Factors affecting fungus-induced larval mortality in Anopheles gambiae and Anopheles stephensi

Abstract

Background: Entomopathogenic fungi have shown great potential for the control of adult malaria vectors. However, their ability to control aquatic stages of anopheline vectors remains largely unexplored. Therefore, how larval characteristics (Anopheles species, age and larval density), fungus (species and concentration) and environmental effects (exposure duration and food availability) influence larval mortality caused by fungus, was studied.

Methods: Laboratory bioassays were performed on the larval stages of Anopheles gambiae and Anopheles stephensi with spores of two fungus species, Metarhizium anisopliae and Beauveria bassiana. For various larval and fungal characteristics and environmental effects the time to death was determined and survival curves established. These curves were compared by Kaplan Meier and Cox regression analyses.

Results: Beauveria bassiana and Metarhizium anisopliae caused high mortality of An. gambiae and An. stephensi larvae. However, Beauveria bassiana was less effective (Hazard ratio (HR) <1) compared to Metarhizium anisopliae. Anopheles stephensi and An. gambiae were equally susceptible to each fungus. Older larvae were less likely to die than young larvae (HR < 1). The effect of increase in fungus concentration on larval mortality was influenced by spore clumping. One day exposure to fungal spores was found to be equally effective as seven days exposure. In different exposure time treatments 0 - 4.9% of the total larvae, exposed to fungus, showed infection at either the pupal or adult stage. Mortality rate increased with increasing larval density and amount of available food.

Conclusions: This study shows that both fungus species have potential to kill mosquitoes in the larval stage, and that mortality rate depends on fungus species itself, larval stage targeted, larval density and amount of nutrients available to the larvae. Increasing the concentration of fungal spores or reducing the exposure time to spores did not show a proportional increase and decrease in mortality rate, respectively, because the spores clumped together. As a result spores did not provide uniform coverage over space and time. It is, therefore, necessary to develop a formulation that allows the spores to spread over the water surface. Apart from formulation appropriate delivery methods are also necessary to avoid exposing non-target organisms to fungus.

Figure 1

Survival curves of mosquito larvae treated with different species and concentration of fungus. Percentage cumulative survival curves of early (L_1-2) and late (L_3-4) larval stages of An. stephensi (As) and An. gambiae (Ag) when treated with different concentrations (2.5 mg, 5 mg, 10 mg and 20 mg) of Metarhizium anisopliae (Ma) and Beauveria bassiana (Bb). The survival curves include the larvae that survived due to pupation.

Figure 2

Survival curves of mosquitoes for different exposure times. Percentage cumulative survival curves of An. stephensi (As) and An. gambiae (Ag) larvae, exposed to Metarhizium anisopliae (Ma) and Beauveria bassiana (Bb) spores for 1, 3, 5 and 7 days. The survival curves include the larvae that survived due to pupation.

Figure 3

Difference in wing lengths of adult mosquitoes. Box plot of wing measurements (mm) for An. stephensi (As) and An. gambiae (Ag) larvae, exposed to B. bassiana (Bb) and M. anisopliae (Ma) spores for 1, 3, 5 and 7 days. Dots and stars represent outliers. Whiskers represent the range. Box limit, the 1^st (lower) and 3^rd (upper) quartiles and box dividing line, the median. The number of adults dissected is shown below each box.

Figure 4

Survival curves of An. gambiae provided with different quantities of food. Percentage cumulative survival curves of An. gambiae larvae, exposed to 10 mg of M. anisopliae (Ma) and B. bassiana (Bb) spores, with different food quantities (F₁, 0.5 mg; F₂, 0.3 mg; F₃, no food). The survival curves include the larvae that survived due to pupation.

Figure 5

Survival curves of An. stephensi provided with different quantities of food. Percentage cumulative survival curves of An. stephensi larvae, exposed to 10 mg of M. anisopliae (Ma) and B. bassiana (Bb) spores, with different food quantities (F₁, 0.5 mg; F₂, 0.3 mg; F₃, no food). The survival curves include the larvae that survived due to pupation.

Figure 6

Survival curves of An. gambiae at different densities. Percentage cumulative survival curves of An. gambiae larvae, exposed to 10 mg of M. anisopliae (Ma) and B. bassiana (Bb) spores, at different densities (D₁, 0.5 larvae/cm²; D₂, 0.3 larvae/cm²; D₃, 0.1 larvae/cm²). The survival curves include the larvae that survived due to pupation.

Figure 7

Survival curves of An. stephensi at different densities. Percentage cumulative survival curves of An. stephensi larvae, exposed to 10 mg of M. anisopliae (Ma) and B. bassiana (Bb) spores, at different densities (D₁, 0.5 larvae/cm²; D₂, 0.3 larvae/cm²; D₃, 0.1 larvae/cm²). The survival curves include the larvae that survived due to pupation.