Hierarchical eco-evo dynamics mediated by the gut microbiome.

The concept of eco-evolutionary (eco-evo) dynamics, stating that ecological and evolutionary processes occur at similar time scales and influence each other, has contributed to our understanding of responses of populations, communities, and ecosystems to environmental change. Phenotypes, central to these eco-evo processes, can be strongly impacted by the gut microbiome. The gut microbiome shapes eco-evo dynamics in the host community through its effects on the host phenotype. Complex eco-evo feedback loops between the gut microbiome and the host communities might thus be common. Bottom-up dynamics occur when eco-evo interactions shaping the gut microbiome affect host phenotypes with consequences at population, community, and ecosystem levels. Top-down dynamics occur when eco-evo dynamics shaping the host community structure the gut microbiome.

Genotype specific and microbiome effects of hypoxia in the model organism Daphnia magna.

The fitness of the host is highly influenced by the interplay between the host and its associated microbiota. The flexible nature of these microbiota enables them to respond swiftly to shifts in the environment, which plays a key role in the host's capacity to withstand environmental stresses. To understand the role of the microbiome in host tolerance to hypoxia, one of the most significant chemical changes occurring in water ecosystems due to climate change, we performed a reciprocal gut transplant experiment with the freshwater crustacean Daphnia magna. In a microbiome transplant experiment, two genotypes of germ-free recipients were inoculated with gut microbiota from Daphnia donors of their own genotype or from the other genotype, that had been either pre-exposed to normoxic or hypoxic conditions. We found that D. magna individuals had a higher survival probability in hypoxia if their microbiome had been pre-exposed to hypoxia. The bacterial communities of the recipients changed over time with a reduction in alpha diversity, which was stronger when donors were pre-exposed to a hypoxic environment. While donor genotype had no influence on the long-term survival probability in hypoxia, donor genotypes was the most influential factor of the microbial community 3 days after the transplantation. Our results indicate that microbiome influencing factors mediate host fitness in a hypoxic environment in a time depending way.

Comparison between the gut bacterial community of laboratory cultured and wild Daphnia.

The fitness of an organism is often impacted by the composition and biological activity of its associated bacterial community. Many factors, including host genetics, diet, and temperature can influence the bacterial community composition. Furthermore, these factors can differ strongly between natural and laboratory environments. Consequently, several studies have highlighted results from laboratory experiments investigating host-associated bacterial communities to be conflicting with those obtained under field conditions. Here, we compared the Daphnia magna gut bacterial communities in natural host populations with those of laboratory cultured hosts. We further analyzed changes in the gut bacterial communities after transferring hosts from natural populations to the laboratory on the short- and long-term. Results show that, in general, the gut bacterial communities from natural populations differ from those of laboratory cultures and that their composition and diversity changed one hour after being transferred to the laboratory. Over the following 14 days, the composition and diversity changed gradually. On the longer term (after two years of rearing hosts in the laboratory) the composition and diversity of the gut bacterial communities was strongly altered compared to the initial state. Our findings indicate that the gut bacterial communities of Daphnia magna in laboratory experiments is not representative for natural field conditions, and that caution should be taken when interpreting results from laboratory experiments for natural settings.

Host-bacteriome transplants of the schistosome snail host Biomphalaria glabrata reflect species-specific associations.

Microbial symbionts can affect host phenotypes and, thereby, ecosystem functioning. The microbiome is increasingly being recognized as an important player in the tripartite interaction between parasitic flatworms, snail intermediate hosts, and the snail microbiome. In order to better understand these interactions, transplant experiments are needed, which rely on the development of a reliable and reproducible protocol to obtain microbiome-disturbed snails. Here, we report on the first successful snail bacteriome transplants, which indicate that Biomphalaria glabrata can accrue novel bacterial assemblies depending on the available environmental bacteria obtained from donor snails. Moreover, the phylogenetic relatedness of the donor host significantly affected recipients' survival probability, corroborating the phylosymbiosis pattern in freshwater snails. The transplant technique described here, complemented by field-based studies, could facilitate future research endeavors to investigate the role of specific bacteria or bacterial communities in parasitic flatworm resistance of B. glabrata and might ultimately pave the way for microbiome-mediated control of snail-borne diseases.

