Hormonal control of tanning by the American cockroach: Probable bursicon mediated translocation of protein-bound phenols

J. Insect Physiol., 1972, Vol. 18, pp. 465 to 469. Pergamon Press. Printed in Great Britain

HORMONAL CONTROL OF TANNING BY THE AMERICAN COCKROACH: PROBABLE BURSICON MEDIATED TRANSLOCATION OF PROTEIN-BOUND PHENOLS JOHN K. KOEPPE” and RICHARD

R. MILLS

Department of Biology, Virginia Commonwealth University, Richmond, Virginia 23220 (Received 17 September 1971)

Abstract-The injection of 2-W-dopamine into the haemocoel of the American cockroach at ecdysis results in the binding of diphenols to blood proteins. These same proteins are apparently translocated to the cuticle where the label becomes incorporated into the hard insoluble matrix. On the other hand, if labelled dopamine is injected during the pre-ecdysis period, the radioactivity is found bound to the protein but it is not incorporated into the cuticle. Further investigations have revealed that translocation occurs during the immediate postecdysial phase, which suggests that the epidermal permeability may be mediated by bursicon, the tanning hormone.

INTRODUCTION

THE HARDinsoluble exoskeleton of insects is composed of a protein-chitin matrix stabilized by quinone crosslinks. These quinones are thought to arise from diphenols via the action of phenoloxidases. The ‘tanned’ proteins resulting from this action have been denoted as sclerotins and the entire process as sclerotization (PRYOR,1940a, b). The diphenol or sclerotization agent which mediates this process in insect cuticles has been identified as N-acetyldopamine (KARLSONet al., 1962; KARLSON and SEKERIS,1962). In the American cockroach, enzymes exist which are capabIe of synthesizing this compound from tyrosine (MILLS et al., 1967) and it appears to be the natural sclerotizing agent (KOEPPE and MILLS, 1970; MURDOCKet al., 1970). Recent work has shown that N-acetyldopamine and other acidic components combine with blood proteins and are subsequently incorporated into the cuticle (KOEPPE et al., 1971; KOEPPE and MILLS, 1971). The present work was undertaken to investigate the possible control or co-ordination of this translocation by hormones. The results suggest that this process may be mediated by bursicon, the tanning hormone. * Present address: Evanston. Illinois.

Department

of Biological 465

Sciences,

Northwestern

University,

466

JOHN K. KOEPPE and

MATERIALS

RICHARDR. MILLS

AND METHODS

American cockroaches Periplaneta americana were held at 25°C in alternating 12 hr periods of light and darkness. Late instar larvae were removed while in the process of shedding the old cuticle (unless otherwise specified) and injections into the haemocoel were performed immediately using a Hamilton Microsyringe apparatus fitted with a 30 gauge needle. The injections consisted of 2 to 3 PC After a preset period of time the blood was removed from a of 2-14C-dopamine. wound in the leg with a capillary tube (MILLS et al., 1966). The proteins to be analysed were separated from other dopamine metabolites on a 1 x 100 cm column of P-30 polyacrylamide. The gel was swollen and the columns were prepared as recommended by the Bio-Rad Laboratory. In all cases, the protein fraction collected and counted was in the void volume as determined by blue dextran. BBS-3 from Beckman was used in a ratio of 1 : 4 (protein fraction : BBS-3) with the addition of 10 ml scintillation fluid cocktail (Liquifluor-toluene). Counting was accomplished on a Beckman LS-200 scintillation counter. To determine the number of counts incorporated into the cuticle 20 min after the injection of 2- 14C-dopamine, the cuticle was scraped, washed several times in water, homogenized in acidified methanol (0.1 N HCl), and the subsequent pellet hydrolysed in 1 ml of 2 N NaOH for a period of 4 hr. After hydrolysis, 4 ml of BBS-2 was added to the hydrolysate along with 10 ml scintillation fluid for countThe stages of cockroach development, intering on a Beckman LS-200 counter. ecdysis, pre-ecdysis, and post-ecdysis have been previously described (MILLS and WHITEHEAD, 1970). RESULTS In previous papers (KOEPPE et al., 1971; KOEPPE and MILLS, 1971) it has been found that dopamine metabolites become bound to proteins and are subsequently incorporated into the cuticle. These experiments were done with freshly ecdysed cockroaches and included animals only in the immediate post-ecdysial stage. In order to ascertain if the dopamine metabolites are bound to proteins and incorporated into the cuticle during other stages in the cycle, 2J4C-dopamine was injected into animals 14 days old and into animals having a silver eyed condition (apolysis stage of the pre-ecdysis period). To analyse for protein binding, haemolymph was chromatographed on a 1 x 100 cm column of P-30 polyacrylamide gel and the fractions corresponding to the void volume (as determined by blue dextran) were counted on a liquid scintillation counter. Fig. 1 shows the comparison of protein-bound 14C-label extracted from the haemolymph during the three different stages of development. Previous work has revealed that the label is binding to the protein since double-labelled polymers (mol. wt. > 20,000) extracted from the haemolymph after 3H-leucine + i4C-dopamine injection cannot be separated (KOEPPE and MILLS, 1971). The results, as illustrated by the data in Fig. 1, show that the diphenols do not bind to proteins during the inter-ecdysial period. On the other hand, protein-bound label reaches a concentration in the pre-ecdysial period (apolysis stage) that is far in

