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.

Participation of Hepcidins in the Inflammatory Response Triggered by λ-Carrageenin in Gilthead Seabream (Sparus aurata).

The role of hepcidins, antimicrobial peptides involved in iron metabolism, immunity, and inflammation, is studied. First, gilthead seabream (Sparus aurata L.) head-kidney leucocytes (HKLs) were incubated with λ-carrageenin to study the expression of hepcidin and iron metabolism-related genes. While the expression of most of the genes studied was upregulated, the expression of ferroportin gene (slc40a) was downregulated. In the second part of the study, seabream specimens were injected intramuscularly with λ-carrageenin or buffer (control). The expression of the same genes was evaluated in the head kidney, liver, and skin at different time points after injection. The expression of Hamp1m, ferritin b, and ferroportin genes (hamp1, fthb, and slc40a) was upregulated in the head kidney of fish from the λ-carrageenin-injected group, while the expression of Hamp2C and Hamp2E genes (hamp2.3 and hamp2.7) was downregulated. In the liver, the expression of hamp1, ferritin a (ftha), slc40a, Hamp2J, and Hamp2D (hamp2.5/6) genes was downregulated in the λ-carrageenin-injected group. In the skin, the expression of hamp1 and (Hamp2A Hamp2C) hamp2.1/3/4 genes was upregulated in the λ-carrageenin-injected group. A bioinformatic analysis was performed to predict the presence of transcription factor binding sites in the promoter region of hepcidins. The primary sequence of hepcidin was conserved among the different mature peptides, although changes in specific amino acid residues were identified. These changes affected the charge, hydrophobicity, and probability of hepcidins being antimicrobial peptides. This study sheds light on the poorly understood roles of hepcidins in fish. The results provide insight into the regulatory mechanisms of inflammation in fish and could contribute to the development of new strategies for treat inflammation in farm animals.

Comparative analysis of miRNAs and mRNAs in large yellow croaker head kidney cells (LYCK) provided novel insights into the redox regulation of fish.

Reactive oxygen species (ROS) over-production and oxidative stress resulted from climate change and environmental pollution seriously endangered global fish populations and healthy development of marine aquaculture. Peroxiredoxins (Prxs), a highly conserved family of thiol-specific antioxidants, can mitigate ROS and protect cells from oxidative stress. We previously demonstrated that large yellow croaker PrxIV (LcPrxIV) could not only regulate the pro-inflammatory responses, but also scavenge ROS. However, the underlying mechanism how LcPrxIV regulated immune response and redox homeostasis remains unknown. MicroRNAs (miRNAs) are non-coding RNAs that play important roles in the regulation of various biological processes. In this study, mRNA and miRNA expression profiles from LYCK-pcDNA3.1 and LYCK-PrxIV cells, with or without oxidative stress stimulated by H2O2 were evaluated using high-throughput sequencing. A series of differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs), as well as DEM-DEG pairs were identified from each two-group comparison, respectively. GO and KEGG functional analyses indicated that most significant DEGs were associated with signaling pathways related to oxidative stress and immune response. Subsequent DEM-DEG interaction analysis revealed that miR-731 and miR-1388 may be involved in both redox regulation and immune response via synergistic effect with LcPrxIV. Interestingly, miR-731 could regulate the expression of different down-stream DEGs under different stimulations of LcPrxIV over-expression, H2O2, or both. Moreover, miR-731 could cause the DEG, γ-glutamyl hydrolase (GGH), to be expressed in opposite ways under different stimulations. On the other hand, the expression of miR-1388 could be negatively or positively regulated under the stimulation of LcPrxIV over-expression with or without oxidative stress, thus regulating gene expression of different mRNAs. Based on these results, we speculate that LcPrxIV may participate in immune response or redox regulation by regulating the expression of different down-stream genes through controlling the expression level of a certain miRNA or by regulating the varieties of expressed miRNAs.

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.

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.

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.

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.

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.

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.

Transcriptome analysis of Chinese sucker (Myxocyprinus asiaticus) head kidney and discovery of key immune-related genes to cold stress after swimming fatigue.

For Chinese sucker (Myxocyprinus asiaticus), passing through a dam with fast flow and cold water are always unavoidable, and this process can cause stress, disease or even death. In this study, comparative transcriptome analysis was conducted to investigate the potential immune mechanism in head kidney of M. asiaticus with swimming fatigue stress and cold stress after fatigue. In general, a total of 181,781 unigenes were generated, and 38,545 differentially expressed genes (DEGs) were identified. In these DEGs, 22,593, 7286 and 8666 DEGs were identified among groups of fatigue vs. cold, control vs. cold, and control vs. fatigue, respectively. Enrichment analysis revealed these DEGs were involved in coagulation cascades and complement, natural killer cell mediated cytotoxicity, antigen processing and presentation, Toll-like receptor signaling pathways, and chemokine signaling pathway. Notably, immune genes including heat shock protein 4a (HSP4a), HSP70 and HSP90α genes were significantly up-regulated in fishes with cold stress after fatigue. Differently, more immune genes in control vs. cold compared with that in control vs. fatigue were significantly down-regulated expression, such as claudin-15-like, Toll-like receptor 13, antimicrobial peptide (hepcidin), immunoglobulin, CXCR4 chemokine receptor, T-cell receptor, complement factor B/C2-A3, and interleukin 8. In this study, the number of DEGs in the head kidney was less than that our previous study in the spleen, which we speculated was more sensitive to changes in water temperature than the head kidney. In summary, lots of immune-related genes in the head kidney were down-regulated under cold stress after fatigue, suggesting that M. asiaticus might have experienced severe immunosuppression in the process of passing through the dam.

