The silkworm (Bombyx mori) feeds exclusively on mulberry leaves; however, the genetic basis underlying its adaptation to mulberry defenses remains unclear. In this study, we identified a silkworm chemosensory protein (CSP), BmCSP4, which is highly expressed in the salivary glands of larvae and secreted onto mulberry leaf wounds during feeding. Biochemical and molecular docking assays revealed that BmCSP4 binds to various mulberry volatiles, particularly cis-3-hexenyl acetate, cis-jasmone, and trans-3-hexen-1-ol. Mulberry leaves chewed by silkworms with exogenous BmCSP4 application or transgenic overexpression showed reduced expression of defense-related genes. Feeding assays showed that while transgenic silkworms overexpressing BmCSP4 exhibited no significant change in food consumption, they displayed significant increases in larval weight, pupal weight, cocoon weight, and fecundity. Transcriptomic analyses showed that mulberry leaves bitten by BmCSP4-expressing silkworms exhibited downregulated transcription of genes involved in defense hormone signaling and anti-insect secondary metabolism. In conclusion, as a salivary effector, BmCSP4 enhances silkworm growth fitness and performance by binding and blocking plant volatile defense signals to suppress host immunity.

Diptera include major agricultural pests and disease vectors, underscoring the need for species-specific population-control strategies. The sterile insect technique (SIT) relies on sterile males that remain sufficiently viable and competitive to mate successfully with wild females without producing viable offspring. This review synthesizes male reproductive genes and regulatory pathways in Diptera according to the reproductive processes they control and the failure modes caused by their disruption. Mechanistic understanding is based largely on studies of Drosophila melanogaster, complemented by evidence from tephritid fruit flies and mosquitoes to assess conservation and operational relevance in pest and vector species. We examine testis morphogenesis, germline maintenance, meiotic and post-meiotic spermatogenesis, sperm function, accessory gland-mediated post-mating responses, and noncoding RNA regulation. The responsible genetic networks are evaluated in relation to sterility penetrance, reproductive specificity, pleiotropic fitness costs, mating performance, residual fertility, and post-mating reproductive effects. Functional studies using RNA interference and genome editing have identified promising targets for male fertility, necessitating species-specific evaluation to determine their suitability for genetic control. By correlating reproductive mechanisms with operational phenotypes, this synthesis can inform target prioritization, strain maintenance, and quality control of irradiated males, thereby enhancing the refinement of SIT and related genetic control strategies.

Stimulator of interferon genes (STING) not only plays an important role in innate immunity, but also acts as a regulator of lipid metabolism in insects. AMP-activated protein kinase (AMPK) serves as a crucial cellular energy sensor that regulates lipid homeostasis. However, the interplay between STING and AMPK remains largely unknown in insects. In this study, STING was cloned from Tribolium castaneum. Spatial-temporal expression analysis revealed that TcSTING was highly expressed in the fat body and in 7-day-old adult females, respectively. Knockdown of TcSTING significantly reduced TAG levels, decreased neutral-lipid storage in fat body lipid droplets, and lowered FAS activity concomitantly with the downregulation of genes encoding sterol regulatory element binding protein (SREBP) and fatty acid synthase (FAS1-5) in 15-day-old larvae and 2-day-old females. Conversely, knockdown of TcAMPK in 2-day-old females significantly increased the levels of TAG and neutral-lipid storage in fat body as well as FAS activity accompanied by the upregulation of TcSREBP and FAS1-5. Mechanistically, knockdown of TcSTING activated AMPK by inactivating TBK1, leading to the upregulation of phosphorylation level of acetyl-CoA carboxylase (ACC) and inhibition of the transcription of TcSREBP, whereas dsTcAMPK treatment suppressed ACC phosphorylation and activated the transcription of TcSREBP. These results demonstrate that TcSTING modulates lipid metabolism via phosphorylation regulation of AMPK, thereby altering the TAG storage.

