Adrien Fónagy

Isolation and primary structure of a novel pheromonotropic neuropeptide structurally related to leucopyrokinin from the armyworm larvae, Pseudaletia separata

A novel pheromonotropic neuropeptide has been isolated from a head extract of the armyworm larvae, Pseudaletia separata, by a seven step purification procedure using an in vivo assay with decapitated female moths of Bombyx mori. Amino acid sequence analysis and comparison with synthetic peptides established the primary structure of the peptide, termed Pseudaletia pheromonotropin (Pss PT), as H-Lys-Leu-Ser-Tyr-Asp-Asp-Lys-Val-Phe-Glu-Asn-Val-Glu-Phe-Thr-Pro-Arg-Le u-NH2. Pss PT is structurally related to leucopyrokinin, an insect myotropic neuropeptide, and possesses the C-terminal pentapeptide, Phe-Thr-Pro-Arg-Leu-NH2, responsible for the biological activity.

Isolation, primary structure and synthesis of neomyosuppressin, a myoinhibiting neuropeptide from the grey fleshfly, Neobellieria bullata.

1. An amidated decapeptide, showing strong inhibitory activity of spontaneous visceral muscle movement was isolated, from head extracts of 42 thousand fleshflies, Neobellieria bullata (Diptera, Sarcophagidae). 2. Amino acid sequencing and verification by peptide synthesis revealed the following primary structure: Thr-Asp-Val-Asp-His-Val-Phe-Leu-Arg-PheNH2. 3. The novel peptide was termed neomyosuppressin or Neb-MS. 4. During the process of consecutive high performance liquid chromatography (HPLC) purifications the biological activity of the samples was monitored using heterologous bioassay system. 5. The threshold level of synthetic Neb-MS was found to be 8.6 +/- 0.5 x 10(-11) M on the Leucophaea hindgut and 3.4 +/- 0.5 x 10(-10) M on the Locusta oviduct bioassay, respectively.

Functional diversity of a pheromonotropic neuropeptide: Induction of cuticular melanization and embryonic diapause in lepidopteran insects by Pseudaletia pheromonotropin

Abstract A pheromonotropic neuropeptide, Pseudaletia pheromonotropin, is an 18 amino acid peptide possessing PheThrProArgLeuNH 2 (FTPRLamide) at the C-terminus. Pseudaletia pheromonotropin and its fragment peptides were synthesized and tested for melanization and reddish coloration hormone (MRCH) activity as well as Bombyx diapause hormone activity by using Pseudaletia separata larvae and Bombyx mori pupae of the polyvoltine strain N 4 , respectively. Pseudaletia pheromonotropin had MRCH activity and the activity resided in the C-terminal FTPRLamide. Pseudaletia pheromonotropin also showed Bombyx diapause hormone activity, but the activity was estimated 1–5% of that of synthetic Bombyx diapause hormone. Based on the physiological function, Pseudaletia pheromonotropin is renamed Pseudaletia MRCH.

Isolation and primary structure of two sulfakinin-like peptides from the fleshfly, Neobellieria bullata

1. Two novel insect myotropic peptides termed neosulfakinin-I (Neb-SK-I) and neosulfakinin-II (Neb-SK-II) were isolated from the heads of 42 thousand fleshflies, Neobellieria bullata (Diptera, Sarcophagidae). 2. A series of four, high-performance liquid chromatographic (HPLC), fractionations performed on columns with different characteristic features yielded two purified biologically active, hindgut motility stimulating fractions, suitable for amino acid sequence analysis. 3. The proposed sequences for the two peptides are: Phe-Asp-Asp-Tyr-Gly-His-Met-Arg-Phe-(NH2), (Neb-SK-I) and X-X-Glu-Glu-Gln-Phe-Asp-Asp-Tyr-Gly-His-Met-Arg-Phe-(NH2), (Neb-SK-II). 4. These sulfakinins exhibit very high homology to putative drosulfakinin sequences which, however, have not yet been isolated, but were deduced from a cloned Drosophila gene encoding these peptides. 5. Here we provide the first evidence for the expression of such peptides present in Dipterans. 6. Insect sulfakinins show structural identities with the hormonally-active portion of vertebrate gastrin II-, cholecystokinin- and caerulin-related peptides and they share common carboxy terminal sequences with invertebrate/vertebrate peptides of the FMRFamide peptide family.

