Integrating fungal entomopathogens in malaria vector control

Widespread and long-term pesticide use has caused a selection and spread of resistance in malaria mosquito populations, which endangers the effectiveness of contemporary malaria control strategies that are based on chemical insecticides. The fungal entomopathogens Metarhizium anisopliae and Beauveria bassiana show potential as alternative and more sustainable malaria vector control agents. These hyphomycetes can effectively infect anophelines and potentially reduce malaria transmission by killing the mosquitoes within several days before they can transmit malaria parasites. <br/> The aim of this thesis was to evaluate the potential of fungal entomopathogens for integration in chemical-based malaria interventions. Its objectives were to evaluate fungal spore application methods, to develop novel field delivery tools with potential for integrated use, to measure fungal efficacy against insecticide-resistant anophelines and to test the compatibility of fungi and chemical insecticides. <br/> <br/> The first part of the work (Chapter 3-5) focused on evaluating fungal spore application methods and developing novel delivery systems that could potentially be effective in tropical field settings and integrated into existing malaria control strategies. Spraying, dipping and coating were effective methods for applying an infective layer of fungal spores on mosquito resting surfaces. A coating method, involving the application of uniform spore layers on papers, was developed to enable accurate laboratory evaluations, and a rotating spray apparatus to standardize the application of oil suspensions inside clay pots. The combination of formulation and substrate was shown to have a high impact on spore infectivity, with viscous suspensions being only effective on porous substrates. Spore application dose, exposure time and type of mosquito contact were key factors of fungal virulence, as they influenced the number of spores effectively picked up by a resting mosquito. Two novel delivery methods were developed and tested in the laboratory. Clay pots showed potential for use as indoor and outdoor point-source objects to target resting mosquitoes with fungal spores. Oil-based Metarhizium suspensions were effective in infecting and killing mosquitoes after spray application inside clay pots and did not affect their attractiveness to resting male and female anophelines. Fungus-impregnated netting showed potential for use as house screens to target host-seeking mosquitoes. Spores of Metarhizium and Beauveria were most effective applied by spraying nets with evaporative suspensions. Fungi were infective on small- and large-meshed polyester and cotton nets, with spores being more viable on cotton. These two fungus delivery systems offer multiple deployment options and could potentially be used complementary to chemical-based malaria control measures such as insecticide-treated bednets (ITNs) or indoor residual spraying (IRS). <br/> The second part of the work (Chapter 6 &amp; 7) focused on evaluating the efficacy of fungi against insecticide-resistant Anopheles mosquitoes and their compatibility with public health insecticides. Metarhizium and Beauveria were highly effective against a diverse suite of insecticide-resistant Anopheles mosquitoes. Four metabolically resistant anopheline strains were equally susceptible to B. bassiana infection as their baseline counterparts. Both fungi were also highly effective in killing a laboratory strain and field population of West African An. gambiae s.s. with genetically conferred knockdown resistance (kdr) to public health insecticides. Moreover, fungi and insecticides were highly compatible and enhanced each other’s efficacy. Fungal infection increased the sensitivity of resistant mosquitoes to the neurotoxic insecticides permethrin and DDT. Fungus-infected mosquitoes with metabolic resistance mechanisms showed a significant increase in mortality after insecticide exposure compared with uninfected control mosquitoes. Reciprocally, permethrin increased subsequent fungus-induced mortality rates in a laboratory and field population of kdr-resistant An. gambiae. Several combinations of insecticide and fungus were shown to induce synergistic effects on mosquito survival. Synergy was highest after simultaneous co-exposure to both agents. These findings suggest that integrated control tools that induce contact to fungi and insecticides within a single feeding episode would have the highest impact on mosquito survival and may enable control at more moderate levels of coverage. Moreover, the synergistic and resistance breaking properties of fungi show potential for augmenting current malaria interventions and managing the further spread of insecticide resistance. <br/> <br/> Several factors still remain to be optimized before fungus-based malaria mosquito control can be realised. Cost-effective field deployment will require the development of high quality, low cost mass-production of mosquito-pathogenic fungi, persistent formulations and efficient delivery systems. The laboratory studies in this thesis provide useful knowledge and tools for future implementation research on these novel biological vector control agents. The potential field delivery systems that were created will, however, still need to be further evaluated in field settings under realistic environmental conditions. <br/> In this thesis it was, for the first time, shown that fungi are effective against insecticide- resistant malaria vectors and induce the highest impact on mosquito survival when used in combination with chemical insecticides. These findings make a compelling case for viewing novel fungus-based and existing chemical-based control measures not as mutually exclusive, but as complementary interventions that would reach the greatest malaria control benefit once successfully integrated.