Metarhizium pingshaense infection reverses insecticide resistance in Anopheles gambiae sensu lato by altering energy reserves and gene expression.

BACKGROUND: Entomopathogenic fungi like Metarhizium are emerging as effective biopesticides against malaria vectors. They reduce mosquito survival, fecundity, and flight ability, and reverse insecticide susceptibility in resistant Anopheles gambiae sensu lato strains. To elucidate the unclear underlying mechanisms, this study investigates the effects of fungal infections and insecticide exposure on the mosquito's energy reserves and the expression of key metabolic and immune genes. METHODS: Three mosquito types: (i) pyrethroid-resistant An. gambiae sensu lato and two laboratory colonies: (ii) pyrethroid-resistant An. coluzzii VKPER and (iii) insecticide-susceptible An. gambiae sensu stricto Kisumu were used. They were infected with Metarhizium pingshaense S10 strain at a concentration of 10⁷ spores/mL (treatment groups) and with solvent only (0.05% Tween® 80; control groups). Live mosquitoes were collected on days 0, 4, and 8 post-infection. They were used to quantify glucose, glycogen, and lipid via Van Handel's protocol and to assess insecticide resistance. For resistance testing, mosquitoes underwent a standard WHO insecticide susceptibility test using deltamethrin (0.05%) or a control. Survival was measured 1 h after exposure, and surviving mosquitoes were analyzed by RT-qPCR for the expression of defensin and CYP6P3, CYP6Z1, and GSTe2. RESULTS: Susceptible An. gambiae Kisumu were eliminated by deltamethrin, while resistant An. coluzzii VKPER and wild An. gambiae s.l. mosquitoes survived. However, deltamethrin exposure following Metarhizium infection significantly reduced survival in these resistant strains compared to the controls. This also resulted in reduced expression levels of defensin, GSTe2, and CYP6Z1 compared to deltamethrin exposure alone, but no difference was found in the expression levels of CYP6P3. These results collectively indicate that Metarhizium infection reduces mosquito survival by impairing their energetic reserves and ability to sustain vital physiological processes, including immune function and metabolic homeostasis. CONCLUSIONS: We demonstrate that Metarhizium infection reverses insecticide resistance in An. gambiae s.l. by depleting energy reserves and suppressing the expression of detoxification genes. This mechanistic insight is crucial for optimizing the future integration of Metarhizium alongside conventional insecticides for malaria vector control.