No title

Advances in

INSECT PHYSIOLOGY

VOLUME 3

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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 3

1966

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.

111

FIFTH AVENUE

NEW YORK, NEW YORK

10003

Copyright 0 1966 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

WRI'ITEN PERMISSION FROM THE PUBLISHERS

Library of' Congress Catalog Card Number: 63-14039

Printed in Great Britain by T. & A. Constable Ltd., Edinburgh

Contributors to Volume 3 E. T . BURTT,Department of Zoology, University of Newcastle upon Tyne, England W . T . CATTON,Department of Physiology, University of Newcastle upon Tyne, England P. S. CHEN,Institute of Zoology and Comparative Anatomy, University of Zurich, Switzerland W . R. HARVEY, Zoology Department, University of Massachusetts, Amherst, Massachusetts, U.S.A. J . A. HASKELL, Zoology Department, University of Massachusetts, Amherst, Massachusetts, U.S.A. A. D. LEES,Agricultural Research Council Unit of Insect Physiology, Zoological Department, University of Cambridge, England P. L. MILLER,Department ofZoolc ,y, University of Oxford, England

V

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Contents CONTRIBUTORS TO VOLUME3 IMAGE

I. Introduction

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V

FORMATION AND SENSORY TRANSMISSION IN THE COMPOUND EYE E. T. BURIT and w. T. CATTON

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11. The Visual Abilities of the Compound Eye

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A. Intensity Discrimination in the Compound Eye . B. Resolving Power of the Compound Eye . C. Recognition of Form . D. Detection of Movement . E. Detection of the Direction of the Plane of Polarization of Light 111. The Optics of the Compound Eye. . A. Image Formation. . B. Properties of the Diffraction Images C. Spacing of Photoreceptors in Relation to Images . D. The Erect Image in the Compound Eye of Lampyris . E. Optical Basis of Movement Detection . . F. Optical Detection of the Plane of Polarization of Light G. Histological Changes AccompanyingLight and Dark Adaptation IV. Electrical Responses in Compound Eye and Optic Lobe . A. Nature of the Responses . B. Potential Profile of Compound Eye and Optic Lobe. . C. Potential Changes in Response to Dark- and Light-adaptation . D. Independent Origin of the Off-Response . . E. Visual Threshold Changes Linked with Potential Variations . F. The Optic Pathway in the Locust . V. The Mechanism of Arthropod Vision . A. The Eye of Lirnulus . B. Excitatory and Inhibitory Systems in the Insect Eye. . C. The Neural Basis of Movement Perception . D. The Optical and Neural Basis of Form Vision. . References

2 5 5 6 8 9 10 10 10 15 16 16 18 18

19 20 20 26 27 31 32 33 38 38 39 42 42 46

AMINOACIDAND PROTEINMETABOLISM IN INSECT DEVELOPMENT P. S. CHEN

I. Introduction . 11. Embryonic Development

53 55

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vii

...

Vlll

111.

1V. V.

VI.

VII.

CONTENTS

A. Changes in Free Amino Acid Pools . B. EnzymePatterns . Larval Development . . A. Amino Acids . B. Peptides and other Amino Acid Derivatives . C. Haemolymph Proteins . Pupal Development . A. Metabolism of Amino Acids and Proteins . B. Changes in Enzyme Activities . Adult . A. Sex-specific Differences in Amino Acids, Peptides and Proteins B. Protein Metabolism in Relation to Reproduction . Some Genetic Aspects of Protein Metabolism in Insects . A. Patterns of Protein Metabolism in Lethal Mutants . B. Synthesis of Enzymes and other Specific Proteins . C. Regulation of Gene Activity . Conclusions . References

55 62 69 69 82 84 89 89 93 96 96 99 102 102 109 112 113 114

METABOLIC CONTROL MECHANISMS IN INSECTS w. R . HARVEY a n d J . A. HASKELL 1. Introduction . 11. Phosphate Acceptor and Substrate Control of Respiration in Isolated Mitochondria . A. History, Definitions and Terms . B. Sarcosomes and their Isolation . C. Energy Requirements of Insect Flight . D. Regulation of Energy Trapping Pathways in Flight Muscle . E. Oxidative Phosphorylation and Respiratory Control . F. Endogenous Uncoupling or Controlling Agents . G. a-Glycerophosphate and Respiratory Control during Flight . H. Biological Factors Influencing Energetics of Mitochondria . . 111. Regulation of Enzyme Levels A. Constant Proportion Enzymes . B. Oxidative Enzymes in Silkworm Development . C. Enzymes of Tanning Reactions . IV. Control at the Chromosome Level . A. Biochemistry of Insect Hormones . . B. Biochemistry of Giant Chromosomes . C. Chromosomal Puffing and its Relation to Development . D. Chromosomal Puffing and its Relation to Synthetic Processes in the Cell . E. Ecdysone and DNA Synthesis . F. Chromosomal Puffs and Transport . . V. Ionic Control of Protein Synthesis and Development A. Ion Control during Development . B. Protein Synthesis Regulated by Ion Concentrations . References .

133 134 134 138 143 144 149 152 154 155 156 157 161 165 166 166 171 174

181 182 182 183 183 186 190

ix

CONTENTS

THECONTROLOF POLYMORPHISM I N APHIDS A. D . LEES

I. Introduction . 11. Aphid Forms and their Terminology . 111. The Fundatrix: Form Changes in Young Clones . . IV. Clonal Variability V. Sex Determination . . VI. The Production of Gamic Females A. Photoperiodic Sensitivity and the Chronology of Embryogenesis B. Response Curves . C. The Site of the Photoperiodic Receptors. . D. Hormones and the Differentiation of Oviparae . E. Interaction of Photoperiod with Temperature . . F. Photoperiodism in Heteroecious Species . . G. Sexual Reproduction in Macrosiphum euphorbiae . H. Aestivation and Gamic Reproduction . 1. Other Environmental Factors . J. Intrinsic Factors: Anholocycly . VII. The Control of Wing Dimorphism . .. A. The Analysis of Crowding . B. Stages Sensitive to Crowding. . C. The Mechanism of Crowding . D. Nutrition . E. Water Content and Ionic Composition of the Host Plant . F. Relationships with Ants . G. Temperature . H. Photoperiod . I. Intrinsic Factors . . J. Developmental Pathways and Wing Dimorphism . K. Endocrine Control of Wing Dimorphism . L. Environmental Regulation of Corpus Allatum Activity . VIII. The Inhibition of Developmental Pathways: Interval Timers . IX. Summary . References

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.

207 209 214 216 219 22 1 222 226 227 23 1 23 I 232 233 235 236 237 238 239 242 244 249 25 1 252 253 253 253 255 257 264 265 27 1 272

THEREGULATION OF BREATHING IN INSECTS P. L. MILLER

I. Introduction . 11. The Control of Ventilation . . A. General Remarks. . B. The Endogenous Nature of the Ventilatory Rhythm. . C. Types of Endogenous Activity Connected with Ventilation D. Co-ordination within the CNS . E. Proprioceptive Input . F. The Effects of Carbon Dioxide and Hypoxia . G. Electrical Stimulation of the CNS . A*

.

279 280 280 282 286 29 I 294 294 297

CONTENTS

X

H. Inspiration through Cuticular Elasticity and Reduced Pressures . 111. The Control of the Spiracles . A. General Remarks. . B. Innervation of the Spiracles . C. Innervated Tracheae . D. Spiracular Activity . E. Control Mechanisms in Two-Muscle Spiracles. . F. Control Mechanisms in One-Muscle Spiracles . . G. Experiments on the Nature of the Chemical Stimulus . H. Synchronized Activity of the Spiracles . I. Independent Activity by the Spiracles . IV. Modifications of the Tracheal System for Flight . A. Functional Morphology of the Tracheal System in the Pterothorax . B. The Locust Pterothorax. C. Movement of Air in the Primary and Secondary Tubes by Ventilation . D. Movement of Gases in the Secondary and Tertiary Tubes by Diffusion. E. Spiracle Behaviour during Flight . F. The Oxygen Supply to the Resting Flight Muscles . V. Summary . References .

298 300 300 301 302 303 304 305 31 1 31 1 316 321 322 329 334 338 340 342 343 344

AUTHORINDEX .

355

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367

SUBJECTINDEX

Image Formation and Sensory Transmission in the Compound Eye E. T . BURTT Department of Zoology, University of Newcastle upon Tyne, England and W. T. CATTON Department of Physiology, University of Newcastle upon Tyna, England I. Introduction. . . II. The Visual Abilities of the Compound Eye . . A. Intensity Discrimination in the Compound Eye . . B. Resolving Power of the Compound Eye . . C. Recognition of Form . . D. Detection of Movement . . E. Detection of the Direction of the Plane of Polarization of Light . 111. The Optics of the Compound Eye . . A. Image Formation . . B. Properties of the Diffraction Images . . C. Spacing of Photoreceptors in Relation to Images . . D. The Erect Image in the Compound Eye of Lampyris . E. Optical Basis of Movement Detection . . F. Optical Detection of the Plane of Polarization of Light . . G. Histological Changes Accompanying Light and Dark Adaptation. IV. Electrical Responses in Compound Eye and Optic Lobe . . A. Nature of the Responses . . . B. Potential Profile of Compound Eye and Optic Lobe . . C. Potential Changes in Response to Dark- and Light-adaptation . D. Independent Origin of the Off-response . . E. Visual Threshold Changes Linked with Potential Variations . F. The Optic Pathway in the Locust . . V. The Mechanism of Arthropod Vision . . A. The Eye of Limulus . B. Excitatory and Inhibitory Systems in the Insect Eye . C. The Neural Basis of Movement Perception . . D. The Optical and Neural Basis of Form Vision . . References . .

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1

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2 5 5 6

8 9 10 10 10 15 16 16

18 18 19 20 20 26 27 31 32 33 38 38 39 42 42 46

2

E. T. B U R T T A N D W. T. CATTON

I. INTRODUCTION The compound eye can be considered from two points of view. First as one of the many types of photoreceptors found in animals and second as a visual system with properties peculiar to itself. Electron microscopy stresses the first approach. Wolken (1963) shows how the eye spots of Protozoa, the simple eyes of Platyhelminthes and the highly specialized eyes of cephalopods, arthropods and vertebrates all have photoreceptors with basically similar patterns. The essential light receptive region (whether it is called a rhabdome, a rod or cone) consists of orientated layers which appear in sections as lamellae or tubes of osmophile material with walls about 100 A thick and 200-500 A apart. The essential photoreceptive pigment retinene is widespread in animal eyes and occurs in insects (Wolken et al. (1960) in Muscu; Goldsmith (1958) in Apis) which also shows the fundamental similarity of the photoreceptor processes in animals. It seems likely that the electrical changes undergone by the retinula cell when the compound eye is illuminated will also show phenomena common to other animal photoreceptors. The second aspect of the compound eye is its peculiar structure when compared with other types of eyes. It would be helpful to the present discussion if stages in the evolution of the compound eye were available from fossil evidence. The eyes of trilobites are of great interest since trilobites occur first in the Cambrian and are very generalized arthropods from many points of view. Harrington (1959) surveys earlier work on trilobite eyes and Clarkson (1964) gives details of the arrangements of lens axes and possible visual fields. Two types of eyes are found : the “holochroal eye” which has a continuous transparent surface with lenses 60-100 p across which may number up to 15 000; and the “schizochroal eye” where the number of lenses is much smaller (up to 300400) and each lens is separated by opaque cuticle while the individual size ranges from 130-500 p and is thus much larger than in the holochroal eye. It would be tempting to suppose, in this ancient and possibly ancestral group of arthropods, that the schizochroal eye gave rise to the holochroal by fusion and reduction in size of the lenses; but the evidence is against this, as the holochroal eye occurs earlier and is more widespread in trilobites than the schizochroal eye. Setting aside any questions of evolutionary primitiveness, the same transition between true compound eyes and multiple simple eyes occurs again and again in different groups of arthropods. Thus Hesse (1901) shows that in the Chilopod Lithobius there are multiple simple eyes with large lenses and shallow photoreceptors, while in Scutigera there is a fused eye with much deeper retinula cells. The same difference

I M A G E F O R M A T I O N A N D S E N S O R Y T R A N S M I S S I O N IN E Y E

3

can be seen in Lepismu and Petrobius in the Thysanura, amongst the apterygote insects. In pterygote insects Lepidoptera show the same contrast. Dethier (1942, 1943) gives quantitative data for the simple eyes of the larvae of the arctiid Isea. Here the eye consists of six simple stemmata ranging in lens diameter from 94 to 138 p with a depth of ca 170 p. The visual fields of the eyes do not overlap and the eye probably gives the larva a simple type of form vision to judge from the work of Hundertmark (1937) on the larva of Lymantriu. Here an erect and an inverted pyramid could be distinguished. The structure of the eye of the larval Lepidoptera is in marked contrast to the typical compound eye of the adult. Yagi and Koyama (1963) give an extensive survey of the form of the compound eye in the families of Lepidoptera. They find the largest facet size of 42 p in the satyrine Melanitis which has an ommatidial length of 420p, while the equivalent figures for the facet and ommatidial length at the other end of the scale for the moth Plutella maculipennis are 17 p and 122 p. These figures show the striking change in form when a visual organ consisting of several separate ocelli is replaced by a typical compound eye. If the same could be said for the trilobites (and the eye may be deeper in the holochroal forms) then, in four groups of arthropods from the most diverse habitats and widely separated in space and time, this transition is accompanied by the following changes. 1. Reduction in size of the single lenses. 2. Greater uniformity in size of the lenses. 3. Marked increase in depth of the photoreceptors in proportion to width of the lenses.

This brings us to one of the functional problems of the compound eye: what is the advantage which the fused compound eye has over an equivalent set of simple eyes? MiiUer’s original observations are relevant at this point as set out in his book on human physiology (1840). He had a wide knowledge of zoology and knew the aggregate eyes of the myriapod Julus, the ocelli of adult insects and of course the compound eyes of Crustacea and Insecta. He realized that the cuticular lens and cone formed an image but thought that the optic nerve extended to the base of the cone and quoted Wagner’s view that it spread out over the surface of the latter. Thus he thought that the photoreceptors lay immediately behind the cones and was not aware of the possibility that light might be received deeper in the eye. His conclusions are as follows : “Each cone receives an aliquot portion of the image which is composed of as many parts as there are cones in the eye and the distinctness of the image increases with the number of cones.

4

E. T. BURTT A N D W. T. CATTON

“The smallest angle of vision under which an insect will be able to distinguish one object from another will be that which is included between the axes of two contiguous cones. “The most perfect insect eye will be that which sees clearly by its absolute size, large numbers of cones and facets, and length of the cones; and has a large field of vision due to the convexity of the eye being such that it forms a great part of a sphere.” These conceptions of the mode of action of the compound eye are still dominant in the minds of entomologists at the present time. The first point which must be made is that Muller’s views are approximately true. Thus the different parts of the visual field receive visual information from the angular projection of the appropriate part of the compound eye. Also high visual powers in insects are associated with large compound eyes composed of many ommatidia, and as a result vision is most acute when the angles between adjacent ommatidia are small. It is not clear, however, from Muller’s point of view why insects should not have evolved large expansions of the head covered with many simple eyes. Indeed Dethier’s (1942, 1943) account of the stemmata in the lepidopterous larva is an almost perfect example of a Mullerian mosaic system. Grenacher’s work (1879) showed that the retinula cell was a deep structure and that the photoreceptive region in some cases stretched from the end of the cones right down to the basement membrane. Exner (1891) brought forward the idea of superposition which explained why (at least in some arthropod eyes) this great depth of photoreceptors was needed. Since Exner, compound eyes have been divided into “apposition eyes” which work in the manner suggested by Muller, and “superposition eyes” in which deeper images are formed, with the additional possibility that many insect eyes can adjust their pigment so as to pass from the apposition to the superpositioncondition. This view explainedwhy the superposition eye is so deep, but the depth of apposition eyes is still a problem. The present review aims at showing how electrophysiologicalstudies, recent behaviouristic work, and anatomy helped with the electron microscope, allow us to reconsider this matter and arrive at a new synthesis. We shall reexamine superposition in terms of physical optics, suggest that it occurs in all true compound eyes, that it necessitates the lenses becoming contiguous and the eye increasing in depth. The single retinula cell rather than the whole ommatidium will be regarded as the functional unit of the eye. The means by which the nervous impulse originatesand is transmitted will then be discussed; aquestionwhichcould hardly be touched on by the earlier authors. Finally the facts of insect

I M A G E F O R M A T I O N A N D S E N S O R Y T R A N S M I S S I O N I N EYE

5

form vision will suggest a modified mosaic theory; one based not on whole ommatidia but on the peculiar eccentric cells which seem widespread in the compound eyes of arthropods. We shall consider work on crustacean eyes where relevant because the structural similarities seem to outweigh the differences. There is always the possibility that the compound eye has originated more than once in arthropod evolution, but for the present the compound eye can be considered as being as general in the arthropods as the single lens eye is in the vertebrates, allowing for the many differences in detail throughout the respective phyla. 11. THEV I S U A L A BI LI TI ES OF

THE C O M P O U N D

EYE

It is essential first to determine the visual abilities of the compound eye. Data showing what arthropods can do using their compound eyes will have a validity independent of any theories as to how the eye works. Such data for colour vision have already been reviewed by Burkhardt (1964). Here the conclusions from behaviour studies and the results of electrophysiological work are in good agreement. The abilities of the compound eye which concern us now are the following:

A. B. C. D. E.

Intensity discrimination in the compound eye. Resolvingpower of the compound eye. Recognition of form. Detection ofmovement. Detection of the direction of the plane of polarization of light.

A. I N T E N S I T Y D I S C R I M I N A T I O N I N T H E C O M P O U N D EYE

The smallest change in illumination (AI) detectable by the eye, expressed as a fraction of the total illumination (I), reveals an essential requirement for stimulating the photoreceptors and generating a visual impulse. Earlier behaviouristic work (e.g. Wolf (1933) on the honeybee) gave values such as 24% for the threshold for intensity discrimination. Burtt and Catton (1962b) using the generation of spikes in the ventral nerve cord (Parry, 1947) as a criterion found a threshold of 7.5%, while Fermi and Reichardt (1963) obtain values in Muscu as low as 0.5%. In their work the stimulus was a cylinder which could be rotated around the insect; and the dark and light stripes on the inner surface of the cylinder used by earlier workers were replaced by a very accurately controlled illumination through vertical slits contrasted with the illumination of the

6

E. T. B U R T T A N D W. T. C A T T O N

inner walls of the cylinder. The insect was attached by the head and thorax to the moving coil of a galvanometer which had a feedbackmechanism so adjusted that when the fly during suspended flight turns about the vertical axis in response to a visual stimulus, the current of the coil alters and keeps the insect stationary. The current in the coil is recorded as the optomotor reaction. This is a delicate method of recording the response of the insect as a whole. It would be of great interest to compare these results with those using the ventral nerve cord spike response. Fermi and Reichardt's results make one hesitate to accept as final the levels of threshold suggested by cruder methods of recording, not only in this but in other types of observation. B. RESOLVING POWER O F T H E C O M PO U N D EYE

An eye must have an imaging system for it to detect pattern in the surroundings. Several types of imaging systems can be constructed (see Rogers (1 963) for a discussion of different systems), but the convex lens is the most familiar. In insects this is seen in the cuticular lens of the stemmata of lepidopterous larvae (Dethier, 1942),in ocelli and in the facets of compound eyes. The image of a point source is not in fact a point but a disc of finite size surrounded by a series of dark and light rings of diminishing intensity. This diffractive effect in the lens (or other imaging system) is directly related to the diameter of its aperture and restricts its resolving power. The usual criterion of the latter is that two small sources can be separated if the angle subtended by them is not less than 8= 1.22 X/d radians, where X is the wavelength of the light and d the diameter of the aperture. Thus for a lens such as the ommatidium of Locustu d = 3 1 p, and the resolving power using light of wavelength 5 500 A (green to the human eye) would be about 1.25"; and for the shortest ultraviolet to which insects are known to respond (about 3 000 A) about 0.75".The size of the lens system thus imposes a limitation on the resolution of the compound eye as was pointed out by Mallock (1922) and Barlow (1952). The imaging system may also have other effects on the image such as distortion or chromatic aberration. We are not now concerned with these, but only with the extent to which the discontinuities in the object will be represented by discontinuities in the image. Thus in some types ofimaging systems, such as a set of apertures or minute lenses, one may obtain an image which shows great departures from the form of the object, nevertheless there may be considerable resolution of the discontinuities of the object. The usual method of testing resolution in the insect eye is to move a series of black and white stripes of known spacing across the visual field ;

IMAGE FORMATION A N D SENSORY TRANSMISSION I N EYE

7

observe some response by the insect such as change in direction of walking or flight; and then reduce the separation of the stripes until the reponse disappears. Such an approach is shown by the work of Gaffron (1934) in Odonata larvae and adult Diptera, Hecht and Wolf (1 929) in the honeybee, and de Bruin and Crisp (1957) in Crustacea. In these examples the animal moved, but in the work of Hassenstein (1951) and Schneider (1956) the insect was held in one central place and thus distances could be easily controlled. Autrum (1961) using Schneider's technique got a value of 2-33"for the minimum stripe separation needed to give a turning response to a flying Culliphoru suspended at the centre of the striped cylinder. The work of Hassenstein is a very ingenious elaboration of this method in that the insect (Chlorophunus, Coleoptera) is held stationary but its turning tendency is shown by the skeleton globe made of straw which it holds in its feet. This allows it to walk (in reality turn the straws through its feet) until it meets a junction, when it has a choice either to turn left or right. Its left or right tendency can be measured statistically by the number of left or right turns as it revolves the globe between its feet. In each of the above cases a threshold is found for a certain separation of stripes. It is important to note that in none of the above work has the subthreshold condition been examined statistically. Thus authors are usually content to take a value for stripe width which gives consistent results, but it would be of very great interest to know the percentage response at a width slightly below this. The values for stripe separation which give consistent results are very varied. Thus the honeybee gives an acuity of about 1O (Hecht and Wolf, 1929), which is comparable both with the separation of the ommatidia in the centre of the eye and the resolution of a small lens with the diameter equal to the single ommatidium. But the large and elaborate crustacean eyes give the very low acuity value of 4.6"(de Bruin and Crisp, 1957). The work of Jander and Voss (1963) suggests that under rather different conditions a much higher level of resolution may be shown. They found that Formica rufu will move towards vertical in preference to horizontal stripes. By reducing the separation of the stripes a point was reached at which the insect no longer showed any preference. Jander and Voss give the threshold as a stripe which subtends 0.5" at the eye. The stripes were alternately black and white in even spacing, so that it is more in keeping with the convention of this review to call this 1O. These authors point out that this is a very high value for visual acuity in insects. Two interesting conclusions can be drawn from this work. The first follows from measurements on the eye: 1" is far less than the ommatidial angular separation, but measurement of the diameter of the ommatidia in Formica rufu shows

8

E. T. BURTT A N D W. T. CATTON

that the resolution to be expected from the formula given earlier would be about 2”.Thus Jander and Voss’s work shows a higher resolution than is to be expected either from the ommatidial separation or the ommatidial diameter. The second conclusion is a statistical one from the dataas presented. If Jander and Voss had taken their threshold at a level where the response to the vertical stripes was lOO%, then the value for the acuity would have been over 5”. This suggests that a more extended statistical treatment might reveal much greater resolution in other behaviouristic experiments. Burtt and Catton (1959a, 1962b) claim an even higher degree of resolution of striped patterns in the locust and in two species of Diptera, using the spike responses which can be detected electrophysiologically in the optic lobe and ventral nerve cord. This gave resolution of stripes with a separation of adjacent black stripes of as little as 0.25-0-3”. It has been suggested by McCann and MacGinitie (1964) that these effects could be due to errors in the patterns causing slight changes in the intensity of light falling on the eye. The high degree of resolution found by Jander and Voss encourages the present authors to think that the resolution is, however, real. A repetition of the Burtt and Catton type of experiment with the delicate means of recording whole-insect movement, as used in McCann and MacGinitie’s or Fermi and Reichardt’s work, would be of very great interest. C. RECOGNITION OF FORM

Hertz (1931) was unable to train bees to separate a black circle, a square, a triangle, and an elongated rectangle. Nor could they separate figures with longer outlines such as an X, Y, four vertical lines, or a hollow square. However, any one of each of these groups could be separated from any one of the other groups. These results are of the greatest interest but they need repeating with the aid of statistical checks to be sure that no slighter preference than “all or none” has been overlooked. Further, MazoxinPorshnyakov and Wischnevskaya (1964) suggest that, in fact, bees can be trained to separate a square from a triangle if each figure is made up of a large number of smaller squares or triangles. Their view is that the larger figures do not allow the insect to appreciate their form when it is close to them. The behaviour of many hunting wasps (Hymenoptera-Sphecoidea) is strongly suggestive of form vision. Thus Hobby (1932) finds a very wide range of species of Diptera constituting the prey of Mellinus arvensis. The colour of the species and the places where they are to be foundvaries very greatly, i.e. orange dung-flies, green flesh-flies, brown and black

l M A G E F O R M A T I O N A N D S E N S O R Y T R A N S M I S S I O N I N EYE

9

Muscidae, etc. The possibility of other senses also being involved cannot be excluded; but it is unlikely that there is a common "dipterous smell". The same selection of a particular form seems also likely in the tropical Sceliphron spirijiex (White, 1962),where the prey consists of many species of spiders of a wide variety of sizes and colours ranging from cryptic to conspicuously marked forms. It would be interesting to know the process of capture in Sceliphron. In Mellinus Hobby observed that the prey was jumped on from a distance of 2-3 cm; the wasp appeared to stalk the flies. It is striking that no one has recorded Mellinus bringing back to its nest prey other than Diptera or attempting to attack the "wrong" sort of insect. They regularly catch syrphid flies which mimic Hymenoptera, which suggests that the form vision is quite accurate. This suggests that the form vision of the hunting wasps is of a much higher order than would be expected by comparison with Hertz' experiments on bees. D. D E T E C T I O N O F M O V E M E N T

It is a common experience of entomologists that insects are very sensitive to motion of objects in their field of vision. Burtt and Catton (1954) tested the threshold for generation of spikes in the ventral nerve cord, and later (1956) in the optic lobe of the locust using a small light source which could be moved over a predetermined distance between rubber stops. Angular displacements of as low as 0.1" could evoke a response in the optic lobe and the latter response seemed significantly more sensitive than that in the ventral nerve cord. Thorson (1964) has obtained even lower values for motion perception in Schistocercu greguriu. He records the torsion in the neck muscles produced by presenting to the eye minute to-and-fro oscillations of a pattern attached to a drum. Angular movements of as low as 0~03"cangiveconsistentresponses. Once again, it would be of the greatest interest to have this delicate technique applied to the problem of the insect's resolution of striped patterns. It will be shown later that the resolution of repeated patterns is of more fundamental interest than the detection of minute point sources or minute movements. Another aspect of movement perception is the very slow angular movements which will evoke responses. Thus, in Chlorophunus, Hassenstein (1958) gets positive reactions to movements as slow as O.O2"/sec. Kunze (1961) in the honeybee in flight gets turning responses to movements of striped patterns of 0.1O/sec, and the same author (1964) finds that in the crustacean Ocypode an angular movement of 0*08"/sec gives consistent reactions and the true threshold is probably lower still.

10

E. T. B U R T T A N D W. T. CATTON

E. D E T E C T I O N OF T H E D I R E C T I O N OF T H E P L A N E O F POLARIZATION OF LIGHT

Starting from the work of von Frisch (1949), a large amount of evidence abundantly confirms the conclusion of Dethier (1963) : “Whatever may be the mechanism the fact remains that many invertebrates behave as though they can detect differences in the plane of polarization of light and utilize this ability in their economy of living”. The mechanism is still far from clear but the reality of the response remains. What concerns us in this section is the evidence for the view that it is the compound eyes as distinct from ocelli which are responsible for this ability. Von Frisch et al. (1960) have produced evidence that it is reception in the compound eye which is essential, by studying the orientation of the bees “waggle dance” where the effect was first observed. When the bees perform their direction-indicating dance on a horizontal surface, the direction of the food place is indicated by the straight “waggle run” in the dance. This can be altered by altering the plane of polarization of the light falling on the bee by interposing a sheet of Polaroid between it and the light from the clear sky. Von Frisch et al. found that if the upper part of the eye was covered the power both to return to the hive and to perform the dance was lost, but bees could with some difficulty still find their way back to the hive and dance perfectly correctly if only the lower half of the eye was covered. The aim of the experiments was to show that the operative factor is light from the sky direct and not reflected from objects in the surroundings; but they also show that the compound eyes rather than the ocelli are the important structures in this case. A full set of references to other arthropods in which responses to polarized light have been confirmed will be found in the above paper, in Jander and Waterman (1960) and in Dethier (1963).

111. THEOPTICSO F T H E COMPOUND EYE A. I M A G E F O R M A T I O N

The single ommatidium of the compound eye admits light from a much wider angle than would be expected from Muller’s theory. This is seen in Locusta where the ventral nerve cord response was studied (Burtt and Catton, 1954). By restricting the exposed part of the eye to a limited number of ommatidia, it was found that the results were consistent with the view that light entered an ommatidium as an effective visual stimulus over an angle of 10”each side of the axis of the ommatidium. Waterman

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(1954a) found independently an even wider angle of acceptance for light in the ommatidium of Limulus. Burtt and Catton found a comparable width of entry of light into ommatidia in isolated eye slices; this is confirmed by Kuiper (1962) in eye slices of Calliphorawhere the light entering individual rhabdomeres was studied. Washizu et al. (1964) find by recording with ultra-microelectrodes from single retinula cells in Calliphora that light enters over a wide angle but that the intensity of response falls off rapidly at angles inclined to the axis of the ommatidium. These observations show the physiological aspect of Exner’s (1891) observation that the image at the apex of the crystalline cone is minute but covers an appreciable area, and that the sense cells have a finite size; further that the images at adjacent cones overlap markedly. These considerations at once raise the problem of the formation of composite images by the compound eye. This does not seem to arise in Limulus with its shallow eye any more than in the single eyes of lepidopterous larvae. The means by which the collection of overlapping images in Limulus could still give orderly information to the central nervous system has been considered by Reichardt (1961) and again by Kirschfeld and Reichardt (1964). It is clear that the overlapping of the fieldspresents no fundamental difficulty provided that the intensity of the light response falls off at each side of the optic axis of the ommatidium. The latter work shows that this is the case in actual recordings from the optic nerve of Limulus. Thus the way seems clear to conclude that in spite of the overlap of the visual fields of adjacent ommatidia, the resolving power of the single lenses is realized. This should be quite high in Limulus since the lenses are relatively large. It is unfortunate that units responding tomovement have not been found in Limulus; perhaps they may be present in the deeper parts of the brain in view of the fact that they are present both in insects (Burtt and Catton, 1960) and in Crustacea (Waterman and Wiersma, 1963). If such units could be studied they might give a direct measure of the finest stripes which could be resolved by the Limulus eye. We now come to the explanation of the high resolution observed in Locusta and Calliphora, using electrophysiological methods, which is about three to four times the resolution to be expected from one ommatidium. Burtt and Catton (1962a, b) and Rogers (1962) attribute this to the diffraction images formed in the eye at depths below that of the familiar first image. Such images can be seen in an eye slice when a striped object is placed in front of the eye. It is convenient to place such an object in the substage of the microscope above the condenser, or to remove the latter. A succession of such images is formed (Fig. I), but the higher members of the series are not functional as they fall behind the pigment-containing

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FIG. 1 . A, Schematic group of ommatidia based on Locusla. B, Retinula cells showing arrangement of rhabdomeres, eccentriccell and rhabdomere structure enlarged. C, Arrange ment of rhabdomeres in Diptera. bm, Basement membrane; c, cornea; cc, crystalline cone; d, axes for maximum resolution; ec,eccentric cell, prf, post-retinal fibres; rh, rhabdome; rh’, rhabdomeres; 1,2, and 3, first, second and third image planes.

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basement membrane. The formatiqn of these images by an array of lenses such as the compound eye is easily shown. Rogers (1962), following Cowley and Moodie (1957), points out that since the lenses are small they will form an array of virtually coherent sources at their foci. This array will reform itself as an array of the same spacing at a distance 2a2/Xfor a square array, and 3b2/h for a hexagonal array, where a and b are the separation of the elements in the array and X is the wavelength of the light. Now calculation shows that the above distances (which we can regard as the focus of this diffractivesystem)aremuchdeeper than could be accommodated in the insect eye. Rogers calculates that even allowing for the curvature of the locust eye, the depth for therepetition of the pattern would be 3.4-3-8 mm and the depth of the basement membrane is at most 0.7mm. Rogers discovered that some of the shorter-distance intermediate images which Cowley and Moodie had considered, far from being more complex and diffuse than the generating system as these authors concluded, were, in fact, quite simple and sharp. His paper gives photographs of such images produced by a simple array of apertures using monochromatic light and they can also be photographed in eye slices (Burtt and Catton, 1962b). We can think of the formation of one of these sharply focused image systems at a given depth as representing the “diffractive focus” of the optical system. A simple question at once follows: if insect eyes are using such a system, what is the effect of increased size on the depth of the eye? Let us suppose that, for example, separation of the ommatidia (equivalent to their diameter) increases whether by growth at different instars of an exopterygote insect or by having facets of one part of the eye larger than in another, as occurs in many insects and Crustacea. Then if the same order of diffraction image is used in any one case, the depth of the eye over which diffraction occurs should increase as the square of the separation of the ommatidia. The results of Burtt et al. (1964b) suggest that this is true. This diffractivefocus of the array gives a means by which images can be produced, and Rogers (1962) figures an image of a fine grating similar to one of those used in experiments on resolution. Burtt and Catton found in eye slices that two (or possibly three) intermediate images might lie between the first image and the basement membrane (see Fig. 1). The second and third images had higher resolving power than the first imageapproximately two and three times respectively-and thus would offer a means of resolving finer patterns than could be resolved by the single lenses of the compound eye alone. Rogers estimates that the minimum aperture required by the system would be 194 p, which suggests an aperture about six ommatidia wide. There is a much more approximate

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approach to the problem of resolution which the reader may find helpful. Thus on Rogers’ model array, each aperture produces a single image in a manner exactly similar to that of a pin-hole camera, but it is also possible to produce an image from a collection of pin-holes as a simple experiment will show. This system has a resolution which approximates to that associated with the diameter of the system even though the form of the image is imperfect. In the same way the increase in the number of lenses contributing to the images, as the deeper images are encountered in the eye slices, also increases the resolution. I There is then in arthropod eyes a diffractive system which could give increased resolution, and measurements on some eyes suggest that they have been modified to make use of such images. There is a further anatomical feature of many eyes, generally described as “superposition eyes” in that the rhabdomes of the retinula cells only occur deep in the eye. This is well seen in the Euphausid crustacean Stylocheiron (Chun, 1896) but is best demonstrated in the moth Erebia (Fernandez-Moran, 1958). Here electromicrographs show the large typically striated rhabdomeres to be restricted to the deeper parts of the eye. In such cases visual microscopy shows that the retinula cells are connected to the cones by a long fine thread (Hesse, 1901;Yagi and Koyama, 1963).Now in moths’ eyes (Burtt and Catton, unpublished) the first image is formed at the apex of the cones, thus (supposing the fine connecting thread to be devoid of rhabdome-like structure) all the photoreceptive structures proper are located not where the first image forms but deep in the eye where the higher order diffractive images occur. The separate nature of the ommatidia in such eyes might also seem to break down. Grant and Sharplin (in Hocking, 1964) suggest in the moth Agrotis that retinula cells may form part of three adjacent ommatidia. This is easy to understand functionally if the retinula cells at this point are a continuous mosaic, but it is against the conception of whole ommatidia acting as separate units. The present authors, however, have claimed that the diffraction images play a part in the action of the eyes of Locusta, CalIiphora and Phormia, which insects (the last two especially) are generally classified as having “apposition eyes” in that the ommatidia are separated by opaque pigment and so lateral spread of light could not occur. In whole eye slices the diffraction images have been seen, but only when the patterns presented to the eyes are rather intensely illuminated. If diffractive effects were restricted to only certain types of compound eyes then the problem of the great depth of all true compound eyes remains. Compared to the nocturnal type of eye, the diurnal eye is faced

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with the problem of cutting down the intensity of light falling on the eye. Both human rods and insect retinula cells contain rhodopsin and, if one may be allowed a subjective observation, note how insupportableit is for the human eye to receive direct sunlight, but insects such as Locustu and Culliphora sit for hours with direct sunlight falling on their eyes. Further, a rough calculation shows that the relative aperture of a Culliphoru ommatidium is about three to four times that of the human eye with the pupil contracted for bright sunlight; thus the unshielded image in the insect eye would be ten times brighter than the image of the sun on the rods of our own retina. It is to be expected, therefore, that pigment is needed to cut down this intensity in the deeper parts of the eye. Another difficultyis this: how do the three images give a unified pattern of information to the central nervous system? Consider the state of affairs in the single rhabdomere; it will tend to be depolarized if the change from a dark to a light stripe passes over it. This seems to be adequate to give information of the type “something is moving in the environment”. It is less easy to understand how the diffraction images could give form vision, but we shall offer another explanation of this later. B. PROPERTIES O F T H E D I F F R A C T I O N IMAGES

The diffraction images (second and third images in Fig. 1) show certain differences from the first (geometric) image which is formed by the lenslets. The second and third images are much influenced by the orientation of the eye. Thus, when stripes of the pattern presented to the eye are at right angles to any one of the axes d in Fig. 1, the stripes are resolved with maximum clarity while at intermediate points the stripes become blurred. Burtt and Catton (1962b) found that there was an effect on the response of the locust eye when the stripes were orientated at different angles to the horizontal. The most important point was that such an effect was only found when the angle subtended by the stripes was less than that resolved by the single ommatidial lenslets, i.e. under conditions when only the second and third images could be operative in the eye. They did not, however, succeed in showing that changes in resolution were simply related to rotations of 60”. Further evidence on this matter comes from the behaviouristic work of Hertz (1931) and Zerrahn (1933) on bees, and Ilse (1934) on butterflies. They found that star-shaped figures were always more attractive to insects than solid ones. Hertz made one particularly interesting observation in which she found that the same elements (dark rods) arranged in parallel lines were much less attractive than when arranged radially. A possible explanation of this on the present theory is that radial patterns will be

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detected by the diffraction images in any orientation, parallel lines only on the average in 50% of cases, i.e. when at right angles to the axes d in Fig. 1. C. S P A C I N G OF P H O T O R E C E P T O R S IN R E L A T I O N T O I M A G E S

Burtt and Catton (1962b) have estimated that the retinula cells in Locusta are spaced in such a way that they could separate the details of the third image at its ljmits of resolution. Both in Locusta and Culliphora each retinula cell has its own nerve fibre which passes to the first synaptic region. This agrees with the earlier work of Cajal and Sanchez (1915) in Calliphora and that of Eltringham in Lepidoptera (1919). Recent electron microscope studies have confirmed this in Lepidoptera (FernandezMoran, 1958)and in Drosophilu (Diptera) (Yasuzumi and Deguchi, 1958; Waddington and Perry, 1960). In addition to this in Diptera, Cajal and Sanchez (1915), Meyer (195 1) and Burtt and Catton (1962b) find that all the fibres except one from any given ommatidium spread out like the roots of a tree and end in different groups in their synaptic association with neurones in the first synaptic region. The exception is the fibre which passes straight through the lamina ganglionaris and ends in the second synaptic region. These facts suggest that there is a neural structure fine enough for the potentiality for resolution of the optic system to be realized. A possible complication should be mentioned. In Diptera, as Dietrich (1909) showed and the recent electron micrographs strikingly confirm, the rhabdomeres are well separated, but in many insects, e.g. Orthoptera (Fernandez-Moran (1958) in Dissosteiru; Burtt and Catton (1962b) in Locusta), the rhabdomeres are closely pressed together. In Apis Goldsmith and Philpott (1957) conclude from electron micrographs that the rhabdomeres are fused in pairs. This might well restrict the resolution possible in such eyes if this appearance of fusion is definitely a physiological union, but until cases are shown where the whole of the rhabdome is continuous the present authors suggest that physiological separation occurs even when the rhabdomeres lie close together. D. T H E E R E C T I M A G E I N T H E COMPOUND E Y E O F Lampyris Exner (1891) figures a photograph of the external world seen through the eye of Lumpyris. This image was erect and surprisingly little distorted. It has been reproduced since (e.g. Wigglesworth, 1953) and a similar photograph was taken by Eltringham (1919). The present authors have seen this type of image in an eye slice of Lampyris, and Nunnemacher (1959) confirmed its occurrence in the closely related eye of Photinus. This image has become so widely known that it is essential to point out

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a number of very puzzling features about it. The eye of Lampyris was taken by Exner as the type for his theory of lens cylinder action but, as Eltringham (1933) makes clear, Kirchhoffer (1908) found that the eyeis of an unusual kind in that the cone is formed from an invagination of the cuticle. This accounts for the ease with which the cones can be separated from the retinula cells and still remain intact. The eye was termed “exocone” in Eltringham’s account. Exner found that when the microscope was focused on an eye slice the following succession of images was seen: (1) just behind the cuticle there was an inverted image which is of general occurrence in this situation in all compound eyes; (2) further back there was an erect image which occurred at the apex of the cones; (3) further back still these images appeared to fuse together into the single erect image which formed the object for Exner’s well-known photograph. Kuiper (1 962) confirms these observations in Lumpyris. The succession of images can readily be seen, but the situation of the superposition image (no. 3 above) was stated by Exner to be at 0.23 mm behind the apex of the cones. From his figures of sections of the Lmnpyris eye one can at once measure the depth of the basement membrane and note that it is only 0.125 mm behind the apex of the cones. Thus the superposition image falls outside the retinula cells altogether! Exner himself was keenly aware of this anomaly and tried to account for it, first by doubting if his section of Lampyris was at the deepest part of the eye and, second, by the possibility of shrinkage in the eye during fixation and embedding. Eltringhammade no estimate of the depth of the image and Exner’s own difficulties have not been noticed by later authors, but Nunnemacher (1959) states that in Photinus the image falls behind the basement membrane. Measurements made on the lampyrid Photorus show the same result, i.e. the single image falls behind the retinula cells. The occurrence of the optical phenomena found by Exner in Lumpyris are not in doubt; the problem is to fit them into our understanding of insect vision. The erect image at the tip of the cones is interesting; Kuiper refers to it but doubts if Exner’s conception of a lens cylinder with layers of higher refractive index in the centre will account for it. He suggests that diffraction theory might offer an explanation. The present authors suggest that the image found by Exner at 0.23 mm from the apex of the cones is a member of the succession of diffraction images found in other insect eyes and, further, that it is too far back to be made use of by the eye. However, the eye may still be making use of earlier members of the series of diffraction images. The fact that the image is erect and not inverted is of optical interest and demands an explanation.

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E. T. B U R T T A N D W . T. CATTON E. OPTICAL BASIS OF MOVEMENT DETECTION

From the foregoing the simplest explanation of movement detection would be to attribute it to movement of images over the photoreceptors. There are, however, a number of possible mechanisms, and the threshold for movement of a point source need not bear any simple relation to the resolving power of the optical system. It is also clear that the smallest single object to which the eye can respond need bear no simple relationship to the resolving power of the system, in much the same way that the human eye can detect single lines of far less angular extent than the limit for resolution. All that is needed is for the movement of the object to effect a change in illumination on the photoreceptors sufficient to initiate an impulse. It is not intended to imply by this that the detection of very small movements is not of value to the insect. It is clearly one of the most striking powers of the compound eye. But the threshold for movement does not allow one to distinguish between mosaic or diffractive theories in explaining its occurrence. F. OPTICAL DETECTION OF THE P L A N E O F POLARIZATION OF LIGHT

It is still not clear by what means these responses are detected by the compound eyes. If the method of detecting changes of direction of polarization is to convert them into changes of intensity, then there is a wide range of structures in the compound eye which might effect this. Partial polarization takes place at both reflecting and refracting surfaces and any such region might conceivably act as an analyser. Kalmus (1958) has even shown a case where the analyser consists of reflecting surfaces outside the insect altogether and the response to polarized light stops when nearby reflecting surfaces are removed. Jander and Waterman (1960) confirm this but show that there is a group of effects which cannot be thus explained and we must look for an analyser in the eye itself. Limulus offers a striking example. Here Waterman (1954b) showed that the characteristic spike response of the optic nerve due to the eccentric cells alters with change in the direction of the plane of polarization of incident light. This effect is most marked when the rays fall on the eye at the greatest inclination to the vertical. This is exactly what would be expected if the cornea of the eye were acting as an analyser and the partial polarization due to the refraction of rays falling obliquely on the cornea were causing changes in intensity in the light falling on the retinula cells. Autrum and Stumpf (1950) showed changes in the form of the ERG of

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Calliphora when a sheet of Polaroid was rotated in front of a small source illuminating the eye, but this hardly allows further conclusions concerning the means by which this change is detected. Later Burkhardt and Autrum ( 1960), Burkhardt and Wendler (1960) and Autrum and Zwehl(l962) give evidence from ultra-microelectrode recording from single retinula cells. These showed potential changes in step with changes in the direction of the plane of polarization of light. This points to the individual ommatidium but does not allow one to discriminate between an analyser present in the lens, cones or the rhabdomes. Earlier Menzer and Stockhammer (1951) claimed that changes occurred in the intensity of the light transmitted by the individual rhabdomeres in the eye of Apis when the plane of polarization was rotated and thus supported the hypothetical scheme of von Frisch (1950), in which each of the rhabdomeres was represented by a sheet of Polaroid. De Vries (1956) failed to confirm Menzer and Stockhammer's findings for eye slices. Recently Giulio (1963) has described an experiment which shows that there is at least a possible analyser in the retinula cell region. The upper part of the eye of Calliphora is cut away and a beam of light is focused with a microscope objective at right angles on to the ommatidia. The ERG is recorded and then the plane of polarization of the light is rotatedwith apolaroid filter. Theresult is to produceawavelike change of potential with the peaks occurring when the electrical vector is at right angles to the long axes of the ommatidia. Giulio relates this to the striated form of the rhabdomeres referred to earlier. These results suggest that the rhabdomes alone could account for the responses of the individual cells noted above. It is interesting to note that records from single units in the arthropod eye (Burtt and Catton (1960) and Horridge et al. (1965) in Locusta; Waterman and Wiersma (1963) in Podophthalmus) have found no response to changes in polarization. These units are, as we shall see later, of third or fourth order neurones and are from arthropods where polarized light responses have not yet been observed. It may well be, however, that polarized light responses are transmitted by smaller neurones which have not so far been detected as spike responses in the optic lobe. G. H I S T O L O G I C A L C H A N G E S A C C O M P A N Y I N G L I G H T A N D DARK ADAPTATION

It has long been known that in the eyes of many insects and crustaceans the change from light to dark is accompanied by alterations in the distribution of pigment. Darkness has the effect of causing the pigment to move distally and this is usually described as converting the eye into a

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superposition eye, while light has the effect of causing the pigment to move down and tend to separate the individual ommatidia. Changes in the position of the nuclei of the retinula cells and of the length of the rhabdomeres are less widely known, although they might have relevance to the changes in d.c. potentials (see Section IV, C). Thus Yagi and Koyama (1963) found in Lepidoptera that in the retinulae of some species the nuclei are immobile, but that in many nocturnal moths the nuclei of the retinula cells begin to move before the pigment, and may move within 30 min from being in a cluster just below the apex of the cone to a position half way down the ommatidium. Ludtke (1953) describes the extension of the rhabdomes in Notonecta during dark adaptation where the rhabdomeres extend between the cells of the crystalline cone. Barnard and Horridge (unpublished observations : see Fig. 2) find a marked difference between the retinula cells as seen under the electron microscope in dark and light. When dark-adapted, the mitochondria move centrally around the rhabdome and the vacuoles are peripheral, while in light adaptation the mitochondria become placed peripherally and the rhabdome is surrounded by vacuoles.

Iv. ELECTRICAL RESPONSES I N COMPOUND EYE A N D OPTICLOBE These have been the subject of many investigations and it is intended here to concentrate on the more recent work, correlating where possible with earlier findings. Three previous reviews may be mentioned, those of Wulff (1956) and of Ruck (1962,1964). A. N A T U R E OF THE RESPONSES

Whether using extracellular or intracellular recording methods (with one exception), the sole type of response obtained from the ommatidial zone has been the complex electrical wave (electroretinogram or illumination potential). Naka and. Eguchi (1962), exceptionally, were able to record spike discharges from impaled retinula cells of the worker honeybee, superimposed on the depolarizing response characteristicallyevoked by illumination in this and other species. Spikes have not yet been recorded with certainty from any vertebrate photoreceptor cell, and records of spikes superimposed on depolarizing waves in Limulus eye are more likely to be derived from eccentric cells than from retinula cells and thus to represent post-synaptic events (Fuortes, 1959; Fuortes and Poggio, 1963). Certainly the insect retinula cell, of diameter not exceeding

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a few micra, presents difficultiesfor intracellular recording, although some workers have obtained steady membrane potentials at normally expected levels (cf. Burkhardt and Autrum, 1960; Naka, 1961; Naka and Eguchi, 1962). Scholes (1964) could record spikes from locust retinula cells only in severely attenuated form, and was troubled by “electrode noise” at normal light levels. This was resolved at very low intensity into discrete miniature potentials of a quanta1 nature, as previously reported for Limulus retinula cells (Fuortes and Yeandle, 1964). It seemed possible that each miniature potential was associated with capture of a single photon. It may be useful to point out that the appearance of spikes in the plateau phase of the depolarizing response in an intracellular record is not a proof that they originate from the impaled cell. In a partly depolarized cell the membrane conductance is increased and action currents from neighbouring active regions may gain access to the recording site (cf. Burkhardt and Autrum, 1960). Spikes recorded in this way are of small amplitude, of random occurrence, and increase in size with progressive depolarization of the cell. On the other hand spikes originating in the impaled cell are regularly spaced, of large and uniform amplitude and tend to decrease in size, whilst increasing in frequency as depolarization progresses. The decrease in spike size is attributed to the increased membrane conductance associated with illumination, and was noted by Naka and Eguchi in their honeybee retinula cells. These workers noted also that when the electrode was withdrawn to a site just outside the cell the sign of the membrane depolarization was reversed (from positive to negative) as expected, but the spikes retained a positive sign. This indicated that the spikes, whilst originating from the same cell, arose from a region other than the site of electrode penetration. They suggested that this site was most likely to be the retinula cell axon, where it passes through the basement membrane. Tomita (1956) had earlier made a similar suggestion for the Limulus retinula cell, and Fuortes (1959) supports this conclusion. Spike initiation from proximal axon rather than from neuron soma is not a novel concept, being established in at least one other case, the mammalian motoneuron (Eccles, 1957). Since however in the majority of cases no spike trains have been observed to originate from retinula cells, one may suppose either that these cells produce only a generator potential, or that present techniques are in some way inadequate to record them. Extracellular recordings of the electrical field potential in the ommatidial zone in response to brief light exposure have been widely studied (e.g. Bernhard, 1942; Autrum, 1950; Autrum and Gallwitz, 1951 ; Burtt

FIG.2. Electron micrographs of transverse sections of ommatidia in Locusta migratoria (P.B.T. Barnard and G. A. Horridge). A, Dark adapted state with mitochondria clustered around rhabdomeres and vacuoles peripheral. B, Light adapted state with vacuoles around the rhabdomeres and mitochondria peripheral. ec, Eccentric cell; m, mitochondria; rh', rhabdomeres; v, vacuoles.

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2. C, Central part of a section under high magnification to show the eccentric cell.

and Catton, 1956; Hassenstein, 1957; Walther, 1958; Ruck, 1958; Goldsmith, 1960), and observations over much longer periods up to 10 min have been reported (Burtt and Catton, 1963, I964b). These investigations have covered many different species and used different electrode arrangements and types of stimulus, so that one may only summarize the results in very general terms. Thus an electrode situated in the ommatidial region records a depolarizing response to light in all species, the form of which is markedly dependent on light intensity. At low intensities the response is a simple negative plateau; at higher intensity there appears an initial fast phase, which overshoots the negative plateau, itself increased in amplitude. I n some cases the initial fast deflexion (the “on-transient”) is simply a rapid rise of negativity, in others it is preceded by a very brief positive wave so that the total deflexion is diphasic, positive-negative. A similar diphasic on-transient is found in many ocellar responses (Ruck, 1958, 1961a, b) and thus it appears to be of some fundamental nature in the

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process of visual excitation. Ruck (1962) sees the initial positive deflexion as a reflexion of a spike-like depolarization initiated in the retinula cell axons. It is recorded in positive sign near the cornea, since this region is acting as a current source during retinula cell axon depolarization. In intracellular records only a simple monophasic transient wave is seen (e.g. some dragonflies and Luciliu, Naka (1961); honeybee, Naka and Eguchi (1962); Culliphora, Burkhardt and Autrum (1960); Limulus, Fuortes (1959)). This may pass smoothly over into the sustained phase of depolarization under continuing illumination, or be separated from it by a more or less well-defined notch (Naka (1961) damselfly and Luciliu; Fuortes and Poggio (1963) Limulus). In the latter cases we can recognize a correspondence with the diphasic on-transient seen in extracellular records, with due allowance for sign reversal. Ruck (1962) draws attention to this feature and quotes more examples. Although spike-like in appearance the on-transient is not an all-ornothing response, but is graded according to stimulus strength. In Limulus it bears a sigmoid relationship to the logarithm of intensity, while the plateau phase bears a linear relationship. Autrum (1950) found similar relations for the eye of Culliphoru. In the locust eye the amplitude of the on-transient is proportional to log [intensity increment], whilst the 5 min adapted level of the plateau phase increases at first and then reaches a saturation level (Burtt and Catton, 1964b). That the on-transient may have spike-likecharacteristics is suggested by the work of Benolken (1961) on single retinula cells of Limulus, where the transient was shown to reverse the membrane potential by 30-40 mV at high intensities. On the other hand Naka (1961) found in Luciliu and a number of dragonfly species that the initial transient reached a level of complete depolarization at high intensities, but did not reverse the membrane potential. The transient shows a fairly sharp threshold in the honeybee ocellus (Goldsmith and Ruck, 1958), as in our observations on Locustu compound eye (Burtt and Catton, 1964b). Again in the ocellus it is followed by a refractory period (Ruck, 1961b), and is abolished by high potassium ion concentration (Ruck, 196la). High potassium (200 mM) has also been shown greatly to attentuate the “retinal action potential” of the crab Eupugurus; substitution of choline for sodium ion appeared to reduce the on-transient differentially, more than the plateau phase of the response (Stieve, 1964). It is thus difficult to assess the status of the on-transient on present evidence; one would suppose that it is unlikely to be a propagated spike. Less consistent behaviour is reported at light-off. In Limulus only a weak off-transient occurs, not seenin all records. Whereas well-developed

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off-transients (positive) have been reported in extracellular records of responses in eyes of certain species (e.g. Calliphora, Aeshna, Autrum and Gallwitz, 1951; Locusta, Burtt and Catton, 1956, 1963, 1964b), such off effects are much less marked in intracellular records taken from retinula cells (e.g. Burkhardt and Autrum, 1960; Naka, 1961; Fuortes, 1959 (Limulus) ). The possibility implied, that the off-transient has an origin distinct from the on-transient, will be further discussed below. Bernhard (1942) first noted that the fast transient deflexions were eliminated from the illumination response in Dytiscus when the optic lobe was severed from the eye, a finding later confirmed for Calliphora by Autrum and Gallwitz (1951) and by the present authors for Locusta (Burtt and Catton, 1963, 1964b). These components of the response to light are the first to disappear in a deteriorating preparation, as noted by Naka (1961) in a damselfly eye, and agree generally with findings in the locust, in which the transient components were specifically eliminated when the optic lobe was crushed or subjected to anoxia (Burtt and Catton, 1964b). Fast transients are either absent or poorly developed in the normal eye in some species, e.g. Dixippus, Tachycines, young larvae of Aeshna (Autrum, 1950); Periplaneta (Burtt and Catton, 1964b). These come into the classification of “slow” eyes (Autrum), characterized also by showing a low flicker fusion frequency, and in contrast with “fast” eyes which show large transients and a high flicker fusion frequency. Ruck (1958) has criticized Autrum’s scheme of classification into these categories. Burkhardt and Autrum (1960) made the interesting observation that transient responses could be recorded intracellularly in retinula cells whose membrane potential had declined to zero. They were led to conclude that the characteristic response of the retinula cell is a simple quasirectangular wave, the transient components being of an extrinsic origin and conceivably from the rhabdomeres. These elements certainly appear to be essential for the depolarizing response, as clearly shown by Eguchi et al. (1962) in the silkworm Bornbyx mori, where during larval development the electrical responses were found to develop in parallel with the differentiation of the rhabdome. Ruck (1964) draws attention to the very limited extracellular space (c 100 A in many species) between the closely packed retinula cells, and questions its sufficiency as an ion store for maintaining a prolonged positive ion entry phase, supposing this to be required to maintain a plateau of depolarization in this region. Certainly in the locust such a partial depolarization may be maintained for periods up to 10 min

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(Burtt and Catton, 1964b), and the same is true for Lirnulus (Hartline et al., 1952). B. POTENTIAL PROFILE OF COMPOUND E Y E A N D O P T I C LOBE

Experiments using Ringer-filled microelectrodes with tip diameters of the order of 10 p have revealed that a characteristic profile of steady potential exists along the axis of the eye and optic lobe in five species investigated : Locusta, Calliphora, Phormia, Periplanata, and Aeshna larva (Burtt and Catton, 1964a). A typical profile in Locusta showed the following features (Fig. 3). Just beneath the cornea was a zone of negativity, which declined sharply as the ommatidial zone was reached, when the 70

I

h

20 200

400

600

J

I

800

1000

I

1200

I

1400

I

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1

1800

I

2000

bm

Depth from corneo

(11)

FIG.3. Axial potential profile of locust eye and optic lobe (based on Burtt and Catton, 1964b). Arrows show direction and magnitude of theon-transient. Solid bars show depth and extent of the three synaptic regions, I, 11,111. bm, Basement membrane.

potential became positive in sign (with respect to a large indifferent electrode in the body). In the first synaptic region of the optic lobe there was a narrow zone of high negativity, 60-70 mV in amplitude. Similar peaks of negativity were often encountered in the second and third synapticregions, with intervening positive zones. The total excursion of potential between the ommatidial zone and first synaptic region was 70-90 mV and extended over a distance of about 350 p. Thus the retinula cells lay in parallel with an electrical gradient of about 0.25 V/mm. The

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27

gradient was a maximum in darkness and declined during illumination, due chiefly to depolarization in the ommatidial zone, light having little effect on the negative peak. It would appear that the retinula cells may be subjected to a depolarizing bias in a manner which would tend to alter their sensitivity to illumination, the bias being larger in the dark. The arrangement is reminiscent of the biasing potential across the cochlea hair cells provided by the endolymphatic potential in the vertebrate ear (von Bekesy, 1951). The negative peak zones in the optic lobe were not believed to be due to impalement of individual cells, whose size was generally comparable to the tip diameter of the electrode used. They were eliminated reversibly when the optic lobe was subjected to a short period of anoxia; it was noted that the synaptic regions were richly supplied with tracheoles (Burtt and Catton, 1964a). C. POTENTIAL CHANGES I N RESPONSE TO D A R K A N D LIGHT-ADAPTATION

In most studies of the electrical response to illumination (electroretinogram) the exposures used were very brief, often not exceeding 1 sec. A study was made (Burtt and Catton, 1963,1964b) of the potential changes in both the eye and the optic lobe of a number of insect species,using much longer exposures (5 min). It seemed more fitting to refer to these longterm records as “adaptational” responses, to distinguish them from the results from brief exposures. Essentially the difference is that in the adaptational response, the full-time course of the negative “plateau” interposed between the on- and off-transients is revealed. A standard period of 5 min was chosen for both light- and dark-adaptation periods, it being found that the potential had nearly stabilized after these times. A typical record from the locust eye is shown in Fig. 4A obtained with a 10 p Ringer-pipette electrode in the ommatidial region together with a large electrode in the body. The on-transient is a simple negative rise, and is followed by a rapid fall of potential, then by a much slower decline; even after 5 min there is still a marked negative deviation from the darkadapted baseline. The off-transient, comparable in size to the on, is of opposite sign and overshoots the baseline. Recovery is almost complete in 5 min. Using this recording technique an initial positive wave was never recorded. Two points of interest arose in the further study of the adaptational response. First, when responses were recorded at different depths in the eye and the optic lobe it was found that the on- and off-transients passed through a null at a level corresponding approximately with the negative B

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peak in the first synaptic region (later work has indicated a better correlation with the basement membrane). Beyond this depth the transient responses reappeared, but reversed in sign, reaching amplitudes equal A

*-

ON

5 min

-

I

OFF

FIG.4. Variation of potential (recorded between extracellular microelectrode and large indifferent electrode) in locust eye during 5 min light-adaptation ( 0 ) followed by 5 min dark adaptation ( 0 ) . A, Electrode in ommatidial region; 0 , response after crushing optic lobe or subjecting insect to oxygen lack. B, Electrode just beyond first synaptic region. (Based on Burtt and Catton, 1964b.)

to or even exceeding those seen in the ommatidial zone (Fig. 4B). The slow plateau component, on the other hand, did not go through a null but was only displaced and followed a similar time-course at all depths. (Reversal of the transient responses with increasing depth of recording

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had been noted earlier (Burtt and Catton, 1956; Naka and Kuwabara, 1959), in Locusta, Calliphora, Aeshna and Chorthippus, the point of reversal being in each case close to the basement membrane; the method of recording did not at that time allow a study of the slow component of the response to light.) The second observation of interest was that the on- and off-transients could be eliminated from the ommatidial zone response by subjecting the optic lobe to trauma by crushing, or to a period of 10-1 5 min of anoxia (Fig. 4A). Theseprocedures did not exclude the slow phase of the response, although it could be greatly reduced by longer periods of anoxia, especially in nitrogen. Perhaps bearing on the relative oxygen requirements of the two processes are the observations of Autrum and Tscharntke (1962), who found that whilst the oxygen consumption of the locust eye was about the same in the steady light- or dark-adapted states, it was markedly raised at the onset of illumination. We conclude that in general the rapid and slow phases of the adaptation responses are of independent origin, and when the method of recording comprises one electrode in eye and a large indifferent contact, they appear to sum algebraically. Two explanations of these phenomena are offered, which are not necessarily mutually exclusive. The first hypothesis will be in passive electrical terms, the second will involve post-synaptic hyperpolarization. On a passive electrical theory one regards the basement membrane as a resistance barrier. No measurements on this have been reported, but one has for comparison the “R-membrane” of the vertebrate eye, associated with the pigment epithelium (Brown and Wiesel, 1959; Tomita et al., 1960) which affords a resistance barrier and across which certain components of the ERG alter in sign. It is proposed that in the insect eye, at light on, there is a large surge of positive current across the basement membrane, and that this transfer of positive charge results in the observed opposite sign of the on-transient recorded on the two sides (Fig. 5). The passage of this current, from ommatidial zone to optic lobe, could be explained by a transient marked increase in conductance of the retinula cell axons where they pass through the pores in the sieve-like membrane. The conductance increase would result from the primary depolarizing action of light on the whole retinula cell, including its axon. The driving EMF would consist of two components, a major one being the standing potential difference of 70-90mVexisting across the membrane(seeabove), and a minor one, the current sink provided by the depolarized axon. Procedures which eliminated the negative peak in the potential profile of the first synaptic region would thus eliminate most of the driving

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EMF, and would abolish or at least strongly attenuate the transient response. This hypothesis agrees with observation. In the dark-adapted eye this standing p.d. is at a maximum, and sudden illumination would produce a maximal on-transient and provide greatest sensitivity. With continuing illumination it would be assumed that the effective conductance of the membrane decreased again, whilst yet remaining somewhat above that in darkness. Much less current now passes through the membrane, and the slow phase of the response, originating by sustained depolarization of the retinula cell bodies, would be recorded as of the same sign on both sides, although somewhat attenuated in the optic lobe. This is observable.

FIG.5. Hypotheses for origin of the deep positive on-transient (for explanation, see text), bm, Basement membrane; ns, neurone in first synaptic region; rc, retinula cell; tr, magnitude of on-transient. Arrow shows direction of postulated current flow.

One must, however, take into account an additional hypothesis to explain the positive on-transient in the optic lobe, derived from a consideration of work on the dragonfly ocellus (Ruck, 1961a-c). Thus, in addition to the depolarizing components recorded in the photoreceptor zone of the ocellus, Ruck described a hyperpolarizing response at the level of the synapses of the retinula cell axons with the ocellar nerve fibres, which was associated with inhibition of the resting discharge in these fibres.If it were assumed that the f i s t synapsein thecompoundeye behaved in the same way, we would expect to record a positive on-wave in the vicinity of the first synaptic region. That post-synaptic hyperpolarization does not appear to offer a complete explanation of the positive on-wave in the optic lobe is seen when we consider the effects of crushing the optic

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lobe in the locust. This should, by destroying the synapses, eliminate such a hyperpolarization, but should not (as is in fact observed) at the same time abolish the negative on-wave in the ommatidial zone. D. I N D E P E N D E N T O R I G I N OF T H E OFF-RESPONSE

It is interesting to note that intracellular records from both Limulus and insect retinula cells fail to show an off-transient; the potential simply bm

1 B

OFF

ON

FIG.6. Potential changes recorded between two microelectrodes in eye of locust @. J. Cosens). A, Position of electrodes; upper electrode tip just inside cornea; lower electrode tip in alternative positions (i) distal and (ii) proximal to the basement membrane, bm. B, Records obtained in response to 3-min period of illumination with lower electrode first at (i), then at (ii).

drifts back to its initial level. Recent work in this laboratory (Cosens, unpublished) has indicated that the off-transient in the locust eye originates in the optic lobe rather than in the ommatidial zone. This was revealed when, instead of using one electrode in the eye and a large indifferent connexion, two electrodes were used in the eye itself (Fig. 6). With the superficialelectrode at earth potential it was found that, whereas the on-transient was present throughout, the off-transient only appeared

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when the deeper electrode passed through the basement membrane. With the original electrode arrangement the two transients appeared at all depths, since they were being recorded in series. Other, less direct evidence for independence of on- and off-responses may be put as follows. (1) Only on-responses occur in Limulus optic nerve (spikes), although off-responses occur in the centrally placed ganglia (Wilska and Hartline, 1941). (2) Ocelli produce an on-transient in the photoreceptor zone and an off-transient in the region of synapses (Ruck, 1961a). (3) The thresh,old for off-spike bursts is consistently lower than that for on-spikes (Burtt and Catton, 1962b). E. V I S U A L T H R E S H O L D C H A N G E S L I N K E D W I T H POTENTIAL VARIATIONS

Recent experiments, brieflyreported(Burtt et al., 1964a),have indicated that the visual threshold of the locust eye, measured as the smallest change of intensity required to evoke a spike discharge in the nerve cord, rises and falls along with the potential recorded in the ommatidial zone, i.e. following approximately the time-course of the adaptational response. When a dark-adapted eye was suddenly illuminated, the threshold for a nerve cord spike response rose by several orders concomitantly with the rise of the negative on-transient. In the steady light-adapted state the visual threshold was higher than in the dark, but to a lesser degree than the initial rise at light-on, in the same way that the negativity in the ommatidial region declines to a steady value during adaptation. Stepwise increments of intensity produced changes of a similar kind quantitatively in both visual threshold and potential. Thus the depolarized state in the photoreceptor zone is linked with a refractoriness to photic stimulation, which is virtually absolute at the height of the on-transient with high levels of illumination. At light-off, however, there was no increase in excitability as a concomitant of the positive off-transient, the visual threshold simply declining to that of the dark-adapted eye. Since the off-transient may well be of separate origin from the on-transient, and develop in the optic lobe, this behaviour could imply that refractoriness was associated chiefly with the photoreceptor elements. The evidence from the visual threshold changes, taken in conjunction with the electrical responses to light- and dark-adaptation, might suggest that the adaptational changes consist of an initial rapid “neural” component (depolarization of retinula cell axon) followed by a delayed slow component probably associated with bleaching and resynthesis of visual

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pigment. Dowling (1963) has described such components in the visual response of the rat, using as a criterion of threshold the evocation of an ERG of constant size. Baker (1963) has summarized the evidence for an early rapid and a delayed slow phase in light-adaptation of the human eye. He concludes that the large initial change in visual threshold is of neural origin, since over a short period of the order of seconds the amount of pigment bleached would be too small to account for the shift in threshold (see also Battersby and Wagman, 1959). The possible role of migration of the screening pigment in the adaptational responses in insect eyes has been suggested by the work of Bernhard and Ottoson (1960a, b) and Bernhard et al. (1963), who studied the timecourse of dark-adaptation in some nocturnal and diurnal Lepidoptera, using again the criterion of a constant-amplitude ERG response. They found a smooth dark adaptation curve in diurnal species (range of 1.5 log units), and a two-stage curve in nocturnal species (first stage 1.5, second stage, 2-3 log units). The second stage, found only in the nocturnal species, was shown to proceed along with migration of the shielding pigment (apposition-superposition transformation). The first stage, complete within about 10 min, they ascribed to resynthesis of visual pigment; it corresponds in the time scale to the slow phase of electrical potential in our adaptational responses. Edwards (1964) has noted in behavioural experiments that dark-adaptation occurs in moths with mutant eyes devoid of screening pigment; this would correspond to the early phase described by Bernhard and Ottoson and to our slow electrical and visual threshold changes, and would be attributed to alterations in visual pigment. F. THE O P T I C P A T H W A Y I N T H E LOCUST

Spike discharges have been recorded from optic lobe and ventral nerve cord of the locust, and have also been recorded in the optic lobes of Calliphora, Chorthippus and Aeshna (Burtt and Catton, 1954, 1956). The stimuli employed have been (1) light on and off and (2) movements of objects in the visual field, e.g. striped patterns and virtual point sources. 1. Spika discharges in the ventral nerve cord Spike responses to on/off and movement stimuli were readily recorded in the thoracic (but not the abdominal) nerve cord in Locusta and Calliphora, but not in Psriglaneta. The spikeswere mostly large, although small spikes would not readily be seen against the high level of background activity normally present in the nerve cord. Sections of the nerve cord connectives showed the presence of a number of large non-myelinated

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fibres, which are likely to be transmitting the spikes observed. These fibres appear to pass through the ganglia without synaptic relays, since they were not blocked by topical application of nicotine. When the two

FIG.7. A, Types of neural connexion in the eye and optic lobe (compiled from Cajal and Sanchez and from Zawarzin). B, Types of neural connexion between the optic lobe and central nervous system (compiled from Burtt and Catton, Satija, Vowles). -Connexions known from histology and electrophysiology; - - - - from histology only. a, Neurone associated with one synaptic region; b, brain; cp, corpora pedunculata; cn, connecting neurones within optic lobe; lvf, long visual fibres; nn, neurones connected to central nervous system ;so, sub-oesophageal ganglion ;svf, short visual fibres ;th’, th”, th’”, thoracic ganglia. I, 11,111, first, second and third synaptic regions.

-

connectives were separated it was found that the responses in each were mainly derived from the opposite eye, and there is anatomical evidence for the crossing of large fibres in the brain stem (Satija, 1957, 1958) (see Fig. 7B). Ipsilateral discharges of smaller spikes were also found. The

35 latency of the large spike discharge in the nerve cord ranged from 53 to 30 msec (mean values), decreasing with increasing stimulus intensity (light flash) (Burtt and Catton, 1959b). Less consistently were seen discharges of smaller spikes with latencies up to 150 msec. Electrical pulse stimuli applied to an electrode deep in the optic lobe evoked non-fatiguing responses in the nerve cord with a latency of 4-10 msec. Conversely, stimulation of the nerve cord evoked responses of similar latency recorded deep in the optic lobe (Burtt and Catton, 1959b).It was concluded that the large diameter crossing fibres system formed a fast conducting final common pathway for visualimpulses reaching the nerve cord, but was not the only available pathway. The ventral nerve cord preparation has been used for the detection of threshold visual responses in tests of the angle of acceptance of single ommatidia (Burtt and Catton, 1954), of visual acuity (Burtt and Catton, 1962a, b) and of visual threshold (Burtt et al., 1964a). For this purpose this preparation is more convenient than the optic lobe with microelectrode, although it was noted (Burtt and Catton, 1956) that the movement sensitivity threshold is higher for the nerve cord than for the optic lobe response. IMAGE FORMATION A N D SENSORY T RA N SM ISSIO N IN EYE

2. Optic lobe spike discharges A glass-shielded silver electrode of tip diameter 5-1 5 p, inserted through the cornea and passed along the axis of eye and optic lobe, was found to pick up spike discharges, partly “spontaneous” but also in response to visual stimuli (Burtt and Catton, 1956b, 1959, 1960). Irregular spikes of small amplitude were encountered throughout the depth of the eye itself and in the outer part of the optic lobe, giving responses to on/off and movement stimuli; there was no evidence of localization of these responses, which were therefore attributed to electrical spread from deeper sites of activity. When the electrode tip reached a depth corresponding to the outer border of the second synaptic region there was an abrupt onset of large spike activity, giving well-defined responses to stimulation. In this region it was often possible to record isolated single unit discharges and study their behaviour (Burtt and Catton, 1960). A little deeper than this, and at a site judged to be within the second synaptic region, there was a zone of intense spontaneous activity, in which it was difficult to pick out visual responses. A little deeper again, level with the inner border of the second synaptic region, the spontaneous activity sharply diminished and clear responses were again obtained. A second active zone was often encountered at the level of the third synaptic region. It was not possible to identify with certainty the structures giving rise B*

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to these spike responses; two of the more likely possibilities are either (a)

the large neuronal masses lying rather to one side of the axis, and sending fibres into the synaptic regions, or (b) these fibres themselves in their transverse course through the synaptic region. Optic lobe responses to stimulation of the contralateral eye were noted, which were abolished by severance of the stalk of the lobe. There was concomitant anatomical evidence for fibre systems connecting the two optic lobes (Fig. 7B). 3. Tima relations of visual impulse transmission Using as a source a brief flash of light of controllable intensity, the latencies for responses at various points along the visual pathway to the nerve cord were measured, choosing two standard intensities, one high (40000 lux) the other much lower (2 000 lux) since it was known that response latency was highly dependent on intensity. The illumination potential, presumably the first recordable electrical event, ranged in latency from 15 down to 6 msec in Locusta (larger value for lower intensity). We may compare this with 8-10 msec in Calliphora (Autrum and Gallwitz, 1951), 10-20 msec in cecropia (Jahn and Crescitelli, 1939), 15-20 msec in Periplaneta (Walther, 1958), 4 msec in Sacophaga and Eristalis (Hassenstein, 1957), and 7-15 msec in Lucilia (Naka, 1961). The latency for a spike response to the test flash in the second synaptic region (the only site in the optic lobe where an accurate estimate could be made) was 34 msec at the lower intensity and 26 msec in the higher. The corresponding values for the nerve cord latency were 53 and 30 msec. Two features emerge : (a) most of the delay in transmission occurs in the optic lobe, presumably in the synaptic regions; (b) the delay in transmission from second synaptic region to nerve cord is severely cut at the higher intensity, amounting then only to 4 msec, avalue about equal to the transmission time in the large fibre system propagating impulses from optic lobe to nerve cord (see above). Anatomical studies at this time revealed a fibre tract passing directly from the second synaptic region into the optic stalk, thus by-passing the third synaptic region and eliminating synaptic delay at that point (Burtt and Catton, 1959b); Fig. 7A, B. Using electrical pulse stimuli applied to the optic lobe electrode, it was possible to evoke spike responses in the nerve cord which fell into four latency groups. The shortest of these (4-10 msec) were able to follow at high frequencies without fatigue and were presumed to be a direct fibre system. The longer latency responses all showed fatigue at lower frequencies, rate of fatigue increasing with latency of group. In general, longer latency was associated with more superficial site of stimulation.

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In some experiments clear threshold minima could be established for the different latency groups, allowing one to localize them at specific depths in the optic lobe. The shortest latency responses could not be localized in this way. Analysis of the data from electrical pulse stimulation yielded approximate values for synaptic delays at the three synaptic regions, which were 11 msec for the first, 19 msec for the second and 7 msec for the third. The total synaptic delay for the optic lobe would then be 37 msec, and adding 4-10 msec for transmission in the fibre system to the nerve cord and 6-1 5 msec for IP latency gives 47-62 msecs for the expected cord latency. The latency measured for the lower intensity source being 53 msec, it is seen that there is fair agreement. Nerve cord latency for highintensity flash, 30 msec, could be explained as due to elimination of delay in the third synaptic region (7 msec) and some reduction in delay at the second, since the by-passingtract takes off from fibres passing transversely through this region. 4. Bshaviour of single unit rasponses in optic lobe

Elements giving rise to large spikes could frequently be distinguished when the electrode tip was sited at the outer margin of the second synaptic region, and since the spikes were of uniform size and tended to discharge with regular frequency they were assumed to originate from single active units, either cells or axons. A study of these responses (Burtt and Catton, 1960) suggested that they could be classified into three types. The most common (about 75%) were of a type which gave brief spike bursts at light-on and off,with a low-frequency dark-discharge partially inhibited by illumination, and responding to movement stimuli. These were termed “D-units”. Less common (“L-units”, 18%)were units silent in darkness, giving a spike burst only at light-on, with a sustained discharge during illumination, and no response to movements. The spike frequency during sustained illumination was an approximately logarithmic function of light intensity. Rarely found (7%) were on-only units, giving a simple spike burst at light-on. The visual field area for each unit was much larger than that for a single ommatidium (20”; Burtt and Catton, 1954) and in many cases covered the whole field of the eye. Presumably these units are at points of convergence in the visual pathway to which most of the retinula cells are able to transmit information. Many of the features of single unit behaviour described above for the locust eye are paralleled in a study of single “optic nerve” fibres in the eye stalk of the crab Podophthalmus (Waterman et al., 1964), pointing to a generality of behaviour in arthropod compound eyes.

