The Tilapia Cyst Tissue Enclosing the Proliferating Myxobolus bejeranoi Parasite Exhibits Cornified Structure and Immune Barrier Function.

Myxozoa, a unique group of obligate endoparasites within the phylum Cnidaria, can cause emerging diseases in wild and cultured fish populations. Recently, the myxozoan Myxobolus bejeranoi has been identified as a prevalent pathogen infecting the gills of cultured hybrid tilapia, leading to systemic immune suppression and considerable mortality. Here, we employed a proteomic approach to examine the impact of M. bejeranoi infection on fish gills, focusing on the structure of the granulomata, or cyst, formed around the proliferating parasite to prevent its spread to surrounding tissue. Enrichment analysis showed increased immune response and oxidative stress in infected gill tissue, most markedly in the cyst's wall. The intense immune reaction included a consortium of endopeptidase inhibitors, potentially combating the myxozoan arsenal of secreted proteases. Analysis of the cyst's proteome and histology staining indicated that keratin intermediate filaments contribute to its structural rigidity. Moreover, we uncovered skin-specific proteins, including a grainyhead-like transcription factor and a teleost-specific S100 calcium-binding protein that may play a role in epithelial morphogenesis and cysts formation. These findings deepen our understanding of the proteomic elements that grant the cyst its distinctive nature at the critical interface between the fish host and myxozoan parasite.

Effects of dietary leucine and valine levels on growth performance, glycolipid metabolism and immune response in Tilapia GIFT Oreochromis niloticus.

An 8-week feeding trial was performed to evaluate the effects of dietary leucine (Leu) and valine (Val) levels on growth performance, glycolipid metabolism and immune response in Oreochromis niloticus. Fish (15.23 ± 0.05 g) were randomly fed four diets containing two Leu levels (1.2% and 2.3%) and two Val levels (0.7% and 1.4%) as a 2 × 2 experimental design (LL-LV, LL-HV, HL-LV and HL-HV). Compared with LL-LV group, the growth parameters (final weight, daily growth coefficient (DGC) and growth rate per metabolic body weight (GRMBW)), feed conversion rate (FCR), the activities of intestinal amylase, lipase, creatine kinase (CK) and Na+, K+-ATPase, liver NAD+/NADH ratio, as well as the expression of SIRT1, GK, PK, FBPase, PPARα, CPT IA, ACO and IL10 all increased significantly in the HL-LV group; however, in the high Val group, final weight, DGC, GRMBW, intestinal enzyme activities, as well as the expression of PEPCK, SREBP1, FAS, IL8 and IL10 of the HL-HV group were significantly lower than those of the LL-HV group, while the opposite was true for the remaining indicators. Significant interactions between dietary Leu and Val were observed in final weight, DGC, GRMBW, plasma IL1ß and IL6 levels, intestinal amylase and CK activities, liver NAD+/NADH ratio, as well as the expression of SIRT1, PK, PEPCK, FBPase, SREBP1, FAS, PPARα, CPT IA, ACO, NF-κB1, IL1ß, IL6 and IL10. The highest values of growth parameters, intestinal enzyme activities and expression of SIRT1, FBPase, PPARα, CPT IA and ACO were observed in the HL-LV group, while the opposite was true for the expression of SREBP1, FAS, PPARα, NF-κB1, IL1ß and IL6. Overall, our findings indicated that dietary Leu and Val can effect interactively, and fish fed with diets containing 2.3% Leu with 0.7% Val had the best growth performance and hepatic health status of O. niloticus.

Impact of high dietary cornstarch level on growth, antioxidant response, and immune status in GIFT tilapia Oreochromis niloticus.

This study was conducted to investigate the relationship between different cornstarch levels in tilapia diet and immune function. All test fish were fed with three cornstarch levels: low-cornstarch (0, LS), medium-cornstarch (18%, MS) and high-cornstarch (36%, HS) diets. Three hundred and sixty fish (initial mean body weight 31.73 ± 1.36 g) were randomly allocated into twelve water-circulated tanks, and thirty fish per tank. Compared with the low and medium cornstarch diets, the results of growth showed that the high cornstarch diet significantly decreased the FBW, WGR, and SGR, and increased the FCR of tilapia (P < 0.05). The high cornstarch diet significantly decreased the content of crude protein and increased the content of crude lipid in whole body composition (P < 0.05). Moreover, the VSI and CF in HS diet were significantly higher than those of LS diet (P < 0.05). The results of blood biochemical index exhibited that the HS diet significantly increased the content of blood glucose, and liver/muscle glycogen (P < 0.05). The results of antioxidant experiments demonstrated that the content of SOD and T-AOC in MS diet were significantly higher than those of HS diet (P < 0.05). Meanwhile, the content of MDA in MS diet was significantly lower than that of HS diet (P < 0.05). The results of immune index test showed that the lysozyme activities in the serum, liver, and gill, and the phagocytic activity and index in MS diet were significantly higher than those of HS diet (P < 0.05). The challenge assay results revealed that the mortality rate of HS diet was higher than those of LS and MS diets, but the difference was not significant (P > 0.05). In conclusion, the overall results suggested that the 36% cornstarch diet reduced not only the growth performance, but also body immunity. Under this experimental condition, GIFT tilapia could tolerate 18% cornstarch, but not 36% cornstarch.

