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Conogethes punctiferalis is a crop and fruit pest that has caused serious economic losses to agricultural production. This pest relies heavily on its sex pheromone to ensure sexual encounters and subsequent mating success. However, the molecular mechanism underlying sex pheromone biosynthesis in this species remains elusive. The present study investigated the detailed mechanism underlying PBAN-regulated sex pheromone biosynthesis in C. punctiferalis by transcriptome sequencing of the C. punctiferalis pheromone glands (PGs) and subsequent functional identification of the target genes. The results showed that female mating started from the first scotophase, and peaked at the second to fifth scotophases in accordance with the release of sex pheromones. PBAN regulated sex pheromone biosynthesis by employing Ca2+ and cAMP as secondary messengers, as demonstrated by RNA interference (RNAi), pharmacological inhibitors, and behavioral assays. Further investigation revealed that calcineurin (CaN) and acetyl-CoA carboxylase (ACC) were activated by PBAN/Ca2+ signaling, and the RNAi-mediated knockdown of CaN and ACC transcripts significantly reduced sex pheromone production, ultimately leading to a significantly reduced ability of females to attract males. Importantly, hexokinase (HK) was found to regulate sex pheromone biosynthesis in response to the PBAN/cAMP/PKA signaling pathway, as demonstrated by RNAi, enzyme activity, and pharmacological inhibitor assays. Furthermore, Far2 and Desaturase1 were found to participate in PBAN-regulated sex pheromone biosynthesis. Altogether, our findings revealed that PBAN regulates sex pheromone biosynthesis through the PBANR/Ca2+/CaN/ACC and PBANR/cAMP/PKA/HK pathways in C. punctiferalis, which enriches our comprehension of the details of sex pheromone biosynthesis in moths.

Helicoverpa zea is a major target pest of pyramided transgenic crops expressing Cry1, Cry2 and/or Vip3Aa proteins from Bacillus thuringiensis (Bt) in the United States. Laboratory-selected Cry1Ac/Cry2Ab cross resistance and field-evolved practical dual resistance of H. zea to these two toxins have been widely reported. Whether the widespread Cry1Ac/Cy2Ab dual resistance of H. zea has resulted from the selection of one shared or two independent resistance mechanisms by pyramided Bt crops remains unclear. Cadherin is a well-confirmed receptor of Cry1Ac and a suggested receptor of Cry2Ab in at least three Lepidopteran species. To test whether cadherin may serve as one shared mechanism for the cross and dual resistance of H. zea to Cry1Ac and Cry2Ab, we cloned H. zea cadherin (HzCadherin) cDNA and studied its functional roles in the mode of action of Cry1Ac and Cry2Ab by gain- and loss-of-function analyses. Heterologous expression of HzCadherin in H. zea midgut, H. zea fat body and Sf9 cells made all three of these cell lines more susceptible to activated Cry1Ac but not activated Cry2Ab, whereas silencing HzCadherin of H. zea midgut and fat body cells significantly reduced the susceptibility to Cry1Ac but not Cry2Ab. Likewise, suppressing HzCadherin with siRNA made H. zea larvae resistant to Cry1Ac. These results clearly demonstrate that HzCadherin is not a receptor for Cry2Ab, and thus it is unlikely to serve as one shared mechanism for the cross and dual resistance of H. zea to Cry1Ac and Cry2Ab.

Helicoverpa armigera (cotton bollworm) is one of the most destructive pests worldwide. Due to resistance to Bacillus thuringiensis and conventional insecticides, an effective management strategy to control this pest is urgently needed. Spinosad, a natural pesticide, is considered an alternative; however, the mechanism underlying the developmental effects of sublethal spinosad exposure remains elusive. In this study, the mechanism was examined using an insect model of H. armigera. Results confirmed that exposure to sublethal spinosad led to reduced larval wet weight, delayed larval developmental period, caused difficulty in molting, and deformed pupae. Further investigation demonstrated that exposure to sublethal spinosad caused a significant decrease in 20E titer and increase in JH titer, thereby leading to the discordance between 20E and JH titers, and consequently alteration in the expression levels of HR3 and Kr-h1. These results suggested that sublethal spinosad caused hormonal disorders in larvae, which directly affect insect development. Our study serves as a reference and basis for the toxicity evaluation of spinosad on molting and pupation in insect metamorphosis, which may contribute to identifying targets for effective control of cotton bollworm.