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.

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.

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.

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.

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

Background: 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. Results: We present a robust genome assembly and a gene set possessing 97.5% of the core proteins known from the holometabolous insects. The ATUMI genome encodes fewer enzymes for plant digestion than the genomes of wood-feeding beetles but nonetheless shows signs of broad metabolic plasticity. Gustatory receptors are few in number compared to other beetles, especially receptors with known sensitivity (in other beetles) to bitter substances. In contrast, several gene families implicated in detoxification of insecticides and adaptation to diverse dietary resources show increased copy numbers. The presence and diversity of homologs involved in detoxification differ substantially from the bee hosts of ATUMI. Conclusions: Our results provide new insights into the genomic basis for local adaption and invasiveness in ATUMI and a blueprint for control strategies that target this pest without harming their honey bee hosts. A minimal set of gustatory receptors is consistent with the observation that, once a host colony is invaded, food resources are predictable. Unique detoxification pathways and pathway members can help identify which treatments might control this species even in the presence of honey bees, which are notoriously sensitive to pesticides.

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, 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 RNA interference (RNAi) has proved to be a powerful tool for studying WCR biology, it has its limitations. Specifically, RNAi itself is transient (i.e. does not result in long-term Mendelian inheritance of the associated phenotype), and it requires knowing the DNA sequence of the target gene. The latter can be limiting if the phenotype of interest is controlled by poorly conserved, or even novel genes, because identifying useful targets would be challenging, if not impossible. Therefore, the number of tools in WCR's genomic toolbox should be expanded by the development of methods that could be used to create stable mutant strains and enable sequence-independent surveys of the WCR genome. Herein, we detail the methods used to collect and microinject precellular WCR embryos with nucleic acids. While the protocols described herein are aimed at the creation of transgenic WCR, CRISPR/Cas9-genome editing could also be performed using the same protocols, with the only difference being the composition of the solution injected into the embryos.

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.

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

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 evolving role of the orphan nuclear receptor ftz-f1, a pair-rule segmentation gene.

Segmentation is a critical developmental process that occurs by different mechanisms in diverse taxa. In insects, there are three common modes of embryogenesis-short-, intermediate-, and long-germ development-which differ in the number of segments specified at the blastoderm stage. While genes involved in segmentation have been extensively studied in the long-germ insect Drosophila melanogaster (Dm), it has been found that their expression and function in segmentation in short- and intermediate-germ insects often differ. Drosophila ftz-f1 encodes an orphan nuclear receptor that functions as a maternally expressed pair-rule segmentation gene, responsible for the formation of alternate body segments during Drosophila embryogenesis. Here we investigated the expression and function of ftz-f1 in the short-germ beetle, Tribolium castaneum (Tc). We found that Tc-ftz-f1 is expressed in stripes in Tribolium embryos. These stripes overlap alternate Tc-Engrailed (Tc-En) stripes, indicative of a pair-rule expression pattern. To test whether Tc-ftz-f1 has pair-rule function, we utilized embryonic RNAi, injecting double-stranded RNA corresponding to Tc-ftz-f1 coding or non-coding regions into early Tribolium embryos. Knockdown of Tc-ftz-f1 produced pair-rule segmentation defects, evidenced by loss of expression of alternate En stripes. In addition, a later role for Tc-ftz-f1 in cuticle formation was revealed. These results identify a new pair-rule gene in Tribolium and suggest that its role in segmentation may be shared among holometabolous insects. Interestingly, while Tc-ftz-f1 is expressed in pair-rule stripes, the gene is ubiquitously expressed in Drosophila embryos. Thus, the pair-rule function of ftz-f1 is conserved despite differences in expression patterns of ftz-f1 genes in different lineages. This suggests that ftz-f1 expression changed after the divergence of lineages leading to extant beetles and flies, likely due to differences in cis-regulatory sequences. We propose that the dependence of Dm-Ftz-F1 on interaction with the homeodomain protein Ftz which is expressed in stripes in Drosophila, loosened constraints on Dm-ftz-f1 expression, allowing for ubiquitous expression of this pair-rule gene in Drosophila.

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

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.

Oxidized phospholipid-induced inflammation is mediated by Toll-like receptor 2.

Oxidative tissue damage is a hallmark of many chronic inflammatory diseases. However, the precise mechanisms linking oxidative changes to inflammatory reactions remain unclear. Herein we show that Toll-like receptor 2 (TLR2) translates oxidative tissue damage into inflammatory responses by mediating the effects of oxidized phospholipids. Intraperitoneal injection of oxidized 1-palmitoyl-2-arachidonyl-sn-3-glycerophosphorylcholine (OxPAPC) resulted in upregulation of inflammatory genes in wild-type, but not in TLR2(-/-) mice. In vitro, OxPAPC induced TLR2 (but not TLR4)-dependent inflammatory gene expression and JNK and p38 signaling in macrophages. Induction of TLR2-dependent gene expression required reducible functional groups on sn-2 acyl chains of oxidized phospholipids, as well as serum cofactors. Finally, TLR2(-/-) mice were protected against carbon tetrachloride-induced oxidative tissue damage and inflammation, which was accompanied by accumulation of oxidized phospholipids in livers. Together, our findings demonstrate that TLR2 mediates cellular responses to oxidative tissue damage and they provide new insights into how oxidative stress is linked to acute and chronic inflammation.

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

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.

Development of intestinal M cells.

Intestinal epithelium contains several specialized cell types including M cells, which can be found in the follicle-associated epithelium (FAE) or occasionally on the villi. M cells are critical for sampling of intestinal flora and for transferring pathogens across the epithelial barrier for recognition by the immune system. Development of M cells on the villi (M(v)) is independent of the presence of lymphocytes, while development of the FAE and M cells within the FAE (M(f)) is dependent on B lymphocytes. Here, the concept is discussed that B cells are not required for induction of M(f) differentiation but are required for transition to and maintenance of the mature M(f) phenotype. Signaling pathways possibly involved in the B-cell-independent stages of M-cell development are also discussed.