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. 2016 Dec 6;205(2):749–759. doi: 10.1534/genetics.116.195172

H2O2-Sensitive Isoforms of Drosophila melanogaster TRPA1 Act in Bitter-Sensing Gustatory Neurons to Promote Avoidance of UV During Egg-Laying

Ananya R Guntur *,1, Bin Gou *,1, Pengyu Gu †, Ruo He *, Ulrich Stern *, Yang Xiang †, Chung-Hui Yang *,2
PMCID: PMC5289849  PMID: 27932542

Abstract

The evolutionarily conserved TRPA1 channel can sense various stimuli including temperatures and chemical irritants. Recent results have suggested that specific isoforms of Drosophila TRPA1 (dTRPA1) are UV-sensitive and that their UV sensitivity is due to H2O2 sensitivity. However, whether such UV sensitivity served any physiological purposes in animal behavior was unclear. Here, we demonstrate that H2O2-sensitive dTRPA1 isoforms promote avoidance of UV when adult Drosophila females are selecting sites for egg-laying. First, we show that blind/visionless females are still capable of sensing and avoiding UV during egg-laying when intensity of UV is high yet within the range of natural sunlight. Second, we show that such vision-independent UV avoidance is mediated by a group of bitter-sensing neurons on the proboscis that express H2O2-sensitive dTRPA1 isoforms. We show that these bitter-sensing neurons exhibit dTRPA1-dependent UV sensitivity. Importantly, inhibiting activities of these bitter-sensing neurons, reducing their dTRPA1 expression, or reducing their H2O2-sensitivity all significantly reduced blind females’ UV avoidance, whereas selectively restoring a H2O2-sensitive isoform of dTRPA1 in these neurons restored UV avoidance. Lastly, we show that specifically expressing the red-shifted channelrhodopsin CsChrimson in these bitter-sensing neurons promotes egg-laying avoidance of red light, an otherwise neutral cue for egg-laying females. Together, these results demonstrate a physiological role of the UV-sensitive dTRPA1 isoforms, reveal that adult Drosophila possess at least two sensory systems for detecting UV, and uncover an unexpected role of bitter-sensing taste neurons in UV sensing.

Keywords: Drosophila egg-laying, UV-sensing, dTRPA1, bitter-sensing neurons

TRPA1 is a member of the evolutionarily conserved TRP family of nonselective cation channels and has been shown to play important roles in sensory functions across species (Clapham 2003; Jordt et al. 2004; Julius 2013). For example, previous studies have shown that TRPA1 can directly sense temperatures as well as chemical irritants such as allyl isothiocyanate, an active ingredient of mustard oil, in both vertebrates and invertebrates (Story et al. 2003; Viswanath et al. 2003; Rosenzweig et al. 2005; Hamada et al. 2008; Chatzigeorgiou et al. 2010; Kang et al. 2010; Cordero-Morales et al. 2011). In addition, TRPA1 can also act downstream of specific sensory receptors in some sensory neurons (Kim et al. 2010; Kwon et al. 2010; Xiang et al. 2010; Shen et al. 2011; Bellono et al. 2013). For example, TRPA1 has been suggested to transduce the chronic itch signal, sensed by other receptors, in the rodent DRG neurons (Morita et al. 2015), as well as to transduce a light signal downstream of the Gr28b receptor in Drosophila polymodal somatosensory C4da neurons (Xiang et al. 2010).

We have recently expanded the repertoire of TRPA1 functions by demonstrating that some of the dTRPA1 isoforms in Drosophila are H2O2-sensitive and can sense blue and UV lights (Guntur et al. 2015). We found that ectopically expressing these dTRPA1 isoforms can confer robust UV-induced calcium responses to light-insensitive cultured HEK 293 cells (Guntur et al. 2015). More strikingly, ectopic expression of these isoforms in two different groups of light-insensitive motor neurons in adult Drosophila enabled muscle contraction in response to UV (Guntur et al. 2015). Further, dTRPA1 isoforms that lacked H2O2 sensitivity could not confer UV sensitivity, and overexpression of catalase (an enzyme that degrades H2O2) significantly reduced UV sensitivity conferred by H2O2-sensitive dTRPA1 (Guntur et al. 2015), suggesting that H2O2-sensitive dTRPA1 isoforms can sense UV due to their capacity to detect UV-induced reactive oxygen species (ROS) production (Hockberger et al. 1999; Bhatla and Horvitz 2015; Guntur et al. 2015). However, despite these results, the physiological purpose of UV sensitivity of H2O2-sensitive dTRPA1 isoforms in animal behavior is not well-understood.

In this work, we provide evidence suggesting that UV sensitivity of the H2O2-sensitive dTRPA1 isoforms plays an important role in promoting avoidance of UV during female egg-laying. We first discovered that, whereas blind (visionless) females were unable to lay eggs away from low-intensity UV as previously reported (Zhu et al. 2014), they exhibited a clear avoidance of UV for egg-laying when the level of UV was elevated but still within the range of natural sunlight. Surprisingly, we also found that such vision-independent UV avoidance was primarily mediated by a group of bitter-sensing neurons on the proboscis. These gustatory neurons expressed the H2O2-sensitive dTRPA1 isoforms and exhibited dTRPA1-dependent UV sensitivity. Importantly, reducing dTRPA1 expression in these neurons, reducing their H2O2 sensitivity, or inhibiting their neuronal activities all significantly reduced blind Drosophila females’ UV avoidance for egg-laying, whereas selectively restoring the H2O2-sensitive isoform in these neurons rescued UV avoidance. Lastly, we showed that optogenetic activation of these dTRPA1-expressing bitter-sensing neurons was sufficient to promote egg-laying avoidance: expressing the red-shifted channelrhodopsin CsChrimson (Klapoetke et al. 2014) in these neurons caused females to lay eggs away from red light, an otherwise neutral stimulus for egg-laying females. Taken together, our results show that UV sensitivity of the H2O2-sensitive dTRPA1 isoforms plays an important role in ensuring that Drosophila females can continue to deposit their eggs away from an aversive and damaging stimulus for their progenies when their vision fails or is temporarily blocked, and that specific dTRPA1-expressing bitter-sensing neurons on the proboscis are recruited to accomplish this task.

