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Evolution of a central neural circuit underlies Drosophila mate preferences

Nature (2018) | Download Citation


Courtship rituals serve to reinforce reproductive barriers between closely related species. Drosophila melanogaster and Drosophila simulans exhibit reproductive isolation, owing in part to the fact that D. melanogaster females produce 7,11-heptacosadiene, a pheromone that promotes courtship in D. melanogaster males but suppresses courtship in D. simulans males. Here we compare pheromone-processing pathways in D. melanogaster and D. simulans males to define how these sister species endow 7,11-heptacosadiene with the opposite behavioural valence to underlie species discrimination. We show that males of both species detect 7,11-heptacosadiene using homologous peripheral sensory neurons, but this signal is differentially propagated to P1 neurons, which control courtship behaviour. A change in the balance of excitation and inhibition onto courtship-promoting neurons transforms an excitatory pheromonal cue in D. melanogaster into an inhibitory cue in D. simulans. Our results reveal how species-specific pheromone responses can emerge from conservation of peripheral detection mechanisms and diversification of central circuitry, and demonstrate how flexible nodes in neural circuits can contribute to behavioural evolution.

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We thank Y. Ding for D. simulans CsChrimson flies; P. Pires-Mussells and J. Marquina-Solis for assistance on initial experiments; E. Clowney, B. Matthews, K. Kistler, J. Petrillo and G. Maimon for technical advice; and E. Clowney, S. Datta, B. Noro, L. Vosshall, S. Shaham, C. McBride and members of the Ruta laboratory for discussion and comments on the manuscript. This work was supported by the New York Stem Cell Foundation, the Pew Foundation, the McKnight Foundation, the Irma T. Hirschl Foundation, the Alfred P. Sloan Foundation and the National Institutes of Health (DP2 NS0879422013) to V.R. and a National Science Foundation and Kavli Fellowship to L.F.S.

Reviewer information

Nature thanks B. Prud’homme and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information


  1. Laboratory of Neurophysiology and Behavior, The Rockefeller University, New York, NY, USA

    • Laura F. Seeholzer
    • , Max Seppo
    •  & Vanessa Ruta
  2. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA

    • David L. Stern


  1. Search for Laura F. Seeholzer in:

  2. Search for Max Seppo in:

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L.F.S. and V.R. conceived and designed the study. L.F.S. and M.S. performed behavioural experiments and generated receptor mutants and fru alleles in D. simulans. D.L.S. generated R71G01, R25E04 and GCaMP strains in D. simulans. M.S. performed immunohistochemistry experiments. L.F.S. performed functional and anatomic tracing experiments. L.F.S. and V.R. analysed data and wrote the manuscript with input from all the authors.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Vanessa Ruta.

Extended data figures and tables

  1. Extended Data Fig. 1 Pheromone regulation of D. simulans courtship.

    Mutant males and males lacking foreleg tarsi still court, but display altered courtship preferences. a, Courtship indices of males with foreleg tarsi intact (+) or surgically removed (−). Data are replotted from Fig. 1b. b, c, Courtship indices of D. melanogaster (b) and D. simulans (c) males with either foreleg tarsi or rear leg tarsi ablated towards D. melanogaster or D. simulans females. d, Schematic of CRISPR–Cas9-induced mutations (top) in Gr32a (left) and ppk23 (right) gene loci. Cas9 was targeted by gRNA to the first exon (cut site) of Gr32a or ppk23 resulting in a 36-bp insertion/2-bp deletion in the Gr32a coding sequence and 90-bp insertion into the ppk23 coding sequence. Both indels generated in-frame stop codons (bottom, asterisk highlighted red in resulting amino acid sequence). Forward (F) and reverse (R) genotyping primers are marked with a line. e, Courtship indices (CI) towards females of different Drosophila species by wild-type (WT), Gr32a−/− and ppk23−/− D. simulans males. The major pheromone carried by each female is listed. f, Courtship indices of D. simulans males towards D. melanogaster and D. simulans females in preference assays. Data are replotted from Fig. 1d. g, Courtship indices of D. simulans males towards D. simulans females perfumed with 7,11-HD (7, green) or ethanol (E, EtOH, blue). Data are replotted from Fig. 1e. The statistical test performed in e was a Kruskal–Wallis test, and different letters mark significant differences. Data are mean and s.d., with individual data points shown. Lines connect courtship indices of the same male towards the different female targets in a preference assay. Because the male can only court one female at a time, the paired points are inherently interdependent on each other, therefore inappropriate for statistical analysis. See Supplementary Table 1 for details of statistical analyses.

