Incorporation of Phosphoproteins and Phenol-Bound Proteins Into the Cuticle of Periplaneta Americana

Virginia Commonwealth University VCU Scholars Compass

Theses and Dissertations Graduate School

1976

Incorporation of Phosphoproteins and Phenol-Bound Proteins into the Cuticle of Periplaneta americana

Joseph Atchison Florence IV

Follow this and additional works at: https://scholarscompass.vcu.edu/etd

Part of the Biology Commons

Downloaded from https://scholarscompass.vcu.edu/etd/4554

This Thesis is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. School of Arts and Sciences Virginia Commonwealth University

This is to certify that the thesis prepared by Joseph Atchison Florence IV entitled Incorporation of phosph::>proteins and phenol-bound proteins into the cuticle of Periplaneta americana has been approved by his committee as satisfactory completion of the thesis requirement for the Master of Science degree in Biology.

Di.rector of Thesis

VYr!!inia Commonwealtfr Incorporation of Phosphoproteins and Phenol-bound Proteins into the Cuticle of Periplaneta americana

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University

THESIS

by Joseph Atchison Florence IV Directors Dr. Richard R, Mills frofessor of Biology Virginia Commonwealth University Richmond, Virginia August, 1976 ii

DEDICATION

This thesis is dedicated to the ubiquitous cockroach. May he rest in peace. iii

ACKNOWLEDGEMENTS

My deepest regards and sincerest appreciation are expressed to Ms. Mary Lou Strickland without whom I would have not completed this work. Her assistance has been invaluable. I would also like to express thanks to Dr. Richard R. Mills, mentor and entomologist par excellence. His ability to communicate his knowledge and share his enthusiasm for living and accomplishing my goals in life have made working with him an experience to remember. My thanks are also expressed to my professors and the professors of the department who have helped me accomplish my work. My compatriots of biology are also to be remembered: John Clore, Ellen Thomen, Nancy Taylor, John Krolak, Danny Custer, Mark Welsh, Greg Brown, and all the other graduate students that have shared their knowledge and experiences with me. Without these friends this work could never have been accomplished. iv

TABLE OF OONTENTS

Page LIST OF TAm..ES...... • . . .. • .. . .• .• • . .• • .. .. • . .• . .. .• • .. . .. • • . vi

LIST OF F"IGURES.••..•••.••••.• ,••••..•••..••••.••••..•.•.••• . vii

ABSTRACT.... •. • •• •• •• . • • •• •. • . .. •• .• .• . •. .• . . .. . •. • . . •. .. •• . • X

INTIDDUCTION. • • • . • . • • . • . • • . • . • • • . • • • • • . • . . • . • . • . • • • • • • • • • . • . . 1

OBJECTIVES.• .• . .• • . . •. • • . .• .• • •• • • • .. .• .• .. • . • . • .• .• • • •• • . • • . 3 LITERATURE REVIEW••••••••••••• , •••• ,...... 4

The Sclerotization Process in Cuticle Fonnation...... 4 Enzymes Involved in the Synthesis of Sclerotization Agent •••••••••••••.•••••••••••••••••••••. 27 Hormonal Control of Moulting and Tanning ••••••••••.•.••. 36 EXPERIMEl'rT.AI.s••••••••••••••• , •• ,• •• • • •• • • • •• • • • •• • • •• •• •• • •• •• l+8

METIDDS AND MATERIALS•••••••••••••••••••••••••••• , •• • •• •• • • •• 49

Phosphate Incorporation into Proteins and Cuticle...... 49 llipamine Metabolites Binding to Cuticle Proteins...... 60 In Vitro Binding of NADA to Lysyl-lysine •••••••••••••••• 62 RESlJI,TS • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 6 5

Phosphate Incorporation into Proteins and Cuticle ...... · ...... 65 llipamine Metabolites Binding to Cuticle Proteins...... 99 In Vitro Binding of NADA to Lysyl-lysine •••••••••••••••. 108 DISCUSSION..• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 142

Phosphate Incorporation into Proteins and Cuticle ...... 142 llipamine Metabolites Binding to Cuticle Proteins...... 1� V

Page In Vitro Binding 0£ NADA to Lysyl-lysine •••••••••••.•.•• 146 REF'ERmilCES.• •• • •• • •• • •. • •• • •• • • • •• • • •• •• • . • • •• • . •• .• • • •• • . •• • 1 l.J8

APP�X •••••.••.•.....•••.•.•.•..••.••.••... ·.. •. . .• • .• • • . . . . 18 5

VITA••• ...... ••...•... � ..•...... •...... •.•.•...... •.. c • • • • • 187 vi

LIST OF TABLES

Table Page 3 1, Computer Program of Zp Activity••••••••••••••••••••••••• .50 2. Statistical Analysis System P:rogram of Phosphate Incorporation••••••••••••••.••••.••••••••••••.. 53

3, Least Squares Standard Curve Program.•••••••.•••••••••••• 57 4. Rf Values of Metabolites of Phosphate Metabolism.•.•••••. 70

5, Incorporation of Labelled :ro4 and Phosphop:rote;i.ns into the Cockroach Cuticle•••••••••••••..••••••••••••.••. 97 6. Rf Values f:rom fupamine Metaboliles when Incubated with Cuticle P:rotein.•••.•...••.•..••.••••••••.••.•.••••• 104

7, Rf Values of Possible fupamine Metabolites f:rom Various Literature Sources.• •• • • .• .• • • • •. •• • • • .• • • • • •• •• . 106 vii

LIST OF FIGURES

Figure Page 1. Sequence of Products Between Quinones and Proteins...... � ...... ·...... 7

2. Proposed Mechanism of Sclerotization Prior to 1974...... 10

J. Modified Scheme of Cuticle Sclerotization••••.••.•. •••.•• 12 4. Andersen's Proposal for Sidechain Sclerotization...... 15 5. Dia.grammatic Representation of a Section of Mature Cuticle and Epidermis....• • • •• • • . • • •• • • •• • • • •• •• . • •• •• . •. 17 6. Representation of Changes in the Integu.�ent During Ecdysis...... ,...... 20

7. Structures of Hyd:rolysates of Possible Sclerotization Agents .••••.•••.• ,...... 24

8. P�thway of Sclerotization Agent ...••••.•••••••..•••••..•. 32 9. Mechanism of Binding of the Sclerotization Agent .•....•.• 34 10. Structure of Ecdysone.••••.•.•..••••.••••••••••••••••••.. 37 11. Two Pathways of Tyrosine Metabolism•••..•••••••••.•...... 41 12. Action of Bursicon on the Haemocyte ..••••••••••••.•••••.. 46 13. Representative Chromatograms of Phosphate Metabolites from the Haemolymph in 2, 4 and 24 hour Incubations...... 66

14. Representative Electropherograms of Phospt>.ate Metabo.lites from Haemolymph.. •• • •• • • •• • •• •• . •• . •• • .. .. •• • 68 15. Two Hour P-JO Gel Chromatograph (1st experiment)•..•.•.•• 72 16. Two Hour P-JO Gel Chromatograph (2nd experiment).••.••.•. 74 17. Two Hour P-JO Gel Chromatograph (3rd experiment) .••••...• 76 18. Two Hour P-JO Gel Chromatograph (composite).••.••••.••••• 78 viii

Figure Page 19. Four Hour P-30 Gel Chromatograph (1st experiment)••.•••.. . 80 20. Four Hour P-30 Gel Chromatograph (2nd experiment)...... 82 21. Four Hour P-30 Gel Chromatograph (3rd experiment)...... 84

22. Four Hour P-30 Gel Chromatograph (composite)...... 86 23. Twenty-four Hour P-30 Gel Chromatograph (1st experiment). 88 24. Twenty-four Hour P-30 Gel Chromatograph (2nd experiment). 90

25. Twenty-four Hour·P-30 Gel Chromatograph (3rd experiment). 92 26. Twenty-four Hour P-30 Gel Chromatograph (composite)...... 94 27. Representative Radiochromatograms of llipamine Metabolite Binding Experiment� with Cuticle Soluble Proteins...... 100

28. Representative Radiochromatograms of llipamine Metabolite Binding Experiments with Cuticle lfomogenate ...... •...... •...... 102

29. Elution -Profile. of Non-labelled N-acetyldopamine, lysyl-lysine and cuticle homogenate...••••.••••••.••••.•• 109 30. Duplicate Elution Profile.•••..••••••.•.••.•••••.••.•••.• 111 31. Elution Profile or14c-N-acetyldopamine, Lysyl-lysine and Cuticle Homogenate (ninhydrin and UV)•••••••••••••••• 113 32. Elution Profile of 14c-N-acetyldopamine, Lysyl-lysine and Cuticle Homogenate (cpm and UV)•••••••••••••••••••••• 115 33. Radiochromatograms of Column Eluant 14c-N-acetyldopamine Lysyl-lysine and Cuticle Homogenate.••••••••••••••••••••• 117, 34. Electropherograms of Column E,l:uant 14c-N-a.cetyldopamine Lysyl-lysine and Cuticle Homogenate•••••••••••.•••••••••• 119 35. Graph of Eluant from P-2 Gel Colllinn on N-acetyldopamine and Lysyl-lysine metabolites (ninhydrin and OD)•••••••.••. 122 36. Chromatograms and Electropherograms of Incubant of N-acetyldopamine and Lysyl-lysine•••••••••.••••••••.•••.• 124 37. Representative UV Spectra of the Oxidized Product of N-acetyldopamine...... •...... 126

38. NMR of Fraction A and B from N-acetyldopamine- Lysyl-lysine Binding••••••••.•••••.• •.•••••.•••••••••••.• 128 ix.:.

Figure Page Dz°, 39. NMR of N-acet!ldopamine in DMS and lysyl-lysine in n2o ...... 131 40. NMR of possible metabolites of the sclerotization agent and di peptides ...... · .. 135 .x

Incorporation of Phosphoproteins and Phenol-bound Proteins into the Cuticle of Periplaneta americana

ABSTRACT

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University

by Joseph Atchison Florence IV Director: Dr. Richard R. Mills Professor of Biology Virginia Commonwealth University Richmond, Virginia August, 1976 xi

The incorporation of phosphoproteins and phenol bound proteins into the cuticle of Periplaneta americana was analyzed. Radioactive monosodium phosphate was injected into newly ecdysed cockroaches, Incorporation into haemolymph proteins as well as tanned cuticle was observed. Phosphoproteins were observed to be combined with the cuticle to a greater extent than monosodium phosphate. This suggests that phosphate is being incorporated into the cuticle more readily than monosodium phosphate. The relevance of these observations is discussed in light of phosphate being a possible cross linking agent between chitin and cuticle protein. Binding of dopamine metabolites to cuticle proteins was investigated. Specific metabolites studied were N-acetyldopamine-J-0-sulphate and dopamine-J-0-sulphate. In an in vitro experiment, these metabolites were readily bound to cuticular, structural proteins while they remained unchanged when incubated with soluble cuticular proteins. The presence of sulphatase activity was also evidenced in the structural proteins but not in the soluble proteins. The binding of N-acetyldopamine to lysyl-lysine was accomplished in vitro. Binding was observed with and without newly ecdysed cuticle homogenate. The products of the binding were separated wia P-2 polyacrylamide gel chromatography, paper chromatography and high voltage electrophoresis. Absorbancy at 280 nm, as well as the xii reaction with ninhydrin were monitored for each fraction. A metabolite (or metabolites) was separated which was both UV positive and ninhydrin positive. This metabolite was analyzed by NMR and no aromatic protons were observed thus indicating complete substitution on the ring. These data support the existing theory that suggests protein binding to the sclerotization agent occurs on the ring. 1

INTIDDUCTION

The general theme of the present work is the hardening of the cuticle or the sclerotization process of insects, The importance of this process to insect viability can hardly be overemphasized,

Existing virtually unchanged for some 2.50 million years, insects have a quite successful life history strategy which is highly dependant on this process,

Sclerotization is the process by which the insect hardens its new cuticle after moulting has occurred, Moulting, or ecdysis, is the developmental stage in which the insect replaces its exoskeleton which enables it to grow. Prior to ecdysis the insect forms a new cuticle which consists largely of proteins, When the old cuticle is shed the new cuticle expands and soon hardens and tans. This process is known as sclerotization. Sclerotization occurs when phenols are bound to the protein matrix of the new cuticle, This process is controlled by bursicon, the tanning hormone.

The phenols which are responsible for insect sclerotization are largely the product of tyrosine metabolism. Tyrosine is not only a major compound in insects but also in mammalian systems including human systems as well. Tyrosine metabolism is implicated in the formation of transmitter substances of the central nervous system (Bjorklund et al., 1970) as well as the etiology of several mental health disorders (Baru, 1968), "Inborn errors of metabolism" such as phenylketonurea (PKU) and albinism are known to be metabolic disorders of phenylalanine and tyrosine metabolism.

The elucidation of the metabolism of tyrosine as is found in insect sclerotization has far reaching effects as tyrosine metabolism is ubiquitous in animal systems.

Sclerotization is a most significant stage of insect development. During this short period of its life the insect is most succeptable to predators and environmental stresses, A thorough understanding of this process may lead to the development of better means of pest control. 3

OBJECTIVES

The objectives for this group of experiments were two-fold.

First, an understanding of the role of phosphate in cuticle sclerotization was to be elucidated. This was done by studying the incorporation of phosphate into haemolymph proteins postecdysis as well as by studying the incorporation of phosphate into the cuticle postecdysis. When phosphoproteins were found in the haemolymph proteins, their incorporation into the cuticle was monitored.

Secondly, a more precise understanding of the binding of phenolic metabolites to cuticle proteins was to be studied. This was initially accomplished by the monitoring of 14C-dopamine metabolites and analyzing their relative binding properties to cuticle proteins either soluble or insoluble. Later, the binding of N-acetyldopamine to lysyl-lysine was studied as an in vitro model for the binding of the sclerotization agent to the protein. Binding was accomplished in vitro and possible mechanisms of binding were postulated. 4

LITERATURE REVIEW

The Sclerotization Process in Cuticle Formation

The insect cuticle is composed largely of chitin and protein bound together in an insoluble matrix, Sclerotization agents are the products of tyrosine metabolism, Tyrosine is a key compound in insect metabolism, Not only is it important in cuticular tanning but also it and its metabolites are essential in chemical defenses (Tschinkel, 1969; Happ, 1968; Ikan et al,, 1970), in protective or attractive body pigmentation (Fuzeau-Braesch, 1972) and as mediators of neuronal transmission in the insect {Klemm and Axelsson, 1973; Klemm and Bjorklund, 1971), Tyrosine is not an essential dietary requirement of insects, Tyrosine may be readily derived from phenylalanine by hydxo:xylation by the enzyme tyrosine hydxo:xylase (Murdock et al,, 1970a), Phenylalanine is essential in all but two insects studied thus far. Blattela gerrnanica, the gerrnan cockroach (Auclair, 1959; Henry, 1962), and Periplaneta (Brunet, 196Ja; b) are known to be able to derive tyrosine from glucose; however; this pathway may be due to bacterial symbionts (Gilmour, 1961; 1965), Tyrosine metabolism in insects has been well investigated and reviewed (Wigglesworth, 1948a; 1957; Richards, 1951; 1958; 1967; Dennell, 1946; 1958; Hackman, 19 59; 1964; Hackman et al , , 1948; Pryor, 1962; Brunet, 196Ja; 1965;. 1967; Cottrell, 1964; Gilmour, 5

1965; Andersen, 1966; Murdock, 1971; Price, 1972). The initial investigations on tyrosine metabolism in insects were largely limited to the identification of possible tyrosine metabolites within the insect. Ibpa ( dihydroxyophenylalanine) and dihydroxy phenylacetic acid were discovered in early experiments (Przibram and Schrnalfuss, 1927; Schrnalfuss, 1937). Protocatechuric acid, dihydroxyphenyllactic acid and dihydroxyphenylacetic acid have been found in various insects (Brunet, 1963b; Pryor et al., 1946;

1947; Hackman et al., 1948) as well as 3,4-dihydroxymandelic acid (Adiyodi, 1969), p-hydroxyphenylpropionic acid and p-hydroxyphenyl acetic acid (Mills and Lake, 1971). Further experimentation showed the presence of a group of compounds known as the benzoquinones (Alexander and Barton, 1943; Roth and Stay, 1958; Pavan, 1959; Chad.ha et al., 1961). Ostlund (1953; 1954) identified the catecholamines (dopamine, norepinephrine and epinephrine) to be present and Dresse et al. (1960) suggested that they played a role in tanning rather than as hormones or neu:rotransmitters. N-acetylated products of catecholamines were found to be present in some insects. N-acetyldopamine was isolated in Calliphora (blow:fly) (Karlson and Sekeris, 1962a) and N-acetyliyramine was found in Bombyx mori (silkworm) (Butenandt et al., 1959). That these two metabolites are involved in sclerotization in Periplaneta was shown by Mills et al. (1967). The phosphate and sulphate metabolites of N-acetyldopamine (N-acetyldopamine-3-0-phosphate and N-actyldopamine-3-0-sulphate) were found in high concentrations in Periplaneta haemolymph postecd.ysis and are thought to be involved in the sclerotization process (Bodnaryk et al., 1974a; Bodnaryk and 6

Brunet, 1974) • Tyrosine-0-phosphate has been found in Drosophila (Hodgetts and Konopka, 1973),

Farly investigations concerning the sclerotization process were quite misleading, Chitin was detennined to be the major factor causing cuticle hardness and melanin was thought to be the darkening factor, It was not until 1929 that Campbell disproved this, Protein content was found to be the factor which causes cuticle hardness,

Unlike crustaceans which have an exoskeleton hardened by calcium salts in a protein-chitin matrix, the insects have a cuticle which is almost mineral free, The fact that few crustaceans are found on land and none fly while insects on the other hand are found on land, iin water, and in the air can be attributed to their protein hardened cuticle, which is associated with an arrangement of fibrous chitin, a polymer of N-actylglucosamine (Brunet, 1967),

The insects' protein hardened matrix of the cuticle aids in protection, durability and water impermeability,

The general process of cuticle sclerotization is caused by the interaction of ortho-quinone with cuticle proteins, This process is well documented (Pryor, 1940a; b; Dennell, 1946; 1947;

Dennell and Malek, 1955b; c; 1956a; b; Hackman, 1953a; b; c; Hackman and Todd, 1953; Mason, 1955; Andersen, 1963; Cottrell, 1964; Brunet and Coles, 1974), A generalized sequence of tanning products is found in Figure 1 ,

The tanning process is an enzymatic process, governed largely by the presence of polyphenol oxidase in the epicuticle (Dennell,

1947), however, the degree of sclerotization is independent of 7

Figure 1, Sequence (I-V) of products formed as a result of the

reaction between quinones and proteins (based on

Hackman and Mason) . The reaction mechanism is shown Figure 8b (Brunet, 1965), 8

R R �NH -protein NH-.protein

oH � V o OII . OH 11 0 R protein-NHP)NH -protein VoH 111 OH

R R protein- NH � NH-protein protein-N,H � NH-protein o � o. o � IV II . V II o 0 protein- N 9 amounts of enzyme (Andersen, 1947b). Orthodihydro:xyJ)henols are oxidized to their re@ective quinones and are then bound to proteins. The general reaction is one between amines and quinones. The quinones first react with the terminal amino group of the cuticular protein chains and becomes an N-catechol protein. The N-catecml protein is then oxidized by excess quinone and becomes an N-quinonoid protein (Mason, 1955). It has been shown that activated phenols can also react with other phenolic groups from tyrosine residues (Andersen, 1972) • The result is a cross-linked covalently bonded network of protein chains joined end to end as well as at intermediate points that makes up the hardened, darkened, insoluble cuticle characteristic of insects (Pryor, 1940a; b; Cottrell, 1964; Hackman, 1953b; c). See Figure 2. Koeppe and Gilbert (1974) modified this scheme for cuticle sclerotization when they traced a phenolic-labelled protein complex across the epidermis and found that it was incorporated into the cuticle. Blood proteins have been shown to be the direct precursors of cuticle proteins and they are immunologically identical (Fox et al., 1972; Koeppe and Gilbert, 1973). The generalized scheme for cuticle sclerotization incorporating the data of Koeppe and Gilbert (1974) is found in Figure 3.

Hackman (1953c) offered another theory for tanning. This theory, known as the auto tanning theory, implicated the oxidized phenolic groups of cuticular proteins as the tanning agents. This theory is not accepted by most investigators; however, the oxidation of tyrosine containing peptides by tyroisinase has been noted (Yasunobu ---et al., 1959). Andersen (1970; 1971; 1974a) and Andersen and Barrett (1971) 10

Figure 2, Proposed mechanism of scle:rotization prior to 1974

(Ko�ppe and Gilbert, 1974).

......

tein tein

N-pro

OH OH

tein

R R

OH OH

o o

N-pro

� �

\ \

/ /

I I

8 8

N·protein N·protein

:N-protein :N-protein

ine ine

dopam

tyl

N-

�o �o

1 1

II II

R R

-----

-� -�

dopamine dopamine

se se

dase dase

o o

oxi

� �

N·protein N·protein

enyl

';;::: ';;:::

ne---

II II

Ph R R

loxidase loxidase

Phenyloxi

yrosi

rophe

p/ p/

protein·N

Cuticle Cuticle

Epidermis Epidermis Haemolymph Haemolymph Figure 3. Modified scheme for cuticle scle:rotization incorporating new data (Koeppe and Gilbert, 1974).

1--" 1--"

\...J \...J

N N

ein

---

">o ">o

N-pr

conjugate conjugate

II II

R R .

