Insect Molecular Biology

Originally from Asia, Drosophila suzukii (Matsumura, 1931, Diptera: Drosophilidae) is presently a global pest of economically important soft-skinned fruits. Also commonly known as spotted wing Drosophila (SWD), it is largely controlled through repeated applications of broad-spectrum insecticides. There is a pressing need for a better understanding of SWD biology and for developing alternative environmentally-friendly methods of control. The RNA-guided Cas9 nuclease has revolutionized functional genomics and is an integral component of several recently developed genetic strategies for population control of insects. Here we have developed transgenic strains that encode three different terminators and four different promoters to express Cas9 in both the soma and/or germline of SWD. The Cas9 lines were evaluated through genetic crossing to transgenic lines that encode single guide RNAs targeting the conserved X-linked yellow body and white eye genes. We find that several Cas9/gRNA lines display very high editing capacity. Going forward, these tools will be instrumental for evaluating gene function in SWD and may provide tools useful for the development of new genetic strategies for control of this invasive species.

Honeybees require a functioning immune system to defend against microbial pathogens. The American foulbrood pathogen, Paenibacillus larvae, is lethal to honeybees and one of the main causes of colony collapse. This study investigated the immune responses of Apis mellifera and Apis cerana honeybees against the bacterial pathogen P. larvae. Both species of honeybee larvae exhibited significant mortalities even at 102 [~] 103 cfu/mL of P. larvae by diet-feeding, although A. mellifera appeared to be more tolerant to the bacterial pathogen than A. cerana. Upon bacterial infection, the two honeybee species expressed both cellular and humoral immune responses. Hemocytes of both species exhibited characteristic spreading behaviors by cytoskeletal extension along with F-actin growth, and formed nodules upon P. larvae infection. Larvae of both species also expressed an antimicrobial peptide called apolipophorin III (ApoLpIII) in response to bacterial infection. However, these immune responses were significantly suppressed by a specific inhibitor to phospholipase A2 (PLA2). Each honeybee genome encodes four PLA2 genes (PLA2A [~] PLA2D), representing four orthologous combinations between the two species. In response to P. larvae infection, both species significantly up-regulated PLA2 enzyme activities and the expression of all four PLA2 genes. To determine the roles of the four PLA2s in the immune responses, RNA interference (RNAi) was performed by injecting gene-specific double stranded RNAs (dsRNAs). All four RNAi treatments significantly suppressed the immune responses, and specific inhibition of the two secretory PLA2s (PLA2A and PLA2B) potently suppressed nodule formation and ApoLpIII expression. These results demonstrate the cellular and humoral immune responses of A. mellifera and A. cerana against P. larvae. This study suggests that eicosanoids play a crucial role in mediating common immune responses in two closely related honeybees.

CRISPR/Cas9 gene drive systems are possible in a few insects and ever expanding. Nonetheless, success in one species and techniques developed for it are not necessarily applicable to other species. As such, the development and expansion of gene drive systems is dependent upon direct experimentation. A critical aspect and potentially limiting factor of gene drive is the ability to induce Cas9-dependent homologous recombination. Here we report our attempts to induce Cas9-dependent homologous recombination and subsequent gene drive in Tribolium castaneum. Utilizing constructs containing one or two target gRNAs in combination with Cas9 under two different promoters and corresponding homology arms, we found a high incidence of CRISPR/Cas9 induced mutations but a complete lack of evidence of homologous recombination and genetic drive. Even though the generated constructs provide new resources for CRISPR/Cas9 modification of the Tribolium genome, our results suggest that Tribolium genome may be refractory towards Cas9-induced homologous recombination and additional modifications will be necessary to increase the potential for homologous recombination.

