Demographic fluctuations and selection during host-parasite co-evolution interactively increase genetic diversity.

Host-parasite interactions can cause strong demographic fluctuations accompanied by selective sweeps of resistance/infectivity alleles. Both demographic bottlenecks and frequent sweeps are expected to reduce the amount of segregating genetic variation and therefore might constrain adaptation during co-evolution. Recent studies, however, suggest that the interaction of demographic and selective processes is a key component of co-evolutionary dynamics and may rather positively affect levels of genetic diversity available for adaptation. Here, we provide direct experimental testing of this hypothesis by disentangling the effects of demography, selection and their interaction in an experimental host-parasite system. We grew 12 populations of a unicellular, asexually reproducing algae (Chlorella variabilis) that experienced either growth followed by constant population sizes (three populations), demographic fluctuations (three populations), selection induced by exposure to a virus (three populations), or demographic fluctuations together with virus-induced selection (three populations). After 50 days (~50 generations), we conducted whole-genome sequencing of each algal host population. We observed more genetic diversity in populations that jointly experienced selection and demographic fluctuations than in populations where these processes were experimentally separated. In addition, in those three populations that jointly experienced selection and demographic fluctuations, experimentally measured diversity exceeds expected values of diversity that account for the cultures' population sizes. Our results suggest that eco-evolutionary feedbacks can positively affect genetic diversity and provide the necessary empirical measures to guide further improvements of theoretical models of adaptation during host-parasite co-evolution.

Experimental inheritance of antibiotic acquired dysbiosis affects host phenotypes across generations.

Microbiomes can enhance the health, fitness and even evolutionary potential of their hosts. Many organisms propagate favorable microbiomes fully or partially via vertical transmission. In the long term, such co-propagation can lead to the evolution of specialized microbiomes and functional interdependencies with the host. However, microbiomes are vulnerable to environmental stressors, particularly anthropogenic disturbance such as antibiotics, resulting in dysbiosis. In cases where microbiome transmission occurs, a disrupted microbiome may then become a contagious pathology causing harm to the host across generations. We tested this hypothesis using the specialized socially transmitted gut microbiome of honey bees as a model system. By experimentally passaging tetracycline-treated microbiomes across worker 'generations' we found that an environmentally acquired dysbiotic phenotype is heritable. As expected, the antibiotic treatment disrupted the microbiome, eliminating several common and functionally important taxa and strains. When transmitted, the dysbiotic microbiome harmed the host in subsequent generations. Particularly, naïve bees receiving antibiotic-altered microbiomes died at higher rates when challenged with further antibiotic stress. Bees with inherited dysbiotic microbiomes showed alterations in gene expression linked to metabolism and immunity, among other pathways, suggesting effects on host physiology. These results indicate that there is a possibility that sublethal exposure to chemical stressors, such as antibiotics, may cause long-lasting changes to functional host-microbiome relationships, possibly weakening the host's progeny in the face of future ecological challenges. Future studies under natural conditions would be important to examine the extent to which negative microbiome-mediated phenotypes could indeed be heritable and what role this may play in the ongoing loss of biodiversity.

First experimental evidence for active farming in ambrosia beetles and strong heredity of garden microbiomes.

Fungal cultivation is a defining feature for advanced agriculture in fungus-farming ants and termites. In a third supposedly fungus-farming group, wood-colonizing ambrosia beetles, an experimental proof for the effectiveness of beetle activity for selective promotion of their food fungi over others is lacking and farming has only been assumed based on observations of social and hygienic behaviours. Here, we experimentally removed mothers and their offspring from young nests of the fruit-tree pinhole borer, Xyleborinus saxesenii. By amplicon sequencing of bacterial and fungal communities of nests with and without beetles we could show that beetles are indeed able to actively shift symbiont communities. Although being consumed, the Raffaelea food fungi were more abundant when beetles were present while a weed fungus (Chaetomium sp.) as well as overall bacterial diversity were reduced in comparison to nests without beetles. Core symbiont communities were generally of low diversity and there were strong signs for vertical transmission not only for the cultivars, but also for secondary symbionts. Our findings verify the existence of active farming, even though the exact mechanisms underlying the selective promotion and/or suppression of symbionts need further investigation.

