Modulation of gut microbiota composition and predicted metabolic capacity after nutritional programming with a plant-rich diet in Atlantic salmon (Salmo salar): insights across developmental stages.

To promote sustainable aquaculture, the formulation of Atlantic salmon (Salmo salar) feeds has changed in recent decades, focusing on replacing standard marine-based ingredients with plant-based alternatives, increasingly demonstrating successful outcomes in terms of fish performance. However, little is known about how these plant-based diets may impact the gut microbiota at first feeding and onwards. Nutritional programming (NP) is one strategy applied for exposing fish to a plant-based (V) diet at an early stage in life to promote full utilisation of plant-based ingredients and prevent potential adverse impacts of exposure to a plant-rich diet later in life. We investigated the impact of NP on gut microbiota by introducing fish to plant ingredients (V fish) during first feeding for a brief period of two weeks (stimulus phase) and compared those to fish fed a marine-based diet (M fish). Results demonstrated that V fish not only maintained growth performance at 16 (intermediate phase) and 22 (challenge phase) weeks post first feeding (wpff) when compared to M fish but also modulated gut microbiota. PERMANOVA general effects revealed gut microbiota dissimilarity by fish group (V vs. M fish) and phases (stimulus vs. intermediate vs. challenge). However, no interaction effect of both groups and phases was demonstrated, suggesting a sustained impact of V diet (nutritional history) on fish across time points/phases. Moreover, the V diet exerted a significant cumulative modulatory effect on the Atlantic salmon gut microbiota at 16 wpff that was not demonstrated at two wpff, although both fish groups were fed the M diet at 16 wpff. The nutritional history/dietary regime is the main NP influencing factor, whereas environmental and host factors significantly impacted microbiota composition in M fish. Microbial metabolic reactions of amino acid metabolism were higher in M fish when compared to V fish at two wpff suggesting microbiota played a role in digesting the essential amino acids of M feed. The excessive mucin O-degradation revealed in V fish at two wpff was mitigated in later life stages after NP, suggesting physiological adaptability and tolerance to V diet. Future studies are required to explore more fully how the microbiota functionally contributes to the NP.

Impact of freshwater rearing history on Atlantic salmon gill response to viral stimulation post seawater transfer.

Land-based recirculating aquaculture systems (RAS) have risen in prevalence in recent years for Atlantic salmon production, enabling intensive production which allows increased growth and environmental control, but also having the potential for reducing water use and eutrophication. The Atlantic salmon has an anadromous life history with juvenile stages in freshwater (FW) and on-growing in seawater (SW), enabled by a transformational process known as smoltification. The timing of smoltification and transfer of smolts from FW to SW is critical under commercial production with high mortalities during this period. The impact of FW rearing system on immune function following seawater transfer (SWT) is not well understood. In this study parr were raised in either RAS or a traditional open-LOCH system until smolting and then transferred to a common marine environment. Two-weeks post-SWT fish were immune stimulated with a viral mimic (poly I:C) for 24 h to assess the ability to mount an antiviral immune response, assessed by whole transcriptome analysis of gill tissue, an important immune organ in fish. We show that unstimulated smolts reared in the LOCH had higher immune gene expression than those reared in RAS as determined by functional analysis. However, following stimulation, smolts reared in the RAS mounted a greater magnitude of response with a suite of immune genes displaying higher fold induction of transcription compared to LOCH reared smolts. We suggest RAS smolts have a lower steady state immune-associated transcriptome likely due to an unvarying environment, in terms of environmental factors and lack of exposure to pathogens, which shows a compensatory mechanism following stimulation allowing immune 'catch-up' with those reared in the LOCH. Alternatively, the RAS fish are experiencing an excessive response to the immune stimulation.

Cell atlas of the Atlantic salmon spleen reveals immune cell heterogeneity and cell-specific responses to bacterial infection.

