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.

Genetic modification via gene editing has become a widely adopted and demonstrably effective method in functional gene research within entomology. However, the optimal efficiency and simplicity of delivering exogenous guide RNA-clustered regularly interspaced short palindromic repeats-associated protein 9 (gRNA-Cas9) complexes into target tissues are crucial for successful gene editing. The Receptor-Mediated Ovary Transduction of Cargo (ReMOT) strategy, which simplifies the delivery process, target site selection, technical requirements, and delivery cost compared with embryonic microinjection, enabling efficient editing at the germline level, is gaining increasing attention. Although the feasibility and advantages of this technique have been demonstrated in various insect species, further optimization of operational details and the overcoming of further bottlenecks are still required. This review focuses on advances in developing ReMOT as a valuable technology, exploring its applicability, rationale for selecting the ovary as a delivery target site, factors influencing its efficiency, and recommendations for improvement. The versatility and effectiveness of ReMOT make it a promising method for researchers looking to make precise genetic modifications with greater ease and efficiency.

In animals, sex-specific social behaviors, such as courtship and aggression, are controlled by sex-specific neural circuits. To understand the neural mechanisms of sex-specific social behaviors from a comparative perspective, it is necessary to understand the sex determination mechanisms of neural circuits and their evolution across animal lineages. To address this, I have focused on the evolution of the neural sex determination system in insects. In the fruit fly Drosophila melanogaster, a model insect with powerful genetics, the molecular mechanisms underlying neuronal sex determination are well understood, and two transcription factor genes, fruitless and doublesex (dsx), have been identified as terminal differentiators. In contrast, those in direct-developing insects, which diverged from the holometabolous lineage, including Drosophila, about 400 million years ago, remain unclear. My previous studies found that the fruitless homolog is unlikely to be integrated into the neuronal sex determination system in direct-developing insects. In this study, I investigated whether the dsx homolog in direct-developing insects contributes to neuronal sex determination to form neural circuits that regulate adult social behaviors. I identified the dsx homolog in the two-spotted cricket Gryllus bimaculatus (Gryllus dsx gene) and revealed that it has a unique isoform configuration, and its encoded proteins contain a cryptic DNA-binding DM domain with accumulated amino acid substitutions. A knockout study revealed that the Gryllus dsx gene functions as an X-linked morphological masculinizing factor but does not determine the sexual orientation or behavioral patterns of adult social behavior in crickets. These results suggest that, at least in crickets, an unknown sex determination factor(s) other than fruitless and dsx plays a central role in neuronal sex determination.

Leishmania spp. are protozoan parasites transmitted by female sand flies (Diptera) that cause a spectrum of devastating human pathologies affecting millions. Control of leishmaniases has proved immensely difficult: vector control strategies remain challenging and available chemotherapeutics imperfect. Gene editing of insect vectors offers prospects to interrupt disease transmission; for example by introducing antiparasite effectors or heritable modifications of genes implicated in fecundity. Here we present convergent evidence for successful gene editing using both CRISPR-Cas9 and PiggyBac approaches in two medically important sand fly species (Lutzomyia longipalpis and Phlebotomus papatasi). Targeted mutagenesis in G0 and G1 generations is supported by three independent lines of evidence, namely observable phenotypic changes, PCR-based detection assays, and confirmatory in silico algorithmic analysis of gene sequences which together provide a robust demonstration of targeted genome editing.

Deformed wing virus (DWV) is a virulent and ubiquitous disease that affects honey bee colonies. DWV infects bees at all life stages, but is most noticeable in adult bees, where clinical symptoms include shriveled, non-functional wings, and a drastically shortened lifespan. DWV is recorded in upwards of 90% of honey bee colonies worldwide, and has been linked to colony loss in symptomatic hives. DWV is primarily spread by Varroa destructor mites, who feed on the fat bodies of honey bee adults and pupae. There is currently no direct treatment or preventative for DWV, with the primary method of reduction being vector control using acaracides. In this study, we tested the effect that vaccinating honey bee queens using killed Paenibacillus larvae bacterin, the causative agent of the honey bee disease American Foulbrood, had on deformed wing virus load in honey bee colonies. We placed vaccinated queens in 200 honey bee colonies, and unvaccinated queens in 200 colonies, and measured quantities of DWV-B in both groups immediately before, and 4 months after vaccination. We found that levels of DWV-B were identical before vaccination, but were significantly reduced in colonies 4 months post vaccination. This change was found despite no difference in mite quantities between groups. Overall, these data provide evidence that vaccination of queens with P. larvae bacterin is an effective method for reduction of DWV-B quantities in honey bee colonies in a commercially relevant field setting.

