Isolation, identification, and pathogenic characteristics of Nocardia seriolae in largemouth bass Micropterus salmoides.

The largemouth bass Micropterus salmoides is an important freshwater aquaculture fish in China. Recently, largemouth bass at a fish farm in Guangdong province experienced an outbreak of a serious ulcer disease. As part of the investigations conducted to identify the aetiology and identify potentially effective control measures, we isolated a pathogenic bacterium (NK-1 strain) from the diseased fish. It was identified as Nocardia seriolae through morphological observation, physiological and biochemical analysis, and molecular identification, and its pathogenicity was verified by experimental infection. Pathological changes in the diseased fish included granulomatous lesions in the liver and spleen, destruction of renal tubules, necrosis of intestinal epithelial cells, infiltration of inflammatory cells in the brain, vacuolation of cells, and swelling and cracking of the mitochondria and endoplasmic reticulum. Bacterial detection using qPCR showed that the spleen and intestine were the main organs targeted by N. seriolae. The mortality of largemouth bass experimentally infected with N. seriolae at 21°C was significantly lower than that in fish infected at higher temperatures between 24 and 33°C; there were no significant differences in the levels of mortality at these higher temperatures. The level of mortality of largemouth bass infected with N. seriolae was lowest at a neutral water pH of 7 but increased significantly at higher and lower pH. Of the tested Chinese herbal medicines, Chinese sumac Galla chinensis and Chinese skullcap Scutellaria baicalensis exhibited the best antibacterial effects. This study lays a foundation for the clinical diagnosis and scientific control of ulcer disease in largemouth bass.

DDX43 recruits TRIF or IPS-1 as an adaptor and activates the IFN-ß pathway in Nile tilapia (Oreochromis niloticus).

DDX43 is one of the members of the DExD/H-box protein family, and emerging data suggest that it may play an important role in antiviral immunity across mammals. However, little is known about DDX43 in the fish immune response. In this study, we isolated the cDNA sequence of ddx43 in Nile tilapia (Oreochromis niloticus). The ddx43 gene was 2338 bp in length, contained an open reading frame (ORF) of 2064 bp and encoded a polypeptide of 687 amino acids. The predicted protein of OnDDX43 has three conserved domains, including the RNA binding domain KH, DEAD-like helicase superfamily DEXDc and C-terminal HELICc domain. In healthy Nile tilapia, the Onddx43 transcript was broadly expressed in all examined tissues, with the highest expression levels in the muscle and brain and the lowest in the liver. After challenge with Streptococcus agalactiae, lipopolysaccharides (LPS) and polyinosinic polycytidylic acid (Poly I:C), the expression level of Onddx43 mRNA was upregulated or downregulated in all of the tissues tested. Overexpression of OnDDX43 in 293 T cells showed that it has a positive regulatory effect on IFN-ß. The subcellular localization showed that OnDDX43 was expressed in the cytoplasm. We performed further pull-down assays and found that OnDDX43 interacted with both interferon-ß promoter stimulator1 (IPS-1) and TIR domain-containing adaptor inducing interferon-ß (TRIF).

R-spondin1 in loach (Misgurnus anguillicaudatus): Identification, characterization, and analysis of its expression patterns and DNA methylation in response to high-temperature stress.

