Advances in Insect Physiology, Volume 2

Advances in

INSECT PHYSIOLOGY

VOLUME 2

This Page Intentionally Left Blank

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 2

AN ACADEMIC PRESS REPLICA REPRINT

This is an Academic Press Replica Reprint reproduced directly from the pages of a title for which type, plates, or film no longer exist. Although not up to the standards of the original, this method of reproduction makes it possible to provide copies of books which otherwise would be out of print.

Copyright 0 1964 By Academic Press Inc. (London) Ltd. Second printing 1968 ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road. London N W 1

US.Edition published by ACADEMIC PRESS, INC.

111 Fifth Avenue, N e w York. N e w York 10003

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

Library of Congress Catalog Card Number: 63-14039

REPRODUCED PHOTOLITHO I N GREAT BRITAIN BY J. W. ARROWRMITH LTD..HRIRTOL 3 808182

9 8 7 6 5 4 3 2

Contributors to Volume 2 SVENDOLAI' ANDERSEN,Zoophysiological Laboratory B, Copenhagen University, Denmark J. W. L. BEAMENT, Department of Zoology, University of Cambridge, England DIETRICH BURSHARDT,Zoological Institute, University of Munich, Germany C. B. COTTRELL, Department of Zoology, University College of Rhodesia and Nyasalsnd, Salisbury, Southern Rhodesia K. G . DAVEY, Institute of Parasitology, McGill University, Montreal, Canada TORKEL WEIS-FOGH, Zoophysiological Laboratory B, Capenhagen University, Denmark V. B. WIGGLESWORTH, Agricultural Research Council Unit of Insect Physiology, Department of Zoology, University of Cambridge, England

V

This Page Intentionally Left Blank

Contents CONTRIBUTORS TO VOLUME2

.

V

RESILIN.A RUBBERLIKE PROTEININ ARTHROPOD CUTICLE SVEN OLAV ANDERSEN

and TORKEL WEIS-FOGH 1

I. Introduction + 11. Identification, Occurrence and Function A. Working Definition . B, Rubberlike Cuticle . C. Function of Resilin 111. Physical Properties of Resilin A. Fundamental Properties , B. Molecular Interpretation . IV. Chemical Properties of Resilin . A. Amino Acid Composition . 73. Swelling in Different Media C. Enzymic Hydrolysis D. Fluorescent Amino Acids V. General Discussion . A. What makes a Protein Rubberlike? B. How are the Networks Formed? . C. Resilin and Insect Cuticle References

.

3 3 4

17 18 20 28 33 33 36 40 41

.

.

.

. .

50 51

53 57 62

.

.

THEACTIVETRANSPORT AND PASSIVE MOVEMENT OF WATER IN

INSECTS

J. W. L. BEAMENT

I. Introduction . 11. The Active Transport of Water A. Basic Premises . B. Active Uptake from the Air . C. Active Transport of Water in the Gut D. Water and the Tracheal System

.

.

vii

.

67 69 69 72 76 78

viii

111.

IV.

V.

VI. VII.

VIII.

IX.

X.

C O N T 11 N TS

E. The Living Cuticle and Liquid Water . F. Conclusions An Interrelation between Grease and Absorbtion . A. A Model Absorbing Water . The Physical Chemistry of the Cuticle . A. The Cuticle other than Lipid Layer . B. The Hydration of Procuticle . C. Control of Procuticle by the Epidermis . The Cuticular Lipid . A. A New Interpretation of Transition in Monolayer Films . B. Monolayer Inversion . C. Conclusions The Asymmetry of Cuticle . A. Thermodynamic Validity of Asymmetry. . Electrical Properties of Cuticular Lipids A. The Electrical Inversion of a Monolayer. B. Mechanical Distortion . C. The Passage of Substances through Monolayers . On Pumps. A. The Electret Ion-pump . B. Continuous-flow Water-pumps . C. Is the Lipid the Water-valve? . Wider Implications . Summary . References.

88 91 92 94 94 95 96 97 98 101 105 107 107 111 111 112 115 115 117 118 120 122 123 124 125

COLOUR DISCRIMINATION IN INSECTS DIETRICH BURKHARDT

. I. Historical Background . 11. Terminology 111. Current State of Knowledge A. Spectral Range Visible for Insects, the Spectral Sensitivity Curves . B. The Question of the Screening Pigments. C. Visual Pigments in Insects D. Wavelength Discrimination and Central Mechanisms of Colour Vision . References.

.

131 135 137

.

137 157 158 159 169

ix

CONTENTS

INSECT ECDYSIS WITH PARTICULAR EMPHASIS ON CUTICULAR

HARDENING AND DARKENING C. B. COTTRELL

. I. Introduction 11. An Outline of Ecdysis . 111. The Hardening and Darkening of Cuticular Arcas prior to Ecdysis . . 1v. The Splitting and Shedding of the Old Cuticle . V. The Mechanism of Expansion VI. The Muscular System Involved in Ecdysis . VII. The Components of thc Sclerotizing System . A. The Tanning Agent . B. The Protein Precursor of Sclerotin . C. Enzymes Concerned in Sclerotization . VIII. The Relationship between Hardening and Darkening . IX. ‘The Control of Various Processes Associated with Ecdysis A , Hardening and Darkening . B. Air Swallowing . C. Mechanical Properties of the Cuticle . D. “Shut off” of Ecdysial Muscles E. Absorption of Fluid from Tracheae . F. Other Processes . X. Some Factors Involved in the Production of the Definitive Body Form at Ecdysis . References.

.

175 175

176 178 179 181 183 184 185 185 199

204 204 208

208 209 209

209 210 21 2

THECONTROL OF V~SCEKAL MUSCLFSIN INSECTS K. G. DAVEY

.

I. Introduction 11. The Heart. A. Muscles of the Heart . B. Pharmacology . C. Nervous Control . D. Endocrine Control . 111. The Ventral Diaphragm . IV. The Muscles of the Alimentary Canal A. General Remarks. B. The Fore-gut . C. The Mid-gut .

219

220 220 22 1

.

223 225 23 1 232 232 233 236

X

CONTENTS

D. The Hind-gut . V. The Muscles of the Malpighian Tubules VI. The Muscles of the Oviducts . VII. The Actonomic Nervous System in Insects References.

236 238 240 240 242

.

.

THEHORMONAL REGULATION OF GROWTH AND REPRODUCTI( IN INSECTS V. B. WIGCLESWORTH

I. Introduction . 11. The Neuro-endocrine System . A. Histology of the Neurosecretory Cells . B. The Role of the Neurosecretory Cells in Moulting . C. The Liberation of the Neurosecretory Product . D. Cycles of Activity in the Neurosecretory Cells. E. The Stimulus to Liberation of the Brain Hormone . F. The Action of the Brain Hormone G . The Chemical Nature of the Brain Hormone . 111. The Thoracic Gland System . A. Anatomy and Histology . B. Activation and Function of the Thoracic Glands . C. Metabolic and Cytological Effects of the Thoracic Gland Hormone . D. Moulting Hormone, Mitosis, Growth and Differentiation E. The Chemical Nature of the Moulting Hormone . IV. Hormones and Diapause . A. Endocrine Organs in Diapause . B. Physiological Changes during Chilling . C. Injury and Diapause . D. The Nature of the Diapause State . E. The Role of Hormones in the Maternal Control of Diapause . V. The Corpus Allatum and the Control of Metamorphosis A. The Corpus Allatum and Juvenile Hormone Secretion B. The Effects of the Juvenile Hormone . C. The Mode of Action of the Juvenile Hormone D. Histology and Histochemistry of the Corpus Allaturn E. The Chemical Nature of the Juvenile Hormone

.

248 248 248 249 250 25 1 252 254 256 258 258 260 263 267 270 27 1 27 1 275 277 278 279 280 280 283 286 29 1 29 1

xi

CONTENTS

VI. Hormonal Control of Reproduction . A. Corpus Allatum and Reproduction . Role of the Nervous System and Neurosecretory Cells B. in Ovarian Development . C. Control of Ovulation and Oviposition VII. Metabolic Hormones and Hormonal Integration . A. Metabolic Hormones B. Homeostasis and Hormonal Action C. Humoral Integration . References. Addenda .

296 296

. .

337 345

.

.

AUTHORINDEX . SUBJECTINDEX .

.

30 1 306 307 308 311 3 14 316 335

This Page Intentionally Left Blank

Resi lin. A Ruhlwrlike Protein in Arthropod Cuticle SVEND OLAV ANDERSEN and TORKEL WEIS-FOGH ZooplzjssiufogicafLaboratory B, Copenhagen University, Denmurk 1. Introduction . . . 11. Identification, Occurrence and Function A. Working Definition . R. Rubberlike Cuticle . C. Function of Resilin I l l . Physical Properties of Resilin . . A. Fundamental Properties . B. Molecular Interpretation . 1V. Chemical Properties of Resilin . A. Aniino Acid Composition . B. Swelling in Different Media . C . Enzymic Hydrolysis . D. Fluorescent Amino Acids . V. General L)iscussion . A. What Makes a Protein Rubberlihe? B. Holv are the Networks Formed'? C. Resilin and Insect Cuticle References .

. .

. .

. .

. . . . .

. .

.

. .

. . . . .

.

1 3 3 4

17 18

20 28 33 33 36 40 41 50 51 53 57 62

I . 1 N T K O 11u C T I O N Resilin is a structural protein whose name is derived from the Latin rcsiliw, to j u m p back. It was discovcrcd recently as a major constituent of

certain clastic hingcs and tendons in the cuticle of locusts and dragonflies (Wcis-Fogh, 1960) but, a$ we shall scc, it occurs widely and exhibits propcrtios which arc unusual and of gcneral interest to students of protcins and elastomers and to biologists. Wc shall therefore discuss the results obtained so far from many dilferent points of view andalso include somc un pu blishcd materia I. Thc wings of locusts are suspcndcd clastically from the strong thoracic box; i t was at f i n t thought that the elastic recoil of a wing which is moved away from its equilibrium position (Fig. 1A) was due exclusively to 'elastic deformations of the solid cuticle of the box, but it turned out that one-quarter to one-third of the energy is stored in the elastic hinge 1

mtitcrial itsclf. It was the cxpcrimcnts illustretcd in big. 1 which led to the discovcry of rcdin ( Weis-Fogh, 1959, 1961c, and unpublishcd). A large part of the suspending ligaments and hinges consists of a colourless

A

D FIG. 1 . The elastic recoil of the forewings in the isolated thorax of a desert locust, drawn from multiple-flashphotographs. (A) Unstrained intact thorax; (B) recoil in intact thorax; (C)recoil after removal of the wing muscles; (D) recoil of the rubberlike wing-hinges. The wings are mutilated and provided with an artificial mass (from Weis-Fogh, 196 1 c).

C

transparent protein, redin, with rubberlike properties (Weis-Fogh, 1960) and with a characteristic amino acid composition (Bailey and Weis-Fogh, 1961). In certain places it is present in pure isotropic form which is suitable for physical and chemical analyses without further purification.

RESILIN

3

In many respects it resembles elastin from vertebrates and may well be called “arthropod elastin” but, as will be shown, it differs from elastin in many important ways and lends itself more easily to studies of the rubberlike state in proteins, the number and nature of cross-links, and the formation of three-dimensional networks in biological structures. N, A N D FUNCTION 11. I D E N T I F I C A T I O OCCURRENCE Resilin is present as an insoluble gel-like component in certain patches of the cuticle of insects and crayfish. In order to identify it we must have some criteria by means of which it can be characterized and distinguished from other members of these compound structures. In view of the detailed treatment in succeeding parts of this review, we shall only give a qualitative working definition here (based mainly on Weis-Fogh, 1960; Bailey and Weis-Fogh, 1961;Andersen, 1961, 1963; Elliott et al., 1964). A . W O R K I N G DEFINITION

1. Both in situ and when dissected free, pure unstrained resilin appears as a mass of optically and mechanically isotropic protein. In the dry state it is hard and brittle and is insoluble in all solvents which do not break peptide bonds. In aqueous media and also in many anhydrous hydrophilic liquids it swells isotropically and reversibly; it then becomes rubbery and exhibits typically long-range deformability and complete elastic recovery. In water the swelling depends on pH, with minimum swclling about pH 4. It is devoid of colour, is transparent and shows no visible structure under the light microscope or electron microscope. 2. Swollen resilin becomes birefringent when strained, being positive in the direction of extension. 3. In water resilin stains with methylene blue and toluidine blue but shows no metachromasia. It stains red with the histological colour reactions of Masson and Mallory. 4. In ultraviolet light (UV-light), resilin fluoresces with a strong bluish colox with maximum intensity of about 420 mp. The intensity increases iri alkaline media. 5. Both in sirir and when free, resilin is easily digested by all proteinases. After complete acid or basic hydrolysis it yields only amino acids, of which fifteen are ordinary amino acids (but no sulphur-containing ones and no tryptophan or hydroxyproline) and two are unusual and specific. The latter are responsible for the characteristic fluorescence both of native resilin and of the hydrolyzate.

4

SVBNU

OLAV ANDERSEN A N D TORKEL WEIS-FOGH

6. So far resilin has only bccn found in specific parts of the cuticle in insects and crustacea, i t . a\ cxtracellular deposits secreted by the epidermis. In certain structures i t is secreted in a pure form but in most cases together with chitin and fibrous proteins. Wher- resilin is mixed with other substances, the bulk properties of the cuticle question may of course differ appreciably from those of the pure material, but in most cases its presence can be established by means of simple tests, such as mechanical behaviour, strain birefringence, colour reactions, swelling and fluorescence. However, it is not safe to use only one or two of these tests. B. R U B B E R L I K E C U T I C L E

Arthropod cuticle is an extracellular product of the single-layered epidermis. The thin epicuticle, the often hard and coloured exocuticle, the softer endocuticle and the flexible membranes are made up mainly of materials in the solid state of matter (chitin, lipoprotein, tanned or fibrous proteins, inorganic crystals; for reviews see Richards, 1958; Wigglesworth, i 957 ; Dennell, 1960). These products are therefore called solid cuticle, but besides water some soluble protein is often present. In contrast to this, La Greca (1947) described the thick wing-hinge ligaments in locusts as being highly elastic, transparent and without colour. An analysis of these and similar structures disclosed that in all cases the elasticity and the great deformability is due to the presence of a large amount of resilin ( Weis-Fogh, 1960) and that swollen resilin is an almost ideal rubber in the physical sense of this word (Weis-Fogh, 1961a). Cuticle which shows long-range elasticity and a large content of resilin is therefore called rubberlike cuticle (Weis-Fogh, 1960). It is easy to demonstrate that the elasticity is caused by resilin since the structures become soft and flabby when treated with proteases and loose their swelling properties, fluorescence and strain birefringence.

I . How to j t i d rubberlike cuticle: a simple colour test The reasons why rubberlike cuticle was not discovered much earlier are undoubtedly due to the smallness of the patches, their transparency, lack of colour and softness-all of which remain unchanged after treatment with heat, alcohols and most other fixatives. However, it is easy to stain resilin almost selectively in fresh cuticle and then apply the other tests for the final identification. A living insect or crayfish is killed by immersion in hot buffered water for a few minutes (pH 6.7, 95-100°C). After opening the body, the soft

TABLF I Colour reactions of locust cuticle dyed in a mikture of 5 mg toltitdine blue and 5 mg light green in 100 ml bufTer solution \ \ l 2 0 ) for 40 h and examined after 6 h washing in pure buffer solutions Type of cuticle Rubberli he : Hinges and ligaments, wing system Transitional : Clypeo-la bra1 spring Outer endocuticle of ocelli and ommatidia Inner endocuticle of hind margin of abdominal tergi tes Tough ligaments: Wing system and mandible Arthrodial membranes: Outer part Inner part Thin inner lamina Tough inextensible tendons: (hardened) Sclerotized cuticle : Exocuticle Outer endocuticle Inner endocuticle Most hairs and bristles:

pH 4.6

Colour at pH 6.1

pH 7.1

Sapphire

Deep sapphire

Deep sapphire

Sky blue Green-blue ( f m t ) Blue

Sapphire Sapphire N o or faint green-blue Faint skj blue Blue Blue

A F

-m

Green

Green

Green. tinge o f blue

Green Green (faint) No or greenish Green

Green Green (faint) Purple Green

No No Purple Green

No or faint N o or blue (faint) No or green Green or green-blue

No N o or blue (faint) Green Green or green-blue

No No or blue (f,iint) Green Green

z

6

S V L N I ) 0 I . A V A N D E K S C N A N D T O R K E I . WEIS-FOGH

parts are removed and the cuticle freed from the epidermis (hypoderm) by means of a thin, strong jet of tap water. The rinsed cuticle is placed for 2 4 4 8 h at room temperature in dilute phosphate buffer (M/20, pH about 7) to which is added 5 mg toluidine blue (British Drug Houses) and 5 mg light green (E. Gurr) per 100 ml buffer. A crystal of thymol is

FIG. 2. Thc tluorcscent rubbcrlikeligarnents in the wing systernof the desert locust, photographed in UV-light through a 420 mp interference filter (A and C ) , as compared with ordinary light ( B and 11). The upper figures show the dorsal cuticle from the outside and the lower figures from the inside.

sufticient to prevent microbial growth. After rinsing in pure buffer for some hours, typical rubberlike cuticle appears as translucent, brilliant blue patches. Table I indicates that the staining reaction of locust cuticle

K I.s i I. I N

7

no! Jcpcnd much o n p!l and that thc typical sapphirc blue colour p t i 0 7 is l.ound o n l y in piltchc\ whcrc additional tcstc 4howcd the prcscncc ,)f' Inrgc amounts of' rcsilin, i.c. in typical ruhbcrlike cuticlc

doe5 ;it

where thcrc is no cover of' tanned exocuticle but only the thin epicuticle. The term frun.\itionu/ cuficle is used only tentatively. In the case of the clypeo-labral spring, which is described later, the thickened part fluoresces strongly and complies with all thc tests for resilin, including the presence of the specific amino acids. The hind margins and the midline of the abdominal tergitcs also fluoresce, they stain blue and contain the two unusual amino acids. There is therefore no doubt about the presence of resilin in these parts. Some of the transparent endocuticle which covers the compound eyes and the ocelli stained faintly blue and showed some elasticity and the usual swelling behaviour, but since it did not fluoresce re!,ilin may not be present or its fluorescence may be quenched by other mate ria Is. Sincc the strong blue colour is contined to the patches which contain resilin, the method is reasonably selective but it should always be supplemented by additional tests. For anatomical work, it is sometimes convenient simply to use a mixture of glycerol and water I : 1 to which is added a few drops of a mcthylene blue solution (British Drug Houses) and a crystal of thymol. The rubberlike parts stain deep blue and the tough ligaments green while the rest remains unstained. The wide distribution of rubbcrlikc pads and ligaments in the wing machinery and tergites of locusts, thus made visible, corresponds in detail to the picture obtained by photographing the untreated cuticle in UV-light through a 420 mp interfercnce filter (Fig. 2). I t is characteristic that resilin is stained selectively by the two basic dyes, methylene blue and toluidine blue (coloured cations), at least from pH 3 to pH 9.5. while basic fuchsin is useless and none of the acid dyes tested staincd selectively if at all (coloured anions; acid fuchsin, eosin, light green). I t is obvims that resilin-containing cuticle is widespread in the locust but, before we discuss the occurrence of the protein in more detail, it is reasonable to describe the three test pieces upon which most of the anidytical work has been done.

2. Thrw t(i.vt piccw As far as resilin is concerned, the simplest structure in Fig. 3 is the elastic tendon of dragonflies (C) and the most complicated is the winghinge (B). It is noticeable that the structures are readily deformed and snap back immediately to their original shape when unloaded. The

x

S V I h i ) 0 l . A V A V I ) I KS1.N A N I )

I O I < K I . l . \YI.IS-l O(it1

description\ arc h s e d upon Weis-Fogh (1960). unless othcrwise stated. ;rnd i n cach C;IX ~ h prcscncc c of resilin has been established by all methods av;iiliiblc, including amino acid :inalyscs (Bailey and Wcis-Fogh, 1961 ; Anderscn, lO(x3). , Ekusric t c ~ i t k w .Most wing muscles in dragonflies run in the dorsalventral ditcction and are attached directly to thc ventral wall. Thcy insert on cuticular tcndons. which are hollow air-filled invaginations from the dorsal cuticle, the so-called cap tendons. The elastic tendon for the C

/

I,.IG.3. The three test pieccs used i n n m t experiments. The prealar arm (A) and the main winghinge of the forewing(E3)from the descrt locust (Sclrisrocercu greguriu). (C') The elastic tcntlon froin the hindwing of a dragonfly (Aeslttr CJYJMJO). All the samples were placcd in dilute buller at pti 7 and are drawn both in the unstrained and in the strained state. (From Weis-Fogh, 1960.)

pleuro-subalar muscle (the third suhalar) is no exception, but in contradistinction to the rcmaining tendons the middle part is swollen like a snusagc and extremely extensible while both ends consist of the usual tough and almost inextcnsible cuticle (see Fig. 6). Being an invagination, the air-filled central canal is lined with a thin folded epicuticle and the periphery is covered by a single layer of the epidermal cells which formed the tendon, apparently in the course of the 3 days prior to the final moult (Neville, 1963a). The tough ends are composed mainly of chitin and

K I.SI I.IN

9

prolcit) clillcrcnt from rc4lin and they rewnblc ordinary lamcllatc ;irthroclial cuticlc (I:ig. 4/11.At thc tran4itioiis bctwccn the tough and thc cl:i\lic p a r t \ . rcsilin hcpiiis t o appear i n thc form of aniorphoub tnasscs hctween rhc other clcment~(13). A I?\v microiis nearer the s\vclling. the othcr protcins disappear aiid u'c arc left w i t h ;I hell-shaped concentric system ol'chitin lamcllac scpar;itcd by and glued togcthcr w i t h isotropic

FIG.4. l'ransvcrsc p:wiitlin scctions ( 4p thich) ofthedragonfly tendon, stained with M;tsson's triplc stain i n WIII~IIrcsilin bcconies rcd (here white). ( A ) Through tough tciitlon dorsiii l o Ihc clii\tic part. (13) l'rarisitional zone in which "flakes" of resilin appear. (< 1 Anclictriny z o ~ i cwith rcsilin atid chitin lamellac. (D) Pure resilin devoid of structure. Thc cpidcrnial ccils ;irc indicated. (From Weis-Fogh, 1960.)

and structurclcss n i x m of rcsilin. This is the anchoring zone (C) and it is confined to the cnds. The major part of the swelling consists of a cylindrical piece of purc rcsilin covered towards the central canal by a thin buckled cpicuticle and by an cqually thin but badly defined cover towards the peripheral cells (D). Neither of these membranes convey any mechanical strength to the tendon, as is easily seen when resilin is removed by digestion with trypsin or othcr proteases. When the cells are stripped

Fit;. 5. Electron micrograph of a .mgitudinal section of an osmium-hxed elastic tendon. The upper part is the central canal filled with the embedding methacrylate and separated from the structureless resilin by the dark wavy epicuticle. (By courtesy of Dr. D. S. Smith.)

FIG.6. Elastic tendon from the forewing of A e . hra cyuticci, dehydrated itr .\i/ii and mounted in Canada balsam. Note the central canal. (From Weis-Fogh, 1960.) FIG.7. A fresh, stripped elastic tendon from Ae.!h u g r c t ~ d i splaced in neutral saline between crossed Nicols in the unstrained state (h = 1) and when stretched up to h = 2.48. Unstrained resilin is optically isotropic, but becomes birsfringent when deforniated, positive in the direction of extension. (From Weis-Fogh, 1960.)

12

svi.Ni) OI.AV

ANIII.RSI.N

ANI)

r o i < ~ t .W ~ ,E I S - I ( ) ( ; t i

oll, the tendon c;in thcrcfc)re bc con\idcrcd a \ ;I cylindrical piccc of purc rcsilin suspended a t either cnd by mean\ o f tough cuticle. Both i n the light and in the electron tnicroscopc (Elliott c / ol., 1964) resilin appears complctcly devoid o f structure (Fig. 5). The cpicuticle is

thin (about 200 mp)and buckled so that it docs not tighten until stretched about three times. Pieces of pure rcsilin can bc broken off. They have sharp edges, chonchoidal surfaces of fracture and fit exactly together when reassembled. Even at break there is thercforc n o sign of flow. Isolated pieces and intact tendons arc optically isotropic at all degrees of swelling but become birefringcnt when strained (Fig. 7). Since the

I

500p A

FIG.8. Frozen scction of thc wing-hinge from a forewing of the dcsert locust. The dark sclerotizcd cuticle is double hatched. the tough fibrillar part is striped, the laniellae of the rubberlikc part are indicated. and the pure pad of resilin is white. (A) Wholchinge. (B)PnI.tofthe1amellarcuticle.asindicated. (FromWeis-Fogh,1960.)

swelling is equal in all directions, pure rcsilin is also mechanically isotropic. The rubbcrlikc part of the tendon is small, 0-7mm long, 0.1 5 mm wide, 5-7 pg of rcsilin in the big Acdzna species. It is present in all dragonfly groups. Prealur arm. In grasshoppers, the anterior edge of the dorsal plate between thc forewings (the mesotergum) is suspended elastically at either side by means of two transparent, hyalinc pegs which extend abruptly from the dark and hard cuticle, the prealar arms (Fig. 3A). The tip continues as a tough flexible ligament attached to the first basalar sclerite

RESILIN

13

of the side wall. In histological transverse sections it is seen tfiat the elastic peg consists of thin concentric lamellae of chitin, less than 0.2 p thick, separated and glued together by 1-3 p thick continuous sheets of pure resilin. As to internal structure, the entire peg resembles the anchoring zone of the elastic tendon (Fig. 32). When the resilin is removed by means of enzymes or by mild acid or alkaline hydrolysis (0.1 N HC1 or NaOH at 96°C for 5-6 h), the continuous chitin lamellae remain as glossy sheets and the structure lacks rigidity in a direction normal to the sheets (indicated by the direction of pull in Fig. 3A). It remains tough however when stretched in a longitudinal direction. Thus during bending and compression, resilin then dominates the elastic behaviour while chitin does so in extension (Jensen and Weis-Fogh, 1962). Under the electron microscope the thin chitin lamellae (about 50 mp thick) are distinct while resilin shows no structure and, as in the case of the tendon, very little contrast (Elliott et al., 1964). The lamellae appear to continue into the solid, dark cuticle and therefore to anchor the peg firmly to its base at the very sharp transition between the two types of cuticle. The wing-hinge. The third structure is the main wing-hinge ligament of grasshoppers. It is more complicated than the other two both with respect to shape and structure (Figs. 3B and 8; see also Neville, 1963a). The major external part consists of a tough ventral ligament with fibrous proteins and chitin which continues dorsally into rubberlike cuticle consisting only of chitin lamellae and resilin, as in the prealar arm. In addition, the part of the ligament closest to the cells is deposited as a pure piece of resilin-the pad. It is particularly interesting that this pad and also some of the lamellar parts are laid down in the course of several days after the final moult, so that it lends itself to studies on the biogenesis of pure resilin (Weis-Fogh, 1960; Neville, 1963b). In addition to these three structures, several other examples are now known and should be mentioned briefly. 3. Miscellaneous examples The cuticles from a number of winged insects and also from a crayfish (Asracus fluviutilis) and some spiders were tested for the presence of resilin. The main results are summarized in Table 11. It is obvious that resilin and rubberlike cuticle may be found in any part of the exoskeleton of insects and presumably also of Crustacea but, according to our very scanty information, it seems to be absent in Arachnida. The different findingsneed some comment because they indicate the biological function of resilin. The main wing-hinge ligumenrs connect the pleural wing process with

TABLE 11 Colour reactions and some other tests for milin in typical rubberlike cuticle performed on rinsed samples from winged insects and from a crayfish(Astacusfiviarilis, Cmtacea, Decapoda) (The insbas were: Aeshna cyanea (Odo~ta).P e r i p k t a Mvricrma CDictyoptcra), Schirtoerm gregaria (Orthoptera), Sphinx spp. (Lepidoptem), Oryctes rhinoceros and Meblontha vdgaris (Caleoptem), Bombus spp. and Apis mellvera (Hymenoptera),Calliphoraeryrhrp cephla (Diptera). So far, we have not found any rubberlike cuticle in the spiders tested (unknown species, cbelicerata, Arachnids).)

Structure, animal

(and systematic group)

pH 4.6

Colour at pH 4 6

pH 7.1

Fluorescence

L

=b

smw3 specific andstrain -o bireacids fringence

Main wing-hinge ligaments: Aeshna cyanea

(Odonata) Perijdaneta americana (Dictyoptera) Schisrocerca gregaria (Orthoptera) Minor wing-hinge ligaments: All insects tested Elastic tendons : Aeshna cyanea (Odonata) calhphora erythrocephala Other structures: Clypeo-labral spring and abdominal tergites of Schistocerca (Orthoptera) Abdominal spring of Oryctes and Melolontha (Coieoptera) Hinge between mems and ischiurn inaphalothoracic leg of

R s C u Z - ~ ffUYiC+lU

Sky blue to sapphire! Sky blue

Sky blue to sapphire Sapphire

Sky blue to sapphire Sapphire

Sapphire

Deep sapphire

Deep sapphire

+ + +

+ + +

>

2 tl

All gradations from sky blue to deep sapphire Outer purple, inner greenish Green-blue

Outer sapphire, Outer sapphire, inner bluegreen inner sky blue Sky blue Sky blue

Sky blue

sapphire

sapphire

Sapphire

Deep sapphire

Deep sapphire

+

+

sapphire

sapphire

+

+

-

+

KESILIN

15

the wing sclerites (second axillary and tip of radial vein). In the three orders in which the plate between the wings (pterotergum) is not rigidly connected with the side walls in front and behmd (Odonata, Dictyoptera, Orthoptera), the ligaments are thick cushions of rubberlike cuticle which act as springy hinges and which take up both compressive and tensile forces. In insects with a strong sclerotized connection (Lepidoptera, some Coleoptera, Hymenoptera), these ligaments are tough, inextensible tension strands which stain green. It is possible that in certain Diptera some of them are rubberlike. When all the ligaments are of the tough type, any elastic recoil must be caused by deformation of the solid thoracic box, in contrast to the rubberlike ligaments of locusts which are known to contribute by significant amounts (see Fig. 1). Most small wing-hinge ligaments are of the tough type, but in all insects studied we have found some rubberlike ones. The elastic ligaments in grasshoppers are too numerous to be listed but their abundance may be judged from Fig. 2. They are often shaped like pads or cushions and can take up both push and pull. It is therefore of particular interest to notice their presence in Calliphora (between Subalar sclerite and second axillary, between subalar sclerite and third axillary), in Bombus (between third and fourth axillaries, and between the basalar sclerite and the humeral wing complex), and in Orvctes (between pleural process and basalar sclerite, along posterior-lateral edge of third axillary). Any interpretation of the wing mechanism must now take the existence of push ligaments into account. Elastic tendons were found in dipteran flies besides the one already described from dragonflies. In Calliphora there are two elastic and rather irregularly shaped tendons to which the “ slow” controller muscles attach (the pleuroaxillary muscle (Bonhag, 1949; No. 35a) and the tergopleural muscle (Bonhag, 1949; No. 42)). There is no doubt about the function of these tendons. In dragonflies and other primitive insects there are no muscles which can control the wing twist during the upstroke (Weis-Fogh, unpublished), but in order to hover and perform similarly refined manoeuvres this is imperative. The dragonflies have solved the problem in two ways (Neville, 1960): (a) by inserting a phasic upstroke muscle at the end of a spring system in such a way that the anterior part of the wing becomes supinated, and (b) by transforming an original downstroke muscle into a slow muscle which is then connected to the posterior part of the wing via the elastic tendon. This means that the muscle does not oscillate in length during flight but only keeps the tendon at a certain average length and tension, the movements being taken up by the spring. Similarly, the rapid

16

S V E N D O L A V A N D E K S E N A N D I ’ O R K E L WEIS-FOGH

oscillations of the diptcran wing do not permit time for a phasic control of the twisting movcmcnts but, by means of similar elastic tendons and “slow” rnusclcs, tlight can be fully controlled in spite of the fact that the main wing muscles are myogenic, i.e. their oscillations bear no dircct rclationship to the nervous impulses which activate them. The dypeo-lubrul spring of grasshoppers is a pair of rib-shaped swellings on the.ora1 aspect of clypeus and labrum. These plates cover the mouthparts anteriorly and the springs serve to keep the labrum against the mandibles when feeding and at rest. The swellings consist of lamellate rubberlike cuticle and a cross-section is seen in Fig. 9.

FIG.9. Frozen section of the clypeo-labral spring of a desert locust, photographed in UV-light to show the fluorescence.

The abdominal springs of beetles are interesting. It is well known that these insects have only expiratory muscles and that the inspiration is due to elasticity. In fact, we found that each pair of tergites and sternites are kept in the inspiratory position relative to each other by means of a pair of elastic ribs which begin as an endocuticular swelling under the exocuticle and continue across the soft arthrodial membrane and end as a similar swelling under the exocuticle of the sternites. These swellings are composed of typical rubberlike cuticle.

RESILIN

17

The elaslie leg-hingc In the crayfish is situated between merus and ischiurn, a hingc which has a flexor muscle but no extensor, so that the pad-likc ligament acts as an extensor. In this case, the resilin is present in the external layer which is flush with the tanned “exocuticie” whereas the innermost layer of the hinge is tough and flush with the calcified “endocuticle”. It is also characteristic that the resilin swells more than in any insect structure when it is placed in alkaline media: the thickness of the rubberlike part increases eight times from pH 1 to 13. Otherwise, all reactions are the same as in insect resilin. Other examples. There is no doubt that resilin will be found in many other places and there are a few additional observations in the literature. Thus Miller (1960) observed that the cuticle of the apparently fused pleura of the pterothorax of locusts dorsal to the second spiracle appears to be rubberlike and is partly responsible for the wide-opening reaction of this valve during flight. An interesting example is offered by Dr. F. S . Edwards (personal communication) who found that the precise spitting of certain predacious bugs is caused by an elastic cuticular pump, partly mzde from rubberlike cuticle. The pump resembles a gastrula in which the invaginated wall is rubberlike and supplied with a muscle which tends to pull the two blades from each other. The saliva is contained between the blades and the volume is controlled by means of the muscle. When it relaxes, the inner blade moves towards the outer and the saliva is driven out by elastic forces alone. Since resilin neither flows nor suffers any permanent deformation, it is particularly interesting that Thurm (1963) recently found that the base of each hair in the sensory hair fields of bees is highly elastic and stains with methylene blue like resilin. In fact, a material like resilin would be an absolute frame of reference for a mechanical sense organ in a way almost no other known material could. A more detailed investigation would obviously be fruitful. So far, it is not known whether resilin is present in the wingless insects (Collembola) although some staining reactions indicate its presence (Noble-Nesbitt, 1963a). We did not find any indication of it in lycosid spiders. C. F U N C T I O N O F R E S I L I N

With a few possible exceptions, resilin has been found to act as an elastic material from which the epidermal cells of arthropods “construct” mechanical springs of great deformability and perfect elastic recovery, i.e. its function is mechanical and is bound up with long-range deformations and the storage and release of mechanical energy. It seems to

18

S V E N D O L A V A N D E R S E N A N D T O R K E L WEIS-FOGH

be metabolically inert but this does not exclude the fact that simple chemical modifications may take place after deposition and thereby alter the mechanical properties slightly (Jensen and Weis-Fogh, 1962). So far, there is no direct proof of this. It is certain, however, that no major changes occur after deposition and this is probably connected with the remarkable stability of resilin compared with other structural proteins. In fact, its physical properties are so unchangeable that they make it an almost ideal object for studies on rubberlike elasticity. Resilin or related proteins may play an optical role in the transparent cuticle over the eyes and ocelli, but nothing is known about this at the time of writing. It is also possible that similar deformable networks are present as a matrix for some parts of the new cuticle when it is being secreted and before it becomes tanned after a moult (Jensen and WeisFogh, 1962), as discussed on p. 62.

RESILIN Organic polymers are often divided into two main groups according to their mechanical properties. The elastomers are rubbery elastic materials in contrast to plastomers which do not exhibit much recovery after deformation, but it should be emphasized that the same material may be rubberlike at certain temperatures and swelling, and either plastic or giasslike under other conditions. We shall now analyse resilin as an elastomer, or as a rubber in the physical sense of the word. Since Meyer et al. (1932) first offered a clear interpretation of the rubberlike state of matter in molecular terms, there has been general agreement about the qualitative description although the quantitative formulations differ as to detail (see Treloar, 1958). The chemical nature is immaterial and a typical unstrained rubber consists of a three-dimensional network of randomly kinked and randomly orientated long-chain molecules. Such a three-dimensional network is optically and mechanically isotropic in the glassy state, in the rubberlike state and at all degrees of swelling in an indifferent liquid (i.e. in a liquid which would be a good indifferent solvent for the chains if they were free). The term rubberlike refers to a specific state of the network. Below a certain temperature or in the absence of plasticizers, the interaction between the chains is so strong that they act as solid isotropic glass but when the bonds are broken or weakened in some way the material may become plastic. If, moreover, rotation is possible between the members of the chain and the interaction between them is insignificant compared with the disruptive forces due to thermal movements, a typical rubber may 111.

PHYSICAL PROPERTIES OF

RESILIN

19

result. It is characterized as follows. The network is highly and reversibly deformable. If the junction points are represented by simple physical entanglements, the strained network is likely to flow with time and to acquire a permanent deformation. If, on the other hand, the major part of the junction points consist of stable chemical cross-links between the linear chains, as in a vulcanizate, perfect elastic recovery is possible. The chains between junction points must be relatively long and possess many freely rotating links so that, in the absence of significant attractive forces, thermal agitation tends to make the chains take up a randomly kinked and constantly changing configuration, i.e. in an unstrained network the configurational entropy of the assembly of chains is maximum. The network owes its great deformability to the fact that the most probable distance between the end points of a long flexible randomly kinked chain is many times smaller than its contour length (extended length). When strained, the chains are removed from their statistically most probable configuration to which thermal agitation tends to bring them back. This means a decrease in entropy and it is the main reason why the mechanical energy used in straining the rubber can be stored as elastic energy and released again, i.e. the elastic forces depend mainly on entropy changes-in the ideal rubber only on entropy changes. In contrast to this, the elastic forces of an ordinary solid are due mainly to the straining of interatomic bonds, i.e. to changes in internal energy. Since the chains between junction points must be long and flexible, the number of cross-links per unit volume must be rather small. If there are too many the material becomes hard like ebonite and tanned cuticle, and if there are too few the material does not recover after deformation but flows to some extent. In typical rubbers theoretical as well as experimental estimates of the elastic modulus indicate a range from 5 to 30 kg cm-2 which is several orders of magnitude less than for most solid materials. Even in a good vulcanized rubber it takes time to reach the final state after loading, deformation or release and this gives rise to the phenomena of creep, stress relaxation and delayed recovery respectively. These viscoelastic properties also cause hysteresis and damping when the sample is subject to an oscillating strain or stress and they are due, in part, to a finite adjustment time which is of the order of minutes in good vulcanizates of natural rubber. Gent (1962) extended such samples to about twice their unstrained length and found that both the creep and the relaxation increased linearly with the logarithm of time, by a few per cent in 10-100 min, until a finite value was reached. In a swollen rubber, damping should also depend on the friction between the chains

S V E N D O L A V A N D E R S E N A N D TORKEL WEIS-FOGH 20 and the liquid and on the movement of liquid in and out of the rubber due to changes in the hydrostatic pressure, but nothing seems to be known about these phenomena. In any case, a good swelling agent is likely to reduce the adjustment time and therefore the damping and hysteresis but at the same time it introduces a new loss factor due to viscous resistance. Most structural proteins do not conform to this picture at all and it was therefore surprising to find that the properties of resilin are in complete agreement with it. In fact, resilin turned out to approach the ideal rubber to a higher degree than any natural or synthetic rubber known so far. Since, moreover, the quantitative theories of rubber elasticity are applicable to this protein, simple mechanical and optical observations offer quantitative information, otherwise difficultto obtain, about the molecular weight of the chains between the junction points, about their flexibility and how it alters with temperature and pH, and about the number of cross-links present. The latter point is particularly interesting because the cross-links are extremely stable and therefore of a co-valent nature, but they are different from the -S-S-bridges commonly found in proteins and must be of a hitherto unknown type (Weis-Fogh, 1960). A. F U N D A M E N T A L PROPERTIES

It is most likely that the macroscopic behaviour of resilin is shared by elastin and the two will be compared, as far as possible.

I . Deformability and stability The elastic tendon shown in Fig. 7 can be extended to almost three times its unstrained swollen length before it breaks at about 40 kg swollen unstrained area (90 kg cm-2 dry cross-sectional area). These figures are somewhat higher than those previously published (WeisFogh, 1961a). As already mentioned, the broken surfaces of the unstrained pieces fit exactly together and show that the material is unchanged even after maximum stress and this extraordinary stability is found under all conditions. In Fig. 10, for example, two pieces plus an intact tendon were placed in 70% ethanol where they were completely rubbery (A). They were then deformed and strained extensively by means of forceps while being placed in absolute ethanol where they quickly became dehydrated and hard, retaining the grotesque forms in (B). However, when returned to 70% ethanol they swelled, became rubbery within a few seconds and resumed their former properties and shapes (C). Such

21

RESILIN

experiments can be repeated indefinitely with the same pieces, also after treatment with protein coagulants and histological fixatives. In fact, no treatment short of chain rupture due to hydrolysis, strong oxidation or temperatures in excess of 140°C have been found to alter the state of swollen resilin. It is not known at which temperature water begins to freeze in the network but, as we have seen, the transition from the swollen rubbery state to the glassy state of the dehydrated material is entirely reversible, with a plastic state in between (Weis-Fogh, 1960). In the following section we are dealing only with resilin swollen in buffered water at different pH where it remains rubbery. It is not known A

6

C

FIG. 10. Samples of elastic tendons: (A) unstrained in 70% ethanol; (B) strained in absolute ethanol, and (C)after recovery in 70% ethanol. (From Weis-Fogh, 196n.)

with certainty whether elastin behaves in a similar way but it is also extremely heat resistant (Partridge et al., 1955) and only rubbery when swollen. Isolated single fibres can then be extended reversibly up to twice the unstrained length (Carton el al., 1962). 2. Elastic recovery and damping Resilin is unusual in showing elastic recovery and no sign of creep or stress relaxation after an imposed change for periods longer than 1 sec (Weis-Fogh, 1961a). It is not known how quickly equilibrium is attained but it must be within the range of milliseconds. A dragonfly tendon may be extended to twice its length under a constant load for months

SVEND OLAV ANDERSEN A N D TORKEL WEIS-FOGH 22 on end without any change of length and it witl snap back to its unstrained length and properties immediately the load is removed. No other biological material is known to be as perfect with respect to elasticity. The ‘fibrous nature of elastin ligaments makes similar tests difficult. The full recovery of locust resilin is also apparent in the composite cuticle of the prealar arm (Fig. 11). In contrast to solid cuticle, the prealar arm does not experience any permanent deformation or change in stiffness although the recovery is slower than in pure resilin. This is attributed to the presence of chitin lamellae made up of a feltlike entanglement of fibrils which can slide relative to each other but which are brought back eventually by the stress set up in the neighbouring resilin (Jensen and Weis-Fogh, 1962).

I

0

I

I

20

I

1

I

40

I

60

I

I

80

I

I

100

Time (mid

FIG.11. The angular deflection of a prealar arm (&-) 88 a function of the duration of the load (minutes).Note the full elastic recovery. (From Jensen and Weis-Fogh, 1962.)

When a rubber is used for mechanical springs in a dynamic system, its fitness can be expressed in terms of damping, i.e. the amount of energy lost as heat in proportion to the amount of energy stored during the deformation and later released as mechanical work. At very low frequencies of deformation, the “ static” experiments just described show that the loss is zero but, as in all rubbers, it increases with the speed of deformation. In the experiments illustrated in Fig. 1, the Wings were suddenly lifted up from the resting position (A) to the upper position and the amount of energy fed into the system was measured. At the top, the mutilated wing (to reduce air resistance) With an artificial mass attached was immediately released and the kinetic energy estimated

23

RESILIN

during the recoil downstroke. The ratio between the kinetic energy and the spent energy is the elastic efficiencyr] of the system. It is a measure similar to the so-called resilience in rubber technology. We may call (1 -7) the loss factor. It can be seen from Table I11 (Weis-Fogh, unTABLE 111 Elastic efficiency of locust thorax at normal conditions of work Intact thorax Empty box Elastic hinges Non-active muscle

0.86 0.02 0.88f 0.02 097 f 002 0.8 f0.2

published) that the intact complex thorax has an elastic efficiency corresponding to the resilience of most rubbers (Buist, 1961) but the complicated rubberlike wing-hinge ligaments have an efficiency as high as 0-97 under normal conditions of operation at 20 c/s. This shows that resilin itself exhibits less damping than other known elastomers. It has

Fnqurmy of altemting rtroln

(a

FIG.12. The elastic loss factor in the prealar arm as a function of the frequency of the sinusiodal strain (c/s). at two different amplitudes (010 and 005 mm). (From Jensen and Weis-Fogh, 1962.)

not been possible as yet to measure 7 in the elastic tendon but only in the isolated preaiar arm of locusts. The results are seen in Fig. 12 (Jensen and Weis-Fogh, 1962). The sample was strained sinusoidalIy and the stress was measured in phase and 90" out of phase with the displacement at two different amplitudes. The biological range of

24

S V E N D OLAV ANDERSEN A N D TORKEL WEIS-FOGH

frequency and deformation is indicated and it is seen that the loss factor is as small as that found in the wing-hinge (2-479, in spite of the fact that the uveruge speed of deformation of the resilin was about 6 lengths per sec when averaged over the entire content and during one complete cycle. In order to compare these low values for the damping with recently published figures for carefully vulcanized natural rubber (Gent, 1962), the ratio FJF, of the imaginary component Fi of the oscillating stress and the real component F, has been plotted as a function of the logarithm of the frequency in Fig. 13. Gent’s results for 0.1 c/s are indicated, sample (A) having the highest cross-link density and (C) the smallest

Frequency (ch)

FIG.13. The same data as in Fig. 12. The ordinate is theratiobetween the imaginary and the real component of the oscillating force (F,/F,) and the abscissa the logarithm of the frequency (log c/s). Gent’s (1 962)results with rubber vulcanizates are shown at the extreme left.

(M,from about 4 0oO in (A) to 10 OOO in (C);Gent, personal communication). Unfortunately there are no values for frequencies higher than 0.1 c/s but it is seen (a) that the damping or the loss factor for natural rubber is significantly higher than for swollen resilin at similar frequencies, and (b) that an increased degree of cross-linking has a tendency to decrease the damping. It will be shown below that the average molecular weight of the chains between junction points, M,, is of the same order of magnitude in sample (A) as in resilin so that the results should be comparable. In spite of being a polyamide with bulky side groups and many potential sites for the formation of hydrogen bonds, swollen resilin

RESILIN

25

behaves as a more ideal rubber than other natural or synthetic materials as far as clastic recovery and damping are concerned. It is not known

how good elastin is in thcsc respects but the results emphasize the desirability of more detailed investigations of this protein and similar polyamides both from a theoretical and from a technological point of view.

3. Thermoelasticity Since the elastic force of a strained ideal rubber is due entirely to thermal agitation of the individual members which make up the flexible chains, in the same way as the pressure of a gas is due to the movements

O

1.0

l

Egtenrion ratio pc

FIG.14. The force-extension diagram of an elastic tendon measured at high and low temperature. (From Weis-Fogh, 1961a.)

of its molecules, the elastic force at a given strain willincrease in proportion to the absolute temperature. No material complies completely with this requirement because, ultimately, the stresses must be borne by the chains and must therefore affect their internal energy. Moreover, in concentrated materials like rubbers there will be some weak forces between the chains or between the groups inside the chain. Nevertheless,

26

S V i NI) 0 1 A V A N I ) L R S t N A N D ’ I O R K C I , W E I S - F O G H

a typical rubber can be shown to behave almost like such an entropy system provided it is not extcnded so much that crystallization begins to occur. All quantitative molecular theories of rubberlike elasticity are based upon this fundamental property which distinguishes rubber from other solids whose elastic behaviour is dominated by changes in internal energy. It is, therefore, important that it has now been shown beyond doubt that both elastin (Hoeve and Flory, 1958) and resilin (Weis-Fogh, 1961a)behave like true rubbers in the thermodynamic sense. The theoretical and experimental difiiculties are due to the fact that both proteins

t

I S 2 (1.19)

<--%--<->-<--------3--

0

+--<--->

>-

1

I

300

320 Temperature

340

360

(OK)

FIG.15. The isometric force of an elastic tendon at different degrees of extension as a function of the absolute temperature. (From Weis-Fogh, 1961a.)

are swollen so that the experimental system is an open one. The fact that the swelling depends on temperature was overlooked by previous investigators on elastin, supercontracted collagen and keratin, but was realized and overcome independently by the above authors. Fig. 14 shows the relationship between the force of an elastic tendon and the degree of stretch expressed as the extension ratio a, i.e. the length in units of the unstrained dry length. The tendon was placed in unbuffered

RESILIN

27

water and it can be seen that it swells slightly when the temperature is increased. Nevertheless, the slope increases with temperature. In the case of elastin placed in a mixture of glycol and water where changes in swelling do not occur, Hoeve and Flory (1958) found a similar increase, the magnitude of which corresponded to theoretical predictions based upon an entropy model. The only deviation concerns the shape of the formextension curve which was linear in elastin in contrast to that of Extension mtio A

Extension ratio Q

no.16. The forceatension

curve of an elastic tendon resolved into its two components, the entropy term and the internal energy term. (From Weis-Fogh, 1961a.)

resilin and other rubbers, but this is probably due to the fact that the elastin preparation consisted of many parallel fibres at different degrees of stretch. However, this fact makes further interpretations difficult. When an elastic tendon was placed in a dilute buffer in which the changes in swelling with temperature are insignificant, it was found that the isometric force (constant length) increased linearly with the absolute temperature over a wide range of extensions and temperatures (Fig. 15), so that the non-linear force-extension curve in Fig. 16 could be resolved into the two components in the Wiegand-Snyder equation, an inkrnal

28

S V E N D O L A V A N D E R S E N A N D T O R K E L WEIS-FOGH

encrgy term and an entropy term (Weis-Fogh, 1961a). It is secn that the shape of the curve is dominated by thc latter over the entire range of extension. There is therefore no doubt that both proteins must be considered true rubbers, as was alrcady claimed for elastin by Meyer and Ferri (1936) on the basis of unconvincing thermoelastic measurements. Whereas the complicated structure of elastin preparations prevent us from going much further, the dragonfly tendon iends itself to more detailed investigations of the only other rubberlike protein known. B. M O L E C U L A R I N T E R P R E T A T I O N

The following discussion is based on Weis-Fogh (1961b). The exact quantitative formulation of the kinetic theory of rubberlike behaviour r

I

4-

Compression

1-

-

h

-

h

Y

; o

I

0

-

-

-

-

c

-u .n c 2

-2

-

-4

0.5

1.o

I

I

1.5

20

25

Extension ratio A

Fro. 17. The relationship between the elastic force of tendon milin and the degree of simple compression or extension. The experimental points and the sequence of measurements are indicated. The smooth curve is drawn according to the simple kinetic theory of rubberlike elasticity (long-chain networks). (From Weis-Fogh, 1961b.)

depends on the assumption made about the properties of the network. If the number of random links in a chain between two junction points is large, the theoretically calculated force increases with extension but

RESILIN

29

with monotonic decreasing slope, as is seen from the unbroken curve in Fig. 17. It has the following form (with slight variations):

I; = NkTY'(X - h-*)

=

N~T(uv'- c'v-'),

wheref, is the force per unit unstrained swollen area, N is the number of chains per unit volume, k is Boltzmann's constant, T is the absolute temperature, Y is the volume fraction and is the ratio between the unswollen and the swollen volume, h is the extension ratio in units of the unstrained swollen length, and (L the extension ratio in units of the unstrained dry length. The product NkT is called the elastic modulus G and depends on the degree of cross-linking, G = pRT/M,, where p is the density of the unswollen material, R the gas constant and M, the number-averagemolecular weight of the chains between junction points. It is seen that an estimation of G gives information about the degree of cross-linking and therefore of M,. It is also seen that if the swelling is isotropic and does not alter the state of the chains, it influences only the geometrical parameter Y so that, if it is known for a single stress-strain diagram, the elastic behaviour can be predicted for any value of v. Similar relationships hold for the photoelastic properties.

1. Cross-links and chain flexibility Figure 17 also shows that the mechanical behaviour of tendon resilin only follows the simple theory up to a certain extension, after which the force increases more steeply than predicted. This is typical of short-chain networks, both in theory and in practice, when the number n of random links per chain decreases. The smaller n is, the steeper the curve becomes but the slope near to the unstrained length is altered only to a small extent. It is, therefore, possible to obtain information both about the number of cross-links and about the flexibility, provided that a suitable specific network model is applied for the latter estimates. In the case of resilin, Treloar's (1954) model of a short-chain network with non-affine displacementhas been adopted since it presupposes that four chains meet at each junction point and that the chains are of about equal length. This would not apply to a rubber vulcanized by some random bulk process, but as we shall see in Section IV,D, the majority of the cross-links are of this type in resilin. Moreover, its very high elastic efficiency points towards the cross-links being introduced at regular distances and not in a random way. The average modulus for tendon resilin thus estimated is 6-7 kg cm-% (against 9 kg cm-2 in locust resilin; Jensen and Weis-Fogh, 1962), corresponding to Me-5 OOO, so that there are about 60 amino acid

S V E N D OLAV A N D E R S E N A N D T O R K E L WEIS-FOGH 30 residues between junction points. As to the flexibility of these chains, the shape of the measured curves indicates that there are more than 5 and less than 25 random links per chain, say about 15. Since it takes at least 3-4 actual links with free rotation to make up for one (ideal) random link, it is seen that there must be about as many links with rotation as there 8 f e amino acid residues in a chain. This means that there can be almost no hydrogen bonds present between any two members of a chain b u s e

I v = 0.52

I

Extension ratio QI

Fro. 18. The relationship between the stress (kg/cml swollen strainod araa) and the extension in a dragonfly tendon swollen at pH 1.8 (v = 0.52), pH 6 7 (v = 0.38) and pH 1 2 3 (v = 0.19). "he smoothcwt8are drawn aocordinoto the simple theory. (From Weis-Po&, 1961b.)

even a few bonds would stiffen that part of the chain lying between the groups which react with each other. The conclusionthen is that the amino acid sequence of the resilin chains is such that intrachain hydrogen bonding is prevented, at least in neutral water. We shall now see that this must also be the case under other conditions of pH and temperature. The evidence derives both from swelling experiments and from photoelastic measurements. Fig. 18 shows that differences

RESILIN

31

in swelling caused by varying pH from 1.8 to 12.3 did not change the initial course in any other way than that predicted from the equation on p. 29. In other words, while v had been altered 2.7 times other parameters had not changed. Similarly, the initial slope of the stress birefringence curves (Fig. 19) remained the same so that the drastic variations in pH had no effect upon the state of the chains, as would have been the case if hydrogen bonds had been formed or broken. The same applies to the effect of increasing temperature on the stress-optical ratio (1.3 x lo-' cm*kg-'

Stress t, (kg/crn'j

FIG. 19. Three stress-birefringence curves from a dragonfly tendon at the same degrees of swelling as in Fig. 18. Each curve is displaced one unit along the ordinate in order to avoid coincidence, the birefringenceof unstrained resilin being zero at all degrees of swelling. (From Weis-Fogh, 1961b.)

at room temperature; for detailed discussion, see Weis-Fogh, 1961b). The photoelastic measurements also confirmed that the number of random links per chain must be somewhere between 5 and 25 (Fig. 20). It is interesting to note that the curve is non-linear and of the shape predicted for the model adopted by Treloar (1 954). Measurements on short-chain natural and synthetic rubbers have resulted in straight lines (Saunders,

32

S V ~ N L )O L A V A N D E R S E N A N D T O R K E L W E I S - F O G H

1956r1, b) but this is probably due to random cross-linking and, again, it points towards resilin being cross-linked at regular intervals.

I

I

I

I

5

10

IS

20

Stress. t,v-2b/G

FIG. 20. The stress-birefringence of dragonfly resilin as compared with that of t w o short-chain networks. (From Weis-Fogh, 1961b.)

2. Sfructurc We have already seen that electron micrographs fail to demonstrate any structure in resilin whatsoever. It was thought that extensive stretching and slow drying would produce some crystallinity in the dragonfly tendon but the best X-ray evidence available is completely negative on this point. Both in relaxed and in highly extended dry tendons, there is no indication of chains being able to take up any regular secondary structure, neither at low-angle nor wide-angle diffraction (Elliott et a!., 1964). Apart from a diffuse ring corresponding to about 4 A at all lengths, there are a pair of equatorial spots in the most extended specimens, corresponding to a spacing between longitudinally oriented parts of the

33

RESI LI N

chains of about 4.5 A. It must bc concluded that the hydrogen bonds formed during drying are located at random and that this protein cannot click into any secondary structure, a result in full agreement with its exceptionally good qualities as a rubber. Since resilin has a characteristic and specific amino acid composition, we must assume that its primary structure, i.e. the sequence of residues, is specific. Its secondary structure is the randomly kinked configuration (if one prefers to call it so) and the cross-links represent the tertiary structure and give it the character of a giant network, or a giant molecule, which may extend in all directions of space like a cross-linked resin.

Iv.

C H E M I C A L P R O P E R T I E S OF

RESILIN

The main features of amino acid composition are the same in the different members of a group of proteins but there may be differences as to detail, depending upon the source from which the protein has been isolated. All collagens, for instance, have a high content of glycyl and prolyl and contain two unusual amino acid residues, hydroxyprolyl and hydroxylysyl, the relative amounts of which may vary appreciably (cf. Gustavson, 1956). We must therefore expect that resilin from different systematic groups will differ to some extent, so that we are dealing not with a single compound but with a group ofclosely related proteins. A . A M I N O A C I D COMPOSITION

The only quantitative analyses made so far have been performed on material isolated from two sources i n the desert locust (Schistocerca gregurin), the prealar arm and the main wing-hinge. Bailey and Weis-Fogh (1961) found no significant differences between these samples so that their results have been pooled and averaged in Table IV in which similar figures are presented for bovine elastin. It is obvious that the gross composition of resilin is quite difTerent from that of other structural proteins, although it bears some resemblance to the collagens, without containing any hydroxyprolyl or hydroxylysyl, however. The amount of glycyl residues is particularly high in resilin (38 out of 100) and the other prominent ones are alanyl ( I l ) , aspartyl (lo), seryl (8) and prolyl (7-6). The complete absence of sulphur-containing residues and of tryptophyl is noticeable and it is important that 34% of the side chains carry hydrophilic polar groups, in contrast to 5 in elastin. The number of asprtrtyl and glutamyl residues carrying amide groups in resilin was calculated to about 120 per 105 g resilin, but this number cannot be very

TABLE IV Amino acid composition of resilin (averaged from Bailey and Weis-Fogh, 1961) compared with that of elastin (averaged from Partridge, 1962) Resilin from locust ligaments residues per residues per 105 g protein 100 residues

Elastin from bovine ligamentum nuchae residues per residues per 10s g protein 100 residues ~-

____.____ ~

Glycine Alanine Valine Leucine Isoleucine Proline Phenylalanine Methionine Serine Threonine Hydroxyproline Tyrosine Aspartic acid Glutamic acid Lysine Histidine Arginine Fluorescent Compound I Fluorescent Compound I1 Total Average residue weight

437 I 26 32 26 19

88 29 Nil 91 35 Nil 31 116 53 6 10

37.9 10.9 2.8 2.3 1.6 7.6 2.5

-

7.9 3.0

-

2.7 1 01

4.6 0 5

09

40

3.5

4 9

0.3

1152 86.8

0.8

99.9

356 239 151 69 29 117 38 Trace 9 9 12 8 8 16

33.2 22.3 14.1 6.4 2.1 10.9 3.5

-

0.8

0-8 1.1 0.7 0.7 I .5 4 0.4 0.6 0.1 7 0.7 Small unknown amounts of fluorescent compounds (Partridge, 1962; Loomeijer, 1961) 1073 99.9 93.2

w

P

RESILIN

35

precise (Bailey and Weis-Fogh, 1961). In addition to the usual amino acids, later analyses disclosed the presence of small amounts of two new amino acids in a full hydrolyzate of resilin, the fluorescent Compounds I and I1 listed at the bottom of column I (Andersen, 1961, 1963). They were not noticed in the quantitative assay because they are strongly adsorbed to the ion exchange material used (Amberlite CG 120). Their properties and their role as cross-linking agents will be discussed in a separate section. The amino acid composition of resilin from dragonflies and from crayfish has been estimated by paper chromatography and we can only state here that they exhibit all the main features of locust resilin, including the presence of the two fluorescent amino acids. The latter compounds are also present in the other resilin samples where we have looked for them (see Table 11) but they have not been found in any part of any arthropod cuticle where the presence of resilin could not be detected by other means. This strongly indicates that the fluorescent amino acids are specific for resilin, but it may of course be that they will be found in some structural proteins which, for other reasons, cannot be classified as resilin. A more extensive search is obviously needed. It appears from Table IV that resilin resembles elastin in having a high content of glycyl, alanyl and prolyl but otherwise they differ in most other respects. It has already been mentioned that resilin has a high proportion of residues with polar side chains whereas elastin has an unusually small amount. Nevertheless, they are both rubberlike and have very similar physical properties. The scarcity of polar groups in elastin has been considered essential for its rubberlike properties (Lloyd and Garrod, 1946; Kendrew, 1954) but since such groups are abundant in resilin, their absence cannot be very important in this respect. Purified elastin preparations are faintly yellow and show fluorescence in UV-light, and several attempts have been made to isolate and characterize the compounds responsible for these properties. Loomeijer (1958) hydrolyzed elastin with papain and found that in thedigest the fluorescent component is strongly bound to peptide material. A similar conclusion has been reached by LaBella (1961) who used pancreatic elastase. Even after prolonged digestion most of the fluorescencewas found to be bound to non-dialyzable peptides. He therefore suggested that the fluorescent component is involved in the cross-linking of the peptide chains. Partridge at al. (1963) have recently succeeded in isolating a fluorescent yellow peptide which per mole contains two different NH,-terminal residues. From this peptide an U V-absorbing component was isolated after strong acid hydrolysis and found to consist of two closely related

36

S V E N U OLAV A N D E R S E N A N D TORKEL WEIS-FOGH

compounds each containing a heterocyclic nucleus, four amino groups and four carboxylic groups. The strong hydrolysis destroyed the yellow colour and may therefore have changed the structure to some unknown extent in contrast to the fluorescent compounds in resilin. The results obtained by Partridge and his collaborators are in strong contrast to the findings of Loomeijer (1961) who concludes that the fluorescent components are lipids with an aliphatic structure, and probably containing a carbonyl group. Although there is some disagreement about the nature of the fluorescent components in elastin, it is reasonable to assume that they act as cross-links between the peptidechains. As will beseenlater, the fluorescent compounds isolated from resilin are distinctly different from those in elastin, but the function of both types may well be the same. €3. S W E L L I N G I N D I F F E R E N T

MEDIA

Resilin is only rubberlike when swollen, i.e. a liquid must be present which solvates the peptide chains and breaks the secondary bonds so that the chains become flexible and free of each other (Weis-Fogh, 1960). It is therefore of interest to investigate the swelling properties since it may offer some information about the chains once the solubility of proteins in anhydrous media becomes better understood (Singer, 1962). We have therefore re-investigated the problem. 1. Swcllitig in aqireoirs media

Although resilin does not dissolve, it swells strongly in water and in water-containing media, and the degree of swelling depends on the pH, as one should expect from the high content of different acid and basic groups which dissociate over a wide range of pH: the carboxyl groups (end groups, non-amidized groups in aspartyl and glutamyl) at pH 2-5, the imidazol group in histidyl at pH 6-7, phenolic groups in the fluorescent Compounds I and I 1 about pH 7, the amino groups (end groups, r-groups in lysyl) at pH 8-9, the phenol group in tyrosyl at pH 10, and the guanidino group in arginyl at pH 11-13. In accordance with the spccttum of pK values, the relative volume of small pieces of native dragonfly rcsilin varies almost smoothly with the pH of the dilute buffers with which they were brought into equilibrium, with a minimum at the isoelectric point about pH 4 (Fig. 21A). It is also understandable that treatments which aimed at blocking the free amino groups have relatively little effect since they are rather few compared with the other protolytic groups, but the shoulder seen at about pH 8-9 in samples treated with

37

RESILIN

dinitrofluorobenzene(FDNB),nitrous acid or benzoquinone is probably due toancliminationofthefreeaminogroups(Fig.21B-D).The blocking experiments are not quite specific, however, and give rise to the following comments. The piece which was treated with FDNB deviated most from the native sample. It had a strong yellow colour and from comparison with similarly treated locust resilin (Andersen, 1963) it can be concluded that both the amino groups and the phenolic groups in tyrosyl residues and in the fluorescent compounds had reacted. Since, moreover, a rather large

1

0

I

I

2

I

I

4

I

I

I

6

I

8

I

I

10

I

I

12

PH

FIG. 21. Swelling of native and modified resilin from Aeshna grandis tendons in dilute buffers at room temperature. The volume is relative to the air-dried samples. (A) Native tendon; (B) tendon treated with dinitrofluorobenzene; (C)tendon treated with nitrous acid, and (D) tendon treated with benzoquinone. (A) and (D) a n the average of two determinations. (B)and ( C ) are single determinations.

hydrop,hobic group has been introduced, the modified protein has become less hydrophilic at all pH values, but it is still rubberlike although it was rather slow in reaching its equilibrium length after a deformation. Tendons treated kith nitrous acid at pH 4 and room temperature became dark red-brown which indicates that the aromatic residues have becn affected besides the transformation of free amino groups into alcohol groups. Apart from the “shoulder”, the swelling behaviour and the elastic properties were unchanged in this case. Treatment of the tendons with benzoquinone was also without any pronounced effects. There is a decrease in the swelling which is more

38

S V E N D O L A V A N D E R S E N A N D T O R K E L WEIS-FOGH

pronounced on the acid side ofthe isoelectric point than on the alkaline side. Similar results were found when pieces cut out from locust prealar arms were treated with benzoquinone. This makes it rather difficult to explain the increase in stiffnessobserved in the prealar arm of locusts after treatment with benzoquinone. It was supposed to be due to a change in the isoelectric point combined with an increased swelling at high pHvalues (Jensen and Weis-Fogh, 1962). It should be remembered that resilin is rubberlike at all pH-values, also at the isoelectric point, so that electrostatic forces cannot be responsible for this property (Weis-Fogh, 1960). This result is corroborated by experiments with water-free media. 2. Swelling in non-aqueous media Resilin is able to swell in many anhydrous solvents which shows that the polypeptide chains have a tendency to dissolve but are prevented from doing so by the cross-links. In agreement with this, most of the solvents which cause resilin to swell can be used as solvents for other proteins. In Table V the different solvents used are listed in two groups, strongly protic and weakly protic (Singer, 1962), and the solubility of the solvents in water are given in the last column. The samples used in Table V were pieces of pure dragonfly resilin which were air-dried before being placed in the solvent. In some (formic acid, formamide), they became fully swollen in the course of a few minutes even at room temperature. In other solvents, the process is much slower and heating to 60-70°C may be necessary (propyleneglycol, glycerol) but they remain swollen also at room temperature. The solvents which are solid at 20°C (trichloracetic acid, acetamide, phenol) were used a few degrees above their melting points. Whether or not resilin swells in a given solvent obviously depends on the combined effect of different factors. In a strongly protic solvent, the protein chains carry a surplus of either positive or negative charges and they will therefore repulse each other, in this way making the swelling more easy. Corresponding to this, most of such solvents turned out to be rather good swelling agents for resilin, acid solvents being better than alkaline ones, but neither diethylamine nor triethylamine caused any swelling. It should be mentioned that concentrated sulphuric acid (98 %) swells resilin strongly and makes it rubbery until it finally degrades. In the weakly protic media, the protein will be near its isoelectric point so that, on average, the chains have no charge. Nevertheless, it is seen that many of them are equally good or better swelling agents than water. The rubberiness does not depend on electrostatic forces but on other

TABLE V Swelling of small pieces of dragonfly resilin in non-aqueous solvents Solvent strongly protic

Formic acid Acetic acid Dichloracetic acid Trichloracttic acid Phenol O-CrcsOl m-crcsOl Diethylamine Triethylamine Ethanolamine E t h y h diamine Fyridine

g solvent soluble in 100 ml water at Swelling of resilin temperature indicated

++ ++ + + ++ (+) (+)

Nil Nil

++ ++

W

03

8-63 120 (25°C) 6.7 (r6"C) 3.1 (40°C) 2-35 (20°C) 81-5 1-5 (20°C) 00 00

(+i

+ + Rcsilin smlls considerably more than it does in neutral water.

Solvent weakly protic

g solvent soluble in Swelling 100 ml water at of resilin temperature indicated

Methanol Ethanol 2-Methox yethanol 2-Chlorethanol Tetrahydrofurfuryl alcohol Ethylene glycol Propylene glycol Glycerol Forrnamide Dimethylf'ormamide Acetamide Chloracctamide Tetrahydrofuran Dioxan Diethylether Acetone Dimethylsulphoxid

+ Swelling is similarto that in water. (+I The pica btcomes plasticized and can be deformated but docs not return to its former shape.

Nil: 'flu piece remains hard and brittk.

Nil Nil Nil

(+I Nil

+ + + ++ + ++ + Nil Nil Nil Nil

+

dD W

m

co 00 00

97.5 (20°C) 10 (24OC)

Very soluble 00

7-5 (20°C) 00

soluble

40

S V E N D O L A V A N D E R S E N A N D T U R K E L WEIS-FOGIf

forccs active between solvent and protein chains. They may dcpend on the specific amino acid sequence and on the location of the cross-links in the chains. This makes it difficult to predict which solvents will be effective for the swelling, but some general features can be seen from Table V. Monovalent alcohols are very poor swelling agents while di- and polyvalent alcohols act well, as do amides and substituted amides. It is of interest that phenol is much more effective than are alcohols and this can hardly be due to phenol being a stronger protic solvent since the cresols are rather poor swelling agents. Like the amides, the phenol molecule seems to fit into the peptide structure (Gustavson, 1956). C. ENZYMIC H Y D R O L Y S I S

Conflicting reports have been published concerning the digestibility of elastin by proteolytic enzymes. It may be due both to impure enzyme preparations and to slight degradation of the elastin preparations used.

Hwrs

FIG.22. Digestion of 10 mg locust resilin by 3 mgsubtilisin in 3 mI 0-2M phosphate buffer, pH 8.0 at 30°C. At intervals samples were withdrawn and the absorption was measured at 280 mp at acid reaction ( x 1 and at 320 mp at alkaline reaction( 0 ) . The measurements were corrected for the absorption due to subtilisin and for the volume changes. The fluorescence (+) was measured at alkaline reaction with activation at 320 mp.

Thus, undegraded elastin cannot be attacked by pure trypsin but when it has been brought into solution by means of elastases, trypsin can split it further down (LaBella, 1961). This shows that bonds are present in elastin which can be split by trypsin but that they are inaccessible in the

R ESI LI N

41

undcgradcd protein. The enzymes from pancreatic juice which readily digest ciastin (elastases) also attack other proteins and are therefore not spccific, but a bactcrial enzyme which is specific has now been isolated from Fkrvoharw-ium elnsrolyticum (Mandl and Cohen, 1960). In contrast to elastin, resilin is readily digested by all proteases investigated so far ( Weis-Fogh, 1960). The list now includes trypsin, chymotrypsin, pepsin, papain, subtilisin, and pancreatic elastase. This difference between the two elastic proteins can be due both to the presence in resilin of more peptide bonds conforming to the specificity of the enzymes in question and to a higher degree of accessibility of the sites. Figure 22 shows the time course of the digestion of resilin with the bacterial protease subtilisin. Samples were withdrawn at intervals and the UV-absorption measured at acid and alkaline reaction. The fluorescence was measured after dilution of the alkaline samples. In spite of the fact that resiiin is present in small lumps which must be attacked from the outside, the digestion is surprisingly rapid. The curve for the absorption at 320 mp in alkaline solution is a measure of the liberation of the fluorescent compounds, whereas the curve for the acid samples measures the fluorescent compounds and tyrosyl residues together. It is seen that the tyrosyl and the fluorescent compounds are liberated at equal rates during the entire period of digestion, and that there is no lag-period in the beginning. This is also the case when other proteases are used and it is therefore clear that fluorescent peptides are being liberated at maximum rate right from the beginning of hydrolysis. In elastin there is a marked lag-phase at the beginning of the digestion. D. FLUORESCENT A M I N O ACIDS

1. Properties and isolation As has been mentioned several times, resilin contains two fluorescent

amino acids not present in other proteins. Their structure is not known in detail as yet but there is a large amount of evidence which indicates that they function as cross-links between the protein chains. They were first discovered as two fluorescent spots on paper chromatograms of resilin hydrolyzate and from their position on the chromatogram they were named Compounds I and 11, Compound I having the smallest Rf value. Later work has shown that this numbering can be a little troublesome, since Compound I1 is the main substance and has the simplest structure (Andersen, 1961, 1963). Both compounds react as a-amino acids and have characteristic absorption and activation spectra (Figs. 23 and 24) which closely resemble

42

SVEND O L A V ANDERSEN A N D TORKEL WEIS-FOGH

miil

w

B FIG.23. (A) UV-absorption spectra for Compound I in (1) acid solution and (2) alkaline solution. (B) Same for Compound II. (From Andemen, 1963.) A

m/l Fig. 24. Activation spectra (uncorrected) for Compound I at pH 8 Compound I1 at pH 1 1 (0). (From Andersen, 1963.)

( 0 ) and

for

RESILIN

43

PH FIG.25. (A) pH-dependence of the fluorescence of Compound I when activated at 285 mp ( c ) , at 325 mp (01, and at 325 mp after blocking the amino groups with methoxycarbonylchloride (+ ). (B) Same for Compound 11. (From Andersen, 1963.)

FIG.26. Separation of Compounds I and I1 on celldosephosphate (1 x 40 cm)at room temperature. The column was equilibrated with 0.2 M acetic acid and elution was perfoxled by a sodiumchloride gradient established by running 500 mll M NaCl into 300 rill 0.2 M acetic acid. Ordinate, left hand side: Absorbancy at 280 mp,right hand side: Conductivity of effluent (lo-' Q-1).

44

S V E N D O L A V A N D E R S E N A N D T O R K E L WEIS-FOGH

those of tyrosine, indicating that they are phenols, but the shift from the acid to the alkaline form of the spectra of Compounds I and I1 occurs near pH 7 (Fig. 25), in contrast to tyrosine where it occurs at about pH 10. The phenciic character of the compounds is confirmed by their ability to coople with diazotized sulphanilic acid, to give colour reaction with Folin’s rcagent for phenols and with nitrosonaphthol. They give no colour with Gibb’s reagent so that the para-position to the phenolic group seems to be blocked (Fearon, 1944). Tests for polyphenols were negative.

mP

FIG.27. Spectra of the fluorescent light emitted from alkaline solutions of Compound I (0) and Compound 11 ( C J ) and from pieces of native resilin (+). In all cases activation was performed at 253.7 mp.(From Andersen, 1963.)

The two compounds can be separated from each other and from the other amino acids present in a resilin hydrolyzate by means of paper chromatography, but for larger amounts it is necessary to use column chromatography on ion-exchange material. Originally Andersen (1 963) used DEAE-cellulose but later he has got more reproducible results with the following method based on cellulosephosphate (Fig. 26). This ion exchanger is brought in equilibrium with dilute acetic acid and the resilin hydrolyzate i s placed on the column in acid solution. The amino acids are elutcd from the column by using a salt-gradient whereby the monoamino acids are eluted first, followed by the basic amino acids, and close

RESILIN

45

to them, Compound I€,while Compound I comes later and well separated from the other amino acids. After Compound I a minor peak is seen which apparently represents a third fluorescent compound of the Same family. Before discussing the possible function of these compounds in resilin, it is important to ascertain whether they occur in the unmodified protein or are artifacts produced during the hydrolysis. All evidence shows that they are present in native resilin, linked to the other amino acids by means of normal pcptide links which became broken during the hydrolysis. The

I't

FIG. 28. pH-dependence of the fluorescence of an enzymic digest of redin, ( x ) when activated at 285 mp,(c) when activated at 320 mp.

best evidence is that (1) the spectrum of the fluorescent light emitted from whole pieces of resilin is identical with the fluorescent spectra of the isolated compounds (Fig. 27) ;(2) the fluorescent peptides released during enzymic digestion have absorption and activation spectra closely resembling those of the isolated compounds and with the same pHdependence (Fig. 28) (Anderscn, unpublished); (3) alkaline hydrolysis liberates the same two fluorescent compounds as does acid hydrolysis (Andersen, 1963). 2. Number of albhatic chains Titration of the isolated compounds shows that both of them contain groups Cissociating near pH 2, 7, and 10; in Compound I1 their relative numbers are 2: 1 :2, while they are 3: 1 :3 in Compound I (Andersen, 1963). The groups which dissociate near pH 2 can be assumed to be carboxyiic and those dissociating near pH 10 to be amino groups. Those which dissociate near neutrality must be identical with the groups

46

SVEND OLAV ANDERSEN A N D TORKEL WEIS-FOGH

responsible for the shift in the absorption spectra and in fluorescence. They are tentatively identified as being phenolic. The relative numbers of the groups strongly indicate that Compound I1 is a dicarboxylic diamino acid, and that Compound I is a tricarboxylic triamino acid. This result is confirmed by experiments with partial dinitrophenylation (Andersen, 1963). When Compound I1 had reacted with FDNB under controlled conditions, the reaction mixture could be separated in three

cm

Fro. 29. Paper electrophoretic separation of the reaction mixtures from partial dinitrophenylation of Compound II (A) and of Compound I 03).The fully drawn lines show the light absorption at 420 mp, and the broken lines show the fluorescence when activation is performed at 285 mp. (From Andersen, 1963.)

components by paper electrophoresis in acid medium (Fig. 29). One of them corresponded to the unmodified fluorescent compound, one reacted as if all amino groups had been blocked, and the third migrated with half the velocity of the unmodified compound, it was yellow but ninhydrinpositive so that one of the amino groups appeared to have become dinitrophenylated but the other was free. For Compound I, the number of reaction products corresponded to the presence of three amino groups since two partially and one fully dinitrophenylated compounds were found. From these results combined with the titration experiments, the molar

RESfLIN

47

absorption coefficient at the wavelength for maximum absorption was calculated to 8 OOO for Compound I and 5 400 for Compound 11. By measuring both the UV absorption and the ninhydrin colour yield of a sample of pure compound, the molar ninhydrin colour yield can be calculated and compared with the yield of some standard substance. Different &-aminoacids have almost the same colour yield in this modification o f t e ninhydrin method (Rosen, 1957). Whenisoleucine was used for compa ison, it was found that Compound I1 reacted as if it contains 1.9 amiris groups per molecule and Compound I 2.6 amino groups, confirming the conclusion that I1 contains 2 and I 3 amino groups (Andersen, unpublished).

!

3. Precursors of fluorescent amino acids In an attempt to get some information about the aromatic nucleus in the fluorescent compounds, we have recently injected immature adult locusts with tyrosine or phenylalanine uniformly labelled with '4c and afterwards isolated the fluorescent compounds and tyrosine from the resilin (Andersen and Kristensen, 1963).The two fluorescent compounds became rzdioactive both with phenylalanine and with tyrosine as precursors so that either of these can be used for the biosynthesis. Also, the relative distribution of the radioactivity between the fluorescent compounds on the one side and tyrosine on the other was independent of whether the parent compound was tyrosine or phenylalanine, so that the synthesis from phenylalanine seems to take place with tyrosine as an intermediate: Phenylalanine -+tyrosine -*Compounds I and 11.

The specific activity of the isolated fluorescent compounds, expressed as counts/min/pmole, was surprisingly high as compared with that of the isolated tyrosine. For Compound I1 it was about three times higher and for Compound I about six times higher than for tyrosine. This was true whether the precursor was given as a single largeinjection or as many small injections spread out over the whole period of resilin deposition (i.e. in the course of 10-14 days). As there seems to be no turnover of resilin when it has been laid down, it is very improbable that this relative enrichment of radioactivity in the fluorescent compounds is due to the existence of different tyrosine pools of which only one is mixed up with the injected tyrosine. A mcch more probable explanation is that Compound I1 is synthesized from three tyrosine molecules. Since the final compound contains two aliphatic chains, each with a carboxyl and an a-amino group, the third

48

S V E N D O L A V A N D E R S E N A N D T O R K E L WEIS-FOCH

tyrosine molecule may have lost this chain during biosynthesis. The structure for Compound 11 may therefore be as follows:

CH- N I

H ~

CH-NH2

COOH

6OOH

It must be stressed, however, that we have no direct evidence for the presence of ether-bridges between the benzene rings other than the fact that the ccnriections between the rings must be very stable. Also, we have no reason for assuming that the free phenolic group is placed on the second ring. Moreover, this structure gives no explanation of the low pK-value of the phenolic group but it is useful as a working model. Preliminary determinations of the molecular weight do not disagree with the model According to the above suggestion, the more complicated Compound I should consist of about six benzene rings derived from tyrosine and contain three end chains. 4. Fluoresccnt amino acids as cross-links

Provided that the amino and the carboxyfic groups are built into the protein chahs by means of peptide links, the fluorescent amino acids are able to function as stable chemical cross-links, in the case of Compound I1 as follows: Compound I1 residue Ri --NH-JH-CO-~

_ _ _ _ - _ _ _ _ _ - _ _ Rz NH-CH-CO

j -NH-CH-co I RI

I

1

ci‘‘

-INH-CH-CO~-NH--CH-CO-

&-&--coII

I

I

___.__ I _ _ _ _ _ _

R4

Similarly, Compound I should be able to link three peptide chains together. To demonstrate the existence of such structures in the native protein it will be necessary to show (1) that neither the amino groups nor the carboxylic groups of the fluorescent compounds are free in the protein, (2) that they are connected to other amino acids through normal peptide links, and (3) that the aliphatic chains from one compound are built into two (three) different protein chains. We have some evidence bearing on the first point but none on the other two. In order to show that the amino groups of both compounds are

R ESI LI N

49

blocked in some way, native resilin was treated with FDNB and it was found that no N-DNP-derivatives of the fluorescent compounds were formed. The failure to react could be due to difficulties for the FDNB molecule to get access to the amino groups, but since all the r-amino groups of lysine and all the phenolic groups in tyrosine as well as in the fluorescent compounds themselves had reacted, this explanation seems unreasonable (Andersen, 1963). As FDNB reacts with phenolic groups, it was impossible to isolate unchanged fluorescent compounds from DNP-resilin. Ninhydrin will react with all free amino groups but should not affect other groups present. if, therefore, the amino groups of the fluorescent compounds are blocked in resilin, it should be possible to isolate the unmodified amino acids after treatment. This turned out to be the case. The ninhydrin colour yield corresponded to about 7 amino groups per 10s g protein, which agrees with about 6 +amino groups present in lysyl and a single NH,-terminal group. From hydrolyzates of ninhydrin-treated resilin, the two fluorescent amino acids could be isolated both by means of paper chromatography and column chromatography. The positions of the compounds had not been changed so their amino groups were intact and therefore not free in native resilin (Andersen, unpublished). It is possible to get some indirect information about the carboxylic groups in Compound I1 and the amino groups in Compound I. From the curves in Fig. 25 showing the pH-dependence of the fluorescence of the free compounds, it can be observed that for Compound I there is a decrease in fluorescence about pH 9 which is due apparently to the presence of a free amino group. Similarly, Compound I1 shows a decrease in fluorescence below pH 3 which could be due to a carboxylic group. The peptide mixture obtained by digesting resilin with proteases shows no decrease at pH 9 and only a very slight decrease below pH 3 in spite of the fact that there is a pronounced increase in fluorescence about pH 7 and some decrease above pH 12 (Fig. 28). This decrease is due to Compound I and its magnitude corresponds to the presence of about half as much Compound I as Compound I1 in locust resilin. The small difference in fluorescent behaviour between the peptides and the free compounds therefore indicate that, in the peptides, at least one amino group in Compound I and one carboxyiic group in Compound 11 arc blocked (Andersen, unpublished). Although all our evidence confirms that the fluorescent compounds are peptide-linked to other amino acids through all their end chains, we have no final proof. The best way to demonstrate it would be to isolate a single fluorescent peptide from an enzymic digest of resilin and show

50

SVEND O L A V A N D E R S E N A N D Y O R K E L WEIS-FOGH

that it ha5 two (three) NH,terminal residues and the same number of COOH-terminal residues. This would indicate that there is some sort of cross-link present in the peptide. Thereafter it should be possible to elucidate t k complete structure of the peptide. From elastin such a peptide has now been isolated (Partridge, 1962), and we have also tried to separate similar fluorescent peptides from resilin digests but, although we have got fractions very enriched as to fluorescence, we are not convinced that any of them consist of a single peptide. 5 . Degree of cross-linking Since the amount of the two fluorescent compounds can be measured by simple spectroscopic measurements, it should be possible to calculate how tightly cross-linked the protein is and compare the determination with the results from the physical measurements. We know that 10s g locust resiiin contains about 4 residues of Compound I and 9 residues of Compound I1 (Andersen, 1963). If the compounds are involved in the cross-linking in such a way that Compound I links three different chains together and Compound I1 two, the average molecular weight of that part of a peptide chain which is located between two neighbouring crosslinks will be 3 300. The average residue wcight of the amino acids in resilin is 87 so, according to this, there are 38 amino acid residues between two cross-links on the average. This result must be a minimum value since any defects in the cross-linking will give higher values for the average distance between cross-links. From the physical measurements it was estimated that there are about 60 amino acid residues between two junction points but these measurements wer:: performed on dragonfly tendons. Since the elastic modulus of locust resilin is about 30% higher than that of dragonflies (Jensen and Weis-Fogh, 1962), the higher elastic modulus corresponds to about 40 amino acid residues between two cross-links in locust resilin. The agreement between the physical and chemical estimates of the crosslinking i s so close that there can hardly be room for many defects in the network, and it strongly indicates that the fluorescent amino acids do in fact act as cross-linking agents in the manner proposed here.

V. G E N E R ADISCUSSION L It has been stressed repeatedly that resilin shares many properties with elastin and these similarities will be treated first followed bya discussioii of problems related to insect cuticle.

RESILIN A. WHAT

51

MAKES A PROTEIN RUBBERLIKE?

Elastin and resilin are both isotropic three-dimensional networks of polypeptide chains held together by means of stable covalent crosslinks. Thsy are both hard, like resin or glass, when dry and become rubberlike when swollen so that the swelling agent must break the weak bonds which give the dry material its solid character. These bonds may be hydrogen bonds and salt linkages (both called secondary bonds in the following account). The problem is how the formation of secondary bonds is prevented when the chains are solvated, since helical chains due to intrachain hydrogen bonding and crystalline areas due to interchain bonding characterize most structural proteins, also in an aqueous environment at room temperature. Much of what follows is speculative but may hclp to guide further thinking. The fact that extended and slowly dried tendon resilin gives a diffuse X-ray diagram with no sign of crystallinity shows that neither the individud polypeptide chains nor the assembly of chains have any tendency to click into regular patterns (Elliott et al., 1964). The same applies to elastin (Cox and Little, 1961) and the conclusion is that the secondary bonds are formed at random. The explanation must be sought in the amino acid sequence which is not known for either of the two proteics, but one may obtain some useful information from Table VI in which the average composition of the chains between the cross-links is set out. In resilin we have seen that the number of junction points corresponds approximately to the number of cross-linking groups so that physical entanglements seem to be reduced in number compared with ordinary rubbers, and we shall also assume that this is the case in elastin. The density of cross-links in elastin has not been estimated as accurately (Partridge, 1962) and may well be somewhat less than indicated in the Table. First of all, it is seen that the number-average molecular weight of the chains M, is relatively small so that the rather bulky cross-linking groups may prevent contact between chains as well as intrachain bonding in their neighbourhood. Since, however, the chains are flexible, being randomly coiled and thermally agitated in the swollen state, the major part of the residues must have the ability to react with each other both within and between chains. As to reaction between chains, it can only be said that flexibility and agitation mean that all possible configurations of bond foimation pust occur so that it is the swelling agent which prevents their actual formation. As to intrachain bonding, the absence of regular order in the dried protein indicates that it is the amino acid

VI

N

TABLE VI Tentative description of the amino acid composition of thz chains between the chemical cross-lin!ts and between the prolyl residues, respectively, ill tesilin arid elastin

--$z 5

Dragonfly tendon

_____ Molecular weight M, of chains between cross-links (aver.) Average number of amino acid residues between cross-links: Total Pro Average number of residues between equally spaced Pro: Total GlY

Ala Val ASP Ser Other

Locust ligaments -~

___~

5 Ooo*

3 soot

60

40 3

c c

Ox ligamentum nuchae ~

2m s

>< >

z z

Tr,

4.5

30 3

0

z

m

z >

3.8

9 3.4

1-1

1

0.3

0.3

1-0 0.8

0.9

-

0.7

0 1

3.0

2,l

2.I

10

.o

7

2.3

1.5 1 .o

-z ci

-!

0

P

x

m

r

s c v, I

Weis-Fogh (l%lb). T 0 t Jensen and Weis-Fogh (1962). 3 According to Partridge (1962) M, is 2 600 as estimated from mechanical tests, but due to the untypical stress-strain diagram (Hoeve $ and Flory, 1958) this figure can only be very approximate.

H I.SI 1 . 1 N

53

i i d l ' wliich i \ the main factor. In resilin the ideal physical propcriich ;LIX h o t cxplititicd i f wc aswine that the network is free of dt.f;.c.ts ;tnd i h c l i a r a c t c r i d by an almost equal spacing of chemical SCC~~ICIICC

crosc-links with no phy\ical entanglements. If this view is adopted and if, furthcrmorc. we assumc that the prolyl residues are distributed almost evenly between the cross-links, there is a marked similarity between resilin and elastin which may be significant (Table VI). Since prolyl cannot bc fitted into the a-helix, it is seen that the average chain between prolyl rcsidues consists of only 7--10 residues of which more than half carry no o r short aliphatic non-polar side groups. It is assumed (Schellman. 1955) that an a-hclix rcyuircs at least 15-20 residues in succession i n order to bc stable, although intrachain hydrogen bonds are formed between -NHand >C=O only three residues away. I t is possible, mnrco\er. that the 2-5 residues other than the simple aliphatic ones are spaced to as to prevent such hydrogen bonding, because we have seen that almost no hydrogen bonds can he present in swollen resilin. Obviously, i n order to go further it is necessary to know the actual amino acid sequence i n both proteins. 1%. IIOW A R I : 'IHF. N E T W O R K S FORMED'.)

Thc precursors of rcsilin and elastin are presumably linear soluble protein\ buf they have not becn isolatcd. Determinations of NH,-end groups in the final product indicate a molecular weight of about 350 OOO for elastin (Partridge and Davis, 1955) and 80-120 OOO for resilin (Andersen, unpublished). I t is therefore clear that each original chain is likely to become cross-linked with other chains at several points (20150), explaining the complete insolubility of both proteins. Although there are terminal NH,-groups present it may be argued, nevertheless, that the rubberlike proteins consist of interwoven polypeptide rings where mcst junction points are simple physical entanglements (as in a ring mail) and, also, that the so-called cross-links either serve to close the rings or are of no real consequence. However, as pointed out by Weis-Fogh !1961b), such a model is untenable because we are dealing with a n isc:ropic network of randomly kinked chains formed in a watery medium. If the chains should be transformed into rings, they can only become interwoven provided that ork end is free to dip through already f,xmed rings and then to react with the other end, but in randomly kinked and agitated chains the probability is zero for the configuration that thc two ends of the chain will occupy the same volume clement. We must, therefore, conclude that the networks are formed

5.1

S V E N D O L A V A N D E R S E N A N D TORKEL WEIS-FOGH

from long Aexiblc chains which become cross-linked while they are in the random1y kin ked configuration. The cross-linking process may proceed either as a bulk process similar to ttic vulcanization of technological rubbers or it may be an additive process similar to growth and crystallization whereby the newly liberated chains bcconie fixed to the surface of already formed crosslinked material. There does not seem to bc any observations bearing upon elastin and speculations arc also hampcrcd by the fact that sonic authors state that the isotropic fibres ;ire, i n fact, made up of submicroscopic fibrils abcut 100 A thick (Gottc ct d., 1963), while other investigators

3

C

F H , . 30. ‘The average incrcase in the total dry weight of a locust wing-hinge (wH) and 0 1 a prealar arm (prA) as ;i function of adult ,rge (A). Similar figures for resilin and chitin in the prenlar a r m (B)and in the wing-hinge (c‘).(From Neville 1963b.)

have failed to see any submicroscopic structure (Cox and Little, 1961). It seems certain, however, that the final product is present (and formed?) outside the cells. As far as resilin is concerned, it is possible to follow the formation as a growth process since it is deposited in the course of several days (Weis-Fogh, f960) and is found as an extracellular product totally devoid of any submicroscopic structure (Elliott et al., 1964). According to Neville (1963b), the deposition of locust resilin starts 3 days before the final moult and is not complete until 15-20 days after (Fig. 30). In spite of sinall variations in the fluorescence which manifest

55

KI-SI 1.1 N

them\clvc\ a\ daily growth layers (Neville, 1963a; see also Fig. 32). there is n o difkrence in the yield of fluorescent material between new and old resilin so that the degree of cross-linking remains the same and specifically fixed (Fig. 31). From experiments with tritiated tyrosine injected at different time intervals. we know that the radioactive tracer is being incorporated in thc course of 2-3 h after the injection and that the radioactivity remains irt srtu for the rest of the animal's life (Fig. 32; Kristensen, unpublished). The secreting epidermal cells in which the

..,,,,.I,

t!,,

.,. ,

I,

'

V'l',Yl,'

1.y;

FIG.31. Optical density at 315 m p of resilin ligament hydrolyzates made alkaline. l h e ordinate is a measure of the amount of the lluorescent compounds since they are the only amino acids prcsent which have absorption at this wavelength. The abscissa gives the iictuitl weight of resilin in samples at known ages. Circles: prealar arms; crosses: wing-hinges; ( I ) and (2) from very young animals, (3) and (4) from fully grow;] animals. (From Neville, 1963b.)

precurscrs pile up do not cxhibit the characteristic blue fluorescence, neither after formaldehyde fixation nor when examined in the fresh statc (unpublished ; cf. Fig. 3 3 ) . Finally, both microdissection, ordinary microscopic investigations ( Weis-Fogh, 1960; Neville, I963b) and electron microscopy (Elliott ef a/., 1964) show that there is only a very thin zone of tran5ition between the cell surface and the fully formed fluorescent resilin. The picture which emerges is that resilin is being secreted in the form of dissolved, large, randomly kinked linear molecules which

Fici. 32. Autoradiograph rnadc from ii section of a prealar arm, photographed both in direct and in UV-light. Thc iiiiinial reccivcd sevcn injections of tritiurnlahellcd tyrosinc with regular intervals o f 2 days. thc lirst injection was given 24 h after thc final moult. Activity froin the first six injections has been incorporated in narrow bands. which coincidc with evcry second of' the brighter fluorescent daily growth layers. (By courtesy of H. Kristcnsen.)

RESlLlN

57

iirc lihcr;iic.tl i t l t o i h c n;irro\v spitcc hctwccn t h e plasmu mcnihranc and I I K prcvi:)u\ly cro\+linhcd rc\ilin. 'l'hc wrfitcc o f the rcsiliti mu\t be

utiutur;itcd w i t h cro\\-link\ ;ind tlic new molcculcs become attached by means o f ;I spccific cross-linking process almost as soon a s they have left the cell. I n othcr words, a l l acnilable evidence points towards the network being formed by a molecular growth process entirely controlled by the rate of liberation of 11ic prccursor. I h i s rnodc o f formation has some features which arc unique as comparcd with technological rubbers and which may explain some of the outstarlding qualities of rcsilin. Being a protein, it is reasonable to assunic that the cross-links are introduced a t specific predetermined sites but this would not in itself prcvcnt network defects. However. if the cross-linking process occurs only at the surface and resilin grows in a way remhiscent of a crystal, electrostatic forces from the charged end groups may prevent the ends from dipping into the already formed material, and a network without physical entanglements will result. So f>r, wc have not discussed the chemical processes involved in cross-linking and it is futile to speculate much further until the chemical structurcs of Compounds I and I I arc known. It should only be mentioned that tho lir:ks seem to be formed between tyrosyl groups on neighbouring chains by means of a modified derivative of tyrosine which may either be free in solution o r attached t o one of the chains. C. R E S l L l N A N D I N S E C T C U T I C L E

One of the most striking features of insect cuticle is its bariability and the exactness with which these extracellular structures are constructed, also at the submicroscopic level. The extremes may be illustrated by the hard dark and sclerotized exocuticle of a mandible on the one sidc and a patch of pure rcsilin on the other, but there are all grades i n between. Moreover. numerous composite structures are known from elab,xitcly built bristles (Slifcr, 1961) to complicated sheets of cuticle with lamellae, pore-canals, ducts and sculpturing (cf. Locke, 1961). It 1\ n o t intcndcd to discuss this enormous complex here (for gencral rcv;cws, bee Hackman. 1959: Richards, 1951, 1958; Wigglesworth, !04X, 1957) but o n l y t o cmph;isir.e some general problems related to thc properties and formation of rcsiiin. Although ;I fair amount of solublc protein is present in some mature cuticles (Wackman, 1953, 1059), tlic characteristic feature of the complex i s that of insolubility brought about hy cross-linking processes, but there must bc it considcrable chemical difl'crcnce between the sclerotization

58

SVI:Nl)

0 1 AC’ A h 1 ) l . H S t . h A N I > ‘ I O K K I . 1 . W F I S - I O G H

of hard cklticle and the similar process in rcsilin. Moreover, next to nothing is hnown about thc endocuticle deposited after moulting and it is likely that othcr types of cross-links will be discovered both i n cuticle and elsewhere (Weis-Fogh, 1963). The sclerctized parts of the exocuticle are secreted in a soft state often in :he course of several days before the moult, extended and stretched during moulting. and fixed and stabilized by the subsequent tanning which is believed to be effected by local liberation of phenolic compounds via the pore canals. These microscopic or submicroscopic canals run normal to the cuticle and begin as extensions of the epidermal cells although they may later become occluded. Unfortunately, as to the tanning process itself, the two best investigated preparations cannot be considered as typical examples of hard cuticle-the ootheca of the cockroach and the puparium of thc ,blowtly. Since, however, the fundamental processes rnay be similar in ordinary “living” cuticle and in these samples, in which tanning occurs uftcr they have lost all direct contact with living cclls, they will be described in some detail. T h c oo;hcc;i of the cockroiich has mainly bcen investigated by Pryor (1940), Brunet and Kent (1955) and Kent and Brunet (1959; see also Pryor, 1962). The left collaterial gland secretes a protein, a phenoloxidase and a /3-glucosidc of protocatechuic acid while the right gland secretes a P-glucosidase so that, when the secretions of the two glands are mixed, protocatechuic acid is liberated from the glucoside and oxidized IG a o-quinone by means of the phenoloxidase present. This o-yuinone apparently reacts with the free amino groups in the protein, i n this way hiking the chains together and transforming the protein into an insoluble hard and dark resin. In the blowfly the metabolism of tyrosine changes just before pupation so that most of the tyrosinc is metabolized according to the scheme: tyrosinc

dopa. +dop;imin

b

N-acetyldopamin

(Karlson and Sckeris, 1962; Sckeris and Karlson 1962). During the pupation, iin inactive I.)rophenoIoxidase becomes activated (Schweiger and kirlson, 1962). It oxidilcs the N-acetyldopamin to an o-quinone which then tans the soft cuticle, forming the hard dark insoluble pu piriuni. In model cxpcrinicti~s,lrcatnicnt o f untanned proteins with quinones results in dark inwlublc products due to cross-linking between free amino groups. Most expcrimcnts on ordinary cuticles indicate that the tanning processes follow a similar course. Thus, various di-phenols and also phenoloxidasc activity are usually present, and it has recently been

RESlLlN

59

ili;.t 'Y-tyrosinc atid ' I ( - d o p arc incurporatcd in the cuticle of S(.hi\/ocwcu Krcgoritr during the first hours after the final moult

found

(Karlson and Schlossbcraer-Kaeche, 1962). Since N-acetyldopamin is also prcsent, liarlson and Sekcrts (1962) propose that the processes in the pupnrium arc also rcprcscntatiw for those in ordinary cuticle. Whatevcr tlic dctails, all cvidencc indicates a bulk-process in which the inaterial t n he tanned is first laid down i n the amount needed and is then impxgnatcd will1 thc tanning agents which react with the free amino groups i n such a way that adjacent protein chains become coniicctcd through \tublc cross-links. The degree of tanning can then be controlled cithcr by thc amount of tannage or by the number of reactive groups or both, hut since the reactions will occur completely at random it seems iiiipos\iblc t o makc regular molecular networks in this way. The most outmnding difrerences between the sclerotization and the formaticn of resilin are (a) that the cross-linking of resilin is a continuous process taking place whenever the precursors are liberated into the extracdlular space. and (b) that it involves the tyrosyl residues and not the free amino groups. However, the systems may be similar in principlc since i n both the cross-linking could depend on sinall reactive molecules in solution. So far, we have not discussed thc uncoloured lamellate endocuticle which is two to three times more abundant i n mature locusts than the sclerotit-cd exocuticlc and which is formed i n the course of several days after thc final moult (Neville, 1963~).According to Jensen and WeisFogh (1902). the clastic modulus in extension is of the same order of magnitude i n thc t w o types, 800 IOW kg/mm2 against 0.2 kg/mm2 in rubbcrlikc cuticle, but endocuticle is undoubtedly the softer in compressior. normal to thc surface. However. i n principle endocuticle behaves a s ;[ solid although it exhibits some properties reminiscent of rubberlike cuticle. I t is devoid of colour, it swclls and shrinks normal to the lamellac to ;in appreciahlc extent when the pH is changed and it exhibits rypical strain-birefringencc when deformed under the polarizing microsccpc. It docs not, however, fluoresce with the typical bluish colour of resilin; it is somewhat plastic and stains blue with Mallory and Masson and grcen with light grccn. As to cross-links there are a t least four possibilities: (a) that they are similar to those found in resilin although slightly modified since Coinpounds I and I1 have not been found; ( h ) that they arc due to the libernlion of small amounts of quinonch without this resulting in any darkening (Dennell, 1958; Malek, 1961 1; ( c ) that specific bonds are formed between chitin and protein as propowl by Hackman (1059); and (d) that the epidermal

ti(;. 33. Section of LL part of the cuticlc from the transition between the dorsal part of the radial vein and the tirst median plate in a forewing of a desert locust. The sample was fixed in 47; neutral formaldehyde and frozen-sectioned (6 p thick). Photographed in UV-light to show fluorescence (A) and in phase-contrast (B). The resilin-containing part is brightly fluorescent and the endocuticle fluoresces only weakly and with another colour. The epidermal cells are non-fluorescent. Notice that resilin has been deposited beneath both dark exocuticle and non-coloured endocuticle.

