This study investigated the temporal dynamics of extracellular signal-regulated kinase (ERK) phosphorylation, together with the expression profiles of its putative upstream ligands and downstream target genes, during embryonic development in Bombyx mori. The results revealed distinct ERK phosphorylation dynamics: significantly higher levels of phosphorylated ERK were observed between Days 1 and 6 after oviposition in developing eggs (non-diapause and HCl-treated eggs), whereas diapause eggs exhibited low levels. The temporal expression patterns of genes in the prothoracicotropic hormone (PTTH)/torso and epidermal growth factor (EGF) signaling pathways-spitz (spi), rhomboid (rho), and Egf receptor (Egfr)-did not differ between diapause and HCl-treated eggs. In contrast, the EGF pathway gene vein (vn) and genes in the transforming growth factor (TGF)-β signaling pathway-including the ligand myoglianin (myo) and its receptor babo-displayed distinct temporal expression patterns. Similarly, Jelly belly (Jeb)/anaplastic lymphoma kinase (Alk), fibroblast growth factor (Fgf)/Fgf receptor (Fgfr), neurotrophin (NT), and Toll-receptor ligands (spätzle family members (spz2, spz4, and spz5)) showed differential expression, with levels significantly elevated during the middle and late stages of embryonic development in HCl-treated eggs. Non-diapause and chilled eggs also exhibited increasing expression patterns of these genes, similar to those observed in HCl-treated eggs. Additionally, ERK target genes pointed and huckebein (hkb) showed higher and broader expression peaks in developing eggs compared to diapause eggs. This study provides a comprehensive analysis of the transcriptional regulation of upstream and downstream ERK signaling components during embryonic development and reveals new insights into the mechanisms underlying embryonic diapause.

Western flower thrips (WFT) Frankliniella occidentalis cause extensive damage to crops either by direct feeding injury or by transmitting diseases. Current management strategies against this pest rely heavily on synthetic insecticides, leading to issues such as resistance development and environmental concerns. Several safer alternatives including RNA interference (RNAi), also referred to as post-transcriptional gene silencing (PTGS), are being extensively researched to develop species-specific molecular pesticides for the management of this pest. However, the success of RNAi depends on the selection of lethal targets and the delivery of double-stranded RNA (dsRNA) to the target pest. In this study, we demonstrate the uptake of dsRNA by WFT through artificial diet feeding and its distribution within the insect. Several candidate gene targets were evaluated, leading to the identification of promising RNAi targets for thrips management. In addition, dsRNA production was successfully standardized using a recombinant bacterial expression system for large-scale applications. We also observed degradation of naked dsRNA following feeding, likely due to nuclease activity associated with thrips feeding processes, highlighting a key limitation for RNAi efficacy. Overall, our findings support the potential of RNAi as a targeted pest management strategy for WFT. The results emphasize the importance of strategic gene selection, improved delivery systems, and scalable dsRNA production for the development of effective RNAi-based molecular biopesticides.

In holometabolous insects, the pupal stage enables the transition from larva to adult and provides a useful system for studying the organization and evolution of ecdysis behavior. However, the motor programs underlying the formation of exarate pupae remain less well characterized than those of obtect and coarctate pupae. Here, we describe the pupal ecdysis sequence in Henosepilachna vigintioctomaculata at phase-resolved behavioral resolution. Pupal ecdysis was subdivided into pre-ecdysis, ecdysis, and post-ecdysis. During pre-ecdysis, prepupae exhibited slight dorso-ventral (D-V) contractions followed by progressively strengthened twitch contractions that loosened the old exoskeleton. During ecdysis, successive anteriorly directed peristaltic posterior-anterior (P-A) contractions, together with D-V contractions and lateral swinging, drove exuvial shedding and the freeing and stretching of pupal wings and cephalothoracic appendages. During post-ecdysis, posteriorly directed anterior-posterior (A-P) and D-V movements restored stretched appendages to their normal sizes and positions. RNA interference against the ecdysis-triggering hormone gene Hveth, delivered by microinjection of in vitro synthesized dsRNA or by oral administration of bacterially expressed dsRNA, impaired pupation. Knockdown of Hveth prolonged pre-ecdysis, reduced twitch frequency, and partially or completely disrupted ecdysis and post-ecdysis movements. Together, these results define the pupal ecdysis sequence of an exarate coleopteran pupa and support a requirement for ETH signaling in the proper execution and coordination of this motor program.