With a well-understood function in mammals, R-spondin1 (Rspo1) is an important regulator of ovarian development via the Wnt/ß-catenin pathway. Rspo1 deficiency causes retardation of ovarian development in XX fish, and increases Rspo1 function induces femininity and sex reversal in XY fish. In this study, Rspo1 was successfully cloned from loach (Misgurnus anguillicaudatus), and its expression profile was analyzed. The full-length cDNA of Misgurnus anguillicaudatus Rspo1 (MaRspo1) comprised 1322 bp and included an open reading frame (ORF) of 795 bp, which encoded a predicted polypeptide measuring 264 amino acids in length. Phylogenetic and gene structure analyses showed a highly conserved sequence of MaRspo1 (identical to the Rspo1 genes of other species), consisting of an N-terminal signal peptide (SP), two furin-like cysteine-rich domains (FU1 and FU2), a thrombospondin type 1 repeat (TSP1) and a C-terminal region. Real-time PCR revealed the female-biased expression profile of MaRspo1, with the highest expression level among tested tissues detected in ovary. Investigation of MaRspo1 expression levels throughout the early development stage (10-60 days post hatching) under three temperature treatments (25 °C, 28 °C, and 31 °C) revealed significantly differential expression of MaRspo1 among the three temperature groups, with decreased MaRspo1 expression in the high-temperature (31 °C) group. The results of DNA methylation analysis indicated that exposure to high temperature during early development can increase the average promoter methylation level of MaRspo1 in both females and males. Taken together, the results of this study provide the basis for the further investigation of the molecular mechanism of Rspo1 in response to temperature.

Molecular characterization and function analysis of three RIG-I-like receptor signaling pathway genes (MDA5, LGP2 and MAVS) in Oreochromis niloticus.

The recognition of microbial pathogens, which is mediated by pattern recognition receptors (PRRs), is critical to the initiation of innate immune responses. In the present study, we isolated the full-length cDNA and genomic DNA sequences of the MDA5, LGP2 and MAVS genes in Nile tilapia, termed OnMDA5, OnLGP2 and OnMAVS. The OnMDA5 gene encodes 974 amino acids and contains two caspase-associated recruitment domains (CARDs), a DExDc domain (DExD/H box-containing domain), a HELICc (helicase superfamily C-terminal) domain and a C-terminal regulatory domain (RD). The OnLGP2 gene encodes 679 amino acids and contains a DExDc, a HELICc and an RD. The OnMAVS gene encodes 556 amino acids and contains a CARD, a proline-rich domain, a transmembrane helix domain and a putative TRAF2-binding motif (269PVQDT273). Phylogenetic analyses showed that all three genes from Nile tilapia were clustered together with their counterparts from other teleost fishes. Real-time PCR analyses showed that all three genes were constitutively expressed in all examined tissues in Nile tilapia. OnMDA5 presented the highest expression level in the blood and the lowest expression level in the liver, while OnMAVS presented the highest expression level in the kidney. The highest expression level of OnLGP2 was detected in the liver. An examination of the expression patterns of these RIG-I-like receptors (RLRs) during embryonic development showed that the highest expression levels of OnMDA5 occurred at 2 days postfertilization (dpf), and the expression significantly decreased from 3 to 8 dpf. The expression levels of OnLGP2 significantly increased from 4 to 8 dpf. The expression levels of OnMAVS mRNA were stable from 2 to 8 dpf. Upon stimulation by intraperitoneal injection of Streptococcus agalactiae, the expression levels of OnMDA5 were first downregulated and then upregulated in the blood, gill and spleen. In the intestine and kidney, the expression of OnMDA5 was first upregulated, then downregulated, and then upregulated again. The expression of OnLGP2 was upregulated in the kidney and intestine, and the expression of OnMAVS was upregulated in the spleen. Overexpression of OnMAVS increased NF-κB activation in 293 T cells (p < 0.05), and after cotransfection with OnMDA5, the OnMAVS-dependent NF-κB activation was slightly increased (p > 0.05), after cotransfection with OnLGP2, the OnMAVS-dependent NF-κB activation was significantly decreased (p < 0.05). These findings suggest that, although the deduced protein structure of OnMDA5 is evolutionarily conserved with the structures of other RLR members, its signal transduction function is markedly different. The results also suggest that OnLGP2 has a negative regulatory effect on the OnMAVS gene. OnMDA5 and OnMAVS were uniformly distributed throughout the cytoplasm in 293 T cells, whereas OnLGP2 was distributed throughout the cytoplasm and nucleus. These results are helpful for clarifying the innate immune response against bacterial infection in Nile tilapia.

