Egg-laying demand induces aversion of UV light in Drosophila females

Abstract

Drosophila melanogaster females are highly selective about the chemosensory quality of their egg-laying sites, an important trait that promotes the survival and fitness of their offspring. How egg-laying females respond to UV light is not known, however. UV is a well-documented phototactic cue for adult Drosophila, but it is an aversive cue for larvae. Here, we show that female flies exhibit UV aversion in response to their egg-laying demand. First, females exhibit egg-laying aversion of UV: they prefer to lay eggs on dark sites when choosing between UV-illuminated and dark sites. Second, they also exhibit movement aversion of UV: positional tracking of single females suggests that egg-laying demand increases their tendency to turn away from UV. Genetic manipulations of the retina suggest that egg-laying and movement aversion of UV are both mediated by the inner (R7) and not the outer (R1-R6) photoreceptors. Finally, we show that the Dm8 amacrine neurons, a synaptic target of R7 photoreceptors and a mediator of UV spectral preference, are dispensable for egg-laying aversion but essential for movement aversion of UV. This study suggests that egg-laying demand can temporarily convert UV into an aversive cue for female Drosophila and that R7 photoreceptors recruit different downstream targets to control different egg-laying-induced behavioral modifications.

Figure 1. Drosophila females prefer to lay eggs away from UV when choosing between a UV-illuminated and a dark site

(A–B) Schematic of the assay we used to test egg-laying preferences of Drosophila. Egg-laying substrates made of 1% agarose are placed at both ends of the egg-laying chamber. A UV LED is placed above one of the two substrates in UV vs. dark egg-laying assays. Grape juice is placed in the middle of the arena to serve as a food source during experiments. See Figure S1 for additional photographs of the experimental setup. (C–D) A representative photograph and egg-laying preference index (PI) of wild type (w¹¹¹⁸) flies when neither substrate is illuminated with UV (dark vs. dark). Egg-laying PI for Canton-S flies in Fig S1. Egg-laying PI is calculated as follows: (N_site1 – N_site2) / (N_site1 + N_site2), where N_site1 and N_site2 represent the numbers of eggs deposited on site1 and site2, respectively. The arrow points to the fly in arena. Egg-laying substrates are outlined in black. Note that females are highly sensitive to the texture/firmness of their egg-laying substrates and rarely lay eggs on the middle hard plastic portion of the chamber. The symbol “&” is used to designate samples that are statistically indifferent from 0 (p > 0.5, one-sample t-test from 0). Error bars represent SEM. Number of animals assayed is labeled above each bar. (E–F) A representative photograph and egg-laying PI of wild type flies when one of the two substrates is illuminated with UV (UV vs. dark). Egg-laying PI is calculated as follows: (N_UV – N_dark) / (N_UV + N_dark), where N_UV and N_dark represent the numbers of eggs on the UV site and the dark site, respectively. p < .0001, one-sample t-test from 0. (G) Egg-laying PI of wild type flies for different UV intensities in UV vs. dark. Gray bar represents the intensity used for Fig 1F and other egg-laying experiments. * p < .05, *** p < .0001, t-test. p < .0001, one-sample t-test from 0. Note that even at the lowest intensity, females still show robust egg-laying aversion of UV. Note that the UV component of sunlight is ~ 25 μW/mm² [34].

