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.

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.

Probabilistic reaction norm reveals family-related variation in the association between size, condition, and sexual maturation onset in Atlantic salmon (Salmo salar).

This study investigated the relationship between the size, condition, year class, family, and sexual maturity of Atlantic salmon (Salmo salar) using data collected in an aquaculture selective breeding programme. Males that were sexually mature at 2 years of age (maiden spawn) have, on average, greater fork length and condition factor (K) at 1 year of age than their immature counterparts. For every 10-mm increase in fork length or 0.1 increase in K at 1 year of age, the odds of sexual maturity at 2 years of age increased by 1.48 or 1.22 times, respectively. Females that were sexually mature at 3 years of age (maiden spawn) have, on average, greater fork length and K at 2 years of age than their immature counterparts. For every 10-mm increase in fork length or 0.1 increase in K at 2 years of age, the odds of sexual maturity at 3 years of age increased by 1.06 or 1.44 times, respectively. The family explained 34.93% of the variation in sexual maturity among 2-year-old males that was not attributable to the average effects of fork length and K at 1 year of age and year class. The proportion of variation in sexual maturity among 3-year-old females explained by the family could not be investigated. These findings suggest that the onset of sexual maturation in Atlantic salmon is conditional on performance (with respect to energy availability) surpassing a threshold, the magnitude of which can vary between families and is determined by a genetic component. This could support the application of genetic selection to promote or inhibit the onset of sexual maturation in farmed stocks.

A versatile nuclei extraction protocol for single nucleus sequencing in non-model species-Optimization in various Atlantic salmon tissues.

The use of single cell sequencing technologies has exploded over recent years, and is now commonly used in many non-model species. Sequencing nuclei instead of whole cells has become increasingly popular, as it does not require the processing of samples immediately after collection. Here we present a highly effective nucleus isolation protocol that outperforms previously available method in challenging samples in a non-model specie. This protocol can be successfully applied to extract nuclei from a variety of tissues and species.

Organized B cell sites in cartilaginous fishes reveal the evolutionary foundation of germinal centers.

The absence of germinal centers (GCs) in cartilaginous fishes lies at odds with data showing that nurse sharks can produce robust antigen-specific responses and affinity mature their B cell repertoires. To investigate this apparent incongruity, we performed RNA sequencing on single nuclei, allowing us to characterize the cell types present in the nurse shark spleen, and RNAscope to provide in situ cellular resolution of key marker gene expression following immunization with R-phycoerythrin (PE). We tracked PE to the splenic follicles where it co-localizes with CXCR5high centrocyte-like B cells and a population of putative T follicular helper (Tfh) cells, surrounded by a peripheral ring of Ki67+ AID+ CXCR4+ centroblast-like B cells. Further, we reveal selection of mutations in B cell clones dissected from these follicles. We propose that the B cell sites identified here represent the evolutionary foundation of GCs, dating back to the jawed vertebrate ancestor.

Human and murine fibroblast single-cell transcriptomics reveals fibroblast clusters are differentially affected by ageing and serum cholesterol.

AIMS: Specific fibroblast markers and in-depth heterogeneity analysis are currently lacking, hindering functional studies in cardiovascular diseases (CVDs). Here, we established cell-type markers and heterogeneity in murine and human arteries and studied the adventitial fibroblast response to CVD and its risk factors hypercholesterolaemia and ageing. METHODS AND RESULTS: Murine aorta single-cell RNA-sequencing analysis of adventitial mesenchymal cells identified fibroblast-specific markers. Immunohistochemistry and flow cytometry validated platelet-derived growth factor receptor alpha (PDGFRA) and dipeptidase 1 (DPEP1) across human and murine aorta, carotid, and femoral arteries, whereas traditional markers such as the cluster of differentiation (CD)90 and vimentin also marked transgelin+ vascular smooth muscle cells. Next, pseudotime analysis showed multiple fibroblast clusters differentiating along trajectories. Three trajectories, marked by CD55 (Cd55+), Cxcl chemokine 14 (Cxcl14+), and lysyl oxidase (Lox+), were reproduced in an independent RNA-seq dataset. Gene ontology (GO) analysis showed divergent functional profiles of the three trajectories, related to vascular development, antigen presentation, and/or collagen fibril organization, respectively. Trajectory-specific genes included significantly more genes with known genome-wide associations (GWAS) to CVD than expected by chance, implying a role in CVD. Indeed, differential regulation of fibroblast clusters by CVD risk factors was shown in the adventitia of aged C57BL/6J mice, and mildly hypercholesterolaemic LDLR KO mice on chow by flow cytometry. The expansion of collagen-related CXCL14+ and LOX+ fibroblasts in aged and hypercholesterolaemic aortic adventitia, respectively, coincided with increased adventitial collagen. Immunohistochemistry, bulk, and single-cell transcriptomics of human carotid and aorta specimens emphasized translational value as CD55+, CXCL14+ and LOX+ fibroblasts were observed in healthy and atherosclerotic specimens. Also, trajectory-specific gene sets are differentially correlated with human atherosclerotic plaque traits. CONCLUSION: We provide two adventitial fibroblast-specific markers, PDGFRA and DPEP1, and demonstrate fibroblast heterogeneity in health and CVD in humans and mice. Biological relevance is evident from the regulation of fibroblast clusters by age and hypercholesterolaemia in vivo, associations with human atherosclerotic plaque traits, and enrichment of genes with a GWAS for CVD.

