Daily crowding stress has limited, yet detectable effects on skin and head kidney gene expression in surgically tagged atlantic salmon (Salmo salar).

To ensure welfare-friendly and effective internal tagging, the tagging process should not cause a long-term burden on individuals given that tagged fish serve as representatives for the entire population in telemetry applications. To some extent, stress is inevitable within regular aquaculture practices, and thus, the consequences of long-term stress should be described in terms of their effects on internal tagging. In fish, stressors activate the Hypothalamus-Pituitary-Interrenal (HPI) and Brain-Sympathetic-Chromaffin Cell (BSC) axes, leading to neuroimmunoendocrine communication and paracrine interactions among stress hormones. The interrelation between wound healing and stress is complex, owing to their shared components, pathways, and energy demands. This study assessed 14 genes (mmp9, mmp13, il-2, il-4, il-8a, il-10, il-12, il-17d, il-1b, tnfa, ifng, leg-3, igm, and crh) in the skin (1.5 cm from the wound) and head kidney over eight weeks. These genes, associated with cell signaling in immunity, wound healing, and stress, have previously been identified as influenced and regulated by these processes. Half of a group of Atlantic salmon (n = 90) with surgically implanted dummy smart-tags were exposed to daily crowding stress. The goal was to investigate how this gene panel responds to a wound alone and then to the combined effects of wounding and daily crowding stress. Our observations indicate that chronic stress impacts inflammation and impedes wound healing, as seen through the expression of matrix metalloproteinases genes in the skin but not in the head kidney. This difference is likely due to the ongoing internal wound repair, in contrast to the externally healed wound incision. Cytokine expression, when significant in the skin, was mainly downregulated in both treatments compared to control values, particularly in the study's first half. Conversely, the head kidney showed initial cytokine downregulation followed by upregulation. Across all weeks observed and combining both tissues, the significantly expressed gene differences were 12 % between the Wound and Stress+ groups, 28 % between Wound and Control, and 25 % between Stress+ and Control. Despite significant fluctuations in cytokines, sustained variations across multiple weeks are only evident in a few select genes. Furthermore, Stress+ individuals demonstrated the most cytokine correlations within the head kidney, which may suggest that chronic stress affects cytokine expression. This investigation unveils that the presence of stress and prolonged activation of the HPI axis in an eight weeklong study has limited yet detectable effects on the selected gene expression within immunity, wound healing, and stress, with notable tissue-specific differences.

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.

Distribution and response strategies of non-specific cytotoxic cell receptor protein 1 in large yellow croaker.

The non-specific cytotoxic cell receptor protein 1 (NCCRP1) is considered the universal marker for teleost non-specific cytotoxic cells (NCCs). However, the specific distribution characteristics and response patterns of NCCRP1, and the confirmed existence of NCCs in fish species remain debatable. In this study, we investigated the distribution of NCCRP1 in the croaker and observed the most dominant abundance in the head kidney. While most common markers of cytotoxicity were localized in the trunk kidney lymphocytes (TKLs) and spleen lymphocytes (SPLs), NCCRP1-positive cells were predominantly detected in head kidney lymphocytes (HKLs) with a positive rate of approximately 10 %, where present a huge amount of macrophages (Mϕ) as well. Furthermore, the remarkable induction evidence of NCCRP1 in HKLs was determined. Collectively, these findings contribute significantly to comprehending the immunological function of NCCRP1 in fish species and enhancing our understanding of its evolutionary development.

Identification and functional analyses of CD4-1+ cells in grass carp (Ctenopharyngodon idella).

In mammals, CD4 is found to be expressed on T cells and innate immune cells, however, teleost cells bearing CD4 have not been well identified and characterized. In this study, we identified two different CD4-1+ cell subsets in grass carp (Ctenopharyngodon idella): CD4-1+ lymphocytes (Lym) and CD4-1+ myeloid cells (Mye), both of which had the highest proportions in the head kidney. The mRNA expression analysis showed that CD4-1, CD4-2, TCRß, CD3γ/δ, and LCK1 are highly expressed in CD4-1+ Lym and also expressed in CD4-1+ Mye. Furthermore, we found that CD4-1+ Lym have a Lym morphology and highly express T-cell cytokines, suggesting that they are CD4+ T cells equivalent to mammalian Th cells. On the other hand, CD4-1+ Mye were found to have a morphology of macrophage and highly express macrophage marker gene MCSFR, indicating that they are macrophages. In addition, functional analysis revealed that CD4-1+ Mye possess phagocytic ability and great antigen-processing ability. Taken together, our study sheds further light on the composition and function of CD4+ cells in teleost fish.

