolysis of di- and triglycerides by insect tissues

Enzyme source

Substrate

Pupal Cecropia haemolymph Adult male Cecropia fat body

l4C-trio1ein 14C-triolein 14C-diolein 14C-triolein '*C-diolein l4C-triolein '4C-triolein l4C-dio1ein

Cecropia flight muscle Cockroach fat body Cockroach midgut

Hydrolytic activity (cts/min in unesterified fatty acid released from substrate/mg protein/hr)

0 35,26 41,46 41,54 290,203 33 973 1016

Run in tris buffer, pH 7.2 at 30". The substrate is actually a mixture of labelled glyceride plus unlabelled carrier glyceride. The enzyme source is the middle layer from a centrifuged homogenate. Assayed according to Chino and Gilbert (1964b). (From Gilbert et a/.,1965.)

important to note that the lipase from Cecropia flight muscle was five times more active against diolein than triolein. This was in contrast to results with other tissues including Cecropia fat body. It is known that calcium ions activate mammalian pancreatic or intestinal lipase (Desnuelle et a/., 1950; DiNella et al., 1960; Chino and Gilbert, 1965b). Our results (Gilbert et a/., 1965) demonstrate that calcium ions stimulate the cockroach digestive lipase threefold but have no effect on the extra-digestive lipases. The data indicate that definite lipolytic action is associated with Cecropia fat body, but the activity is low. To investigate the possibility that an endogenous lipase inhibitor resides in the fat body, attempts were made to remove this hypothetical inhibitor by preparing acetone powders of the fat body lipase and dialyzing fresh

114

LAWRENCE I. GILBERT

I

I

4

8

I I2

Tlme ( h r )

FIG. 10. Time course of hydrolysis of triolein by adult male H. cecropia fat body. Lipolytic activity is expressed in cts/min in FFA released per mg protein per hour. p H 7.2 in tris buffer. (From Gilbert et a/., 1965.)

I

I

6

7

I

I

I

5

8

9

P"

FIG. 1 I . Effect of p H on the hydrolysis of triolein by the fat body lipase of adult H. FFA released per rng protein per hour. (From Gilbert e f a/., 1965.)

cecropia. Lipolytic activity is expressed in cts/rnin in

L I P I D METABOLISM A N D F U N C T I O N I N I N S E C T S

1 I5

extract against water. In neither case was the enzymic activity enhanced. Furthermore, addition of fat body homogenate to incubating mammalian pancreatic lipase did not diminish the activity of the mammalian enzyme. It thus appears that no endogenous inhibitor is present. Thus we have shown that extra-digestive lipases do exist in insect tissues but that their activity is not measurable (at least in H . cecropia) when conventional techniques are used. Techniques such as manometry do, however, uncover esterase activity when substrates such as triacetin and tributyrin are used. The distinction between lipases and esterases that hydrolyze short chain fatty acid glycerides is certainly not clear, but several criteria can be used to differentiate between these two classes of glycerol ester hydrolases. In general, a lipase has a preference for long chains in the acyl groups of the glycerol ester (as in triolein, tripalmitin, etc.) whereas an esterase has a preference for short chains in the acyl groups (as in triacetin, tributyrin, etc.). When the ester substrate is soluble in water it is not attacked by pancreatic lipase (Desnuelle and Savary, 1963). These authors assume that an emulsion contains multimolecular aggregates estranged from the aqueous medium by an interface. The lipase molecule is absorbed to this interface and can thus exert its effect. In aqueous solution, the lipase would be inactive. In fact, in mammalian adipose tissue, different enzymes are responsible for the hydrolysis of long and short chain fatty acid glycerides, one of which has been termed a “tributyrinase” (Schnatz and Williams, 1962). Finally, the use of substrates such as triacetin and tributyrin has little physiological significance since these substances are not usually present in animal tissues, although the rate of hydrolysis of these esters may be an indicator of general metabolic activity . In Cecropia, esterase activity in the tissues studied increases steadily during the pupal-adult transformation and reaches its highest level in the adult moth (Gilbert et a/., 1965). This increase during development is most likely a reflection of the general increase in metabolic rate during adult development. The sexual dimorphism in esterase activity is another example of biochemical differences between males and females of this species (see Section I I ) , and again may be related to the more active flying conducted by the male moth. The physiological significance of the ability of these tissues to hydrolyze triacetin and tributyrin remains obscure. Our results on the flight muscle esterase are in agreement with those of Fodor (1948) who showed that locust flight muscle could not hydrolyze olive oil but didcleave the fatty acid moiety from monobutyrin and methylbutyrate. Cecropia flight muscle

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LAWRENCE I . GILBERT

is capable of hydrolyzing tributyrin at about the same rate as S.gregaria flight muscle (George et al., 1958) and honey bee flight muscle (George and Bhakthan, 1963). It is possible that the enzymes in various insect tissues responsible for the hydrolysis of these short chain fatty acid glycerol esters are in reality primarily concerned with completely different reactions and that glycerol ester hydrolase activity is only a secondary effect. Desnuelle and Savary (1963) suggest that the hydrolytic activity of some of the so-called pancreatic esterases merely corresponds to side activities of well known proteolytic enzymes. The most significant results on this extra-digestive lipase of Cecropia are those indicating that Cecropia flight muscle preferentially hydrolyzes DGL whereas the other lipases examined preferentially attack TGL. This question of lipase in flight muscle and fat body is an integral part of the general problem of lipid transport. We must assume that the lipase action of the fat body results in DGL that are then transported in the hemolymph as discussed previously, to sites of utilization such as the flight muscle. We must further assume that the DGL are separated from their carrier protein as they move into the tissue just as fatty acids disengage from their carrier protein as they enter mammalian tissue (Fredrickson and Gordon, 1958). The data suggest that DGL enters the flight muscle intact, whereupon the fatty acid is cleaved from the glycerol ester and rapidly oxidized in the flight muscle sarcosomes. Thus, ingested lipid makes its way to the tissue where it can finally be completely oxidized and contribute to the energy currency of the cell. The next question is how this lipid catabolism takes place. D . FATTY ACID CATABOLISM

The major portion of our knowledge regarding fatty acid oxidation in insects comes from the study of flight muscle. Because of this, flight muscle will serve as our example of an active tissue capable of converting fatty acids to CO, and water. Before discussing this wide subject, let us look at some other processes where fatty acid oxidation may play a major role. 1. Embryos

Almost 30 years ago, Ditman and Weiland (1938) proposed that reduced body water, increased lipid and increased saturation of the lipid are associated with the ability of insects to withstand hibernating conditions (i.e., diapause). This has been consistently confirmed in the last decades and indicates that fatty acid oxidation may play a large

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117

role in supplying the energy required for the relative long diapause periods of some insects. In several insects at least, free lipids are the primary source of energy for the larval-pupal moult as in Tenebrio molitor (Moran, I959), Tribolium confusum (Villeneuve and Lemonde, 1963) and possibly Bombyx (Niemierko, 1959). In addition, as we have seen previously (Section II), lipids are a prime source of energy for insect embryogenesis. In the case of the Cecropia egg for example, there is a dramatic reduction in lipid content between the time of egg laying and the hatching of the first instar larva. Lipids are known to be catabolized to a greater extent in cleidoic eggs than in acleidoic eggs (cf. Babcock and Rutschky, 1961). In 1885, Tichomirov showed that there is a 20% decrease in the free lipid from the termination of diapause to hatching in the eggs of B. mori. Similar studies have been made on other insect eggs, notably M . diferentialis which combined with manometric measurements suggested that about 90% of the oxygen consumed by Melanoplus embryos was used for lipid catabolism (Boell, 1935; Hill, 1945; Slifer, 1930). Further studies on other species led to similar conclusions although only a small drop in lipid was noted during the embryonic development of Oncopeltus (cf. Babcock and Rutschky, 1961). For example, Allais et a/. (1964) reported a 32% decrease in the lipid content during the 18 days of embryogenesis of L. migratoria, much of which was due to the catabolism of glycerides. In Dixippus, about two-thirds of the energy used for embryogenesis is attributed to lipid. About one-half of the original lipid is oxidized to CO, and water during embryonic development as compared to onefourth of the protein (Lafon, 1950). Recent studies on the ovoviviparous cockroach, Leucophaea maderae (Gilbert, unpublished observations) reveal a dramatic increase in the lipid content of the ovary of an adult female prior to oogenesis (about 0-5 mg) to approximately 80 mg in an ovary containing mature eggs just prior to ovulation. This is associated with a concomitant decrease in the lipid content of the fat body indicating transport of lipid to the active ovary. After fertilization, the embryos are enclosed within an egg case held in the female’s uterus and embryogenesis proceeds for about two months. During this period, the content of lipid decreases from about 80 mg per egg case to approximately 40 mg per egg case, indicating that lipid is one major substrate for embryogenesis in this insect. This supposition is supported by our radiotracer studies in which ovaries undergoing oogenesis were found to possess a profound capacity to incorporate [l*C]palmitate into egg .lipids (L. I . Gilbert, unpublished observations).

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LAWRENCE I . GILBERT

There have been relatively few studies on the lipid composition of insect eggs or embryos. One of the exceptions is the recent work of Strong (1963a, b, 1964) who demonstrated that 13.9% of the fresh weight of the aphid egg is lipid. Of this, 85% is neutral lipid, 2% FFA, 4.75% sterol ester and 7.5% is made up of sterols, hydrocarbons and miscellaneous lipids. Of the fatty acids composing the neutral lipid fraction, he has demonstrated a preponderance of myristoleic (C,,,,), myristic and oleic acids. Our studies on the developing eggs of L. maderae (L. I. Gilbert, unpublished observations) demonstrated that the egg lipid is made up of 9O-92% “neutral lipid” and 8-10% phospholipid. In the former fraction, about 90% is TGL, 2 - 3% FFA, 1 - 2% sterol and about 2% sterol ester and hydrocarbon. The remaining lipid is MGL and DGL. During embryogenesis, an increasing percentage falls into the phospholipid fraction (up to 16% of the total lipid) but no drastic qualitative changes occur in the neutral lipid fraction. The mechanism of lipid utilization by embryos has not been comprehensively studied with biochemical techniques, although a number of histochemical investigations have been reported. For example, Nath et al. (1958) demonstrated the presence of three types of lipid bodies in the oocyte of P . americana. One was believed to be wholly phospholipid, one TGL surrounded by an incomplete sheath of PL, while the third is composed of TGL. One can speculate that the TGL may be an energy source for oogenesis while the PL may be used as basic material for membrane formation and microcompartmentalization of the cell. While on the subject of insect eggs, it is of interest to note that in 1952, Hsu reported that mitochondria containing large quantities of unsaturated lipid pass from the Drosophilu nurse cell to the oocyte. According to this author, these mitochondria then transform into lipid yolk bodies. As we shall see subsequently, if any subcellular organelle is to be a lipid body precursor, the mitochondria containing a large quantity of lipid are almost ideal candidates. The notion that the mitochondria may act as an ultimate source of energy as well as an energy converter is a novel (and perhaps far-fetched) idea. As seen from the foregoing, the role of lipid in insect development poses some intriguing questions. Inroads have been made but the fundamental questions remain unanswered. This is in contrast to the more recent attempts to elucidate the role of lipids in flight muscle metabolism. 2. Flight muscle metabolism The flight muscle of locusts, moths, flies and bees has contributed greatly to many aspects of our understanding of insect biochemistry

LIPID METABOLISM A N D F U N C T I O N I N INSECTS

119

(cf. Gilbert and Schneiderman, 1961a). This is especially true regarding the oxidation of fatty acids, in which case muscle appears to be the sole object of contemporary experimentation. A great deal of the research on insect flight muscle has been concerned with the a-glycerophosphate dehydrogenase system in those insects that use carbohydrate as flight fuel (Chance and Sacktor, 1958; Sacktor and Dick, 1962; Van den Burgh and Slater, 1962; Zebe et al., 1959). Although the data are of great general interest, they need not concern us here in our discussion of muscle that preferentially oxidizes lipid. However, it should be pointed out that a-glycerophosphate dehydrogenase can mediate the reduction of dihydroxyacetone phosphate to a-glycerophosphate which in turn can be converted to various complex lipids (see Section IV). In addition, a-glycerophosphate may play a special role in the so-called “a-glycerophosphate cycle” (Klingenberg and Biicher, 1960). This cycle is a mechanism whereby hydrogen generated into the extra-mitochondria1 pool of NADH by the EmdenMeyerhof pathway is made available to the respiratory chain in the mitochondria. The hydrogen is transferred to dihydroxyacetone phosphate by the action of an NAD-dependent extra-mitochondria1 aglycerophosphate dehydrogenase. The a-glycerophosphate diffuses readily into the mitochondrial compartment where a mitochondrialbound a-glycerophosphate dehydrogenase is located. This enzyme is not coupled to NAD but enters directly into the respiratory chain (Biicher et al., 1959; Sacktor, 1959; Klingenberg and Biicher, 1960). Intensive study into lipid utilization by insect flight muscle essentially began with the comprehensive investigation of Weis-Fogh (1952) on flight in Schistocerca gregaria. He found that 80-85% of the total energy expended in the first five hours of flight was derived from lipid. The fat body was responsible for delivering 85-90% of the energy expended by the muscles. Previously, Krogh and Weis-Fogh (1951) demonstrated a decline in RQ from 0.82 to 0.75 during the first 90 min. of flight (see also Bode and Klingenberg, 1964). An RQ approximating 0.7 is indicative of almost exclusive lipid catabolism. Weis-Fogh’s experiments were well conceived, used insects raised under near natural conditions and left little doubt that lipid was the main fuel for locust flight (see also Fulton and Romney, 1940). As Weis-Fogh points out, lipid is an ideal substrate for flying insects since the hydration of glycogen would make isocaloric quantities eight times heavier than lipid (and result in an extremely heavy flying machine). Since Weis-Fogh‘s original observations, a number of publications have appeared that deal with some aspect of the relation of lipids to 6+A.I.P.