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%).

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.

Comparative Transcriptomics in Atlantic Salmon Head Kidney and SHK-1 Cell Line Exposed to the Sea Louse Cr-Cathepsin.

The development of vaccines against sea lice in salmon farming is complex, expensive, and takes several years for commercial availability. Recently, transcriptome studies in sea louse have provided valuable information for identifying relevant molecules with potential use for fish vaccines. However, the bottleneck is the in vivo testing of recombinant protein candidates, the dosage, and the polyvalent formulation strategies. This study explored a cell-based approach to prospect antigens as candidate vaccines against sea lice by comparison with immunized fish. Herein, SHK-1 cells and Atlantic salmon head kidney tissue were exposed to the antigen cathepsin identified from the sea louse Caligus rogercresseyi. The cathepsin protein was cloned and recombinantly expressed in Escherichia coli, and then SHK-1 cell lines were stimulated with 100 ng/mL cathepsin recombinant for 24 h. In addition, Atlantic salmons were vaccinated with 30 ug/mL recombinant protein, and head kidney samples were then collected 30 days post-immunization. SHK-1 cells and salmon head kidney exposed to cathepsin were analyzed by Illumina RNA sequencing. The statistical comparisons showed differences in the transcriptomic profiles between SHK-1 cells and the salmon head kidney. However, 24.15% of the differentially expressed genes were shared. Moreover, putative gene regulation through lncRNAs revealed tissue-specific transcription patterns. The top 50 up and downregulated lncRNAs were highly correlated with genes involved in immune response, iron homeostasis, pro-inflammatory cytokines, and apoptosis. Also, highly enriched pathways related to the immune system and signal transduction were shared between both tissues. These findings highlight a novel approach to evaluating candidate antigens for sea lice vaccine development, improving the antigens screening in the SHK-1 cell line model.

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.

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.

Response of immunoglobulin M in gut mucosal immunity of common carp (Cyprinus carpio) infected with Aeromonas hydrophila.

Immunoglobulin (Ig) M is an important immune effector that protects organisms from a wide variety of pathogens. However, little is known about the immune response of gut mucosal IgM during bacterial invasion. Here, we generated polyclonal antibodies against common carp IgM and developed a model of carp infection with Aeromonas hydrophila via intraperitoneal injection. Our findings indicated that both innate and adaptive immune responses were effectively elicited after A. hydrophila infection. Upon bacterial infection, IgM+ B cells were strongly induced in the gut and head kidney, and bacteria-specific IgM responses were detected in high levels both in the gut mucus and serum. Moreover, our results suggested that IgM responses may vary in different infection strategies. Overall, our findings revealed that the infected common carp exhibited high resistance to this representative enteropathogenic bacterium upon reinfection, suggesting that IgM plays a key role in the defense mechanisms of the gut against bacterial invasion. Significantly, the second injection of A. hydrophila induces strong local mucosal immunity in the gut, which is essential for protection against intestinal pathogens, providing reasonable insights for vaccine preparation.

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.

CD4-1 and CD8α T lymphocytes subsets in spotted sea bass (Lateolabrax maculatus) and comparison on antigenicity of T lymphocytes subsets in other three marine fish species.

CD4 and CD8 molecules play an important role in the identification of T lymphocytes, and diverse among fish species. In this study, CD4-1 and CD8α gene of spotted sea bass (Lateolabrax maculatus) were cloned, polyclonal antibodies against CD4-1 (CD4-1 pAbs) and CD8α (CD8α pAbs) were produced, respectively. And the variations in CD4-1+ and CD8α+ T-lymphocytes in spotted sea bass and the cross-reactivity with leukocytes in pearl gentian grouper (Epinephelus fuscoguttatus x E. lanceolatus), schlegel's black rockfish (Sebastes schlegelii) and flounder (Paralichthys olivaceus) were investigated using CD4-1 pAbs and CD8α pAbs. The results showed that CD4-1 molecule ORF was 1413 bp and CD8α was 690 bp, both molecules are transmembrane glycoproteins with high amino acid homology to grouper. The CD4-1 pAbs specifically recognized both the CD4-1 recombinant and natural proteins, as does the CD8α pAbs to CD8α molecule, and no cross-reactivity between the two antibodies. CD4-1+ and CD8α+ T lymphocytes were detected in peripheral blood, spleen and head kidney leukocytes in spotted sea bass. In cross-reactivity assay with other three fish, CD4-1 pAbs could recognize the lymphocytes from pearl gentian grouper and schlegel's black rockfish, both with highest proportions in the spleen leukocytes, 5.3 ± 0.4% and 2.6 ± 0.3%, respectively, and CD8α pAbs could only recognize the lymphocytes in pearl gentian grouper, and no cross-reactivities to lymphocytes of flounder. These data suggested that the CD4-1 and CD8α molecules varied by fish species in the genes features and antigenicity, which might result in the diversities of T lymphocytes subpopulations. This will be a key to elucidating the classification and evolution of T lymphocytes in fish.