The spleen is a conserved secondary lymphoid organ that emerged in parallel to adaptive immunity in early jawed vertebrates. Recent studies have applied single cell transcriptomics to reveal the cellular composition of spleen in several species, cataloguing diverse immune cell types and subpopulations. In this study, 51,119 spleen nuclei transcriptomes were comprehensively investigated in the commercially important teleost Atlantic salmon (Salmo salar L.), contrasting control animals with those challenged with the bacterial pathogen Aeromonas salmonicida. We identified clusters of nuclei representing the expected major cell types, namely T cells, B cells, natural killer-like cells, granulocytes, mononuclear phagocytes, endothelial cells, mesenchymal cells, erythrocytes and thrombocytes. We discovered heterogeneity within several immune lineages, providing evidence for resident macrophages and melanomacrophages, infiltrating monocytes, several candidate dendritic cell subpopulations, and B cells at distinct stages of differentiation, including plasma cells and an igt + subset. We provide evidence for twelve candidate T cell subsets, including cd4+ T helper and regulatory T cells, one cd8+ subset, three γδT subsets, and populations double negative for cd4 and cd8. The number of genes showing differential expression during the early stages of Aeromonas infection was highly variable across immune cell types, with the largest changes observed in macrophages and infiltrating monocytes, followed by resting mature B cells. Our analysis provides evidence for a local inflammatory response to infection alongside B cell maturation in the spleen, and upregulation of ccr9 genes in igt + B cells, T helper and cd8+ cells, and monocytes, consistent with the recruitment of immune cell populations to the gut to deal with Aeromonas infection. Overall, this study provides a new cell-resolved perspective of the immune actions of Atlantic salmon spleen, highlighting extensive heterogeneity hidden to bulk transcriptomics. We further provide a large catalogue of cell-specific marker genes that can be leveraged to further explore the function and structural organization of the salmonid immune system.

Mapping the cellular landscape of Atlantic salmon head kidney by single cell and single nucleus transcriptomics.

Single-cell transcriptomics is the current gold standard for global gene expression profiling, not only in mammals and model species, but also in non-model fish species. This is a rapidly expanding field, creating a deeper understanding of tissue heterogeneity and the distinct functions of individual cells, making it possible to explore the complexities of immunology and gene expression on a highly resolved level. In this study, we compared two single cell transcriptomic approaches to investigate cellular heterogeneity within the head kidney of healthy farmed Atlantic salmon (Salmo salar). We compared 14,149 cell transcriptomes assayed by single cell RNA-seq (scRNA-seq) with 18,067 nuclei transcriptomes captured by single nucleus RNA-Seq (snRNA-seq). Both approaches detected eight major cell populations in common: granulocytes, heamatopoietic stem cells, erythrocytes, mononuclear phagocytes, thrombocytes, B cells, NK-like cells, and T cells. Four additional cell types, endothelial, epithelial, interrenal, and mesenchymal cells, were detected in the snRNA-seq dataset, but appeared to be lost during preparation of the single cell suspension submitted for scRNA-seq library generation. We identified additional heterogeneity and subpopulations within the B cells, T cells, and endothelial cells, and revealed developmental trajectories of heamatopoietic stem cells into differentiated granulocyte and mononuclear phagocyte populations. Gene expression profiles of B cell subtypes revealed distinct IgM and IgT-skewed resting B cell lineages and provided insights into the regulation of B cell lymphopoiesis. The analysis revealed eleven T cell sub-populations, displaying a level of T cell heterogeneity in salmon head kidney comparable to that observed in mammals, including distinct subsets of cd4/cd8-negative T cells, such as tcrγ positive, progenitor-like, and cytotoxic cells. Although snRNA-seq and scRNA-seq were both useful to resolve cell type-specific expression in the Atlantic salmon head kidney, the snRNA-seq pipeline was overall more robust in identifying several cell types and subpopulations. While scRNA-seq displayed higher levels of ribosomal and mitochondrial genes, snRNA-seq captured more transcription factor genes. However, only scRNA-seq-generated data was useful for cell trajectory inference within the myeloid lineage. In conclusion, this study systematically outlines the relative merits of scRNA-seq and snRNA-seq in Atlantic salmon, enhances understanding of teleost immune cell lineages, and provides a comprehensive list of markers for identifying major cell populations in the head kidney with significant immune relevance.