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5 . Association of on- and off-transient waves with spike bursts in optic lobe There was a close time correlation between the on- and off-transient deflexions and the on- and off-bursts of spikes from the common D-units in the second synaptic region (Burtt and Catton, 1960). Whilst it seemed evident that the transients gave rise to spike bursts, nothing is at present known about the way in which the wave-responseis coupled to the spikegenerating mechanism, except to say that it seems unlikely on present evidence that spikes originate directly from retinula cells. Indeed it is not possible to be certain that spike discharges arise in the first synaptic region; present techniques have not been adequate to decide this question. O F ARTHROPOD VISION V. THEMECHANISM A. T H E E Y E OF Limulus The lateraleye of Limulus has been the object above allothers for studies of visual excitation in arthropods. Limulus is one of those arthropods (Chelicerata or Arachnida) which diverge markedly in structure from the rest of the phylum. No arachnid has a typical compound eye, and while the eye of Limulus is generally described as “compound”, it is certainly very different from the compound eyes of insects and crustaceans. It consists of about 1 000 ommatidia each of which has 8-20 radially arranged retinula cells. The ommatidia are spaced about 150-200 p apart and the retinula cells are about 120 p deep (measured from the figures of Lankester and Bourne, 1883).Thus their dimensions are comparable with the simple eyes of caterpillars (Dethier, 1942), but unlike the latter the cuticle covering the ommatidia is a continuous transparent sheet. Apart from this last feature, the eyes seem comparable with the schizochroal eyes of trilobites, but the number of ommatidia is much larger than that in any living arthropod possessing aggregates of simple eyes. The above facts suggest that, owing to the large size of the facets and shallowness of the eye as a whole, it is unlikely that diffraction images are formed. Neurologically each ommatidium has a basal eccentric cell which differsfrom theotherretinulacells in having no rhabdomere. The processes of all the retinula cells form the elongated “optic nerve’’ which passes to the synaptic regions of the brain. Only the fibres from the eccentric cells give rise to spike discharges in the optic nerve (Waterman and Wiersma, 1954), and since the former have no rhabdomeres they are presumably not sensitiveto light, although their electrical response(judged from intracellular recording) is similar to that of the other retinula cells. It may be

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(Ruck, 1962) that the eccentric cell has its distal process so close to the retinula cells and their rhabdomeres that the generator current produced by the latter passes through the cell membrane of the eccentric cell and so causes graded depolarization and discharge of spikes. On the other hand Fuortes (1959) and Rushton (1959) argue that chemical mediation occurs at the retinula/eccentric cell junction. In both cases the eccentric cell acts like a second order neurone. There is a further peculiar feature of the Limulus eye : the presence of a plexus of fibrils connecting the retinula cells across the ommatidia. This has not been described in insect and crustacean compound eyes; perhaps it is the functional homologue of the first synaptic region of other arthropods. This layer of basal synapses is responsible for lateral inhibition, i.e. inhibition of the discharge of one ommatidium (through its eccentric cell fibre) due to illumination of adjacent ommatidia. This phenomenon has been widely studied (Hartline and Ratliff, 1957, 1958; Tomita, 1958; Fuortes, 1958, 1959; Ratliff et al., 1963), and its possible function in sharpening the retinal image has been treated quantitatively by Reichardt (1961). Thus in Limulus there is a dual excitatory/inhibitory system; the messages passing in the eccentric cell fibres to the brain are frequency coded, and the discharge in each fibre is a balance between the excitatory action of the light falling on the ommatidium from which it originates and whatever inhibitory action is being exerted on the fibre by neighbouring ommatidia. B. E X C I T A T O R Y A N D I N H I B I T O R Y S Y S T E M S I N T H E I N S E C T EYE

Ruck (1962) considers the inhibitory synapse as a primitive condition and points to comparable behaviour in the insect ocellus. Here illumination typically causes inhibition of the post-synaptic discharge in the fibres of the ocellar nerve. This is associated with hyperpolarization of the postsynaptic neurone. Is there evidence for such a dual excitatory/inhibitory system in the typical compound eye? The first point is whether cells comparable to the Limulus eccentric cells occur in insects. Information is scanty and the situation is complicated by the wide variety of ommatidial structure found in insect eyes (see Ruck, 1964, for review), but the occurrence of one retinula cell which is distinct from the others is widespread in insects. Hanstrom’s account (1927) gave examples from Diptera, Coleoptera, Hemiptera and Lepidoptera and later work has extended this. In Diptera Dietrich (1909) found that one rhabdomere of the radially arranged group is displaced centrally. This is made very clear by electron microscope

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studies, e.g. of Woken et al. (1957), Yasuzumi and Deguchi (1958), and WaddingtonandPerry (1960)on Drosophila, andBurttand Catton (1962b) on Calliphora. In the silkworm Bombyxmori, Eguchi (1962) has described a centrally placed cell having a distal process similar to that in the Limulus eccentric cell but revealing a rhabdomere of characteristic structure under the electron microscope. He comments on the general similarity to the situation in Limulus. In many insect eyes, however, the rhabdomeres are closely packed with no axial space, but even here one or two may be unequal in size to the rest and occupy a basal position. Such cells have recently been identified in the locust eye (Horridge and Barnard, unpublished) as depicted in Fig. 2. There is a strong suggestion that the basal cells have different central connexions via their post-retinal fibres. Thus Cajal(l909) and more fully Cajal and Sanchez (1915 ) have found long visual fibres, one (occasionally two) of which arises from each ommatidium and passes right through the first synaptic region without forming a synapse, to end in the second synaptic region. In Calliphora they claim that these fibres arise from the centrally placed retinula cell. Fibres of a similar form occur both in Lepidoptera and in the honeybee, and it would be interesting to relate this finding to the form of the rhabdomeres noted by Goldsmith and Philpott (1957). Cajal and Sanchez suggested that such fibres would occur in all insects, but Zawarzin (1914) denied their occurrence in Aeslzna. It is easy to confirm their presence in Calliphora, but in Locustu the present authors have not yet been able to follow individual fibres over a long enough distance to decide the matter either way. Earlier workers speculated on the function of these long and short visual fibres. Cajal and Sanchez suggested that they were related to colour vision in insects; the long fibres being analogous to rods and the short ones to cones. Hanstrom (1927) reversed this role concluding that the long fibres were equivalent to cones and the short to rods. He noted that several short fibres synapsed at the same level with one second-order neurone in.the first synaptic region while the long fibres pursued a more single path and that this was more consistent with his interpretation. He noted that in deep-sea Crustacea only short fibres occur (i.e. the rod type), suggesting a comparison with the retina of deep-sea fish where only rods are present. He pointed out that at great depths in the sea the light is virtually monochromatic. Recent work on insect colour vision makes this explanation of the functions of these cells much less likely because there is no correlation with the presence of these long visual fibres and colour vision. Burkhardt (1962) finds that the upper part of the eye in Calliphora is monochromatic while wavelength discrimination occurs in the lower

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part; however, the long fibres occur in both. Burkhardt’s data gives a ratio of 5 : 1 : 1 for his three types of receptors, which gives a total of 7. This is equal to the total number of fully formed rhabdomeres in the eye of Calliphora; if this is so, then colour discrimination is between different retinulacells and not concentrated in anyone type. Mazoxin-Porshnyakov (1959) also finds in Libellula that the upper part of the eye is monochromatic and only the lower part can discriminate wavelength. However, the presence or absence of long fibres has not yet been demonstrated in Odonata. If the basal cells are really comparable to the eccentric cells of Limulus, then it should be possible to record a continuous spike discharge from them when the eye is illuminated. The only observations showing spike discharge from the retinula cell region of the insect eye is that of Naka and Eguchi (1962) in the honeybee drone. Their own interpretation of this observation was given above. The possibility remains that they had penetrated one of the eccentric cells of the insect eye and so got a spike discharge. A visual unit which responds in a very similar way to the eccentric cell of Limulus has been described by Burtt and Catton (1960) in the optic lobe of the locust. Out of 31 single units, 5 (i.e. about one in six) were of a type described as L-units. These did not respond to movements in the visual field but gave a continuous spike response on illumination, where rate of discharge was directly proportional to the log of light intensity. These units are located in the second synaptic region and it seems more likely that they are second-order neurones than the terminations of basal cells. The size of the spikes seems too large for them to be first-order cells. The frequency of occurrence of the L-units suggests that they might be oneto-one second-order neurones, connected to a single basal cell in each ommatidium. A tentative scheme for the dual excitatory/inhibitory system of the insect eye might be as follows. The normal retinula cells send their fibres to the first synaptic region, but do not generate spikes but only a wave response, which spreads electrotonically to the second-order neurones situated in this region. These second-order neurones show a steady discharge in the dark which is inhibited by light (D-units of Burtt and Catton (1960); comparable units in Bombyx (Ishikawa, 1962)).The post-retinal fibres in CalIiphora disperse widely before entering the first synaptic region, and the same may be true of Locusta, so that these units may receive information from wide areas of the visual field. The basal cells are the excitatory system and these give rise ultimately

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(after one or more synapses) to the L-units of Burtt and Catton and comparable units described in Bombyx by Ishikawa (1962), which were silent in darkness but gave a train of spikes on illumination. In the latter case there might be continuation of the discharge for a period of several seconds after the end of illumination. A theory concerning the functional significance of these two systems will be put forward in the next section. C. THE N E U R A L BASIS O F MOVEMENT PERCEPTION

The normal type of retinula cell would on the above view be responsible for initiating on and off bursts of spikes and hence allow the detection of light-dark boundaries as they move across the receptor mosaic. Because of the rapid adaptation shown by these units, it is unlikely that they will assist in the recognition of the form of stationary objects, in the absence of eye movements as in the human (Ditchburn, 1959), i.e. stationary patterns would soon be adapted away. The diffractive images of the compound eye would be detected by such a system and one can imagine that an eye could serve a useful function even if it consisted of such elements alone. D. THE OPTICAL A N D N E U R A L BASIS OF FORM VISION

1. A modijied mosaic theory The problem of form vision remains. Diffraction images alone might account for the level of form discrimination found by Hertz (1931), but form vision in bees is probably better than this (Mazoxin-Porshnyakov and Wischnevskaya, 1964), and the powers of recognition of their prey shown by sphecoid wasps suggest form vision of a high order. Further, the simple eyes of lepidopterous larvae certainly, and the eye of Limulus very probably, do not produce diffractive images yet both are functional visual organs. Reichardt (1961) and Kirschfeld and Reichardt (1964) have put forward a theory of vision in Limulus taking the eccentric cell spike responses as the basis for building up avisualmosaic. Ifweimagine the Limulus type of eye to be either a precursor or a derivative of the typical compound eye, then we are left with the problem as to how the Limulus type of structure is represented in the compound eyes of insects and crustaceans. The system of eccentric cells (one per ommatidium) would bring us back to a simple spatial representation of visual objects and realize Muller’s original conclusion from his examination of the compound eye. Thus these cells would be responsible for form recognition, reproducing light and dark areas of the field on a mosaic based on ommatidial units. Stationary patterns would not be adapted away. It is

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interesting to note that Waterman et al. (1964) have found single fibre discharges in the optic nerve of the crab Podophthalmus which are evoked by stationary patterns. Further, in Calliphora Cajal and Sanchez (1 91 5) found that the long visual fibres of the eccentric cells pass to the deeper part of the second synaptic region and thus they might form a distinct neural system. This view shows a departure from that of an earlier paper (Burtt and Catton, 1961) where the possibility of the occurrence of any single image in the compound eye was doubted. The visual acuity for perception of moving patterns would on this hypothesis be higher than that for perception of stationary patterns; the former being based on a fine mosaic consisting of almost the whole population of retinula cells, while in the latter case the functional unit would be the eccentric cells spaced at a distance apart represented by a whole ommatidium. The “light compass” response of insects could be understood on this basis; thus von Buddenbrock (1935) presents very striking data for a wide range of arthropods in which the minimum angle of displacement of a light needed to induce turning is correlated with the angular separation of the ommatidia. 2. Geometrical interference in the insect 0ye Hassenstein’s results (1 951)’ examining the beetle Chlorophanus by the method described earlier, seem to demand a mosaic theory for their explanation. His workraises thepossibility of another typeof phenomenon in insect vision, i.e. geometrical interference between, for example, the set of stripes used in visual experimental work and the array ofommatidia. The phenomenon is best understood by describing a simple model. The eye is represented by a sheet of metal with holes in it arranged as a hexagonal lattice each hole 2.5 mm from the next; this is in fact a sample of commercial perforated zinc. The stripes used were an extensive array with a periodicity of 1 mm. The two arrays were put in the divergent beam from a projector with the array of stripes nearest the source. Together the two patterns produce geometrical interference patterns with the following properties; if the projection of both the patterns on the screen has the same spacing, then movement of one relative to the other only produces an increase or decrease in total illumination. The same is true if it is exactly double, half or a quarter, but at intermediate stages movement of the two patterns produces broad stripes (similar to moire‘ fringes) which may move either in the direction of the outer stripes or in the opposite direction. This point of reversal is the essential matter. Hassenstein (1951) finds that in Chlorophanus the minimum stripe width to which turning re-

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sponses are made is 6.8"in general agreement with the angular separation of the ommatidia morphologically. The important experiment from the present point of view is one in which the insect is suspended and separated from the cylinder of vertical stripes by a horizontal slit of such a width that two rows of ommatidia are projected through it. With this arrangement beetles show alternating directions of turning as the stripe width is altered; thus for a width of stripe below 4-5" there is no response; but a positive response (moving with the stripes occurs over the range 5-6", dropping down to zero at about 6-7". With larger stripes the response becomes negative, then positive and zero again at about 1.5 times the value of 6.8".His results show this alteration up to 2.5 times the basic angle. The results seem in very good agreement with those predicted from geometrical interference between the spacing of the stripes and the spacing of the ommatidia regarded as separate apertures. If this is so then this type of response would be one which, on the theory put forward above, would be mediated by the eccentric cells. Geometrical interference might be suggested as a possibility for explaining the high resolution of insect eyes discussed above (Section 11, B), since stripes with a width below the ommatidial separation can still produce alternating dark and light bands by geometrical interference. While we cannot exclude this possibility it seems to us doubtful for the following reasons. Let us suppose that each ommatidium has a system whereby all but the exact centre of the field is cut off; this might allow alignment of each ommatidium of a row on a separate stripe. What happens below the level of resolution of the individual lenslets? Now in Hassenstein's experiment the stripes are well within the resolving power of the single lenslets, but in Burtt and Catton's observation the width of the stripe is below what could be resolved by a single lenslet and the images of the stripes would be blurred. The model described above is misleading in that the stripes are projected on to the screen while in the living eye image formation by a lens is essential. If the image cannot be resolved, the system becomes unworkable.

3. Alternative theories of form vision in compound eyes Several workers have been impressed by the neural complexity of the compound eye and have been driven to abandon the simplicityof Muller's original mosaic theory. Thus Vigier (1907) noted that the retinula fibres in Diptera spread out in such a way that those from one ommatidium do not make synapses with the same second-order neurone. He suggested that the visual sensations from the single retinula cells were reconstructed as a "neuromosaic" by the optic lobe. Eltringham (1919) and recently

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Kuiper (1962) regard the single retinula cell as the visual unit and suggest that there is resolution of the image in each ommatidium by the individual retinula cells. Yagi and Koyama (1963) also consider the single retinula cell as the functional unit and think of vision being due to the juxtaposition of images from neighbouring ommatidia. 4. The role of the nervous system

The problem here seems to be twofold. First how far can one expect the nervous system (presumably essentially the neurones in the optic lobe) to “unscramble” the information coming to the ommatidia and sort out from it a coherent set of data about the outside world? Second, supposing this is not possible, how much information about the outside world can an arthropod get from its compound eye provided it gives some sort of pattern capable of stimulating the photoreceptors. In other words, even if the image of the object is very distorted and confused could it not be useful purely as a means of recognition, provided always the resolving power of the optical system gives sufficient detail? If the problem were no more than the insect responding to a single pattern in the environment then this might be enough. But in the case of hunting wasps the recognition can be that of a common form. Thus a representational imaging system seems to be demanded. Note, however, that the theory outlined in the last section is not form vision based on an optical image but rather on the results of nervous integration. The present theory comes therefore nearer to the neuromosaic of Vigier (1907), but as the ommatidia are acting as single units it gets over the difficulty of how the dispersed stimuli are sorted out into a representational image. We return therefore to the role of the central nervous system in insect vision. Vowles’ (1955) account of the connexions of the corpora pedunculata in the bee shows that a variety of nerve tracts join the 2nd and 3rd synaptic regions (i.e. outer and inner optic glomeruli) to the calyces and the lobes of the corpora pedunculata, and the latter organs to one another. Typically the nerve cells arising within the corpora pedunculata send a fibre to the calyx, another to the A lobe and issue as a motor fibre from the B lobe. The latter fibres pass to the rest of the body. We know very little concerning what might happen to visual information once it passes from the optic lobe, but there is an abundance of neural pathways by which the central nervous system might make use of it. ACKNOWLEDGMENTS We are greatly indebted to Dr G. A . Horridge and Mr P. B. T. Barnard for the generous loan of the electron micrographs from which Fig. 2 was

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made; and to Mr D. J. Cosens for allowing us to quote from his unpublished work.

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Kalmus, H. (1958). Responses of insects to polarised light in the presence of dark reflecting surfaces. Nature, Lond. 182, 1526-7. Kirchhoffer, 0. (1908). Untersuchungen iiber die Augen pentamerer Kafer. Arch. Biontol. 2, 237-287. Kirschfeld, K. and Reichardt, W. (1964). Die Verarbeitung stationarer Nachrichten im Komplexauge von Limulus. Kybernetik 2, 43-61. Kuiper, J. W. (1962).The optics of the compound eye. Symp. SOC.exp. Biol. 16,58-71. Kunze, P. (1961). Untersuchung des Bewegungssehen fixiert fliegender Bienen. 2. vergl. Physiol. 44, 656-84. Kunze, P. (1964). Eye stalk reactions of the ghost crab Ocypode. Zn “Neural Theory and Modeling” (R. F. Reiss, ed.), pp. 293-305. Stanford University Press. Lankester, E. R. and Bourne, A. G. (1883). The minute structure of the lateral and central eyes of Limulus and Scorpio. Q. JI microsc. Sci. 23, 177-212. Ludtke, H. (1953). Retinomotorik und Adaptationsvorgange im Auge des Ruckenschwimmers (Notonecta glauca L.). Z . vergl. Physiol. 35, 129-152. Mallock, A. (1922). Divided composite eyes. Nafure, Lond. 110, 770. Mazoxin-Porshnyakov, G. A. (1959). Colorimetric study of colour vision in the dragon-fly. Biofizika 4,427-436. Mazoxin-Porshnyakov, G. A. and Wischnevskaya, T. M. (1964). Beweise der Fahig Keit der Insekten den Kreis, das Draeck und andere einfachen Figuren zu unterscheiden. Znt. Congr. Ent. 12, 340. McCann, G. D. and MacGinitie, G. F. (1965). Optomotor response studies of insect vision (in press). Menzer, G. and Stockhammer, K. (1951). Zur Polarisationsoptik der Fazeltenauger von Insekten. Naturwisserschaften 38, 190-191, Meyer, G. F. (1951). Versuch einer Darstellung von Neurofibrillen im zentralen Nervensystem verschiedener Insekten. Zool. Jb., Abt. Anat. 71, 41 3-426. Miiller, J. (1 840). “Handbuch der Physiologie des Menschen.” Coblenz. Naka, K. (1961). Recording of action potentials from single cells in the insect compound eye. J . gen. Physiol. 44, 571-584. Naka, K. I. and Eguchi, E. (1962). Spike potentials recorded from the insect photoreceptor. J . gen. Physiol. 45, 663-680. Naka, K. and Kuwabara, M. (1959). Electrical responses from the compound eye of Lucilia. J. Insect Physiol. 3, 41-49. Nunnemacher, R. F. (1959). The retinal image of arthropod eyes. Anat. Rec. 134, 618-619. Parry, D. A. (1947). The function of the insect ocellus. J. exp. Biol. 24, 211-219. Ratliff, F., Hartline, H. K. and Miller, W. H. (1963). Spatial and temporal aspects of retinal inhibitory interaction. J. opt. SOC.Am. 53, 110-120. Reichardt, W. (1961). Uber das optische Auflosungsvermogen der Facettenaugen von Limulus. Kybernetik 1, 57-69. Rogers, G. L. (1962). A diffraction theory of insect vision. 11. Theory and experiments with a simple model eye. Proc. R. SOC.B, 157, 83-98. Rogers, G. L. (1963). The process of image formation as the re-transformation of the partial coherence pattern of the object. Proc. phys. SOC.Lond. 81, 323-331. Ruck, P. (1958). A comparison of the electrical responses of compound eyes and dorsal ocelli in four insect species. J. Insect Physiol. 2, 261 -274. Ruck, P. (1961a). Electrophysiology of the insect dorsal ocellus. I. Origin of components of the electroretinogram. J. gen. Physiol. 44, 605-627.

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Ruck, P. (1961b). Electrophysiology of the insect dorsal ocellus. 11. Mechanisms of generation and inhibition of impulses in the ocellar nerve of dragonflies. J. gen. Physiol. 44,629-639. Ruck, P. (1961~).Electrophysiology of the insect dorsal ocellus. 111. Responses to flickering light of the dragonfly ocellus. J. gen. Physiol. 44,641-657. Ruck, P. (1962). On photoreceptor mechanisms of retinula cells. ,Biol. Bull. Mar. biol. Lab., Woods Hole 123, 618-634. Ruck, P. (1964). Retinal structure and photoreception. A. Rev. Ent. 9, 83-102. Rushton, W. A. H. (1959). A theoretical treatment of Fuortes’s observations upon eccentric cell activity in Limulus. J. Physiol. 148, 29-38. Satija, R. C. (1957). Visual paths in the insect nervous system. J. Physiol. 136,27~. Satija, R. C. (1958). A histological and experimental study of nervous pathways in the brain and thoracic nerve cord of Locusta migratoria migratorioides (R & F). Res. Bull. Panjab Univ. Sci. 138, 13-32. Schneider, G. (1956). Zur spektralen Empfindlichkeit des Komplexauges von Calliphora. Z. vergl. Physiol. 39, 1-20. Scholes, J. H. (1964). Discrete subthreshold potentials from the dimly lit insect eye. Nature, Lond. 202, 572-573. Stieve, H. (1964). Das Belichtungspotential der Isolierten Retina der Einsiedlerkrebses (Eupagurus Bernhardus L.) in abhangigkeit den Extracellularen Ionenkonzentrationen. Z. vergl. Physiol. 47, 457-492. Thorson, J. (1964). Dynamics of motion perception in the desert locust. Science 145, 69-7 1 . Tomita, T. (1956).The nature of action potentials in the lateral eye of the horseshoe crab as revealed by simultaneous intra and extra cellular recording. Jap. J. Physiol. 6, 327-340. Tomita, T. (1958). Mechanism of lateral inhibition in the eye of Limulus. J. Neurophysiol. 21, 419-429. Tomita, T., Murakami, M. and Hashimoto, Y.(1960). On the R membrane in the frog’s eye: Its localisation and relation to the retinal action potential. J . gen. Physiol. 43, 8 1-94. Vigier, P. (1907). Sur les terminaisons dans les yeux composes des Insectes, en particulier chez les Muscides. C.r. hebd. Seanc. Acad. Sci., Paris 145, 633-636. Vowles, D. M. (1955). The structure and connexions of the corpora pedunculata in bees and ants. Q. JI microsc. Sci. 96, 239-255. Vries, H. de (1956). Physical aspects of the sense organs. Prog. Biophys. biophys. Chem. 6, 207-264. Waddington, C. H. and Perry, M. M. (1960). The ultra structure of the developing eye of Drosophila. Proc. R. SOC.B, 153, 155-178. Walther, J. B. (1958). Untersuchungen am Belichtunspotential des Komplex auges von Periplaneta mit farbigen Reizen und selectiver Adaptation. Biol. Zbl. 77, 63-104. Washizu, Y., Burkhardt, D. and Streck, D. (1964). Visual field of single retinula cells and interommatidial inclination in the compound eye of the blowfly Calliphora erythrocephala. Z. vergl. Physiol. 48, 41 3-428. Waterman, T. H. (1954a). Directional sensitivity of single ommatidia in the compound eye of Limulus. Proc. natn. Acad. Sci. U.S.A. 40, 252-257. Waterman, T. H. (1954b). Polarised light and angle of stimulus incidence in the compound eye of Limulits. Proc. natn. Acad, Sci, U.S.A. 40, 258-262.

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Waterman, T. H. and Wiersma, C. A. G. (1954). The functional relation between retinal cells and optic nerve in Limulus. J. exp. Zool. 126, 59-85. Waterman, T. H. and Wiersma, C. A. G. (1963). Electrical responses in Decapod Crustacean visual systems. J. cell. comp. Physiol. 61, 1-16. Waterman, T. H., Wiersma, C. A. G. and Bush, B. M. H. (1964). Afferent visual impulses in’the optic nerve of the crab, Podophthalmus. J. cell. comp. Physiol. 63, 135-156. White, E. (1962). Nest building and provisioning in relation to sex in Sceliphrcm spirifex L. (Sphecidae). J . Anim. Ecol. 31, 317-329. Wigglesworth, V. B. (1 953). “The Principles of Insect Physiology.” Methuen, London. Wilska, A. and Hartline, H. K. (1941). The origin of “off responses” in the optic pathway. Am. J. Physiol. 133, 4 9 1 ~ . Wolf, E. (1933). The visual intensity discrimination of the honey bee. J. gen. Physiol. 16, 407-422. Wolken, J. J. (1963). Structure and molecular organization of retinal photoreceptors. J . opt. SOC.Am. 53, 1-19. Wolken, J. J., Mellon, A. D. and Contis, G. (1957). Photo receptor structures. 2. Drosophila melanogaster. J. exp. Zool. 134, 383-406. Wolken, J. T., Bowness, J. M. and Scheer, I. J. (1960). The visual complex of the insect-retinene in the housefly. Biochim. biophys. Actu 43, 53 1-537. Wulff, V. J. (1956). Physiology of the compound eye. Physiol. Rev. 36, 145-163. Yagi, N. and Koyama, N. (1963). “The Compound Eye of the Lepidoptera.” Shinkyo-Press, Tokyo. Yasuzumi, G. and Deguchi, N. (1958). Submicroscopic structure of the compound eye as revealed by the electron microscope. J. Ultrastruct. Res. 1, 259-270. Zawarzin, A. (1914). Die optischen Ganglien der Aeshna larven. Z. wiss. Zool. 108, 175-257. Zerrahn, G. (1933). Formdressur und Formunterscheidung bei der Honigbiene. Z . vergl. Physiol. 20, 117-150.

Amino Acid and Protein Metabolism in Insect Development P. S . CHEN Institute of Zoology and Comparative Anatomy, University of Zurich, Switzerland I. Introduction . 11. Embryonic Development . A. Changes in Free Amino Acid Pools . B. Enzyme Patterns . . 111. Larval Development . A. Amino Acids . B. Peptides and other Amino Acid Derivatives . C. Haemolymph Proteins . IV. Pupal Development . A. Metabolism of Amino Acids and Proteins . B. Changes in Enzyme Activities. . V. Adult. . A. Sex-specific Differences in Amino Acids, Peptides and Proteins B. Protein Metabolism in Relation to Reproduction . VI. Some Genetic Aspects of Protein Metabolism in Insects . A. Patterns of Protein Metabolism in Lethal Mutants . B. Synthesis of Enzymes and other Specific Proteins . C. Regulation of Gene Activity . . VII. Conclusions . References

53 55 55

62

.

69 69 82 84 89 89 93 96 96 99 102 102 109 112 113 I I4

I. INTRODUCTION

Insect ontogeny consists of both embryonic and post-embryonic development. One outstanding feature of the post-embryonic development is the striking change of forms occurring at successive ontogenetic periods. This is especially true of the so-called holometabolous insects where a pupal stage is interposed between the larva and the adult. The phenomenon of metamorphosis is known to be complex. Furthermore, according to evidence available, embryogenesis may vary greatly in different species (cf. recent reviews by Counce, 1961; Krause, 1961; Seidel, 1961). Despite such a complexity there is apparently no principal 53

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difference between the development of insect and that of other animals. The basic mechanism is the transformation of a developing organism from an immature to an adult state by the process of cell differentiation. As differentiation involves the formation of proteins, the primary importance of protein metabolism in insect development appears evident. From a number of brilliant investigations in various laboratories it is now well established that the major events during the post-embryonic development, such as growth, moulting and pupation, are under hormonal control (see reviews by Williams, 1952; Bodenstein, 1953a; Wigglesworth, 1954, 1964; Gilbert, 1964). However, much less is known about the mode of action of the hormones. It has previously been suggested by Thomsen (1952) and Bodenstein (1953b) from their experiments ofextirpatingand transplanting theendocrineglands, as wellas by Wigglesworth (1957) from his cytological observations, that it is the synthesis of protein which is primarily concerned. More direct evidence supporting this advanced hypothesis has been provided by subsequent work designed to study the biochemical action of the hormones (for references, see Gilbert and Schneiderman, 1961). In a more recent paper Burdette and Coda (1963) reported that ecdysone, the hormone of the prothoracic gland, even enhances the synthesis of hepatic protein in Mammalia. We shall discuss the biochemical effects of insect hormones in more detail in later sections. It may only be mentioned here that strong evidence has been obtained suggestingthat the hormonal action is directly or indirectly linked to protein metabolism. Therefore, extensive studies on the metabolic changes in proteins and related compounds would be of great help in elucidating the basic processes which underlie insect development. Various aspects of the protein metabolism in insect development, such as the patterns of free amino acid pools, the intermediary pathways of individual amino acids and their derivatives, qualitative and quantitative changes in lymph proteins as well as the synthesis and the metabolic activity of specific cxymes, have attracted the interest of many insect biochemists. Although great progress has been made in these fields during recent years, there is no doubt that many gaps still exist. Difficulties are encountered in putting the available information into an integrated account, especially when one attempts to relate one biochemical change to a particular morphogenetic event. Firstly, work has been done with a wide variety of insect species and, owing to differences in the nutritional requirement and the habit of life, shows large variations so that it is impossible to draw ageneral conclusionfrom thedataobtained. Secondly, the morphogenetic state of the materials used by various workers has not always been clearly indicated. The developing organism represents a

55 dynamic system which changes continuously in its physiological and biochemical properties as morphogenesis proceeds. The same is true for the different organ-systems each of which has its own ontogenetic pattern. Thus the results could be quite different if insects raised under other conditions and of other ages are used. Thirdly, since in most studies homogenates of whole insects have been used, the biochemical properties of individual tissues and organs are unknown. Owing to the lack of adequate culture medium only a few in vitro experiments have so far been performed. Consequently the observations reported do not enable us to understand the metabolic interrelationships between the different organ-systemsand themorphogeneticmeaning ofa particular biochemical process remains obscure. In spite of these difficulties,valuable information about the normal pattern and indications concerning the nature of several basic events of protein metabolism have been obtained, as we shall see later. These results demonstrate certain features which are of importance in characterizing the different stages of insect development. In this article only recent work which has been carried out from the morphogenetic point of view will be considered, for it is impossible to give a general survey of protein chemistry in insects within such a short space. It would also be beyond the present scope to deal with other major fields of insect biochemistry, such as carbohydrate and fat metabolism. Energy metabolism and nucleic acids will be briefly mentioned because of their close connections with protein synthesis. Enzyme proteins will be included, however, because of their functional significance. In the last section recent studies on the protein metabolism in several lethal mutants and some genetic implications concerning enzyme synthesis are presented in order to illustrate the genic control of protein metabolism as well as insect development in general. The author is aware that the present review is by no means complete, since emphasis has to a certain extent been determined by the author’s own interest. A general consideration of the protein metabolism in insects is given by Gilmour (1961). AMINO A C I D A N D P R O T E I N METABOLISM

11. E M B R Y O N IDEVELOPMENT C A. C H A N G E S I N F R E E A M l N O A C I D P O O L S

There is a large body of literature on the free amino acids in insects (cf. Chen, 1962), but most studies are limited to post-embryonic and adult stages and much less work has been done on the developing egg. During embryogenesis an intensive protein metabolism takes place which involves mainly the breakdown of pre-existing yolk reserves and the

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P. S. CHEN

conversion of these into tissue- and organ-specific proteins. Thus it is reasonable to anticipate that the rate of such metabolic changes varies as development proceeds. A detailed analysis of the amino acid pattern at different embryonic stages would provide us with valuable information about the biochemical processes which accompany morphogenetic events. In general, the content of free ninhydrin-reacting components in the fertilized egg is very low although the number and the quantities of amino acids increase in the course of development. For example, Drilhon and Busnel(l950) detected in the eggs of Bombyx mori only four amino acids (glutamic acid, serine, alanine, valine) at the time of fertilization, and eight additional ones (tyrosine, leucine, glycine, tryptophan, proline, hydroxyproline, cystine, histidine) at the end of development. In Drosophila melanoguster von der Crone-Gloor (1959) reported the presence of sixteen amino acids in developing embryos, but ,8-alanine and y-aminobutyric acid occur only at the time of hatching. The recent work of Indira (1963) on the freshwater bug Sphaerodema molestum demonstrated also that among the fifteen free amino acids valine, alanine, glycine, tyrosine and threonine become detectable only beginning from 72 to 120 h of embryonic life. Therefore, for the identification of the free amino acid pattern in eggs and embryos, the developmental age has to be carefully controlled. The results thus far obtained for several insect eggs are summarized in Table I. The above conclusion has been confirmed by quantitative studies which, in addition, demonstrate the close relationship between yolk utilization and morphogenesis. Recently Chen and Briegel(l965) undertook an extensive analysis of the changes of free amino acids in the embryo of the autogenous (Culexpipiens var. molestus) and anautogenous (Culex pipiens var. fatiguns) mosquitoes. The development lasts about 41-43 h at 25°C. Together with the morphological characters, which have been described in detail by Idris (1960) and Oelhafen (1961), the results of this study can be summarized as follows. The first part of embryogenesis (up to 16 h) is accompanied by a sharp rise in the total concentration of free amino acids, resulting from a rapid breakdown of the yolk reserves for the initiation of protein synthesis (Fig. 1). The main morphogenetic process at this'period is the formation of the blastoderm followed by the separation of the germ layers (gastrulation) and the elongation of the germ band. With the exception of two temporary drops at 24 and 32 h respectively, the total content of amino acids remains high in the subsequent period of development (16-36 h). This is the time when the growth of the mesenteron and the formation of the dorsal organs (dorsal closure) take place.

AMINO ACID A N D PROTEIN METABOLISM

57

In the last part of development (from 36 h onwards) when the differentiation process is in full swing, a slight decline in the amino acid concentration can be recognized, especially in the anautogenous embryos. Apparently at the present period the rate of yolk proteolysis can no more keep place with the progress of protein synthesis. However, the final concentration is stillmuch higher than that at the beginning of development. TABLE1 Occurrence of free amino acids in insect embryos

Amino acids

a-Alanine ,!?-Alanine y-Aminobutyric acid Arginine Asparagine Aspartic acid Citrulline Cysteine Cystine Glutamine Glutamic acid G1ycine Histidine Leucines Lysine Methionine Methionine sulphoxide Phenylalanine Proline Serine Taurine Threonine Tryptophan Tyrosine Valine

+ + + + +-

+ + + + - - - + + + - + + + + + + + + + + + - + + + + + + + + + + + + + + + + + + + - + -- +- +- +- + + - + + ++ + +- ++ + + + + + - - + + i + + + + -

'

+ + + +- +- ++ + + - - + + + + + - + - + + + - + + + + + + + + + + , + + + + + + +- + + - + + + + + + + -+ +- ++ + + + + + + + + + +

+

+

58

P. S. CHEN

I t is of interest to notice that in the locust Schistocerca gregaria the pattern of variation of the free amino acid pool is markedly similar to that found in the mosquito eggs: it rises rapidly at the period of germ-band formation, keeps at a high level during blastokinesis, and falls off when tissue differentiation is most intensive (Colombo et al., 1961).The same is true for the developing embryos of Sphaerodema mot'estum (Tndira, 1963). For the grasshopper Chortophaga viridifasciata Shaw (1955) also observed an increase of all amino acids as development advances, although no quantitative data were given. L

0

8

16

24

32

40

,

B

Hours of development

FIG.1. Changes in total concentrationof free amino acids and peptides during embryonic development of Culexpipiens ( 0 ) and Culex futiguns ( 0 ) . Amino acid content of fertilized eggs laid by autogenous females after a blood meal ( A ) is indicated by an arrow. The vertical lines express standard deviation. (From Chen and Briegel, 1965.)

That the level of total free amino acids reflects the utilization of yolk on the one hand and the synthesis of embryonic proteins on the other is clearly shown in Sphaerodema by the work of Indira (1963). Based on the determination of protein-bound amino acids she concluded that the decrease of these components in the yolk coincides with an increase of them in the yolk-free embryos (Fig. 2). The same finding has been reported for the locust eggs (Colombo et al., 1961). Of course, fluctuations due to interconversions and other metabolic pathways of the amino acids have also to be considered, but as yet no study has been carried out to support this.

AMINO A C I D A N D PROTEIN METABOLISM

59

With regard to individual amino acids glutamic acid and its amide glutamine, which are considered to be of general importance in protein metabolism, occur usually in high concentrations. In both Drosophila (von der Crone-Gloor, 1959) and Tenebrio (Po-Chedley, unpublished) it has been shown that during the increase of glutamine there is a concomitant drop of glutamic acid (Fig. 3). This fact suggests the interconversion of these two compounds.

29

48

72

96

120

144

166

Hours of development

FIG.2. Quantitative distribution of protein-bound amino acids in the yolk and yolk-free embryos during development of Sphaerodema molesfurn. (From Indira, 1963.)

Tyrosine is the only amino acid which exhibits large fluctuations in the insect eggs. The recent work of Chen and Briegel(l965) demonstrated that there is a clear correlation between the variation of tyrosine and the pigmentation of the developing embryo. At the time of oviposition the shell of the mosquito egg is soft and white in colour, and becomes hard and dark within 1-2 h after laying. The process of hardening and darkening is reported to bemainly due to tanning of the protein in the exochorion (see Clements, 1963, pp. 24-29). Within the first 4 h of development the content of tyrosine declines to a minimum (Fig. 4). Apparently at the initial period this amino acid is largely utilized for the tanning process of C

P. S. CHEN

Drosophila

Tenebrio

I

2t 0

6

12

18

2L

L

8

3

n

n

8

s

1

2

3

L

5

6

7

8

Days of deveiopment

Hours of development

FIG.3. Changes in the concentration of glutamic acid (GLU,0 ) and glutamine (GLUNHa, 0 ) during embryonic development of Drosophih mehogaster (von der Crone-Gloor, 1959) and Tenebrio molitor (Po-Chedley, unpublished.)

EE

0025

0

8

16

Hours of

24

32

40

development

FIG.4. Variations in the concentration of free tyrosine during embryonic development of Culex pipiens ( 0 ) and Culexfutiguns ( 0 ) .(From Chen and Briegel, 1965.)

AMINO A C I D A N D PROTElN METABOLlSM

61

the egg shell. Its subsequent rise is obviously due to further proteolysis of the yolk. The second drop at 28 h coincides with the time when the bristles, mandibular teeth and other cuticular structures become dark in colour. The eyes, being bright-red coloured at 24 h, are now covered by a layer of dark pigment. Shortly before hatching even the epidermis appears somewhat brownish (cf. Idris, 1960). Tyrosine derivatives such as polyphenols and quinones are known to be directly responsible for the hardening and darkening of the cuticular proteins (see p. 73). In the Chortophaga eggshaw (1 955) reported the occurrence of ethanolamine phosphoric acid which is probably related to the metabolic pathways of serine and glycine. Citrulline is also present. Since ornithine could not be detected with certainty, its relation to the tricarboxylic acid cycle is doubtful. Sulphur-containing amino acids such as methionine and cystine are of common occurrence in insect eggs. Their metabolic changeshave been analysed in detail by Fu (1957) in the developing embryo of the grasshopper Melanoplus diferentialis. The total content of sulphur remains constant during the whole period of embryogenesis. In pre- and post-diapause methionine and cystine/cysteine show distinct variations. Detailed analysis of the various fractions indicate the dissociation of methionine from the yolk and its uptake into embryonic proteins. As the increase in cystine/cysteine takes place with a concomitant decline in methionine, there is obviously a transfer of sulphur between these compounds, a phenomenon similar to that found for glutamic acid and glutamine. During the period of diapause, when the development becomes temporarily blocked, the values of these amino acids remain strikingly constant. In the late post-diapause they show a distinct drop, suggesting their degradation through oxidation and their incorporation into -SH or S-S containing compounds like coenzymes. According to unpublished work of Po-Chedley, among the free amino acids present in the developing embryo of the mealworm Tenebrio molitor aspartic acid, glutamic acid, glycine, leucine and phenylalanine occur in high concentrations. Characteristic fluctuations of the amino acid pool have been detected in X-ray irradiated embryos. This touches the question as to the roles of protein metabolism, tissue differentiation and other physical and physiological factors involved in radiosensitivity of the developing organism. A detailed discussion of this interesting problem would go, however, beyond the bounds of the present paper. At least four peptides have been noted in the egg of Drosophila (von der Crone-Gloor, 1959) and Culex (Chen and Briegel, 1965), and five in that of Schistocerca (Colombo et al., 1961). Since no detailed

62

P. S. CHEN

biochemical data of these compounds are available, their metabolic and morphogenetic significance remains unknown. There is no significant change in total nitrogen as embryogenesis proceeds (Chen and Briegel, 1965). Studies hitherto have shown that the major source of energy in insect embryos is fat oxidation which, according to Boell(1955), amounts to at least 75% of the total oxygen uptake. In general the amino acid pool in the developing egg is quite similar to that in the adult. This is to be expected since these components can be taken up by the oocyte from the haemolymph, and, as has been shown by Telfer (1954, 1960) in Hyalophora cecropia and by Hill (1962, 1963) in Schistocerca gregaria, blood proteins may be directly deposited in the yolk of the egg (p. 101). B. ENZYME PATTER N S

Work has been carried out by both histochemical and straight biochemical methods to map the enzyme patterns in the developing insect embryo. That the activities of enzymes are closely associated with the morphogenetic processes is self-evident. For instance, during the period of growth and differentiation one would expect to find enzymes which are more concerned with such activities of the cells as maintenance, substrate transport and tissue formation, whereas at the end of embryogenesis enzymes catalysing specific functions of the various organsystems, such as contraction, conduction, secretion, digestion and excretion, might be expected. In the following sections the results of several studies of this type will be summarized to illustrate this point. 1. Phosphatases The wide occurrence of phosphatases in animal tissues is thought to be associated with (a) transport of metabolites, (b) metabolism of phospholipids, phosphoproteins, nucleotides and carbohydrates, and (c)synthesis of proteins. The importance of these enzymes in embryonic tissues has also been repeatedly pointed out (cf. Moog, 1946; Boell, 1955). An interesting observation has been made by Tawfik (1957) on the egg of Apanteles glomeratus. The oocyte and mature ovarian egg of this parasitic hymenopteran insect possess a cytoplasmic inclusion at the posterior pole, the so-called germ-cell determinant (Fig. 5). This inclusion is stainable with iron haematoxylin and apparently of cytoplasmic origin. It breaks down into granules shortly after oviposition or, as in other species, at the early cleavage stage. The resulting material appears to spread around the nuclei which migrate into the posterior polar cytoplasm and

AMINO ACID A N D P R O T E I N METABOLlSM

63

give rise to the pole cells, the primordia of the germ cells.* Tawfik (1957) could show that the germ-determinant contains alkaline phosphatase and suggested that it is the activity of this pre-existing enzyme which causes the breakdown of the cytoplasmic inclusion, and that the products

FIG.5. Longitudinal sections through the posterior pole of the egg of Apanteles glonierafus. Left: mature ovarian egg showing the germ-cell determinant (gcd). Right: newly laid egg showing the germ-cell determinant undergoing histolysis and the extruded granules (ex.gr). (From Tawfik, 1957.)

of this process are responsible for transforming the cleavage nuclei into the primordial germ cells. If this is true, we have a clear example that one particular enzyme is directly involved in an important morphogenetic step.

* That the pole cells give rise to primordial germ cells has been demonstrated beyond doubt by Geigy (1931) and Poulson (1947) in Drosophila, and more recently by Oelhafen (1961) in Culex. But according to Poulson and Waterhouse (1958, 1960) some of the pole cells also migrate into the gut and take a part in the formation of the midgut epithelium. This means that the fate of these cells is not yet determined. In any event, as pointed out by Bodenstein (1955), there is no doubt that some factors in the polar cytoplasm “endow the pole cells with the potentialities necessary for the formation of germ cells”.

64

P. S. CHEN

In this connection one can further question if the site of alkaline phosphatase demonstrated by Tawfik (1957) is in some way related to the socalled activation centre which, according to Seidel (1936), also has its location in the posterior polar region of the egg. The reaction between the cleavage nuclei and the pole-plasm leads to the production of some agent which diffuses in the anterior direction and thus initiates the formation of the embryonic anlage. Seidel (1960) is of the opinion that the basic

29

48 72 96 120 14L 166 Hours of development

FIG.6. Activity of alkaline phosphatase during embryonic development of Sphaeroderna molesturn. (From Indira, 1963.)

mechanism involves the activation of the cleavage nuclei by some cytoplasmic factors at the posterior pole. Possibly it is the activity of the alkaline phosphatase which is concerned in this activation process. However, it would appear unwise to speculate too far on the finding of Tawfik until more biochemical data are available. Quantitative determinations of alkaline phosphatase in the developing egg of Sphaerodema molesturn have been made by Indira (1963). As can be seen on Fig. 6, the enzyme activity is very low at the onset of develop-

A M I N O A C I D A N D PROTEIN M E T A B O L I S M

65

ment. With the exception of a brief lag between 48 and 72 h, it rises rapidly until 96 h of age, and thereafter remains rather constant. The first increase corresponds to the period of gastrulation and elongation of the germ band and the second one occurs at the period of intensive histo-differentiation. The implication that this enzyme is concerned with protein synthesis is further supported by the parallel increase in both amino acids and ribonucleic acid (RNA) at the corresponding periods. In other insects the occurrence of alkaline phosphatase can be detected only at relatively late embryonic stages. For example, using the histochemical technique Yao (1950a) reported that in Drosophilu acid phosphatase is present at all stages but shows no apparent changes throughout embryogenesis. By contrast alkaline phosphatase cannot be demonstrated until the time when the germ band contracts. Thereafter it spreads rapidly to all parts of the embryo, but disappears again in most tissues except the gut epithelia, salivary glands and Malpighian tubules at the time of hatching. The site of the first appearance of alkaline phosphatase has been shown to be in the ventral ectoderm of the future thorax and corresponds probably to the so-called differentiation centre of Seidel (1936). The author concluded that the alkaline phosphatase is mainly concerned with histo-differentiation, whereas the acid phosphatase plays a role in both the synthesis and the degradation of yolk. Fitzgerald (1949) claimed that in the grasshopper Melanoplus dzflerentiulis alkaline phosphatase is located principally in the extra-embryonic fluid and appears in developing tissues only shortly before hatching. Since this author’used the biochemical method for enzyme analysis, the results of both studies are not directly comparable. In connection with his studies on the carbohydrate metabolism in the silkworm Bombyx mori Chino (1961) performed biochemical analyses of the phosphatases in both diapause and non-diapause eggs. The occurrence of acid phosphatase, which splits glycerol phosphate and sorbitol-6phosphate, can be detected at all embryonic stages. The activity appears consistently higher when the former is used as the substrate, but the increase is more rapid with the latter substance. On the other hand, alkaline phosphatase becomes detectable only one and a half days before hatching. It is believed that the late appearance of this enzyme has a correlation with the initiation of the function of the digestive tract (cf. Drilhon and Busnel, 1945; Horie, 1958). Similarly Sridhara and Bhat (1963), who also worked on Bombyx, found that acid phosphatase occurs during the whole period of embryogenesis and exhibits a steady increase as development proceeds, whereas alkaline phosphatase can be traced only 2-3 days before hatching. Likewise they attribute the sudden

66

P. S. CHEN

appearance of this alkaline enzyme to the functional differentiation of the gut. It can be summarized that studies on the activity of both acid and alkaline phosphatases in developing embryos of various insects have furnished evidence for the different metabolic roles of these two types of enzymes : the acid phosphatase has a more general distribution and is involved in such processes as yolk and substrate utilization, whereas the alkaline phosphatase shows stage- and tissue-specificpatterns indicating its role in differentiation and other physiological functions, in particular digestion. 2. Pro teases There are only a few papers dealing with the occurrence of proteolytic enzymes in the developing egg of insects. In the Moroccan locust Dosiostaurus marocanus glycerine extracts from diapause eggs have been found to be active in hydrolysing peptone, leucylglycine,leucylglycylglycine and chloroacetyltyrosine, but neither casein nor gelatine is attacked by such extracts (Lichtenstein et al., 1949). On the other hand, extracts from eggs in active development and shortly before hatching are able to digest casein. Thus the pattern of proteolytic enzymes of the egg in diapause differs distinctly from that in post-diapause. In a more recent study of Shulov etal. (1957) the presence of at least two kinds of endopeptidases* has been reported in the developing eggs of the locust Locusta migratoria migratorioides. A cathepsin-like endopeptidase, which splits casein at pH 5.6, appears already about 5 days after the beginning of development and increases in activity in the later period. It seems, however, that this enzyme concerns mainly transpeptidation rather than protein hydrolysis, since the egg homogenate has a pH value of 6.0-6.6. A trypsin-like enzyme, which attacks casein at pH 7.8, becomes detectable only on the 8th day of development and reaches a peak at the time of hatching. Its first occurrence coincides with the beginning of development of the midgut after blastokinesis. A third enzyme which acts on the tripeptide leucylglycylglycinehas been observed in embryos aged 4-5 days, but its activity remains rather low throughout development.

* The proteolyticenzymesconsist of two main groups:proteinaseswhichcausethedegradation of large protein molecules into smaller fragments, and peptidases which split the peptides and thus lead to the liberation of free amino acids. However, Bergmann and Fruton (1941) and Bergmann (1942) showed that such typical proteinases as pepsin, trypsin and chymotrypsin also act upon relatively simple peptides if the proper peptide linkages are present. For this reason Bergmann suggested the term exopeptidases for the hithertocalled peptidases which can only act upon peptide links between terminal amino acid residues and the main chain, and endopeptidases for the so-called proteinases which are able to break the peptide bonds remote from the terminal residues.

A M I N O A C I D A N D P R O T E I N METABOLISM

67

Cathepsin-like endopeptidase has also been found in the eggs of the housefly Musca domestica (Greenberg and Paretsky, 1955). In this connection it is of interest to notice that during the embryogenesis of Melanoplus, as reported by Norman (1954), there is an increase of non-protein SH, probably glutathione, whichis known to be an activator ofcathepsins. In both Bombyx (Lichtenstein, 1947) and Schistocerca (Kuk-Meiri et al., 1954) a maximal activity of proteolytic enzymes at pH 8 was recorded only at the end of embryogenesis. From these examples it is clear that enzymes which take a part in the synthesis of tissue-specific proteins appear earlier in development, whereas those which effect protein hydrolysis are formed only in association with the functional differentiation of the digestive system. 3. Respiratory enzymes Enzymes involved in the biological oxidation of insects have been studied by numerous workers (see review by Gilrnour, 1961,pp. 106-119). Since all typical cytochromes are identified in insect tissues, there is obviously no principal difference in the pathway of electron transport between insects and other organisms (see also Stegwee and van KammenWertheim, 1962). But most of these studies concern mainly the respiratory processes during post-embryonic and adult life. It is thus questionable if the same mechanism is also operative in the developing egg. That cytochrome oxidase is present in insect eggs has been demonstrated by the earlier work of Bodine and Boell(l934, 1936) on Melanoplus. Embryos in active development are sensitive to cyanide and carbon monoxide, the powerful inhibitors of this enzyme. Parallel to the increase in respiration there is an increase in the content of cytochrome oxidase. On the other hand, eggs in diapause are insensitive to such inhibitors. This fact has been interpreted as indicating the occurrence of two distinct and separate fractions of respiration, and only the cyanide-insensitive one remains during the diapause period. It is possible that instead of cytochrome oxidase a certain other oxidizing enzyme resistant to the inhibitory agents is operative in the blocked eggs, as once suggested for a similar phenomenon in the diapause pupa of the cecropia silkworm (Williams, 1951; Schneiderman and Williams, 1954). A more critical examination of the cytochrome system in such eggs will prove profitable, especially in view of the recent findings on the diapause pupa that it is the excessive amount of cytochrome oxidase relative to cytochromes b and c, which is actually responsible for its insensitivity to cyanide or carbon monoxide. We shall discuss this point in more detail in a later section dealing with pupal development (p. 94). C*

68

P. S. C H E N

Important results on the respiratory enzymes of the egg of Bornbyx have recently been reported by Chino (1963). In the diapause eggs it has been noted that sorbitol and glycerol are formed from glycogen. The latter is again synthesized from the accumulated polyhydric alcohols when diapause is terminated. The conversion of glycogen to sorbitol and glycerol is due to the action of polyol dehydrogenases in the presence of reduced NAD or NADP* delivered from the pentose phosphate cycle and glycolysis (Chino, 1957, 1958, 1960). In a search of the metabolic relation of this conversion process to the electron transport system quite unusual characters of the respiratory enzymes in early embryonic stages (including diapause) have been disclosed. For instance, in homogenates of prediapause eggs cytochrome c is not detectable. The major enzymes are cytochrome oxidase and cytochrome b,, both of which occur mainly in the lipid-rich particles. This point is noteworthy because in a typical cytochrome system cytochrome oxidase is located in the mitochondria whereas cytochrome b, has been shown to be present in the microsomes (cf. Chance and Pappenheimer, 1954; Shappirio and Williams, 1957). Further analyses by fractionating the homogenates including the artificial non-diapause eggs treated with dilute HC1 have yielded the following results. NADH, oxidase as well as NADH, and NADPH, cytochrome c reductases, which occur largely in the soluble fraction at the beginning of embryogenesis, are present in either mitochondria or microsomes shortly before hatching. Succinate cytochrome c reductase is found in the mitochondria at the end of development, but is apparently absent in all fractions soon after egg-laying. The latter fact suggests that the tricarboxylic acid cycle may not function in the early embryonic stages. Finally, at the initiation of development all cytochrome enzymes are resistant to antimycin A which is known to inhibit the electron transport between cytochromes b and c, but they become sensitive to this antibiotic at the time of hatching. As emphasized by Chino (1963) the above results indicate that the basic mechanism of the electron transport in early embryonic stages differs from that in later development. As embryogenesis proceeds, a typical cytochrome system similar to that found in insect and mammalian tissues is built up. Apparently there are changes in the activity and distribution of the respiratory enzymes in relation to the differentiation of particular tissues, especially the muscles. One point which should be mentioned is that the mitochondria, the

* NAD, Nicotinamide adenine dinucleotide (formerly known as DPN); NADHz,reduced nicotinamide adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; NkDPHz, reduced nicotinamide adenine dinucleotide phosphate.

AMINO ACID A N D P R O T E I N METABOLISM

69

sites of the major respiratory enzymes, are very easily damaged by such drastic procedures as homogenization, fractionation and extraction, so that the patterns found in these preparations could be quite different from that in intact tissues. Thus stage-specificvariations of the enzyme pattern detected by such procedures may possibly reflect nothing more than changes in the structures of cellular components during the differentiation of the embryonic cells. In any event, such technical difficulties have to be considered in the evaluation of the results.

111. LARVALDEVELOPMENT A. A M I N O A C I D S

Studies on the metabolism of amino acids in insect larvae have yielded a wealth of data. Especially since the advent of such new techniques as microbiological assays, paper and ion-exchange chromatography and isotopic labelling, it has become possible to follow quantitative changes in these amino compounds which are present even in extremely low concentrations. It has also been demonstrated by many authors that the amino acids, in addition to their function as protein constituents, enter into diverse metabolic pathways and participate in many other physiological activities. In view of the fact that a large part of our knowledge about amino acids in insects derives from studies dealing with larval development, the inclusion in the following discussion of a short account of some general features of amino acid metabolism in insects seems to be justified. 1. Occurrence and morphogenetic significance offree amino acids in insects Insects are known to contain an unusually large amount of free amino acids whose total concentration in some species has been estimated to be more than thirty times higher than that in other groups of animals (Florkin, 1960, p. 331). A survey of the patterns thus far reported for various insects belonging to seven different orders has been presented in a previous paper (Chen, 1962). In general, all amino acids which are commonly contained in the proteins have been identified, either in tissue extracts or in the haemolymph. Among these the aliphatic amino acids have a dominant part, as indicated by their large quantities and the constancy of their occurrence. Several amino acids such as /3-alanine, taurine, ornithine, a- and y-aminobutyric acid, which do not occur in the protein molecules, are found in quite a number of species. On the other hand, some amino acid derivatives have so far been recorded only

70

P. S. CHEN

in a single species. These include S-methylcysteine in Prodenia eridania (Irreverre and Levenbook, 1960), tyrosine-0-phosphate in Drosophila melanogaster (Mitchell et al., 1960), thyroxine in Rhodnius prolixus (Harington, 1961a), phenylglutamic acid and methylhistidine in Agria uJtffnis (Villeneuve, 1962), as well as homoarginine in Attacus ricini (Pant and Agrawal, 1964). Vereshtchagin et al. (1961) reported that both ,Balanine and yaminobutyric acid depress the bioelectrical activity of the larval nerve ganglion of the pine moth Dendrolimus pini. Otherwise nothing definite is known about the physiological significance of these substances. While abundant information is available on the amino acids in the haemolymph (cf. Wyatt, 1961), very little is known about their distribution in tissues. The available data indicate that differences do exist. In Bombyx it is found that the concentration of dialysable N is twice as high in the intracellular fluid than in the blood (Bricteux-GrCgoireand Florkin, 1959). There is also an unequal distribution of individual amino acids even though the total concentration appears to be the same. In Prodenia larvae the total content of free amino acids is about the same in the fat body and haemolymph, although that in the gut is much lower (Levenbook, 1962). Glutamine plus glutamate amount to only about 10-20 yo of the total concentration in the former, whereas the value is closer to 50 yo in the latter. Significant differences have also been noted in the relative concentration of the two components in these tissues. Somewhat similar results have been obtained for the salivary gland in Drosophila larvae (Chen and Baumann, unpublished). As can be seen in Table 11, the concentration of glutamate is more than ten times higher in the salivary gland tissue than in the haemolymph, although the values for glutamine are much closer to each other. The differences for many other amino acids are also very evident. On the other hand, the total quantity of peptides accounts for about 29% of all free ninhydrin-positive compounds in the haemolymph, the corresponding value for the salivary gland being only about 5%. The in vitro experiment of Hines and Smith (1963) demonstrated clearly that the ability of homogenates of the leg muscle, fat body and head of the locust Schistocerca varied significantly in the incorporation of 14C from labelled glucose, acetate and succinate into amino acids and other metabolic intermediates. Such variations reflect no doubt the differences in the specific metabolic activities of the tissues and organs concerned. Let us now consider briefly to what extent are the amino acids related to the morphogenetic events during insect development. One outstanding function is no doubt their support of growth. Using the conventional

71

A M I N O A C I D A N D P R O T E I N METABOLISM

TABLEI1 Relative distribution (yototal content) of amino acids and peptides in salivary gland and haemolymph of Drosophitu larvae aged 96 h at 25°C Amino acids and peptides

Salivary gland

Cystine Aspartic acid Glutamic acid Glutamine Serine Glycine Taurine Lysine Arginine Tyrosine Threonine a-Alanine p-Alanine Histidine Valine Leucine Peptides Unknown substances

Haemolymph ~-

~~

34 6-5 45.2 11.5

2.5 1 -2

-

9.4 0.4 7.7 0.1

-

1.7 1.2 54

3.6

__

__

0.2 0.1

4.3 18.1 5.8

2.4 0.3 6.1 1.9 2.7 4.8 14.5 0.3

4.2 2. I I .2 28.6 2.2

method of growing the larva on chemically defined medium together with the deletion procedure, it is found that the ten amino acids (arginine, histidine, lysine, tryptophan, phenylalanine, methionine, threonine, leucine, isoleucine, valine), which are known to be necessary for the growth of mammals, have been proved to be also essential for insects (Lipke and Fraenkel, 1956;House, 1962).More recently this has indirectly been confirmed by following the incorporation of 14C from uniformly labelled glucose into the non-essential amino acids in the larvae of Phormia regina, Agrotis orthogonia, Ctenicera destructor (Kasting and McGinnis, 1958, 1960, 1962; Kasting et al., 1962) as well as Neodiprion pratti (Schaefer, 1964). However, some exceptions have been noted. For example, tyrosine seems to be essential for the green peach aphid Myzus persicae (Strong and Sakamoto, 1963). Drosophila needs glycine (Hinton et al., 1951) and Aedes glycine and cystine (Golberg and de Meillon, 1948), while methionine, phenylalanine and threonine are obviously not required for Blattella germanica (House, 1949; Hilchey, 1953). It has been claimed that a number of amino acids, such as arginine, cystine, glycine, proline, tryptophan, tyrosine and phenylalanine, are especially concerned with either moulting. differentiation, pupation or

72

P. S. CHEN

emergence of the adult (for references, see Chen, 1962). We are, however, completely ignorant about the mechanism of action of these substances in the ontogenetic process. There is no doubt that the nutritional requirements for growth and development vary in different insects. The present state of our knowledge is yet too fragmentary to draw a general conclusion. 2. Qualitative and quantitative changes during growth and moulting Both qualitative and quantitative changes in the contents of free amino acids have been followed during the post-embryonic development of a large variety of insect species. The more extensive investigations are those on Calliphora erythrocephala and Phalera bucephala (Agrell, 1949), Galleria mellonella (Auclair and Dubreuil, 1952), Culex quinquefasciatus and Aedes aegypti (Micks and Ellis, 1952), Macrothylacia rubi (Drilhon, 1952), Bombyx mori (Florkin, 1937, 1959; Sarlet et al., 1952; Amanieu et al., 1956; Wyatt et al., 1956), Anomala orientalis (Po-Chedley, 1956), Drosophila melanogaster (Hadorn and Mitchell, 1951;Chen and Hadorn, 1955; Benz, 1957), Corethra plumicornis (Chen and Hadorn, 1954), Calliphora augur (Hackman, 1956) and Ephestia kuhniella (Chen and Kuhn, 1956). Similar studies have recently been carried out on the desert locust Schistocercu gregaria (Benassi et al., 1961), the bug Rhodnius prolixus (Harington, 1961a), the southern armyworm Prodenia eridania (Levenbook, 1962), and the rice moth Corcyra cephalonica (Ganti and Shanmugasundaram, 1963). As work on Drosophila can be carried out under more precise nutritional and genetic control than is probably possible for many other insects, it is felt appropriate to present at first some of the results on this insect and then to compare them with those reported for other species. During the larval development of Drosophila a total of at least twentyone free ninhydrin-positive compounds have been identified on the twodimensional paper chromatogram ; these include aspartic acid, asparagine, a-alanine, /3-alanine, arginine, y-aminobutyric acid, glutamine acid, glutamine, glycine, histidine, leucine, lysine, methionine, proline, serine, threonine, tyrosine,valineand34peptides(ChenandHadorn,1954,1955). In general glutamic acid, glutamine and a-alanine occur in the highest concentrations, especially at earlier stages. The total quantity per larva increases rapidly from 48 to 72 h after oviposition at 25"C, remains rather constant for the subsequent 24 h, and then drops steadily near the time of pupation. If the values are calculated per unit body weight, a maximum is shown at 72 h, corresponding to the period of most intensive growth as determined by both total N and wet weight (Chen, 1960). On the other

AMINO A C I D A N D PROTEIN METABOLISM

73

hand, the total concentration per unit volume haemolymph decreases rapidly as development proceeds (Hadorn and Stumm-Zollinger, 1953). As we shall see later (p. 85), there is a parallel increase in the concentration of blood proteins. This suggests that a large part of the amino acids is utilized for protein synthesis. No general pattern can be recognized in the variation of individual amino acids: many of them decline steadily as development proceeds, while others exhibit a temporary increase. Some of the minor changes probably reflect merely nutritional variations. Tyrosine and proline are however two exceptions :they show a continuous increase when the values are expressed per unit body weight or unit volume of haemolymph. This is especially true during the time approaching puparium formation. It is known that the larval cuticle increases considerably in thickness shortly before pupation (Dennell, 1946). According to Hackman (1953a) the cuticular proteins have a high content of proline and tyrosine. Furthermore, as mentioned previously, tyrosine serves as the precursor for the formation of polyphenols and quinones necessary for the darkening and hardening of the larval cuticle which gives rise to the puparium (cf. Dennell, 1947;Pryor et ul., 1947; Fraenkel and Rudall, 1947; Hackman, 1953b). Karlson (1960) reported that when uniformly labelled tyrosine was injected into Culliphoru larvae, up to 80% of the total activity could be recovered in the puparium. The accumulation of these two amino acids suggests therefore the preparation of the larvae for the synthesis of cuticular proteins and the associated tanning. Various aspects of tyrosine metabolism in insects have been considered by Brunet (1963). The results described above have been confirmed by more recent studies of Chen and Hanimann (1965) using the technique of ion-exchange chromatography according to Spackman et ul. (1958). As shown in Fig. 7, in addition to the amino acids previously identified phenylalanine was detected, and leucine and isoleucine could be separated distinctly. There is also a considerable amount ofammonia. It is known that free ammonia occurs naturally in the haemolymph of some insects, but as pointed out by Levenbook (1950), it may also be formed rapidly from certain unknown precursors when the blood samples stand in air. Of special interest is the occurrence of a large number of unknown ninhydrin-positive fractions which have hitherto escaped our detection on paper chromatogram because much more material has been used in the present analysis than is possible with paper chromatography. Preliminary investigations indicate that these consist of mainly acidic peptides and some other amino acid derivatives which we shall deal with in more detail in the next section (p. 82; see also Addenda, p. 131).

74

P.

20 L 10

05

4'

--

02

-

01

-

Glycinc

8

I

Threonine Proline

4

8

I

I

I

60

30

Arpvtlc acid

I

I

90

120

150 Effluent

z2

9

a-Alrninc

213

=-

-

1 0.5 03 8

S. CHEN

180

210

ZLO

270

(m0 150 cm column

1

05 O I

I

f 03

Lcucine

Tyrorine

Isohcine Valine Methionint

3w

330

360

p-Almine

390

0 0 WO 480 Etflutnt (ml) 150 cm coiumn

510

540

5m

2.0 1.0

05

;04

<

03

9

02

I

01

FIG. 7. Chromatographic analysis (automatic analyser) of free ninhydrin-reacting components in the methanol extract (0.4g wet weight/2 ml) of Drosophila larvae aged 4 days at 25°C. Solid lines indicate absorbance at 570 mp; broken lines at 440 mp. Fractions containing peptides and other unknown amino acid derivatives are numbered according to their order of elution from the column. (From Chen and Hanimann, 1965.)

AMINO ACID A N D PROTEIN METABOLISM

75

The pattern of free amino acids in the larvae of the mosquito Culex pipiens is quite similar to that in Drosophila (Chen, 1958a). The increase in total quantity parallels larval growth. However, in contrast to Drosophila, the values calculated per unit body weight or unit volume of blood remain essentially unchanged during the whole period of larval development (see also Chen, 1960). It seems that this difference is at least partly due to the different habits of feedingbetween these two insects :Drosophila larvae leave the culture medium prior to pupation, whereas Culex larvae keep on taking up food and have thus a continuous supply of amino acids from the nutritional source. In Culex, as in Drosophila, there is a distinct increase of both tyrosine and proline, substances whose connections with the formation of cuticular proteins have already been mentioned. Reference to studies on other insects indicate that, as in the case of Culex, a rather constant total concentration is maintained in the larval blood of Ephestia (Chen and Kiihn, 1956), Schistocerca (Benassi et al., 1961) and Rhodnius (Harington, 1961a). On the other hand, a rapid decrease in the total concentration similar to Drosophila has been reported for Bombyx (Legay, 1960)and the rice moth Corcyra cephalonica (Ganti and Shanmugasundaram, 1963). But in no case is there any significant qualitativevariation in the pattern of freeamino acids as larval development proceeds. Larval growth is in close association with moulting. One major procedure of this involves the breakdown of the endocuticle. Passonneau and Williams (1953) observed that the early moulting fluid of the silkworm Hyalophora contains more proteins and less non-protein nitrogen, while the reverse is true for the late moulting fluid. The presence of Nacetylglucosamine was also identified. In the moulting fluid of the fourth instar larvae and prepupae of Bornbyx Zielinska and Laskowska (1958) reported the occurrence of seventeen amino acids as well as N-acetylglucosamine and glucosamine. All these substances are no doubt derived from the degradation of protein and chitin; the latter is known to be built up of N-acetylglucosamine residues (cf. Fristrom, 1965). The enzymatic digestion of endocuticle is further indicated by the high proteolytic and chitinolytic activity of the moulting fluid (Jeuniaux and Amanieu, 1955). 3. Metabolic interrelationships of amino acids It is certainly an oversimplification to interpret the amino acid pattern merely in terms of protein synthesis and degradation. The amino acids are interrelated from the metabolic viewpoint. Moreover, in contrast to the egg and pupa, which can be considered as closed systems, the larva cannot survive and grow without external nutritional sources which may

76

P. S. CHEN

influence to a great extent its composition and level of the free amino acid pool. In the following account we shall consider more precisely some of these factors which have a bearing on our understanding of the metabolic pattern of these components in the growing larvae. a. Nutrition and absorption. That nutrition has a direct effect on the amino acid pattern of insect larvae has been demonstrated by both starvation and feeding experiments. When Drosophila larvae are totally deprived of food at about 65 h, there is a gradual decrease in both total nitrogen and total content of free ninhydrin-positive compounds. Most amino acids drop rapidly within the first 24 h after starvation and remain at a low level during the subsequent 3-4 days (Chen and Hadorn, 1955; Chen, 1958~). Some amino acids such as glycine and serine do not seem to be affected. At least one peptide, probably derived from the breakdown of tissue proteins, exhibits a steady increase during the starvation period. The degradation of body proteins which leads to the increase in amino nitrogen at prolonged starvation has been suggested by the work of Ludwig and Wugmeister (1953) on Popillia japonica and that of PoChedley (1958) on Anornala orientalis. When Drosophila larvae are fed with casein alone, there is a tremendous increase of all amino acids (Chen and Hadorn, 1955). On the other hand, if sucrose is given as the only diet, most amino acids decrease in their concentration, but alanine increases, indicating its synthesis from pyruvate, the direct product of glycolysis. A similar phenomenon occurs in the sucrose-fed aphid Myzus persicae, where the total concentration of free amino acids is reduced to 50-70% of the normal value at 48 h after the beginning of nitrogen deprivation (Strong, 1964). Although most amino acids decline, threonine remains unchanged and cystine shows even an 11-fold increase. Furthermore, as many as thirty-one unidentified ninhydrin-reacting compounds accumulate as the starvation period lengthened. Even though the nature of these compounds is still not known it seems that some of them must be peptides or other amino acid derivative; resulting from the proteolysis of body tissues. Interesting observations have been made by Auclair (1959) in which starved cockroaches Blattella germanica were fed with individual amino acids. He was able to show that amino acids, such as a-aminobutyric acid, hydroxyproline, phenylalanine and taurine, which are normally undetectable in this insect, appear in the blood if given in the diet. Other amino acids show a profound effect on the quality and concentration of the already existing ones, suggesting their interconversion possibilities. Differences in the blood composition of Agria aflnis between larvae fed on pork liver and those on synthetic medium have been noted by

A M I N O ACID A N D P R O T E I N M E T A B O L I S M

77

Villeneuve (1962). Individuals grown on the latter have a markedly low content of glutamine and asparagine, indicating a certain block in the synthesis of these two amides. ' Irreverre and Levenbook (1960) demonstrated that, in Prodenia, irrespective of the amino acid content in the diet (kale or potato), amino. acids are selectively accumulated in the larval haemolymph, while others remain at a low level, being either metabolized, excreted or not absorbed. Somewhat similar results have been obtained by Schaefer (1964) for the Virginia pine sawfly Neodiprion pratti. In order to understand the dietary effect on the blood amino acid composition the problem of absorption cannot be ignored. As demonstrated by Treherne (1959) in Schistocerca, labelled glycine and serine are rapidly absorbed from the caeca in the midgut region, by establishing a diffusion gradient through the net movement of water into the haemolymph. This would mean that, in contrast to mammals, active transfer of amino acids either does not operate in insects or plays only a minor role. Furthermore, as suggested by the work of Auclair (1959) on Blattella, the configuration of the amino acid molecule seems to have a definite influence on the uptake process: L-glutamic acid is much more easily absorbed than its D-isomer, and cystine is readily absorbed though not its homologue homocystine. But Nuorteva and Laurema (1961) reported that both D- and L-isomers of valine can readily be taken up by Dolycoris baccarum. More work is needed to explain such differences, for that reported by Treherne (1959) appears to be the only information available on the mechanism of amino acid transport in insects. b. Excretion. It has been demonstrated by Ramsay (1958) that in the stick insect Dixippus morosus amino acids from the haemolymph can enter the Malpighian tubules by passive diffusion. Some of them are probably reabsorbed in the rectum, but significant quantities may be eliminated with the faeces. There are only a few reports dealing with the amino acids in insect excreta (cf. review by Craig, 1960). Their presence has been found in the excretory products of the webbing clothes moth Tineola bisselliela and the carpet beetle Attagenus piceus by Powning (1953) and in that of Bombyx by Yoshitake and Aruga (1950). In Aedes, Anopheles and Culex about 5% of the total nitrogen in excreta, which contain mostly uric acid (Terzian et al., 1957), is represented by amino acids (Irreverre and Terzian, 1959). Harington (1961b) found in Rhodnius cystine and cysteic acid in urine and histidine, histamine, taurine, glycine, valine, phenylalanine, alanine and at least two peptides in the pigmented excreta. In all probability histidine, which is derived from the globin of haemoglobin, gives rise to histamine by decarboxylation.

78

P. S. CHEN

A large number of amino acids are also present in the excreta of both the larva and the fresh adult of Attacus ricini (Pant and Agrawal, 1963). The presence of arginine, homoarginine and one unknown derivative suggests the existence of guanidine metabolism in this insect. Recently Mitlin et al. (1964) detected a total of twenty-three free and bound non-protein amino acids in the excreta of the boll weevil, Anthonomus grandis. Since there is an increase in the concentration after hydrolysis, some of them must exist in the form of peptides or other derivatives. That these amino acids actually represent the metabolic products of this insect is shown by the absence of free amino acids in the food. However, as noted by the above authors, the possibility that at least a part of these compounds are derived from the micro-organisms in the gut is not excluded. c. Specificfunctions other than protein formation. The high titre of free amino acids in insect haemolymph is not well understood. They certainly occur in excess of the demand for protein synthesis. There is some evidence, though rather indirect, that the amino acids take a part in buffering and osmo-regulation (cf. Florkin and Morgulis, 1949, p. 22; Buck, 1953). This appears to be more probable in the aquatic Sialis larva (Beadle and Shaw, 1950). Otherwise many of such suggestions are based merely on analogous results obtained for other brackish-water and marine invertebrates such as crustaceans (Duchiiteau and Florkin, 1956) and molluscans (Allen, 1961).A more critical proof of this point in insects is eagerly awaited. Several experiments indicate that the free amino acids play an important role in detoxication. In Locusta (Friedler and Smith, 1954), Aedes (Casida, 1955) and more recently in Bombyx (Shyamala, 1964) glycine is found to conjugate with benzoic acid to form hippuric acid, a detoxication mechanism similar to that in higher animals. The site of hippuricase which regenerates glycine from hippurate has been detected in both fat body and silk gland (cf. Shyamala, 1964). Limpel and Casida (1957a, b) were able to show that radioactive iodine injected into the cockroach Periplaneta americana was excreted as monoiodohistidine. When labelled iodine was given in the form of monoiodohistidine, diiodohistidine appeared in the excreta. This points out that histidine like glycine also serves as a detoxicating agent in insects. Under particular conditions proline can be mobilized as energy reserve. In DDT-poisoned cockroaches Corrigan and Kearns (1963) reported that there is distinct depletion of proline. Injection of labelled proline into these animals revealed a threefold increase in the oxidation of this amino acid to CO, compared to controls. Apparently there is an

AMINO ACID AND P R O T E I N M E T A B O L I S M

79

inhibition of certain glycolytic enzymes by the insecticide and the demand for oxidizable carbon is shifted to proline. In this connection it should be mentioned that a sharp drop of the proline content in the thoracic muscle during flight of the tsetse fly Glossina morsitans has been observed by Bursell (1963). As suggested by Bursell the most likely mechanism is that proline is converted to glutamic acid by proline oxidase (Fig. 8). After deamination the ketoglutaric acid is taken up by the Krebs’ CH2 + 1202 = :20

+

Cop

+

energy

n

Oxaloacet ic acid

cycle

Alanine Ketoglutari c acid

J

acid Pyruvic acid Glutamic acid

FIG. 8. A hypothetical explanation for variations in the contents of proline, glutamate, alanine and a-ketoglutarate during flight in the tsetse fly GIossina morsituns. (From Bursell, 1963.)

cycle and thus joins the main chain of the oxidative process. Its conversion to pyruvic acid through the intermediate step of oxaloacetic acid is indicated by the concomitant increase in alanine. Some details of the intermediary pathways of amino acids will be discussed in the following section. d. Intermediarypathways. A preliminary step in the intermediary metabolism of amino acids is deamination which is catalysed by deaminases by

80

P. S. CHEN

cleaving oxidatively the a-amino group from the amino acid molecule. The resulting keto acids may act as acceptors for the transfer of amino groups from other amino acids, serve as substrates for fat and carbohydrate synthesis, orjoin the main channel of oxidation via the tricarboxylic acid cycle, the operation of which has been demonstrated in various insects including Prodeniu (Levenbook, 1961). Both D- and L-amino acid oxidases have been reported in insects and transaminase activities have 006

E

-

005

E'

P I n c 0

"G

4 .

I

E w3 01

d .

-55

002

0

g

If

-"

001

0

1

2

3 Incubation

L

5

6

time (h)

FIG.9. Synthesis of glutamate by fat body homogenates of Drosophila larvae from ar-ketoglutarate and either (1) DL-alanine, (2) DL-aspartate, (3) asp leu cine, (4) DL-threonine, (5) L-arginine, (6) glycine, or (7) DL-valine. (From Chen and Bachmann-Diem, 1964.)

also been demonstrated in various tissues, such as the fat body, Malpighian tubules, gut, muscle and haemolymph (for references, see Gilmour, 1961, pp. 238-242). The most extensive studies on the transamination reactions are that of Kilby and Neville (1957) on the locust Schistocerca and Desai and Kilby (1958) on the blowfly Culliphora. Essentially the same results have been obtained by Chen and Bachmann-Diem (1964) from their recent work on Drosophila larvae. Using homogenates of fat body it was found that the formation of glutamate from a-ketoglutarate and DL-alanine or DLaspartate proceeds most rapidly (Fig. 9). The transfer of the amino group

81

AMINO A C I D A N D PROTEIN METABOLISM

from other amino acids has always a much slower rate. From the results of these authors the transamination process in insects can be briefly characterized as follows. The most active reaction always involves glutamate, aspartate and alanine with the corresponding keto acids. Pyridoxal phosphate is used as the coenzyme and the pH optimum has a valueof about 7.5. There isalso anactive synthesisof glutaminefromglutamate and ammonia in the presence of ATP and Mg. The enzyme glutamine synthetase which catalyses this reaction has been prepared from internal larval tissues of Prodenia and characterized by Levenbook and Kuhn (1962). In general, the transaminase system in insects including its range of synthetic ability is essentially the same as that known in bacterial and mammalian tissues (cf. Baldwin, 1952; Meister, 1957; Leuthardt, 1963). A general sketch illustrating the key role of glutamate in the intermediary metabolism of amino acids is given in Fig. 10. ,,Amino

acids, acids

I

Amino acid acid Amino oxidases

Keto acids

\

\

Amin'o acid oxidases

Glutamate

Keto acids

i

Glutamine

FIG.10. A general scheme to illustrate the central role of glutamate in the intermediary metabolism of amino acids.

Although transaminase activity has been demonstrated in various insect tissues, it seems that the major part of the transamination process takes place in the fat body, which has been shown to exhibit many other synthetic activities and fulfil a large variety of metabolic functions similar to the midgut gland (hepatopancreas) in Mollusca and Crustacea and the liver in vertebrates (Urich, 1961; Kilby, 1963). There is evidence that all these reactions occur mainly in the peripheral globules of the fat body (Nair and George, 1964). It may be added here that Wang and Dixon (1960) detected a decrease of the transaminase activity in the muscles of allatectomized Periplaneta. According to McAllen (cited in Bheemeswar, 1959) the activity of these enzymes increases during larval development and adult differentiation parallel to the increase of protein synthesis. Such findings indicate

82

P. S. C H E N

clearly the close connection between enzyme synthesis on the one hand and hormonal control and morphogenesis on the other. B. P E P T I D E S A N D OTHER AMINO A C I D D E R I V A T I V E S

Paper chromatographic studies on the free amino acids in insects have frequently revealed unknown ninhydrin-positive spots on the chromatogram. Many of these are probably polypeptides, as indicated by their disappearance after hydrolysis and the corresponding increase in amino acids. Until now very little work has been done to follow the pattern and nature of such peptides in the developing larvae. The main handicap is the limited absorbing capacity of the filter paper. Atconcentrations not overloading the paper only a few components of low concentrations can be recognized. For instance, in Drosophila not more than three to four peptides have so far been detected on the two-dimensional chromatogram (Chen and Hadorn, 1954; Stumm-Zollinger, 1954; Benz, 1957). On the other hand, as reported by Mitchell and Simmons (1962) by using column chromatography, at least 600 peptides and other amino acid derivatives are present in Drosophila larvae. Estimation of the total balance of amino acids indicates that about half the amino acids are protein-bound and a large part of the other half occurs in the form of peptides and related compounds. There is also evidence that peptides may exist among the lipid-amino acid conjugates (Wren and Mitchell, 1959). Following the analytical procedure of Spackman et al. (1958) a preliminary search for the occurrence of some of these substances at various stages of Drosophila development has been carried out by Chen and Hanimann (1965). Acidic peptides and other ninhydrin-positive components which appear before the aspartic acid fraction are summarized in Fig. 11. It is clear that there is no significant qualitative change in the pattern of these compounds as development proceeds, although quantitative variations are quite evident. That at least some of these fractions are peptides is shown by the study of Shotwell et al. (1963) on the larval haemolymph of Popillia japonica, while the work of Mitchell and Simmons (1962) on Drosophila revealed that many of them are very similar in their amino acid composition and some differ only in the number of repeats of glutamic acid. The predominant components are the common non-essential amino acids. This is understandable because peptides consisting of amino acids which can be synthesized by the larvae would be expected to have a greater accumulation. Labelled glutamic acid injected into the larvae is taken up rapidly by peptides but enter comparatively slowly into proteins, whereas the reverse is true for labelled essential amino acids such as leucine and valine (Simmons and Mitchell, 1962).

83

AMINO A C I D A N D PROTEIN METABOLISM

The meaning of such a difference is not clearly understood, but it could only be a consequence of the relative pool sizes of essential and nonessential amino acids. Of course, the fact that the injected amino acids go first into peptides does not mean that there is no direct incorporation.

-7

~~

-Larva ( 2 days)

10

-1

Larva ( 4 days1

:: 0 4

8

Aspartic acid

I 30

60

11

120

:: 0.4

01

30

60 90 Effluent (ml) 150cm column

120 Effluent (rnl) 150cm column

FIG. 11. Patterns of acidic peptides and other amino acid derivatives at various developmental stages of Drosophila. (For further explanations, see text in Fig. 7.) (From Chen and Hanimann, 1965.)

In the absence of crucial evidence the possibility that the peptides are hydrolysed before being taken up by the proteins can not be excluded. Further work is needed to elucidate the synthesis mechanism. A number of complex peptides conjugated with lipids and carbohydrates are found in the haemolymph of Bornbyx (Sissakian, 1959). In Ephestia Chen and Kuhn (1956) observed that there is a much higher

84

P. S. CHEN

concentration of peptides in the haemolymph than in the tissue of the developing larvae. Two peptides which have a high content in early larval stages decrease in the course of development and disappear almost completely in newly hatched adults. Whether or not these peptides serve as intermediates in the synthesis of lymph or tissue proteins must await further investigation. As already mentioned (p. 76), the peptide concentration increases as the starvation period becomes prolonged. This has been clearly shown in at least two insects, Drosophilu (Chen and Hadorn, 1955) and the aphid Myzus (Strong, 1964). In all probability such an accumulation reflects the progressive disintegration of tissue proteins. In summary, according to more accurate information the pool of peptides and related amino acid derivatives in insects is large and complex. As pointed out by Mitchell and Simmons the large variation in the concentration and the marked similarity in the composition of individual compounds make the isolation of pure materials and their subsequent identification a tedious and laborious job. Even by using such a recent technique as the automatic amino acid analyser many problems in the analytical procedure still have to be solved, as can be seen from the work of Zacharius and Talley (1962) who identified 122 naturally occurring non-protein ninhydrin-positive compounds. There is no doubt that an extensive exploration of these substances would be very useful in order to understand their morphogenetic meaning as well as their potential roles in protein synthesis. C. HAEMOLYMPH PROTEINS

From the morphogenetic point of view investigations of the haemolymph proteins are of particular interest because they provide us with an adequate background to judge the synthetic activity associated with the differentiation processes in the developing organism. Different methods, mainly paper, starch- and agar-gel electrophoresis, have been employed to separate the blood proteins. Both species- and stagespecific patterns have been reported (see van Sande and Karcher (1960) for species differentiation, for example). As almost nothing is known about the properties of the insect blood proteins, the fractions separated are usually classified as albumin or globulins according to their isoelectric point and electrophoretic mobility. Such a classification is certainly insufficient. More specific studies using the immunological, enzymological and histochemical techniques must prove profitable. A list of the electrophoretic pattern for various insects is given in a review paper by Wyatt (1961).

AMINO A C I D A N D PROTEIN METABOLISM

85

1. Total content

Various studies agree in showing that the total content of haemolymph proteins increases during larval development. The increase is most rapid during the time approaching pupation. In Drosophila larvae the total protein concentration is low at earlier stages, but increases fourfold within 24 h prior to puparium formation (Chen, 1956). A similar increase is found in Culex(Chen, 1959b). In Bombyx Wyatt et a/. (1956)agree with the previous findings of Florkin (1937), and report that the blood protein rises from 1.2%,in early third instar to 5.3"/,, in the late fifth instar. Apparently the same is true for Samia Cynthia whose protein concentration, according to Laufer (1 960b), increases rapidly from the third instar to a maximum in the spinning fifth larval instar. Quite similar changes have been noted for the beetle Popillia japonica (Ludwig, 1954) and for the wax moth Galleria mellonella (DenucC, 1958). 2. Ontogenetic patterns More detailed analyses indicate that the rise in total concentration is not due toa general increase in all proteincompounds. Inother words,the relative content of individual components varies with the advance of development. In Drosophila the ratio of the two fractions B to A separated by paper electrophoresis is about 12.3 in larvae aged 72 h, whereas the corresponding ratio at 96 h has a value of ca 4.5, indicating a much more rapid increase of fraction A (Chen, 1956). As shown in Fig. 12, the variation of individual components is even more impressive by using starchgel electrophoresis which separates the blood proteins into at least seven fractions. Both protein bands designated as Al, A, and B, appear only at the time approaching puparium formation. The immunological study on the blood proteins in the silkworm Hyalophora led Telfer and Williams (1953) to the disclosure of nine proteins. Among the seven proteins followed by them five are present in all stages of development, while a sixth one appears first in the late fifth larval instar. They were able to show that all six proteins increase in concentration in the last larval stage and decrease during the period from pupa to adult, but they differ from each other in the time of change of concentration. A seventh protein which is specific for the female appears first in the prepupa. Electrophoretic patterns with definite and specific protein bands at various developmental phases have been further reported for Bornbyx and Galleria (DenucC, 1958), Tenebrio (Po-Chedley, 1959)and the scarabs Lichnanthe rathvoni (Stephen and Steinhauer, 1957). The fact that in all

86

P. S. C H E N

these cases each protein component appears at a definite stage and possesses its own pattern of change in concentration suggests that the synthetic process is under individual genetic control. Strong evidence has been provided by the recent study of Pantelouris and Duke (1963) in Drosophifu showing that the formation of each lymph protein fraction is under

1

0

20

,

1

40

,

,

60

,

,

80

,

,

100

1

J

120 mm

FIG. 12. Separation of haemolymph proteins by starch-gel electrophoresis (below) and changes of quantities (above) in Drosophila larvae aged 72 (a), 84 (b) and 96 (c) h at 25°C.