Strong selection and high mutation supply characterize experimental Chlorovirus evolution.

Characterizing how viruses evolve expands our understanding of the underlying fundamental processes, such as mutation, selection and drift. One group of viruses whose evolution has not yet been extensively studied is the Phycodnaviridae, a globally abundant family of aquatic large double-stranded (ds)DNA (dsDNA) viruses. Here we studied the evolutionary change of Paramecium bursaria chlorella virus 1 during experimental coevolution with its algal host. We used pooled genome sequencing of six independently evolved populations to characterize genomic change over five time points. Across six experimental replicates involving either strong or weak demographic fluctuations, we found single nucleotide polymorphisms (SNPs) at sixty-seven sites. The occurrence of genetic variants was highly repeatable, with just two of the SNPs found in only a single experimental replicate. Three genes A122/123R, A140/145R and A540L showed an excess of variable sites, providing new information about potential targets of selection during Chlorella-Chlorovirus coevolution. Our data indicated that the studied populations were not mutation-limited and experienced strong positive selection. Our investigation highlighted relevant processes governing the evolution of aquatic large dsDNA viruses, which ultimately contributes to a better understanding of the functioning of natural aquatic ecosystems.

Honey Bee Larval and Adult Microbiome Life Stages Are Effectively Decoupled with Vertical Transmission Overcoming Early Life Perturbations.

Microbiomes provide a range of benefits to their hosts which can lead to the coevolution of a joint ecological niche. However, holometabolous insects, some of the most successful organisms on Earth, occupy different niches throughout development, with larvae and adults being physiologically and morphologically highly distinct. Furthermore, transition between the stages usually involves the loss of the gut microbiome since the gut is remodeled during pupation. Most eusocial organisms appear to have evolved a workaround to this problem by sharing their communal microbiome across generations. However, whether this vertical microbiome transmission can overcome perturbations of the larval microbiome remains untested. Honey bees have a relatively simple, conserved, coevolved adult microbiome which is socially transmitted and affects many aspects of their biology. In contrast, larval microbiomes are more variable, with less clear roles. Here, we manipulated the gut microbiome of in vitro-reared larvae, and after pupation of the larvae, we inoculated the emerged bees with adult microbiome to test whether adult and larval microbiome stages may be coupled (e.g., through immune priming). Larval treatments differed in bacterial composition and abundance, depending on diet, which also drove larval gene expression. Nonetheless, adults converged on the typical core taxa and showed limited gene expression variation. This work demonstrates that honey bee adult and larval stages are effectively microbiologically decoupled, and the core adult microbiome is remarkably stable to early developmental perturbations. Combined with the transmission of the microbiome in early adulthood, this allows the formation of long-term host-microbiome associations. IMPORTANCE This work investigated host-microbiome interactions during a crucial developmental stage-the transition from larvae to adults, which is a challenge to both, the insect host and its microbiome. Using the honey bee as a tractable model system, we showed that microbiome transfer after emergence overrides any variation in the larvae, indicating that larval and adult microbiome stages are effectively decoupled. Together with the reliable vertical transfer in the eusocial system, this decoupling ensures that the adults are colonized with a consistent and derived microbiome after eclosion. Taken all together, our data provide additional support that the evolution of sociality, at least in the honey bee system tested here, is linked with host-microbiome relationships.

Varroa destructor: A Complex Parasite, Crippling Honey Bees Worldwide.

The parasitic mite, Varroa destructor, has shaken the beekeeping and pollination industries since its spread from its native host, the Asian honey bee (Apis cerana), to the naïve European honey bee (Apis mellifera) used commercially for pollination and honey production around the globe. Varroa is the greatest threat to honey bee health. Worrying observations include increasing acaricide resistance in the varroa population and sinking economic treatment thresholds, suggesting that the mites or their vectored viruses are becoming more virulent. Highly infested weak colonies facilitate mite dispersal and disease transmission to stronger and healthier colonies. Here, we review recent developments in the biology, pathology, and management of varroa, and integrate older knowledge that is less well known.