0 0

cose cose

I I

p�enolic p�enolic

chitin chitin

· ·

em em

t t

, ,

0 0

c..._ c..._

N·pro N·pro peptide peptide

.,,

II II

0 0

R R

e e

---?

� �

njug

eptid

"· "·

n n

protem·N·

complex complex

ote

� �

ein ein

r. r.

phenolic phenolic

H H

--;�------1-----

R R

e e

---

d d

x1 x1

\ \

phenoloxidase phenoloxidase

dopamine dopamine

:---t--

{ {

olymph olymph

cle cle

Cuti

Epidermis Epidermis Haem 14 have presented convincing evidence for the possibility of another mechanism involved in sclerotization. This mechanism involves the side chain binding of ketocatechols (Figure 4) and is reported to be found in hardened but untanned cuticle.

The relationship between hardening and darkening of insect cuticle has not been completely elucidated. Originally it was thought that melanin was the contributing factor to the color of the cuticle. However, Hackman (1953c) disproved this conclusion upon spectral analysis of melanin and tanned cuticle protein by showing that there were distinct differences between the two. Mills and

Fox (1972) show that melanin is at best a secondary sclerotization agent in Periplaneta and in point of fact, its production is inhibited by the tanning hormone, bursicon. However, its presence is of importance in Musca, the muscid flys, (Cima and Grigolo, 1968),

Sarcophaga, the flesh flies, (Fogal and Fraenkel, 1969) and Lucilia, the blowfly, (Hackman, 1967). Andersen and Ba=ett (1971) present evidence for the color of the cuticle to be directly co=elated to the ratio of quinone tanning to side chain tanning.

Before any further discussion on the biochemistry of scleroti zation, it is necessary to review the morphological development of the cuticle (see reviews by Fraenkel and Rudall, 1940; 1947;

Wigglesworth, 1948a; b; 1965; 1969). The cuticle consists of five layers which have been studied by means of light microscopy as well as electron microscopy (Dennell and Malek, 1954; 1955a; Gouin, 1970;

Filshie, 1970; Locke, 1964; 1967). See Figure 5. These layers consist of the epicuticle, the exocuticle, the endocuticle, the epidermal cell layer and the basement membrane. The epicuticle is 15

Figure 4. Andersen's proposal for side chain sclerotization

(Andersen, 1974a), 16

OH H O O protein - protein cI - CH2 NHCCH3 II 17

Figure 5. Dia.gra.m.matic representation of a section of mature cuticle and epidennis (Chapman, 1975). cement epicuticle {layer of wax . -. / · · ... cuticulin 1 I -- • h--,. ,1 .I1 , ...... 1 • . · · · . · .. ,, .. · ,; ·, ,. · , . · /. 1� 1 r·r. (tann�d protei -u:1�· ��.:::·/ ... t ,· ·� ..• : .. : • · m • • . t ... :··, �endocuticretchitin �1 11-t• t,1 •t:t: •'. ��. 1 \ _. :I .. :-)..�i·· � { l .. _,itt�•.1 1, m·�:�� tMIW:¢1',, ,i·i��t· il !,�ii.i .. r�tnum.f JL .. ll__ll_ 1- end utiq:l.e_ . · �untannedchi tin Prlli!tein .. I l!,lt;-Schm1dt 's la.ye"· llll 1111 I I I 111111 11111111111 1 u111 1 -�..:----- .. -�-···· ..... , ...... , ....,. 1, , •: ,,• ,, . ..!, 7:-r;.:;: •...'" 1... , ...... :.! f • ,., ._,.J-.,;,-. •,••-.•11o•,:,..,,l,I .. �,. , ,, I 1,-.c.,•;,�-;•..,\,r•11,P .... - I\'.�•-, - " � ' "'""' .,;,;,r ' ' •:, d... ,.,,. ,••I- :>.<.,..-;--:;-.,,. I':, • •••e · _,,,·,,,·.;•.I) • \!!..!&,-_1 · -•- t s 1f1. , ' epidermal ce layer e ...... --... c.:> --- � --- �

basement membrane

..... ()) 19 ma.de up of an inner cuticulin layer, then a.layer of wax frequently covered by a layer of "cement". The cuticulin layer is divided into two components: an outer, thin dense layer, the sensu strictu (Locke, 1961; 1964; 1966) and a thicker dense homogenous layer beneath (Kramer and Wigglesworth, 19.50; Bruck and Stockem, 1972a; b). The wax layer is ma.de of long chain hydrocarbons and the esters of fatty acids and alcohols packed closely so as to provide a waterproof layer (Beament, 1948; 1951; 1955; 19.58; 1960; 1961; 1964; 1965; Beament et al., 1964). The exocuticle consists of the tanned protein and chitinous cuticle. The endocuticle is a matrix of protein and chitin but is not tanned nearly to the extent of the exocuticle. Between the endocuticle and the epidermis is an amorphous layer known as the subcuticle or Schmidts layer. The epidermal cell layer is the layer of cells which is responsible for the secretion of the cuticle. The basement membrane is the membrane below the epidermal cell layer. The cuticle is a limiting factor for growth and in order for any increase in size the animal must shed its old cuticle and form a new cuticle. The process of moulting and cuticle formation may be divided into several different stages. See Figure 6. The first stage is the separation of the old cuticle from the underlying epidermal cells. This is called apolysis (Jenkin and Hinton, 1966). The second stage is the shedding of the remnants of the old cuticle. This is known as ecdysis. This process occurs after a new cuticulin layer has been produced, the old endocuticle has been digested and the moulting fluid has been resorbed (Wigglesworth, 1973) which is facilitated by a moulting chitinase (Ba.de, 1974) and protease (Ba.de and Shoukimas, 20

Figure 6. Di.agra.mma.tic representation of the changes occurring

in the integument during the moulting cycle (Chapman, 1975), 21

A MATURE cuncu 8 AP'OLYSIS

maximal development of oenocvtes C NEW CVTICUUN LAYER PRODUCED D ENDOCUTICLE DIGE5nD

discharged oenocyte E MOULTING FLUID F REMAINS OF OLD RESORBED CUTICLE CAST-OFF

ld .;xocuticle -a-o

ecdysial ---- - =- � ------� membrane 22

1974). The shedding of the old cuticle is facilitated by the increase in blood volume and the swallowing of air or water which swells the gut so that haemolymph pressure is increased {Cottrell, 1962d; e).

Aided by the muscular activities of the insect, the old cuticle is split along the ecdysial lines and the cuticle is shed (Bernays, 1972),

At this stage the insect is white (untanned). The new cuticle expands

(Bennet-Clark, 1963) and hardens by the processes previously described (Hackman and Goldberg, 19.58; Wigglesworth, 1933; 1945;

1947; 1959; Beament, 1959; Dennell, 1946; 1947).

The identification of the tanning agent has been extensively!_· investigated. The tanning agent is a tyrosine metabolite which is incorporated into the insect cuticle at ecdysis and pupation. Pryor

(1940b) proposed a mechanism in which tyrosine was hydro:xylated to dopa and subsequently to dihydro:xyphenylacetic acid which was then transported to the cuticle and oxidized to quinone. Tyrosine titer was shown to peak one half hour prior to pupation in blowfly larvae and decrease sharply at ecdysis with a corresponding rise of non protein material of hardened puparial cuticle (Fraenkel and Rudall,

1947; Karlson et al., 1962). Using l4c-tyrosine it was shown by incorporation studies that tyrosine was a definite precursor of the tanning agent {Karlson, 1960; Srivasta, 1971) and that it was incorporated at a steady rate for approximately eight hours postecdysis (Fox and Mills, 1971), Later it was shown that the tanning agent was an amine derivative rather than an acetic derivative as was previously assumed {Pryor et al., 1947),

N-acetyldopamine was identified as the amine derivative and was found to be the primary tanning agent of puparium in Calliphora 23

(Karlson et al., 1962; Karlson and Ammon, 1963; Ammon and Karlson,

1964; Karlson and Sekeris, 1962a). Utilizing incorporation experiments,

Karlson and his coworkers found that 14e-1eucine was only 5-10% incorporated into the cuticle while 14c-tyrosine, dopa, dopamine and tyramine were incorporated about 80% (Karlson et al., 1962). Moreover,

N-acetyldopamine was completely incorporated into the puparium

(Karlson and Sekeris, 1962b) and is now thought to be the major sclerotization agent in insects. The incorporation of label is attributed to N-acetyldopamine being completely incorporated into the cuticular proteins. N-acetyl tyramine as well as N-acetyldopamine were shown to be sclerotization agents in Periplaneta (Mills ---et al., 1967) and in Bombyx .(Butenandt et al., 1959). 14c-noradrenaline has been shown to be incorporated into the cuticle of Periplaneta (Koeppe. and Mills, 1970) and 3,4-dihydroxyphenylacetic acid may also be a minor sclerotization agent (Koeppe and Mills, 1974).

Evidence from acid hydrolysates of insect cuticle suggests that there may be a beta-hydroxy-N-acetyldopamine metabolite which causes cuticle sclerotization (Andersen, 1970; Karlson et al., 1962).

Arterone (2-amino-3 ,4-dihydroxyacetophenone), 2-hydroxy-3,4-dihydroxy acetophenone, and 3,4-dihydroxyphenylglyoxal which can be reduced to

3,4-dihydroxyphenylglycol (See Figure 7) have been isolated from cuticle homogenates suggesting there may be more than one tanning agent (Andersen, 1971; Andersen and Barrett, 1971).

Storage forms of the tanning agent have been identified in the haemolymph prior to ecdysis. Gamma-L-glutamyl-L-phenylalanine as well as Beta-alanyltyrosine has been suggested to play a major part in puparium formation in � by providing a large phenylalanine 24

figure 7. Structures of hyd.rolysates of possible sclerotization agents. I. arterenone (2-amino-J,4-dihyd.roxyaceto phenone), II. 2-hyd.roxy-J,4-dihyd.roxyacetophenone, III • J, 4-dihyd.roxyphenylglyo:xa.l, IV. 3, 4-dihyd.roxy phenylglycol (Andersen and Barrett, 1971), 25

OH OH H H O O O O C=O C=O CH2 NH2 CH20H

OH OH H H O I O . O O C=O CHOH CHO CH20H

111 IV reservoir (Bodnaryk and Levenbook 1963; Bodnaryk, 1970; Bodnaryk and McGirr, 1973; Bodnaryk and Skillings, 1971; Bodnaryk et al-., 1974a; b) which is later incorporated into the cuticle (Bodnaryk, l97la; b). Ibpa.mine-3-0-sulphate, N-acetyldopa.mine-3-0-sulphate and N-acetyldopa.mine-3-0-phosphate have been shown to be probable storage or transport forms of the tanning agent in Periplaneta (Bodnaryk and Brunet, 1974; Bodnaryk et al., 1974a). Tyrosine-0- phosphate may be a storage reservoir in Drosophila (Lunan and Mitchell, 1969; Mitchell and Lunan, 1964). Glucosides of the phenols have also been shown to be possible storage and transport forms of tanning agents (Brunet and Coles, 1974; Brunet and Kent, 1955; Lake et al., 1975). 27

Enzymes Involved in the Synthesis of the Sclerotization Agent

In order to show- the intermediate steps in the pathway from tyrosine to N-acetyldopamine and its metabolites, the enzymes for conversion must be identified and their presence in the insect determined. Forms of the enzymes in insect development have been reviewed by Laufler (1961). There are many pathways of tyrosine as tyrosine is a major branching point in intermediary metabolism in many animal systems. Only those enzymes specifically relating to the formation of possible sclerotization agents will be discussed,

The conversion of pheny]alanine to tyrosine by the enzyme tyrosine hydroxylase was found in insects (Murdock et al,, 1970a). Al though this enzyme may be important it is only essential if the diet of the insect is quite limited. Tyrosine is present in most protein sourses.

There are two major pathways for the conversion of tyrosine to other metabolites. The first pathway is found mainly in mammals and is found to some lesser extent in insects. The pathway converts tyrosine to acidic metabolites such as parahydroxyphenylpyruv.ate, parahydroxyphenylpropionate (Karlson and Sekeris, 1966b; Sekeris and

Karlson, 1966) or parahyd:roxyphenylacetate (Aerts et al., 1960).

However, this pathway is thought to be largely for the control of excretory products in insects, although some may be in9orporated into the cuticle as lipid antioxidants (Atkinson et al., 1973).

The second major pathway is the conversion of tyrosine to 28

catecholamines. This pathway is :found in both mammals and insects

and is reported to be the one directly related to the sclerotization

agent, In this pathway tyrosine is converted to dopa and then dopa

to dopamine to norepinephrine to epinephrine. This pathway has been

reviewed extensively (Axelrod, 1959; Daly and Witkop, 1963; Udenfriend,

1966b; Iverson, 1970),

The conversion of tyrosine to dopa is governed by tyrosine

hydroxylase or tyrosinase, both of which are mixed function hydroxy

lases. This is the rate limiting step in mammals (Nagatsu et al.,

1964; Udenfriend, 1966a; b). It is present in insect haemolymph

(Kawase, 1960) as a non-specific enzyme having a pH optimum of 6.8

and requires copper as a cofactor (Mills et al., 1968) and it has

been shown to be activated by polyanions and salts (Katz et�. 1976;

Brennan and Beck, 1972).

D:>padecarboxylase (the enzyme necessary to convert dopa to

dopamine) has been partially purified in Calliphora (Sekeris and

Karlson, .1966), localized in Aedes (Schlaeger and Fuchs, 1974), and

analyzed spectrophotometrically in Drosophila (Sherald et al., 1973).

It has a pH optimum of 6 .8 and is dependent on pyridoxal phosphate and

ferrous ions (Sekeris and liarlson,r1966). D:>padecarboxylase. is

- inhibited by N-acetyldopamine which supports the exil![ltence of a

negative feedback mechanism. Its activity increases as ecdysone titer

increases (Karlson and Sekeris, 1962b; 1966b; Shaaya and Karlson,

1965; Fuchs and Schlaeger, 1973). Its presence has been found in

Sarcophaga (Chen and Hodgetts, 1974) and in Periplaneta (Murdock

et al., 1970b), In inhibition studies it has been shown that

dopadecarboxylase is necessary for normal sclerotization to occur 29

(Schlaeger-and Fuchs,-1974)� Dopadecarboxylase has_been found in

the haemolymph and haemocytes of flies (Whitehead, 1970).

Beta-hydroxylase has been found in cockroach haemolymph (Lake

et al•, 1970). It hydroxylates dopamine to norepinephrine (Levin

et al., 1960).

N-acetylation of amines has been reviewed by Daly and Witkop

(1963). Acetylcoenzyme A transactylase has been found in several

species of insects (Karlson and Ammon, 1963). This enzyme is necessary

for the acetylation of dopamine. It is also specific for the acety lation of tyramine and histamine but not to tyrosine, dopa or glucosamine. This enzyme is not aff'ected by ecdysone. It is assumed that N-acetylation protects the amine against -indole formation.

N-acetyldopamine glucoside has been implicated in the storage of N-acetyldopamine until tanning occurs (Okubo, 19.58; Karlson et al.,

1962). The presence of a glucoside of ortho-aminophenol has been found in Periplaneta (Dutton and Duncan, 1960), and in Schistocerca

(Trivelloni, 1960). Glucosides have also been implicated in the pathways of defense secretions. Odoriforous metabolites of quinone and diphenols are stored as glucosides and are enzymatically cleaved when needed (Roth and Eisner, 1962; Happ, 1968; Ikan et al., 1979).

N-acetyldopamine-3-0-phosphate and N-acetyldopamine-3-0-sulphate have been isolated in newly ecdysed cockroaches and are incorporated into the cuticle, but not as intact esters (Bodnaryk and Brunet, 1974;

Bodnaryk et al., 1974a). Although cuticle phosphatases or sulphatases have not been reported in the literature their presence is hypothesized if the phosphate and sulphate metabolites are storage JO moities.

The oxidation of the diphenols is accomplished by phenoloxidase which occurs as inactive prophenoloxidase in haemocytes (Evans, 1967;

Sin and Tromson, 1971; Th>hke, 1973) and is activated by a lipid moiety (Heyneman and Vercauteren, 1968; Tromson and Sin, 1970). Epidermal cell phenoloxidase which catalyzes the oxidation of ortho-diphenols with amine groups but not ortho-diphenols with acidic side chains has been studied (Karlson and Sekeris, 1962a). The best substrates for this reaction have been found to be N-acetyldopamine (Km=Jxl0-3), dopamine (Km=2xl0-4) and dopa (Km=8x10-3). Phenoloxidase has been reported to be found in both blood and cuticle (Locke and Krishnan,

1971; Yamazaki, 1969; 1972). The_blood phenoloxidase is non-specific and oxidizes both mono and diphenols. However, cuticle diphenoloxidase has been shown to be specific only catalyzing the oxidation of di phenols with amine side chains (Hackman and Goldberg, 1967; Firtel and Saul, 1967). Phenoloxidase has been found in numerous insects

(Whitehead et al., 1960; Karlson and Liebau, 1961; Sekeris and

Karlson, 1966; Karlson and Sekeris, 1962a; Sekeris and Mergenhagen,

1964; Karlso.n et _al., 1964; Mills et al., 1967; Hackman and Goldberg,

1967; Aerts and Vercauteren, 1964; Evans, 1968b; Ishaaya, 1972;

Krishnam and Ravindranath, 1973; Warner et al., 1974). The binding of phenols to proteins in the haemolymph prior to sclerotization has been shown to occur in Periplaneta (Koeppe and

Mills, 1972) and in Manduca (Koeppe and Gilbert, 1974). It has been suggested that these proteins enter the new cuticle via the epidermal cells and are cross-linked to other proteins in the cuticle by phenoloxidase (Koeppe and Gilbert, 1974). '.31

The probable pathway for the production of the sclerotization agent and its ·storage metabolites is diagrammed in Figure 8 and a proposed mechanism for the binding of the sclerotization agent to cuticular proteins is diagrammed in Figure 9. , .32

Figure 8. Pathway for the production of the sclerotization agent and its storage metabolites. 33

NH CHCOOH 1'JH CHCOOH NH CHCOOH 2CH 2 2 2 CH2

OH 0 0OH 0OH phenflalanine tyrosine dopa

COCH3 NH2 NH CH 2 CH 2

OH I OH dopamine acetyldopamine

COCH3 COCH 3 \ COCH3 NH NH NH CH CH CH 2 CH22 2 � CH; O O OH V0Po20H � oso20H H O OH OH � { H� OH N-acetyldopamine N-acetyldopamine N·acetyldopamine -0-glucoside -3-0-phosphate -3-0-sulphate 34

Figure 9. Mechanism of binding of scle:rotization agent to proteins with amino terminal groups (RNH2). a::: J: J:Z 0 a:Q5 J:a::: "'·· Z J:Z J:Z� a::: .. + 0 I I a::: -b J:Z0 a:::

... :c J: 0 0 ...... O :CJ::C ozo0 O1/ \ J:0

:CZ a::: 36

Hormonal Control of Moulting and Tanning

Hormonal controls of ecdysis and sclerotization have been reviewed (Karlson, 19.56; Gilbert and Schneiderman, 1961; Williams,

1952; Wigglesworth, 19.54; 1957; 1959; 1965; Hikino and Hikino, 1970).

The pupal moult has been smwn to be initiated by a mrmone secreted by the brain (Kopec, 1922; Wigglesworth, 1933; 1934). Later it was smwn that larval moulting and pupation are initiated by a mrmone secreted by the protmracic gland. This mrmone has been called growth, pupation or moulting hormone and more recently has been called ecdysone (Fraenkel, 1935; Fukuda, 1940) .

Ecdysone has been isolated and its structure identified (Figure

10) (Karlson and Shaaya, 1964; Karlson et al,, 1965; Huber and Hoppe,

1965). It was found that brain hormone activates the protmracic gland which produces ecdysone (Williams, 1942; 1946; 1947; Wigglsworth,

1951; i952a; b) and ecdysone is secreted in an area in the abdominal region (Chang, 1972), The mode of action of brain mrmone is directly correlated with intense nuclear RNA synthesis which is coupled with the appearance of cytoplasmic RNA and consequently to protein synthesis, The production of.these proteins presumably corresponds to enzymes for production of ecdysone and possibly ecdysone binding proteins (Butterworth and

Berendes, 1974). The production of ecdysone is coupled with the renewal of growth of epidermal cells, the deposition of new cuticle 37

Figure 10. Structure of ecdysone (Karlson and Sekeri.s, 1966b). OH

HO HO 39

and mow.ting,

The action of ecdysone is modified by juvenile hormone or neotinin

which is secreted from the corpus allatum (Wigglesworth, 19.54� Williams,

1961). The physiological effects depend on the relative presence_of

these two oo:rmones. In homometabolous insects (egg-larvae-pupa-

. adul.t) ecdysone together with small amounts of juvenile hormone when

incubated with larval epidermal cells produced a pupal cuticle,

However, with large amounts of juvenile hormone, larval cuticle was

produced, In hemimetabolous insects ( egg-nymph-adul.t) ecdysone and

small amounts of juvenile hormone produced a more mature cuticle while

in the absence of juvenile hormone produced an adul.t (Gilbert and

Schneiderman, 1961). These studies show the exi.stance of an inverse

relationship between juvenile hormone and the amount of flow of fresh

genetic information from nucleus to cytoplasm at the time of ecdysis

(Williams, 1961). Juvenile hormone permits growth but inhibits

maturation.

