Drosophila TRPA1 channel is required to avoid the naturally occurring insect repellent citronellal

Abstract

Plants produce insect repellents, such as citronellal, which is the main component of citronellal oil. However, the molecular pathways through which insects sense botanical repellents are unknown. Here, we show that Drosophila use two pathways for direct avoidance of citronellal. The olfactory coreceptor OR83b contributes to citronellal repulsion and is essential for citronellal-evoked action potentials. Mutations affecting the Ca(2+)-permeable cation channel TRPA1 result in a comparable defect in avoiding citronellal vapor. The TRPA1-dependent aversion to citronellal relies on a G protein (Gq)/phospholipase C (PLC) signaling cascade rather than direct detection of citronellal by TRPA1. Loss of TRPA1, Gq, or PLC causes an increase in the frequency of citronellal-evoked action potentials in olfactory receptor neurons. Absence of the Ca(2+)-activated K(+) channel (BK channel) Slowpoke results in a similar impairment in citronellal avoidance and an increase in the frequency of action potentials. These results suggest that TRPA1 is required for activation of a BK channel to modulate citronellal-evoked action potentials and for aversion to citronellal. In contrast to Drosophila TRPA1, Anopheles gambiae TRPA1 is directly and potently activated by citronellal, thereby raising the possibility that mosquito TRPA1 may be a target for developing improved repellents to reduce insect-borne diseases such as malaria.

Figure 1

Avoidance to airborne citronellal vapor. (A) Variation of DART assay (Figure S1B) used to assay repulsion of female Anopheles gambiae to citronellal and benzaldehyde (10% each). (B) Avoidance to 0.1 – 10% citronellal vapor. (C) Time-dependence for avoidance to 1% citronellal. (D) Requirement of antenna and Or83b-expressing ORNs for citronellal repulsion. The asterisks indicate significant differences from the wild-type control (ANOVA, p<0.05). (E) Survey of SSR responses of the chemosensory neurons in the 3^rd antennal segment to 10% citronellal. (F) Cartoon indicating location of novel ab11 and ab12 sensilla (green box). (G and H) Representative SSR traces of spontaneous activity demonstrating presence of 3 ORNs (top) and unique odorant response profiles for ab11 (G) and ab12 (H) sensilla to 1% odorants (bottom). Mean ±SEMs are shown. The number (n) of experiments for the SSRs shown in Figures 1E, 1G and 1H are indicated in Table S1.

Figure 2

Requirement for trpA1 for avoidance to airborne citronellal vapor. (A) Avoidance of trp mutants to 1% citronellal. (B) Wild-type and trpA1¹ responses to 0.1 – 10% citronellal. (C) Rescue of the trpA1¹ phenotype with either Drosophila (dtrpA1-A and dtrpA1-B) or Anopheles gambiae (agtrpA1-A and agtrpA1-B) trpA1 transgenes using the GAL4/UAS system. (D) Wild-type and trpA1¹ responses to 0.01 – 10% benzaldehyde. (E) Expression of UAS-trpA1 in ORNs using the Or83b-GAL4 restored citronellal repulsion in trpA1¹. (F – H) Expression of the trpA1 reporter in 2^nd and 3^rd antennal segments using the trpA1-GAL4 and UAS-GFP (green). (F) Staining in a coronal plane of an antenna from a late-stage pupae. GFP positive neurons in the 3^rd antennal segment are indicated with arrowheads. (G) High magnification view of the inset in panel (F). (H) Staining in a transverse plane of an antenna from a late-stage pupae. Error bars represent ±SEMs. The asterisks indicate significant differences from the wild-type control (ANOVA, p<0.05). The pound signs denote significant differences between the indicated flies (unpaired Student t-test, p<0.05).

Figure 3

Expression of TRPA1 in Xenopus oocytes and requirement for PLC and Gqα for avoiding 1% citronellal. Expression of Drosophila TRPA1 (A) and Anopheles TRPA1 (B) in oocytes. (A and B) Filled bars indicate the duration of a stimulus and open bars indicate washouts with stimulus-free buffers. (A) Representative effects of 1 mM citronellal (Mean ΔI SEM= −0.16±0.03 μA, n=6) and a heat shift (~25 to 39 °C, ΔI = −11.0±2.7 μA, n=3) on Drosophila TRPA1. (B) Activation of Anopheles gambiae TRPA1 by increasing concentrations of citronellal (10 μM, ΔI= −3.00±0.68 μA, n=7; 100 μM, ΔI= −5.48±0.78 μA, n=10; and 1 mM, ΔI= −12.0±3.4 μA, n=5). (C) Requirement for PLC (NORPA) and dGqα for citronellal avoidance. (D) Expression of UAS-norpA using the trpA1-GAL4 or Or83b-GAL4 rescued the norpA^P24 phenotype. (E) Requirement of NORPA and dGqα in ORNs for citronellal avoidance. RNAi knockdown of norpA and dGqα using the trpA1-GAL4 or Or83b-GAL4. Asterisks indicate statistically significant differences from wild-type unless indicated with a bracket (ANOVA, p<0.05). The pound sign indicates significant differences between the indicated measurements (unpaired Student t-test, p<0.05). Error bars represent ± SEMs.

Figure 4

Increased action potential frequency in trpA1¹. (A and B) Representative SSR traces from ab11 sensilla stimulated with 10% citronellal. (C) Quantification of deactivation defect observed in ab11a ORNs during 200 ms immediately following citronellal stimulus (n=10). (D) Impairment of citronellal avoidance by mutation or RNAi knockdown of slo. (E) Reduction of citronellal avoidance in the or83b¹ mutant. (F) Dose response to benzaldehyde in Or83b¹ flies. (G) Representative SSR traces from ab11 sensilla in control and Or83b¹. Error bars represent ± SEMs. The asterisks indicate significant differences from the wild-type control (ANOVA, p<0.05). The pound signs denote significant differences between the indicated flies (unpaired Student t-test, p<0.05).