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  • Brief Communications Arising
  • Published:

Ir40a neurons are not DEET detectors

Nature volume 534, pages E5–E7 (2016)Cite this article

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ARISING FROM P. Kain et al. Nature 502, 507–512 (2013); doi:10.1038/nature12594

N,N-Diethyl-meta-toluamide (DEET) is the most widely used insect repellent, but it requires repeated application at high, potentially harmful, concentrations, which is prohibitively impractical and costly in the countries suffering most from insect vector-borne diseases1; understanding DEET’s mode of action might help identify improved alternatives. Kain et al.2 characterized ionotropic receptor 40a (Ir40a)-expressing olfactory sensory neurons (OSNs)—located in the sacculus of the Drosophila antenna3—as comprising a key pathway mediating aversion to both DEET and chemoinformatically predicted ‘DEET-like’ compounds, using calcium imaging, a transcription-based neural activity reporter, Ir40a RNA interference and behavioural experiments. We are unable to reproduce evidence for physiological activation of Ir40a OSNs by these repellents, and find that Ir40a mutant flies avoid DEET. These results call into question the importance of this sensory pathway in DEET detection. There is a Retraction accompanying this Brief Communication Arising by Kain, P. et al. Nature 534, http://dx.doi.org/10.1038/nature18613 (2016).

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Figure 1: Ir40a neurons are activated by ammonia but not DEET, and avoidance of DEET does not require Ir40a or the co-receptor Ir25a.

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Author information

Author notes

  1. Carolina Gomez-Diaz

    Present address: † Present address: Department of Biology, University of Konstanz, 78457, Konstanz, Germany.,

Authors and Affiliations

  1. Center for Integrative Genomics, Faculty of Biology and Medicine, University of Lausanne, Lausanne, CH-1015, Switzerland

    Ana F. Silbering, Rati Bell, Steeve Cruchet, Carolina Gomez-Diaz & Richard Benton

  2. Department of Biology, University of Konstanz, Konstanz, 78457, Germany

    Daniel Münch, Thomas Laudes & C. Giovanni Galizia

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  1. Ana F. Silbering
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  2. Rati Bell
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  5. Carolina Gomez-Diaz
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  8. Richard Benton
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Corresponding author

Correspondence to Richard Benton.

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Extended data figures and tables

Extended Data Figure 1 Odour screen for Ir40a neuron agonists.

Top, representative wide-field fluorescence image of the antennal lobes of an Ir40a-Gal4;UAS-GCaMP3 fly, highlighting the region of interest (marked by a black dashed circle) used for quantification of stimulus-evoked calcium responses in Ir40a neuron axon termini in the ‘column’ glomerulus. Bottom, mean response amplitude ± s.e.m. (n =3–6 flies) for the indicated odours (10% v/v in paraffin oil or water solvent, presented for 1 s as in Fig. 1d). GCaMP3 fluorescence changes during these recordings were not bleach-corrected; thus, the decreases in fluorescence intensity quantified for the vast majority of odours do not reflect stimulus-evoked inhibitory responses.

Extended Data Figure 2 Generation and characterization of an Ir40a mutant.

a, Schematic of the Ir40a locus (exons in green), showing the position of the CRISPR target sequence, and the sequence of the Ir40a1 allele in this region, which contains a 10-base-pair frameshift deletion. PAM, protospacer adjacent motif. b, Immunofluorescence with anti-Ir40a (green) and anti-Ir25a (magenta) on antennal cryosections from wild-type (w1118) and Ir40a1 mutant flies. The merged channels are shown on a bright-field background to provide anatomical landmarks. The arrowhead marks the sensory dendrites where Ir40a and Ir25a co-localize in wild-type flies. The same result was obtained in at least five independent experiments. The scale bar is 20 μm. c, Top: Calcium responses of Ir40a neurons to 3% ammonia in the antennal lobes of control (Ir40a-Gal4;UAS-GCaMP3/+), Ir40a mutant (Ir40a-Gal4,Ir40a1;UAS-GCaMP3/+) and Ir40a rescue (Ir40a-Gal4,Ir40a1;UAS-GCaMP3,UAS-Ir40a/+) animals (n = 9, 11 and 8 flies for control, mutant and rescue, respectively). Bottom: Calcium responses of Ir40a neurons to 3% ammonia in the antennal lobes of control (Ir40a-LexA,LexAop-GCaMP3), Ir25a mutant (Ir25a2;Ir40a-LexA,LexAop-GCaMP3) and Ir25a rescue (Ir25a2,Ir25a-BAC;Ir40a-LexA,LexAop-GCaMP3) animals (n = 8, 7 and 7 flies for control, mutant and rescue, respectively). The bar amplitude corresponds to the median percentage ΔF/F of the peak of the response across flies; the error bars indicate the range between the first and third quartiles of the distribution. All pairwise comparisons were performed using the Wilcoxon Test corrected for multiple testing with Benjamini and Hochberg’s method; *P < 0.05.

