The tobacco beetle, Lasioderma serricorne (F.) (Coleoptera: Ptinidae), is a highly adaptable generalist insect pest that inflicts billions of dollars in damage to stored food and tobacco products worldwide. Despite the neurotoxic effects of nicotine, L. serricorne thrives on tobacco, likely due to specific adaptations; however, the underlying molecular mechanisms remain uncharacterized. To investigate, we performed a comparative transcriptomic analysis of L. serricorne larvae reared on tobacco versus an artificial diet. Bioinformatic analysis revealed a significant upregulation of genes involved in metabolic detoxification in tobacco fed larvae, including 92 cytochrome P450 (CYPs), 74 ATP-binding cassette (ABC) transporters, 37 glutathione S-transferases (GSTs), 30 UDP-glycosyltransferases (UGTs) and 21 carboxylesterases (CarEs). Spatiotemporal profiling identified several ABC transporters and CYPs as midgut-enriched, indicating roles in xenobiotic metabolism. RNAi-mediated knockdown of LsCYP6TC1, a tobacco-induced CYP, reduced transcript abundance and decreased larval survival to 36.1% compared to controls. Notably, co-silencing of LsABCA1 and LsABCG1 significantly reduced larval survival (77.1%). These results indicate that both Phase I (oxidation via CYPs) and Phase III (efflux via ABC transporters) detoxification play critical roles in tobacco tolerance. Our findings offer valuable insights into insect adaptation to dietary shifts and suggest the specific detoxification genes could be targeted for developing innovative pest management strategies.

[Objective]Gram-negative binding proteins(GNBPs),also known as β-1,3-glucan recognition proteins,represent a class of crucial pattern recognition receptors(PRRs)in insects and play pivotal roles in the innate immune response.This study aimed to systematically identify members of the PxGNBP gene family in the diamondback moth(Plutella xylostella),analyze their structural characteristics and expression patterns,as well as screen and validate key target genes.The findings are expected to reveal the innate immune mechanisms and evolutionary adaptations of P.xylostella in response to pathogenic infection,thereby providing theoretical foundations and potential targets for the development of novel biological control.[Method]Based on the whole-genome data of P.xylostella,members of the PxGNBP gene family were identified.Bioinformatic approaches were comprehensively employed to analyze their structural characteristics and evolutionary relationships,and AlphaFold3 was used to predict their three-dimensional structures.In addition,combined with public transcriptome data and quantitative real-time polymerase chain reaction(RT-qPCR)technology,the expression patterns of these family members in different tissues and post-infection with Beauveria bassiana and Metarhizium anisopliae were detected.Recombinant M.anisopliae strains carrying pSilent-PxGNBP3 were constructed.The expression levels of PxGNBP3 and downstream antimicrobial peptide genes post-infection were determined via RT-qPCR,and the pathogenicity of different strains against P.xylostella was evaluated using bioassays.[Result]A total of 10 PxGNBP members were identified in P.xylostella.Among them,PxβGRP4 is located on chromosome 22 and belongs to the glucanase subfamily,while the remaining 9 members are located on chromosome 29 and belong to the PRR subfamily.Phylogenetic and chromosome location analyses suggested the occurrence of tandem duplication events within this gene family.Conserved motif analysis indicated that the N-terminal domain of PxGNBP exhibited lower conservation compared to the C-terminal domain.Except for PxβGRP4,the key catalytic sites of glucanase in other members were mutated.Three-dimensional structure predictions revealed that all members,except PxβGRP4 and PxβGRP3,possessed the typical GNBP protein structure;the C-terminus of PxβGRP3 contained a structural fragment that was similar but not identical to Carbohydrate-binding module 39(CBM39).Expression profile analysis demonstrated that most members exhibited a time-series expression pattern of first increasing and then decreasing after infection with the two fungi.RNA interference(RNAi)assays showed that the recombinant M.anisopliae strains could effectively suppress the expression of PxGNBP3,leading to a significant reduction in antimicrobial peptide expression levels and a decrease in host survival rate.Moreover,the virulence of recombinant strains was significantly higher than that of the wild-type strain and enhanced with increasing concentration.[Conclusion]Ten members of the GNBP gene family were identified in P.xylostella,with PxβGRP3 and PxGNBP3-2 showing structural specificity.This gene family exhibited a time-series regulatory expression pattern in response to fungal infection.In vivo functional validation of PxGNBP3 via RNAi was successfully achieved using the constructed recombinant M.anisopliae strains.The results provide important insights for elucidating the innate immune mechanisms of P.xylostella and developing novel targets for biological control.

