Insect Biochemistry and Molecular Biology最新文献

{"title":"Corrigendum to “Cuticular proteins: Essential molecular code for insect survival” [Insect Biochem. Mol. Biol. 184 (2025) 104402]","authors":"Huitang Qi, Tian Liu","doi":"10.1016/j.ibmb.2025.104458","DOIUrl":"10.1016/j.ibmb.2025.104458","url":null,"abstract":"","PeriodicalId":330,"journal":{"name":"Insect Biochemistry and Molecular Biology","volume":"187 ","pages":"Article 104458"},"PeriodicalIF":3.7,"publicationDate":"2025-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145695742","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genome-wide identification of cuticular protein genes in the social aphid Pseudoregma bambucicola and the functional role of PbamCPR-54 in soldier hindleg development","authors":"Fenglan Li,&nbsp;Jianjun Lu,&nbsp;Zhixiang Liu,&nbsp;Xiaolei Huang","doi":"10.1016/j.ibmb.2025.104455","DOIUrl":"10.1016/j.ibmb.2025.104455","url":null,"abstract":"<div><div>Cuticular proteins (CPs) constitute the primary structural components of the insect cuticle and play crucial roles in cuticle formation. In the social aphid <em>Pseudoregma bambucicola</em>, sterile soldiers cease molting and evolve specialized defensive structures, including enlarged forelegs and elongated hindlegs, whereas normal nymphs lack these features. However, it remains unclear whether soldier caste-specific CPs are involved in regulating reproductive sterility, suppression of molting, and the formation of defensive leg structures. To explore this, we identified 89 CP genes in the <em>P. bambucicola</em> genome. Chromosomal mapping revealed multiple gene duplication events. Transcriptomic analysis demonstrated distinct temporal expression patterns of CP genes between soldiers and normal nymphs. Cluster II genes are associated with molting and development of normal nymphs, while soldiers exhibit the opposite pattern for these genes. Cluster I genes show transient expression at the early stage of post-embryonic development in soldiers, whereas Cluster III genes maintain stable expression levels throughout postnatal development. Among these, <em>PbamCPR-54</em>, a pro-resilin-like gene containing the RR-2 motif, exhibits upregulated expression from 2 to 72 h during post-embryonic development of soldiers, with the highest levels detected in hindleg tissues. To investigate its function, RNA interference (RNAi) knockdown of <em>PbamCPR-54</em> was performed, resulting in deformities and bending of soldier hind tibiae. Eosin Y staining further revealed that gene silencing altered the structural integrity of hindleg cuticle. These findings provide novel insights into the roles of CP genes in caste-specific development and morphological differentiation in social insects.</div></div>","PeriodicalId":330,"journal":{"name":"Insect Biochemistry and Molecular Biology","volume":"186 ","pages":"Article 104455"},"PeriodicalIF":3.7,"publicationDate":"2025-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145675931","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"UGT35B1 is the principal enzyme mediating nicotine glycosylation in adult Drosophila melanogaster","authors":"Luke J. Pfannenstiel ,&nbsp;Rachel H. Norris ,&nbsp;Tobias Ziemke ,&nbsp;Christophe Duplais ,&nbsp;Nicolas Buchon ,&nbsp;Jeffrey G. Scott","doi":"10.1016/j.ibmb.2025.104456","DOIUrl":"10.1016/j.ibmb.2025.104456","url":null,"abstract":"<div><div>Nicotine is a plant-derived pyridine alkaloid with potent neurotoxic properties. A major pathway for detoxification of nicotine in mammals is via glucuronidation to produce nicotine <em>N</em>-glucuronide, but this process in insects remains poorly understood. Using mass spectrometry, we demonstrate that <em>Drosophila melanogaster</em> detoxifies nicotine through glycosylation, producing nicotine <em>N</em>-glycoside. Given that many new agrochemicals contain pyridine rings, we also investigated the metabolism of flonicamid and imidacloprid. We detected glycosylation of flonicamid, but not imidacloprid. A targeted RNAi screen across 21 UDP-glycosyltransferases (<em>Ugt</em>s) identified <em>Ugt35B1</em> as important for survival of nicotine exposure. CRISPR-based knockout of <em>Ugt35B1</em> increases sensitivity to nicotine and flonicamid, but not to imidacloprid, nor to a structurally distinct neonicotinoid (thiamethoxam). Mass spectrometry of knockout and control flies confirms that <em>Ugt35B1</em> glycosylates nicotine, its metabolite cotinine, and flonicamid. Together these findings establish <em>Ugt35B1</em> as an important UGT mediating nicotine detoxification in adult <em>D. melanogaster</em>, revealing a previously uncharacterized insect glycosylation pathway with potential implications for herbivory, insecticide detoxification and toxicology.