Supplementary information

Supplementary Information

This file contains Supplementary Methods, Supplementary References and Acknowledgements. (PDF 252 kb)

Video 1: Responses of IR40a neurons to air stimulation

Raw changes in fluorescence of IR40a OSN soma expressing GCaMP3 (imaged in the antenna by confocal microscopy at 4 Hz) in response to a puff of air (blank control) delivered manually. The ROI is marked by a red circle and the time of stimulation is indicated by the label appearing in the bottom right corner of the image during the corresponding frames. Reproduction rate is 7 fps. (MOV 2341 kb)

Video 2: Responses of IR40a neurons to DEET stimulation

Raw changes in fluorescence of IR40a OSN soma expressing GCaMP3 (imaged in the antenna by confocal microscopy at 4 Hz) in response to a puff of 100% DEET delivered manually. The ROI is marked by a red circle and the time of stimulation is indicated by the label appearing in the bottom right corner of the image during the corresponding frames. Reproduction rate is 7 fps. (MOV 2284 kb)

Video 3: Responses of IR40a neurons to DMSO stimulation

Raw changes in fluorescence of IR40a OSN soma expressing GCaMP3 (imaged in the antenna by confocal microscopy at 4 Hz) in response to a puff of DMSO delivered manually. The ROI is marked by a red circle and the time of stimulation is indicated by the label appearing in the bottom right corner of the image during the corresponding frames. Reproduction rate is 7 fps. (MOV 2243 kb)

Video 4: Responses of IR40a neurons to BA stimulation

Raw changes in fluorescence of IR40a OSN soma expressing GCaMP3 (imaged in the antenna by confocal microscopy at 4 Hz) in response to a puff of 50% BA delivered manually. The ROI is marked by a red circle and the time of stimulation is indicated by the label appearing in the bottom right corner of the image during the corresponding frames. Reproduction rate is 7 fps. (MOV 2266 kb)

Video 5: Responses of IR40a neurons to EA stimulation

Raw changes in fluorescence of IR40a OSN soma expressing GCaMP3 (imaged in the antenna by confocal microscopy at 4 Hz) in response to a puff of 50% EA delivered manually. The ROI is marked by a red circle and the time of stimulation is indicated by the label appearing in the bottom right corner of the image during the corresponding frames. Reproduction rate is 7 fps. (MOV 2314 kb)

Video 6: Responses of IR40a neurons to MDA stimulation

Raw changes in fluorescence of IR40a OSN soma expressing GCaMP3 (imaged in the antenna by confocal microscopy at 4 Hz) in response to a puff of 50% MDA delivered manually. The ROI is marked by a red circle and the time of stimulation is indicated by the label appearing in the bottom right corner of the image during the corresponding frames. Reproduction rate is 7 fps. (MOV 2263 kb)

Video 7: Responses of IR40a neurons to H2O stimulation

Raw changes in fluorescence of IR40a OSN soma expressing GCaMP3 (imaged in the antenna by confocal microscopy at 4 Hz) in response to a puff of H2O delivered manually. The ROI is marked by a red circle and the time of stimulation is indicated by the label appearing in the bottom right corner of the image during the corresponding frames. Reproduction rate is 7 fps. (MOV 2392 kb)

Video 8: Responses of IR40a neurons to ammonia stimulation

Raw changes in fluorescence of IR40a OSN soma expressing GCaMP3 (imaged in the antenna by confocal microscopy at 4 Hz) in response to a puff of 10% ammonia delivered manually. The ROI is marked by a red circle and the time of stimulation is indicated by the label appearing in the bottom right corner of the image during the corresponding frames. Reproduction rate is 7 fps. (MOV 2285 kb)

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Silbering, A., Bell, R., Münch, D. et al. Ir40a neurons are not DEET detectors. Nature 534, E5–E7 (2016). https://doi.org/10.1038/nature18321

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