Aphids (Hemiptera: Aphididae) are important agricultural pests and exhibit strong ecological adaptability, allowing them to persist under stress, disperse to new habitats, and rapidly increase population size. Recent advances in functional genomics have identified microRNAs (miRNAs) as key post-transcriptional regulators involved in these processes, yet their roles have remained fragmented across studies. Here, we synthesize current evidence into a "three-stage framework", encompassing population maintenance under stress, dispersal to new habitats, and population expansion upon establishment. We highlight how miRNAs regulate detoxification pathways (e.g., P450s, UGTs, ABC transporters), mediate interactions with host plants and symbionts, and integrate hormonal signaling networks including insulin, juvenile hormone, and ecdysteroid pathways. This framework identifies candidate miRNAs, target genes, and signaling pathways that may recur across different ecological contexts, including stress responses, dispersal-related plasticity, and reproductive regulation. However, direct evidence demonstrating that candidate shared miRNA regulators coordinate multiple life-history stages remains limited and requires further experimental validation. We critically evaluate the strength of functional evidence, distinguishing experimentally validated miRNA-target interactions from prediction- or expression-based associations. Finally, we discuss emerging applications of miRNA-based pest control, including artificial miRNAs, RNAi technologies, and nanocarrier delivery systems. By linking molecular mechanisms with ecological outcomes, this review provides a synthesis and highlights miRNAs as important regulators of aphid adaptation and candidate targets for sustainable management strategies.

We previously demonstrated that ornithine decarboxylase (ODC) deficiency critically impairs nitrogen metabolism and survival in Aedes aegypti. To further examine the role of the polyamine pathway in Ae. aegypti nitrogen metabolism, we evaluated the expression of three additional genes encoding proteins involved in the biosynthetic pathway: S-adenosylmethionine decarboxylase, spermidine synthase (SdS), spermine synthase (SmS), and seven genes encoding proteins involved in the catabolic pathway in fat body, midgut and Malpighian tubules by qPCR. Distinct transcriptional profiles were observed in mosquito tissues during the first gonotrophic cycle. SdS and SmS showed a differential protein expression pattern in fat body of sugar- and blood-fed mosquitoes. Genetic silencing of SdS, SmS or SdS and SmS by RNA interference (RNAi) decreased female survival. Mosquitoes with SdS or SmS deficiency exhibited a reduction of 5G1 trypsin level in the midgut at 24 h post-blood meal (PBM) , a delay in blood digestion, and a decrease in uric acid concentration in the excreta at 48 h PBM. RNAi-mediated SdS knockdown also caused a decrease in SmS protein level and vice-versa, RNAi-driven SmS deficiency resulted in a decrease in SdS protein abundance. Notably, ODC knockdown reduced SdS, SmS, xanthine dehydrogenase-1 protein levels, and decreased specific metabolite concentrations in fat body at 24 h PBM. In addition, RNAi-mediated ODC, SdS and SmS knockdown impacted transcript levels of genes involved in polyamine and purine pathways in fat body at 24 h PBM. Our findings uncover unique crosstalk regulations within the polyamine pathway and between polyamine and purine pathways.

The two-spotted spider mite Tetranychus urticae rapidly evolves resistance to acaricides, frequently through the upregulation of detoxification enzymes such as cytochrome P450 monooxygenases. Among these, CYP392A16 and CYP392D2 have repeatedly been implicated as major resistance determinants, yet their in vivo contributions remain incompletely resolved. Here, we used efficient CRISPR/Cas9-mediated genome editing to generate stable knockouts of CYP392A16 and CYP392D2 in a multi-resistant genetic background. Contrary to expectations, loss of either gene resulted in surprisingly limited effects on acaricide susceptibility, with increased sensitivity observed only for a subset of compounds. Experimental evolution assays revealed a moderate, host-dependent fitness cost associated with disruption of CYP392D2, but not CYP392A16. Transcriptomic analyses further uncovered stark contrasts between the knockouts, with CYP392D2 loss triggering extensive regulatory reprogramming suggestive of compensatory detoxification pathways. Together, our results demonstrate that even resistance-associated detoxification genes that are considered functionally validated in vitro can have strong context-dependent phenotypic effects in vivo, underscoring the complexity of resistance gene networks and highlighting the need to interpret CRISPR/Cas9 knockout phenotypes within the broader genetic and regulatory context.