Materials and Methods

Fly stocks

Animals were raised in standard molasses/cornmeal fly food and kept in a Darwin Chamber with temperature typically set at 25° and humidity level at 60–65%. The following stocks were used in this work: w1118, norpA36 (BL-9048), dTRPA1KO (Hamada et al. 2008), HdcJK910 (Burg et al. 1993), UAS-catalase (BL-24621), UAS-dTRPA1(A)10a (Guntur et al. 2015), UAS-dTRPA1-RNAi (Hamada et al. 2008), Gr66a-Gal4 (Wang et al. 2004), Gr66a-lexA (Thistle et al. 2012), dTRPA1-Gal4 (Petersen and Stowers 2011), lexA-op2-FLP (BL-55819), tub > Gal80> (Gordon and Scott 2009), UAS-GCaMP6s (BL-42746), Gr5a-lexA (Gordon and Scott 2009), lexA-op2-GCaMP6(s) (BL-44589), UAS-Kir 2.1 (Baines et al. 2001), and UAS-CsChrimson (BL-55134).

Reverse transcription PCR

RNA was extracted from whole adults or from dissected labellum using TRIzol and an RNeasy plus micro kit (QIAGEN, Valencia, CA). First-strand complementary DNA was synthesized by using a SuperScript III RT kit (Invitrogen, Carlsbad, CA) with oligo dT primer. EmeraldAmp Max HS PCR Master Mix (TAKARA) was used for amplifying the dTRPA1 isoform-specific products. The PCR program was set as below: 94° for 5 min, 94° for 20 sec, 55° for 20 sec, and 72° for 2 min, repeat steps 2–4 29 times (30 cycles in total), followed by 72° for 7 min. For discriminating different dTRPA1 isoforms, the primer pairs used were as followed: (i) dTRPA1(A)10a: A-F/10a-R and (ii) dTRPA1(A)10b: A-F/10b-R. Sequences of the primers described are as follows: A-F: 5′-GCCGGAACAGCAAGTATT-3′; 10a-R: 5′-CCATGTGTTACCATGGTATTCAAAG-3′; and 10b-R: 5′-TGTTACCATGGTGTTCACCA-3′.

Egg-laying assay

To assess how females respond to UV when selecting for an egg-laying site, we assayed their preference via a UV vs. dark assay in custom egg-laying chambers that were fitted with light-emitting diode (LED) holders. The general design of the egg-laying chambers and UV light control were as previously described (Zhu et al. 2014) but with two modifications. First, the UV (380 nm) intensity was set at 6 µW/mm2 instead of < 0.5 µW/mm2 (note that we chose 6 µW/mm2 because it was the highest level of UV that our controller could deliver with the LEDs that we had purchased). Second, to maintain an adequate amount of egg-laying, we directly added 150 mM sucrose (a food source) into the 1% agarose as opposed to placing a drop of grape juice into a hole in the middle of the chamber. For the Red LED vs. dark assay, we replaced the UV LED in the light holder with a red light LED (part # C503B-RAN-CA0B0AA1; Mouser Electronics) to assess how females respond to red light when expressing CsChrimson in specific groups of neurons. The intensity of red light was set at 2.3 or 4.5 µW/mm2.

To maintain adequate levels of egg-laying rate, we typically prepared the females by depriving them of egg-laying using the previously described method (Zhu et al. 2014; Gou et al. 2016). Briefly, every ∼30 females were paired with ∼15 males and put in a regular food vial that was supplemented with some active yeast paste (made by mixing 3 g of active yeast with 5 ml of 0.3% propionic acid); the females were allowed to mate and lay eggs in the vial for ∼4–5 d. Females would lay many eggs initially, due to the presence of yeast paste, and thus caused the surface of the food vial to become very mushy, a texture that deters egg-laying and thus causes females to start withholding eggs. After withholding eggs for several days, these females would readily lay eggs when placed in our egg-laying chambers. For females to be assayed in the red LED vs. dark assay, we supplemented the active yeast paste and fly food with 500 µM all-trans retinal (Sigma [Sigma Chemical], St. Louis, MO), the chromophore for activating CsChrimson. We typically let the females lay eggs overnight in our egg-laying chambers and calculated the preference index (PI) according to the following formula: (NLED − NDark) / (NLED + NDark). NLED and NDark represent the number of eggs on the LED and dark sides, respectively.

Behavioral tracking

Females to be tracked were fed active yeast paste and deprived of egg-laying, as described previously, so that they were in “egg-laying state” (Gou et al. 2014, 2016). They were then placed in egg-laying chambers that contained two sucrose substrates (prepared as described earlier), but UV-LEDs were mounted below the egg-laying chamber so that cameras could be mounted above to videotape females’ behaviors (see also Zhu et al. 2014). We typically recorded the females for 2 hr and then analyzed the periods in which they did not lay eggs. This is because we wanted to determine whether females in an egg-laying state showed signs of positional avoidance of UV. Selected videos were then tracked using Ctrax (Branson et al. 2009), and the tracked trajectories analyzed using a custom MATLAB code (available upon request). PI was calculated according to the following formula: (NLED − NDark) / (NLED + NDark). NLED and NDark represent the number of frames females spent on the LED and dark sides, respectively.

Ca2+ imaging

To image calcium responses of gustatory neurons on the labellum, we severed the proboscises and placed them in a custom-built imaging chamber (Gou et al. 2014) that was filled with imaging buffer. The imaging buffer contains the following chemicals: NaCl (120 mM), KCl (3 mM), MgCl2 (4 mM), CaCl2 (2 mM), NaHCO3 (10 mM), trehalose (10 mM), glucose (10 mM), 2-[(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid (TES, 5 mM), sucrose (10 mM), and HEPES (10 mM), with the final pH adjusted to 7.25. We used an X-cite lamp as the light source and we filter set # 49 from Zeiss [Carl Zeiss] (Thornwood, NY) (300–400 nm, peak at 365 nm) to produce UV. Light intensity was measured with a Thorlabs PM100USB optical power meter.