The epidermal growth factor receptor (EGFR) signaling pathway is an important regulator of insect reproduction, yet its function in parasitoid wasps remains largely unknown. Here, we investigated the role of EGFR in the reproductive development of the parasitoid wasp Leptopilina drosophilae, an effective natural enemy of the globally invasive pest Drosophila suzukii. We cloned the LdEGFR gene and confirmed its structural conservation through phylogenetic analysis. Using RNA interference (RNAi), we demonstrated that LdEGFR is essential for ovarian development but dispensable for testis development. Intriguingly, this sexually dimorphic functional requirement contrasts with its expression pattern, as LdEGFR transcript levels were approximately 171-fold higher in testes than in ovaries. Transcriptomic analysis of ovaries following LdEGFR knockdown revealed significant down-regulation of genes involved in transporter, DNA replication, cell cycle, and oocyte meiosis pathways, providing a molecular basis for the observed ovarian defects. Our findings establish LdEGFR as a critical regulator of female reproduction in L. drosophilae and highlight a notable decoupling between transcript abundance and functional essentiality. This study not only advances the understanding of reproductive regulation in parasitoid wasps but also identifies LdEGFR as a potential target for enhancing the mass rearing of this promising biocontrol agent.

The wheat stink bug, Aelia rostrata (Hemiptera: Pentatomidae), is a major pest of wheat in Central Anatolia. This species has an extended adult dormancy period lasting approximately 9 months. This period encompasses summer estivation and winter diapause in mountainous dormancy areas. Despite its economic importance and unusual dormancy biology, the molecular basis of seasonal dormancy in A. rostrata remains unexplored. Here, we combined RNA-seq, proteomics, and lipid content measurements to provide the first molecular insights into estivation and winter diapause responses in the fat body of the wheat stink bug. Transcriptomic enrichment analyses suggested that genes associated with protein synthesis, cytoplasmic translation, and ribosomal activity were enriched during estivation, whereas pathways related to catabolic processes, fatty acid metabolism, and pathogen response were associated with winter diapause. Complementary proteomic analyses identified mitochondrial and chitinase-like proteins with higher abundance during estivation, while proteins related to energy metabolism, including acyl-CoA dehydrogenases, were more abundant during winter diapause. Lipid measurements indicated significantly lower lipid reserves in post-diapause individuals compared to actively feeding adults, consistent with the utilization of lipid stores during dormancy and/or the transition to post-dormancy activity. Overall, this study provides the first transcriptomic and proteomic resource for A. rostrata and offers initial molecular insights into the physiological signatures associated with seasonal dormancy in this economically important pest.

The ectoparasite Varroa destructor (Mesostigmata: Varroidae) poses a major threat to honey bee colonies due to its role as a viral vector and its capacity to develop resistance to treatments. While physiological resistance and target-site mutations are well documented, behavioral mechanisms remain largely unexplored. This study investigated the potential existence of behavioral resistance in V. destructor, defined as a modification of behavior in response to an acaricidal molecule. Using Y-maze olfactometric assays, we evaluated mite responses to three acaricides-amitraz, tau-fluvalinate, and oxalic acid-across five French populations with distinct treatment histories. Forty groups of five mites were exposed to "acaricide/solvent" pairs, and choices were recorded after 18 h. Results revealed marked heterogeneity among populations. Two population exhibited significant avoidance of tau-fluvalinate, providing the first case of avoidance behavior in V. destructor, but no coinciding phenotypic resistance. No avoidance was detected for amitraz and oxalic acid. These findings suggest that behavioral resistance may exists in France but is not systematic. Identifying this phenomenon opens new perspectives on the parasite's adaptation to treatments and underscores the need to integrate behavioral aspects into control strategies.

