Report. Egg-Laying Demand Induces Aversion of UV Light in Drosophila Females

Transcription

1 Current Biology 24, , December 1, 2014 ª2014 Elsevier Ltd All rights reserved Egg-Laying Demand Induces Aversion of UV Light in Drosophila Females Report Edward Y. Zhu, 1 Ananya R. Guntur, 2 Ruo He, 2 Ulrich Stern, 3 and Chung-Hui Yang 2, * 1 Department of Pharmacology and Cancer Biology 2 Department of Neurobiology Duke University Medical Center, Durham, NC 27710, USA 3 Durham, NC 27705, USA Summary Drosophila melanogaster females are highly selective about the chemosensory quality of their egg-laying sites [1 6], 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 [7 13], but it is an aversive cue for larvae [14 17]. 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 [12], 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. Results Drosophila Females Avoid Laying Eggs on UV Sites in a UV versus Dark Paradigm To investigate how egg-laying females respond to UV, we first examined their egg-laying preference between UV-illuminated and nonilluminated (dark) substrates. We constructed a highthroughput apparatus that houses 30 chambers (Figures S1A and S1B available online). Each chamber contains two egglaying substrates, one of which can be illuminated by a lightemitting diode (LED) from above (Figures 1A, 1B, S1C, and S1D). Females show no preference when neither substrate is illuminated (Figures 1C and 1D), but they prefer to lay eggs on dark sites when one of the two substrates is illuminated with UV (Figures 1E 1G and S1E S1G). They also prefer to lay eggs away from blue, green, and white light (Figures S1H and S1I), but are most sensitive to UV, and will lay eggs away from UV when choosing between UV versus white-illuminated substrates (Figures S1J and S1L). These results suggest that females prefer to deposit eggs away from UV. However, UV does not inhibit egg laying in general, as females lay *Correspondence: yang@neuro.duke.edu comparable amounts of eggs in UV-only and dark-only conditions (Figures S1K and S1M). Egg-Laying Demand Induces Movement Aversion of UV in a UV versus Dark Paradigm Given that Drosophila prefer to lay eggs away from UV but are generally phototatic toward UV, we hypothesized that egglaying demand may temporarily cause females to avoid UV. To test this hypothesis, we recorded individual females as they explored and laid eggs in the UV versus dark paradigm for 8 hr (Figures 2A and S2A S2C). We then manually annotated the times of individual egg-laying events (ELEs) and tracked the position of each female using a modified version of the open-source tracking software Ctrax (Figures 2A, S2D, and S2E) [19, 20]. To examine whether egg-laying demand triggers UV aversion, we first analyzed the relative amount of time spent on UV versus dark sites (Figure 2B, left panel). We found that (egg-laying) mated females spend less time on UV sites compared to (non-egg-laying) virgin females (Figure 2C), suggesting that increased egg laying correlates with emergence of positional aversion of UV. However, after each egg laying, females often rest on the substrate where an egg was laid (Figure 2H, arrows) [5]. Therefore, relative time spent is not the best measurement for active aversion of UV prior to egg laying, as resting on dark after egg laying will increase time spent on dark. To overcome this problem, we reexamined recorded trajectories, and we found instances where females would reverse moving direction when they are in the middle portion of the chamber. Specifically, females would often leave and return to the UV site (UV return) without visiting the dark site (Figure 2B, right panel). Similarly, females would leave and return to the dark site (dark return) without visiting the UV site (Figure 2B, right panel). We propose that these returns reflect a decision to change direction of movement and that UV returns and dark returns are indicative of UV attraction and aversion, respectively. Therefore, the relative occurrences of these two returns can be used to assess whether a female is exhibiting UV attraction or aversion in a given time period. We next analyzed the return preference index of (non-egglaying) virgins and (egg-laying) mated females. We found that virgins show UV attraction, whereas mated females show neither attraction nor aversion of UV (Figure 2D). The lack of UV attraction by mated females suggests that egg laying may increase the number of dark returns they execute. To test this idea, we compared the return index of mated flies when they are versus are not laying eggs. Because the temporal pattern of ELEs was unevenly distributed during each 8 hr video, we identified many hour-long periods with no egg laying, as well as hour-long periods with high egg laying (Figure 2E). We found that during periods of no egg laying, mated females exhibit UV attraction (Figures 2F and 2G) but that they switch to exhibit UV aversion during high-egg-laying periods (Figures 2F and 2H). Moreover, analysis of the return index in the minute immediately before each ELE suggests that UV aversion is present prior to egg deposition (Figure 2F). Together, these results suggest that egg-laying demand turns UV into an aversive cue for Drosophila. We show that