To acquire GCaMP fluorescence change, we used a Zeiss LSM700 confocal microscope equipped with a 40 ×/0.8 NA water immersion objective and the live-series ZEN image acquisition software. Briefly, baseline GCaMP fluorescence was acquired by scanning the cells with a 488 nm laser at 128 × 128 pixels at 8-bit dynamic range. The GCaMP6 signal acquired before light stimulation (baseline) was used as F0 and the signal after 5′ light stimulation was used as F′. ΔF / F was then calculated as (F′ − F0) / F0 to reflect the changes in GCaMP signals before and after stimulation. For each experiment, we examined GCaMP responses from 4 to 10 animals. Because each animal has multiple GCaMP-positive cells (∼7–18 cells), we calculated the GCaMP response for each experiment by first obtaining an averaged ΔF / F for each animal. For analyzing caffeine and H2O2-induced responses, ImageJ software (NIH) was used to register images and calculate fluorescence in assigned regions of interest. The GCaMP6 signal acquired before addition of caffeine and H2O2 was used as F0 and the peak change in fluorescence after addition of H2O2 was used as F′. ΔF / F was then calculated as (F′ − F0) / F0 to reflect the change in GCaMP6 after H2O2 stimulation.

Data availability

Drosophila lines are available at the Bloomington Drosophila Stock Center or upon request. The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article.

Results

Blind females avoid laying eggs on a UV site when the level of UV is close to that of sunlight

We have previously shown that Drosophila females robustly avoided laying eggs on the UV-illuminated site when given a choice between an unilluminated (dark) option and a UV-illuminated one in our two-choice egg-laying paradigm (Zhu et al. 2014). This avoidance of UV for egg-laying critically depended on phototransduction in the compound eyes: animals that lacked the phototransduction molecule phospholipase C (PLC) (norpA mutants) no longer avoided UV, whereas selective rescue of norpA function in the UV-sensing R7 photoreceptors restored UV avoidance (Zhu et al. 2014). However, the intensity of UV used in that study (< 0.5 μW/mm2) was significantly lower than that of the UV component of sunlight received on the Earth’s surface (∼25 μW/mm2), as most studies on UV-induced behaviors in adult flies employ very low-intensity UV (Gao et al. 2008; Karuppudurai et al. 2014). Therefore, we wondered whether Drosophila females continue to rely exclusively on their visual system to sense and avoid UV during egg-laying when the level of UV intensity is closer to that of sunlight. Notably, a previous study has suggested that Drosophila larvae use the Bolwig organ (the primitive larval eye) to detect low levels of light, but recruit additional sensors (the polymodal somatosensory C4da neurons on the body wall) to detecting high levels of blue and UV lights (Xiang et al. 2010).

To test the idea that adult Drosophila may recruit extraocular UV sensors to avoid higher but physiologically relevant levels of UV during egg-laying, we first assessed the egg-laying preference of blind (norpA36) females in a new UV vs. dark two-choice task, where we raised the UV intensity to 6 μW/mm2 (Figure 1A). Interestingly, we found that whereas blind (norpA36) females failed to avoid very low levels of UV (< 0.5 μW/mm2) as reported previously (Zhu et al. 2014), they consistently showed a moderate but significant avoidance of a higher level of UV (6 μW/mm2) for egg-laying (Figure 1, B–D). To further confirm this result, we examined the egg-laying preference of another blind mutant HdcJK910 that cannot produce histamine, the critical neurotransmitter by which photoreceptors in the eyes signal their targets (Burg et al. 1993). Again, Hdc mutants showed a moderate but significant avoidance of UV for egg-laying (Figure 1D). These results suggest that adult Drosophila indeed possess “extraocular UV sensors” that enable them to avoid laying eggs on UV sites (where the level of UV is higher than that used in our earlier study but is still physiologically relevant).

Figure 1.

Figure 1

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Blind females prefer not to lay eggs on UV sites. (A) A schematic diagram of the two-choice behavioral assay used to test the egg-laying preferences of Drosophila. Each fly was given two agarose-containing egg-laying options (that were separated by hard plastic). One of the options was illuminated with UV. (B) A representative picture of the egg-laying result of a single w1118 female. (C) A representative picture of the egg-laying result of a single norpA36 female. Note that there are fewer eggs deposited on the UV site. (D) Egg-laying preference index (PI) of w1118, norpA36, and HdcJK910. Note that both vision mutants are null alleles. **P < 0.01, ***P < 0.001, one-sample t-test from 0. The number of flies used for each experiment performed in this work is labeled directly on each graph.

Blind females’ preference to lay eggs away from UV is the result of positional avoidance of UV

We next assessed the behavioral mechanism that underlies blind females’ avoidance of laying eggs on sites illuminated with high UV. There are at least two possible explanations: first, that egg-laying females avoid approaching a site illuminated with UV; second, that egg-laying females readily approach the UV site but selectively withhold from depositing eggs while visiting it. To distinguish between these two possibilities, we tracked the behaviors of egg-laying females as they explored the UV vs. dark chamber. Plotting and analyzing the trajectories of egg-laying wild-type (w1118) females revealed that they tended to spend times away from the UV site (Figure 2, A and C). Further, analysis of trajectories of blind (norpA) egg-laying females revealed that they also exhibited a moderate but significant positional avoidance of UV, despite their lack of visual input (Figure 2, B and C). Importantly, because positional avoidance of UV of these animals was evident even during periods when there were no egg-laying events (Figure 2, A and C), despite these animals being in an “egg-laying state” in general (Gou et al. 2014), such avoidance cannot be trivially explained because (1) the physical act of egg-laying takes time, and (2) females tended to stay immobile for a while on the site where they had just deposited an egg. Instead, this result suggests that females in an egg-laying state tend to avoid spending time on sites exposed to higher levels of UV and that extraocular UV sensors play a role in promoting such UV avoidance.