Catch of the Day: New Serum Amyloid A (SAA) Antibody Is a Valuable Tool to Study Fish Health in Salmonids.

Serum amyloid A (SAA) proteins belong to a family of acute-phase reactants, playing an integral role in defending the organism from pathological damage. Despite a wealth of data on the regulation of SAA transcripts in teleosts, there is only limited information on these proteins' abundance in fish. The aim of this study is to characterise SAA protein levels in salmonids using a newly developed antibody specific to salmonid SAA. The salmonid SAA antibody detected SAA and accurately discriminated between stimulated and control specimens from rainbow trout macrophage cell line (RTS-11) in vitro, as well as rainbow trout challenged with Aeromonas salmonicida- or flagellin-stimulated Atlantic salmon in vivo. The presence of SAA protein was analysed in RTS-11 cell line supernatants, liver, and spleen samples using ELISA, immunoblotting, and immunohistochemistry. This study is the first to characterise SAA protein levels in salmonids in vivo and in vitro. The newly developed salmonid SAA antibody was able to discriminate between stimulated and unstimulated specimens, showing that it can be used to study the acute-phase response in salmonids with the potential to be further developed into assays to monitor and evaluate health in wild and farmed fish.

Microbiomes in the context of developing sustainable intensified aquaculture.

With an ever-growing human population, the need for sustainable production of nutritional food sources has never been greater. Aquaculture is a key industry engaged in active development to increase production in line with this need while remaining sustainable in terms of environmental impact and promoting good welfare and health in farmed species. Microbiomes fundamentally underpin animal health, being a key part of their digestive, metabolic and defense systems, in the latter case protecting against opportunistic pathogens in the environment. The potential to manipulate the microbiome to the advantage of enhancing health, welfare and production is an intriguing prospect that has gained considerable traction in recent years. In this review we first set out what is known about the role of the microbiome in aquaculture production systems across the phylogenetic spectrum of cultured animals, from invertebrates to finfish. With a view to reducing environmental footprint and tightening biological and physical control, investment in "closed" aquaculture systems is on the rise, but little is known about how the microbial systems of these closed systems affect the health of cultured organisms. Through comparisons of the microbiomes and their dynamics across phylogenetically distinct animals and different aquaculture systems, we focus on microbial communities in terms of their functionality in order to identify what features within these microbiomes need to be harnessed for optimizing healthy intensified production in support of a sustainable future for aquaculture.

Conserved and divergent arms of the antiviral response in the duplicated genomes of salmonid fishes.

Antiviral innate immunity is orchestrated by the interferon system, which appeared in ancestors of jawed vertebrates. Interferon upregulation induces hundreds of interferon-stimulated-genes (ISGs) with effector or regulatory functions. Here we investigated the evolutionary diversification of ISG responses through comparison of two salmonid fishes, accounting for the impact of sequential whole genome duplications ancestral to teleosts and salmonids. We analysed the transcriptomic response of the IFN pathway in the head kidney of rainbow trout and Atlantic salmon, which separated 25-30 Mya. We identified a large set of ISGs conserved in both species and cross-referenced them with zebrafish and human ISGs. In contrast, around one-third of salmonid ISG lacked orthologs in human, mouse, chicken or frog, and often between rainbow trout and Atlantic salmon, revealing a fast-evolving, lineage-specific arm of the antiviral response. This study also provides a key resource for in-depth functional analysis of ISGs in salmonids of commercial significance.

Palliative Nursing in Africa: Scoping the Landscape of Evidence Focusing on Cancer Care.

AIM: The aim of this study was to describe the research output of African's nurses in the field of palliative care from January 1, 2012 to December 31, 2021. METHODS: A scoping review was conducted. The key words Africa and nursing in combination with palliative care, end of life, terminal care, hospice, and supportive care were used to search the databases Cumulative Index to Nursing and Allied Health Literature, PsychINFO, PubMed, Scopus, and Web of Science. Only studies authored by a nurse with an African affiliation focusing on issues related to advanced cancer were included. The data were captured onto an extraction sheet and analyzed by means of descriptive statistics and content analyses. RESULTS: Of the 522 articles identified, only 16 met the inclusion criteria. The work originated from eight African countries was primarily qualitative and focused on the family and caregivers. Pain was the only symptom investigated. CONCLUSIONS: Studies focusing on symptoms, psychosocial, spiritual, end of life care as well as studies testing nursing interventions are urgently needed. Interregional research could also assist with building the current evidence.