  2. Extended Data Fig. 2 Anatomic and functional conservation of Fru+ neurons.

    a, Left, schematic of chromosomal location of fruattP and fru−/− integration sites in D. simulans and previously generated fruGal4 and fruLexA transgenes in D. melanogaster; right, schematic of attP oligonucleotide integrated into the fru intron to generate fruattP allele and subsequent integration of attB plasmids (right). ExF and ExR are primers located in the genome and InR is a primer located inside the transgene. bg, Maximum intensity confocal (bd) and two-photon stacks (eg) of anatomically defined regions of Fru+ neuropil in D. melanogaster fruGal4>UAS-GCaMP and D. simulans fruGFP males: LPC (b, c), SEZ (d), antennal lobe (e), lateral horn (f) and mushroom body γ-lobes (g). Scale bars, 10 μm. h, D. simulans fru−/− was generated by integrating an oligonucleotide that deleted codons 1 and 2 of the first exon, introducing a frame-shift mutation. i, Male–male chaining indices of wild-type (+/+) and fru−/− males. A paired t-test was used, data are mean and s.d. and individual data points are shown. See Supplementary Table 1 for details of statistical analyses.

  3. Extended Data Fig. 3 Conserved anatomy and functional tuning of Ppk23+Fruitless+ foreleg sensory neurons.

    ac, ppk23 promoter expression in D. melanogaster and D. simulans males in forelegs (a), ventral nerve cord (VNC, b) and brain (c). GFP, green; differential interference contrast, grey; neuropil counterstain, magenta. a, Forelegs (top left) with paired soma (bottom left) and quantification of number of Ppk23+ sensory neuron soma in the first three tarsal segments of the foreleg (right). d, Ppk23 neuron innervation in the first thoracic ganglion of the VNC of D. simulans females. Ppk23+ sensory neurons display a characteristic sexually dimorphic expression pattern in the VNC, where they do not cross the midline in females, but do in males. e, Schematic of VNC imaging preparation (left); functional responses evoked by individual taps of a female abdomen in Fru+ neurons (middle) and Ppk23+ neurons (far right) in the VNC of wild-type and ppk23−/− D. melanogaster and D. simulans males. Data replotted from Fig. 3b–d. f, Schematic of Ppk23+ somatic imaging preparation (left) and functional responses of the paired neurons (cell A and B, see Methods) within a sensory bristle stimulated with 7,11-HD or ethanol (middle); comparison of 7,11-HD responses in Ppk23+ soma across species (far right). Statistical tests used were an unpaired t-test (a), a Kruskal–Wallis test, for which different letters mark significant differences (e), and paired and unpaired t-tests (f). Scale bars, 10 μm. See Supplementary Table 1 for details of statistical analyses.

