Insect Science

The tick Ixodes ricinus is the main vector of human and animal pathogens in Europe. Despite its importance in epidemiology and medicine, our understanding of physiological mechanisms controlling blood feeding, osmoregulation, or development are still limited. Here, we identify novel neuropeptide invertebrate parathyroid hormone-like peptide (iPTH) and its two receptors - PTHR1 and PTHR2 in I. ricinus. Functional aequorin-based assay confirmed specific activation of both receptors by iPTH. Using RT-qPCR we detected the PTHR1 transcript in the synganglion, while increased expression levels of PTHR2 were found in the salivary glands, hindgut and female gonads. RNA-mediated knockdown of iPTH receptors in nymphs resulted in delayed blood feeding, and a high incidence of defects in adult ecdysis. Consistent with observed phenotypes, iPTH is expressed in multiple neurons of the synganglion which project arborizing axons to the salivary glands, rectal sack and skeletal muscles. iPTH was colocalized with orcokinin-immunoreactivity (OK-IR) in all neurons that innervate these peripheral tissues. iPTH is further colocalized with tachykinin (TK) in Pd1DL1 neurons, suggesting coordinated action with other neuropeptides. Our findings indicate that iPTH signaling is required for normal feeding, development and successful ecdysis.

In many insect species, mating is coordinated through multimodal signaling, yet less obvious channels are often overlooked. In the burying beetle Nicrophorus vespilloides, chemical communication is well-documented, but the role of substrate-borne vibrational signals (stridulations) during courtship remains unknown. We investigated whether stridulation is essential for mating success through two sets of experiments. First, we found a positive correlation between the frequency of stridulations and both the number and duration of copulation events. Second, we employed a silencing experiment to test the necessity of these signals by silencing males, females, or both partners. We found no significant differences between silenced and control groups regarding the frequency or duration of physical contact and mounting events, suggesting that stridulation is not required for mate recognition or the initiation of courtship. However, the proportion of successful copulations relative to mounting events was significantly lower when females were silenced. These results suggest that while N. vespilloides relies on a redundant multimodal system that likely utilizes chemical cues to initiate mating, vibrational signals, particularly from the female, may play a critical role in facilitating successful copulation. This study provides the first evidence for the role of stridulation in the mating behavior of N. vespilloides and highlights the potential for female-mediated vibrational signaling in burying beetle courtship.

Many social insects, such as ants, change their behavior stereotypically from nursing to foraging as they age, a concept known as temporal polyethism. Biogenic amines are associated with these specific behaviors, but much about them remains poorly understood in these animals. Many aminergic systems lack anatomical characterization, and little is known about the expression of genes required for the aforementioned behavioral phenotypes. Here, we studied Pogonomyrmex barbatus brains and identified the neurons that produce two relevant amines, dopamine and octopamine, by detecting their synthesis enzymes, tyrosine hydroxylase and tyramine beta-hydroxylase, and their transcripts. We also compared the expression of both genes in young nurses and mature foragers by measuring the fluorescence intensity. Dopaminergic and octopaminergic neurons are predominantly located in the protocerebrum and in the subesophageal zone. Neurons that produce dopamine are also present in the optic lobes, whereas octopamine-producing ones show clusters in the antennal lobes. Both genes are downregulated as the organism ages. A reduction in the expression of these genes might be correlated with the mature workers increased propensity to perform extranidal tasks, such as foraging.

