An epoxide hydrolase involved in the biosynthesis of an insect sex attractant and its use to localize the production site

Abstract

Epoxide hydrolases (EHs) are enzymes occurring in virtually any living organism. They catalyze the hydrolysis of epoxide containing lipids and are involved in crucial mechanisms, such as the detoxification of xenobiotics or the regulation of inflammation and blood pressure. Here, we describe a function of a putative EH gene in the biosynthesis of a sex attractant in the jewel wasp Nasonia vitripennis and use this gene to localize the site of pheromone production. Males of this parasitic wasp release a mixture of (4R,5R)-(Athreo-) and (4R,5S)-(Aerythro-)5-hydroxy-4-decanolide (HDL) to attract virgin females. Using a stable isotope labeled precursor, we demonstrated that vernolic acid (Aerythro-12,13-epoxy-octadec-9Z-enoic acid) is converted by N. vitripennis males to threo-HDL. This suggested the involvement of an EH in hydrolyzing the fatty acid epoxide under inversion of the stereochemistry into the respective diol, which might be further processed by chain shortening and lactonization to HDL. We cloned a putative N. vitripennis EH gene (Nasvi-EH1) encoding 470 amino acids and localized its transcripts in the male rectal papillae by in situ RT-PCR. Chemical analyses and histological studies confirmed that males synthesize the sex attractant in the rectal vesicle and release it via the anal orifice. Involvement of Nasvi-EH1 in HDL biosynthesis was established by RNAi-mediated gene silencing. Injection of Nasvi-EH1 dsRNA into male abdomens inhibited pheromone biosynthesis by 55% and suppressed the targeted gene transcripts in the rectal vesicle by 95%.Nasonia vitripennis ͉ pheromone biosynthesis ͉ pheromone gland

How parasitoid females produce sexy sons: a causal link between oviposition preference, dietary lipids and mate choice in Nasonia

“…Incorporation of 13 C was indicated by mass shifts of the four diagnostic ions m/z 86, 101, 115 and 186 in the mass spectra of (4R,5R)-and (4R,5S)-HDL ( [38], figure 1). The incorporation rate was 36 per cent each.…”

“…Abdomens of 2-day-old N. vitripennis males reared on these 13 C-LA-labelled hosts were extracted with 25 ml of dichloromethane and used for GC-MS analysis using the equipment and conditions described below. Incorporation of 13 C-LA into the two HDL stereoisomers was concluded from the appearance of diagnostic ions in the mass spectra as described elsewhere [38].…”

“…The response is shown by virgin females only [34][35][36] and is synergized by the trace component 4-methylquinazoline [37]. The HDLs are biosynthesized in the rectal vesicle of males [38] and released via the anal orifice by dabbing movements of the abdominal tip [39,40]. Females have been shown to orient along HDL concentration gradients thereby avoiding the danger of mating with sperm-depleted males that release less of the attractive chemicals than males of sufficient fertility [41].…”

“…Females have been shown to orient along HDL concentration gradients thereby avoiding the danger of mating with sperm-depleted males that release less of the attractive chemicals than males of sufficient fertility [41]. Vernolic acid (¼ erythro-12,13-epoxy-octadec-9Z-enoic acid) is a precursor of HDL [38], suggesting that pheromone biosynthesis starts from linoleic acid (LA), which is a possible epoxidation substrate of cytochrome P450 enzymes [42]. LA is an essential nutrient for most animals and is, like other PUFAs, important for a number of metabolic processes in animals including sperm production [21].…”

Precise RNAi-mediated silencing of metabolically active proteins in the defence secretions of juvenile leaf beetles

“…Another RNAi target was the production of pheromone in jewel wasps, Nasonia vitripennis. Silencing an epoxide hydrolase in these insects resulted in pheromone reduction by 55 per cent and suppressed the targeted gene transcripts by 95 per cent [52]. Freshly emerged males were injected and 2 days later levels of transcript and pheromones were analysed.…”