Insect Biochemistry and Molecular Biology

The Lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) superfamily (LU super family) of proteins are involved in diverse biological processes. In Drosophila melanogaster, members of the LU superfamily have undergone lineage-specific gene duplication and acquired specialized functions in distinct tissues. A glycosylphosphatidylinositol (GPI)-anchored LU family protein Belly roll (Bero) has recently been shown to regulate larval escape behavior; however, its cellular expression profile and potential roles remain incompletely understood. In this study, we generated a bero-GAL4T2A transgenic line to delineate endogenous bero expression. This analysis revealed that bero is expressed in the peptidergic neurons in the central nervous system (CNS) that had not been documented in previous studies, as well as in the peripheral nervous system (PNS) and non-neuronal tissues, such as the anal pad and epidermis. Reanalysis of publicly available single-cell RNA sequencing (scRNA-seq) datasets demonstrated that bero is expressed in several peptidergic neurons. These findings suggest that Bero is specifically expressed in diverse peptidergic neurons and may play important roles in coordinating hormonal and neural regulation in D. melanogaster.

The European honey bee (Apis mellifera L.) is a key agricultural pollinator frequently exposed to pesticide residues, yet the molecular basis of its chemical adaptation, particularly glutathione S-transferases (GSTs) involved in xenobiotic detoxification, remain incompletely understood. In this study, AmGSTO1 was structurally and functionally characterized to evaluate its role in agrochemical interaction and protection against oxidative stress. The crystal structure of AmGSTO1 in complex with glutathione revealed its 3D architecture and key active-site residues were identified by structural analysis and site-directed mutagenesis. Fluorescence binding assays demonstrated measurable affinity for multiple agrochemicals, including TCP, fenoprop, 2,4-D, tetramethrin, nicotine, and 3-phenoxybenzaldehyde. However, HPLC analysis showed no detectable substrate depletion, suggesting ligand binding to AmGSTO1 without catalytic turnover. AmGSTO1 exhibited antioxidant activity toward cumene hydroperoxide, hydrogen peroxide, and paraquat, as well as dehydroascorbate reductase activity. These findings indicate that AmGSTO1 may contribute to agrochemical tolerance through ligand sequestration and redox protection mechanisms.

It is commonplace in East Africa for 100% of cassava fields to be infected with Cassava mosaic disease (CMD) and/or Cassava brown streak disease (CBSD), resulting in annual losses of more than US$1.25 billion and reduced food and economic security for farming households. The vector of both diseases is the African cassava species of the whitefly Bemisia tabaci. Since the late 1990s, there has been an unprecedented increase in whitefly populations, to the extent that they are referred to as "super-abundant". Research efforts since the late 1990s has focused mainly on developing plant resistance to the viral pathogens and paid scant attention to understanding the root causes of disease epidemics or the control of whitefly infestation. Here, we aimed at developing long-term whitefly-control solutions using an in-planta RNA interference (RNAi) approach. First, transcriptome analysis identified candidate genes that play key roles in whitefly biology: osmoregulation, sugar metabolism and transport, symbiosis with endosymbiotic bacteria and detoxification of phytotoxins. Then, fifteen RNAi inverted repeat constructs were produced, designed to target the candidate genes and 140 independent transgenic lines were generated in cassava variety NASE 13. Whole plant bioassays showed insecticidal activity of transgenic plants, reaching 58% lethality for adults within 7 days and 75-90% lethality of nymphs after 25 days, compared to control plants. Target genes were confirmed to be downregulated by up to 2.5-fold in adult whiteflies and nymphs. We used population dynamics modelling to predict the potential of the RNAi technology to control whiteflies under field conditions in East Africa.

The MAT_HMGbox domain in the MAT1-1-1 protein and the HMG-box_ROX1-like domain in the MAT1-2-1 protein play essential roles in DNA binding and regulating the transcription of genes that control sexual reproduction in Ophiocordyceps sinensis. Previous studies have documented differential occurrences, differential transcription, and alternative splicing of the MAT1-1-1 and MAT1-2-1 and pheromone receptor genes in Hirsutella sinensis (Genotype #1 among 17 genome-independent O. sinensis genotypic fungi). This study further revealed that the DNA-binding domains of the paired MAT1-1-1 and MAT1-2-1 proteins derived from each of the 20 O. sinensis strains exhibit heteromorphic tertiary structures, as predicted by AlphaFold 3D structural modeling. The differentially paired mating proteins, characterized by different truncations, 1-4 amino acid substitutions at distinct sites, altered hydrophobicity and secondary structures, and heteromorphic tertiary structures, indicate divergency in the fungal origins of the mating proteins within the mycologically and genetically impure O. sinensis strains. These findings support the hypothesis of O. sinensis self-sterility and are likely ensure the fidelity and genetic diversity of heterothallic or hybrid reproduction throughout the lifecycle of the Cordyceps sinensis insect-fungal complex.

