Genomic and functional characterization of the Atlantic salmon gut microbiome in relation to nutrition and health.

To ensure sustainable aquaculture, it is essential to understand the path 'from feed to fish', whereby the gut microbiome plays an important role in digestion and metabolism, ultimately influencing host health and growth. Previous work has reported the taxonomic composition of the Atlantic salmon (Salmo salar) gut microbiome; however, functional insights are lacking. Here we present the Salmon Microbial Genome Atlas consisting of 211 high-quality bacterial genomes, recovered by cultivation (n = 131) and gut metagenomics (n = 80) from wild and farmed fish both in freshwater and seawater. Bacterial genomes were taxonomically assigned to 14 different orders, including 35 distinctive genera and 29 previously undescribed species. Using metatranscriptomics, we functionally characterized key bacterial populations, across five phyla, in the salmon gut. This included the ability to degrade diet-derived fibres and release vitamins and other exometabolites with known beneficial effects, which was supported by genome-scale metabolic modelling and in vitro cultivation of selected bacterial species coupled with untargeted metabolomic studies. Together, the Salmon Microbial Genome Atlas provides a genomic and functional resource to enable future studies on salmon nutrition and health.

Sampling fish gut microbiota - A genome-resolved metagenomic approach.

Despite a surge in microbiota-focused studies in teleosts, few have reported functional data on whole metagenomes as it has proven difficult to extract high biomass microbial DNA from fish intestinal samples. The zebrafish is a promising model organism in functional microbiota research, yet studies on the functional landscape of the zebrafish gut microbiota through shotgun based metagenomics remain scarce. Thus, a consensus on an appropriate sampling method accurately representing the zebrafish gut microbiota, or any fish species is lacking. Addressing this, we systematically tested four methods of sampling the zebrafish gut microbiota: collection of faeces from the tank, the whole gut, intestinal content, and the application of ventral pressure to facilitate extrusion of gut material. Additionally, we included water samples as an environmental control to address the potential influence of the environmental microbiota on each sample type. To compare these sampling methods, we employed a combination of genome-resolved metagenomics and 16S metabarcoding techniques. We observed differences among sample types on all levels including sampling, bioinformatic processing, metagenome co-assemblies, generation of metagenome-assembled genomes (MAGs), functional potential, MAG coverage, and population level microdiversity. Comparison to the environmental control highlighted the potential impact of the environmental contamination on data interpretation. While all sample types tested are informative about the zebrafish gut microbiota, the results show that optimal sample type for studying fish microbiomes depends on the specific objectives of the study, and here we provide a guide on what factors to consider for designing functional metagenome-based studies on teleost microbiomes.

Intestinal microbial profiles of wild Alaskan rainbow trout (Oncorhynchus mykiss) characterized by 16S rRNA amplicon data.

Rainbow trout (Oncorhynchus mykiss) is a dominant aquaculture species of the Salmonidae family, native only to the North Pacific. Recently, the gut microbiome has been shown to reflect the health status and responses to environmental changes in farmed fish. In this analysis we investigated the microbiome composition of the intestinal tract in 20 wild-caught rainbow trout specimens sampled in Alaska, USA. The targeted 16S rRNA gene (V3-V4 region) was sequenced on the Illumina NovaSeq 6000 platform. After quality control, demultiplexing and adapter trimming reads were analyzed using the DADA2 pipeline to obtain Amplicon Sequencing Variants (ASVs) which were subsequently taxonomically assigned. We found two phyla dominating the gut ecosystem present in every sample, Firmicutes and Fusobacteria, followed by lower abundances of Cyanobacteria, Proteobacteria and Bacteroidetes. At the genus level, we found high relative abundances of Cetobacterium and Clostridium sensu stricto 1. Interestingly, we did not identify often dominant genera Mycoplasma, Pseudomonas or Weisella which were prevalent in numerous studies previously, in cultured rainbow trout. Wild fish are exposed to a plethora of unpredictable environmental challenges, ranging from fluctuating water temperatures to variable food availability, as opposed to controlled conditions in production facilities. Examining and comparing the gut ecosystem of wild and reared individuals holds great potential in optimizing management practices for commercially important species. Microbiome studies can provide novel ways to enhance the overall welfare of fish, strengthen disease prevention and increase sustainability in aquaculture production.

Telomere dynamics as mediators of gut microbiota-host interactions.

The highly proliferative gut tissue exhibits rapid telomere shortening with systemic effects on the host organism. Recent studies have demonstrated a bidirectionality in interactions between intestinal telomere length dynamics and the composition and activity of the gut microbiome thus linking processes of inflammation, dysbiosis and aging across different vertebrate species.

