As a major arbovirus vector, Aedes aegypti relies on high fecundity, which is governed by nutrient metabolism and vitellogenesis via the PI3K/Akt/TOR pathway. However, the upstream regulators linking nutrition to this pathway remain unclear. Here, we found translation elongation factor EF1A2 (AAEL017096) is a key regulator of reproduction. Knockdown via RNAi and CRISPR/Cas9-mediated knockout of EF1A2 led to complete arrest of ovarian development and sharply reduced egg production-a conserved phenotype also observed in Aedes albopictus and Culex quinquefasciatus. Mechanistically, loss of EF1A2 disrupted mobilization of lipid and glycogen reserves from the fat body, causing nutrient retention and suppressed catabolism. Additionally, EF1A2 binds to the PI3K regulatory subunit p85α to promote Akt phosphorylation, thereby activating the PI3K/Akt/TOR pathway and driving vitellogenin (Vg) transcription, essential for oocyte maturation. These findings identify EF1A2 as a novel upstream regulator of the PI3K/Akt/TOR pathway, linking nutrient metabolism to mosquito reproductive output. This study reveals a non-canonical function of an EF1A family member in upstream pathway regulation and provides new insights into how mosquitoes integrate nutritional signals with reproduction, potentially informing future vector control strategies.

Adult females of the two-spotted spider mite, Tetranychus urticae Koch, enter a photoperiodically induced diapause to overwinter. Diapause in T. urticae is accompanied by reproductive arrest and the orange body coloration that arises from the accumulation of astaxanthin esters. How these two traits are coordinated at the molecular level remains poorly understood. Here, we compared the proteomes of adult females reared under diapause-inducing (long-night) and non-diapause-inducing (short-night) photoperiods using liquid chromatography-tandem mass spectrometry, followed by RNA interference (RNAi) to validate the function of candidate genes. The carotenoid biosynthesis enzymes phytoene desaturase (TuPDS) and lycopene cyclase/phytoene synthase (TuLCPS), both encoded by genes horizontally transferred from fungi, were more abundant in diapausing females than in non-diapausing females. RNAi of TuPDS, TuLCPS, and TuCYP384A1 (a candidate β-carotene ketolase) markedly reduced orange pigmentation as well as β-carotene and astaxanthin contents, demonstrating that these enzymes are required for diapause-associated pigmentation. Our proteomic analysis further identified a single PLAT (Polycystin-1, Lipoxygenase, Alpha-toxin) domain protein, TuPLAT10, as one of the most strongly upregulated proteins in diapausing females. The PLAT domain is a lipid-binding module, suggesting a role for TuPLAT10 in lipid metabolism. In addition to the suppression of orange pigmentation, RNAi of the TuPLAT10 gene resumed oviposition even under diapause-inducing conditions and reduced TuPDS, TuLCPS, and TuCYP384A1 protein levels, despite the absence of sequence similarity to their genes. We propose that TuPLAT10 acts as a lipid-allocation switch that, in response to photoperiodic information, partitions fatty acids between astaxanthin esterification and yolk lipid supply, thereby coupling reproductive arrest and carotenoid pigmentation during diapause in T. urticae.

Extreme weather events can profoundly alter the transmission dynamics of dengue virus (DENV) by its primary vector, Aedes aegypti. Understanding how mosquitoes respond to heat stress and how this affects viral transmission is critical for informing global public health strategies. RNA cytosine methylation is an evolutionarily conserved stress-response mechanism in A. aegypti that links environmental perturbations to shifts in RNA metabolism and gene expression. Here, we show that a 5-day heatwave initiated at the time of DENV2 acquisition increases the lifespan of surviving mosquitoes, thereby extending the window of potential virus transmission. Heatwave-exposed mosquitoes exhibit elevated global 5-methylcytosine RNA (5mC-RNA) levels and suppressed antiviral gene expression, facilitating efficient viral replication. Pharmacological disruption of 5mC-RNA with azacytidine counteracts heatwave-induced increases in transmission potential by enhancing antiviral transcription, reducing DENV2 infection prevalence from 78% to 37%, and restoring mosquito late-life survival to rates comparable to non-stressed controls. This integrative approach reveals an epitranscriptomic mechanism by which climate extreme scenarios like heatwaves can amplify dengue transmission risk, and highlights RNA methylation as a potential target for mitigating DENV transmission.

