Research Article: Metarhizium brunneum Blastospore Pathogenesis in Aedes aegypti Larvae: Attack on Several Fronts Accelerates Mortality

Date Published: July 7, 2016

Publisher: Public Library of Science

Author(s): Abeer M. Alkhaibari, Aline T. Carolino, Sare I. Yavasoglu, Thierry Maffeis, Thalles C. Mattoso, James C. Bull, Richard I. Samuels, Tariq M. Butt, Elizabeth Ann McGraw.


Aedes aegypti is the vector of a wide range of diseases (e.g. yellow fever, dengue, Chikungunya and Zika) which impact on over half the world’s population. Entomopathogenic fungi such as Metarhizium anisopliae and Beauveria bassiana have been found to be highly efficacious in killing mosquito larvae but only now are the underlying mechanisms for pathogenesis being elucidated. Recently it was shown that conidia of M. anisopliae caused stress induced mortality in Ae. aegypti larvae, a different mode of pathogenicity to that normally seen in terrestrial hosts. Blastospores constitute a different form of inoculum produced by this fungus when cultured in liquid media and although blastospores are generally considered to be more virulent than conidia no evidence has been presented to explain why. In our study, using a range of biochemical, molecular and microscopy methods, the infection process of Metarhizium brunneum (formerly M. anisopliae) ARSEF 4556 blastospores was investigated. It appears that the blastospores, unlike conidia, readily adhere to and penetrate mosquito larval cuticle. The blastospores are readily ingested by the larvae but unlike the conidia are able infect the insect through the gut and rapidly invade the haemocoel. The fact that pathogenicity related genes were upregulated in blastospores exposed to larvae prior to invasion, suggests the fungus was detecting host derived cues. Similarly, immune and defence genes were upregulated in the host prior to infection suggesting mosquitoes were also able to detect pathogen-derived cues. The hydrophilic blastospores produce copious mucilage, which probably facilitates adhesion to the host but do not appear to depend on production of Pr1, a cuticle degrading subtilisin protease, for penetration since protease inhibitors did not significantly alter blastospore virulence. The fact the blastospores have multiple routes of entry (cuticle and gut) may explain why this form of the inoculum killed Ae. aegypti larvae in a relatively short time (12-24hrs), significantly quicker than when larvae were exposed to conidia. This study shows that selecting the appropriate form of inoculum is important for efficacious control of disease vectors such as Ae. aegypti.

Partial Text

Aedes aegypti is the vector of a wide range of viral diseases (e.g. yellow fever, dengue, Chikungunya and Zika) [1–5]. Dengue fever annually affects 284 to 528 million people around the world [6]. The range of this pest appears to be expanding due to global warming [7]. Of major concern is the establishment of Ae. aegypti and Aedes albopictus throughout Europe with the latter now firmly established in Southern Europe [7]. The success of these two species is partly due to their ability to readily adapt to urban environments and the tolerance of the eggs to desiccation [8]. Current control is still heavily dependent upon the use of chemical pesticides, which should be discouraged because of the risks they pose to human health and the environment [9, 10]. Moreover, mosquitoes are also rapidly developing resistance to chemical insecticides as well as to the biological larvicide Bacillus thuringiensis [11–14]. Much attention is currently being focussed on the use of entomopathogenic fungi (EPF) such as Beauveria bassiana and Metarhizium anisopliae for the control of mosquito adults and larvae [15–24] as they are considered to be environmentally friendly and highly versatile [25].

Blastospores of M. brunneum strain ARSEF 4556 were significantly more virulent than conidia in killing Ae. aegypti larvae. This study shows for the first time that blastospores have specific characteristics which explain why they are more aggressive to mosquito larvae than conidia. These attributes include the ability to readily adhere to the cuticle surface, production of copious mucilage and the ability to infect without the differentiation of appressoria or excessive production of the proteases Pr1 and Pr2. Blastospores infected larvae through integument and gut, retaining the dynamic blastospore form both on the host surface and during invasion of the haemocoel. This study also shows that the host immune and stress management responses rapidly kick in but are inadequate as they fail to prevent infection.




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