Metabolic, neurotoxic and immunotoxic effects of PFAAs and their mixtures on the proteome of the head kidney and plasma from rainbow trout (Oncorhynchus mykiss).

PFAAs (Perfluoroalkyl acids) are a class of bioaccumulative, persistent and ubiquitous environmental contaminants which primarily occupy the hydrosphere and its sediments. Currently, a paucity of toxicological information exists for short chain PFAAs and complex mixtures. In order to address these knowledge gaps, we performed a 3-week, aqueous exposure of rainbow trout to 3 different concentrations of a PFAA mixture (50, 100 and 500 ng/L) modeled after the composition determined in Lake Ontario. We conducted an additional set of exposures to individual PFAAs (25 nM each of PFOS (12,500 ng/L), PFOA (10,300 ng/L), PFBS (7500 ng/L) or PFBA (5300 ng/L) to evaluate differences in biological response across PFAA congeners. Untargeted proteomics and phosphorylated metabolomics were conducted on the blood plasma and head kidney tissue to evaluate biological response. Plasma proteomic responses to the mixtures revealed several unexpected outcomes including Similar proteomic profiles and biological processes as the PFOS exposure regime while being orders of magnitude lower in concentration and an atypical dose response in terms of the number of significantly altered proteins (FDR < 0.1). Biological pathway analysis revealed the low mixture, medium mixture and PFOS to significantly alter (FDR < 0.05) a number of processes including those involved in lipid metabolism, oxidative stress and the nervous system. We implicate plasma increases in PPARD and PPARG as being directly related to these biological processes as they are known to be important regulators in all 3 processes. In contrast to the blood plasma, the high mixture and PFOA exposure regimes caused the greatest change to the head kidney proteome, altering many proteins being involved in lipid metabolism, oxidative stress and inflammation. Our findings support the pleiotropic effect PFAAs have on aquatic organisms at environmentally relevant doses including those on PPAR signaling, metabolic dysregulation, immunotoxicity and neurotoxicity.

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.

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.

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.

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.

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.

Expression Analysis of Moritella viscosa-Challenged Atlantic Salmon Identifies Disease-Responding Genes, MicroRNAs and Their Predicted Target Genes and Pathways.

Moritella viscosa is a bacterial pathogen causing winter-ulcer disease in Atlantic salmon. The lesions on affected fish lead to increased mortality, decreased fish welfare, and inferior meat quality in farmed salmon. MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional regulation by guiding the miRNA-induced silencing complex to specific mRNA transcripts (target genes). The goal of this study was to identify miRNAs responding to Moritella viscosa in salmon by investigating miRNA expression in the head-kidney and the muscle/skin from lesion sites caused by the pathogen. Protein coding gene expression was investigated by microarray analysis in the same materials. Seventeen differentially expressed guide-miRNAs (gDE-miRNAs) were identified in the head-kidney, and thirty-nine in lesion sites, while the microarray analysis reproduced the differential expression signature of several thousand genes known as infection-responsive. In silico target prediction and enrichment analysis suggested that the gDE-miRNAs were predicted to target genes involved in immune responses, hemostasis, angiogenesis, stress responses, metabolism, cell growth, and apoptosis. The majority of the conserved gDE-miRNAs (e.g., miR-125, miR-132, miR-146, miR-152, miR-155, miR-223 and miR-2188) are known as infection-responsive in other vertebrates. Collectively, the findings indicate that gDE-miRNAs are important post-transcriptional gene regulators of the host response to bacterial infection.

Comparative Transcriptome Analysis of Head Kidney of Aeromonas hydrophila-infected Hypoxia-tolerant and Normal Large Yellow Croaker.