Many insects enter diapause, a programmed state of developmental arrest that enables survival under adverse environmental conditions. In Drosophila melanogaster Meigen, 1830, diapause is characterized by reproductive arrest and reduced intestinal growth, accompanied by suppressed intestinal stem cell (ISC) activity. Juvenile Hormone (JH) promotes ISC proliferation under favorable conditions, but its capacity to modulate stem cell dynamics during cold-induced diapause remains unclear. Here, we investigated whether JH signaling can reactivate midgut remodeling in adult females maintained at 11. At this temperature, flies exhibited pronounced gut atrophy and elevated Phospho-histone H3 (PH3+) cell abundance, consistent with temperature-dependent G2/M phase arrest JH treatment significantly increased the proportion of Delta-positive progenitor cells in the anterior (R2) and posterior (R5) midgut regions at both 11 and 25, demonstrating that JH acts as a conserved mitogen for the ISC pool irrespective of thermal environment. A trend toward reduced PH3+ accumulation in the posterior midgut following JH treatment (p = 0.061) suggests possible facilitation of mitotic exit, though this effect did not reach statistical significance. Despite cellular-level changes, JH treatment did not restore overall gut size, indicating that the 72-84 hour exposure window was insufficient for subsequent tissue hypertrophy. Additionally, we identified a recurrent cold-induced pathology of gut distension, provisionally termed Lumen Obstruction Syndrome (LOS), which was independent of JH signaling. These findings reveal an uncoupling of JH-driven stem cell expansion from gross organ growth under diapause conditions, highlighting the selective sensitivity of the ISC compartment to endocrine signaling during environmental stress.

Fat accumulation and use is critical for sustaining life. Most insects show a typical response to feeding where fat is accumulated when sufficient sugars and other carbohydrates are consumed. Parasitoid insects are an exception, because most species do not accumulate fat when feeding on a sugar-rich diet. Studies on fat metabolism generally measure fat content early in life without considering lipid metabolism as a dynamic process that is expected to change as life progresses. In this paper, we compared fat accumulation and use throughout the lives of adult female Drosophila melanogaster and females of 5 inbred lines of the parasitoid wasp Leptopilina heterotoma. We expected that fat accumulation would take place irrespective of teneral fat content in D. melanogaster. We found that fat D. melanogaster initially used fat reserves, while lean flies economized on fat stores. Both lean and fat flies started accumulating fat after 7 days of life, indeed showing a typical response for insects. Unlike other parasitoids, L. heterotoma populations differs in fat accumulation patterns that we expected to observe also between inbred lines. In none of the inbred lines, however, did fat accumulation take place. Our results did reveal that inbred lines differed in the rate at which fat was used mainly later during life. We further confirmed that D. melanogaster pupal size was highly correlated with adult female size for both D. melanogaster and L. heterotoma. Overall, our findings for L. heterotoma provide strong evidence that genetic background has a major impact on the rate at which fat is used over a lifetime.

The multivoltine non-native box tree moth, Cydalima perspectalis (Lepidoptera: Crambidae), was accidentally introduced in several continents and exhibited a very rapid expansion in its invaded ranges, colonising around 40 countries in less than twenty years. Initially observed in urban areas, it quickly spread to forest stands, causing significant damage to boxwood trees. If the role of the ornamental plant trade in its fast invasion has been investigated, the insects flight capabilities has thus far been overlooked, limiting estimations of its natural dispersal. In this context, we assessed the flight performance of the adults and investigated potential effects of sex, age and generation using computer-monitored flight mills. Under these controled conditions, we estimated the distance flown per night and over the adults lifespan in both sexes for each generation. The adults were able to fly on average 18 km within their lifespan, even though distances flown were highly variable, including several females capable of performing long-distance flights (up to 150 km). The distances covered were partly correlated with the age and body mass of the adults. Mated females flew longer distances than virgin ones, but long dispersal seemed to limit their fecundity. Finally, the overwintering generation presented the highest flight capabilities with individuals able to cover 22 km on average while the late summer generation covered only 10 km. The box tree moth showed good dispersal abilities that likely played a significant role in its rapid local expansion, while human-mediated dispersal favoured its long-distance dispersal.

