Insect Molecular Biology

CRISPR-Cas genome editing toolkits have expanded the scope of genetic studies in various emerging model organisms. However, their applications are limited mainly to knockout experiments due to technical difficulties in establishing knock-in strains, which enable in vivo molecular tagging-based experiments. Here, we investigated knock-in strategies in the harlequin ladybug Harmonia axyridis, a model insect for evolutionary developmental biology, which shows more than 200 color pattern variations within a species. We tested several knock-in strategies using synthetic DNA templates. We found that ssDNA templates generated founder knock-in strains efficiently (2.5-11%), whereas the 5 regions of ssDNA templates were frequently deleted when the insert length exceeded [~]40 bases. To overcome this limitation, we designed several 3 extended DNA templates. Fast-annealed 3-extended double-stranded DNA templates, which were designed for tagging endogenous proteins with epitope tags, showed high founder generation efficiency (9.9-20.9%) and accuracy (30.8-85.7%). This strategy is also applicable to the two-spotted cricket Gryllus bimaculatus, suggesting that the fast-annealed 3-extended dsDNA template is a versatile DNA template for generating knock-in strains in emerging model insects for developmental genetic studies. Summary statementFast-annealed 3-extended dsDNA templates facilitate efficient CRISPR-Cas9-mediated knock-in in emerging model insects.

Drosophila suzukii (Matsumura, 1931, Diptera: Drosophilidae) is a globally invasive pest of soft-skinned fruits that is currently controlled largely through the use of broad-spectrum insecticides. Increasing resistance to pesticides and regulatory pressures have motivated the development of genetic control strategies. We previously developed a CRISPR/Cas9-based homing gene drive targeting the coding sequence of the female-specific exon of the sex-determination gene doublesex, achieving highly efficient inheritance (94-99%) in both male and female germlines. A major limitation of homing gene drives is the formation of resistant alleles that evade cleavage yet retain gene function. Multiplexing guide RNAs (gRNAs) could reduce the formation of such functional resistance alleles. Here, we generated and tested homing constructs expressing one, two, or three gRNAs targeting different regions of the female-specific exon of doublesex, including a splice-junction target site. A single gRNA targeting the splice junction supported high inheritance in males but showed reduced efficiency in females. Combining this gRNA with a coding sequence-targeting guide further reduced drive efficiency, particularly in the female germline. Constructs expressing two gRNAs performed similarly whether guides were linked by transfer RNA (tRNA) sequences or expressed from independent promoters. Constructs expressing three gRNAs using tRNA processing showed consistently low drive inheritance in both sexes, likely a consequence of reduced cleavage efficiency due to inefficient gRNA production. Inheritance was significantly higher in male than female germlines for several constructs, indicating that germline context strongly influences drive performance. Our findings highlight that the approach used for multi-gRNA expression, target site choice and sex-specific germline environments can impact gene drive efficiency, and emphasize the need to optimize construct design within the biological context of the target species.

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.

Gene model for the ortholog of tango (tgo) in the Sep. 2015 (UC Berkeley ASM127793v1/DbusGB1) Genome Assembly (GenBank Accession: GCA_001277935.1) of Drosophila busckii. 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.

BACKGROUNDThe soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a pest of soybean in North America that can cause significant yield loss when outbreaks are not managed. Current management tactics primarily rely on inexpensive pyrethroids, but the sustainability of this option is threatened by insecticide-resistance in A. glycines populations across the Upper-Midwest United States. Field-evolved resistance is associated with mutations in the voltage-gated sodium channel subunit h1 (vgsc-h1) gene. RESULTSFour double-stranded RNA (dsRNA) molecules, each matching the sequence of a vgsc-h1 transcript variant ("Specific dsRNAs"), were topically applied to aphids with a genotype carrying the corresponding allele. The mortality of pyrethroid resistant aphids exposed to a Specific dsRNA increased in a dose-dependent manner when applied alone or with a constant concentration of lambda-cyhalothrin, plateauing at 1000 ng ul-1. Synergism was detected between two of four combinations of the Specific dsRNAs and lambda-cyhalothrin. These results were mirrored by the topical application of a single dsRNA with the consensus sequence of all vgsc-h1 variants ("Combined dsRNA"). Mortality was consistently higher in aphids treated with either Specific dsRNA or the Combined dsRNA, alone or with lambda-cyhalothrin, compared to insecticide alone. The number of nymphs produced per female treated with the Specific or Combined dsRNA alone decreased significantly compared to untreated controls. CONCLUSIONThis study demonstrates that the topical application of dsRNAs targeting vgsc-h1 increases the susceptibility and reduces the reproductive capacity of pyrethroid resistant soybean aphids, potentially providing a novel tool for the management of insecticide-resistant aphid populations.

