African Water Storage Pots for the Delivery of the Entomopathogenic Fungus Metarhizium anisopliae to the Malaria Vectors Anopheles gambiae s.s. and Anopheles funestus Marit Farenhorst, Daniela Farina, Ernst-Jan Scholte, Willem Takken, Richard H. Hunt, Maureen Coetzee, and Bart G. J. Knols* Laboratory of Entomology, Wageningen University and Research Centre, Wageningen, The Netherlands; Plant Protection Service, Wageningen, The Netherlands; Vector Control Reference Unit, National Institute for Communicable Diseases, Johannesburg, South Africa; Animal, Plant and Environmental Sciences, University of the Witwatersrand, Johannesburg, South Africa; School of Pathology, University of the Witwatersrand, Johannesburg, South Africa Abstract. We studied the use of African water storage pots for point source application of Metarhizium anisopliae against the malaria vectors Anopheles gambiae s.s. and An. funestus. Clay pots were shown to be attractive resting sites for male and female An. gambiae s.s. and were not repellent after impregnation with fungus. M. anisopliae was highly infective and virulent after spray application inside pots. At a dosage of 4 × 10 10 conidia/m 2 , an average of 95 ± 1.2% of An. gambiae s.s. obtained a fungal infection. A lower dosage of 1 × 10 10 conidia/m 2 infected an average of 91.5 ± 0.6% of An. gambiae s.s. and 91.8 ± 1.2% of An. funestus mosquitoes. Fungal infection significantly reduced mosquito longevity, as shown by differences between survival curves and LT 50 values. These pots are suitable for application of entomopathogenic fungi against malaria vectors and their potential for sustainable field implementation is discussed. INTRODUCTION Human malaria, transmitted by female mosquitoes of the genus Anopheles, is a major public health challenge in devel- oping countries. Malaria is Africa’s major cause of mortality in those younger than 5 years of age and constitutes 10% of the continent’s overall disease burden. 1 Anopheles gambiae s.s. Giles and An. funestus Giles are two of the most important malaria mosquito vectors in sub-Saharan Africa in terms of human morbidity and mortality, with high susceptibility to Plasmodium infection and high degrees of anthropophily. 2 An. gambiae s.s. has a continent-wide distribution, occupies temporary aquatic habitats, and is considered to be one of the most efficient species to develop and transmit malaria para- sites. 3,4 An. funestus is widely distributed throughout tropical Africa, occupies a wide range of ecological niches, and ex- tends its activity even into the dry season when other malaria mosquito vectors are present in much reduced densities. 5,6 Current vector control methods against adult mosquito populations are mainly based on insecticide application, par- ticularly indoor residual spraying (IRS) and insecticide- treated bednets (ITNs). However, insecticide resistance poses a serious threat to sustainable insecticide-based vector control in many African countries. 7 Both An. gambiae and An. fu- nestus populations are showing increasing levels of resistance to one or more of the insecticide classes used in vector con- trol. 1 There is a pressing need, therefore, to develop novel vector control methods that can complement or replace ex- isting intervention tools. Metarhizium anisopliae and Beauveria bassiana are hypho- mycetous insect-pathogenic fungi of which the conidia can infect insects by penetrating the cuticle, without the need for ingestion, and kill them within several days. 8 Metarhizium spp. and Beauveria spp. are endemic worldwide and are not harmful to birds, fish, or mammals, 9 and because of their opportunistic nature, lack of association with humans, and inability to survive at human body temperatures, are consid- ered to cause no human health risks (S. De Hoog, personal communication). The US Environmental Protection Agency has declared no risk to humans when using products contain- ing M. anisopliae, based on toxicity tests. 10 Hyphomycetous fungi can be cost-effectively mass-produced, 11 and several strains are already commercially available 12,13 and in use for the control of agricultural and veterinary pests. 14,15 Several studies have shown that conidia of M. anisopliae and B. bassiana are pathogenic to adult Anopheles, Aedes, and Culex mosquitoes, because fungal infection significantly reduces their longevity. 16–18 This implies a high potential for use as biological control agents against vectors transmitting malaria, dengue, Chikungunya, and other mosquito-borne diseases. Besides a reduction in daily survival rates, fungal infection has also shown other effects that are likely to affect malaria transmission. First, infection of Anopheles with these fungi has an inhibitory effect on the development of malaria parasites in mosquitoes. 19 Second, fungal infection reduces a mosquito’s feeding propensity and fecundity. 20 Another im- portant potential benefit of biological vector control with en- tomopathogenic fungi is that fungi are slow-killing agents, which is considered to reduce the selection pressure on resis- tance formation by allowing adult mosquitoes partial repro- ductive success. 21 An international consortium of seven research institutes was formed in 2005, with the main aim of reducing the burden of mosquito-borne diseases through the development and de- livery of novel and sustainable approaches of adult mosquito control based on fungi. 22 Although research on fundamental aspects regarding fungus production and fungus–mosquito in- teractions is underway, gaps remain between the scientific laboratory discoveries and a successful implementation of fungi in the field. Practical issues concerning the conidial vi- ability, infectivity, formulation, and delivery of fungi need to be resolved before progress toward field research can be made. 23 The choice of delivery method will be an important determinant of the overall effectiveness of a fungus-based vector control method. An optimal delivery system requires * Address correspondence to Bart G. J. Knols, Laboratory of Ento- mology, PO Box 8031, 6700 EH, Wageningen University and Re- search Centre, Wageningen, The Netherlands. E-mail: bart.knols@ wur.nl Am. J. Trop. Med. Hyg., 78(6), 2008, pp. 910–916 Copyright © 2008 by The American Society of Tropical Medicine and Hygiene 910