Figure 2.

Figure 2

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Blind females exhibit positional avoidance of UV. (A) Trajectory of a single w1118 female as it explored the UV vs. dark chamber for 2 hr. x-axis represents time. y-axis represents the “y position” of the animal in the chamber. It denotes the relative distance to the edges of the substrates. Further, each black circle denotes an “aversive return” toward UV. It indicates an event where the fly had changed its direction from moving toward UV to moving away from UV. The red square denotes an “attractive return” toward UV. It indicates that the fly had changed its direction from moving away from UV to moving toward UV. (B) Trajectory of a single norpA36 female as it explored the UV vs. dark chamber for 2 hr. (C) Positional preference index (PI) of w1118 and norpA36 flies. **P < 0.01, ***P < 0.001, one-sample t-test from 0.

Proboscises of adult flies houses neurons that express H2O2-sensitive isoforms of dTRPA1

We next set out to determine the identity of the extraocular UV-sensitive neurons that promote the egg-laying avoidance of UV in blind females. Because H2O2-sensitive isoforms of dTRPA1 are UV-sensitive, we hypothesized that the extraocular UV-sensitive neurons may rely on these isoforms to sense UV. Previous findings have suggested that one of the H2O2-sensitive TRPA1 isoforms is expressed in the Gr66a-expressing bitter-sensing neurons on the proboscis (Kang et al. 2012). To further confirm this result, we performed RT-PCR experiments and found that H2O2/UV-sensitive isoforms were indeed present on the proboscis (Figure 3, A and B). These results suggest that Gr66a-expressing bitter-sensing neurons may exhibit dTRPA1-depedent UV sensitivity.

Figure 3.

Figure 3

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H2O2/UV-sensitive dTRPA1 isoforms are present on the proboscis. (A and B) RT-PCR showing that the H2O2/UV-sensitive isoforms dTRPA1(A)10a and 10b are present on the labellum of the proboscis. Note that the nomenclature of dTRPA1 isoforms that we adopted in this work and in Guntur et al. 2015 was conceived and proposed to us by Paul Garrity and his student Vincent Panzano. A, whole adult; B, blank control with no DNA template; G, genomic DNA control; L, labellum.

Gr66a-expressing neurons on the proboscis exhibit dTRPA1-dependent UV sensitivity

We next tested whether functional H2O2-sensitive isoforms of dTRPA1 are indeed present in the Gr66a-expressing bitter-sensing neurons on the proboscis and confer these neurons with UV sensitivity. We first used Gr66a-Gal4 to express the genetically-encoded calcium indicator GCaMP6 (Chen et al. 2013) in these neurons, and found that these neurons showed a clear response to both UV and H2O2 (Figure 4, A–C). Importantly, illuminating even very strong UV onto the Gr5a-expressing sweet-sensing neurons on the proboscis did not induce any responses, suggesting that the UV-induced calcium response of Gr66a neurons that we observed was not a nonspecific injury-induced response (Figure 4C). Two additional experiments also support the notion that H2O2-sensitive isoforms of dTRPA1 are present in the Gr66a neurons on the proboscis. First, in addition to exhibiting a clear sensitivity to UV and H2O2, we found that proboscis neurons labeled by dTRPA1-Gal4 (Petersen and Stowers 2011), a reporter for dTRPA1 expression, showed a clear sensitivity to caffeine (Figure 4G, black bar), a known bitter agonist of Gr66a neurons (Moon et al. 2006; Lee et al. 2009). Second, colabeling experiments showed that most, if not all, of the neurons labeled by the dTRPA1-Gal4 on the proboscis also expressed Gr66a (Figure 4, D–F and Supplemental Material, Figure S1 and File S1).

Figure 4.

Figure 4

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H2O2/UV-sensitive dTRPA1 isoforms are expressed in the Gr66a-expressing neurons and confer them with UV sensitivity. (A [before UV] and B [after UV]) Representative calcium responses of Gr66a neurons illuminated with UV. (C) Quantification of calcium responses of Gr66a neurons to UV and H2O2. In contrast, Gr5a neurons did not respond to UV even when its intensity was elevated to 35 mW/mm2 (note that the response of Gr66a neurons to 35 mW/mm2 UV was significantly higher than their response at 6 mW/mm2, data not shown). *P < 0.05, **P < 0.01, one-sample t-test from 0. (D–F) Representative expression patterns of Gr66a-Gal4 alone (D), Gr66a-Gal4 plus dTRPA1-Gal4 (E), and dTRPA1-Gal4 alone (F) on the proboscis. Note that the numbers of neurons labeled on the proboscis by Gr66a-Gal4 alone vs. Gr66a-Gal4 plus dTRPA1-Gal4 were comparable. (G) Quantification of calcium responses of dTRPA1-Gal4-labeled neurons to caffeine, UV, and H2O2. Note that UV and H2O2 responses both reduced significantly when these neurons lacked dTRPA1 or when they overexpressed catalase (cat). For a−b: *P < 0.05, **P < 0.01, one-sample t-test from 0. For c: *P < 0.05, ***P < 0.001, one-way ANOVA followed by Tukey’s multiple comparison test. For d: **P < 0.01, unpaired t-test.

Does the UV sensitivity exhibited by dTRPA1/Gr66a-expressing neurons depend on H2O2-sensitive dTRPA1? We did two experiments to address this. First, we eliminated dTRPA1 function from these neurons (by examining them in the dTRPA1KO mutant background) and found that they no longer responded to UV or H2O2 but still responded to caffeine (Figure 4G). Second, we overexpressed the H2O2 degrading enzyme catalase (cat) in the dTRPA1-expressing neurons and found their UV response to be significantly reduced (Figure 4G). Taken together, our collective results so far suggest that the bitter-sensing dTRPA1/Gr66a-Gal4-expressing gustatory neurons on the proboscis are UV-sensitive, and that their UV sensitivity critically depends on light-induced H2O2 production and H2O2-sensitive dTRPA1 isoforms.

