Insect Molecular Biology

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.

RNA interference (RNAi) is a widely exploited reverse-genetics tool with potential uses for disease control. Successful RNAi has been reported in trematode-vectoring snails, but the composition of RNAi effector-encoding gene complements, a key driver for RNAi efficiency, remain unstudied in these species. Using bioinformatics and comparative genomics, we searched for orthologues of 115 RNAi effector sequences in genomes or transcriptomes of four snail vectors: Biomphalaria glabrata, B. pfeifferi, Bulinus truncatus, and Lymnaea staginalis. Gene expression patterns of selected RNAi effectors were then examined across developmental stages and tissues of the model B. glabrata snail. At least 74 RNAi-related proteins were conserved across all four species, including core components known to be essential for gene silencing. Classical systemic RNAi-deficient (SID) genes that facilitate systemic RNAi in other systems were absent, suggesting that alternative pathways may compensate for dsRNA uptake and transport. Core effectors of secondary RNAi amplification and heritable RNAi were not detected. Expressions of Dicer-1, Argonaute-2, and the exonuclease Eri-1 did not vary significantly with snail size. A putative RNAi-inhibiting Staufen orthologue showed elevated expression in the ovotestis, while another putative cholesterol-interacting gene was overexpressed in the trunk tissue and may partly contribute to RNAi import. Altogether, our results present the most comprehensive overview of RNAi pathway effectors in major intermediate snail hosts for trematodes. The findings underscore the likely broad potential for RNAi use in trematode intermediate hosts as an experimental tool and potential control method.

AO_SCPLOWBSTRACTC_SCPLOWEndogenous viral elements (EVEs) are widespread across animal genomes, yet the processes governing EVE evolution and diversification remain poorly understood. Here, we characterize the evolution of densoviral EVEs and exogenous densoviruses across the aphid tribe Macrosiphini, an agriculturally important group in which exogenous densoviruses and their endogenous derivatives have been linked to the plastic production of wings. Using new genome assemblies, transcriptomics, and phylogenetic analysis, we find that EVE content varies extensively across species. Moreover, we discovered a novel densovirus that is vertically transmitted, but phylogenetic incongruence between other densoviruses and their hosts suggests that horizontal transmission may also occur. Finally, we show that EVE-mediated regulation of wing plasticity extends across species that use different environmental signals to induce winged offspring. Our study shows that in this system, the evolution of EVEs is highly variable and lineage-specific, generating genomic patterns that cannot be predicted from host evolutionary relationships.

Nesidiocoris tenuis is an important zoophytophagous mirid bug used as a biological control agent in agriculture, and breeding efforts based on genomic information aim to increase its utility. Visible eye-color mutants are useful genetic markers because they are easily distinguishable and are therefore widely used in insect genetics and genome editing studies. Here, we investigated the genetic basis of a spontaneous red-eye mutant identified in a laboratory strain of N. tenuis. Classical crossing experiments suggested that the red-eye phenotype is controlled by a single recessive locus. RNA-seq and RNA interference (RNAi) analyses identified scarlet and cinnabar as the primary candidate genes associated with the phenotype. Further genomic analysis revealed a large deletion and insertion within exon 5 of the mutant scarlet allele, potentially causing exon skipping and disrupting transporter structure. The insertion pattern is consistent with a microhomology-mediated break-induced replication (MMBIR)/fork stalling and template switching (FoSTeS)-like event that may have been generated through polymerase{theta} -mediated repair. Together, these findings identify the causative mutation underlying the red-eye phenotype and provide a useful visible marker for future functional genetic studies and genome-assisted breeding in N. tenuis.

