The effect of symbiosis on symbiont fitness – interactions within a simple metaorganism

Organisms and their resident microbial communities form a complex and mostly stable ecosystem. It is known that the specific composition and abundance of certain bacterial species affect host health and Darwinian fitness, but the processes that lead to these microbial patterns are unknown. We investigate this by deconstructing the simple microbiome of the freshwater polyp Hydra. We contrast the performance of its two main bacterial associates, Curvibacter and Duganella, on germ free hosts with two in vitro environments over time. We show that interactions within the microbiome but also host modulation lead to the observed species frequencies and abundances. More specifically, we find that rare microbiome members are essential for achieving the observed community composition, which ultimately sets the maximum carrying capacity. Bacterial fitness strongly depends on the environment: while Duganella performs better than Curvibacter in a non-host habitat, Curvibacter benefits through the host association. This is of particular interest because Curvibacter and its host show a history of co-evolution, as inferred from phylogenies, whereas the colonization with Duganella seems to be a recent event. Our findings oppose the assumption that bacteria always benefit through the association with the host and poses questions regarding the long-term maintenance of such relationships.


Introduction
Growth rates of Curvibacter did not significantly differ between the host and the 151 microcosm environments. This is in marked contrast to Duganella, where significantly 152 higher growth rates were observed in the non-host as compared to the host 153 environment. In all environments, except for the host, Duganella achieved a significantly 154 higher growth rate than Curvibacter

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We observed r < 0 for all environments and frequencies, indicating that Curvibacter 201 performs worse than Duganella when they are in direct competition (Fig. 6). There is no 202 overall effect of the initial frequency -only in the mixed environment differences can be 9 detected with the lowest performance of Curvibacter at equal frequencies of the two 204 competitors (Generalized linear model: where ecological interactions can experimentally be dissected, allows for hypothesis 218 testing. For this, a relatively simple system, such as Hydra and its microbiome is ideal for 219 'deconstructing' a metaorganism and its interactions (24). This strategy is novel in 220 meta-organism research but has yielded exciting results regarding rules that determine 221 community assembly and stability in non-host associated microbial communities (35, 222 36). In microbiome research, the added advantage of including a host provides the 223 opportunity to study the performance of the individual microbiome members in their 224 natural environment. In addition, the comparison of the in vivo to in vitro environments 225 provides information on host effects in regulating its microbiome.

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Carrying capacity is defined as the largest population size that an ecosystem can 227 sustainably support without degrading the ecosystem. Therefore population size is significance of host carrying capacity has been largely overlooked until very recently 231 where a link between host health and microbiome density has been reported (8).

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Bacterial levels have been quantified for only a few model organisms such as in the gut 233 of larval zebrafish (37) or the gut of Drosophila melanogaster (38). The host's carrying 234 capacity ultimately sets the upper boundary for bacterial fitness relative to any 235 competitors, and provides a reference for investigating the interactions within the host 236 microbiota at spatial and temporal scales.

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We here show that wild-type Hydra is characterized by a specific carrying 238 capacity of about 10 5 bacteria per polyp that is stable in adult polyps and can be 239 artificially assembled through the re-population of germ-free animals with the natural Duganella reaching up to 10 8 CFUs/ml and Curvibacter up to 10 5 CFUs/ml. Interestingly 280 we were not able to replicate the in vivo dynamics between Curvibacter and Duganella in 281 vitro, suggesting that the host mediates the interaction between the two bacterial 282 species. Previous work has suggested that continued coexistence between bacterial 283 species is dependent on their relative growth. Coexistence is maintained if the slower-12 growing species is released from its dependence on the other (41), which in our case 285 could be compensated by the host. This aspect will be subject of further investigation. whereas it was only present at very low frequencies in both in vitro habitats due to the 295 high continuous growth rates of Duganella (see also (43) for homogeneous 296 environment). This pattern seems to be more pronounced in the static environment, 297 fitting to the general assumption that extinctions are more likely to occur in spatially 298 structure environments. In terms of cross-feeding interactions one could reason that the 299 faster growing-species is released from its dependence on the other, resulting in the 300 slower-growing species being lost (a hypothesis that is termed 'feed the faster grower') 301 (41).

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Many host-associated microbial communities live in highly competitive 303 surroundings, in which they struggle to persist. According to Ghoul and Mitri (44)

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When comparing the bacterial growth rates in mono-associations we observe 313 that Curvibacter performs equally well in all habitats, whereas Duganella shows a 314 reduced growth rate compared to Curvibacter on the host and performs best in the non-315 host habitat. This suggests that Duganella is not particularly adapted to the host niche.

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Interestingly the two species also differ in their phylogenetic association patterns with 317 different Hydra species (45). Whereas different lineages of Curvibacter have been shown 318 to be associated with the majority (N=6) of Hydra species analysed (N=7), Duganella 319 was detected in less than half (N=3). This indicates that Duganella is a more recent 320 member of the Hydra microbiome and that it is not as co-evolved as it has been 321 suggested for one of the keystone species of Hydra's microbiome, Curvibacter (43).

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Nevertheless, the specific microbiome of Hydra vulgaris, which is studied here, has been 323 shown to be stable as animals living in the wild were colonized by a similar microbiome 324 as compared to the ones that had been maintained in the laboratory for a relatively long 325 time (25). One of the few metaorganisms, where symbiont performance within the host 326 relative to a non-host environment has been contrasted, is the squid-Vibrio system. For 327 this obligate association Wollenberg and Ruby (19) demonstrated that symbionts have 328 an increased reproductive capacity and higher fitness when associated with the host. In 329 combination with the results from this study, this indicates that more specifically 330 associated symbionts indeed benefit from growing in the host habitat.

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The response variable was 'selection rate constant Curvibacter' and the explanatory 472 variables were 'environment', 'starting density' and 'environment' x 'starting density'.

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Sample size was chosen to maximise statistical power and ensure sufficient 474 replication. Assumptions of the tests, that is, normality and equal distribution of 475 variances, were visually evaluated. Non-significant interactions were removed from the 476 models. All tests were two-tailed. Effects were considered significant at the level of P < 477 0.05. All statistical analyses were performed with JMP 9. Graphs were produced with