CRISPR/Cas9-mediated genome editing of Frankliniella occidentalis, the western flower thrips, via embryonic microinjection.

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-colour 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-colour phenotype. Specifically, while young larvae exhibit orange-coloured eyes, the colour transitions to bright red as they age. Unexpectedly, loss of Fo-w function also altered body colour, with Fo-w mutants having a lighter coloured 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 function 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.

A draft Diabrotica virgifera virgifera genome: insights into control and host plant adaption by a major maize pest insect.

BACKGROUND: Adaptations by arthropod pests to host plant defenses of crops determine their impacts on agricultural production. The larval host range of western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), is restricted to maize and a few grasses. Resistance of D. v. virgifera to crop rotation practices and multiple insecticides contributes to its status as the most damaging pest of cultivated maize in North America and Europe. The extent to which adaptations by this pest contributes to host plant specialization remains unknown. RESULTS: A 2.42 Gb draft D. v. virgifera genome, Dvir_v2.0, was assembled from short shotgun reads and scaffolded using long-insert mate-pair, transcriptome and linked read data. K-mer analysis predicted a repeat content of ≥ 61.5%. Ortholog assignments for Dvir_2.0 RefSeq models predict a greater number of species-specific gene duplications, including expansions in ATP binding cassette transporter and chemosensory gene families, than in other Coleoptera. A majority of annotated D. v. virgifera cytochrome P450s belong to CYP4, 6, and 9 clades. A total of 5,404 transcripts were differentially-expressed between D. v. virgifera larvae fed maize roots compared to alternative host (Miscanthus), a marginal host (Panicum virgatum), a poor host (Sorghum bicolor) and starvation treatments; Among differentially-expressed transcripts, 1,908 were shared across treatments and the least number were between Miscanthus compared to maize. Differentially-expressed transcripts were enriched for putative spliceosome, proteosome, and intracellular transport functions. General stress pathway functions were unique and enriched among up-regulated transcripts in marginal host, poor host, and starvation responses compared to responses on primary (maize) and alternate hosts. CONCLUSIONS: Manual annotation of D. v. virgifera Dvir_2.0 RefSeq models predicted expansion of paralogs with gene families putatively involved in insecticide resistance and chemosensory perception. Our study also suggests that adaptations of D. v. virgifera larvae to feeding on an alternate host plant invoke fewer transcriptional changes compared to marginal or poor hosts. The shared up-regulation of stress response pathways between marginal host and poor host, and starvation treatments may reflect nutrient deprivation. This study provides insight into transcriptomic responses of larval feeding on different host plants and resources for genomic research on this economically significant pest of maize.

Structural and functional insights into the ATP-binding cassette transporter family in the corn planthopper, Peregrinus maidis.

The corn planthopper, Peregrinus maidis, is an economically important pest of maize and sorghum. Its feeding behaviour and the viruses it transmits can significantly reduce crop yield. The control of P. maidis and its associated viruses relies heavily on insecticides. However, control has proven difficult due to limited direct exposure of P. maidis to insecticides and rapid development of resistance. As such, alternative control methods are needed. In the absence of a genome assembly for this species, we first developed transcriptomic resources. Then, with the goal of finding targets for RNAi-based control, we identified members of the ATP-binding cassette transporter family and targeted specific members via RNAi. PmABCB_160306_3, PmABCE_118332_5 and PmABCF_24241_1, whose orthologs in other insects have proven important in development, were selected for knockdown. We found that RNAi-mediated silencing of PmABCB_160306_3 impeded ovary development; disruption of PmABCE_118332_5 resulted in localized melanization; and knockdown of PmABCE_118332_5 or PmABCF_24241_1 each led to high mortality within five days. Each phenotype is similar to that found when targeting the orthologous gene in other species and it demonstrates their potential for use in RNAi-based P. maidis control. The transcriptomic data and RNAi results presented here will no doubt assist with the development of new control methods for this pest.

Development of transgenic corn planthopper Peregrinus maidis that express the tetracycline transactivator.