The deployment of insect-resistant rice cultivars is a sustainable strategy for pest control, while the adaptation of pest insects to resistance limits the efficiency of resistant rice varieties. The cytochrome P450 gene CYP4C61 was previously identified as a key locus underlying brown planthopper (BPH, Nilaparvata lugens) adaptation to the resistant rice variety IR36, but its metabolic function remained unknown. Here, we integrated RNAi-mediated gene silencing, untargeted metabolomics, and transcriptomics to elucidate the metabolic role of CYP4C61 in the BPH population virulent to resistant rice IR36. CYP4C61 silencing significantly impaired BPH fitness, including reduced body weight, increased mortality, disrupted feeding behavior, and a progressive body darkening of BPH reared on IR36 rice, reflecting dopamine accumulation entering the melanization branch. Metabolomic analysis identified 240 differentially abundant metabolites in silenced BPH on IR36, revealing a pattern of precursor reduction and product accumulation in the dopamine pathway. Transcriptomic analysis also revealed that CYP4C61 knockdown altered gene expression in the dopamine pathway in a host-dependent manner. Enzyme-linked immunosorbent assay validated dopamine accumulation after CYP4C61 knockdown exclusively in the IR36 background. Our integrated multi-omics evidence indicates that CYP4C61 contributes to dopamine homeostasis in the virulent BPH, providing a mechanistic link between a P450 gene and dopamine-mediated insect adaptation to resistant host plants.

The red imported fire ant, Solenopsis invicta Buren, is a highly invasive eusocial insect pest that threatens native biodiversity, agriculture, and human health. The innate immune system and intricate social immune responses of S. invicta pose challenges to the development of effective control strategies. MicroRNAs (miRNAs) play critical roles in the post-transcriptional regulation of gene expression, which influences various biological processes, including immunity and host-pathogen interactions. While the miRNA-mediated response of insects to pathogens has been extensively studied in solitary insects, little is known about the innate immune responses of individual members within a colony. To address this gap, we constructed small RNA libraries from Metarhizium anisopliae-infected S. invicta workers and investigated the temporal dynamics of miRNA-mediated immune responses to the entomopathogen. Several differentially expressed miRNAs were identified, and they were found to regulate genes involved in the Toll, IMD, and melanization immune pathways. Quantitative real-time PCR (qRT-PCR) was employed to analyze the spatiotemporal dynamics of key miRNAs/target genes, specifically miR-71/ModSP1-Relish and miR-7/Lysozyme2-Serine protease7. A dual luciferase assay (in vitro) was performed to validate the interactions between miRNAs and their target genes. Overexpression of miR-71 and miR-7 (via miRNA mimics) efficiently suppressed their target genes, impaired the antifungal immune response of S. invicta and increased the susceptibility to M. anisopliae infection compared to controls. Furthermore, RNA interference-based gene silencing elucidated the roles of these immune genes in regulating fungal susceptibility, thus providing vital clues for developing virulent and effective mycoinsecticides using modern genetic engineering tools.