Complex II inhibitors (METI-II) are important acaricides for controlling the two-spotted spider mite, Tetranychus urticae, but resistance to this relatively new mode of action is rapidly emerging. Here, we identified a novel sdhB substitution, S212N, in a highly resistant Turkish field strain, while a different substitution at the same position, S212I, has previously been associated with resistance, but without functional validation. We therefore used CRISPR-Cas9 gene editing to introduce S212N and S212I into a susceptible genetic background and revert the resistant-field S212N allele to the susceptible S212 genotype. Functional validation revealed mutation- and compound-specific effects. Introduction of S212I conferred high resistance to all tested METI-II acaricides (resistance ratios >200), whereas S212N caused limited resistance to cyflumetofen and pyflubumide and no detectable resistance to cyenopyrafen. In contrast, reversion of the S212N allele in the resistant field strain significantly increased susceptibility to pyflubumide and cyenopyrafen, demonstrating that the phenotypic effects of sdhB mutations depend strongly on genetic background. However, resistance levels remained elevated after reversion, indicating additional resistance mechanisms. Transcriptomic comparisons between the susceptible GSS strain and the resistant field strain revealed overexpression of detoxification genes previously implicated in METI-II metabolism, suggesting that metabolic detoxification acts synergistically with target-site resistance. Together, our results provide functional validation of two sdhB resistance mutations and show that their phenotypic effects can vary by chemical compound and genetic background. More broadly, our findings demonstrate that fully resolving resistance mechanisms requires not only introducing candidate mutations into susceptible backgrounds but also removing them from resistant populations.

Drug-resistant bacteria pose an escalating threat to global public health, underscoring the urgent need for novel antibacterial agents. Natural green control strategies offer sustainable solutions against environmental pathogenic bacterial infections. Among them, natural antibacterial molecules derived from eukaryotic sources are undoubtedly the most compelling, owing to their safety, biocompatibility and resistance to drug resistance. The black soldier fly (Hermetia illucens, BSF) is recognized as a valuable reservoir of antibacterial factors due to its exceptional resistance to dense microbial environments. In this study, three previously uncharacterized antibacterial proteins were systematically identified from black soldier fly larvae (BSFL) using high-performance liquid chromatography-mass spectrometry (HPLC-MS). Hemoglobin (Hb) exhibited the strongest antibacterial activity in vitro, with low cytotoxicity, thermal stability, and acid resistance. Mechanistic analyses indicate that Hb compromises bacterial cell membrane integrity via biofilm disruption and direct membrane interaction, leading to nucleic acid and protein leakage, increased intracellular reactive oxygen species (ROS), and impairment of ATP-dependent energy metabolism. Furthermore, HiHb was structurally predicted to target intracellular components, including DNA topoisomerase and ribosomal proteins. Together, these multi-target effects accelerate bacterial cell death. Notably, ectopic expression of Hb markedly enhanced antibacterial ability in silkworms, suggesting that its antibacterial function is transferable across species. These findings highlight Hb derived from BSFL as a potent antibacterial protein, offering a promising resource for the development of novel therapeutics against bacterial infections and reduce the application of harmful bactericide.

Wing scales are essential for insect flight, thermoregulation, camouflage, and mating behavior. During routine rearing of the bivoltine silkworm Qiufeng, a scaleless wing mutant, designated P33, was identified, in which adult wing scales were severely reduced or absent. However, the gene responsible for the scaleless wing mutation in the silkworm remains unknown. Here, Genetic analysis indicated that this phenotype is controlled by a single autosomal recessive locus. Positional cloning mapped the responsible locus to a 0.24 Mb interval on chromosome 13 of the Bombyx mori genome containing 21 predicted genes. qRT-PCR analysis of these candidate genes in the wing primordia of Qiufeng and P33 at pupal day 3-4 and pupal day 6-7 revealed that, among the 21 genes, BmILRUN(KWMTBOMO08006) displayed a stable upregulation expression in P33 compared to Qiufeng. Sequence analysis further revealed that the BmILRUN gene in P33 carries four synonymous substitutions in the coding region, together with a 284bp deletion in the third intron and a 633bp insertion in the fourth intron. 5' and 3' RACE results revealed that both the 5'-UTR and 3'-UTR regions of BmILRUN were truncated in P33 relative to Qiufeng. CRISPR-Cas9-mediated knockout of BmILRUN resulted in a significant reduction in its expression and a marked decrease in wing scales. Together, these results demonstrate that BmILRUN is essential for wing scale formation in Bombyx mori providing insight into the molecular regulation of wing scale development.