Rhodnius prolixus is a primary insect vector of Trypanosoma cruzi, the causative agent of Chagas disease, a neglected parasitosis endemic in Latin American countries. It has been estimated that Chagas disease affects 7-8 million people worldwide and is responsible for approximately 1000 deaths per year. Genetic and molecular studies in this species remain challenging due to its life cycle and feeding habits, thus hindering the development of new strategies to control their populations and reduce the diffusion of Chagas disease. Recently, two stable cell lines - RPE/LULS53 and RPE/LULS57 - were derived from Rhodnius embryos, which represent promising new tools to investigate the genetics of this insect vector. Here, we describe their gene expression landscapes through transcriptomic approaches. We show that 8,968 expressed genes are shared between the two cell lines, whereas 391 and 1,088 genes are uniquely expressed in RPE/LULS53 and RPE/LULS57, respectively. Although key components of primary developmental, immune and redox signaling pathways are expressed in both cell lines, some genes such as Frizzled-10-a-like and catalase show marked differences in expression. Our results strongly suggest that RPE/LULS53 and RPE/LULS57 likely represent two different cell phenotypes. Consistent with this, gene ontology analysis reveals that RPE/LULS53 is enriched for animal organ morphogenesis and stress response, while RPE/LULS57 for DNA-directed RNA polymerase activity, among others. Despite these differences, both cell lines express comparable levels of transcripts from resident transposable elements, including the highly abundant Mariner and LINE/I elements, as well as horizontally transferred transposons. Our findings shed light on the nature of the RPE/LULS53 and RPE/LULS57 embryo-derived cell lines and provide valuable transcriptomic resources for future genetic and functional studies in Rhodnius and other triatomine insect vectors. Author summaryRhodnius prolixus is a blood-feeding insect and a major vector of Chagas disease, a parasitosis endemic in Latin America and affecting millions of people worldwide. In the absence of effective drugs and vaccines, the control of the insect population represents a promising strategy to reduce the diffusion of the disease. Yet, genetic and functional studies in Rhodnius are extremely challenging due to its feeding habit and life cycle. To overcome these limitations, researchers have previously developed two stable cell lines derived from Rhodnius embryos. In this study, we provide the first characterization of the genes expressed in these cell lines. We found that, while the two cell lines share many expressed genes, each of them also has distinct gene expression patterns pointing to two different cell types with specialized functions. These differences likely affect the way they respond to stress and regulate biological processes. Our findings provide an important resource for researchers studying Rhodnius prolixus and other insect vectors, helping advance our understanding of the genetic and molecular mechanisms that control the insect development and mediate the interactions between insect vectors and the parasites they transmit

Although all insects are segmented, the genes that control this process vary across species. Many of the pair-rule (PR) genes that direct segment formation in Drosophila are similarly utilized in other holometabolous insects, but more distantly related species use different genes for PR-patterning. Previously, we showed that Lepidoptera lack a highly conserved PR-gene, paired. Here, we used the painted lady butterfly Vanessa cardui as a lepidopteran model to explore the expression and function of PR-genes in this large clade of moths and butterflies. Orthologs of four Drosophila PR-genes are expressed in PR-like stripes and at least one displays PR-like function. Neither of the two genes that have PR-function in Hemiptera but not in Drosophila have PR-roles in Vanessa. Rather, the hemipteran PR-gene Blimp1 functions in a novel fashion in abdominal segmentation in Vanessa. Thus, while butterflies appear to share PR-patterning mechanisms with other insects, they utilize only a subset of the Drosophila PR-gene orthologs and have not taken on hemipteran PR-orthologs for this process. These findings suggest extensive rewiring of the segmentation gene regulatory network in Lepidoptera.

