Genomic responses to the socio-sexual environment in male Drosophila melanogaster exposed to conspecific rivals

Abstract

Socio-sexual environments have profound effects on fitness. Local sex ratios can alter the threat of sexual competition, to which males respond via plasticity in reproductive behaviors and ejaculate composition. In Drosophila melanogaster, males detect the presence of conspecific, same-sex mating rivals prior to mating using multiple, redundant sensory cues. Males that respond to rivals gain significant fitness benefits by altering mating duration and ejaculate composition. Here we investigated the underlying genome-wide changes involved. We used RNA-seq to analyze male transcriptomic responses 2, 26, and 50 h after exposure to rivals, a time period that was previously identified as encompassing the major facets of male responses to rivals. The results showed a strong early activation of multiple sensory genes in the head-thorax (HT), prior to the expression of any phenotypic differences. This gene expression response was reduced by 26 h, at the time of maximum phenotypic change, and shut off by 50 h. In the abdomen (A), fewer genes changed in expression and gene expression responses appeared to increase over time. The results also suggested that different sets of functionally equivalent genes might be activated in different replicates. This could represent a mechanism by which robustness is conferred upon highly plastic traits. Overall, our study reveals that mRNA-seq can identify subtle genomic signatures characteristic of flexible behavioral phenotypes.

Keywords: RNA-seq; sexual selection; sperm competition; subsampling normalization.

FIGURE 1.

Summary of the number of differentially expressed genes. The number of genes showing consistent differential expression (>|log₂ 1.5| ∼ 0.5 OFC) across replicates in the (A) HT and (B) A body parts, following 2, 26, and 50 h of exposure of males to no rivals versus rivals. Up-regulated (+ rivals > no rivals) or down-regulated (+rivals < no rivals). The number of up (down)-regulated genes in the HT at 2, 26, and 50 h, respectively, was 325 (10), 8 (59), 13 (19) and for the A was 4 (1), 4 (8), and 4 (12).

FIGURE 2.

Differentially expressed genes in two example functionally enriched gene categories. Examples of the frequency density of expression levels for rivals–no rivals for two functional keyword gene groups: (A) G-protein coupled receptor “GPCR” for the HT (94 genes), and (B) “Immune” for the abdomen (Abd) (43 genes). A shift in the positive direction indicates higher expression in the presence of rivals, a shift to the left lower expression. In pink are all the genes corresponding to the keyword category and in green a random control distribution of the same number of genes drawn from the total set of 15,513 genes. For each keyword, the frequency density of gene expression for both replicates is shown for the 2, 26, and 50 h time periods.

FIGURE 3.

Summary model for the responses of males to rivals. Exposure to rivals initiated a rapid early increase at 2 h in the expression of sensory genes, which was shut down by 26 h and then off by 50 h. The pattern in the A was contrasting, with far fewer genes involved and the number of genes changing in expression increased, rather than decreased, over time. In the A there was an early up-regulation in immune genes and by 50 h, changes in seminal fluid protein and hormone genes. Many changes in seminal fluid protein gene expression were inconsistent, and each replicate appeared to switch on different sets of ejaculate component genes.

FIGURE 4.

qRT-PCR validation of mRNA-seq gene expression levels. Variation in expression level in the qRT-PCR and the corresponding mRNA-seq sequencing expression levels are shown for four example loci (no rivals, light gray; rivals, dark gray); Fbgn0015586 (Acp76A), Fbgn0036110 (Cpr67Fb), Fbgn0000276 (CecA), and Fbgn0028396 (TotA). Shown are the normalized expression levels (RNA-seq) or normalized ΔCt expression levels (qRT-PCR) for A (Acp76A, Cpr67Fb, CecA) and HT (TotA) body parts. Sample labels: 02, 26, or 50 h of exposure, rep 1 or 2. Error bars show ± 10% expression level.