Chemical characterization of cytoplasmic lipid droplets in the pheromone-producing cells of the silkmoth, Bombyx mori

Accumulation of lipid droplets within the cytoplasm is a common feature of the pheromone gland cells of many lepidopteran species. The cytoplasmic lipid droplets in the pheromone-producing cells of the silkmoth, Bombyx mori, were effectively extracted by dipping the trimmed glands in acetone for 10 min. In order to analyze the components originating from the lipid droplets, we separated the acetone extracts prepared before and after adult eclosion using HPLC, and specified the peaks showing a similar pattern of stage-dependence to that in the morphological change of the lipid droplets previously reported by Fónagy et al. (Arthropod Struct. Dev. 30 (2001) 113). Finally, we specified the peaks #1-5 and #1a-4a separated by reversed-phase HPLC as lipid droplet contents. Structure elucidation using FAB-MS and MS-MS analyses confirmed that they were triacylglycerols (TGs), and 12 species of TGs were identified as lipid droplet contents. Fatty acyl groups contained in these TGs were limited to five unsaturated C16 and C18 fatty acyl groups (delta 11-hexadecenoate, delta 10,12-hexadecadienoate, delta 9-octadecenoate, delta 9,12-ocatadecadienoate, and delta 9,12,15-ocatadecatrienoate), including the pheromone precursor delta 10,12-hexadecadienoate as a major component. Digestion with porcine pancreatic lipase confirmed that three major TGs eluted in the peaks #3-5 all contained C18 fatty acyl groups at the sn-2 position, indicating that the pheromone precursor is sequestered preferentially at the sn-1 and/or sn-3 position. Present results combined with the fact that the morphological change of the lipid droplets is under the control of PBAN indicate that the role of the cytoplasmic lipid droplets in the pheromone-producing cells is to store the pheromone precursor in the form of TGs and to provide it for pheromone production in response to the external signal of PBAN.

INVOLVEMENT OF CALCINEURIN IN THE SIGNAL TRANSDUCTION OF PBAN IN THE SILKWORM, BOMBYX MORI (LEPIDOPTERA)

In several moth species sex pheromone production in the pheromone gland is regulated by a neurohormone, pheromone biosynthesis activating neuropeptide (PBAN). In Bombyx mori it is suggested that PBAN, after binding to the cell-surface receptor, primarily activates a plasma membrane receptor-activated Ca2+ channel to increase cytosolic levels of Ca2+, and Ca2+/calmodulin complex directly or indirectly activates a phosphoprotein phosphatase, which in turn elicits activation of acyl CoA reductase (the key enzyme under PBAN control) through dephosphorylation, resulting in pheromone (bombykol) production. The effect of cyclosporin A (CsA) and FK 506, specific inhibitors of calcineurin (phosphoprotein phosphatase 2B) was studied on the sex pheromone production, in B. mori. The in vitro experiments showed that both chemicals exerted a dose-dependent inhibitory action when they were co-incubated with TKYFSPRL amide (Hez-PBAN fragment peptide). Practically, no difference was detected between the two chemicals in the tested doses (0.025-1250 microM). When effects of CsA or FK 506 were studied on cell-free production of bombykol by using microsomal fraction no inhibition was detected. Since microsomal fraction contains the acyl CoA synthetase, the rate-limiting acyl CoA reductase and the precursor, bombykol is produced if supplied with CoA, ATP and NADPH. Thus, the inhibitory action of CsA and FK506 under in vitro conditions should occur before the step of acyl group reduction and the effect is likely to be attributable to the inhibition of calcineurin in the signal transduction cascade mechanism of PBAN, in B. mori. The existence of calcineurin in the pheromone gland by using Western blot analysis is also demonstrated.

Isolation and characterization of calmodulin in the pheromone gland of the silkworm, Bombyx mori

Production of the sex pheromone bombykol in the silkworm, Bombyx mori, is regulated by a neurohormone termed pheromone biosynthesis activating neuropeptide (PBAN). It has been suggested that the external signal of PBAN in this species is transmitted to the intracellular cascade reactions consisting of Ca2+/calmodulin (CaM) complex and phosphoprotein phosphatase. To demonstrate the molecular mechanisms regulated by PBAN, we attempted to characterize CaM in the pheromone gland of B. mori. By using ion-exchange and RP-HPLC, B. mori CaM was purified from the cytosolic fraction of the pheromone gland. The primary structure was deduced by composition/sequence analysis and mass spectrometric analysis of the fragment peptides obtained from enzymatic and chemical fragmentations. The amino acid sequence of B. mori CaM was identical with Drosophila CaM deduced from the CaM gene of D. melanogaster, suggesting that insects have well conserved the molecule of CaM.