BackgroundSynonymous mutations, once considered neutral, can affect translation efficiency through mRNA folding and splicing, generating codon usage bias. This bias is often linked to genomic GC content, which also influences gene regulation. In the parasitoid wasp Lysiphlebus fabarum, GC content was previously shown to shift between developmental stages, with larvae showing higher GC than adults. Whether this phenomenon is widespread among insects remains unknown. ResultsTranscriptomic data from six insect species spanning Diptera, Hymenoptera, and Lepidoptera was used to compare GC content between expressed genes in larvae and adults. In five species, larval transcripts exhibited higher GC content than adult transcripts. Differential expression analysis revealed that stage-biased genes displayed consistent GC shifts, and orthologous gene families with representatives across species showed particularly GC-rich larval-biased genes in Hymenoptera and Diptera. At the genome scale, modeling in 317 insect species demonstrated an association between parasitic lifestyle and reduced mean GC content in Hymenoptera and Diptera, providing a possible ecological explanation for AT-rich genomes. ConclusionsOur results show that GC content is dynamic across developmental stages, independent of overall genome composition. Stage-specific GC enrichment may reflect adaptive codon usage optimizing translation during energetically demanding life-history stages such as larval development. Furthermore, the association between parasitism and reduced genomic GC highlights how ecological lifestyle might with genome content and evolution. Lastly, this work identifies candidate genes underlying stage-specific GC bias and provides new insights into the interplay between molecular evolution, development, and parasitic adaptation in insects.

Insects show extreme diversity and have long intrigued biologists. Recent technological advancements, such as gene editing and transgenesis, should in principle enable the use of almost any insect species for biological research. However, in practice, species-specific challenges remain and conditions must be optimized carefully. Here, using the milkweed bug Oncopeltus fasciatus, we first generate a useful eye- and body-color mutant strain by using CRISPR/Cas9-mediated genome editing. Then we use this strain to develop an efficient piggyBac-mediated transgenesis system using a nuclear targeting approach. We show that incorporating 1x and 3x nuclear localization signals (NLS) into piggyBac mRNA substantially enhances overall transformation efficiency in O. fasciatus. Taking advantage of both the useful mutant strain and the efficient transgenesis system, we successfully integrated multiple expression cassettes ranging from 1.8 to 7.2 kb, including attP strains for phiC31-mediated site-specific integration and histone-labelled strains for live fluorescence imaging. We further provide evidence that the Q system, a binary expression system, is functional in this species, paving the way for future sophisticated genetic manipulations including functional assays for cis-regulatory elements. Together, our results not only expand the genetic toolkit of O. fasciatus as a comparative model insect, but also provide a practical framework for developing efficient transgenesis in other non-traditional model organisms.

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.

The migratory endoparasitic pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease, causing significant economic and ecological losses in conifer forest ecosystems in Europe and Asia. Understanding the molecular mechanisms regulating PWN parasitism-related genes may lead to new sustainable solutions for control. Based on previous PWN transcriptomic datasets from the pre-parasitic and parasitic stages and from the pharyngeal gland cells (GC), an in silico analysis was performed to identify transcription factors (TF) highly expressed in the GC. Seven candidates TF genes were selected, and their spatial expression validated by in situ hybridisation. From those, two GC-expressed TFs, BXY_079 and BXY_022, each encoding zinc finger domains, were successfully knocked down by RNA interference. Transcriptomic data from silenced BXY_079 and BXY_022 TFs, analysed with existing life cycle specific transcriptomic data, showed that both TFs control genes expressed at similar times, by repressing male-related genes while activating genes expressed during the J3 and D3 stages, yet each represents the extreme of the others minor function. In addition to these common roles, BXY_079 also activates parasitism-related genes in the J2 stage. These BXY_079-activated parasitism-related genes predominantly encode proteins with lytic functions, including secreted peptidases and glycoside hydrolases. Consistent with their proposed role in parasitism, these genes are highly expressed during the parasitic juvenile stages and are likely involved in nematode feeding, tissue penetration, and migration within the host. In contrast, BXY_022 also represses the expression of several genes related to the reproduction system, such as major sperm proteins and cytosolic motility proteins, particularly in the adult male stage. Taken together, both dual-functional TFs work together, non-redundantly, to regulate gene expression across the life cycle, while each is additionally specialised to regulate diverse and distinct gene sets: ranging from genes implicated in lytic parasitic functions to sexual dimorphism.

