Complete metamorphosis of insects is regulated by mutually interacting juvenile hormone (JH) and ecdysteroid signals. The holometabolous pupa is a stage at which a natural absence of JH permits adult morphogenesis. Administration of exogenous JH or synthetic JH mimics at this stage disrupts development, but details of the ectopic JH action are not well understood. Here, using time-resolved imaging in the red flour beetle (Tribolium castaneum) and the honey bee (Apis mellifera), we show that treatment with the native hormone JH III not only blocks the pupa-adult transition, but also accelerates the appearance of morphological traits and shortens the total duration of the pupal stage. As assessed in T. castaneum, these effects were accompanied by altered expression of genes regulating metamorphosis and by reduced ecdysteroid levels, thus likely causing heterochronic developmental shifts resulting from endocrine imbalance. Pupae of both species were equally affected by direct application of JH III on the pupal cuticle surface and by contactless exposure to JH III vapors. Like the topical application, the vapor-phase hormone activated the established JH signaling pathway hallmarked by transcriptional induction of the anti-metamorphic gene Krüppel-homolog 1 (Kr-h1). This gaseous JH action is relevant to experimental settings and to applications in insect pest management, apiculture, and insect mass rearing. Our findings broaden understanding of JH impact on metamorphosis, address the mechanism of volatile JH effect, and suggest that JH might play a role as an airborne endocrine signal acting at short distances.
Insect herbivores employ many adaptive mechanisms to successfully colonize host plants. Transcriptional plasticity is one mechanism that enables broad host use in polyphagous insect herbivores; however, whether specialist herbivores use transcriptional plasticity or undergo epigenome modifications to cope with host shifts remains largely unexplored. We measured the transcriptome and DNA methylome of the specialist aphid herbivore, Daktulosphaira vitifoliae, after transfer to new hosts using RNA-seq and whole-genome bisulfite sequencing. We found the transcriptome of D. vitifoliae is highly plastic and responsive to host genotype – 3706 genes (28% of expressed genes) are significantly differentially expressed in at least one contrast comparing host genotypes. Most host-responsive genes (2667 genes) were upregulated when feeding on the non-native host Vitis vinifera and enriched for genes encoding predicted secretory effectors, detoxification enzymes, and cuticle- and chitin-associated proteins. We found gene co-expression modules that correlated with variation in fitness suggesting plasticity in transcriptional networks contributes to D. vitifoliae adaptation to host plants. We also identified a previously undescribed family of proline-rich secretory proteins conserved across the Aphidomorpha infraorder of insects. Our analysis of DNA methylation revealed a negative correlation between gene-level CpG methylation and host-induced transcriptional plasticity, suggesting a lack of gene body methylation may enable transcriptional plasticity when insects move to new host environments. Together, these results provide new insight into the molecular mechanisms of host use in insect herbivores.
Although cytochrome P450-mediated xenobiotic detoxification has been well characterized, comparatively little is known about how naturally occurring host-plant phytochemicals regulate the expression of insect detoxification genes. Capsaicin, a defensive alkaloid produced by chili pepper (Capsicum annuum), deters many vertebrate and invertebrate herbivores, whereas the generalist crop pest Helicoverpa armigera tolerates and metabolizes this compound. In this species, the cytochrome P450 enzyme CYP6AN1 contributes to capsaicin metabolism, and the corresponding gene is strongly induced by capsaicin; however, the regulatory mechanism underlying this induction has remained unknown. Using dual-luciferase reporter assays, we identified several cis-regulatory regions with positive or negative effects on the capsaicin responsiveness of the CYP6AN1 promoter. An element essential for capsaicin induction of CYP6AN1, designated the xenobiotic response element to capsaicin (XRE-Cap), was fine-mapped to positions −57 to −44 and found to consist of a direct repeat of the CAAAGTC motif. Internal deletions and site-directed mutagenesis demonstrated that both the nucleotide sequence and direct-repeat architecture of XRE-Cap contribute to maximal induction. Conversely, converting a single distal CAAAGTC motif into a second XRE-Cap copy significantly enhanced capsaicin induction, thereby providing gain-of-function evidence for the regulatory importance of this direct-repeat architecture. Electrophoretic mobility shift assays showed that XRE-Cap is specifically bound by nuclear proteins from Helicoverpa zea fat body cells. Together, these findings identify and functionally characterize XRE-Cap as a cis-regulatory element required for capsaicin-inducible expression of CYP6AN1 and provide mechanistic insight into phytochemical-responsive regulation of detoxification genes in lepidopterans.