Involvement of mitochondrial fatty acid ß-oxidation in the antiviral innate immune response in head kidney macrophages of large yellow croaker (Larimichthys crocea).

As a vital pathway for cellular energy production, mitochondrial fatty acid ß-oxidation (FAO) is essential in regulating immune responses to bacterial pathogens and maintaining intracellular homeostasis in vertebrates. However, the specific role of FAO in antiviral innate immune response in macrophages remains insufficiently understood. In this study, virus infection simulated by poly(I:C) inhibited FAO, as indicated by the reduced expression of FAO-related genes and proteins in the head kidney of large yellow croaker, with similar results observed in poly(I:C)-stimulated macrophages. Then, inhibition of FAO by supplementary mildronate in vivo and etomoxir treatment in vitro revealed varying increases in the mRNA expression of antiviral innate immune response genes after stimulated by poly(I:C) in the head kidney and macrophages. Notably, etomoxir significantly facilitated the transcriptional up-regulation of the IFNh promoter by IRF3. Moreover, inhibiting FAO by knockdown of cpt1b promoted antiviral innate immune response triggered by poly(I:C) in macrophages. Conversely, activating FAO through overexpression of cpt1b or cpt2 significantly reduced the mRNA levels of antiviral response genes in macrophages stimulated by poly(I:C). Unlike etomoxir, cpt1b overexpression inhibited the transcriptional up-regulation of the IFNh promoter by IRF3. Furthermore, in vivo dietary palm oil feeding and in vitro exposure to palmitic acid inhibited the antiviral innate immune response triggered by poly(I:C) in the head kidney and macrophages, respectively. These effects were partly associated with FAO activation, as evidenced by etomoxir. In summary, this study elucidates FAO's critical role in regulating antiviral innate immune response in head kidney macrophages. These findings not only deepen insights into the interaction between metabolic remodeling and host immune responses, but also offer valuable guidance for developing nutritional strategies to improve antiviral immunity in aquaculture.

Extracellular ATP- and adenosine-mediated purinergic signaling modulates inducible nitric oxide synthase (iNOS) gene expression, enzyme activity and nitric oxide production in common carp (Cyprinus carpio) head kidney macrophages.

Inducible nitric oxide (NO) synthase (iNOS) is a key immune mediator for production of inflammatory mediator NO from l-arginine. Tight regulation of iNOS expression and enzyme activity is critical for proper NO productions under inflammation and infection conditions. However, the regulatory mechanism for iNOS expression and enzyme activity in fish remains largely unknown. Here, we show that extracellular ATP treatment significantly up-regulates iNOS gene expression and enzyme activity, and consequently leads to enhanced NO production in Cyprinus carpio head kidney macrophages (HKMs). We further show that the extracellular ATP-induced iNOS enzyme activity and NO production can be attenuated by pharmacological inhibition of the ATP-gated P2X4 and P2X7 receptors with their respective specific antagonists, but enhanced by overexpression of P2X4 and P2X7 receptors in grass carp ovary cells. In contrast, adenosine administration significantly reduces iNOS gene expression, enzyme activity and NO production in carp HKMs, and these inhibitory effects can be reversed by pharmacological inhibition of adenosine receptors with the antagonist XAC. Furthermore, LPS- and poly(I:C)-induced iNOS gene expression, enzyme activity, and NO production are significantly attenuated by blockade of P2X4 and P2X7 receptors with their respective specific antagonists in carp HKMs, while overexpression of P2X and P2X7 receptors results in enhanced iNOS gene expression, enzyme activity and NO production in LPS- and poly(I:C)-treated grass carp ovary cells. Taken together, we firstly report an opposite role of extracellular ATP/adenosine-mediated purinergic signaling in modulating iNOS-NO system activity in fish.