Juvenile hormone activity is widespread in nature. ILis found

in invertebrates other than insects (Schneiderman and Gilbert, 19.58),

in plants, bacteria and yeasts (Schneiderman et al., 1960) and ifl

vertebrates including man (Williams, 19:.$6; 1959),

There has been shown an intense interrelationship between brain

hormone, juvenile hormone and ecdysone (Schneiderman and Gilbert, 1964).

The prothoracic glands are controlled by brain hormone and juvenile

hormone. Low levels of juvenile hormone and ecdysone are necessary

for normal tissue growth throughout the larval life of' insects and are

continuously found in low concentrations during most of this period.

Periodically there is a release of brain hormone which causes ecdysis, 40

The brain hormone superactivates the prothoracic glands and therefore increases ecdysone level (Wigglesworth, 1936; 1940a; b; Scharrer,

1952).

The mode of action of ecdysone has been experimentally determined

(Karlson and Sekeris, 1962a; b; 1966a; Sekeris and Karlson, 1962; 1966;

Congote et al., 1969; Fraenkel and Hsiao, 1967; Fraenkel et al., 1972;

Nijhout, 1976), During inte:moult stages the major pathway of tyrosine metabolism is one of transamination and reduction -(tyrosine.-para hydro:xyphenylpyruvic acid.-parahydro:xyphenyllactic acid-parahydroxy phenylpropionic acid) (Murdock et al., 1970a). However, one day prior to pupation the pathway shifts to one of N-acetyldopamine production

(Karlson and Herrlich, 1965; Hopkins et al,, 1971) (See Figure 11).

The rise in ecdysone titer is largely responsible for this, by activation of dopadecarboxylase activity (Karlson and Sekeris 1962b; Shaaya and Karlson, 1965; Shaaya and Sekeris, 1965), Dopadecarboxy lase activation is accomplished by induction (Fuchs and Schlaeger,

1973; Fragoulis and Sekeris, 1975) and follows the Jacob and Monod

(1961) gene repression model. Ecdysone is the genetic inducer which activates mRNA production by releasing a histone repressor from the

DNA, The mRNA then travels to the cytoplasm where it attaches to ribosomes and the formation of protein take-s place, One such protein is dopadecarboxylase (Sekeris and Karlson, 1964; Sekeris, 1965;

Sekeris and Lang, 1964), The production of structual proteins is also probable by this mechanism, This model has been reviewed by O 'Malley and Schrader (1976), Although most of this work has been done on Calliphora, the blowflies, convincing data for induction of dopadecar boxylase by ecdysone in other insects have been shown (Sekeris, 1964). 41

Figure 11, The two pathways of tyrosine metabolism in the blowfly

(Calliphora) and the locust (Schistocerca), After

Karlson, Sekeris and others, The left-hand (acid)

metabolism takes place early in the inte:rmoult period.

The right-hand (amine) metabolism takes place before a

moult and results in the synthesis of the tanning

agent N-acetyldopamine. The change-over is brought

about by a rise in concentration of the hormone,

ecdysone, in the blood (Brunet, 1967), 42

NH 2 CH 2C CO � H OH O H � tyrosine

NH2 CH CCII OH HO CH CHCOOH O f0Y 2 I 2 HO O )V HO h p·hydroxyphenyl- dopa pyruvic acid I l OH CH2 C OH

. HCO

H O O p-hydroxyphenyl- lactic acid

3 I COHCH H CH CH CH2 CH2 O H 2 N 2 C O O � � HO HO � � p-hydroxyphenyl N acetyldopamine propionic acid 43

Recently there have been t wo tYJ)es of ecdysone isolated in Calliphora: a.1.pha-ecdysone being responsible for initiation of pupariwn :fomation and beta-ecdysone being responsible for normal growth and development (Young and Young, 1976).

There is a definite relationship between ecdysone and the

formation of the tanning agent. However, the actual tanning was

shown to be controlled by another blood-borne factor which is

released within a few minutes after ecdysis (Fraenkel, 1962; Cottrell,

1962b). It has been shown that tanning is under nervous control

(Fraenkel, 1935; Clarke and Langley, 1963). However, it has been

found that nervous stimulation initiates hormonal release. This experimentation was accomplished by decapitation and ligation

experiments postecdysis. It was shown that decapitation after

emergence prevented tanning (Cottrell, 1962a; b; c). Head ligation

postecdysis was also shown to prevent tanning (Fraenkel and Hsiao.

1962). However, when blood was taken 3-30 minutes after emergence

and then injected into ligated untanned animals, nomal hardening

and darkening was accomplished (Cottrell, 1962c; Fraenkel and Hsiao,

1965). It was known that ecdysone titer was greatly decreased by

this time (Karlson, 1965; Karlson and Sekeris, 1962b; Sekeris, 1965).

To prove that ecdysone did not induce tanning an experiment was conducted which showed that blood with high ecdysone activity as well

as purified ecdysone did not promote tanning when injected into

ligated or decapitated insects (Fraenkel and Hsiao, 1962; 1965; Mills,

1965). Ecdysone did not inhibit tanning which would have allowed

tanning when the ecdysone levels dropped (Cottrell, 1962b; Fraenkel

-and Hsiao, 1965). Nomal tanning was induced in ligated animals 44 with·extracts of neu:rosecretory cells of the pars intercerebralis of the brain and also with extracts of thoracic ganglion (Fraenkel and Hsiao, 1963), Therefore the honnonal nature of tanning was proved, Fraenkel and Hsiao (1965) named this tanning hormone bursicon after the Greek bursikos which means "related to tanning", It has been shown ·that bursicon release was localized in the terminal abdominal ganglion in Periplaneta (Mills et al,, 1965) and localized to the abdominal nervous system in Manduca (Truman, 1973). There it was postulated that bursicon is made in the nervous system (Provansal et al., 1974), stored in the posterior ganglion and released upon stimulus from brain tissue (Fraenkel and Hsiao, 1965; Mills, 1967; Delachambre et al., 1972; Grillot et al., 1971). The physical properties of bursicon have been characterized (Cottrell, 1962c; Fraenkel and Hsiao, 1965; Mills, 1966). It is a protein honnone with a molecular weight of approximately 40,000 daltons (Fraenkel --et al., 1966; Mills and Lake, 1966; Mills and Nielsen, 1967). Its release and activity in the haemolymph during ecdysis and postecdysis has been analyzed (Srivastava and Hopkins, 1975). Bursicon is thought to increase membrane penneability (Mills and Whitehead, 1970; Si vasubramanian et al • , 1974) • It has been suggested that bursicon influences cuticular dehydration postecdysis (Vincent, 1971; 1972), Honnonal control of water excretion has been widely studied {Maddrell, 1963; 1964a; b; Mills and Nielsen, 1967; Goldbard et al., 1970), Antidiuretic honnone activity increases preecdysis and then drops at ecdysis while diuretic activity increases post ecdysis and parallels bursicon activity (Mills and Whitehead, 1970). It has been proposed that diuretic honnone and bursicon are identical (Mills, 1967; Mills and Whitehead, 1970). Tyrosine decarbo:xylase action increases in haemocytes at ecdysis in Periplaneta with the increased bursicon titer (Whitehead, 1969). In Sarcophaga., bursicon induces melanization of the outer layers of the cuticle and deposition of post-emergent endocuticle (Fogal and Fraenkel, 1969).

Bursicon has been shown to increase- tyrosine permeation into insect haemocytes (Mills and Whitehead, 1970; Post, 1972; Post and deJong,

1973) and it has been suggested that bursicon is involved with the control of the activation of tyrosine hydroxylation (Seligman et al.,

1969). Epidermal permeability has been mediated by bursicon during the immediate postecdysial phase in Peripla.neta in the translocation of protein-bound phenols (Koeppe and Mills, 1972).

Dibutyryl cyclic AMP mimics the action of bursicon. It is speculated that bursicon activates membrane adenylcyclase which produces cyclic AMP which would subsequently increase membrane permeability ana/or tyrosine hydro:xylase activity (Berridge and

Price, 1972; VandenBerg and Mills, 1974; 1975). See Figure 12. 46

Figure 12. Hypothetical model showing the action of' bursicon on the haemocyte and the involvement of' an adenylcyclase system (VandenBerg and Mills, 1974). 47

Tyrosine Dopa ..---- �amine

. Dopamine,\ �tyldopamme 48

EXPERIMENTAL

The experiments were divided into three categories, These

are: (1) phosphate incorporation into proteins and cuticle,

(2) dopamine metabolites binding to cuticle proteins, (3) in vitro binding of N-acetyldopamine to lysyl-lysine, 49

METH)DS AND MATERIALS

Phosphate Incorporation into Proteins and Cuticle Animals

Insects used in this experiment were newly ecdysed (white)

Periplaneta americana, either late instar nymphs or white adults, which were raised on a diet consisting of Purina dog chow and water and were maintain-ed - at :room temperature and controlled photoperiod

(12 hours light/ 12 hours dark).

Animals were injected with 10 ul of (32!>) monosodium phosphate

(New England Nuclear 500 me/mm lot 3764) using a Hamilton microsyringe, The activity of 32p was dete:rmined at time of count by utilyzing a computer program to dete:rmine activity of 32!> for any day when the activity initially is known. This program is found in Table 1.

All .cpm were standardized by taking cpm per activity injected (uCi).

Animals were incubated at room temperature for either 2 hours, 4 hours, or 24 hours. Ten minutes prior to sacrifice a'rl injection of 15 ul

0 .3M sucrose O .OlM diethylthiocarbamate was made; ·Haemolymph was removed by capillary method (Mills et al., 1966) and centrifuged on a Beckman microfuge, model 152, for 5 minutes, Paper chromatography and high voltage electrophoresis

(32P)-phosphate metabolites were separated by descending paper chromatography using a solvent system of n-butanol:glacial acetic acid: deionized water (4:1:1) and by high voltage paper electro- Table 1. Computer program for determination of J2p activity when initial activity and change of time in days are known. This program is written in Basic and was run on the Interactive Terminal Facility (ITF). 51

LOGON JOEIV LOGON JOEIV THANK YOU. DATE 76.215 TIME 1-1.48. 07 READY EDIT ACT BASIC EDIT LIST 0001.0 PRINT 'DETERMINATION OF 32P ACTIVITY AT ANY KNOWN' 00 020 PRINT "TIME GIVEN INITIAL ACTIVITY' 00 030 PRINT USING 00040 00040 'ORIGINAL ACTIVITY CHANGE IN TIME 00 050 INPUT A,T 00 0 60 B=A*&E**CC-0.693*T)/l4.3) 00070 PRINT 'ORIGINAL ACTIVITY',A 00080 PRINT 'ACTIVITY AT',T, 'DAYS =',B 00090 END EDIT 52 phoresis using a pH 3.5 buffer system of pyridine: glacial acetic acid: deionized water (1: 10: 89) • Whatman 3mm chromatograpey paper was used :for both chromatography and electrophoresis. Authentic standards were chromatographed with the phosphate metabolites and

Rf' values were obtained and compared to unknowns.

Fingerprinting technique was utilized by :first subjecting the haemolymph supernate to electrophoresis and th en d etermining areas of radioactivity using a Packard Model 7201 Radiochromatogram

Scanner. Peak areas of activity were cut out and sewn into new papers and rechromatographed. Column chromatography

Haemolymph was separated on a 0.6x 40 cm P-30 polyacrylamide gel column (Bio-Rad) 100-200 mesh. Forty fractions of 0.5ml. w ere collected. Protein was monitored at 280nm on a Coleman Model 124D double.beam spectrophotometer. Radioactivity was determined by placeing .50 ul of each fraction into vials containg 10 ml of Aquasol II (NEN) and counting on a Beckman L-250 Liquid Scintillation counter.

Calculations concerning each fraction such Ve/Vo, Kav, Cpm/uCi, etc. were aided by use of IBM 370 computer utilyzing a packaged program known as Statistical Analysis System (SAS). A r epresentative program for these calculations is found in Table 2. Concentration of protein was determined from known concentrations of bovine serum albumin upon which OD values w ere obtained as above. These values and the known concentrations were used to determine the best standard curve utilyzing a statistical analysis known as least squares and was faciliated by using the Interactive Terminal Facility (ITF) and a 53

Table 2, Computer p:rogram :for analysis of' data f:rom :fractions f:rom

a gel column (]:'-2). This p:rogram computes elution volume

(Ve), Kav, Ve/Vo, predicted molecular weight of molecules

in a :fraction (MW), corrected cpm (CCPM), cpm per

:fraction (CPFRACT), cpm per :fraction per uCi injected

(CPFPAI), mg p:rotein (MGPRTN) and cpm per fraction per

uCi injected per mg protein (CPFPAIPP). The program

graphs the following: UV vs, :fraction, UV vs. VeVo,

CCPM vs, :fraction, CCl?M vs. VeVo, CPFRACT vs. fraction,

CPFRACT vs, Ve Vo, CPFPAI vs, fraction, CPFPAI vs, . VeVo,

MQP!iTN-:vs� :f:ractian• . Mfil'RTN vs. VeVo, CPFPAIPP vs. :fraction

a.nd CPFPAIPP vs, VeVo, 'PlDSPHATE TITLE INCDRroRATION. IN'lO HAEIDLYMPH MACIDIDLECULES'; DATA RAW-DATA; EXP INPUT $1�4'FRACTION 6-e'ABsiIv'10�14 Bimn 16-23 em 2.5-32; VT =11.31; V{)=4.0. VF=O.5� VE=VF+VE; VEVO=VE/VO; KAV=(VE-VO)/(VT-V{); . PJO_S1: IF KAV 0.93 THEN MW=25Q'0; F30_S2: IF KAV =0.93 & KAV =0.00 THEN MW=10**«-1.2947527)*KAV�G10. (40000»; CCPM=CPM-BKGD; IF CCPM 6 THEN CCPM=O CPFRACT=CCPM*O .5/0.05; CPFPAI=CPFRACT/(20*0.1625704); MGFRTN=(ABSUV=1.18308�02)/5.521189�02; CPFPAIPP=CPFPAI/MGFRTN; IF MGPRTN 0 THEN MGFRTN=O AND CPFPAIPP=O; CARDSj FIDC Pf.J0T IDWS=49; VAR ABSUV FRACTION; TITLE • ABSUV VS. FRACTION 00.';

FIDC PIDT IDWS=49; VAR ABSUV VEVO; TITLE 'ABSUV VS. VEVO':

FIDC PIDT IDWS=49; VAR CCPM FRACTION; TITLE • CDRRECTED CPM VS. FRACTION 00.';

FIDC PIDT IDWS=49; VAR CCPM VEVO; TI.TLE ' CD RRECTED CPM VS. VEVO1 ;

FIDC PIDT IDWS=49; VAR CPFRACT FRACTION; TITLE ' CCPM FER FRACTION VS. FRACTION';

FIDC PIDT IDWS=49; VAR CPFRACT VEVO; TITLE ' CCPM FER FRACTION VS. VEVO';

FIDC PIDT IDWS=49; VAR CPFPAI FRACTION; TITLE" CCMP FER FRACTION FER ACTIVITY INJECTED VS. FRACTION';

FIDC PIDT IDWs=49; VAR CPFPAI VEVO; TITLE ' CCMP FER FRACTION FER ACTIVITY INJECTED VS. VEVO';

FIDC PIDT IDWS=49; VAH MGFRTN FRACTION; TITLE • MG FIDTEIN VS. FRACTION '; 55

PFOC PI.DT FOWS=49; VAR MGPRTN VEVO; TI'll,E I MG PFOTEIN vs. VEVO'; PFOC PIDT FOWS=49; VAR CPFPAIPP FRACTION; TI'll.E ' CCPM PER FRACTION PER ACTIVITY INJECTED PER PFOTEIN VS. FRAC- TION'; PFOC PI.DT FOWS=49; VAR CPFPAIPP VEVO; TI'll.E ' CCPM PER FRACTION PER ACTIVITY INJECTED PER PFOTEIN VS. VEVO'; computer program which is found in Table 3.

Incorporation of (3�)-phosphate into cuticles was established by counting the cuticle hyd:rolysate. Cuticle was removed from the abdominal region at the time of sacrifice, scraped and homogenized in O.lM IDa in methanol. The homogenate was centrifuged at 10,000 g for 10 minutes on a Beckman J-21 centrifuge, before hydrolysis in 1ml of lM NaOH for 24 hours. The sample was placed in 10 ml

Biofluor (NEN) and counted for radioactivity.

Phospoorylated proteins, separated by column chromatography, were concentrated by lyophilization and reinjected into newly ecdysed cockroaches. These incubated for 24 hours and the incorporation of

3�-p:roteins into the cuticle was analyzed as described above. 57

Table 3. Computer program for determination of the best straight

line for any given set of points (linear least squares

statistical analysis). LIST 01 03 . INV CMD EDIT LIST DD DID PRINT 'LEAST SQUARES--LINEAR REGRESSION' 00011 PRINT 'THIS PROGRAM DETERMINES A BEST LINEAR' ODD 12 PRINT 'FIT FOR KNOWN DATA SUCH AS STANDARD' DD D 13 PRINT 'CURVE DATA FROM PROTEIN AND SUGAR TESTS' ODD14 PRINT 'IT WILL ALSO GIVE EXTRAPOLATED X VALUES' DD D 15 PRINT 'IF Y IS KNOWN.' 00020 PRINT 'NUMBER OF DATA PAIRS' 00 030 INPUT N 00033 IF N>O GOTO 39 00034 LET N=-N 00035 LET Q=-1 00036 GOTO 40 00039 LET Q=I 00040 DIM XC45>, YC45> 00 050 PRINTUSING46 0. 00 060 FOR l=l TO N 00 070 INPUT XCI>, YCI> 000 75 IF Q>O GOTO 80 000 76 LET YCI>=LGTCYCI>> 00 080 NEXT I 00090 PRINT 'ERROR IN PAIR # ?' 00100 INPUT A 0011 0 IFA,YCA> 00135 IF Q>O GOTO 140 00 136 LET YCA>=LGTCYCA>> 00140 GOT090 00 150 LET 51=0 00160 LET 52=0 DO I 70 LET x1; .0 00 180 LET Yl.i .0 00190 LET Rl=O 0020 0 FOR 1=1 TO N 00 21 0 LET Sl=Sl+XCI> 00220 LET Xl>•Xl+CXCI>*XCI» 00230 LET S2=S2+YCI> 00240 LET Yl=Yl+CXCI>*YCI>> 00250 NEXT I 00260 LET S2=S2/N 00270 LET Sl=Sl/N 00275 IF Q>O GOTO 280 00276 PRINT/ ____ _ �EAST SQUARES FIT USING BASE 10 LOG OF Y DATA' 5'3

00280 PRINT .AVG x=·,s1,· AVG Y=',S2 00290 LET DO=N*CX1-CN*Sl*S1)) 00300 LET AO=N*CS� w Xl-Yl*Sl)/DO 0031� LET Ali4N*Yl-N*N*Sl�S2)/DO Ot;J20 LET S5= 0 00330 LET S6=0 00340 FOR I:::i '1-· ,, 00350 LET S5=S5+CCS1-XCI>>**2> 0036G w�, �6=S6+CCS2-YCI>>**2> 00370 LET Rl =Rl+CYC1)-S2)*CXCI>-Sl> 00360 NEXT I 00390 LET S5=SQRCS5/CN-l>> 00400 LET S6=SQRCS6/CN-l>> LET R=Rl/ CN- >*S?* 2 FRINI 51 b DE ¢ X= , �g , STD DEV Y= ,S6 88��8[�430 .�ET S5=S5/Sl 0044ij �ET S6=S6/S2 00450 P�INT 'REL STD DEV x=·, S5,' REL STD DEV Y=·,s6 00460 CXJ [YJ 00470 PRiNT 'LiNEAR LEAST-SQUARES FIT. 00480 PRINT 'SLOPE=',Al, • Y INTERCEPT=',AO 00481 PRINT 'THE EQUATION FOR THE LINE IS' 00482 PRINT' Y=',A1,·x + ',AO 00490 PRINT 'CORRELATION COEFFICIENT=',R 00491 PRINT ·oo YOU WISH TO FIND X FOR KNOWN Y?' 00492 PRINT •YES=l, N0=2· 00493 INPUT T 00494 LET U=2 00495 IFU=T GOT0501 00496 PRINT 'Y=?· 00497 INPUT V 00496 LET W=/Al 00499 PRINT 'IF y�·, v�·THEN x=·,w 00500 GOTO 491 00501 PRINT .AGAIN? YES=l, N0=2· 00510 INPUT A 00520 LET B=l 00530 IFA=BGOT020 00540 END 60

ll:>pamine Metabolites Binding to Cuticle Proteins

Insects used in this experiment were similar to those described in the phosphate incorporation methods and materials. Animals were injected with 10 ul (ZUCi) of 14e-dopamine (New England Nuclear (ethylamine-2-14c) dopamine hydrochloride Code CFA.314 Batch 24 specific activity 60 mCi/mmol mixed in methanol 0.25 ml yielding ZUCi/lOul), using a Hamilton microsyringe. Animals were incubated for 20 minutes then injected with 15 ul O.JM sucrose O.OlM diethylthiocarbamate. Ten minutes after this injection animals were bled and haemolymph microfuged as previously described. Supernate was chromatographed in BAW paper chromatography system (n-butanol:glacial acetic acid: water; 4:1:1). Chromatographs were scanned for radioactivity as before. Areas of peak radioactivity were ·cut out and sewn onto another chromatogtaph paper. Two newly ecdysed cockroaches were obtained and their abdominal cuticles removed and scraped. These cuticles were homogenized in approximately 4 ml �.lM tris acetate buffer (6.8 pH) and O.lM Cu (II) sulphate. The homogenate was centrifuged at 10,000 g for 10 minutes using a Beckman J-21 centrifuge. The supernate was sprayed onto the radioactive areas of the chromatogram. The chromatogram was incubated for 1 hour in a humid area to inhibit drying of the sprayed area. This procedure was duplicated except that the cuticle homogenate was not centrifuged. In this case the whole homogenate was brushed on the 61 spot of radioactivity.

