Stress-induced preference for antioxidants by Drosophila

Abstract

In an ever-changing environment, animals make optimal decisions to ensure their well-being. Faced with limited food sources, they often seek foods that provide the nutrients they require to maintain homeostasis. Under extreme circumstances (e.g., infection), they may exhibit ingestive behaviors, such as seeking out substances (e.g., toxins) that suggest self-medication. Few studies, however, have investigated the mechanisms that ensue self-medication. Here, we report the selective intake of antioxidants by Drosophila melanogaster during a period of heat stress or sleep deprivation. This preference was alleviated by prefeeding them vitamin C or dehydroascorbic acid (DHA) before exposure to stress. Heat stress led to an increase in reactive oxygen species (ROS) levels in the gut, which was alleviated by the intake of vitamin C. Heat stress reduced vitamin C in hemolymph, whereas the consumption of vitamin C or DHA increased it. Furthermore, the intake of vitamin C ameliorated the intestinal barrier dysfunction and extended the survival of flies that had been exposed to chronic heat stress. The heat-induced preference for vitamin C appears to develop independently of the known peripheral chemosensory receptors for this micronutrient. We propose that fruit flies possess an interoceptive mechanism that mediates the detection of vitamin C to overcome environmental challenges.

Keywords: Drosophila; reactive oxygen species (ROS); self-medication; stress-induced attraction to antioxidant; vitamin C.

Fig. 1.

Drosophila develops a preference for antioxidants when exposed to stress. (A) Schematic illustration of the two-choice assay. (B and C) Behavioral preferences for antioxidants in heat-stressed flies. (B) Flies were given a choice between d-glucose and d-glucose with denoted antioxidant (2 mM) after exposure to heat stress (n = 8 to 12). Non: no heat shock, HS: heat shock (used in this and subsequent figures). (C) Development of flies’ behavioral preferences in a two-choice assay conducted at 10, 20, 30, or 120 min after exposure to 1 h heat shock (n = 11 to 12). Filled circles: treated; open circles: untreated (used in this and Fig. 2). (D) The percentage of flies that consumed glucose-containing food only, vitamin C-containing food only, both foods, or none in (C) (n = 290 to 342). (E and F) Sleep profile of male flies with activated (E) GMR11H05+ neurons (n = 46 to 76) or (F) GMR60D04+ neurons (n = 34 to 58), assayed on the 7th day of DAM assay. The white bar represents the light-on phase, and the black bar represents the light-off phase. Quantifications of (E and F) are shown in SI Appendix, Fig. S3 A and B. (G) Two-choice preferences of sleep-deprived male flies between d-glucose and d-glucose with 2 mM vitamin C, assayed on the 7th day (n = 8 to 9). Data are presented as mean ± SD for panels B–D, and G, and mean ± SEM for panels E and F. Statistical analyses were performed as follows: unpaired two-tailed t test for panel B; Welch’s ANOVA with Dunnett’s T3’s post hoc test for panel C; and one-way ANOVA with Tukey’s post hoc test for panel G. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 2.

Heat-induced preference for vitamin C is blunted by prefeeding vitamin C or DHA. (A) Two-choice preferences (d-glucose versus d-glucose with 2 mM vitamin C) of heat-stressed flies that had been prefed with vitamin C (0, 10, 20, and 40 mM) for 4 d (n = 5 to 6). (B) Two-choice preferences for sleep-deprived male flies that had been prefed 10 mM vitamin C, or none for 2 d, assayed on the 7th day (n = 6 to 8). (C) Measurement of antioxidant capacity of 2 mM vitamin C and 2 mM DHA using Trolox equivalent antioxidant capacity (TEAC) assay (n = 3). (D and E) Two-choice preferences of heat-stressed flies (n = 9) (D), and heat-stressed flies that had been prefed 40 mM DHA, or none for 4 d (n = 9) (E). (F and G) LC–MS measurement of vitamin C levels in hemolymph of heat-stressed and control flies (n = 8) (F), and heat-stressed flies that had been prefed 40 mM vitamin C, 40 mM DHA, or none for 4 d (n = 6) (G). Data are presented as mean ± SD. Statistical analyses were performed as follows: one-way ANOVA with Tukey’s post hoc test for panels A, B, and E; Welch’s ANOVA with Dunnett’s T3’s post hoc test for panel G; and unpaired two-tailed t test for panels C, D, and F. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 3.

Vitamin C intake reduces the stress-induced increase in ROS levels of the gut. (A) Representative images of the gut of heat-stressed flies that had been prefed 40 mM vitamin C, 40 mM DHA, or none for 4 d, stained with DAPI (gray) and H₂DCF (green). (B) Quantifications of H₂DCF intensities (n = 19 to 40). (C) Representative images of the gut of sleep-deprived male flies that had been prefed 10 mM vitamin C or none for 2 d, stained with DAPI (gray) and H₂DCF (green) on the 7th day. (D) Quantifications of H₂DCF intensities (n = 10 to 21). Data are presented as mean ± SD One-way ANOVA with Tukey’s post hoc test is used. **P < 0.01; ***P < 0.001; ****P < 0.0001. (Scale bar, 500 μm.)

Fig. 4.

The intake of vitamin C or DHA alleviates gut leakage and extends the survival of flies exposed to chronic heat stress. (A) Proportions of Smurf flies that received heat shock, heat shock with postfeeding of 2 mM vitamin C or DHA, or none for 3 d (n = 15). Flies were subjected to a 30 min heat shock once daily for 4 consecutive days. (B) Survival curves of heat-stressed flies and those with postfeeding of the denoted antioxidant at 2 mM or none (n = 180). (C) Individual plots of (B) (n = 180). Flies were subjected to a 30 min heat shock once daily throughout the course of the survival assay. Data are presented as violin plots representing the full distribution of the data for panels A and C; no error bars are included. Statistical analyses were performed as follows: one-way ANOVA with Tukey’s post hoc test for panel A; Kaplan–Meier with log-rank test (Mantel–Cox) for panel B; and Welch’s ANOVA with Dunnett’s T3’s post hoc test for panel C. *P < 0.05; **P < 0.01; ****P < 0.0001.

Fig. 5.

Heat-induced preference for vitamin C is independent of taste and olfactory inputs. (A) Two-choice preferences of taste receptor (Ir76b², Gr64b^LexA, Gr64c^LexA) mutants and odorant receptor (Orco¹) mutant flies after exposure to heat stress (n = 9). Ir76b² and Gr64b^LexA flies were subjected to a 30 min heat shock, and Gr64c^LexA and Orco¹ were subjected to a 45 min heat shock. (B) PER response of heat-stressed flies to 1 M sucrose, HCl (pH 3.2 to 3.5), and 2 mM vitamin C (n =18). Flies received heat shock for 30 min before conducting the PER assay. (C) The intake amount of HCl (pH 3.2 to 3.5) and 2 mM vitamin C by control flies (n = 20 to 22). (D) The intake amount of HCl (pH 3.2 to 3.5) and 2 mM vitamin C by heat-stressed flies, which had been subjected to a 30 min heat shock before the MAFE assay (n = 19 to 25). Data are presented as mean ± SD Statistical analyses were performed as follows: unpaired two-tailed t test for panels A, C, and D; Welch’s ANOVA with Dunnett’s T3’s post hoc test for panel B. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.