A pleiotropic chemoreceptor facilitates the production and perception of mating pheromones

Abstract

Optimal mating decisions depend on the robust coupling of signal production and perception because independent changes in either could carry a fitness cost. However, since the perception and production of mating signals are often mediated by different tissues and cell types, the mechanisms that drive and maintain their coupling remain unknown for most animal species. Here, we show that in Drosophila, behavioral responses to, and the production of, a putative inhibitory mating pheromone are co-regulated by Gr8a, a member of the Gustatory receptor gene family. Specifically, through behavioral and pheromonal data, we found that Gr8a independently regulates the behavioral responses of males and females to a putative inhibitory pheromone, as well as its production in the fat body and oenocytes of males. Overall, these findings provide a relatively simple molecular explanation for how pleiotropic receptors maintain robust mating signaling systems at the population and species levels.

Keywords: Biochemistry; Biological sciences; Ecological biochemistry.

Graphical abstract

Figure 1

Gr8a is a chemosensory receptor with sexually dimorphic expression in abdominal tissues (A–F) Gr8a-Gal4 labels cells in the proboscis (A and B) and prothoracic legs (C and D) of both males (top) and females (bottom), but only labels cells in the abdomen of males (E and F). (G) Gr8a has sexually dimorphic mRNA expression in the bodies of flies. Relative mRNA levels were measured by real-time quantitative RT-PCR. Head: p = 0.215, Student’s t test; Appendages: p = 0.377, Student’s t test; Body: p = 0.008, Student’s t test. Depicted as boxplots with inner points plotted, whiskers represent the minimum and maximum values, n = 3/group. (H–L) Axonal projection patterns in the ventral nerve cord (VNC) (dorsal view) and brain (anterior view) of a Gr8a-GAL4>UAS-CD8::GFP (green) male (H, J, and L) and female (I, K, and M). Magenta, neuropil marker (nc82). Scale bars in H-I = 100 μm. Scale bars in J-M = 25 μm. (N and O) Gr8a-Gal4 labeled GRNs are distinct from ppk23 and fru pheromone sensing neurons. (N) Confocal z stack of a male fruP1-LexA>LexAop-myrGFP (green); Gr8a-GAL4>UAS-Red-Stinger (magenta) prothoracic leg. Scale bar = 50 μm (O) Confocal z stack of a male ppk23-LexA>LexAop-CD8::GFP (green); Gr8a-GAL4>UAS-Red-Stinger (magenta) prothoracic leg. Scale bar = 50 μm.

Figure 2

Gr8a is enriched in the male reproductive system and abdominal cells (A) Depiction of the male reproductive system. (B and C) Gr8a-Gal4 labels cells in the seminal vesicles. Confocal z stack images of a Gr8a-GAL4>UAS-Red-Stinger (red) male reproductive system. Turquoise, nuclear marker (DAPI). Scale bar in B = 100 μm. Scale bar in C = 50 μm. (D–G) Gr8a-Gal4 labeled cells and the GR8A protein are enriched in the ejaculatory bulb. Confocal z stack image of the ejaculatory bulb in a Gr8a-GFP; Gr8a-GAL4>UAS-CD4::tdTomato male: (D) Gr8a-Gal4 (magenta); (E) GFP-tagged Gr8a (green); (F) Merge. GR8A protein is enriched where the ejaculatory duct meets the ejaculatory bulb (white arrow). Yellow arrow, duct leading from the ejaculatory bulb to the male genitalia. Scale bar = 50 μm. (G) Inset of F. GR8A protein is enriched along the basal surface of ejaculatory bulb columnar cells. White dashed lines, outline of individual cells; green arrow, GR8A protein sub-cellular localization; white arrow, nucleus of cell; blue, nuclear marker (DAPI). (H–J) Gr8a-Gal4 labels oenocytes and other abdominal cells. Confocal z stack images of oenocytes in a Gr8a-GAL4>UAS-CD8::GFP; desat1>luciferase male: (H) desat1 (green); (I) Gr8a (magenta); (J) Merge. Blue, nuclear marker (DAPI). Scale bar = 50 μm. (K) GR8A protein is enriched in abdominal cells. Confocal z stack of a GFP-tagged Gr8a allele in male abdominal cells; green, anti-GFP; blue, nuclear marker (DAPI). Scale bar = 50 μm.