HORMONAL

CONTROL

OF TANNING

BY

of that found during post-ecdysis. apolysis, these protein(s) failed to enter cumulated in the blood. The following elucidate this question.

THE

AMERICAN

467

COCKROACH

It was surmised at this point that during the new cuticle and are, therefore, acseries of experiments was designed to

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FIG. 1. Protein-bound label extracted from the blood at various times during the cycle. Haemolymph was chromatographed on a 1 x 70 cm column of P-2 polyacrylamide to remove non-protein-bound label. In each case, 3 PC of l-‘*Cdopamine was injected, and 20 min later the blood was removed. Inter-ecdysial (I) animals were 14 days from the previous ecdysis and animals denoted as ecdysis were in the process of shedding the old cuticle. Pre-ecdysial animals were in apolysis and were characterized by the ‘silver eyed’ appearance. The column elution was with deionized water which was mixed with Bio-solv (Beckman) before counting in a toluene-Liquifluor cocktail. Radioactivity was determined with a Beckman LS-200 liquid scintillation counter.

During post-ecdysis, 2 J4C-dopamine and/or metabolites are rapidly bound to protein and subsequently incorporated into the cuticle. Analysis of the cuticle taken at apolysis (after prior extraction with acidified methanol) revealed the absence of bound label, thus confirming the above supposition. The comparison of incorporated label during the three stages is depicted in Fig. 2. DISCUSSION

The results from these experiments show that dopamine and/or its metabolites are bound to haemolymph proteins during the immediate pre-ecdysial stage (apolysis) and during post-ecdysis. During inter-ecdysis, little protein-bound material can be detected. The reason for this is unknown but it could be speculated that the protein-receptor may not be present at this time or the enzyme(s) responsible for the attachment may be absent. An analysis of the bound label in the cuticle during the three different developmental stages shows that translocation does not occur during inter-ecdysis and

JOHN K. KOEPPE and RICHARDR. MILLS

468

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FIG. 2. Incorporation of protein-bound label into the cuticle at various times during the cycle. Cuticle was scraped to remove adhering tissue and ground in 0.1 N HCl in methanol to remove unbound label. The residue was hydrolysed in 2 N NaOH, neutralized with Beckman BBS-2 Biosolv, and counted in a tolueneLiquifluor cocktail using a Beckman LS-200 liquid scintillation counter. Proteinbound label was extracted from post-ecdysial animals previously injected with I-“C-dopamine as in Fig. 1. The effluvium from the void volume of the P-2 gel column was lyophilized, taken up in water, and injected into the stages as indicated. Cuticle was removed 20 min later.