Gene model for the ortholog of raptor in the D. eugracilis Apr. 2013 (BCM-HGSC/Deug_2.0) (DeugGB2) Genome Assembly (GenBank Accession: GCA_000236325.2) of Drosophila eugracilis. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

Many insects rely on intimate interactions with bacterial symbionts housed in specialized cells called bacteriocytes. While host and symbiont gene expression in bacteriocytes appears highly integrated, the underlying regulatory mechanisms remain largely unknown. MicroRNAs regulate gene expression and are emerging as mediators of cross-kingdom communication. Here, in the pea aphid, Acyrthosiphon pisum, and the green peach aphid, Myzus persicae, we demonstrate cross-kingdom translocation of aphid miRNAs into their obligate endosymbiont, Buchnera aphidicola. miRNA fluorescence in situ hybridization in aphid embryos provides direct evidence that four out of five candidate miRNAs (miR-1, miR-10, miR-29, and miR-927) localize inside Buchnera cells. To investigate how these miRNAs may regulate Buchnera gene expression, we further demonstrated the cross-kingdom translocation of aphid argonuate 1 protein (Ago1), into Buchnera using immunolocalization. Given the translocation of both Ago1 and miRNAs, we applied a eukaryotic miRNA target prediction framework and found that all cross-kingdom-translocated miRNAs are predicted to target Buchnera genes involved in symbiotic functions. Although the precise in vivo functions of translocated miRNAs remain challenging to determine, our findings suggest a previously unrecognized layer of regulation between insect host and its obligate endosymbiont, offering new insight into the molecular dialogue that supports insect-microbe interactions, highlighting potential targets for miRNA-based pest management. Significance statementMany insects have evolved bacteriocyte cells to house essential bacterial endosymbionts. However, how bacteriocytes are specified and how gene expression is coordinated between host and symbiont remain largely unknown. Here, we identify that aphid microRNAs, along with an argonaute protein, are translocated into the obligate bacterial endosymbiont Buchnera, revealing a previously unrecognized regulatory layer mediating host and obligate endosymbiont interactions. This discovery provides evidence of miRNA cross-kingdom movement into an obligate endosymbiont, filling a key knowledge gap in how eukaryotic host small RNAs can influence microbial partners. Given the critical role of Buchnera in aphid survival and reproduction, these findings not only advance fundamental knowledge of insect-microbe interactions but also point to new molecular targets for innovative pest management strategies.

Precise knock-in of fluorescent reporters is a powerful tool for studying the dynamic cellular and molecular processes of embryogenesis. However, conventional CRISPR-Cas9 knock-in of large inserts, such as full-length fluorescent proteins, is inefficient. This has limited its application in many emerging model systems, including sea urchins. Here, we overcome this barrier using a transgenic Lytechinus pictus line that constitutively and ubiquitously expresses a large fragment of mNeonGreen (mNG3K1-10). In this line, fluorescence is only reconstituted when CRISPR-mediated knock-in delivers mNG211, the 11th beta strand of the fluorescent protein, to complement the constitutively expressed fragment. Because this strategy requires integrating only the short 11th-strand, together with short homology arms (~130 nt total), by homology directed repair, it circumvents the size constraints that limit conventional full-length reporter knock-ins using CRISPR. Using this approach, we achieved integration efficiencies of 14-22%, roughly an order of magnitude higher than those obtained with full-length fluorescent protein knock-ins. This provides a streamlined, scalable method for endogenous protein visualization in echinoderm embryos and a valuable resource for studying gene function, morphogenesis, and toxicant response in this classic developmental model.

Planning husbandry tasks and experiments with Drosophila melanogaster requires converting a target date into development times that depend on the rearing temperature. This calculation needs to be done for each cross, genotype, and temperature, and the risk of error grows quickly. Available laboratory management tools let users register stocks, crosses, and track them, but they do not create schedules based on a clear, adjustable thermal model. To fill that gap, we developed DrosoTracker, a self-contained web application that works offline and predicts Drosophila development with a thermal summation model recalibrated through regression on data from Powsner (1935) (T0 = 11.78 {degrees}C, DD = 116.38 {degrees}C{middle dot}days, R{superscript 2} = 0.997). The model offers an optional two-level calibration driven by user observations. A wild-type strain first adjusts the model to the laboratorys own conditions. Then each genotype is calibrated against that reference using a random-effects shrinkage estimator that accounts for measurement error and between-batch variability. The model creates schedules for husbandry tasks, evaluates adult cohort survival with the Kaplan-Meier estimator and the log-rank test, and calculates sample size for lifespan studies using Schoenfelds formula. The quantitative components were checked against independent references, including Rs survival package and manual calculations. Ongoing work is focused on validating the calibrated model using cohorts specifically bred for this purpose. DrosoTracker runs entirely in the browser, stores data locally, and is available in English and Spanish.