</div></div>","PeriodicalId":330,"journal":{"name":"Insect Biochemistry and Molecular Biology","volume":"187 ","pages":"Article 104456"},"PeriodicalIF":3.7,"publicationDate":"2025-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145675883","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Nuclear receptor ERR contributes to imidacloprid resistance by upregulating cytochrome P450 in Nilaparvata lugens","authors":"Woo-Ram Park ,&nbsp;Byungyoon Choi ,&nbsp;Nanthini Sadasivam ,&nbsp;Hui-Jin Bae ,&nbsp;Sunmin Kim ,&nbsp;Eunae Kim ,&nbsp;Hueng-Sik Choi ,&nbsp;In-Hong Jeong ,&nbsp;Don-Kyu Kim","doi":"10.1016/j.ibmb.2025.104457","DOIUrl":"10.1016/j.ibmb.2025.104457","url":null,"abstract":"<div><div>Insecticides are widely used in pest control; however, the increasing development of resistance in pests poses a significant global challenge. Nuclear receptors (NRs), ligand-dependent transcription factors, regulate the expression of genes involved in diverse metabolic processes, including development, detoxification, and innate immunity. However, the role of NRs in the detoxification processes of the brown planthopper, <em>Nilaparvata lugens</em> (<em>N. lugens</em>), remains poorly understood. Here, we show that estrogen-related receptor (NlERR) is a novel regulator of cytochrome P450 (CYP) genes for imidacloprid (IMD) resistance in <em>N. lugens</em>. Interestingly, the NlERR was significantly overexpressed in IMD-resistant strains. Genome-wide transcriptome analysis of NlERR-deficient <em>N. lugens</em> showed a positive correlation between the NlERR and the transcription of genes associated with detoxification metabolism. In addition, IMD treatment significantly increased the gene expression of phase I P450 enzymes in suspectable <em>N. lugens</em>, which was reversed by NlERR knockdown. Moreover, chromatin immunoprecipitation analysis confirmed that the NlERR directly binds to the ERR-response elements on the promoter of the CYP4CE1 gene. Consequently, NlERR knockdown in IMD-resistant strains significantly reduced the survival rate following IMD treatment and decreased expression of its target genes, CYP4CE1 and CYP6CW1. Finally, silencing either CYP4CE1 or CYP6CW1 in IMD-resistant strains significantly decreased the survival rate of the strains treated with IMD treatment. These findings establish NlERR as a key genetic factor for conferring IMD resistance in the <em>N. lugens</em>. Selectively controlling NlERR activity with a specific modulator will provide critical insights for developing new strategies to combat insecticide resistance in the <em>N. lugens</em>.</div></div>","PeriodicalId":330,"journal":{"name":"Insect Biochemistry and Molecular Biology","volume":"187 ","pages":"Article 104457"},"PeriodicalIF":3.7,"publicationDate":"2025-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145658832","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Temperature-responsive spatial-temporal regulation underlies phenotypic plasticity of body pigmentation in Eastern honey bee","authors":"Shuai Wang ,&nbsp;Lifei Qiu ,&nbsp;Yashuai Wu ,&nbsp;Zhenfang Li ,&nbsp;Zhengwei Wang ,&nbsp;Zhenhua Chen ,&nbsp;Shanlin Liu ,&nbsp;Xin Zhou","doi":"10.1016/j.ibmb.2025.104454","DOIUrl":"10.1016/j.ibmb.2025.104454","url":null,"abstract":"<div><div>Temperature-induced variation in body coloration is a widespread form of phenotypic plasticity among insects, often mediated by the differential expression of pigmentation-related genes. Despite extensive documentation of this phenomenon, the molecular mechanisms by which environmental cues modulate pigmentation pathways remain largely unresolved. In this study, we investigated seasonal darkness plasticity in <em>Apis cerana</em>, the Eastern honey bee exhibiting a characteristic “yellow-black” abdominal pattern that responds dynamically to ambient temperature. Individuals reared under lower temperature conditions developed a markedly darker phenotype compared to those maintained at higher temperatures, highlighting a temperature-dependent shift in cuticular pigmentation. Exposure to low temperature also resulted in thicker