2 Current Biology Vol 24 No Figure 1. Drosophila Females Prefer to Lay Eggs away from UV When Choosing between a UV-Illuminated and a Dark Site (A and B) Schematic of the assay that we used to test egg-laying preferences of Drosophila. Egglaying 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 versus dark egg-laying assays. Grape juice is placed in the middle of the arena to serve as a food source during experiments. Top view (A) and side view (B) are shown. See Figure S1 for additional photographs of the experimental setup. (C and D) A representative photograph (C) and egg-laying preference index (PI; D) of wild-type (w 1118 ) flies when neither substrate is illuminated with UV (dark versus dark). Egg-laying PI for Canton-S flies is shown in Figure 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 site 1 and site 2, respectively. The arrow points to the fly in the arena. Egg-laying substrates are outlined in black. Note that females are highly sensitive to the degree of firmness of their egg-laying substrates and rarely lay eggs on the middle hardplastic portion of the chamber. The ampersand designates samples that are statistically indifferent from 0 (p > 0.5, one-sample t test from 0). All error bars in this work represent the SEM. The number of animals assayed is labeled above each bar. (E and F) A representative photograph (E) and egg-laying PI (F) of wild-type flies when one of the two substrates is illuminated with UV (UV versus 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 < , one-sample t test from 0. (G) Egg-laying PI of wild-type flies for different UV intensities in UV versus dark. The gray bar represents the intensity used for (F) and other egglaying experiments. *p < 0.05, ***p < , t test. p < , 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 w25 mw/mm 2 [18]. See also Figure S1. females (1) avoid laying eggs on UV sites and (2) tend to turn away from UV sites prior to egg laying. The rest of this report will refer to the former as egg-laying aversion of UV and the latter as movement aversion of UV. R7 Photoreceptors Are Required for Egg-Laying and Movement Aversion of UV Next, we started to investigate the circuit mechanism that regulates egg-laying and movement aversion of UV. We first examined egg-laying preferences of blind mutants. Mutants that lack physical eyes (GMR-hid [21]), the phototransduction molecule phospholipase C (norpa 36 [22]), or the ability to produce histamine (hdc JK910 [23]) the neurotransmitter released by photoreceptors (PRs) all no longer show egg-laying aversion of UV (Figure 3A and S3A). Moreover, restoration of norpa to the PRs of norpa 36 mutants using the GAL4/UAS system [24] restores egg-laying aversion of UV (Figure 3A). To test whether vision is also required for movement aversion of UV, we analyzed the trajectories of norpa 36 mutants, and we found that these mutants do not exhibit either attraction or aversion of UV regardless of their egg-laying state (Figure 3B). Collectively, our data suggest that visual input is required for both egg-laying and movement aversion of UV. We next sought to identify the PRs that regulate egg-laying and movement aversion of UV. The PRs of the Drosophila eye can be divided into two anatomically and functionally distinct groups: the inner (R7 and R8) and the outer (R1 R6) PRs [9]. R7 and R1 R6 both express UV-sensitive rhodopsins (Rh3 and Rh4 in R7 and Rh1 in R1 R6 [9]). Moreover, R7s mediate UV spectral preference, whereas R1 R6 s promote UV attraction during fast phototaxis [8, 10, 12, 25]. Thus, both groups of PRs could contribute to UV aversion. We examined the roles of R7s and R1 R6s in regulating egg-laying aversion of UV. Removal of R7s (sev 14 )[10, 26] and inhibition of their output (R7-GAL4 driving the synaptic inhibitor UAS-TNT [27] or the hyperpolarizing UAS-Kir2.1 [28]) both reduce egg-laying aversion, whereas selective rescue of norpa in R7s in norpa 36 mutants restores egg-laying