Association between melanin deposits in gill tissue and microbiome across different hatchery reared Atlantic salmon.

AIMS: To investigate the relationship between microbial community profiles and gill pathology during a production cycle of Atlantic salmon in two commercial hatcheries. METHODS AND RESULTS: Relationships between gill histology, environmental conditions, and microbiome were determined using high-throughput data, including 16S rDNA amplicon sequencing data, histopathology data, and water quality parameters. Hatchery A used riverine water and operated a mixed system of recirculation aquaculture system (RAS) and flowthrough. Hatchery B was used bore water and operated a RAS. Melanin deposits, hyperplastic, and inflammatory lesions were observed histologically in the gills. A higher prevalence of melanin deposits was detected and correlated to a change in beta diversity of bacterial communities in early time points (fingerling and parr stages). High abundance of Sphaerotilus sp.,Pseudomonas sp.,Nitrospira sp.,Exiguobacterium sp.,Deinococcus sp.,and Comamonas sp. was correlated with a high prevalence of melanin in filaments. Bacterial diversity increased as the fish cohort transitioned from RAS to flowthrough in hatchery A. CONCLUSIONS: Under commercial conditions, the commensal community of gill bacteria was related to melanin prevalence.

Single cell genomics as a transformative approach for aquaculture research and innovation.

Single cell genomics encompasses a suite of rapidly maturing technologies that measure the molecular profiles of individual cells within target samples. These approaches provide a large up-step in biological information compared to long-established 'bulk' methods that profile the average molecular profiles of all cells in a sample, and have led to transformative advances in understanding of cellular biology, particularly in humans and model organisms. The application of single cell genomics is fast expanding to non-model taxa, including aquaculture species, where numerous research applications are underway with many more envisaged. In this review, we highlight the potential transformative applications of single cell genomics in aquaculture research, considering barriers and potential solutions to the broad uptake of these technologies. Focusing on single cell transcriptomics, we outline considerations for experimental design, including the essential requirement to obtain high quality cells/nuclei for sequencing in ectothermic aquatic species. We further outline data analysis and bioinformatics considerations, tailored to studies with the under-characterized genomes of aquaculture species, where our knowledge of cellular heterogeneity and cell marker genes is immature. Overall, this review offers a useful source of knowledge for researchers aiming to apply single cell genomics to address biological challenges faced by the global aquaculture sector though an improved understanding of cell biology.

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.

Prevalence of epitheliocystis in freshwater Atlantic salmon reared in flow-through and recirculation aquaculture systems.

Epitheliocystis, an intracellular bacterial infection in the gills and skin epithelium, has been frequently reported in Atlantic salmon (Salmo salar) during freshwater production in a number of countries. This study describes the prevalence and intensity of a natural epitheliocystis infection present in the gills of two strains of Atlantic salmon reared in either a flow-through (FT) or a recirculation aquaculture system (RAS) in Ireland. Repeated sampling of gills prior to and throughout seawater transfer, histology and quantitative real-time PCR were used to determine infection prevalence and intensity. Despite no clinical gill disease, and minor histopathological changes, epitheliocystis lesions were identified in histology at all time points. Specific PCR confirmed the presence of Candidatus Clavichlamydia salmonicola in both strains and its number of copies was correlated with intensity of epitheliocystis lesions. A significant interaction between hatchery system and fish strain on the prevalence and intensity of gill epitheliocystis was found both using histological and molecular methods. Specifically, fish from FT had higher prevalence and intensity than RAS reared fish and within FT, the Irish cohort were more affected than Icelandic.