The corn planthopper, Peregrinus maidis, is a vector of several maize viruses and is consequently a significant agricultural pest in many tropical and subtropical regions. As P. maidis has developed resistance to insecticides, the aim of this study was to develop transgenic P. maidis strains that could be used for future genetic biocontrol programs. To facilitate the identification of transgenic P. maidis, we isolated and characterized the promoters for the P. maidis ubiquitin-like and profilin genes. Transient expression assays with P. maidis embryos showed that both promoters were active. Transgenic lines were established using piggyBac vectors and fluorescent protein marker genes. The lines carried an auto-regulated tetracycline transactivator (tTA) gene, which has been widely used to establish conditional lethal strains in other insect species. The transgenic lines showed low levels of tTA expression but were viable on diet with or without doxycycline, which inhibits the binding of tTA to DNA. We discuss possible modifications to the tTA overexpression system that could lead to the successful development of conditional lethal strains. To our knowledge, this is the first report of a transgenic Hemiptera. The approach we have taken could potentially be applied to other Hemiptera and, for P. maidis, the technology will facilitate future functional genomics studies.

New Technologies for Studying Negative-Strand RNA Viruses in Plant and Arthropod Hosts.

The plant viruses in the phylum Negarnaviricota, orders Bunyavirales and Mononegavirales, have common features of single-stranded, negative-sense RNA genomes and replication in the biological vector. Due to the similarities in biology, comparative functional analysis in plant and vector hosts is helpful for understanding host-virus interactions for negative-strand RNA viruses. In this review, we will highlight recent technological advances that are breaking new ground in the study of these recalcitrant virus systems. The development of infectious clones for plant rhabdoviruses and bunyaviruses is enabling unprecedented examination of gene function in plants and these advances are also being transferred to study virus biology in the vector. In addition, genome and transcriptome projects for critical nonmodel arthropods has enabled characterization of insect response to viruses and identification of interacting proteins. Functional analysis of genes using genome editing will provide future pathways for further study of the transmission cycle and new control strategies for these viruses and their vectors.

Structural and functional insights into the Diabrotica virgifera virgifera ATP-binding cassette transporter gene family.

BACKGROUND: The western corn rootworm, Diabrotica virgifera virgifera, is a pervasive pest of maize in North America and Europe, which has adapted to current pest management strategies. In advance of an assembled and annotated D. v. virgifera genome, we developed transcriptomic resources to use in identifying candidate genes likely to be involved in the evolution of resistance, starting with members of the ATP-binding cassette (ABC) transporter family. RESULTS: In this study, 65 putative D. v. virgifera ABC (DvvABC) transporters were identified within a combined transcriptome assembly generated from embryonic, larval, adult male, and adult female RNA-sequence libraries. Phylogenetic analysis placed the deduced amino-acid sequences of the DvvABC transporters into eight subfamilies (A to H). To supplement our sequence data with functional analysis, we identified orthologs of Tribolium castaneum ABC genes which had previously been shown to exhibit overt RNA interference (RNAi) phenotypes. We identified eight such D. v. virgifera genes, and found that they were functionally similar to their T. castaneum counterparts. Interestingly, depletion of DvvABCB_39715 and DvvABCG_3712 transcripts in adult females produced detrimental reproductive and developmental phenotypes, demonstrating the potential of these genes as targets for RNAi-mediated insect control tactics. CONCLUSIONS: By combining sequence data from four libraries covering three distinct life stages, we have produced a relatively comprehensive de novo transcriptome assembly for D. v. virgifera. Moreover, we have identified 65 members of the ABC transporter family and provided the first insights into the developmental and physiological roles of ABC transporters in this pest species.

Effects of targeting eye color in Tenebrio molitor through RNA interference of tryptophan 2,3-dioxygenase (vermilion): Implications for insect farming.

The gene vermilion encodes tryptophan 2,3-dioxygenase, part of the ommochrome pathway, and is responsible for the dark pigmented eyes in some insects, including beetles. Using RNA interference, we targeted the vermilion gene ortholog in embryos and pupae of the yellow mealworm, Tenebrio molitor, resulting in larvae and adults, respectively, that lacked eye pigment. RNA-Seq was used to analyze the impact of vermilion-specific RNA interference on gene expression. There was a 425-fold reduction in vermilion gene expression (p = 0.0003), as well as significant (p < 0.05) differential expression of 109 other putative genes, most of which were downregulated. Enrichment analysis of Gene Ontology terms found in the differentially expressed data set included genes known to be involved in the ommochrome pathway. However, enrichment analysis also revealed the influence of vermilion expression on genes involved in protein translocation to the endoplasmic reticulum, signal transduction, G-protein-coupled receptor signaling, cell-cycle arrest, mannose biosynthesis, and vitamin transport. These data demonstrate that knockdown of vermilion in T. molitor results in complete loss of eye color (white-eyed phenotype) and identify other interrelated genes in the vermilion metabolic pathway. Therefore, a dominant marker system based on eye color can be developed for the genetic manipulation of T. molitor to increase the value of mealworms as an alternative food source by decreasing negative traits, such as disease susceptibility, and increasing desired traits, such as protein content and vitamin production.