The burgeoning global plastic crisis necessitates transformative solutions beyond current recycling and disposal methods. This study investigates the ability of wax moth larvae (Achroia grisella) to biodegrade low-density polyethylene (LDPE) and polylactic acid (PLA), emphasizing the complex interactions between the physiology of larvae, their gut microbiome, and the plastic degradation process. Using 16S ribosomal RNA sequencing, Seahorse bioassays, and advanced metabolomic and lipidomic profiling, we demonstrate that plastic consumption is associated with microbial and metabolic restructuring in larvae. LDPE-fed larvae displayed elevated microbial diversity, dominated by Bacillus spp., which correlated with shifts in carbohydrate metabolism and amino acid biosynthesis pathways critical for energy production and detoxification. Conversely, PLA-fed larvae were enriched with Enterococcus spp., linked to oxidative stress mitigation and nucleotide turnover. These diet-induced adaptations, such as the proliferation of Bacillus spp. in LDPE‑fed larvae, known to express alkane‑hydroxylase enzymes that initiate polyethylene depolymerization, and enrichment of Enterococcus spp. in PLA‑fed larvae, linked to ester bond hydrolysis, underscore a symbiotic co-metabolism that may play a contributory role in plastic processing, albeit at the cost of reduced larval growth and suppressed mitochondrial function. By unraveling these complex biological interactions, this study establishes a foundation for harnessing insect-microbiome ecosystems to develop scalable and eco-friendly strategies for plastic waste management. Future research should explore the genetic and enzymatic mechanisms underpinning plastic metabolism in insect-microbiome ecosystems.

Phloem-feeding insects such as aphids, planthoppers, and whiteflies use salivary proteins to aid feeding, adapt to hosts, and manipulate plant defenses, shaping a dynamic plant-herbivore conflict. Among them, the sweet potato whitefly, Bemisia tabaci, is a major global pest that threatens food security by causing feeding damage and transmitting plant viruses. The salivary secretory proteins of B. tabaci remain poorly characterized. We conducted a proteome-wide hypothesis-based screening of the B. tabaci MEAM1/B-biotype reference proteome using secretion, membrane-topology, localization, and effector-likelihood filters to prioritize candidate secreted proteins. Among 1404 proteins screened in EffectorP 3.0, 884 were identified as putative effector candidates and reported as computationally predicted rather than a validated effectorome. Most of these candidates were cytoplasmic (741), with fewer following the classical extracellular secretory pathway (143). Functional annotation showed strong enrichment of proteases (cathepsin B, cathepsin L, and serine proteases) and thioredoxin-related proteins, some of which resemble proteins implicated in feeding systems among hemipteran insects, supporting their prioritization for validation. Protein-association analyses highlighted protease-related candidates as central predicted network hubs. Transient validation of one prioritized candidate, BtApe, showed attenuation of chitin-associated oxidative staining and normalized defense-marker responses in tomato, supporting BtApe as an immune-modulatory candidate and defining a focused framework to validate salivary gland expression and secretion, as well as loss of function effects.

Spermatogenesis is a conserved process across animals, involving the maintenance and differentiation of germ stem cells, haploid spermatid production via meiosis, and morphologically distinct sperm formation during spermiogenesis. We previously demonstrated that Slmap (Sarcolemma-associated protein) knockdown causes male sterility with no sperm in the seminal vesicle. Here, we show that Slmap knockdown in the early Drosophila melanogaster germline led to severe spermiogenesis defects, including aberrant paracrystalline material deposition in both mitochondrial derivatives, fusion of the two derivatives and of spermatids, loss of the central pair of axonemal microtubules, and axoneme clustering. These phenotypes were supported by transcriptomic data showing that the most prominently affected genes were those involved in cuticle development and plasma-membrane adhesion, including 20 cuticular protein genes. In parallel, we detected upregulation of individualization complex genes (chic, ctp, WASp, Lasp) and significant association with the oxidative phosphorylation pathway. Consistently, KEGG analysis and functional assays showed that the sustained damage activated germ cell apoptosis, as indicated by increased TUNEL signals, elevated Hid levels, and reduced Diap1 expression. Together, these findings place Slmap at the intersection of structural integrity and cell survival, linking its loss to both morphogenetic failure and activation of the Hid-Diap1 apoptotic pathway.