R E S 11-1 N

61

cells arc. ablc tu produce a third, unknown system for cross-linking proteins. I t may be instructive to consider the relationship between the three types of cuticle just discussed as illustrated in Fig. 33. I t is a transverse frozen section (6 p thick) of formaldehyde-fixed cuticle from the transition between the dorsal part of the radial vein and the first median plate in a forewing of a desert locust (the resilin fluoresces strongly and it is just visible in Fig. 2). The fluorescing rubberlike cuticle is lamellate throughout and, although its outer part is formed before the final moult simultaneously with the prospective exocuticle (Neville, 1963c), there is a sharp bomdary between the two types. After the moult, the endocuticle is laid down together with rubberlike cuticle but, again, the boundaries remain sharp and distinct. It is also seen that the rubberlike cuticle lacks pare canals (Weis-Fogh, 1960) although they are present in neighbouring endocuticle. Finally, in spite of the sharp lines of separation, the epidermal cells in the region of overlap must have produced exocuticie, endocuticle and rubberlike cuticle in succession. This is another demonstration of the almost omnipotent qualities of these cells (Wigglesworth, 1961), and it indicates that the same cell may be able to cross-link proteins in two or three different ways, but perhaps not at the same time. The lzck of pore canals and the sharp boundaries between rubberlike cuticle and the other types may be due to the small meshes of the network combined with the fact that resilin is cross-linked while being formed. Thus, the amount of fluorescent amino acids seen in Table 1V corresponds to an average distance between neighbouring cross-links of about 2%A in normally swollen locust resilin (v=0.4). This means that ordinary protein molecules cannot diffuse through it, particularly not since a considerable part of the volume is occupied by the network chains themselves. In other words, after the network has been formed resilin acts as a barrier for the passage of ordinary macromolecules. This restriction also concerns chitin which, when present, is arranged in regular continuous lamellae in rubberlike cuticle. It is obvious that a dense network starting at the cell surface will prevent mixing of chitin molecules with resilin and resist the intrusion of chitin crystallites. Since chitin lainellae seem to be formed by simultaneous excretion over large areas (Noble-Nesbitt, 1963b) and i n discontinuous fixed amounts (quanta1 growth; Neville, 1963b), it is understandable that the chitin in rubberlike cuticle forms thin continuous sheets (Elliott et al., 1964) of long crystallites oriented at random in the planes of the sheets (Clark and Smith, 1936). The lamellae do not touch each other and the chitin

62

S V E N I ) 0 I . A V A N D E R S E N A N D TORKLL. W E i S - F O G H

rcmains confined lo discrcte laycrs and not distributed among the protein, as in certain farms of solid cuticle (Lockc, 1961 ; Neville, 1963d). While a resilin network can exclude other macromolecules from entering, i.; may of course also function as a matrix for soluble protein molecules which become entrapped in the meshes during secretion and which are tanned later (Jensen and Weis-Fogh, 1962). Scales (Picken, 1949), hairs, bristles (Slifer, 1961 : Noble-Nesbitt, 1963b) and other "critical" structures are formed early during ecdysis and undergo tanning before the rest of the cuticle (Cottretl, 1968, cited from NobleNesbitt, 1963b; and our own observations on locusts). They are characterized by x i extraordinarily refined molecular architecture which may be understcod not only as a result of spontaneous crystallization but also of the formation of such mixed networks growing by addition from the surfaccs and moulded by the cell membranes due to an exact determination of time and amount of secretion. REFERENCES Andersen, S 0. (1961). Fluorescent components from resilin. Abstr. Commun. V. in/. Congr. Biorheni. 21. Andersen, S. 0. (1963). Characterization of a new type of cross-linkage in resilin, a rubber-like protein. Biothim. biophys. Actu 69, 249-262. Andersen, 5. 0.and Kristensen, B. (1963). Incorporation of phenylalanine and tyrosine in the cross-linkages of a protein, resilin. Acta physiol. srund. 59, Suppl. 213, 15. Bailey, K. and Weis-Fogh. T. (1961).Amino acid composition of a new rubber-like protein, resilin. Biochini. hiophys. Artu 4 8 , 4 5 2 4 9 . Bonhag, P. F. (1949). The thoracic mechanism of the adult horsefly (Diptera: Tabanidae). Cornell Utaiv. AKric. Exp. SIU.Mem. 285, 1-39. Brunet, P. C. J. and Kent, P. W. (1955). Observations on the mechanism of a tanning reaction in Peripbrieta and Bluttu. Pror. m y . Sor. B 144, 259-274. Buist, J. M . (1961). Physical testing of rubber. In "The Applied Science of Rubber" (W. J. S. Naunton. ed.), pp. 709-776. Arnold, London. Carton, R . W., Dainauskas. J. and Clark, J. W. (1962). Elastic properties of single elastic fibres. J. uppl. Physiol. 17, 547-551. Clark, G. L. and Smith, A. F. (1936). X-ray diffraction of chitin, chitosan and derivatives. J. phvs. Chem. 40, 863-879. Cotterell, C . B. ( I 960). Thesis, Cambridge University. Cited from Noble-Nesbitt (1963b). Cox, R. C. an.? Little, K. (1961). An electron microscope study of elastic tissue. Pror. roy. S x . B 155, 232-242. Dennell, R. 11958). The hardening of insect cuticle. Biol. Rev. 33, 178-196. Dennell, R. (1960). Integument and exoskeleton. /ti "The Physiology of Crustacea" (T. H. Waterman. ed.), Vol. I , pp. 449-472. Academic Press. New York and London.. Elliott, Ci. U., Huxley, A. F. and Weis-Fogh. T. (1964). In preparation.

RESILIN

63

Fearon, W. it. (1944).The detection and estimation of uric acid by 2:6-dichloroquinone-cbloroimide. Biochem. J. 38, 399402. Gent, A. N. (1962).Relaxation processes in vulcanized rubber. 1. Relation among stress relaxation, creep, recovery. and hysteresis.J. appl. Polymer Sri. 6,433-441. Gotte, L., Stern, P., Elsden. D. F. and Partridge, S. M. (1963).The chemistry of connective tissues. 8. The composition of elastin from three bovine tissues. Biochem. J. 87. 344-35I . Gustavson, K. H. (1956).“The Chemistry and Reactivity of Collagen”. Academic Press, New York. Hackman, R. H. (1953).Chemistry of insect cuticle. I. The water-soluble proteins. Bioche.71. J. 54, 362L367. Hackman, R. H. (1959). Biochemistry of the insect cuticle. I n “Biochemistry of Insects”, Proc. I V . int. Congr. Biochem., Vienna (L.Levenbook, ed.), pp. 48-72. Pergamon Press, London. Hoeve. C . A. J. and Flory, P. J. (1958). The elastic properties of elastin. J. Amer. chcm. S,K. (10. 6523-6526. Jensen, M. and Weis-Fogh, T. ( 1962).Biologyand physics oflocust flight. V. Strength and elasticity of locust cuticlc. Phil. Truns. B 245, 137-169. Karlson, P. and Schlossberger-Raecke. I. ( 1962). Zum Tyrosinstoffwechsel der Insektrn---VIll. Die Sklerotisierung der Cuticula bei der Wildform und der Albinomutante von Schistocercu greguria Forsk. J. Insect Physiol. 8, 441-452. Karlson, P.and Sekeris, C. E. ( I 962).N-acetyl-dopamine as sclerotizing agent of the insect cuticle. Nctture. Lond. 195, 183-1 84. Kendrew, J. C.(1954).Structure proteins. 1. In “The Proteins, Chemistry, Biological Acti./lty. and Methods” (H. Neurath and K. Bailey, eds.), Vol. 2,B, pp. 845-950. Academic Press, New York. Kent, P. W. and Brunet. P. C. J. (1959).The occurrence of protocatechuic acid and its aOB-D-glucoside in Blartu and Periplaneta. Tetrahedron 7 , 252-256. LaBella, F. S. ( 1961 1. Studies on the soluble products released from purified elastic fibers by pancreatic elastase. Arch. Biochem. Biophys. 93, 72-79. La Cireca, hl. ( 1947).Morfologia funzionale dell’articolazione alare degli Ortotteri. Arch.

70d.

(itirl.) Napoli 33,

271-327.

Lloyd, D. J . and Garrod, M.(1946).The rubber-like condition of the fibres of animal skin. Fihruus proteir/s. I n “The Society of Dyers and Colourists Symposium”, 1946,rsp. 24-29. I-ocke. M.(1961).Pore canals and related structures in insect cuticle. J. biophys. biocl/c,:n.C)ftnl.10. 5 8 9 4 1 8. Loonieijer. F. J. (1058). A yellow fluorescent pigment in elastin. Nature, Lond. 182, I 82-1 83. Loomeijer, F. J . (1961). The lipid compound of elastin. J. Atheroscler. Res. 1,62-66. Malek. S. R. A . (1961). Polyphenols and their quinone derivatives in the cuticle of the deser! locust. Schistocercu Kreguria (Forskill). Comp. Biochem. Physiol. 2,35-50. Mandl, I. and Cohen, B. B. (1960). Bacterial elastase. 1. Isolation, purification and propcr:ics. Arch. Biuchem. Biophys. 91,47-53. Meyer, K. t4. and Ferri, C. (1936).Die elastischen Eigenschaften der elastischen und der kollaaenen Fasern und ihre molekulare Deutung. PjYiig. Arrh:ges. Physiol. 238. 18 -90 Meyer. K. H., Susich, G. v. and Valko, E. 11932). Dieeiastischen Eigenschaften der organischen Hochpolymeren und ihre kinetische Deutung. Kolhidzschr. 59, 208 -216.

64

SVENII O L A V A N D E H S E N A N D T O R K E L WEIS-FOGH

Miller, P. L. (1960). Respiration in the desert locust. I I . The control of the spiracles. J . cxp. B i d . 37, 237-263. Neville, A. C. (1960). Aspects of flight mechanics in anisopterous dragonflies. J . exp. Bb!. 37, 631-656. Neville, A. C. (1963a). Daily growth layers in locust rubber-like cuticle influenced by an extcrnal rhythm. J. Irtwct Physiol. 9. 177-186. Neville. A . C. (1963b). Growth and deposition of resilin and chitin in locust rubberlike cuticl:. J. Irrsrc/ Phjsiol. 9. 265-278. Neville, A. C . (1963~).Daily growth layers for determining the age of grasshopper populations. Oikos 14, 1--8. Neville, A. C . (1963d). Daily growth zones in insect skeletons. Acta ph.vsio/. scund. 59, Suppl. 213. 107. Noble-Neshitt. J. { 1963a). The fully formed intermoult cuticle and associated structures of Poduru uyuu/ic~u(C’ollembola). Quart. J . micr. Sci. 104, 253-270. Noble-Nesbitt, J. (1963b). The cuticle and associated structures of Poduru aquatica at the mcult. Quar./. J . 1nic.r. .‘%.104, 369-391. Partridge, S. M. (1962). Elastin. Adranc. Protein Chern. 17, 227-302. Partridge, S. M. and Davis, H. F. (1955). The chemistry of connective tissues. 3. Composition of the soluble proteins derived from elastin. Biochem. J. 61, 2 I -30. Patridge. S. M., Davis, H. F. and Adair, G . S. (1955). The chemistry of connective tissues. 2 . Soluble proteins derived from partial hydrolysis of elastin. Biochem. J. 61, I I -21. Partridge, S. M., Elsden, D. F. and Thomas, J. (1963). Constitution of the crosslinkages in elastin. Nature. Lond. 197, 1297-1298. Picken, L. E. R. (1949). Shape and molecular orientation in lepidopteran scales. Phil. Trwrs. B 234. 1-28. Pryor. M. G . M. (1940). On the hardening of the ootheca of Bluttu orientulis. Proc. roy. Soc. B 128. 378-393. Pryor, M. C. kl. (1962).Sclerotization. In “Comparative Biochemistry“ (M. Florkin and H. S. Mason, eds.). Vol. 4, pp. 371--396. Academic Press, New York and Londo,i. Richards, A. G . (1951). “The Integument of Arthropods”. University of Minnesota Press, Minneapolis. Richards, A . G. I 1958). The cuticle of arthropods. Erg&. Biol. 20, 1-26. Rosen, H. (1957). A modified ninhydrin colorimetric analysis for amino acids. Arch. Biochtvn. Biophys. 67, 10--I5 . Saunders, D. W. (l956a). The photo-elastic properties of cross-linked amorphous polymers. 1. Natural rubber and gutta-percha. Trans. Furaday Soc. 52, 14141425. Saunders, L). W. ( I956b). The photo-elastic properties of cross-linked amorphous polymers. 2. Polythene and polymethylene. Trans. Faraduy Soc. 52, 1425-1431. Schellman, J. A. (1955). The stability of hydrogen-bonded peptide structures in aqueoiss solution. C. R. Lub. Curlsberg 29, 230-259. Schweiger, A. and Karlson, P. (1962). Zum Tyrosinstoffwechsel der Insekten. X. Die Aktivierung der PrCphenoloxydase und das Aktivator-Enzym. Hoppe-Seyl. Z. 329, 210-221. Sekeris, C. E. and Karlson. P. (1962). Zum Tyrosinstoffwechsel der Insekten. VII. Der katabolische Abbau des Tyrosins und die Biogenese der Sklerotisierungssubstam N-Acetyl-dopamin. Rioc~hini.hioph,~..\.Actu 62. 103-1 13.

R ESI 1. I N

65

Singer, S. J. (1062). Thc propertics of proteins in nonaqueous solvents. Advanc. Prokin (‘hcm. 17. 1 68. Slifer, F,. H . (1961j. The finc structure of insect sense organs. In!. Rev. Cytol. 11, 125 159. Thurm, L‘. (1963). Ilie Bezeihungen zwischen mechanischen Reizgrossen und stationiren E.rregungszustanden bci Borstenfeld-Sensillen von Bienen. Z . w r g l . Physiol. 46, 351-382. Treloar, L. R. G . (1954). Photoelastic properties of short-chain molecular networks. 7”. Furciclay SOC.50, 88 1-896. Treloar, t. R . G. (1958). “The Physics of Rubber Elasticity”, 2nd ed. Clarendon Press. 3xford. Weis-Fogn, T. ( 1959). Etlasticity in arthropod locomotion : a neglected subject, illustrated by the wing system of insects. Proc. XV. itit. Congr. Zool. (1958), 393-;95. Weis-Fogh, T. ( 1960). A rubber-like protein in insect cuticle../. exp. Biol. 37,889-907. Weis-Fogh, T. (1961a). Thermodynamic properties of resilin, a rubber-like protein. J . mol. Biol. 3. 520-53 1 . Weis-Fogh, T. ( I 961 b). Molecular interpretation of the elasticity of resilin, a rubberlike protein. ./. mol. B i d . 3, 648-667. Weis-Fogh, T. (1961~).Power in flapping flight. In “The Cell and the Organism” (J. A . Ranisay and V. B. Wigglesworth, cds.). pp. 283-300. Cambridge University Press. Weis-Fogh, T. (1963). Resilin, a rubber-like protein, and its significance. In “Aspects of Protein Structure” (G. N. Ramachandran,ed.), pp. 337-341. Academic Press, London and New York. Wiggleswxth, V. B. (1948). The insect cuticle. B i d . Rev. 2 3 . 4 0 8 4 1 . Wiggles*ucrth.V. B. (1957).The physiology of insect cuticle. Annu. Rev. Ent. 2.37-54. Wigglesworth. V. B. (1961). The epidermal cell. In “The Cells and the Organism” (J. A. Rainsay and V. B. Wigglesworth, eds.), pp. 127-143. Cambridge University Presc.

This Page Intentionally Left Blank

The Active Transport and Passive Movement of Water in Insects J. W. L. BEAMENT Department of Zoology, University of Cambridge,

England I. Inkduction . . . 11. The Active Transport of Water . A. BasicPremises . B. Active Uptake from the Air . C. Active Transport of Water in the Gut . D. Water and the Tracheal System . E. The Living Cuticle and Liquid Water . F. Conclusions . 111. 1.n Interrelation between Grease and Absorbtion . A. A Model Absorbing Water . IV. The Physical Chemistry of the Cuticle . A. The Cuticle other than Lipid Layer . B. The Hydration of Procuticle C. Control of Procuticle by the Epidermis . V. Tne Cuticular Lipid . A. A New Interpretation of Transition in Monolayer Films B. Monolayer Inversion . C. Conclusions. . VI. The Asymmetry of Cuticle A. Thermodynamic Validity of Asymmetry . V11. Eirctrical Properties of Cuticular Lipids . A. The Electrical Inversion of a Monolayer . B. Mechanical Distortion C. The Passage of Substances through Monolayers . VIII. On Pumps A. The Electret Ion-pu.np B. Continuous-flow Water-pumps , C. Is the Lipid the Water-valve? . 1X. N'ider Implications . X. Summary . References

.

. . . . . . . . . . . . . . . . * . . . . .

. .

. . .

. . . .

67 69 69 72 76 78 88 91 92

9 4 94 95 96 97 98 101

I05 107 107 111 111 112 115 115 117 118 120 122 123 124 125

I. I N T R O D U C T I O N Water is actively transported by insects. Direct evidence for this statement relies on a small number of experiments involving only some half-dczen species and is especially associated with the external cuticle 67

68

J. W. L. B E A M E N T

and the gut linings. However a critical evaluation of this work will show that the evidence is beyond reasonable doubt. As a statement of physiological fact this discovery is of enormous importance. An active transport ~f ions and of unionized material is a well substantiated phenomer-on in all kinds of living material, and it is commonplace to assume such processes in explaining the functioning of nerve, of muscle, of digestive, excretory and osmoregulatory devices and of every kind of cellular traisporting phenomenon. The movement of water, however, seems always to be assumed to follow an osmotic gradient, to be incidental to the active transport of a hydrated particle, or it is neglected altogether. Sometimes even the possibility of a n active transport of water per se is bluntly denied. Yet it would be most surprising if living systems which have devised methods of transporting so many biological substances against concentration gradients had not found a means of handling &becommonest of all biological chemicals-water. If it can be shown bejjond reasonable doubt that any one organism can actively transport water, then an independent mechanism for water-transport may not be eliminated from studies of physiological systems simply on grounds of minimum hypothesis. In most physiological systems a redistributim of water accompanies the transport of other materials, so that a discussion of water transport must include some consideration of mechanisms which would transport ionized particles and unionized materials; it is even possible that some substances could be passively redistributed as a result of an active transport of water. It is the author’s belief that the literature on the tracheal system contains evidztice suggesting an active transport of water; when these findings are considered in conjunction with some hitherto anpublished experimental evidence briefly reported here, one may make a case for the active transport of water in many insects and perhaps sugest that this is a basic ability of the class, if not of the Arthropoda as a whole. The first section of this paper aims to make this case. The water movements under discussion all take place through an integumental membrane of some kind. In recent years we have obtained some insight into the physicrt-chemical nature of these membranes, especially into their relationship to water. Organized lipids play the most important role in the passive regulation of water movement and in modifying the adhesion of water to the surface of cuticular membranes; the molecular arrangement within thc lipid may change and in doing so greatly modify the relationship of the membrane with water. Tbere is reason for believing that some part of the essential mechanism for regulating and perhaps transporting water involves organized lipids.

A C T I V E TRANSPORT O F WATER I N INSECTS

69

The most recent development in this field of investigation concerns the clectric dipoles of polar lipid and the consequent presence of an electrostatic field of great strength, associated with the cuticular membrane. The physiological consequences of such a system are by no means fully realized; two possibilities which are apparent are: (a) the separation of two solutions of different ionic composition without the continiicus expenditure of energy even though the solutions are in diffusive communication, and (b) the facilitation of the movement of ions of particular charge in a particular direction. In other words, this electrostaticfield can be the basis of an ion pump. Another problem in this field has frequently been called the “asymmetry” of insect cuticle. We must examine whether it is physically reasonable for the cuticle membrane to have greater permeability to water in one direction through it than another. New experimental evidence about the physicochemical construction of the lipid layer makes possible the development of a theory that the lipid can act as a “valve” or “rectifier”, whence one can outline a mechanism which will actively pump water. Finally one must consider whether such a pump has chamteristics similar to any of the systems which appear to be transporting water actively in the insect. We have not yet reached the stage where one mechanism provides a universal answer to all these transport phenomena. Nevertheless it should be borne in mind when reading this paper that all cell membranes contain, as an essential central constituent, a bi-molecular leaflet of organized lipid. While the utmost caution must be exercised in extending any analogy between the cuticle of the insect and the cell membrane, it must be apparent that this research on the cuticle could be useful as a model when thinking about similar processes which occur at the membrane of every cell, and even within cells. 11. THEA CT I V ET R A N S P OOF R TW A T E R A. B A S I C P R E M I S E S

Unless energy is supplied from some external source, materials tend to migrate from a higher into a lower concentration; the movement may

be by simple diffusion or, in more complex systems, by special transporting mechanisms commonly called “activated” or “facilitated” diffusion (e.g. Bayliss, 1959; Davson, 1959; Harris, 1960). But when a material passes from a place where it is at lower concentration into a place where it is at a higher concentration involving, as this must, a

70

J . W . L. B E A M E N T

supply of energy, then the process is called a c t k transport. The term is a physiological one. Transport may bc achieved, for example, by a temporary reversal of the gradient which is not observable. Movement may thcn bc achieved by simple or complex diffusion down the temporary gradient, but work is done in changing the gradient temporarily. Alternately, models have been proposed in which chains of molecules are pushed or pulled through specific channels connecting the two concentrations. Energy is fed into this transport system and the measurable concentratioit difference is not changed. The gradient to be considered is not necessarily simple : an ion may move up a concentration gradient if, at the same time, it is moving down an electro-potential gradient, and energy is provided in setting up the potential gradient. But, whatever actual physical processes are at work, if one can establish that a substance moves from a lower to a higher concentration, then one can automatically assume that energy is provided to move that substance. Nevertheless this is a limited view of the use of physical forces in moving materials in biological systems. There are occasions where substances move down a concentration gradient, but against other kinds of force or at rates much higher than can be accounted for by the concentration difference. Bath these phenomena also require a supply of energy, and it is only the conventional use of the term “active transport” which prevents the inclusion of all kinds of similar processes, probably based upon similar physical mechanisms. In this review we shall examine movement 9f water in this wider sense. Physiological studies to date on active transport have almost exclusively considered the passage, for example, of specific ions across the cell membrane of nerve or muscle, or similar cellular systems. The actual flux and gross amount of the ion which is moved is usually minute and the transport is a reversible process depending on the state of a cell membrane; indeed the cell membrane itself is usually regarded as the actual physical site of the pump mechanism (e.g. reviews by Hodgkin, 1951, 1958; Nachmansohn, 1961). Although eventually it may prove that the sodium pump of a membrane like that of a nerve or muscle has molecular mzchanics very similar to the sodium pump of a system involved in thz osmoregulation of the body fluids of a fresh-water animal, there is a difference of degree between these two kinds of phenomena which could allow their mechanisms to be fundamentally different. In an osmoregulatory device, such as those associated with the gill structures, active transport maintains a concentration difference between an ion in the fresh-water environment continuously in one direction with a relatively high flux. In colloquial terms, we could postulate the same

A< I I V F 7HANTI’OKJ

0 1

W A f b R I N INSECTS

71

mechanism inside the cell membranes causing sodium to move into a nerve and into a gill epithelial cell. In the nerve the mechanism can be turned off, and the sodium return through the cell membrane again; the same amount of sodium shuttles back and forth. But in osmoregulation, either the mechanism can never be turned off, or if it must be turned off for re-activation and for transferring sodium into the blood, something must prevent sodium from returning to the outside world. The sodiumattractiiig device of an osmoregulator is only one of the essential components; a continuous-flow system needs additional components to those basically required by a nerve. The situation we shall discuss in relation to water transport is generally more comprrable with the osmoregulatory device. Many suggestions can be made, and have been made, of mechanisms capable of causing water tc move into a cell (e.g. Harris, 1960), ranging from simple ideas of osrwsis, hydrostatic pressure or electro-osmosisto anomolous osmosis and counter-flow systems, or marginal processes based on minute thermal differences to the folding and unfolding of proteins and “colloidal pressures”. The terminologies are various and in some cases incomprehensible either in the context of the period in which they were advacced, or in the light of later developments. Therefore amongst our basic prexises it must be clearly understood that the key to continuousflow active transport which has so far been overlooked is the means of maintainifig the attracting system despite uptake, and/or preventing a reversal of flow when the attracting system is regenerated. As with osmoregulation, the reason for postulating an active transport of water in an arthropod usually derives from a consideration of a concentration difference between the blood and the external medium : that is to say, active transport takes place across a cellular layer (two cell membranes and the cell content) together with an inert integumental or extracellular membrane. But whether transport occurs across a single cell membrane, or a more complex series of membranes, most of the physiological circumstances in which an active transport of water might occur include the possibility of moving the water in association with some other substance, as water of hydration of an ion, for example, thus preventing the establishment of a clear-cut case. This is where the terrestrial animal is of critical importance. Consider an insect confined in air, without food or accessible liquid. The air has a relative humidity substantially lower than that which would be in equilibrium with the insect’s blood fluids. By respiration the insect will lose some weight. But the insect gains in total weight. The only substance it can obtain is water from the air, and this water must

72

J. W. I.. B E A M E N T

have been ac!ively transported from thc air into the blood. The use of the blood as thc intcrnal refcrencc level in this type of experiment is particulariy significant when the animal is an insect. Many animals exercise careful control over the composition of their blood, but it is well known that the insect uses its haemolymph as a variable reservoir for water 2nd for many other substances. Any change in the water balance of an insect is generally reflected as a change in the concentration of the blood fluids. Physiologically there is the implication in this statement that the insect has the general ability to exercise control over water transport into and out of the blood. Physically it is necessary to consider thz activity level of water in the blood as the ultimate level of the gradient up which the water is pumped. B. A C T I V E U P T A K E F R O M THE A I R

There are many reports of insects and their eggs gaining weight when in saturated or almost saturated air, or when in contact with soil, blotting paper etc. Eggs often show the interesting property of being able to live for days, submerged in distilled water, without changing in volume, then to take up water and perhaps even double their volume, later to stup expanding and maintain constant volume for several days; and these various conditions are not accompanied by any demonstrable change in the physical permeability of the membrane of the egg-shell. In all probability there are all kinds of processes involving water regulation in the insect which are based upon mechanisms similar to those which are t k direct object of our discussion, but at present they must be discounted as evidence for active uptake of water. It is extremely difficult to obtain and to maintain an air system at 99% relative humilet alone at saturation point, and be sure that no particles dity (R.H.), of liquid water are ever present. The margin of activity difference between the ends of the gradient we consider, must be large enough to exclude any possibility that small thermal cycles could promote humidity cycles, precipitation or any similar objection. The earliest work on the uptake of atmospheric moisture is that of Buxton (1930) on the mealworm Tenebrio molitor, subsequently confirmed by Mellanby (1932), Browning (1957) and Edney (1957). All these worbers agree that in humidities at or above 88% R.H. starved mealworm larvae gain weight, and it has been shown by direct measurement that an increase in real body water-content has occurred. The equilibrium value for their blood (and that of insects in general) may be taken as 99 % R.H. Mellanby believed that equilibrium between the

A< I

ivr. I H A N S I V ) I ~ Ioi.

WA*IF:R

IN INS~CTS

73

animal and atmospheric water was established at 88% R.H. over a wide biological range of temperature. Should it prove that equilibrium is a function of relative humidity rather than of saturation deficit, this is an important discovery and it niust be a characteristic of any “pump” mechanism which may be proposed. Similar experiments have been performed on other arthropods: Chorrophaga gains weight when without food and in humidities above 82% R.H. (Ludwig, 1937); a number of species of Ixodid (Lees, 1946, 1947) and Argasid (Browning, 1954) ticks obtain water down to 92% R.H., while the pre-pupa of the flea Xenopsylla can show this ability with the remarkably low level of 50% R.H.(Edney, 1947). Very recently, the author heard from Dr. A. D. Lees of a conference at which a number of workers reported similar findings in various mites, while the apterygote Thermobia can certainly take up water from sub-saturated air (Beament et al., 1964). The list of experimenta! animals given there shows a wide distribution of this property amongst the terrestrial arthropods and it will be surprising if further investigation does not reveal many more examples. But the species listed there also show that an active water transporting mechanism is not entirely confined to animals in which obtaining or retaining water is a matter of extreme necessity. An alternative kind of experimental method comprises the confining of an insect in a small sealed volume of air which has a known initial humidity, subsequently measuring the humidity of that air when it comes into equilibrium with the living animal. Kalmus (1936) showed that TeneErio larvae bring their surrounding air to approximately 90% R.H. regardless of whether it starts well beiow or above this level of moisture, but the air becomes nearly saturated when the animal dies. The rat-flea pre-pupa, whether starting in dry air or in water-saturated air, is said to produce 50% R.H. in its micro-environment for so long as that Darticular stadium lasts; if it dies or changes to the pupa, the humidity rises to a value close to saturation (Edney, 1947). It may not be irrelevant that pupae have generally a far more impermeable cuticle than other instars (Beament, 1959). We have to scrutinize the experimental evidence most carefully, but it is dificult to find any flaw in the simple procedure of weighing an animal to determine its increased water content; artificial control of a constant relative humidity is difficult and humidity gradients can occur, but almost all the experiments reported used the reliable method of confining the specimens in air over saturated solutions of various salts, with an excess of solid salt present (e.g. Buxton and Mellanby, 1934). 3 is also difficult to measure relative humidity accurately over

74

J. W. L. D E A M E N T

any range of humidity (see Beament, 1958b), but there are simple and reliable ways of calibrating 3 hygrometer for one particular relative humidity and wing it to detect with great sensitivity any very small departure from the calibration value. This method has been employed in the second category of experiments described above. Differences between 94%. 88%, and 50% R.H. and the blood equilibrium figure of 99 % R.H. cannot be challenged on grounds of experimental error or thermal and random fluctuation of environmental conditions. The two facts which emerge are that certain insects can actively take up water, and that they can control the humidity of the air in a small space around them to levels in striking contrast with the humidity which is in equilibrium with the dead body, or with the same animal in a different physiological state. Now another interesting characteristic of all the many insects which have so far been investigated (Ramsay, 1935; Wigglesworth, 1945, etc.) is that there is no significant difference between the rate at which they evaporate water into completely dry air when they are alive or when they are dead. Tenebrio and Ixodes were included in these experiments. Davies and Edney (1952) do suggest that there i s a difference between water loss in dry air from dead and from living spiders; the explanation might for example lie in the appearance of free water over the lung books of dead spiders. But no one has published any information which suggests that spiders are capable of that kind of active uptake of water which we are at present discussing. So we must accept another curious feature of the arthropod water-pump: it can direct water inwards from air of humidities down to a level which would seem to be species-specific, but the pump has no “purchase” when trying to work against humidities substantially below the equilibrium figure. Put another way, the pump does not seem capable of reducing the rate of outward flow of water when high transpiration is experienced. We have next to consider whether we can associate the water-pump with any specific anatomical part of the animal. Buxton (1930) originally attributed the increased water content of Tenebrio to “metabolic water ”-water produced by the metabolism of food. Mellanby (1932) demonstrated that this could not account for the increase, and that water must be taken up from the atmosphere. He believed that absorbtion occurred through the spiracles via the respiratory system. While Mellanby’s suggestion of an uptake of atmospheric water seems abundantly proved, it is shown from first principles in Section 11, D that uptake through the tracheal system is impossible. In most instances attempts to demonstrate the role of the tracheal system by blocking the

A C T I V E T R A N S P O R T O F W A T E R I N INSECTS

75

spiracles have failed (Browning, 1954; Locke, private communication). It is diFcult in such circumstances to differentiate between the effects of anoxia and internally accumulated carbon dioxide. Browning did, however, show that in Ornithodorus a large concentration of C o t which produces direct anaesthesia also prevented water uptake in any case with spiracles open. But Ixodes can apparently respire sufficiently through the cuticle when the spiracles are blocked and Lees demonstrated thvt this did not interfere with active uptake. On the other hand, the animai must be healthy and active: cyanide, oxygen-lack and severe desiccation all prevent water from being absorbed. Also, a number of workers have reported that uptake is “spasmodic” giving the impression that in some instances there is a mechanism which is capable of being switched ‘‘on and off ”. All this evidence is consistent with the existence of an inzegral metabolic process. Lees (1947) has calculated (admittedly on a basis of complete efficiency) that the tick could easily afford to burn fat at the rate necessary to provide the energy for water uptake from 94% R.H. into its blood. If we can eliminate the tracheal system, we must next consider the gut, with which certain forms of active transport are certainly associated; in Section 11, C, it will appear that active transport of water does occur in the hind-gut of certain insects. Consequently, one possible mechanism for obtaining atmospheric water would be for an animal to produce hygroscopic faeces, to re-ingest these after exposure to the air, and use the hind-gut to obtain the water. However, in the experiments we have been considering, mealworms increase in weight when they do not eat their f3ecal material (Wigglesworth, unpublished), ticks are incapable of taking in any solid material through their sucking mouthparts, and the flea pre-pupa does not feed at all. No evidence has been produced, and no suggestion made, that any specialized region of the animal‘s surface is specifically responsible for water uptake, so at the present time w:: must implicate the whole of the external integument. Lees (1947) showed that uptake in Ixodes ceases if the cuticle is subjected to minute abrasion sufficient only to interrupt the superficial wax layer and only over a limited area of the animal’s surface. Two interpretations of this discovery have been put forward. Either the great water loss through the abraded area masks any actual uptake which is occurring elsewhere, or the whole epidermis (which is well known to act as a unit in the arthropods) stops its pump for the period of time during which repair of the wax is taking place. In either eventuality it is the integument-the cuticular membrane plus its epidermis-which is likely to be the seat of the active transport.

76

J. W. L. B E A M E N T @. A C T I V E T R A N S P O R T O F W A T E R IN T H E G U T

There i s ample evidence that insects can produce a urine hypertonic to their blood. Experiments giving such indications start from the work of Wigglesworth (1934), and centre upon the enormous body of evidence presented by Ramsay (e.g. 1950, 1954) supplemented by such recent workers as SutclSe (1 360). But whatever the true state of affairs, almost all of the events taking place in these insect rectal systems could be explained on the basis of the absorbtion of salts. The exception, which pointed the way to the need for different kinds of investigation, was the demonstration by Ramsay (1954) that the stick insect Dixippus rnorosus could secrete against a hydrostatic pressure of 20 cm of water. The thesis of Phillips (1961), however, clearly proposes an active secretion of water. Briefly, the rectum of the locust Schistocercu gregariu is exceedingly impermeable to small water-soluble molecules, including amino acids, monosaccharides and dyes (Treherne, 1957a, b, 1958). An uptake which is almost certainly an active absorbtion of sodium and potassium goes on through the hind-gut wall, but by feeding the locust on a diet uf pure water and by washing out the rectum it is possible to reduce the zmount of ionic material there to negligible proportions. T h e rectum can then be ligatured anteriorly in such a way as to prevent any ionic material secreted into the gut from the Malpighian tubules from reaching it. Fluids are introduced into this sealed sac through the anus. Phillips reports that the animals remained in good physiological condition throughout these experiments, and he made careful checks on the composition of the blood which remained normal. Solutions of material known not to penetrate the hind-gut wall were introduced into the rectum at known concentration and volume; some of these were isotope-labelled as an additional check on any absorbtion of substances other than water. The rate of decrease in volume of the sealed hind-gut content at various osmotic pressures was obtained by sampling.The locust can certainly absorb water against osmoticpressures of 11 atm without taking in any other substance, and osmotic gradients of twice this value were recorded between the rectal content and the blood at the end of an experiment in which the animal had concentrated the solute from dilutions below the osmotic pressure of the blood. Phillips 'concluded that when the blood exceeded the rectal content in osmotic activity by 0.5 osmoles there could be a large exchange of water with no net transfer; when there was no osmotic pressure difference, water was transferred into the blood at a rate of 17 mg water/cma/h. (These are not the units used by Phillips; the values are given in this

A(

’r ivf

’I R A N S P O R I I

oi

WATE‘R I N iNsr,cTs

77

form to enable direct comparison with othcr rates of transport given in thi\ rcvicw.) With certain rcscrvations, the system appcarcd to behave a5 though the active uptake of water occurred at a fixed rate independent of the actual osmotic pressure prevailing, including the condition in which both the active process and the osmoticactivitymoved water in the same direction. At very high and probably completely unphysiological gradients af osmotic pressure this apparent relationship did break down. This work cstablishes another irrefutable example of the active uptake of water independent of the movement of another material, and it is important to note the apparent independence of the active transport from the diffusive (osmotic) exchange, regardless of the latter’s direction. If we atLempt to explain this phenomenon in terms of “pores” we may have to postulate two kinds of pores: one for osmosis and another for active transport. This situation might be no different to the alternative postulate of Lees (1947) mentioncd above, except that the rate of uptake by the tick was too small to be measured in the presence of evaporative loss through abrasions. Phillips measurcd t hc hydrostatic pressure in the hind-gut where there is a cyclical change of about 4 cm watcr pressure and a maximum value of 12 cm watcr (0.02 atm). This is insignificant in comparison with the pressurcs theoretically needed to move water against thc osmotic pressures encountered i n thc rcctnl system. In experiments with the isolated rectal mcm branc, comparable rates of transmission of water were obtained with 2 cm hydrostatic water pressure and with a n osmotic gradient. equivalent to 56 cm water pressure, while a hydrostatic pressure of 56 cm water produced four times the rate of flow of its osmotic equivalent. This result must be compared with the theory of Pappenheimer (1953) that osmotic and hydrostatic flow should be identical for pressurcs opcrating across large pores, but that hydrostatic pressure should have the greater effcct across small pores; further experiments bearing on this subject are rcported and discussed in Section VI. Phillips, with an extreme caution characteristic of his whole thesis, is not preparcd to attach great significance to the differences he measured betwecn hydrostatic and osmotic cffects through the isolated rectal preparation. He believed he was working with a system of ‘‘large’’ pores by Pappcnheimer standards. But if the membrane is as impermeable to small water-wlublc moleculcs as the work of Treherne suggests, and if Phillips cxpcrimcnts were carried out as carefully as one has zvcry reason to know they wcrc, it does really look as though this is a “small” pore systcm. Somc indication of the mcaning to be attached to the terms “large” and “small” in this context may be

78

J. W. I.. B E A M E N T

obtained f';om t h c work of Rcnkin (1954), who studied diffusion through porous cellulo~emcnibtanes. If a pore is 40 A in radius, there is only a factor of tW(J bctwccn its pcrmcability to water and to a much larger mc/!cc.uk like sucrosc; if the pore has only a 10 A radius, its permeability to watcr is an order greater than to sucrose. There is every possibility i h a t in the rectal membranc, and in the other membranes we are discussing in this paper, which are virtually impermeable to sucrose and similar s i ~ e dmoleculcs, the pores will be much smaller than 10 A. There is ;r staiiding potential of the order of 30 mV across the hindgut membrane, bctwccn blood and rectal content. Active uptake is not afrected i n any way by balancing out or by augmenting this potential. We agree with Phillips that the application of an external voltage source may not have more than a proportional effect on any potentials actually across the walls of the rectal epithelial cells. Nevertheless, it does not seem that electro-osmosis can be the primary cause of the movement of water in this system. Finally, Phillips calculated on a basis of only ZCS'.!, efficicncy, that the observed rate of transfer of water is metabolically feasible against thc gradients theoretically present, and he points to the rich trachciition of the hind-gut as evidence of high nietabolism involved i i i the process of water secretion. From the point of view of the present rcvicw, his subscquent denionstration that prccisely similar events occur i n the hind-gut of the blowfly Culliphoru crythrocephala is especially interesting because of thc very different nature of the diets and environments of thew two animals, and because of the taxonomic separation of this dipteran from the Orthoptera. It is to be hoped that other workers will now re-invcbtigatc the process of production of hypertonic urine-not only i n the insects--in cases where events can be explained on a basis of reabsorbtion of salts, to see whether such a doctrine of minimum hypothesis has not been misapplied. I).

W A l r K A N D T H E T R A C ' H E A L SYSTEM

Does an active l r a n q m r t of water occur in the tracheal system? This question is directly rclcvaiit in two ways: watcr uptake from the atmosphere (Sectior: I f , 13) could occur through the tracheal system and, again, active transport may bc involved i n the normal process of moving the fuid i n r h r trdcheole. Our discussion will start at a more elementary level than might seem necessary because no one has directly demonstratcd the precise nature of the moving substance in the tracheole of insect \. Consider threc moleculcs : oxygen, water and carbon dioxide. Water

A('l I V k 'IRANSPORT 01. WAlER

IN INSECTS

79

is the sniallcst in si/e whilc oxygcn lies about midway between water and C ' 0 2 . So far a \ ;ictual physical dill'usion is conccrncd it would heem a scnsiblc working a~wtnptiontllilt through membranes, biological or othcrwisc, water should pass about ten times as quickly as oxygen, while C O , would move correspondingly more slowly, when equivalent concentration gradients are disposed across a given membrane. We should be able to make useful inferences about the functioning of the tracheal system by piecing together isolated knowledge about one or other of these molecules into a general picture of the permeability of the whole system. But we run into anomalies from the start. As we have already noted, any attempt to demonstrate directly the respiratory function of the tracheal system by sealing the spiracular openings usually fails. Experiments such as those of Schneiderman (1953), who showed that the diapausing cecropia pupa opens its spiracles only occasionally to release accumulated CO,, similarly imply that the external cuticle is thoroughly impermeable to CO,. If the cuticle by virtue of its wax layer is extremely impermeable to water, which seems so very well established, then it ought to be two orders more impermeable to CO?. Yet there are some surprising exceptions. We can of course discount the generalization, which is not at all uncommon in elementary texts, that "insects lose their C'O, through the general body surface", and which almost certainly stenis from some early cxperimcnts on aquatic insects whose cuticles (t-!oldgatc, 1956; Bcument, 1961a) are now known to have thoroughly watcr-pcrmcablc areas through which oxygen and CO, must be able t o cxchttngc cfticicntly. H u t Wigglesworth ( I 954) summarizes data abcut a number of insects with quite high CO, transfer through the body surface of terrestrial forms including animals believed t o be really waterproof. Thorpe (1928) says that adult beetles loose CO, more through thc intcr-segmental membranes than the dark sclerotized regions; i!' both regions are covered by the epicuticular lipid laver one wonders w5y any <:02 at all should escape there. The author has found that one can rub a stag-beetle with coarse emery paper without getting detectable water-loss from it in dry .-if! Clearly, some fresh and very critical work is needed to elucidate this picture we have o f CO, physiology, but fortunately no such paradoxes mar thc correlation hctween oxygcn and water permeabilities. The supcr1ici:tl wax hycr is impcrmc:iblc to both. while the underlying bulk of the cuticlc is watcr-saturated, and will no doubt allow both oxygen and ('0,t o pas'; Ihrough it frccly. N o w the tracheal system is ;I modified and iiillcctcd cuticle. I-ockc ( 19%) has cxarnincd its relationship to cxtcrnal cuticle in grcat dctail :tiid one may conclude from the homology

ur A M E N T that the bulk of the tracheal tube is composed of protein, partly tanned 80

J.

w.

I..

and intercrystallized with some chitin. Thc layer immediately next the lumcn of the tube is an “epicuticle”; this has a “cuticulin” laminaa tanned and polymeriscd lipoprotein. There are no dermal cementforming glands such as are associated with the external integument, so that if a wax layer is present which is at all comparable with that of the main cuticle it will be exposed at the surface of the lumen. In the absence of any direct method for demonstrating this we must proceed by inference. Onc of the very few ways of showing the site of oxygen exchange in a tracheal system involves the injection of reduced methylcne blue into the haenlocoel (Martin, 1893); it is re-oxidized only at the stellate end cclls containing the trachcoles, and remains reduced even when very close to the tracheal tubcs themselves. From this we infer that water can readily pass through the tracheolc but not through a trachea. (The trachea is extracellular and formed similarly to the general cuticle; the tracheole is intracellular and is not moulted. Little is known of the chemistry of the tracheolar membrane but it is apparently not chitinous and it may very well consist of a single layer of protein.) It is true that claims have been made for experiments demonstrating the exchange of gases through the trachea, but all relate to aquatic insects or to dipterans which seem able to provide exceptions to every entomological generalization. On grounds of oxygen permeability it does look as though the tracheal tubes could have a wax layer which must be absent from the tracheole. Tt is most difficult to persuade liquid water to enter the tracheal system through thc spiracles. This fact has not only been demonstrated repeatedly in all kinds of experiments, i t is also an essential feature of the success of tracheal respiration with open spiracles in tlic aquatic medium (e.g. Brocher, I9 IS ; Beamcnt, 1962). Many aquatic insect larvae have cxternal cuticles which are readily wetted by water and some of them are also rcadily pcrmcated by water, but their spiracles and trachea1 tubes arc cxtrcmely hydrofuge. The nature of these surfaces, which retain a very low adhesion to water although continually submerged in it, can only bc explained by assuming a molecular surface of closely packed --(--I I:, groups of paraflinic matcrial (see Section V, B). One must equally presume that such a surface of highly organized lipid must be present on the luminal surface of tracheal tubes of terrestrial insects so as to give them thcir wetting propcrtics, especially since the air in these tubes may bc permanently saturated with water vapour. If watcr is forccd into the tracheal system it is not possible to observe the form of the meniscus, in part because of the corrugation of the tubes,

A C ' I I V E T R A N S P O R T OF W A ' I E R I N I N S E C T S

81

so that the rtdhcsion of the surface cannot thus be directly measured. An alternative approach is however posGble, starting from an interesting and hitherto uncxplained phenomenon first reported by Pal (1950). If a drop of watcr is placed, for example, on the external cuticle of the leg of a cockroach and in close proximity to a large tracheal trunk, minute water droplcts appear inside the trachcal tube near the external drop. Such explanation as has so far been offered seems to suggest that watcr is transferred through the cuticlt, across the blood and then secreted into the nearest trachea-as remarkable a case of the active transport of water as could be envisaged. Leaving any of the implicd mcmbranc phenomena on one side, the assimilation of the water into thc blood would in itself prevent any localized action at the nearest trachea. Unpublishcd experirncnts by the author indicate a rather different explanation. Droplets can be produccd in trachcal tubes by placing methyl or ethyl alcohd, acetone, ether, or any of these in dilution with water on an area of external cuticle adjacent to a trachea. Some of these substances :ire admittedly efficient wax solvents when i n the pure state, but they h a w little effcct on wax when diluted with a n equal quantity of water. The phenomenon is most marked when very volatile liquids are used : many droplets are formed in the tube and they drip into the lumen. On the oth'yr hand, neither glycerine nor liquid paraffin produce detectable effect Wow if a cockroach is so held that one limb can be inserted into a thiii-bhl;illcd glass tube which has one side flattened, reasonable obscrvation by microscope of thc tracheal trunks in the leg may be made through the flattened region even if the tubc is filled with water. In thcse experiments, the obscrvation tube was connected through a Y-junctim and taps to two rcservoirs each containing water whose tcmpcraiurc was c;ircl'ully cnntrollcd. Thc tcnipcrature of the animal's limb couid thus he :tdjustcd by Ilowing watcr through the surrounding jacket. Idc;il cxpcrimcntal circirmstanccs obtain if one vcssel of water is at 0.2 C'cntigradc clcgrec above mnbicnt, thc otlier a similar interval of tcmpcr;tturc bclow ambicnt. If thc coldcr watcr is run i n t o the glass tube, droplcts imtnediiitcly appear throughout thc tr;1cheiil trunks of the leg ;ind drip continuously. If' the flow is switchcd to the hotter supply, all droplcts disippcar at once. The proccss can be reversed and repeated at will. Thc evidcncc suggcsts that precipitation in a tracheal tube can be produced by ;I very small reduction in temperature-by applying a cooler fuid or through thc latent heat of cvaporation of fluid on the surface. Liquids at the same tcmpcrature as the insect which are not volatile do not produce cooling and thus no condensation.

82

J. W. L. 1 3 E A M E N T

Of course the temperature of thc tracheal lumen will not change to exactly that of thc surrounding water jacket so that a very small decrease indeed suficcs to produce condensation. From dew-point tables one finds that the reduction in temperature at 2 0 T , needed to cause condensation from 99 R.H., is 0.13 Centigrade degree. The experiments make it clear that for all practical purposes the air in the tracheal system is williin 1 Po of saturation with water vapour. Several points of interest follow from these experiments. Since many minute droplets form and remain discrete, dripping rather than coalescing, the surface against which they form must have a high contact angle; the maintenance of this high contact angle in the presence of saturated or near-saturated air in turn requires something like the system of packed -CH, groups which one postulated on the evidence of the difficulty of introducing water into the spiracle. The general picture we have developed matches the scheme proposed (Beament, 1962) for the evolution of hydrofuge wax layers and for the physiological emptying of the tracheal systcni by overturning an inverted monolayer while the lumen of the tubc is still filled with water (see Section V, B). Next, if the walls of the tracheal tubes are so waxed and thus so impermeable to water, then elsewhcre in the respiratory system there must bc some surface sufficiently permeable to provide the saturated conditions throughout. The only source for such evaporation of water is the tracheoles, but before wc consider them further, we have already established a most importmt point in relation to Section 11, B above. In a purely physical system, water vapour will not have a net rate of transfer by diffusion into the near-saturated air of the tracheal lumen, except from air of an even higher humidity, whatever may go on in the tracheole. In other words, even if experiments such as those of Govaerts and Leclercq (1946) showed that there is a very high rate of exchange between atmospheric water and the body fluids of insects, almost certainly through the tracheal system, there is no possibility that the tracheal system can act as part of a mechanism for the active uptake of atmospheric water from the air. We must therefore concentrate our attention an thc gcncral external cuticle when we subsequently come to discuss pumps working against the atmosphere. The tracheole wall must bc freely permeable to oxygen and thus correspondingly we now bclicve t o water; its walls must therefore be saturated with water and hc readily wetted by water. Is the substance which is seen to movc in the trachcole liquid water? The external diameter of the largest tracheole which can be examined by the light microscope is well below Ip, and the lumen will be still smaller. Thc external

<:

A ( ' I I V I . 'I I < h N S I ' O K ' I 0 1 . WA'I E R I N I N S E C T S

83

di;ttiictcr 01 ;i typicid tubc I s 0.4 0.2 1 4 tapering down to 0.05 p, with corres~~c~ritliri~ly \iniillcl- internal diitmctcrs. The resolving power of the light microwope is 0.2 p , and no othcr mcthod of direct observation can be uscJ on living tiiatcrial; greater magnification requires the use of dehydratcd 1ni~tcri;iI.What one usually sees under the light microscope i s ;in interfc'rcncc pattern: air from the trachea continucs into the tracheole so that it contains distally a niedium of refractive index very dill'erent to the surrounding ccllular material. There is a sharp boundary between the air and tltc content of thc portion of the tracheole towards its inner end, whcrc the substance has a refrxtive index close to that of water or cell content, and when so filled the tracheole cannot necessarily be clearly discerned. The sharp boundary is seen to movc under conditions of respiratory necd and obvious!! the length of the air column in the tube incrcascs, but it will never be possible to see the meniscus or the appearance o f the natural fluid content. This tracheolar Huid must be aqueous and if it is in equilibrium with at least 90'';j K . f 1 . i t cannot contain more dissolved substance than would givc i \ n osmotic prcssure of about 10 atm. I t may be even more dilute. 'I'hc lluid i n the tritchcolc moves in a rational manner correlated with thc oxygen rcquircnient o f thc animal and with a n increase in the osmotic pressure of the ilnit11;tl's blood through thc accumulation of n~ctitholitcs(Wigglcswort h, 1954). Now however thc force moving the fluid is produced, i t will only move ifthat force isgreater than thecapillary forcc acting u p thc tube; for any given static position within the tube, thcrc m u a t be a i i cquilibrium between thc forcc inwards and the capillary forcc ;it that particular intcrnal diameter of tube. The pressure up a c:ipill;iry i4 given h!, tlic rrlationship:

p

2 TcosO -:

j.

wherc P is pressure 7' is surface tension (/ is angle of contact I' is riidius of tubc.

I t wnuld appear jusiilicd (see Adam, 1948; Champion and Davy, 1941) to use this rclationship to sires of tube down to 0.05 p diameter, but with tubes of thc order of041 p diaincter there will be circumstances in which the h~hitviourof individual niolccules enters into the considerat i o n ol' tlic incniscus ;IS it htatisticiil assemblage. There is no simple trc:il n i w t ol'tlic movcniont of capilhry lluids in an annularly corrugated fiihc hut twtain awtnptions c;in I ~ Cmade (Fig. 1). For angles ofcontact /c'ro and c l o x to K I - O . tlicrc will bc t w o positions wifiiin each cycle of cc)rrtipiltion iii whit% tlic \urt'acc tcn\iori forcc will be acting along the

84

J . W. L. B E A M E N T

direction of the tube: wherc thc angle of contact is zero with respect to the axis of t11e tube. One of these will be of maximal force (minimum diamctcr) and the other of smaller force (maximum diameter). The part of the tube between maximum and minimum radius will behave just as a tapering tube. When the fluid is being caused to retreat down the tube, it will be thc maximum forcc which has to be overcome, and the fluid will retrcat in a series of minute steps corresponding to annuli. Provided thc angle of contact is low or tero, the effective capillary force will therefore be that in a parallel-sided tube of diameter equal to the minimum intcrniil radius of the annular constrictions. Because the proteinaccous walls are freely pcrmcable to water and saturated with it thc angle of contact must be closc to zcro, and because we are dealing

I-IG. I . Pixitions of ;I meniscus in an annularly corrugated tube, seen in longitudinal section. (A) Contact angle: 0 . Maximum capillary force at minimum diameter; lower capillary forcc at iiiaximiim diametcr. (B)Contact angle small.

with a cosine function thc cRect of small departures from zero will have a ncgligiblc clrccl o n t h s ciipillary force in any case. What is the moving substance in the tracheole? Bult (1939) made the suggcstion that thc tracheolc could be open at its inner end, from which it could follow that thcrc could he a pseudopodium working by protoplasmic or amoeboid movcmcnt. As the so-called surface tension of a naked cell is 0.2 dync/cni (cf. some 72 dync/cm for water) this would solve all problems. Hut the original electron micrograph (Fig. 2) shows no protoplasmic or other solid inclusion in the tube, and the tracheole tcrminatcs blindly in the closing of a single annulus. This picture of a tracheole of R h d n h s also suggests that the annuli are equally spaced regardless of diameter and are very regular, and it shows the extent of

A < . I I V t T R A N S P O R T O F W A I E R 1'4 I N S E C T S

85

the restriction of the lumen by the annular folds. The end and side wall are not visibly diffcrcntiatcd so that we must assume equal activity in the transport of fluids across the whole tracheolar wall. If the tracheole is intracellular, there is no cell membrane outside it. This is important when considering the speed at which fluid passes through the walls, for ordinary cell membranes can be comparatively impermeable to water. Thus if we convert the units of values obtained by Kitching (1954) for the cell wall of protozoa to rng water transferred through 1 cm2 surfa'ce per h we find : Amoeba (fresh water: say 10 atm 0.p. difference) Ciliates (from figures quoted per atm) Impermeable insect (Rhodnius) in dry air Tracheole emptying in 6 sec

0.3 units 0.72 units 0-1 units 6.0 units

I'Ici. 2. A n original electron micrograph of the end of a tracheole of Rhodnius, showing the blind ending and annular corrugation. Taken on an electron microscope in thc Cavcndish Laboratory, Cambridge. x 37500.

This docs not mean to say that a cell membrane is comparable in permeability with an insect cuticlc; but it does mean either that a trachcole wall i s an order more permeable than a protozoan cell

86

J. W . I . . I S 1 A M I N ' I

tiicnihraiic o r t h a t tlic l'otcc n i o ~ i t i pwater through i t is ,)f'thc order of I00 at I l l . tact L I t~ i n t siipposc [ h i i t ;I simple ori m ) l i c' systcni i \ working: thiit tlic tr;ichcolc yall i 4 sciiii-pet nicablL-. 'Then for i i n y position i n thc tubc there will be a n equilibrium bctwcen t h e t//#crcwc.t~in intcrnal i L t I C 1 rxtcnial osmotic prcssurcs (ccll contcnt and fluid i n tube) ~ i n dthe capillary forcc up the tube. If, when the tube is full, it contains f u i d of osmotic pressure cqual to t h a t i n the cell, therc bill be no net osmotic pressure to meet the capillary force. Such a systcni will require a lower osmotic pressure in thc tube even at equilibrium with the tube full. Next, if only water is removed from tlie tube its contents will concentrate. Considering only tlie situation i n ;I pnrallel-sided tubc. and hence constant capillary

force. a niassivc increase in celluliir osniot ic pressure due t o accumulation of metabolites, such a s from 10 to 17 a t n i osmotic pressure. would only ;illow n small percent;ige of traclicoltir water to be removed before equilibrium wcrc re-established: X",, if the tracheole fluid starts a t 10 atm. 5 0 " , if the fluid has as little osmotic pressure as 2 atm when the tube is full. Therefore, t o obtain the observed degree of rcmoval of fluid from a parallel-sided tracheolar tube, it must contain effectively pure wdter. If it contains pure water, we must then see if that transport must be active. Suppose, on tlie other hand, that there is a n aqueous solution which is pumped. solute and solvent together, through the walls so that there is no concentration effect. This rules out classical osmosis. So long as surface-active agents are not present, pure water o r a solution of salts of the order of I p'; concentration will have a surface tension of the order of 72 dynes/cm. O n the assumption of contact angles close t o zero, the capillary pressure for tubes of various sizes will have orders: Diameter ( p ) 0.4 0.2 0.1

0.05

Capillary force (atm) 7.5 15

30 60

As the effective diameter of the corrugated capillary is its minimal internal diameter, these figures for pressures may have t o be doubled. O n the other hand. a surface active agent could reduce the surface tension by a factor of two or three at most (see suggestion made by the author, reported in Wigglesworth, 1953). Such an agent would have to work without reducing water and oxygen permeability and without any risk of interfering with waterproofing waxes. But even supposing a

,A(

I IVI., I R A N S I * O Rr O I W A T C K I N I N S I ; C ~ S

87

surface-activc agcnt, the c:ipilliiry force in thc fine endings of the Lrachcolc will t,c at Icast twicc thc osmotic pressure of the cell. The othcr fcaturc pre\cnted by the Table is even morc significant: whatever the surface tension, in a tapering tube the change in capillary force covers an eight- or ten-fold range, so that the cellular force must also change over this range, regardless of any concentration erect discussed in the previous paragraph. Continuing with the suggestion that a solution occurs in the tube, the solute will collide randomly with the walls. If only one molecule of solute in every million collisions is actively removed through the wall, then 9 5 % of solute will be removed from a tube 0-5 p diameter in 1 msec and from a tube 0.05 p diameter in 0.1 msec. There would be no difficulty in visualizing the active removal of the solute. The demonstration by Bult (1939) that substances interfering with cell membrane ionic transport prevent tracheoles from emptying is only very indirectly relevant; it may indicate the importance of the tracheolc cell-wall pumping into the blood, but there is no suggestion that such materials could interfere with the permeability of the tracheole wai I. We conclude that the tracheole cannot work by classical osmosis, and that the minimal force, assuming the best of all circumstances, which must be provided in the tracheole cell to remove the content of the tube must vary between 2 and 20 atm, with the possibility of being three times this. The limited concept of “active transport” defined earlier required that water moved from a place where it was at a lower concentration to a place where it was at a higher concentration. This is an “energy” concept-the energy could be put in by moving a second substance which passively carried the water. In the tracheole, water either moves from a higher concentration into a lower one, or the levels of activity are identical on both sides of the membrane, but in moving great capillary forces are overcome. So far as the specification of an energy consuming process is concerned, the idea of “active transport” is equally applicable to any case in which either the movement of water takes place contrary to the physical forces working in the system, or even where the rate at which water moves is greater than that calculated from the physical forces in the system. Within this context, water is moved against forces varying between wide limits, and certainly against 20 atm capillary pressure. If the liquid in the tracheole is pure water, the case for active transport is absolute. If a solute is present, it cannot occur in greater quantity than say one molecule (or ion) to every 100 of water, so that in the most

xx

J . W. 1.. B I : A M E N T

favourablc circumstawcs cach unit of' actively transported solute would havc to movc ICK) niolecules of water against capillary pressures of 10 or 20 atm. At the end of this Section we shall briefly consider the codiffusion system of Diamond (1962), but even if we take the most favourable conditions, co-diffusion will not transport water on this scale against such forces. Until proved otherwise, it is sensible to adopt the attitude that the movement of fluid through the wall of the tracheole involves a n active transport of water. Wigglesworth's conclusions in 1951, arrived at by other arguments, were not as outspoken but may be interpreted as implying the same thing. What is of more general import is that the movement of fluid in tracheoles is probably commonplace in insects. Wigglesworth (1954) states that there are insects in which the tracheoles are always found full, or always empty; but this in itself must mean that osmosis cannot be playing any part in the mechanism.

E. T H E L I V I N G C U T I C L E A N D L I Q U I D WATER

Before we turn to physical experiments and possible mechanisms for directing the movement of water, some simple biological experiments which have not previously been reported are relevant. They form a direct link between the biological and physical aspects of this discussion. The physico-chemical nature of cuticular lipid is reviewed below in some detail, and in order to understand these new experiments it is necessary only to know that the cuticle of the living cockroach has, exposed at its surface, a layer of grease of the order of 0.2-03 p thick, i.e. several times thicker than a vertically orientated monolayer of that grease, and that the grease will spread over the surface of water at biological temperatures (Ranisay, 1935). The grease molecules have one paraffinic ending which is very hydrofuge, and one polar ending attracting water strongly. If a drop of water is placed on a suitable impermeable solid surface, and grease applied to it, a film spreads over the drop (Beament, 1958a). This film is probably one monolayer thick, but tightly packed and organized, so that it has great impermeability to water. As a result, the droplet takes a very long time to evaporate in dust-free dry air at temperatures below 30"C, compared with an ungreased droplet: a drop of volume 0.5 mm3 would requirc hours to evaporate completely instead of a fcw minutes. Thus when similar drops of water are directly applied to the surface of a living cockroach, grease spreads over the water; the drop is sitting on a layer of grease which from experiments on rates

A C T I V E TRANSPORT OF WATER I N INSECTS

89

of evaporation from living or dead cockroaches (Ramsay, 1935; Wigglesworth, 1945; Beament, 1945, 1958a) is presumed to be very impermeable to water. Thus, as Ramsay found in 1935, drops can remain in dry air for several hours, while the water evaporates slowly. But some of these droplets do disappear in about 10 min: this will happen with a drop of 0.5 mm3 at laboratory temperatures of about 15°C.

If this water travels into the animal (and one can prove that this is what happens), the actual rate of transfer through the cuticle is between lo3 and lo4 times as rapid as the rate of transfer outwards when the animal is losing water into dry air. So either the permeability of the cuticle changes in some way, or the actual forces moving the water must be enormously greater than those governing evaporative transpiration, or something more complex occurs. Perhaps the most striking indication that vital activities are involved in this phenomenon is given by the discovery that there is an anatomical pattern of areas with different rates of uptake : identical sized droplets placed simultaneously on one living cockroach disappear in times varying from 10 min to 2 or 3 h, depending on the part of the cuticle they cover. The pattern is relatively consistent from one individual to another, and it may be described most concisely by saying that water disappears most quickly through the hard, dark, thick parts of the cuticle, most slowly where the cuticle is pale, soft and thin. The same principle appears to hold whether nymphs or adults are used; intersegmental membranes of nymphs and the thin dorsal abdominal cuticle of adults behave similarly, while the tegmen, and the head or dorsal sclerites of nymphs and adults, are areas where water disappears quickly. At first sight the correlation between the nature of the underlying cuticle and the rate of removal of water is completely contrary to expectation. But the living epidermis must be involved in the phenomenon, for droplets disappear at the same rate regardless of site on anaesthetized animals. If one uses an animal whose two cuticles have separated prior to ecdysis, the droplets shrink so slowly that one can account for their disappearance byevaporation into the air. This last observation confirms the proposal that a droplet placed on the animal is completely waterproofed on its external surface, just as is a greased droplet on an impervious plate. The spreading of grease over such a small surface is accomplished in a fraction of a second. A naive investigation into the nature of a force causing water to move inwards from a droplet on the surface of the living animal has produced much of interest if little in explanation. If the inward force is

90

I W . I-. I % I . A M I Y I

“osmotic” ,ind arises from the internal concentration of solutes in cell or blood or even inner cuticlc, thcn one should be able to place a solution of such strength on the animal’s surface that a droplet remains constant in sim, and indeed a droplet of such concentration that it would swell by drawing water out of the animal. Surprising as is the discovery that a droplet of 1 ”/, NaCl disappears from the cuticle in a time sensibly the same as that in which a droplet of pure water would have been removed from such a site, it is not as difficult to comprehend as the culminating experiment in which water is removed from saturated salt solutions through the hard dark cuticle, leaving solid crystals of sodium chloride on the outside of the animal-circumstances in which the animal is working against “osmotic pressures” of the order of 300 atm. A very significant fact emergcs from this kind of experiment: cockroaches will remove water from saturated solutions of sodium cyanide and sodium fluoride placed on their cuticles, again leaving the salt on the surface, in circumstances where the quantities of these poisons which were applied far exceeded the lethal dose. The animals showed no harmf u l symptoms. Solutions of sodium azide did produce rapid death but this azide is appreciably oil-soluble. Therefore, from the point of view of function as an ion-filter, the grease film under the droplet must be an almost perfect semi-permeable membrane, even if the nature of the transporting machinery acting through that film is completely outside the range of conventional osmosis. Once again we have an example here of the uptake of water by an insect which must unquestionably involve the expenditure of energy from an outside source. In circumstances which are far simpler for us to appreciate, it outlines much of the phenomena we have suspected in the tracheole, and demonstrates even more clearly the need for the extended view of “active” transport. When pure water is placed on the animal it moves down a concentration gradient, but it does so at rates which vary considerably, and the fastest of these is much greater than the physical circumstances would predict. When there is no concentration difference, defined in terms of osmotic pressures of the external solution and the blood, the water still moves quickly; when there is an enormous concentration gradient against which the water moves, we have active transport in its narrowest sense unaccompanied by ions or other transport. Obviously the same forces and the samevital mechanism arc acting in all instances, and all merit the description “active transport” even if only the experiments using strong solutions do on inspeclion justify the description active transport of water itself.

A C T I V E T K A N S P O R ’ I Of I,.

W A T E R I N INSECTS

91

cot-.<; L u s I O N S

A nunibcr o f terrestrial arthropods show the facility to move water by thc expenditure of cnergy under circumstances which grade continuously from simple examples of the movement of water alone up a concentration gradient, through examples where the rates of movement arc high in comparison with expectcd diffusive and osmotic movement, to those whcrc salts are also involved, or where large forces other than those represented by concentration difference are easily overcome. In most Circumstances movement takes place through a cuticular type of membrane backed by a cellular layer such as an epidermis. Expressed in conventional terms the cuticle extracting water from saturated salt solutions or from air of less than 70% R.H. exhibits “osmosis” equivalent to 300 atm; the tracheole may produce 60-100 atm pressure and the hind-gut up to 20 atm pressure. The blood and cellular osmotic pressure of insects is of the order of 10 atm. It remains to be seen whether all these different phenomena could be explained in terms of one mechanism. As Diamond (1962) clearly points out, the demonstration that water will move up a concentration gradient is not necessarily a demonstration of the active transport of water itself if anything else is moved at the same time. In the roach (the fish Rirfilusrutilus) water is carried through the wall of the gall-bladder against a concentration gradient. Diamond shows that thc transport of water could not be accounted for by filtration, classical osmosis or electro-osmosis. He then applies the irreversible thermodynamics of Kedem and Katchalsky (1 958) and concludes that his observed rate of water movement could be accounted for by co-diffusion, the energy being provided in the transport of ions in the water. This carries onc stage further the general contention that if physiological plicnomena can be explained without assuming a n active transport of water itself, then an active transport of water per se is ruled out of consideration. Howevcr, we claim in the preceding discussion to have established two kinds of instance-atmospheric absorbtion and rectal absorbtionin which water moves up a concentration gradient without being associated with other transported substance. Here, active transport of water in its own right surely cannot he denied! Closely parallel with these eases we have examples of water transport involving ions, but some have perfectly semi-permeable membranes in the system: others do not. The circumstances fall outside the Kedem-Katchalsky system and merge into them. Consequently, we are prepared to argue that not only in all

92

J . W. L. REAMENT

thcse systems, active transport of water must be considered, but also that others who have so far eliminated active water transport on a basis of minimum hypothesis should be prepared to reconsider. We shall now turn to a discussion of the mechanisms involved in the active transport of water.

IIr. A N

I N T E R R E L A T I O N B E T W E E N G R E A SAE ND

ABSORBTION

This short section outlines evidence suggesting a very clear relationship between water uptake through the cuticle and the behaviour of grease molecules. The contact angle between water and a surface gives a direct measure of .the adhesion of water to that surface. Thus, both theoretically and practically, when water is placed on the exposed monolayer of an aquatic insect (Holdgate, 1955 as interpreted by Beament, 1960a) and therefore against a packed array of -CH3 groups of organized lipid, there is a contact angle of the order of 130" (Fig. 3C). If a

I

B

A

D

E

C

F

FIG.3. Behaviour of small water-droplets on a living cockroach, interpreted in terms of rearrangement of the polar lipid. The convention used for a lipid molecule is shown on the extreme left. For further explanation, see text.

surface is made up entirely of the polar groups (Fig. 3B) it should attract so milch water that the contact angle should be at or below 30". The grease on a cockroach is thick, and in molecular terms thermal agitation produces a random arrangement in the bulk of the grease above a basal monolayer. Statistically, therefore, the molecules in the outermost surface onto which a water-droplet is applied should present some hydrofuge -CH3 endings, some hydrophil polar endings, and some chains sideways on. This ought to have a contact angle intermediate between the extreme cases, and indeed the contact angle on immediate application of a waterdroplet is about 80"(Fig. 3A).

A C T I V E T R A N S P O R T OF W A T E R IN INSECTS

93

However, the contact angle does not remain constant; over the following 3 min or so (depending on tcmperature) from the moment of application, the contact angle falls to around 30', indicating that in the presence of water the hydrophilic endings are preferentially held in the interface, so that an "inverted" monolayer is formed under the droplet (Fig. 3B). Now from the profile of the droplet its volume can also be determined, and it is important to observe that while this change in the underlying surface is taking place the drop does not change in volume. Only after the surface beneath the drop has reached maximum hydrophilic property does it appear that water can move into the animal at the surprisingly high rate described in Section 11, E. It has already been suggested that grease spreads over a water-droplet on the cuticle and forms a packed and orientated monolayer (Fig. 3D). Thus, immediately a droplet has been absorbed by the animal, the molecular surface of the area of absorbtion should be this collapsed monolaycr surface (Fig. 3E). If one adds a further droplet to this site immediately, the contact angle is at first 130" (Fig. 3F). More grease spreads from the surrounding area onto the droplet outer surface. Now the contact angle over 3 min or so falls to 80" indicating a breakdown in the erect monolayer under the water, and, over a further interval of time, it falls to 30 ' indicating complete inversion of the lipid molecules. Throughout this preliminary period of 6 or 7 min, the drop does not decrease in volume. On the other hand, if one places a droplet on a fresh surface, allows grcasc orientation under it to occur, and the droplet to be almost completely absorbed, and then introduces a second volume of water into the rapidly disappearing droplet, absorbtion continues unabated and the contact angle remains steady at around 30". Whatevcr preparation for fast absorbtion is necessary, it includes the formation of an inverted monolayer at the interface between the water and the rest of the animal. Also, because of the interval of time for monolayer inversion, the period of 10 min on a fast absorbing area, from the time of application of the drop to its disappearance, contains only perhaps 7 min for actual uptake. So far as can be seen, over the areas of the animal where absorbtion is very slow, the molecular rearrangements are nevertheless complcted in about 3 min; it is the actual process of absorbtion which is slow. Temperaturc has only a small cffect on the rate of overturning of the grease molecules, presumably because the basic process is a kinetic one of random movement. But over the range 15-25"C, there is Q,,, of the order of three-suggesting a high temperature sensitivity of the absorbtion mechanism. This need not indicate a "biological" reaction as is

94

J. W. L. ISEAMENT

sometimes automatically claimed from such evidence. Danielli and Davson (1952) show that high temperature coefficients may be associated with, for cxamplc, transport through relatively impermeable lipid films which involve activated diffusion : where only those molecules of sufficiently high energy amongst the difiusant can pass through a resistant barrier in a membrane. A. A MODEL A B S O R B I N G WATER

A model of the system described in the previous section can be set up by removing the epidermis from a thoracic tergite of a cockroach and mounting it as a membrane, with its grease layer intact. The epidermis side is completely enclosed with a small volume of air, containing a pellet of phosphorus pentoxide, and then the whole preparation placed in a desiccator so as to remove all free water from the membrane. With the dry membrane, a water-droplet placed on the greasy side is waterproofed by grease spreading over its surface. It remains for so long that its disappearance can be accounted for by evaporation. If a little water is now sprayed against the epidermis-side, a droplet on the grease disappears very quickly indeed. On adding fresh droplets of water, similar phenomena to those observed on the living cuticle occur; after initial molecular reorganization water is absorbed, but the rate of absorbtion falls as the cuticle membrane becomes saturdted with water. However interesting the anomolous behaviour of this model when the cuticle is completely dry, that is outside the range of biological possibility. Water deficient cuticle in the dead state can provide sufficient force to absorb liquid water through a thick grease film at rates which are comparable with the living integument.

I V . T H EP H Y S I C ACLH E M I S T ROYF THE CUTICLE A certain amount is known about the materials in the bulk of the insect cuticle, and a great deal has been discovered about the epidermis (e.g. reviews by Wigglcsworth, 1948, 1957; Richards, 1951, 1958). Much has recently been discovered about the waterproofing lipids on thecuticle (c.g. review by Bcamcnt, 1961b). Little indeed is known in comparison about the tracheole or the gut-linings. The external cuticle obviously represents the bcst matcrial for invcstigating possible mechanisms of water regulation and active transport, and offers the possibility that discoveries or principles could be applied to the other systems.

A < ” I I V f : ‘I H A N S P O K ’ I 0 1 ; W A T E R I N I N S E C T S

95

A . ’rI1E C I J I I C L oTHtK ~ THAN LIPID LAYER

Watcr is to be transferred from the outside environment through a layer of lipid, layers of epicuticle, layers of protein-chitin complex, the cell membrane and cytoplasm of the epidermal cell, another cell membrane, and thence to the blood (Fig. 4). Whatever energy-consuming process we postulate for active transport must be spatially associated with the living material. Thus (e.g. Beament, 1960b), an actual physical gradient of water-activity, down which water will move by normal physical process, must be created through the non-living layers which are extracellular. We can now specify this prerequisite in addition to molecular rearrangement of the lipid in the example of uptake from liquid water against the surface. }Lipid 0 2p ]Lipoprotein epicuticle

1

.

1 1

0

1 A

0

1

0

o 2~

Procuticle 1-2O/r -Pore conols

Cellular

epidermis

A-.-

FIG.4. A diagrammatic section through an insect cuticle indicating the main layers referred to in the text, and their order of thickness.

The lipoprotein epicuticle or “cuticulin” (Wigglesworth, 1947) is an extremely inelastic substance (Bennet-Clark, 1962) corresponding to the physico-chemical properties one would predict from its composition: the protein is so tightly bound and cross-linked with both lipid and poly-phenolic tanning materials that it cannot swell, and most of the hydratable groups of the original protein will have been used up in the cross-iinking proccss. Therefore if a gradient is to be created in the purcly physical part of the system, it must be produced by lowering the watcr activity in the protein-chitin complex of exo- and endo-cuticle (rcfcrrcd to below by Richards’ term “procuticle”) and this in turn must be mediatcd through the action of the living cells upon it. In this connection, the curious disposition of that characteristic structure of many insect cuticles, the pore canals, may be significant. Although many functions have been ascribed to pore canals (see reviews

96

J. W. L. BEAMENT

quoted above), cuticles devoid of such structures, as, for example, those of the silkworm (Ito, 1953), are entirely morphologically homologous and very largely physiologically analogous with cuticles containing pore canals. But pore canals with cytoplasmic filling are present in the cuticles of animals showing active uptake of water, and would enable the living material to make the most intimate contact throughout the thickness of the chitin-protein complexes, to modify it for the creation of the necessary physical gradient. This view is supported by the evidence of Lees (1947) who showed that the ageing adult tick, which can no longer extract water from sub-saturated atmospheres, has filled its pore canals with solid and probably chitinous material ;it also tallies with the discovery reported earlier that liquid water is not absorbed by the cockroach when in the pre-ecdysial state of having two cuticles and when the pore canal content has been withdrawn from the outer cuticle. B. T H E H Y D R A T l O N O F P R O C U T I C L E

Both the characteristic protein arthropodin, and the polysaccharide chitin, have many hydratable side-chains and groupings. They differ in that those of chitin are not usually considered to be strongly ionizable, so that water associated with chitin will be bound by van der Waals forces, whereas the protein with its strong acidic and basic groups binds water through electrical forces as well as through London and similar forces. The indications of experiments with models (Beament, 1954; Section 111, A) are that a comparatively small degree of dehydration will produce great imbibitional forces with respect to water; all the water which is associated with the chitin-protein complex is strongly attracted. Of course arthropodin (see Rudall, 1963) is very different from the kinds of protein which have recently been suggested as models for the active transport of sugars, ions etc., and in mechanisms such as chelation (e.g. Christensen, 1962; Harris, 1960; Bayliss, 1959). Cellular proteins, perhaps even the special class of cell-membrane proteins, may change their properties by actually folding and unfolding their secondary organization cyclically, thus presenting different configurations and templates for adsorption and desorption, or as carrier substrates. Such proteins may be further unfolded or rearranged, often by quite small changes in circumstances, so as to destroy their biological function entirely in the process commonly called denaturation. The late Sir William Astbury once referred to arthropodin as a protein “always in the denatured state”, which anachronism may be interpreted in modern terminology as meaning that it does not occur in a complexly folded

A C T I V E T R A N S P O R T 01 W A T E R I N I N S E C T S

97

secondary structure. Arthropodin is intercrystallized with chitin, in a very stable association with both sets of chains straight, and can, for example, be compared with Pauling and Corey's (1951) model for &keratin. It is exceptionally stable; it does not appear to be changed in any way when, for example, it is heated by a 70 kV beam in the electron microscope or when the dead cuticle is stored for a long period. Chitin is likewise a most stable substance. For these reasons, membrane preparations of insect cuticle have the great advantage that they may be used for several days without any apparent decay of property. C. C O N T R O L O F P R O C U T l C L E B Y THE E P I D E R M I S

The elasticity of the chitinous cuticle of Rhodnius is under the control of the epidermis (Bennet-Clark, 1961, 1962). In preparation for the plastic expansion of the cuticle by the blood-meal, the animal can convert the chitin-protein part of the cuticle from a material resisting an inflationary pressure of some atmospheres to one which is expanded by an excess abdominal internal pressure of a fraction of an atmosphere. The nature of the control mechanism, which is brought about in life through the action of a plasma dialysate on the epidermal cell, is not fully understood; but in model experiments such changes in the procuticle can be produced in a number of ways, and can be explained on the general assumption that the hydration of the cuticle is altered with a consequent change in the intermolecular binding of the untanned protein and chitin complex. The cuticulin layer is not involved in these changes; it is complexly folded before expansion, and is unfolded when plastic extension of the underlying material takes place. Furthermore, it is the tensile strength of the inelastic cuticulin which sets the ultimate limit on the expansion of the abdomen by blood, despite the minute thickness of the epicuticle. Tanned lipoprotein is thus not under the control of the epidermal cell in this system, and Bennet-Clark's findings agree with thc suggcstions about the properties of this layer made in Section TV, A. It would now appear justified to propose that an epidermal cell can create changes in parts of the cuticle with which it makes direct contact, and that these changes can include control of hydration. Dehydration of the cuticle under the lipid can produce sufficient force to cause an inward current of water at a high rate. We have now to distinguish two effects rather carefully. The condition of hydration of a protein can be modified without nccessarily adding or extracting water; for example least water is bound by a protein at its iso-electric point, and the amount of water

98

J . W. L. B E A M E N T

associated with it incrcases rapidly as its pH is changed in either direction. A strong force, attracting water, can thus be produced or destroyed. Clearly an enormous force is nceded to remove the water bound onto protein by siniplc dehydration-thc force with which dehydrated protein attracts water. Thus one explanation which may be offered for the different rates at which water is taken up by thc different areas of the cockroach (Section 11, E) is that the underlying protein has different degrees of hydration. Indeed, if thc animal were actively making the hard, mechanically rigid cuticlc least plastic, we might expect it to keep the water-content of these areas at a low level while it maintained a very high level of hydration in the soft elastic cuticle for maximum plasticity. Such vital activity would-and does-disappear with anaesthesia or death. But we must not visualize such a mechanism as a simple removal of water, keeping the whole of the protein identical in property; the animal is more likely to adjust the hydration by adjusting the environment of the protein. Even if we have found a mechanism producing sufficient force to transport water inwards through the lipid, we still have not the basis of a continuous-flow active transport mechanism. Any force attracting water inwards through the lipid must act simultaneously in likewise drawing water out of the epidermal cell. We do not know the permeability of the epidermal cell membrane to water, or even if, in the pore canal, there is anything corresponding to a cell membrane-the pore canal is very small to contain a double cell membrane. Should the cell membrane prove as impermeable to water as that of a protozoan (Section IT, D), it is still perhaps one order more permeable than the outer lipid layer, and we must remember that transpiration from the whole animal appears unchanged when the insect dies (Section 11, B). Unless we can find an additional mechanism in the cuticle system, we can only propose the means of discontinuous uptake.

V. T H EC U T I C U L ALRI P I D Much of the evidence for the physico-chemical state and behaviour of cuticular lipid has been given in recent papers by the author (Beament, 1955, 1958a, 1959, 1960a, 1961a, b, c, d, 1962). Summarizing briefly, there is a sharp transition phenomenon in the relationship between tempcrature and permeability for many intact insect cuticles (Fig. 5). Using insects such as the cockroach, where the lipid is a mobile grease, the form of thc tcmperaturelpermeability relationship can be explained

A C T I V E T R A N S P O R T O F W A T E R IN INSECTS

99

if one assumcs: one monolayer of lipid molecules against the cuticulin surface, which are so organized that the chains are tightly packed; overlying this monolayer, grease 20-30 times in thickness but with a random arrangement of molecules due to thermal agitation; the monolayer is five times as impermeable to water as is the randomly arranged grease; the monolayer is stable because of the special adhesion of its polar groups to its cuticulin substrate. When transition obtains (c. 30°C in Peripluneta umericana), the particular packing of the monolayer which produces its great impermeability is thermalfy destroyed and water traverses this region at approximately the same rate as through randomly arranged molecules. But it must be noted that, contrary to

Temperature ---t F~G. 5. Graph showing the form of the relationship between permeability of a monolayer of inscct grease on cuticle and its temperature. previous explanations which have been advanced (Beament 1958a, 1961b), evidence is given below suggesting that the change produced in the monolayer by heat is not its random disorganization. By comparison, an aquatic insect whose surface is extremely and permanently hydrofuge (such as Gyrinus) shows a transition increment with temperature, and an actual permeability, compatible with there being only one monolaycr of grease altogether on its cuticle surface. By extracting thc cuticular grease with solvents, and compressing it on a Langmuir Trough, thc area corrcsponds to the expected surface area of

100

J. W. L. B E A M E N T

the insect. The irregular surface of an insect is very difficult to assess (see also Lockey, 1960) but there is every possibility that the cuticle of a hydrofuge insect is covered by only one monolayer, and that the area of the extracted grease on a Trough is by far the most accurate assessment of the true surface area of an insect. Now it is the outer surface of this layer of grease molecules, with closely packed -CH,groups exposed, which gives the property of low adhesion to water on an aquatic insect. The presence of such a monolayer on a cockroach can be directly demonstrated by washing the animal (Fig. 6) with a spray of waterdroplets. Each droplet falling on the animal is coated with grease,

0

i

FIG.6. Method of washing acockroachwith a water-spray which (a) collects grease uncontaminated by solvents, and (b) produces an extremely hydrofuse surface covered by one monolayer.

which is carried away when the droplet falls off. After a time the whole of the thick grease is carried away; the animal then has a permanent high contact angle such as we associate with an insect like Gyrinus, an actual permeability similar to that of Cyrinus, a very large and sharp transitional break in its permeabilityltemperature relationship which we associate with one monolayer alone, and one can strip from it with solvents a quantity of grease which has the order of one cockroach surface-area when spread as a monolayer on a Trough. By washing with water one can similarly prepare dead cuticle membranes of the cockroach. These preparations are extremely important when considering the special physical properties of monolayers, which will be described below.

A C I I V I : T R A N S P O R T 0 1 W A T E R IN INSECTS

101

A. A NE W I N T E R P R E T A T I O N O t T K A N S l T l O N I N M O N O L A Y E R FILMS

The theory put forward (Beamcnt, 1958a, 1961b) to explain the transition phenomenon of sudden increase in thc permeability of the cockroach cuticle at about 30T, suggested that the organization in the monolayer was thermally destroyed : it became a random assemblage of molecules like the grease which is not in contact with an organizing substrate. Using the preparation described in the previous paragraph, this theory can be directly tested: the monolayer exposed by washing has a contact angle of 130" to a drop of clean water. But if such a preparation is heated complete with water-drop, to a temperature above transition (c. 35"C), the contact angle ought to fall at least to a value of about 80" as the grease molecules become disorganized, and perhaps to 30" through reorientation of the polar groups towards the water, just as happens when water-droplets are placed on the thick grease of the animal (Section 111; Fig. 3A-C). But no such change occurs. The contact angle falls by only perhaps some 10 degrees from 130".Transit.ion therefore occurs without the disorganization of the monolayer, at least in so far as that the -CH3 ends remain externally exposed, and the polar groups are still tied down to the substrate. Now different terrestrial arthropods display transition temperatures covering a very wide range (Beament, 1958, 1959, 1961), from as low as 22°C for Djvtiscus to values around 70°C for resistant pupae and some Argasids. There is a general correlation between the melting point of extracted lipid, the probable length of the paraffinic chain, and the transition temperature: the longer the chain, the higher the transition temperature. The stability of the monolayer depends on the van der Waals forces between the adjacent chains, and the link between the polar groups and the substrate. The link of the polar groups must be very strong-stronger for example than the affinity of a polar group for water; we would expect the polar groups of the waxes from many insects to be very similar, and hence to be alike in their affinity for a cuticulin type of substrate. Hence if thermal agitation were to overcome the adhesion of the polar group to the substrate we should expect all cuticular lipids to show transition at approximately the same temperature. We must however expect the much weaker van der Waals forces to be disrupted first, and the correlation indicates that the shorter chains with less inter-chain forces do go through transition at much lower temperatures than thc long-chain lipids. How can the disruption

102

J . W. L. D P A M E N T

of only the van der Wads forces give rise to a fivefold increase in permeability to water ? The conventional drawing of a monolayer of polar lipid (Fig. 7) shows the molccules as cigar-shaped cylinders with bulbous polar groups, standing vertically with respect to their long axes. Surface chemistry gave the first direct measurements of molecular dimensions and these representations are satisfactory diagrammatizations of the impressions thus obtained of molecules. The dangers of making too literal use of such models is no less great in the field of surface chemistry than in, for example, the contemporary plethora of models which give the principle but not the mechanism of the replication of protein by R.N.A. Nevertheless most surface chemists believe that molecules in a

FIG.7. Conventional drawing of molecules of a polar monolayer; cf. models in Fig. 8.

monolayer do stand exactly vertically, and whenever alternative suggestions have been made in the past, the pioneer of the science, N. K. Adam, always defended the principle of verticality. But it is obvious from these simple models that if the polar groups are larger in crosssection than the parafin chains, the chains cannot be tightly packed. Consider now a more up-todate molecular model of such a molecule (Fig. 8a) which is mainly based upon X-ray crystallography, electronbeam diffraction and similar techniques, and uses dimensions calculated by such workers as Pauling and his colleagues. The paraffin chain is a zig-zag of carbon atoms. The polar groups, especially if surrounded by hydration shells, are certainly larger in cross-section than the chains. But if one manipulates models of these molecules, one can find an arrangement with the axis of the chains at about 244' to the vertical, where the carbon atoms of adjacent chains inter-fit, with the chains off-set by one pair of carbons with respect to their neighbours (Fig. 8b)

A('1 IVI

I R A N S P O R T O F W A T F R IN I S S E C T S

103

F K , . 8. Models o f polar lipid. (a) A polar niolcculc. (b) A row o f molecules i n closcst packing inclined at 24; t o the vcrtical: suggested form below transition. (c) '1-hc samc molecules above transition, uitli iiiean vertical spacing.

104

J . W. 1,. I%! A M L N I

and with the polar group\ in equally closc contact. Such an arrangement will achievc closc packing o f chains, and bring the atoms within an atomic diameter of each other, so that strong van der Wads forces will bc dcveloped. On this basis, impermeability would be expected to be very high, and the arrangement still leaves the -CH, groups exposed at the outer surface. On the basis of a model of this kind it is also much easier to understand the wide differences in the permeability of monolayers of different substances, which differ only by small substitutions in the polar groups (e.g. Adamson, 1960). In conjunction with the radical suggestion made in the previous paragraph, it may be significant that saturated n-paraffins in bulk do crystallize with the long axis of the chain at an angle of 63' 20' to the plane passing through corresponding carbon atoms in adjacent chains, i.e. at an angle very similar indeed to that we propose from our models (Miiller, 1927); while the inter-fit of the zig-zag chains allows such a form of crystallization, it is not necessarily straightforward to see why this form of obliqueness occurs in the absence of polar groups. For clarity, a description has been given of a proposed molecular packing in one particular plane perpendicular to the substrate of the monolayer; a full treatment of this subject is ohviously beyond the scope of the present article (Beament, unpublished work): it must suffice to say that the crystal form in the monolayer must be monoclinic, and that thc model shown in Fig. 8 illustrates the principle. We must now consider what may happen when this newly proposed system is heated. The van der Waals forces along the chains are the weakest in the structure, and thermal agitation will break them first. The molecules can then display their increased thermal energy by vibrating the chains on their fixed polar ends, occupying a tnean vertical position (Fig. 8c) but statistically occupying the space between adjacent chains. Only the fixed nature of the substrate linkage would differentiate this state from a compressed liquid monolayer film on a water surface: the state could be referred to as a one-dimensional liquid. Between the chains there will therefore be much greater space, statistically, for the migration of water, as compared with the tily-packed solid state of the film. Yet the -CH8 groups continue to be exposed at the outer surface to give a high contact angle, and the maintenance of the position of the polar groups ensures immediate return to the solid state on cooling below transition-a characteristic feature of the grease-covered cuticle. A simplified treatment of the change in permeability to be expected when transition occurs from the tilt-packed system to the meanvertical system could be based on the size of the holes between the

AC"I I V I 3 H A N S I * O W I

oi

wA'rI:H

I N iNsEc'is

I05

chains of thc niodcl in the two sfatcs. A water molcculc in such a hole is travclling i n ;I porc o f i t \ o w n ordcr of dimcnsions and according lo Barrcr (194X) the pcrmcability might be governed by an equation of the general form suggcstcd by Knudsen (1909).This states that the rate of flow will be proportional to the cube of the radius of the hole. Such calculations give mean radii of holes of 0.338 and 0.59 A, and therefore a ratio of permeabilities of I :4.8.This is a remarkably close fit with experimentally determined values of fivefold increases for monolayers on water and on cuticles with monolayers, (Beament, 1958a, 1961a, and unpublished work) and the deduced increase in permeability of the monolayer beneath the thick grease of the intact cockroach. In truth, a relationship adequately describing the transferrence of a molecule through a system of this kind might very well contain a term involving the cubc of the radius of the pore, measured when the long-chain molccules arc i n a mean static position, but the idea that a water molecule does actually travel through a hole smaller than itself or even precisely of its own dimensions is hardly in keeping with modern ways of thinking. Molecules and their component atoms are not static in the solid state; water is likely to traverse the monolayer through holes temporarily occurring bCCdUSe of kinetic activity : holes which in the immediate vicinity of the water molecule will be bigger than the water. An individual hole may have only a transient existence, but provided we consider a sufficient area of monolayer we can say that a t any instant of time below transition holes will occur with a certain frequency per unit area. Above transition holes will occur with a greater frequency, and somc of these holes may be significantly larger. But the holes will occur with greater frequency because the van der Waals forces have decreased, and although the mean separation of the chains might have increased by a very small amount, we must remember that van der Waals forces are inversely proportional to the order of the sixth power , o f the distance separating the atoms, so that a small increment in separation may have a surprisingly large effect.

R. M O N O L A Y E R I N V E R S I O N

Based upon permeability measurements, insects such as Dytiscus appear to have one orientated monolayer on their surface (Beament, 1961a). If this is a normal monolayer, the whole surface should be permanently hydrofuge : the respiratory reservoirs are so covered but regions such as the outer surfaces of the elytra are readily wetted by

106.

J . W. I.. 1 1 f A M E N T

watcr. Thc only aolution t o this apparent paradox is the proposal that wettable areas have an inverted monolayer, with the polar groups on the outsidc (Beament, 1960a). The physical demonstration that a monolayer can be produced in the inverted state and is stable is illustrated ir! Fig. 9. The argument is that if the stability of a normal monolayer depends on the high attraction’of its polar groups to their substrate, then a suitable substrate for an inverted monolayer would be a saturated n-paraffin itself which would attract the -CH, endings and not the polar

FIG.9. Right: a ball of pure paraffin wax, coatcd in cockroachpolar grease,washed until no further grease spreads away, dried and dipped in pure water. Low contact angle indicates an inverted monolayer with polar groups outwards. Left: a control of pure paraffin wax without grease, otherwise similarly treated. High contact angle.

groups. The Figurc \bows a ball of dry paraffin wax, coated with cockroach grease and washed in water until all unattached grease has been removed. Thc prcparation has been dried and photographed immediately after placing i n a water surface. It has a very low contact angle, compared with the control of dry paraffin wax. Since the polar groups arc the only wcttable parts of the entire preparation, they must be disposed on the outside. From this follows the suggestion that changes in the natural substrate on the cuticle in the form of the introduction of

A C T I V E 1 K A N S P O R T O F W A T E R IN INSECTS

107

tanning quinoncs into the lipoprotein of the epicuticle causes a monolaycr of lipid, first laid down in the inverted state because of the attraction of (’I I , to lipoprotein, to turn over and form a hydrofuge normal monolaycr (Dcament, 1961b). flccause the removal of water from the tracheal system at hatching and moulting coincides with the tanning of the tracheal cuticle, and because there are parallel systems in the emptying of the respiratory sponge of eggshells (Wigglesworth and Beament, 1950) and in plastrons (Thorpe, 1950), it has been suggested that monolayer inversion provides the mechanism and perhaps gives evidence suggesting that insects may have become air-breathing before they became terrestrial animals (Beament, 1962). Experiments showing for the first time that a monolayer of stearic acid could be inverted on a mica substrate were published in the same issue of the journal carrying the preliminary note on the inversion of insect monolayers (Gaines, 1960). C. CONCLUSIONS

In comparison with the relatively low resistance to the passage of water presented by all other cuticular components, the resistance of the lipid is high and for all ordinary purposes is the controlling factor in the passivc permeability of the cuticle. But the lipid itself can exist in a number of different molecular arrangements which may have very different permeabilities and adhesions, and some lipids are so mobile that their rearrangement in vivo is possible and could even be integral to the regulation of water movement through the cuticle.

VI. THEASYMMETRY OF C U T I C L E Experiments by Hurst (1941, 1948) and Beament (1945, 1948) indicated that under certain conditions water would pass through an isolated piece of insect cuticle more rapidly in one direction than the other and this phenomenon has attracted the slightly unfortunate description : the “asymmetry” of insect cuticle. It is neither an unusual nor a surprising thing that a compound membrane (one consisting of two or more laminae of different property) should allow water or any other diffusant to pass at different rates in the two directions when the driving gradients are identical at the outsides; this has been described in detail for other biological and synthetic systems and the evidence is reviewed by Bcament (1961b). There is, however, one feature of the phenomenon manifest by the insect cuticle which distinguishes it from

108

J . W. L. B E A M E N T

all other such systems which have been investigated, and which therefore requires special attention. An cxplanation of the usual phenomenon encountered in compound membranes has been offered by Hartley (1948), and for brevity this will be referred to as the “Hartley Effect”. Consider (Fig. 10A) a membrane consisting of two components: one lamina is of hydratable material which does not obey Fick’s Law; the other lamina does not hydrate and does obey Fick’s Law. At dynamic equilibrium, the outer surface of the hydratable lamina will have a water-content determined by the

Water

Dry

air

D‘Y

air

*-

A B FIG.10. Diagram representing the “Hartley” effect in a compound membrane, consisting of a swelling phase with large “pores” and a lamina of high resistance to water movement, which obeys Fick’s Law (hatched region). (A) With swollen phase against pure water: large pores and higher rate of flow into dry air. (B) With desiccated phase against dry air: small pores and low rate of flow. Insect cuticle appears to have this form of construction, but the measured rates of flow are the inverse of expectation.

environment with which it is in contact. In general, the degree of hydration will be higher when this side of the membrane faces a higher concentration of water, and lower when next to a lower concentration. The greater the degree of hydration, the larger the pores in the material and the lower the resistance to water; the permeability of the whole membrane is thus related to the swelling of the hydratable lamina. Therefore if we create a gradient through the membrane by disposing a particular concentration of water on one side, and a lower concentration on the other side, the rate of flow through the membrane will be highest if the hydratable lamina faces the high concentration: it will be lower when it faces the low concentration. The resistance of the second lamina will remain constant throughout.