AMINO A C I D A N D PROTEIN METABOLISM

87

the control of a separate gene. We shall deal more with this point in Section VI, B. 3. Function One relevant problem is the physiological activity and the morphogenetic meaning of the blood proteins. There is evidence indicating that they may function as enzymes. Laufer (1960a, b, 1961), who employed starch-gel electrophoresis for analysing the blood proteins in both cecropia and Cynthia silkworm, reached a similar conclusion to that reported by Telfer and Williams (1953) in regard to changes in the ontogenetic pattern. Moreover, he could demonstrate that many blood proteins in these two lepidopteran insects act as specific enzymesincluding esterase, phosphatase, carbohydrase, sulphatase, tyrosinase, chymotrypsin and dehydrogenase. Since Laufer was able to correlate the enzymesidentified by their substrate specificityinthe histochemical staining and their antigen-antibody reaction in agar diffusion to definite protein bands separated by electrophoresis, it seems that the possibility of contamination of blood samples from the leakage of cellular tissues is excluded. The occurrence of specific enzymes in insect haemolymph has been noted by previous authors. These include tyrosinase in Bombyx (Ito, 1953,1954; Kawase, 1960), Calliphora (Sekeris and Mergenhagen, 1964), Drosophila and Musca (Ohnishi, 1953, 1958, 1959), phosphatase in Gastrophilus (Levenbook, 1950) and trehalase in Phormia (Friedman, 1960, 1961). In addition, three enzymes involved in the carbohydrate metabolism (a hexose-1-phosphatase, a TPN-linked L-malic enzyme and a TPN-linked polyol dehydrogenase) have been reported to occur in Bombyx haemolymph (Faulkner, 1955,1956,1958). The possibility that some blood proteins may play a more direct role in the ontogenetic process is suggested by the work of Steinhauer and Stephen (1959). These two authors identified three lymph proteins in the cockroach Periplaneta and found that one of these was detectable only during moulting, but was absent at the intermoult period. It seems that the occurrence of this particular fraction is in some way linked to the moulting process even though its precise role is still unknown. 4. Site and mechanism of synthesis Finally it appears pertinent to inquire in which tissue and by what mechanism the blood proteins are formed. Evidence has been obtained suggesting that the fat body may be the major site of synthesis. Convincing results have been reported by Shigematsu (1958, 1960), who demon-

88

P. S. C H E N

strated that incubation of larval fat body of Bombyx with labelled amino acid resulted in a net synthesis of proteins, as shown by both increase in quantity and high radioactivity of the proteins secreted into the medium. Their identity to blood proteins have been checked by paper electrophoresis. That the fat body plays a key role in the metabolism of insects has been demonstrated by many other authors (see review by Kilby, 1963). There is almost no information concerning the synthetic mechanism of proteins in insects. As already mentioned (p. 82), according to Simmons and Mitchell (1 962) it seems that in Drosophila the amino acids are first incorporated into peptides and later enter into proteins (cf. Weinmann, 1964). More extensive data have been provided by the work of Faulkner and Bheemeswar (1960) on Bombyx. It is found that 14C-glycineinjected into the larvae is rapidly incorporated into proteins of the blood, silk gland, fat body and gut. The incorporation rate depends on the stage of development: it is low in the fourth moult, increases in the fifth larval instar and drops again shortly before spinning. Stage-dependent incorporation has also been reported by Demyanoskii et al. (1952) for the uptake of 35S-methionine into blood proteins of larvae and nondiapause pupae of the oak silkworm Antheraea pernyi. In order to get more insight into the incorporation mechanism Faulkner and Bheemeswar (1960) carried out in vitro experiments by using silk gland tissues and enzyme preparations. Incorporation of labelled glycine into tissue proteins was increased in the presence of such bivalent ions as Mg++ or Ca++ and the addition of various intermediates of the tricarboxylic acid cycle such as malate, citrate, succinate, fumarate and a-glycerophosphate. That the incorporation process requires energy was indicated by the inhibitory effect of anaerobiosis or the addition of cyanide. As is known from the work of Hoagland, Zamecnik and their collaborators, for the biosynthesis of proteins a so-called pH 5 enzyme is necessary to catalyse the activation of the amino acid by ATP to form the aminoacyl AMP compound and the subsequent condensation of the latter with soluble RNA (see Hoagland et al., 1958; Zamecnik el al., 1958). Using the posterior part of the silk gland Faulkner and Bheemeswar prepared a pH 5 extract and found that the incorporation of 14Cglycine into this extract could be stimulated by Mg++and either ATP or GTP. The nucleoprotein nature of the complex formed was indicated by its decomposition by heating at pH 7.8, treating with hot TCA as well as by the action of ribonuclease. Of particular interest was the finding that the pH 5 extract purified by ammonium sulphate showed a twentyfold increase in the activation of glycine, but its incorporation ability was

AMINO A C I D A N D P R O T E I N METABOLISM

89

almost completely lost. This indicates the complex nature of the crude extract prior to purification and, moreover, that separate enzymes are involved in the activation and incorporation processes in contrast to that found in other organisms. Quite unexpected results have also been obtained by Heller et al. (1959), who showed that glycine, which is a major component of fibroin, could be activated merely to a limited extent by the pH 5 enzymes, whereas tyrosine and tryptophan showed a much higher activation even though there are only traces of tryptophan in the silk protein. There is apparently no correlation between the rate of activation of one amino acid and its relative amount in the protein molecule. Whether or not the activation of the carboxyl group forms a necessarily preliminary step for the incorporation of amino acids into insect proteins is thus open to question. The in vitro experiment of Takeyama et al. (1958) demonstrated that inhibition of the uptake of 14C-oroticacid into RNA does not affect the incorporation of l4C-g1ycine into fibroin. This means that renewal of R N A is not necessary for fibroin synthesis. However, the importance of RNA was indicated by showing that no protein synthesis took place when ribonuclease was added to the reaction system. Further investigations will probably reveal more features which might be peculiar to insects. But at the present state of our knowledge there is yet no reason to assume that the mechanism of protein synthesis in insects forms an exception to the conventional scheme already established in micro-organisms and vertebrates.

IV. PUPALDEVELOPMENT Metamorphosis includes a series of ontogenetic events through which the insect transforms from the larva to the adult. The transformation process involves mainly the destruction of most larval tissues and organs (histolysis) and the differentiation of imaginal structures (histogenesis). In the following account we shall see to what extent such changes are reflected in the metabolism of amino acids and proteins as well as in the activity of certain enzymes. A. M E T A B O L I S M O F A M I N O A C I D S A N D P R O T E I N S

Considering the profound changes which the developing insect undergoes at the time of metamorphosis, it is perhaps surprising that only minor changes in the pool size of free amino acids during this period have been detected. In Calliphora slight reductions in the total concentration of

90

P. S. C H E N

these compounds occur at the initiation and during the later half of pupal life (Agrell, 1949). It is thought that the first decline is related to the histogenesis of the hypodermal tissue, especially the formation of the imaginal buds, and that the second one coincideswith the differentiation of muscles in the thorax. The highest concentration is found at about the early middle of the pupal development, at which stage the breakdown of larval tissues reaches a maximum. Similar results have been obtained for Ephestia (Chen and Kuhn, 1956) and Culex (Chen, 1958a). In both cases a faint increase in the total content of free amino acids takes place in the early pupae when the histolytic process is in full swing, whereas a gradual

t

4 0 L - L 0

. .-I

L .-I 50

Pupal duration (%)

100 (Adult)

FIG.13. Changes in the content of free ninhydrin-positivesubstances (free amino acids plus “peptides”) before and after hydrolysis during metamorphosis of Prodenia eridania. (From Levenbook, 1962.)

decrease is characteristic for the later period. Likewise no major variations in the total free a-amino nitrogen have been observed in the metamorphosing mealworms Tenebrio (Patterson, 1957). After an initial drop it remains constant until the 7th day of pupal life, though it increases again thereafter. In a more recent contribution Levenbook (1962) presented quantitative data on the changes in total free amino acids, glutamic acid, glutamine as well as peptides during the pupal development of Prodenia. There is a sharp decrease in all of them during the first 12 h after larvalpupal ecdysis which is followed by rapid increase to almost the initial level (Figs. 13 and 14). Thereafter the values decline again but at a more gradual rate. The relative changes for both glutamic acid and glutamine

91

AMINO A C I D A N D PROTEIN METABOLISM

are somewhat greater, indicating that these two components may contribute to a large part of the variation of the total content. The fact that the concentration ratio from glutamine to glutamic acid is maintained at a constant level suggests their metabolic interrelationships. One possible explanation of these changes is again the reflection of ontogenetic events : the initial drop results probably from the formation of new cuticular protein, the subsequent rise and decline being related to the breakdown of larval tissues and the onset of adult differentiation at the corresponding periods. I’

,’

, 7 ‘ 1

/ GLutamate

c

GLutamine

0

E

3

L

0

1 -

L

L

__LI

I I - . . L

50

Pupal duration (7.)

100 (Adult)

FIG. 14. Changes in free glutamate and glutamine during metamorphosis of Prodeniu eriduniu. (From Levenbook, 1962.)

As Levenbook has pointed out, it must be admitted that no direct evidence has so far been brought forward to show that such variations are really in association with the process of metamorphosis. It is true that the developing pupa represents a closed system and thus its concentration levels of free amino acids could be considered as indicating the balance between histolysis and histogenesis. In general, however, the fluctuation is too small to account for such drastic morphological changes, unless it is assumed that the amino acids produced by histolysis are immediately used for synthetic purpose. In many cases the variation of the amino acid concentration also does not fit the time axis of the major morphogenetic events. As a matter of fact there is no experimental proof that the decomposition of larval proteins actually proceeds as far as the production of amino acids prior to their being utilized for the formation of adult

92

P. S. CHEN

proteins. As noted by Agrell (1952), some tissue proteins of the larval structures in Calliphora may be decomposed only to the level of peptides which are bound with phospholipids and enter probably directly into cellular components of the adult. A close parallelism between the peptide fraction and the morphological changes has been emphasized (Agrell, 1964). It is true that tracer studies on both Sphinx ligustri (BricteuxGrCgoire et al., 1957) and Hyalophora (Skinner, 1960) indicate a high incorporation of amino acids into pupal tissues. But these results show only the ability of developing pupa to take up free amino acids and do not necessarily mean that it is the major pathway of protein synthesis during histogenesis.It is also clear that besides histolysis and histogenesis variations due to interconversion and other metabolic connexions of amino acids cannot be neglected. The causal relationships between changes in free amino acids and metamorphosis are still open to question. More convincing experiments have been performed showing that the rate of protein synthesis is closely related to the developmental state of the pupa. Working with the cecropia silkworm Telfer and Williams (1960) were able to demonstrate that the rate of incorporating injected glycine l-14C into blood proteins was four times higher in individuals at the beginning of adult development than that in diapausing pupae. The difference increased to nearly twentyfold at the time of adult emergence. Moreover, exposure to CO, which has been shown to block the pupal development.and to reduce the rate of oxygen uptake (Schneiderman and Williams, 1954), inhibited the rate of incorporation in post-diapausing pupae, but had no such effectin diapausing individuals, paralleling to their metabolic resistance to such treatment. This fact indicates clearly the endergonic nature of the incorporation process. Quite similar results have been reported by Stevenson and Wyatt (1962) using the in vitro technique. According to these authors fat body from Hyalophora pupae aged 2 days of adult development incorporated leucine-1-14Cinto tissue proteins about thirty times faster than that from animals in diapause. As protein synthesis is closely associated with RNA, it is not unexpected that at theinitiationof adultdevelopment RNAsynthesis also increases. In Tenebrio Patterson (1957) reported that there is a high ratio of RNA to deoxyribonucleicacid (DNA) at the beginning of pupal development and at the time of emergence, corresponding to the periods of formation of adult tissues and adult cuticle respectively. A threefold increase in the incorporation of 32Pinto RNA of the fat body has also been observed in Hyalophora (Wyatt, 1962). Similarly, in both Gryllus (Krishnakumaran, 1961) and Rhodnius (Wigglesworth, 1963) there is an accumulation of

AMINO A C I D A N D PROTEIN METABOLISM

93

RNA in the cytoplasm and nucleolus at the time of action of ecdysone. All these studies point out that the processes of RNA and protein formation parallel the progress of metamorphosis, and thus suggest indirectly the hormonal control of cellular activities. In an attempt to detect to what extent the morphological alterations at the time of metamorphosis are reflected at the molecular level, Kominz et al. (1962) investigated the physicochemical properties of purified muscle proteins (tropomyosin, myosin and actin) of the blowfly Phormia regina. They found that larval tropomyosin exists in a more polymerized state than the corresponding adult protein as shown by its higher values of sedimentation rate, molecular weight and intrinsic viscosity. Furthermore, fingerprint patterns of the tryptic digests suggested the possible occurrence of structural differences between larval and adult tropomyosins though no significant variation in the amino acid composition could be detected. We have so far dealt with the metabolic changes in amino acids and proteins in general. Let us now consider the synthesis of specific proteins, (i.e. enzymes) which play an active part in the metabolic machinery of the developing organism and are thus virtually responsible for both maintenance and development. B. C H A N G E S IN E N Z Y M E A C T I V I T I E S

'

I . Respiratory enzymes It is known that during pupal development the rate of oxygen uptake follows a U-shaped curve (for references, see Chen, 1951;Wigglesworth, 1954; Agrell, 1964). In a number of insects it has been shown that the activity of respiratory enzymes parallels precisely the respiratory pattern. We need only mention the work of Agrell (1948) on the hydrogen activating enzymes in Calliphora and that of Bodenstein and Sacktor (1952) on the cytochrome c oxidase in Drosophila. In both cases the enzyme activity decreases to a minimum during the middle part of pupal life and rises abruptly near the time of hatching. The same has been shown to be true for the corresponding enzymes during the metamorphosis of Tenebrio and Musca (Ludwig and Barsa, 1959a, b). Such a variation may reflect realquantitative changes of the respiratory enzymes in connection with the disintegration of larval tissues and the reconstruction of imaginal structures. It is also equally possible, however, that it merely indicates the changes in enzyme activity under the influence of other physiological and biochemical factors in the developing pupa. A clear-cut answer to this question is by no means easy. D

94

P. S. CHEN

Extensive work has been done on the cytochrome system in the silkworm Hyalophora, mainly by Williams and his collaborators. There are several recent reviews dealing with this subject (Gilbert and Schneiderman, 1961;Harvey, 1962; Wigglesworth, 1964)and no further discussion is necessary. It may be summarized that the situation in the blocked diapause pupa is a substantial excess of cytochrome oxidase relative to cytochromes b and c. This explains why the respiration of these pupae is insensitive to CO and cyanide, but becomes sensitive to the inhibitors under experimental conditions (injury, exposure to low oxygen tension, and injection of dinitrophenol). The main chain of terminal electron transport is thus still the cytochrome system and does not involve any other alternate pathways. From their recent studies on the phosphate pattern in pupal tissues Carey and Wyatt (1963) suggested that the limit factor of the metabolic rate in the diapause pupae is most likely the supply of phosphate (see also Wyatt, 1963). Although further information is necessary to determine to what degree the phosphate levels are involved in the initiation and termination of diapause, it is clear that at the renewal of adult development synthesis of enzymes becomes accelerated and the whole cytochrome system is rebuilt. For our present purpose the most significant point is that the changes both in quantity and in turnover rate of the respiratory enzymes correlate precisely with the morphogenetic process. In accordance with this conclusion it has been found by Wigglesworth (1957, 1963) in Rhodnius and by Shappirio and Williams (1957) in Hyalophora that the mitochondria increase in both number and size in response to the action of ecdysone. For the mosquito Aedes Lang (1959, 1961) showed that the activity of DPN-, TPN- and succino-cytochrome c reductases drops to a low level during pupal development. At the time of adult emergence the activity of DPN- and succino-cytochrome c reductases rises rapidly to a high level, but that of TPN-cytochrome c reductase remains low. This finding is of interest in showing that the relative specific activities of individual enzyme components vary with developmental stages and alternate pathways may be used for terminal respiration. 2. Proteases A detailed analysis of the proteases in the developing pupa would be of interest because these enzymes may be directly involved in the histolytic process. In Calliphoru Agrell (1951) found only a faint change in the activity of both proteinase and dipeptidase during pupal development. In order to correlate the proteolytic activity with the morphogenetic events, it is suggested that the enzyme action is probably under the

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control of tissue pH which has been shown to follow a U-shaped curve and has a direct relation to energy metabolism (Agrell, 1948). In other words, a lowering of pH would activate the proteinase and thus leads to the proteolysis of the larval tissues, whereas the reverse process would favour the synthesis of adult proteins. In the housefly Musca it is found that a dipeptidase, which splits alanylglycine, drops abruptly in its activity at the beginning of metamorphosis, rises again and declines thereafter until the time of hatching (Russo-Caia, 1960, cited in Urbani, 1962). The activity of an acid proteinase (pH 4.7-4.9) remains high and rather constant during the whole period of metamorphosis, while that of an alkaline proteinase (pH 8.6) becomes practically undetectable. The relative importance of these enzymes in the pupa is, however, unknown. 3. Phosphatases In his histochemical exploration of alkaline phosphatase during the post-embryonic life of Drosophila Yao (1950b) reported that pupation is accompanied by a considerable increase of this enzyme. The high enzyme activity is maintained for the first 24 h after head eversion, at which period histolysis and ,histogenesis proceed rapidly. Subsequently the enzyme activity declines until the time of adult emergence. The overall results suggest that alkaline phosphatase takes an active part in the degradation and resynthesis of tissue proteins. By contrast Sridhara and Bhat (1963) found that in Bombyx alkaline phosphatasedrops abruptly to almost undetectable levelsat the beginning of pupation, whereas the acid phosphatase remains very active during metamorphosis. As already mentioned in Section 11,B, alkaline phosphatase is thought to be concerned with the transport of materials in connection with digestion which is most active in the fifth larval instar. On the other hand, the presence of the corresponding acid enzyme is probably related to an active glycogen metabolism with dephosphorylation at the acid level. The decrease of glycogen during pupal development suggests such a possibility. 4. Tyrosinase Tyrosinase is another enzyme which has received the attention of a number of investigators, mainly because of its role in the hardening and darkening of the insect cuticle (see p. 73). In general the activity of tyrosinase increases rapidly at the time of pupation and thereafter declines again to a rather low level. This has been found, for instance, for Drosophila (Ohnishi, 1953) and Bombyx (Kawase, 1960). In Calliphora

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P. S. C H E N

Karlson and Wecker (1955) detected also a distinct increase in the activity of this enzyme during the transformation from larva to pupa. There is evidence for the existence of an activating enzyme whose synthesis is apparently under the control of ecdysone (Karlson and Schweiger, 1961; Mitchell and Weber, 1965). The increase of tyrosinase activity during the time of pupation is doubtless related to the tanning of the pupal cuticle. Since both enzyme and substrate are present in each stage, the question arises why there is no or only a limited reaction prior to the initiation of metamorphosis. Various hypotheses have been advanced to explain the control mechanism of the tyrosine activity, including the occurrence of an inhibitor or activator, the level of redox potential, the direct action of pupation hormone as well as changes in the cytochrome system. We shall not, however, go into this interesting problem in detail since it has been adequately reviewed by Mason (1955) and Cottrell (1964). V. ADULT In addition to maintenance the dominant phenomenon in the life of adult insects is reproduction. In the female there is a continuous deposit of yolk at the time of egg production, whereas in the male probably different proteins are synthesized in connection with spermatogenesis and secretion of the accessory glands. For example, a characteristic composition of free and bound amino acids has been found in the semen of honeybee drones (Novak et al., 1960). It therefore seems reasonable to expect some differences in the protein metabolism between the two sexes. We shall first consider briefly the general pattern of amino acids, peptides and proteins in the adult life and then examine in more detail the mechanism of egg formation, particularly the synthesis of yolk proteins. A. S E X - S P E C I F I C DIFFERENCES I N FREE AMINO ACIDS, PEPTIDES A N D PROTEINS

In Culex it is found that there is a distinct sex-specific difference in the composition of free amino acids (Chen, 1958b, 1963). Male mosquitoes aged 4-6 days contain about seven times more /3-alanine than the females. The reverse is true for methionine sulphoxide whose content is about five times higher in females than in males. These differences persist even when the body size is taken into consideration. Further analyses proved that the two amino acids are not derived from the reproductive glands because a similar situation has been observed in the haemolymph as well as in body tissues. It is of interest that such a difference is hardly detectable in newly emerged individuals, but becomes more obvious in

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97

the course of adult life, suggesting that the accumulation of these compounds is in some way related to the process of reproduction. Strong evidence has been provided by Geiger (1961), who demonstrated that in the autogenous form (C. pipiens) a parallelism exists between the increase in methionine sulphoxide and the progressive ovarian development, whereas in the anautogenous form (C. fatigans), whose ovaries remain undeveloped in the absence of blood meal, there is no accumulation of this amino acid (Fig. 15). The precise role of this compound is, however, still not clear, neither is there any information on the significance of p-alanine in the male adults.

€I- -

i 1

2

3

h

5

Days after adult emergence

FIG.15. Correlation of changes in methionine sulphoxide and ovarian development in Culex pipiens (0) and Cufexfutiguns ( 0 ) .(From Geiger, 1961 .)

In Drosophila Kaplan et al. (1958) reported that female flies aged 2-3 days contain twice the amount of methionine as males. Methionine is one of the essential amino acids needed by insects including Drosophila, and has been demonstrated in various biological systems to serve as a methylating agent in intermediary pathways. In both Aedes (Lea et al., 1956; Dimond et al., 1956)and the boll weevil Anthonomusgrandis (Vanderzant, 1963) it is an important component in the synthetic medium and has the ability to promote egg production. Thus its connection with the reproductive process is again indicated.

98

P. S. CHEN

These examples may be sufficient to show that male and female insects possess a differential ability to metabolize amino acids. Sexual difference has been also disclosed at the peptide level. In Drosophilu Fox (1956a, b) found a peptide in males but not in females. Working with the same species Chen and Diem (1961) detected the presence of a peptide in the accessory glands (paragonia) in the male adults. Hydrolysis of this peptide revealed the presence of aspartic acid, glutamic acid, glycine, a-alanine, valine, leucine and traces of at least two other unidentified components. Judging from its Rf values on the paper chromatogram it corresponds obviously to the sex peptide discovered by Fox. There is also a marked similarity in its amino acid composition which, according to Fox et al. (1962), includes serine, methionine, ethanolamine and one unknown component in addition to the amino acids mentioned above. Parallel to growth of the accessory glands, Chen and Diem showed that the content of the paragonia substance increases in the course of adult life. The appearance of this substance in female flies, which have received male genital disks in the larval stage by transplantation, indicates its autonomous formation. This is in agreement with the presence of this peptide in transformed sterile males of the mutant “transformer” (Fox et al. (1959) and unpublished data of Chen and Diem). The most interesting point is, of course, the functional significance of this peptide. In Drosophilu there is so far no satisfactory explanation of the role of the accessory glands in reproduction. It has been suggested that their secretion is essential for fertilization (Gottschewski, 1937) or probably has a stimulating effect on oviposition (Kummer, 1960). Recently, work has been done by Garcia-Bellido (1964) in our laboratory to test these possibilities. It is known that fecundity of females increases rapidly after mating. Garcia-Bellido was able to show that there is no correlation between the number of eggs laid and the reserve of sperms. For instance, females with interrupted copulation or after mating with exhausted males had an initial increase in fecundity which, however, fell off rapidly even though they still contained sperms in their receptacula and spermathecae. Transplantation of paragonia into virgin females resulted in a distinct increase in fecundity, but there was no stimulating effect when either vesicula seminis or fat body of males was implanted (Fig. 16). A similar increase was observed by injecting only the fluid from the accessory glands. There is, of course, still no direct proof that the stimulating effect is actually due to the sex peptide and further experiments using purified extract are needed to confirm this point. With regard to proteins, studies on adult insects have revealed that

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99

protein concentration in the blood can be greatly influenced by nutrition. As has been clearly shown in the blowfly Phormia by the recent work of

Orr (1964a, b), during enforced starvation it may drop to extreme low levels. Thesameis true for both CeIerioeuphorbiae(Hel1erand Moklowska, 1930) and Sialis lutariu (Beadle and Shaw, 1950). These results indicate that the haemolymph proteins serve as an important reserve. Sex-specificdifferencesin the antigenic pattern of adult Drosophila have been reported by Fox (1958). From his genetic and immunological analyses it is concluded that the X-chromosome dosage controls the synthesis of the polypeptide chain, whereas the presence of a “maternal” Y

Days after adult emergence

FIG. 16. Fecundity of females of Drosophila melanogaster under various conditions: fertilized females ( 0 ) and virgin females after implantation of either paragonia (A ), male fat body ( A ) , filled vesicula seminis (O), or without implantation ( 0 ) .The arrow indicates the time of implantation or copulation. (From Garcia-Bellido, 1964.)

chromosome affects the gross structure (folding, cross-linkage,etc.) of the protein molecule. We shall consider in more detail the genetic control of the synthesis of some enzyme proteins in Section VI, B. B. P R O T E I N METABOLISM I N R E L A T I O N T O R E P R O D U C T I O N

The hormonal control of reproduction has been repeatedly reviewed (see, for example, Wigglesworth, 1964; de Wilde, 1964b), and will not be considered here. It may only be mentioned that, in addition to hormones, the quality of the diet may have also a definite influence on ovarian development, a point which has hitherto received rather little attention. A variety of amino acids included in the synthetic diet are found to be indispensable for egg formation. It is further known that in many

100

P. S. CHEN

insects egg maturation does not occur when the adult females are kept solely with sugar and water but without protein (Fraenkel, 1940; Rasso and Fraenkel, 1954; Harlow, 1956; House, 1962). Indeed, it has been demonstrated that the activation of the neurosecretory cells in Calliphora (Strangways-Dixon, 1962)and the corpus allatum in Phormia (Orr, 1964a, b) depend on the amount of dietary proteins ingested by the females, and there is a close correlation between the secretory activity of the glands and the concentration level of the protein metabolites in blood. Thus nutrition does not merely supply the raw materials necessary for yolk synthesis but also exerts a fundamental effect on the control system. Ovarian development is, after all, a problem of growth, and a series of observations is in line with the conclusion that interference with the function of the endocrine system generally results in a deficiency in the production of proteins. For instance, chemical analyses of Dixippus females deprived of their corpora allata revealed a definite decrease in the protein content with a corresponding increase in the free amino acids of the tissue (L'HClias, 1953a, b, 1956). Extirpation of the neurosecretory cells in the brain of Culliphora led to a reduced activity of the intestinal proteinase (Thomsen and Mraller, 1959, 1963). Exactly the same phenomenon has been reported for the allatectomized Tenebrio (Dadd, 1961). Furthermore, Roller (1962) showed that in Galleria larvae there was no more synthesis of body proteins and specific enzymes following allatectomy. In Schistocerca it was found by Hill (1962) that a high content of haemolymph protein was always correlated with an active neurosecretory system. Moreover, destruction of the neurosecretory cells in the brain by cautery led to a reduction of the protein concentration in the haemolymph and implantation of the corpora cardiaca, which released the secretion of the neurosecretory cells, resulted in an elevation of the blood protein concentration. Parallel but reversed variations of the haemolymph amino acid concentration were also observed, indicating that it is the synthesis of proteins which is under the neurosecretory control. The study of Wang and Dixon (1960), who demonstrated a diminished activity of transaminases in adult Periplaneta deprived of corpora allata, has been already mentioned in Section 111, A, 3, d (p. 81). The biosynthesis of protein is known to be RNA-dependent. In confirmation of this L'HClias (1953a, b, 1956) noted that in allatectomized Dixippus the RNA content of various tissues was reduced. Likewise Berreur (1961) has provided evidence that RNA metabolism in Culliphora is under endocrine.contro1. The results of all these investigations support the view that the corpus allatum and the neurosecretory cells play a part in regulating different

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101

phases of protein synthesis and are thus directly or indirectly involved in egg development. There is considerable difference of opinion concerning the mode of yolk formation in insects. This situation obviously arises from speciesspecificdifferences in the structure of the ovarioles and in the composition of the yolk (mainly proteins, carbohydrates and fats). Cytochemical findings indicate that yolk proteins can be synthesized within the ovary. Proteins provided by the follicular cells and the trophocytes are transferred to the oocyte where the final elaboration of the yolk spheres takes place (see reviews by Bonhag, 1958; de Wilde, 1964a). On the other hand, several investigations have demonstrated beyond doubt that egg proteins may be synthesized outside the ovary and then transferred without change to the oocyte. According to an earlier observation of Wigglesworth (1943) in the blood-sucking insects like Rhodnius, Cimex and Pediculus, a part of the haemoglobin can be taken up by the haemolymph without being digested and is deposited in the yolk of the growing egg; In Hyalophora Telfer and Williams (1953) and Telfer (1954) found, as already mentioned (p. 85), a blood protein which is limited to the female and appears initially in the pre-pupal stage. This so-called female protein is later accumulated in the oocyte during egg development with a corresponding decrease of its blood concentration in the female. A more recent study of Telfer (1960) showed that another protein, a carotenoid protein which normally occurs in the blood, is also accumulated in the developing egg. hdeed, according to Telfer nearly all haemolymph proteins of this silkworm can be detected in the oocyte. This has been shown to occur even with proteins normally not present in the insect when artificially introduced into the pupae. The accumulation process is highly selective, and the blood proteins are deposited in the yolk spheres. A close correlation has been further found between the growth rate of the oocyte and the amount of female protein available in the pupal blood (Telfer and Rutberg, 1960). The mechanism of protein uptake is thought to have a great similarity to pinocytosis in Amoeba (Holter, 1959). Adsorption on the cell surface and the intracellular structure as well as permeability of the cellular wall are possible factors responsible for the selective accumulation. The transfer of blood proteins to the egg is by no means unique for Hyalophora. Using paper electrophoresis Hill (1962) concluded that in Schistocerca at least one protein component, and possibly another one, may be removed from the haemolymph by the growing oocyte. The same possibility has been suggested for Calliphora (Strangways-Dixon, 1962) and Phormia (Orr, 1964a, b). In all these cases the blood proteins decrease D"

102

P. S. C H E N

during the time of ovarian development, whereas in ovariectomized females they accumulate to a high concentration. The site of protein synthesis is probably the fat body (cf. Hill, 1963, and p. 87). From the preceeding account our present state of knowledge about protein synthesis during egg maturation can be outlined as follows. Yolk proteins are synthesized both within the ovary and in tissues outside it. In the ovary protein synthesis is initiated in the trophocytes and the follicle cells. Outside the ovary proteins are synthesized in the fat body, released into the haemolymph and taken up by the female gonads. Of course these results do not rule out the possibility of de novo synthesis within the oocyte itself, though its distinction from the formation of other cytoplasmic proteins may not be easy. VI. S O M EGENETIC ASPECTSO F P R OTEIN I N INSECTS METABOLISM Progress from recent genetic and biochemical investigations on both micro-organisms and higher animals has provided unequivocal evidence that specific genes control the synthesis of enzymes and other specific proteins (cf. Chen, 1961 ; Wagner and Mitchell, 1964). There is now an increasing interest in similar problems in insects. Studies on the biochemical properties of lethal mutants as well as those on the synthesis of isozymes have already yielded promising results. Furthermore, cytochemical approaches to the study of hormone action have provided experimental evidence for the links between gene activation, RNA synthesis and protein formation. A brief survey of some of these results may serve to illustrate the sort of genetic problems which are involved in the study of protein metabolism in insects. A. P A T T E R N S O F P R O T E I N M E T A B O L I S M I N L E T H A L M U T A N T S

Nobody would disagree with the statement that development is under genic control. The morphogenetic achievemknts and the metabolic activities are different at various developmental periods. At those stages where the ontogenetic processes are more elaborate and intensive, apparently a higher activity and a co-operative action of a larger number of genes are necessary. This has clearly been shown from studies on lethal mutants whose development is altered, due to either chromosomal changes or gene mutations, and the individual dies before it reaches the adult reproductive stage. From their analysis of a total of fifty-nine lethal factors in the second chromosome of Drosophila melanogaster Hadorn and Chen (1952) found that deaths of these mutants do not have a random distribution, but are more frequent at four distinct periods : the

AMINO A C I D A N D PROTEIN METABOLISM

103

time of embryonic hatching, the beginning of the third larval instar, the onset of puparium formation and during pupal development. Thus the manifestation of the lethal effect reflects specific functions of genes at definite ontogenetic phases. Since the basic mechanism underlying growth and differentiation is protein synthesis, it would be of particular interest to analyse in more detail the protein, metabolism of the lethal mutants. The results of such analyses provide us with valuable information about the causal relationships of the events which lead to the developmental failure and, moreover, the roles of genes in the metabolic processes. Unfortunately, among the numerous lethal mutants registered by geneticists only a few have been studied from a biochemical viewpoint. There are, however, more data reported for lethal factors in Drosophila, mainly by Hadorn and his collaborators. In the following we shall limit our discussion to some recent results of these studies. For further information the comprehensive book by Hadorn (1 96 1) should be consulted. 1. Lethal-translucida (Itr, 3-20 f 0.8) Homozygotes of the Itr mutant pupate regularly, but with a delay of about 24 h at 25°C. During larval development there is a large accumulation of haemolymph in the lethals which appear quite transparent and can be easily distinguished from the normal heterozygotes. Their transparent appearance is partly due to the reduction of fat body. In general, the lethal larvae remain at the pre-pupal stage without further development. However, some of them can undergo imaginal differentiation in the head and thorax, but never hatch (Hadorn, 1948,1949). It was first demonstrated by Hadorn and Mitchell (1951) by paper chromatography that the haemolymph of the Itr lethal larvae has a much higher concentration of free ninhydrin-reacting components than that of the normal genotype. This was later confirmed by Hadorn and StummZollinger (1953) who showed that the lethals accumulate four to eight times more amino acids and peptides than the corresponding controls during larval development. Furthermore, they discovered that, in contrast to normal individuals, the total concentration of these substances does not fall off, but stays at a high level. At the time of pupation the concentration in the Itr homozygotes is at least four times higher than in normals. The more detailed analyses of Stumm-Zollinger (1954) revealed that the mutational effect on individual ninhydrin-reacting compounds is not the same : serine, glycine, lysine, ornithine, threonine and glutamine have an abnormally high content, whereas proline, tyrosine, a-alanine and the peptides are markedly reduced. These studies

104

P. S. C H E N

demonstrate clearly the following two points : (1) the excessive accumulation of haemolymph in the Itr lethals is not simply a result of the dilution of body fluid, and must be due to some specific changes in the metabolic processes; and (2) in the Itr homozygotes there is no general increase of all free ninhydrin-positive components whose pattern of distribution appears locus-specific. More recently, using column chromatography, Mitchell and Simmons (1962) also detected characteristic differences in peptides and other amino acid derivatives between the ltr mutant and the wild type. One significant difference is the high concentration of tyrosine-0-phosphate in the lethal larvae with a corresponding reduction of free tyrosine. As mentioned on page 70, this phosphate ester has so far been found only in Drosophila and there is no information as to its function. In view of the tremendous changes in free amino acids, one would naturally inquire about the protein concentration in the lethal haemolymph. In an earlier brief report Gloor (1949) noted that the Itr larvae have a very low blood protein content. This has been confirmed by later studies using both paper and starch-gel electrophoresis (Wunderly and Gloor, 1953; Chen 1956, 1959a). As illustrated in Fig. 17, the protein concentration in the lethals is markedly reduced as compared to the normal form, although the relative reduction for individual components may not be the same. The total concentration of the mutant larvae has been estimated to be only 5 1 3 % of that of the controls at the time of pupation. The wild-type larvae, as already mentioned, exhibit a rapid increase in blood proteins with a corresponding decrease in free amino acids as development proceeds. Apparently the situation is entirely different in the lethals: there is almost no increase in proteins, whereas the amino acid concentration remains high. On the other hand, the total nitrogen is about the same in both. In a recent study Weinmann (1964) was able to show that the ability to incorporate 14C-valineinto proteins is greatly diminished in the mutant larvae. All these results point to the conclusion that protein synthesis in the ltr lethals is blocked. In an attempt to search for the blocking mechanism Metzenberg (1962) carried out a detailed investigation on the nucleic acid metabolism of this mutant. There is neither qualitative nor quantitative difference in RNA between Itr and wild type larvae. However, from a closer analysis of the acid-soluble phosphorus pool he discovered that the relative content of adenine nucleotides differ significantly between the two genotypes. As shown by the data summarized in Table 111, ADP and 5’-AMP are extremely reduced in the lethals. Even if corrections are made for the excessive amount of Itr haemolymph and the values are compared on a

I05

AMINO A C I D A N D PROTEIN METABOLISM

dry weight or total N basis, the levels of 5'-AMPstill remain at least twice as low in the mutant as the wild type. This result could be interpreted as being due to a diminished rate of conversion of ATP to A D P or AMP. It 0-34

0-26

GC

gm 0.18 X

Y

0.12

r /tr//tr

'

'

0

'

20

I

I

,

40

*

60

,

1

8

,

1

1

120 mm

100

80

a

FIG. 17. Separation (below) and content (above) of haemolymph proteins in normal ( +/+) and

lethal ( l t r i l t r ) larvae of Drosophilu melunoguster.

TABLE111 Levels of adenine nucleotides in wild type and Itr larvae of Drosophilu (from Metzenberg, 1962)

mrmoles of nucleotide per g (wet weight) of larvae Nucleotide

5'-AMP ADP ATP

.

~.

Wild type 9214 h

Wild type I l6*4 h

11614 h

79.5 259 27 I

92.5 307 367

12.0 77.7 314

Itr

106

P. S . C H E N

is known that the first step in protein biosynthesis involves the activation of amino acids in the presence of ATP and soluble enzymes to form the enzyme-bound aminoacyl-5-AMP. The latter is converted to the amino acid ester derivative of transfer RNA and 5'-AMP is released (cf. Cohen and Gros, 1960). From such considerations we may speculate that the reduced protein synthesis in /tr mutant may be linked to a block in the energy transfer system. Many other interpretations and possibilities also exist however, and we must await further investigation for more direct evidence. 2. Lethal-meander ( h e , 2-7 1 to 73) Homozygous larvae of the present mutant can develop to the early third instar, but, in contrast to Itr, they never pupate. There is a marked reduction in larval growth, as shown by measurements of body length, total weight and total nitrogen (Schmid, 1949; Chen, 1951, 1958~). Among various organs the salivary glands are most affected and only amount to 30(y0 of the normal size at the end of larval life. According to Schmid( 1949) the morphological pattern of the /me lethalscan be phenocopied by starving normal larvae. Biochemical analysis of the /me mutant by Chen and Hadorn (1955) disclosed that the lethal homozygotes have an extremely low amino acid pool. Many amino acids, such as valine, lysine, leucine, methionine, histidine and isoleucine, which are known to be essential to Drosophilu, become either markedly reduced or undetectable. By contrast, glycine is gradually accumulated to an abnormally high concentration, especially at the prolonged larval period. Since Schmid (1949) found that the growth pattern of the lme mutant can be phenocopied by total starvation, it would be of interest to compare the amino acid pattern between the lethals and starved normal individuals. The results indicate that, although many amino acids behave similarly in both, there are significant differences (Chen, 1958~).For example, glycine does not increase during starvation as in lethals. On the other hand, one peptide rises rapidly in the phenocopies, but remains quite constant in the /me homozygotes. These results suggest that the lethal effect is apparently not the consequence of totalstarvation. In fact, feeding experiments showed that the /me larvae are able to utilize carbohydrates and fats. The in vitro study of Chen and Hadorn ( I 955) revealed that homogenates of the midgut from lethal larvae have a striking low activity of proteolytic enzymes. In accordance with this observation it was found that the epithelial cells of the /me midgut show a much reduced secretory activity (Meyer-Taplick and Chen, 1960). All these facts support the

107

AMINO ACID A N D PROTEIN METABOLISM

view that a reduced synthesis or inactivation of the proteolytic enzymes in the digestive tract could be responsible for the lethality of this mutant. Relevant information on the nucleic acid metabolism in the Ime lethals is also available. Changes in the contents of DNA and RNA in the salivary gland cells of both normal and lethal individuals during larval development have been followed by Chen et al. (1963). As can be seen from the data summarized in Table IVYthe total quantities of both DNA TABLE1V Content and ratio of RNA and DNA in the salivary glands o f normal and Ime larvae of Drosophila (summarized from Tables 2 and 3 in Chen et al., 1963)

(h) RNA/gland (pgx DNA/gland (pgx RNA/DNA

72 96 72 96 72 96 a

b

na

Normal M f S.E.b

27 142 & 9.4 27 351 &15.2 27 13.2f 1.2 27 4 0 . 1 1 2.1 -

10.8 8.8

Ime n

22 25 22 25

MfS.E.

35.355.9 29.5&3.8 .10.4&0.63 13.4f0.72 3.4 2.2

lme

normal

x

loo

24.8 8.4 78.8 33.4 31.4 25.0

Number of determinations. Mean standard error.

and RNA are strikingly reduced in the lethal salivary gland. Of special interest are the RNA/DNA ratios which amount to only 2.2-3.4 in the mutant compared to values of 8.8-10.8 in the wild type of corresponding ages. This indicates that per unit amount of DNA the lethal larvae synthesize three to four times less RNA than normals. Although we still do not know the reason why there is such a disproportion of the two nucleic acids, it suggests again a deficient protein synthesis which may account for the inhibition of growth and enzyme synthesis in the Ime homozygotes. Their low incorporation rate of 14C-valine into body proteins is in favour of this conclusion (Weinmann, 1964). 3. Lethal giant larvae (Igl, 2-0 f ) Judging from their external appearances it seems that individuals homozygous for the Igl factor develop normally until the third larval instar. Puparium formation may be much delayed or absent. Those which undergo pupation form the so-called pseudopupae without imaginal differentiation. Metamorphosis does not take place because among other defects there is a deficient function of the ring gland (Hadorn, 1937,1948).

108

P. S. CHEN

The detailed analysis of the lgl mutant by Faulhaber (1959) demonstrates again that protein synthesis in the lethal larvae is retarded. Similarly, as described for ltr, she found that in the course of larval life the free amino acids rise gradually to an abnormally high concentration, whereas the protein content in the haemolymph remains at a low level. From the work of Welch (1957) it is known that DNA synthesis in the lethals is greatly reduced. For instance, nuclear DNA in the salivary gland cells was estimated to be only 20% of the normal value. Since the synthesis of RNA is DNA dependent, it is highly possible that the RNA content in this mutant is also reduced. The inhibition of protein formation, as revealed by Faulhaber, supports such a possibility. 4. Other lethal mutants Comparable results have been obtained by Benz (1957) for two other lethal mutants, “lethal-bluter” (Ibl, 2-43.8*) and “lethal-polymorph” (lprn, 2-30.3*). The 161 lethals lose much haemolymph during adult emergenceand die fromunsuccessful hatching movements. One important feature of this mutant is the abnormally thick cuticle. A larger demand for the synthesis of cuticular protein could possibly account for the low concentration of amino acids found in the mutant larvae. The lpm homozygotes exhibit strong muscular dystrophy and consequently do not contract prior to the formation of puparium. As reported by Benz, there is a high content of amino acids and peptides in the lethals, which seems to be linked with their subnormal formation of muscular proteins. Since many other differences in the relative distribution of individual amino acids have been noted in these two mutants, the situation is probably more complicated than it appears. Further analysis of the protein composition of the cuticle and muscle tissues and their patterns of formation by using labelled amino acids would be helpful to clear up the situation. Characteristic differences in the amino acid pattern have also been detected in a melanoma-producing lethal mutant 1 ( I ) 7 in Drosophila by Lewis (1954). Cystine is not present in the tumour larvae which have, however, a higher content of alanine, arginine, glycine, methionine, serine and tyrosine than the normal genotype. The absence of cystine is considered to be linked with certain metabolic disturbance during tumour formation, possibly an accelerated utilization and uptake of this amino acid into tissue proteins. In summary, there is now an adequate amount of information about the lethal mutants just quoted. We have seen that in all cases the developmental failure has been traced back to a deficient or abnormal pattern of

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protein synthesis which is in turn related to a certain block in the formation of RNA or DNA. This certainly does not mean that we have found the primary cause of the lethal effect. Indeed, there is reason to believe that many of the physiological and biochemical properties observed are of only secondary nature. The complex pattern of damage and the close interrelationships of the different organ-systems in the mutants make the analysis on the biochemical basis of lethality extremely complicated. We still know much too little about the primary action of genes. B. SYNTHESIS OF ENZYMES A N D OTHER SPECIFIC PROTEINS

There is only a limited information on the synthesis of enzymes in lethal mutants. As already mentioned (p. 107), in the /me homozygous larvae it seems that the activity of the proteolytic enzyme in the midgut is inhibited. In the Notch deficient embryo of Drosophila an abnormally high content of cholinesterase has been observed, which, according to Poulson and Boell (1946), is related to the hypertrophy of the nervous system. In the Minute lethal M (2) l2 it is found that its cytochrome oxidase activity is twice as high as that in the wild type (Farnsworth, 1956). From their analysis of chromosome interchange stocks of two different strains Ward and Bird (1963) concluded that cytochrome oxidase is utilized as the terminal electron carrier in Drosophila, and that the enzyme activity is under the control of some factors in the second chromosome. Interesting work has been done with isozymes, that is, enzymes which catalyse the same reaction but have different physical, chemical and kinetic properties (cf. Markert and Mraller, 1959; Kaplan et ul., 1960). This is now known to be a widespread phenomenon as multiple enzyme forms have been found for malate dehydrogenase, isocitrate dehydrogenase, ribonuclease, peroxidase, hexokinase, esterase and alkaline phosphatase (Wroblewski, 1961).Extensive studies of the lactate dehydrogenase by Markert and his colleagues led to the conclusion that the isozymes are composed of tetramers of two kinds of polypeptide sub-units, each of which is probably under the control of a separate gene (cf. Appella and Markert, 1961; Markert, 1962; Markert and Ursprung, 1962). In recent years multiple forms of enzymes have been detected in various insects, the synthesis of which has been shown to be under genic control. According to Kikkawa (1960, 1963) in Drosophila melanogaster seven amylase bands can be found on a zymogram using agar-gel electrophoresis; the formation of these isoenzymes are controlled by allelic

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genes located on the second chromosome. In D. virilis there are only two amylase bands which are likewise under the control of two allelic genes on the fifth chromosome. Crosses between two strains containing only one enzyme form gave rise to offspring which exhibited both bands of the parent type, indicating that each gene produced its own amylase product. Wright (1963) detected two forms of esterase, Esterase 6Fand Esterase 6', occurring in natural populations of D. rnelanogaster. The two enzyme forms, which differ in electrophoretic mobility, in heat stability and in sensitivity to an organophosphate inhibitor, are under the control of a pair of codominant alleles located at 36.8* on the third chromosome. Similar to the finding of Kikkawa (1963) for amylase, zymograms of the heterozygotes Est 6"/Est 6' showed both esterase bands of the parent pattern. Exactly the same results have been obtained for D. simulans; and evidence has been brought forward to show that the two pairs of alleles in both species are homologous (Wright and Macintyre, 1963). Isoenzymes of the esterase group have been further reported to occur in Hyalophora and Samia by Laufer (1960a, 1961),in Musca by Oppenoorth, and van Asperen (1960), Velthuis and van Asperen (1963) and Menzel et al. (1963), as well as in Bombyx by Yoshitake (1963). The existence of multiple esterase patterns is apparently a general phenomenon in many other insects (cf. Afsharpour and O'Brien, 1963). For the synthesis of the enzyme xanthine dehydrogenase in Drosophila it is known that at least two genes are involved : the rosy gene (ry)on the third chromosome and the maroon-like gene (ma-l)on the X chromosome (Forrest et al., 1956; Hadorn, 1958; Glassman and Mitchell, 1959). Using starch-gel electrophoresis it has been shown that two or possibly three isozymes of this enzyme can be detected in the wild type (Smith et al., 1963). It is postulated that two different types of polypeptides may possibly be produced by two genes and these peptide chains assemble in groups of three or four to form the isoenzymes. Multiple forms of xanthine dehydrogenase have also been reported by Keller et al. (1963). The genetic control of the synthesis of another enzyme system, tryptophan pyrrolase, has been extensively investigated. According to Baglioni (1959, 1960) the activity of tryptophan pyrrolase in the allelic mutants v1 and v36f of D. melanogaster is considerably lower than that in the wild type. No enzyme activity has been detected in the mutant f18b of D. virilis, but that in the mutant cdseems to be normal. Analyses of the suppressors of v su2-s, su3-s and suS2-s showed that they increase the tryptophan pyrrolase activity of v1 but not that of v36f, suggesting that there is a difference in the alternation of the enzyme system between these two mutants. The

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suppressors act probably by changing the cell conditions and thus modifying the gene products, that is, enzyme proteins. The detailed studies of Lewis and Lewis (1961, 1963) on the dopa oxidase of Drosophilu have revealed the following facts. The crude extracts are complex, including the proenzyme associated with other protein factors known as activators or inactivators as well as inorganic inhibitors. The genetic control of the enzyme system is multigenic. At least one gene located at 52.4 of the second chromosome is involved in determining the qualitative nature, that is, the structure of the enzyme molecule. Several other genes located on the second and third chromosomes are found to influencethe level of enzyme activity. Genetic analyses of the interaction of these factors suggest the existence of structural and control genes, analogous to that disclosed in bacteria (cf. Lewis, 1962). Finally, in addition to enzymes, cases are also known which illustrate that the synthesis of tissue proteins is likewise under genic control. We have briefly mentioned the work of Pantelouris and Duke (1963) on the blood proteins in Drosophilu on page 86:Among the seven protein fractions separated on starch-gel electrophoresis three have been followed for their inheritance. It is found that the synthesis of each fraction is under the control of a separate gene and its presence depends on the dominant allele, whereas its absence is the recessive dependent. One of the three genes is sex-linked and the other two are autosomal and show linkage. An interesting example concerning the synthesis of cytoplasmic proteins in Bombyx has been reported by Tsujita and Sakurai (1963a, b). They isolated chromogranules from the hypodermal cells of the normal larvae and the mutants lemon (/em)and dilute-lemon (d-lem).In the wild type the granules contain only isoxanthopterin, whereas those in the mutants contain in addition yellow pigments resulting from the block of transforming dihydropterin to tetrahydropterin by the /em gene. The protein components of the granules from the three genotypes differ in their electrophoretic mobility. Furthermore, using the fingerprint technique two supernumerary peptides have been found in the trypsin hydrolysate of the chromoprotein from the /em mutant and only one in that from the d-/em mutant. There is thus a distinct difference in the peptide composition. These results are interpreted as showing that under the action of the +d-lem gene a protein is produced which combines with the yellow pigments, whereas in the presence of the d-/em gene an altered protein is formed which fails to retain yellow pigments. Consequently the latter become lost from the epidermal cells and the larvae have thus a dilute yellow appearance.

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We have already seen that in the lethal mutants alterations of the genetic constitution have a profound effect on the synthesis of nucleic acids and proteins. Cases have also been quoted suggesting that the formation of specific enzymes as well as blood and tissue proteins can be traced to the control of definite genes. There is no doubt that such major morphogenetic events like growth and differentiation are directly related to the synthetic activity of the genic system. Indeed hypotheses have been advanced suggestingthat an increasing number of genes come into action as development proceeds (Hadorn, 1948), or that certain genes are activated whilst others are inactivated in different parts of the developing organism (Waddington, 1956). Unfortunately, no direct evidence for changes in gene activity during development is available except that from studies on “puffs” in the salivary gland chromosomes during the development of the dipteran insects (Beermann, 1952, 1956; Breuer and Pavan, 1955). In the opinion of Beermann (1963) puffing patterns, which are organ- and phase-specific, represent the pattern of gene activity during development. It is now known that the puffing regions are the sites of active synthesis of messenger RNA (Pelling, 1959; Sirlin, 1962; Edstrom and Beermann, 1962), which conveys the genetic information to the specificproteins formed. Recently Laufer et al. (1964) demonstrated that puffing of the chromosomal loci, enzyme activity of the salivary gland, and development itself could be inhibited by actinomycin D which is known to be a specificinhibitor of mRNA synthesis. As the hormones play a deciding role in insect development, it appears reasonable to assume that these substances might be of primary importance in the regulation of the genic activity. Clever and Karlson (1960) have, in fact, reported that characteristic puffing patterns could be induced by injecting purified ecdysone into the larvae of Chironornus tentans, especially in the chromosomal sections I-18-C and IV-2-B (cf. Clever 1961a, b, 1963b). Since these puffs appear within 30-60 min after injection and their sizes depend on the hormone concentration, it seems that ecdysone acts on the chromosomal loci in a direct way. A similar conclusion has been reached by Becker (1962) from studies on the puffing patterns of Drosophila larvae. From his study on the tanning of puparium at the time of metamorphosis Karlson (1959,1963) is also in favour of the opinion that ecdysone probably acts at the gene level in activating or synthesizing the necessary enzymes. Clever (l962,1963a, b) has gone even so far to suggest that the action of ecdysone could be similar to that of an effector in the regulator-operator system proposed by Jacob and Monod

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(1961) from their studies on bacteria. It is of course still open to question whether the regulating model of the bacterial system could be applied to higher organisms. Moreover, according to Kroeger (1963a, b), who worked on Chironomus thummi, similar puffing patterns can be produced by many other substances like Zn++,Cd++ and narcotics. It seems that such hormone imitators do not act directly on the genetic loci, but indirectly through a control system which regulates the Na+/K+ balance in
VII. CONCLUSIONS From the present review it is evident that profound changes in protein metabolism take place at various periods during insect development. The major morphogenetic events, such as determination of the organ anlagen during embryogenesis, growth and moulting during larval life, as well as transformation from larva to pupa at the time of metamorphosis, are accompanied by characteristic variations in the patterns of amino acids, peptides and proteins. Physiological and biochemical studies on insect development have now provided us with a reasonable picture of the causal relationships of hormone action, protein synthesis, growth and differentiation. We have to admit, however, that we are still a long way from understanding the precise mechanism involved and the present state of our knowledge still does not permit us to offer a biochemical hypothesis of insect inorphogenesis. The genetic aspect of protein metabolism has been reviewed to a very limited extent by citing a few selected examples. There is no doubt that equally important contributions have been made by many other workers. We have only to recall investigations on the biochemical properties of various mutants in Drosophila, Bombyx, Ephestia and Habrobracon. That all these studies have yielded valuable information on the specific functions of genes in the ontogenetic process needs no further comments. It is hoped that the present review will stimulate further contributions to our understanding of the complex problem of insect development. ACKNOWLEDGEMENTS

I am greatly indebted to Drs L. Levenbook, M. L. Dinamarca, T. Indira and D. S. Po-Chedley for permission to quote from their un-

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published work and to reproduce figures from it. The original work reported in this article was supported by grants from the Swiss National Science Foundation and the Karl Hescheler-Stiftung.

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Addenda Page 73: The unknown fractions numbered in Figs. 7 and 11 have been identified as containing the following components: 1, phosphoserine; 2, tyrosine phosphate; 3(4), glycerophosphoethanolamine; 6, phosphoethanolamine; 8, taurine; 13, 7aminobutyric acid; 14, ornithine; 15, ethanolamine. Evidence has been obtained indicating the presence of peptides in the remaining peaks (cf. Chen and Hanimann, 1965). Recently similar findings have been reported by Levenbook et ul. (1965) for the blowfly Phormia regina. Page 72: The intermediary metabolism of amino acids in insects has been extensively reviewed in two recent contributions by Gilmour (1965) and Chefurka (1965). Page 82: In the larval haemolymph of the blowfly Phormiu reginu a minimum of nineteen peptides have been found (Levenbook, in preparation). Nine of these have been isolated and analysed; unlike the acidic insect peptides described heretofore, these are all basic di- or tripeptides containing either lysine for the most part, or histidine. Their concentration changes remarkably during larval growth, being maximal at the early third instar, and virtually non-existent in the late larva shortly before pupation. Page 88: By following the incorporation of 3zP-labelledpyrophosphate into ATP, Howells and Birt (1964) analysed the rate of amino acid activation at various developmental stages of the blowfly Luciliu cuprina. They showed that the total rate of activation was high during larval life, dropped to a minimum at the mid-pupal stage, and increased again until the time of emergence. Such variations reflect apparently the synthesis of proteins during both larval growth and adult differentiation. Page 92: Studies by Dinamarca and Levenbook (in preparation) on the kinetics of alanine and lysine metabolism in the mature larva, during metamorphosis and in adult flies of Phormiu regina demonstrated that alanine was more rapidly and extensively oxidized than lysine throughout development. The rate of lysine turnover was relatively constant and low at all stages, whereas alanine turnover was maximal at the white pupa stage, declined rapidly to a minimum halfway along metamorphosis, and increased somewhat in the adult. Based on rigorous kinetic criteria they found that the incorporation rates into protein were identical for both amino acids, and followed a markedly U-shaped curve during metamorphosis. However, lysine was incorporated more rapidly than alanine in the adult fly.

REFERENCES Chefurka, W. (1 965). Intermediary metabolism of nitrogenous and lipid compounds in insects. In “The Physiology of Insecta” (M.Rockstein, ed.), Vol. 2, pp. 669-768. Academic Press, New York.

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Gilmour, D. (1965). “The Metabolism of Insects”. Oliver and Boyd, Edinburgh and London. Howells, A. J. and Birt, L. M. (1964). Amino acid-dependent pyrophosphate exchange during the life cycle of the blowfly Lucilia cuprina. Comp. Biochem. Physiol. 11, 61-83. Levenbook, L., Dinamarca, M. L. and Lucas, F. (1965). Unusual free amino acids and determination of ninhydrin-positive substances during morphogenesis of the blowfly Phormia regina. Fed. Proc. 24, 471.

Metabolic Control Mechanisms in Insects W. R. HARVEY and J. A. HASKELL Zoology Department, University of Massachusetts, Amherst, Massachusetts, U.S.A. 1. Introduction. . 11. Phosphate Acceptor and Substrate Control of Respiration in Isolated Mitochondria . A. History, Definitions and Terms . B. Sarcosomes and their Isolation. . C. Energy Requirements of Insect Flight . D. Regulation of Energy Trapping Pathways in Flight Muscle E. Oxidative Phosphorylation and Respiratory Control F. Endogenous Uncoupling or Controlling Agents . G. a-Glycerophosphate and Respiratory Control during Flight. H. Biological Factors Influencing Energetics of Mitochondria . 111. Regulation of Enzyme Levels . A. Constant Proportion Enzymes . B. Oxidative Enzymes in Silkworm Development . C. Enzymes of Tanning Reactions . IV. Control at the Chromosome Level A. Biochemistry of Insect Hormones . B. Biochemistry of Giant Chromosomes . C. Chromosomal Puffing and its Relation to Development D. Chromosomal Puffingand its Relation to Synthetic Processes in the cell E. Ecdysone and DNA Synthesis . F. Chromosomal Puffs and Transport . . V. Ionic Control of Protein Synthesis and Development A. Ion Control during Development . B. Protein Synthesis Regulated by Ion Concentrations . References

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133 134 134 138 143 144 149 152 154 155 156 157 161 165 166 166 171 I 74

181 182 182 183

183 186 190

I. INTRODUCTION The flight of a bumble bee or the metamorphosis of an organism challenge the creativity of the human mind. Rimsky-Korsakov responded by writing an instrumental piece; Ovid by writing a collection of tales; and a host of scientists by writing papers. The analysis of these papers reveals a symmetry of design amidst diversity of function. An example is the hierarchy of metabolic control. 133

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The rates of enzyme-catalyzed reactions are controlled by the concentration of substrates, ions, and other co-factors, as well as by enzyme concentrations. For example, the rate of oxidative phosphorylation of flying insects presumably depends on the concentration of phosphate acceptor as deduced from studies on mitochondria isolated from flight muscle (Van den Bergh and Slater, 1962; Lehninger, 1964). Enzyme concentrations are presumably controlled by repression of genetic material in micro-organisms(Jacob and Monod, 1961a, b) and in mammalian tissues (e.g. Potter and Ono, 1961).Less wellunderstood is the control of enzyme levels in insect tissues ( e g Bucher and Pette, 1963;Shappirio and Harvey, 1965). Chromosomal RNA synthesis in the large polytene chromosomes of flies may be controlled by hormones (Clever, 1963a), by undetermined mechanisms involving other small molecules or ions (Kroeger, 1963a-c), or by other mechanisms perhaps involvingproteins. Finally, the compartmentation of ions and molecules within the hemolymph, within cells, and within intracellular compartments such as mitochrondria, is controlled by limiting membranes (Shaw and Stobbart, 1963; Stobbart and Shaw, 1964; Narahashi, 1963). We shall attempt to describe experiments and theoretical concepts relating to each level of control mentioned above. These four levels were chosen arbitrarily because their analyses have been more rewarding and are more intelligible than those in other areas. 1 1 . P H O S P H A TACCEPTOR E AND SUBSTRAT CONTROL E OF RESPIRATION I N ISOLATED MITOCHONDRIA A. H I S T O R Y , D E F I N I T I O N S A N D TERMS

I . Factors regulating respiration Harden and Young (1906) showed that the concentration of inorganic phosphate could regulate the intensity of anaerobic fermentation in cellfree yeast extracts. Engelhardt (1932) and Kalckar (1937) showed that respiration similarly is dependent on the concentration of inorganic phosphate (Pi). That an energy requiringprocess, such as muscle activity, might regulate respiration was suggested by Meyerhof. The accumulation of lactic acid in muscle was accompanied by a corresponding rise in respiration, whereas the gradual oxidative elimination of lactic acid was accompanied by a corresponding gradual lowering of respiration. However, lactic acid itself could not be responsible for the respiratory enhancement because isolated muscle poisoned with iodoacetate exhibited elevated respiration during work despite the block in lacticacid production

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(Meyerhof and Boyland, 1931). Engelhardt (1932) suggested that the factor enhancing respiration during work might be the breakdown products of muscle phosphagen, creatine, or Pi, and in 1939 Belitzer stimulated respiration of a muscle pulp by adding creatine, thereby demonstrating phosphate acceptor control for the first time. Recognizing the importance of adenosine triphosphate in endergonic cellular processes, Lardy and Wellman (1952) proposed that dephosphorylation of adenosine triphosphate (ATP) might regulate the catabolic processes of cells by a direct action on energy-trapping reactions. Either a drop in ATP or an increase in either Pi or phosphate acceptor could serve this role. Regulation by inorganic phosphate level was invoked by Lynen (1941), Johnson (1941) and others to account for the inhibition of fermentation by respiration in micro-organisms (Pasteur effect). Under aerobic conditions respiration is postulated to compete successfully with fermentation for Pi (Lynen et af., 1959). Chance (1959) emphasized the disadvantages of Pi for regulating respiration in isolated mitochondria on the grounds that the affinity of mitochondria1 systems for adenosine diphosphate (ADP) is some fifty-six times higher than that for Pi. More recently, Chance and Hollunger (1961) and Klingenberg and Schollmeyer (1960, 1961a, b) showed that oxidative phosphorylation can be reversed and that in tightly coupled mitochondria (see Section 11, A, 4) respiration can be controlled by the concentration of ATP. Klingenberg and Schollmeyer conclude that the respiratory rate of isolated mitochondria is controlled not by just ADP alone but by the ratio [ATPI/ [ADP] [Pi], the so-called phosphate potential. Alternatively, Chance and Sacktor (1958) suggested that in the case of the transition from rest to flight in insect muscle a-glycerophosphate (aGP) may play the critical regulatory role (see Section 11, G). Finally, “there is evidence that inorganic ion transport may be energized directly by an intermediate of oxidative phosphorylation rather than by ATP” (Lehninger et al., 1963). In this case the possibility arises that respiration might be controlled by the concentration of the transported ion. 2. Chance and Williams steady states To simplify and standardize the analysis of phosphate acceptor as a metabolic regulator Chance and Williams (1955c, 1956) defined five steady state conditions for the study of isolated mitochondria. These steady state conditions are summarized in Table I to facilitate later discussions. The values for percent reduction of pyridine nucleotides and electron transport components are derived from spectrophotometric E*

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studies on rat liver mitochondria with /3-hydroxybutyrate as substrate (Chance and Williams, 1955b). TABLEI Steady states in isolated mitochondria (Modified from Chance and Williams, 1955c.) Characteristics

Oxygen ADP level Substrate level

State 1 vaguely starved

State 2 starved

Excess Low Lowendogenous Slow

Excess High Approaching 0 Slow Substrate

Respiratory Phosphate Oxygen acceptor chain

Respiration rate Rate-limiting Phosphate component acceptor Percent reduction of: ca 90 NAD Flavoprotein 21 Cytochromes 0-17

0 0

State 3 active

State 4 acceptorless

State 5 anaerobic

Excess High High

Excess Low High

None High High

Fast

Slow

53 20 4-1 6

99 40

0-35

100 150 100

State 1 (vaguely starved) is characterized by the omission of ADP and substrate. The state has limited usefulness because endogenous concentrations of ADP and substrate are difficult to fix. State 2 (starved) endogenous substrate is depleted by adding large amounts of ADP. The slow respiration is thereby substrate limited and the cytochromes, flavoprotein, and NAD are consequently completely oxidized. Such mitochondria are fragile, presumably due to the lack of ATP. State 3 (active) with an excess of oxygen, ADP, and substrate, the oxidation is rapid and the reduction of electron transport components moderate, these components supposedly being rate-limiting. The prospect that permeability of mitochondria to substrate or ADP may be rate-limiting must be considered. State 4 (acceptor-less)high substrate levels are introduced but the slow respiration is rendered ADP-limited by the exhaustion of all available ADP by oxidative phosphorylation. The cytochromes are more oxidized than in the active state. State 5 (anaerobic) characterized by excess ADP and substrate but oxygen is excluded thereby limiting the respiration and placing the respiratory chain components in a completely reduced state.

3. Respiratory control coeBcients Qland Q2 If respiratory control is defined as the effect of ADP on respiration of isolated mitochondria, then the transitions from the active state (3) to

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the acceptor-less state (4) are the ones in which respiratory control can be evaluated. It is more difficult to decide on the conditions best suited for the quantitative determination of control ratios because the ATPase activity of isolated mitochondria depends on the source of mitochondria and the method of isolation. The degree of control has been described empirically by either of two control ratios which were designated Q , and Q, by Klingenberg and Bucher (1959).

Q,,ofmitochondria after the addition ofADP = "=

Q,, of mitochondria before addition of ADP Q,, of mitochondria with a small amount of ADP Q,, of mitochondria after the ADP is phosphorylated

Because fresh, intact mitochondria from mammalian sources appear to have little ATPase activity (Low, 1959; Siekevitz et al., 1958), Chance and Baltscheffsky (1958) emphasized the advantages of the second measure (Q2) as a test for integrity of mitochondria1 preparations. However, in systems with appreciable ATPase activity the final value of Q, would be determined not only by the rate of coupling of respiration to oxidative phosphorylation but also by changing ADP levels due to endogenous ATPase and myokinase activity (Gregg et al., 1960; Hatefi et ul., 1961). In the absence of a separate test for ATPase and myokinase activities, the first coefficient (Q,) would seem to be a more reliable index of respiratory control. 4. Postulated intermediates in oxidative phosphorylation

From a consideration of the effects on the electron transport system of ADP and uncoupling agents such as dinitrophenol, Chance et al. (1955) have proposed a mechanism for oxidative phosphorylation. Similar schemes have been proposed by many other workers (cf. Lehninger et al., 1959). The scheme of Chance and Williams (1956) is arbitrarily reproduced and defined here as a basis for discussions in later sections. A,e,+B,x+I + A,x+Br,--I (1) Br,-I+X + Bred+X-1 (2) X-I+Pi + X-Pi+I (3) X-Pi+ADP + ATP+X (4) A and B represent two adjacent components of the electron transport chain, I and X are hypothetical intermediates, and indicates an energy rich bond (dG of hydrolysis M 12 kcal/mole, Klingenberg,

-

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1961). X-I is hypothesized to be hydrolyzed in the presence of dinitrophenol to form free X and free I. DNP

X-I+H,O

s X+I

The scheme accounts for respiratory control in “tightly coupled” systems, i.e. those in which respiration is stimulated by ADP with the retention of high P/O ratios as follows: “Accumulation of X-I limits the rate of reaction 1 by lowering the concentration of free I, which is an obligatory component of reaction 1. Addition of Pi liberates I from X-I. Similarly,removal of ADP will lead to an accumulation of X-Pi, leaving no free X available for reaction 2. In ‘loosely coupled’ systems the respiration rate is unaffected by the concentration of ADP, but the P/O ratio may well be dependent on it.” The scheme for oxidative phosphorylation may well account for loose coupling. “If we assume that both pathways for ‘unloading’ X-I are available-reaction 3+4 and reaction 5-then reactions 3+4 are favoured in loose coupled preparations and reaction 5 is favoured in uncoupled preparations” (Van den Bergh, personal communication). In uncoupled preparations X-I is broken down by reaction 5 thereby speeding up respiration (reactions 1 and 2) while preventing phosphorylation (reactions 3 and 4). The P/O ratio is the ratio of ions of inorganic phosphate disappearing from the incubation medium to the atoms of oxygen consumed. The disappearance of phosphate from the medium is presumably in exact stoichiometric equivalence to the amount of inorganic phosphate undergoing anhydride formation with ADP (often incorrectly described as esterification). B. S A R C O S O M E S A N D T H E I R I S O L A T I O N

The contemporary view that mitochondria are the structural sites of oxidative phosphorylation and its control stems from Michaelis’s oxidation-reduction studies on mitochondria in living cells and Warburg’s finding that the capacity of cells to consume oxygen resides in the particulate elements. Claude (1 946) fractionated cells and described the size, structure, osmotic properties, and oxidative abilities of isolated mitochondria. The most recent comprehensive treatment of the subject is Lehninger’s monograph (1964) on the integration of structure, function, and control of biological oxidation by mitochondria. Flight muscle mitochondria were first described by Aubert in 1853 and were called sarcosomes by Retzius in 1890. Keilin (1925) considered the flight muscle of the honeybee to be the object of choice for the spectral examination of the rediscovered cytochromes. The functional significance

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of sarcosomes was not revealed until Watanabe and Williams (195 1,1953) demonstrated that they are giant mitochondria measuring as much as 4 p in diameter. Sarcosomes in dipterous insects account for about onethird of the dry weight of the muscle (Levenbook and Williams, 1956). Watanabe and Williams (1953) used phase contrast optics to confirm the observations of Kolliker (1888) that living sarcosomes possessa selectively permeable, osmotically active membrane. They showed, moreover, that serum albumin is able to preserve mitochondria1 structure during isolation. These properties of mitochondria have subsequently engaged investigators of both insect and mammalian mitochondria (see Lehninger, 1964). 1. Methodsfor isolating sarcosomes The intensity of oxidation, the P/O ratio, and the extent of respiratory control in isolated mitochondria are dependent on not only the substrate added to the incubation medium but also the method of isolating the sarcosomes and the composition of the isolation medium. A typical procedure, closely resembling the one developed by Watanabe and Williams (1951), was used by Van den Bergh and Slater (1962). “The flies were immobilized by cooling below 4” for 30 min and placed on an ice-cooled glass plate in the cold room. The heads and abdomens were removed and 200 thoraces were gently pounded in a mortar with 5 ml of isolation medium. The resulting brei was filtered by suction through two layers of muslin, previously saturated with isolation medium, into a tube immersed in an ice bath. The filtrate was centrifuged at 4” for 3 min at 150 g and the supernatant again centrifuged for 8 min at 3 000 g. The sedimented sarcosome pellet was rinsed with two portions of isolation medium and then suspended in 4 ml of isolation medium with the help of a plastic pestle fitting closely into the centrifuge tube. This procedure gave sarcosomal suspensions containing 6-8 mg of protein/ml.” The most important deviation from this procedure is the use of carbon dioxide for anesthesia, sometimes at room temperature, initiated by Watanabe and Williams (1951) and continued by Sacktor (1953). The possible consequences of narcotization and its attendant anoxia are discussed in Section 11, E.

2. Isolation media The isolation medium used by Van den Bergh and Slater (1962) was simply 250 mM sucrose and 1 mM ethylenediaminetetra-acetic acid (EDTA) at pH 7.4. Variations such as the substitution of KCI for the

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sucrose (Sacktor, 1953) and the addition of a substrate mixture (Gregg et al., 1959) are discussed in the appropriate sections. a. EDTA. Lehninger (1949) and Slater and Cleland (1952) found that calcium ions inhibit oxidative phosphorylation in isolated mitochondria and Slater (1952) was able to stabilize oxidative phosphorylation in mammalian heart muscle sarcosomes by adding a calcium chelating agent, Versene (EDTA). Klingenberg and Bucher (1959) found that sarcosomes from Locusta migratoria exhibit respiratory control only when the isolation medium contained at least 1 mM EDTA. b. KC1 v. sucrose. Van den Bergh and Slater (1962) replaced the sucrose in their isolation medium with 154 mM KC1. The P/O ratio and respiratory control index were virtually the same following isolation with either sucrose or KC1. Sarcosomes isolated in the KCl medium had slightly higher respiratory activity with aGP and much higher activity with pyruvate+malate as substrate whereas the low activity of succinate and NADH were depressed even further following isolation in the KClEDTA medium (however, see Section 11, B, 3 , b). c. Tris and phosphate*. Using their substrate-fortified medium discussed below, Gregg et al. (1960) found that including either 0.05 M Tris or 0.1 M phosphate buffer in the isolation medium gave greatly increased respiratory activity and even greater enhancement of phosphate “esterified” with correspondingly high P/O ratios. Moreover, sarcosomes isolated without Tris or phosphate required a co-factor solution containing NAD, TPP, NADP, CoA, and cytochrome c, whereas sarcosomes isolated with Tris or phosphate had no such requirement. d. Serum albumin. Watanabe and Williams (1953) prevented the “fuzzy degeneration” characteristic of sarcosomes isolated in 320 mM sucrose by using instead 2.5% bovine plasma albumin or human serum albumin in 160 mM potassium phosphate buffer. Sacktor (1954) subsequently reported that serum albumin was needed to demonstrate oxidative phosphorylationininsects. Gregg et al. (1960), Van den Bergh and Slater (1962), Cochran (1963) and others have been able to obtain rapid oxidation with high P/O ratios in sarcosomes exhibiting respiratory control without adding serum albumin. Cochran (1963)found that serum albumin had a stimulatory effect on oxidative phosphorylation of cockroach sarcosomes whether it was used only in the isolation medium or only in the incubation medium. As discussed in Section 11, F, the role of the

* The following abbreviationsare used in this section. Tris, Tris (hydroxymethyl)aminoethane; NAD, nicotinamide adenine dinucleotide; TPP, thiamine pyrophosphate; NADP, nicotinamide adenine dinucleotide phosphate; CoA, coenzyme A. NADH is the reduced form of NAD.

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serum albumin is to bind and thereby inactivate free fatty acids released by lipolytic enzymes from mitochondria and other cell fractions during the isolation procedure (Wojtczak and Wojtczak, 1959, 1960; Wojtczak, 1963). e. Substrates. Presumably to protect mitochondria from degenerative phenomena during isolation Gregg et al. (1959) used an isolation medium consisting of 250 mM sucrose, 50 mM Tris, 100 mM phosphate, 3 mM EDTA, 3 mM MgC12,and 6 mM each of citrate, succinate, and pyruvate at pH 7.4. Judging from the high P/O ratio and respiratory control with pyruvate+ fumarate as substrates, their medium is about as successful as the simpler procedure (Van den Bergh and Slater, 1962) in which substrate is generated in the soluble part of the thoracic extract. 3 . Damage to mitochondria during isolation a. Loss of NAD. Following the report by Chance and Sacktor (1958) that sarcosomes from the housefly could oxidize aGP at least ten times faster than succinate and about fifty times faster than pyruvate or isocitrate, Bucher et al. (1959) suggested that the mitochondria isolated by Chance and Sacktor may have lost pyridine nucleotide which would diminish oxidation of Krebs cycle intermediates but would have little or no effect on aGP oxidation. Indeed, Chance and Sacktor (cited in Chance and Williams, 1955b) found no spectroscopic evidence of pyridine nucleotides in fly sarcosomes. However, isolated mitochondria generally contain substantial amounts of pyridine nucleotide. Thus, Birt (1961) isolated sarcosomes from Musca and Lucilia and found about 2.53.0 pm-moles of total coenzymes/mgof protein, almost all in the oxidized form. About 95% of the total was NAD+. Slightly higher amounts were found in locust mitochondria (Klingenberg and Bucher, 1959) and in the blowfly (Price and Lewis, 1959; see also Klingenberg and Bucher, 1960). b. Criteria for healthy sarcosomes. Isolated mitochondria gradually lose their characteristic biochemical properties. Endogenous substrates are important in preserving mitochondrial integrity (Klingenberg and Bucher, 1959) but can largely be replaced by exogenous substrates (Gregg et al., 1959, 1960). Klingenberg and Bucher (1959) listed several criteria of mitochondrial integrity in order of decreasing sensitivity. The best preserved mitochondria still retain endogenous substrate. Gradually respiratory control with aGP is lost, NAD-reduction with aGP, respiratory control with pyruvate-t malate, and loss of NAD following in order. Finally, the loss of cytochrome c is a sign of considerable mitochondrial damage. More important physiologically are such properties indicating integrity of isolated sarcosomes as-high

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enough Q,, with phosphate acceptor present to account for in vivo respiration, and P/O ratios near the theoretical value. The difficulty of applying criteria for integrity of mitochondria in practice is illustrated by Van den Bergh and Slater (1962). In spite of the high respiration rates, P/O ratios, and respiratory control, their preparations in KC1-EDTA medium may have lost cytochrome c. When this enzyme was added to the incubation medium, the Q,, of aGP oxidation was increased to well over 1 000 whereas the Q,, with pyruvate+malate was unaffected by the cytochrome c (Van den Bergh, 1962). In the presence of added cytochrome c, saline isolated sarcosomes oxidized aGP about twice as rapidly as pyruvate+malate (see Section 11, G). Although the P/O ratios reported by Gregg et al. (1960) and Van den Bergh and Slater (1962) and others are high, they do not approximate the expected theoretical ratios as closely as do the preparations from mammalian and avian heart muscle (Hatefi et al., 1961). Possibly the use of Hagihara's bacterial proteinase (1960) for isolating the heart mitochondria accounts for the success of Hatefi et al. The present consensus seems to be that a completely satisfactory isolation procedure for insect mitochondria remains to be developed. 4. Composition of incubation media

A typical medium for studies of oxidation, P/O ratio, and respiratory control in isolated sarcosomes (Van den Bergh and Slater, 1962) contained 15 mM KC1,2 mM EDTA, 5 mM MgCl,, 50 mM Tris buffer, 30 mM potassium phosphate buffer (pH 7.5), 1 mM ADP, 30 mM glucose, 150180 Cori units of hexokinase, and substrate in concentrations ranging from 10 to 100 mM. The pH of the reaction mixture was brought to 7.5 by the addition of HCl, the temperature was 25"C, the reaction volume was 1 ml, and the reaction period 30 min. For determinations of respiratory control the ADP, glucose, and hexokinase were replaced by 0.1 mM ATP. When oxygen uptake was constant for 6 min, 3-6 pmoles of ADP were added from the side arm, and the respiratory control taken as the respiratory rate before and after the addition of ADP Magnesium is characteristically included in the incubation medium. Chance and Williams (1956) and Klingenberg and Bucher (1960) review its effects on respiratory control. In general Mg++ antagonizes the uncoupling and disruptive effects of Ca++.In liver mitochondria, magnesium stabilizes or reconstitutes respiratory control of loosely coupled preparations. However, magnesium may abolish respiratory control in mitochondria from heart, kidney, and brain, apparently because of the high magnesium-activated ATPase activity of these mitochondria. A mag-

(el).

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nesium requirement has been demonstrated on digitonin-treated particles from mammalian mitochondria. 5. Measurement of oxygen uptake Most studies on insect mitochondria have employed manometric measurements of oxygen uptake. Chance and Sacktor (1958; see also Chance and Williams, 1955a) used a vibrating platinum electrode which has the advantage that changes in oxygen uptake can be made almost immediately after the addition of substrate, phosphate acceptor, or inhibitors. Moreover, by use in conjunction with the double-beam spectrophotometer or the split-beam spectrophotometer (Chance and Williams, 1956), simultaneous kinetic and spectral observations are possible. The main disadvantage is that the vibrating electrode is susceptible to contamination. C. E N E R G Y R E Q U I R E M E N T S O F I N S E C T F L I G H T

The metabolic cost of insect flight has been reviewed extensively by Chadwick (1953) and Weis-Fogh (1961). Its relationship to the biochemistry of flight muscle is discussed by Sacktor (1965). Recently WeisFogh (1964) has emphasized the importance of lift in determining metabolic rate in full-grown, sexually immature desert locusts (Schistocerca gregaria Forskil). The metabolic rate (P)was determined from separate measurements of the net rate of heat production in the working thorax, the heat dissipated by evaporation of water in the respiring thorax, and the net amount of aerodynamic power transferred from the animal to the air in forms other than heat. The heat production due to metabolism outside the wing muscles amounted to only 1-27, of the metabolic rate and could be ignored. The metabolic rate expressed in kcal/kg/h was 41 at a lift of 50% of the animal’s weight, 65 at 100yo,110 at 170%, and (by extrapolation) 127 at 200%. The rate at zero aerodynamic work was estimated by extrapolation to be 12. The metabolic rate therefore increases about 1 1-fold from zero lift to a lift twice the weight of the insect. By comparison, the oxygen consumption at rest was 10 pl/g/min and increased 15-50-fold to 166500 during tethered flight. The average oxygen uptake was 250 pl/g/min or 70 kcal/kg/h (Krogh and Weis-Fogh, 1951) which agrees well with the 65 kcal/kg/h mentioned above as the metabolic rate necessary for level continuous flight (i.e. 100yolift). For a P/O ratio of 3 and a AF’ for the breakdown of ATP to ADPfPi of 7 kcal/mole, about 150 p moles ATP/g/min are required at 100yolift. The close agreement between metabolic rate estimated from aerodynamic parameters and from oxygen uptake suggests that energy

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production and expenditure are closely regulated during flight. Sacktor (1 965) reviews cases where the oxygen consumption of insects increased

over 100-fold during the transition from rest to flight, but we could find no record of the 1 000-fold increase suggested by Lehninger (1964). Both the high rate of respiration in vivo and the large transition from rest to flight pose fundamental questions regarding metabolism of sarcosoma1preparations. (1) Does the oxygen uptake of isolated mitochondria occur fast enough to account for the rate in vivo during flight? (2) Can one induce in isolated mitochondria an increase in oxidation rate large enough to account for the increase during flight? We will return to these two fundamental questions after a brief consideration of the oxidative pathways in isolated sarcosomes. D. R E G U L A T I O N O F E N E R G Y T R A P P I N G P A T H W A Y S I N F L I G H T MUSCLE

Glycolysis in insect tissues utilizes the well-known Embden-Meyerhof pathway. The details of the pathway in insect tissues are reviewed comprehensively by Gilmour (1961) aud Sacktor (1965). For our present purpose it will be sufficient to describe briefly the steps crucial to an understanding of its metabolic regulation. The cleavage products of fructose- 176-diphosphate,namely glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone-phosphate(DHAP) can be isomerized and subsequently handled in either of two ways. The DHAP can be reduced to aGP with the concomitant oxidation of NADH +H+ to NAD+ (von Euler-Baranowski enzyme) or the GAP can be oxidized in five enzymatic steps to pyruvic acid with the concomitant reduction of NAD+ to NADH+H+. The NADH produced must be reoxidized or the pyruvate pathway will be closed. However, both mammalian and insect mitochondrial membranes are virtually impermeable to pyridine nucleotide. The impasse is overcome by the first alternative because mitochondria are freely permeable to both aGP and DHAP. aGP diffuses into the mitochondria where an active oxidase (MeyerhofGreen enzyme) reconverts it to DHAP which can diffuse back into the extramitochondrial compartment. Its subsequent reduction to aGP thereby reoxidizes the extramitochondrial NADH. This so-called aglycerophosphate cycle transfers reducing equivalents from extramitochondrial to mitochondrial compartments thereby assuring a continuous supply of extramitochondrial NAD+ as long as there are trace amounts of DHAP, the NAD-linked extramitochondrial dehydrogenase, and the mitochondrial oxidase present (for references, see Klingenberg and Bucher, 1960; Sacktor, 1965).