Glutathione S-transferases (GSTs) constitute a critical enzyme superfamily responsible for xenobiotic detoxification in insects. The green peach aphid, Myzus persicae, is a globally significant agricultural pest that has exhibited declining field susceptibility to λ-cyhalothrin (LCT), a widely applied pyrethroid insecticide. While elevated GST activity has been correlated with LCT tolerance in various aphid populations, the precise molecular mechanisms driving this process have not been fully elucidated. In this study, we functionally characterized a delta-class GST gene (designated MpGSTd1) from M. persicae. The encoded protein possesses hallmark structural motifs of the GST family, including distinct glutathione-binding (G-site) and hydrophobic substrate-binding (H-site) pockets. Transcriptional profiling revealed maximal MpGSTd1 expression during the late nymphal to adult stages, and its transcript levels were markedly induced following LCT challenge. Biochemical analysis of the recombinant MpGSTd1 protein confirmed its catalytic competency toward the model substrate CDNB, exhibiting a Vmax of 0.91 µmol/min/mg and a Km of 2.73 mM. Notably, LCT exposure inhibited the enzyme's activity in vitro with an IC50 of 0.02 mM, and high-performance liquid chromatography (HPLC) assays demonstrated that MpGSTd1 can directly deplete LCT by 11.4%. Furthermore, RNA interference (RNAi)-mediated suppression of MpGSTd1 in vivo significantly elevated aphid mortality upon subsequent LCT exposure. Collectively, these findings delineate an essential role for MpGSTd1 in modulating LCT susceptibility in M. persicae and underscore its potential utility as a molecular target for future aphid management strategies.

Lectins, the glycan-binding proteins that are known to function as essential pattern-recognition receptors (PRRs) in the innate immune system of insects, also act as strategic modulators of physiological stress responses. An attempt is made here to demonstrate how chronic stress affects the lectin from the larvae of Zophobas morio, an adapter molecule in physiological response. The haemagglutination (HA) titer used to measure the activity of lectin revealed discrepancies in the larval serum of Z. morio after exposure to various stress conditions. Among the stress-induced treatment groups, except the control, the treatments exhibited a marked reduction in the HA activity from the larval serum of Z. morio. Examination of the haemagglutination inhibition (HAI) activity, which is used to assess the ability of specific sugars that inhibit this process, clearly showed that lectin from the larval serum of stress-induced groups had deteriorated in its binding ability to d-fucose and lactose. Pyriproxyfen (a juvenile hormone analog) mimics hormones exclusive to insects and is extremely target-specific. It induces physiological and cellular alterations in insects. Although the effects of pyriproxyfen on insect lectins are indirect, it acts through hormonal signaling to affect lectins. The relative quantitation of d-fucose-binding lectin (FBL) mRNA under various stress conditions in the larval fat body of Z. morio, analyzed through RT-qPCR, clearly indicated significantly lower levels of its expression, except for pyriproxyfen treatment. Molecular docking and simulation were performed to recognize the stability of the interaction between FBL and pyriproxyfen. These results revealed a significant binding characterized by high affinity through a strong amide bridge, and other non-covalent interactions, showing evidence for increased expression of lectin at doses of pyriproxyfen treatments as a measure of neutralization. On the whole, stressful environments bring swift and dynamic changes in the activity of lectin, including its expression in Z. morio larvae, revealing lectin as a vital constituent in response to physiological challenges.

Reproductive behavior refers to a series of activities involved in animal reproduction, including intersexual attraction, courtship, mating, oviposition, and parental care. It is the most common behavior in the biological world and plays a vital role in population propagation. The in-depth study of insect reproductive behavior is fundamental to understanding species' sexual selection and evolution, with important practical significance for pest control and the utilization of beneficial insects. Reproductive behavior is a complex biological process regulated by genes. Research on such genes can offer new ideas and target genes for novel pest control strategies based on reproductive behavior, pheromones, and RNAi. Presently, control methods established using insect sex pheromones and mating behavior patterns have been successfully applied in monitoring and controlling some significant agricultural pests. This review synthesizes current knowledge on genes regulating insect reproductive behavior from three interconnected perspectives: intrasexual regulation (with emphasis on neuropeptides such as tachykinins and natalisin), intersexual regulation (focusing on male accessory gland proteins and female post‑mating responses), and sex pheromone synthesis control (including both female‑autonomous PBAN/PBANR signaling and male‑derived pheromonostatic factors). While major progress has been made in model insects, functional validation in non‑model species and the integration of multi‑omics data remain key challenges. We also discuss emerging tools such as CRISPR/Cas9 and bacterial‑mediated RNAi, which hold promise for translating mechanistic insights into behavior‑based pest management strategies.