The two arms of innate immunity consist of the cell-mediated cellular defenses and the systemic humoral immune responses. Drosophila humoral immune defenses in the context of antimicrobial immunity, particularly the regulation and activation of antimicrobial peptide secretion from the fat body, have been studied extensively. How Drosophila regulates humoral immunity against another major natural enemy, the parasitoid wasp, is less well-characterized. In this study, we focused on a gene crucial in anti-parasitoid immunity, lectin-24A, which is specifically induced following parasitization. We found that a fluorescent reporter driven by the region upstream of lectin-24A showed localized posterior expression in the larval fat body, the Drosophila tissue mediating humoral immunity. Furthermore, with RNA sequencing of the anterior and posterior fat body sections, we found that components of JAK/STAT, GATA, and Toll pathways were regulated differentially in the anterior-posterior axis of the fat body and/or by infection. Predicted binding motifs for transcription factors in all three of these pathways were identified in the 444bp upstream region of the lectin-24A gene, where scrambling these motifs leads to reduced basal or induced expression of the fluorescent reporter. Investigating each of these pathways, we found that JAK/STAT, the GATA factor Pannier, and the NF-{kappa}B factor dorsal all modulate the expression of lectin-24A. The binding motifs associated with these transcription factors were also enriched in the upstream sequences of parasitism-induced genes in the fat body. Taken together, these results indicate that JAK/STAT, Pannier, and NF-{kappa}B signaling are involved in the regulation of lectin-24A and, more generally, Drosophila humoral anti-parasitoid immunity after infection.

Entomopathogenic nematodes (EPNs) from the genus Steinernema and Heterorhabditis form mutualistic relationships with symbiotic bacteria from the genus Xenorhabdus and Photorhabdus, respectively. Together, these nematode-bacterium pairs infect and kill insect hosts--primarily larvae from the orders Lepidoptera and Coleoptera. This tripartite interaction provides a powerful model for investigating the molecular mechanisms underlying mutualism and parasitism. A key step toward this goal is the development of a genetically tractable EPN. While RNAi has been applied in some EPN species, stable, transgenerational genetic tools remain limited. Here, we establish a robust CRISPR-Cas9 system in the emerging model Steinernema hermaphroditum, a species that is easily cultivated in both in vivo and in vitro conditions and amenable to gonadal microinjection. Notably, its hermaphroditic reproduction simplifies the generation of genetically stable mutant lines. We present a detailed protocol for efficient, targeted gene knockout via microinjection in S. hermaphroditum. As a proof-of-concept, we knocked out a conserved homologue, unc-22, which causes a twitching phenotype. The CRISPR-Cas9 based genome editing in S. hermaphroditum has potential to be used to express transgene, or to be adapted to other EPN species that are applicable to benefit agriculture. SUMMARYThis article demonstrates CRISPR-Cas9 mediated genome engineering in Steinernema hermaphroditum, an entomopathogenic (EPN: insect-parasitic) nematode and an emerging genetic model. The described technology is useful for creating mutants allowing for the elucidation of gene functions in the nematode biology that is relevant to mutualistic and parasitic symbiosis.

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.

Conditional sex transformation systems are promising tools in the fight against insect pests. In this study, we developed and tested CRISPR-based, tetracycline-repressible sex transformation strains in the Australian sheep blowfly, Lucilia cuprina. Two CRISPR effector molecules, Cas9 and dCas9, were employed to target the sex-determining gene transformer with the goal of turning female blowflies into males. The Cas9 version of the system induced robust knockout of a visual marker gene but failed to trigger sex transformation without external provision of transformer-targeting sgRNAs. Furthermore, we found that dCas9 expression was linked to several deleterious phenotypes, including developmental delays, reduced body weight, and death. Our study provides the first proof-of-concept conditional CRISPR systems in L. cuprina, and suggests that while dCas9 is toxic at high levels in this species, Cas9 is well-tolerated and may be able to induce sex transformation with minor modifications to the system.

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.

