Effects of allatotropin and allatostatin on in vitro production of juvenile hormones by the corpora allata of virgin females of the moths of Heliothis virescens and Manduca sexta
Introduction
Insect juvenile hormones are critical developmental hormones that have direct effects on both larval development and adult reproductive competence. Most insect orders appear to synthesize a single JH homolog, methyl (2E,6E)-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate (JH III) but Lepidoptera [17] and at least some Diptera [24] synthesize additional homologs. The usual homologs produced by the corpora allata of adult female Lepidoptera include JH III, JH II (methyl (2E,6E,10cis)-(10R,11S)-10,11-epoxy-3,7,11-trimethyl-2,6-tridecadienoate) and JH I (methyl (2E,6E,10cis)-(10R,11S)-10,11-epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate) [1], [7], [14], [19], [20]. Interestingly, the corpora allata of male Lepidoptera appear to lack the methyl transferase required to produce JH and, instead produce the acid analogs of JH [2], [8]. Presumably, the acids are transported to the accessory glands where they are esterified to JH [2]. JH that is present in the male accessory glands of several species of Lepidoptera is transferred to the female during mating [2], [13], [18], where it may induce significant physiological and behavioral changes.
Although not well documented, regulation of JH production in adult female Lepidoptera is complicated and involves endogenous neural, neuroendocrine signals and, in some cases, male produced exogenous regulators transferred to the female during mating [6], [9], [13], [17], [20], [23]. Among virgin females, JH is required for vitellogenesis [6], [12], [15], [16], [21], [25] and, thus, females do not become reproductively competent until JH production is stimulated. To date, only two neuropeptides that regulate JH biosynthesis in adult Lepidoptera have been identified. These were identified from the tobacco hornworm moth (Manduca sexta) (THW) and are an allatotropin (Manse-AT, Gly-Phe-Lys-Asn-Val-Glu-Met-Met-Thr-Ala-Arg-Gly-Phe-NH2) [10] and an allatostatin (Manse-AST, pGlu-Val-Arg-Phe-Arg-Gln-Cys-Tyr-Phe-Asn-Pro-Ile-Ser-Cys-Phe-COOH) [11]. Although both neuropeptides have significant influence over JH biosynthesis by adult THW females surprisingly little work has been conducted on the effects of these neuropeptides. Studies by Kataoka et al. [10] demonstrated that the C-terminus of Manse-AT must be amidated and that truncated fragments having as few as the eight C-terminal amino acids were as effective in inducing JH biosynthesis as the intact peptide. Their work also showed that Manse-AT was not effective in stimulating JH biosynthesis in TBW larvae or pupae and adult females of Tenebrio molitor, Schistocerca nitens, or Periplaneta americana, but induced JH biosynthesis in adult females of the tobacco budworm moth, Heliothis virescens (TBW). Unni et al. [23] showed that analogs formed by substitution of Met by Nle at positions six and seven and acetylation of the N-terminus of the C-terminal nine amino acid fragment of Manse-AT had significant allatotropic activity. Their results suggested that Manse-AT stimulates a rate limiting enzyme in the biosynthesis of farnesoate but does not affect methylation or epoxidation of the acid [23]. Manse-AST is active in both TBW adults and larvae and in adult TBW females, but is inactive in adults of T. molitor, P. americana and Melanoplus sanguinipes [11]. Additionally, the results suggested that Manse-AST acts prior to the conversion of farnesoic acid to JH [11]. Although both Manse-AT and Manse-AST affect JH biosynthesis in adult THW and TBW females, no detailed studies have been conducted on how the effects are manifest. The following reports the results of studies in which the retrocerebral complexes (corpora cardiaca plus corpora allata) (RC) of adult females were incubated with either Manse-AT or Manse-AST or both. Biosynthesis of JH I, II and III in the presence of these neuropeptides was compared to those from unstimulated retrocerebral complexes.
Section snippets
Chemicals
Capillary GC-MS grade ethyl acetate, hexane and methanol were from Burdick and Jackson and 18 megohm water was obtained from a Milli Q UVplus® water purification system. Tissue culture medium 199 containing Hank’s salts and glutamine was obtained from Gibco. Synthetic Manse-AT was purchased from Sigma and Manse-AST was custom synthesized at the Interdisciplinary Center for Biotechnology Research, Protein Core Facility (University of Florida) using methods described elsewhere [11]. Both peptides
In vitro production of JH
Analyses of extracts obtained from in vitro incubations of RCs from both species, in medium containing no supplemental propionate, indicated the presence of JH I, JH II and JH III (Fig. 1). No other homologs of JH were found despite analysis of pooled extracts containing as much as 1 ng of JH III and the fact that the lower limit of sensitivity for full spectra (60–350 amu) for the JH homologs is ca. 3.0 pg (see [22]). JH II and JH III were present in approximately equal amounts. Amounts of JH
Discussion
In initial analyses data were obtained on the effect of age of adult females on in vitro biosynthesis of JH by isolated RCs of female TBW. As was found in earlier work with RCs of female THW [22] only three homologs, JH I, JH II and JH III, were identified from extracts from TBW females. This was expected because other in vivo and in vitro studies reported only JH I, JH II and JH III [12], [20]. However, our results with TBW indicated that the ratio of the three homologs was different from that
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