Mating-Induced Transcriptome Changes in the Reproductive Tract of Female Aedes aegypti

Abstract

The Aedes aegypti mosquito is a significant public health threat, as it is the main vector of dengue and chikungunya viruses. Disease control efforts could be enhanced through reproductive manipulation of these vectors. Previous work has revealed a relationship between male seminal fluid proteins transferred to females during mating and female post-mating physiology and behavior. To better understand this interplay, we used short-read RNA sequencing to identify gene expression changes in the lower reproductive tract of females in response to mating. We characterized mRNA expression in virgin and mated females at 0, 6 and 24 hours post-mating (hpm) and identified 364 differentially abundant transcripts between mating status groups. Surprisingly, 60 transcripts were more abundant at 0 hpm compared to virgin females, suggesting transfer from males. Twenty of these encode known Ae. aegypti seminal fluid proteins. Transfer and detection of male accessory gland-derived mRNA in females at 0 hpm was confirmed by measurement of eGFP mRNA in females mated to eGFP-expressing males. In addition, 150 transcripts were up-regulated at 6 hpm and 24 hpm, while 130 transcripts were down-regulated at 6 hpm and 24 hpm. Gene Ontology (GO) enrichment analysis revealed that proteases, a protein class broadly known to play important roles in reproduction, were among the most enriched protein classes. RNAs associated with immune system and antimicrobial function were also up-regulated at 24 hpm. Collectively, our results suggest that copulation initiates broad transcriptome changes across the mosquito female reproductive tract, "priming" her for important subsequent processes of blood feeding, egg development and immune defense. Our transcriptome analysis provides a vital foundation for future studies of the consequences of mating on female biology and will aid studies seeking to identify specific gene families, molecules and pathways that support key reproductive processes in the female mosquito.

Fig 1. Transcripts that are significantly differentially expressed after mating.

(A) Venn diagram showing the number of down-regulated (left) and up-regulated (right) transcripts across the three post-mating time points. The up- and down-regulated gene counts are not mutually exclusive, such that transcripts that are both significantly up- and down-regulated are counted in both sets. (B) Histogram of log2 fold-change for transcripts with 2-fold or higher difference in abundance between virgin and post-mating samples (divided into 0.5 log2 bins). Each bin is partitioned into the time-point in which differential expression is detected (DE stage). The histograms are cumulative, such that transcripts that are differentially expressed in multiple time-points are represented multiple times.

Fig 2. Expression patterns of seven genes using quantitative RT-PCR.

Each sample was obtained from the female reproductive tract minus the ovaries at different time points after mating. Each sample represents three different biological replicates, two of them using the expression of the gene RpS17 for normalization and a third using actin expression. Black bars show the results of the quantitative PCR and gray bars show the results of the RNAseq data. Error bars represent standard deviation. A Pearson correlation coefficient shows a positive correlation between qRT-PCR and RNAseq results (R² = 0.912, p-value = 4.88E-6).

Fig 3. Expression profile of transcripts that are differentially expressed between the virgin and 0hpm sample, including their behaviors at 6 and 24hpm.

The color scale represents the median-centered log2 RPKM values. Each row is a transcript and the samples are indicated at the bottom (ordered chronologically from left to right). The top 16 transcripts represent the down-regulated set, while the remaining 60 are those with higher abundances at 0hpm relative to virgin. Transcripts found to be up-regulated in male reproductive organs (MRO)[29], as well as known Sfp genes (Sfp)[39], are indicated on the left by blue (n = 20) and purple markers (n = 33), respectively.

Fig 4. Transfer of eGFP mRNA during mating.

GFP mRNA content in wild type females was measure by qRT-PCR. Each sample was obtained from either wild type Thai females (4 or 5 individuals) mated to AAEL010824-GFP transgenic males or wild type Thai females (4 or 5 individuals) mated to non-transgenic males. Relative expression values were calculated by normalizing the expression with RpS17. This graph represents three technical replicates, with the error bars representing the standard deviation between those three replicates.

Fig 5. Transcripts that are significantly up- or down-regulated at 6 and 24hpm.

(A) Merged clusters from the K-means clustering analysis depicting the four mean expression profiles (red line) among transcripts differentially expressed between virgin and later time-points (6hpm and 24hpm). (B) Pie charts of GO terms associated with up-regulated transcripts (C1 and C2) and down-regulated transcripts (C3 and C4). Only ancestral GO terms are shown for the three ontology classes.