  4. Extended Data Fig. 4 Behavioural and functional analysis of P1 neurons.

    af, Anatomy (a) and optogenetic behavioural manipulations (bf) of P1 neurons using R71G01-Gal4 to drive the expression of CsChrimson. b, Courtship indices (top, right) towards a rotating magnet by D. melanogaster males pre-, during, and post-P1 neuron optogenetic stimulation. Fraction of male flies courting (bottom; grey boxes indicate bright light illumination, see Methods). c, Courtship indices towards a magnet moving at different speeds during optogenetic P1 neuron stimulation in D. simulans males. df, Comparison of courtship indices towards magnet (d), D. simulans female (e), or D. melanogaster female (f) by D. simulans males of denoted genotypes, either fed or not fed retinal. g, Experimental set-up used to measure pheromone responses in the P1 neurons in vivo (top left) and overlay of the Fru+ (green) neurons and fasciculated P1 neuron processes (magenta). The white boxes indicate the approximate ROI used to image P1 responses. hj, Functional responses evoked by individual taps of a female abdomen in P1 neurons (h) and Fru+ neurons in the LPC (i, j) of wild-type and ppk23−/− D.melanogaster and D. simulans males (data replotted from Fig. 4e, f). Statistical tests used were the Kruskal–Wallis test (b, df, hj) and a one-way ANOVA (c). Data are mean and s.d., with individual data points shown. See Supplementary Table 1 for details of statistical analyses.

  5. Extended Data Fig. 5 Anatomy of P1, vAB3 and mAL neurons in D. simulans and D. melanogaster males.

    ac, Detailed anatomic images of P1 neurons (a), vAB3 neurons (b) and mAL neurons (c). Schematic of neural anatomy (left), Texas-Red dextran dye-fill (red) in D. simulans fruGFP (green) males (middle-left), magnified view of labelled neurons in the LPC showing dye-filled neurons (red) and Fru+ neurons (green) or just dye-filled neurons (black, middle-right) and photoactivated neurons in D. melanogaster LPC (black, right). d, Antibody staining of D. simulans Fru+ neurons (anti-GFP, green) with anti-GABA (red) in the SEZ and the LPC demonstrating that mAL neurons are GABAergic and thus inhibitory. Scale bars, 10 μm.

  6. Extended Data Fig. 6 Pheromone responses in central neurons of D. melanogaster and D. simulans males.

    a, Schematic of in vivo preparation used to measure pheromone responses in vAB3 processes in the brain (top). Representative fluorescence increase of vAB3 responses in a D. melanogaster male evoked by tapping a D. melanogaster female (bottom left). GCaMP was expressed in vAB3 neurons using the AbdB-Gal4 driver. Anatomy of fasciculated vAB3 processes co-labelled by fruGal4 (green) and AbdB-Gal4 (magenta) in the same in vivo preparation used for imaging (right). The white box indicates the approximate ROI analysed for functional imaging. bd, Functional responses evoked by the taste of female pheromones in the vAB3 processes of wild-type and ppk23−/− males. b, Functional responses evoked by individual taps in vAB3 neurons in D. melanogaster labelled using AbdB-Gal4. c, Average responses of vAB3 neurons in ppk23−/− D. melanogaster and D. simulans males in response to the taste of D. melanogaster (m) and D. simulans (s) females. GCaMP was expressed in vAB3 neurons using fruGal4. d, Functional responses evoked by individual taps in vAB3 neurons in wild-type and ppk23−/− mutant males. Data replotted from Fig. 5c and Extended Data Fig. 6c. e, Expression of 25E04-Gal4>UAS-GCaMP (green) with neuropil counterstain (magenta) in the brains of D. melanogaster (left) and D. simulans (right) males. f, Courtship indices towards conspecific females during optogenetic activation of mAL neurons in D. simulans males with parental controls. g, Functional responses evoked by individual taps in mAL neurons. Data replotted from Fig. 5d. h, Average ΔF/F responses in Fru+ neurons of the LPC evoked by the taste of a female before and after local injection of picrotoxin, a GABAA receptor antagonist, into the LPC. In males of both species, application of picrotoxin increased responses only to D. melanogaster female stimuli. Lines connect average functional responses in the same male towards the different female targets. Statistical tests used were the Kruskal–Wallis test, in which different letters mark significant differences (b, d, f, g), and the paired t-test (c, h). Data are mean and s.d., with individual data points shown. See Supplementary Table 1 for details of statistical analyses.

