Cooperation and cheating orchestrate Vibrio assemblages and polymicrobial synergy in oysters infected with OsHV-1 virus.

Polymicrobial infections threaten the health of humans and animals but remain understudied in natural systems. We recently described the Pacific Oyster Mortality Syndrome (POMS), a polymicrobial disease affecting oyster production worldwide. In the French Atlantic coast, the disease involves coinfection with ostreid herpesvirus 1 (OsHV-1) and virulent Vibrio. However, it is unknown whether consistent Vibrio populations are associated with POMS in different regions, how Vibrio contribute to POMS, and how they interact with OsHV-1 during pathogenesis. By connecting field-based approaches in a Mediterranean ecosystem, laboratory infection assays and functional genomics, we uncovered a web of interdependencies that shape the structure and function of the POMS pathobiota. We show that Vibrio harveyi and Vibrio rotiferianus are predominant in OsHV-1-diseased oysters and that OsHV-1 drives the partition of the Vibrio community observed in the field. However only V. harveyi synergizes with OsHV-1 by promoting mutual growth and accelerating oyster death. V. harveyi shows high-virulence potential and dampens oyster cellular defenses through a type 3 secretion system, making oysters a more favorable niche for microbe colonization. In addition, V. harveyi produces a key siderophore called vibrioferrin. This important resource promotes the growth of V. rotiferianus, which cooccurs with V. harveyi in diseased oysters, and behaves as a cheater by benefiting from V. harveyi metabolite sharing. Our data show that cooperative behaviors contribute to synergy between bacterial and viral coinfecting partners. Additional cheating behaviors further shape the polymicrobial consortium. Controlling cooperative behaviors or countering their effects opens avenues for mitigating polymicrobial diseases.

Genome-wide DNA methylation predicts environmentally driven life history variation in a marine fish.

Epigenetic modifications are thought to be one of the molecular mechanisms involved in plastic adaptive responses to environmental variation. However, studies reporting associations between genome-wide epigenetic changes and habitat-specific variations in life history traits (e.g., lifespan, reproduction) are still scarce, likely due to the recent application of methylome resequencing methods to non-model species. In this study, we examined associations between whole genome DNA methylation and environmentally driven life history variation in 2 lineages of a marine fish, the capelin (Mallotus villosus), from North America and Europe. In both lineages, capelin harbor 2 contrasting life history tactics (demersal vs. beach-spawning). Performing whole genome and methylome sequencing, we showed that life history tactics are associated with epigenetic changes in both lineages, though the effect was stronger in European capelin. Genetic differentiation between the capelin harboring different life history tactics was negligible, but we found genome-wide methylation changes in both lineages. We identified 9,125 European and 199 North American differentially methylated regions (DMRs) due to life history. Gene ontology (GO) enrichment analysis for both lineages revealed an excess of terms related to neural function. Our results suggest that environmental variation causes important epigenetic changes that are associated with contrasting life history tactics in lineages with divergent genetic backgrounds, with variable importance of genetic variation in driving epigenetic variation. Our study emphasizes the potential role of genome-wide epigenetic variation in adaptation to environmental variation.

Functional Diversification of Oyster Big Defensins Generates Antimicrobial Specificity and Synergy against Members of the Microbiota.

Big defensins are two-domain antimicrobial peptides (AMPs) that have highly diversified in mollusks. Cg-BigDefs are expressed by immune cells in the oyster Crassostrea gigas, and their expression is dampened during the Pacific Oyster Mortality Syndrome (POMS), which evolves toward fatal bacteremia. We evaluated whether Cg-BigDefs contribute to the control of oyster-associated microbial communities. Two Cg-BigDefs that are representative of molecular diversity within the peptide family, namely Cg-BigDef1 and Cg-BigDef5, were characterized by gene cloning and synthesized by solid-phase peptide synthesis and native chemical ligation. Synthetic peptides were tested for antibacterial activity against a collection of culturable bacteria belonging to the oyster microbiota, characterized by 16S sequencing and MALDI Biotyping. We first tested the potential of Cg-BigDefs to control the oyster microbiota by injecting synthetic Cg-BigDef1 into oyster tissues and analyzing microbiota dynamics over 24 h by 16S metabarcoding. Cg-BigDef1 induced a significant shift in oyster microbiota ß-diversity after 6 h and 24 h, prompting us to investigate antimicrobial activities in vitro against members of the oyster microbiota. Both Cg-BigDef1 and Cg-BigDef5 were active at a high salt concentration (400 mM NaCl) and showed broad spectra of activity against bacteria associated with C. gigas pathologies. Antimicrobial specificity was observed for both molecules at an intra- and inter-genera level. Remarkably, antimicrobial spectra of Cg-BigDef1 and Cg-BigDef5 were complementary, and peptides acted synergistically. Overall, we found that primary sequence diversification of Cg-BigDefs has generated specificity and synergy and extended the spectrum of activity of this peptide family.