The papers were ch:romatographed and scanned for radioactivity as was previously described. Rf values were obtained and compared to standard Rf values from previous experimentation or from values obtained in the literature. In vitro Binding of N-acetyldopamine to Lysyl-lysine

Insects used in this experiment were similar to those described

in the phosphate incorporation methods and materials. Two newly ecdysed

nymphs were obtained and their abdominal cuticle removed, scraped�.a.nd -4 homogenated in 1.0 ml pH 6,8 tris acetate buffer 10 M Cu(II)ro4. The homogenate was placed together with approximately 30 umoles N-acetyl

dopamine monohydrate (Regis) and 30 umoles L-lysyl-L-lysine dihydro chloride (Sigma) into a test tube and incubated in a water bath at ° 37 c for three hours.

The mixture was centrifuged at 12,000g for 10 minutes using a

Beckman J-21 centrifuge. The supernate was chromatographed on a P-2

(200-400 mesh) polyacrylamide gel column (Bio-Rad) 0,6 x 60cm.

Seventy fractions were collected each containing 0,5 ml. Separation of products was monitored via UV absorption at 280 nm and by dotting small portions of each fraction on chromatography paper and spraying . 0 with ninhydri.n solution (0,2% in acetone) and developing at 100 C in a chromatography drying oven. Assignments were made as to the degree of reaction with the ninhydri.n by using a scale from Oto 5 with O being no reaction and 5 being a very quick reaction which was deep purple in color, Assignments from 1 to 4 were made corresponding to the degree of reaction of each spot,

Peaks of high absorbance were concentrated and chromatographed using a BAW solvent system and electrophoresis at pH 3.5 as-was 6J described before.

Radioactive N-acetyldopa.mine was prepared by a method described 14 by Bodnaryk et al. (1974a) using ethyla.mine-1- c-dopa.mine hydro chloride (Ame:rsham/Searle 52 mCi/mmol specific activity Code CFA,

295 Batch 10), Fifty ul of this solution was added to the solution described above and the experiment was repeated. Radioactivity was monitored by placing 10 ul aliquots into vials containing 10ml

Aquasol II (NEN) and counted on a Beckman 1-250 liquid scintillation counter,

Peaks of high UV absorbance were grouped and concentrated and then chromatographed using paper chromatography (BAW system) and electrophoresed.-.(pH 3,5),

Similar experiments were completed using only N-acetyldopamine

(non-labelled) and lysyl-lysine with•no cuticle present. 0,1 mmoles of N-acetyldopa.mine and 0,2 mmoles of lysyl-lysine were mixed in 1.0 ml tris acetate (O,lM and ph 6.8) and Cu (II)slil,.phate (10-4). This mixture was stirred with a magnetic stirrer at room temperature until the solution became dark brown (approximately 24 hours), The solution was then subjected to paper chromatography and high voltage electrophoresis as was previously described. The remaining portion was chromatographed on a 0.6 x 50 P-2 pol1,acrylamide gel column and

70 fractions of 0,5 ml were collected •. These fractions were analyzed for UV absorbance at 280 nm, reactivity with ninhydrin and with Pauley's reagent.

UV positive fractions were grouped--'. and concentrated to dryness using a freeze dryer. The remaining precipitate was mixed with methylsulfoxide in 1% TMS (Diaprep) and analyzed on NMR using 64 a Varian A-60 NMR. RESULTS

Phosphate Incorporation into Proteins and Cuticle

Chromatography and Electrophoresis The ch:romatogra.phs and electropherograms of this experiment are found in Figures 13 and 14. Rf' s are found in Table 4. As one can readily ascertain the ch:romatographs and electropherograms of the 2

hour and 4 hour incubations are relatively the same. There are two major peaks present •. The first p�ak was electrophoretically neutral and had an Rf of 0.010 which probably was made up largely

of proteins. The second peak was electrophoretically negative and

had an Rf of O ,19. This peak was identical to 32P-phosphate standard,

The chromatograms and electorpherograms of the 24 hour

incubations yielded the same peaks as the 2 hour and 4 hour

incubations with one major exception, There existed a large

electrophoretically neutral peak which had an Rf value of O ,94, This

indicates a phosphate compound with two neutral side cha.ins R-R>4H-R. Columns

Data obtained from the elution fractions are graphed in Figures

15 through 26 with composite graphs for the 2 hour, 4 hour and 24 hour

incubation being found in Figures 18, 22, 26 respectively. There is

little significant difference between the 2 hour and the 4 hour

incubations. However, once again, there was a notable difference

between the 24 hour incubations and the 2 or 4 hour incubations, In 66

Figure 13, Representative chromatograms of phosphate metabolites from haemolymph obtained in 2 hour, , 4 hour and 24 hour incubations illustrated in chromatograph I, chromatograph II, and chromatograph III, respectively. "0" denotes the origin, while "SF" denotes the solvent front. Spots are UV positive areas. "A", "B" and "C" denote, radioactive areas. Note the presence of peak "C" in the 24 hour incubation. This corresponds to the increase of radioactivity found at the origin in the electrophe:oo,""' grams for the 24 hour incubation (See Figure 14).

°'

--.J

� �

.. 5 ..

f f

0. 0.

�

� �

0.25

A A

i i

e,.

E E

ll ll

1 1 1 68

Figure 14. Representative electropherograms of phosphate metabolites from haemolymph. Electropherogram I is haemolymph from the 2 hour incubation. Electropherogram II is haemolymph f:rom the four hour incubation while electropherogram III is haemolymph from the 24 hour incubation., D::>tted eyots represent UV positive areas. "0" is the origin. "A" and "B" represent pea.ks of radioactivity. Note the increase "A" in the 24 hour incubation electropherogram. 69

+ +

wli\

I 70

Table 4. Rf' s of metabolites of phosphate metabolism in haemolymph. 71

2 hour incubation R R R Rf fA fB fA(ave) B(ave) 0.017 0.209 0.020 0.207 0.019 0.221 0.024 0.192

4 hour incubation R R R Rf f A fB fA(ave) B(ave) 0.010 0.197 0.010 0.193 0.021 0.200 0.000 0.182

24 hour incubation R Rf Rfc( RfA RfB Rfc fA(ave) B(ave) ave) 0.937 0.015 0.182 0.951 0.015 0.185 0.020 0.189 0.949 0.011 .0.183 0.911 72

Figure 15, Graph of eluant from polyacrylamide gel chromatography

P-30 ( 0 • 6 x 40 cm.) of haemolymph obtained from 2 hour

incubation (1st experiment). Open circles denote optical

density at 280 nm while closed circles denote cpm/uCi.

"A" denotes fraction with molecular weight of 40,000

daltons while "B" denotes fraction with molecular weight

of 2500 daltons, 7.3

osz00

___ o ___ -o ---0 ------o==: -- -- 0 / ------0 - ---- o:---- ;;;.�---·/· 0..o' ... � • -...- - . o __ _ �,, c:J9 • ' ,, ...... cS, ...... _ _::o ... �,... •I.· o:_ - /• -o... • -o ...... -o. "' • '9 \

o_ .... _ --o

en [OT x !3"/ wd:, 74

Figure 16. Graph o:f eluant :from polyacrylamide gel chromatography

P-JO (0.6 x 40 cm.) o:f haemolymph obtained :from 2 hour incubation (2nd experiment). Open squares denote optical density at 280 nm while closed �quares denot� cpm/uCi. "A" denotes :fraction with molecular weight o:f 40,000 daltons while "B" denotes :fraction with molecular weight o:f 2500 daltons. 75

osz0o

OIi":�-__.

;· �-0 . ,,," . •/9 ,I /o/ \ ·�.. . ' . -__ a . m ------·�.--0 / ,�" •, D,b �.,,,,,,.. 0''• /;a • ,I

t\0. ... .11

I I O') CD 76

figure 17. Graph of eluant from polyacrylamide gel chromatography

P-JO ( 0 , 6 x 40 cm.) of haemolymph obtained from 2 hour

incubation (Jrd experiment) • Open triangles denote optical

density at 280 nm while closed triangles deoote cpm/uCi.

"A" denotes fraction with molecular weight of 40,000

da.ltons while "B" denotes fraction with molecular weight

of 2.500 da.ltons. 77

osz00

, -c::> � I ' c:c 78

Figure 18, Composite graph of eluant from polyacrylamide gel

chromatography P-30 (o.6 x 40 cm,) of haernolymph obtained

from 2 hour incubations, Open characters denote optical

density at 280 nm while closed characters denote cpm/uCi,

"A" denotes fraction with molecular weight of 40,000

daltons while "B" denotes fraction with molecular weight

of 2,500 daltons, 79 os z00 80

Figure 19. Graph of elua:ht from polyacryla.mide gel chromatography P-.30 (o.6 x 40 cm.) of haemolymph obtained from 4 hour incubation (1st experiment). Open circles denote optical density at 280 nm while closed circles denote cpm/uCi. "A" denotes fraction with molecular weight of 40,000 daltons while "B" denotes fraction with molecular weight of 2500 daltons. ,, ... o

I0 0 \ 0 •

\,,/.-:, :s:

Io', :, • / 'o . ;'' o .//4 - .:o ,.," � C .c ------o- o ------o - . -�. ----e "·- "',e K \ ,,,,,,. �,� 0'"" I 0,. 9 o...... 82

Figure 20. Graph of eluant from polyacrylaroide gel chromatography

P-JO (0 . 6 x 40 cm.) of haemolymph obtained from 4 hour

incubation (2nd experiment). Open squares denote optical

density at 280 nm while closed squares denote cpm/uCi.

"A" denotes fraction with molecular weight of 40,000

daltons while "B" denotes fraction with molecular weight

of 2500 daltons. 83

1) 8Zoo

a I I a, aI I / I / 0/

/ ,/q / ..,... er" a--- /. g._____ . __....,....-• .cr:_- .-. ,. cca C) � C: .5?- u -,,_

-C)

\ )\ •'

X 84

Figure 2l. Graph o:f eluant :from polyacryla.mi.de gel chromatography

P-JO ( 0 • 6 x 4o cm.) o:f haemolymph obtained :from , 4 hour

incubation (3rd experiment). Open triangles denote optical

density at 280 nm while closed triangles denote cpm/uCi.

"A" denotes :fraction with molecular weight of' 40,000

dal tons while "B" denotes f'raction with molecular weight

of' 2.500 daltons. osz00

( .c .. ----

�� \ .... �, «=- '\ "<] '' �

Figure 22, Composite graph of eluant from polyacrylamide gel chromatography P-30 (0.6 x 4o cm.) of haemolymph obtained from 4 hour incubations. Open characters denote optical density at 280 nm while closed characters denote cpm/uCi. "A" denotes :fraction with molecular weight of 4o,OOO daltons while "B" denotes fraction

with molecular weight of 2500 dal tons. 87

osz00

a:. - C"' .._ C C _____ - -o �...3 • ·--a. • • • r•• �- •• • '. -- ' � :;:;u ""\•...... C � E -. ---0-- --�• .--�. \-: ------�j �------�' � o•• ·. I :, � o.: �t S:: "\

EOl x ��n;wd:> 88

Figure 23. Graph of eluant from polyacrylamide gel chromatography

P-JO (0 • 6 x 40 cm.) of haemolymph obtained from 24 hour incubation-(1st experiment). Open circles denote optical density at 280 nm while closed

circles denote cpm/uCi. "A" denotes fraction with

molecular weight of 40,006 daltons while "B" denotes

fraction with molecular weight of 2500 dal tons. OSZoo

-' o----' ' ,o ,o' o' I 0 .,'o o" --_-_....,_.,,,.ei,:.....------.J_J/ . Cl0 ------o C: - ---- ,.,• 0 ------.,,,.- .2- -- ' � --�---o ------• -----o� -- ·�o, ·��."r ., 0 , "'• b • ,.oe I ,' ., o \ c::, ., I -

Figure 24. Graph of eluant from :polyacrylamide gel chromatography P-30 (0.6 x 40 cm.) of haemolymph obtained from 24 hour incubation- (2nd experiment). Open squares denote optical density at 280 nm while closed squares

denote cpm/uCi. "A" denotes fraction with molecular weight of 40,000 daltons while "B" denotes fraction with molecular weight of 2500 daltons. 91

osz00

0 0.... b I I I 0/ / ., cY ' --0 a------D / ------0 ------� ------o:-- -- C: ------I C D - ·-u • -ca '\ A.- 0-- - • ------o ------o ----b ,\ ' \ I• I I 0 / /

[01 X �D/Wd3 92

Figure 25, Graph of. eluant from polyacrylamide gel chromatography P-30 (0,6 x 40 cm.) of haemolymph obtained from 24 hourincubation· (3rd experiment), Open triangles denote optical density at 280 nm while closed

triangles denote cp�/uCi. "A" denotes fraction with molecular weight of 40,000 daltons while "B" denotes fraction with. molecular weight of 2500 daltons, 93

os z00

.. ,, ... - <1' ,.I � \'

'�\ \._ '\ '

en 94

Figure 26. Composite graph of eluant from polyacrylamide gel

chromatography P-JO (0.6 x 40 cm.) of haemolymph

obtained from 24 hour incubations� Open characters

denote-optical density at 280 nm while closed

characters denote cpm/uCi. "A" denotes fraction with

molecular weight of 40,000 daltons while "B" denotes

fraction with molecular weight of 2500 daltons. 95

osz00

/ �, _..P····• • �··<>-·- a � l ""o . , ' ' D. a--\--I/ � � // � /.,,, . � ,0 -...... 0 .Yt \ ______d,o ----0 f------p/ J------/j .. ··/· -=-=----=-..,,._0 --- o:[-:.=.-:=---:-=------u / ..; ,.... ------_ •.Ji... a - -a._ ___ ------�···-,: "'"'...... II ' - ·------a------...... �� ... ��1' a- •...... 'o,··· ' � :\ o�'l?lt, k � o.1..1,.� o'' . -= . O"...... 'c i'

rOI x !:Jll/wd:, 96 the".2 .and 4 hour incubations there was a large amount of radioactivity incorporated into the molecules less than 2,500 daltons, This

radioactivity was not present in the 24 hour incubation profile.

There also .was a corresponding decrease in absorbance in the UV

range for molecules less than 2,500 daltons. The large values for.

absorbance in the small, molecular range immediately postecdysis and during the following 4 hours are due mainly to the high titer of phenolic groups present in the blood which are incorporated into the cuticle as sclerotization agents, The corresponding loss of radioactivity in this range of molecules may indicate that phosphate is being incorporated into the cuticle.

Phosphate incorporation into proteins is found in all investi gations., However, phosphate is not incorporated into large molecular weight proteins (those greater than 40,000 daltons). Most of the phosphate incorporated into molecules of molecular weight greater than 2,500 daltons was into molecules of molecular weight between ·

15,000 and 2,500 daltons.

Cuticles Phosphate incorporation into cuticles is given in Table 5,, This incorporation is definite proof that phosphate is a part of the cuticular matrix,

The fact that protein bound phosphate is incorporated into the cuticle, although not to a great extent, leads one to speculate as to

the reasons for incorporation. It is noted that phosphate is

incorporated to a greater extent when it is on the proteins. This is evidence for the phosphate to be incorporated to a greater extent

as phosphoproteins rather than as phosphoric acid. 97

Table .5. I. IncorporatiOll• of labelled phosphate into newly ecdysed cockroach cuticle·.,

II. Incorporation o•f phosphoproteins into cock.roach cuticle, 98

I. _g_ hour incubation 320,78 cpm/uCi/mg cuticle

4 hour incubation 832.5 cpm/uCi/mg cuticle

24 hour incubation 285,55 cpm/uCi/mg cuticle

II. incorporation of labelled proteins in to cuticle 18,013 cpm/uCi/mg cuticle 99

fupa.mine Metabolites Binding to Cuticle Proteins

The chromatograms from this experiment are found in Figures 27 and 28 with the Rf' values for the radioactive peaks being found in Table 6. '1-'.hese Rf' values may be compared to known values f'rom the literature and values obtained from standards, These values are found in Table 7 (for a more complete table see Appendix). The differences between the chromatograms and electropherograms of the incubation with the supernate compared to the whole homogenate are obvious. The supernate incubation yielded a great change in the metabolites indicating that the soluble proteins did not bind to the metabolites or metabolize them. However, the whole cuticle incubation yielded a great change in the metabolites showing binding to occur to molecules which were immobile and which were probably proteins, Also a peak which corresponded to dopamine was indicated, This would suggest the presence of a sulphatase in the structural proteins which fo:rm the cuticle matrix,. as the �opamine metabolite is reported to be a sulphate ester (Bodnaryk and Brunet, 1974). 100

Figure, 27, Representative radiochromatograms of dopamine .metabolite

binding experiments performed with cuticle soluble proteins,

I. Radiochromatogram of haemolymph of 14C-dopa,!ld.ne

m.et&bolites after 30 minutes incubation in newly

ecdysed cockroach,

II, Radio chromatogram of peak "B" of I subjected to incuba

tion with soluble proteins of the cuticle, No change

in the position of radioactivity is noted indicating

no binding or change in the metabolite occurred.

III, Radiochromatogram of peak "C" of I subjected to

- incubation with soluble proteins of· the cuticle, No

change in the position of radioactivity is noted

indicating ·no binding or change in the metabolite

occurred. I DOPAMINE C A (\ D E F �1� � -- � I 0,25 o.�- 0.75 0 R, s� II SPOT B B

l �1 0.5 0.75 0 SF Rt Ill SPOT C C

� - � 1--- ...... , __ -.- - ...,.,...... , - ,.. I 0 O:.t �,�-...... v••v ..., ...... ---...... Kf . �t 102

Figure,28. Representative radiochromatogra.ms of dopamine metabolite

binding experiments performed with cuticle homogenate. 14 I. Radiochromatogra.m of haemolymph of c-dopamine

metabolites after JO minute incubation in newly

ecdysed cockroach.

II. Radiochromatogram of peak "B" of I subjected to

incubation with cuticle homogenate. "A" is p:robably

p:rotein bound label, while "C" is most likely.

dopamine. III. Radiochromatogram of peak "C" of I subjected to

incubation with cuticle homogenate. "A" is p:robably p:rotein bound labei. I DOPAMINE C A B D E F I (\ E=- /\ u, .,..., ....., ,...... I -o.'2s ��. T.75 0 . �f SF II SPOT B B C

I I 0.25 0.75 0 I; SF Ill SPOT C A B C t � E u=- L - --- 0 If SF I-' 104

Table 6. Rf values f'rom dopa.mine metabolites when incubated with cuticle proteins. I. Rf values of' dopa.mine metabolites. II. Rf values of' dopa.mine metabolites when incubated with soluble proteins. III. Rf values of' dopa.mine metabolites when incubated with whole homogenate. 105

I. soluble Eroteins whole homogenate SEOt Rf Max CEm -1!L. Max cpm A 0.166 180 0.173 150 B 0.237 410 0,246 470 C 0.339 650 0.343 590 D 0.438 250 0.452 230 E 0.576 130 0.662 120 F 0,801 160 0,804 230

II. starting material starting material spot sEot from above· Rf spot SEOt from above Rf A B 0.287 A B 0.042 B B 0.263 C B o.424

III. ------A C 0.396 A _C 0.055 B C 0,415 C C 0.846 106

Table 7, Rf' s of possible dopamine metabolites from various literature sources. 107

Metabolite _Rf_ Source N-acetyldopamine-J-0-phosphate 0. 28 Bodnaryk et al. , 1974a N-acetyldopamine-J-0-sulphate O. 37 Bodnaryk et al. , 1974a Dopamine-J-0-sulphate 0.2 Bodnaryk and Brunet, 1974 N-acetylhydroxytyramine-glucoside 0.5 Okubo, 1958 N-acetyldopamine 0,8 Okubo, 1958 Dopamine o.4 Okubo, 1958 Dopa 0,2 Sekeris, 1964 Phosphate (monosodium) 0.18 Florence, unpublished . 108

In vitro Binding of N-actyldopamine to Lysyl-lysine

The first experiments involved the in vitro incubation of

N-actyldopamine, lysyl-lysine and untanned cuticle homogenate. The column chromatograph elution profile showing UV absorption and ninhydrin activity in each f'raction is found in Figures 29 and 30,

14e-N-acetyldopamine was added to the mixture to aid the elucidation of where the N-acetyldopamine metabolites were eluted of'£. Graphs of this column eluant are found in Figures 31 and 32. Radioactive incorporation, UV absorption as well as ninhydrin reactivity were monitored and are shown in these figures. One readily notes that there are two peaks which are ninhydrin reactive, UV positive and also have some radioactivity found within th peak. These peaks correspond to N-acetyldopamine bound lysyl-lysine.

