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

Abstract

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.

Highly specific host-pathogen interactions influence Metarhizium brunneum blastospore virulence against Culex quinquefasciatus larvae

“…Differences in virulence between spore types was first observed in Ae. aegypti infected with conidia or blastospores as reported by Butt et al [13] and Alkhaibari et al [12], respectively. Blastospores attack Ae.…”

“…This novel study demonstrated some of the possible reasons why Culex larvae infected with blastospores take longer to die (LT 50  = 2.68 days) when compared with those exposed to conidia (LT 50  = 1.27 days for wet conidia and 1.45 days for dry conidia). We have recently shown that blastospores of this fungus are highly virulent to A. aegypti larvae, rapidly penetrating the larval integument with the aid of copious amounts of mucilage [12]. Therefore, we were somewhat surprised to find a totally different infection pattern for Culex larvae exposed to M. brunneum [3].…”

“…No gut penetration. Accumulation in the gut [13]More susceptible to blastospores than conidia [3,12]Upregulated prior to infection [12]Upregulated prior to invasion [12]Little or no role in pathogenesis [12]High levels of cuticle attachment and penetration. Gut penetration [12] Cx.…”

“…Accumulation in the gut [13]More susceptible to blastospores than conidia [3,12]Upregulated prior to infection [12]Upregulated prior to invasion [12]Little or no role in pathogenesis [12]High levels of cuticle attachment and penetration. Gut penetration [12] Cx. quinquefasciatus Conidia were more virulent than blastospores [3] and present paperSignificant up- regulation within 12h post-infection (present paper)Adhesion gene (Mad 1) and penetration genes (Pr1, Pr2) were upregulated.…”

“…Elucidation of the underlying pathogenicity mechanisms is crucial as this will help identify specificity and virulence determinants, host barriers to infection and ultimately help increase EPF efficacy in the control of insect pest species. For example, Alkhaibari et al [12]. recently showed that Aedes aegypti larvae were significantly more susceptible to blastospores of M. brunneum than conidia of the same isolate.…”