Although honey bee responses to pathogens have been systematically described in the past decades, antiviral signalling pathways mechanisms are not thoroughly characterized. To decipher direct antiviral roles of an immune pathway, we firstly used the infectious clone of Israeli acute paralysis virus (IAPV) to screen 42 immune genes involved in mTOR, MAPK, Toll, Endocytosis, Jak-STAT pathway and homeobox protein, heat shock protein, as well as antimicrobial peptides (AMPs), and found that Toll pathway was a potential predominant immune pathway in Apis mellifera. Consistent with this, only dsRNA-PGRP-S2 treated A. mellifera significantly exhibited impaired activation of Toll pathway, promoting susceptibility to the IAPV infection. Finally, immunofluorescence results confirmed that the Toll pathway was initiated by peptidoglycan recognition protein S2 (PGRP-S2) interacting with Toll protein. Co-immunoprecipitation findings also further preliminarily confirmed PGRP-S2 directly interacting with viral capsid protein IAPV-VP3 to induce the activation of the Toll pathway in A. mellifera. These findings highlight that the Toll pathway is demanded efficient inhibitions of IAPV replication as a specific antiviral pathway in A. mellifera, and PGRP-S2, acting as a pattern recognition receptor, could be a new approach for control of the viral disease. Author summaryHoney bee viruses, particularly IAPV, had been implicated in the colony decline with a global distribution resulting in insufficient pollination services. However, little is known about the antiviral mechanism of honey bee. In this study, we found that the Toll pathway was required for A. mellifera against IAPV infection and initiated by PGRP-S2. We also confirmed that dsRNA-PGRP-S2 treated A. mellifera exhibited impaired Toll pathway activation and promoted susceptibility to the IAPV infection. As a result, we employed co-immunoprecipitation technique to identify the interaction between the PGRP-S2 with Toll. Moreover, it was found the PGRP-S2 directly recognized IAPV-VP3 to activate the immune pathway against IAPV infection. Our work provides novel evidence that honey bees own a specific antiviral immune pathway and suggests that targeting PGRP-S2 could be a new approach for controlling the viral disease.

Epoxyoctadecamonoenoic acids (EpOMEs) are epoxide derivatives of linoleic acid (9,12-octadecadienoic acid: LA). They are metabolized into dihydroxyoctadecamonoenoic acids (DiHOMEs) in mammals. Unlike in mammals where they act as adipokines or lipokines, EpOMEs act as immunosuppressants in insects. However, the functional link between EpOMEs and pro-immune mediators such as PGE2 is not known. In addition, the physiological significance of DiHOMEs is not clear in insects. This study analyzed the physiological role of these C18 oxylipins using a lepidopteran insect pest, Spodoptera exigua. Immune challenge of S. exigua rapidly upregulated the expression of the phospholipase A2 gene to trigger C20 oxylipin biosynthesis, followed by the upregulation of genes encoding EpOME synthase (SE51385) and a soluble epoxide hydrolase (Se-sEH). The sequential gene expression resulted in the upregulations of the corresponding gene products such as PGE2, EpOMEs, and DiHOMEs. Interestingly, only PGE2 injection without the immune challenge significantly upregulated the gene expression of SE51825 and Se-sEH. The elevated levels of EpOMEs acted as immunosuppressants by inhibiting cellular and humoral immune responses induced by the bacterial challenge, in which 12,13-EpOME was more potent than 9,10-EpOME. However, DiHOMEs did not inhibit the cellular immune responses but upregulated the expression of antimicrobial peptides selectively suppressed by EpOMEs. The negative regulation of insect immunity by EpOMEs and their inactive DiHOMEs were further validated by synthetic analogs of the linoleate epoxide and corresponding diol. Furthermore, inhibitors specific to Se-sEH used to prevent EpOME degradation significantly suppressed the immune responses. The data suggest a physiological role of C18 oxylipins in resolving insect immune response. Any immune dysregulation induced by EpOME analogs or sEH inhibitors significantly enhanced insect susceptibility to the entomopathogen, Bacillus thuringiensis. Author summaryUpon immune challenge, recognition signal triggers insect immunity to remove the pathogens by cellular and humoral responses. Various immune mediators propagate the immune signals to nearby tissues, in which polyunsaturated fatty acid (PUFA) derivatives play crucial roles. However, little was known on how the insects terminate the activated immune responses after pathogen neutralization. Interestingly, C20 PUFA was detected at the early infection stage and later C18 PUFAs were induced in a lepidopteran insect, Spodoptera exigua. This study showed the role of epoxyoctadecamonoenoic acids (EpOMEs) in the immune resolution at the late infection stage to quench the excessive and unnecessary immune responses. In contrast, dihydroxy-octadecamonoenoates (DiHOMEs) were the hydrolyzed and inactive forms of EpOMEs. The hydrolysis is catalyzed by soluble epoxide hydrolase (sEH). Inhibitors specific to sEH mimicked the immunosuppression induced by EpOMEs. Furthermore, the inhibitor treatments significantly enhanced the bacterial virulence of Bacillus thuringiensis against S. exigua. This study proposes a negative control of the immune responses using EpOME/DiHOME in insects.