Development and use of a piggyBac-based jumpstarter system in Drosophila suzukii.

Spotted wing drosophila, Drosophila suzukii, is an invasive pest that primarily attacks fresh, soft-skinned fruit. Although others have reported successful integration of marked piggyBac elements into the D. suzukii genome, with a very respectable transgenesis rate of ∼16%, here we take this work a step further by creating D. suzukii jumpstarter strains. These were generated through integration of a fluorescent-marked Minos element carrying a heat shock protein 70-driven piggyBac transposase gene. We demonstrate that there is a dramatic increase in transformation rates when germline transformation is performed in a transposase-expressing background. For example, we achieved transformation rates as high as 80% when microinjecting piggyBac-based plasmids into embryos derived from one of these D. suzukii jumpstarter strains. We also investigate the effect of insert size on transformation efficiency by testing the ability of the most efficient jumpstarter strain to catalyze integration of differently-sized piggyBac elements. Finally, we demonstrate the ability of a jumpstarter strain to remobilize an already-integrated piggyBac element to a new location, demonstrating that our jumpstarter strains could be used in conjunction with a piggyBac-based donor strain for genome-wide mutagenesis of D. suzukii.

Genes related to mitochondrial functions are differentially expressed in phosphine-resistant and -susceptible Tribolium castaneum.

BACKGROUND: Phosphine is a valuable fumigant to control pest populations in stored grains and grain products. However, recent studies indicate a substantial increase in phosphine resistance in stored product pests worldwide. RESULTS: To understand the molecular bases of phosphine resistance in insects, we used RNA-Seq to compare gene expression in phosphine-resistant and susceptible laboratory populations of the red flour beetle, Tribolium castaneum. Each population was evaluated as either phosphine-exposed or no phosphine (untreated controls) in triplicate biological replicates (12 samples total). Pairwise analysis indicated there were eight genes differentially expressed between susceptible and resistant insects not exposed to phosphine (i.e., basal expression) or those exposed to phopshine (>8-fold expression and 90 % C.I.). However, 214 genes were differentially expressed among all four treatment groups at a statistically significant level (ANOVA, p < 0.05). Increased expression of 44 cytochrome P450 genes was found in resistant vs. susceptible insects, and phosphine exposure resulted in additional increases of 21 of these genes, five of which were significant among all treatment groups (p < 0.05). Expression of two genes encoding anti-diruetic peptide was 2- to 8-fold reduced in phosphine-resistant insects, and when exposed to phosphine, expression was further reduced 36- to 500-fold compared to susceptible. Phosphine-resistant insects also displayed differential expression of cuticle, carbohydrate, protease, transporter, and many mitochondrial genes, among others. Gene ontology terms associated with mitochondrial functions (oxidation biological processes, monooxygenase and catalytic molecular functions, and iron, heme, and tetrapyyrole binding) were enriched in the significantly differentially expressed dataset. Sequence polymorphism was found in transcripts encoding a known phosphine resistance gene, dihydrolipoamide dehydrogenase, in both susceptible and resistant insects. Phosphine-resistant adults also were resistant to knockdown by the pyrethroid deltamethrin, likely due to the increased cytochrome P450 expression. CONCLUSIONS: Overall, genes associated with the mitochondria were differentially expressed in resistant insects, and these differences may contribute to a reduction in overall metabolism and energy production and/or compensation in resistant insects. These data provide the first gene expression data on the response of phosphine-resistant and -susceptible insects to phosphine exposure, and demonstrate that RNA-Seq is a valuable tool to examine differences in insects that respond differentially to environmental stimuli.

The genome of the model beetle and pest Tribolium castaneum.

Tribolium castaneum is a member of the most species-rich eukaryotic order, a powerful model organism for the study of generalized insect development, and an important pest of stored agricultural products. We describe its genome sequence here. This omnivorous beetle has evolved the ability to interact with a diverse chemical environment, as shown by large expansions in odorant and gustatory receptors, as well as P450 and other detoxification enzymes. Development in Tribolium is more representative of other insects than is Drosophila, a fact reflected in gene content and function. For example, Tribolium has retained more ancestral genes involved in cell-cell communication than Drosophila, some being expressed in the growth zone crucial for axial elongation in short-germ development. Systemic RNA interference in T. castaneum functions differently from that in Caenorhabditis elegans, but nevertheless offers similar power for the elucidation of gene function and identification of targets for selective insect control.