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The anaerobic production of aGP by yeast was demonstrated by Neuberg. Embden and Meyerhof first described an active, extramitochondrial, NAD-linked aGP dehydrogenase in muscle (Embden and Deuticke, 1934a,b; Meyerhof and McEachern, 1933).The dehydrogenase was reported in insect muscle by Kubista (1958) and by Bucher and associates (Zebe et al., 1957; Bucher and Klingenberg, 1958). The mitochondrial oxidase, a flavoprotein, bypasses the first phosphorylating step in the respiratory chain, with a resultant theoretical P/O ratio of but two (for references, see Sacktor, 1965). 1. Triose phosphate oxidation

Insect tissues can be divided roughly into two groups. One group, characterized by grasshopper jumping muscle, contains low aGP dehydrogenase activity and high lactic dehydrogenase activity. The second group, characterized by flight muscle, contains the reverse (i.e. low lactic dehydrogenase and high aGP dehydrogenase activity). Under anaerobic conditions tissues of the jumping muscle group produce lactic acid whereas tissues of the flight muscle group produce roughly equimolar amounts of aGP and pyruvate. In air both jumping muscle and flight muscle cells at rest remain aerobic. Pyruvic acid is oxidized via acetyl CoA and the mitochondria1 tricarboxylic acid (TCA) cycle to CO, and water with its reduced mitochondrial pyridine nucleotide and direct dehydrogenase reducing equivalents serving as electron donors for electron transport chain phosphorylations. However, during intense activity the differing dehydrogenase activities together with a difference on tracheation lead to the following results. The tracheation of the jumping muscle is relatively poor and the explosive contractions leave the cells essentially anaerobic. Extramitochondrial NADH is reoxidized by the NAD-linked lactic dehydrogenase with a resultant production of lactic acid. On the other hand, the tracheation of flight muscle is so extensive (tracheolar endings are in virtual contact with sarcosomes) that the tissue remains completely aerobic. Extramitochondrial NADH is reoxidized by the uGP pathway with a concomitant enhancement of mitochondrial aGP oxidation. 2. Relative roles of aGP andpyruvate oxidation duringfright In early amperometric studies of oxidative phosphorylation in sarcosomes isolated from housefly flight muscle, Chance and Sacktor (1958) calculated that the rate of aGP oxidation exceeded that of succinate by about ten times and that of other TCA cycle intermediates and of pyruvate by as much as fifty times. Moreover, the absolute Q,, for uGP

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oxidation was about 215 p1 O,/mg sarcosomal protein/h and could be increased to 580 by the addition of an uncoupling agent such as dibromophenol. This latter value was large enough to account for the in vivo value for Q,, of about 500 calculated by Chance and Sacktor (1958). On the basis of this and other similar evidence compiled in his 1965 review, Sacktor has continued to emphasize the role of aGP as the principal substrate for the activation of the respiratory chain upon initiation of flight, and to minimize the role of the TCA cycle, relegating its principal function to prolonged flight and to periods of rest. Sacktor’s position has led to a very lively controversy. The Chance-Sacktor calculation has been questioned by Van den Bergh (personal communication) who calculated from the Chance and Sacktor data a minimum in vivo Q,, of 1 000. This value is considerably greater than the in vitro value of 580 mentioned above. Moreover, whereas housefly. sarcosomes isolated in sucroseEDTA by Van den Bergh and Slater (1962) oxidized pyruvate+malate at only 36% of the rate that they oxidized aGP, the sarcosomes isolated in KCI-EDTA medium oxidized pyruvate+malate about 86% as fast as aGP, and the absolute rate (647) was great enough to account for oxygen uptake in the living housefly in flight. There is disagreement about the relative importance of succinate and aGP oxidation rates in locusts and cockroaches, Zebe et al. (1959) and Fukami (1961) finding relative rates of 71 and 92%, respectively, and Hess and Pearse (1961) and Cochran and King (1960) finding rates of 8 and 26%, respectively. Stegwee and van Kammen-Wertheim (1962) found aGP oxidation at a more rapid rate than succinate in flies, locusts, and cockroaches but about equal rates in Lep t inotarsa. Much of the controversy over the relative roles of aGP and pyruvate oxidation seem to have stemmed from the poor quality of sarcosomes originally isolated by Chance and Sacktor. Recent work in Sacktor’s laboratory is aimed at resolving the conflict. During the first 10-15 sec of flight in Musca the breakdown products of glycogen and trehalose are stoichiometrically accounted for by the accumulation of glucose and pyruvate, and of alanine which is derived from pyruvate. The glucose is derived mainly from trehalose. After the first minute of flight pyruvate oxidation becomes much more significant (Sacktor, 1964). The low early rate of pyruvate oxidation is attributed to the absence of oxaloacetate in mitochondria during rest. Upon initiation of flight oxaloacetate is presumably produced (from proline) and TCA cycle oxidation of pyruvate ensues (Sacktor, personal communication). However, Van den Bergh (1964) was able to measure pyruvate oxidation at maximal rates by isolated housefly mitochondria with no demonstrable lag period even

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when respiration was measured with the oxygen polarograph. He also demonstrated that freshly isolated mitochondria do contain appreciable amounts of Krebs cycle intermediates. Possibly some step used by Sacktor in the preparation of his mitochondria leads to the loss of Krebs cycle intermediates. 3. aGPpathway in vivo Although the oxidation of aGP or pyruvate can be studied independently of one another in vitro, exclusive use of one or the other pathway in vivo would be limited to the oxidation of any aGP or pyruvate which might have accumulated from non-glycolytic reactions. The in vivo glycolysis of compounds such as trehalose, glucose, or glycogen involves an obligatory link between the two pathways. The products of anaerobic glycolysis in insects are equimolar amounts of aGP and pyruvate (KubiSta, 1958; Chefurka, 1958).No aGPcan be formed without NADH and NADH is formed during the production of pyruvate. For this reason no aGP can be formed without an equivalent amount of pyruvate being formed at the same time. Similarly, because the DHAP+GP reaction is the only available way to reoxidize NADH produced during the formation of pyruvate, no pyruvate can be formed without the production of an equivalent amount of aGP. Therefore the exclusive use of one or the other pathway is impossible. The two pathways are obligatorily linked in anaerobic glycolysis. Turning to aerobic glycolysis the pathways are similarly linked. The aGP cycle is not the pathway for the oxidation of any substrate. It is only a mechanism for the reoxidation of cytoplasmic NADH. In aerobic glycolysis, as in all types of glycolysis, one glucose unit is converted to two molecules of pyruvate. During this reaction two molecules of NAD are reduced. They are reoxidized by two full turns of the aGP cycle. Because only catalytic amounts of DHAP or aGP are required to keep this cycle going, glycogen is converted quantitatively to pyruvate. Aerobic breakdown of carbohydrate is therefore described by the following reactions.

Glucose+2 NAD++2 H3P0,+2 ADP-+2 Pyruvate+ 2 NADH+ 2 ATP+2 H+ 2NADH+2H++2DHAP+2NAD++2 aGP 2 aGP+0,+4 H3P0,+4 ADP+2 DHAP+4 ATP 2 Pyruvatef5 02+30 H3P0,+30 ADP+6 c0,+30 ATP (Sum) GlucoseS6 0,+36 H,PO4+36ADP-t6CO,+36 ATP

(6) (7) (8) (9)

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a. Oxygen and energy. By comparison of reactions 8 and 9 it is clear that pyruvate is the more important respiratory substrate for flight muscle metabolism :five times on an oxygen basis, 7Q times on an energy basis, and 19 times on a P/O ratio basis. But it is also clear that both oxidation reactions must occur simultaneously and can never be separated save for the oxidation of aGP or pyruvate which may have accumulated in differing amounts during some prior state due, for example, to non-glycolytic utilization or production of pyruvate. b. Respiratory quotient. Whereas the r.q. for glycogen utilization via the pyruvate-TCA pathway is theoretically one (6 C02/60,),there is no cycle. Therefore CO, produced during a transient initial use of the CLGP the theoretical initial r.q. would be zero. In long duration experiments Chadwick (1947) reported an average r.q. for Drosophila of 1.23 before flight, of 1 during flight (when the oxygen uptake was 14 times and the CO, production 11 times the previous resting rate) and frequently less than 1 after flight. Zebe (1954) reported average r.q. values during rest of 0.65,0.69 and 0.59 and during flight of 0-73,0-75and 0-74for Odonestis pruni, Antheraea pernyi, and Vanessa io (all Lepidopterans) respectively. By comparison Polacek and Kubigta (1960) have reported r.q. values of about 0.6 during the early stages of cockroach flight. This finding is in accord with Sacktor's view that CLGPoxidation predominates during the first minute of flight. 4. Compartmentation of oxidative reactions The impermeability of the mitochondrial membrane to NAD was first

reported by Lehninger (1951). Sacktor (1960,1961)reviews evidence for a mitochondrial barrier to reduced coenzyme in insects. To test the prospect that insect mitochondria are impermeable to NADH and other substrates Van den Bergh and Slater (1962) treated a sarcosome preparation' from houseflies in a sonic disintegrator for 2.5 min. The oxidation of pyruvate+malate was reduced slightly, and that of aGP reduced to about three-quarters of its original value. By contrast, the oxidation of NADH increased 12-fold, that of succinate 7-fold and that of other TCA intermediates to a lesser degree. The absolute value of DPNH oxidation (381) and succinate oxidation (257) now approached the estimated in vivo value for housefly flight (500). These results seem to indicate that the mitochondrial membrane is relatively impermeable not only to NADH but also to most of the TCA cycle intermediates. The aGP cycle has been found in normal mammalian tissues such as liver, brain, and muscle. The cycle fails to operate in most malignant tumor cells due to the extremely low activity or absence of aGP dehydro-

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genase (Boxer and Devlin, 1961). In this case the reoxidation of extramitochondria1 NADH by pyruvate with the production of lactate is a necessity because otherwise glycolysis and ATP formation would cease (Warburg, 1956). E. O X I D A T I V E P H O S P H O R Y L A T I O N A N D RESPIRATORY CONTROL

Sacktor (1954) found no effect of phosphate and Sacktor and Cochran (1958) found no effect of ADP in concentrations from 0 to 25 pmoles/2-5 ml on the oxygen uptake of mitochondria isolated from Musca domestica. The sarcosomes were described as lacking respiratory control. However, phosphate uptake increased with increasing ADP concentrations, reaching a maximum at about 10 pmoles ADP/2.5 ml. Therefore the P/O ratio increased upon addition of ADP. Chance and Sacktor (1958), using a vibrating platinum electrode, which enabled them to measure initial reaction rates, substantially confirmed the lack of respiratory stimulation with ADP or dinitrophenol. Cochran and King (1960) in manometric studies on sarcosomes isolated from Periplaneta americana similarly found no respiratory control but contrary to the work on Musca found phosphate uptake (and therefore P/O ratios) independent of ADP concentration as well, obtaining consistent P/O ratios of about 1.6 provided at least 0.5 pmoles/2.5 ml of ADP was present. Subsequently, Sacktor and Packer (1959) found respiratory control in minces of housefly muscle, reporting 2-5-fold stimulations of aGP oxidation. In Sacktor’s original description of his sarcosome isolation procedure he says, “A known number of flies, usually 200, were anesthetized with carbon dioxide and the thoraces obtained by removing heads and abdomens. The thoraces, as acquired, were placed in approximately 1.5 ml of ice-cold 0.2 M sucrose.” Except for increasing the sucrose to 0.25 M (Sacktor, 1954), he evidently has used the same procedure in subsequent experiments. Assuming that all the flies are exposed to pure CO, during the thorax isolation period, the average fly must be narcotized for as much as 5 min at room temperature. The effect of CO, anesthesia of flies at room temperature on the subsequent behaviour of isolated sarcosomes appears not to have been studied. However, Harvey and Nedergaard (unpublished results) were unable to detect electrical potentials in Hyalophora cecropia midguts isolated after as little as 5 min of carbon dioxide anesthesia at room temperature whereas, if the larvae were chilled and subsequently placed under frozen COz, which chills as it anesthetizes, potentials ranging from 70 to 140mV were obtained (Harvey and Nedergaard, 1964).

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Haskell et al. (1965) have demonstrated a loss of the transepithelial potential in isolated midguts exposed either to 25% CO, or anoxia. The effect is reversible for 1 or 2 min and irreversible soon thereafter. Whether or not the CO, anesthesia has anything to do with the lack of respiratory control in Sacktor's early experiments is not known but it may be significant that in the experiments to be described below the insects were in most cases immobilized by chilling at from 1O to 4°C. Recently Sacktor (personal communication) reports that when he eliminates the CO, anesthesia by inactivating the flies in the cold room, values for parameters of oxidative phosphorylation and respiratory control are the same as those found with CO, anesthesia. Gonda et al. (1957) immobilized mosquitoes by cooling and isolated mitochondria in sucrose-EDTA media. From their data using oxoglutarate or fumarate as substrate one can calculate respiratory control ratios (Q,) of 2.7 and 2.3 respectively. The control ratio with oxoglutarate was increased to 5.7 in mitochondria which were allowed to stand for 15 min at 30°C. Homogenization of the original preparation at 0" in a Virtis homogenizer did not change the ratios appreciably. Klingenberg and Bucher (1959; see also Bucher et al., 1959) isolated mitochondria from Locusta migratoria flight muscle. With aGP, pyruvate malate, and endogenous substrate they demonstrated respiratory control (Q,=2.5, 4.0 and 22 respectively and Qz=2-5, 3.5 and 5.0 respectively). P/O ratios of 2.6 with pyruvate+ malate were reported. Their mitochondria were isolated on 0.30 M sucrose, 2 mM EDTA and 10 mM triethanolamine buffer (TRAP) at pH 7-2 with all operations conducted at 0°C. Gregg et al. (1959, 1960) isolated sarcosomes from 4-day-old houseflies using a special substrate-fortified medium, and measured oxygen uptake manometrically. With pyruvate the oxidation rate was relatively high (100) and the P/O ratio 2.65. Acceptor ratios determined by comparing flasks with ATP to those without (all flasks contained hexokinase, glucose, and MgCl,) were about 12 with pyruvate+fumarate and 1-73 with aGP. With aGP as substrate added serum albumin led to higher control ratios, oxidation rates, and P/O ratios although substantial oxidative phosphorylation and highacceptor ratios were obtained without serum albumin. The protein had little beneficial effect when pyruvate was the substrate. Birt (1 96 1) studying Lucilia cuprina and Musca domestica reported respiratory control (Q,) with glutamate, pyruvatefmalate, and aGP but not with succinate. The respiratory stimulation was not markedly affected by the concentration of mitochondria in the flask but tended to

+

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decreasewith age of flies, althoughwith glutamateand pyruvatet-malate respiratory control existed in 25-day-old flies. The experiments stressed how greatly detection of respiratory control by ADP depends on the conditions used in the experiment, with particular emphasis on substrate used and the age of the flies. Van den Bergh and Slater (1960) reported that the supernatant fraction from the thoracic muscles of Musca stimulated sarcosomal respiration 10-20-fold with the stimulated rates approaching those calculated for in vivo flight respiration. Subsequently, Van den Bergh and Slater (1962) traced the supernatant effect to the continuous formation of aGP and pyruvate in this fraction. Using aGP and pyruvate as substrates Q,,s of 673 and 245 (plOl/mgprotein/h), P/O ratios of 1-38and 2.23, and respiratory control ratios (QJ of 1-67and 8.76, respectively, were reported. By contrast TCA cycle intermediates and NADH gave little respiration, low P/O ratios, and no respiratory control. As prepared, the supernatant fraction contained but little aGP or pyruvate but when ADP, ATP, Pi, and Mg++ were added these substrates were rapidly formed by glycolysis in the supernatant fraction, thereby accounting for the supernatant effect. The low activity of sarcosomes towards Krebs cycle intermediates and NADH was shown to be due to the impermeability of the mitochondria1 membrane to these substrates. After sonication the oxidation of succinate, NADH, a-oxoglutarate, isocitrate, glutamate, and malate increased by 7-, 12-, 5-, 14-, 2- and 3-fold respectively. Mitochondria isolated in KC1EDTA instead of their standard sucrose-EDTA medium gave higher oxidation of aGP and pyruvate+malate, the enhanced values being high enough to account for calculated in vivo rates. Respiratory control by the phosphate concentration was also demonstrated. The control ratios with pyruvatefmalate in the absence of added phosphate ranged from 17 to 48 with an average of 24 in seven experiments. Neither age of flies (cf. Lewis and Slater, 1954), nor the presence of serum albumin (cf. Sacktor, 1954), nor exogenous oxidizable substrate (cf. Gregg et a/., 1959, 1960), nor unusual buffering procedures (cf. Klingenberg and Bucher, 1959) in the isolation medium were found to be necessary for controlled oxidative phosphorylation. In short Van den Bergh and Slater showed that housefly sarcosomes are very similar to mammalian mitochondria in P/O ratios and respiratory control. What is significantly different is the unusually high respiration rate with aGP and pyruvate as substrate and the relative impermeability to all other substrates with resultant low oxidation rates for these substrates (see, however, Section 11, B, 3).

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F. ENDOGENOUS U N C O U P L I N G O R CONTROLLING AGENTS

Living cells contain uncoupling agents which are released during the preparation of cell fractions. Such agents have been isolated from insect homogenates, from nuclear and microsomal fractions of mammalian tissues, and directly from mammalian mitochondria. Whether or not the agents are present in intact cells and what role they may play in metabolic regulation is not entirely clear. Nuclear and microsomal fractions from mammalian sources themselves respire feebly, but greatly enhance the oxygen consumption by mitochondrial systems containing no phosphate acceptor (Schneider and Potter, 1949; Potter et al., 1951). Pressman and Lardy (1952) obtained from such microsomes a heat stable, acetone soluble substance which enhances respiration and uncouples phosphorylation by mitochondria. The active substances were identified as a mixture of long chain fatty acids with unsaturated fatty acids especially active (Pressman and Lardy, 1955, 1956).

Wojtczak and Wojtczak (1959) found that the ATP-Pi32exchange reaction (see Section 11, A, 4, equations 3 and 4) in isolated mitochondria from larvae of Galleria mellonellu L. was exhibited only in the presence of serum albumin. They could wash out the serum albumin and still observe the exchange reaction. The washing fluid was heated for 5 min at 100°C to coagulate the albumin. After centrifugation the activity was found in the coagulated protein. Both ethanol and benzene extracts of the precipitated albumin were able to prevent the exchange reaction, and the protein after extraction was inactive. Subsequently, Wojtczak (1963) identified the uncoupling substance to be a mixture of non-esterified long chain fatty acids obtaining as much as 500 pmoles per g of mitochondrial protein. Examination of the supernatant (from the precipitated protein or from the original mitochondrial preparation) revealed “highly active lipolytic enzymes which can hydrolyze egg and mitochondrial phosphatides, triglycerides,Tweens and endogenous lipids”. Wojtczak postulates that the uncoupling non-esterified fatty acids found in insect mitochondria are formed by these lipolytic processes and absorbed by mitochondria during homogenization and isolation. In contrast to the larval homogenates, the supernatant from rat liver and heart homogenates contain much less fatty acid. A knowledge of the uncoupling activity of fatty acids from insect and mammalian tissues is of supreme importance in preparing phosphorylating mitochondrial systems but the prospect that they serve a role in metabolic control in vivo appears dim. Perhaps more physiologically relevant are uncoupling agents from

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mitochondria themselves. Well-washed mitochondria which are allowed to stand for a few hours at room temperature exhibit uncoupling of oxidative phosphorylation. Pullman and Racker (1956) demonstrated and Polis and Shmukler (1957) isolated from such aged mitochondria a substance which uncouples oxidative phosphorylation in fresh mitochondria. They named the substance “mitochrome” because it has the spectrum of a haem compound. Apparently, however, the uncoupling activity of the preparation does not stem from its haemoprotein, mitochrome, but from an associated lipid component which is extractable with isooctane (Hiilsmann et al., 1958). The isooctane-extracts uncouple phosphorylation, stimulate ATPase activity, and inhibit the ATP-PiS2 exchange reaction. Hulsmann et al. suggest that lipid normally attached to cytochrome in its native state is released during a postulated transformation of cytochrome to mitochrome. The uncoupling activity of both the unextracted mitochrome preparation of Polis and Shmukler and that of the lipid extracted by isooctane is counteracted by the addition of albumin which apparently binds the substance. The prospect that these or other uncoupling substances may play a role in regulating respiration in vivo is discussed by Slater and Hulsmann (1959) who elaborate on Lardy’s suggestion (1952) that an endogenous uncoupling agent might function in metabolic control. However, like the fatty acids from other cell fractions discussed above, these mitochondria1 lipids may not exist as such in the living cell. More promising is the report of Remmert and Lehninger (1959) concerning the isolation of a substance which they suggest might be a more native form of mitochrome or its isooctane soluble activity but probably is a mixture of factors affecting in vivo energy coupling. They disrupted fresh rat liver mitochondria with sonic oscillation and extracted the debris with dilute sucrose-KC1 solutions. Following centrifugation at 115 000g and dialysis, a protein was precipitated from the supernatant by adjusting the pH to 5.5. This substance, which they termed “R factor”, is similar in some of its actions to the agents discussed above and to 2,4dinitrophenol (DNP) but different in ways which appear significant for metabolicregulation in living cells. When DNP at a concentration high enough to release (stimulate) respiration maximally in the absence of ADP is added to a test system containing ADP, virtually complete uncoupling of phosphorylation results (P/O ratios approach zero). By contrast R factor at a concentration just sufficient to maximally release respiration in the absence of ADP has little effect on the P/O ratio when ADP is present. R factor stimulates respiration to a limiting level which is exactly the maximum stimulation obtainable with ADP. Thus the R factor has more accurately

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been called a “respiration releasing” factor (Lehninger et al., 1959). This substance accounts for the so-called “loose coupling” of Hoch and Lipmann (1954) in which mitochondria from thyroxinized animals show elevated respiration and no stimulation of respiration with ADP but have normal P/O ratios. The attractiveness of Lehninger’s R factor is that it is an endogenous agent from fresh sonicated mitochondria which is capable of speeding up respiration with a concomitant increase in phosphorylation. This is the sort of agent which might account for metabolic control in vivo. Numerous other factors with uncoupling or regulatory effects on mitochondria have subsequently been isolated (Lehninger, 1964). G. a-GLYCEROPHOSPHATE AND RESPIRATORY CONTROL D U R I N G FLIGHT

In Section 11, D, we reviewed some evidence that isolated insect sarcosomes oxidize aGP more rapidly than other substrates, allegedly differing in this respect from mammalian mitochondria, that aGPDH in insect flight muscle is far more active than lactate dehydrogenase (LDH) and that the products of anaerobic glycolysis in many insect tissues, including flight muscle, are equimolar amounts of aGP and pyruvate. Chance and Sacktor (1958) proposed an hypothesis of metabolic regulation by the direct activation of aGP oljidation. The hypothetical activation of aGP in vivo was attributed to inorganic cations postulated to be released during muscle activity. According to this hypothesis aGP-OX is in an inhibited statein resting muscle due to the chelation of divalent cation by an hypothetical endogenous chelating agent thought to function in vivo as Versene did in the in vitro study of Estabrook and Sacktor (1958). Maintenance activity and the low level of respiration is underwritten by the TCA cycle. During flight, the aGP inhibition is reversed and the high respiratory rate characteristic of aGP oxidation is released. The reversal of such an inhibited state is attributed to either the accumulation of substrate or the release of divalent cation by nervous stimulation of the muscle. The hypothesis has been modified substantially (Sacktor, 1965). Birt (1961) observed in mitochondria from Lucilia and Musca that the rate of aGP oxidation decreased with time and eventually became very low. He attributed the decrease to the approaching of an equilibrium between aGP and DHAP in which the ratio of aGP/DHAP is about 3. Cochran (1963) obtained similar results with cockroach sarcosomes. However, Van den Bergh and Slater (1962) found that aGP oxidation continues until all the substrate is used up. Possibly the results of Birt and those of Cochran are attributable to the use of impure substrates.

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H. B I O L O G I C A L FACTORS I N F L U E N C I N G ENERGETICS OF M I T O C H O N D R I A

There is evidence that developmental state and nutritional condition of the donor insect influence the bio-energetics of isolated sarcosomes. A priori one might expect that oxidative phenomena in a primitive insect such as a cockroach might differ from those in an advanced insect such as a housefly. In fact, however, the oxidative metabolism of flight muscle from fly and cockroach is remarkably similar but both differ markedly from leg (walking or jumping) muscle of either fly or cockroach. Among developmental states one must consider progressive changes during tissue differentiation, for example to flight muscle or limb muscle and, in general, the developmental changes characteristic of molting, metamorphosis, and post-emergent maturation in adults (Rockstein, 1957). At any stage there might be mitochondria1 changes associated with feeding or nonfeeding and with developmental arrest (diapause). The age of post-emergent adult flies has been considered a significant factor in the control of oxidative metabolism but at present the data are conflicting. Levenbook and Williams (1956) showed that both the wing beat frequency and the sarcosomal cytochrome c content of blowflies increased in post-emergent adults. They pointed out that the increased cytochrome c level might either be a genetically programmed characteristic of adult maturation or a physiological adaptation to the enhanced wing beat frequency. Lewis and Slater (1954) found lower P/O ratios with sarcosomes isolated from blowflies younger than 10 days. Klingenberg and Bucher (1959) could find respiratory control only in sarcosomes isolated from locusts in the 10th to 15th day of adult life. By contrast Van den Bergh and Slater (1962) found no systematic difference in respiratory rates, P/O ratios, and respiratory control indicesin housefly mitochondria from 24 h to 27 days after adult emergence. However, flies 24 h after emergence had but 40% of the sarcosomal protein normally found in preparations from flies at least 9 days old. The only systematic study of oxidative phosphorylation during metamorphosis is that of Michejda (1963) on mitochondria isolated from the thoracic muscles of Hyalophora cecropia from about the 15th day of adult development, when the flight muscle starts to differentiate, to about the 8th day of post-emergent adult life. At the early stages the oxidation of aGP, pyruvate+fumarate, and pyruvate+malate was twice as high as that of succinate. Between the 19th and 21st days, succinate oxidation increased concomitantly with a drastic decrease in the oxidation of

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pyruvate malate. The inversion from aGP and NAD-linked substrates to succinate oxidation occurred on the 20th day. Phosphorylation was equally efficient at all developmental stages with respectable P/O ratios of 1.3 (succinate), 1.5 (a-keto-glutarate), 2.3 (glutamate), 2.4 (glutama te fumarate), 2.6 (glutamate mala te), and 2.5 (glutamate+pyruvate+fumarate). The net result of the changes in oxidation with the constant phosphorylation was that the total “energic potency” (Q,, x P/O) decreased during the late stages of metamorphosis. Respiratory control with ADP was low on the 15th day, increased to a maximum of 4-5 with glutamate on about day 21-22 (the day the adult moth emerges) and decreased thereafter so that there was virtually no controlin mitochondria from older adults. Michejda’s sarcosomes showed some symptoms of physiological damage in that respiration was stimulated by exogenous cytochrome c and NAD. He reported less stimulation when he prepared sarcosomes with serum albumin than with the Gregg et al. (1959) medium. Michejda anesthetized his insects in CO, and sometimes performed the dissections at room temperature (see Section 11, B). A possible complication in such studies is the difficulty of isolating muscle free of contamination by other tissues, particularly at earlier stages of differentiation. Assurance on this point is necessary in order to be sure that the changes described are not of histological origin rather than reflecting a change in the properties of the mitochondria. Michejda discussed differences in control bioenergetics between adult Cecropia moths which do not feed and are sluggishfliers and adult blowflies which feed and are active fliers. In blowfly sarcosomes we have seen that aGP is most rapidly oxidized in short-term amperometric experiments (Chance and Sacktor, 1958), whereas in long-term manometric experiments either pyruvate was most actively oxidized (Gregg et al., 1960) or aGP and pyruvate were both more rapidly oxidized than other substrates (Van den Bergh and Slater, 1962). In both types of experiment the oxidation of succinate was slow. By contrast sarcosomes from adult Cecropia moths, both in short-term (amperometric) and long-term (manometric) experiments, oxidized succinate much more rapidly than pyruvate. In Cecropia sarcosomes endogenous phosphorylation is high (P/O = 24-3.0) throughout imaginal life (Michejda, 1963).

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111. REGULATION OF E N Z Y M E LEVELS

In 1958 Dixon and Webb listed 660 enzymes known to occur in living cells. Although no single cell is able to make all 660 enzymes many cells

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have the genetic capability to make a few hundred enzymes. On the other hand as much as 5-8 yoof the protein synthesized in microbial cells may be a single enzyme (Davis, 1961). Similarly, patches of pure resilin are produced by wing-hinge tissue in Schistocerca (Weis-Fogh, 1960). Clearly, in both bacteria and metazoan animals the level of both enzyme and structural protein must be closely regulated. The concept of genetic regulation of protein synthesis was clearly formulated by McClintock (1956) from studies on maize genetics and brought to 2 high level of sophistication by Jacob and Monod and others from studies on bacteriophage and on E. coli. Two classes of enzymeswere generally recognized, constitutive enzymes, present in substantial amounts in cells at all times, and repressible enzymes whose concentrations are closely regulated by the presence of small molecules (e.g. Jacob and Monod, 1961a, b). The distinction appears to be reaching the limit of its usefulness (Pardee and Beckwith, 1963). Hand-in-hand with the mid-twentieth century triumphs in understanding the mechanism of protein synthesis has come an understanding of.the control mechanisms (e.g. Umbarger, 1963; Maas and McFall, 1964; Ames and Martin, 1964) in micro-organisms. Less rewarding have been attempts to reveal either the mechanism or the controlling factors in metazoan protein synthesis (Weber, 1964). A great many studies on insects have some bearing on the regulation of protein synthesis but most of them lack the freedom from conflicting interpretations or the adequacy of experimental design to allow definitive conclusions to be drawn. In this section we will select for discussion three potentially fruitful lines of evidence: the work of Biicher’s group on constant proportion enzymes, the work of Williams’ group on oxidative enzymes in silkworm development, and the work of Karlson’s group and Wolsky’s group on tyrosine metabolism. A. C O N S T A N T PROPORTION ENZYMES

Bucher and associates have described ratios of activities of enzymes which fall into two types. In one type there is a constant proportion between the activities of each member of the enzyme group even though the absolute activity level of the group as a whole may vary by orders of magnitude in different tissues or in the same tissue from different animals. Enzymes of the other type vary independently-perhaps according to the requirements of the animals-with no relationship to each other. Moreover, enzymes in one constant proportion group may vary in activity from those of a different constant proportion group (Biicher and Pette, 1963).

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1. The phosphotriose-glyceratephosphate(PTG) group

For these studies the soluble enzymes of 1 g of tissue were extracted and the activity ofthe enzymes ofthe Embden-Meyerhof chain determined under standard conditions. In effect this procedure represents a partial purification of the enzymes so that the values for activities are to some extent representative of the actual concentrations of the enzymes. At least one can conclude that the enzymes are extracted in constant proportions. Presumably these observations reflect enzyme concentrations in constant proportions in the insect tissues. The activities of the extracted enzymes were plotted on a logarithmic scale and patterns from different tissues and species were compared. The group of enzymes best worked out are those (listed below) which regulate the oxidation of phosphotriose in the Embden-Meyerhof sequence and are referred to as the PTG group. dihydroxyacetone phosphate f triosephosphate isomerase ( T I M ) 3-phosphoglycericaldehyde 1 glyceraldehyde-phosphatedehydrogenase (GAP D H ) 1-3-diphosphoglyceric acid 1 phosphoglycerate kinase (PGK) 3-phosphoglycericacid 1 phosphoglycerate mutase (GPM) 2-phosphoglycericacid 1 enolase ( E N ) 24eno1)phosphopyruvicacid The activity of each of the five PTG enzymes was compared in three types of muscle from the desert locust. The absolute activity of the five enzymes varied by orders of magnitude between flight muscle, extensor (jumping) muscle, and flexor (tonal) muscle. However, the proportions of activity of these five enzymes was the same within narrow limits in the three muscles. Thus the ratio of activity to that of GAPDH varied less than from 6 to 12 for TIM,O.6 to 1.2 for PGK and GPM, and0-15 to0.3 for EN. By contrast the ratio of activity of the NAD-linked (uGPDHbore a constant relationship neither to the PTG group just mentioned nor to the activity of LDH (Vogell et al., 1959; Pette et al., 1962). The activity of aGPDH was high in flight muscle and low in leg muscle and the activity of LDH was oppositely distributed as reported some time ago by Zebe (1956) and others (see reviews by Klingenberg and Bucher, 1960; Sacktor, 1961).

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The PTG enzymes represent the longest unbranched portion of the Embden-Meyerhof chain. Were proportions between the levels of these five enzymes to differ widely without compensating differences in concentrations of substrates or affinitites for substrates, intermediates would accumulate and the glycolytic chain would not operate efficiently. On the other hand a particularly high activity of aGPDH is thought to be essential for insect flight and a high activity of LDH is thought to be essential for the explosive contractions of jumping muscle (Vogell et al., 1959). Aside from the teleological attractiveness of constant proportion grouping, the linking of a series of enzymes in an activity group lessens the number of enzymes that must be considered for a comprehensive understanding of metabolic control. Constant proportions in PTG enzymes are by no means restricted to insect muscle tissues. A comparison was made between PTG enzymes of both muscles and other tissues from a variety of organisms (Biicher and Pette, 1963; Pette et al., 1962b). Insmoothand heart muscle from beef, in red, white, and mixed skeletal muscle from rabbit, in flight muscle from pigeon, in brain, liver, heart, and skeletal muscle from rat, and even in Baker’s yeast the ratio of activity to GAPDH varied within about the same limits as that in flight, jumping, and tonal muscle from locusts.

2. Mitochondria1 enzymes Not only cytoplasmic enzymes but mitochondria1 enzymes as well revealed constant proportion groups (Pette et al., 1962a). Soluble mitochondrial enzymes-malate dehydrogenase (MDH), glutamate dehydrogenase (GLUDH), NADP-specific isocitrate dehydrogenase (IDH), and glutamate-oxaloacetate transaminase (GOT)-were extracted from 1 g of tissue after the previous washing out of the extramitochondrial enzymes and assayed according to procedures worked out by Delbruk et al. (1959 a, b) and Vogell et al. (1959). The residue from this extraction was resuspended and the activity of succinate dehydrogenase (SDH) and aGP oxidase (aGP-OX) were measured photometrically. Finally, pyruvate oxidase was measured amperometrically as the respiratory activity of isolated mitochondria in the presence of pyruvate and malate. To express these results the absolute enzyme activities as well as the molar ratio of cytochrome a were compared to the cellular level of cytochrome c. The tissues studied were brain, muscle, liver, and heart from rat and locust flight muscle. For all these tissues the molar ratio of cytochrome c to cytochrome a showed a range of but 1-1-1.3. The ratio of activities of MDH, pyruvate oxidase (PYR-OX), and SDH showed but small variation when referred to the level of cytochrome c. The constancy of proportion of F

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these enzymes of pyruvic oxidation, the TCA cycle, and theelectron transport chain is emphasized by the large degree of variation in the range of their corresponding absolute activities. This result confirms the earlier reports of Chance and Hess (I 959) and Schollmeyer and Klingenberg (1962) that respiratory pigments are present in 1 :1 proportions in mitochondria isolated from various tissues. Although levels of cytochromes have never been quantitatively estimated in silkworm tissues, by contrast Shappirio and Williams (1957a, b) found different ratios of activities and different ratios of intensities of absorption bands of respiratory pigments in wing tissue and isolated mitochondria in larvae, dormant pupae, and developing adults of Hyalophora cecropia, and Shappirio and Harvey (1965) report different ratios of activities of enzymes in non-phosphorylating mitochondria isolated from wing epithelium of dormant and injured pupae of Hyalophora. 3 . Pyridine nucleotides Turning to pyridine nucleotides, Klingenberg and Pette (1962) found that mitochondrial NAD (sum of oxidized and reduced forms) bears a constant ratio to cytochrome c in brain, skeletal, and heart muscle, and liver from rat and in flight muscle from locusts. For all these tissues there were about 6-12 molecules of NAD to 1 molecule of cytochrome c. This result implies that all the enzymes with constant proportions to cytochrome c discussed in the preceding paragraph also have a constant proportion to NAD. By contrast NADP is claimed to have no constant relationship to cytochrome c or enzymes of theTCA cycle such as MDH. However, NADP exhibits constant proportions to GLUDH of rat liver, brain, and kidney and to IDH of rat skeletal and heart muscle and insect flight muscle. The prospect that constant NADP-GLUDH ratios reflect co-participation in reductive syntheses is suggested by Klingenberg and Pette. 4. Multilocated enzymes

In Section 11, D, we reviewed evidence that two enzymes, an extramitochondrial NAD-linked aGPDH and a mitochondrial aGP-OX, COoperate to form an aGP cycle. These two enzymes, linked across the mitochondrial membrane by their common pair of substrates, show a constant ratio of their activities in differenttypes of insect muscle (Vogell, et al., 1959) and the same ratio fits the pattern found in the rat brain but not that in rat liver, or cardiac or skeletal muscle. GOT and MDH have both intra- and extramitochondrial locations, The distribution of their activities between intra- and extramitochondrial compartments is not the

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same in different tissues. Thus, 30% and 20% respectively of the activity of these enzymes are mitochondrial in rat smooth muscle whereas 60% and 80% are mitochondria1 in insect flight muscle. However, the ratio of intra- to extramitochondrial activities of MDH is paralleled by the ratio of the corresponding activities of GOT in these tissues. Further discussion of correlations between activities of multilocated enzymes is beyond the scope of the present discussion (Pette and Luh, 1962). Pette et al. (1962b) suggest, particularly from their analysis of the PTG glycolytic enzymes, that the nearly constant proportions may result from co-ordination at the molecular level and point out the similarity to the co-ordinate pleiotropy at the level of enzyme patterns found in microorganisms. Experimental confirmation that constant proportion enzymes are a manifestation of an “operon” is awaited. In passing we note that the future of the operon concept itself is questionable unless convincing support of its generality is provided (cf. Stent, 1964). B. O X I D A T I V E E N Z Y M E S I N S I L K W O R M D E V E L O P M E N T

The oxidative metabolism of giant silkworms is high during actively developing stages and after injury but low in uninjured diapausing (dormant) pupae. Both activity and concentration of respiratory enzymes change. The topic has been reviewed several times, particularly from the viewpoint of hormonal regulation of development (Shappirio, 1960; Gilbert and Schneiderman, 1961; Harvey, 1962; Wyatt, 1963; Agrell, 1964; Gilbert, 1964; Wigglesworth, 1964; Shappirio and Harvey, 1965). We will take a fresh look from the point of view of metabolic control. Mainly from work of Williams’ group the problem has come to be analyzed in terms of contrasts betweendormant(diapausing) pupae, early developing adults, and injured pupae. Stated simply, are there systematic, reproducible changes in oxidative metabolism between these three stages? 1. Is there a changefrom aerobic to partially anaerobic existence ?

In recent reviews (e.g. Gilbert, 1964) the suggestion has been discussed that dormant pupaeare tosomeextent anaerobicand attention isdirected to the formation of glycerol (Wyatt and Meyer, 1959; Wilhelm et al., 1961) in dormant pupae of Hyalophoracecropia, Samia cynthia,and other saturniids. Moreover, polyol accumulation and its enzymatic basis have been described in diapausing eggs of Bombyx mori. Nevertheless, it is virtually certain that dormant pupae are in no respects anaerobic. Schneiderman and Williams (1954) showed that pupae are killed by complete anoxia with an LD5,,of about 3 days, and Harvey and Williams

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(1958a) showed similarly that the heartbeat required oxygen with a 50% diminution in rate in ca 2 h of anoxia. The dormant pupae could derive but little ATP from glycerol production because the energy yield is but 1 ATP per hexose from glycogen and none per hexose from glucose or trehalose. Moreover, as we shall discuss below, the dormant pupa contains all the components of the classical respiratory chain albeit with low activities, particularly of cytochrome c. Finally the ATP/ADP ratio is high (Carey and Wyatt, 1963). This fact coupled with the low capability of the pupa to make ATP glycolytically suggests that there is no defect in the cytochrome system, a claim supported by the large DNP effect (Harvey and Shappirio, cited in Kurland and Schneiderman, 1959) which is more likely a consequence of low ADP concentration. A more likely explanation for the. accumulation of polyols is that the respiration is low for lack of ADP (due to curtailed endergonic activity of dormancy). Substrates for ADP-controlled steps such as mitochondrial NADH, succinate, and aGP would be expected to accumulate. This would lead to the accumulation of extramitochondrial aGP which in the presence of phosphatase would break down to glycerol and Pi, accounting for the accumulation of polyol. If the mitochondria of pupal tissues are impermeable to pyridine nucleotide and the extramitochondrial oxidation of NADH requires the aGP cycle, the accumulation of extramitochondrial aGP would slow the conversion of DHAP to aGP with a corresponding slowing of the oxidation of extramitochondrial NADH. Pyruvate production would become limited by the lowered concentration of extramitochondrial NADH which would be determined both by the lowered mitochondrial production of ADP and the extramitochondrial hydrolysis of aGP. The situation could be viewed as being analogous to flight muscle at rest in which all the factors for glycerol production are present save the presence of phosphatase. In brief we would be confronted with the extramitochondrial consequences of an acceptor-less state (state 4) of mitochondria in vivo. Evidence required to substantiate the key assumptions of this hypothesis would be: (1) Is endergonic activity curtailed in the dormant pupa? (2) Is the concentration of ADP low or, better, is the ratio of [ATPI/ [ADP] [Pi] high? (3) Does aGP accumulate in pupal tissues? (4) Is there phosphatase present in the cytoplasm of pupal tissues? (5) Is there an accumulation of Pi stoichiometrically equivalent to the accumulation of glycerol?

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(6) Are pupal mitochondria impermeable to pyridine nucleotides? (7) Does the aGP cycle operate in pupal mitochondria? Recently Shappirio (personal communication) has found that there is no accumulation of reduced pyridine nucleotide during diapause. There is a considerable fall in level of pyridine nucleotide in the transition from active growth to diapause, but the relative proportion of NADH to NAD does not showa great change. The vast preponderance of pyridine nucleotide is in the oxidized form. These data argue against the proposal that the tissues of the diapausing pupae are anaerobic. 2. Do the absolute activities and concentrations of cytochrome enzymes change ? Shappirio (1960) has reviewed evidence that the level of cytochrome enzymes shows systematic shifts in epidermis, fat body, heart, midgut, gonads, and Malpighian tubules of larvae, diapausing pupae, early developing adults and late developing adults of Hyalophora cecropia. Using low temperature spectroscopy, Shappirio and Williams (1957a) found that larval tissues contain moderate to high concentrations of cytochromes byc, a+a,, and b,. In contrast the tissues from diapausing pupae show a spectrum in which components b and c are not detectable spectroscopically whereas cytochromes a+a3 and b, persist in low, although detectable, concentration. When growth and metamorphosis resume in response to ecdysone, cytochromes b and c again become detectable. All cytochrome components then undergo a progressive increase in concentration during adult development. Neither somatic muscles nor brain tissue (Shappirio, 1960) participate in these changes, retaining characteristic concentrations of cytochromes b and c as well as a+a3 throughout the pupal diapause. Several oxidative enzyme systems, including NADH-oxidase, NADH-cytochrome c reductase, succinatecytochrome c reductase, and cytochrome c oxidase show changes in activity associated with the onset and termination of pupal dormancy which Shappirio and Williams (1957b) interpret as being essentially parallel to the spectroscopic changesjust described. Absorption spectra and mitochondria1 enzyme activities in injured pupae are approximately the same as in early developing adults in that absorption bands attributable to cytochromes b and c and enzyme activities reappear following integumentary injury and to some extent subside after recovery from injury (Shappirio, 1960; Shappirio and Harvey, 1965).

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3. Do the ratios of cytochrome activity change ? From Shappirio and Williams’ spectroscopic data the ratios of cytochromes b : c : a+a3 appear not to be 1 : 1 : 1. Whether or not the ratios of these cytochromes remain the same while the activities of oxidative enzyme systems change during adult development is not clear. These results differ from the results of Chance and Hess (1 959), Schollmeyer and Klingenberg (1962), and Pette et at. (1962) discussed in Section 111, A. 4. Does the terminal oxidase change ? Despite the evidence for the presence of cytochromes a+a3 and substantial cytochrome c oxidase activity in mitochondria isolated from tissues of dormant pupae, it appeared for some time that cytochrome oxidase was not functioning in dormant pupae. The principal evidence was that the respiration (Schneiderman and Williams, 1954) and heartbeat (Harvey and Williams, 1958a, b) are remarkably resistant to such inhibitors of cytochrome oxidase activity as cyanide and carbon monoxide. Moreover, the lack of spectroscopic evidence for cytochrome c and the vanishingly low NADH-oxidase activity of mitochondria from dormant pupae (save in skeletal muscle) suggested that this classical electron donor for cytochrome oxidase might limit its function. However, Harvey and Williams (1958b) showed that at low oxygen pressures the heartbeat could be inhibited by carbon monoxide in the dark with reversal in the light by wavelengths corresponding to the absorption maxima of cytochrome oxidase. Similarly, Kurland and Schneiderman (1 959) were able to inhibit the respiration of pupae in the dark at low oxygen pressures using carbon monoxide. These findings are in accord with theoretical interpretations first formulated by Warburg (1927) that inhibition of respiration with carbon monoxide can be expected in yeast only when the cells are saturated with substrate (electrons). In modern times this would be equivalent to saying that such inhibition can be expected only if the oxidase is saturated with electrons. This analysis tacitly assumes that there is only one terminal oxidase exhibiting light reversible carbon monoxide inhibition, an assumption which must now be re-evaluated in view of the report by Bonner (1964) and Ikuma et al. (1964) of a second oxygen and CO-binding cytochrome component in plant tissues. 5 . Are there changes in organic phosphate levels and Pi levels ? Carey and Wyatt (1963) found ATP/ADP ratios of 12 in dormant pupae and 17 in developing adults. Thus the pupa is able to synthesize

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ATP efficiently during diapause. Confirming early reports by Heller (1949), Carey and Wyatt found that inorganic phosphate is accumulated in the gut of diapausing pupae. The levels of Pi in the blood showed a corresponding drop. These data support Heller’s (1949) suggestion that levels of inorganic phosphate may play a role in regulating oxidation in diapausing pupae. 6. Are thejive changes discussed above the cause or eflect of developmental changes ? A large body of theory and data support the regulatory role of ADP in controlling respiration (Lardy and Wellman, 1952; Chance and Williams, 1956; Lehninger, 1964). An example is the large phosphate acceptor coefficient in housefly mitochondria (e.g. Van den Bergh and Slater, 1962). Mitchell (1962) and Harvey (1962) proposed that such endergonic processes as muscle contraction, active transport, and biosynthesis control respiration through the ADP that they release rather than the endergonic events being controlled by oxidative processes. C. E N Z Y M E S O F T A N N I N G R E A C T I O N S

I . Pupariumformation in Calliphora Karlson (1963a, b) discusses enzyme synthesis induced by ecdysone. As was shown by Fraenkel(1935), one of the effects of ecdysone is the promotion of puparium formation in the blowfly, Calliphora erythrocephala (see Section IV, A). Karlson and Sekeris (1962a) showed that puparium formation is accomplished in part by the incorporation of quinones into the cuticle. Tyrosine metabolism is shifted to yield Nacetyldopamine which is oxidized in the cuticle to the corresponding quinone. The quinone reacts immediately with the cuticular proteins in a quinone tanning reaction (Sekeris and Karlson, 1961). Ecdysone promotes an increase in the level of two enzymes of this pathway, dihydroxyphenyl alanine (DOPA) decarboxylase (Karlson and Sekeris, 1962b) and one of the phenoloxidase enzymes (Karlson and Schweiger, 1961). Karlson’s group conclude that the activation of this enzyme system is probably due to de novo synthesis of enzyme proteins through the mechanism of gene activation (Karlson, 1963a).

2. Tyrosinase metabolism in ebony mutants Wolsky and Kalicki (1959) propose a sequence of events leading from the ebony gene to its expression in the external character of the puparium

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in ebony mutants of Drosop&la melanogaster. They propose that the primary gene action is to increase the degree of polyteny in the nuclei of the corpus allatum. As evidence they report that the nuclear diameter of corpora allata cells of ebony at the time of pupation is 6-25 p compared to 4.60 p in the wild type. They calculate that this corresponds to a nuclear volume of 122.1 p3 and 48-7 p3 respectively (Wolski et al., 1961). Taking the nuclear volume to be a measure of polyteny (cf. Kurnick and Herskowitz, 1952), they note that the values for ebony and wild type are very close to integral multiplying factors of 5 and 2 of the nuclear volume of normal diploid nuclei. The higher degree of polyteny, if it really exists, is proposed to lead to increased synthesis of juvenile hormone because cytologists are said generally to agree that polyteny is an expression of high metabolic activity in non-dividing cells. The extra juvenile hormone is thought to promote a higher rate of respiratory metabolism. The average oxygen uptake of ebony mutants was 22.6 k 1.1 in ebony compared to 18.5 f 1-5 in the wild type. Juvenile hormone is thought by several workers to promote oxygen consumption directly as discussed in Section IV, A. The high respiratory metabolism is thought to inhibit tyrosinase activity because Ohnishi (1954a, b) has shown that at the time of pupation, whereas tyrosine concentration is the same in ebony and wild type, tyrosinase activity (measured as oxygen uptake of homogenate) was lower in ebony (19 plo,/h/mg) than in the wild type (43 pl/h/mg). Dennell(l946, 1949) and Karlson and Wecker (1955) have similarly proposed that the higher the oxidative metabolism the lower the tyrosinase activity. Finally the lowered tyrosinase activity is argued to lead to imperfect (juvenile) tanning characterized by lighter pigmentation and greater softness of the ebony puparism. Pryor (1940a, b) has discussed the role of products of tyrosinase activity such as dihydroxyphenols in the process of sclerotization. The uncertainties in the series of steps proposed illustrate the difficulty in attempting to account for gene function in molecular terms in metazoans.

IV. CONTROL AT

THE

CHROMOSOMAL LEVEL

A. B I O C H E M I S T R Y OF I N S E C T H O R M O N E S

1. Brain hormone An oily substance with brain hormone activity was extracted with ether from 8 500 surgically isolated brains of Bombyx mori by Kobayashi and Kirimura (1958). Water soluble extracts with brain hormone activity have

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also been prepared (Ichikawa and Ishizaki, 1961,1963). These seem to be protein in nature. Kobayashi et al. (1962) and Kirimura et al. (1962) after analyzing the crystalline substance from an extract of 220 000 brains of silkworm pupae reported that the brain hormone is probably identical to cholesterol. Schneiderman and Gilbert (1964), who review the biochemistry of brain hormone, give evidence of the effectiveness of cholesterol as a minic for brain hormone, but also state that the concentration of cholesterol has no clear correlation to the degree of hormone activity it produces in brainless pupae of Samiu Cynthia. They also point out that it is difficult to reconcile that cholesterol could be the brain hormone when cholesterol levels in the blood are quite high (Goodfellow and Gilbert, 1964)and have calculated that the amount of cholesterol alleged to cause molting is only about 0.0001 the amount normally present. Williams (1947) first demonstrated that brain hormone stimulates the prothoracic glands to secrete ecdysone. According to Oberlander and Schneiderman (1963) the initial effect of the brain hormone on the prothoracic glands of many silkworms is to enhance nuclear RNA synthesis. This is followed by an increased synthesis of RNA and protein in the cytoplasm. These workers hypothesize that these events reflect enzyme synthesis necessary for the production of ecdysone. Indirect evidence that the production of ecdysone is associated with the RNA metabolism of the prothoracic glands of Drosophilu was given by el-Wahab and Sirlin (1 959). 2. Juvenile hormone The first extracts ofjuvenile hormone were prepared from abdomens of adult male Hyalophora cecropia by Williams (1956). Subsequently, active extracts were reported from other insects, invertebrates, various organs of vertebrates, and from plants and micro-organisms (Schneiderman and Gilbert, 1958; Gilbert and Schneiderman, 1958; Wigglesworth, 1958; Williams et al., 1959; Schneiderman et al., 1960). The active principle from meal worm excreta (Karlson and Schmialek, 1959) was shown to be composed of two substances, the open-chain terpene alcohol farnesol and its aldehyde farnesal (Schmialek, 1961). Farnesol copies both the action of juvenile hormone and the action of the gonadotropin secreted by the corpora allata which promotes egg development (Wigglesworth, 1961) but requires milligram or tenths of a milligram amounts whereas purified preparations of juvenile hormone are active in millimicrogram amounts. Yamamoto and Jacobsen (1962) found the biological activities of farnesol and related terpenes to require the transconfiguration at the F*

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d6or middle linkage of the molecule. Two terpene derivativeswith greater juvenile hormone activity than farnesol have recently been uncovered (Wigglesworth, 1963b; Karlson, 1963b). Karlson reports that these compounds have 200 times the activity of farnesol or of Cecropia extracts; however, Schneiderman and Gilbert (1964) find them to be only about ten times as active as purified all trans farnesol. Reviews of the current concepts of the mode of action of juvenile hormone are given by Gilbert and Schneiderman (1961) and Wigglesworth (1962,1964). Most of the hypotheses concerning the corpora allata suggest that they affect the general metabolism (Thomsen, 1949;de Wilde and Stegwee, 1958;Novak et ul., 1960)and more specifically the metabolism of proteins (Thomsen, 1952; Bodenstein, 1953; Wang and Dixon, 1960; Dadd, 1961; and Vanderberg, 19@a, b). That these results are due to juvenile hormone is unresolved. Most of the experimental evidence in the field was gained by in vivo studies concerned with the ablation or implantation of corpora allata. It has not been established whether or not more than one hormone is secreted by this gland. Another source of complication arises when one considers that most extracts of juvenile hormone and also farnesol and its derivatives can also induce the prothoracic glands to secrete ecdysone (Williams, 1959; Gilbert and Schneiderman, 1959; Krishnakumaran and Schneiderman, 1963). To resolve the extensive and complex literature relating to the mode of action of juvenile hormone is beyond the scope of the present review. Recent papers in the field are Doane (1962), Highnam et al. (1963), Stegwee (1963), Thomsen and M ~ l l e r(1963), Wigglesworth (1963a), Davis (1 965), Nair (1963), and Orr (1964).

3. Ecdysone At the end of larval development, the integument of Calliphora erythrocephulu hardens to form a puparium within which the animal molts to a pupa which in turn develops into an adult fly. Fraenkel(l935) was the first to compile convincing evidence that the “molting processes” beginning with the formation of a puparium were due to a blood-borne agent. By ligating Culliphoru larvae before a critical period Fraenkel showed that only the anterior portions of the larvae formed puparia while the posterior portions remained permanently juvenile. When blood from larvae that had passed the critical period was injected into these larval abdomens they too formed puparia and underwent the pupal molt. It was concluded by Fraenkel that the blood from animals after the critical period must contain a puparium-forming hormone. Fraenkel’s “Culli-

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phora assay” for the molting hormone was used by Becker and Plagge (1939), in their attempts to purify the hormone. Becker (1941) succeeded in producing an extract with relatively potent hormone activity. The molting hormone was first crystallized by Butenandt and Karlson (1954) and named ecdysone. In this first isolation only 25 mg of material was obtained from 500 kg of Bornbyx pupae. Karlson and his co-workers (1963) now conclude that ecdysone is a steroid with the empirical formula C!&406. Ultraviolet, infrared, and proton resonance spectra indicate that the molecule has a typical steroid configuration with a keto group at carbon atom 12 conjugated to a double bond between carbon atoms 9 and 11, an hydroxal group on the side chain at carbon atom 25, and four other hydroxal groups at undetermined locations (see Fig. 1). 4-OH

FIG. 1 . The structural formula of ecdysone.

It is perhaps surprising that ecdysone is a steroid because insects are unable to synthesize the entire steroid nucleus. In all cases studied the dietary requirement for sterols can be satisfied by cholesterol. For reviews of sterol utilizationand synthesisin insects, see Lipke and Fraenkel( 1956), Levinson (1955, 1962), Clark and Bloch (1959), Clayton (1964) and Fast (1964). Extracts of pupae of Culliphora erythrocephulu, previously injected with tritium labeled cholesterol, co-crystallized with pure hormone yield ecdysone with a constant specific activity. These results suggest the biogenesis of ecdysone from cholesterol (Karlson and Hoffmeister, 1963). The significanceof cholesterol having brain hormone activity, of juvenile hormone probably being related to an intermediate of cholesterol (farnesol), and of the biogenesis of ecdysone from cholesterol is discussed by Schneiderman and Gilbert ( 1 964).

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Mode of action ofecdysone. Gilbert and Schneiderman (1961), Wigglesworth (1962, 1964), Burdette (1964) and Schneiderman and Gilbert (1964) present comprehensive reviews of many facets of the control of metabolism by insect hormones. Schmidt and Williams (1953) found that isolated spermatogonia and spermatocytes from testes of dormant pupae of silkworms, Hyalophora cecropia and Samia Cynthia, form well-developed spermatids when cultured in the presence of blood from animals at stages when ecdysone appears to be in high titer in the blood. Wigglesworth (1957, 1962) describes histological effects of ecdysone in fat body, ventral intersegmental muscle, and epidermal cells of Rhodnius prolixus. Within 6 h after the administration of ecdysone to intermolt (dormant) nymphs of Rhodnius visible changes are induced in epidermal cells. The nucleoli enlarge, nucleolar RNA synthesis increases, and later RNA begins to accumulate in the cytoplasm around the nucleus. Concurrently the mitochondria change from fine granules and slender filaments to enlarged oval vesicular bodies. Wigglesworth regards these changes as indicating a renewal of protein synthesis. The early effects of ecdysone in stimulating the synthesis of enzymes and other proteins is discussed more thoroughly in Section 111. Cleveland (1959) was among the first to demonstrate that nuclear processes could be regulated by hormones. He reported a profound effect of injected crystalline ecdysone on the reproductive cycle of protozoan flagellates in the hind-gut of the woodfeeding roach Cryptocercus punctulatus. Normally sexual reproduction occurs in these symbionts only at the time of molting of the host roach. During the prolonged intermolt period of the roach, when ecdysone is presumably absent, all reproduction is asexual and asynchronous. Sexual cycles of the flagellates could be induced in adults or intermolt nymphs by the injection of exogenous ecdysone. Gametogenesis was induced almost synchronously in all haploid individuals of a species, and both meiosis and gametogenesis in diploids. The time required to induce gametogenesis varied in different genera of the protozoans by as much as 2 days with various dosages of hormone administered, but was never 40-50 days as in control animals (Cleveland et al., 1960). When ecdysone was injected during the normal molting period of the host, the sexual cycles of the protozoans were greatly accelerated and the host molted much sooner than was expected. With advances in understanding both the mechanisms of protein synthesisand its geneticregulation in micro-organisms(Jacob and Monod, 1961a) the attention of developmental biologists has beendirected to the prospect that a hormone might fulfil its function by regulating the syn-

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thetic activity of genes (Gilbert and Schneiderman, 1959).This problem is now being widely attacked by several groups studying the patterns of puffing in the giant chromosomes of Diptera. B. B I O C H E M I S T R Y OF G I A N T CHROMOSOMES

Larval growth in many insect tissues is accomplished by cell enlargement. In some cases DNA replicates, but chromatids do not separate, and cells do not divide. Large cells with giant nuclei containing polytene chromosomes result. This tendency is expressed most completely in the giant polytene chromosomes of the salivary glands of dipterous insects (Trager, 1935; Bodenstein, 1943). 1. Location and size of chromosomes A comprehensive review of early literature on giant chromosomes is given by Alfert (1954). Giant chromosomes occur in the highly differentiated giant cells of larval salivary glands, intestines, and Malpighian tubules of many Diptera (Mainx, 1949; Makino, 1938). Pavan and Breuer (1 952) reported giant chromosomes in salivary glands, Malpighian tubules, and seminal vesicles in Rhynchosciara; Bier (1960) in nurse cells of ovaries of Calliphoru; Laufer (1963) in salivary cells of Phormia; and Whitten (1964) in footpads of developing adults of Sarcophaga. Makino (1938) also found some degree of polyteny in muscle, tracheal, adipose, and isolated ganglion cells of Drosophila virilis. The giant chromosomes of Chironomus larvae attain a length of at least ten times and a cross-section up to 10 OOO times that of normal univalent interphase chromosomes (Beermann, 1963). These chromosomes are considered to be “polytene” (i.e. they are multivalent in a cable-like fashion, because of progressive replication of chromatids without mitotic splitting) (Beermann, 1963; Koltzoff, 1934; Bauer, 1935). Drosophila chromosomes contain as many as 4 096 chromatids and Chironomus as many as 32 768 (Beermann, 1959).

2. Chromosomal bands The most conspicuous feature of the polytene chromosomes is their banding pattern. Bridges (1935) calculated that there are about 1 800 bands in chromosomes of Drosophila. He judged this figure to be in good agreement with the calculated totals of known genic loci. Homologous chromosomes in different tissues of the same species show the same banding pattern. Beermann (1963) states that the ultimate unit composing

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each band of the polytene chromosome is considered to be a tightly folded portion of the constituent DNA-histone fiber of the chromatid. However, variations may occur in the fine structure of many of the individual bands. A particular band may vary from a sharply defined disk of high optical density containing concentrated DNA through intermediate degrees of puffing to a huge ball-like diffusely staining region now called a Balbiani ring. “Puffing”, a swelling of a particular band of the chromosome first described by Balbiani in 188 1, is now hypothesized to be a visible indication of genic activity (Beermann, 1963). In terms of a single chromomere, puffing is the unfolding of the DNA-histone fiber into a long loop-like thread. Beermann and Bahr (1954), by means of electron microscopy, have observed this to be the case in Balbiani rings of Chironomus. The total solid material in the bands and interbands of chromosomes of larval Chironomus salivary glands has been estimated from refractive indices of the various regions (Berendes and Ross, 1963). Chromosomal bands contain not less than 25% solid material, and interband regions not more than 15%. Nuclear sap contains 12% solid material. Chromosomes isolated in saline had lower refractive indices, the interband regions being proportionally lower than the banded regions. Chromosomes isolated in a 12% isotonic saline-protein solution of the same refractive index as the nuclear sap had higher concentrations of solid material. These workers conclude that isolated chromosomes can regulate entrance of protein molecules from mounting medium or egress of some of the protein associated with the chromosome, depending on the protein concentration gradients established in the artificial system. 3. Analysis of nucleic acids in bands and interbands When treated with Feulgen’s reagent, compact bands of the chromosomes stain heavily; the intensity of staining diminishes as puffing of a band increases. Peripheral regions of large puffs where progessive unfolding of the DNA molecules has occurred no longer show visible Feulgen staining (Beermann, 1963). Incorporation studies with tritiated thymidine show radioactivity to be concentrated primarily in regions that correspond to the bands (Woods et al., 1961). Pelling (1959) studying the incorporation of tritiated uridine as a precursor of RNA in Chironomus tentans found that large amounts of RNA are produced in puffs. Short pulses of labeled nucleotide result in almost exclusive labeling of the puffs and nucleoli of salivary cells, indicating that these regions are active sites of RNA synthesis (Beermann, 1963). Similar results were obtained by Rudkin and Woods (1959) in Drosophilu and

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Sirlin (1960) in Smittia. The rapidly labeled RNA of puffs and nucleoli appears to have no structural significance (Beermann, 1963). Edstrom and Beermann (1962) determined by microelectrophoresis the base composition of RNA from samples of individually isolated giant chromosomes, puffed chromosome segments, nucleoli, and cytoplasm of Chironomus tentans and the adenine-guanine ratio of the chromosomal DNA. Their results indicated that although nucleolar and cytoplasmic RNA‘s are both rich in adenine and uracil, they are significantly different from each other and from chromosomal RNA. The RNA of three different regions of chromosome 4, each containing a Balbiani ring, differfrom each other and from the RNA of chromosome 1. None of the chromosomal RNAs investigated show base symmetry, and the guanine-cytosine content is different from that of DNA. These workers conclude that their data “excludes the possibility that chromosomal RNA is a complete copy of both strands of the chromosomal DNA and that in spite of the difference in guanine plus cytosine content between the two nucleic acids the RNA may still partly or completely be a single strand copy, dependingupon how representative the DNA values are for the synthetically active DNA”. Messenger RNA has recently been hypothesized to be a copy of only a portion of a single strand of the DNA molecule (Hayashi et al., 1963a, b, 1964). Puffing appears to be a reversible reaction except in the polytene chromosomes of Rhynchosciara angelae, a Sciarid fly (Breuer and Pavan, 1955; Rudkin and Corlette, 1957). In this case in some loci the puffing is accompanied by a permanent accumulation of large amounts of DNA. Stich and Naylor (1958) working on Glyptotendipes larvae at 6 8 ° C have also reported that a few puffed regions accumulate a Feulgen-positive globule (DNA) when larvae are incubated at 18°C for 2-3 days. This globule gradually disappears when the larvae are returned to the former temperature. 4. Proteins associated with pufled regions

A large accumulation of proteins occurs at each active chromosome locus. These proteins associated with puffs are not histones (Beermann, 1963)and it is not known whether the proteins of particular puffed regions are the same or are different. Autoradiographs of salivary cells of larval Smittia 5 min after injection of tritiated leucine show most of the radioactivity to be localized in the cytoplasm of the cells. The nucleoli show a lesser degree of incorporation and no label is found in Balbiani rings. One hour after injection most of the label is seen in the cytoplasm, but chromosomes have more radioactivity than nucleoli (Sirlin, 1960). In

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larvae of Chironomus tentans autoradiographs show heavy labeling in the cytoplasm, but no incorporation of radioactivity in salivary cell nuclei 1 h after injection of tritiated leucine (Beermann, 1963).Clever also notes no incorporation of triated leucine in nuclei 2 h after simultaneous injection of ecdysone although rather large amounts of protein have already accumulated in newly induced puffs (Clever andBeermann, 1963). No preferential incorporation of leucine, tryptophan, or lysine is found at the puff level either when the puff is functioning or when the puff is forming in chromosomes of Drosophifa buskii (Ritossa and Pulitzer, 1963). It would seem then that the puffing pattern of the polytene chromosomes usually represents the pattern of RNA synthesis along the chromosome, and that the synthesis of new proteins does not occur in puffed regions. C . CHROMOSOMAL P U F F I N G A N D ITS R E L A T I O N TO D E V E L O P M E N T

1 . Tissue and stage specificity In Chironomus about 10% of the chromosomal bands in each tissue

appear in a more or less puffed condition regardless of developmental stage (Beermann, 1963). Although the banding pattern is the same for all tissues, the bands which are puffed may be dissimilar in different tissues. Even in loci which puff in a particular tissue, phases of puffing alternate with phases of non-puffing (Beermann, 1956, 1959). Bodenstein in 1943 showed that the developmental changes that occur in Dipteran salivary glands are under hormonal control and that the salivary glands grow only by cell enlargement. Drosophila salivary glands contain about 1 15 cells at hatching and retain the same number throughout larval life (Makino, 1938). Becker (1959) recorded seventy different puffed regions in Drosophila chromosomes. Puffs were most numerous 6 h before, but also during and slightly after the last larval or pupal molt. At this time he suggested that puffing must be closely related to molting. Chromosomes of nurse cells of ovaries of Cuffiphoraalso undergo a series of developmental changes including alterations of the puffing pattern (Bier, 1960). The first investigation of the temporal relationships between puffing of chromosomal loci and changes in hormone concentrations regulating larval development was performed by Clever and Karlson (1960). Clever (1962b, 1963b) distinguished two main types of puffs in salivary gland cells of Chironomus tentans. The first group contains loci which form puffs, regardless of the developmental stage of the larva. Clever regards these loci as representing genes whose activities take part in processes of basalor functionalmetabolism. The second group which form

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puffs only during certain phases of molting are interpreted as being loci of genes whose activities are specificfor that phase of development. Very soon after the induction of the processes involved with the pupal molt the puffing pattern alters from type one to include type two. 2. Induction by ecdysone Clever and Karlson (1960) noted that after injection of ecdysone into last instar larvae of Chironomus tentans specific loci of the salivary gland chromosomes puffed. At the same time other puffs disappeared. Changes in two gene loci, I-18-C and IV-2-B, can be detected very soon after the injection of ecdysone. Locus I-18-C reacts 15-30 min after injection and IV-2-B about 30 min later. In both regions the duration of the puff and the size attained by it are dependent upon the amount of ecdysone injected. More ecdysone gives larger puffs of longer duration (Clever and Beermann, 1963; Clever, 1963a, b). Clever states that 2 h after injection of 2x 1C5pg of ecdysone, both puffs are present, but they have disappeared after 8 h. However, after an injection of 2x pg, small puffs are present for 24 h. Both puffs will reappear if ecdysone is injected again immediately after their disappearance. Clever has found the reaction pg and that of locus IV-2-B to be threshold of locus 1-18-C to be It7 pg of ecdysone per mg larval weight. The maximum size of the former locus is reached at pg per mg larval weight. The appearance of these puffs is apparently due to the injected ecdysone, and the regression of the puffs is due to its elimination. During normal development both puffs appear during the molt from third to fourth instar larvae and disappear after the molt is completed. They reappear as soon as the pupal molt processes have begun-again indicating their dependence on the presence of ecdysone. However, loci I- 18-C and IV-2-B have slightly different reactions during normal development and following the injection of ecdysone. In third stage larvae puff I-18-C develops 1-2 days before puff IV-2-B and at the end of the .larval molt it regresses much more slowly. During the pupal molt it does not disappear but reaches its maximal size in older prepupae. Puff IV-2-B reaches its maximal size in the middle of the prepupal period and then becomes smaller and finally disappears (Clever and Beermann, 1963). Clever (1963a) concludes that the concentration of ecdysone rises gradually during normal development and the activity pattern of these loci is dependent on the increasing hormone concentration. He estimates that ecdysone concentration varies from lC7pg per mg larval weight in young larvae to pg in old prepupae. Clever and Beennann (1963) investigated the hormone titer of the

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hemolymph of larvae in different stages of development by using larvae as donors of hemolymph for injection into young larvae. They concluded that the alterations in puff I-18-C are due solely to increasing hormone concentrations. Because puff IV-2-B loses its activity during the pupal molt while large amounts of ecdysone are still present, it appears with regard to this gene locus that some other process is at work which is antagonistic to that of ecdysone. 3. Larval v. pupal molt Chromosomal puffing during a larval molt was compared with puffing during metamorphosis by Clever (1962b, 1963b). The pattern ofpuffing in salivary gland chromosomes of third instar Chironomus prior to the initiation of the larval molting processes does not differ from that of last (fourth) instar larvae. During the larval molt there is no change in the frequency of puffing in loci not specific for molting, nor is there any increase in size of these p f f s . In the dormant period of the larva before the initiation of the pupal molt, the lower metabolism of the animal is reflected in a reduction in the frequency of puffs (Clever, 1962a). Puffs specific for metamorphosis have not yet appeared and but few of the puffs present in the growing larvae remain. Following the initiation of the pupal molt (at the time of ecdysone secretion) besides the appearance of molting-specific puffs, there is an increase in size of some of the nonmolting-specific puffs. This seems to indicate that some parts of the nonmolting-specific metabolism are enhanced during the initial stages of metamorphosis. During the course of metamorphosis the frequency of puffing of most of these loci decreases but rarely do the puffs disappear entirely (Clever, 1962b). Puffs I-18-C and IV-2-B lead a procession of molting specific puffs. The majority of these puffs do not occur during a larval molt. There are, however, a few puffs which occur only during a larval molt or at least become larger than at any other time. Clever seems to feel that most of the effects of ecdysone described for the pupal molt are characteristic not of molting in general but only of metamorphosis. Metamorphosis of the salivary gland in the Diptera consists of histolysis of the larval gland and differentiation of the new imaginal salivary gland (Bodenstein, 1943). Clever concludes that only those metabolic processes which are included in cell autolysis remain active during metamorphosis, and that the effect of juvenile hormone in the salivary cells is to prevent ecdysone from inducing the catabolic processes which lead to the breakdown of the gland. Hadorn (1964) has reported that larval salivary glands of Drosophila explanted into adults which have non-functional pro-thoracic glands, not only continue to

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live, but form super giant chromosomes that are about four times the size of the normal polytene chromosomes. A further conclusion by Clever is that the primary effect of ecdysone in the salivary gland cells is identical during the larval and pupal molts and that its effect is independent from any action of juvenile hormone. The function of juvenile hormone, he feels, is the prevention of induction of cellular breakdown by ecdysone and, as a consequence of this action, the maintenance of the general metabolism. Williams (1961)hypothesized that the action of juvenile hormones is “associated with its ability to block the de-repression and decoding of fresh genetic information without interfering with the use and re-use of information already at the disposal of the cell.” Wigglesworth (1962) suggested that the action of juvenile hormone is the maintenance in action of those genes which favor the production of enzymes necessary for larval syntheses. 4. Efect on pufing of inhibition of RNA or protein synthesis Clever (1964) temporarily inhibited RNA and protein synthesis in salivary gland cells of Chironomus tentans to determine more specifically the effect on gene activation by ecdysone. Larvae were incubated in medium containing actinomycin C for 6 h, then transferred to medium without the inhibitor. Larvae were removed at various intervals, the salivary glands removed, and incubated for 30 min in a sucrose medium containing tritiated uridine. Glands were then squashed and autoradiographs made. RNA synthesis was decreased by 4-6 h exposure to actinomycin. It had stopped completely 1-2 h after removal of the larvae from the actinomycin containing medium but begin to recover, with high variability, 15-20 h after the transfer. Disappearance of puffs and Balbiani rings paralleled the disappearance and reappearance of the RNA synthesis. Puffing patterns were identical in uninjected larvae and in those injected with ecdysone 16-24 h after transfer from actinomycin medium. However, the injection of hormone immediately after the 6 h actinomycin treatment elicited no response for 15-20 h following the injection. The appearance of all puffs was delayed for approximately the same length of time as RNA synthesis was inhibited. Because the normal puffing pattern was merely delayed by the cessation of RNA synthesis and initial puffing stages were not omitted, Clever concludes that “some inducible genes must participate in the processes which lead to the activation of later-appearing puffs”. Puromycin at 100-120 pg/ml in the Chironomus larvae almost completely inhibited protein synthesis for 4-5 h. During periods when protein synthesis was inhibited by puromycin, injection of ecdysone did

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not effect the induction of puffs I-18-C and IV-2-B. Apparently protein synthesis is not required for induction of puffing in these regions. Simultaneous injection of puromycin and ecdysone causes the delay of puffs which normally occur 15-20 h after ecdysone injection. The period of puff cessation approximately parallels the period of protein synthesis inhibition. Clever concludes from these experiments that although the synthesis of new proteins apparently is not necessary for the induction of puffs I-18-C and IV-2-B it must be part of the process which induces later appearing puffs. Clever summarizes his view of induction of genic activity by ecdysone in the following diagram.

He further states that his work “disproves the hypothesis that all the genes which change their activity in the course of a molt are regulated by a single control system, and that it is this system (the ratio of K+ to Na+ in the nucleus sap) which is affected by ecdysone” (Kroeger, 1963a, b). Clever gives no evidence that the (?)in his scheme cannot be the K/Na ratio of the nuclear sap (see Section V, B). 5 . Induction of pu$ing by hormone imitators Kroeger (1963a, b) finds that implantation of salivary glands of third instar Chironomus thummi into prepupae which have a high ecdysone titer effects a limited progress in the puffing patterns exhibited by the chromosomes. The advanced puffing pattern induced when glands are implanted into prepupae containing high ecdysone titer is mimicked by

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injection of ZnCl, (23 mM in the hemolymph) or rather high concentrations of CdC12or the narcotics, chloroform, butanol, and urethane. These agents do not induce metamorphosis and seem to act regardless of the amount of hormone present. In other words they do not seem to increase the sensitivity of the animal to existing ecdysone. Kroeger believes that what is seen after explantation of salivary glands into their own hemolymph is identical to the effects of juvenile hormone. The puffing pattern is changed within 30 min to that of the previous instar larvae. Puffing patterns in explanted salivary glands of younger than third instar larvae reversed while those of older than third instar larvae proceeded in a normal developmental sequence. At a critical period in the third instar some reversed and some advanced. Bodenstein (1943) found that salivary glands of young Drosophilu react much more slowly to ecdysone than glands of older larvae. Glands had to be of a certain developmental stage before they were able to respond to the hormone by metamorphosis. Kroeger terms this reversal of the puffing pattern “rejuvenation” and states that it is “violent” in that although the pattern of puffing is identical to stage 1 larvae, the puffs are considerably oversized; they seem to overshoot their normal size during their rapid build up. Rejuvenation appears to be an unspecific response by cells to any wound stimulus, including X-ray, mechanical injury, and oxygen poisoning. It can be induced in all stages except stage 1 (which already has the puffing pattern characteristic of rejuvenation) and in salivary cells which are degenerating after pupation. Rejuvenation can be prevented in a cell of any developmental stage if any ecdysone imitator is administered immediately after the wounding stimulus is applied. However, once the puffing pattern of wound metabolism is established no amount of ecdysone imitator can reverse it. Because ecdysone is a comparatively small molecule and because identical effects are produced by so diverse and unspecific agents as heavy metal ions and narcotics, Kroeger feels that ecdysone does not have a direct effect on the DNA molecule. He believes that instead an intermediate system exists somewhere in the cell that links the hormone action to the chromosome effect. By surgically removing portions of the chromosomes he has shown that the puffing patterns of rejuvenation are not regulated by interactions with other portions of the genome excluding those regions immediately beside the puffed loci which cannot be surgically removed. He has found that nuclei rejuvenate in a typical way when they are stripped of their cytoplasm and kept alive in a totally synthetic medium or in the content of Drosophila eggs, and that glands explanted in oil with no hemolymph also react in the same manner as those surrounded by

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hemolymph. By elimination he suggests that a control system upon which ecdysone acts exists in the nuclear sap. The effects of ecdysone and juvenile hormone on protein synthesis in cell free systems have not been studied. For further analysis of the control system induced by the hormones Kroeger is forced to use the rejuvenation process. Microsurgery seems to induce such an excessive wounding in the cell that it can no longer respond to ecdysone or its imitators. Kroeger argues that it is sufficient to locate the mechanism of action on only one of the two hormones because a locus can be activated in two ways-during normal development by ecdysone or during rejuvenation by juvenile hormone imitators. He deems it unlikely that two independent control mechanisms exist, one of which would produce a puffing pattern during normal development, and the other which would produce the same puffing pattern during rejuvenation. Kroeger (1963~)in a recent article states that stepwise replacement of sodium ions by potassium ions in saline into which salivary glands of Chironomus thummi have been explanted induces a series of puffing patterns which is identical with the sequence of puffing patterns during the transition of last stage larvae to middle prepupae. In contrast “rejuvenation” appears to be caused by an influx of sodium ions into the nucleus (see Section V, B). 6. Induction of pufing by uncouplers of oxidative phosphorylation Evidence that metabolic variation can produce primary inducing stimuli for puffing, and that the production of puffs requires a functional energy producing system, is given by Ritossa (1964). Temperature shock induces a specific puffing pattern in chromosome 2 of salivary glands of larval Drosophila buskii. When larvae reared at 25°C or explanted salivary glands maintained at 25°C are placed for 30 min at 30°C or higher, puffs occur in regions 2L-14, -1 5, and-20(RitossaY1962).The same puffing pattern can be induced by methods which are known to depress the level of high energy phosphate bonds in the cell (see Section 11, A). Chromosomes of salivary glands explanted under mineral oil into saline containing sodium azide (3 mM), dicumarol (1 mM), dinitrophenol (1 mM), or sodium salicylate (10 mM) exhibit puffs which are identical to those induced by temperature shock (Ritossa, 1964). Many of these inhibitors also induce puffs on chromosomes of Drosophila melanogaster (Ritossa, 1963). DNP at the concentration mentioned above does not interfere with the RNA or protein synthesis in salivary cells of D . buskii (Ritossa, 1964). Anaerobiosis, produced by submersion of whole larvae in mineral oil at 25°C for 1 h causes various results, but larvae subjected to anaero-

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biosis and then returned to air for 10 min. display the same puffed regions on their chromosomes as animals treated with uncouplers of oxidative phosphorylation. Concentrations of DNP higher than 1 mM, iodoacetate (1 mM), and sodium fluoride (10 mM) inhibit puff formation induced by temperature shock, but simultaneously block the synthesis of RNA and proteins. D. C H R O M O S O M A L P U F F I N G A N D I T S R E L A T I O N TO SYNTHETIC PROCESSES I N THE CELL

1. Secretory granules Beermann (1963) has correlated the formation of a specific Balbiani ring with the synthesis of cytoplasmic granules. In larvae of Chironornus pallidivittatus a small specialized section of the salivary gland produces a secretion which is granular while the secretion of the major portion of the gland is clear. The granular component is not present in the equivalent cells of Chironomus tentans, the closest relative of C. pallidivittatus, although the details of the fine structure of these cells is identical in both species. Located on the small fourth chromosome are three Balbiani rings which are shared in both species. An additional Balbiani ring is found on the fourth chromosome of the specialized cells in C. pallidivittatus. In C. tentans the loss of the Balbiani ring, the distinguishing feature on the chromosomal level, is concomitant with the loss of the distinctivefunction of the cells. Beermann has found that the inability to produce secretion granules is a recessive trait which is inherited as a simple Mendelian factor. Its location, confirmed by crossing over tests, coincides with the lobespecific Balbiani ring. In heterozygotes the allelic segments furnished by C. pallidivittatus formed Balbiani rings, but the homologous segments from C. tentans did not. In some rare instances the formation of the lobe specific ring was suppressed by modifying genetic factors. In these cases parallel decrease in the amount of the secretion granules in the lobe was observed. Beermann concludes that these results demonstrate the possibility of an “operational mutation” as opposed to an informational one. No visible deficiency is present at the locus of the mutant which distinguished C. tentans from C. pallidivittatus, and he believes it is conceivable that the mutation does not involve the information content of the locus but involves instead its operational properties. If this is true these operational properties might be controlled by a segment immediately adjacent to the informational one just as the operation of genes in bacteria is controlled by the special “operator” site (Jacob and Monod, 1961a).It will be recalled, however, that Kroeger has seemingly ruled out all

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chromosomal sites except those immediately adjacent to the puffed regions as having any influence on the puffing pattern of rejuvenation in chromosomes of the salivary gland of larval Chironomus thummi. E. E C D Y S O N E A N D D N A S Y N T H E S I S

Recent work suggests that DNA synthesis may also be close to the primary action of ecdysone. Ecydsone is evidently required for the activation of epidermal cells and intersegmental muscle cells but not for other cells of Rhodnius (Wigglesworth, 1963a). Similarly, ecdysone is required for DNA synthesis in hypodermis and abdominal intersegmental muscle cells in HyuZophora(Bowers and Williams, 1964).Krishnakumaran and Schneiderman (1964) suggest that ecdysone might be required for the production of a key enzyme such as DNA polymerase or thymidylate kinase. In response to ecdysone epidermal cells of larvae and pupae of several saturniids synthesize DNA at each molt. These workers conclude that this DNA synthesis must occur before the cells can engage in further syntheses that characterize molting. For example, they have found that chilled Polyphemus pupae (about to initiate development), when injected with 3 pg/g of mitomycin C at 2-day intervals, do not initiate development as long as the injections are continued (35 days), although they remain healthy and active. By contrast the injection of mitomycin fails to block cuticle synthesis and molting in pupae which have just initiated adult development. F. CHROMOSOMAL P U F F S A N D T R A N S P O R T

About a decade ago Telfer (1954) concluded from an immunological study of blood proteins in the Cecropia silkworm that a particular blood protein which he called antigen 7 “is secreted into the blood by some tissue other than the ovaries, and that during the period of egg formation the ovaries remove the antigen from the blood and deposit it in the yolk”. This view received substantial support from Telfer’s subsequent finding (1961) that fluorescent antibodies, prepared against blood proteins, appear in yolk spheres within the oocyte, in association with the oocyte brush border, and in the spaces between follicular cells but not in the follicle cells themselves. He suggested that proteins might pass from blood to oocyte by pinocytosis. Anderson (1964) found abundant evidence of pinocytotic activity in oocytes of Peripluneta. Moreover, the pinosomes of early oocytes were notably empty whereas those of later stages in oocyte formation were filled with a dense material thought to be precursors of yolk. Similarly, Laufer and Nakase (1964) studied the movements of carbon-

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14labeled blood proteins into the lumen of salivary glands of Chironomus. Apparently the glands do not synthesize any of the major secretory proteins but instead select, concentrate, and transport proteins from the blood to the lumen. Because the presence of Balbiani rings in the salivary chromosomes is correlated with the secretory process, these workers suggest that the presence of particular Balbiani rings reflects the functioning of special genes concerned with the synthesis of special “transport proteins” or “permeases” rather than with the synthesis of tissue specific secretion proteins. V. IONIC CONTROLOF PROTEINSYNTHESIS AND DEVELOPMENT A. ION C ONTROL D U R I N G D E V E L O P M E N T

Buck (1953), Wyatt (1961), and Florkin and Jeuniaux (1964)reviewthe chemical composition of insect hemolymph. Because of extreme differences in the pattern of cation distribution, Duchiteau et al. (1953) identified two major types of insect blood. One type, illustrated by the Odonata, a primitive insect order, has high sodium, low potassium, and low magnesium concentrations. The other type, which occurs in the more advanced orders such as Coleoptera and Lepidoptera, has low sodium, high potassium, and high magnesium. Duchiteau and Florkin feel that all insects can be classified into one or the other of these types as a function of the degree of their evolution and of their diet. BonC (1944) first pointed out that regardless of phylogenetic position, zoophagous insects tend to have much higher Na/K ratios in their hemolymph than do phytophagous insects. The ionic composition of insect tissues other than blood has been studied much less intensively. 1. Active potassium transport by the Cecropia mid-gut Harvey and Nedergaard (1964) report a potassium regulatory mechanism in mid-guts isolated from larval Hyalophora cecropia. These phytophagous larvae eat food containing very high concentrations of potassium. Leaves of a typical food plant, Viburnum notatum, contain 153 mM potassium and 4 mM sodium. An even lower Na/K ratio occurs in mid-gut contents of Cecropia larvae which have average potassium and sodium concentrations of 208 mM and 0.7 mM respectively. Hemolymph of these animals is rich in potassium, but does not reflect the extreme conditions of the mid-gut. Hemolymph potassium averages about 27 mM and hemolymph sodium about 6.0 mM. Harvey and Nedergaard (1964) find that mid-guts of fourth and fifth instar Cecropia larvae, isolated and bathed in aerated physiological solution, are capable of

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transporting large quantities of potassium from the blood side to the lumen side of the tissue. In vivo, this transport of potassium would be an effective device for the removal of excess potassium from the blood. When isolated and perfused on both sides with a solution containing 30 mM potassium chloride, 5.0 mM magnesium chloride, 4.5 mM calcium chloride, 2 mM potassium bicarbonate, and 164.5 mM sucrose, mid-guts of fifth instar larvae demonstrate an average electrical potential of 84 mV with the lumen side positive to the blood side. Potassium diffusion potentials contribute to but do not account for the transepithelial potential. The average value of the mid-gut potential increases moderately after the animal evacuates its gut prior to spinning; thereafter the potential diminishes gradually and is usually lost during the first 24 h of spinning. Mid-guts of prepupae and recently formed pupae have no electrical potential in the physiological solution described above (Harvey and Haskell, unpublished results). When the potential is “short circuited” mid-guts exhibit currents with an average value of 614 PA. Flux measurements with radioisotopes indicate that at least 83% of this current is caused by the active transport of potassium from blood side to lumen side of the mid-gut. Neither the potential nor the current of the isolated mid-gut requires the presence of sodium (Harvey and Nedergaard, 1964). The short circuit current is unaffected by ouabain, cholinesterase inhibitors, adrenalin, pituitary hormones, or small changes in pH, but is inhibited by anoxia, 2,4-dinitrophenol (DNP), iodoacetate, 25% carbon dioxide in the aerating gas, and by the carbonic anhydrase inhibitors, Cardrase and sodium sulfide. This system may employ a potassium-hydrogen ion linked mechanism rather than a potassium-sodium mechanism. The former prospect is supported by observations that the mid-gut contents of fifth instar Cecropia larvae have an average pH of 6-2 compared to a pH of 9.7 in the blood (Haskell et al., 1965). The universality of potassium ion regulation by the mid-gut of phytophagous insects remains to be demonstrated. However, sizable electrical potentials have also been found in isolated mid-guts of saturniids, Anthereae pernyi, Automeris io, and the sphyngid, Protoparce quinquemaculata (Harvey and Haskell, unpublished results). 2. Potassium secretion by labial glands of Saturniids The anterior portion of the glands which secrete silk in larvae of Anthereaepolyphemus, A . pernyi, and A . mylitta redifferentiate, with little if any mitosis, during adult development to form labial glands which secrete an aqueous solution of 0.15 M potassium bicarbonate (Kafatos

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and Williams, 1964). This solution acts as a solvent for “cocoonase” and is buffered at precisely the pH optimum of this potent proteinase which allows the adult moth to locally hydrolyze and separate the silk fibers and subsequently escape from the cocoon. In developing adults, the secretion of the labial glands contains potassium in concentrations four or five times that of the hemolymph, has little or no sodium, and only traces of calcium. The major anion of the secretion is bicarbonate whereas chloride ions are present in low concentrations similar to those in the blood. Kafatos and Williams suggest that potassium is actively transported from the hemolymph to the lumen of the labial glands. They believe that this phenomenon is followed by the passive flow of water and anions, at least chloride ions. Preliminary experiments on isolated silk glands of fifth instar Cecropia larvae disclosed average transepithelial potentials of 3.4 mV whereas labial glands of Anthereaepernyi on the 19th or 20th day of development have average potentials of 4.7 mV (lumen side positive) (Ryan and Harvey, unpublished results). Neither of these potentials is influenced by the concentration of extracellular sodium. Increasing potassium ion concentrations from 30 to 60 mM on the blood side of the glands doubles the potential whereas lowering the potassium ion concentration from 30 to 2 mM drops the potential to zero and often reverses the sign. The above experiments indicate that the electrical potential of the silk and labial glands as measured in vitro is due at least partially to a potassium diffusion potential (Ryan and Harvey, unpublished results). These results neither support nor disprove the suggestion by Kafatos and Williams that the potassium movement is active. 3. Compartmentationof alkali metal ions Sanborn (1957) reported reversible swelling of testes of diapausing pupae of Cynthia explanted into ionic salt solutions of 0.25-0.30 M total ionic concentration and into 0.15 M sucrose. Swelling did not occur when testes were placed in hemolymph or sucrose more concentrated than 0.25 M. Michejda and Thiers (1963) find no ion barriers in diapausing pupae of H . cecropia. They state that adult development in this animal does not appear to be triggered by membrane selectivity, but that permeability changes in membranes occur only after the 2nd day of development. Sodium, magnesium, calcium, and many amino acids appear to be evenly distributed between the hemocoel and testicular compartments in diapausing pupae. There is, however, a slight accumulation of potassium in the mid-gut, and hind-gut lumen, and the testes cavity at this time

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(54 mEq/l compared to 45 mEq/l in the hernolymph). During development potassium accumulated in the testes cavity to twice the amount found in the blood. Exogenous sodium and potassium ions injected into the animal during diapause appear to distribute evenly between the testes and hemocoel, but if injected after the 2nd day of development cause a strong accumulation of potassium in the testes cavity. Injection of 0.35 M sucrose induces similar results.

