SUMMARY Malaria exerts a heavy public health burden, and the emergence of insecticide-resistant mosquitoes and drug-resistant parasites, along with the lack of a broadly effective vaccine, necessitates the development of new methods for disease control. While Wolbachia has been successfully developed for arbovirus control and shown a promising potential for malaria control, the limited choices of Wolbachia-transinfected Anopheles mosquitoes and the lack of knowledge on how external environmental factors and both mosquito and parasite genotypes may influence Wolbachia-mediated parasite-blocking, along with a lack of understanding of the blocking-mechanism, are remaining hurdles for the development of effective Plasmodium transmission- blocking approaches. Compared to other mosquito biological control strategies, Wolbachia-based population replacement is advantageous because it has the potential to provide sustainable disease control at a relatively low cost. To date, only a single Wolbachia transinfected anopheline line has been published, with the establishment of the wAlbB strain in Anopheles stephensi, and this transinfection confers a greater resistance to infection with Plasmodium. We aim to develop novel Wolbachia transinfected lines with enhanced Plasmodium suppressing effect (Aim 1), tolerance to high temperature, and minimal fitness costs, and assess how external environmental factors influence both Wolbachia infection and its ability to block Plasmodium infection (Aim 2) and how both mosquito and parasite genotypes may influence Wolbachia-mediated parasite- suppression and whether the mosquito’s REDOX system is a determining blocking-mechanism (Aim 3). Importantly, this project will develop new tools such as novel Wolbachia transinfected Anopheles lines, and further our knowledge on crucial factors for Wolbachia symbiosis with the Anopheles mosquito vector, and mechanisms involved in Wolbachia-mediated Plasmodium suppression, thereby facilitating the development of Wolbachia-based biocontrol strategies.