ACTIVE T R A N S P O R T O F WATER IN INSECTS

109

In terms of our previous discussion, the insect pro-cuticle is a hydratable lamina, and the lipid (and probably lipoprotein tanned cuticulin) is not. So long as it does not indulge in molecular rearrangement, we would expect the lipid to obey Fick’s Law. Thus if distilled water is placed against the procuticle, and dry air against the lipid, we would expect much higher transpiration than if there is pure water against the lipid and the proteinaceous inside cuticle is air-dried. But the inverse is usually found. Water passes inwards more readily than outwards in an evaporating system, using cuticles of the cockroach, Rhodnius or the blowfly (Beament, 1948, and unpublished work; Hurst, 1941, 1948; Richards et al., 1953). This we shall call “anomalous asymmetry”. The asymmetry ratio (giving the inwards figure first) reported in the early work varied between 100: 1 and 2 : 1, and all the work was carried out using the gradient between pure liquid water and dry air. The very high vslues of the ratio can almost certainly be explained by bad experimental technique, but values of between 1.2 :1 and 2 :1 have been obtained with the most stringent modern methods (Beament, unpublished work), using desiccation (Fig. 10B). Some experiments by Richards and Fan (1949), using heavy water as a tracer, suggested that when water on both sides of the cuticle was in the liquid phase (instead of one gas phase-air) there was no asymmetry. These experiments were not amenable ;o the calculation of flux rates, and it is doubtful if the results justified so firm a conclusion. But on the same cuticle of a blowfly larva, using the evaporation method, they not only demonstrated very significant anomolous asymmetry, but suggested that the whole of the “lipid epicuticle” had mechanically to be destroyed before anomalous asyrnmctry disappeared. This could prove a very important experiment: our difficulty is that there is no precise homology between the sarcophagan cuticle and that of other orders of insect; from the work of Wolfe (1954) and of Dennell (1946) the dipteran epicuticle cannot be treated as having separatc laminae of cuticulin and free lipid, such as we believe to be the state of affairs in other insects. Now the Hartley effect is real: it can be readily demonstrated with artificial membranes, and we can scarce envisage a situation which would produce a greater effect than the one which contrasts a membrane with its hydratable phasc against distilled water, and that membrane with its hydratable phase against dry air, especially when the lipid lamina cf the membrane has a high water-resistance. Thus there has got to be a Hartlcy effect present in the permeability ratios of these insect cuticle preparations. We have no idca of the relationships to employ in combining a Hartley effect simultaneously with an anomalous asym-

I10

J. W. L. B E A M E N T

metry, but it is safe to say that the true size of the anomalous asymmetry ratio must be higher than we ever mcasure by the evaporation method, and that a membrane which shows no asymmetry must have anomolous properties sufficient to cancel the Hartley effect. We must in fact be surprised if any compound membranc shows no asymmetry at all. Whether the mechanism which gives rise to anomalous asymmetry will prove an important factor in cuticular water regulation or transport only the future can tell; at the present time it has served to draw our attention to phenomena which indicate even greater complication. Just as we have suggested that dry air should desiccate the protein phase strongly and produce an extreme Hartley effect, so also we can suggest (supported by the argument in Section IV, B) that a solution of sucrose with 10 atm osmotic pressure ought not to produce much dehydration of protein in contact with it. Therefore in some recent experiments (Beament, 1964) using dead cockroach cuticle as the membrane in an osmometer, the Hartley effect will be small. Unfortunately, whether salt or sugar solutions arc used to provide an osmotic gradient, using xembranes which do not transmit isotopes and are sensibly perfectly semi-permeable, and taking experimental readings over periods of days, ?he asymmetry ratio can be as high as 10: 1. This ratio is substantially higher than that obtaincd on the self-same pieces of cuticle using ar, evaporation method. Argument as to how much of the experinental values should be attributed to a Hartlcj effect can be avoided by a simple expedient; if we place distilled water on both sides of the membrane and apply small hydrostatic pressures, there is unlikely to be any change in the state of hydration of the protein whichever way the water is moving, in so far as its swelling and permeability is concerned. But there can be very significant differences between the rates of 8ow inwards and outwards through the cuticle, corresponding with the etkct determined by osmosis. We are obliged to infer that the cuticle contains a valve: a device which offers greater resistance to the passage nf watcr i n one direction than the other. There may well be more than one valve; it is impossible at the present time to obtain quantitative relationships betwecii the effects of osmotic pressure, hydrostatic pressure and evaporation gradients, but the behaviour of many pieces of cuticle is consistent. Much difficult experimental work must ensue before this situation is disentangled, but one clue already seems reasonably well established : however the measurements are made, the asymmetry is greatly reduced and may even be reversed in sense by the removal of the free lipid. We have already seen how readily lipid molecules can be reorganized, with consequent very real changes in

AC1 IVE

I RANSI’OR

01. W A I

EK IN lNSECTS

111

permeability. Does thc lipid act as a ‘‘molecular valve”? It certainly can be used to act as a molecular valve to ions (Section VIII, A). H.

T H E R M O D Y N A M I C V A L I D I T Y OF A S Y M M E T R Y

It cannot bc stressed too strongly that there is nothing in principle about the concept that a membrane can have greater permeability in one direction through it than in the other, which is at variance with thermodynamic consideration. To say that there is a “valve” or “rectifier” present in the cuticle instead of talking about asymmetry merely replaces one word by another, and contributes nothing to mechanism. But one cannot make a perpetual motion machine with such a valve, any more than one can with such analogues as a friction band on a pulley or an electronic vacuum tube. But the experiments which indicate :hat the lipid molecules may rcally be capable of acting as a valve stem from the discovery of strong fixed electrical fields in the lipid laycr of the cuticle (Reament, 1961~). V I I. E L E C T R I C PARLO P E R T I E OF S C U T I C U L ALIPIDS R

Since polar molcculcs such as long-chain paraffinic alcohols have slightly displaced electron ficlds, through the presence at one end only of the parafin chain, of the polar group, the molecules carry an electrical dipole moment, which is the causc of the well-known electrical phenomena at the surface of a monolayer on water. The great difficulty of experimenting with monolayers at an air-water interface arises from having to use techniques involving such things as ionizing air-electrodes. If, however, as we have seen, the lipid monolayer is so firmly bound by the links of the polar groups to their substrate that it remains stable even when liquid water is placed at the non-polar end of the monolayer, then the grcat problcm of liquid-air interface surface electro-chemistry is overconic: one should be able to measure surface potential simply by introducing suitable electrodes into aqueous conducting phases on cithcr sidc of .in in\cct cuticle prcpriration. It proved (Beament, 1961c) that between identical solutions of I ‘4, NaCl on cither side of a piece of dead cockroach cuticlc: tlicrc was often a potential difference of the order of 150-200 m V , and that this voltage could be detected on preparations which had been soaked in sodium cyanide and had been dead fcr 110 days. Thc idcntification o f this vollagc with the lipid is readily demonstrated by the simplc expcdicnt of rcmoving the grease. Voltages have also been

112

J. W. L. B E A M E N T

produced on artificial mcmbrancs spread with grease from the cockroach. Similar voltagcs are present on an intact membrane having 0.2 p of grease and those washcd down to a single monolayer : we hduce both that one monolayer only is present in the grease when thick, and that thc monolayer is the source of the potential difference. The original report includes reference to the “electret” or permanently electrostatically charged object, often produced in the past by allowing beeswax or a plant wax in the molten state to solidify in a strong electric field. Such waxes show strong crystallization in powder X-ray diagrams when they have had no special treatment and show no net electretic effect; a reconciliation of these two facts is obtained by using the principle of the “domain”, first derived in the theory of magnetism. Instead of visualizing randomness at the level of individual molecules, one cons i d m supra-molecular aggregations : crystallites with aligned molecules, which havc magnetic, or in this case “electretic”, moments, and randomness occurs at this level of organization. Such devices may be important when considering the effect of applying a strong electrical field across the thick grease of intact cockroach cuticle; 6 V applied momentarily across 0.2 p of grease produces residual electretic voltage of 4 V, fitting closely with the proposed 200 mV per monolayer, on the basis that the whole of the grease molecules have now been aligned. But over a space of some 10 min from the time of removal of the polarizing voltage, the system decays back to 200 mV. Decay is exponential, which is what we would expect if the randomization had been produced thermally. Whether this randomization is at molecular level or domain levcl remains to be seen: the actual phenomenon and the time scale of events parallel closely the suggested process (Section III) for the reorientation of grease molecules under a water droplet. Thick grease can be temporarily polarized into an electret of either sign, according to the sense of the polarizing voltage, but always decays back to the condition of the single monolayer of natural cuticle. A. T H E E L E C T R I C A L I N V E R S I O N OF A M O N O L A Y E R

It will already be apparent that the electrical properties of the cuticular lipid are going to provide a powerful tool for further investigation into the various problems of the control of cuticular transport. One obvious application (Beament, unpublished work) is to test the relatively novel proposition of Section V, B that a monolayer can be inverted. Fig. 11 shows a rcsult of a typical expcriment on a dead cockroach cuticle washed down to a monolayer. As the applied electrical field, tending to

A C T I V E T R A N S P O R T O F WATER IN I N S E C T S

113

FICAI 1. Current flowing through a monolayer-cuticle preparation mounted betwecn identical solutions of NaCI, when potential differences are applied across the preparation. Below each graph, an interpretation of events in the monolayer. (A) Applied voltage tending to invert the monolayer: current rises, falls as though packing is increased, then rises when the electretic voltage indicates monolayer inversion. (B) The preparation with inverted monolayer, subject to an applied potential tending to revert the monolayer. Similar events occur, but the reversion voltage is much smaller than the inversion voltage.

114

J. W. L. B E A M E N T

invert the monolayer, is increased, the electric current passed by the prcparation does not increase proportionally; thc preparation appears to increasc its resistance..Assuming that the resistance of the grease will be several orders higher than that of the rest of the preparation, the local potential gradient tending to invert the monolayer may be more than 1 MV/cm. Up to a certain value of applied voltage the reduction in electret voltage is temporary; after removing the applied voltage the preparation will recover. But beyond this range of polarizing field, the current falls sharply and then rises in a fashion more resembling an Ohm's Law: the permanent electretic voltage has now been reversed and the monolayer has been inverted. If this preparation with an inverted monolayer is now subjected to an applied field tending to turn the polar molecules back into normal orientation, a similar kind of phenomenon occurs: the current rises, then falls sharply and rises again, and an electretic voltage of normal sense reappears; the monolayer has been restored to normal state but only about one-fifth of the applied voltage needed to invert the monolayer is required to turn it back again.

FIG.l'!. Indicating that if monolayer molecules were most stable when vertical, an applied force acting perpendicular to the monolayer would produce no turning movement and inversion would be sudden; if molecules are tilted in most stable position, the applied force will produce increasing turning movements.

This kind of experiment gives independent confirmation of some of the theoretical principles upon which the whole of these ideas about waterproofing have been based. Far greater electrical force is needed to dislodge the polar group from its substrate than to dislodge the -CH, group of the inverted monolayer. rndecd with refinement of technique it might be possible directly to measure the strength of the polar bond, and of the adhesion of -CH, in this fashion. More important however may be the implication of the increase in impedance as the applied voltage is increased. Suppose (Fig. 12) the most stable position of a molecule in a natural monolayer had been with the long axis perpendicular to the substrate plane. One would not have expected any change in the Properties of the monolayer until inversion occurred suddenly.

A C T I V E T R A N S P O R T O F W A T E R IN I N S E C T S

115

Suppow o n the other hand, as is cssential to our general theory, that thc mol:culcs arc packed tilted; the resolved part of the applied field acting at right-angles to the axis of the lipid will produce a turning moment tending to increase the packing. Packing, electrical impedance

and permeability are obviously interrelated properties, and the experiment is best interpreted on the assumption of molecules initially tilted. B. M E C H A N I C A L DI S TO RTIO N

The monolayer preparation shows electrical changes with mechanical distortion, i.e. piezo-electric phenomena. This must follow from the proposed construction of the monolayer, since mechanical displacement of the dipole of the lipid molecule will change the electretic field. At the present time this phenomenon is primarily an experimental hazard ; in the long-term view a molecular mechanism linking together mechanical distortion, electrical changes in a membrane, and permeability changes, may be helpful in interpreting the functioning of senseorgans and perhaps even of sensory nerve-endings. C. T H E P A S S A G L OF S U B S T A N C E S T H R O U G H M O N O L A Y E R S

Assuming a monolayer to have thickness of the order of 50 A, an object passing through it must experience a potential gradient of the order of 2-300000 V/cm. While this would be expected to produce certain effects o n a particle carrying a single charge, such as an ion, inspection of the molecular model suggests that, difficult as it may be to produce a space for the translation of a water molecule, the chance that there would be a hole big enough for a hydrated ion to pass between packed lipid molecules is remote indeed. This suggestion is borne out by the physiological experiments of Section 11, E, and by experiments (Beament, 1961c, and unpublished work) using the cuticle as the semipermeable membrane in osmometers, with isotope-labelled ions. In fact, membrane preparations fall into two categories: those with lower electrical impedance which under certain circumstances will transmit ions, and those of higher electrical impedance which are permeable to water but show exceptional resistance to the passage of ions and larger particles. If we are practical, we cannot assume perfect organization of a monolayer over the area of a membrane preparation 0.25 cm2, and if the rnoleculcs are packcd tilted, it is even more remote to think that over the wholc ;ma they will tilt the ~aiiieway! One interpretation of the

116

J. W. L. B E A M E N T

idea of domains is to consider the existence of micro-areas where all the molecules point the same way, with discontinuities at the junctions of differently orientated domains. This will give rise to pores of greater permeability and areas of high impermeability. Thus if the mechanism for actively transporting water is associated with the monolayer, it will always work in parallel with pores for free diffusion. Such a scheme fits with much of the available evidence: a water-pump and independent osmotic or diffusive leakage. Phillips’ (1964) work on the locust hindgut for example illustrates this. The possibility of an ion pump based upon the electret potential is discussed in thc next section. Here, we must consider theoretically the effect on a polar molecule-water, of the electret field; a qualitative discussion is appropriate. For its size, the unionized water molecule has a remarkably high dipole moment; this contributes substantially to the whole range of unexpected physical properties of water which are otherwise so out of keeping with the simple formula H,O. In the ordinary liquid state, these dipoles produce statistical aggregations. When a molecule passes through a space between two orientated lipid molecules, its freedom of movement is so restricted that in the plane at right-angles to the length of the pore it cannot exhibit the properties of a liquid or gas. It is from this kind of argument, as well as from experimental evidence, that one refers to water in transit through certain forms of membrane as “ice”, though it may be more correct to visualize a twodimensional solid-one-dimensional liquid : a state perhaps analogous to the anchored monolayer molecule above its transition temperature. Experiments indicating “ single-file” water molecules in a membrane are described, for example, by Diamond (1962). Consider however the condition of a diffusing molecule of high dipole meeting with a pore in an electret field. There will be two separate additional factors because of the dipole (Fig. 13). The water molecule arriving at the lipid with appropriate dipole orientation will be attracted into the electret field, whercas the water molecule arriving in reverse orientation will experience a repulsion. There will also be a rotational effect: the electret field will tend to orientate the water molecule so as to attract it into the field. Thus the selection of appropriate molecules and the regimentation of partly orientated molecules will result in a higher probability of linked single-file molecules of water in a pore through the grease. The importance of this concept of a “string” of water molecules can be seen in relation to any mechanism for its active transport. In all the systems where the diffusant moves down the concentration or activity

ACTIVE TRANSPORT OF W A T E R IN INSECTS

117

gradient, the ultimate source of the energy is kinetic. In simple diffusion more molecules randomly move down the gradient than up it; in activated diflusion more molecules of higher energy move down the gradient than up it; in facilitated diffusion, more molecules of higher energy get onto the higher end of the carrier system and displace molecules at the lower end of the carrier, than get onto the lower end of the carrier and displace molecules at the higher end. (“Carrier” is used in the simple sense of a static substrate providing a preferential linkage for the d8usant : the diffusant moves along it, and there is no suggestion in this paper of a “carrier” which moves or oscillateswithin amembrane.) All three varieties of passive transport contain the analogue of a pressure pushing molecules through the membrane, and therefore requiring no attraction between the diffusing molecules. Active transport in the sense used in this paper visualizes an energy-producing system at the higher

t

0”

- 1

+0-

9+

I-

FIG.13. Suggesting the effect on a polar diffusingmolecule of water (circle with line through dipole axis) of the electret field of a monolayer, tending to produce single-file. water crystals in a “pore”. For further explanation, see text.

end of a gradient (within the cell with respect to its membrane: deep in the integument with respect to the monolayer of lipid) and requires the analogue of pulling the translated molecule from the inside of the membrane. Jt is obvious therefore that the active removal of molecules from one erd of a “string” tends to produce an overall inward movement in the string, and that processes increasing inter-molecular attraction of the diffusant can be important. VIII.

O N PUMPS

Harris (1960) in his book on transport in biological systems surveys knowr! methods of producing a movement of water. These include hydrostatic pressure, diffusion promoted by osmosis, anomolous osmosis, electro-osmosis, transfer by ions and “pumping by contractile

118

J. W. L. B E A M E N T

structures”. From the systems we have been discussing we can eliminate hydrostatic pressure and simple osmosis ; anomolous osmosis is in general 2 transient phenomenon while counter-flow requires a second diffusant. Electro-osmosis would require water to have a net charge instead of a dipole, together with a continuous supply of electrical current. Under the heading of “pumping movements” he describes the contractilc vacuole and admits that the reason for its filling with water is unknowii; he includes Kitching’s (1954) suggestion that a secretory mechanism must be involved. Harris’s novel suggestion of pumping by changing the shape of a cell, exposing first a greater area to one solution and then to another, cannot apply to a system including rigid membranes in any case. It does seem that any attempt to provide a mechanism for the active transport of water per se must involve some new concepts or principles. A. T H E E L E C T R E T I O N - P U M P

Beament (1961~)points out that two solutions of different ionic composition, separated by a. permeable barrier, will inter-diffuse to produce two solutions of the same composition, but (Fig. 14) in this diffusion cell the potential difference between the two sides decreases to zero in the process. If, however, the products of diffusion are removed by, for example, perfusion, thus maintaining the composition of the two sides, the potential difference is maintained. The energy is provided by the perfusion process. If the potential difference is balanced out by an externally connected source of electrical potential (not of energy) ’10 current flows in the system: this is an equilibrium state in which no energy can be derived from the difference between the solutions. The source of balancing potential can be the electret voltage across the membrane. Since this state of affairs represents equilibrium, it follows that when two identical solutions are placed on either side a membrane having such an electrostatic potential, they will tend to change towards the equilibrium state: two solutions of different composition. The device will therefore tend to transport ions of opposite charge in opposite directions, and this is substantiated in experiments with membranes reported in that paper. In the light of further experiments, it appears that pumping of ions is achieved with membranes which have a relatively lower electrical impedame; they probably have larger pores or imperfections in the monolayer, through which the actual transport of the ions occurred. Membranes of high impedance do not transmit ions. In a whole series

A C T l V E T R A N S P O R T OF WATER IN I N S E C T S

m

119

Perneobie membrane

1.,A

lhe product$ of

ddfu',!on

Or

Permeable charged membrone

m

Moximum entropy

FIG. 14. The clcctret ion pump. Upper diaflms: a concentration cell, with a permeable but uncharged membrane, will run down until solutions on either side arc of identical concentration, when there is no volate act- the membrane. This is accelerated if the solutions are stirred by internal perfmion. If the two sides arc continuously perfused with new solutions, the voltage is maintained; energy is provided by removing the products of diffusion. Lower diagrams: if the membrane has a permanent electrostatic potential across it, this will appear on a volt.meter of suitably high impedance. One may not draw energy continuously from this system.If one introduces a source of potential to balance out the static membrane potential. the solutions remain identical on either side. If one stirs (introducing energy) selective diffusion will occur, giving two different solutions whose concentration potentiel balances out the membrane potential. If om continually remom the products of diffusion by perfusing with fresh solution, energy is supplied, and on0 may use the maintained membrane potential to perform work.

I20

J. W. L. B E A M E N T

of experiments with such mcmbrancs (Beament, unpublished work), using NaCl labcllcd iis sodium or as chloride, the transport with the electrct voltage and the diffusion against the electret voltage was measured. There was no significant transfer of ions over periods of 24 h. Potentials of up to 4 V were then placed across the membrane preparation, and the movement of sodium and of chloride separately determined in either direction. Only in two conditions were significant quantities of ion moved from one side of the membrane to the other. Sodium and chloride ions each respectively moved down the electro-potential gradient of 4 V when this also had inverted the monolayer. These experiments confirm the remarkable impermeability of the monolayer to larger particles; they indicate that the lipid may be made very significantly more permeable by rearranging the lipid monolayer. But they also indicate that when water is moved through this system it cannot be in association with an ion because an ion will not be transmitted. B. CONTINUOUS-FLOW WATER-PUMPS

We have already seen that the proteinaceous cuticle may be under the control of the epidermal cell, and that its hydration may be modified by vital action (Section TV, C). By dehydration sufficient force attracting

A

E

FIG. 15.

B

C

F

G

D

H

An active-transport water-pump. For explanation, see text.

water may be developed in cuticular protein to parallel the circumstances of active uptake (Section IV, B). Complex as the events may be, the study of the “asymmetry” of the cuticle shows the presence of one or more “valves”--devices which have a greater resistance to the passage of water in one direction than the other (Section VI), and molecular rearrangement of the lipid monolayer is associated with a change in

ACTIVE TRANSPORT OF WATER I N INSECTS

121

rate of water absorbtion (Section 111). Can we combine these properties to form a continuous-flowwater-pump ? Consider (Fig. 15) a sheet of protein separating two identical aqueous solutions. The protein is at its iso-electric point and water is in equilibrium distribution between the protein and the solutions. By the application of energy the circumstances within the protein are changed (Fig. 15B) so that it is moved away from its iso-electric point; its hydration potential increases and it takes up water with considerable force equally through both sides from the solutions. The protein is now returned ?o its iso-electric point; equilibrium is restored by passage of water back into the solutions (Fig. 1SC). There is no net change over the cycle. Now (Fig. l5D) add to w e surface of the protein, a valve of such a kind that its resistance to water passing into the protein is less than its resistance to water passing from the protein back into the solution. The hydration-dehydration cycle of the protein is now repeated. As the hydration potential of the protein is increased, water is drawn in from both solutions; because even in its “open” state the valve still represents a resistance not present in the situation of Fig. ISA-C, more water may be drawn in from the solution on the free side of the protein (Fig. 15E). But on returning the prQtein to its initial state, water endeavouring to pass back through the valve encounters a high resistance, and even more water is transferred back into the solution on the free side (Fig. 15F). The amount transferred in each cycle will, inter alia, be proportional to the back-to-front ratio, i.e. the asymmetry ratio, of the valve. Of course an extremely efficient pump could be produced by having a valve on either side of the protein membrane, and by multiplying this in a laminar system (Fig. 15G and H), but the principle has already been made plain. We must confine. our attention to the simplest model outlined above which has one particular property similar to the specification of the transporting devices we were considering in Section 11. The model pump (as with all suction pumps) cannot draw water in through the valve unless the pressure inside (the activity level of the water) is reduced below the level outside, for otherwise there will be no gradient between the outside world and the protein. If the activity level of the outside world is not reached, the whole of the dehydration of the protein must be satisfied by the transference of water from the solution of free access. On the other hand, the valve can still allow water to travel outwards, and when the protein is re-hydrated on the sccond half of the cycle there could be a quite substantial leakage into a phase with very low activity-dry air for example. Thus this pump would have an

122

J . W. L. R E A M E N T

equilibrium condition in which it would establish, for example, a certain humidity in surrounding air; if thc level of activity of the water outside the valve werc very low, thc amount of leakage outwards might not be significantly different whether the protein was going through a cycle or not. Thus one would not expect to find a difference in the rates of evaporation from live animals with a pump trying to work, compared with that from similar dead animals, in such an extreme condition as desiccation in dry air. C. I S THE L I P I D THE W A T E R - V A L V E ?

A great deal of work must be done before one could be sure that the pump outlined in the previous section existed in the material of the cuticle. There are interesting pointers ; they could have entirely different explanations. Thus in Section 11, B we noted the proposal, originally of Mellanby (1932) taken up by others, that when one determines the equilibrinm condition of an animal regulating the water-content in the air surrounding it at different temperatures, equilibrium is established at a particular relative humidity rather than at a particular saturation deficit. Now the passage of water through a lipid is almost certainly an activated diffusion (e.g. Danielli and Davson, 1952) and is therefore exponentially related to temperature. The amount of water vapour in saturated air is also an activation process, similarly exponentially related to temperature. Thus even though the cuticle, and the physicochemical processes going on in it, are complex, if the lipid does occupy a dominant place in the pump-mechanism we should not be surprised to find this particular relationship to relative humidity. With a theory of this kind, one is tempted to make short-cuts, so that the follcwing observation could be meaningless or it could be most significant. In Section 111 we discussed the uptake of liquid water and noted the disappearance of a differential pattern of absorbtion when a cockroach was anaesthetized. If a cockroach is subjected to a very heavy dose of carbon dioxide, we would expect the pH of its tissues and of the cuticle to be shifted considerably. When one does this the animal “sweats”-small droplets of water appear on the surface of the cuticle, and are resorbed when it recovers. Has this brought the protein to the lowest hydration-potential, or is the whole situation far more complex? As the author pointed out first in 1945, if the rates of passage of water through the cuticle are different in the two directions and if the structure has asymmetric permeability, it must itself be asymmetric in construction. With our present knowledge, no single structure is more

A C T I V E T R A N S P O R T O F W A T E R I N INSECTS

123

asymmetric than the monolayer itself; water experiences an entirely different sequence of events passing through a monolayer in one direction than in the other. Another obvious feature of the phenomena discussed in Section 111 is that liquid water is not absorbed until the monolayer beneath it is inverted. Very little has been said in this review about experimental difficulties or experimental methods; some of these were examined critically in another paper (Beament, 1961b). It is, however, worth recording that dynamic flow methods using osmotic forces in non-equilibrium conditions are exceptionally difficult. Much has been written about the experimental uncooperativeness of semi-permeable membranes ;preparations of cockroach cuticle have none of these drawbacks, and with them one may verify most elegantly that osmotic solutions obey the gas laws when under static conditions, counterbalancing osmotic pressure by a hydrostatic force. But however carefully and efficiently one perfuses surfaces, a minute hydrostatic pressure will move far more water through a cuticle than a theoretically large osmotic force. This is why, in the discussion above, great emphasis was placed on the future value of the use of hydrostatic pressure as a research tool ;preliminary experiments suggest that in one direction through the cuticle transfer is proportional to pressure, but in the other direction it cedinly is not. This again is a characteristic of a valve. XI. WIDERIMPLICATIONS

In our opening paragraphs, and in another paper (Beament, 1961c), we suggested that the cuticle might prove a model from which to develop theories of importance to the general functioning of the cell membrane. The lipid in cell membrane may be chemically very different to the lipid on cuticle, and it almost certainly occurs in bimolecular leaflets. But in principle the idea that potentials and ion-distributions can arise from a net electretic potential still holds. Such systems are piezo-electric as in the broadest sense are mechano-receptors. An imposed electrical field brings about rearrangement in the lipid and a change in permeability to ions, sometimes with a consequent transfer of ions; conversely a change in the organization of the lipid produces a change in the electropotential across the monolayer membrane. These suggestions alone should be sufficient to convince the reader that the study of the permeability of insect cuticle could prove to have wide implications for general physiology and biophysics.

124

1. W.

L. BEAMEN?

X. S U M M A R Y 1. Insects transfer water into their blood (R.H. equivalent 99 %) from air of relative humidity as low as 70%. Water can be taken up through the rectum against large concentration gradients. In both instances there is an active transport of water without transport of other materials. 2. Uptake of water from the tracheole and from solutions in contact with the mticle require a supply of energy. The cuticle can be perfectly semi-permeable. The circumstances grade continuously from active transport in the narrowest sense into those where energy must be supplied although an activity gradient is not established. 3. The facility to transport water actively may be a common property of insects; the particular examples which are discussed are free from objection on grounds that complementary processes could hate promoted the movement of water passively. 4. The absorbtion of liquid water into the cuticle is accompanied by temporary reorganization of lipid molecules. Water-uptake can be imitated with models of cuticle; the force moving the water is produced by dehydrating protein. 5. A careful distinction is drawn between an absorbtion process which stops when saturated, and a continuous-flow transporting mechanism. 6. The cuticular protein is under an active control by the epidermis and there is specific evidence that it can modify the hydration of the cuticle. The pore canals may be important in this process. This may allow temporary absorbtion of water, but does not provide a continuousflow pump. 7.Changes in permeability of cuticular lipids and especially of monolayers are brought about by thermal agitation. A new explanation of the phase transition in monolayers is advanced, based upon the theory that monolayers are constructed with long-chain molecules inclined at about 25” to the vertical. 8. Monolayers can be inverted in natural circumstances, and by the application of electrical forces. Natural inversion may account for the prcduction of hydrofuge areas in insect respiratory systems. The experiments with electric fields confirm the arrangement of the cuticular lipids which has been postulated from experiments on permeability. 9. The evidence for “asymmetric” permeability of insect cuticle is discussed and new experiments are described. The cuticle contains a valve having a greater resistance to water in one direction than the other.

A C T I V E TRANSPORT OF WATER IN INSECTS

125

10.The monolayer is an electret; this may be used as the basis of an ion pump, and may have an effect on the state of water traversing the lipid. 11. The various elements now known to be present in the integument may be combined together to produce a pump actively transporting water. The working of this pump is described. Some of its properties correspond with the general phenomenon of active uptake of water by an insect: it works down to a specific IeveI of water-activity, which would be proFortiona1 to relative humidity rather than saturation deficit, and though endeavouring to work, it would not have any effect against very low activity levels such as dry air. 12. Some of the wider implications of the cuticle model upon the general field of cellular transport and electrical phenomena are briefly discussed. I have received advice and criticism from many colleagues during these researches and I am grateful for the privilege of referring to the unpublished work of others. I wish especially to acknowledge the advice of Professor H. C. Longuet-Higgins, F.R.S.,and the many hours of discussion I have spent with Dr. K. E. Machin. In this review I have made xithout reference, a number of generalizations about insect physiology, and usually these will have stemmed from Professor Wigglesworth’s textbooks, if not from his conversations and own researches. I am grateful to him for this, as well as for the freedom with which he has allowed me to pursue in his Sub-Department, a field which has sometimes seemed far removed from classical insect physiology. REFERENCES Adam, N. K.(1948).In the reported discussion of papers. Disc. Furuduy Soc. 3,lO. Adamson, A. W. (1 960).“Physical Chemistry of Surfaces”. Interscience, New York. Barrer, R. M. (1 948). Fluid flow in porous media. Disc. Faruhy Soc. 3,61-72. Bayliss, L.E.(1959).“Principles of General Physiology”. London, Longmans. Beament, J. W. L. (1945).The cuticular lipoids of,insects. J. exp. Biol. 21,115-131. Beament, J. W. L. (1948). The role of wax layers in the waterproofing of insect cuticle and eggshell. Disc. Faraday SOC.3, 177-182. Beament, J. W. L. (1954).Water transport in insects. Symp. SOC.exp. Bfol.8.94-117. Eeament, J. W. L. (1955).Wax secretion in the cockroach. J. exp. Biol. 32,514-538. Beament, J. W.L. (1958a).The effect oftemperature on the waterproofing mechanism of an insect. J. exp. B i d . 35,494-519. Beament, J. W. L. (1958b).The measurement and control of humidity. I n “Electronic Apparatus for Biological Research”. Butterworth, London. Beament, J. W. L. (1959). The waterproofing mechanisms of arthropods. I. The effect of temperature on cuticle permeability in terrestrial insects and ticks. J. exp. Biol. 36, 391-422.

126

J. W.L. B E A M E N T

Beament, J. W. L. (1960a). Wetting properties of insect cuticle. Nature, Lond. 186, 4084. Bearnent, 4. W. L. (1960b). Physical models in biology. Synrp. SOC.exp. Biol. 10,14. Beament, J. W. L. (1961a). The waterproofing mechanism of arthropods 11. The permeability of the cuticle of some aquatic insects. J. exp. Biol. 38,277-290. Bearnent, J. W. L. (1 961b).The water relations of insect cuticle. Biol. Rev. 36,281-320. Beament, J. W. L. (1961~).Electrical properties of orientated lipid on a biological membrane: an electrostatic diffusion barrier and ion pump. Nature, Lond, 191, 217-221. Beament, J. W. L. (1961d). The role of physiological mechanism in animal competition. Syrnp. SOC.exp. Biol. 15, 61-71. Beament, J. W. L. (1962). The surface properties of insects-some evolutionary and ecological implications. Proc. Linn. SOC.Lond. 173, 115-119. Beament, J. W. L., Noble-Nesbitt, J. J. and Watson, J. A. L. (1964). The waterproofing mechanisms of arthropods IV. J. exp. Biol. (in press). Bennet-Clark, H.C. (1961). The physiological mechanisms of feeding in bloodsucking bug, Rhodnius prolixus. Thesis, University of Cambridge. Bennet-Clark, H. C. (1962). Active control of the mechanical properties of insect cuticle. J. Insect Physiol. 8, 227. Brocher, F. (1915). Recherches sur la respiration des insectesaquatiques. Rev. suisse Zool. 23,401-438. Browning, T. 0.(1 954). Waterbalance in the tick OrnithodorusmoubufaMurray with particular reference to the influence of carbon dioxide on the uptake and loss of water. J. exp. Bid. 31, 331-340. Browning, T. 0.(1957). Quoted by Edney (1957). Bult, T. (1939). Over de Beweging der Vloeistof in de Tracheolein der Insecten. Thesis,University of Groningen. Bwton, P.A. (1930). Evaporation from the mealworm and atmospheric humidity. Proc. TOY. SOC.B 106, 560-577. Bwton, P. A. (1931). The law governing the loss of water from an insect. Proc. roy. enf. SOC.Lond. 6, 27-3 1. Buxton, P. A. and Mellanby, K. (1934). The measurement and control of humidity. Bull. ent. Res. 25, 171-175. Champion, F. C. and Davy, N. (1941). “Properties of Matter”, pp. 99-164. Blackie, London. Christensen, H. N. (1962). “Biological Transport”. Benjamin, New York. Danielli, J. F. and Davson, H. (1952). “The Permeability of Natural Membranes”. Cambridge University Prus. Davies, M.E. and Edney, E. B. (1952). The evaporation of water from spiders. J. exp. Biol. 29, 571-582. Davson, H. (1 959). “Textbook of General Physiology”. Churchill, London. Dennell, R. (1946). A study of an insect cuticlc: the larval cuticle of Surcophugu falculata Pand. (Dipt.). Proc. roy. SOC.B 133, 348-373. Diamond, J. M . (1962). The reabsorptive function of the gall-bladder. J. Physiol 161,474-502. Edney, E. B. (1947). Laboratory studies of the bionomics of the rat fleas, Xenopsylla brasiliensis Baker and X . cheopis Roths. 11. Bull. ent. Res. 38, 263-280. Edney, E. B. (1957). ‘“TheWater Relations of Terrestrial Arthropods”. Cambridge University Press.

A C T I V E T R A N S P O R T OF WATER IN INSECTS

127

Gaines, G. L. Jr. (1960). Overturning of stearic acid molecules in monolayers. Nutwe, Lond. 186, 3114385. Govaerts, J. and Leclercq, 1. (1946).Waterexchangebetween insects andair moisture. Nature, Lond. 157, 483. Harris, E. J. (1960). “Transport and Accumulation in Biological Systems”. Butterworth, London. Hartley, Ci. S . (1948).Contribution to a discussion on asymmetry. Disc.Faraday Soc. 3, 223. Hodgkin, A. L. (1951). The ionic basis of electrical activity in nerve and muscle. Biol. Rev. 26, 339-409. Hodgkin, A. L. (1958).Ionic movements and electrical activity in giant nerve fibres. Proc. roy. SOC.B 148, 1-37. Holdgate, M. W. (1955). The wetting of insect cuticles by water. J. exp. Bwf. 32, 591-617. Holdgate, M.W. (1956).Transpiration through the cuticles of some aquatic insects. J. ex9. Biol. 33, 107-1 18. Hurst, H. (1941). Insect cuticle as an asymmetric membrane. Nature, Lond. 147, 388-389. Hurst, H. (1948). Asymmetrical behaviour of insect cuticle in relation to water permeability. Disc. Furaday Sac. 3, 193-210. Ito, T. (1953). Studies on the integument of the silkworm, Bombyx mori, 7 . Biol. Rev. 105, 308-3 15. Kalmus, H. (1936). Die Venvendung der Tracheenblasm dcr Corethralarve als Mihohygrometer. 2. wiss. Mikr. 53, 215-219. Kedem, 0.and Katchalsky, A. (1958).Thermodynamic analysis of the permeability of bidogical membranes to nonelectrolytes. Biochim.biophys. Acta 27,229-236. Kitching, J. A. (1954). The physiology of contractile vacuoles IX. Effects of sudden changes in temperature on the contractile vacuoles of a suctorian. J. exp. Biol. 31,68-75. Knudsen, M. (1909). Die Gesetze der Molekulantromung und der inneren Reibungsstromung der Gase durch Rohren. Ann. Physik. 28, 75. Lees, A. D. (1 946). The water balance in Ixodes ricinus L. and certain other species of ticks. Purisitology 37, 1-20. Lees, A. D. (1947). Transpiration and the structure of the epicuticle in ticks. J. exp. Biol. 23, 379410. Locke, M. (1958). The formation of tracheae and tracheoles in Rhodnius prolixus. Quart. J. mirr. Sci. 99, 2 9 4 6 . Lockey, Y.H. (1960). The thickness of some insect epicuticular wax layers. J. exp. Biol. 37, 3 16429. Ludwig, D. (1937). The effect of different relative humidities on respiratory metabolism and survival of the grasshopper Chortophaga viridifasciata. Physiol. 2001. 10, 342-35 1. Martin, J. (1893).Les tracheeset respiration trachenne. C. R. SOC.Philomat. Paris6.3. Mellanby, K. (1932).The effect of atmospheric humidity on the metabolism of the fasting mealworm Genebrio molitor). Proc. roy. SOC.B 111, 376390. Milllet, A. (1927). An X-ray investigation of certain longchain compounds. Prm. roy. SOC.A 114, 542. Nachmansohn, D. (1961). “Molecular Biology”. Academic Press, New York and London.

I28

J.

W. L. BEAMENT

Pal, R.(1950). The wetting of insect cuticle. Bull ent. Res. 41, 121-139. Pappenheimer, J. R. (1953). Passage of molecules through capillary walls. Physiol. Rev. 33, 387-423. Pauling, L. and Corey, R. B. (1951). The structure of keratin. Proc. not. Acad. Sci., Wash. 37, 729. Phillips, J. E. (1964). Rectal absorbtion in the Desert Locust. I. J. exp. Biol. 41,15-38. Phillips, J. E.(1961). Studies on the rectal absorbtion of water and salts in the locust Schistorerca gregaria and the blowfly Calliphora erythrocephala. Thesis, University of Cambridge. Ramsay, J. A. (1935). (2). The evaporation of water from the cockroach. J. exp. Biol. 12,373-383. Ramsay, J. A. (1950). Osmotic regulation in mosquito larvae. J. exp. Biol. 27, 145-1 57.

Ramsay, J. A. (1954). Active transport of water by the Malpighian tubules of the stick insect Dixippus morosus. J. exp. Biol. 31, 104-1 13. Renkin, E. M. (1954). Filtration diffusion and molecular sieving through porous cellulose membranes. J. gen. Physiol. 38, 225-243. Richards, A. G. (1951). “The Integument of Arthropods”. University of Minnesota press. Richards, A. G. (1958). The cuticle of arthropods. Ergebu. Biol. 20, 1-26. Richards, A. G. and Fan, H.Y.(1949). Studies on arthropod cuticle V .J. cefl.comg. Physiol. 33, 177-198. Richards, A. G., Clawen, M.B. and Smith, M. N. (1953). Studies on arthropod cuticle. X. The asymmetrical penetration of water. J. cell. romp. Physiol. 42, 395413. Rudall, K. M. (1963). The chitin/protein complexes of insect cuticle. I n “Advances in Insect Physiology” (J. W. L.Beament, J. E. Treherne and V.B.Wigglesworth, eds.), vol. 1, pp. 257-313. Academic Press, London and New York. Schneiderman, H. A. (1953). The discontinuous release of carbon dioxide by diapausing pupae insects. Anat. Rec. 117, 540. SutclifTe D. W.(1961). Studies on salty water balance in Caddis larvae. I. Osmotic and ionic regulations of body fluids in Limnephilus uffnis. J. exp. Biol. 38, 501-519.

Thorpe, W.H. (1928). The elimination of carbon dioxide in the insecta. Science, 68,433443.

Thorpe, W. H.(1950). Plastron respiration in aquatic insects. Biol. Rev. 25,344-390. Treherne, J. E. (1957a). The diffusion of non-electrolytes through the isolated cuticle of Schistocerca gregaria. J . Insect Physiol. 1, 178-186. Treherne, J. E. (1 957b). Glucoseabsorbtion in thecockroach. J. exp. Biol.34,478485. Treherne, J. E.(1958). The absorbtion of metabolism of some sugars in the locust Schistocerca gregaria. J. exp. Biol. 35, 611-625. Treherne, :. E. (1961). The kinetics of sodium transfer in the central nervous system of the cockroach Periplaneta americana. J. exp. Biol. 38, 737-746. Wigglesworth, V. B. (1931). The physiology of excretion in a blood-sucking insect, Rhodnius prolixus. J. exp. Biol. 7, 41 1451, Wigglesworth, V. B. (1934). The function of the anal gills of the mosquito larva. J. exp. Biol. 10, 1626. Wigglesworth, V. B. (1938). The regulation of osmotic pressure and chloride concentration in the haemolymph of mosquito larvae. J. exp. Biol. 15,235-247.

ACTIVE TRANSPORT O F WATER I N INSECTS

129

Wigglesworth, V. B. (1945). Transpiration through the cuticle of insects. J. exp. Biol. 21,97-114. Wigglesworth, V. B. (1947). The epicuticle of an insect, Rhodniusprolixus. Proc. roy. SOC.B 134, 163-181.

Wigglesworth, V. B. (1953). Surface forces in the trachael system of insects. Quurr. J. micr. Sci. W,507-522. Wigglesworth, V. B. (1954). “The Principles of Insect Physiology”. Methuen, London. Wigglesworth, V. B. (1957). The physiology of insect cuticle. Annu. Rev. f i t . 2,37-54. Wigglesworth, V. B. and Beament, J. W. L. (1950). The respiratory mechanisms of some insect eggs. Quart. J . micr. Sci. 91, 429-452. Wolfe, L. S. (1954). The deposition of the third instar larval cuticle of Calliphora erythrocephala. Quart. J . micr. Sci. 95,49.

This Page Intentionally Left Blank

Colour Discrimination in Insects DIETRICH BURKHARDT Zoological Institute, University of Munich, Germany 131 135 137 137 157 158

I. Historical Background

11. Terminology. . 111. Current State of Knowledge

.

Spectral Range Visible for Insects, the Spectral Sensitivity Curves The Question of the Screening Pigments . Visual Pigments in Insects . Wavelength Discrimination and Central M e c h a n h s of Colour 159 Vision . 169 References . A. B. C. D.

I. H I S T O R I C ABACKGROUND L

The ebility of insects to discriminate between different colours was established beyond doubt just fifty years ago. Scientists and amateurs had investigated colour vision, or discussed it, before this but the final proof was lacking. Thus, for example, the Munich ophthalmologist von Hess in 1913 still claimed that all invertebrates and the fishes were d o u r blind. According to his theory these animals responded to different colours not by discriminating wavelengths but by determining the relative brightness of the different stimuli. The decisive proof was an experiment in which the possibility of brightness-discrimination was excluded. In 1913 von Frisch solved this problem by presenting a set of grey papers of differing reflectances with one coloured paper among them to bees. The bees were trained to search for food placed in a small bowl on the coloured paper. When the bowl with food was removed, the searching bees always gathered on the coloured paper but never on any of the grey papers, one of which must have had the same brightness as the coloured paper. Colour vision was thus proved beyond all doubt for the bee. The same basic method of examining colour vision within the animal kingdom still holds: that is to determine whether the experimental subject is able to discriminate between two visual stirnuti of different wavelengths-or wavelength composition-but independently of their brightness. Unfortunately, there is no similar conclusive proof for colour blindness in animals. If an animal does not react to different colours in any experimental situation, this does not reveal anything about 131

132

DIETRICH BWRKHARDT

colour blindness. It might well be, for example, that the same species could react to colours in some other situation. Several cases are known where animals do react to colours only under very special conditions. Following the pioneer work of von Frisch several other principles were developed to examine colour vision in insects. While the bee is a favourable experimental animal, because it is easily conditioned, other insects are not conditionable. Different methods, therefore, have to be applied using, for example, the optomotor responses. Schlieper (1927) developed a rotating drum with alternating vertical stripes of coloured papers and greys of a certain reflectance. He found that for each given colour a grey of a certain reflectance could be selected so that the animal no longer responded by optomotor reactions (running or making movements with its limbs or body appendages to follow the turn of the drum). Schlieper investigated crustaceans and concluded that for this type of response all animals tested behaved as though colour blind. Von Buddenbrock and Friedrich (1933) improved the method and investigated crabs. First a grey was selected which evoked no alteration in response with a given colour. Next, a second colour was selected of a brightness such that no response occurred if this colour was used in alternation with the grey already given from the first set of experiments. Finally, they used a pattern of alternating stripes of the two colours which gave no response if each of them was used against the reference grey. In spite of the fact that the two colours were equal in brightness in respect to the reference grey, a response could be observed. Obviously, the animals did distinguish different colours from each other despite their equal brightness. This improved method proved to be a powerful tool in examining colour vision in lower animals. While the first experiments of this type were done with crustaceans, Schlegtendahl (1934) and Moller-Racke (1952) showed that several species of insects were able to discriminate colours. Knoll (1921, 1922, 1925, 1927) and later Ilse (1928, 1934, 1937) made use of the spontaneous preference of some insects for certain colours. These insects visited flowers or dummy flowers of the preference colour more frequently than those of any other colours or of greys of different reflectances. Yet another method was used by Hamilton in 1922. He utilized first the phototactic response and second the principle of selective adaptation (the method itself was fist introduced by von Frisch and Kupelwieser (1913) in investigating Crustacea). Drosophilu were presented with two light stimuli of different wavelengths but so matched in brightness that they attracted equal numbers of individuals. Adapting the

C O L O U R D I S C R I M I N A T I O N , I N INSECTS

133

animals before the test to one of the colours resulted in a preference for the colour to which they were not adapted. Evidently, this could not have happened if the animals were colour blind. This basic method of wavelength-dependent adaptation was later modified by other authors. The method most recently introduced is the electrophysiological approach. While Jahn and Crescitelli (1939) did not find any differences in the shape of action potentials elicited by differing wavelengths in the eye of :he moth Cecropia, Walther (1958a, b) was able to prove wavelength-specific time courses of action potentials in the cockroach eye. These differences in the shape of the mass-response were considered to be due to the action of specific receptors for different regions of the spectrum. Walther also showed that in the cockroaqh the electrical response changed in a specific manner due to wavelength-dependent adaptation. Autrum and Stumpf (1 953) combined the electrophysiological method with heterochromatic flickering. Two light sources flickering out of phase and being controlled independently from each other in intensity and wavelength were used. For many combinatiops of wavelengths it proved to be impossible to match the intensities of two intermittent stimuli so that they would be equivalent to a continuously lasting stimulus. In summary it can be said that measurements of heterochromatic flicker-responses, wavelength-selective adaptation and wavelengthdependent shapes of action-potentials may be an indication of colour visioh-if certain methodological limits are kept in mind. In general most of the electrophysiological approaches to this problem do not show whether colour vision is present or not, but they do give information regarding the extent of the visible spectrum and the effectiveness of different stimuli in eliciting responses. With these refinements in methods our knowledge of colour discrimination in insects increased rapidly. In 1886 Lubbock (cited in Lubbock, 1929)found that in ants the visible spectrum did not end in the violet, as in man, but extended further down into the ultraviolet range. This ultraviolet-sensitivity of insects was investigated carefully in bees by Kuhn and Pohl(1921), who also introduced the use of pure spectral lights to replace the broad-band reflected light from coloured papers. Von Frisch demonstrated that bees can distinguish at least two hues (namely those regions of the spectrum apparent to us as orange, yellow and green from those we call blue and violet) and Kiihn and Pohl subsequently demonstrated a much finer wavelength-discrimination. According to their results the bee distinguishes ultraviolet, also bluish green, as separate qualities and

134

1 ) I i l RICH IilJRKHARDT

von Frisch had iipparently neglected the ultraviolet reflectance of the pigment papers used and was therefore misled in his interpretations. Ultraviolet is the complementary region of the spectrum to bluish green for the bee. The paper appearing to us as bluish green must accordingly have appeared nearly colourless to the bee, as proved by M. Hertz (1937). Using pure spectral lights K u h n and Pohl trained the bees to distinguish the blue-green region of the spectrum from other stimuli as a distinct category of colour. I n 1956 Daumer (a co-worker of von Frisch) reinvestigated the bee using an ingenious colorimeter. He found that there are two additional qualities of colours distinguishable by the bee, namely violet and “bees’ purple”, a mixture of yellow and ultraviolet. Thus, the bee is capable of distinguishing at least six major categories of colours : yellow, blue-green, blue, violet, ultraviolet and “bees’ purple”. It is thus established that colour vision in bees is highly developed and is somewhat comparable to colour vision in man. This finding cannot be emphasized enough, for some physiologists and psychologists, experimenting chiefly with humans and higher mammals, are inclined to deny that invcrtebrates may possess such capabilities (cf. Bartley, 1959). The bee’s colour vision is comparatively well known, certainly better than that of other insects. During the last forty years colour discrimination per se has been investigated for many species of several orders of insects, yet very rarely was any additional information gained. Thus we do know that these species are able to discriminate colours but we do not know the hues discriminated, only in a few cases the major categories of colours have been described, but in no single case does the amount of information available compare with that in the case of the bee. In general it is evident that the visible spectrum of insects is shortened at long wavelengths as compared to man but does extend further down in the short wavelength region, i.e. the ultraviolet. Perhaps a few exceptions from this general rule are possible; certain butterflies and possibly the firefly Photinus are for example reported to visualize red stimuli (cf. Kiihn and Ilse, 1925; Ilse, 1928; Buck, 1937). A new field for research was opened very recently, when it proved possible to trace the activity of single photoreceptor cells by means of intracelldar electrodes. The first to do this were workers from a Japanese group (Naka and Kuwabara, 1959a). Almost simultaneously Burkhardt and Autrum (1960) and Burkhardt and Wendler (1960) succeeded in penetrating single photoreceptor units in insects. It therefore became possible to investigate the spectral sensitivity of singlevisual cells in animals which are able to discriminate colours (Autrum and

C O L O U K D I S C R I M I N A T I O N IN I N S E C T S

135

Burkhardt, 1960, 1961 ; Liurkhardk, 1962; Autrum and von Zwehl, 1962; Autrum, 1063). The results of furthcr work along these lines may make a thorodgh understanding of the basic mechanism of colour vision possible at the level of the receptor cells and associated neurons. 11. T E R M I N O L O G Y

In the past much misunderstanding has resulted from the improper use of terms in the field of d o u r vision. A few of the more frequently used ones will therefore be defined here. Intensity. The term light intensity refers to the physical energy-content of the photo-stimulus applied. Intensity may be quantified either in terms of energy (watts) or numbers of quanta, or it may be defined in multiples of a given reference-intensity. In the first case it is referred to as the intensity level, in the second as the quantum level, in the third as the relative intensity level. Since the equation E = h .c / hgives the energy, of any quantum of given wavelength it may readily be calculated that spectral lights of equal energy content mean increasing numbers of quanta with increasing wavelength ;conversely, spectral stimuli of equal quanta decrease in energy content with increasing wavelength. Most modern work refers to stimuli of equal quanta. Brightness is distinguished from intensity of a given stimulus and is measured subjectively in man or behaviourally by an animal's reaction. At any given wavelength the reaction increases normally with intensity, and the relation is a logarithmic one in most cases. Brightness (as indicated by the magnitude of the reaction) therefore increases with the logarithm of intensity. Furthermore, when stimuli of differing wavelengths are compared, the brightness depends not only on intensity, but on the wavelength used. A highly effective wavelength elicits a stronger reaction than a less effective one. At equal physical intensities the more effective stimulus is therefore the brighter one. Wavelength-content. Distinction must also be made between colow and wavelength-content of the stimulus. The wavelength-content describes the physical nature of the stimulus, while the colour is judged by the animal's reaction. Two stimuli of differing wavelength-content may be equal in appearance to the animal (for example, a pure spectral light of 580 mp looks equally yellow to us as does an appropriate mixture of 520 and 640 mp). On the other hand, two stimuli of differing wavelengths may he perceived as similar or equal colours (as, for example, 650 and 750 mp both elicit the sensation red in man). Colour. The term colour is used here chiefly to describe a range of

136

DIETRICH B U R K H A R D T

differing hues, which are nevertheless related. For convenience, in this paper regions of the spectrum are named by their appearance to the human cye. Thus, words like yellow, red and so on are frequently used to describe certain regions of the spectrum. If exceptions are made and the term yellow does not refer to wavelengths between 510 and 575 mp but to the appearance of a composite stimulus it is expressly referred to as yellow-sensation. Furthermore, if any name for a colour does not refer to man but to an animal, it is expressly stated; for example, the word “bees’ purple” does not refer to purple as perceived by man but to a mixture of yellow and ultraviolet, which has stimulating effects to the bee similar to those of a mixture between red and violet to our eye. Spectiul eficiency. The term spectral efficiency (or action spectrum) is described by the following curve. The size of response is plotted against wavelength, while the energy-(or quantum-) level is kept constant. The term spectral sensitivity refers to the curve obtained by noting a constant response and plotting the reciprocal of the intensity necessary to elicit this response against the wavelength used. The spectral sensitivity curve may be constructed also from a set of curves showing the size of response as dependent on intensity at several wavelengths. Finally, this curve may be constructed from the spectral efficiency-curve and one response-intensity-curve for a certain wavelength but this only under some conditions 1 the system dealt with must be a homogeneous population of receptors or a single receptor cell. If this is the case, the size of response to any one wavelength is transferred from the efficiency curve to the response-intensity-curve and the corresponding intensity is read off. These corresponding intensities are replotted versus wavelength and thus reveal the sensitivity curve. If receptors differing in their characteristics are involved, obviously this method is not applicable: the response-intensity-curvesmay differ with wavelength. Colour vision. An animal which does not react in a specific manner to different wavelengths independently from the stimulus-intensity is called colour blind (in the experimental situation, cf. p. 132). Correspondingly an animal which is able to discriminate differing wavelengths or mixtures of them is said to have colour vision. Some physiologists and psychologists are inclined to restrict the term colour vision to man and here to the reactions within the highest cerebral levels. According to their opinion reactions of animals ought to be called spectral vision, and finally reactions within the retinal levels ought to be referred to as spectral responses (cf. Bartley, 1959). But this proposal is not thoroughly useful. Experimental situations not only deal with pure spectral lights, but with mixtures of them in addition. These mixtures may give rise to

C O L O U R DISCRlMINATlON IN INSECTS

137

the same reactions as a pure spectral light of wavelengths quite different from any of the components of the mixed stimulus. Hence the term spectral vision or spectral response does not apply to the topics under consideration. Certainly the term colour as used in general refers to the appearance of wavelength-regions or mixtures of spectral lights to the human eye and under no circumstances can we state anything about the animal’s impressions. But keeping this in mind, the term colour discrimination may well be applied to the ability of animals to distinguish differing spectral lights or mixtures of them from each other and independently from intensity. Even the term colour vision might be applied if the experimental material indicates strongly enough that integrative actions are taking place within the central nervous system of the animal which are comparable to the events in the human brain. As will be discussed later in full detail, at least in the honeybee this is definitely the case. While discussing these matters, it might be useful to indicate where some of the misunderstandings arise. Investigating the range of spectral lights visible to an insect or determining the spectral sensitivity (or efficiency) curve does not tell anything about colour vision. But some authors entitle their work on range of visible spectrum as investigations on colour vision. If the animal does visualize red light this does not tell anythiiig about colour vision and the ability of this species to discriminate red light as a quality of colour, but only indicates that the eyes of this animal are sensitive to long wavelengths. 111.

C U R R E N T S T A T E OF

KNOWLEDGE

A. SPECTRAL RANGE VISIBLE FOR INSECTS, THE SPECTRAL SENSITIVITY CURVES

1, General considerations

Sevzral methods have been applied to determine the spectral range visualized by insects : phototactic reactions, behavioural preference of a colour, optomotor responses, conditioning and electrophysiological methods. It turned out that all insect species investigated in this respect arecapable of visualizing ultraviolet within the range from 300 to 400 mp or even below, cf. Bertholf (1932) (data are listed in papers by Goldsmith, 1961; Weiss et at., 1942, 1943). The values of long wavelength limits sre not so clearly defined. In general, the long end of the spectrum visible to insects seems to be shifted 100 mp towards shorter wavelengths as compared with the spectrum visible to man. The bee does not react

138

DIETRICH R U K K H A R D T

to wavelengths above about 600 mp (cf. Daumer, 1956), nor do most other insects investigated unless extreme intensities are applied. Some exceptions to this state of affairs are certain in diurnal butterflies (which are reported to be attracted by red-coloured natural or experimental stimuli), a firefly and a few other species (cf. von Frisch, 1960; Autrum, 1960). How uncertain this question may be is shown by the results obtained from several authors on Vespidae: while Armbruster (1922) and Molitor (1939) claim that Vespa vulgaris and V. germanica are able to distinguish red as a colour (and hence their visible spectrum must extend to this region), Schremmer in 1941 found that V.rufu confused red and black pigmented papers. The red-sensitivity of flies is also questionable even though often reported (Autrum and Stumpf, 1953; Walther and Dodt, 1959; Mazokhin-Porshniakov, 1960a, b) and it is considered to be an indication for a special red-receptor-system. This problem will be discussed in more detail on page 143. It appears likely however, that these species are not any more sensitive at the long-wavelengths-end of the spectrum than the bee, i.e. the range of the spectrum visible to them .i cut off at approximately 650 mp (Autrum, 1955; Hoffmann and ,anger, 1961;Burkhardt, 1962). While each method will give valid results in determining the cut-off ends of the visible spectrum, only a very few methods are adequate to determine the exact shape of the spectral sensitivity (or efficiency) curve. To do this, a response must be chosen as a test, which is dependent in a graded fashion on the effectiveness of the stimulus. This may be a reaction graded either by itself or by the statistical probability of all-or-none responses in single animals or in a population. Two possible cases can be distinguished. (a) The response of the animal may be mediated by a receptor system containing only one type of receptor in respect to spectral sensitivity. In this casc the effects of wavelength and intensity of the stimuli applied will be interchangeable, so that alternation of each of the two parameters will result in a change of brightness and nothing else. Any reaction depending in a graded fashion on intensity will give in this case an undistorted picture of the spectral sensitivity, if the wavelength is changed. (b) In the second type of system under consideration the receptorsystem mediating the reaction consists of two or more different types of receptor elements with differing spectral characteristics. In principle such a system is capable of colour discrimination. Stimuli of different wavelengths and intensities are not interchangeable. At a given wavelength, for example, there will be a certain ratio between the sizes of ill response of the two differing receptors. Increasing the intensity w increase the responses of both the receptors, while the ratio between them

C O L O U R D I S C R I M I N A T I O N IN INSECTS

139

will stay the same or similar. Alternating the wavelength of the stimulus while its intensity is kept constant will change the sizes of individual responscs and in addition the ratio between them. A reaction chosen for theexperimental test may depend not only on the intensity, but inaddition or solely on the spectral characteristics of the stimulus. It might well be., that only the excitation of one type of receptor-element elicits a certain response of the animal. If this reaction is used, the spectral sensitivity of the eye as a whole is not measured, but the spectral sensitivity of this particular receptor type. Furthermore, quite complex central integrative mechanisms may introduce distortions in the shape of the responsecurve. For example, it is reported that the cabbage-butterfly, Pieris brassicae, neglects green stimulus-sources in its feeding behaviour, while the same wavelengths are strongly preferred by females trying to deposit eggs (Ilse, 1937). Daumer (1956) states that in the honeybee brightness and saturation determine the efficacy of coloured lights as stimuli. Also in Drosophila phototactic responses are qualitatively different with differing wavelength (MCdioni, 1961). Conclusively, behavioural experiments reveal data about the cut-off ends of the visible spectrum, which are below 300 mp in the ultraviolet and at approximately m 6 5 0 mp in the red in the case of most insects. On account of general considerations behavioural experiments will not reveal in each case the spectral sensitivity curve of the eye as a whole, especially in animals which have colour vision.

2. Behavioural experiments Thus one cannot expect to get conclusive results in animals which are colour-susceptible, if methods are applied which may depend on central integrative mechanisms. First of all, a peak of preference for any wavelength region offered as stimulus does not necessarily mean that these wavelengths are the brightest in appearance to the animal. Fig. 1, for example, shows several sensitivity curves as obtained for honeybees by different authors using different methods. The large scatter clearly indicates that behavioural work done on colour-susceptible species often reveals not the true spectral sensitivity of the eye but rather gives a measure for the power of certain wavelengths to elicit the reaction tested. Critical and careful evaluation of curves thus obtained is necessary. On the other hand, much useful information is easily gained by such methods. Using phototactic responses, Weiss and co-workers (1942, 1943, 1945, 1946) investigated the spectral sensitivity curves of more than forty species of insects from many orders. From this extensive work and others (cf. Goldsmith,

140

DIETRICH BURKHARDT

1961) the generalization can be made that regions of the spectrum which are especially efficient in eliciting visual reactions in insects are positioned on the average around 350 mp and 490-500 mp. The species of insects which are reported to be sensitive to red light

I -

300

500

400

600

mll

FIG. 1. Relative effectiveness of different wavelengths to elicit behavioural responses in the worker bee as obtained by several authors. Ordinates: relative magnitudc of response as related to the maximum response, scale indicated on bottom figure, applies to all others. (A) According to Weiss cr al. (1942); (B)from Bertholf (1941a, b), the parts of the curve with filled and open circles are from different experiments; (C) based on data given by Daumer (1956) calculated from Goldsmith (1961); @) from Heintz (1959); (E)according to Sander (1933). (Modified and completed from Goldsmith, 1961.)

above 600 mp have not been checked with regard to their ultraviolet sensitivity. It would therefore be of some interest to know whether these species do have much broader range of visible wavelengths than other insects or whether they are comparable to most of the vertebrates in lacking ultraviolet-sensitivity.

COLOUR DISCRIMINATION I N INSECTS

141

3. The mass-response As behavjoutal studies do not in every case give a clear picture of the spectral sensitivity of the receptors, the question arises whether electrophysiological research can give more meaningful results. With this

250

200 h

6

' 3

.E

150

U

al X

.-al -0 U

d

100

50

C

I

I

300

400

500

600

m/l

FIG.2. Relative extinction of the red screening pigments, ommochromes (-), and the yellow screening pigments, pteridines (.-----),of Calliphora erythrocephulu. Ordinate :extinction as related to the maximum extinction within the visible spectrum. For details compare Langer and Patat (1961). (Courtesy of H.Langer.)

method the contributions of central integrative mechanisms are eliminated or restricted, depending on whether or not ganglionic layers are adjacent to the receptor structures and may contribute to the recorded response. In most of the work published so far the impulses of the optic nerve were not recorded but rather the so-called ERG, i.e. the massresponse of the eye as a whole. Usually it consists of several components,

142

DIET R I C H €3 U H K H A R DT

A

B

FIG.3. Schematic illustration of the means by which light rays reach the photosensitive structures of a compound eye. (S) Stimulating light source; (L) corneal lens; (P)crystal cone or-as in flies-pseudocone; (Sc,) primary pigment cells; (Sc,) wondary pigment cells, both containing screening pigments; (V)visual cell; (Rh) rhab domercs, the light-sensitivesubdivision of the receptor cell. Inset shows a cross-section below the pseudocone. (A) Short wavelength stimulus. Ommatidia directed towards the light source are strongly stimulated, oblique light rays are absorbed in the accessory pigment cells. (B) Long wavelength stimulus. Ommatidia directed towards the light source are stimulated additionally by oblique light rays penetrating the accessory pigment cells, whose pigments fail to absorb light of this wavelength. Ommatidia not opening towards the light source are stimulated in the same way, as indicated on right. (Modifiedfrom Burkhardt, 1962.)

C O L O U R DISCRIMINATION IN INSECTS

143

which are the graded responses of the receptor layer and in addition graded responses from adherent synaptic and ganglionic layers. Several investigations deal with the question of analysing these components, for example Autrum (1950, 1958), Hartline et al. (1952), Naka and Kuwabara (1959b). A mass-response will increase if intensity is raised, due to the fact that each of the contributing elements will give a graded response of larger amplitude; it will also increase if the number of contributing elements rises, for example if a larger area of receptors is stimulated or elements of higher threshold are recruited by raising the intensity. These properties of the mass-response can cause wavelength-dependent effects which conceal the true sensitivity curve of the eye. In Calliphora at least the screening- or Nter-pigments do not absorb equally well all the wavelengths to which the receptors are sensitive. The same is probably true for other species with brightly coloured eyes. Data for the absorption of the screening pigments are given for Calliphora in Fig. 2. If the screening pigments within the pigment-cells surrounding the receptor-cells do not absorb at particular wavelengths, additional light by-passing through adjacent ommatidia may stimulate the receptor cells (Fig. 3). The response of receptor cells within ommatidia directed towards the light-source will be enhanced in this way (i.e. above the level expected from the sensitivity curve due to the photosensitive substance within the receptor cell). Furthermore, ommatidia not opening towards the light-source may be stimulated under these conditions. Altogether, the number of contributing elements is increased as well as the size of the individual contributions. The mass-response in regions of the spectrum where the screening-pigments do not absorb is not a true measure of the sensitivity of the receptor cells. An example is found in Calliphora. While investigating the mass-response of the corn. pound eye of normal flies, Autrum and Stumpf (1953), Walther and Dodt (1 959) and Mazokhin-Porshniakov (1960a, b) found three peaks of sensitivity, the first around 350 mp, the second around 500 mp and a third one at 620 mp. It was postulated from their results, that in the Calliphoru eye a receptor system was present which was maximally sensitive around 620 mp. But in 1955 Autrum found that the peak at 620 mp was absent in the mutant white-apricot of Calliphora, which is lacking the brownish-red screening pigments of the ommochrome-group. Obviously, if the screening pigments are absent, in all regions of the spectrum more light will reach the receptor cells than in the normal eye. In fact, a larger amplitude of the mass-response is observed and a 620 mp peak is not present.

144

DIETRICH B U R K H A R D T

These experiments were repeated in 1961 by Hoffmann and Langer together with observations on the “chalky” mutant. This new mutant lacks all the screening pigments, the ommochromes as well as the yellowish pteridines. The spectral sensitivity curves obtained from these mutants show that the red peak is not present (Fig. 4). That the red peak as observed in the mass response is an artifact also became evident from intracellular recording of the receptor cells (Autrum and Burkhardt, 1960, 1961; Burkhardt, 1962; Burkhardt and Hoffmann, 1962). In these experiments the graded response of a single

FIG.4. Spectral efficiency c w e s for two mutants of Calliphora erytluocephah. Ordinate: relative height of the mass response; (-) chalky mutant, lacking ommochromes and pteridines; (--------) white-apricot mutant, lacking the ommochromes. {Modified from Hoffmann and Langer, 1961.)

visual cell was observed so that all area-effects which occur in the massresponse were excluded. In the cells of ommatidia directed towards the stimulating light-source the stimulus passing the dioptric apparatus is much more powerful than scattered light. With such directed ommatidia a sensitivity curve is obtained which lacks the red peak and fits closely the curve obtained investigating the mass-response of the “chalky” eye. However, in cells giving only very small action potentials a red peak is observed. Apparently, these are cells within ommatidia not opening towards the light source and therefore stimulated only by a small amount of scattered light. In such a case, light of long wavelength

COLOUR DISCRIMINATION I N INSECTS

145

penetrating the pigment cells and reaching the rhabdomeres in an oblique direction may contribute considerably to the total stimulus. Hence a hump or a clear peak around 616 mp is observed, as shown in Fig. 5. In the case of Calliphora because the accessory pigment sells do not absorb red light, the red peak clearly is a pseudopeak. Two additional phenomena observed in this species support this conclusion. When the intensity-level was gradually raised, Autrum (1955) observed a shift of

mtL

FIG.5. Spectralefficiency for individual receptor cells of Calliphora erythrocephala, intracellular recording. For comparison the maximum response in each curve was set as 100% (ordinate). (-1 Strongly illuminated cell, placed within an ommatidium directed towards the light source; (------I more weakly stimulated cell, placed ) scarcely stimulated within an ommatidium facing the edge of the light source; ( cell, placed within an ommatidium only illuminated by scattered light. (From Burkhardt, 1962.)

the peak within the green region from roughly 490 to 540 mp simultaneously with the progressive growth of the peak at 620 mp. Obviously, not only the extreme end of the curve is affected but in addition the part of the curve falling from the green peak towards the red. Thus, besides the appearance of the pseudopeak, the screening pigments most probably affect the measured location of the true peaks. Schneider (1956), in behavioural experiments, investigated the visual acuity of Calliphora at varying wavelengths. He found that the visual acuity declined at the long wavelength end of the spectrum. This is to be ex-

146

DIETRICH B U R K H A R D T

pected, if the screening pigments do fail to isolate the ommatidial units from each other, which must be the case within the red region of the spectrum . These findings point to two conclusions. (a) Not every peak observed in the sensitivity curve while recording electrical responses from the eye needs to be due to maximum absorption of the light-sensitive pigments. (b) While recording the mass-response the properties of the screening pigments and hence area-effects may interfere with effects due to intensity or wavelength of the stimulus. This problem will be discussed further in a later section dealing with adaptation to selected wavelengths. Figure 6 summarizes results of several authors on the mass-response in insect-eyes of several species. The results were similar to those from behavioural work, for in all cases where the ultraviolet was included as stimulus a sensitivity peak was found in the region of 350 mp. With each species at least one more peak was observed, situated in the visible range between 440 and 540 mp; in most cases this peak was located around 490 mp, seldom at 440 or 540 mp. Finally, in only a very few cases were two peaks observed simultaneously within this region. In some cases a third (or a fourth) peak occurred at about 620 mp, as in Calliphora, Macroglossurn, Carabus (Hasselmann, 1962), Libelluh (Mazokhin-Porshniakov, 1959b) and Notonecta (Ludtke, 1954). Whether the conclusions drawn concerning the red peak in Calliphora apply to other species is still an open question. In conclusion, it can be stated that electrophysiological as well as behavioural measurements of spectral sensitivity curves need careful and critical evaluation before data concerning the receptors themselves may be accepted. 4. The spectral sensitivity curve of single receptor types The spectral sensitivity curve of any eye consisting of more than one receptor type will be built up by the individual contributions of the different receptors. Hence the question arises of how to obtain the data for the spectral sensitivities of isolated receptor types. The occurrence of more than one peak within the overall-sensitivityis often regarded as indicating the peaks of individual receptors. Obviously this does not hold true. Even if the individual receptors do have smooth and single peaked sensitivity curves, and their peaks differ widely in location, the overall-sensitivity of the eye still may be a smooth and single peaked curve. If one receptor type predominates in number, in the massresponse and in certain behavioural situations the excitation of the deviating receptors may contribute only in a very small amount and

COLOUR DISCRIMINATION IN INSECTS

147

may not be visible. On the other hand, the sensitivity curve of a single receptor might be in itself double-peaked. This is the case in vertebrates, where most of the retinenes exhibit besides the a-absorption above 500 m p a second absorption-peak at below 380 mp (cf. Wald, 1959). Insects are also similar in this respect, as shown by the investigation on single receptor cells in Culliphoru where, besides a peak within the visible range, situated between 430 and 540 mp, all types of receptor cells have a second sensitivity peak located at 350 mp and of equal height (Burkhardt, 1962). How then can one distinguish between sensitivity curves obtained from a homogeneous population of receptors and those originating from more than one receptor type? The latter type is the only one of interest in the context of d o u r vision. Differences in spectral sensitivities among different receptors may be due to both physical and chemical phenomena. To the first group belong structures separating or filtering certain wavelength of the spectrum. An example of this mechanism is that in birds, where coloured oil-bubbles are placed in front of the receptor-elements. Similar effects may arise from the filtering absorption of light by light-sensitive pigments within the receptors, the concentration of which may vary from cell to cell. Furthermore, screening pigments will cause such effects as proved for insects (cf. p. 143). Finally, wavelength-dependent diffraction has been proposed as a possible mechanism. Up to now there is no evidence in insects for such physical modes, except for some possible influence of the screening pigments. The other possible basis for different wavelength sensitivities is a chemiv cal one. Different receptors may cgntain light-sensitive substances of differing chemical nature and hence differing absorption properties. If so, it ought to be possible to exhaust individual receptors by monochromatic adaptation to their particular wavelength. Such wavelengthselective adaptation has been widely employed to prove the existence of several types of receptors. Another method is changing the stimulusintensity. If the receptors do differ in threshold, it ought to be possible to get the isolated sensitivity curve of the most sensitive one by lowering intensity sufficiently.In vertebrates, the threshold is different for chromatic and achromatic vision, the sensitivity-peaks differ also in wavelength -the phenomenon known as Purkinje-shift: the peak wavelength is shifted towards shorter wavelength with decreasing intensity. Several authors claim to have found a comparable Purkinje-shift in inseds and conclude that colour vision exists in these species (e.g. Fingerman and Brown, 1953; Liidtke, 1954; Hasselmann, 1962). In a careful investigation Schneider (1956) examined optomotor

148

DIETRICH B U R K H A R D T

responses of CalZiphora at very low intensities, near the threshold level of dark-adapted humans. He found a sensitivity peak around 480 mp, which is in good agreement with the sensitivity peak of the eye as a whole and in agreement with the location of the peak of the most common type of receptor cells as revealed by intracellular investigation. There is not much evidence for a “Purkinje-shift” in CaZZiphora. On the other hand, Hasselmann (1962) found evidence for a shift of the sensitivity-peak in the eye of Carabus and of Macroglossurn. In both species at higher levels of intensity a prominent peak is observed at 500 mp, while at low levels the most prominent peak is observed around 430 mp (cf. Fig. 6). In the butterfly Deilephilu Zivornic colour vision is present to below intensities of human achromatic vision (Knoll, 1925). Several authors, for example Walther (1958a, b) and Goldsmith (1960), used wavelength selective adaptation to separate receptors. By adaptation with red light Walther found in the cockroach a decrease in sensitivity to long wavelengths and no change at short wavelengths, as far as the dorsal part of the eye is concerned. Adaptation to violet light did not reveal any changes in the sensitivity curve. In the ventral part of the eye wavelength-dependent effects were absent completely. Walther concluded, therefore, that the ventral part of the eye consists of only one receptor type, maximally sensitive to green, while in the dorsal half at least two types of receptors are apparently present, one maximally sensitive in the green region of the spectrum, the other in the near ultraviolet. In 1959 Walther and Dodt investigated this matter further and included ultraviolet stimulation. The ventral part of the eye which was thought to contain only one type of receptor, exhibited in addition to the green peak another peak of sensitivity around 360 mp, nearly as high as the green peak (cf. Fig. 6). Thus the so-called green receptor also seems to be sensitive to ultraviolet. The sensitivity curve obtained from the upper part of the eye was of quite similar shape, but the relative height of the ultraviolet and the green peaks changed after adaptation either to ultraviolet or to red light. This strongly suggests that the receptor type common to both halves of the eye has a double-peaked sensitivity curve. Furthermore the second receptor type present only in the dorsal half does not contribute very much to the shape of the overall sensitivity curve, but just enough to cause changes in the relative height of the two peaks observed, when investigated with the mass-response. Comparable results are reported by Goldsmith (1960) for the bee. In this careful investigation of the compound eye of workers a doublepeaked sensitivity curve for the whole eye was obtained, one peak situated around 350 mp, the other at 535 mp (cf. Fig. 6). Adaptation with

C O L O U R DISCRIMINATION IN INSECTS

149

I

4013

500.

600

700

my.

FIG.6. Spectral sensitivity curves of severalinsectsas revealed by the mass-response of the eye. Ordinates not calibrated but indicating the relative sensitivity on a logarithmic scale. (A) Worker bee adapted to red light, from Goldsmith (1960); (B) cockroach, Periplaneta americana, from Wdther and Dodt (1959); (0 groundbeetle Carabus aurutus, from Hasselmann (1962). (C,) high intensity; (C,) the same but at low intensity ;(D) Macroglossumstellafarum,sphingid moth,from Hasselmann (1962); (DJ high intensity; (D2) low intensity; (E)blowfly, Culriphora erythrocephalu, from Walther and Dodt (1959). Note grouping of the peJs from all the curvesaround certain wavelengths, namely 350 rnp, 440 mp, 500 mp, 540 mp, and 620 mp. The same peak positions are also seen in Figs. 4,5,9 and 10. (Modified from Hasselmann, 1962.)

150

DIETRICH B U R K H A R D T

red light enhanced the height of the ultraviolet peak relative to that of the green one. On the other hand, ultraviolet adaptation did not affect the shape of thc sensitivity curve as tested with the mass-response. Goldsmith concluded that at least two receptor systems are present, an ultraviolet receptor sensitive only to short wavelengths (and therefore not affected by red conditioning light) and a green receptor which is also sensitive to short wavelengths (and therefore affected similarly to the ultraviolet receptor by adaptation to short wavelengths). In the drone bee evidence was found for a receptor system maximally sensitive at 440 mp (Goldsmith, 1958a, b). Finally, in the ocellus of workers a receptor was found with a maximum at 590 mp (Goldsmith and Ruck, 1957/58).As will be discussed later, these findings are strongly supported by results of intracellular investigations carried out by Autrum and von Zwehl(l962) and Autrum (1963). One objection to be made against experiments based on differential adaptation can be stated as follows. Wavelength specific effects of adaptation might not only be due to the different absorption by the visual pigments, but could also result from differential absorption within the screening pigments. If any wavelength is not absorbed jy the screening pigments, this light will stimulate a larger retinal are than other wavelengths. Since the size of the recorded mass-response is dependent on the number of contributing elements, equal sized respolises at two different wavelengths are obtained, in one case from a large number of comparable weakly stimulated elements, in the other from a small number of strongly stimulated elements. If conditioning and testing lights are of different wavelengths, one has to expect wavelength-dependent changes within the recorded sensitivity curve. As will be reported later, in Calliphora there is no proof for wavelength selective effects of adaptation to monochromatic lights when single visual cells are investigated. Yet while recording the mass-response of a pure retina of the mutant white-apricot preparation wavelength selective effects similar to those reported for the bee and the cockroach have been observed (Ch. Hoffmann, personal communication). Certainly this does not mean that in the bee and the cockroach such effects occur, but they must be considered. A second example is the eye of Notonectu gluucu. As known from several investigations these eyes have two parts which differ in morphological structure, distribution of screening-pigments and in physiological properties. According to Rokohl(l942) the dorsal part of the eye discriminates colours while the ventral half is colour blind. The dorsal part contains a brownish screening pigment, the ventral a crimson one. Ludtke (1954) investigated the spectral sensitivities and found differentcurves for the two parts. In addition, he observed different

COLOUR D I S C R I M I N A T I O N IN INSECTS

151

changes of these curves while bright-adapting the eye with white light: in the dorsal part the sensitivity for short wavelengths was increased markedly while the sensitivity to 575 mp (the sensitivity-peak) and long wavelengths is not changed much. In the colour blind ventral half the sensitivity was most prominently changed at 644 mp, where a sharp peak arises. It is difficult to interpret these curves shown in Fig. 7 in regard to the spectral sensitivity of the receptors, but it appears likely that the

I

01

450

I

I

1

535

57s

I

644

7 1 0

w FIG.7. Spectral efficiency of the eye of Notonecta glauca. Ordinates: magnitude of the massresponse in mV. (A) dorsal half of theeye, (1) after 2min darkadaptation; (2) after 90 min dark adaptation. (B) Ventral half of the eye, (1) 2 rnin dark adaptation; (2) 30 min dark adaptation. (According to Lildtke, 1954.)

screening pigments are involved in the observed changes of spectral sensitivity: during the first 2 min only minor changes were observed, while the significant changes occurred in a later, long-lasting phase of adaptation. This agrees fairly well with the time-course of the well known migration of screening pigments and of retinomotor movements, which have been described in detail in Notonectu by Ludtke (1953) and

152

DIETRICH B U R K H A R D T

earlier by Bedau (1911). According to these experiments pigment migration is complete after 90min. In summary, it appears very likely that the screening pigments have a profound influence on the shape of the sensitivity curve of the eye as recorded with the mass-response. Investigation of the absorption properties of the screening pigments can be helpful in evaluating the properties of the receptors themselves, and unless this is done the results obtained with the mass-responses remain inconclusive. 5. Recordingfrom single receptor cells Very recently it has proved possible to record transmembrane action potentials from visual cells in the insect eye. Some of these investigations dealt with the general properties of the photoreceptor cell (Burkhardt

ml*

FIG.8. Distribution of maximum responses of individual receptor cells in the compound eye of Calliphoru erythrocephalu. Ordinate : number of cells exhibiting their maximum sensitivities(ultraviolet and visible peak) at a particular filter location. Abscissa: filter locations in mp. AJI data from intracellular recording. The material shown consisted of 108 cells in total. (Burkhardt, unpublished data.)

and Autrum, 1960; Naka 1961; Naka and Eguchi, 1962), and with its reaction to polarized light (Naka and Kuwabara, 1959a; Burkhardt and Wendler, 1960), but others have given the first information concerning the spectral sensitivity of individual photoreceptor cells (Autrum and Burkhardt, 1960,1961 ; Burkhardt, 1962;Burkhardt and Hoffmann, 1962; Autrum and von Zwehl, 1962; Autrum, 1963). This type of

C O L O U R DISCRIMINATION I N INSECTS

153

experiment gives direct information about the spectral properties of the visual cells themselves. The spectral efficiency curves of the majority of visual cells investigated in the normal coloured eye of Cdiphora agree exactly with the spectral efficiency curve of the chalky mutant, where there is no influence of the screening pigments (Hoffmann and

mP

FIG.9. Spectral sensitivity curves of three individual cells in the compound eye of Cuffiphoru.(A) A cell representative for the blue type; (B) for the yellow-green type; (C)for the most numerous green type. Ordinates in (B) omitted, but the same as A and C: sensitivity is related to the maximum sensitivitywithin the visible spectrum. (Modified from Burkhardt, 1962.)

Langer, 1961). Comparison of the action spectra obtained by intracellular investigation from the normal eye with those from the whiteapricot mutant does not reveal any significant differences. Finally, as already discussed on page 144, comparing cells from ommatidia differing in position in respect to the light source show that in well illuminated

154

DIETRICH B U R K H A R D T

cells there is no influence from the screening pigments on the spectral response of the visual cell, although the influence may be observed in cells not directed toward the light source. Figs. 8 and 9 summarize some data from Burkhardt (1962). In the blowfly Culliphora eryfhrocephalu all the receptor cells so far investigated exhibited two peaks in their spectral efficiency-curves (and hence also in their sensitivitycurves). One of the peaks is situated in the visible range, the other in the near ultraviolet at 350 mp. Although the position of the ultraviolet peak is close to the filter-location of 345 mp in nearly all the cells, the positions of the peaks within the visible spectrum are widely scattered. Individual cells may show their maximum at between 431 and 542 mp. If the number of peaks found for individual cells at any given filter-location is plotted against wavelength, the peak of this distribution is situated at 496 mp. If one compares the broad distribution of the peaks within the visible range to that in the ultraviolet range, where no such scatter is observed, the following explanation seems to be the most likely: the population of cells investigated is not a homogeneous one, but is composed of several, most probably three, groups. Cells within each of the groups show a location of the visible maximum at roughly the same wavelength but there are differences from group to group, while the position of the maximum within the ultraviolet range is the same for all the groups. Several facts indicate that in this case one is dealing most probably with three groups or types of cells. One type with the sensitivity maximum around 490 mp predominates in number over the two other groups. Because of the location of the sensitivity peak within the visible range at 490 mp this type of cell is called the green type. The deviating types have their maxima around 470 mp and 521 mp, the numbers of cells within each of these groups being only one-fifth of the major group. According to the location of the peaks within the visible range of the spectrum these cells are called the blue type or the yellow-green type respectively. The numerical relation of the cells belonging to the three types fits the hypothesis that within each ommatidium composed of seven visual cells five cells belong to the predominating green type receptor while only one cell belongs to each of the two deviating groups. Therefore in this fly there is no evidence for a separate green and a separate ultraviolet receptor system often postulated for different species of insects. But there are three types of cells, each of them having an astonishing high ultraviolet-sensitivity and in addition a peak of comparable height within the visible range. Furthermore, the majority of cells do have their visible peak within the green, at around 490 mp. Burkhardt and Hoffmann (1962) attempted to determine whether there

COLOUR DISCRIMINATION IN INSECTS

155

was any wavelength dependent adaptation within the individual receptor cell. If stimuli at 350 and 490 m p were adjusted to elicit equal height response for any impaled cell of the green type, the size of the action potentials was equally affected when either wavelength was used for light adaptation, whether tested with ultraviolet or green. Each of the four possible combinations of conditioning and test-stimuli revealed the same depression in sensitivity and the same time-course of recovery. The same held true for cells of the blue type, which showed their peaks at 350 m p and below 470 mp. There is thus no wavelengthdependent adaptation in single cells of Calliphora. This again indicates that there is no separate ultraviolet-receptor system in the eye of Calliphora,but that all receptor cells present have a double peaked sensitivity curve. In addition to the peak in the visible range there is in each case a second one in the ultraviolet range. This second peak is most probably not due to another photosensitive pigment, for it cannot be adapted selectively, but appears to be due to a double peaked absorption of the photosensitive pigments. Thus in Calliphora, apparently, colour vision is not mediated by a s p 5 c ultraviolet (a green and another receptor type) but by three types of receptors each of them sensitive to ultraviolet but each with a second peak within the visible range and differing in location. Furthermore, the three types of receptors are not sharply separated, but do scatter around three mean positions, not very much separated from each other. These results are to some extent comparable to the situation in vertebrates as demonstrated recently by MacNichol(l963). Similar intracellularinvestigationswere made subsequentlyby Autrum and von Zwehl(1962) and Autrum (1 963), on the drone and worker of the honeybee. In the worker the most common type of receptor-cells exhibits its maximum sensitivity around 540 mp, the flanks of the efficiency curve being steep towards longer wavelength, more flattened to short wavelength. Some individual cells show in addition a slight hump in the ultraviolet at 350 mp. Less commonly found is another trpe of receptor cell with a sensitivity peak at roughly 350 mp sometimes showing a hump around 540 mp (which is thought not to be an essential feature of the curve itself but rather a product of the experimental procedure due to some influence of the mass-response superimposed on the intracellular record). A third type of visual cell exhibitsthe sensitivity maximum at 440 mp, the curve declines steeply at both flanks. These results are in general agreement with the findings of Goldsmith (1960), who claimed one receptor system maximally sensitive to 350 mp, a second to 440 mp and a third to 540 mp, On the other hand, the results

156

DIETRICH B U R K H A R D T

are comparable to those obtained from Calliphora, if it is assumed that in the fly the second peak is more pronounced than in the bee, where it is merely indicated by a slight hump. Fig. 10 shows the sensitivity curves as described for the drone. In the drone the dorsal part of the eye apparently contains only the ultraviolet and the blue receptor, while according to new findings the ventral part of the eye contains in addition green receptors (Autrum and von Zwehl, 1963). The significance of these findings in reference to colour vision will be discussed in Section 111, C.

green

FIG.10. Spectral efficiency curves from three individual cells in the compound eye of the drone bee, each cell regarded as representative for one receptor type. Ordinate: magnitude of response as related to the maximum. (A) Ultraviolet recep tor; @) blue receptor; (C)green receptor. Intracellular recording. Similar receptor types were found in the worker bee, but some deviations as described in the text (p. 164) may occur. (From Autrum and von Zwehl, 1963.)

In summary it can be stated that there is good evidence that in insects the visible spectrum of most species investigated extends from 300 to 650 mp.Peaks of sensitivity occur at 350,430-470,490-500 and 540 mp. In all species investigated the peak at 350 mp is much sharper than the peaks within the visible range. In addition a peak at 620 mp is observed in some species, but in blowflies which are the most frequently used experimental animals, this peak must be regarded as an artifact due to the transparency of the screening pigments which fail to isolate the

COLOUR DISCRIMINATION I N INSECTS

157

ommatidial units from each other within this region of the spectrum. Investigations involving recordings of the mass-response or effects of selective adaptation have led most authors to conclude that the peaks in the overall-sensitivity curve reflect the sensitivity peaks of several individual types of receptors, each showing a single peaked sensitivity curve. In the case of the Culfiphoru this concept is not valid; here all the receptors exhibit in addition to a maximum within the visible range another maximum in the ultraviolet. In the case of the bee, there are indications of several receptors with a less pronounced secondary maximum or single peaked sensitivity curves. Thus one has to face the situation that on the one hand there is evidence for similar positions of the sensitivity peaks as observed in the mass-response of many species of insects (cf. Fig. 6), while on the other these peaks might be due to double-peaked sensitivity curves of several single receptors as in the blowfly and cockroach (cf. p. 148) or to single peaked sensitivity curves of different receptors as in the bee. Behavioural investigations show roughly the same peaks of sensitivity, but they do not tell in every case exactly what the relative heights of these peaks are. B. T H E QUESTION OF THE SCREENING PIGMENTS

The screening pigments of the insect eye play an important role in isolating optically the ommatidia of the compound eye from each other and in enhancing visual acuity. Furthermore, they may be involved in adaptation to light. Their properties do not determine colour vision, but they have to be considered for they may mask the true sensitivity curves of the receptors in the course of experiments. In addition, several authors have proposed the hypothesis that the ultraviolet sensitivity of the insect eye is not mediated by a photosensitive substance absorbing ultraviolet light, but by the screening pigments (Forrest and Mitchell, 1954; Egelhaaf, 1956). To the group of screening pigments belong the pteridines, yellowish pigments unstable to light. If irradiated with ultraviolet they fluoresce and re-emit blue-green light. It was suggested that this fluorescent light in turn stimulates the green receptor system. As clearly pointed opt by Goldsmith (1961) this cannot be the case. Such a system could not mediate an ultraviolet sensitivity higher than that of the green, the ultraviolet and the green sensitivity would thus be coupled in every respect, which clearly is not the case. Finally, ultraviolet could not be a genuine and separate colour for insects as it is in bees. Experimentally this fluorescence hypothesis was excluded for the Calliphora

158

DIETRICH BURKHARDT

by Hoffmann and Langer (1961), who found that the ultraviolet sensitivity of the chalky mutant is even higher than in the wild type. Clearly, in this mutant the pteridines are absent and the higher sensitivity as observed in the mass-response is due to the lack of screening action of these substances. On the other hand, the so-called red sensitivity of the fly as revealed by the electrical mass-response of the eye has been proved to be caused by the ommochromes which fail to isolate the optical units at the red end of the spectrum. (References to papers dealing with the chemical nature of screeningpigments are given by Butenandt and coworkers, 1958,1960; Ziegler-Gunder, 1956; and Autrum, 1961). C. V I S U A L PIGMENTS IN INSECTS

As far as the light-sensitive pigments are concerned, only a very few data are available in insects. The first to extract a photosensitive pigment in insects was Goldsmith (1958). He gave evidence for a retinene in the head of worker bees, the maximum absorption being at 440 mp. This agrees with the results from investigating the mass-response of drones and certain individual receptor cells in the worker as done by Goldsmith (1960) and Autrum and von Zwehl(l962). Somewhat later Briggs (1961) also found retinenes in several species of insects in the orders Odonata, Hymenoptera, midoptera, Coleoptera and Orthoptera. In spite of the fact that there has been little chemical work done on the photosensitive substances in insects, it is most likely that in general they belong to the retinenes. Up to now, in all invertebrates carefully investigated retinenes were found, as for example in squids and Crustacea (for references, see Milne and Milne, 1959; Autrum, 1961). Furthermore, the sensitivity curves of individual receptors in the bee and the blue-bottle fly are indicative for retinenes. Comparable to the a-absorption in the vertebrate pigments there are sensitivity peaks within the region between 430 and 540 mp and similar to the secondary 8-absorption of all vertebrate pigments and derivatives of them there is a secondary sensitivity peak in the near ultraviolet. While the position of the &absorption is located within a small part of the near ultraviolet, the position of the a-absorption varies widely with the specific vertebrate pigment investigated. The a-peak position of rhodopsin or rod pigment varies among species with differences in the protein (opsin). Furthermore, the three conepigments in the same animal and with the same retinene but different proteins do vary in position of the a-peak. Comparably, the position of the ultraviolet peak

159 is the same in all insects investigated, while the peaks within the visible part vary considerably with the insect investigated and individual receptor within the same animal. These similarities are a remarkable example of evolutionary convergence. Apparently in arthropods, molluscs and vertebrates the same chemical modes have been developed independently to mediate colour vision, namely to couple retinenes as a prosthetic group to different proteins and thus to vary the spectral sensitivity of individual receptors. COLOUR DISCRIMINATION IN INSECTS

D . WAVELENGTH DISCRIMINATION A N D CENTRAL MECHANISMS OF COLOUR VISION

1. Electrophysiological evidence in the blowjy

Wavelength discrimination means the ability to distinguish between spectral lights of differing wavelengths and independently of intensity. The smaller the wavelength difference between two adjacent spectral lights may be, the better the wavelength discrimination. It can be tested by electrophysiological studies or by behavioural investigations. Up to now electrophysiologicalexperiments have been done in insects only by recording the mass-response of the eye, the ERG. Unfortunately this method is not a very satisfactory approach. According to the considerations made on pages 143 and 150 the mass-response is influenced by the efficiency of the stimulus and the stimulated area, and the latter may depend on wavelength. This interference may result in misinterpretation of the results obtained, as will be discussed below. Another objection to this method is as follows. Recent investigations have shown that the compound eye is most likely composed of a large number of uniform elements and only a small number of visual cells differs from the majority in regard to spectral sensitivity. While the mass-response will reflect chiefly the properties of the predominating type of cells, the important input to the neuronal network mediating colour discrimination will be delivered from the few deviating cells. Thus it would be better to investigate second or higher order neurons to get data for colour discrimination by electrophysiological methods. While in vertebrates this has been done to a large extent (cf. p 164), in insects no experiments of this type have been undertaken. The mass-response of the Culliphoru eye was investigated by Autrum and Stumpf (1952), using heterochromatic flicker as a stimulus. The intensity of the light from a filament bulb was matched against monochromatic lights or of two monochromatic lights against each other as described on p. 133. They found a response to flickering the

160

DIETRICH B U R K H A R D T

filament bulb light against any wavelength except 580 mp and concluded that a stimulus of 580 mp is perceived as uncoloured. Since the ultraviolet sensitivity of the eye was not taken into consideration, this finding should be accepted with some caution. Red stimuli between 620 and 690 mp gave a clear response if with any wavelength outside of this region of the spectrum intermittent. It was thought that red light was a separate category of colour for this insect. One objection to be made against this is that, especially in this region of the spectrum, the screening pigments are transparent and hence a large area of the eye was stimulated intermittently with a smaller area illuminated by the stimulus complementary in time and of shorter wavelength. Obviously it must be difficult to match these stimuli to give no response under such conditions. The region between 630 and 580 mp was found to be a quality distinguished from other colours and of a low threshold for wavelength discrimination. Wavelengths around 480 mp are apparently of another quality and highly saturated, but the wavelength discrimination in this region is not as good as between 580 and 630 mp. Several insects were studied with similar techniques by MazokhinPorshniakov (1959a, b, 1960a, b, 1962a, b) but the intermittent stimuli were obtained from a colorimeter, so that mixing of wavelengths was possible. Like Autrum and Stumpf he claimed a red receptor system for the fly Calliphoru; the reasons why the existence of such a receptor is unlikely have already been discussed. Since investigations on the responses of neurons are lacking, the most valid data concerning colour discrimination and colour vision come from the study of the behaviour of insects. 2. Colour vision in the bee The bee is the insect best investigated in respect to colour vision. Our knowledge was extended by the work of Daumer (1956, 1958). He developed a colorirneter for mixing pure spectral lights, including ultraviolet. The stimulating lights were projected on small figures to which the bees had been trained to search for food offered in small bowls. When offered four such possible feeding places the bees were forced to make the right choice. While Kuhn and Pohl(l921) proved at least four major qualities to be distinguishable for the bee, namely yellow (650500 mp), blue-green (500-480 mp), blue and violet (480-400 mp) and ultraviolet (400-300 mp), Daumer found two more qualities. Mixing violet and ultraviolet stimuli of around 440 and 360 mp resulted in a colour clearly distinguishable from others, that colour was called “bees’ violet”. Pure spectral lights around 400 rnp fall in the same category.

C O L O U R DISCRIMINATION I N INSECTS

161

Similarly, another new quality resulted from mixing yellow and ultraviolet stimuli and was called “bees’ purple”, in accordance with a mixture of stimuli from the ends of our spectrum which results in the impression purple for the human being. Thus one result of these investigations was the finding that the ends of the spectrum visible for the bee can be connected to a circle, on which the different qualities distinguishable for this species can be arranged in a closed sequence. Fig. 11 shows this colour circle for the bee as compared to that for man. Daumer proved that for any colour positioned on this circle, there is always another colour complementary to it. If these two colours are mixed in appropriate quantities the mixture will be completely interchangeable with the composite spectrum of the xenon arc or the sun. Hence it is likely that with a proper mixture one is dealing with light Ultraviolet

Blue-green

Honeybee

Blue-violet

Yellow

Man

FIG. 1 1 . Colour circle of man and honeybee. Hatched parts: primary coloun, intermediate position the secondary colours. (From D a m , 1956.)

appearing white or uncoloured to the bee. The existence of the category “white” for the bee is likely, for training the bee to coloured stimuli is more easily accomplished than to white as defined by the sun’s spectrum (cf. Hertz, 1937; Daumer, 1956). Pairs of stimuli complementary to kach other are positioned on opposite sides of the circle. The wavelength discrimination is not equally good in all parts of this circle. In the orange, yellow, yellow-green and green region the discrimination is poor, but it is high around 490 mp in the bluish green and around 400 mp in the violet region and within the bees’ purple. Concepts such as the white content and the saturation of a colour as appropriate for colour vision in man are applicable if applied in the right way for the bee. The saturation of any colour may be proved by mixing a small amount of a coloured light to another one. Adding 2 %

162

DIETRICH B U R K H A R D T

ultraviolet to yellow proved to be already distinguishable from pure yellow for the bee. This indicates, that ultraviolet is a highly saturated hue for’these animals. Less saturated are the colours of the blue-violet and the yellow region, least saturated is the blue-green region. Daumer’s work leads to the conclusion that a trichromatic theory for colour vision is valid for the bee as well as for man. The chromaticity diagram is shown in Fig. 12. Any hue on the periphery may be evoked by this stimulus itself or by an appropriate mixture of adjacent colours. Any

FIG.12. Tentative chromaticity diagram of the honeybee. (W) White point, the equivalent of the emission spectrum of the sun or a xenon arc; (P) “bees purple”, complementary to 440 mp. (Drawing modified from Goldsmith (1961), based on data given by Daumer (1956).)

hue within the triangle is less saturated and might be mixed by appropriate amounts of three primary colours positioned at the edges. The laws of Grassmann are valid for the bee although the exact numerical equations are somewhat different from those valid for man. Altogether, this work suggests strongly that colour vision in the bee is nearly as highly developed as in man and quite similar basic mechanisms in the central nervous system are involved in colour vision in man and bee. Daumer (1958) attempted to determine what flowers look like to

COLOUR DISCRIMINATION I N INSECTS

163

bees. He took three sets of photographs from flowers which were frequently visited by bees. One photograph was exposed with an ultraviolet filter in front of the camera, the next with a blue, the third with a yellow filter, so that the filters corresponded to the three primary colours for the bee. On each of the exposures a grey scale was pictured simultaneously with the flower. It was thus possible to calibrate the reflection of the flower within the spectral range investigated. Green leaves turned out to be reflectant in all major regions of the spectrum visible to the bee-thus appearing greyish to the animal-while many flowers looking green to us do not reflect blue and ultraviolet, therefore they are distinguishable from green leaves for the bee. In general, flowers of orange, yellow and yellow-green d o u r fall into the categories of “bees’ yellow” or “bees’ purple”, according to whether they reflect ultraviolet or not. Many flowers looking white to us, some pink or lilac, reflect yellow and blue, but absorb ultraviolet. Their appearance is for the bee similar to the complementary colour of ultraviolet (i.e. bluegreen region of the spectrum). Most of the red flowers reflect ultraviolet, thus appearing in this colour to the bee. Finally, the flowers which look blue or violet fall into at least four categories for the bee, depending again on their ultraviolet reflectance. Thus flowers, looking quite similar to man may look quite different to the bee; on the other hand, flowers looking very different to us may have the same appearance to the bee. Many of the flowers photographed exhibited only in the ultraviolet a pattern around their centre, corresponding to the well known honey-guide of flowers within the visible range of the spectrum.On the border between this ultraviolet pattern and the rest of the petal the bees regularly protruded their proboscis, thus searching for nectar. Experiments indicate that this behaviour is mediated by an inherent neuronal mechanism.