4. Ionic regulation induced by a hormone It is well established that the ionic environment of insect tissues can be partially regulated via the insect excretory system (Krogh, 1939; Wigglesworth, 1931a-c, 1953; Roeder, 1953; Ramsay, 1952, 1954, 1958, 1961;Phillips, 1964a-c). In at least one case this ionic regulation has been demonstrated to be mediated by the elaboration of a diuretic hormone (Maddrell, 1962, 1963, 1964). During the lengthy intervals between feedings, larvae of Rhodnius prolixus conserve water. However, large quantities of hypotonic urine are excreted within 3 min after feeding begins (Maddrell, 1963). At the end of the diuretic period the insect has usually lost about 40% of the weight of the blood meal, and the osmotic concentration of its hemolymph is approximately the same as it was before feeding. Maddrell(l962, 1963) finds that diuresis is triggered by a hormone probably released from neurosecretory cells in the posterior region of the fused ganglionic mass in the mesothorax. Maddrell(l963) assumes that the rate at which urine is voided from the insect is a direct measure of the rate of secretion of the Malpighian tubules, although in other cases the production of urine has been attributed to the dual process of secretion in the Malpighian tubules and resorption in the hind-gut (Ramsay, 1958) and in particular in the rectum (Phillips, 1964a-c). Higher temperatures result in an increase in the rate of excretion as well as an increase in the concentration of the urine (Maddrell, 1964). Sodium, the principle cation in the urine (Ramsay, 1952), behaves similarly to the total osmotic concentration. Diuresis at a high temperature leaves the animal with a relative excess of water whereas diuresis at a low temperature leaves the animal with a relative excess of salts. If animals fed at both temperature extremes are brought to an average temperature, during subsequent excretions the former produces a smaller quantity of a much more concentrated urine. B. P R O T E I N S Y N T H E S I S R E G U L A T E D B Y ION

CONCENTRATIONS

Walwick and Main (1 962) after studying the effects of monovalent cations on theincorporation of tritiated thymidine into DNA in a multi-

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enzyme system derived from rat thymus wrote, “One is encouraged to investigate the possible influence of these monovalent cation concentrations and of their regulation within the cell on intracellular DNA and protein synthesis”. The possible sites for the regulation of ions are epithelial membranes such as the mid-gut and silk glands just discussed, plasma membranes, membranes surrounding cell organelles, and the nuclear envelope. Whether or not the passive or selective qualities of these membranes are influenced by the hormonal environment of the animals is an open question. 1. Intracellular potassium concentration of Escherichia coli B mutants

Theconcentration of intracellular po tassium regulates the rate ofprotein synthesis in Escherichia coli (Lubin and Ennis, 1964). Mutant strains of this organism which lack the normal capacity for potassium regulation have been studied to elucidate the mechanism by which the intracellular potassium affects the synthesis of proteins. When cells are depleted of potassium, by incubation in potassium-free medium, cell division and protein synthesis stop, but RNA synthesis continues. In cell-free systems the rate of protein synthesis at low potassium concentrations is found to be limited by the transfer of amino acids from the aminoacyl soluble RNA to polypeptides. In the intact animal this step in protein synthesis occurs at the transfer level in the cytoplasm. More specifically the requirement for potassium is in a “priming reaction”, involving ribosomes and messenger RNA which precedes polypeptide synthesis (Lubin and Ennis, 1963; Lubin, 1963). Apparently the priming reaction is the formation of a complex of aminoacyl-s-RNA, ribosomes, and messenger RNA requiring high concentrations of potassium or ammonium ions reported by Nakamoto et al. (1964). Lubin and Ennis (1963, 1964) find that in the cell-free system the substitution of sodium or lithium for potassium in the medium markedly inhibits the rate of incorporation of (2-14 labeled phenylalanine into polypeptides. Rubidium shows an activity about equal to that of potassium in the cell-free system, but normally contributes to the stimulation of protein synthesis in the intact animal to only a small degree. To explain the high levels of potassium ions in all cells, Lubin and Ennis suggest “the possibility that cell K may regulate protein synthesis in other cell species and may account for the persistence of high levels of cell K i n evolutionary development”. If cells of higher organisms resemble E. coli, a direct effect of cell potassium on protein synthesis may be one of the mechanisms that link extracellular regulators to cell growth (Lubin, 1964).

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2. Zinc deficiency in Euglena Wacker (1962) reports that zinc deficiency in Euglena gracilis results in an impairment of protein synthesis and RNA synthesis. As a result cells increase in size and their DNA content doubles, but mitosis is arrested. 3. The eflect of sodium and potassium ions on chromosomal puJtfng in Chironomus At inactive gene loci “it is assumed that the DNA is complexed with different histone molecules which block the respective DNA segment ; activation of a gene would then mean the temporary or permanent abolishment of the blocking action of the respective histone molecule” (Kroeger, unpublished). The addition of thymus histones to isolated thymus nuclei inhibits RNA synthesis as well as other biosynthetic reactions. By selectively removing the histones from the nucleus, the rate of RNA synthesis is increased. The newly synthesized RNA occurs in the “messenger”-RNA complement of the thymus (AMrey et al., 1963). Huang and Bonner (1962) showed that the DNA from pea embryo chromatin is present in at least two forms :free DNA, and DNA complexed into nucleohistones. The DNA fully complexed with histone is inactive. The activity of puffed regions of salivary gland chromosomes of larval Chironomus thummi, as measured by autoradiography, can be increased threefold by the injection of buffered trypsin solution into the nuclei. Non-puffed bands respond but slightly if at all (Robert and Kroeger, 1965). These workers state that because trypsin is known to preferentially attract denatured proteins, the histone molecules at active gene loci must be in a structurally changed state. In this state they cannot block DNA and are rendered more sensitive to trypsin. Clever (1964) has shown in Chironomus that some gene loci seem to be much more directly influenced by ecdysone than others, and that protein synthesis appears to be necessary for the activation of certain loci. However, Kroeger (1963b) argues (because the same puffing patterns can be induced by such diversified agents as heavy metal ions and narcotics, as well as ecdysone) that a molecular interaction of the hormone with the histone or DNA molecule is hard to envision, and has hypothesized an intermediate system which is responsible for gene activation. Kroeger (1963c, and unpublished results) has compiled evidence that sodium and potassium ions of the nucleus are implicated in the activation of genetic loci in salivary glands of larval Chironomus thummi. His data indicate that ecdysone and juvenile hormone control the sodium and

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potassium ratio in the nuclear sap; ecdysoneincreasing the potassium ion concentration and juvenile hormone maintaining a high sodium ion concentration. Most somatic cells possess mechanisms for the extrusion of sodium and the accumulation of potassium. This active movement results in a highly asymmetric distribution of ions; high potassium-low sodium ion concentration inside the cell as opposed to low potassiumhigh sodium ion concentration in the extracellular fluid. The overall result of the asymmetry of ion distribution across the plasma membrane is an electrical potential difference from inside to outside the cell (Ussing, 1960). The puffing “pattern of rejuvenation” which Kroeger describes when salivary glands are explanted or injured by various techniques is seen by him as being due to an irritation or severing of cell membranes which subsequently allows the flooding of sodium ions into the cell accompanied by the outpouring of potassium ions. By explanting glands into sodium-free medium Kroeger can partially control the rejuvenation phenomenon. Increasing amounts of sodium in the medium force the activity pattern of the chromosomes back to that of young larvae. By replacingmore and more sodium with potassium, rejuvenation is reversed. When the potassium concentration in the medium is raised beyond that of thecell, the puffingpattern of the chromosomes progresses to that which normally occurs at a later stage of development. Kroeger believes that this is caused by exogenous potassium being forced into the cell and subsequently into the nucleus. Utilizing microelectrodes Kroeger finds that the electrical potential across the cell membrane becomes larger as development advances. At the stage of puffing pattern 1 the mean potential is 12.5 mV. This increases to a mean of 2 3 5 3 5 . 6 , and finally to a mean of 46.7 mVat puffing pattern 4 (the inside of the cell is negative to the outside). Assuming that the cellular potassium concentrations are increasing, that hemolymph potassium and sodium are constant, and that C. thummi cells are more permeable to K ions than Na ions, the increasing intracellular negativity is to be expected. Kroeger (unpublished results) also has evidence that puffs which form after the middle prepupal stage are not sensitive to changes in the Na/K ratio and has found at least one puff which is specificallysensitive to Mg ions. ACKNOWLEDGEMENTS

We express our great debt to Drs. S. Van den Bergh, D. G . Shappirio, and H. Kroeger for many critical suggestions which were incorporated into the manuscript. We are grateful to all those who answered our request

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for reprints, especially Drs. B. Sacktor and I. Agrell; and to Mr. Fredric Oltsch, Mr. Sunil Datta, and the staffs of the University of Massachusetts Library and The Marine Biological Laboratories Library at Woods Hole for help with the bibliography. The writing of this paper was supported in part by a research grant (AI-04291)from the National Institute of Allergy and Infectious Diseases, U.S. Public Health Service, and a grant from the University of Massachusetts Research Council. REFERENCES

Agrell, I. (1964). Physiological and biochemical changes during insect development. In “The Physiology of Insecta” (M. Rockstein, ed.), Vol. 1, pp. 91-148. Academic Press, New York. Alfert, M. (1954). Composition and structure of giant chromosomes. Int. Rev. Cytol. 3, 131-176. Allfrey, V. G . , Littau, V. C. and Mirsky, A. E. (1963). On the role of histones in regulating ribonucleic acid synthesis in the cell nucleus. Proc. natn. Acad. Sci. U.S.A. 49, 414-421. Ames, B. N. and Martin, R. G. (1964). Biochemical aspects of genetics: the operon. Ann. Rev. Biochem. 33, 235-258. Anderson, E. (1964). Oocyte differentiation and vitellogenesis in the roach Periplaneta americana. J . Cell Biol. 20, 13 l - 155. Aubert, H. (1853). Ueber die eigenthumliche Structur der Thorazmuskeln der Insekten. Z . wiss. Zool. 4, 388-399. Balbiani, E. G. (1881). Sur la structure du noyau des cellules salivaires chez les larves de Chironomus. Zool. Anz. 4, 637-641. Bauer, H. (1935). Der Aufbau der Chromosomen aus den Speicheldriisen von Chironomus thummi Kiefer (Untersuchungen an den Riesenchromosomen der Dipteren I). Z . Zellforsch. mikrosk. Anat. 23, 280-313. Becker, E. (1941). u b e r Versuche zur Anreichung und physiologischen charakter; isierung des Wirkstoffs der Puparisierung. Biol. Zbl. 61,360-388. Becker, E. and Plagge, E. (1939). Uber das die Pupariumbildung auslosende Hormon der Fliegen. Biol. Zbl. 59, 326-341. Becker, H. J. (1959). Die Puffs der Speicheldrusenchromosomen von Drosophila melanogaster. I. Beobachtungen zum verhalten des Puffmusters im Normalstamm und bei zwei Mutanten, giant und lethal-giant-larvae. Chromosoma 10, 654-678. Beermann, W. (1956). Nuclear differentiation and functional morphology of chromosomes. Cold Spring Harb. Symp. quant. Biol. 21, 217-232. Beermann, W. (1959). Chromosomal differentiation in insects. In “Developmental Cytology” (D. Rudnick, ed.), pp. 83-103. Ronald Press, New York. Beermann, W. (1963). Cytological aspects of information transfer in cellular differentiation. Am. Zool. 3, 23-32. Beermann, W. and Bahr, G. F. (1954). The submicroscopic structure of the Balbianiring. ExpI Cell Res. 6, 195-201. Belitzer, V. (1939). La regulation de la respiration musculaire par les transformations du phosphaghe. Enzymologia 6, 1-8.

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The Control of Polymorphism in Aphids A. D. LEES Agricultural Research Council Unit of Insect Physiology, Zoological Department, University of Cambridge, England

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I. Introduction . 11. Aphid Forms and their Terminology . 111. The Fundatrix: Form Changes in Young Clones . . IV. Clonal Variability . V. Sex Determination . . VI. The Production of Gamic Females . . A. Photoperiodic Sensitivity and the Chronology of Embryogenesis B. Response Curves . C. The-Site of the Photoperiodic Receptors . . D. Hormones and the Differentiation of Oviparae . . E. Interaction of Photoperiod with Temperature . . F. Photoperiodism in Heteroecious Species. . . G. Sexual Reproduction in Macrosiphum euphorbiae . . H. Aestivation and Gamic Reproduction . . I. Other Environmental Factors . . J. Intrinsic Factors: Anholocycly . . VII. The Control of Wing Dimorphism . . A. The Analysis of Crowding . . B. Stages Sensitive to Crowding . . C. The Mechanism of Crowding . . D. Nutrition . . . E. Water Content and Ionic Composition of the Host Plant . F. Relationships with Ants . . G. Temperature . . H. Photoperiod . . I. Intrinsic Factors . . . J. Developmental Pathways and Wing Dimorphism . . K. Endocrine Control of Wing Dimorphism . . L. Environmental Regulation of Corpus Allatum Activity . . VIII. The Inhibition of Developmental Pathways: Interval Timers . IX. Summary . . . References .

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I. INTRODUCTION The study of polymorphism in aphids can be said to date from the discovery of parthenogenesis by Bonnet (1745). His detailed and vivid observations on the reproductive behaviour of viviparous aphids quickly 207

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led to the realization that both sexual and asexual females occur in the same species, and that the life cycle follows a complex seasonal pattern of parthenogenetic and sexual generations. Nineteenth-century observers were more concerned with the intricacies of the life cycle; nevertheless, writers such as Kyber (1815) and Slingerland (1893) found that under suitable conditions a long succession of parthenogenetic generations could be reared without the appearance of the sexual forms. However, despite the realization of some early experimentalists of the importance of temperature in form changes in aphids (Loeb, 1906),the significanceof the environment as a regulator was not fully appreciated until 1924 when Marcovitch demonstrated that the appearance of the sexual forms in the strawberry root aphis (Aphisforbesi) was controlled by length of daythe first recorded instance of a photoperiodic response in animals. The nature of the causal factors influencing the development of winged and wingless aphids has also aroused endless speculation-the most favoured being that control is exercised through the food plant. It was only in 1951 that Bonnemaison called attention to the essential part played by interactions between the aphids themselves. Early in the present century observers like Tannreuther (1907) and Klodnitski (1912), who were influenced by Weismann’s doctrine of the segregation of the soma and the germ plasm, developed the view that the succession of aphid forms, and even the number of generations, was to a great extent immutable. Although these investigators underestimated the role of the environment (which they believed affected only the soma) there is probably more than an element of truth in their theory. For it is now known that a number of form changes, sometimes spanning several generations, is regulated by endogenous timing mechanisms that operate in partial independence of the environment (Bonnemaison, 1951; Lees, 1961). Several types of polymorphism are recognized in aphids. Some kinds (e.g. male/female ;sexual/parthenogenetic ; apterous/alate) are examples of “alternative” polymorphism in which the developing organism is confronted by a choice of developmental pathways. .The precocious development of the embryos in the viviparous aphid, resulting in the “telescoping” of generations (Uichanko, 1924; BruslC, 1962), has favoured the evolution of such mechanisms since the embryo is accessible to maternal influences for the entire period of embryogenesis. A further dimension is added by the operation of “interval timers” mentioned above, that temporarily restrict one or more of the available paths. In addition, aphids show “successive” polymorphism in which morphological or physiological changes of varying magnitude are seen in different

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generations. Larval/adult transformations must also be considered. Each morph has its own pattern of growth and differentiation and in the alate morphs this may culminate in a striking “metamorphosis”. These different kinds of polymorphism generate a broad series of forms and “near forms”. Some are morphologically distinct, some are recognizable with difficulty, while others are morphologically alike but are physiologically different. It is scarcely surprising that the nomenclature of aphid morphs is sometimes confusing. For this reason some remarks on terminology have been included in this article. Genetic variability provides a further possible source of differences between individuals and between lineages. In experimental work on the influence of environment on parthenogenetic lines, the assumption is usually made that the materialis genetically uniform and constitutes a clone. But this, also, has recently been questioned. The study of aphid polymorphism embraces the environment, the endocrine system of the insect and the morphological characters it controls. But since our knowledge of the morphogenetic processes is extremely meagre, detailed descriptions of morphs need not be given here. These may be found in the textbooks of Borner and Heinze (1957) and Bodenheimer and Swirski(1 957). A comprehensivesurvey of the older literature on aphid form determination has been given by Bonnemaison (1 951). The biological adaptations of aphid morphs have been discussed recently by Kennedy and Stroyan (1959). Some physiological aspects of form control have been previously considered by the present writer (Lees, 1961). This article is, of necessity, concerned with aphids that have served as material for experimental work. These species are few in number and belong almost entirely to the most specialized family, the Aphididae. It is probable that future work on more primitive families (the Pemphigidae, for example)will reveal considerable differencesin the mechanisms of form determination or may place a different emphasis on the relative importance of environmental and “endogenous” factors. 11. APHID FORMS AND

THEIR

TERMINOLOGY

The subject of aphid polymorphism is wider in scope than the terms form or morph would suggest. To those forms that are clearly separable on morphological grounds, and which provide the raw material for taxonomic research, one must add variants with a similar or identical morphology that are physiologically dissimilar. These concealed differences can be recognized only by studying the behaviour or examining the

210 A. D. L E E S reproductive potentialities of the aphid in controlled environments. The nomenclature of aphid forms is indeed based on all these criteria :morphology, behaviour (particularly “ecological” behaviour) and reproductive capacity. But even if “differences” can be demonstrated, it is often a matter of opinion as to whether this justifies the use of a separate term. On the other hand, many well-established aphid terms are used only as a convenient shorthand for describing the various phases of the aphid life cycle and do not refer to any morphological or physiological differences. In most aphids from temperate climates both sexual and parthenogenetic forms are found (holocycly), but there are also many anholocyclic species in which the capacity to form sexuales has been lost. Some species alternate between the primary, woody, host plant and one or more species of summer herbaceous plants (heteroecy); others are confined to a perennial host (monoecy). The parthenogenetic generations are also viviparous (except in the families Adelgidae and Phylloxeridae which are not usually considered as aphids in the strict sense). These forms can therefore be called viviparae or, if it is wished to acknowledge their mode of egg maturation, virginoparae. Nevertheless, since the aphids of the first parthenogenetic generation have a very distinctive morphology, they have received a special name-the fundatrix-and “virginopara” is usually reserved as a more general term for the later parthenogenetic descendants of the fundatrix. The fundatrix is a relatively sessile form, often with reduced antennae, legs and compound eyes. Only a few genera have alate fundatrices. This morph is often highly adapted to the primary host and is gall-forming in the Eriosomatidae. Unlike the virginoparae of the later parthenogenetic generations, the fundatrix is monomorphic. Virginoparae of the second generation exhibit a pronounced form change. The legs and antennae are longer and the sense organs better developed. They are more agile but less fecund. Some aphids show wing dimorphism in this generation whilst others are either alate or apterous according to species. The later parthenogenetic generations usually have the ability to produce both apterous and alate virginoparae. In heteroecious aphids the viviparae on the primary host are referred to as ,fundatrigeniae (Borner and Heinze, 1957). But as they are difficult to distinguish from viviparae belonging to later generations, this term of convenience is sometimes restricted to the first post-fundatrix generation. The terms migruntes and aZienicoZae are useful for describing virginoparae that are colonizing the secondary host plants but are of little significance when considering polymorphism.

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The sexuales comprise the egg-laying oviparae and the males. The latter may be apterous or alate according to species and are occasionally dimorphic within a species (e.g. in some clones of Acyrthosiphum pisum and Aulacorthum solani). The oviparae have a number of distinctive features. The ovaries differ in structure from those of parthenogenetic morphs: the adults are usually apterous (but are winged in the Callipterinae) and may be larviform (in the Pemphiginae). Their sense organs are sometimes reduced; and the tibia of the hind leg contains a number of structures (“scent plaques”) opening by pores on to the cuticle surface”. Most confusion arises in assessing the status of the generation of virginoparae that precedes the sexuales. The terms commonly employed are sexupara (producer of both sexes), gynopara (producer of oviparae) and andropara (male-producer). The difficulty is as follows. If the parent virginopara has a dual capacity for producing gamic and parthenogenetic offspring, its sexuparous function may represent only one of the reproductive potentialities, expressed in particular environmental conditions. On the other hand, if its ability as a virginopara producer has become restricted, this will be evidence that these more specialized individuals differ from “ordinary” virginoparae. The reproductive capacities of several species that have been tested in a wide range of environments are shown schematically in Fig. 1. In monoecious species such as Brevicoryne brassicae or Megoura viciae apterous parents can produce all four morphs-apterous and alate virginoparae, oviparae and males (Fig. 1A) (Bonnemaison, 1951 ; Lees, 1959). Alate virginoparae, produced by crowding, can also give birth to these same morphs although in very different proportions. The inability of alate parents to form many alate daughters is one notable difference (see p. 269), but there are many others. Bonnemaison (1951) refers to wingless and winged parents that are producing sexuales as sexuparae apterae and sexuparae alatae respectively. Yet these are purely descriptive terms, since the production of sexual offspring is only one of the potentialities of the “ordinary” apterous and alate virginopara. On the other hand, Hille Ris Lambers (1960) justifiably restricts this term to virginoparae that are incapable of producing virginoparous daughters. An example is found in the Eriosomatidae (e.g. Pemphigus bursurius, Fig. 1B), where the winged migrants returning to the primary host plant give birth to oviparae and males, never to virginoparae.

* In the past these organs have usually been regarded as sensilla of unknown function. But the manner in which the hind legs are waved about before copulation occurs, and the excitatory effect upon the male, suggest that these are glands dispensing an aphrodisiac (Stroyan, 1958, p. 686).

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In many heteroecious aphids of the family Aphididae (e.g. Aphis fabae, Sappaphis plantaginea) the sexuales are produced by different parents and indeed by different generations (Fig. 1C). Males and winged parthenogenetic females (gynoparae) produced by apterous parents in short day conditions are the migrant forms. The oviparae, which are born on the primary host plant, are the daughters of these individuals. Although the gynoparae do not ever appear to produce males, they can nevertheless give birth to virginoparae if the environment is suitable. In this respect they resemble the alatae of monoecious species. Unfortunately Parent

Fl A

Parent

5 F2

D

C

Apteraus vlrglnoparae

@ Alute virginoparae

0

Gynoporae

@ Oviporae

0

Males

FIG.1. The reproductive capacities of four species of aphids. A, Megoura oiciae; B, Pemphigus bursarius; C , Aphis fabae; D, Myzuspersicae.

the reproductive potentialities of alatae that have been produced by crowding do not seem to have been studied in any heteroecious aphid; but until it is proved that such alatae cannot be ovipara-producers this characteristic should not be regarded as the sole perquisite of the gynopara. What are the other special features of gynoparae? Morphological differences are slight in the extreme. Gynoparae of A . fabae have more antenna1 plate organs (secondary rhinaria) than alate virginoparae produced by crowding, but the difference is only statistical (Kennedy and Booth, 1954). There is also a distinct shift in the feeding preference.

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Although their discrimination is not complete, alate virginoparae of A . fubue prefer broad bean (Viciufubu) and gynoparae spindle (Euonymus) when offered a choice of foliage in an alternative chamber. A third criterion involves the response to crowding (see p. 239). Whereas alate virgiiioparae are usually produced in response to contacts with other aphids, gynoparae are formed in short photoperiods even if all crowding is avoided. However, this circumstance does not necessarily prove that two types of alate daughters are involved. One could postulate, for example, that alternative control systems were present, one actuated by crowding, the other by length ofday, and that both systemswere capable of channelling morphogenesis towards the same “alate” developmental path. However, this interpretation appears improbable. And in the absence of evidence to the contrary, one must conclude that the gynopara constitutes a distinct form in these species. The situation is more complex in certain heteroecious aphids with less specialized host relationships. Mucrosiphum euphorbiue is sometimes regarded as a species with host alternation, but in the strain studied by MacGillivrayand Anderson (1964) all the morphs, including the oviparae, fed and reproduced on the secondary host plant (potato). The criterion of host plant preference was therefore of no value in deciding the form of a given alate individual. Further, the range of forms produced by apterous or alate parents was wider than in A .fubue. For example, parent apterae produced alate offspring in large numbers in response to short photoperiods. These alatae resemble gynoparae of heteroecious species in that they are mainly ovipara-producers. However, unlike A . fubue, oviparae are also formed by apterous parents. Since ovipara-production is obviously much less restricted than in A.fubue, this function does not provide the means of deciding whether a given alate aphid is a “gynopara”. Neither Shull(1930a, b) nor MacGillivray and Anderson (1964) have in fact used this term. Although the distinctions between alata and gynopara have become blurred, it would be of interest to determine whether differences in the proportion of the various types of progeny can be demonstrated under controlled environmental conditions, using alate parents produced by crowding on the one hand, and alate parents produced by short day regimes on the other. The reproductive potentialities of the heteroecious Myzuspersicue are even more extensive in certain respects (Fig. 1D). Normal alatae (presumably produced by crowding) have green larvae whereas gynoparalike alatae tend to be pink in colour (Bonnemaison, 1951).Pink and green alatae can both produce the whole range of forms with the apparent exception of oviparae (e.g. apterae, alate virginoparae, gynoparae and

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males). But the gynoparae can in turn give rise to apterae, alatae, gynoparae and males, in addition to oviparae. X : CHANGES 111. THEF U N D A T R IFORM

IN

YOUNG CLONES

Reference has already been made to some of the distinctive morphological features of the fundatrix (p. 210). Although these morphs develop from fertilized eggs, the differentiation of the typical “fundatrix facies” does not seem to be associated with this mode of origin.” Thus in the adelgid Sacchiphantes abietis the overwintering larvae are derived both from fertilized eggs and from parthenogxetic mothers. The former develop into a fundatrix, the latter into a “pseudofundatrix“ which is morphologically similar and produces exactly the same type of gall (Mordvilko, 1924; Schneider-Orelliet al., 1938). On the other hand, there is evidence that low temperature may be implicated. It is known, for example, that aphids inhabiting Arctic regions often have shortened leg and antennal joints (Hille Ris Lambers, 1955; Stroyan, 1960; Prior and Stroyan, 1960). This possibility has not yet been tested experimentally

since viviparae of temperate species cannot be reared at temperatures lower than the developmental threshold (about S O C ) , nor can fertilized e g g s be hatched at high temperatures since this is incompatible with the completion of diapause. The first generation of “fundatrigeniae” are to some extent transitional between the fundatrix and her later descendants, although they resemble the latter more closely. In this type of “progressive” polymorphism, the morphological changes in successive generations proceed in only one direction. The transformation cannot be stabilized by manipulating the environment . The transition seems to involve the “carry over” of morphological features from one generation to the next. The fundatrix itself may, for example, possess characters (e.g. scent plaques on the hind tibiae) that are typical of the oviparae in the previous generation (Hille Ris Lambers, 1960). Similarly, a close examination shows that the progeny of the fundatrix are usually more fundatrix-like than the later descendants. This statement can be illustrated by measurements made on two clones of Megoura viciae (Fig. 2). It will be noted that the increase in the length of the legs and antennae, as well as in the number of antennal plate

* Hille Ris Lambers (1 960) mention; the occurrenceof a male larva of Clyphina schrankiuna in circumstancesthat suggested its origin from a winter egg. It would be interestingto know whether this rarity also possessed fundatrix-likecharacters.

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21 5

organs, was largely accomplished in one generation. Indeed, in one clone (B) the transition was apparently complete in the first generation of fundatrigeniae. But in clone A small increments were still observed in the third and fourth generations. The dark pigmentation of the legs and 5.0

5.c A

0

4-0

41c

- 15 3.0

- 10

3.c

-5

2.0

u

1

2

3

Genero tion

2 .o

-0

4

Generation

FIG.2. Progressive morphological changes in two young clones of Megoura viciae. Leg and antennal measurements (left ordinate) are in mm and refer to the combined lengths of femur and tibia of the hind leg and to the four distal segments of the antenna respectively. The right ordinate shows the number of plate organs on the third antennal segment. About twenty apterae were examined in each generation except the first which comprised only one individual-the fundatrix.

antennae, which is typical of the fundatrix, also suffers a gradual reduction in intensity; but the rate characteristics of this process are different, for even in the tenth generation viviparae are still appreciably darker than individuals taken from a clone severalyears old. Nothing is known of the developmental factors regulating these progressive transformations. Nevertheless, they are probably a function of time since the first-born daughters of the fundatrix are often more fundatrix-like than the later progeny (see p. 270).

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IV. CLONALVARIABILITY As the product of different fertilized eggs, clones may be expected to exhibit considerable genetic heterogeneity. But it has generally been accepted that no variability attributable to the recombination of recessive genes is likely to occur in the clonal descendants of a single fundatrix, maintained permanently in the parthenogenetic condition by exposure to a suitable environment. This expectation agrees with the apparent stability of such lineages. In Acyrthosiphum pisum, for example, clones have been described which differ in the mean weight of the aphids, in their rate of reproduction and in the readiness with which these insects drop from the plant when disturbed (Harrington, 1945; Cartier, 1959). Yet all these characters have remained constant over many generations. Colour variants of the pea aphid-green, pale yellow and salmon pinkhave been maintained for severalyears without change (Lees, unpublished results). Early cytological studies on diploid parthenogenesis in aphids seemed to have accounted for the observed stability of clonal characters. During the formation of the female parthenogenetic ’egg the chromosomes appear in the diploid number during prophase of the single maturation division. But synapsis was thought to be reduced or even lacking altogether. For example, in Tetraneura ulmi pairing fails completely (Schwartz, 1932), and in Aphis rosae (Macrosiphum rosae) and A . palmae (Cerataphis Zataniae) synapsis appeared to be perfunctory, homologous chromosomes separating before metaphase (de Baehr, 1920; Paspaleff, 1929). Division then takes place, one set of daughter chromosomes passing into the polar body. The transient nature of synaptic pairing has suggestedthatcrossingoverandgene segregationareunlikely to takeplace. This view has recently been challenged by Cognetti (1961a, b, 1962), whose cytological studies on the parthenogenetic eggs of Macrosiphum rosae, Brevicoryne brassicae and Myzuspersicae have led him to conclude that chromosome synapsis is sufficientlyprolonged to permit chiasmata to form in the bivalents and for crossing over to occur. After the chromosomes have fallen apart, without the dissolution of the nuclear membrane, the diploid number is restored. Cognetti regards this process of “endomeiosis” as the first maturation division, and the subsequent chromosome division and polar body formation as the second. He further considers that endomeiosis is thermosensitive. At high temperatures (28°C) this mechanism is replaced by ameiotic parthenogenesis which does not involve chromosome pairing and crossing over (see Boschetti and Pagliai, 1964).

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Cognetti maintains that endomeiosis is responsible for much intraclonal variability. He has supported this contention by conducting selection experiments in which the character chosen is the ability of the aphid to produce apterous or alate progeny (Cognetti and Dallari, 1961; Cognetti and Pagliai, 1962).In experiments with Myzuspersicae selection towards “apterousness” was carried out by starting each fresh generation with a number of apterae which were allowed to develop in large colonies under uncontrolled physical conditions. There appeared to be some reduction in the percentages of alatae formed over ten generations, but the results were somewhat irregular (Cognetti and Dallari, 1961). In later observations on Brevicoryne, each selection line was begun with a single individual (a fundatrix), and all subsequent generations with twenty parent apterae. The cultures were also exposed to two different environments, one relatively favourable to the production of apterae, the other less favourable. In the first instance the percentage of apterae rose from 70 to 100% in five generations; in the latter from 51 to 98% over twelve generations. Selection for “alateness” was less successful, but this was attributed to the fact that “selection” could only be practised in alternate generations, since alate parents tend to produce few alate progeny (see p. 253) (Cognetti and Pagliai, 1962). The principal objection to using aptera- or alata-production as a genetic character is the difficulty of furnishing an environmental stimulus of known intensity. In Megoura, for example, it is necessary to exercise careful control over a variety of factors that influence the crowding response. These include the growth stage of the aphids, the time available for mutual stimulation, the crowding density, as well as such physical factors as temperature (see p. 244). An experiment designed primarily to test the constancy of response in a line reproducing by parthenogenesis has been carried out in Megoura using the following procedure. In each generation twenty apterous aphids were reared in isolation from the third larval instar. Three days after the final moult the adults were removed from their host plants and were placed together in an empty 2 x 1 in. specimen tube for 24 h at a density of 10 per tube. The first few offspring deposited after returning the aphids to their plants indicate whether they have become alata-producers (p. 240). It is usual to find that after this treatment most parents produce mixed families of alatae and apterae, whilst a few produce no alatae at all. Apterae are therefore always available to serve as parents for the next generation. And it is also possible to run two selection lines, one employing apterae derived from parents that, despite the crowding treatment, produced no alate progeny (“apteraproducers”) ; the other using the apterous progeny of alata-producing

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parents (“alata-producers”). An additional line was also maintained, again using apterous parents in each generation, but without taking into consideration the performance of the grandparents as aptera- or alataproducers. All three lines originated from a single alata, taken from a long-established parthenogenetic clone. The selection of “alataproducers” and “aptera-producers” was begun in the third generation. Figure 3 shows that the results were entirely negative. The production of alate offspring is suppressed in the first two generations (this phenomenon is referred to later, p. 269); but over the next sixteen generations there was no permanent declinein the ability to form alate progeny inany of the three lines, although random variations were fairly considerable.

FIG.3. The response of successive generations of Megoura apterae to a standard crowding stimulus. With the exception of generation 1, which consisted of a single alata, all three lineages were propagated through apterous individuals. Note that this “selection” does not reduce their ability to form alate offspring.

Cognetti and Pagliai (1963) have recently selected different populations of Brevicoryne brassicae for their ability to reproduce parthenogenetically under conditions that would be expected to promote sexual reproduction (short days, low temperature). Virginoparae were obtained in August from different climatic zones in Italy. Stocks from high altitudes in the foothills of the Appenines could not be maintained in the test environment for more than three or four generations since all the females became oviparae. In contrast, aphids collected from Livorno, which enjoys a Mediterranean climate, produced hardly any oviparae. Those from a temperate locality (Modena) yielded a different result. The proportion of parthenogenetic offspring fell sharply during the first few generations, then increased again to reach 80%) after ten generations. Cognetti and

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Pagliai claim that intra-clonal genetic variability is being generated by endomeiosis and that the upward trend in the curve is caused by the selection of gene combinations that favour parthenogenetic reproduction in short days. However, as each selection line was started with fifty virginoparae, there is no reason why the material should not have included several clones with different response patterns. The falling section of the curve would be accounted for by the conversion of the holocyclic aphids into oviparae, the rising section by the increasing preponderance of anholocyclic clones. V. SEX DETERMINATION

It has long been accepted that sex determination in aphids is chromosomal, the mechanism being of the common XX-XO type. The chromosome number in the somatic cells of all female morphs is constant, whereas male cells have one chromosome fewer (de Baehr, 1920; Morgan 1909, 1910, 1915; Schwartz, 1932; Lawson, 1936; Ris, 1942). An exception to this rule has recently been reported by Shibata (1954)in Myzocallis kuricola where the diploid chromosome number in both sexes is 12. The implications of this finding are still uncertain. The maturation of male parthenogenetic eggs has been described by Schwartz (1932) in Tetraneura ulmi. The X chromosomes, unlike the autosomes, enter prophase in the undivided condition. They proceed to pair at zygotene but eventually fall apart without division or chiasma formation. One X chromosome then passes into the polar body. It will be recalled (p. 216) that in the female parthenogenetic egg the X chromosome divides before prophase begins so that both egg and polar body retain an XX constitution. Since the sex determining mechanism does not involve a chance process such as the random association of chromosomes, it is perhaps less surprising to find that the sex ratio often departs widely from a 1 : 1 relationship. There is frequently a marked deficiencyin males even under environmental conditions that favour their appearance. For example, the apterous virginopara of Megoura produces about 10 males and 80 female offspring at 15°C. In the alate morph the incidence of males is still lower-usually only 2-4 in a total progeny of 70-80. Additional evidence of an intrinsic regulation of males is afforded by the sequence in which the male progeny is born. As the embryos in different ovarioles appear to keep in step during their development, the time of parturition probably reflects with some accuracy the sequence in which the eggs were ovulated. I n Megoura the males are usually born in the middle of the reproductive

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life of the parent virginopara. But this pattern is a species characteristic. In Acyrthosiphumpisum, for example, males tend to be born last. Under short day conditions this aphid often gives birth to a sequence of 50 or so females (oviparae), followed by an unbroken run of 20-30 males (Kenten, 1955;Lees, unpublished results). Some “androparae” in Metapolophium dirhodum are unusual in that they produce only males (Hille Ris Lambers, 1960). There is no reason to believe that abnormal sex ratios are ever caused by the differential mortality of embryos. Degenerative changes are easily recognized, yet in Megoura where the first twenty or so ovulations are always female no such changes can ever be detected. The characteristic distribution of male and female eggs within the entire egg sequence is better regarded as an intrinsic property of the ovary which differs according to the morph and the species. It is possible that a spatial factor, such as the position of the oogonia within the ovary, is involved. The production of males is sometimes influenced by the immediate ancestry. For instance, the formation of male embryos is entirely inhibited in virginoparae that have recently descended from a fundatrix (see p. 268). When this “interval timer” has operated, the sex ratio comes under environmental control. Although the proportion of males cannot be increased by any known means beyond the maximum characteristic of the species and morph, male production is readily suppressed by exposing the parent to the appropriate environment. In species where the sexual females are photoperiodically determined, this factor may also completely prevent the appearance of males (e.g. in Acyrthosiphumpisum; Kenten, 1955). Sometimes, however, the control is less rigid and a few males develop under long day conditions (Brevicoryne brassicae; Bonnemaison, 1951). In other species males are differentiated regardless of photoperiod, provided the temperature is suitable (Megoura viciae; Lees, 1959). The role of temperature is influential but complex. In Aphis chloris (Wilson, 1938), Myzuspersicae, and Brevicoryne brassicae (Bonnemaison, 1951) constant high temperatures are particularly effective in eliminating males. In Acyrthosiphum pisum and Megoura viciae both high and low temperature treatments produce few males, the most favourable temperatures being in the 15-20”C range (Kenten, 1955; Lees, 1959).In Myzocalliskuricolait is believed that male production is promoted by fluctuating temperatures (Shibata, 1952, 1954). There is still little to indicate how these agencies control chromosome behaviour. It is conceivable that temperature influencesthe chromosomal mechanism of the oocyte directly, although this is less probable in the

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22 1

case of photoperiod. An alternative possibility is that both the meiotic chromosome behaviour and the differentiation of older embryos as virginoparae or oviparae are controlled by the same maternal hormone. This suggestion is compatible with the chronology of embryogenesis. In most aphids, as mentioned earlier, males are born in the middle or towards the end of parturition. Their sex would therefore be decided during the early larval development of the mother, that is, at the time when the female determiner is operating(seep. 222). A similar explanation will also account for the occurrence of “androparae” in Metopolophium dirhodum (Hille Ris Lambers, 1960). Since these viviparae produce no daughters, the first offspring must have been determined as males during the prenatal development of the mother, that is, at the time when ovulation is starting (see Fig. 6). But it is now known that the photoperiodic centre also begins to function in the embryo and is triggered by light transmitted through the body wall of the grandparent (Lees, 1964). It follows that the control mechanism need not necessarily be looked for in the grandparent. V I . T H E P R O D U C T ~ OONF G A M I CFEMALES The ability to form sexual females (oviparae), as well as parthenogenetic virginoparae, is widespread in aphids from temperate climates. A number of factors, both exogenous and endogenous, influence these dual capacities. Environmental photoperiod and temperature are of very general significance. The parental type and the operation of timedependent inhibitors are important innate factors. As in the case of males, both parentage and the more remote antecedents must therefore be taken into account. Photoperiodic control will be considered first. The connexion between day length and the seasonal appearance of sexual females was discovered by Marcovitch (1924) in Aphis forbesi. Oviparae were induced to develop prematurely in summer by restricting the normal daylight; and gamic reproduction was postponed by extending the daily light period. Many other species have since been shown to have similar photoperiodic responses. They include Aphis chloris (Wilson, 1938), Brevicoryne brassicae (Bonnemaison, 195l), Acyrthosiphumpisum (Kenten, 1955) and Megoura viciae (Lees, 1959,1960,1963).These are all monoecious species without host alternation. The heteroecious Aphis fabae (Davidson, 1929; de Fluiter, 1950), Myzuspersicae (Bonnemaison, 1951)and Sappaphisplantaginea (Bonnemaison, 1958)are also responsive to photoperiod. So also is Macrosiphum euphorbiae ( M . solanifolii), a species showing some intermediate features. Since the photoperiodic

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relationships of monoecious aphids appear to be less complex, they will be described first, with special reference to Megoura. A. P H O T O P E R I O D I C S E N S I T I V I T Y A N D T H E C H R O N O L O G Y OF E M B R Y O G E N E S I S

Short day photoperiodic treatment in Megoura has no overt effect on the parent aptera, even if the exposure is maintained from birth. The end effect is only observed in the progeny which may become oviparae. Although this observation shows that the final target of photoperiod is the developing embryo, it is important to discover whether the latter is influenced by light passing through the abdominal cuticle of the parent or whether its differentiation is subordinate to a maternal photoperiodic centre. There is no doubt that the second possibility is correct. When Megoura is allowed to develop in a “neutral” photoperiod of intermediate length both virginoparae and oviparae are sometimes produced by the same mother. If the embryos responded independently to photoperiod their determination as oviparae or virginoparae would be a random process. Since embryos of similar age keep in step during development, batches of larvae should contain both morphs. But serial collections of the progeny show that the two morphs are not born in random sequence, but occur in alternating groups, as if the system controlling their production were in unstable equilibrium. A second and more direct proof of the maternal control of photomorphogenesis can be derived from experiments on localized illumination, described on p. 228. Nevertheless, it is worth noting at this juncture that photoperiod can influence the embryos directly; but it does so at a later developmental stage, some time after their determination as virginoparae. The switching action of the maternal photoperiodic mechanism has been further examined by transferring aphids from long to short days and vice versa at different stages in their ontogeny. Some experimental results are given in Fig. 4. The histograms show that the fate of the embryos as virginoparae or oviparae is decided progressively. There is indeed a close relationship between the time of reversal of the photoperiod and the time of appearance of the alternative morph in the progeny sequence. We see, for example, that when first instar larvae are exposed to long photoperiods until the third instar they begin their reproductive lives as virginoparaproducers and this morph makes up 60% of the entire offspring. If the transfer to short days is delayed until the final moult, a longer series of virginoparae is produced (86% of the total). And 3 days later, as parturi-

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tion is beginning, all the embryos are found to be immune from photoperiodic influence ; in other words, their determination as virginoparae has been completed. This finding introduces a second important component in the system controlling embryonic determination, namely the competence of the embryos to respond to maternalcontrol. Embryos that have reached a certain point in morphogenesis (certainly by the stage of incipient eye pigmentation) can no longer respond, even though the maternal centre is still causing the younger embryos to differentiate as virginoparae.

20 40

Virginoparae

.*+..

Oviporae

FIG.4. Progressive determination of the embryos in growing apterae of Megoura uiciae. Theeffect is demonstrated by reversing the photoperiod at differentstages in the development of the parents. S, Short day; L, long day.

An additional fact to be taken into account is that while the maternal centre is functioning in the larva, the number of embryos in the ovarioles is increasing (Fig. 5). The newly born larva has only two, the third instar four, the adult six. Since the modal number of ovarioles in Megoura is 18, we can infer the positions that the progeny occupied as embryos (assuming, of course, that the embryos in different ovarioles develop synchronously and that no embryos degenerate). A reconstruction of this kind is given in Fig. 6. It depicts the ultimate fate of the embryos in four different switching experiments. We see that when the long day treatment precedes the short (a, b) only the youngest embryo remains undetermined H

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in the teneral adult. Yet there is no doubt that this embryo, which is destined to become an ovipara, is already present while the older embryos are undergoing determination as virginoparae. It seems then that the youngest embryos are also unresponsive to the maternal regulator. The ovarioles of newborn larvae already contain two embryos each and these develop in conformity with the photoperiod maintained during postnatal development of the parent. This circumstance suggests that the maternal photoperiodic centre does not begin to function until birth. But this can be disproved by exposing the grandparent to long or short r

0.2 mrn i

B 4

FIG.5. Outline drawings of ovarioles in Megouru viciue at different stages in the development of the apterous parent. (a) First instar larva; (b) third instar larva; (c) teneral adult; (d) adult at inception of parturition; (e) older adult which has already given birth to eightytwo young. I, Germarium; 11, degenerating embryo. (From Lees, 1959.)

photoperiods for several days before the birth of the parent and subsequently applying a weakly antagonistic photoperiod during the postnatal phase. The response is reversed more easily in one direction than in the other. A prenatal short day treatment appears to exert no effect and reversal is prompt. On the other hand, a long day prenatal regime is not readily cancelled by a later weak short photoperiod so that the parent invariably gives birth to a short initial sequence of virginoparae before switching over to oviparae. This is interpreted as meaning that the photoperiodic mechanism is activated by long but not by short photoperiods, and that it first becomes functional several days before birth (Lees, 1963). Experiments with localized light beams have shown that the embryo is

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influenced directly, by light passing through the integument of the grandparent. These ideas are also consistent with another feature shown in Figs. 4 and 6 . In this series of experiments all the parent apterae received a prenatal long day treatment. It is obvious from the progeny sequences that reversal is much more immediate if, during the postnatal period, the short photoperiod precedes the long (Fig. 6 , cf. b and d). The reason may be that the maternal control centre can easily be “turned on” by long days at any point in larval development, particularly if it has already been partially activated during the prenatal period. But it may be more difficult to “turn off” the activated centre with short day exposures.

FIG.6. The effect of different photoperiodic regimes on the sequence of female embryos in the ovarioles of Megouru uiciue (apterous parent). Hatched areas, embryos destined to become virginoparae; dotted areas, future oviparae; black areas, degenerating embryos. Note that embryos at growth stages 5 and 6 have already begun to form the next generation of embryos. Stages ofdevelopment of the mother are: (1) first instar larva; (2) third instar larva; (3) teneral adult; (4) female near end of reproductive life. (From Lees, 1959.)

The slightly different photoperiodic response of the alate virginopara of Megouru can also be explained along these lines. This morph can produce exclusively oviparous daughters if maintained in short days from the prenatal period (Lees, unpublished). But when short days are given only from the time of birth, the alate mothers invariably produce an initial series of virginoparae before switching over to oviparae. Reversal of a previous long day response is even more difficult to achieve in Acyrthosiphum pisum (Kenten, 1955 ; Lees, unpublished). In some individuals three or four long day cycles during the prenatal period is sufficient to ensure that the parent will remain a virginopara-producer throughout the reproductive period, even though the entire larval and adult life has been spent in short photoperiods.

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Megoura apterae that have developed under different photoperiodic regimes can be classified in three ways, as virginopara-producers, as ovipara-producers or as producers of both forms. The response to different 24 h cycles of light and darkness is shown in Fig. 7. In all cases the grandparents had been taken from cultures maintained in long 16 h days. But in one set of curves (solid lines) the parents only received the experimental exposures from birth onwards. In the second set (interrupted + v)

n 50

2 50

z ............

'

Sl P0Op,; 50

-..._....

0

...-.-.--.-...--

2

4

i,.,1

.. .. -._ *: d.G%@ .............................

s

0

,

8

12

14015 16

24

I4 Photoperiod (h light per 24 h)

FIG. 7. Photoperiodic response curves for apterae of Megoura oiciue. Parents either produce oviparae only (A), virginoparae only (B), or mixed families (C). Two prenatal treatments were employed.Apterae were either exposed during their late embryonicdevelopment to a long 16 h photoperiod (-) or to the same photoperiodic regime that was given after their birth (. ..).

lines) the grandparents had also been exposed to the indicated photoperiods for 4 days before the birth of the parent. With photoperiods lying within the range 4-14 h (combined, of course, with complementary dark periods of 1G20 h) all the parents developed as ovipara-producers (Fig. 7A), and only virginopara-producers were obtained in photoperiods longer than 15Ij h. But mixed families were frequently met with when parents had been reared in photoperiods

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intermediate between long and short; and this also occurred in ultrashort photoperiods or in permanent darkness. It will be seen that the critical photoperiod comes at about 146 h in aphids that have received only postnatal treatment, whereas it lies at about 14 h 45 min when the experimental photoperiod has also been given during the prenatal period. The reason is as follows : if prenatal long days are followed by postnatal short days, a reversal of the photoperiodic switch mechanism is involved. “Strong” photoperiods are adequate to reverse this incipient effect promptly and the parent produces only oviparae. But photoperiods that fall just short of the critical length exert weak short day effects. Reversal is then delayed, and the run of oviparous daughters is usually preceded by a short sequence of virginoparae. This results in a slight displacement of the response curve. From the standpoint of experimental design it is obviously preferable to maintain the photoperiod constant throughout the period of photoperiodic sensitivity. Nevertheless, when this is done, a high proportion of aphids still give birth to mixed families when exposed to critical photoperiods. The tendency of aphids reared in intermediate photoperiods to produce alternating batches of virginoparae and oviparae has been referred to previously (p. 222). It seems that under these finely balanced conditions the maternal determiner is switched “on” and “off” irregularly. It is near this point of unstable equilibrium that slight changes in the response are most easily detected. A 15min difference is easily perceptible in a population of aphids. The previous paragraphs have carried the implication that the photoperiod, and not the intervening dark phase, is the essential element in time measurement. In fact, the reverse is true. This can be shown by combining a dark period that exceeds the critical length of 9a h (i.e. 24- 144 h) with very extended photoperiods of up to 40 h in length. With such cycles ovipara-producers are still formed, indicating that the significant part of the cycle is the long night and not the short day. Light appears to act by cancelling out the dark-induced timing reaction. C. T H E SITE OF T H E P H O T O P E R I O D I C RECEPTORS

This problem has been approached in Megoura by illuminating small areas of the aphid’s body (Lees, 1964). The localized light supplements a preceding short day period of general illumination. If the light is perceived, the photoperiod is effectively extended (the dark period reduced) beyond the critical length required to produce a long day effect. A positive response is shown if the aphid, previously reared in short photo-

228 A. D. LEES periods, “switches over” to the production of virginoparae. About eight daily cycles of photostimulation are required. During the period of localized illumination the young adult apterae are attached to the illuminators. Fortunately, this short period of starvation does not interfere with the photoperiodic response. And since the host plants are not exposed to the supplementary photoperiod, a positive reaction also shows that the plants can be excluded as possible photoperiodic “receptors”.

FIG.8. Sensitivity of Megouru to localized photoperiodic stimulation. The areas covered by the capillary illuminators are shown as circles. The numbers of parents tested, and those responding to the supplementary light, are also given. (From Lees, 1964.)

In the micro-illuminators devised for this purpose the beam from a small source was contained in a short length of metal capillary. Aphids could be attached to the free end of the capillary by suction or, in the case of very small diameter tubes, by means of a polyvinyl alcohol adhesive. When very small areas were illuminated plastic fibres were used to conduct light to the required area. Some results are shown in Figs. 8 and 9. Although the abdomen of the adult insect is packed with embryos, none were ever caused to develop

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as virginoparae by direct abdominal illumination. On the other hand, when the head and thorax were exposed to a long day and the abdomen to a short one, all the parents responded (Fig. 8A). Stimulation with the capillary illuminators of smaller diameter showed that light sensi-

I

,'

L

~. -

~

Anterior lobe of salivary gland

Pars intercerebralis

----__

- - - Thoracic

ganglia

FIG.9. A-D, Illumination of the head of Megouru, using light-conducting fibres. Positive and negative responses to photoperiod are indicated by plus and minus signs. E, Dissection of the head and prothorax to show the disposition of some of the major internal organs. (From Lees, 1964.)

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tivity was restricted to the head capsule. When the light beam was normal to the integument, the centre of the dorsum provided a particularly favourable site (Fig. 8C). Fewer responses were secured when the capillaries were placed laterally over one compound eye. These results suggest that the compound eye and optic nerve is not essential for the perception of photoperiodic light stimuli. This has been confirmed by destroying the eyes with a cautery. Since the response persists in many blinded insects, the light pathway and receptors must still be intact. Further exploration with the fibres of smaller diameter indicated that if the beam was sufficientlybright, a response could be elicited from almost any part of the head-a possible consequence of light scattering (Fig. 9A). But when the conducted light approached threshold intensity, the sensitive spots were usually confined to areas near the midline of the dorsum, slightly anterior of centre (e.g. Fig. 9D). The apterous aphids studied do not possess ocelli; and as no specialized receptor structures could be discovered in the cuticle or epidermis of this region, it was concluded that the photoreceptors must be located in the underlying brain. The pars intercerebralis of the protocerebrum is perhaps the most probable area. The precise nature of the receptors is still in doubt. Since there are at least two different groups of neurosecretory cells in the pars cerebralis of aphids, it was suggested as a hypothesis that these cells might function both as receptors and as humoral effectors. However, it is equally possible that other sensory elements are involved. As the photoperiodic reaction is many times more sensitive to blue light than to longer wavelengths in the green, orange and red part of the spectrum, it is apparent that an absorbing pigment must be present. L'HClias (1962a, b) considers that these pigments are photolabile pteridines. Fluorimetric estimations of pteridines in the head and body of Sappaphis plantaginea indicated that these substances diminished in concentration in the head after exposure to long days (16 h) or permanent illumination, and that they accumulated after treatment with short 12 h photoperiods. But it seems likely that most of this pigment may have been located in the compound eyes which, as we have seen, are not involved in the photoperiodic response. L'HClias also states that some oviparae were produced after injecting biopterin or folic acid. But it is difficult to understand why this treatment should have required three generations to take effect. Hille Ris Lambers (1960), on the other hand, has drawn attention to the resemblance of the aphin pigments to the plant toxin hypericin. Both these substances are quinonoids with marked photodynamic action. The fact that the aphin pigments are mainly concentrated in the fat body and embryos, and not in the head of the

THE CONTROL OF POLYMORPHISM IN APHIDS

23 1

aphid, does not of course rule out the possibility that these or related substances are concerned in photoperiodism. Whatever its nature, the pigment seems to be present in low concentrations, and perhaps may be localized in a few cells, for the brain tissue appears to be entirely colourless when viewed under the high power binocular microscope. D . H O R M O N E S A N D T H E D I F F E R E N T I A T I O N OF O V I P A R A E

Since the maternal photoperiodic centre is remote from the ovarioles (which are probably without innervation), it seems likely that embryonic differentiation is controlled by a hormone liberated by the brain. This idea receives some support from the finding that aphids exposed to long day rigimes can sometimes be caused to produce oviparae by damaging the brain with a cautery. This suggests that the release of the hormone is induced by long day photostimulation. If this interpretation is correct, it seems that the embryos of appropriate age will become determined as oviparae if the secretion is withheld. The later differentiation of the oviparae is doubtless under intrinsic endocrine control. But of this very little is known. It is worth noting, however, that in Megouru the ovipara is a more extreme neotinic than the apterous virginopara. Its apterous condition, the lack of abdominal pattern, the reduced number of antenna1plate organs (about ten) and the light pigmentation of legs and antennae-these are all larval features. It would not therefore be surprising if this metathetely was associated with the hyperactivity of the corpus allatum. In fact, it has been found that the topical application of juvenile hormone extract to developing oviparae results only in a further slight reduction in the pigmentation of the legs and pronotum. This result is, of course, to be expected if an excess of juvenile hormone is already present in the blood. E. I N T E R A C T I O N O F P H O T O P E R I O D W I T H T E M P E R A T U R E

High temperatures seem always to act in unison with long photoperiods in suppressing the appearance of oviparae. In Brevicoryne brussicue a temperature of 25°C cancels the effect of a short photoperiod so that neither oviparae nor males are formed. At 22°C some males appear, but no oviparae; at lower temperatures both morphs are differentiated. The same relationships hold good for the differentiation of gynoparae in Myzus persicae (Bonnemaison, 1951). Low temperature, on the other hand, does not prevent long photoperiodic regimes from promoting the development of virginoparae. The photoperiodic response in Megouru is virtually temperatureH*

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independent within the range 10-20°C (Lees, 1963). The only consequence is that the critical photoperiod shortens by about 15 min for each 5°C rise of temperature within this range. This shift is caused by the action of higher temperatures in cancelling the weak influence of photoperiods that are slightly shorter than the critical length. The overall effect is then equivalent to a long day. Above 20°C temperature compensation fails rapidly, so that at 23°C parents either become virginopara-producers under short day conditions or yield mixed families of virginoparae and oviparae. F. PHOTOPERIODISM IN HETEROECIOUS SPECIES

In migratory aphids two generations are required for the production of the gamic females. The first stage at least of this process is photoperiodically controlled. When apterae of Aphis fabae are reared in isolation from the first instar, in a 12 h photoperiod at 15"C, they give birth almost exclusively to winged daughters (gynoparae) and males, although a few apterae may also be formed. The gynoparae show a feeding preference for the primary host, Euonymus, and in continuing short days, and under conditions of moderate temperature, produce only oviparous offspring (de Fluiter, 1950; Lees, unpublished results). In Sappaphis plantaginea where the production of gynoparae is also induced by short photoperiods, Bonnemaison (1958) has shown that the short day effect can be cancelled by high temperatures. It is not yet known whether the differentiation of embryos as gynoparae is maternally controlled; and the time of determination of the embryos as gynoparae is also uncertain. Since they are almost indistinguishable morphologically from alate virginoparae induced by crowding, it appears possible that both these forms are determined relatively late in embryonic development, perhaps just before birth. In this event, the developmental potentialities of the embryos could be represented by a three branched pathway, with different routes leading to the apterae, alate virginoparae and gynoparae respectively. Gynoparae also respond to the environment. Shull(l928, 1942) found that at temperatures above 24°C oviparae were replaced by apterae in the progeny of alate Macrosiphum solanifolii ( M . euphorbiae). This experiment was repeated by de Fluiter (1950) on authentic gynoparae of A .fabae with similar results. According to Bonnemaison (1958) gynoparae of Sappaphis plantaginea are responsive both to temperature and photoperiod. Long photoperiods and high temperatures are equally effective in causing them to switch over to the production of parthenogenetic daughters.

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G. S E X U A L R E P R O D U C T I O N I N MACROSIPHUM EUPHORBIAE

Shull’s (1928, 1929, 1930a, b) extensive researches on this species require special comment. The assessment of this work has been greatly assisted by the careful and detailed studies of MacGillivray and Anderson (1964) on the same aphid. As these writers have pointed out, some of the difficulties of interpretation are due to the fact that Shull worked with colonies of aphids and not with individuals, so that no lines of descent could be traced. Moreover, it does not appear that the conditions of maintenance of his stock cultures were such as to suppress gamic reproduction completely. The result may have been that the sexuales-inducing stimuli had already begun to operate in some of his insects. But many of the ambiguities may certainly be ascribed to the complexity of this species as experimental material, and in particular to the difficultyin distinguishing “virginoparae alatae” from “gynoparae”, if indeed there is any essential difference in this species (see p. 213). In Shull’s experiments it is impossible to estimate the relative numbers of his “winged forms” that have arisen as a result of crowding rather than from exposure to reduced day lengths. MacGillivray and Anderson (1964) have traced the progenies of individual aphids through several generations under constant environmental conditions. As the parent aphids were transferred to a fresh excised potato leaf at daily intervals, leaving only very small larval clusters, it is unlikely that crowding was an important factor. However, for the reasons discussed earlier, these writers use the neutral term “alate viviparae” rather than “gynoparae” for the alatae produced in their experiments. Some of their results are given in Fig. 10. Four factors were of significance. Photoperiod. First generation apterous parents, when exposed to 11-13 h photoperiods over the temperature range 10-21 ”C, produced both alate daughters and oviparae, with the former predominating. These second generation alatae yielded only oviparae in generation 3 (Fig. 1OA). With the photoperiod extended to 13i h, parent apterae produced apterous daughters in significant numbers (13%), although alate viviparae again made up the bulk of the progeny (Fig. 10B). On testing the second generation, the alatae once more proved to be mainly oviparaproducers, whilst the apterae, having “escaped” the consequences of low temperature and a short or intermediate photoperiod, responded in the

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same way as their parents, producing mixed families of alate (54%) and apterous daughters. Temperature. Exposure to a higher temperature (16°C) reduced the proportion of oviparae in the first generation progeny (Fig. 1OC). When combined with a long photoperiod, high temperature strongly promoted

Apterous wirginoporoe

.Alate virginoporae

enerotion 2 Generation 3

Oviparoe

? h photoperiod, ll°C

Moles

I

Generotion 2

Generotion 3

Generation 4

\ Generation 4

FIG.10. The forms produced by Macrosiphum euphorbiae when this aphid is exposed over several generations to different combinations of photoperiod and temperature. The parents and offspring in successive generations are connected by arrows. (Based on data from MacGillivray and Anderson, 1964.)

parthenogenetic reproduction. Indeed, the progenies of three successive generations were mainly composed of apterae, with no oviparae and few alatae (Fig. 10D). But very high proportions of males sometimes occurred in the later generations. Parentage was also significant. For example, apterous parents tended to produce more alate viviparous females and males; while alate parents

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gave a higher proportion of apterous viviparae and oviparae. The occurrence and incidence of a particular form in the progeny was also sometimes influenced by the generation to which the parent belonged. This was particularly noticeable in connexion with the differentiation of males (Fig. 10D). Whilst it would be unwise to underrate the obvious complexities of form determination in M . euphorbiae, this aphid evidently possesses features that are common to both monoecious and heteroecious species. Under conditions that promote the production of gamic forms (short photoperiods and low temperatures) many “alate viviparae” are produced by uncrowded apterous parents. Since these alates yield only oviparae they would be classed as gynoparae in a species with well defined host alternation. At the same time, the ability of these same parent apterae to produce oviparae is characteristic of monoecious aphids such as Brevicoryne or Megoura. It is possible that some of the effects of parentage are due to the relative importance of these two pathways in generating oviparae. It is perhaps to be expected that most “gynoparae” would be formed by apterous parents (although the response of normal “crowd induced” alatae to short photoperiods hardly seems to have been investigated in any migratory species). At the same time, if the two-stage “gynopara pathway” takes precedence over the one-stage route, we should expect most of the oviparae to stem from alate “gynoparous” mothers. Setting aside the uncertainties surrounding Shull‘s work, and regarding his alatae as “gynoparae”, many of his findings are seen to be in agreement with more recent work on monoecious species. The response curve for “wing production” in different environmental photoperiods is almost identical with the corresponding curve in Megoura relating photoperiod and the formation of oviparae (Fig. 7) (Shull, 1929). Shull also noted several other important features of the response which have since been confirmed in other species. For instance, he observed that the induction of alatae required daily exposure to a continuous dark phase of about 12 h; and he emphasized that the response was virtually independent of light intensity. H. AESTIVATION A N D GAMIC REPRODUCTION

A few species of aphids which remain on the woody host during the summer instead of migrating to herbaceous plants, undergo a lengthy period of summer diapause. Photoperiod controls both the course of aestivation and the subsequent development of the sexuales. The aestivating first instar larvae of Periphyllus testudinatus are distinct morphs,

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the body and legs being fringed with leaf-shaped chaetae. Aestivation begins in the third post-fundatrix generation and continues from midMay until late August. The larvae then develop as sexuparae, giving birth to oviparae and males. As it has proved impossible to postpone or suppress the appearance of the aestivating larvae by manipulating the environment, an “endogenous” mechanism has been proposed (Bonnemaison, 1956). But the length of diapause is photoperiodically controlled. In 16 h photoperiods its duration may exceed 160days, whereas in a short 8 h photoperiod it lasts only 12-44) days. The larvae then develop into apterous virginoparae and under outdoor conditions some eight more generations of virginoparae may be interposed before the sexuparae are formed in September. Adults of Drepanosiphum platanoides undergo a reproductive diapause in summer. Prolonged crowding seems to be responsible for inducing this state, but long photoperiods are again necessary for its maintenance (Dixon, 1963). Exposure to short photoperiods causes reproductive activity to be resumed and also induces the production of sexuales. This is associated with an increase in the size of the corpus allatum. I. O T H E R E N V I R O N M E N T A L F A C T O R S

Although day length and temperature are clearly of major significance, other external agencies may sometimes participate in controlling the seasonal development of the sexual forms. Nutrition seldom seems of importance and certainly the appearance of the sexuales of Brevicoryne brassicae or Megoura viciae is not induced by the maturity or senescence of the host plant, provided the insects are otherwise exposed to long day conditions (Bonnemaison, 1951; Lees, 1959). One positive instance described by Bonnemaison relates to the reproductive behaviour of Myzus persicae when reared in a physical environment that is causing gynoparae to develop. Under these circumstances the substitution of cabbage for the drier peach foliage is said to cause the resumption of parthenogenetic reproduction. Particular interest attaches to form determination in subterranean aphids. As a result of his work on the strawberry root aphid, Aphis forbesi, Marcovitch (1 924) was inclined to believe that the effect of photoperiod must be transmitted through the plant. Nevertheless, this species is not wholly subterranean and tends to feed at or just below soil level, so that access to light might occur. However, it is most unlikely that this could happen in such entirely subterranean root-feeders as the Eriosomatidae. Yet many of these species are holocyclic, forming sexuparae

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which, on leaving the roots of herbaceous plants, migrate to the primary host. Nutrition may perhaps play some part. According to Schwartz (1932) colonies of Tetraneuru ulmi are wholly converted to sexuparae by late summer if the host is an annual grass (oats), while many individuals continue to reproduce parthenogenetically during the winter if the host is the perennial Lolium. In England sexuparae of Pemphigus bursarius are formed in June on the roots of April-sown lettuce. But when plants are overwintered in a glasshouse, sexuparae are produced as early as March (Dunn, 1959). This again suggests a nutritional factor and militates against the possibility that photoperiod is acting through the plant. But more experimental work is required. Crowding. Bonnemaison (1951) concluded that under short day conditions the proportion of gynoparae in Myzus persicae was increased if the population density was high. But he did not distinguish between the performance of alatae produced by crowding and gynoparae induced by short days. J. I N T R I N S I C F A C T O R S : A N H O L O C Y C L Y

A few European species which remain throughout the year on the primary host plant, without aestivation, produce their sexual forms in midsummer. Examples are Dysaphis devecta which has four generations a year on apple, the fundatrix being followed by two generations of viviparae and a final generation of sexuales. Aphis farinosa ( A . suliceti), Mindarus abietinus and Lachniella costata have similarly abbreviated life cycles (Hille Ris Lambers, 1960). Sexual reproduction therefore occurs at a season when temperatures are relatively high and the day length long. Some boreal aphids even produce their sexual forms in continuous daylight. Although experimental work is lacking, it seems improbable that these species are in fact responding to external cues. The sequence of forms may indeed be predetermined, and geared to a timing mechanism of some sort (see p. 265 et seq.). Tropical aphids frequently lack the ability to produce sexuales, or do so sporadically. A number of species from temperate latitudes are also entirely anholocyclic and survive the winter as parthenogenetic virginoparae in situations sheltered from low temperatures (e.g. Myzus asculonicus, Rhopalosiphon latysiphon, Aulacorthum circumflexurn). In other species both anholocyclicand holocyclic strains are found, sometimes side by side in the same locality (e.g. Myzus persicae, Acyrthosiphum pisum). The origin of anholocycly is obscure. Bonnemaison (1951) considered that the ability to produce sexual forms could be impaired by a protracted

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phase of parthenogenetic reproduction. This conforms withhis theory that sexual reproduction in aphids requires the presence of an internal factor (“facteur interne”) which can be dissipated in old clones. Bonnemaison believed that he had observed this phenomenon occurring in 19-monthold stocks of Brevicoryne brassicae and Myzus persicae. Unfortunately, these clones (which were started not from a fundatrix but from overwintering virginoparae) appear to have been virtually anholocyclic at the outset of the experiment. In contrast, clones of Sappaphis plantaginea and Megoura viciae have been propagated parthenogenetically for 8 and 14 years, respectively, without losing their powers of producing sexual forms (Bonnemaison, 1958;Lees, unpublished). It seems more probable that the anholocyclic character arises in the fertilized egg as a gene mutation which is subsequently preserved in all members of that clone. Certainly, anholocycly is a stable condition and, once lost, the ability to produce sexual forms seems never to be regained. (This has been tested by exposing an anholocyclic clone of A . pisum to short photoperiods for 3 years (Lees, unpublished).) Such mutants may well be comparatively rare. When anholocyclic clones are found in localities where virginoparae cannot have overwintered successfully, one would suspect that the aphids have arrived as migrants and that the anholocyclic character is not necessarily of recent origin. Nothing is known of the physiology of anholocyclic reproduction. The “defect” may lie either in the maternal determiner or in the competence of the embryos to respond to it. VII. THECONTROLO F W I N G DIMORPHISM Many biotic and physical variables have been regarded-often with little justification-as being influential in regulating the development of apterous and alate viviparae. Most of these are listed below under headings 1-4. In addition, the environmental response is often modified or abolished by “intrinsic” factors which can be defined in relation to the form of the parent or even to that of the more remote ancestors. 1. The density and behaviour of aphids on the plant (“crowding”). 2. The condition of the food plant: (a) Nutritiousness of the plant; the quantity of the food; plant maturity; starvation effects. (b) Water relations and salt composition. 3. Relationships with ants. 4. Physical agencies; temperature and photoperiod.

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5. Intrinsic factors: the ancestry of the aphid. (a) Form of the parent. (b) Clonal differences. In general, most authors have believed that the nutritional state or the water balance in the aphid was decisive. And although the appearance of alatae has long been associated with the growth of populous colonies, the condition of the host plant and not the population density has usually been regarded as the prime mover. Unfortunately, however, one essential factor had been overlooked, namely the sensory perceptions of the insects themselves and their ability to recognize one another. The role of group effects (“effet de groupe” and “effet de mass$’) in regulating various activities in insect populations had previously been stressed by Grass6 (1946). But the relevance of this behavioural approach to the study of aphid wing polymorphism was first pointed out by Bonnemaison (1951). The importance of this discovery is considerable. Since anything that affects the aggregation or dispersal of aphids also influences the type and frequency of the contacts that develop between individuals, rigorous experimental methods are required to exclude the possibility that other variables, nutritional or otherwise, may be acting through the crowding response. This circumstance makes the interpretation of much of the older work difficult or even impossible. A. THE A N A L Y S I S O F C R O W D I N G

Several earlier authors had succeeded in rearing alata-free generations-usually by keeping their aphid colonies sparse-and had noted that alatae again appeared as soon as the population density was allowed to rise (Slingerland, 1893;Reinhard, 1927). The correlation between population density and the incidence of alatae has since been demonstrated many times by authors who were careful to standardize both the numbers of aphids and the size of their experimental plants or excised leaf segments (e.g. Schaefer, 1938; Noda, 1958). Nevertheless, the effect of high aphid densities on the appearance of alatae was generally attributed to partial starvation. In Bonnemaison’s (1951) experiments on Brevicoryne brassicae this possibility was guarded against by transferring the aphids in their small leaf cages to a fresh leaf surface each day. Although damage to the plant had been virtually excluded, and food could no longer be limiting, there was still a pronounced density effect. Similar conclusions have recently been reached by Johnson (1965). In his extensive studies on Aphis craccivora punched leaf disks floating in a nutrient solution were used for confining the aphids to standard leaf areas.

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In some aphids the interactions between individuals can be studied in complete isolation from the plant. In Megouru, for example, parent apterae removed from the host bean plants and confined singly in a 2 x 1 in. specimen tube can survive for 3 days at 15°C. If the apterae have previouslybeen reared in isolation, only apterous daughters are born when they are returned to the plant. But if similar apterae are confined in groups of two or more (10 per tube being the standard number), many Botch 1

A

B

C

2

3

4

I

Off plont for 24 h crowded

D

Crowded

E

Crowded

F

Apterous virginoporoe

Botch

2

3

4

Off plont for 24 h isolated

lsoloted

Isolated

Alate virginoporae

Moles

FIG. 1 1 . The crowding response in Megoura uiciae. The histograms indicate the composition of successive batches of offspring produced by single apterae. Aphids A, B and C were taken from the plants after a single batch of (apterous) larvae had been deposited, and they were then exposed to contacts with other aphids. The controls, D, E and F, were also temporarily removed from the plant but were kept in isolation.

start producing alate daughters immediately they are replaced on a plant. In this instance it is clearly the presence of other aphids and not the effects of starvation that provide the stimulus for the formulation of alate progeny (Lees, 1961). The progeny sequences of individual apterae given in Fig. I 1 illustrate various features of the response in Megouru. All these aphids had developed in isolation on separate bean shoots and, when parturition began, were transferred serially to fresh plants after each batch of larvae had

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24 1

been born. The control apterae, which had no contacts with other aphids, produced only apterous offspring, as mentioned above (Fig. 1ID-F). However, such controls were hardly necessary as apterae prepared for crowding could be allowed to produce one or more batches of larvae before the crowding stimulus was applied. This experience was provided by placing the aphids in a 2x 1 in. tube for 24 h at 15°C at a density of 10 per tube. Figure 11B and C shows that the crowded apterae usually produce a mixture of apterous and alate daughters. The graded response contrasts with the all-or-nothing threshold effect observed in the determination of embryos as virginoparae and oviparae. This leads to a greater variability. One extreme is shown by apterae that produced several successive batches consisting almost exclusively of alate daughters (Fig. 11A); on the other hand, it is common to find that even with an intense crowding stimulus one or two parent apterae from each group of ten are unaffected by the proximity of other aphids and continue to produce only apterous daughters. Since the control of wing dimorphism in Megoura is clearly maternal, it is appropriate to assess the effectivenessof a given treatment in terms of the performance of the parent. For most purposes, then, it is convenient to classify the parents as aptera- or alata-producers and to ignore the precise proportion of alatae in the progeny. Since the response is graded, the percentage of alate daughters does provide some measure of the reaction intensity. But the variability between parents is such that it is often a less useful indicator of the efficacy of a given crowding treatment. We have already seen that, after crowding, almost the first larvae deposited can be alate (Fig. 11). The development of embryos can therefore be switched by the maternal regulator shortly before their birth. The persistence of the response is also of some interest. In some cases alatae continue to be formed (sometimes exclusively) throughout the parent’s reproductive life, even though the crowding stimulus has long since been withdrawn (Fig. 11A). Taken together these observations seem to cast doubt on Kitzmiller’s (1950) suggestion that the time interval from the withdrawal of the wing-inducing stimulus to the birth of the last alate daughters is indicative of the time of determination of the youngest embryos. The sequence of alate daughters probably signifies that the hormonal state induced in the mother by crowding may be strikingly persistent. Decapitation experiments, in which the maternal centre responsible for control of wing dimorphism is removed, have shown that embryos do not become competent to respond to alata-promoting influences until a few hours before birth (p. 265).