3 Egg Laying Induces UV Aversion in Drosophila 2799 aversion (Figures 3C and S3B), suggesting that R7s are necessary and sufficient for egg-laying aversion of UV. In contrast, removal of R1 R6 function (ninae 17 [29]) or inhibition of their output (Rh1-GAL4 driving UAS-TNT or UAS-Kir2.1) does not reduce egg-laying aversion of UV (Figures 3E and S3C). Curiously, selective rescue of norpa in R1 R6s results in females preferring UV sites for egg laying (Figure 3E). This suggests that R1 R6s promote egg-laying attraction of UV but that such promotion is suppressed in the presence of functional R7s, as both wild-types and animals with norpa rescued in both R7s and R1 R6s exhibit egg-laying aversion of UV (Figure 3E). We next assessed the roles of R7s and R1 R6s in movement aversion of UV. We found that inhibition of R7s eliminates movement aversion of UV during high-egg-laying periods, whereas rescue of norpa function in R7s in norpa 36 mutants restores it (Figure 3D). In contrast, inhibition of R1 R6s does not impair movement aversion during highegg-laying periods, and rescue of norpa in R1 R6s in norpa 36 mutants fails to rescue it (Figure 3F). It has been suggested that TNT may not be effective in blocking R1 R6 function [30]; we also analyzed Rh1>Kir2.1 flies, and we found that they did not show significant impairment of movement aversion either (Figure S3F). Together, our results suggest that egg-laying and movement aversion of UV are mediated by R7 and not R1 R6 PRs. It is interesting that although inhibition of R7s or R1 R6s did not reduce movement attraction of UV during no-egg-laying periods (Figure S4A), rescue experiments suggest that both PRs might have a role in promoting it. First, selective rescue of R7s is sufficient for movement attraction during no-egglaying periods (Figure 3D). Moreover, selective rescue of R1 R6s leads to more time spent on UV during both noand high-egg-laying periods (Figure S3E). This constitutive positional UV preference suggests that R1 R6s do promote some UV attraction (although it is not evident in our return analysis). Together, these data hint at the possibility that R7 and R1 R6 might act in parallel to promote movement attraction for UV when females are not laying eggs. Dm8 Neurons Are Required For Movement Aversion, but Not Egg-Laying Aversion, of UV We next assessed how egg-laying and movement aversion of UV are regulated by circuit components downstream of R7s. We focused on examining the role of Dm8 amacrine neurons as they are synaptic targets of R7s and each can pool information from multiple R7 PRs [12, 25]. Moreover, they mediate UV attraction during spectral preference assays [12, 25]. To test the role of Dm8s in UV aversion, we used a split-gal4 combination (ort C2 XvGlut) to specifically inhibit their output (Figure S4E) [12]. Inhibition of Dm8s does not reduce egg-laying aversion of UV (Figure 4A). This is corroborated by the results that animals with their ort C1-4 -GAL4 neurons inhibited or lack one (ort 1 or ort 5 ) or both (HisCl1 134, ort 1 ) histamine receptors still exhibit egg-laying aversion of UV (Figure 4A). (Dm8 neurons express ort and are labeled by ort C1-4 -GAL4 [12].) These results suggest Dm8s are not essential for egg-laying aversion of UV. However, females with inhibited Dm8s no longer exhibit movement aversion of UV during high-egg-laying periods (Figure 4B). Interestingly, they still show movement attraction during periods of no egg laying (Figure S4A). Inspection of their trajectories (Figures 4C and 4D) reveals that although Dm8-inhibited females lay eggs away from UV, they continue to exhibit UV returns during high-egg-laying periods (Figure 4D). In contrast, wild-type animals exhibit mostly dark returns during high-egg-laying periods (Figure 2H). This result suggests that movement aversion is not causal for egg-laying aversion of UV. Instead, egg-laying and movement aversion of UV are under separate circuit control downstream of R7s, as the former does not require Dm8s, whereas the latter does. Discussion In this report, we show that egg-laying demand induces UV aversion in female Drosophila. Egg-laying females avoid laying eggs on UV sites and tend to turn away from UV during highegg-laying periods. Our results suggest that the R7, and not the R1 R6, PRs regulate both features of UV aversion and that R7s recruit different second-order neurons to promote egg-laying versus movement aversion of UV (Figure 4E). Although UV attraction is thought to contribute to the openspace response [31] and is perhaps an advantageous trait in general, we propose that avoiding UV during egg laying may protect eggs and larvae from exposure to temperature extremes [32] and reduce the predation risk of egg-laying females. Are R7s the only PRs that mediate UV aversion? While inhibition of R7s abolishes movement aversion of UV, it only partially reduces egg-laying aversion of UV. One possibility is that the motor program that controls egg-laying preference is more sensitive to UV, and our manipulations left a few functional R7s that are sufficient to drive some egg-laying aversion. Alternatively, R8s may contribute to egg-laying aversion of UV, as animals with norpa function restored in R8s do exhibit minor egg-laying aversion of UV (Figure S3D). What are the second-order neurons downstream of R7s that mediate egg-laying and movement aversion of UV? Our results suggest these two aversions are controlled by separate second-order neurons, as Dm8s are required