Locally adapted gut microbiomes mediate host stress tolerance.

While evidence for the role of the microbiome in shaping host stress tolerance is becoming well-established, to what extent this depends on the interaction between the host and its local microbiome is less clear. Therefore, we investigated whether locally adapted gut microbiomes affect host stress tolerance. In the water flea Daphnia magna, we studied if the host performs better when receiving a microbiome from their source region than from another region when facing a stressful condition, more in particular exposure to the toxic cyanobacteria Microcystis aeruginosa. Therefore, a reciprocal transplant experiment was performed in which recipient, germ-free D. magna, isolated from different ponds, received a donor microbiome from sympatric or allopatric D. magna that were pre-exposed to toxic cyanobacteria or not. We tested for effects on host life history traits and gut microbiome composition. Our data indicate that Daphnia interact with particular microbial strains mediating local adaptation in host stress tolerance. Most recipient D. magna individuals performed better when inoculated with sympatric than with allopatric microbiomes. This effect was most pronounced when the donors were pre-exposed to the toxic cyanobacteria, but this effect was also pond and genotype dependent. We discuss how this host fitness benefit is associated with microbiome diversity patterns.

The Role of Microbiome and Genotype in Daphnia magna upon Parasite Re-Exposure.

Recently, it has been shown that the community of gut microorganisms plays a crucial role in host performance with respect to parasite tolerance. Knowledge, however, is lacking on the role of the gut microbiome in mediating host tolerance after parasite re-exposure, especially considering multiple parasite infections. We here aimed to fill this knowledge gap by studying the role of the gut microbiome on tolerance in Daphnia magna upon multiple parasite species re-exposure. Additionally, we investigated the role of the host genotype in the interaction between the gut microbiome and the host phenotypic performance. A microbiome transplant experiment was performed in which three germ-free D. magna genotypes were exposed to a gut microbial inoculum and a parasite community treatment. The gut microbiome inocula were pre-exposed to the same parasite communities or a control treatment. Daphnia performance was monitored, and amplicon sequencing was performed to characterize the gut microbial community. Our experimental results showed that the gut microbiome plays no role in Daphnia tolerance upon parasite re-exposure. We did, however, find a main effect of the gut microbiome on Daphnia body size reflecting parasite specific responses. Our results also showed that it is rather the Daphnia genotype, and not the gut microbiome, that affected parasite-induced host mortality. Additionally, we found a role of the genotype in structuring the gut microbial community, both in alpha diversity as in the microbial composition.

A methodological framework to analyse determinants of host-microbiota networks, with an application to the relationships between Daphnia magna's gut microbiota and bacterioplankton.

The past 30 years have seen both a surge of interest in assessing ecological interactions using tools borrowed from network theory and an explosion of data on the occurrence of microbial symbionts thanks to next-generation sequencing. Given that classic network methods cannot currently measure the respective effects of different environmental and biological drivers on network structure, we here present two methods to elucidate the determinants of bipartite interaction networks. The first method is based on classifications and compares communities within networks to the grouping of nodes by treatment or similar controlling groups. The second method assesses the link between multivariate explanatory variables and network structure using redundancy analyses after singular value decomposition. In both methods, the significance of effects can be gauged through two randomizations. Our methods were applied to experimental data on Daphnia magna and its interactions with gut microbiota and bacterioplankton. The whole network was affected by Daphnia's diet (algae and/or cyanobacteria) and sample type, but not by Daphnia genotype. At coarse grains, bacterioplankton and gut microbiota communities were different. At this scale, the structure of the gut microbiota-based network was not linked to any explanatory factors, while the bacterioplankton-based network was related to both Daphnia's diet and genotype. At finer grains, Daphnia's diet and genotype affected both microbial networks, but the effect of diet on gut microbiota network structure was mediated solely by differences in microbial richness. While no reciprocal effect between the microbial communities could be found, fine-grained analyses presented a more nuanced picture, with bacterioplankton likely affecting the composition of the gut microbiota. Our methods are widely applicable to bipartite networks, can elucidate both controlled and environmental effects in experimental setting using a large amount of sequencing data and can tease apart reciprocal effects of networks on one another. The twofold approach we propose has the advantage of being able to tease apart effects at different scales of network structure, thus allowing for detailed assessment of reciprocal effects of linked networks on one another. As such, our network methods can help ecologists understand huge datasets reporting microbial co-occurrences within different hosts.

