A modular circuit architecture coordinates the diversification of courtship strategies in Drosophila

Abstract

Identifying a mate is a central imperative for males of most species but poses the challenge of distinguishing a suitable partner from an array of potential male competitors or females of related species. Mate recognition systems are thus subject to strong selective pressures, driving the rapid coevolution of female sensory cues and male sensory preferences. Here we leverage the rapid evolution of female pheromones across the Drosophila genus to gain insight into how males coordinately adapt their detection and interpretation of these chemical cues to hone their mating strategies. While in some Drosophila species females produce unique pheromones that act to attract and arouse their conspecific males, the pheromones of most species are sexually monomorphic such that females possess no distinguishing chemosensory signatures that males can use for mate recognition. By comparing several close and distantly-related Drosophila species, we reveal that D. yakuba males have evolved the distinct ability to use a sexually-monomorphic pheromone, 7-tricosene (7-T), as an excitatory cue to promote courtship, a sensory innovation that enables D. yakuba males to court in the dark thereby expanding their reproductive opportunities. To gain insight into the neural adaptations that enable 7-T to act as an excitatory cue, we compared the functional properties of two key nodes within the pheromone circuits of D. yakuba and a subset of its closest relatives. We show that the instructive role of 7-T in D. yakuba arises from concurrent peripheral and central circuit changes: a distinct subpopulation of sensory neurons has acquired sensitivity to 7-T which in turn selectively signals to a distinct subset of P1 neurons in the central brain that trigger courtship behaviors. Such a modular circuit organization, in which different sensory inputs can independently couple to multiple parallel courtship control nodes, may facilitate the evolution of mate recognition systems by allowing males to take advantage of novel sensory modalities to become aroused. Together, our findings suggest how peripheral and central circuit adaptations can be flexibly linked to underlie the rapid evolution of mate recognition and courtship strategies across species.

Figure 1.. Sexually ambiguous pheromones do not preclude courtship in the dark.

a, Phylogeny of 99 Drosophila species where cuticular pheromones have been characterized (left) and the primary sex pheromones of the species of the D. melanogaster subgroup (right) (14, 15). 7-T: 7-tricosene; cVA: cis-Vaccenyl acetate; 7,11-HD: 7,11-heptacosadiene; 7,11-ND: 7,11-nonacosadiene; 9,23-TTCD: 9,23-tritriacosadiene. b, Courtship as captured by the inter-fly distance (IFD) between a D. melanogaster (mel), D. erecta (ere), D. simulans (sim), D. yakuba (yak), D. eugracilis (eug), or D. ananassae (ana) male with a conspecific female in the dark. (Left) IFD traces over time for a single representative pair as courtship proceeds. (Middle) Heatmaps for 6 pairs, aligned to courtship initiation for all species except D. eugracilis where initiation of courtship in the dark was never observed and purple dotted line marks the time of first interaction. (Right) Histograms of the time as a function of IFD for same 6 courting pairs. c, Average percent time pairs from b spent at IFD<8mm after first interaction (eug) or after courtship initiation (all other species). Each data point represents the individual pairs shown in the heatmaps in (b). d, Histograms and average percent time D. yakuba males spent at IFD<8mm with oenocyte-less females mock perfumed (oe−) or perfumed with the D. yakuba pheromone 7-T in the dark. Statistical tests performed were ANOVA with Tukey’s post-hoc (c) or unpaired Mann-Whitney (d). Data points represent individual males and error bars are mean±SEM. Letters above data sets denote statistically different groups (p<0.05). Asterisks denote **P<0.01.

Figure 2.. P1 neurons of D. yakuba males share conspecific tuning pattern of dimorphic species.

a, P1 neurons labeled by 71G01>CD8::GFP (D. melanogaster, D. erecta), 71G01>GCaMP6s (D. simulans), or SplitP1>CD8::GFP (D. yakuba) expression, stained for GFP (green) and neuropil counterstain (magenta). Images were masked to remove glial fluorescence from ie1 marker and non-P1-specific labeling for clarity. b, (Left) Representative traces of P1 responses in the LPC in males of each species evoked in response to the taste of a conspecific or heterospecific female (black ticks indicate time of foreleg taps). (Middle) Averaged tap-evoked functional responses (ΔF/F₀, black ticks) of the P1 neurons. (Right) Average peak response (ΔF/F₀) for each male evoked by a given female target. Statistical tests performed were ANOVA with Tukey’s post-hoc. Data points represent individual males and error bars are mean±SEM. Letters above data sets denote statistically different groups (p<0.05).

