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.

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.

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

[Figure: see text].

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.

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.

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.

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.

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.

Loss of Integrin αvß8 in Murine Hepatocytes Accelerates Liver Regeneration.

Recent fate-mapping studies in mice have provided substantial evidence that mature adult hepatocytes are a major source of new hepatocytes after liver injury. In other systems, integrin αvß8 has a major role in activating transforming growth factor (TGF)-ß, a potent inhibitor of hepatocyte proliferation. We hypothesized that depletion of hepatocyte integrin αvß8 would increase hepatocyte proliferation and accelerate liver regeneration after injury. Using Itgb8flox/flox;Alb-Cre mice to deplete hepatocyte αvß8, after partial hepatectomy, hepatocyte proliferation and liver-to-body weight ratio were significantly increased in Itgb8flox/flox;Alb-Cre mice compared with control mice. Antibody-mediated blockade of hepatocyte αvß8 in vitro, with assessment of TGF-ß signaling pathways by real-time quantitative PCR array, supported the hypothesis that integrin αvß8 inhibition alters hepatocyte TGF-ß signaling toward a pro-regenerative phenotype. A diethylnitrosamine-induced model of hepatocellular carcinoma, used to examine the possibility that this pro-proliferative phenotype might be oncogenic, revealed no difference in either tumor number or size between Itgb8flox/flox;Alb-Cre and control mice. Immunohistochemistry for integrin αvß8 in healthy and injured human liver demonstrated that human hepatocytes express integrin αvß8. Depletion of hepatocyte integrin αvß8 results in increased hepatocyte proliferation and accelerated liver regeneration after partial hepatectomy in mice. These data demonstrate that targeting integrin αvß8 may represent a promising therapeutic strategy to drive liver regeneration in patients with a broad range of liver diseases.

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.

Single-cell transcriptome analyses reveal novel targets modulating cardiac neovascularization by resident endothelial cells following myocardial infarction.

AIMS: A better understanding of the pathways that regulate regeneration of the coronary vasculature is of fundamental importance for the advancement of strategies to treat patients with heart disease. Here, we aimed to investigate the origin and clonal dynamics of endothelial cells (ECs) associated with neovascularization in the adult mouse heart following myocardial infarction (MI). Furthermore, we sought to define murine cardiac endothelial heterogeneity and to characterize the transcriptional profiles of pro-angiogenic resident ECs in the adult mouse heart, at single-cell resolution. METHODS AND RESULTS: An EC-specific multispectral lineage-tracing mouse (Pdgfb-iCreERT2-R26R-Brainbow2.1) was used to demonstrate that structural integrity of adult cardiac endothelium following MI was maintained through clonal proliferation by resident ECs in the infarct border region, without significant contributions from bone marrow cells or endothelial-to-mesenchymal transition. Ten transcriptionally discrete heterogeneous EC states, as well as the pathways through which each endothelial state is likely to enhance neovasculogenesis and tissue regeneration following ischaemic injury were defined. Plasmalemma vesicle-associated protein (Plvap) was selected for further study, which showed an endothelial-specific and increased expression in both the ischaemic mouse and human heart, and played a direct role in regulating human endothelial proliferation in vitro. CONCLUSION: We present a single-cell gene expression atlas of cardiac specific resident ECs, and the transcriptional hierarchy underpinning endogenous vascular repair following MI. These data provide a rich resource that could assist in the development of new therapeutic interventions to augment endogenous myocardial perfusion and enhance regeneration in the injured heart.

MicroRNA annotation of plant genomes - Do it right or not at all.

MicroRNAs are non-coding regulators of gene expression and key factors in development, disease, and targets for bioengineering. Consequently, microRNAs have become essential elements of already burgeoning draft plant genome descriptions where their annotation is often particularly poor, contributing unduly to the corruption of public databases. Using the Citrus sinensis as an example, we highlight and review common failings of miRNAome annotations. Understanding and exploiting the role of miRNAs in plant biology will be stymied unless the research community acts decisively to improve the accuracy of miRNAome annotations. We encourage genome annotation teams to do it right or not at all.

Effects of an unprecedented summer heatwave on the growth performance, flesh colour and plasma biochemistry of marine cage-farmed Atlantic salmon (Salmo salar).

