Identification of Differentially Expressed Genes in the Pheromone Glands of Mated and Virgin Bombyx mori by Digital Gene Expression Profiling

Background

Mating decreases female receptivity and terminates sex pheromone production in moths. Although significant progress has been made in elucidating the mating-regulated inactivation of pheromone biosynthesis-activating neuropeptide (PBAN) secretion, little is known about the mating induced gene expression profiles in pheromone glands (PGs). In this study, the associated genes involved in Bombyx mori mating were identified through digital gene expression (DGE) profiling and subsequent RNA interference (RNAi) to elucidate the molecular mechanisms underlying the mating-regulated gene expression in PGs.

Results

Eight DGE libraries were constructed from the PGs of mated and virgin females: 1 h mating (M1)/virgin (V1) PGs, 3 h mating (M3)/virgin (V3) PGs, 24 h mating (M24)/virgin (V24) PGs and 48 h mating (M48)/virgin (V48) PGs (M48 and V48). These libraries were used to investigate the gene expression profiles affected by mating. DGE profiling revealed a series of genes showing differential expression in each set of mated and virgin female samples, including immune-associated genes, sex pheromone synthesis-associated genes, juvenile hormone (JH) signal-associated genes, etc. Most interestingly, JH signal was found to be activated by mating. Application of the JH mimics, methoprene to the newly-emerged virgin females leaded to the significant reduction of sex pheromone production. RNAi-mediated knockdown of putative JH receptor gene, Methoprene tolerant 1 (Met1), in female pupa resulted in a significant decrease in sex pheromone production in mature females, suggesting the importance of JH in sex pheromone synthesis.

Conclusion

A series of differentially expressed genes in PGs in response to mating was identified. This study improves our understanding of the role of JH signaling on the mating-elicited termination of sex pheromone production.     

Identification of lipases involved in PBAN stimulated pheromone production in Bombyx mori using the DGE and RNAi approaches

Background

Pheromone biosynthesis activating neuropeptide (PBAN) is a neurohormone that regulates sex pheromone synthesis in female moths. Bombyx mori is a model organism that has been used to explore the signal transduction pattern of PBAN, which is mediated by a G-protein coupled receptor (GPCR). Although significant progress has been made in elucidating PBAN-regulated lipolysis that releases the precursor of the sex pheromone, little is known about the molecular components involved in this step. To better elucidate the molecular mechanisms of PBAN-stimulated lipolysis of cytoplasmic lipid droplets (LDs), the associated lipase genes involved in PBAN- regulated sex pheromone biosynthesis were identified using digital gene expression (DGE) and subsequent RNA interference (RNAi).

Results

Three DGE libraries were constructed from pheromone glands (PGs) at different developed stages, namely, 72 hours before eclosion (−72 h), new emergence (0 h) and 72 h after eclosion (72 h), to investigate the gene expression profiles during PG development. The DGE evaluated over 5.6 million clean tags in each PG sample and revealed numerous genes that were differentially expressed at these stages. Most importantly, seven lipases were found to be richly expressed during the key stage of sex pheromone synthesis and release (new emergence). RNAi-mediated knockdown confirmed for the first time that four of these seven lipases play important roles in sex pheromone synthesis.

Conclusion

This study has identified four lipases directly involved in PBAN-stimulated sex pheromone biosynthesis, which improve our understanding of the lipases involved in releasing bombykol precursors from triacylglycerols (TAGs) within the cytoplasmic LDs.     

The release of a pheromonotropic neuropeptide, PBAN, in the turnip moth Agrotis segetum, exhibits a circadian rhythm

In the female turnip moth, Agrotis segetum, a pheromone biosynthesis activating neuropeptide (PBAN) stimulates sex pheromone biosynthesis which exhibits a daily rhythm. Here we show data supporting a circadian rhythm in PBAN release from the corpora cardiaca, which we propose regulates the endogenous rhythm in sex pheromone biosynthesis. This conclusion is drawn as the observed daily rhythm in PBAN-like immunoreactivity in the hemolymph is persistent in constant darkness and is phase-shifted by an advanced light:dark cycle. PBAN-like immunoreactivity was found in the brain, the optic lobe, the suboesophageal ganglion and in the retrocerebral complex. In each hemisphere ca. 10 immunopositive neurons were observed in the pars intercerebralis and a pair of stained somata in the dorso-lateral protocerebrum. A cluster of cells containing PBAN-like immunoreactive material was found in the tritocerebrum and three clusters of such cells were found in the SOG. Their processes reach the corpora cardiaca via nervi corporis cardiaci and the dorsal surface of the corpora allata via the nervi corporis allati.     

A novel domain-duplicated SlitFAR3 gene involved in sex pheromone biosynthesis in Spodoptera litura

Fatty acyl reductases (FARs) are key enzymes that participate in sex pheromone biosynthesis by reducing fatty acids to fatty alcohols. Lepidoptera typically harbor numerous FAR gene family members. Although FAR genes are involved in the biosynthesis of sex pheromones in moths, the key FAR gene of Spodoptera litura remains unclear. In this work, we predicted 30 FAR genes from the S. litura genome and identified a domain duplication within gene SlitFAR3, which exhibited high and preferential expression in the sexually mature female pheromone glands (PGs) and a rhythmic expression pattern during the scotophase of sex pheromone production. The molecular docking of SlitFAR3, as predicted using a 3D model, revealed a co-factor NADPH binding cavity and 2 substrate binding cavities. Functional expression in yeast cells combined with comprehensive gas chromatography indicated that the SlitFAR3 gene could produce fatty alcohol products. This study is the first to focus on the special phenomenon of FAR domain duplication, which will advance our understanding of biosynthesis-related genes from the perspective of evolutionary biology.     