for movement, but not egg-laying, aversion of UV. Given that Dm8s promote UV spectral preference [10, 12, 25], we suspect that they control the motor programs that orient the flies during UV encounters (i.e., promote movement away from UV during egg-laying and toward UV during spectral preference). Because females still visit the UV site during high-egg-laying periods (see the trajectories in Figures 2H and 4D), there must be mechanism in place that prevents females from laying eggs during these UV visits. Recent reports have identified additional targets of R7s, including Mi9, Dm2 [33], Rh3TmY, and Rh4TmY [34]; each could contribute to egg-laying aversion of UV. Moreover, because egg-laying aversion requires histamine production and HisCl1 134,ort 1 double mutants still exhibit significant aversion, we hypothesize that the second-order neurons that regulates egg-laying aversion of UV may express an unidentified histamine receptor. What mediates movement attraction toward UV when females are not laying eggs? We hypothesize that R7s and R1 R6s may act in a redundant manner to promote UV attraction in our paradigm, in keeping with earlier reports that show that both PRs can promote UV attraction [8, 10, 12]. Interestingly, Dm8s, the critical mediator for spectral UV preference, are dispensable for movement attraction of UV. However, it is worth noting that spectral preference assesses whether flies move toward UV or green light on the order of minutes, whereas our paradigm assesses how often females turn toward or away from UV during the course of 8 hr. Moreover,

4 Current Biology Vol 24 No 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. The bright spot in the chamber is the UV LED illuminating the substrate from below. The red line that follows the animal is part of the position trajectory generated by Ctrax [19]. 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 versus dark chamber. The y axis denotes the y position, and the x axis denotes time. The 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 versus dark (positional PI). The right panel depicts a UV return and a dark return in a trajectory, which were used to calculate the index for relative returns toward UV versus 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 versus dark site, respectively. ***p < , t test. & p > 0.05, 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 (legend continued on next page)

5 Egg Laying Induces UV Aversion in Drosophila 2801 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 (norpa 36 ), histamine production mutants (hdc JK910 ), and norpa 36 mutants with norpa selectively rescued in all PRs (norpa 36 ; GMR>norpA). & p > 0.05, one-sample t test from 0. (B) Return PI of norpa 36 mutants during periods of no and high egg laying. & p > 0.05, one-sample t test from 0. (C) Egg-laying PI of flies with defective R7 PR function (sev 14 and R7>TNT) and flies with only R7 PRs functional (norpa 36 ; R7>norpA). ***p < , 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 (norpa 36 ; R7>norpA). One-way ANOVA, Bonferroni post hoc for R7>TNT. ***p < , t test. & p > 0.05, one-sample t test from 0. (E) Egg-laying PI of flies with defective R1 R6 PR function (ninae 17 and Rh1>TNT), flies with only R1-R6 PRs functional (norpa 36 ; Rh1>norpA), and flies with both functional R1 R6 and R7 PRs (norpa 36 ; Rh1+R7>norpA). ***p < , t test. One-way ANOVA, Bonferroni post hoc for Rh1>TNT. (F) Return PI of flies with defective R1-R6 function (Rh1>TNT) and flies with only R1-R6 functional (norpa 36 ; Rh1>norpA). & p > 0.05, one-sample t test from 0. See also Figures S3 and S4. our UV intensity is significantly stronger than the intensity typically used for spectral preference (even though it is much less compared to sunlight) [12, 18, 25]. Because Dm8s are required for movement aversion and not movement attraction of UV, this suggests that there are other second-order neurons that orient flies toward UV and that movement aversion and attraction in our paradigm are controlled by different neurons. the numbers of UV returns and dark returns in a given trajectory, respectively. ***p < , t test. & p > 0.05, one-sample t test from 0. (E) Temporal pattern of ELEs from two wild-type flies. Blue lines represent individual ELEs. Red bars depicts a 1 hr period of no egg laying (zero eggs laid), whereas green bars depicts a 1 hr period of high egg laying (seven or more eggs laid). (F) Return PI for 1 hr periods of no egg laying (no EL), 1 hr periods of high egg laying (high EL), and 1 min prior to each ELE in mated flies. ***p < , t test. (G and H) Representative 30 min trajectories of a period with no egg laying (G) and a period with high egg laying (H). The x axis denotes time, and the y axis denotes the y position of the fly. Purple boxes outline UV returns, whereas 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. See also Figure S2.

