Aspects of research on insect growth hormones*

Bull. Org. iniond. Sante 1 1971, 44, 397-398Bull. Wld Hlth Org.J Aspects of Research on Insect Growth Hormones* LAWRENCE I. GILBERT,1 S. APPLEBAUM,2 T. A. GORELL,3 J. B. SIDDALL 4 & Y. C. SIEW 5 Current research on insect growth hormones includes studies on the binding ofhormones to receptor molecules, probably proteins. Evidence has been obtainled that this process does in fact occur and may be the means whereby the hormones " recognize - target tissues. Other studies on the possibility of a feedback effect when growth hormones are used for insect control suggest that there is a positive feedback relationship between the hormllone titre and the activity of prothoracic glands and corpora allata, blut the details are not yet clear. Cyclic adenosine 3',5'-monophosphate has an important role as " second messenger " in vertebrate endocrinology and may also be important in insects. Studies have shown that adenyl cyclase is present in pupal epidermis and the preliminary results have shown that it can be stimulated by a steroid hormone. Several current projects in our laboratory may have practical significance if insect growth hormones and their analogues are to be used as agents for insect control. The design of the experiments is based on the premise that basic research on the mode of action of insect hormones will yield information necessary for the proper design of specific hormone analogues. BINDING PROTEINS It is well established that several vertebrate steroid hormones (e.g., oestrogen) are bound to cytosol receptor proteins and are then transported to the nucleus, where they presumably exert their effects. This may be the means by which hormones in the general circulation are able to " recognize" target tissues. Since ecdysone is a steroid, and is found in essentially all tissues of an insect after a dose * From the Department of Biological Sciences, North- western University, Evanston, Ill. USA. This work was supported by grant No. AM-02818 from the US National Institutes of Health. 1 Professor of Biological Sciences, Northwestern Uni- versity, Evanston, Ill., USA. 2 Senior Lecturer, Hebrew University of Jerusalem, Rehovot, Israel. 3 Post-Doctoral Fellow, Ben May Institute for Cancer Research, University of Chicago, Ill., USA. 'Director of Research, Zoecon Corporation, Palo Alto, Calif., USA. 6 Senior Lecturer, Department of Zoology, University of Malaya, Kuala Lumpur, Malaysia. has been injected (unpublished data), we thought that one major difference between cells that respond to ecdysone and those that do not might be the presence or absence of specific hormone receptors. Our model system was the hepatopancreas of a crustacean, Orconectes virilis, which, as we have shown, responds to moulting hormone. High-specific- activity a-ecdysone (a-ecdysone-23,24-3H) was in- jected into the animal or incubated with the hepato- pancreas in vitro, and the cytosol portion of the organ was subjected to gel chromatography. Appro- priate fractions containing both protein and radio- active material were analysed by stepwise, linear, sucrose-gradient centrifugation. The analysis revealed the presence of 2 peaks with radioactivity, indicating that complexes had formed between the radioactive material and hepatopancreatic proteins. (Unbound labelled material remained at the top of the gradient.) Using enzyme markers, we showed that the 2 proteins had approximate molecular weights of 250 000 (11.3 S) and 130 000 (6.35 S). Further experiments suggested that the heavier component wvas an aggre- gate of the lighter one. Chemical analyses conducted to elucidate the nature of the labelled material demonstrated that it was neither a-ecdysone nor any known phytoecdy- sone or ecdysone metabolite. Since more than 50% of the administered a-ecdysone is converted to this product in a relatively short time, we feel that it may have important significance. Perhaps the a-ecdy- sone is a pro-hormone that must be converted to an 2654 - 397- L. I. GILBERT AND OTHERS active form (possibly the unknown metabolite) before hormonal effects are elicited. In any event, if " receptor " substances are needed for hormone action, it may be possible to control insect populations by saturating the receptors with inactive analogues. Other devices that would inter- fere with the binding process or the synthesis of receptor molecules could also conceivably prevent normal insect growth. FEEDBACK If hormones are to be used as " third generation" pesticides it is important to know what effects these exogenous agents have on the insect's endo- crine glands. From a theoretical point of view it is important that we gather data on the feedback relationships between the hormone titre and gland activity, since many experiments in insect endocrino- logy include hormone injections or gland implant- ations. Phiilosamia cynthia and Hyalophora cecropia pupae were given injections of ,B-ecdysone or juvenile hormone (JH) and the activity of the prothoracic glands and corpora allata was measured over a 4-day period by radioautographic analysis of uridine-3H uptake into nuclear RNA. When pupae with inactive glands received /3-ecdysone injections, the insects' own prothoracic glands were stimulated within 3 hours. Activity then slowly decreased, reaching control levels at the end of 24 hours. Surprisingly, the corpora allata were also activated by moulting hormone, the peak of activity lagging behind that of the prothoracic glands by 3 hours. The activity of the corpora allata decreased quickly thereafter; 12 hours after the injection of JH the corpora allata were activated, while the prothoracic glands reached peak activity 12 hours later. Thus, both moulting hormone and JH stimulate both the prothoracic glands and the corpora allata, though in reverse order. Although these data suggest a positive feedback relationship between the hormone titre and gland activity, we feel that the situation is more complex and that the quantitative relation- ships are most important. For example, when /3-ecdysone is injected into young pharate adults that have active prothoracic glands, the glands are shut off in 6-12 hours. This agrees with a basic tenet of vertebrate endocrinology-namely, that a negative feedback relationship exists between the hormone titre and gland activity. CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE The importance of cyclic adenosine 3',5'-mono- phosphate in endocrinology cannot be overesti- mated and its role as a " second messenger " in vertebrate endocrinology is undisputed. We demon- strated in 1970 that when ecdysone is injected into diapausing pupae, the cyclic adenosine 3',5'-mono- phosphate level rose from less than 0.5 x 10-7 mmoles per g of tissue to more than 3.5 x 10-7 mmoles per g of tissue and that JH appeared to anta- gonize this effect (E. Whitmore, L. Gilbert & R. W. Butcher, unpublished data). We are now following up this initial finding by using less cumbersome assay conditions. We have recently shown that adenyl cyclase is present in pupal epidermis, using an assay based on the incorporation, from a pre-labelled pool of nucleotides into cyclic adenosine 3',5'-monophos- phate. Adenyl cyclase can be stimulated either in vivo or in vitro by ,B-ecdysone administration in less than 1 hour and this appears to be the earliest discernible effect of the moulting hormone. If this is a direct effect of the moulting hormone, then our data would be the first to demonstrate adenyl cyclase stimu- lation by a steroid hormone. 398