Landscape genomics of the American lobster (Homarus americanus).

In marine species experiencing intense fishing pressures, knowledge of genetic structure and local adaptation represent a critical information to assist sustainable management. In this study, we performed a landscape genomics analysis in the American lobster to investigate the issues pertaining to the consequences of making use of putative adaptive loci to reliably infer population structure and thus more rigorously delineating biological management units in marine exploited species. Toward this end, we genotyped 14,893 single nucleotide polymorphism (SNPs) in 4190 lobsters sampled across 96 sampling sites distributed along 1000 km in the northwest Atlantic in both Canada and the USA. As typical for most marine species, we observed a weak, albeit highly significant genetic structure. We also found that adaptive genetic variation allows detecting fine-scale population structure not resolved by neutral genetic variation alone. Using the recent genome assembly of the American lobster, we were able to map and annotate several SNPs located in functional genes potentially implicated in adaptive processes such as thermal stress response, salinity tolerance and growth metabolism pathways. Taken together, our study indicates that weak population structure in high gene flow systems can be resolved at various spatial scales, and that putatively adaptive genetic variation can substantially enhance the delineation of biological management units of marine exploited species.

Genomic signatures of thermal adaptation are associated with clinal shifts of life history in a broadly distributed frog.

Temperature is a critical driver of ectotherm life-history strategies, whereby a warmer environment is associated with increased growth, reduced longevity and accelerated senescence. Increasing evidence indicates that thermal adaptation may underlie such life-history shifts in wild populations. Single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) can help uncover the molecular mechanisms of temperature-driven variation in growth, longevity and senescence. However, our understanding of these mechanisms is still limited, which reduces our ability to predict the response of non-model ectotherms to global temperature change. In this study, we examined the potential role of thermal adaptation in clinal shifts of life-history traits (i.e. life span, senescence rate and recruitment) in the Columbia spotted frog Rana luteiventris along a broad temperature gradient in the western United States. We took advantage of extensive capture-recapture datasets of 20,033 marked individuals from eight populations surveyed annually for 14-18 years to examine how mean annual temperature and precipitation influenced demographic parameters (i.e. adult survival, life span, senescence rate, recruitment and population growth). After showing that temperature was the main climatic predictor influencing demography, we used RAD-seq data (50,829 SNPs and 6,599 putative CNVs) generated for 352 individuals from 31 breeding sites to identify the genomic signatures of thermal adaptation. Our results showed that temperature was negatively associated with annual adult survival and reproductive life span and positively associated with senescence rate. By contrast, recruitment increased with temperature, promoting the long-term viability of most populations. These temperature-dependent demographic changes were associated with strong genomic signatures of thermal adaptation. We identified 148 SNP candidates associated with temperature including three SNPs located within protein-coding genes regulating resistance to cold and hypoxia, immunity and reproduction in ranids. We also identified 39 CNV candidates (including within 38 transposable elements) for which normalized read depth was associated with temperature. Our study indicates that both SNPs and structural variants are associated with temperature and could eventually be found to play a functional role in clinal shifts in senescence rate and life-history strategies in R. luteiventris. These results highlight the potential role of different sources of molecular variation in the response of ectotherms to environmental temperature variation in the context of global warming.

Thermal adaptation rather than demographic history drives genetic structure inferred by copy number variants in a marine fish.

Increasing evidence shows that structural variants represent an overlooked aspect of genetic variation with consequential evolutionary roles. Among those, copy number variants (CNVs), including duplicated genomic regions and transposable elements (TEs), may contribute to local adaptation and/or reproductive isolation among divergent populations. Those mechanisms suppose that CNVs could be used to infer neutral and/or adaptive population genetic structure, whose study has been restricted to microsatellites, mitochondrial DNA and Amplified fragment length polymorphism markers in the past and more recently the use of single nucleotide polymorphisms (SNPs). Taking advantage of recent developments allowing CNV analysis from RAD-seq data, we investigated how variation in fitness-related traits, local environmental conditions and demographic history are associated with CNVs, and how subsequent copy number variation drives population genetic structure in a marine fish, the capelin (Mallotus villosus). We collected 1538 DNA samples from 35 sampling sites in the north Atlantic Ocean and identified 6620 putative CNVs. We found associations between CNVs and the gonadosomatic index, suggesting that six duplicated regions could affect female fitness by modulating oocyte production. We also detected 105 CNV candidates associated with water temperature, among which 20% corresponded to genomic regions located within the sequence of protein-coding genes, suggesting local adaptation to cold water by means of gene sequence amplification. We also identified 175 CNVs associated with the divergence of three previously defined parapatric glacial lineages, of which 24% were located within protein-coding genes, making those loci potential candidates for reproductive isolation. Lastly, our analyses unveiled a hierarchical, complex CNV population structure determined by temperature and local geography, which was in stark contrast to that inferred based on SNPs in a previous study. Our findings underline the complementarity of those two types of genomic variation in population genomics studies.