The tobacco beetle, Lasioderma serricorne, is a globally distributed pest that inflicts major economic losses on stored products due its exceptional dietary adaptability. Understanding host-microbe-diet interactions is crucial for developing sustainable biological control strategies targeting insect-associated microorganisms. Here, we employed comparative metagenomic sequencing to characterize gut microbial communities in L. serricorne larvae reared on four dietary treatments i.e., artificial diet; Artemisia argyi; Nicotiana tabacum and Angelica sinensis. Taxonomic diversity, functional profiles, carbohydrate-active enzymes (CAZymes), and antibiotic resistance genes (ARGs) were analyzed using comprehensive bioinformatic pipelines. Our analysis showed that diet profoundly shaped the gut microbiome architecture, with natural plant substrates supporting significantly higher microbial diversity than artificial diet. Proteobacteria dominated across all groups, with Enterobacteriaceae as the predominant family and Enterobacter as the most abundant genus. Notably, Enterobacter cancerogenus was consistently present in all dietary groups. Linear discriminant analysis effect size (LEfSe) identified diet-specific distinct microbial biomarkers: N. tabacum-fed larvae showed enrichment of Enterococcus spp. (Lactobacillales: Enterococcaceae), potentially facilitating alkaloid detoxification, while A. sinensis-fed larvae exhibited remarkable fungal diversity, particularly Ascomycota spanning multiple classes. Functional analysis revealed diet-specific enzymatic specialization, with A. sinensis-fed larvae exhibiting diverse CAZyme profiles including polysaccharide lyases and lignin-modifying enzymes. A. argyi-fed larvae showed moderate CAZyme abundance, while simplified artificial diet reduced both microbial diversity and functional complexity. KEGG pathway analysis showed N. tabacum-fed larvae exhibited comparatively enhanced oxidative metabolism with enriched respiratory chain components and cellular transport proteins, indicating metabolic adaptation to alkaloid detoxification. Co-occurrence analysis identified significant correlations between ARGs and specific microbial genera (Enterobacter, Escherichia, Enterococcus), suggesting potential resistance gene reservoirs within the gut microbiome. Our findings demonstrate that dietary substrate drives distinct microbial adaptations underlying the polyphagous lifestyle of L. serricorne, with diet-specific dependencies offering targets for microbiome-based biological control strategies.

【Objective】Chitinases are essential for chitin metabolism and the growth and development of insects. This study systematically identified the chitinase gene family members in the red fire ant (Solenopsis invicta), analyzed their molecular characteristics and expression patterns, and explored their potential functions during development. The findings provide a theoretical basis and genetic resources for novel green control strategies targeting chitin metabolism.【Method】Based on the whole genome data of S. invicta, members of the chitinase gene family were identified. Bioinformatics analysis were performed to characterize the physicochemical properties, chromosomal localization, and phylogenetic relationships of their encoded proteins. The expression patterns of these genes across different developmental stages (egg, larva, pupa, and adult) and in various tissues were further examined using RT-qPCR.【Result】A total of six chitinase genes were identified in S. invicta, including five SiCht genes and one SiIDGF gene. The corresponding encoded proteins exhibited lengths ranging from 444 to 3 050 amino acids, most of which were predicted to be stable and acidic in nature. These genes were distributed across five chromosomes, with SiCht6-1 and SiCht6-2 both located on chromosome 6. Collinearity analysis suggested that the chitinase gene family is evolutionarily conserved, and phylogenetic analysis grouped them into seven distinct clades. Expression profiling revealed that SiCht1, SiCht2, and SiCht7 were predominantly expressed at high levels during eggs, larvae, and pupae stages, whereas SiIDGF3 displayed the highest expression in adults. Tissue-specific expression analysis further demonstrated that SiCht7 and SiIDGF3 were significantly enriched in the epidermis, suggesting their potential key roles in cuticular development and remodeling.【Conclusion】This study successfully identified six members of the chitinase gene family in S. invicta, which exhibit diverse molecular features and evolutionary conservation. Expression patterns suggest that SiCht genes are involved in molting and growth-related processes of S. invicta, with SiCht7 and SiIDGF3 emerging as potential key targets for cuticle remodeling. These findings provide insights into the molecular mechanisms contributing to the invasiveness of S. invicta and provide a theoretical basis for the development of environmentally friendly control strategies, such as RNAi or chitinase inhibitors targeting chitin metabolism.