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B. STAGES SENSITIVE TO C R O W D I N G

Responsiveness to crowding varies with the stage of development and with the species. In some aphids the treatment the parent receives is decisive; in others the period of sensitivityis postnatal; in others, again, both the parents and the young larvae can respond to crowding. Megoura belongs to the first category, the control being strictly maternal. This can be demonstrated by confining insects of uniform or mixed ages in the usual 2 x 1 in. tube “arena”. The adult apterae were prepared by isolating them at the third instar. Apteriform larvae were obtained by allowing similarly isolated adults to deposit a small group of larvae overnight (not more than 10-15). Some of the latter were then grown on in isolation to provide third and fourth instar apterae. Adults were tested by permitting them to deposit a small batch of larvae whose apterous or alate condition was checked at the third instar or later (Lees, unpublished results). Table I, A, shows that when the first instar larvae are crowded together, they never develop into alatae. With third and fourth instar larvae we are not concerned with the form of the adult-they all become apterae-but with their progeny. It will be seen that adults derived from larvae crowded for 24 h in the third instar did not produce any alatae, whereas those crowded in their fourth larval instars became alata-producers in significant numbers (27%; Table I, A-C). The response is fully developed in the teneral female and appears to persist without much change throughout adult life. TABLE I Development of the crowding response in Megoura viciae Material and treatment

No. tested

A. 100 first instar larvae (presumptive apterae) crowded together B. 50 third instar apterae crowded together C. 10 fourth instar apterae crowded together D. 10 adult apterae crowded together E. One adult aptera crowded with 100 first instar larvae. F. One adult aptera crowded with 50 third instar larvae

X,‘ becoming alatae (A)

yo becoming alata-producers (B-F)

45

0

31

0

22

27

80 20

86

20

100

95

T H E CONTROL O F POLYMORPHISM IN A P H I D S

243

These results suggest that sensitivity to crowding develops in the fourth instar, but this does not exclude the possibility that any effects exerted by crowding on younger larvae may damp out during the subsequent period of isolation (9 days in the case of the third instar) before parturition begins. Although the “crowds” in these experiments consisted of individuals of like age and instar, it is improbable that the insensitivity of young larvae is due to the fact that they have been denied contacts with older individuals. Apteriform larvae of Megoura will not in fact respond to the presence of older larvae or of adults. Conversely, Table I, E, F, shows that adult apterae will respond readily when crowded with first or third instar larvae. For this reason it is desirable when testing adult apterae to remove them very soon after parturition has begun-the adults sometimes respond to their own offspring if more than thirty or so have been deposited. In Brevicoryne brassicae the controlling system is also maternal but the parent aptera appears to require the stimulus of her larvae. When adult apterae were crowded at a density of 5 or 20 per 12 mm leaf cell and their offspring removed daily, less than 7% of the latter developed into alatae (Bonnemaison, 1951). Similarly, when the progenies of isolated apterae were collected in the first instar and crowded together until they had reached the fourth instar, very few became alate. But a high proportion did so when the larvae were allowed to remain in contact with the parent apterae during the entire period. The interaction between parent and offspring takes 3 or 4 days to develop. This was investigated by withdrawing larvae from the parents after periods of 1,2,3 and 4 days. The results were as follows : Duration of contact (h) 1-24 148 1-73

1-96

yo alatae 0-1.5

0-4 8-34 13-74

Although these observations do not entirely exclude the possibility that the larvae are influenced by the parents and not vice versa, it seems that many of the alatiform first instar larvae from the 3- and 4-day series were born relatively late in the sequence of offspring. If so, their actual duration of contact with the parent will have been shorter than 24 or 48 h; yet these periods had proved ineffective in the first two treatments. Some further evidence of a maternal effect was that a 3-day period of parent-progeny crowding varied in effectiveness during the reproductive life of the mother. If parturition is reckoned to begin on day 0, very few

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larvae born during days 0- 1 or 3-6 become alate, even though the rate of parturition was very high at this time, and dense larval populations were built up. But those born during days 6-9 and 9-12, though fewer in numbers, became alate in considerable numbers. An experiment designed as a direct test of adult sensitivity was unsuccessful. When previously isolated parent apterae were placed with large numbers of young larvae for 6 days, no alate offspring were born after the larval population was removed. But it is possible that the response fails to persist once the crowding stimulus is withdrawn. Other aphids are only sensitive to crowding during the early larval instars. First stage larvae of Myzus persicae react to crowding within 24 h of their birth, the intensity of the response increasing as the duration of crowding is extended from 24 to 72 h. Second instar larvae are less responsive and parent apterae are completely unaffected (Bonnemaison, 1951). Maximum sensitivity to crowding is even more sharply defined in Rhopalosiphum prunifoliae (Noda, 1958). Larval interactions are most effective 21 h after birth, late in the first instar. Responsiveness has only just begun to develop in the newborn larva and has already vanished by the beginning of the second instar. Aphis craccivora resembles Megoura in that adult apterae are extremely responsive to crowding and other environmental stimuli (Johnson and Birks, 1960;Johnson, 1965).But a striking effect can also be demonstrated on the first instar larvae if the alata-inducing stimuli are temporarily relaxed. Sensitivity is even retained in the second larval instar, though not in the third. Johnson and Birks (1960) have suggested that the period of sensitivity may, in general, be more extended than had been suspected ;and that this may have escaped attention because the “active” stimuli are apteraproducing rather than alata-producing. However, this idea has been tested in Megoura with negative results. Presumptive alatae in the first instar cannot be deflected from the alate course of development either by isolating them or by raising the temperature. These factors (or their opposites) are only effective before birth. C. T H E M E C H A N I S M OF C R O W D I N G

Aphids living in populous colonies are evidently aware of the presence of their neighbours. And this form of recognition will no doubt have a sensory basis. In order to study this question, previously isolated adult apterae of Megoura were crowded in conditions that would be expected to eliminate certain sensory functions. To secure a constant response it is necessary to take these individuals from a line maintained permanently

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in long day conditions and propagated at each generation through apterous parents (see p. 217). Some apterae had their compound eyes covered with an opaque cellulose paint. After crowding the blinded insects under standard condition (24 h at 15°C at a density of 10 per 2. 1 in. tube) 68% became alataproducers. And 60% did so when the crowding took place in permanent darkness instead of in alternating light and dark (Table 11, A, B). Vision does not therefore appear to be of great consequence. There are also convincing reasons for rejecting odour as a possibility. Tests were conducted by confining previously isolated apterae singly in small containers made from lengths of glass tubing with the open ends covered with voile. TABLEI1 The effect of sundry treatments on the production of alate progeny by parent apterae of Megoura Treatment

.No* % alata-producers tested

A. Eyes blackened, crowded 10 per tube 30 B. Crowded in darkness at a density of 10 per tube 40 C. Contact with other aphids prevented by a gauze 26 barrier 16 D. In contact with dead aphids E. In contact with immobile, living aphids with their 20 appendages amputated F. In contact with stationary aphids with motile 24 appendages G . Normal controls, isolated from third instar 53 H. Normal controls, crowded 10 per tube 270

68 60 0

0 0 21

0 84

These were introduced into the standard 2 x 1 in. specimen tubes which, in turn, contained a “crowd” of ten aphids. This arrangement prevented the isolated test insect from establishing any form of contact with the group of aphids outside, but would not have presented a barrier to the diffusion of odours. Yet the sequestered apterae invariably failed to become alata-producers (Table 11, C). The following negative evidence is also relevant. (a) The crowding response is not prevented from developing if a brisk air stream is passed through the tube containing the group of aphids. (b) Very high density crowding leads to a reduction in the response (see below). This would perhaps be unlikely if olfactory stimulation was involved. (c) Insects belonging to taxonomic groups other than aphids can stimulate Megoura to produce alate offspring (p. 247).

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The possibility that the maternal response involves the exchange of a pheromone transmitted by contact can also be dismissed in Megoura. Previously isolated apterae are not affected if they are placed in intimate contact with the bodies of other aphids freshly killed by a brief exposure to -20°C (Table 11,D). It is also possible to provide the test aphid with a “carpet” of living aphid bodies rigidly fixed to the substratum with a polyvinyl alcohol adhesive. When complete immobility is achieved by amputating the legs and antennae, no response is ever given by previously isolated apterae, even though an intimate and prolonged contact occurs. But when the insects forming the “carpet” are allowed to retain their appendages, test aphids walking over them are sometimes stimulated to produce alatae (Table 11, E, F). This can no doubt be attributed to the fact that the lower aphids, even though unable to walk, can still move their legs and antennae. The arguments set out above under (b) and (c) in connexion with odour are of course, equally cogent when considering pheromones. High density crowding should favour rather than inhibit the response if this involved the transmission by contact of a chemical substance. Moreover, we shall see that entirely alien insects such as beetles and cockroaches can evoke the response. In view of the known specificity of pheromones, it is hardly probable that materials with comparable effects would be produced by such diverse species. These considerations suggest that the interaction between aphids is dependent on touch. Johnson (1965) has independently arrived at similar conclusions in his recent studies of A . craccivora. The location of the relevant tactile receptors is not known with certainty. Bonnemaison (1951) noted some lessening of the crowding response after the antennae of Brevicoryne brassicae had been amputated. And the antennae of aphids certainly bear a variety of tactile and chemosensory sensilla (Slifer et al., 1964). However, antenna1 amputation has almost no effect in A . craccivora (Johnson, 1965), and in tests with Megoura 68 of the antennaless insects became alata-producers after crowding under the usual standard conditions. This compared with a value of 85% in the intact controls. These results are perhaps understandable, as tactile bristles are widely distributed on the body and are particularly plentiful on the legs. It has not yet proved possible to eliminate the tactile bristles. But an alternative method of investigating the aphid’s sense of touch is to provide substitute stimuli of a tactile nature. Johnson (1965) succeeded in eliciting a mild response by repeatedly stroking the legs, antennae and body of A . craccivora with a fine hair. Attempts to produce alatae by

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touching Megouru apterae with a hair or by revolving them slowly in a tube containing fine cork particles have not yet proved successful. Despite the failure of artificial stimuli, many living insects can act as “aphid substitutes”, even though the degree of stimulation is often weak. We have already noted that Megouru adults respond to the relatively minute first instar larvae. They are also stimulated by contacts with different morphs of their own species (alate virginoparae and oviparae; Table 111, A, B). Adult apterae of other active aphid species such as Acyrthosiphum pisum are also quite effective, the relatively sluggish Aphis fubue rather less so (Table 111, C, D). Some results with other quite unrelated insects are included in this Table. Drosophilu flies, the adult TABLE111 The effect of contacts with different morphs and with other insect species on the production of alatae by parent apterae of Megoura viciae Treatment. One aptera crowded with A.

B. C. D. E. F. G. H. 1. J. K. L.

10 Megouraalatae 10 Megoura oviparae 20 Aphis fabae apterae 10 Acyrthosiphum pisum apterae 10 Drosophila adults 30 Drosophila adults 10 Stegobium paniceum adults 10 Tribolium conjusum adults 20 Pieris brassicae, first instar larvae 100 Pieris brassicae larvae 10 Periplaneta americana, first instar larvae 10 Ptinus tectus adults

No*

”/o alata-producers

20

90 80

tested 20 20 21 10 10

20 22 20 5 11

20

50

73 0 10 15

0 5

20 45 65

beetles Stegobium paniceum and Tribolium confusum, young caterpillars of Pieris brussicue-all these provoked only slight responses and a high crowding density was sometimes required. On the other hand, the stored products beetle Ptinus tectus and young cockroaches proved to be almost as effective as another aphid. It seems likely that for tactile stimulation to occur the aphid must be repeatedly jostled in a particular way. The pattern of stimulation is not particularly specific, although an “aphid” stimulus is usually superior to those imparted by other insects of comparable size and activity. The temporal component of crowding can, of course, be examinedby following the development of the response in a standard aphid population. It is

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remarkable that in A . craccivoru alata-production can be elicited by a series of encounters lasting no more than 2 min (Johnson, 1965). The response appears to develop more slowly in Megoura. When ten apterae were placed in the usual 2 x 1 in. tube arena, none became alata producers after 1 h and only 25% after 6 h. However, the number of encounters was left to chance and no “recovery time” was allowed after the aphid had been removed from the plant. The time relations of the crowding response can also be studied by varying both the stocking density and the volume of the “arena”. These s

-0

0

0

8 -5

E

: -10 -15

.-g

z

E

-20 -25

Density 2

P

5 10 20 40

2

5 I0 2 0 4 0

2 5 10 2 0 4 0

2 5 10 2 0 4 0

FIG.12. The effect of population density on the crowding response in Megoura viciae. The frequency of contacts between apterae has been varied both by changing the population size and by selecting containers of different volume (thereby altering the surface area available for perambulation).

changes will naturally influence the frequency of the encounters between aphids. In the experiments illustrated in Fig. 12 the population densities ranged from 2 to 40 and the size of the arenas from specimen tubes I3i in. in diameter to glass jars of nearly 1 gal capacity. Figure 12 shows that all the populations-even those consisting of only two individuals-constituted a “crowd”. This point has also been emphasized by Johnson (1965). Moreover, even with a population of two, none of the vessels was so large that mere spatial separation prevented the response from developing. Even in the largest arena mutual stimulation was observed in 30% of the trials. It hardly seems likely that the number

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249

of aphis encounters during this 24 h experiment would have been very numerous. The crowding response also diminishes in intensity if the space available for normal walking is much reduced. When the aphids are packed so tightly that locomotion is prevented (e.g. with the smallest arenas and a stocking density of 40) almost no response is forthcoming, although the aphids are not injured by this treatment. We may conclude that there is an optimum, crowding density for the initiation of alata-production. If the populations are sparse, the contacts with other aphids may be too infrequent to allow the response to develop fully. If the density is high, the response is inhibited by the prolonged enforced contacts which ensue. Since the crowding response is readily elicited when only two aphids meet, it has been possible to study their encounters in detail. Johnson (1965) followed the behaviour of A . craccivoru apterae walking in opposite directions round the rim of a plastic vial 2 cm in diameter. The two insects usually recoil from one another when they first meet; they then stop and probe, and finally one aphid crawls over the other before proceeding on its way. Many of these aphids become alata-producers. Enforced encounters were also arranged by releasing an aphid immediately behind a second stationary insect so that it was compelled to climb over it. When this manoeuvre was repeated ten times within a period of 1-2 min, the lower aphid usually began to produce alate progeny while the “top” aphid did not. Contrived encounters which involve the handling of the aphids seem to be less effective in Megouru. Apterae confined in a tube walk about freely and apparently encounter one another at random. Freedom of movement (and perhaps the absence of extraneous tactile stimulation) seems to be required for the development of the response. Thus aphids that are suspended by suction tubes applied to their backs always fail to stimulate one another when their legs and antennae are allowed to touch for 24 h. It is also uncertain whether there is the same element of submissiveness in the response. Apterae temporarily immobilized by attaching them to a filter pzper substratum do not becomealata-producers when freely moving aphids are’ allowed to climb over their backs for 24 h; and we have already seen that the active “top” insects are sometimes stimulated by stationary aphids, provided their legs and antennae are free (Table 11, F). D. N U T R I T I O N

This factor perhaps more than any other has been invoked as a controlling agent in the production of alatae. The state of growth of the host

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plant will certainly influence the concentration of nutrients in the sap. This variant is believed to be of significance by Pintera (1957) who reared an anholocyclic strain of Myzus persicae on stinging nettles-a plant with well-defined growth stages. It was observed that more alatae were produced on plants in flower than on the young or post-flowering stages. Paschke (1959) found no effect of alfalfa leaf age on Theriouphis muculata but attributed the increased numbers of alatae at high population densities to plant damage induced by the injection of the toxic saliva. The host species itself may have some influence (e.g. Rhopalosiphum prunifoliae grown on Triticum and Hordeum; Noda, 1954). It has also been thought that other factors, such as temperature, influenced the development of the embryos through their nutritional requirements. For example, Smith (1937) showed that the number of honeydew droplets excreted by Hyalopterus pruni at high temperatures was smaller relative to the number of young born. And he therefore argued that under these conditions the latter would be partially starved. The possible connexion of alata-production with malnutrition has often been put to the test by subjecting aphids to periods of complete starvation off the plant. The results have been diverse. For example, Pintera (1957) detected some effect when parent aptera of Myzuspersicae were deprived of food for 4 days. And starvation treatments were apparently successful in producing alatae in Acyrthosiphum pisum (Schaefer, 1938)andin Rhopalosiphumprunifoliae (Noda, 1956). On the other hand, neither larvae nor parent apterae of Therioaphis maculata responded to starvation (Paschke, 1959). Unfortunately, many authors conducting starvation experiments do not state explicitly whether their experimental insects were isolated from one another after removal from the plant or whether they were kept in a group. This apparently trivial detail may well account for some discrepancies in the literature since mutual stimulation is most extreme if the insects are deprived of food and are in active movement. When the crowding response has been taken into account or has been eliminated, it often seems that nutrition has little or no direct influence on alata-production. Thus Bonnemaison (1951) recorded only slightly increased numbers of alate progeny when the leaf cages containing the aphids (Brevicoryne) were placed on yellowing cabbage foliage or on leaves damaged by the feeding punctures of other aphids. Adult apterae that were subjected to a 16i h daily period of starvation produced no alatae; and shorter periods of inanition were also ineffective when applied to presumptive first instar apterae. On the other hand, the nature of the food plant does seem to exert a measure of direct control in A .

THE CONTROL O F POLYMORPHISM IN A P H I D S

25 1

craccivora for apterae reared on mature bean foliage tend to produce more alate offspring than those maintained on germinating seedlings (Johnson and Birks, 1960; Johnson, 1965). We have already noted (p. 240) that in Megoura periods of starvation lasting up to 3 days do not cause isolated apterae to become alataproducers. And intermittent starvation is similarly ineffective. Nevertheless, nutrition still plays a most important indirect role. When previously isolated Megoura apterae are caged in pairs on juvenile, mature or senescent bean leaves, many begin to give birth to alatae, and the proportion of alata-producers is highest on senescent leaves and lowest on the more nutritionally favourable juvenile bean shoots. However, this relationship with the nutritional qualities of the food is only secondary, since aphids caged singly on the same leaves never produce alatae. The explanation is not far to seek. On senescent foliage the insects are visibly restless, whereas on young leaves or shoots they tend to feed tranquilly side by side. Clearly, one would expect the number of encounters, and hence the crowding response, to be greatly influenced by this behaviour. Since aphid crowding reactions are both complex and variable, it is no easy matter to decide whether, in any situation involving a group of aphids, heightened activity alone is sufficient to account for the degree of stimulation observed. The present indications are that the tactile response is usually the prime mover. Unequivocal evidence that starvation or malnutrition, or specific changes in the quality of the plant sap, can cause isolated aphids to become alatae or alata-producers is still lacking. E. WATER C O N T E N T A N D I O N I C COMPOSITION OF THE HOST P L A N T

The appearance of alate viviparae has often been associated by observers with temporary water stress or wilting of the host plant. For example, Rivnay (1937) noted that many larvae of Toxoptera aurantii developed into alatae if kept on partially dried out citrus twigs or if stored at low humidities in tubes off the plant. Schaefer (1938) also believed that the production of alate offspring in the pea aphid Acyrthosiphum pisum was induced by the ingestion of more concentrated plant sap or by rapid transpiration from aphid itself. La1 (1952, 1955) assumed that the physiological processes controlling wing form determination in the mustard aphid Rhopalosiphum pseudobrassicae (Lipaphis erysimi) were influenced by the dehydration of the parent aphid and hence by the moisture content of the plant. Kennedy et al. (1958) noticed that water shortage in the host plants (broad beans) sometimes led to an increase in

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the proportion of alate offspring in Aphis fabae even though the phloem sap was simultaneously enriched by premature senescence. Nevertheless, it seems doubtful whether a direct connexion has yet been demonstrated between the water relations of the aphid and the wing determining mechanism. Populations have always been employed and population densities have often been large and uncontrolled. Aphids feeding on wilting plants become increasingly restless, eventually withdrawing their stylets and moving to other positions on the plant. As the condition of the plant deteriorates, the number of encounters between wandering aphids will increase. Eventually the aphids may walk off the plants altogether, or they form dense aggregations in relatively favourable sites. This kind of behaviour will, of course, have considerable, but largely unpredictable, effects on alata-production in the population. The claim has sometimes been made that the proportion of alatae developing in aphid colonies can be influenced by the chemical composition of the nutrient solution in which the cut plants are placed. The evidence, which has been reviewed by Bonnemaison (1951), is inconclusive. F. R E L A T I O N S H I P S W I T H A N TS

When Aphis fabae is tended by the ant Lasius niger, the rate of multiplication is increased, while the production of winged forms is delayed (El-Ziady and Kennedy, 1956; El-Ziady, 1960). In A . craccivora, which has a similar relationship with the ant Paratrachina baveri, the suppression of alatiform structures takes place in the first and second larval instars (Johnson, 1959a). It is well known that feeding and excretion are stimulated in ant-attended colonies (Banks and Nixon, 1959).Accelerated parturition may therefore be the direct result of improved nutrition, although Banks suggests that it arises from the delay in the dispersal of young aphids from the more nutritious growing points of the bean plants. The way ant attendance influences form determination is less certain. Neither Johnson (1959a) nor El-Ziady (1 960) consider that apterousness is related to increased food uptake. In Johnson’s experiments the ant-attended aphids did not grow more rapidly or attain a larger size than the unattended controls, and nutrition did not therefore appear to be limiting. An alternative suggestion advanced by Johnson is that the ant can influence the endocrine system of the aphid, perhaps through the sensory nervous system. The behaviour of feeding aphids in the presence of soliciting ants is so striking that it is easy to believe that a changed pattern of tactile stimulation could be linked with a neuroendocrine switch mechanism governing

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form determination. However, it is also clear that ants profoundly affect the movements of aphids on the host plant. For example, the “tranquilizing” effect of ants on larval aphids has been emphasized both by El-Ziady (1960) and by Banks (1958). It seems certain then that the frequency of contacts between aphids in the colony will be influenced by the presence of ants. The possibility therefore remains that the apterising effect of ant attendance is mediated through the normal crowding response. G. T E M P E R A T U R E

This factor influences the production of alatae in Macrosiphoniella sanborni (White, 1946), Aphis craccivora (Johnson and Birks, 1960) and Megoura viciae (Lees, unpublished results). In all these species higher temperatures tend to suppress the production of alatae. Since these conditions would be expected to increase rather than to diminish locomotor activity, it may well be that the action of temperature is direct. In Megoura this can be proved by isolating previously crowded apterae during the subsequent temperature treatment. The output of alate offspring subsides more rapidly at 26°C than at 15°C. H. P H O T O P E R I O D

In the monoecious aphids Brevicoryne brassicae and Megoura viciae, length of day controls the development of the gamic females but is without influence on the production of alatae (Bonnemaison, 1951;Lees, unpublished results). But in some anholocyclic species, which lack the ability to produce sexual morphs, photoperiod appears to have assumed a new role. Some measure of photoperiodic control of alata-formation seems to occur in Macrosiphoniella sanborni (White, 1946) and in A . craccivora (Johnson, 1965), although it is clear that in the latter species this factor is of minor significancein comparison with the tactile response. In heteroecious species with host alternation day length often plays a prominent part in the production of alate gynoparae. Although the latter may be difficult to distinguish from “ordinary” alate virginoparae produced by crowding (e.g. in Macrosiphum euphorbiae, see p. 213), this response is best considered in the context of sexual form determination (p. 232). I. I N T R I N S I C F A C T O R S

1 . Form of the parent and grandparent It has long been recognized that alate aphids of many species are much more sparing in their production of alate daughters than are parent

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apterae. This generalization, moreover, holds good for species in which sensitivity to crowding is confined to the young larva as well as to species with maternal form control. This means that the larvae of alate and apterous mothers must be physiologically different at birth even though both develop into apterae. Indeed, Noda (1960) believes that in Rhopalosiphum prunifolii parentage affects many larval characteristics other than the sensitivity to crowding. These include the length of larval life, the resistance to starvation and ability to survive on wilting plants. More remarkable still, the inhibitory effect of alate-parenthood may extend beyond the immediate progeny into the following generation. The nature of this inhibition, which seems to be governed by a long range timing mechanism, is discussed in greater detail elsewhere (p. 269). The potentialities of other viviparous morphs as alata-producers have hardly been explored. It may be worth noting, however, that the (apterous) fundatrix of Megoura responds to crowding stimuli in precisely the same way as do her apterous descendants (Lees, unpublished results). 2. Species and clonal diflerences Although standardized crowding techniques have not yet been used in species comparisons, it is clear that some aphids form alatae much more readily than others. For example, Bonnemaison (1951) noted that although Myzus persicae appears to produce alatae with greater ease than Brevicoryne brassicae it is also less resistant to overcrowding. These two species differ strikingly in the types of aggregations formed. Colonies of Brevicoryne are densely packed, the progeny tending to remain aggregated round the immobile parent. Young larvae of Myzus, on the other hand, at first remain grouped round the parent but disperse by the end of the first instar, appearing then to distribute themselves evenly over the available leaf surface. Some species with this habit, such as the nut aphid Myzocallis coryli, space themselves out with almost mathematical precision. It is tempting to infer that the latter may prove to be particularly sensitive to the enforced proximity of other aphids. Megoura viciae, which forms alatae with moderate ease, is intermediate in its behaviour. Adult apterae commonly deposit a small group of twenty or so larvae, which subsequently remain loosely aggregated ;but the parent then leaves the group and settles down elsewhere on the plant to deposit another cluster of larvae. In some species there are also well marked clonal differences. For example, certain clones of Aulacorfhums o h i produce alatae abundantly in laboratory cultures whereas others have never been known to do so (MacGillivray and Anderson, 1958).

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J . DEVELOPMENTAL P A T H W A Y S A N D W I N G DIMORPHISM

Although natural intermediate winged aphids are not uncommon, virginoparae normally develop along one of two alternative routes-the apterous or the alate. The nature and direction of this form change has been the subject of argument. Johnson and Birks (1960) have suggested that all aphids (virginoparae) begin development as presumptive alatae ; and that environmental and other stimuli conducive to the formation of apterae act by suppressing these incipient alatiform tendencies. This process is referred to as “diversion”. Their formal representation of this idea is given in Fig. 13.

FIG.13. Johnson and Birks’ (1960) diagram (slightly modified) illustrating their theory of “diversion” in Aphis crucciuoru. Development is regarded as proceeding towards the alate condition, unless it is diverted by various apterising factors. This process can occur both before and after birth.

Johnson and Birks (1960) have drawn attention to a number of points that bear on their theory. It is well known from the work of Shull(l938) that all late embryos and first instar larvae of Macrosiphum euphorbiae develop small thoracic epidermal thickenings irrespective of whether they are destined to develop into alatae or apterae. Similarwing rudiments are found in A . craccivora and can be first recognized about 1 day before birth at the time of the embryonic moult. During the first and second instars the wing adage of future apterae diminish in size and finally disappear. They measure only 7 p in first instar apterae, compared with 15 p in alatiform larvae. “Diversion” from the alate course of development takes place over an extensive period in A . craccivora, namely from the prenatal period a day or so before birth to the end of the second larval instar (see p. 244). This I

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period of time is so protracted that these authors consider it inappropriate to refer to this process as form “determination”. The continued growth of these wing rudiments can be suppressed at any time during the responsive phase by introducing a suitable aptera-promoting stimulus (which may be either environmental or intrinsic). Although the condition of the wing rudiments in first and second instar larvae of A . craccivora was not fully described, it appears probable that the size of the anlage will be larger if the apterising agency is applied relatively late. Johnson and Birks have concluded that the aphid always develops towards the alata condition unless “diverted” by a stimulus favouring winglessness. In experiments designed to test the progress of “diversion” the parent aphids and young larvae were fed on mature (alata-promoting) leaves except for the more limited periods when they were provided with young (aptera-promoting) foliage. Since the latter appeared to be equallly effective whether it was given early or late during the responsive phase, they concluded that the process of “diversion”, once begun, is irreversible. Johnson and Birks were at first inclined to emphasize the special significance of aptera-promoting factors. In terms of the crowding response, control would then be exercised by isolation rather than by contacts with other aphids. This was in agreement with their hypothesis that the environment acts undirectionally and in opposition to the “inherent” course of development (towards alateness). More recently, however, Johnson (1965) has concluded that aphids can be positively influenced by factors favouring alate development, including crowding, and the occurrence of “diversion” is regarded as depending on the integration of stimuli of several different kinds. The view held by most previous authors is that the overriding factors are those which induce alata-formation, and it hardly seems that this possibility has yet been excluded. We could suppose, for example, that in A . craccivora development can be switched to the alate pathway by suitable stimuli, but that stimulation must be sustained through most of the sensitive phase in order to generate a maximal response. Any relaxation in the environment (e.g. isolation), even though of limited duration, might suffice to annul the effect. We have already noted elsewhere (p. 244) that Megoura cannot be “diverted” by isolating larvae born immediately after a brief alata-promoting (crowding) stimulus has been given to the parent. The morphological evidence also lends itself to other interpretations. The embryonic wing rudiments do not always appear to be as prominent as in A . craccivora or M . euphorbiae. For example, White (1946) and Kitzmiller (1950) were unable to locate them in Macrosiphoniella

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sunborni. In Megouru they are present in some presumptive apterae but not in others (Lees, 1961). The occurrence of these rudiments certainly accords with the generally accepted view that the primitive phylogenetic condition of aphids is winged. But one wonders whether their significance as developmental signposts is any greater than that of gill pouches in the embryonic mammal. The latter is not regarded as a “fish” at this stage of its development. It may be preferable to look upon the embryonic aphid merely as a “virginopara” and not as an incipient alata or aptera. This view is incorporated in the simple pathway diagrams shown in Fig. 18. Certain physiological evidence is also difficult to reconcile with “diversion”, since this concept implies that if the embryo could be deprived of the normal extrinsic regulators it would continue along the alate course of development. This can virtually be done in A . cruccivoru (Johnson and Birks, 1960) and in Megouru (see p. 265) by decapitating the parent aphids. By this means the source of (maternal) control is removed. Yet the embryos invariably develop as apterae, not as alatae. It may also be worth emphasizing that “determination”, as it is usually understood, is by no means an instantaneous morphogenetic process. The fact that A . cruccivoru remains competent to respond to environmental stimuli over a relatively long period does not seem to preclude its usage. In Megoura where prenatal determination occupies only a few hours, and is then irreversible, the term is particularly appropriate and conforms with general usage in other polymorphic insects (e.g. termites).* K . E N D O C R I N E C O N T R O L OF W I N G D I M O R P H I S M

The environmental regulators controlling the differentiation of apterous and alate morphs no doubt act through the insect’s endocrine system. Since apterous aphids have usually been regarded as natural neotinics, it would not be surprising if the same endocrine components are involved as in the control of metamorphosis in other insects; and the corpus allatum might have a special significancein this respect (Kennedy and Stroyan, 1959; Johnson, 1959b; Lees, 1961). This view can be supported by observations on the juvenilizing effects of parasites, and also by recent work involving the direct administration of materials with juvenile hormone activity. But before considering this evidence, the principal morphological differences in larvae and adults of the two morphs may be noted briefly. Megouru will serve as an example. Alate and apterous larvae cannot be distinguished externally until the

* In this article “determination” is used both in the sense of form (whole organism) determination and in the sense of organ determination.

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third instar when the small mesothoracic wing thickenings become visible. These structures develop into sizeable wing pads in the final larval instar (the fourth). But apart from this feature, the changes in form during larval growth are relatively slight. The legs preserve the same, rather stout, proportions; and both thorax and abdomen remain green and devoid of pigmentation patterns. Adult aphids, whether alate or apterous, have certain anatomical features in common. The genital plate and the open genital duct are new structures associated with reproductive maturity (Fig. 15C, D, E). The shape of the cauda differs from that in the larva; and the legs are longer in proportion to the body. Both adult morphs develop plate organs (secondary rhinaria) on the third antennal segment. And the prothorax and head become sclerotized. Moulting ceases. But adult alatae also differ in many respects from apterae. Apart from their wings, the alatae have a complex sclerotized pterothorax and a striking abdominal pattern consisting of two segmentallyarranged rows of pigment spots (the marginal sclerites) (Fig. 14A, B). Unlike the aptera, the alata also possesses ocelli, and the antennal plate organs on the second segment are more numerous (over thirty compared with about fifteen). It will be seen that the adult aptera differs from the adult alata principally in the suppression of those sensori-motor structures (wings, pterothorax, sense organs) characteristic of many adult insects (Fig. 14C) (Kennedy, 1956). These are larval or “juvenile” features. It follows that the larval-adult “metamorphosis” is very much less striking than in the alata. It isinteresting to note, however, that not all the adult apteriform characters result from the suppression of normal “adult” features. In some species new structures make their appearance. For example, in Aphis craccivora (Johnson, 1959b) or A . cerasi the dorsal surface of the abdomen consists of a rigid sclerotized “shield” quite different from the characteristic abdominal pattern of the alata, or indeed from the soft, patternless abdomen of the larva. FIG.14. The effect of juvenile hormone extract on the differentiation of alate and apterous morphs of Megouru viciue. A, Normal alate virginopara; arrow indicates marginal sclerites. B, Ventral aspect of normal alata, to show the anatomy of the mesosternum (arrow). C, Normal apterous virginopara. D, Natural intermediate, with small, asymmetrical wing rudiments and little pterothoracic development. E, Aptera treated with juvenile hormone extract in the third instar, showing the unsclerotized pronotum (arrow). F-H, Alatae treated with juvenile hormone extracts in the third instar. The alata in F shows slight wing reduction and the removal of marginal sclerites. G and H are more severely affected; the pterothorax is only partly sclerotized and the individual plates are confluent; wings are markedly reduced and abdominal sclerites missing (with the exception of the presiphuncular sclerites).

FIG.14 (Legend opposite)

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1. Form reversal in parasitized aphids When presumptive alate larvae of Aphis craccivora are parasitized by the braconid wasp Aphidius platensis, alatiform structures are partially

or completely suppressed (Johnson, 1959b). The suggestion that this apterizing effect is caused by the diffusion of juvenile hormone from the parasite eggs appears very plausible in view of the marked influence of juvenile hormone extracts on developing alate aphids (see below). The morphogenetic processes were studied by controlling the time of the parasite attack. Presumptive alate larvae parasitized in the first instar survived until the fourth instar when they exhibited complete form reversal. Aphids parasitized in the second instar became intermediate adults. The ocelli were rudimentary ; the antennal plate organs were either lacking or reduced in number; the pterothorax was only slightly developed and vestigial wing evaginations were sometimes present. When the introduction of the parasite egg was delayed until the third instar, ocelli, antennal plate organs and wing rudiments were always present but the adults failed to develop any abdominal pigmentation. Only a slight degree of metathetely was noted in adult aphids parasitized in the fourth instar. These results show that the distinctive characters separating apterae and alatae are determined progressively. Determination of the antennal plate organs and ocelli takes place in the first two instars. The abdominal pigment pattern is decided relatively late, perhaps in the third instar. Organs of large size and complex structure, such as the wings and pterothorax, remain sensitive to the presence of parasite eggs over a much longer period (the first three instars). In all cases the process of determination results in the suppression of these adult characters. But the interesting feature is that the final expression of the character is delayed until the adult moult, some two to four instars later.

2. The eflect of juvenile hormone on the diflerentiation of apterous and alate characters The action of materials with juvenile hormone activity on the differentiation of alatiform structures has recently been studied in Megoura (Lees, 1961, and unpublished). A concentrated hormone extract prepared from the abdomen of adult male Cecropiamoths* (Williams, 1956; Schneiderman and Gilbert, 1959) and the trans-trans isomer of farnesol (Wigglesworth, 1958) were tried with very similar results. Small droplets were delivered on the surface of thorax or abdomen from a fine pipette.

* Kindly provided by Professor H . A. Schneiderman.

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Penetration occurred readily without abrasion of the cuticle and the effects were systemic. Some of the most striking results were obtained by treating third instar alatae. The insects undergo the normal two moults and become reproductive. The genital duct, genital plate, cauda and legs are of the normal adult type. And the ocelli and plate organs are also unaffected, being appropriate to a normal adult alata. However, the mesonotum at the site of the hormone application is often completely unsclerotized, revealing the green colour of the underlying fat body. The general pigmentation of the mesothorax is also lighter and the sutures between neighbouring sclerites tend to become obliterated. The wings are reduced in size and are usually crumpled. The marginal sclerites on the abdomen often disappear entirely (Figs. 14F, G, H, 15A, B). Different results are seen when the other instars are exposed to the hormone. In the fourth instar the effect is confined to the weakening or removal of a few abdominal sclerites. With first and second instar larvae it is necessary to compare the treated insects with an untreated control series, since the young alatiform larvae cannot be recognized by eye. Such a comparison has suggested that complete form reversal was never achieved. When the same individuals received hormone applications in each of the first three instars, some extremely apteriform adults were produced, but these intermediates always retained the normal alate complement of antennal plate organs as well as normal ocelli. If wing dimorphism is regulated by the concentration of juvenile hormone in the blood, developing apterae should already contain the secretion in some quantity. The addition of exogenous hormone might therefore have little influence on differentiation. This expectation is partly fulfilled. Third instar apterae receiving topical applications of Cecropia extract moulted twice and became almost normal adults, although the pale colour of the legs and the unsclerotized pronotum indicated that their neoteny had been slightly enhanced (Fig. 14E). However, a much more interesting effect is obtained if hormone is applied in each of the first three instars. A few insects then show abnormalities affecting the juvenile/adult characters. These aphids can be recognized as adults from the condition of the legs, cauda and antennal plate organs. But other features remain larval : for example, the genital plate sometimes fails to differentiate and the genital duct remains closed. These individuals often undergo an additional moult (Fig. 15F). Megoura therefore resembles A . craccivora in that the various characters that distinguish adult alata from aptera have different, but sometimes broadly overlapping, periods of determination. Moreover, the presence

FIG. 15 (Legend opposite)

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ofjuvenile hormone at this time also results in their differentiation being suppressed at the adult moult. Again, as in A . craccivora, the wings and pterothorax can readily be influenced by hormone in the third instar; and the abdominal pattern even later. But the fact that the ocelli and antennal plate organs cannot be suppressed by treating the first three instars with juvenile hormone suggests that these structures may be determined before birth, presumably just after the embryo has become committed to the alate course of development through the action of the environment and the maternal switching mechanism. We have noted also that structures that differ in the larva and adult, but which are not concerned in aptera/alate dimorphism, are also influenced during early larval development by juvenile hormone. The precocious determination of both larval/adult and aptera/alate characters contrasts with the type of humoral control observed in many other insects. In Rhodnius, for example, metamorphosis is controlled by the level of juvenile hormone in the blood immediately prior to the final moult (Wigglesworth, 1954). It is well known that high concentrations of hormone at this time can induce supernumerary moults by preventing the degeneration of the thoracic glands. If the same relationship exists in aphids, the programme of secretion of the thoracic glands must be decided by the concentration of juvenile hormone several instars before the terminal moult. Even the most massive hormone applications to third and fourth instar larvae have failed to cause extra moulting. How then does the same secretion-the juvenile hormone-apparently control both aptera/alate and larval/adult transformations? These dual functions might perhaps be reconciled if the determination of the different structures was segregated in time. But this cannot be so, as some features common to both adult morphs (genital plate, genital opening, number of moults) will respond to hormone administered during the first three larval instars, that is, at a time when the aptera/alate characters are also undergoing determination. A more probable explanation is that these various organs differ in their response thresholds. With certain adult structures FIG.15. A and B, Dorsal and ventral aspects of an alata treated with juvenile hormone extract in the third instar. Note the “erosion” of the sclerotized cuticle in the mesonotum and mesosternum. C, Cleared cuticle of normal fourth instar aptera, to show the form of the anal plate (a.p.)and cauda (c.). D, Cleared cuticle of normal adult alata showing the cauda (c), anal plate (a.p.),genital opening (g.op.) and genital plate (g.p.). E, The same structures in the adult aptera. Note that the genital plate is rather less emphasized. F, Cleared cuticle of an aptera treated with juvenile hormone in each of the first three instars. This aphid has adult legs, antennal plate organs and cauda but is undergoing a supernumerary moult (the cast cuticle of a siphunculus is visible on the right). There is no genital plate and the genital pore is still closed (the position of the opening is marked by the black spot (arrow) which has been caused by the exudation of haemolymph).

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(cauda, legs) the threshold cannot apparently be reached. Other adult structures or processes (genital plate, genital opening, number of moults) are somewhat less resistant, but it is probably significant that the only positive effectswere achieved with apterae-which perhaps already have a high titre of hormone in the blood. In contrast, the structures involved in wing dimorphism all appear to be relatively susceptible to changes in the level of juvenile hormone-changes to which other larval structures may be immune. We may recall (p. 258) that in some species of aphids the adult aptera has special features not possessed by either adult alatae or by larvae. There are indications that response thresholds are important here also. Johnson (1 959b) has shown that the presence of parasite eggs in the third instar larva of A . craccivora causes the normal sclerotized abdominal “shield” to be replaced by patternless larval cuticle. The application of juvenile hormone extract to third instar larvae of Myzus cerusi has much the same effect (Lees, unpublished). It seems possible that there are three types of response to different hormone levels. If the concentration is low, the alata abdominal pattern will be differentiated; if it is somewhat higher the aptera pattern arises; and if the hormone level is raised still further, both patterns are suppressed and larval cuticle is laid down. Experiments involving the application of materials with juvenile hormone activity have also been carried out by von Dehn (1963), using Aphis fabae. When larvae in their first and second instars were wiped daily with a thin film of farnesol, the percentage of alate adults developing varied from 0 to 51 yo(mean 23%), as compared with a value of 8090% in the untreated controls. Complete form reversal seems therefore to have been achieved. Farnesol vapour was also effective. In addition, alata-production was slightly inhibited if the bean plants on which the aphids were developing were immersed in a dilute solution of DLmevalonic acid lactone-a possible farnesol precursor. Von Dehn is inclined to believe that the synthesis of natural farnesol precursors is stimulated in plants damaged by aphids, and that this may directly influence the production of alatae. However, this suggestion is not applicable to Megoura since there is no evidence that nutrition plays any part in form determination. The decisive factor, namely the crowding response, functions normally when the aphid is separated from the plant. L. E N V I R O N M E N T A L R E G U L A T I O N O F C O R P U S ALLATUM ACTIVITY

If the environmental control of wing dimorphism is exercised through the agency of the corpus allatum, we should expect that aptera-promoting

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conditions would increase, and alata-promoting conditions diminish its secretory activity. Since environmental cues are often quite transient (a few minutes of contact with another aphid), it seems probable that the pattern of secretion can be “set”-perhaps for several instars aheadat the appropriate level (see Johnson, 1959b).This process can be equated with the act of form determination referred to earlier (p. 257). Natural intermediates are quite common in most aphids (Fig. 14D). Since many of these can be recognized as such as early as the third instar, it seems that their whole postnatal development has been of an intermediate nature. The probable cause is that the corpus allatum is only partially “turned on”. But the possibility cannot entirely be dismissed that the gland may sometimes suffer spontaneous shifts in activity during early larval development with the result that some alate structures are differentiated whilst others are suppressed. When the environment acts on the embryo through the medium of the parent, an additional controlling mechanism is present. This may well be humoral also. Since the small wing vestiges formed in presumptive apterae undergo a phase of rapid regression it would seem that the parent may be secreting a corpus allatum activating factor. However, this interpretation is not in agreement with certain observations by Johnson and Birks (1960) on decapitated aphids. Although in the intact insect no larvae are born if feeding is interrupted, this restraint disappears after decapitation. In the experiment in question apterae of A . craccivora were prepared as alata-producers. But after decapitation almost all the progeny proved to be apterous. This result has been confirmed in Megoura. The switch-over to aptera production takes place with surprising rapidity and may be reflected in the first larva produced by the headless aphids. Apart from indicating that embryonic determination is confined to a very restricted period just before birth, these results support the idea that the corpus allatum activity is controlled by a maternal hormone, probably coming from the head. But it seems that this secretion may in fact be inhibitory.

VIII.

THEI N H I B I T I O NO F DEVELOPMENTAL PATHWAYS : INTERVAL TIMERS

Since gene segregation appears either to be non-existent or considerably hindered during the parthenogenetic phase of the aphid’s life cycle, nuclear processes hardly seem to provide favourable raw material for the evolution of polymorphic control systems. It is not surprising, therefore,

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to find that other, presumably cytoplasmic, mechanisms play a prominent role in form determination. These mechanisms, which are sometimes virtually endogenous, are often coupled with environmentally-driven switching systems. They commonly take the form of “interval timers” which act by restricting the number of available developmental pathways. The best known timing mechanisms are those controlling the differentiation of the sexual forms. Marcovitch noted in 1924 that both the fundatrices of A . forbesi and their immediate descendants failed to produce sexuales when exposed to short spring photoperiods, whereas the summer virginoparae did so readily if the day length was artscially curtailed. In his studies on Aphis chloris Wilson (1938) noted that this inhibitory post-fundatrix phase lasted for about nine generations at 21“Cwhen the parents of each successive generation of virginoparae were taken from among the first-born progeny of the previous generation. Since parturition continued for almost as long as the whole developmental period, Wilson estimated that lineages constructed from the “lastborn” progeny of each generation would have only reached the fifth generation at this time. And he made the important suggestion that the recovery of the photoperiodic response mechanism might be governed by a “time factor” that functioned independently of the number of intervening generations and was passed from each generation to the next by some form of somatic inheritance. This hypothesis was put to the test by Bonnemaison (1949, 1951) who reared “accelerated” and “retarded” lines of Brevicoryne brassicae in short day conditions. His evidence showed that Wilson’s prediction held good for males and possibly did so for oviparae. Experiments of similar design have confirmed that in Megoura the refractory phase is time- but not generation-dependent. The specimen clone shown in Fig. 16 indicates the progress towards sexuality of two lineages, both derived from the same fundatrix, when exposed continuously to a short 12 h photoperiod at 15°C.We see that in the “first-born” lineage, with a generation time of about 17 days, the first adult oviparae appeared some 96 days after the fundatrix had deposited her first larva (day 0). They belonged to the sixth generation (counting the fundatrix as the first). Since several sister virginoparae were tested in each generation, it is possible also to express these results in terms of the maternal response. In fact, all the fifth generation parents examined proved to be ovipara-producers. Turning now to the “last-born” lineage we see that the generation time was about 30 days and that adult oviparae first developed in the fourth generation after about 93 days. It so happened that the parental virginoparae were available for testing when the “time

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interval" was running out, for only 20% of the third generation parents actually produced oviparae. But the process had, of course, gone to completion after a further period of 30 days, so that all the fourth generation parents yielded only oviparae. A comparison of the two lineages

"First - born" lineage

Time

"Lost-born"

lineage

(days)

FIG.16. The operation of the interval timers controlling the production of the sexual forms in a young clone of Megoura viciae. The two lineages, which were derived from the first- and last-born offspring of a single fundatrix, were continued by retaining only the oldest (left side) and youngest (right side) offspring in each generation. The time of appearance of the different adult morphs (virginoparae, oviparae and males) is indicated ;in addition, the sector diagrams show the proportion of parent virginoparae in each generation that were ovipara(stippled) or male-producers (crosses). The environmental photoperiod and temperature were 12 h and 15"C, respectively.

shows that the ability to produce ovipara is regained almost synchronously, occurring between the 66th and 76th day after zero time, regardless of generation. The functioning of the ovipara inhibitor is unaffected by photoperiod : thus under long day conditions the ability to produce these morphs is I*

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restored at the normal time, but the potentiality remains latent unless evoked by short days. The timing process is not disturbed if the lineage is transmitted through alate rather than apterous parents. It is, nevertheless, conspicuously temperature-sensitive, higher temperatures reducing the duration of the refractory period. The production of males is also totally inhibited in young clones. It is interesting that the males invariably reappear before the oviparae, suggesting perhaps that their formation is governed by a second regulator with a different “time” characteristic. Bonnemaison (1951) attributes the temporary suppression of sexual forms to the presence of a “facteur fondatrice” which is gradually dispersed, revealing the innate tendency towards amphigonic reproduction (“facteur interne”). However, as we are clearly concerned with a type of biological clock, it seems desirable to emphasize this function. The term “interval timer” has been proposed (Lees, 1961). The assumption is that the clock is first “wound up” and set running (perhaps in the fertilized egg), but that only one timing cycle is carried through before the mechanism requires reactivation. A simple hour-glass model inwhich a cytoplasmic inhibitor is progressively partitioned out among the progeny will not of course account for the known properties of the clock. In particular, the failure ofthegeneration number to influence timekeeping indicates that the clock determinants must be self-replicating. It seems that the full complement of oogonia are formed at roughly the same time in the germarium of the virginopara. Yet the cell which chances to become an early embryo and a “fist-born” larva will have undergone all the cell divisions associated with cleavage, blastoderm formation and the differentiation of the “germ cylinder” before the last oocyte has even been ovulated. Evidently, then, the information transmitted to daughter cells after mitosis is not influenced by the number of previous cell divisions. These considerations do not suggest that timing can be due to the imperfect replication of the clock determinants at cell division. A more appropriate model would consist of a mechanism capable of registering the passage of time during the intermitotic period but equally capable of duplicating this information accurately when the cell divides. Systems of this type recall the loss of kappa activity in Paramecium when the particles are introduced by autogamy into a clone of incompatible genotype (Beale, 1954). There are undoubtedly many other long range interval timers in aphids. It has long been recognized that alate aphids tend to produce fewer alate daughters than parent apterae. Hyalopterus pruni (Smith, 1937), Rhopalosiphum prunifolii (Noda, 1954) and Aphis gossypii (Rein-

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hard, 1927) are a few examples chosen from many. The inhibition of alata-production is particularly severe in Megoura viciae and embraces not only the parent alata but also her apterous daughters (Lees, unpublished). This has been tested by exposing both the parent and offspring to the standard crowding procedure described on p. 217. In the examplegiven in Fig. 3, the original parent alata was crowded (with nine other alatae) for 24 h in a 2 x 1 in. tube “arena” but no alate daughters were formed; similarly, when twenty of the second generation daughter apterae were “First-born“

“Lost- born”

r

z0

FIG.17. The alata interval timer in Megoura viciae. The operation of this timing mechanism, which inhibits the production of alate progeny, was tested by crowding the “first-” and “lastborn” daughters of alatae. It will be seen that although these apterae belonged to the same generation, the later individuals almost invariably proved to be more responsive to crowding.

crowded together at a density of 10 per tube, none became an alataproducer. But nearly all the third generation apterae produced alate progeny abundantly, as did the apterous parents of succeeding generations. To determine whether this is an effect of “generation”, the ten first-born and the ten last-born apterae of the second generation were crowded separately. The results showed that in nearly every instance the last-born daughters responded much more readily to the crowding stimulus than the daughters born some 2 weeks earlier (Fig. 17). Evidently, this ability is governed by the passage of time and not by generation. The duration of the reproductive period is such that the parent alata will sometimes produce a few alate daughters at the end of her life. Parent and offspring are then found to regain the function of oviparaproduction almost synchronously.

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In other species of aphids alate virginoparae may often produce daughters in significant, and sometimes large, numbers. Nevertheless, it appears to be a general rule that the first alate offspring are preceded in the progeny sequence by a series of apterae (e.g. Aphis craccivora; Johnson and Birks, 1960). It seems that in these aphids the interval timer preventing alata-production under crowded conditions operates (or ceases to operate) rather sooner than in Megoura. Viewed in this light, alata-production appears as an ephemeral rather than a stable character. There are probably still other long term timing mechanisms that influence the formation of alatae. Bonnemaison (1951) has recorded that some long established (12 months) clones of Myzus persicae begin to produce alatae much more freely. And he is of the opinion that alatae are even produced spontaneously, in the absence of crowding stimuli. Similar changes might also be sought among the fundatrigeniae of holocyclic aphids such as Sappaphis plantaginea. During the spring reproductive phase when Suppaphis is on the primary host, the entire population is converted into winged migrants over the course of three or four generations, notwithstanding the fact that the population density, and presumably the intensity of crowding, must ultimately be reduced by emigration. Other interval timers appear to control progressive morphological changes. The transitional status of the fundatrigeniae has already been noticed (p. 214). The lack of dependence on “generation” is again suggested by the fact that in some clones of Megoura the first-borndaughters of the fundatrix are distinctly more fundatrix-like than the last-born (Lees, unpublished). All these timing mechanisms have important biological functions. The ovipara and male interval timers serve to prevent the premature appearance of the sexual forms during the short days of spring. These clocks do not apparently require the accuracy conferred by a temperature-independent mechanism. It is sufficient if the formation of sexuales is delayed until the photoperiod exceeds the critical length in early summer. Populations will, of course, be built up more rapidly if the colonizing alata produces few alate daughters. And it may even be that the exploitation of a newly discovered food plant requires that the full response to crowding should be postponed still further. Finally, the processes controlling form changes in incipient clones ensure the rapid replacement of a relatively inactive but fecund morph (the fundatrix) by more active viviparae that are better adapted for competing with other aphids of the same species for the available food supplies.

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IX. SUMMARY Polymorphism in aphids is an expression of the multiple developmental potentialities inherent in the egg, the embryo and the young larva. Polymorphism is frequently of the “alternative” type. Competence to differentiate along different alternative routes often arises sequentially during

FIG.18. “Possible” developmental pathways in aphids, as related to the stage of egg or embryonic development. A-D, Megoura uiciae : A, apterous virginoparae;B, alate virginopara; C, fundatrix; D, apterous virginopara from a young clone in which the ability to form males has just been restored. E, Aphisfabae (hypothetical).

development. In monoecious aphids the successive“decisions” comprise the determination of sex (presumably at the oocyte stage); the determination of young female embryos as virginoparae or oviparae ; and finally, the determination of alate embryos as alate or apterous virginoparae (Fig. 18A). In heteroecious aphids the production of oviparae takes place in two stages and requires the intermediary of an additional morph, the gyno-

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para. Although the full range of morphs is therefore wider, the embryonic potentialities arein some respects more restricted. It seems probable that the developmental routes confronting the first generation female embryos are not two- but three-branched, and terminate in the aptera, the alata, and the gynopara respectively (Fig. 18E). The choice of pathway depends on the operation of a series of switch mechanisms which are often maternal (invariably so in the case of oviparae) but are sometimes postnatal. These are in turn controlled by the environment. Photoperiod and, to a lesser extent, temperature play an important role in regulating the development of gamic and agamic forms. Tactile stimulation by other aphids is the most significant component of the crowding response which governs the differentiation of apterae and alatae. The maternal regulation of both sexual and wing form dimorphism seems to depend on hormone-producing centres in the head of the parent insect. The photoperiodic light receptors are located in the brain. Timing mechanisms are also of great importanceinformdetermination. Long range “interval timers”, sometimes acting over many generations, temporarily prevent development from proceeding along certain developmental pathways. The formation of oviparae and of alatae may both be inhibited in this fashion (Fig. 18B, C, D). The timekeeping of these “biological clocks” is unaffected by the number of preceding aphid generations and is also independent of the number of cell generations in the lineage. In addition, aphids exhibit morphological transformations that are continued over several successive generations (“progressive polymorphism”). Here, too, timing processes seem to be involved. The final target both of the environmental cues and of the maternal switch mechanisms is the endocrine system of the embryo or young larva. Evidence is only available for wing dimorphism. But the effect of juvenile hormone extracts, and also of certain hymenopterous parasites in suppressing alatiform characters, and inducing metathetely, strongly suggests that differentiation of these forms is controlled by the level of activity of the corpus allatum early in larval life. REFERENCES Baehr, W. B. de (1920). Recherches sur la maturation des oeufs parthenogenktiques dans 1’Aphis palmae. Cellule 30, 317-349. Banks, C. J. (1958). Effects of the ant, Lasius niger (L), on the behaviour and reproduction of the black bean aphid, Aphis fabae Scop. Bull. ent. Res. 49, 701-714.

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Banks, C. J. and Nixon, H. L. (1959). The feeding and excretion rates of Aphisfabae Scop. on Vicia faba L. Entomologia exp. appl. 2, 77-81. Beale, G. H. (1954). “The Genetics of Paramecium aurelia.” Cambridge University Press. Bodenheimer, F. S. and Swirski, E. (1957). “The Aphidoidea of the Middle East.” Weizmann Science Press of Israel. Bonnemaison, L. (1949). Sur l’existence d’un facteur inhibitant l’apparition des formes sexuCes chez les Aphidinae. C.r. hebd. Skanc. Acad. Sci., Paris 229, 386-388. Bonnemaison, L. (1951). Contribution a l’etude des facteurs provoquant l’apparition des formes ailCes et sexuCes chez les Aphidinae. Annls Epiphyt. 2, 1-380. Bonnemaison, L. (1956). DCterminisme de l’apparition des larves estivales de Periphyllus (Aphidinae). C.r. hebd. S6anc. Acad. Sci., Paris 243, 1166-1168. Bonnemaison, L. (1958). Facteurs d’apparition des formes sexupares ou sexukes chez le puceron cendrC du pommier (Sappaphis plantaginea Pass.). Annls Epiphyt. 3, 331-355. Bonnet, C. (1745). “Trait6 d’insectologie ou observations sur quelques espkces de vers d’eau douce et sur les pucerons.” Paris. Borner, C. and Heinze, K. (1957). In “Sorauer’s Handbuch der Pflanzenkrankheiten,” 5th ed. (0. Appel, H. Blunck and H. Richter, eds.), Bd. V (4). Paul Parey, Berlin and Hamburg. Boschetti, M. A. and Pagliai, A. M. (1964). L‘azione della temperatura sull’ovogenesi partenogenetica di Macrosiphum rosae (Homoptera, Aphididae). Caryologia 17, 203-218. BruslC, S. (1962). Chronologie du dCveloppement embryonnaire des femelles parthCnogCnCtiques de Brevicoryne brassicae (Aphididae, Homoptkes). Bull. SOC.zool. Fr. 87, (4), 396-410. Cartier, J. J. (1959). Recognition of three biotypes of the pea aphid from southern Quebec. J. econ. Ent. 52, 293-294. Cognetti, G. (1961a). Endomeiosis in parthenogenetic clones of aphids. Experientia 17, 168. Cognetti, G. (1961b). Citogenetica della partenogenesi negli Afidi. Archo. zool. ital. 46,89-122. Cognetti, G. (1962). La partenogenesi negli Afidi. Boll. Zool. 29, 129-147. Cognetti, G. and Dallari, L. (1961). Effetti diversi della selezione su due linee partenogenetiche di Myzodes persicae in ambiente non costante. Monitore zool. ital. 69, 3-7. Cognetti, G. and Pagliai, A. (1962). Nuove experienze per le forme attere e alate di Brevicoryne brassicae. Atti Accad. naz. Lincei Rc. 32, ser. 8, fasc. 3, 403-407. Cognetti, G. and Pagliai, A. M. (1963). Razze sessuali in Brevicoryne brassicae L. (Homoptera Aphididae). Archo. zool. ital. 48, 329-337. Davidson, J. (1929). On the occurrence of parthenogenetic and sexual forms in Aphis rumicis L. with special reference to the influence of environmental factors. Ann. appl. Biol. 16, 104-134. Dehn, M. von (1963). Hemmung der Fliigelbildung durch Farnesol bei der schwartzen Bohnenlaus, Doralis fabae Scop. Naturwissenschaften 50 (17), 578-579. Dixon, A. F. G. (1963). Reproductive activity of the sycamore aphid, Drepanosiphum platanoides (Schr.) (Hemiptera, Aphididae). J. anim. Ecol. 32, 33-38. Dunn, J. A. (1 959). The biology of the lettuce root aphid. Ann. appl. Biol. 47,475-491.

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The Regulation of Breathing in Insects P. L. MILLER Department of Zoology, University of Oxford, England I. Introduction. . 11. The Control of Ventilation . A. General Remarks . B. The Endogenous Nature of the Ventilatory Rhythm . C. Types of Endogenous Activity Connected with Ventilation . D. Co-ordination within the CNS E. Proprioceptive Input . F. The Effects of Carbon Dioxide and Hypoxia . G. Electrical Stimulation of the CNS . H. Inspiration through Cuticular Elasticity and Reduced Pressures . 111. The Control of the Spiracles . A. General Remarks . B. Innervation of the Spiracles . C. Innervated Tracheae . D. Spiracular Activity . E. Control Mechanisms in Two-muscle Spiracles . F. Control Mechanisms in One-muscle Spiracles . G. Experiments on the Nature of the Chemical Stimulus . . H. Synchronized Activity of the Spiracles I. Independent Activity by the Spiracles . IV. Modifications of the Tracheal System for Flight . A. Functional Morphology 07 the Tracheal System in thePtero;hora; B. The Locust Pterothorax . C. Movement of Air in the Primary and Secondary Tubes by Ventilation . D. Movement of Gases in the Secondary and Tertiary Tubes by Diffusion . . E. Spiracle Behaviour during Flight . F. The Oxygen Supply to the Resting Flight Muscles . V. Summary . References

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I. INTRODUCTION Much of the success of insects is due to their effective solution of the two major physiological problems of water retention and oxygen supply. The potentially rapid water loss of small organisms which are active in dry environments is prevented by a highly specialized cuticle (Beament, 1961), and by spiracles which accurately regulate gasexchange by using a 219

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mechanism which recent work has shown to be remarkably complicated. The oxygen supply to flight muscles, which may metabolize aerobically at rates equalled only by certain bacteria (Weis-Fogh, 1961), is brought in through an elaborate system of tracheae and tracheoles which in many species terminate alongside the mitochondria by means of indenting the cell walls. Several reviews of various aspects of insect respiration have appeared recently (Wigglesworth, 1960; Edwards, 1960; Buck, 1962; Keister and Buck, 1964; Miller, 1964d), and in this chapter there is a call only for the treatment of those parts of the subject where new developments have taken place in the last five years or so, or those which were covered only in outline by the reviewers. I shall deal therefore with the control of ventilation, with the regulation of spiracle movements, and with the specializations of the tracheal system to meet the enormous demands in flight. The interesting results of Wigglesworth (1954, 1959) and Locke (1958a, b) on the morphogenesis of the tracheal system, the control of its distribution, and its responses to areas deficient in oxygen do not appear to have been followed up and so will not be dealt with here. Nor have we yet any direct measurements of tracheal and tracheolar permeability to gases. Further information about the movement of liquids within tracheal systems (Wigglesworth, 1953b) is still lacking, although Beament (1964) has argued convincingly that water movement in tracheoles is an active process brought about by the surrounding cytoplasm. Recent electron microscope studies by Shafiq (1963), following on from the earlier work of Keister (1948) and Wigglesworth (1954), have added to our knowledge of the intracellular fevelopment of tracheoles, but many problems of tracheal growth and development remain, not least those posed by the enormously hypertrophied abdomen of the mature queen termite (Macrotermes), where the conventional tracheal system of the young winged queen is entirely abandoned and a new system, supported by a unique form of taenidium, replaces it (Miller, unpublished). 11. T H E CONTROL OF VE NTI LATI ON A. G E N E R A L R E M A R K S

Gases are moved along tracheae under a gradient either of pressure (ventilation) or of concentration (diffusion). Ventilation may be taken to include mechanisms in which the elasticity of tracheal walls or body segments, together with controlled spiracle activity, creates a draught inwards : the more obvious pumping movements will here be considered first.

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The study of ventilation may be divided into three phases. In the first the pumping movements are described and catalogued, and here information exists about a large number of species (Plateau, 1884; Wigglesworth, 1931). In the second the volume of air pumped is measured and compared with the total volume of the system, and with that in other species. There is a certain amount of information about a few species here (Krogh, 1920; Fraenkel, 1932) and much about the locust (WeisFogh, 1964a, b, and in preparation). The third stage, hardly broached, is concerned with the underlying neuromuscular mechanisms which bring about pumping. The need for the pterothorax to have a rigid box-like structure for the operation of the flight mechanism has limited pumping in most insects to the more flexible abdomen. In a few species pumping occurs in the segments anterior to the pterothorax, but it is then an auxiliary form only (Miller, 1960a). Some apterous insects such as Dixippus (Buddenbrock and Rohr, 1922) or Lentula (Ewer, 1958) may pump with the thorax, and it is likely that large apterous ancestors of present-day insects normally performed such movements. Abdominal pumping strokes are either in the longitudinal axis of the insect (cockroach) or in the vertical axis (dragonfly); they may occur in both in a few types (locust) when ventilation is strong. Recordings of ventilation are often made with a mechanical spirogram in which a lever is attached to the moving sclerites by a thread (Plateau, 1884; Miller, 1960a; Myers and Fisk, 1962). Although an adequate method for comparative results, it may lead to distortion of the abdomen and a compensating alteration of pumping frequency. Similar difficulties may be met when strain gauge transducers are used (Myers and Retzlaff, 1963). A different method, which has been employed by Weis-Fogh (in preparation) on Schistocerca, makes use of pressure changes in a chamber enclosing the abdomen ;air expired from the posterior spiracles is led out separately and other spiracles are sealed. This method largely overcomes the difficulties mentioned above and allows simultaneous measurements to be made of the total volume pumped and of the magnitude of the uni-directional airstream. The need to seal spiracles 4-9 may slightly decrease the stroke volume, but the effect is small. By such means Weis-Fogh (1956a, and in preparation) has shown that the desert locust pumps 40 l/kg/h in normal resting ventilation, and 250 litres when maximally stimulated by CO, and hypoxia. An additional 50 litres may be provided by head and prothoracic strokes which occur during strong ventilation in phase with the abdominal movements (Miller, 1960a). Weis-Fogh has shown that the amount of air pumped uni-

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directionally through a locust does not increase much during hyperventilation. The increase is therefore in the tidal component and the spiracle valves become increasingly inefficient as one-way valves. A similar tidal component is superimposed on the one-way stream when honeybees are stimulated to ventilate strongly (Bailey, 1954)and the same may again occur in adult dragonflies (Miller, 1962). Hughes (1958) has investigated the swimming movements performed by the abdomen of dragonfly nymphs (Aeshnidae) using cinephotography with simultaneous pressure recordings from the branchial chamber. Synchronization of events in the two records was achieved by means of a rotating wire which simultaneously came into focus on the film and altered the brightness of the oscilloscope screen. Mill and Hughes (1965) have used a similar combination of pressure and photoelectric recording techniques to study the nervous control of ventilation in the same species. Experiments on neuromuscular mechanisms responsible for ventilation usually involve destruction of parts of the body wall. The effectiveness of the pumping stroke may thereby be considerably reduced since pressure cannot be built up and haemolymph movements will not occur in parts of the insect remote from the pump: only the abdomen will be ventilated effectively while the thorax and head may suffer from oxygen shortage and produce in consequence hyperventilation. Attempts to overcome the difficulties may be made by perfusing the larger tracheal trunks with air or oxygen (cf. Wilson, 1961), or by minimizing cuticular damage. Further difficulties arise when lateral nerves or connectives are cut, or ganglia are halved, since tracheae are likely to be damaged, and subsequent changes in activity may result as much from oxygen starvation as from the dissociation of parts of the central nervous system (CNS)-an important consideration when long term changes in activity are being measured. Moreover such operations allow mixing of intraand extra-ganglionic fluids which are known to be different in composition (Treherne, 1962). Attempts to record from units by splitting connectives may avoid some of these problems since the neurilemma is broken in areas remote from synapses, but damage to tracheae must still be minimized since, in vertebrates at least, nervous conduction fails as quickly as synaptic transmission with oxygen lack (Bronk et al., 1948). B. THE ENDOGENOUS N A T U R E OF THE VENTILATORY RHYTHM

In some insects the separated abdominal segments can initiate respiratory movements independently (Alverdes, 1926). Moreover, Adrian (1931) recorded rhythmic bursts of nervous activity from the isolated

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cord of Dytiscus which he believed to be ventilatory in nature. The formation of endogenous bursts which might produce pumping in the intact insect seems to be acommon feature of the isolated CNS of many species, and the abundant proprioceptive input available (Hughes, 1952) seems not to be essential for ventilation. For example, Case (1961a) has described records which were taken from the lateral nerve stumps of the isolated cord of Blaberus; they comprised bursts of impulses of similar frequency and character to those recorded from intact ventilatory nerves. The rhythmic activity is confined to the anterior end of the abdominal cord, and, for the rhythm to appear, at least the first abdominal ganglion plus either adjacent ganglion are needed ; the more posterior ganglia produce only tonic activity. Likewise, in the Cuban burrowing cockroach, Byrsotria fumigata, the first abdominal ganglion is principally responsible for initiating ventilatory bursts and they occur at 6/min (Myers and Retzlaff, 1963); this activity continues when the ganglion is isolated. The sixth abdominal ganglion (the last) of the same insect may give rise to burstsat a much lower frequency (0.5-0.12/min), while intervening ganglia produce only tonic activity or irregular and infrequent bursts (Fig. 1). In locusts the metathoracic ganglion complex contains the first three embryonic abdominal ganglia (Albrecht, 1953), while in cockroaches the first two, and in dragonflies only the first is joined in this way. These structural differences may explain ventilatory differences between the insects, if the embryonic third abdominal ganglion always contains the most anterior and important pacemaker for ventilation. In the locust (Miller, 1960a) and in Dytiscus (Adrian, 1931), rhythmic activity can be recorded from the isolated metathoracic ganglion, as well as from those in the abdomen; when intact it can be seen to cause ventilation in the anterior segments, and this continues after removal of the whole abdominal nerve cord. In Periplaneta (Schreuder and de Wilde, 1952) and dragonflies (Miller, 1962) such rhythmic activity is confined to the abdominal cord which includes the third embryonic ganglion. Both Case (1961a) in Blaberus and Periplaneta, and Myers and Retzlaf (1963) in Byrsotria have described this ganglion as playing a major part in ventilation. When isolated the abdominal ganglia of the locust may each produce rhythmic bursts. Sometimes in air they are tonically active, but bursts are formed when CO, is applied (Miller, 1960a).In Periplaneta, however, CO, may do no more than raise the level of tonic activity, and burst forming centres seem to be absent from more posterior ganglia (Roeder and Roeder, 1939; Boistel and Coraboeuf, 1954; Case, 1961a). Rhythmic movements, co-ordinated with ventilation, may also take

FIG.1. Simultaneous records from a lateral nerve of the first abdominal ganglion ( A l ) and of thc sixth abdominal ganglion (A6) of Byrsotriufurnigufu after removal of the third and fourth ganglia. The different r h y t h m of each are shown, and in addition the periodic pattern of A l can be seen. (From Myers and Retzlaff, 1963.)

FIG.2. Intracellular records from the Br giant nerve cell of the visceral ganglion of Aplysiu. In oxygen the cell generates spcntancous bursts of spikes; with COP(lower record) the t i n t frequency increases although the frequency of spikes in a burst remains much the same. (From Chalazonitis, 1963.)

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place in more anterior thoracic segments. In the locust they occur in all thoracic spiracles and at times in the head and prothorax; they are caused by the metathoracic ganglion, and there is no evidence for the existence of more anterior pacemakers (Miller, 1960a, b). However, Hoyle (1959) reported the occurrence of rhythmic fallsin tension, at about l/sec, in the muscle of spiracle 2 of Schistocerca, after section of the connectives between meso- and metathoracic ganglia, whereas Miller (1960b) recorded only continuous trains of impulses in the two motor axons to this spiracle under similar conditions. Hoyle concluded that the mesothoracic ganglion initiated a rhythm while Miller, who stated that it did not, attempted to reconcile the conflicting results on the basis of the occurrence of “beats” in the two motor axons, or periods when they fired in phase alternaLing with periods when they were out of phase. Outof-phase firing was seen to correspond sometimes to a smooth tetanus, while in-phase firing gave rise to flutters (Section 111, H). However, this explanation does not seem adequate to account for Hoyle’s published records, and another must be sought. One possibility is that an additional nerve (perhaps inhibitory) supplies the spiracle from the metathoracic ganglion and that its presence and activity have so far been overlooked; but this seems unlikely in view of the exhaustive examination that spiracle 2 has received (Hoyle, 1959, 1960, 1961), and the problem must await further investigation. Ventilatory activity is endogenous in so far as it is not dependent on extra-ganglionic afference. Facilitation or excitation from within the central nervous system is probably needed, however, excitation being in part derived from “receptors” which respond to intra-ganglionic changes of the COz and oxygen Ievels (Section 11, F). In some insects, e.g. wasps (Fraenkel, 1932) and cockroaches (Schreuder and de Wilde, 1952), ventilation is seen only during and for a short time after periods of activity, the insects being otherwise apnoeic. In mature locusts and dragonflies, however, pumping continues indefinitely in quiescent insects at normal environmental temperatures, although it may be much retarded in streams of oxygen: in these therefore the ventilation pacemakers appear to have much lower thresholds. While it is likely that some degree of tonic input from ganglionic receptors is essential for pacemaker activity in all these insects, phasic restimulation of the pump every cycle is not needed. The formation of bursts of spikes, not related to external movements, is a common pattern of interneurone activity (Fielden and Hughes, 1962; Hughes and Tauc, 1962; Biederman, 1964; Vowles, 1964). Even isolated axons may discharge rhythmically under certain conditions (Arvanitaki,

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1943). In a study of ventilation, it is therefore essential to distinguish between spurious forms of activity in the CNS and those which are involved in the rhythmic efferent flow to the pumping muscles. Activity in lateral nerves which supply these muscles may be recognized as ventilatory, after distal section, by comparison of the frequency and phasing of the firing units before and after the operation. The identification of interneurones however on the basis of their characteristic activity is fraught with difficulties. Rhythmic bursts which continue in the cord after de-afferentation and which are synchronized with bursts in the lateral nerves, though their component spikes are not on a 1 :1 basis, may occur in ventilatory pre-motor or command interneurones :but the widespread occurrence of “irradiation” in the insect CNS makes their identification less certain. Irradiation occurs, for example, in operated locusts where rhythmic movements of mouthparts, legs and even wings are often seen in phase with expiration. Moreover, by leading off from chronically implanted electrodes in locust leg muscles, Hoyle (1964) has shown that single impulses or small bursts are superimposed on the resting discharge with every ventilatory stroke. They cause no movement in the intact insect, but they show that “irradiation” may be a normal occurrence. That they are not caused by proprioceptive reflexes is shown by their perpetuation after abolition of all pumping movements (Miller, in preparation). Hoyle suggests that they may be due to internal electric field effects in the neuropile or to widely distributed interneurones. They clearly underline the “need to distinguish between the accidental and the functionally important features of a biological system” (Gregory, I958), if we are to understand the part played by command interneurones (Section 11, D). Additional methods, such as electrical stimulation (Section 11, G), must be employed for their recognition. C. TYPES OF E N D O G E N O U S A C T I V ITY C O N N EC TED WITH VENTILATION

Three types of endogenous activity play a part in ventilation: (1) continuous repetitive firing by neurones, with a periodicity of a few milliseconds; (2) the firing of bursts, with a periodicity of a few seconds; (3) phases of burst formation separated by long quiescent periods, which may last for many minutes. 1. Repetitive firing This is a widespread phenomenon in central nervous systems (Bullock, 1961; Roeder, 1963). It is characterized by a depolarizing drift of the

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membrane of a nerve cell towards the threshold for firing, then spike formation followed by repolarization, whereupon the cycle is repeated. Neurones may fire trains of impulses indefinitely, ceasing only when inhibited by other cells. Much information about such pacemaking cells in arthropods has been derived from a study of the crustacean heart ganglion (Hagiwara, 1961), where more than one pacemaker locus has been found in a single cell. For a useful summary of knowledge about interaction within ganglion cells, the reader is referred to Horridge (1963). 2. Burstformation Two hypotheses have been put forward to account for burst formation in central nervous systems. In the first, two neurones or groups of neurones are envisaged each with similar properties, the most important being repetitivefiring,accumulation of refractoriness (or accommodation) and reciprocal inhibition. Thus one group fires, slows and then stops, thereby lifting inhibition from the other which then repeats the cycle. Evidence for such a mechanism has been obtained from the study of respiratory centres in the mammalian medulla with intracellular electrodes (Salmoirhagi and Burns, 1960; Salmoirhagi and Baumgarten, 1961; Salmoirhagi, 1963). Separate recordings from either inspiratory or expiratory neurones have shown typically a burst which slows as the threshold for spike initiation rises and is followed by a barrage of inhibitory post-synaptic potentials (IPSPs) presumed to come from the antagonistic cells. Records were made after the animal (a cat) was paralysed with succinyl choline so that proprioceptive input played no part. In the second hypothesis, two neurones or groups of neurones with dissimilar properties are postulated. The first produces bursts and these rhythmically inhibit the tonic activity of the second. Thus, while the actual mechanism of burst formation is not accounted for, it may well be the property of a single cell, which spontaneously produces undulatory potentials with spikes occurring on a particular part of the wave, not uecessarily the peak (cf. Hagiwara, 1961). This in outline is the theory supported by Wang and Ngai (1963 ;see also Wang et al., 1957)to account for respiratory activity in the mammalian pons, where it is thought that rhythmic inhibitory activity derived from a pontile pneumotactic centre periodically interrupts the tonic activity of an apneustic centre. Intracellular recordings made from the spontaneously active giant nerve cells in the visceral ganglion of Aplysiu have demonstrated properties which might also be found in respiratory neurones (Arvanitaki and Chalazonitis, 1961; Chalazonitis, 1961, 1963). The characteristic

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activity of one of these, the Br cell, is burst formation, and the failure to record IPSPs or excitatory post-synaptic potentials (EPSPs) strongly indicates that it is spontaneously derived. Slow waves of potential change occur, with spiking on the crest of depolarization; within each burst the spikes accelerate and then decelerate. Electrical stimulation of a neighbouring cell shortens the interburst period by reducing the level of repolarization reached after a burst. CO, or hypoxia have a similar effect, the actual duration and spike frequency of the burst remaining more or less unchanged (Fig. 2). The little evidence available about insect ventilatory centres suggests that the second, Wang and Ngai, type of mechanism operates in locusts and cockroaches. Inspiratory bursts in lateral nerves from the metathoracic ganglion of a locust are regular in frequency and they wax and wane during a burst; moreover they are of more or less constant duration at widely different frequencies of ventilation. The duration of expiratory bursts, on the other hand, from the same ganglion varies directly with the overall frequency of ventilation. In other words the frequency of ventilation is altered largely by adjustment of the length of the expiratory burst (Fig. 3). When ventilation ceases temporarily, expiratory firing continues until the next cycle commences (Miller, in preparation). These observations suggest that a burst-forming cell, perhaps similar in properties to the Br cell of the Aplysiu ganglion, rhythmically inhibits the tonic activity of expiratory cells, and simultaneously excites inspiratory motor cells. The burst-forming unit may itself be an inspiratory motor neurone. Such a mechanism may be repeated serially in each abdominal ganglion, although the metathoracic pacemaker normally overrides more posterior centres (Section 11, D). In Bluberus too the mechanism may be similar(Case, 1961a). Here the tonic expiratory activity of more posterior abdominal ganglia is rhythmically interrupted by the first abdominal ganglion, as already mentioned, and the same may be true in Byrsotriu (Myers and Retzlaff, 1963), although this is less clear. Finally it may be noted that Adrian (1931) recorded slow waves of potential change from Dytiscus ganglia in phase with the ventilatory bursts, which might be those of burst-forming cells. 3. Periods of burst formation The endogenous nature of the third type of rhythm has only recently come to light. A pattern in which there are periods of ventilation interrupted by pauses of variable length has been described in a number of insects (e.g. Babhk, 1921; du Buisson, 1924; Miller, 1960a), and it

A.

GIII-ABDC

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FIG.3. Records from an expiratory lateral nerve from the metathoracic ganglion of Schistocercugreguriu. A, With the m e n - and prothoracic ganglia removed: B, with the metathoracic ganglion completely isolated from the insect: C, as in B treated with CO,.

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explains, for example, the periodic output of CO, from teneral locusts during the first 24 h of adult life (Hamilton, 1959, 1960, 1964.). Miller (1964d) suggested that it was due to the periodic washing out from the blood of stimuli which excited ventilation and that it was therefore comparable to Cheyne-Stokes or intermittent ventilation in man. In some cockroaches, however, it has been shown to arise endogenously in the CNS and to continue when actual pumping movements have been arrested. By recording the ventilatory movements of intact Byrsotria over long periods, Myers and Retzlaff (1963) have shown that the typical pattern comprises groups of 3-15 pumping strokes at a frequency of 5/min, separated by apnoeic intervals lasting about 7 min. In every hour, therefore, the cockroach might ventilate for only 4 min. After decapitation the movements changed to continual pumping at increased amplitude, and these persisted for at least 1 week so that injury effects were probably not involved. The subsequent removal of the sixth abdominal ganglion restored the pattern of periodic ventilation with a similar frequency to that in the intact insect. Moreover records from the isolated first abdominal ganglion (Fig. 1) continued to show a ChcyneStokes pattern with 10-20 bursts separated by silent periods lasting 5-10 min. Thus even when pumping no longer occurs, the CNS is able to produce a pattern of periodic ventilation. Myers and Retzlaff suggest that the last abdominal ganglion has an excitatory effect which causes periodic to be replaced by continual ventilation. In the presence of the head the excitatory effect of the last ganglion is counteracted so that periodic ventilation appears in the intact insect. In another cockroach, Blaberus discoidalis, a similar type of periodic ventilation can be recorded in the intact insect, and at increased amplitude after decapitation. Subsequent removal of the last abdominal ganglion has no effect in this species. Electrical records from the first abdominal ganglion, in contact only with the more anterior parts of the cord, show periods of bursts and silent phases, the former occurring at a frequency, duration and amplitude similar to those of the spirogram records from the intact insect. As in Byrsotria, the pattern therefore can be derived endogenously without reference to the periphery. Perfusion of Blaberus with CO, does not abolish periodic ventilation, although the previously silent periods may become filled with shallow fast ventilation of a different nature. The rhythmic clock may then be independent of the chemical situation, although other parts of the ventilatory mechanism respond to it (Miller, unpublished). No functional interpretation of periodic ventilation has yet been made, but the mechanism may be comparable to that in the silkmoth pupae and other insects where the periodic

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release of C 0 2is thought to help to conserve water (Buck, 1962). Unlike the pupa, however, the cockroach bursts are not triggered by an increase in CO, or hypoxia. A similar type of periodic ventilation has been reported in Periplaneta americana at 29°C(Paulpandian, 1964),although it has not been observed at lower temperatures (Schreuder and de Wilde, 1952). Both in burst formation and in periodic ventilation we are concerned with latencies which may be several thousand times greater than the known time constants of synaptic potentials (Horridge, 1963); in periodic ventilation hormonal mechanisms may be responsible. Longterm physiological effects are well known in central nervous systems: for example Kandel and Tauc (1964) have shown in Aplysia that test EPSPs may be augmented by up to 40 min after the delivery of paired stimuli, and other long-lasting after-effects have been demonstrated in the crustacean nerve cord (Kennedy and Preston, 1963). Their mechanism is not understood but secretory processes affecting the presynaptic fibre may be involved. D. C O - O R D I N A T I O N W I T H I N THE C N S

The pumping strokes of the locust are not peristaltic in nature; they comprise synchronized contractions throughout the length of the abdomen. Although abdominal ganglia can independently initiate movements in their own segments, an overriding mechanism co-ordinates the activity in the intact insect. Recording and stimulation experiments have revealed something of this mechanism (Miller, in preparation); some of the difficulties in this approach have already been outlined (Section 11, A and B) and conclusions must be treated with caution. In the locust a burst of impulses travels posteriorly from the metathoracic ganglion to the end of the cord, slightly in advance of the expiratory bursts recorded in lateral nerves. The burst may comprise a few units firing at aconstant frequency, but with stronger pumping more units participate. The impulses travel through the intervening ganglia without delay, so that probably singleinterneurones extend down the cord. That they make contact with motor cells on both sides of each ganglion, possibly through a second transverse interneurone, is shown by the continuation of bilateral pumping after section of one connective (Fig. 4A). Impulses take about 20 msec to reach the last ganglion, a delay which is less than 10%of the duration of even the shortest expiratory strokes and therefore unlikely to be significant. Inspiratory bursts cannot be seen in records from the cord and the

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command interneurones (Fig. 4, cells B) may set the cycle by initiating an expiratory stroke. They fire throughout expiration, and continue to be active at a reduced frequency during the ventilatory pause which some-

SPIMCLES

AUX. PUMP

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\ ABDL.G

A

FIG.4. A. Suggested organization of the command interneurones on one side of the abdominal nerve cord of Schistocerca. They are shown running from the metathoracic pacemaker and supplying the expiratory motor cells ineach ganglionvia a transverse interneurone. B. Suggested organization of the metathoracic pacemaker. Cell A produces spontaneous undulatory potentials and bursts of impulses: these rhythmically inhibit the tonic activity of cell B. Cell B is the command interneurone which runs down the cord and excites cells C (via an interneurone, E), which are the expiratory motor neurones in each ganglion. The activityof a cell C inhibits thetonic firingofcell D, the inspiratory motor neurone. Theanterior connexions of the pacemaker with thoracic spiracles and with auxiliary pumping centres (aux. pump) is also indicated.