Defensins are evolutionarily conserved antimicrobial peptides (AMPs) considered a cornerstone of innate immunity. However, the rapid emergence of resistance in pathogens like Staphylococcus aureus challenges our understanding of defensin efficacy in vivo. We used the blowfly Calliphora vicina-an insect adapted to pathogen-rich environments-to investigate S. aureus resistance dynamics. During an active immune response, defensin concentrations in larval hemolymph reach 11-16 μM. At these levels, purified defensin effectively eradicates both planktonic cells and pre-formed S. aureus biofilms (MBEC90 = 50 μg/mL). Crucially, S. aureus develops high-level genetic resistance to the defensin within only nine passages, and the total larval AMP complex fails to prevent this adaptation. Since defensin accounts for over 90% of the hemolymph's anti-Gram-positive activity, this rapid resistance renders the entire systemic humoral response ineffective from an evolutionary perspective. Our findings challenge the traditional view of defensins as a primary "first line of defense." Instead, we propose a "Final Clearance" hypothesis, suggesting that systemic AMPs act as a secondary precision tool to eliminate pathogens already decimated by cellular and enzymatic responses. This shift is vital for understanding innate immunity stability and developing sustainable antimicrobial strategies.

Reproduction in insects is a tightly regulated process that relies on the accumulation of yolk protein precursors (YPPs) to support embryonic development. Vitellogenin, the main YPP, is synthesized in the fat body-analogous to the liver and adipose tissue of vertebrates-and transported through the hemolymph to the ovarian follicles, where it is internalized by developing oocytes via the vitellogenin receptor (VgR). Despite its essential role in vitellogenesis, the functional characterization of insect VgR remains incomplete. Triatomines are hematophagous insects and vectors of Trypanosoma cruzi, the causative agent of Chagas disease. In this study, we characterized the VgR of Dipetalogaster maxima (DmaxVgR), a triatomine species, using biochemical, molecular, and bioinformatics approaches. The DmaxVgR gene was cloned, sequenced, and annotated, revealing a highly conserved protein sequence. Phylogenetic analysis clustered VgR amino acid sequences by taxonomic groups. Structural modeling of DmaxVgR showed a conserved folding pattern, enabling docking analyses with a modeled vitellogenin and indicating a stable interaction between the two proteins. DmaxVgR gene silencing disrupted follicular architecture and reduced vitellogenin uptake by the oocytes, resulting in increased vitellogenin levels in the hemolymph. Concurrently, elevated vitellogenin transcript levels and vitellogenin amounts in the fat body suggest a feedback mechanism regulating YPP production. Together, our findings provide new insights into the role and molecular regulation of VgR in triatomine reproduction and offer a framework for future studies exploring its potential relevance for vector control strategies.

Aedes aegypti is a major vector of arboviruses that pose major threats to global public health. In female mosquitoes, the fat body governs nutritional metabolism and vitellogenesis after a blood meal, with its functional state directly determining fecundity. Although endocrine and nutritional signals regulating mosquito reproduction have been widely studied, the role of non-coding RNAs (ncRNAs) and competing endogenous RNA (ceRNA) networks in fat body metabolic homeostasis remains poorly understood. To address this, we performed whole-transcriptome sequencing of fat bodies from sugar-fed and blood-fed females and identified 30 circRNAs, 142 miRNAs, 874 lncRNAs, and 4362 differentially expressed mRNAs, with significant enrichment in nutrient metabolism, energy homeostasis, and mTOR/MAPK signaling. Based on these data, we constructed ceRNA networks centered on blood-responsive miRNAs. Focusing on the downregulated, high-abundance aae-miR-283, we generated a transgenic overexpression line and found that elevated aae-miR-283 markedly impaired ovarian development, oviposition, and egg hatching. Mechanistically, dual-luciferase reporter assays confirmed that aae-miR-283 directly binds to the 3'UTR of ApoLp-Ⅱ/Ⅰ, supporting ApoLp-Ⅱ/Ⅰ as a direct target of aae-miR-283. The lncRNA MSTRG.88121.1 also showed binding to aae-miR-283 in the dual-luciferase reporter assay, suggesting that it may participate in an aae-miR-283-associated regulatory network. Functional studies revealed that both aae-miR-283 overexpression and ApoLp-Ⅱ/Ⅰ knockdown disrupted lipid homeostasis, increasing triacylglycerol accumulation in the fat body while depleting ovarian lipid stores, supporting a role for aae-miR-283-ApoLp-Ⅱ/Ⅰ regulation in lipid transport from the fat body to the ovaries. Our findings reveal a novel aae-miR-283-centered regulatory mechanism controlling reproductive energy allocation in Ae. aegypti and provide potential molecular targets for metabolic intervention-based vector control strategies.