The primary sequence of the Arthropod neurohormone neuroparsin is so variable that so far no orthologs from moths and butterflies have been characterized, even though classical neurosecretory stains identify cells that are homologous to those producing this hormone in other insect species. Here Lepidopteran cDNAs showing limited sequence similarity to other insect neuroparsins are described. That these cDNAs do indeed code for authentic neuroparsins was confirmed by in situ hybridization in the wax moth, Galleria mellonella, which labeled the neuroparsin neuroendocrine cells. Although in virtually all genome assemblies from Lepidoptera a neuroparsin gene could be identified, the genome assembly from the silkworm, Bombyx mori, has a neuroparsin gene containing a 16 nucleotide deletion that renders this gene nonfunctional. Although only a small number of all silkworm strains carry this deletion, it suggests that the domestication of the silkworm has rendered the function of this neurohormone dispensable.

Current methods for gene editing in insects rely on embryonic microinjection, which can be challenging for non-specialist laboratories. Recently, an alternative method known as "direct parental" CRISPR (DIPA-CRISPR) was developed. This method involves injecting commercial Cas9 protein and single-guide RNA into adult females, which can efficiently introduce mutations into developing oocytes. However, its versatility has not been fully explored, particularly in insects that have the most derived, polytrophic meroistic ovaries. In this study, we successfully applied DIPA-CRISPR to the yellow fever mosquito Aedes aegypti, which has polytrophic meroistic ovaries. Following adult injection of Cas9 ribonucleoproteins (Cas9 RNPs) targeting the kynurenine 3-monooxygenase gene, we recovered gene-edited G0 individuals. Injection at 24 h after blood-feeding resulted in the highest gene editing efficiency (3.5%), confirming that a key parameter of DIPA-CRISPR is the stage in which the adult females are injected. Together with our previous study, we demonstrated that DIPA-CRISPR is applicable to all three types of insect ovaries (i.e., panoistic, telotrophic, and polytrophic), which indicates that DIPA-CRISPR is a generalizable approach for insect gene editing.

The distinct honey bee (Apis mellifera) worker and queen castes have become a model for the study of genomic mechanisms of phenotypic plasticity. Prior studies have explored differences in gene expression and methylation during development of the two castes, but thus far no study has performed a genome-wide analysis of differences in RNA processing. To address this here we performed a Nanopore-based direct RNA sequencing with exceptionally long reads to compare the mRNA transcripts between honey bee queen and workers at three points during their larval development. We found thousands of significantly differentially expressed isoforms (DEIs) between queen and worker larvae. Most DEIs contained alternative splicing, and many of them contained at least two types of alternative splicing patterns, indicating complex RNA processing in honey bee caste differentiation. We found a negative correlation between poly(A) length and DEI expression, suggesting that poly(A) tails participate in the regulation of isoform expression. Hundreds of isoforms uniquely expressed in either queens or workers during their larval development, and isoforms were expressed at different points in queen and worker larval development demonstrating a dynamic relationship between isoform expression and developmental mechanisms. These findings show the full complexity of RNA processing and transcript expression in honey bee phenotypic plasticity.