Gr66a-expressing neurons on the proboscis are critical for UV avoidance of blind females

We next addressed whether the bitter-sensing Gr66a neurons on the proboscis play a role in promoting blind females’ egg-laying avoidance of UV. First, we surgically removed the proboscis from two different blind mutants (norpA36 and HdcJK910 mutants) and found that these “blind and proboscisless” animals no longer avoided UV for egg-laying in the UV vs. dark assay (Figure 5A). We next directly assessed the role of the Gr66a neurons in promoting UV avoidance in blind females. Because there is no Gal4, as far as we know, that labels only the Gr66a bitter-sensing neurons on the proboscis (and nowhere else in the nervous system), we used two different Gal4s [dTRPA1-Gal4 (Petersen and Stowers 2011) and Gr66a-Gal4 (Wang et al. 2004)] to silence the activities of these neurons. These two Gal4s both labeled H2O2/UV-sensitive neurons on the proboscis (Figure 4, D–F and Figure S1) and, more importantly, outside of the proboscis their expression patterns were sparse and rather distinct (Figure 5, B and C and Figure S2). Expressing the hyperpolarizing Kir2.1 channel using either Gal4 caused the blind females to reduce their egg-laying avoidance of UV (Figure 5D). Thus, both the “surgery experiment” and the neuron-silencing experiment support the idea that Gr66a/dTRPA1-expressing neurons on the proboscis play a critical role in promoting vision-independent egg-laying avoidance of UV.

Figure 5.

Figure 5

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Gr66a-expressing neurons are critical for UV avoidance in blind females. (A) Egg-laying PI of proboscis-less norpA36 and proboscis-less HdcJK910 females. Neither is significantly different from 0, one-sample t-test. Note that proboscis-less females laid fewer eggs. For example, the average numbers of eggs laid overnight by individual proboscis-less norpA and Hdc females were 18.1 ± 1.5 and 18.3 ± 1.8, respectively. While the egg-laying numbers were low, they were still sufficient for us to calculate the PI; our typical cutoff for PI was 10 eggs. (B and C) Expression pattern of dTRPA1-Gal4 and Gr66a-Gal4 in the adult CNS. Arrows point to the axonal termini (in the SEG) of the gustatory neurons on the proboscis. (D) Egg-laying PIs of blind (norpA) flies whose dTRPA1/Gr66a neurons were silenced by Kir2.1 overexpression. *P < 0.05, **P < 0.01, one-way ANOVA followed by Tukey’s multiple comparison test. CNS, central nervous system; PC, proboscis cut; PI, preference index; SEG, suboesophageal ganglion.

Gr66a-expressing bitter-sensing neurons rely on H2O2-sensitive dTRPA1 to promote UV avoidance in blind females

Do the Gr66a neurons on the proboscis rely on H2O2-sensitive dTRPA1 to promote avoidance of UV? To address this, we first reduced dTRPA1 expression in these neurons by employing a commonly used UAS-dTRPA1-RNAi tool (that was designed against all dTRPA1 isoforms) (Hamada et al. 2008) and found that this manipulation significantly reduced blind females’ egg-laying avoidance of UV (Figure 6A). Second, we reduced these neurons’ UV sensitivity by overexpressing cat in them and found that this also significantly reduced UV avoidance (Figure 6B). Lastly, we reintroduced the H2O2-sensitive isoform of dTRPA1 into the Gr66a neurons in the norpA; dTRPA1 double-mutants (that were completely incapable of avoiding UV) and found that it restored animals’ UV avoidance (Figure 6C). Importantly, the ability of these rescued animals to avoid UV disappeared if we surgically removed their proboscis (Figure 6C), suggesting that a major “source of the rescue” likely resides on the proboscis. Taken together, these results suggest strongly that the Gr66a neurons on the proboscis rely on H2O2-sensitive dTRPA1 to promote vision-independent egg-laying avoidance of UV.

Figure 6.

Figure 6

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Expression of H2O2/UV-sensitive dTRPA1 isoforms in Gr66a-expressing neurons promotes egg-laying avoidance of UV in blind females. (A) Egg-laying PIs of blind (norpA) flies whose Gr66a neurons overexpressed dTRPA1-RNAi. ***P < 0.001, one-way ANOVA followed by Tukey’s multiple comparison test. (B) Egg-laying PIs of blind (norpA) flies whose Gr66a neurons overexpressed catalase (cat). *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA followed by Tukey’s multiple comparison test. (C) Egg-laying PIs of norpA; dTRPA1KO double-mutant (left bar), norpA36; dTRPA1KO double-mutant with isoform dTRPA1(A)10a rescued in their Gr66a neurons (middle bar), and the rescued flies with their proboscis severed (right bar). **P < 0.01, ***P < 0.001, one-way ANOVA followed by Tukey’s multiple comparison test. PI, preference index.

Optogenetic activation of the Gr66a-expressing neurons on the proboscis is sufficient to promote egg-laying avoidance

Finally, we set out to ask whether artificially activating the Gr66a-expressing neurons on the proboscis is sufficient to drive egg-laying avoidance. If so, this would lend further support to the idea that detection of UV by these neurons promotes egg-laying avoidance. We addressed this by taking advantage of the recently made available red-shifted channelrhodopsin CsChrimson (Klapoetke et al. 2014) (Figure 7A). We found that, whereas control females showed no innate avoidance of red light for egg-laying in our red LED vs. dark assays (Figure 7B), animals that overexpressed CsChrimson in their dTRPA1-Gal4- or the Gr66a-Gal4-expressing neurons showed a clear preference to lay eggs away from red light when given a choice between a red light-illuminated option and an unilluminated one, likely because egg-laying females positionally avoided red light (Figure 7B).