Reporters are widely used in Drosophila genetics to visualize gene expression and cell lineages. However, uncharacterized limitations in specific reporter lines can lead to data misinterpretation. Here, we identify a consistent, driver-independent tdTomato signal in the adult proventriculus from the widely used lexAop-tdTomato.nls reporter line. This signal was observed across multiple lexA driver combinations and was directly detectable in lexAop-tdTomato.nls responder-alone adult proventriculi lacking any lexA driver and without antibody staining. In contrast, no comparable native red fluorescence was detected in larval proventriculi under the same no-antibody imaging condition. Mouse and rabbit anti-RFP immunostaining further supported the presence of proventriculus-associated tdTomato/RFP antigen in adult responder-alone animals. In larval responder-alone proventriculi, antibody-amplified staining was antibody-source-dependent: a detectable signal was observed only with rabbit anti-RFP, whereas mouse and rat anti-RFP produced no reliable detectable signal under the same staining condition. A driver-matched comparison using lexAop-RFP.nls did not reproduce the proventricular signal, arguing against detectable ectopic activity of the tested lexA driver in this tissue. However, because lexAop-tdTomato.nls and lexAop-RFP.nls differ in reporter/transgene architecture and possibly genomic insertion context, the underlying cause cannot be assigned specifically to the lexAop sequence. Our findings highlight the necessity of including driver-negative and no-antibody controls when using this reporter line in adult Drosophila proventriculus and gut studies.

BackgroundHalyomorpha halys (brown marmorated stink bug) is an invasive polyphagous pest causing significant agricultural damage worldwide and is an emerging target for RNAi-based pest management. Despite growing interest in dsRNA-based biocontrol, progress is constrained by the lack of tissue-resolved transcriptomic resources covering key biological processes such as feeding, detoxification, and reproduction. Furthermore, our understanding of how RNAi machinery expression varies across tissues remains limited, which impairs both target gene selection and predictions of RNAi efficacy. Critically, the transcriptional response of H. halys to haemolymph-delivered non-specific dsRNA represents a key knowledge gap for evaluating potential non-target immune reactions of dsRNA-based approaches. ResultsField-collected adult males were injected with either nuclease-free water or dsRNA targeting GFP (dsGFP), and transcriptomes were generated from the brain, midgut, salivary glands, and testes. Sequencing produced high-quality datasets with clear tissue-level separation and tight clustering of biological replicates. As expected in targeting a non-endogenous gene, differential expression analysis revealed a limited transcriptional response to dsGFP. Baseline profiling of RNAi pathway genes in controls showed broad expression of core siRNA and miRNA components across all tissues, yet with marked specialisation: two additional Argonaute-2 isoforms and multiple piRNA factors were testes-specific, whereas salivary glands showed strong, restricted expression of nuclease-encoding genes, including a T2 ribonuclease and a non-specific endonuclease. Expression atlases also revealed pronounced tissue partitioning for other protein families. Consistent with their respective functions, secreted trypsins and chymotrypsins are salivary-enriched while the cathepsins for intracellular protein catabolism are midgut-enriched, with brain-centred neuropeptide expression. However, we also uncovered unexpected nuance, such as closely related subfamilies of Cytochrome P450s, which generally function as detoxification enzymes, being partitioned between the midgut, brain or testes. ConclusionsThis work delivers the first tissue-resolved transcriptomic atlas of adult male H. halys, providing a high-resolution resource on compartmentalization of proteolysis, detoxification, and neuroendocrine signalling, as well as for candidate gene discovery in RNAi-based pest control. The modest, tissue-restricted transcriptional response to non-specific dsRNA, together with strong tissue-specific enrichment of some components, offers mechanistic insight into tissue-dependent RNAi efficiency and supports rational dsRNA target selection in H. halys.

Gene model for the ortholog of Density regulated protein (DENR) in the Apr. 2013 (BCM-HGSC/Deug_2.0) (DeugGB2) Genome Assembly (GenBank Accession: GCA_000236325.2) of D. 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.

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.