Triacylglycerol (TAG) is the main lipid reserve in insect eggs and represents a critical energy source for embryonic development. In the hematophagous vector Rhodnius prolixus, oocyte growth relies heavily on lipids transported from the fat body through the hemolymph, which are delivered mainly as diacylglycerol and free fatty acids. Therefore, the ovary must catalyze the final esterification step converting these substrates into TAG to ensure proper lipid storage in developing oocytes. This reaction is mediated by diacylglycerol acyltransferases (DGATs), yet the specific roles of DGAT1 and DGAT2 in insect reproduction remain poorly understood. Here, we investigated the functions of RpDGAT1 and RpDGAT2 in ovarian physiology and reproduction in R. prolixus. RNAi gene knockdown efficiently reduced transcript levels of both enzymes without affecting blood digestion, survival, or oviposition rates. However, RpDGAT1 knockdown caused pronounced reproductive defects. Females produced morphologically abnormal eggs with collapsed appearance and reduced size, leading to an approximately 70% reduction in hatching rates. Ultrastructural analyses revealed alterations in eggshell organization as seen by SEM, but no changes in the hydrocarbon contents of the eggshell were detected. Lipid analyses showed that RpDGAT1 knockdown reduced ovarian TAG levels at day 10 after blood feeding, but not at day 5, while increasing ovarian phosphatidylethanolamine at day 10. Ovarian free fatty acid levels remained unchanged at both time points. In phenotypic eggs, TAG levels were reduced and free fatty acids were increased. Consistently, oocytes from RpDGAT1-knocked down females exhibited smaller lipid droplets, reflecting impaired neutral lipid storage. Hemolymph lipid composition and ovarian incorporation of radiolabeled palmitate were unchanged, indicating that reduced ovarian TAG was not associated with detectable changes in circulating lipid availability or overall ovarian fatty acid uptake. In contrast, RpDGAT2 knockdown produced no detectable reproductive phenotype. Together, our findings demonstrate that RpDGAT1, but not RpDGAT2, is essential for TAG accumulation, proper oocyte development, and egg hatchability in R. prolixus, highlighting the importance of lipid metabolism in insect reproductive physiology.
The olfactory system is essential for insect survival and behavior, with odorant-binding proteins (OBPs) playing a key role by binding and transporting chemical signals to olfactory receptors. Although MaltOBP9 from Monochamus alternatus, a primary vector of pine wilt disease, has been linked to olfactory functions in M. alternatus, its regulatory mechanisms and specific roles are not well understood. In this study, we show that MaltOBP9 is highly expressed in the antennae of both male and female adults after sexual maturation. Using fluorescence spectroscopy, molecular docking, and site-directed mutagenesis, we identified the amino acid Phe54 as essential for MaltOBP9's ability to bind 18 different volatile compounds. RNA interference (RNAi) and behavioral tests revealed that MaltOBP9 is necessary for sexually mature adults to detect the pheromone 2-methoxy-4-vinylphenol (2M4VP). Additionally, dual-luciferase reporter assays, electrophoretic mobility shift assays, and RNAi experiments demonstrated that the transcription factor MaltEms positively regulates MaltOBP9 expression, thereby influencing the detection of 2M4VP. By combining insights into transcriptional regulation and protein-ligand interactions, this study clarifies the MaltEms-MaltOBP9-2M4VP axis as a mediator of sex-specific olfactory behavior—where females avoid and males are attracted to the pheromone—in sexually mature M. alternatus. These findings provide a theoretical basis for developing pest control methods that target MaltOBP9 and its critical residue Phe54 through behavior-based strategies.