The large yellow croaker (Larimichthys crocea) is one of the most economically important marine fish on the southeast coast of China and much of its yield is usually lost by hypoxia. To address this problem and lay a foundation for culturing a new strain of large yellow croaker with hypoxia tolerance, our research group screened a hypoxia-tolerant population of L. crocea. Surprisingly, we also found that hypoxia-tolerant population exhibited higher survival when infected with pathogens compared to the normal population during the farming operation. In order to understand the mechanism underlying the higher survival rate of the hypoxia-tolerant population and enrich the head kidney immune mechanism of L. crocea infected with pathogens, we compared and analyzed the head kidney transcriptome of the hypoxia-tolerant and normal individuals under Aeromonas hydrophila infection. We obtained 159.68 GB high-quality reads, of which more than 87.61% were successfully localized to the reference genome of L. crocea. KEGG analysis revealed differentially expressed genes in the signaling pathways involving immunity, cell growth and death, transport and catabolism, and metabolism. Among these, the toll-like receptor signaling pathway, Nod-like receptor signaling pathway, cytokine-cytokine receptor interaction, phagosome, apoptosis, and OXPHOS pathways were enriched in both groups after infection compared to before, and were enriched in infected tolerant individuals compared to normal individuals. In addition, we found that the expression of hif1α and its downstream genes were higher in the hypoxia-sensitive group of fish than in the normal group. In conclusion, our results showed some signaling pathways and hub genes, which may participate in A. hydrophila defense in the head kidney of two populations, and may contribute to the higher survival rate in the hypoxia-tolerant population. Overall, these findings increase our understanding of the defense mechanism within the head kidney of L. crocea under A. hydrophila infection, and suggest a preliminary hypothesis for why hypoxia-tolerant individuals may exhibit a higher survival rates after infection. Our study provides scientific evidence for the breeding of a new hypoxia-tolerant strain of L. crocea for aquaculture.

Chitosan-Ocimum basilicum nanocomposite as a dietary additive in Oreochromis niloticus: Effects on immune-antioxidant response, head kidney gene expression, intestinal architecture, and growth.

Several studies have looked into the use of basil, Ocimum basilicum (L.) in aquaculture as a dietary additive; however, more research is needed to see the possibility of it's including in nanocarriers in aquafeeds. An experiment was undertaken to highlight the efficacy chitosan-Ocimum basilicum nanocomposite (COBN), for the first time, on Nile tilapia (Oreochromis niloticus) growth, stress and antioxidant status, immune-related parameters, and gene expression. For 60 days, fish (average weight: 23.55 ± 0.08 g) were fed diets provided with different concentrations of COBN (g/kg): 0 g [COBN0], 1 g [COBN1], 2 g [COBN2], and 3 g [COBN3], where COBN0 was kept as control diet. Following the trial, the fish were challenged with pathogenic bacteria (Aeromonas sobria) and yeast (Candida albicans) infection. In comparison to the control (COBN0), a notable increase in growth parameters (weight gain, feed intake, and specific growth rate) and intestinal morphometric indices (average intestinal goblet cells count, villous width, and length) in all COBN groups was observed, where COBN2 and COBN3 groups had the highest values. The COBN diets significantly (p < 0.05) declined levels of serum triglycerides, glucose, cholesterol, and hepatic malondialdehyde. Moreover, the higher levels of serum biochemical biomarkers (growth hormone, total protein, globulin, and albumin), immunological parameters (phagocytic activity%, nitric oxide, and lysozyme), and hepatic antioxidant parameters (superoxide dismutase, total antioxidant capacity, and glutathione peroxidase) were obvious in the COBN2 and COBN3 groups followed by COBN1. The immune-antioxidant genes (TNF-α, IL-10, IL-1ß, TGF-ß, GPx, and SOD) were found to be considerably up-regulated in all COBN groups (COBN2 and COBN3 followed by COBN1). Fifteen days post-challenge with A. sobria and C. albicans, the highest survival rate was recorded in the COBN2 group (83.33 and 91.67%) followed by the COBN3 group (75 and 83.33%), respectively. The findings showed that a dietary intervention with COBN can promote growth, intestinal architecture, immunity, and antioxidant markers as well as protect O. niloticus against A. sobria and C. albicans infection. As a result, the COBN at a dose of 2 g/kg could be used as a food additive for the sustainable aquaculture industry.

