Development of a Germline Transformation System for the Western Corn Rootworm, diabrotica virgifera virgifera

The western corn rootworm (WCR) is a major pest of maize and is notorious for rapidly adapting biochemically, behaviorally and developmentally to a variety of control methods. Despite much effort, the genetic basis of WCR adaptation remains a mystery. Transformation-based applications such as transposon tagging and enhancer trapping have facilitated the genetic dissection of model species such as Drosophila melanogaster. Following this model, we are developing a genome-wide mutagenesis system for use in WCR. However, we must first establish a germline transformation system for use in this beetle. Here we report results from our first sets of experiments. These include: 1) testing marker genes (EGFP and DsRed); 2) testing heterologous promoters (Tc-alpha-Tubulin and Dm-heat-shock-70); and 3) testing transposable elements (piggyBac and Minos). Since our overall goal is to create a jumpstarter mutagenesis system that is analogous to that used in another coleopteran, the red flour beetle, Tribolium castaneum, we are also using the CRISPR/Cas9 system to: 1) create a white-eyed mutant strain; and 2) specifically target transposon insertions to precise genomic locations (i.e. locations analogous to those known to enable efficient piggyBac remobilization in Tribolium). Establishing transgenic technologies in WCR is the first step towards bringing a wide-range of transformationbased tools to bear on understanding WCR biology, which can then be extended to other rootworm species. Development of a Germline Transformation System for the Western Corn Rootworm, diabrotica virgifera virgifera