  7. Extended Data Fig. 7 Functional responses of Fru+ neurons to direct vAB3 stimulation in D. melanogaster and D. simulans males.

    a, Schematic and representative image depicting direct stimulation of vAB3 neurons by iontophoresis of neurotransmitter onto their dendrites within the VNC. Maximum intensity z-projection (top) and single z-plane (bottom) showing electrode placement in the VNC. The electrode is filled with neurotransmitter and Texas-red dye (red) to enable precise targeting in the Fru+ neuropil (green). b, Representative multi-plane fluorescence increase of Fru+ brain neurons in D. simulans males when the VNC is stimulated with acetylcholine (left), glutamate (middle) and saline (right) iontophoresis. c, To test the necessity of vAB3 in propagating signals from the VNC to the higher brain, we compared response profiles in the brain before severing vAB3 (black), after severing a nearby Fru+ axon (mock control, blue) and then after severing vAB3 axons (orange) in D. melanogaster males (top) and D. simulans males (bottom). Representative multi-plane fluorescence increase of Fru+ neurons (left) shows the loss of evoked functional responses in the brain after severing vAB3. The graph (middle) depicts the relationship between average ΔF/F responses in vAB3 and mAL. Average ΔF/F responses of vAB3 (green) and mAL (red) neurons evoked by vAB3 stimulation, before and after severing vAB3 (right). Responses were lost in both neural populations in both species after vAB3 was severed but not in the mock control. d, The relationship of functional responses in the SEZ and LPC of D. melanogaster and D. simulans males evoked by direct vAB3 stimulation. The dots on the graph represent different stimulation intensities and the trend lines relate the responses of individual D. melanogaster (green) and D. simulans (blue) males. e, Left, the response of vAB3 (top) and mAL (bottom) axonal tracts in response to vAB3 stimulation in D. melanogaster (green) and D. simulans (blue) males. Coloured lines represent single stimulations and black lines represent the average. Right, peak ΔF/F plotted with coloured dots representing the average response per fly and data are mean and s.d. Individual data points are shown. f, g, Comparison of vAB3-evoked responses in D. melanogaster and D. simulans males in vAB3 neurons (f) and P1 neurons (g) before (+) and after (−) mAL severing. Statistical tests used were the Kruskal–Wallis test, in which different letters mark significant differences (c, f, g), and an unpaired t-test (e). Scale bars, 10 μm. See Supplementary Table 1 for details of statistical analyses.

Supplementary information

  1. Supplementary Tables

    This file contains Supplementary Table 1 which contains the genotypes for all experiments and details of the statistical analyses, and Supplementary Table 2 containing a list of primers used for cloning, genotyping and CRISPR mutagenesis.

  2. Reporting Summary

  3. Video 1

    Optogenetic activation of P1 neurons in D. simulans (71G01-Gal4 >UAS-CsChrimson) is sufficient to induce courtship of a rotating magnet.

  4. Video 2

    In vivo imaging of the lateral protocereberal complex (LPC) in a D. melanogaster fruGal4>UAS-GCaMP male in response to the taste of a D. melanogaster female.

  5. Video 3

    In vivo imaging of the lateral protocereberal complex (LPC) in a D. melanogaster fruGal4>UAS-GCaMP male in response to the taste of a D. simulans female. Male is the same as in Supplementary Video 2.

  6. Video 4

    In vivo imaging of the lateral protocereberal complex (LPC) in a D. simulans fruGal4 >UAS-GCaMP male in response to the taste of a D. simulans female.

  7. Video 5

    In vivo imaging of the lateral protocereberal complex (LPC) in a D. simulans fruGal4>UAS-GCaMP male in response to the taste of a D. melanogaster female. Male is the same as in Supplementary Video 4.

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