Figure 3

Gr8a activity contributes to the perception and production of an inhibitory signal associated with mating decisions in males and females (A) Blocking neural activity in female Gr8a-labeled sensory neurons (Gr8a>TNT) shortens copulation latency relative to wild-type controls (Gr8a>TNT^inactive), p = 0.008, Student’s t test. (B) Homozygous Gr8a null females show shortened copulation latency relative to wild-type controls, p = 0.009, Mann Whitney Rank-Sum Test. (C) Homozygous and hemizygous Gr8a null females show shortened copulation latency relative to wild-type controls, p = 0.0006, Kruskal-Wallis Test. Letters above bars indicate statistically significant Dunn’s Test with FDR p value adjustment contrasts between groups. Df(1)BSC663 is a deficiency that covers the Gr8a locus. Df(1)BSC754 was used as a control. (D) Expression of Gr8a cDNA with the Gr8a promoter (Gr8a-;Gr8a^tg) rescues the copulation latency phenotype in Gr8a mutant females, p = 0.022, Kruskal-Wallis Test. Letters above bars indicate statistically significant Dunn’s Test with FDR p value adjustment contrasts between groups. (E) Wild-type females exhibit shortened copulation latency when courted by Gr8a mutant males relative to wild-type males, p = 0.048, Mann Whitney Rank-Sum Test. (F) Gr8a mutant males do not recognize the mating status of females, and have a reduced effect on female post-mating attractiveness, p = 0.004, Kruskal-Wallis Test. Letters above bars indicate statistically significant Dunn’s Test with FDR p value adjustment contrasts between groups. Female, female genotype; Sperm donor, genotype of males mated first with focal females; Focal male, genotypes of experimental males presented with mated females. All assays performed under red light conditions. All data depicted as boxplots with inner points and outliers plotted, whiskers represent the minimum and maximum values, n > 12/group. See Data S1 “P-value data” for exact p values.

Figure 4

Gr8a mutation and knock-down affect the pheromone profiles of males (A and B) Wild-type (wt) and Gr8a mutant (Gr8a⁻) males differ in the relative abundance of individual CHCs. (A) CHCs found in low amounts in males. (B) CHCs found in high amounts in males. Only affected CHCs are shown. See Table 3 for the complete list and exact p values. ∗, p < 0.05, ∗∗, p < 0.01, Student’s t test or Mann Whitney Rank-Sum Test, n = 6 (Gr8a-) or 7 (wt). (C) The Gr8a mutation affects the expression level of several desaturase genes in male abdomens. Only affected genes are shown. See Table 4 for the complete list and exact p values. ∗, p < 0.05, Student’s t test, n = 4/group. (D) Control (desat1 > GFP-RNAi) and oenocyte-specific Gr8a knockdown (desat1 > Gr8a-RNAi) males differ in the relative abundance of individual CHCs. Only affected CHCs are shown. See Table 6 for the complete list and exact p values. ∗, p < 0.05, ∗∗, p < 0.01, Student’s t test or Mann Whitney Rank-Sum Test, n = 10. All data depicted as boxplots with inner points and outliers plotted, whiskers represent the minimum and maximum values. Alkanes denoted as C_n, where n denotes the number of carbon atoms in the chain. Alkenes denoted as N-C_n, where N denotes the location of carbon-carbon double bond in chain. Methyl-branched alkanes denoted as NMe-C_n, where N denotes the carbon in the chain at which the methyl branch occurs.