4

120

LAWRENCE I. GILBERT

flight muscle metabolism. For the most part, they are concerned with the study of rates of oxidation of fatty acids by flight muscle, flight muscle homogenates or sub-cellular fractions and the assay of particular enzymes of the oxidative pathway. Let us now consider the final step 0

II

:--------,R-CH,-CH,C-OH

+

A

CoASH ATP

0 II R- CH,CH,C-SCoA

HP,O?- + A M P

G FAD

0

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$".

FmH,

R-CH=CHC-SCoA

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JI Krebs' cycle FIG.12. The 8-oxidation of fatty acids. The enzymes involved are: a. acyl thiokinase b. acyl dehydrogenase c. enol hydrase d. 8-hydroxyacyl dehydrogenase e. thiolase

in the utilization of lipid by the insect, namely the oxidation. of fatty acids to carbon dioxide and water. Figure 12 outlines the known steps of the Boxidation of fatty acids as we know them from the classic work with microbial and vertebrate systems. It can be readily seen that a relatively enormous quantity of

LIPID METABOLISM A N D FUNCTION I N INSECTS

121

energy is made available to the cell when long chain fatty acids are completely oxidized. Although one ATP is sacrificed in the “sparking reaction” (reaction a in Fig. 12), only one is utilized regardless of the chain length of the fatty acid. Once the process of /?-oxidationis initiated within the mitochondrion, it usually runs to completion by the removal of C, segments from the original fatty acid, which then enter the Krebs’ cycle as acetyl CoA. For each C, unit the cell gains the potential energy inherent in 1 reduced FAD, 1 reduced NAD and that released in one complete turn of the Krebs’ cycle. For each mole of a saturated C18fatty acid that is completely oxidized, the cell gains more than 140 moles of ATP. As an example of the work conducted on the flight muscle oxidation of fatty acids, let us again consider the Cecropia moth (Domroese and Gilbert, 1964). During the second and fifth day of adult life, male Cecropia moths show a sudden utilization of total lipid which had remained more or less a constant quantity during adult development (see Section 11). This sudden drop in lipid was thought to be due to the rather abrupt assumption of flight activity in young adults. Prevention of flight results in a conservation of lipid, suggesting that flight is the major causative factor in the sudden decrease in lipid content of the moth. The adult moths do not feed and of necessity, the metabolic substrates of the adult are limited to those available after the requirements of metamorphosis have been satisfied. The carbohydrate store of the male moth is almost completely exhausted during adult development leading to the hypothesis that lipid must be the main substrate for adult life (namely flight muscle metabolism; see Table IX). By measuring the endogenous Qo, of flight muscle homogenates we were able to obtain a reproducible estimate of their metabolic rate. Dialysis of these homogenates resulted in a large reduction in the Qoa due to the loss of requisite cofactors. By adding cofactors back to the dialyzed homogenate we were able to determine which were necessary for normal cellular metabolism. These results show that ATP, Mg+ and citrate are required to restore the endogenous respiration of dialyzed homogenates to the level of the non-dialyzed control. This infers that these same cofactors are necessary for fatty acid oxidation. The stimulation of respiration by citrate may be due to some extent to its oxidation as an added substrate or perhaps to its stimulation of oxidation of endogenous lipid by priming the citric acid cycle. To test these two alternatives the oxidation of [14C]palmitate by dialyzed flight muscle homogenate was measured in the presence of added cofactors. The results (Table X) clearly indicate that added citrate stimulates fatty +

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LAWRENCE I . GILBERT

acid oxidation and verify the need for ATP and Mg . The stimulatory effect of citrate is not specific and other citric acid cycle intermediates can also stimulate fatty acid oxidation. The pH optimum for the oxidation of [l4CJpalmitate by the dialyzed homogenate with added cofactors coincides with that of endogenous respiration of undialyzed homogenate (Fig. 13). Malonate was an effective inhibitor of fight muscle respiration and together with the demonstration that citric acid cycle intermediates stimulate fatty acid oxidation, indicates that the pathway of fatty acid +

+

TABLE IX Oxidation of various labelled substrates by homogenate of male H. cecropia flight muscle

Substrate (1.1 x los cts/min) Acetate Alanine Glucose Pyruvate Glycerol Succinate Butyrate Decanoate Palmitate

Acid insoluble dry weight (ms) 10.20

10.10 8.43 8.19 7.85 7.92 8-34 10.40 10.30

1 4 ~ 0 ,

total cts/min 60 min

53,410 16,853 4,063 115 1,581 55,032 43,042 56,071 113,510

cts/min mg acid insol. dry weight

14C02

5,236.3 1,668.6 482-0 14.0 201.4 6,948.5 5,160-9 5,391.4 1 1,020.4

Homogenate prepared in 0 . 1phosphate ~ buffer, pH 7. (From Domroese and Gilbert, 1964.)

oxidation is via the citric acid cycle. Additional evidence for the participation of this pathway was obtained by measuring the production of 14C02from a fatty acid labelled in different numbered carbons. Studies on flight muscle sarcosomes (mitochondria) showed that the same cofactors were necessary if fatty acids were to be oxidized at a maximum rate. [It should be noted that not all insect mitochondria can oxidize fatty acids as can be seen from the numerous studies on flight muscle sarcosomes from carbohydrate utilizing insects and from the study of mitochondria isolated from the silkworm intestine (Sridhara, 1965).] In addition, we studied the fatty acid activating enzyme of the Cecropia flight muscle and found that it was maximally active only when CoA, ATP and fatty acid were present. That relatively high con-

LIPID METABOLISM A N D FUNCTION IN INSECTS

123

centrations of coenzyme A exist in insect tissues is no longer in doubt (Boccacci and Bettini, 1956). The above results showed that fatty acids are actively oxidized by fight muscle homogenates whereas glucose, pyruvate and glycerol were oxidized only to a limited extent, supporting the original observations 17 16

15 14

13 12 L

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400

350

300

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6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6

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FIG.13. Effect of pH on endogenous respiration and oxidation of palmitate-l-'*C by flight muscle homogenates from H. cecropia moths. (From Domroese and Gilbert, 1964.)

of Zebe (1954) on a feeding moth. The underlying basis for this inactivity presents an intriguing problem in the comparative physiology of insect muscle. Zebe (1959a, b) has already suggested that insect flight muscle is of three physiological types : the exclusive carbohydrate users, the flies; the exclusive lipid users, the moths and butterflies; and those that

124

LAWRENCE I . GILBERT

can use both fat and carbohydrate, the locusts. Studies on the biochemistry of fly and locust muscle have shown no qualitative differences correlated with the type of fuel used (Zebe, 1959a; Boettiger, 1960). That some differences do exist between the above groups of insects is suggested by the observation that after flight there is a continued elevation of respiration for an hour or more in locusts and moths whereas this does not occur in the Diptera (Chadwick and Gilmour, TABLE X Cofactors necessary for the oxidation of [ l-14C]palmitic acid by dialyzed homogenate of male H. cecropia flight muscle Reaction medium Complete -ATP Cit r at e -MgCL No cofactors Undialysed control Boiled control

-

Acid insol. dry weight (mg)

14C02cts/min 60 min

13.03 13.10 13-04 13.65 12.4 1

67,030 6,220 21,533 9,453 6,376

5,144.3 474.8 1,651.3 692-5 513.8

16-12 12.05

74,150 126

4,599.3 10.5

14COacts/min mg acid insol. dry wt.

The complete reaction medium received 1 ml of dialyzed homogenate, 1.2 x lo5ctslmin of [l-14C]palmiticacid, and 1 p o l e of each of ATP, citrate, and MgCla added in a total ~ buffer, pH 7. Flasks lacking one or more cofactors received of 0-3 ml. of 0 . 1 phosphate an equivalent volume of buffer. Control flasks received 0.3 ml of buffer in addition to the homogenate indicated. (From Domroese and Gilbert, 1964.)

1940; Krogh and Weis-Fogh, 1951; Zebe, 1954). Our findings (Domroese and Gilbert, 1964) that flight muscle of Cecropia cannot appreciably oxidize glucose and glycolytic end products indicates that further experiments relating to the biochemical pathways of this tissue may reveal sigmficant quantitative differences when compared to flight muscle from insects of other orders. In respect to the requisites for fatty acid oxidation, the system we studied shows similarities to the requirements for fatty acid oxidation by rat liver mitochondria (Kennedy and Lehninger, 1949). The insect system differs from the mammalian system by the lack of cytochrome c dependence. This may be due to the naturally high concentration of cytochrome c in moth flight muscle sarcosomes. E. Margoliash (personal

L I P I D METABOLISM A N D F U N C T I O N I N INSECTS

125

communication) found that moth flight muscle contains the highest concentration of cytochrome c yet described. Because of the naturally high concentrations, it may persist in sufficient quantities during tissue preparation, eliminating the necessity of an exogenous supply. The requirements for the flight muscle fatty acid oxidizing system of Cecropia are also similar to those known in other invertebrates. At present the only well characterized invertebrate systems have been described in the hepatopancreas of Carcinus (Munday and Munn, 1962) and the thoracic muscles of the locust (Meyer et al., 1960). The Cecropia system is comparable to the locust particulate system in that both require ATP and magnesium for optimum fatty acid oxidation. It differs, however, in that addition of citric acid cycle intermediates enhances the oxidative capacity of the Cecropia flight muscle. The action of the intermediate in Cecropia is similar to that of the intermediate necessary as a primer in rat liver mitochondria, whereas the primer in the oxidation of higher fatty acids by the locust thoracic particle is butyrate. Butyrate is unable to replace the citric acid cycle intermediate in our system. The pH optimum for oxidation of fatty acids in Cecropia flight muscle was identical with that in the locust (6.4). This is somewhat lower than that found in mammalian systems but is in keeping with the fact that the pH of the hemolymph of various Lepidoptera and locusts has been reported to fall on the acid side of neutrality. The complete &oxidation scheme has not been worked out in detail in any one insect. However, recent work has identified individual enzymes in insect tissue that are similar to those found in mammalian tissue, suggesting the presence of the /3-oxidative pathway. The acetate activating enzyme was detected in the flight muscle of the honey bee (Hoskins et al., 1956). Nelson (1958) showed the presence of fatty acid activating enzymes in the fat body of the cockroach and several species of Lepidoptera. In 1960, Zebe identified the condensing enzyme that leads activated acetate into the citric acid cycle and /3-ketoacyl thiolase that leads /3-ketoacyl into the fatty acid cycle in locust flight muscle. In comparing several enzymes from leg and flight muscle of a carbohydrate utilizing insect (bee) and one utilizing lipid (locust), Beenakkers (1963a) demonstrated that although lactic dehydrogenase activity was high in the leg muscle, it was only slight in the flight muscles of both insects. In all cases, the locust flight muscle homogenate oxidized fatty acids at a greater rate than isolated sarcosomes. The addition of carnitine however, raised the activity of the mitoehondria in relation to that of the homogenate while the addition of palmityl camithe

126

L A W R E N C E I . G I L B ER T

resulted in the mitochondria exhibiting 50% more activity than the homogenate. Beenakkers postulates that under normal physiological conditions, carnitine may activate the fatty acids in insect flight muscle. Carnitine transacetylase mediates the reaction : Acetylcarnitine

+ CoASH +carnitine + CoASAc

resulting in activated acetate. Although 5,150 enzyme units were located in the locust flight muscle, that of the bee was devoid of activity (Beenakkers and Klingenberg, 1964). The enzyme appears to be present in the soluble matrix of the mitochondria since on disruption of these organelles, the enzymic activity becomes associated with the soluble fraction. As Beenakkers and Klingenberg suggest, carnitine transacetylase is not an obligate intermediate for fatty acid oxidation (and possibly not needed in the Cecropia flight muscle) but may function in the transport of fatty acids into and within the mitochondria. Further work is needed to prove this supposition but it gains some support from the recent work of Bressler and Friedberg (1964) who demonstrated that carnitine not only stimulates the oxidation of palmityl CoA but also augments the rate of palmitate incorporation into mitochondrial phospholipids. Carnitine may thus function as an acyl carrier from extramitochondrial to intramitochondrial sites. This entire problem of fatty acid relocation within the mitochondrion is an important one and will undoubtedly receive more attention in the future. Recently, Beenakkers (1963b, c, 1965) reported on the fatty acid content of the fat body, hemolymph and flight muscles of L. migrutoriu before and after fight. He calculates that these tissues contain enough lipid reserve for five hours of flight and if the substrates of other body parts are utilized, the locust can stay aloft for seven to eight hours. During flight, the esterified fatty acid concentration of the hemolymph increases from 4.0 to 16.9 mg fatty acid per ml while the FFA concentration remains the same. There is a simultaneous 30% increase in the fatty acid content of the fight muscles, half of which is due to linolenate. This suggests a transport of fatty acids from the hemolymph to the flight muscle. The fat body contains 43.5% of its dry weight as fatty acid of which 42% is palmitate. The hemolymph yields 37% palmitate while the flight muscle contains 24% of its fatty acids in the form of palmitate. The decreasing order of concentration supports the contention of transport in that direction and final oxidation in the flight muscles. Beenakkers believes that palmitate and oleate are preferentially oxidized by the flight muscle and calculates that during flight the flight muscle consumes some 23 mg fatty acids per gram fresh weight per

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127

hour and that this fatty acid ultimately is derived from the fat body. These data agree with those discussed previously, indicating the use of fatty acids for flight fuel in some insects. Of particular interest is Beenakker's finding that it is the esterified fatty acids which appear to play the predominant role in lipid transport, although he did not elucidate the nature of these esters. It is probable that they are diglycerides as discussed in Section 111. Thus in insects that ingest lipid, we can now trace with some authority the phases of digestion, storage, release from storage centres, transport and finally oxidation of the fatty acids in tissues such as fight muscle. However, it would be rare indeed for an animal to ingest enough lipid to establish a lipid storage supply that may equal 50% of its entire weight. More commonly we would expect the insect to have the capacity to convert non-lipid substrates into lipid and to synthesize long chain fatty acids and then glycerides from simpler precursors (e.g. acetate). These two processes of substrate interconversion and lipid biosynthesis probably play a more important role in supplying lipid than that of lipid digestion. This is particularly true in phytophagous insects since most plants contain only minute quantities of lipid relative to the amount of carbohydrate (i.e., starch and cellulose). IV. LIPID BIOSYNTHESIS A . GENERAL MECHANISM OF FATTY A C I D SYNTHESIS