Gene model for the ortholog of Density regulated protein (DENR) in the Apr. 2013 (BCM-HGSC Dpse_3.0/DpseGB3) Genome Assembly (GenBank Accession: GCA_000001765.2) of Drosophila pseudoobscura. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

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.

Satellite DNA is long arrays of tandem repetitive DNA located often near the centromeres of chromosomes, whose function, or lack of, has been debated since its discovery. Although situated in heterochromatin, satellite DNA may be expressed as long noncoding RNAs (lncRNAs). Although there are a few examples of satellite lncRNAs being characterized, and functions suggested, how widespread and functionally important they may be for developmental processes is not understood. Here, we take an evolutionary approach to investigate satellite lncRNA expression in Drosophila spp. ovaries, a tissue whose development is well-characterized but where satellite expression has only been minimally explored. Using a publicly-available total RNAseq dataset, we find that 118/156 surveyed satellite DNAs were expressed across 10 species, with 33 satellites having high expression over 20 RPM. However, all but two of these expressed satellites (AAACTAC in D. virilis and ACAGACAGACAGG in D. ananassae) had higher read counts in a sister smallRNA dataset, suggesting that most satellite transcripts primarily serve as precursors for piRNA biogenesis. The two "stand-alone" lncRNAs were highly strand-biased, with 96-97% of the total reads coming from one strand. We further investigated AAACTAC expression with RNA FISH and found the transcript is specifically present in the oocyte nucleus following a dynamic spatiotemporal pattern, with the highest expression in stage 3-5 oocytes. The transcription pattern of AAACTAC is conserved in the three other virilis clade species that contain this satellite DNA. Further, we found expression of unrelated satellites in more distantly related D. borealis and littoralis both in the oocyte and the nurse cells. Overall, our work identifies a novel lncRNA AAACUAC found in the early oocyte nucleus, which is conserved across ~5 MY of evolution, and is therefore a strong candidate for the discovery of novel functions of satellite lncRNAs in development.

Insects of the subfamily Triatominae, commonly known as kissing bugs, are obligate blood-feeding vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Rhodnius prolixus is among the most epidemiologically important vectors in Latin America, whereas Triatoma rubida frequently invades homes and is a potential vector in the southern United States and northern Mexico. Triatomines likely evolved from predatory reduviid assassin bugs through a transition from feeding on arthropods associated with vertebrate hosts to feeding directly on vertebrate blood. To investigate the genomic basis of this ecological and dietary shift, we generated highly contiguous, near chromosome-level genome assemblies and structural gene annotations for R. prolixus and T. rubida. The new R. prolixus assembly improves scaffold N50 more than 40-fold over the current reference genome, from 1.1 to 43.9 Mb, while reducing assembly gaps by several orders of magnitude. Both assemblies exceed 97% BUSCO completeness. Comparative analyses with representative hemipteran genomes revealed expansions of gene families associated with chemosensation and metabolism, including detoxification, protein degradation, and digestion, together with signatures of positive selection in genes involved in digestive and sensory functions. These assemblies represent the most contiguous and complete genomic resources available for Triatominae and provide a robust foundation for investigating vector biology, host adaptation, and the evolutionary origins of blood feeding within Reduviidae. Interpretive summaryKissing bugs are insects that are known for feeding on blood. They can spread a disease called Chagas disease because they transmit a parasite called Trypanosoma cruzi. To understand how kissing bugs evolved and which genes facilitate blood feeding of vertebrates, a collaboration between scientists at USDA-ARS, University of Georgia, and University of South Bohemia sequenced the genome of two kissing bugs: Rhodnius prolixus and Triatoma rubida. By comparing the genes with those of other insects in the order Hemiptera, scientists discovered that kissing bugs have more genes involved with detecting environmental chemical stimuli and metabolism as well as positive selection for genes involved with digestion and sensory-related proteins. These genome assemblies will help scientists learn more about how these insects evolved, and this research is important for understanding insect feeding biology which can be used to develop methods to control the kissing bugs and the spread of Chagas disease.