Chemical cues play a critical role in driving key insect behaviors and their detection is mediated by proteins such as binding proteins and chemosensory receptors. While holometabolous insects exhibit distinct morphologies, physiologies and ecologies across developmental stages, most research on chemical detection has focused on adults. Here, we analyze the larval chemosensory transcriptome of the polyphagous moth Spodoptera littoralis, the life stage responsible for plant damage. We identified expression of 39 previously unreported chemosensory genes, substantially expanding the known larval repertoire. Functional analyses of larval odorant receptors (ORs) revealed two receptors responding to terpenes and aromatic compounds emitted by host plants, and behavioral assays indicated that some of these ligands elicited attraction, demonstrating ecological relevance. A larval OR response profile perfectly mirrored that of an unrelated OR expressed in adults, suggesting that these two ORs converged on similar function in larvae and adults. Our results highlight stage-specific chemosensory equipment and identify promising targets for modifying larval behavior, with potential applications for improved insect control.

The oriental fruit fly Bactrocera dorsalis is a globally invasive pest with increasing insecticide resistance that threatens sustainable crop production. ATP-binding cassette (ABC) transporters are key mediators of xenobiotic detoxification, but the molecular and regulatory basis of ABCC-type transporters in this species remains unclear. Here, we present the first systematic reannotation and functional analysis of the ABCC gene family in B. dorsalis and identify a novel post-transcriptional regulatory pathway involving miR-980. Using updated genomic and transcriptomic resources, we accurately annotated eight BdABCC genes with conserved nucleotide-binding domains (NBD) and transmembrane domains (TMD) and strong phylogenetic conservation with dipteran homologs. Spatiotemporal expression profiling showed constitutive expression across development, elevated transcription in the midgut, Malpighian tubules and fat body, and transcriptional responsiveness of several members, particularly BdABCC1, BdABCC2, BdABCC4, and BdABCC6, to multiple insecticides. RNA interference and MK-571 inhibition demonstrated that BdABCC1 contributes to avermectin tolerance. Furthermore, luciferase reporter assays, RNA pull-down and fluorescence in situ hybridization confirmed that miR-980 directly targets the BdABCC1 coding sequence, with miR-980 overexpression suppressing BdABCC1 and increasing avermectin-induced mortality. Together, these findings identify a previously uncharacterized miR-980-BdABCC1 regulatory axis that modulates avermectin tolerance and highlights post-transcriptional regulation of ABC transporters as a potential molecular target for future RNAi-based pest management strategies.

Wing polyphenism-the developmental switch between flight-capable long-winged and flightless short-winged morphs-is a central example of phenotypic plasticity, yet its genomic and regulatory architecture remains poorly resolved outside a few acridoid models. Here we report a chromosome-level genome for the pygmy grasshopper Bolivaritettix circocephalus (Tetrigidae), spanning 1.27 Gb across seven pseudochromosomes (scaffold N50 = 284 Mb; 96.0% complete BUSCOs) and encoding 21,985 protein-coding genes. Phylogenomic analysis of nine orthopterans placed B. circocephalus as sister to Eucriotettix oculatus (divergence ∼14.4 Ma), and gene-family analysis revealed 205 expanded families enriched for chromatin assembly and sensory perception, alongside 131 contracted families enriched for juvenile-hormone and terpenoid metabolism. A genome-wide association study of 50 individuals (30.9 million SNPs) across seven morphometric traits and wing morph identified 2084 candidate genes and recurrent pleiotropic loci, notably a Bonferroni-significant cluster on BoCi001 containing the lipid-storage gene Seipin (BSCL2) and a chemosensory/opsin cluster on BoCi005. A developmental transcriptome (six nymphal instars and both adult morphs) validated these candidates, localized the principal transcriptional switch to the instar3-instar4 transition, and-through inflection-network and WGCNA analyses-converged on a wing-morph-specific module enriched for lipid and energy metabolism. FAR-17a/AIG1-like (BoCi004-g2346) and the respiratory subunit NDUFV1 emerged as genes simultaneously supported by genetic association, differential expression, inflection response and module hubness. These results establish the first comprehensive genomic resource for wing-polyphenic Tetrigidae and nominate lipid/energy metabolism, coordinated by transcriptional and chromatin regulators, as a core axis of the dispersal-reproduction trade-off in a deeply divergent orthopteran lineage.

