Molecular characterization, expression pattern and immunologic function of CD82a in large yellow croaker (Larimichthys crocea).

Visceral white spot disease (VWND) caused by Pseudomonas plecoglossicida poses a major threat to the sustainable development of large yellow croaker (Larimichthys crocea) aquaculture. Genome-wide association analysis (GWAS) and RNA-seq research indicated that LcCD82a play an important role in resistance to visceral white spot disease in L. crocea, but the molecular mechanism of LcCD82a response to P. plecoglossicida infection is still unclear. In this study, we cloned and validated the Open Reading Frame (ORF) sequence of LcCD82a and explored the expression profile of LcCD82a in various tissues of L.crocea. In addition, two different transcript variants (LcCD82a-L and LcCD82a-S) of LcCD82a were identified that exhibit alternative splicing patterns after P. plecoglossicida infection, which may be closely related to the immune regulation during pathogenetic process of VWND. In order to explore the function of LcCD82a, we purified the recombinant protein of LcCD82a-L and LcCD82a-S. The bacterial agglutination and apoptosis function analysis showed that LcCD82a may involve in extracellular bacterial recognition, agglutination, and at the same time participate in the process of antigen presentation and induction of cell apoptosis. Collectively, our studies demonstrate that LcCD82a plays a crucial role in regulating apoptosis and antimicrobial immunity.

Gill Transcriptome Analysis Revealed the Difference in Gene Expression Between Freshwater and Seawater Acclimated Guppy (Poecilia reticulata).

Guppy (Poecilia reticulata) can adapt to a wide range of salinity changes. To investigate the gene expression changes in the guppy exposed to seawater, we characterized its gill transcriptome using RNA sequencing. Experimental fish were exposed to salinity increase from 0 to 30‰ within 4 days, while control fish were cultured in freshwater (0‰ salinity). Seven days after salinity exposure, the gills were sampled and the mortality within 2 weeks was recorded. No significant difference in the cumulative mortality at the second week was found between the two groups. Transcriptomic analysis identified 3477 differentially expressed genes (DEGs), including 1067 upregulated and 2410 downregulated genes. These DEGs were enriched in several biological processes, including ion transport, ion homeostasis, ATP biosynthetic process, metabolic process, and immune system process. Oxidative phosphorylation was the most activated pathway. DEGs involved in the pathway "endoplasmic reticulum (ER)-mediated phagocytosis," "starch and sucrose metabolism," and "steroid biosynthesis" were mainly downregulated; chemokines and interleukins involved in "cytokine-cytokine receptor interaction" were differentially expressed. The present results suggested that oxidative phosphorylation had essential roles in osmoregulation in the gills of seawater acclimated guppy, during which the decline in the expression of genes encoding V-ATPases and calreticulin had a negative effect on the phagocytosis and immune response. Besides, several metabolic processes including "starch and sucrose metabolism" and "steroid biosynthesis" were affected. This study elucidates transcriptomic changes in osmotic regulation, metabolism, and immunity in seawater acclimated guppy.

Development and characterization of a continuous cell line (EL) from the liver of European eel Anguilla anguilla.

In the present study, a new hepatic tissue-origin cell line from European eel Anguilla anguilla has been developed and characterized. This cell line designated EL has been maintained in Leibovitz L-15 supplemented with 10% fetal bovine serum over 72 months, and subcultured more than 90 times. The EL cell line consisted predominantly of fibroblast-like cells, which could survive over 100 days in vitro, and could grow at 15-32°C. The optimum temperature for growth was 27°C. The chromosome analysis revealed a modal diploid karyotype of 2n = 38. The origin of this cell line was confirmed by the 18S recombinant (r)RNA sequencing. The susceptibility test indicated significant cytopathic effects in the EL cells with regard to the Rana grylio virus and the Herpesvirus anguillae. The viral replication was confirmed by transmission electron microscopy and polymerase chain reaction analysis. Following poly (I:C) exposure, the expression levels of the immune-related molecules interferon regulatory factor-7 (irf7) and transforming growth factor-ß (TGF-ß) were downregulated in EL cells, whereas the expression levels of the rf3 and the cytochrome P450 (CYP450) were upregulated. All four genes were significantly upregulated following inflammation by lipopolysaccharide (LPS). These data suggested the application of EL cell line for viral identification, as well as for immunodiagnosis and pharmacological targeting.

Development and characterization of a new cell line derived from European eel Anguilla anguilla kidney.

A new cell line derived from the kidney of European eel, Anguilla anguilla, has been established and characterized. This cell line, designated as EK (eel kidney), has been maintained in Leibovitz's L-15 supplemented with 10% fetal bovine serum for over 24 months, and subcultured more than 60 times. This cell line consists predominantly of fibroblast-like cells, and can grow at 15-37°C under an optimum temperature of 26°C. The origin of this cell line was confirmed by polymerase chain reaction (PCR) amplification and 18s recombinant (r)RNA sequencing. The chromosome analysis of EK cells at passage 58 revealed an ananeuploid karyotype. The EK cells were successfully transfected with the Pegfp-N1 plasmid, suggesting its potential in genetic studies. The susceptibility test showed a significant cytopathic effect (CPE) in EK cells for Rana grylio virus, and the viral replication was evidenced with quantitative real-time PCR (qRT-PCR) assay. After poly (I:C) stimulation, the expression of the immune-related molecules including interferon regulatory factor-3 (irf3), interferon regulatory factor-7 (irf7) and cytochrome P450 (CYP450) were significantly upregulated in EK cells, while the expression of transforming growth factor (TGF-ß) was downregulated. These results suggested the potential of EK cell line as a model in gene engineering, virus identification and environmental toxicology.

Essential role of TRPC channels in the guidance of nerve growth cones by brain-derived neurotrophic factor.

Brain-derived neurotrophic factor (BDNF) is known to promote neuronal survival and differentiation and to guide axon extension both in vitro and in vivo. The BDNF-induced chemo-attraction of axonal growth cones requires Ca2+ signalling, but how Ca2+ is regulated by BDNF at the growth cone remains largely unclear. Extracellular application of BDNF triggers membrane currents resembling those through TRPC (transient receptor potential canonical) channels in rat pontine neurons and in Xenopus spinal neurons. Here, we report that in cultured cerebellar granule cells, TRPC channels contribute to the BDNF-induced elevation of Ca2+ at the growth cone and are required for BDNF-induced chemo-attractive turning. Several members of the TRPC family are highly expressed in these neurons, and both Ca2+ elevation and growth-cone turning induced by BDNF are abolished by pharmacological inhibition of TRPC channels, overexpression of a dominant-negative form of TRPC3 or TRPC6, or downregulation of TRPC3 expression via short interfering RNA. Thus, TRPC channel activity is essential for nerve-growth-cone guidance by BDNF.