The overlooked biodiversity loss.

As most life-forms exist as holobionts, reduction of host-level biodiversity drives parallel habitat losses to their host-adapted microorganisms. The holobiont concept helps us to understand how species are habitats for - often ignored - coevolved microorganisms also worthy of conservation. Indeed, loss of host-associated microbial biodiversity may accelerate the extinction risks of their host.

Genomic context determines the effect of DNA methylation on gene expression in the gut epithelium of Atlantic salmon (Salmo salar).

The canonical view of DNA methylation, a pivotal epigenetic regulation mechanism in eukaryotes, dictates its role as a suppressor of gene activity, particularly within promoter regions. However, this view is being challenged as it is becoming increasingly evident that the connection between DNA methylation and gene expression varies depending on the genomic location and is therefore more complex than initially thought. We examined DNA methylation levels in the gut epithelium of Atlantic salmon (Salmo salar) using whole-genome bisulfite sequencing, which we correlated with gene expression data from RNA sequencing of the same gut tissue sample (RNA-seq). Assuming epigenetic signals might be pronounced between distinctive phenotypes, we compared large and small fish, finding 22 significant associations between 22 differentially methylated regions and 21 genes. We did not detect significant methylation differences between large and small fish. However, we observed a consistent signal of methylation levels around the transcription start sites (TSS), being negatively correlated with the expression levels of those genes. We found both negative and positive associations of methylation levels with gene expression further upstream or downstream of the TSS, revealing a more unpredictable pattern. The 21 genes showing significant methylation-expression correlations were involved in biological processes related to salmon health, such as growth and immune responses. Deciphering how DNA methylation affects the expression of such genes holds great potential for future applications. For instance, our results suggest the importance of genomic context in targeting epigenetic modifications to improve the welfare of aquaculture species like Atlantic salmon.

A practical introduction to holo-omics.

Holo-omics refers to the joint study of non-targeted molecular data layers from host-microbiota systems or holobionts, which is increasingly employed to disentangle the complex interactions between the elements that compose them. We navigate through the generation, analysis, and integration of omics data, focusing on the commonalities and main differences to generate and analyze the various types of omics, with a special focus on optimizing data generation and integration. We advocate for careful generation and distillation of data, followed by independent exploration and analyses of the single omic layers to obtain a better understanding of the study system, before the integration of multiple omic layers in a final model is attempted. We highlight critical decision points to achieve this aim and flag the main challenges to address complex biological questions regarding the integrative study of host-microbiota relationships.

Unraveling host regulation of gut microbiota through the epigenome-microbiome axis.

Recent studies of dynamic interactions between epigenetic modifications of a host organism and the composition or activity of its associated gut microbiota suggest an opportunity for the host to shape its microbiome through epigenetic alterations that lead to changes in gene expression and noncoding RNA activity. We use insights from microbiota-induced epigenetic changes to review the potential of the host to epigenetically regulate its gut microbiome, from which a bidirectional 'epigenome-microbiome axis' emerges. This axis embeds environmentally induced variation, which may influence the adaptive evolution of host-microbe interactions. We furthermore present our perspective on how the epigenome-microbiome axis can be understood and investigated within a holo-omic framework with potential applications in the applied health and food sciences.

Host-gut microbiota interactions shape parasite infections in farmed Atlantic salmon.

Animals and their associated microbiota share long evolutionary histories. However, it is not always clear how host genotype and microbiota interact to affect phenotype. We applied a hologenomic approach to explore how host-microbiota interactions shape lifetime growth and parasite infection in farmed Atlantic salmon (Salmo salar). Multi-omics data sets were generated from the guts of 460 salmon, 82% of which were naturally infected with an intestinal cestode. A single Mycoplasma bacterial strain, MAG01, dominated the gut metagenome of large, non-parasitized fish, consistent with previous studies showing high levels of Mycoplasma in the gut microbiota of healthy salmon. While small and/or parasitized salmon also had high abundance of MAG01, we observed increased alpha diversity in these individuals, driven by increased frequency of low-abundance Vibrionaceae and other Mycoplasma species that carried known virulence genes. Colonization by one of these cestode-associated Mycoplasma strains was associated with host individual genomic variation in long non-coding RNAs. Integrating the multi-omic data sets revealed coordinated changes in the salmon gut mRNA transcriptome and metabolome that correlated with shifts in the microbiota of smaller, parasitized fish. Our results suggest that the gut microbiota of small and/or parasitized fish is in a state of dysbiosis that partly depends on the host genotype, highlighting the value of using a hologenomic approach to incorporate the microbiota into the study of host-parasite dynamics.IMPORTANCEStudying host-microbiota interactions through the perspective of the hologenome is gaining interest across all life sciences. Intestinal parasite infections are a huge burden on human and animal health; however, there are few studies investigating the role of the hologenome during parasite infections. We address this gap in the largest multi-omics fish microbiota study to date using natural cestode infection of farmed Atlantic salmon. We find a clear association between cestode infection, salmon lifetime growth, and perturbation of the salmon gut microbiota. Furthermore, we provide the first evidence that the genetic background of the host may partly determine how the gut microbiota changes during parasite-associated dysbiosis. Our study therefore highlights the value of a hologenomic approach for gaining a more in-depth understanding of parasitism.

