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APPENDIX 2
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Performance and its relation with immune response
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The observed negative covaration between upregulated PO levels and spore load can be
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explained by two mechanisms not related to immunocompetence. First, this negative covariation
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of PO level and spore load may reflect that animals with higher spore load have lower PO levels
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because they suffer more from the infection. Then we would expect that animals with a lower
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performance when infected, hence that suffer more from the infection, have lower PO levels, and
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that animals with a higher spore load (hence should suffer higher costs) have a lower
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performance. Second, small differences in condition/quality, with the good-quality individuals
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having less intense infections and a better immune response, may be driving the observed
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patterns. Then we would expect that animals with a higher performance when not-infected have
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higher PO levels and lower spore loads after infection. To evaluate both alternative mechanisms
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we determined performance of each clonal line both in the absence and in the presence of
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parasites and quantified the above-mentioned relationships with PO levels and spore load.
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(a) Methods
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For each set of 10 individuals we determined performance based on intrinsic growth rate as
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calculated by the Euler equation(see Pauwels et al. 2010). The effects of clone and parasite
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treatment on performance were tested using a two-factor ANOVA. The relationships with
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performance (both baseline and 21 days after infection) were tested at the clonal line level using
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ANCOVAs with clone as random categorical factor, performance as covariate, and PO levels
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and spore load (log-transformed) as dependent variables.
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(b) Results
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Infected animals had a considerably lower performance (F1,22=37.5; p<0.0005, figure 1). Clonal
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lines with a higher performance in the absence of parasites did not have higher PO levels
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(ANCOVA, slope 0.00002, SE = 0.00003, t11= 0.51, p = 0.62, figure 2a) or a lower parasite load
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when infected (ANCOVA, slope -0.050, SE = 0.030, t10= -1.66, p = 0.13, figure 2b).
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Furthermore, clonal lines with a lower performance when infected did not have lower PO levels
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when infected (ANCOVA, slope 0.00006, SE = 0.00025, t11= 0.25, p = 0.81, figure 2c) or a
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higher spore load (ANCOVA, slope -0.34, SE = 0.21, t11= -1.61, p = 0.14, figure 2d).
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(c) Discussion
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In our study, higher PO levels in infected hosts were associated with lower spore loads. One may
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argue that this negative covariation of PO level and spore load reflects that animals with higher
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spore load have lower PO levels because they suffer more from the infection. This explanation
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is, however, unlikely. Under that scenario, animals with a lower performance when infected,
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hence that suffer more from the infection, should have lower PO levels which was not true.
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Moreover, then we would also expect a negative covariation between spore load and
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performance as animals with a higher spore load should bear higher costs. This also was not the
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case indicating that spore loads were not driving life history and PO patterns. Similarly, Auld et
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al. (2010) reported a higher upregulation of another immune component, hemocyte numbers, in
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D. magna that became infected with P. ramosa indicating that also hemocyte upregulation was
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not lower in animals that suffer more from the infection. Further, it is unlikely that small
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differences in condition/quality, with the good-quality individuals having less intense infections
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and a better immune response, are driving the observed patterns. This is because such good-
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quality individuals should have a higher performance, which was, however, not related to
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parasite load and immune response. In short, our results indicate that D. magna that up-regulate
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PO to a higher level when infected with P. ramosa have a higher resistance to that parasite.
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Obviously, very high doses of pathogens may succeed to overcome the immune system and then
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we may no longer find any relationship between spore load and PO levels. In current experiment
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the observed spore loads were all within the range observed when exposing Daphnia to
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sediments from natural ponds (e.g. Decaestecker et al. 2007). Further, it remains to be tested to
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what extent this observation can be generalized to other parasites or other host species.
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(d) References
Auld, S. K. J. R., Scholefield, J. A. & Little, T. J. 2010 Genetic variation in the cellular response of Daphnia
magna (Crustacea: Cladocera) to its bacterial parasite. Proc. Roy. Soc. B, -.
(doi:10.1098/rspb.2010.0772)
Decaestecker, E., Gaba, S., Raeymaekers, J. A. M., Stoks, R., Van Kerckhoven, L., Ebert, D. & De Meester,
L. 2007 Host-parasite 'Red Queen' dynamics archived in pond sediment. Nature 450, 870-U16.
(doi:10.1038/nature06291)
Pauwels, K., Stoks, R. & De Meester, L. 2010 Enhanced anti-predator defence in the presence of food
stress in the water flea Daphnia magna. Funct. Ecol. 24, 322-329. (doi:10.1111/j.13652435.2009.01641.x)
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Figure captions
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Figure 1. Performance for four D. magna clones after 21 days of infection with the parasite P.
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ramosa. Given are least square means ± 1 standard error.
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Figure 2. Relationships between PO levels in infected hosts and (a) performance in not-infected
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hosts (b) and performance in infected hosts; and the relationships between (log transformed)
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spore loads and (c) performance in not-infected hosts and (d) performance in infected hosts.
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Symbols represent the different clones, and clonal means are plotted ± SE (full symbols).
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