Behavioural effects of fungal infection by Metarhizium anisopliae in adult malaria mosquitoes

Abstract

Malaria remains a major global health problem with the burden of disease greatest in Sub-Saharan Africa. The strategies for malaria control differ throughout the world according to levels of endemicity and the magnitude of disease but the focus remains either to control malaria parasites or vectors. A high degree of drug resistance and the absence of malaria vaccines are a major hindrance to control of the disease. In such circumstances, vector control becomes an alternative and has remained the most effective means to prevent malaria transmission. Contemporary adult mosquito control is almost exclusively based on indoor application of chemical insecticides in the form of indoor residual spraying (IRS) of walls and ceilings and insecticide-impregnated bed nets. However, sustainable use of chemicals is undermined by problems of insecticide resistance in mosquito populations, environmental contamination and risks to human health. Biological control based on fungal pathogens has shown potential to complement existing vector control methods. The entomopathogenic fungi (EPF) Metarhizium anisopliae and Beauveria bassiana have demonstrated ability to infect, kill and reduce the survival of malaria vectors. However, the effect of EPF on the behaviour of malaria vectors has not been fully addressed. This thesis was designed to provide baseline information on mosquito-fungus interaction focusing on the efficacy of entomopathogenic fungus M. anisopliae ICIPE 30 on the important life-history behaviours of the African malaria vector Anopheles gambiae Giles sensu stricto under laboratory and semi-field conditions. The information is important to facilitate the further development of malaria vector control based on biological control agents. Host-seeking, sugar-feeding, mating and oviposition were the behaviours investigated. Since mosquito-fungus contact is crucial for infection with EPF, a paper sheet (28.6 × 14.3 cm) lined inside a plastic cylinder (9-cm diameter and 15-cm height) was developed as a cost effective method of infection. Moreover, 0.1 g (approx. 1011 conidia/ m2) of dry conidia and 6 hr exposure time sufficient for An. gambiae to pick up large numbers of conidia were established to cause high pathogenicity (Chapter 3). As the impact of EPF on insect behaviour was reported to occur at least three days post-exposure to fungal pathogen (Chapter 2), all experiments were conducted with a special focus on mosquitoes three days post-exposure to fungus. It is, however, important to mention that on average 50% of the mosquitoes died on the third day after fungal exposure (Chapter 3) and only those that survived were used for behavioural assays. The host-seeking capability of An. gambiae mosquitoes is an important parameter in the vectorial capacity equation. At short-range (1 m from host) assessment using a dual-choice olfactometer under semi-field conditions, infection with EPF strongly reduced the host-seeking response of mosquitoes, but did not impair their olfactory-based capability to discriminate between hosts (Chapter 4). At medium-range, using experimental cages (3 x 3 x 2 m) under laboratory conditions, fungal infection reduced the host-seeking response and feeding propensity of female An. gambiae mosquitoes (Chapter 7) whereas at long-range (7 m from host) inside a semi-field enclosure, infection with EPF sharply reduced the house-entry response and the hourly human-biting responses of host-seeking mosquitoes indoors and outdoors (Chapter 5). Plant sugar feeding is an important component in the biology of mosquitoes and is the main priority for both sexes at emergence. Infection with fungal pathogen strongly reduced the survival and sugar-feeding propensity of both sexes of the malaria vector An. gambiae but did not affect their potential to feed and digest meals (Chapter 6). Mating behaviour plays a key role in population growth. The activity takes place after sugar feeding and thereafter, the females search for their blood meal host. Infection with M. anisopliae strongly reduced multiple mating propensity and the mating performance of adult male An. gambiae mosquitoes in a large arena such as a screenhouse. Although this resulted in a reduction in the number of females inseminated, it facilitated the transfer of fungal conidia to conspecific healthy females during mating (Chapter 8). Finally, after blood meal intake, the females prepare to lay eggs. Infection with M. anisopliae reduced the oviposition propensity of female An. gambiae mosquitoes although the number of eggs laid remained unaffected (Chapter 7). In conclusion, these findings demonstrate that the entomopathogenic fungus M. anisopliae alters the major life history behaviours of An. gambiae mosquitoes. This is possible because the fungus strongly impairs flight performance of mosquitoes that makes the insect less able to fly and engage in hostseeking, sugar-feeding, mating and oviposition behaviours. The high mortalities observed in the early days of infection prior to conducting behavioural assays, mortalities observed while conducting behavioural assays and a reduction in behavioural response of M. anisopliae-infected mosquitoes collectively are likely to have a significant impact in suppressing a vector population. The susceptibility of male mosquitoes to fungal conidia opens a new strategy for mosquito vector control. Overall, this thesis has demonstrated that EPF may be a good complement to other mosquito vector control tools for the reduction of mosquito bites, and transmission of malaria and other mosquitoborne diseases.