Lipid biosynthesis can be thought of as a two stage process. First, fatty acids must be synthesized and second, the fatty acids must enter the pathway leading to the synthesis of glycerides. In both cases, we shall use schemes derived from work on organisms other than insects to present the overall picture; and then attempt to discuss those investigations dealing with insect material. Figure 14 reveals the essentials of what is now understood to be the main pathway for the biosynthesis of fatty acids in living things. In an excellent study, Wakil et al. (1964) have demonstrated that a heat-stable protein called an acyl carrier protein (ACP) participates in fatty acid synthesis as well. This ACP acts by accepting acetyl and malonyl groups from their CoA derivatives forming covalently linked acetyl and malonyl derivatives. Acetyl-ACP and malonyl-ACP then react to yield acetoacetyl-ACP. In the presence of NADPH, they react to form long chain fatty acids. When an enzyme fraction is incubated with acetyl CoA, malonyl CoA, NADPH and ACP, a mixture of I5-hydroxyacyl-ACPs of even numbered carbon chain 6'

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LAWRENCE I . GILBERT

length are formed. These range from C8 to C14.On further incubation with NADPH, enzyme and malonyl CoA, Wakil and his colleagues demonstrated the conversion of the &hydroxyacyI derivatives to their saturated homologues or palmitic acid. The final product was palmitic acid but in the presence of stoichiometric quantities of ACP, they were able to isolate palmityl-ACP. On the basis of these results they propose the following scheme for fatty acid synthesis:

+

(1) CH,COSCoA+ HSACP * CH3COSACP COASH (2) HOOCCHzCOSCoA HSACP HOOCCHZCOSACP COASH (3) CH3COSACP HOOCCHZCOSACP CH3CHOHCHZCOSACP COZ + HSACP (4) CH3COCHzCOSACP + NADPH + H + ---j CH3CHOHCHZCOSACP + NADP" (5) CH3CHOHCHzCOSACP * CH,CH=CHCOSACP Ha0 (6) CH3CH=CHCOSACP + NADPH + H * CHSCHZCHZCOSACP + NADP

+

+

+

+

+

+

+

Repetition of steps (3) to (5) six more times will yield palmityl-ACP which is then hydrolyzed to palmitic acid and ACP. There may be two systems for the biosynthesis of fatty acids. One is a mitochondrial system that may essentially be the reverse of &oxidation and is primarily concerned with the elongation of existing fatty acids by two carbon units (e.g. Cle to C18).The second pathway resides in the cytoplasm outside of the mitochondria and here one can demonstrate the conversion of acetyl CoA to palmitate (see Wakil, 1961, 1963 for an excellent review of this subject). This extra-mitochondria1system appears to function as in Fig. 14. Not all investigators agree that the main fatty acid biosynthetic system lies soleIy in the soluble fraction of the cell. Hulsmann (1962), for example, believes that mammalian heart sarcosomes incorporate acetate into long chain fatty acids by a mechanism similar to that of the soluble system. He goes so far as to suggest that the mitochondria are solely responsible for lipogenesis and that activity of the soluble system is due to mitochondrial enzymes that have escaped from their subcellular enclosures during fractionation. Lung tissue mitochondria also appear to be capable of synthesizing fatty acids (Tombropoulos, 1964). Thus, even in the more numerous experiments involving mammalian material, some doubt exists regarding the biosynthesis of fatty acids. As we shall see subsequently, information is sparse regarding the means by which insects synthesize unsaturated fatty acids and the situation is not greatly improved when we consider other animal forms

LIPID METABOLISM A N D F U N C T I O N I N INSECTS

129

(cf. Langdon and Philips, 1961). This problem is of significance to the insect since some insects require one or more unsaturated fatty acids in their diet (see Section D), and they constitute a large percentage of 0

II

CH3C-OH

\ / CoASHf ATP

CH~C-SC~A

CO,

+ ATP

3ADP +HP,O,+

O It C-OH

I

CHZ

I C-SCoA

0

ti

II

0

CH3C- SCoA

+ 2 NADPH +H+

CH,CH~CH,C--SCOA I

:a .G

C, Fatty acid

+CO,

FIG.14. Simplified scheme of fatty acid biosynthesis. a. Formation of acetyl CoA. b. Formation of malonyl CoA in the presence of COa through a biotin-COa complex. c. Condensation of several reactions whereby malonyl CoA acetyl CoA yield butyryl CoA COa. d. Butyryl CoA+malonyl CoA yields a Cg fatty acid through a series of reactions. Once rnalonyl CoA is present in sufficient quantities, acetyl CoA is needed only in the formation of the C4 fatty acid. After this, the process proceeds by the addition of rnalonyl CoA and the evolution of COa resulting in the lengthening of the molecule by one Ca unit at a time. For the formation of stearic acid the overall reaction would be: acetyl CoA 8 rnalonyl CoA 16 NADPH H stearyl CoA 16 NADP 8 COa 8 CoASH

+

+

+

+

+

+

-

+

+

+

the total fatty acids present in others (see Section B). Bloomfield and Bloch (1960) have demonstrated that anaerobic yeast possesses a mechanism for desaturation of palmitate to palmitoleic acid and stearate to oleate. In their view, the fatty acids are first converted to CoA thioesters and then desaturated by a mechanism involving formation

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LAWRENCE I . GILBERT

and dehydration of hydroxyl intermediates. If this mechanism applies to insects as well, then desaturation occurs subsequent to synthesis of the carbon chain. Perhaps, in those reports where insects incorporated labelled precursors into saturated fatty acids only, the experiments were not allowed to proceed for a long enough period of time. Mead (1963) suggests that the pathways of biosynthesis of monoenoic acids in animals may be quite different from those leading to saturated fatty acids. If this proves true, then we are at present completely ignorant of this phenomenon in insects. In a more recent investigation, Schroepfer and Bloch (1965) demonstrated the stereospecificity of the reaction responsible for the conversion of stearic to oleic acid in bacteria. The desaturating enzyme removes only one of each pair of hydrogens at Cg and Clo. The precise mechanism of desaturation is still unknown although it appears that hydrogen removal at C9 occurs prior to the corresponding desaturation at Clo. B . FATTY A C I D BIOSYNTHESIS I N INSECTS

That insects can synthesize long chain saturated fatty acids from simple precursors has been shown numerous times in various species (see for example, Bloch et al., 1956 and Rajalakshmi et al., 1963). In the course of these experiments that utilize labelled precursors, unsaturated fatty acids also exhibit the label. When the green peach aphid (Myzus persicae) is fed through an artificial membrane on a diet containing 18% sucrose solution with the addition of either [1-l4C]acetate or [U-14C]glucose,the insect was found to incorporate 75% of the label into stearic, palmitoleic and oleic acids (Strong, 1963b). Small quantities of myristic, linoleic and linolenic acids also contained label although there was no significant incorporation of these precursors into short chain fatty acids. It is of interest to note that both aseptically reared and non-aseptic adult boll weevils (Anthonornus grandis) synthesize long chain fatty acids from acetate at about the same rate (Lambremont, 1965). Palmitic, oleic and stearic were the major fatty acids synthesized but myristate, palmitoleate and oleate also were labelled when [14C]acetatewas injected. In addition to demonstrating the biosynthetic capacity of these insects, Lambremont’s results also suggest that the animal’s symbiotic flora make no significant contribution to fatty acid biosynthesis. The inability of Anthonornus to synthesize linoleate is in accordance with this fatty acid’s important role in the nutrition of several insects.

, ‘

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131

By extracting whole larvae and pupae of B. mori, Sridhara and Bhat (1964) studied the fate of [l-14C]acetate ingested by, or injected into fifth instar larvae. As one would expect, the more metabolically active larvae incorporated almost twice as much label into lipid as did the pupae, and the radioactivity in the saponiiiable fraction was double that in the unsaponifiable fraction. The major component of the latter mixture was hydrocarbon and it is possible that this was due to the synthesis of cuticular lipids since whole insects were extracted. The most heavily labelled fatty acids in this case were the saturated molecules, predominantly palmitate and stearate. The most heavily labelled unsaturated fatty acid was oleate and no label was detected in either linoleate or linolenate. It appears from this work and that on Prodenia by Zebe and McShan (1959) that saturated acids are synthesized at a higher rate in Lepidoptera than the unsaturated series. This contrasts with much of the work on the Orthoptera and Diptera, although Tietz’s (1961) in vitro studies suggest that palmitate is the major fatty acid synthesized by locust fat body. Recently, Bade (1964) demonstrated that after the injection of [l-14C]acetate,EurycotisJloridana showed a labelling pattern consistent with the classical view of the biosynthesis of fatty acids by the condensation of C2 units. Oleate composed about one half of the total newly synthesized fatty acids while palmitate was the most abundant saturated fatty acid. This is in agreement with the data of Sedee (1961) on Calliphora erythrocephala. Bade detected only traces of fatty acids having more than 18 carbons, and arachidonic acid was absent. From her data, Bade postulates that oleate is synthesized by desaturation of stearate. This is in accordance with the views of Bloomfield and Bloch (1958) but disagrees with Sedee’s (1961) supposition that insects use two different mechanisms for the synthesis of saturated and unsaturated fatty acids. Although this work of Bade’s was conducted in vivo rather than on individual tissues, it is significant because xenic and aseptic cultures were utilized. Louloudes et al. (1961) had demonstrated the synthesis of linolenate by another cockroach, Blatella, but this multiple desaturation may have been a consequence of gut symbionts. The main difference between the fatty acids of Eurycotis and those of higher animals is the virtual absence of fatty acids with a chain length greater than CI8 in the insect. Although the techniques of injection of labelled precursors, followed by the extraction of the whole insect (aseptic or non-aseptic) and radioassay of the resulting fatty acids, do tell us if the insect possesses the biosynthetic capability of producing fatty acids, they give us relatively

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little information regarding the pathway of synthesis, the enzymes involved or cofactors required. To elucidate these latter points, we must turn to in vitro studies. This is in no manner intended to deprecate in vivo experiments, for even while using homogenates or sub-cellular fractions, we must still strive to extrapolate the results back to the living insect. One of the pioneering in vitro studies on fatty acid biosynthesis in insects was that of Zebe and McShan (1959) on the fat body of the adult lepidopteran, Prodenia eridania. Acetate was readily incorporated into long chain fatty acids but required malonate, ATP, CoA and a reducing agent such as glutathione or cysteine (containing SH groups) for maximum incorporation. The major fatty acid produced was palmitic although small amounts of stearate, oleate, myristate and laurate were also identified. Homogenates had less than one-third the activity of intact fat body in synthesizing fatty acids. Zebe and McShan also demonstrated a slight conversion of [14C]glucoseinto fatty acid and showed that muscle was only one-fifth as effective as fat body in synthesizing fatty acids. In an elegant series of studies on the capacity of locust fat body homogenates and sub-cellular fractions to synthesize fatty acids, TietzDevir (1963) showed that homogenates were optimally effective when the incubation medium was supplemented with ATP, MgC12, glutathione, KHC03 and malonate. Addition of CoA and NADPH further stimulated the synthesis (Table XI). Although the use of citric acid cycle intermediatesdid not relieve the dependency of the system on malonate, they resulted in even greater stimulation when added in the presence of malonate. Of these intermediates, a-ketoglutarate was most effective. When the particulate matter was removed from the homogenate by centrifugation at 20,000 x g for 20 min, the supernatant proved to be as effective as the original homogenate. The newly formed fatty acids were esterified with glycerol almost immediately after synthesis and the FFA did not accumulate to any sigtllficant degree. Whereas in the homogenate 18% of the newly synthesized fatty acids were in the form of phospholipid and 8 1% as glycerides, in the supernatant 10% were in the phospholipid fraction and 90% were in the form of glycerides. Of the fatty acids synthesized, more than half was palmitate. The optimal conditions cited above were similar to those utilized in mammalian and avian systems (PopjAk and Tietz, 1954; Porter et al., 1957). The function of the malonate is not known but Tietz-Devir believes that the bicarbonate is utilized in the conversion of acetyl CoA to malonyl CoA. Kallen and Lowenstein (1962) had shown with non-

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133

insect material that stimulation of fatty acid synthesis by isocitrate and malonate is not linked to NADPH generation but is more likely connected with the synthesis of malonyl CoA. Using 14C02, Tietz-Devir also demonstrated CO, fixation by a combined particulate and supernatant system from the locust fat body. 91% of the label was recovered in malonate, 9% in acetate but none in the newly synthesized fatty acids. The particulate fraction was necessary for C02 fixation and for activation and decarboxylation of malonate. Thus in the supernatant fraction, malonate and bicarbonate TABLE XI Requirements for fatty acid synthesis by fat body homogenates of Locusta migratoria Cofactor omitted None1 ATP CoA Glutathione NAD NADP MgCL MnS04 Malonate a-Ketoglutarate KHC03

Acetate incorporated into fatty acid (pmoles) 0.33 0.05

0.14 0.17 0.33 0.19 0.01 0.32 0.01 0.20 0.08

Complete system contained (in pmoles); acetate (5); ATP (5); CoA (0.1 mg); Glutathione (5); NAD (0.5); NADP (0.5); MgCla (10); MnSOI (0.5); Malonate (20); a-Ketoglutarate (10); KHCOa (lo), and 0-6 ml (2 mg protein) homogenate in a total volume of 1 ml. pH 7-0. (From Tietz, 1961.)

stimulate fatty acid synthesis but yet the supernatant cannot metabolize these compounds. This contradiction is unexplained at present. The above, although by no means an inclusive survey, does support the proposition that many insects can synthesize -longchain fatty acids from acetate, presumably by the same means employed by both their inferiors (micro-organisms) and superiors (vertebrates) on the evolutionary scale. Once these FFA are synthesized, they are shunted into TGL and PL as can be seen by the relatively small quantities of FFA present. Since the biosynthetic pathway of the glycerides and phospho-