Oviposition site selection is critical for mosquito population dynamics. Gravid mosquitoes rely on chemical cues to identify suitable breeding habitats. However, the sensory mechanisms governing this behavior in Anopheles stephensi remain poorly understood. Here, we examined the role of indole, a microbial volatile associated with aquatic environments, in oviposition site choice and assessed the involvement of sensory organs in its detection. In two-choice oviposition assays, water conditioned with first-instar larvae attracted gravid females (OAI = 0.56), whereas water from fourth-instar larvae was repellent (OAI = -0.20), consistent with avoidance of suboptimal, resource-depleted habitats. Indole elicited strong oviposition attraction across a broad concentration range (0.1-50 {micro}M), with no clear dose-response relationship. Surgical ablation of antennae and maxillary palps did not abolish indole-mediated preference but significantly reduced behavioral variability, suggesting that these structures modulate, rather than solely mediate, indole detection. Reanalysis of transcriptomes of antennae, maxillary palps, and legs in An. gambiae and An. coluzzii, along with quantitative RT-PCR in An. stephensi, revealed the expression of chemosensory genes (including Obp1, Obp13, Obp25, Obp71, Or2, and Or10) in the legs, indicating a potential role for leg chemosensation in oviposition decisions. These findings underscore the complexity of chemoreception and chemoperception in mosquito habitat assessment.

Animals sense and integrate complex external cues to make developmental decisions that help them better survive and adapt to their natural habitats. Under environmental adversity, nematodes can enter an alternative developmental pathway to form a diapautic and stress-resistant stage, termed the dauer larvae. While dauer formation has been well characterized in Caenorhabditis elegans, how environmental factors influence analogous stages in other nematode species remains largely unexplored. This study examines how symbiotic bacteria, temperature, and pheromones affect the formation of the infective juvenile (IJ), a dauer-like stage, of the insect-parasitic nematode Steinernema hermaphroditum. In contrast to C. elegans, where dauer entry is promoted by heat, IJ development in S. hermaphroditum development is enhanced by reduced temperature. Moreover, the presence and absence of live symbiotic bacterium Xenorhabdus griffiniae functions as an ON-and-OFF switch that regulates the host IJ formation. Crude pheromone extracts from S. hermaphroditum liquid culture do not robustly induce IJ formation in a dose-responsive manner, unlike the potent pheromone-driven dauer entry observed in C. elegans. Nutrient-rich liver-kidney media that mimics host insect environment showed IJ entry induction in a pheromone-dependent manner. These data suggest that external cues, such as temperature, microbial diet, and pheromone, are perceived differently by S. hermaphroditum in comparison to that of C. elegans, reflecting species-specific adaptations to distinct ecological niches and life history strategies.

Isoflavone synthase (IFS), a cytochrome P450 monooxygenase of the CYP93C subfamily, catalyzes the conversion of flavanones into isoflavones, the first committed step in the biosynthesis of isoflavonoid phytoalexins. In pea (Pisum sativum L.), the phytoalexin pisatin plays a pivotal role in defense against pathogens. However, the molecular basis underlying IFS function in pea remains poorly understood. In this study, we performed a comprehensive genome-wide identification and characterization of IFS genes in pea. Three IFS candidates, PsIFS7A, PsIFS7B, and PsIFS7C, were identified that reside on chromosome 7, each harboring all conserved cytochrome P450 signature motifs. PsIFS genes exhibited predominant expression in root tissue, with transcript levels induced rapidly upon Aphanomyces euteiches infection. Enzymatic assays confirmed their catalytic activity in converting the flavanones naringenin and liquiritigenin into the isoflavones genistein and daidzein, respectively, both in vitro and in planta systems. Furthermore, all three PsIFS genes were found in close proximity to quantitative trait loci (QTL) associated with Aphanomyces root rot resistance. Together, these findings provide novel insights into the IFS gene family in pea and lay a foundation for metabolic engineering or molecular breeding strategies to enhance disease resistance through targeted modulation of pisatin biosynthesis.