The bacterioplankton community composition and a host genotype dependent occurrence of taxa shape the Daphnia magna gut bacterial community.

The assembly of host-associated bacterial communities is influenced by a multitude of biotic and abiotic factors. It is essential to gain insight in the impact and relative strength of these factors if we want to be able to predict the effects of environmental change on the assembly of host-associated bacterial communities, or deliberately modify them. The environmental pool of bacteria, from which the host is colonized, and the genetic background of the host are both considered to be important in determining the composition of host-associated bacterial communities. We experimentally assessed the relative importance of these two factors and their interaction on the composition of Daphnia magna gut bacterial communities. Bacterioplankton originating from natural ponds or a laboratory culture were used to inoculate germ-free Daphnia of different genotypes. We found that the composition of the environmental bacterial community has a major influence on the Daphnia gut bacterial community, both reflected by the presence or absence of specific taxa as well as by a correlation between abundances in the environment and on the host. Our data also indicate a consistent effect of host genotype on the occurrence of specific bacterial taxa in the gut of Daphnia over different environments.

Diet and Genotype of an Aquatic Invertebrate Affect the Composition of Free-Living Microbial Communities.

In spite of the growing interest in the role of the gut microbiome (GM) in host physiology and health, the mechanisms governing its assembly and its effects on the environment are poorly understood. In this article, we show that the host genotype and the GM of Daphnia influence the community structure of the surrounding bacterioplankton (BPK). When Daphnia genotypes were placed in an identical environment, both the GM and BPK showed a genotype and diet-dependent taxonomic composition. Overall, the GM strongly differed from the BPK in taxonomic composition and was characterized by a lower α-diversity, suggesting a selective rejecting of bacteria from the regional species pool. In a microbiome transplant experiment, the assembly of both the GM and BPK was strongly affected by the host genotype and the inoculum to which germ-free Daphnia were exposed. The combination of these results suggests a strong interaction between the host genotype, its GM and free-living microbial communities. Currently, it is generally assumed that an animal's diet has a strong effect on the animal's GM, but only a negligible (if any) effect on the surrounding environment. However, our results indicate that the diet/microbiome inocula have a small effect on the gut community and a large effect on the community in the surrounding environment. This structuring genotype × microbiome × environment effect is an essential prerequisite that could indicate that microbiomes play an important role in eco-evolutionary processes.

Urbanization drives cross-taxon declines in abundance and diversity at multiple spatial scales.

The increasing urbanization process is hypothesized to drastically alter (semi-)natural environments with a concomitant major decline in species abundance and diversity. Yet, studies on this effect of urbanization, and the spatial scale at which it acts, are at present inconclusive due to the large heterogeneity in taxonomic groups and spatial scales at which this relationship has been investigated among studies. Comprehensive studies analysing this relationship across multiple animal groups and at multiple spatial scales are rare, hampering the assessment of how biodiversity generally responds to urbanization. We studied aquatic (cladocerans), limno-terrestrial (bdelloid rotifers) and terrestrial (butterflies, ground beetles, ground- and web spiders, macro-moths, orthopterans and snails) invertebrate groups using a hierarchical spatial design, wherein three local-scale (200 m × 200 m) urbanization levels were repeatedly sampled across three landscape-scale (3 km × 3 km) urbanization levels. We tested for local and landscape urbanization effects on abundance and species richness of each group, whereby total richness was partitioned into the average richness of local communities and the richness due to variation among local communities. Abundances of the terrestrial active dispersers declined in response to local urbanization, with reductions up to 85% for butterflies, while passive dispersers did not show any clear trend. Species richness also declined with increasing levels of urbanization, but responses were highly heterogeneous among the different groups with respect to the richness component and the spatial scale at which urbanization impacts richness. Depending on the group, species richness declined due to biotic homogenization and/or local species loss. This resulted in an overall decrease in total richness across groups in urban areas. These results provide strong support to the general negative impact of urbanization on abundance and species richness within habitat patches and highlight the importance of considering multiple spatial scales and taxa to assess the impacts of urbanization on biodiversity.