Microplastics have evolved as widespread contaminants in terrestrial and aquatic environments, raising significant environmental concerns due to their persistence and bioaccumulation. In this study, we investigated the toxicity of polyethylene microplastics (PE-MPs) on the agricultural insect, Spodoptera frugiperda. Maize leaves containing three sizes (0.5 μm, 5 μm, and 50 μm) of PE-MPs were fed to fall armyworm larvae for 12 days at concentrations of 1.25 g/ L, 5 g/L, and 20 g/L. The results showed that smaller size and higher concentration of microplastics led to increased toxicity. Furthermore, different sizes and maximum concentrations of PE-MPs were selected for subsequent experiments to observe changes in histological and enzymatic biomarkers, midgut microbiome, and metabolic responses. Following PE-MPs exposure, inflammation signs and oxidative stress were detected in the midgut. Significant changes were also observed in midgut microbiota and metabolomes, most related with oxidative stress, inflammatory disorders, and energy metabolism. These results provide evidence of midgut damage and alterations in the microbiota and metabolome of S. frugiperda because of PE-MPs exposure, highlighting the harm that microplastics can inflict on agricultural insects. Additionally, the study lays a theoretical foundation for future research on the transmission of microplastics through the food chain in agricultural ecosystems.

Plutella xylostella represents a significant agricultural pest affecting cruciferous crops globally. The extensive use of synthetic insecticides has resulted in environmental contamination and resistance development, necessitating research into environmentally sustainable biopesticides. Serine protease inhibitors (serpins) serve essential functions in melanization during innate immunity, reproduction, and metamorphic development. Through proteomic analyses conducted across developmental stages of P. xylostella, serpin15 was identified as a crucial member of the typical inhibited serpin family, though its precise function remained undetermined. RT-qPCR analyses of gene expression patterns across tissues and developmental stages demonstrated that the serpin15 gene exhibits high expression in male adult gonads and reaches maximum levels in hemolymph. The serpin15 mRNA levels showed dynamic regulation in the midgut following Serratia marcescens (PS-1) infection, characterized by an initial decline followed by upregulation. CRISPR/Cas9-mediated knockout of serpin15 in homozygous lines led to decreased oviposition and embryonic hatching rates in offspring. Functional analyses confirmed that serpin15 inhibits phenoloxidase activity, while exogenous supplementation with recombinant serpin15 protein effectively suppressed hemolymph melanization, establishing its regulatory role in countering PS-1 through immune melanization. These findings demonstrate serpin15’s dual functionality in regulating both fecundity and immunity against PS-1 in P. xylostella. This research establishes a theoretical foundation for developing biocontrol strategies targeting insect immune and developmental systems.

The degradation and transformation of nicotine-containing environmental contaminants into harmless substances have become essential processes. In this study, we identified and characterized Pseudomonas alloputida strain Pa_12 (GenBank: CP158025), a bacterium with significant potential for the bioremediation of nicotine-contaminated environments. Comprehensive morphological, physiological and genomic analyses, including 16S rRNA sequencing, phylogenetic analysis, complete genome sequencing, and average nucleotide identity (ANI) comparison-confirmed the identity and metabolic capabilities of Pa_12. Under optimized conditions (pH 7, 30 degrees C), Pa_12 exhibited efficient nicotine catabolism. Absolute RT-qPCR and fluorescence in situ hybridization (FISH) demonstrated that Pa_12 successfully colonized tobacco leaves, promoted plant growth, and significantly reduced nicotine content in the foliage. LC-MS/MS analysis identified a nicotine catabolic pathway in Pa_12, distinct from the canonical pyrrole route, culminating in 2,5-dihydroxypyridine, thereby highlighting the unique capabilities of this strain. These findings advance our understanding of microbial nicotine degradation and present a viable solution for tobacco waste management and the improvement of flue-cured tobacco quality. Further investigation and development of Pa_12 may enable transformative applications in environmental remediation and sustainable tobacco cultivation.