3. Colour discrimination in other species of insects Nothing comparable to the knowledge of the chromaticity diagram of the bee has yet been achieved for other insects. While for most of the speciesinvestigatedit is obvious that they are able to discriminatecolours, detailed data are lacking. Autrum (1960) and Goldsmith (1961) have listed species reported to have colour vision. More recent papers not listed there include Suzuki (1961), Tsuneki (1961), and MazokhinPorshniakov (1962a, b). In short, colour vision is present in the following orders : Hymenoptera, Diptera, Coleoptera, Lepidoptera, H d p tera, Homoptera, Neuroptera, Orthoptera. For in many of the investigations ultraviolet stimuli have not been included nor has appropriate physical calibration of the stimuli been done, hence they do not reveal

164

D i I??’ R I C M B (IR K HA R D T

much more than the bare fact that the insect under consideration is colour susceptible, so that the conclusions drawn by these authors have to be accepted with caution. However, one fact of general interest emerges. Several species have eyes divided into two portions: one mediating a colour sense, the other colour blind. The first to report a case like that was Rokohl (1942) who found the dorsal part of the eye of Notonecta glauca colour blind, the ventral one colour discriminating. These Gndings were later confirmed byLudtke(1954).For thecockroach Walther (1958a, b)foundonly one type of receptor in the ventral half of the eye, while at least two are present in the dorsal part. Also in Libellula Mazokhin-Porshniakov (1959) claimed one d o u r blind and one colour susceptible part of the eye. Finally, investigations of Autrum and Burkhardt (1960, 1961) and Autrum and von Zwehl (1962) proved the existence of more than one receptor type only in certain regions of the eyes from Calriphora and from the drone bee. In both the latter experiments there is a possibility that the investigation of a larger number of cells may alter the picture somewhat. 4. Central mechanisms

In the vertebrate retina second and third order neurons and the optical pathways within the highest centres have been investigated thoroughly with electrophysiologicaltechniques (cf. Granit, 1955,1959; Donner, 1950; Svaetichin, 1956; MacNichol and Svaetichin, 1958; Wagner et al., 1960; De Valois and Jones, 1961;Motakawa et al., 1962). No investigations of this type have been possible up to now in insects. Thus the only information available about central integrativemechanisms is the outcome of behavioural studies and of theoretical considerations giving certain limitations to speculations about possible mechanisms. If in any eye two or more receptors are present, differing in their spectral sensitivity curves, colour vision becomes possible. In Fig. 13 two hypothetical receptors are assumed, both with a single peaked sensitivity curve. Five distinct regions of the spectrum are distinguishable. In the region AB only the receptor 1 is excited; in the region EF only receptor 2. In the regions BC and DE both the assumed receptors will be excited, and if the wavelength is changed at a given intensity the excitation of both the receptors will increase or decrease in a similar manner. Within the region CD changing the wavelength will cause one receptor to be more excited, the other less. Changing the wavelength within the region AB and EF will obviously have the same effect as when intensity is increased: the excitation of the single receptor func-

C O L O U R DISCRIMINATION

IN INSECTS

165

tionin within this range will be increased in either case. Thus colour shod not change within these regions, but should only become brighter or less bright if the wavelength of the stimulus is altered. Quite a similar situation holds true in the regions BC and DE. Both receptors are excited, but changing wavelength will cause changing the excitation of both receptors in the same direction. Similar effects would be obtained too if intensity is changed at a constant wavelength. Again one has to predict that within these regions the stimulus must result in a colour changing only slightly in hue but chiefly in brightness. Altogether, one has to expect a highly graded wavelength discrimination only around the wavelengths B and E and in the region CD.

d

A

C

0

E

Wavelength

FIG.13. Hypothetical and schematic: two receptors of slightly differing spectral sensitivitiesare thought to mediate colour discrimination. Regions of good and bad wavelength discrimination are indicated; for details compare text.

According to these considerations it appears possible to predict from the shape of known receptor-sensitivity curves the accuracy of wavelength discrimination and the regions of the spectrum where colours change markedly or do not change very much, but become only brighter. Fig. 10 illustrates the sensitivity curves of three types of receptor cells found in the compound eye of the drone. Below are indicated the ranges of the spectrum corresponding to a certain quality of colour perceived by the worker bees as evaluated by the work of Daumer. Ranges of good wavelength discrimination and rapidly changing hues are the violet and the blue-green regions. The sensitivity curves of the receptors of the worker are similar to those of the drone, but sometimes

166

DIETRICH B U R K H A R D T

the following deviations are observed (Autrum, personal communication): (a) the side of the bluc receptor curve at long wavelengths is less steep and extends sometimes to 600 mp;(b) the ultraviolet receptors often have a secondary maximum or a hump at 540 mp; (c) the curves are not as sharp as in the drone but are mostly more flattened. If one takes these differences into consideration, the picture is as follows. In the range indicated as yellow, the sensitivity curves run in the same direction as in the range DE, EF in Fig. 13, the wavelength discrimination is poor. In the blue-green range the curves of the green and the blue receptor cross, similarly in the violet range both of them cross the ultraviolet receptor curve. Both regions show a good wavelength discrimination. In contrast, in the blue and the ultraviolet region the curves of all three receptors run a similar course, and as in the yellow range wavclength discrimination is poor and the colour does not change very much. TIius the results obtained by studying the behaviour of the worker bees agree fairly well with the predictions made from the receptor response. Furthermore, one would expect that brightness would increase with shorter wavelength within the yellow range, which according to Daumer’s work is-the case. Another conclusion is possible about the distribution of the receptors within the retina. Besides the fact that in some insects parts of the eye may contain only one type of receptor and other large areas more than one, the question arises as to how the receptors differing in spectral sensitivity are distributed within the ommatidia. It is unlikely that in each single ommatidium only receptors of one type are assembled, in another ommatidium receptors of the next type and so on. If this were true, visual acuity should depend on the wavelength used as stimulus, but there is no evidence that visual acuity in insects depends on other parameters than the brightness of light. In Culliphora it was found that five-sevenths of all cells investigated belong to the most common green type and only one-seventh belong each to the blue and the yellow-green type. Since the ommatidium is composed of seven receptor cells it is likely that each ommatidium contains five cells of the common green type and only one each of the two deviating types. This is probably comparable to the situation at the level of ganglion cells within the vertebrate retina, where the majority of cells belong to the dominator type, obviously mediating brightness sensation, and only a small number of cells are modulators responsible for colour vision. Finally, the question will be discussed as to the way in which the information input from visual cells might be processed by the following neurons. In the vertebrate retina Svaetichin (1956), MacNichol and

167

COLOUR DISCRIMINATION IN INSECTS

Svaetichin (1958), Wagner et al. (I 960) have described second and third order neurons exhibiting excitation patterns which change with wavelength, by either graded action potentials or impulse frequencies. In the first case it is observed that when certain regions of the spectrum are applied as stimulus to the retina, the sign of the potentials changes. Stimuli below or above this critical wavelcngth elicit either depolarization or hyperpolarization of the neuronal membrane. In the second case the resting activity of the neurons is changed when the eye is stimulated, the frequency of firing is increased or decreased. The most likely explanation would seem to be that there are simultaneously excitatory and inhibitory synaptic inputs delivered to the neurons under consideration. Whether the excitatory or inhibitory input is stronger depends on the wavelength of the light stimulatingthe receptor cells.

!RJ-R"

I

I

4

I

I

A2

FIG.14. Hypothetical scheme of neuronal interaction to produce colour vision. (A), (B)Receptor cells differing in spectral sensitivity (S)(compareinset); (0interneuron; (N) second order neuron, inhibited via (I by) receptor cell (B), excited by (A),

spontaneously active in the dark. At the wavelength Aa the frequency of the neuron is accelerated, at A, slowed down. (From Burkhardt, 1%3.)

The simplest assumption is that two receptor cells differing in spectral sensitivity are driving one higher order neuron. In Fig. 14 such a scheme is pictured. The receptor cell A is connected to the neuron directly by an excitatory synapse, while the cell B is connected via an interneuron to inhibit the same ganglion cell. The spontaneously firing gangliw cell will lower or increase its frequency, depending on whether the inhibitory or the excitatory input predominates. Since the two receptor cells are thrown into differing states of excitation by stimuli of

168

DIETRICH B U R K H A R D T

equal intensity but differing wavelength, the excitation of the ganghon cell will depend strongly on wavelength. In this way it becomes possible that the output of the ganglion cell reflects the difference between the sensitivity curves of the two receptor cells involved. Fig. 15 shows the calculated difference between sensitivity curves of a green receptor cell and a blue receptor in Calliphora.For comparison, a curve of the spectral

+so 0

-50

-100

m/l

FIG.15. Upper half: response of a yellow-green neuron cell in the fish retina, re-drawn from MacNichol and Svaetichin (1 958). Ordinate (right): hyperpolarization (negative) and depolarization (positive), as related to the maximum response. Lower half: algebraic difference between the excitation of an individual green-type receptor cell and an individual blue-type cell in Culliphoru, calculated from data given by Burkhardt (1%2). Ordinate (left): difference in excitation as related to the maximum difference.

response from the ganglion cell of a fish retina is given above. Typically there are two types of colour responses observed in fish neurons, a yellow/blue response and a red/green response. These two curves are similar to the set of curves calculated for the hypothetical ganglion cell of the insect if driven by a blue and green receptor or a green and yellow-green receptor respectively. The response curves of the k h neurons are shifted approximately 100 m p towards longer wavelengths

COLOUR DISCRIMINATION IN INSECTS

169

as compared with the calculated curves for Culliphuru,but in general the spectrum visible for insects is shifted 100 mp towards shorter wavelengths as compared to that of vertebrates. While these considerations are highly speculative, they may be of some value in planning further experiments. Experimental information about the activity of neurons within the optical pathway of insects while the eye is undergoing stimulation with spectral lights is needed and correspondingly more information about the spectral properties of the vertebrate receptor cells. When this material becomes available, some of the questions about the peripheral mechanisms mediating colour vision can be solved. There are many indications that the basic mechanisms of colour vision in vertebrates and insects are not so different as one might expect at first glance. The chemical mechanisms causing the differential spectral sensitivity in single receptors are similar in insects and vertebrates. In both groups of animals the retina is divided in colour blind areas and areas sensitive to colours. In vertebrates and insects a predominant number of receptors of one type are obviously mediating brightness sensation while only a comparable small number of cells deviating in spectral sensitivity mediate the colour response. Finally there are indications that within the level of higher order neurons similar integrative mechanisms take place. The probable existence of three types of receptors results in trichromatic colour vision in man and the bee. Small differences in the spectral sensitivity curves of the receptors may be enhanced by excitatory and inhibitory input to the neurons which in turn will exaggerate the spectral response curves. REFERENCES Armbruster, L. (1922). Uber das Farbensehen der Wcspen. Nutwwiss. Wschr. N.F. 21,419-422. Autrum, H. (1950). Die Belichtungspotentiale und das &hen der Insekten (Untersuchungen an Calliphora und Dixippus.) 2.vergl. Physiol. 32, 176227. Autrum, H. (1955). Die spektrale Empfindlichkeit der Augermutation whiteapricot von Calliphora erythrocephala. Biol. Zbl. 14, 515-524. Autrum, H. (1958). Electrophysiological analysis of the visual systems in insects. Exp. Cell. Res., Suppl. 5 , 426-439. Autrum, H. (1960). Vergleichende Physiologie des Farbcnsehens. Fortschr. Zool.

N.F.12, 176205. Autrum, H. (1961). Physiologie des Sehens. Fortschr. Zool. N.F. 13,257-302. Autrum, H. (1963). Wie nimmt das Ange Farben wahr? Umschau63,332-336. Autrum, H. and Burkhardt, D. (1960). Die spektrale Empfindlichkeit einalner Sehzellen. Naturwissenschuften 47, 527. Autrum, H. and Burkhardt, D. (1961). Spectral sensitivity of single visual cells. Nature, Lmd. 190, 639.

170

DIETRICH BURKHARDT

Autrum, H. and Stumpf, H. (1953). Eiektrophysiologische Untersuchungen iiber das Farbensehen von Calliphora. Z. vergl. Physiol. 35,71-104. Autrum, H. and Zwehl, V. von (1962). Zur spektralen Empfindlichkeit eimlner Sehzellen der Drohne (Apis mellifca&. Z. vergl. Physiol. 46,8-12. Autrum, H. and Zwehl, V. von (1963). Ein Grtinreceptor irn Drohnenarge (Apis mellificad). Naturwissenschqften50,698. Bartley, S. H. (1959). In “Handbook of Physiology” (J. Field, ed.), Sect. I, Vol. 1, . pp. 71 3-740. American Physiological Society, Washington, D.C. Bedau. K. (1911). Das Facettenauge der Wasserwanzen. Z. wiss. Zool. 91,417456. Bertholf, k. MI (1931a). Reactions of the honeybee to light. J. agric. Res. 42, 379-41 9. Bertholf, L. M. (1931b). The distribution of stimulative efficiency in the ultraviolet spectrum for the honeybee. J. agric. Res. 43, 703-713. Bertholf, L. M. (1932). The extent of the spectrum for Drosophilu and the distribution of stimulative efficiency in it. 2,vergl. Physiol. 18, 32-64. Briggs, M. H. (1961). Retinene -I in insect tissues. Nature, Lond. 192, 874-875. Buck, J. B. (1937). Studies on the firefly. 11. The signal system and color vision in Photinus pyralis. Physiol. 2001.10, 412419. Buddenbrock, W. von and Friedrich, H. (1933). Neue Beobachtungen Ubei die kompensatorischen Augenbewegungenund den Farbensinn der Taschenkrabben (Curcinus maenas). Z. vergl. Physiol. 19, 747-761. Burkhardt, D. (1962). Spectral sensitivity and other response characteristicsof single visual cells. Symp. SOC.exp. Biol. 16, 86109. Burkhardt, D. (1963). Lichtrezeptoren imTierreich. Naturwksenschaftsn 50,586590. Burkhardt, D. and Autrum, H. (1 960). Die Belichtungspotentialeeinzeher Sehzellen von Calliphora erythrocephala Meig. Z . Natauf. 15B,612-616. Burkhardt, D. and HoffmaM, C. (1962). Untersuchungen zur spektralen Empfindlichkeit des Insektenauges. Zool. Anz. 25 Suppl. Bd., 181-185, Burkhardt, D. and Wendler, L. (1960). Ein direkter Beweis fiir die Fahigkeit einzelner Sehzellen des Insektenauges, die Schwingungsrichtung polarisierten Lichtes zu analysieren. Z. vergl. Physiol. 43, 687-692. Butenandt, A., Biekert, E. and L i m n , B. (1958). uber Ommochrome, XIV. Zur Verbreitung der Ommine im Tierreich. Hoppe-Seyl. Z. 313, 251-258. Butenandt, A,, Biekert, E., Kiibler, H.and Linzen, B. (1960). uber ommochrome XX. Zur Verbreitung der Ommatine im Tierreich. Neue Methoden N ihrer Identifizierung und quantitativen Bestimmung. Hoppe-Seyl. 2.319.238-256, De Valois, R. L. and Jones, A. E. (1961). In “Neurophysiologie und Psychophysik des visuellen Systems”. Symposium Freiburg. pp. 178-191. Springer-Verlag, Berlin, Gottingen, Heidelberg. Daumer, K. (1956). Reizmetrische Untersuchung des Farbansehens der Bienen. 2. vergl. Physiol. 38, 413-478. Daumer, K. (1958). Blumenfarben, wie sie die Bienen when. 2.wrgl. Physwl. 41, 49-1 10. Donner, K. 0. (1950). The spike frequencies of mammalian retinal elements as a function of wavelength of light. Acta physiol. scursd. 21, Suppl. 72, 1-59. Egelhaaf,A. (1956). PhotolabileFluoreszerzstoffebeiEphestiukhtiella. Natuwissenschaften 43, 309. Fmgermann, M. and Brown jr. F. A. (1953). Color discrimination and physiological duplicity of Drosophila vision. Physiol. Zool. 26, 59-67.

C O L O U R DISCRIMINATION I N INSECTS

171

Forrest, H. S. and Mitchell, H. K. (1954). Pteridinca from Drosophifa. I. Isolation of a yellow pigment. 11. Structure of the yeilow pigment. J. Amer. chem. SOC.76, 5656-5658. Frisch, K. von (1913). Zur Frage nach dern Farbensinn der Tiere. Verh. Ges. dtsch. Naturf. & f e85, 112-1 15. Frisch. K. von (1960). In “Mechanisms of Colourdiscrimination”, pp. 19-28. Pergamon Press. London. Frisch, K. von and Kupelwieser, H.(1913). Uber den Einfluss der Lichtfarbe aufdie phototaktischen Reaktionen niederer Krebse. Biol. Zbl. 33. 517-552. Goldsmith, T.H. (1958a). The visual system of the honeybee. A.m. nur. A d . ScL, Wash. 44, 123-126. Goldsmith, T. H. (1958b). On the visual system of the bee ( A p k mellifra). Ann. N.Y. Acad. Sci. 74, 223-229. Goldsmith, T. H.(1960). The nature of the retinal action potential, and the spectral sensitivities of ultraviolet and green receptor systems of the compound eye of the worker honeybee. J. gen. Physiol. 43, 775-799. Goldsmith, T. H. (1961). In “Light and Life” (W. D. McElroy and B. Glass,eds.). pp. 771-794. Johns Hopkins Press, Baltimore. Goldsmith, T. H. and Ruck, P. R. (1958/58). The spectral sensitivities of the dorsal ocelli of cockroaches and honeybees. J . gen. Physiol. 41, 1171-1185. Granit, R. (1955). “Receptors and sensory perception. A discussion of aims. means, and results of electrophysiological research into the process of reception”. 366 pp. Yale University Press. New Haven. Granit, R. (1959). In “Handbook of Physiology” (J. Field, 4.1, Sect. 1. Vol. 1, pp. 693-7 12. American Physiological Society, Washington, D.C. Hamilton, W. F.(1922). A direct method of testing color vision in lower animals. Proc. nat. Acad. Sci., Wash. 8,350-353. Hartline, H . K.,Wagner, H.G. and MacNichol jr., E. F. (1952). The paipherd origin of nervous activity in the visual system. Cold Spr. Hmb. Symp. quant. Biol. 17, 125-141. Hasselmann, E.-M. (1962). Ober die relative spektraleEmfindlichkeitvonKifcr-und Schmetterlingsaugen bei vaschiedcnen Helligkeiten. 2001.Jb,, Phydol. 69, 537-576. Heintr, E. (1959). La question de la smsibilite des abeilles A l’ultra-violet. Insectes sociaux 6, 223-229. Hertz, M. (1937). Reitrag zum Farbensinn und Formensinn der Biene. 2.wrgl. Physiol. 24,4 13421. Hess, C. (1 9 13). Neue Untersuchungen zur vergleichendenPhysiologie des Gesichtssinnes. 2001.Jb., Physiol. 33, 387-440. Hoffmann, Ch. and Langer. H. (1961). Die spektrale Augcreupfindlichkeit der Mutante “chalky” von Calliphoru erythrocephala. Natwwissensc~ten48, 605. Ilse, D. (1928). Ober den Farbensinn der Tagfalter. 2.vergl. Physlol. 8,658492. Ilse, D. (1934). Ober das %hen der Insekten, beeronders der Tagfalter. S.B. Ges. naturf. Fr. Berlin 1/3, 1-16. Ilse, D. (1937). New observations on responses to colours in egglaying butterflies. Nature, Lo&. 140, 544. Jahn, T. L. and Crescitelli, F. (1939). The electrical reepanse of the Cecropia moth eye. J. cell. comp. Physiol. 13, 113-119.

172

DIETRICH BURKHARDT

Knoll, F. (1921). Insekten und Blwnen. Experimentelle Arbciten zur Vertiefung unserer Kenntnisse Uber die Wechselbeziehungenzwischen Pflanzen und Tieren. Abh. zoo1.-bot. Ges. Wien 12, 1-1 16. Knoll, F. (1922). Inserten und Blumen II. Lichtsinn und Blummbesuch dea Falters von Macroglossurn stellatarum. Abh. zoo1.-bot. Ges. Wien 12,123-377. Knoll, F. (1925). Lichtsinn und BIUtenbesuch des Falters von Deilcphih livornica. 2.vergl. Physiol. 2, 329-380. Knoll, F. (1927). Uber Abendschwtirmer und Schwhnerblumen. &r. dtsch. bot. Ges. 45, 510-518. Kilhn, A. and Ik, D. (1925.) Die Anlockung von Tagfaltern durch Pigmentfarbcn. Bi d. Zbl. 45, 144-149. Kiihn, A. and Pohl. R. W. (1921). Dressurfahigkeit dor Biene auf Spe-en. Natwwissenschaften 9, 738-140. Langer, H. and Patat, U.(1962). Uber die Bcdeutungeiner neum Augdarbmutante von Call@hora erythroccphala Meig. for die Untersuchung der Funktion des Facettenauges. Zool. Anr. 25, Suppl. Bd. 174-180. Lubbock, Sir J. (1929). “Ants, bees and wasps, a record of observation on the habits of the social Hymenoptera”, pp. 152-168. E. P.Dutton and Co.,New York. Lildtke, H. (1953). Retinomotorik und Adaptationsvorgiinge im Augc des Rtkkcnschwimmers (Notonecta gl&ucaLa).Z . vergl. Physwl. 35, 129-152. Lildtke, H. (1954). Dunkeladaptation und Verschicbung dcr Hclligkeitswcrt im Augc von Notonecta glauca L. Z . N a tu d , Tubingen 9B,159-163. MacNicholjr. E. J. (1963). Int. Congr. Psychol. Washington, D.C. MacNichol, jr., E. J. and Svaetichin, G. (1958). Electrical responses from the isolated retinas of fishes. Amer. J. Ophthalmol.46,2646. Mazokhin-Porshniakov, G . A. (1959a). Green, yellow and o m colour discriination in bees. (Russian.) Akad. Nauk. SSSR. Bwfiika 44-54. Mazokhin-Porshniakov, G. A. (1959b). Colorimetric study of colour Vision in the dragon By. Akad. Nauk SSSR Biofzika 4,427-436. Mazokhin-Porshniakov, G . A. (196Oa). Colorimetric study of the properties of the housefly. Akad. Nauk SSSR Bwjizika 5, 295-303. Mazokhin-Porshniakov, G. A. (1960b). System of colour vision of the fly Calliphora Akad. Nauk SSSR Blofizika 5,697-703. Mazokhin-Porshniakov, G. A. (196%). Colouring colorimetric demonstration of trichromatism of the colour vision of bees (e.g. Bumblebees.) (Russian.) Akad. Nauk. SSSR Biofzika 7,211-217. Mazokhm-Porshniakov, G. A. (1962b). About the colour vision of the rosechafer Coleoptera, Cetoniini). Dokl. Akad. Nauk SSSR 143, 12.08-1210. MCdioni. J. (1961). “Contribution it l’ttude psychophysiologique et ghetique du phototropisme d’un insecte: Drosophila melanogaster Meigen”. Thesis, Univcrsity of Strasbourg. Milne, L. J. and Milne, M. (1959). In “Handbook of Physiology” (J. Field, cd.), Sect. 1, Vol. 1, 621-645. American Physiological Society, Washington, D.C. Molitor, A. (1939). Zum Farbensinn der Faltenwespm. Zool. Anz, 126,259-264. Moller-Racke, I. (1952). Farbensinn und Farbenblindheit bci Insekten. 2001. Jb. Physiol. 63, 231-214. Motakawa, K., Taira, N. and Okuda, J. (1962). Spectral responses of single units in the primate visual cortex. Tohoku J. exp. Med. 78, 32&327. Naka, K. (1961). Recording of retinal action potentials horn single cells in the insect compound eye. J. gen. Physiol. 44, 571-584.

COLOUR DISCRIMINATION IN INSECTS

173

N a b , K. and Eguchi, E. (1962). Spike potentials recordad from the insect photoreceptor. J . gen. Physiol. 45, 663-680. Naka, K. and Kuwabara. M. (1959a). Response of a sin& retinufa cell to polarized l i b t . Nature, bnd. 184,455456. Naka, K. and Kuwabara, M. (1959b). Electrical response from the compound eye of Lucilia. J. Insect Physiol. 3, 4 1 4 9 . Rokohl, R. (1942). ober die regionale Verschiedenheit der Farbentilchtigkeit im zusammengesetzten Auge von Notonecta glauca. 2.vergl, Physiol. 29,638-676. Sander, W. (1933). Phototaktische Reaktionen der Bienen auf Lichter verschiedener Wellenlange. Z. vergl. Physiol. 20, 267-286. Schlegtendal,A. (1934). Beitrag zum Farbensinn der Arthropoden. 2.vergl. Physiol. 20, 545-581.

Schlieper, C. (1927). Farbensinn der Tiere und optomotorische Reaktionen. 2.vergl. Physiol. 6, 453472. Schneider, G. (1956). Zur spektralen Empfindlichkeit des Komplexauges von Calliphora. 2. vergl. Physiol. 39, 1-20. Schremmer, F. (1941). Versuche zum Nachweis der Rotblindheit von Vespa rufa L. 2. vergl. Physiol. 28, 457466. Suzuki, K. (1961). The colour sense of a mosquito, Culexpipienspallens Coquillet. Jap. J . Zool. 13, 185-197. Svaetichin, G. (1956). Spectral response curves from single cones. A m phy8iol. scand. 39,Suppl. 134, 1746. Tsuneki, K. (1961). Mem. Fac. Lib. Arts, Fukui Univ. Ser. 11.. Nat. Sci. 1 I , 103-160. Wagner, H. G., MacNichol jr., E. F. and Wolbarsht, M.L. (1960). The response properties of single ganglion cells in the goldfish retina. J. gen. Physlol. 43, Suppl. 2, 45-62. Wald,G. (1959). 1n"Handbookof Physiology"(J.Field,ed.).Sect. 1,Vol. 1,671-692. American Physiological Society, Washington, D.C. Walther, I. B. (1958a). Untersuchungen am Belichtungspotentialdes Komplexauges von Periplaneta mit farbigen Reizen und selektiver Adaptation. Biol. Zbl. 77, 63-104.

Walther, J. B. (1958b). Changes induced in spectral sensitivity and form of retinal action potential of the cockroach eye by selectiveadaptation. J. Insect Physiol. 2, 142-1 5 1. Walther, J. B. and Dodt, E. (1959). Die Spektralsensitivitiit von Insekten-komplexaugen im Ultraviolett bis 290 mp. Elektrophysiologische untersuchungen an Calliphora und Periplaneta. 2.Naturf. 14B,273-278, Weiss, H. B. (1945). Insect response to colors. Sci. mon. N.Y.61, 61-56. Webs, H. €3. (1 946). Insects and the spectrum. J. N. Y.mi. Suc. 54, 17-30. Weiss, H. B., Soraci, F. A. and McCoy, jr,, E. E. (1942). The behavior of certain 50, 1-35. insects to various wavelengths of light. J. N. Y.en?. SOC. Weiss, H. B., Soraci, F. A. and McCoy, jr., E. E. (1943). Insect behavior to various wavelengths of light. J, N.Y.ent. SOC.51, 117-131. Ziegler-Gilnder, I. (1956). Pterine: Pigmente und Wirkstoffe im Tierreich. Biol. Rev. 31, 313-348.

The following reviews on colour vision in insects have also appeared: Buddenbrock, W. von (1 952). "Vergleichende Physiologie". Bd. 1 , Sinnesphysiologie, 504 S. Verlag Birkhauser, Basel. Wulff, V. J. (1956). Physiology of the compound eye. Physiol. Rev. 36,145-163.

This Page Intentionally Left Blank

Insect Ecdysis with Particular Emphasis on Cuticular Hardening and Darkening C. B. COTTRELL

Department of Zoology, University College of Rhodesia and Nyasaland I. Introduction . . 175 11. An Outline of Ecdysis . . 175 III. The Hardening and Darkening of Cuticular Areas prior to Ecdysis . 176 IV. The Splitting and Shedding of the Old Cuticle . 178 V. The Mechanism of Expansion . . 179 VI. The Muscular System involved in Ecdysis . . 181 VII. The Components of the Sclerotizing System . . 183 A. TheTanningAgent . . 184 B. The Protein precursOr of Scleroth . . 185 C. Enzymes Concernad in Sclerotization . 185 VILI. The Relationship between Hardening and Darkening . . 199 IX. The Control of Various Proctssts Associated with Ecdysis . .204 A. Hardening and Darkening . 204 B. Air Swallowing . .208 C. Mechanical Properties of the Cuticle . 208 D. “Shut off” of Ecdysial Muscles . m E. Absorption of Fluid from Tracheae . .209 F. Other Processes , . 209 X. Some Factors Involved in the Production of the Definitive Body Form at M y s i s . . 210 References 212

.

.

. .

.

I. INTRODUCTION

In the past thirty years the exciting discoveries which have been made in the field of the hormonal control of growth and moulting seem virtually to have eclipsed interest in ecdysis itself. It is the purpose of this article, not to review the vast literature relevant to ecdysis but rather, even at the risk of being inconclusive and controversial, to direct attention to some of the imperfectly investigated aspects of this spectacular phenomenon. 11. AN O U T L I N E OF ECDYSIS

At a normal insect ecdysis, it is convenient to distinguish three processes: (1) the splitting and shedding of the old cuticle; (2) the expansion of the new cuticle; (3) the hardening and darkening of the new cuticle. 175

176

C . B. C O T T R E L L

The splitting of the old cuticle occurs along predetermined lines (Henricksen, 1931 ; Snodgrass, 1947) where the normal sclerotized exocuticle is wanting and the endocuticle extends right up to the epicuticle (Duarte, 1939; Plotnikow, 1904). When the endocuticle has been dissolved away by the action of the moulting fluid these ecdysial lines become extremely fragile and only a very slight pressure is required to rupture them. The pressures necessary for this purpose may be transmitted to the old cuticle by the undifferentiated new cuticle or by means of special structures such as spines or eversible bladders; they are created, at least in part, by contractions of the body muscles (Kunkel d’Herculais, 1890; Brocher, 1919), but these contractions are assisted in the vast majority of insects by the swallowing of quantities of air (Jousset de Bellesme, 1877; Knab, 1909, 1911;Eidmann, 1924) or water (Blunck, 1923). This swallowing of the surrounding medium also plays an important part in the expansion of the new cuticle so that in general the two processes are initiated almost simultaneously and the new cuticle begins to expand even as it is being withdrawn from the old. After the old cuticle has been shed, further swallowing is usually still necessary in order to complete the process of expansion. In a few insects however, the splitting and shedding of the old cuticle is independent of air or water swallowing. Thus at the imaginal moult of cyclorrhaphous Diptera, no air is swallowed during emergence and the pupal and puparial cuticles are split by means of a special device, the ptilinum, which enables a localized pressure to be brought to bear against them (Laing, 1935). In such cases the insect has become at least potentially capable of delaying its expansion until a considerable time after the shedding of its old cuticle. In any event, the cuticle of the newly emerged insect is, except for certain specialized areas, quite soft and usually in addition colourless; only later does it harden and darken. The tanning and darkening agents are ultimately derived from the amino acid tyrosine which occurs in the blood. The remainder of this article will be devoted to the consideration of certain topics selected from this outline. 111. THEH A R D E N I NAGN D D A R K E N I NOF G CUTICULAR A RE A SP R I O R TO ECDYSIS

Almost all insects exhibit some areas of the new cuticle which are at least partially hardened before the old cuticleis shed, but the extent of this hardening is extremely variable. In the early stages of the Exopterygota

INSECT ECDYSIS

177

and in the soft bodied larvae of Endopterygota, prehardening is at a minimum being confined to areas such as the tarsal claws, margins of the head capsules and sometimes the mandibles. This may be correlated with the fact that in these cases there is (i) no delay between the shedding of the old cuticle and the expansion of the new one; (ii) little or no locomotion at the time of ecdysis, and ( 5 ) few organs or structures of complex form. At the imaginal moult of Exopterygota, although parts of the genitalia may be involved, prehardening is only slightly more extensive than in the earlier stages and it is only at the last moult of certain Endopterygota that it becomes really prominent. Correlated with the acquisition of a quiescent pupal stage, the Endopterygota have had to develop ways of escaping from the protective cocoon or cell in which the pupa is normally enclosed (see Hinton, 1946, 1949). Broadly, they accomplish this in three ways. 1. The escape is made before the shedding of the pupal cuticle. This occurs in all the primitive Endopterygota (Neuroptera, Mecoptera, Trichoptera, Zeugloptera and Dacnonypha) as well as in the Monotrysia and the more primitive Ditrysia and in the Nematocera and Brachycera-Orthorrhapha. In these cases it is the pupal skin which protects the soft new cuticles and carries the adaptive structures which make escape possible. Thus there is little or no need for extensive prehardening of the new cuticle. 2. The escape is made only after the new cuticle has been expanded and hardened and darkened. Again there is no need for extensive prehardening of the new cuticle. For obvious reasons this form of escape occurs only in those relatively short winged forms (Coleoptera, Strepsiptera and Hymenoptera) in which the adult is also equipped with mouth parts or other structures suitable for breaking open the cocoon. 3. The escape is made after the old cuticle has been shed but before the beginning of expansion. This is the method used by all the Brachycera-Cyclorrhaphaand many of the higher Ditrysia. The prehardened cuticular areas of a newly emerged blowfly (Culliphoru) fall roughly into three classes: those involved in locomotion or movement in the newly emerged fly (apodemes, apophyses, hardened areas of the legs and internal proboscis sclerites), those concerned in prQtectingthe soft cuticle (bristles and microtrichia) and those occurring in regions of complex shape or bearing sensillae (antennae, halteres, genitalia, pteralia) (Cottrell, 1962a). In this as in other cyclorrhapha, where expansion may involve an increase in volume of some 120%, the distribution of the prehardened areas can be seen as a compromise between the need to provide a sound mechanical basis for the various

178

C. B. COTTRELL

movements and at the same time protection for the soft cuticle and the need to allow for subsequent expansion of the general body surface. In the Lepidoptera, where there is little or no increase in total volume during expansion, such a compromise is much less evident. For instance, in Pieris brussicue parts of the head (the antennae and proboscis), almost the whole of the thorax, the legs and pteralia, the tergites of the first two abdominal segments and the genitalia are all sufficiently hardened before emergence from the pupa to prevent subsequent expansion or change in shape. After expansion these areas become much harder and darker and hardening begins in the remaining parts of the head, the wings, abdominal sternites 1 and 2 and abdominal segments 3-7 which are completely soft at emergence. Little, if any, of the prehardening of a newly emerged lepidopteran is visible at emergence because the whole body is covered with scalesand bristles which have been pigmented in the pupa. Although prehardening in general has seldom received more than a passing mention, the pre-ecdysial colouring of the bristles of the cyclorrhaphous Diptera, which was noticed by Weismann (1864), has been used as a means of ageing tsetse pupae (l'otts, 1933; Bursell, 1959). In contrast to the post ecdysial hardening and darkening which occurs apparently simultaneously over the whole body surface, the process of hardening and darkening the bristles seems to occur in a wave which originates in the neighbourhood of the mesothorax and spreads backwards and forwards along the body and outwards along the appendages(Cottrell, 1962a). At the present time the occurrence, mechanism and control of preecdysial hardening and darkening remain almost entirely uninvestigated.

Iv. THESPLITTING

A N D S HE D D I N G OF THE O L D CUTICLE

The superficial mechanisms involved in this aspect of ecdysis have been described for numerous insects and are adequately reviewed in most texts. However, the forces involved and the mode of their production remain almost totally unknown. That they may in some cases be relatively large can be assumed from measurements of the internal hydrostatic pressures produced during digging by the newly emerged blowfly. Ptilinal digging in these insects is brought about by a mechanism virtually identical to that by which the pupal and puparial cuticles are split (Lewis, 1934; Fraenkel, 1935b). Each digging cycle corresponds with a characteristic cycle of internal pressure changes which in Samphaga barbata reach maxima of from 6 to 12 cm of mercury in excess of

INSECT ECDYSIS

179

atmospheric pressure. In agreement with this finding individuals confined within a glass tube (which limited the effective area of application of the ptilimum to its cross sedonal area of 0-13 cmq were found to be capable of exerting forces ranging from 9 to 23 g (Cottrell, 1962d).

v. THEMECHANISM O F EXPANSXON Expansion, like the splitting and shedding of the old cuticle, is generally considered to involve both the contraction of the body muscles and the swallowingof the surrounding medium. Although many authors have assumed the occurrence of positive internal pressures at this time (Knab, 1909, 1911; Fraenkel, 1935b), there have been few attenhpts to determine their magnitude. Shafer (1923) used as a measure of the internal pressure the height to which the haemolymph rose in a capillary inserted into the insect. In this way he wag able to show an increase of 3-5 cm of water at the imaginal ecdysis of A m x and Aeshna but such wide fluctuations were recorded for larvae that it is impossible to decide whether a similar increase occurs at larval moults. Continuous records obtained with an almost isometric condenser manometer show two different phenomena at the imaginal ecdysis of the blowflies Calliphora erythrocephala and Sarcophaga barbata. First there is a gradual rise and fall in the basic haemolymph pressure which reaches a maximum (of about 6 cm of mercury in CaZZiphora and 9-5 cm in Sarcophuga) a few minutes after the moment of full wing extension. Second, superimposed on the basic rise, there is a series of brief rhythmic pressure pulses which gradually decline and them cease at about the time of full wing extension. By blocking the probosces or denervating the abdominal muscles of newly emerged blowflies it is possible to show that the gradual rise in haemolymph pressure is due to airswallowing while the pressure pulses are due to the performance of “muscular efforts” (i.e. simultaneous contractions of both ptilinal and abdominal muscles) (Cottrell, 1962d). Although air-swallowing plays no part in the splitting and shedding of the pupal and puparial cuticles of cyclorraphous Diptera, it is of major importance in expansion which fails to occur if the proboscis is blocked or ligated (Evans, 1935; Fraenkel, 1935b). “Muscular efforts” on the other hand, seem merely to have a regulatory and guiding function and their elimination leads only to limited abnormalities in the usual process (Cottrell, 1962d). It is of interest to 6ee how far these conclusionscan be applied to other insects.

180

C. B. COTTRELL

Air swallowing at ecdysis was first reported at the imaginal moult of a libellulid dragonfly by Jousset de Bellesme in 1877. Since then it has been reported for numerous insects including agrionid dragonflies (Brocher, 1919), acridids (Kunkel d’Herculais, 1890), Locusts (Duarte, 1939), Periplaneta, Dixippus, Limnotrechis and Drosophila (Eidmann, 1924), Bombyx larvae (Wachter, 1930), butterflies (Prell, 1914) and mosquitoes (Marshall and Staley, 1932) as well as in various muscoid Diptera by authors who have already been mentioned. Water swallowing at ecdysis has been reported for several aquatic insects such as Anax larvae (Shafer, 1923) and Dyrisnts larvae (Blunck, 1923). As was noted by Brocher (1919) and Dmrte (1939) in their excellent accounts of ecdysis, air swallowing in the dragody and the locust occurs both before and after the splitting and shedding of the old cuticle. In confirmation of this, Clarke (1958) has published X-ray photographs showing that at the moment of ecdysis the gut of Locusra already contains a considerable quantity of air. During the next 10 min the gut becomes very greatly distended but 1 h later has collapsed completely, the space which it previously occupied now being iilled by the air sacs. Similarly Eidmann (1924) found that if the crop of Periplaneta or Limnotrechis was opened to the exterior before ecdysis, splitting and shedding of the old cuticle was prevented. If it was opened after the insect had freed itself from the old cuticle, normal expansion of the new one did not occur. Thus in contrast to the condition in cyclorrhaphous Diptera, air swallowing in these cases is necessary for both the splitting and shedding of the old cuticle and the expansion of the new one. “Muscular efforts” in connection with the splitting of the old cuticle have been universally noted and most authors have also appreciated the importance of peristaltic movements of the abdomen in the shedding of the old cuticle. However, the part played by “muscular efforts” in the expansion of the new cuticle has not always been understood. Prell (1914) appreciated the importance of abdominal contractions in the expansion of the wings of Pyrameis and Duarte (1939) noted that the locust continues “muscular efforts” after shedding the old cuticle in order to “unfold the hypodermis to its full extent before the cuticle hardens”. Brocher (1919) considered that during emergence an agrionid dragonfly employs “muscular efforts” in three ways. First they are used to distend the thorax and split the old cuticle, then after an interval of air swallowing, to expand the new cuticle and finally to extend the abdomen. This brief survey indicates that both air pumping and muscular efforts are of common occurrence in the ecdysis of a wide variety of insects.

INSECT ECDYSIS

181

However it would be unwise to conclude that the two activities always have the same relative importance for expansion. According to Brocher (1919) expansion of the wings of agrionid dragonfliesis very slight during the bout of air swallowing which follows shedding of the last larval cuticle, but is rapid during the phase of ‘‘muscular efforts” which succeeds it. This suggests that in the emerging dragonfly muscular efforts are at least as important for wing expansion as is air swallowing. Experiments with Pieris brassicae indicate that here air swallowing has even less importance (Cottrell, unpublished). In this butterfly, air swallowing begins at the moment of splitting the old pupal cuticle and ceases when the wings are fully expanded and stiff enough to remain upnght: at the time of full wing extension the crop is only about one quarter full of gas. When the proboscis was blocked just prior to emergence, seven out of ten insects were able to expand normally while three showed only very slight deformities of the wings although dissection confirmed that none of the insects had taken in any air. In a single specimen the neck was ligated and the head removed just after the pupal cuticle had been split. Nevertheless the abdominal muscles continued to contract and, with the insect suspended from a pin waxed to the thorax, normal expansion of the wings was achieved. It thus seems clear that in Pieris, unlike Calliphora and Sarcophaga, air swallowing plays only a minor role in the process of expansion. Wigglesworth (personal communication) has similarly found that in Rhodnius prolixus decapitated individuals or those in which the rostrum has been removed will occasionally extricate themselves from the old cuticle and at the adult moult expand their wings in a more or less normal fashion. Whether or not air (or water) swallowing is essential to expansion seems to depend (like the extent of cuticular prehardening) largely on the change in volume which accompanies the particular ecdysis.

VI. THEMUSCULAR SYSTEM I N V O L V E D

IN ECDYSIS

In blowflies many of the activities which occur characteristically or uniquely in the newly emerged or expanding insect are brought about by the action of a special musculature in the head (Laing, 1935)or abdomen (Cottrell, 1962d) which is carried over from the pupa and which undergoes involution during the first few days of adult life. Finlayson (1956) has proposed the term “imaginicaducous” for muscles like these which degenerate during the life of the imago. He describes how certain segmental units (in 3, 4, 5 and 6) of the three main longitudinal muscle bands in the abdomen of the larvae of Galleria and certain saturnids are

182

C.

B. C O T T R E L L

similarly carried through the pupal stage and only degenerate during the first two days of adult life. In Pieris (Cottrell, unpublished) there is a similar, though not idcntical set of muscles in segments 3-6 which degenerate within 3 days of emergence. As in the blowfly these muscles all belong to the internal layer of the body musculature and the fibres are of segmental length and collected together to form discrete muscles. The buckling and compressing action of the imaginicaducous muscles of Pieris is clearly responsible for the abdominal contractions which are visible in segments 3, 4, 5 and to a lesser extent in 6 during the first few minutes after emergence. It is of some interest that these muscles are absent from segments I and 2 where the tergites are hardened before emergence. In the Exopterygote, Rhodnius,the ventral intersegmental muscles of the abdomen undergo a cycle of development and involution during the course of each larval stage. They disappear a few days after ecdysis and begin to develop again after the ingestion of a blood meal (Wigglesworth, 1956). Whether this sort of cycle in the abdominal muscles will be found to be widespread in other Exopterygotes can at present only be guessed at. However, among the Orthoptera there are several records of thoracic muscles which persist throughout the larval stages and disappear only after the adult moult. Thus van Schreven (1938) showed that a pair of thoracic dorsomedian muscles in GryZZus domesticus begins to break down within 2 days of the imaginal moult. Thomas (1955) showed that in Locusta the tergo-pleural muscles and a pair running nearly vertically up ,the pleuron are present in all instars and in young adults but are absent or only represented by traces in older adults. She thought that from their position the contraction of these muscles could cause a slight outward bulging in the region of the tergo-pleural junction and this might “assist in rupturing the skin along the middorsal line and perhaps also in the emergence of the individual from its old skin”. Ewer (1954) found that in the pterothorax of the last instar nymphs of Acanihacris, Phymateus and Locusfa certain muscles occur which are not found in fully mature adults. At 25°C histolysis of most of these muscles was completed by 168 h after the last moult. Ewer suggests that although these muscles may assist in ecdysis or in special nymphal respiratory movements a third possibility is that they might maintain the form of the soft pterothorax which would otherwise be disturbed by contraction of the muscles moving the coxae. Whatever the true explanation in this particular case, it seems probable that muscles concerned wholly or partially with ecdysis will be found to be fairly widespread among insects.

INSECT ECDYSIS

183

At ecdysis the Endopterygota, and probably also the Exopterygota, depend at least partially on a hydrostatic or combined hydrostatic and pneumatic skeleton. Indeed the ecdysial muscles of imaginal Endopterygoa (whose larvae are themselves largely dependent on a hydrostatic skeleton) are either carried over from the larva through the pupa to the adult (Lepidoptera) or are at least of larval form (Cyclorrhapha). Shortly after the imaginal ecdysis these insects convert (by hardening of the cuticle) to an exoskeleton and this conversion requires that the ecdysial muscles, which act by buckling the cuticle, should be put out of action. In the blowfly muscular efforts cease and digging movements can no longer be elicited at about the point in the air pumping cycle when the wings would be fully extended whether or not expansion is permitted to occur (Cottrell, 1962d). The mechanism of this “shut off” of the ecdysial muscles has not been investigated : possibly it is the first step in their degeneration which is completed during the first few days of adult life (Laing, 1935; Finlayson, 1956; Cottrell, 19626). Because of the possibility of mechanical damping of activities such as wing movements, the high blood volume associated with ecdysis would also ’be a disadvantage in combination with an exoskeleton. In the blowfly (Cottrell, 1962d) and Drosophih (Wigglesworth, 1963) the blood volume is considerably reduced after the final ecdysis. This is certainly also true of Pieris which can be seen to eliminate large quantities of an almost clear fluid, quite different from the coloured meconium which is produced soon after emergence. VII. T H E COMPONENTS

OF T H E

SCLEROTIZING SYSTEM

The literature on insect sclerotization is voluminous, but it has been much reviewed (Hackman, 1959; Richards, 1951, 1958; Mason, 1955; Wigglesworh, 1948,1957)and it will only be necessary to deal here with immediately relevant topics. The scheme which seemed to be generally accepted at the time of the last review was as follows. At sclerotization, dihydroxyphenols diffuse outwards through the cuticle and on reaching the epicuticle become oxidized to oquinones by a phenol oxidase. The quinones then diffuse inwards and, as was first recognized by Pryor (194Oa, b), tan the cuticular proteins forming the hard epicuticle and exocuticle. The peptide effect between amines and quinones (Mason, 1955) makes it probable that the quinones react first with the terminal amino groups of the protein chains forming N-catechol proteins which, in the presence of

184

C.

D. C O T T R E L L

excess quirlone, are then converted to N-quinonoid proteins. The N quinonoid proteins then react with the terminal amino groups of other proteins and later with the e-amino groups of lysine which occur along the chains. In this way a cross-linked structure is formed in which the protein chains are joined end to end as well as at intermediate points. A. T H E T A N N I N G A G E N T

The tanning agent is derived from tyrosine which occurs free in the blood. Tyrosine is known to build up as ecdysis approaches and to fall abruptly before hardening commences (Dennell, 1947;Hackman, 1959; Ohnishi, 1954b). Fraenkel and Rudall (1947) have shown that in Sarcophugu the gain in weight of the larval cuticle at puparium formation can readily be accounted for by the loss of free tyrosine from the blood. Karlson (1960) found that when uniformly labelled tyrosine was injected into Calliphoru larvae, the activity of the cuticle increased remarkably at the time of visible puparium formation and quickly reached up to 80% of the total recoverabie radioactivity. During the further pupal period almost no alteration in radioactivity occurred. In ligated abdomens incorporation occurred only after injection of ecdysone. Since in parallel experiments injection of radioactive leucine did not lead to an increase in cuticle activity, it is clear that tyrosine metabolism and not cuticular protein synthesis was being observed. The exact nature of the diphenol which is finally oxidized to the tanning quinone was for long uncertain. Several diphenols (3,Cdihydroxyphenylacetic acid, 3, 4-dihydroxyphenyllactic acid and 3, 4-dihydroxybenzoic acid) had been isolated from insects (Hackman et a!., 1948) and because these substances can be derived by a known or probable sequence of reactions from 3, 4-dihydroxyphenylalanine(dopa) which is itself produced by the action of Tenebrio phenolase on tyrosine (Raper, 1926), it was thought that one or more of these substances or substances like them must be involved. However, it now seems to be definitely established that in Culliphoru puparium formation the diphenol concerned is N-acetyl-3,4-dihydroxy-/3-phenylethylamine(N-acetyldopamine) (Karlson et ul., 1962) and this substance has also been detected in Tenehrio molitor and Schistocerca gregaria (Karlson and Sch1ossbe:gcr-Raecke, 1962; Karlson and Sekeris, 1962). Dennell's (1958) hypothesis that the sclerotizing quinones are produced by nonspecific hydroxylation and side chain elimination has not been confirmed (Karlson, 1960; Sekeris and Karlson, 1962).

INSECT ECDYSIS

185

B. T H E PROTEIN PRECURSOR O F SCLEROTIN

Hackman (1959) has reviewed what is known of the cuticular proteins but the characteristics of the protein which is tanned remain unknown. There have been several suggestions that the sclerotin precursor may itself be :he phenoloxidase responsible for producing the tanning quinones. Karlson and Liebau (1961) point out that such a mechanism would explain the rapid fall in enzyme activity which occurs at pupation and draw a?tention to the aggregation of soluble enzyme which is brought about by o-quinones. C. ENZYMES C O N C E R N E D I N SCLEROTIZATION

The enzymes most generally mentioned in connection with the sclerotization process are phenol oxidases but others, such as dopadecarboxylase (Sekeris and Karlson, 1962) and acetyl-Co A-transacetylase (Karlson and Ammon, 1963)are also concerned. 1. Phenol oxidases

Phenol oxidases occupy a central position in sclerotization yet, despite a very considerable literature and some intensive and definitive work on particular preparations, our understanding of their overall role is far from clear. In reviews of insect ecdysis the part played by these enzymes is frequently glossed over but as has already been shown by the work of Karlson and his collaborators, their characterization will undoubtedly provide the keys to many aspects of hardening and darkening which are at present obscure. Accordingly they are allotted an unusual amount of space in the present article. There are several reviews dealing with tyrosinase or tyrosine metabolism as well as with other phenolases (Nelson and Dawson, 1944; Sussman, 1949; Dawson and Tarpley, 1951; Lerner, 1953; Mason, 1955) but most of the fundamental work has been done with enzymes from plant or vertebrate sources and it is difficult to know how far such information can be applied to the enzymes from insects. This is particularly so when it is realized that phenolases from different sources may differ considerably in properties while even those from the same source may have their specificity greatly altered depending on the method of purification (Dawson and Tarpley, 1951). Mason (1955) has partly avoided the difficulty by speaking of the “phenolase conplex”. This he defines as “that pair of enzyme activities occurring together, associated with copper-protein and responsible for both a-hydroxylation of phenols and the dehydrogenation of o-diphenols”.

186

C. B. C O T T R E L L

However, there seems little reason to doubt that phenolases from certain sources are incapable of at lcast the first of these activities. Almost all the work on insect phenolases has been done with breis from whole inaects and little or no regard has been paid to the original location of the extracted enzymes. Phenolases have been demonstrated in both the blood and the cuticle of insects but in the present state of our knowledge it is by no means clear whether the enzyme in the blood plays any part in the normal sclerotizing process. When the blood is shed it will oxidize tyrosine first to 3, 4-dihydroxyphenylalanine(cf, Raper, 1926) and then to the corresponding quinone eventually producing melaliic pigments. By analogy, it has been suggested (Dennell, 1947) that the dihydroxyphenol concerned in the tanning reaction may also be prodaced in the blood. On the other hand, in the intact insect this step might equally occur in the epidermis or even, perhaps in the cuticle, and at present we have no decisive evidence. Further, it is by no means clear why, if the dihydroxyphenol is produced by a mechanism similar to that which occurs in shed blood, the oxidation of tyrosine should stop at the dihydroxyphenol stage. Nevertheless, in any consideration of the sclerotizing system we must bear in mind that in addition to the cuticular oxidase, other phenolases in other tissues may also be involved. In Calliphora puparium formation the specificity of the enzyme which oxidizes N-acetyldopamine to the tanning quinone (Karlson and Liebau, 1961) is such that it almost certainly cannot be involved in the hydroxylation of tyrosine to dopa (Sekeris and Karlson, 1962). The enzymes obtained from breis of whole insects or from blood often show strong monophenolase activity and are generally referred to as tyrosinases (Raper, 1926; Bodine and co-workers, 1935 et seq.; Ohnishi, 1953zt seq.; Ito, 1953; Horowitz and Fling, 1955). Howeve:, Whitehead et al. (1960) have shown that the phenolase responsible for the tanning of the oothecal proteins of Periplunetu americana has no activity towards L-tyrosine (ortho or para) or towards other monophenols. It promotes the oxidation of p-phenylanediamine and of several 0- and pdihydroxyphenols but not DL-3:4dihydroxyphenylalinine or DL-3, 4dihydroxyphenylethylamine which were the only two tested which contained amino groups in the side chain. In the sense of Keilin and Mann (1938) and of Dawson and Tarpley (1951) the enzyme shows the properties of a laccase and not of a tyrosinase. The in vifro tanning experiments of Fraenkel and Rudall(l947) suggested that the cuticular phenolase of the blowfly larva might show similar properties. Karlson and Liebau ( I 961) have now demonstrated, using a purified pr2paration obtained in crystalline form, that it is in fact a true

INSECT ECDYSIS

187

odiphenol oxidase which does not attack monophenolswith measwable velocity. Unlike the enzyme of Whitehead et al. it does not attack odiphenols with acidic side chains like protocatechuic acid, 3,4dihydroxyphenylaceticacid and 3,4-dihydroxyphenylpyruvic acid. The best substrates are N-acetyldopamine, dopamine and dopa. In both these cases, enzymes which can reasonably be considered as homogeneous and which are both definitely concerned with sclerotization have been shown to differ significantly from the more widely studied “tyrosinase ” preparations. Similarly Ohnishi (1954a) has shown that a phenofase from the cuticle of white prepupae of Drosophila differs from the blood phenolase of the same insect in being able to oxidize dimethylp-phenylenediamine and also in being more stable to heat treatment. Ito (1953) noted that isolated larval and pupal cuticles of Bombyx mori were capable of oxidizing p-phenylenediamine. He obtained an active phenol onidase in the supernatant liquid from homogenates of larval integuments which, besides being active towards diphenols, showed weak monophenolase activity. However, it is not clear that any steps were taken to remove endogenous substrate so that the results may not represent &hetrue enzyme specificity. Bearing in mind that in extracts of whole insects any peculiar properties of the cuticular (i.e. sclerotizing) phenoloxidase may be masked by the presence of the better known tyrosinase, we may now pass on to consider some of the properties of such extracts. Insect tyrosinases, like those from other sources, are strictly aerobic and will only catalyse the transfer of hydrogen to molecular oxygen. However, the 3,4-quinone products of their reaction with o-dihydroxyphenolswill oxidize ascorbic acid to dehydroascorbic acid (Allen and Bodine, 1940) becoming reconverted into dihydroxyphenols in the process. This cycle will repeat itself un51 all the ascorbic acid has been used up, and until this point is reached the formation of the red indolequinone intermediate product of melanin will be prevented. Similar reactions probably occur with other substances such as amino acids (Keilin and Mann, 1938). Like plant tyrosinases, some at least of the insect phenolases are soluble and are not bound to particles as is the enzyme from vertebrate melanomas (Lerner, 1953). However, possibly as a result of interaction with their reaction products (Karlson and Liebau, 1961), they show a strong tendency to aggregate after activation (Allen et al., 1943; Horowitz and Fling, 1955; Karlson and Liebau, 1961) and may then appear to be particulate because they can be sedimented by centrifugation (Karlson and Sckmid, 1955; cf. Whitehead et al., 1960). A peculiar characteristic of plant tyrosinases is that they are inever-

I88

C. B . C O T T R E L L

sibly inactivatcd while catalysing the oxidation of phenols (Dawson and Tarplcy, 1951). The rate of destruction is higher for some substrates than for others, phenol, catechol and methyl catechol having a strong effect but tyrosine and tyramine hardly any. There appears to have been no clear-cut demonstration of a similar effect for insect tyrosinase and while some of Pryor’s (1955) results are suggestive, Karlson and Schmid (1955) note that their preparation displayed much greater stability towards substrates than did plant tyrosinases. Ray and Bodine (1939) described a different type of artificial inactivation for tyrosinase from Melanoplus eggs. This was traced to the use of tyramine hydrochloride as substrate. Plant tyrosinases are capable of oxidizing a wide variety of tyrosine derivatives including proteins (Sizer, 1953 ;Mason, 1955). In the absence of acconipanying free phenols their action is slow but rather specific. Thus the pressor, oxytocic and melanophore hormones may be completely inactivated apparently by destruction of some 20% of their tyrosyl residues while insulin, trypsin and chymotrypsin are unaffected. Enzymes from other sources do not seem to be able to attack proteins in this way, but when simple phenolic compounds are present, all preparations can produce relatively rapid and unspecific inactivation. In some cases at least, it appears to be the quinones which are responsible and their action on the proteins seems to be equivalent to sclerotization. The blood and tissues of many insects contain both tyrosinase and tyrosine (or some other suitable substrate) and when the blood is shed or the tissues are damaged the two react together to form melanic pigments. However, it is a matter of common observation that this reaction does not occur in the intact animal and in seeking to explain this, a number of hypotheses havc been advanced. These are of interest because they have also been invoked to explain why the tanning of the puparium does not occur before the ontogenetically appropriate time (Dennell, 1947, 1949). It is to be noted that in this context the idea depends on the assumption that the blood tyrosinase does in fact play a part in the sclerotization process. an assumption which is as yet far from proi.en. The hypotheses which have been advanced may be divided into two classes, those which postulate the presence of some sort of inhibition and those wrlich postulate that the tyrosinase system requires some sort of activation to bring it into action. The type of inhibition postulated varies from direct chemical inhibition to spatial separation of one or more of the components. Recent definitive work on an insect phenolase

INSECT ECDYSIS

189

(Schweiger and Karlson, 1962a) does not support the concept of inhibition but the idea still occurs in the literature and indeed an inhibitory mechanitm was tentatively suggested by Fraenkel and Hsiao as recently as 1962. Although certain of the early hypotheses have now been disproved, it seems too early to discard in toto the idea of some type of inhibition perhaps due to spatial separation. This seems particularly to apply to the blood tyrosinase. Graubard (1933) was the first to put forward a theory involving an inhibitor of tyrosinase. He found that the activity of extracts prepared from living larvae and pupae of Drosophila was variable. Active extracts resulted when the insects were crushed in water but much of the activity was lost if they were ground with sand. From this he concluded that there must be some inhibitor of tyrosinase present in the tissues and that this is set free by grinding. Incubating the material for 6 h in chloroform vapour seemed to destroy the inhibitor so that grinding with sand now yielded active extracts. Fraenkel and Rudall (1947) with the use of methyl alcohol were able to simulate to a limited extent the pigmentation and weight changes which occur in normal puparium formation. The mechanism involved in the release of this pigmentation process was not clear t u t they suggested that methyl alcohol might inhibit the action of enzymes which normally prevent the oxidation of phenols. Dennell (1947) suggested on the basis of its destruction by chloroform that the inhibitor might be a dehydrogenase and later (1949) he affirmed this. He claimed “that tyrosinase activity in the blood of the blow-fly larva is held in check by the low-oxidation reduction potential produced by the activity of a dehydrogenase” and that “the liberation of the pupation hormone coincides with the complete and abrupt termination of dehydrogenase activity which leads to tyrosinase activity and the formation of the puparium”. Pryor (1955) reviewed the literature concerning inhibitors of tyrosinase and re-evaluated the techniques and results of previous workers. He suggested that the lack of tyrosinase activity in Drosophila ground with sand is due to the destruction of the enzyme as it oxidizes naturally occurring aromatic substrates. He provided evidence indicating that tyrosinase is in fact destroyed by its reaction with a naturally occurring substrate which is present in the “skins” of CaNiphora larvae and which might therefore be expected to be set free by grinding. Further, he found that provided they are tested soon after grinding, Drosophila and Calliphora larvae yield more active extracts when ground than when they are simply crushed, and that the tyrosinase activity of such extracts could not be increased by previous treatment with chloroform or

190

C. B. COTTRELL

methanol. He also points out that the interpretation of classical tests for dehjdrogenases is complicated by the presence of the tyrosinase system. The compound formed between o-quinones and amino acids is capable of oxidizing ascorbic acid or excess amino acid without the aid of enzyme and of simultaneously reducing methylene blue. This reaction, rather thar the activity of dehydrogenases, is probably responsible for most of the ability of damaged insect tissue to reduce methylene blue. Previous observations on fluctuations in the oxidation-reduction potential of the blood of Calliphoru larvae with age are considered to be due to changes in the rate at which oxygen is consumed by the blood after it is she+. Pryor concludes that there appears to be no valid evidence that tyrosinas2 is inhibited in vivo by the action of dehydrogenases and that “the absence of tyrosinase activity in undamaged tissues is probably due to the structure of the cytoplasm which keeps enzyme and substrate apart”. Ohnishi (1954~)has also criticized Dennell’s hypothesis. He considers that thc inactivity of tyrosinase in vivo is better explained by means of the proenzyme-activator principle and interprets Graubard’s chloroform effect as being due to the release of an activator rather than the destruction of an inhibitor. Karlson and Wecker (1955) found that the cyanide insensitive respiration (which is equivalent to Dennell’s dehydrogenase activity) was the least variable component of respiration during the course of puparium formation in Calliphoru erythrocephub. Measured absolutely, it drops to half in the brown puparium but measured as a percentage of the total respiration (minus tyrosinase activity) it rises to 78 %. Thus the metabolic processes which represent the cyanide insensitive respiration have a greater role in the puparium and pupal stage than in the larva. This is clearly at variance with the views of Dennell. The foregoing discussion indicates that none of the hypotheses involving chemical inhibition has been confirmed. There remain those which postulate inactivity due to spatial separation. Dennell (1947) demonstrated tyrosinase activity in a class of the haemocytes of larval Sarcophagu fulculutu and Jones (1958) found similar activity in the “sphaerule cells” of S. bullatu. Rizki (1957) recognized a class of haemocytes in D. melunogaster which he named crystal cells. They were characterized by the presence of crystal-like rodlets in the cytoplasm and Rizki and Rizki (1959) showed that in shed blood, blood treated with methanol, hot ethanol or hot water they underwent a characteristic darkening, the melanization being heavily localized in tne crystals though it also occurred in the cytoplasm. In

INSECT ECDYSIS

191

preparations incubated with dopa, blackening was confined to the cytoplasm and in auto-radiographs of crystal cells from larvae fed on a-C”-tyrosine the reaction foci were almost exclusively located in the crystals. In preparations not treated with methanol, the cells quickly ruptured, the crystals disappearing into the haemolymph and leaving only an empty vesicle or ghost containing the remains of the nucleus. These observations are consistent with the idea that the substrate is localized in the crystals and the enzyme in the cytoplasm and to this extent they support the idea that the inactivity of the tyrosinase system in vivo is duc to separation of its components. The release of enzymatic oxidation by lipid solvents might suggest that the disruption of a cytoplasmic membrane containing lipid components is involved. A peculiar feature of many fresh insect extracts is that they appear to be devoid of activity towards both mono- and diphenols and only become active after standing. This “activation” process can apparently occur in the absence of any phenolic substrate but when a substrate is present it may give the appearance of a lag or induction period not unlike that seen when certain mammalian or plant tyrosinases act on monophenols (see Lerner, 1953 ; Dawson and Tarpley, 1951). However, the latter phenomenon seems to have an entirely different basis because it is skown only towards monophenols and can be eliminated by the addition of small amounts of diphenol. According to Lerner (1953) in mammalian tyrosinase the lag period is due to the fact that the enzyme must pe “activated” by oxidizing a diphenol (L-dopa) before it can act on a monophenol (L-tyrosine). A well documented account of the activation of an insect tyrosinase has been provided by Bodine and his co-workers. Bodine et al. (1937) found that in the eggs of Melanoplus diferentialis tyrosinase is present in an inactive form which they termed “protyrosinase”. Stable extracts of protyrosinase could be prepared by grinding eggs in 0.9% NaCI, centrifuging at 544 g for 5-10 min and removing the upper lipoidal layer. Further purification could then be achieved by fractionation with ammonium sulphatc, redissolving in 0.9 % NaCl and dialysing against the same solution. The resulting extract had no activity towards tyrosine, tyramine or similar substrates but it could be activated by mixing with the lipoidal layer (termed the “natural activator”), a process which would normally occur if the lipoid was not removed by centrifugation (cf. Bodine and Boell, 1935). The protyrosinase could also be transformed to active tyrosinase by treatment of the saline solution with a variety of compounds such as acetone, chloroform, ether, urea, ethyl urethane, sodium taurocholate, thymol, and sodium oleate

192

C. B. COTTRELL

(Bodine and Allen, 1938a). Treating the protyrosinase with the anionic detergents aerosol OT (dioctyl sodium sulphosuccinate) and dupanol (sodium dodecyl sulphate) led to activation but treatment with cationic detergents (alkylamine hydrochlorides) was ineffective. This difference between the effects of oppositely charged ions also seemed to apply for the hydrogen and hydroxyl ions. Activation did not occur in the acid pH range but it occurred at pH's above 9.30 (Allen et al., 1942). Heating for 10 min at temperatures between 60 and 70°C brought about activation (Bodhe et al., 1944) as did the addition in certain definite concentrations of salts of mercury, gold, platinum and palladium (Bodine and Tahmisian, 1943). Shaking of the saline solution of protyrosinase caused it to change irreversiblyinto definiteproportions of protyrosinasp, tyrosinase and inactive products (Allen et al., 1943). Dialysing agaj.ist distilled water brought about activation in 8 h, the active enzymc becoming insolbble (in the sense that it could now be brought d r m by low speed centrifugation) as activation proceeded. The actiie enzyme produced by grinding whole eggs in distilled water is alsc thrown down by centrifugation (Ray and Bodine, 1939). Activatior. produced by dialysis is reversible in that it can be destroyed by thP addition of electrolytes or non-electrolytes to a final concentration of 0.1 M. Finally, Bodine and Hill (1945) have also noted that protyrosinase can be activated by adsorption onto filter paper. As Bodine and Allen (1938b) point out the activating agents are all known to act on proteins, denaturing them. Activation occurs only within specific concentration ranges and although with some substances (i.e. the "natural activator", sodium oleate, aerosol OT and dupanol) larger amounts have no effect, others (such as urea and ethyl urethane) are toxic to the active enzyme when present in greater than activating concentrations. With many others (heavy metal salts, acetone) activation is paralleled by destruction. At temperatures greater than 80°C complete destruction occurs in a 10 min exposure. On the basis of their experiments with detergents and with pH, Allen et al. (1942) conclude that it is probable that a positive charge is borne by the part of the protyrosinase which is primarily affected in the process of activation. The method of activation of the protyrosinase can to some extent cause variation in the ratio of catecholase to cresolase activity. Detergents and heat tend to produce both mono- and diphenol activity whereas urea and mercuric chloride tend to produce high cresolase activity (Bodine, 1945a). Enzymes produced by different activating agents also differ in their resistance to temperature (Bodine et al., 1944) and in their susceptibility to mercuric chloride (Bodine, 1945b). Bodine and Hill (1945)

INSECT ECDYSIS

193

have put forward evidence which suggests that dupanol acts on protyrosinase by causing a fragmentation of the enzyme protein complex. Bodine et al. (1944) considered that their results suggested that the conversion at activation of protyrosinase to tyrosinase is associated with molecular rearrangements or conversions and that the modified protein molecules possess physical properties related to the type of damage produced by the activating agent. Melanoplus egg protyrosinase is rather stable to heat and little, if any, activation occurs with 5-10 min exposures at temperatures below W C , and it is more sensitive to pH changes than is tyrosinase (Allen et al., 1942). Allen and Bodine (1941) showed that removal of copper results in an inactive material which can no longer be converted into tyrosinase by the action of detergents. Treatment with copper sulphate solution restores the pqotyrosinase. Little is krlown of the “natural activator”. It appears within the first 3 days of embryonic life (when there is no protyrosinase present) and at different developmental stages its potency bears no relation to the volume of the lipoidal material with which it is associated (Bodine et al., 1939). Apparently it is fairly stable to heat (Bodine and Carlson, 1940). Bodine and M e n (1941) point out that all the classical methods of preparing tyrosinase involve the use of chloroform, acetone or of other procedures which might allow activation to occur. Using the methods which had been developed for Melanoplus eggs, they found that mushrooms and potatoes furnished only active tyrosinases but that like Melanoplus eggs, Tenebrio larvae yielded an inactive proenzyme. The mealworm protyrosinase differed from that of the grasshopper eggs (i) in the form of its activator (sodium oleate) concentration function; (ii) in that it could not be activated by heat treatment due to destruction; (iii) in that dialysis against water neither decreased its “solubility” nor resulted in activation, and (iv) in its solubility in ammdnium sulphate. Nevertheless the two preparations resembled one another in that their potential ability to oxidize phenolic compounds was associated with a particular form of copper and that both could be activated by egg or mealworm oils, sodium oleate, chloroform, acetone, urethane or urea. Ohnishi (1953), in studying tyrosinase activity at puparium formation in P. melanogaster and later in D. virilis (1954a), found that tyrosinase -occurred as an inert proenzyme which could readily be activated by incubating it with tissue brei. Most of the potential tyrosinase activity was found to be located in the body fluid and when dissections to release body fluid were made in water, much higher activity was obtained

194

C.

B. C O T T R E L L

than whcn they were made in saline. Ohnishi interpreted this as indicating that the distilled water caused cytolysis and partial release of an activator contained in the tissues. In a later paper (19%) he showed that the activation process was inhibited by the presence of sodium chloride in concentrations higher than about 0.25 M and that potassium chloride, sodium nitrate and calcium chloride were also effective. This inhibition was not due to destruction of the proenzyme and similar concentrations had no effect on the activated enzyme. He also found that activation occurred most effectively at about neutrality and that it is inhibited at pH’s greater than 9.0. It was suppressed by 2 M urea and could not be inhibited by monoiodoacetate or heavy metals. Horowitz and Fling (1955) worked with adult (6 h old) Drosophilu melunognster. Fresh extracts (prepared by grinding in ice-cold 0.1 M phosphate buffer at pH 6, centrifuging for 5 min at 20 OOO g and using the supernatant consisting of a clear aqueous layer and a thin lipid layer) were found to be devoid of activity. However, they became active towards both tyrosine and dopa on standing at 0°C: at higher temperatures, lower final yields were obtained. Activation, unlike tyrosinase activity, can occur in the absence of oxygen and tests with copper binding agents, competitivt inhibitors and metabolic inhibitors showed that inhibition of activation does not parallel inhibition of enzyme activity. After activation the enzyme tends to aggregate and 70-80’;/, of the activity can be centrifuged down at 40 OOO g for 10 min. The activator is not tyrosinase itself since it can be separated from the latter. It appears to increase in concentration as activation proceeds, is non-dialysable and is precipitated by 35% ammonium sulphate. On the basis of kinetic findings the authors propose that activation occurs by a modified autocatalytic reaction in which tyrosinase is a product of the reaction and has no role in the process : precursor i- activator

-

tyrosinase + 2 activator

They were not able to obtain stable preparations of the tyrosinase precursor by centrifugation nor were they able to detect any activating effect of sodium oleate, sodium lauryl sulphate or urea and they therefore conclude that the Drosophilu system differs at least superficially from the grasshopper one. Ohnishi (1958) obtained results similar to those of Horswitz and Fling with pupae of Musca vicina. He found that the activating factor was thermolabile (being destroyed by exposure to 65°C for 7 min), nondialysable and precipitable by 60 ”/, ammonium sulphate. It would pass down a column of Hyflo Super-Cel on which most of the proenzyme

INSECT ECDYSIS

195

was absorbed. In a later paper (1959) using the same insect he confirmed the observation of Horowitz and Fling (1955) that the final yieid of tyrosinase is linearly reduced as activation is allowed to take place at increasing temperatures between 0 and 40°C, and showed that this effect was not due to inactivation of the proenzyme or the enzyme. In support of Horowitz and Fling's hypothesis he found a close relation between the production of activator and that of tyrosinase. Finally, Karlson and Liebau (1961). working with Culliphoru larvae, have obtained in crystalline form a pure homogeneous phenoloxidase having a molecular weight of 530000. This enzyme, which is an odiphenoloxidase is concerned in the production of the sclerotiziig quinone and is present as an inactive proenzyme which is converted to the active form by an activator enzyme (Karlson and Schweiger, 1961). The fact that the prophenoloxidase occurs without significant concentrations of activator enzyme in larvae which have had their ring glands removed (Karlson and Schweiger, 1961) enabled its preparation free from activator and low molecular weight substrates (Schweiger and Karlson, 1962a) and hence the development of a test for the activator enzyme. This latter could be separated from low molecular weight substrates as well as from the proenzyme and active phenoloxidase and was partially purified by fractionation with ammonium sulphate. In raw honogenates activation followed an S-shaped course similar to that seen in Drosophilu by Horowitz and Fling, but the curve obtained from the interaction of purified prophenoloxidase and partially purified activator enzyme suggested only the normal action of an enzyme on its substrate without any hint of autocatalysis (Schweiger and Karlson, 1962a). Activation of the prophenoloxidase is assumed to involve a limited proteolysis since various protease preparations were found to have the same effect. Curiously, the kinetics of activation by a-chymotrypsin were the same as for whole homogenates. The activator itself has the properties of a protein, is non-dialysable and is inactivated by heat (Schwciger, unpublished quoted in Schweiger and Karlson, 1962a). Schweiger and Karlson suggest that the S-shaped curve might be due to the presence of contaminating proteins in the raw prophenoloxidase preparations but they do not rule out the possibility that the activator enzyme may itself exist in the form of an inactive precursor. As Horowitz and Fling have noted, there seem to be considerable differencesbetween the phenolase system of Melunoplus eggs and that of larvae of the higher Diptera and this applies both to the specificity of the final enzyme and to its known activator. To what extent this is due to the presence of two or more different systems is difficult to assess. It seems

196

C.

B. C O T T R E L L

probable that (apart from the time of ecdysis) phenolase activity is developed only in damaged tissues. The fact that breis are inactive when first prepared indicates that the absence of melanin formation in intact tissues is not due simply to the prevention of the final oxidation and polymerization of the aromatic substrate but to the inactivity of the enzyme itself. It is just possible that the very process of extraction might bring about inactivation of an active enzyme but this is made improbable by certain observations of Bodine and Allen (I 941) on intact grasshopper eggs. In 1938a these authors showed that heating saline extracts of protyrosinase for 5 min periods at temperatures between 60°C and 85°C leads to the production of an active tyrosinase. With increasing temperature, protyrosinase is activated while tyrosinase is destroyed. Consequently the tyrosinase activity of treated extracts increases from 60 to 75°C but declines from 75 to 90°C. In their 1941 paper they showed that the relative vdues for the velocity of oxygen uptake of batches of 100 intact post diapause eggs exposed for 5 min to temperatures from 25 to 90°C showed a similar differential effect of heat. Since protyrosinase and a naturally occurring substrate can be extracted from such eggs (Bodine et al., 1937) it seems highly probable that the increase in velocity of oxygen uptake of the intact egg must be due to heat-induced oxidation of the native substrate. It was also noticed that eggs which had been exposed 6 h previously to temperatures between 60 and 80°C changed from pale lemon yellow to a dark olive green, this change being apparently due to the presence of a brown pigment in the space between the serosa and cuticle. Eggs heated at temperatures below 62°C did not show the effect and the R.Q. of eggs heated at 75°C (where optimum activation of tyrosinase occurs) was 0.1 or 0.2 agreeing with the idea that oxidation of phenolic sxbstances was taking place. These observations seem to make it very clear that in the intact egg tyrosinase is inactive but can be activated by processes identical to those which bring about its activation in tissue breis. Again, Sussman (1952) has found that the injection df small quantities of aromatic substances which would be oxidized by tyrosinase, or inhib4tors of tyrosinase, has very little effect on the oxygen consumption of intact pupae of Platysamia cecropia although both kinds of substances have their expected effect when added to breis. At ecdysis one or more phenolases are clearly active in the absence of tissue damage. Ohnishi has found that in Drosophila melanogaster (1953) and D. virilis (I 954a) potential phenolase activity increases significantly at the time of puparium formation, and Karlson and Schweiger (1961) by comparing mature crop-full larvae, crop-empty larvae and white puparia showed a similar increase in CaZlijdtoraeryihrocephala.The experiments of Karlson

INSECT ECDYSIS

197

(1960) and of Karlson and Ammon (1963) leave no doubt that at puparium formation tyrosine metabolites are oxidized and incorporated into the cuticle. According to Karlson and Ammon (1963) the oxidation of N-acetyldopamine must occur in the cuticle because the oxidized radioactive N-acetyldopamine is located in the outer layers of the cuticle and oxidation is prevented by ultraviolet irradiation. Because of its substrate specificity,there can be little doubt that the enzyme involved is in fact that obtained by Karlson and Liebau (1961) in crystalline form and hence that it is located in the cuticle. This enzyme is not a tyrosinase but a diphenolase and the whole question of whether the tyrosinase systems of the blood and tissues are involved in normal sclerotization remains unsettled. This is not the place to consider the possible functions of an inactive tyrosinase in the economy of the insect. It is sufficient to point out that there must t e a considerable selective advantage attached to the possession of a system which in the event of damage is capable of producing metabolites (quinones) which have a strong antibiotic effect, are capable of rapidly and effectively inactivating biologically active proteins and other substances and can assist in the formation of tough insoluble sheaths around foreign bodies (such as parasites) or over wounds (Jones, 1958).The fact that these or similar metabolites are also used in thenatural tanning of the cuticle does not necessarily mean that in this instance they are produced by an identical system. However, it is possible to imagine that if dihydroxyphenols and quinones were produced as a consequence of a damage reaction at a time when the cuticle was ready to undergo hardening and darkening they might also become involved in the tanning of cuticular proteins. Tanning produced in this way might be perfectly normal in its appearance and chemistry but its initiation could scarcely be described as natural. As regards the inactivity in vivo of the tyrosinase system the hypotheses involving separation of the components and the need for activation of an inactive precursor need not necessarily be regarded as contradictory. They seem rather to represent different aspects ofthe same phenomenon : indeed each can be expressed in terms of the other. Thus, as Ohnishi has suggested, the activator or activators, which are released by damage, may be considered to be separated from the proenzyme by some structural organization. On the other hand, if the proenzyme is inactive because its active centre is masked this in itself amounts to the effective separation of enzyme and substrate. AU the information on the protyrosinase of Melanoplus eggs suggests that activation in this case is associated with changesin the carrier protein

198

C.

B. C O T T R E L L

(or proenzyme) which resemble those occurring at denaturation, and the observations of Bodine and Hill (1945) on the action of sodium dodecyl sulphate suggest that fragmentation of the carrier protein may also play a part. This idea gains further support from Schweiger and Karlson’s (1962a) evidence that activation of Culliphoru phenol oxidase involves limited proteolysis. Their data from purified preparations do not confirm the autocatalytic mechanism proposed by Horowitz and Fling for Drosophilu “tyrosinase”, but it is of some interest that Bodine and Hill (1945) noted that a filtrate derived from protyrosinase activated by sodium dcdecyl sulphate and thought to contain the fragments of the original carrier protein had a marked activating effect on further protyrosinase suggesting an autocatalytic action. Fragmentation and partial denaturation of proteins are both processes which might reasonably be expected to occur in autolysing or damaged tissues. In some respects the activation of insect phenoloxidases is reminiscent of activation of certain extracellular proteolytic enzymes in vertebrates. Thus pepsin, rennin, trypsin, chymotrypsin and carboxypeptidase are secreted as pepsinogen, prorennin, trypsinogen, chymotrypsinogen and procarboxypeptidase respectively. Prorennin is activated by hydrogen ions, pepsinogen by hydrogen ions and pepsin itself, chymotrypsinogen and procarboxypeptidase by trypsin and trypsinogen by trypsin or by enterokinkse. Where information is available (see Dixon and Webb, 1958) the process appears to consist of the breaking of peptide linkages, with or without the removal of free peptides. Perhaps more relevant to the present subject is the fact that certain of the factors associated with the formation of the mammalian blood clot also occur physiologically as inactive precursors. Unfortunately little is known of the process of activation in these cases. This discussion of insect phenolase systems has had of necessity to be long because, in the present state of our knowledge, it is impossible to know how much of the available information is relevant to sclerotization. However, if it has done nothing else, it will have served to emphasize that many of the present ideas about sclerotization are based on extraordinarily slight evidence. In particular the following points seem currently to be under-emphasized :

I . that the systems concerned are of very considerable complexity; 2. that the cuticular phenolase is not a tyrosinase in the true sense; 3. that there is at least as much information against the assumption that blood and tissue tyrosinase is involved in normal sclerotization as there is for it;

INSECT ECDYSIS

199

4. that whether or not the blood tyrosinase is concerned in normal

sclerotization, it can, in response to damage (whether caused chcmically or mechanically), produce metabolites (such as dopa) sin;ilar to those which occur as intermediates in the normal tanning of rhe cuticle. Thus there is the possibility that if such damage occurs at a time when the cuticle is ready to undergo sclerotization, the normal control processes may be short circuited and cuticular tanning may occur without being initiated by the normal physiological means.

2. Other enzymes In the late third instar larva of Calliphora eryfhrocephah,preparatory to puparium formation, Sekeris and Karlson (1962) believe that tyrosine is hydrorylated to dopa by particle-bound system which could be either a phenol oxidase with cresolase and catecholase activity or a pure hydroxylating system. Dopa is then decarboxylatedto dopamine and the latter acetylated to give N-acetyldopamine, the diphenolic precursor of the tanning agent. This step is brought about by an acetyl-&A-transacetylase (Karlson and Ammon, 1963) which is capable of acetylating tyramine, dopamine, histamine and serotonin but not amino acids (including tyrosine and dopa) or glucosamine. At the time of writing, a paper by Sekeris (1963) on the decarboxylating system has not yet appeared. In the early third instar larva the path of tyrosine metabolism is different. Here it is mainly catabolized by transamination to p-hydroxyphenylpJfruvicacid and thence to p-hydroxyphenylpropionic acid or it is decarboxylated to tyramine (Karlson, 1962; Karlson and Sekeris, 1962; Sekeris and Karlson, 1962). It seems probable that the diphenolic substances discussed by Hackman ef al. (1948) are derived from this catabolic pathway and are not in fact concerned in normal sclerotization. VIII. T H E RELATIONSHIP BETWEEN HARDE NI NG AND DARKENING In the present paper the terms hardening and darkening have been used as if they referred to different aspects of the same process. This attitude requires some amplification since the relationship between hardness and darkness is still very imperfectly understood and has been the subject of much inconclusive debate. The cuticle of a newly moulted insect is at first pale and soft; later it becomes harder and darker. Similar changes accompany the quinone

200

C. B. COTTRELL

tanning of proteins and there can be little doubt that the process of tanning or sc1e;otization plays a very important part in the formation of the mature cuticle. What is much less clear is the extent of the contributions made by other processes. Fraenkel and Rudall (1940) in their study of puparium formation in Calliphora erythrocephala noted several physical changes in addition to increasing insolubility of the larval cuticular proteins which is now known to be cauced by quinone tanning. In the larval cuticle the chitin crystallites can be rotated in any direction by the application of forces of compression or extension, but this freedom of rotation is lost in the puparium presumably as a result of the stabilization of the protein components in consequence of tanning. Again, the chitin crystallites in the larval cuticle are randomly orientated in planes parallel to the surface but at the time of the puparial contraction they become aligned approximately parallel to the transverse dxis of the larva. Finally, the water content decreases from about 70 % in the larval cuticle to about 40% in the 36-h-old puparium. From these facts Fraenkel and Rudall concluded that the hardness of the mature puparium depends at least in part on (i) the higher degree of chitin orientation, and (ii) the closer packing, by dehydration, of the materials of the cuticle. Kroon et al. (1952) after studying the X-ray patterns of wings of butterflies before and after extension have expressed the rather extreme opinion that the change in the packing of the micelles observed by them provides an explanation for the permanent hardness and stiffness of the wing. They consider that the orientation of the chitin micelles is not merely an accompanying phenomenon but (as with silk) is an essential factor in the hardening process. Despite this opinion there is no doubt at all that a very considerable degree of hardening can occur in the absence of any forces of extension or of compression. Thus Fraenkel and Rudall (1940) state that in larvae ligated after the critical period, the muscles of the posterior parts are paralysed so that there is no puparial contraction. Presumably in such uncontracted cuticle there would be no orientation of the chitin crystallites but the authors note that the hardening and darkening developed is apparently normal though it may not achieve the same degree of hardness as that found in the contracted cuticles. This is in agreement with the observation that hardening and darkening take place in an apparently normal manner in adult blowflies with blocked probosces. Such flies cannot swallow air and therefore do not expand (Fraenkel, 1935b; Cottrell, 1962a). Here, although hardening is considerable, it is dficult to be sure that it is as great as that achieved in expanded cuticle. In any

INSECT ECDYSIS

201

case the complex folding of the unexpanded integument would appear to preclude any but the most general comparison of mechanical properties. The opinion of Kroon et ul. (1952) does not seem to accord with the known facts of butterfly emergence. Ifthe stiffnessof the mature wing was largely due to the orientation of chitin micellae which OCCUTS at expansion, then one would hardly expect that there should be any considerable period bctween the apparent full extension of the wing and the develop ment of stiffness. However, in Pieris brussicue emerging at room temperature, full wing extension is generally achieved in about 10 min but the wings do not become stiffenough for the insect to walk upright until some 30 min later. Conversely, wings which are only half expanded due to some accident will harden at about the same time as fully expanded ones. Petersen et ul. (1956) have shown that in Pieris napi the hardness of the wings increases greatly during the first 3 days after emergence and thereafter may continue to increase slightly up to the 5th day. In the light of these observationsit seems reasonable to conclude that, whatever the contribution of miceliar orientation to the hardness of the fully developed cuticle, it is likely to be less than that due to sclerotization. The contribution due to the loss of water is more difficult to assess. Fraenkel and Rudall (1940) state that the decrease in water content which takes place in the first 36 h after puparium formation (i.e. while the body is still in contact with the cuticle) will occur even in a saturated atmosphere so that it cannot be due to evaporation. Such drying is difficult to separate from the effects of sclerotization since it probably depends on the changes which have taken place in the properties of the cuticular proteins. Later, purely physical drying could presumably add to the stiffness of the cuticle, and it is possible that the relatively long-term changes seen in butterfly wings might be due to this cause. Protocatechuic acid, in the absence of other chromogens, will react with protein to give brown sclerotin derivatives; indeed, sclerotin formation in general seems to be associated with the formation of brown colours. Same authors (Pryor, 1948) have thought that hardening of the insect cuticle probably always involves the formation of compounds colowed brown or black, but others (Brunet and Kent, 1955) have pointed out that such structures as the lateral pronotal extensions of BZuberus are parchment coloured and more or less transparent yet are of considerable hardness. Similar considerationsapply to the cuticle covering the eyes of insects and have led to the idea that sclerotization may be of more than one kind or that insects may possess ways of hardening the cuticle which do not involve quinone tanning. In the absence of any exact chemical knowledge concerning the sclerotization reaction it is

202

C. B. C O T T R E L L

impossible to do more than mention such suggestions. For instance we have at present no idea to what extent the colour associated with sclerotization is directly due to thc cross-links responsible for the stabilization of the pzoteins or how far it is due to the polmerization of phenols not attached to the protein chains. In this connectionit is of some interest that the introduction (by blood transfusion) of the “darkening factor” into newly emerged blowflies some 35 min earlier than it would normally have been released brought about sufficient hardening to prevent expansion (Cottrell, 1962b). At the time when the flies were trying to expand themselves their cuticles were still either quite pale or were only just beginning to show the first signs of darkening. Clearly a considerable degree of hardening can occur without the development of much colour. Nevertheless it is an undoubted fact that the mandibles which are among the hardest parts of insects (Bailey, 1954) are invariably brown or darker. It is perhaps worth mentioning that contrary to one or two statements in the literature, artificial melanins (i.e. melanins not associated with proteins) can be formed just as easily by polymerization of phenols without amino containing side chains (i.e. catechol) as they can by 3,4-dopa (Mason, 1953, 1959). In any case as Hackman and Todd (1953) have suggested, after o-quinones have reacted with the free amino groups of proteins, indole formation followed by exhaustive polymerization may occur provided there is a second disubstitutable position available on the aromatic nucleus. The question has sometimes been asked whether there is a formation of true melanin alongside sclerotization (cf. Wigglesworth, 1948). This is not an easy question to deal with because, although Hackman (1953) has shown that the absorption spectra of artificial melanins derived from tyrosine or 3,4-dopa are quite different from those obtained from protein tanned by o-quinones or from hardened protein of Aphodius elytra, these sclerotins would themselves fall under the category of natural melanins AS the term is understood by Mason (1959). It seems better to try to avoid this verbal impasse by asking whether there is any form of melanin pigmentation which does not involve cross-linking of proteins since anj such pigmentation can hardly be due to sclerotization. In trying to decide this, it is relevant to point out that the Culliphora transfusion experiments mentioned above have shown that a degree of sclerotization insufficient to produce more than a very faint colour will completely prevent expansion. In Schistocerca gregaria the dark pattern of markings although not fully developed is already distinct at the time of ecdysis. Its presence seems to have no adverse effect on expansion so that it is difficult to believe that the formation of this pigment can

203 involve the cross-linking of any of the cuticular proteins. It appears to be a form of pigmentation different from sclerotization. This view finds support in the occurrence of an albino strain of Schisfocerca which lacks the black patterriing as wcll as almost all the normal insectorubin, although tkc h:irdncss of tlic cuticlc sccins to he little affected (Katlson and Schlossbergcr-Kuecke, 1962). Mulck (1957) also conddtrcd that in Schistocerca, “ melanization ” and sclerotization are independent processes and that each appears to involve its own copper-protein phenolase. Unfortmately, his attempts to distinguish critically between the two enzymes were onty partially successful. He also noted (inagreement with the observation on expansion mentioned above) that the two processes are consecutive, sclerotization being delayed until after melanization had been completed. Other instances of dark pigmentations which do not interfere with expansion are probably to be found in the darkening of Drosophila cuticle which is seen before emergence (Waddington, 1941) and in the dull brown thoracic stripes of newly emerged Surcophagu. Mason (1959) considers that all natural melanins are in fact melanoproteins, and as the black pigment (in Schistocerca for instance) can be seen to develop in the cuticle it is of interest to speculate as to why it should not be accompanied by cross-linking of cuticular proteins. When phenols (such as 3,4dopa) having amino groups in their side chains are oxidized to quinones, the increase in reactivity of position 6 towards amino groups leads to imrrediate indob formation thus preventing the attachment of the free amino groups of cuticular proteins to the quinone nucleus. Now in the oxidation of 3,4-dopa to melanin several quinonoid intermediates occur (see Mason, 1953,and it would at first sight appear possible that the formation of dopa melanoproteins might take plcce by coupling between one or more of these quinones and the reactive groups of proteins. It is of particular interest therefore that Mason (1959) has studied the enzymatic oxidation of dopa, 5,Cdihy.in the presence of a droxyindole and 2-carboxy-5,6-dihydroxyindole variety of amino acids and peptides. There was no interaction between dopa quinone, dopachrome, indole-5,6-quinone and any of the amino groups found in proteins. Dopa quinone alone was capable of combining with certain reactive amines not found in proteins such as p-aminobenzoic acid, anthranillic acid, kynurenine and 3-hydroxytryptamine. However Mason and Petersen (quoted in Mason, 1959) have found that simple substances and proteins which contain sulphydryl groups do react readily with indole-5,6-quinone. Apparently natural dopa melanin is “a bipolymer in which polymeric pigment is bound to protein by INSECT ECDYSIS

204

C. B. C O T T W E L L

primary valence bonds involving sulphur’’ (Mason, 1959). Now it is a well known fact that in the insect cuticular proteins sulphydryl groups are absent. Hackman (1953) has suggested that this might be because they inhibit the action of polyphenol oxidases. However it seems equally possible that the absence of these, the only groups contained in proteins which are known to be capable of reacting with any quinonoid derivative of dopa, may be important because it will allow the formation of a category of melanin pigments which is not associated with crosslinking of proteins. In Calliphoru larvae the phenolic precursor at puparium formation is N-acetyldopamhe and this is produced by acetylation of dopamine derived from dopa. Karlson et ul. (1962) believe that the significance of this acetylation is that it prevents dihydroxyindole formation so that the quinone is left to react with the cuticular proteins. The quinones formed from catechol and from phenols without amino groups in their side chains are of course all capable of combining directly not only with sulphydryl groups of proteins but also with N-terminal proline as well as with N-terminal and e-lysyl amino groups. The melanoproteins (sclerotins) so formed are thus inevitably associated with cross-linking. The occurrence of melanization not associated with sclerotin formation is certainly detectable at present when it takes place before expansion. In the adult Culliphora all parts which are darkened before emergence appear also to be hard and certainly they do not subsequently expand. Darkening in blowflies has never been observed without some concurrent hardening and it seems simplest, for the present, to consider the darkening to be a by-product of sclerotization. The final pigmentation is very dark however, and it may be that concurrent with sclerotization there is some formation of dopa melanin; on the other hand the dark colour could equally be due to the polymerization of deaminated phenols.

Ix. THEC O N T R O L

OF V A R I O U S P R O C E S S E S W I T H ECDYSIS

ASSOCIATED

A. H A R D E N I N G A N D D A H K E N I N O

Fraenkel(l935a) demonstrated that puparium formation in CuIripAora erythrocephala can be induced in the posterior portions of ligated larvae by transfusing them with blood from pupating individuals. The source of the active principle in the blood was traced to Weismann’s ring by

INSECT ECDYSIS

205

Burtt (1938) and when the substance was obtained in crystalline form (Butenandt and Karlson, 1954) it was already clear that it was the prothoracic gland hormone, ecdysone. Since pure ecdysone is all that is necessary to induce puparium formation in the isolated hind parts of maggots and pupation in the iscilated abdomina of Lepidoptera (see Karlson, 1956), it is evident that in addition to its many other actions it can bring about sclerotization. This has led a number of authors to seek some direct effect on the sclerotizing system. The views of Dennell (1949) concerning the connection between the liberation of the “pupation hormone ” and the termination of supposed dehydrogenase activity have already been mentioned. Karlson and Schmid (1955) failed to show any in vitro effect of ecdysone on an activated phenoloxidase preparation. More recently, Karlson and Schweiger (1961) showed that in the mature third instar larva, at the time when the ecdysone titre is known to be rising, both phenoloxidaseand its activator enzyme increase strongly in amount, reach a maximum just before the white puparhn stage and then decrease rapidly. If the ring gland is destroyed in the late third instar, the concentration of prophenoloxidase remains unaltered; that of the activator enzyme at first remains constant, but after about 10 days, decreases sharply to almost zero. Following injection of 5-10 Calliphora units of ecdysone, the activator concentration returns to its former level in 24-36 h. Evidently synthesis of the activator enzyme is under control of ecdysone. Karlson and Sekeris (1962) have also shown that the change in tyrosine metabolism which occurs between the early and mature third instar larva is similarly controlled by ecdysone. In young larvae tyrosine is mainly transaminatmi while in older larvae conversion to dopa and decarboxylation to dopamine predominate. Again,ecdysone appears to act by promoting the synthesis of the decarboxylase. By extrapolation from the observations of Clever and Karlson (1960) on the effect of ecdysone on “puff” production in the polytene chromosomes of Chironomus tentuns, Schweiger and Karlson (1962b) suggest that ecdysone brings about these results by directly activating the genetic material which controls the synthesis, by the ribosomes, of these particular enzyme proteins. In the examples mentioned above, sclerotization would appear merely as one of the last steps in the whole series of processes which are initiated and carried through by an increasing titre of ecdysone (cf., for instance, Karlson and Hanser, 1953; BGckmann, 1959). However, it seems probable that in some instances at least the events which occur at ccdysis are independently controlled. An example of this is found in the imaginal ecdysis of blowflies. Under favourable conditions at 22”C,

206

C. B. C O T T R E L L

adult Calliphora erythrocephala begin to swallow air about 10 min after emergence, complete expansion at 30 min and cease air-swallowing 10 min later. The first signs of darkening appear about 30 min after the completion of expansion : darkening then progresses rapidly and uniformly so that within 40 min of its first appearance the cuticle of the intact insect appears fully dark while within 2 h the fly is capable of buzzing (Cottrell, 1962a). Fraenkel (1935b) showed that if flies are forced to keep digging through sawdust (or some similar medium) from the moment of their emergence, they will delay expansion, hardening and darkening for periods several times in excess of those normally required for the completion of these processes. On the contrary, if they are lightly anaesthetized with ether before being placed in the sawdust, there is no delay. Clearly the nervous system must be involved. If the flies are kept digging for long periods they weaken and the percentage which are able to expand normally after 5 h of digging is small (Cottrell, 1962a; Fraenkel and Hsaio, 1962). If digging is prolonged beyond this time, the flies may react in two ways. A few swallow air and harden and darken normally; most show an aberrant, patchy and slow-developing form of darkening which is not associated with air-swallowing and has been called “secondary darkening” (Cottrell, 1962a). By means of transfusion and ligature experiments, it has been shown that normal hardening and darkening is brought about by the release into the blood of an active factor (Cottrell, 1962a; Fraenkel and Hsaio, 1962). The factor is released into the blood some 45 min before the appearance of first signs of darkening and between 3 and 15 min after the fly has reached conditions suitable for expansion, that is, at about the time of initiation of air-pumping (Cottrell, 1962b). Some 10 h later it has disappeared from the blood (Cottrell, 1962~).Decapitation (Cottrell, 1962b, c) or ligaturing off the head at emergence (Fraenkel and Hsiao, 1962) will prevent the initiation of normal hardening and darkening, but not secondary darkening. Severance in the neck of both the stomatogastric and the central nervous systems at emergence likewise prevents the initiation of normal hardening and darkening as does removal of both the lateral and medial groups of neurosecretory cells in the brain (Fraenkel and Hsiao, 1962). These results may indicate that control is due to the release of a factor from the brain in response, in part, to nervous stimuli from the thorax (but cf. also Fraenkel and Hsiao, 1962). The factor which appears in the blood will withstand boiling for 10 min or drying at 120°C for 20 min; is non-dialysable, relatively

INSECT E C D Y S I S

207

insoluble in organic solvents and is inactivated by ethyl alcohol and the bacterial protease subtiiisin (Cottrell, 1962~).It is not active inlbringing about puparium formation in the posterior portions of ligatured maggots (Cott:ell, 1962c; Fraenkel and Hsiao, 1962) or in inducing moulting in decapitated, fed, fifth instar larvae of Rhodnius (Cottrell, 1962~). Concentrated preparations of ecd ysone do not bring about darkening in flies in which the head has been ligated at emergence (Fraenkel and Hsiao, 1962). There can be little doubt then that the darkening factor is not ccdysone, nor does it seem likely to be the prothoracotropic hormone or the juvenile hormone, both of which seem to be ether soluble (Kobayashi and Kirimura, 1958; Karlson, 1956). Its thermostability appears to preclude the possibility that it is a phenol oxidase or even a prophenoloxidase and this, together with its poor solubility in organic solvents, precludes comparison with either the prophenoloxidase activator of higher Diptera (Ohnishi, 1958; Schweiger, quoted in Schweigei and Karlson, 1962a) or the activator of Melanoplus prophenoloxidase (Bodine and Allen, 1941). It is nondialysable and does not show the properties of any of the known tanning agent precursors. Many insect cuticular proteins are known to be stable to denaturation by boiling (Hackman, 1953) so that it is possible that the darkening factor could perhaps represent the protein substrate mhich is tanned. However, Cottrell (1 962c), using starch-gel electrophoresis could detect no difference between the protein patterns of active and inactive blood. The occurrence of “secondary” darkening in the isolated abdomen in the absence of normal initiation suggests that all the components of the sclerotizing system are in fact present in or near the cuticle at the time of emergence, but that for some reason they are not able toreact together readily and that the function of the “darkening factor” is to bring about their rapid and complete reaction. It is difficult to avoid the conclusion that the general hardening and darkening which occurs in the newly emerged blowfly is controlled independently from other processes involved in the moult. Possibly this ecdysis is an exceptional case but, in addition to showing that the darkening factor is effective among several genera and species of cyclorrhaphous Diptera, both Fraenkel and Hsiao (1962) and Cottrell (1962~) have found substances with darkening factor activity in other insects, namely, Periplaneta americana, Tenebrio molitor and Schistocerca gregaria at the time of ecdysis but not after hardening and darkening had been completed. It may be that the imaginal ecdysis of blowflies, where air-swallowing and expansion are not initiated until after the

208

C. B. COTTRELL

attainment of suitable conditions, is merely a particularly favourable instance for the demonstration of this type of control. B. AIR SWALLOWING

The air swallowing which is the major factor in bringing about expansion in the newly emerged b l o d y is rather independent of external stimuli. It starts at about the time of release of the darkening factor and ceases after some 30-40 min whether or not expansion has occurred (Cottrell, 1962a; Frankel, 1935b). Transfusion of active blood from donors engaged in air swallowing into digging flies does not bring about air swallowing in the recipients (Cottrell, unpublished) so that the centre responsible for the control of air-pumping is not activated directly by the release of the darkening factor. Nevertheless, in the intact fly the two activities seem never to be initiated independently so that they must be controlled and co-ordinated through a common centre itself capable of being effected by external conditions. Fraenkel (1935b) working with flies anaesthetized with ether found that if the abdominal cuticle and gut were pricked so as to abolish the internal pressure at the moment of wing expansion, air-pumping continued fOk’ only a few minutes longer than in the controls. In normal flies the usual curve obtained when the frequency of air-swallowing is plotted against time is fairly sharply “peaked” with the maximum rate (about 160/min in Calliphora) occurring shortly after the moment of full wing extension (Cottrell, 1962a). In flies with ligated probosces the curve is “flat-topped”, the highest rates being maintained for several minutes and the whole cycle prolonged for from 10 to 15 min. Similar curves are obtained if the abdomen and gut are slit at any time between emergence and full wing extension and with flies in which the cuticle has been prehardened by transfusion with active blood. Shortly after full wing extension, slitting of the cuticle no longer prolongs the maximum rate of air swallowing (Cottrell, unpublished). There is thus no doubt that although air-pumping in the blowfly occurs only during a very limited part of its ontogeny, the fly is to some extent capable of regulating both the frequency and the duration of its pumping. The sensory mechanisms involved in this regulation remain unknown. C. MECHANICAL PROPERTIES OF THE CUTICLE

By subjecting blowflies (with the proboscis and anus blocked at emergence) to artificial internal pressures similar in magnitude to those

INSECT ECDYSIS

209

which bring about expansion, it has been shown (Cottrell, 196%) that expansion of the cuticle of presuniptive sclerite areas can only occur during a brief period while air-swallowing movements are Wing performed. Before and after air swallowing, application of identical artificial pressures merely results in the temporary distention (not expansion) of areas destined to remain unsclerotized and in the unfolding of the wing membranes. This change in the properties of the (as yet) unsclero t k d cuticle which occurs during air-pumping could perhaps be a consequencl: of the early stages of sclerotization but its mechanism is unknown. The inexpansibility of the sclerite cuticle after air swallowing is clearly due to sclerotization even though at this time no darkening may yet be visible. An analagous phenomenon has been described by Bennet-Clark (1962) during the feeding of larvae of Rhodniusprolixus. D. “SHUT OFF” OF ECDYSLAL MUSCLES

Both digging movements and the muscular efforts which occur during expansioil are produced by a special musculature which degenerates during early adult life. Muscular efforts cease after the moment of full wing extension and from this time digging movements can no longer be elicited by appropriate stimuli (Cottrell, 1962a, d). This “shut off” of the ecdysial muscles is not due to the expansion of the cuticle since it occurs in flies in which expansion is prevented; nor is it due to hardening of the cuticle or to release of the darkening factor since it is still possible to induce recognizable digging movements in flies in which the cuticle has been artificially hardened during digging by transfusion with active blood (Cottrell, 1962d). Probably, the mechanism of the “shut-off’ is nervous. E. A B S O R P T I O N O F F L U I D FROM TRACHEAE

The mechanism by which fluid is absorbed from the tracheal system shortly before ecdysis has not been adequately investigated (Keister and Buck, 1949) but Wigglesworth (1938) has shown that absorption in the newly hatched larva of Aedes may be temporarily suppressed by narcotization with chloroform. Since the tracheal cells are not innervated it seems likely that some humoral control is involved. F. O T H E R PROCESSES

There are a great many processes which occur rapidly all over the body just before and during ecdysis. Amongst these may be mentioned

2 10

C. B. C O T T R E L L

the absorption of the moulting fluid beneath the old cuticle, the secretion of wax onto the surface of the new cuticle and the production of the epicuticular cement, as well as the initiation of muscular efforts associated with splitting and shedding the old cuticle, the diminution of the blood volume after expansion and the “shut-off” of the ecdysial muscles. The co-ordination of these many processes at ecdysis suggests a fairly complex control system. Wigglesworth (1954) cites the observation of Arvy and Gabe (1953) that there is a sudden discharge of neurosecretion from the neurosecretory cells along the axons at the moment of ecdysis in Ephemeroptera and Odonata and writes “It may be that there is a series of hormonal signals given out as one phase of growth succeeds another.”

x. SOME FACTORS INVOLVED THE

I N THE PRODUCTION DEFINITIVE BODYFORMA T ECDYSIS

OF

It is not yet possible to give an integrated account of the many factors which interact to producc the final body form of a newly moulted insect. Leaving aside questions of embryological determination and of the inductive influences of different parts of the body, the present article seems to provide a basis for briefly discussing certain of the factors which are operative at ecdysis. At expansion the external shape of the insect is largely limited by the inherent form of the different parts and more particularly by the extent to which the epicuticle can be unfolded (cf. Bennet-Clark, 1963). The unfolding of the epicuticle and the stretching of the underlying endocutide which accompanies it, is brought about by hydrostatic pressures which are produced by two different activities, the swallowing of air or water and the performance of muscular efforts. The relative importance of these two activities may differ in different insects but both can be reckoned as among the factors which affect the final form of the body. Although hydrostatic pressures are responsible for the expansion of the soft parts of the body, the form of other parts is fixed before ecdysis by prehardening, and the pressures developed at ecdysis may then serve not only to expand the soft parts but also to ensure that they are held in correct alignment with respect to the prehardened areas until general hardening has proceeded sufficiently to keep them there. Hydrostatic pressures may be involved in morphogenesis in other ways. According to Kiihler (1932) the eversion of the wing buds during the prepupal quiescent period at the end of the last larval instar of Ephestia is brought about by hydrostatic pressures combined with the action of

INSECT ECDYSIS

211

muscles which draw open the mouths of the peripodial cavities. Fraenkel (1938) bas shown that in Culfiphora the eversion of the pupal head is brought about by the peristaltic contractions of the abdomen which occur at 27°C some 30 h after puparium formation. Waddington’s (1940) description of the development of Drosophila indicates that the wing buds periodically become swollen and increase in thickness while at the same time spaces appear between the basal ends of the cells. Such bulging occurs particularly during phases PP4 and P1, that is just before and during the phase when the head is everted. At this time (Pl) the wing buds are almost circular in cross section and the surface membrane is stretched very thin. In the next stage of development (P2)the wings again assume the form of a thin blade. The coincidence of this bulging of the wing bud with the eversion of the head suggests that the former may alss be due to an increase in hydrostatic pressure. Both BytinskiSalz (1936) and Kahler (1932) consider that hydrostatic pressure is important in expanding the pupal appendages and bringing them together to complete the armour of the Lepidopterafi pupa. The smooth sclerotized outer surface of an obtect pupa is a mosaic formed partly by the cuticle of the body and partly by the cuticle of the appendages (the maxillae, antennae, wings and legs), but the cuticle of parts of the body surface covered by pupal appendages, as well as that of parts of the pupal organs which are not exposed on the surface, remains soft and membranous. Since the areas which are to become hard and sclerotized are determined before the 3rd instar (Bytinski-Salz, 1936)the insect must be capable of bringing the various appendages together at pupation sufficieritly accurately to complete the definitivepupal armour. BytinskiSalz considers that this assembling of the appendages is brought about by the combined action of numerous factors which include (i) the displacement ventrally of the pupal appendages during shedding of the old cuticle; (ii) the stretching of the sheaths by pressure of haemolymph, and (iii) the reciprocal mechanical influences of the various organs as they are pressed together. If the anlage of a leg or part of a leg is removed in the larval stage the insect can often, by mechanical regulation, maintain the “closed” structure of the mature pupa so that no membranous areas are visible. Thus in spite of the limitations of the epicuticle and of whatever other factors are responsible for the inherent form of the organs, a certain amount of regulation in form is still possible. However, if the anlage removed is that of an organ which contributes a large area to the outer shell of the pupa (such as a wing or the proboscis), the insect is incapable of sufficientregulation and membranous cuticular areas are left exposed. As might be expected, an adequate volume of

212

C. B. COTTRELL

haemolymph is necessary for the production of the “closed” pupal form. Thus Bytinski-Salz has found that if a pupating larva or young pupa is injured and haemolymph is pressed out, the full expansion of the appendages is prevented and in the mature pupa areas of membranous cuticle are left exposed. Muscles play a part in creating the pressures necessary for expansion, but they may also affect the final body form in more direct ways. For example, during the expansion of a blowfly the ptilinal musculature prevents the eversion of the ptilinum and maintains the head in its definitive shape so that in spite of the internal pressure, the head does not become hardened in its expanded form. Again, according to Maloef (1935) the formation of apodemes may also be influenced by muscular contraction. Not all the changes in form which occur at the time of ecdysis are assisted by muscular action, many are solely dependent on differential growth at an earlier stage in the moulting cycle which becomes expressed only at expansion. A particularly good example of this has been provided by Sellier (1950). In gryllids (as in other Orthoptera) at a particular larval stadium, often the penultimate one, the wing buds rotate through about 260 degrees so that the ventral face comes to lie dorsally and the hindwings cover the forewings. Sellier confirmed that this movement is due to differential cell growth by means of sections and by implantation experiments. Wing buds freed from their muscle attachments and implanted into the prothorax rotated dorsally at the same time as did those of the host and at the imaginal moult returned to the normal position. This brief Consideration of the factors concerned in the production of the definitive body form at ecdysis is by no means complete but it will at least serve to show that a very considerable number of factors is involved. REFERENCES Allen, T. H. and Bodine, J. H. (1940). Enzymes in ontogenesis. XIII. Activation of protyrosinasc and the oxidation of ascorbic acid. J. gen. Physiol. 24,99-103. Allen, T. H. and Bodine, J. H. (1941). Enzymes in ontogenesis. XVII. The importance of copper for protyrosinase. Science 94,443444. Allen, T. H., Otis, A. B. and Bodine, J. H. (1942). The pH stability of protyrosinase and tyrgsinase. J . gen. Physiol. 26, 151-155. Allen, T. H., Otis, A. B. and Bodine, J. H. (1943). Changes in the properties of protyrDsinase due to shaking. Arch. Biochem. 1, 357-364. Arvy, L. ard Cabe, M, (1953). Donnks histophysiologiques sur la neuroskcrbtion chez les PalCoptkres (EphCm6ropdreset Odonates). 2.Zellfosch. 38, 591-610. Bailey, S. W. (1954). Hardness of arthropod mouthparts. Nature, Lmd, 173, 503.