The tropical house cricket Gryllodes sigillatus is a major species used in the edible insect farming industry. Despite the rapid expansion of this sector, diagnostic tools for detecting infections in these species remain limited. The lack of validated reference genes compromises the reliability of RT-qPCR-based gene expression analyses, which are essential for the development of molecular tools for disease diagnosis and health monitoring in insect production systems. To address this gap, we evaluated the expression stability of six candidate reference genes (ACTB, EF1, GAPDH, HisH3, RPL5, and 18SrRNA) across four body parts (abdomen, head, legs, and whole body) using a combination of complementary statistical approaches, including geNorm, NormFinder, BestKeeper, the {Delta}Ct method, the R statistical environment, and the integrated RefFinder tool. Candidate genes were identified and annotated using the recently published G. sigillatus genome, through sequence comparisons with closely related insect species using BLAST and reciprocal BLAST analyses, multiple sequence alignments. All procedures complied with MIQE 2.0 guidelines to ensure methodological rigor and transparency. The results showed that ACTB, EF1, RPL5, and 18SrRNA exhibited stable and consistent expression across all analyzed tissues, whereas GAPDH and HisH3 displayed high variability and were generally unsuitable for normalization, except in head tissue where GAPDH remained stable. This study provides the first validated set of reference genes for G. sigillatus, establishing a robust foundation for accurate, reproducible, and comparable gene expression analyses. Furthermore, these findings support the development of RT-qPCR-based diagnostic tools, contributing to improved health monitoring and biosafety in insect production systems.

The western honey bee (Apis mellifera) is an essential pollinator facing unprecedented threats from pesticide exposure. While pesticide resistance evolution is well documented in agricultural pests, our understanding of genetic variation in honey bee detoxification systems remains limited. This represents a missed opportunity, as harnessing naturally occurring detoxification diversity could provide new avenues for pollinator protection. Cytochrome P450 monooxygenases (CYPs), which are central to xenobiotic metabolism, offer a promising starting point. Here, we present the first comprehensive analysis of CYP genetic diversity in A. mellifera. We analysed the CYPome of 1,467 individuals representing 18 A. mellifera subspecies from 25 countries and identified 5,756 single-nucleotide polymorphisms (SNPs) in 46 CYP genes. Imputed McDonald-Kreitman testing revealed that 56% of non-synonymous CYP substitutions were driven by positive selection. Of the 1,302 haplotypes identified, 84% resided in CYP3, concentrated in the CYP9 and CYP6AS subfamilies implicated in xenobiotic detoxification. Population-level analysis of nucleotide diversity, Tajimas D selection signatures, FST-based differentiation, and McDonald-Kreitman testing pointed to CYP3 clan genes as the primary locus of adaptive variation. This work provides the first step toward building a comprehensive pharmacogenomic resource for honey bees, enabling the prediction of population-specific pesticide vulnerabilities and leveraging naturally occurring detoxification variants to enhance pollinator resilience - a critical step toward sustainable pollinator management.

In yeast, transcriptional adaptor 2 (ADA2; SAGA complex subunit ADA2), a member of histone acetyltransferase (HAT) complex, regulates transcription through cell signalling, but its precise role in cellular metabolism remains unclear. In this study, genetic loss of ADA2 (ada2{Delta}) induces squalene (SQ) accumulation, indicating aberrant triterpene metabolism, coupled with endoplasmic reticulum (ER)/nuclear ER (nER) expansion. Lipid analyses of ada2{Delta} revealed elevated phosphatidic acid (PA) and phosphatidylcholine (PC) levels, indicating disrupted phospholipid metabolism. The expanded ER causes basal autophagy elevation, cellular recycling, and nER phagy, suggesting a regulatory role for ADA2 in autophagy. Downregulation of phosphatidate cytidylyltransferase (CDS1) and inositol-3-phosphate synthase (INO1), coupled with elevated PA and PC in ada2{Delta}, points to a significant disruption in cytidine-diphosphate-diacylglycerol and phosphatidylinositol pathway. Overexpression of CDS1 or INO1, or the inositol supplementation, in ada2{Delta} restores SQ, basal autophagy and ER phagy. The observed target of rapamycin Ser/Thr kinase complex (TORC1) activity in ada2{Delta} is due to the high PA content. Rapamycin-mediated inhibition of TORC1 reduced SQ, PA and ER expansion while increasing lipid droplets. In contrast, a rapamycin-treated ada2{Delta}pah1{Delta} strain retained high PA, SQ and ER expansion, underscoring the functional role of TORC1-nuclear envelope morphology protein 1 (Nem1)/sporulation-specific protein SPO7 (Spo7)-Pah1 axis. Notably, SQ levels remained unchanged in a rapamycin-treated ada2{Delta}atg39{Delta} strain, suggesting that loss of nER-phagy receptor, Atg39, impairs the effectiveness of TORC1 inhibition. In conclusion, our data unveiled a critical role for Ada2 in maintaining the intricate relationship between lipid and triterpene/sterol metabolism and connecting autophagy and ER homeostasis.