Comprehensive expression datasets were constructed for Apis mellifera and Apis cerana japonica. Post-oviposition day 6 to day 58 samples of A. mellifera workers (larva to adult); day 9, 10, 12, and 13 samples of A. mellifera queen (larva to pupa); and day 9 to day 18 samples of A. cerana japonica workers (larva to adult) were prepared, and RNA-Seq data were obtained. For A. cerana japonica, reference transcript sequence data, predicted amino acid sequence data, and functional annotation data were generated based on the genome sequence and RNA-Seq data. Using the transcript sequence and RNA-Seq data, comprehensive expression data for all transcripts of A. mellifera and A. cerana japonica were prepared. Hierarchical clustering analyses and the used sample preparation method ensured that both sets of expression data were reliable for use as comprehensive reference expression datasets. Therefore, these data are applicable for honey bee research or for comparative or evolutionary studies on insect species or social insect species at the genetic and molecular levels.

Insects often establish long-term relationships with intracellular symbiotic bacteria, i.e. endosymbionts, that provide them with essential nutrients such as amino acids and vitamins. Endosymbionts are typically confined within specialized host cells called bacteriocytes that may form an organ, the bacteriome. Compartmentalization within host cells is paramount for protecting the endosymbionts and also avoiding chronic activation of the host immune system. In the cereal weevil Sitophilus oryzae, bacteriomes are present as a single organ at the larval foregut-midgut junction, and in adults, at the apex of midgut mesenteric caeca and at the apex of the four ovarioles. While the adult midgut endosymbionts experience a drastic proliferation during early adulthood followed by complete elimination through apoptosis and autophagy, ovarian endosymbionts are maintained throughout the weevil lifetime by unknown mechanisms. Bacteria present in ovarian bacteriomes are thought to be involved in the maternal transmission of endosymbionts through infection of the female germline, but the exact mode of transmission is not fully understood. Here, we show that endosymbionts are able to colonize the germarium in one-week-old females, pinpointing a potential infection route of oocytes. To identify potential immune regulators of ovarian endosymbionts, we have analyzed the transcriptomes of the ovarian bacteriomes through young adult development, from one-day-old adults to sexually mature ones. In contrast with midgut bacteriomes, immune effectors are downregulated in ovarian bacteriomes at the onset of sexual maturation. We hypothesize that relaxation of endosymbiont control by antimicrobial peptides might allow bacterial migration and potential oocyte infection, ensuring endosymbiont transmission.

AO_SCPLOWBSTRACTC_SCPLOWSexually dimorphic phenotypes are thought to arise primarily from sex-biased gene expression during development. Major changes in developmental strategies, such as the shift from hemimetabolous to holometabolous development, are therefore expected to have profound consequences for the dynamics of sex-biased gene expression. However, no studies have previously examined sex-biased gene expression during development in hemimetabolous insects, precluding comparisons between developmental strategies. Here we characterized sex-biased gene expression at three developmental stages in a hemimetabolous stick insect (Timema californicum): hatchlings, juveniles, and adults. As expected, the proportion of sex-biased genes gradually increased during development, mirroring the gradual increase of phenotypic sexual dimorphism. Sex-biased genes identified at early developmental stages were generally consistently male- or female-biased at later stages, suggesting their importance in sexual differentiation. Additionally, we compared the dynamics of sex-biased gene expression during development in T. californicum to those of the holometabolous fly Drosophila melanogaster by reanalyzing publicly available RNA-seq data from third instar larval, pupal and adult stages. In D. melanogaster, 84% of genes were sex-biased at the adult stage (compared to only 20% in T. californicum), and sex-biased gene expression increased abruptly at the adult stage when morphological sexual dimorphism is manifested. Our findings are consistent with the prediction that the dynamics of sex-biased gene expression during development differ extensively between holometabolous and hemimetabolous insect species.