INSECT ECDYSIS

213

Bennct-Clark, H. C. (1962). Active control of the mechaniml properties of insect endocuticle. J. Insect Physiol. 8, 627-633. Bennet-Clark, H. C. (1963). The relation between epicuticular folding and the subsequent size of an insect. J. Insect Physiol. 9,4346. Blunck, H. (1923). Die Entwicklung des Dytiscus marginalis L. vom Ei bis sur Imago. Z. wiss. Zool. 121, 171-391. Bodine, J. H. (1945a). Tyrosinas and phenols. Action of diversely activated tyrosinase on monohydric and o-dihydric phenols. Proc. Soc. exp. Biol., N.Y. %, 205-209. Bodine, J. H. (1945b). Action of mercuric chloride upon variously activated tyrosinase. Arch. Biochem. 6, 479. Bodine, J. H. and Allen, T. H. (1938a). Enzymes in ontogenesis (Orthoptera). Iv. Natural and artificial conditions governing the action of tyrosinase. J. cell. comp. Physiol. 11, 409423. Bodine, J. H. and Allen, T. H. (1938b). Enzymes in ontogenesis (Orthoptera). v. Further studies on the activation of tyrosinase. J. cell. comp. Physiol. 11, 409423. Bodine, J. H. and Allen, T. H. (1941). Enzymes in ontogenesis (Orthoptera). XV. Some properties of protyrosinases. J . ceN. comp. Physiol. 18, 151-160. Bodine, J. H. and Boell, E. J. (1935). Enzymes in ontogenesis (Orthoptera). I. Tyrosinase. J. cell. comp. Physiol. 6, 263-215. Bodine, J. H. and Carlson, L. D. (1940). Enzymes in ontogenesis (Orthoptera). X. The effects of temperature on the activity of the naturally occurring and other activators of protyrosinase. J . cell. comp. Physiol. 16, 71-83. Bodinc, J. H. and Hill, D. L. (1945). The action of sfithetic detergents on protyrosinase. Arch. Biochem. 7 , 21-32. Bodine, J. H. arid Tahmisian,T. N. (1943). Theeffect of heavy metals on the activation and irjury of the enzyme tyrosinase. Arch. Biochem. 2, 40341 1. Bodine, J. H., Allen, T. H. and Boell, E. J. (1937). Enzymes in ontogenesis (Orthoptera). 111. Activation of naturally occurring enzymes (Tyrosinase). Proc. SOC. exp. Biol., N . Y. 37, 450-453. Bodine, J. H., Ray, 0. M.,Allen, T. H. and Carlson, L. I). (1939). Enzymes in ontogenesis (Orthoptera). VIIl. Changes in the propertics of the natural activator? of protyrosinasc during the course of embryonic development. J. cell. comp. Physiol. 14, 173-1 8 1. Bodine, J. H., Tahmisian, T. N. and Hill, D. L. (1944). Effect of heat on protyrosinase. Arch. Biochem. 4,403412. Brocher, F. (1919). Le mkanisme physiologique de la dernikre mue des larva des Agrionides (transformation en imago). Ann. Biol. locust. 9, 183-200. Brunet, P. C. J. and Kent, P. W. (1955). Observations on the mechanism of a tanning reaction in Periplaneta and Blatta. Proc. roy. SOC.B 144, 259-274. BUckmann, D. (1959). Die Auslosung der Umfarbung durch des Hautungshormon bei Cerura vinula L. J. Insect Physiol. 3, 159-1 89. Bursell, F.(1959). Determination of age of tsetse puparia by dissection. Prof. R. ent. SOC.Lond. A 34, 23-24. Burtt, E. T. (1938). On the corpora allata of dipterous insects. 11. Proc. roy. SOC. B 126, 210-223. Butenandt, A. and Karlson, P. (1954). Uber die Isolierung eines MetamorphoseHormons der Insekten in kistallisierter Form. 2,Nuturforsch. %, 389-391.

214

C. B. COTTRELL

Bytinski-Salz, H. (1936). Die Ausbildung des Chitin panzers in der Schmetterlingspuppe. Biol.Zbl. 56, 35-61. Clarke, K. U. (1958). Studies on the relationship between changes in the volume of the treacheal system and growth in Locusta migratoria L. Proc. 10th inr. Congr. Ent. (1 956) 2, 205-21 1. Clever, U. and Karlson, P. (1960). lnduktion von Puff-Veranderungen in den Speicheldrusenchromosomen von Chironomus tentans durch Ecdyson. Exp. Cell Res. 20, 623-626. ‘Cottrell, C. B. (1 962a). General observations on the imaginal ecdysis of blowflies. Trans. R. ent. Soc. Lond. 114, 317-333. Cottrell, C. B. (1962b). The imaginal ecdysis of blowflies. The control of cuticular hardening and darkening. J. exp. Biol. 39, 395-41 1. Cottrell, C. B. (1 962c). The imaginal ecdysisof blowflies. Detection of the blood-born darkening factor and determination of some of its properties. J. exp. Biol. 39, 41 3-430. Cottrell, C. B. (1962d). The imaginal ecdysis of blowflies. Observations on the hydrostatic mechanisms involved in digging and expansion. J. exp. Biol. 39, 431-448. Cottrell, C. B. (1962e). The imaginal ecdysis of blowflies. Evidence for a change in the mechanical properties of the cuticle at expansion. J. exp. Biol. 39,449-458. Dawson, C. R. and Tarpley, W. E. (1951). Copper oxidases. In “The Enzymes” (J. B. Sumner and K. Myrback, eds.), vol. 2, pp. 454491. Academic Press, New York. Dennell, R. (1947). A study of an insect cuticle: the formation of the pupanurn of Sarcophagafalculata Pand. (Diptera). Proc. roy. SOC.B 133,348-373. Dennell, R. (1949). Weismann’s ring and the control Of tYrOSiMSC activity in the larva of Carliphora erythrocephala. Proc. roy. SOC.B 136,94-109. Dennell, R. (1958). The hardening of insect cuticles. Biol. Rev. 33, 178-196. Dixon, M.and Webb, E. C. (1958). ‘‘Enzymes’’. Longmans, Green and Co., London. Duarte, A. J. (1939). On the ecdysis ofthe African migratory locust. 1, 22-40. Eidmann, H. (1924). Untersuchungen iiber Wachstum und Hiiutung der Insekten. Z . Morph. Okol. Tiere 2, 567-610. Evans, A. C. (1935). Some notes on the histology and physiology of the sheep blowfly Lucilia sericata Meigen. Bull. ent. Res. 26, 115-122. Ewer, D. W. (1954). On the nymphal musculature of the pterothorax of certain Acrididae (Orthoptera). Ann. Natal Mus. 13, 79-89. Finlayson, L. H.(1956). Normal and induced degeneration of abdominal muscles during metamophosis in the Lepidoptera. Quart. J. micr. Sci. 97,215-233. Fraenkel, G . (1935a). A hormone causing pupation in the blowfly Culliphora erythrocephala. Proc. roy. SOC.B 118, 1-12. Fraenkel, G. (1935b). Observations and experiments on the blowfly (Culliphora erythrocephala) during the first day after emergence. Proc. 2001. SOC.Lond. 893-904. Fraenkel, G . (1 938). The evagination of the head in the pupa of syclorrhaphous flies (Diptera). Proc. R. ent. SOC.Lond. A 13, 137-139. Fraenkel, G. and Hsiao, C. (1962). Hormonal and nervous control of tanning in the fly. Science 138,27-29. Fraenkel, G. and Rudall, K. M.(1940). A study of the physical and chemical properties of t t e insect cuticle. Proc. roy. SOC.B 129, 1-35. Fraenkel, G . and Rudall, K.M. (1947). The structure of insect cuticles. Proc. my. SOC.B 134, 111-143.

INSECT ECDYSIS

215

Graubard, M.A. (1933). Tyroiinase in mubnts of Drosophila mekanogaster. J. Genet. 27, 119-218.

Hackman, R. H. (1953). Chemistry of inscct cuticle. I. The water soluble proteinr. Biochem. J . 54, 362-367. Hackman, R. H.(1959). Biochemistry of the insect cuticle. Proc. 4th int. Congr. Biochem., 1958, 12,4842. Hackman, R. H. and Todd, A. R. (1953). Some observations on the reaction of catechol derivatives with amines and amino acids in prescnce of oxidising agents. Biochem. J. 55, 63 1-637. Hackman, R. H., Pryor, M. G. M. and Todd,A. R. (1948). The OcEurrem of phenolic substances in arthropods. Biochem. J. 43,474477. Henricksen, K. L. (1931). The manner of moulting in Arthropoda. Notula Entomol. 11, 103-127.

Hinton, H. E. (1946). A new classification of insect pupae. Proc. zool. SOC.Lond. 116, 282-382.

Hinton, H. E. (1949). On the function, origin and classification of pupae. Trans. S. Lond. ent. nut. Hist. Soc. 1947-48, 1 1 1-1 54. Horowitz, N. H.and Fling, N. (1955). The autocatalytic production of tyrosinase in extracts of Drosophila melanogaster. In “Amino Acid Metabolism” (W. D. McElroy and B. Glass, eds.), pp. 207-218. John Hopkins Press, Baltimore. Ito, T. (1953). Blood phenoloxidase in Bumbyx mori. I. Effect of inhibitors. Annot. zool. jap. 26, 176-180. Jones, B. M. (1958). Enzymatic oxidation of protein as a rate-determining step in the formation of highly stable surface membranes. Proc. roy. Soc. B 149, 263-277.

Jousset de Bellesme ( I 877). PhCnombnes qui accompagnent la metamorphose chez la Libellule dbprimbe. C. R. SOC.Biol., Paris 85,448450. Karlson, P. (1956). Biochemical studies on insect hormones. Vitam. & Horm. 14, 227-266.

Karlson, P. (1960). Zum Tyrosinstoffwechsel der Insekten. 111. Ubet den Einbau von Tyrosin-Umwandlungsprodukten in das Puppcntonnchen dcr Schmeissfliege Calliphora erythrocephala. Hoppe-Seyl. Z . 318, 194-200. Karlson, P. (1962). N-acttyl-dopamine a8 sclerotizing agent of the insect cuticle. Nature, Lond. 195, 183-184. Karlson, P. and Ammon, H. (1963). Zum Tyrosinstoffwechsel der Insekten. XI. Biogenese und Schisksal der Acetylgruppe des N-acetyl-dopamins. Hoppe-Seyl.

Z.330 161-168. Karlson, P. and Hanser, G. (1953). Bildungsort und Erfolgsorgan dcs Puparisicrungshormons der Fleigen. Z. NaturJ 8b, 91-96. Karlson, P. and Liebau, H. (1961). Zurn Tyrosinstoffwechsel der Insekten. V. Reindarstellung, Kristallisation und Substratspezifitat der 0-Diphenoloxydase ans Calliphora crythrocephala. Hoppe-Seyl. Z . 324, 135-143. Karlson, P. and Schlossberger-Raecke, I. (1962). Zum Tyrosinstoffwechsel der Insekten. VIII. Die Sklerotisierung der cuticula bei der Wildform und der Aljinomutante von Schistocera gregaria Forsk. J. Insect Physiol. 8,441452. Karlson, P . and Schmid, H. (1955). Uber die Tyrosinaee der CallijJhorularven. Hoppe-Seyl. Z. 300,35-41. Karlson, P. and Schweiger, A. (1961). Zum Tyrosinstoffwechsel dcr Insekten. IV. Das Phenoloxydase-System von Calliphora und seine Beeinflussing durch das Horman M y s o n . Hoppe-Seyl. Z. 323, 199-210.

216

C. B. C O T T R E L L

Karlson, P. and Sekeris, C. E. (1962). Zum Tyrosinstoffwechsel der Insekten. IX. Kontrolle des TyrosinstofTwechsels durch Ecdyson. Biochim. biophys. Acta 63, 489495.

Karlson, P. and Wecker, E. (1955). Die Tyrosinaseaktivitat wahrend der puparium bildung von Calliphora erythrocuphala. Hoppe-Seyl. Z. 300,42-48. Karlson, P., Sekeris, C. E. and Sekeri, K. E. (1962). Zum Tyrosinstoffwechsel der Insekten. VI. ldentifizierung von N-Acetyl-3, 4-dihydrozy-p-phenathylamin (N-Acetyl-dopamin) als Tyrosinmetabolit. Hoppe-Seyl. Z. 327, 86-94. Keilin, D. and Mann, T. (1938). Polyphenol oxidase. Purification, nature and properties. Proc. roy. SOC.B 125, 187-204. Keister, M. L. and Buck, J. B. (1 949). Tracheal filling in Sciuru larvae. Biol. Bull. 97, 323-330.

Knab, F. (1909). The rble of air in the ecdysis of insects. Proc. ent. SOC.Wash. 11, 68-73.

Knab, F. (1911). Ekdysis in Diptera. Proc. ent. SOC.Wash. 13, 3242. Kobayashi, M. and Kirimura, J. (1958). The brain hormone of the silkworm, Bombyx mori L. Nature, Lond. 181, 1217. Kohler, W.(1 932). Die Entwicklung der Fliigel bei der Mehlmotte Ephesria kuhnielh Zeller mit besonderer Berilcksichtung der Zeichmusters. Z. Morph. OkoI. Tiere 24, 582-68 1.

Kroon, D. B., Veerkamp, T. A. and Loeven, W. A. (1952). X-ray analysis of the process of extension of the wing of the butterfly. Proc. Acad. Sci. A m t . 55,209-214. Kunkel d‘Herculais (1 890). D u r61e de l’air dans le mcCanisme physiologique de I’eclosion, des mues et de la mttamorphose chez les Insectes Orthopteres de la famille des Acidids. C.R. SOC.Biol., Paris 110, 807-809. Laing, J. (1935). On the ptilinum of the blow-fly (Calliphoru eryfhrocepliala). Quart. J . micr. Sci. 77,497-521. Lerner, A. B. (1935). Metabolism of phenylalanine and tyrosine. Advanc. Enzymol. 14, 73-124.

Lewis, D. J. (1 934). Movements of the ptilinum in newly emerged tsetse-flies. Proc. R. ent. SOC.Lond. 9, 78. Malek, S. R A. (1957). Sclcrotisation and melanisation: two independent processes in the cuticle of the desert locust. Nature, Land. 180, 237. Maloef, S. R. A. (1935). The rble of muscular contraction in the production of configurations in the insect skeleton. J. Morph. 58,4146. Marshall, J. F. and Staley, J. (1932). On the distribution of air in the oesophageal diverticula and intestine of mosquitoes. Parasitology 24, 368-381. Mason, H. S. (1953). The structure of melanins. I n “Pigment Cell Growth” (M. Gordon, ed.), pp. 277-303. Academic Press,New York. Mason, H. S. (1955). Comparative biochemistry of the phenolase complex. Advanc. Enzymol. 16, 105-183. Mason, H. S . (1959). Structure of melanins. I n “Pigment Cell Biology” (M.Gordon, ed.), pp. 563-581. Academic Press, New York. Nelson. J. M. and Dawson. C. R. (1 944). Tyrosinase. Advanc. Entyrnol. 4,99-I 52. Ohnishi, E. (1953). Tyrosinase activity during puparium formation in Drosophilu melanopasler. Jap. J. Zool. 11,6!3-74. Ohnishi, E. (1 954a). Tyrosinase in Drosophila virilis. Annot. 2001. jap. 27, 33-39. Ohnishi, E. (1954b). Melanin formation in the mutant “ebony” of DrosophUa melanogaster. Annot. 2001. jap. 27, 33-39. Ohnishi, E.(19%). Activation of tyrosinme in Drosophila virilis. Annot. zoof.jup. 27, 188-193.

INSECT E C D Y S I S

217

Ohnishi. E. (1958). Tyrosinase activation in the pupae ofthe housefly Musca vicina M. Jap. J . 2001.12, 179-188. Ohnishi, E. (1959). Studies on the mechanism of tyrosinase activation in the howfly Musca vicina Macq. J . Insect Physiol. 3,219-229. Petersen, B., Lundgren, L. and Wilson, L. (1956). The development of flight capacity in a butterfly. Behaviour 10, 324-339. Plotnikow, W. (1904). Uber die Hautung und iiber einige Elemente der Haut bei den Insekten. Z. wiss. Zool. 76, 333-366. Potts, W.H.(1933). Observations on Glossina morsitans Westw. in East Africa. Bull. ent. Res. 25, 293-Mo. Preli, H. (1914). Die Beteiligung des Dames an der Enffaltung der Fliigel bei Schmetterlingen. Z. wiss. InsektBiol. 10, 345-349. Pryor, M.G. M. (194Oa). On the hardening of the 00th- of Bhtta orientalis. Proc. roy. Soc. B 128, 378-393. Pryor, M.G . M.(1940b). On the hardening of the cuticle of insects. Proc. roy. Soc. B 128, 393-407. Pryor, M.G .M.(1948). Hardness and colour of insectcuticle.Proc. R. ent. Soc.Lond. A 23,9697. Pryor, M. G. M. (1955). Inhibitors of tyrosinase. J. exp. Biol. 32,46844. Raper, H. S. (1926). The tyrosinase-tyrosine reaction. 5. Production of L-3, Cdihydroxyphenylalanine from ty-rosine. Biochem. J. 20, 735-742. Ray, 0. M. and Bodine, J. H. (1939). Enzymes in ontogenesis (Orthoptera). VII. Effects of dialysis on the activity of the enzyme tyrosinase. J. cell. comp. Physiol. 14,43-59. Richards, A. G . (1951). “The lntegument of Arthropods.”University of Minnesota Press, Minneapolis. Richards, A. G. (1958). The cuticle of Arthropods. Ergebn. Biol. 20, 1-26. Rizki, M.T. (1957). Alterations in the haemocyte population in Drosophila mehnogaster. J . Morph. 100, 437-457. Rizki, M. T. and Rizki, R. M.(1959). Functional significanceof crystal cells in the larva of Drosophila melanogaster. J. biophys. biochem. Cytol. 5,235-240. Schweiger, A. and Karlson, P. (1962a). Zum Tryosinstoffwechsel der 1nsekt.cn. X. Die Aktivierung der Praphenoloxydase und das Aktivator-Enzym. H o p p S e y l . z.329, 210-221. Schweiger, A. and Karlson, P. (1962b). Zur Wirkungsweise des ProthorakaldrusenHormons Ecdyson. Symposia Genefica et Biologica Ztalica 10, 12-21. Sekeris, C. E. (1963). Zum Tyrosinstoffwechsel der Insekten XII. Reinigung, Eigenschafter und Substratspezifitiitdes decarboxylierenden Enzyms. Hoppe-Seyl. Z. (in press). Sekeris, C. E. and Karlson, P.(1962). Zum Tyrosinstoffwechsel der Insekten. VII. Der Katabolische Abbau des Tyrosins und die Biogenese der Sklerotisierungsubstanz, N-Acetyl-dopamin. Biochim. biophys. Acta 62, 103-1 13. Sellier, R. (1950). Sur le retournement des ptkrothodques chez les Orthopteres Gryllides. C. R. SOC.Biol., Paris 231, 923-924. Shafer, G. D. (1923). The growth of dragonfly nymphs at the moult and between moults. Stanf. Univ. Publ. Biol. Sci. 3, 307-338. Sizer, 1. (1 953). Oxidation ofproteins by tyrosinaseand peroxidase. Advunc. Enzymol. 14. 129-161. Snodgrass, R. E. (1947). The insect cranium and “epicranid suture”. Smithon. mhc. CON.107, 1-52.

218

C . U. C O I T K E L L

Susaman, A. S. (1949). The functions of tyrosinase in insects. Quart. Rev. Biol. 24, 328-341.

Sussman, A. S. (1952). Tyrosinase and the respiration of pupae of Phtysamia cecropia L. Biol. Bull. 102, 39-47. Thomas, J. G. (1955). The post-embryonic development of the pterothoracic skeleton and certain muscles of Locusta migratorioides. Proc. zool. Soc. Land. 124, 229-238.

van Schreven, A. C. (1938). Ober die sogenanten Tracheenlungen von Grylfus domesticus. Zool. Anz. 121, 263-264.

Wachter, S. (1930). The moulting of the silkworm and a histological study of the moulting gland. Ann. ent. SOC.Amcr. 23,381-389. Waddington, C. H. (1940). The genetic control of wing development in Drosophila. J. Genet. 41, 75-140.

Waddingtm, C. H. (1941). Body colour genes in Drosophila. Proc. mi.Soc. Lond. A 111, 173-180.

Weismann, A. (1864). Die Nachembryonale fitwicklung der Musciden nach Beobachtungen an Musca vomitoria und Sarcophaga carnaria.2.wiss. Zml. 13, 107-220.

Whitehead, D. L., Brunet, P. C. and Kent, P. W. (1960). Specificity in vitro of a phenoloxidasc system from Periplaneta nmericana. Nature, Lond. 185,610. Wigglesworth, V. B. (1938). The absorption of fluid from the tracheal system of mosquito larvae at hatching and moulting. J. exp. Biol. 15, 248-254. Wigglesworth, V. B. (1948). The insect cuticle. Bioi. Rev. 23.408451. Wigglesworth, V. B. ( I 954). “The Physiology of Insect Metamorphosis”. Cambridge University Press. Wigglesworth, V. B.(1 956). Formation and involution of striated muscle f i h during the growth and moulting cycles of Rhodnius prolixus (Hemiptera). Q w r . J. micr. Sri. 97, 465-480. Wigglesworth, V. B. (1957). The physiology of the insect cuticle. Annu. Rev.Ent. 2, 37-54.

Wigglesworth, V. B.(1 963). A further function of the air sacs in insect$.Nature, b n d . 198,106.

Note added in Proof Subsequent to the completion of this contribution two papers of particular interest have become available. Brunet (1963) has reviewed all aspects of tyrosine metabolism in insects. Fraenkel and Hsaio (1963) have reported further on the “darkening factor” of adult blowflies. Active extracts can be obtained both from the brain and the compound ganglion of the thorax but virtually all the activity of the brain appears to be contained in the median neurasecretory cells of the pars intercerebralis. There can be little doubt that the “darkening factor” is in fact a neurohormone. REFERENCES Brunet, P. C. J. (1963). Tyrosine metabolism in insects. Ann. N.Y.Acad. Sci. 100, 1020-1034.

Fraenkel, G . and Hsaio, C. (1963). Tanning in the adult fly: a fbnction of neurosecretion in the brain. Science 141,1057-1058.

The Control of Visceral Muscles in Ineects K.G.DAVEY Institute of Parasitology, McGill University, Montreal, Canada I. Introduction . II. TheHeart . . A. MusclesoftheHeaut B. Pharmacology . C. NervousControl . D. Endocrine Control. . 111. The Ventral Diaphragm . IV. ~he~uscleoftbe~lknentaryCar;al A. GeneralRcmarks . B. TheForagut . C. The Mid-gut D. TheHind-gut . V. The Muscles of the Malpighian Tubules VI. TheMusclesofthcOviducts MI. The Autonomic Nervous System in Insects RefmnCes

.

:

.

.

.

219 2#)

220 221 223 225 231 232 232 233 236 236

238 240 240 242

I. INTRODUCTION The somatic musculature of insects has been intensively studied; the morphology of the various muscles has been described in elegant detail for many species, and for some somaticmuscles detailsof the finestructure are known. The muscles of the viscera are not 80 well known, possibly because of their superficial similarity in structure to the somatic muscles. Among the vertebrates the visceral or involuntary muscles form a dietinct structural group in that most of them lack striations,while the visceral muscles of insects are, like the somaticmuscles, striated. Neverthdess, the distinction between muscles performing automatic, involuntary actions, and the somatic or voluntary muscles is possibly a valuable one in insects and it is on this basis that the muscles considered hen have been chosen. Most of the visceral muscles contract in a rhythmic fashion; this review considers what is known of the control of these movements in the heart, ventral diaphragm, gut, Malpighian tubes and oviducts. Other visctral muscles are not considered either because they have been insuBiciently studied or because, like the spiracular muscle, they require a separate review. 219

220

K. G . D A V E Y

11. T H EH E A R T A. MUSCLES O F THE H E A R T

There are two anatomically distinct types of muscle involved in the action of the heart of insects. The dorsal vessel is a tube running along the dorsal mid-line and contractile throughout most of its length. It is made up of a single layer of muscle cells, usually with circular fibrils, although some species are reported to exhibit less ordered arrangements (Zawarzin, 1911). For at least part of its length the aorta has a segmental organization, each segment containing a pair of ostia and separated from adjacent segments by a pair of valves consisting of flap-like extensions of the muscle cells. The second set of muscles involved in the pumping action of the heart is represented by the alary muscles which are inserted on the wall of the dorsal vessel and on the lateral part of the dorsum on each side. In most insects these alary muscles are segmentally arranged in fan-like groups with the broad part of the fan inserted on the heart. The alary muscles, along with connective tissue and pericardial cells, make up the dorsal diaphragm which separates the pericardial sinus from the perivisceral sinus. It is important to realize that the pumping action of the heart is p r o b ably the result of the contractions of these two sets of muscles. The nature of the resulting beat will depend in part on the relative timing of the two contractions. De Wilde (1947) investigated the roles of the two muscles, and a review of the earlier observations bearing on this problem can be found in his paper. Using the mechanographic recording technique of Yeager (1938), De Wilde found that the alary muscles of each side of a particular segment contracted synchronously but out of phase with the heart in a variety of Lepidoptera. Thus, the alary muscles contributed to the diatole of the heart. In the heart of Hydrophiluspiceus the situation was more complex. The alary muscles of each segment contract alternately and out of phase with the heart, the frequency of contraction being about one-tenth that of the heart muscle. De Wilde also observed periodic changes in the amplitude of contraction of the alary muscles. Most of the discussion which follows is concerned with the effect of various materials on the heart rate, i.e. with the resultant of two components, the alary muscles and the muscles of the heart. It is conceivable that some materials might affect these muscles differentially,producing for instance a greater dilatation of the heart if the alary muscles contracted mare vigorously, but studies in this direction have not yet appeared.

T H E C O N T R O L O F V I S C E R A L MUSCLES I N INSECTS

221

Throughout the review reference will be made to isolated hearts. This preparation normally consists of the dorsum of the abdomen together with the dorsaldiaphragm containing the heart, alary muscles, pencardial cells, some fat body, and of course, some hypodermal tissue. B. P H A R M A C O L O G Y

It is clear from the relatively few systematic studies that have been made that there is considerable species-variation in the response of insect hearts to various drugs. Part of the variation, of course, is due to differeEt methods of application of the drug in question; the effects of topical application of material to the intact insect can hardly be compared to the effects of adding the material directly to the fluid bathing a partially isolated heart. On the other hand, there are differences which cannot easily be explained in this way. Whether or not differences of this sort represent fundamental differencesin the control of the heart remains to be seen. Since the control of the heart will ultimately involve the effect of chemicals, released locally from nerve endings or more centrally from endocrine organs, a knowledge of the classes of compounds which affect the heart rate, and their mode of action, is important to an understanding of the control of the heart. Chlorinated hydrocarbon insecticides, whether applied externally or injected, have little effect on the rate of beating of the heart of intact or decapitated Periplanetu. Rotenone, on the other hand, steadily depresses the rate (Orser and Brown, 1951). Similar results with rotenone have been obtained using the isolated heart of Periplamtu (Krijgsman and Krijgsman-Berger, 1951). DDT and its relatives are well hown for their ability to disrupt the functioning of the somatic nervous system. If the failure of the insecticides to affect the heart rate represents a real immunity of the nerves supplying the heart (see below), and not a failure of the insecticides to reach the tissues in question, then these experiments reveal a fundamental difference between the somaticand visceral nervous systems in insects. A number of studies have revealed that the partially isolated hearts of cockroaches are sensitive to acetylcholine. The threshold for the effect lies in the region of 10-*M, and concentrationsup to about 10-5M bring about a pronounced increase in frequency of contraction (Krijgsman and Krijgsman-Berger, 1951; Naidu, 1955; Davey, unpublished). This effectis antagonized by curare (Krijgsman and Krijgsman-Berger, 1951) and by atropine, but not by the vertebrate ganglionic blocking agent hexamethonium (Naidu, 1955). Nicotine, a classical blocking agent for

222

K. G . D A V E Y

the sympathetic and parasympathetic ganglia of vertebrates, brings about an initial increase in rate followcd by a gradual return to the normal rate when applied to isolated cockroach hearts. At this point further applications of the drug have no effect (Naidu, 1955). Presumably the initial increase signals the stimulation by nicotine of the ganglia which are known to exist on the cockroach heart (see below), and the gradual return to the normal rate indicates the blocking of the ganglia by the drug, thus preventing any reaction to further additions of nicotine. Now prolonged exposure to nicotine has no effect on the sensitivity of the cockroach heart to acetylcholine (Naidu, 1955), which suggests that the primary site of action of acetylcholine may be on the muscles ofthe heart rather than on the ganglia. This reveals a difference between the muscles of the heart and the somatic muscles, which are insensitive to acetylcholine. Further evidence that acetylcholine may be important in the normal control of the heart in cockroaches is provided by studies with anticholinesterases. Thus, TEPP will potentiate the action of added acetylcholine (Krijgsman and Krijgsman-Berger, 195I), and the addition of eserine alone will bring about a sustained acceleration of the rate (Naidu, 1955; Davey, unpublished). These results suggest that cholinesterase may be functioning in the isolated heart preparation. Similar results have been obtained with the partially isolated hearts of other orthopteroid species : the cricket Stenopelmatus and the grasshopper Melanoplus both have hearts which are sensitive to acetylcholine. That of Melanoplus is particularly sensitive, with a threshold for reaction at about lO-l*M (Davenport, 1949; Hamilton, 1939). Most of the orthopteroid species probably possess ganglion cells on the heart, but the hearts of some other insects do not have an intrinsic innervation. Indeed, that of larval Anopheles quadrimaculatus is totally devoid of innervation (Jones, 1954). It is not therefore surprising that acetylcholine and eserine are both without effect on the heart rates of intact larvae or on the rate of beating of the partially isolated heart (Jones, 1956). On the other hand, acetylcholine accelerates the heart of intact larvae of another Diptera, Corethra (Kopenec, 1949). The lack of intrinsic innervation in the heart of Prodenia has been invoked to explain the relative lack of response of this heart to nicotine, when compared to the heart of Periplaneta (Yeager and Gahan, 1937). Relatively few of the biogenic amines have been tested on insect hearts. Adrenalin accelerates the isolated heart of Periplaneta, with a threshold for excitation at not more than lO-'M (Krijgsman and KrijgsmanBerger, 1951; Naidu, 1955; Davey, unpublished). This effect is often transitory, and hearts which have been exposed to adrenalin soon become

223 erratic (Naidu, 1955). This effect of adrenalin is antagonized by ergotamine, which is a recogrued antagonist for the action of adrenalin on mammalian smooth muscle. That the excitatory effect of adrenalin is not mediated by the nervous system present in the heart of cockroaches is demonstrated by the insensitivity of the response to antagonists of acetylocholinelike atropine, hexamethonium and nicotine (Naidu, 1955). In contrast, however, the heart in intact Anopheles is unaffected by topical application of adrenalin, and the isolated heart shows only a slight stimulation in the presence of the drug (Jones, 1956). The hearts of intact larvae of Corethra are accelerated in the presence of adrenalin (Kopenec, 1949). The heart of Stenopelmatus appears to be unique in that it is dcpressed by adrenalin (Davenport, 1949). Other amines with a phenolic nucleus such as tyramine and dopamine also excite the isolated heart of Periplaneta (Davey, 1963b). Histamine is entirely without effect on the isolated heart of Peripluneta (Davey, unpublished). The indolalkylamines, serotonin and tryptamine, are potent stimulators of the heart of Periplanetu.Serotonin has a threshold at about 104M (Davey, 1961b) and tryptamine is only shghtly higher (Davey, unpublished). ‘fie hallucinogenic drug, lysergic acid diethylamide (LSD), is well known in vertebrate pharmacology for its ability to antagonize some of the peripheral effects of serotonin while it mimics others. In Periplanetu, LSD at all concentrations brings about an increase in heart rate leading to systolic collapse at high concentrations. This effect disappeals only after repeated washings (Davey,unpublished). The 2-bromo derivative of LSD (BOL) is, on the other hand, a good antagonist for the action of the indolalkylamines on the isolated heart of Periplanetu (Davey, 1961b). Furthermore, the action of dopamineis also antagonized by BOL (Davey, 1963b), suggesting perhaps that the phenylalkylamines and the indolalkylamines may act at the same receptors on the heart muscle. Certain amino acids, precursors of their corresponding biogenic amines, are also capable of stimulating the isolated heart of Periplanetu. Their action will be considered in detail later. It is important to point out, however, that the sensitivity of the heart to these materials renders the cockroach heart almost useless for the indentification and assay of materials in tissue extracts unless suitable precautions are taken. T H E CONTROL OF VISCERAL MUSCLES I N INSECTS

C. N E R V O U S CONTROL

Relatively few species have been examined, but it is already apparent that the innervation of the heart varies from species to species. Many insects such as Periplanefa and Blutta exhibit a pair of lateral cardiac

224

K. G. D A V E Y

nerves which pass from the frontal ganghon and recurrent nerve along either side of the aorta (Alexandrowicz, 1926; McIndoo, 1945). These lateral cardiac nerves have a number (30-40 in Per@Zuneta) of large ganglia cells scattered along their length, and it is probably the stimulation of these cells which accounts for the action of substances like nicotine on the isolated heart. In addition to this nerve supply from the retrocerebra1 complex, cockroach hearts also possess an innervation directly from the segmental ganglia of the ventral nerve cord (Alexandrowicz, 1926; McIndoo, 1945). Some of the fibres from the segmental nerves running from the ganglia to the dorsal body wall enter the lateral cardiac nerves. The fibres of the lateral nerves eventually end both on the alary muscles and on the dorsal aorta (Alexandrowicz, 1925). The basic pattern of innervation outlined above appears with only minor variations in a number of species. The hearts of honeybees (Rehm, 1939) and stick insects (Opoczynska-Sembratowa, 1936) are innervated in the same way as described for cockroaches. On the other hand, many species exhibit various degrees of reduction of the innervation. Zawarzin (1911) demonstrated segmental and lateral nerves in the nymphs of the dragonfly Aeschna, but was unable to find ganglion cells along the lateral nerves. Maloeuf (1935) confirmed these observations using nymphs of Anax, and larvae of the silkworm also lack these cells (Kuwana, 1932; McIndoo, 1945). In at least one other lepidopteran, Prodenia, lateral nerves are entirely absent (McIndoo, 1945).The hearts of larval Anopheles quadrimadatus are devoid of any innervation, an observation which is supported by the pharmacological evidence (Jones, 1954, 1956). The heart of Chaoborus (Corethra) appears to possess lateral cardiac nerves (Gersch, 1955) ;this dipteran is, as we have seen, sensitive to acetylcholine (Kopenec, 1949). To what extent the potentialities for control presented by the innervation of the heart are realized in the normal physiology of the insect is uncertain. Unsystematic observations on intact, immobilized adults of Periplaneta demonstrate that although the heart rate over any 1 min interval is approximately constant, there are short-term fluctuations which may be controlled by nerves. Touching the cerci of a cockroach, for instance, produces an instantaneous and short-lived acceleration of the heart (Davey, unpublished). Stimulation of the brain of stag-beetles results in stoppage of the heart (Lasch, 1913), but this can hardly be regarded as physiologically significant. More sophisticated experiments showed that faradic stimulation of the ventral nerve cord in the cervical region accelerated the heart in headless specimens of Periplanefa. The acceieratory impulses were shown to travel along the ventral nerve

T H E CONTROL OF VISCERAL MUSCLES IN INSECTS

225

cord and segmental cardiac nerves to the lateral cardiac nerves (Steher, 1932). D. ENDOCRINE CONTROL

1. The corpus curdiacum Cameron (1953a, b) demonstrated that extracts of the corpus cardiacum possessed powerful cardiostimulatory properties. A concentration of 10pairs of corpora cardiaca in 100ml of the Ringer bathing the isolated heart resulted in an increasein rate of 100%. Onvarious grounds Cameron was led to the tentative conclusion that the active principle in the corpus cardiacum was an o-diphenol similar to, but distinct from, adrenalin. It is well known that the corpus cardiacum of insects consists of at least two functional units: a storage portion made up of the endings of neurosecretory fibres from other parts of the central nervous system, and a secretory portion made up of cells which elaborate their secretion within the gland. Using histochemical techniques Cameron demonstrated that the reaction for o-diphenols was confined to the central, secretory portion of the carpus cardiacum. Furthermore, he showed that pharmacological activity of the corpus cardiacum was undiminished 3 months after the nervi corpori cardiaci had been severed. The two portions of the gland are so closely associated in Peripluneta as to make physical separation impossible.In Schistocercu, on the other hand, the two portions are quite distinct and can be separated fairly easily (Highnam, 1961). When these two parts are assayed separately, the pharmacological activity is found to reside in the secretory portion alone (Highnam, 1961;Davey, 1963a). The observation that the corpus cardiacum contains an adrenalin-like material has been confirmed by various workers (Davey, 1961b; BartonBrowne et QZ., 1961a). On the other hand, if one assumes that the material in the corpus cardiacum is sufficiently like adrenalin to have the same threshold concentration, then the quantitative studies of Barton-Browne et aZ. (1961a) show that the titre of the material in the corpus cardiacum is too low to account for the observed pharmacological activity (cf. Davey, 1963a). Furthermore, it is not possible to detect the presence of the o-diphenol which Cameron studied in simple aqueous extracts of the corpus cardiacum of the sort which are normally used to demonstrate pharmacological activity (Davey, 1961b). The pharmacological activity of aqueous extracts of the corpus cardiacum is destroyed by the action of trypsin and pepsin, and unaffected or increased by heating (Davey, 1961b; Ralph, 1962). On this basis it has been suggested that the cardio-accelerator normally studied is a pcptide.

226

K. G. D A V E Y

Treatment designed to reveal the presence of the o-diphenol (Cameron, 1953a) results in the destruction of the peptide (Davey, 1961b). The corpus cardiacum is also known to contain serotonin (Gersch el ul., 1961;Colhoun, 1963). There are then, three known cardio-accelerators in the corpus cardiacum: a peptide, serotonin, and an unidentified adrenalin-like material. There is, of course, the possibility that the last two factors are identical, but because Barton-Browne and his co-workers excluded the possibility of serotin being involved, this is held to be unlikely. I[ the functional cardio-accelerator is the peptide, then what are the possible functions for the other two materials? They may, of course, simply be metabolic intermediates. Another possibility is that they may function as synaptic transmitters ;this possibility is particularly likely in the case of serotonin, which has been shown to occur in small amounts in other parts of the central nervous system (Gersch et ul., 1961; Colhoun, 1963). Again, the corpus cardiacum has been shown to contain a powerful hyperglycaemic factor, and it has been suggested that the adrenalin-like material may be responsible for this activity (Steele, 1961). It is worth noting that Vanucci (1953), apparently independently of Cameron, demonstrated pharmacological activity in the corpus cardiacum of an unnamed grasshopper. Her assay preparations consisted of pieces of epithelio-musculartube of the earthwormPontoscofexcorethrus, but she used very large concentrations of the hormone-in the region of 300 pairs of glands per 100 ml of Ringer-to obtain the effect. Which of the various factors in the corpus cardiacum was responsible for the excitation of the preparation from the earthworm is difficult to say. Vannucci found no distinct evidence of chromafiin cells in the corpus cardiacum. The peptide from the corpus cardiacum appears to act indirectly on the heart. Evidence obtained by Davey (1961a) suggests that some element in the isolated heart preparation can be exhausted by prolonged exposure to the hormone. On various grounds, the element in the assay preparation through which the corpus cardiacum operates is held to be the pericardial cells. Thus, blocking the pericardial cells with ammonia carmine, indian ink, or trypan blue also blocks the response of the heart to the corpus cardiacum (Davey, 1961a). Histological changes can be observed in pericardial cells exposed to the hormone: the cells enlarge and produce abundant large vacuoles in the area around the nucleus (Davey, 1962c).Pharmacological activity can be demonstrated in extracts of the pericardial cells (Davey, 1961a). On various pharmacological grounds, the material elaborated by the pericardial cells w a s originally

T H E CONTROL OF VISCERAL MUSCLES I N INSECTS

227

tentatively identified as an o-dihydroxyindolakylamine.Thus the action of the corpus cardiacum was antagonized by bromolysergic acid diethylamide, an indication that an indolalkyamine was perhaps involved (Davey, 1961a). Extracts of the pericardial cells blocked neuromuScular transmission in the locust (Usherwood, personal communication), a property which is shared by various indolallcylamines (HU and Usherwood, 1961). The pericardial cells of cockroaches exposed to the corpus cardiacum are more strongly argentailin than those from CockrOBches which have been deprived of their corpora cardiaca (Davey, 1962~); argentaffn-positivecells are often associated with tissues which elaborate amines. Finally extractsof pericardial cells gave spots on paper chromatograms which fluoresced blue-green in UV light after treatment with ninhydrin-acetic (Davey, 1961b). These methods are however, indirect, and as pointed out by Colhoun (personal communication) dihydroxy derivatives of tryptamine are extremely unstable and would be unlikely to survive the extraction procedures used for the studies with paper chromatography. Furthermore, Colhoun (1963) has examined various tissues in the cockroach for indolalkylamines. Although he found serotonin in various parts of the nervous system, he could find no trace of indolalkylamines in the tissues of the heart, including pericardial cells. Finally, the excitatory action of dopamine on the isolated cockroach heart is antagonized by bromolysergic acid diethylamide (Davey, 1963b). It is therefore unlikely that the active factor from the pericardial cells is an indolalkylamine. The action of the corpus cardiacum on the isolated heart of Per@Zmefa is inhibited by semicarbazide: this inhibition is at least partially reversed by pyridoxal phosphate. The presence of semicarbazidc appears to lower the percentage of argentaffin-positive pericardial cells in hearts which have been exposed to the corpus cardiacum. These considerations have led to the suggestion that the action of the corpus cardiacum on the pericardial cells might involve an amino acid decarboxylase. Some support for this hypothesis comes from the observation that an amino acid, dopa, will excite the isolated heart of Periphneta. This excitation occurs through the intervention of the pericardial cells and is inhibited by semicarbazide.The author suggests that the process Controlled by the hormone might be either the decarboxylation of an amino acid to form a pharmacologically active amine or the production of the appropriate amino acid which would then be decarboxylated to form the amine (Davey, 1963b). The form in which the cardiac stimulator is stored in the corpus

228

K. G . D A V E Y

cardiacum has been examined by Evans (1962), who found that a good deal of the pharmacological activity of aqueous extracts was associated with particulate material sedimenting at 11 0o0 g. Evans, on the basis of the light b!ue colour of the centrifuged material (the neurosecretory portion of the corpus cardiacum is distinguished by its blue colour) and the size of the granules in the pellet, suggests that the particulate material with its associated pharmacological activity is neurosecretory in origin As we have already seen, there is evidence to show that the pharmacological activity resides in the secretory part of the corpus cardiacum. It is worth pointing out that the concentration of corpora cardiaca used by Evans in his assays appears to be very high indeed. It is therefore difficult to say what relation the particulate material isolated by Evans bears to the pharmacological activity normally studied in aqueous extracts of the corpus cardiacum. The evidence in the preceding paragraphs describes a system which is potentially capable of regulating the rate of beating of the heart in the intact insect. There is some evidence to suggest that this potential is realized when the insect feeds. Immediately after an intact specimen of Periplaneta feeds on a 10% solution of glucose, the heart rate rises and remains elevated for about 2 h. Removal of the corpus cardiacum, or blockage of the pericardial cells with trypan blue prevents this increase in rate (Davey, 1962a). This study did not rule out the possibility of nervous control via the recurrent nerve-lateral cardiac nerve system, since the recurrent nerve would almost certainly be damaged during the removal of the corpora cardiaca. However, the involvement of the pericardial cells suggests that the corpus cardiacum was responsible for the increase in rate. Surgical manipulation showed that the sense organ involved in the release of the hormone by feeding on sugar was located on the posterolateral borders of the inner surface of the labrum. The impulses travelled to the brain in the labral nerve, left the brain via the frontal connectives to the frontal ganglion, and travelled along the recurrent nerves to the corpus cardiacum. There was some evidence that the suboesophageal ganglion was involved in the process (Davey, 1962b).

2. Extracts of other tissues Various workers have found that extracts of other tissues, chiefly from the central nervous system, contain substances which affect the beating of the isolated heart. In some cases claims have been made that these substances are important in the control of the heart; in no case, however, is there good evidence to substantiate these claims.

1 H E C O N 1 R O 1 0 1 . V I S C E R A L M U S C L E S IN I N S E C T S

229

Gersch and his collaborators have isolated, in addition to acetylcholine, four substances from the central nervous system of various insects which they designate as neurohormones. Unger (1957) isolated two materials which he called neurohormones C and D. Both substances occurred in the corpora cardiaca, the brain, the suboesophageal ganglion, and the ventral nerve cord, while substance C occurred in the corpora allata as well. The two materials were differentiated not only on the basis of their behaviour on paper chromatograms, but also by their differential action on the isolatcd heart. As a preparation for assay, Unger used the isolated heart of Pcriplaneta orientalis in which the alary muscles had been severed, and which had been brought to a complete standstill by repeated washings with Ringer. Substance C brings about an increase in frequency accompanied by a decrease in amplitude. Very high concentrations result in systolic collapse. Substance Dalso promotes an increase in frequency but with an increase in amplitude leading to diastolic standstill. The two substances were further distinguished by their differential effect on the frog heart: substance D increases the amplitude in low concentrations and decreasesit at higher concentrations, while substance C brings about a very slight increase in amplitude (Gersch and Deuse, 1957). Substance D has been crystallized (Gersch, 1959). The two substances also have an effect on the migration of the pigment in the hypodermis of Dixippus and the melanocytes of Corethra. Substance C promotes darkening of the two animals, while D brings about paling in low concentrations and darkening in high concentrations (Gersch, 1959). A further refinement of the separatory procedures has led to the discovery that substances C and D are each made up of two materials. Substance C1 increases the frequency of beating of the insect heart, and decreases the amplitude, bringing about systolic collapse at high concentrations. Substance D1also increased the frequency, but increased the amplitude. C, brought about darkening of isolated pieces of hypodermis from Dixippus, while D, caused them to pale. Substance Cpincreased the frequency of beating of the heart, while Dt decreased it; neither of these two substanccs affected the amplitude of the heart or the migration of pigment (Gersch et al., 1960). As evidence that some at least of these factors have functional Significance in the intact insect, Gersch (I 958) quotes the following evidence. The frequency of beating of the heart of Corethru is increased by stimulation of one of the ventral ganglia, even when that ganglion is isolated from its neighbours. This, coupled with the observations that substances C and D increase the heart rate when injected into intact larvae of

230 K. G. D A V E Y Corethru and that the threshold for effect of thc extract from the corpus allatum is near 1 in 1014,leads Gersch to suggest that these neurohormonal factors must be important in the regulation of the heart. There are, however, reservations in the mind of this reviewer, at least, about the functional implications of this work; these reservations are shared, with respect to the chromatophorotropic effects, by Raabe (1959). Some light may be thrown on the results of the experiments involving ganglionic stimulation by a consideratiom of the so-called “ stress” phenomenon in insects. Evidence is accumulating that any event which involves massive stimulation of the nervous system, including electrical stimulation, resultsin the release of a number of materials, some of which are cardiac stimulators (Sternburg, 1963; Davey, 1963a). Stimulation of the isolated nerve cord of Periplunefa brings about the release of a pharmacologically potent material (Sternburg et al., 1959). There is, however, no evidence that these materials play a role in the normal physiology of the insect. It is possible that ganglionic stimulation in Corethru similarly involves the release of such materials, which may or may not be related to the compounds isolated by Gersch and his coworkers. Gersch and his collaborators are alone in claiming a cardio-regulatory effect from the corpus allatum. Cameron (1953a, b) detected no phannacological activity in breis of the corpora allata. It is difficult, as Raabe (1959) has pointed out, to separate completely the corpus allatum and the corpus cardiacum, but the minimal concentration reported by Gersch for an effect on the heart of Corefhru is loh1*.An effect resulting from such a dilution could hardly be a result of contamination from the corpus cardiacum. The cardiac stimulator from the corpus cardiacum appears to be a peptide (Davey, 1961b). There is some evidence that this material can be changed chemically without losing its activity. Thus, boiling breis of the corpus cardiacum increases their activity (Davey, 1961b; Ralph, 1962), and certain extraction procedures, while destroying the original cardio-regulator, may reveal other compounds which are of unknown functional significance (Davey, I961b). Furthermore, while the relationship between the cardio-regulatory and chromatophorotropic actions of Gersch’s factors is considered in detail by Raabe (1959), it is worth pointing out here that the so-called “A substance”, a chromactivator occurring, inter a h , in the corpus cardiacum, will disintegrate into two materials, both also possessing chromatophorotropic activities (Knowles ef ul., 1955). In view of this evidence it would be unwise to ascribe any functional significance to materials which, like those isolated by Gersch

THE C O N T R O L OF V I S C E R A L MUSCLES I N INSECTS

231

and his co-workers, may very easily be the products of disintegration of larger molecules. Another factor which causes some uneasiness about the significance of substances isolated from tissues and assayed on the inscct heart is the lack of Tpccificity of that orgm in its responses to added materials. Thus the isolated heart of Periplunetn is sensitive not only to those substances like acetylcholineand adrenalin which are normally regarded as pharmacologically active, but also responds to various other materials, like certain amino acids which are not in themselves pharmacologically potent (Davey, 1963b). Ralph (1962) extracted a number of fractions with pharmacological activity from various tissues in Periplaneta. He found as many as six different accelerators, separableon the basis of their solubilitiesin various organic solvents, in the corpus allatum-cardiacumcomplex, and another factor soluble in saline in all parts of the nervous system. Up to five decelerators could be extracted from the central nervous system. Ralph also found that the heart, gut, male reproductive glands, and the haemolymph contained excitatory substances when ground up in saline. Other workers have also found pharmacological activity in the gut (Koller, 1948; Barton-Browne et al., 1961b; Davey, 1962a), the heart (Davey, 1961a), the male reproductive glands @avey, 1960) and the blood (Barton-Browne et al., 1961b). It is important to emphasize that none of these organs has been shown to have a cardio-regulatory function in the intact insect. 111. T H EVENTRAL DIAPHRAGM

Analogous to the dorsal diaphragm, or septum, which carries the heart and associated tissues, is the ventral diaphragm, a more or less continuous sheet of connective tissue and muscle fibres separating the perineural sinus from the perivisceral sinus. In some insects this structure is well developed and contractile, while in others it may be reduced or absent. In those groups in which it is well developed (Orthoptera, Odonata, Ephemeroptera, Hymenoptera, and Lepidoptera) the whole sheet of tissue undulates rhythmically; this movement is probabIy responsible for the irrigation of the ventral nerve cord by driving the haemolymph posteriorly (Wigglesworth, 1950). The ventral diaphragm of Schistocerca has been examined in some detail by Guthrie (1962). The diaphragm consists of segmental bundles of 3 0 4 anastomosing muscle fibres resting on a membrane of connee tive tissue. The diaphragm is innervated from the segmentalventral

232

K. G. D A V E Y

nerve of the appropriate ganglion. These nerves lead to sensory nerves with the morphological characteristicsof stretch receptors; motor fibres are also present. The intact diaphragm in Schistocerca undulates from anterior to posterior at about 8-12 beats per min. Interruption of the nerve supply results in an increased frequency of beating; Guthrie considers that this constitutes evidence for the existence of inhibitory fibres from the central nervous system. There is no evidence for an acceleratory response to nervous stimulation, although direct mechanical or electrical stimulation of the diaphragm increases the frequency.

Iv. T H E MUSCLESOF

THE

ALIMENTARY CANAL

A. G E N E R A L R E M A R K S

A number of observations have been made on the movements of the alimentary canal in intact insects. From these studies it is clear that although there are rhythmical or cyclical movements, these movements are generally very complex. Furthermore it is apparent that each of the three main divisions of the gut functions more or less independently of the other two. Thus Jones (1957, 1960) observed rhythmic contractions of the gut in larvae of Anopheles, but found that contractions of one portion were unrelated to those of another. Roughly similar observations have been made by Knight (1962) on Phormiu reginu. Because these movements are complex it might be expected that nervous control is important. Relatively little is known of the innervation of the gut, but the following brief account describes what seems to be the generally accepted pattern. The pharynx, oesophagus, crop, proventriculus and possibly parts of the mid-gut are supplied by nerves from the frontal ganglion. In Periplaneta, for instance, five pairs of very short nerves from the frontal ganglion supply some of the mouthparts and the muscles of the pharynx. The recurrent nerve passes posteriorly from the frontal ganglion, gives off branches to the retrocerebral glands, and continues over the oesophagus as the oesophageal nerve to the ingluvial ganglion. A pair of ingluvial nerves from the ingluvial ganghon descends to the proventriculus where each ends in a proventricular ganglion. The entire fore-gut is well supplied with nerves from this complex, and fibres from the proventricular ganglia reach the more anterior parts of the mid-gut (Willey, 1961). The fore-gut is well supplied with sense organs (Orlov, 1924). The hind-gut of insects receives nerves from the last abdominal ganglion which ramify over the organ in a much less ordered fashion than in

T H E C O N T R O L O F V I S C E R A L M U S C L E S I N INSECTS

233

the fore-gut. Sensory cells are abundant, and a network of nerves and ganglion cells Is apparent in preparations stained with methylene blue (Orlov, 1924; Davey, 1963a). With this general background in mind, it will be convenient to consider the control of each of the main divisions of the alimentary canal in turn. B. T H E FORE-GUT

The fore-gut includes the pharynx, the oesophagus, the crop, and the proventriculus. Beard (1960) was able to make recordings of the electrical activity in the fore-guts of intact, but paralysed, larvae of Galleria melonella. He recognized four distinct types of activity: type P was a pulsation of the proventriculus with a frequency of 2 or 3 per sec. Type V also occurred at the proventriculus, spreading from the posterior to the anterior of that structure; this activity was intermittent. Type X originated at the junction between the crop and the proventriculus, spreading feebly forwards and more strongly backwards. Type E activity took the form of pulsations in the oesophagus. Thus, even in specimens in which the somatic motor system has been paralysed, the fore-gut continues to show considerable activity. Whether this activity, recorded as it is from paralysed animals, has any relationship to the normal physiology of the animal is difficult to say. It is worth pointing out, however, that in Periplaneta interruption of the nerve supply to the proventriculus results in complete paralysis of that organ (Davey and Treherne, 1963b). Beard’s work might therefore be regarded as a demonstration that the control of visceral muscles differed in some quite fundamental way from the control of somatic muscles. The pharynx of many insects, particularly among the Heteroptera and Diptera, is highly specialized to form a pump with which to suck up juices. When active, the muscles of the pharyngeal pump contract rhythmically (see, for instance, Bennet-Clark, 1963). It is perhaps not generally appreciated that most insects must have a similar structure, albeit not so well developed or highly specialized, in order to ingest the food. Thus, cockroaches deprived of most of their mouthparts are still able to ingest fluids (Davey, 1962b). Swallowing of fluids must involve some pumping action, presumably in the pharynx or oesophagus, and may correspond to the E type activity recorded by Beard (1960). The control of these pumping movements awaits detailed investigation. A series of papers by Davey and Treherne (1963a, b, c) illustrates the complexity of the phenomena involved in the control of the fore-gut. Periplaneta has a capacious crop in which the ingested food,mixed with

234

K. G.

DAVEY

the salivary secretions, is stored. For a variety of solutes and over a wide range of concentrations, the rate of emptying of the crop (i.e. passage of food into the mid-gut) is related to the osmotic pressure of the meal, so that meals of higher osmotic concentration are emptied into the mid-gut more slowly than more dilute meals (Treherne, 1957). The release of fluid from the crop into the mid-gut is mediated by the proventriculus ; contraction of the longitudinal muscles in that organ opens the lumen and allows the fluid to pass. The rate of flow of fluid through an orifice is described by a modified Poiseuille equation in which the rate of flow is directly related to the radius of the orifice, the pressure difference across the orifice, the duration and frequency of opening of the orifice, and inversely related to the length of the orifice and the viscosity of the fluid involved. On the other hand, Treherne (1957) showed that the rate of flow from the crop into the mid-gut was an exponential process; it is clear, therefore, that one or more of the parameters in the modified Poiseuille equation must vary with time in a non-linear manner. The frequency of opening of the proventricular valve decays exponentially with time, but this variation alone is not sufficient to account for the observed differences in the rate of emptying. A further complication resulted from the observation that cockroaches appeared to be compensating in some way for very viscous meals; the rate of emptying of such meals was very much faster than would be expected on the basis of the modifled Poiseuille equation, even when the effects of osmotic pressure were taken into account. At least part of this relative increase in rate resulted from a slower rate of decay in the frequency of opening of the proventricular valve; but again this effect alone did not account for all of the observed increase in the rate of emptying (Davey and Treherne, 1963a). X-rays of cockroaches taken at various intervals after the ingestion of a radio-opaque meal revealed that the bubble of air contained in the crop did not decrease very markedly duaing the period of emptying of the crop. It was suggested that the cockroach swallowed air during this period, thereby maintaining a gradient of pressure across the valve. This suggestion received support from experiments in which blocking the mouth after feeding resulted in a lower rate of emptying (Davey and Treherne, 1963a). The nature of the nervous control of the proventricular valve emerged in a series of experiments in which various nerves were severed and the rate of emptying of the crop determined for the operated animals. The nervous pathway involved a pair of small nerves (N, of Willey, 1961) from the pharynx to the frontal ganghon. Impulses travelled from the

THE C O N T R O L O F V I S C E R A L M U S C L E S I N INSECTS

235

frontal ganglion, along the recurrent nerve, through the various ganglia on the crop to the proventriculus. The sensory end of the system was represented by a new sense organ in the pharynx. The authors suggest that this organ might function as a receptor for osmotic pressure (Davey and Treherne, 1963b). Measurements of hydrostatic pressure within the crops of feeding and recently fed insects demonstrated that the pressure increased to about 6 cm of water above the resting level on feeding. This rise in pressure involved the release of air from the crop, although as emptying of the crop proceeds air is again swallowed. Transient increases in hydrostatic pressure occurred periodically during the period of emptying ; these increases were dependent on the same nervous pathway as the movements of the proventriculus. The inference is that these transient increases are co-ordinated with the opening of the proventricular valve and represent the effective pressure across the valve. Both the general and transient pressures declined in an approximately exponential way, but there was no difference in the magnitude or rate of decay of these pressures between animals fed on meals of different osmotic pressures. When the insects were fed on viscous meals, the transient pressures decayed rapidly at first, but remained relatively high throughout the remainder of the period of crop emptying. In other words, although the hydrostatic pressure may contribute to the observed differences in the rate of emptying, its effect does not explain all of the differences (Davey and Treherne, 1963c). The authors were unable to observe directly the other characteristics of the proventricular valve which might be expected to vary, namely the extent of opening and the duration of opening. However, a quantitative analysis of the results suggested that the extent of opening of the valve increases during the period of emptying, and that the rate of increase is greater with more concentrated meals (Davey and Treherne, 1963~). Thus the control of the proventricular valve which regulates the flow of food from the crop into the mid-gut is mediated by the retrocerebral system. This control involves both the extent and frequency of opening of the valve as well as transient pulses of hydrostatic pressure within the crop. The sensory end of the system detects the osmotic pressure of the ingested meal, and perhaps the viscosity, of the ingested meal, although the role of stretch receptors known to be present in the foregut (Clarke and Langley, 1962) may be an important one. It is perhaps significant that this degree of sophistication of control can be achieved entirely within the retrocerebral nervous system, which can function in isolation from the central nervous system (Davey and Treherne, 1963b).

236

K. G . D A V E Y

The endocrine control of the fore-gut has not been extensively investigated. Cameron (1953b) mentioned that the isolated fore-gut is inhibited by breis of the corpus cardiacum. On the other hand, preliminary observations with the isolated fore-gut revealed a slight excitation of the contractions under the influence of breis of the corpus cardiacum (Davey, unpublished). It is to be emphasized that the fore-gut is very delicate and that conventional mechanographic techniques, which have so far been employed, may not yield useful results. C. THE MID-GUT

Little or no information is available concerning the movements of the mid-gut. In larval Anopheles quadrimculatus, the mid-gut executes a complex cycle of peristaltic and antiperistaltic contractions which cease when the entire gut is isolated in a Ringer which maintains the contractions of the fore- and hind-gut (Jones, 1957, 1960). Gerscb (1955) noted that electrical stimulation of various ventral ganglia resulted in an increase in frequency of contraction of the mid-gut. This excitation occurred even when the stimulated &on was isolated from its neighbours. Gersch suggests that this indicates that a neurohormonal agent is released on stimulation of the ganglion. Whether such a system operates in the intact insect is, however, problematical. D. THE H I N D - G U T

The hind-gut of most insects is more robust than the other portions of the alimentary canal; it is probably for this reason that much of the work on isolated guts has been performed on it. The discussion which followsincludes some observationson isolated, entire guts, because most of the contractions which were recorded were probably due to the hindgut. 1. Pharmacology Acetylcholine at concentrations as low as lo-* M stimulates the contractions of whole alimentary canals (Kooistra, 1950; Gersch, 1955). This effect is potentiated by anticholinesterases like eserine, TEPP, and parathion (Kooistra, 1950). Eserine alone stimulates the contrao tion of isolated guts of Chaoborus (Gersch, 1955), and Periplaneta (Kooistra, 1950). Cholinesterase has been demonstrated in the intestine of Periplaneta (Kooistra, 1950). These observations suggest that acetylcholine may be important in the control of the contractions of the gut. Whether the acetylcholine is effective on the nerves which are

T H E C O N T R O L O F V I S C E R A L MUSCLES I N INSECTS

237

known to exist on the hind-gut or directly on the muscle, or both, is uncertain. Indolalkylamines like tryptamine, S O H tryptamine and 5,6 di-OH tryptamine also excite the hind-gut. The action of these compounds is antagonized by 2-bromolysergic acid diethylamide (Davey, 1962d;Colhoun, 1963). Lysergic acid diethylamide has not been tested on isolated guts, but if it behaves as it does on the heart, that is if it produces marked stimulation which is reversible only with great difficulty, these effects might explain the results of Siva Sunkar et ul. (1960). These workers found that including LSD in the diet of larval Tenebrio brought about a reduction in growth which did not cease on withdrawal of the drug. The fact that the indolalkylamines excite the isolated gut emphasizes the difference between the visceral and somatic nervous systems of insects, for Hill and Usherwood (1961) demonstrated that indolalkylamines paralyse the myoneural junction of leg muscle of locusts.

2. Endocrine control of the hind-gut Cameron (1953a, b) noted that extracts of the corpus cardiacum stimulated the contractions of the isolated hind-gut of Periplunetu. The stimulation was complex; the amplitude normally increased, as did the general tonus of the preparation, but the frequency sometimes increased and sometimes decreased. The most characteristic response to the corpus cardiacum was an increased co-ordination of the preparation as evidenced by the tendency of the contractions to occur in groups. These results have been confirmed (Davey, 1962d). On the other hand, Koller (1954) claimed that extracts of the corpus cardiacum inhibited the contractions of the isolated gut of Tenebrio. Koller used the entire gut, and, as we have already seen, there are reports that the fore-gut is inhibited by extracts of the corpus cardiacum. Perhaps a more likely explanation of these results lies in the fact that Koller used the frequency of contraction as the sole criterion for activity. As mentioned above the frequency of contraction often decreases under stimulation from the corpus cardiacum. It is worth noting that the concentration of Koller’s extracts was very high-in the region of 25 glands per 100 ml of Ringer. Excitation of the gut can be obtained very readily with concentrations ten times more dilute. Koller (1954) recognized that the gut itself possessed remarkable myotropic properties, an observation which has been confirmed by other workers (Barton-Browne et al., 1961b; Davey, 1962a). The lower colon of Peripluneta contains argentaffin-positive cells. The presence of the region oontaining these cells is necessary for the action of the corpus

238

K. G. D A V E Y

cardiacum on the hind-gut. Thus, the isolated rectum is insensitive to breis of the corpus cardiacum, but will exhibit contractions when the colon is attached to it. This evidence has led to the suggestion that the corpus cardiacum acts on the hind-gut in a way analogous to its action on the heart, that is by stimulating the cells in the hind-gut to produce and release an amine. In the case of the gut, however, it is suggested that the active material produced by the cells in the gut does not act directly on the muscles, but through the peripheral nervous system in the hind-gut (Davey, 1962d). The active material from the hind-gut was tentatively identified as an o-dihydroxyindolalkylamine, using the indirect methods already described in the section on the heart (Davey, 1962d). However, as already pointed out, Colhoun (1963) has been unable to find any evidence of an indolalkylamine in the tissues of the heart. On this basis, it is unlikely that the active material from the gut is an amine with an indole nucleus. Barton-Browne et ul. (1961b) have characterized a substance from the gut and blood of Periplunetu which stimulates the isolated uterus of the rat; this material is non-dialysable and destroyed by exposure of 50°C for 10 min. The material was differentiated from serotonin, acetylcholine and histamine. Whether this material is the same as that which is active on the gut of Periplanetu is uncertain. It has been assumed by most workers that the material from the corpus cardiacum which acts on the heart is identical with the one which acts on the gut. There is no direct evidence to support this supposition, but the observation that the contractions of the hind-gut increase after feeding when the cardiac stimulator is released (Davey, 1962a) suggests that the substances may indeed be identical. Extracts of other tissues are also reported to affect the contractions of the gut. Koller (1954) found that extracts of the corpus allatum increased the frequency of contractions, while extracts from the brain decreased the frequency. Extracts of the suboesophageal ganglion increased the frequency at low concentrations and decreased it at higher concentrations. Gersch (1955) found that extracts of the nervous system of Corefhruand Periplunetu stimulated the isolated gut of Corethra.

V. THEMUSCLESOF

MALPIGHIAN TUBULES In some insects the Malpighian tubules are supplied with muscles, the contractions of which produce various movements in the tubules. Palm (1346) examined a large number of individuals from a wide variety of species and concluded that insectscould be dividedinto four groups on the basis of the musculature of the Malpighian tubules. The Thysanura, THE

T H E C O N T R O L O F V I S C E R A L MUSCLES I N I N S E C T S

239

Dermaptera and Thysanoptera lack muscle fibres on the tubules and comprise the first group. The second, made up of the Lepidoptera, Diptera, Trichoptera and some Hemiptera have muscles only at the base of the tubule. These muscles are possibly extensions of the musculature of the gut. Another group of insects, represented by the Coleoptera and Neuroptera, possesses a more or less continuous coating of muscles over the tubes. Contraction of these muscles may be quite local, producing constructions at the point of contraction. The type of musculature familiar to most entomologists is that found in the Orthopteroid orders and the Odonata where one or two muscle fibres are helically wound about the tubule along its entire length. Contraction of these muscles throws the Malpighian tubules into a tight helix. Like the third type mentioned above, these muscles may also contract locally, so that one portion of the tubule may be tightly coiled, while an adjacent portion is extended. Presumably because of the difficulty of working with such delicate material, the movements of the Malpi&an tubules have been studied only superficially in the last two groups. In both groups, the muscles will exhibit rhythmic contractions even when the tubules are completely isolated in a suitable Ringer’s solution (Koller, 1948). The functionalsignificance of these movements is by no means clear. It has been suggestedthat they contribute to the flow of haemolymph over the tubule, and possibly to the movements of materia within the tubule (Wigglesworth, 1950). On the other hand, such movements may produce changes in pressure within the tubule large enough to affect the rate of flow of substances across the cells of the tubule. Control of the contractions could in this way offer a potential mechanism for the control of excretory processes. According to Palm (1946), the contractions are unaffected by chloroform, curare, cocaine or DDT; this evidence supports the direct observation that the muscles are not innervated. The rate of contraction o f . the tubules of PeripIuneta is increased by adrenalin (Cameron, 1953b), serotonin and 5,6-dihydroxytryptamine (Colhoun, 1963). Breis of the corpus cardiacum also exert a stimulatory effect on the muscles; at very high concentrations, however, the frequency of contraction is depressed (Cameron, 1953b). Which of the three pharmacologically potent materials that have thus far been demonstrated in the corpus cardiacum (see Section IT, D above) is responsible for these effects is uncertain. If the peptide were involved, then it would be reasonable to expect to find cells in the Malpighian tubules analogous to the pericardial cells. Preliminary examination has thus far failed to reveal argentaffin-positive cells (Davey, unpublished data) in the tubules.

240

K. G . D A V E Y

v1. T H EM U S C L E SOF THE OVIDUCTS The muscles of the oviducts have several tasks to perform. In some insects, at least, they are responsible for transporting the semen within the female (Davey, 1958); their contractions carry the eggs from the ovary to the ovipositing apparatus; contractions of the muscles of the spermathecae are presumably responsible for the transfer of semen from the spermatheca into the micropyles of the eggs. This variety and complexity of movements suggests that a nervous system might be involved in the control of these contractions. The oviducts of Rhodnius are served by a network of nerves, possibly containing nerve cells, which is connected to the central nervous system through the fused abdominal ganglia. Evidence has been presented which demonstrates that the movements of the oviducts are responsible for the transport of semen from the bursa copulatrix to the spermathecae. These movements are initiated by the opaque accessory secretion of the male, acting through the nervous system in the oviducts (Davey, 1958). It has been suggested that the active material in the opaque accessory glands is an indolalkylamine, identical to the material from the pericardial cells and hind-gut of the cockroach (Davey, 1961a), but Colhoun (1963) has been unable to detect indolalkylamines in this tissue. Extracts of various tissues have been found to increase the rate of contraction of isolated oviducts. Thus, Koller (1954) showed that extracts of the brain of Carausius stimulated the contractions of the oviducts of Tenebrio, and Nayar (quoted in Raabe, 1959) demonstrated that the pars intercerebralis has a stimulatory effect. Highnam (1962) reports that the blood of ovipositing females of Schistocerca contains a tryptamine derivative, and that serotonin will excite the isolated oviducts of that species. Whether or not these phenomena, as has been suggested, are related to the release of neurosecretory material from the pars intercerebralis and corpus cardiacum which is associated with oviposition is uncertain (Nayar, 1958; Highnam, 1962). The control of the muscles of the male reproductive system awaits investigation. VII. T H EAUTONOMIC NERVOUS SYSTEM

I N INSECTS

The evidence presented in this review supports the view that insects possess a visceral nervous system which can be distinguished from the somatic nervous system. There is an extensive peripheral network of cells and nerves on many of the organs which, while it can function in

T H E C O N T R O L O F V I S C E R A L MUSCLES I N INSECTS

241

isolation from the central nervous system, is connected to it at various points. These networks of cells, and the muscles which they control, appear to be functionally different from the somatic neuromuscular system as it is currently understood. Thus, the various indolalkylamines excite the rhythmic activities of the visceral muscles, but paralyse the somatic neuromuscular system (Hill and Usherwood, 1961). The work of Beard (1960) has shown that insects paralysed by various venoms still exhibit rhythmic contractions of the viscera. The networks on the organs are connected to the central nervous system by one or more of three main pathways. Perhaps the most familiar is the retrocerebral system which has been described in a number of species. It mediates, by straightforward nervous control, some of the functions of the fore-gut (Davey and Treherne, 1963a, byc), it governs the release of the cardio-regulator from the corpus cardiacum (Davey, 1962c), and, as is well known, it carries neurosecretory material from the brain to the storage portion of the corpus cardiacum. It is probably also involved in a number of sensory mechanisms, like that described by Clarke and Langley (1962), which are mediated by the various nerves leading into the frontal ganglion. The frontal ganglion in Periplunetu is connected to the brain by the frontel connectives and the nervus connectivus. These nerves become involved with almost every region of the brain, including the suboesophagea1 ganglion (Willey, 1961). This system, along with the various other nerves which lead more directly from the brain to the retrocerebral glands, presents a formidable potential for the interaction of the somatic and visceral systems. The other two pathways have been less extensively studied. One is the caudal or proctadeal system which innervates the hind-gut and reproductive system from the last abdominal ganglion; the other is the system of segmental nerves which travel to the muscles of the heart. The influence of the visceral nervous system may be much wider than supposed at present. Johnson (1 962) has described in various aphids an extensive system of nerves carrying fuchsinophilic neurosecretory material from the brain and corpus cardiacum to various organs. The observation that neurosecretory material appears to be carried directly to various somatic muscles leads to the speculation that the autonomic system of insects might influence the behaviour of somatic muscles. More direct evidence for the influence of the visceral nervous system on a somatic muscle is provided by the work of Voskresenskaya and Svidersky (1961) who found that the tymbal muscle of cicadas had adual innervation from the mesothoracic ganglion. Stimulation of the tymbal nerve, representing the somatic side of the system, resulted in a single

242

K. G. D A V E Y

muscle spike for every nerve pulse, whereas stimulation of the tymbal nerve and the “sympathetic nerve” together yielded a number of muscle spikes for every nerve spike. The influence of the sympathetic nerve could be blocked by injecting sympathotoleneinto the muscle. There have been suggestions that secretions from the retrocerebral system may influence the behaviour of insects (Hodgson and Geldiay, 1959; Milburn et al., 1960). This is in many ways an attractive hypothesis, but it is important to point out that much of the evidence rests on the supposition that the cardio-accelerator from the corpus cardiacum is released from the storage portion of that organ. As we have seen it is a product of the secretory portion. The effects on nervous transmission which Milburn et al. (1960) obtained with extracts of the corpus cardiacum are often quoted as supporting the hypothesis that behavioural changes in insects may be mediated by the effects of neurosecretory material on the central nervous system. The demonstration of this effect required massive concentrations of corpora cardiaca in the bathing medium, concentrations which it would be difficult to envisage as occurring in nature. REFERENCES Alexandrowin, J. S. (1926). The innervation of the heart of the cockroach. J. c o w . Neurol. 41,291-310. Barton-Browne, L., Dodson, L. F., Hodgson, E.S. andKiraly, J. (1961a). Adnnergic properties of the cockroach corpus cardiacum. Gen. comp. Ehdocr. 1,232-236. Barton-Browne, L., Hodgson, E.S. and Kiraly, J. K.(1961b). Stimulation of uterine contractions by extracts of the cockroach, Pertplaneta americana. Science 134, 669-670. Beard, R.L. (1960). Electrographicrecording of foregut activity in larvae of Galleria melonellu. Ann. ent. Soc. Amer. 53,346351. &Met-Clark, H. C. (1963). Negative pressures produced in the pharyngeal pump of the blood-sucking bug, Rhohius prolixus. J. exp. Biol. 40,223-230. Cameron, M . L. (1953a). Secretion of an o-diphenol in the corpus cardiacum of insects. Nature, Lond. 172, 349-350. Cameron, M. L. (1953b). Some pharmacologicallyactive substancesfound in insects. Ph.D. dissertation, University of Cambridge. Clarke, K.U. and Langley, P. (1962). Factors concerned in the initiation of growth and moulting in Locusta migrutoria L. Nature, Lond. lW,16&162. Colhoun,E. H. (1963). Synthesisof 5-hydroxytryptamine in the Amencancockroach. Experientia 19,9-10. Davenport, D. (1949). Studies in the pharmacology of the heart ofthe orthopteron, Stmopelmatus. Physiol. Zool. 22, 35-44. Davey, K. G. (1958). The migration of spermatozoa in the female of Rkodnius prolixus Stal.J. exp. Biol. 35, 694-701. Davey, K. G. (1960). A pharmacologically active agent in the reproductive system of insects. Canad. J. Zool. 38,3945.

T H E CONTROL OF VISCERAL MUSCLES IN INSECTS

243

Davey, K. 0.(1961a). The mode of action of the heart accchatingfador from the corpus cardiaaun of insects. Gen.comp. Endocr. 1,24-29. Davey, K.G. (1961b). Substances controlling the rate of beating of the heart of Periplaneta. Nature, Lond. 192, 284. Davey, K. G. (1962a). The release by fceding of a phamadogically active factor from the corpus cardiacum of P e r i p h t a americona. J. I-t Phydol. 8, 205208. Dany, K.G. (1962b). The nervous pathway involved in tbo release by feeding of a pharmacologically active factor from the corpus cardiocum of Pe.r’f J. Insect Physiol. 8, 579-583. Davey, K. G. (1962~).Changea in the pericardd c e b of Pcrfplaneia antericana induced by exposure to homogenates of the corpus cardiaaun Quart. J. micr. ScL 103, 349-358. Davey, K. G. (1962d). The mode of action of the corpus cardiacum on the hind-pt in Periplaneta americana. J. exp. Biol. 39, 319-324. Davey, K. G. (1963a). The release by enforced activityof the cardiacacceleratorfrom the corpus cardiacum of Periplaneta americana. J. Insect Physiol. (in p-). Davey, K. G. (1963b). The possible involvement of an amino acid dccarboxylasc in the stimulation of the pericardial cells of Periplaneta by the corpus cardiacum. J. exp. Biol. (in press). Davey, K. G. and Treherne, J. E.(1963a). Studies on crop M o n in the cockroach (Periplaneia americana). I. The mechanism of crop emptying. J. exp. Bbl. (in press). Davey, K. G. and Treherne, J. E.(1963b). Studies on crop function in the cockroach (Periplaneta americana).11. The nervous control of crop emptying. J. exp. Biol. (in press). Davey, K. G. and Treherne, J. E.(1963~).Studies on crop function in the cockroach (Periplanetaamericana).111. Pressure changesduring fndinp and cropemptying. J. exp. Biol. (in press). Evans, J. J. T. (1962). Insect neurosccretory material #puated by diffaential centrifugation. Science 136, 314-315. Gersch, M. (1955). Untersuchungen Uber Ausliisung und Stcuerung da Darmbewegungen bei det Larve von Chaoborus. Biol. Zbl. 74,601628. Gersch, M. (1958). Neurohumoral Beeinflussung der H d t i g k e i t bei der Larve von Corethra. J. Insect Physbl. 2, 281-297. Gersch, M.(1959). Untersuchungen Ober Ncurohormoncn bei Insekten. Proc. XV int. Cmgr. 2001. 493-497. Gersch, M. and Deuse, R.(1957). Die Wirkung von Ncurohormoncn aus Insekten auf das Froschherz. Biol. Zbl. 76, 436-442. Gersch, M.,Fischer, F., Unger, H.and Koch, H.(1960). Die Isolierung newohormonaler Factoren aus dem Nervensystem der KWhcn8chabe Periplaneta umericana. 2.Nuturf. 15, 319-322. Gasch, M., Fischer,F., Unger.H.andKabitza, W.(l%l).VorkommenvonSerotonin in Nervensystem von Periplaneta americana L. (Insects). Z. NaturJ 16,351-352. Guthrie, D. M.(1962). Control of the ventral diaphragm in an insect. Nuture, Lond. 1%. 1010-1012. Hamilton, H. K. (1939). The action of acetylcholine, atropine and nicotine on the heart of the grasshopper (Melanoplus diflerenthlis). J. cell. comp. Physiol. 13, 91-103.

.

244

K. G. D A V E V

Highnam, K. C. (1961). Induced changes in the amounts of material in the neurosecretory system of the desert locust. Nature, Lo&. 191, 199-200. Highnam, K.C. (1962). Variation in neurosecretory activity during oocyte development in Schistocerca gregaria. J. Endocr. 24, iv-v. Hill, R. B. and Usherwood, P. N. R. (1961). The action of 5-hydroxytryptamine and related compounds on neuromuscular transmission in the locust Schistocerco gregaria. J. Physiol. 157, 393-401. Hodgson, E. S. and Geldiay, S. (1959). Experimentally induced release of neurosecretory material from roach corpora cardiaca. Biol. Bull. 117,275-283. Johnson, B. (1962). Neurosecretion and the transport of secretory material from the corpora cardiaca in aphids. Nature, Lond. 196,1338-1 339. Jones, J. C. (1 954). The heart and associated tissues of AnophelesquadrimaculatusSay (Diptera: Culicidae). J . Morph. 94, 71-1 16. Jones, J. C. (1956). Effects of drugs on Anopheles heart rates. J . exp. Zool. 133, 573-588.

Jones, J. C.(1957). Cyclic movements of the mid-gut of Anophefesquadrimaculatus larvae. Anat. Rec. 128,570-571. Jones, J. C. (1960). The anatomy and rhythmical activities of the alimentary canal of Anopheles larvae. Ann. ent. SOC.Amer. 53,459-474. Knight, M. R. (1962). Rhythmic activities of the alimentary canal of the blow fly, Phormia regina (Diptera Calliphoridae). Ann. ent. SOC.Amer. 55, 38&382. Knowles, F. G. W., Carlisle, D. B. and Dupont-Raabe, M. (1955). Studies on pigment activating substances in animals. I. The separation by paper electrophoresis of chromactivating substances in arthropods. J. Mar. biol. Ass. U.K. 34, 611-635.

Koller, G. (1948). Rhythmische Bewegung und hormonale Steuerung bei den Malpighischen Gefiissen der Insekten. Biol. Zbl. 67,201-21 1. Koller, G. (1954). Zur Frage der horrnonaler Steuerung bei der rhythmischen Eingeweidebewegungen von Insekten. Verh. dtsch. zool. Ges. 27, 417422. Kooistra, G. (1950). Contribution to the knowledge of the action of acetylcholine on the intestine of Periplaneta americana L. Physiol. comp. 2,75-80. Kopenec, A. (1949). Farbwechsel der Larve von Corethra plumicornis. Z. vergl. Physiol. 31,490-505. Krijgsman, B. J. and Krijgsman-Berger, N. E. (195 1). Physiological investigations into the heart function of arthropods. The heart of Periplaneta americana. Bull. ent. res. 42, 143-155. Kuwana, F. (1932). Morphological studies of the nervous system of the silkworm Bombyx mori Lime. 1. The innervation of the dorsal vessel. Bull. imp. Seric. Sta. Japan 8, 109-120. Lasch, W. (1913). Einige Beobachtungen am Herzen der Hirschk5ferlarve. Z . allg. Physiol. 14,3 12-3 19. Maloeuf, N. S. R. (1935). The myogenic automatism of the contraction of the heart of insects. Ann. ent. SOC.Amer. 28,332-337. McIndoo, N.E. (1945). Innervation of insect hearts. J. comp. Neurol. 83, 141-155. Milburn, N., Weiant, E. A. and Roeder, K. D. (1960). The release of efferent nerve activity in the roach Periplaneta americana by extracts of the corpus cardiacum. Biol. Bull. 118,1 1 1-1 19. Naidu, M. B. (1955). Physiological action of drugs and insecticides on insects. Bull. ent. Res. 46, 205-220.

T H E C O N T R O L O F V I S C E R A L M U S C L E S I N INSECTS

245

Nayar, K. K. (1958). Studies in the neurosecrctory system of lphita limhutu Stal. Part V . Prohahle endocrinc basis of oviposition in the female insect. Proc. Indirrn AcnJ. Sd.I3 41, 233 251. Opocynska-Sembratowe. 2:. ( I 936). Rechcrchcs sur I’anatomie ct L‘innervation du coeur dr: (brrtuslur tiiutosits. Hull. int. Akud Crocovie B 5. 41 1 - 4 6 . Orlov, J. (1924). Die Innervation dcs Darinesder Insekten. Z.wius. ZOOI.122.405-522. Orser, W. B. and Brown, A. W. A. (1951). Thecffect of insecticides on the heart beat of Periplaneta. Cantid. J. Zool. 29, 54-64. Palm, N. B. (1946). Studies in the peristalsis of malpighian tubules in insects. Acta Univ. lund. 42, 1-39. Raabe, M. (1959). Neurohormones chez les insectes. Bull. Soc. Zool. Fr. 84,272-316. Ralph, C. L. (1962). Heart accelerators and decelerators in the nervous system of Periplaneta americana. J . Insect PhyJiol. 8, 431439. Rehm, E. (1939). Die Innervation der innern Organe von Apis mellifica. Zugleich ein Beitrag zur Frage des sog. Sympathetischen Nervensystems der Insekten. Z. Morph. dkol. Tiere 36, 89-122. Siva Sunkar, D. V., Kopelman, A., Schilder, P. and Gold, E.(1960). Effect of lysergic acid diethylamide on larval growth. Nature, Lond. 187, 153-154. Steele, J. S. (1961). Occurrence of a hyperglycaemic factor in the corpus cardiacum of an insect. Nature, Lond. 192, 680-681. Steiner, G. (1932). Die Automatie und die zentrale Beeinflussung des Herzens von Periplaneta americana. Z. vergl. Physiol. 16, 290-304. Sternburg, J. (1 963). Autointoxication and some stress phenomena. Annu. Rev. Ent. 8, 19-38. Sternburg, J.. Chang, S. C. and Kearns, C. W. (1959). The release of a neuroactive agent by the American cockroach after exposure to DDT or electrical stimulation. J . ec‘on. Ent. 52. 1070-1076. Treherne, J. E. ( I 957). Glucose absorption in the cockr0ach.J. exp. Biol.34,478485. Unger, H. ( I 957). Untersuchungen zur neurohormonalen Beeinflussung der Herztitigkeit bei Schaben ( P . orientalis, P. americana, Phyllodroma germanica). Biol. Zbl. 16, 204-225. Vannucci, M. ( 1953).The function of the corpora cardiaca of a grasshopper. Dusenia 4, 439-442. Voskresenskaya, A. K. and Svidersky, V. L. (1961). The role of the central a sympathetic nervous system in the function of the tymbla muscles of cicadas. J. Insect Physiol. 6, 26-35. Wigglesworth, V. B. (1950). “The Principles of Insect Physiology”. Methuen, London. Wilde, J. de (1947). Contribution to the physiology of the heart of insects with special reference to the alary muscles. Arch. nker. Physiol. 28, 530-542. Willey, R. B. (I961 ).The morphology of the stomodeal nervous system in Periplanetu umericana (L.)and other Blattaria. J. Morph. 108, 219-261. Yeager, J. F. (1938). Mechanographic method of recording insect cardiac activity, with reference to the effect of nicotine on isolated heart preparations from Periplaneta orientalis. J. Agric. Res. 56, 267-276. Yeager, J. F. and Gahan, J. B. (1937). Effects of alkaloid nicotine on rhythmicity of isolated heart preparations from Periplaneta americana and Prodenia eridania. J. Agric. Res. 55, 1-19. Zawarzin, A. (191 1). Histologische Studien Uber Insekten. I. Das Herz der Acschnalarven. Z. w i n . Zool. 97,481-510.

This Page Intentionally Left Blank

The Hormonal Regulation of Growth and Reproduction in Insects V. B. WIGGLESWORTH

Agricultural Research Council Unit of Insect Physiology, Department of Zoology, University of Cambridge, England I. Introduction . 11. The Neuro-endocrine System.

111.

1V.

V.

VI.

VII.

248 248 A. Histology of the Neurosecretory Cells . 248 B. The Role of the Neurosecretory Cells in Moulting . 249 250 C. The Liberation of the Neurosecretory Product D. Cycles of Activity in the Neurosecretory Cells 25 1 E. The Stimulus to Liberation of the Brain Hormone . 252 F. The Action of the Brain Hormone . 254 G. The Chemical Nature of the Brain Hormone . 256 258 The Thoracic Gland System . A. Anatomy and Histology . 258 260 B. Activation and Function of the Thoracic Glands . C. Metabolic and Cytological Effects of the Thoracic Gland Ho*mok 263 267 D. Moulting Hormone, Mitosis, Growth and Differentiation . E. The Chemical Nature of the Moulting Hormone . 270 271 Hormones and Diapause . 27 1 A. Endocrine Organs in Diapause . B. Physiological Changes during Chilling . 275 C. Injury and Diapause . 277 D. The Nature of the Diapause State . . 218 . 279 E. The Role of Hormones in the Maternal Control of Diapause The Corpus Allatum and the Control of Metamorphosis . 280 A. The Corpus Allatum and Juvenile Hormone Secretion . 280 B. The Effects of the Juvenile Hormone . 283 C. The Mode of Action of the Juvenile Hormone 286 D. Histology and Histochemistry of the Corpus Allatum . 29 1 E. The Chemical Nature of the Juvenile Hormone. . 29 1 Hormonal Control of Reproduction . 296 A. Corpus Allatum and Reproduction . 296 B. Role of the Nervous System and Neurose&eto& Cells in Ovarian Development . 301 C. Control of Ovulation and Oviposition . 306 Metabolic Hormones and Hormonal Integration . 307 A. Metabolic Hormones 308 B. Homeostasis and Hormonal Action 31 1 C. Humoral Integration . 314 References . 316 Addenda . 335

.

.

241

248

V. B. W I G G L E S W O R T H

1. I N T K O I ) I J C T I O N

Many reviews have been published on the regulation of insect growth: the monograph on the physiology of metamorphosis by the present writer (Wigglesworth, 1954a) and a more discursive survey of the same subject (Wigglesworth, 1959a); the detailed books by Pflugfelder (1958) and Novak (1959); the monograph on insect diapause by Lees (1955); and review articles on various aspects of the subject by Van der Kloot (1960) on neurosecretion, Harvey (1962) on metabolism in diapause, de Wilde (1962) on photoperiodism and diapause, Karlson (1963) on the chemistry and biochemistry of hormones. The present review will not cover all this ground again; work published before 1954 will be quoted only when this is necessary for the argument. The subject has reached the point where the comparatively consistent hypotheses which were earlier accepted are being obscured by a multitude of apparent exceptions. We need to distinguish between discrepancies resulting from the varying experimental procedures used, and real differences in the physiology of different species. The objective will therefore be to seek the maximum number ofcommon factor sinthegrowth control system of insects; to define as clearly as possible such differences as do exist; to review current ideas about the nature of the action of the hormones at the cellular level; and in so doing to separate these immediate effects from the secondary consequences of their action. Needless to say, the outcome of this attempt is far from resulting in a definitive account of the action of growth hormones in insects. It represents a mere “stock-taking”-with the formulation of provisional conclusions as a basis for renewed experiment.

I r . THEN E U R O - E N D O C R ISN Y SET E M A . H I S T O L O G Y OF T H E N E U R O S E C R E T O R Y C E L L S

It is now well recognized that the cell body of the neurone in insects, as in other animals, is in a state of more or less continuous secretory activity. The secretory product is the axoplasm; and there is some histological evidence that the strands of axoplasm, each about half a micron in diameter, which are bundled together to form the large axons of insects, are the product of the dictyosomes or Golgi bodies (Wigglesworth, 1960). But nerve cells with distinctive secretory activities, as judged by histological coloration, exist in all the nerve ganglia of insects. They fall into classes recognized by differences in size and in staining properties

liOKh10NI:S I N (IROWTH

A N D REPRODUCTION

249

( C k i w d by Nayar (1955) as types A and B, by Johansson (1958) as types A, 13, Cand I>) which will not bcrcviewcd hcre(seeals0 Highnam,l96lb).