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times intervenes before the movement is complete (Section 111, H). When the inspiratory motor cells in abdominal ganglia participate, they fire regular trains of impulses like those of the metathoracic ganglion. Their tonic activity may be periodically inhibited either by the expiratory cells or by the command fibres. Bursts in the expiratory motor nerves, on the other hand, comprise irregular discharges in which many units participate. This hypothesis is summarized in Fig. 4B. The endogenous activity of the abdominal pacemakers apparently does not contribute to the output in the intact insect, and the system cannot be compared to a hierarchy of relaxation oscillators, each triggering the activity of the next. Attempts to de-synchronize abdominal ganglia by localized gassing with C 0 2do not make them break away from metathoracic domination. In intact locusts, however, which are pumping weakly, two superimposed rhythms may at times be recorded on a spiro- . gram, the faster belonging to the more anterior segments (Miller, 1960a). Likewise Myers and Retzlaff ( 1963) have reported the coincidence of two ventilatory rhythms in records from a lateral nerve of Byrsotria after treatment with C 0 2 ,and similar records have been obtained from locust nerves after cutting one connective. Under these conditions the dominant pacemaker continues to drive the rhythm of the whole abdomen, but it fails to suppress the endogenous activity of other ganglia. In the cricket a comparable system of command fibres has been recognized and it is responsible for the co-ordination of pumping in the whole abdomen (Huber, 1960). After an abdominal ganglion was halved, Huber found that pumping continued in the more posterior segments, but ceased in the operated segment. He suggested that command fibres travel through every ganglion, but that the integrity of transverse interneurones is essential for the activity of each. Bursts of activity, synchronized with ventilatory strokes, have been recorded also from the cords of dragonfly larvae (Mill, 1963), and here too they continue after de-afferentation and are possibly part of a command system. Few fibres are involved when ventilation is shallow, but many participate during strong pumping (Fielden, 1960). In general, command fibres concerned with ventilation run from the dominant pacemaker to more posterior ganglia where they act on the expiratory motor cells, perhaps via transverse interneurones. They are therefore comparable to the integrative follower neurones in the crustacean heart ganglion (Hagiwara, 1961)which also run between pacemakers and motor cells. Such command fibres probably figure largely in arthropod central nervous systems where activity in different segments must be co-ordinated (Huber, 1962).

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De-afferentation experiments have shown that the pumping rhythm continues to be produced by the CNS in the total absence of phasic input. Nevertheless, proprioceptors probably play a part in the maintenance of frequency, and possibly in other ways. Hughes (1952) described sensory activity associated with ventilation in Dytiscus and Locusta. Some units responded with expiration, others with inspiration and others again were silenced during the inspiratory stroke. While some of the responses may arise from chordotonal organs, the principal receptors involved are probably stretch receptors (Finlayson and Lowenstein, 1958; Osborne and Finlayson, 1962). After dissection of the abdomen, Hughes found that the ventilatory frequency in Dytiscus fell from 10-1 5/ min to 6/min, presumably due to the inactivation of receptors. In other insects,however, suchasByrsotriu(Myers and Retzlaff, 1963)andBZaberus, de-afferentation does not necessarily lead to a fall in frequency. Again in Schistocerca the burst frequency may be maintained after deafferentation if the insect is perfused with 5% C 0 2 throughout. There may indeed be a considerable “hyperventilation” after such an operation, arising from the increased hypoxia in the head which follows on the absence of pumping. This will therefore offset any effect produced by the cessation of proprioceptive input. However, anarchy, as Hoyle (1964) remarks, often follows from the use of a multiplicity of species and in no case has the part played by proprioceptors in ventilation been thoroughly examined. Bullock (1961) has drawn a clear distinction between rhythmic forms of activity which must be phasically variable, such as walking in which the steps are continually adjusted to match an irregular terrain, and those which need be only tonically variable, such as swimming,flying and ventilation, where the medium is uniform. Some similarities between the central nervous mechanisms which co-ordinate flight and ventilation would therefore be expected and such are found: both, for example, are produced by endogenous rhythms, and both may receive phasic input from stretch receptors which have only a tonic effect on the rhythm (Wilson, 1964b). A sequence of reflexes, each triggered by proprioceptors or by gas receptors, would probably entail greater neural complexity than does a built-in pattern which needs only a tonic stimulus for its release. F. T H E EFFECTS O F CARBON DIOXIDE A N D H Y P O X I A

The responses of ventilation to changes in the 0 2 / C 0 2ratio have been studied in many insects, but the exact location of the reaction is not

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known, nor are the receptor mechanisms understood. Fraenkel (1932) postulated the presence of secondary thoracic centres with a low threshold to C 0 2in the locust, while primary abdominal centres were much less responsive. A similar sensitivity of thoracic ganglia has been shown to exist in Dixippus (Stahn, 1928) and in Periplaneta (Schreuder and de Wilde, 1952; Case, 1961a). In Byrsotria a rise in the threshold of the response.to C 0 2 follows decapitation, but sensitivity is restored when the last abdominal ganglion has been removed (Myers and Retzlaff, 1963). This last supports the hypothesis of an antagonistic relationship between the head and last abdominal ganglion in this species, which affects not only the response to COZY but also the appearance of periodic ventilation (Section 11, C, 3). The removal of other ganglia does not alter the threshold further, and the isolated first abdominal ganglion responds much as in the intact insect. Experiments in which connectives are cut may not only cancel the effect of specific C 0 2and hypoxia ganglionic receptors, but also inactivate more general excitatory and inhibitory pathways (cf. Rowell, 1964); changes in the threshold of the ventilatory response may arise from both causes. Moreover, decapitation may also produce effects through the removal of endocrine glands whose secretions are in some cases known to alter central nervous activity (Milburn and Roeder, 1962). For these reasons experiments in which the intact ganglia are separately perfused with gas mixtures through their tracheae will indicate more clearly the part played by C 0 2 and hypoxia receptors in the CNS. Local perfusion of the head and thoracic ganglia of Schistocercu has shown that each is able to promote hyperventilation, and that the head receptors are the most responsive. Stimulation of the abdominal ganglia, however, when the thoracic ganglia are present, does not alter ventilation (Miller, 1960a). The receptors involved may be long interneurones with dendrites in several ganglia through which they respond to the prevailing C 0 2 and oxygen tensions. Experiments to date do not exclude the possibility that additional receptors lie outside the CNS, perhaps in tracheae, and that they also contribute to the control of ventilation. However, localized perfusion of tracheae was found to stimulate ventilation with a latency more or less proportional to the distance from the nearest ganglion, suggesting that the action was only on the CNS. Huber (1960) has shown that the head of the cricket contains centres controlling ventilation. When the mushroom bodies in the brain were eliminated, he recorded a permanent increase in frequency, as well as a general excitation of other motor activities. Removal of the brain caused K

296

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a slight temporary disturbance of the rhythm, whereas after the suboesophageal ganglion had been cut out, there was a permanent slowing. Likewise, removal of the locust sub-oesophagealganglion or, separately, of the abdominal ganglia depresses ventilation, although when continuously perfused with 5% C 0 2 such operations may have little or no effect. Subsequent removal of other ganglia in these insects does not affect ventilation further. Both the sub-oesophageal and abdominal ganglia seem therefore to be needed for the maintenance of the normal ventilatory frequency : while the former may act on the pacemaker through receptors and other pathways, the abdomen seems to contribute only a general excitatory effect. The locust and cricket stand in contrast to some species of cockroach where decapitation is followed by a permanent increase in ventilation (Myers and Retzlaff, 1963). Strong stimulation of ventilation in Hydrocyrius and Schistocerca produces not only hyperventilation by the abdomen, but also auxiliary pumping by the head and prothorax (Miller, 1960a, 1961a).For the movements to appear in the locust, the metathoracic and the sub-oesophageal ganglia must be present. Units, which arise in the metathoracic ganglion and initiate pumping in anterior segments, appear to remain insensitive to the ventilatory pacemaker until excited by other cells which respond to chemical stimulation in the head. The situation recalls Horridge's (1961) hypothesis of interaction in the neuropile in which anatomical connexions are thought to be of less importance than are the graded and variable thresholds of different units to a widely diffusing transmitter. We may now consider in more detail the possible modes of action of COP and hypoxia on nerve cells. Both are able to depolarizeneurones(Cha1azonitis, 1963), but whereas 5-25% C 0 2 may be required to produce an appreciable effect on non-respiratory cells (cf. Boistel and Coraboeuf, 1954; Boistel et al., 1957), as little as 1-2% may stimulate ventilation. The ganglionic receptors may therefore be interneurones particularly sensitive to changes in the 0 2 / C0 2ratio. Chalazonitis has described the effect of C 0 2 and of hypoxia on spontaneous units in the ganglion of Aplysiu (cf. Section 11, C, 2) :for example the frequency of steadily firing cells is increased; cells which fire in bursts have the interburst interval shortened; previously silent cells may be made to fire repetitively; cells showing a slow barrage of EPSPs but no spiking may be induced to fire once every few EPSPs, while IPSPs are antagonized by C02. All these effects are attributable to an increased rate of depolarization, and any might provide the receptor cell with a suitable mechanism. Nothing is known of the nature of the stimulus acting on the cell membrane: both C 0 2 and hypoxia might act through pH changes as

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suggested for the flea spiracle by Wigglesworth (1935). Changes in the pH are thought to be responsible for altering ventilation in Blaberus where Case (1961b) has shown that any one of thirteen weakly dissociated acids applied to the cord produced hyperventilation. The more highly dissociated acids have this effect only at lower pH values, and Case postulated the presence of a barrier surrounding the CNS which is more permeable to undissociated molecules than to ions. Hence weak acids penetrate more readily, but once within, the effect takes place through the pH. Oxygen lack may have a direct action on nerves, causing inactivation of the mechanism which maintains the selective permeability of the membrane. Metabolic inhibitors, such as indoleacetic acid (IAA), also produce depolarization (Narahashi, 1963), and this may be brought about by the accumulation of K+ outside the membrane which can no longer retain the ions. At the spiracle, in contrast, it appears that the muscle is specifically sensitiveto C 0 2and not to pH effects (Hoyle, 1960), while hypoxia has either no action, or an antagonistic one (Section 111, G). G. ELECTRICAL STIMULATION OF THE C N S

,.

Stimulation of the protocerebrum of a cricket produces one of four different effects on ventilation according to the area selected: an increase or decrease in the rate of pumping without other detectable movements, or an increase or decrease of pumping and general motor activity (Huber, 1960). Huber concluded that there are specific areas in the brain for controlling ventilation, but it is not clear if these respond normally to gas tensions or are part of a general excitatory or inhibitory mechanism with an overriding effect on ventilation. After hyperventilation, Huber noted a period of depression before the usual frequency was resumed, perhaps due to a temporary lowering of the blood C 0 2level. In a decapitated locust with all lateral thoracic nerves cut, tonic stimulation of the connectives between pro- and mesothoracic ganglia produces hyperventilation, sustained for as long as the stimulus is applied. With increased intensity the frequency of pumping rises up to a maximum of about 120/min, presumably as more units are stimulated. Stimulation of the cord between the metathoracic and first abdominal ganglion has a similar effect, but after the metathoracicganglion has been extirpated, stimulation then produces only a tonic contraction of expiratory muscles. Under these conditions ventilation can be driven at up to 200/sec by stimulating phasically with short high-frequency bursts, and the command fibres, described in Section 11, D, are probably responsible since the movements are co-ordinated throughout the abdomen. Tonic

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stimulation, therefore, drives the pacemakers of the metathoracic ganglion, whether it is applied anteriorly or posteriorly, whereas the pacemakers in the isolated abdominal ganglia do not respond in this way (Miller, unpublished). The control of the swimmerets on the crayfish abdomen shows several similarities with that of ventilation in the cricket and the locust. Hughes and Wiersma (1 960) found little evidence for an endogenously produced rhythmic output since, after de-afferentation, bursts either failed to appear or were of a different nature from those in the intact animal. More recently, however, Ikeda and Wiersma (1964) claimed that normal bursts of motor impulses to the swimmerets do arise endogenously after isolation of the cord. A posterior pacemaker normally sets the rhythm, but in its absence more anterior pacemakers take over. The first convincing evidence for the function of a command fibre was obtained from this preparation by Hughes and Wiersma (1960) by electrical stimulation of single units in the circum-oesophageal commissures. Such stimulation produced a pattern of bursts in the efferent nerves to the swimmerets when they were previously silent. The command fibres follow a route in the CNS similar to that of locust and cricket ventilatory command fibres, except that in the anterior abdominal segments each crayfish command fibre divides and travels in both connectives (Wiersma and Ikeda, 1964). The technique employed by Hughes and Wiersma of stimulating small bundles of fibres from split connectives is much superior to that of Miller who stimulated the whole connective of the locust CNS. In the crayfish a delay is interposed between the excitation of motor cells in one ganglion and the next so that the swimmerets are caused to beat metachronally. In the locust and cricket, however, a nearly simultaneous excitation of expiratory motor cells in all the abdominal ganglia is achieved. The main features of the control systems in crayfish and insect are similar, therefore, both being responsible for integrating separate ganglionic reflexes into a pattern meaningful for the whole animal. H. I N S P I R A T I O N T H R O U G H C U T I C U L A R ELASTIClTY A N D REDUCED PRESSURES

In many insects expiration depends on active muscular contractions while inspiration is brought about by cuticular elasticity which produces negative pressures. In Oryctes adults, the tergites and sternites of the abdomen tend to be kept in the inspiratory position by means of an elastic pair of ribs composed of resilin (Andersen and Weis-Fogh, 1964). It seems likely that resilin will be found to play a part in the ventilatory systems of other insects where inspiratory muscles are small or lacking.

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For example, in dragonfly nymphs the dorso-ventral muscles are expiratory but the principal inspiratory mechanism is cuticular elasticity aided by the transverse muscles (Mill and Hughes, 1965) and perhaps by resilin ligaments. Tracheae may be ventilated not only by abdominal pumping, but also by controlled spiracle activity and cuticular elasticity. For example, in silkmoth pupae and some other insects there may be a continuous uptake of oxygen, but C 0 2 is released in a series of intermittent and at times widely separated bursts (Schneiderman and Williams, 1953, 1955; Buck and Keister, 1955). Several recent reviews have covered this peculiar form of respiration adequately (Schneiderman, 1960; Buck, 1962; Miller, 1964d) and here only some new developments will be described. Buck (1958) suggested that a negative pressure is maintained in the tracheae as a result of the rate of oxygen uptake exceeding its rate of replacement from the outside. The negative pressure produces a mass inflow through a spiracle which makes small fluttering movements near the fully closed position (Schneiderman, 1960), but which does not open sufficiently to allow atmospheric pressure to be restored. Direct measurements in Hyalophora pupae have shown that a negative pressure of about - 2 to - 10 mm Hg is maintained in the tracheae for over an hour, and smaller pressure fluctuations of - 0.025 to - 0.4 mm Hg are superimposed (Levy and Schneiderman, 1958a, b; Brockway and Schneiderman, 1962). Simultaneous measurements of the pupal length show that it shortens as the pressure drops; when the spiracles open fully, the pressure rises and the pupa lengthens again. Intersegmental elasticity, as well as tracheal resilience, probably plays a part, although when pupal shortening is prevented a larger negative pressure is measured after the spiracles reclose. A maintained negative pressure of about - 5.0 cm water has been recorded in the haemolymph of Periplaneta (Davey and Treherne, 1964) and in this insect too there may be a discontinuous release of COz (Wilkins, 1960). The conditions for a continuous uptake of oxygen through slightly open spiracles may therefore exist, although measurements have not yet shown if this takes place. In order to learn more about the interburst fluttering of the spiracle valves in Hyalophora pupae, Brockway and Schneiderman (1961) studied the spiracles in atmospheres in which nitrogen was replaced with helium or with sulphur hexafluoride (SF,). During the flutter period of the spiracle there are irregular pressure falls in the tracheae which in air have a duration of 23.711.4 sec; in helium and oxygen mixtures the average duration was 146.917.1yoof that in air; in SF6and oxygen it was 69.0k 3.9% of that in air. It follows that helium diffuses out of the tracheal K*

300 P. L. MILLER system more quickly than nitrogen would : more gas is therefore sucked in and the trachealpo, is kept up for a longer period. SFBon the other hand diffuses out more slowly than nitrogen and less gas is sucked in: the popfalls more rapidly and the next flutter follows sooner. Not only each full opening of the spiracle, corresponding to the release of COB, but also each flutter of the valves is determined chemically, the latter by a fall inpO,. Hence the “indifferent” component is not after allindifferent, since its rate of diffusion outwards is one factor determining the flutter interval. Some non-ventilating insects are known where reduced intratracheal pressures may be responsible for sucking in air when the spiracle opens. For example, in the flea cyclical collapse and re-inflation of certain tracheae has been observed to coincide with closing and opening of the spiracles (Herford, 1938), the movements occurring at from 0.27 to 12 cycles/min. Extension in the long axis of the tracheae accompanies reinflation. Likewise in the mosquito larva the longitudinal tracheal trunks gradually collapse during a dive and suddenly re-inflate when the spiracles make contact with air at the water surface. The movement is most marked within the siphon. Intersegmentally placed bristles which project into the lumen of the trunks (Richards and Anderson, 1942) may help to prevent their complete collapse and so keep open a diffusion pathway when the tracheal volume has been reduced.

111. THECONTROL OF

THE

SPIRACLES

A. G E N E R A L REMARKS

Many attempts have been made to classify spiracles according to their location, or on the type of closing mechanism present, or on their general morphology (Keilin, 1944; Hassan, 1950; Tonapi, 1958). From the point of view of their mechanism, however, they can most conveniently be divided firstly into functional and non-functional spiracles (Keilin, 1944); functional spiracles are then divided into those with closing mechanisms and those without. The latter includes the spiracles of the Apterygota, many aquatic larvae, and a few aquatic adults (e.g. water bugs), and in some cases those of insects inhabiting damp situations, for example the Peloriidae, which live in damp mosses or caves and have valveless posterior spiracles (Pendergrast, 1962). Spiracles with closing mechanisms are finally divided into those with two valve muscles, an opener and a closer, and those with one only, a closer. Reports may be found of spiracles with more complex musculature (Maki, 1938), but nothing is known of their physiology.

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Considerable information about valveless spiracles is available, especially about those with a sieve plate guarding the aperture (Nunome, 1951; Miller, 1961a; Lotz, 1962). One-muscle spiracles have been investigated intensively during the last few years (e.g. Hoyle, 1959, 1960, 1961), but little is known of the operation of two-muscle spiracles, although the antagonistic action of the opener and closer presents some interesting problems. In a few cases there is indirect evidence about their activity, for example with respect to reduced pressures and water loss in beetles (Monro et al., 1962; Sharplin and Bhambhani, 1963), and something is known of the activity of such spiracles in Schistocerca (Miller, 1960b). B. I N N E R V A T I O N OF T H E S P I R A C L E S

Where it has been examined, the motor innervation of the spiracle muscles has been found to be derived from the median nerve of the segmental ganglion, or of the next anterior ganglion (Schmitt, 1961). The course of the nerve has been described in detail in Periplaneta (Case, 1957b), in various Acrididae (Ewer, 1954, 1957; Huber, 1960; Miller, 1960b; Campbell, 1961), in Sphinx larvae (Newport, 1834), Hyalophoru larvae(Libby, 1959),pupae (Beckel, 1958;van derKloot, 1963)andadults, (Libby, 1961), and in dragonfly adults (Miller, 1962). Typically the median nerve leaves the mid-dorsal surface of a thoracic ganglion and then divides into right and left transverse nerves. In locusts and dragonflies it supplies the closer muscles with two motor nerves whose axons divide at the bifurcation: each spiracle of a segment therefore receives the same pattern of motorimpulses. The median nerve may be concealed in lateral nerves in some insects (Beckel, 1958; van der Kloot, 1963) and this may explain its apparent absence in Dytiscus, for example (Wigglesworth, 1953a, p. 128). Most accounts describe a branch from a lateral nerve of the next posterior ganglion which joins the transverse nerve peripherally : in some species it has been shown to contain sensory nerves which run from the neighbourhood of the spiracle to the CNS and give rise to a valve-closing reflex when stimulated (Case, 1957b; Hoyle, 1959; Miller, 1962). In spiracle 1 of Schistocerca (Section 111, E) and in Hyalophora (van der Kloot, 1963) similarly situated nerves supply additional motor axons to the spiracle muscles. The innervation of abdominal spiracles is more complex. The abdominal median nerves have been thoroughly examined in dragonfly larvae (Zawarzin, 1924; Mill, 1964), and although in this case they supply unpaired transverse muscles and participate in the innervation of the branchial basket (Mill, 1965), their course is probably similar to that in

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other species where they do run to spiracles. They arise from a pair of large nerve cells in a ganglion and send their axons down to the next ganglion in the connectives; the axons then turn anteriorly to enter the median nerve which subsequently divides as in the thorax. In the cockroach, abdominal median nerves follow a similar course (Case, 1957b) . Many functions have been ascribed to median nerves in addition to supplying spiracles. As well as those mentioned above, they include trophic or regulatory roles, analogous to those performed by the vertebrate autonomic system (Voskresenskaya, 1950) and neurosecretory functions (Whitten, 1963b).In some cases they are thought to be responsible for ventilatory movements in the thorax (Ivanova, 1956), or to supply muscles which resist thoracic inflation due to abdominal expiration in soft teneral adults (Miller, 1960b). Such suggestions, however, await critical examination. C. I N N E R V A T E D T R A C H E A E

Many anatomical descriptions speak of nerves ending on parts of the tracheal system, and in some cases they are associated with sensory cells (Snodgrass, 1935; Ivanova, 1956; Imms, 1957). In no case, however, has a specific function been determined. In the abdomen of Periplaneta (Case, 1957b) and of the crickets, Acheta and Gryllus (Huber, 1960), ganglion cells occur as thickenings in the median nerve near its division into transverse branches. In Schistocerca similar nerve cells occur in the transverse nerve of spiracle 2 where a branch proceeds dorsally to the pleuro-subalar muscle : they give rise to slender branches which appear to terminate on air sacs (Miller, 1960b). Whitten (1963a) has described multipolar neurones (type 11) in Sarcophaga larvae which send branches to epithelium, to muscles and to the walls of the collapsed tracheae which run to the nonfunctional spiracles (stigmatic cords). Although the tracheae supplied do not contain air, Whitten points out that the nerves terminate close to the air-filled longitudinal trunks and she suggests that they may act as C 0 2 receptors. She argues that neither the physiological experiments of Hoyle (1959,1960) nor the morphological examinations of Beckel (1958)exclude this possibility in other insects. In another cyclorrhaphan larva, Phormia, Osborne (1963) concluded that similar multipolar cells with endings on tracheae might be no more than part of the general sub-epidermal nerve network, drawnin on the trachealepithelium. But more recently (Osborne, 1964)he has shown that the multipolar cell sends an axon into the median nerve in each abdominal segment, and he inclines to the view that it may participate in more specific respiratory reflexes. The abdominal median

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303

nerves of Phormia larvae contain four axons, of which only two divide (they may therefore be motor), so that altogether three axons pass down each transverse nerve, the third supplying the peripheral multipolar cell. They therefore differ from the thoracic median nerves of other species which do not seem to contain sensory nerves (Case, 1957b; Hoyle, 1959). Spiracle movements have been reported as giving rise to proprioceptive input only in Hyalophora (van der Kloot, 1963):bursts of impulses accompany valve opening, and they may arise in the scolophorous organ which lies close to the spiracle (Beckel, 1958). In no insect has a discharge been detected following treatment of the spiracle with CO,. In some species, chordotonal organs are attached to tracheae (Larsen, 1955; Rowell, 1961;Parsons, 1962) and at least in aquatic insects a hydrostatic function may be served. D. S P I R A C U L A R A C T I V I T Y

Spiracle movements are either controlled separately and sometimes locally by chemical means, or they come under a central nervous coordinating mechanism which may command them to march in step with ventilation. A spiracle may be subject to either or both mechanisms at one time. Flea spiracles (Wigglesworth, 1935) and silkmoth pupal spiracles (Schneiderman, 1960) are of the first type; the flea spiracles open in response to oxygen lack, but the duration of their opening is determined by CO, tensions. Likewise in Hyalophora each flutter in the interburst is determined by low oxygen tensions, whereas full spiracular opening at the beginning of a burst is initiated by internal CO,. In Periplaneta (Hazelhoff, 1926) and in some adult dragonflies(Miller, 1962) a central synchronizing mechanism takes over from a predominantly peripheral control at certain CO, tensions, and it produces synchronized movements of some spiracles after activity. The interaction of peripheral and central mechanisms is seen in the dragonfly Hadrothemis where in the presence of CO, the spiracles become synchronized with ventilation without a change in the pattern of motor impulses arriving at the closer muscle. This is achieved by CO, acting peripherally and preventing the muscle from responding to the lower frequency of motor impulses in an undulating barrage. The higher frequencies,corresponding to expiration, still close the valve fully (Miller, 1961b). In locusts and some other Acrididae the spiracles are normally synchronized with ventilation : local control may, however, play a part within the context of synchronized movements. For example, spiracles may open more widely or close for a shorter duration in the presence of high CO, concentrations (Miller, 1960b).

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In many insects therefore spiracle synchrony is facultative; it may produce uni-directional ventilation, which, compared with tidal pumping, probably results in less water loss (Buck, 1962). In restinginsects pumping may continue through only one or two spiracles, or it may cease and diffusion through peripherally controlled spiracles then operates. The mechanisms involved will be considered in detail in the ensuing sections. E. C O N T R O L M E C H A N I S M S I N T W O - M U S C L E S P I R A C L E S

The following applies only to spiracle 1 in Schistocerca, since few other two-muscle spiracles have been studied. In this spiracle the opener muscle is in series with a cuticular spring which allows the muscle to shorten even when the closer remains contracted and the valve closed. Tension on the spring makes the valve open more widely when the closer relaxes. The closer muscle receives two motor axons from the median nerve of the prothoracic ganglion. Two motor axons travel to the opener in the same nerve, and a third reaches it from the mesothoracic ganglion in the anterior lateral nerve (cf. Ewer, 1954; Campbell, 1961). Opener contractions are normally slower and longer lasting than those of the closer, and correspondingly the opener receives slower frequencies of impulses. The prothoracic nerves to the opener (median nerve supply) may fire during expiration when the closer axons are also active: with increasing COz they extend their period of activity into the inspiratory #hase. The mesothoracic nerve to the opener, however,is silent for part of the expiratory phase and, when active, fires throughout inspiration. The combined activity of pro- and mesothoracic nerves may, therefore, maintain a tetanus in the opener. A reduction of the Oz/C02ratio in either the head or prothoracic ganglion stimulates the prothoracic opener nerves ; likewise similar reduction in the mesothoracic ganglion stimulates its opener nerve. In the resting insect all three nerves may contribute to the activity of the opener, whereas in flight at times only the mesothoracic nerve is active and this has important consequences for the control of abdominal ventilation (Section IVYE). When not strongly stimulated the motor impulses to opener and closer muscles cease simultaneously in the prothoracic ganglion. Peripheral delays, however, ensure that the opener contraction outlasts that of the closer, even when the mesothoracic nerve is silent. These take the form of a slower conduction speed in the opener nerves and a five times slower rate of relaxation by the opener muscle. Thus the valve still opens widely at the commencement of inspiration under these conditions (Miller, 1965). The actual opening and closing of the valve is brought about therefore

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through the relatively simple action of the closer; the degree of opening, however, is controlled by the opener which responds to stimulation in the head, pro- and mesothoracic ganglia. This separation of power and control systems is reminiscent of that found in the flight muscles of more advanced insects (Pringle, 1957) and of dragonflies (Neville, 1960).

F. CONTROL MECHANISMS IN ONE-MUSCLE SPIRACLES

Many spiracles are of the one-muscle type and both power and control systems must operate through the closer. Sensitivity of this muscle to C02,which has been elegantly demonstrated by Hoyle (1960) in the locust spiracle 2, may be a general property of all such spiracles; there is, for example, evidence for it in dragonfly and fly spiracles (Miller, 1962). Hoyle (1959) showed that spiracle 2 in Schistocerca is supplied by two motor axons, 6 and 7pin diameter, and that every muscle fibre seems to be doubly innervated. An inner core of slender fibres with sarcomeres of up to 10 p in length seems not to have functional properties different from those of the stouter peripheral fibres. Compared with the nerves of leg muscles, both axons would be considered “fast”, but the action of each is distinguishable and for convenience Hoyle terms them “fast” and “slow”. Stimulation of the fast axon alone produces end-plate potentials which vary from 10 to 60 mV. Facilitation and summation are seen between smaller potentials and tensions of up to 0.14 g can be obtained. Stimulation of the slow axon alone produces potentials two-thirds the size of those of the fast axon, but the time course of each is similar, and a maximum tension of 0.05 g has been recorded (Fig. 5). The only other spiracle to have received comparable electrophysiological treatment is the first abdominal spiracle of Hyalophora (van der Kloot, 1963). This spiracle is opened by an elastic strand similar in situation to the opener muscles of other Lepidoptera (Beckel, 1958). Its closer muscle receives at least two motor axons in the median nerve (ALN) and possibly two from a lateral nerve (MLN). In air one axon in the median nerve fires continually, while another fires periodically at lower frequencies. During treatment with C 0 2much of the median nerve activity ceases although impulses may continue to reach the muscle through the lateral nerve. Electrical stimulation of the median nerve produces twitches of the muscle, as in the locust, whereas stimulation of the lateral nerve apparently affects the spontaneous pacemaker of the closer (Section 111, I).

w

0 Q\

in P FIG.5. The mechanical responses recorded from the apodeme of spiracle 2 of Schistocerca, when only the “fast” axon is stimulated (first seven responses), and when only the “slow” axon is stimulated (remaining responses). (From Hoyle, 1959.)

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1. Peripheral control

The hypothesis that C 0 2acts on or close to the spiracle has been put forward by many authors (Hazelhoff, 1926; Stahn, 1928; Schreuder and de Wilde, 1952; Case, 1956a, 1957b; Schneiderman, 1960), but the site of action was not identified until Hoyle (1960) showed that CO, almost certainly acts on the neuromuscular junction of spiracle 2 in the locust. C 0 2lowers the tension in the closer muscle so that the valve opens partially despite the maintained barrage of motor impulses in the median nerve. While 1-5% COBproduces slight drops in tension, higher concentrations are needed to give a detectable opening which is usually accompanied by fluttering movements. The muscle responds very quickly when the gas is injected into air sacs from which small tracheae run into the muscle (Fig. 6). On the other hand, injections of gas into tracheae farther from the spiracle, or bubbling the gas through saline surrounding the muscle, produce responses only after much longer delays. Simultaneous recordings of the resting and action potentials showed no change when 5% CO, was used, although a tension drop was measured. Higher concentrations of C 0 2 produced a much greater tension drop and an attenuation of the end-plate potentials; when pure CO, was used the resting potential of at least some of the fibres was also reduced. Because of the separation of tension and e.p.p. effects, Hoyle postulated that CO, has two sites of action, one at the junction and one beyond it at some stage in the coupling between membrane and fibrils. Hoyle suggested that CO, acted at the junction both by blocking the transmitter and by increasing the conductance of the membrane, and he pointed out the similarity of the C 0 2 effects to those of peripheral inhibitory nerves of Crustacea. 2. Central control The sensitivity of the reaction described above is determined in part by the frequency of motor nerve impulses arriving at the closer muscle. In the dragonfly, Ictinogomphusferox, oxygen shortage, water balance and temperature have all been shown to affect the motor frequency to spiracle 2 (Miller, 1964a, b). In 10% oxygen in nitrogen the frequency starts to fall, and it may be extinguished in 2%. Other motor acts, such as ventilation and leg movements, continue in this concentration and the spiracle touch reflex can still be evoked. On the other hand, high concentrations of C 0 2(20%) are needed to produce an appreciable decline in frequency. In contrast to ventilatory mechanisms which may respond to 1-2% C 0 2 (Miller,

308

P. L. M I L L E R

1960a), the central control of dragonfly spiracles seems therefore to depend on a specifically hypoxia-sensitive mechanism (cf. Section 11, F). However, as Schneiderman (1960) emphasizes, the concentration of gases acting at the receptor sites in such experiments is quite unknown. Schneiderman (1960) concluded that hypoxia acts on the spiracles

slrgle JUnCtaol polentlal (I(

L d i - L d u A &

-'kkk\+-

n

Total

'

L

L

A

U

I

L

' '

4 4 4 * * W & h

co2

ISOC L! --c-L.--h

-L-L,-

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tmsm

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FIG. 6. Simultaneous records of the electrical and mechanical events in spiracle 2 of Schistocerca when a pulse of COz is injected into air sacs close to the spiracle. The motor nerve is stimulated at regular intervals throughout. (From Hoyle, 1960.)

of Hyalophora pupae through the central nervous system, whereas CO, affects them directly, and both in Hyalophora and in some flies (Case, 1956a) the CO, and hypoxia effects interact so that sensitivity to COP is increased with hypoxia. Perfusion with mixtures containing more than 21 yo oxygen may further decrease the CO, sensitivity of Hyalophora

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309

spiracles; but in dragonflies there is no corresponding increase in motor frequency to the spiracles. However, a clear-cut division into a central hypoxia effect and a peripheral CO, effect in Hyalophora is not possible since van der Kloot (1963) has shown that CO, acts both centrally and peripherally. Since both CO, and hypoxia depolarize nerve cells and might therefore tend to increase their rate of endogenous firing (Chalazonitis, 1963), both gases probably act indirectly on spiracle motor cells through an inhibitory interneurone with a threshold lower than that of the motor cells. In hypoxic dragonflies, regular firing is usually replaced with a slower irregular discharge, suggestingthe synaptic interaction of several units. Likewise in the motor cells of the closer muscle of the locust spiracle 1, free running (Section 111, H) is probably inhibited by an interneurone sensitiveto 0 2 / C 0 2ratio changes in the head. This has been demonstrated by localized perfusion with gas mixtures (Miller, 1960b, 1965). After decapitation free running continues, but it is now remarkably insensitive to hypoxia and CO,. The motor cells of the opener act in the reverse direction, their spontaneous firing being increased in frequency by such stimulation: here therefore the action may be a direct one on the motor cells. The frequency of motor impulses to spiracle 2 of Ictinogomphus is affected by the water balance of the insect. In desiccated insects spiracle control is tighter, as a result of an increase in motor frequency. This effect can also be produced by perfusing the insect with hypertonic saline solutions. It does not take place by osmotic changes, but it is apparently dependent on the concentration of a specific ion. Moreover, an increase in the threshold to hypoxia accompanies the rise in frequency, and dragonflies perfused with double-strength saline show very little response until less than 1% oxygen is used (Fig. 7). Raising the temperature provides another means of increasing the frequency: the Qlofor the reaction is 2-7 (15-25"C), but above 35°C the frequency declines rapidly probably as a result of hypoxia, since at high temperatures the cells remain just as sensitive to hypoxia. Increased frequency and a raised threshold to hypoxia, which appear in desiccated insects, are not therefore necessarily linked. Excessive hydration of dragonflies, on the other hand, produces a decline in the motor frequency and the spiracles open, thereby increasing the rate of water loss. Insects living in habitats normally deficient in oxygen and with abundant CO, may keep their spiracles permanently open. For example, the larvae, pupae and adults of the Cerambycid, Orthosoma brunneum,

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live in rotting logs where there may be 2% oxygen and 15% C02(Paim and Beckel, 1963a). In air they release C 0 2discontinuously in a series of bursts, as do silkmoth pupae (Schneiderman and Williams, 1953,1955), but in their normal gaseous environment C 0 2 is released continuously because the spiracles remain wide open. The environment is moist so that permanently open spiracles do not lead to excessive water loss (Paim and Beckel, 1963b). It is interesting to note that the larvae survive in 1% oxygen, but die in 0.6%, while they are able to recover after narcotization

'01 L60-

A

U

x

50-

3

z

40-

?! 303

C

20

-

10-

t

I

I

I

2

4

6

I

I

10 /2 14 Oxygen In nitrogen (%)

I

I

I

1

16

18

20

22

FIG.7. The frequency of motor impulses to spiracle 2 of Zctinogomphus ferox, with the insect in a chamber through which various concentrationsof oxygen in nitrogen are passed. A, Insect perfused with double-strengthsaline; B, perfused with normal saline; C, perfused with half-strengthsaline. (Based on Miller, 1964b.)

for 10 days in 50% C 0 2(Paim and Beckel, 1964). Two-muscle spiracles are therefore controlled from the CNS whereas much of the control of one-muscle spiracles depends ultimately on a peripheral reaction to C 0 2 ,even though this may be modified through the CNS. If two-muscle spiracles, which occur commonly on abdominal segments but rarely on the thorax, are considered to be more primitive, then the evolution of a peripheral controlling system may have allowed the CNS to control the spiracles through only two motor neurones rather

T H E RE G U L A T I O N OF B R E A T H I N G I N INSECTS

31 1

than the four or fiveneededfor two-muscle spiracles. But whether natural selectionhas ever favoured the simplificationof a mechanism by reducing the number of its central neurones is debatable (cf. Vowles, 1961; Fielden, 1963; Hughes, 1965). G. EXPERIMENTS O N THE N A T U R E O F THE CHEMICAL S T I M U L U S

Wigglesworth (1935) believed that changes in the 02/C 02 ratio affected spiracle activity in the flea through alterations of the pH. Work on other species, however, has indicated that C 0 2may act more directly. Case (1957a) perfused different solutions through Sarcophaga adults and showed that solutions in which pH was as low as 4.2, but which contained no dissolved C02,did not cause the spiracles to open. During such perfusion he could open the spiracles at any time by blowing C02into them. On the other hand, perfusion with solutions which did contain dissolved C02caused immediate reversible opening. The mechanism in fly spiracles, which are controlled by one muscle (Hassan, 1944),may be similar to that in spiracle 2 of locusts. Hoyle (1960) showed that C 0 2and not pH was probably the immediate stimulus in the locust, since HC1 or ammonia vapour when blown into opened air sacs close to the spiracle.muscle was without effect. There may alternatively be a selective barrier with a preferred permeability for C 0 2between the exterior and the neuromuscular junction and that within the barrier the effective stimulus is a change of pH. Such changes would provide a workable mechanism since insect haemolymph is poorly buffered around the normal pH, while buffering progressively increases either side (Levenbook, 1950). However the failure of the spiracles to respond to hypoxia, except through the CNS (Miller, 1962), indicates that the mechanism may be specific for COB.Moreover Sears and Eisenberg(196 1) have produced evidenceto show that C02has adirect effect on cell membranes, increasing their permeability to ions. H. S Y N C H R O N I Z E D ACTIVITY OF THE SPIRACLES

Synchronized movements are a common feature of the activity of the spiracles of many large insects, the valves opening or closing in phase with each other and with abdominal pumping strokes. In locusts (Fraenkel, 1932; Weis-Fogh, to be published), cockroaches (Kitchel and Hoskins, 1935 ;Myers, 1959), bees (Bailey, 1954) and dragonflies (Tonapi, personal communication) this has been shown to produce uni-directional

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P. L. M l L L E R

ventilation, but in addition other functions may be served, for example the improvement of haemolymph circulation in connexion with a pressure phase when all spiracles are temporarily closed. Synchronized activity is brought about by motor patterns which are derived from the ventilatory centres. In adult dragonflies, the efferent flow to the closer muscles of the thoracic spiracles can be divided into two components. The first is produced in the segmental ganglion and could result either from endogenous activity in the two spiracle motor cells, or in pacemakers which drive them; anti-dromic stimulation of the axons, as carried out by Preston and Kennedy (1962) in Crustacea, has recently shown that the motor cells are themselves endogenously active. This activity, termed “free running”, comprises regular independent trains of impulses in the two axons at similar but not identical frequencies, and in quiescent dragonflies it continues indefinitely. Differences between the behaviour of spiracles 1 and 2 are achieved by different frequencies of free running: for example, in Acanthagyna the spiracle 1 nerves fire at about 15-17/sec/axon, whereas in spiracle 2 they fire at up to 60-70/sec/ axon. In consequence spiracle 2 remains closed while spiracle 1 flutters between 0 and 25% open, and resting respiration takes place through the latter, and through spiracle 10 which also remains active. The second component which appears in thoracic spiracle nerves when ventilation is strong is derived from the abdominal ganglia. I t consists of high-frequency bursts, or in different species temporary inhibitions, synchronized with expiration and superimposed on free running. High-frequency bursts are found, for example, in Ictinogomphus and Hadrothemis, whereas expiratory inhibitions occur in some Aeshnidae (e.g. Anax and Anaciaeshna). In Pantala and some other Libellulids a high-frequency burst and a subsequent inhibition occur, both during expiration. Now the factors described in Section 111, F, 2, affect free running so that in Ictinogomphus, for example, after activity, highfrequency bursts close the spiracle during expiration, while during inspiration the spiracle opens because hypoxia,reduces the frequency of free running. In the Anax pattern the inhibitions allow the spiracle to open during expiration, while in the more complex Puntala-type of synchronization, the thoracic spiracles may at first play an inspiratory and later an expiratory role as recovery proceeds. A functional interpretation of this last type is hard to make, but Weis-Fogh (196410) has pointed out that dragonfly flight muscles consume nearly as much oxygen as the theoretical maximum which the tracheal system can supply; marginal improvements may therefore be significant, particularly immediately after flight when the thoracic pump stops.

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Bursts of impulses can be recorded in the connectives of Pantala in phase with the spiracular high-frequency bursts and also with the inhibitions. The impulses, which proceed anteriorly from the abdominal ganglia to the thorax, are probably in interneurones which couple the spiracle motor cells to the ventilation pacemakers since they persist after denervation of the ganglion. Effective synchronization of spiracles necessitates the accurate timing of events in widely separated segments, and fast-conducting interneurones would be expected. Indirect measurementson comparable units in the locust suggest that they carry impulses at over 1 m/sec (25"C), and the large spikes in the cord of Pantala suggest that here too synchronization is brought about by rapidly conducting interneurones (Miller, 1962). In Schistocerca synchronized activity of the thoracic spiracles is produced by interneurones which arise in the metathoracic ganglion. Free running does not usually play a part in the efferent flow to the spiracles, and in this respect locusts differ markedly from dragonflies. Free running can, however, be recorded after the metathoracic ganglion has been surgically removed, andit then usually proceeds at a higher frequency to spiracle 1 than to spiracle 2. Locusts with the connectives between meso- and metathoracic ganglia sectioned have been kept alive for several weeks and free running continues in them throughout. In intact Schistocerca the activity recorded from the spiracle closer nerves is normally one of three types. These are: (1) inhibitions, corresponding to inspiration and spiracle opening; (2) high-frequency bursts during expiratory movements; (3) a maintained pattern corresponding to ventilatory pauses which often divide the expiratory stroke into two parts. In the second and third types, both motor axons fire at the same frequency either synchronously or one after the other; driven activity is therefore distinguishable from free running in which the two motor cells fire independently of each other. During high-frequency bursts (2) the firing of the motor cells in adjacent ganglia is not coupled, whereas during the maintained activity (3) a considerable proportion of the spikes appears synchronously in the two median nerves (Fig. 8). In flight the nerve to spiracle 2 is silent whereas spiracle 1 continues synchronized activity (Miller, 1960~).The closer motor cells of spiracle 2 are probably continuously inhibited since otherwise they would revert to free running. These facts suggest that separate inhibitory and highfrequency burst interneurones supply spiracles 1 and 2, whereas the lower frequency activity (3), which does not occur in flight, may be brought about by an interneurone shared by all thoracic spiracles. The gradual application of a cold block to the connectives between pro-

FIG.8. Simultaneous records from the motor nerves to the closer muscles of spiracles 1 (top) and 2 (lower) of Schistocerca gregariu during a ventilatory pause. Each ‘‘large’’ spike represents the simultaneous occurrence of an impulse in each motor axon of one spiracle nerve. Note the occurrence of quartets of impulses in one spiracle nerve and their synchronization with quartets in the other nerve. The coupling is not at all rigid.

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315

and mesothoracic ganglia causes the three types of activity in the spiracle 1 nerve to drop out in the sequence, high-frequency bursts, lower maintained frequency, inspiratory inhibitions. As the driven patterns disappear they are smoothly replaced by free running, which is unaffected by the block. On re-warming, the patterns reappear in the reverse sequence. This again suggests that at least three interneurones are involved, and that their combined activity normally obliterates free running, although such activity has occasionally been recorded from intact locusts (Miller, 1965). 1. The occurrence ofpaired motor impulses in spiracle nerves Hoyle (1959) showed by electrical stimulation of the motor nerve of spiracle 2 of Schistocerca that paired shocks separated by as little as 3-4 msec produced a much greater tension in the muscle than the same overall frequency evenly spaced. The tension continued to increase for 20-30 sec after the start of stimulation, and achieved a peak 24 times normal (Fig. 9). He drew attention to the natural occurrence of paired spikes in records from the intact median nerve, and also to those which appeared in the records of Case (1957b) from Periplaneta. Up to 90% of all the spikes were sometimes paired, with a separation within a pair of less than 9 msec. In the locust, the two motor cells of the closer of spiracle 1, when driven from the metathoracic ganglion, often fire in duets comprising one spike in each axon (Miller, 1960b).In addition, during the maintained discharge accompanying a ventilatory pause, each axon sometimes fires in pairs, so that altogether a quartet of spikes proceeds to the spiracle, two in each axon (Fig. 8). Moreover this patternmay beduplicatedin the spiracles of the adjacent segment. Under these circumstances simultaneous facilitation and spatial summation may occur at the neuromuscular junctions of every fibre, as all appear to be dually innervated (Hoyle, 1959). During high-frequency bursts the normal spike separation is no more than 4-5 msec and pairing has not been observed. Double firing has been reported in a few instances elsewhere. Wiersma and Adams (1950) recorded paired motor impulses in crustacean motor nerves, and they showed that much greater tensions were produced than when the same frequency occurred equally spaced. It is also seen in motor nerves to the flight muscles of the locust where paired impulses, separated by 4-8 msec, may occur with each wing beat: they increase the work done by the flight muscles by two or three times (Wilson and Weis-Fogh, 1962; Neville and Weis-Fogh, 1963). In spiracle muscles, however, where no great expenditure of energy is required, pairing of the impulses may serve a different purpose. When they take place at a low frequency, the valve

316

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flutters and each closing movement corresponds to a tension peak brought about by a pair of impulses (Hoyle, 1959). Fluttering, which occurs at times in many species (cf. Schneiderman, 1960), may allow the valve to maintain on average a slightly open position, which, having "fast" motor innervation, it could not do by other means. During free running the valve sometimes flutters during the in-phase firing of the two axons, but closes fully when they fire out of phase; hence, without a change in

+

r

L

L

-

L

-

-

L

L -

L

~

--. ..--.-\

FIG.9. Simultaneous electrical records from a single muscle fibre (upper trace in each set) and tension records from the whole muscle (middle trace) of spiracle 2 of Schistocercu. The nerve is first stimulated with single shocks; from the arrow onwards, it receives closely paired shocks, which produce a continuing increase in amplitude of the mechanical records. Lower trace=l sec marker. (From Hoyle, 1959.)

frequency of either unit, the spiracle may open and close rhythmically (Miller, 1960b; cf. Section 11, B). I. I N D E P E N D E N T A C T I V I T Y B Y T H E S P I R A C L E S

Many observations in the past have suggested that some spiracles remain active after they have been denervated (Hazelhoff, 1926; Wigglesworth, 1935,1941),and this has been confirmed by Beckel and Schneider-

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317

man (1956, 1957) in Hyalophora pupae, and by Case (1956a, 1957b) in Periplaneta adults. In both, the spiracle remains closed after denervation, and limited sensitivity to C 0 2persists, although as much as 15-20% may be required to open Periplaneta spiracles. No sign of a ganglion cell or a peripheral nervous system has been found. The cockroach spiracle usually closes 24 h after denervation, and after 3-5 days it starts “fasciculating”, an activity distinguished by alternate contractions and relaxations of small groups of muscle fibres. Spiracle 2 of Schistocerca closes after peripheral or central section of its transverse nerve, often with a lag of more than 1 day. Having closed, it remains sensitive to C02, sometimes opening in as little as 1-2% (Miller, 1960b). Hoyle (1961) has shown that the spontaneous activity results from a potassium contracture for which a minimum haemolymph level of 30 mM/1 is required. Locusts fed on bran contain less potassium than normal and their spiracles may remain open after nerve section; conversely after a diet of abundant green food and no water, spiracle closing may take place immediately after the operation. According to Miller (1960b) the lag between nerve section and closure does not correspond to an increase in haemolymph potassium concentration resulting from water loss through the open spiracles; there may alternatively be an increase in excitability of the muscle following denervation. Once closed, Hoyle found that small injections of saline near the spiracle caused it to re-open momentarily, while larger doses resulted in maintained opening. When bathed in a solution containing more than 30 mM/1 of potassium, he showed that there was a rapid onset of tension and that the contracture was maintained for at least several hours; indeed, spiracle 2 may remain closed for several weeks after its denervation in intact locusts. The greatest tensions were produced in 70 mM/1, and they exceeded the maximum tensions which Hoyle had recorded from the intact muscle (Fig. 10). He therefore concluded that all the fibres were involved, and not merely the inner core with long sarcomeres, as Miller (1960b) had suggested. Contracture was initiated when the membrane potential fell from - 60to - 34 mV, and the maximum tension corresponded to a drop to - 18 mV. According to Hoyle the contracture could be maintained in calcium-free solutions, but Aidley (1965) has suggested that this was due to a failure to remove all the calcium ions from the extracellular spaces. Potassium contractures in the mesothoracic extensor tibialis of Schistocerca take place only in the presence of calcium ions (Aidley, 1963). A short pulse of C02,injected into opened air sacs close to the muscle, reduced the tension by about one-third after a delay of only 1-2 sec.

318

P. L. MILLER

Hoyle found that the subsequent recovery was faster than in the intact spiracle and suggested that this was because CO, now acted only at one site, the coupling mechanism, whereas in the intact spiracle it acted also at the neuromuscular junction. Since the intact spiracle muscle would also be expected to be affected by high potassium concentrations, it is puzzling to know how in locusts

I

60

1

1

40

1

I

1

29

18

I

I

0 rnv

FIG. 10. The tension developed by the denervated closer muscle of spiracle 2 of Schismcerca greguriu plotted against the potassium concentration of the bathing fluid. The mean membrane potential of muscle fibres is shown on the lower horizontal scale. (From Hoyle, 1961.)

fed on a rich green diet, the muscle relaxes when motor impulses cease in the transverse nerve. Hoyle suggested that the preceding tetanus produced enough CO, to weaken the contracture long enough to allow the spiracle to open with inspiration, and he pointed out that during the first few inspirations after a pause in ventilation the spiracle hardly opened. There may in addition be an increased excitability of the muscle after denervation, as already suggested, so that only in locusts with unusually high haemolymph potassium would closure follow denervation immediately.

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319

Hoyle showed that potassium contractures produced in other locust muscles were similar to those in the spiracle muscle, but generally they were not so long-lasting, nor were they so sensitive to C 0 2 .The spiracle does not, therefore, operate through a wholly new mechanism, but depends on the accentuation of properties shared by many muscles. Spontaneous activity by the pupal spiracle of Hyalophora depends on a different mechanism. Intracellular recordings made by van der Kloot (1963) from denervated fibres of the closer muscle show a slow regular depolarization followed by a spike and then a rapid repolarization, after which the cycle starts again. Muscular contraction coincides with the slow depolarization. The frequency of the cycle is determined by the maximum repolarization reached : for example, a spike occurred every 1-8 sec when the membrane repolarized to - 54 mV; when it reached - 81 mV a spike appeared every 2.2 sec. C 0 2 affected the membrane by increasing the level of repolarization reached, thereby slowing the cycle. For example, 10% COBslowed the cycle from 34 to 27 spikes/min (Fig. 11). Higher concentrations of C 0 2hyperpolarized the membrane to - 85 mV where it stayed and the muscle relaxed. The action of C 0 2 on this spiracle is therefore different from its action on the locust spiracle and on nerve cells, where it produces a depolarization (Chalazonitis, 1963); a hyperpolarizing effect of C 0 2 has, however, been reported from frog muscle fibres (Meves and Volkner, 1958). Hyalophora spiracles resemble crustacean heart ganglia in several ways (Bullock, 1961;Hagiwara, 1961).For example,in neither is the repolarization necessarily initiated by spiking and the slow wave of potential change is able to continue in the absence of spikes. The spike may occur before the maximum depolarization is reached although repolarization always starts at -29 mV. The pacemaker locus in the spiracle has not been identified but it seems clear from the careful morphological examination of Beckel (1958) that no nerve cell is present and that the activity is myogenic. In the intact spiracle, according to van der Kloot, pacemaker activity continues although the muscle receives regular trains of impulses from the CNS. The electrical activity is therefore found to be compounded from four sources. These are, local pacemaker potentials, end-plate potentials arising from spikes in the median nerve, small potentials of unknown origin and small hyperpolarizations which may appear during pacemaker potentials and return the membrane to the resting level. The last two may result from activity in the lateral nerve branch which supplies the spiracle (MLN). Stimulation of this nerve at high frequencies gives rise either to an increase or a decrease of the spontaneous firing, depend-

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ing on the intensity, and van der Kloot suggested that this nerve contributes both excitatory and inhibitory axons to the pacemaker. Spiracle activity in the intact pupa results therefore from the interaction of the CNS and of the local pacemaker. Van der Kloot suggested that during winter the activity of the CNS of diapausing Hyalophora pupae may reach low levels, although at least some activity persists (Schoonhoven, 1963), and that the spiracular myogenic mechanism is then responsible for maintained closure of the valve.

ab

c

c

c

c

/

c -

0 0

I SEC. FIG. 1 1 . Action potentials from the denervated closer muscle of an abdominal spiracle of the pupa of Hyalophoru. Trace c, spiracle exposed to air; traces a and b, spiracle exposed to 10% CO1.CO, increases the membrane potential which is achieved following the action potential. (From van der Kloot, 1963.)

Evidence for the presence of peripheral inhibitory nerves in Periplaneta is now available (Usherwood and Grundfest, 1964). If further studies confirm the presence of excitatory and inhibitory regulatory nerves to the pacemakers of spiracles of Hyalophora pupae, then the resemblance with the crustacean heart ganglion will be closer. The complexity of control mechanisms in this spiracle is perhaps not surprising in view of the long diapause through which the pupa passes without replenishment of fuel. If insects’ eggs were equipped with spiracles, their control would doubtless be as complex.

THE R E G U L A T I O N O F B R E A T H I N G I N I N S E C T S

IV. MODIFICATIONS OF

THE

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T R A C H E ASYSTEM L FOR FLIGHT

While there are many accounts of the general morphology of the tracheal system, few of these consider the detailed arrangement of the supply to the flight muscles. Yet it is here that the system is taxed most heavily and the most interesting modifications are found. In both the fine structure and the gross morphology of the major tubes the system is highly adapted to meet the enormous demands for flight. As Weis-Fogh (1964a) has pointed out, oxygen must be supplied at two very different rates: at rest the consumption is about 0.02 ml/g muscle/ min while in the flight of the bee, for example, it may increase by about four hundred times to 7.3 ml (Hocking, 1953). Representative values are given in Table I. They show the tracheal system to be one of the most efficient delivery systems in the animal kingdom, standing comparison with the vertebrate blood system. TABLE 1 Oxygen uptake of insect flight muscle during steady-state flight. In addition the amount of air needed per stroke is shown assuming that 25% of the oxygen present is consumed (i.e. 5 % of the total air). (From Weis-Fogh, 1964b.)

Insect Schistocerca Aeshna Butterflies and moths Aphis Drosophila Vespa crabro Lucilia Apis rnellifera

ml O,/g muscle/min 1 +2*8 1.8 1 +3.5

1.4-1.8 2.0-2.3 2.6-3.3 5.6

7.3

100 x ml air/ g/stroke 26-5.2 2.0 0.3-0.4 0.9-1' 1 1 .o

The metabolic rate of a sexually immature migrant locust in level flight (1OOyo lift) is 65 kcal/kg/h, which corresponds to an oxygen uptake of 14 l/kg/h. This rises to 127 kcalat 200% lift, or to an oxygen consumption of about 27 litres (Weis-Fogh, 1964~).Power output is proportional to lift, not to wing beat frequency (Weis-Fogh, 1956b), increases in power output being achieved by the employment of more motor units or by increasing the amount of double firing of motor nerves with each stroke (Wilson and Weis-Fogh, 1962). It follows that the thoracic ventilating

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mechanism, which depends on the flight movements, does not pump in proportion to the demand, and must therefore normally work at a rate adequate to meet the highest consumption. A. F U N C T I O N A L M O R P H O L O G Y OF T H E T R A C H E A L SYSTEM I N T H E P T E R O T H O R A X

Weis-Fogh (1964b), in a thorough investigation of the mechanism of oxygen supply to the flight muscles in locusts, dragonflies and other large insects, has divided the route from the spiracles to the flight muscles into three parts; the primary, secondary and tertiary pathways. Supply type:

A

( 1 ) Ccntro-radial

FIG. 12. The three types of tracheal supply to the wing muscles of locusts. The primary supply runs along the long axis of the muscle, either centrally or on the outside, while secondary tracheae leave it at right angles and penetratethrough the muscle.Shunt systems are indicated in broken lines. (From Weis-Fogh, 1964a.)

1. Primary pathways

The first part of the route leads in from a spiracle on the thorax and runs either axially through the centre of a fight muscle, or else along one side of it. Beyond the muscle it swells into an air sac, either blind-ending or sometimes confluent with sacs from other muscles. Alternatively the primary tube takes the form of a sac attached to the outside of the muscle. In small insects of the size of Drosophila no appreciable ventilation takes place and the whole system depends on diffusion; in larger species the

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primary tubes are ventilated either by flight movements or by an abdominal pump (Section IV, C ) . Bulky flight muscles may have more than one "axial" primary (e.g. the first sub-alar depressor in Aeshna) and their ventilation is often assisted by additional sacs.

2. Secondary pathways Secondary tracheae leave from the primary usually at right angles and they travel through the muscle to end near its periphery. An axial primary with radiating secondaries is described by Weis-Fogh as centroradial ; when the primary is a peripherally situated tube, the arrangement is termed latero-radial; and when it is a peripheral sac, it is known as latero-linear (Fig. 12). The locust exemplifies all three types in different muscles, whereas nearly all dragonfly flight muscles are supplied by the centro-radial type. Small insects, in which diffusion plays an important part in the primaries, normally have a latero-linear supply, the wide primary path offering less resistance to diffusing gases. In larger species the centro-radial type is common and it offers the shortest diffusion path in the secondary tubes. In very large insects, however, even this becomes too long, and both the secondaries and the primaries are ventilated. In Melolontha the proximal ends of the secondaries are expanded into air sacs (Straws-Diirckheim, 1828), but their ventilation would protlably have little effect on distal parts of the tubes. However, when the sac is situated at the peripheral end of the secondary, the whole tube can be ventilated. This is found, for example, in the flight muscles of giant water bugs (Belostomatidae) where the secondaries of the dorsal longitudinal and dorso-ventral muscles are flattened tubes which arise from an axial primary and extend beyond the periphery of the muscle (Moller, 1920; Weis-Fogh, 1964a). In the dorso-ventral muscles of Hydrocyrius and Limnogeron, the terminal few secondaries, furthest from the spiracle, are expanded peripherally into larger air sacs, which probably serve further to boost ventilation of the tubes. These are the only air sacs of conventional shape found in Belostomatidae (Fig. 13). The dorsal oblique and basalar muscles, also fibrillar in these insects (Barber and Pringle, in preparation), are supplied with the latero-radial arrangement, although the secondaries do not pass through the muscles, but instead encircle it. The muscle is therefore surrounded by an envelope of air from which a well-developed tertiary system invades the fibres. The envelope may serve not only a ventilatory function, but it may also reduce mechanical damping and heat losses from the fibres, functions ascribed to air sacs elsewhere (Weis-Fogh, 1956a; Church, 1960). Belostomatid spiracles are valveless, but they are covered with a sieve L

FIG.13 (Legend opposite)

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plate which prevents the entry of water into the tracheal system even under considerable pressures (Miller, 1961a). Although such sieve plates d o not obstruct diffusion (Nunome, 1944), they present a considerable frictional drag on the bulk movement of air through them and might thus impede respiratory exchanges during flight. However, compared with other spiracles, the sieve plate covering spiracle 3 allows air to pass through a hundred times more rapidly per unit area under a pressure of 10 cm water; this spiracle therefore probably contributes much of the oxygen consumed i n flight. It communicates directly with the primary tubes of the dorso-longitudinal and oblique muscles, and via a large lateral trunk to those of the dorso-ventral and basalar muscles. Spiracles 1 and 2 also open into the lateral trunk and contribute to the oxygen intake.

3. Tertiary pathways Tertiary branches leave the secondaries at right angles, and after branching several times they terminate in tracheoles. In a lobe of the tergo-sternal muscle of Aeshna, for example, Weis-Fogh ( 1964b) has shown that each secondary tube gives off about twenty-four tertiaries in all, one every 20 p, and that they taper from a diameter of about I .O p to one of 0.2 p at the tip of the tracheole. Each tertiary trachea splits into 20-30 tracheoles and these “grasp” the muscle fibres about every 4-5 p along their length. In dragonflies the tracheoles do not indent the muscle fibres (Smith, 1961b) (Fig. 14A). The plan in other insects is generally similar but in fibrillar flight muscles (Edwards et a/., 1958), and also in some non-fibrillar examples (Vogell eta/., 1959), the tracheoles indent the muscle fibres and become functionally if not morphologically intracellular (Fig. 15). Gaseous oxygen can therefore be brought extremely close to the mitochondria. The tracheole always remains enclosed in several layers, but these are thin and probably do not offer much diffusion resistance. The layers are: the membrane on which the intima was deposited ; the tracheoblast cytoplasm ; the tracheoblast plasma membrane; and the muscle plasma membrane (Smith, 1961a). The tracheoFIG.13. Hydrocyriits colCm:hiue.A, Primary and secondary tracheae to the oblique dorsal flight muscle. B, Same t o the basalar flight muscle. In both examples, the secondary tracheae are wrapped round the outside of the muscle and are ventilated in flight. C, A much simplified diagram of the left side of the pterothorax, exploded to show the main tracheal supply routes to the fibrillar flight muscles. In all cases the compressible secondary tracheae can be ventilated since they are either entirely outside the muscle, or, in the dorsal longitudinal and dorso-ventral muscles, they extend beyond the periphery. Expansion of the secondaries into small balloon-shaped air sacs occurs in a few cases at the far ends of the two primary tubes which supply the dorso-ventral muscle.

FIG.14A (Legend on page 329)

FIG. 14B (Legend on page 329)

FIG.14C (Legend opposite)

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blast outer membrane appears to be continuous with the inner membrane surrounding the tracheolar intima; the tracheole is therefore extracellular and surrounded by a cell whose shape is a hollow cylinder. The tracheole may nevertheless be formed intracellularly (Shafiq, 1963). The tracheole taenidium takes the form of annulets, helices or series of helices (cf. Buck, 1962) or in some tissues more complex patterns (Fig. 14B). The different patterns, which have probably no separate functional significance, may provide evidence as to the mode of taenidial formation (Locke, 1958a). The tracheae and tracheoles persist after flight muscles have degenerated (Fig. 16) in many insects (Smith, 1964) and they give rise to the so-called “lungs” of some species (Hamilton, 1931). It is interesting to note that diffusion may even be facilitated in the smallest tracheoles due to the adsorption of gas molecules on to the walls and their instant replacement by lateral movements of other molecules (Weis-Fogh, 1964b). To describe the amount of tracheation a tissue receives, Weis-Fogh (1964b) has coined the term hole fraction, i.e. the ratio of the summed cross-section of all the air tubes in a given tissue surface to the total surface of the tissue. In the secondary tubes of flight muscle the hole fraction is 10-1to lo-,; for the tertiary system it is lo-, to It follows that the rate of diffusion of oxygen into the muscle is lo3 to lo5times faster than if diffusion were all in the liquid phase. Because of the greater permeability of CO,, it is only 5 x lo1to 5 x lo3times faster for this gas. B. T H E L O C U S T P T E R O T H O R A X

Weis-Fogh (1964a) has made a thorough examination of the tracheal system in the pterothorax of the locust. The following is no more than a summary of his work, and for a full understanding reference should be made to his paper with its beautiful illustrations. He divides the system into three parts (Fig. 17). 1. A tergo-pleural system which is connected to the first three spiracles FIG.14A. Electron micrograph of a longitudinal section of a flight muscle of a damselfly (Odonata). The section passes through the peripheral regions of two adjacent fibres and shows tracheoles lying between but not indenting them. (Reproduced by kind permission of D. S. Smith.) x 22 500. B. Electron micrograph of a section through a tracheole situated between two photocytes in the light organ of Photurispennsyluanica. Note the considerable complexity of the taenidium and the proximity of tracheoles to mitochondria in the photocytes. (From Smith, 1963.) x 32000. C. Electron micrograph of negatively stained tracheoles from the flight muscle of Calliphora. Note the two helical taenidia coiled apparently like the famous staircases at Chambord Chlteau. (Reproduced by kind permission of D. S . Smith.) x 36 OOO.

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FIG. 15A

FIG.15. Electron micrographs of longitudinal sections of fibrillar flight muscles showing tracheoles which have indented the muscle cell membranes and lie along mitochondria. A. The wasp, Polistes. (Reproduced by kind permission of D. S . Smith.) x 17 400.

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FIG. 15B B. The carabid beetle, Hurpuhs aeneus. Compare with Fig. 16. (From Smith, 1964.) x 13000.

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FIG.16. Electron micrograph of a section of the vestige of the flight muscle of the carabid, Ophonus puhestens. The flight muscles in this species do not normally develop beyond the

stage shown here, but the tracheal system is fully developed and the abundant tracheoles lie incontactwithoneanother.(FromSmith, 1964.) x 16000,

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and provides the main flight muscle supply. It comprises the large central pair of air sacs from which branches supply the dorsal longitudinal muscles, an intermediate group of sacs on either side of the elevator muscles and a lateral group lying between the pleural walls and the controller depressor muscles. There are no connexions between the two sides of this system and only small connexions with other tracheae.

FIG.17. A narrow transverse section of the right side of the pterothorax of Scliistocerca greguriu, exploded laterally, t o show the tracheal supply t o the flight muscles. Arrows indicate the movements of the wings and notum. Three muscles are shown, a depressor, an elevator and a controller depressor. The tergo-pleural (Tt) and sternal (Ts) tracheae are shown with transverse hatching, while the pleuro-coxal tracheae (Tpl) are additionally marked with longitudinal broken lines. Air sacs 1 and 2 belong t o the mesial group of the tergo-pleural system, and 3,4, 5 , 6 and 7 to the intermediate group. Sac 9 represents the lateral subdermal group, and sac 1 1 the ventral subdermal system. Sac 10 represents the coxal group of the pleuro-coxal system. The only anastomosis between sternal and pleuro-coxal systems shown is blocked by liquid. (From Weis-Fogh, 1964a.)

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2. A pleuro-coxal system (the supra-ventral trunks of Albrecht, 1953), which communicates with spiracles 1 and 2 and connects with the intermediate group of air sacs of the tergo-pleural system. It also sends branches to the legs. In the first larval instar this system anastomoses with the sternal system at the level of the mesothoracic and metathoracic ganglia. In subsequent instars these anastomoses become blocked with liquid and in consequence the tracheal system to the flight muscles is largely isolated from other tracheae. Comparable anastomoses are blocked in the prothorax and head at the same time and short-circuiting of the cephalic tracheal system by the uni-directional airstream is thereby prevented (Miller, 1960~). 3. A sternal system which comprises the longitudinal ventral trunks which run through the thorax supplying the ganglia. The main features of the pterothoracic system in the locust are therefore its separation into right and left halves, and its almost total isolation from the remainder of the tracheal system. Similar but less complete isolation occurs in the pterothorax of Belostomatids and dragonflies (Miller, 1962). Such isolation probably improves the gaseous exchange between the pterothoracic tracheae and the outside during thoracic pumping. C. M O V E M E N T O F A I R I N T H E P R I M A R Y A N D S E C O N D A R Y TUBES BY VENTILATION

Respiration in flight is entirely aerobic (Krogh and Weis-Fogh, 1951). Hyperventilation, which continues for a short time after flight, may be then caused by a temporarily high level of haemolymph CO, or by other factors. By comparing the amount of oxygen consumed in the flight of a locust with the volume pumped by the abdomen, Weis-Fogh (1956a, and to be published) has shown that abdominal ventilation alone is quite inadequate; thoracic pumping must therefore contribute most of the oxygen. He measured the pressure and volume changes in the pterothorax during one wing stroke and the pressure-flow characteristics of the tracheae concerned, and by this means proved that thoracic autoventilation took place and provided adequately for the known consumption. An intuitive inference held for a long time (e.g. Burmeister, 1832) was thereby given a quantitative basis. The meso- and metathoracic nota of a locust rise and fall with each wing stroke and, together with small lateral movements, produce volume changes of 20 p1 per stroke, when the wing amplitude is 68".(This is equivalent to 575 I/kg/h for a locust weighing 2.5 g and flying with a wing beat frequency of 20/sec). Only about one-third of this, 7 pl, is available for pterothoracic respiration, since the remainder

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affects large anterior abdominal sacs which are contiguous but not confluent with the central pair of thoracic sacs. In Schistocerca the total tracheal volume is between 500 and 950 pl (Weis-Fogh, 1964a). Air sacs in the pterothorax of sexually immature migrants contain 100-1 50 pl, of which 90 pl is in the large central pair of sacs; a stroke volume of 7 pi therefore can exchange 5-77" of the air in the pterothorax. Isolation of this system ensures that the exchange takes place only with the outside. In the dragonfly, Weis-Fogh has shown that lateral movements of the pleura are more important than the dorso-ventral excursions of the nota. The pleural walls are loosely joined at the carina anterior to the wings and they separate when the wings are in the mid position, but come together when the wings are up or down. A pumping stroke may therefore take place twice every complete wing beat. For part of the cycle, notal and pleural movements may act in opposition, but this is insignificant and the mechanism is able to pump 2 000 litres air/kg/h. The primary tubes therefore probably remain filled with fresh air in flight, and the conditions for diffusion in the secondaries are optimum (WeisFogh, 1964a, b). Thoracic pumping probably plays an important part in the flight of most large insects, particularly when the increased length of secondaries means that they too must be ventilated. Weis-Fogh has shown that the nota of hawk-moths and of the beetles Oryctes and Geotrupes perform large movements with each wing stroke, while in the giant Scarabaeid, Heliocopris co/ossus (with a wing span of 180 mm) the metanotum moves through over 1 mm with each beat and sweeps out a volume of nearly 300 pl (Miller, in preparation). This species has a complex arrangement of secondary tubes associated with air sacs, the tubes being stacked so that they form a curtain of air between almost every row of fibres and the next in the flight muscles (Fig. 18). Again in Hydrocyvius the thoracic stroke volume is about 80 pl and altogether about 1 000 litres of air/kg/h may be provided. Since air sacs are few, the tracheal volume is probably comparatively small and much of the contained air may therefore be exchanged with each wing stroke (cf. Section IV, A, 2). In the large Cerambycid beetle, Petrognatha gigas, air may be blown directly through the primary tubes in flight (Amos and Miller, 1965).Two large primary tracheae run through the metathorax between spiracles 2 and 3 on either side (Fig. 19). Sheaves of secondary tracheae radiate out from them and supply the flight muscles, while further secondaries leave directly from the expanded atria of spiracles 2 and 3. They pass between the fibres of all the flight muscles, and are themselves probably ventilated by the flight movements. The primary tubes are not associated

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with air sacs and their secondarily thickened taenidia probably enable them to resist compression ; hence volume changes in the pterothorax are unlikely to ventilate them directly. By suspending a dead beetle, with wings and elytra opened, in a wind and by releasing smoke or dust in front of the insect, a current ofair entering spiracles 2 and leaving from spiracles 3 has been demonstrated : it probably renews the air in the primaries when the insect flies. A comparable mechanism has been thought to operate

FIG.18. Section through the pterothorax of the Scarabaeid, Heliocopris co/ossus, to show the arrangement of secondary tracheae separating almost every row of flight muscle fibres from the next.

in the flying locust (Stride, 1958), but here the tracheal morphology does not suggest that a through draught is created. Abdominal pumping takes place well below maximum rates during flight in many insects and it may even stop, at least temporarily, in some (Fraenkel, 1932). In locusts, Weis-Fogh ( 1956a, and in preparation) measured an initial abdominal pumping rate of about 180 l/kg/h which drops to 150 litres after the first 5 min of flight ; maximally stimulated ventilation on the other hand can pump 250 litres. Similarly auxiliary

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forms of ventilation by head and prothorax do not appear in normal flight. Although the pterothoracic sacs of Schistocerca contain 5';/, CO, (Weis-Fogh, unpublished), these gases apparently do not reach the ventilatory centres; the independent pterothoracic tracheal system may therefore allow abdominal pumping to maintain a supply of fresh air to the head and thoracic ganglia, entering through spiracle I and proceeding expanded atria

FIG.19. View of the left half of thepterothorax of the Cerambycid, fetrognuthagigus, from the posterior. The diagram is much simplified to show the flight muscles and the two large primary tubes which arise from spiracle 2 on each side and proceed directly through the metathorax t o spiracle 3. Air is probably blown through these tubes during the flight of the beetle.

along the ventral trunks (Miller, 1960c; Section IV, C). Similarly, flying Hydrocyrius pumps at about 1 abdominal stroke/sec, whereas during preparation for flight it spends about 7 min pumping vigorously with combined movements of abdomen and prothorax at frequencies of up to 4/sec; this accompanies a considerable rise in pterothoracic temperature (Miller, 1961a). Again in Oryctes and Geotrupes pre-flight pumping rates are much in excess of those seen during flight (Weis-Fogh, 1964a).

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Diptera share with Hymenoptera a type of flight mechanism which involves only small volume changes with each wing beat. In consequence thoracic pumping is insignificant for respiration during flight. Small species (e.g. Drosophila; Weis-Fogh, 1964b) depend on diffusion, while in larger ones abdominal ventilation plays a major role. The presence of abundant air sacs in Drosophila not only assists diffusion but may also allow a reduction in haemolyrnph volume to take place after the teneral adult has expanded and hardened, with a concomitant increase in haemolymph fuel concentration (Wigglesworth, 1963). Indeed Weis-Fogh suggests that problems of fuel supply to active flight muscles may be more limiting than those of oxygen supply. In wasps and bees abdominal ventilation is much increased in flight, and in the latter a uni-directional stream through the thorax persists, air enteringvia spiracle 1 and leaving from spiracle 3 (Bailey, 1954).Although the worker wasp ( Vespa germmica) pumps at 250 strokes/min in tethered flight,itcontinues to fly for at least 8 min at apparently normal frequency and amplitude after removal of the abdomen. Thus diffusion alone through the broad sacs of the latero-linear supply may be able to meet the demands of insects of this size, although normally abdominal pumping is important (Miller, unpublished). The stroke volume of the thoracic pump is small compared with the total capacity of the voluminous pterothoracic tracheal system which has broad connexions with abdominal air sacs. This means that thoracic ventilation probably produces no exchange with the outside, but only mixing within the system. This may be more significant for the fuel supply than for the delivery of oxygen (Weis-Fogh, 1964a,b). In Eristalis tenax there are similar broad connexions between the pterothoracic latero-linear system and abdominal air sacs. In tethered flights of up to 13 min duration, however, no abdominal pumping can be seen. Strong abdominal ventilation commences as soon as flight ceases and it takes the form of ventral flexing movements, which might affect the stability of the insect when flying (Miller, unpublished). Apparently diffusion provides the major part of the oxygen consumed, and again thoracic pumping will be more significant for intra-thoracic mixing than for respiratory exchanges. According to Weis-Fogh (1964b) insects up to the size of a locust may be able to depend on diffusion alone, if they possess a latero-linear supply. D. T H E MOVEMENT OF GASES I N T H E S E C O N D A R Y A N D T E R T I A R Y T U B E S BY D I F F U S I O N

Krogh ( 1920), and later Nunome ( 1944) and Thorpe and Crisp ( I 947), measured the pathways between spiracles and tissues and showed that

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oxygen could be supplied even to the ends of long extremities in resting insects by diffusion alone. The means for supplying oxygen in flight, however, were not explored until Weis-Fogh (1964b) made a painstaking analysis of the gas movements in secondary and tertiary tubes, and in the tissues beyond. In addition he has provided formulae for calculating diffusion rates in many different situations, which should have wide applicability. Weis-Fogh has shown that the oxygen consumption of Aeshna in flight is 1.8 ml oxygen/g muscle/min; the amount of oxygen in the primary tubes would therefore be adequate for 1-2 strokes only, and it must be renewed every wing beat. Measurements on a lobe from the second basalar muscle show that an oxygen partial pressure drop of 0.05 atm would be expected between the start of the secondary and the tip of the tertiary tube. In another example, taken from the tergo-sternal muscle, TABLE11 Maximum radius (p) for the diffusion of dissolved oxygen from the surface into the axis of a resting and ail active muscle fibre when the partial pressure between the ends of the air diffusion pathway is 0.05 atm. (From Weis-Fogh, 1964b.)

Metabolic rate (ml O,/g muscle/min) At rest In flight 0.02

1

2

118 p

16.7

11.8

4 8.4

8 5.9

which had the longest diffusion path found, a drop of 0.31 atm was calculated. Regarding the muscle as virtually surrounded by air, WeisFogh determined the maximum possible radius for a fibre dependent on tissue diffusion of oxygen from the periphery, at various rates of oxygen consumption (Table 11). In the dragonfly the measured fibre radius is 10 p: there are no indenting tracheoles (Smith, 1961b), and the radius therefore comes close to the theoretical maximum of 11.8 p, when the oxygen consumption is 2 ml/g muscle/min. These figures emphasize the necessity for the diffusion path to start from almost fresh air, which in turn demands an efficient thoracic ventilation (Section IV, C). This may lead to higher rates of water loss than in other flying insects, but dragonflies feed often on the wing and they may drink periodically (Miller, 1964c) so that only during migrations is it likely that water will be short (cf. Corbet, 1962). The dragonfly system is therefore pushed to the limit M

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of its capabilities, and Weis-Fogh suggests that factors such as the remarkable radial arrangement of mitochondria and fibrils in the fibre (Smith, 1961b) may be functionally significant. Higher metabolic rates can be achieved only by decreasing the size of the fibres or through tracheoles indenting the cells : both modifications are found in other orders. The high permeability of COzin tissues means that normally it can be shunted through them as well as passing along tracheae. However, in flight muscle the large hole fraction (Section IV, A, 3) probably obviates the tissue shunt so that most of the CO, escapes through the tracheal system. Weis-Fogh has extended his analysis to a number of other insects drawn from four orders; in all, diffusion is shown to account for the oxygen consumed when the primary tubes are adequately ventilated. It appears therefore that oxygen is delivered to the flight muscles of insects through a system no different in principle from that which supplies other organs and tissues. Hypertrophy, extensions and fusions have enabled it to meet the enormous demands made by flight, but ventilation plus diffusion remain the only means for transporting oxygen. E. SPIRACLE B E H A V I O U R D U R I N G FLIGHT

The thoracic pump can work efficiently in flight only if the appropriate spiracles are kept open, and this has been observed to take place in a number of insects. In others, however, such as the bee (Bailey, 1954), the thoracic pump does not contribute to the oxygen intake (Section IV, C) and the thoracic spiracles remain synchronized with abdominal ventilation. Spiracles 2 and 3 of the locust open widely when flight starts and they remain open throughout (Fig. 20). When thewing beat frequency declines, they may both commence to make small closing movements, synchronized with abdominal expiration, but these do not significantly impede the airflow. The size of the closing movements is proportional to the wing beat frequency and not to the metabolic rate; it is therefore probably of no functional significance. The movements reflect an incomplete inhibition of the synchronizing bursts which arise in the metathoracic ganglion and normally drive the spiracles (Section 11, H ; Miller, 1960~). Spiracle 2 opens more widely than at rest due to a cuticular lever which is actuated indirectly by the contractions of the flight muscles. Spiracle 3 opens fully as a result of a strong contraction of its opener muscle. Spiracle 1, however, maintains synchronized activity and this probably

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ensures adequate ventilation of the head and central nervous system as already described (Section IV, C; Miller, 1960~). Stimulation of the mesothoracic ganglion with C 0 2causes the opener muscle of spiracle 1 to contract as a result of excitation of its mesothoracic nerve (Section 111, E), and this seems to be a normal occurrence in fight. Provided the prothoracic nerve to the opener is silent, the opener and closer do not contract simultaneously and in consequence the ventral orifice of the spiracle is not constricted. Therefore abdominal expiration can blow pterothoracic gases across the atrium of the closed

A

t

Flight

C

B

starts

D

E

u 5 sec

FIG.20. Activity of spiracle 2 of Schistocercagregaria redrawn from mirror recordings of the valve movements. A, Before flight; B, 5 sec after the start of flight; C, 5 min after the start of flight; D, 30 min after the start of flight; E, 90 min after the start of flight. At rest the spiracle opens only about 30%. In flight it opens 100% as the result of the actuation of a cuticular lever. Small closing movements, synchronized with expiration, commence after 5 min, but they probably do not significantly impede ventilation. (From Miller, 1960~).

spiracle and into the cephalic tracheae; from there they reach the CNS where they can excite both ventilation and the prothoracic nerves to the opener. Excitation of the latter constricts the ventral orifice and cuts off the leakage of gases into the cephalic tracheae. Thus spiracle 1 provides the CNS with a mechanism for sampling pterothoracic gases without becoming swamped by them. Abdominal ventilation can therefore be varied in flight to meet the demands, while the thorax continues to pump a fixed amount (Miller, 1965). In flying dragonflies, spiracles 1-3 are held open (Fraenkel, 1932) and M*

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tidal thoracic ventilation pumps air through them. Abdominal ventilation continues and is responsible for supplying the head through spiracle 1 (dorsal orifice). Spiracle opening may precede the first wing beat by 100 msec and it is produced by stimuli which also normally give rise to flight, although it can appear in the absence of wing movements. Electrical stimulation of certain regions of the metathoracic ganglion can produce instantaneous spiracle opening without other detectable movements (Miller, 1962). F. THE OXYGEN S U P P L Y TO THE RESTING FLIGHT MUSCLES

Weis-Fogh (1964a) has called attention to the parallel between the design of the bird lung and the tracheal supply to locust flight muscle. In both systems the areas of diffusion exchange are ventilated by air sacs situated beyond them, and both have a shunt running parallel to but outside the exchange regions (Fig. 13). Contrary to some other interpretations (cf. Salt, 1964),Weis-Fogh suggests that the bird shunt is concerned with evaporative heat loss only when not in flight, the normal areas for heat loss being then less effective. In resting locusts the shunt tracheae are believed to supply oxygen by diffusion to the part of the muscle farthest from the spiracle. In other insects, where no shunt is found, oxygen may reach this part by different means; for example through fusions of the primary air sacs in dragonflies, or from other spiracles in Belostomatidae. Limited ventilation of the pterothorax in resting locusts is brought about by abdominal pumping. Spiracles 1-3 open during inspiration, and air entering them may pass into the central pair of air sacs and thence anteriorly to the cephalic trunks. Alternatively expiration may drive air across the closed atria of spiracles 1 and 3 and so into the cephalic or abdominal systems. Blind-ending primary tubes which adjoin this route may be ventilated tidally. Synchronized movements of all thoracic spiracles usually appear for a short period after flight in libellulid dragonflies. Coupled with abdominal pumping they probably give rise to air currents which pass directly through the axial primary tubes of the flight muscles, and then leave the thorax posteriorly in the visceral tracheal trunk which proceeds directly to the expiratory spiracle 10. The dorsal confluence of primary tube air sacs communicates both with the visceral trunk and with the dorsal abdominal trunk. The latter is connected to spiracles 4-9, which are inspiratory in function. There may therefore be a second airstream which enters through spiracles 4-9, travels anteriorly to the dorsal confluence and then posteriorly to spiracle 10 in the visceral trunk (Fig. 21A). In

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other dragonflies, such as Anax, the first stream may proceed in the reverse direction and the spiracular roles are reversed. Finally in some crepuscular Aeshnidae (e.g. Acunthugyna, Heliaeshna), the thoracic spiracles have never been seen to make synchronized movements and after flight they remain open so that ventilation takes place tidally through them. Simultaneously the abdominal spiracles stay closed and the abdominal strokes are therefore able to ventilate the thorax more effectively. As recovery proceeds and the thoracic spiracles re-close, spiracle 10 again becomes active (Fig. 21B; Miller, 1962). PRIMARIES

VISCERAL TRUNK

A

B FIG.21. Hypothetical directions followed by ventilating airstreams in the dragonflies, Hadrothemis defecta (A), and Acanthagyna villosa (B), immediately after flight. (Based on Miller, 1962.)

