Sex Determination Disruption and Intersexuality in Aedes Hybrids

Manipulation of the sex determination pathway can be used for efficient sex separation and effective reduction of target mosquito populations. Understanding the sex determination in mosquitoes need contributions from different perspectives. When male hybrids of Aedes aegypti females and Ae. mascarensis males are backcrossed to Ae. aegypti females, one part of the backcross progeny consists of abnormal males. Our study reveals that male abnormality exhibits three types of morphology at the pupa stage: weak, medium, and strong feminization. At the adult stage of backcrossing generations, there are five types of reproductive organs with varying levels of male abnormalities. By investigating reproductive organs in adults that developed from abnormal pupae, we discovered a significant correlation between the feminization of pupae and the resulting adults. All abnormal individuals were genetic males as they expressed a male determining factor, Nix. We used RT-PCR to examine sex-specific splicing of doublesex and fruitless genes and found that the sex determination pathway was disrupted in abnormal males, resulting in intersex individuals. Through analyzing RNA-seq data from reproductive organs and carcasses of normal males and females of Ae. aegypti and Ae. mascarensis, we have identified distinct gene groups associated with sex, tissue, and species specific and biased expression. Furthermore, our comparison of normal and abnormal individuals revealed a consistent misexpression of male- and female-associated genes in reproductive organs and carcasses of intersexual males. Our study reveals that hybridization between Ae. aegypti and Ae. mascarensis can lead to the disruption of sex determination in backcross males and misregulation of genes associated with sexual development of somatic and germline tissues. which can be utilized for the vector control. These findings provide new insights into the mechanisms of reproductive isolation in species with homomorphic sex chromosomes.