Insecticide resistance has led to the failure of chemical pest control and threatens global food security, with transcriptional regulation of detoxification genes by xenobiotic-response transcription factors (TFs) playing a key role. However, the regulation mechanism remains unclear in most crop insect pests. This study characterized the role of the nuclear receptor NlNR2E in response to insecticides in the brown planthopper Nilaparvata lugens, a destructive rice pest. Using DAP-seq, we identified detoxification gene promoters targeted by NlNR2E. NlABCG23, encoding an ATP-binding cassette (ABC) transporter, showed significantly reduced expression after the RNA interference (RNAi) knockdown of NlNR2E. Dual-luciferase reporter, yeast one-hybrid, and electrophoretic mobility shift assays confirmed that NlNR2E directly binds to NlABCG23 promoter, thus activating NlABCG23 expression. RNAi knockdown of NlABCG23 significantly reduced resistance to imidacloprid, dinotefuran, pymetrozine, and chlorpyrifos in N. lugens. These findings illustrated how xenobiotic-induced TF regulates the ABC transporter, conferring resistance to multiple insecticides in planthoppers.

Peptidoglycan recognition proteins (PGRPs) are essential for innate immune recognition and regulation from insects to mammals. However, the specific role of PGRPs in responding to Bacillus thuringiensis (Bt) infection and maintaining midgut microbial homeostasis in Plutella xylostella remains poorly understood. In this study, we identified and characterized a PGRP gene from P. xylostella, designated PxPGRP4. The spatiotemporal expression analysis revealed that PxPGRP4 is predominantly expressed in the midgut of naïve larvae and at adult stages. A homozygous mutant strain featuring a four-base pair nucleotide deletion was successfully generated through CRISPR/Cas9-mediated knockout of PxPGRP4. The bioassay results indicated that the susceptibility of P. xylostella larvae to Cry1Ac protoxin was significantly increased by the loss of PxPGRP4 expression. Furthermore, 16S rRNA sequencing and qPCR analysis revealed that the PxPGRP4 mutants exhibited a significantly reduced total bacterial load and altered microbiota composition in the midgut compared to the wild-type strain, with a shift in the dominant bacterial family from Enterobacteriaceae to Enterococcaceae. Additionally, the knockout of PxPGRP4 resulted in significant alterations in the expression of midgut immune-related genes. These findings highlight the crucial role of PxPGRP4 as a modulator of midgut microbiota and immune responses and provide valuable insights into Bt resistance management.

Insecticide resistance in pest control poses a threat to agricultural production and human health. Numerous insect species express genes coding for detoxification enzymes that have broad substrate promiscuity thus conferring resistance to various insecticides. However, whether the homologs of these genes play similar roles in resistance phenotypes of closely related species remains largely unclear. Therefore, this study compares the resistance profiles of three major rice planthopper species (Delphacidae) (Laodelphax striatellus, Nilaparvata lugens, and Sogatella furcifera) based on the metabolic activity of their cytochrome P450s. Genome-wide analyses resulted in 68, 70, and 64 P450 genes in L. striatellus, N. lugens, and S. furcifera, respectively. Phylogenetic analyses among these genes found that most resistance-related genes in one species had homologs in other planthopper species. The most resistance-relevant orthogroup (CYP6ERs) showed higher evolutionary instability than most other groups. RNAi and in vitro metabolism assays revealed that CYP6ERs confers more divergent insecticide resistance profiles among planthopper species than the other two major resistance-related P450 subfamilies (CYP6AYs and CYP4C61s). Alphafold-based structural predictions and alignments suggested that P450 orthogroups with higher phylogenetic instability tended to have less structural similarities, resulting in more divergent metabolic profiles. This relationship was also in silico validated on Aphidae aphids and Lepidoptera noctuids. This study proposes combined phylogenetic and toxicogenomic analyses for understanding CYPome-based insecticide resistance convergency and divergency among closely related pests. These findings may improve the accuracy and rationality of chemical pest control.