Wolbachia is a bacterial endosymbiont that is primarily transmitted from mother to offspring. To increase their transmission, some strains manipulate their hosts reproduction to favor female offspring, such as by inducing parthenogenesis. Here, we assess whether Wolbachia induces parthenogenesis in recently discovered parthenogenetic populations of the ladybug Nephus voeltzkowi by treating females with an antibiotic. Females from sexual populations, which we show are not infected by Wolbachia, serve as a control. Our results demonstrate that the treatment decreases Wolbachia load, subsequently reducing egg production and development among parthenogenetic females, while having no effect on sexual females. Wolbachia load and reproduction then rebound when the treatment is removed. This suggests that the Wolbachia infection is necessary for successful reproduction in the parthenogenetic females, and it may play a two-part role by facilitating egg laying and late embryo development. The cooccurrence of Wolbachia infection and parthenogenesis in N. voeltzkowi, as well as Wolbachias manipulation of host reproduction makes a likely candidate of Wolbachia-induced parthenogenesis outside of haplo-diploids. Understanding how Wolbachia can impact diverse insect reproductive systems can shed a light on the extent in which these bacteria can manipulate hosts for their own gain.

Today, the deformed wing virus (DWV) can be considered as one of the major causes of global elevated western honey bee colony losses (Apis mellifera). Virus transmission may occur horizontally between individuals of the same generation, but also vertically from parents to offspring. The recently defined heritable suppressed in ovo virus infection (SOV) trait describes the absence of viruses in pooled drone eggs of a queen, associated with significant lower DWV prevalence and viral loads in the subsequent developmental offspring stages. By definition, the trait reflects the absence of vertical virus transmission from SOV-positive (SOV+) queens themselves to their offspring. However, the genetic basis influencing this heritable virus resilience has not been identified yet. In this study, we aimed to identify SOV-associated genetic marker(s) or loci in the honey bee genome through genome-wide variant comparison of 44 DWV-positive and 44 DWV-negative drone pupae descendent from an artificially created hybrid SOV+/SOV- colony. After whole genome sequencing (WGS), variant calling, and genotype-phenotype association analysis by means of single marker tests and elastic net regression, one variant in a locus of 241.246 bp on chromosome 7 that contained 17 other highly SOV-associated variants classified 68,2% of the drone phenotypes correctly. These results may support the potential application of marker-assisted selection (MAS) strategies targeting reduced vertical virus transmission in honey bees.

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.

Gene model for the ortholog of Lst8 (Lst8) in the May 2011 (WUGSC dyak_caf1/DyakCAF1) Genome Assembly (GenBank Accession: GCA_000005975.1) of Drosophila yakuba. 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.

We sequenced the nearly complete mitochondrial genome of the hammerhead flatworm Bipalium nobile Kawakatsu and Makino, 1982 using short-read sequencing technology, yielding a 16,018 bp genome comprising 12 protein-coding genes, 22 tRNA genes, and 2 rRNA genes. The composition and order of genes were consistent with those observed in the closely related species Bipalium kewense and Diversibipalium multilineatum, except for the position of tRNA-Glu. Phylogenetic analysis based on all mitochondrial proteins from species within the family Geoplanidae supports the monophyly of a clade comprising B. nobile, B. kewense, and D. multilineatum. The mitochondrial genome sequence obtained in this study provides a valuable resource for investigating the genetic diversity and population structure of B. nobile, a soil-dwelling predator with the potential for global spread as an invasive organism.

Gene model for the ortholog of raptor in the May 2011 (Agencourt Dere_CAF1/DereCAF1) Genome Assembly (GenBank Accession: GCA_000005135.1) of Drosophila erecta. 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.

Target enrichment methods have provided unprecedented advances in phylogenomics. Targeting hundreds of conserved regions has proven to be a good tradeoff between cost and efficiency, while being useful for museomics and diversified non-model clades. Unfortunately, current methods used for identifying such regions involve high degrees of conservation within targeted elements, usually pushing researchers to rely on flanking data with little guarantee for homology. With a growing number of high quality genomes available throughout the Tree of Life emerges new opportunities to improve marker selection. In this study, we introduce GABBI, a new method for designing target capture probes by taking advantage of genome alignments, avoiding the selection of a single reference genome that can cause notable biases. We compare GABBI-derived markers to the most commonly used probe design method, PHYLUCE, at two taxonomic scales, the weevil superfamily Curculionoidea and the tribe Pachyrhynchini. At both taxonomic scales, results show that our new method allows identifying more variable loci that prove to be more phylogenetically resolutive than the PHYLUCE-derived ones. Doing so, we provide the first probe set specifically designed for weevils, targeting a wide set of 4,255 shared homologous regions, encouraging future research on systematics and macroevolution of one of the most diverse and economically important groups of insects. By providing GABBI as an automated and open-access pipeline, we hope to open new probe design opportunities to other taxonomic groups that face similar phylogenetic obstacles.