Electropherograms and chromatograms were obtained on concentrated portions of fractions 27-33, f'ractions Y-l-37, and f'ractions 4.5-49,

These are represented in Figures 33 and 34, Nbte that £raction "D"

(fractions 4.5-49), corresponds to N-acetyldopamine. Both fractions

"B" (fractions 27-33) and "C" (fractions Jl.!-37) are undoubtedly

N-acetyldopamine metabolites and are possibly N-acetyldopamine-lysyl lysine metabolites, Fraction "A" (fractions ll.!-18) is probably N-acetyldopamine bound to protein or a polymer of' N-acetyldopamine and lysyl-lysine. In vitro·incubations were carried out on lysyl-lysine and 109

Figure 29. Elution profile of an aliquot from the incubation of non-labelled N-acetyldopamine, lysyl-lysine and cuticle homogenate. Dotted lines represent absorbance at 280 nm while continuous lines represent relative reaction with ninhyd.rin. Note that samples 25-27 were pink in color indicating quinone formation. r\n / II I 4� ,� I I \ � .c � g� 'ii= I � 2 t [ I ,J I r, I J � '- jI ' l l:::::.. ,,., -.. 15 30 45 60 fraction ...... 0 111

Figure JO. This graph is an elution profile obtained when the ·

previous experiment was duplicated. 11£

osz00

I I . ,,,� ______,.,,. ,-- ---·- -:-- ---_;-·-,-·------�-- -- ·- I I ftt I c:> I ""' \

," \ ' .... � 113

Figure 31. Elution p:rofile of aliquot f:rom the incubation of 14 c-N:acetyldopamine, lysyl-lysine and cuticle· homogenate. Aliquot was placed on a P-10 polyacryiamide gel (200-400 mesh) 0.6 x 60 cm. and 0.5 ml fractions were collected. Wavy line represents absorbance at 280 nm while straight line represents relative reaction with ninhydrin. Note that samples 26 and 27 were pink in color, a color that is characteristic of quinone. 114

osz ... 00

C c:> � -ca- 115

Figure 32, Elution profile of an aliquot from the incubation of 14 e-N-acety1.dopamine, lysyl-lysine and cuticle homogenate, An aliquote was place on a P-10-polyacrylamide gel (200- 400 mesh) 0,6 x 60 cm. and 0,5 ml fractions were collected.· Dotted line represents absorbance at 280 nm while continuous line represents cpm in each sample. 116

osz00

, I

CD (------/ = ....- - - = ' ' _ c.-:______/ "I '\ I I

zOI x wd3 117

Figure 33. Representation of radiochromatograms obtained from various fractions of eluant of column chromatography of labelled N-acetyldopamine, lysyl-lysine and cuticle homogenate after being incubated for 3 hours at ° 37 c. I. Radiochromatogram of fraction B (fractions 27-33 of

figure 31). II. Radiochromatogram of fraction C (fractions .34-37 of

figure 31). III. Radio chromatogram of fraction D (fractions 45-49

of figure 31) . Note the presence of a relatively high radioacti_ve peak which is UV positive in fraction D. This is N-acetyldopamine. ,,

SF II � l.o- - -l 0 Ill

N-acetyldopamine

...... 0 SF (X) 119

Figure 34. Representations of electropherogra.ms obtained from various fractions of eluant of column chromatography of labelled N-acetyldopamine, lysyl-lysine and cuticle ° homogenate af'ter being incubated at J7 C for 3 hours. I • El.ectropherogra.m of fraction B (fractions 27-33 of figure 32), II • El.ectropherogram of fraction C (fractions 34-37 of figure 32). III • El.ectropherogram of fraction D (fractions 45-49 of figure 32). Note the presence of a relatively high radioactive peak which is UV positive in fraction D.,. This :is N-acetyldopamine. . FRACTION B + � I- * � •b+ j G O R FRACTION C

e + ft G 0 R FRACTION D

UY + e ft C 0 R 121

N-acetyldopamine without cuticle homogenate and analyzed as before.

Elution profile from column chromatography is found in Figure 35.

Chromatographs and electropherograms of the incubant are diagrammed in Figure 36, One should note the similarity of these analyses with the analyses which were made utilyzing cuticle homogenate.

Oxidation of this mixture occurred at room temperature and the total product was analyzed spectrophotometrically at the visible and UV range. This was compared to the oxidation product of an equimolar amount of N-acetyldopamine. The spectra are shown in Figure

37. Note that there was a large increase in the absorptivity in the visible light region from 600 run to 370 nm and in the UV region from

370 nm to 296 nm when compared to the oxidation product of N-acetyl dopamine alone. This suggests that the binding of the di peptide to the N-acetyldopamine also adds to the color of the product.

Two portions of the fractions from the column (fractions 19-26 and fractions 34-41) were concentrated to dryness, The remaining precipitate was disolved in dimethylsulphoxide and analyzed on NMR.

The resulting NMR spectra are found in Figure 38. These NMR spectra can be compared to the NMR's of the reactants which are found in

Figure 39. Additional NMR' s of possible metabolites of the sclerotization agent as well as the other dipeptides are found in Figure 40. 122

Figure 35. Graph of eluant from polyacrylamide P-2 gel column of N-acetyldopamine and lysyl-lysine metabolites. Fractions 18-26 designated "A" were grouped and analyzed by NMR (see Figure 38). Fractions Y+--41 designated "B" were grouped and analyzed by NMR (See Figure 38). Ibtted line denotes OD at 280 nm while continuous line denotes ninhydrin positive fractions. I' I \ I I 4 I I I I : I I = I � 't: I I N -=>,, Cl -= I I c:, = I I 'E 2 I \

(\ ( I I ' 1_,�,' \' - / ...... 15 30 45 60 A • fraction6 r \. 124

Figure 36. Chroma.tographs a.nd elect:rophe:rogra.ms of incuba.nt of

N-acetyldopa.mine a.nd-lysyl-lysine, Spots are indicated

by either Pauley' s reagent, ninhydrin, · or UV. Note the

spots which are both UV a.bsorptive a.nd ninhydrin positive. pauley's

.. • UV • 14 ninhydrin • 0.25 0.5 0.75 ;, 0 Rf SF J.: .. , · pauley's • •I 7 :: a • 1e '" UV - ••• • •I' Al . ninhydrin ,, ., '5 1, ••• • • ••. I • I

I\).... G 0 R V, 126

Figure 37. I. Representative UV spectra of' the oxidized product

of' N-acetyldopamine.

II. Representative UV spectra of' the oxidized product

of' N-acetyldopamine and lysyl-lysine. F.quimolar

quantities of' N-acetyldopamine were used in each

experiment. Note the increase of' absorption in the

visible light region f'rom 600 run to 370 run and in

the UV region f'rom 370 run to 296 run. 127

- - - -E ·E= = -� -J:: -ta,) -ta,) =Cl) �= -Cl) Cl) "'> "'> 31: 31:

=- ...-0

..-0 --�: 00 00 128

Figure 38. NMR of :fractions A and B f'rom N-a.cetyldopamine lysyl-lysine binding,

N N

� �

_ _

flt flt

'>H• '>H•

i; i;

q q

,1 ,1

C C

i., i.,

� �

1.0 1.0

______

T/\\S T/\\S

c..-

IUII IUII

?10 ?10

0 0

I I

2.0 2.0

:O��':.o :O��':.o

REMARKS: REMARKS:

b b

• •

3.0 3.0

/!,/s,J(�,.,,t..u /!,/s,J(�,.,,t..u

.� .�

:,1111 :,1111

� v-•'\....__ �

.• , t ,

.P .P

NT: NT: /'\.,

uv,�-

,:SP

,.o ,.o

SAMPLE: f,

SOLV

2) 2)

OPERATOR OPERATOR

PPM(I) PPM(I)

(.05) (.05)

( (

(250) (250)

(500) (500)

(/) (/)

AUTO

50 50

:ton :ton

;.v' ;.v'

.. ..

�_ �_

0 03 03 0

� �

7/,-2/)t.

80 80

{HzJ· {HzJ· u It1

(SEC): (SEC):

�"iTiTiTi) �"iTiTiTi)

MANUAL MANUAL

LEVEL: LEVEL:

__ __

TIME TIME

WIDTH WIDTH

DATE: DATE:

POWER POWER

,oo ,oo

RF RF SWEEP SWEEP

FILTER: FILTER:

SWEEP SWEEP

60T) 60T)

7.0 7.0

(TPV (TPV

..!..O ..!..O

=1,

.J:!c.,. .J:!c.,.

-1)_ -1)_

.,.,,,...... ,..,,,,.. .,.,,,...... ,..,,,,..

. .

60T) 60T)

(RPS): (RPS):

(Hz): (Hz):

AMPLITUDE: AMPLITUDE:

(WCV (WCV

RATE RATE

AMPLITUDE: AMPLITUDE:

8.0 8.0

OFFSET OFFSET

500 500

,.:.._,., ,.:.._,.,

WILMAO WILMAO

THOMPSON THOMPSON PACKARD

SPINNING SPINNING

INTEGRAL INTEGRAL

SPECTRUM SPECTRUM SWEEP SWEEP

0 0

I-' I-'

w w

t t

, ,

,.,. ,.,.

., .,

"-'/

1="\ 1="\

I I

l l

e,;,

-(!JI� -(!JI�

. .

I I

'/ '/

1 1

I}_ I}_

-· -·

q q

b b

C C

d d

,.o ,.o

s s

� �

NO. ______NO.

-r

c..

I I i i

I I

,oa ,oa

NMR NMR

':1 ':1

�" �"

t, t,

1 1

2.0 2.0

SPECTRUM SPECTRUM

IJOMH, IJOMH,

rd rd

�-�� -��

C. C.

REMARKS: REMARKS:

,d ,d

� �

::i ::i

c c t

,\e ,\e

I I

:;,.l

3.0 3.0

,... ,...

CM�

I I

/ /

I, I,

�

1' 1'

.5.:;

. .

_ _

<. <.

.,( �.,,...,.... .,(

·5 ·5

f)I f)I

A F F A

T: T:

.:r .:r

1 1

"1{JP "1{JP

40 40

SAMPI.E:

SOLVEN

OPEAATOR OPEAATOR

06) 06)

PPMIII PPMIII

I I 21

mo1 mo1

I I

AUTOO

I I

i i

I I

,. ,.

60 60

! !

W W

11 11

,1,) ,1,)

/? /?

'i 'i

•

)

� � (0001 1

�

7/,� 7/,�

>)•

-�--- I

)

•

60 60

I I

IH.t)· IH.t)·

l&J

MANUAL MANUAL

LEVEL: LEVEL:

. .

· ·

WIDTH WIDTH

DATE: DATE:

POWER POWER

· ·

I I

i i

RF RF

SWEEPTIME(SEC):

FILTER: FILTER:

SWEEP

0 0

· ·

_l----

60T) 60T)

(TPV (TPV

� �

0 0

60T) 60T)

(Hi):.

PACKARD PACKARD

AMPLITUDE:

(WCV (WCV

80 80

OFFSET OFFSET

I I

THOMPSON THOMPSON

WILMAO WILMAO

SWEEP SWEEP

��:� :;t��:� :;t��:� ��:� SPECTRUM SPECTRUM 131

e . NMR spectra o:f N-acetyldopamine in n o . Figur 39 2 NMR spectra o:f N-acetyldopamine in dimethylsulphoxide.

NMR spectra o:f lysyl-lysine in Dz°. 500 ,oo Hi I ···:'?>{•" I >H• ·: :100. · :·:1 . ·:- �t) ·: ;;,;i�. :,[,�, '[.·:1::·!g:f,:? ?f ]j;(''!If :···' 'T '::>...,.,.P.·· �.,..,. 2h!- lI ft .. . ,:.,. . '...... · .. :�:�::IS'w""'1' .Jt'1.:l!r/J 1 r,,.,' ! . �-l -�i.L bl11f1 }">· .. �\ ' ·. . ! ., . . . : F� ' ·;c ::·:· .\ . i t:1 . · , · ! i i �·,' j' :::_· � Ir I ..:: ··i.,'· :\:r_:_\r.

'.j r' ·,I e.o 5.0 Pl'M l 6 I 4 0 3.0 2.0 1.0 ,J-Au.½IJof"'...,..;�,_ MANUAL AUTOO SAMPLE· REMARKS: c..._C-,4.,,.,,,.lc.M.5 " II ort�: 3 SWEEP TIME (SEC): (33 l SWEEP OFFSET (Ht): -�- . (25�1 - � , : t , I?.. U /J �f-.,1 J SPECTRUM AMPUTUOE: .ur SWEEPWIDT�� ,500) o C. ,z. : • l TEEE) I d INTEGRAL AMPLITUDE: .5. FILTER: • > • l 21 61\ 0: .. /¥ I 3 ]} {),.-12_ ... I SPINNING RATE (RPS): .!i!J__ RF POWERLEVEi.: 6 o3 l .051 SOLVENT: �0 + f) .;, � ·1,.J e. t 90MHzNMR DATE: :JvJ,F/,/- 1 l�1t OPERATORI TA,Fu,1'E'IJCI' SPECTRUM NO. ______! THOMPSON PACKARD (TPV 60T) i WILMAD (WCV 60T) �-_..,_,_.,_cc._..__.._.....•�·�· -·•�-c.=..��=--�-� ····•- ··�···- ��-· ,, ..... \..,.) N

>H• >H•

0 0

0 0 Hz

� �

-� -�

,-g ,-g

b b �

Li! .:!

12., q

C C 'l

cl cl

1.0 1.0

" "

3 3

�

:-��-

l-1

�

I I

100 100

t�TMS t�TMS

I I

2.0 2.0

��'!.o. ______��'!.o.

l.J

REMARKS: REMARKS:

d d

<,,' -�·"-'-- <,,'

\ .. .. \

-=-°'<'l -=-°'<'l

3.0 3.0

( (

1 1

C. C.

200 200

�l-l

F F

N/l.'":>;. N/l.'":>;.

,...., ,1-{..

fl fl

-S -S

Of'ERATOR Of'ERATOR

300 300

'f/26 'f/26

, ,

7p 7p

b b

DATE: DATE:

I I

\11 \11

I I

' '

400 400

60T) 60T)

7.0 7.0

(TPV (TPV

J,__ J,__

.JiQ. .JiQ.

_2.2., _2.2.,

....£1..._ ....£1..._

60T) 60T)

(RPS): (RPS):

(Hz\: (Hz\:

AMPLITUDE: AMPLITUDE:

(WCV (WCV

RATE RATE

AMPLITUDE: AMPLITUDE:

8.0 8.0

OFFSET OFFSET

WILMAO WILMAO

THOMPSON THOMPSON PACKARD

SPINNING SPINNING

INTEGRAL INTEGRAL

SPECTRUM SPECTRUM SWEEP SWEEP r

500 400 300 200 100 OH,

' l)H• ' _,', " , ,,,.- - :,;: I 'i - Ii ! I,--_:._ ___:--r-·-· . ---:-r. I - ! I I . - .. - .

I I I i I \ 1· c.___ • ____ : --·-·J ·-· ---t-----�I : - ·-·-··-···-�: ----�---·- ---·--:·····------· --,· .i . - , - . -- ! -. :, . .. --. - I • ! �.fiI �i.',,fWf�tNt�·,.,' ,�J .

8.0 7.0 60 50 PPMl&I 40 30 2.0 1.0 0 "'"'1.�M·'·N1Ctj,tCOOW � SWEEP OFFSET (Hz): SWEEP TIME (SEC): � 12SO) ,0.11>�-,/.J .. ,.....,.. "' �... .. b ·-·°-- O f�"\I �,s,nc. f SPECTRUM AMPLITUDE: Jlj_ SWEEP WIDTHMANUAL !HtJ· � AUT 1500) SAMPLE REMARKS: �:: i: C INTEGRALAMPLITUDE: _s_ FILTER: (D�.J�l�lil£1!..J I I 2)DI �11, �.. 0t)t)!t cl O 2."/, ...... 90MHz NMR SPINNING RATE (RPS}: _.�_I!_ RF POWER LEVEL: �-- ( OSI SOlVENT -p� .. DATE: f'.\11'!71. THOMPSON PACKARD (TPV 60T) :r,. .... WILMAD (WCV 60T) OPERATOR J. A."f" ...... _,,1<« SPECTRUM NO. ______

I-' 't 1.35

Figure 40. NMR spectra of possible metabolites of the scle:rotization agent and NMR spectra of dipeptides. ,... ,-..,...... ,...... -,--,--,--,--,-.,...... ,...... ,...... ,...... ,...... ,_,-.-...... __...... ,...... ,...... ,...... ,...... ,...... ,...... "'T""""'T""""'T""""'T""""'T""""'T"""T- ··1u:::1�['ri'illil!i "liiil'i·. I : '.iI iii i{'i " :Ii J�\ 1 J:!J T · · : ... ! F' .; 1 ; t I :� ·. :. . I ••• i .....i - '.'�- I . 1 �. .I : : . .. .·,. .... i .... j ... � .. l .. J i ··: ; . i ET] , . . ... :::.-! ... .I .. �-!��:_"! ..... I • ' • ·- .. : -� : �. \ ·- . · ..:1 .1.11.t'...�.: . ... ·. ··•• .r ��"! , �·'fYY"�� .., I ...... ' . I . ', .... ,. \ .(···; 8.0 6.0 3.0 I MANUAL 7.0 5.0 PPM ( I I 4.0 'I \ i (2SOJ AUTO(SOO) O SAMPLE /J•,s<1"il -f' _,;.,. REMARKS: 0 11 �™ SWEEP OFFSET (Hz): _0_ SWEEP TIME tSEC): I• I •I, I• I I ( 2) t 1�.00 ... /...Ju,...., I, 'i ( 1 SPECTRUM AMPLITUOE: .!.!.'!tE SWEEP WIDTH Hzf D,:,.o .. 2 % 1>t>.s C.I It INTEGRA.LAMPLITUD E: L FILTER: (ijit' ) 11 SOMHzNMR SPINNING RATE (RPS): --le_ RF POWER LEVEL, �.:i.__ ( 05) SOI.VENT. THOMPSOl'IPACKARO (TPV 60T) 1 J'tll, 0. WILMAO (WC� bOT) OATE: :J"'\!lll.- ,t OPERATOR -:J. . $" C..•&C; !'Ec::R SPECTRUM NO. ______I-----

I-' \..,J 300 �·oo !ll)O "'"' ' ,uu "' I I I >H• \' ,a I\ 1.:\1' t ' ;110 ·.,,· ·Ii' ·" .,1 .,. I I ' I I ., .•·, -:;,.,UI" Ot',:s,;r ZO+J i Sw,J.t,1-\ ..::t.so i ; I Fi!-;/._ ! I .: ...... : ···:·· . .. -·--· � . s-u r, .... So·-- I . . h... L. ____ .._ ..... _,_,,· I ·- .··------. --�--·-·-· - \ . ! : __ : I I I __V?i .

i I� iI I . : . . i J/? (.. I ·---!-... i ·-·········. ·--- ·- . -·i··--·------··- . . I ·,·--,· ----...... •.,. . ..· ' I I i l I · I l�W•/,#,\�MJ ·�1�Mll,��1W� ·. i��),fl��l\11<1/!Ml ' I M . . .. ! I I ! i

8.0 7.0 eo 5.0 PPMl&I 4.0 30 2.0 1.0 AUTO SAMPLE REMARKS: c ...... MANUAL D �-�«,..,,,',,, � a. ,,, : .s $WE,t_p OFFSET !Hz). -� SWEEP TIME (SEC): (250) fJ7,1,_,4i.-k ,I, t>/ : ... SPEC"l�UM AMPLITUDE: .U� SWEEP WIDTH (Hz\. � (500} /3,1!i-,t-' ...... INTEG"'l AMPLITU ETIIillD] I C 5: I DE· ._?._ FILTER: QJ?I.! I 2) �- SPINNl�tlRATE (RPS): ..i:!- RF POWER LEVEL: .-£.i.2.J.___ ( 051 SOLVENT. J>.,, o + ..l% J)J)S 90MHz NMR f CATE, _:2;:;n� 1,/, OPERATORI :r:A. <-otU.Jc• SPECTRUM NO. ______THOMPSON •�CKARO (TPV 60T) 1 111(, WILMAD (WCV bOT) I'

wj,,,>, -..J ,,...... ,...... ,.--,-,--,---,---,-,--,--,--,-,--,-..,...-,-,r-' I ' 5'r1'. i 4f · . 3'j'O Hz >H• i !, :i ,,!;f /� .. i · Lllj�ilij�i·f j:/illf�i·f l�@ii;2,�•i 1iiil ::i;:f� r!:;ffli\ ]/f f .J.liht:·f ..

ii.. i .fti�I • i !· lj ; , .•. ! i

8.0 1.0 8.0 5.0 PPMIII 40 3.0 2.0 1.0 AUTO SAMPI.E e"•"" MANUAL O T.,,..,,.,;,.,_ . REMARKS: • ... z.J>:4 SWEEP C:it=FSET (Hz): O SWEEP TIME !SEC): � (250) /1 .f,..,,�/.r,.lc I, zo :'I SPECT 7 RUM A.MPLITUOE SWEEPWIOTH (Hz)· (500) . s.r....,J. : .@ '™ 11.•11. � /.J Q C. q', INTEGRAL •M>U FILTER: I 2) TUOE: 2- 0!'.'.U.l!lil.iEW ( SPINNING "''• o + ..2"!. ::PD5 !RPS): _f!!!"_?" RF POWER LEVEL: �...1._ ( 051 SOLVENT :P,). e0MHzNMR DATE::l'""v,.,.,- I./,{"f?J. OPERATOI R ..Z:A. £.o2£Af�.,C SPECTRUM NO. ______THOMPSON PAt""RO (TPV 60T) WILMAO (WCV 61n\

...... l..,J 0) �

\()

I..,)

·o

� cl

•••

,.o

______

....

.....

�·- �··

<; .. t•"N�NCOOt4 .. <;

NO.

fr,llf

NMR

2.0

SPECTRUM

80MHz

REMARKS:

3.0

• .l3/. DD-'

""3/..J••'•-"

"°

"·

/1•,'""•�,;_._

4.0

SAM=

SOlVENT

)

OPERATOR�

fi •

(500) I 21 (0$)

125

1� 1.