The most characteristic type of these so-called “ neurosecretory cells” elaborates an acidophil product, staining with acid fuchsin or with phloxin, which becomes strongly basophil after permanganate oxidation, and then stains deeply with chromehaematoxylin (Gomori, 1941). It stains strongly also with the paraldehyde-fuchsin of Gomori (Pearse, 1953; Gabe, 1953b; Ewen, 1962b), and moderately, to give a brown tint, with osmium and ethyl gallate (Wigglesworth, 1959b). As was first shown by Thomsen (1954) the neurosecretory cells, and the axons coming from them, when examined under dark-ground illumination, have a luminous blue appearance. This was clearly a “physical colour ” (Tindall’s blue) and indicated that the material was made up of fairly uniform minute colloidal particles capable of scattering the shorter wavelengths of light. More recent electron microscope studies have shown that in all animals thus far examined, including the stick insect Curuusius (Meyer and Pflugfelder, 1958), the neurosecretory material is in the form of perfect minute spheres ranging from about 1 OOO to 3 500 A in diameter (Scharrer, 1962). In the Phasmids, the multilayered Golgi membranes in association with mitochondria and endoplasmic reticulum, seem to be concerned in the formation of these submicroscopic granules, which become aggregated in clusters to form the material visible with the light microscope (Stiennon and Drochmans, 1961). In LRucophueu and Peripluneta the bulbous endings of the nerve axons in the corpus cardiacum form sacs filled with packed granules of this type (Willey and Chapman, 1960). B. T H E ROLE O F T H E NEUROSECRETORY CELLS IN MOULTING

The most conspicuous neurosecretory celIs in the insect lie superficially in the pars intercerebralis of the protocerebrum. These were recognized by Hanstrom (1938) in Rhodnius; and following this demonstration they were proved by implantation to be the source of the hormone which initiates moulting in this insect (Wigglesworth, 1939, 1940b). This observation was of some general interest because it was the first experimental demonstration of a specific function, and an endocrine function, for the neurosecretory cells of any animal. The role of the neurosecretory cells in growth and moulting has been confirmed in a number of insects: by Williams (1946) in Hyulophora cecropia; by Possompbs (1953) in Culliphora; by Nayar (1956), who showed that extirpation of the neurosecretory cells in larvae of Iphitu

250

V. B. WIGGLESWORTH

(Hemipt.) early in the 5th instar prevented their moulting to the adult stage. Williams (1948) had reached the conclusion that in the Hyulophoru pupa, adult development is initiated only if the secretion from the medial group of neurosecretory cells is able to mix with secretion from the lateral group. This conclusion has been supported by Van der Kloot (1960,1961) who burned the neurosecretory cells in the brain of Hyalophoru in situ with a microcautery and found that the hormone was produced as long as one medial and one lateral group remained intact. No evidence of this sort has yet been obtained in other insects; but such an interaction might provide the ruison $&re for the different histological types of neurosecretory cell. Further evidence for the involvement of the neurosecretory cells of the dorsum of the brain in the initiation of growth and moulting will be found under other headings of this section. They will be considered again in relation with reproduction (p. 301). C. THE L I B E R A T I O N OF THE NEUROSECRETORY P R O D U C T

It was later shown by Hanstrom (1940) that the secretion from the neurosecretory cells which lie above the dorsum of the brain in Petrobius maritimus passes down the axons to the corpus cardiacum, which seems to be the main site of release and sometimes of storage. This movement of neurosecretory material was more conclusively proved by Scharrer (1952a) in Leucophaeu maderue: if the nerve to the corpus cardiacum on one side was cut, the neurosecretory substance accumulated above the point of section, and disappeared from the corpus cardiacum on that side. In Bombyx mori the neurosecretory material was visible along the nerves to both corpus cardiacum and corpus allatum (Arvy et al., 1953) and the same was seen in the larva of Leptinotarsa (Arvy and Gabe, 1954). Among other insects, in which transfer along the axons, and storage in the corpus cardiacum have been observed, are Euroleon (Neuropt.) (Arvy, 1956), HyCirous @e Lerma, 1956), the earwig Anisolubis (Ozeki, 1958), Rhodnius (Wigglcsworth, 1956), Schistocercu (Highnam, 1961a, b). Although the product of the neurosecretory cells is conducted to the corpus cardiacum along the nerve axons, it is commonly believed that it then passes through the connective tissue sheath of this organ in a dispersed form and is carried around the body in the circulating haemolymph (Scharrer, 1962). But Johnson (1962) has shown that in Aphids the material from two large neurosecretory cells in the antero-dorsal surface of the protocerebrum is widely disseminated throughout the

HORM0NF.S I N GROWTH A N D REPRODUCTION

25 1

body along nerve axons: nerves leave the corpus cardiacum and run to the aorta and pericardial cells and to various muscles; other neurosecretory axons pass along the nerve cord to the hind-gut. He suggests that direct transport to the tissues may have been overlooked in other insects. Fiiller (1960) in fact noted that in the larva of Corethruplwnipennis stainable neurosecretion can be seen in all parts of the vegetative nervous system (although in this insect it is not visible in the corpus allatum) and two secretory tracts run through the entire ventral cord. In most of these records the neurosecretory material was demonstrated by staining methods. In the adult Calliphoruthe movement of the material along the nerves to the corpus cardiacum, and its arrest and damming back by ligature of the nerves, was clearly shown by Thomsen (I 954) by means of dark ground illumination. If the corpus cardiacum nerves were severed in transplanted brains of Hyulophoru pupae, the cut ends grew out to form a tangled mass of fibres mixed with neuroglia cells, an attempted “regeneration” of the corpus cardiacum (Stumm-Zollinger, 1957). It was noted by Johansson (1957) that extirpation of the ten neurosecretory cells (A cells) of the pars intercerebralis of Oncopeltus did not prevent larval moulting: but it was assumed that the neurosecretory material which, in this insect, is stored in the wall of the aorta, was sufficient for this purpose.

D. C Y C L E S O F A C T I V I T Y IN T H E N E U R O S E C R E T O R Y CELLS

Although the experimental evidence proves that the neurosecretory cells in Rhodnius are liberating their product at or soon after the time of ingestion of the blood meal (Wigglesworth, 1957),it has not been possible to observe distinct histological changes either in the cells themselves or in the corpus cardiacum. This may have been due to inadequate observations. In many insects clear-cut cycles of activity have been reported. In Ephestiu there is a very evident discharge of neurosecretory material in the pars intercerebralis during the critical period of the prepupal stage, and again during early pupal development; not in the older pupa or in the adult (Rehm, 1951). At the beginning of the last larval stage, at the time when neurosecretory material is disappearing from the neurosecretory cells, the corpora cardiaca of Ephestiu show a very large increase in volume (Rehm, 1951); and a similar increase in volume is seen in Bombyx after each burst of secretion from the neurosecretory cells (Arvy et ul., 1953). In Leptinotarsa, also, &heneurosecretory cells show maximal

25 2

V. R. WIGGLESWORTH

activity at prepupation, with a fresh outburst at the imaginal moult (Arvy and Gabe, 1954). In the stick insect Bacillus rossii the secretion accumulates in the corpus cardiacum from the time of moulting until the middle of the intermoult period. It is then discharged into the blood and accumulates again during the second half of the intermoult period (Boisson, 1948). In Carausius during the intermoult period, there seem to be two neurosecretory outbursts in the brain, associated with two invasions of the corpus cardiacum by the product: (i) during the phase of elaboration and (ii) during the phase of discharge (Herlant-Meewis and Paquet, 1956). Thus at the time of moulting the corpus cardiacum contains shrunken cells, but clumps of neurosecretory material remain in the centre of the organ. For 6 7 days thereafter the secretory cells of the corpus cardiacum itself contain large vesicles; at this same time the neurosecretory cells of the brain secrete actively and “reservoirs” in the axons of the corpus cardiacum nerves are filled with large drops. At the middle of the intermoult period (10 days) the neurosecretory cells are reduced; the material staining with paraldehyde-fuchsin is discharged into the aorta. By about 16 days there is a fresh accumulation of neurosecretory substance in the brain (Herlant-Meewis and Paquet, 1956). Likewise in the Lepidoptera, Ephestia, Galleria and Pieris, the most striking accumulation of stainable material in the cytoplasm of the neurosecretory cells takes place after the period of active liberation of the secretion (Rehm, 1955).

It would thus appear that, in general, the periods when the neurosecretory cells are laden with secretion are those in which the hormone is not being liberated. The same conclusion has been reached from observation of the neurosecretory cells during diapause (p. 273) and during reproductive activity (p. 305). E. T H E S T I M U L U S TO L I B E R A T I O N OF T H E B R A I N H O R M O N E

It was carly shown that in the blood-sucking bug Rhodnius, which takes large and infrequent meals of blood, it is the degree of distension of the abdomen which provides the stimulus for liberation of the moulting hormone; and that section of the nerve cord in the prothorax interrupted the conduction of this stimulus to the brain (Wigglesworth, 1934). Van der Kloot (1960, 1961) extended these observations and demonstrated the existence of stretch receptors in the abdomen of Rhodnius (one on each side of each abdominal segment) which lead to the appearance of impulses in the nerves to the corpus cardiacum. These are perhaps the

H O R M O N E S IN G R O W T H A N D REPRODUCTION

253

chordotonal organs which have been observed in this position (Wigglesworth, unpublished). These receptors adapt hardly at all; they continue to discharge as long as the abdomen is expanded. In Locustu migratoriu, Clarke and Langley (1962) could observe no sign of cyclical changes in the medial neurosecretory cells, or in the corpus cardiacurn, during the 3rd. 4th and 5th stages; they suggest that there may be continuous secretion of the hormone; but the appearance of a peak of mitotic activity in the thoracic glands just about the time of ecdysis, suggests that there may be a discharge of secretion from the brain at that point (Clarke and Langley, 1963b). Excision of the frontal ganglion early in the stadium or section of both connectives of the frontal ganglion with the brain, arrests growth and moulting. Section of the recurrent nerve, or of a single connective, is without effect (Clarke and Langley, 1963b). The frontal ganglion has two pairs of antero-lateral nerves which run to stretch receptors in the pharyngeal wall. These authors suggest that stimulation of these stretch receptors during feeding gives a continuous stimulus to the brain which brings about release of the hormone (Clarke and Langley, 1963d, e). It may be that stretch receptors will prove to play a larger part in the liberation of the brain hormone than has been supposed ;but many other factors are involved. The larva of the wheat stem sawfly Cephus cinctur is reluctant to begin post-diapause development once it has been removed from the stub of the wheat straw; hormone secretion is influenced by the ‘‘feel” of the surroundings (Church, 1955). Likewise in the larva of the honeybee, contact with the silk cocoon (or with a similar cell of gauze) provides a necessary stimulus to the brain for the initiation of pupation (Fyg, 1956). Mellanby (1938) recorded similar effects in Lucilia sericatu. In the squash fly Zeugodugus depressus, the high level of carbon dioxide (4-6%) in the cavity of the fresh fruit suppresses pupation, which does not take place until, after 6 or 7 months of storage, the carbon dioxide content has fallen to 0-8-1.4 % (Takaoka, 1960). Low oxygen (2.5%) prevents moulting in Rhodnius, presumably through failure of secretion of the brain hormone (Wigglesworth, 1952b); 1 % or 5 % oxygen has the same effect on the pupae of Bombyx mori (Kobayashi and Nakasone, 1959). High temperature (36°C) prevents moulting in Rhodnius by inhibiting either the secretion or the action of the brain hormone, although all other functions appear normal (Wigglesworth, 1952b). One of the most important factors influencing the liberation of the brain hormone is the regencration that follows injury. If a limb of a Bluttella larva is removed early in the instar, moulting is delayed until the limb has been regenerated (O’Farrell and Stock, 1953, 1954). The

254

V. B. WIGGLESWORTH

nature of this inhibition is unknown. It may perhaps have something in common with the inhibition of moulting which follows the injection into the blood of particulate matter that is taken up by the haemocytes (Wigglesworth, 1955b; cf. Gilbert and Schneiderman, 1961). This type of arrest is certainly due to the failure of the brain to secrete, or to inactivation of the hormone; but the nature of the action is unknown. F. THE ACTION O F THE B R A I N HORMONE

The demonstration of the role of the neurosecretory cells in initiating moulting, substantiated the belief of KopeE (1917, 1922) that the hormone responsible for the pupal moult in the Lymuntriu caterpillar came from the brain. But subsequent work on other Lepidoptera by Hachlow (1931) had indicated the existence of some kind of growth centre in the thorax, the existence of which was proved by Fukuda (1940a, b) when he showed that the “prothoracic glands” in larvae and pupae of Bornbyx were the immediate source of the moulting hormone. Piepho (1942) suggested that the thoracic glands of Galleria might be stimulated to secrete their hormone by the secretion from the neurosecretory cells of the brain ;and that was proved experimentally to be the case by Williams (1947) in the diapausing pupa of Hyulophoru cecropia. The distribution and characters of the thoracic glands and their homologues will be considered in the next section (p. 258). Experiments on other groups of insects have confirmed the foregoing results: that the chief action of the brain hormone of the young stages is to activate the thoracic glands (commonly termed “ventral glands” when they remain in the head; sometimes termed “peritracheal glands” in the young stages of Diptera). That has been shown in Rhodnius (Wiglesworth, 1952a), in Siulis (Rahm, 1952), in Culliphoru (Possompts, 1953), and in Cephus (Church, 1955). In the Culliphoru larva the brain exerts its action upon the thoracic gland (which here forms the sides of the “ring gland ” of Weismann) only when the nervous connection between brain and ring gland is intact (Possompb, 1953). In the other insects the hormone reaches the thoracic gland via the circulating blood. But perhaps this difference is not very great; for in Cull@horuthe corpus cardiacum is an integral part of the “ring gland”, and according to Fraser (1960) the active material, after reaching the corpus cardiacum via the ncrvcs, is then liberated into the blood, as in other insects. The nature of the action of the brain hormone on the thoracic gland has been little studied. It is no brief “triggering” action; the thoracic gland must be exposed to the hormone continuously until the moulting

H O R M O N E S IN G R O W T H A N D REPRODUCTION

255

process is well established. In Rhodnius the first signs of renewed growth within the epidermis are apparent within a few hours after feeding. But the process stops and reverts to the resting state if the head is removed before about 4 days after feeding in the 4th-stage larva, before about 7 days in the 5th-stage larva (Wigglesworth, 1934). These times are what is commonly referred to as the “critical period”. Likewise in the larva of Cephus, the brain continues to influence the prothoracic glands even after they have begun to secrete; and if the production of brain hormone is halted by high temperature (35°C) the larva reverts to the resting state (Church, 1955). On the other hand, in the larva of Philosamia Cynthia the thoracic gland seems to release the pupation hormone continuously once it has been stimulated by the brain hormone (Ichikawa and Nishiitsutsuji, 1952). The suggestion has been made that the neurosecretory product of the brain may perhaps provide the labile raw material from which hormones manufactured by different parts of the endocrine system may be derived (Wigglesworth, 1954b). A similar suggestion has been made by Ichikawa and Nishiitsutsuji-Uwo (1960) that the brain hormone in Philosamia may be converted into the moulting hormone in the thoracic gland. But there is no real experimental evidence to support these ideas. As we have seen, there is histological evidence that the neurosecretory product from the brain finds its way aIong the axons to the corpus allatum also. In Leptinotarsa larvae the corpora allata increase in volume before the final moult, and fine droplets of secretion can be seen in the nerves ramifying within them (Arvy and Gabe, 1954). The same is to be seen in the larva of Bombyx (Arvy ef al., 1953) and of Odonata (Arvy and Gabe, 1952b). And a study of the corpus allatum of Leucophaea with the electron microscope has revealed the axon terminals from the neurosecretory cells with only a thin membrane separating the neurosecretory granules from the corpus aUatum cells (Scharrer, 1962). These observations are of interest because it was found by Williams (1959) that implantation of active corpora allata will sometimes cause diapausing pupae of Hyalophora (in which the brain had been extirpated) to resume their development. The same result was obtained by Kobayashi and Yamashita (1959) when corpora allata from 5th-instar larvae of Bombyx mori were implanted into brainless pupae (“Dauerpupae”) of the same species, and by Ichikawa and Nishiitsutsuji-Uwo (1959) in Philosumia. The Japanese authors interpreted this to mean that one function of the corpus allatum is, like that of the corpus cardiacum, to store and relcasc neurosecretory materials that have originated in the brain.

256

V. B.

WIGGLESWORTH

Certain authors have claimed that the brain hormone can sometimes exert its action of initiating moulting, without the intervention of the thoracic glands. Williams (1947) had got no development in the isolated abdomens of sixty Hya/ophoru pupae after the implantation of brains known to be active. But Ichikawa and Nishiitsutsuji-Uwo (1960) found that brains from larva, pupa or adult of Philosamia Cynthia would cause moulting when implanted into the isolated pupal abdomen. They suggest that the brain hormone may be convertzd to the “moulting hormone” not only by the thoracic gland but by some unknown agency when these glands are absent. And Kobayashi and Burdette (1961) claim that low concentrations of the purified moulting hormone (ecdyson) which alone produce no action on isolated abdomens of Calliphora,will induce pupation if brain hormone is added. They infer that this hormone has a “synergistic action” on the tissues as well as a “tropic action” on the thoracic gland. But these experiments are subject to the doubt that some non-specific stimulus may have set going the growth process. For example, Kobayashi and Nakasone (1 960b) have observed that when brainless pupae of Bombyx mori in “artificial diapause” were exposed to a high oxygen concentration (60 % O2for 8 days) some of the pupae resumed develop ment and became moths within 30 days. And Kobayashi ef al. (1960) noted that although Bombyx pupae deprived of the brain immediately after pupation showed no signs of imaginal differentiation for 3 months at 25”C, all pupae which survived became moths in about 10 months after pupation. Thus, eventually, adult development does take place, in the absence of the pupal brain, if normal corpora allata and prothoracic glands are present. In a few insects, notably Ephemeroptera and Odonata, there is an anatomical connection between the ventral gland (the homologue of the thoracic gland) and neurosecretory cells in the suboesophageal ganglion. It has been suggested by Gabe (1953a) that this may represent a functional connection comparable with the relation between the neurosecretory cells of the protocerebrum and the thoracic gland of Lepidoptera, Hemiptera, Diptera etc.

G . T H E CHEMICAL N A T U R E OF T H E B R A I N H O R M O N E

In a study of neurosecretion in Lepidoptera, Rehm (1955) observed a striking increase in volume of the nucleolus of the neurosecretory cells during formation of the secretory substance. There was no associated

H O R M O N E S IN GROWTH AND REPRODUCTION

257

increase in ribonucleic acid content in the cells; she concluded that the material accumulating was mainly protein. The histochemical characters of the neurosecretory substance are variable. It does not contain detectable lipids (Arvy and Gabe, 1962) but is rich in protein-bound sulphydryl groups which will reduce ferric iron (Sloper, 1957) and which are probably responsible for the staining reaction with osmium tetroxide and ethyl gallate (Wigglesworth, 1963~). In most vertebrate animals the active product of the neurosecretory cells seems generally to consist of small polypeptides associated with “inactive” large protein molecules (Scharrer and Scharrer, 1958). It was therefore a matter for surprise when Kobayashi and Kirimura (1958) reported the extraction with methanol from the pupal brain of B. mori of an oily substance which on injection would induce adult development in “Dauer-pupae” produced by removal of the brain immediately after pupation. They have followed this up with the claim that the purified brain hormone of Bornbyx is in fact cholesterol itself (Kirimura et al., 1962). As pointed out above, the isolated corpus allatum of Hyalophoru (Williams, 1959) or of Bombyx (Kobayashi and Yamashita, 1959) will bring about activation of the thoracic gland in these insects; and the same result can be obtained by the injection of the oily extracts of Hyalophora with juvenile hormone activity (p. 293). That is true not only of the crude extracts, but of the most highly concentrated and purified preparations of juvenile hormone (Williams, 1959). That means that the brain hormone is either the juvenile hormone itself (which seems improbable) or that its solubility properties agree so closely with those of the juvenile hormone that the two keep together throughout the fractionation procedures. Meanwhile the situation has been further complicated by the fact that Ichikawa and Ishizaki (1961) have prepared aqueous extracts from the brain of B. mori (either methanol extracts, later shaken up with water, or direct extracts in 2 % sodium chloride) which are highly active in inducing adult development when injected into test pupae of the same species from which the brain had been removed. The active material is believed to be a protein. Tt is non-dialysable, inactivated by heat (90°C in acid, pH 4-8, or alkali, pH 8.8). precipitated by ammonium sulphate, acetone and trichloroacetic acid. Though not inactivated by pepsin or trypsin it is inactivated by various bacterial proteinases (Ichikawa and Ishizaki, 1963). At the present time the only way of reconciling these apparently contradictory results is to suppose that the active principle from the

258

V. B. W I G G L E S W O R T H

brain may be associated, according to the method of preparation, with either lipid soluble or water soluble carriers. (Berger (1963) found that the neurohormone factor D of Gersch extracted from the brain of Cailiphora will activate the ring gland and induce puparium formation; and it also has a weak effect in the absence of the ring glands, that is, acting directly upon the epidermis. He suggests that this material is the activating hormone from the brain that is concerned in moulting. The results of Cottrell, reviewed in this volume (p. 175), suggest other possible explanations of these results.) The problem of the chemical nature of the brain hormone is still further complicated by the fact that the neurosecretory product from the brain is important in reproduction in the adult insect (p. 297). There is at present no evidence that the secretion from the adult brain will activate the thoracic glands of the young insect (e.g. in Rhodnius: Wigglesworth, 1963b) but little exact work has been done along these lines. Granules of different types arc visible in the neurosecretory cells of the glow-worm Lampyris noctiluca and some attempt has been made by Naisse (1962) on histological grounds to ascribe different functions to these products. Some go to the corpus allatum which appears to be activated by them; most go to the corpus cardiacum where they are said to mix with the secretory product of the corpus cardiacum cells, and then pass through the bounding membrane to the haemolymph. 111. THETHORACIC G L A N DSYSTEM A. A N A T O M Y A N D H I S T O L O G Y

What may be conveniently called the “thoracic gland” takes its origin, as was shown by Toyamo (1902) in the embryo of the silkworm, from an ingrowth of ectodermal cells at the base of the second maxilla. In some insects, such as Thysanura (Gabe, 1953c), Ephemeroptera (Gabe, 1953a), Odonata (Gabe, 1Y53a), Tsoptera (Pflugfelder, 1947) Dermaptera (Ozeki, 1959), Acridiidae (Strich-Halbwachs, 1959), Phasmida (Pflugfelder, 1938), the glands remain as compact structures in the lower part of thc head and are called “ventral glands”. Whereas in other insects, such as Blattidae (Bodenstein, 1953), Hemiptera (Wigglesworth, 1952a), Coleoptera (Srivastava, 1960; Nliiiez, 1954) Lepidoptera (Lee, 1948), Hymenoptera (Fyg, 1956), the cells are carried backwards into the thorax, perhaps by the growth of the salivary glands (Wells, 1954), and here the components form loose chains of cells or lace-like meshworks. In the larval stages of the higher Diptera the corresponding

H O R M O N E S IN GROWTH A N D REPRODUCTION

259

cells remain in close association with the corpus allatum and corpus cardiacum, immediately behind the cephalic lobes, to form the lateral arms of “Weismann’s ring” around the aorta (Thomsen, 1942; Vogt, 1943;Possompks, 1953). In Lepidoptera the thoracic glands are closely connected with the tracheal system ; in Rhodnius, where they form a single layer of cells, dispersed over the surface of certain lobes of the fat-body, they have an exceedingly rich supply of tracheae, in striking contrast to the surrounding fat body (Wigglesworth, 1952a). In the honeybee the thoracic glands are effective only when supplied with abundant air (Fyg, 1956). But in the Carabid beetle Anisotarsus they are not richly supplied with tracheae (Ndiiez, 1954). It has often been pointed out that the tissues must be exposed to an adequate supply of oxygen if growth processes are to occur. interruption of tracheal supply may be responsible for some of the effects of ligation experiments (Fraenkel, 1935; Takaoka, 1957,1959; Bodenstein, 1939; Wigglesworth, 1952b). The nerve supply to the thoracic glands is curiously variable. It is very conspicuous in Lepidoptera (Lee, 1948), Blattidae (Scharrer, 1948a), Acridiidae (Pflugfelder, 1958); but appears to be entirely absent in Rhodnius and many other Hemiptera (Wigglesworth, 1952a; Wells, 1954)and in Coleoptera (NGiiez, 1954).On the other hand a nerve supply has been demonstrated in the alfalfa plant bug Adelphocoris lineolutus (Ewen, 1962a). No physiological differences have been found to be associated with these differences in nerve supply. The cells of the thoracic gland undergo a conspicuous cycle of secretory activity during each instar, when exposed to the brain hormone. In Rhodnius, for example, the nuclei become greatly enlarged and lobulated during the “critical period” when moulting is beginning; the cytoplasm also becomes more extensive and more basophil. Later in the moulting stage, and well before the old cuticle is shed, the cells revert to their resting condition (Wigglesworth, 1952a). In the thoracic gland of Anisuf a r . wthcrc is likewise increased basophil staining and vacuolation during the critical period (Nriiiez, 1954), as had indeed been observed by Vogt (1 942a) in the lateral cells of the ring gland in Drosophila (cf. Tenebrio (Srivastava, 1960)). In Ephmiu, a s the larva approaches maturity, the cells of the thoracic gland become dktcnded and large, mainly as the result of increase of cytoplasm. Radial striations appear in the cytoplasm, with elongated secrctory droplets between them (lchikawa and Nishiitsutsuji, 1955b). In the larva and pupa of Bombyx mori the secretion, which is produced about two-thirds through the moulting stage, is positive to periodic

260

V. B. WIGGLESWORTH

acid-Schiff (PAS) staining and “seems to be a mixture of mucoprotein, glycoprotein, glycolipids, unsaturated lipids and phospholipids” (Kobayashi, 1956). In fact this author concludes that the hormone seems to be contained “in a mucus-like substance”. B. ACTIVATION A N D FUNCTION O F THE THORACIC GLANDS

As described in the previous section (p. 254) the thoracic gland is normally activated by the brain hormone. But in experiments on Rhodnius (Wigglesworth, 1936) it was shown that a decapitated 4th-stage larva, induced to start moulting by joining in parabiosis to a similar larva which had reached the critical period, would itself induce moulting when separated from its first partner and joined to another decapitated larva. This was interpreted as meaning that the moulting tissues could themselves provide a humoral stimulus to moulting. At that time, however, the role of the thoracic glands in moulting was not known. Williams (1952) carried out this type of experiment in a far more elegant manner by joining headless pupae of Hyalophoru in series and implanting an active brain into the first pupa. These experiments showed quite clearly that once the thoracic gland of one pupa had been activated, it could activate the gland in the next pupa, and so on for the whole chain of six or more pupae, all of which were caused to moult. If theexperiment was repeated with a chain of isolated abdomens, this did not happen: only the next adjacent member of the chain was caused to moult. Thus, although the thoracic gland cells are normally activated by the hormone from the brain, they can be brought into action by the secretion from other thoracic glands. Experiments by Possompis (1953) on the larva of Culliphoru substantiated the earlier work of Burtt (1938) on CulZiphoru and of Vogt (1942a) on Drosophiln, in showing that extirpation of the thoracic gland (“peritracheal gland”) results in persistent larvae which fail to pupate; and that implantation of the thoracic gland into such permanent larvae induces pupation and further development. The role of the thoracic glands in Lepidoptera as the immediate source of the hormone inducing moulting in the larva or pupa, was first proved by the implantation experiments of Fukuda (1940a, b, 1941) on the silkworm. This was confirmed by Williams (1947) in Hjwlophoru. Extirpation of the ventral glands in Locustu before the critical period causes permanent arrest of moulting (Joly et ul., 1956); and implantation of the corresponding glands in the earwig Anisdubis (Ozeki, 1959, 1960) proved them to be the source of the moulting hormone. Likewise in

H O R M O N E S IN G R O W T H A N D REPRODUCTION

26 1

Aeschna, implantation of ventral glands will induce moulting in larvae rendered “permanent” by their previous extirpation (Schaller, 1960). Further evidence for this function will be considered in connection with diapause (p. 271). Piepho (1948) obtained moulting in Galleria larvae and pupae in which the thoracic glands were considered to have been completely extirpated; and Chadwick (1956) got similar results in Periplaneta, which led these authors to conclude that the thoracic gland could not be the only source of the moulting hormone. In these experiments the complete removal of the gland was established with great care; it will therefore be well to bear these results in mind. Strich-Halbwachs (1959) confirmed that extirpation of the ventral glands in Locusta retards or suppresses moulting, while implantation of glands will accelerate moulting. But she failed to induce moulting in “permanent larvae”, from which the glands had been removed, or in adults. On the other hand, in Rhodnius, both permanent (decapitated) larvae, and adults, can be caused to moult by implanting thoracic glands removed from moulting larvae during the height of their secretoryactivity (Wigglesworth, 1952a). When injecting the posterior halves of ligatured larvae of CuZZiphora, as a method of assay in the isolation of the moulting hormone, Karlson and Hanser (1953) used as their unit dose the amount of material needed to induce puparium formation in larvae ligated near the critical period. These larvae had doubtless secreted already almost enough hormone to induce puparium formation; the test dose was needed only to complete the process. But if the injection was delayed, “activation” of the tissues was gradually lost, and then large doses of the hormone were needed. Corresponding observationshave been made on Rhodnius. Implantation of active glands, or injection of hormone extracts, has usually been done in larvae decapitated at one day after feeding. But by that time a certain amount of hormone has already been secreted. The activated epidermis reverts to the dormant state in larvae kept decapitated for a week or so (Wigglesworth, 1955b). The hormone of the thoracic gland is commonly called the “moulting hormone”, and this is probably the most appropriate term for it. The epidermis of the insect secretes over its surface a continuous cuticle to which the cells remain attached. Growth in insects therefore takes place in cycles, during the periods immediately before a new cuticle is laid down. Between these periods the epidermal cells remain dormant. They are aroused from this state of dormancy by the moulting hormone when the cuticle has to be renewed. In Rhodniur it is the epidermal cells and the

262

V. B. WIGGLESWORTH

cells which make up the intersegmental muscles of the abdomen which are activated by the moulting hormone. The growth of the fat body, for example, shows normal growth changes in the absence of the hormone (Wigglesworth, 1963b).The moulting hormone is not an essential element for catalysing growth in general. It is a “messenger” which calls forth the appropriate growth responses in cells which otherwise would be expected to remain dormant. Bodenstein (1957) noted that the imaginal discs in Drosophila continue to grow during the larval intermoult periods. He ascribed this to the presence of thoracic gland hormone at a titre sufficient for growth of the discs, but inadequate for the induction of moulting. Likewise Eassa( 1953) observed that mitotic activity in the epidermis of Lepidoptera larvae is cyclic, but the discs seem to grow continuously. Bodenstein even goes so far as to suggest that the growth response of cells around a wound in the epidermis of Rhodnius is dependent on a low titre of thoracic gland hormone. But since this response occurs equally . well in the adult insect which has no thoracic glands that cannot always be so (Wigglesworth, 1937).

It is possible to argue that the raison d‘&e of the imaginal discs of holometabolous insects is to relieve these parts of the body from the necessity of taking part in cuticle formation at each moult, and thus to free them from the cycles of growth that are tied to moulting and cuticle formation, and to enable them to continue their growth independently; and that means independently of the moulting hormone. But it should be recalled that Bodenstein (1946) found that the imaginal discs of the last larval stage of Aedes are unable to grow and differentiate if implanted into the adult. (We shall discuss further examples of the independent growth of imaginal discs later, under regeneration.) The growth of insect tissues in the absence of the moulting hormone is most obvious in embryonic development. In the egg of Locusiana pardalina the burst of mitoses at the end of diapauseisinitiated before the ventral head glands are formed. The ventral glands come into action, and show their maximum activity at the time when the embryonic cuticle is being moulted. Indeed, ligaturing experiments prove the need for the secretion of the ventral glands in initiating the embryonic moult and for the full activity of the pleuropodia, which are needed to complete the moulting process that is ended at hatching (Jones, 1956). Although the primary function of the thoracic gland hormone is the induction of the moulting process, it may be responsible for bringing about other changes which precede moulting in certain insects. The most

HORMONES I N GROWTH A N D REPRODUCTION

263

familiar cxamplc is the hardening and darkening of the larval cuticle of higher flies to form the puparium before moulting begins (Fraenkel, 1935). The catcrpillar C’c~rirruvinukr changes colour from green to’dark red when it ceases to feed and migrates down the tree trunk before preparing the cocoon. This change results from the accumulation of a red ommochrome pigment in the epidermal cells and later in the fat body, and it is brought about by small amounts of the thoracic gland hormone. 66 “CuNiphoru units” of the purified hormone “ecdyson” will cause reddening of the epidermis; 330 units reddening of the fat body; whereas 3 000-6 OOO units will induce the pupal moult in a ligated larva directly without any colour change (Biickmann, 1959). C. M E T A B O L I C A N D C Y T O L O G I C A L EFFECTS O F T H E THORACIC GLAND HORMONE

During the pupal stage of insects the course of oxygen consumption follows a U-shaped curve. This curve is associated with a corresponding curve in the level of oxidative (including dehydrogenase) enzymes (for literature, see Zwicky and Wigglesworth, 1956; Sulkowski and Wojkczak, 1958; Ludwig and Barsa, 1959a, b). Agrell(l952) was inclined to consider the co-rse of enzyme concentration as being the cause of the U-shaped curve of oxygen uptake. But the oxygen consumption of the body at any time is determined by metabolic activity-whether this be muscular or secretory activity on the one hand, or the energy required for endothermic chemical syntheses on the other. The level ofenzymespresent is commonly adapted secondarily to the metabolic needs (Zwicky and Wigglesworth, 1956; Wyatt, 1962). Williams and his colleagues (see Williams, 1951) studied the cytochrome system during the change from dormancy to active development in the overwintering pupa of Hyulophoru cecropia. Not only was the cytochrome system very small in amount in most of the tissues of the dormant insect, but cytochrome c appeared to be completely absent and the insect showed extraordinary tolerance to carbon monoxide and to cyanide. Soon after the renewal of growth cytochrome c reappeared and the usual sensitivity to cyanide was restored. It was therefore suggested that synthesis of cytochrome c was a key process initiated by the moulting hormone. More recently it has been shown that respiration, even in the dormant Hyulophora pupa, is mediated by cytochrome oxidase; the apparent resistance of the system to inhibitors is due to the great excess of cytochrome oxidase in relation to the small amount of cytochrome c present (Shappirio and Williams, 1957; Harvey and Williams, 1958; Kurland

264

V.

B. W 1 L G L f . S W O R T I . I

and Schneiderman, 1959). Morcovcr, nicchanical injury to the dormant pupa will riil\C: the metabolic activity t o ;L high level and cause extensive cytochromc synthesis, but it docs not induce adult development (Schneiderman, 1953). Thc hypothesis that the synthesis of cytochrome c is the essential change that restores growth in the dormant insect has therefore been abandoned (Shappirio, 1960; Harvey, 1962). The rising co~isumptioriof oxygen which accompanies the moulting process is doubtless due to many causes (Slama, 1960; Janda, 1961). But it must be due mainly to endergonic syntheses-partly of food material for storage, largely of the materials needed by the growing body. When the cytological changes during the earliest stages of moulting are observed in the epidermal cells of the Rhodnius larva, they follow a constant pattern. Enlargement of the nucleolus is detectable within a few hours after exposure to the hormone; this is soon followed by enlargement and multiplication of the mitochondria (which must indicate a synthesis of cytochrome components and other enzyme systems) and the appearance of ribonucleic acid (RNA), especially around the nucleus (Wigglesworth, 1957, 1963b). These changes of nucleolar enlargement, mitochondria1 increase, and RNA synthesis, which are commonly rcferred to as “activation”, point to an active renewal of growth with the synthesis of protein. It was therefore suggested that the prime effect of the moulting hormone is the restoration of protein synthesis in thosc tissues that are concerned with growth (Wigglesworth, 1957). Similar observations have been made by others. In discussing the re-synthesis of oxidative enzymes during metamorphosis, Agrell (1952) pointed out that one limiting factor could be the protein component of the enzymes to be incorporated into the mitochondria. Wyatt (1958, 1959)found that the ratio RNA/DNA in the diapausing Hyalophora pupa was 5.6. It increased to 7.4 during early adult development. This indicated an early synthesis of RNA in response to the moulting hormone. Later the ratio fell once more as DNA was synthesized in preparation for mitosis. Krishnakumaran (1961) observed the accumulation of RNA in the cytoplasm and nucleolus of Gryllus at the time of action of the thoracic gland hormone. Moultingin the larva of Rhodnius is initiated by the meal of blood. This has three immediate effects: stretching of the abdomen, nutrition of the tissues, and liberation of moulting hormone. When these effects are separated expcrimentally (Wigglesworth, 1963b)it can be shown that abundant nutrition alone has almost no effect on the cells of the epidermis and of the intersegmental muscles (the tissues actively concerned in moulting); these

H O R M O N E S I N G K O W T H A N D REPRODUCTION

265

cells show no signs of activation. But the cells of the fat body show identical changes in ribonucleic acid synthesis and mitochondria1 increase whether they are exposed to nutrition alone or nutrition plus hormone. The renewal of protein synthesis is certainly one of the earliest effects detectable in those cells that are concerned with moulting. But it cannot be regarded as a process for which the moulting hormone is always necessary: that is true only of the epidermal cells and some other cells that are concerned in moulting; the effect of the moulting hormone is to arouse these particular cells from their dormant state and thus bring about the renewal of growth. Results pointing to precisely the same conclusion have been reported by Telfer and Williams (1960) in the dormant pupa of Hyalophora. Glycine labelled with lacis incorporated into protein four times more rapidly when injected into developing pupae than it is in dormant pupae. Similar results were obtained with labelled glycine in Sphinx ligustri pupae (Bricteux-GrCgoire et al., 1957) and with labelled leucine in Saturniid silk moths (Stevenson and Wyatt, 1962). The Iow rate of incorporation in the dormant insect parallels the low rate of respiratory metabolism. This supports the view mentioned above (Zwicky and Wigglesworth, 1956) that the oxygen uptake in the developing pupa is a measure of the supply of energy for endergonic syntheses. If the dormant pupa is injured (by excision of the epidermis or the brain) this causes a prompt increase in both respiratory rate and glycine incorporation (Harvey and Williams, 1961), or leucine incorporation (Stevenson and Wyatt (1962), up to levels equal to or above those produced by development. As Telfer and Williams (1960) point out, inability to synthesize protein is clearly not the cause of the persistence of dormancy. But what is clear from the observations on Rhodnius summarized above is that it is not the capacity for protein synthesis in the body as a whole that is important, but the activation or lack of activation in those tissues responsible for growth and moulting. We still lack knowledge of the nature of the change brought about by the moulting hormone. One suggestion (Wigglesworth, 1957) was that the hormone might act by influencing permeability relations within the cells and thus allowing access of enzymes to their substrates. A similar view is put forward by Telfer and Williams (1960). During the past few years the belief has been gaining acceptance that the moulting hormone may be acting, not upon some cytoplasmic mechanism but upon the nuclear genes themselves. According to Becker (1962) the hormones can act in two ways : (i) they may trigger the activation of the whole accessible gene pool of a cell, as defined by its state of

266

V.

B. W I G G L E S W O R T H

differentiation, or (ii) the milieu, that is, perhaps, the relative amounts of different hormones present, may select to a certain degree from the available gene pool. This conception of hormone action is based partly on the suggestive model of induced enzyme formation developed by Jacob and Monod (1961) from their extensive studies on bacteria (Clever, 1962a)and partly on direct observation of the activation of particular genes or groups of genes by the hormone (Clever, 1961a, b, 1962a, b, c, 1963; Becker, 1962). Jacob and Monod (1961) distinguish between “structure genes” which convey “information” by means of “messenger RNA” from the chromosomes to the synthetic system in the cytoplasmic ribosomes, and “regulating genes” which control the activity of the structure genes by means of “repressor substances”. Such genes may either facilitaterepressor action, or they may hinder it and thereby act as “inductors”. In this way, materials can control gene activity without themselves taking any part in cell metabolism. Although, as applied to multiellular organisms, this cell model is almost wholly hypothetical, it provides such a helpful tool for thought that it is in some danger of being accepted almost too readily. So far as direct observation of gene activation is concerned, it was shown by Clever (1961a, b) that the larva of Chironornus can be caused to moult into the pupa early in the final instar by injection of the purified moulting hormone ecdyson. The moulting process, whether occurring naturally, or whether induced precociously in this way, is characterized by a succession of “puffs” appearing at different loci of the polytene chromosomes, each at a particular stage of development. After the injection of ecdyson the first recognizable reaction is a new puff which appears within 15-30 min in the region 18-C of chromosome I. The enlargement of this puff is complete within 2 h, and the size of the puff (a measure of the activity of the gene concerned) is determined by the hormone concentration in the haemolymph. Indeed, the size of this puff in the normal insect can be used as an index of hormone concentration at any given period. Clever therefore suggests that the primary action of the moulting hormone is to activate a small number of specific genes, or perhaps the single gene corresponding to the locus 1-184, which can be activated by ecdyson in precisely the same manner in the chromosomes of salivary glands, rectum, and Malpighian tubules. Several other puffs, not specific for development, become active at a later stage; indeed a whole chain of gene activities in the salivary gland chromosomes is initiated by the moulting hormone.

H O R M O N E S IN GROWTH A N D R E P R O D U C T I O N

267

The visible effect of ecdyson in inducing the puff I-18-C in the salivary glands can be brought about by a very small amount of the hormonesomething like 10 molecules of ecdyson per haploid set of chromosomes As judged by the size of the puffs, Clever (1963) estimates (Clever, 1962~). that a late prepupa of Drosophifuprobably contains lo-%pg of ecdyson per mg of larval weight, or even more. It is worth noting that this same quantity (lOpg/g) is the smallest amount of crystalline ecdyson which will induce moulting in Rhodnius (Wigglesworth, 1955b). By ligaturing Drosophifu larvae at the appropriate moment, Becker (1962) has shown that only those salivary gland cells that are exposed to the moulting hormone undergo puffing. The hormone seems to act on the genes direct and to activate the complete gene pool that is needed for temporary salivary gland function. On the other hand, if the salivary gland of an early prepupa is transplanted to the abdomen of a third instar larva, the puffing pattern of the implant is quite different and shows the characters of the host. Likewise, Karlson (1963) interprets the action of ecdyson in inducing tanning of the puparium of higher flies as an effect at the gene level resulting in synthesis of the necessary enzymes. Further evidence on the effect of the moulting hormone on metabolism, and on the relation between the moulting hormone and wound “hormones” will be considered under the heading “ Hormones and diapause” (p. 271). D. MOULTING HORMONE, MITOSIS, G R O W T H A N D DIFFERENTIATION

The most obvious effect of the moulting hormone is to induce “activation” in the epidermal cells as described in the preceding section. This same activation can be induced by chemical products diffusing from injured cells, the so-called “wound hormones”. The mitosis that occurs around a wound is a consequence of the migration of epidermal cells to the site of the injury; for this creates a surrounding zone in which the cells are unduly sparse; and the stimulus to mitosis was shown to be the “mutual separation of activated cells” (Wigglesworth, 1937). Precisely the same relation is seen in the epidermal cells of Rhodnius when these are activated by the moulting hormone (Wigglesworth, 1W b , 1963b). If the insect is unduly stretched by occluding the anus after feeding, there is excessive mitosis in the epidermis. If the insect is induced to moult without feeding, so that the cells are closely packed, moulting may occur with almost no mitosis at all. It is evident that the moulting hormone cannot be described as causing mitosis directly. Mitosis occurs in the absence of the moulting hormone during wound

268

V. B. W I G G L E S W O R T H

healing (Wigglesworth, 1937), in the early embryo of Locustuna (Jones, 1956) and in thedevelopment of the ovaries, which continues in “permanent larvae” of Locusts after removal of the ventral glands (StrichHalbwachs, 1959). In the preceding section it was pointed out that the cells of the fat body in Rhodnius become activated by nutrition alone; if the activated cells are widely separated by the stretching of the abdomen they also will go on to mitosis in the absence of the moulting hormone; for example, after decapitation (Wigglesworth, 1963b). The abdomen of Rhodnius is in some ways a special case, since the cuticle is extensible and the density of nuclei will vary with the degree of distension of the abdomen. The situation will be different when the epidermal cells are attached to an inextensible sclerotized cuticle. In Locustu, for example, the number of nuclei was found to be 7 811 per mm2 before the burst of mitosis 3 - 4 days after moulting; after mitosis there were 12 180 per mm2 (P. Joly, 1955). Here it seems that a wave of mitosis is an integral part of the moulting process. Strich-Halbwachs (1959) regards the ventral glands in Locustu as inducing moulting, acting on the detachment of the cuticle and the rhythm of cellular divisions in the epidermis, but as affecting neither “growth” nor “differentiation”. The relation between moulting and growth is a subject that has been little studied in recent years. According to Dyar’s law and Przibram’s rule the increasc in size of the body, and so of the cuticle, follows certain empirical laws. But there are many circumstances under which these laws break down. When the insect is poorly nourished the amount of growth achieved at a particular moult is reduced. This adjustment of growth to the needs of the body as a whole is an example of the homeostatic character of growth and moulting which will be considered later (p. 31 1). Much more consideration has been given to the question whether the moulting hormone controls the diffcrcntiation of the tissues. B. Scharrer (1948b, 1952b) has gone so far as to describe the moulting hormone as the “growth and differentiation” hormone, and some authors have referred to it as the “metamorphosis hormone”, regarding it as responsible for the differential growth of different parts of the body which characterizes metamorphosis. It is now generally recognized, however, that a single substance acting upon a uniform substrate cannot produce differential growth; the capacity for differentiation is a property of the tissues themselves. This inference is supported by experimental observations. An injection of ecdyson into nymphs of Kuloterincs will cause moulting to take place in the absence of growth. They give rise to normal nymphs again; there

IiORMONES

IN G R O W T H A N D R E P R O D U C T I O N

269

is no “differentiation” (Liischer and Karlson, 1958). But the most striking evidence is afforded by regeneration. If a limb is amputated in a young larva of Bluttellu it is regenerated; and during regeneration the secretion of the moulting hormone is inhibited (O’Farrell and Stock, 1953, 1954). If the imaginal discs of the hind wings in an Ephestia larva are extirpated, pupation isdelayed for 1Odays.And if the brain and thoracic gland are extirpated at an early stage along with the wing discs, a “permanent” larval stage is produced, but the wings are regenerated as usual. If the wing discs are removed at a later stage in moulting, pupation occurs without any regeneration. It is therefore evident that regeneration resembles wound healing: it does not require the presence of the moulting hormone (Pohley, 1961). Indeed, during the period of regeneration the activity of the brain and thoracic gland is suppressed. There is, however, another way in which the moulting hormone does have a morphogenetic effect on differentiation. In an early discussion on the mode of action of the juvenile hormone in inhibiting metamorphosis (Wigglesworth, 1936) it was suggested that its action consisted in accelerating the deposition of the new cuticle and thus limiting the amount of differentiation towards the adult stage which could take place at that moult. The juvenile hormone was supposed t o sustain the status quo by precipitating cuticle formation. Experiments were described in support of this interpretation. If Rhodnius larvae of the 4th-instarYat a fairly advanced stage in moulting (7 days after feeding) were joined to the abdomens of other 4th-instar larvae, at 24 h after feeding, these younger insects were caused to moult unduly rapidly, and they developed characters in wings and genitalia which were intermediate between those of the 4th and 5th instar. These effects were attributed to the juvenile hormone (Wigglesworth. 1952b). But Halbwachs et al. (1957) obtained the same results in Locusta by implanting vcntral glands at the beginning of the 4th instar. Moulting was accelerated, the instar was shortened, and there was a consequent reduction in the length of the femur of the mctathorax, and of the pterothecac. Since the growth of the pterothcca is strongly allometric, its size is very greatly reduced by this acceleration. The principle o n which the earlier experiments on Rhodnius were interprcted appears to be correct. but the hormone responsible for the accelerated deposition of the cuticle is the moulting hormone and not the juvenile hormone. In Lepidoptera it has been shown that large overdoses of purified moulting hormone (ccdyson) lead simiIarly to excessively rapid and atypical development, the new cuticle being secreted before bristles, scales or primordia of genitalia are fully developed (Williams,

270

V. B. W I G G L t S W O R T i i

see Karlson, 1956). We shall return to this topic when discussing the role of the juvenile hormone in the control of metamorphosis (p. 286). E. f H E C H E M I C ‘ A L N A T U K I : O F T l I E M O U L T I N G H O R M O N E

Methods for isolating the active factor which will induce puparium formation in the posterior halves of ligatured Culliphoru larvae were worked out by Butenandt and Karlson (1954). The same methods applied to silkworm pupae eventually gave a crystalline preparation of what appeared to be the same material. The biological unit adopted was the quantity of material that would induce puparium formation in the isolated abdomen of the Calliplioru larva, the so-called “ Culliphora unit”. This was contained in about 0-0075pg of the crystalline material. About 5 mg, or some 600000 Culliphora units, were obtained from 100 kg of Bornbyx pupae. This material, named “ecdyson ”, was believed to have a molecular weight of 310 and an empirical formula of C,,H,,O,. It appeared to be a two-ringed structure, an unsaturated ketone, with two or more hydroxyl groups, one ethyl group and probably one methyl group (Karlson, 1959). As obtained from Bornbyx pupae it contains two components termed a-ecdyson and 8-ecdyson, both of which are active, though p-ecdyson is considerably less so. They diKer notably in their partition coefficients, p-ecdyson being much more lipid soluble (Karlson, 1956). The application of X-ray crystallographic methods directly to the crystals of pure ecdyson have recently enabled W. Hoppe to modify these conclusions; and Karlson has supported the new results by classical chemical means. It now appears that the molecular weight is 464 and the empirical formula C,,H,,O,. Considering that a keto group and a double bond in conjunction with it are present and in the molecule, the composition of the fundamental saturated substance was taken to be C27H48 and ecdyson is therefore probably a steroid (Xarlson, 1963). The structure is not yet fully known ‘but Karlson (1963) suggests the following as a partial representation; the position of the remaining four hydroxyl groups being unknown. 0

-l

J It seems likely that this substance will be derived from cholesterol;

HORMONES IN G R O W T H A N D R E P R O D U C T I O N

27 1

and Karlson suggests that the cholcsterol extracted by Kobayashi and his colleagues (p. 257) from the brain of the silkworm may have appeared to have moulting hormone activity because it served as a precursor substance for ecdyson synthesis. Ecdyson is non-specific in its action. Besides causing puparium formation in Calliphora, and continued development of the pupa, it will act on the diapausing pupa of Hyalophora cecropia: 5pg/g is needed to initiate development; lopgig for complete development (Williams, in Butenandt and Karlson, 1954). 0.25pg will activate the epidermal cells in the decapitated 4th-stage larva of Rhodnius (weight about 80 mg) and will lead to the development of striated muscle fibrils in the vzntral abdominal muscles. 0.5pg causes more development, but growth in the epidermis comes to an end before the new cuticle is laid down. 0.751.Opg will lead to complete formation of the new cuticle. This is a dose of about 10 pg/g (Wigglesworth, 1955b). Since purified ecdyson will bring about puparium formation in Calliphora larvae deprived of the ring gland (Karlson and Hanser, 1953) and will induce moulting in the isolated pupal abdomen of Hyalophora (Williams, in Butenandt and Karlson, 1954) and in the isolated abdomen of Rhodnius, it is believed to be the product of the thoracic gland. But it is rather surprising that it can be extracted from adult female silkworm moths (in which the thoracic gland has disappeared) in quantities about one quarter of those obtained from the pupa (something like 16OO00 Calliphora units, or about 1.3 mg, being got from 100 kg) (Karlson and Stamm-MenCndcz, 1956). a- and p-ecdyson have been similarly extracted from adult locusts Dociostaurus (Stamm, 1959). Since the cytological effects of the chemical factors from injured cells (“wound hormones”) are identical with those of the moulting hormone, it will be well to bear in mind the possibility that the injured cells may be liberating a substance that is similar to that produced by the thoracic glands; it is even possible that some of the active “moulting hormone” obtained in extracts may have come from the tissue cells throughout the body. Nothing is known about the chemical nature of the wound hormones of insects. Of the various materials tested the most active in reproducing the wound effect were certain commercial samples of proteose (Wigglesworth, 1937). IV. HORMONES A N D DIAPAUSE A. E N D O C R I N E O R G A N S I N D I A P A U S E

The unfed larva of Rhodniusis in a state resemblingdiapause; and larvae

272

V. B. W I G G L E S W O R T H

which have received too small a meal to provoke secretion of the moulting hormone, or have been deprived of the neurosecretory cells of the pars intercerebralis by decapitation, are in a similar state. These observations prompted the suggcstion that the immediate cause of natural diapause was probably a lack of moulting hormone (Wigglesworth, 1934). This idea was confirmed by the clear-cut experiments of Williams (1946) on the diapausing pupa of Hyalophora cecropia. Since then the study of the changes which characterize the onset and termination of diapause has thrown much light on thc hormonal control of growth. Much of this work has already been discussed in connection with the metabolic effects of the moulting hormone (p. 263). In most non-diapausing Lepidoptera either the brain and the thoracic glands remain active in the pupa, or the brain may cease producing its secretion, but the thoracic glands retain their activity. In either case adult development proceeds in the pupa from which the brain has been removed. On the other hand, i n the polyvoltine Arctius sclene the brain is active and the thoracic glands inactive. Hence if the brain is removed immediately after pupation, the pupae are caused to enter diapause (Williams, 1952). Likewise in Luciliu, the thoracic glands are inactive during diapause owing to failure of activation by the brain. Growth of the nuclei halts and the gland cells shrink; and at the same time development of the imaginal rudiments ceases (Fraser, 1960). In the larval diapause of the rice stem borer Chilo (Fukaya, 1955) and in the pupal diapause of Luehdorfa japonica (Ichikawa and Nishiitsutsuji, 1955a) the arrest is ascribed solely to the dormant state of the brain. In the over-wintering larva of the wheat stem sawfly Cephw cinctus, visible imaginal development continues very slowly even during diapause. For example, spermatogonial mitoses continue to take place more rapidly at 10°C than at temperatures rather higher or rather lower. This change runs parallel with “diapause development” (p. 278). But the moulting hormone must be present (or present in high concentration) before meiosis and spermatid formation can occur. If moulting hormone is absent thc spermatocytes are sloughed off and replaced by new ones from other spermatogonia (Church, 1955). These observations recall the extensive growth and maturation observed by Schneider (1950,1951) in the imaginal tissues of dormant Syrphid larvae. In Cephus larvae the brain hormone is needed to sustain the thoracic glands even after they have begun to secrete (cf. Vogt (1942a) in Drosophila; Ketchel and Williams (1953) in Hyulophora ; Wigglesworth (1955b) in Rhodnius). And if the secretion is insufficient, a high temperature (35°C) will cause the post-diapause larva to revert to diapause.

I I O K M O N L S IN C R O W T I { A N D R E P R O D U C T I O N

273

This heat treatment halts thoracic gland activity and the gland returns to the inactive state (Church, 1955). Lees (1955) has pointed out that in the “artificial diapause” produced by removing the brain in full-grown larvae of Lyntuntriu (Kopec, 1922), the larvae survive only 31 days. They seem to lack the mechanism required for reducing metabolism to a low level; whereas the unhatched larva (the diapause stage in Lymuntria) has this faculty and survives as long as 14 months. But this difference may be one of physical activity and waterproofing, combined with a high levcl of “injury metabolism” (p. 277) in the insects with the brain removed. Decapitated Rhodnius can survive for more than a year. What is the nature of the arrest of activity in the neuro-endocrine system? In the diapausing larva of the European corn borer Pyrausta (Ostriniu) nubilalis the neurosecretory cells contain stored secretion ; but there seems to be some “barrier”, perhaps the absence of impulse transmission in the neurosecretory axons, which prevents the escape of the hormone. If diapausing brains are taken out and implanted into diapause larvae, diapause is terminated in the recipients and they promptly go on to pupation (Cloutier et al., 1962). In the dormant pupa of Hyalophora, the secretory arrest in the twentysix neurosecretory cells is associated with far-reaching physiological changes in the brain. Spontaneous and evoked electrical activity are absent and the brain is quite inexcitable. This is regarded by Van der Kloot (1954, 1955, 1961) as the immediate cause of the failure to release the hormone. The entire central nervous system (including the brain) retains its normal metabolic rate and cyanide sensitivity. But cholinesterase is below the level of detection. During diapause acetylcholine accumulates in the brain; perhaps cholinesterase function is evoked when a critical level has been reached; cholinergic activity then decreases to the level characteristic of the active brain; electrical activity reappears and in a few days, the neurosecretory cells release the brain hormone, and diapause comes to an end. The changes in the brain characteristic of diapause do not take place if the brain is removed from a larva just before pupation and implanted into a brainless pupa (Williams, 1952). It seems likely that these changes in the brain are not present in all diapausing insects. Schoonhoven (1962) has shown that, in contrast to Hyalophoru, thc brain in the diapausing pupa of Bupalus piniurius still shows spontaneous activity; neurosecretory material is produced in the medial neurosecretory cells, but chilling is needed to make them competent to release it. The activity of cytochrome c oxidase

274

V. B. WIGGLESWORTH

during diapause is at an average of about 10% of thelevel just before emergence. From time to time the suggestion has been made that the corpus allatum secretion may be responsible for keeping the insect in diapause. Rahm (1952) showed that the thoracic gland in Siulis is already activated by the brain in the autumn, but secretion is arrested during the winter. This was ascribed to the corpus allatum suppressing the liberation of the hormone-but this suggestion appears to have been based only on the supposed antagonism between the secretions of the corpus allatum and thoracic gland. More distinct evidence was obtained by Fukaya and Mitsuhashi (1957, 1958, 1961) in the rice stem borer Chilo. They showed that if the head of the diapausing larva is ligatured in such a way that the brain goes to the prothorax while the corpora cardiaca and corpora allata remain in the head, development is accelerated, diapause is brought to an end and pupation occurs without chilling. They interpret this experiment as proving that the corpus allatum is the source of an active principle which maintains larval diapause. But tying off the brain from the corpora cardiaca and corpora allata clearly breaks the nerves to the corpora cardiaca, that convey the neurosecretory material. If this operation were to allow the escape of this material directly into the blood, and even more, if the operation served as a stimulus to its secretion from the brain, this treatment might be expected occasionally to bring diapause to an end-just as implantation of an isolated diapause brain into a diapausing Pyausla larva will induce development (Cloutier et al., 1962). This interpretation is supported by the fact that Fukaya and Mitsuhashi (1961) showed likewise that brains from diapausing larvae of Chilo, implanted into brainless larvae pf the same age, led them to pupate; and that if the corpora cardiaca and corpora allata are removed, leaving the brain intact, termination of diapause occasionally occurs. The Japanese authors interpret this experiment as showing that the corpus allatum hormone keeps the brain and thoracic gland inactive. They show that the corpora allata are highly vacuolated during diapause and regard this as evidence of activity; the vacuoles disappear in the last few days before pupation and they regard the gland in this state as inactive. But the vacuolated gland agrees exactly in its appearance with the inactive condition in Rhodnius, while the non-vacuolated gland, with dense cytoplasm (as illustrated in their photographs) agrees with the active condition in Rhodnius (Wigglesworth, 1934, 1936). Waku (1960a, b) noted that the corpus allatum in the larva of Plodh interpunctella became enlarged during diapause. He regards this

H O R M O N E S IN GROWTH AND REPRODUCTION

275

as a sign of increased secretory activity, and therefore supports the conclusions of Fukaya and Mitsuhashi. He showed also that there are distinct changes in the larva well before diapause begins: those larvae which are later going to enter diapause have a lower level of activityof cytochromeoxidase and succinicdehydrogenase, and the neurosecretory cells and thoracic glands show histological evidence of reduced activity. Mochida and Yoshimeki (1962) noted that the gonads of hibernating larvae of Chilo begin to grow when the corpora cardiaca and corpora allata begin to decrease in size. This led them to suggest that the corpus allatum may secrete a substance which restrains development of the gonads during diapause. But further evidence of this is clearly needed. B. P H Y S I O L O G I C A L C H A N G E S D U R I N G C H I L L I N G

The developmental process which takes place during diapause and which (in most insects in temperate latitudes) can go forward only at low temperatures, has been termed by Agrell (1947) “latent development” and by Andrewartha (1952) “diapause development”. The nature of this process is unknown; it may well be different according to the stage of the life cycle at which diapause supervenes. But in the postembryonic diapause with which we are concerned here, there is obvious physiological activity in progress. The remarkable cycle of changes in the brain of the diapausing pupa of Hyaiophora (Van dcr Kloot, 1961) has already been described. It is a more common feature for the thoracic gland, icactive at the beginning of diapause, to be reactivated during the period of chilling. That is seen in the pupal diapause of Luehdorfa juponica, so that after chilling these pupa will develop to adults even though the brain is removed. The brain hormone is assumed to be secreted during the cooling period (Ichikawa and Nishiisutsuji-Uwo, 1956). But the rapidity with which the brain hormone is released varies from species to species. In Hyalophora as we have seen, it is very slow and docs not take place until diapause is ended (Williams, 1947). The same applies to the larva of Cephus (Church, 1955). In the diapausing pupa of the Sphingid Mimas tiliue the process is more rapid. During the first 3 weeks at 3°C the neurosecretory material is passed to the corpora cardiaca, and by the end of the low temperature period the neurosecretory cells have finished their job and are again inactive (Highnam, 1958). When the diapausing pupae of Antheraea are chilled at 7”C, they show1 a progressive increase in the rate of oxygen uptake which follows their

276

V. B. W I C G L E S W O R T q

transfer to 28°C. After short periods of chilling, this transfer does not lead directly to adult development and emergence, but to a gradual fall in oxygen uptake and even (after very short periods of chilling) a return to the diapause level (Waku, 1957). This same temporary increase was shown by pupae with brain and thoracic gland extirpated. A possible explanation of these results would be that a small amount of moulting hormone has been set free during the period of chilling; and the longer the chilling the more hormone. These amounts of hormone are sufficient to start development but not enough to complete it-as is seen after small injected doses of ecdyson (p. 271).

Time

FIG. 1. Diagram showing the course of oxygen uptake in pupae of Antherueu pernyi, measured at 28°C. (------)Results in pupae qhilled at 7°C for differing lengths subsequent course of uptake in pupae transferred t0 28°C after of time; (-)

different periods of chilling. The topmost circles show oxygen uptake at emergenp. (After Waku, 1957.)

In the hibernating larva of Mormoniellu 2 days exposure to 25°C undid most of the physiological effect of 7 days of chillipg at 5°C; and 7 days at 25°C completely undid these effects. It looks as though at low temperatures hormone accumulates, at high temperatures it may be broken down, But anaerobiosis in the chilled larvae completely inhibited the physiological process leading to the termination of diapause ;and when warming was conducted anaerobically it failed to undo the effect of cl-$llingindeed, the termination of diapause was accelerated. It may be that low temperature slows down an aerobic breakdown reaction within the brain of the insect, and permits synthesis of a substance necessary for neurosecretory activity (Schneiderman and Horowitz, la%), This same

HORMONES I N GROWTH A N D REPRODUCTION

277

phenomenon of hormone inactivation at high temperature is described by Church (1955) in Cephus. It is seen in Rhodnius at temperatures above the normal physiological range (Wigglesworth, 1952b, 1955b). In the reactivation of embryos of Bombyx (Ducleaux, 1869) or Melanoplus (Robbie et al., 1938), the low temperature is effective although no brain or other endocrine organ is yet differentiated. Indeed, in Melanoplus, the embryo, or fractions of embryos, can be caused to resume development by explantation into hanging drops of Ringer’s solution (Bucklin, 1953). In the hibernating pupa of Papilio xuthus chilling at 0 4 ° C for 30 days restores the ability to develop. But this happens equally well if the pupa is decapitated at the time of pupation. So here also the brain is not concerned (Ozeki, 1954). C. INJURY A N D DIAPAUSE

It is well known that some insects in diapause may be aroused, and development re-started, by pricking or burning; but the question remains unanswered whether the wound hormones liberated at the site of injury have excited general growth, or whether the injury has provided a stimulus to the endocrine system. If the larva of the parasite Dipluzonpectorutorius is injected into the posterior compartment of ligated diapausing Syrphid larvae (Epistophe bifasciata), this fragment forms a puparium, while the anterior half remains larval. Here pupation has been induced, not by activation of the endocrine centres of the host, but by direct action of the parasite on the epidermis of the host, apparently through the operation of moulting hormone diffusing through the body wall (Schneider, 1950, 1951). In the case of the pupa of the moth Bupalus piniarius the synchronization of development of the host and its Tachinid parasite Eucarcelia seems to depend on the great sensitivity of the parasite to the thoracic gland hormone of the host (Schoonhoven, 1962). In the diapausing pupa of Hyalophora the oxygen consumption is only 1.4% of that in the mature larva, and 5 % of that in the adult just before emergence. By the first day of visible adult development the oxygen consumption has increased to 3a times that of the diapausing pupa. After extensive injury, metabolism may rise 14-fold and it may require 10weeks to return to the control level (Schneiderman and Williams, 1953). It may be that it is the very great increase in haemocytes, that takes place in injured Lepidoptzra, which is responsible for the rise in oxygen consumption. Whatever the “injury factor” may be, it certainly evokes a generalized metabolic response; for injury to one member of a pair of pupae joined in parabiosis, will induce a high rate of “injury metabolism”

278

V. B. W I G G L E S W O R T H

in the other pupa. t h t no evidcncc has been obtained that ecdyson is concerned-or cnough ccdyson t o iriitiatc visible adult dcvelopment (Shappirio, 1960; Harvey and Willianis, 1961). D. T H E N A T U R E Of, T I l E D I A P A U S E S T A T E

Diapause is essentially a physiological mechanism for survival during an adverse season, most commonly the winter cold. The mechanism seems to consist in the separation of physiological processes, some of which require a low temperature, while others require a high temperature. It seems that certain processes (called “latent” or “diapause” development) have become adapted by selection to proceed only at some low temperature range characteristic of the species ; while the main process of development will go forward only in the upper range of temperature. (The foregoing is the usual state of affairs. But if the diapause of a given species occurs in the hot dry season, “diapause development” may require a high temperature for its completion. The European corn borer, Ostrinia nubilalis, has a faculative diapause induced by a short-day photoperiod. But the removal of the block to secretion of the brain hormone does not require a period of chilling. It can occur at 30°C and is accelerated by a succession of long days (McLeod and Beck, 1963).) There is no reason to suppose that the same physiological processes are always involved in “diapause development ’*. In post-embryonic diapause it seems usually to be some proccss in the brain, which restores competence to the neurosecretory cells, that is responsible. But it must be something different in diapausing embryos in which neither nervous system nor endocrine organs have been differentiated (Bucklin, 1953). In the larva of Rhodnius, as soon as deposition of the cuticle is complete, the mitochondria in the epidermis are rapidly reduced in number. The same applies to the intersegmental muscles of the abdomen. If these tissues were studied in isolation it is likely that they would show the same pwperties as the whole diapausing pupa of Hyulophoru-with a great reduction in the cytochrome systerr and perhaps the virtual disappearance of cytochrome c. But in the intact larva of Rhodnius these dormant tissues are dominated by the active muscles, nervous system. digestive system etc. which remain fully functional. The same is true of the diapausing adult of the sweet-clover weevil Sitona (Davey, 1956). The question arises as to what is the cause of the dormant state in the epidermal cells and other dormant tissues. Harvey (1962) raises the question whether the substitution of permeability barriers in place of active regulation may not be used as an energy conserving device that

t l 0 K M O W l 5 I N ( , V O W I i’

1 Y I I Rtl’RO1)L‘CTlOh

279

contributes to the low metabolic rate of diapausing insects. This suggestion was put forward by Tclfcr and Williams (1960) who compared the condition with that supposed to exist in dormant seeds; and a similar suggestion had been made by the writer (1957). But perhaps the situation can be described equally well by saying that the dormant tissues of the Hyulophoru pupa or the resting larva of Ritodttius are so constituted that in the absence of the moulting hormone they do not become activated and grow (Wigglesworth, 1963b). At the present time we are completely ignorant of the adaptations within the cell that are responsible for this condition. E. THE R O L E O F H O R M O N E S I N T H E M A T E R N A L CONTROL OF DIAPAUSE

The occurrence of diapause in the egg of the silkworm is controlled by the conditions of temperature and photoperiod to which the mother was exposed during the earliest stages of her development-in the egg or the first stage larva (Lees, 1955). The immediate cause of diapause in the egg is the exposure of the ovaries of the mother to a “diapause hormone” secreted in the suboesophageal ganglion (Hasegawa, 1952; Fukuda, 1953). Secretion of this hormone may be inhibited by nervous impulses from the brain, and the female then lays non-diapause eggs. Hasegawa (1952, 1957) has extracted this “diapause hormone” from the suboesophageal ganglion of Bombyx pupae, and considerable concentration of the active substance has been achieved. When injected into a female pupa it leads to the production of hibernatingeggs by the resulting moth, even in insects that would otherwise have produced non-hibernating eggs. Yoshitake (quoted by Hinton, 1957) claims that the diapause hormone has the effect of increasing the permeability of the ovary to a number of unrelated substances, both natural derivatives of tryptophane (kynurenine and 3-hydroxykynurenine) which are normally translocated from the haemolymph to the ooplasm, and injected dyes such as neutral red, Nile blue and Bismarck brown. It may be some component, whose entry to the egg is controlled by this mechanism, which converts a developing egg into a diapause egg. These observations on the control of diapause in the silkworm egg illustrate a feature which adds to the difficulty of understanding the physiology of diapause : the fact that environmental stimuli may bring about diapause in a developmental stage widely separated in time from the stage on which they act. If females of Mormoniellu are exposed to low temperatures during oogenesis, their progeny will enter diapause at

280

V. B. W I G G L E S W O R T H

the end of the last larval stage. This effect on the female wears off after few days and then she begins to produce nondiapausing offspring again. A similar effcct is seen in Trifnopfis-but here the low temperature must act on the larva itself between the second and the final instar if it is to provoke diapause in that generation (Schneiderman and Horwitz, 1958). The question of hormones and diapause in the adult will be deferred until we come to consider hormones and reproduction (p. 312).

3

v. T H EC O R P U S

ALLATIJM A N D THE CONTROL OF

M ETAMOKPHOSIS A. T H E C O R P U S A L L A T U M A N D J U V E N I L E H O R M O N E

SECRETION

The first indication that metamorphosis” is controlled by some hormonal influence from the head was the precocious development of adult characters that occurred when 3rd- or 4th-stage larvae of Rhodnius were decapitated immediately after the critical period of moulting. The source of the hormone which normally prevents the appearance of adult characters in these young larval stages was readily traced to the corpus allatum: (i) removal of the anterior parts of the head containing the brain, but leaving the corpus allatum intact, did not lead to precocious metamorphosis (Wigglesworth, 1934); (ii) implantation of isolated corpora allata from the younger larval stages into the 5th-stage larva induced a further larval moult (Wigglesworth, 1936). This hormone, which inhibits the realization of the latent adult form, was at first termed the “inhibitory hormone”, but when it was shown that, under some circumstances, the epidermal cells of an adult insect, that was caused to moult, could be induced by the corpus allatum hormonc to lay down partially larval cuticle, it seemed more appropriate to refer to the active substance as the “juvenile hormone” or “neotenin” (Wigglesworth, 1940b). The effect of the corpus allatum, and the juvenile hormone secreted by it, in controlling metamorphosis, has been confirmed in all the main groups of insects (review by Wigglesworth, 1954a). During the larval stages the development of the gonads is suppressed; they normally become capable of maturation only at the time of metamorphosis to the adult form. “It is impossible not to be reminded of the metamorphosis that occurs in man at puberty” (Wigglesworth, 1959a); and it may well “

I I O K h 4 0 h T S I Y C , R O W T t i AND R E P R O D U C T l O h

28 1

be that thc control ot'mctamorpho~isby the corpus allatum is a secondary cf'fect, and t h a t the primary stcp i n cvolution was the control of gonad dcvclopment (Wells and Wells, 1959).Thc parallel between metamorphosis and puberty is pointed out also by L. Joly (1960) and Girardie (1962). The corpus allatum in Rhodnius secretes the juvenile hormone throughout the first four larval stages. It appears not to secrete it in the 5th stage, which moults to the adult; and it reappears, as we shall see (p. 296), in the mature adult. Direct estimates on extracts from Hyulophoru cecropia at all stages of development have been made by Gilbert and Schneiderman (1961). These show that the hormone is already present in the egg; in part this is derived from the mother, but from about the 7th day onwards the hormone is produced by the developing embryo itself. The hormone is present throughout the larval stages, but is absent prior to the moult from the pupa to the adult. In holometabolous insects, notably in Lepidoptera, it is generally supposed, following Piepho (1951), that for larval moults a large amount of juvenile hormone is needed; for the pupal moult much less; and for the imaginal moult still less or none. When corpora allata were taken from the adult silkmoth Philosamiu cynthia or from the 4th-stage larva, and implanted into the pupa, this resulted in an extra pupal moult (that is, juvenile hormone was being secreted). Glands taken from another pupa led to adult development. Glands taken from the 5th-stage larva gave variable results, indicating a gradual change from activity to the temporary inactivity which leads to metamorphosis (Ichikawa and Nishiitsutsuji-Uwo, 1959). These results confirm those of Fukuda (1944) in Bombyx mori. Fukuda (1 962), by using allatectomized 4th-instar larvae of Bombyx as recipients, showed that the activity of the corpus allatum in secreting juvenile hormone, is absent or very low at the beginning of the pupal stage; but during the second half of the pupal stage the activity rises and reaches a high level shortly before emergence of the adult moth. He showed also that (as in Rhodnius) the isolated corpus allatum retains its activity and may, indeed, regain its activity, even when completely isolated from the brain. As he points out, this does not support the view that secretion of the juvenile hormone by the corpus allatum is governed by the neurosecretory cells of the brain. By means of a standard pupal assay, Williams (1961b) had obtained similar results on Hyalophora cecropia : the corpora allata were least active just before each larval moult and most active soon after the moult. Hc suggests that the declining titre of juvenile hormone during the larval moults may be responsiblc for the changes in morphology in

282

V . B. W I G G L E S W O R T H

successive larval stages in the cecropia silkworm-but there is as yet no experimental proof of this. The corpora allata show a low persistent level of activity at pupation-if removed, the resulting pupa shows partial adult development (prothetely). Immediately after the pupal moult they become completely inactive and remain so during the resting pupal stage; but during the final week of development (that is, at twothirds through the developing period) they again become active, reaching maximal activity at the time of emergence of the adult moth. The next question is the mechanism by which the secretory activity of the corpus allatum is controlled. In Locusta, metamorphosis seems to be decided in the first day after the 4th moult: if “adultoids” are to be produced an active corpus allatum must be implanted in the 4th stage or at the very beginning of the 5th stage. At the beginning of the 5th stage some factor inhibits the corpus allatum. L. Joly (1960) postulated some unknown organ secreting an inhibitor substance. But it was early shown in Rhodnius (Wigglesworth, 1934) that the isolated corpus allatum of a 4th-stage larva will continue to secrete juvenile hormone when implanted into a 5th-stage larva, so that this will moult not only to a 6th-stage larva but may moult again to a 7th-stage larva. It was inferred that secretion is normally inhibited by nervous control from the brain. This conclusion was supported by Scharrer (1952a) after similar experiments on Leucophaea. As Liischer and Engelmann (1955, 1960), who likewise produced additional larval moults in Leucophaea by cutting the nerves from the brain to the corpora allata, point out, this indicates that the corpus allatum secretion is controlled in the same way, both during normal metamorphosis and during the reproductive cycle (p. 302). From a survey of the literature, Scharrer (1958) concludes that the restraining influence on the corpus allatum is nervous, the stimulating effect is by some substance in the neurosecretory product from the brain (cf. Fukuda, 1962; seep. 255 above). It has been found by Noviik and CervenkovA (1961) that if the corpus allatum is removed from the 5th-stagelarva of Pyrrhocoris 5-6 days after moulting, and implanted into newly moulted 5th-stage larvae, a number of these develop partial or complete larval characters when they moult; and they observed similar results in Galleria, using larvae a day or two before pupation. Novhk and Cervenkovh regard these results as evidence that the corpus allatum is producing juvenile hormone continuously during the last larval instar. But they might be explained by release of the corpus allatum from inhibition by the brain when its nerve supply is severed.

H O R M O N E S IN GROWTH A N D REPRODUCTION

283

Some further details of the nerv~uspathways of the control have been obtained by Ozeki (1961) studying the earwig Anisolubis. Implantation of active corpora allata normally causcs the corpora allata of the adult host to cease secreting juvenile hormone; but the implantation does not have this effect if the frontal ganglion or the pars intercerebralis of the brain are removed, or if the recurrent nerve and the nerves to the corpora cardiaca are cut. In Ephemeroptera the corpora allata are innervated from neurosecretory cells in the suboesophageal ganglion (Arvy and Gabe, 1952a); activity in the neurosecretory cells ceases at the end of larval life and at the same time the corpora allata show signs of atrophy (Arvy and Gabe, 1953). B. T H E EFFECTS OF T H E J U V E N I L E H O R M O N E

The obvious effect of the juvenile hormone during the early stages of the insect is the maintenance of juvenile or larval characters, which make possible the continued growth of the larval form. This general effect entails certain special effects which may be considered separately. An important effect is the maintenance of the thoracic gland which will then secrete the hormone necessary for renewed moulting and growth. In Rhodnius the thoracic gland goes through a conspicuous cycle of secretion in each moulting stage. This happens also in the 5thstage larva; and by the end of this stage the cells have reverted to the resting state and appear quite normal; but within 24 h after moulting to the adult the nuclei are undergoing chromatolysis, and by 48 h the nuclei have disappeared completely (Wigglesworth, 1952a). In Ephestia some of the cells undergo involution at the time of pupation; others survive to produce the moulting hormone for the imaginal moult, and undergo rapid degeneration in the freshly metamorphosed moths (Rehm, 1951; Ichikawa and Nishiitsutsuji, 1955s). In Periplaneta, Dixippus etc. the disappearance of the thoracic glands is much delayed (see review by Wigglesworth, 1954a). In the Carabid beetle Anisotarsus cupripennis the disappearance of the gland does not occur in the newly formed adult, as in most insects, but during the critical period of the pupa rather more than 24 h old; that is, when pigmentation of the eyes is beginning (Nciiez, 1954). Williams (1952) had shown that the thoracic gland of Hyalophora could likewise be dispensed with when pigmentation of the eyes begins. If extra larval stages are produced experimentally by the implantation of active corpora allata, they retain their thoracic glands and are therefore able to moult again (Wigglesworth, 1952a). Retention of the

284

V. B. W I G G L E S W O R T H

thoracic gland\ is one of the larval characters for which the juvenile hormone is responsible. This has been shown also in the pupa of Anlhcrucu (Gilbert, 1962). I n Rhohius, if the larva (for example, the 5th-stage larva) has gone through its moulting cycle in the absence of juvenile hormone, the thoracic gland breaks down in the young adult within 24 h. But if the gland is removed from a 5th-stage larva shortly before moulting and is implanted into a young adult it undergoes no breakdown. On the other hand, if the gland is removed immediately after moulting, it will break down in any environment, even in a 4th-stage larva with juvenile hormone present. From these and other experiments it was concluded that when the thoracic gland goes through a secretory cycle in the absence of the juvenile hormone, it will break down. But some further humoral factor, to which the gland is exposed at or immediately after moulting to the adult, is necessary to precipitate this breakdown (Wigglesworth, 1955a). The nature and source of this second factor are unknown. When termite larvae moult into supplementary reproductives, the thoracic glands (ventral glands) immediately degenerate (Liischer, 1957). In this case there is no evidence that the breakdown is bound to a preceding phase of inactivity in the corpora allata. Indeed the corpora allata increase four-to-five times in volume immediately after the determination of the larvae to develop into supplementary reproductives (Liischer, 1958a). This increase in size does not prove, however, that juvenile hormone is being secreted (cf. Scharrer, 1952a). There is some evidence that the juvenile hormone may be playing a part in some other types of polymorphism besides the normal metamorphosis. Kaiser (1955) concluded that the termite soldier could be regarded as a “superlarva” which appeared after prolonged action of the juvenile hormone, and Liischer (1958b) confirmed that implantation of extra corpora allata into termite larvae causes the appearance of “ pre-soldiers” and soldiers. Whereas the thoracic glands (ventral glands) degenerate rapidly in the adult of Calotermes, they persist in soldiers (Pflugfelder, 1947). In the Apterygota, in which moulting continues in the adult insect, and the moults alternate with phases of reproduction, the thoracic glands again persist (p. 299). In Lepisma saccharina there is a constant correlation between the body size and the length of the genital appendages. This relation is scarcely upset if the corpora allata are extirpated: it is not possible to produce adults of unduly small size as can be done in other insects (Richter, 1962).

H O R M O N E S IN G R O W T H A N D REPRODUCTION

285

Joly and Joly (1953), P. Joly (1956) and L. Joly (1960) obtained evidence that extra juvenile hormone may be responsible, or partially responsible, for the phase change in locusts. Corpora allata transplanted from gregarious adults of Locustu into gregarious larvae of the same culture led to the appearance of green coloration as in the solitary phase, by a direct action in the epidermis (L. Joly, 1955), and sometimes to biometrical changes in the resulting adults, comparable with those characteristic of solitaria. These same colour changes had been obtained by Pfeiffer (1945b) in Melanoplus, an observation which led Joly and Joly (1953) to suggest that the phase change in migratory locusts might be a modification of some universal effect. The inhibition of wing development in the presence of juvenile hormone results from action during the period of intense cell division soon after moulting; development of green colow is related with activity of the implanted corpus allatum at the moment of moulting (L. Joly, 1955). But there are other differences between the locust phases which cannot be reproduced by a simple increase in the supply of juvenile hormone (Joly et al., 1956; Staal, 1961). The transformation can be described as the result of environmental changes which provoke a genetic switch mechanism (Kennedy, 1961; Wigglesworth, 1961a); the juvenile hormone itself doubtless plays only a subsidiary role in the resulting change. But the fact remains that both the morphological and behavioural characters of the solitary forms of locusts can be regarded as more “juvenile” than those of the gregarious forms (Kennedy, 1961). This interpretation is borne out by the persistence of the ventral glands in the solitary female of Locustu and Schistocercu for 6-8 weeks after metamorphosis, even after fertile eggs have been laid (Carlisle and Ellis, 1959). According to this way of looking at the matter the solitary adult locust may be regarded as a partially neotenous form. The same applies to the apterous forms of virginoparous Aphids. In general morphology and colour pattern the apterous female resembles the larval form; and the application of juvenile hormone to 5th-stage larvae of the vetch Aphid Megouru viciue, that have been determined to produce winged forms, results in the development of wingless adults similar to the normal apterous form (Lees, 1961). These observations lead on to the question of brachypterous forms in other insects. These again are controlled by genetic factors (Cousin, (1956) in Gryllus, Brinkhurst (1959, 1963) in Gerroidea) but the suggestion has been made that the mechanism which producesthe short wingsis an excessive secretion of juvenile hormone (Southwood, 1961). However,

286

V.

A. W I G ( ~ I . r S W O K T H

as Rrinkhurst (1963) has pointed out, in the case of Gerroidca the general morphology of the brachyptcrous forms, both in the wing and in the abdomen, is that of the adult. An alternative suggestion, therefore, is that the short wings may result from over-activity of the moulting hormone, which causes accelerated deposition of the cuticle over the wings as already discussed (p. 269). If a 5th-stage larva of Rhohius (A) at 1 day after feeding, is joined in parabiosis with a second 5th-stage larva (B) at 9 days after feeding, the moulting of “A” is accelerated and completed in 17 days (as compared with the normal 21 days) and the wings are greatly reduced in size (Wigglesworth, 1961a, and unpublished). This matter will be discussed further under the heading of “humoral integration” (p. 314). Finally, there are certain incidental effects of the juvenile hormone which may be mentioned here. Throughout the larval life of Bombyx mori the silk gland is restrained by the corpus allatum as though it were an imaginal organ (Bounhiol, 1953). Likewise the behaviour of the animal may be under the direct control of the juvenile hormone. Larvae of Galleria spin a slight flat web before they make a larval moult; before moulting to the pupa the full-grown larva spins a tough cocoon. If active corpora allata are implanted into the last stage larva, so that it moults again into a larva, it will spin a larval type of web or an intermediate type (Piepho, 1950). In the Sphingid Mimas tiliue, if the juvenile hormone is above a threshold concentration the larval behaviour is that which is normal before a larval moult. If the hormone is below this threshold the larva migrates down the tree trunk to the soil as before a pupal moult. In this case there was no intermediate type of behaviour, as in Galleria (Piepho et al., 1960). These observations serve to illustrate the close relation that exists between behaviour and growth.

C. T H E M O D E O F ACTION O F T H E JUVENILE H O R M O N E

According to the interpretation of metamorphosis that was put forward by Aristotle and later elaborated by William Harvey, the process is essentially a reversion to embryonic development, the pupa being compared with the egg. In recent years this idea has been advocated particularly by Henson (1946). But in fact the same claim can be made for every larval moult. During each moult in Rhodnius the epidermis undergoes a process of differentiation with the formation of new organs, notably dermal glands and sensilla, which is exactly comparable with differentiation in the embryo. This process includes even the new forma-

HORMONES I N GROWTH A N D REPRODUCTION

287

tion of primary sense cells whose axons grow inwards to join the central nervous system (Wigglesworth, 1953a, b). The idea that metamorphosis is a renewal of embryonic growth was encouraged by the discovery of the imaginal discs by Weismann in the middle of the last century. These he regarded as nests of embryonic cells. In Hymenoptera and in higher Diptera the entire epidermis of the adult is derived from embryonic cells set aside for this purpose. But in most parts of most holometabolous insects (Lepidoptera, Coleoptera, etc.) and in all parts of hemimetabolous forms, the adult cuticle is laid down by the same cells or by the daughter cells of those that have previously laid down the larval cuticle. The clearest example is that of the abdominal hairs in Rhodnius, the formative cells of which persist from one instar to the next, and can be caused to lay down hairs of larval, aault, or intermediate type according to the amount of juvenile hormone present (Wigglesworth, 1934).

It would seem that the chief function of the imaginal discs is to free certain ectodermal cells from the task of cuticle formation, and thus to make possible the evolution of a larval form with characters fully adapted to the larval mode of life and in no way influenced or deflected by the form of the future adult. In the caterpillar of Pieris there is a region of epidermis, between segments 2 and 3 of the leg, where cell division is very active after the final moult. This appears to be a “differentiation centre” for the adult leg, controlling development of the entire limb. This region requires a high oxygen supply and probably absorbs more moulting hormone. The active outgrowth and differentiation of the adult limb is to some extent separable from the realization of adult characters in the epicuticle laid down, but both are controlled by the juvenile hormone (Kim, 1959). A similar process, differing only in certain details, occurs in Galleria (Kuske er al., 1961) and in Antheruea (Kuske, 1963). As Kim (1960) points out, if the legs of caterpillars had no function at all (like the leg rudiments in the insect embryo) they would be regarded as “imaginal buds”. But since they function as limbs, they are not commonly thought of in this way. The difference consists solely in the fact that the cells in question arc occupied in laying down cuticle during the larval stages. The idea of renewed embryonic development carries with it the suggestion that the insect is differentiating progressively towards the adult state. This conception is opposed to the idea of “metamorphosis” involving a switch to some line of development which is qualitatively different from

288

V.

B. W I G G L E S W O R T H

that in the larva. Thesc two ways of regarding the transformation of insects are reflected in the two ways of regarding the action of the juvenile hormone. Williams (1956b) regards the corpus allatum as acting merely as a “brake on development” and thus maintaining the status quo-rather than actively evoking the realization of larval or juvenile characters. The observation which led the author to abandon the status quo interpretation, which had been adopted earlier (Wigglesworth, 1934, 1936), was that the pupal integument of Galleria (Piepho, 1939) or the adult integument of Rhodnius (Wigglesworth, 1939) could be caused to resume larval characters if induced to moult in the presence of the corpus allatum of a young insect. This result has been observed in few insects; and in few parts of the body, even in Rhodnius; but a new example was reported by Ozeki (1 959). He showed that adult earwigs (Anisoldis) retaining their own corpora allata, and caused to moult by the implantation of active ventral glands, developed once more the ecdysial line on the thorax. This reversal of metamorphosis does not occur if the moulting adults have been deprived of the source of juvenile hormone by rcmoving the corpora allata. An interesting example of reversal of metamorphosis has been described in the endodermal tissues of Galleria (Piepho and Holz, 1959) where the juvenile hormone acts also upon the basal cells of the mid-gut. If the atrophied mid-gut of the adult is implanted into the body cavity of a larva, which moults again, the basal cells will grow and multiply once more and develop a mid-gut of larval type. These facts are perhaps best described by comparing the different forms of the insect at different stages in its life cycle, on the one hand with the different form of the parts 9f the body that results from differentiation, and on the other with environmentally controlled polymorphism in the individuals of a species4iffcrences in form which are brought about by the combination of genetic and environmental influences. The insect is pictured as containing within its gene system the potentiality to produce three different forms: the larva, the pupa and the adult. The components of the gene systep responsible for these three forms are brought into action by the concentration and timing ofjuvenile hormone secretion in the moulting insect (Wigglesworth, 1954a). This comparison between metamorphosis, differentiation and environmentally controlled polymorphism has been developed in several publications (Wigglesworth, 1957, 1959a, 1961a). In earlier writings (Wigglesworth, 1953b), the juvenile hormone was pictured as interveningin specificsyntheticreactionsin the cytoplasm. More recently the bor-

H O R M O N E S I N GROWTH A N D REPRODUCTION

289

mone has been pictured as operating upon the gene-system-although the cytoplasm might clearly serve as an intermediary in the process. The nature of the local effect, that is, the extent of cell division and outgrowth, and the character of the synthetic products of single cells, as provoked by the hormone, will be different in each part of the body. The mechanism of this difference is the problem of differentiation which is beyond the scope of this review. But one is reminded of the model proposed by Jacob and Monod (1961) by which a single hormone, acting upon a series of different allosteric proteins in different tissues, could bring about a variety of different effects of enzyme synthesis or enzyme activation. It is usually a characteristic of metamorphosis that there is an exaggeration of local growth, with enlarged outgrowths; but in some insects there are outgrowths of this kind provoked by the juvenile hormone in the larva. A mild example of this is seen in the abdomen of Rhodnius where cell division is much greater in the presence of the juvenile hormone than in its absence (Wigglesworth, 1940b, 1963b). Novik (195 1) differentiates between “harmonic growth” during larval development, and “allometric growth” which characterizes metamorphosis. This is another way of saying that metamorphosis is associated with great enlargement of the wings, legs and external genitalia. He ascribes this excessive growth to the presence of an intrinsic growth factor within the cells concerned. Having made this assumption it necessarily follows that the observed differences in growth activity of the different parts can be described in terms of this hypothesis. This so-called “gradient-factor ” hypothesis has naturally varied in the details of its presentation since it was first put forward by Novik in 1951. It is often expressed by the statement that the presence of an intrinsic growth factor (the “gradient factor”) is characteristic of adult cells; whereas the larval cells lack this intrinsic growth factor and are dependent on a circulating growth factor (the juvenile hormone) to activate their growth. Expressed in this way the hypothesis did not describe adequately the fact that in most parts of most insects the same cells (or their daughter cells) produce first larval characters and then adult characters. But in its most recent expression the “gradient factor” hypothesis comes much closer to the usual hypotheses of growth and differentiation. It is recognized by Novak (1961), and by all other writers on the subject, that the common feature of morphogenesis is “allometric growth” or “gradient growth”. It is this that determines the changing form of the body. This description is applicable both to the different parts

290

V. I). W I C J G L E S W O R T H

of the body and to different parts of one and the same cell. He accounts for the striking outgrowths of the body which characterize metamorphosis by supposing that this differentiated growth activity is dependent upon the differential distribution of “ a hypothetical tissue growth factor . . . a simple substance or group of substances (a physiological, not necessarily chemical individual).” Or again, the gradient factor is perhaps “a single chemical substance . . . perhaps more probably. . . it depends on the common disposition of a number of related substances such as nucleic acids.” The juvenile hormone is said to stop this process and convert it into “one universal process of isometric . . . growth without any change of form”. Such a description does not differ very much from other attempts to describe morphogenesis that have been current during the past half century. Indeed, if one likes to regard the gene system as generating the appropriate “hypothetical tissue growth factor” in response to the hormonal environment of the cells, the description hardly differs from that given above (p. 288). There is another aspect of the control of metamorphosis which must be mentioned. Cells seem to acquire a certain “inertia” such that once they have begun to form pupal or adult structures they resist the action of the juvenile hormone in causing them to revert to the larval state. Even in the abdomen of Rhodnius the return to the larval condition is far from complete. And there is a similar resistance to precocious metamorphosis. Kaji (1953) showed that when legs of caterpillars of Philosamia Cynthia are transplanted to other hosts of the same species, some made a larval moult when the host pupated, and then pupated when the host became adult, whereas Piepho (1951 ) had generally found synchronous metamorphosis in host and implant in Galleria. In young larvae of Rhodnius induced to transform precociously to the adult, there are many more persistent bristles on the abdomen than in the normal adult (Wigglesworth, unpublished). Likewise in Dixippus (Pflugfelder, 1937) and Leucophaea (Scharrer, 1948b),two moults are needed, after removal of the corpora allata, before the fully adult form is developed. This was attributed (Wigglesworth, 1948) to the persistence of juvenile hormone in the tissues; but it was pointed out by NovBk (1959) that since juvenile hormone is being continually removed, perhaps by the Malpighian tubules (Bounhiol, 1943) and certainly by metabolism (Gilbert and Schneiderman, 1960; Wigglesworth, 1963a)some other explanation of this inertia, or persistent action within the cells, must be found. Perhaps it can be compared with those changes in the cytoplasm which cause “ paramutational” alterations in the chromosomes and so lead to semipersistent hereditary effects in those

IIOKMONES IN GROWTH A N D REPRODUCTION

29 1

parts of the soma concerncd ;the sort of change that occurs in differentiation (Mather, 1961). It was shown long ago by Piepho (1939) that this inertia was soon eliminated in the regenerating cells responsible for wound repair. These cells, undergoing repeated division, became de-erentiated and highly sensitive once more to the juvenile hormone. Finally it may be well to emphasize that there is no evidence that the juvenile hormone has an anti-ageing effect. It is a morphogenetic hormone. D. H I S T O L O G Y A N D H I S T O C H E M I S T R Y OF THE C O R P U S ALLATUM

The active corpus allatum in Rhodnius consists of closely packed cells with a relatively large amount of homogeneous cytoplasm; the inactive gland is often smaller, but the main change is in the shrinkage of the cytoplasm with the appearance of vacuolated spaces between the cells (Wigglesworth, 1934). In Leucophaea the active gland likewise shows increased cytoplasm with separation of the nuclei; with the cessation of activity during pregnancy (p. 302) the cell number falls and pycnotic nuclei can be seen (Scharrer and von Marnack, 1958). As seen in sections under the electron microscope, the inhibited glands show closely packed nuclei with clumped chromatin, and little cytoplasm, with deep infolding of the (;ell boundaries. These stellate membranes straighten out as the cytoplasm e3pands during activity; the nuclei enlarge and the mitochondria and ribosomes in the cytoplasm increase (Scharrer, 1961, 1962). The presence of neurosecretory substance in nerve axons extending into the corpus allatum has been mentioned earlier (p. 250). This neurosecretory material has been observed also in Periplaneta (Khan and Fraser, 1962) and in Leucophaea (Scharrer, 1961). The packed granules (100-350 mp in diameter) can be seen with the electron microscope in the nerves that enter the corpus allatum in Celerio lineata (Schultz, 1960). The enlarged corpora allata in castrated Leucophaea females contain progressively increasing amounts of saliva-resistant granules staining with PAS, and presumably a glucoprotein of some kind; no such effect is seen in castrated malcs (Scharrer and von Harnack, 1961). E. THE C H E M I C A L N A T U R E O F T H E JUVENILE HORMONE

Since the corpus allatum of the young larval stages in Rhodnius will induce yolk production in the adult female, and the corpus allatum of the

292

V. B. W I G ( J L E S W O R T H

adult will maintain larval characters in the young stages (Wigglesworth, 1948) it seems likely that the suggesrion of Pfeiffer (1945a) that a single hormone is used for both purposes is probably correct. This hypothesis found practical application when Williams (l956a) showed that the adult ffyalophora,and particularly the adult male, is an exceedingly rich source of juvenile hormone. Simple extraction of the abdominal lipids with ether gives a deep orange oil which shows strong juvenile hormone activity. This discovery made possible the development of simple assay methods for the juvenile hormone. (i) Application to the abraded cuticle of the 5th-stage larva of Rhodnius. (ii) Application to punctures in the abdorninal sternites of Tenebrio pupae (Wigglcsworth, 1958). (iii) The application in solution in wax after excision of small areas of pupal cuticle in Hyalophora (Schneiderman and Gilbert, 1958). The so-called “wax test” on Hyalophora, which takes advantage of the increased sensitivity of regenerating cells, is the most sensitive; it gives positive results with a 1 : 2 0o0 dilution of the crude extract (Schneiderman and Gilbert, 1958); and in a later communication Gilbert and Schneiderman (1960) claim to have detected activity when a mixture containing 0.3pg of crude extract was applied in 20 OOO parts of wax. But the Tenebrio test, although it has not been possible to adopt it as a reliable quantitative test (Karlson and Nachtigall, 1961), has proved the most generally useful on account of its rapidity and simplicity (Schmialek, 1961). There are various factors which influence the susceptibility of the tissues in these tests. (i) The hormone is readily inactivated. If injected too long a time before the initiation of adult development the hormone is ineffective in the pupa of ffyalophora (Gilbert and Schneiderman, 1960); and the same quantity of extract produces much greater effects if it is dissolved in a large volume of vegetable oil and thus protected from rapid inactivation. Likewise, emulsified preparations are inactive. Inactivation is presumably a chemical process. It takes place more slowly at a low temperature. Pupae with their corpora allata removed inactivate the juvenile hormone at the same rate as normal pupae (Gilbert and Schneiderman, 1960). (In an earlier paper the writer had wrongly attributed inactivation in the normal insect at metamorphosis to the corpus allatum itself (Wigglesworth, 1948).) (ii) There is a constant sequence or pattern in the sensitivity of the various organs in the Hyalophora pupa (Williams, 1961b): the development of pigment in the eyes is most difficult to suppress; this requires the most active extracts (Gilbert and Schneiderman, 1960). A different

H O R M O N E S IN G R O W T H A N D R E P R O D U C T I O N

293

sequence was described in Galleria by Piepho (1942), who was implanting living corpora allata. But Piepho was implanting the gland into last stage larvae, whereas thc extracts of Gilbert and Schneiderman were injectcd into pupae. Likewise in the last stage larva of Aeschna, the different organs show different degrees of sensitivity to the juvenile hormone (Schaller, 1962). In Rhodnius the order of susceptibility is: general cuticle of the abdomen > lateral pleats > genitalia (the female being more susceptible than the male) > wings. It is those structures which show most cell division that fail to react to small amounts of the juvenile hormone. It may be the rapid cell division which enables certain tissues to escape the action of the hormone. For complete suppression of adult characters in all organs there must be abundant juvenile hormone available from the outset. The last stage larva of Lepidoptera is highly resistant to the juvenilizing effect of active extracts. This is attributed by Gilbert and Schneiderman (1960) in part to a powerful inactivation mechanism, in part to relative insensitivity of the cells at this stage. But it might also be due in part to the extensive cell division that accompanies metamorphosis. It is suggested by Slama (1961) that the apparent juvenile hormone activity of the extract from the adult male Hyulophoru is not due to the juvenile hormone itself but is a “regressive metathetely”, the result of injury and the suppression of development of imaginal cells, comparable with the imperfect adult cuticle seen in Pyrrhocoris and Galleria after injections of colchicine or surface applications of olive oil plus oleic acid. In Rhodnius, also, there can be some resemblance between the cuticle of adults after the repair of severe injuries, and that resulting from the action of the juvenile hormone. But such doubtful cases are exceptional; they can be regarded as equivocal and discarded. In most experiments the distinction between the larval and adult types of cuticle is unequivocal. The well-formed larval type has been obtained only after the implantation of corpora allata, the application of ether extracts from Hyulophora and from some other insects, and the use of preparations containing farnesol or closely related compounds (Wiggesworth, 1958, 1961b). The apparent occurrence in the extracts from Hyulophoru, of an agent which will activate the thoracic glands and induce a pupal moult, even in the brainless pupa, has already been discussed. Massive amounts of the extract are necessary for this purpose: 30 mg has no effect; 100 mg or more are needed to induce moulting (Schneiderman, 1961). Once the method of extraction had been discovered and methods of

294

V. 18. W l ( r O l , f ! S W O R T H

assay had been devised, it became possible to define the various sources of material with juvenile hormone activity. It was extracted from other insects (Wigglesworth, 1958) and from many other invertebrates; the crustacean eyestalk and the heads of polychaetes being particularly active (Schneiderman and Gilbert, 1958). It was demonstrated in the suprarenal glands of cattle (Gilbert and Schneiderman, 1958b), in many other tissues of vertebrates, including man (Williams et ul., 1959), and even in some plants, bacteria and yeasts (Schneiderman et al., 1960). Its recognition in these materials was greatly helped by the adoption of countercurrent separation in various solvents, which enabled the American workers to obtain highly concentrated extracts. Among the sources discovered were the excreta of the mealworm Tenebrio (Karlson and Schmialek, 1959). The active principle in this material, and in the extract from the suprarenal gland, and from yeast, was shown by Schmialek (1361) to be a mixture of the open chain terpene alcohol farnesol and its aldehyde farnesal. Synthetic trunstrans-farnesol was equally active. This material was traced during the fractionation procedures, by means of the Tenebrio test; but it was equally active when applied to the surface of the abraded cuticle in Rhodnius. Here it led to the production of giant larvae which retained the thoracic gland and could moult again; it induced egg production in the adult female deprived of the corpus allatum ;and it caused partial reversal of metamorphosis in the moulting adult (Wigglesworth, 1961b). Farnesol has not proved very effective in reproducing the effects of the juvenile hormone when injected into the body cavity of Rhodnius (Wigglesworth, 1963a). A series of farnesol derivatives were examined with the Tenebrio test by Schmialek (1963~).He found that the activity is reduced by methylation at C-10, by hydrogenation of the double bond between C-10 and C-11 (dihydrofarnesol), and by extending the length of the carbon chain at the polar end beyond nineteen carbon atoms (geranyl-geraniol). Activity is increased by blocking the C-1 end of the molecuIe as farnesyl acetone or as farnesyl methyl ether, ethyl ether or butyl ether. Schmialek (1963b) further showed that farnesyl methyl ether applied to punctures on the pupa of Tenebrio has a powerful juvenile hormone effect throughout the body; the compound is active also in the food of the larva of Tenebrio provided it is given immediately after the larval moult. Some of these compounds were tested on Rhodnius, and again, farnesyl methyl ether was among the most active. Like the natural extract from Hyulophora it is completely inactive in large doses if injected in the form of a fine emulsion. But like the natural extract it is much

HORMONES IN G R O W T H A N D REPRODUCTION

295

more active whcn injectcd in solution (0.1, 1 or lo”/,) in paraffin or triglyceride oil. Given in this way 5pml (i.e. 25pg/g) will induce a completely larval moult in a 5th-stage larva; 2pml (i.e. 8pg/g) will ensure continued larval activities of epidermal cells that are due to persist into the next instar. This results in adult insects with larval abdomen. Like the natural extract, this material is circulated by the insect and will affect a second insect in parabiosis; a small dose is more effective near the time of cuticle formation (Wigglesworth, 1963a). Among naturally occurring substances, farnesyl pyrophosphate is quite inactive (presumably it is too easily metabolized); “dendrolasin” (the furan of farnesal, which was isolated from the mandibular glands of Lasius by Quilico et a!. (1956)) has weak juvenile hormone activity (Wigglesworth, 1963a); the vitamin K fractions, Ka(30), 2-methyl-3 (all-trans-farnesyl-geranyl-geranyl)-1,4-naphthoquinone,and Ka(35), 2-methyl-3-farnesyl-farnesyl- 1,4-naphthoquinone, kindly put at my disposal by Dr. 0. Wiss of Messrs. Hoffmann-La Roche, Basel, gave entirely negative results (Wigglesworth, unpublished). Yamamoto and Jacobson (1962) have confirmed that the juvenile hormone activity of farnesol depends on the trans-trans configuration. Negative results were obtained with geraniol, citronellol, squalene, and mevalonic acid. Fukaya (I 962) found that farnesol applied to the cuticle of rice stem borer larvae in the last stage produced a high degree of metathetely, and in the last stage larva of Periplanetu it led to the appearance of short wings in the adult. Slhma (1962) observed suppression of normal wing growth in Pyrrhocoris larvae when either short chain and unsaturated fatty acids, or farnesol or nerolidol were applied to the cuticle of the wing pad. He regards this as a metathelic effect and therefore considers that this effect is not specific for the juvenile hormone. But it would seem that what is being observed in these experiments is a nonspecific toxic action which interferes with normal cell multiplication and growth. This is something different from the morphological changes and the persistence or breakdown of the thoracic gland controlled by the presence or absence of the juvenile hormone. The question naturally arises whether the active principle in the insect is actually farnesol (or some closely related compound) or something quite different. The farnesol and farnesal isolated by Schmialek (1961) from the excreta of Tenebrio could have been derived from the food. Williams (196Ia) reports that preliminary infrared and nuclear magnetic resonance studies on the concentrated material from adult male HyuZophora “suggest that the active principle is a water-insoluble lactone rich in methyl and methylene functions”.