Figure 7.

Figure 7

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Optogenetic activation of Gr66a-expressing neurons on the proboscis promotes egg-laying avoidance of red light. (A) A schematic diagram of the red LED vs. dark egg-laying assay. (B) Egg-laying PIs of flies whose Gr66a neurons overexpressed the red-shifted channelrhodopsin CsChrimson (CsC). ***P < 0.001, one-way ANOVA followed by Tukey’s multiple comparison test. (C) Expression pattern of Gr66a-Gal4 and Gr66a-lexA driving GFP and nls-Cherry, respectively, on the proboscis. Note that while Gr66a-Gal4 typically labels ∼22 neurons on the proboscis, only seven of them were colabeled by Gr66a-lexA. This was the reason why only a few neurons were labeled when we used Gr66a-lexA to intersect with dTRPA1-Gal4. (D) Representative images of CsChrimson labeling in the brain and VNC of the “intersected” females. Note that the only processes that were labeled in these animals were the axons of bitter-sensing neurons in the SEG. “>”: FRT site. (E) Egg-laying PIs of the intersected females that were or were not fed with retinal-supplemented food. ***P < 0.001, unpaired t-test. FRT, ; LED, light-emitting diode; PI, preference index; SEG, suboesophageal ganglion; VNC, ventral nerve cord.

To further assess whether the avoidance of red light for egg-laying we observed was mediated specifically by the activation of Gr66a-expressing neurons on the proboscis (as opposed to by ones residing on the leg, for example), we attempted to restrict CsChrimson expression to just the proboscis neurons by using an intersectional approach. We typically found that about five of the dTRPA1-Gal4-expressing neurons on the proboscis were colabeled by the Gr66a-lexA (it is worth pointing out that the lack of substantial overlap between these two drivers is consistent with the lack of significant overlap between Gr66a-lexA and Gr66a-Gal4 expression on the proboscis, Figure 7C). By using a combination of transgenes (dTRPA1-Gal4, Gr66a-lexA, tub > GAL80>, lexA-FLP, and UAS-CsChrimson), we indeed limited CsChrimson expression to just a few neurons on the proboscis (Figure 7D). These animals showed a clear avoidance of red light for egg-laying (Figure 7E). However, their level of avoidance was reduced as compared to the nonintersected animals, most likely because they had fewer neurons that expressed CsChrimson. Importantly, these animals no longer showed red light avoidance if we did not supplement their diet with retinal (a critical chromophore for CsChrimson activation, Figure 7E), suggesting that the red light avoidance we observed was not an artifact. Taken together, these results suggest that ectopically expressing CsChrimson in the Gr66a-expressing gustatory neurons on the proboscis is sufficient to promote egg-laying avoidance of red light, further supporting the notion that these neurons play a role in promoting egg-laying avoidance of UV, a natural stimulus for them.

Discussion

In this work, we addressed the question of whether the H2O2-mediated UV sensitivity of specific Drosophila TRPA1 isoforms (Guntur et al. 2015) has a physiological role in animal behavior. By using a combination of behavioral assays, calcium imaging, and neuronal activity manipulation experiments, we found that H2O2/UV-sensitive dTRPA1 isoforms act in a group of bitter-sensing gustatory neurons on the proboscis to promote egg-laying avoidance of strong UV, an aversive and damaging stimulus for young larvae. We found that animals that lacked vision (norpA or Hdc mutants) were still capable of sensing and avoiding UV for egg-laying, but these blind females no longer avoided UV if they lacked dTRPA1 or if their bitter- and UV-sensitive gustatory neurons on the proboscis were rendered UV-insensitive (by cat overexpression or removal of dTRPA1 function) or hyperpolarized (by Kir2.1 overexpression). Additionally, ectopic expression of the red-shifted channelrhodopsin CsChrimson in these bitter-sensing gustatory neurons enabled females to lay eggs away from red light, an otherwise neutral cue for egg-laying females, suggesting that these neurons are capable of promoting egg-laying avoidance when activated. Taken together, these results support the hypothesis that UV sensitivity of the H2O2-sensitive dTRPA1 isoforms is physiologically relevant, and that these isoforms act in the bitter-sensing gustatory neurons as an extraocular backup to promote UV avoidance during egg-laying.

What is the advantage of having an extraocular system for UV sensing? UV is a known aversive stimulus for young larvae and may incur damage to them (Xiang et al. 2010). Moreover, laying eggs on UV-illuminated substrates, as opposed to laying eggs in the dark, may expose the egg-laying females to increased predation risk also (Yang et al. 2008, 2015). Thus, having a second UV-sensing system should confer some advantage to the species in case animals’ vision fails due to genetic defects, injuries, or temporary blockage or desensitization. It is worth noting that such “dual UV sensor” design is not unprecedented. As mentioned earlier, a previous report has shown that Drosophila larvae employ two different systems, the Bolwig organ and the C4da neurons, for light sensing (Xiang et al. 2010). Interestingly, reminiscent of the dual light-sensing systems we describe here, the Bolwig organ has a high light sensitivity and uses the conventional Rhodopsin-PLC pathway to sense light (Busto et al. 1999; Hassan et al. 2000; Malpel et al. 2002), whereas the C4da neurons have a low light sensitivity and use the Gr28b-dTRPA1 pathway to specifically sense strong blue and UV lights (Xiang et al. 2010). For both larvae and adults, the extraocular UV sensors are recruited from sensory neurons that are known to promote behavioral avoidance (Xiang et al. 2010).

It is also worth pointing out that different isoforms of dTRPA1 appear to act in different subsets of Gr66a neurons in adult flies. For example, our results suggest that H2O2-sensitive isoforms of dTRPA1 are present in the Gr66a neurons on the labellum (at the tip of the proboscis) and can act to drive positional avoidance of UV in egg-laying females. On the other hand, a recent report has suggested that Gr66a neurons that reside on the esophagus express both the H2O2-sensitive and -insensitive dTRPA1 isoform and can act to suppress ingestion of the bacterial toxin lipopolysaccharide, another candidate agonist of dTRPA1 (Soldano et al. 2016). These results again highlight the notion that, in Drosophila, dTRPA1 appears to primarily act as a sensor for aversive stimuli and is often housed in different avoidance-promoting sensory neurons.