Most organisms have evolved mechanisms to reduce the negative effects of toxins in their diet. Some animals that are toxin specialists, such as dendrobatid poison frogs, have amino acid substitutions in proteins targeted by the toxins that prevent or limit the toxins ability to bind and exert its bioactive effects. The Phantasmal poison frog, Epipedobates tricolor, has amino acid substitutions in its neuronal nicotinic acetylcholine receptors that were previously shown to provide resistance to the highly potent neurotoxin epibatidine in vitro. However, it is unclear whether E. tricolor resists the physiological effects of epibatidine in vivo. To investigate this, we examined the effects of epibatidine exposure on the locomotion and behavior of E. tricolor. We performed whole-animal performance assays and behavioral evaluations at multiple time points following the administration of high yet biologically relevant levels of epibatidine. These assays were followed by alkaloid quantification to track chemical concentrations in the skin. Epipedobates tricolor exhibited similar locomotor performance and behavior at both epibatidine doses compared to controls and regardless of the quantity of alkaloid accumulated into the skin. However, we observed an impact of low-percentage ethanol solutions on behavior when compared to water controls, as well as general impacts of handling stress (regardless of the administered solution type), which should be considered in future experimental designs. Overall, we demonstrate that E. tricolor likely avoids a physiological fitness trade-off between toxin ingestion and the defensive benefits of epibatidine sequestration. Our study suggests that the ability to ingest toxins involves multi-faceted resistance mechanisms. Highlights- Whole-animal performance assay shows no impact of epibatidine on frog behavior - Epipedobates tricolor is 20-5000 times more resistant to epibatidine than mice - Assays demonstrate that frogs are sensitive to 6% ethanol and handling stress

Healthspan, the period of life where organisms are without frailty and/or disease, is a major focus of biogerontological research. To understand late-life decline and increased mortality risk, short-lived organisms such as nematode worms are commonly used. Pristionchus nematodes are established models for evolutionary developmental genetics research and show promise as systems for comparative and experimental study of ageing. To support this, we developed phenotypic ageing profiles for the evo-devo model Pristionchus pacificus and its little-studied congener Pristionchus fissidentatus. We find that various life history traits differ between P. pacificus and P. fissidentatus (lifespan, brood size, and reproductive period), demonstrating their utility for studying divergent ageing trajectories. Further, several traits are consistently impacted by age, including intestinal barrier function, body size, and locomotory ability. Additionally, in P. pacificus, rupture avoidance, cuticle integrity, and feeding rate decline with age, indicating dysregulation across many tissue types. Several age-linked patterns resemble those documented for Caenorhabditis elegans despite considerable evolutionary distance, suggesting conserved senescent processes across the Rhabditida family of nematodes. This work highlights similarities and differences in the impact of ageing in two Pristionchus nematodes and supports their development as models for evolutionary genetic study of senescence.

Animal venoms are a rich source of bioactive molecules, yet their diversity remains incompletely characterized in many species. Here we present the first long-read transcriptomic analysis of venom glands from Philippine tarantulas (Theraphosidae), a highly endemic but understudied group. Using Oxford Nanopore sequencing, we reconstructed near full-length venom gland transcriptomes across multiple species and identified extensive repertoires of toxin-encoding peptides. Venom glands were enriched in cysteine-rich inhibitor cystine knot (ICK) peptides, which dominated the toxin landscape and are known modulators of ion channels. Cross-species comparative analyses revealed a distinct transcriptional signature separating venom from non-venom tissues, driven by coordinated expression of toxin-associated and regulatory gene families. Phylogenomic reconstruction based on orthologous peptides recovered expected taxonomic relationships while revealing potential lineage-specific diversification and potential cryptic taxa. Despite a conserved core set of toxin families, substantial variation in toxin composition was observed among species, consistent with rapid evolution driven by gene duplication and functional divergence. Analysis of highly expressed ICK peptides showed a conserved cysteine framework alongside marked sequence variability in inter-cysteine regions, supporting a model in which structural stability is maintained while functional diversification proceeds. Together, these findings establish the first long-read transcriptomic resource for Philippine theraphosid spiders, reveal a conserved molecular signature underlying venom gland specialization, and provide new insights into the diversification of ICK toxin repertoires that may facilitate future evolutionary and functional studies, including the discovery and characterization of bioactive venom peptides.

