h2secl Biochem. Vol. 16, No. 1, pp. 109 113, 1986 0020-1790/86 $3.00 +0.00 Printed in Great Britain. All rights reserved Copyright ~ 1986 Pergamon Press Ltd ECDYSTEROID CONJUGATION BY TISSUES OF ADULT FEMALES OF DROSOPHILA MELANOGASTER ANDREAS DUBENDORFER I and PETER MAROY 2 qnstitute of Zoology, University of Z/irich, Winterthurerstrasse 190, CH--8057 Z/irich, Switzerland and 2Institute of Genetics, Biological Research Center, P.O. Box 521, H-6701 Szeged, Hungary Abstract--In order to determine whether female adults of Drosophila can synthesize enzymatically cleavable ecdysteroid conjugates, we injected radiolabelled ecdysone into flies and cultured various organs m vitro in the presence of labelled ecdysteroids. Within 1 hr most of the injected ecdysone was metabolized to 20-hydroxyecdysone, high polarity products (HPP), low polarity products (LPP) and some highly apolar compounds (AP). Incubation of organs from axenically reared flies (abdominal carcass, gut, Malpighian tubules, ovaries) in a culture medium containing tritiated ecdysteroids revealed tissue-specific conjugate synthesis. HPP was found in all tissues, but was by far the most prominent metabolite in ovary cultures. All tissues except the ovaries also contained labelled LPP which appeared bound to the cells, especially of the carcass and the gut. HPP and LPP were synthesized from both ecdysone and 20-hydroxyecdysone in vitro. They were shown to be ecdysteroid conjugates by enzymatic hydrolysis: HPP was cleaved by Helix pomatia hydrolases, and LPP was digestible with porcine liver esterase. Whether any of these conjugates have specific functions in supplying the eggs with ecdysteroid precursors is not yet known. Key Word Index: Drosophila melanogaster, ecdysteroid conjugates, conjugated ecdysteroids, ovaries, Malpighian tubules INTRODUCTION The presence of ecdysteroid hormones has been demonstrated not only in larval, pupal and adult stages, but also in the eggs and embryos of many insects (for review see Lagueux et al., 1981). In many instances embryonic ecdysteroids could be found well before ecdysial glands were functional, which sug- gested that the hormones in freshly laid eggs and early embryos were of maternal origin. Since in the course of any insect's development ecdysteroids are required in pulses and not as a constant supply, the maternal hormone should be deposited in the egg in an inactive form which can later be activated by the developing embryo. The pioneering work of the last few years on Locusta migratoria (Goltzen~ et al., 1978; Hoffmann et al., 1980; Lagueux et al., 1981; Sail et al., 1983) and Schistocerca gregaria (Dinan and Rees, 1981; Isaac and Rees, 1985) has revealed that adult females of orthopteran insects indeed synthesize ecdysteroids which they deposit in their eggs in the form of highly polar conjugates (HPP = high polarity products). From these the em- bryos liberate active ecdysteroids (Sail et al., 1983), a process which can be experimentally reproduced in vitro by hydrolysis with a crude enzyme preparation from Helix pomatia. High polarity conjugates of ecdysteroids have also been found in ovaries of Sarcophaga bullata (Briers et al., 1983) but whether these compounds also repre- sent the embryonic ecdysteroid precursors in this species is not known. The eggs of Nauphoeta cinerea are reported to contain virtually no cleavable HPP (Zhu et al., 1983), nor could the presence of HPP in the eggs of Drosophila melanogaster be demonstrated (Mar6y, Kaufmann and Diibendorfer, unpublished results). Recently a class of ecdysteroid conjugates oI lower polarity than ecdysone (LPP = low polarity products) has been found in a number of arthropods, such as the tick Ornithodoros moubata (Connat et al., 1984) and Boophilus microplus (Wigglesworth and Rees, pers. commun.; Crosby, pers. commun.). Simi- lar (or identical) low polarity conjugates are also present in some species of spiders, myriapods and insects (Connat and Diehl pers. commun.) including Teleogryllus commodus (Greenwood pers. commun.), Gryllus rnaculatus (Hoffmann, pers. commun.) and Drosophila melanogaster (Dfibendorfer, unpublished results). In ticks, LPPs have been identified as 20-hydroxyecdysone esterified at C22 with long-chain fatty acids (Diehl et al., 1985). Typically, these conju- gates are cleaved by esterases into free hormone and conjugating moiety. Drosophila embryos already contain high levels of ecdysteroids (Kraminsky et al., 1980), at a stage when the ring gland is not yet assembled (Poulson, 1950). In the adults, however, the titre of free ecdysteroids is low, as shown by radioimmunoassay (Kraminsky et al., 1980; Handler, 1982; Bownes et al., 1984). Therefore, if the female is the source of the embryonic ecdysteroids, these must be present in the adult in the form of poorly immunoreactive conjugates. In the present study, we investigated whether fe- male adults of Drosophila can synthesize conjugates of ecdysone and 20-hydroxyecdysone, and whether free hormone can be liberated from any conjugate by enzymatic digestion. MATERIALS AND METHODS All experiments were done at least twice. The absolute counts recovered per experiment sometimes varied consid- I09