Nociception is an essential response for organisms to avoid potential harm and promote survival. Its molecular determinants are largely conserved across Eumetazoa. TRPV receptors are polymodal ion channels exhibiting selective peripheral expression and functional coupling that underpins nociception and pain modulation in complex organisms. However, the execution of protective behaviours triggered by TRPVs is also found in species with a simpler nervous organisation, thus encouraging their investigation in invertebrate model organisms to increase understanding of animal nociception. Cephalopods represent an interesting invertebrate phylum with respect to the evolution of the nervous system, whose complexity suggests it might support pain-like states that exist in vertebrates. This possibility is reflected by the inclusion of cephalopods in the UK and EU animal welfare legislations. Despite this, there is poor characterisation of cephalopod molecular nociceptors. For this reason, we used in silico analysis to identify two TRPV channels in Octopus vulgaris genome (Ovtrpv1 and Ovtrpv2). We validated the putative transcript sequences and highlighted prevalent expression in sensory tissues. We investigated the functional competence of these TRPVs by heterologously expressing Ovtrpv1 and Ovtrpv2 cDNA into Caenorhabditis elegans null mutants of the orthologous genes, ocr-2 and osm-9 respectively. Ovtrpvs successfully rescued the aversive response to chemical and mechanical noxious stimuli in the C. elegans mutants, suggesting these receptors are polymodal nociceptors. Additionally, complementary investigation using Xenopus laevis oocytes showed Ovtrpv1 and Ovtrpv2 form an active heteromeric channel gated by nicotinamide. This study highlights Ovtrpvs as an important route to better understand nociceptive detection in cephalopods.

Gene Model for Insulin-like peptide 4 (Ilp4) in the D. simulans DsimGB2 assembly (GCA_000754195.3). The characterization of this ortholog was carried out as part of a larger, ongoing dataset designed to explore the evolution of the insulin/insulin-like growth factor signaling (IIS) pathway across the genus Drosophila, utilizing the Genomics Education Partnership gene annotation protocol within Course-based Undergraduate Research Experiences.

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 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 -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.

The cockroach Blattella germanica possesses panoistic ovaries, in which oocytes lack nurse cells and therefore need to rely on their own transcriptional activity to support embryogenesis. Ovarian development in this species involves the development of a single basal ovarian follicle (BOF) per gonadotropic cycle, a process strictly regulated by endocrine signals, primarily juvenile hormone and ecdysone, which act at both the transcriptional and translational levels. In addition, transcriptional activity in these ovaries is necessary for both regulating and genome protection, and at this level, PIWI-interacting RNAs (piRNAs) play an essential role. Although insect ovaries are known to be particularly rich in piRNAs, their function in ovary maturation is still not well defined. For this purpose, we characterize the piRNA expression dynamics across seven key developmental and reproductive stages, ranging from late nymphal instars to post-vitellogenic adults. piRNA expression in B. germanica shows coordinated fluctuations. Expression remains stable in previtellogenic ovaries, whereas vitellogenic ovaries show pronounced changes. Moreover, vitellogenic ovaries exhibit reduced piRNA diversity due to strong enrichment of a subset of highly expressed piRNAs. Our data show that although piRNAs predominantly map to transposable elements, particularly LINEs, there is a notable increase in gene-derived piRNAs toward the end of the cycle. Our results suggest regulatory roles of piRNAs in modulating both TEs and mRNAs during BOF maturation, likely related to changes in the follicular cell program.