Lastly, while our data suggest the dTRPA1-expressing gustatory neurons play a critical role in enabling females to lay eggs away from UV in the absence of vision, it is likely that they serve additional roles. First, these neurons are also present on the proboscis of males, and norpA males did show weaker but significant positional avoidance of UV (PI ∼ −0.16 ± 0.02, n = 22). Moreover, one important function of bitter-sensing gustatory neurons is to suppress feeding. It is conceivable that expression of H2O2-sensitive dTRPA1 in these neurons may act to suppress feeding when levels of UV are stronger and closer to that of sunlight. While the specific advantage of feeding suppression in response to high levels of UV is unclear, Bhatla and Horvitz (2015) have recently shown that, in Caenorhabditis elegans, short-wavelength blue and UV lights can indeed inhibit feeding via activation of a specific group of pharyngeal neurons. Curiously, blue and UV lights activate these neurons via light-induced H2O2 production, but the G protein-coupled receptors LITE-1 and GUR3, not TRPA1, are suggested to be the molecular sensors for H2O2 in this case (Bhatla and Horvitz 2015). UV-induced feeding suppression aside, the presence of the H2O2-sensitive dTRPA1 isoforms in the gustatory neurons may also serve to prevent feeding on ROS-containing chemicals that can activate the H2O2-sensitive dTRPA1 in these neurons (Kang et al. 2012; Du et al. 2016), as the ingestion of some of ROS-containing chemicals may cause harm to the animals. Future work is needed to fully elucidate the roles of UV/H2O2-sensitive dTRPA1 in animal behavior.

Supplementary Material

Supplemental material is available online at www.genetics.org/lookup/suppl/doi:10.1534/genetics.116.195172/-/DC1.

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Acknowledgments

We thank S. Stowers, K. Scott, P. Garrity, and the Bloomington Drosophila Stock Center for providing us with fly stocks. We also thank members of the Yang and Xiang labs, and A. Bellemer for comments and suggestions this work. This work was supported by National Institutes of Health (NIH) grant R01GM100027 to C.-H.Y. and NIH grant R01NS089787 to Y.X.

