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Interleukin-10 (IL-10) is a pleiotropic cytokine involved in the regulation of innate and adaptive immunity. In this study, IL-10 from big-belly seahorse (Hippocampus abdominalis) (HaIL-10) was characterized based on its molecular and functional aspects. The coding sequence of HaIL-10 is 570 bp in length and encodes a 189-amino acid residue protein (calculated molecular weight, 21.89 kDa). The deduced amino acid sequence comprises a typical signal peptide and a mature peptide domain sequence carrying four conserved Cys residues and two additional Cys residues specific to fish. Phylogenetic analysis indicated an evolutionary relationship between HaIL-10 and its counterparts in other vertebrates, with close clustering to the fish-specific homologs. Recombinant HaIL-10 (rHaIL-10) significantly reduced nitric oxide (NO) production by lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells in a concentration-dependent manner but had no effect on cell viability, suggestive of its involvement in immune response. The protein expressions of iNOS and COX-2 were significantly reduced by rHaIL-10 in LPS-induced murine macrophages RAW 264.7 cells. HaIL-10 mRNA expression was observed in all analyzed tissues, with the maximum expression being noted in the kidney and ovary. However, transcriptional levels of HaIL-10 were significantly higher in the blood, gill, and intestine upon in vivo induction with LPS, polyinosinic:polycytidylic acid [poly (I:C)], and Streptococcus iniae. To summarize, our findings help in the improved understanding of the biological functions of HaIL-10 and modulation of HaIL-10 mRNA expression in response to immune stress., (Copyright © 2018 Elsevier B.V. All rights reserved.)

• Two ferritin like subunits (LhFerH & LhFerM) were identified from Liza haematocheila. • Recombinant ferritin subunits showed detectable iron deprivation activity. • Both recombinant subunits could inhibit the growth of E.coli in LB media. • Both LhFerH and LhFerM expressed universality in all selected fish tissues. • In-vivo expression of both LhFerH and M was induced post immune stimulation. Iron (Fe) is considered as an essential micronutrient due to its diverse functions in living systems. However, regulation of free iron levels is essential because free Fe ions, in excess, induce biological toxicity through different routes, including production of reactive oxygen species. Ferritin proteins play a vital role in controlling free Fe ion homeostasis by sequestering excess iron in the body. Ferritins comprise an H subunit with a ferroxidase center and an L subunit with a Fe nucleation site. However, lower vertebrates such as fish harbor an additional subunit termed ferritin M, which shows the characteristic features of both H and L. In this study, two ferritin subunits (H and M) with ferroxidase centers were identified and characterized from red-lip mullet (Liza haematocheila). The open reading frames of red-lip mullet ferritin H (LhFerH) and ferritin M−like (LhFerM) subunits comprise 534 and 531 bps, which encode for putative polypeptides of 177 and 176 amino acids, respectively. LhFerH and LhFerM were found to retain well-conserved residues, including seven ferroxidase di-iron centers, characteristic domains, and signatures of their known homologs. We cloned the open reading frames of the two ferritin subunits to overexpress the corresponding proteins in Escherichia coli and subsequently demonstrated their iron sequestration activity along with antibacterial activity against E. coli using respective purified recombinant proteins in vitro. A basal expression analysis of two LhFer genes in selected tissues using qPCR assays showed pronounced expression in blood cells with respect to both genes. A relatively high expression level of LhFerH was also detected in muscle tissues. The expression level of LhFer in the head kidney was significantly up-regulated following lipopolysaccharides (LPS) and Lactococcus garvieae injection. The resulting gene expression pattern upon immune stimulation suggests that ferritin may contribute to the defense against harmful pathogen infection. Collectively, our results indicate that both LhFerH and LhFerM potentially participate in the homeostasis of free Fe ions and in the host immune defense of red-lip mullet. [ABSTRACT FROM AUTHOR]

Viperin is an important virus-induced protein in animals that negatively participates in RNA viral replication and transcription. The reactive machinery of viperin suggests that it produces a regulatory molecule ddhCTP, which may affect immune regulation. In this study, we investigated the expression pattern of viperin in larval and adult stages of zebrafish by whole-mount in situ hybridization and reverse transcription-quantitative PCR (RT-qPCR). To elucidate the function of viperin, we generated a zebrafish knockout model using the CRISPR/Cas9 method and evaluated the mutation's effects under viral hemorrhagic septicemia virus (VHSV) infections. In zebrafish larvae, viperin was expressed in the brain region, eye, and pharynx, which was confirmed by cryosectioning. In adult zebrafish, blood cells showed the highest levels of viperin expression. In 5 dpf fish challenged with VHSV, the expression of the viral NP protein was significantly enhanced in viperin−/− compared to wild-type fish. In vitro VHSV propagation analysis indicated comparatively higher levels of virus propagation in viperin−/− fish. Mortality analysis confirmed higher mortality rates, and interferon gene expression analysis showed a strong upregulation of interferon (ifn)φ1 and 3 gene in viperin−/− fish infected with VHSV. This study describes the successful generation of a viperin-knockout model and the role of viperin during VHSV infections. [Display omitted] • A zebrafish knockout model (KO) for the viperin gene was constructed by CRISPR/Cas9-mediated genome editing. • Zebrafish viperin expression was analyzed by in situ hybridization and cryosectioning. • Fish morphology, virus tropism and titer were analyzed in VHSV-infected KO fish. • Interferon system and neutrophile migration were studied. [ABSTRACT FROM AUTHOR]