MicroRNAs (miRNAs) play essential roles in the posttranscriptional regulation of gene expression in organisms. In the process of synthesizing mature miRNAs from miRNA precursors, the miRNA precursors are cleaved via Dicer at their loop structure, after which the miRNA precursors become mature and regulate transcription. However, the consequences of altering the loop sequence are not fully understood. The silkworm Bombyx mori is a lepidopteran insect with many genetic strains. We identified a mutant of the miRNA miR-3260 whose the part of the loop structure was lacking in a silkworm strain with translucent larval skin. Here, we aimed to analyze the role of wild-type miR-3260 and the influence of the mutation of the loop structure in B. mori. First, we identified the genomic region responsible for the translucent larval skin phenotype and determined that the mutated miR-3260 nucleotide sequences. Then, we predicted the binding partners of wild-type miR-3260 using the RNA hybrid tool and found two juvenile hormone (JH)-related genes as targets of wild-type miR-3260. Next, we assessed the relationships between miR-3260 and JH and found that miR-3260 was highly expressed in the Corpora allata and its expression responded to JH treatment. Meanwhile, miR-3260 mimic and inhibitor did not induce the typical phenotypes associated with JH in B. mori. Then, we compared the dicing products from wild-type and mutant miR-3260 precursors and observed that neither form underwent Dicer-mediated cleavage when the loop structure was altered. These results suggest that loop mutations in the miR-3260 precursor may not influence dicing activity, consistent with the lack of observable phenotypic effects.

BACKGROUNDThe two-spotted spider mite, Tetranychus urticae Koch, is a major agricultural pest with a rapid propensity for developing acaricide resistance. Bifenazate targets mitochondrial cytochrome b (CYTB). While the G126S mutation is frequently associated with resistance, its independent role remains unclear as it often occurs with other substitutions. This study explores the molecular basis of bifenazate resistance in a Russian laboratory strain derived from a St. Petersburg greenhouse population. RESULTSDisruptive selection with increasing bifenazate concentrations generated resistant and susceptible isofemale lines. AlphaFold2 structural modeling of CYTB indicated that G126S causes a steric clash, leading to conformational destabilization, whereas other reported mutations primarily affect the ligand-binding pocket. Oxford Nanopore sequencing revealed a very low initial frequency of the G126S allele (<1%; 226/35,895 reads) in the unselected population. After one year of stepwise selection (0.00005-0.031% a.i.), the mutant allele frequency surged to 90% (7,272/8,056 reads). No other known resistance-associated mutations were found in the analyzed cytb fragment. CONCLUSIONWe report the first identification of the G126S mutation in a Russian T. urticae population and demonstrate its rapid fixation under bifenazate selection. Within this genetic background, G126S alone appears sufficient to confer high-level resistance, emphasizing the population-specific nature of resistance evolution and the critical need for local monitoring.

The Drosophila adult central brain contains 240 circadian neurons, of which there are more than 25 different neuron subtypes based on connectomic data. Recent single cell RNA-seq (scRNAseq) characterization of these neurons "around the clock" also indicates a similar number of molecular subtypes of circadian neurons, but other conclusions from these transcriptomic studies warranted verifying and extending with other approaches. To this end: 1) We used a genetic multiplexing strategy to profile the transcriptomes of circadian neurons from multiple time points in a single experiment, reducing confounding technical variation between timepoints; 2) Large numbers of single nuclei were sequenced (snRNA-seq), which was enabled because the new method EL-INTACT purifies nuclei from frozen heads; 3) We assayed 12 time points under both light-dark (LD) and constant darkness (DD) conditions. These approaches showed dramatic transcriptional differences between time points in many circadian neuron types and enhanced time-of-day gene expression analysis. The data indicate that most of this regulation is transcriptional and circadian. There were however a small number of light-dependent transcripts, including a few that correspond to mammalian immediate-early genes. They probably play a role in the light-regulation of gene expression and behavior in specific neurons, perhaps circadian entrainment or phase-shifting. The results taken together provide a more comprehensive picture of gene expression heterogeneity within adult Drosophila circadian neurons including how intrinsic clock mechanisms and light cues are integrated across circadian neuron subtypes.