Figure 3.. Altered pheromone sensitivity in D. yakuba sensory neurons.

a, (Left) Cartoon showing the organization of paired Ppk23+ sensory neurons within chemosensory bristles and bright field image showing expression of CD8::GFP (green) in the soma of the Ppk23+ sensory neurons in the foreleg tarsal segments of D. melanogaster and D. yakuba males. Images are distal end up. (Middle) Cartoon showing the Ppk23+ sensory afferents in the ventral nerve cord (VNC) targeted for recording pheromone responses and anatomic images of Ppk23+ sensory afferents expressing CD8::GFP (green) with neuropil counterstain (magenta). Images are anterior side up. (Right) Representative functional responses of Ppk23+ sensory afferent in a D. melanogaster or D. yakuba male evoked by the taste of a D. melanogaster (green) or D. yakuba (purple) female (black tick marks indicate time of foreleg taps). b, Average courtship bout length in the 10 minutes following courtship initiation for wildtype or ppk23 mutant D. yakuba males towards D. yakuba or D. melanogaster females in the light. c,d, Average courtship bout length of ppk23 mutant D. melanogaster (c) or D. yakuba (d) males towards a conspecific females in the dark. e, (Left) Average functional responses (ΔF/F₀) aligned to time of a tap of indicated conspecific or heterospecific female of the foreleg sensory afferents and (Right) average peak response (ΔF/F₀) per male for a given female target. f, Average courtship bout length of wildtype and ppk25 mutant males toward D. yakuba females in the dark (left) and D. melanogaster females in the light (right). g, Percent time courting prior to (Pre) or during (Stim) periods of optogenetic stimulation of D. yakuba males expressing CsChrimson in Ppk23+ sensory neurons (ppk23>CsChrimson) or control animals (>CsChrimson) paired with a D. melanogaster female. h, Proposed model summarizing the inferred or observed changes in the sensitivity of peripheral sensory populations and the resulting effect on P1 neuron activity. Dotted line represents signaling inferred from previously reported behavioral data (52). Shading in response traces represents mean±SEM. Data points represent individual males and error bars are mean±SEM. Statistical tests performed were ANOVA with Tukey’s post-hoc (e), unpaired Mann-Whitney (b-d, f), or paired t-test (g). Letters above data sets denote statistically different groups (p<0.05). Asterisks denote **P<0.01, ***P<0.001, ****P<0.0001, ns, not significant.

Figure 4.. Sensory specialization of Fru+ and Dsx+ P1 subpopulations in D. yakuba.

a,b, (Left) Maximum intensity projections of neurons labeled by intersection of Fru (a) or Dsx (b) and the P1-driver 71G01 (Fru⋂P1 and Dsx⋂ P1, respectively) in D. yakuba, registered to a common template brain (see methods). (Middle) Averaged functional responses (ΔF/F₀) aligned to time of a tap of indicated conspecific or heterospecific female and average peak response (ΔF/F₀) per male for a given female target. (Right) Total percent time pursuing (top) or performing unilateral wing extensions (bottom) toward a D. melanogaster female target prior to (Pre) or during (Stim) optogenetic stimulation of Fru⋂P1>CsChrimson or Dsx⋂P1>CsChrimson males and the proportion of flies engaging in these behaviors over the course of the experiment. c,d, Functional responses of the Fru+ (c) or Dsx+ (d) neurons innervating the LPC where P1 neurons reside. (Left) Averaged functional responses (ΔF/F₀) aligned to time of a tap of indicated conspecific or heterospecific female and (Right) average peak response (ΔF/F₀) per male for a given female target. e, Functional responses of Dsx+ neurons in the LPC (as in d) in ppk23 mutant males. f,g,h, Courtship in the dark as captured by inter-fly distance (IFD) of a D. yakuba male with constitutively silenced P1 subsets (Fru⋂P1>Kir (g), Dsx⋂P1>Kir (h), or genetic control (f; 71G01-DBD; UAS-Kir))towards a conspecific female in the dark. (Left) IFD traces over time for a single representative pair as courtship proceeds. (Middle) Heatmaps for 6 pairs, aligned to courtship initiation (red dotted line). (Right) Histograms of the time as a function of IFD for same 6 courting pairs. i, Percent time males spent at IFD<8mm after courtship initiation for 6 pairs in f-h, and percent time courting for 10 minutes after first initiation and average bout length for a larger (n=25) manually scored sample of each genotype. Shading in response traces represents mean±SEM. Data points represent individual males and error bars are mean±SEM. Statistical tests performed were ANOVA with Tukey’s post-hoc (a-e, i) or paired t-test (a,b). Letters above data sets denote statistically different groups (p<0.05). Asterisks denote *P<0.05, **P<0.01, ns, not significant.

Fig. 5.. Subspecialization of P1 neuron subtypes.

a, Diagram summarizing proposed sensory specializations of Fru⋂P1 and Dsx⋂P1 in D. yakuba and D. melanogaster. Pheromone inputs inferred from functional imaging and behavioral data in Fig. 4 & Fig. S6 are indicated by black arrows. Additional proposed sensory inputs and behavioral outputs are indicated by gray arrows. D. melanogaster (Mel), D. yakuba (Yak), D. erecta (Ere). b, Model for the diversification of courtship behaviors by neural subspecialization. Independent retuning of sensory inputs to behaviorally redundant but molecularly distinct P1 subtypes may facilitate the rapid evolution of sensory signals that control a male’s sexual arousal and courtship.