The head of chelicerates, such as spiders, scorpions and mites, is composed of the ocular, chelicerae and pedipalp segments and is considered to be homologous to the procephalon of insects which comprises the ocular, antennal and intercalary segments. Head segmentation in the spider, Parasteatoda tepidariorum, is a dynamic process in which a single stripe of expression of the P. tepidariorum hedgehog (hh) gene splits twice to form three separate stripes, which help pattern the three spider head segments. This dynamic hh stripe splitting process is dependent on spider homologues of the transcription factors orthodenticle (otd) and odd-paired (opa). Here we investigate the conservation of this dynamic patterning mechanism in two insect models: the hemimetabolous pea aphid, Acyrthosiphon pisum, and the holometabolous red flour beetle, Tribolium castaneum.. We show that insect hh, otd and opa homologues are expressed in a highly conserved temporal and spatial pattern during procephalon development in these insects. Our data are consistent with an ancestral insect state in which a single hh stripe splitting event underpins patterning of the ocular and antennal segments, followed by de novo formation of the intercalary hh stripe. Using parental RNAi in T. castaneum, we show that hh, otd and opa homologues exhibit striking similarities in their regulatory interactions during spider and insect head/procephalon segmentation. Our data suggest that hh, otd and opa homologues contribute to an ancient and largely conserved gene network controlling head/procephalon patterning in arthropods. We discuss the implications of these data for our understanding of the origin and evolution of the arthropod head, and propose a new model for the evolution of anterior patterning in holometabolous insects.

Biting behavior is an important natural defense mechanism in honeybees (Apis mellifera) against Varroa destructor. Significant variation in this behavior exists across genetic lines of honeybees, with certain colonies exhibiting higher mite-biting activity than others. Selective breeding for enhanced biting behavior provides a promising strategy for sustainable mite control and colony resilience. However, successful implementation of such breeding programs requires a comprehensive understanding of the genomic mechanism underlying this trait. In this study, RNA-seq analysis of mandible transcriptomes of 1-day and 8-day old worker honeybees from high mite biting (HB) and low mite biting (LB) colonies were performed. A total of 9,345 genes (97.30%) showed a significant differential expression between LB and HB honeybees across different ages (one-way ANOVA, FDR < 0.05). Comparison of LB vs. HB workers collected on day 1 detected 166 down-regulated and 403 up-regulated genes, whereas workers collected on day 8 identified 82 down-regulated and 46 up-regulated genes. Furthermore, the Weighted Gene Coexpression Network Analysis (WGCNA) exhibited the brown and yellow modules with significantly higher expression in HB compared with LB on both day 1 (FC = 1.52, FDR = 0.0019 and FC = 1.47, FDR = 2.7 x 10-4, respectively) and day 8 (FC = 1.27, FDR = 0.056 and FC = 1.16, FDR = 0.073, respectively). Gene Ontology (GO) enrichment analysis identified over-representation of biological processes involved in muscle contraction, chitin binding, neural signaling, oxidative phosphorylation, neuron development, electron transport chain, mitochondrial ATP synthesis, stress response, sensory perception and metabolic processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis identified significant enrichment of various pathways including oxidative phosphorylation, cytoskeleton-related pathways, carbon metabolism, motor proteins, ribosome-associated pathways, and citrate cycle (TCA cycle). The present findings demonstrate mite-biting behavior is associated with coordinated activation of neural, energetic and muscular system rather than a single molecular mechanism. These findings provide a basis to improve honeybee health, enhance resistance to Varroa and other ectoparasite, and eventually support sustainable beekeeping and agricultural pollination systems.