adult cuticles and a concomitant reduction in water loss rate. The co-occurrence of darkened pigmentation and cuticle thickening corresponds with the colder and drier conditions characteristic of winter in the natural habitat of <em>A. cerana</em>. Furthermore<em>,</em> the Hedgehog (Hh) pathway was found to be enriched during early development stages. Notably, its target gene <em>optomotor-blind</em> (<em>omb</em>), a key temperature-sensitive regulator, was upregulated under decreased temperature, establishing the spatial arrangement of the black stripe at the posterior end of each abdominal tergite. Moreover, transcriptional suppression of <em>omb</em> induced upregulation of the pigmentation gene <em>ebony</em>, whose intrinsic thermally responsive expression directly enhances phenotypic plasticity in epidermal pigmentation, serving as a regulatory amplifier for environment-dependent melanin patterning. At the Pb (black eyes with an unpigmented body) in the mid-pupal period, temperature significantly influences 20-hydroxyecdysone (20E) titers. Integrated analyses combining <em>in vivo</em> 20E injection and LC-MS-based hormone quantification revealed that elevated developmental temperature upregulates 20E titers during the temperature-sensitive window, thereby suppressing the expression of pigmentation-related genes. These findings reveal a two-phase mechanism in which spatial patterning and hormonal signaling are temporally decoupled: <em>omb</em> acts early to establish the pigmentation boundary, while 20E acts later to adjust pigment intensity in a temperature-dependent manner. This layered control system allows <em>A. cerana</em> to fine-tune both the position and extent of abdominal pigmentation, providing a flexible strategy for thermal adaptation.</div></div>","PeriodicalId":330,"journal":{"name":"Insect Biochemistry and Molecular Biology","volume":"186 ","pages":"Article 104454"},"PeriodicalIF":3.7,"publicationDate":"2025-11-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145652935","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CSP15 attenuates chlorpyrifos toxicity through sequestration and bioactivation suppression in Nilaparvata lugens","authors":"Mengqing Deng,&nbsp;Xiyue Xu,&nbsp;Zhiming Yang,&nbsp;Kai Lu","doi":"10.1016/j.ibmb.2025.104453","DOIUrl":"10.1016/j.ibmb.2025.104453","url":null,"abstract":"<div><div>The escalating problem of insecticide resistance in agricultural pests highlights the need to identify non-canonical resistance mechanisms. In this study, comparative transcriptional analysis identified five chemosensory protein (CSP) genes significantly overexpressed in a chlorpyrifos (CPF)-resistant strain of <em>Nilaparvata lugens</em>. RNA interference and heterologous expression confirmed their contribution to resistance, with <em>CSP15</em>, exhibiting 42.98-fold overexpression, identified as a key mediator. <em>CSP15</em> knockdown increased CPF susceptibility 1.76-fold, while its bacterial expression raised tolerance 5.47-fold. Competitive fluorescence binding assays showed that CSP15 binds CPF (<em>K</em>_i = 3.32 μM) with higher affinity than its toxic metabolite chlorpyrifos-oxon (CPO; <em>K</em>_i = 6.00 μM). Structural modeling revealed a CPF-binding pocket mediated by Arg84 and Lys118. CPO binding utilized four hydrogen bonds with Lys28; the K28A mutation induced structural rearrangement through neo-formed interactions with Arg84 and Lys118, reducing the binding free energy by −40.95 kcal mol⁻¹. Mutagenesis revealed that R84A/K118A double mutants lost 62.85–64.37 % of CPF-binding capacity, whereas the K28A variant increased CPO affinity by 22.65 %. <em>In vivo</em> metabolic profiling indicated that <em>CSP15</em> knockdown promoted CPF degradation and CPO accumulation, supporting its role in suppressing bioactivation. Yeast two-hybrid assays confirmed a direct interaction between CSP15 and CYP6BD12, the P450 enzyme responsible for CPF bioactivation. Additionally, <em>CSP15</em> expression was regulated by the transcription factor Lim1β through two conserved promoter <em>cis</em>-elements. Together, these findings reveal a novel dual resistance mechanism wherein CSP15 acts synergistically by sequestering CPF and binding CYP6BD12, thereby effectively suppressing bioactivation and identifying promising targets for pest management.