Developing Transposon Helper Strains for Two Agriculture Pests; Western Corn Rootworm and Spotted Wing Drosophil

Both western corn rootworm (WCR), Diabrotica virgifera virgifera, and spotted wing drosophila (SWD), Drosophila suzukii, are invasive species that have become important agricultural pests around the world. WCR is a major pest of maize that has invaded every corn-growing region in the US, and has shown a great ability of adapting to different control methods. Unlike WCR, SWD is a recently-introduced invasive species with a wide range of hosts including many important crops, which makes the control of this pest very complicated. A lack of basic molecular research has slowed down not only progress in understanding these two pests, but also the development of novel control methods. The goal of this dissertation is to develop molecular tools using transgenic techniques in these pest species. To do this, I created and established transgenic "helper strains" (i.e. express piggyBac transposase) for both pests. These strains will help in future transposon-based experiments and could open the door for more functional genetic research, or the development of genetic pest management based control methods in the future.;I report the first germline transformation of WCR in chapter 1, with detailed tests of the newly created transgenic strains. The transgenic WCR were produced using a Minos transposable element carrying both a marker gene and a piggyBac transposase gene. During the course of this work we developed a protocol for using digital-droplet PCR to ascertain the number of Minos elements integrated into a beetles' genome. Moreover, I cloned and sequenced at least one side of each insertion-site junction from all single insertion strains, identifying nine unique insertion-sites generated from this experiment. Finally, I confirmed piggyBac transposase expression through reverse-transcription PCR for each of the different strains. These strains will serve as helper strains for future transformation-based experiments.;In Chapter 2, I describe the microinjection protocol for WCR and the rearing system I designed and optimized to fit my experimental needs. Although WCR has been reared in the lab for decades, the efficiency of the standard system is low. In fact, many molecular labs request samples for short-term experiments rather than establishing their own colonies. The optimized rearing system I developed will help in setting up stable, small-scale colonies that require very little space. I also discuss related experimental setups, like single-pair crosses, rearing and screening methods in this chapter.;SWD transgenesis experiments are described in Chapter 3. I created three helper strains of SWD, and tested piggyBac transposase expression levels in each, along with their transformation efficiencies. These results indicate that one strain (H7) is highly efficient, with a transformation rate of 80%. I further tested the H7 strain for its ability to integrate piggyBac elements of different sizes and demonstrated that this strain retains high efficiency, even with large inserts (10kb). Finally, I tested the ability of the H7 strain to remobilize an already integrated piggyBac element by hybridizing H7 with different "Donor strains", and achieved remobilization rates of 6% to 25.6%.;Finally, I conclude my work for this dissertation in the Conclusions Chapter by presenting results from other experiments which were based on the work of preceding chapters, along with some future research directions.