The red palm weevil, Rhynchophorus ferrugineus, is the most economically destructive palm pest worldwide, threatening livelihoods, food security, and ecosystems across 49 countries. Weevil management currently relies predominantly on chemical insecticides, raising significant environmental and public health concerns. Despite its global agricultural importance, genetic approaches to pest management and the mechanistic basis of genome-editing strategies in Rhynchophorus remain largely unexplored. Here, we employed CRISPR/Cas9 genome editing to disrupt the R. ferrugineus ommochrome biosynthetic pathway -- a multi-enzymatic metabolic cascade that converts tryptophan into ommochrome pigments, including brown, yellow, and red pigments. We targeted two key pathway components: the ATP-binding cassette transporter white and the heme peroxidase cardinal. Both genes were ubiquitously expressed, with peak expression levels in the gut, fat body, and head. Elevated transcript levels were observed across early, mid, and late pupal stages and in 0-, 1-, and 2-day-old adult males and females, consistent with the progression of eye pigmentation throughout the R. ferrugineus life cycle. Embryonic microinjection of a single guide RNA (sgRNA)-Cas9 ribonucleoprotein complex targeting white produced in the Generation-0 (G0) adults with a distinct, white-eyed phenotype with a brownish outer margin, in contrast to the black eyes of wild-type adults. Genome-edited cardinal mutant adults displayed a translucent, brownish-white-eyed phenotype, with white streaks that gradually transitioned to a persistent translucent reddish-brown eye coloration. Mutations in both genes were confirmed in G0 adults by genomic DNA sequencing. Mutant adults were crossed to generate heterozygous G1 (+/-), G2 (-/-, -/+, and +/+), and G3 lines (-/-) with genotypes verified as carrying 2-, 3-, 9-, and 13-nucleotide deletions. A stable, heritable eye-color phenotype was established in homozygous knockout (-/-) G3 lines for both white and cardinal, confirmed by unambiguous indel (insertions/deletions) genotyping. Inheritance analysis revealed that both genes are X-linked, following a classical Mendelian sex-linked pattern: paternal alleles are transmitted exclusively to daughters, while maternal alleles are inherited equally by both daughters and sons. This study establishes the first fully homozygous knockout strain in R. ferrugineus and, by characterizing sex-linked inheritance in a coleopteran system, advances our understanding of how CRISPR/Cas9 can be efficiently applied to destructive palm weevil species. The present study represents the first report of CRISPR/Cas9 genome editing in any weevil (Curculionidae), using white and cardinal as marker genes. These findings provide a valuable platform for functional genomics and genome engineering in R. ferrugineus and offer a translational framework for genome editing in the invasive South American palm weevil, R. palmarum, laying a solid foundation for the development of gene-drive strategies aimed at sustainable palm weevil population control.

Spodoptera frugiperda is one of the most important agronomical pests due to its migratory capacity and broad host range. Since it is resistant to several insecticides, novel control strategies are being explored to control it. In this way, Spodoptera frugiperda Multiple Nucleopolyhedrovirus, a natural pathogen, has been proposed for its biocontrol. In this work, we performed a small RNA-seq on uninfected larvae and larvae infected with SfMNPV to identify expressed miRNA, characterize them, and identify differentially expressed (DE) miRNA in the infected condition. We identified several known and putative novel miRNAs, some of which are encoded in multiple copies and may be expressed within miRNA clusters. We also found 13 DE miRNA, most of them previously reported, two of them are putative novel miRNAs identified in this work. We predicted miRNA targets and found that their putative biological role could be related with processes relevant to the infection such as proliferative and apoptotic pathways, cell cycle regulation, autophagy, DNA damage response (DDR), vesicle transport, cytoskeleton remodelling, JAK/STAT and Toll signaling pathway, and immune response activation, among others. Moreover, we observed that several of the putative targets were hub genes in a predicted protein - protein interaction network. Finally, we found DE miRNA putatively associated with the regulation of viral gene expression, suggesting they might have a role in modulating the infection. Our results contribute to better understanding the miRNA landscape in S. frugiperda, and their putative role upon SfMNPV infection.