Evaluation of the Infectious Potential of Neoparamoeba perurans Following Freshwater Bathing Treatments.

Freshwater bathing for 2-3 h is the main treatment to control amoebic gill disease of marine-farmed Atlantic salmon. Recent in vitro studies have demonstrated that amoebae (Neoparamoeba perurans) detach when exposed to freshwater and that some eventually reattach to culture plates when returned to seawater. Here, we evaluated the potential for gill-detached N. perurans to survive a commercially relevant treatment and infect AGD-naïve fish and whether holding used bathwater for up to 6 h post treatment would lower infectivity. AGD-affected fish were bathed in freshwater for 2 h. Naïve salmon were exposed to aliquots of the used bathwater after 2, 4, 6 and 8 h. The inoculation was performed at 30 ppt for 2 h, followed by gradual dilution with seawater. Sampling at 20 days post inoculation (dpi) and 40 dpi confirmed rapid AGD development in fish inoculated in 2 h used bathwater, but a slower AGD development following exposure to 4 h bathwater. AGD signs were variable and reduced following longer bathwater holding times. These results suggest that viable amoebae are likely returned to seawater following commercial freshwater treatments, but that the risk of infection can be reduced by retention of bathwater before release.

MIR503HG Loss Promotes Endothelial-to-Mesenchymal Transition in Vascular Disease.

[Figure: see text].

Seawater transmission and infection dynamics of pilchard orthomyxovirus (POMV) in Atlantic salmon (Salmo salar).

The Tasmanian salmon industry had remained relatively free of major viral diseases until the emergence of pilchard orthomyxovirus (POMV). Originally isolated from wild pilchards, POMV is of concern to the industry as it can cause high mortality in farmed salmon (Salmo salar). Field observations suggest the virus can spread from pen to pen and between farms, but evidence of passive transmission in sea water was unclear. Our aim was to establish whether direct contact between infected and naïve fish was required for transmission, and to examine viral infection dynamics. Atlantic salmon post-smolts were challenged with POMV by either direct exposure via cohabitation or indirect exposure via virus-contaminated sea water. POMV was transmissible in sea water and direct contact between fish was not required for infection. Head kidney and heart presented the highest viral loads in early stages of infection. POMV survivors presented low viral loads in most tissues, but these remained relatively high in gills. A consistent feature was the infiltration of viral-infected melanomacrophages in different tissues, suggesting an important role of these in the immune response to POMV. Understanding POMV transmission and host-pathogen interactions is key for the development of improved surveillance tools, transmission models and ultimately for disease prevention.

Palivizumab Prophylaxis for infants 29 to 32 weeks gestation at birth: A 10-year audit from Vancouver Island using BC Guidelines.

Background: After initially recommending palivizumab (PVZ), a monoclonal antibody against respiratory syncytial virus (RSV) for all infants 29 to 32 weeks at birth if <6 months age at season start, the American Academy of Pediatrics (AAP) and Canadian Paediatric Society (CPS) guidelines were revised. British Columbia was the only jurisdiction in North America to restrict eligibility for this group to those with additional risk factors, long before the change in national recommendations. Objectives: To determine the risk for first season RSV admission for 29 to 32-week gestational age (GA) infants admitted to Victoria Neonatal Intensive Care Unit (NICU) that either received or were denied PVZ prophylaxis. Methods: Descriptive cohort study of infants eligible for prophylaxis according to earlier CPS guidelines. Instead, BC guidelines for prophylaxis were applied and data for Vancouver Island infants were collected over 10 consecutive RSV seasons. Results: We followed 423 infants. Three hundred and thirty-six (79%) did not receive prophylaxis, of which 10 (3.0%; 95% confidence interval [CI] 1.4% to 5.4%) had an RSV hospitalization before the end of April during their first RSV season versus 3 admissions from 87 (3.5%; 95% CI 0.7% to 10%) infants who received prophylaxis. Conclusions: Our risk factor approach to RSV prophylaxis for infants born at 29 to 32 weeks GA resulted in a low (average incidence=3.1%) rate of RSV hospitalization. Our approach would offer considerable cost savings to RSV prophylaxis programs that continue to offer routine prophylaxis beyond 28/29 weeks GA at birth.