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L A W R E N C E I . GILBERT

lipids is in part a common one, we will discuss these two lipid types together. C . P H O S P H O L I P I D A N D TRIGLYCERIDE

1. General mechanism of synthesis

Again we must call on data derived from investigations of non-insect material to describe both the biosynthetic pathway of the glycerides and phospholipids as well as the function of PL. Figure 15, derived mainly Glycerol

ATp

a-Glycerophosphatef ATPI

Di-hydroxyacetone phosphate

NADH

1 2 Acyl-CoA

Phosphatidic acid /

Phosphatidyl inositol

Phosphatidylcholine

7

YCDP-choline

inositol

CDP-diglyceride

ie

a-Glycerophosphate

Phosphatidylglycerophosphate AcYI-COA

Phosphatidylglycerol CDP-diglyceride

riglyceride

Cardiolipin

FIG.15. The biosynthesis of phospholipids and triglycerides. (From Kennedy, 1961, 1963.)

from the classic experiments of Kennedy and his colleagues, reveals the close interrelationship between the synthesis of these two types of lipid. In addition to the pathway via a-glycerophosphate to TGL, another subordinate pathway via monoglyceride has been suggested (Hubscher, 1963). Monoglyceride

acetyl CoA

+ diglyceride

acetyl CoA

t

triglyceride

In this discussion we will lean heavily on the work of Kennedy’s laboratory and will allude to aspects of TGL synthesis as well (Kennedy,

LIPID METABOLISM A N D F U N C T I O N I N I N S E C T S

135

1961, 1963; Weiss and Kennedy, 1956; Weiss et al., 1960). For a complete review of the phospholipids, the reader should consult Ansell and Hawthorne (1964). It now appears that phosphatidic acid is the key compound in the synthesis of PL and TGL (Fig. 15). This compound is derived from a-glycerophosphatewhich itself results either from the phosphorylation of glycerol or reduction of dihydroxyacetone phosphate. It was at first believed that the glycerophosphate was mainly a consequence of the glycolysis of glucose. Recent data indicate the possibility of free glycerol acting as a precursor for the a-glycerophosphate through interaction with ATP (Isselbacher, 1965; Fig. 16). In at least some insects, there is a high glycerol titre in the hemolymph during the pupal stage and this becomes drastically reduced during adult development (Wyatt and Meyer, 1959; Wilhelm et al., 1961). It is possible that this glycerol may be used for the formation of a-glycerophosphate which then enters the pathway for the synthesis of PL or TGL, both of which are required for the development of the adult insect. Of importance is the requirement for the cytidine coenzymes. For the cell to obtain CDP-ethanolamine, ethanolamine must react with ATP to yield phosphorylethanolamine which in the presence of CTP gives rise to CDP-ethanolamine. Analogous reactions occur with choline finally to produce CDP-choline. Phosphatidic acid can be dephosphorylated in the presence of phosphatidic acid phosphatase to yield a D-a,&diglyceride which in turn can be converted to TGL, phosphatidylethanolamhe (PTE) or phosphatidylcholine (PTC). The DGL can also be rephosphorylated to phosphatidic acid by diglyceride kinase and in some instances this may be an important pathway (Hokin and Hokin, 1959, 1960; Strickland, 1962). Phosphatidic acid and the compounds derived from it contain fatty acids of varying degrees of unsaturation and of different chain lengths. As Kennedy (1963) suggests, these fatty acids bound to PL can be an important source of reserve energy for the cell. The scheme outlined above points out the close relationship between glycolysis, PL synthesis and TGL synthesis (see also Fig. 17). If for example, glycolysis was inhibited prior to the formation of pyruvate, there is a possibility that C3 units from glucose might be converted to lipid in the form of glycerides or PL. If there is an uninhibited flow from glucose to acetate, the acetate may give rise to fatty acids and join that moiety of the PL or glyceride molecule. It is now generally believed that the biosynthesis of choline and PL proceeds from the decarboxylation of serine and includes three methyla-

136

LAWRENCE I . G I L B E R T

CH,OH

I

CHOH

HOCH

I

0

CH,0P03H, 0 0

II

R-CH,C-OCH

II

II

2 R-CH,C-SCoA 2 CoASH

CH,OC-CH,-R

I I

CH,OPO,H,

0

II

CHZOC-CHZ-R

0

II

R-CH,C-0-CH

CH,&-CH,-R

I I

0

II

CH,OC-CH,-R FIG.16. Triglyceride biosynthesis from glycerol. a. Glycerol + ATP yields L-a-glycerophosphate in the presence of glycerokinase. b. a-Glycerophosphate+ 2 fatty acyl CoA derivatives yields phosphatidic acid. c. Phosphatidic acid is converted to a diglyceride in the presence of phosphatidic acid phosphatase. d. Diglyceride is converted to triglyceride.

137

LIPID METABOLISM A N D F U N C T I O N IN INSECTS

/Phospholipid

Glycerides

\

Carbohydrates

1 1

Phosphatidic acid COASH Sphingolipids *.-T Triosephosphate + a-Glycerophosphate+ ir Long-chain acyl-CoA2

t f

P

M

a

l

o

\Fatty acid

nyI-CoA

";" '.t\

Amino --f Acetyl-CoA A A c e t o a c e t y l - C o A *Methyl glutaryl-CoA acids J bcitrate Acetoacetate Terpenes Acetylations Oxalacetate 2 coz Sterols

3

J

\

+

FIO.17. Some interrelationships between lipid, carbohydrate and protein metabolism. (From Lynen et al., 1963.)

tion reactions (cf. Wagner and Folkers, 1964). Choline itself can be incorporated into PL in the mitochondrion while serine can be incorporated into phosphatidylserine prior to any decarboxylation. This latter reaction is thought to proceed in the microsomes. Thus, amino acids can become an integral part of PL synthesis.

2. General functions of phospholipids Phospholipids play a variety of roles in the life of the cell. Their presence in cell membranes has been unequivocally accepted. For example, the human erythrocyte membrane has about 59% PL of which about 35% is PTC and 25% PTE (Wolfe, 1964). The RNA-free microsomal fraction of neuronal membranes contains about 65% phosphatides. Petrushka et al. (1959) believe that PL is responsible for the spatial configuration of the mitochondrion, since these organelles rupture when PL is removed. Their low solubility in water makes the PL ideal substances for use in membranes that are vital for both the partitioning of separate cells and the microcompartmentalization of the cytoplasm. Their importance in oxidative phosphorylation is well known (Fleischer et al., 1961; Garbus et al., 1963; Green, 1962). Wojtczak et al. (1963) postulate that the phosphatidic acid formed during mitochondria1 contraction may be involved in tlfe mechanism of the active extrusion of water from the mitochondria. In fruits, it is

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LAWRENCE I . GILBERT

suggested that the initiating signal for senescence may be triggered by ethylene acting on the permeability of the mitochondria1 membrane (Biale, 1964) and may be affecting phospholipid in the membrane. A large amount of data has accumulated that links PL with transport of sodium ions (cf. Wolfe, 1964). A possible intermediate for this cationic transport is a labile phosphate attached to specific phosphatides or a lipo-phosphoprotein complex. This compound is believed to be rapidly phosphorylated by ATP through the activity of membrane ATP’ase. Subsequent dephosphorylation may lead to changes in the configuration of membrane molecules and this perhaps controls cation transport. The main proponents for the role of PL in active transport are L. and M. Hokin (Hokin and Hokin, 1955, 1959, 1960, 1961). Their original observations were on the effects of acetylcholine and various mammalian hormones on PL turnover in several tissues. A few of these substances increased the turnover rate of phosphatidic acid. In their view, ATP and DGL act at the interior of the cell membrane to form phosphatidic acid. The sodium salt of phosphatidic acid then diffuses through the membrane, is hydrolyzed on the exterior surface and results in the release of sodium and phosphate ions. In this way, sodium ions are actively transported. As Ansell and Hawthorne (1964) point out, much of the evidence for PL involvement in the sodium pump is circumstantial and there are many valid objections to the theory. However, it is still an open question. The adult mammalian brain contains between 20-25% of its dry weight as PL. With all the numerous papers published on the analysis and metabolism of PL in the brains of higher animals “no precise function has been attributed to any single component” (Ansell and Hawthorne, 1964; p. 278). Notwithstanding the absence of precise information, PL have been implicated in ageing of the nervous system, regeneration and in various diseases affecting the nervous system. The insect nervous system contains quantities of PL of the same order of magnitude as those reported for other animals (Heslop and Ray, 1961). The lipids of the bee brain for example, contain PTE (12% of total lipid), PTC (2%) and sphingomyelin (1.5%) (Patterson et al., 1945). It is thus probable that PL may play a comparable role in the nervous integration of insects as they do in higher animals. Beef heart mitochondria contain about one fourth of their weight as lipid, of which 90% is PL (the major constituents being PTC and PTE). Biochemical and electron microscopic studies leave no doubt of the central role played by lipids in electron transfer and oxidative phosphorylation within the mitochondrion (Green and Fleischer, 1963). In

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insects for example, NADH-cytochrome c reductase, cytochrome b, and cytochrome oxidase are for the most part located in the “lipid rich particle fraction” of centrifuged homogenates of Bombyx embryos (Chino, 1963). The mitochondria lose much of their biochemical activity after acetone extraction but this can be restored by the addition of coenzyme Q (CoQ) (see Section D) and PL. Green and Fleischer now visualize the mitochondrion as having a PL requirement at three points along the electron transfer chain : succinate

Q:QHa

-

cytochrome c:cytochrome c reduced

-

O2

They suggest that PL interacts with the mitochondria1 proteins in two types of reactions. One is ionic between the acidic PL and basic proteins like cytochrome c while the other is hydrophobic between the PL and structural protein. One of the properties of the PL which make them indispensable to the mitochondrion is the fact that they orient in water to form micelles. The amphipathic character of the PL molecule allows it to act as a bridge between water and protein. Green and Fleischer propose that the PL provides a non-aqueous medium (giving it a low dielectric constant) in which specific reactions can take place. The oxidationreduction reactions along the electron transfer chain appear to be oriented in such a way so as to be in contact with or actually lay within the PL layer that envelops the complex. Without PL, the mitochondrion ceases to function. In addition to the PL and ubiqcinone content of mitochondria, lipids may be important in another aspect of mitochondria1 function. Lewis and Fowler (1960) showed that in blow-fly flight muscle mitochondria, an endogenous lipid uncoupling agent was generated enzymatically. This had previously been suggested from the work of Lehninger and Remmert (1959) in rat liver mitochondria. It is proposed that this lipid may be a crucial factor in the reversible swelling and contracting of mitochondria; a phenomenon that may be vital to mitochondria1 regulation. 3. Metabolism and function in insects Having denoted some of the functions of PL we can now turn to PL and TGL in insects. Little can be tabulated regarding the synthesis of TGL in insects other than what we have discussed previously. No work has been reported on this biosynthetic pathway in insects although we know the fat body readily esterifies FFA intu TGL (Section 11). The elucidation of the role of the cytidine cofactors in the insect would

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be of real interest but little has appeared on this subject as of this time. In a histochemical study of the larval fat body of the dermestid beetle, Anthrenus uorax, Nair and George (1964) showed that the peripheral globules of the fat body cells are composed of a PL-protein complex, whereas the centrally located globules are composed of glycerides. On the basis of this microscopical study, they propose that the peripheral globules are lipopoietic centres where dietary amino acids are deaminated and converted into specific lipids. The lipids are then supposed to be transported to the centre storage globules. Although an interesting hypothesis, there is little or no biochemical evidence for or against it. It is at best, difficult to draw conclusions regarding the temporal sequence of a biochemical process on the basis of microscopical observations. As mentioned previously, several insects have large concentrations of glycerol in their hemolymph (Wyatt and Kalf, 1957; Wyatt and Meyer, 1959; Salt, 1957; Wilhelm et al., 1961). Salt (1959) has shown that larvae of the sawfly Bracon sephis contain concentrations of glycerol as high as 25% (5 molal) in the hemolymph. In H . cecropia, glycerol may constitute up to 3.5% of the hemolymph and tissues of diapausing pupae. This glycerol appears to be derived from glycogen. In diapausing embryos of B. mori, both glycerol and sorbitol are present (Chino, 1958). It is possible that this glycerol may be a precursor of PL and TGL in these insects in addition to acting as an “anti-freeze” (see Touster and Shaw (1962) for a review of the biochemistry of polyhydric alcohols). Our results (Habibulla and Gilbert, 1965) indicate a high rate of incorporation of labelled glycerol into the phospholipids of various tissues of developing adults of H. cecropia. As part of a survey of invertebrate phospholipids, Hack et al. (1962) studied insects representative of five orders. All contained PTE, PTC, monophosphoinositide, polyglycerophosphatide, ethanolamine plasmalogen and traces of choline plasmalogen. Thus, insects appear to contain the same general types of PL as the vertebrates. A great deal of the work on PL in Lepidoptera has been conducted on Galleria mellonella with a view towards explaining the mechanism of wax digestion. Galleria larvae possess a remarkably high concentration of PL in the hemolymph (about 22% of the hemolymph lipid) and on a percentage basis contain twice the amount present in mammalian plasma (Wlodawer and Wisniewska, 1965). There appears to be a discrepancy regarding the PL constitution of waxmoth larval hemolymph with Lenartowicz (1961 ; Lenartowicz and Niemierko, 1964;