Characterization of the impact of dietary immunostimulant CpG on the expression of mRNA biomarkers involved in the immune responses in Atlantic salmon (Salmo salar).

Infectious diseases have significantly impacted Atlantic salmon aquaculture worldwide. Modulating fish immunity with immunostimulant-containing functional feeds could be an effective strategy in mitigating disease problems. Previously, we characterized the impact of polyriboinosinic polyribocytidylic acid (pIC) and formalin-killed typical Aeromonas salmonicida bacterin on miRNA expression in Atlantic salmon fed a commercial diet with and without immunostimulant CpG. A set of miRNA biomarkers of Atlantic salmon head kidney responding to pIC and/or bacterin immune stimulations was identified (Xue et al., 2019) [1]. Herein, we report a complementary qPCR study that investigated the impact of the pIC, bacterin and dietary CpG on the expression of immune-relevant mRNAs (n = 31) using the same samples as in the previous study (Xue et al., 2019) [1]. Twenty-six of these genes were predicted target transcripts of the pIC- and/or bacterin-responsive miRNAs identified in the earlier study. The current data showed that pIC and/or bacterin stimulations significantly modulated the majority of the qPCR-analyzed genes involved in various immune pathways. Some genes responded to both stimulations (e.g. tnfa, il10rb, ifng, irf9, cxcr3, campb) while others appeared to be stimulation specific [e.g. irf3, irf7a, il1r1, mxa, mapk3 (pIC only); clra (bacterin only)]. A. salmonicida bacterin stimulation produced a strong inflammatory response (e.g. higher expression of il1b, il8a and tnfa), while salmon stimulated with pIC showed robust interferon responses (both type I and II). Furthermore, the current data indicated significant down-regulation of immune-relevant transcripts (e.g. tlr9, irf5, il1r1, hsp90ab1, itgb2) by dietary immunostimulant CpG, especially among pre-injection and PBS-injected fish. Together with our prior miRNA study, the present research provided complementary information on Atlantic salmon anti-viral and anti-bacterial immune responses and on how dietary CpG may modulate these responses.

Tenacibaculum dicentrarchi produce outer membrane vesicles (OMV) that are associated with the cytotoxic effect in rainbow trout head kidney macrophages.

Tenacibaculum dicentrarchi is the second most important pathogen in Chilean salmon farming. This microorganism causes severe skin lesions on the body surface of farmed fish. The bacterium can also adhere to surfaces and form biofilm, survive in fish skin mucus, and possess different systems for iron acquisition. However, the virulence mechanisms are still not fully elucidated. Outer membrane vesicles (OMV) are nanostructures released by pathogenic Gram-negative bacteria during growth, but none has been described yet for T. dicentrarchi. In this study, we provide the first reported evidence of the fish pathogen T. dicentrarchi producing and releasing OMV from 24 h after incubation, increasing thereafter until 120 h. Analyses were conducted with T. dicentrarchi TdCh05, QCR29, and the type strain CECT 7612T . The OMV sizes, determined via scanning electron microscopy, ranged from 82.25 nm to 396.88 nm as per the strain and incubation time point (i.e., 24 to 120 h). SDS-PAGE revealed that the number of protein bands evidenced a drastically downward trend among the T. dicentrarchi strains. In turn, the OMV shared five proteins (i.e., 22.2, 31.9, 47.7, 56.3, and 107.1 kDa), but no protein pattern was identical. A heterogeneous amount of protein, RNA, and DNA were obtained, depending on the time at which OMV were extracted. Purified OMV were biologically active and induced a cytotoxic effect in macrophage-enriched cell cultures from rainbow trout (Oncorhynchus mykiss) head kidneys. This is the first step towards understanding the role that OMV could play in the pathogenesis of T. dicentrarchi.

Evaluation of stress in farmed Atlantic salmon (Salmo salar) using different biological matrices.