296

V. B. WIGGLESWORTH

In all its biological properties as studied on Rhodnius, farnesyl methyl ether is extremely similar to the natural hormone. If it were suitably formulated (as seems to happen in the insect when it is taken up by the haemocytes from the injected oily solutions) it would be indistinguishable. Schmialek (1963a) extracted the juvenile hormone from a number of large species of Saturniids (Hyalophora cecropia, Platysamia Cynthia, Antheraea pernyi) and concentrated the active material on an alumina column. The material was then further fractionated by thin layer chromatography, the various bands being eluted and used for the Tenebrio test. The juvenile hormone activity appeared at two points, with RP values corresponding with those of farnesol and farnesal. Further, mevalonic acid (the precurosr substance of farnesol and cholesterol in the biosynthesis of these materials) when labelled with 14Cand injected into male silkmoths (Platysamia Cynthia) was shown (by thin layer chromatography and gas chromatography) to become converted into farnesol, farnesal and nerolidol. The farnesol was the trans-trans isomer; the nerolidol showed no juvenile hormone activity. It appears, the?efore, that the main substances with juvenile hormone activity present in the Saturniid moths are indeed farnesol and farnesal; and these substances are synthesized by the insect itself. The possibility remains, of course, that other materials with juvenile hormone activity may be present, but these have not so far been detected. As introduced into the insect under experimental conditions, farnesol is not a very effective material. It is doubtless rendered effective in the insect by suitable formulation, presumably in association with some protein carrier. This is a well known phenomenon in the endocrinology of vertebrates, where thyroxin and oestrone are relatively ineffectual in simple solution or suspension.

VI. H O R M O N ACONTROL L O F REPRODUCTION A. C O R P U S ALLATUM A N D R E P R O D U C T I O N

It was found in Rhodnius that the corpus allaturn again becomes active in the adult stage, and its secretion is then necessary for the later development of the oocytes in the female, that is, for the deposition of yolk, and for the full activity of the accessory glands in the male (Wiggles-

H ( J H M 0 l r ; k S IN G R O W T H A N D R E P R O D U C T I O N

297

worth, 1936). This was confirmed in the grasshopper Melanoplus, where the corpus allatum hormone is ncedcd also for the secretory activity of the lower part of the oviduct, which produces the ootheca (Pfeiffer, 1939); and in Cimex after fertilization (Mellanby, 1939). Other insects in which the corpus allatum is necessary for yolk formation are LRptinotarsa (de Wilde, 1954) and the earwig Anisolubis (Ozeki, 1958). In the mosquito Culex molestus the ovaries will develop in the absence of a meal of blood ;whereas in Culex pipiens and Aedes aegypti a blood meal is always needed. This difference seems to depend upon differences in the secretory activity of the corpora allata, for transplantation of C. molestus corpora allata into C. pipiens hosts will induce egg development in these even in the absence of a blood meal; and transplanted ovaries of C. molestus will develop in C. pipiens or A . aegypti only when these hosts are given a meal of blood (Larsen and Bodenstein, 1959; and cf. Geiger, 1961). In Rhodnius and in most other insects studied the oocytes develop normally in the absence of the corpus allatum until they reach the stage when yolk is deposited. But in Dytiscus the corpus allatum secretion is necessary at a much earlier stage of oocyte development (P.Joly, 1945). As we have already seen, metamorphosis, which is controlled by the corpus allatum, is comparable with puberty: the ovaries are capable of producing ripe oocytes only when it has taken place. In Locusta, if an active corpus allatum is implanted too late in the 5thinstar to affect metamorphosis, it may accelerate ovarian development. If the corpora allata are extirpated early in the 5th-instar the oocytes in the resulting adult develop up to a length of 0.9 mm and by then contain an appreciable amount of yolk. If extirpated in the adult at 8 days, the oocytes reach a length of 1.4 mm before they cease growing (L. Joly, 1960). That applies also to Periplaneta (Girardie, 1962). If the corpora allata are extirpated in the last larval stage, growth in the ovaries is arrested only after the beginning of yolk formation. But if extirpated in the 9th or 8th larval stage, arrest occurs before yolk formation, because in the precocious adults produced the ovaries are still in a very juvenile condition. T h e activity of the corpus allatum and the ripening of the oocytes are highly dependent upon nutrition. In unfed Rhodnius adults new oocytes develop continuously but each dies when it reaches the stage when yolk formation should occur, and both oocyte and the investing follicle cells undergo involution (Wigglesworth, 1936). In the pupa of Bombyx mori the nutrient materials available for development of the ovaries normally exceed 30 % of the pupal weight. If they are less than this, many of the

298

V. B. W J G G L E S W O R T H

ova undergo degeneration in the devzloping ovaries (Hasegawa, 1943, 1947). The role of nutrition in the activity of the corpus allatum is particularly striking in Oncopeltus. In the normally fed adult female the corpus allatum increases in volume 12 times during the 2 weeks after moulting; in insects given water alone it increases only 2.5 times. And if the corpus allatum is removed from a normally fed female and implanted into a female that has been given water alone, she will start egg production despite the lack of food (Johansson, 1954). The same result is seen in Leucophueu where again starvation inhibits ovarial development not by a direct effect, but through the corpora allata: starved females will develop eggs if corpora allata from fed donors are implanted (Johansson, 1955; von Harnack, 1958). During starvation the corpus allatum seems to be inhibited by the brain, for severance of the corpus allatum nerves results in activationof the gland and the beginning of egg maturation (Johansson, 1958). In the female Andrena, parasitized by Stylops, the ovaries are defective and reproduction fails. Brandenburg (1955) suggeststhat this results kom an indirect action via the corpora allata, which are much smaller in size in the parasitized insects. This again may well be a nutritional effect. Adult females of the blowfly Calliphora ingest preferentially a fluid rich in protein during the early stages of egg growth, while during yolk formation protein ingestion declines, carbohydrate feeding increases, and the volume of the corpus allatum is reduced (Strangways-Dixon, 1961a, by 1962). But here it is difficult to decide which is cause and which is effect. A subject of controversy has been whether the hormone necessary for yolk formation is identical with the juvenile hormone. It was originally assumed'in the case'of Rhodnius that two different hormones were concerned. But it was pointed out by Pfeiffer (1945b) that the corpus allatum of the Melunaplus adult continues to secrete the juvenile hormone; she suggested that the same hormone might be concerned in yolk formation. And later it wasproved in Rhodnius that the corpus allatum of young larvae will induce yolk formation, and the corpus allatum of the adult will prevent metamorphosis ; which strongly suggested that a single hormone was involved (Wigglesworth, 1948). In the earwig Anisdubis the corpora allata of the adult and of young larvae are said to prqduce both juvenile hormone and yolk-forming hormone, while the corpora allata of last stage larvae produce only the latter (Ozeki, 1958)-but Anisolubis may complete its metamorphosis at either the 4th,5th or 6th moult. Luscher and Springetti (1960) formed the opinion that in Culorermes

299 flavicollis the juvenile hormone and a gonadotropic hormone are concerned in caste determination and in yolk development. But it is possible that their results may be explicable by a higher concentration of a single factor (the juvenile hormone) being necessary for yolk formation. The fact that the methyl ether of farnesol has both a juvenile hormone effect and a yolk promoting effect, renders it almost certain that a single hormone is responsible for both activities (Wigglesworth, 1961b, 1963a); but considerably larger quantities of the active principle are needed for yolk formation. In Schisrocercu, as in Rhodnius etc., the secretion from the corpus allatum has a direct effect on the ovary: it seems to ensure the transfer of nutrients by the follicular cells from the haemolymph to the yolk. In the absence of the corpora allata the oocytes are resorbcd. This resorption can be at least partially prevented by smearing the insects with farnesol (Highnam et a)., 1963a, b). Both cecropia extract and farnesol applied to the surface of the integument of allatectomized females of Peripluneta will induce yolk formation (Chen et al., 1962). The question arises as to why a hormonal stimulus should be needed by most insects for the full activity of the reproductive system. The reason may well be the same as that suggested for the existence of a moulting hormone (p. 261), that is, because egg production, like moulting, is a cyclical process. It is desirable that it should be initiated only at those times when (i) nutrition, (ii) the phase of the reproductive cycle, i.e. the presence or absence of eggs lower down the tract, and (iii) the season, are all appropriate. The insect must have some mechanism for restraining ovary development. In Thysanuruwhere a moulting cycle alternates with a reproductive cycle throughout adult life, hormonal regulation is clearly necessary (Watson, 1962). In the parthenogenetic Dixippus where feeding and egg production are continuous processes in the adult female, there seems to be no call for hormonal regulation, and removal of the corpora allata does not influence yolk formation (Pflugfelder, 1937). Particular interest attaches to the Lepidoptera. Bounhiol(l938) showed that in Bombyx mori, as in Dixippus, removal of the corpora allata does not prevent the development of eggs; and Williams (1952) observed that the isolated abdomen of Hyolophoru cecropia, induced to undergo metamorphosis by implantation of thoracic glands, will produce eggs. The corpus allatum appears to play no part in the egg development of these insects. But even in B. mori extirpation of the corpora allata from the 5th-stage larva always results in a decrease of some 12% in the number of eggs produced (Yamashita et al., 1961). And in Galleria the corpora allata are clearly necessary for egg production. If they are removed at the end of the last larval stage the normal number of eggs is produced; H O R M O N E S IN G R O W T H A N D R E P R O D U C T I O N

300

V. B. W I G G L E S W O R T H

if rcmoved at the bcginning of the last stage, the number of eggs falls to 38 ‘%, of the normal; if removed at thc beginning of the penultimate larval stage, thcre is a further reduction to 13 of the normal (Roller, 1962). Thcsc efrccts can be reversed if other corpora allata are implanted. And similar eti’ects arc produced if the corpora allata are inactivated by starvation (“pseudoallatectomy ” of Johansson (1958)). These are species of Lepidoptera in which all the eggs are produced in one large batch which is developed during pupal life. It is possible that in other Lepidoptera, in which egg development can depend upon feeding or on water drinking by the adult, the corpus allatum may prpve to be involved. L. Joly (1960) suggests that in those Lepidoptera which have been studied, the puberty change which renders the ovaries competent to develop, takes place while juvenile hormone is still present in the moulting larva or pupa. In the mealmoth Ephesriu kuehnieila the corpora allata undergo renewed growth in the pupa, and this growth is much greilter in the male. During imaginal life there is prolonged secretory activity in the male; in the much smaller corpora allata of the female the secretory process is much less marked (Rehm, 1951). This sexual difference in the size of corpora allata occurs also in Hyutophora adults (in the pupa of either sex the corpus allatum weighs 30kg; in the adult female, about 65pg; in the adult male, about 400pg) and it is reflected in the amount of juvenile hormone accumulating in the tissues. The adult male contains five times as much ether extractable lipid per gram wet weight as the female; and the oil extracted from the female has only one-eighth of the juvenile hormone activity of the male oil. The total content in the male is therefore about forty times that in the female (Gilbert and Schneiderman, 1959, 1961). During the period of chilling in the pupa the small amount of juvenile hormone present disappears ; the corpora allata are re-activated during the later days (17th-20th day) of adult development-as in Ephestiu (Rehm, 1951). If ovaries are implanted into the male pupa the extractable juvenile hormone is markedly decreased. It does appear, therefore, that the geveloping ovaries consum’ejuvenile hormone (Gilbert and Schneiderman, 1957,1958a). But the large corpora allata and the very large amounts of hormone in the adult male suggest that it has an important function in this sex. In the male Rhodnius it is needed for the activity of the accessory glands (Wigglesworth, 1936), which are responsible for secreting the spermatophorq. Adult Lepidoptera produce great numbers of spermatophores in rapid succession (Norris, 1932). It is probable that that is where the function of the corpus allatum secretion in male Lepidoptera lies.

x,

H O R M O N E S XN G R O W T H A N D R E P R O D U C T I O N

30 1

B. R O L E OF T H E N E R V O U S SYSTEM A N D N E U R O S E C R E T O R Y C E L L S IN O V A R I A N DEVELOPMENT

It was shown by Thomsen (1942) that the corpus allatum secretion plays an important part in the deposition of yolk in the oocytes of Culliphoru; but occasional females developed their eggs fully even when the corpus allatum was removed. Possomphs (1955) found that if the corpus allatum was extirpated in the Culliphoru larva, the resultant females developed their ovaries and laid viable eggs. And in 1952 Thomsen proved that the source of the hormone concerned was the medial group of neurosecretory cells in the brain. If these were extirpated, the egg chambers failed to grow beyond about 0.17 mm. Subsequent implantation of corpora allata induced some slight further growth-but not beyond the initial stage of yolk deposition ; re-implanted medial neurosecretory cells led to complete development. The corpus cardiacum serves as a store for the product of the neurosecretory cells and can therefore replace them in the implantation experiments. Gillett (1957) in studying egg development in the mosquito Aedes aegypti agrees with Clements (1956) who worked with Culex, in showing that development is initiated within minutes of the blood meal. The effect (due perhaps to hormone released from storage) will carry the oocytes from the resting stage (stage 2) as far as stage 3. But complete development up to maturity (stage 5 ) is dependent on a hormone released during a critical period 8-14 h later, presumably secreted by the neurosecretory cells of the brain. Transfusion of haemolymph from mosquitos in the middle of this critical period, to mosquitos decapitated just before the critical period, results in a greater number of them developing their oocytes to maturity (Gillett, 1958). There is good evidence, therefore, that in these insects the neurosecretory cells in the brain arc the chief source of the hormone necessary for yolk formation. But in Rhodnius there is no reduction in the number of eggs produced when the brain is removed, provided the corpus allatum remains (Wigglesworth, 1963a). Tn the stick insects Dixippus and Clitumnus eggs are produced in the absence of the medial neurosecretory cells, but in smaller numbers (Dupont-Raabe, 1952,1954). The same is true of Oncopelrus. Hcrc there arc four typcs of large neurones in the pars intercerebralis. The most conspicuous are the A cells which give rise to the medial nerves carrying ncurosecretory material to the corpus cardiacum. But this material is stored, not in the corpus cardiacum, but in the walls of the aorta. If thesc nerves are cut, the neurosecretory material accumulates above the point of section. Fecundity is reduced after removal of the

302

V.

B. W I G G L E S W O R T H

A cells, and the corpus allatum is smaller than normal; but mature eggs are developed. The essential soprce of the yolk-forming hormone seems to be the corpus allatum, but this gland is dependent on connection with the neurosecretory cells for its full activity, and these cells in turn are dependent upon nutrition (Johansson, 1958). We are thus concerned with a complex system comprising neurosecretory cells in the protocerebrum and suboesophageal ganglion, the corpus allatum and corpus cardiacum, and the ovary. These components influence one another by nervous stimuli, by neurosecretions carried along nerve pathways, and by humoral factors in the circulating blood. The relative parts played by the different elements in this system vary from one insect to another. They have been most closely studied in the Orthoptera. In Leucophueu maderue the brain is the final regulator of the function of the corpus allatum throughout the reproductive phase. The species is ovoviviparous and the ovaries become inactive during pregnancy. This inactivity results from the arrest of secretion by the corpus allaturn. If active corpora allata from larvaeare implanted into pregnant females the ovaries resume their development, the oocytes ripen, and yolk is deposited. The corpus allatum (and thus the ovary) can be activated during pregnancy by cutting the corpus allatum nerve or the medial corpus cardiacum nerve, or by destroying a certain part of the protocerebrum. This is effective even when the neurosecretory ceus and the pathways for the neurosecretion are untouched. It appears that during pregnancy a nerve centre in the brain inhibits the corpus allatum; but activation of the corpus allatum by these operations takes place only when the nervous connection between the suboesophageal ganglion and the corpus allatuxq remains intact. The corpus allatum cannot continue to function if all nervous connections are severed (Liischer and Engelmqnn, 1955 ; Engelmann, 1957). It will be recalled that a similar inhibition of the corpus allatum comes into operation during the metamorphosis of the last larval stage (p. 282). The mechanism by which the presence of developing eggs in the lower part of the genital tract brings about this nervous inhibition from the brain has been a subject of controversy. In Latcophueu this inhibition is relieved and the corpora allata become active again if the ootheca is removed from the brood chamber. But inhibition is said to persist if the developing eggs are implanted in the body cavity. That led to the suggestion that normally a humoral influence is exerted on the brain by some substance from the eggs ir, the blood sac (Engelmann, 1957). Roth and Stay (1959) working with the cockroaches BIuttellu and

1IORMONT.S I N G R O W T H A N D R E P R O D U C T I O N

303

Pycnoscelus produced evidence that the inhibition of development in the colleterial glands and ovarian eggs by the presence of an ootheca in the brood chamber is effected by the mechanical stimulus of stretching. For the inhibition can be maintained by the insertion of a dummy ootheca made of glass. Removing the ootheca or cutting the nerve cord reactivates the corpora allata and restores egg development. The same conclusion was reached in Diploprera (Roth and Stay, 1961). In unmated females of Blattellu w g a , which have long carried an ootheca with undeveloped eggs, the corpora allata may eventually become active again. Presumably there is some central or peripheral “adaptation” to the sensory stimuli (Roth and Stay, 1962a, b). Engelmann (1 960) supported these conclusions in renewed experiments on Leucophaea where severance of the ventral nerve cord in the pregnant female results in activation of the corpora allata. The picture is still further complicated by the fact that the totally isolated corpus allatum in Leucophaea shows cyclic activity correlated with the periodic cycles of egg growth. The fact that this cyclic activity is released by severance of the ventral nerve cord suggests that a mechanism involving a nervous pathway from the posterior abdominal region normally restrains the corpora allata (Engelmann, 1962). If larval prothoracic glands are implanted into adult females of Leucophaea, or if ecdyson is injected, and they are thus induced to moult, the eggs do not ripen. This was attributed by Engelmann (1959a) to a direct inhibition of the corpus allatum by the thoracic gland hormone; but it is possible that, as a result of the induction of moulting, the growing tissues compete for nutrients with the ovaries and in this way prevent the ripening of the eggs. That was the explanation suggested for similar observations in Rhodnius (Wigglesworth, 1952a). Another example of a similar competition for nutrients was described in mosquitos by Larsen and Bodenstein (1959). A young ovary fails to develop if it is transplanted into a female with older ovaries; but if the implanted ovary is the older, then the maturation of the ovaries of the host is depressed. A further stimulus that can intervene in the hormonal control of reproduction is the act of mating. In the viviparous cockroach Diplopteru mating seems to be cssential for the normal rate of egg growth; and since allatectomy aftcr mating prevents egg maturation, the stimulus connected with mating appears to activate the corpus allatum (Engelmann, 1958). The stimulus to the corpus allatum is probably nervous, for section ofthe abdominal nerve cord prior to mating also stops egg growth. On the other hand, if the corpora allata are separated from the brain in virgin

304

V. B. W I G G L E S W O R T H

females they become prematurely active and several successive batches of eggs may be developed. It appears that the corpora allata are normally restrained by the brain and that mating stimuli counteract this restraining influence. During pregnancy the corpora allata are similarly restrained and they resume their activity only in response to renewed afferent nervous stimuli, normally provided by the act of parturition. Egg maturation can also be induced by inserting an artificial spermatophore in the form of a glass bead into the bursa copulatrix or by excising the gonapophyses of virgin females (Engelmann, 1958).Thus mating(in the virgin female) or parturition in the pregnant female can provide an adequate stimulus, by way of the ventral nerve cord, brain, and corpus allatum, to induce the ripening of the eggs (Engelmann, 1959b). It may be recalled here that in Cinier also the corpus allatum does not become active until the sperm, after passage through the body cavity, have reached the correct part of the fernale genital tract; and that soon after the sperm have disappeared from the spermathecae, the ovary reverts to the virgin condition (Mellanby, 1939).Another example of a peripheral nervous stimulus to ovarian development is seen in the mosquitos Culex pipiens and Aedes aegypti, in which prolonged distension of the gut and abdomen by sealing the anus will lead to egg development even in the absence of a blood meal (Larsen and Bodenstein, 1959). The hormonal control of reproductive physiology has been analysed in considerable detail in locusts. In Locusta each batch of eggs represents the terminal oocytes. These batches succeed one another at intervals of 6-7 days. But the interval between successive oocytes is only 24 days. Clearly the presence of ripe eggs inhibits the ovary until they are discharged (L. Joly, 1960). In normal females of Locusta development of the ovaries is promoted by copulation even with castrated males. After removal of the terminal abdominal ganglia, development of the ovaries is normal, but the female does not lay and the eggs are reabsorbed (Quo Fu, 1959). As Highnam (1962a) points out, the linkage of the brain, by way of the corpus allatum, with ovarial development, is common to many insects, but the participation of the brain in this process may be either humoral or nervous. In Schisrocerca, cauterization of the neurosecretory cells, or removal of the corpora cardiaca, prevents growth of the terminal oocytes; re-implantation of neurosecretory cells to such females gives rise to normal development (Highnam, 1962b). The secretion from the brain is evidently important in ovarial growth. In Locusta, removal of the frontal ganglion will prevent sexual maturation in both sexes. As in the

HORMONES I N G R O W T I I A N D R E P R O D U C T I O N

305

larval stages (p. 253) it seems to serve as a relay station between the intake of food and the release or synthesis of neurosecretory material in the brain (Clarke and Langley, 1962, 1963a). Hodgson and Geldiay (1959) ,had shown that in Biaherus electrical stimulation of the brain causes depletion of the neurosecretory material in the protocerebrum. The same change occurs in Schistocerca if the optic nerves are stimulated with high frequency electric shocks. Emptying of neurosecretory cells and corpora cardiaca of stainable material can also be brought about by rotating the female in a flask, by drastic wounding or by copulation-and all these treatments lead to accelerated development of the oocytes (Highnam, 1961a). I t had been shown by Norris (1953) that copulation is necessary if Schistocerru is to lay the full complement of eggs; and repeated copulation is needed for successive batches--3lthough a single copulation will supply all the spermatozoa that are required. It seems likely that copulation provides the normal stimulus for the liberation of the neurosecretory material. Moreover, all the evidence suggests that neurosecretory cells which are laden with stainable secretion are in the inactive state, partially emptied cells are active (Highnmi, 1962a, b). The fact that DL-cystine labelled with 35Sis taken up by the neurosecretory cells much more strikingly in females reared with mature males, supports the conclusion that when the system contains little stainable material the hormone is being released most rapidly ( Highnani, 1962a, b). For the neurosecretory system contains little stainable material in females whose terminal oocytes are developing rapidly in the presence of mature males, but contains a large amount whcn the terminal oocytes are mature, or in females reared without males. whose terminal oocytes are developing very slowly (Highnam and Lusis, 1962; cf. Dupont-Raabe, 1951, 1954, 1956). Likewise, in the Chrysomelid beetle G'ufrruca tanaceti the rate of turnover of r:idio;iclivc cystine in the ncurosecretory cells (A cells) is increased about tenti)ld whcn the female goes from diapause to oviposition, and there is a corresponding increase in the rate of discharge from storage in thc corpus cardiacum (Sicw, 1963). Thus, rearing fcniales of Schisrocvvm without males has three effects: the ovarics of some feinalcs do not start developing; or the rate ofdevelopment of terminal oocytcs is low; and the percentage of resorbed oocytes increases (Sirigh, 1958; Highnnm and Lusis, 1962). Loher (1960) found that certain colour changes in the mature male of Scliistocerca, notably the appearance of a bright yellow pigment in the epidermis, are brought about by the secretion from the corpus allaturn.

306

V. B. W I L G L E S W O R T H

These changes arc associated with the production of a volatile lipophilic secrction from thc epidermis which servcs as an olfactory stimulus that accelerates maturation in both males and females. It may be that this external chemical factor, or “ pheromone”, from the male, lowers the general sensory threshold of the female, and so favours the discharge of material from the neurosecretory system (Highnam and Lusis, 1962). In Dermestes maculatus there are three groups of neurosecretory cells in the brain, of which the large A type cells of the medial group seem to be concerned in reproduction. Ovarial development is totally inhibited in young females deprived of this group of neurosecretory cells. In those deprived of the corpora allata, the eggs develop up to the point where yolk deposition is due to begin. In mature females deprived of the neurosecretory cells, oviposition and further oocyte development are prevented, and the corpora allata atrophy. All these processes appear to depend on the continued activity of the medial neurosecretory cells (Ladduwahetty, 1962). It seems that the neurosecretory cells, the corpora cardiaca and the corpora allata act as a unit, and the relative importance of the components in this unit seem to vary in different insects. Thomsen (1 952,1954) showed that in Culliphora the neurosecretory system may influence ovarian development both directly and by the production of a hormone acting on the corpus allatum. The neurosecretory system provides a link between the nervous and endocrine moieties of the co-ordination mechanism (Highnam, 1962a, b). Perhaps there are two factors involved : the oocytes must be kept alive; and they must be furnished with the protein and other reserves needed for yolk formation. In Rhodnius and in Oncopeltus the juvenile hormone seems essential for the survival of the oocytes (Wigglesworth, 1936; Johansson, 1958). Perhaps the nutritional requirements demand the activity of the neurosecretory cells (Thomsen, 1952; Hill, 1962, 1963). This will be considered in the section dealing with the metabolic effects of growth hormones (p. 308). C. C O N T R O L O F O V U L A T I O N A N D O V I P O S I T I O N

Humoral factors, probably derived from the neurosecretory cells in the brain, are involved also in ovulation and oviposition. The blood of fecundated Bomhyx mori will induce oviposition when injected into unfertilized females (Mokia, 1941) and it is at the time of oviposition that the neurosecretory material in the adult female Bombyx is most actively discharged along the axons (Arvy et al., 1953). In Dermestes, mating acts as a stimulus to oviposition and appears to hasten final maturation

H O R M O N E S I N G R O W T H A N D REPRODUCTION

307

of the eggs (Roth and Willis, 1950); and in Aedes aegypti females, also, ovulation seems to be provoked by some humoral factor which is liberated when the spermathecae are filled with sperm (Gillett, 1956). In the Pyrrhocorid bug Iphitu fimbuta the neurosecretory cells of the pars intercerebralis seem to be concerned in oviposition. Immediately before oviposition no visible neurosecretory material can be seen entering the corpus allatum, from which the supply seems to be cut off; but instead it flows into the anterior end of the aorta close to the corpus cardiacum. Meanwhile the material in the neurosecretory cells becomes depleted; and in females which have started oviposition the cells are almost devoid of stainable material. Blood from a laying female will induce oviposition when injected into a female not yet fully gravid; but blood from earlier stages, or from a female which has started mating again, has not this effect. It may be that an “ovarian hormone” is concerned in this response, for injection of a watery extract of the ripe ovary will likewise stimulate the neurosecretory cells to discharge their secretion and so induces immediate oviposition. The role of the neurosecretory cells in oviposition has been confirmed by implanting clusters of these cells into nearly gravid females: they likewise induced immediate oviposition (Nayar, 1958). In the normally mated female of Dermestes maadatus, oviposition is continuous and the secretion from the A cells is continually released; these cells are in a state of “high release”. In the unmated females, oviposition never occurs and the secretion is released only in small quantities : accumulated secretion is present in the cell bodies, which are in a state of “low release”. Alternation of these two states is seen in females mated at wide intervals. Mature eggs are ovulated throughout the “high release” condition. They are resorbed throughout the “low release ” condition and form “resorption bodies”. Virgin females joined in parabiosis with mated females begin to oviposit, and their A cells attain a state of “high release”. Further experiments led to the conclusion that the spermathecae containing active spermatozoa release a “ spermathecal factor” which provokes oviposition by stimulating the A cells to release more secretion (Ladduwahetty, 1962). VII. METABOLIC HORMONES AND INTEGRATION

HORMONAL

A single hormone can produce widespread effects on morphogenesis and metabolism. But during normal growth several growth hormones may be operating, as happens with the brain hormone, thoracic gland

308

V. B . W I G G L C S W O R T H

hormone, and corpus allatum hormone during larval moults, or the brain hormone and corpus allatum hormone during reproduction. In the course of growth and moulting the cellular processes take place in a regular sequence, which is correlated with changes in metabolism throughout the body. We are confronted. therefore, with two problems: (i) are the hormones concerned directly in starting particular processes of metabolism, or are these processes a consequence of changes elsewhere in the body, which have been set going by the hormones; (ii) by what mechanisms, humoral or otherwise, is the co-ordination of the processes of growth in different parts of the body secured? A. M F T A B O L l C t l O R M O N E S

The action of insect hormones is comnionly associated with a rise in oxygen consumption. As we saw earlier ( p . 263) this is largely the result of the heightened synthesis which growth entails, including the synthesis of additional respiratory enzymes to support the processes of growth. In adult females of C’ulliphora the corpora allata are necessary to maintain full oxygen consumption (Thomsen, 1949);and this oxygen consumption is not reduced by castration-which suggests that the endergonic syntheses responsible are taking place outside the ovary, presumably in the fat body (Thomsen and Hamburger, 1955). Some of the clearest evidence of a direct efkct of the corpus allatum on metabolic processes outside the ovary was given by Pfeiffer (1945a). In each maturation period in female M i k m p l r t s there is a great increase in the size of the fat body. At the close of this period, yolk production begins and hypertrophy of the fat body ceases. This change is not caused by the ovaries, for it occurs also in castrated females which retain their corpora allata. But if the corpora allata are removed, the fat body continues to increase and becomes enormously hypertrophied. The corpora allata seem to affect fat metabolism in two ways: (a) by indueing utilization of stored fat in a process independent of the ovaries; (b) by facilitating production of yolk, which in turn affects fat metabolism. At maturation the fat stores are reduced: in the normal female, to a low level at which they are stabiliLed; i n the castrated female, to an extremely low level with no stabilization. The metabolic change at maturation In Mt~1ai:oplusis also followed by great increases in non-fatty material (probably glycogen) and water. In the normal female these changes are associated with yolk formation; in the castrated female both the accumulation in the fat body and the enormous increase in blood volume may be even more marked. These

HORMONES I N G R O W T H A N D R E P R O D U C T I O N

309

changes again are the result of corpus allatum activity: they do not occur in females with the corpora allata removed, whether castrated or not. Pfieffer (1945a) therefore concluded that the corpora allata control egg production principally, if not entirely, through the agency of a metabolic hormone; and the primary function of this hormone is .o facilitate mobilization or production of materials necessary for egg growth. Vogt (1948), who observed that extirpation of the corpus allatum in either sex of Drosophifu leads to a great increase in fat storage, likewise favours the view that the corpus allatum hormone has both gonadotropic and metabolic effects. Comparable results were obtained in Leucophaea by Sagesser (1960). Active corpora allata (from donors which had deposited their eggs four days earlier) implanted into castrated females with the accessory glands removed, caused increased oxygen consumption. He describes the corpus allatum secretion as “ a hormone which stimulates respiratory metabolism’’-but gives no indication of the nature of the process that is being stimulated. In the larva of Galleria, well-marked changes in metabolism follow removal of the corpora allata (Raller, 1962). Although the mid-gut cells continue to secrete proteinases and allow amino acids to be absorbed, no intrinsic body protein is synthesized. In addition, these allatectomized larvae are unable to build up carbohydrates and fats. All these effects are ascribed to a failure in the synthesis of enzyme proteins. In recent years the metabolic effects of hormones in reproduction have been complicated by the discovery of the role played by the neurosecretory cells. The secretion from these cells is necessary for yolk production in female Culliphoru (Thornsen, 1952). An important effect of the hormone is to stimulate the production of proteolytic enzymes in the mid-gut of the fly that has been fed on meat (Thomsen and Maller, 1959, 1963). The protease activity in females deprived of the medial neurosecretory cells is only about onequarter to one-third of that in normal females on the same diet. The corpus allatum has only a minor effect on protease activity-perhaps as the result of its stimulating the neurosecretory cells. Implantation of the corpus cardiacum (presumably containing neurosecretory material) into flies with normal neurosecretory cells, will increase protease activity about 50 %. Since proteases are themselves proteins, Thomsen and Msller suggest that the hormone from the neurosecretory cells may be involved in the regulation of protein synthesis in general. The importance of the product of the medial neurosecretory cells in the protein metabolism of locusts has been made very clear by the work

310

V. 9. W I G G L E S W O R T H

of Hill (1963) and Highnam et al. (1963a, b). We saw that in Schistocerca the corpus allatum hormone appears to act directly upon the growing oocytes and the follicular cells of the ovary; the neurosecretory system acts upon the protein metabolism of the body. When the neurosecretory system releases material into the haemolymph, the blood protein rises; when the material is accumulating in the system, the blood protein falls. The neurosecretory material induces the fat body to synthesize blood protein from the circulating amino acids: if labelled glycine is injected I

Hours after injection

Fro. 2. The rate of incorporation of 14C glycine into the proteins of fat body, haemolymph, and oocytes of Srhistorerca. (After Hill, 1963.)

(Fig. 2) it appears in the form of protein, first of all in the fat body cells, then in the haemolymph, and finally it begins to appear in the oocytes (Hill, 1963). It is the formed proteins of the haemolymph which are transferred by the follicular cells into the developing oocytes (Telfer, 1954, 1960).When progressively larger amounts of the ovary were removed from a series of Schislocerca females, the concentration of protein in the haemolymph rose from around 3 % in the normal females, to 7.3 % when two-thirds of the ovaries had been removed, and to 13.4 % when ovariectomy was nearly complete (Hill, 1963; and cf. Telfer, 1954). Cauterization of the neurosecretory cells results in a low level of haemolymph protein (1-1.5 %). Whereas all those factors which induce liberation of neurosecretory material and accelerate maturation of the ovary (hyperactivity, rearing alongside mature males, copulation etc.,

t i O R M O N E S IN G R O W T H A N D R E P R O D U C T I O N

31 I

p. 305) also increase the level of haemolymph protein. The implantation of corpora cardiaca containing the stored secretion produces a rapid but transitory rise. The neurosecretory material Seems to be required continuously throughout the synthetic process. Three protein fractions were demonstrated by electrophoresis of the haemolymph; one of these in particular increased greatly after ovariectomy and during yolk deposition (Hill, 1962,1963). We saw earlier (p. 297) that in the absence of juvenile hormone the oocytes are resorbed. The same applies to Schistocerca, in which resorption of oocytes takes place (i) as the result of competition between oocytes for corpus allatum hormone, and (ii) as the result of competition for available protein, the product of neurosecretory activity (Highnam et al., 1963a, b). The corpora allata appear to exert an activating effect upon the neurosecretory cells : if they are removed in Schistocerca the neurosecretory system becomes inactive. As Hill (1963) points out, the observed decrease in oxygen consumption which follows removal of the corpora allata may be due to the resulting cessation of protein synthesis and not to any direct effect of the corpus allatum hormone. L'Helias (1953) had found that removal of corpora allata from Dixippus resulted in a failure to synthesize protein.

B. H O M E O S T A S I S A N D H O R M O N A L ACTION

The evidence reviewed above strongly supports the belief that insect hormones can act directly upon tissues, such as the fat body or the gut wall, which are concerned in intermediary metabolism, and thus produce the materials that are needed for the growth processes that the hormones have set going. But it is necessary always to exclude the possibility that these metabolic effects are homeostatic responses (Wigglesworth, 1954a). When the neurosecretory material in adult female Caliiphora causes increased protease secretion in the mid-gut, is it certain that this is not a feed-back response via the haemolymph from the fat body which is demanding amino acids for protein synthesis? And is it certain that the synthetic activity of the fat body is not a feed-back response from the developing ovaries which are demanding protein? It is possible that both processes are operating at the same time; and it is probable that the relative importance of the two processes (direct action and feed-back effects) may differ in different insects.

312

V. B. W 1 G G L E S W O R T H

Thc incrcascd consumption of oxygen in the adult female of Pyrrhowhich follows implantation of active corpora allata (in contrast to the rcsults in Culliphora described by Thomsen and Hamburger, 1955) does not take place in castrated specimens. This seems not to be a direct effect on metabolism but is a consequence of ovarian development. Novak et al. (1961) therefore conclude that the juvenile hormone does not stimulate total metabolism but, in the presence of demand from the ovaries, it has a catalytic or activating effect on protein synthesis. Likewise, Novrik and Sliima (1 962) concluded that in the earlier stages of growth, the influence of the juvenile hormone on oxygen consumption depends on its increasing the amount of tissue that participates in the active metabolism. The same problem appears in Leptinotursa where extirpation of the corpora allata in the adult female induces the diapause state, with a reduced level of oxygen consumption. Since diapause is characterized by cessation of egg production it might be supposed that the fall in respiration was secondary to this effect. But the oxygen consumption of tissue homogenates of diapausing Leptinotarsa is increased by the addition of active corpora allata or by the active extract of juvenile hormone from Ifyalophora cecropia; de Wilde and Stegwee (1958) and Stegwee (1960) therefore favour the view that the hormone is exerting a direct action at the subcellular level, perhaps between succinate and cytochrome I'. On the other hand, Neugebauer (1961) could obtain no definite cffect on oxygen consumption in adult stick insects Dixippus (Carausius) after extirpation of the corpora allata or implantation of as many as five pairs of glands. In this insect the corpus allatum is not needed for oocyte development (p. 299). In Rhodnius females deprived of the corpus allatum by decapitation, the oocytes fail to develop yolk and the large meal of blood in the stomach is digested very slowly. (The fat body in Rhodnius is relatively inconspicuous ;it is important in intermediary metabolism, but the main reserve for growth is the undigested blood in the stomach.) If the fore part of the head, containing the brain and neurosecretory cells, is cut away, leaving the corpus allatum [and corpus cardiacum) intact, the blood meal is rapidly digested and the full complement of eggs is developed. The accelerated digestion in the presence of the corpus allatum was attributed to a feed-back effect from the developing ovary (Wigglesworth, 1948). This conclusion is supported by the fact that if the ovaries are removed from a female larva in the 4th stage, so that castrated adult females are obtained which have completely recovered from the injury of the operation, there is the same delay in the digestion of blood; and coris

H O R M O N E S IN G R O W T H A N D R E P R O D U C T I O N

313

this happens whether the corpus allatum is retained or not (Wigglesworth, unpublished). The hypertrophy of the fat body in castrated females of Oncopeltus (Johansson, 1958) may well be a feed-back response of the same kind, operating on the storage organ, which in this case is the fat body. Likewise in the fat body of silkworm pupae, ligatured to prevent development, the glycogen content remains for long unchanged, whereas in the developing pupa it falls to one-fifth of the initial value (It0 and

Horie, 1957). Wang and Dixon (1960) noted that allateatomy will decrease transaminase activity in the muscles of cockroaches. There is evidently a reduction in protein synthesis; but this may well be the result of feed-back from the ovary upon the neurosecretory system. In the adult female of Calliphora the homeostatic effect of the developing ovaries seems to influence the selection of food by the fly. During the early stages of egg growth the female of Calliphora ingests preferentially a fluid rich in protein, whereas during the later stages of yolk formation a diet of sugar is preferred. And if the ovaries are extirpated, the flies select a high carbohydrate and low protein diet. The normal cycle of ovarian growth thus seems to be responsible for the succession on feeding cycles (Strangways-Dixon, 1961a, b, 1962). Feed-back effects may produce visible changes in the endocrine organs themselves. In many insects (Calfiphora (Thomsen, 1942), Drosophilu (Vogt, 1948), Rhodnius (Wigglesworth, 1948)) the corpus allatum hypertrophies if the female is castrated (cf. Scharrer and von Harnack, 1961). This looks like retention of unused secretion. Likewise in the “female sterile adipose” (adpladp) mutant of Drosophila, in which the ovaries fail to ripen, the corpus allatum hypertrophies just as in castrated females. But it remains normal in size if wild-type ovaries are implanted. (The sterility of the ovaries of adpladp females is autonomous; wild type ovaries develop normally in these flies.) Similarly, the fat body of adp/ adp females, which ordinarily stores excessive quantities of lipid in this mutant, does not do so if wild type ovaries are implanted. This also appears to be a homeostatic response (Doane, 1961). Such results are to be contrasted with those obtained in Melanoplus (Pfeiffer, 1945a) and Calfiphora (Thomsen and Hamburger, 1955) in which the corpus allatum exerts an effect on storage metabolism even in the absence of the ovaries. A similar feed-back effect is seen in the neurosecretory cells. Allatectomy in Schistocerca, as we have seen, prevents yolk deposition and is followed by complete resorption of the developing oocytes. Meanwhile the level of protein in the haemolymph rises much above that of the

314

V. R . WIGGLESWORTH

normal femaie, and secretory material accumulates in the neurosecretory cells of the brain and in the corpus cardiacum (Highnam et al., 1963a, b). Lea and Thonisen (1962) describe cyclical changes in the medial neurosecretory cells of Culliphora, and parallel cycle of changes in size in the corpus allatum. Experiments strongly suggested that the changes in the neurosecretory cells are consequent upon those in the corpus allatum; and that the active corpus allatum secretes a hormone which stimulates synthesis of secretion by the neurosecretory cells. But it is possible that here again the corpus allatum may act on the ovary and it is the needs of the ovary which provoke by homeostasis the resumed activity of the neurosecretory cells. A feed-back response of a different kind can be observed in the corpus allatum in certain experiments. It was noted by Johansson (1958) that in Oncopelfus the presence of additional, implanted, corpora allata depressed the growth of the corpus allaturn of the host. That is doubtless the explanation of the curious results described in Rhodnius when the corpus allatum of a 5th-stage larva or of a young adult was implanted into a 4th-stage larva. Certain of these larvae, instead of moulting into 5th-stage larvae, developed slightly adultoid characters (Wigglesworth, 1948). It was suggested that this was due to active removal of juvenile hormone by the corpus allatum of the insect at the time of metamorphosis; but it is more iikely that it was the result of partial suppression of the activity of the corpus allatum of the host insect. A somewhat similar example of the unpredictable results which follow transplantation of corpora allata between different instars and different sexes is given by Fukuda (1 963). C . H U M O R A L INTEGRATION

The means by which the growth and differentiation of the various parts of the body are co-ordinated is the most difficult and obscure part of hormone physiology. In this scction it will be possible to do no more than indicate some of the problems that exist. In Blattella (O’Farrell et al., 1956) and Leucophaea (Engelmann and Luscher, 1956) the amputation of legs, or the infliction of extensive epidermal damage, lead to extra larval moults. This effect is attributed by Pnhley (1959, 1960, 1961, 1962) to the intensity of the developmental process, that is associated with regeneration, reducing the level of the moulting hormone and thus causing the activity of the corpus allatum to increase above the normal level, with increased production of juvenile hormone. But perhaps one could simply say that the active regeneration and the feed-back action which this exerts upon the neurosecretory cells

HORMONES IN GROWTH A N D REPRODUCTION

315

(p. 269) upset the normal process in the brain which ‘‘countsthe instars”; and the corpus allatum is thus permitted to continue juvenile hormone secretion. This effect of regenerating tissues upon the central organs of internal secretion is just one example of the way in which the organs, including the endocrine organs, are continually receiving and responding to chemical messages carried presumably by the body fluids. An example of a different kind is seen in the formation of muscles in a regenerating limb, where, as Steinberg (1958) points out, one must conclude that in the determined regions of the epidermis there appear certain influences which induce the approach of myoblasts and their fixation at certain points. But these considerations go beyond the bounds of this review. Hormones are operating upon tissues whose pattern of growth is determined by genetic constitution. Many hormones doubtless act largely by evoking or suppressing the realization of genetic potentialities. In a recent review Karlson (1961) has emphasized the role of hormones in activating genes, but he omitted the classic example of the juvenile hormone which, as has long been evident, controls metamorphosis by action of this kind (p. 288). The change in form of the insect at metamorphosis is commonly said to be controlled by a balance between the moulting hormone and the juvenile hormone. These hormones do two quite different things; the moulting hormone initiates and maintains the growth process that leads on to moulting; the juvenile hormone ensures the operation of those components of the gene system which give rise to larval structures. And yet partial deficiencies in the amount of juvenile hormone will give rise to insects with mixed characters intermediate between larva and adult. There is evidence, as we have seen, that the pupa is a natural intermediate of this kind. In Leyisma the scales on the integument normally make their first appcarance after the third moult, but they will develop in the integument of thc first instar if this is implanted into the body cavity of the adult and allowed to moult along with the host (Piepho and Richter, 1959). It is not known whether the humoral difference responsible for this change concerns the juvenile hormone. Deficiency of juvenile hormone (resulting in prothetely) or excess of juvenile hormone (resulting in metathetely) may be due to many causes (see Wigglesworth, 1954a). A well known cause is the presence of parasites of one sort or another. Recent examples are the upsets in metamorphosis in Aeschna associated with heavy infestation by the trematode Prosotocus (Schaller, 1962); and the appearance of apterous Aphids (regarded as naturally occurring neotenics) as the result of parasithation

316

V.

B. W I G G L E S W O R T H

by Aphidius; it has been suggested that this may result from juvenile hormone escaping from the Aphidius embryo (Johnson, 1959). This morphogenetic role of hormoncs is well illustrated in the polymorphism of insects (Kennedy, 1961). I n some cases body form is determined automatically by genetic constitution; the differences in form in different individuals represent diflerent mutations; and the relative numbers of the various forms present in a population is regulated by the balance of selective pressures acting on the several mutants. In other cases the gene system is responsive to changes in the environment. Examples ofthis are the castes in termites (Luscher, 1959) and Hymenoptera, the phases of locusts, the polymorphism of Aphids, and seasonal varieties in which the presence or absence of diapause may be correlated with striking changes in form. It is highly probable that hormones form an essential link in the chain leading from external stimulation to the activation of particular genes responsible for body form. We have seen some examples of the juvenile hormone itself being involved in this action (p. 284); but no attempt will be made to deal comprehensively with the subject in the present review. REFERENCES Agrell, 1. (1947). Some experiments concerning thermal adjustment and respiratory metabolism in insects. Ark. Zool. 39A, No. 10 1-48. Agrell, 1. (1952). The aerobic and anaerobic utilization of metabolic energy during insect metamorphosis. Acta physiol. srand. 28, 303-335. Andrewartha, H. G . (1952). Diapause in relation to the ecology of insects. Biol. Rev. 27, 50-107. Arvy, L. (1956). Neuroskretion et glandes endocrines ritrdrebrales chez Euroleon nostrus Fourcroy. Bull. Sor. tool. Fr. 81, 167. Arvy, L. and Gabe, M. (19524. Particularites morphologiques des formations endocrines rktrocirkbrales chez Ecdyonurus &spar (Curt) et chez Ecdyonurus torrentis Kimmins. Bull. Soc. zool. Fr. 77, 225-300. Amy, L. and Gabe, M. (1952b). D o n n k s histophysiologiques sur les formations endocrines retrdrebrales de quelques Odonates. Ann. Sci. nut. Zool. 14, 345-374. Arvy, L. and Gabe, M. ( I 953). Donnks histophysiologiques sur la neuroskrktion chez quelques Ephimeropthres. Cellule 55, 203-222. Arvy, L. and Gabe, M. (1954). Modifications de la neuroskcrbtion protocirkbrales et des glandes endocrines ckphaliques de Leptinotarsa decemlineata Say au cours de la metamorphpse. 7P Congr. SOC.savantes. 189-196. Arvy, L. and Gabe, M. (1962). Histochemistry of the neurosecretory product of the pars intercerebralis of pterygote insects. Mem. SOC.Endocr. 12,331-344. Arvy, L., Bounhiol, J. J. and Gabe, M. (1953). D6roulement de la neurodcrktion protodrkbrale chez Bombyx mori L. au cours du developpement post-embryomire. C.R. Acad. Sci.,Paris 236, 627-629.

HORMONES IN GROWTH A N D REPRODUCTION

317

Ekcker, H. J. (1962). Die Puffs der SpeicheldrUsenchromosomen von Drosophih melanogaster. 11. Die Auslijsung der Puffbildung, ihre Spezifitiit und ihrc Betiehung zur Funktion der RhgdrUe. Chromosoma 13,341-384. Berger, H, (1963).Die Wirkung der Neurohormone auf das Metamorphosegeschehen bei Calliphora erythrocephaln Meig. Zoof. Jb., Physiol. 70, 245-265. Bodenstein, D.(1939).I m a m differentiationinaugurated by oxygen in Drosophila pupae. Proc. nat. Acad. Sci.. Warh. 25, 1416. Bodenstein, D. (1946). A study of the relationship between organ and organic environment in the post-anbryonic development of the yellow fever mosquito. Bull. Conn. Erp. Sta. 501, 100-114. Bodenstein, D.(1953).Studiea on the humoral mechanisms in growth and metamorphosis of the cockroachPeriphnetaamericanu. 11. The functionof the prothoracic gland and the corpus cardiacum. J. exp. Zool.123,413433. Bodenstein, D.(1957).Humoral dependence of growth and differentiationin insects. In “Recent Advances in Invertebrate Physiology” (Scheer, ed.)pp. 197-211. Oregon University. Boisson, C. (1949). Recherches histologiques sur le complexe allatocardiaque de Bacillus rossii (Fabr.) Bull. biol. Suppl. 34, 1-92. Bounhiol, J. J. (1938).Recherchesexpkrimentalessur le dkterminismede la mc5tmorphose chez l a Upidoptkres. Bull. biol. Suppl. 24, 1-199. Bounhiol, J. J. (1943).Nymphose (partielle) loCali&, chez des vers B soie div& en Vois parties par deux ligatures. C.R. Acad. Sci., Poris 217,203-204. Bounhiol, J. J. (1953). R61e du corpus allatum dans la metamorphose des insect=. Trans. IXth int. Congr. Ent. 2, 63-72. Brandenburg, J. (1955). Corpora allata, Gonaden und Stylophation. Naturwisssns c w t e n 42, 260-261. Bricteu-Gr&goire, S.,Verly, W. G. and Florkin, M.(1957).Protein synthesis in Sphinx ligustri pupae. Nature, Lond. 179,678679. Brinkhurst, R. 0.(1959). Alary polymorphism in the Gerroidea (HemipteraHeteroptera). J. unim. Ecol. 28,211-230. Brinkhurst, R. 0.(1963). Observations in wing-polymorphism in the Heteroptera. Proc. R. ent. SOC.Lond. A 38, 15-22. Bucklin, D. H. (1953).Termination of diapause in grasshopper embryos cultured In vitro. Anat. Rec. 117,539. Btlckmann, D. ( I 959). Die Auslosung der Umfarbung durch das Hiiutungshormon bei c‘erura vinula L. (Lepidoptera, Notodontidae). J. Insect Physiol. 3, 159-189. Burtt. E.T.(1938).On the corpora allata of dipterousinsects 11. Proc. roy. SOC.B 126, 210-223. Butenandt, A. and Karlson, P. (1954). ober die Isoleirung eines MetamorphoseHormons der Insekten in kristallisierter Form.2.Nuturf. 9b,389-391. Carlisle, D. B.and Ellis, P.E. (1959). La persistence des glandes ventrales dphaliquea chez les criquets solitaires. C.R. Acad. Sci., Paris 249, 1059-1060. Chadwick, L.E.(1956). Removal ofprothoracicglands from the nymphal cockroach. J. exp. Zool. 131,291-306. Chen, D. H., Robbins, W. E. and Monroe,R. E.(1962).The gonadotropicaction of cecropia extracts in allatectomized American cockroaches. Experientiu 18, 577. Church, N.S.(1955).Hormones and the termination and reinduction of diapause in Cephus cinctw Nort. (Hymenoptera: Cephidae). Canud. J. 2001. 33, 339-369.

318

V. B. W I G G L E S W O R T H

Clarke, K. U. and Langley, P. A. (1 962). Factors concerned in the initiation of growth and moulting in Locusta rnigratoria L. Cen. comp. Endocr. 2,625-626. Clarke, K. U. and Langley, P. A. (1963a). Effect of the removal of the frontal ganglion on the development of the gonads in Locusta migratoria L. Nature, Land. 198, 811-812. Clarke, K. U. and Langley, P. A. (1963b). Studies on the initiation of growth and moulting in Locustu migratoria migrutorioides R. and F. I. The time and nature of the initiating stimulus. J. Insect Physiol. 9, 287-292. Clarke, K. U. and Langley, P. A. (1963~).Studies on the initiation of growth and moulting in Locusta migratoria migratorioides R. and F. 11. The role of the stomatogastric nervous system. J. Insect Physiol. 9, 363-374. Clarke, K. U. and Langley, P. A. (1963d). Studies on the initiation of growth and moulting in Lacusta migratoriu migrulorioides R. and F. 111. The role of the frontal ganglion. J. Insect Physiol. 9, 41 1-422. Clarke, K. U. and Langley, P. A. (1963e). Studies on the initiation of growth and moulting in Locustu migratoria migrutorioides R. and F. IV. The relationship between the stomatogastric nervous system and neurosecretion. J . Insect Physiol. 9, 423-430. Clements, A. N. (1956). Hormonal control of ovary development in mosquitoes. J. exp. Biol. 33, 21 1-223. Clever, U. (1961a). o b e r das Reaktions-system einer hormonalen Induktion. Untersuchungen an Chironomus tentans. Verb. dtsch. zool. Ges. (Saarbriicken) 1961, 75-92. Clever, U. (1961b). Genaktivitaten in den Riesenchromosomen von Chironomus tentans, und ihre Beziehungen zur Entwicklung. i. Genaktivierung durch Ecdyson. Chromosoma 12, 607-675. Clever, U. (19624. Genaktivitiiten in den Riesenchromosomen von Chironomus tentans und ihre Beziehungen zur Entwicklung. ii. Das Verhalten der Puffs wahrend das letzten Larvenstadium und der Puppenhautung. Chromosoma 13, 385436. Clever, U. (1962b). Genaktivitiiten in den Riesenchromosomen von Chironomus tentans. iii. Das Aktivitatsmuster in Phasen der Entwicklungsruhe. J. Insect Physiol. 8, 357-376. Clever, U.(19624. Quantitative relationships between hormone dosage and gene activity patterns in the development of Chironomus tentans. Gen. comp. Endocr. 2, 604. Clever, U. (1963). Von der Ecdysonkonzentration abhangige Genaktivitiitsmuster in den Speicheldrilsenchromosomen von Chironomus tentans. Develop. Blol. 6, 73-98. Cloutier, E. J., Beck, S. D., McLeod, D. G. R. and Silhacek, D. L. (1962). Neural transplants and insect diapause. Nature, Lond. 195, 1222-1224. Cousin, G. (1956). Biomktrie et dkfinitions de morphologie quantitative des e s p k e s et de leurs hybrids. Bull. Soc. zool. Fr. 81, 247-288. Davey, K. G. (1956). The physiology of dormancy in the sweet-clover weevil. Canad. J. Zool. 34, 86-98. Doane, W. W. (1961). Developmental physiology of the mutant female sterile (2) adipose of Drosophilu melunogaster. iii. Corpus allatumcomplex and ovarian transplantation. J. exp. 2001. 146, 275-298. Ducleaux, E. (1896). De l'influence du froid de l'hiver sur le dkveloppement de

HORMONES I N G R O W T H A N D REPRODUCTION

319

I’embryon du ver h soie et sur l’klosion de la graine. C.R. Acad. Sci., Park 69, 1021-1022.

Dupont-Raabe, M. (1951). Etude morphologique et cytologique du cerveau de quelques Phasmides. Bull. SOC.zool. Fr. 76, 386-397. Dupont-Raabe, M. (1952). Contribution l‘etude du role endocrine du cerveau et notamment de la pars intercerebralis chez l a phasmides. Arch. Zooi. exp. gkn. 89, 128-138.

Dupont-Raabe, M. (1954). Repartition des activitb chromatiques dam le ganglion susoesophagien des Phasmides; mise en evidence d’une rkgion &r&iore dans la partie deuto et tritdrkbrale. C.R. Acad. Sci., Paris 238, 95@-951. Dupont-Raabe, M.(1956). Quelques d o n n h relatives aux phhomknes & neuro&retion chez les phasmides. Ann. Sci. nut. Zool. 18, 293-304. Eassa, Y.E. E. (1953). The development of imaginal buds in the head of Pieris brassicae Linn. (Lepidoptera). Trans. R. ent. SOC.Lomi. 104,39-50. Engelmann, F. (1957). Die Steuerung der Ovarfunktion bei der ovoviviparen Schabe Leucophaea maderae (Fabr.) J . insect Physiol. I, 257-278. Engelmann, F. (1 958). The stimulation of the corpora allata in Diplopferupunctafu (Blattaria). Anat. Rec. 132, 432433. Engelmann, F. (1959a). Ober die Wirkung implantierter Prothoraxdriisen im adulten Weibchen von Leucophaea maderae (Blattaria). 2. vergl. Physiol. 41, 456470.

Engelmann, F. (1 959b). The control ofreproductionin Diplopterupuncruru(Blattar’a). Biol. Bull., Woods Hole 116, 406419. Engelmann, F. (1960). Mechanisms controlling reproduction in two viviparous cockroaches (Blattaria). Ann. N. Y.Acad. Sci. 89,516539. Engelmann, F. (1962). Further experiments on the regulation of the sexual cycle in females of Leucophaea muderae (Blattaria). Gen. comp. Endocr. 2, 183-192. Engelmann. F. and Luscher, M. (1956). Die hemmende Wirkung des Gehirns auf die Corpora allata bei Leucophaea maderae (Orthoptera). Verh. dtsch. 2001. Ges., Hamburg, 1956, 215-220.

Ewen, A. B. (1962a). Histophysiology of the neurosecretory system and retrooerebral endocrine glands of the alfalfa plant bug Adelphocoris lineolufus (Goeze) (Hemiptera: Miridae). J. Morph. 111. 255-269. Ewen, A. B. (1962b). An improved aldehyde fuchsin staining technique for neurosecretory products in insects. Trans. Amer. micr. SOC.81, 94-96. Fraenkel, G. (1935). A hormone causing pupation in the blowfly Culliphoru erythrocephola. Proc. roy. SOC.B 118, 1-12. Fraser, A. (1959). Neurosecretion in the brain of the larva of the sheep blowfly, Luciliri cuesar L. Quart. 1. micr. Sci. 100, 377-394. Fraser, A. (1960). Humoral control of metamorphosis and diapause in the larvae of certain Calliphoridae (Diptera: Cyclorrhapha). Proc. roy. Sac. Edinb. B 67, 127- 140.

Fukaya, M. ( I 955). The role of the brain in the metamorphosis of the rice stem borer, Chilo mppressalis. Jap. 1. appl. Zool. 20, 179-183. Fukaya, M. (1962). The inhibitory action of farnesol on the development of the rice stem borer in post-diapause. Jap. J. appl. Ent. Zool. 6,298. Fukaya, M. and Mitsuhashi, J. (1957). The hormonal control of larval diapause in the rice stem borer, Chilo suppressalis. i. Some factors in the head maintaining larval diapause. Jap. J . appl. Ent. Zool. 1, 145-154.

320

V. B. W I G G L E S W O R T H

Fukaya, M. and Mitsuhashi, J. (1958). The hormonal control of larval diapause in the rice stenr borer, Chilo suppressalis. ii. The activity of the corpora allata during the dinpausing period. Jup. J. uppl. Enr. Zool. 2 223-226. Fukaya, M.and Mitsuhashi, J. (1961). Larval diapause in the rice stem borer with special reference to its hormonal mechanisms. Bull. nut. Insf. agric. Sci. (Jopan) (C) NO. 13, 1-32. Fukuda, S . (1940a). Induction of pupation in silkworm by transplanting the prothorack gland. Proe. imp. Acud. Tokyo 16, 414-416. Fukuda, S. (1940b). Hormonal control of moulting and pupation in the silkworm. Proc. imp. Acud. Tokyo 16,417-420. Fukuda, S . (1941). Role of the prothoracic gland in differentiation of the imaginal characters in the silkworm pupa. Annot. iool. japon. 20, 9-13. Fukuda, S. (1944). The hormonal mechanism of larval molting and metamorphosis in the silkworm. J . Fac. Sci. Tokyo imp. Utriv. Sec. lV, 6,477-532. Fukuda, S. (1953). Alteration of voltinism in the silkworm following transection of pupal oesophageal connectives. Proc. imp. Acud. Japan 29, 389-391. Fukuda, S. (1962). Secretion of juvenile hormone by the corpora allata in pupae and moths of the silkworm, Bombys mori. Annot. zool. jup. 35, 199-212. Fukuda, S. (1963). Induction of supernumerary molting in silkworm larvae by implantation of corpora allata from female moths. Annot. 2001. jup. 36, 1420. Fuller, H. B. (1 960). Morphologische und experimentelle Untersuchungen iiber die neurosekretorischen Verhaltnisse im Zentralnervensystem von Blattiden und Culiciden. Zool. Jb., Pbysiol. 69, 223-250. Fyg, W. (1956). Experimentelle Untersuchungen iiber die Entwicklung der Honigbiene. Miff.scbweiz. enf. Ges. 29, 404-416. Gabe, M. (1953a). Quelques acquisitions r k n t e s sur les glandes endocrines des arthropodes. Experirnfiu 9, 352-356. Gabe, M. (1953b). Sur quelques applications de la coloration par la fuchsineparaldehyde. Bull. micr. appl. 3, 153-162. Gabe, M. (1953~).Donnks histologiques sur les glandes endocrines ckphaliques de quelques Thysanoures. Bull. SOC.zool. Fr. 78, 117-193. Geiger, H. R. (1961). Untersuchungen uber freie Aminosauren wahrendder Adultentwicklung von Culex pipiens und Culex fufigans und deren Einfluss auf die Eireifung. Rev. suisse Zool. 68, 583-626. Gilbert, L. I. (1962). Maintenance of the prothoracic gland by the juvenile hormone in insects. Nufure, Lond. 193, 1205-1207. Gilbert, L. I. and Schneiderman, H. A. (1957). The quantitative assay of the juvenile hormone of insects. Anat. R e . 128, 5 5 5 . Gilbert, L. I. and Schneiderman, H. A. (1 958a). The inactivation of juvenile hormone extracts by pupal silkworms. Anar. Ree. 131,557-558. Gilbert, L. I. andschneiderman, H. A. ( I 958b). Occurrence of substances withjuvenile hormone activity in adrenal cortex of vertebrates. Science 128, 844. Gilbert, L. I. and Schneiderman, H. A. (1959). Sexual dimorphism in lipid content, juvenile hormone content and size of corpora allata in Lepidoptera. Anuf. Rec. 134, 569. Gilbert, L. I. and Schneiderman, H. A. (1960). The development of a bioassay for the juvenile hormone of insects. Truns. Amer. micr. SOC.79, 38-67. Gilbert, L. I. and Schneiderman, H. A. (1961). The content ofjuvenile hormone and

H O R M O N E S IN GROWTH A N D REPRODUCTION

321

lipid in Lepidoptera: sexual differencesand developmentalchanges. Gen. comp. Endocr. 1, 453-472. Gillett, J. D. (1956). Initiation and promotion of ovarian development in the mosquito Aedes (Stegomyia) aegypti (Linnaeus). Ann. trop. Med. Parasit. 50 375-380. Gillett, J. D. (1957). Variation in the time of release of the ovarian development hormone in Aedes aegypti. Nature, Lond. 656-657. Gillett, J. D. (1958). Induced ovarian development in decapitated mosquitoes by transfusion of haemolyrnph. J. exp. Biol. 35,685493. Girardie, A. ( I 962). etude biometrique de la croissance ovarienne a p r b ablation et implantation de corpora allata chez Peripkmeta americana.J. Insect Physiol. 8, 199-204. Gomori, G. (1941). Observations with differential stains on human islets of Langerhans. Amer. J. Path. 17, 395-406. Hachlow, V. (1931). Zur Entwicklungsmechanik der Schmetterlinge. Roux Arch. EnrwMech. Organ. 125, 2649. Halbwachs, M. C., Joly, L. and Joly. P. (1957). Rbultats &implantations de“glandes ventrales” a Locusta migraroria L. J . Insect Physiol. 1, 143-149. Hanstrom, B. (1938). Zwei Probleme betreffs der hormonalen Localization im Insektenkopf. Acta Univ. lund. N.F., Avd. 2, 39, 1-17. Hanstrom, B. (1940). Inkretorische Organe, Sinnesorgane und Nervensystem des Kopfes einiger niederen Insektenordnungen.K. svenska VetensAkad. Handl. 18, 3-265. Harnack, M. von (1958). The effect of starvation on the endocrine control of the ovary by the corpus allatum in the insect Leucophaea maderae. Anat. Rec. 130, 446. Harvey, W. R. (1962). Metabolic aspects of insect diapause. Annu. Rev. Ent. 7 , 57-80. Harvey, W.R. and Williams, C. M. (1958). Physiology of insect diapause. xii. The mechanism of carbon monoxide-sensitivityand -insensitivity during the pupal diapause of the cecropia silkworm. Biol. Bull., Woods Hole 114, 36-53. Harvey, W. R. and Williams, C. M. (1961). The injury metabolism of the cecropia silkworm. i. Biological amplification of the effects of localized injury. J . Insect Physiol. 7 , 81-99. Hasegawa, K. (1943). Studies on the development of the ovum in the silkworm, Bombyx mori L. i. On the development of the ovary during the pupal stage. Bull. seric. exp. Sta. 11, 359-377. Hasegawa, K. (1 947). Studieson the developmentof the ova in the silkworm, Bombyx mori. ii. On the degeneratedovum. Bull. seric. exp. Sta. 12.481492. Hasegawa, K. (1952). Studies on the voltinism in the silkworm Bombyx mori, L., with special reference to the organs concerning determination of voltinism. J. Fac. Agric. Tottori Univ. 1, 84-124. Hasegawa, K.(1 957). The diapause hormone of the silkworm, Bombyx mori.Nature, Lond. 179, 13Wl301. Henson, H. (1946). The theoretical aspect of insect metamorphosis. Biol. Rev. 21, 1-14. Herlant-Meewis, H. and Paquet, L. (1956). Neurokrdtion et mue chez Carausius morosus Brdt. Ann. Sci. nat.. Zool. 18, 164-169. Highnarn, K. C. (1958). Activity of the braidcorpora cardiaca system during pupal diapause “ break” in Mimas tiliae (Lepidoptera). Quart. J. micr. Sci. 99,73-88.

322

V. B. WIGGLESWORTH

Highnam, K. C. (1961a). Induced changes in the amounts of material in the neurosecretory system of the desert locust. Nature, Lond. 191, 199-200. Highnam, K. C.(1961b). The histology of the neurosecretory system of the adult female desert locust, Schistocerca gregrrria. Quart. J. micr. Sci. 102, 27-38. Highnam, K. C. (1962a). Neurosecretorycontrol of ovarian development in Schistocerca gregaria. Quart. J. micr. Sci. 103, 57-72. Highnam,K. C. (1962b). Neurosecretorycontrol of ovarian development in the desert locust. Mem. SOC.Endocr. 12, 379-390. Highnam, K. C. and Lusis, 0. (1962). The influence of mature males on the neurosecretorycontrol of ovarian development in the desert locust. Quart. J. micr. Sci. 103, 73-83. Highnam, K. C., Lusis, 0. and Hill, L. (1 963a). The role of the corpora allata during oocyte growth in the desert locust, Schistocerca gregaria Forkdl. J . Insect Physiol. 9, 587-596. Highnam, K. C., Lusis, 0. and Hill, L. (1963b). Factors affecting oocyte resorption in the desert locust Schistocerca gregaria Forskill. J. Insect Physiol. 9, 827-838. Hill, L. (1962). Neurosecretorycontrol of haemolymph protein concentrationduring ovarian development in the desert locust. J. Insect Physiol. 8, 609-619. Hill, L. (1963). “The Endocrine Control of Oocyte Development in the Desert Locust, Schistocerca gregaria, ForskAl”. Thesis, University of Sheffield. Hinton, H. E. (I 957). Some aspects of diapause. Science Progr. No. 178.308-320. Hodgson, E. S. and Geldiay, S. (1959). Experimentally induced release of neurosecretory materials from roach corpora cardiaca. Biol. Bull., Woods Hole 117, 275-283. Ichikawa, M. and Ishizaki, H. (1961). Brain hormone of the silkworm Bombyx mori. Nature, Lond. 191, 933-934. Ichikawa, M. and Ishizaki, H. (1963). Protein nature of the brain hormone of insects. Nature, Lond. 198, 308-309. Ichikawa, M. and Nishiitsutsuji, J. (1952). Studies on insect metamorphosis. ii. Determination of the critical period for pupation in the eri-silkworm,Philosamia Cynthia ricini. Annot. zool. jap. 25, 143-148. Ichikawa, M. and Nishiitsutsuji, J. (1955a). Studies on insect metamorphosis. v. Implantation of larval brains into the pupae of Luehdorfiajaponica. Mem. Coll. Sci. Univ. Kyoto B 22, 11-1 5. Ichikawa, M. and Nishiitsutsuji, J. (1955b). Studies on insect metamorphosis. iv. Prothoracic glands of Ephestia cautella. Mem. Coll. Sci. Univ. Kyoto B 22, 1-9. Ichikawa, M. and Nishiitsutsuji-Uwo, J. (1956). Studieson the insect metamorphosis. vi. Effect of low temperature on the morphogenesis of Leuhdorfia pupae. Mem. Coll. Sci. Univ. Kyoto B 23, 19-26. Ichikawa, M. and Nishiitsutsuji-Uwo. J. (1959). Studies on the role of the corpus allatum in the eri-silkworm,Phihamica Cynthia ricini. Biol. Bull., Woods Hole 116, 88-94. Ichikawa, M. and Nishiitsutsuji-Uwo,J. (1 960). Studies on insect metamorphosis. vii. Effect of the brain hormone on the isolated abdomen of the eri-silkworm, Philosamia Cynthia ricini. Mem. CON.Sci. Univ. Kyoto 27,9-15. Ito, T. and Horie, Y. (1957). Glycogen in the ligated silkworm pupa (Bombyx mori). Nature, Lond. 179, 1 136-1 137. Jacob, J. and Monod, J. (1961). On the regulation of gene activity. Cold Spr. Harb. Symp. quant. Biol. 26, 193-21 1.

H O R M O N E S I N G R O W T H A N D REPRODUCTION

323

Janda, V. (1961). The total metabolism of insects. 12. Metabolism during the intermoulting period of Neodiprion sertqer Geoffr. larvae (Hym. Tenthredhoidea). Mem. SOC.z p l . tchbcosl. 25, 306-317. Johansson, A. S. (1954). Corpus allatum and egg production in starved m e e d bugs. Nature, Land. 174. 89. Johansson, A. S. (1955). The relationship between corpora allata and reproductive organs in starved female kucophaea nraderx (Blattaria). Biol. Bull., Woods Hole 108, 40-44. Johansson, A. S. (1957). Neurosecretion and nietamorphois in the milkweed bug, Oncopeltus fasciatus. Experientia 13. 410. Johansson, A. S. (1 958). Relation of nutrition to endocrine-reproductive functions in the milkweed bug Oncopelfusfasciatus (Dallas) (Heteroptera: Lygwidae). Nyt. Mag. Zool. 7, 5-132. Johnson, B. (1 959). Effect of parasithation by Aphidius platensis Brethes on the developmental physiology of its host, Aphis craccivora Koch. Ent. exp. appl. 2, 82-99. Johnson, B. (1962). Neurosecretion and the transport of secretory materid from the corpora cardiaca in Aphids. Nature, Lond. 196, 1338-1339. Joly, L. (1955). Analyse du fonctionnement des corpora allata chez la larve & Locusra mipatoria L. C.R. SOC.Biol., Paris 149, 584. Joly, L. (1 960). ” Fonctions des corpora allata chez Locusta migratoria (L.)”. Thesis, Strasbourg, 103 pp. Joly, P. (1945). La fonction ovarienne et son contrble humoral chez les DytiscidcS. Arch. 2001.exp. gPn. 84, 49-164. Joly, P. (1955). Chronologie de la mitose chez Locusta migratoria (L.) Arch. Anat., Strasbourg 37, 87-96. Joly, P. (1956). Croissance et indices de gregarisation chez Locusfa migratoria (L). Insectes sociuux 3, 17-24. Joly, P. and Joly, L. (1953). Rksultats de greffes de corpora allata chez Locurta migratoria L. Ann. Sci. Nut. Zool. 15, 331-345. Joly. P., Joly, L. and Halbwachs, M. (1956). ContrBle humoral du developpement chez Locusra migratoria. Ann. Sci. nut., Zool. 18. 2 5 6 2 6 1 . Jones, B. M. (1956). Endocrine activity during insect embryogenesis. Function of the ventral head glands in locust embryos (Locustuna pardalina and Locusfa migratoria, Orthoptera. J. exp. Biol. 33. 174185. Kaiser, P. ( I 955).Die Inkretorganeder Termiten in Kastendifferenzierungsgeschehen. Nuturwissenschafrm 42, 303-304. K i i , S. (1953). Transplantation of thoracic legs of the eri-silkworm, Philosamia Cynthia ricini. Annot. :ooL japon. 26, 224-227. Karlson, P. (1956). Biochemical studies on insect hormones. Vitam. & Horm. 14, 227-266. Karlson, P. ( I 959). Zur Chemie und Wirkungsweise der Insektenhormone. Proc. ZVth inr. CoitAv. Hiochem. 12 (Biochemistry of insects), 37-47. Karlson, P. ( 1 963). Chemistry and biochemistry of insect hormones. Angew. G e m . 2, 175-182. Karlson, P. and tlanser, G. (1953). Bildungsort und Erfolgsorgan des Puparisierungshormons der Fliegen. Z. Nuturf. 8b, 91-96. Karlson, P. and Nachtigall, M. (1961). Ein biologischer Test zur quantitativen

324

V. B. W I G G L E S W O R T H

Bestimmung der Juvenilhormon-activitat von Insektenextrakten. J. Insect Physiol. 7 , 210-215. Karlson, P. and Schmialek, P. (1 959). Nachweis der Exkretion von Juvenilhormon. Z. Naturf. 14b, 821. Karlson, P. and Stamm-Menendez, M. D. (1956). Notiz uber den Nachweis von metamorphosehormone in den Imagines von Bombyx mori. Hoppe-Seyl. Z. 306,110-111. Kennedy, J. S. (Ed.) (1961). “Insect Polymorphism”. Symposium No. 1, Roy. Ent. Soc. London. 115 pp. Ketchel, M. M. and Williams, C. M. (1953). The prothoracic gland hormone as a sustained stimulus for the growth and differentiation of insect tissues. Anat. Rec. 117, 542. Khan, T, R. and Fraser, A. (1962). Neurosecretion in the embryo and later stages of the cockroach (Periplanetu americanu L.) Mem. SOC.Endocr. 12, 349-369. Kim, C. W. (1959). The differentiation centre inducing the development from larval to adult leg in Pieris brassicae (Lepidoptera).J . Embryol. exp. Morph. 7,572-582. Kim, C . W. (1960). On the use of the terms “larva” and “nymph”. Proc. R . ent. SOC. Lond, A 35, 61-64. Kirimura J., Saito, M. and Kobayashi. M. (1962). Steroid hormone in an insect Bombyx mori. Nature, Lond. 195, 729-130. Kobayashi, M. (1956). Cytohistological studies on the prothoracic gland of the silkworm, Rombyx mori. iii. On the histochemical changes of carbohydrates and the physiological significance of the secretions during larval and pupal moulting. Bull. seric. Exp. Sta. 14, 451-469. Kobayashi, M. and Burdette, W. J. (1961). Effect of brain hormone from Bombyx mori on metamorphosis of Calliphora erythrocephala. Proc. SOC.exp. Biol., N . Y. 107, 240-242. Kobayashi, M. and Kirimura, J. (1 958). The brain hormone in the silkworm, Bombyx mori L. Nafure, Lond. 181. 1217. Kobayashi, M. and Nakasone, S. (1959). Inhibitory effects of low oxygen tension and carbon monoxide on the secretion of brain hormone in the silkworm, Bombyx mori. J. seric. Sci..Tokyo 28, 12-76. Kobayashi, M. and Nakasone, S. (1960a). Inhibitoryeffect of low temperature on the secretion of the brain hormone in the silkworm pupa (Bombyx mori L.). J. seric. Sci., Tokyo 29, 203-205. Kobayashi, M. and Nakasone, S. (1960b). Imaginal differentiation in artificially induced diapausing brainless pupae of the silkworm, Bombyx mori, by the treatment of carbon monoxide or oxygen. Bull. scrie. exp. Sta. Japan 16, 100-1 12. Kobayashi, M. and Yamashita, Y.(1957). Effects of temperature and oxygen tension on the imaginal differentiation of the silkworm Bombyx mori L.J . seric. Sci., Tokyo 26, 274-278. Kobayashi, M. and Yamashita, Y. (1959). A function of corpus allatum in neurosecretory system in the silkworm Bombyx mori. J. seric. Sci., Tokyo 28,335-339. Kobayashi, M., Fukaya, M. and Mitsuhashi, J. (1960). Imaginal differentiation of “Dauer-pupae” in the silkworm, Bombyx mori. J. seric. Sci., Tokyo 29,337-340. KO+, S . (1 9 17). Experiments on metamorphosis of insects. Bull. int. Acad. Crucovie B, 57-60. KopeE, S. (1922). Studies on the necessity of the brain for the inception of insect metamorphosis. Biol. Bull., Woods Hole 42, 322-342.

t i o R M o N t s I N C;Kow-rIi A N D R E P R O D U C ~ I O N

325

Krishnakumaran, A. (1961). Ribonucleicacidsin themoultcycleofan insect. Narure, LAml. 189, 243- 245. Kurland, C. C i . and Schneidcrman, H.A. (1959). The respiratory enzymes of diapausing silkworm pupae: a new interpretation of carbon monoxide-insentitive respiration. Bid. Bull., Woods Hole 116, 136-161. Kuske, G. (1963). Untersuchungen zur metamorphose dcr Schmetterlingsbeine. Roux. Arch. EntwMech. O r g m . 154, 354-377. Kuske, G., Penner, M.L. and Piepho, H. (1961). Zur metamorphose des Schmetterlingsbeines. B i d . Zbl. 8, 347-351. Ladduwahetty, A. M.(1962). “The reproductive cycle and neuro-endocrine relations in Dermestes maculatus (Coleoptera: Dermestidae).” Thesis, University of London. Larsen, J. R. and Bodenstein, D. (1959). The humoral control of egg maturation in the mosquito. J. exp. Zool. 150, 343-381. Lea, A. 0. and Thomsen. E. (1962). Cycles in the synthetic activity of the medial neurosecretory cells of Calliphora eryrhrocephala and their regulation. Mem. SOC.Endocr. 12, 345-347. Lee, H. Tsui-Ying (1948). A comparative morphological study of the prothoracic glandular bands of some Lepidopterous larvae with special reference to their innervation. Ann. ent. Soc. Amer. 41, 200-205. Lees, A. D. (1 955). “The Physiology of Diapause in Arthropods’’. 15 I pp. Cambridge University Press. Lees, A. D. (1961). Clonal polymorphism in aphids. In “Insect Polymorphism”. Symposium No. I . Royal Entomological Society of London (J. S. Kennedy, ed.) pp. 68-79. L’Helias, C. (t953). etude comparke de l’azote total et de I’azote non proteinique chez le phasme Dixippus morosus aprks ablation des corpora allata. C.R. Acad. Sci., Paris 236, 2489-2491. Lerma, B. de (1956). Corpora cardiaca et neuroskrdtion protockribrale chez le colioptere Hydrous piceus L. Ann. Sci. nat., Zool. 18,235-250. Loher, W. (1960). The chemical acceleration of the maturation process and its hormonal control in the male of the desert locust. Proc. roy. SOC.B 153,380-397. Ludwig, D. and Barsa, M. C. (1959a). Activities of respiratory enzymes during the metamorphosisof the housefly, Musca domestica L.J. N . Y.ent. SOC.67,151-156. Ludwig, D. and Barsa, M. C. (I 959b). Activity of dehydrogenase enzymes during the metamorphosis of the mealworm, Tenebrio molitor L. Ann. ent. SOC.Amer. 51, 31 1-314. Liischer, M.(1957). Ersatzgeschlechtstiere bei Termiten und die Beeinfliissung ihrer Entstehung durch die Corpora allata. Verh. drsch. Ges. angew. Entom. 1957, 144-150.

LUscher, M. (1958a). Experimentelle Erzeugung von Soldaten bei der Termite Kalotermes jlavicollis Fabr. Naturwissenschqflen 45.69-70. Liischer, M . (1958b). Ober die Entstehung der Soldaten bei Termitten. Rev. suisse ZOO[. 65,372-377. Lllscher. M. (1959). Die Physiologie der Differenzierung der Kasten bei der Termite Kalotrrmes Pavicollis (Fabr.). In ” Ontogeny of Insects ” (Symposium in Prague, 1959) pp. 161-166. LUscher, M. and Engelmann, F. (1955). Uber die Steurerung der Corpora allataFuriktion bei der Schabe hucophaea maderae. Rev. suisse 2001.62,649-657.

326

V. B. WIGGLESWORTH

Lilscher, M. and Engelmann, F. (1960). Histologische und experimentelk Untersuchungen ilber die Auslosung der Metamorphose bei Leucophaea maderae (Orthoptera). J . Insect Physiol. 5. 240.-258. LUscher, M. and Karlson, P.(1958). Experimentelle Auslosung von Hautungen bei der Termite Kalotermesjavicollis (Fabr.). J. Insect Physiol. 1, 341-345. LUscher, M. and Springhetti, A. (1960). Untersuchungen Uber die Bedeutung der Corpora allata filr die Differenziening der Kasten bei der Termite Kalotermes ji’avicollis F. J. Insect Physiol. 5 , 190-2 12. McLeod, D. G. R. and Beck, S. D. (1963). Photoperiodic termination of diapause in an insect. Biol. Bull., Woods Hole 124, 84-96. Mather, K. (1961). Nuclear materials and nuclear changes in differentiation. Nature, Lond. 190,404408. Mellanby. K. (1938). Diapause and metamorphosis of the blowfly Lucifia sericata Meig. Patmitology 30, 392-402. Mellanby, K. (1939). Fertilization and egg production in the bed-bug, Cimex Iectularius L. Parasitology 31, 193-199. Meyer, G. F. and Pfiugfelder, 0. i1958). ElektronenmikroskopischeUntersuchungen an den Corpora cardiaca von Carausius morosus Br. 2.Zellforsch. 48, 556-564. Mochida, 0. and.Yoshimeki, M. (1962). Relations with development of the gonads, dimensional changes of the corpora allata and duration of post-diapause period 6, 114-123. in hibernating larva of the rice stem borer. Jap. J . appl. Ent. 2001. Mokia, G. G. (1941). Contribution to the study of hormones in insects. C.R. Acad. Sci. U.R.S.S.30,311-313. Monod, J. and Jacob, J. (1961). Telconomic mechanisms in cellular metabolism, growth and differentiation. Cold Spr. Harb. Symp. quanr. Biol. 26, 389-401. Naisse, J. (1962). Neurosecretion and corpora cardiacacorpora allata during postembryonic development in Lumpyris noctiluca L. (Coleoptera). Gen. comp. Endocr. 2, 63043 I. Nayar, K. K. (1955). Studies on the neurosecretory system of Iphita limbata Still. i. Distribution and structure of the neurosecretory cells of the nerve ring. Biol. Bull., Woods Hole 108, 296-307. Nayar, K. K. (1956). Effect of extirpation of neurosecretory cells on the metamorphosis of Iphita limbata Still. Current Sci. 25, 192-193. Nayar, K. K. (1958). Studies on the neurosecretory system of Iphita limbata Still.: v. Probable endocrine basis of oviposition in the female insect. Proc. Indian Acad. Sci. 67, 233-249. Neugebauer, W. (1 961). Wirkungen der Extirpation and Transplantation der Corpora allata auf den Sauerstoffverbrauch, die Eibildung und den Fettkorper von Carausius (Dixippus). morosus Br. Roux Arch. EntwMech. Organ. 153, 314353. Norris, M. J. (1932). Contributions towards the study of insect fertility. i. The structure and operation of the reproductive organs of the genera Ephestia and PIodia (Lepidoptera, Phycitidae). Proc. tool. SOC.Lond. 1932, 595-611. Norris, M. J. (1954). Sexual maturation in the desert locust Schistocerca gregaria Forskill. Anti-locust Bull. 18, 1-44. Novik. V. J. A. (1951). New aspects of the metamorphosis of insects. Nature, Lond. 167, 132-133. Novhk, V.J. A. (1959)“Insektenhormone”. 283 pp. Verlagder tschechoslowakischen Akademie der Wissenschaften.

f 1 0 K M 0 N ES

I N CJ R 0 W ‘ I 11 .4 N I) H E P R 0 0 I; C ‘r I O N

327

NovAk, V. J. A . (1901). Juvenile hormone and morphogenesis. X I . int. Kongr. Enlom. Wien 1960, I , 371-377. Novak, V. J. A. and Ccrvcnkovi, ti. (1961).The function of corpus allatum in the last larval iristar of metabolic insects. In ‘* ‘The Ontogeny of Insects” (Symposium in Prague 1959), pp. 152-156. Novak, V. J. A. and Sbma, K. ( I 962). The influence ofjuvenile hormone on the oxygen consumption of the last larval instar of Pyrrhocoris apterus L. J . Insect Physiol. 8, 145-153. NovAk, V. J. A., S l i m , K . and Wenig, K. (1961). Influence of implantation of corpus allatum on the oxygen consumption of Pyrrhocwis apterus. I n “The Ontogcny of insects” (Symposium in Prague 1959), pp. 147-151. Nliiiez, J. A. (1954). Uber das Vorkoininen von Prothoraxdrilsen bei Anisotarsus cupripenrris (Coleoptera, Carabidse). B i d . Zbl. 73, 602-6 10. O’Farrell, A. F. and Stock, A. (1953). Regeneration and the moulting cycle in BluttelIa germanica L. i. Single regeneration initiated during the first instar. Aust. J. hid. Sci. 6, 485-500. O’Farrell, A. F. and Stock, A. (1954). Regeneration and the moulting cycle in Blattella germmica L. iii. Successive regeneration of both metathoracic legs. Aust. J . b i d . Sci. I, 525-536. O’Farrell, A. F., Stock, A. and Morgan, J. (1956). Regeneration and the moulting cycle in Blattellu germariira L. iv. Single and repeated regeneration and metamorphosis. Aust. J . hid. Sci. 9, 406-422. Ozeki, K. (1954). Experiments on the formation of imaginal structures in the pupae of the swallowtail, Pupilio xuthus Linnaeus. Sci. Pupers Coll. gen. Educ. Univ. Tokyo 4,4746. Ozeki, K.(1958). Neurosecretion of the earwig, Anisolabis maritima. Sci. Papers Coll. gen. Educ. Univ. Tokyo 8, 85-92. Ozeki, K.(1959). Secretion of molting hormone from the ventral glands. Sci. Papers Coll. gen. E h c . Univ. Tokyo 9, 256-262. Ozeki. K. (1960). Hormonal control of molting in the earwig Anisoiabis maritima. Sci.Papers Coll. gen. Educ. Univ. Tokyo 10, 88-97. Ozeki, K. (1961). Secretion of the juvenile hormone in the imago of the earwig Anisolahis maritima. Sci. Papers CON.gen. m u c . Univ. Tokyo 11, 102-107. Pearse, A. G. E. (1953). “Histcchemistry”, 530 pp. Churchill, London. Pfeiffer, I. W. (1939). Experimental study of the function of the corpora allata in the grasshopper, Melanoplus differenrialis. J. exp. 2001.82, 439-461. Pfeiffer, I. W. (1945a). Effect of the corpora allata on the metabolism of adult female grasshoppers. J. exp. Zool. 99, 183-233. Pfeiffer, I. W. (1945b). The influence of the corpora allata over the development of nymphal characters in the grasshopper Melanoplus differentialis. Trans. Conn. Acarl. Arts Sci. 36, 489-5 IS. Pflugfelder, 0. (1937). Rau, Entwicklung und Funktion der Corpora allata und cardiaca von Di.vippus morosus Br. Z. wiss. Zool. 149,477-512. Pflugfelder, 0. (1938). Weitere experimentelle Untersuchungen ilber die Funktion dcr Corpora allata von Dixippus morosus. Z. wiss. 2001.151, 149-191. Pflugfelder, 0. (1947). Ober die Ventraldriisen und einige andere inkretorische Organe des Insektenkopfes. Biol. Zbl. 66, 21 1-235. Pflugfelder, 0. (1958). “Entwicklungsphysiologieder Insckten”, 2nd ed. Stuttgart.

328

V. B. W I G G L E S W O R T H

Piepho, H. (1939). Raupenhautungen bereits verpuppter Hautsttkcke bei der wachsmotte Galleria mellonella L. Naturwissenschaften 27, 301-302. Piepho, H. (1 942). Untersuchungen zur Entwicklungsphysiologie der Insektenmetamorphose. Uber die Puppenhautung der Wachsmotte Galleria mellonella L. Roiu Arch. EntwMech. Organ. 141, 500-583. Piepho, H. (1948). Zur Frage dtr Bildungsorgane dea Hautungswirkstoffs bei Schmetterlingen. Naturwissenschaf~en35, 94. Piepho, H. (1950). Horrnonale Grundlagen der Spinntiitigkeit bei Schmetterlingsraupen. Z. Tierpsychol. 7 , 424-434. Piepho, H. (1951). Uber die Ltnkung der Insektenmetamorphose durch Hormone. Verh. dtsch. zool. Ges. Wilhelmshaven, 1951, 62-75. Piepho, H. and Holz, 1. (1959). Verjiingung des Mitteldarms von Schmetterlingen. Biol. Zbl. 78, 4 17424. Piepho, H. and Richter, A. (1959). Zur Entwicklungsphysiologie des Schuppenkleides bei Ur-Insekten, Untersuchungen an1 Silberfischchen Lepisma saccharina L. Biol. Zbl. 78, 855-861. Piepho, H., Boden, E. and Holz, I. (1960). Uber die Hormonabhangigkeit des Verhaltens von Schwlrmerraupen vor den Hautungen. Z. Tierpsychol. 17,261-269. Pohley, H. J. (1959). Experimentelle Beitriige zur Lenkung der Organentwicklung, des Haustungsrhythmus und der Metamorphose bei der Schabe Periplaneta americana L. Roux Arch. EntwMech. Organ. 151, 323-380. Pohley, H. J. (1960). Experimentelle Untersuchungen uber die Steuerung des Hautungsrhythmus bei der Mehlmotte Ephestia kiihniella Zeller. Roux Arch. EntwMech. Organ. 152, 182-203. Pohley, H. J. (1961). Interactions between the endocrine system and the developing tissue in Ephestia kiihniella. Roux Arch. EntwMech. Organ. 153, 443458. Pohley, H. J. (I 962). Untersuchungen iiber die Veranderung der Metamorphoserate durch Antennenamputation bei Periplaneta americana. Roux Arch. EntwMech. Organ. 153, 492-503. Possompks, B. (1953). Recherches expkrimentales sur le dkterminisme de la m&amorphose de Calliphora erythrocephala Meig. Arch. Zool. exp. gPn. 89,203-364. Possornpts, B. (1955). Corpus allaturn et developpement ovarien chez Calliphora erythrocephala Meig. (Dipthe). C. R. h a d . Sci.. Paris 241, 2001-2004. Quilico, A., Piozzi, F. and Pavan, M. (1956). Sulladendrolasina. Ric.sci.26,177-180. Quo Fu (1959). Studies on thereproduction of the oriental migratory locust: the physiological effects of castration andcopulation. Acta ent. sinica, Peking 9,464-476. Rahm, U. H. ( I 952). Die innersckretorische Steuerung der posternbyonalen Entwicklung von Siulis lutaria L. (Megaloptera). Rev. suisse Zool. 59, 173-237. Rehm, M. (195 I ) Die zeitliche Folge der Tiitigkeitsrhythmen inkretorischer Organe von Ephestia kijhniella wahrend der Metamorphose und des Imaginal-lebens. Roux Arch. EntwMech. Organ. 145, 205-248. Rehm, M. ( 1955). Morphologische und histologische Untersuchungen an Neurosekretorischen Zellen von Schmetterlingen. Z. Zellforsch. 42, 19-58. Richter, A. (1962). Ober die Entwicklung der Schuppenorgane und der Genitalanhange in Abhangigkeit vorn Hormonsysteni bei Lepisma saccharina L. Roux Arch. EntwMech. Organ. 154, 1-28. Robbie, W. A,, Boell, E. J. and Bodine. J. H. (1938). A study of the mechanism of cyanide inhibition: i. Effect of concentration on the egg of Melanoplus diflerentialis. Physiol. 2001. 11 (1938), 54-62.

I I O K h l O N E S I N G K O W T H AND R E P R O D U C T I O N

329

Riillcr, H. ( 1962). uber den Einfluss der Corpora allata auf den Stoffwechsel der Wachsmotte. h’rciitr~~issc~tisc~huJicti 49, 524. Roth, L. M . and Stay, B. (lY59). Control of o k y t e development in cockroaches. Scicwc,e 130, 271-272. Roth, L. M. and Stay. B. (1961). Oocyte dcveloprnent in Diploptera punctuta (Eschscholtz) (Blattaria). J. Iuscct /‘/i,v.wl. 7 , 186-102. Roth, L.M. and Stay, €3. ( l 9 6 w . A comparativestudy of oiicytedevelopment in false ovoviviparous cockroaches. fsydie 69. 165-208. Roth, L. M. and Stay, B. (1962b). OZicyte developnient in Bluttclla germanica and Bluttrlla vuKa (Blattiaria). Ann. etit. So(. Amer. 55, 633-642. Roth, L. M. and Willis. E. R. (1950). The oviposition of Dermestes ater Degeer, with notes on bionomics under laboratory conditions. Amer. Midl. Nut. 44, 427-47. SPgesser, H. (1960). Uberdie Wirkungder CorporaallataaufdenSauerstoffverbrauch bei der Schabe Lrucophueu modeme (F). J . Insect Physiol. 5,264-285. Schaller, F. (1960). Etude du ddveloppement post-embryonaire d’Aeschna cyanea Mull. Ann. Sci. nut. Zool. I t , 755-868. Schaller, F. (1962). Phenomhes d’inhibition de la metamorphose chez des larva ilgdes d’Arschnci cyutrea Mull. (Insecte Odonate). Bull. Sor. zool. Fr. 87,582-600. Scharrer, 8. ( 1948a). The prothoracic glands of Leucophaea mderae (Orthoptera). Biol. Bull., Woods Hole 95, 186-198. Scharrer, B. (194th). Hormones in insects. I n “The Hormones” (G. Pincus and K. V. Thimann, eds.), Vol., 1, pp. 121-158. Academic Press, New York. Scharrer, B. (1952a). Neurosecretion. xi. The effects of nerve section on the intercerebralis-cardiacum-allatum system of the insect Leueophaea maderae. Bid. Bull., Woods Hole 102, 261-272. Scharrer, B. (1952b). Uber neuroendokrine Vorgange bei Insekten. Pjugers Archiv. 255, 154-163. Scharrer, B. (1958). Neuro-endocrine mechanisms in insects. I n “Proc. I1 int. Symp. Neurosecretion” (W. Bargrnann, B. Hanstrom, B. and E. Scharrer, eds.), pp. 79-84. Springer, Berlin. Scharrer, B. (1961). Functional analysis of the corpus allatum of the insect Leucophnra muderac., with the electron microscope. Biol. Bull., WoodsHole 121, 370. Scharrer, B. (1962). The finc structure of the neurosecretory system of the insect Leircnphaeu rtiutlrruc.. Mcm. SOC.Eiidocr. 12, 89-97. Scharrcr, R. and Harnack. M. von (1958). Histophysiological studies on the corpus itllattlnl of Lcucophum nradertre. i. Normal life cycle in male and female adults. Biol. Bull., u”(JOl/.V Ilulc~1.15, 508-520. Scharrer, H. and Harnack, M. von (1961). Histophysiological studies on the corpus allatum of L a u c ~ p h u wmadcrttc. iii. The effects of castration. Biol. Bull., Woods Hole 121, 193-208. Scharrer, E. and Scharrer, B. (1958). Neurosecretion. Science 127, 1396-1398. Schmialek, P.(1961). Die ldentifizierung zweier im Tenebriokot und in Hefe vorkommender Substanzcn mit Juvenilhormonewirkung. Z. Nuturf. 16b, 461-464. Schmialek, P. ( 1 963a). Uber die Bildung von Juvenilhormonen in Wildseidenspinnem. 2. NuturJ. lflb, 462-465. Schmialek, P. (I 963b). Metamorphose henunung von Tenebrio molitor durch Farnesylmethylather. Z . Nuturf, 18b, 5 13-5 15.