6 Current Biology Vol 24 No 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 (vglutxort C2 > TNT), with their ort neurons inhibited (ort C1-4 > TNT), or lacking one or both histamine receptors (ort 1, ort 5, and HisCl1 134,ort 1 ). **p < 0.001, ***p < , t test. (B) Return PI during periods of high egg laying (high EL) in flies without functional Dm8 amacrine neurons (vglutxort C2 > TNT). *p < 0.05, ***p < , t test. & p > 0.05, one-sample t test from 0. (C and D) Representative 30 min 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 toward UV in our paradigm. The R7-Dm8 pathway mediates spectral preference [12, 25], but R7s (legend continued on next page)

7 Egg Laying Induces UV Aversion in Drosophila 2803 In conclusion, our results suggest that the Drosophila visual system contains several parallel UV-processing pathways, some of which may be preconfigured to direct specific actions (Figure 4E). Such circuit design highlights the various influences UV has on Drosophila behaviors and perhaps allows different contexts and reproductive states to bias action selection during early stages of sensory processing. Experimental Procedures Details of the UV setup, behavioral analysis, and procedures are described in the Supplemental Experimental Procedures. Fly Stocks The following fly stocks were used: CS WU, w 1118, GMR-hid [21], norpa 36 (BL 9048), hdc JK910 [23], GMR-GAL4 (BL 8605), UAS-norpA (BL 26267), sev 14 (BL 5691), R7-GAL4 (BL 8604), UAS-TNT (BL 28838), ninae 17 (BL 5701), Rh1-GAL4 (BL 8691), ort 1 [12], ort 5 (BL 29637), HisCl1 134,ort 1 [12], ort C1-4 -GAL4 [12], vglut-dvp16ad;ort C2 -GAL4DBD [12], norpa 36 ;Rh5- norpa (gift from Chi-Hon Lee), and UAS-Kir2.1 [28]. Behavior Assays Egg-Laying Preference A total of newly eclosed females were collected together with males in yeasted vials for 4 5 days before being assayed. Thirty minutes before the experiment, we loaded 1% agarose into the bottom plate and flies into the top portion of the apparatus (Figure S1). To begin the experiment, we assembled the two parts of the apparatus and allowed flies to lay eggs overnight. The next morning, we took pictures of the bottom plate and manually counted the eggs laid. Positional and Return Index Preference Flies were handled as just described. However, for these experiments, we used an inverted setup in which UV is illuminated from below so that cameras can be placed on top of the apparatus (Figure S2). After 8 hr of recording, we manually annotated each egg-laying event in the videos, tracked females positions using a modified Ctrax [19, 20], and analyzed time spent on/off UV and movement attraction/aversion of UV using custom MATLAB code. Supplemental Information Supplemental Information includes Supplemental Experimental Procedures, four figures, and one movie and can be found with this article online at Acknowledgments We thank Julie Simpson, Barry Dickson, Craig Montell, Chi-Hon Lee, Claude Desplan, and Hugo Bellen for sharing fly stocks and Tom Clandinin for advice on some of the behavioral analysis. We thank the Duke Physics shop, in particular Phil Lewis, for making the egg-laying chambers, LED holders, and camera holders and for input on their designs. We also thank members of the C.-H.Y. lab for reading the manuscript. This work is supported by NIH grant R01 GM Received: February 1, 2014 Revised: August 4, 2014 Accepted: September 29, 2014 Published: October 30, 2014 References 1. Schwartz, N.U., Zhong, L., Bellemer, A., and Tracey, W.D. (2012). Egg laying decisions in Drosophila are consistent with foraging costs of larval progeny. PLoS ONE 7, e Azanchi, R., Kaun, K.R., and Heberlein, U. (2013). Competing dopamine neurons drive oviposition choice for ethanol in Drosophila. Proc. Natl. Acad. Sci. USA 110, Joseph, R.M., Devineni, A.V., King, I.F., and Heberlein, U. (2009). Oviposition preference for and positional avoidance of acetic acid provide a model for competing behavioral drives in Drosophila. Proc. Natl. Acad. Sci. 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