Functional analyses of two interferon-stimulated gene 15 (ISG15) copies in large yellow croaker, Larimichthys crocea.

In this study, we conducted functional analyses for two ISG15 homologues of Larimichthys crocea (LcISG15-1 and LcISG15-2). Our results of qRT-PCR showed that both LcISG15-1 and LcISG15-2 were significantly changed in head kidney and peripheral blood, after poly (I:C) stimulation. Western blot analyses with prepared polyclonal antibodies suggested that LcISG15-1 and LcISG15-2 both could be secreted by primary head kidney lymphocytes into the extracellular milieu. The purified recombinant LcISG15-1 (rLcISG15-1) and LcISG15-2 (rLcISG15-2) could both activate primary macrophages as extracellular cytokines and significantly enhance macrophage respiratory burst, NO production and bactericidal activity and induce the expression of proinflammatory cytokine genes of the cells. Moreover, rLcISG15-2 exhibited much stronger cytokine-like activities than those of rLcISG15-1, indicating the ISG15-2 gene copy evolved enhanced activity after gene duplication of ISG15 in sciaenid fishes. These results indicated important roles of LcISG15-1 and especially LcISG15-2 in immune regulation and host immune defense of large yellow croaker against viral and bacterial infection.

Molecular identification and functional exploration of interleukin-20 in snakehead (Channa argus) involved in bacterial invasion and the proliferation of head kidney leukocytes.

As an inflammatory cytokine of the interleukin-20 (IL-20) subfamily, IL-20 has various functions in immune defenses, inflammatory diseases, tissue regeneration, cancer, and metabolism. Although the characteristics and functions of mammalian IL-20 have been clarified, those of fish IL-20 remain unclear. In this study, the IL-20 gene from the snakehead Channa argus (shIL-20) was cloned and functionally characterized. Similar to the IL-20 homologues of other species, the shIL-20 has a five exon/four intron structure in the coding region. The open reading frame of shIL-20 consists of 528 base pairs and encodes 175 amino acids (aa), including a signal peptide (aa 1-24) and a mature peptide (aa 25-175). The mature shIL-20 protein has six conserved cysteine residues, which occur in the IL-20 proteins of all species analyzed, and an additional cysteine residue (Cys-82) found only in the IL-20 proteins of several teleosts. The modeled tertiary structure of shIL-20 is similar with that of Homo sapiens IL-20. The shIL-20 was expressed constitutively in all the tissues analyzed, and its transcription was induced in the spleen and head kidney by Aeromonas schubertii and Nocardia seriolae in vivo and in head kidney leukocytes (HKLs) by lipoteichoic acid, lipopolysaccharide, and polyinosinic-polycytidylic acid in vitro. The recombinant shIL-20 protein induced the transcription of tumor necrosis factor α1 (TNF-α1), TNF-α2, IL-1ß, and endogenous shIL-20, and promoted the proliferation of HKLs. In conclusion, these findings demonstrate that shIL-20 participates in the immune response to bacterial invasion and promotes leukocyte proliferation, offering new insights into the functions of fish IL-20 during pathogen invasion.

Role of miR-451 in mediating cadmium induced head kidney injury in common carp via targeting cacna1ab through autophagy pathways.

Cadmium (Cd) is a common environmental pollutant, which leads to Cd residue in aquatic animals. The Cd in aquatic animals will be enriched into the human body through the food chain and seriously harm human health. The research aims to investigate the molecular mechanism of Cd poisoning in common carps. Our previous studies have confirmed that 23 differentially expressed miRNAs were potential biomarkers for Cd exposure in common carp head kidney lymphocytes. Herein, based on the prediction of the website and previous studies, miR-451 and cacna1ab were selected and their targeting relationship was verified again by dual-luciferase. Subsequently, we established the miR-451 overexpression/knockdown models and miR-451 inhibitor, cacna1ab co-knockdown models in common carp head kidney lymphocytes respectively. Immunofluorescence staining, MDC staining, calcium staining, qRT-PCR (Quantitative Real-time PCR) and western blot were used to detect the levels of autophagy. Our results demonstrated that Cd significantly decreased the expression of miR-451, miR-451 suppression thereby induced increased cacna1ab and the expression of ATG5, LC3-I, LC3-II and Beclin 1, while significantly inhibiting the expression of mTOR, P62 and Bcl-2, which indicated that autophagy was triggered. Moreover, the miR-451 knockdown group activated the expression of autophagy related factors as well as the Cd group. However, cacna1ab knockdown can reduce autophagy activation induced by miR-451 knockdown. Our results indicated that Cd induced autophagy in head kidney lymphocytes through the inhibition of miR-451 and the excitation of cacna1ab.

