The in vitro secretion of ecdysteroids from the prothoracic glands of last instar larvae of Spodoptera littorulis was detected and analysed by HPLC-RIA. The primary product was identified as 3-dehydroecdysone (= 82%), with lesser amounts of ecdysone (= 18%). Interconversion of ecdysone and 3-dehydroecdysone by prothoracic glands was not detectable.3-Dehydroecdysone 3P-reductase activity was demonstrated in the haemolymph. Ecdysone, the endproduct, was characterised by reverse-phase and adsorption HPLC, chemical transformation into ecdysone 2, 3-acetonide, and mass spectrometry. The conditions for optimal activity were determined. The enzyme requires NADPH or NADH as cofactor and K,,, values for NADPH and NADH were determined to be 0.94 pM and 22.8 pM, respectively. Investigation of the kinetic properties of the enzyme, using either NADPH or NADH as cofactor, revealed that it exhibits maximal activity at low 3-dehydroecdysone substrate concentrations, with a drastic inhibition of activity at higher concentrations (> 5 pM). The results suggest that the 3-dehydroecdysone 3P-reductase has a high-affinity (low K,) binding site for 3-dehydroecdysone substrate, together with a lower-affinity inhibition site.The 3P-reductase enzyme was purified to homogeneity using a combination of poly(ethy1ene glycol) 6000 precipitation and successive FPLC fractionation on Mono-Q, phenyl Superose (twice), and hydroxyapatite columns. The native enzyme was shown to be a monomer with molecular mass of 36 kDa by SDSPAGE and gel-filtration chromatography. Furthermore, the activity of the enzyme during the last larval instar was found to reach a peak prior to that of the haemolymph ecdysteroid titre, supporting a role for the enzyme in development.Keywords: 3-dehydroecdysone 3P-reductase; ecdysone ; prothoracic gland; Spodoptera littoralis.In immature stages of insects, the prothoracic glands are the primary source of ecdysteroid (moulting hormone), generally ecdysone (Scheme 1, I) in most species. However, recently it has been shown that in most lepidopteran species studied, the major product of the glands is 3-dehydroecdysone (Scheme 1, U), together with varying proportions of ecdysone [l, 21. After release from the prothoracic glands, the 3-dehydroecdysone is reduced to ecdysone by an NAD(P)H-linked 3-dehydroecdysteroid 3p-reductase in the haemolymph [2-41. The level of reductase activity in different insect species would appear to be related to the proportion of 3-dehydroecdysone secreted by the prothoracicThe exact details of ecdysteroid biosynthesis are not fully elucidated, although the early and late stages are the best understood [5-81. There is substantial evidence for the intermediacy of 2,22,25-trideoxyecdysone (5p-ketodiol ; Scheme 1, 111) in the biosynthesis of ecdysone [5,8, 91. This labelled substrate is effi-

Vitellogenin synthesis by the fat body has been monitored using in vitro culture and immunoprecipitation. This system was found to be efficient for measuring vitellogenin production in both non-infected Tenebrio molitor and those infected with Hymenolepis diminuta. In fat bodies from infected beetles, vitellogenin production was decreased by up to 75% (day 24 post-infection) and, at all times investigated, vitellogenin synthesis was significantly below control levels (days 3-30 post-infection). Incubating fat bodies from control insects with isolated metacestodes indicated that this may be a direct effect by the parasite which is developmental stage-specific. Stage II, but not Stage III-IV, not heat-killed parasites could bring about this decrease in vitellogenin. In addition, these effects may be density dependent within the range of 2-20 parasites per fat body; only 2 metacestodes were necessary to cause a significant decrease. Since metacestodes do not take up vitellogenin, nor limit the amount of [14C]leucine available to the fat body for vitellogenin production, it is conceivable that the parasite produces a potent inhibitor of vitellogenin synthesis, or a molecule which induces cells within the fat body.