Evaluation of Peregrinus maidis transformer-2 as a target for CRISPR-based control.

The corn planthopper, Peregrinus maidis, is an economically important pest of corn and sorghum. Here we report the initial steps towards developing a CRISPR-based control method, precision guided sterile insect technique (pgSIT), for this hemipteran pest. Specifically, we evaluated the potential of transformer-2 (tra-2) as a target for sterilizing insects. First, we identified tra-2 transcripts within our P. maidis transcriptome database and performed RNA interference (RNAi) to confirm functional conservation. RNAi-mediated knockdown of Pmtra-2 in nymphs transformed females into pseudomales with deformed ovipositors resembling male claspers. While males showed no overt difference in appearance, they were indeed sterile. Importantly, the results were similar to those observed in another planthopper, Nilaparvata lugens. We also used CRISPR/Cas9 genome editing to assess the impact of tra-2 knockout in injectees. CRISPR-mediated knockout of Pmtra-2 had lethal effects on embryos, and hence not many injectees reached adulthood. However, mosaic knockout of Pmtra-2 did impact female and male fertility, which supports the use of tra-2 as a target for pgSIT in this hemipteran species.

The Genome of Arsenophonus sp. and Its Potential Contribution in the Corn Planthopper, Peregrinus maidis.

The co-evolution between symbionts and their insect hosts has led to intricate functional interdependencies. Advances in DNA-sequencing technologies have not only reduced the cost of sequencing but, with the advent of highly accurate long-read methods, have also enabled facile genome assembly even using mixed genomic input, thereby allowing us to more easily assess the contribution of symbionts to their insect hosts. In this study, genomic data recently generated from Peregrinus maidis was used to assemble the genome of a bacterial symbiont, Pm Arsenophonus sp. This ~4.9-Mb assembly is one of the largest Arsenophonus genomes reported to date. The Benchmarking Universal Single-Copy Orthologs (BUSCO) result indicates that this Pm Arsenophonus assembly has a high degree of completeness, with 96% of the single-copy Enterobacterales orthologs found. The identity of the Pm Arsenophonus sp. was further confirmed by phylogenetic analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicates a major contribution by Pm Arsenophonus sp. to the biosynthesis of B vitamins and essential amino acids in P. maidis, where threonine and lysine production is carried out solely by Pm Arsenophonus sp. This study not only provides deeper insights into the evolutionary relationships between symbionts and their insect hosts, but also adds to our understanding of insect biology, potentially guiding the development of novel pest control methods.

Functional analysis of the ATP-binding cassette (ABC) transporter gene family of Tribolium castaneum.

BACKGROUND: The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins that have important physiological functions in all living organisms. Most are integral membrane proteins that transport a broad spectrum of substrates across lipid membranes. In insects, ABC transporters are of special interest because of their role in insecticide resistance. RESULTS: We have identified 73 ABC transporter genes in the genome of T. castaneum, which group into eight subfamilies (ABCA-H). This coleopteran ABC family is significantly larger than those reported for insects in other taxonomic groups. Phylogenetic analysis revealed that this increase is due to gene expansion within a single clade of subfamily ABCC. We performed an RNA interference (RNAi) screen to study the function of ABC transporters during development. In ten cases, injection of double-stranded RNA (dsRNA) into larvae caused developmental phenotypes, which included growth arrest and localized melanization, eye pigmentation defects, abnormal cuticle formation, egg-laying and egg-hatching defects, and mortality due to abortive molting and desiccation. Some of the ABC transporters we studied in closer detail to examine their role in lipid, ecdysteroid and eye pigment transport. CONCLUSIONS: The results from our study provide new insights into the physiological function of ABC transporters in T. castaneum, and may help to establish new target sites for insect control.

An Optimized Small-Scale Rearing System to Support Embryonic Microinjection Protocols for Western Corn Rootworm, Diabrotica virgifera virgifera.

