Premeiotic and meiotic failures lead to hybrid male sterility in the Anopheles gambiae complex

Hybrid male sterility contributes to speciation by restricting gene flow between related taxa at the beginning stages of postzygotic isolation. However, we have limited knowledge about cellular processes and molecular mechanisms that come into play when fertility is first affected. Hybrids between closely related species of the Anopheles gambiae complex offer opportunities to identify spermatogenic errors that arise early during speciation. To investigate possible cellular causes of hybrid male sterility, we performed crosses between sibling species of the An. gambiae complex. Our results demonstrate that testes are severely underdeveloped in hybrids between male An. merus and female An. gambiae or An. coluzzii. No meiotic chromosomes are identified in these hybrid males. However, testes have nearly normal morphologies and sizes but produce mostly nonmotile spermatozoa in hybrids from the reciprocal crosses. Using chromosome X– and Y-specific fluorescent probes, we followed the process of meiosis in each species and their F1 hybrids between female An. merus and male An. gambiae or An. coluzzii. Unlike for pure species, sex chromosomes in meiotic prophase I of F1 hybrids are largely unpaired and all chromosomes show various degrees of insufficient condensation. Instead of entering the reductional division in meiosis I, primary spermatocytes undergo an equational mitotic division producing abnormal diploid sperm. Meiotic chromosomes of some F1 hybrid individuals are involved in de novo genome rearrangements. Yet, the germline-specific genes β2-tubulin, Ams, mts, and Dzip1l express normally in these hybrid males. Thus, our study identified cytogenetic errors in hybrids that arise during the early stages of postzygotic isolation. This knowledge will inform the development of innovative mosquito control strategies based on population suppression by manipulating reproduction via genetic technologies. Author Summary The genetic basis and molecular mechanisms of hybrid male sterility are of considerable interest as they inform our understanding of both speciation and normal fertility function. Studies of sterility in male hybrids between recently evolved species offer opportunities to identify developmental errors that arise early in speciation. We performed crosses between sibling species of the Anopheles gambiae complex to gain insights into a cellular basis of postzygotic isolation. We demonstrate that hybrid male sterility in the malaria mosquitoes is caused by two processes in reciprocal crosses: premeiotic arrest in germline stem cells and the failure of the reductional meiotic division in primary spermatocytes. The meiotic abnormalities also include unpairing of sex chromosomes, chromatin decompaction, and, in some cases, de novo genome rearrangements. The failure of the reductional division in meiosis I results in the production of diploid nonmotile sperm. Despite these meiotic errors, tested germline-specific genes express normally in these hybrid males. Thus, our study identified cellular errors in hybrids that arise during the early stages of postzygotic isolation. Studying molecular mechanisms of the developmental abnormalities in testes of hybrids between closely related species of mosquitoes will improve our knowledge of speciation and empower the sterile insect technique.