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.

Gene model for the ortholog of raptor in the D. grimshawi May 2011 (Agencourt dgri_caf1/DgriCAF1) Genome Assembly (GenBank Accession: GCA_000005155.1) of Drosophila grimshawi. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

One of the most devastating threats to global honey bee health is the ectoparasitic mite and viral vector Varroa destructor, yet the transmission dynamics of viruses carried by mites are poorly understood. RNA interference (RNAi) is a major antiviral defence mechanism in invertebrates including Varroa, where actively replicating viruses are degraded into virus-derived small interfering RNAs (vsiRNAs). Insects typically produce 20-22 nt vsiRNAs with sense and antisense polarity, however established viral infections in V. destructor lead to the production of 24-nt antisense vsiRNA fragments, which could indicate the presence of secondary siRNA synthesis. To better understand viral infection and transmission dynamics in V. destructor, we conducted small RNA sequencing of male and female mites throughout development, from egg to reproductive stages. Viral community structure was largely driven by developmental stage, with younger and older life stages clustering separately. We identified five viruses that are consistently degraded into antisense 24-nt vsiRNA across all developmental stages, suggesting that these viruses are transmitted vertically and form part of Varroas core virome. This includes the highly diverse Varroa destructor virus 2 (VDV-2), for which we observe eight distinct VDV-2 strains that simultaneously co-infect individual mites throughout development. In contrast, sense and antisense 23-nt vsiRNA fragments are generated in response to the honey bee pathogen, Iflavirus aladeformis (deformed wing virus A, DWV-A) in eggs, but the vsiRNA size profile transitions to 24-nt antisense fragments at later life stages. Our results suggest that once a virus is first acquired by Varroa, a primary 23-nt sense and antisense antiviral response precedes the production of secondary 24-nt antisense vsiRNAs as the infection progresses. We confirm this observation using synthetic dsRNA, which show both primary and secondary siRNA processing, revealing how exogenous dsRNA processing occurs in Varroa. These results show distinct primary and secondary antiviral RNAi responses across V. destructor life stages and demonstrate how vsiRNA profiles can be used to infer virus transmission routes and long-term persistence within vector populations.

Phenotypic plasticity, the ability of a single genotype to produce alternative phenotypes in response to environmental cues, is a key driver of evolutionary change. In the water strider Microvelia longipes, males display remarkable continuous variation in hindleg length, a sexually selected trait used as a weapon in male/male contests for access to females. To determine whether DNA methylation mediates this environmentally induced phenotypic variation, we used three inbred lines of M. longipes that differ in mean hindleg length, body size, and allometric coefficients. We performed whole-genome bisulfite sequencing on adult males and females from all lines, and tested the effect of nutritional treatment on DNA methylation patterns. Our analysis identified 12,684,876 CpG 12% of which were methylated. This global level of DNA methylation is among the highest reported in insects. DNA methylation was predominantly concentrated within or near gene bodies (77% of methylated CpGs), consistent with patterns observed in other insects. Unsupervised clustering and principal component analyses revealed that methylation patterns differed significantly between genetic lines but showed minimal differences between sexes, indicating a strong genetic influence. Most surprisingly, despite nutrition having a pronounced effect on leg length, we observed no significant changes in DNA methylation in response to dietary treatment. These results show that in M. longipes, DNA methylation patterns are largely stable across environmental conditions and primarily determined by genetic background. This challenges the common assumption that DNA methylation universally mediates environmentally induced phenotypic plasticity and suggests that other epigenetic mechanisms, such as histone modifications or non-coding RNAs, may play a more direct role in regulating continuous plastic traits. Our study underscores the complexity of epigenetic regulation and highlights the need for broader investigation of molecular pathways to fully understand the molecular basis of phenotypic variation in natural populations.