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FIG. 3. Comparison of bound label in the cuticle and protein-bound label in the blood at various times during the cycle. Protein-bound label in the blood and cuticle was determined as in Figs. 1 and 2. It can be seen that label only enters the cuticle during and after ecdysis while blood protein label builds up during apolysis.

HORMONALCONTROLOF TANNING BY THE AMERICANCOCKROACH

469

apolysis (see Fig. 3). Only during the actual sclerotization process, does the protein-bound material enter and become part of the hard insoluble matrix. This, of course, is a necessity for the survival of the insect since prior hardening would prevent expansion and subsequent shedding of the old cuticle. The control of this process is open to question but it is interesting to speculate that the tanning hormone, bursicon, may be responsible. This hormone is secreted from the terminal abdominal ganglia during the immediate post-ecdysial period (MILLS et al., 1965) and is present during the ensuing 8 hr (MILLS, 1965). Thus, the influx of protein-bound substances and the release of the hormone occur at the same time. The fact that bursicon has been suggested to be identical with the diuretic hormone and that their mode of action may be to control permeability (MILLS and WHITEHEAD,

1970)

supports

this contention.

Acknowledgements-Appreciation is extended to Drs. J. R. SEED and F. R. Fox for helpful discussions and to Dr. K. RAO, JAIME GEIGER, and GARY LEVISON for a critical reading of the manuscript. J. GEIGERalso helped with the figures. This work was supported by N.S.F. grants GB-17490 and GB-7428, and by a Cell Biology Training Grant U.S.P.H.S.N.I.H.-GM-669 which provided a predoctoral fellowship to J. K. K. REFERENCES KARLSON P. and SEKERISC. E. (1962) N-acetyl dopamine as sclerotizing agent of the insect

cuticle.

Nature, Lond. 195, 183-184. SEKERIS C. E., and SEKERIS K. E. (1962) Zum Tyrosinestoffwechsel der Identifizierung von N-acetyl-3,4-dihydroxy-B-phenathylamin (NInsekten-VI. acetyl-dopamine) als Tyrosinmetabolit. Hoppe-Seyler’s 2. physiol. Chem. 327, 86-94. KOEPPEJ. K. and MILLS R. R. (1970) A possible new sclerotization agent in the cockroach cuticle. Biochem. J. 119, 66. KOEPPEJ. K. and MILLS R. R. (1971) Cuticle sclerotization: identification and transport of the cuticle phenol 3,4_dihydroxyphenylacetic acid by the American cockroach. In press. KOEPPE J. K., MILLS R. R., and BRUNETP. C. J. (1971) High molecular weight carriers of phenolic beta-glucosides by the American cockroach. In press. 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., GREENSLADEF. C., and COUCH E. F. (1966) Studies on vitellogenesis in the American cockroach. J. Insect Physiol. 12, 767-779. MILLS R. R., LAKE C. R., and ALWORTH W. I,. (1967) Biosynthesis of N-acetyl dopamine by the American cockroach. J. Insect Physiol. 13, 1539-I 546. MILLS R. R., MATHUR R. B., and GUERRAA. A. (1965) Studies on the hormonal control of Release of an activation factor from the terminal tanning in the American cockroach-I. abdominal ganglion. J. Insect Physiol. 11, 1047-1053. 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. 16, 331-340. MURDOCK L. I~., HOPKINS T. L., and WINTZ R. -4. (1970) Tyrosine metabolism in vivo in teneral and mature cockroaches, Periplaneta americana. J. Insect Physiol. 16, 555-560. PRYORM. G. M. (1940a) On the hardening of the ootheaca of Bluttu orient&. Proc. R. Sot. (B) 128, 378-393. PRYOR M. G. M. (194Ob) On the hardening of the cuticle in insects. Proc. R. Sot. (B) 128, 393-407. KARLSON P.,