Parasites opt for the best of both worlds.

The fungal parasite Podosphaera plantaginis employs both sexual and asexual reproduction to increase its chances of infecting the plant Plantago lanceolata.

Microbiota inoculum composition affects holobiont assembly and host growth in Daphnia.

BACKGROUND: Host-associated microbiota is often acquired by horizontal transmission of microbes present in the environment. It is hypothesized that differences in the environmental pool of colonizers can influence microbiota community assembly on the host and as such affect holobiont composition and host fitness. To investigate this hypothesis, the host-associated microbiota of the invertebrate eco(toxico)logical model Daphnia was experimentally disturbed using different concentrations of the antibiotic oxytetracycline. The community assembly and host-microbiota interactions when Daphnia were colonized by the disturbed microbiota were investigated by inoculating germ-free individuals with the microbiota. RESULTS: Antibiotic-induced disturbance of the microbiota had a strong effect on the subsequent colonization of Daphnia by affecting ecological interactions between members of the microbiota. This resulted in differences in community assembly which, in turn, affected Daphnia growth. CONCLUSIONS: These results show that the composition of the pool of colonizing microbiota can be an important structuring factor of the microbiota assembly on Daphnia, affecting holobiont composition and host growth. These findings contribute to a better understanding of how the microbial environment can shape the holobiont composition and affect host-microbiota interactions.

The impact of human activities and lifestyles on the interlinked microbiota and health of humans and of ecosystems.

Plants, animals and humans, are colonized by microorganisms (microbiota) and transiently exposed to countless others. The microbiota affects the development and function of essentially all organ systems, and contributes to adaptation and evolution, while protecting against pathogenic microorganisms and toxins. Genetics and lifestyle factors, including diet, antibiotics and other drugs, and exposure to the natural environment, affect the composition of the microbiota, which influences host health through modulation of interrelated physiological systems. These include immune system development and regulation, metabolic and endocrine pathways, brain function and epigenetic modification of the genome. Importantly, parental microbiotas have transgenerational impacts on the health of progeny. Humans, animals and plants share similar relationships with microbes. Research paradigms from humans and other mammals, amphibians, insects, planktonic crustaceans and plants demonstrate the influence of environmental microbial ecosystems on the microbiota and health of organisms, and indicate links between environmental and internal microbial diversity and good health. Therefore, overlapping compositions, and interconnected roles of microbes in human, animal and plant health should be considered within the broader context of terrestrial and aquatic microbial ecosystems that are challenged by the human lifestyle and by agricultural and industrial activities. Here, we propose research priorities and organizational, educational and administrative measures that will help to identify safe microbe-associated health-promoting modalities and practices. In the spirit of an expanding version of "One health" that includes environmental health and its relation to human cultures and habits (EcoHealth), we urge that the lifestyle-microbiota-human health nexus be taken into account in societal decision making.

Back to the future in a petri dish: Origin and impact of resurrected microbes in natural populations.

