Intraspecific genetic variation in host vigour, viral load and disease tolerance during Drosophila C virus infection

Abstract

Genetic variation for resistance and disease tolerance has been described in a range of species. In Drosophila melanogaster, genetic variation in mortality following systemic Drosophila C virus (DCV) infection is driven by large-effect polymorphisms in the restriction factor pastrel (pst). However, it is unclear if pst contributes to disease tolerance. We investigated systemic DCV challenges spanning nine orders of magnitude, in males and females of 10 Drosophila Genetic Reference Panel lines carrying either a susceptible (S) or resistant (R) pst allele. We find among-line variation in fly survival, viral load and disease tolerance measured both as the ability to maintain survival (mortality tolerance) and reproduction (fecundity tolerance). We further uncover novel effects of pst on host vigour, as flies carrying the R allele exhibited higher survival and fecundity even in the absence of infection. Finally, we found significant genetic variation in the expression of the JAK-STAT ligand upd3 and the epigenetic regulator of JAK-STAT G9a. However, while G9a has been previously shown to mediate tolerance of DCV infection, we found no correlation between the expression of either upd3 or G9a on fly tolerance or resistance. Our work highlights the importance of both resistance and tolerance in viral defence.

Keywords: Drosophila C virus; resistance; tolerance; viral infection.

Figure 1.

Effects of pst, genetic variation, sex and viral dose on survival up to 78 days post-infection. Flies were sham treated (control) or infected with one of five doses (10³, 10⁵, 10⁶, 10⁸, 10⁹) of Drosophila C Virus. (a) Survival in resistant (R) and susceptible (S) line types. (R lines: RAL-59, RAL-75, RAL-379, RAL-502, RAL-738; S lines: RAL-138, RAL-373, RAL-380, RAL-765, RAL-818). Uninfected resistant lines have a survival advantage in comparison to susceptible lines. Survival tends to improve later in life at low to intermediate infection intensities, but this effect is nearly absent at high DCV doses. (b) Each Kaplan–Meier curve represents the cumulative survival of 20 individuals. Viral dose is logged for ease of interpretation. (c) Heatmaps showing mean lifespan for female (i) and male (ii) flies, where DGRP lines are arranged according to mean total survival time of males and females. There were differential effects of both line and dose and line and sex on survival after viral infection (b) and (c). R lines are shown in black and S lines are shown in dark orange. For statistics, see table 1.

Figure 2.

Drosophila melanogaster resistance to DCV. (a) DCV viral load (DCV copies per fly) in R and S DGRP lines, measured 3 days post-infection (3 DPI). R lines: RAL-59, RAL-75, RAL-379, RAL-502, RAL-738; S lines: RAL-138, RAL-373, RAL-380, RAL-765, RAL-818. DCV load is generally lower in resistant DGRP lines. (b) DCV load measured at 3 DPI differs as a function of sex and line and increases as dose increases. Each data point (n = 5, line × sex × dose) represents the viral load from a single fly. Values are plotted on log₁₀ transformed x- and y-axes. (c) Variation in mean viral load for each level of line and dose. Viral load is logged for clarity. R lines are shown in black and S lines are shown in grey. For statistics, see table 2.

Figure 3.

Mortality tolerance in DCV-infected flies shows evidence of genetic variation and nonlinearity. (a) Lifespan in resistant (R) and susceptible (S) DGRP lines. Resistant lines tend to live longer than susceptible lines and are equally tolerant to DCV infection. R lines: Ral-59, Ral-75, Ral-379, Ral-502, Ral-738; S lines: Ral-138, Ral-373, Ral-380, Ral-765, Ral-818. (b) Reaction norms are plotted for each line and split by sex. We use dose in place of titre (i.e. [42,46]) to estimate variation in tolerance. (c) Integrals for each DGRP line, split by sex. The y-intercept of each function was standardized at 0 to account for differences in general vigour (e.g. [5]) before integration. Bars are ordered from least tolerant (Ral-373) to most tolerant (Ral-765).

Figure 4.

DCV-infected DGRP lines show evidence of genetic variation in fecundity tolerance. (a) Cumulative fecundity in R and S DGRP lines. Susceptible lines have fewer offspring than resistant lines regardless of infection status but are equally as tolerant as R lines (similar slopes). R lines: Ral-59, Ral-75, Ral-379, Ral-502, Ral-738; S lines: Ral-138, Ral-373, Ral-380, Ral-765, Ral-818. (b) Reaction norms are plotted for each DGRP line. Each data point represents the cumulative fecundity of a single fly during its lifetime. (c) Slopes ± s.e. of reaction norms plotted in (b). Bars represent the fecundity tolerance of each DGRP line. Lines are ordered from the least tolerant (Ral-379) to most tolerant (Ral-138). For statistics, see table 3.

Figure 5.

pst has differential effects on gene expression between uninfected and infected flies. (a) G9a expression relative to rp49 in the absence of infection is not significantly affected by pst. (b) Infected flies G9a expression is higher in (S) susceptible DGRP lines but is unaffected by sex. (c) upd3 expression relative to rp49 is higher in (R) resistant DGRP lines and tends to be lower in males. (d) Sex affects infected expression of upd3. R lines: Ral-59, Ral-75, Ral-379, Ral-502, Ral-738; S lines: Ral-138, Ral-373, Ral-380, Ral-765, Ral-818. For statistics, see table 4.