Ruvbl1 (also known as TIP49, Pontin) encodes an ATPase of the AAA+ protein superfamily involved in several cellular functions, including chromatin remodeling, control of transcription, and cellular development (motility, growth, and proliferation). Here, we used an in-vivo RNA interference (RNAi) approach to evaluate the effect of Ruvbl1 silencing on the physiology of the corn planthopper, Peregrinus maidis. Silencing of P. maidis Ruvbl1 (PmRuvbl1) was correlated with visible morphology changes in female individuals with significant increases in body mass observed at 8 and 12 days after double strand RNA (dsRNA) injection. Ovary morphology was significantly affected in adult females with PmRuvbl1 silenced, with no mature oocytes observed at 8 and 12 days after gene silencing. Whereas no significant difference in egg laying was observed 4 days after dsRNA injection, significantly fewer eggs were laid in plants at 8 and 12 days after dsRNA treatment. Furthermore, dramatic reductions in egg hatching were observed at all time points after PmRuvbl1 silencing, compared to dsGFP-injected controls. These results extend PmRuvbl1 functions as a putative regulator of P. maidis reproduction and demonstrate the potential of Ruvbl1 to be further exploited as a target for RNAi-mediated insect control.

Insects comprise the majority of all described animal species and dominate the terrestrial habitats. The evolution of insect metamorphosis played a profound role in their successful adaptation and radiation. Insect metamorphosis is dependent on hormones ecdysteroids and sesquiterpenoids such as juvenile hormone. Despite the fact there are genuine differences between sexes during insect metamorphosis which facilitate their successful mating, how such sexual dimorphism in metamorphosis is being controlled is poorly known. We first generated transcriptomic profiles of male and female flies in late larvae and early pupae stages. Using a combination of genome-wide prediction and in vitro dual-luciferase validations, members of a microRNA cluster miR-277/34 were found to potentially regulate the neuropeptide receptor (AstC-R1) that when activated inhibits the sesquiterpenoid pathway and a juvenile hormone-dependent transcription factor (Kr-h1) in fly Drosophila melanogaster. Loss-of-function mutants were created deleting either miR-277 or miR-34, and expression levels of both AstC-R1 and Kr-h1 as well as ecdysteroid and sesquiterpenoid hormone titres were altered. Further comparison of transcriptomes of the late larvae and early pupae of both sexes revealed differential gene pathways being regulated by members of miR-277/34 between sexes during metamorphosis. This study highlights how members of a microRNA cluster control hormonal and developmental gene pathways in different sexes of insects during metamorphosis.

Drosophila melanogaster has one of the best characterized antiviral immune responses among invertebrates. However, relatively few easily-transmitted natural virus isolates are available, and so many Drosophila experiments have been performed using artificial infection routes and artificial host-virus combinations. These may not reflect natural infections, especially for subtle phenotypes such as gene expression. Here, to explore the laboratory virus community and to better understand how natural virus infections induce changes in gene expression, we analyse seven publicly available D. melanogaster transcriptomic sequencing libraries that were originally sequenced for projects unrelated to virus infection. We find ten known viruses--including five that have not been experimentally isolated--but no previously unknown viruses. Our analysis of host gene expression found numerous genes were differentially expressed in flies that were naturally infected with a virus. For example, flies infected with nora virus showed patterns of gene expression consistent with intestinal vacuolization and host attempted repair via the upd3 JAK/STAT pathway. We also found marked sex-differences in virus-induced differential gene expression. Our results show that natural virus infection in laboratory Drosophila does indeed induce detectable changes in gene expression, suggesting that this may form an important background condition for experimental studies in the laboratory.

Genetic manipulation of Aedes aegypti is key to developing a deeper understanding of this insects biology, vector-virus interactions and makes future genetic control strategies possible. Despite some advances, this process remains laborious and requires highly skilled researchers and specialist equipment. Here we present two improved methods for genetic manipulation in this species. Use of transgenic lines which express Cre recombinase allowed, by simple crossing schemes, germline or somatic recombination of transgenes, which could be utilized for numerous genetic manipulations. PhiC31 integrase based methods for site-specific integration of genetic elements was also improved, by developing a plasmid which expresses PhiC31 when injected into early embryos, eliminating the need to use costly and unstable mRNA as is the current standard.