141 Lenartowicz et al., 1964) demonstrating large quantities of phosphorylethanolamine and phosphorylglycerol in the larval lipids, and more phosphorylethanolamine than phosphorylcholine in the hemolymph. These investigators assert that the concentration of phosphorylcholine in the hemolymph is the highest reported for any animals. Both the phosphorylcholine and phosphorylethanolamine concentrations exceed those reported for B. mori, H. cecropia, A . polyphemus and P. sexta (Wyatt et al., 1963). On the other hand, Wlodawer and Wisniewska (1965) maintain that PTC makes up 60% of the hemolymph PL while PTE constitutes only 20%. Sphingomyelin was not detected. The possibility exists however that phosphorylcholine which is a precursor of CDP-choline and PTC, is present in higher quantities due to a block leading to PTC, while the titre of phosphorylethanolamine remains low because it is rapidly converted to PTE. This is speculation however, and the discrepancy has not yet been resolved. The means by which these compounds come to be in the hemolymph in such great quantities has not been adequately explained. In their studies of H. cecropia, Wyatt’s laboratory (Carey and Wyatt, 1963; Wyatt et al., 1963) showed that the concentrations of phosphorylcholine and phosphorylethanolamineare higher in the hemolymph than in the fat body and the enzymes necessary for the synthesis of these PL appear to be absent from the hemolymph. With this circumstantial evidence, they suggest a selective secretion of these compounds from the fat body into the hemolymph. The role of PL in the hemolymph is obscure but they may be involved in lipid transport as they are in mammals (see Section 11). Our data on H. cecropia (Habibulla and Gilbert, 1965) reveal that although PTC and PTE are the predominant PL of the fat body and other tissues, phosphatidic acid is the major PL component in the pupal hemolymph. In fact, phosphatidic acid and cardiolipin constitute 80% of the pupal hemolymph PL but this decreases during adult development to reach a low of 3%. The hemolymph phosphatidic acid is utilized during development as a precursor for TGL and PL synthesis in the fat body. It is of interest in this regard that histochemical data have implicated the oenocytes as the major depot for PL in the locust rather than the fat body cells (Coupland, 1957). In waxmoth larvae, the hemolymph is suggested to be a phospholipid “depot” (Lenartowicz and Niemierko, 1964). This would have no counterpart in higher animals but Galleria appears to be an atypical animal on the basis of its diet. These same authors suggest that phosphorylethanolamine is utilized to a higher degree than”its choline counterpart during starvation and that its metabolism is independent of L I P I D METABOLISM A N D FUNCTION I N INSECTS

142

LAWRENCE I . G I L B E R T

phosphorylcholine. On the other hand, Wlodawer and Wisniewska (1965) found little qualitative change during the period of food deprivation. The question remains open. In addition to the PL discussed above, some Lepidoptera contain a minor component containing alanine but its nature has not yet been resolved (Hodgson, 1965). In a developmental study of the PL content of B. mori, Niemierko et al. (1956) showed that the quantity of PL as percentage of total lipid reaches a maximum at the end of metamorphosis. They postulate synthesis of PL toward the latter part of adult development. Our work on Cecropia supports this contention and suggests that it is due mainly to the morphogenesis of the giant mitochondria in the developing flight muscle (Habibulla and Gilbert, 1965). We must take care in evaluating differences between synthesis and turnover. For example, the fatty acid portion of PTC is known to turn over without either synthesis or breakdown of the remainder of the molecule (Lands, 1958). Thus incorporation of label into PL does not necessarily imply synthesis. Only quantitative evaluation by gravimetric procedures will allow a true differentiation between these two alternatives. The study of Carey and Wyatt (1963) suggests a steady turnover of PL in the Cecropia fat body. Radiotracer studies also indicate a rapid turnover of hemolymph PL but this in turn is most likely due to turnover within other tissues and subsequent release into the hemolymph. In contrast to the results cited above on Bombyx, Sridhara and Bhat (1965a) reported that 23% of the larval lipid of this insect is PL while the lipid of pupae and moths is only 18 to 19% PL. Of the various larval body parts analysed, silk gland had the highest concentration of PL (68% of total lipid) whereas about half the total lipid extracted from the hemolymph and intestine was PL and only 3% of the fat body lipid was PL. Of interest is the finding by these investigators (1962) that the PL of Bornbyx contains about 20 times more choline than ethanolamine. This is in contrast to the PL constitution reported for Diptera and Orthoptera which will be discussed subsequently. In addition, a considerable amount of sphingosine was found whereas it is absent from the Diptera. These results are similar to those of Chojnacki (1961; Chojnacki and Korzybski, 1962, 1963) working on another moth Acta caia. In the adult there is about twice as much PTC as PTE with a relatively high concentration of sphingomyelin. It appears that these PL in Acta are utilized during adult life as the quantity of lipid phosphorus diminishes with the age of the moth. Using 32Porthophosphate, these workers demonstrated a higher rate of incorporation into PTE of Celerio eurphorbiae adults than into the PTC. On the basis

LIPID METABOLISM A N D FUNCTION I N INSECTS

143

of specific activities, they suggest that PTC is incorporated in toto into PTE. According to present information however (Fig. 15) it is more likely that phosphorylcholine is first converted to CDP-choline which combines with diglyceride to yield PTC. Investigations of the PL of Diptera have been primarily concerned with the analysis and incorporation of nutrient precursors into these compounds. In contrast to higher animals, the major PL fraction has been identified as PTE (Bieber et al., 1961) and in A . aegypti this PL comprises 86% of the total extractable PL (Fast and Brown, 1962). Bieber et al. (1961) demonstrated that when larvae of Phormia regina were reared on a carnitine containing medium, a new “lecithin” was recovered. This compound constituted 20% of the total PL and was eluted prior to PTC on silicic acid columns. It contained 8-methylcholine rather than choline and was absent from larvae raised on a choline containing medium rather than on one having carnitine. It is thought that the larvae have the ability to decarboxylate carnitine, and under conditions where choline is limited, other quaternary ammonia compounds can be utilized for structural purposes, such as the manufacture of membranes (Bieber ef al., 1963; see however, Agarwal and HOUX,1964). In 1963, Bieber and Newburgh demonstrated that dimethylaminoethanol and dimethylaminoisopropanol are incorporated into the PL of Phormia when they replace choline in the diet. Under these conditions the quantity of choline detected in the PL fraction was drastically reduced. It appears then, that the quality of the PL is not of great importance to the functioning of the organism and agrees with the contention of Fleischer et al. (1962) that although the respiratory activity of mammalian mitochondria depends upon the presence of lipids, it is not dependent on one specific lipid organization. Following the work on Phormia, it was found that Musca domestica larvae bred on a chemically defined diet under aseptic conditions could also incorporate 8-methylcholine into PL when the dietary source of choline is replaced by either carnitine or 8-methylcholine (Bridges et al., 1965). Although a low level of PTC was present in larvae grown on this choline-deficient diet, it was absent from the adult flies. It may be that the ability to decarboxylate carnitine is lost during metamorphosis. If this is truly a juvenile characteristic, it may be a proper mechanism for the study of the effect of insect hormones on the synthesis or activation of specfic enzymes or enzyme systems. Though grown on a diet containing carnitine, these flies were unable to synthesize phosphatidyl carnitine (see also Agarwal and Houx, 1964). In none of the studies discussed above was the function of PL in

144 LAWRENCE I . GILBERT insects dealt with. They were nutritional studies which showed that the insect was capable of certain biochemical feats. In general, the extraction of whole insects is not the most efficient method of studying physiological mechanisms. In an attempt to use specific sub-cellular organelles, Crone (1964; see also Price and Lewis, 1959) analysed the PL content of the flight muscle sarcosomes of M. domestica. The sarcosomal lipid phosphorus comprises about 7% of the total lipid phosphorus in the adult female or about 210 mg of PL/g of protein. This figure is close to that reported for beef heart mitochondria (Fleischer et al., 1962). Relatively, the proportion of total sarcosomal PL existing as PTC is much lower than that of the whole fly (Crone and Bridges, 1963). After injection of 32P,the PTC was shown to have a greater specific activity than the PTE. Of the total lipid phosphorus in the sarcosomes, 69% was due to PTE; 9% to PTC and 6% to polyglycerolphosphatide. Most of the polyglycerolphosphatide of the fly was located in the thorax and as in mammalian tissues, was associated with the mitochondria. It is most likely that PL in insect mitochondria play a role similar to that played in mammalian mitochondria. The function of PL in the insect sarcosome is certainly an open question and one well worth answering. In addition to their most certain role in the post-embryonic development of insects, PL play an important role in insect embryogenesis. The egg lipid of L. migratoriu, for example, is composed of about 20% PL, which actually constitutes about 5% of the total dry weight of the egg (Allais et al., 1964). PTE predominates but phosphatidylserine, PTC and a sphingomyelin-like lipid were also identified. There was little variation in PL during embryogenesis although blastokinesis occurred simultaneously with an 11% decrease in PTC. Perhaps the energy for this vital embryonic process was supplied by PTC. Allais and his colleagues also demonstrated synthesis of PL at the termination of blastokinesis. This phenomenon of PL synthesis during embryonic development has also been reported for Bombyx (Niemierko et al., 1956). It is likely that as cell differentiation progresses more PL is needed for the construction of cellular and sub-cellular membranes. Insects then, can synthesize many of the long-chain fatty acids and esterify these into PL and TGL. Fatty acid biosynthesis is most likely a process very similar to that found in other organisms. The data regarding the synthesis of PL and TGL are sparse but until contrary evidence is forthcoming, we must postulate that here again, the insect does not break biochemical regulations. Can insects, however, synthesize all the fatty acids necessary for life?

L I P I D METABOLISM A N D F U N C T I O N I N INSECTS

145

D. FATTY ACIDS I N NUTRITION

Although oleic acid can be synthesized by vertebrates, linoleate and linolenate are thought to be dietary requirements. Linoleate in fact is believed to be a primary precursor for essential fatty acid biosynthesis in mammals (cf. Wagner and Folkers, 1964). A fatty acid deficiency in mammals is characterized by low survival rate, subnormal growth, scaly skin, skin lesions and impaired ability to reproduce. One can draw a close analogy between these nutritional defect symptoms and those reported in insects. For reviews of insect nutrition see Friend (1958) and House (1962, 1965). More than twenty years have passed since Fraenkel and Blewett (1945, 1946) demonstrated that wheat germ oil was needed for the normal development of Ephestia kuhniella. The critical substance in the wheat germ oil was identified as linoleic acid. This unsaturated fatty acid is needed for the normal formation of adult scales and is perhaps necessary for either the production of moulting fluid or the proper functioning of the moulting fluid. Linolenate was equally effective but oleate was an inadequate substitute, indicating that this insect cannot convert oleate to linoleate. In 1947, these same investigators showed that Tenebrio larvae do not require linoleate, indicating that linoleate or linolenate can be synthesized by Tenebrio (or its symbionts). Since this early work there have been numerous reports on the necessity in the diet of various fatty acids if the normal physiological processes of the insect are to continue. For example, Gordon (1959) demonstrated that the progeny of Blattella germanica that were raised on a linoleate deficient diet showed deficiency symptoms (see also Pant and Pant, 1961). Dadd (1960a, b) corroborated the need for linoleate in L. rnigratoria and S. gregaria. Although larval growth of GalZeria is not affected by a diet devoid of linoleate, successful emergence of the adults from the pupal cuticle depends on the presence of linoleate or linolenate in the diet (Dadd, 1964; see also Vanderzant et al., 1957). In Argyrotaenia velutinana, linoleic or linolenic acids are needed for normal growth. Gas chromatographic analysis revealed that pupae of this species grown on deficient diets did not contain either linoleate or linolenate (Rock et al., 1965) indicating a biosynthetic block. From the vast nutritional literature one can conclude that either several insects cannot synthesize linoleate or linolenate from less complex precursors and so are similar to mammals, or the proper precursors are not yet known and are thus omitted from the defined diets. However, the questions as to how these fatty acids promote normal growth and where the metabolic blocks exist, are still

146

LAWRENCE I. GILBERT

unanswered. While on the question of fatty acids and nutrition, it is of interest to note that in the locust, the maternal diet influences the lipid content of young locusts (Blackith and Howden, 1961); the reasons for this are unknown. One problem regarding this nutritional work concerns the purity of the fatty acids. Until fatty acid analyses could easily be conducted with gas chromatography, the investigator had to trust his commercial source. In our experience, many of the so-called pure lipids produced commercially are grossly contaminated and perhaps these contaminants have a nutritional value. The data of Chippendale et al. (1964) support this contention. Their work on the dietary requirements of the cabbage looper, Trichoplusia ni, suggest that linolenate is an essential nutrient and that linoleate cannot replace it. This is the reverse of the situation in the rat and many of the insect studies. These investigators used fatty acids of extremely high purity and point out that many of the past effects attributed to linoleate may have been the responsibility of contaminating linolenate. This infers a critical re-evaluation of all the past work where commercial fatty acids not analysed by the investigator were used. As Dadd (1963) points out, the dietary requirements for unsaturated fatty acids that have been demonstrated, were demonstrated on only a few species of Lepidoptera and Orthoptera. Thus we are led to the possibility that some insects may indeed be able to synthesize all required fatty acids. The lipid content of the insect’s diet may have physiological consequences far removed from those cited above. By increasing the lipid content of the larval diet of the pink bollworm, the insect can be induced to enter diapause at a greater rate (Bull and Adkisson, 1962). This finding is in agreement with those of Squire (1940) and Vanderzant and Reiser (1956). Since diapause is usually thought of as a hormonal deficiency (cf. Gilbert, 1964), the presence of a critical amount of lipid in the digestive tract, hemolymph or fat body may influence the synthesis and/or release of a hormone (perhaps through nervous stimuli acting on the neurosecretory cells of the brain). The large lipid store is of course important to diapausing insects as a ready energy source for this non-feeding stage. As we shall see subsequently, several of the insect hormones are lipoidal and lipid appears to be the precursor for their synthesis. In addition to possessing a growth promoting effect, fatty acids may also have a growth inhibiting effect in insects. A recent report claims that particular fatty acids can prevent the normal growth and development of young larvae of the orthopteran, Gryllodessigillatus (McFarlane

