Lysine deficiency impaired growth performance and immune response and aggravated inflammatory response of the skin,spleen and head kidney in grown-up grass carp (Ctenopharyngodon idella)

This dissertation was primarily focused on the immune response,inflammatory response and molecular mechanisms in the skin,head kidney and spleen of grown-up grass carp(Ctenopharyngodon idella).Six iso-nitrogen diets differing in lysine concentrations(5.6,8.5,11.6,14.4,17.5 and 20.7 g/kg)were fed to 540 grass carp(164.85±0.79 g)for 60 d.After that,grass carp were challenged by Aeromonas hydrophila for 6 d.This study revealed that lysine deficiency(1)suppressed the growth performa,nce of the fish and decreased their ability to resist skin lesion morbidity,(2)impaired the immune organ s immune response by decreasing the gene expressions of mucin,liver-expressed antimicrobial peptide(LEAP)-2B,β-defensin-1 and LEAP-2A and the production of antibacterial compounds of grown-up grass carp,and(3)aggravated the inflammatory response of immune organs in the fish by increasing the gene expressions of pro-inflammatory cytokines(interferon y2[IFN-γ2],tumor necrosis factorα[TNF-α],interleukin[IL]-15,IL-17D,IL-12p40,IL-6 and IL-8)and down-regulating anti-inflammatory cytokines(IL-11,transforming growth factorβ1[TGF-β1],IL-10 and IL-4/13A),which were tightly correlated with signal transducer and activator of transcription(STAT)1 and STAT3 signaling pathway,respectively.The different phenomenon in the skin,spleen and head kidney of fish may be correlated with the difference in gene subtype.In addition,using quadratic regression analysis of percent weight gain(PWG),skin lesion morbidity,and the lysozyme activities in the spleen and head kidney,the dietary lysine requirements for grown-up grass carp were estimated to be 13.58,13.51,14.56 and 14.18 g/kg,respectively.

Cloning of catalase gene and antioxidant genes in Scophthalmus maximus response to metalloprotease of Vibrio anguillarum stress

Metalloproteases represent a class of extracellular proteases found in Vibrio anguillarum that can generate toxic and pathogenic eff ects in turbot(Scophthalmus maximus).The toxicological eff ect partly results from oxidative damage due to the production of excessive reactive oxygen species(ROS).Catalase(CAT),superoxide dismutase(SOD),and glutathione peroxidase(GPx)are major antioxidant enzymes induced by various oxidative stresses and can scavenge peroxides generated in cells.To evaluate the eff ects of metalloprotease-induced ROS on the antioxidation defense mechanism of S.maximus head kidney cells,the cDNA of CAT gene(designated as SmCAT)was cloned and characterized.SmCAT comprises a 1584-bp coding sequence that encodes a protein containing 527 amino acids with a poly(A)tail.Bioinformatics analysis revealed an active site signature sequence,a heme-ligand signature sequence,and three catalytic amino acid residues.The deduced SmCAT amino acid sequence shares a sequence similarity of 66.1%-92.4%with those of other species.Phylogenetic analysis revealed that SmCAT is classifi ed with CAT of other fi shes.Quantitative real-time PCR analysis showed that SmCAT was extensively expressed in all tested tissues,especially in blood.The expression of SmCAT,SmMnSOD,and SmGPx were inhibited signifi cantly in head kidney cells treated with metalloprotease from 12 to 24 h.In 6 to 24 h metalloprotease-treated groups compared to that of the untreated group,it was found that the production of ROS was markedly increased,and the mitochondrial membrane potential was decreased considerably.Hoechst 33342 staining revealed the presence of apoptotic bodies when the cells were incubated with 8.0 or 40.0μg/mL metalloprotease for 12 and 24 h.Hence,the toxic eff ects of metalloprotease are associated with the down-regulation of antioxidant enzyme expression and increased ROS levels,which trigger the activation of apoptosis in the head kidney cells of turbot.Our fi ndings provide a better understanding on the mechanism of metalloprotease-induced apoptosis in fi sh.