Targeting the breeding sites of malaria mosquitoes: biological and physical control of malaria mosquito larvae

Malaria causes an estimated 225 million cases and 781,000 deaths every year. About 85% of the deaths are in children under five years of age. Malaria is caused by the Plasmodium parasite which is transmitted by the Anopheles mosquito vector. Mainly two methods of intervention are used for vector control, i.e. insecticide-treated bed nets and indoor residual spraying. Both involve the use of insecticides and target Anopheles adults indoors. A rising increase in resistance against these insecticides plus the fact that some Anopheles mosquitoes blood-feed outdoors can result in malaria transmission despite the currently extensively deployed interventions. By targeting the aquatic stages, larval control can complement these interventions. The main aim of this study was to develop non-chemical larval control tools for Anopheles mosquitoes and to evaluate their potential under field conditions. Two biological and one physical control tool were evaluated: the entomopathogenic fungi, Beauveria bassiana (Bals.-Criv.) Vuillemin and Metarhizium anisopliae (Metchnikoff) Sorokin and a silicone-based monomolecular film, Aquatain®. These tools were evaluated in the laboratory against the primary African and Asian malaria vectors Anopheles gambiae and An. stephensi, respectively, and mainly against An. gambiae under field conditions. Beauveria bassiana and M. anisopliae conidia are known for their potential to control Anopheles adults. However, their ability to control larval stages of Anopheles, especially under field conditions, remains largely unknown. Laboratory bioassays showed that, after ingestion or attachment to the cuticle, conidia of both B. bassiana and M. anisopliae caused high mortality of An. gambiae and An. stephensi larvae. Both An. gambiae and An. stephensi were equally susceptible to each fungus. Early stage (L1-2) larvae were more susceptible compared to late (L3-4) stage larvae. Higher concentrations of fungal conidia did not cause increased mortality as the conidia clumped together. Larvae exposed to fungal conidia for one day were found to be equally affected as those exposed for seven days. Fungal infection was detected in pupae and adults that developed from the surviving larvae. Larval mortality was higher when larval density increased and a higher amounts of food did not reduce the efficacy of fungal conidia against Anopheles larvae. Results of this laboratory study also indicated the necessity to formulate fungal conidia prior to application. Formulation was required not only to prevent conidia from clumping but also to improve their persistence as the pathogenicity of fungal conidia rapidly declined after application on the water surface. Beauveria bassiana and M. anisopliae conidia, when formulated in ShellSol T (a synthetic oil), were easy to apply on the water surface and effective against Anopheles larvae. In addition, ShellSol T improved the persistence of the conidia. Under field conditions in Kenya, B. bassiana and M. anisopliae conidia, formulated in ShellSol T, reduced the percentage pupation of An. gambiae 39 - 50 % more than the unformulated conidia. This is the first report of the efficacy of fungal conidia against An. gambiae larvae under field conditions. Monomolecular films prevent mosquito larvae and pupae from breathing by reducing the surface tension of water. The reduced surface tension also prevents adults from emerging and ovipositing on the treated water surfaces. Compared to previously tested monomolecular films, Aquatain® has a better spreading ability and flexibility on the water surface. Tests were initially carried out with Aquatain mosquito formulation (AMF®), which contained 2% eucalyptus oil in addition to the original Aquatain® formulation. In the laboratory, AMF® showed larvicidal, pupicidal, and oviposition repellent effects against both An. stephensi and An. gambiae. In contrast with fungal conidia, late stage larvae were more susceptible to Aquatain® treatment than early stage larvae. Larvae from treatments showed no pupation at all and pupae died within two hours. When provided with a choice between an Aquatain-treated cup and an untreated oviposition cup, female mosquitoes avoided the treated cup and laid their eggs in the untreated cups. In a no-choice situation the lowered water surface tension caused most females to drown while attempting to oviposit. Aquatain® (without the eucalyptus oil) did not have a repellent effect as even in the presence of an untreated cup, females attempted to oviposit in the treated cups and drowned. Therefore, to prevent ovipositing females from searching and depositing eggs at untreated sites, it was recommended to use Aquatain® without eucalyptus oil for the efficacy field trials. Field trials with Aquatain® were done in rice paddies at the Ahero rice irrigation scheme in western Kenya. In general, rice paddies are difficult to treat with a mosquito control agent because of their large size, soft mud and vegetation. It is also important for the mosquito control agent to be safe for a variety of non-target organisms that are found in the rice paddies. After Aquatain® application on the rice fields, there was a significant reduction in the densities of early and late stage Anopheles larvae. Adult emergence was reduced by 93%. Aquatain® had no negative effect on a variety of non-target organisms except backswimmers (Heteroptera: Notonectidae). Moreover, Aquatain® had no adverse effect on the growth and development of rice plants, and did not affect the rice yield. This field study thus shows that Aquatain® can be used as an effective control agent against Anopheles mosquitoes in rice-agro ecosystems. The study described in this thesis identifies new candidate tools for the control of Anopheles larvae with additional negative effects on pupae and adults in contact with the breeding sites. However, follow-up studies are required before these fungi and Aquatain® can be used operationally. For application of the fungi the crucial step is to develop a cost-effective system to mass-produce fungal conidia. It is also important to develop a formulation that reduces the quantity of conidia required to significantly reduce anopheline densities in natural aquatic habitats. In the case of Aquatain®, the next step is to carry out a field trial that determines the impact of Aquatain® application on Anopheles mosquito density as well as on malaria transmission. Development and availability of biological and physical tools is necessary for sustainable and environmentally safe integrated malaria vector management.