The feedback between selection and demography shapes genomic diversity during coevolution.

Species interactions and coevolution are integral to ecological communities, but we lack empirical information on when and how these interactions generate and purge genetic diversity. Using genomic time series data from host-virus experiments, we found that coevolution occurs through consecutive selective sweeps in both species, with temporal consistency across replicates. Sweeps were accompanied by phenotypic change (resistance or infectivity increases) and expansions in population size. In the host, population expansion enabled rapid generation of genetic diversity in accordance with neutral processes. Viral molecular evolution was, in contrast, confined to few genes, all putative targets of selection. This study demonstrates that molecular evolution during species interactions is shaped by both eco-evolutionary feedback dynamics and interspecific differences in how genetic diversity is generated and maintained.

Quantifying the efficiency and biases of forest Saccharomyces sampling strategies.

Saccharomyces yeasts are emerging as model organisms for ecology and evolution, and researchers need environmental Saccharomyces isolates to test ecological and evolutionary hypotheses. However, methods for isolating Saccharomyces from nature have not been standardized, and isolation methods may influence the genotypes and phenotypes of studied strains. We compared the effectiveness and potential biases of an established enrichment culturing method against a newly developed direct plating method for isolating forest floor Saccharomyces spp. In a European forest, enrichment culturing was both less successful at isolating Saccharomyces paradoxus per sample collected and less labour intensive per isolated S. paradoxus colony than direct isolation. The two methods sampled similar S. paradoxus diversity: The number of unique genotypes sampled (i.e., genotypic diversity) per S. paradoxus isolate and average growth rates of S. paradoxus isolates did not differ between the two methods, and growth rate variances (i.e., phenotypic diversity) only differed in one of three tested environments. However, enrichment culturing did detect rare Saccharomyces cerevisiae in the forest habitat and also found two S. paradoxus isolates with outlier phenotypes. Our results validate the historically common method of using enrichment culturing to isolate representative collections of environmental Saccharomyces. We recommend that researchers choose a Saccharomyces sampling method based on resources available for sampling and isolate screening. Researchers interested in discovering new Saccharomyces phenotypes or rare Saccharomyces species from natural environments may also have more success using enrichment culturing. We include step-by-step sampling protocols in the supplemental materials.

Measuring microbial fitness in a field reciprocal transplant experiment.

Microbial fitness is easy to measure in the laboratory, but difficult to measure in the field. Laboratory fitness assays make use of controlled conditions and genetically modified organisms, neither of which are available in the field. Among other applications, fitness assays can help researchers detect adaptation to different habitats or locations. We designed a competitive fitness assay to detect adaptation of Saccharomyces paradoxus isolates to the habitat they were isolated from (oak or larch leaf litter). The assay accurately measures relative fitness by tracking genotype frequency changes in the field using digital droplet PCR (DDPCR). We expected locally adapted S. paradoxus strains to increase in frequency over time when growing on the leaf litter type from which they were isolated. The DDPCR assay successfully detected fitness differences among S. paradoxus strains, but did not find a tendency for strains to be adapted to the habitat they were isolated from. Instead, we found that the natural alleles of the hexose transport gene we used to distinguish S. paradoxus strains had significant effects on fitness. The origin of a strain also affected its fitness: strains isolated from oak litter were generally fitter than strains from larch litter. Our results suggest that dispersal limitation and genetic drift shape S. paradoxus populations in the forest more than local selection does, although further research is needed to confirm this. Tracking genotype frequency changes using DDPCR is a practical and accurate microbial fitness assay for natural environments.

A systematic forest survey showing an association of Saccharomyces paradoxus with oak leaf litter.