Myeloid differentiation primary response protein-88 (MYD88) is an essential adaptor molecule in pathogen-related pattern recognition signaling pathways. Toll-like and interleukin receptors recognize numerous signals and are funneled through MyD88 to express genes responsible for the innate and adaptive immune systems. In the present study, the relevance of MyD88 in viral hemorrhagic septicemia virus (VHSV) was investigated by generating myd88 −/− zebrafish. The model was challenged with VHSV, and viral propagation was quantified by evaluating clinical symptoms, mortality, and VHSV copy number. The infected fish showed abnormal morphologies, such as subcutaneous hemorrhages, abdominal swelling, and bulging eyes, which were comparatively more intense in myd88 −/− fish than in the wild-type. An injury infection experiment conducted in zebrafish larvae indicated a substantial spread of VHSV in the wound site. The number of neutrophils and macrophages recruited to the wounded area were markedly reduced in myd88 −/− fish. According to gene expression analysis, VHSV NP gene expression was considerably upregulated in myd88 −/− fish. Substantial gene expression and immune cell marker modulation were observed in the mutant model compared to that in the wild-type. These results suggest that the lack of a significant adaptor protein for immune signal transduction results in enhanced VHSV replication. • A myd88 ablated zebrafish with a 4 bp frameshift mutation was generated by CRISPR/Cas9 genomic engineering. • myd88 KO fish had lower survival and higher VHSV titers during the VHSV infection experiment. • A lack of myd88 impairs the rapid immune responses to VHSV. • The tail injury model of myd88 ablated fish had higher virus penetration. • myd88 ablation significantly impaired the immune cell dynamics. [ABSTRACT FROM AUTHOR]

Aquaporin 8 (Aqp8) is a transmembrane protein that is selectively permeated by water and some small solutes, and physiologically contributes to acid-base equilibrium in the gastrointestinal tract. Here, we described the characterization and spatiotemporal expression pattern of zebrafish aqp8 ( zaqp8 ) gene family, including zaqp8a.1 , zaqp8a.2, and zaqp8b , during the early developmental stages. The expression of zaqp8a.1 started first in the lateral plate mesoderm at the 12-somite stage (ss) and then expanded sequentially to the dorsal aorta, intersegmental blood vessels and then to the dorsal longitudinal anastomotic vessel at 24 h post fertilization (hpf). At 28 hpf, expression of zaqp8a.1 was also detected in the embryonic heart tube. Four days post fertilization (dpf), strong zaqp8a.1 expression was detected in the gastrointestinal tract and liver. By 72 hpf, the expression of zaqp8a.2 was first detected in the primitive gut region but not detected in the liver. The expression of z a qp8b was first detected in the intermediate mesoderm at 10 ss. From 24 hpf to 6 dpf, the proximal convoluted segment of the embryonic kidney was marked by zaqp8b expression Overall, these differential expression patterns of aqp8a.1 , aqp8a.2, and aqp8b suggest that they possibly play distinct roles throughout the embryonic development by controlling or maintaining organ-specific cellular water homeostasis. Our study provides new evidence that organogenesis requires differential roles of Aqp8 proteins in zebrafish. [ABSTRACT FROM AUTHOR]

In this study, fluorene (FL), FL-1-carboxylic acid (FC-1), and FL-9-carboxylic acid (FC-9) were investigated to understand their acute toxicity by measuring inhibitory effects on hatching rates and developmental processes of zebrafish embryos (Danio rerio). For exposure concentrations up to 3000 μg/L, FC-1 alone showed acute toxicity at 1458 μg/L for LC 50 value. FC-1 caused yolk sac and spinal deformities, and pericardial edema. Molecular studies were undertaken to understand FC-1 toxicity examining 61 genes after exposure to 5 μM (equivalent to LC 20 value of FC-1) in embryos. In the FC-1-treated embryos, the expression of the cyp7a1 gene, involved in bile acid biosynthesis, was dramatically decreased, while the expression of the Il-1β gene involved in inflammation was remarkably increased. In addition to these findings, in FC-1-treated embryos, the expression of nppa gene related to the differentiation of the myocardium was 3-fold increased. On the other hand, cyp1a , cyp3a , ugt1a1 , abcc4 , mdr1 , and sult1st1 responsible for detoxification of xenobiotics were upregulated in FC-9-treated embryos. Taken together, carboxylation on carbon 1 of FL increased acute toxicity in zebrafish embryos, and its toxicity might be related to morphological changes with modification of normal biological functions and lowered defense ability. Image 1 • Fluorene-1-carboxylic acid exhibited potent acute toxicities on zebrafish embryos, including high mortality rate. • Fluorene-1-carboxylic acid induced abnormal developments such as yolk sac, spinal deformities, and pericardial edema. • Fluorene-1-carboxylic acid down-regulated cyp7a1 gene expression involved in bile acid biosynthesis. • Carboxylation on 1-carbon in fluorine ring core might mediate metabolic modifications when compared to fluorine. [ABSTRACT FROM AUTHOR]