Coupling between tolerance and resistance differs between 1 related Eimeria parasite species: implications for coevolution with 2 their mouse hosts 3

23 Resistance (host capacity to reduce parasite burden) and tolerance (host capacity to 24 reduce impact on its health for a given parasite burden) manifest two different lines of 25 defence. Tolerance can be independent from resistance, traded-off against it, or the 26 two can be positively correlated because of redundancy in underlying (immune) 27 processes. We here tested whether this coupling between tolerance and resistance 28 could differ upon infection with closely related parasite species. We tested this in 29 experimental infections with two parasite species of genus Eimeria . We measured 30 proxies for resistance (the (inverse of) number of parasite transmission stages 31 (oocysts) per gram of feces at the day of maximal shedding) and tolerance (the slope 32 of maximum relative weight loss compared to day of infection on number of oocysts 33 per gram of feces at the day of maximal shedding for each host strain) in four inbred 34 mouse strains and four groups of F1 hybrids belonging to two mouse subspecies, 35 Mus musculus domesticus and M. m. musculus . We found a negative correlation 36 between resistance and tolerance against E. falciformis , while the two are uncoupled 37 against E. ferrisi. We conclude that resistance and tolerance against the first parasite 38 species might be traded off, but evolve more independently in different mouse 39 genotypes against the latter. We argue that evolution of the host immune defences 40 can be studied largely irrespective of parasite isolates if resistance-tolerance coupling 41 is absent or weak ( E. ferrisi ) but host-parasite coevolution is more likely observable 42 and best studied in a system with negatively correlated tolerance and resistance 43 ( E. falciformis ). 44


Introduction
Host defence mechanisms evolve to alleviate the detrimental effect of parasites. They can be categorised into two components: resistance and tolerance (Råberg et al.,  81 Nevertheless, resistance and tolerance can also be genetically and physiologically 82 independent, involving different proximate mechanisms. Lack of correlation between 83 both defences was shown for example in monarch butterflies (Danaus plexippus) 84 infected by the protozoan parasite Ophryocystis elektroscirrha. This study found 85 genetic variation in resistance between butterflies families, but a fixed tolerance 86 (Lefèvre et al., 2010). Similarly, no correlation could be found between resistance and 87 tolerance for the fish Leuciscus burdigalensis in response to infection with its parasite 88 Tracheliastes polycolpus. The authors explain the decoupling of both defences by the 89 fact that, in this system, tolerance likely involves wound repair rather than immune 90 regulation, making resistance and tolerance mechanisms independent (Mazé-Guilmo 91 4 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 27, 2020. Sheldon & Verhulst, 1996). 103 We have seen that depending on the system studied resistance and tolerance can be 104 (1) uncoupled (independent), (2) positively correlated (involving same genes and 105 mechanisms), or (3) negatively correlated (traded-off). Theoretical models show that 106 coupling between resistance and tolerance (or absence thereof) could depend not 107 only on the host but also on the parasite (Carval & Ferriere, 2010). Here we tested 108 this hypothesis. More precisely, we asked whether there could be differences in the  117 The evolutionary history of these different Eimeria species in the two house mouse 118 subspecies is unknown and it is unclear whether subspecies-specific adaptation 119 exists in one or the other. 120 We tested if coupling between resistance and tolerance differs between both parasite 121 species and discussed the implication for parasite-host coevolution.  Table S1). . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 27, 2020. ; https://doi.org/10.1101/2020.01.24.918144 doi: bioRxiv preprint analyses were consistent with those obtained on the main data set. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 27, 2020. ; This weight (loss) can be expected to be a very conservative estimate for our 228 analyses (rendering tolerance conservatively low for these animals, which might have 229 lost more weight if not sacrificed). 230 Tolerance is usually defined as a reaction norm, i.e. the regression slope of host fitness   For each of our model, we also asked within each parasite isolate if the response 248 differed between mouse groups using likelihood ratio tests (G) as described above. Of 249 11 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 27, 2020. ; https://doi.org/10.1101/2020.01.24.918144 doi: bioRxiv preprint note, four mice infected byE. falciformis isolate Brandenburg88 did not shed any 250 oocysts as death occurred at or one day before the peak of oocysts shedding in other 251 mice. For this reason, we modelled maximum OPG for mice infected with this parasite 252 using a zero-inflated negative binomial (ZINB) generalised linear model, after verifying 253 that it provided a better fit than the simple negative binomial based on log likelihood 254 and AIC criteria.   268 We tested coupling between resistance and tolerance for E. ferrisi and E. falciformis 269 using the isolates Brandenburg64 and Brandenburg88 and our eight mouse groups. 270 12 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 27, 2020. Graphics were produced using the R package ggplot2 (Wickham, 2016) and compiled 290 using the free software inkscape (https://inkscape.org). 291 13 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 27, 2020.