Regulation of cortisol production during chronic social stress in rainbow trout.

Chronic stress resulting from social interactions impacts the endocrine stress response in many vertebrates, including teleost fishes. Juvenile rainbow trout held in pairs form a dominance hierarchy with the subordinate individual exhibiting chronic elevation of plasma cortisol and an attenuated cortisol response to an additional acute stressor. The current study investigated the mechanisms underlying this apparent dichotomy in cortisol production at the level of the head kidney (adrenal homolog). Following four days of chronic social stress, subordinate rainbow trout exhibited elevated plasma cortisol levels that correlated with basal cortisol production by the head kidney in vitro. Subordinate trout had higher transcript abundances of steroidogenic acute regulatory protein and cytochrome p450 side chain cleavage enzyme, which facilitate key steps in steroidogenesis, as well as two paralogs of steroidogenic factor 1. Despite elevation of basal steroidogenesis, acute cortisol production in response to ACTH (in vivo and in vitro) was lower in subordinate trout. Transcript abundances of the ACTH receptor accessory proteins were elevated in subordinate fish, but head kidney cortisol production in response to a cAMP analogue was lower than in dominant fish. Together, the data suggest that the attenuated acute cortisol response of subordinate trout reflects limitations on cortisol production downstream of cAMP signalling in steroidogenic cells of the head kidney, despite the increased basal abundance of key components of the steroidogenic pathway.

Astilbin attenuates apoptosis induced by cadmium through oxidative stress in carp (Cyprinus carpio L.) head kidney lymphocyte.

As a kind of environmental pollutant, heavy metal Cadmium (Cd) exists widely in the environment. It is well known that Cd can accumulate and cause damage in liver, kidney and other organs. However, there are few studies on the immune cytotoxicity of Cd to fish. In particular, there are few studies on the toxicity of Cd to the head kidney lymphocytes of common carp. In order to further explore these mechanisms, we established an Cd exposure model in vitro. At the same time, we used the natural antioxidant astilbin (AST) to treat the cells to study its antagonistic effect on the toxicity of Cd. After exposure to Cd, the level of oxidative stress in head kidney lymphocytes increased, and the mRNA and protein expression of apoptosis-related markers Fas, FADD, Caspase8 and Caspase3 increased significantly (P < 0.05), which led to lymphocytes apoptosis. Hoechst staining and AO/EB staining also showed that the level of apoptosis increased after exposure to Cd. This is consistent with our previous research results. AST treatment reduced oxidative stress and apoptosis induced by Cd. In addition, oxidative stress inhibitor NAC could also reduce head kidney lymphocytes apoptosis induced by Cd, indicating that oxidative stress was involved in this process. Our results suggested that AST can alleviate the apoptosis of carp head kidney lymphocytes induced by Cd through oxidative stress. This study enriches the theoretical mechanism of Cd toxicity to fish head kidney lymphocytes, and puts forward a method to solve the toxicity of Cd, which provides a theoretical and research basis for the in vivo study of animal models in the future.

Short-Term Immune Responses of Gilthead Seabream (Sparus aurata) Juveniles against Photobacterium damselae subsp. piscicida.

