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.

Characterization and evaluation of an oral vaccine via nano-carrier for surface immunogenic protein (Sip) delivery against Streptococcus agalactiae infection.

Streptococcus agalactiae causes systemic disease in a variety of wild and farmed fish, resulting in high levels of morbidity and mortality, as well as serious economic losses to the Nile tilapia aquaculture industry. The development of economic and applicable oral vaccines is therefore urgently needed for the sustainable development of Nile tilapia aquaculture. In this study, mesoporous silica nanoparticles (MSNs) were fabricated using sol-gel synthesis technology, and the antigens of surface immunogenic protein (Sip) was loaded into MSNs to develop a nanovaccine MSNs-Sip@HP55. The results showed that the prepared nanovaccine exhibited pH-controlled release, which could survive in the simulated gastric environment (pH 1.5), and release antigens in the simulated intestinal environment at pH 7.4. The nanovaccine could induce innate and adaptive immune responses in Nile tilapia. When the challenge doses were 1.5 × 106, 1.18 × 106, and 0.88 × 106 CFU/mL, the relative protection rates in immunized Nile tilapia were 63.33 %, 64.23 %, and 76.31 %, respectively. Taken together, the nanovaccine exhibited a high antigen utilization rate and was easily administered orally via feeding, which could protect Nile tilapia against challenge with S. agalactiae in large-scale farms. Oral vaccine based on MSNs carriers is a potentially promising strategy for the development of fish vaccines.

Identification and Expression Analysis of LncRNAs Reveal the Immune Mechanism of Visceral White-Nodules Disease Resistance in Large Yellow Croaker.

Long non-coding RNAs (lncRNAs) have several known functions in fish growth processes and signal transduction, but their possible roles in response to bacterial diseases remain largely unresolved. In this study, we report a comprehensive cold-water bacterial disease-responsive lncRNA expression profile for understanding the transcriptional regulatory mechanisms of visceral white-nodules disease resistance in large yellow croaker. A total of 2534 high-confidence lncRNAs were identified by a rigorous filtering pipeline as a basic sequence set for comparative transcriptional analysis. In addition, a total of 10,200 lncRNA-mRNA pairs with high correlation coefficients were identified by expressions level correlation analysis, including non-redundant 381 DE lncRNAs and 2590 differential expressed genes. MSTRG_11084_1 and MSTRG_20402_1 were linked to a large number of target genes and may be involved in important functions in immune regulation. We further revealed the conserved and idiosyncratic features of the disease response process between the technical control strain (TCS) and the resistant strain (RS). Immune-related pathways were enriched in GO terms and KEGG pathways, among which cytokine-cytokine receptor interaction, MAPK signaling pathway, and NF-kappa B signaling pathway may play a key role in VWND resistance in large yellow croaker. Protein-protein interaction network (PPI) analysis revealed that immune-related target genes such as il-10, met, acta2, myc, cav1, and ntrk1, as well as growth and metabolism-related target genes such as pik3r2, igf1, sc5d, hmgcr, and lss were considered the main hub genes. This study represents the first characterization of lncRNAs involved in VWND resistance in large yellow croaker and provides new clues for elucidating the disease response mechanism of large yellow croaker.

Integrative analysis of GWAS and transcriptome reveals p53 signaling pathway mediates resistance to visceral white-nodules disease in large yellow croaker.

Visceral white-nodules disease (VWND), caused by Pseudomonas plecoglossicida, is one of the primary causes of morbidity and mortality in large yellow croaker aquaculture. Host disease resistance is a heritable trait that involves complex regulatory processes. However, the regulatory mechanism of bacterial resistance in large yellow croaker is still unclear. This study attempted to systematically evaluate the major genetic loci and transcriptional regulatory mechanisms associated with the resistance to VWND in large yellow croaker by crossover method studies. A large population of large yellow croaker was challenged with P. plecoglossicida, with survival time recorded and samples were taken for genotyping. Meanwhile, spleen samples that were used for RNA-seq to compare their transcriptomic profiles before and after infection were taken from resistant populations (RS) and susceptible control populations (CS) bred using the genomic selection (GS) technique. Genome-wide association analyses using 46 K imputed SNP genotypes highlighted that resistance is a polygenic trait. The integrative analysis results show the co-localization of the cd82a gene between disease resistance-related genetic loci and comparative transcriptional analysis. And functional enrichment analysis showed differential enrichment of the p53 signaling pathway in RS and CS groups, suggesting that there may be cd82a-mediated p53 signaling pathway activation for VWND resistance. This large-scale study provides further evidence for the heritability and transcriptional regulatory mechanisms of host inheritance of VWND resistance.

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.

Early immune response in large yellow croaker (Larimichthys crocea) after immunization with oral vaccine.