Anoxia is a major stress for most vertebrates and frequently accompanies harsh winter conditions, particularly in species that spend much of the season frozen solid. North American freeze-tolerant wood frogs (Rana sylvatica) can survive several months without oxygen and endure whole-body freezing for up to eight months of the year, with [~]70% of total body water frozen as extracellular ice, yet revive when temperatures rise in spring. Survival depends on multiple adaptations, including tolerance of prolonged oxygen deprivation while frozen, when breathing and circulation are halted. A key strategy involves hepatic glycogen mobilization, producing large amounts of glucose that are distributed to tissues where it functions both as a cryoprotectant and as a substrate for anaerobic ATP production. The present study examines the role of histone lysine methylation and demethylation in regulating liver proteins under anoxic conditions. Relative protein expression of seven histone methyltransferases (ASH2L-S, ASH2L-L, RBBP5, SETD8, SMYD2, ESET, SETD1), six lysine demethylases (KDM1A, KDM3B, KDM4A, KDM4B, KDM5A, KDM5C), and eight histone marks (H3K4me1, H3K4me2, H3K9me3, H3K27me3, H3K36me3, H3K79me3, H4K20me1, H4K20me3) were evaluated in wood frog liver under control, 4-hour, and 24-hour anoxia exposures. The data indicate that histone lysine methylation and demethylation contribute significantly to transcriptional regulation under anoxia. Specifically, H3K4, H3K36, and H3K79 methylation were associated with transcriptional activation, whereas H3K9, H3K27, and H4K20 methylation correlated with transcriptional repression. These findings highlight the dynamic role of epigenetic regulation in supporting hypometabolism and stress adaptation in freeze-tolerant wood frogs.

Survival analysis is a workhorse assay in Drosophila research to evaluate somatic fitness. It is indispensable in the study of ageing and insightful in immunity, metabolism, radiobiology, toxicology, ecology, and others. While conceptually simple, lifespan measurement is labour-intensive because it requires the continuous manual maintenance of large experimental cohorts. Here, we describe Drosben, an approach that combines a 3D-printed device to transfer flies from several vials simultaneously, a paper system for quick data recording and accompanying software that automatically digitalises life tables for analysis. We show that using Drosben reduces the time investment to perform lifespan assays by ~85%, with improved speed regardless of experience handling Drosophila vials. Using Drosben, we address the effects on longevity of chronic feeding of indole-acetic acid (IAA), naphthalene-acetic acid (NAA) and trimethoprim (TMP) -- compounds used to control heterologous targeted protein degradation systems. We find that IAA and NAA have noticeable deleterious effects while TMP has a small protective effect specifically in females. We further show that strong static magnetic fields do not affect Drosophila lifespan. Our work suggests that Drosben can cheaply accelerate research where lifespan is used as a life history trait.

Crown-of-thorns sea star (CoTS), Acanthaster cf. solaris, outbreaks are a major cause of hard coral cover decline across the west Pacific, threatening coral reefs. Coral taxa vary in susceptibility to CoTS predation from preferred (Acropora spp.) to non-preferred (Porites spp.), yet the mechanisms underlying these differences are poorly understood. We investigated coral defenses during an ongoing CoTS outbreak in Mo'orea, French Polynesia by examining gene expression (including putative toxin genes) in healthy and actively predated colonies of a preferred (Acropora hyacinthus) and a non-preferred (Porites sp.) coral prey species. During predation, A. hyacinthus exhibited molecular signatures of cellular stress responses involving oxidative stress signalling, inflammation, and tissue proteolysis. In contrast, Porites sp. showed enrichment of genes involved in mitochondrial metabolic adjustment and aerobic metabolism, suggesting metabolic compensation to maintain cellular function. Furthermore, A. hyacinthus demonstrated a reactive defense behaviour by differentially expressing toxins (e.g., kunitz-type neurotoxins) while Porites sp. employed constitutive expression of all putative toxins regardless of active predation, suggesting a proactive defense strategy. Together, these findings suggest that preferred and non-preferred coral prey exhibit fundamentally different molecular and defensive strategies during CoTS predation, shedding light on the evolutionary arms race between corals and their predators.

