Identification of an aggression-promoting pheromone and its receptor neurons in Drosophila

Abstract

Aggression is regulated by pheromones in many animal species. However, in no system have aggression pheromones, their cognate receptors and corresponding sensory neurons been identified. Here we show that 11-cis-vaccenyl acetate (cVA), a male-specific volatile pheromone, robustly promotes male-male aggression in the vinegar fly Drosophila melanogaster. The aggression-promoting effect of synthetic cVA requires olfactory sensory neurons (OSNs) expressing the receptor Or67d, as well as the receptor itself. Activation of Or67d-expressing OSNs, either by genetic manipulation of their excitability or by exposure to male pheromones in the absence of other classes of OSNs, is sufficient to promote aggression. High densities of male flies can promote aggression by the release of volatile cVA. In turn, cVA-promoted aggression can promote male fly dispersal from a food resource, in a manner dependent on Or67d-expressing OSNs. These data indicate that cVA may mediate negative-feedback control of male population density, through its effect on aggression. Identification of a pheromone-OSN pair controlling aggression in a genetic organism opens the way to unravelling the neurobiology of this evolutionarily conserved behaviour.

Figure 1. Synthetic cVA promotes aggression

(a-f) Number of (a) lunges, (b) wing threats, (c) tussles, (d) chases, (e) unilateral wing extensions and (f) circling behaviors (per 20 minutes) performed by pairs of Canton-S (CS) males in the presence of solvent alone or 500 μg cVA (n=28-30). (a′-f′) The temporal distribution of behaviors is shown in raster plots. Each row of spikes represents one fly pair, and each spike represents one occurrence of the behavior. The histogram integrates the occurrences of each behavior in 1-minute bins. (g) Illustration (to scale) of the behavior chamber used for experiments shown in Fig. 1-3. (h,i) Number of all aggressive (h) and courtship (i) actions, based on the data in (a-f). (j) Walking distance of pairs of (n=28-30) or individual (n=16) CS males in the presence of solvent alone or 500 μg cVA. (k,l) Cumulative latency of CS males to copulate with virgin CS females in the presence of solvent (acetone) alone or 500 μg cVA (k; n=29), or solvent alone or 5 mg cVA (l; n=30). (m) Number of lunges performed by pairs of C-S males in the presence of solvent alone, 100 μg cVA, 500 μg cVA or 5 mg cVA (n=20-24). * P<0.05, ** P<0.01 and *** P<0.001. Error Bars are s.e.m. in this and all figures.

Figure 2. Or67d-expressing OSNs mediate the aggression-promoting effect of synthetic cVA

(a,c,d,f) Number of lunges (per 20 minutes) performed by pairs of males of the indicated genotype, in the presence of solvent alone or 500 μg cVA (a,d; n=18-20), or with no added pheromone (c,f; n=20-26). * P<0.05, ** P<0.01 and *** P<0.001. (b,e) Cumulative latency of males of the indicated genotype to copulate with virgin females in the presence of solvent alone or 5 mg cVA (n=20-28). Note that silencing of Or67d-expressing OSNs impairs the suppression of mating by cVA (green open circles in b). *** P<0.001.

Figure 3. Or67d-expressing OSNs are sufficient to mediate the aggression-promoting effect of endogenously produced cVA

In all graphs, the number of lunges (per 20 minutes) performed by pairs of males of the indicated genotype is shown (n=20-34). For this experiment, single-housed male flies, which exhibit a higher baseline level of aggression were used in order to more readily detect the decreased aggression caused by the Or83b mutation. Note that restoration of Or83b expression to Or67d-expressing neurons rescues the loss of aggressiveness in Or83b^-/- flies (c). * P<0.05, ** P<0.01 and *** P<0.001.

Figure 4. cVA promotes aggression at high fly densities and dispersal of male flies from a food resource

(a) Illustration (to scale) of the behavior chamber used for the experiments shown in (b,c). (b) Number of lunges (per 20 minutes) performed by pairs of “tester” Canton-S males together with the indicated number of caged male “donor” flies (n=15); (c) Number of lunges (per 20 minutes) performed by pairs of “tester” males of the indicated genotype, in the presence or absence of 100 male “donor” flies (n=15). *** P<0.001. (d) Illustration (to scale) of the behavior chamber used for the experiments shown in (e-h). (e-h) Number of flies of the indicated genotype on the food cup in the presence of solvent only (blue line) or 500 μg cVA (green line) (n=8). Note gradual dispersal of flies from the food cup in the presence of cVA. Blue bars represent data sets that are significantly different (P<0.05). (h) Flies of the indicated genotype tested in the absence of solvent or cVA (n=10). (i) Model illustrating hypothetical negative feedback regulation of fly population density by cVA-promoted aggression.