The route of infection determines Wolbachia antibacterial protection in Drosophila

Abstract

Bacterial symbionts are widespread among metazoans and provide a range of beneficial functions. Wolbachia-mediated protection against viral infection has been extensively demonstrated in Drosophila. In mosquitoes that are artificially transinfected with Drosophila melanogaster Wolbachia (wMel), protection from both viral and bacterial infections has been demonstrated. However, no evidence for Wolbachia-mediated antibacterial protection has been demonstrated in Drosophila to date. Here, we show that the route of infection is key for Wolbachia-mediated antibacterial protection. Drosophila melanogaster carrying Wolbachia showed reduced mortality during enteric-but not systemic-infection with the opportunist pathogen Pseudomonas aeruginosaWolbachia-mediated protection was more pronounced in male flies and is associated with increased early expression of the antimicrobial peptide Attacin A, and also increased expression of a reactive oxygen species detoxification gene (Gst D8). These results highlight that the route of infection is important for symbiont-mediated protection from infection, that Wolbachia can protect hosts by eliciting a combination of resistance and disease tolerance mechanisms, and that these effects are sexually dimorphic. We discuss the importance of using ecologically relevant routes of infection to gain a better understanding of symbiont-mediated protection.

Keywords: Drosophila; Wolbachia; infection tolerance; invertebrate immunity; sexual dimorphism; symbiont protection.

Figure 1.

Fly survival after systemic oral infection with P. aeruginosa PA14. OreR^Wol− (black) and OreR^Wol+ (grey) were either (a) pricked with a needle dipped in PA14 culture (OD = 1), or (b) left to feed on a PA14 culture (OD = 25) or on a control solution of 5% sugar for 12 h. Survival was monitored for 24 h (systemic infection) or daily (oral infection). In systemic infections, 100% of control flies survived over the 24 h period. In orally exposed flies, control flies are shown as dotted lines. Each data point shown is the mean of 10 replicate groups of 10 flies; these data were analysed using a Cox proportional hazard model.

Figure 2.

Within-host microbe loads. The number of viable within-host CFUs was quantified in five to seven individual live flies following 12 h of oral exposure, and then every 24 h for a week. Males and females are plotted separately for OreR^Wol− (black) and OreR^Wol+ (grey) flies. Data shown are means ± s.e.m.

Figure 3.

Disease tolerance. To measure disease tolerance, we analysed the relationship between host health and microbe loads. For each time point, we plot the survival probability (as a measure of health) against the microbe load (number of CFU per fly) for five biological replicates per sex and Wolbachia combination. Here, we show the fit of a four-parameter logistic model to the data (see electronic supplementary material, table S1 for model fits, and accompanying text for analysis details). The x-axis is reversed to read from beginning to the end of the infection (only clearance occurred).

Figure 4.

Gene expression relative to rp49 control gene in infected flies relative to uninfected flies. The expression of genes involved in IMD-mediated antimicrobial immunity were measured: (a) PGRP-LC, a peptidoglycan pattern recognition molecule in the anterior fly midgut; (b) PGRP-LE, an intracellular peptidoglycan active in the posterior midgut; and (c) Attacin A, an AMP activated during infection by Gram-negative bacteria. We also measured the expression of GstD8—involved in ROS detoxification (d) and other genes involved in tissue damage repair (gadd45) (e) as well as and a component of the peritrophic matrix (CG32302) (f). Wolbachia-positive flies are shown in grey, and Wolbachia-negative flies in black. Data show the mean ± s.e. of pooled technical duplicates for three biological groups of five flies for each sex/Wolbachia combination, exposed orally to P. aeruginosa infection.