Periodic Reporting for period 1 - GeEpiAdaptation (Identifying genetic and epigenetic changes underlying adaptation in the invasive species Drosophila suzukii)
Okres sprawozdawczy: 2023-02-01 do 2025-01-31
Adaptation is a critical process for the success of biological invasions. These invasions occur when species are introduced to new environments outside their native range and establish themselves permanently. Invasive species can threaten biodiversity and cause economic damage, making it essential to understand the ecological and evolutionary processes behind invasions.
A prominent example of invasive species is Drosophila suzukii, a fruit fly pest that damages fruit crops by laying eggs in unripe fruits. Native to Southeast Asia, it has rapidly invaded North America and Europe, demonstrating impressive adaptability. D. suzukii is thus an excellent model for studying the mechanisms underlying adaptation during invasions.
Phenotypic plasticity, the ability of a genotype to express different phenotypes depending on environmental conditions, is thought to facilitate invasions, especially when genetic variation is low. In D. suzukii, phenotypic plasticity has been studied mainly concerning adaptation to new environmental conditions like temperature changes and nutritional sources. However, little is known about the genetic and epigenetic modifications driving these adaptations.
Recent studies have identified candidate genomic regions related to adaptation, with evidence suggesting transposable elements (TEs) may contribute to rapid adaptation by altering gene structure and expression. Also, tolerance to oxidative stress is linked to longer lifespan, which may be under selection during invasions.
Epigenetic modifications, such as histone changes, can also promote gene expression changes and TE activation. Oxidative stress is known to induce such modifications, and there is evidence suggesting transgenerational epigenetic inheritance may contribute to adaptation.
This project aims to uncover the genetic and epigenetic mechanisms underlying adaptation in D. suzukii through two specific objectives:
1. Functional validation of candidate genes and TEs:
- Use CRISPR/Cas technology to delete candidate genes and TEs in natural D. suzukii populations.
- Establish causal links between these regions and their fitness effects.
2. Transgenerational epigenetic effects of oxidative stress:
- Assess whether oxidative stress induces epigenetic changes passed to subsequent generations.
- Analyze these effects at both the molecular (transcriptomic) and organismal (phenotypic) levels.
- Investigate whether TEs are enriched for these epigenetic modifications.
This comprehensive study will provide valuable insights into the genetic and epigenetic mechanisms promoting the successful adaptation of D. suzukii to new environments. The findings could inform new strategies for controlling invasive species.
A prominent example of invasive species is Drosophila suzukii, a fruit fly pest that damages fruit crops by laying eggs in unripe fruits. Native to Southeast Asia, it has rapidly invaded North America and Europe, demonstrating impressive adaptability. D. suzukii is thus an excellent model for studying the mechanisms underlying adaptation during invasions.
Phenotypic plasticity, the ability of a genotype to express different phenotypes depending on environmental conditions, is thought to facilitate invasions, especially when genetic variation is low. In D. suzukii, phenotypic plasticity has been studied mainly concerning adaptation to new environmental conditions like temperature changes and nutritional sources. However, little is known about the genetic and epigenetic modifications driving these adaptations.
Recent studies have identified candidate genomic regions related to adaptation, with evidence suggesting transposable elements (TEs) may contribute to rapid adaptation by altering gene structure and expression. Also, tolerance to oxidative stress is linked to longer lifespan, which may be under selection during invasions.
Epigenetic modifications, such as histone changes, can also promote gene expression changes and TE activation. Oxidative stress is known to induce such modifications, and there is evidence suggesting transgenerational epigenetic inheritance may contribute to adaptation.
This project aims to uncover the genetic and epigenetic mechanisms underlying adaptation in D. suzukii through two specific objectives:
1. Functional validation of candidate genes and TEs:
- Use CRISPR/Cas technology to delete candidate genes and TEs in natural D. suzukii populations.
- Establish causal links between these regions and their fitness effects.
2. Transgenerational epigenetic effects of oxidative stress:
- Assess whether oxidative stress induces epigenetic changes passed to subsequent generations.
- Analyze these effects at both the molecular (transcriptomic) and organismal (phenotypic) levels.
- Investigate whether TEs are enriched for these epigenetic modifications.
This comprehensive study will provide valuable insights into the genetic and epigenetic mechanisms promoting the successful adaptation of D. suzukii to new environments. The findings could inform new strategies for controlling invasive species.
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.
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.
Our results provide the first evidence of the role of TE insertions in the stress response of the crop pest D. suzukii. Specifically, we demonstrated that two TE insertions alter the expression of nearby genes when flies are exposed to oxidative stress, highlighting their potential regulatory impact.
From a methodological perspective, we also found that using plasmids to deliver gRNAs and Cas9 protein for CRISPR/Cas9 genome editing in natural D. suzukii populations results in very low efficiency. This underscores the need for alternative approaches, such as developing D. suzukii lines that constitutively express Cas9 or optimizing more efficient genome editing tools.
Establishing a robust CRISPR/Cas9 system in D. suzukii is crucial for advancing functional genomics research and understanding adaptive processes. This, in turn, could contribute to the development of novel strategies for managing and controlling invasive pest species.
From a methodological perspective, we also found that using plasmids to deliver gRNAs and Cas9 protein for CRISPR/Cas9 genome editing in natural D. suzukii populations results in very low efficiency. This underscores the need for alternative approaches, such as developing D. suzukii lines that constitutively express Cas9 or optimizing more efficient genome editing tools.
Establishing a robust CRISPR/Cas9 system in D. suzukii is crucial for advancing functional genomics research and understanding adaptive processes. This, in turn, could contribute to the development of novel strategies for managing and controlling invasive pest species.