Targeting the highly invasive malaria vector, Anopheles stephensi using yeast RNAi pesticides

Apart from widespread resistance of malaria mosquitoes to insecticides, Plasmodium parasite resistance to frontline anti-malaria drugs, and challenges in malaria diagnosis, the World Health Organization (WHO) has described the highly invasive Anopheles stephensi mosquito as a major threat to malaria control. New classes of insecticides are vitally needed for integrated control of this dangerous malaria vector that continues to spread across African countries. Yeast RNAi-based technologies offer a versatile, modular platform capable of providing a comprehensive toolkit for integrated vector management (IVM). Here we explore the multi-functional applications of RNAi yeast technologies for comprehensive control of this significant vector across three fronts: achieving broad-spectrum population suppression through larvicides, adult control using attractive targeted sugar baits (ATSBs), and through the use of female-specific larvicides to facilitate male-only production for use in sterile insect technique (SIT) programs or other population control strategies. Sh.463, a modified Saccharomyces cerevisiae baker’s yeast RNAi pesticide corresponding to the A. stephensi Shaker (Sh) gene, was evaluated in A. stephensi . A scalable ATSB-based system for delivery of Sh interfering RNA pesticides (IRPs) to adult A. stephensi under insectary conditions is examined, and a yeast-based system for delivery of Sh IRP to larvae is developed and evaluated. Additionally, female-specific yeast RNAi larvicides targeting putative A. stephensi female-specific genes for male mosquito sorting are also developed and evaluated in laboratory assays. We demonstrate that the treatment of A. stephensi larvae or adults with Sh.463-56.10R yeast silences the mosquito Sh gene, resulting in high levels of both larval and adult mortality in laboratory studies. Additionally, this study revealed female-specific RNAi yeast larvicides which resulted in significant female mortality in cup bioassays, leading to significantly higher male: female ratios in the resulting offspring. The results of these studies demonstrate that the RNAi yeast expression system provides a versatile insecticide production platform that can support integrated pest control by promoting the production of RNAi yeast larvicides for juvenile control, RNA interference- (RNAi-) yeast-based ATSBs for adult control, and female-specific larvicides to be used to facilitate male separation in support of population control strategies involving male-only releases.