Plant immunity against herbivorous insects is often triggered by insect-derived salivary elicitors, yet the molecular mechanisms underlying their perception and signaling remain limited understood. Here, we identified a protein disulfide isomerase, MsPDI3, from the salivary glands of the oriental armyworm (Mythimna separata), which functions as an elicitor of plant defense. Transient expression of MsPDI3 in tobacco induced hypersensitive response-like necrosis and elevated salicylic acid (SA) levels. Mechanistically, MsPDI3 is secreted into maize plant through feeding, silencing MsPDI3 in larvae attenuated maize defense and enhanced larval growth, indicating its role in activating plant immunity. Using yeast two-hybrid screening and bimolecular fluorescence complementation, we demonstrated that MsPDI3 interacts with a maize Src homology 3 domain-containing protein (ZmSH3), at the plasma membrane. Virus-induced gene silencing of ZmSH3 abolished MsPDI3-triggered SA accumulation and compromised plant resistance, suggesting ZmSH3 as a key component in the defense signaling cascade. These findings established a novel link between SH3-mediated signal transduction in mediating insect-derived elicitor perception and SA-dependent defense activation, providing new insights into the molecular dialogue between plants and chewing insects.

Insecticide resistance is threatening malaria control. While the evolution and spread of resistance has been linked to scale-up in the distribution of public health insecticides, the role of environmental pollutants such as the polyaromatic hydrocarbons (PAHs) from industrial and agricultural use remains largely uncharacterized. The PAHs are potent ligands of the aryl hydrocarbon receptor (Ahr) transcription factors involved in the regulation of xenobiotic metabolizing enzymes, and potentially involved in insecticide resistance. Here, using field insecticide-resistant (Auyo) An. coluzzii and a laboratory-susceptible colony (Ngousso), we conducted a multi-generational selection experiment using naphthalene, fluorene and a mixture of both PAHs. After ten generations, the changes in susceptibility to insecticides were monitored using WHO bioassays and whole-transcriptome analysis (RNASeq) was conducted. Compared with the non-selected colony lines, PAH exposures significantly reduced pyrethroid and DDT resistance in the field population, suggesting a fitness cost associated with established resistance. In contrast, Ngousso showed a significant increase in DDT resistance (p = 0.01) at the tenth generation. A significant increase in permethrin resistance was also observed at the seventh generation (p = 0.03). Several candidate genes from the major detoxification classes were overexpressed in the selected lines (including GSTe2, CYP6Z1, and CYP6P4); the most consistent were CYP6M4 and CYP4C27, as well as those from the Ahr pathway. Heterologous expression of CYP6M4 revealed its ability to metabolise pyrethroids, including permethrin, deltamethrin, and α-cypermethrin, as well as PAHs (naphthalene and fluorene). These findings establish the role of environmental pollutants as additional drivers of metabolic insecticide resistance in An, coluzzii.

The gap (apterous and rudimentary gonads) mutation is a spontaneous mutation that causes defects in adult organs in the silkworm Bombyx mori. Although gap larvae exhibit normal external morphology, gap mutants exhibit severe defects in adult organs, suggesting that the gap gene is required for the development of imaginal discs and the primordia of adult organs. Genetic analysis of the gap mutation provides an opportunity to understand the molecular mechanisms underlying the development of adult organs. In this study, we performed RNA-seq analysis of ovaries from gap mutants. De novo assembly of RNA-seq reads identified the insertion of a retrotransposon in the gene encoding the Bombyx ortholog of protein-cysteine N-palmitoyltransferase (Rasp), which is required for Hedgehog signaling in Drosophila. PCR and subsequent sequencing analyses revealed that an LTR transposon is inserted into exon 1 of the BmRasp gene. To verify the involvement of BmRasp in the gap phenotype, we generated a knockout allele of BmRasp (BmRasp[KO]) using the CRISPR/Cas9 system. Both homozygous knockout mutants and F1 pupae with the gap/BmRasp[KO] genotype exhibited gap-like phenotypes characterized by apterous pupa, indicating that BmRasp corresponds to the gap locus. To investigate why zygotic BmRasp expression is dispensable during embryogenesis but essential for the development of adult organs, we analyzed BmRasp expression levels in eggs and larval tissues. Furthermore, testis transplantation experiments demonstrated that BmRasp is dispensable for spermatogenesis, despite the fact that the testis exhibits the highest expression level of BmRasp among the tissues analyzed. Finally, we discuss the physiological roles of Hedgehog signaling in embryogenesis and in the development of imaginal discs in B. mori.

