Microinjection of Western Corn Rootworm, <em>Diabrotica virgifera virgifera</em>, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although ...

Microinjection of Corn Planthopper, <em>Peregrinus maidis</em>, Embryos for CRISPR/Cas9 Genome Editing

The corn planthopper, Peregrinus maidis, is&#160;a pest of maize and a vector of several maize viruses. Previously published methods describe the ...

North Carolina State University

Though initially identified as necessary for neural migration, Disconnected and its partially redundant paralog, Disco-related, are required for proper head segment identity during Drosophila embryogenesis. Here, we present evidence that these genes are also required for proper ventral appendage development during development of the adult fly, where they specify medial to distal appendage development. Cells lacking the disco genes cannot contribute to the medial and distal portions of ventral appendages. Further, ectopic disco transforms dorsal appendages toward ventral fates; in wing discs, the medial and distal leg development pathways are activated. Interestingly, this appendage role is conserved in the red flour beetle, Tribolium (where legs develop during embryogenesis), yet in the beetle we found no evidence for a head segmentation role. The lack of an embryonic head specification role in Tribolium could be interpreted as a loss of the head segmentation function in Tribolium or gain of this function during evolution of flies. However, we suggest an alternative explanation. We propose that the disco genes always function as appendage factors, but their appendage nature is masked during Drosophila embryogenesis due to the reduction of limb fields in the maggot style Drosophila larva.

The appendage role of insect disco genes and possible implications on the evolution of the maggot larval form.

The adult Drosophila melanogaster body develops from imaginal discs, groups of cells set-aside during embryogenesis and expanded in number during larval stages. Specification and development of Drosophila imaginal discs have been studied for many years as models of morphogenesis. These studies are often based on mutations with large developmental effects, mutations that are often lethal in embryos when homozygous. Such forward genetic screens can be limited by factors such as early lethality and genetic redundancy. To identify additional genes and genetic pathways involved in leg imaginal disc development, we employed a Genome Wide Association Study utilizing the natural genetic variation in leg proportionality found in the Drosophila Genetic Reference Panel fly lines. In addition to identifying genes already known to be involved in leg development, we identified several genes involved in pathways that had not previously been linked with leg development. Several of the genes appear to be involved in signaling activities, while others have no known roles at this time. Many of these uncharacterized genes are conserved in mammals, so we can now begin to place these genes into developmental contexts. Interestingly, we identified five genes which, when their function is reduced by RNAi, cause an antenna-to-leg transformation. Our results demonstrate the utility of this approach, integrating the tools of quantitative and molecular genetics to study developmental processes, and provide new insights into the pathways and networks involved in Drosophila leg development.

New components of Drosophila leg development identified through genome wide association studies.

In Drosophila melanogaster, each of the three paralogous ABC transporters, White, Scarlet and Brown, is required for normal pigmentation of the compound eye. We have cloned the three orthologous genes from the beetle Tribolium castaneum. Conceptual translations of Tribolium white (Tcw), scarlet (Tcst), and brown (Tcbw) are 51, 48, and 32% identical to their respective Drosophila counterparts. We have identified loss-of-eye-pigment strains that bear mutations in Tcw and Tcst: the Tcw gene in the ivory (i) strain carries a single-base transversion, which leads to an E → D amino-acid substitution in the highly conserved Walker B motif, while the Tcst gene in the pearl (p) strain has a deletion resulting in incorporation of a premature stop codon. In light of these findings, the mutant strains i and p are herein renamed white(ivory) (w(i)) and scarlet(pearl) (st(p)), respectively. In addition, RNA inhibition of Tcw and Tcst recapitulates the mutant phenotypes, confirming the roles of these genes in normal eye pigmentation, while RNA interference of Tcbw provides further evidence that it has no role in eye pigmentation in Tribolium. We also consider the evolutionary implications of our findings. 

The ABCs of eye color in Tribolium castaneum: orthologs of the Drosophila white, scarlet, and brown Genes.

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.

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

The small hive beetle (Aethina tumida; ATUMI) is an invasive parasite of bee colonies. ATUMI feeds on both fruits and bee nest products, facilitating its spread and increasing its impact on honey bees and other pollinators. We have sequenced and annotated the ATUMI genome, providing the first genomic resources for this species and for the Nitidulidae, a beetle family that is closely related to the extraordinarily species-rich clade of beetles known as the Phytophaga. ATUMI thus provides a contrasting view as a neighbor for one of the most successful known animal groups.

Genome of the small hive beetle (Aethina tumida, Coleoptera: Nitidulidae), a worldwide parasite of social bee colonies, provides insights into detoxification and herbivory.

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.

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

The plant viruses in the phylum , orders  and , 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.

Nathaniel Grubbs

Department of Entomology and Plant Pathology

North Carolina State University

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

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

Nathaniel Grubbs

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Entomology and Plant Pathology

North Carolina State University

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

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

Department of Entomology and Plant Pathology