We found two distinct phases of infection over the five days that defense was assessed. The acute phase (Days 1–3) was characterized by a high pathogen burden, severe mortality, and a reduction in fecundity. The chronic phase (Days 4–5) consisted of a still substantial but stable pathogen burden that was in no way predictive of mortality or fecundity. We found genetic variation for resistance, mortality tolerance, and fecundity tolerance in the acute phase of the infection, but only for resistance in the chronic phase. Experimental diet significantly influenced resistance and mortality tolerance during the acute phase of the infection. However, fecundity tolerance was not sensitive to diet. That is, even though diet altered both pathogen load and fecundity, it did so proportionally such that the relationship between load and fecundity (and hence fecundity tolerance) was unchanged. Using overall estimates of resistance and tolerance in the acute phase of infection, we found a positive relationship between resistance and tolerance, although the strength of this relationship was diet-dependent. We also demonstrate a weak evolutionary cost of resistance, but fail to detect any cost of tolerance.
During the chronic phase of infection, pathogen levels remained constant. This finding suggests either that the host and pathogen have reached an equilibrium where the immune system is killing bacteria at the same rate that the bacteria are replicating or that the bacteria have stopped replicating and are no longer exposed or sensitive to host immunity. We speculate that the latter may be occurring, because mortality and fecundity both return to near uninfected levels during the chronic phase. Further understanding the patterns of infection from the perspective of both host tolerance and pathogen virulence will clarify how both players interact to create host tolerance.
Current theoretical models describing variation of resistance and tolerance in the context of host-pathogen coevolution assume a single state of infection [2–4, 6, 7, 14]. The empirical distinction between the acute and chronic phases of infection demonstrated here emphasizes the importance of looking at disease dynamics over time before extrapolating predictions about the spread of disease and evolution of defense in the wild. If transmission of P. rettgeri were to occur during the chronic phase of infection, then our results could be interpreted as supporting a model where tolerance alleles have fixed in the population. Infection tolerance through static chronic infection, which may not have major fitness consequences, should be integrated in future theoretical work on defense and infectious disease dynamics.
Consistent with previous studies (e.g., [29–31], we found genetic variation for resistance. We also found genetic variation for fecundity tolerance and for mortality tolerance. Previous studies in animals have found genetic variation in tolerance but see [10, 11, 15] and genetic variation for fecundity tolerance has been repeatedly observed in both the plant and animal literature [15, 32, 33], observations which we recapitulate in the acute (but not chronic) phase of infection. The continued presence of genetic variation for tolerance is inconsistent with basic theoretical models predicting tolerance alleles to be more likely to fix in the population than resistance alleles [3, 4]. Non-independence between resistance and tolerance, or genotype-by-environment interactions may explain this inconsistency.
Although diet altered all three phenotypes measured (bacterial load, survival, and fecundity), it did not alter fecundity tolerance. In other words, the relationship between fecundity and pathogen load did not change across the two diets used in this experiment. However, mortality tolerance was very sensitive to diet. We expected that both fecundity and mortality tolerance would be sensitive to diet because both egg production and survival are regulated by metabolic pathways such as insulin-like signaling, which are responsive to alteration in dietary sugar (e.g. ). We observed that host females do not completely stop egg production under infection conditions, nor do we see any evidence for “egg dumping” – a rapid increase in egg output to maximize short-term fitness upon infection . Instead, we observe only a minimal reduction in fecundity after infection, implying that females continue to invest in short-term reproductive output even though it may increase the risk of dying from infection and hence potentially limit lifetime fecundity.
We observed genotype-by-diet effects on mortality tolerance and fecundity tolerance, but saw no significant genotype-by-diet interaction for resistance. While increasing dietary glucose increased pathogen burden, there was little evidence for genetic variation in immunological sensitivity to dietary sugar in this study. Since there are significant genotype-by-diet effects on tolerance but not resistance during acute infection, we infer that the genes underlying tolerance are likely to function outside of the canonical resistance pathways.
In general, we found a positive relationship between resistance and tolerance, which supports the hypothesis that resource allocation to defense contributes to both resistance and tolerance . Although, conceptually, a tradeoff between resistance and tolerance is easy to imagine and has some theoretical support (, but see [7, 8, 36, 37]), the empirical evidence is very mixed. Several high-impact papers have demonstrated a negative relationship between the two strategies [11, 12, 18]. However, a meta-analysis of 31 ecological and agricultural studies of herbivore defense strategies did not find a significant relationship between resistance and tolerance , and several studies have demonstrated a positive relationship between the two strategies [39, 40]. This lack of consensus may be a result of idiosyncrasies of specific host and pathogen combinations , as well as to employment of different measures of tolerance and resistance across studies. Our observation that the strength of the relationship between resistance and tolerance is diet-dependent suggests that environmental heterogeneity could decouple these two traits and allow them to evolve independently.
The observed tradeoff between pre-infection fecundity and bacterial load was driven by two genotypes that both were derived from line RAL-832. After the experiment was performed, it was discovered that this genotype contains a premature stop codon in the antimicrobial peptide gene, Diptericin. Diptericin is a downstream effector of the IMD pathway, which is responsible for resistance against Gram-negative pathogens. Although traditional life-history theory would suggest that an evolutionary tradeoff may be a result of limited resources between maintenance of the immune system and reproduction, we speculate from our results that it is more likely driven by direct costs of immunity, such as collateral damage from active antimicrobial activity. Other work has demonstrated a tradeoff between immunity and lifespan by showing that constitutive activation of the Gram-negative bacterial recognition protein, PGRP-LE, reduces lifespan. This phenotype can be rescued in flies with a homozygous loss-of-function mutation in the NF-kB transcription factor, Relish . These results, combined with our study, suggest that the downstream effectors are the modulators of these tradeoffs, because if the tradeoff was simply based on resource availability, functional downstream effectors would play a smaller role than recognition and signaling molecules.
We did not observe any costs of tolerance. This suggests that resistance is more costly than tolerance, at least in terms of early-life reproduction. It is possible that under different conditions, increased power, or through measurements of different life-history traits, such as longevity, we may have found a cost of tolerance. Although this is the first attempt to identify costs of tolerance in animals as uninfected fitness, many studies in plants have found costs of tolerance [7, 13], but some have failed to detect these costs [37, 43, 44].
We have empirically demonstrated that defense as tolerance and resistance is a dynamic process with multiple phases of infection, and that these defense strategies are differentially affected by diet and genotype-by-diet interactions. Future theoretical work should aim to incorporate these complexities into disease models, while empirical studies should acknowledge these alternative, yet synergistic, strategies and how they change over time and environment.