Increased auxin exposure in adult Drosophila decreases survival and alters feeding behavior.

(A) Cartoon schematic of auxin exposure feeding protocol. Cartoon was created using BioRender. (B,C) Dose response curves for males and females exposed to increasing concentrations of auxin in the VK00040 transgene injection line (B) or the AID transgenic line (C). (D) The total number of feeding events for females in the VK00040 or AID fly line exposed to 0 mM or 10 mM auxin. (E) The median time of feeding activity of females in the VK00040 or AID fly lines exposed to 0 mM or 10 mM auxin. (F) The total number of events for males in the VK00040 or AID fly lines exposed to 0 mM or 10 mM auxin. (G) The median time of feeding activity of males in the VK00040 or AID fly lines exposed to 0 mM or 10 mM auxin. Data shown as mean ± SEM. *P<0.05, Mann-Whitney U-test.

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Benchmark dose of male and female Drosophila exposed to auxin.

Average feeding events and durations in male and female Drosophila.

Auxin exposure decreases acylcarnitine, fatty acid, and triglyceride levels in adult Drosophila.

(A,B) Box plots illustrating the relative abundance of acylcarnitine or fatty acid metabolites in adult females (A) or males (B) exposed to 0 mM or 10 mM auxin. All box plots were generated using MetaboAnalyst 5.0 as described in the methods. Black dots represent individual samples, the horizontal bar in the middle represents the median, and the purple diamond represents the mean concentration. For all box plots, the metabolite fold-change was >2-fold and P < 0.05. (C,D) TAG contents (mg TAG/mg protein) in whole adult females (C) or males (D) in the VK00040 or AID fly lines exposed to 0 mM or 10 mM auxin. Data shown as mean ± SEM. *P<0.05, **P<0.01, two-tailed Student’s t-test.

Lipid metabolism and detoxification transcripts are significantly altered in adult Drosophila in response to auxin exposure.

(A-D) Volcano plots of differentially expressed genes graphing the statistical significance [-log10(padj)] against the magnitude of differential expression (log2 Fold Change) in females (A,B) or males (C,D) of the VK00040 or AID line. (E) Significantly up-regulated genes involved in “drug metabolism” in adult females or males of the VK00040 or AID lines exposed to 10 mM auxin and compared to the 0 mM control. (F) Significantly up-(purple) and down-regulated (green) genes involved in fatty acid metabolism in adult females or males of the VK00040 or AID lines exposed to 10 mM auxin and compared to the 0 mM control.

Auxin exposure does not significantly influence processes of oogenesis.

(A) Cartoon schematic of the adult Drosophila ovariole showing the anterior germarium followed by developing egg chambers, which consist of 16 germ cells (15 nurse cells and one oocyte; green) that are surrounded by follicle cells (purple). (B) Schematic of the germarium, which contains 2-3 germline stem cells (GSCs, dark green) and somatic cells (gray and purple). Each GSC divides asymmetrically to self-renew and generate a cystoblast that divides to form a 16-cell cyst. Early germline cysts are surrounded by follicle cells (purple) to bud a new egg chamber. GSCs and their progeny are identified based on the position and morphology of the fusome (pink), a germline-specific organelle. (C) Germaria from adult females exposed to 10 mM auxin for 10 days. α-spectrin (magenta), fusome; LamC (red), cap cell nuclear lamina; Vasa (green), germ cells; DAPI (blue), nuclei. GSCs are outlined. Scale bar, 10 µm. (D,E) Average number of GSCs per germarium over time in females exposed to 0 mM or 10 mM auxin in the VK00040 (D) AID (E) lines. Data shown as mean ± SEM. No statistically significant differences, two-way ANOVA with interaction. (F) Germaria from adult females exposed to 10 mM auxin. ApopTag (green), dying cells; DAPI (blue), nuclei). (G,H) Average percentage of germaria containing ApopTag-positive germline cysts in adult females in the VK00040 (G) or AID (H) lines exposed to 0 mM or 10 mM auxin. Data shown as mean ± SEM; Student’s t-test. 100 germaria were analyzed for each genotype and condition. (I) Ovarioles exposed to 10 mM auxin for 10 days showing a healthy ovariole (top) and an ovariole with a dying vitellogenic egg chamber (bottom). Arrowheads point to healthy or dying vitellogenic egg chambers. DAPI (white), nuclei. Scale bar, 100 µm. (J,K) Average percentages of ovarioles containing dying vitellogenic egg chambers in females exposed to 0 mM or 10 mM auxin in the VK00040 (J) or AID (K) lines. Data shown as mean ± SEM, Student’s t-test. 100 ovarioles were analyzed for each genotype and condition.