·i-"or

I• I I•

('!11,

I•

i't,

� !t•l'°l'!*?'?l*'f

e,.,,,

I•

IHz

.,!\�l;:rt

:G,,,

0.,

MANUAL

WIDTH

DATE:

. i ! -� ! I, ji�j:;!';11[u�jrn;t

,:.-.

POWER LEVEL:

SWEEPTIME(SECJ

FILTER:

SWEEP

)

it;

i<"

60T

1 20

1:/:-1

1.0

�

(TPV

I! . I!

•

)

...Q__

60T

(Hzl:

. ;:-:

.:.j� --:;) ... --:;) .:.j�

PACKARD

AMPLITUDE: �

___ -! __ .'._ ___ ,_·_] ,_·_] ___ .'._ __ -! ___

(WCV

RATE {RPS): ...±.A, RATE {RPS):

�--�1· ;;1 ·: ;;1 �--�1·

'""

OFFSET

:�::,r

�-,�-i-;'

25 '

1 j

WJLMAD

THOMPSON

SPINNING

SPECTRUM INTEGRAL AMPLITUDE: 2_

SWEEP

_ffi_i

�j�-�-t

I 500 ,oo 300 200 100 Hz ! I .>·,r ' ' i I H• -�.,' "' -IV I .•,·,

1· Ii I I ····---·· ----__ J i I i i . . : I �! ·.. .,· . .. I i ' .· i I . 11,--....,.+-/ ··-·------�----·--····- ...... ! ·-···· l I -- :_ -- -�-i-�c------! .,,,,'.i,111ui.hJ, ,, j . ;: !, J�l IL ,.MIit !j . 11IJ1 f)_ !, ,J\M.,,, ,,,._,,,,1! b1 • ,J/1 �, Iii r'l•il/Pll\1 •,yn' ¥ �llt"''f'f'fi''/rf'�ivrt�I ,lr',V1\l r,\'1¥(�',\'f\!�r ��fi;1f,rr I ! �� i�o�\1 r ��-;\' I . . ! ...... I . . . .· .

80 7.0 80 SO PPM(61 40 30. 2.0 1.0 c., "r. MANUAL AUTO SAMPLE 1'1"!:I'::l/'j REMARKS: SWEEP OFFSET :Hz): ___9.__ SWEEP TIME (SEC): � (2501 ld.01. ""'� /1...0: ..,-� ""•�"t.,.i�C4o� �,...L .. SPECTRUMINTEGRAL AMPLITUDE: AMPLITUDE: .,;;;�_o$.. _ SWEEPFILTER: WIDTH[i] (Hz1·f , J��•I,,.,, I•I (SOO)I 2)DI <•, <•1 . SPINNING RATE JRPSI: � RF POWERLEVEL: d> • 03 I C OSI SOlVtNT i::,.:>o,. :i. �'J:,.p.S...... :;TA· 'Fu> IZcN <:le 80MH1NMR DATE, t+ '/71, OPERATOR SPECTRUM NO. ______THOMPSON PACKARD (TPV 60T) P_,. .1 WILMAD (WCV 60T)

I-' g ��_,.....,rut,..--,,...... ,.-,--,--,.--,--,--,,...... ,.-,--,-....--,--,--,e--"T"".. I r,

100 OH, ""' ""' 3'?° ""' ' ...

..II' , I I I ; : i . I . j''• - I, --···· ...... · ·• ... \ : _: .:·-·· i ___: -- L 1 · . I -�---I, __ . I : I I I . : I

, I : I. , . . . :' ; . , l• · I ! · · ·· \.I · . i. . , _, · . • I ·. ; . : .: .: : .

I : ,I . ! .. i

80 70 1.0 0 ··�·::�·".- .. M"-.�� Hl\�WCOON .1 t. ( 60 � 5.0 PPM ( 6) 4.0 '.[�· . •.· :r:-. 30 2.0 CM.., SWEEP OFFSET Hz!: ·--�- SWEEPJIME!SECl• 12SOI � /l«�./).5, SPINNING RATE (RPS), _!,Le_ RF POWER LEVEL. ,L,12..i,__ I .OSI ' SOLVENT: 2 �...... &"., e0MH1NMR DATE: 14,{!'ft SPECTRUM NO. ______THOMPSON PACKARD (TPV bOT) :J:.�,, oPERAroR :re F"""11.ti1Jc.# W!LMAD (WCV bOT)

� ....+' DISCUSSION

Ph:>sphate incorporation into proteins a.nd cuticle

There has been little investigation into the phosphate role in scle:rotization, Bod.naryk et al. (1974a) indicated that there was an N-acetyldopamine-J-0-phosphate moiety found in the haemolymph of post ecdysial white nymphs of Peripla.neta america.na. No ·such metabolite was found in our investigations as is evidenced by comparing Rf values of known N-acetyldopamine-J-0-phosphate with the peaks of radioactivity obtained with labelled phosphate. Tyramine phosphates have also been isolated in Drosophila (Hodgetts and

Konopka.•. J-973), a�d have been implicated as possible storage agents of precursors to the sclerotization agents, however, there was no indication of the presence of tyrosine phosphate in our experiments.

Phosphate incorporation into proteins has been widely investi gated (Taborsky, 1974). There is indication of the existence of phosphate cross-links involving hydroxyl groups of complex polysaccharides and amino acid side chains. In Cladosporium werneckii, a diester bridge is :proposed to provide branches in complex polysaccharide chains which are glycosidically bound to serine a.nd threonine side chains of the phosphoglycoprotein (Lloyd, 1972). This may well apply to the chitin (glucosamine) linkages to cuticular proteins. There is evidence for the existence of seryl and U1.ceonyl-glycopeptides in association to oligosaccharides in the 143 sclerotized cuticle of Periplaneta americana (Lipke, 1971), The presence of phosphate-mediated cross-linkages in hard tissue collagens has been shown in bovin dentine collagen (Veis and Schluet, 1963).

The binding of chitin and proteins of the cuticle may well take place with phosphate bonds.

The fact that a major precentage of phosphate is not incorporated into the cuticle does not hinder this theory. The reactions occuring during the period of sclerotization which involve phosphate are many.

Indeed, all of the energy reactions which occur in intermediary metabolism require the use of phosphate as nucleotide tripoosphates.

The production of chitin is well documented, This pathway involves the use of phosphate as part of intermediary metabolites

N-acetylglucosamine-phosphate (Benson and Friedman, 1970). It is hypothesized that this metabolite may well be shunted off to another pathway involving the binding to a seryl or threonyl groups of cuticular proteins.

The fact that there was a greater tendency for phosphoproteins to be incorporated into the cuticle rather than monosodium phosphate is significant. Phosphate may be an important cross-link in the structural matrix of the .cuticlB of insects, 144

Dopamine Metabolites Binding to Cuticle Proteins

l The identificati_on of aJ.l of the metabolites from the �C-dopamine

incubation is uncertain. However, the Rf vaJ.ues obtained are similar

to those found in the liberature for dopamine metabolites (see Table

6 and Table 7) • In examination of the dopamine metabolite chromato gram (Figure 28, I and Figure 29, I) .one can hypothesize the 14 identification of the peaks. Peak "A" is likely to be a c-dopamine metabolite bound to protein. Peak "B" is likely to be dopamine-J-O sulphate. Peak "C" is likely to be N-acetyldopamine-J-0-sulphate.

Peak "D" is likely to be dopamine. Peak "E" is unidentifiable while peak "F" is N-acetyldopamine.

B0th peak "B". .and peak "C" contained a great amount of radio activity. These peaks, dopamine-J-0-sulphate and N-acetyldopamine

J-0-sulphate, were incubated with enzyme, either soluble or structural.

The soluble enzyme fraction showed no influence on these peaks, however, the structural proteins changed the peaks greatly. The dopamine-J-O sulphate was shown to be incorporated into protein. This was indicated by an Rf value of O.042. Also action of a sulphatase was evidenced. This is concluded by the presence of a peak corresponding to dopamine (Rf=O.424). The remaining peak is undoubtedly the starting product. yielded similar results. The N-acetyldopamine-J-0-sulphate peak was indicated by the Rf value The binding of the metabolite to protein 145 of O .055 while the presence of sulphatase is indicated by a peak of

N-acetyldopamine (Rf 0.84).

N-acetyldopamine-3-0-sulphate and dopamine-3-0-sulphate ·have been postulated to be :possible storage agents of the scle:rotization agent

(Bodnaryk and Brunet, 1974a). The data presented. here support this contention. The presence of cuticle sulphatase has not been reported in the literature. However, its presence is obvious by the data presented here. 146

In vit:ro Binding of N-acetyldopamine to Lysyl-lysine

The binding of N-acetyldopamine to lysyl-lysine was accomplished

both with and without the use of cuticle enzyme, The oxidation of

N-acetyldopamine and its subsequent binding to lysyl-lysine occurs

readily.at :room temperature in the presence of oxygen :f':rom the

atmosphere, There are many possible binding sites of' the dipeptide to

N-acetyldopamine. Andersen suggests a side chain binding (Andersen,

1974a). See Figure 4. Evidence from NMR however, supports a binding

to the ring. The occum:nce of' the aromatic ring in fraction l in the NMR experiment is evidenced by the high optical density as

well as the incorporation of radioactive N-acetyldopamine. The

occurrence of' the lysyl-lysine is evidenced by the positive reaction

with the ninhydrin, The NMR of this fraction showed no aromatic

protons which suggests that all of the protons have been replaced

by other moieties. .This would follow the theory that binding occurs on the ring via a mechanism as outlined in Figure 9, with one

exception. This exception is that binding occurs at all locations rather than just the two ortho positions,

The occunmce of lysyl-lysine as a component of proteins has been taken as common knowledge, Lysine is known to be a component of high concentration in cuticle protein (Karlson et al,, 1969), ·Its occurrence as a part of naturally occurring low-molecular-weight peptides from the blowfly, Phormia regina, has been shown in the identification of glycyl-L-lysyl-1-lysine in young larvae (Bodnaryk and Levenbooki. 1963). The occurrence of N-acetyldopamine in insects is common knowledge as it is considered to be the major sclerotization agent, The binding of these two moieties via ring-amine interactions supports the proposed mechanism of binding of proteins to the proposed mechanism of binding of proteins to the sclerotization agent, REFERENCES 149

REFERENCES

Adiyodi, K.G. (1969) 3,�-Dihydroxy-mandelic acid in the mushroom-gland of cockroaches. Naturwissen schaften, .2,21373. Aerts, F. and Vercauteren, R. (1964) .Specificity and mode of action of phenoloxidase from larvae of Tenebrio molitor. Enzymologia, 2811-20. Aerts, F,, Vercauteren, R. and Decleir, W. (1960) Enzymes in the metabolism of phenolic acids in insects. Proc. 11th Intern. Congr, Entomol,, ]:

Alexander, P, and Barton, D.H.R. (1943) The excretion of ethylquinone by the flour beetle. Biochem, J., .:rz.1463-465, Ammon, H. and Karlson, P. (1964) Zurn Tyrosinstoffwechsel der Insekten - XIV. Radioautographische Lokalisation der Sklerotisierungssubstanz in Puppentonnchen von Calliphora erythrocephala. J. Insect Physiol., 101 525-527, Andersen, s.o. (1963) Characterization of a new type of cross-linkage in resilin, a rubber-like protein. Biochim, Biophys, Acta., .£2.:249-262, Andersen, s.o. (1966) Covalent cross-links in a 150

structural protein, resilin. Acta Physiol, Scand., 6611-81. Andersen, S.O. (1970) Isolation of arterenone (2-amino- 3', 4 '-dihydroxyacetophenone) from hydrolysates of sclerotized insect cuticle, J, Insect Physiol., 1611951-1959, Andersen, S.O. (1971) Phenolic compounds isolated from insect hard cuticle and the relationship to the sclerotization process, Insect Biochem., J.1157-170, Andersen, S.O. (1972) An enzyme from locust cuticle involved in the formation of crosslinks from N-acetyldopamine, J. Insect Physiol. 181527-540. Andersen, S.O. (1974a) Evidence for two mechanisms of sclerotization in insect cuticle. Nature, _gjl: 507-508. Ander s en • SO• • (1974b) Cuticular sclerotization in larval and adult locusts, Schistocerca gregaria. J. Insect Physiol., 20:1537-1552, -Andersen, S.O. and Barrett, F.lVI,- (1971) The isolation of ketocatechols from insect cuticle and their possible role in sclerotization, J. Insect Physiol., 11169-83, Atkinson, P.W., Brown, W.V., and Gilby, A.R. (1973) Phenolic compounds from insect cuticles identification of some lipid antioxidants. Insect

Biochem,, J:309-315, 151

Auclair, J.L. (1959) The influence of dietary amino acids ori-the bloe>d- amirro--ac±-a.s- --of-the ·German cockroach, Blattella germanica. J. Insect Physiol., ]1127-131, Axelrod, J. (1959) Metabolism of epinephrine and other sympathomimetic amines, Physiol.-Revs, 391751-776, Bade, M.L. (1974) Localization of moulting chitinase in insect cuticle, Biochimica et Biophysica Acta, 3721474-477, Bade, M.L. and Shoukimas, J.J. (1974) Neutral metal chelator-sensitive protease in insect moulting fluid. J, Insect Physiol., 201281-290, Baru, A.M. (1968) Catecholamines in maniac-depressive psychosis. Zh Nevropatol Psikhiat Im S S Korsakova, 6811358-1361. Beament, J.W.L. (1948) The role of wax layers in the waterproofing of insect cuticle and egg-shell. Discuss. Faraday Soc,,1,:177-182. Beament, J.W�L. (1951) Wax secretion in insects. Nature, 16z :652-653. Beament, J.W.L. (1955) Wax secretion in the cockroach, J. Exp. Biol., 32: 514-538, Beament, J.W.L. (1958) The effect of temperature on the waterproofing mechanism of an insect, J. Exp. Biol., 35:495-51 9 · Beament, J ..W L (1959)- The waterproofing mechanisms of arthropods, 1. The effect of temperature on 152

cuticular permeability in terrestrial insects and ticks. J, Exp. Biol., l&i391-422. Beament, J.W.L. (1960) Wetting properties of insect cuticle, Nature, 1861408-409, Beament, J.W.L. (1961) The water relations of insect cuticle. Biol, Rev. 361281-320. Beament, J.W.L. (1964) The active transport and passive movement of water in insects. Advs. Insect Physiol., g:67-129, Beament, J.W.L. (1965) The active transport of water, evidence, models and mechanisms. Syrop, Soc. Exp. Biol. , 1.2.:273-298, Beament, J.W.L., Nobel-Nesbitt, J, and Watson, J.A.L. (1964) The waterproofing mechanism of arthropods III. Cuticular permeability in the firebrat, Thermobia domestica (Packard) J. Exp. Biol., 41: 323-330. Bennet-Clark, H.C. (1963) The relation between epicuticular folding and the subsequent size of an insect. J. Insect Physiol., 9:43-46. Benson, R.L. and Friedman, S. (1970) Allosteric control of glucosamine phosphate isomerase from the adult housefly and its role in the synthesis of glucosamine-6-phosphate. J. Biol. Chem., 245:2219- 2228. Bernays, E.A. ( 1·972) The intermediate mault ( first ecdysis) of Schistocerca gregaria (Forskal). 153

z. Morphol. riere, .211160-179, Berridge, M.J. and Price, W.T. (1972) The role of cyclic AMP in the control of fluid secretion. Adv. Cyclic Nucleotide Res., 11137-147, Bjorklund, A., Cegrell, L., Falck, B., Mitzen, M. and Rosengren, E. (1970) Dopamine-containing cells in sympathetic ganglia. Acta Physiol. Scand. 781 334-338. Bodnaryk, R.P. (1970) Biosynthesis of gamma-L-glutaroyl L-phenylalanine by the larva of the housefly, Musca domestica. J. Insect Physiol., 16:919-929, Bodnaryk, R.P. (1971a) N-Terminal B-alanine in the puparium of the fly, Sarcophaga bullata, Evidence from kinetic studies of its release by partial acid hydrolysis. Insect Biochem., 1:181-227, Bodnaryk, R.P. (1971b) Studies on the incorporation of B-alanine into the puparium of the fly, Sarcophaga bullata. J. Insect Physiol., 17:1201- 1210. Bodnaryk, R.P. and Brunet, C.J. (1974) 3-0-Hydrosulphato- 4-hydroxyphenethylaroine (dopamine-J-0-sulphate), a metabolite involved in the sclerotization of insect cuticle. Biochem. J. U§.:46J-469, Bodnaryk, R.P. and Levenbook, L. (1963) Naturally occurring low-molecular-weight peptides from the blowfly Phormia regina. Biochem. J., 110:771-773, Bodnaryk, R.P. and McGirr, L. (1973) Purification, 1.54

properties and function of a unique )"-glutamyl cyclotransferase from the housefly, Musca domestica L. Biochimica et Biophysic� Acta, fil1352-362, Bodnaryk, R.P. and Skillings, J.R. (1971) t-Glutamyl transpeptidase catalyses the synthesis of t" -glutamylphenylalanine in the larva of the housefly Musca domestica. Insect Biochem., 1_1467, Bodnaryk, R.P., Brunet, P.C,J. and Koeppe, J.K. (1974a) On the metabolism of N-acetyldopamine in Periplaneta americana. J. Insect Physiol., 20:911-923, Bodnaryk, R.P., Bronskill, J.F. and Fetterly, J,R, (1974b) Membrane-bound t-glutamyl transpeptidase and its role in phenylalanine absorption-reabsorption in the larva of Musca domestica. J. Insect Physiol., 20:167-181, Brennan, J,J, and Beck, S.D. (1972) Activation of tyrosine hydroxylase in haemolymph from diapausing larvae-of the-European- corn borer, Ostrinia nubilalis, Insect Biochem,, �1451-459, Bruck, E. and Stockem, W. (1972a) Morphologische Untersuchungen an der Cuticula von Insekten. I. Die Feinstruktur der larvalen Cuticula von Blaberus trapezoideus Burm, z. Zellforsch, Mikrosk, Anat,, 1321403-416, Bruck, E. and stockem, W. (1972b) Morphologische Untersuchungen an der Cuticula von Insektem. II, Die Feinstruktur der larvalen Cuticula von 155

Periplaneta americana (L.) Z, Zellforsch. Mikrosk. Anat., 1321417-430, Brunet, P.C.J. (1963a) Tyrosine metabolism in insects. Ann, N.Y. Acad, Sci., 1.QQ:1020-1034, Brunet, P.C.J. (1963b) Synthesis of ari aromatic ring in insects, Nature (Lond) 199:492-493, Brunet, P.C.J, (1965) The metabolism of aromatic compounds, Aspects of Insect Biochemistry, 25:49-77, Brunet, P.C.J, (1967) Sclerotins. Endeavour, £Q_:68-74, Brunet, P.C.J. and Coles B.C. (1974) Tanned Silks. Proc. R. Soc. Lond, B., 1.§1.:133-170, Brunet, P.C.J. and Kent, P.W. (1955) Mechanism of sclerotin formation: the participation of a Beta-glucoside. Nature, 175:819-820, Butenandt, A,, Groschel, U., Karlson, P. and Zillig, W. (1959) Uber N-acetyltyramine seine isolierung aus Bombyx-puppen und seine chemischen und biologischen eigen schaften, Arch, Biochem, Biophys., 83:76-83. Butterworth, F.M. and Berendes, H.D. (1974) Ecdysone- binding proteins in haemolymph and tissues of Drosophila hydei larvae, J. Insect Physiol., 20:2195-2204. Campbell, F.L. (1929) The detection and estimation of insect chitin; and the relation of "chitinization" of to hardness and pigmentation of the cuticule the American cockroach P. americana. Ann. Ent. Soc. Am. �1401-440. Chadha, M.S., Eisner, T. and Meinwald, J. (1961) Defence mechani.sms of arthropods--IV. Para-benzoquinones in the secretion ofEleodes longicollis Lee. J. Insect Physiol. 1146-50. Chang, F. (1972) The effect of ligation on tanning in the larva of a tachinid parasite, Lespesia archippivora. J. Insect Physiol., 18:729-735, Chapman, R.F. (1975) -The- -Insects--- Structure------and Function---- AmericanElsevier Publishing Co., Inc. New York. Chen, T.T. and Hodgetts, R.B. (1974) The appearance of dopa decarboxylase activity in imaginal discs of Sarcophaga bullata undergoing development in vitro. Dev. Biol., 38:271-284. Cima, L. and Grigolo, A. (1968) Biosynthesis of melanin in Musca domestica L. 1 study of the activity of o-diphenol-oxidase in the different developmental stages. Redia, 51:251-257, Clarke, K.U. and Langley, P.A. (1963) Studies on the initiation of growth and moulting in Locusta migratoria migratorioides R. and F. IV. The relationship between the stomatogastric nervous system and neurosecretion. J. Insect Physiol . .2_: 423-430, · Congote, L.F., Sekeris, CE· , , Karlson, P. (1969) On the mechanism of hormone action. XIII. Stimulation 157

effects or-ecdysone, juvenile h -ormone and ions on on RNA-synthesi-s · in·fat-body- cell nuclei from Calliphora erythrocephala isolated by a filtration technique. Exp. Cell Res. , 56 1 338-346. Cottrell, C,B. (1962a) General observations on the imaginal ecdysis of blowflies, Trans. R. Ent. Soc. Lend., 114:317-333, Cottrell, C.B. (1962b) The imaginal ecdysis of blowflies. The control of cuticular hardening-and darkening. J, Exp. Biol.,J2:395-411. Cottrell, C.B. (1962c) The imaginal ecdysis of blowflies, Detection of the blood-borne darkening factor and determination of some of its properties. J. Exp,

