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- Materials and methods
Parasites are ubiquitous and frequently cause considerable harm to their hosts. However, harm during the infection process may have more than one source. First, parasites directly cause harm through their own activities that damage host tissue or alter host behaviour. Secondly, parasitism indirectly harms because immune responses may self-harm. Such a cost of immunity may be due to the immune response damaging host tissue (typically called immunopathology), or because immune systems monopolize resources that could have been used for other important functions. Depending on the magnitude of their fitness effects, costs of immunity may play a crucial part in the evolution of virulence or how host–parasite coevolution impacts population genetic structure or breeding systems (e.g. Haldane 1949; Lively 1987; Hamilton, Axelrod & Tanese 1990; Jokela, Schmid-Hempel & Rigby 2000).
It is important to distinguish the two main types of costs that are commonly discussed in the coevolutionary literature. The first cost is associated with genetic differences among hosts: some genotypes invest heavily in defence systems at the expense of other functions. Genetic-based costs are a form of pleiotropy and are evident as reduced performance of resistant compared with susceptible genotypes in the absence of parasitism. The second cost is the cost of launching an immune response, which is simply the loss in performance suffered due to the energetic requirements or immunopathological effects of immune responses. This type of cost is detected by comparing the fitness of hosts that have deployed their immune systems to those that have not.
The present study concerns this second cost of immunity – the cost of launching an immune response. A significant challenge when trying to detect such a cost is to stimulate an immune response and measure the consequences of that response while not measuring direct costs of parasitism. To circumvent this problem, studies have, for example, stimulated immune responses by exposing hosts to compounds that mimic parasites (Moret & Schmid-Hempel 2001) or used tissue grafts to investigate self-reactivity (Sadd & Siva-Jothy 2006). An alternate approach, pioneered by Hasu, Valtonen & Jokela (2006) and that the present study adopts, is to study live parasite strains, but include noninfective host–parasite combinations. The major assumption here is that resistance is due to a host immune response, and if true, reductions in host fitness will be due to the costliness of that immune response. Although making this assumption in the absence of explicit measurement of an immune reaction is not faultless, this approach has advantages over the use of pathogen mimics or dead pathogens because these may not qualitatively or quantitatively stimulate immune responses to the extent a natural invasion route would.
We studied host mortality when the crustacean Daphnia magna was exposed to one of four parasite strains of the bacterium Pasteuria ramosa. Two of these strains are not aggressive, being essentially noninfective on the host clone studied, while the other two strains are highly infective. We assumed that the cases where an infection failed to establish (essentially always with the noninfective strains, occasionally with the infective ones) were attributable to an immune response. Our experimental designs tested this assumption. In particular we predicted that immune responses, and thus costs of immunity, should intensify as treatments moved from single exposures with relatively non-aggressive strains, to double exposures with non-aggressive strains. Resisting aggressive strains should see the cost of immunity rise still higher, with higher doses of aggressive strains inducing greater costs than lower doses. These predictions were met and, further, hosts that resisted infection showed higher mortality than those that succumb to infection.
- Top of page
- Materials and methods
When comparing hosts expected to have activated their immune systems to differing degrees, we found that mortality was highest among hosts expected to have launched a stronger response. This pattern might be attributable to direct damage caused by parasites that tried but failed to establish infection, but then infected hosts would be expected to suffer the most parasite-mediated damage, and thus the most mortality. Infected hosts, however, showed relatively low mortality. Our observations are compatible with the hypothesis that fighting off infection compromises longevity because deployment of the immune response comes with costs. This cost could be due to immune-mediated damage to host tissues (immunopathology), but it could equally be due to the energetic cost of launching an immune response, i.e. energy devoted to fighting parasites is unavailable for growth or maintenance. This cost of immunity is largely evident in the short term (30 or fewer days); over longer time frames, infection also compromises longevity (Fig. 3) (Jensen, Little, Skorping & Ebert 2006).
We studied costs of immunity with respect to mortality because we reasoned that patterns of mortality would be relatively easy to interpret. Reproduction, for example, is confounded because the principal effect of P. ramosa infection is fecundity reduction, while at the same time hosts may show fecundity compensation during the pre-patent period (Ebert, Carius, Little & Decaestecker 2004). The present study was not designed to disentangle these nuances. In addition, reproduction itself can compromise longevity, and our work provided evidence that short-term levels of mortality experienced by uninfected hosts is partly attributable to reproductive effort: having more offspring increased mortality. This relationship between reproduction and longevity is surprising because longer lived hosts have more opportunity to reproduce, and thus our estimate of how reproduction compromises longevity is likely conservative.
The cost of immunity described here – the cost of deploying an immune response – should not be confused with other types of costs, such as those associated with evolving greater resistance. For example, lines of Drosophila melanogaster artificially selected for greater parasitoid resistance also have reduced levels of larval competitive ability (Kraaijeveld & Godfray 1997; Fellowes, Kraaijeveld & Godfray 1998; Fellowes, Kraaijeveld & Godfray 1999; Kraaijeveld, Limentani & Godfray 2001), while artificial selection for phenoloxidase activity in dung flies revealed a trade-off with survivorship (Schwarzenbach & Ward 2006). There is no evidence for such a genetic-based cost of resistance in D. magna. In particular, a comparison of a range of susceptible and resistant genotypes in terms of mortality, life-history traits and competitive ability in the absence of parasites found no evidence for standing costs of resistance (Little, Carius, Sakwinska & Ebert 2002). It remains conceivable, however, that different Daphnia genotypes would show quantitatively or qualitatively different immune responses upon exposure to parasites, and this could be a focus of future experiments. A further focus of future experiments could be studying costs (pleiotropic or costs of launching the immune response) under a range of environmental and host conditions, because these have clearly been shown to effect the capacity of hosts to resist infection (Krist, Jokela, Wiehn & Lively 2004; Mitchell, Rogers, Little & Read 2005) and they ought to similarly impact immunopathology or energy use by the immune system.
Studies of fitness variation (which, requiring the study of a substantial number of individuals, may not always feasibly include detailed immunological assays) are required to determine how immune responses mediate host and parasite evolution. Nevertheless, studies of the fitness costs of immunity in Daphnia would be enhanced by immune assays that can verify that immune responsiveness mediates mortality. This is a challenging task with the Daphnia–Pasteuria system, which is practical for the efficient measurement of fitness parameters, but for which knowledge of immune mechanisms is lacking. The simple immune measures commonly used in other systems (e.g. Kraaijeveld et al. 2001; Moret & Siva-Jothy 2003) might be adapted to the Daphnia system, or, alternatively, the recent sequencing of a Daphnia genome may help advance knowledge of the immune-related genome to the stage where a large range of immune molecules can be assayed. Overall, knowledge of the mechanistic basis of either costs of immunity or costs of evolving resistance in invertebrates is scarce (reviewed in Kraaijeveld, Ferrari & Godfray 2002; Rolff & Siva-Jothy 2003), compared with, for example, the related field of costs of resistance to insecticides where extremely detailed knowledge is available (e.g. Labbé, Lenormand & Raymond 2005). This situation seems likely to change given the increasing interest in invertebrate immune systems (Kurtz 2004; Little, Hultmark & Read 2005).