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

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; bitter-sensing neurons; dTRPA1.

Figure 1

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 Hdc^JK910. 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.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

Gr66a-expressing neurons are critical for UV avoidance in blind females. (A) Egg-laying PI of proboscis-less norpA³⁶ and proboscis-less Hdc^JK910 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.

Figure 6

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; dTRPA1^KO double-mutant (left bar), norpA³⁶; dTRPA1^KO 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.

Figure 7

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.