Experimental evolution of D. simulans with increased OA resistance.

A Phylogeny of the drosophilid trio studied in this work. Mya: million years ago. B Schematic of octanoic acid (OA) resistance tube assay (see Materials and Methods) and selection procedure. This panel was created using BioRender.com. C Species differences in resistance of D. simulans 04 and D. sechellia 07 strains to 3 μl OA. D Schematic illustrating the generation of the base populations for Evolve-and-Resequence (see Materials and Methods). This panel was created using BioRender.com. E Changes in survivability to 3 μl OA of the ten D. simulans populations over generations 0-25. Box plots represent the interquartile ranges of the survivability for all the replicates; blue lines shows the generalized additive model (GAM) regression; grey shading represents the 95% confidence interval for the mean responses at each time point. F As in E for the 10 populations combined. G OA resistance for the combined 10 populations with increasing OA volumes during generation 25-31. H As in E, with 9 μl OA during generations 31-50. I As in H for the 10 populations combined. J Comparison of OA resistance of unevolved D. simulans (i.e., the original 10 outcrossed populations maintained on standard fly food; see Materials and Methods), evolved (generation 50 (G50)) D. simulans, and D. sechellia. K-N Phenotypic comparison of unevolved and evolved (G50+) D. simulans populations 1-3 for K noni resistance (G54), L fecundity (G54), M developmental viability (G57), and N lifespan (G57). For K-M, significance was assessed using the unpaired t-test correcting for multiple testing. NS p>0.05, ***p<0.001. For L-N, tests were run on standard media. No significant differences between unevolved and evolved flies were detected using the Cox regression model. See Materials and Methods for details and Data S1 for raw data.

Dynamic directional selection of allele frequencies during experimental evolution.

A Schematic of the analysis. B Identity-by-state analysis. The heatmap depicts the proportion of shared alleles between populations, with values ranging from 0.8 (blue, lowest shared proportion) to 1 (red, identical alleles). C,D Manhattan plot showing the probability of allele frequency changes between C generations 0 and 25, and D generations 0 and 50. Top panel shows the results of the CMH test; orange dots indicate significant variants at a threshold of -log10(p) ≥40, a conservative approximation of the top 0.000005% simulated SNPs under neutrality (Fig. S1B). Bottom panel shows the absolute log-Bayes factors estimated by Bait-ER; orange dots indicate significant variants at the conservative threshold log(0.99/0.01). Raw data in Data S2. E Circular plots illustrating overlap of predicted genomic regions under directional selection between the two statistical approaches at generation 25 (left) and generation 50 (middle), or between generation 25 and 50 (right). Raw data in Data S3.

A genome-wide pooled CRISPR screen for OA susceptibility and resistance genes in D. melanogaster cultured cells.

A Left: images of S2R+ cells cultured in the absence or presence of OA. Scale bar, 50 μm. Right: dose-dependent effects of OA on S2R+ cell viability, through measurement of ATP levels with a CellTiter-Glo assay (see Materials and Methods). B Schematic of CRISPR screen design. C Top 5% “susceptibility” genes (see Materials and Methods and Data S4). Box plots represent the interquartile ranges for all the replicates of the Z-scores obtained through maximum likelihood estimation to test for selection. The inset (bottom-right) displays the gene-ontology analysis of these genes. D As in C, for the top 5% “resistance” genes. E Venn diagram showing the overlap between all the genes identified in the experimental evolution at G25 and G50, and genes associated with susceptibility and resistance in the CRISPR screen. F Subportion of the top panel (CMH test) of the Manhattan plot from Fig. 2C for chromosome arms 2L and 3L, with the red arrows highlighting the regions magnified on the right showing the significant variants and linkage disequilibrium blocks (triangles) for kraken and Alkbh7 (CG14130).

Sequence and expression analyses of kraken and Alkbh7.

A AlphaFold protein models for D. melanogaster Kraken (AF-O18391-F1-model_v4) and Alkbh7 (AF-Q9VTP1-F1-model_v4) (Jumper, et al. 2021; Varadi, et al. 2022), highlighting a conserved putative catalytic serine in the active site in Kraken, and the mitochondrial targeting sequence in Alkbh7 (predicted by MitoFates (Fukasawa, et al. 2015)). See also Fig. S2A,B. B Tissue-specific expression of D. melanogaster kraken and Alkbh7 from bulk RNA-sequencing data from the Fly Atlas 2.0 (Krause, et al. 2022). C tSNE plots illustrating D. melanogaster kraken expression in single-cell transcriptomes of the Malpighian tubules and gut from the Fly Cell Atlas (Stringent 10x datasets) (Li, et al. 2022). D Left: RNAscope detection of kraken (green) transcripts in the gut and Malpighian tubules, with nuclei counterstained with DAPI (blue). Right: higher-magnification images showing kraken transcript expression in the indicated tissues. Scale bars, 200 μm. This expression pattern was observed in tissues from >20 individuals. E Expression levels of the kraken and Alkbh7 in the indicated laboratory strains (left panel) and wild caught strains (right panel) of adult female D. sechellia and D. simulans measured by qPCR (Data S6). Expression is represented as calibrated and normalized relative quantities (CNRQ). Significance was assessed using the unpaired t-test correcting for multiple testing and represented using letter codes. F Expression levels of kraken and Alkbh7 in the indicated strains and species at larval or adult stages as indicated from published RNA-seq data. (References: 1, {Watanabe, 2019 #6929}, 2 {Ma, 2018 #8721}, 3 {Kalra, 2024 #10088}). Significance was assessed using the unpaired t-test correcting for multiple testing and represented using letter codes when multiple replicates were present in the original data. G Expression levels of the candidate genes at generation 0, 25 and 50 of the experimentally-evolved D. simulans populations measured by qPCR (Data S6). Significance was assessed using the paired t-test correcting for multiple testing and represented using letter codes.

Functional validation of the contribution of kraken and Alkbh7 to OA resistance.

A-C Survival curves of adult flies of the indicated genotypes tested in the plate assay with 2 μl OA for kraken and Alkbh7 RNAi (A), mutants (B) and overexpression (C). Each replicate consisted of 10 2-7 day-old female flies. N replicates ≥6, n flies ≥60. Shading indicates 95% confidence intervals. Genotypes: A act-Gal4/+;UAS-krakenRNAi/+, UAS-krakenRNAi/+, act-Gal4/+, act-Gal4/+;UAS-Alkbh7RNAi/+, UAS-Alkbh7RNAi/+, act-Gal4/+ B kraken1, Alkbh71, Canton-S (wild-type genetic background control for both mutants), C act-Gal4/+;UAS-krakeno/e/+, UAS-krakeno/e/+, act-Gal4/+, act-Gal4/+;UAS-Alkbh7o/e/+, UAS-Alkbh7o/e/+, act-Gal4/+. Significance is based on the resulting mixed effect Cox regression model. NS p>0.05, *p<0.05, **p<0.01, ***p<0.001. The same data for D. sechellia are shown in both plots in C. D Survival curves of wild-type (Dsec07), and kraken (Dsec\krakenRFP) and Alkbh7 (Dsec\Alkbh7RFP) mutant D. sechellia in the plate assay with 30 μl OA. Statistics as in A. E Long-term survivability of the same genotypes as in D (n = 60 per condition) in control conditions (no OA) (left) and food supplemented with 0.9% OA (right). Statistics as in A. Raw data in Data S7.

Drosophila strains.

Effective population size estimates from the experimentally-evolved populations.

qPCR primer efficiency.