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.
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 (BmRaspKO) using the CRISPR/Cas9 system. Both homozygous knockout mutants and F1 pupae with the gap/BmRaspKO 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 svT2A-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 prosT2A-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, atkGAL4. 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.
Pine wilt disease (PWD) is one of the most destructive global forest diseases, causing extensive mortality in pine trees and severe economic and ecological damage. PWD is caused by the pinewood nematode Bursaphelenchus xylophilus, which is transmitted by Monochamus beetles. Despite the critical role of M. alternatus in PWD epidemiology, molecular and cellular studies of this species have been severely limited by the lack of a continuous cell line suitable for functional analyses. In this study, the first continuous insect cell line derived from M. alternatus, designated NARO-Moal, was produced from prepupal fat bodies. The cell line has been stably maintained for over 50 passages and adapted to the commercially available Grace's Insect Medium. To provide a molecular framework for functional studies, de novo transcriptome sequencing of NARO-Moal cells generated an assembly of 14,141 genes with 89.5% completeness, based on BUSCO analysis. The NARO-Moal cells supported transient gene expression following plasmid transfection and showed efficient RNA interference in response to simple soaking with double-stranded RNA. Moreover, this cell line responded to juvenile hormone at the gene expression level and supported stable infection with Wolbachia. These results revealed that the NARO-Moal cell line is a versatile molecular and cellular platform for gene functional analysis, endocrine signaling, and symbiotic research in M. alternatus, providing new opportunities for mechanistic studies and the development of innovative strategies to control pine wilt disease.
Eye-color mutants provide easily distinguishable visual markers for genetic analysis and genome editing, yet the molecular basis of naturally occurring eye-color variation in Nilaparvata lugens remains unclear. Here, we combined classical genetic analysis, candidate-gene screening, and CRISPR/Cas9 genome editing to elucidate the genetic basis of a stably inherited orange-eye phenotype and evaluate the editing performance of different sgRNA strategies. The orange-eye phenotype was inherited as a single autosomal recessive trait, and the mutant strain showed only limited fitness differences from the WT strain. A 1287-bp insertion spanning exon 13 and the adjacent intronic region of Nlscarlet introduced a premature stop codon, truncating NlScarlet after the fifth transmembrane helix. The insertion completely cosegregated with the orange-eye phenotype, and CRISPR/Cas9-mediated disruption of Nlscarlet recapitulated the phenotype, demonstrating that this insertion underlies the natural mutation. A separate in-frame deletion of lysine 535 produced a dark-orange-eye phenotype, and structural modeling suggested that this residue may contribute to the interaction between the NlWhite/NlScarlet transporter complex and 3-hydroxykynurenine. Using Nlscarlet as a visually scorable marker, two multiple-sgRNA strategies, each using four sgRNAs, increased the proportions of G0 individuals with complete loss-of-function phenotypes from 30.97% to 84.73% and 92.47%, respectively, and of mutant-eyed G1 progeny from 37.33% to 81.33% and 90.67%, respectively. Heritable mutations generated by both strategies were predominantly large deletions. These findings identify the genetic basis of a natural orange-eye mutation in N. lugens and establish multiple-sgRNA editing as an efficient strategy for rapid functional screening in insects.
Reproductive condition can strongly influence animal behavior, yet the genetic and endocrine pathways that connect mating status with sensory perception remain insufficiently understood. In the greater wax moth, Galleria mellonella, sexual signaling is reversed: males emit pheromones while females actively search for mates. Here, we show that mating selectively reduces female behavioral attraction and electroantennogram (EAG) responses to the primary male pheromone component, nonanal. Transcriptomic profiling of female heads identified the sex peptide receptor (SPR) as significantly upregulated after mating. Using CRISPR/Cas9-mediated knockout, disruption of the SPR gene eliminates the normal mating-induced decline in pheromone attraction. Mated females lacking SPR gene behave to stronger attraction to nonanal and elevated electroantennogram responses compared to normal mated wild type females. Further analyses indicate that SPR influences this process through the juvenile hormone (JH) pathway. Several genes involved in JH synthesis and metabolism show altered expression in mated females as well as in SPR mutants, and SPR protein is detected in the corpus allatum, the endocrine gland responsible for JH production. Treatment with a JH analog restores the typical post-mating reduction in pheromone attraction in SPR mutant females, indicating that JH signaling operates downstream of SPR. In addition, preliminary antennal transcriptome comparison also revealed differential expression of several olfactory receptor genes in SPR mutants. Together, our results identify an SPR-JH signaling axis that links mating status to plasticity in pheromone perception in a species with reversed sexual communication, revealing how a conserved neuropeptide receptor coordinates reproductive physiology with sensory modulation.
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.
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.
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 carotenoid 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.
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.
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.
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.