Intestinal epigenotype of Atlantic salmon (Salmo salar) associates with tenacibaculosis and gut microbiota composition.

It remains a challenge to obtain the desired phenotypic traits in aquacultural production of Atlantic salmon, and part of the challenge might come from the effect that host-associated microorganisms have on the fish phenotype. To manipulate the microbiota towards the desired host traits, it is critical to understand the factors that shape it. The bacterial gut microbiota composition can vary greatly among fish, even when reared in the same closed system. While such microbiota differences can be linked to diseases, the molecular effect of disease on host-microbiota interactions and the potential involvement of epigenetic factors remain largely unknown. The aim of this study was to investigate the DNA methylation differences associated with a tenacibaculosis outbreak and microbiota displacement in the gut of Atlantic salmon. Using Whole Genome Bisulfite Sequencing (WGBS) of distal gut tissue from 20 salmon, we compared the genome-wide DNA methylation levels between uninfected individuals and sick fish suffering from tenacibaculosis and microbiota displacement. We discovered >19,000 differentially methylated cytosine sites, often located in differentially methylated regions, and aggregated around genes. The 68 genes connected to the most significant regions had functions related to the ulcerous disease such as epor and slc48a1a but also included prkcda and LOC106590732 whose orthologs are linked to microbiota changes in other species. Although the expression level was not analysed, our epigenetic analysis suggests specific genes potentially involved in host-microbiota interactions and more broadly it highlights the value of considering epigenetic factors in efforts to manipulate the microbiota of farmed fish.

Co-diversification of an intestinal Mycoplasma and its salmonid host.

Understanding the evolutionary relationships between a host and its intestinal resident bacteria can transform how we understand adaptive phenotypic traits. The interplay between hosts and their resident bacteria inevitably affects the intestinal environment and, thereby, the living conditions of both the host and the microbiota. Thereby this co-existence likely influences the fitness of both bacteria and host. Whether this co-existence leads to evolutionary co-diversification in animals is largely unexplored, mainly due to the complexity of the environment and microbial communities and the often low host selection. We present the gut metagenome from wild Atlantic salmon (Salmo salar), a new wild organism model with an intestinal microbiota of low complexity and a well-described population structure, making it well-suited for investigating co-evolution. Our data reveal a strong host selection of a core gut microbiota dominated by a single Mycoplasma species. We found a clear co-diversification between the population structure of Atlantic salmon and nucleotide variability of the intestinal Mycoplasma populations conforming to expectations from co-evolution between host and resident bacteria. Our results show that the stable microbiota of Atlantic salmon has evolved with its salmonid host populations while potentially providing adaptive traits to the salmon host populations, including defence mechanisms, biosynthesis of essential amino acids, and metabolism of B vitamins. We highlight Atlantic salmon as a novel model for studying co-evolution between vertebrate hosts and their resident bacteria.

Influence of African Swine Fever Virus on Host Gene Transcription within Peripheral Blood Mononuclear Cells from Infected Pigs.

African swine fever virus (ASFV) has become a global threat to the pig production industry and has caused enormous economic losses in many countries in recent years. Peripheral blood mononuclear cells (PBMCs) from pigs infected with ASFV not only express ASFV genes (almost 200 in number) but have altered patterns of host gene expression as well. Both up- and down-regulation of host cell gene expression can be followed using RNAseq on poly(A)+ mRNAs harvested from the PBMCs of pigs collected at different times post-infection. Consistent with the time course of changes in viral gene expression, only few and limited changes in host gene expression were detected at 3 days post-infection (dpi), but by 6 dpi, marked changes in the expression of over 1300 host genes were apparent. This was co-incident with the major increase in viral gene expression. The majority of the changes in host gene expression were up-regulation, but many down-regulated genes were also identified. The patterns of changes in gene expression within the PBMCs detected by RNAseq were similar in each of the four infected pigs. Furthermore, changes in the expression of about twenty selected host genes, known to be important in host defence and inflammatory responses, were confirmed using high-throughput microfluidic qPCR assays.

