Dietary Administration of Novel Multistrain Probiotics from Healthy Grouper Intestines Promotes the Intestinal Immune Response against NNV Infection.

Epinephelus lanceolatus (giant grouper) is a high-value cultured species in the Asia-Pacific region. However, nervous necrosis virus (NNV) is an infectious viral disease that affects over 120 species of marine cultured species and causes high mortality, ranging from 90-100% in the grouper industry. Probiotics isolated from the intestines of healthy individuals have provided insight into novel approaches involved in the defense against viral pathogens. In this study, we isolated three strains of bacteria as candidate probiotics from healthy grouper intestines and a 28-day feeding trial was performed. At day 21, the nervous necrosis virus (NNV) challenge test was conducted for 7 days to evaluate the antiviral effect of candidate probiotics. The results showed that candidate probiotics could improve growth conditions, such as weight gain (WG) and specific growth rate (SGR), and increase the utilization of feed. Furthermore, the candidate probiotic mixture had the ability to protect against NNV, which could decrease the mortality rate by 100% in giant grouper after NNV challenge. Subsequently, we analyzed the mechanism of the candidate probiotic mixture's defense against NNV. A volcano plot revealed 203 (control vs. NNV), 126 (NNV vs. probiotics - NNV), and 5 (control vs. probiotics - NNV) differentially expressed transcripts in intestinal tissue. Moreover, principal components analysis (PCA) and cluster analysis heatmap showed large differences among the three groups. Functional pathway analysis showed that the candidate probiotic mixture could induce the innate and adaptive immunity of the host to defend against virus pathogens. Therefore, we hope that potential candidate probiotics could be successfully applied to the industry to achieve sustainable aquaculture.

The Alteration of Intestinal Microbiota Profile and Immune Response in Epinephelus coioides during Pathogen Infection.

Epinephelus coioides, or grouper, is a high economic value fish species that plays an important role in the aquaculture industry in Asia. However, both viral and bacterial diseases have threatened grouper for many years, especially nervous necrosis virus, grouper iridovirus and Vibrio harveyi, which have caused a bottleneck in the grouper industry. Currently, intestinal microbiota can provide novel insights into the pathogenesis-related factors involved in pathogen infection. Hence, we investigated the comparison of intestinal microbiota communities in control group and pathogen-infected grouper through high-throughput sequencing of the 16S rRNA gene. Our results showed that microbial diversity was decreased, whereas microbial richness was increased during pathogen infection. The individuals in each group were distributed distinctly on the PLSDA diagram, especially the GIV group. Proteobacteria and Firmicutes were the most abundant bacterial phyla in all groups. Interestingly, beneficial genera, Faecalibacterium and Bifidobacterium, predominated in the intestines of the control group. In contrast, the intestines of pathogen-infected grouper had higher levels of harmful genera such as Sphingomonas, Atopostipes, Staphylococcus and Acinetobacter. Additionally, we investigated the expression levels of innate and adaptive immune-related genes after viral and bacterial infection. The results revealed that immunoglobulin T and proinflammatory cytokine levels in the intestine increased after pathogen infection. Through these unique bacterial compositions in diseased and uninfected fish, we could establish a novel therapeutic approach and bacterial marker for preventing and controlling these diseases.

Secretory Production of Functional Grouper Type I Interferon from Epinephelus septemfasciatus in Escherichia coli and Bacillus subtilis.

Nervous necrosis virus (NNV) results in high mortality rates of infected marine fish worldwide. Interferons (IFNs) are cytokines in vertebrates that suppress viral replication and regulate immune responses. Heterologous overexpression of fish IFN in bacteria could be problematic because of protein solubility and loss of function due to protein misfolding. In this study, a protein model of the IFN-α of Epinephelus septemfasciatus was built based on comparative modeling. In addition, PelB and SacB signal peptides were fused to the N-terminus of E. septemfasciatus IFN-α for overexpression of soluble, secreted IFN in Escherichia coli (E-IFN) and Bacillus subtilis (B-IFN). Cytotoxicity tests indicated that neither recombinant grouper IFN-α were cytotoxic to a grouper head kidney cell line (GK). The GK cells stimulated with E-IFN and B-IFN exhibited elevated expression of antiviral Mx genes when compared with the control group. The NNV challenge experiments demonstrated that GK cells pretreated or co-treated with E-IFN and B-IFN individually had three times the cell survival rates of untreated cells, indicating the cytoprotective ability of our recombinant IFNs. These data provide a protocol for the production of soluble, secreted, and functional grouper IFN of high purity, which may be applied to aquaculture fisheries for antiviral infection.

The microbiota profile and transcriptome analysis of immune response during metamorphosis stages in orange spotted grouper (Epinephelus coioides).

Metamorphosis is a transformation process in larval development associated with changes in morphological and physiological features, including the immune system. The gastrointestinal tract harbors a plethora of bacteria, which might affect the digestion and absorption of nutrients, immunity, and gut-brain crosstalk in the host. In this study, we have performed metagenomic and transcriptomic analyses on the intestines of grouper at the pre-, mid- and post-metamorphosis stages. The sequencing data of 16S rRNA gene showed drastic changes in the microbial communities at different developmental stages. The transcriptomic data revealed that the leukocyte transendothelial migration and the phagosome pathways might play important roles in mediating immunity in grouper at the three developmental stages. This information will increase our understanding of the metamorphosis process in grouper larvae, and shed light on the development of antimicrobial strategy during larval development.

Cloning and expression of Malabar grouper (Epinephelus malabaricus) ADAR1 gene in response to immune stimulants and nervous necrosis virus.

ADARs are RNA editing catalysts that bind double-stranded RNA and convert adenosine to inosine, a process that can lead to destabilization of dsRNA structures and suppression of mRNA translation. In mammals, ADAR1 genes are involved in various cellular pathways, including interferon (IFN)-mediated response. However, the function of fish ADAR1 remains unclear. We report here the cloning of ADAR1 in Malabar grouper (Epinephelus malabaricus) (MgADAR1) and its response to various immune stimulants. The MgADAR1 cDNA is 5371-bp long, consisting of an open reading frame encoding a putative protein of 1381 amino acids, a 235-nt 5'-terminal untranslated region (UTR), and a 990-nt 3'-UTR. The deduced amino acid sequence exhibits signature features of a chitin synthesis regulation domain, two Z-DNA-binding domains (Z alpha), three dsRNA binding motifs (DSRM) and one tRNA-specific and dsRNA adenosine deaminase domain (ADEAMc). MgADAR1 mRNA expressed ubiquitously in tissues of healthy Malabar grouper, with elevated levels in the brain, gills and eyes. In response to poly (I: C), the MgADAR1 mRNA level was significantly up-regulated in the brain and spleen, but not head kidney. Upon nervous necrosis virus (NNV) infection the level of MgADAR1 increased in the brain, whereas Mx increased in the brain, spleen and head kidney. Induction of MgADAR1 by poly (I: C) and NNV was also observed in vitro. Additionally, the expression of MgADAR1 was upregulated by recombinant grouper IFN in grouper cells. These data indicate an intricate interplay between ADAR1 and NNV infection in grouper as MgADAR1 might be regulated in a tissue-specific manner.