147 and Henneberry, 1965). In these experiments, methyl oleate and methyl palmitate were most effective. Curiously, they were only effective when applied to the exterior of the insect, presumably since this method of application bypassed any detoxication mechanism that may be present in the gut. Although the effects may be real, their significance remains obscure since one does not usually find insects wading through pools of fatty acid esters. As far as this author is aware, no data exist that conclusively demonstrates any nutritional requirements for the fat soluble vitamins. Perhaps any such requirements are met by the synthetic capabilities of symbiotic micro-organisms (see Henry, 1962 for a discussion of micro-organisms and insect nutrition). Notwithstanding the criticisms cited above, it may be that dietary fatty acids will be ultimately shown to have profound effects on insect growth. This is certainly the case for sterols (see Section VI), but insects cannot synthesize any sterol. LIPID METABOLISM A N D FUNCTION I N INSECTS

E. SUBSTRATE INTERCONVERSION

A final topic in connection with the build-up of lipid stores in insects is that of substrate interconversion. Not only are insects capable of utilizing ingested lipid and synthesizing fatty acids from acetate, but like most animals they are capable of converting nonlipoidal material into lipid. Indeed, in some insects this may in fact be the main source of body lipid. One of the most striking examples of the interconversion of carbohydrate to lipid in the animal kingdom, is the involuting mammary gland of the rat in which almost 100% of [14C]glucoseadministered, is recovered in the lipid (McLean, 1964). In this case the C2 units for lipid synthesis arise exclusively from glycogen. In lung tissue, glucose is converted to fatty acids and PL (Tombropoulos, 1964) while glucose is the precursor for glycerol in the mammary gland, and the glycerol is then used for the synthesis of milk lipids (Popjhk et al., 1952; Luick and Kleiber, 1961). In fact, the hydrogen atoms of glucose are utilized in the synthesis of mammary gland fatty acids by a pathway distinct from that which leads to the incorporation of the carbon atoms (Levy, 1963). The conversion of amino acids to lipid in mammalian systems is well known (see for example Feller and Feist, 1959). Figure 16 summarizes some of these general relationships. Although insects are devoid of mammary glands, it has been known for many years that they can convert non-lipid substances to lipid. Many of the earlier experiments consisted of feeding an insect a nonlipid diet and noting the increase in lipid with time by either extraction

148

LAWRENCE I . GILBERT

procedures or histochemical techniques (Abderhalden, 1925;Hofmann, 1872, MacCay, 1938; Melampy and Maynard, 1937; Mishikata, 1922; Weinland, 1908). For example, when mosquitoes were depleted of their lipid reserve by food deprivation and subsequently fed on a diet containing protein, amino acids and glucose, lipid reappeared in the fat body suggesting conversion of one or more of the above mentioned substrates into lipid (Wigglesworth, 1942). At pupation, B. mori contained twice as much lipid as could be accounted for by the leaves it had eaten, indicating conversion of other substances to lipid (Manunta, 1935; Uvarov, 1928). Another technique utilized was manometry where the RQ of the insect indirectly suggested the conversion of carbohydrates to lipid (Fulton and Chamberlain, 1934; Johannson, 1920; Timon-David, 1927). More recent investigations utilizing modern biochemical methodology support the proposition presented above. In an excellent in iiitro study of the locust fat body, Clements (1959) found that acetate, glycine, leucine and glucose are in part converted to lipid. Using labelled precursors, he showed that acetate was the most efficient lipid precursor and that less 14C02was recovered with acetate. The carbon dioxide was the result of the acetate passing through the citric acid cycle (Hines and Smith, 1963). A most interesting series of experiments on the mosquito Aedes was reported by Van Handel and Lum (1961). The female mosquito can be starved until virtually no TGL remains in the body but resumes neutral lipid synthesis when fed glucose. In contrast to the male mosquito or both sexes of housefly, the female mosquito could readily synthesize TGL when fed only glucose. The lipid synthesizing system of Aedes females is so efficient that the quantity of TGL may in some cases exceed the lean dry weight. Of the fatty acids identified in these TGL synthesized from glucose, palmitate, palmitoleate and oleate constitute 92% of the total. Polyunsaturated fatty acids were not synthesized under the experimental conditions utilized. These experiments dramatize the fact that at least some insects have a profound capacity to convert carbohydrate to lipid. We (Chino and Gilbert, 196%) have studied the interconversion of carbohydrate and fatty acid in Cecropia pupae and developing adults using [14C]labelled glucose, acetate and protein-bound palmitate. As seen in Table XI, when [14C]glucosewas injected, considerable radioactivity was recovered in the glycogen fraction in both chilled pupae and developing adults. A significant amount of radioactivity was also demonstrable in the glyceride fraction and as much as 2-2-5% of the label was recovered in glycerides extracted from developing pupae.

TABLE XI1

z z U

The fate of (U-14C)glucose injected into pupae a n d developing adults of H. cecropia Glycerides Stage

Control (chilled Pupa)

Free fatty acid

A

I

Sex

Male Female

Chilled Female Female (3-5

Female

co,

Glycogen A

>&r

Amount % ctslmin % ctslmin % Amount % recovery recovery (mg) fresh (mg) fresh wt. Wt.

s

I -

ctslmin

%

ctslrnin

recovery

%

m

recovery

0

479 471

-

113 115

0.01 0.01

8 7

0.00 0.00

78 65

-

2,550 2,700

0.25 0.27

-

-

364 366 291 342 332 349 341 249 334 349 316 273

7.8 7.7 6.5 5.7

2,410 2,115 4,250 3,006 23,760 21,055 17,830 17,840 22,9W 19,840 25,500 23,900

0.24 0.21 0.43 0.30 2.4 2.1 1.8 1.8 2.3 2.0 2.6 2.4

162 150 200 242 303 382 328 249 194 270 348 273

0.02 0.02 0.02 0.02 0.03

101 49 31 57 48 40 87

2.2 1.0 0.5 0.9 1-2

120,800 75,400 118,040 107,400 52,550 33,000 64,300 34,700 4,730 13,730 78,780 58,900

12.1 7.5 11.8 10.7 5.2 3.3 6.4 3.4 0.47 1.4 7.9 5.9

47,470 51,620 37,460 36,550 122,530 142,750 108,490 109,900 335,180 239,980 300,330 230,950

4.7 5.2 3-7 3.7 12.3 14.3 10.8 11.0 33.5 24.0 30-0 23.1

8.0

8.1 5.4 5.9 8.6 8.4 4.7 4.2

0.04

0.03 0.02 0.02 0.03 0.03 0-03

44 2 4 30 45

0.9 1.6 1.0 0.04 0.08 0.05 0.8

2 W

t:

-

~

E

~-

From Chino and Gilbert (1965~). Animals were sacrificed one day after injection of loEctslmin. The control consisted of injecting the ['*C]glucose into a pupa and sacrificing the animal immediately by freezing in a bath of dry ice and acetone. These animals were then analysed in an identical manner to the experimentals.

2 2 cl

=] 2

5

.-cl 2 CI

P

W

150

LAWRENCE I . GILBERT

The radioactivity found in the long chain FFA fraction was low but significant. Although a considerable amount of the label from [“CIpalmitate was incorporated into glycerides, we could detect no sign%cant incorporation into glycogen (Table XIII). Similar results were obtained with labelled acetate; that is, high incorporation into lipid, a large quantity combusted to C02, but no appreciable amount found in carbohydrate. These results support our previous contention that when FFA are synthesized they are readily incorporated into glycerides ; but more relevant to the present section is the demonstration that Cecropia can convert carbohydrate to lipid, but not the reverse. There have been several suggestions that insects possess the mechanism for converting lipid to carbohydrate, but for the most part this evidence has been circumstantial or only suggestive (Nair and George, 1964; Hitchcock and Haub, 1941 ; Levinson and Silverman, 1954). As far as this author is aware, the conversion of lipid to carbohydrate has not been unequivocally demonstrated in any metazoan although it is a reality in plants and certain microorganisms. In a careful study of the larval-pupal moult in Cecropia, Bade and Wyatt (1962) found that it was unnecessary to invoke a lipid to carbohydrate conversion to explain the changes in nutrient reserves during pupation, although by examining the changes in substrate one may have assumed such a conversion. It appears that the seeming increase in carbohydrate can be explained by a transfer of material from the cuticle to the interior. In fact, the glyoxylate cycle could not be demonstrated in Cecropia and this is prerequisite for such a substrate interconversion (Bade, 1962). It should be noted however, that isocitric lyase activity has been detected in pre-pupae and young pupae of Prodenia (Carpenter and Jaworski, 1962). According to these authors, the enzyme which converts isocitrate to succinate and glyoxylate is most likely maximally active at those developmental stages when the insect is consuming its lipid store. It may be that the past failure to demonstrate this enzyme in insect material is due to utilization of the wrong stage in the life history of the insect. The complete glyoxylate cycle still remains to be demonstrated in insects. Taking a teleological view, one can find a number of reasons why an insect, or any animal for that matter, would find it more beneficial to convert carbohydrate to lipid rather than the converse. The reasons have been stated by many and include the fact that lipid contains more energy per unit weight and yields more metabolic water than carbohydrate. Only if the insect lives in an environment of low oxygen tension or is dependent on anaerobic metabolism, would carbohydrate be

+?

21

7

Irr

r

P

U

TABLEXI11

z1

The fate of (l-14C)palmitate injected into pupae a n d developing adults of H. cecropia Glycerides Stage

Sex

Control (chilled pupa) Chilled pupa

Developing adult (3-5 days) Developing adult (1517 days)

Male Female Male Male Female Female Male Male Female Female Male Male Female Female

i

I {

From Chino and Gilbert (196%).

Amount % (mg) fresh wt. 403 336 386 371 298 333 349 439 274 247 345 356 201 232

8.7 8.0 5.5

5.8 7.1 8.6 4.8 4.7 8.8 9.2 4.3 4.2

ctslmin

)

% recovery

2,635 1,805 346,400 345,000 277,500 323,000 352,700 377,500 379,000 379,800 278,100 382,300 467,000 322,300

coz

Glycogen

A

i

U

0.03 0.02 34.6 34.5 27.8 32.3 35.3 37.8 39.9 38.0 27.8 38.2 46-7 32.2

zm

A

I

Amount % (mg) fresh wt. 85 98 76 84 85 120 55

107 60 59 0 0 21 53

-

1.7 1.8 1.5

2.0 1.4 2.0 1.0 1.1 0.0 0.0 0.45 1.0

cts/min

%

j-2

recovery 1,824 1,955 1,210 1,025 850 1,170 525 1,320 845 810 350 645

0.18 0.19 0.12 0.10 0.09 0.12 0.05 0.13 0.08 0.08 0.03 0.06

recovery 0

!2

89,930 76,620 . 55,230 76,090 56,790 63,170 61,790 80,720 194,000 148,900 226,400 138,900

-

-

8.9 7.7 5.5 7.6 5.7 6.3 6.2 8.1 19.4 14.9 22.6 13.9

E 9

1:

z

2 cI

2: c1

1: 0 +I vl

152

LAWRENCE I . GILBERT

preferred. Glycolysis, although inefficient, will yield enough energy for life processes to continue up to a point, but the oxidation of fatty acids will not occur in the absence of oxygen. Perhaps an intensive investigation of insects inhabiting an ecological niche where the pOz is low would uncover instances of a complete glyoxylate cycle and demonstrate lipid to carbohydrate conversion. Until such data are forthcoming, we must assume that this conversion does not take place in the Insecta. Thus far we have not discussed waxes, hydrocarbons, or the so-called cuticular lipids. These compounds do not really fit into the topic under consideration but are still lipids and are vital to the existence of the insect.

V. HYDROCARBONS A N D WAXES A . CUTICLE

It is now taken for granted that one or more layer of lipid on the exterior surface of the cuticle is in part responsible for the resistance of the insect to desiccation. This relationship between the cuticular lipid and its role in minimizing transpiration was demonstrated more than thirty years ago (Ramsay, 1935). In his review of the insect cuticle, Beament (1964) notes that “organized lipid plays the most important role in the passive regulation of water movement and in modifying the adhesion of water to the surface of cuticular membranes” (p. 68). According to Beament, the physico-chemical properties of the cuticular lipid layer suggest that the lipid acts as a “valve” or “rectifier”. He visualizes the organization of the lipid of the cockroach (P. americana) cuticle as consisting of a tightly packed single monolayer of lipid molecules up against the cuticular surface. Overlaying this monolayer is a “grease” twenty to thirty times as thick as the monolayer but arranged in a random manner. It is the monolayer, however, that is more impermeable to water. This arrangement probably varies in different insects but little research effort has been dedicated to the problem in insects other than cockroaches. In 1955, Beament suggested that shorter chain molecules may act as solvents and promote the tightly packed arrangement of the waterproofing layer. Unfortunately, very little is known of the biochemistry of this lipid, although it is a most promising area of investigation. There have however, been several chemical analyses of insect cuticular lipids and some may shed light on the mechanism by which this material is synthesized. The total hydrocarbon content of M . domestica is composed of unsaturated, cyclic and straight chain compounds (Louloudes

L I P I D METABOLISM A N D FUNCTION I N INSECTS

153

et al., 1962). These range in chain length from cl6 to CS5and are composed of both odd and even numbers of carbons although the odd numbered compounds predominate. This appears to be true for the Mormon cricket (Anabrus simplex) as well (Baker et al., 1960). The cuticular hydrocarbons of Musca differed from the total body hydrocarbons by having a higher concentration of lower molecular weight compounds. This finding appeared to corroborate Beament’s (1955) suggestion regarding the presence of “solvents ” in cuticular lipid. In 1963, Gilby and Cox reported on their analysis of the wax of the cast cuticle of P . americana (the same insect investigated by Beament). By utilizing column and gas chromatography as well as infra-red spectrometry, they demonstrated that hydrocarbons compose 75% of the cuticular wax. Fatty acids, esters and aliphatic aldehydes are present in roughly equal amounts and together with a trace of sterol account for the remaining 25%. The major component was identified as a C2, unconjugated diene, probably heptacosa-9,18-diene (see also Baker et al., 1963). The largest percentages of saturated hydrocarbons were C25and c 2 6 although most of the hydrocarbons were unsaturated. Table XIV reveals that the main fatty acids present are c16 and C18 while the predominant aldehydes are C14 and CI6. Of great interest is the absence of alcohols, except for sterols that comprise less than 1% of the ‘total wax. This finding negates the argument that the cuticular lipids are similar to other waxes. The results are remarkably similar to those reported for the Mormon cricket (Baker et al., 1960). The main points of difference are that the cricket wax contained more varieties of hydrocarbons and these were saturated; the cricket wax contained polyunsaturated C18 fatty acids whereas they are absent from the cockroach wax; the cricket wax was devoid of aldehydes but contained polymeric resins. As Gilby and Cox suggest, it is possible that these cricket resins are derived from aldehydic precursors. In 1962, Gilby reported the absence of natural volatile substances in the cockroach wax and suggests (Gilby and Cox, 1963) that the fluidity of the “grease” can be accounted for by involatile liquid constituents of the hydrocarbons and free acid fractions. Although the cuticular wax hardens when removed from the cuticle, it remains fluid in vivo. The Australian investigators propose that in vivo, an inhibitor prevents the chemical reaction leading to wax hardening or that physical factors are responsible. As noted previously, Beament suggested that a monolayer was largely responsible for prevention of water loss. If alcohols are absent from the wax, what molecules are present that would form a monolayer with