Current natural populations face new interactions because of the re-emergence of ancient microbes and viruses. These risks come from the re-emergence of pathogens kept in laboratories or from pathogens that are retained in the permafrost, which become available upon thawing due to climate change. We here focus on the effects of such re-emergence in natural host populations based on evolutionary theory of virulence and long-term studies, which investigate host-pathogen adaptations. Pathogens tend to be locally and temporally adapted to their co-occurring hosts, but when pathogens from a different environment or different time enter the host community, the degree to which a new host-pathogen interaction is a threat will depend on the specific genotypic associations, the time lag between the host and the pathogen, and the interactions with native or recent host and pathogen species. Some insights can be obtained from long-term studies using a resurrection ecology approach. These long-term studies based on time-shift experiments are essential to obtain insight into the mechanisms underlying host-pathogen coevolution at several ecological and temporal scales. As past pathogens and their corresponding host(s) can differ in infectivity and susceptibility, strong reciprocal selective pressures can be induced by the pathogen. These strong selective pressures often result in an escalating arms race, but do not necessarily result in increased infectivity over time. Human health can also be impacted by these resurrected pathogens as the majority of emerging infectious diseases are zoonoses, which are infectious diseases originating from animal populations naturally transmitted to humans. The sanitary risk associated with pathogen emergence from different environments (spatial or temporal) depends on a combination of socioeconomic, environmental, and ecological factors that affect the virulence or the pathogenic potential of microbes and their ability to infect susceptible host populations.

Early transcriptional response pathways in Daphnia magna are coordinated in networks of crustacean-specific genes.

Natural habitats are exposed to an increasing number of environmental stressors that cause important ecological consequences. However, the multifarious nature of environmental change, the strength and the relative timing of each stressor largely limit our understanding of biological responses to environmental change. In particular, early response to unpredictable environmental change, critical to survival and fitness in later life stages, is largely uncharacterized. Here, we characterize the early transcriptional response of the keystone species Daphnia magna to twelve environmental perturbations, including biotic and abiotic stressors. We first perform a differential expression analysis aimed at identifying differential regulation of individual genes in response to stress. This preliminary analysis revealed that a few individual genes were responsive to environmental perturbations and they were modulated in a stressor and genotype-specific manner. Given the limited number of differentially regulated genes, we were unable to identify pathways involved in stress response. Hence, to gain a better understanding of the genetic and functional foundation of tolerance to multiple environmental stressors, we leveraged the correlative nature of networks and performed a weighted gene co-expression network analysis. We discovered that approximately one-third of the Daphnia genes, enriched for metabolism, cell signalling and general stress response, drives transcriptional early response to environmental stress and it is shared among genetic backgrounds. This initial response is followed by a genotype- and/or condition-specific transcriptional response with a strong genotype-by-environment interaction. Intriguingly, genotype- and condition-specific transcriptional response is found in genes not conserved beyond crustaceans, suggesting niche-specific adaptation.

Host-genotype dependent gut microbiota drives zooplankton tolerance to toxic cyanobacteria.

The gut microbiota impacts many aspects of its host's biology, and is increasingly considered as a key factor mediating performance of host individuals in continuously changing environments. Here we use gut microbiota transplants to show that both host genotype and gut microbiota mediate tolerance to toxic cyanobacteria in the freshwater crustacean Daphnia magna. Interclonal variation in tolerance to cyanobacteria disappears when Daphnia are made germ-free and inoculated with an identical microbial inoculum. Instead, variation in tolerance among recipient Daphnia mirrors that of the microbiota donors. Metagenetic analyses point to host genotype and external microbial source as important determinants of gut microbiota assembly, and reveal strong differences in gut microbiota composition between tolerant and susceptible genotypes. Together, these results show that both environmentally and host genotype-induced variations in gut microbiota structure mediate Daphnia tolerance to toxic cyanobacteria, pointing to the gut microbiota as a driver of adaptation and acclimatization to cyanobacterial harmful algal blooms in zooplankton.

Conserved Transcription Factors Steer Growth-Related Genomic Programs in Daphnia.