Transcriptome Response of Atlantic Salmon (Salmo salar) to a New Piscine Orthomyxovirus.

Pilchard orthomyxovirus (POMV) is an emerging pathogen of concern to the salmon industry in Australia. To explore the molecular events that underpin POMV infection, we challenged Atlantic salmon (Salmo salar) post-smolts in seawater via cohabitation. Tissue samples of the head kidney and liver were collected from moribund and surviving individuals and analyzed using transcriptome sequencing. Viral loads were higher in the head kidney compared to the liver, yet the liver presented more upregulated genes. Fish infected with POMV showed a strong innate immune response that included the upregulation of pathogen recognition receptors such as RIG-I and Toll-like receptors as well as the induction of interferon-stimulated genes (MX, ISG15). Moribund fish also presented a dramatic induction of pro-inflammatory cytokines, contributing to severe tissue damage and morbidity. An induction of major histocompatibility complex (MHC) class I genes (B2M) and markers of T cell-mediated immunity (CD8-alpha, CD8-beta, Perforin-1, Granzyme-A) was observed in both moribund fish and survivors. In addition, differential connectivity analysis showed that three key regulators (RELA/p65, PRDM1, and HLF) related to cell-mediated immunity had significant differences in connectivity in "clinically healthy" versus "clinically affected" or moribund fish. Collectively, our results show that T cell-mediated immunity plays a central role in the response of Atlantic salmon to the infection with POMV.

Transcriptional dynamics of pluripotent stem cell-derived endothelial cell differentiation revealed by single-cell RNA sequencing.

AIMS: Pluripotent stem cell-derived endothelial cell products possess therapeutic potential in ischaemic vascular disease. However, the factors that drive endothelial differentiation from pluripotency and cellular specification are largely unknown. The aims of this study were to use single-cell RNA sequencing (scRNA-seq) to map the transcriptional landscape and cellular dynamics of directed differentiation of human embryonic stem cell-derived endothelial cells (hESC-EC) and to compare these cells to mature endothelial cells from diverse vascular beds. METHODS AND RESULTS: A highly efficient directed 8-day differentiation protocol was used to generate a hESC-derived endothelial cell product (hESC-ECP), in which 66% of cells co-expressed CD31 and CD144. We observed largely homogeneous hESC and mesodermal populations at Days 0 and 4, respectively, followed by a rapid emergence of distinct endothelial and mesenchymal populations. Pseudotime trajectory identified transcriptional signatures of endothelial commitment and maturation during the differentiation process. Concordance in transcriptional signatures was verified by scRNA-seq analysis using both a second hESC line RC11, and an alternative hESC-EC differentiation protocol. In total, 105 727 cells were subjected to scRNA-seq analysis. Global transcriptional comparison revealed a transcriptional architecture of hESC-EC that differs from freshly isolated and cultured human endothelial cells and from organ-specific endothelial cells. CONCLUSION: A transcriptional bifurcation into endothelial and mesenchymal lineages was identified, as well as novel transcriptional signatures underpinning commitment and maturation. The transcriptional architecture of hESC-ECP was distinct from mature and foetal human EC.

The interaction between temperature and dose on the efficacy and biochemical response of Atlantic salmon to hydrogen peroxide treatment for amoebic gill disease.

Hydrogen peroxide (H2 O2 ) is a commonly used treatment for a range of parasitic diseases of marine finfish, including amoebic gill disease (AGD). While this treatment is partially effective at reducing parasite load, H2 O2 can have detrimental effects on the host under certain conditions. Treatment temperature and dose concentration are two factors that are known to influence the toxicity of H2 O2 ; however, their impact on the outcome of AGD treatment remains unclear. Here, we investigated the effects of treatment temperature (8, 12 or 16°C) and dose concentration (750, 1,000, 1,250 mg/L) on the efficacy of H2 O2 to treat AGD. We demonstrated that a 20-min bath treatment of H2 O2 at all doses reduced both parasite load and gross gill score significantly. Parasite load and gross gill score were lowest in the 1,000 mg/L treatment performed at 12°C. At the high dose and temperature combinations, H2 O2 caused moderate gill damage and a significant increase in the plasma concentration of electrolytes (sodium, chloride and potassium). Taken together, our study demonstrates that higher H2 O2 treatment temperatures can adversely affect the host and do not improve the effectiveness of the treatment.