Atlantic salmon were subjected to an acute crowding scenario, and their subsequent stress responses were observed under three distinct swimming speed/water flow (WF) conditions: 0.5, 1, and 1.5 body lengths per second (BL/s). Feces, dermal mucus, and plasma were collected for analysis at 1, 6, and 24 h (h) post-stress. Additionally, the head kidney and two regions of the brain (pituitary and POA) were collected for transcript expression analysis. Fish swimming at 0.5 BL/s exhibited higher pre-stress (baseline) cortisol levels. Across all groups and matrices, the highest cortisol/cortisol metabolites (CM) levels were observed at the 1 h post-stress sampling point. At 6 h (second sampling time point), a clear decline toward baseline levels were observe in all groups. Significant increases in mean plasma glucose levels were observed at 1 h post-stress for all groups. The mean plasma lactate levels varied based on WF treatments, with a significant increase observed at 1 h only for the 1.5 BL/s group. Additionally, significant decreases in mean plasma lactate were noted at 6 and 24 h post-stress for some groups. The mRNA abundances of the tested genes (star, cyp17a1, hsd11ß2, srd5a1) increased following the stress events. These changes were not uniform across all groups and were tissue dependent. In summary, the results indicate that mucus and feces can be used as potentially less invasive matrices than blood for evaluating stress and, consequently, the welfare of Atlantic salmon in captivity.

Alternative splicing in Atlantic salmon head kidney and SHK-1 cell line during the Piscirickettsia salmonis infection: A comparative transcriptome survey.

Piscirickettsia salmonis, an intracellular bacterium in salmon aquaculture, is a big challenge because it is responsible for 54.2% of Atlantic salmon mortalities. In recent years, the high relevance of Alternative Splicing (AS) as a molecular mechanism associated with infectious conditions and host-pathogen interaction processes, especially in host immune activation, has been observed. Several studies have highlighted the role of AS in the host's immune response during viral, bacterial, and endoparasite infection. In the present study, we evaluated AS transcriptome profiles during P. salmonis infection in the two most used study models, SHK-1 cell line and salmon head kidney tissue. First, the SHK-1 cell line was exposed to P. salmonis infection at 0-, 7-, and 14-days post-infection (dpi). Following, total RNA was extracted for Illumina sequencing. On the other hand, RNA-Seq datasets of Atlantic salmon head kidney infected with the same P. salmonis strayingwase used. For both study models, the highest number of differentially alternative splicing (DAS) events was observed at 7 dpi, 16,830 DAS events derived from 9213 DAS genes in SHK-1 cells, and 13,820 DAS events from 7684 DAS genes in salmon HK. Alternative first exon (AF) was the most abundant AS type in the three infection times analyzed, representing 31% in SHK-1 cells and 228.6 in salmon HK; meanwhile, mutually exclusive exon (MX) was the least abundant. Notably, functional annotation of DAS genes in SHK-1 cells infected with P. salmonis showed a high presence of genes related to nucleotide metabolism. In contrast, the salmon head kidney exhibited many GO terms associated with immune response. Our findings reported the role of AS during P. salmonis infection in Atlantic salmon. These studies would contribute to a better understanding of the molecular bases that support the pathogen-host interaction, evidencing the contribution of AS regulating the transcriptional host response.

Involvement of CD27 in innate and adaptive immunities of Nile tilapia (Oreochromis niloticus).

CD27 is a member of the TNF-receptor superfamily and plays various roles in immunities. However, the detailed information and mechanism of CD27 in bony fish immunity remain unclear. Therefore, in this research, certain interesting roles of CD27 in Nile tilapia (On-CD27) were determined. On-CD27 was largely expressed in the immune organs, head kidney, and spleen, and was sharply induced during bacterial infection. The in vitro tests suggested On-CD27 was involved in regulating inflammatory responses, activating immune-related signal pathways, and inducing apoptosis and pyroptosis progress. The scRNA data and in vivo experiments indicated that On-CD27 is mainly expressed in CD4+ T cells and involved in both innate and adaptive immunities. The present data provide a theoretical principle for further research on the mechanisms of CD27 in the innate and adaptive immunities of fish.

Integrative mRNA-miRNA interaction analysis associated with the immune response in the head kidney of rainbow trout (Oncorhynchus mykiss) after infectious hematopoietic necrosis virus infection.