Global seawater temperatures are increasing and becoming more variable, with consequences for all marine animals including those in food production systems. In several countries around the world,arming of Atlantic salmon (Salmo salar) occurs towards the upper end of the thermal tolerance window for this species, and marked effects on salmon production during summers have been experienced but never empirically investigated. This project tracked the effects of an extreme summer heatwave on two different cohorts of fish stocked into farm cages either during early winter (EW) or late winter (LW). The farm site experienced an unprecedented high water temperature event, with a peak water temperature of 22.9 °C and 117 days above 18 °C. Fish in both EW and LW cohorts experienced a temperature-induced cessation of voluntary feed intake as well as inefficient osmoregulatory, liver and renal function during high temperature periods. Flesh colour declined primarily in the dorsal and ventral regions of the fillet and secondarily along the midline, with over 20% of fish demonstrated a complete loss of flesh colour during the months of March and April. A return to feeding in autumn occurred faster in some fish and caused a marked bimodal size distribution to appear within both the EW and LW cohorts as autumn progressed. However, the LW cohort returned to feeding at seawater temperatures of 20.2 °C, compared with 18.6 °C for the EW cohort. There was a strong positive relationship between fillet colour recovery and residual condition index (RCI). These findings identified alkaline phosphatase as a potential marker to non-destructively track individual fish for signs of recovery after a thermal stress event, and shed light on the physiological consequences of marine heatwaves on fishes. This study also identified that supporting feed intake or promoting a return to feeding may help mitigate the negative impacts of climate warming on cultured Atlantic salmon.

Inferring selection in instances of long-range colonization: The Aleppo pine (Pinus halepensis) in the Mediterranean Basin.

Teasing apart the effects of natural selection and demography on current allele frequencies is challenging, due to both processes leaving a similar molecular footprint. In particular, when attempting to identify selection in species that have undergone a recent range expansion, the increase in genetic drift at the edges of range expansions ("allele surfing") can be a confounding factor. To address this potential issue, we first assess the long-range colonization history of the Aleppo pine across the Mediterranean Basin, using molecular markers. We then look for single nucleotide polymorphisms (SNPs) involved in local adaptation using: (a) environmental correlation methods (bayenv2), focusing on bioclimatic variables important for the species' adaptation (i.e., temperature, precipitation and water availability); and (b) FST -related methods (pcadapt). To assess the rate of false positives caused by the allele surfing effect, these results are compared with results from simulated SNP data that mimics the species' past range expansions and the effect of genetic drift, but with no selection. We find that the Aleppo pine shows a previously unsuspected complex genetic structure across its range, as well as evidence of selection acting on SNPs involved with the response to bioclimatic variables such as drought. This study uses an original approach to disentangle the confounding effects of drift and selection in range margin populations. It also contributes to the increased evidence that plant populations are able to adapt to new environments despite the expected accumulation of deleterious mutations that takes place during long-range colonizations.

microRNAs and the evolution of complex multicellularity: identification of a large, diverse complement of microRNAs in the brown alga Ectocarpus.

There is currently convincing evidence that microRNAs have evolved independently in at least six different eukaryotic lineages: animals, land plants, chlorophyte green algae, demosponges, slime molds and brown algae. MicroRNAs from different lineages are not homologous but some structural features are strongly conserved across the eukaryotic tree allowing the application of stringent criteria to identify novel microRNA loci. A large set of 63 microRNA families was identified in the brown alga Ectocarpus based on mapping of RNA-seq data and nine microRNAs were confirmed by northern blotting. The Ectocarpus microRNAs are highly diverse at the sequence level with few multi-gene families, and do not tend to occur in clusters but exhibit some highly conserved structural features such as the presence of a uracil at the first residue. No homologues of Ectocarpus microRNAs were found in other stramenopile genomes indicating that they emerged late in stramenopile evolution and are perhaps specific to the brown algae. The large number of microRNA loci in Ectocarpus is consistent with the developmental complexity of many brown algal species and supports a proposed link between the emergence and expansion of microRNA regulatory systems and the evolution of complex multicellularity.

Atlantic Salmon (Salmo salar L.) Gastrointestinal Microbial Community Dynamics in Relation to Digesta Properties and Diet.

To better understand salmon GI tract microbial community dynamics in relation to diet, a feeding trial was performed utilising diets with different proportions of fish meal, protein, lipid and energy levels. Salmon gut dysfunction has been associated with the occurrence of casts, or an empty hind gut. A categorical scoring system describing expressed digesta consistency was evaluated in relation to GI tract community structure. Faster growing fish generally had lower faecal scores while the diet cohorts showed minor differences in faecal score though the overall lowest scores were observed with a low protein, low energy diet. The GI tract bacterial communities were highly dynamic over time with the low protein, low energy diet associated with the most divergent community structure. This included transiently increased abundance of anaerobic (Bacteroidia and Clostridia) during January and February, and facultatively anaerobic (lactic acid bacteria) taxa from February onwards. The digesta had enriched populations of these groups in relation to faecal cast samples. The majority of samples (60-86 %) across all diet cohorts were eventually dominated by the genus Aliivibrio. The results suggest that an interaction between time of sampling and diet is most strongly related to community structure. Digesta categorization revealed microbes involved with metabolism of diet components change progressively over time and could be a useful system to assess feeding responses.