Footnotes

Communicating editor: M. F. Wolfner

Literature Cited

  1. Baines R. A., Uhler J. P., Thompson A., Sweeney S. T., Bate M., 2001.  Altered electrical properties in Drosophila neurons developing without synaptic transmission. J. Neurosci. 21: 1523–1531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bellono N. W., Kammel L. G., Zimmerman A. L., Oancea E., 2013.  UV light phototransduction activates transient receptor potential A1 ion channels in human melanocytes. Proc. Natl. Acad. Sci. USA 110: 2383–2388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bhatla N., Horvitz H. R., 2015.  Light and hydrogen peroxide inhibit C. elegans feeding through gustatory receptor orthologs and pharyngeal neurons. Neuron 85: 804–818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Branson K., Robie A. A., Bender J., Perona P., Dickinson M. H., 2009.  High-throughput ethomics in large groups of Drosophila. Nat. Methods 6: 451–457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burg M. G., Sarthy P., Koliantz G., Pak W., 1993.  Genetic and molecular identification of a Drosophila histidine decarboxylase gene required in photoreceptor transmitter synthesis. EMBO J. 12: 911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Busto M., Iyengar B., Campos A. R., 1999.  Genetic dissection of behavior: modulation of locomotion by light in the Drosophila melanogaster larva requires genetically distinct visual system functions. J. Neurosci. 19: 3337–3344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chatzigeorgiou M., Yoo S., Watson J. D., Lee W.-H., Spencer W. C., et al. , 2010.  Specific roles for DEG/ENaC and TRP channels in touch and thermosensation in C. elegans nociceptors. Nat. Neurosci. 13: 861–868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen T.-W., Wardill T. J., Sun Y., Pulver S. R., Renninger S. L., et al. , 2013.  Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499: 295–300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clapham D. E., 2003.  TRP channels as cellular sensors. Nature 426: 517–524. [DOI] [PubMed] [Google Scholar]
  10. Cordero-Morales J. F., Gracheva E. O., Julius D., 2011.  Cytoplasmic ankyrin repeats of transient receptor potential A1 (TRPA1) dictate sensitivity to thermal and chemical stimuli. Proc. Natl. Acad. Sci. USA 108: E1184–E1191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Du E. J., Ahn T. J., Wen X., Seo D.-W., Na D. L., et al. , 2016.  Nucleophile sensitivity of Drosophila TRPA1 underlies light-induced feeding deterrence. eLife 5: e18425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gao S., Takemura S.-y., Ting C.-Y., Huang S., Lu Z., et al. , 2008.  The neural substrate of spectral preference in Drosophila. Neuron 60: 328–342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gordon M. D., Scott K., 2009.  Motor control in a Drosophila taste circuit. Neuron 61: 373–384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gou B., Liu Y., Guntur A. R., Stern U., Yang C.-H., 2014.  Mechanosensitive neurons on the internal reproductive tract contribute to egg-laying-induced acetic acid attraction in Drosophila. Cell Rep. 9: 522–530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gou B., Zhu E., He R., Stern U., Yang C.-H., 2016.  High throughput assay to examine egg-laying preferences of individual Drosophila melanogaster. J. Vis. Exp. 109: e53716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Guntur A. R., Gu P., Takle K., Chen J., Xiang Y., et al. , 2015.  Drosophila TRPA1 isoforms detect UV light via photochemical production of H2O2. Proc. Natl. Acad. Sci. USA 112: E5753–E5761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hamada F. N., Rosenzweig M., Kang K., Pulver S. R., Ghezzi A., et al. , 2008.  An internal thermal sensor controlling temperature preference in Drosophila. Nature 454: 217–220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hassan J., Busto M., Iyengar B., Campos A. R., 2000.  Behavioral characterization and genetic analysis of the Drosophila melanogaster larval response to light as revealed by a novel individual assay. Behav. Genet. 30: 59–69. [DOI] [PubMed] [Google Scholar]
  19. Hockberger P. E., Skimina T. A., Centonze V. E., Lavin C., Chu S., et al. , 1999.  Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells. Proc. Natl. Acad. Sci. USA 96: 6255–6260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jordt S.-E., Bautista D. M., Chuang H.-h., McKemy D. D., Zygmunt P. M., et al. , 2004.  Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1. Nature 427: 260–265. [DOI] [PubMed] [Google Scholar]
  21. Julius D., 2013.  TRP channels and pain. Annu. Rev. Cell Dev. Biol. 29: 355–384. [DOI] [PubMed] [Google Scholar]
  22. Kang K., Pulver S. R., Panzano V. C., Chang E. C., Griffith L. C., et al. , 2010.  Analysis of Drosophila TRPA1 reveals an ancient origin for human chemical nociception. Nature 464: 597–600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kang K., Panzano V. C., Chang E. C., Ni L., Dainis A. M., et al. , 2012.  Modulation of TRPA1 thermal sensitivity enables sensory discrimination in Drosophila. Nature 481: 76–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Karuppudurai T., Lin T.-Y., Ting C.-Y., Pursley R., Melnattur K. V., et al. , 2014.  A hard-wired glutamatergic circuit pools and relays UV signals to mediate spectral preference in Drosophila. Neuron 81: 603–615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kim S. H., Lee Y., Akitake B., Woodward O. M., Guggino W. B., et al. , 2010.  Drosophila TRPA1 channel mediates chemical avoidance in gustatory receptor neurons. Proc. Natl. Acad. Sci. USA 107: 8440–8445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Klapoetke N. C., Murata Y., Kim S. S., Pulver S. R., Birdsey-Benson A., et al. , 2014.  Addendum: independent optical excitation of distinct neural populations. Nat. Methods 11: 972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kwon Y., Kim S. H., Ronderos D. S., Lee Y., Akitake B., et al. , 2010.  Drosophila TRPA1 channel is required to avoid the naturally occurring insect repellent citronellal. Curr. Biol. 20: 1672–1678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lee Y., Moon S. J., Montell C., 2009.  Multiple gustatory receptors required for the caffeine response in Drosophila. Proc. Natl. Acad. Sci. USA 106: 4495–4500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Malpel S., Klarsfeld A., Rouyer F., 2002.  Larval optic nerve and adult extra-retinal photoreceptors sequentially associate with clock neurons during Drosophila brain development. Development 129: 1443–1453. [DOI] [PubMed] [Google Scholar]
  30. Moon S. J., Köttgen M., Jiao Y., Xu H., Montell C., 2006.  A taste receptor required for the caffeine response in vivo. Curr. Biol. 16: 1812–1817. [DOI] [PubMed] [Google Scholar]
  31. Morita T., McClain S. P., Batia L. M., Pellegrino M., Wilson S. R., et al. , 2015.  HTR7 mediates serotonergic acute and chronic itch. Neuron 87: 124–138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Petersen L. K., Stowers R. S., 2011.  A gateway multisite recombination cloning toolkit. PLoS One 6: e24531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rosenzweig M., Brennan K. M., Tayler T. D., Phelps P. O., Patapoutian A., et al. , 2005.  The Drosophila ortholog of vertebrate TRPA1 regulates thermotaxis. Genes Dev. 19: 419–424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Shen W. L., Kwon Y., Adegbola A. A., Luo J., Chess A., et al. , 2011.  Function of rhodopsin in temperature discrimination in Drosophila. Science 331: 1333–1336. [DOI] [PubMed] [Google Scholar]
  35. Soldano A., Alpizar Y. A., Boonen B., Franco L., López-Requena A., et al. , 2016.  Gustatory-mediated avoidance of bacterial lipopolysaccharides via TRPA1 activation in Drosophila. eLife 5: e13133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Story G. M., Peier A. M., Reeve A. J., Eid S. R., Mosbacher J., et al. , 2003.  ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 112: 819–829. [DOI] [PubMed] [Google Scholar]
  37. Thistle R., Cameron P., Ghorayshi A., Dennison L., Scott K., 2012.  Contact chemoreceptors mediate male-male repulsion and male-female attraction during Drosophila courtship. Cell 149: 1140–1151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Viswanath V., Story G. M., Peier A. M., Petrus M. J., Lee V. M., et al. , 2003.  Ion channels: opposite thermosensor in fruitfly and mouse. Nature 423: 822–823. [DOI] [PubMed] [Google Scholar]
  39. Wang Z., Singhvi A., Kong P., Scott K., 2004.  Taste representations in the Drosophila brain. Cell 117: 981–991. [DOI] [PubMed] [Google Scholar]
  40. Xiang Y., Yuan Q., Vogt N., Looger L. L., Jan L. Y., et al. , 2010.  Light-avoidance-mediating photoreceptors tile the Drosophila larval body wall. Nature 468: 921–926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Yang C.-H., Belawat P., Hafen E., Jan L. Y., Jan Y.-N., 2008.  Drosophila egg-laying site selection as a system to study simple decision-making processes. Science 319: 1679–1683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yang C.-H., He R., Stern U., 2015.  Behavioral and circuit basis of sucrose rejection by Drosophila females in a simple decision-making task. J. Neurosci. 35: 1396–1410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zhu E. Y., Guntur A. R., He R., Stern U., Yang C.-H., 2014.  Egg-laying demand induces aversion of UV light in Drosophila females. Curr. Biol. 24: 2797–2804. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

Click here for additional data file. (44.9KB, docx)
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Data Availability Statement

Drosophila lines are available at the Bloomington Drosophila Stock Center or upon request. The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article.

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