Systematic review of psychosocial interventions for adult cancer patients and their family caregivers in Sub-Saharan Africa.

Cancer is becoming a public health issue in the Sub-Saharan Africa (SSA). This systematic review aims to synthesise psychosocial interventions and their effects on the health outcomes of adult cancer patients and their family caregivers in SSA. We identified eligible publications in English language from PubMed, Cumulative Index of Nursing and Allied Health Literature Plus with Full Text, Embase, APA PsycInfo, Scopus, and African Index Medicus databases. We included psychosocial interventions targeted adult cancer patients/survivors or their family caregivers in SSA. This review identified five psychosocial interventions from six studies that support adult cancer patients and their family caregivers in SSA. The interventions focused on providing informational, psycho-cognitive, and social support. Three interventions significantly improved quality of life outcomes for cancer patients and their caregivers. Significant gaps exist between the rapidly increasing cancer burdens and the limited psychosocial educational interventions supporting adult cancer patients and their families in SSA. The reviewed studies provide preliminary evidence on development and testing interventions that aim to improve patients' and caregivers' quality of life.

Psychosocial experiences of cancer survivors and their caregivers in sub-Saharan Africa: A synthesis of qualitative studies.

OBJECTIVE: To synthesize and examine current literature on survivorship experiences and psychosocial oncologic care programs of individuals affected by cancer in sub-Saharan Africa (SSA). METHODS: This was a systematic review guided by the PRISMA 2020 guidelines. We searched 6 databases for articles published from inception to 21 October 2021. Articles were appraised using the Johanna Briggs Institute's Checklist for Qualitative Research. For data synthesis, we used the World Health Organization (WHO) quality of life framework [WHOQOL] to organize experiences into 6 domains/themes. RESULTS: Twenty-five qualitative studies were included in the synthesis. Studies focused on psychosocial care of adults (>18 years) affected by cancer in SSA. The common WHOQOL domains were social relations, spirituality/religion/personal beliefs, and psychological. CONCLUSION: Findings echo need for individuals with cancer and their caregivers. Healthcare professionals are an essential resource for information and support services that can be tailored to individuals need. This synthesis highlighted caregiver stress and stressors from the community that could impact care of individuals with cancer. A holistic approach is needed that incorporates professional and social aspects of care.

Fur removal promotes an earlier expression of involution-related genes in mammary gland of lactating mice.

Peak lactation occurs when milk production is at its highest. The factors limiting peak lactation performance have been subject of intense debate. Milk production at peak lactation appears limited by the capacity of lactating females to dissipate body heat generated as a by-product of processing food and producing milk. As a result, manipulations that enhance capacity to dissipate body heat (such as fur removal) increase peak milk production. We investigated the potential correlates of shaving-induced increases in peak milk production in laboratory mice. By transcriptomic profiling of the mammary gland, we searched for the mechanisms underlying experimentally increased milk production and its consequences for mother-young conflict over weaning, manifested by advanced or delayed involution of mammary gland. We demonstrated that shaving-induced increases in milk production were paradoxically linked to reduced expression of some milk synthesis-related genes. Moreover, the mammary glands of shaved mice had a gene expression profile indicative of earlier involution relative to unshaved mice. Once provided with enhanced capacity to dissipate body heat, shaved mice were likely to rear their young to independence faster than unshaved mothers.

Gut Associated Lymphoid Tissue (GALT) primary cells and stable cell lines as predictive models for intestinal health in rainbow trout (Oncorhynchus mykiss).