BACKGROUNDSpecies that experience outbreaks and those that display density-dependent phase polymorphism demonstrate density-dependent prophylaxis (DDP) by increasing their immune investment in response to increasing densities. Despite this phenomenon, the mechanisms of DDP remain largely unexplored.RESULTSHere, we showed that Spodoptera litura exhibited heightened cuticular melanization and enhanced cuticular immune responses when reared at higher population density. Transcriptomic analysis identified differentially expressed genes (DEGs) associated with immune responses, nutritional metabolism, and cuticular synthesis in the cuticle, revealing the molecular underpinnings of density-dependent plasticity in larval cuticles. Gregarious S. litura larvae exhibited significant up-regulation of immune-related genes, particularly those in the Toll and immune deficiency (IMD) signaling pathways and tyrosine metabolism, suggesting a strategic enhancement of immune defenses. Concurrently, a reduced lipid metabolism was observed in the cuticle of gregarious larvae, with suppressed expression of key genes in the fatty acid synthesis, leading to a decrease in integument triglyceride content. The immune defense of gregarious larvae was further amplified by increased expression of cuticle- and melanin formation-related genes, and reduced chitin degradation, reinforcing the cuticle as a physical barrier against pathogens.CONCLUSIONOur findings clarify that shifts in phenotypic plasticity, metabolic pathways, and immune response mechanisms underscore the adaptability of insects to population density changes and their consequent vulnerability to pathogens, offering new directions and insights for uncovering the mechanisms underlying pest outbreaks and for enhancing the effectiveness of biological control measures by targeting gregarious immunity. (c) 2025 Society of Chemical Industry.

Insects, the most numerous and diverse group of animal species on Earth, have important interactions with humans through providing resources, transmitting diseases and damaging agricultural cultivars. Cytochrome P450 monooxygenases (P450s) are one of the most important protein families in insects implicated in the endogenous metabolism and detoxification of xenobiotics, including allelochemicals, insecticides and environmental pollutants. To better understand the evolution and function of insect P450s and support the development and application of insecticides for pest control, an integrated bioinformatics platform is highly desirable. Here, we present the Insect Cytochrome P450 database (ICPD, http://www.insectp450.net/), which contains 66,477 P450s collected from public databases and predicted from the genomes of 682 insect species using a standardised bioinformatics protocol. Phylogenetic relationships between P450 genes are constructed for each species. The structures of all P450 proteins in the database are predicted using ESMFold, then visualised using WeView. Web services, such as BLAST, homogeneous modelling and molecular docking, are provided for determining the catalytic activities of P450 proteins. The ICPD will facilitate systematic investigations of the evolution and functions of the complete insect P450 complement, and represents a powerful tool for guiding insecticide design and application.

The meprin and TRAF-C homology (MATH) family of proteins plays essential roles in diverse biological processes across eukaryotes. Fecundity is a key determinant underlying the rapid outbreaks of agricultural insect pests. Nevertheless, the potential involvement of MATH proteins in the regulation of fecundity in agriculturally important insects, particularly planthoppers, remains largely uncharacterized. This study identified key members of the MATH protein family that are conserved in planthoppers and involved in the regulation of insect fecundity. A total of 121 identified MATH proteins from 31 insect species were classified into five distinct clades based on protein structures, predominantly represented by the MATH-BTB, MATH-USP7, and MATH-Zf-Box subtypes, which are largely conserved across most agricultural insect species. In planthoppers, the MATH-BTB subtype gene cluster SfMATH1-NlMATH3-LsMATH3 constitutes a tripartite collinear gene set conserved across all three species. Among the four ovary-specific expressed MATH genes, NlMATH3 exhibited the highest expression level in the ovary. Moreover, silencing NlMATH3 significantly impaired ovarian development in adult females and reduced both the number of deposited and hatched eggs, which was associated with downregulation of vitellogenin (Vg) and its receptor VgR, as well as elevating activity in metabolic and immune signaling pathways. In summary, this study provides novel insights into the evolutionary dynamics of the MATH family in agricultural insects, particularly planthoppers, and elucidates the critical regulatory role of the planthopper conserved MATH-BTB protein NlMATH3 in insect fecundity. The conservation of NlMATH3 homologs across planthoppers highlights their potential as targets for RNAi-based pest control strategies.

Macroautophagy/autophagy is a conserved process in eukaryotic cells to degrade and recycle damaged intracellular components. Higher level of autophagy in the brain has been observed, and autophagy dysfunction has an impact on neuronal health, but the molecular mechanism is unclear. In this study, we showed that overexpression of Toll-1 and Toll-7 receptors, as well as active Spätzle proteins in Drosophila S2 cells enhanced autophagy, and Toll-1/Toll-7 activated autophagy was dependent on Tube-Pelle-PP2A. Interestingly, Toll-1 but not Toll-7 mediated autophagy was dMyd88 dependent. Importantly, we observed that loss of functions in Toll-1 and Toll-7 receptors and PP2A activity in flies decreased autophagy level, resulting in the loss of dopamine (DA) neurons and reduced fly motion. Our results indicated that proper activation of Toll-1 and Toll-7 pathways and PP2A activity in the brain are necessary to sustain autophagy level for DA neuron survival.