Although we understand the genetics of the laboratory model yeast Saccharomyces cerevisiae very well, we know little about the natural ecology and environment that shaped its genome. Most isolates of Saccharomyces paradoxus, the wild relative of S. cerevisiae, come from oak trees, but it is not known whether this is because oak is their primary habitat. We surveyed leaf litter in a forest in Northern Germany and found a strong correlation between isolation success of wild Saccharomyces and the proximity of the nearest oak. We compared the four most common tree genera and found Saccharomyces most frequently in oak litter. Interestingly, we show that Saccharomyces is much more abundant in oak leaf litter than on oak bark, suggesting that it grows in litter or soil rather than on the surfaces of oaks themselves. The distribution and abundance of Saccharomyces over the course of a year shows that oak leaf litter provides a stable habitat for the yeast, although there was significant tree-to-tree variation. Taken together, our results suggest that leaf litter rather than tree surfaces provide the better habitat for wild Saccharomyces, with oak being the preferred tree genus. 99.5% of all strains (633/636) isolated were S. paradoxus.

The native microbiome of the nematode Caenorhabditis elegans: gateway to a new host-microbiome model.

BACKGROUND: Host-microbe associations underlie many key processes of host development, immunity, and life history. Yet, none of the current research on the central model species Caenorhabditis elegans considers the worm's natural microbiome. Instead, almost all laboratories exclusively use the canonical strain N2 and derived mutants, maintained through routine bleach sterilization in monoxenic cultures with an E. coli strain as food. Here, we characterize for the first time the native microbiome of C. elegans and assess its influence on nematode life history characteristics. RESULTS: Nematodes sampled directly from their native habitats carry a species-rich bacterial community, dominated by Proteobacteria such as Enterobacteriaceae and members of the genera Pseudomonas, Stenotrophomonas, Ochrobactrum, and Sphingomonas. The C. elegans microbiome is distinct from that of the worm's natural environment and the congeneric species C. remanei. Exposure to a derived experimental microbiome revealed that bacterial composition is influenced by host developmental stage and genotype. These experiments also showed that the microbes enhance host fitness under standard and also stressful conditions (e.g., high temperature and either low or high osmolarity). Taking advantage of the nematode's transparency, we further demonstrate that several Proteobacteria are able to enter the C. elegans gut and that an Ochrobactrum isolate even seems to be able to persist in the intestines under stressful conditions. Moreover, three Pseudomonas isolates produce an anti-fungal effect in vitro which we show can contribute to the worm's defense against fungal pathogens in vivo. CONCLUSION: This first systematic analysis of the nematode's native microbiome reveals a species-rich bacterial community to be associated with C. elegans, which is likely of central importance for our understanding of the worm's biology. The information acquired and the microbial isolates now available for experimental work establishes C. elegans as a tractable model for the in-depth dissection of host-microbiome interactions.

The interaction of Saccharomyces paradoxus with its natural competitors on oak bark.

The natural history of the model yeast Saccharomyces cerevisiae is poorly understood and confounded by domestication. In nature, S. cerevisiae and its undomesticated relative S. paradoxus are usually found on the bark of oak trees, a habitat very different from wine or other human fermentations. It is unclear whether the oak trees are really the primary habitat for wild yeast, or whether this apparent association is due to biased sampling. We use culturing and high-throughput environmental sequencing to show that S. paradoxus is a very rare member of the oak bark microbial community. We find that S. paradoxus can grow well on sterile medium made from oak bark, but that its growth is strongly suppressed when the other members of the community are present. We purified a set of twelve common fungal and bacterial species from the oak bark community and tested how each affected the growth of S. paradoxus in direct competition on oak bark medium at summer and winter temperatures, identifying both positive and negative interactions. One Pseudomonas species produces a diffusible toxin that suppresses S. paradoxus as effectively as either the whole set of twelve species together or the complete community present in nonsterilized oak medium. Conversely, one of the twelve species, Mucilaginibacter sp., had the opposite effect and promoted S. paradoxus growth at low temperatures. We conclude that, in its natural oak tree habitat, S. paradoxus is a rare species whose success depends on the much more abundant microbial species surrounding it.