Improved efficacy of an arthropod toxin expressing fungus against insecticide-resistant malaria-vector mosquitoes

Abstract

The continued success of malaria control efforts requires the development, study and implementation of new technologies that circumvent insecticide resistance. We previously demonstrated that fungal pathogens can provide an effective delivery system for mosquitocidal or malariacidal biomolecules. Here we compared genes from arthropod predators encoding insect specific sodium, potassium and calcium channel blockers for their ability to improve the efficacy of Metarhizium against wild-caught, insecticide-resistant anophelines. Toxins expressed under control of a hemolymph-specific promoter increased fungal lethality to mosquitoes at spore dosages as low as one conidium per mosquito. One of the most potent, the EPA approved Hybrid (Ca⁺⁺/K⁺ channel blocker), was studied for pre-lethal effects. These included reduced blood feeding behavior, with almost 100% of insects infected with ~6 spores unable to transmit malaria within 5 days post-infection, surpassing the World Health Organization threshold for successful vector control agents. Furthermore, recombinant strains co-expressing Hybrid toxin and AaIT (Na⁺ channel blocker) produced synergistic effects, requiring 45% fewer spores to kill half of the mosquitoes in 5 days as single toxin strains. Our results identify a repertoire of toxins with different modes of action that improve the utility of entomopathogens as a technology that is compatible with existing insecticide-based control methods.

Figure 1

(a) LT50 values for mosquitoes treated with 1 × 10⁵, 1 × 10⁶ and 1 × 10⁷  M. pingshaense conidia/ml suspensions resulting in ~1, ~3 and ~6 conidia/mosquito, respectively, of M. pingshaense WT or M. pingshaense expressing Hybrid, AaIT or both AaIT and Hybrid. LC50 values are reported along the x-axis as the inverse of the estimated spore inoculum. Lettering represents statistical differences (p < 0.05) based on a log-rank test comparing the Kaplan-Meier survival curves. The LC50 dose for untreated mosquitoes was fixed at zero and reported for all spore concentrations for comparison. (b) Schematic representing infection timing (beige) and LT50s of mosquitoes treated with conidial suspensions and exposed to cloth impregnated with toxin expressing strains or WT. Lettering groups toxins by statistical significance (p < 0.05) based on a log-rank test comparing the Kaplan-Meier survival curves.

Figure 2

Survival following WHO tube exposure: these three graphs represent survival curves for RFP expressing Metarhizium (Met-RFP) and Hybrid toxin-expressing Metarhizium (Met-Hybrid) against two wild-caught, insecticide-resistant (An. coluzzii and An. gambiae s.s.) and one lab-reared (An. gambiae Kisumu) human malaria vector mosquitoes. There are no significant differences in time to kill insecticide resistant and susceptible strains, but Met-Hybrid is more effective on all mosquito strains.

Figure 3

(a) Impact of fungal infection on blood-feeding at 1–5 days post-infection with either Met-Hybrid or Met-RFP. Mosquitoes were placed in a choice chamber with the guinea pig host outside of the chamber and just out of reach (Supplementary Figure 1a,b). Host-seeking (blood feeding) interest was quantified as the percentage of the mosquito population choosing the chamber closest to the host. The symbol “*” denotes no significant differences in mosquito choices with or without a host: 30 ± 3.05% of the mosquitoes chose the “host” chamber even in the absence of a host. (b) Mortality and transmission of mosquitoes exposed to fungus, the light area represents the percent survival of mosquitoes for each treatment, while the dark area shows the proportion of mosquitoes in each treatment that are alive and would seek a host to blood feed (capable of malaria transmission). The upper dashed line represents the LT50 while the lower dashed line represents the 80% control threshold suggested by the World Health Organization (WHO) for a successful vector control agent.

Figure 4

The number of spores infecting each mosquito after spraying with three different spore concentrations (1 × 10⁵, 1 × 10⁶, and 1 × 10⁷ spores/mL in 0.01% Tween80) plotted against the probability of death. The mean number of spores delivered with each suspension is marked with a blue crossbar. Assuming that mosquitoes with a higher dose are more likely to die, we calculated the chance of death for each mosquito based on our survival data for each treatment at each concentration. Mosquitoes with an estimated 100% chance of death are colored in red, and 0–99% is represented with a green to orange gradient. The red dashed line indicates the estimated LD100 in each treatment (10 spores for WT, 8 spores for AaIT, 7 spores for Hybrid and 6 spores for Hybrid/AaIT).