3 30

V. B. W I G G L E S W O R T H

Schmialek, P. (I 9634. Uber Verbindungen mit Juvenilhormonwirkung.2.Naturf. 18b, 516-519. Schneider, F. (1950). Die Entwicklung des Syrphiden parasiten Diplazon fissorius Grav. (Hym. Ichneum). Mitt. schweiz. ent. Ges. 23, 156194. Schneider, F. (195 I). Einige physiologische Beziehungen zwischen Syrphidenlarven und ihren Parasiten. Z. angew. Ent. 33, 150-162. Schneiderman, H. A. (1953). Metabolic effects of localized injury to the integument of the cecropia silkworm. Anat. Rec. 117, 640-641. Schneiderman,H. A. (196l).Thejuvenile hormone and other insect growth hormones. Acta Soc. ent. Bohern. (Cs’l.)58, 12-28. Schneiderman, H. A. and Gilbert, L. I. (1958). Substances with juvenile hormone activity in Crustacea and other invertebrates. Biol. Bull., Woo& Hole 115, 530-535. Schneiderman, H. A. and Horowitz, J. (1958). The induction and termination of facultative diapause in the chalcid wasps Mormoniella vitripennis (Walker) and Tritneptis klugii (Ratzeburg). J. exp. Biol. 35, 520-551. Schneiderman,H. A. and Williams, C. M. (1953). The physiology of insect diapause. vii. The respiratory metabolism of the cecropia silkworm during diapause and development. Biol. Bull., Woods Hole 105,32&334. Schneiderman, H. A., Gilbert, L. I. and Weinstein, M. J. (1960). Juvenile hormone activity in micro-organisms and plants. Nature, Lond. 188, 1041-1042. Schoonhoven,L. M. (1962).Diapause and the physiology of host-parasite synchronization in Bupalus piniarius L. (Geometridae)and Eucarcelia rutilla Vill. (Tachinidae). Arch. nker. Zool. 15, 111-174. Schultz, A. L. (1960). Electron microscope observations of the corpora allata and associated nerves in the moth Celerio lineata. J. Ultrastructure Res. 3, 320-327. Shappirio,D. G. (1960). Oxidative enzymes and the injury metabolism of diapausing cecropia silkworms. Ann. N.Y.Acad. Sci. 89, 537-547. Shappirio,D. G. and Williams, C. M. (1957). The cytochrome system of the cecropia silkworm. Proc. roy. SOC.B 147, 218-246. Siew, Yow Cheong (1963). “Some physiological aspects of adult reproductive diapause in the Chrysomelid beetle, Galeruca tanaceti (Linn)”. Thesis, London University. Sin&, T. (1958). Ovulation and corpus luteum formation in Locusta migratoria migratorioides R. and F. and Schistocerca gregaria (Forskal). Trans. R. ent. SOC. Lond. 110, 1-19. S h a , K. (1960). Physiology of sawfly metamorphosis. (i). Continuous respiration in diapausing prepupae and pupae. J. Insect Physiol. 5, 341-348. SlAma, K. (1961). Pseudo-juvenilizingeffect in insects. Acta SOC.ent. Bohern. (C31.) 58, 117-120. SlAma, K. (I 962). The juvenile hormone-like effect of fatty acids, fatty alcohols, and some other compounds in insect metamorphosis. Acta Soc. ent. Bohem. (CX) 59, 322-340. Sloper, J. C. (1957). Presence of a substance rich in protein-bound cystine or cysteine in the neurosecretory system of an insect. Nature, Lond. 179, 148-149. Southwood, T. R. E. (1961). A hormonal theory of the mechanism of wing polymorphism in Heteroptera. Proc. R. ent. Soc. Lond. A 36,63-66. Srivastava, U. S. (1960). Secretory cycle and disappearance of the prothoracic glands in Tenebrio molitor L. (Coloeptera: Tenebrionidae). Experientia 16,445

H O R M O N E S IN G R O W T H A N D R E P R O D U C T I O N

33 1

Staal, CJ.R. ( 196 L ), “Studies on the physiology of phase induction in Locusla migratoriu migrutorioides H. and F.” 125 pp. Thesis, Wageningen, Veenman and

Zonen. Stamm, M. D. (1959). Estudios sobre hormonas de invertebrados. ii. Aislamiento de hormonas de la metamorphosis en el ortoptera Dociostawus marcoccanw. An. Fis. Quim. B Madrid, 55, 171-178. Stegwee, D. (1960). Metabolic effect of a corpus allatum hormone in diapausing kptinotarsa decemlineata Say. XI. int. Kongr. f i t . Wien,,19W, 3,218-221. Steinberg, D. M. (1958). Analyse morphogbnetique du dkveloppement des organa imaginaux chez les insectes holombtaboles. Proc. Xth int. Congr. Ent. Montreal, 1956,3, 261-266. Stevenson, E. and Wyatt, G. R. (1962). The metabolism of sikmoth tissues. i. Incorporation of leucine into protein. Arch. Biochem. Biophys. 99.65-71. Stiennon, J. A. and Drochmans, P. (1961). Electron microscope study of neurosecretory cells in Phasmidae. Gen. comp. Endocr. 1,286294. Strangways-Dixon, J. (1961a). The relationship between nutrition, hormones and reproduction in the blowfly Calliphoraerythrocephala(Meig.). i. Selectivefeeding in relation to the reproductive cycle, the corpus allatum volume and fertilization. J. exp. Biol. 38,225-235. Strangways-Dixon, J. (1 961b). ii. The effect of removing the ovaries etc. J. exp. Biol. 38, 637-646. Strangways-Dixon, J. (1962). iii. The corpus allatum in relation to nutrition etc. J. exp. Biol. 39, 293-306. Strich-Halbwachs, M.C. (1959). Controle de la mue chez Locusta migratorh. Ann. Sci. nut., Zool. Ser. 12, 1, 483-570. Stumm-Zollinger, E. (1957). Histological study of regenerative processes after transsection of the nervi corporis cardiaci in transplanted brains of the cecropia silkworm (Platysamia cecropia L.). J . exp. Zool. 134.3 15-326. Sulkowski, E.and Wojkczak, L. (1958). Studies on the biochemistry of the waxmoth (Galleria mellonella.).I xviii. Succinoxidase system in metamorphosis. Acra Biol. exp., Varsovie 18, 239-248. Takaoka, M. (1957). Studies of the metamorphosis in insects. ii. Relation between pupation and the tracheal system in Drosophila melanogaster. Embryologla 3, 343-353. Takaoka, M. (1959). Studies of the metamorphosis in insects. iii. Relation between pupation and oxygen tension in Drosophila melanogaster.Embryologia4.231-246. Takaoka, M. (1960). Studies of the metamorphosis in insects. v. Factors controlling the larval period of the squash fly Zeugodugus depressus Shiraki. Etnbryologia 5, 259-269. Telfer, W. H. (1954). lmmunological studies of insect metamorphosis. ii. The role of a sex-limited blood protein in egg formation by the cecropia silkworm. J. gen. Physiol. 37, 539-558. Telfer, W. H. (1960). The selective accumulation of blood proteins by the oocytes of saturniid moths. Biol. Bull., Woods Hole 118, 338-351. Telfer, W. H. and Williams, C. M.(1960). The effects of diapause, development, and injury on the incorporation of radioactive glycine into the blood proteins of the cecropia silkworm. J. Insect Physiol. 5, 61-72. Thomsen, E. ( I 942). An experimental and anatomical study of the corpus allatum in

332

v. B.

W1GGLCSWORTH

t h e blowfly Ccllliphoro crythrorcphulti Meig. Vidensk. Medd. clunsk. nuturh. JiJrcn. Khh. 106. 319-405. Thoirisen, E. ( 1949). Influence of the corpus allatum o n the oxygen consumption of adult C'ulliphoru crythrocephulu Meig. J . c x p . Biol. 26, 137- 149. Thomsen, L:. ( 1952). Functional significance of the neurosecretory brain cells and the corpus cardiacum in the female blowfly Cnlliphora erythrocephalu Meig. J . exp. Biol. 29, 137-172. Thomsen, E. (1954). Studies on the transport of neurosecretory material in Calliphoru erythrocephuln by means of ligaturing experiments. J . exp. Biol. 31, 322-330.

Thomsen, E. and Hamburger, K. (1955). Oxygen consumption of castrated females of the blowfly, Culliphoru erythrocephalu Meig. J . exp. Biol. 32, 692-699. Thomsen, E. and Moller, I. (1959). Neurosecretion and intestinal protease activity in an insect, Culliphorri erJ,rhrocrp!iuluMeig. Nature, Lond. 183, 1401-1402. Thomsen, E. and Moller, 1. (1963). Influence of neurosecretory cells and of corpus allaturn on intestinal protease activity in the adult Calliphoru eryrhrocephrrfrr Meig. J . cxp. Biol. 40.301-321. Toyama, K . (1902). Contributions to the study of silkworms. i. On the embryology of the silkworm. Bull. Coll. Agric. Tokyo 5 , 73-1 17. Van der Kloot, W. G . (1954). Neurosecretion and brain activity in the cecropia silkworm. Annt. Rec. 120, 46. Van der Kloot, W. G. (1955). The control of neurosecretion and diapause by physiological changes in the brain of the cecropia silkworm. Biol. Bull., Woods Hole 109, 276-294. Van der Kloot, W. G. (1960). Neurosecretion in insects. .4nnu. Rev. Ent. 5 , 35-52. Van der Kloot, W. G. (1961). Insect metamorphosis and itsendocrine control. Amer. Zoologisi 1, 3-9. Vogt, M. (l942a). Die " Puparisierung" als Ringdrusenwirkung. Biol. Zbl. 62, 149- 1 54. Vogt, M. (1942b). Weiteres zur Frage dcr Artspezifitat gonadotroper Hormone. Untersuchungen an Drosophilu-Arten. Roux Arch. EntwMech. Organ. 141,424454. Vogt, M. (1943). Zur Produktion und Bedeutung metamorphosefordernder Hormone wahrend der Larvenentwicklun von Drosophila. Biol.Zbl. 6 3 , 3 9 5 4 6 . Vogt, M. (1948). Fettkorper und nocyten der Drosophilu nach Extirpation der adulten Ringdruse. 2.Zellfurscli. 34, 160-1 64. Waku, Y. (1957). Pupal oxygen uptake in the Chinese oak-silkworm Antheraeu pernyi Guirin and breaking of its diapause. Sci. Rep. TJhoku Univ. 23, 143-1 55. Waku, Y. (1960af. Studies o n the hibernation and diapause in insects. iv. Histological observations of the endocrine organs in the diapause and non-diapause larvae of the Indian mealmoth, Plodiu i~iterpunc~tellu Hubner. Sci. Rep. Tbhoku Univ. 26, 327-340. Waku, Y. (1960b). Studies on the hibernation and diapause in insects. v. Respiratory metabolism and enzyme activity in the diapause and non-diapause larvae of the Indian mealmoth. Plodia intcrpunctdlu Hubner. Sci. Rep. T6hoku Univ. 26, 341-352. Wang, Shu-Yiand Dixon,S. E. (1960). Studies on the transaminase activity of muscle tissue from allatectomized roaches, Prriplmeta umericana. Canad. J. 2001.38, 275-283. Watson, J. A. L. (1962). " Moultingand reproduction in the adult firebrat, Thermobiu

d

H O R M O N E S IN GROWTH AND REPRODUCTION

333

domesrica (Packard) (Thysanura, Lepismatidae)". Thesis, University of Cambridge. J. micr. Wells, M.J. (1954). The thoracic glands of Hemiptera Heteroptera. Qwr. Sci. 95, 231-244. Wells, M.J. and Wells, J. (1959). Hormonal control of sexual maturity in Octopus. J. exp. Biol. 36, 1-33. Wigglesworth, V. B. (1934). The physiology of d y s k in Rhodniusprolixus ( H e h p tera). 11. Factors controlling moulting and metamorphosis. Qwr. J. nticr. Sci, 77,191-222. Wigglesworth, V. B. (1936). The function of the corpus allatum in the growth and reproduction of Rhodnius prolixus (Hemiptera). Quart. J. micr. Sci. 79,91-121. Wigglesworth, V. B.(1937). Wound healing in an insect (Rhodniusproliixus, Hemip tera). J. exp. Biol. 14, 364381. Wigglesworth, V.B. (1939). Hiiutung bei Imagines von Wanzen. Nafurwissenschaften 27, 301. Wigglesworth, V. B. (194Oa). Local and general factors in the development of "pattern" in Rhodniusprolfxus (Hemiptera). J. exp. Biol. 17, 180-200. Wigglesworth, V. B. (1940b). The determination of characters at metamorphosis in Rhodnius prolixus (Hemiptera). J . exp. Biol. 17,201-222. Wigglesworth, V. B.(1948). The functions of the corpus allatum in Rhodniusprolixus (Hemiptera). J. exp. Biol. 25, 1-14. Wigglesworth, V. B. (1952a). The thoracic gland in Rhodnius prolixus (Hemiptera) and its role in moulting. J. exp. Biol. 29,561-570. Wigglesworth, V . B. (1952b). Hormone balance and thecontrol of metamorphosis in Rhodnius prolixus (Hemiptera). J. exp. Bid. 29, 620-631. Wiggksworth, V. B. (1953a). The origin of sensory neuronca in an insect, M i u s prolixus (Hemiptera). Quart. J . micr. Scl. 94, 93-1 12. Wigglesworth, V. B. (1953b). Determination of cell function in an insect. J. Embryol. exp. Morph. 1 , 269-277. Wigglesworth, V. B. (19%). "The Physiology of Insect Metamorphosis". 152 pp. Cambridge University Press. Wiggleaworth, V. B. (1954b). Neurosecretion and the corpus cardiacum of insects. Pubbl. Stat. zool. Napoli 24, (Suppl.), 41-45. Wigglesworth, V. B.(1955a). The breakdown of the thoracic gland in theadult insect, Rhodnius prolixus. J. exp. Biol. 32, 485-491. Wigglesworth. V. B. (1955b). The role of the haemocytes in the growth and moulting of an insect, Rhodnius prolixus (Hemiptera). J. exp. Biol. 32, 649-663. Wigglesworth, V. B. (1956). The haemocytes and connective tissue formation in an insect, Rhodnius prolixus (Hcmiptera). Quart. J. Micr. Sci. 97, 89-98. Wigglesworth, V. B. (1957). The action of growth hormone in insects. Symp. SOC. exp. Biol. 11, 204-227. Wigglesworth, V. B. (1958). Some methods for assaying extracts of the juvenile hormone in insects. J. Insect Physiol. 2,734. Wigglcaworth, V. B. (1959a). "The control of growth and form". 140 pp. Cornell University Press,Ithaca. Wigglesworth, V. B. (1959b). The histology of the nervous system of an insect, RhodniusproNxus, (Hemiptera). ii. The central ganglia. Quart.J. micr. Sci. 100, 299-313.

334

V. B. W I G G L E S W O R T H

Wigglesworth, V. B. (1960). Axon structure and the dictyosomes (Golgi bodies) in the neuron- of the cockroach, Periplaneta americana. Quart. J. mu.Scf. 101, 381-388. Wigglaworth, V.B. (1961a). Insect polymorphism-a tentative synthesis. In “Insect Polymorphism”. Symposium No. 1. Royal Entomological Society of London (J. S. Kennedy, ed.), pp. 104-113. Wigglesworth, V. B. (1961b). Some observations on the juvenile hormone effect of famesol in RhodniusprolixusStAl. J. Insect Physiol. 7.73-78. Wigglesworth, V. B. (1963a). The juvenile hormone eflect of famesol and some related compounds: quantitative experiments. J . Insect Physiol. 9, 105-119. Wigglesworth, V. B.(1963b). The action of moulting hormone and juvenile hormone at the cellular level in Rhodniusprollxus. J. exp. Biol. 40,231-245. Wigglesworth, V. B. (1963~).The union of protein and nuclcic acid in the living cell and its demonstration by osmium staining. Quart. J. micr. Sci. 105, 113-122. Wilde, J. de (1954). Aspects of diapause in adult insects,with special regard to the Colorado beetle, Leptinotarsa decemlineata Say. Arch. nkerl. 2001. 10,375-385. Wilde, J. de (1962). Photoperiodism in insects and mites. Annu. Rev. Ent. 7, 1-26. Wilde, J. de and Stegwee, D. (1958). Two major effects of the corpus a h t u m in the adult Colorado beetle. Arch. nkerl. Zool.l3,277-289. Will9, R.B. and Chapman, G. B. (1960). The ultrastructure of certain components of the corpora cardiaca in Orthopteroid insects. J. Ultrastructure Res. 4, 1-14. Williams, C. M.(1946). Physiology of insect diapause: the role of the brain in the production and termination of pupal dormancy in the giant silkwormPlatysamia cecropia. 8101. Bull., W o d Hole 90, 234-243. Williams, C. M.(1947). Physiology of insect diapause. ii. Interaction between the pupal brain and prothoracic glands in the metamorphosis of the giant silkworm, Platysamia cecropia. Biol Bull., Woodr Hole 93,89-98. Williams, C. M. (1948). Extrinsic control of morphogenesis as illustrated in the metamorphosis of insects. Growth Symposium 12, 61-74. Williams, C. M.(1 95 1). Biochemical mechanisms in insect growth and metamorphosis. Fed. Proc. 10, 546-552. Williams, C. M.(1952). Physiology of insect diapause. iv. The brain and prothoracic glands as an endocrine system in the cecropia silkworm. Biol. Bull., Woods Hole 103, 120-138. Williams,C. M.(1956a).Thejuvenilehormoneof insects.Nature,Lond. 178,212-213. Williams, C. M.(1956b). Insect metamorphosis. Proc. R. ent. Six.Lond. C 21,20; 27-28. Williams, C. M.(1959). The juvenile hormone. i. Endocrine activity of the corpora allata of the adult cecropia silkworm. Biol. Bull., Woodr Hole 116, 323-338. Williams, C. M. (1961a). Present status of the juvenile hormone. Science 133, 1370. Williams, C. M.(1961b). The juvenile hormone. ii. Its role in the endocrine control of moulting, pupation, and adult development in the cecropia silkworm. Biol. Bull., Woods Hole 121, 572-584. Williams, C. M.,Moorhead, L. V. and Pulis, J. F. (1959). Juvenile hormone in thymus, human placenta and other mammalian organs. Nature, Lond. 183, 405. Wyatt, G. R. (1958). The metabolic control of insect development. “Symposium on the chemical basis of Development” (W. McElroy and B. Glass,eds.), pp. 807-812. Baltimore, Johns Hopkins Press.

H O R M O N E S I N G R O W T H A N D REPRODUCTION

335

Wyatt, G. R. (1959). Phosphorus compounds in insect development. Proc. IVth inr. Congr. Biochem. VlcnM, 1958, 12, 161-184. Wyatt, G. R. (1962). Metabolic regulation in the development of insects. J. Ben. Physiol. 45, 622. Yamamoto, R. T. and Jacobson, M. (1962). Juvenile hormone activity of isomers of farnesol. Nature, b n d . 196, 908-909. Yamashita, Y., Tani, K.and Kobayashi, M.(1961). ”%e Mects of allatectomy on the number of eggs in the silkworm Bombyx mod. Acta sericologica39, 12-15. Zwicky, K. and Wigglesworth, V. B. (1956). The course of oxygen consumption during the moulting cycle of Rhuhius prollxrucr. Proc. R. ent. Soc.Lond. A 31, 153-160.

Aden& The following supplementary notes are taken from recent publications. Page 250:Electron microscope studiesof the corpus cardiacum offmcophaeaconfirm the belief that this serves both as a storage and release centre for extrinsic neurosecretory substances, and as an endocrine organ with its own neuroglandular cells with axon-like processes (Scharrer, 1963). Page 257: Cholesterol in 10% ethanol will induce development in brainless pupae of cecropia (Edwards and Krishnakumaran in Schneiderman and Gilbert, 1964); and tritium-labelled cholesterol injected into Call&hora larvae gives rise to radioactive ecdyson; it is therefore concluded that cholesterol can serve as a p r e c m r of ecdyson (Karlson and Hoffmeister, 1963). Page 265 : Ecdyson will increase the rate of protein synthesis in mammalian cells (Burdette and Coda, 1963). p. 270: ecdyson from Bombyx pupae has been separated by counter current partition into five fractions a,p, and three new ones (Burdettc and Bullock, 1963). Puge 266: Since it is possible to induce the enlargement of different series of puffs in the salivary glands of Chironomus by changing the Na/K balance, Kroeger (1963) suggests that hormones may exert their effects on the chromosomes by regulating the sodium: potassium ratio in the nuclear sap. Page 273: In Ostrinia nubilalis spccialiwd cells of the hind-gut appear to set free into the blood a hormone (“proctodone”) which is mxesary to activate the neurosecretory cells of the brain and thus bring diapause to an end (Beck and Alexander, 1964). Schoonhoven (1963) has found that the lack of electrical activity during diapause does not extend to all parts of the brain of the cecropia pupa; and in several other insect pupae spontaneouselectrical activity does not disappear during diapaw. Page 279: The diapause hormone in Bombyx is effective only in the young pupa, at the time when the ovarioles have started to grow vigorously (Hasagawa, 1963). Puge 281 : The corpora allata in larvae of Anisolabis are inactivated by removal of the brain and by section of the recurrent nerve and the nervi corpori cardiaci. They are not affectedby removal of the frontal ganglion of the pars intercerebralis(Ozeki, 1962).

Page 293: Williams (1963) and Schneiderman and Gilbert (1964) give further evidence that the juvenile hormone itself will cause moultiag in brainlesspupae of the large silkmoths, and Krishnakumaran (in Schneiderman and Gilbert, 1964) has shown that farnesol also has this effect in cecropia. Attempts to repeat these experiments in Rhodnius have not so far proved successful.

336

V. B. WIGGLESWORTH

Page 295: Prospective winged larvae of Aphis f a k e give rise to wingless adults if smeared with farnesol, or if exposed to the vapour of f a m e d , or if allowed to feed on plants placed in a solution of mevalonic acid ladone (von h h n , 1963). Page 299: Farnesol will induce yolk formation in the ovary of Calliphora (Weirich, 1963). Page 300: Extirpation of the corpus allatum either in the fifth-stage larva or the newly emerged adult of Pieris brassicae, in which the ovaries mature after emergence, arrests the deposition of yolk in the eggs (Karlinsky, 1962, 1963). Page 305: In the grasshopper Gomphocerusthe corpus allatum secretion is necessary

in the female for singing and copulating; whereas in the male singing, courting, and copulation are not affected by allatectomy (Loher, 1962). REFERENCES Beck, S. D. and Alexander, N. (1964). Hormonal activation of the insect brain. Science 143,478-479. Burdette, W. C. and Bullock, M. W. (1963). Ecdysone: five biologically active fractions from Bombyx. Science 140, 131 1. Burdette, W. C. and Coda, R. L. (1963). Effect of ecdysone on incorporation of C14-leucineinto hepatic protein in vitru. Proc. Sac. exp. Biol., N. Y. 112,216-21 7. Dehn, M. von (1963). Hemming der FlUgelbildung durch Farnesol bei der schwanen Bohnenlaus, Doralis fabae Scop. Naturwissenschaften50, 578-579. Hasegawa, K. (1963). Studies on the mode of action of the diapause hormone in the silkworm, Bombyx mori. J. exp. Biol. 40,517-529. Karlinsky, A. (1962, 1963). Effets de l’ablation dm corpora allata larvaries sur le developpement ovarien de Pieris brashicue. (Upidopt&). C.R. Acad. Sci., Paris 255,191-193; 256,4101-4103. Karlson, P. and Hoffmeister, H. (1963). Zur Biogenese des Ecdysons. i. Umwandlung von Cholesterin in Ecdyson. Hoppe-Seyl. 2.331,298-300. Karlson, P.,Hoffmeister, H., Hoppe, W. and Huber, R. (1963). Zur Chemie des Ecdysons. Liebigs Ann. Chem. 662, 1-20. Kroeger, H. (1963). Chemical nature of the system controlling gerle activities in insect cells. Nature, Lond. 200, 1234-1235. Loher, W. (1962). Die Kontrolle des Weibchengesanges von Gomphocerus r u f i L. (Acridinae) durch die Corpora allata. Naturwissenschqfien49,402. Ozeki, K. (1962). Studies on the secretion of the juvenile hormone in the earwig Anisolabis mariiima. Sci.Papers Coll. gen. Educ. Univ. Tokyo 1 2 , 6 7 2 . Scharrer, B. (1963). Neurosecretion. xiii. The ultra structure of the corpus cardiacum of the insect Leucophaea maderae. 2.Zellforsch. 60,761-796. Schneiderman, H. A. and Gilbert, L. I. (1964). Control of growth and development in insects. Science 143, 325-333. Schoonhoven, L. M. (1963). Spontaneous electrical activity in the brains of diapausing insects. Science 141, 173-1 74. Weirich, G. (1963) “Zur Frage der horrnonalen Regulation der Eireifung bei Insekten”. Thesis, Munich. Williams, C. M. (1 963). The juvenile hormone. iii. Its accumulation and storage in the abdomens of certain male moths. Biol. Bull., Woods Hole 124, 355-367.

Author Index Numbers in italics refer to the pages on which references are listed in bibliographies at the end of each articte. A Adair, G. S.,2 I , 64 Adam, N. K., 83, 102,125 Adamson, A. W., 104,125 Agrell, I., 263, 264, 275, 316 Alexander, N., 335, 336 Alexandrowicz, J. S.,224, 242 Allen,T. H., 186,187, 191, 192,196,207, 212, 213 Ammon, H., 185, 197, 199, 215 Andersen, S.O., 3, 8, 35, 37, 41,42,43, 44,45, 46, 47, 49, 50, 62 Andrewartha, H. G., 275, 316 Armbruster, L., 138. 169 Arvy. L., 210, 212, 250, 251, 252, 255, 257, 283, 306,316 Autrum,H., 133,134,135,138,143,144, 145, 150, 152, 155, 156, 158, 159, 163, 164, 166, 169, 170 B Bailey, K., 2, 3. 8, 33, 34, 35, 62 Bailey, S. W.,202, 212 Barrer, R. M., 105, I25 Barsa, M. C., 263, 325 Bartley, S. H., 134, 136, 170 Barton-Browne, L., 225, 226, 231, 237, 238, 242 Bayliss, L. E., 69, 96,125 Beament, J. W. L., 73, 74,79, 80, 82,88, 89, 92, 94, 95, 96, 98, 99, 101, 104 105,106,107, 109, 110, 111,112, 115, 118, 120, 122, 123. 125, 126, 129 Beard, R. L., 233, 241. 242 Beck, S. D., 213,274,278,318,326,335, 336 Becker, H. J., 265, 266, 267, 317 Bedau. K., 152, 170 Bennet-Clark, H. C., 95, 97, 126, 209, 210, 213, 233, 242 Berger, H., 258, 317 337

Bertholf, L. M.,137, 140, 170 Biekert, E., 158, 170 Blunck, H., 176, 180,213 Baden, E., 286,328 Bodenstein, D., 258, 259, 262, 272, 297, 303, 304,317,325 Bodine, J. H., 186,187,188,191,192,193, 196, 198,207,212,213,217,277, 328 Boell, E. J.. 186, 191, 196, 213,277,328 Boisson, C., 252, 317 Bonhag, P. F., 15, 62 Bounhiol, J. J., 250, 251, 255, 286, 290, 299, 306, 316, 317 Brandenburg, J., 298,317 Bricteux-Grbgoire, S., 265, 317 Briggs, M.H., 158, 170 Brinkhurst, R. O., 285, 286,317 Brocher, F., 80, 126, 176, 180, 181,213 Brown, A. W. A., 221, 245 Brown, F. A. Jr., 147, 170 Browning, T.0.. 72, 73, 75, 126 Brunet, P. C., 186, 187, 218, 218 Brunet, P. C. J., 58, 62, 63, 201, 213 Buck, J. B., 134. 170, 209, 216 Bucklin, D. H.,277, 278, 317 Bilckmann, D., 205,213,263.317 Buddenbrock, W.von, 132,170, 173 Buist, J. M., 23, 62 Bullock, M.W.,335, 336 Bult, T.,84, 87, 126 Burdette, W. C., 335, 336 Burdette, W.J., 256, 324 Burkhardt, D., 134, 135, 138, 142, 144, 145, 147, 152, 153, 154, 164, 167, 168, 169, 170 Bursell, E., 178, 213 Burtt, E. T., 205, 213, 260, 317 Butenandt, A., 158, 170, 205, 213, 270, 271, 317 Buxton, P. A., 72, 73, 74, 126 Bytinski-Salz, H.,211, 212, 214

338

A U T H O R INDEX

Cameron, M. L., 225,226,230,236,237, 239.242 Carlislc, D. B., 230, 244, 285, 317 Carlson, L. D., 186, 193, 213 Carton, R. W., 21, 62 Cervenkova, E.. 282, 327 Chadwick, L. E., 261, 317 Champion, F. C., 83, 126 Chang, S. C., 230,245 Chapman, G. B., 249,334 Chen, D. H., 299, 317 Christensen, H. N., 96, 126 Church, N. S., 253, 254, 255, 272, 273, 215, 211, 317 Clark, G. L., 61, 62 Clark, J. W., 21, 62 Clarke, K. U., 180, 214, 235, 241, 242, 253, 305, 318 Clausen, M. B., 109, 128 Clements. A. N., 301,318 Clever, U., 205, 214 266, 261, 318 Cloutier, E. J., 273, 274, 318 Coda, R. L., 335,336 Cohen, B. B., 41, 63 Colhoun, E. H.. 226, 227, 237, 238, 239, 240, 242 Corey, D. B., 97, 128 Cottrell, C. B., 62,62, 177, 178, 179. 181, 182, 183, 200, 202, 206, 207,208,209, 214 Cousin, G., 285, 318 Cox, R. C., 51, 54, 62 Crescitelli, F., 133, 171 D Dainauskas, J., 21, 62 Danielli, J. F., 94, 121, 126 Daumer, K.,134, 138, 139, 140, 160, 161, 162, 165, 166, 170 Davenport, D., 222, 223, 242 Davey, K. G., 221, 222, 223, 224, 225, 226, 227, 228, 230, 231. 233, 234, 235, 236, 237, 238,239,240,241, 242,243, 278, 318 Davies, M. E., 74, 126 Davis, H. F., 21, 53, 64 Davson, H., 69, 94, 126, 126 Davy, N., 83, 126

Dawson, C. R., 185, 186, 188, 191, 214, 216 Dehn, M. von, 335, 336 Dennell, R., 4,59,62, 109,126. 184,186, 188, 189, 190,205, 214 Deuse, R.,229, 243 De Valois, R. L., 164, 170 Diamond, J. M.,88, 91, 116, 126 Dixon, M., 198, 214 Dixon, S. E., 313, 332 Doane, W. W., 313, 318 Dodson, L. F., 225, 242 Dodt, E., 138, 143, 148, 149, 173 Donner, K. O., 164, I70 Drochmans. P., 249,331 Duarte, A. J., 176, 180, 214 Ducleaux, E., 277,318 Dupont-Raabe, M., 230, 244, 301, 319

E Eassa, Y.E. E., 262, 319 Edney, E. B., 72, 73, 74, 126, 127 Egelhaaf, A., 157, I70 Eguchi, E., 152, 173 Eidmann, H.,176, 180, 214 Elliott, G. F., 3, 12, 13, 32, 51, 54, 55, 61, 62 Ellis, P. E., 285, 317 Elsden, D. F., 35, 36, 54, 63, 64 Engelmann, F., 282, 302, 303, 304, 314, 319, 325, 326 Evans, A. C., 179, 214 Evans, J. J. T.,228, 243 Ewen, A. B., 249, 259,319 Ewer, D. W., 182,214

F Fan, H. Y.,109,128 Fearon, W. R.. 44,63 Fem, C., 28,63 Fingermann, M.,147, 170 Finlayson, L. H., 181, 183, 214 Fischer, F., 226, 229, 243 Fling, N., 186, 187, 194, 195, 198, 215 Florkin, M., 265, 317 Flory, P. J., 26, 27, 52.63 Forrest, H. S., 157, 170

AUTHOR INDEX Fraenkel, G.. 178. 179, 184, 186, 189, 200.201,204,206,207,208,211,214, 218, 218, 259, 263, 319 Fraser, A., 254,272, 291, 319,324 Friedrich, H., 132, 170 Frisch, K. von, 131, 132, 133, 134, 138, 171 Fukaya. M.,256,272,274,275,295,319, 320,324 Fukuda, S.,254,260,279,281,282,314, 320 Fuller, H. B., 251, 320 Fyg, W., 253, 258, 259, 320

339

Hanstrorn, B., 249, 250, 321 Harnack, M. yon, 271, 298, 313, 321, 329 Harris, E. J., 69, 71, 96, 117, 127 Hartley, G. S., 108, 127 Hartline, H. K.,143, 171 Harvey, W. R.,248, 263, 264, 265, 278, 321 Hasegawa, K., 279, 298, 321,335, 336 Hasselmann, E.-M., 146, 147, 148, 149, 171 Heintz, E., 140, 171 Henricksen, K. L., 176,215 Henson, H., 286,321 G Herlant-Meewis, H., 252, 321 Gabe, M., 210, 212, 249, 250, 251, 252, Hertz, M., 134, 161, 171 255, 256, 257, 258, 283, 306, 316, 320 Hess, C., 131,171 Gahan, J. B., 222, 245 Highnam, K. C., 225,240,244,249,250, Gaines, G. L. Jr., 107, 127 275,299,304, 305,306,310,311,314, Garrod, M.,35, 63 321, 322 Geiger, H. R.,297, 320 Hill, D. L., 186, 192, 193, 198, 213 Geldiay, S.,242, 244, 305, 322 Hill, L., 299, 306, 310, 311, 314,322 Gent, A. N., 19, 24,63 Hill, R. B., 227, 237, 241, 244 Gersch, M.,224,226,229,230,236,238, Hinton, H. E., 177, 215,279, 322 243 Hodgkin, A. L., 70, 127 Gilbert, L. I., 254, 281, 284, 290, 292, Hodgson, E. S., 225,231,237,238,242. 293, 294, 300, 320, 330, 335, 336 242, 244,305, 322 Gillett, J. D., 301, 307, 321 Hoeve. C. A. J., 26, 27, 52. 63 Girardie, A., 281, 297, 321 Hoffrnann, C., 144, 152, 154, 170 Gold, E., 237. 245 Hoffmann, Ch., 138, 144, 150, 153,158, Goldsmith. T. H., 137, 139, 140. 148, I71 149, 150, 155, 157, 158, 162, 163, 171 Hoffmcister, H..335, 336 Gomori, G., 249.321 Holdgate, M.W., 79, 92, 127 Gotte, L.,54, 63 Holz, I., 286, 288, 328 Govaerts, J., 82, 127 Hoppe. W., 336 Granit, R., 164, 171 Horie, Y., 313, 322 Graubard, M. A.. 189, 215 Horowitz, J., 276, 280, 330 Gustavson, K. H., 33, 40,63 Horowitz, N. H., 186, 187, 194,195,198, Guthrie, D. M.,231, 232, 243 215 Hsiao, C.. 189. 206, 207, 214, 218, 218 H Huber. R., 336 Hachlow, V., 254, 321 Hurst, H., 107, 109, 127 Hackman, R. H., 57, 59, 63, 183, 184, Huxley, A. F.,3, 12, 13, 32, 51, 54, 55, 185, 199, 202, 204, 201, 215 61, 62 Halbwachs, M. C., 260. 269, 285, 321 Hamburger, K., 308, 312, 313, 332 I Hamilton, H. K., 222, 243 Ichikawa, M.,255, 256, 257, 259, 272, Hamilton, W. F., 132, 171 275,281, 283,322 Hanser. G., 205, 215, 261, 271, 323 Ilse, D., 132, 134, 139, 171, 172

340

AUTHOR INDEX

Ishizaki, H., 257, 322 Ito, T., 96, 127, 183, 187, 215. 313, 322 J Jacob, J., 266, 289, 322, 326 Jacobson, M., 295,335 Jahn, T, L., 133, 171 Janda, V.,264, 323 Jensen, M.,13, 18, 22,23, 29,38,50,52, 59,62,63 Johansson, A. S., 249,251,298,300 302, 306, 313, 314, 323 Johnson, B., 241, 244, 250, 316,323 Joly, L., 260, 269, 281, 282, 285, 297, 300, 304, 321, 323 Joly, P., 260, 268, 269, 285, 297, 321, 323 Jones, A. E., 164, 170 Jones, B. M., 190, 197, 215, 262, 268, 323 Jones, J. C., 222, 223,224,232,236,244 Jousset de Bekesme, 176. 180, 215 K Kabitza, W., 226, 229, 243 Kaiser, P., 284, 323 Kaji, S.,290, 323 Kalmus, H., 73, 127 Karlinsky, A., 335, 336 Karlson, P., 58, 59,63,64, 184, 185, 186, 187, 188, 189, 190, 195, 196, 197, 198, 199, 203, 204,, 205, 207, 213, 214, 215, 2I6, 217, 248, 261, 267, 269, 270, 271, 292, 294, 315, 317, 323, 324, 325,335,336 Katchalsky, A., 91, 127 Kearns, C. W., 230. 245 Kedem, O., 91, 127 Keilin, D., 186, 187, 216 Keister, M. L., 209, 216 Kendrew, J. C., 35, 63 Kennedy, J. S.,285, 316, 324 Kent, P. W., 58, 62, 63, 186, 187, 201, 213, 218 Ketchel, M. M., 212, 324 Khan, T. R., 291, 324 Kim, C. W., 287, 324 Kiraly, J., 225, 231, 237, 238, 242 Kirimura, J., 207, 216, 257, 324

Kitching, J. A., 85, 118, I27 Knab, F., 176, 179, 216 Knight, M. R., 232, 244 Knoll, F., 132, 148, 171, 172 Knowles, F. G. W., 230,244 Knudsen, M., 105, 127 Kobayashi, M., 207, 216, 253, 255, 256, 257, 260, 299, 324, 335 Koch, H., 229, 243 Kohler, W., 210, 21 1, 216 Koller, G., 231, 237, 238, 239, 240, 244 Kooistra, G., 236, 244 KO?&, S.,254, 273, 324 Kopelman, A., 237, 245 Kopenec, A., 222, 223, 224, 244 Krijgsman, B. J., 221, 222, 244 Krijgsman-Berger, N. E., 221, 222, 244 Krishnakumaran, A., 264, 325 Kristensen, B., 47, 62 Kroeger, H., 335,336 Kroon, D. B., 200, 201, 216 Kubler, H., 158, 170 Kuhn, A., 133, 134, 160, 172 Kunkel d’HercuIais, 176, 180, 216 Kupelweiser, H., 132,171 Kurland, C. G., 263, 325 Kuske, G., 287, 325 Kuwabara, M., 134, 143, 152, 173 Kuwana, F., 224, 244

L Laklla, F. S., 35, 40, 63 Ladduwahetty, A. M., 306, 307,325 La Greca, M., 4, 63 Laing, J., 176, 181, 183, 216 Langer, H., 138, 141, 153, 158, 171, 172 Langley, P., 235, 241, 243 Langley, P. A,, 253, 305, 318 Larsen, J. R.. 297, 303, 304, 325 Lasch, W., 224, 244 Lea, A. 0.. 314, 325 Leclercq, J., 82, 127 Lee, H. Tsui-Ying, 258. 259, 325 Lees, A. D., 73,75,77,96,127,248,273, 279, 285, 325 Lerma, B. de, 250, 325 Lerner, A. B., 185, 187, 191, 216 Lewis, D. J., 178, 216 L’Helias, C., 311, 325

34 1

AUTHOR INDEX Liebau, H., 185, 186, 187, 195. 197,215 Linzcn, B.. 158, 170 Little, K.,51, 54,62 Lloyd, D.J., 35, 63 Locke, M., 57, 62,63,75, 79,127 Lxckey, K.H.,100, 127 Loeven, W.A., 200,201,216 Loher, W.,305,325,336, 336 Loomeijer, F. J., 34, 35, 36, 63 Lubbock, S i r J., 133.172 LUdtke, H., 146,147, 150, 151, 164,172 Ludwig, D.,73, 127,263,325 Lundgren, L., 201,217 Lilscher, M., 269, 282, 284, 298, 302,

314, 316,319,325,326 Lusis, 0.. 299,305,306,310,311,314322 M McCoy, E. E. Jr., 139, 140,173 McIndoo, N. E.,224,244 McLeod, D.G.R., 273,274, 278,318,

326 MacNicho1.E. F. Jr., 155, 164,166,167,

168,171, 172,173, Malek, S. R. A., 59,63,203, 216 Maloef, S. R. A., 212, 216 Maloeuf, N. S. R., 224,244 Mather, K.,291. 326 Mandl, I., 41.63 Mann, T.,186, 187.216 Marshall, J. F., 180,216 Martin, J.. 80,127 Mason, H.S., 183, 185, 188, 202, 203, 204,216 Mazokhin-Porshniakov, G.A,, 138,143, 146, 160, 163, 164, I72 MMioni, J., 139,172 Mellanby, K.,72, 73, 74, 122, 126, 127, 253,297, 304,326 Meyer, G.F.,249,326 Meyer, K.H.,18, 28, 63 Milburn, N., 242, 244 Miller, P. L.,17,64 Milne, L. J., 158,172 Milne, M., 158,172 Mitchell, H.K.,157, 170 Mitsuhashi, J., 256, 274,275, 319,320, 324 Mochida, O., 275,326

Mokia, G. G.,306,326 Molitor, A.,138, 172 Maller, I., 309, 332 Moller-Racke. I., 132, 172 Motakawa, K.,164, 172 Monod, J.. 266,289,322.326 Monroe, R. E.,299,317 Moorhead, L.V., 294,334 Morgan, J., 314,327 MUller, A., 104,127

N Nachmansohn, D., 70,127 Nachtigall, M.,292,323 Naidu, M. B., 221,222,223, 244 Naisse, J., 258,326 Naka, K.,134, 143, 152,172,273 Nakasone, S., 253,256,324 Nayar. K. K.,240,245,249,307,326 Nelson, J. M., 185,216 Neugebauer, W.,312,326 Neville, A. C.,8, 13, 15, 54, 55, 59, 61,

62,64 Nishiitsutsuji, J., 255, 259,272, 283,322 Nishiitsutsuji-Uwo, J., 255, 256, 275,

281,322 Noble-Nesbitt, J., 17,61,62, 64 Noble-Nesbitt, J. J., 73, 126 Noms, M. J., 300, 305,326 Novak, V. 3. A., 248,282,289,290,312,

326,327 Nlilkz, J. A., 258,259,283,327

0 O'Farrcll, A. F.,253,269, 314,327 Ohnishi, E.,184,186,187,190,193,194, 195, 196, 197,207,216,217 Okuda, J., 164, 172 Opocynska-Sembratowa, Z.,224,245 Orlov, J., 232, 233,245 Orser, W.B., 221,245 Otis, A, B., 187, 192, 193,212 Ozeki, K.,250, 258, 260, 277, 283, 288, 297,298,327, 335,336

P Pal, R.,81, I28

Palm, N.B., 238,239,245

342

AUTHOR INDEX

Pappenheimer, J. R., 77, I28 Paquet, L.,252, 321 Partridge, S. M.,21, 34, 35, 36, 50, 51, 52, 53, 54. 63, 64 Patat, U., 141, 172 Pauling, L.,97, 128 Pavan, M.. 295, 328 Pearse, A. G. E., 249,327 Penner, M. L., 287, 325 Petersen, B., 201, 217 Pfeiffer, I. W., 285, 297, 298, 308, 309, 313, 327 Pflugfelder, O., 248, 249, 258, 259, 284, 290,299,326,327 Phillips, J. E., 76, 77, 78, 116, 128 Picken, L. E. R., 62, 64 Piepho, H., 254,261,281,286,287,288, 290, 291, 293, 315,325, 328 Piozzi, F., 295, 328 Plotnikow, W., 176, 217 Pohl, R. W., 133, 134, 160, I72 Pohley, H. J., 269, 314, 328 Possomph, B.,249,254,259,260,301,328 Potts. W. H.,178,217 Prell, H., 180, 217 Pryor, M.G. M.,58, 64, 183, 184, 188, 189, 199, 201, 215,217 Pulis, J. F., 294, 334

Q

Quilico, A., 295, 328 Quo Fu, 304,328 R Raabe, M., 231, 241, 246 Rahm, U. H., 254, 274,328 Ralph, C.L., 225, 230, 231, 245 Ramay, J. A., 74, 76, 88, 89, 128 Raper, H. S., 184, 186, 217 Ray, 0.M.,186, 188, 192, 193,213,217 Rehm, E., 224,245 Rehm, M., 251, 252, 256, 283, 300, 328 Renkin, E. M., 78. 128 Richards, A. G., 4, 57, 64, 94, 109, 128, 183,217 Richter, A., 284, 315, 328 Rizki, M.T., 190, 217 Rizki, R. M.,190,217 Robbie, W.A., 277, 328

Robbins, W. E.,299,317 Roeder, K. D.. 242, 244 Rokohl, R., 150, 164,173 Roller, H., 300,309,329 Rosen, H..47, 64 Roth, L. M.,301, 302, 303, 307, 329 Ruck, P. R., 150, 171 Rudall, K. M., 96, 128, 184, 186, 189, 200, 201,214 S

Sagesser, H., 309,329 Saito, M., 257, 324 Sander, W., 140, 173 Saunders, D. W., 31,64 Schaller, F., 261, 293, 315, 329 Scharrer, B., 249,250,255,257,259,268, 282, 284, 290, 291, 313, 329, 335, 336 Scharrer, E.,257, 329 Schellman, J. A,, 53, 64 Schilder, P., 237, 245 Schlegtendal, A., 132, 173 Schlieper, C., 132,173 Schlossberger-Raecke, I., 59, 63, 184, 203, 215 Schmialek, P., 292, 294, 295, 296, 324, 329, 330 Schmid, H., 187, 188, 205, 215 Schneider, F., 272, 277, 330 Schneider, G., 145, 147, 173 Schneiderman, H.A., 79, 128, 254, 264, 276,277,280,281,290,292,293,294, 300, 320, 325, 330, 335,336 Schoonhoven, L. M.,273,277,330,335, 336 Schremmer, F., 138, 173 Schultz, A. L., 271, 330 Schweiger, A., 58, 64, 189, 195, 196, 198, 205, 207, 215, 217 Sekeri, K., 184, 204, 216 Sekeris, C. E.,58, 59, 63, 64, 184, 185, 186, 199, 204, 205, 216, 217 Sellier, R., 212, 217 Shafer, G. D., 179, 180, 217 Shappirio, D. G., 263,264, 278,330 Sew, Yow Cheong, 305,330 Silhacek, D. L., 273, 274, 318 Singer, S. J., 36, 38, 65 Singh, T., 305. 330

AUTHOR INDEX Siva Sunkar, D. V., 237, 245 Sizer, I., 188, 217 Slifer, E. H., 57, 62, 65 SlAma, K., 264, 293, 295, 312, 327, 330 Sloper, J. C., 257. 330 Smith, A. F., 61, 62 Smith, M. N., 109, 128 Snodgrass, R. E., 176,217 Soraci, F. A., 139, 140, 173 Southwood, T. R. E., 285, 330 Springhetti, A., 298, 326 Srivastava, U. S.,258, 259, 330 Staal, G. B., 285, 331 Staley, J., 180, 216 Stamm, M.D., 271, 331 Stamm-Menendez, M. D., 271,324 Stay, B.,302. 303, 329 Steele, J. S..226, 245 Stegwee, D., 312,331, 334 Steinberg, D. M.,315,331 Steiner, G., 225, 245 Stern, P.,54, 63 Sternburg, J., 230, 245 Stevenson, E., 265,331 Stiennon, J. A., 249, 331 Stock, A., 253, 269, 314,327 Strangways-Dixon, J., 298, 313, 331 Strich-Halbwachs, M. C., 258,261,268, 331 Stumm-Zollinger, E., 251, 331 Stumpf, H., 133, 138, 143, 159, 170 Sulkowski, E., 263,331 Susich, G. v., 18, 63 Sussman, A. S., 185, 196, 218 Sutcliffe, D. W., 76, 128 Suzuki, K.,163, I73 Svaetichin, G., 164, 166, 167, 168, 173 Svidersky, V. L.. 241, 245

T Tahmisian, T. N., 186, 192, 193, 213 Taira. N., 164, 172 Takaoka, M.,253, 259, 331 Tani, K.,299,335 Tarpley, W. E., 185, 186, 188, 191, 214 Telfer. W. H., 265, 279, 310, 331 Thomas, J., 35, 36. 64 Thomas, J. G., 182,218

343

Thomsen, E., 249, 251, 259, 301, 306, 307, 309, 312, 313, 314, 325, 331,332 Thorpe, W. H., 79, 106,128 Thum, U., 17.65 Todd, A. R., 184, 199,202,215 Toyama, K.,258, 332 Treherne, J. E.. 76, 128, 233, 234, 235, 241, 243, 245 Treloar, L. R. G., 18, 29, 31,65 Tsuneki, K.,163, 173 U Unger, H., 226, 229, 243, 245 Usherwood, P.N. R., 227,237,241,244 V Valk6, E., 18, 63 Van der Kloot. W. G., 248, 250, 252, 273,275, 332 Van Schreven, A. C., 182. 218 Vannucci, M.,226, 245 Veerkamp, T. A., 200, 201, 216 Verly, W. G.,265, 317 Vogt, M., 259,260,272,309, 313,332 Voskresenskaya, A. K.,241,245 W Wachter, S., 180, 218 Waddington, C. H., 203, 21 I, 218 Wagner, H. G., 164, 167, 171,173 Waku, Y., 274,276,332 Wang, Shu-Yi, 313,332 Wald, G., 147, 173 Walther, J. B., 133, 138, 143, 148, 149, 164,173 Watson, J. A. L., 73,126, 299,332 Webb, E. C., 198, 214 Wecker, E., 190,216 Weiant, E. A., 242. 244 Weinstein, M.J., 294, 330 Weirich, G., 336,336 Weis-Fogh, T., I , 2, 3.4, 8,9, 11, 12, 13, 15, 18,20,21,22,23,25,26,27,28,29, 30,31,32,33,34,35,36,38,41,50,51,

52, 53, 54, 55,58,59,61,62,62,63,65 Weismann, A., 178, 218 Weiss, H. B., 137, 139, 140, 173 Wells, J., 258, 259, 281, 333 Wells, M.J., 281, 333

344

A U T H O R INDEX

Wendler, L., 134, 152, 170 Wenig, K., 312, 327 Whitehead, D. L., 186, 187, 218 Wigglesworth, V. B., 4, 57, 61, 65, 74, 75, 76, 79, 83, 86, 88, 89, 94, 95, 107, 128, 129, 181, 182,183,202, m . 2 1 0 , 218, 231, 239, 245, 248, 249, 250, 251, 252, 253,254,255,257,258, 259,260, 261,262,263,264,265,267,268,269, 271, 272, 274, 277, 279, 280, 282, 283, 284, 285, 286,287,288,289,290,291, 292, 293,294, 295, 297,298, 299, 300, 301, 303, 306, 311, 312, 313, 314, 315, 333,334, 335 Wilde, J. de, 220, 245,248,297, 312, 334 Willey, R. B., 232, 234, 241, 245, 249, 334 Williams, C. M.,249,250, 254, 255. 256, 257,260,263,265,269,271,272,273, 275, 277, 278, 279, 281, 283, 288, 292, 294, 295, 299, 321, 324,330,331,334, 335,336

Willis, E. R.,307, 329 Wilson, L., 201, 217 Wojknak, L., 263, 331 Wolbarsht, M. L., 164, 167, 173 Wolfe, L. S., 109, 129 Wulff, V. I., 173 Wyatt, G. R., 263, 264, 265, 331, 334, 335 Y Yamamoto, R. T.,295, 335 Yamashita, Y.,255, 257, 299,324,335 Yeager, J. F., 220, 222, 245 Yoshimeki, M., 275,326

z Zawarzin, A., 220, 224, 245 Ziegler-GUnder, I., 158, 173 Zwehl, V. von, 135, 150, 152, 155, 156, 158, 164. 170 Zwicky, K., 263, 265, 335

Subject Index A Abdomen resilin in cuticle of, 14, 16 stretch receptors, 252, 253, 264, 268, 303

Aeschna cyanea, resilin in wing cuticle,

I I , 14 Aeschna grandis, resilin in wing cuticle,

11,37 Aeschna sp.

resilin in cuticle, 12 (see also Dragonfly) Air-swallowing control of, 208 role in expansion, 179-181 Alanine, in milin, 34,52 Alary muscles, and heart action, 220, 22 1,224 Alfalfa plant bug (seeAdelphocoris) Alimentarycanal active transport of water, 76-78 innervation of, 232,233 musculature of, 232,238 scnse organs,232 Amines effect on insect heart, 222,223 effect on Malpighian tubules, 239 reactions with dopa quinone, 203 Amino acids composition of resilin, 14, 29, 3 4 33-36,52 effect on heart rate, 223 Amino acid decarboxylase, corpus cardiacum active agent, 227 c-Amino groups, role in tanning, 184 Amoeba, diffusion rate ofcell membrane,

Acanthacris

histolysis of larval musculature, 182 Acetylcholine accumulation in brain during diapause, 273 control over hind-gut, 236.237 effect on heart rate, 221,222,229 Acetyl-CoA-transacetylase, in sclerotization, 185,199 N-Acetyldopamine as tanning agent, 59,184,186,204 enzymic oxidation of, 58, 186, 187, 197 formation, 58,199 Acrididae air-swallowingat ecdysis, 180 ventral glands, 258 Active transport basic premises, 69-72 definition, 69,70,87,90 of water (seeWater, active transport) Adelphocoris lineolarus, thoracic glands, 259 Adrenalin effect on heart rate, 222,223 effect on Malpighian tubules, 239

85

Aedes

Anax

absorption of tracheal fluid, 209 growth of imaginal diks, 262,272 Aedes aegypti, ovulation and ovarian development,297,301,304,307

ecdysis, 179,180 innervationof heart, 224 Adrena, parasitism and reproduction, 298

Aeschna

Anisolabis

effect of parasitism on metamorphosis, 315 innervation of heart, 224 internal ecdysial pressure, 179 sensitivityto juvenile hormone, 293 ventral glands, 261

corpus allatum, 283, 288, 297, 298, 335 storage of neurosecretory substance, 250 ventral glands, 260,288 Anisotarsus, thoracic glands, 259

345

346

SUBJECT INDEX

Anisotarsus cupripennis,thoracic glands, 28 3 Anopheles effect of adrenalin on heart rate, 223 rhythmic contractions in gut, 232 Anophelesquadrimacuiatus effect of acetylcholine on heart rate, 222 innervation of heart, 222,224 mid-gut contractions, 236 Antheraea, effects of juvenile hormone, 284,287 Antheraeupernyi chilling during diapause, 275,276 extraction ofjuvenile hormone, 296 Anticholinesterases effect on heart rate, 222 effect on hind-gut, 236 Ants, colour vision, 133 Aorta, and heart action. 220, 224 Aphidius, 3I6 Aphodius, hardened protein in elytra, 202 Aphids hormones and polymorphism, 3I 5, 316 transport of neurosecretory product, 250 Apis mellifera, resilin in cuticle, 14 Arachnida, resilin in cuticle, 13, 14 Arctias selene, brain activity and diapause, 272 Arginine, in resilin, 34 Arthropod, resilin in cuticle, 1-62 Arthropodin, cuticular protein, 96.97 Ascorbic acid, enzymic oxidation of, 187 Aspartic acid, in resilin, 34,52 AstacusJluviatalis resilin in cuticle, 13, 14, (see also Crayfish) Atropine, effect on heart rate, 221, 223 Autonomic nervous system. 240-242

B Bacillus rossli, neurosecretory cells, 252

Bee colour vision, 131 ff, 160-163, 164 ff compared with man, 134, 137, 161163 sensory hair fields, 17

Behaviour, in colour discrimination, 131, 139-141, 160-163, 166 Blaberus hardening of cuticle, 201 neurosecretory material, 305 Blatta, innervation of heart, 223,224 Bluttella corpus allatum and ovarian develop ment, 302,303 regeneration and hormone activity, 253,269,314,315 regeneration and moulting, 253 Blattella vaga, corpus allatum and ovarian development, 303 Blattidae, thoracic glands, 258,259 Blindness, for colour, 131-1 33, 136, 150,164,169 Blood meal, and ovarian development, 297,301 Blowfly, tanning of cuticle, 58,59 Bombus sp., resilin in cuticle, 14, 15 Bombyx air-swallowingat ecdysis, 180 brain hormone, 256,257,277,335 extraction of “diapause hormone”, 279 role of neurosecretory product, 255 source of moulting hormone, 254, 270,335. Bombyx mori brain hormone, 253,256,257 corpus allatum and juvenile hormone, 281,286,297,299 cuticular enzymes, 187 neurosecretory material, 250, 255, 306 thoracic gland hormone, 259,260 Brachyccra-Cyclorrhapha, m o n escape, 177 Brachycera-Orthorrhapha, c m o n escape, 177 . Brain electrical activity and ovarian development, 306 during diapause, 273 role in corpus allatum activity, 255, 280-283,293,301-304 Brain hormone action of, 253-260,272-275,307

SUBJECT I N D E X

Brain hormone--con(. chemical nature, 256-258 liberation of, 252-254,275,278 Bupalus piniarius, diapausing pupa, 273, 274,277 C Calliphora N-acetyldopamine in puparium formation, 184, 186,204 action of brain hormone, 254,258 colour vision, 141, 143-149, 152-160, 166,168,169 corpus allatum and reproduction, 298, 301,306,308,309 crystalline o-diphenoloxidase from, 195 eversion of pupal head, 21 1 hardening without darkening, 202 hormones and metabolism, 308, 309, 311-314 neurosecretory cells, 249, 251, 306, 309.31 I , 314 prehardened cuticular areas, 177 redox potential of blood, 190 role of air-swallowing in expansion, 181,208 thoracic glands, 254,260,261,272 tyrosinase extracts from, 189 tyrosine metabolism in cuticle, 184 “units” and ecdysone assay, 263, 270,271 Calliphora erythrocrphalu active transport of water, 78 cyanide insensitiverespiration, 190 internal ecdysial pressure, 179 phenolase activity, 196, 198 postemergence development, 206 puparium formation, 200,204 resilin in cuticle, 14, 15 Capillary force in trachcdes, 83,84 Carabus, spectral sensitivity, 146, 148, 149 Carausius (see Dixippus) Carbon dioxide, permeability of cuticle, 78,79 Cardiac nerves, 224 Cardiac stimulator. from corpus cardiacum, 225-228

347

Cardio-regulatory substances in central nervous system, 228-230 in corpus allatum, 230 in corpus cardiacum, 225-228 in other tissues, 23 I Catechol, effect on tyrosinase, 188 N-Catechol proteins formation in cuticle, 183 Cderio lineata, corpus allatum, 291 Cell “inertia”, 290,291 Cephus, action of brain hormone, 254, 255,275,277 Cephus cinctus, hormones and diapause, 253,272 Cerula viriula, thoracic gland hormone and colour change, 263 Chaoborus (see Corethru) Chelicerata, resilin in cuticle, 14 Chilling, physiological changes during, 276277,300 Chilo, hormones and diapause, 272,274, 275 Chironomus, moulting of, 266,335 Chironomus tentans, polytene chromosomes, 205 Chitin, and resilin, 4,9, 13, 22, 54, 59, 61,62 micelle and crystallite orientation, 61, 200,201 physicochemical propertie, 96,97 Cholesterol, and brain hormone, 257, 270,271,335 Cholinesterase, level in brain, 273 Chorfophaga,atmospheric water uptake, 73 Chromatography column ofjuvenile hormone, 296 resilin composition, 44,49 gas and thin layer in juvenile hormone assay, 296 Paper neurohormones, 229 pericardial cell extracts, 227 resilin composition, 41, 42, 44,49 a-Chymotrypsin, 195 Cimex, corpus allatum and reproduction, 297,304 Clilumnus, ovarian development, 30t

348

SUBJECT I N D E X

Cockroach colourvision, 133, 148-150, 164 tanning of cuticle, 58 Cocoon escape, 177 Coleoptera cocoon escape, 177 colour vision, 163 resilin incuticle, IS, 16 thoracic glands, 258, 259 Collagen composition, 33 swelling of, 26 Collembola. resilin in cuticle, 17 Colorimeter. use in colour vision studies, 134,160 Colour blindness, 131-133, 136, 150, 164,169 Colour change, hormonal control of, 263,285,305,306 Colour discrimination (see also Colour vision) behaviour in, 131, 139-141, 146, 160-163 history of research in, I3 1-135 in ants, 133 inbees, 131 ff, 160-163,164 ff compared with man, 134, 161-163 in Calliphora, 141, 143-149, 152-160, 166169 in Curabus, 146,148,149 in cockroach, 133, 148-1 50, i64 in Deilephila livornicz, 148 in Drosophila, 132,139 in general, 131-169 in insect orders, 163 in Libellula, 146, 164 in Macroglossum, 146,148,149 inNotonecta, 146, 150,151, 164 in Photinus, 134 in Pieris brassicae, 139 in Vespa, 138 range and sensitivity behaviour, 139-141, 146, 157, 160163 mass-response of the eye, 133, 141146.149, 150, 151, 157-159 methods, 137-139,140,143, I44 sensitivity curves, 136-157, 164168

Colour discrimination-conr. single reccptor cells, 133, 152-157, 164-169 Colour reactions, rcsilin in cuticle, 6 7 , 14.44 Colour vision and wavelength discrimination, I3 I, 159-164 central mechanisms, 134, 135, 139, 141-143,159,162-169 definition of, 131, 136, 137 measurement of, colorimeter, 134,160 electrophysiological, 133, 137, 141, 143-157,159,160,164 flicker responses, 133, 159, 160 intracellular, 134,135,150,152-155 optomotor responses, 132,137, 148, 149 phototactic response, 132. 137, 139 spontaneous preference, 132, 137, 139 presence in insect orders, 163 screening pigments, 141-147, 150154,156-158 terminology, 135-1 37 trichromatic theory, 162 vertebrates and invertebrates, 131, 134, 137, 147, 148, 155, 158, 159, 161-163, 166, 168, 169 visual pigments, 142-147, 150, 155, 158,159 Corethra heart rate effect of acetylcholine, 222 effect of adrenalin, 223 raeurohormonal effect, 229, 230 gut stimulating substances,236,238 innervation of heart, 224 neurohormonal effect on melanocytes, 229 Corethra plumipennis, neurosecretion, 25 1 Corpus allatum and control of metamorphosis, 280296 and diapause, 274,275 and juvenile hormone, 280-291, 297 and neurosecretory cells, 250, 251,

S U B J E C T INDEX

Corpus allatum-conr. 255, 258, 274, 291, 301-304, 306, 307.3 1 I , 314 cardio-regulatoryeffects,230 chemical nature of juvenile hormone, 29 1-296 control of secretion, 282,283,303,304 effect of nutrition on activity of, 297300,302,336 effect on metabolic processes, 308-3 I3 histology and histochemistry, 291 role in reproduction, 291, 296.304, 308,309 role of brain in activity of, 255, 280283,293,301-304 staiof, 291 Corpus cardiacum and diapause, 274,275,305 and neurosecretion, 249-255, 258, 301,302,305,306,309,311,314 behavioural influence, 242 control over fore-gut, 236 control over heart, 225-228 control over hind-gut, 237,238 control over Mdpighian tubules, 239 nervouscontrol of, 228 structure, 225 Crayfish, resilin in cuticle, 3, 4, 13, 14, 17,35 Crop air content, 234,235 electricalactivity, 233 emptying ratea, 234 hydrostatic preasm, 235 Culex, ovarian development,301 Culex molesfus, corpus allatum and ovarian development,297 Culex pipiens, corpus allatum and ovarian development,297,304 Curare, effect on heart rate, 221 Cuticle absorption of water when d a d , 94 activetransport water-pump, 122 active uptake of water, 90 and resilin, 1 ff, 57-62 Arthropod, 1-62 aSYInnIt?tryOf, 107-1 11,120 dehydration and chitin orientation, 200

349

Cuticle-conr. &positionof,269,286 epidermalcontrol, 97,98 expansion of, 176, 179-181. 208, 209 hardening and darkening, 58, 59, 62, 175-212,262,263,267 impermeability to poisons, 90 of Diptera, 109 of tracheal system, 80.82 outer gmme layer, 88 permeability to simple molecules, 79,

80 physical chemistry of, 94-98 "rubber-like," 4-29, 31, 33, 51, 57-61 splitting and shedding of, 176, 178, 179 stainingof types, 4-7,9,15,59 total surface measurement, 100 water absorption, 89,90 water m o m t s , 109-1 11 water relations, 88-90 Cuticular lipid electricalproperties of, 111-1 17 monolayer electricalproperties of, 11 1-1 17 inversion, 105- 107 ion transfer, 119,120 passage of ions, 115 passage of water, 105.115-1 17 thermal destruction, 99,101 molecular arrangement, 1027105, 107, 116 physical chemistry of, 98-107 role 8s water-valve, 122,123 temperature / permeabiIity relationships, 98,W thermal rearrangement, 101-105 transition of, 99,101-105 Cuticular proteins resilin, I 4 2 (.we also Protein) ,-t 58,59,62, 181,182, 198,267 Cuticulin, 95 cyclonhapha ecdysial muscles, 183 postecdysial expansion, 177, 178 Cytochrome system, and development, 263,264,274,27s, 278 Cytology, effects of thoracic gland hormone, 263-267

350

SUBJECT INDEX

D Dacnonypha, cocoon escape, 177 Darkening factor, of cuticle, 206-208 (seealso Hardening and Darkening) DDT, effect on musculature, 221 Dehydrogenase, inhibition of tyrosinase,

Diploptera, egg development,303 Diptera colour vision, 138,163 imaginal moult, 176 resilin in cuticle, 15,16 thoracic gland, 258,259 189,190 Ditrysia, w w o n escape,177 Deilephila livornicz, colour vision, 148 Dixippus Dermaptera, ventral glands, 258 activity of brain extract, 240 Dermestes, oviposition and ovulation, air-swallowingat acdysis, 180 306 cell “inertia”, 290 Dermestes maculatus, neurosecretory corpus allatum, 299.3 11,3 12 cells and reproduction, 306, 307 hypodermal pigment migration, 229 Desert locust neurosacretory material, 249,252,301 resilin in wing cuticle, 8, 12,60,61 thoracic glands, 283 (see also Locust) Dixippus morosus, active secretion of Detergents, activation of tyrosinasc, water, 76 192 Dociostaurus, extraction of euiysone, Diapause 271 accumulation of acetylcholineduring, Dopa 273 effect on heart rate, 227 and hormones enzymic oxidation of, 58,187,203 brain hormone, 273-275 production from tytosine, 58, 184, chilling, 274-277,279,300 199 effect of injury, 277,278 Dopa-decarboxylasc, in sclerotization, endocrine organs, 271-275, 282, 185,199 312 Dopa melanin, chemical structure, 203, maternal control, 279,280 204 moulting hormone, 271,272,276 Dopamine neurosecretory cells, 252, 273-275 acetylation, 199,204 changes in brain during, 273 effect on heart rate, 223,227 effect of temperature, 275-280 enzymic oxidation, 187 hormone, 279 formation, 58,199 in egg and larva, 278-280 Dormancy, and cytochrome system, nature of, 278-280 263,264,278 Dictyoptera, reailin in cuticle, 15 Dorsal diaphragm, 220,221 Diffusion Dragonfly relative and comparative rates, 79, 85 air-swallowingat ecdysis, 180,181 typcs of, 69,117 resilin in cuticle, 1, 7-12, 15, 21, 28, Dihydroxyphenols 30,3 1,35-39,50,52 as tanning quinone precursors, 184, Drosophila 186 air-swallowingat ecdysis, 180 in cuticular sclerotization, 183-187 colour vision, 132,139 o-Diphenol, active principle in corpus corpus allatum and metabolism, 309, 313 cardiacum, 225,226 o-Diphenol oxidase, specific activity of, darkening of cuticle, 203 187 euiysone content, 267 Diplazon pectoratorius, effect on host, hydrostatic pressure in development, 277

211

SUBJECT I N D E X

Drosophila-cont.

reduction of blood volume after ecdysis, 183 ring gland, 259 thoracic glands, 260,262,272 tyrosinase extracts from, 189 tyrosinase inhibitor in, 189 Drosophila melamgaster phenolase activity, 196,198 potential tyrosinase, 193-195

tyrosinase activityin haemolymph, 190 Drosophila virilis different phenolases, 187 phenolase activity, 196 potential tyrosinase, 193 Dyar's Law, 268 Dytiscus

corpus allatum, 297 cuticular monolayer, 105, 106 lipid transition temperature, 101 Dyriscus larva, water-swallowing at ecdysis, 180

351

Elasth-cont. digestion of, 35,40,41 Elastomer, and resilin, 18-20 Electro-osmosis, role in water transport, 78

Elect rophoresis Paper resilin composition, 46 Electrophysiology, colour vision studies, 133, 137, 141, 143-157, 164

159, 160,

Endocrine organs, role in diapause, 269273

Endocuticle, 5,59-61, 176,210 Endopterygota cocoon escape, 177 prehardening of cuticle, 177 Enzyme activity,and hormones,263,264, 309

Enzymes, of sclarotization proctss, 185199 Ephemeroptera, corpus allatum, 283

E Earwig (see Anisolobfs) Ecdysial lines, 176 Ecdysial muscles action duringecdysis, 181-183 "shut-or' and degeneration, 183, 209

Ecdysis control mechanisms, 204-210 cuticular hardening and darkening, 175-21 2

outline of, 175,176 production of definitive body form, 210-212

Ecdysone (Ecdyson) effect on polytene chromosomes, 205 in regulation of growth and reproduction, 256. 263, 266271. 276, 278, 303,335

role in sclerotization, 205 role in tyrosine metabolism. 205, 207 Elasticity of resilin in cuticle, 1-4, 11-1 3, 15, 17, 18, 20-32, 51, 52

Elastin compared with resilin, 3, 20-28, 3 4 36,50-54

neurosecretorycells, 256 thoracic glands,258 Ephestia eversion of wing buds, 210,211 neurosecretorycells, 25 1,252 thoracic glands,259,269,283 Ephestia kuehniella, corpus allatum secretion, 300 Epicuticle,95,176,183,210 Epidermal cells activation of, 264,265, 267, 268, 271, 280

dormancy of, 278,279 Epidermis, control over cuticular elasticity, 97 Epistophe b(f&sciata, puparium formation, 277 Ergotamine, effect on heart rate, 223 Eserine effect on heart rate, 222 effect on hind-gut, 236 Eucarcelia, sensitivity to hormones of host, 217 Eurolen, neurosecretorycells, 250 European corn borer (see Pyrausta) Exocuticle, 5,5761, 176,183

352

SUBJECT INDEX

Exopterygota, prehardening of cuticle, 176,177

Eye compound colour discrimination, 131-166 division of, 148, 150,156,144 mass-response, 141-146, 149, 150, 152,157,158

photoreception, 134, 135, 142-144, 152-157,159,164,166

singlereceptor response, 1 4 6 157 ocellus, 150 presence of resilin, 5,7,18 pigmentation and thoracic gland disappearance, 283

hormonal control of, 283,292 pigments screening, 141-147, 150-154, 156158

Visual, 142-147, 150, 155, 158, 159

F Farnesol, juvenile hormone activity, 293-296,299,335,336

Fat body, effect of hormones on, 262, 263,265,308,309,311-313

Feed-back effect, homeostatic, 311-314 FIavobacteriumelastolyticum, proteolytic enzyme. 41 Flight, and resilin in cuticle, I , 2, 15-17 Fluorescence,of resilin in cuticle, 3,6,7, 14, 16, 35, 36, 40-50, 60,61 Fluorescent compound, in resilin of cuticle, 3435,41-51,55,57 Foregut electrical activity, 233 endocrine control, 236 innervation of, 232 nervous control of, 234,235 stretch receptors, 235

Galleria-cont.

juvenile hormone, 282,286288,293 neurosecretorycells, 252 thoracic glands, 261 Galleria melonella, electricai activity in foregut, 233 Gene activation, and moulting hormone, 265-267,3 15

Gene system, effect of juvenile hormone, 288-290.3 15,3 16,

Genitalia,preecdysial hardening, 177 Gerroidea, brachypterous form, 285, 286

Glutamicacid, in resilin, 34 Glycine, in resilin, 34,52 Golgi bodies, 248 Gonadotropic hormone, 299 Gonads, development of, 275,280,281 Grasshopper, resilin in cuticle of, 12, 13, 15,16

Grease effect on water uptake, 92-94 on cuticle, 88,89 Growth role of moulting hormone, 261-265, 267,268,271

role of neurosecretory cells, 249, 250, 253

Growth and differentiation, hormonal control, 267-270, 314-316 Gryllus

brachypterous form, 285,286 thoracic gland hormone, 264 Gryllus domesticus

thoracic dormmedian muscles in ecdysis, 182 Gut (seeAlimentary canal) Gyrinus, hydrofuge cuticle, 99 H Haemocytes, tyrosinase activity in, 190, 191

G Galeruca tanaceti, neurosecretory cells, 305 Galleria

corpus allatum and metabolism, 299, 309

imaginicaducous muscles, 181

Haemolymph circulation of neurosecretory product, 250,254,301,310,311

synthesis of dihydroxyphenols, 186 tyrosinase activity in, 190 Heart endocrine control,225-231

SUBJECT INDEX

353

Hcart-ront. Hormonal regulation innervation, 222-224 and diapause, 271,280 musculature, 220,221 and metamorphosis,280-296 Heart rate hind-gut, 237,238 effect of acetylcholine, 221,222,229 Malpighiantubules, 239 effect of acetylcholine antagonists, metabolism, 207-3 16 221,223 mid-wt, 236 effect of anticholinesterases,222 neuro-cndocrinesystem, 248-258 effect of biogenic amines, 222,223 of heart rate, 225-231 effect of chlorinated hydrocarbons, reproduction,296307 22 f thoracic gland system,258-271 effect of nicotine, 221-224 Hormone effect of rotenone, 221 brain (see Brain hormone) inhibition of corpus cardiacum effect, diapause, 279 225-228 inactivation at high temperature, 276, nervous control, 223-225 277 neurohorrnonal control, 228-230 juvenile (see Juvenilehormone) role of pericardial cells, 226 moulting (see Moulting hormone) Hemiptera prothoracotrophic, 207 colour vision, 163 thoracic gland thoracic gland, 258,259 metabolic and cytological effects, Hexamethonium, effect on heart rate, 263-267 221,223 “wound”, 267,268,271,277 Hind-gut Hormones contractions and diapause effect of acetylcholine,236 chilling, 274-277 effect of indolalkylamines,237 effect of injury, 277,278 effect of LSD and BOL, 237 maternal control, 279,280 endocrine control, 237,238 nature of state of, 278,279 innervation of, 232,233 role of endocrine organs, 271-275 pharmacology, 236,237 control of reproduction,296307 Histamine, effect on heart rate, 223 metabolic Histidine, in resilin, 34 homeostasis, 31 1-314 Histochemistry humoral integration, 286, 3 14-3 I6 of neurosecretory substance, 257 t m S , 308-3 1 1 of corpus allatum, 291 Hyalophora Histology brain hormone, 256,272,273 of corpus allaturn, 291 changes in brain during diapause, 27 of neurosecretory cells, 248, 249, 258 corpus allatum and juvenile hormone, of thoracic gland system, 258-260, 255,257,292-295,300 275 corpus cardiacum, 25 I History, colour vision. 13 1 - I 35 dormant tissues, 278,279 Holometabola, cocoon escape, 177 injury and diapause, 277 Homeostasis, and hormonal action, neurosecretory cells, 250, 251, 255, 311-314 273 Honeybee thoracic gland hormone, 260, 265, initiation of pupation, 253 272,283 innervation of heart, 224 Hyalophora cecropia thoracic glands, 259 colour vision, 133

354

SUBJECT INDBX

Hyalophora cecropia-cont.

corpus allatum and juvenile hormone, 281,282,296,312 corpus allaturn and reproduction, 299,300 cytochrome system, 263, 264 effect of ecdysone on diapause, 271, 272 neurosecretory cells, 249,254 tyrosinase activity in, 196 Hydrophilus piceus, contraction of heart muscles, 220 Hydrostatic pressure eversion of wing buds, 210,211 in crop, 235 relation to rate of active transport, 77 role during ecdysis, 183, 210, 211 role in water movement, I 17, 1 18 Hydrous, neurosecretory cells, 250 Hydroxy proline, in resilin, 3.34 Hymenoptera cocoon escape, 177 colour vision, 163 resilin in cuticle, 15 thoracic glands, 258 I Imaginal discs, action and function of, 262,269,287,293 Irnaaisal moult. 176 Imaginicaducous musculature, I8 I f Indoalkylamina effect on heart rate, 223 excitation of hind-gut, 237,238 in opaque accessory glands, 240 in pericardial cells, 227 IIljurY

and diapause, 277,278 and liberation of brain hormone, 253,254 and metabolism, 264,265,273,3 14,315 and production of juvenile hormone, 293,314,315 “wound” hormones, 267, 268, 271, 277 Insecticides, effect on heart musculature, 22 1 Integument, atmospheric water uptake, 75

Iphira, neurosecretory cells, 249,250 Iphira limbata, neurosecretory cells and

oviposition, 307 Isoleucine, in resilin, 3447 Isoptera, thoracic glands, 258 Ixodes atmospheric water uptake, 75 evaporation rate, 74

J Juvenile hormone and metamorphosis, 280, 281, 288290,297,315 and ovarian development, 298-300, 311,312 chemical nature of, 257,291-296 effects of, 283-288, 290-293,298, 315 extraction and assay, 292-296 in hardening and darkening of cuticle, 207 mode of action, 269,270,286-291 secretion of, 280-283,314,315 K Kalotermes effect of ecdysone, 268,269 effect of juvenile hormone, 284 Kalofermesfivicollis, juvenile hormone and yolk formation, 298,299 Keratin, swelling of, 26

L Lampyris nocriluca, neurosecretory cells,

258 Lasius, isolation of “dendrolasin”, 295 Leg, resilin in cuticle, 14, 17

Lepidoptera colour vision, 138, 148, 163 ecdysial muscles, 183 juvenile hormone, 281, 293, 299, 300 moulting hormone, 269 neurosecretory cells, 256 postecdysial expansion, 178 resilin in cuticle, 15 thoracic glands, 258-260,262,272 Lepisma, metamorphosis, 315 Lppisma saccharinu,moulting, 284 Leptinotarsa

corpus allaturn, 297,312 neurosecretory cells, 251, 252,255

SUBJECT INDEX

Leucine, in resilin, 34 Leucophaea corpus allatum, 255, 282, 290, 291, 298,303,309,314,315

neurosecretorycells, 249,25S thoracic glands, 303 Leucophaea maa2rae corpus allatum, 302 neurosecretorymaterial, 250,335 Lcuhdqrjia japonica, djapause, 272. 275 Likllula, spectral sensitivity, 146,164 Limotrechis. air-swallowing at ecdysis, 180

Locust, resilin in cuticle, 1, 2, 4-7, 17, 22, 23, 29, 38, 40,47, 49, 50, 52, 54, 59,61

Loccrsra air-swallowing, 180 corpus allatum secretion, 282,285,297 histolysis of larval musculature, 182 hormonal control of reproduction, 304 tergo-pleural muscles in ecdysis, 182 ventral glands and moulting, 260,261,

Mating, and hormonal control of reproduction, 303-307 Mecoptera, camon escape, 177 Megowa viciae, effect of juvenile hormone, 285 Melanin, formation of, 202,203 Melanoplus corpus allatum, 285,297,298,313 effect of acetylcholine on heart rate, 222

embryonic reactivation, 277 inactivation of tyrosinase, 188 prophenol oxidase. 207 Melanoplus diflerentialis, tyrosinase activityineggs, 191, 193, 195, 197 Melolontha vulgaris, resilin in cuticle, 14 Membranes electrostatic potential, 118 pore size, 78 Metabolism hormonal regulation and injury, 277,278,3 14.3 15 effect of thoracic gland hormone,

268,269

Locusta gregaria, effect of juvenile hormone, 285 Locusta migratoria, neurosecretorycells, 253

Locustana, ventral glands, 268 Locustana pardalina, ventral glands, 262 Lucilia, thoracic gland, 272 Lymantria brain removal and diapause, 273 thoracic gland, 254 Lysergic acid diethylamide (LSD) effecton gutcontraction, 237 effect on heart rate, 223,227 Lysine, in cuticular proteins, 34,49, 184

M Macroglossurn, spectral sensitivity, 146, 148,149

Macroglossurnstellatarum, 149 Mdpighian tubuiea hormonal control of, 239 muscles of, 238,239 Mandible prehardening of cuticle, 177 resilin in, 57

355

263-267

homostasis, 3 11-3 14 humoral integration, 314-316 metabolic hormones, 308-3 15 role of resilin, 17, 18 Metamorphosis and puberty, 280,281,297 as embryonicdevelopment,286-288 hormonal control of, 268-270, 280299,3 15

Methionine, in resilin, 34 Methyl catechol, effect on tyrosinase, 188

Methylene blue, reduction by insect tissues, 190 Mid-gut innervation of, 232 neuro-hormonal control, 236 Mimm tiliae effect ofjuvenile hormone, 286 neurosecretorycells, 275 Mites, atmospheric water uptake, 73 Mitosis, and moulting hormone, 267270

Molecular weight elastin, 53

356

SUBJECT INDEX

Molecular weight--cont. phenol oxidase, 195 resilin, 53 Monolayer films, of cuticular lipids, 98-107,112-117,119,120 Monophenolase activity, 186,187 Monotrysia, cocoon escape, 177 Mormoniella, hibernating larva, 276, 279 Moulting and brain hormone, 253-260,307 and nutrition, 264, 265,268, 271, 272 and resilin deposition, 13, 18, 54, 56, 58,59,61 “puff” formation and polytene chromosomes, 205,266,267 role of juvenile hormone, 280, 281, 283,284 role of neurosecretory cells, 249-256 role of thoracic glands, 256,259-263, 265,266,268,269 Moulting fluid, 176 Moulting hormone and brachypterous forms, 286 and metamorphosis, 268,308,315 and mitosis, growth and differentiation, 261,262,267-270.286 chemical nature of, 270,27 1 effect on chromosomes, 266, 267 induction of pupation, 277 liberation of, 252,256256,264,276 metabolic and cytological effects, 263-267 source of, 260,261 Mouth parts, resilin in cuticle, 5 , 7, 14, 16.57 Musra vicina. potential tyrosinase, 195, 196 Musculature autonomic nervous control, 241 causing hydrostatic pressure, 2 1 I, 212 eversion of wing buds, 21 1 involuntary (see Musculature, visceral) of alimentary canal, 232-238 of foregut endocrine control, 236 nervous control, 234,235

Musculahue-cont. of hind-@, endocrine control, 237,238 pharmacology, 236,237 of heart, 220,22 I of Malpighian tubules, 238,239 of oviducts, 240 of pharynx, 233 of proventriculus, 234 of ventral diaphragm, 23 1,232 roleinecdysis. 181,183,210,212 roleinexpansion, 180,181 somatic, 220 visceral, 220-243 Mutants of Calliphoraerythrocephala “chalky”, 144,153,158 “white-apricot”, 143, 144 N Nematocera, cocoon escape, 177 Neotenin (see Juvenile hormone) Nervous system autonomic connections to central system, 241 control over musculature, 241, 242 differences from somatic, 240, 241 control over heart muscles, 223,225 control over intestinal muscles, 232235 role in ovarian development, 301-306 Neurohormone factor D, 258 Neurohormones effect on heart, 228-230 effect on melanocytes. 229 effect on mid-gut, 236 Neurophysiology,colour discrimination, 134,l35,139,141-143,l59,162-169 Neuroptera cocoon escape, 177 colour vision, 163 Neurosecretion, effect on behaviour, 242 Neurosecretory cells, brain hormone (seeBrain hormone) chemical nature, 257 cycles of activity, 251, 252, 275, 276, 314 histology of, 248,249,258 role in moulting, 249-254

357

SUBJECT INDEX

Neurosecretory cells--cont. role in ovarian development, 301307, 309, 313 role in protein metabolism, 309-3 1 I , 313 role in reproduction, 301, 302, 304307,309,313,314 staining of, 248,249,252 Neurosecretory product, liberation of, 250,25 1,273 Nicotine effect on heart rate, 221-224 effect on nervous system, 222 Notonecta, spectral sensitivity, 146, 151 Notonecta gluuca, colour discrimination, 150,151,164 Nutrition and corpus allatum activity, 272, 297, 300,302 and moulting, 264,265,268,271,272 and ovarian development, 302, 303, 306.3 13

Osmotic prtssure-conr. receptor organ,235 relation to rate of active transport, 76,77 role in crop emptying rate, 234,235 Ostrinia (see Pyrausta) Ovarian development and nutrition, 302,303,306,307 role of corpus allatum, 291,297-304, 306,309,310,312-314 role of neurosecretory cells, 301-307, 309,3 13 Oviducts, control of musculature, 240 Oviposition hormonal control, 240,306,307 role of neurosecretory cells, 306, 307 Ovulation, role of neurosecretory cells, 306,307 Oxidation of tyrosine, 58,59, 186 Oxygen, relative permeability, 78,79 Oxygen consumption, and hormone activity, 263-265, 275, 276, 308, 309, 312

0 Odonata neuro-endocrine system, 255,256,258 resilin in cuticle, 15 Oesophagus, electrical activity, 233 Ommatidia, 142-145. 153, 154, 157, 166 Ommochrome pigment, 263 Ommochromes in colour vision, 141, 143,144,158 Oncopeltus

corpusallatum, 298,313,314 neurosecretory cells, 251 Opaque accessory glands, role of secretion, 240 Ornithodorus, COz effect on water uptake, 75

Orthoptera colour vision. I63 resilin in cuticle, IS resilin in cuticle, 14,15 Osmosis role in tracheolor fluid movement, 86 role in water movement, 117.118 Osmotic pressure equilibrium with capillary force, 86 Oryctes rhinoceros,

P Papilio xuthus, hibernating pupa, 277

Parasitism, and secretion of juvenile hormone, 298,315,316 Peptide, active principle in corpus cardiacum, 225,226,230 Pericardial cells associated with heart, 220,221 o-dihydroxyindolalkylamine,227 effect on heart rate, 226 Pericardial sinus, 220 Periplaneta

air-swallowing at ecdysis, 180 anatomy of corpus cardiacum, 225 argentaffin cells in gut, 237 asymmetry of cuticle, 109 cholinesterase in intestine, 236 contraction of Malpighian tubules, 239 corpus allatum and reproduction, 297,299 crop emptying rates, 234 cuticular grease layer, 88 effect of corpus cardiacum on heart, 227,228

358

SUBJECT INDEX

Periplaneta-cont. effectof farnesol, 295 effect on heart rate of adrenalin, 222 amine, 222,223 amino acid, 223 indolalkylamine, 223 insecticide, 221 nicotine, 222 effect on hind-gut of corpus cardiacum, 237 eserine, 236 frontal ganglion connections, 241 gut stimulating substances, 238 innervation of gut, 232,233 innervation of heart, 223,224 nerve supply to proventriculus, 233 nervous control of heart rate, 224, 225 neurosecretory cells, 249,291 thoracic glands, 261,283 water absorption through cuticle, 89,90 water droplets on grease layer, 88.89, 92,93 Periplaneta americana darkening factor activity, 207 phenolase, 186 resilin in cuticle. 14 spectral sensitivity, 149 thermal destruction of monolayer, 99 Periplaneta orientalis, neurohormonal effect on heart, 229 Perivisceral sinus, 220 Petrobius maritimus,neurosecretorycells, 250 Pharmacology of heart, 221-223 of intestine, 236,237 of Malpighian tubule contraction, 239 Pharynx, musculature of, 233 Phasmida neurosecretory cells, 249 thoracic glands, 258 Phenolase, 184ff Phenol oxidase activator enzyme of, 195 as sclerotin precursor, 185 autocatalytic activation, 195,198

Phenol oxidase-cont. catecholase activity, 199 control by ring gland, 205 cresolase activity of, 199 crytallhe, 195 inactive proenzyme, 58,195 molecular weight, 195 proteolytic activation of, 195, 198 role in sclerotization, 58, 183, 185199 Phenylalanine, in resilin, 34,47 p-Phenylene diamine, oxidation of, 186 Philosamia Cynthia action of brain hormone, 255,256 cell “inertia”, 290 effect of juvenile hormone, 281 extraction of juvenile hormone, 296 Phormia regina, muscular contractions in gut, 232 Photinus,colour vision, 134 Photoperiod, and diapause, 278,279 Phymafeus, histolysis of larval musculature, 182 Pieris embryonic cells, 287 neurosecretory cells, 252 reduction of blood volume after final ecdysis, 183 role of imaginicaducous muscles, 182 Pieris brassicae air-swallowingat ecdysis, 181 expansion of wings, 181 pre-ecdysial hardening of cuticle, 178 spectral sensitivity, 139 wing extension and hardening, 201 Pieris napi, wing hardening, 201 Pigmentation, hormonal control of, 263, 283 Pigments screening, 141-147, 150-154, 156158 visual, 142-147, 150, 155,158, 159 Platysamia (see Hyalophora) Plodia interpunctella, corpus allatum, 274-275 Pneumatic skeleton, role during ecdysis, 183 Poiseuilleequation, 234 Polymorphism, role of hormones, 288, 315,316

359

SUBJECT INDEX

Polytene chromosomes, “puff” formation at moulting, 205,266,267 Pore canals and cuticle, 57.58 in relation to water uptake, 95,96 P r d y s i a l hardening of cuticle, 176178 Procuticle, 95-98 Prodenia effact of nicotine on heart rate, 222 innervation of heart, 222,224 Proline in resilin, 34 Prosotocus, 3 15 Protocatechuicacid,darkeningofcuticle, 58,201 Protein N-catechol protein formation in cuticle, 183 d i n and insect cuticle, 4,5742 resilin in cuticle chemical properties, 14, 17, 33-50 function, 13,17,18 identification, 1-7,9,10,12,14,35 occurrence, 7-17,35 rubber-like character, 4-29, 31, 33, 51-53,5761 physical properties, 1 1,18-33 Rotein metabolism, hormonal regulation of, 264,265,309-313 Protmlytic enzymes, 41,I98 Proteose, wound effect of, 271 Prothoracotrophichormone, 207 Protyrosinase, 191-193 Proventricular valve nervous control, 234,235 role in food movement, 234,235 Proventriculus electricalactivity, 233 paralysis of, 233 role in crop emptying, 234 Przibram’s rule, 268 pttridincs, as eye pigments, 141, 144, 157,158 Ptilinial digging, 178 Ptilinum, 176,179 Pumps in water movement activetransport pump, 120,121 continuous-flow, 120-122

Pumps-conr. in water movement-cont. contractile vacuole, 117.1 18 electret ion-pump, 1 18,120 in cuticle, 122 lipid water-valve, 122,123 model, 120-122 Puparium, tanning of, 58, 59, 184, 186, 200,267 Pupation hormone. 255 Pupation, initiation of, 251, 253, 258, 260,261,270,277 Purkinjeeffect, 147,148 Pycnoscelus, inhibition of egg develop ment, 303 Pyrameis, expansion of wings, 180 Pyrausta. diapaue, 274 Pyrausra nubilalis, diapause, 273, 278, 335 Pyrrhocoris, corpusallatum, 282,293,312

Q

N-Quinonoid proteins, formation incuticle, 184 Quinones biological significance,197 effect on proteins, 58,59,188,203 formation of, 203 reaction with amines, 203 reaction with proteins, 203 0-Quinones,in cuticle, 58,182 R Radioactive carbon (W) incorporation into cuticle, 59 famesol, 296 protein, 265,310 resilin, 47 Radioactive sulphur (35S), uptake by neurosecretorycells. 305 Rectum, active transport of water,76 Regeneration, and hormonal activity, 253,254,269,293,314,315 Regulation, hormonal (see Honnoml regulation) Relative humidity effect on water uptake, 72-75 measurement accuracy, 14 oftracharrllumen, 82,83

360

SUBJECT I N D E X

Reproduction hormonal control of ovulation and oviposition, 306, 307 role of corpus allatum, 291, 296304,308.309 role of nervous system and neurosecretory cells, 258, 301-307, 309,313,314 Resilin and chitin, 4, 9, 13, 22, 54, 59, 61, 62 and cuticle, 57-62 as a rubber, 4, 18-20, 22-29, 31, 33, 51,57 chain network, 18,28 ff chemical properties amino acid composition, 2, 3, 14, 29,30,33-36,41-52 enzymic hydrolysis, 40,41 fluorescent amino acids, 3, 6, 7, 14, 16,34-36,40-50,60,61 swelling, 3. 12, 14, 17, 24, 25, 30, 31, 3 6 4 0 colour reactions, 4-7, 14,44 compared with elastin, 3, 20-28, 3436,5654 digestion of, 3,40,41 discussion of properties, 50-57 in cuticle abdomen, 14-16 Aeschna, 11,12, 14,37 and flight, 1,2, 15-1 7 Apis, 14 Arthropod orders, 13-1 7 Bombus, 14,15 Calliphora, 14,15 Collembola, 17 crayfish, 3,4,13,14,17,35 dragonfly, 1,7-12,15,2 1,28,30,3 1, 35-39,50,52 elastic tendon, I , 2, 7-12, 14-16, 20,21,25-32,37,50-52 eye, 5.7, 18 function of, 13. 17, 18 grasshopper, 12, 13, IS, 16 hinge-ligaments, 1, 2, 4, 8, 13-15, 23,54,55 identification of, 1-7,14 leg, 14-17

Resilin-cunt. in cuticlwont. locust, 1, 2, 4-7, 17, 22, 23, 29, 38, 40,47,49,50,52,54,59,61 Melolontha, 14 metabolic role, 17, 18 mouth parts, 5,7, 14, 16,57 occurrence, 7-1 7 Oryctes, 14-15 Periplaneta, 14 prealar arm, 12, 13, 15, 22-24, 38, 54-56 Schistocerca, 14,33-35 Sphinx, 14 strain birefringence, 4, 12, 14, 31, 32,59 thorax, 1,2,17,22,23 physical properties deformability and stability, 3, 11, 12,20,21 molecular interpretation, 28-33 optics, 3, 12, 18,55 recovery and damping, 3,19-25 thermoelasticity, 25-28 precursors of, 53-57 staining of, 3-7,9, 15, 17,59 structure of, 3,32,33,54 Retina, types of receptors, 148, 150, 156, 164,166,169 Retinene, 147, 158, 159 Retrocerebral nervous system, role in food movement, 235 Rhodnius action of brain hormone, 253-255, 272,273 activity of moulting hormone, 252, 264,265,267-269,271,272,279,283 assay of juvenile hormone, 292-294, 296,335 asymmetry of cuticle, 109 corpus allatum, 274, 280-282, 288, 291,296,301, 303,312,313 cuticular elasticity, 97 cyclical development of muscles,182 cytological changes during moulting, 264 diapause, 278,279 diffusion rate through integument, 85 effect of ecdysone, 267,271

SUBJECT INDEX

Rhodnius-cont. effect of juvenilc hormone, 269, 280, 283, 286,287,289,290,293,306 experimentally introduced moulting, 207 fine-structure of trdcheole, 84,85 hormone inactivation, 277 hormones and homeostasis, 312, 313 innervation of oviducts, 240 neurosecretory cells, 249-25 1,272,3 12 thoracic glands, 259-262, 271-273, 283,284 wing reduction, 286 Rhodniusprolixus expansion of cuticle, 209 expansion of decapitated insect, 18I Rice stem borer (see Chilo) Ring gland, 205,254,258,262,271 Rotenone, effect on heart rate, 221 Rubber and resilin, 4, 18-20. 22-29, 31, 33,51,57 Rutilis rutilis, water transport through bladder, 91

361

Schistocerca gregaria-cont. darkening factor activity, 207 melanization, 202-203 resilin in cuticle of, 14,33-35 Sclerotin, protein precursor of, 185 Sclerotization, and resilin formation, 57-59 enzymes, 185,199 summary of problems, 198 Sclerotizing system, components of, 183-199 Serine in resilin, 34,52 Serotonin effect on heart rate, 223 in central nervous system, 226 in corpus cardiacum, 226 Sialis action of brain hormone, 254 thoracic gland secretion, 274 Silkmoth, extraction of ecdysone, 271 metabolic effect of moulting hormone, 265 Silkworm diapause in egg, 279 S innervation of heart, 224 Sarcophaga thoracic gland, 258,260 darkening of cuticle, 203 Silkworm pupa role of air-swallowing, 181 hormones and homeostasis, 313 tyrosine incorporation in cuticle, 184 isolation of ecdysone, 270,271 Sarcophaga barbata, ptilinial pressure, Sirona, diapausing adult of, 278 178,179 Sodium azide, permeability of cuticle to, Sarcophaga bullata, tyrosinase activity, 90 190 Sodium pump, different mechanisms, Sarcophagafafculata,tyrosinase activity, 70,71 190 Spectral efficiency, definition of, 136 Sawfly, wheat stem (see Cephus) Sphinx ligustri, metabolic effect of Schistocerca moulting hormone, 265 action of neurosecretory cells, 250. Sphinx spp., resilin in cuticle of, 14 304,305,310,311,313 Spitting of predacious bugs, 17 colour change, 305,306 Squash fly (see Zeugodugus) . corpus allatum and reproduction, 299, Staining 311,313 of corpus allatum, 291 corpus cardiacum, 225 of neurosecretory cells, 248, 249,252, ventral diaphragm, 23 1,232 257 ventral glands, 285 of resilin, 3-7,9, 15, 17,59 Schistocerca gregaria of thoracic gland,259,260 N-acetyldopamine in, 184 Stenopelmatus active secretion of water, 76 effect ofacetylcholineonheart rate, 222 albino form, 203 effect of adrenalin on heart rate, 223

362

SUBJECT INDEX

Stick insect (seealso Dixippus) innervation of heart, 224 Storage of neurosecretory material, 250, 255,301 Strain birefringence, of resilin in cuticle, 4,12,14,31,32,59 Sfrepsiptera,cocoon escape, I77 Stretch receptors, 235, 252, 253, 264, 268,303 SfyIops,298

Thoracic gland system activation and function, 254-256, 259-264,271,272,215,293,303 anatomy and histology, 258-260 moulting hormone chemical nature of, 270-271 mitosis, growth and differentiation, 261,262,264-270,310 thoracic gland hormone metabolic and cytological effects, 263-267 Thorax,resilin in cuticle of, 1, 2, 17, 22, T 23 Tanning, of cuticle, 58, 59,62, 183, 184, Threonine in resilin, 34 Thysanura 186,200,204 Tarsal claws, prehardening of cuticle, hormonal regulation of reproduction, 177 299 ventral glands, 258 Temperature Trachea, and thoracic glands, 259 and elasticity of resilin, 25-28 and hormone activity, 253, 255, 273. Tracheal system active transport of water, 87, 88 276-280 effect on grease orientation, 93,94 atmospheric water uptake, 74.75, 78, Tendon 82 composition of tracheal cuticle, 79,80, elastic resilin in, 1, 2, 7-12, 14-16, 20, 21, 82 internal water condensation, 81, 82 25-32,37,50-52 internal wax layer, 80,82 Tenebrio monolayer inversion, 107 effect of corpus cardiacum, 237 effect of LSD,237 movement of water vapour, 82 evaporation rate, 74 R.H.in lumen, 82,83 site of oxygen transfer, 80 extraction and assay of juvenile water exchange, 82 hormone, 292,294-296 Tracheole metabolic water, 74 active transport of water, 87,88 protyrosinase in larva, 193 ring gland, 259 capillary force, 83,84,87,88 concentration of fluid, 83,87,88 stimulation of oviduct muscles, 240 diameters, 82,83,86,87 Tenebrio molitor effect of annular corrugation, 83-85 N-acetyldopamine in, 184 fluid movement in, 78 atmospheric water uptake, 72.73 darkening factor activity, 207 internal surface properties, 80 m h a n i s m of fluid movement, 86-88 TEPP,affect on heart rate, 222 Terminology of colour vision, 135-1 37 microstructure, 83-85 permeability of wall, 82,85,86 Thermobia, atmospheric water uptake, Transport, of product of neurosecretory 73 cells, 250, 251, 254, 310, 317 Thoracic gland maintenance by juvenile hormone, Trichoptera, cocoon escape, 177 283,284, Trichromatic theory of colour vision, role in hardening and darkening of 162 cuticle, 262,263 Tritium, incorporation into resilh, 55,56 '

SUBJECT INDEX

Tritneptls, temperature and diapause, 280

Tryptamine, effect on heart rate, 223 Tryptophan, in readin, 3 Tymbal muscle, nervous control, 241 Tyramine, effect on heart rate, 223 Tyramine hydrochloride, inactivation of tyrosinase, 188 Tyrosinase activation, 191-1 93 activity, 187-189 concentration of activating agents, 192 effect on proteins, 188 enzyme kinetics, 195,196 intissues, 187,188,190 inactive protyrosinase, 191 inhibition and activation, 188-198 monophenolaseactivity, 186,187 of plants, 187 properties of, 187 stabilitytowards substrates, 188 structural separation, 191,197 Tyrosine concentration in crystal cells, 191 decarboxylationof, 199 hydroxylation of, 199 in haemolymph, 188 in resilia, 34,42,4749,55-59 oxidation, 186 precursor of tanning agent, 184 role in tanning, 58,176 transaminationof, 199

U Ultraviolet light elastin under, 35.36 resilin under, 3,6,7,16,41,49,55, 56, 60.61 sensitivity, 137, 148, 150, 154-156, 161-163 Urine, hypertonicto blood, 76 V Valine in resilin, 34,52 Ventral diaphragm, 231,232 Ventral glands, role in moulting, 254, 258,260-262,268,269,285

363

Vertebrates, compared with inverb brat-, 131, 134, 137, 147, 148, 155, 158, 159,160-163,166,168,169 Yespa germanica, colour vision, 138 Vespa rufa, colour vision, 138 Vespa vulgaris, colour vision, 138 Visceral musculature, control of, 219242 Vision acuityof, 145,157,166 CO~OW blindness, 131-133, 136, 150, 164,169 colour discrimination (see Colour discrimination and Colour vision) role of resilin, 18

W Water active transport and passive movement, 67-125 basic premises, 69-72 conclusiveargument, 91,92 effect of pore size, 78 in gut, 7678 in termtrial insects,71.72 in tracheal system, 79-88 metabolic feasibility,78 osmotic and hydrostatic pnssures, 77,78 through living cuticle, 89,W active uptake by eggs, 72 active uptake from air inhibition with anaesthesia, 75 metabolic energy supply, 75 relative humidity equilibrium, 73 role of alimentary canal,75 role of integument, 75 role of tracheal system, 74,7582 adsorption of chitin, 96 adsorption to cuticdar protein, 96 asymmetricalmovement, 107-1 1 1 contact angles with surfaces, 92.93 droplet behaviour on grease,93 electricalproperties of, 1 16 in tracheal system, 79-88 mechanism of transport, 71 molecular behaviour, 1 16 movement activateddiffusionthroughlipid, 122

364

S U B J E C T INDEX

Water-cont. movement-cont. anomolous osmosis, 117,118 contractile structures, 117, 118 electret ion-pump, 118.120 electro-osmosis, 1 17, I 18 experimental methods, 123 hydrostatic pressure, 117, I 18 in electrical field, 118, 119 membrane valves, 110,111,120 methods, 117,118 osmosis, 117, 118 pump in cuticle, 122 through asymmetrical membrane, 107-1 10

through lipid monolayers, 105, 118 through membrane pores, 116, 117 transfer by ions, 117,118 passage through cuticle effect of carbon dioxide, 122,123 relations to living cuticle, 88-90 reIative permeability, 78,79 Water vapour, movement in trachea, 82 Wavelength -dependent adaptation, 132, 133, 143, 148,150,155

discrimination, 159-1 64 -selective adaptation, 147,148,150

Wax, cuticular, in trachea, 80, $2 Weismann's ring, role in pupation, 204 Wiegand-Snyder equation, 27 Wing development effect of farnesol, 295 effect of hormones,285,286 discs, and moulting hormone, 269 expansion, 181 extension, 201 resilin in cuticle of elastic tendon, 1, 2, 7-12, 14-16, 20,21,25-32,37,50-52

hinge-ligaments, 1, 2, 4-8, 13-15, 23,54,55

prealar arm, 12, 13, 15 ,2244, 38, 54-56

X Xenopsytta

atmospheric water uptake, 73

Z Zeugloptera, cocoon escape, 177 Zeugodugus depressus, brain hormone secretion, 253