Daphnia: from ecological to biomedical microbiome model in the context of hypoxia

Host-associated microbiomes relate to the microbiota that live inside and on the surfaces of hosts. The microbial community is not a random collection of organisms but is the result of complex interactions between microbiota within the host and between the host and the microbiota. These interactions are mediated by host physiology and environmental factors. The host-associated microbiota is currently considered as a novel factor in host health and disease with the host-associated microbiota being linked with disease states such as gastric diseases in humans and host tolerance to environmental stressors. Unfortunately, research towards finding causal contributions of the host-associated microbiota to disease states or stress-linked phenotypical states is faced with a lot of limitations due to the complex interactions between the host and its microbial environment as well as the microbiota interactions within the host-associated environment. Vertebrate animal model systems used to dominate the microbiome research field, but recently there has been an increase in interest in the potential use of alternative models, such as invertebrate models, due to their potential to be used in large-scale high-throughput studies in a cost-, labor- and resource-effective manner without ethical constraints. The aim of this PhD was to investigate the use of Daphnia as an interdisciplinary test model for host-microbiota interactions. More specifically, we focused on hypoxia, an abiotic factor whose microbiome-induced changes are both understudied in ecology and are known to be linked to human diseases.

In the first two chapters, we investigated the use of Daphnia as an ecological model system to test host-microbiota interactions in the context of hypoxia. In the first chapter, we compared the effect of normoxia with an environmental relevant hypoxia concentration on Daphnia performance, Daphnia microbiome and the bacterioplankton community  that surrounded two Daphnia magna genotypes. We discovered that hypoxia adversely affected Daphnia performance, with a significant genotype-specific effect being shown for Daphnia body size, but not for other performance traits. Hypoxia induced shifts both in the composition and structure of the bacterioplankton and Daphnia-associated microbial communities. A decreasing trend towards a lower alpha diversity within the Daphnia microbiome was associated with a higher alpha diversity in the bacterioplankton. This caused a strong hypoxia induced difference in the microbial community between the surrounding bacterioplankton and the Daphnia microbiome. Building further on chapter one, the role of the microbiome and Daphnia genotype in phenotypic plasticity with respect to hypoxia tolerance in Daphnia magna was tested through a reciprocal gut transplant in the second chapter of the thesis. Two genotypes of germ-free recipient Daphnia magna were inoculated with gut microbiota from donors of their own genotype or from the other genotype, that had been either pre-exposed to normoxic or hypoxic conditions. Daphnia had a higher survival probability in hypoxia when they received a microbiome that had been pre-exposed to hypoxia, however pre-exposure of the microbiome did not influence body size. In addition, we found evidence for a time dependent response of Daphnia microbiota to hypoxia that is only partly translated in host performance. A time-dependent shift in the microbial diversity of recipient Daphnia when exposed to hypoxia was found, with the strongest decrease in recipients who got a hypoxia pre-exposed donor. Time was also an important factor influencing the impact of the donor inoculum characteristics on the recipients. While early in the transplant, the donor genotype was an important influencing factor of the microbial communities of the recipients, donor pre-exposure was the most influencing factor at the end of the transplant.

To expand the use of Daphnia as a biomedical model system to test host-microbiota interactions, we adapted two commonly used biomedical techniques to test such interactions for their use in Daphnia in the last chapters. In chapter three, a broad pool of human microbiota was transferred to Daphnia individuals with or without a microbiome via the use of fecal microbiota transplantations. We build proof-of-concept that Daphnia can take-up human fecal donor microbiota and that the impact of fecal microbiota transplantations on Daphnia performance is dose dependent. The fecal concentrations that resulted in the highest survival could be linked with the highest unique ASVs in the recipient gut of the Daphnia. Moreover, a higher concentration was needed for Daphnia with a microbiome compared to germ-free Daphnia to take-up the donor microbiota, most likely due to stronger priority effects in Daphnia with microbiomes in comparison with germ-free individuals. The transplant success rate of these firstly performed fecal microbiota transplants in Daphnia was low and the protocol should be optimized to further increase the bacterial uptake upon human bacterial strain exposure.

In the last chapter, a human bacterial strain (E. coli RCC443) was inoculated into Daphnia with or without microbiome via mono-association experiments. We were able to build proof-of-concept that causal effects induced by specific facultatively anaerobic bacteria can be tested in germ-free Daphnia via mono-association experiments. And this even without adding algae to the diet avoiding contamination by the food source of Daphnia in the investigation of causal relationships. Indications for interspecific competition between the bacterial strains in the Daphnia gut and the administered human strain were present when evaluating the effect of the E. coli strain on Daphnia performance in the presence or absence of a microbiome. E. coli had a stronger adverse effect on Daphnia survival when Daphnia had a microbiome compared to germ-free Daphnia. Further, the influence of a hypoxic environment, which is favorable for the facultative anaerobic bacterial E. coli strain, was found to negatively affect Daphnia magna performance when exposed to the E. coli strain.

From this doctoral thesis, we conclude that hypoxia affects host performance in a mostly genotype independent way. We showed that host-associated microbiota and bacterioplankton communities shift in hypoxia and that the microbiome mediated responses to hypoxia in Daphnia is only partially translated to host performance in hypoxia but are time-dependent. These findings are relevant in the context of host acclimatization and evolutionary potential upon climate change, which is the primary cause of hypoxia and is predicted to worsen over the next decade. We further provided the foundation for implementing Daphnia as a model system in the biomedical research field to explore causal host-microbiota interactions in the last chapters. This was done by describing -the first to our knowledge- protocol for transplanting human fecal microbiota into Daphnia and by conducting mono-association experiments with human facultative anaerobic bacteria. This might be expanded in the future to examine how particular bacteria affect the expression of conserved genes important for both ecological and biomedical research. To conclude, Daphnia can be used as a model system to test host-microbiota interactions in hypoxia but more research is required before Daphnia can be implemented as a model system in biomedical research.