Single-cell profiling of Anopheles gambiae spermatogenesis defines the onset of meiotic silencing and premeiotic overexpression of the X chromosome

Understanding development and genetic regulation in the Anopheles gambiae germline is essential to engineer effective genetic control strategies targeting this malaria mosquito vector. These include targeting the germline to induce sterility or using regulatory sequences to drive transgene expression for applications such as gene drive. However, only very few germline-specific regulatory elements have been characterised with the majority showing leaky expression. This has been shown to considerably reduce the efficiency of current genetic control strategies, which rely on regulatory elements with more tightly restricted spatial and/or temporal expression. Meiotic silencing of the sex chromosomes limits the flexibility of transgene expression to develop effective sex-linked genetic control strategies. Here, we build on our previous study, dissecting gametogenesis into four distinct cell populations, using single-cell RNA sequencing to define eight distinct cell clusters and associated germline cell–types using available marker genes. We reveal overexpression of X-linked genes in a distinct cluster of pre-meiotic cells and document the onset of meiotic silencing of the X chromosome in a subcluster of cells in the latter stages of spermatogenesis. This study provides a comprehensive dataset, characterising the expression of distinct cell types through spermatogenesis and widening the toolkit for genetic control of malaria mosquitoes

Hybrid incompatibilities in the anopheles gambiae species complex

Malaria is an infectious disease caused by parasites of the genus Plasmodium which is responsible for approximately 400,000 deaths annually, primarily in sub-Saharan Africa. Malaria is transmitted by mosquitoes belonging to the Anopheles gambiae species complex. While progress has been made to reduce the incidence of malaria, the emergence of insecticide resistance necessitates the development of novel vector control strategies. Gene drive technologies have seen significant advances in recent years, providing hope for their implementation in the near future. While gene flow has been identified between sibling species of the An. gambiae species complex, they are reproductively isolated by both pre- and post-zygotic isolation mechanisms. Interspecific crosses between most member species produce sterile hybrid males, in accordance with Haldane’s rule of speciation. The aim of this project was to support the development of gene drive technologies by investigating hybrid incompatibilities between two of the most significant vector species, Anopheles gambiae and Anopheles arabiensis. The potential for the introgression of genomic regions from one species into the genetic background of the other was investigated to help inform models regarding the spread of gene drives between sibling species. In addition, the identification of genetic elements involved in hybrid male sterility could provide potential targets for vector control strategies. Large autosomal regions were found to introgress and persist in interspecific genomes without a detectable fertility cost. In addition, the introduction of distinct autosomal regions of conspecific DNA into otherwise heterospecific genomes of hybrid males was found to overcome hybrid incompatibilities and partially restore fertility. While no specific genetic factors involved in hybrid incompatibilities could be identified, the results indicate that such factors are present at least on the X chromosome. Furthermore, the evidence suggests that asynapsis between interspecific homologous autosomes during gametogenesis plays a role in the manifestation of hybrid male sterility.Open Acces

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

Understanding development and genetic regulation in the Anopheles gambiae germline is essential to engineer effective genetic control strategies targeting this malaria mosquito vector. These include targeting the germline to induce sterility or using regulatory sequences to drive transgene expression for applications such as gene drive. However, only very few germline-specific regulatory elements have been characterised with the majority showing leaky expression. This has been shown to considerably reduce the efficiency of current genetic control strategies, which rely on regulatory elements with more tightly restricted spatial and/or temporal expression. Meiotic silencing of the sex chromosomes limits the flexibility of transgene expression to develop effective sex-linked genetic control strategies. Here, we build on our previous study, dissecting gametogenesis into four distinct cell populations, using single-cell RNA sequencing to define eight distinct cell clusters and associated germline cell–types using available marker genes. We reveal overexpression of X-linked genes in a distinct cluster of pre-meiotic cells and document the onset of meiotic silencing of the X chromosome in a subcluster of cells in the latter stages of spermatogenesis. This study provides a comprehensive dataset, characterising the expression of distinct cell types through spermatogenesis and widening the toolkit for genetic control of malaria mosquitoes

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

Understanding development and genetic regulation in the Anopheles gambiae germline is essential to engineer effective genetic control strategies targeting this malaria mosquito vector. These include targeting the germline to induce sterility or using regulatory sequences to drive transgene expression for applications such as gene drive. However, only very few germline-specific regulatory elements have been characterised with the majority showing leaky expression. This has been shown to considerably reduce the efficiency of current genetic control strategies, which rely on regulatory elements with more tightly restricted spatial and/or temporal expression. Meiotic silencing of the sex chromosomes limits the flexibility of transgene expression to develop effective sex-linked genetic control strategies. Here, we build on our previous study, dissecting gametogenesis into four distinct cell populations, using single-cell RNA sequencing to define eight distinct cell clusters and associated germline cell–types using available marker genes. We reveal overexpression of X-linked genes in a distinct cluster of pre-meiotic cells and document the onset of meiotic silencing of the X chromosome in a subcluster of cells in the latter stages of spermatogenesis. This study provides a comprehensive dataset, characterising the expression of distinct cell types through spermatogenesis and widening the toolkit for genetic control of malaria mosquitoes