The use of functional feeds for farmed fish is now regarded as a key factor in improving fish health and performance against infectious disease. However, the mechanisms by which these nutritional components modulate the immune response are not fully understood. The present study was undertaken to identify the suitability of both primary gut-associated lymphoid tissue (GALT) leucocyte cells and established rainbow trout cell lines as potential alternative methods to test functional feed ingredients prior to full fish feeding trials that can take months to complete. In addition to the primary GALT culture cells, the two rainbow cell lines RTS11 and RTgutGC which are from macrophage and gut epithelial cells, respectively. The cells were stimulated with a variety of pathogen associated molecular patterns (PAMPs) (PHA and Poly I:C) and recombinant rainbow trout IL-1ß (rIL-1ß), a proinflammatory cytokine, additionally two forms of ß-glucan, a prebiotic commonly used aquafeeds were used as stimulants. From this, the suitability of cell models as a health screen for functional feeds was assessed. GALT leucocytes were deemed most effective to act as a health screen over the 4hr time point demonstrating responses to Poly I:C, PHA, and rIL-1ß. RTS11 and RTgutGC also responded to the stimulants but did not give a strong T-cell response, most likely reflecting the nature of the cell type as opposed to the mixed cell populations from the primary GALT cell cultures. When stimulated with both forms of ß-glucan, GALT leucocytes demonstrated a strong proinflammatory and T-cell response.

Transcriptomic response to ISAV infection in the gills, head kidney and spleen of resistant and susceptible Atlantic salmon.

BACKGROUND: Infectious Salmon Anaemia virus (ISAV) is an orthomyxovirus responsible for large losses in Atlantic salmon (Salmo salar) aquaculture. Current available treatments and vaccines are not fully effective, and therefore selective breeding to produce ISAV-resistant strains of Atlantic salmon is a high priority for the industry. Genomic selection and potentially genome editing can be applied to enhance the disease resistance of aquaculture stocks, and both approaches can benefit from increased knowledge on the genomic mechanisms of resistance to ISAV. To improve our understanding of the mechanisms underlying resistance to ISAV in Atlantic salmon we performed a transcriptomic study in ISAV-infected salmon with contrasting levels of resistance to this virus. RESULTS: Three different tissues (gills, head kidney and spleen) were collected on 12 resistant and 12 susceptible fish at three timepoints (pre-challenge, 7 and 14 days post challenge) and RNA sequenced. The transcriptomes of infected and non-infected fish and of resistant and susceptible fish were compared at each timepoint. The results show that the responses to ISAV are organ-specific; an important response to the infection was observed in the head kidney, with up-regulation of immune processes such as interferon and NLR pathways, while in gills and spleen the response was more moderate. In addition to immune related genes, our results suggest that other processes such as ubiquitination and ribosomal processing are important during early infection with ISAV. Moreover, the comparison between resistant and susceptible fish has also highlighted some interesting genes related to ubiquitination, intracellular transport and the inflammasome. CONCLUSIONS: Atlantic salmon infection by ISAV revealed an organ-specific response, implying differential function during the infection. An immune response was observed in the head kidney in these early timepoints, while gills and spleen showed modest responses in comparison. Comparison between resistance and susceptible samples have highlighted genes of interest for further studies, for instance those related to ubiquitination or the inflammasome.

Single cell transcriptomics of Atlantic salmon (Salmo salar L.) liver reveals cellular heterogeneity and immunological responses to challenge by Aeromonas salmonicida.

The liver is a multitasking organ with essential functions for vertebrate health spanning metabolism and immunity. In contrast to mammals, our understanding of liver cellular heterogeneity and its role in regulating immunological status remains poorly defined in fishes. Addressing this knowledge gap, we generated a transcriptomic atlas of 47,432 nuclei isolated from the liver of Atlantic salmon (Salmo salar L.) contrasting control fish with those challenged with a pathogenic strain of Aeromonas salmonicida, a problematic bacterial pathogen in global aquaculture. We identified the major liver cell types and their sub-populations, revealing poor conservation of many hepatic cell marker genes utilized in mammals, while identifying novel heterogeneity within the hepatocyte, lymphoid, and myeloid lineages. This included polyploid hepatocytes, multiple T cell populations including γδ T cells, and candidate populations of monocytes/macrophages and dendritic cells. A dominant hepatocyte population radically remodeled its transcriptome following infection to activate the acute phase response and other defense functions, while repressing routine functions such as metabolism. These defense-specialized hepatocytes showed strong activation of genes controlling protein synthesis and secretion, presumably to support the release of acute phase proteins into circulation. The infection response further involved up-regulation of numerous genes in an immune-cell specific manner, reflecting functions in pathogen recognition and killing, antigen presentation, phagocytosis, regulation of inflammation, B cell differentiation and T cell activation. Overall, this study greatly enhances our understanding of the multifaceted role played by liver immune and non-immune cells in host defense and metabolic remodeling following infection and provides many novel cell-specific marker genes to empower future studies of this organ in fishes.