Copy number variants outperform SNPs to reveal genotype-temperature association in a marine species.

Copy number variants (CNVs) are a major component of genotypic and phenotypic variation in genomes. To date, our knowledge of genotypic variation and evolution has largely been acquired by means of single nucleotide polymorphism (SNPs) analyses. Until recently, the adaptive role of structural variants (SVs) and particularly that of CNVs has been overlooked in wild populations, partly due to their challenging identification. Here, we document the usefulness of Rapture, a derived reduced-representation shotgun sequencing approach, to detect and investigate copy number variants (CNVs) alongside SNPs in American lobster (Homarus americanus) populations. We conducted a comparative study to examine the potential role of SNPs and CNVs in local adaptation by sequencing 1,141 lobsters from 21 sampling sites within the southern Gulf of St. Lawrence, which experiences the highest yearly thermal variance of the Canadian marine coastal waters. Our results demonstrated that CNVs account for higher genetic differentiation than SNP markers. Contrary to SNPs, for which no significant genetic-environment association was found, 48 CNV candidates were significantly associated with the annual variance of sea surface temperature, leading to the genetic clustering of sampling locations despite their geographic separation. Altogether, we provide a strong empirical case that CNVs putatively contribute to local adaptation in marine species and unveil stronger spatial signal of population structure than SNPs. Our study provides the means to study CNVs in nonmodel species and highlights the importance of considering structural variants alongside SNPs to enhance our understanding of ecological and evolutionary processes shaping adaptive population structure.

Shared ancestral polymorphisms and chromosomal rearrangements as potential drivers of local adaptation in a marine fish.

Gene flow has tremendous importance for local adaptation, by influencing the fate of de novo mutations, maintaining standing genetic variation and driving adaptive introgression. Furthermore, structural variation as chromosomal rearrangements may facilitate adaptation despite high gene flow. However, our understanding of the evolutionary mechanisms impending or favouring local adaptation in the presence of gene flow is still limited to a restricted number of study systems. In this study, we examined how demographic history, shared ancestral polymorphism, and gene flow among glacial lineages contribute to local adaptation to sea conditions in a marine fish, the capelin (Mallotus villosus). We first assembled a 490-Mbp draft genome of M. villosus to map our RAD sequence reads. Then, we used a large data set of genome-wide single nucleotide polymorphisms (25,904 filtered SNPs) genotyped in 1,310 individuals collected from 31 spawning sites in the northwest Atlantic. We reconstructed the history of divergence among three glacial lineages and showed that they probably diverged from 3.8 to 1.8 million years ago and experienced secondary contacts. Within each lineage, our analyses provided evidence for large Ne and high gene flow among spawning sites. Within the Northwest Atlantic lineage, we detected a polymorphic chromosomal rearrangement leading to the occurrence of three haplogroups. Genotype-environment associations revealed molecular signatures of local adaptation to environmental conditions prevailing at spawning sites. Our study also suggests that both shared polymorphisms among lineages, resulting from standing genetic variation or introgression, and chromosomal rearrangements may contribute to local adaptation in the presence of high gene flow.

Comparing Pool-seq, Rapture, and GBS genotyping for inferring weak population structure: The American lobster (Homarus americanus) as a case study.

Unraveling genetic population structure is challenging in species potentially characterized by large population size and high dispersal rates, often resulting in weak genetic differentiation. Genotyping a large number of samples can improve the detection of subtle genetic structure, but this may substantially increase sequencing cost and downstream bioinformatics computational time. To overcome this challenge, alternative, cost-effective sequencing approaches, namely Pool-seq and Rapture, have been developed. We empirically measured the power of resolution and congruence of these two methods in documenting weak population structure in nonmodel species with high gene flow comparatively to a conventional genotyping-by-sequencing (GBS) approach. For this, we used the American lobster (Homarus americanus) as a case study. First, we found that GBS, Rapture, and Pool-seq approaches gave similar allele frequency estimates (i.e., correlation coefficient over 0.90) and all three revealed the same weak pattern of population structure. Yet, Pool-seq data showed F ST estimates three to five times higher than GBS and Rapture, while the latter two methods returned similar F ST estimates, indicating that individual-based approaches provided more congruent results than Pool-seq. We conclude that despite higher costs, GBS and Rapture are more convenient approaches to use in the case of species exhibiting very weak differentiation. While both GBS and Rapture approaches provided similar results with regard to estimates of population genetic parameters, GBS remains more cost-effective in project involving a relatively small numbers of genotyped individuals (e.g., <1,000). Overall, this study illustrates the complexity of estimating genetic differentiation and other summary statistics in complex biological systems characterized by large population size and migration rates.