(Supplementary Fig. S8) confirming that most of the genes required for the late stages are transcribed before meiosis, as previously observed also in other model organisms and human spermatogenesis 24,55,56 .

I-PpoI is a homing endonuclease that has a high cleavage activity and specificity for a conserved sequence within the ribosomal rDNA repeats, located in a single cluster on the Anopheles gambiae X chromosome. This property has been exploited to develop a synthetic sex ratio distortion system in this mosquito species. When I-PpoI is expressed from a transgene during spermatogenesis in mosquitoes, the paternal X chromosome is shredded and only Y chromosome-bearing sperm are viable, resulting in a male-biased sex ratio of >95% in the progeny. These distorter male mosquitoes can efficiently suppress caged wild-type populations, providing a powerful tool for vector control strategies. Given that malaria mosquito vectors belong to a species complex comprising at least two major vectors, we investigated whether the sex distorter I-PpoI, originally integrated in the A. gambiae genome, could be transferred via introgression to the sibling vector species Anopheles arabiensis. In compliance with Haldane’s rule, F1 hybrid male sterility is known to occur in all intercrosses among members of the Anopheles gambiae complex. A scheme based on genetic crosses and transgene selection was used to bypass F1 hybrid male sterility and introgress the sex distorter I-PpoI into the A. arabiensis genetic background. Our data suggest that this sex distortion technique can be successfully applied to target A. arabiensis mosquitoes.

Reference list, where references 4-9 are incorrectly numbered as references 8, 7, 4, 9, 5 and 6 respectively. References 4-9 are correctly numbered below: 4. Galizi, R. et al. A synthetic sex ratio distortion system for the control of the human malaria mosquito. Nat. Commun. 5, 3977 (2014). 5. Galizi, R. et al. A CRISPR-Cas9 sex-ratio distortion system for genetic control.

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.