Many types of viruses infect insects and other arthropods. In contrast, little is known about how arthropods sense viruses, although several innate immune pathways including Toll have antiviral functions. Large DNA viruses in the family Baculoviridae are used to control a number of pest insects. Here, we studied Spodoptera litura and Autographa californica multiple nucleopolyhedrovirus (AcMNPV) to test the hypothesis that one or more myeloid differentiation-like (ML) proteins and Toll family members sense baculoviruses. We identified 11 ML and 12 Toll genes in the S. litura genome. A series of experiments indicated that S. litura ML protein 11 (SlML-11) binds the budded form of AcMNPV and partners with S. litura Toll5 (SlToll5). SlML-11 also bound sphingomyelin (SPM), which is a component of the virion envelope. Disabling SlML-11 and SlToll5 increased susceptibility to infection, while priming larvae with SPM reduced susceptibility as measured by increased survival to the adult stage and clearance of AcMNPV from individuals that emerged as adults. We conclude that SPM is a pathogen- associated molecular pattern molecule while SlML-11 and SlToll5 interact to function as a pattern recognition receptor that senses AcMNPV.

Crystal (Cry) toxins, produced by Bacillus thuringiensis, are widely used as effective biological pesticides in agricultural production. However, insects always quickly evolve adaptations against Cry toxins within a few generations. In this study, we focused on the Cry1Ac protoxin activated by protease. Our results identified PxTrypsin-9 as a trypsin gene that plays a key role in Cry1Ac virulence in Plutella xylostella larvae. In addition, P. xylostella miR-2b-3p, a member of the micoRNA-2 (miR-2) family, was significantly upregulated by Cry1Ac protoxin and targeted to PxTrypsin-9 downregulated its expression. The mRNA level of PxTrypsin-9, regulated by miR-2b-3p, revealed an increased tolerance of P. xylostella larvae to Cry1Ac at the post-transcriptional level. Considering that miR-2b and trypsin genes are widely distributed in various pest species, our study provides the basis for further investigation of the roles of miRNAs in the regulation of the resistance to Cry1Ac and other insecticides.

Peptidoglycan recognition proteins (PGRPs) are a class of pattern recognition receptors (PRRs) that activate the innate immune system in response to microbial infection by detection of peptidoglycan, a distinct component of bacterial cell walls. Bioinformatic studies have revealed four PGRPs in the red imported fire ant Solenopsis invicta; nonetheless, the mechanism of the immune response of S. invicta induced by pathogens is still poorly understood. The peptidoglycan recognition protein full-length cDNA (designated as SiPGRP-S1/S2/S3/L) from S. invicta was used in this investigation. According to the sequencing analysis, there was a significant degree of homology between the anticipated amino acid sequence of SiPGRPs and other members of the PGRPs superfamily. Molecular docking studies demonstrated that SiPGRPs show strong binding affinity for a variety of PGN substrates. Additionally, tissue distribution analysis indicated that SiPGRPs are primarily expressed in several tissues of naïve larvae, including fat body, hemocytes, head, and thorax, as detected by quantitative real-time PCR (RT-qPCR). Microbial challenges resulted in variable changes in mRNA levels across different tissues. Furthermore, the antibacterial effects of antimicrobial peptides (AMPs) produced by major ants infected with Metarhizium anisopliae were assessed. These AMPs demonstrated inhibitory effects against M. anisopliae, Staphylococcus aureus, and Escherichia coli, with the most pronounced effect observed against E. coli. In conclusion, SiPGRPs act as pattern recognition receptors (PRRs) that identify pathogens and initiate the expression of AMPs in S. invicta, this mechanism contributes to the development of biopesticides designed for the targeted control of invasive agricultural pests.