Fig. 3: Phenotypic changes in gene expression, metabolism and ovariole development in the evolved lines.

a UMAP of single-nuclei transcriptomes of stage 5 embryos (see Fig. S6 for details). The colored clusters show yolk nuclei. Wild-type is VK33. The evolved line is population 2-6-1 A at Gen. 20. b Representative marker genes of yolk nuclei. c Representative marker genes of yolk nuclei that were differentially expressed in the evolved line. P-values were based on a two-sided Wilcoxon Rank Sum test followed by Bonferroni correction. d Changes in expression of metabolic genes across cell types between the evolved line and the other two samples. Only significant changes (adjusted p-value < 0.05) are shown. ( + ) Reg., positive regulators; (-) Reg., negative regulators. FC, fold change. Images of marker gene expression in (b) and (d) are from BDGP in situ database⁸⁰. e Enzymatic determination of triglyceride levels in stage 5 embryos (Gen. 50 for population 2-6-1 A). Points represent values from independent homogenates made from 50 embryos each. P values are from a two-sided Student’s t-test. f MALDI-IMS of wild-type (w1118) and evolved (2-6-1 A from Gen. 42) ovaries. Left, middle sections from ovaries employed in MALDI-IMS. Scale bar = 100 μm. Middle, spatial distribution of a representative triglyceride, TG(40:1) at m/z = 715.5846. Right, spatial distribution of a representative glycerophosphocholine, PC(32:1) at m/z = 732.5537. Abundance was normalized to TG(44:3) at m/z = 767.6159 which showed constant levels across all experiments. g Enrichment analysis based on the abundance values for 122 lipids detected through MALDI-IMS (same experiments as f). N = 9 oocytes for wild-type, N = 10 for the evolved line. The vertical solid line indicates a cutoff at FDR q-value of 0.05. Triacylglycerols were highlighted in red. h Ovaries of wild-type (w1118) and evolved (2-6-1 A, Gen. 39) lines, stained with DAPI. The solid red bracket indicates an ovariole, and the dashed red bracket indicates a mature oocyte. Scale bar = 200 um. i Ovariole number and (j) length of mature oocytes of wild-type and the evolved lines (Gen. 39). The horizontal dashed lines represent the mean of all wild-type/evolved lines aggregated (p = 9.783e-09 for ovariole number and p < 2.2e-16 for oocyte length, two-sided Wilcoxon test). N = 8–20 ovaries for ovariole counts. N = 27-72 oocytes for oocyte length. All boxplots in this work are defined as follows: center line, median; box limits, the first and third quartiles; whiskers, 1.5x interquartile range. Source data are provided as a Source Data file.