A strategic model of a host-microbe-microbe system reveals the importance of a joint host-microbe immune response to combat stress-induced gut dysbiosis.

Microbiomes provide key ecological functions to their host; however, most host-associated microbiomes are too complicated to allow a model of essential host-microbe-microbe interactions. The intestinal microbiota of salmonids may offer a solution since few dominating species often characterize it. Healthy fish coexist with a mutualistic Mycoplasma sp. species, while stress allows the spread of pathogenic strains, such as Aliivibrio sp. Even after a skin infection, the Mycoplasma does not recover; Aliivibrio sp. often remains the dominant species, or Mycoplasma-Aliivibrio coexistence was occasionally observed. We devised a model involving interactions among the host immune system, Mycoplasma sp. plus a toxin-producing pathogen. Our model embraces a complete microbiota community and is in harmony with experimental results that host-Mycoplasma mutualism prevents the spread of pathogens. Contrary, stress suppresses the host immune system allowing dominance of pathogens, and Mycoplasma does not recover after stress disappears.

Microbiome "Inception": an Intestinal Cestode Shapes a Hierarchy of Microbial Communities Nested within the Host.

The concept of a holobiont, a host organism and its associated microbial communities, encapsulates the vital role the microbiome plays in the normal functioning of its host. Parasitic infections can disrupt this relationship, leading to dysbiosis. However, it is increasingly recognized that multicellular parasites are themselves holobionts. Intestinal parasites share space with the host gut microbiome, creating a system of nested microbiomes within the primary host. However, how the parasite, as a holobiont, interacts with the host holobiont remains unclear, as do the consequences of these interactions for host health. Here, we used 16S amplicon and shotgun metagenomics sequencing to characterize the microbiome of the intestinal cestode Eubothrium and its effect on the gut microbiome of its primary host, Atlantic salmon. Our results indicate that cestode infection is associated with salmon gut dysbiosis by acting as a selective force benefiting putative pathogens and potentially introducing novel bacterial species to the host. Our results suggest that parasitic cestodes may themselves be holobionts nested within the microbial community of their holobiont host, emphasizing the importance of also considering microbes associated with parasites when studying intestinal parasitic infections. IMPORTANCE The importance of the parasite microbiome is gaining recognition. Of particular concern is understanding how these parasite microbiomes influence host-parasite interactions and parasite interactions with the vertebrate host microbiome as part of a system of nested holobionts. However, there are still relatively few studies focusing on the microbiome of parasitic helminths in general and almost none on cestodes in particular, despite the significant burden of disease caused by these parasites globally. Our study provides insights into a system of significance to the aquaculture industry, cestode infections of Atlantic salmon and, more broadly, expands our general understanding of parasite-microbiome-host interactions and introduces a new element, the microbiome of the parasite itself, which may play a critical role in modulating the host microbiome, and, therefore, the host response, to parasite infection.

Disentangling host-microbiota complexity through hologenomics.

Research on animal-microbiota interactions has become a central topic in biological sciences because of its relevance to basic eco-evolutionary processes and applied questions in agriculture and health. However, animal hosts and their associated microbial communities are still seldom studied in a systemic fashion. Hologenomics, the integrated study of the genetic features of a eukaryotic host alongside that of its associated microbes, is becoming a feasible - yet still underexploited - approach that overcomes this limitation. Acknowledging the biological and genetic properties of both hosts and microbes, along with the advantages and disadvantages of implemented techniques, is essential for designing optimal studies that enable some of the major questions in biology to be addressed.

Invasion genomics uncover contrasting scenarios of genetic diversity in a widespread marine invader.

Invasion rates have increased in the past 100 y irrespective of international conventions. What characterizes a successful invasion event? And how does genetic diversity translate into invasion success? Employing a whole-genome perspective using one of the most successful marine invasive species world-wide as a model, we resolve temporal invasion dynamics during independent invasion events in Eurasia. We reveal complex regionally independent invasion histories including cases of recurrent translocations, time-limited translocations, and stepping-stone range expansions with severe bottlenecks within the same species. Irrespective of these different invasion dynamics, which lead to contrasting patterns of genetic diversity, all nonindigenous populations are similarly successful. This illustrates that genetic diversity, per se, is not necessarily the driving force behind invasion success. Other factors such as propagule pressure and repeated introductions are an important contribution to facilitate successful invasions. This calls into question the dominant paradigm of the genetic paradox of invasions, i.e., the successful establishment of nonindigenous populations with low levels of genetic diversity.

