Transcriptome profiling reveals sex-specific gene expressions in pupal and adult stages of the mosquito Culex pipiens

Abstract

Understanding the development process of male and female mosquitoes provides important basic information for sterile insect release programmes and is important for improving other vector control strategies. However, little is known about the molecular mechanisms that distinguish male from female-specific developmental processes in this species. We used IlluminaRNA-seq to identify sex-specific genes during pupal and adult stages. One hundred and forty-seven genes were expressed only in pupal males, 56 genes were expressed in adult males and another 82 genes were commonly expressed in both male samples. In addition, 26 genes were expressed only in the pupal females, 163 genes were found in the adult females and only one gene was expressed in both female samples. A further quantitative real-time PCR validation of selected genes from the RNA sequencing (RNA-seq) analysis confirmed upregulation of those genes in a sex-specific manner, including: fibrinogen and fibronectin, a zinc finger protein, phospholipase A(2) and a serine protein for female pupae; venom allergen 3, a perlecan, testis-specific serine/threonine-protein kinase 1, testis-specific serine/threonine-protein kinase 6 and cytochrome c-2 for male pupae; a salivary protein, D7 protein precursor, trypsin 7 precursor, D7 protein and nanos for female adults; and tetraspanin F139, cytosol aminopeptidase, testis-specific serine/threonine-protein kinase 1, a testis-specific serine/threonine-protein kinase 6 and a C-type lectin for male adults. These findings provide insight into the development and physiology of Culex mosquitoes, which will help in the development of more effective control methods for these disease vectors.

Keywords: Culex pipiens; RNA-sequencing; gene expression; gene ontology; genomics; quantitative real-time PCR; vector biology.

Figure 1.

Summary statistics of sex-specific genes from female and male Cx. pipiens (A) Venn diagram of common and unique male-specific genes of pupae and adult Cx. pipiens. (B) Venn diagram of common and unique female-specific genes of pupal and adult Cx. pipiens.

Figure 2.

Functional annotation clustering for sex-specific genes in pupae and adults of Cx. pipiens. (A) Functional clusters of the male-specific genes (B) Functional clusters of the female-specific genes.

Figure 2.

Functional annotation clustering for sex-specific genes in pupae and adults of Cx. pipiens. (A) Functional clusters of the male-specific genes (B) Functional clusters of the female-specific genes.

Figure 3.

Female-specific expression of genes in pupae and adults of the mosquito Cx. pipiens (A) Five genes expressed only in male pupae. (B) Four genes expressed only in female pupae. A student’s t test was performed on the expression levels of male and female replicates for each gene. (*, P< 0.05), (**, P< 0.01). Error bars represent the 95% confidence interval of the average relative expression, n = 4 groups (each group contains 10 individuals). The box indicates the 25th and 75th percentiles and the whiskers caps represent the maximum and minimum values. A center line across the boxes indicates the median. The x in the box represents the mean.

Figure 3.

Female-specific expression of genes in pupae and adults of the mosquito Cx. pipiens (A) Five genes expressed only in male pupae. (B) Four genes expressed only in female pupae. A student’s t test was performed on the expression levels of male and female replicates for each gene. (*, P< 0.05), (**, P< 0.01). Error bars represent the 95% confidence interval of the average relative expression, n = 4 groups (each group contains 10 individuals). The box indicates the 25th and 75th percentiles and the whiskers caps represent the maximum and minimum values. A center line across the boxes indicates the median. The x in the box represents the mean.

Figure 4.

Male-specific expression of genes in pupae and adults of the mosquito Cx. pipiens (A) Five genes expressed only in male adults. (B) Five genes expressed only in female adults. A student’s t test was performed on the expression levels of male and female replicates for each gene. (*, P< 0.05), (**, P< 0.01). Error bars represent the 95% confidence interval of the average relative expression, n = 4 groups (each group contains 10 individuals). The box indicates the 25th and 75th percentiles and the whiskers caps represent the maximum and minimum values. A center line across the boxes indicates the median. The x in the box represents the mean.

Figure 4.

Male-specific expression of genes in pupae and adults of the mosquito Cx. pipiens (A) Five genes expressed only in male adults. (B) Five genes expressed only in female adults. A student’s t test was performed on the expression levels of male and female replicates for each gene. (*, P< 0.05), (**, P< 0.01). Error bars represent the 95% confidence interval of the average relative expression, n = 4 groups (each group contains 10 individuals). The box indicates the 25th and 75th percentiles and the whiskers caps represent the maximum and minimum values. A center line across the boxes indicates the median. The x in the box represents the mean.