Susceptibility of adult female Aedes aegypti (Diptera: Culicidae) to the entomopathogenic fungus Metarhizium anisopliae is modified following blood feeding

<p>Abstract</p> <p>Background</p> <p>The mosquito <it>Aedes aegypti</it>, vector of dengue fever, is a target for control by entomopathogenic fungi. Recent studies by our group have shown the susceptibility of adult <it>A. aegypti </it>to fungal infection by <it>Metarhizium anisopliae</it>. This fungus is currently being tested under field conditions. However, it is unknown whether blood-fed <it>A. aegypti </it>females are equally susceptible to infection by entomopathogenic fungi as sucrose fed females. Insect populations will be composed of females in a range of nutritional states. The fungus should be equally efficient at reducing survival of insects that rest on fungus impregnated surfaces following a blood meal as those coming into contact with fungi before host feeding. This could be an important factor when considering the behavior of <it>A. aegypti </it>females that can blood feed on multiple hosts over a short time period.</p> <p>Methods</p> <p>Female <it>A. aegypti </it>of the Rockefeller strain and a wild strain were infected with two isolates of the entomopathogenic fungus <it>M. anisopliae </it>(LPP 133 and ESALQ 818) using an indirect contact bioassay at different times following blood feeding. Survival rates were monitored on a daily basis and one-way analysis of variance combined with Duncan's <it>post-hoc </it>test or Log-rank survival curve analysis were used for statistical comparisons of susceptibility to infection.</p> <p>Results</p> <p>Blood feeding rapidly reduced susceptibility to infection, determined by the difference in survival rates and survival curves, when females were exposed to either of the two <it>M. anisopliae </it>isolates. Following a time lag which probably coincided with digestion of the blood meal (96-120 h post-feeding), host susceptibility to infection returned to pre-blood fed (sucrose fed) levels.</p> <p>Conclusions</p> <p>Reduced susceptibility of <it>A. aegypti </it>to fungi following a blood meal is of concern. Furthermore, engorged females seeking out intra-domicile resting places post-blood feeding, would be predicted to rest for prolonged periods on fungus impregnated black cloths, thus optimizing infection rates. It should be remembered that lowered susceptibility was only a temporary phenomenon and this may not necessarily occur when mosquitoes are infected with other fungal isolates. These results may have implications for field testing of entomopathogenic fungi by our group and further studies should be carried out to better understand the insect-fungus interaction.</p

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

<i>Metarhizium brunneum</i> Blastospore Pathogenesis in <i>Aedes aegypti</i> Larvae: Attack on Several Fronts Accelerates Mortality

<div><p><i>Aedes aegypti</i> 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 <i>Metarhizium anisopliae</i> and <i>Beauveria bassiana</i> 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 <i>M</i>. <i>anisopliae</i> caused stress induced mortality in <i>Ae</i>. <i>aegypti</i> 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 <i>Metarhizium brunneum</i> (formerly <i>M</i>. <i>anisopliae</i>) 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 <i>Ae</i>. <i>aegypti</i> 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 <i>Ae</i>. <i>aegypti</i>.</p></div

Scanning electron microscopy of <i>Aedes aegypti</i> larvae infected with <i>Metarhizium brunneum</i> blastospores.

<p>Larvae were inoculated with 1X10⁷ blastospores ml^-1 and prepared for SEM 20 hrs post inoculation. (A): Head of <i>Ae</i>. <i>aegypti</i> larva showing blastospores (BS) attached to the surface of the cuticle. (B): Blastospores at different stages of germination attached to surface of abdomen. (C): Germinating and non-germinating blastospores surrounded by a mucilaginous matrix (M). (D). Cross section of infected larva showing blastospores of <i>M</i>. <i>brunneum</i> occluding the gut lumen (GL).</p

Transmission electron microscopy of <i>Aedes aegypti</i> larvae infected with <i>Metarhizium brunneum</i> blastospores 24 hr post-inoculation.

<p>(A) Blastospore (BS) firmly adhering to surface of the cuticle (CU). The blastospore has dense cytoplasm, a relatively thin wall and a coating of mucilage (MU) which extends beyond the fungus. The mucilage consists of heterogeneous electron opaque material. (B) Penetration of the larval cuticle. Vacuoles (V) containing electron dense material evident in the penetration hyphae. Cuticle readily distorted by penetration hypha. (C) Blastospores in gut lumen penetrating the peritrophic membrane (PM). (D) One blastospore has penetrated the midgut epithelium and has entered the haemocoel (H).</p

Survival of <i>Aedes aegypti</i> larvae exposed to blastospores in the presence of different protease inhibitors.

<p><i>Ae</i>. <i>aegypti</i> larvae (n = 72) were exposed to <i>M</i>. <i>brunneum</i> blastospores with and without the addition of the protease inhibitors chicken egg white (CEW, a Pr1 specific inhibitor) and α2-macroglobulin (global protease inhibitor). The Kaplan-Meier method was used to plot survival curves of larvae; Log-rank test was used to assess difference in survival between treatments. Controls consisted of either distilled water or distilled water with protease inhibitors. Error is represented as SE.</p

Cross section of <i>Aedes aegypti</i> larvae 24 hrs post infection with <i>Metarhizium brunneum</i> blastospores.

<p>(A) Blastospores of <i>M</i>. <i>brunneum</i> mostly confined to gut lumen. (B) Blastospores adjacent to the peritrophic membrane are swollen with some having penetrated the peritrophic membrane and midgut epithelium. Cells colonizing the haemocoel consisted of short filaments or hyphal bodies as well as yeast like cells. There was no evidence of branched filamentous hyphae. EP: Epithelium, PM: peritrophic membrane, MV: Microvilli, N: Nuclei, BS: Blastospores, GL: Gut lumen.</p