Western corn rootworm (WCR), a major pest of corn, has been reared in laboratories since the 1960s. While established rearing methods are appropriate for maintaining WCR colonies, they are not optimal for performing germline transformation or CRISPR/Cas9-based genome editing. Here we report the development of an optimized rearing system for use in WCR functional genomics research, specifically the development of a system that facilitates the collection of preblastoderm embryos for microinjection as well as gathering large larvae and pupae for downstream phenotypic screening. Further, transgenic-based experiments require stable and well-defined survival rates and the ability to manipulate insects at every life stage. In our system, the WCR life cycle (egg to adult) takes approximately 42 days, with most individuals eclosing between 41 and 45 days post oviposition. Over the course of one year, our overall survival rate was 67%. We used this data to establish a quality control system for more accurately monitoring colony health. Herein, we also offer detailed descriptions for setting up single-pair crosses and conducting phenotypic screens to identify transgenic progeny. This study provides a model for the development of new rearing systems and the establishment of highly controlled processes for specialized purposes.

Genome and Genetic Engineering of the House Cricket (Acheta domesticus): A Resource for Sustainable Agriculture.

Background: The house cricket, Acheta domesticus, is one of the most farmed insects worldwide and the foundation of an emerging industry using insects as a sustainable food source. Edible insects present a promising alternative for protein production amid a plethora of reports on climate change and biodiversity loss largely driven by agriculture. As with other crops, genetic resources are needed to improve crickets for food and other applications. Methods: We present the first high quality annotated genome assembly of A. domesticus from long read data and scaffolded to chromosome level, providing information needed for genetic manipulation. Results: Gene groups related to immunity were annotated and will be useful for improving value to insect farmers. Metagenome scaffolds in the A. domesticus assembly, including Invertebrate Iridescent Virus 6 (IIV6), were submitted as host-associated sequences. We demonstrate both CRISPR/Cas9-mediated knock-in and knock-out of A. domesticus and discuss implications for the food, pharmaceutical, and other industries. RNAi was demonstrated to disrupt the function of the vermilion eye-color gene producing a useful white-eye biomarker phenotype. Conclusions: We are utilizing these data to develop technologies for downstream commercial applications, including more nutritious and disease-resistant crickets, as well as lines producing valuable bioproducts, such as vaccines and antibiotics.

BeetleBase in 2010: revisions to provide comprehensive genomic information for Tribolium castaneum.

BeetleBase (http://www.beetlebase.org) has been updated to provide more comprehensive genomic information for the red flour beetle Tribolium castaneum. The database contains genomic sequence scaffolds mapped to 10 linkage groups (genome assembly release Tcas_3.0), genetic linkage maps, the official gene set, Reference Sequences from NCBI (RefSeq), predicted gene models, ESTs and whole-genome tiling array data representing several developmental stages. The database was reconstructed using the upgraded Generic Model Organism Database (GMOD) modules. The genomic data is stored in a PostgreSQL relatational database using the Chado schema and visualized as tracks in GBrowse. The updated genetic map is visualized using the comparative genetic map viewer CMAP. To enhance the database search capabilities, the BLAST and BLAT search tools have been integrated with the GMOD tools. BeetleBase serves as a long-term repository for Tribolium genomic data, and is compatible with other model organism databases.

The Genome of Rhyzopertha dominica (Fab.) (Coleoptera: Bostrichidae): Adaptation for Success.

The lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), is a major global pest of cereal grains. Infestations are difficult to control as larvae feed inside grain kernels, and many populations are resistant to both contact insecticides and fumigants. We sequenced the genome of R. dominica to identify genes responsible for important biological functions and develop more targeted and efficacious management strategies. The genome was assembled from long read sequencing and long-range scaffolding technologies. The genome assembly is 479.1 Mb, close to the predicted genome size of 480.4 Mb by flow cytometry. This assembly is among the most contiguous beetle assemblies published to date, with 139 scaffolds, an N50 of 53.6 Mb, and L50 of 4, indicating chromosome-scale scaffolds. Predicted genes from biologically relevant groups were manually annotated using transcriptome data from adults and different larval tissues to guide annotation. The expansion of carbohydrase and serine peptidase genes suggest that they combine to enable efficient digestion of cereal proteins. A reduction in the copy number of several detoxification gene families relative to other coleopterans may reflect the low selective pressure on these genes in an insect that spends most of its life feeding internally. Chemoreceptor genes contain elevated numbers of pseudogenes for odorant receptors that also may be related to the recent ontogenetic shift of R. dominica to a diet consisting primarily of stored grains. Analysis of repetitive sequences will further define the evolution of bostrichid beetles compared to other species. The data overall contribute significantly to coleopteran genetic research.

