Metarhizium anisopliae Pathogenesis of Mosquito Larvae: A Verdict of Accidental Death

Metarhizium anisopliae, a fungal pathogen of terrestrial arthropods, kills the aquatic larvae of Aedes aegypti, the vector of dengue and yellow fever. The fungus kills without adhering to the host cuticle. Ingested conidia also fail to germinate and are expelled in fecal pellets. This study investigates the mechanism by which this fungus adapted to terrestrial hosts kills aquatic mosquito larvae. Genes associated with the M. anisopliae early pathogenic response (proteinases Pr1 and Pr2, and adhesins, Mad1 and Mad2) are upregulated in the presence of larvae, but the established infection process observed in terrestrial hosts does not progress and insecticidal destruxins were not detected. Protease inhibitors reduce larval mortality indicating the importance of proteases in the host interaction. The Ae. aegypti immune response to M. anisopliae appears limited, whilst the oxidative stress response gene encoding for thiol peroxidase is upregulated. Cecropin and Hsp70 genes are downregulated as larval death occurs, and insect mortality appears to be linked to autolysis through caspase activity regulated by Hsp70 and inhibited, in infected larvae, by protease inhibitors. Evidence is presented that a traditional host-pathogen response does not occur as the species have not evolved to interact. M. anisopliae retains pre-formed pathogenic determinants which mediate host mortality, but unlike true aquatic fungal pathogens, does not recognise and colonise the larval host

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

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

T helper type-1 (Th1)/Th2 profiles of peripheral blood mononuclear cells (PBMC); responses to antigens of Chlamydia trachomatis in subjects with severe trachomatous scarring

Increased stimulation of Th2 cytokines may contribute to the development of persistent ocular chlamydial infection, resulting in the blinding pathological changes of trachoma. Proliferation and cytokine production profiles of PBMC in response to stimulation with antigens of Chlamydia trachomatis were compared in 30 patients with severe conjunctival scarring due to trachoma and in 30 age-, sex- and location-matched controls. Interferon-gamma (IFN-γ) and IL-4 were detected at the single-cell level by ELISPOT assay. Transcription of the genes encoding IFN-γ, IL-4 and IL-10 was detected in mRNA isolated from parallel cultures of PBMC using reverse transcriptase-polymerase chain reaction (RT-PCR). Incubation with the chlamydial heat shock protein (hsp)60 resulted in increased numbers of IL-4-producing cells in PBMC isolated from patients with scarring disease and increased secretion of IFN-γ from PBMC of control subjects. Incubation with the chlamydial major outer membrane protein (MOMP) increased the number of IFN-γ-producing cells in the control group only. Messenger RNA encoding IL-4 was only detected in PBMC of patients with scarring disease after in vitro stimulation with chlamydial antigens, but IFN-γ mRNA and IL-10 mRNA were also more frequently detected in this group. Thirty-eight subjects were HLA-DRB1 and -DQB1 typed. Associations were observed between certain HLA class II alleles and cellular immune responses to chlamydial antigens. No HLA associations were found with clinical status, and overall we found no evidence of strong associations and the type of immune response. These data are consistent with a role for Th2 cells and cytokines in the pathogenesis of trachomatous scarring