Identification of African Swine Fever Virus Transcription within Peripheral Blood Mononuclear Cells of Acutely Infected Pigs.

African swine fever virus (ASFV) has become widespread in Europe, Asia and elsewhere, thereby causing extensive economic losses. The viral genome includes nearly 200 genes, but their expression within infected pigs has not been well characterized previously. In this study, four pigs were infected with a genotype II strain (ASFV POL/2015/Podlaskie); blood samples were collected before inoculation and at both 3 and 6 days later. During this period, a range of clinical signs of infection became apparent in the pigs. From the blood, peripheral blood mononuclear cells (PBMCs) were isolated. The transcription of the ASFV genes was determined using RNAseq on poly(A)+ mRNAs isolated from these cells. Only very low levels of virus transcription were detected in the PBMCs at 3 days post-inoculation (dpi) but, at 6 dpi, extensive transcription was apparent. This was co-incident with a large increase in the level of ASFV DNA within these cells. The pattern of the virus gene expression was very reproducible between the individual pigs. Many highly expressed genes have undefined roles. Surprisingly, some genes with key roles in virus replication were expressed at only low levels. As the functions of individual genes are identified, information about their expression becomes important for understanding their contribution to virus biology.

Genome-resolved metagenomics suggests a mutualistic relationship between Mycoplasma and salmonid hosts.

Salmonids are important sources of protein for a large proportion of the human population. Mycoplasma species are a major constituent of the gut microbiota of salmonids, often representing the majority of microbiota. Despite the frequent reported dominance of salmonid-related Mycoplasma species, little is known about the phylogenomic placement, functions and potential evolutionary relationships with their salmonid hosts. In this study, we utilise 2.9 billion metagenomic reads generated from 12 samples from three different salmonid host species to I) characterise and curate the first metagenome-assembled genomes (MAGs) of Mycoplasma dominating the intestines of three different salmonid species, II) establish the phylogeny of these salmonid candidate Mycoplasma species, III) perform a comprehensive pangenomic analysis of Mycoplasma, IV) decipher the putative functionalities of the salmonid MAGs and reveal specific functions expected to benefit the host. Our data provide a basis for future studies examining the composition and function of the salmonid microbiota.

Salmon gut microbiota correlates with disease infection status: potential for monitoring health in farmed animals.

BACKGROUND: Infectious diseases cause significant production losses in aquaculture every year. Since the gut microbiota plays an essential role in regulating the host immune system, health and physiology, altered gut microbiota compositions are often associated with a diseased status. However, few studies have examined the association between disease severity and degree of gut dysbiosis, especially when the gut is not the site of the primary infection. Moreover, there is a lack of knowledge on whether bath treatment with formalin, a disinfectant commonly used in aquaculture to treat external infections, might affect the gut microbiome as a consequence of formalin ingestion. Here we investigate, through 16S rRNA gene metabarcoding, changes in the distal gut microbiota composition of a captive-reared cohort of 80 Atlantic salmon (Salmo salar L.), in consequence of an external bacterial skin infection due to a natural outbreak and subsequent formalin treatment. RESULTS: We identified Tenacibaculum dicentrarchi as the causative disease pathogen and we show that the distal gut of diseased salmon presented a different composition from that of healthy individuals. A new, yet undescribed, Mycoplasma genus characterized the gut of healthy salmon, while in the sick fish we observed an increase in terms of relative abundance of Aliivibrio sp., a strain regarded as opportunistic. We also noticed a positive correlation between fish weight and Mycoplasma sp. relative abundance, potentially indicating a beneficial effect for its host. Moreover, we observed that the gut microbiota of fish treated with formalin was more similar to those of sick fish than healthy ones. CONCLUSIONS: We conclude that external Tenacibaculum infections have the potential of indirectly affecting the host gut microbiota. As such, treatment optimization procedures should account for that. Formalin treatment is not an optimal solution from a holistic perspective, since we observe an altered gut microbiota in the treated fish. We suggest its coupling with a probiotic treatment aimed at re-establishing a healthy community. Lastly, we have observed a positive correlation of Mycoplasma sp. with salmon health and weight, therefore we encourage further investigations towards its potential utilization as a biomarker for monitoring health in salmon and potentially other farmed fish species.