The Role of miRNAs in Drosophila melanogaster Male Courtship Behavior

Abstract

Drosophila melanogaster courtship, although stereotypical, continually changes based on cues received from the courtship subject. Such adaptive responses are mediated via rapid and widespread transcriptomic reprogramming, a characteristic now widely attributed to microRNAs (miRNAs), along with other players. Here, we conducted a large-scale miRNA knockout screen to identify miRNAs that affect various parameters of male courtship behavior. Apart from identifying miRNAs that impact male-female courtship, we observed that miR-957 mutants performed significantly increased male-male courtship and "chaining" behavior, whereby groups of males court one another. We tested the effect of miR-957 reduction in specific neuronal cell clusters, identifying miR-957 activity in Doublesex (DSX)-expressing and mushroom body clusters as an important regulator of male-male courtship interactions. We further characterized the behavior of miR-957 mutants and found that these males court male subjects vigorously, but do not elicit courtship. Moreover, they fail to lower courtship efforts toward females with higher levels of antiaphrodisiac pheromones. At the level of individual pheromones, miR-957 males show a reduced inhibitory response to both 7-Tricosene (7-T) and cis-vaccenyl acetate, with the effect being more pronounced in the case of 7-T. Overall, our results indicate that a single miRNA can contribute to the regulation of complex behaviors, including detection or processing of chemicals that control important survival strategies such as chemical mate-guarding, and the maintenance of sex- and species-specific courtship barriers.

Keywords: 7-T; Genetics of Sex; behavior; cVA; male courtship; miRNA; pheromones.

Figure 1

Selection of miRNA KO strains and results summary for original screen. (A) Eighty miRNA KO strains (Chen et al. 2014) in the w1118 background were selected for a screen to evaluate effects of miRNA KO alleles on male courtship behaviors. After removing those strains that did not survive as homozygotes (20 strains) or had low viability as homozygotes (3 strains), 57 strains were used to compare courtship parameters with those of a control strain. (B) Number of strains showing significant differences in the behavioral parameters tested. CI was calculated for only three strains, out of which one strain had significantly altered CI. CI, courtship index; KO, knockout; miRNA, microRNA.

Figure 2

tCCI in grouped miR-957 KO males. (A) tCCI for miR-957 KO mutants, miR-957 KO/Df transheterozygotes, and flies with neuronal rescue of miR-957 KO compared with respective controls. Plotted tCCI values are the average of tCCI values for three groups of 10 males each for every genotype. tCCI values were compared between mutants and controls using Student’s t-tests. (B) miR-957 KO males displaying pairwise courtship or chaining. Error bars represent mean ± SE (SEM). Df, chromosomal deficiency; KO, knockout; tCCI, total courtship and chaining index.

Figure 3

tCCI (total courtship and chaining index) for grouped males with tissue-specific reduction of miR-957. Ubiquitous (actin-gal4), pan-neuronal (elav-gal4), or neuron subpopulation (via fru-gal4, dsx-gal4, or D52H-gal4) expression of UAS-miR-957.sponge. Plotted tCCI values are the average of tCCI values for three groups of 10 males each for every genotype. tCCI values for each miR-957.sponge and gal4 combination were compared to the respective scramble control using Student’s t-tests. Error bars represent mean ± SE (SEM).

Figure 4

miR-957 KO courtship and elicitation assays. (A) Courtship of miR-957 KO (green thorax) or control males toward decapitated Canton-S male subjects. (B) Courtship elicited by control or miR-957 KO males from Canton-S males. The possibility that miR-957 mutant males have altered pheromone profiles is indicated by the purple coloration of the pheromone. CI values for experimental and control groups were compared using the Mann–Whitney U-test. N = 25–30 for each genotype. Error bars represent mean ± SE (SEM). CI, courtship index; KO, knockout.

Figure 5

miR-957 mutant males prefer females. The proportion of miR-957 knockout and control males that choose to court females or males for the majority of a 10-min two-choice assay. Results were compared using a χ² test. N = 18 (control) and N = 21 (miR-957).

Figure 6

miR-957 mutant males do not significantly reduce courtship efforts toward mated D. melanogaster females. (A) The proportion of miR-957 KO or control males that court decapitated D. melanogaster mated or virgin females during the last 5 min of a 30-min assay. (B) CI of miR-957 KO or control males toward decapitated D. melanogaster mated or virgin females during the last 10 min of a 30-min assay. CI values were compared using Mann–Whitney U-tests. N = 28–30 for each genotype. Error bars represent mean ± SE (SEM). CI, courtship index; CS, Canton-S; KO, knockout.

Figure 7

miR-957 mutant males do not significantly reduce courtship efforts toward virgin D. simulans females. (A) The proportion of miR-957 KO or control males that court decapitated virgin D. simulans or D. melanogaster females during the last 5 min of a 30-min assay. (B) CI of miR-957 KO males or control males toward decapitated virgin D. simulans or D. melanogaster females during the last 10 min of a 30-min assay. CI values were compared using Mann–Whitney U-tests. N = 28–30 for each genotype. Error bars represent mean ± SE (SEM). CI, courtship index; CS, Canton-S; KO, knockout.

Figure 8

miR-957 mutant males show reduced inhibitory response to 7-T and cVA. (A) CI of miR-957 KO males or control males toward 7-T- or hexane-treated females during the last 10 min of a 30-min assay. N = 28–32 for each genotype. (B) CI of miR-957 KO males or control males toward cVA- or ethanol-treated females during the last 10 min of a 30-min assay. N = 27–31 for each genotype. CI values were compared using one-way ANOVA followed by post hoc Tukey–Kramer tests. Error bars represent mean ± SE (SEM). CI, courtship index; cVA, cis-vaccenyl acetate; KO, knockout; n.s., not significant; 7-T, 7-Tricosene.

Figure 9

Predicted targets of miR-957. (A) Top six weighted targets from TargetScan Fly predictions. (B) Total courtship and chaining index (tCCI) for grouped males expressing UAS-miR-957.sponge in gustatory receptor neurons. tCCI values for each miR-957.sponge and gal4 combination were compared to the respective scramble control using Student’s t-tests. Error bars represent mean ± SE (SEM).