</div></div>","PeriodicalId":330,"journal":{"name":"Insect Biochemistry and Molecular Biology","volume":"186 ","pages":"Article 104453"},"PeriodicalIF":3.7,"publicationDate":"2025-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145577095","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Herbivore-induced plant root volatiles phthalic acid mediate host selection for Holotrichia parallela larva","authors":"Jiarui Tao ,&nbsp;Xiao Wang ,&nbsp;Changhong Xia ,&nbsp;Xingyuan Wang ,&nbsp;Yifan Zhang ,&nbsp;Jianan Liu ,&nbsp;Juhong Zhang ,&nbsp;Jun Wang ,&nbsp;Jinghui Xi ,&nbsp;Shang Wang","doi":"10.1016/j.ibmb.2025.104452","DOIUrl":"10.1016/j.ibmb.2025.104452","url":null,"abstract":"<div><div>The soil-dwelling grubs of <em>Holotrichia parallela</em> inflict substantial damage to the root systems of key cereal crops, including maize and peanuts, thereby posing a serious threat to agricultural productivity. While the olfactory mechanisms through which above-ground insects utilize odorant receptors (ORs) and odorant co-receptors (Orco) to detect plant volatiles for host localization are well established, the role of OR-mediated chemoreception in the host-seeking behaviors of underground pests remains poorly understood. To address this gap, we systematically compared the volatile compounds released by rhizospheric and aerial plant tissues. Through antennal transcriptome analysis and sequence structure prediction, we identified an odorant receptor with biased olfactory expression—<em>HparOR48</em>. Tissue expression analysis revealed that <em>HparOR48</em> is predominantly expressed in the larval antennae across developmental stages. The functional characterization of <em>HparOR48</em> was performed using the <em>Xenopus laevis oocyte</em> expression system, which demonstrated its broad responsiveness to phthalic acid, a root-emitted acidic volatile. Electroantennogram (EAG) assays further confirmed that phthalic acid elicited significant electrophysiological responses in the larval antennae. Moreover, behavioral assays utilizing a four-arm olfactometer indicated that phthalic acid acts as an attractant for the larvae. Our findings provide clear evidence that H. parallela larvae rely on olfactory recognition of rhizosphere-specific acidic volatiles to guide host-seeking behavior, thereby extending our understanding of chemical interactions between soil-dwelling pests and their host plants.</div></div>","PeriodicalId":330,"journal":{"name":"Insect Biochemistry and Molecular Biology","volume":"186 ","pages":"Article 104452"},"PeriodicalIF":3.7,"publicationDate":"2025-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145577096","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Master genetic switches controlling sexual development in Leptinotarsa decemlineata","authors":"Jiangjie Li ,&nbsp;Yimeng Chen ,&nbsp;Jing Wang,&nbsp;Clauvis NT. Taning,&nbsp;Kristof De Schutter","doi":"10.1016/j.ibmb.2025.104451","DOIUrl":"10.1016/j.ibmb.2025.104451","url":null,"abstract":"<div><div>Sex determination is a fundamental process during early embryonic development that shapes the morphological, physiological, and behavioral sexual dimorphism of an organism. Insects, the most diverse group of animals on earth, have evolved a wide range of sex determination systems. This study investigates the sex determination and differentiation in the Colorado potato beetle (CPB), a species with an X0 sex chromosome system, by identifying key regulatory genes involved in these processes. Our findings reveal that the genes <em>Sex-lethal</em> (<em>Sxl</em>), <em>transformer-2</em> (<em>tra2</em>), <em>doublesex</em> (<em>dsx</em>), and <em>fruitless</em> (<em>fru</em>) are highly conserved and all contribute to fertility. Among them, <em>dsx</em> and <em>tra2</em> emerge as key genetic switches of sex differentiation in CPB: Disruption of <em>dsx</em> leads to feminization of male traits and abnormal female development. Additionally, <em>tra2</em> appears to regulate the alternative splicing of <em>dsx</em>, promoting the production of the female-specific transcripts. This study highlights the pivotal roles of <em>dsx</em> and <em>tra2</em> in CPB sex determination and provides valuable insights into the evolution and diversity of sex determination mechanisms in insects.