Figure 5

Gr8a-associated alkenes inhibit normal courtship behaviors (A and B) Perfuming males with a biologically relevant amount of 7-C₂₅ does not affect copulation latency with wild-type females, p = 0.537, Mann Whitney Rank-Sum Test (A) or Gr8a mutant females, p = 0.691, Mann Whitney Rank-Sum Test (B). Perfuming males with a biologically relevant amount of 7-C₂₇ does not affect copulation latency with wild-type females, p = 0.463, Mann Whitney Rank-Sum Test (A), but does affect copulation latency with Gr8a mutant females, p = 0.008, Mann Whitney Rank-Sum Test (B). Depicted as boxplots with inner points and outliers plotted, whiskers represent the minimum and maximum values, n = 15/group. See Data S1 “P-value data” for exact p values.

Figure 6

Sexually dimorphic Gr8a expression across the Drosophila genus may contribute to species-specific differences in male CHC profiles (A) Phylogenetic tree of Drosophila Gr8a proteins. Substitution rate = 0.2. (B) Gr8a mRNA expression is enriched in males relative to females across Drosophila. Black, males; white, females. ∗, p < 0.05; ∗∗, p < 0.01; Mann Whitney Rank-Sum Test, n = 4/group. Live D. grimshawi was not analyzed because live specimens were not available at the Drosophila Species Stock Center (DSSC). (C) Multiple aligned amino acid sequences of Gr8a protein sequences from 12 species across Drosophila. The magenta dashed box highlights a putative hypervariable protein domain. Numbers on top of the alignment indicate amino acid number. Black, 100% identical; Dark Gray, 80–100% similar; Light Gray, 60–80% similar; White, less than 60% similar (Blosum62 score matrix, threshold = 1). Bars below consensus represent overall level of amino acid conservation. (D) Gr8a protein topology. Boxes, transmembrane domains; Red lines, intracellular domain; Blue lines, extracellular domains. (E) In female choice assays, D. melanogaster males court females from most other Drosophila species first at an equal proportion as D. melanogaster females, but court D. mojavensis females first at a lower proportion than D. melanogaster females. Assays performed under red light. ∗, p < 0.05, Pearson’s Chi-squared test. (F and G) Gr8a mutant D. melanogaster males with oenocyte-specific D. melanogaster Gr8a rescue differ in the relative abundance of many CHCs from Gr8a mutant D. melanogaster males with oenocyte-specific D. mojavensis Gr8a rescue. (F) CHCs found in low amounts in males. (G) CHCs found in high amounts in males. Only affected CHCs are shown. See Table 8 for the complete list and exact p values. ∗, p < 0.05, ∗∗, p < 0.01, Student’s t test or Mann Whitney Rank-Sum Test, n = 10. Depicted as boxplots with inner points and outliers plotted, whiskers represent the minimum and maximum values. Gr8a^-; desat1 > Gr8a^melanogaster, D. melanogaster Gr8a oenocyte rescue; Gr8a^-; desat1 > Gr8a^mojavensis, D. mojavensis Gr8a oenocyte rescue. Alkanes denoted as C_n, where n denotes the number of carbon atoms in the chain. Alkenes denoted as N-C_n, where N denotes the location of carbon-carbon double bond in chain. Methyl-branched alkanes denoted as NMe-C_n, where N denotes the carbon in the chain at which the methyl branch occurs. See Data S1 “P-value data” for exact p values.

Figure 7

Model for the pleiotropic action of Gr8a in the perception and production of pheromones (A) Drosophila male. The location of CHC-producing oenocytes is shown in magenta. (B) Gr8a-expressing GRNs are located at the last tarsal segment of the prothoracic legs. (C) Gr8a functions as an inhibitory pheromone receptor in a specific subset of leg GRNs. (D) Oenocytes are the primary CHC-producing cells in the male abdomen. (E) Gr8a functions as an autoreceptor in oenocytes, which regulates CHC synthesis [I-II] and/or CHC secretion [III] via signaling feedback loops.