Resistance and tolerance to E. ferrisi isolate Brandenburg64
328 are uncoupled 329 We tested coupling between resistance and tolerance for E. ferrisi isolate 330 Brandenburg64 in our eight mouse groups. First, we tested whether our proxies for 331 resistance, impact on weight and tolerance were different between the mouse groups. 332 We found the maximum number of OPG and relative weight loss to be statistically 333 different between mouse groups (LRT: maximum number of OPG: G=26.6, df=7, 334 P<0.001; Figure 4A; maximum relative weight loss: G=21.5, df=7, P<0.01; Figure   335 4B). Tolerance was not found to significantly differ between mouse groups for this 336 15 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 27, 2020. ; https://doi.org/10.1101/2020.01.24.918144 doi: bioRxiv preprint parasite isolate (LRT: G=6.8, df=7, P=0.45; Figure 4C). 337 We found a non significant positive correlation between resistance (inverse of maximum 338 number of OPG) and impact on health (maximum weight loss) (Spearman's ρ=0.69, 339 P=0.07, N=8; Figure 4D). Eventually, we did not find a correlation between resistance 340 (inverse of maximum number of OPG) and tolerance (inverse of slope of maximum 341 weight loss on maximum OPG) (Spearman's ρ=0, P=1, N=8; Figure 4E). 342 In conclusion, we did not find indications of resistance-tolerance coupling for E. ferrisi   346 We then tested coupling between resistance and tolerance for E. falciformis isolate 347 Brandenburg88 in our eight mouse groups. First, we tested if our proxies for 348 resistance, impact on weight and tolerance were different between the mouse groups. 349 We found the maximum number of OPG and relative weight loss to be statistically 350 different between mouse groups (LRT: maximum number of OPG: G=28.6, df=14, 351 P=0.012; Figure 5A; maximum relative weight loss: G=21, df=7, P<0.01; Figure 5B). 352 Finally, contrary to our results on E. ferrisi isolate Brandenburg64, the tolerance 353 slopes for E. falciformis isolate Brandenburg88 were different between mouse groups 354 (LRT: G=13.9, df=7, P=0.05; Figure 5C). 355 We detected a strong negative correlation between (inverse of) resistance (maximum 356 number of OPG) and tolerance (inverse of slope of maximum weight loss on 357 maximum OPG) (Spearman's ρ=-0.95, P=0.001; Figure 5E). We conclude that this 358 16 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 27, 2020. ; https://doi.org/10.1101/2020.01.24.918144 doi: bioRxiv preprint correlation is unlikely a statistical artifact, as (1) mouse groups present statistically 359 different values of resistance and tolerance and (2) we found a (non significant) 360 negative correlation between resistance (inverse of maximum number of OPG) and 361 impact on health (maximum weight loss) (Spearman's ρ=-0.5, P=0.22; Figure 5D), 362 indicating that mouse groups losing more weight also shed less parasites. 363 We conclude that our results indicate the presence of negative resistance-tolerance 364 coupling for E. falciformis isolate Brandenburg88.

366
In this study, we assessed resistance and tolerance to two closely related parasites, 367 E. ferrisi (two isolates) and E. falciformis (one isolate), in four mouse strains and their 368 intra-and intersubspecific hybrids.
Understanding this coupling has two major 369 implications. 370 From a practical "measurement" perspective we can ask whether tolerance can be 371 predicted from resistance, as the latter is easier to measure (e.g. in field sampling). 372 Many studies assess the impact of parasites on host fitness based on resistance. If, 373 as we found in the present study, resistance and tolerance are decoupled this can be 374 missleading. In our host system, the house mice, for example, it has been shown that  . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 27, 2020.   (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 27, 2020. ; https://doi.org/10.1101/2020.01.24.918144 doi: bioRxiv preprint and absence of correlation between maximum oocysts per gram of feces used as a 618 proxy for (inverse of) resistance and tolerance (E); Grey error bars represent 95% 619 confidence intervals. Our results do not support coupling between resistance and 620 tolerance E. ferrisi isolate Brandenburg64.

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. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 27, 2020.  Figure 3: Comparison of resistance, impact on weight and tolerance between mouse strains for both Eimeria ferrisi isolates.

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. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted June 27, 2020. ; https://doi.org/10.1101/2020.01.24.918144 doi: bioRxiv preprint