Mesoporous silica nanoparticles (MSNs) have been used in the field of biomedicine as antigen carriers and adjuvants for protective antigens. In the present study, an oral nanovaccine against Vibrio alginolyticus was prepared employing MSNs as carriers. The uptake of the dihydrolipoamide dehydrogenase (DLDH) antigens in the intestine of large yellow croaker was evaluated using an immunohistochemistry assay. Additionally, the effects of the nanovaccine on the early immune response in large yellow croaker were investigated via oral vaccination. The presence of the antigens was detected in the mucosa and lamina propria of the foregut, midgut, and hindgut of large yellow croaker at 3 h following oral immunization. The expression levels of cytokines (i.e., lysozyme, IFN-γ, IFITM, TNF-α, IL-1ß, IL-2, IL-4, IL-10, and IL-13) in the intestine, spleen, and head kidney tissues of large yellow croaker before and after the immune challenge were determined via RT-qPCR assay. The obtained results revealed that the expression levels of lysozyme, IFN-γ, IFITM, TNF-α, IL-1ß, IL-2, IL-4, IL-10, and IL-13 in the intestine and head kidney of the vaccinated large yellow croaker, as well as the expression of lysozyme, IL-1ß, and IL-10 in the spleen, exhibited time-dependent oscillation regulation patterns. Notably, the nanovaccine immunization could induce early (6 h) and high expression of IFN-γ in the spleen and kidney tissues after the bacterial infection. The current study supplements the available data on the early immune response to fish nanovaccines. It also provides a valuable theoretical basis for the future development of large yellow croaker oral vaccines.

Infectivity and genes differentially expressed between young and aging theront cells of the marine fish parasite Cryptocaryon irritans.

The ciliated protozoan Cryptocaryon irritans infects a wide range of marine fish and causes the highly lethal white spot disease. This parasite possesses three morphologically and physiologically distinct life stages: an infectious theront, a parasitic trophont, and an asexually reproductive tomont. In the past few years, several attempts have been made to help elucidate how C. irritans transforms from one stage to another using transcriptomic or proteomic approaches. However, there has been no research studying changes in transcription profiles between different time points of a single C. irritans life stage-the development of this parasite. Here we use RNA-seq and compare gene expression profiles of theront cells collected by 1 and 10 hrs after they emerged from tomonts. It has been shown that infectivity of theront cells declines 6-8 hours post-emergence, and we used this characteristic as a physiological marker to confirm the aging of theront cells. We identified a total of 41 upregulated and 90 downregulated genes that were differentially expressed between young and aging theront cells. Using Blast2Go to further analyze functions of these genes, we show that genes related to energy production are downregulated, but quite surprisingly many genes involved in transcription/translation processes are upregulated. We also show that expression of all nine detectable agglutination/immobilization antigen genes, with great sequence divergence, is invariably downregulated. Functions of other differentially expressed genes and indications are also discussed in our study.

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.

System for the heterologous expression of NS1 protein of H9N2 avian influenza virus in the ciliate Tetrahymena thermophila.

Tetrahymena is commonly used as an alternative eukaryotic system for efficiently expressing heterologous genes. In this study, we inserted the non-structural (NS) 1 gene of avian influenza virus (AIV) into the shuttle vector pD5H8 and transformed conjugating T. thermophila with the recombinant plasmid pD5H8-NS1 by particle bombardment. Positive transformants were selected with paromomycin. We demonstrated that the NS1 protein could be expressed steadily following induction with cadmium in this Tetrahymena system. An enzyme-linked immunosorbent assay detection method was preliminary established using the expressed protein as coating antigens for serodiagnosis. This is the first study in which a Tetrahymena expression system was employed for the expression of the AIV NS1 protein, and it provides a good basis for the development of differential diagnostic kits and vaccines for the prevention and control of avian influenza.

High genetic diversities between isolates of the fish parasite Cryptocaryon irritans (Ciliophora) suggest multiple cryptic species.

The ciliate protozoan Cryptocaryon irritans parasitizes marine fish and causes lethal white spot disease. Sporadic infections as well as large-scale outbreaks have been reported globally and the parasite's broad host range poses particular threat to the aquaculture and ornamental fish markets. In order to better understand C. irritans' population structure, we sequenced and compared mitochondrial cox-1, SSU rRNA, and ITS-1 sequences from 8 new isolates of C. irritans collected in China, Japan, and Taiwan. We detected two SSU rRNA haplotypes, which differ at three positions, separating the isolates into two main groups (I and II). Cox-1 sequences also support the division into two groups, and the cox-1 divergence between these two groups is unexpectedly high (9.28% for 1582 nucleotide positions). The divergence is much greater than that detected in Ichthyophthirius multifiliis, the ciliate protozoan causing freshwater white spot disease in fish, where intraspecies divergence on cox-1 sequence is only 1.95%. ITS-1 sequences derived from these eight isolates and from all other C. irritans isolates (deposited in the GenBank) not only support the two groups, but further suggest the presence of a third group with even greater sequence divergence. Finally, a small Ka/Ks ratio estimated from cox-1 sequences suggests that this gene in C. irritans remains under strong purifying selection. Taken together, the C. irritans species may consists of many subspecies and/or syngens. Further work is needed to determine if there is reproductive isolation between the groups we have defined.