Photobacteriosis is a septicaemic bacterial disease affecting several marine species around the globe, resulting in significant economic losses. Although many studies have been performed related to the pathogen virulence and resistance factors, information regarding the host defence mechanisms activated once an infection takes place is still scarce. The present study was designed to understand innate immune responses of farmed juvenile gilthead seabream (Sparus aurata) after Photobacterium damselae subsp. piscicida (Phdp) infection. Therefore, two groups of seabream juveniles were intraperitoneally injected with 100 µL of PBS (placebo) or 100 µL of exponentially growing Phdp (1 × 106 CFU/mL; infected). The blood, plasma, liver, and head kidney of six fish from each treatment were sampled immediately before infection and 3, 6, 9, 24 and 48 h after infection for the broad screening of fish immune and oxidative stress responses. Infected animals presented marked anaemia, neutrophilia and monocytosis, conditions that are correlated with an increased expression of genes related to inflammation and phagocytic activity. Similar studies with different fish species and bacteria can be useful for the definition of health biomarkers that might help fish farmers to prevent the occurrence of such diseases.

Evaluation of blood cell viability rate, gene expression, and O-GlcNAcylation profiles as indicative signatures for fungal stimulation of salmonid cell models.

Fungal diseases of fish are a significant economic problem in aquaculture. Using high-throughput expression analysis, we identified potential transcript markers in primary head kidney and secondary embryonic cells from salmonid fish after stimulation with the inactivated fungi Mucor hiemalis and Fusarium aveneacium and with purified fungal molecular patterns. The transcript levels of most of the 45 selected genes were altered in head-kidney cells after 24 h of stimulation with fungal antigens. Stimulation with the inactivated fungus M. hiemalis induced the most pronounced transcriptional changes, including the pathogen receptor-encoding genes CLEC18A and TLR22, the cytokine-encoding genes IL6 and TNF, and the gene encoding the antimicrobial peptide LEAP2. In parallel, we analyzed the total GlcNAcylation status of embryonic salmonid cells with or without stimulation with inactivated fungi. O-GlcNAcylation modulates gene expression, intracellular protein, and signal activity, but we detected no significant differences after a 3-h stimulation. A pathway analysis tool identified the "apoptosis of leukocytes" based on the expression profile 24 h after fungal stimulation. Fluorescence microscopy combined with flow cytometry revealed apoptosis in 50 % of head-kidney leukocytes after 3 h stimulation with M. hiemalis, but this level decreased by > 5% after 24 h of stimulation. The number of apoptotic cells significantly increased in all blood cells after a 3-h stimulation with fungal molecular patterns compared to unstimulated controls. This in vitro approach identified transcript-based parameters that were strongly modulated by fungal infections of salmonid fish.

Zinc Alleviates Arsenic-Induced Inflammation and Apoptosis in the Head Kidney of Common Carp by Inhibiting Oxidative Stress and Endoplasmic Reticulum Stress.

Arsenic (As) pollution is ubiquitous in water, which shows immunotoxicity to aquatic organisms. As an indispensable regulator of gene transcription and enzymatic modification, zinc (Zn) may play a preventive and therapeutic effect on As toxicity. The purpose of this study was to investigate the interactions of As and Zn on the head kidney of common carp Cyprinus carpio. Herein the carp were treated alone or in combination with waterborne As3+ (2.83 mg/L) and/or Zn2+ (1 mg/L). Results suggested a head kidney-toxic effect of As exposure, which was manifested by the histopathological damage of the head kidney, elevation of nuclear translocation of pro-inflammatory nuclear factor-kappa light chain enhancer of B cells (NF-κB), and blockage of the anti-oxidative nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. The global activation of three endoplasmic reticulum (ER) stress pathways led to the execution of programmed cell death, including ER apoptosis mediated by C/EBP-homologous protein (CHOP), death receptor-mediated exogenous cell apoptosis, and the endogenous apoptosis executed by Caspases9. The combined application of Zn can significantly improve the histopathological damage of the head kidney, the imbalance of the antioxidant system, and the apoptosis outcomes due to ER stress. In conclusion, this study indicates that Zn has an antagonistic effect on the head kidney injury of common carp induced by sub-chronic As exposure. The results of this study provide basic data for the risk assessment of As accumulation in an aquatic environment and a reference for the use of Zn preparation in aquaculture.