Rainbow trout (Oncorhynchus mykiss) is an important cold-water fish widely cultivated in China. The frequent occurrence of viral diseases caused by infectious hematopoietic necrosis virus (IHNV) seriously restricted the healthy development of the rainbow trout farming industry. However, the immune defense mechanism induced by IHNV in rainbow trout has not been fully elucidated. In the present study, we detected mRNA and miRNA expression profiles in rainbow trout head kidney after IHNV infection using RNA-seq and identified key immune-related genes and miRNAs. The results showed that a total of 7486 genes and 277 miRNAs were differentially expressed, and numerous differentially expressed genes (DEGs) enriched in the immune-related pathways such as Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, NOD-like receptor signaling pathway, cytokine-cytokine receptor interaction, and JAK-STAT signaling pathway were significantly up-regulated, including LGP2, MDA5, TRIM25, IRF3, IRF7, TLR3, TLR7, TLR8, MYD88, and IFN1. Integration analysis identified six miRNAs (miR-141-y, miR-200-y, miR-144-y, miR-2188-y, miR-725-y, and miR-203-y) that target at least six key immune-related genes (TRIM25, LGP2, TLR3, TLR7, IRF3, and IRF7). Further, we verified selected immune-related mRNAs and miRNAs through qRT-PCR and confirmed the reliability of the RNA-seq results. These findings improve our understanding of the immune mechanism of rainbow trout infected with IHNV and provide basic data for future breeding for disease resistance in rainbow trout.

Butyrate induces STAT3/HIF-1α/IL-22 signaling via GPCR and HDAC3 inhibition to activate autophagy in head kidney macrophages from turbot (Scophthalmus maximus L.).

As one of short-chain fatty acids, butyrate is an important metabolite of dietary fiber by the fermentation of gut commensals. Our recent study uncovered that butyrate promoted IL-22 production in fish macrophages to augment the host defense. In the current study, we further explored the underlying signaling pathways in butyrate-induced IL-22 production in fish macrophages. Our results showed that butyrate augmented the IL-22 expression in head kidney macrophages (HKMs) of turbot through binding to G-protein receptor 41 (GPR41) and GPR43. Moreover, histone deacetylase 3 (HDAC3) inhibition apparently up-regulated the butyrate-enhanced IL-22 generation, indicating HDACs were engaged in butyrate-regulated IL-22 secretion. In addition, butyrate triggered the STAT3/HIF-1α signaling to elevate the IL-22 expression in HKMs. Importantly, the evidence in vitro and in vivo was provided that butyrate activated autophagy in fish macrophages via IL-22 signaling, which contributing to the elimination of invading bacteria. In conclusion, we clarified in the current study that butyrate induced STAT3/HIF-1α/IL-22 signaling pathway via GPCR binding and HDAC3 inhibition in fish macrophages to activate autophagy that was involved in pathogen clearance in fish macrophages.

Micro-algal astaxanthin ameliorates polystyrene microplastics-triggered necroptosis and inflammation by mediating mitochondrial Ca2+ homeostasis in carp's head kidney lymphocytes (Cyprinus carpio L.).

Polystyrene microplastics (PM) is a pressing global environmental concern, posing substantial risks to aquatic ecosystems. Microalgal astaxanthin (MA), a heme pigment, safeguards cells against oxidative damage induced by free radicals, which contributes to various health conditions, including aging, inflammation and chronic diseases. Herein, we investigated the potential of MA in ameliorating the immunotoxicity of PM on carp (Cyprinus carpio L.) based on head kidney lymphocytes treated with PM (250 µM) and/or MA (100 µM). Firstly, CCK8 results showed that PM resulted in excessive death of head kidney lymphocytes. Secondly, head kidney lymphocytes treated with PM had a higher proportion of necroptosis, and the levels of necroptosis-related genes in head kidney lymphocytes were increased. Thirdly, the relative red fluorescence intensity of JC-1 and MitoSox showed decreased mitochondrial membrane potential and increased mtROS in head kidney lymphocytes treated with PM. MitoTracker® Green FM fluorescence analysis revealed enhanced mitochondrial Ca2+ levels in PM-treated lymphocytes, corroborating the association between PM exposure and elevated intracellular Ca2+ dynamics. PM exposure resulted in upregulation of calcium homeostasis-related gene (Orail, CAMKIIδ and SLC8A1) in lymphocytes. Subsequent investigations revealed that PM exposure reduced miR-25-5p expression while increasing levels of MCU, MICU1, and MCUR1. Notably, these effects were counteracted by treatment with MA. Furthermore, PM led to the elevated secretion of inflammatory factors (IFN-γ, IL-1ß, IL-2 and TNF-α), thereby inducing immune dysfunction in head kidney lymphocytes. Encouragingly, MA treatment effectively mitigated the immunotoxic effects induced by PM, demonstrating its potential in ameliorating necroptosis, mitochondrial dysfunction and immune impairment via regulating the miR-25-5p/MCU axis in lymphocytes. This study sheds light on safeguarding farmed fish against agrobiological threats posed by PM, highlighting the valuable applications of MA in aquaculture.

