Wolbachia enhances the survival of Drosophila infected with fungal pathogens

Abstract

Background: Wolbachia bacteria of arthropods are at the forefront of basic and translational research on multipartite host-symbiont-pathogen interactions. These vertically transmitted microbes are the most widespread endosymbionts on the planet due to factors including host reproductive manipulation and fitness benefits. 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. 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.

Results: 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. Finally, RNA sequencing results show altered expression of many immune and stress response genes in the context of Wolbachia and fungal infection, suggesting host immunity may be involved in the mechanism.

Conclusions: This study demonstrates Wolbachia's protective 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. It also is a critical step forward to understanding how symbionts can protect their hosts from a variety of pathogens.

Keywords: Drosophila; Wolbachia; Filamentous fungi; Immunity; Stress response; Yeast.

Fig. 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 (Fig. 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 Fig. 2a because they were performed concurrently in the same background

Fig. 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.

Fig. 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 (Fig. 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

Fig. 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 Percentage 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 25th (lower) and 75th (upper) percentiles and the middle line indicates 50th 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

Fig. 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 25th (lower) and 75th (upper) percentiles and the middle line indicates 50th 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

Fig. 6

Wolbachia infection alone and Wolbachia-B. bassiana co-infection associate with altered expression of many host stress- and immune-response genes. Male flies were pierced with a needled dipped in either 20% glycerol (sham control) or B. bassiana infection with RNA extracted after 24 h. a Volcano plot depicting genes differentially expressed comparing only Wolbachia-positive to Wolbachia-negative control flies. Yellow indicates that the absolute value of the log₂ fold change is greater than one, blue indicates that the adjusted P-value is less than 0.01, and red indicates that both thresholds are met. b Expression of turandot genes in all conditions. Triangles indicate B. bassiana infections, whereas circles represent sham controls; dark blue indicates Wolbachia infected, while light blue indicates Wolbachia uninfected. c Plot showing all genes with a significant Wolbachia x B. bassiana interaction effect (red dots). The two axes show the log₂fold change for B. bassiana infected flies over sham control for Wolbachia-infected flies (X axis) and Wolbachia uninfected flies (Y axis). d Expression of all genes with a significant Wolbachia x B. bassiana interaction in all conditions. Triangles indicate B. bassiana infections, whereas circles represent sham controls; dark blue indicates Wolbachia infected, while light blue indicates Wolbachia uninfected

Fig. 7

Toll and Imd antimicrobial peptides as classes broadly show different expression patterns with Wolbachia infection. a Expression of antimicrobial peptides in sham control flies (glycerol) with vs without Wolbachia. b Expression of antimicrobial peptides in B. bassiana-infected flies, comparing those with Wolbachia to those without. Green: Toll-mediated peptides. Purple: Imd-mediated peptides. Gray: Peptide mediated by both pathways. Note that the maximum for the X axis increases by an order of magnitude in the infected condition, indicating an overall induction of AMPs