Single-cell profiling of mosquito spermatogenesis defines the onset of meiotic silencing and pre-meiotic overexpression of the X chromosome.

Abstract

Understanding of the Anopheles gambiae germline has been essential to acquiring effective genetic control strategies targeting this malaria mosquito vector. This includes targeting germline genes to induce sterility or using regulatory sequences to drive transgene expression for applications such as gene drive. However, only very few germline regulatory elements have been characterised with the majority showing leaky expression. This has been shown to significantly reduce the efficiency of current genetic control strategies, which rely on regulatory elements that are more tightly restricted in their spatial and/or temporal expression. Additionally, meiotic silencing of the sex chromosomes during spermatogenesis limits the flexibility of transgene expression for design of novel genetic control strategies. In this work, we build on our previous study that dissected gametogenesis into four distinct cell populations. We used single-cell RNA sequencing to further discriminate these populations and define distinct germline cell-types. In doing so, we revealed an overexpression of X-linked genes in the germline stem cells (GSCs) and were able to pinpoint the onset of meiotic silencing of the X chromosome in the spermatogonia/primary spermatocytes. This study provides a comprehensive dataset to identify genes expressed at specific stages of spermatogenesis, widening the toolkit for genetic control of malaria mosquitoes.

scRNA-seq reveals novel genetic pathways and sex chromosome regulation inTriboliumspermatogenesis

“…We observed that expression of Xchromosome genes is already extremely depressed in GSC and rises in expression until secondary spermatocytes. The main difference with other species here is the scale, as we only observed an X-Autosome expression ratio of 0.2% while other insect species note minima never crossing below 20% Page et al, 2023). This implies an overall suppression of the X chromosome and MSCI more absolute than other species and localized entirely to meiotic cells unlike what has been observed in Drosophila (Mikhaylova & Nurminsky, 2011;Meiklejohn et al, 2011;Vibranovski et al, 2012;Landeen et al, 2016;Witt et al, 2021;Mahadevaraju et al, 2021;Prince et al, 2010).…”

“…This atlas gives us the ability to directly compare to other studies in Drosophila and mosquito Page et al, 2023). We did not observe separate clusters for germline and cyst cells, likely due to a lack of known beetle markers for cyst cells for the weighted principal components, however the mosquito study also did not identify separate clusters for cyst cells.…”

“…We found little correlation between Drosophila or Anopheles markers of spermatogenesis and marker expression in Tribolium (Table 1, Supplementary Figure 1k-m) Page et al, 2023). This is because essential markers for sperm staging in the two dipterans such as vasa, fzo, or bam , does not seem to play the same role in beetle spermatogenesis.…”

“…The fact that Tribolium was missing or lacked expression of key Drosophila spermatogenesis genes like fzo, esg, and tj made the overall analysis difficult but highlights the rapid evolutionary dynamics of male gamete development. Many of these genes are shared in mosquitoes which, like fruit flies, belong to the order Diptera (Page et al, 2023). The fact that these genes are not expressed in Coleoptera spermatogenesis suggests that critical genes that have been well studied in Drosophila, and suggested to be critical in insect sperm development, may not actually be reflective of the larger class and phylum.…”

“…MSCI occurs when unsynapsed X and Y chromosomes are silenced during the meiotic stages of spermatogenesis and is thought to be related to a phylogenetically widely conserved surveillance system that protects against aneuploid gametes by condensing unsynapsed chromatin (MSUC) (Turner et al, 2005). This has been observed in insects through high-throughput single cell analysis experiments of both Drosophila melanogaster and Anopheles gambiae Page et al, 2023). This type of analysis provides high level detail of cell specific differentiation in tissue maps but is limited by the need for quality molecular evidence usually associated with well-studied model organisms (Alfieri et al, 2022).…”