Low temperature is a key abiotic factor shaping tick-associated microbial communities, which in turn influence host physiology, vector competence, and environmental adaptation. However, the impact of prolonged cold exposure, such as overwintering conditions, on the microbiome of the invasive tick Haemaphysalis longicornis remains insufficiently characterized in terms of its microbial response. In this study, unfed adult ticks were subjected to a gradient of low temperatures (8°C, 4°C, 0°C, -4°C) for 7 days, while a control group was maintained at 27°C. The bacterial communities of whole ticks were characterized using Illumina NovaSeq-based 16S rRNA gene sequencing, followed by comprehensive bioinformatics analyses to evaluate alpha diversity, beta diversity, taxonomic composition, and differentially abundant taxa. The results showed that cold exposure markedly reshaped the microbial community structure, with an overall increase in alpha diversity (Shannon index) observed in several treatment groups. Across all samples, the dominant bacterial phyla included Proteobacteria, Firmicutes, and Bacteroidota. Notable shifts were detected at the genus level, particularly in Coxiella endosymbiont and Pseudomonas, whose relative abundances changed substantially under low-temperature conditions. In addition, microbial responses exhibited clear sex-specific patterns: Escherichia-Shigella and Serratia were enriched in certain cold-treated groups, whereas Staphylococcus showed a reduction in males exposed to low temperatures. The endosymbiont Coxiella was significantly enriched in male ticks at 8°C (p = 0.009). Beta diversity analysis further demonstrated distinct clustering of the -4°C male group relative to all other groups. Collectively, these findings indicate that sustained low temperatures drive pronounced and sex-dependent restructuring of the bacterial microbiome in H. longicornis. The enrichment of specific taxa, including putative nutritional symbionts such as Coxiella, under cold stress conditions suggests a potential role for microbial communities in facilitating host adaptation to low-temperature environments, thereby providing new insights into the ecological dynamics and adaptive capacity of this invasive vector species.

The red palm weevil (RPW), Rhynchophorus ferrugineus (Oliver), is a major pest of palm plants. Entomopathogenic fungi (EPF) are considered promising biocontrol agents against RPW. This study investigated the changes in immune-related gene expression and gut microbiota of RPW larvae infected with Beauveria bassiana (Bb-NCHU-155). In infected larvae, fungal genome copy numbers in the midgut and hindgut were lower than those detected in the fat body, suggesting that infection primarily occurs via the cuticle rather than the digestive tract. Immune-related gene expression in the fat body increased steadily from 3 days post-inoculation (dpi), reflecting a typical host response, whereas digestive tissues exhibited fluctuating patterns. The midgut showed peak induction of C-type lysozyme at 3 dpi and C-type lectin at 6 dpi, while the hindgut displayed the highest expression of serine protease-like protein at 3 dpi. Despite an overall downregulation of immune-related genes, these results indicate tissue-specific immune responses. Although B. bassiana may not infect hosts through the digestive system, gut microbiota composition differed significantly between infected and control groups, with higher relative abundances of Acetobacteraceae, Lactobacillaceae, and Streptococcaceae in the infected larvae. These shifts co-varied with the expression of immune-related genes, such as defensin and C-type lectin, suggesting potential functional links between gut microbiota and host immunity. This study provides fundamental insights into the effects of EPF on gut microbiota and immune gene expression in RPW, supporting further research into the complex interactions underlying microbial control.

The extensive use of pesticides poses a significant threat to sericulture, yet effective control strategies are still limited. Trehalose, the primary sugar in insect hemolymph, is a key player in abiotic stress responses and its metabolism is regulated by trehalase. Despite this, the role of the Bombyx mori trehalase 2 (BmTreh2) in the response to the pyrethroid insecticide remains unexplored. In this study, comparative analysis revealed significant differences in BmTreh2 expression among silkworm (Bombyx mori) strains with varying resistance levels to fenpropathrin, suggesting a potential role of this gene in fenpropathrin resistance. At the cellular level, overexpression of BmTreh2 increased tolerance to fenpropathrin, whereas RNAi-mediated knockdown of BmTreh2 decreased cell viability under fenpropathrin exposure. These results demonstrate that BmTreh2 plays a critical role in mediating fenpropathrin resistance. Furthermore, correlation analysis between BmTreh2 and key energy metabolism-related genes, including lactate dehydrogenase (LDH), alanine aminotransferase (ALT), malate dehydrogenase (MDH), and isocitrate dehydrogenase (IDH), revealed a significant positive correlation with MDH and IDH expression, and a negative correlation with LDH and ALT. This study elucidates key molecular mechanisms underlying silkworm resistance to fenpropathrin, establishing a theoretical framework for molecular breeding of resistant silkworm strains and informing integrated pest management strategies.