Adaptation to enemy shifts: rapid resistance evolution to local Vibrio spp. in invasive Pacific oysters

One hypothesis for the success of invasive species is reduced pathogen burden, resulting from a release from infections or high immunological fitness of invaders. Despite strong selection exerted on the host, the evolutionary response of invaders to newly acquired pathogens has rarely been considered. The two independent and genetically distinct invasions of the Pacific oyster Crassostrea gigas into the North Sea represent an ideal model system to study fast evolutionary responses of invasive populations. By exposing both invasion sources to ubiquitous and phylogenetically diverse pathogens (Vibrio spp.), we demonstrate that within a few generations hosts adapted to newly encountered pathogen communities. However, local adaptation only became apparent in selective environments, i.e. at elevated temperatures reflecting patterns of disease outbreaks in natural populations. Resistance against sympatric and allopatric Vibrio spp. strains was dominantly inherited in crosses between both invasion sources, resulting in an overall higher resistance of admixed individuals than pure lines. Therefore, we suggest that a simple genetic resistance mechanism of the host is matched to a common virulence mechanism shared by local Vibrio strains. This combination might have facilitated a fast evolutionary response that can explain another dimension of why invasive species can be so successful in newly invaded ranges

Persistence, seasonal dynamics and pathogenic potential of Vibrio communities from pacific oyster hemolymph

Bacteria of the genus Vibrio occur at a continuum from free-living to symbiotic life forms, including opportunists and pathogens, that can contribute to severe diseases, for instance summer mortality events of Pacific oysters Crassostrea gigas. While most studies focused on Vibrio isolated from moribund oysters during mortality outbreaks, investigations of the Vibrio community in healthy oysters are rare. Therefore, we characterized the persistence, diversity, seasonal dynamics, and pathogenicity of the Vibrio community isolated from healthy Pacific oysters. In a reciprocal transplant experiment we repeatedly sampled hemolymph from adult Pacific oysters to differentiate population from site-specific effects during six months of in situ incubation in the field. We characterized virulence phenotypes and genomic diversity based on multilocus sequence typing in a total of 70 Vibrio strains. Based on controlled infection experiments we could show that strains with the ability to colonize healthy adult oysters can also have the potential to induce high mortality rates on larvae. Diversity and abundance of Vibrio varied significantly over time with highest values during and after spawning season. Vibrio communities from transplanted and stationary oysters converged over time, indicating that communities were not population specific, but rather assemble from the surrounding environment forming communities, some of which can persist over longer period

Genomic variation among closely related Vibrio alginolyticus strains is located on mobile genetic elements

Background: Species of the genus Vibrio, one of the most diverse bacteria genera, have undergone niche adaptation followed by clonal expansion. Niche adaptation and ultimately the formation of ecotypes and speciation in this genus has been suggested to be mainly driven by horizontal gene transfer (HGT) through mobile genetic elements (MGEs). Our knowledge about the diversity and distribution of Vibrio MGEs is heavily biased towards human pathogens and our understanding of the distribution of core genomic signatures and accessory genes encoded on MGEs within specific Vibrio clades is still incomplete. We used nine different strains of the marine bacterium Vibrio alginolyticus isolated from pipefish in the Kiel-Fjord to perform a multiscale-comparative genomic approach that allowed us to investigate [1] those genomic signatures that characterize a habitat-specific ecotype and [2] the source of genomic variation within this ecotype. Results: We found that the nine isolates from the Kiel-Fjord have a closed-pangenome and did not differ based on core-genomic signatures. Unique genomic regions and a unique repertoire of MGEs within the Kiel-Fjord isolates suggest that the acquisition of gene-blocks by HGT played an important role in the evolution of this ecotype. Additionally, we found that ~ 90% of the genomic variation among the nine isolates is encoded on MGEs, which supports ongoing theory that accessory genes are predominately located on MGEs and shared by HGT. Lastly, we could show that these nine isolates share a unique virulence and resistance profile which clearly separates them from all other investigated V. alginolyticus strains and suggests that these are habitat-specific genes, required for a successful colonization of the pipefish, the niche of this ecotype. Conclusion We conclude that all nine V. alginolyticus strains from the Kiel-Fjord belong to a unique ecotype, which we named the Kiel-alginolyticus ecotype. The low sequence variation of the core-genome in combination with the presence of MGE encoded relevant traits, as well as the presence of a suitable niche (here the pipefish), suggest, that this ecotype might have evolved from a clonal expansion following HGT driven niche-adaptation

