Abstract
Mosquito immunity studies have focused mainly on characterizing immune effector mechanisms elicited against parasites, bacteria and more recently, viruses. However, those elicited against entomopathogenic fungi remain poorly understood, despite the ubiquitous nature of these microorganisms and their unique invasion route that bypasses the midgut epithelium, an important immune tissue and physical barrier. Here, we used the malaria vector Anopheles gambiae as a model to investigate the role of melanization, a potent immune effector mechanism of arthropods, in mosquito defense against the entomopathogenic fungus Beauveria bassiana, using in vivo functional genetic analysis and confocal microscopy. The temporal monitoring of fungal growth in mosquitoes injected with B. bassiana conidia showed that melanin eventually formed on all stages, including conidia, germ tubes and hyphae, except the single cell hyphal bodies. Nevertheless, melanin rarely aborted the growth of any of these stages and the mycelium continued growing despite being melanized. Silencing TEP1 and CLIPA8, key positive regulators of Plasmodium and bacterial melanization in A. gambiae, abolished completely melanin formation on hyphae but not on germinating conidia or germ tubes. The detection of a layer of hemocytes surrounding germinating conidia but not hyphae suggested that melanization of early fungal stages is cell-mediated while that of late stages is a humoral response dependent on TEP1 and CLIPA8. Microscopic analysis revealed specific association of TEP1 with surfaces of hyphae and the requirement of both, TEP1 and CLIPA8, for recruiting phenoloxidase to these surfaces. Finally, fungal proliferation was more rapid in TEP1 and CLIPA8 knockdown mosquitoes which exhibited increased sensitivity to natural B. bassiana infections than controls. In sum, the mosquito melanization response retards significantly B. bassiana growth and dissemination, a finding that may be exploited to design transgenic fungi with more potent bio-control activities against mosquitoes.
Highlights
Melanization is an immediate immune response in arthropods leading to the physical encapsulation of pathogens in a dense melanin coat, and to the generation of toxic metabolites that can harm certain pathogens
Melanization is an important immune response and wound healing mechanism in arthropods that leads to melanin formation and deposition on microbial and wound surfaces, respectively
In the Anopheles gambiae mosquito that transmits the malaria parasite Plasmodium, melanization is dispensable for parasite killing
Summary
Melanization is an immediate immune response in arthropods leading to the physical encapsulation of pathogens in a dense melanin coat, and to the generation of toxic metabolites that can harm certain pathogens. In the dipterans Drosophila and Anopheles gambiae, the absence of SPN43Ac, called necrotic, [4] and SPRN2 [5], respectively, resulted in the appearance of spontaneous melanotic pseudotumors in adult tissues and a reduced life span, suggesting that aberrant control of melanization imposes a fitness cost on the host. This process is regulated spatially which ensures that melanin formation occurs exclusively on microbial surfaces minimizing collateral damage to the host
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