We identify PAN4 (GAYTFKIRRK), an AI-designed host-directed immunomodulatory peptide, as a modulator of intestinal immune homeostasis in Drosophila melanogaster. PAN4 lacks detectable bactericidal activity against Pseudomonas aeruginosa in vitro but improves host survival following infection, supporting a host-directed mode of action rather than direct pathogen killing. Genetic analyses indicate that PAN4-associated protective phenotypes require PGRP-SC1a function, a negative regulator of the Imd pathway. Consistently, PAN4 attenuated infection-induced suppression of PGRP-SC1a-associated responses and maintained immune-related homeostasis without broadly inducing antimicrobial peptide expression. PAN4 expression was also associated with multiple physiological alterations in the intestine, including changes in mitochondrial signal, luminal acidification, calcium-associated responses in enteroendocrine cells, and microbiota composition. These observations suggest that PAN4 activity is associated with broad intestinal physiological changes, although the causal relationships among these phenotypes remain to be determined. Functional assays further show that PAN4 reduces stress-associated intestinal barrier dysfunction and suppresses abnormal intestinal stem cell overproliferation, linking immune regulation with tissue homeostasis. Together, these findings identify PAN4 as an AI-designed host-directed immunomodulatory peptide that contributes to intestinal immune homeostasis and tissue resilience in Drosophila. Consistent with these observations, oral PAN4 administration in honeybees was associated with a context-dependent protective effect following bacterial challenge, suggesting that PAN4-associated protective effects may extend beyond Drosophila under appropriate delivery conditions.

Non-neuronal acetylcholine (ACh) has been shown to elevate intracellular calcium levels in the Malpighian tubules (MTs) of Mythimna separata. However, the physiological functions and mechanisms of this ACh-mediated signaling in MTs remain largely unknown. In this study, pharmacological analyses suggest that muscarinic receptors trigger ACh-induces Ca2+ signal via the phospholipase C (PLC) and inositol trisphosphate receptor (IP3R) pathway in M. separata MTs. This Ca2+ signal activates a Ca2+-dependent nitric oxide synthase (NOS1), which is specifically localized within the principal cells. NADPH diaphorase and pharmacological analyses confirmed that ACh stimulates NOS activity and elevates intracellular NO levels. This effect was inhibited by the NOS inhibitor L-NAME and by inhibitors of the mAChR-PLC-IP3R cascade. The resulting NO production led to elevated intracellular cGMP levels. Ramsay assay revealed that ACh significantly stimulated fluid secretion, and this effect was suppressed by inhibiting any component of the mAChR-PLC-IP3R-NOS-cGMP signaling pathway. These findings indicate that non-neuronal ACh triggers NO release in the principal cells of M. separata MTs primarily through PLC-IP3R pathway, and that this NO signal in turn promotes fluid transport of the tubules. This study provides new evidence for a functional link between non-neuronal ACh and intracellular NO production in insects.

Climate change threatens ectotherms, yet the cellular mechanisms underlying thermal injury remain poorly characterized. Here, we used an integrative approach combining acclimation treatments, transcriptional profiling of cell-death-related genes, and fluorescence-based cellular markers to compare cold- and heat-induced cell death pathways in third-instar Drosophila melanogaster larvae. Individuals were exposed to cold (-3°C) or heat (38°C) stress across three acclimation phenotypes. Cold acclimation enhanced cold tolerance but reduced heat tolerance, and vice versa, revealing clear acclimation trade-offs. Melanized lesions (black spots) appeared during recovery, predominantly after cold stress, and strongly predicted mortality; cold acclimation reduced their incidence from 82% to 13%. Cold stress induced upregulation of pro-apoptotic genes (reaper, grim, sickle) and caspases (Dronc, Dcp-1, Damm), whereas heat stress predominantly induced Hsps genes (upregulation over 4000-fold) and some autophagy-related genes (Atg1, Atg6). Larvae with black spots exhibited hallmarks of necrosis, including low cell viability, severe cytoskeletal collapse, and nuclear loss. Heat-stressed larvae showed elevated cytosolic Ca[2+], while all stressed groups exhibited increased reactive oxygen species. No autophagy-associated acidification was detected under our conditions. We propose that thermal stress induces a continuum of cellular responses: moderate stress activates protective mechanisms, greater damage triggers apoptosis, and severe injury in susceptible phenotypes leads to necrosis with secondary melanization in sensitive tissues. By distinguishing the respective signatures of apoptosis, necrosis, and autophagy under contrasting thermal stresses, our study identifies necrosis as a major and previously underappreciated driver of thermal mortality in insects. Thermal acclimation mitigates cell death but only when matching subsequent stress, highlighting physiological trade-offs.

