During the fellowship, the work conducted and the key achievements were as follows:
Selection of candidate genomic regions based on evidences in D. melanogaster
To better identify candidate genes and TE insertions involved in the oxidative stress response in Drosophila suzukii, we first reviewed the literature for evidence of their roles in the model species D. melanogaster. For some candidate genes, we also used CRISPR/Cas9 to generate deletions in D. melanogaster, followed by oxidative stress survival assays and expression analysis.
Based on the literature, we identified Mrp4 and inaE as genes associated with oxidative stress response in D. melanogaster. Furthermore, using CRISPR/Cas9 to delete part of the coding region of Mrp4, we confirmed that mutant flies exhibited reduced Mrp4 expression and increased sensitivity to oral exposure to the oxidative agent paraquat, as expected.
Gene expression analysis in natural D. suzukii populations
To functionally validate candidate genomic regions, we analyzed three natural D. suzukii populations from Japan (S29), France (MT47), and the USA (W120). The S29 strain from Japan carries a TE insertion in the first intron of both Mrp4 and inaE genes. We hypothesized that this TE insertion could alter gene expression and influence the phenotypic response to oxidative stress.
To test this, we measured Mrp4 and inaE expression levels under both control and oxidative stress conditions. Our results showed that flies with the TE insertion exhibited increased expression of both genes, but only under oxidative stress conditions. This suggests that the presence of the TE insertion is associated with transcriptional activation of Mrp4 and inaE in response to oxidative stress.
Allele specific expression in natural D. suzukii populations
To further validate the association between TE insertions in the first intron of Mrp4 and inaE and their increased expression under oxidative stress, we performed allele-specific expression analysis. We generated heterozygous flies carrying one allele with the TE insertion and one without, allowing us to measure Mrp4 and inaE expression separately for each allele.
Our results showed that, for both genes, the allele containing the TE insertion was more highly expressed than the allele without the insertion, in both control and oxidative stress conditions. This suggests that these TEs may act in cis to modulate the expression of nearby genes.
Oxidative stress survival assays in natural D. suzukii populations
To determine whether the increased expression of Mrp4 and inaE has a phenotypic impact on oxidative stress survival, we conducted survival assays using paraquat exposure in three D. suzukii populations: S29 (Japan), MT47 (France), and W120 (USA).
Strikingly, S29 flies, which carry the TE insertions, exhibited significantly reduced survival under paraquat-induced oxidative stress compared to MT47 and W120, which lack the insertion. This suggests an unexpected potential association between the TE insertions and increased susceptibility to oxidative stress.
However, this difference in survival could also be influenced by genetic background effects, as the three strains not only differ in the presence of the TE insertion but also harbor numerous other genomic variations. Further experiments would be necessary to disentangle the specific contribution of the TE insertion from other genetic factors.
Genome editing with CRISPR/Cas9 in D. suzukii
To establish a definitive link between Mrp4 and inaE function and the phenotypic response to oxidative stress—or to demonstrate that TE insertions in their introns enhance their effects—we aimed to use CRISPR/Cas9 genome editing to mutate both genes and remove the TE insertions.
We employed different approaches, including homology-directed repair CRISPR/Cas9, where we tried to introduced a visual marker into the mutated region to facilitate screening, and a co-CRISPR strategy, in which we tried to target both the gene of interest and the white gene responsible for eye pigmentation, using the latter as a visual marker.
Despite applying these strategies to Mrp4, inaE, and their respective TE insertions, the efficiency was too low to generate successful target mutations. However, we did observe mutations in white, suggesting that our approach was technically functional but with low efficiency. To improve success rates, we plan to conduct further experiments using Cas9-expressing flies to enhance editing efficiency.
