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The tumour necrosis factor (TNF) super-family is a group of important cytokines involved in inflammation, apoptosis, cell proliferation, and the general stimulation of the immune system. The TNF gene has been cloned in some bony fish; however, its counterparts are still unidentified in the majority of fish species. In this study, we cloned gTNF-1 and gTNF-2 from the orange-spotted grouper (Epinephelus coioides), an economically important farmed fish. Both genes include 4 exons and 3 introns and encoded 253 and 241 amino acid proteins with a molecular weight of approximately 27 and 26 kDa, respectively. The identity of the putative amino acid sequences between gTNF-1 and gTNF-2 was only 38%. The positions of cysteine residues, a protease cleavage site, and a transmembrane domain sequence derived from gTNF-1 and gTNF-2 were similar to those in other fish and mammalian TNF-α. The mRNA expression levels of the 2 gTNF molecules were evaluated in unstimulated/stimulated peripheral blood leukocytes, various organs, and fish larvae. Following lipopolysaccharide (LPS) treatment, gTNF-2 was expressed at higher levels, was up-regulated more quickly, and was more sensitive to the immune response than gTNF-1. gTNF-1 was constitutively expressed in the thymus, brain, and spleen, but it was also expressed in the heart, head kidney, and trunk kidney after LPS stimulation. gTNF-2 was constitutively expressed in the thymus, head kidney, trunk kidney, spleen, and intestine; further, gTNF-2 was highly expressed in all organs post-LPS stimulation. Finally, the gTNF expression levels were evaluated at various developmental stages in grouper larvae. A higher variation of gTNF expression levels was observed in fish larvae from a contaminated hatchery. This study revealed the different expression patterns of gTNF-1 and gTNF-2. In addition, gTNF-2 was more sensitive to pathogens than gTNF-1; therefore, it may be an appropriate marker for pathogen invasion and the evaluation of the larval rearing environment.

Interleukin 6 (IL-6) is a protein secreted by T cells and macrophages and plays an important role in immune responses. Include regulated inflammatory reactions, proliferation and differentiation lymphocyte, regulation in both innate and adaptive immune responses; these responses will help to eliminate the invaded pathogens in mammal. In our previous research, the grouper (Epinephelus coioides) IL-6 is proven to enhance the production of immunoglobulin, antimicrobial peptide, acute phase protein, increasing phagocyte phagocytosis and lysozyme activity; that was also increase the survival rate of recombinant IL-6 injected fish under bacterial or viral pathogen challenge. These results may represent the IL-6 might be a good immune immunestimulatant candidate for fish to prevent or treatment disease. However, an optimal administrated route of the recombinant IL-6 is important for its efficacy and used friendly. In this study, we try to evaluated minimal effective dose in the various administration routes include injection, oral, immersion and jet injection by detect immune gene expression and survival rate in the challenge test. The results shown injection administration with the lowest dose can achieve the protection; even with the higher dose, the oral administration can also achieve the same protection level; but it is not achieve the protection level in immersion or jet injection administration method. These tentative results shown the injection administration route was still the most efficient method, however oral application was the potential to be the most convenience method if we can overcome the protein digestion problem in the GI tract.