Hexokinase Is Required for Sex Pheromone Biosynthesis in Helicoverpa armigera

Acetyl-CoA, the precursor of sex pheromone biosynthesis in Helicoverpa armigera, is generated from glycolysis. As the first speed-limited enzyme in glycolysis, Hexokinase (HK) plays an important role in acetyl-CoA production. However, the function of HK in sex pheromone production remains unclear. This study employed H. armigera as material to explore the role of HK in sex pheromone production. Results demonstrated that the transcription profile of HaHK in female moth pheromone glands (PGs) was consistent with the release fluctuation of sex pheromone. Interference of HaHK prevented the increase of acetyl-CoA content induced by PBAN. Therefore, knockdown of HaHK in female PGs caused significant decreases in (Z)-11-hexadecenal (Z11-16:Ald) production, female capability to attract males, and mating rate. Furthermore, sugar feeding (5% sugar) increased the transcription and enzymatic activity of HK. Pheromone biosynthesis activating neuropeptide (PBAN) signal phospho-activated HaHK in PGs and Sf9 cells via protein kinase A (PKA), as shown by pharmacological inhibitor analysis. In general, our study confirmed that PBAN/cAMP/PKA signal activated HaHK, in turn promoted glycolysis to ensure the supply of acetyl-CoA, and finally facilitated sex pheromone biosynthesis and subsequent mating behavior.

Transcriptome Analysis of Ostrinia furnacalis Female Pheromone Gland: Esters Biosynthesis and Requirement for Mating Success

Female moths use sex pheromones to attract males, and corresponding regulatory mechanism underlying sex pheromone biosynthesis is species-dependent. However, the detailed mechanism involved in sex pheromone biosynthesis in Ostrinia furnacalis has not yet been fully addressed. In the present study, transcriptome sequencing of O. furnacalis pheromone glands screened a serials of candidate genes involved in sex pheromone biosynthesis. Our analysis showed that sex pheromone release in O. furnacalis females arrives its peak at the 2nd scotophase, consistent with its mating behavior. Pheromone biosynthesis-activating neuropeptide (PBAN) was confirmed to regulate sex pheromone biosynthesis, and Ca2+ is the secondary messenger of PBAN signaling in O. furnacalis. The functional analysis of candidate genes demonstrated that the decreased mRNA levels or activities of calcineurin (CaN) and acetyl-CoA carboxylase (ACC) led to significant decrease in sex pheromone production and female capability to attract males, as demonstrated by RNAi-mediated knockdown and pharmacological inhibitor assay. Most importantly, the activities of CaN and ACC depend on the activation of PBAN/PBANR/Ca2+. Furthermore, fatty-acyl reductase 14 was involved in PBAN-mediated sex pheromone biosynthesis. Altogether, our results demonstrated that PBAN regulates sex pheromone biosynthesis through PBANR/Ca2+/CaN/ACC pathway to promote sex pheromone biosynthesis in O. furnacalis and provided a reference for non-model organism to study neuropeptide signal transduction.

Pyruvate Kinase Is Required for Sex Pheromone Biosynthesis in Helicoverpa armigera

Pyruvate kinase (PYK) is a speed-limited enzyme of glycolysis that catalyzes the formation of pyruvate, and plays an important role in acetyl-CoA synthesis. The acetyl-CoA is the precursor of sex pheromone biosynthesis in Helicoverpa armigera. However, the role of PYK in sex pheromone biosynthesis remains elusive. Here, PYK in H. armigera (HaPYK) was found to be highly expressed in the pheromone glands (PGs). The developmental expression profile of HaPYK was consistent with the fluctuation of sex pheromone release. Function analysis revealed that the knockdown of HaPYK led to a decrease in the levels of pyruvic acid and acetyl-CoA in PGs, which in turn caused a significant decrease in cis-11-hexadecenal (Z11-16: Ald) production, female capability to attract males, and mating frequency. Further study demonstrated that sugar feeding (5% sugar) increased the transcription and enzyme activity of HaPYK, thereby facilitating sex pheromone biosynthesis. Moreover, pheromone biosynthesis activating neuropeptide (PBAN) upregulated HaPYK activity through protein kinase C (PKC), as shown by PKC-specific inhibitor analysis. Altogether, our results revealed that PBAN activated HaPYK by Ca2+/PKC, thereby regulating the synthesis of pyruvate and subsequent acetyl-CoA, ensuring the supply of sex pheromone precursor, and finally facilitating sex pheromone biosynthesis and mating behavior.

Supplemental Sugar Is Required for Sex Pheromone Biosynthesis in Mythimna separata

Supplemental nutrients of adult moths maximize moth fitness and contribute to the pollination of many plants. Previous reports have revealed that sugar feeding promotes to sex pheromone biosynthesis by increasing the haemolymph trehalose concentration in mating moths. Here, Mythimna separata adults were employed as a model to investigate the effect of sugar feeding on sex pheromone biosynthesis. Results showed that in virgin females, sugar feeding markedly increased the concentrations of trehalose, pyruvic acid, and acyl-CoA in pheromone glands (PGs), which in turn led to an increase in sex pheromone titer, female ability to attract males and successfully mating frequency in sugar-fed females. Consistently, sugar-fed females laid more eggs than water-fed females. Furthermore, the refeeding of starved females also caused significantly increase in the concentrations of trehalose, pyruvic acid, and acyl-CoA in PGs, thus facilitating a significant increase in sex pheromone production. Most importantly, RNAi-mediated knockdown of trehalase (leading to PG starvation) resulted in an increase in trehalose content, and decrease in the concentrations of pyruvic acid, and acyl-CoA in PGs, which in turn led to a decrease of sex pheromone titer, female ability to attract males and successful mating efficacy. Altogether, results revealed a mechanism by which sugar feeding contributed to trehalose utilization in PGs, promoted to significantly increased sex pheromone precursor by increasing the concentrations of pyruvic acid and acyl-CoA, and facilitated to sex pheromone biosynthesis and successful mating.