Ecological genomics aims to understand the functional association between environmental gradients and the genes underlying adaptive traits. Many genes that are identified by genome-wide screening in ecologically relevant species lack functional annotations. Although gene functions can be inferred from sequence homology, such approaches have limited power. Here, we introduce ecological regulatory genomics by presenting an ontology-free gene prioritization method. Specifically, our method combines transcriptome profiling with high-throughput cis-regulatory sequence analysis in the water fleas Daphnia pulex and Daphnia magna. It screens coexpressed genes for overrepresented DNA motifs that serve as transcription factor binding sites, thereby providing insight into conserved transcription factors and gene regulatory networks shaping the expression profile. We first validated our method, called Daphnia-cisTarget, on a D. pulex heat shock data set, which revealed a network driven by the heat shock factor. Next, we performed RNA-Seq in D. magna exposed to the cyanobacterium Microcystis aeruginosa. Daphnia-cisTarget identified coregulated gene networks that associate with the moulting cycle and potentially regulate life history changes in growth rate and age at maturity. These networks are predicted to be regulated by evolutionary conserved transcription factors such as the homologues of Drosophila Shavenbaby and Grainyhead, nuclear receptors, and a GATA family member. In conclusion, our approach allows prioritising candidate genes in Daphnia without bias towards prior knowledge about functional gene annotation and represents an important step towards exploring the molecular mechanisms of ecological responses in organisms with poorly annotated genomes.

A historical perspective of nutrient change impact on an infectious disease in Daphnia.

Changes in food quality can play a substantial role in the vulnerability of hosts to infectious diseases. In this study, we focused on the genetic differentiation of the water flea Daphnia magna towards food of different quality (by manipulating C:N:P ratios) and its impact on the interaction with a virulent infectious disease, "White Fat Cell Disease (WFCD)". Via a resurrection ecology approach, we isolated two Daphnia subpopulations from different depths in a sediment core, which were exposed to parasites and a nutrient ratio gradient in a common garden experiment. Our results showed a genetic basis for sensitivity towards food deprivation. Both fecundity and host survival was differently affected when fed with low-quality food. This strongly impacted the way both subpopulations interacted with this parasite. A historical reconstruction of nutrient changes in a sediment core reflected an increase in organic material and phosphorus concentration (more eutrophic conditions) over time in the studied pond. These results enable us to relate patterns of genetic differentiation in sensitivity towards food deprivation to an increasing level of eutrophication of the subpopulations, which ultimately impacts parasite virulence effects. This finding was confirmed via a dynamic energy budgets (DEB), in which energy was partitioned for the host and the parasite. The model was tailored to our study by integrating (1) increased growth and a fecundity shift in the host upon parasitism and (2) differences of food assimilation in the subpopulations showing that a reduced nutrient assimilation resulted in increased parasite virulence. The combination of our experiment with the DEB model shows that it is important to consider genetic diversity when studying the impact of nutritional stress on species interactions, especially in the context of changing environments and emerging infectious diseases.

Bisulfite Sequencing with Daphnia Highlights a Role for Epigenetics in Regulating Stress Response to Microcystis through Preferential Differential Methylation of Serine and Threonine Amino Acids.

Little is known about the influence that environmental stressors may have on genome-wide methylation patterns, and to what extent epigenetics may be involved in environmental stress response. Yet, studies of methylation patterns under stress could provide crucial insights on stress response and toxicity pathways. Here, we focus on genome-wide methylation patterns in the microcrustacean Daphnia magna, a model organism in ecotoxicology and risk assessment, exposed to the toxic cyanobacterium Microcystis aeruginosa. Bisulfite sequencing of exposed and control animals highlighted differential methylation patterns in Daphnia upon exposure to Microcystis primarily in exonic regions. These patterns are enriched for serine/threonine amino acid codons and genes related to protein synthesis, transport and degradation. Furthermore, we observed that genes with differential methylation corresponded well with genes susceptible to alternative splicing in response to Microcystis stress. Overall, our results suggest a complex mechanistic response in Daphnia characterized by interactions between DNA methylation and gene regulation mechanisms. These results underscore that DNA methylation is modulated by environmental stress and can also be an integral part of the toxicity response in our study species.