Characterization and expression of Na+/K+-ATPase in gills and kidneys of the Teleost fish Oreochromis mossambicus, Oreochromis urolepis hornorum and their hybrids in response to salinity challenge.

Tilapia (Oreochromis mossambicus, O. urolepis hornorum, their hybrids O. mossambicus♀ × O. hornorum♂ and O. hornorum♀ × O. mossambicus♂) were exposed to a high salinity environment to evaluate their osmoregulatory responses. The plasma osmolality of all the tilapia species were elevated with the salinity challenge. The activities of Na+/K+-ATPase (NKA) in both the gill and kidney showed a similar increased change tendency compared with the control. The distribution of NKA α1 mRNA in all the examined tissues suggested that NKA α1 has a possible housekeeping role for this isoform. The amount of NKA α1 mRNA in the gill and kidney was elevated in the four fishes with similar expression patterns after transfer from freshwater to seawater. The NKAα1 mRNA expression levels in the gill reached their peak level at 24 h after transfer (P < 0.01) compared to the freshwater group, following decreases in the pretreatment level at 48 h (P > 0.05). However, the NKAα1 mRNA expression levels in the kidney were not significantly affected with increasing environmental salinity (P > 0.05). The differences in the responses to saltwater challenge may be associated with differences in saltwater tolerance between the four tilapia. The drastic increase in the plasma osmolality, NKA activities and mRNA expression suggested that the hybrids (O. mossambicus♀ × O. hornorum♂) possess heterosis in salinity responsiveness compared to that of both the parents, indicating a maternal effect on the salinity tolerance of the tilapia hybrids. This study provides a theoretical basis to further study the mechanism of fish osmoregulation response to salinity challenge.

Identification of novel MYO18A interaction partners required for myoblast adhesion and muscle integrity.

The unconventional myosin MYO18A that contains a PDZ domain is required for muscle integrity during zebrafish development. However, the mechanism by which it functions in myofibers is not clear. The presence of a PDZ domain suggests that MYO18A may interact with other partners to perform muscle-specific functions. Here we performed double-hybrid screening and co-immunoprecipitation to identify MYO18A-interacting proteins, and have identified p190RhoGEF and Golgin45 as novel partners for the MYO18A PDZ domain. We have also identified Lurap1, which was previously shown to bind MYO18A. Functional analyses indicate that, similarly as myo18a, knockdown of lurap1, p190RhoGEF and Golgin45 by morpholino oligonucleotides disrupts dystrophin localization at the sarcolemma and produces muscle lesions. Simultaneous knockdown of myo18a with either of these genes severely disrupts myofiber integrity and dystrophin localization, suggesting that they may function similarly to maintain myofiber integrity. We further show that MYO18A and its interaction partners are required for adhesion of myoblasts to extracellular matrix, and for the formation of the Golgi apparatus and organization of F-actin bundles in myoblast cells. These findings suggest that MYO18A has the potential to form a multiprotein complex that links the Golgi apparatus to F-actin, which regulates muscle integrity and function during early development.

The PDZ-containing unconventional myosin XVIIIA regulates embryonic muscle integrity in zebrafish.

Myosin XVIIIA, or MYO18A, is a unique PDZ domain-containing unconventional myosin and is evolutionarily conserved from Drosophila to vertebrates. Although there is evidence indicating its expression in the somites, whether it regulates muscle function remains unclear. We show that the two zebrafish myo18a genes (myo18aa and myo18ab) are predominantly expressed at somite borders during early developmental stages. Knockdown of these genes or overexpression of the MYO18A PDZ domain disrupts myofiber integrity, induces myofiber lesions, and compromises the localization of dystrophin, α-dystroglycan (α-DG) and laminin at the myotome boundaries. Cell transplantation experiments indicate that myo18a morphant myoblasts fail to form elongated myofibers in the myotomes of wild-type embryos, which can be rescued by the full-length MYO18A protein. These results suggest that MYO18A likely functions in the adhesion process that maintains the stable attachment of myofibers to ECM (extracellular matrix) and muscle integrity during early development.