Variation in antiviral protection mediated by different Wolbachia strains in Drosophila simulans

Abstract

Drosophila C virus (DCV) is a natural pathogen of Drosophila and a useful model for studying antiviral defences. The Drosophila host is also commonly infected with the widespread endosymbiotic bacteria Wolbachia pipientis. When DCV coinfects Wolbachia-infected D. melanogaster, virus particles accumulate more slowly and virus induced mortality is substantially delayed. Considering that Wolbachia is estimated to infect up to two-thirds of all insect species, the observed protective effects of Wolbachia may extend to a range of both beneficial and pest insects, including insects that vector important viral diseases of humans, animals and plants. Currently, Wolbachia-mediated antiviral protection has only been described from a limited number of very closely related strains that infect D. melanogaster. We used D. simulans and its naturally occurring Wolbachia infections to test the generality of the Wolbachia-mediated antiviral protection. We generated paired D. simulans lines either uninfected or infected with five different Wolbachia strains. Each paired fly line was challenged with DCV and Flock House virus. Significant antiviral protection was seen for some but not all of the Wolbachia strain-fly line combinations tested. In some cases, protection from virus-induced mortality was associated with a delay in virus accumulation, but some Wolbachia-infected flies were tolerant to high titres of DCV. The Wolbachia strains that did protect occurred at comparatively high density within the flies and were most closely related to the D. melanogaster Wolbachia strain wMel. These results indicate that Wolbachia-mediated antiviral protection is not ubiquitous, a finding that is important for understanding the distribution of Wolbachia and virus in natural insect populations.

Figure 1. Wolbachia strain wMel provides antiviral protection in D. simulans.

(A) Graph shows survival of flies infected with DCV (black line) or mock infected (grey line). wMel-infected (circle and plus sign) or uninfected (triangle and cross) flies. The survival of DCV infected flies with and without Wolbachia is significantly different (p<0.0001). Error bars represent SEM calculated from three replicate vials. This is a representative experiment which was repeated twice more with similar results. (B) Graph showing accumulation of infectious DCV in wMel infected (grey bars) or uninfected (white bar) flies. Bars represent means from two replicates with SEM shown, and * indicates a significant difference between the means of day 2 samples (p<0.05, unpaired t test).

Figure 2. Antiviral protection of different Wolbachia strains in D. simulans.

Graphs show survival of flies infected by wAu (A), wRi (B), wHa (C), and wNo (D) challenged with DCV (black line) or mock infected (grey line). Flies with Wolbachia (circle and plus sign) and without Wolbachia (triangle and cross). Error bars represent SEM calculated from three replicates. The survival of DCV infected flies with and without Wolbachia is significantly different for wAu (p<0.0001), wRi (p<0.0001), and wHa (p<0.01), using log rank test on Kaplan-Meier curves. Experiments were replicated on at least two additional independent cohorts of flies, and the results for all respective replicates of experiments shown in panel A, B and D were similar, however the replicates for panel C varied (see Results).

Figure 3. The effect of different Wolbachia strains on the accumulation of DCV in D. simulans.

Graphs show accumulation of infectious DCV in flies with (grey bar) or without (white bar) wAu (A), wRi (B), wHa (C), and wNo (D). Bars represent means from two replicates with SEM shown, and * indicates a significant difference between the means of day 2 samples (p<0.05, unpaired t test).

Figure 4. The effect of different Wolbachia strains on the accumulation of FHV in D. simulans.

Graphs show survival of flies infected by wAu (A), wRi (B), wHa (C), and wNo (D) challenged with FHV (black line) or mock infected (grey line). Wolbachia infected (circle and plus sign) and uninfected (triangle and cross) flies. Error bars represent SEM calculated from three replicates. The survival of FHV infected flies with and without Wolbachia is significantly different for wAu and wRi (p<0.0001, log rank test on Kaplan-Meier curves). For each fly line a similar result was recorded in a replicate experiment.

Figure 5. Relative-density of Wolbachia strains in D. simulans.

For each fly line the graph shows the relative abundance of Wolbachia to host genomic DNA estimated using quantitative PCR. Bars represent the mean of 10 replicates and error bars are SEM.