BackgroundThe cabbage stem flea beetle (CSFB, Psylliodes chrysocephala) is a major threat to oilseed rape crops. Management of CSFB has become increasingly challenging due to the European Unions ban on neonicotinoids and the emergence of pyrethroid-resistant populations. Recently, RNA interference (RNAi) has shown potential as an environmentally friendly alternative for the management of CSFB, and proteasome subunits have been identified as very effective RNAi targets. However, the mechanism of action of proteasome-targeting RNAi strategies remains to be fully characterized at the molecular level in CSFB and other pests. Here, we used CSFB to investigate the mechanism of action of dsPros{beta}1, which is a double-stranded RNA targeting a proteasome subunit. ResultsRNA degradome sequencing identified siRNA-mediated cleavage events in the target transcript, with cleavage events occurring at higher rates between uracil-guanine and adenine-adenine pairs. RISC-bound small RNA sequencing confirmed the presence of mature siRNAs guiding these cleavage events while revealing discrepancies between siRNA abundance and cleavage patterns. Proteomics analysis identified changes in protein levels caused by proteasome inhibition, including an increase in mitochondria- and cytoskeleton-related proteins and a decrease in central dogma-associated proteins. ConclusionThis study demonstrates that combining RNA degradomics, RISC-bound sRNA-seq, and proteomics is an insightful approach to investigating the mechanism of RNAi-based pest control at the molecular level. The insights gained from these methods can be used to enhance proteasome-targeting RNAi strategies against insect pests. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=177 HEIGHT=200 SRC="FIGDIR/small/642439v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@79f29org.highwire.dtl.DTLVardef@62773borg.highwire.dtl.DTLVardef@81ecbeorg.highwire.dtl.DTLVardef@1ddce01_HPS_FORMAT_FIGEXP M_FIG C_FIG RNA degradomics revealed dsPros{beta}1-derived siRNA-mediated mRNA cleavage events, mainly at uracil-guanine and adenine-adenine pairs. Proteasome inhibition via dsPros{beta}1 increased mitochondrial and cytoskeletal proteins while reducing translation-related and mRNA-binding proteins.

RNA interference (RNAi) regulates gene expression in most multicellular organisms through binding of small RNA effectors to target transcripts. Exploiting this process is a popular strategy for genetic manipulation in invertebrates and has applications that includes control of pests. Successful RNAi technologies are dependent on delivery method. The most convenient method is likely feeding which is effective in some animals while others are insensitive. Thus, there is a need to develop RNAi technology on a per-species basis, which will require a comprehensive approach for assessing small RNA production from synthetic nucleic acids. Using a biochemical and sequencing approaches we investigated the metabolism of ingested RNAs using the two-spotted spider mite, Tetranychus urticae, as a model for RNAi insensitivity. This chelicerae arthropod shows only modest response to oral RNAi and has biogenesis pathways distinct from model organisms. To identify RNAi substrates in T. urticae we characterized processing of synthetic RNAs and those derived from plant transcripts ingested during feeding. Through characterization of read size length and overlaps of small RNA reads, visualization methods were developed that facilitate distinguish trans-acting small RNAs from degradation fragments. Using a strategy that delineates small RNA classes, we found a variety of RNA species are gated into spider mite RNAi pathways, however, potential mature trans-acting RNAs appear very unstable and rare. This suggests spider mite RNAi pathway products that originate as ingested materials may be preferentially metabolized instead of converted into regulators of gene expression. Spider mites infest a variety of plants, and it would be maladaptive to generate diverse gene regulators from dietary RNAs. This study provides a framework for assessing RNAi technology in organisms where genetic and biochemical tools are absent and benefit rationale design of RNAi triggers.