154

LAWRENCE I . GILBERT

TABLE XIV Composition of the cuticular wax of Periplaneta americana Chemical Class

% of wax

Components of class

Hydrocarbons

75-77

C27 n-alkene (heptacosa-9, 18diene) C25, C26, C27 n-alkanes C17-C20, C28, C29 n-alkanes C14, C16, C18 n-alkanoic acids C13, C15, C17 n-alkanoic acids C15, C16, C17 branched alkanoic acids C18 n-alkenoic acid (octadec-9enoic acid) unknown unsaturated C14, C15, C16 n-alkanals C13, C17-C25 n-alkanals C16, C18 branched alkanals C18 n-alkenal (octadec-9-enal)

Fatty acids

Aldehydes

Esters Sterols

7-1 1

8-9

Relative abundance of components weight % of class 66 15, 14, 12 each < 1 5 , 24, 23 each ,1 each 1-2 35 6 16, 10, 14 each 2-8 0.5, 3 3

3-5 <1

Unaccounted for 2-3 From Gilby and Cox (1963).

low permeability to water? Gilby and Cox argue that fatty acid monolayers would be ineffective in controlling water evaporation and suggest that either some unusual and uninvestigated property of the wax causes the formation of these hypothetical monolayers or that a three dimensional network exists in the wax which imparts water impermeability characteristics to the cuticle. This latter alternative may be due to associations of the aldehydic components of the wax, but both of these suggestions are without experimental proof. At least one insect does contain a large amount of alcohol (albeit solid) exterior to its cuticle. For those of us who have worked with the larvae of S. cynthiu, the nature of the crystalline white powder on the cuticle has proved somewhat of a mystery. Very recently, Bowers and Thompson (1965) have demonstrated that this material is composed

LIPID METABOLISM A N D F U N C T I O N I N INSECTS

155

of saturated alcohols. The major component is a C30alcohol (n-triacontanol) with traces of a C28alcohol (n-octacosanol). The triacontanol actually comprises about 93% of the total cuticular lipid. This finding is in sharp contrast to the composition of the cuticular lipids of the cockroach and points out how difficult it is to generalize in this research area. Injection of [l4C]acetateinto the Cynthia larva resulted in a small but significant amount of label in these alcohols suggesting that the larvae synthesize these long chain saturated lipids rather than the alcohols being a product of leaf ingestion. If these alcohols are secreted by the epidermal cells, then it is possible that such secretions may be under endocrine control and may find parallels in the work of Locke (1964) who has described a most intriguing phenomenon in larvae of Calopedes ethlius. After the moult to the fifth instar, the larva secretes wax in the form of cylinders, 0.5 p in diameter, through two patches of modified integument. This process seems to be under endocrine control. Although much important information has been gleaned regarding the morphology of the secretion and cellular elements responsible (cf. Locke, 1964), little or no biochemical work has been conducted on either the nature of the wax or its biosynthesis. If indeed the process of wax secretion is under endocrine influence, the biosynthesis of the wax may be an ideal subject for the study of hormones on biochemical processes. B . EXTRA-CUTICULAR

Hydrocarbons do exist in tissues of the insect other than those associated with the cuticle. For example, the scent secretion of the hemiptperan Rhoecocoris sulieventris contains n-tridecane as the major hydrocarbon component along with n-dodecane and n-undecane (Waterhouse and Gilby, 1964), and Baker et al. (1963) showed that the hemolymph of the Mormon cricket contains saturated hydroCzs with traces of C41to carbons with chain lengths of C25,CZ6,C27, C43. Acree et ul. (1965) have reported on their gas chromatographic analysis of the principal hydrocarbonsin the hemolymph of P. americuna and B. germanica. As an example of the almost unmatched flexibility of the gas chromatograph, these investigators were able to analyse the supernatant of centrifuged hemolymph by injecting it directly into the instrument. The three main hydrocarbons of male Periplaneta were pentacosane, methyl pentacosane, and heptacosadiene ; the latter constituting more than 50% of the total at all stages studied. This finding agrees with that of Baker et al. (1963) as does the observation that

156

LAWRENCE I . GILBERT

twenty-four-day old adults show a sexual dimorphism in hydrocarbon content. That is, the female contains more than the male, although the same relative concentration exists in both sexes of younger adults. It would have been of interest to determine whether this sexual difference also existed in castrated animals. The older females are presumably undergoing oogenesis and it is possible that this involves a decrease in the water content of the hemolymph. Since these investigators analysed a constant quantity of hemolymph without determining the blood volume, the possibility exists that the rise in the quantity of hydrocarbons per volume of hemolymph is an indirect result of “ desiccation” rather than a direct result of hydrocarbon synthesis. Perhaps the total quantity of hydrocarbon in the hemolymph remains the same in both sexes of older cockroaches, but greater loss of water occurs in the female. This problem of water loss assumes great importance in experiments dealing with hemolymph whether it concerns lipid, protein or any other chemical constituent. Many “alterations ” in concentration may be more apparent than real. The function of these hydrocarbons in the hemolymph is not known with certainty but they are most likely involved in the synthesis of new cuticular lipid after each moult. A novel problem relating to the biosynthesis of lipids by insects is that regarding the wax secreted by the honey bee (Apis rnelliferu) which is used for housing the brood and the storage of nutrient (honey). As Young (1963) points out, it is the young worker bees who secrete the major portion of the colony’s wax. By feeding bees heavy water and [1-l4C]acetate, Piek (1961) found that the acetate is incorporated into wax acids and hydrocarbons but not into the esters. He suggested that the FFA were synthesized from acetate in a tissue distinct from that whose function was the synthesis of esters and alcohols. Piek then postulates that the hydrocarbon portion of the wax could then be formed from the FFA by oxidation to ketones and subsequent reduction. By autoradiographic analysis, Piek (1964) demonstrated that the oenocytes take up labelled acetate whereas the fat body cells fail to do so. This is rather unusual in itself since acetate is readily taken up by the fat body of many other species of insects during fatty acid biosynthesis (see Section IV). Piek‘s supposition is that the oenocytes synthesize the wax acids and hydrocarbons from acetate derived from the fat body. The fat body cells derive the acetate from the glycolytic pathway and are responsible for providing the acetate precursor to the oenocytes, as well as synthesizing the esters and their component acids and alcohols from acetate. The suggestion that the oenocytes participate in the production of wax is not new since Wigglesworth (1947, 1948) has

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157

presented evidence that the oenocytes are responsible for the production of cuticular lipids. Further work is needed to substantiate this hypothesis but it is nevertheless an attractive theory.

VI. ISOPRENOID COMPOUNDS A . N U T R I T I O N A L STUDIES

Isoprenoid compounds are of vital importance to the insect. These include the steroids and terpenes as well as other compounds. [Terpenes are compounds chemically related to the simple isoprene, C5H8 and are usually classified as monoterpenes (C,,) ; sesquiterpenes (C15) ; diterpenes (C2,); triterpenes (C,,) (Nicholas, 1963)l. Since in most living things (and insects appear to be one exception), sterols and terpenes utilize the same biosynthetic pathway, these important molecules will be discussed together. It is a vast topic for a general review and several aspects will be omitted. Because of the importance of some of these substances as insect hormones or mimics of insect hormones, certain endocrine aspects will be alluded to although the reader should consult Gilbert (1964) for a comprehensive survey of this topic. An excellent review on the utilization of sterols by insects is that of Clayton (1964). For general reviews of cholesterol and isoprenoid metabolism, see Cornforth (1959), Wright (1961), Popjiik (1963), Goodwin (1963), Garattini and Paoletti (1963), Talalay (1957), Staple (1963), Nicholas (1963), Bloch (1965). See Lederer (1964) for a review of the origin and function of some methyl groups in branched chain fatty acids, plant sterols and quinones. Unfortunately there is no work discussed in which insects have been utilized. Since carotenoids and insect pigments will not be discussed the reader is referred to the reviews of Olson (1964), Cromartie (1959) and Fuseau-Braesch (1963). The formulae for several of the more important steroids relating to the current topic and that will be alluded to in the present discussion, are given in Fig. 18. One of the basic tenets in insect biochemistry is the inability of insects to convert simple precursors to sterol. This was first demonstrated by Hobson (1935a, b), who showed that the fleshfly Lucilia sericata, required an exogenous source of sterols for normal growth. This observation has been corroborated in a multitude of nutritional experiments on various insect species (cf. Clayton, 1964). There is no rule as to when a sterol deficiency will manifest itself, but varies in different insects. In some cases the nutritional deficiency can be clearly seen shortly after hatching from the egg, in Gthers just prior to pupation, and in still others, only in the process of oogenesis (Robbins and

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L A W R E N C E I . GILBERT

Cholesterol

Cholestanol

< 7-Dehydrocholesterol

< A5-Cholestanone

A5-Cholestene

7-H ydroxycholesterol

,k?-Sitosterol

Ergosterol

FIG.18. The structure of some sterols important to insects.

L I P I D METABOLISM A N D F U N C T I O N I N INSECTS

159

OH

0 Zymosterol

Ecdysone FIG.18 continued.

Shortino, 1962). In some instances, sterols are requisite for normal embryonic development and hatching (Chauvin, 1949;Monroe, 1959,1960). To explain this sterol requirement in the diet of insects, experiments over the past two decades have fallen into four main categories. First, a nutritional survey of many sterols and sterol precursors that can replace or “spare” cholesterol, the main insect sterol. Second, biochemical investigations aimed at proving that insects can or cannot synthesize sterols from simple precursors. Third, experiments on how insects (particularly phytophagous insects) can structurally modify the dietary sterols. Finally, more recent investigations have attempted to define some physiological role for these sterols. Noland (1954) found that various derivatives of cholesterol can be utilized by B. germanica. Nutritional studies on flies (Silverman and Levinson, 1954; Levinson and Bergmann, 1957), Dermestes larvae (Fraenkel et d.,1941, Clark and Bloch, 1959a, b, c), Cdobruchus larvae (Ishii, 1955) and a host of other insects (Table XV) conclusively show that a whole range of sterols can substitute for, or partially spare, cholesterol in the diet. In a classic paper on sterol utilization in Dermestes uulpinis, Clayton and Bloch (1963) demonstrated that cholestanol had a full cholesterol sparing activity. They postulate that any sterol that can fully replace cholesterol in the diet must be closely allied to cholesterol in structure so that it can fit into the correct “spaces”. The substituting molecule must be 3-/?-hydroxy and must have essentially the planar structure of cholesterol itself. The introduction of a A5 bond restores some of the activity lost as a result of the removal of successive carbon atoms from the side chain. The requirements for cholesterol sparing activity in insects as suggested by Clayton and Bloch are: (1) The molecule must have a generally planar nuclear stfucture. (2) It must have a side chain of the cholestane type. (3) It must possess a /? oriented 3-hydroxyl group. 7.

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LAWRENCE I . GILBERT

It appears that some insects can at least preferentially extract from their medium that sterol most closely resembling cholesterol. Even if several A5,3 8-hydroxy sterols are present in the defined medium of the housefly, it will selectively take up or retain that sterol most closely approximating cholesterol (Thompson et al., 1963). In a detailed investigation of the nutrition of L. rnigratoria and S. gregaria, Dadd (1960a, b) has shown that cholesterol will satisfy the sterol requirement of these insects. These orthopterans can also utilize cholesteryl acetate, /I-sitosterol and dihydrocholesterol but not stigmasteryl acetate, ergosterol, 7-dehydrocholesterol, cholestenone or 7-oxocholesteryl acetate. Dadd also concludes that an hydroxyl group at C3 that can be esterified is required but that a ketone group at C, prevents sterol utilization (see also Levinson, 1955, 1960, 1962). The ability of insects to live on sterol free diets has been reported, but without exception has been accounted for by the presence of digestive tract symbionts that do have the capacity to convert acetate to sterol (cf. Pant and Fraenkel, 1954). While on the subject of nutrition, it should be noted that in addition to their role as dietary factors, certain terpenoid derivatives including sterols appear to be important as phagostimulants (cf. Thorsteinson, 1960). A great deal of this work has utilized the commercial silkworm due to the persistent efforts of the Japanese in their search for a synthetic substitute for mulberry leaves. fi-Sitosterol has been implicated as both a phagostimulant and “biting factor ” (Hamamura, 1959; Hamamura et al., 1961; Hamamura and Naito, 1961; Ito et al., 1964). Hamamura believes that separate chemicals attract the silkworm larva to the food, cause them to bite it, and finally induce swallowing. Several of these may be terpenes in accordance with Watanabe’s (1958) data indicating that hexenol and hexenal act as attractants. In addition to j3-sitosterol, stigmasterol and campesterol may also act as phagostimulants (It0 et al., 1964). However, in grasshoppers it appears that the phagostimulatory action of wheat germ oil is a consequence of the PL present (Thorsteinson and Nayer, 1963). Waldbauer and Fraenkel(l961) are of the opinion that plant odours will cause insect larvae to bite on any leaf. Dadd (1963) suggests that biting appears to follow orientation to olfactory stimuli common to many plants and that it is an automatic process. This may be true since even Bornbyx larvae will feed on plants other than mulberry (Legay, 1958). Though the basic questions remain open, the role of isoprenoids as phagostimulants presents a most intriguing and challenging problem. It now appears that certain plant monoterpenoids may be in part

