The potential of Clonostachys rosea as a biolarvicide against Anopheles gambiae.

Mosquitoes vector multiple disease-causing pathogens detrimental to humans, imposing a high public health and economic burden. Chemical and biological larvicides are used in some settings to reduce mosquito populations as a supplement to adult population control. In this study, we assessed the larvicidal potential of a newly isolated strain of the hypocrealean fungus, Clonostachys rosea , by modifying a previously established laboratory-based experimental design. This design allowed us to distinguish between the larvicidal impact of the fungal culture supernatant and conidia. Our data show that C. rosea efficiently kills larvae of the African malaria mosquito, Anopheles gambiae , at an LC50 of 7.78 x 10 6 for culture supernatant and 7.13 x 10 7 conidia/mL for washed conidia. Fungal culture supernatant retained over 95% of its activity upon autoclaving, suggesting a temperature stable non-proteinaceous mycotoxin is responsible for larvicidal activity in the supernatant. Conidia were readily ingested, remained in the gut lumen, and did not germinate or translocate to the ectoperitrophic space nor the hemocoel. UV and heat-killed conidia retained larvicidal activity, demonstrating that no active secretion of toxins, fungal growth, or infection is required for killing mosquito larvae. However, autoclaving conidia abolished larvicidal activity, suggesting that larvae may not be killed via a mechanical mechanism, such as occlusion. Instead, our data demonstrate that C. rosea conidia kill An. gambiae larvae via the passive release of intracellular mycotoxins and have potential as a fungal biolarvicide to control An. gambiae populations. Highlights: Clonostachys rosea is a potent larvicide of Anopheles gambiae mosquitoes. Larvicidal activity from C. rosea culture supernatant is heat stable. C. rosea conidia are ingested but remain in the gut lumen and do not germinate. C. rosea conidia passively release toxins ultimately leading to larval death.