RNA interference of PBAN receptor suppresses expression of two fatty acid desaturases in female Plutella xylostella

Pheromone biosynthesis-activating neuropeptide (PBAN) stimulates sex pheromone biosynthesis by activating PBAN receptor (PBANr), which triggers a specific signal transduction in the pheromone gland cells. We have shown that RNA interference (RNAi) of PBANr of Plutella xylostella significantly suppressed pheromone biosynthesis and subsequent mating behavior. In order to assess molecular events occurring downstream of PBAN signaling, we cloned partial sequences of Δ9 and Δ11 fatty acid desaturases of P. xylostella. Phylogenetic analysis indicated that these two desaturase genes were highly clustered with other desaturases associated with sex pheromone biosynthesis in other insects. RT-PCR analysis showed that Δ9 desaturase was dominantly expressed in adult females, whereas Δ11 desaturase was expressed in all P. xylostella developmental stages. When PBANr expression was suppressed by PBANr-RNAi, the treated females also showed significant suppression of expression of both desaturases. These results suggest that expressions of the two desaturases are controlled by PBAN and that the two desaturases may be involved as downstream components in sex pheromone biosynthesis of P. xylostella.     

Age-dependent plasticity of sex pheromone response in the moth, Agrotis ipsilon: Combined effects of octopamine and juvenile hormone

Male moths use sex pheromones to find their mating partners. In the moth, Agrotis ipsilon, the behavioral response and the neuron sensitivity within the primary olfactory centre, the antennal lobe (AL), to sex pheromone increase with age and juvenile hormone (JH) biosynthesis. By manipulating the JH level, we previously showed that JH controls this age-dependent neuronal plasticity, and that its effects are slow (within 2 days). We hypothesized that the hormonal effect might be indirect, and one neuromodulator candidate, which might serve as a mediator, is octopamine (OA). Here, we studied the effects of OA and an OA receptor antagonist, mianserin, on behavioral and AL neuron responses of mature and immature males during stimulation with sex pheromone. Our results indicate that, although OA injections enhanced the behavioral pheromone response in mature males, OA had no significant effect on behavior in immature males. However, mianserin injections decreased the behavioral response in mature males. AL neuron sensitivity increased after OA treatment in immature males, and decreased after mianserin treatment in mature males. Determination of OA levels in ALs of immature and mature males did not reveal any difference. To study the possible interactive effects of JH and OA, the behavioral pheromone response was analyzed in JH-deprived mature males injected with OA, and in immature males injected with fenoxycarb, a JH agonist, and mianserin. Results show that both JH and OA are necessary to elicit a behavioral response of A. ipsilon males to sex pheromone.     

Feeding and hemolymph trehalose concentration influence sex pheromone production in virgin Heliothis virescens moths

Previously, we demonstrated that sex pheromone production in mated female Heliothis virescens moths is dependent upon hemolymph trehalose concentration (HTC), which is influenced by activities such as the feeding of adults on sucrose. In this paper we demonstrate, for the first time, that this effect also occurs in starved (i.e., sugar-stressed) virgin females. Females allowed to feed on sugar for 6 days, following eclosion, had significantly greater titers than females that had fed only on water (i.e., were starved). No differences in pheromone titer were observed between sugar- and water-fed females at shorter (1 or 3 days) periods following eclosion. The relatively short-term effects of HTC on sex pheromone titer of virgins, were demonstrated by feeding experiments, in which starved (for 4 days) virgins fed on 10% sucrose solution had significantly greater HTC and pheromone titers than ones fed only on water; an increase in HTC was apparent within an hour, while the increase in pheromone titer was apparent within 2.5 h, of sugar feeding. Starvation also showed similar effects on titers of pheromone gland fatty acids (pheromone intermediates) and HTC. Over 6 days of starvation, fatty acid titers and HTC declined gradually. After feeding on sucrose, titers of hexadecanoic, (Z)-9-hexadecanoic, (Z)-11-hexadecanoic and (Z)-9-octadecanoic, acids, as well as HTC, increased significantly 24 h later, but titers of octadecanoic and (Z,Z)-9,12-octadecanoic (linoleic) acids did not. Lepidoptera cannot biosynthesize polyunsaturated acids, but the lack of change in octadecanoic acid titer suggests this acid may not participate in pheromone biosynthesis. In addition to these short-term changes in pheromone and fatty acid production, mediated by HTC, a longer-term effect of age, regardless of HTC, on pheromone titer was observed. Overall, these results are consistent with hemolymph trehalose and glandular fatty acids acting as twin metabolite reservoirs for pheromone biosynthesis. Hemolymph trehalose, able to be refilled through feeding on exogenous sugars, has a one-way flow of metabolites for synthesis of glandular free fatty acids (FFAs) and pheromone, while glandular glycerolipids provide a reversible reservoir for metabolites, accepting surplus FFAs when glandular concentrations are high, and providing FFAs for pheromone biosynthesis when concentrations are low.