The maternal-effect, selfish genetic element Medea is associated with a composite Tc1 transposon.

Maternal-Effect Dominant Embryonic Arrest ("Medea") factors are selfish nuclear elements that combine maternal-lethal and zygotic-rescue activities to gain a postzygotic survival advantage. We show that Medea(1) activity in Tribolium castaneum is associated with a composite Tc1 transposon inserted just downstream of the neurotransmitter reuptake symporter bloated tubules (blot), whose Drosophila ortholog has both maternal and zygotic functions. The 21.5-kb insertion contains defective copies of elongation initiation factor-3, ATP synthase subunit C, and an RNaseD-related gene, as well as a potentially intact copy of a prokaryotic DUF1703 gene. Sequence comparisons suggest that the current distribution of Medea(1) reflects global emanation after a single transpositional event in recent evolutionary time. The Medea system in Tribolium represents an unusual type of intragenomic conflict and could provide a useful vehicle for driving desirable genes into populations.

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing.

The corn planthopper, Peregrinus maidis, is a pest of maize and a vector of several maize viruses. Previously published methods describe the triggering of RNA interference (RNAi) in P. maidis through microinjection of double-stranded RNAs (dsRNAs) into nymphs and adults. Despite the power of RNAi, phenotypes generated via this technique are transient and lack long-term Mendelian inheritance. Therefore, the P. maidis toolbox needs to be expanded to include functional genomic tools that would enable the production of stable mutant strains, opening the door for researchers to bring new control methods to bear on this economically important pest. However, unlike the dsRNAs used for RNAi, the components used in CRISPR/Cas9-based genome editing and germline transformation do not easily cross cell membranes. As a result, plasmid DNAs, RNAs, and/or proteins must be microinjected into embryos before the embryo cellularizes, making the timing of injection a critical factor for success. To that end, an agarose-based egg-lay method was developed to allow embryos to be harvested from P. maidis females at relatively short intervals. Herein are provided detailed protocols for collecting and microinjecting precellular P. maidis embryos with CRISPR components (Cas9 nuclease that has been complexed with guide RNAs), and results of Cas9-based gene knockout of a P. maidis eye-color gene, white, are presented. Although these protocols describe CRISPR/Cas9-genome editing in P. maidis, they can also be used for producing transgenic P. maidis via germline transformation by simply changing the composition of the injection solution.

Characterization, expression patterns, and transcriptional responses of three core RNA interference pathway genes from Ostrinia nubilalis.

RNA interference (RNAi) is commonly used in the laboratory to analyze gene function, and RNAi-based pest management strategies are now being employed. Unfortunately, RNAi is hindered by inefficient and highly-variable results when different insects are targeted, especially lepidopterans, such as the European corn borer (ECB), Ostrinia nubilalis (Lepidoptera: Crambidae). Previous efforts to achieve RNAi-mediated gene suppression in ECB revealed low RNAi efficiency with both double-stranded RNA (dsRNA) injection and ingestion. One mechanism that can affect RNAi efficiency in insects is the expression and function of core RNAi pathway genes, such as those encoding Argonaut 2 (Ago2), Dicer 2 (Dcr2), and a dsRNA binding protein (R2D2). To determine if deficiencies in these core RNAi pathway genes contribute to low RNAi efficiency in ECB, full-length complementary DNAs encoding OnAgo2, OnDcr2, and OnR2D2 were cloned, sequenced, and characterized. A comparison of domain architecture suggested that all three predicted proteins contained the necessary domains to function. However, a comparison of evolutionary distances revealed potentially important variations in the first RNase III domain of OnDcr2, the double-stranded RNA binding domains of OnR2D2, and both the PAZ and PIWI domains of OnAgo2, which may indicate functional differences in enzymatic activity between species. Expression analysis indicated that transcripts for all three genes were expressed in all developmental stages and tissues investigated. Interestingly, the introduction of non-target dsRNA into ECB second-instar larvae via microinjection did not affect OnAgo2, OnDcr2, or OnR2D2 expression. In contrast, ingestion of the same dsRNAs resulted in upregulation of OnDcr2 but downregulation of OnR2D2. The unexpected transcriptional responses of the core machinery and the divergence in amino-acid sequence between specific domains in each core RNAi protein may possibly contribute to low RNAi efficiency in ECB. Understanding the contributions of different RNAi pathway components is critical to adapting this technology for use in controlling lepidopteran pests that exhibit low RNAi efficiency.