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161

responsible for sexual maturation of the desert locust (S. gregaria). A low titre of these essential substances is present in senescent leaves and may explain why sexual maturation is delayed in those locusts feeding upon older leaves (Ellis et al., 1965). B . ISOPRENOID BIOSYNTHESIS

When labelled acetate is administered to a vertebrate or incubated with vertebrate tissues, isotopically labelled cholesterol can usually be extracted from the incubation mixture. In higher animals we now know that every carbon atom in cholesterol is derived from either the methyl or carboxyl carbon of acetic acid (cf. PopjAk, 1963). The reason that insects require a sterol in their diet is they are unable to perform this same synthesis, owing to one or more metabolic blocks between acetate and cholesterol (Clark and Bloch, 1959a, b, c; Kodicek and Levinson, 1960; Robbins, et al., 1960; Schaeffer et al., 1965; Goodfellow and Gilbert, 1965). This failure to use acetate as a building block in cholesterol synthesis is not restricted to the Insecta but exists in crustaceans, araneideans and diplopods (Zandee, 1962, 1964), as well as other invertebrates (Wootton and Wright, 1960). Precursors other than acetate have been unsuccessfully tried in the hope that the metabolic block exists prior to the synthesis of the precursor. For example, farnesol could not substitute for cholesterol in the diet of the rice stem borer (Ishii and Hirano, 1961) and [14C]mevalonatewas not converted to sterol by the housefly (Kaplanis et al., 1961), Cecropia silkmoth (Goodfellow and Gilbert, 1965) and several other insects (cf. Clayton, 1964). Sedee (1961) has demonstrated that in aseptically reared Calliphora, squalene cannot replace cholesterol as a growth factor and that in the presence of labelled acetate, the cholesterol of the insect remains devoid of isotope. Figure 19 shows the biosynthetic scheme leading to cholesterol as we now understand it for higher animals and micro-organisms. This scheme is important because it shows the close relationship between sterols and non-sterolic isoprenoids as well as demonstrating a series of reactions that the insect is incapable of conducting. The first step in sterol biogenesis is the activation of acetate and subsequent condensation of acetyl CoA and acetoacetyl CoA to form 3-hydroxy-3-methylglutaryl CoA. This five carbon compound is then reduced by NADPH to form mevalonic acid. Mevalonic acid in the presence of mevalonic kinase and ATP yields mevalonic acid pyrophosphate. Decarboxylation and dehydration of this compound gives rise to isopentenyl pyrophosphate

c

Q\ h)

TABLE XV Sterols as growth factors in various species of insects'

Species

7DehydroCholes- choles- Choles- Ergosterol terol tan01 tan01

Lucilia sericata Dermestes vulpinus

-

++

-

+

0

0 0

0 0

0 0

++ ++ ++ ++ ++ + ++f +++ 0 +++ ++ ++f ++

+++

+ + ++ + 0

+++ +++ ++ +++ +++ ++

-

-

-

-

++f ++f +++ +++ 0 Tribolium confusum + + + + + + Lasioderma sericorne + + -k + + + Silvanus surinamensis + + f + + + Ptinus tectus + + + + + + Stegobium peniceum ++f +++ Ephesiia kuhniella Attagenus piceus

EpiCholest- CholestSito- Stigma- Stigma- choles- Copros- Zymo- 4-en4-en- Ergosterol sterol Stan01 tan01 tan01 sterol 3-one 38-01 sterol

Phormia regina ++f Musca vicina + + f Locusta migratoria

+++

0

+++

+++ ++ +++ +++

0

0

+

+++ ++

0

(0)

-

++ +

0

+++

+++

-

0

._

Schistocerca gregaria + Blatella germanica Gryllus domesticus Bombyx mori Aedes egypti Tenebrio molitor -t Cdosobruchus chinensis Pyrausta nubilalis Drosophila melanogaster Hylotrupes baJulus ++

++ +++ +++ +++ +++ + ++ ++ +++ ++

+++

+++ +

-

++ 0

+++ 0 ++ ++ ++ + +++

++ +-+

-

+++ +++ +++ +++ +++ +++

+++ +++ +

++

+++

-

-

-

-

0 -

tt+

++

0

+(0) 0

++ +++ 0 + +++ -

+++

+++ +

The symbols indicate good ( +), moderate (+ +), poor (+), or no (0) utilization of a sterol by a given species. Some negative results obtained with the sterol acetates are placed in parenthesis. (-) indicates “not studied”. (From review by Clayton, (1964); See Clayton’s article for original reference.)

I 64

LAWRENCE I . GILBERT

which is converted to its isomer y,ydimethylallyl pyrophosphate. [Isopentenyl pyrophosphate, sometimes known as "active isoprene" is not only a key intermediate in the biosynthesis of many terpenoids, but also in the synthesis of carotenoids (Goodwin, 1963). Since some insects appear unable to synthesize the requisite carotenoids for normal visual reception (Goldsmith et al., 1964), the metabolic block is most likely prior to isopentenyl pyrophosphate.] Condensation of two of these 5-carbon molecules yields geranyl pyrophosphate which, together with

C~ASH 0

OH

II

0

I

II

HOC-CH2C-CH2-C-SCoA 2 NADPH

OH

+H+

2 NADP

0

II

CH"C-0-

I

I1

xATP

2-03POCH2CH,C-CH,C-OAH, OH

JO~ADP II

I I

'-O8P20CH2CH2C- CH2C- 0CH3 s G 3-OJ'@CH2CHzC=CH,

I

~

+

C

O

~

. 'A

CH3C=CHCH20P,0,3C H S HP@?- CH, I CH,C=CHCH,CH,C= CH, I

CHCH,OP,0,3~ = ~ H , C H , O P z O ~ -

kH3

HPzO?-

H3

CH3-C=CHCH2CH2C=CHCH2CH,C=CHCH20P20~-

I

CH,

I

CH,

I

CH,

L I P I D METABOLISM A N D F U N C T I O N I N IN SECTS

CH,

2 HP,O,S-

165

CH3

CH, CH,

FIG.19. The biosynthesis of cholesterol. a. Acetoacetate + acetyl CoA combine to yield hydroxymethylglutarate in the presence of a condensing enzyme. b. Hydroxymethylglutarate NADPH yields mevalonate. c. Mevalonate ATP yields 5-phosphomevalonate in the presence of mevalonic kinase. d. 5-Phosphomevalonate + ATP yields 5-pyrophosphomevalonate in the presence of phosphomevalonic kinase. e. 5-Pyrophosphornevalonate ATP yields isopentenyl pyrophosphate COZ. f. Isopentenyl pyrophosphate + dirnethylallyl pyrophosphate combine to form geranyl pyrophosphate. Dimethylallyl pyrophosphate is an isomerization product of isopentenyl pyrophosphate. g. Geranyl pyrophosphate + isopentenyl pyrophosphate yield farnesyl pyrophosphate. h. Reductive dimerization end to end of two molecules of farnesyl pyrophosphate yields squalene in the presence of NADPH. i. Cyclization of squalene to yield lanosterol in the presence of oxygen. j. A series of reactions whereby lanosterol is converted to cholesterol via 1Cdesmethyllanosterol, zyrnosterol and desmosterol. These latter compounds have been omitted from the scheme for the sake of simplicity.

+

+

+

+

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LAWRENCE I . GILBERT

another molecule of dimethylallyl pyrophosphate, gives rise to farnesyl pyrophosphate. The condensation reaction to form farnesyl pyrophosphate is mediated by a farnesyl synthetase extracted from liver and yeast (Lynen et al., 1958). Condensation of two of these C15molecules leads to the formation of the dihydrotriterpene squalene (C3,). Folding of this molecule yields lanosterol and finally cholesterol. Along the pathway, dephosphorylation may yield such endocrinologically active molecules as farnesol (see Section VI). Before continuing our discussion of the sterols, let us briefly discuss another most important molecule containing isoprene units. This is coenzyme Q (CoQ) which plays a crucial role in electron transport and shares part of the biosynthetic pathway leading to sterol. The coenzymes Q (CoQ, to CoQ,,) differ from one another only in the length of the isoprenoid side chain, the quinone portion of the molecule being identical. Gloor and Wiss (1958, 1959) have shown that mevalonate is a precursor of the isoprenoid chain but is not involved in the synthesis of the quinoid nucleus. The entire isoprenoid side chain appears to be synthesized by a stepwise incorporation of C5 units derived from mevalonate. The mechanism by which mevalonate is converted to the C5 unit is now understood (Lynen, 1959). Mevalonate is decarboxylated and diphosphorylated to isopentenyl pyrophosphate. This molecule then isomerizes to form 3,3-dimethylallyl pyrophosphate which is subsequently alkylated by another molecule of active isoprene, yielding allylic pyrophosphate. Further alkylations by active isoprene increase the chain by one unit at a time. The final reaction is thought to be an electrophilic aromatic substitution on an unknown precursor by the isoprenoid allylic pyrophosphate (cf. Wagner and Folkers, 1964).

0 0

CH,O CH30

f"'

CH,

CH,CH=CCH,

) H

0 Coenzyme Q

That CoQ is an important constituent of the mitochondria is seen from the fact that its concentration in this organelle is higher than any of the cytochromes. Although Laidman and Morton (1962) demonstrated the presence of CoQB,CoQ9 and CoQ,, in larvae of the blowfly and suggest that they may have been products of digestion (see also Lester and Crane,

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167

1959; Heller et al., 1960), almost nothing is known regarding the possibility of CoQ biogenesis in insects. One can assume that insects most likely cannot synthesize this molecule if a metabolic block exists prior to active isoprene. We do not really know whether CoQ is as crucial to the proper functioning of the insect mitochondrion as it is to beef heart mitochondria, although we must assume for the present that it is. It would appear that an almost ideal situation for the study of both function and synthesis of CoQ in insects, would be tissues such as developing flight muscle where the giant mitochondria are undergoing morphogenesis. This would be especially true in some Lepidoptera where this morphogenesis occurs in a “closed system” and one need not worry about ingestion of materials containing exogenous CoQ. To return to the main topic of sterol biosynthesis, we have stated that insects cannot synthesize sterols. However, in the past several years there have been reports that either conclude or suggest that certain insects can convert acetate to sterol. Most of these data have been shown to be due to symbiotic micro-organisms and not to the insect’s ability. Because of the importance of this point we will discuss a few recent examples. When the silverfish Ctenolepisma sp. was reared on a diet containing [‘*C]acetate, some radioactivity was recovered in the sterol fraction (Clayton et al., 1962). These investigators did suggest that any sterol biosynthesis may be due to symbionts in this notoriously infected insect. When a closely related insect, Thermobia domestica, was grown aseptically and fed or injected with [l*C]acetate, only about 0.001% of the label was recovered in cholesterol (Kaplanis et al., 1963a, b). These authors doubt that the firebrat has the capacity to synthesize sterols from acetate and we must agree. When 1 to 2 pc of labelled acetate is injected into silkworm larvae or pupae, some radioactivity is recovered in the sterol fraction (Saito et al., 1963a). However, this is extremely low and ranges from 1-69-56.88 cts/min per mg of the digitonide. Although theseauthors argue that acetate is a sterol precursor in Bombyx, the percentage of the initial isotope recovered was extremely low. It was, in fact, of the same order of magnitude as that obtained by Sridhara and Bhat (1965b) in the same insect injected with labelled mevalonate, but they conclude “that the sterol biosynthetic pathway is absent in this insect”. However, even this extremely low quantity of incorporation could be evidence of synthesis. The major objection is that even this is most likely to be due to symbiotic micro-organisms. We have also investigated this problem of sterol biosynthesis in another silkworm, H. cecropia (Goodfellow and Gilbert, 1965). An

168

LAWRENCE I . GILBERT

aqueous solution of ~ ~- [ 2 - ~ ~C] me v a l onafe was injected into pupae and developing adults and the lipids analysed after adult emergence. About 8% of the label was incorporated into lipid and 93% of this was recovered in the non-saponifiable fraction. Column chromatography yielded a sterol fraction that was precipitated with digitonin. The digitonide was cleaved and the cholesterol purified. Only 0.011% of the radioactivity was associated with cholesterol. This low level of activity and our inability to obtain constant specific activity during the purification process suggests that there is no significant incorporation of mevalonate into cholesterol in this insect. C. I S O P R E N O I D C O N T E N T

It is almost 30 years since Bergmann (1934) reported that chrysalis oil (lipid extract of B. mori pupae) contains sterols composed of 85% cholesterol and 15% sitosterol (as we shall see, other sterols exist in this insect in addition to these two). Since that time, numerous insects and insect tissues have been analysed. Prior to the use of gas chromatography, large amounts of sterol were necessary for analysis. For instance, in their work on the sterols of M . domestica, Agarwal et al., 1961) extracted 65,000 houseflies and obtained 319 mg of crystalline sterols. Today, analyses can be accomplished on the tissue of a single insect (Goodfellow and Gilbert, 1965). Numerous isoprenoids have been identified in insect tissues. Some of these have their.source in the insect’s food, some result from the insect’s capacity to modify dietary sterols, while others are the result of the metabolic processes of the symbionts. The more common sterols will be discussed in detail subsequently but we may mention some of the more recent findings now. The major sterol of pupal and adult Colorado potato beetles is the common phytosterol p-sitosterol (Schreiber et al., 1961). Mass spectrometric analysis of the sterols of Calotermesflavicollis revealed the major body sterol to be cholesterol with lesser amounts of stigmasterol and p-sitosterol plus an as yet unidentified sterol (Duperon et al., 1964). Queen bee larvae, as well as the royal jelly upon which they feed, contain 24-methylene cholesterol (Barbier and Bogdanowsky, 1961; Pain et al., 1962; Weaver et al., 1964). It