Prostaglandin F2α drives female pheromone signaling in cichlids, revealing a basis for evolutionary divergence in olfactory signaling

Abstract

Pheromones play essential roles in reproduction in many species. Prostaglandin F_2α (PGF_2α) acts as a female reproductive hormone and as a sex pheromone in some species. An olfactory receptor (OR) for PGF_2α was recently discovered in zebrafish, but this signaling pathway is evolutionarily labile. To understand the evolution of signals that attract males to fertile females, we used the African cichlid Astatotilapia burtoni and found that adult males strongly prefer fertile female odors. Injection of a prostaglandin synthesis inhibitor abolishes this attractivity of fertile females, indicating these hormones are necessary for pheromonal signaling. Unlike zebrafish, A. burtoni males are insensitive to PGF_2α, but they do exhibit strong preference for females injected with PGF_2α. This attractiveness is independent of the PGF_2α hormonal receptor Ptgfr, indicating that this pheromone signaling derives from PGF_2α metabolization into a yet-undiscovered pheromone. We further discovered that fish that are insensitive to PGF_2α lack an ortholog for the OR Or114 that zebrafish use to detect PGF_2α. These results indicate that PGF_2α itself does not directly induce male preference in cichlids. Rather, it plays a vital role that primes females to become attractive via an alternative male OR.

Keywords: cichlid; hormone; olfactory receptor; pheromone; prostaglandin.

Fig. 1.

(A) Depiction of the tank setup for olfaction preference test. Stimulus odors are delivered by peristaltic pumps to opposing sides of a tank, and fish locations are tracked over time. Note that the two focal individuals in the arena are separated by a transparent divider so that they can see but will not be able to physically interact with each other. (B) Representative fish positions before and during odor delivery. In these plots, dots represent the position of fish in every video frame during the trials. Note that the distribution of the dots is skewed to the side of stimulus odor, indicating the fish are attracted to this odor cue.

Fig. 2.

Adult males, but not females or sexually immature juvenile males, show an olfactory preference for reproductive female cues. (A) Male A. burtoni exhibit a strong side preference for fertile female odors over nonfertile female (control) odors, suggesting that they can perceive the differences between these two types of females solely by odor cues. (B) Adult female and (C) sexually immature juvenile male cichlids do not prefer fertile female odors. (D) Male cichlids contact the perforated divider associated with fertile female odors more frequently than the divider associated with nonfertile female odor. Contact on the central divider between males and mutual aggressive acts are similar between the preodor and odor stages. Mutual aggressive attack is counted when the pair of focal fish exhibit aggressive display simultaneously across the central divider. Gray squares and lines represent individual behavior performance, while black squares and lines represent mean ± SD. Mann–Whitney U test, n = 10 fish per experiment. n.s., nonsignificant in statistical analysis.

Fig. 3.

Prostaglandin signaling is necessary for production of attractive fertile female cue. (A) IM inhibits the prostaglandin synthesis pathway by antagonizing cyclooxygenases (COX1 and COX2), thereby preventing the conversion of arachidonic acid to prostaglandin H₂, precursor for PGF_2α, PGD₂, and PGE₂. (B) An experimental flowchart for testing the necessity of prostaglandins in mediating female reproductive pheromone signaling. (C) In fertile females, IM treatment significantly reduced waterborne PGF_2α to a level similar to that of nonfertile females 4 h after injection. Mean ± SEM; the ANOVA test followed by Tukey’s test for post hoc analysis. (D) Before IM injection, male cichlids show strong preference to fertile female odors; after IM injection, this preference is significantly reduced. Gray squares and lines represent individual behavior performance, while black squares and lines represent mean ± SD. Mann–Whitney U test, n = 10 fish per experiment. n.s., nonsignificant in statistical analysis.

Fig. 4.

PGF_2α induces attractivity but is not itself attractive. (A) Males do not prefer PGF_2α, three major known metabolites individually, or the mixture of PGF_2α plus five commercially available metabolites. Control is DMSO vehicle. 13,14-DH PGF_2α: 13,14-dihydro PGF_2α; 15K-PGF_2α: 15-keto PGF_2α; PGFM: 13,14-dihydro-15-keto PGF_2α. (B) Males prefer odors from wild-type, nonfertile females which are injected with PGF_2α but not odors from females which are injected with PGD₂ or PGE₂. (C) Males do not prefer odor from nonfertile female-conditioned water spiked with PGF_2α. (D) Males show a mild preference for the odor from the PGF_2α hormonal receptor knockout (ptgfr^−/−) females which were injected with PGF_2α. Gray squares and lines represent individual behavior performance, while black squares and lines represent mean ± SD. Mann–Whitney U test, n = 10 fish per experiment. n.s., nonsignificant in statistical analysis.

Fig. 5.

Phylogeny of teleost fish reveals that olfactory sensitivity to PGF_2α arose after the split of modern teleosts from the basal Lepisosteiformes (gar) species, and was subsequently lost, as inferred by olfactory responses to PGF_2α and the presence of an or114 gene paralog. Fish in six orders, underlined, have been previously tested for an olfactory response to PGF_2α. Colored bars at right indicate the number of gene paralogs for each β OR. Zero indicates that no paralog was detected. Absence of data indicates either no genome sequence available or sequences were not tested. Order names and their lineages are colored based on the presence (red) or absence (green) of an or114 gene paralog. For species that were tested for PGF_2α response, the presence of an or114 gene paralog successfully predicted a positive response. Dotted lineage refers to incomplete or ambiguous data. Paralog count is determined for representative species for each order; for details, see Table 1. Phylogeny adapted from ref. .

Fig. 6.

Genomic search for putative PGF_2α ORs. We identified β ORs through BLASTp searches of vertebrate genomes and then assembled phylogenetic trees. Teleost ORs clustered into OR clades corresponding to Or112, Or113, and Or114 proteins named per zebrafish nomenclature. Basally branching fishes (Reedfish, Erpetoichthys calabaricus, a polypteriform, and sturgeon, Acipenser ruthenus, an acipenseriform) carry ORs that do not cluster with Or113 or Or114, consistent with a model in which Or114 arose following the divergence of their lineages from teleostei. We observed that each species demonstrated to be sensitive to PGF_2α by EOG carries a predicted protein sequence similar to zebrafish Or114. Conversely, no species with an olfactory system shown to be insensitive to PGF_2α has an Or114 ortholog. (Scale bar, 0.2 substitutions per residue.)