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.

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.

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.

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.

Food availability affects the strength of mutualistic host-microbiota interactions in Daphnia magna.

The symbiotic gut microbial community is generally known to have a strong impact on the fitness of its host. Nevertheless, it is less clear how the impact of symbiotic interactions on the hosts' fitness varies according to environmental circumstances such as changes in the diet. This study aims to get a better understanding of host-microbiota interactions under different levels of food availability. We conducted experiments with the invertebrate, experimental model organism Daphnia magna and compared growth, survival and reproduction of conventionalized symbiotic Daphnia with germ-free individuals given varying quantities of food. Our experiments revealed that the relative importance of the microbiota for the hosts' fitness varied according to dietary conditions. The presence of the microbiota had strong positive effects on Daphnia when food was sufficient or abundant, but had weaker effects under food limitation. Our results indicate that the microbiota can be a potentially important factor in determining host responses to changes in dietary conditions. Characterization of the host-associated microbiota further showed that Aeromonas sp. was the most prevalent taxon in the digestive tract of Daphnia.

Effects of host plant on life-history traits in the polyphagous spider mite Tetranychus urticae.

Studying antagonistic coevolution between host plants and herbivores is particularly relevant for polyphagous species that can experience a great diversity of host plants with a large range of defenses. Here, we performed experimental evolution with the polyphagous spider mite Tetranychus urticae to detect how mites can exploit host plants. We thus compared on a same host the performance of replicated populations from an ancestral one reared for hundreds of generations on cucumber plants that were shifted to either tomato or cucumber plants. We controlled for maternal effects by rearing females from all replicated populations on either tomato or cucumber leaves, crossing this factor with the host plant in a factorial design. About 24 generations after the host shift and for all individual mites, we measured the following fitness components on tomato leaf fragments: survival at all stages, acceptance of the host plant by juvenile and adult mites, longevity, and female fecundity. The host plant on which mite populations had evolved did not affect the performance of the mites, but only affected their sex ratio. Females that lived on tomato plants for circa 24 generations produced a higher proportion of daughters than did females that lived on cucumber plants. In contrast, maternal effects influenced juvenile survival, acceptance of the host plant by adult mites and female fecundity. Independently of the host plant species on which their population had evolved, females reared on the tomato maternal environment produced offspring that survived better on tomato as juveniles, but accepted less this host plant as adults and had a lower fecundity than did females reared on the cucumber maternal environment. We also found that temporal blocks affected mite dispersal and both female longevity and fecundity. Taken together, our results show that the host plant species can affect critical parameters of population dynamics, and most importantly that maternal and environmental conditions can facilitate colonization and exploitation of a novel host in the polyphagous T. urticae, by affecting dispersal behavior (host acceptance) and female fecundity.

Local mate competition mediates sexual conflict over sex ratio in a haplodiploid spider mite.

In haplodiploids, females pass their genes on to all their offspring, whereas a male's genes are only passed on to his daughters. Hence, males always benefit from female-biased sex ratios, whereas for females the optimal offspring sex ratio depends on the level of local mate competition (LMC), ranging from highly female-biased under strict LMC to unbiased in Panmixia. This generates a sexual conflict over sex ratio, the intensity of which depends on the LMC level, with most intense conflict in Panmixia. Such conflict might lead to an evolutionary arms race, with persistence traits evolving in males and resistance traits in females. Although this prediction is theoretically straightforward, it remains untested empirically. We addressed this by crossing spider mites that evolved under varying intensities of LMC (hence of sexual conflict), to mates from inbred lines. Under high levels of sexual conflict, both sexes evolved manipulative traits to shift the sex ratio to their own advantage. In females, this was partly achieved through changes in egg size. We thus show that (1) LMC levels modulate sexual conflict over sex ratio in haplodiploids, driving the evolution of manipulative traits, and (2) fathers can affect sex ratio, challenging conventional assumptions.

Sex-ratio adjustment in response to local mate competition is achieved through an alteration of egg size in a haplodiploid spider mite.

Sex-ratio adjustments are commonly observed in haplodiploid species. However, the underlying proximate mechanisms remain elusive. We investigated these mechanisms in Tetranychus urticae, a haplodiploid spider mite known to adjust sex ratio in response to the level of local mate competition (LMC). In this species, egg size determines fertilization probability, with larger eggs being more likely to be fertilized, and thus become female. We explored the hypothesis that sex-ratio adjustment is achieved through adjustment of egg size. By using spider mites from a large population, we found that females produced not only a higher proportion of daughters under high levels of LMC, but also larger eggs. Moreover, in populations experimentally evolving under varying levels of LMC, both the proportion of females and the egg size increased with LMC intensity. These results suggest that sex-ratio adjustment in spider mites is mediated by egg size, although the causal relationship remains to be tested.

Mating modifies female life history in a haplodiploid spider mite.

Mating usually modifies females' resource allocation pattern, often as a result of conflicts between male and female partners. Can such a switch occur even in the absence of sexual conflicts? We addressed this issue in the haplodiploid spider mite Tetranychus urticae, whose biology and population structure considerably reduce conflicts between males and females over reproductive decisions. Comparing virgin and mated females, we tested the hypothesis that mated females modify their allocation pattern so as to maximize their probability of producing daughters. Mated females produced fewer but larger eggs, resulting in an overall similar reproductive effort but an increased probability of producing daughters, since in this species larger eggs are more likely to be fertilized and thus to become female. Moreover, mated females concentrated their reproduction early in life. Again, this might be a way to produce more daughters, since sperm is more abundant early in life. For virgins, spreading reproductive investment might be a way to save resources to extend life span, thus increasing their probability of encountering a sexual partner. Females with multiple opportunities for mating produced fewer eggs and a less female-biased sex ratio than once-mated females, raising the question of why multiple mating often occurs in this species.

Experimental evolution of reduced sex ratio adjustment under local mate competition.

Theory predicts that local mate competition (LMC) favors the evolution of female-biased sex ratios. Empirical support of this prediction is indirect and comes from comparative studies or from studies showing that individuals can adjust their offspring sex ratio in response to varying LMC intensities. Replicate lines from a population of the spider mite Tetranychus urticae were selected under three LMC intensities for up to 54 generations. Within each selection regime, the final sex ratio matched theoretical predictions. Furthermore, the ability of individuals to adjust their offspring sex ratio diminished in females evolving under strict LMC, but not in females evolving under relaxed LMC levels. These results provide direct experimental evidence for the evolutionary process by which LMC modifies sex-allocation strategies and suggest that evolution under strict and constant LMC may lead to a loss of phenotypic plasticity.

Sex allocation in haplodiploids is mediated by egg size: evidence in the spider mite Tetranychus urticae Koch.

Haplodiploid species display extraordinary sex ratios. However, a differential investment in male and female offspring might also be achieved by a differential provisioning of eggs, as observed in birds and lizards. We investigated this hypothesis in the haplodiploid spider mite Tetranychus urticae, which displays highly female-biased sex ratios. We show that egg size significantly determines not only larval size, juvenile survival and adult size, but also fertilization probability, as in marine invertebrates with external fertilization, so that female (fertilized) eggs are significantly larger than male (unfertilized) eggs. Moreover, females with on average larger eggs before fertilization produce a more female-biased sex ratio afterwards. Egg size thus mediates sex-specific egg provisioning, sex and offspring sex ratio. Finally, sex-specific egg provisioning has another major consequence: male eggs produced by mated mothers are smaller than male eggs produced by virgins, and this size difference persists in adults. Virgin females might thus have a (male) fitness advantage over mated females.