Heavy metal bioaccumulation triggers oxystress, genotoxicity and immunomodulation in head kidney macrophages of Channa punctatus Bloch.

Studies on heavy metal induced toxicity have been conducted in many water bodies across the globe and such effects have been evaluated in various fish species. The present study was designed to determine the load of some heavy metals in select sites in Southern Assam, India, along with estimating their concentration in tissues of Channa punctatus Bloch. inhabiting those niches. The effect of heavy metals in oxystress generation, genotoxicity and subsequent immune response in fish was also evaluated. In all of these sites, the concentration of Hg, Cd, Pb and Cr were above the permissible ranges while their concentrations were several folds higher in the piscine tissues due to bioaccumulation and possible biomagnification. Kidney showed the highest metal pollution index followed by liver and gills. Generation of ROS was significantly elevated and that in turn triggered oxystress, as is evident from enhanced lipid peroxidation, protein carbonylation and respiratory burst activity. These were in association with the compromised antioxidant enzyme levels with concomitant damage to DNA as evident from Comet parameters. The innate immune potential was significantly impaired as evident from the compromised cell adhesion, phagocytosis, intracellular killing activity in head kidney macrophages (HKM) along with decreased release of nitric oxide (NO) and myeloperoxidase (MPO). Immunosuppression was further validated at protein levels where compromised release of cytokines viz. TNF-α, IL-1ß, IL-6, IL-10 and IL-12 and cell signaling molecules iNOS and NF-κß were noted. Thus the present study indicates genotoxicity along with a compromise in immune status of Channa punctatus Bloch. living in a habitat laden with heavy metals.

Differential Expression Genes of the Head Kidney and Spleen in Streptococcus iniae-Infected East Asian Fourfinger Threadfin Fish (Eleutheronema tetradactylum).

Streptococcus iniae is a Gram-positive bacterium and is considered a harmful aquaculture pathogen worldwide. In this study, S. iniae strains were isolated from East Asian fourfinger threadfin fish (Eleutheronema tetradactylum) reared on a farm in Taiwan. A transcriptome analysis of the head kidney and spleen was performed in the fourfinger threadfin fish 1 day after infection using the Illumina HiSeq™ 4000 platform for RNA-seq to demonstrate the host immune mechanism against S. iniae. A total of 7333 genes based on the KEGG database were obtained after the de novo assembly of transcripts and functional annotations. Differentially expressed genes (DEGs) (2-fold difference) were calculated by comparing the S. iniae infection and phosphate-buffered saline control group gene expression levels in each tissue sample. We identified 1584 and 1981 differentially expressed genes in the head kidney and spleen, respectively. Based on Venn diagrams, 769 DEGs were commonly identified in both the head kidney and spleen, and 815 and 1212 DEGs were specific to the head kidney and spleen, respectively. The head-kidney-specific DEGs were enriched in ribosome biogenesis. The spleen-specific and common DEGs were found to be significantly enriched in immune-related pathways such as phagosome, Th1, and Th2 cell differentiation; complement and coagulation cascades; hematopoietic cell lineage; antigen processing and presentation; and cytokine-cytokine receptor interactions, based on the KEGG database. These pathways contribute to immune responses against S. iniae infection. Inflammatory cytokines (IL-1ß, IL-6, IL-11, IL-12, IL-35, and TNF) and chemokines (CXCL8 and CXCL13) were upregulated in the head kidney and spleen. Neutrophil-related genes, including phagosomes, were upregulated post-infection in the spleen. Our results could offer a strategy for the treatment and prevention of S. iniae infection in fourfinger threadfin fish.