Biol ., 39:413-430, Cottrell, C.B. (1962d) The imaginal ecdysis of blowflies. Observations on the hydrostatic mechanism involved in digging and expansion. J. Exp. Biol., 39:431- 448. Cottrell, C-.B, . ( 1962e) ·- · The 0 imaginal.-ecdysi-s-0L bLowflies. Evidence for a change in the mechanical properties of the cuticle at expansion. J. Exp, Biol. , 39: 449- 458, Cottrell, C.B. (1964) Insect ecdysis with particular emphasis on cuticular hardening and darkening. Advanc, Insect Physiol., 2_:175-281, Daly, J.W. and Witkop, B. (1963) Recent studies on the centrally active endogenous amines, Angew, 1.58

Chem. Interriat. �dit.··_g_:4·21-440. Delacharobre, J. (1971) Le tannage de la cuticule adulte de Tenebrio molitor: mise en �vidence d'une action hormonale induite par la region cephalique. J, Insect Physiol., 12:2481-2490, Delacharobre, J,, Provansal, A. and Grillot, J.P. (1972) Mise en evidence de la liberation d'un facteur de tannage assimilable a la bursicon par les organes perisympathiques chez Tenebrio molitor L. C.R. Acad. Sci. Paris, Ser., D., 275:2703-2706. Denneil, R. (1946) A study of an insect cuticle. Proc. Roy. Soc. B., 133:348-373, Dennell, R. (1947) A study of an insect cuticle: The formation of the puparium of Sarcophaga falculata. Proc. Roy. Soc, (Land,) B., .!...21=79-110, Dennell, R. (1958) The hardening of insect cuticle, Biol. Rev., 33:178-196, Dennell, R. and Malek, S,R.A, (1954) The cuticle of the cockroach, Periplaneta americana, I. The appearance and hl..stologit:al structure of the cuticle of the dorsal surface of the abdomen. Proc. R. Soc. B., 143:126-136. Dennell, R. and Malek, S.R.A. (1955a) The cuticle of the cockroach, Periplaneta americana, II. The epicuticle, Proc. R, Soc, B., 1431239-257, Dennell, R. and Malek, S.R.A, (1955b) The cuticle of the III, The cockroach, Periplaneta americana, 159

hardening of the cuticle: impregnation preparatory to phenolic tanning. Proc. R. Soc. B., 1431414-426, Dennell, R. and Malek, S.R.A. (1955c) The cuticle of the cockroach, Periplaneta americana, IV. The hardening of the cuticle: phenolic tanning. Proc. R. Soc, B., 143:427-434, Dennell, R, and Malek, S.R.A. (1956a) The cuticle of the cockroach, Periplaneta americana. V. The chemical resistance of the impregnating material of the cuticle, and the 'self-tanning' of its protein component. Proc. R. Soc. B., 144:545-556, Dennell, R. and Malek, S.R.A. (1956b) The cuticle of the cockroach, Periplaneta americana, VI. The composition of the cuticle as determined by quan titative analysis. Proc. R, Soc. B., 1451249-258, Dohke, K. (1973) Studies on prephenoloxidase-activating enzyme from cuticle of the silkworm Bombyx mori (Lep., Bomb.ycidae), II. Purification and characterization of the enzyme, Arch, Biochem. Biophys., 157:210-221, Dresse, A., Jeniaux, C, and Florkin, M. (1960) Contribu tion a la biochimie du ver a soie, XIV. Variations de concentration des catecholamines au cours de la mue nymphale, Arch, Intern. Physiol, Biochem., 68:196-202, D u t ton, G, J . d an Duncan, A.M. (1960) Mechanism of conjugation of a-amino-phenol in plants and an 160

insect. Biochem. J., 77:18. Evans, J.J.T. (1967) The-activation of prophenoloxidase during melanization of the pupal blood of the Chinese oak silkmoth, Antheraea pernyi. J. Insect Physiol., !.J:1699-1711. Evans, J.J.T. (1968a) The distribution of prophenoloxidase and its activator in pupae of the Chinese oak silkmoth, Antheraea pernyi. J. Insect Physiol., 14:107-119. Evans, J.J.T. (1968b) Natural substrates of the phenol oxidase in the pupal blood of the sillanoths, Antheraea pernyi and A. eucalypti. J. Insect Physiol., 14:277-291. Filshie, B.K. (1970) The fine structure and deposition of the larval cuticle of the sheep blowfly, Lucilia cuprina (Dipt., Calliphoridae). Tissue and Cell, l.:479-498. Firtel, R.A. and Saul, G.B. (1967) Characteristics of phenol oxidases in Mormoniella vitripennis (Walker) J. Insect Physi�l., U:1197-1206. Fogal, w. and Fraenkel, G. (1969) The role of bursicon in the melanization and endocuticle formation in the adult fleshfly Sarcophaga bullata. J. Insect Physiol., 15:1235-1247- Fox, F ..R d an M"lli s, R.R. (1971) Cuticle sclerotization by the American cockroach: Differential incorporation y of leucine and tyrosine during post-ecd sis. J. Insect Physiol., 17:2363-2374. Fox, F.R., Seed, J.R. and Mills, R.R. (1972) Cuticle sclerotization by the American cockroach: Immuno logical evidence for the incorporation of blood proteins into the cuticle. J, Insect Physiol., �: 2065-2070 .- Fraenkel, G. (1935) Observations and experiments on the blowfly (Calliphora erythrocephala) during the first day after emergence, Proc. Zool. Soc, (Lond.), §.Z:89J-904. Fraenkel, G. (1962) Tanning of the adult fly. A new hormonal action, Amer. Zool., �:524-547. Fraenkel, G. and Hsiao, C. (1962) Hormonal and nervous control of tanning in the fly, Science, l.J§:27-29, Fraenkel, G. and Hsiao, C. (1963) Tanning in the adult fly, A new function of neurosecretion by the brain, Science, 141:1057-1058, Fraenke�, G. and Hsiao, C. (1965) Bursicon, a hormone which mediates tanning of th cuticle in the adult fly and other insects, J, Insect Physiol., 11:513-556, Fraenkel, G. and Hsiao, C, (1967) Calcification, tanning, and the role of ecdyson in the formation of the puparium of the facefly, Musca autumnalis, J, Insect Physiol., 13:1387-1394, Fraenkel, G. and Rudall, K.M. (1940) A study of the physical and chemical properties of the insect cuticle, Proc. Roy. Soc. B., 129:1-35, · Fraenkel;· - G. and Rudall, K.M. (1947) The structure of insect cuticles. Proc. Roy. Soc. B., 134:111-143. Fraenkel, G., Hsiao, C, and Seligman, M. (1966) Properties of bursicon: An insect protein hormone that controls cuticular tanning. Science, 151:91-93, Fraenkel, G,, Zdarek, J. and Sivasubramanian, P. (1972) Hormonal factors in the CNS and haemolymph of pupariating fly larvae which accelerate puparium formation and tanning, Biol. Bull. Mar. Biol.Lab. Woods Hole, 143:127-139, Fragoulis, E.G. and Sekeris, C.E. (1975) Induction of dopa (3,4-dihydroxyphenylalanine) decarboxylase in blowfly integument by ecdysone. Biochem. J., 146: 121-126. Fuchs, M.S. and Schlaeger, D.A. (1973) The stimulation of dopa decarboxylase activity by ecdysone and its.enhancement by cyclic AMP in adult mosquitoes, Biochemical and Biophysical Research Communications, 54:784-789. "' Fukuda, s. (1940) Hormonal control of moulting and pupation in the silkworm, Proc. Imp, Acad. Japan, �:417-420. Fuzeau-Braesch, s. (1972) Pigments and color changes. A. Rev, Biochem., 17:403-424. Gilbert, L.I. and Schneiderman, H.A. (1961) Some biochemical aspects of insect metamorphosis. Am.

Zoologist., .!.11- : 51, 163

Gilmour, D. (1961) The Biochemistry of Insects, Acad. Press, New York. Gilmour, D. (1965) The Metabolism of Insects, W.H. Freeman and Co •. , San Francisco. Goldbard, G., Sauer, J.R., and Mills, R.R. (1970) Hormonal control of excretion in the American cockroach. II. Preliminary purification of a diuretic and antidiuretic hormone. Comp. and Gen. Pharm., 1:82-86. Gouin, F.J. (1970) Morphology, histology, and development in >Insecta and Myriapoda. VI. Cuticle; basal membrane; connective-, sheath-, and supporting-tissues. Fortschr. Zool., 20:300-317. Grillot, J.P., Delachambre, J. and Provansal, A. (1971) Les organes perisympathetiques de Tenebrio molitor. C.R. Acad .. Sci. Paris, Ser. D., 273:2599-2602. Hackman, R.H. (1953a) Chemistry of insect cuticle. I. The water-soluble proteins. Biochem. J., 54: 362-367. Hackman, R.H. (1953b) Chemistry of insect cuticle. II. The water-insoluble proteins of the cuticle of Aphrodius howitti Hope. Biochem. J . .2!:_:367-370. Hackman, R.H. (1953c) Chemistry of insect cuticle. III. Hardening and darkening of the cuticle. Biochem. J. 54:371-377• Hackman, R.H. (1959) Biochemistry of the insect cuticle. In: Levenbook, L., (ed.) Biochemistry 164

of Insects. Proc. IV Int. Congr. Biochem. Vienna, 12, London, Pergamon Press,48-72, Hackman, R.H. (1964) Chemistry of the insect cuticle. In: Rockstein, M. (ed.) Physiology of the Insecta, ]., Academic Press, London. Hackman, R.H. (1967) Melanin in an insect, Lucilia cuprina (Wied.), Nature, 216:163. Hackman, R.H. and Goldberg, M. (1958) Proteins of the larval cuticle of Agrianome spinicollis. J. Insect Physiol., Z 1221-231. Hackman, R. H. and Goldberg, M. (1967) The o-diphenol oxidases of fly larvae. J. Insect Physiol., 1J.: 531-544. Hackman, R.H. and Todd, A.R. (1953) Some observations on the reaction of catechol derivatives with amines and amino acids in presence of oxidizing agents. Biochem. J, 55:631-637, Hackman, R.H., Pryor, M.G.M. and Todd, A.R. (1948) The occurrence of phenolic substances in arthropods. Biochem. J. ,!U:474-477, Happ, G. M. (1968) Quinone and hydroxarbon production in the defensive glands of Eleodes longicollis and Tribolium castaneum. J, Insect Physiol., 14: 1821-1837, Henry, S.M. (1962) The significance of microorganisms in the nutrition of insects. Trans. N.Y. Acad. Sci., 24:676-683. Heyneman, R.A. and -vercauteren, R.E. (1968) -Evidence for-a lipid activator of prophenolase-in Tenebrio molitor. J. Insect Physiol., 14,409-415, Hikino, H. and Hikino, Y. (1970) Arthropod molting hormones. Fortschr, Chrm. Org. Naturst., 28: 256-321. Hodgetts, R,B. and Konopka, R.J. (1973) Tyrosine and catecholamine metabolism in wild-type Drosophila melanogaster and a mutant, ebony. J. Insect Physiol . .!2:1211-1220 . Hopkins, T.L., M urdock, L.L. and Wirtz, R.A. (1971) Tyrosine side-chain metabolism in larval and adult cockroaches, Periplaneta americana: post-ecdysial patterns. Insect Biochem., _1:97-101. Huber, R. and Hoppe,-W. (1965) Bestimmung des sterin skeletts und seiner orientierung mit diffuser rontgenstreuung in kristallen von ecdyson. Chem. Ber. 98:2353-2360. -Ikan,-cR,; Cohen,"-E. andShuiov, A, __ (1970)- Benzo- and hydroquinones in the defense secretions of Blaps sulcata and Blaps wiedemanni. J. Insect Physiol., 16:2201-2206. Ishaaya, I. (1972) Studies of the haemolymph and cuticular phenoloxidase in Spodoptera littoralis larvae. Insect Biochem,, �:1972, Iverson, L.L. (1970) Metabolism of catecholamines. In: Handbook of Neurochemistry, IV. Lajtha, A. (ed.) 166

197-220,

Jacob, F, and Monad, J, (1961) Genetic regulatory mechanisms in synthesis of proteins. J, Mol, Biol., ] 1318-365, Jenkin, P.M. and Hinton, H.E. (1966) Apolysis in arthropod moulting cycles. Nature, 2111871-, Karlson, P. (1956) Biochemical studies on insect hormones. Vitam. and Horm., 14:227-266, Karlson, P. (1960) Zum tyrosinstoffwechsel der insekten. V. Mitteilung, Reindarstellung, Kristallisation und substratspezifitat der phenoloxydase aus Calliphora erythrocephala. Hoppe-Seyler's, 326: 135-143. Karlson, P. (1965) Biochemical studies of ecdysone control of chromosomal activity. J. Cell, and Comp. Physiol,, 66:69-76, Karlson, P, and Ammon, H. (1963) Zum tyrosinstoffwechsel der insekten,. XI. . Biogenese .und .schisksal der acetylgruppe des N-acetyl-dopamins, Hoppe-Seyler's 330 :161-168. Karlson, p, and Herrlich, P, (1965) Zum tyrosinstoff wechsel der Insekten. XVI. Der Tyrosinstoffwechsel der Heuschrecke Schistocerca gregaria Forsk, ·J, Insect Physiol, , 11:79-.89, Karlson, p, and Liebau, H. (1961) Zum tyrosinstoff wechsel der Insekten, V. Reindarstellung, Kristallisation und Substratspezifitat der 167

0 -Diphenoloxydase aus Calliphora erythrocephala. Hbppe-Seyler's z. Physiol, Chem., 3261135-143, Karlson, P. and Sekeris, C.E. (1962a) N-acetyl-dopamine as sclerotizing agent of the insect cuticle. Nature (Land,), 1951183-184. Karlson, P. and Sekeris, C.E. (1962b) Zurn tyrosinstoff wechsel der insekten. IX. Kontrolle des tyrosin stoffwechsel durch ecdyson. Biochim Biophys. Acta., �:489-495, Karlson, P. and Sekeris, C.E. (1966a) Biochemical mechanisms of hormone action. Acta Endocrinologica. 53:505-518. Karlson, P. and Sekeris, C.E. (1966b) Ecdysone, an insect steroid hormone, and its mode of action. Rec, ·Prag. Hor. Res., 221473-499, Karlson, P. and Shaaya, E. (1964) Der Ecdysontiter wahrend der Insektenentwicklung. I. Eine Methode zur Bestimmung des Ecdysongehalts. J, Insect Physiol., 101797-804. Karlson, p,, Hoffmeister, H., Hummel, H., Hocks, P. and Spiteller, G. (1965) Reaktionen des ecdysonmolekuls. Chem. Ber. 98:2394-2402, Karlson, P., Mergenhagen, D. and Sekeris, C.E. (1964) xv. Zum tyrosinstoffwechsel der insekten, Weitere untersuchungen uber das o-diphenoloxydase system van C a 11. ip hora ervthrocephala Hoppe-Seyler's, 338:42--49, 168

Karlson, P., Sekeri, K.E., Richards, A.G. and Richards, P. A. (1969) The amino acid composition of the various types of cuticle of Limulus polyphemus, J, Insect Physiol., J.i,495-507, Karlson, P., Sekeris, C.E. and Sekeri, K.E. (1962) Zurn Tyrosinstoffwechsel der Insekten, VI. Identifizierung von N-Acetyl-3,4-dihydroxy-B phenathylamin (N�Acetyl-dopamin) als Tyrosinmetabolit. Z. Physiol. Chem., J..sZ,86-94, Katz, I.R., Yamauchi, T. and Kautman, S. (1976) Activation of tyrosine hydroxylase by polyanions and salts an electrostatic effect. Biochimica et Biophysica Acta. 429:84-95, Kawase, S. (1960) Tyrosinase in the silkworm during the pupation period. J, Insect Physiol., i_:335-340, Klemm, N. and Axelsson, S. (1973) Detection of dopamine, noradrenaline and 5-hydroxytryptamine in the cerebral ganglion of the desert locust, Schistocerca gregaria Forsk, Brain Res,, 57:289-298, Klemm, N., and Bjorklund, A. (1971) Identification of dopamine and noradrenaline in nervous structures of the insect brain, Brain Res., 26:459-464. Koeppe, J.K. and Gilbert, L.I. (1973) Immunochemical evidence for the transport of haemolymph protein into the cuticle of Manduca sexta. J. Insect Physiol,, 19,615-624. Ko eppe, J. K , and Gilbert, L.I. (1974) Metabolism and protein transport of a possible pupal cuticle tanning agent in Manduca �. J, Insect Physiol., 201981-992, Koeppe, J.K. and Mills, R.R. (1970) A possible new sclerotization agent in the cockroach cuticle. Biochem. J., ill:66. Koeppe, J.K. and Mills, R.R. (1972) Hormonal control of tanning by the American cockroach: Probable bursicon mediated translocation of protein-bound phenols. J. Insect Physiol., 18:465-469. Koeppe, J.K. and Mills, R.R. (1974) Possible Involvement of 3,4-dihydroxyphenylacetic acid as a sclerotization agent in the American cockroach. J. Insect Physiol., 20:1603-1609. Kopec, S. (1922) Studies on the necessity of the brain for the inception of insect metamorphosis. Biol. Bull. , 42 1 322-342. Kramer, S. and Wigglesworth, V.B. (1950) The outer layers of the cuticle in the cockroach Periplaneta americana and the function of the oenocytes. Q. Jl. Microsc. Sci., 91163-72. Krishnan, G. and Ravindranath, M.H. (1973) Phenoloxidase in the blood cells of millipedes, J. Insect Physiol., 19:647-654, Lake, C.R., M·11i s, R.R. and Brunet, P.C.J. (1970) B-Hydroxylation of tyramine by cockroach haemolymph. Biochimica et Biophysica Acta, 2151226-228. 170

Lake, C.R., Mills, R.R. and Koeppe, J.K. (1975) In vivo conversion of noradrenalin to 3-hydroxy-4-0-B-D glucosidobenzoic acid by the American cockroach. Insect Bio chem. , 2..1223-229. Laufler, H. (1961) Forms of enzymes in insect development. Anh, N.Y. Acad. Sci., 94:825-835, Levin, E.Y., Levenberg, B. and Kaufman, S. (1960) .The enzymatic conversion of 3,4-dihydroxyphenylethylamine to norepinephrine, J, Biol. Chem., 235:2080-2086. Lipke, H. (1971) Conjugates of glucosamine in cockroach cuticle. Insect Biochem., .l_:189, Lloyd, K.O. (1972) Biochemistry,11:3884. Locke, M. (1961) Pore canals and related structures in

insect cuticle. J. Biophys, Biochem. Cytol., 10:--r 589-618. Locke, M, (1964) The structure and formation of the integument in insects, The Physiology of Insecta (IVI. Rockstein,. ed.), 1=379-470, Academic Press, New York, Locke, IVI. (1966) The structure and formation of the cuticulin layer in the epicuticle of an insect, Calpodes ethlius (Lepidoptera, Hesperiidae). J. Morph,, 118 :461-494, Locke, M, (1967) The development of patterns in the integument of insects, Adv. Morph,,.£, Locke, M, and Krishnan, N, (1971) The distribution of phenoloxidases and polyphenols during cuticle- 171

formation (Calpodes ethlius1 Lep., Hesperiidae), Tissue and Cell, ]:103-126. Lunan, K.D. and Mitchell, H.K. (1969) The metabolism of tyrosine-0-phosphate in Drosophila (melanogaster). Arch. Biochem. Biophys., 1321450-456. Maddrell, S.H.P. (1963) Excretion in the blood-sucking bug, Rhodnius prolixus (Stal.). I. The control of diuresis. J. Exp. Biol., 40:247-256. Maddrell, S.H.P. (1964a) Excretion in the blood-sucking bug, Rhodnius prolixus (Stal.), II. The normal course of diuresis and the effect of temperature. J. Exp. Biol., 41:163-176, Maddrell, S.H.P. (1964b) Excretion in the blood-sucking bug, Rhodnius prolixus (Stal.). III. The control of the release of the diuretic hormone. J. Exp. Biol., 41:459-472, Mason, H.S. (1955) Comparative biochemistry of the phenolase complex, Adv. Enzymol. 1£:105-184. Mills,- R.R. (1965) Hormonal control of tanning in the American cockroach, II. Assay for the hormone and the effect of wound healing, J. Insect Physiol., 11:1269-1275. Mills, R.R. (1966) Hormonal control of tanning in the American cockroach, III, Hormone stability and post-ecdysial changes in hormone titre. J. Insect

Physicl., 12:275�280, in the Mills, R.R. (1967) Control of cuticular tanning 172

cockroach: Bursicon release by nervous stimulation. J, Insect Physiol,, .!].1815-820. Mills, R.R. and Fox, F.R. (1972) The sclerotization process by the American cockroach: Contribution of melanin, Insect Biochem., _g_:23-28, Mills, R.R. and Lake, C.R. (1966) Hormonal control of tanning in the American cockroach. IV. Preliminary purification of the hormone. J, Insect Physiol., 12:1395-1401, Mills, R.R. and Lake, C.R. (1971) Metabolism of tyrosine to p-hydroxyphenylpropionic acid and to p-hydroxy phenylacetic acid by the haemolymph of the American cockroach, Insect Biochem., .!_:264-270, Mills, R.R. and Nielsen, D.J. (1967) Hormonal control of tanning in the American cockroach, V. Some properties of the purified hormone. J. Insect Physiol., ll= 273-280. Mills,-R.R. and Whitehead, D.L. (1970) Hormonal control of tanning in the American cockroach:- Changes in blood cell- permeability during ecdysis. J. Insect Physiol., 161331-340. Mills, R.R., Androuny, S. and Fox, F.R. (1968) Correlation of phenoloxidase activity with ecdysis and tanning hormone release in the American cockroach, J, Insect