The regulation of δ11-desaturase gene expression in the pheromone gland of Mamestra brassicae (Lepidoptera; Noctuidae) during pheromonogenesis

Cabbage moth (Mamestra brassicae) females produce sex pheromones to attract conspecific males. In our M. brassicae colony, the pheromone blend is composed of Z11-hexadecenyl acetate (Z11-16Ac) and hexadecyl acetate (16Ac) in a 93:7 ratio. A fatty acyl Δ11-desaturase is involved in the production of the main pheromone component. The release of Pheromone Biosynthesis Activating Neuropeptide (PBAN) regulates the pheromone production in the pheromone gland (PG). We cloned a cDNA encoding the MambrΔ11-desaturase and analyzed its expression profile over time in M. brassicae tissues. Transcript levels of the Δ11-desaturase in larvae, pupal PGs, fat body, brain and muscle tissues were <0.1% of that in female PGs, whereas expression in male genitalia was 2%. In the PGs of virgin females the expression level increased continuously from eclosion to the end of the 1st day when it reached a plateau without further significant fluctuation up to the 8th day. In contrast, we recorded a characteristic daily rhythmicity in pheromone production with a maximum around 200 ng Z11-16Ac/PG. In some experiments, females were decapitated to prevent PBAN release and thereby inhibit pheromone production, which remarkably increased after treatment with Mambr-Pheromonotropin. Further experiments revealed that mating resulted in a significant suppression of pheromone production. However, expression of the Δ11-desaturase was not affected by any of these interventions, suggesting that its not regulated by PBAN. Fluorescent microscopy was used to study the potential role of lipid droplets during pheromone production, however, no lipid droplets were identified indicating that pheromonogenesis is regulated via de novo fatty acid synthesis.

Further Studies of Lipid Droplets in the Bombykol‐Producing Pheromone Gland of Bombyx mori

Abstract: Lipid droplets are abundant in the pheromone‐producing cells of B. mori at adult eclosion, followed by daily fluctuations in both their size and number. Their dynamics are related to PBAN‐stimulated de novo bombykol production. To elucidate associated events, we performed the following: (1) extraction, purification, and partial characterization of lipid droplet‐associated proteins found on their surface since their function could possibly be to transport and/or dock putative lipases that are responsible for the lipolysis of triglycerides in them; (2) separation, purification, and initial analysis of lipids carried by lipophorins and lipid transfer particles originating from pupal and adult hemolymph because of their role in the formation and accumulation of lipid droplets.

Studies of sex pheromone production under neuroendocrine control by analytical and morphological means in the oriental armyworm, Pseudaletia separata, Walker (Lepidoptera: Noctuidae).

Most female moths produce species-specific sex pheromone blends in the modified epidermal pheromone gland (PG) cells generally located between the 8 and 9th abdominal segments. The biosynthesis is often regulated by pheromone biosynthesis activating neuropeptide (PBAN) either in or prior to de novo fatty acid synthesis or at the formation of oxygenated functional group. In Pseudaletia separata, information about life span, calling, PG morphology, daily fluctuation of pheromone production and its hormonal regulation is limited. We measured pheromone titer daily (16:8; L:D) at 2h intervals in scotophase. Blend ratio stabilized during the 2nd day (till 4-5th) at 6th hour of scotophase, with the ratio of 27.5:12.8:44.4:15.3 for Z-11-16OH:16OH:Z-11-16Ac:16Ac, respectively. Females showed calling behavior from this time. We found with light and fluorescence microscopy that PG consisted of intersegmental membrane (A part), and dorso-lateral region of 9th abdominal segment (B part), encountering for ∼ 35% of total production revealed by gas chromatography. Ratios did not reveal difference. We did not find precursor (triacylglycerols) accumulation in form of lipid droplets, implying that PBAN stimulates de novo biosynthesis of 16:acyl precursors. In vivoHez-PBAN injections (1-3 × 5 pmol, 2h intervals) into 3 days old 16-18 h decapitated females stimulated pheromone production, both in A and B parts. Blend analyses including ratios suggest stimulation of the initial phase of synthesis, but desaturation of fatty acyl intermediates do not follow proportionally. More saturated fatty acid is converted from the available pool to the final OH and Ac, compared to females kept intact in scotophase. In vitro studies (PGs incubated 4-6h in the presence of 0.25 or 0.5 μM Hez-PBAN, especially with surplus 2mM malonyl-CoA) revealed higher saturated component ratio than the unsaturated, compared to natural blend or in vivo injections.