Draft Genome Sequence of Vibrio splendidus DSM 19640

Here, we present the draft genome sequence of Vibrio splendidus type strain DSM 19640. V. splendidus is an abundant species among coastal vibrioplankton. The assembly resulted in a 5,729,362-bp draft genome with 5,032 proteincoding sequences, 6 rRNAs, and 117 tRNAs

Filamentous phages reduce bacterial growth in low salinities

Being non-lytic, filamentous phages can replicate at high frequencies and often carry virulence factors, which are important in the evolution and emergence of novel pathogens. However, their net effect on bacterial fitness remains unknown. To understand the ecology and evolution between filamentous phages and their hosts, it is important to assess (i) fitness effects of filamentous phages on their hosts and (ii) how these effects depend on the environment. To determine how the net effect on bacterial fitness by filamentous phages changes across environments, we constructed phage–bacteria infection networks at ambient 15 practical salinity units (PSU) and stressful salinities (11 and 7 PSU) using the marine bacterium, Vibrio alginolyticus and its derived filamentous phages as model system. We observed no significant difference in network structure at 15 and 11 PSU. However, at 7 PSU phages significantly reduced bacterial growth changing network structure. This pattern was mainly driven by a significant increase in bacterial susceptibility. Our findings suggest that filamentous phages decrease bacterial growth, an indirect measure of fitness in stressful environmental conditions, which might impact bacterial communities, alter horizontal gene transfer events and possibly favour the emergence of novel pathogens in environmental Vibrios

Fitness benefits to bacteria of carrying prophages and prophage‐encoded antibiotic‐resistance genes peak in different environments

Understanding the role of horizontal gene transfer (HGT) in adaptation is a key challenge in evolutionary biology. In microbes, an important mechanism of HGT is prophage acquisition (phage genomes integrated into bacterial chromosomes). Prophages can influence bacterial fitness via transfer of beneficial genes (including antibiotic-resistance genes, ARGs), protection from superinfecting phages, or switching to a lytic lifecycle which releases free phages infectious to competitors. We expect these effects to depend on environmental conditions because of, for example, environment-dependent induction of the lytic lifecycle. However, it remains unclear how costs/benefits of prophages vary across environments. Here, studying prophages with/without ARGs in Escherichia coli, we disentangled effects of prophages alone and adaptive genes they carry. In competition with prophage-free strains, benefits from prophages and ARGs peaked in different environments. Prophages were most beneficial when induction of the lytic lifecycle was common, whereas ARGs were more beneficial upon antibiotic exposure and with reduced prophage induction. Acquisition of prophage-encoded ARGs by competing strains was most common when prophage induction, and therefore free phages, were common. Thus, selection on prophages and adaptive genes they carry varies independently across environments, which is important for predicting the spread of mobile/integrating genetic elements and their role in evolution

Population specific genotype x genotype x environment interactions in bacterial disease of early life stages of Pacific oyster larvae

The consequences of emerging marine diseases on the evolutionary trajectories of affected host populations in the marine realm are largely unexplored. Evolution in response to natural selection depends on the genetic variation of the traits under selection and the interaction of these traits with the environment (GxE). However, in the case of diseases, genotypes of pathogens add another dimension to this interaction. Therefore, the study of disease resistance needs to be extended to the interaction of host genotype, pathogen genotype and environment (GxGxE). In the present study we used a full-sib breeding design crossing two genetically differentiated populations of the Pacific oyster Crassostrea gigas (Thunberg, 1793), to determine the influence of host genotype, pathogen genotype and temperature on disease resistance. Based on a controlled infection experiment on two early life stages, i.e. D-larvae and Pediveliger larvae at elevated and ambient water temperatures we estimated disease resistance to allopatric and sympatric Vibrio sp. by measuring survival and growth within and between genetically differentiated oyster populations. In both populations survival was higher upon infection with sympatric Vibrio sp. indicating that disease resistance has a genetic basis and is dependent on host genotype. In addition we observed a significant GxGxE effect in D-larvae, where contrary to expectations, disease resistance was higher at warm than at cold temperatures. Using thermal reaction norms, we could further show, that disease resistance is an environment dependent trait with high plasticity, which indicates the potential for a fast acclimatization to changing environmental conditions. These population specific reaction norms disappeared in hybrid crosses between both populations which demonstrates that admixture between genetically differentiated populations can influence GxGxE interactions on larger scales

Co-transfer of functionally interdependent genes contributes to genome mosaicism in lambdoid phages

