Pathogen-microbiome interactions and the virulence of an entomopathogenic fungus.

Bacteria shape interactions between hosts and fungal pathogens. In some cases, bacteria associated with fungi are essential for pathogen virulence. In other systems, host-associated microbiomes confer resistance against fungal pathogens. We studied an aphid-specific entomopathogenic fungus called Pandora neoaphidis in the context of both host and pathogen microbiomes. Aphids host several species of heritable bacteria, some of which confer resistance against Pandora. We first found that spores that emerged from aphids that harbored protective bacteria were less virulent against subsequent hosts and did not grow on plate media. We then used 16S amplicon sequencing to study the bacterial microbiome of fungal mycelia and spores during plate culturing and host infection. We found that the bacterial community is remarkably stable in culture despite dramatic changes in pathogen virulence. Last, we used an experimentally transformed symbiont of aphids to show that Pandora can acquire host-associated bacteria during infection. Our results uncover new roles for bacteria in the dynamics of aphid-pathogen interactions and illustrate the importance of the broader microbiological context in studies of fungal pathogenesis. IMPORTANCE: Entomopathogenic fungi play important roles in the population dynamics of many insect species. Understanding the factors shaping entomopathogen virulence is critical for agricultural management and for the use of fungi in pest biocontrol. We show that heritable bacteria in aphids, which confer protection to their hosts against fungal entomopathogens, influence virulence against subsequent hosts. Aphids reproduce asexually and are typically surrounded by genetically identical offspring, and thus these effects likely shape the dynamics of fungal disease in aphid populations. Furthermore, fungal entomopathogens are known to rapidly lose virulence in lab culture, complicating their laboratory use. We show that this phenomenon is not driven by changes in the associated bacterial microbiome. These results contribute to our broader understanding of the aphid model system and shed light on the biology of the Entomophthorales-an important but understudied group of fungi.

Variation in density, immune gene suppression, and coinfection outcomes among strains of the aphid endosymbiont Regiella insecticola.

Many insects harbor heritable microbes that influence host phenotypes. Symbiont strains establish at different densities within hosts. This variation is important evolutionarily because within-host density has been linked to the costs and benefits of the symbiosis for both partners. Studying the factors shaping within-host density is important to our broader understanding of host-microbe coevolution. Here we focused on different strains of Regiella insecticola, a facultative symbiont of aphids. We first showed that strains of Regiella establish in pea aphids at drastically different densities. We then found that variation in density is correlated with the expression levels of two key insect immune system genes (phenoloxidase and hemocytin), with the suppression of immune gene expression correlating with higher Regiella density. We then performed an experiment where we established coinfections of a higher- and a lower-density Regiella strain, and we showed that the higher-density strain is better able to persist in coinfections than the lower-density strain. Together, our results point to a potential mechanism that contributes to strain-level variation in symbiont density in this system, and our data suggest that symbiont fitness may be increased by establishing at higher density within hosts. Our work highlights the importance of within-host dynamics shaping symbiont evolution.