V. SUMMARY 1. Ventilation arises from an endogenously produced rhythm in the CNS. In several insects the third embryonic abdominal ganglion may act as a pacemaker for the rhythm, regardless of whether it migrates to the thorax or remains in the abdomen. 2. Various forms of spontaneous activity in nerve cells are considered. The available evidence suggests that burst formation in the locust CNS may depend ultimately on the properties of a single pacemaking cell; its activity is relayed throughout the abdomen by command interneurones, and synchronized pumping strokes are thereby brought about. 3. The action of proprioceptive and chemoreceptiveinput on the pacemaker is variable in different species; in some, pacemaker activity is not

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strictly endogenous and ceasesin the absence of input. On the other hand, in certain cockroaches, not only burst formation but also a pattern of periodic ventilation is endogenously derived. 4. New experiments on the discontinuous release of CO, from diapausing silkmoth pupae indicate that a negative intratracheal pressure normally occurs, and that the rate of diffusion of nitrogen outwards determines the interval between flutters. The mechanism proposed by Buck (1958) is still tenable with small modifications. 5. In one-muscle spiracles activity is dually controlled from the CNS and at the periphery. The CNS acts largely by grading the response of the spiracle muscle to CO,. In two-muscle spiracles the opener muscle may be controlled by a central nervous mechanism which spans several segments, and peripheral control plays no part. 6. Movements of spiracles, synchronized with ventilation, are brought about by interneurones which run from ventilation centres and superimpose various patterns of activity on the free running or spontaneous behaviour of the spiracle motor neurones. The latter still plays alarge part in, for example, dragonfly spiracles, but becomes insignificant in locust spiracles. 7. After denervation, one-muscle spiracles remain active and continue to respond to CO,. In the locust the activity depends on a potassium contracture, but in the pupa of Hyalophora there is apparently a myogenic pacemaker present which is active before and after denervation. 8. The pterothoracic tracheal system is much modified for flight. The pathway from a spiracle to the tracheoles is divided into primary, secondary and tertiary sections, and the relative parts played by ventilation and diffusionin these sections in insects of different sizes is discussed. Some specializations in very large insects are noted. 9. Finally some of the methods employed for ensuring that the resting flight muscles receive an adequate supply of oxygen are discussed, with particular emphasis on the period immediately following flight. ACKNOWLEDGMENTS It is a great pleasure to acknowledge the assistance of Drs. D. J. Aidley and A. C. Neville who read this chapter and made many valuable comments and criticisms. REFERENCES Adrian, E. D. (1931). Potential changes in the isolated nervous system of Dytiscus marginalis. J . Physiol. 72, 132-151.

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Author Index Numbers in italics refer to the pages on which references are listed in bibliographies at the end of each article.

A

Balbiani, E. G., 172, I90 Baldwin, E., 81, 114 Baltscheffsky, M., 137, 192 Banks, C. J., 252, 253, 272, 273 Barlow, H . B., 6, 46 Barsa, M . C., 93, 124 Bartsch, K., 159, 193

Adams, R. I., 315, 353 Adrian, E. D., 282, 283, 288, 344 Afsharpour, F., 110, 114 Agrawal, H. C., 70, 78, 126 Agrell, I., 72,90,92,93,94,95,114, 161, 190 Aidley, D.J . , 317, 345

Battersby, W. S., 33, 46

Albrecht, F. O., 283, 334, 345 Alfert, M., 171, 190 Allegri, G., 57, 58, 61, 72, 75, 115, I18 Allen, K., 78, 114 Allende, J. E., 187, 199 Allfrey, V. G., 188,190 Alverdes, F., 282, 345 Amanieu, M., 72, 75, 114, 122 Ames, B. N., 157, 190 Amos, W. B., 335, 345 Andersen, S. O., 298, 345 Anderson, E., 182, 190 Anderson, G. B., 213,233,234,254,275 Anderson, T. F., 300, 351 Appella, E., 109, I14 Aruga, H., 77, 130 Arvanitaki, A., 285, 287, 345 Asperen, K . van, 110, 125 Asperen, K . K . van, 110,129 Aubert, H., 138, 190 Auclair, J. L., 72, 76, 77, 114 Autrum, H., 7 , 18, 19,21, 24,25, 29,36, 46, 47 Avi-Dor, Y.,150, 194

Bauer, H., 171, 190 Baumgarten, R. von, 287,351 Beadle, L. C., 78, 99, 114 Beale, G. H., .268, 273 Beament, J. W . L., 279, 280,345 Beckel, W . E., 301, 302, 303, 305, 310, 316, 319, 345,350,351 Becker, E., 168, 190 Becker, H . J., 112, 114, 174, 190 Beckwith, J. R., 157, 199 Beermann, W., 112, 114, 115, 118, 171, 172, 173, 174, 175, 181, 190, 193 Bekesy, G. von, 27, 46 Belitzer, V., 135, 190 Benassi, C. A., 57, 58, 61, 72, 75, 115, 118 Bennett, G. A., 82, 127 Benolken, R. M., 24, 46 Benz, G., 72, 82, 108, 115 Berendes, H . D., 172, 191 Bergmann, M., 66, 115 Bernhard, C. G., 21, 25, 33, 46 Berreur, P., 100, 115 Bhambhani, H . J., 301,352 Bhat, J . V., 65, 95, 127 Bheemeswar, B., 81, 88, 115, 119 Bieber, R. E., 109, 122 Biederman, M . A., 285,345 Bier, K., 171, 174, 191 Bird, M . A., 109, 129 Birks, P. R., 244,251, 253,255, 257,265, 270, 274 Birt, L. M., 131, 132, 141, 150, 154, 191

B Babak, E., 288, 345 Bachmann-Diem, C., 80, 116 Baehr, W . B. de, 216, 219, 272 Baglioni, C., 110, 114 Bahr, G. F., 172, 190 Bailey, L., 282, 31 1, 338, 345 Baker, H . D., 33, 46

355

356

AUTHOR INDEX

Bischai, F. R., 158, 159, 160, 203, 325, 352 Bloch, K., 169, 192 Blum, M. S., 96, 125 Bodenheimer, F. S., 209, 273 Bodenstein, D., 54, 63, 93, 115, 168, 171, 174, 176, 179, 191 Bodenstein, F. S., 66, 124 Bodine, J. H., 67, 115 Boell, E. J., 62, 67, 109, 115, 126 Boistel, J., 283, 296, 345 Bonk, G. J., 183, 191 Bonhag, P. F., 101, 115 Bonnemaison, L., 208, 209, 21 1, 213, 220, 221, 231, 232, 236, 237, 238, 239, 243,244, 246,250,252, 253, 254, 266, 268, 270, 273 Bonner, J., 188, 195 Bonner, W. D. Jr., 164, 191, 195 Bonnet, C., 207, 273 Booth, C. O., 212, 251, 274 Borner, C., 209, 210, 273 Boschetti, M. A., 216, 273 Bourne, A. G . , 38, 50 Bowers, B., 182, 191 Bowness, J. M., 2, 52 Boyland, E., 135, 198 Boxer, G. E., 149, 191 Breuer, M. E., 112, 115, 171, 173, 191, 199 Bricteux-Grkgoire, S., 70, 92, 115 Bridges, C. B., 171, 191 Briegel, H., 56, 57, 58, 59,60,61, 62,116 Brockway, A. P., 299, 345 Bronk, D. W., 282,345 Brown, K. T., 29, 46 Bruin, G. H. P. de, 7, 46 Brunet, P. C. J., 73, 115 Bruslk, S., 208, 273 Biicher, Th., 134, 137,140, 141, 142, 144, 145, 146, 150, 151, 155, 157, 158, 159, 160, 161, 164,191,193,196,199,203, 205, 325, 352 Buck, J., 280, 291, 299, 304, 329, 346, 348 Buck, J. B., 78, 115, 183, 191 Buckland, C. T., 301, 350 Buddenbrock, W. von, 43,46 Buddenbrook, W. R., 281, 346

Buisson, M. du, 288, 346 Bullock, T. H., 286, 294, 319, 346 Burdette, W. C., 54, 116 Burdette, W. J., 170, 191 Burke, A. W. Jr., 170, 192 Burkhardt, D., 5, 11, 19, 21, 24, 25, 40, 46, 47, 51 Burmeister, H., 334, 346 Burns, B. D., 287,351 Bursell, E., 79, 116 Burtt, E. T., 5, 8, 9, 10, 11, 13, 15, 16, 19, 21, 24, 25, 26, 27, 28, 29, 32, 33, 35, 36, 37, 38, 40, 41,43, 47 Bush, B. M. H., 37, 43, 51 Busnel, R. G . , 56, 65, 118 Butenandt, A., 169, 191

C Cajal, Ramon y, 16, 40, 43, 47 Campbell, J. I., 301, 304, 346 Carey, F. G., 94, 116, 162, 164, 191 Cartier, J. J., 216, 273 Case, J. F., 283, 288, 295, 297, 301, 302, 303, 307, 308, 311, 315, 317, 346 Casida, J. E., 78, 116, 124 Catton, W. T., 5, 8, 9, 10, 11, 13, 15, 16, 19, 23, 24, 25, 26, 27, 28, 29, 32, 33, 35, 36, 37, 38, 40, 41, 43, 47 Chadwick, L. E., 143, 148,191, I92 Chalazonitis, N., 284,287,296, 309,3 19, 345,346 Chance, B., 68, 116, 135, 136, 137, 141, 142, 143, 145, 146, 149, 154, 156, 160, 164,165,192 Chefurka, W., 131, 132, 147, 192 Chen, P. S., 55, 56, 57, 58, 59, 60, 61,62, 69, 70, 72, 73, 74, 75, 76, 80, 82, 83, 84, 85, 90, 93, 96, 98, 102, 104, 106, 107, 116, 117, 120, 125, 131 Chino, H., 65, 68, 117 Chun, C., 14,47 Church, N. S., 323, 346 Ciotti, M. M., 109, 122 Clark, A. J., 169, 192 Clarkson, E. N. K., 2, 47 Claude, A. J., 138, 192 Clayton, R. B., 169, 192 Cleland, K. W., 140, 202 Clements, A. N., 59, 117

357

AUTHOR INDEX

Clemons, R. D., 150, 184,195 Cleveland, L. R., 170, 192 Clever, U., 112, 117, 118, 134, 174, 175, 176, 177, 188,192,193

Cochran, D. G., 140, 146, 149, 154,193, 201

Coda, R. L., 54, 116 Cognetti, G., 216, 217, 218, 273 Cohen, G. N., 106, 118 Colombo, G., 57,58,61,72, 75,115,118 Contis, G., 40, 52 Conway, T. W., 187, 199 Coraboeuf, E., 283, 296, 345 Corbet, P. S., 339, 346 Corlette, S. L., 173, 200 Corrigan, J. J., 78, 118 Cosens, D. J., 31, 32, 35, 47 Cottrell, C. B., 96, 118 Counce, S. J., 53, 118 Cowley. J. M., 13, 47 Craig, R., 77, 110, 118, 125 Crescitelli, F., 36, 49 Crisp, D. J., 7,46, 338, 352 Crone-Gloor, U. von der, 56,57,59,60, 61, 118

D Dadd, R. H., 100,118, 168,193 Dallari, L., 217, 273 Daniel, L. S., 161, 204 Daumer, K., 10, 48 Davey, K. G., 299, 346 Davidson, J., 221, 273 Davis, B. D., 157, 193 Davis, G. R. F., 71, 122 Davis, N. T., 168, 193 Deguchi, N., 16, 40, 52 Dehn, M. von, 264,273 Delbriick, A., 145, 146, 159, 193, 205 De Long, D. M., 97,118,123 Demyanoskii, Ja. S., 88, 118 Dennell, R.,73, 118, 166, 193 DenucB, J. M., 85, 118 Desai, R. M., 80, 118 Dethier, V. G., 3, 4, 6, 10, 38, 47, 48 Deuticke, J., 145, 193, 194 Devlin, T. M., 149, 191 Diem, C., 98, 116 Dietrich, W., 16, 39, 48

Dimond, J. B., 97,118,123 Dinamarca, M. L., 131,132 Ditchburn, R. W., 42, 48 Dixon, A. F. G., 236, 273 Dixon, M., 156, 168, 193 Dixon, S. E., 81, 100, 129, 168,203 Doane, W. W., 168,193 Dowling, J. E., 33, 48 Drilhon, A., 56, 65, 72, 118 Dubreuil, R., 72, 114 Duchlteau, G., 72, 78, 114, 118, 127, 183, 193

Duke, E. J., 86, 111, 126 Dumas, T., 301, 350 Dunn, J. A., 237,273

E Eccles, J. C., 21, 48 Edstrom, J. E., 112, 118, 173, 193 Edwards, D. K.,33, 48 Edwards, G. A., 280, 325,346 Eguchi, E., 20,2 1, 24, 25,40,4 1,48,50 Eisenberg, R. M., 311,352 el-Wahab, A., 167, 193 El-Ziady, S., 252, 253, 274 Elliott, W. B., 153, 195 Ellis, J. F., 71, 121 Ellis, J. P., 71, 72, 125 Eltringham, H., 16, 17, 44,48 Embden, G., 145, 193, 194 Engelhardt, W., 134, 135, 194 Ennis, H. L., 187, 198 Ernster, L., 137, 202 Estabrook, R.W., 154,194 Etten, C. H. van, 82, 127 Ewer, D. W., 281, 301, 304,346,347 Exner, S., 4, 1 I , 16, 48

F Farinella-Ferrum, N., 107, 117 Farnsworth, M. W., 109, 119 Fast, P. G., 169, 194 Faulhaber, I., 108, 119 Faulkner, P., 87, 88, 119 Fermi, G., 5, 48 Fernandez-Moran, H., 14, 16, 48 Fielden, A., 285, 293, 311, 347 Finlayson, L. H., 294, 347, 350 Fisk, F. W., 281, 350

358

A U T H O R INDEX

Fitzgerald, L. R., 65, 119 Florkin, M., 69, 70, 72, 78, 85, 92, 114, 115,118,119,127, 183, 193,194 Fluiter, H. J. de, 221, 232, 274 Forrest, H. S., 110,119 Fox, A. S., 98, 99, 119 Fraenkel, G., 71, 73, 100, 119, 124, 126, 165, 168, 169, 194, 198, 281, 285, 295, 311, 336, 341,347 Friedler, L., 78, 119 Friedman, S., 87, 119 Frisch, K. von, 10, 19, 48 Fristrom, J. W., 75, 119 Frumin, M. H., 287, 352 Fruton, J. S., 66, 115 Fu, Y.Y., 61, 120 Fukami, J., 146, 194 Fuortes, M. G. F., 20, 21, 24, 25, 39, 48

G Gaffron, M., 7, 48 Gallwitz, U., 21, 25, 36, 46 Ganti, Y.,72, 75, 120 Garcia-Bellido, A., 98, 99, 120 Gay, H., 172, 205 Gaynor, J. B., 282, 345 Geiger, H. R., 97, 120 Geigy, R., 63, 120 George, J. G., 81, 125 Gilbert, L. I., 54, 94, 120, 161, 167, 168, 169, 171, 194, 201, 260, 276 Gilmour, D., 55, 67, 80, 120, 131, 132, 144, 194 Giulio, L., 19, 48 Glassman, E., 110, 119, 120, 122 Gloor, H., 104,120, 130 Golberg, L., 71, I20 Goldsmith, T. H., 2, 16, 23, 24, 40, 48, 49 Gonda, O., 150,194 Goodfellow, R. D., 167, 194 Gottschewski, G., 98, 120 GrassC, P., 239, 274 Greenawalt, J. W., 135, 197 Greenberg, B., 66,120 Gregg, C. T., 137, 140, 141, 150, 151, 156,194 Gregory, R. L., 286, 347

Grenacher, H., 4,49 Grillo, R., 166, 205 Gros, F., 106, I18 Grundfest, H., 320, 352 GuCrin, J., 296, 345 H Haavik, A. G., 137, 142, 195 Hackman, R. H., 72, 73,120 Hadorn, E., 70, 72, 73, 76, 82, 84, 102, 103, 106, 107, 110, 112, 117, 120, 121, 125, 176,194 Hagihara, B., 142,194 Hagiwara, S., 287, 293, 319, 347 Hahnert, W. F., 97, I18 Hall, H. H., 82, 127 Hamilton, A. G., 290, 347 Hamilton, M. A., 329, 347 Hamolsky, M., 109, 122 Hanimann, F., 73, 74, 82, 83, 117, 131 Hanstrom, B., 39, 40, 49 Harden, A., 134, 194 Harington, J. S., 70, 72, 75, 77, 121 Harlow, P. M., 100, 121 Harrington, C. D., 216, 274 Harrington, H. J., 2, 49 Hartline, H. K., 26, 32, 39, 49, 50, 52 Hartman, G., 135, 198 Harven, E. de, 325, 346 Harvey, W. R., 94, 121, 134, 149, 150, 160, 161, 163, 164, 165, 183, 184, 194 Hashimoto, Y.,29, 51 Haskell, J. A., 150, 184, 195 Hassan, A. A. G., 300, 31 1, 347 Hassenstein, B., 7, 9, 23, 36, 43, 49 Hatefi, Y. 137, 142, 195 Hayashi, M., 173, 195 Hayashi, M. N., 173, 195 Hazelhoff, E. H., 303, 307, 316, 347 Hecht, L., 88, 121, 131 Hecht, S., 7, 49 Hedin, P. A., 78, 125 Heinze, K., 209, 210, 273 Heisler, C. R., 137, 140, 141, 150, 151, 156,194 Heller, J., 99, 121, 165, 195 Heller, J. R., 89, 121 Herford, G. M., 300, 347 Herskowitz, I. H., 166, 197

359

AUTHOR INDEX

Hertz, M., 8, 15, 42, 49 Hess, B., 160, 164, 192 Hess, R., 146, 195 Hesse, R., 2, 14, 49 Higgins, J., 137, 192 Highnam, K.tC., 168,195 Hilchey, J. D., 71, 121 Hill, L., 62, 100, 101, 102, 121, 168, 195 Hille Ris Lambers, D., 211, 214, 220, 221, 230, 237, 274 Hines, W. J. W., 70, 121 Hinton, T., 71, 121 Hoagland, M., 88, 121 Hobby, B. M., 8,49 Hoch, F. L., 154, 195 Hocking, B., 14, 49, 321, 347 Hochman, B., 97,122 Hoffmeister, H., 169, 196 Hoglund, G., 33, 46 Holden, J. T., 97, 122 Hollunger, G., 135, 192 Holmes, W. F., 137, 192 Holter, H., 101, 121 Hoppe, W., 169, 195 Horie, Y.,65, 121 Horridge, G. A,, 19, 40, 49, 287, 291, 296, 347 Hoskins, W. M., 110, 125, 311, 348 House, H. L., 71, 100, 121 Howells, A. J., 131, 132 Hoyle, G., 285, 286, 294, 297, 301, 302, 303, 305, 306, 307, 308, 311, 315, 316, 317, 318,347,348 Huang, R. C., 188,195 Huber, F., 293, 296, 297, 301, 302, 348 Huber, R., 169, 196 Hughes, G. M., 282, 283,285, 294, 298, 299, 31 1,347, 348, 349 Hiilsmann, W. C., 153, 154, 195, 202 Hundertmark, A., 3, 49

I Ichikawa, M., 166, 195 Idris, B. E. M., 56,61,121 Ikeda, K., 298,348,353 Ikuma, H., 164,195 Ilse, D., 15, 49 Imms, A. D., 302,348 Indira, T., 56, 57, 58, 59, 64, I21

N*

Irreverre, F., 70, 77, 121, 122, 128 Ishikawa, S., 41, 42, 49 Ishizaki, H., 166, 195 Ito, H., 89, 128 Ito, T., 87, 122 Ivanova, T. S., 302, 348

J Jackson, R. W., 82, 127 Jacob, J., 112, 122, 134, 157, 170, 181, 195 Jacobson, M., 167, 205 J a b , T. L., 36, 49 Jander, R., 7, 10, 18, 49 Jeuniaux, C., 72, 75, 114, 122, 183, 194 Johnson, B., 239, 244, 246, 248, 249, 251, 252, 253,255,256,257,258,260, 264,265,270,274 Johnson, M. J., 135, 195 Jurtshuk, P., ,137, 142, 195

K Kafatos, F. C., 184, 195 Kalckar, H., 134, 195 Kalicki, H., 165, 166, 205 Kalmus, H., 18, 50 Kammen-Wertheim, A. R. van, 67, 128, 146, 202 Kandal, E. R., 291,348 Kaplan, N. O., 109, 122 Kaplan, W. C., 97, 122 Karcher, D., 84, 127 Karlson, P., 73, 96, 112, 118, 122, 165, 166, 167, 168, 169, 170, 174, 175,191, 192, 193, 195, 196, 201 Kasting, R., 71, 122 Kawase, S., 87, 95, 122 Kearns, C. W., 78, 118 Keilin, D., 138, 196, 300, 348 Keister, M. L., 280, 299, 346, 348 Keller, E. C., 110, 122 Kennedy, D., 291, 312,348,351 Kennedy, J. S., 209, 212, 251, 252, 257, 258, 274 Kenten, J., 220, 221, 225, 274 Kikkawa, H., 109, 110,122,123 Kilby, B. A., 80, 81, 88, 118, 123 King, K. W., 146, 149,193 Kirchhoffer, O., 17, 49

360

AUTHOR INDEX

Kirimura, J., 166, 167, IY6 Kirschfeld. K., 11, 42, 49 Kitchel, R. L., 31 1, 348 Kitzmiller, J. B., 241, 256, 274 Klingenberg, M., 135, 137, 140, 141, 142, 144, 145, 150, 151, 155, 158, 159, 160, 164,191, 196, 199, 201, 203, 325, 352 Klodnitski, L., 208, 274 Kloot, W. G. van der, 301, 303, 305, 309, 319, 320,348 Kobayashi, M., 166, 167, 196 Kolliker, A., 139, 196 Koltzoff, N., 171, 196 Kominz, D. R., 93,123 Konikova, A. S., 88, 118 Koyama, N., 3, 14, 20, 45, 52 Krause, G., 53, 123 Krishnakumaran, A., 92, 123, 168, 182, 196, 197 Kroeger, H., 113, 123, 134, 178, 180, 188,197,200 Krogh, A., 143, 186, 197, 281, 334,338, 348 KubiZSta, V., 145, 147, 148, 197, 199 Kiihn, A., 72, 75, 83, 90, 117 Kiihn, J., 81,124 Kuiper, J. W., 11, 17, 45, 50 Kuk-Meiri, S., 66, 67, 123, 127 Kummer, H., 98, 123 Kunze, P., 9, 50 Kurland, C. G., 162, 164, 197 Kurnick, N. B., 166, 197 Kuwabara, M., 25, 29, 48, 50 Kyber, J. F., 208, 274

L Lal, R., 251, 274 Lamb, K. P., 251, 274 Lang, C. A., 94,123 Lankester, E. R., 38, 50 Lardy, H. A., 135, 152, 153, 165, 197, 199,200 Larrabee, M. G., 282, 345 Larsen, O., 303, 349 Laskwoska, T., 75,131 Laufer, H., 85, 87, 110, 112, 123, 171, 182,197 Laurema. A., 77, 125 Lawson, C. A., 219, 274

Lea, A. O., 97, 118, 123 Leclercq, J., 183, 193 Lees, A. D., 208,209,211,220,221,224, 225, 227, 228, 229, 232, 236, 240, 246, 257, 260, 268, 269, 274, 275, 277 Legay, J. M., 57, 75, 123 Lehninger, A. L., 134, 135, 137, 138, 139, 140, 144, 148, 153, 154, 165,197, 200 Leuthardt, F., 81, 123 Levenbook, L., 70, 72, 73,77,80,81,87, 90, 91, 93, 121, 122, 123, 124, 131, 132, 139, 155, 197, 31 1, 349 Levinson, Z. H., 169, 198 Levy, R., 299,349 Lewis, H. N., 108, 124 Lewis, H. S., 1 11, 124 Lewis, H. W., 111, 124 Lewis, M., 93, 123 Lewis, S. E., 141, 151, 155, 198, 200 L'HBlias, C., 100, 124, 230, 275 Libby, J. L., 301, 349 Lichtenstein, N., 66, 67, 123, 124, 127 Limpel, L. E., 78, 124 Lindauer, M., 10, 48 Lindberg, O., 137, 202 Lipke, H., 71, 124, 169, 198 Lipmann, F., 154, 187, 195, 199 Littau, V. C., 188, 190 Liuzzo, J. A., 96, I25 Locke, M., 280, 329,349 Loeb, J., 208, 275 Lotz, G., 301, 349 Loughheed, I. C., 72, 85, 130 Low, H., 137,198, 202 Lowenstein, O., 294, 347 Lubin, M., 187, I98 Lucas, F., 131,132 Ludwig, D., 76, 85, 93, 124 Liidtke, H., 20, 50 Luh, W., 161,199 Lusis, O., 168, 195 Lyle, G. G., 152, 199 Lynen, F., 135, 198

M Maas, W. K., 157,198 McCann, G. D., 8,50 McClintock, B., 157, 198

361

A U T H O R INDEX

Mchchern, D., 145,198 McFall, E., 157, 198 MacGillivray, M. E., 213, 233, 234, 254, 275

MacGinitie, G. F., 8, 50 McGinnis, A. J., 71, 122 Macintyre, R. J., 110, 130 MacNichol, E. F., 26, 49 Maddrell, S. H. P., 186, 198 Main, R. K., 186, 203 Mainx, F., 171, 198 Maki, T., 300,349 Makino, S., 171, 174, 198 Mallock, A., 6, 50 Marcovitch, S., 208, 221, 236, 266, 275 Markert, C. L., 109, 114, 124, 125 Martin, R. G., 157, 190 Maruyama, K., 93, 123 Mason, H. S., 96, 125 Mazoxin-Porshnyakov, G. A., 8,41,42, 50

Mead, C. G., 98, 119 Meillon, B. de, 71, 120 Meister, A., 81, 125 Mellon, A. D., 40, 52 Menzel, D. B., 110, 125 Menzer, G., 19, 50 Mergenhagen, D., 87, 127 Metzenberg, R. L., 104, 105, 125 Meves, H., 319, 349 Meyer, G. F., 16, 50 Meyer, W. L., 161, 205 Meyer-Taplick, T., 106, 125 Meyerhof, C., 135, 145, 198 Michejda, J., 155, 156, 198 Michejda, J. W., 185, 198 Micks, D., 72, 125 Milburn, N., 295, 349 Mill, P. J., 282, 293, 299, 301, 349 Miller, P. L., 280, 281, 282, 283, 285, 288, 290, 293, 295, 296, 299, 301, 302, 303, 304, 305, 307, 309, 310, 311, 313, 315, 316, 317, 325, 334, 337, 339, 340, 341, 342, 343, 345, 349, 350 Miller, W. H., 39,50 Mirsky, A. E., 188, 190 Mitchell, H. K., 70,72,82,88,96,102,103, 104, 110, 119, 120, 125, 127, 129, 130 Mitchell, P., 165, 198

Mitlin, N., 78, 125 Miura, Y.,89, 128 Moklowska, A., 99, 121 Merller, F., 109, 124 Moller, H., 323, 350 Msller, I., 100, 128, 129, 168, 202 Monod, J., 112, 122, 134, 157, 170, 181, 195

Monroe, H. A. U., 301,350 Moodie, A. F., 13, 47 Moog, F., 62, 125 Moore, S., 73, 82, 127 Moorhead, L. V., 167, 204 Mordvilko, A., 214, 275 Morgan, T. H., 219,275 Morgulis, S., 78, 119 Muller, J., 3, 50 Munyon, I. L., 98, 119 Murakami, M., 29, 51 Myers, T. B., 281, 283, 284, 288, 290, 293,294,295,296, 3 1 I , 350

N Nair, K. M., 168, 199 Nair, K. S. S., 81, 125 Naka, K., 20, 21, 24, 25, 29, 36, 41, 48, 50 Naka, K. I., 22, 24, 41, 50 Nakamoto, T., 187, 199 Nakase, Y.,112,123, 182, 197 Narahashi, R., 134, 199 Narahashi, T., 297, 350 Naylor, J. M., 173, 202 Nedergaard, S., 149, 183, 184, 194 Netter, K. F., 135, 198 Neville, A. C., 305, 315, 350 Neville, E., 80, 123 Newport, G., 301, 350 Ngai, S. H., 287, 352 Nixon, H. L., 252, 273 Noda, I., 239, 244, 250, 254, 268, 275, 276

Norman, C., 67,125 Novak, A. F., 96,125 Novak, V. J. A., 168,199 Noyes, D. T., 71,122 Nunnemacher, R. F., 16, 17.50 Nunome, Z., 301, 325, 338, 350 Nuorteva, P.,77, 125

362

AUTHOR INDEX

0 OBrien, R. D., 110, 114 O’Neill, J. J., 149, 201 Oberlander, H., 167, 199 Oelhafen, F., 56, 63, 125 Oelhafen-Gandolla, M., 107, 117 Ohnishi, E., 87, 95, 125, 166, 199 Ono, T., 134, 199 Oppenoorth, F. J., 110, 126 Orr, C. W. M., 99, 100, 101, 126 Orr, C. W. N., 168, 199 Osborne, M. P., 294, 302, 350 Ottoson, D., 33, 46

Polacek, I., 148, 199 Polis, B. D., 153, 199 Potter, V. R., 134, 152, 199, 201 Poulson, D. F., 63, 109, 126 Powning, R. B., 77, 126 Pressman, B. C., 152, 199, 200 Preston, J. B., 291, 312, 348, 351 Price, G. M., 141, 200 Pringle, J. W. S., 305, 351 Prior, R. N. B., 214, 276 Pryor, M. G. M., 73, 126, 166, 200 Pulis, J. F., 167, 204 Pulitzer, J. F., 174, 200 Pullman, M. E., 153, 200

P Packer, L., 149, 201 Pagliai, A., 217, 273 Pagliai, A. M., 216, 218, 273 Paim, U., 310,350, 351 Pant, R., 70, 78, 126 Pantelouris, E. M., 86, 111, 126 Pappenheimer, A. M., 68,116 Pardee, A. B., 157, 199 Paretsky, D., 67, 120 Parry, D. A., 5,50 Parsons, M. C., 303, 351 Paschke, J. D., 250, 276 Paspaleff, G. W., 216, 276 Passonneau, J. V., 75, 126 Patterson, D. S. P., 90, 92, 126 Paulpandian, Al., 291, 351 Pavan, C., 112,115, 171, 173,191,199 Pearse, A. G. E., 146, 195 Pelling, C., 172, 199 Pelling, G., 112, 126 Pendergrast, J. G., 300, 351 Penefsky, Z. J., 200 Pener, M. P., 66, 127 Perry, M. M., 16,40, 51 Pette, D., 134, 157, 158, 159, 160, 161, 164,191,196,199, 325,352 Phillips, J. E., 186, 199 Philpott, D. C., 16, 40,48 Pintera, A., 250, 276 Plagge, E., 168, 190 Plateau, F., 281, 351 Po-Chedley, D. S., 57, 59, 60, 61, 72, 76, 85, 126 Poggio, G. F., 20, 48

R Racker, E., 153, 200 Ramsay, J. A., 77, 126, 186, 200 Rasso, J. C., 100, 126 Ratliff, F., 39, 49, 50 Reichardt, W., 5, 11, 39, 42, 48, 50 Reinhard, A. J., 239, 268, 276 Remmert, L. F., 137, 140, 141, 150, 151, 153, 154, 156, 194, 197, 200 Retzlaff, E., 281, 283, 284, 288,290,293, 294, 295, 296,350 Richards, A. G., 300,351 Ris, H., 219, 276 Ritossa, F., 174, 180, 200 Rivnay, E., 25 1, 276 Robert, M., 188, 200 Rockstein, M., 155, 200 Roeder, K. D., 186, 200, 283, 286, 295, 349,351 Roeder, S.,283, 351 Rogers, G. L., 6, 11, 13, 47, 50 Rohr, G. V.,281, 346 Roller, H., 100, 127 Roos, K., 214, 276 Ross, K. F. A., 172, 191 Rossi, C. S.,135, 197 Rowell, C. H. F., 295, 303, 351 Ruck, P. R., 20, 23, 24, 25, 30, 32, 39, 49, 50, 51 Rudall, K. M., 73, 119 Rudkin, G. R., 172, 200 Rudkin, G. T., 173, 200 Rudney, H., 153, 195 Rushton, W. A. H., 39, 51

A U T H O R INDEX

Ruska, H., 325,346 Russell, P. B., 73, 126 Rutberg, L. D., 101, 128

S Sacktor, B., 93, 115, 135, 139, 140, 141, 143, 144, 145, 146, 148, 149, 151, 154, 156, 158,192,194,201 Saito, M., 167, 196 Sakamoto, S. S., 71, 128 Sakurai, S., 111, 129 Salmoirhagi, G. C., 287, 351 Salt, G. W., 342, 352 Sanborn, R. C., 185, 201 Sanchez, D., 16, 40, 43, 47 Sande, M. van, 84,127 Sarlet, H., 72, 127 Satija, R. C., 34, 51 Saverance, P., 1 10, 122 Schaefer, C. H., 71, 77, 127 Schaefer, C. W., 239, 250, 251, 276 Scheer, I. J., 2, 52 Schirnassek, H., 159. 293

363

Sengun, A., 172, 205 Shafiq, S. A., 280, 329, 352 Shanmugasundaram, E. R. B., 72, 75, I20 Shappirio, D. G., 68, 94, 127, 134, 160, 161, 163, 201 Sharplin, J., 301, 352 Shaw, E. I., 57, 58, 61, 127 Shaw, J., 78, 99, 114, 134, 202 Shaw, S., 19, 49 Shibata, B., 219, 220, 276 Shigematsu, H., 87, 127 Shmukler, H. W., 153, 199 Shotwell, 0. L., 82, 127 Shull, A. F., 213,232,233,235,255,276, 277 Shulov, A., 66, 67, 123, 124, 127 Shyamala, M. B., 78, 127 Siekevitz, P., 137, 202 Simmons, J. R., 82, 88, 104, 125, 127 Singh, 1. P., 98, 119 Sirlin, J. L., 112, 127, 167, 173, 193, 202

Schindler, F. J., 164, 195

Sissakian, N.M., 83, 127

Schmialek, P., 167, 196, 201 Schmid, W., 106,127 Schmidt, E. L., 170, 201 Schmitt, J. B., 301, 351 Schneider, G., 7, 51 Schneider, W. C., 152, 199, 201 Schneider-Orelli, O., 214, 276 Schneiderman, H. A., 54, 67, 92, 94, 120,127, 161, 162, 164, 167, 168, 169, 171, 182, 194, 196, 197, 199, 201, 204, 260, 276, 299, 303, 307, 308, 310, 316, 345,349, 351 Scholes, J. H., 19, 21, 49, 51 Schollmeyer, P., 135, 160, 164, 196, 201 Schoonhoven, L. M., 320,351 Schreuder, J. E., 283, 285, 291,295,307, 351 Schvegraf, A., 135, 198 Schwartz, H., 216, 219, 237, 276 Schweiger, A., 96, 122, 165, 196 Scott, J., 88, 121 Sears, D. F., 31 1,352 Seidel, F., 53, 64, 65, 127 Sekeris, C. E., 87, 127, 165, 196, 201 Sekhon. S. S.. 246, 277

Skinner, D. M., 92, 127 Slama, K., 168, 199 Slater, E. C., 134, 139, 140, 141, 142, 146, 148, 151, 154, 155, 156, 165, 198, 202, 203 Slifer, E. H., 246, 277 Slingerland, M. V., 208, 239, 277 Smith, D. S., 325, 329, 331, 332, 339, 340,352 Smith, J. N., 78, I19 Smith, K. D., 110,127 Smith, L. M., 250, 268, 277 Smith, M. J. H., 70, 121 Snodgrass, R. E., 302, 352 Spackman, D. H., 73, 82,127 Spiegelman, S., 173, 195 Spyrides, G. J., 187, 199 Sridhara, S., 65, 95, 127 Stahler, N., 77, 128 Stahn, I., 295, 307, 352 Stegwee, D., 67, 128, 146, 168, 202, 204 Stein, W. H., 73, 82, 127 Steinhauer, A. L., 85, 87, 128 Stent, G. S., 161, 202 Stephen, W. P., 85, 87, 128

364

AUTHOR INDEX

Stephenson, M., 88, I21 Stephenson, M. L., 88, I31 Stevenson, E., 92, 128 Stich, H. F., 173, 202 Stieve, H., 24, 51 Stobbart, R. H., 134, 202 Stockhammer, K., 19, 50 Strangways-Dixon, J., 100, 101, 128 Strauss-Durckheim, H . E. G., 323,352 Streck, D., 11, 51 Stride, G. O., 336, 352 Strong, F. E., 71, 76, 84, 128 Stroyan, H. J . G., 209, 257, 274 Stroyan, H. L. G., 211, 214, 276, 277 Stumm-Zollinger, E., 73, 82, 103, 121, 128 Stumpf, H., 18, 46 Sulkowski, E., 89, 121 Sweeney, E. A., 98, I19 Swirski, E., 209, 273 Sytinsky, I. A., 70, 129 Szafranski, P., 89, 121

T Taber, S., 96, 125 Takeyama, S., 89, 128 Talley, E. A., 84, 130 Tannreuther, G. W., 208, 277 Tauc, L., 285, 291, 348 Tawfik, M. F . S.,62, 63, 64, 128 Telfer, W. H., 62, 85, 87, 92, 128, 182, 202 Terzian, L. A., 77, 122, 128 Thiers, R. E., 185, 198 Thomsen, E., 54, 100, 128, 129, 168, 202 Thorpe, W. H., 338, 352 Thorson, J., 9, 51 Todd, A. R., 73, 126 Tomita, T., 21, 29, 39, 51 Tonapi, G. T., 300, 31 1, 352 Trager, W., 171, 202 Traub, A., 150, 194 Treherne, J . E., 77, 129, 282, 299, 346, 352 Tscharntke, H., 29, 46 Tsujita, M., 111, 129 Tunstall, J., 19, 49 Tyshchenko, V. P., 70, I29

U Uichanko, L. B., 208,277 Umbarger, H . E., 157, 202 Urbani, E., 95, 129 Urich, K., 81, 129 Ursprung, H., 109, 110,125, 127 Usherwood, P. N . R., 320, 352 Ussing, H. H., 189, 203 V Van den Bergh, S. G., 134, 138, 139, 140, 141, 142, 146, 148, 151, 154, 155, 156, 165, 203 Vanderberg, J., 112, 123 Vanderberg, J. P., 168, 203 Vanderzant, E. S.,97, 129 Vasil'ova, N. V., 88, 118 Velthuis, H. H . W., 110, 129 Vereshtchagin, S. M., 70, 129 Verly, W. G., 92, 115 Vickers, D. H., 78, 125 Vigier, P., 44, 45, 51 Villeneuve, J. L., 70, 77, 129 Vogell, W., 158, 159, 160, 203, 325, 352 Volkner, K. G., 319, 349 Voskresenskaya, A. K., 302,352 voss, c.,7, 49 Vowles, D. M., 45, 51, 286, 31 1, 352 Vries, H. de, 19, 51 W Wacker, W. E. C., 188, 203 Waddington, C. H., 16, 40, 51, 112, 129 Wadkins, C. L., 137, 154, 197 Wagman, I. H., 33, 46 Wagner, H. G., 26, 49 Wagner, R. P., 102, 129 Walther, J. B., 23, 36, 51 Walwick, E. R., 186, 203 Wang, S., 81, 100,129, 168,203 Wang, S. C., 287, 352 Warburg, O., 149, 164, 203 Ward, C. L., 109, 129 Washizu, Y., 11, 51 ' Watanabe, M. I., 139, 140, 203 Waterhouse, D. F., 63, 126 Waterman, T. H., 10, 11, 18, 19, 37, 38, 43, 49, 51, 52 Webb, E. C., 156,193

365

AUTHOR INDEX

Weber, G., 157, 203 Weber, U. M., 96, 125 Wecker, E., 96, 122, 166, 196 Weinmann, H. P., 88, 104, 107, 129 Weinstein, M. J., 167, 201 Weis-Fogh, T., 143, 157, 197, 203, 280, 281, 298, 311, 315, 321, 322, 323, 325, 329, 333, 334, 335, 336, 337, 338, 339, 342,345,348, 350, 353, 354 Welch, R. M., 108,129 Wellman, H., 135, 165, 197 Wendler, L., 19, 47 Wenig, K., 168, 199 White, E., 9, 52 White, W. S., 253, 256, 277 Whitten, J. M., 171, 203, 302, 353 Wiersma, C. A. G., 11, 19, 37, 38, 43, 52,298, 315,348,353 Wiesel, T. N., 29, 46 Wiesrnann, R., 214,276 Wigglesworth, V. B., 16, 52, 54, 92, 93, 94, 99, 101, 129, 130, 161, 167, 168, 170, 177, 182, 186,203,204,260, 263, 277, 280, 281, 297, 301, 303, 311, 316, 338,353 Wilde, J. de, 99, 101, 130, 168, 204, 283, 285,291, 295, 307,351 Wilhelrn, R. C., 161, 204 Wilkins, M. B., 299, 353 Williams, C. M., 54, 67, 68, 75, 85, 87, 92, 94, 101, 126, 127, 128, 130, 139, 140, 155, 160, 161, 163, 164, 165, 167, 168, 170, 177, 182, 185, 191, 194, 195, 197, 201, 203, 204, 260, 277, 299, 310, 351 Williams, G. R., 135, 136, 137, 141, 142, 143, 192 Wilska, A., 32, 52 Wilson, D. M., 282, 294, 315, 321, 353, 354

Wilson, F., 220, 221, 277, 277 Wischnevskaya, T. M., 8,42, 50 Wolf, E., 5, 7, 49, 52 Wolken, J. J., 2, 40, 52 Wojtczak, A. B., 141, 152, 204 Wojtczak, L., 141, 152, 204 Wolsky, A., 165, 166, 205 Woods, P. S., 172, 205 Woods, Ph. S., 172, 200 Wren, J. J., 82, 130 Wright, T. R. F., 110, 127, 130 Wroblewski, F., 109, I30 Wugmeister, M., 76, 124 Wulff, V. J., 20, 52 Wunderly, Ch., 104, 130 Wyatt, G. R., 70, 72, 84, 85, 92, 94, 116, 128, 130, 161, 162, 164, 183, 205 Wyatt, S. S., 72, 85, 130

Y yagi, N., 3, 14, 20,45, 52 yamarnoto, R.T., 167, 205 yao,T., 65, 95, 130 Yasuzumi, G., 16,40,52 Yeandle, s., 21, 48 Yoshitake, N., 77, 130 Young, W. J., 134, 194

Z Zacharius, R. M., 84, 130 ' Zamecnik, P., 88, 121, 131 Zawarzin, A., 40, 52, 301, 354 Zebe, E., 141, 145, 146, 148, 150, 158, 159, 191, 193, 205, 325, 352 Zerrahn, G., 15, 52 Zielinska, Z. M., 75, 131 Zwehl, V. von, 19, 46

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Subject Index A Acanthagyna, spiracle activity, 312 Acanthagyna villosa, tracheal modifications for flight, 343 Acheta, innervation of tracheae, 302 Acrididae, spiracles, 301, 303 Acyrthosiphum pisum, polymorphism clonal variability, 216 gamic female production anholocycly, 237, 238 day length, 221, 225 polymorphic forms, 21 1 sex determination, 220 wing dimorphism, crowding, 247 host plant, 251 nutrition, 250 Adaptation, effect on compound eye histological changes, 19-20 potential changes, 27-3 1 Adult, amino acid and protein metabolism reproduction, 99-102 sex-specific differences, 96-99 Aedes amino acids essential, 71 excretion, 77 methionine, 97 detoxication, 78 Aedes (pupa), respiratory enzymes, 94 Aedes aegypti, amino acids and growth, 72 Aeshna eye, and central nervous system, 33 post-retinal fibres, 40 transients, 25, 29 flight, oxygen consumption, 321 tracheal modifications, 323, 325, 339 Aeshna (larva), eye, 25, 26

Age, and energetics of mitochondria, 155 Agria afinis, amino acids, 70 Agria afinis (larva), amino acids and nutrition, 76-77 Agrotis, image formation, 14 Agrotis orthogonia (larva), amino acids, 71 Amino acids (see also Development) and proteins, metabolism during development, 53-13 1 in adult, 96-102 in blood, 62, 69, 70, 71, 73, 75, 77, 78 in embryo, 55-62 essential, 71 excretion, 77-78 in growth and moulting, 72-75 intermediary pathways, 79-82 interrelationships, 75-82 in larva, 69-82 in lethal mutants, 106 nutrition and absorption, 76-77 occurrence and significance, 69-72 osmoregulation, 78 other specific functions, 78-79 in pupa, 89-93 sex specific differences, 96-99 Amoeba, pinocytosis, 101 Anaciaeshna, spiracle activity, 3 12 Anax spiracle activity, 312 ventilation and flight, 343 Anomala orientalis, amino acid metabolism, 72,76 Anopheles, amino acid excretion, 77 Ants, effect on aphid polymorphism, 252-253 Anthereae mylitta, potassium secretion, 184- 185 Anthereae pernyi potassium secretion, 184-185 protein synthesis, 88 r.q. in flight, 148

367

368

SUBJECT I N D E X

Anthonomus grandis, amino acids, 78.97 Antigens, sex-specific differences, 99 Apanteles glomeratus, phosphatases in egg, 62 Aphids, control of polymorphism in (see Polymorphism) Aphidius platensis, and aphid polyrnorphism, 260 Aphis, oxygen consumption in flight, 321 Aphis cerasi, hormones and wing dimorphism, 258 Aphis chloris, polymorphism gamic females and day length, 221 interval timers, 266 sex determination, 220 Aphis craccivora, polymorphism control of wing dimorphism, crowding, 239,244, 246, 248, 249 developmental pathways, 255-256, 257 effect of ants, 252 hormones, 258, 260, 261, 263, 264, 265 nutrition, 251 photoperiod, 253 temperature, 253 interval timers, 270 Aphis fabae, polymorphism control of wing dimorphism crowding, 247 effect of ants, 252 hormones, 264 host plant, 251-252 developmental pathways, 27 1 gamic females, 221 gynoparae, 232 polymorphic forms, 212, 213 Aphis farinosa, gamic females, 237 Aphis forbesi, polymorphism gamic females, 221, 236 interval timers, 266 photoperiodic response, 208 Aphis gossypii, interval timers in polymorphism, 268 Aphis palmae, clonal variability, 216 Aphis rosae, clonal variability, 216 Aphis saliceti (see Aphis farinosa) Apis, vision eye, 2

Apis-cont. polarized light, 19 spacing of photoreceptors, 16 Apis mellifera, breathing in flight, 321 Aplysia, visceral ganglion, 287,288,291. 296 Aquatic insects, spiracles, 303 Arthopoda, vision excitatory and inhibitory systems, 3942 form vision, 42-45 light compass response, 43 in Limulus, 38-39 mechanism of vision, 38-45 of movement, 42 Attacus ricini, amino acids, 70, 78 Attagenus piceus, amino acids, 77 Aulacorthum circumflexurn, gamic females and anholocycly, 237 Aulacorthum solani, polymorphism polymorphic forms, 21 1 wing dimorphism, 254 Automeris io, potassium regulation, 184

B Bee flight, tracheal modifications, 338, 340 vision and corpora pedunculata, 45 diffraction images, 15 of form, 8,42 polarized light, 10 Blaberus, ventilation, 283, 288, 290, 294, 297 Blattella, amino acids, 77 Blattella germanica, amino acids, 7 1, 76 Blowfly control bioenergetics, 156 mitochondria, 141 oxidative metabolism and age, 155 Bombyx amino acids excretion, 77 in tissues, 70 D and L units in eye, 41, 42 detoxication, 78 enzymes isoenzymes, 110 proteolytic, 67

369

SUBJECT INDEX

Bombyx-cont. haemolymph carbohydrate metabolism enzymes, 87 protein synthesis, 87-88 tyrosinase, 87 peptides, 83 protein synthesis, 88, 1 13 respiratory enzymes in egg, 68 Bombyx (larva) amino acids, 75 haemolymph proteins, 85 moulting fluid, 75 Bombyx (pupa) alkaline phosphatase, 95 ecdysone, 169 tyrosinase, 95 Bombyx mori amino acids in embryo, 56 in growth and moulting, 72 brain hormone, 166-167 eccentric cells of eye, 40 enzymes in egg oxidative, 161 phosphatases, 65 eye development, 25 Breathing, regulation, 279-354 spiracles, 300-321 (see Spiracle) tracheal modifications for flight, 321343 (see Tracheae) ventilation, 280-300 (see Ventilation) Brevicoryne brassicae, polymorphism clonal variability, 216, 217, 218 forms and terminology, 21 1 gamic females, 221, 23 1, 236, 238 interval timers, 266 sex determination, 220 wing dimorphism crowding, 239, 243, 246 intrinsic factors, 254 nutrition, 250 photoperiod, 253 Butterfly oxygen consumption in flight, 321 vision, 15-16 Byrsotria, ventilation, 288, 290, 293, 294, 295 Byrsotria fumigata, ventilation, 283

C CalIiphora chromosomes giant, 171 puffing, 174 eye eccentric cells, 40, 43 illumination potential, 36 image formation, 16 polarized light, 18-19 post-retinal fibres, 40, 41 potential profile, 26 resolving power, 7, 1 I , 14, 15 rhabdomere, 11 transients, 24, 25, 29 wavelength discrimination, 40-41 and ventral nerve cord, 33 haemolymph proteins, 101 Calliphora (larva) tanning of cuticle, 73 tissue proteins, 92 Calliphora (pupa) amino acids, 89-90 proteases, 94 respiratory enzymes, 93 Calliphora augur, amino acids and growth, 72 Calliphora erythrocephala amino acids and growth, 72 ecdysone, 165, 168, 169 Carbon dioxide and hypoxia effect on spiracles control of activity, 303-304, 305311, 317-321 mechanism of stimulation, 31 1 effect on ventilation, 294-297 electrical activity, 288. periodic ventilation, 290-29 1 pumping, 281 receptors, 285 role of abdominal ganglion, 283-293 Celerio euphorbiae, proteins, 99 Cerataphis lataniae (see Aphis palmae) Chironomus, chromosome puffs and transport enzymes, 183 Chironomus(larva), giant chromosomes, 171, 172 Chironomus tentans chromosome puffing

370

SUBJECT I N D E X

Chironomus tentans-cont. and development, 174-178 and synthetic processes, 181 giant chromosomes, 172, 173, 174 Chironomustentans (larva), chromosome puffs and ecdysone, 112 Chironomus thummi chromosome puffing and development, 178-180 and Na+ and K+, 188-189 and Zn++, 113 Chironomuspallidivattatus, chromosome puffing and synthetic processes, 181 Chlorophanus, vision, 7, 9, 43-44 Choline, effect on transients in eye, 24 Chorthippus, electrical activity of eye, 29,33 Chortophaga, amino acids in egg, 61 Chortophaga viridifasciata, amino acids in embryo, 58 Chromosomes giant, biochemistry, 171-174 hormones, biochemistry brain hormone, 166-167 ecdysone, 168-171 juvenile hormone, 167-168 metabolic control at level of, 166183 puffing and development, 174-181 (see also Development) and Na+ and K+, 188-189 and synthetic processes, 112, 181182 and transport enzymes, 188, 189 Cimex, haemoglobin in egg, 101 Cockroach effect of crowding, 247 energy reserve, 78 flight oxidative metabolism, 146, 155 r.q., 148 tracheal modifications, 155 pumping, 281 spiracles, innervation, 302 ventilation, 283, 285, 288, 290, 291, 296, 298 Colour vision, 5 , 40

Compound eye evolution, 2-4 and optic lobe, electrical responses, 20-38 adaptation, 27-3 1 in locust, 33-38 nature of, 20-26 off-response, independent origin of, 31-32 potential profile, 26-27 threshold changes, 32-33 optics of, 10-20 adaptation, 19-20 diffraction images, 11-17 erect image in Lampyris, 16-17 image formation, 10-15 movement detection, 18 polarized light, 10, 18-19 spacing of photoreceptors, 16 theories of vision, 42-45 visual abilities, 5-10 and form, 8-9 and intensity, 5-6 and movement, 9 and polarized light, 10 and resolving power, 6-8 visual mechanism, 38-45 excitation and inhibition, 39-42 and form, 42-45 in Limulus, 38-39 and movement, 42 Cones, 3, 4, 14, 17, 20 Corethra pulmicornis, amino acids and growth, 72 Corcyra cephalonica, amino acids and growth, 72 Corcyra cephalonica (larva), amino acids, 75 Corpora allata and aphid photoperiodism, 236 and aphid wing dimorphism, 257, 264-265 and nutrition, 100 and RNA, 100 and transaminases, 81, 100 Crayfish, control of swimmerets, 298 Cricket, ventilation, 293, 295, 297 Crowding, and aphid polymorphism effect on gamic females, 237

SUBJECT INDEX

Crowding-cont. effect on wing dimorphism, 239-249, 269-270 analysis, 239-241 mechanism, 244-249 sensitive stages, 242-244 Crustacea and relevance to insect breathing, 287, 293, 307, 312, 315, 319-320 vision, 7, 11, 40 Cryptocercus punctulatus, ecdysone, 170 Ctenicera destructor (larva), amino acids, 71 Culex amino acids excretion, 77 sex-specific differences, 96 peptides in egg, 61 pole cells, 63 Culex (larva), haemolymph protein, 85 Culex (pupa), amino acids, 90 Culex fatigans, sex-specific differences in amino acids, 97 Culex pipiens, sex-specific differences in amino acids, 97 Culex pipiens (larva), amino acids, 75 Culex pipiens var. fatigans, amino acids in embryo, 56 Culex pipiens var. molestus, amino acids in embryo, 56 Culex quinquefasciata, amino acids and growth, 72 Cuticle elasticity and ventilation, 298-300 and eye in Lampyris, 17 larval, 73, 75 resilin and, 299

D D-units in eye, 37, 38, 41 Damselfly tracheal modifications for flight, 326 transients in eye, 25 Dendolimus pini, amino acids, 70 Development and amino acid and protein metabolism adult

371

Development-cont. proteins and reproduction, 99- 102 sex-specific differences, 96-99 embryo enzymes, 62-69 free amino acids, 55-62 genetic aspects enzymes, 109-1 11 gene regulation, 112-113 lethal mutants, 102-109 larva amino acids, 69-82 amino acid derivatives, 82-84 haemolymph proteins, 84-89 and chromosome puffing induction by ecdysone, 175-176 induction by hormone imitators, 178-180 induction by uncoupling agents, 180-181 inhibition of RNA or protein synthesis, 177-178 larval v. pupal moult, 176-177 tissue, and stage specificity, 174175 Detoxication, and amino acids, 78 Dissosteira, spacing of photoreceptors, 16 Dixippus breathing, 281, 295 corpora allata and RNA, 100 protein metabolism, 100 transients in eye, 25 Dixippus morosus, amino acid excretion, 77 Dolycoris baccarum, amino acid absorption, 77 Dosiostaurus morocanus, proteases in egg, 66 Dragonfly flight muscles, 305 flight and tracheal modifications, 322, 323, 325, 334, 335, 339-340, 341-342, 343 spiracles activity, 303, 312 control mechanisms, 307-308, 309 innervation, 301 ventilation, 281, 282, 283

372

SUBJECT INDEX

Dragonfly-conf. vision, 24, 30 Dragonfly (larva) innervation of spiracles, 302 ventilation, 293 Dragonfly (nymph), ventilation, 282, 285. 299 Drepanosiphum platanoides, aestivation, 236 Drosophila amino acids in embryo, 59 essential, 71 chromosomes giant, 171, 172 puffs, 174, 176, 179 crowding, 247 ecdysone and RNA, 167 eye eccentric cells, 40 spacing of photoreceptors, 16 flight oxygen consumption, 321 r.q., 148 tracheal modifications, 322, 338 genes and enzyme synthesis, 109-111, 113 lethal mutants, 109 peptides in egg, 61 phosphatases, 65 pole cells, 63 protein synthesis, 88 sex-specific differences in amino acids, 97 in antigens, 99 in peptides, 98 tyrosinase, 87 Drosophila (larva) amino acids and nutrition, 76 chromosome puffs, 112 haemolymph proteins, 85, 86 peptides, 82, 84 transamination reactions, 80 Drosophila (pupa) alkaline phosphatase, 95 respiratory enzymes, 93 tyrosinase, 95 Drosophila buskii chromosome puffs, 180

Drosophila buskii-cont. giant chromosomes, 174 Drosophila melanogaster amino acids in embryo, 56 in growth and moulting, 72-75 chromosome puffing, 180 isoenzymes, 109-110 lethal mutants, protein metabolism 102-109 lethal giant larvae, 107-108 lethal meander, 106-107 lethal translucida, 103-106 others, 108-109 tyrosinase, in ebony mutants, 165-166 tyrosine-0-phosphate, 70 Drosophila simulans, isoenzymes, 110 Drosophila virilis giant chromosomes, 171 isoenzymes, 110 Dysaphis devecta, gamic females and anholocycly, 237 Dytiscus spiracles, 301 transients in eye, 25 ventilation, 283, 288, 294

E Ecdysone biochemistry, 168-171 and chromosome puffs, 175-180 and DNA synthesis, 182 and gene activation, 112-113 and hepatic protein, 54 and RNA in pupa, 92-93 and tanning, 165 Embryo, amino acid and protein metabolism amino acids, changes in, 55-62 enzyme patterns phosphatases, 62-66 proteases, 66-67 respiratory enzymes, 67-69 Embryogenesis, and amino acids, 5657

Enzymes cytochrome enzymes, 163 genetic control, 109-111

373

SUBJECT INDEX

Enzymes-cont. in haemolymph, 87 in mitochondria, 159-160 multilocated, I60 phosphatases in embryo, 62-66 in pupa, 95 phosphotriose glyceratephosphate group, 158-159 proteases in embryo, 66-67 in pupa, 94-95 in pupal development, 93-96 pyridine nucleotides, 160 regulation of levels, 156-165 respiratory in embryo, 67-69 in pupa, 93-94 in silkworm development, 161-165 transport enzymes, 182-183 tyrosinase, 95-96, 165-166 Ephestia, protein synthesis, 113 Ephestia (larva) amino acids, 75 peptides, 83 Ephestia (pupa), amino acids, 90 Ephestia kiihniella, amino acids and growth, 72 Erebia, image formation, 14 Eristulis, illumination potential, 36 Eristalis tenax, image formation, 14 Escherichia coli, potassium in, 187 Euglena gracilis, zinc deficiency, 188 Eupagurus, retinal action potential, 24 Excretion, and amino acids, 77-78 Eye aggregate, in Julus, 3 apposition, 4, 14 compound (see Compound eye) diurnal, 14, 33 fast, 25 holochroal, 2 nocturnal, 14, 33 oxygen consumption, 29 schizochroal, 2 simple, 2, 3, 4, 38 slow, 25 visual pigment, 11, 19-20, 32-33, 23023 1

F Flea, breathing, 296, 300, 303, 311 Flight amino acids during, 79 energy requirements, 143-144 modifications of tracheae (see Tracheae) muscle compared with jumping muscle, 145 energy trapping pathways, 144-149 fly and cockroach compared, 155 oxidation in, 145-148 respiratory control, 154 and ventilation, similarities, 294 Fly oxidation rates, 146 sarcosomes, 139 spiracle control, 305, 309, 3 1 1 Formica rufa, vision, 7-8 Frog, carbon dioxide and hyperpolarization, 319

G Galleria rnellonella amino acids and growth, 72 haemolymph protein, 85 uncoupling agents, 152 Galleria (larva) corpora allata and proteins, 100 haemolymph proteins, 85 Gamic females, production in aphids aestivation, 235-236 anholocycly, 237-238 heteroecious species, 232-233 hormones, 231 intrinsic factors, 237-238 in Macrosiphum euphorbiae, 233-235 other environmental factors, 236-237 photoperiodic receptors, 227-23 1 photoperiodic sensitivity, 222-226 response curves, 226-227 sexual reproduction, 233-235 temperature, 23 1-232 Ganglion abdominal, third embryonic as pacemaker in ventilation, 283284

374

SUBJECT INDEX

Ganglion-cont. metathoracic and spiracle activity, 313-316 and ventilatory rhythms, 283, 285, 289, 293, 297 Gustrophilus, phosphatase in haemolymph, 87 Genes and protein metabolism in development, 102-113 and chromosome puffs, 1 12 and enzyme synthesis, 109-111, 112 and hormones, 112-113 mutation (see Mutants) and ontogenetic phases, 102-103 regulation of activity, 112-113 Geometrical interference, in eye, 43-44 Geotrupes, tracheal modifications for flight, 335, 337 Germ-cell determinant, 62-63 Glossina rnorsituns, proline in muscle, 79 Glutamic acid, interconversion with glutamine, 59, 60, 91 Glyphinu schrankiunu, polymorphism, 214 Glyptotendipes, giant chromosomes, 173 Grasshopper, jumping muscle, 145 Growth and moulting, amino acids, 7275 Gryllus, innervation of tracheae, 302 Gryllus (pupa), ecdysone and RNA, 92-93

H Hubrobrucon, protein synthesis, 113 Hudrothernis, spiracles, 303,3 12 Hudrothernis defectu, trachea! modifications for flight, 343 Haemolymph amino acids in, 62, 69, 70, 73, 75, 77, 78 ammonia in, 73 enzymes in, 87 and flight muscles, 338 in lethal mutants, 103-4, 108 proteins in genetic control of synthesis, 111 function, 87 and nutrition, 100

Haemolymph-cont. ontogenetic patterns, 85-87 synthesis, 111, 87-89 total content, 85 in pupa, 92 sex-specific differences, 96 and spiracle activity, 3 11, 3 17 transaminase activities, 80 and yolk, 101-102 Harpalus aeneus, trachea and flight, 33 1 Hawkmoth, tracheae and flight, 335 Heliaeshna, tracheae and flight, 343 Heliocopris colossus, tracheae and flight, 335, 336 Honeybee amino acids, 96 flight muscles, 138 pumping, 282 vision electrical responses, 20, 23, 24 movement perception, 9 post-retinal fibres, 40 resolution, 7 Hormones brain hormone, 166-167 ecdysone (see Fxdysone) and ionic regulation, 186 juvenile hormone (see Juvenile hormone) mode of action, 54 and sex determination in aphids, 221 and wing dimorphism in aphids, 257265 Housefly oxidative metabolism, 155 respiratory control, 150, 165 sarcosomes, 141, 145, 146, 148 Hunting wasps (see Wasps) Hyulophoru ecdysone and DNA, 182 and mitochondria, 94 haemolymph proteins in egg, 101 isoenzymes, 110 moulting fluid, 75 spiracles and tracheae, 303, 305, 309 Hyulophora (larva) haemolymph proteins, 85 spiracles, 301

375

SUBJECT I N D E X

Hyafophora (pupa) amino acids, 92 cytochromes, 94 protein synthesis, 92 RNA, 92 spiracles control, 308-309 independent activity, 3 17, 3 19-321 ventilation, 299-300 Hyalophora cecropia alkali metal ions, 185 amino acids in embryo, 62 bioenergetics, 156 chromosome puffs, 187 ecdysone, 170 enzymes, 160, 161, 163-165 eye, 36 hormones effect on aphids, 260-261 juvenile, 167-168 K+ regulation, 183-184 Hyafophora cecropia (larva) haemolymph proteins, 87 potassium secretion, 185 Hyalophora cecropia (pupa) protein synthesis, 92 respiratory enzymes, 67 Hyafophora Cynthia, alkali metal ions, 185 Hyalophora Cynthia (larva), haemolymph protein, 87

I Ictinogomphus ferox control mechanisms of spiracle, 307308, 309, 310 synchronized activity of spiracles, 3 12 Images, in compound eye, 1-52 (see also Compound eye) diffraction images, 11-16, 38, 42 erect image in Lymantria, 3 first, 14, 15 formation, 10-15 in Lampyris, 16-17 movement over photoreceptors, 18 overlapping, 11 size, 11 spacing of photoreceptors in relation to,

16

Imaging systems in compound eye, 6 Interneurones, " command ", 286 and central nervous co-ordination, 291-293, 297-298 Interval timers, in aphid polymorphism, 220 Ions concentration and protein synthesis, 186-189 control of protein synthesis and development 183-189 (see Protein synthesis) of host plant and aphid polymorphism, 251-252 regulation by a hormone, 186 Isea (larva), eye, 3 Isoenzymes, 109-111

J Julus, eye, 3 Juvenile hormone and aphid polymorphism, 231, 260264 biochemistry, 167-168 and chromosome puffing, 177 and polyteny, 166

K Kappa activity in Paramecium, 266

L L-units in compound eye, 37,41,42 Lachniella costata, gamic females and anholocycly, 237 Larva, amino acid and protein metabolism amino acids in growth and moulting, 72-75 metabolic interrelationships, 75-82 occurrence and significance, 69-72 haemolymph proteins function, 87 ontogenetic patterns, 85-87 synthesis, 87-89 total content, 85 peptides and other amino acid derivatives, 82-84 Lens, resolving power, 6 Lentula, pumping, 28 1

376

SUBJECT INDEX

Lepisma, eye, 3 Leptinotarsa, relative oxidation rates, 146 Libellula, wavelength discrimination, 41 Lichnanthe rathvoni, haemolymph protein patterns, 85 Limnogeton, tracheal modifications for flight, 323 Limulus vision eccentric cells, 39, 40, 41 image formation, 11 light acceptance, 11 mechanism, 38-39 partial depolarization, 26 polarized light, 18 spike discharges, 20, 23 theory of vision, 42 transients, 24-25, 31, 32 Lipaphis erysimi (see Rhopalosiphum pseudobrassicae) Lithobius, eye, 2 Locust amino acids in egg, 58 diffraction images in eye, 15 electrical responses of eye adaptation, 27-29 eccentric cells, 41 fast transients, 25, 31 hyperpolarization, 3 1 optic pathway, 33-38 oxygen consumption, 29 partial depolarization, 25 visual threshold, 32 enzymes, 158-159 flight motor units, 298 muscles, 315-316 tracheal modifications, 321-322, 323, 329, 334-335, 336, 342 mitochondria, 141 oxidation rates, 146 post-retinal fibres, 40 resolving power of eye, 8 respiratory control, 150 spiracles activity, independent, 317, 318, 319 activity, synchronized, 313, 315 control, 305, 307, 309, 311 innervation, 301

Locust-cont. ventilation, 283, 285, 286, 288, 290 291, 293, 295, 296, 297, 298 Locusta detoxication, 78 eye illumination potential, 36 image formation, 16 ommatidium, 10 polarized light, 19 post-retinal fibres, 41 potential profile, 26 resolution, 6, 11, 13, 14, 15 transients, 24-25, 28-29 and ventral nerve cord, 33 Locusta migratoria, respiratory control, 150 Locusf a migratoria migratorioides, endopeptidases in egg, 66 Lucilia illumination potential, 36 isolation of mitochondria, 141 oxygen consumption in flight, 321 respiratory control in flight, 154 transients in eye, 24 Lucilia cuprina, respiratory control, 150 Lymantria (larva), erect image in eye, 3

M Macrosiphoniella sanborni wing dimorphism, 253, 256 Macrosiphum euphorbiae,polymorphism effect of crowding, 253 forms and terminology, 213 gamic female production effect of day length, 221 gynoparae, 232 sexual reproduction, 233-235 wing dimorphism, developmental pathways, 255, 256 Macrosiphum rosae (see Aphis rosae) Macrosiphum solanifolii (see Macrosiphum euphorbiae) Macrotermes, tracheal system, 280 Macrothylacea rubi, amino acids and growth, 72 Magnesium, effect on respiratorycontrol, 142-143

S U B J E C T INDEX

Malpighian tubules and alkaline phosphatase, 65 and blood amino acids, 77 Mammals, spike initiation in eye, 23 Meal worm, active principle in excreta, 167 Megoura viciae, polymorphism clonal variability, 217, 218 developmental pathways, 27 1 fundatrix, 214, 215 gamic females, production anholocycly, 238 day length, 221, 222-225 hormones and photoperiodism, 23 1 other environmental factors, 236 oviparae, 235 photoperiodic receptors, 227 photoperiodic response curves, 226227 temperature and photoperiodism, 23 1-232 interval timers, 266-268, 269, 270 polymorphic forms, 21 1, 212 sex determination, 219, 220 wing dimorphism developmental pathways, 257 effect of crowding, 239-249,256,269 hormones, 257-258, 260-263, 264, 265 intrinsic factors, 254 nutrition, 250 photoperiod, 253 ' temperature, 253 Melanitis, eye, 3 Melanoplus cytochrome oxidase in egg, 67 non-protein-SH in embryo, 67 Melanoplus differentialis alkaline phosphatase, 65 S-containing amino acids in embryo, 61 Mellinus arvensis, recognition of form, 7-8 Melolontha, tracheal modifications for flight, 323 Metabolism of amino acids and proteins during development, 53-13 1 (see Development and Amino acids)

377

Metabolism-cont. control mechanisms, 133-205 control at chromosomal level 166183 (see Chromosomes) control of respiration in mitochondria 134-156 (see Respiration) ionic control of protein synthesis and development 183-189 (see Protein synthesis) regulation of enzyme levels, 156-166 (see Enzymes) Metapolophium dirhodum, sex determination, 220, 221 Mitochondria enzymes, 159-160 phosphate acceptor and substrate control of respiration in, 134-156 (see Respiration) Mindarus abietinus, gamic females and anholocycly, 237 Mosquito, respiratory control, 150 Mosquito (larva), ventilation, 300 Moth image formation, 14 . oxygen consumption in flight, 321 visual threshold, 33 Mullerian mosaic system, 4 Musca eye, 2, 5 isoenzymes, 110 isolation of mitochondria, 141 oxidation during flight, 146 proteases in metamorphosis, 95 respiratory control in flight, 154 respiratory enzymes in metamorphosis, 93 tyrosinase in haemolymph, 87 Musca domestica endopeptidases in egg, 67 respiratory control, 150-151 Mutants of Drosophila, protein metabolism enzymes, 109 haemolymph, 103-104, 108 lethal giant larvae, 107-108 lethal meander, 106-107 lethal translucida, 103-106 muscles, 108 nucleic acid, 104ff

378

SUBJECT INDEX

Mutants-cont. nutrition, 106-107 sex peptide, 98 tyrosinose in ebony mutants, 165-166 Myzocallis coryli, wing dimorphism, 254 Myzocallis kuricola, sex determination, 219 Myzus, peptides, 84 Myzus ascalonicus, gamic females and anholocycly, 237 Myzus cerasi, hormones and wing dimorphism, 264 Myzus persicae amino acids, 71, 76 polymorphism clonal variability, 216, 217 forms and terminology, 212, 213214 gamic females, 221, 231, 236, 237, 238 interval timers, 270 sex determination, 220 wing dimorphism, 244, 250, 254

N Neodiprion pratti (larva), amino acids, 71, 77 Notonecta, histological changes in eye, 20 Nucleic acids, metabolism in lethal mutants, 104ff Nutrition and amino acids, 76-77 in lethal mutants, 106-107 and reproduction, 99-100

0 Ocypode, perception of movement, 9 Odonata (larva), resolving power of eye, 7 Odonestis pruni, r.q. in flight, 148 Ommatidium angle of light acceptance, 10-1I angles between, 4 angular separation, 43 diffraction images, 13 electrical activity, 23-25, 28-29, 31, 32 and excitation and inhibition, 40

Ommatidium-cont, length, 3 in Limulus, 38-39, 42 and the nervous system, 45 potential profile, 26 and retinula cell, 4 and theories of form vision, 42-45 visual field area, 37 Ophonus pubescens, tracheal modifications for flight, 332 Optic lobe, and compound eye, electrical responses, 20-38 adaptation, 27-3 1 nature, 20-26 off-response, 31-32 potential profile, 26-27 single-unit responses, 37 spike discharges (see Spike discharges) threshold changes, 32-33 and time relations of impulse transmission, 36 Optic pathway, in locust, 33-38 Orthosoma brunneum, control of spiracle activity, 309 Oryctes tracheal modifications for flight, 335, 337 ventilation, 298 Osmoregulation and amino acids, 78

P Pacemakers and central co-ordination, 291-293 in crustacean heart, 293 metathoracic ganglion as, 283, 285, 289 and proprioceptive input, 294 and spiracle activity, 3 13, 3 19-320 and stimulation of CNS, 297-298 third embryonic abdominal ganglion as, 283-284 Pantula, activity of spiracles, 312, 313 Paramecium, kappa activity, 266 Pediculus, haemoglobin in egg, 101 Pelariidae, spiracles, 300 Pemphigus bursarius, polymorphism, 211, 212, 237 Peptidases, definitions, 66

SUBJECT I N D E X

Peptides and amino acid derivatives in larva, 82-84 in egg, 61-62 and isoenzyme formation, 110 in larval development, 82-83 in pupal development, 92 sex-specific differences, 98 Periphyllus testudinatus, gamic females and aestivation, 235-236 Periplaneta breathing regulation independent activity of spiracles, 317 innervation of spiracles, 301 innervation of tracheae, 302 spiracular activity, 303 synchronized activity of spiracles, 315 ventilation, 283, 295, 299 chromosome puffing, 182 lymph proteins and moulting, 87 transamination, 8 1 vision illumination potential, 36 potential profile, 26 transients, 25 ventral nerve cord, 33 Periplaneta americana detoxication, 78 periodic ventilation, 291 respiratory control, 149 Periplaneta (larva), and wing dimorphism in Megoura viciae, 247 Petrobius, eye, 3 Petrognatha gigas, tracheal modifications for flight, 335-336, 337 Phalera bucephala, amino acid changes in growth, 72 Phormia blood proteins in egg, 101 giant chromosomes, 17 1 image formation, 14 potential profile in eye, 26 proteins and nutrition, 99 Phormia (larva), innervation of tracheae, 302-303 Phormia regina, amino acids and protein, 71, 93

379

Phosphate acceptor and substrate control of respiration, 134-156 (see Respiration) Photinus, erect image in eye, 16, 17 Photuris pennsylvanica, tracheal modifications for flight, 327 Photoreceptors (see also Compound eye and Eye) characteristics, 2 requirements for stimulation, 5 Pieris brassicae, effect of crowding, 247 Pieris brassicae (larva), and wing dimorphism in Megoura viciae, 247 Pigment and tanning, 59-61, 73, 95-96 visual, 1 1 , 19-20, 32-33, 230-231 Plutella maculipennis, eye, 3 Podophthalmus eye, 37 perception of stationary objects, 43 and polarized light, 19 Polarized light, detection in compound eye, 10, 18-19 Polistes, tracheal modifications for flight, 330 Polymorphism in aphids clonal variability, 216-219 control of wing dimorphism, 238-265 (see Wing dimorphism) forms and terminology, 209-214 fundatrix, 214-21 6 interval timers, 265-270 production of gamic females, 221-238 (see Gamic females) sex determination, 219-221 Polyphemus (pupa), ecdysone and DNA, 182 Popillia japonica amino acids and nutrition, 76 haemolymph protein, 85 Popillia japonica (larva), peptides, 82 Potassium active transport in Hyalophora cecropia, 183-184 andcompartmentationof ions,185-186 in Escherichia coli B mutants, 187 in labial glands of Saturniids, 184-185 and sodium, and chromosome puffing, 188-189

380

S U B J E C T INDEX

Prodenia, intermediary pathways of amino acids, 80, 81 Prodenia (larva), amino acids, 70, 77 Prodenia eridania amino acid changes in growth, 72 S-methyl cysteine, 70 Proprioceptors, role in ventilatory rhythm, 294 Protein and amino acid metabolism during development, 53-131 (see Amino acids and Development) Protoparce quinquemaculata, K+ regulation, 184 Ptinus tectus, effect of crowding, 247 Pupa amino acids and proteins, 89-93 enzymes, changes phosphateses, 95 proteases, 94-95 respiratory enzymes, 93-94 tyrosinase, 95-96

Rhodnius (pupa), ecdysone and RNA, 92-93 Rhodnius prolixus amino acids and growth, 72 ecdysone, 170 ionic regulation by hormone, 186 thyroxine, 70 Rhopalosiphon latysiphon, gamic females and anholocycly, 237 Rhopalosiphon pseudobrassicae, wing dimorphism and host plant, 251 Rhopalosiphon prunifolii, polymorphism interval timers, 268 wing dimorphism crowding, 244 intrinsic factors, 254 nutrition, 250 Rhynchosciara angelae, giant chromosomes, 171, 173 Rhythms in ventilation, 282-291

R

Sacchiphantes abietis, fundatrix, 214 Samia Cynthia ecdysone, 170 haemolymph proteins, 85 isoenzymes, 110 Samia Cynthia (pupa) brain hormone, 167 oxidative enzymes, 161 Sappaphis plantaginea, polymorphism gamic females, 221, 230, 232,238 interval timers, 270 terminology of forms, 212 Sarcosomes, isolation, 138-143 composition of incubation media, 142-143 damage, 141-142 media, 139-141 methods, 139 Sarcophaga giant chromosomes, 171 illumination potential, 36 spiracle control, 3 11 Sarcophaga (larva), innervation of tracheae, 302 Sceliphron spirifex, recognition of form, 9

Rat, visual threshold, 33 Resilin, 157, 299 Respiration, control in isolated mitochondria, 134-156 biological factors, 155-156 definitions, 134-138 energy trapping pathways in flight muscles, 144-149 during flight, 143-144 and a-glycerophosphate in flight, 154-155 and oxidative phosphorylation, 149152 sarcosomes, 138-143 uncoupling agents, 152-154 Retinene, distribution, 2 Rhodopsin, 15 Rhodnius amino acid excretion, 77 ecdysone and DNA, 182 ecdysone and mitochondria, 94 haemoglobin in egg, 101 juvenile hormone and metamorphosis, 263 Rhodnius (larva), amino acids, 75

S

38 1

S U B J E C T INDEX

Schistocerca amino acids, 70, 77 blood proteins and egg, 101 flight, oxygen consumption, 321 neurosecretory cells and blood protein, 100 peptides in egg, 61 proteolytic enzymes in embryo, 67 pumping, 281 resilin, 157 spiracles control, 304-305, 306, 308 independent activity, 317, 318 innervation, 301 synchronized activity, 3 13-316 tracheae, 302, 337 transamination reactions, 80 ventilation, 285, 292, 294-296 Schistocerca (larva), amino acids, 75 Schistocerca gregaria amino acids in embryo, 58, 62 amino acids during growth, 72 flight, energy requirements, 143 flight and tracheae, 333, 335 motion perception, 9 Scutigera, eye, 2 Sensory transmission and image formation in compound eye, 1-52 (see Compound eye) Sex, differences in amino acids, peptides and proteins, 96-99 Sexual reproduction in Macrosiphum euphorbiae, 223-235 parentage, 234-235 photoperiod, 233-234 temperature, 234 Sialis (larva), functions of amino acids, 78 Sialis lutaria, proteins and nutrition, 99 Silkmoth (pupa) spiracle control, 303, 310 ventilation, 290, 299 Silkworm, giant, oxidative enzymes in development, 161-165 Silkworm (pupa), brain hormone, 167 Smittia, giant chromosomes, 173 Sphaerodema rnolesturn alkaline phosphatase in egg, 64 amino acids in embryo, 56, 58

Sphinx (larva), spiracle innervation, 301 Sphinx ligustri (pupa), amino acids, 92 Spike discharges during spiracle activity, 313-316, 319 during ventilation, 286-291 burst formation, 287-288 periods of bursts, 288-291 repetitive firing, 286-287 during vision in optic lobe, 6, 9, 19, 33, 35-36, 38 in retinula cell, 20, 21, 24 in ventral nerve cord, 5,8,9,32,3335, 36 Spiracles, control of, 300-321 activity, 285, 303-304 chemical stimulus, nature, 3 1 1 during flight, 340-342 independent activity, 317-321 innervation of spiracles, 301-302 innervation of tracheae, 302-303 one-muscle spiracles, 305-311 synchronized activity, 31 1-317 two-muscle spiracles, 304-305 Stegobium paniceurn, crowding, 247 Stylocheiron, image formation, 14 Synchronization of spiracle activity, 31 1317

T Tanning enzymes puparium formation in Calliphora, 165 tyrosinase in ebony mutants, 165166 and tyrosine, 59-61, 73, 95-96 Tachycines, transients in eye, 25 Tenebrio amino acids in embryo, 59 corpora allata and proteinase, 100 respiratory enzymes during metamorphosis, 93 Tenebrio (larva), haemolymph protein, 85 Tenebrio (pupa) amino acids, 90 RNA: DNA ratio, 92 Tenebrio molitor, amino acids in embryo, 61

382

SUBJECT I N D E X

Tetraneura ulmi, polymorphism, 216, 219, 237 Theriouphismaculata, wing dimorphism, 250 Tineola bisselliela, amino acid excretion, 77 Toxoptera aurantii, wing dimorphism, 25 1 Tracheae, modifications for flight, 321343 gas movement by diffusion, 338-340 gas movement by ventilation, 334-338 morphology in pterothorax, 322-334 oxygen and resting flight muscles, 342-343 spiracle behaviour, 340-342 Transients in eye absence of, 25 off-transients, 24-25, 27-3 1, 38 on-transients, 23-24, 27-3 1, 38 in retinula cells, 24, 25, 26 Tribolium confusum, crowding, 247 Trilobites, eye, 2, 3 Tyrosine, and tanning, 59!61,73,95-96, 165-166 I

U Uncoupling agents, and metabolic regulation, 152-154

V Vanessa io, r.q. in flight, 148 Ventilation, control of associated endogenous activities, 286-291 co-ordination in CNS, 291-294 . cuticular elasticity, 298-300 definition, 280 effects of carbon dioxide and hypoxia, 294-297

Ventilation-cont. during flight, 334-338 proprioceptive input, 294 rhythm, 282-286 stimulation of CNS, 297-298 Ventral nerve cord, and vision, electrical activity, 5, 8, 9, 32, 33-35, 36 Vertebrate, potentials in ear, 27 Vespa crabro, oxygen and flight, 321 Vespa germanim, 338 Vision (see Compound eye and Eye)

W Waggle dance in bees, 10 Wasps hunting, vision, 8-9, 45 tracheal modifications for flight, 338 Water, effect on spiracular activity, 309 Water bugs, giant, tracheal modifications for flight, 323-325, 334, 342 Water bugs, spiracles, 300 Wing dimorphism in aphids, control crowding, 239-249 developmental pathways, 255-257 effect of ants, 252-253 endocrine control, 257-265 host plant, 251-252 intrinsic factors, 253-254 photoperiod, 253 temperature, 253

Y Yolk and blood proteins, 62,101-102 formation, 96, 101-102 utilization, 55-61, 66

Z Zinc deficiency in Euglena, 188