Lambdoid (or Lambda-like) phages, are a group of related temperate phages that can infect Escherichia coli and other gut bacteria. A key characteristic of these phages is their mosaic genome structure which served as basis for the "modular genome hypothesis". Accordingly, lambdoid phages evolve by transferring genomic regions, each of which constitutes a functional unit. Nevertheless, it is unknown which genes are preferentially transferred together and what drives such co-transfer events. Here we aim to characterize genome modularity by studying co-transfer of genes among 95 distantly related lambdoid (pro-)phages. Based on gene content, we observed that the genomes cluster into twelve groups, which are characterized by a highly similar gene content within the groups and highly divergent gene content across groups. Highly similar proteins can occur in genomes of different groups, indicating that they have been transferred. About 26% of homologous protein clusters in the four known operons (i.e., the early left, early right, immunity, and late operon) engage in gene transfer, which affects all operons to a similar extent. We identified pairs of genes that are frequently co-transferred and observed that these pairs tend to be in close proximity to one another on the genome. We find that frequently co-transferred genes are involved in related functions and highlight interesting examples involving structural proteins, the CI repressor and Cro regulator, proteins interacting with DNA, and membrane-interacting proteins. We conclude that epistatic effects, where the functioning of one protein depends on the presence of another, plays an important role in the evolution of the modular structure of these genomes

Ecology and Evolution of Invasive Pacific Oysters in Response to Pathogen Infection and Rising Temperatures

Nature is a highly complex system that is subject to competition from several factors, which can be of physical, chemical, biotic but also anthropogenic origin. Nevertheless, we commonly consider only a few of those factors in our experiments. However, to understand the bigger picture, we have to test the synchronous effects of multiple factors. One great opportunity to do so comes from the direct interplay between bioinvasions and climate change. Bioinvasions constitute a natural experiment in evolution: when invasive species colonize new habitats they experience strong selection pressures from novel abiotic and biotic stressors. For a successful invasion, adaptation to those stressors is essential for survival. Additional threats may result from current climate change scenarios that further challenge the adaptive potential of invaders. Major threats of global change, such as emerging diseases are caused directly and indirectly by rising temperatures. A combined approach addressing direct effects of global change on host-parasite interactions of invasive species has rarely been taken. However, there is growing evidence that such multiple factors interact in complex ways. Furthermore, the way invasive species cope with novel parasites is still a black box. Using invasive Pacific oysters Crassostrea gigas and their opportunistic pathogens of the genus Vibrio as model organisms, this thesis addresses the evolution of an invasive species to novel sympatric parasites and combines this with additional challenges imposed by rising temperatures that are expected to occur in the habitat. C. gigas independently invaded and successfully colonized the Southern and the Northern area of the European Wadden Sea. The successful invasion of C. gigas‘ is mainly attributed to a lack of natural enemies and high propagule pressure. While Southern populations have occasionally been subjected to extensive mortalities resulting from a complex interaction of high temperatures, oyster genetics and parasite infections, Northern populations have been spared from this fate so far. Those differences in invasion and disease selection history were the starting point of my thesis. Based on a set of controlled infection experiments at contemporary and future water temperatures, I demonstrated that invasive oysters have the potential to adapt rapidly to novel sympatric Vibrio spp. (Chapter III). By using lab-bred hybrids of the two invasion waves, I determined that the rapid adaptation is facilitated by dominant inheritance of disease resistance alleles. This adds a further factor, apart from lack of natural enemies and high propagule pressure in explaining C. gigas‘ enormous invasion success: rapid adaptation to enemy shifts. This adaptation to sympatric pathogens could only be observed at high temperatures (21°C in contrast to 17°C). Indeed, I could show that Vibrio infection and temperature add additively to disease outbreaks in adults (Chapter I). At high temperatures adult oysters have a reduced ability to clear out infectious strains efficiently (Chapter I), while simultaneously the likelihood of encountering pathogenic strains from the surrounding environment correlates positively with temperature (Chapter II). However, in contrast to adults, susceptibility to disease of larvae was reduced at high temperatures, i.e. 23°C in contrast to 19°C (Chapter IV). I assume, that nowadays, selection is less pronounced on adults when average water temperatures remain below 20°C. However, if average summer temperatures will rise as predicted by global change scenarios, selection pressure on adult oysters will increase while simultaneously decrease on larvae by favoring successful recruitment and disease resistance (Chapter IV). Such opposing temperature-mediated effects between life-stages will play a major role in determining this species‘ persistence in the invaded area in the face of global warming. In summary, by studying the evolution of an invasive species to novel parasites in the context of climate change I unveiled another facet explaining the outstanding success of C. gigas‘ invasion in the European Wadden sea: rapid adaptation to enemy shift. In concert with this finding, I suggest, that in the context of rising temperatures, the persistence of C. gigas underlies on the one hand the opposite response of temperature-mediated infection outcome between larvae and adults, and on the other hand the equilibrium between the rate of rising temperatures and the rate at which the populations will be able to catch up