During complete metamorphosis, holometabolous insects remodel their entire anatomy, including the gut and must control their microbiota to avoid infectious disease. High activity of antimicrobial peptides and proteins in the gut during metamorphosis has been best described in several Lepidoptera. The immune system of the dipteran Drosophila melanogaster also controls the number of bacteria during metamorphosis. However, little is known about the regulation of immune genes during the larval-adult moult in Hemimetabola which undergo less drastic metamorphic changes. Different patterns of immune effector expression during metamorphosis were shown in a study comparing the lepidopteran Galleria mellonela and the orthopteran Gryllus bimaculatus, where G. mellonella showed a strong up-regulation of antimicrobial peptides in the gut at the larval-pupal moult but not G. bimaculatus. Whether these findings reflect general patterns within holometabolous versus hemimetabolous insects remains unclear. Using RNAseq, we compare the expression of immune effector genes in the gut during metamorphosis in two holometabolous (Calliphora vicina and Tenebrio molitor) and a hemimetabolous insect (Pyrrhocoris apterus). We found high read count abundances of differentially expressed immune effectors in the gut at the larval-pupal moult in C. vicina and T. molitor; no such high abundances were observed at the larval-adult moult in P. apterus. Our findings confirm that only complete metamorphosis elicits a prophylactic immune response as an adaptive response in holometabolous insects, which controls the microbiota during gut replacement.

Immune self-tolerance is the ability of a hosts immune system to recognize and avoid triggering immune responses against self-tissue. This allows the host to avoid self-directed immune damage while still responding appropriately to pathogen infection. A breakdown of self-tolerance can lead to an autoimmune state in which immune cells target healthy self-tissue, leading to inflammation and tissue damage. In order to better understand the basic biology of autoimmunity and the role of the innate immune system in maintaining self-tolerance, we have recently characterized the Drosophila melanogaster tuSz autoimmune mutant. This mutant strain can serve as a model of innate immune mediated self-tolerance, and here we identify transcripts that are deregulated in flies experiencing a loss of self-tolerance. We find that these changes include the activation of the Relish/NF{kappa}B transcription factor, alterations in transcripts encoding proteins predicted to mediate organismal metabolism, and a downregulation of transcripts linked to developmental processes. We further find that NF{kappa}B signaling plays a protective role against loss of self-tolerance in Drosophila. Our findings provide insight into the transcriptional and physiological changes underlying self-tolerance and autoimmunity.

The western flower thrips, Frankliniella occidentalis, poses a significant challenge in global agriculture as a notorious pest and a vector of economically significant orthotospoviruses. However, the limited availability of genetic tools for F. occidentalis hampers the advancement of functional genomics and the development of innovative pest control strategies. In this study, we present a robust methodology for generating heritable mutations in F. occidentalis using the CRISPR/Cas9 genome editing system. Two eye-color genes, white (Fo-w) and cinnabar (Fo-cn), frequently used to assess Cas9 function in insects were identified in the F. occidentalis genome and targeted for knockout through embryonic microinjection of Cas9 complexed with Fo-w or Fo-cn specific guide RNAs. Homozygous Fo-w and Fo-cn knockout lines were established by crossing mutant females and males. The Fo-w knockout line revealed an age-dependent modification of eye-color phenotype. Specifically, while young larvae exhibit ivory-colored eyes, the color transitions to bright red as they age. Unexpectedly, loss of Fo-w function also altered body color, with Fo-w mutants having a lighter colored body than wild type, suggesting a dual role for Fo-w in thrips. In contrast, individuals from the Fo-cn knockout line consistently displayed bright red eyes throughout all life stages. Molecular analyses validated precise editing of both target genes. This study offers a powerful tool to investigate thrips gene functions and paves the way for the development of genetic technologies for population suppression and/or population replacement as a means of mitigating virus transmission by this vector.

The larvae of the greater waxmoth, Galleria mellonella, are gaining prominence as a versatile non-mammalian in vivo model to study host-pathogen interactions. Their ability to be maintained at 37{degrees}C, coupled with a broad susceptibility to human pathogens and a distinct melanisation response that serves as a visual indicator for larval health, positions Galleria as a powerful resource for infection research. Despite these advantages, the lack of genetic tools, such as those available for zebrafish and fruit flies, has hindered development of the full potential of Galleria as a model organism. In this study, we describe a robust methodology for generating transgenic Galleria using the PiggyBac transposon system and for precise gene knockouts via CRISPR/Cas9 technology. These advances significantly enhance the utility of Galleria in molecular research, opening the way to its widespread use as an inexpensive and ethically compatible animal model for infection biology and beyond.