Tenebrio molitor larvae meal inclusion affects hepatic proteome and apoptosis and/or autophagy of three farmed fish species.

Herein, the effect of dietary inclusion of insect (Tenebrio molitor) meal on hepatic pathways of apoptosis and autophagy in three farmed fish species, gilthead seabream (Sparus aurata), European seabass (Dicentrarchus labrax) and rainbow trout (Oncorhynchus mykiss), fed diets at 25%, 50% and 60% insect meal inclusion levels respectively, was investigated. Hepatic proteome was examined by liver protein profiles from the three fish species, obtained by two-dimensional gel electrophoresis. Although cellular stress was evident in the three teleost species following insect meal, inclusion by T. molitor, D. labrax and O. mykiss suppressed apoptosis through induction of hepatic autophagy, while in S. aurata both cellular procedures were activated. Protein abundance showed that a total of 30, 81 and 74 spots were altered significantly in seabream, European seabass and rainbow trout, respectively. Insect meal inclusion resulted in individual protein abundance changes, with less number of proteins altered in gilthead seabream compared to European seabass and rainbow trout. This is the first study demonstrating that insect meal in fish diets is causing changes in liver protein abundances. However, a species-specific response both in the above mentioned bioindicators, indicates the need to strategically manage fish meal replacement in fish diets per species.

Genome-Wide Reconstruction of Rediploidization Following Autopolyploidization across One Hundred Million Years of Salmonid Evolution.

The long-term evolutionary impacts of whole-genome duplication (WGD) are strongly influenced by the ensuing rediploidization process. Following autopolyploidization, rediploidization involves a transition from tetraploid to diploid meiotic pairing, allowing duplicated genes (ohnologs) to diverge genetically and functionally. Our understanding of autopolyploid rediploidization has been informed by a WGD event ancestral to salmonid fishes, where large genomic regions are characterized by temporally delayed rediploidization, allowing lineage-specific ohnolog sequence divergence in the major salmonid clades. Here, we investigate the long-term outcomes of autopolyploid rediploidization at genome-wide resolution, exploiting a recent "explosion" of salmonid genome assemblies, including a new genome sequence for the huchen (Hucho hucho). We developed a genome alignment approach to capture duplicated regions across multiple species, allowing us to create 121,864 phylogenetic trees describing genome-wide ohnolog divergence across salmonid evolution. Using molecular clock analysis, we show that 61% of the ancestral salmonid genome experienced an initial "wave" of rediploidization in the late Cretaceous (85-106 Ma). This was followed by a period of relative genomic stasis lasting 17-39 My, where much of the genome remained tetraploid. A second rediploidization wave began in the early Eocene and proceeded alongside species diversification, generating predictable patterns of lineage-specific ohnolog divergence, scaling in complexity with the number of speciation events. Using gene set enrichment, gene expression, and codon-based selection analyses, we provide insights into potential functional outcomes of delayed rediploidization. This study enhances our understanding of delayed autopolyploid rediploidization and has broad implications for future studies of WGD events.

Gill Transcriptomic Responses to Toxin-producing Alga Prymnesium parvum in Rainbow Trout.