The tumor suppressor protein p53 is extensively characterized as a regulator of genome integrity, apoptosis, and metabolic homeostasis in mammalian systems. In early-diverging metazoans, however, p53 metabolic functions remain poorly understood. Here, we investigated the metabolic responses of BME26 cells, an embryonic cell line derived from the cattle tick Rhipicephalus microplus, exposed to PRIMA-1. Pharmacological treatment using PRIMA-1 reduced BME26 cell viability in a dose- and time-dependent manner, exhibiting similar effects to those observed in human breast cancer cells, which were utilized as comparative reference models displaying distinct PRIMA-1 response profiles. Annexin V/7-AAD staining and morphological analyses showed that the decrease in BME26 viability was primarily caused by apoptosis. Metabolomic profiling by [1]H-[13]C HSQC NMR revealed coordinated changes in central carbon metabolism, amino acid pathways, and choline-derived metabolites, indicating broad metabolic remodeling upon PRIMA-1 exposure. Despite showing no effect on glucose uptake and glycogen content, PRIMA-1 selectively reduced the expression of key glycolytic genes, including hexokinase (HK) and pyruvate kinase (PK), and significantly reduced glucose-6-phosphate dehydrogenase (G6PDH) gene expression. This was accompanied by decreased intracellular NADPH levels, while isocitrate dehydrogenase-1 (IDH-1) gene expression remained unaffected, suggesting a pathway-specific metabolic modulation rather than global energetic collapse. Collectively, our findings demonstrate that PRIMA-1 treatment in tick embryonic cells restricts glycolytic and pentose phosphate pathway flux, limiting NADPH production and reshaping redox balance to favor apoptosis. These results support the potential conservation of metabolic pathways sensitive to PRIMA-1, which in mammalian systems are linked to modulation of the p53-G6PDH-NADPH axis, thereby expanding our understanding of metabolic regulation and stress responses in arthropod biology.

Queens of eusocial insects maintain their reproductive monopoly through queen pheromones. We recently identified the sesquiterpene alcohol (3R,6E)-nerolidol (RNERO) as the queen pheromone in the termite Embiratermes neotenicus and characterized a terpene synthase, EneoC, that produces RNERO in vitro. Here, we aimed to (i) determine whether EneoC is responsible for RNERO biosynthesis in vivo, (ii) analyze its expression and protein abundance patterns across castes in the context of terpenoid biosynthesis and endocrine regulation, (iii) estimate the relative investment into queen signaling based on EneoC protein abundance compared to other queen proteins, (iv) assess whether eggs actively produce RNERO, and (v) localize RNERO biosynthesis in queens. We report that EneoC is strongly upregulated in queens at both transcript and protein levels, and that it ranks among the most abundant queen proteins. Using RNA interference, we demonstrate that EneoC produces RNERO in vivo. We show that EneoC is exclusively expressed in queen epidermis all over the body surface, specifically within structures derived from mitochondria in class 1 secretory cells. We identify an enrichment of additional terpenoid biosynthetic enzymes in queens, including the farnesyl diphosphate synthase EneoB, expected to supply the substrate for EneoC. Both enzymes contain mitochondrial targeting sequences, suggesting their co-localization within modified mitochondria in class 1 cells. The absence of EneoC in eggs indicates that eggs acquire RNERO from queens rather than via endogenous biosynthesis. Taken together, our findings establish EneoC as a key element underlying the reproductive dominance of queens within the complex breeding system of E. neotenicus.