In sharp contrast to biological control that seeks control of pests below economic levels, eradication programs seek total extermination of pest species. However, eradication programs are often undertaken without knowledge of the pests potential geographic distribution and relative abundance (i.e., geographic risk assessment), often leading to eradication efforts conducted across areas much greater than the potential permanent range of the target species. To demonstrate this, we review eradication efforts against the tropical New World screwworm (Cochliomyia hominivorax) in the SE USA and Mexico, the exotic sub-tropical tephritid Mediterranean fruit fly (Ceratitis capitata, medfly) in California, and the exotic tropical pink bollworm (Pectinophora gossypiella, PBW) in the SW USA. We use mechanistic physiologically based demographic models (PBDMs) that are physiologically based time varying life tables to decompose the systems and to assess risk under extant and climate change scenarios.

Flower-visiting insect populations are declining since the 1990s, especially because of the decrease of floral resources in agricultural settings. Mass flowering crops can help increase resource availability, and plant breeding can be directed towards selecting varieties attracting more flower-visiting insects. This requires the implementation of an automated high-throughput phenotyping tool for assessing the attractiveness of plant genotypes to flower-visiting insects. In this study, (i) we present a procedure to take standardized images of sunflower heads with camera traps continuously at day and night in the field; (ii) we trained two versions of a deep learning model, named PolliCrop, to automatically detect and identify three classes of the main insects visiting sunflower on these images (non-Bombus bees, bumble bees, lepidopterans); (iii) we assessed and validated the ability of PolliCrop to correctly predict the true visitation frequencies of the insect classes on three sunflower genotypes; (iv) we presented two statistical approaches to compare the insect visitation frequencies between plant genotypes, one including weather variables, and the other one without. One PolliCrop version yielded satisfying performance to correctly detect the three insect classes. In particular, it correctly predicted the insect visitation frequencies on two sunflower genotypes in a range of {+/-}10%. The other PolliCrop version can be useful in certain contexts of images and objectives. PolliCrop can be extended in the future to other crop species by training PolliCrop on new images captured in these crops. The field experimental design to set up for comparing the attractiveness between genotypes is also discussed.

Environmental warming is generally expected to increase metabolic demand in ectotherms. However, facultatively endothermic insects such as bumblebees regulate body temperature and may reduce thermogenic investment under warm conditions, potentially altering physiological performance and responses to climate change. We combined flow-through respirometry and infrared thermography to test how elevated ambient temperature (25 vs 35{degrees}C) affects feeding energetics and postprandial metabolism in the bumblebee Bombus terrestris. Bees maintained substantially lower thoracic temperature excess at 35{degrees}C than at 25{degrees}C, both before and during feeding. Feeding metabolic rate was also lower at 35{degrees}C and was strongly positively associated with thoracic temperature excess, indicating that feeding energetics were primarily explained by thermoregulatory state rather than ambient temperature alone. Elevated temperature reduced both the probability and energetic magnitude of specific dynamic action (SDA), including total SDA expenditure, early postprandial metabolism, and peak metabolic amplitude. In contrast, SDA duration and time to peak response showed little temperature dependence. Our results demonstrate that warming can suppress energetic expenditure in facultatively endothermic pollinators by limiting thermogenic investment and postprandial metabolic responses, potentially constraining the energetic flexibility underpinning foraging performance under climate warming.