Distribution of nephrons in the head kidney of three species of Sebastes (Teleostei: Scorpaenidae).

Nephrons are generally not distributed in the head kidney of teleost. Nonetheless, in this study, the nephron structure was identified in the head kidney of three species of Sebastes (Sebastes inermis, Sebastes schlegelii and Sebastes thompsoni). The kidney is Y shaped, with the development in the head kidney. The nephron structure was confirmed in the head kidney and body kidney. In addition, the nephron consisted of renal corpuscles and tubules, and the renal corpuscle consisted of the Bowman's capsule and glomerulus. Histologically, previous studies reported that the nephron structure is similar to that of other marine teleost. The renal tubule is a simple columnar epithelial layer with microvilli and cilia on the free surface, which is observed as a brush border. The Rrk (relative area ratio of kidney to body surface) was 5.14%, 7.58% and 5.17% in S. inermis, S. schlegelii and S. thompsoni, respectively. The Gar (glomerular area ratio of the head kidney) was higher in the central area than in the peripheral area, and species, which showed significant difference (P < 0.05), were in the following order: S. thompsoni (1.60%) > S. schlegelii (0.90%) > S. inermis (0.66%).

Validation of a Liquid-Liquid Extraction Method to Study the Temporal Production of D-Series Resolvins by Head Kidney Cells from Atlantic Salmon (Salmon salar) Exposed to Docosahexaenoic Acid.

A simple and rapid method for the extraction of D-series resolvins (RvD1, RvD2, RvD3, RvD4, RvD5) released into Leibovitz's L-15 complete medium by head kidney cells from Atlantic salmon and the further determination of liquid chromatography triple quadrupole mass spectrometry is proposed. A three-level factorial design was proposed to select the optimal concentrations of internal standards that were used in the evaluation of the performance parameters, such as linear range (0.1-50 ng mL-1), limits of detection and quantification (0.05 and 0.1 ng mL-1, respectively), and recovery values ranging from 96.9 to 99.8%. The optimized method was used to determine the stimulated production of resolvins by head kidney cells exposed to docosahexaenoic acid, and the results indicated that it is possible that the production was controlled by circadian responses.

Acute stress response on Atlantic salmon: a time-course study of the effects on plasma metabolites, mucus cortisol levels, and head kidney transcriptome profile.

Farmed Atlantic salmon (Salmo salar) welfare and performance can be strongly influenced by stress episodes caused by handling during farming practices. To better understand the changes occurring after an acute stress response, we exposed a group of Atlantic salmon parr to an acute stressor, which involved netting and transferring fish to several new holding tanks. We describe a time-course response to stress by sampling parr in groups before (T0) and 10, 20, 30, 45, 60, 120, 240, 300, and 330 min post-stress. A subgroup of fish was also subjected to the same stressor for a second time to assess their capacity to respond to the same challenge again within a short timeframe (ReStressed). Fish plasma was assessed for adrenocorticotropic hormone (ACTH), cortisol, and ions levels. Mucus cortisol levels were analyzed and compared with the plasma cortisol levels. At 5 selected time points (T0, 60, 90, 120, 240, and ReStressed), we compared the head kidney transcriptome profile of 10 fish per time point. The considerably delayed increase of ACTH in the plasma (60 min post-stress), and the earlier rise of cortisol levels (10 min post-stress), suggests that cortisol release could be triggered by more rapidly responding factors, such as the sympathetic system. This hypothesis may be supported by a high upregulation of several genes involved in synaptic triggering, observed both during the first and the second stress episodes. Furthermore, while the transcriptome profile showed few changes at 60 min post-stress, expression of genes in several immune-related pathways increased markedly with each successive time point, demonstrating the role of the immune system in fish coping capacity. Although many of the genes discussed in this paper are still poorly characterized, this study provides new insights regarding the mechanisms occurring during the stress response of salmon parr and may form the basis for a useful guideline on timing of sampling protocols.

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.