</div></div>","PeriodicalId":330,"journal":{"name":"Insect Biochemistry and Molecular Biology","volume":"186 ","pages":"Article 104451"},"PeriodicalIF":3.7,"publicationDate":"2025-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145577126","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Hypoxanthine accumulation caused by mutations of Bombyx mori xanthine dehydrogenase triggers female sterility","authors":"Wen-qing Lai ,&nbsp;Zhan-peng Lu ,&nbsp;Qiang Zhang ,&nbsp;Zuo-min Shao ,&nbsp;Han Gao ,&nbsp;Xia Sun ,&nbsp;Sheng Qin ,&nbsp;Xue-yang Wang ,&nbsp;Mu-wang Li","doi":"10.1016/j.ibmb.2025.104450","DOIUrl":"10.1016/j.ibmb.2025.104450","url":null,"abstract":"<div><div>Insect reproductive capacity is a key determinant of population fitness, though its regulatory mechanisms remain limited. Here, we identified an op50 silkworm mutant (derived from p50 strain) exhibiting female-specific reproductive defects, which provided an important model for studying insect reproductive regulation mechanism. We found that <em>Bombyx mori xanthine dehydrogenase 1</em> (<em>BmXDH1</em>) in op50 contained a single-base insertion causing premature termination, deleting 154 C-terminal amino acids and losing catalytic activity. <em>ΔBmXDH1</em> mutants recapitulated the op50 phenotype with 50 % reduced oviposition and 90 % lower hatchability, attributed to severe structural impairments of the eggshell surface and egg surface pore. In addition, female reproductive defects initiated at the pupal stage, characterized by oviduct malformation and impaired oviposition. This phenotype was associated with excessive accumulation of hypoxanthine, leading to ovarian and embryonic developmental disruption, including extracellular matrix remodeling, cellular signaling pathways, and oxidative stress regulation. The results uncovered how <em>BmXDH1</em> regulates hypoxanthine metabolism to control silkworm reproduction, advancing understanding of insect reproductive networks and providing targets for pest control/economic insect breeding.</div></div>","PeriodicalId":330,"journal":{"name":"Insect Biochemistry and Molecular Biology","volume":"186 ","pages":"Article 104450"},"PeriodicalIF":3.7,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145562280","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Field-deployable CRISPR-Dx for BmNPV and Nosema bombycis: DNA-extraction-free one-pot RPA-Cas12a and Cas12a/Cas13a dual-gene assays with handheld devices","authors":"Na Zhang ,&nbsp;Xuemin Zhou ,&nbsp;Xinhao Jiao ,&nbsp;Zi Liang ,&nbsp;Wenwen Jiang ,&nbsp;Shuyi Liu ,&nbsp;Ping Wu","doi":"10.1016/j.ibmb.2025.104449","DOIUrl":"10.1016/j.ibmb.2025.104449","url":null,"abstract":"<div><div>Simple, accurate, sensitive, and rapid pathogen diagnosis is crucial for effective control of silkworm diseases. Although CRISPR-based nucleic acid detection systems show great potential for on-site detection of silkworm pathogens, their practicality is hindered by complex workflows and reagent-storage constraints. To address these limitations and enhance field suitability, we developed a DNA extraction-free one-pot RPA-CRISPR/Cas12a (DEORC) system and a dual-gene assay for detecting <em>Bombyx mori nucleopolyhedrovirus</em> (BmNPV) and <em>Nosema bombycis</em> using a handheld device. The DEORC assay detects BmNPV in hemolymph samples as early as 6 h post-infection (hpi) and <em>N. bombycis</em> at 10³ spores/mL in spore suspensions. The entire process from sampling to visual readout is completed in approximately 70 min without requiring sophisticated equipment. To further enable off-grid deployment, we lyophilized the Cas12a detection reagents using 1 M betaine as a lyoprotectant, which retained measurable activity for at least one month at 4 °C under our test conditions, facilitating short-term refrigerated transport and field storage. Additionally, the dual-gene assay detects 10³ copies/μL of a double-reference plasmid and simultaneously detects both BmNPV and <em>N. bombycis</em> in a single tube from midgut samples at 48 hpi; when combined with extraction-free techniques, it enables simultaneous detection of both pathogens in hemolymph samples at 72 hpi. Collectively, these advancements provide sensitive and portable tools for on-site sericulture disease management, offering faster and more practical workflows than two-step single-gene and traditional approaches.</div></div>","PeriodicalId":330,"journal":{"name":"Insect Biochemistry and Molecular Biology","volume":"186 ","pages":"Article 104449"},"PeriodicalIF":3.7,"publicationDate":"2025-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145533966","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}