Final Report for GS14-132
Drosophila suzukii, aka spotted-wing drosophila, is an invasive pest that attacks soft-skinned fruit. D. suzukii's presence posses a substantial risk to the fresh-fruit industry because, unlike most Drosophila species, D. suzukii larvae feed on sound/ripening fruit rather than overripe/rotting fruit. Therefore, the presence of D. suzukii reduces the chance of meeting zero infestation thresholds required for marketing fresh or processed fruit. Moreover, D. suzukii is highly polyphagous and has been recorded feeding on many economically important crops throughout the United States. Since the first US sighting in 2008, D. suzukii has rapidly spread and is now known to be present in 33 states, including several Southern region states. The dramatic expansion of D. suzukii's range has been accompanied by significant crop losses and large increases in pesticide usage. Here we propose to bring a novel eco-friendly control method to bear on sustainable agricultural by producing a conditional sterile-male strain. Unlike traditional Sterile Insect Technique (SIT), our method will utilize the conditional expression of a designer nuclease exclusively within the male testis. Our goal is to generate flies with “toxic sperm” (i.e. sperm that carry a nuclease that targets a highly repetitious sequence within D. suzukii chromosomes). This approach has the potential of being even more powerful than traditional SIT since transgenic sperm may degrade wild-type sperm, thus having an increased impact on population size if D. suzukii mate more than once.
Objective 1: Identify target sequences and test digestion with suitable endonuclease.
We mined the D. suzukii genome sequence for candidate endonuclease sites, as well as sites that could be targeted by guide RNAs using the CRISPR/Cas9 nuclease system. We tested a variety of endonucleases and guide RNAs via microinjection into D. suzukii embryos. Of these, a guide that targeting a highly-repetitious sequence (>100 copies) was the most damaging to D. suzukii chromosomes. Therefore the CRISPR/Cas9 system was selected for use in downstream transgenic constructs.
Objective 2: Design and construction of D. suzukii transformation vectors.
The D. suzukii beta 2-tubulin (Dsb2tub) promoter was identified early on and placed upstream of a red fluorescent marker gene (DsRed). Males carrying this transgene show strong testis-specific DsRed expression (Figure 1). To avoid false positives that could be caused by auto fluorescence, we also visualized DsRed expression using a GFP filter set (see Figure 1B). The GFP filter set results in green, rather than red auto fluorescence, so all reddish/orange fluorescence is due to DsRed expression. As seen in Figure 1B, the cloned Dsb2tub promoter is not only sufficient for driving testis-specific expression of our transgene, but it drives very high levels of transgene expression. Importantly, DsRed expression was never seen in testis from wild-type males (data not shown).
Objective 3: Establish sterile-male D. suzukii strain.
Although we were not able to establish a sterile-male strain, we were able to establish several valuable transgenic D. suzukii strains during the award period. The first of these carries a piggyBac transposase gene driven by the D. melanogaster heat shock 70 protein (hsp70) promoter. We knew from our findings in the red flour beetle (Lorenzen et al. 2007) that microinjecting new piggyBac-based donor constructs into embryos derived from a “helper strain” (i.e. a strain expressing the required transposase) should greatly increase transformation efficiency. And indeed this was the case in D. suzukii as well. Many new piggyBac-base strains have resulted from use of this strain, and we have already shared this important tool with two other NCSU research groups (Dr. Hannah Burrack and Dr. Max Scott). We also generated a donor strain. When used together this helper/donor system can be used for genome-wide mutagenesis thus helping researcher gain a greater understanding of D. suzukii biology. A manuscript describing these strains is in progress and we expect to submit it this fall.
Another important D. suzukii strain resulting from this award is one that carries the Cas9 nuclease gene driven by the D. suzukii polyubiquitin (PUb) promoter. Similar to how endogenous expression of piggyBac transposase increases transformation efficiencies, endogenous expression of Cas9 nuclease increases the efficiency of CRISPR/Cas9 genome editing. Another graduate student in the lab will be microinjecting guide RNAs targeting the transformer (tra) gene in an effort to create a “daughterless strain”. Since tra is not required for male development, males carrying a mutation in tra are expected to be fertile, however, without an intact copy of tra, chromosomal females are expected to develop as “phenotypic males” – i.e. will look and act like males, but should be sterile. Moreover, it is possible to build gene drive into this system. Because fertile males would push the tra mutation into a population, we would expect an imbalance in the male to female ratio to occur over a fairly short period of time. This would result in population suppression due to having too few fertile females. Importantly, we will not be adding gene drive to our tra mutation at this time, rather we will only be testing the system for its ability to produce a “daughterless strain”.
While the above mentioned strains are of immense value, to date neither has enabled us to create a useful strain for use in Genetic Pest Management. The problem, while super interesting, is also extremely frustrating. We do indeed have strains that carry our “effector” genes, however, despite having active marker genes (e.g. eye-specific EGFP expression), the effector genes, despite being in tandem with the marker, are being silenced. We have seen a similar phenomena in other species. For example, transgenes designed to produce hairpin RNAs have also been inexplicitly silenced in the red flour beetle. Our initial assumption was that this was due to our use of a constitutively active promoter. Since this would cause continual activation of the RNAi pathway, we switched to a more restrictive promoter, but that too resulted in transgene silencing. Despite issues caused by silencing of our effector genes, we have established useful D. suzukii strains and are in the process of writing manuscripts that detail creation, use and efficiency of the piggyBac helper strain, and we are currently using the Cas9 strain in ongoing experiments.
Educational & Outreach Activities
Manuscript in progress, draft title = piggyBac transposon remobilization in Drosophila suzukii using helper/donor hybrids.
We expect additional manuscripts to come from this work within the next year or two.
As mentioned above, our piggyBac helper strain has already been shared with two research groups at NCSU - Dr. Hannah Burrack and Dr. Max Scott. Additional labs will very likely request this strain once it has been published.
We also expect our Cas9 strain to be highly sought after, however, we are not sharing this strain until it has been published since we are currently using it to develop a daughterless strain.
Areas needing additional study
- We would like to understand what molecular mechanism causes our effector genes to be silenced.
- Determine if targeting the transformer gene will result in chromosomal females developing as sterile males. It is possible that this type of effector gene will also be silenced since it is not in the evolutionary interest of the organism.