Spatial emanators (SE) are a promising complement to existing tools for preventing mosquito transmitted diseases. In 2025, the WHO updated the WHO Guidelines for Malaria to include a conditional recommendation for the indoor use of prequalified SE products in malaria control. Both prequalified, and many other SE products contain the volatile pyrethroid transfluthrin, which shares the same target site as other (contact/solid phase) pyrethroids. Therefore, an assessment of cross resistance is critical to predict effectiveness against mosquitoes with existing pyrethroid resistance. Our results show that resistance to solid phase pyrethroids is correlated with resistance to transfluthrin in Anopheles and Aedes species. Moreover, commonly-selected resistance mechanisms including target site mutations and over-expression of P450 detoxification enzymes can confer resistance to transfluthrin. Furthermore, we show that resistant mosquitoes are less impacted by transfluthrin in terms of flight activation (irritancy) and reduced blood feeding inhibition, with the response correlating with resistance strength. Transfluthrin did not elicit an electroantennography response in Anopheles gambiae and surgically ablating mosquitoes antennae did not result in differences in flight activation upon transfluthrin exposure, suggesting the antennae are not required for transfluthrin to elicit behavioral responses. These results provide new insight regarding the mode of action of transfluthrin and the risk of resistance reducing transfluthrins efficacy in vector control interventions.

Iron is an essential nutrient for all types of organisms, including insects and the microbes that infect them. We predicted that insects fed an iron-supplemented diet would accumulate more iron in their hemolymph, and, because infectious microbes acquire iron from their hosts, that this extra iron would increase the severity of bacterial infections. To test this hypothesis, we studied the effects of dietary iron supplementation on infection outcomes in Manduca sexta (tobacco hornworm). Larvae were fed an artificial diet, with or without antibiotics, or the same diets supplemented with 10 mM iron. Control and iron-treated larvae were inoculated with non-pathogenic Escherichia coli or the entomopathogenic Enterococcus faecalis, and bacterial load and larval survival were measured. We found that dietary iron supplementation increased the iron content of hemolymph by approximately 20 fold; however, contrary to our prediction, this increase in iron did not result in an increase in the bacterial load of either E. coli or E. faecalis. The effect of iron supplementation on survival was more complicated. As expected, for larvae inoculated with nonpathogenic E. coli, iron supplementation had no effect. For larvae inoculated with E. faecalis, the effect of iron supplementation depended on whether antibiotics were present in the diet. Without antibiotics, iron supplementation prolonged larval survival; with antibiotics, iron supplementation decreased larval survival. The results of this study do not support the hypothesis that dietary iron supplementation increases infection severity in M. sexta. Instead, the results support the viewpoint that the relationship between dietary iron and infection outcome is complex.

Benzoylphenylurea (BPU) insecticides disrupt insect chitin formation, yet their molecular target is debated. Although resistance-associated mutations map to the Chitin Synthase (CHS) gene, direct inhibition of CHS has not been demonstrated. Given the structural similarity of BPUs to mammalian glycogen phosphorylase (GP) inhibitors, GP has been proposed as a potential target controlling chitin precursor flux. We characterized Plutella xylostella GP (PxGP) and found that the human GP inhibitor GPI potently inhibits both recombinant PxGP (IC = 2.96 nM) and native larval GP activity (57.5% reduction), while the BPU diflubenzuron showed no effect. Despite this biochemical inhibition, neither GPI treatment nor RNAi-mediated PxGP knockdown (87.6% suppression) caused mortality or developmental defects. Mechanistic analysis revealed coordinated downregulation of glycogenolysis genes (trehalase 69%, hexokinase 77%) and robust upregulation of gluconeogenolysis genes (PEPCK 3.95-fold, G-6-Pase 3.34-fold). Critically, protein catabolism (29.4% decline at 72 h) provided substrates for gluconeogenolysis by releasing amino acids. Metabolite profiling validated the full substrate-to-product pathway: transient glucose shortfall was followed by massive accumulation of trehalose (7.4-fold) and glucose-6-phosphate (6.5-fold), confirming a quantitatively sufficient metabolic rescue via de novo glucose synthesis. These findings demonstrate that BPUs do not target GP, and that GP inhibition alone is not lethal due to metabolic compensation. This highlights a fundamental metabolic plasticity that must be considered in future target-based insecticide design.