BackgroundHuman head lice (Pediculus humanus capitis de Geer) are known to harbor diverse maternally inherited bacterial symbionts. These endosymbiotic bacteria may contribute to insecticide degradation, potentially helping lice withstand particular environmental pressures. Using next-generation sequencing (NGS), this study investigated the bacterial symbionts present in wild head-louse populations and characterized their phylogenetic relationships in mutant and permethrin-susceptible strains. MethodsHead lice specimens were collected from 10 locations across Fars Province, Iran. Following DNA extraction, the samples were analyzed using polymerase chain reaction (PCR), and the resulting amplicons were sequenced to detect mutations in specimens from each location. Lice were then classified according to the presence or absence of mutations and subjected to NGS to characterize the symbiotic bacterial communities in mutant and putatively permethrin-susceptible strains. ResultsMutant strains were detected at only three sampling stations. Bioinformatic analysis of the nucleic acid sequences revealed three exons and two introns, with an expected amplicon length of 582 bp. NGS analysis showed that Candidatus Riesia pediculicola (Arsenophonus), belonging to the phylum Proteobacteria, was the predominant bacterial genus. Actinobacteria and Firmicutes were the second- and third-most abundant phyla, respectively. Most of the remaining 25 bacterial taxa were associated with the mutant strain. Additionally, two previously unreported bacterial genera were deposited in GenBank. ConclusionsThe distinct distribution of Arsenophonus species between susceptible and mutant head-lice strains--along with the greater abundance of Escherichia, Shigella, Lawsonella, and Megamonas in mutant strains--highlights the need for advanced metagenomic analyses to determine how these endosymbionts may help their hosts withstand specific environmental disturbances.

AbstractMetarhizium anisopliae is an important entomopathogenic fungi used in biological control of agricultural pests, but its commercial application is limited by relatively slow host mortality. This study aimed to engineer a chimeric protease (cPr1A) with enhanced binding affinity and protease activity against insect cuticle. We hypothesized that stronger cuticle binding would increase local enzyme concentration at the cuticle surface and thereby enhance cuticle degradation. To achieve this, the Bombyx mori chitin-binding domain (BmCBD) was fused to the C-terminus of the Pr1A protease from M. anisopliae. Recombinant Pr1A and cPr1A were expressed in Escherichia coli, purified by Ni-NTA affinity chromatography. Binding and protease activity were assayed in triplicate using Samia ricini cuticle powder as substrate. Results are presented as mean +/- SEM. The chimeric protease cPr1A showed a 28.9% increase in cuticle binding compared to wild-type Pr1A (15.81 +/- 1.97 vs. 12.27 +/- 2.13 g bound protein/mg cuticle powder; p < 0.002) and a 35% increase in protease activity (0.343 +/- 0.08 U/mg vs. 0.254 +/- 0.06 U/mg; p < 0.03). These results indicate that cPr1A is a promising candidate for overexpression in M. anisopliae to enhance cuticle degradation and potentially improve fungal virulence against insect pests.

The generalist Helicoverpa armigera and the specialist Helicoverpa assulta are closely related noctuid pests and are among the few insect herbivores capable of feeding on and damaging hot pepper fruits, which contain the defensive compound capsaicin. Cytochrome P450 monooxygenases (P450s) contribute to the metabolism of plant defensive compounds and can facilitate insect adaptation to chemically defended host plants. Here, we identified CYP6AN1 in H. assulta (HassCYP6AN1) and comparatively characterized the CYP6AN1 orthologs from H. armigera (HarmCYP6AN1) and H. assulta. RACE identified one full-length HarmCYP6AN1 transcript and three full-length HassCYP6AN1 transcript isoforms. The HassCYP6AN1 isoforms contained distinct 5' UTRs generated by alternative transcription initiation and splicing but shared an identical coding sequence. Sequence comparisons and phylogenetic analysis supported their assignment as orthologs. Constitutive CYP6AN1 expression was higher in the H. assulta midgut, whereas dietary capsaicin significantly induced CYP6AN1 expression in the H. armigera midgut. Recombinant CYP6AN1 proteins were co-expressed with NADPH-cytochrome P450 reductase in Escherichia coli, and their substrate-metabolizing activities were evaluated using HPLC-based depletion assays. Both orthologs metabolized capsaicin, but HarmCYP6AN1 exhibited an approximately 2.3-fold higher depletion activity than HassCYP6AN1 under the conditions tested. HarmCYP6AN1 also showed P450-content-dependent xanthotoxin depletion, whereas no detectable xanthotoxin metabolism was observed for HassCYP6AN1. These findings establish CYP6AN1 as a component of the capsaicin-metabolizing repertoire of both species and reveal substantial divergence between the orthologs in transcript organization, expression regulation, catalytic activity, and detectable substrate range.