Engineering translational delay mechanisms in the malaria mosquito Anopheles gambiae

Malaria is a severe global health issue that causes a significant burden on human populations. In 2021, the World Health Organization estimated that there were 619,000 deaths from malaria, with 96% of these occurring in Sub-Saharan Africa (WHO report, 2022). Although traditional methods to control malaria have saved millions of lives so far, new technologies are needed to address the arising resistances towards insecticides and drugs. It has been demonstrated that inducing an extreme reproductive sex ratio is an effective way to suppress caged populations of Anopheles gambiae, the main malaria vector. This strategy relies on a synthetic sex distortion system that can produce a strong male bias in the progeny of Anopheles mosquitoes. The system works by targeting an X-linked locus present in multiple copies with site-specific endonucleases, such as I-PpoI or CRISPR/Cas9, during male meiosis, which results in the production of unviable X-bearing spermatozoa and unaffected Y-bearing spermatozoa, this leads to a strong male bias in the progeny. It has been theorized that expressing the endonuclease that targets the X chromosome, from the Y chromosome, could result in the clonal transmission of the sex distortion system to all males in the progeny, making this strategy self-sustainable. However, in mosquitoes, both sex chromosomes are subject to a silencing mechanism called meiotic sex chromosome inactivation (MSCI) during male meiosis, which, so far, has hampered the possibility to drive the expression of an X-targeting endonuclease from the Y chromosome. Previous attempts to overcome MSCI were based on expressing the Y-linked endonuclease at an early stage of spermatogenesis when MSCI hasn’t been established yet (Galizi et al., 2014). These attempts have resulted in a toxic effect on the diploid meiotic cell progenitors, leading to the sterility of the male mosquitoes. Here, we developed a follow-up project based on engineering an endogenous mRNA translation control mechanism capable of delaying the translation of mRNAs. In principle, the expression of an endonuclease targeting the X chromosome from the Y chromosomes could happen at an early stage of spermatogenesis, and a delay in the translation of the endonuclease mRNA could be used to overcome toxicity in the meiotic cell progenitors. It has been demonstrated (Barckmann et al., 2013) that protamine genes in D. melanogaster are regulated by translation delay mechanisms. While in D. melanogaster there are multiple protamine and protamine-like genes, in A. gambiae we were able to identify only one orthologous. The role of this gene is currently under investigation through knockout experiments. In this thesis work, we investigated if the protamine sequences that regulate delays of translation in Drosophila, were conserved in the A. gambiae protamine gene. We identified those sequences and designed genetic constructs for the generation of transgenic strains to test the possibility of delaying the expression of genes of interest. Preliminary results point to a great potential for the use of this mechanism in A. gambiae. This could be used in the context of developing a Y drive system or designing alternative strategies for vector control.