In the midgut cytosol of Lepidoptera, ecdysteroids undergo inactivation by transformation via the 3-dehydro derivative to the corresponding 3-epiecdysteroid (3 alpha-hydroxy) and by phosphate conjugation. The oxygen-dependent oxidase catalyses formation of 3-dehydroecdysteroid, which can be reduced either irreversibly by 3-dehydroecdysone 3 alpha-reductase to 3-epiecdysteroid, or by 3-dehydroecdysone 3 beta-reductase back to the initial ecdysteroid. Furthermore, these ecdysteroids undergo further inactivation by phosphorylation. These ecdysteroid transformations have been investigated in last instar larvae of the cotton leafworm, Spodoptera littoralis. The products of the phosphorylation have been characterized as predominantly ecdysteroid 2-phosphate accompanied by smaller amounts of the corresponding 22-phosphate. The phosphotransferases require Mg2+ and ATP. Whereas the 3-dehydroecdysone 3 alpha-reductase has a clear preference for NADPH rather than NADH, the corresponding 3 beta-reductase markedly favours NADH. The physiological significance of the latter enzyme is unclear. The profiles of the various enzymic activities in dialysed midgut cytosol supplemented with appropriate cofactors were determined throughout the last larval instar. All activities were detectable throughout the instar, but the respective enzymes exhibited maxima at different times. Ecdysone oxidase showed a peak early in the instar, with 3-dehydroecdysone 3 alpha-reductase increasing to a peak as the former activity declined. The 3-dehydroecdysone 3 beta-reductase exhibited peak activity late in the instar, a profile similar to that observed for the corresponding haemolymph enzyme involved in reduction of the 3-dehydroecdysone product of the prothoracic glands to ecdysone. Thus, the significance of the midgut 3 beta-reductase may be related to production of active hormone. Both ecydsteroid 22- and 2-phosphotransferases showed high activities early in the instar and then declined. The physiological significance of the profiles for the ecdysone oxidase, the 3-dehydroecdysone 3 alpha-reductase and phosphotransferases is unclear.

Reproductive output of female Tenebrio molitor beetles is reduced upon infection with metacestodes of the rat tapeworm, Hymenolepis diminuta. We are using this as a model to investigate the adaptive signi¢cance of parasite-induced curtailment of insect reproduction. Production of the yolk protein vitellogenin (Vg) in the insect fat body is signi¢cantly reduced both in vitro and in vivo by metacestodes. Synthesis can be measured by using [14 C]L-leucine incorporation, followed by immunoprecipitation. In this paper we demonstrate that a signi¢cant decrease in [14 C]Vg can be produced by an acetic acid extract of the parasite. Conclusive evidence is presented that the active component(s) originate from the metacestodes: an extract of parasites grown entirely axenically has similar deleterious e¡ects. The developmental stage of the metacestode is important: immature (stage I^II) parasites had greater capacity to suppress Vg synthesis than mature ones (stage V^VI). Examination of the chemical nature of the e¡ector molecule(s) revealed that acetic-acid-extractable, boiling-resistant, pronase-sensitive agents in the molecular mass range 10^50 kDa reduced Vg synthesis by 47.4%. These data suggest that metacestodes produce a modulator molecule that directly a¡ects insect vitellogenesis and, therefore, that reduction of host ¢tness may confer a selective advantage upon the parasite.

Oogenesis in the codling moth, Cydia pomonella, and the role of juvenile hormones (JHs) were addressed. Rudimentary ovarian structures were recognisable in day 3–4 pupae, when haemolymph JH was still undetectable by coupled gas chromatography‐mass spectrometry in the selected ion mode (GC‐MS/SIM). The presence of developing oocytes was observed by light microscopy on day 8, coincident with very low JH titres (0.74 ± 0.05 ng/ml JH II). Chorionation was only evident upon emergence, following an increase in JH in the pharate adult (0h old: 4.71 ± 0.34 ng/ml JH II). Analysis of haemolymph from virgin and mated females indicated that JH II was predominant, with approximately equal and lower quantities of JHs I and III (3.3‐ to 5.0‐fold less). When pupae or newly emerged adults were treated with JH homologues, no alteration in ovarian protein content was apparent, but the JH mimetic, fenoxycarb, depressed the number of oocytes filling ≥ 50% follicular volume. Chorion deposition was stimulated by JHs I, II, or III (10 μg), but not by fenoxycarb (0.05 μg, 10 μg). Mating provided correct stimuli for enhanced choriogenesis and egg laying, and, since haemolymph JH titres were concomitantly elevated (approximately 2‐fold), it was postulated that the rise in JH elicited both these events. Application of JHs to virgin females, however, could not mimic mating; only increases in choriogenesis were induced: JH‐treatment of virgins (or mated insects) significantly decreased oviposition rates over 24 and 48 h and markedly reduced the life‐time total number of eggs. Arch. Insect Biochem. Physiol. 41:186–200, 1999. © 1999 Wiley‐Liss, Inc.