The gill of teleost fish is a multifunctional organ involved in many physiological processes, including protection of the mucosal gill surface against pathogens and other environmental antigens by the gill-associated lymphoid tissue (GIALT). Climate change associated phenomena, such as increasing frequency and magnitude of harmful algal blooms (HABs) put extra strain on gill function, contributing to enhanced fish mortality and fish kills. However, the molecular basis of the HAB-induced gill injury remains largely unknown due to the lack of high-throughput transcriptomic studies performed on teleost fish in laboratory conditions. We used juvenile rainbow trout (Oncorhynchus mykiss) to investigate the transcriptomic responses of the gill tissue to two (high and low) sublethal densities of the toxin-producing alga Prymnesium parvum, in relation to non-exposed control fish. The exposure time to P. parvum (4-5 h) was sufficient to identify three different phenotypic responses among the exposed fish, enabling us to focus on the common gill transcriptomic responses to P. parvum that were independent of dose and phenotype. The inspection of common differentially expressed genes (DEGs), canonical pathways, upstream regulators and downstream effects pointed towards P. parvum-induced inflammatory response and gill inflammation driven by alterations of Acute Phase Response Signalling, IL-6 Signalling, IL-10 Signalling, Role of PKR in Interferon Induction and Antiviral Response, IL-8 Signalling and IL-17 Signalling pathways. While we could not determine if the inferred gill inflammation was progressing or resolving, our study clearly suggests that P. parvum blooms may contribute to the serious gill disorders in fish. By providing insights into the gill transcriptomic responses to toxin-producing P. parvum in teleost fish, our research opens new avenues for investigating how to monitor and mitigate toxicity of HABs before they become lethal.

Sampling the fish gill microbiome: a comparison of tissue biopsies and swabs.

BACKGROUND: Understanding the influence of methodology on results is an essential consideration in experimental design. In the expanding field of fish microbiology, many best practices and targeted techniques remain to be refined. This study aimed to compare microbial assemblages obtained from Atlantic salmon (Salmo salar) gills by swabbing versus biopsy excision. Results demonstrate the variation introduced by altered sampling strategies and enhance the available knowledge of the fish gill microbiome. RESULTS: The microbiome was sampled using swabs and biopsies from fish gills, with identical treatment of samples for 16S next generation Illumina sequencing. Results show a clear divergence in microbial communities obtained through the different sampling strategies, with swabbing consistently isolating a more diverse microbial consortia, and suffering less from the technical issue of host DNA contamination associated with biopsy use. Sequencing results from biopsy-derived extractions, however, hint at the potential for more cryptic localisation of some community members. CONCLUSIONS: Overall, results demonstrate a divergence in the obtained microbial community when different sampling methodology is used. Swabbing appears a superior method for sampling the microbiota of mucosal surfaces for broad ecological research in fish, whilst biopsies might be best applied in exploration of communities beyond the reach of swabs, such as sub-surface and intracellular microbes, as well as in pathogen diagnosis. Most studies on the external microbial communities of aquatic organisms utilise swabbing for sample collection, likely due to convenience. Much of the ultrastructure of gill tissue in live fish is, however, potentially inaccessible to swabbing, meaning swabbing might fail to capture the full diversity of gill microbiota. This work therefore also provides valuable insight into partitioning of the gill microbiota, informing varied applications of different sampling methods in experimental design for future research.

Immunologic Profiling of the Atlantic Salmon Gill by Single Nuclei Transcriptomics.

Anadromous salmonids begin life adapted to the freshwater environments of their natal streams before a developmental transition, known as smoltification, transforms them into marine-adapted fish. In the wild, smoltification is a photoperiod-regulated process, involving radical remodeling of gill function to cope with the profound osmotic and immunological challenges of seawater (SW) migration. While prior work has highlighted the role of specialized "mitochondrion-rich" cells (MRCs) and accessory cells (ACs) in delivering this phenotype, recent RNA profiling experiments suggest that remodeling is far more extensive than previously appreciated. Here, we use single-nuclei RNAseq to characterize the extent of cytological changes in the gill of Atlantic salmon during smoltification and SW transfer. We identify 20 distinct cell clusters, including known, but also novel gill cell types. These data allow us to isolate cluster-specific, smoltification-associated changes in gene expression and to describe how the cellular make-up of the gill changes through smoltification. As expected, we noted an increase in the proportion of seawater mitochondrion-rich cells, however, we also identify previously unknown reduction of several immune-related cell types. Overall, our results provide fresh detail of the cellular complexity in the gill and suggest that smoltification triggers unexpected immune reprogramming.