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Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in glycolysis, is commonly used as an internal reference gene in humans, mice, and insects. However, the function of GAPDH in insect development, especially in metamorphosis, has not been reported. In the present study, Helicoverpa armigera and Spodoptera frugiperda ovarian cell lines (Sf9 cells) were used as materials to study the function and molecular mechanism of GAPDH in larval metamorphosis. The results showed that HaGAPDH was more closely related to GAPDH of S. frugiperda and Spodoptera litura. The transcript peaks of HaGAPDH in sixth instar larvae were 6L-3 (epidermal and midgut) and 6L-1 (fat body) days, and 20E and methoprene significantly upregulated the transcripts of HaGAPDH of larvae in qRT-PCR. HaGAPDH–GFP–His was specifically localized in mitochondria in Sf9 cells. Knockdown of HaGAPDH by RNA interference (RNAi) in sixth instar larvae resulted in weight loss, increased mortality, and decreases in the pupation rate and emergence rates. HaGAPDH is directly bound to soluble trehalase (HaTreh1) physically and under 20E treatment in yeast two-hybrid, coimmunoprecipitation, and colocalization experiments. In addition, knockdown of HaGAPDH increased the Treh1 activity, which in turn decreased the trehalose content but increased the glucose content in larvae. Therefore, these data demonstrated that GAPDH controlled the glucose content within the normal range to ensure glucose metabolism and metamorphosis by directly binding with HaTreh1.

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.

Helicoverpa armigera can develop resistance to Bacillus thuringiensis (Bt), which threaten the long-term success of Bt crops. In the present study, RNAseq was employed to investigate the midgut genes response to strains with different levels of resistance (LF5, LF10, LF20, LF30, LF60, and LF120) in H. armigera. Results revealed that a series of differentially expressed unigenes (DEGs) were expressed significantly in resistant strains compared with the LF-susceptible strain. Nine trypsin genes, ALP2, were downregulated significantly in all the six resistant strains and further verified by qRT-PCR, indicating that these genes may be used as markers to monitor and manage pest resistance in transgenic crops. Most importantly, the differences in DEG functions in the different resistant strains revealed that different resistance mechanisms may develop during the evolution of resistance. The immune and detoxification processes appear to be associated with the low-level resistance (LF5 strain). Metabolic process-related macromolecules possibly lead to resistance to Cry1Ac in the LF10 and LF20 strains. The DEGs involved in the “proton-transporting V-type ATPase complex” and the “proton-transporting two-sector ATPase complex” were significantly expressed in the LF30 strain, probably causing resistance to Cry1Ac in the LF30 strain. The DEGs involved in binding and iron ion homeostasis appear to lead to high-level resistance in the LF60 and LF120 strains, respectively. The multiple genes and different pathways seem to be involved in Cry1Ac resistance depending on the levels of resistance. Although the mechanisms of resistance are very complex in H. armigera, a main pathway seemingly exists, which contributes to resistance in each level of resistant strain. Altogether, the findings in the current study provide a transcriptome-based foundation for identifying the functional genes involved in Cry1Ac resistance in H. armigera.