Figure 2. Egg-laying demand induces movement aversion of UV

(A) A representative frame of a video where the position of an egg-laying fly is being tracked. Bright spot in the chamber is the UV LED illuminating the substrate from below. Red line that follows the animal is part of the position trajectory generated by Ctrax [18]. The dark specs on the dark site are eggs. (B) Schematic of the parameters we used for analyzing females’ trajectories as they explored and laid eggs in the UV vs. dark chamber. Y-axis denotes y position and x-axis denotes time. Left panel depicts time-spent on the UV site (time_UV) and time-spent on the dark site (time_dark), which were used to calculate the index for relative time spent on UV vs. dark (Positional PI). Right panel depicts a “UV return” and a “dark return” in a trajectory, which were used to calculate the index for relative returns towards UV vs. dark sites (Return PI). (C) Positional PI of virgin (non-egg-laying, average eggs laid = 0) and mated (egg-laying, average eggs laid = 44) flies. Positional PI was calculated as (T_UV – T_Dark)/(T_UV + T_Dark), where T_UV and T_Dark represent times spent on UV vs. dark site, respectively. *** p < .0001, t-test. & p > .05, one-sample t-test from 0. (D) Return PI of virgin (non-egg-laying) and mated (egg-laying) flies. Return PI was calculated as (R_UV – R_Dark)/(R_UV + R_Dark), where R_UV and R_Dark represent the numbers of UV returns and dark returns in a given trajectory, respectively. *** p < .0001, t-test. & p > .05, one-sample t-test from 0. (E) Temporal pattern of egg-laying events (ELEs) from two wild type flies. Blue lines represent individual ELEs. Red bars depicts a 1-hour periods of no egg-laying (0 eggs laid) while green bars depicts a 1-hour periods of high egg-laying (7 or more eggs laid). (F) Return PI for 1-hour periods of no egg-laying (no EL), 1-hour periods of high egg-laying (high EL), and 1 min prior to each ELE in mated flies. *** p < .0001, t-test. (G–H) Representative 30-minute trajectories of a period with no egg-laying (G) and a period with high egg-laying (H). X-axis denotes time and Y-axis denotes the y position of the fly. Purple boxes outline UV returns while black circles outline dark returns. Red arrows point to the stereotypical “rest periods” that follow individual ELEs. Black, vertical lines on trajectories represent the occurrence of an ELE.

Figure 3. R7 photoreceptors are required for egg-laying and movement aversion of UV

(A) Egg-laying PI of structural eye mutants (GMR-hid), phototransduction mutants (norpA³⁶), histamine production mutants (hdc^JK910), and norpA³⁶ mutants with norpA selectively rescued in all photoreceptors (norpA³⁶; GMR>norpA). & p > .05, one-sample t-test from 0. (B) Return PI of norpA³⁶ mutants during periods of no and high EL. & p > .05, one-sample t-test from 0. (C) Egg-laying PI of flies with defective R7 photoreceptor function (sev¹⁴ and R7>TNT) and flies with only R7 photoreceptors functional (norpA³⁶; R7>norpA). *** p < .0001, t-test. One-way ANOVA, Bonferroni post-hoc for R7>TNT. (D) Return PI of flies with defective R7 function (R7>TNT) and flies with only R7 functional (norpA³⁶; R7>norpA). One-way ANOVA, Bonferroni post-hoc for R7>TNT. *** p < .0001, t-test. & p > .05, one-sample t-test from 0. (E) Egg-laying PI of flies with defective R1-6 photoreceptor function (ninaE¹⁷ and Rh1>TNT), flies with only R1-6 photoreceptors functional (norpA³⁶; Rh1>norpA), and flies with both functional R1-6 and R7 photoreceptors (norpA³⁶; Rh1+R7>norpA). *** p < .0001, t-test. One-way ANOVA, Bonferroni post-hoc for Rh1>TNT. (F) Return PI of flies with defective R1-6 function (Rh1>TNT) and flies with only R1-6 functional (norpA³⁶; Rh1>norpA). & p > .05, one-sample t-test from 0.

Figure 4. Dm8 amacrine neurons are required for movement aversion of UV but not egg-laying aversion of UV

(A) Egg-laying PI of flies with their Dm8 amacrine neurons inhibited (vGlut∩ort^C2>TNT), their ort neurons inhibited (ort^C1-4>TNT), or lack one or both histamine receptors (ort¹, ort⁵, and HisCl1¹³⁴,ort¹). ** p < .001, *** p < .0001, t-test. (B) Return PI during periods of high egg-laying (high EL) in flies without functional Dm8 amacrine neurons (vGlut∩ort^C2>TNT). * p < .05, *** p < .0001, t-test. & p > .05, one-sample t-test from 0. (C–D) Representative 30-minute trajectories of a period with no egg-laying (C) and a period with high egg-laying (D) of flies without functional Dm8 neurons. Note that these flies still lay eggs on dark sites, but exhibit frequent UV returns. (E) Model of the roles of R7 and Dm8 neurons in regulating UV-driven behaviors. When flies are not actively laying eggs, they show spectral preference for UV in regular phototaxis experiments and movement attraction towards UV in our paradigm. The R7-Dm8 pathway mediates spectral preference [12, 24], but R7s recruit neurons other than, or in addition to, Dm8s to promote movement attraction towards UV in our paradigm. Once active egg-laying begins, flies show egg-laying aversion of UV when given a choice between UV vs. dark options, and exhibit movement aversion of UV in our paradigm. The R7-Dm8 pathway promotes movement aversion of UV, but is dispensable for promoting egg-laying aversion of UV. There are unidentified pathways that mediate egg-laying aversion of UV.