Wolbachia enhances the survival of Drosophila infected with fungal pathogens

Abstract

Wolbachia bacteria of arthropods are at the forefront of basic and translational research on multipartite host-symbiont-pathogen interactions. These microbes are vertically inherited from mother to offspring via the cytoplasm. They are the most widespread endosymbionts on the planet due to their infamous ability to manipulate the reproduction of their hosts to spread themselves in a population, and to provide a variety of fitness benefits to their hosts. Importantly, some strains of Wolbachia can inhibit viral pathogenesis within and between arthropod hosts. Mosquitoes carrying the wMel Wolbachia strain of Drosophila melanogaster have a greatly reduced capacity to spread viruses like dengue and Zika to humans. Therefore, Wolbachia are the basis of several global vector control initiatives. While significant research efforts have focused on viruses, relatively little attention has been given to Wolbachia-fungal interactions despite the ubiquity of fungal entomopathogens in nature. Here, we demonstrate that Wolbachia increase the longevity of their Drosophila melanogaster hosts when challenged with a spectrum of yeast and filamentous fungal pathogens. We find that this pattern can vary based on host genotype, sex, and fungal species. Further, Wolbachia correlates with higher fertility and reduced pathogen titers during initial fungal infection, indicating a significant fitness benefit. This study demonstrates Wolbachia's role in diverse fungal pathogen interactions and determines that the phenotype is broad, but with several variables that influence both the presence and strength of the phenotype. These results enhance our knowledge of the strategies Wolbachia uses that likely contribute to such a high global symbiont prevalence.

Figure 1.. Wolbachia increases the longevity of flies of the w^k background line infected with several filamentous fungal pathogens.

Flies of each given background and sex were systemically infected with the indicated pathogen. Infections were performed with either (a) Aspergillus fumigatus, (b) Aspergillus flavus, (c) Fusarium oxysporum, or (d) Fusarium graminaerum. Infections of all groups were performed side-by-side, along with those of the w¹¹¹⁸ background line (Figure S1), with at least two blocks of infections performed on different days. Each line represents a total of 60 flies. Sham controls were performed with sterile 20% glycerol. Full statistics, available in Table S1, were done with a Cox mixed effects model. Controls are the same in all panels and in Figure 2a because they were performed concurrently in the same background.

Figure 2.. Wolbachia increases the longevity of flies of the w^k background line infected with certain filamentous fungal entomopathogens.

Flies of each given background and sex were systemically infected with the indicated pathogen. Infections were performed with either (a) Beauveria bassiana, (b) Metarhizium anisopliae, (c) Clonostachys rosea, or (d) Trichoderma atroviride. Infections of all groups were performed side-by-side, along with those of the w¹¹¹⁸ background line (Figure S2), with at least two blocks of infections performed on different days. Each line represents a total of 60 flies. Sham controls were performed with sterile 20% glycerol. Full statistics, available in Table S1, were done with a Cox mixed effects model. Controls for panel 2a are the same for Figure 1, and the panels in 2b–d are the same because they were performed concurrently in the same background.

Figure 3.. Wolbachia increases the longevity of flies of the w^k background line infected with yeast pathogens.

Flies of each given background and sex were systemically infected with the indicated pathogen. Infections were performed with either (a) Candida auris, (b) Candida glabrata, or (c) Galactomyces pseudocadidus. Infections of all groups were performed side-by-side, along with those of the w¹¹¹⁸ background line (Figure S3), with at least two blocks of infections performed on different days. Each line represents a total of 60 flies. Sham controls were performed with sterile 20% glycerol. Full statistics, available in Table S1, were done with a Cox mixed effects model. Controls are the same in all panels and because they were performed concurrently in the same background.

Figure 4.. Wolbachia increases the number of eggs laid but not the percentage of eggs hatched post-B. bassiana infection in the w^k background line.

Female flies were systemically infected with B. bassiana or treated with a sham control. The flies then laid eggs for 3 days post-infection. (a) Numbers of eggs laid. (b) Proportion of eggs hatched. Each dot represents the total offspring of a single female, with an overall mean of 35 eggs laid. The boxes indicate the interquartile range. Outer edges of the box indicate 25^th (lower) and 75^th (upper) percentiles and the middle line indicates 50^th percentile (median). Whiskers represent maximum and minimum ranges of data within 1.5 times the interquartile range of the box. Statistics are based on a logistic regression (Table S1). The entire experiment was performed twice, and graphs represent a combination of data from both blocks.

Figure 5.. Wolbachia associates with reduced pathogen titer after infection with no significant change in Wolbachia titer in w^k flies.

Female flies were systemically infected with the indicated fungal pathogen and pathogen titers were measured both immediately after infection and 24 h post-infection. Dots represent pools of 3 infected females. (a) Wolbachia titers. (b) B. bassiana titers. The boxes indicate the interquartile range. Outer edges of the box indicate 25^th (lower) and 75^th (upper) percentiles and the middle line indicates 50^th percentile (median). Whiskers represent maximum and minimum ranges of data within 1.5 times the interquartile range of the box. Statistics are based on a logistic regression (Table S1). The entire experiment was performed twice, and graphs represent a combination of data from both blocks.