Solute carriers (SLCs), the largest human transmembrane transporter family regulating cellular metabolism, mediate drug resistance in mammals but remain understudied in insects. While several SLCs are known to contribute to drug resistance evolution in mammals, their functions are comparatively underexplored in insects. The brown planthopper (Nilaparvata lugens) is a major sucking mouthparts rice pest and causes severe damage to rice growth and food production in Asia. Presently, field populations of N. lugens have developed resistance to many insecticides. However, the underlying resistance mechanisms are complex and variable, and elucidating them is essential for effective management of resistant populations. In this study, we show that NlSLC23A1 overexpression confers resistance to insecticides in field N. lugens populations. Its expression is significantly induced by neonicotinoids; transgenic Drosophila melanogaster expressing NlSLC23A1 exhibit reduced neonicotinoid susceptibility. Knocking down NlSLC23A1 increases N. lugens' sensitivity to imidacloprid (but not other neonicotinoids), and imidacloprid specifically upregulates NlSLC23A1 in the pest's brain. These findings identify NlSLC23A1 as a key mediator of imidacloprid resistance, providing mechanistic insights and a potential target for pest management.

In the Drosophila antenna, olfactory sensilla are the fundamental unit of odor detection and processing. Sensilla house one to four olfactory neurons enwrapped by three distinct support cells: thecogen, trichogen, and tormogen cells. While olfactory neurons and their odor receptors have been extensively studied in Drosophila, and increasingly in other insect species, much less is known about the support cells and their functional roles within the mature sensillar unit. A critical barrier to studying their function has been the lack of validated markers to distinguish and manipulate these cells in adult flies. Here, we systematically identify and characterize tools to differentially label specific olfactory support cells in the antenna, including sv[T2A-GAL4] as a broad support cell marker. First, we confirm that ASE5-GAL4 labels olfactory tormogen cells, which are also strongly labeled by an anti-Su(H) antibody. Next, we demonstrate that a prospero antibody broadly and exclusively marks thecogen cells, unlike the previously used marker nompA-GAL4. The pros-positive cells are also labeled by a recently developed pros[T2A-GAL4] line, but surprisingly, they are distinct from those labeled by a widely used pros-GAL4 line, which instead labels olfactory neurons. Third, we generate and validate the first reporter line for olfactory trichogen cells, atk[GAL4]. Finally, we provide evidence that each tool may function similarly in the maxillary palp. Together, our findings create a framework for distinguishing olfactory support cell classes in the adult fly antenna, a crucial step towards understanding their individual contributions to sensillar function.

CPEB (Cytoplasmic Polyadenylation Element Binding protein) comprises a highly conserved family of RNA-binding proteins that serve as key post-transcriptional regulators in diverse biological processes. In Drosophila melanogaster, the CPEB family member Orb2 plays vital roles in spermatogenesis, particularly during the flagellar axoneme elongation. Bombyx mori, like other lepidopterans, produces dimorphic sperm: eupyrene sperm and apyrene sperm. However, whether CPEB functions in this spermatogenesis process remains unknown. In this study, we demonstrate that B. mori orb2 (Bmorb2) is critical for the proper progression of eupyrene spermatogenesis, particularly at the elongation stage. We found that Bmorb2 is predominantly expressed in testis, specifically in spermatocytes and early elongating eupyrene sperm, but absent from late eupyrene sperm and apyrene sperm. Functional analysis reveals that Bmorb2 depletion severely disrupts eupyrene sperm development, causing aberrant nuclear positioning and shortened flagella. Consequently, these defective eupyrene sperms fail to migrate to the spermatheca after mating, leading to complete male sterility. Partial rescue of male sterility was achieved through sequential mating of females with Bmorb2 and Sex-lethal mutant males, demonstrating that apyrene sperm from Bmorb2 mutants retain normal function. In addition, our data suggest that the spermatogenesis defects in mutants are potentially associated with disrupted energy metabolism, impaired chromatin remodeling, and dysregulated assembly of motor protein and IFT complexes. Our findings demonstrate that Bmorb2 functions as a crucial stage-specific regulator of late spermiogenesis in B. mori.