In the last 20 years, Pseudomonas entomophila (Pe) has emerged as a model to explore insect immunity to bacterial intestinal pathogens. Laboratory studies evidenced multiple detrimental effects of Pe on Drosophila melanogaster. However, these effects require that the bacteria are ingested in extremely high concentrations of 1010 - 1011 CFU per mL (OD600 20 - 200), questioning the relevance of this pathogen in nature. Here, we tested whether the need for such high doses may be due to protective effects of the food preservative methylparaben (Nipagin), a standard ingredient of artificial Drosophila diets. While significant mortality in flies fed diet containing standard methylparaben concentration required doses of >1010 CFU per mL, when methylparaben was absent we could observe mortality using 500,000x lower doses. Consistent with these results, we demonstrated strong bactericidal properties of methylparaben on Pe in vitro. In the absence of methylparaben even the smallest inocula (105 CFU per mL) led to high bacterial loads (106 CFU per fly) after several days, indicating the ability of Pe to grow and overcome the flies defenses. We also demonstrate that in the absence of methylparaben, infected flies could easily transmit the pathogen to other adults and to offspring, resulting in high mortality and thus highlighting the potential of Pe as a pathogen of Drosophila in nature. Our study also underscores that careful consideration should be given to food additives used in standard diets in laboratory research on host-pathogen interaction. ImportanceAccurate characterization of pathogen infections requires appropriate experimental methodologies. Infections of insects with Pe are frequently studied using fruit flies as a model organism, with laboratory cultures typically maintained on artificial media containing various food preservatives. In this study, we show that one commonly used preservative, methylparaben, significantly influences the outcome of oral infections with Pe. We found that minimal infection doses, far below the standards of the field, could be still lethal to flies raised on media without methylparaben. This increased virulence was also associated with increased transmission of the pathogen, both from infected adult flies to their offspring and to uninfected adults. Our findings show how subtle variations in experimental conditions can profoundly affect how we perceive pathogenic threats.

Selecting an appropriate target gene is critical to the success of feeding RNA interference (f-RNAi)-based pest control. Gene targets have been chosen based on their ability to induce lethality. However, lethality induction by f-RNAi is slow-acting and crop damage can progress during this time. Here, we show that f-RNAi of death-associated inhibitor of apoptosis protein 1 (diap1), but not two conventional targets vacuolar ATPase subunit A and E, induces acute feeding cessation in the solanaceous pest, Henosepilachna vigintioctopunctata during 24-48 hours. We also found that the feeding cessation by diap1 f-RNAi has species-specificity and occurs with only 1.6 ng dsRNA. Our results suggest that diap1 is an appropriate target in the context of rapid reduction of crop damage. We propose that acute feeding disorder should be assessed as a novel criterion for selecting appropriate target genes for RNAi-based pest control in addition to the conventional criterion based on lethality.

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.

Molecular tool development in traditionally non-tractable animals opens new avenues to study gene functions in the relevant ecological context. Entomopathogenic nematodes (EPN) Steinernema and their symbiotic bacteria of Xenorhabdus spp are a valuable experimental system in the laboratory and are applicable in the field to promote agricultural productivity. The infective juvenile (IJ) stage of the nematode packages mutualistic symbiotic bacteria in the intestinal pocket and invades insects that are agricultural pests. The lack of consistent and heritable genetics tools in EPN targeted mutagenesis severely restricted the study of molecular mechanisms underlying both parasitic and mutualistic interactions. Here, I report a protocol for CRISPR-Cas9 based genome-editing that is successful in two EPN species, S. carpocapsae and S. hermaphroditum. I adapted a gonadal microinjection technique in S. carpocapsae, which created on-target modifications of a homologue Sc-dpy-10 (cuticular collagen) by homology-directed repair. A similar delivery approach was used to introduce various alleles in S. hermaphroditum including Sh-dpy-10 and Sh-unc-22 (a muscle gene), resulting in visible and heritable phenotypes of dumpy and twitching, respectively. Using conditionally dominant alleles of Sh-unc-22 as a co-CRISPR marker, I successfully modified a second locus encoding Sh-Daf-22 (a homologue of human sterol carrier protein SCPx), predicted to function as a core enzyme in the biosynthesis of nematode pheromone that is required for IJ development. As a proof of concept, Sh-daf-22 null mutant showed IJ developmental defects in vivo (in insecta). This research demonstrates that Steinernema spp are highly tractable for targeted mutagenesis and has great potential in the study of gene functions under controlled laboratory conditions within the relevant context of its ecological niche.

Most gene functions were detected by screens in very few model organisms but it has remained unclear how comprehensive these data are. Here, we expanded our RNAi screen in the red flour beetle Tribolium castaneum to cover more than half of the protein-coding genes and we compared the gene sets involved in several processes between beetle and fly. We find that around 50 % of the gene functions are detected in both species while the rest was found only in fly (~10%) or beetle (~40%) reflecting both technical and biological differences. We conclude that work in complementary model systems is required to gain a comprehensive picture on gene functions documented by the annotation of novel GO terms for 96 genes studied here. The RNAi screening resources developed in this project, the expanding transgenic tool-kit and our large-scale functional data make T. castaneum an excellent model system in that endeavor.