1With the increasing availability of genomic and transcriptomic information within Formicidae, the investigation of gene function has become possible within ants. However, eusocial life history renders the generation of transgenic strains of ants difficult outside of specific ant lineages that can induce reproductive behavior in workers. RNA interference (RNAi) remains a practical option to investigate gene function within their genomic context in ant lineages that do not exhibit reproductive behavior in workers. This method can be leveraged to investigate the large odorant receptor (OR) gene families present in ant clades. However, ORs tend to be expressed in a tissue-specific fashion, and the capability of dsRNA to achieve knockdown of genes exhibiting localized expression remains uncertain. In this study, we use fluorescently labelled dsRNA to track the spread of dsRNA through the worker body, qPCR to verify that dsRNA can knockdown gene expression in the antennae, and behavioral trials to verify that knockdown of ORs affects nestmate recognition. We found that orally-administered dsRNA is capable of being spread systemically and knocking down tissue-specific genes. Additionally, we found that RNAi may be useful in investigating the influence of ORs in eusocial insect nestmate recognition.

There are several explanations for the extinction of ancient giant insects. Here, we present a new hypothesis suggesting that the innate immune system limits the body sizes of insects. In this study, we co-cultured bacteria, fungi, and insect blood cells, and performed single-cell RNA sequencing analyses of insect blood cells to determine their division of labor. In the innate immune system, prohibited molecules are listed as signals of invasion. The increasing diversity of organisms makes this list extensive. The burden of managing such an extensive list leads to a division of labor among blood cells and reduces the effective number of blood cells. Our simulation indicates that a reduced number of effective blood cells cannot protect a giant body from invaders.

Trichogramma, a genus of egg parasitoid wasps, are widely used as biological control agents and serve as model organisms in parasitoid research. Despite their significance, the understanding of RNA interference (RNAi) in Trichogramma remains very limited. In this study, we investigated RNAi-associated genes by bioinformatic approaches and experimentally assessed the feasibility of RNAi and the susceptibility of environmental RNAi in Trichogramma. We found that Trichogramma genomes contain a complete set of genes in the RNAi pathway and exhibit extensive gene expansion of dsRNase, which may influence RNAi efficiency by degrading dsRNA. We demonstrated successful RNAi through pupal microinjection in T. dendrolimi Matsumura, providing a technical approach for future gene functional studies. In addition, we observed no evidence of susceptibility to environmental RNAi in either T. dendrolimi adults or larvae, which might be attributed to the extensive expansion of dsRNase. This low environmental RNAi sensitivity in Trichogramma could suggest a reduced risk of RNAi-based pest management strategies affecting nontarget Trichogramma populations. Overall, this study presents a technical approach for conducting gene functional studies in Trichogramma and provides a foundation for evaluating the nontarget effects of RNAi-based pest control strategies on Trichogramma.

Transposon-mediated transgenesis has been widely used to study gene function in Lepidoptera, with piggyBac being the most commonly employed system. However, because the piggyBac transposase originates from a lepidopteran genome, it raises concerns about endogenous activation, remobilization, and silencing of transgenes, thus questioning its suitability as an optimal tool in Lepidoptera. As an alternative, we evaluated the dipteran-derived Minos transposase for stable germline transformation in the pantry moth, Plodia interpunctella. We injected syncytial embryos with transposase mRNA, along with donor plasmids encoding 3xP3::EGFP and 3xP3::mCherry markers of eye and glial tissues. Across multiple experiments, we found that G0 injectees could transmit Minos transgenes through the germline even in the absence of visible marker expression in the soma, and that large mating pools of G0 founders consistently produced transgenic offspring at efficiencies exceeding 10%. Using these methods, we generated transgenic lines with a dual expression plasmid, using 3xP3::mCherry for driving red fluorescence in eyes and glial tissues, as well as the Fibroin-L promoter expressing the recently developed mBaoJin fluorescent protein in the silk glands. This demonstrated the feasibility of screening two pairs of promoter activity in tissues of interest. Collectively, these results--along with previous findings in the silkworm Bombyx mori--demonstrate that Minos achieves robust germline integration of transgenes in Lepidoptera, offering a valuable pathway to the genetic modification of species where the remobilization or suppression of piggyBac elements might be rampant.