Physiol., 14:60 3-611, Mills, R.R., Greenslade, F.C. and Couch, E.F. (1966) Studies on vitellogenesis in the American cockroach, 173

J, Insect Physiol., 12:767-779, Mills, R.R., Lake, C.R. and Alworth, W.L. (1967) Biosynthesis of N-acetyldopamine by the American cockroach. J. Insect Physiol., 13:1539-1546. Mills, R.R., Mathur, 'R.B. and Guerra, A.A. (1965) Studies on the hormonal control of tanning in the American cockroach, I. Release of an activation factor from the terminal abdominal ganglion, J, Insect Physiol., 11: 1047-1053, Mitchell, H.K. and Lunan, K.D. (1964) Tyrosine-0-phosphate in Drosophila, Arch, Biochem. Biophys. 106:219-222, Murdock, L.L. (1971) Catecholamines in arthropods: a review, Comp. Gen. Pharmacol., .f:254-274, Murdock, L.L., Hopkins, T.L. and Wirtz, R.A. (1970a) Phenylalanine metabolism in cockroaches, Periplaneta americana: tyrosine and benzoyl-B-glucoside biosynthesis (Diet., Blattidae). Comp. Biochem. Physiol,, 1§.:535-545, Murdock, L.L., Hopkins, T.L. and Wirtz,- R.A. (1970b) Tyrosine metabolism in vivo in teneral and mature cockroaches, Periplaneta americana. J. Insect Physiol., 16:555-560, Nagatsu, T., Levitt, M. and Udenfriend, S. · (1964) The J, initial step in norepinephrine biosynthesis. Biol. Chem,, 239:2910-2917, Nijhout, H.F. (1976) The role of ecdysone in pupation of Manduca �· J, Insect Physiol., 22:453-463. 174

Okubo, S. (1958) Occurrence of N-acetylhydroxytyramine glucoside in Drosophila melanogaster. Med. J. Osaki Univ. 2= 327-337, . O'Malley, B.W. and Schrader, W.T. (1976) The receptors of steroid hormones. Sci. Amer., ZJ!±:32-43. Ostlund, E. (1953) Adrenaline; noradrenaline, and hydroxytyramine in extracts from insects. Nature, 172 :1042-1043. Ostlund, E. (1954) The distribution of catechol amines in lower animals and their effect on the heart. Acta Physiol. Scand., 31:1-67. Pavan, M. (1959) Biochemical aspects of insect poisons. Proc. 4th Intern. Cong. Biochem., 1£:15-36, Post, L.C. (1972) Bursicon: its effect on tyrosine permeation into insect haemocytes. Biochem. Biophys. Acta., 2901424-428. Post, L.C. and deJong, B.J. (1973) Bursicon and the metabolism of tyrosine in the moulti�g cycle of Pleris larvae. J. Insect Physiol. 19:154h1546. Price, G.M. (1972) Tyrosine metabolism in the larva of the blowfly, Calliphora erythrocephala. Insect Biochem. z_:175, P rovansa 1 A , • , Gr1.·11ot, J.P. and Delachambre, J. (1974) Dynamique de la secretion de la bursicon dans l'ensemble du syst�me nerveaux de Tenebrio molitor L. (Col�opt�re). C. R. Acad. Sci, Paris D., 278:

2193-2196. 175

Pryor, M.G.M. (1940a) On the hardening of the ootheca of- B-latta--ori-ental·i·-s, - �nro c. Roy., __ S oc� .B -- ,. ., ___128 , 378-393, Pryor, M.G.M. (1940b) On the hardening of the cuticle of insects, Proc. Roy. Soc. Ser. B,, �:393-407, Pryor, M.G.M. (1962) Sclerotization. In: Florkin, M. and Mason, H.S. (eds,), Comparative Biochemistry, New York, Acad. Press, �1371-396, Pryor, M.G.M., Russell, P.B. and Todd, A.R. (1946) Protocatechuic acid, the substance responsible for the hardening of the cockroach ootheca. Biochem. J., 40: 627-628. Pryor, M.G.M., Russell, P.B. and Todd, A.R. (1947) Phenolic substances concerned with the hardening of insect cuticle. Nature, 159:399-400. Przibram, H. and Schmalfuss, H_. (1927) Das dioxyphenyl alanin in der kokons des nachtpfauenanges Samia cecropia (L.) (Saturnidae). Biochem,, Z,, 1871467-469, Richards, A.G,_{19.,51)__.The Integument of Arthropods, Minneapolis, University of Minnesota Press, 4f1. R±chard·s, -A.G. (1958) The-cuticle of arthropods, Ergebn. Biol., 20:1-26. · Richards, AG.. - (1967) Sclerotization and the localization of brown and black colors in insects. Zool. Jb. (Anat), 84:25-62, Roth, L.M. and E.isner, T, (1962) Chemical defenses of arthropods, Ann, Rev. Ent,, 7:107-136, 176

Roth, L.M. and Stay, B. (1958) The occurrence of para quinones in some arthropods, with emphasis on the quinone-secreting tracheal glands of Diploptera punctata (Blattaria), J. Insect Physiol., .!_1305-318, Scharrer, B. (1952) Neurosecretion. XI, The effects of nerve section on the intercerebralis-cardiacum allatum system of the insect Leucophaea maderae, Biol. Bull. 102:261-272, Schlaeger, D.A. and Fuchs, M.S. (1974) -Effect of dopa decarboxylase inhibition on� aegypti eggs: evidence for sclerotization. J. Insect Physiol., 20:349-357, Schmalfuss, H. (1937) Uber das vorkommen und den nachweis von 3,4-dioxyphenylessigsaure in den rosenkafern Cetonia aurata (L.) und Potosia cuprea (F.) und im maikafer Melolontha hippocastani (F,). Biochem, Z., 2941112-119, Schneiderman, .H.A. and Gilbert, L.I. (1958) Substances with juvenile.hormone activity in crustacea and other invertebrates, Biol, Bull,, 155:530-535, Schneiderman,-H.A.- and-Gilbert, L.I. (1-964) Contro-1 of growth and development in insects, Several growth hormones appear to be isoprenoid derivatives and some may act upon the cell nucleus. Science, 143: 325-333, M. (1960) Schneiderman, H.A., Gilbert, L.I. and Weinsterin, sms and Juvenile hormone activity in microorgani 177

plants. Nature, 188:1041-1042. Sekeri-s,- C.E. --(1964} · Scl€rotiza-tion in the blowfly imago, Science, Wash., 1441419-420, Sekeris, C.E. (1965) Action of ecdysone on RNA and protein metabolism in the blowfly, Calliphora erythrocephala. Ina Mechanisms of Hormone Action, P. Karlson (editor). Academic Press. New York. Sekeris, C.E. and Karlson, P. (1962) Zum Tyrosinstoff wechsel der Insekten, VII. Der Katabolsiche Abbau des Tyrosins und die Biogenese der Sklerotisierungs substanz, N-Acetyl-dopamin. Biochim.Biophys, Acta, 62:103-113, Sekeris, C.E. and Karlson, P. (1964) On the mechanism of hormone action, II. Ecdysone and protein bio synthesis, Arch, Biochem. Biophys,, 105:483-487, Sekeris, C.E. and Karlson, P. (1966) I. Biosynthesis of catecholamine in insects. Pharmacological Reviews, 18:89-94, Sekeris,_ C.E. and -Lang, N. (1964) Induction of Dopa- '- decarboxylase activity-by insect messenger RNA in an 2:!! vitro amino acid incorporating system from rat liver, Life Sciences, .2..:625-632.

Sekeris, C.E. and Mergenhagen, D. (1964) Phenoloxidase system of the blowfly, Calliphora erythrocephala, Science, 145:68-69, G, (1969) Seligman, M., Friedman, $, and Fraenkel, ion during Bursicon mediation of tyrosine hydroxylat 178

tanning of the adult cuticle of �he fly, Sarcophaga bullata. J. Insect Physiol., 15:55.3-562. Shaaya, E. and Karlson, P. (1965) Der Ecdyson titer Wahrend der Insektenentwicklung--II. Die Postembryonale Entwicklung der Schmeissfliege Calliphora erythrocephala Meig. J. In.sect Physiol., 11=65-69. Shaaya, E, and Sekeris, C.E. (1965) Ecdysone during insect development--III. Activities of some enzymes of tyrosine metabolism in comparison with ecdysone titre during the development of the blowfly, Calliphora erythrocephala Meig. Gen. Comp,. Endocr., 2.:.35-.39 . Sherald, A.F., Sparrow, J.C. and Wright, T.R.F. (197.3) A spectrophotometric assay for Drosophila dopa decarboxylase (Dipt. ,, Drosophilidae). Anal. Biochem., 56:.300-.305. Sin, Y.T. and-,Thomson,,J.A,, (1971) DevelopmentaLchanges in_,the. prophenoloxidases of larval haemolymph in the fly, Calliphora. Insect Biochem., �156. Sivasubramanian, P., Friedman, S, and Fraenkel, G. (1974) Nature and role of proteinaceous hormonal factors acting during puparium formation in flies, Biol. Bull., 147:16.3-185, Srivasta, R.P. (1971) Autoradiographic study of glycine and tyrosine uptake by integument of different developmental stages of Galleria mellonella. 179

J. Insect Physiol., .11,261-267. Srivastava, B.B.L. and Hopkins, T.L. (1975) Bursicon release and activity in haemolymph during metamorphosis of the cockroach, Leucophaea maderae. J. Insect Physiol., _il:1985-1994, Taborsky, G. (1974) Phosphoproteins. Advances in Protein Chemistry, g§,11-210. Thomson, J.A. and Sin, Y�T. (1970) The control of prophenoloxidase activation in larval haemolymph of Calliphora. J. Insect Physiol., 16:2063-2074. Trivelloni, J.C. (1960) Biosynthesis of glucosides and glycogen in the locust. Arch. Biochem. Biophys., .§.2I 149-150, Truman, J.W. (1973) Physiology of insect ecdysis. III. Relationship between the hormonal control of eclosion and of tanning in the tobacco hornworm, Manduca sexta. J. Exp. Biol., 58:821-829, Tschinkel, W.R. (1969) Phenols and quinones from the defensive secretions of the Tenebrionid beetle, Zophobas rugipes, J, Insect Physiol., .!..2_:191-200. Udenfriend, s. (1966a) Tyrosine hydroxylase, Pharm. Rev., 18:4)-52, Udenfriend, s. (1966b) Biosynthesis of the sympathetic neurotransmitter norepinephrine. Harvey Lectures, 1964-1965:57-84, . VandenBerg, R.D. and Mills, R.R. (1974) Hormonal control of tanning by the American cockroach: 180

cyclic AMP as a probable intermediate. J. Insect Physiol., 20:623-627, VandenBerg, R.D. and Mills, R.R. (1975) Adenylcyclase in the haemocytes of the American cockroach, J. Insect Physiol., 211221-230. Veis, A. and Schlueter, R.J, (1963) Presence of phosphate-mediated cross-linkages in hard tissue collagens. Nature (Lond,), fil:1204. Vincent, J.F.V. (1971) Effects of bursicon on cuticular properties in Locusta migratoria migratorioides, J. Insect Physiol., 11.:625-636, Vincent, J,F.V. (1972) The dynamics of release and the possible identity of bursicon in Locusta migratoria migratorioides, J. Insect Physiol,, 1..§.:757-780. Warner, C.K., Grell, E.H. and Jacobson, K.B. (1974) Phenol oxidase activity and the lozenge locus of Drosophila melanogaster, Biochem. Gen., 11:359-365, Whitehead, D.L. (1969) New evidence for the control mechanism -of"'scl-erotization·-in insects, Nature, 224:721-723, Whitehead�-D.L,- (1970) L-Dopa--decarboxylase in the haemocytes of Diptera, FEBS Lett,, l_:263-266. · Whitehead, D.L., Brunet, P.C.J. and Kent, ,P.W.- (1960) Specificity in vitro of a phenoloxidase system from Periplaneta americana (L.). Nature (Lond.), 1.§..i:610, Wigglesworth, V.B. (1933) The physiology of the cuticle and of ecdysis in Rhodnius prolixus with special 181

reference to the function of the oenocytes and of the dermal glands. Q.J. Microsc. Sci.,2£1269-318. Wigglesworth, V.B. (1934) The physiology of ecdysis in Rhodnius prolixus. II. Factors controling moulting and "metamorphosis." Quart. J. Mier. Sci., 11=191-222. Wigglesworth, V.B. (1936) The function of the corpus allatum in the growth and reproduction of Rhodnius prolixus. Quart. J. Mier. Sci., 79:91-121. Wigglesworth, V.B. (1940a) Local and general factors in the development of "pattern" in Rhodnius prolixus. J. Exp. Biol., 121180-200. Wigglesworth, V.B. (1940b) The determination of characters at metamorphosis in Rhodnius prolixus. J. Exp.Biol., 17:201-222. Wigglesworth, V.B. (1941) Permeability of insect cuticle. Nature (Lend.), 147:116, Wiggle-sworth, V.B. (1945) Transpiration throught the cuticle of insects. J. Exp. Biol., 21:97-114. Wigglesworth, _V.B. (1947) The epicuticle in an insect, Rhodnius prolixus, Proc. R. Soc. B., 134:163-181. Wigglesworth, V.B. (1948a) The structure and deposition of the cuticle in the adult mealworm, Tenebrio molitor L. Quart, J. Mier, Sci,, 89:197-217, Wigglesworth, v.B. (1948b) The insect cuticle, Biol. Rev. , 23 1408-451, . Wigglesworth, v.B. (1948c) The insect epicuticle Proc. Int. Congr, Ent., Stockholm, �:J07-J09, Wigglesworth, V.B. (1951) Source of moulting hormone in Rhodnius. Nature, 1681558, Wigglesworth, V.B. (1952a) The thoracic gland in Rhodnius prolixus and its role in moulting. J. Exp, · Biol., 29:620-6J1. Wigglesworth, V.B. (1952b) Hormone balance and the control of metamorphosis in Rhodnius prolixus. J. Exp. Biol., .f.2_:620-6J1. Wigglesworth, V.B. (1954) The physiology of insect metamorphosis. (Cambridge roomographs in experimental biology, Vol. _!). Cambridge U.P. , London. Wigglesworth, V.B. (1957) The physiology of insect cuticle. Annu. Rev. Ent,, �:J7-54, Wigglesworth, V.B. (1959) The control of growth and form. Cornell University Press. Ithaca, N.Y. Wigglesworth, V.B. (1960) The epidermal cell and the metamorphos_is of insects. Nature (Land.), 188:J58-J59, Wigglesworth, V.B. (1965),The principles of insect physiology. Methuen and Co. Ltd. London. Wigglesworth, V.B. (1969) The insect cuticle. Mem. Soc. Ent. Ital., 48:167-178, Wigglesworth, V.B. (197J) Haemocytes and basement membrane formation in Rhodnius, J. Insect Physiol., 19:8J1-844. Williams, C.M. (1942) The effects of temperature of gradients on the pupal-adult transformation 183

silkworm . Biol. Bull,, 82 1 347_355, Williams, C.M. (1946) Continuous anesthesia for insects. Science, 1-Ql:57. 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., 93 1 89-98, Williams, C.M. (1952) Morphogenesis and metamorphosis of insects, The Harvey Lectures, � 1 126-155, Williams, C.M. (1956) The juvenile hormone of insects. Nature, 11§ 1 212-213, Williams, C.M. (1959) The juvenile hormone. I. Endocrine activity of the adult corpora allata of the Cecropia silkworm. Biol. Bull., 116:323-338. Williams, C.M. (1961) Insect metamorphosis: An approach to the study of growth, In: Growth in Living Systems,(M.X. Zarrow, ed.), Basic Books, Inc., New York. 1.· Yamazaki, H.I. (1969) 'The cuticular phenoloxidases in Drosophila virilis, J. Insect Physiol,, l,i12203-2211, Yamazaki, H.I. (1972) Cuticular phenoloxidase from the silkworm Bombyx mori1 Properties, solubilization and purificatim, Insect Biochem,, £1431. Yasunobu, K.T., Peterson, E. and Mason, H. (1959) The oxidation of tyrosine-containing peptides by tyrosinase, J. Biol, Chem,, 234:3291-3295, 184

Young, N.L. and Young, P.R. (1976) Biochemical control of moulting hormone titer in Calliphora erythrocephala during puparium formation. Insect Biochem., 61 169-177. APPENDIX COMPOUND Rf Rf Rf Rf Rf Rf Rf ave.

2-5-dihydroxy-l,4-benzoquinone .530 .519 (475 .528 .528 T--- .516 Hydroxyhydroquinone .676 .67� .686 .698 .673 .674 .680 Dopa .193 .218 .185 .219 .181 .206 .198 1-tyrosine .276 .341 ;281 .3�7 .364 .340 .327 ------5-hydroxydopamine .297 .286 .252 {270 T .276 Dopamine .428 .410 .411 .447 .444 .437 .434 Tyramine .600 .637 .631 .597 .564 ---- .604 Octopamine .507 .505 .511 .497 .515 .529 .510 Methyldopa .372 .376 .365 .377 .374 ,388 .374 h-hydroxy-3-methoxymandelic acid .712 • 724 . 707 .701 .699 .no .706 J,4-dihydroxymandylic acid .537 .525 .515 .517 .530 .543 .527 N-acetyldopamine .795 �808 .780 . 780 .820 .tlOl .tl04 L��ysyl ;-L-1ysine ;o:n .028 .031 .033 .034 .031 1-lysyl-L-alanine .139 .142 .137 .1�3 . .L55 ---- .142 1-lysyl�L-glycine .077 .070 .070 .074 .Otll ---- .074 N-acetyltyrosine .843 .840 .tl28 . tl2tl ------.835 Hydroxytyrptamine �reatine sulfate .439 .452 .457 .446 .443 .447---- .447 3-hydroxytryptamine hydrochloride .436 .443 .437 .457 .425 ---- .440 6-hydrocydopamine hydrochloride .327 .324 .327 .343 .326 ---- .329 N-carbobenzoxyl 1-tyrosine .932 .924 .932 ,939 .911---- .928 Homogentisic acid .766 .760 .760 .743 ---- .757 Catechol .8'.:>7 .847 .844 .838 .853 .842 .847 p-hydroxyephedrine hydrochloride .668 .656 .677 .613 .619 .b77 .645 Hydroquinone .827 .818 .838 .835 .839 .840 .833 p-hydroxybenzaldehyde .910 .899 .912 .913 .908 .906 .908 p-hydroxyphenyl pyruvic acid .874 .896 .874 .882 ------• tltl2 p-hydroxybenzylacetone .913 .925 .909 .920 .917 .922 .9ltl

Chromatography solvent system: Butanol:acetic acid:water;4:l:l. QOMPOUND jf' Ef ID' SI' Ef p;f Ef ·ave·,

2-5:dihydroxy-l,4-benzoquinone -1.470 -1�630 -0,086 -2.230 -1.500 ,-0.118 -1.172 Hydroxyhydroquinone •0,050 +0,103 +0.159 +0,084 +O.v�8 +0,061 +0,086 Dopa +0,089 +0;109 +0.094 +0.109 ------+0,115 +o.ie2------+0.106 1-tyrosine +0.079 +0,109 +0.044 +0.013 ------+0.084 5-hydroxydopamine +0,853 +0,904 +0,959 +0.892 ------+0,902 Tyramine +0.51� +0,406 +1.119 +0.509 ------+0,924 ------+0.693 Octopamine +0,992 +0.102 +0.816 ------+u.94J ------+0,702 MM,hyldopa -0.082 -0.107 -0.009------0.066 4-hydroxy-3-methoxymandelic acid --0,950 -1.030 ------0.990 3,4-dihydroxymandelic acid -1.000 -1.140 ------1.070 Dopamine +0,437 +0.390 ------+U.622 N-acetyldopamine +0.066 +0.104 +U,097 +0.101 +0,043 ------+0,050 +U.078 L-lysyl-1-lysine +1.210 +1.250 +l.26u +1.170 +1.010 +l.180 L-lysyl-L-alanine +0,979 +0.625 +0.838 +0,877 +0,954 +0.728 +0.834 L�lysyl-L-glycine +0.986 +0.616 +1.090 +U,9J4 +0,978 +0,978 +0.921 N-acetyltyrosine -0.764 -0.923 -1.250 -0.t:lOO------1.090------0.------763 --0,932 5-hydroxytryptamine creatine sulfate +0.500 +1.400 +0.866 ------+0.922 3-hydroxytryptamine hydrochloride' +0.633 +1.600 +1.030 ------+1.088 6-hydroxydopamine hydrochloride +0.667 +1.400 +U,709 +0.686 ------+0,866 N-acetyl-L-phenylalanine -0.637 -0,365 -1.460------1.030------U.t:l73 N-carbobenzoxyl L-tyrosine --0.545 -0.2�0 ------0.352 Homogeritisic acid -0.182 -0,182 ------l.t:l20 Catechol +0,050 +0.053 +U,737 +0,737 ------+0,052 Hydroxyphenylpyruvic acid -1.720 -1.310 -1.210 ------1.413 Ef values determi�ed by reference to Methyl-green (positive) and Phenol Red (negative)...... Buffer system was Pyridine:acetic acid:water;l:10:89 pH=3,5, VITA