The effect of dietary supplementation with Clostridium butyricum on the growth performance, immunity, intestinal microbiota and disease resistance of tilapia (Oreochromis niloticus).

This study was conducted to assess the effects of dietary Clostridium butyricum on the growth, immunity, intestinal microbiota and disease resistance of tilapia (Oreochromis niloticus). Three hundreds of tilapia (56.21 ± 0.81 g) were divided into 5 groups and fed a diet supplemented with C. butyricum at 0, 1 x 104, 1 x 105, 1 x 106 or 1 x 107 CFU g-1 diet (denoted as CG, CB1, CB2, CB3 and CB4, respectively) for 56 days. Then 45 fish from each group were intraperitoneally injected with Streptococcus agalactiae, and the mortality was recorded for 14 days. The results showed that dietary C. butyricum significantly improved the specific growth rate (SGR) and feed intake in the CB2 group and decreased the cumulative mortality post-challenge with S. agalactiae in the CB2, CB3 and CB4 groups. The serum total antioxidant capacity and intestinal interleukin receptor-associated kinase-4 gene expression were significantly increased, and serum malondialdehyde content and diamine oxidase activity were significantly decreased in the CB1, CB2, CB3 and CB4 groups. Serum complement 3 and complement 4 concentrations and intestinal gene expression of tumour necrosis factor α, interleukin 8, and myeloid differentiation factor 88 were significantly higher in the CB2, CB3 and CB4 groups. Intestinal toll-like receptor 2 gene expression was significantly upregulated in the CB3 and CB4 groups. Dietary C. butyricum increased the diversity of the intestinal microbiota and the relative abundance of beneficial bacteria (such as Bacillus), and decreased the relative abundance of opportunistic pathogenic bacteria (such as Aeromonas) in the CB2 group. These results revealed that dietary C. butyricum at a suitable dose enhanced growth performance, elevated humoral and intestinal immunity, regulated the intestinal microbial components, and improved disease resistance in tilapia. The optimal dose was 1 x 105 CFU g-1 diet.

Simultaneous Administration of Boesenbergia pandurata Extract and Vaccination to Stimulate Immune Response in Tilapia, Oreochromis niloticus.

BACKGROUND AND OBJECTIVE: The use of adjuvants or immunostimulants is often necessary to increase vaccine efficacy, in this study we evaluated the improvement of the immune response in tilapia treated by either oral and immersion administration with vaccine and Boesenbergia pandurata extract (BPE). MATERIALS AND METHODS: The initial concentration of BPE and the cell density of vaccine were 900 mg L-1 and 104 CFU mL-1 for oral administration while 106 CFU mL-1 for immersion, respectively. The extract and vaccine were mixed homogeneously in a ratio of 1:1. Further, the mixture was supplemented to feed at 1 mL g-1 feed. Tilapia with average initial body weight of 15 g were fed containing vaccine and BPE 3 times a day. The other group of fish was immersed with vaccine and BPE for 20 min. After 7th (d7), 14th (d14) and21th (d21) days of treatment, a challenge test was conducted by intramuscularly injection of 0.1 mL of Aeromonas hydrophila and Pseudomonas fluorescens mixture (1:1) at a density of 105 CFU mL-1. Antibody levels, total white blood cell (WBC) and phagocytic activity (PA) were evaluated to determine the immune improvement of the fish. Furthermore, relative percent survival (RPS) and the survival rate (SR) were evaluated at week 2 and 4 after challenge test. RESULT: Results indicated that the all parameters of tilapia immune system were increased (p<0.05) after 2-4 weeks of both administration methods. Meanwhile, the efficacy of the vaccine has increased by combining BPE treatment using immersion method better than oral method. The RPS of vaccination plus extract by immersion was 83-100% and by oral administration was 83-87%. CONCLUSION: The present results implied that B. pandurata extract boost the efficacy of the Pseudomonas sp. vaccine by increasing the immune system and diseases resistance in tilapia.

Streptococcus agalactiae infection kills red tilapia with chronic Francisella noatunensis infection more rapidly than the fish without the infection.

In this study we examined the effect that a Francisella noatunensis (Fno) infection had on hybrid red tilapia (Oreochromis niloticus × Oreochromis mossambicus) subsquently infected with Streptococcus agalactiae. A variety of hemato-immunological parameters (haematocrit, total red blood cell count, mean corpuscular volume, total white blood and differential cell counts, total plasma protein, plasma lysozyme and plasma peroxidase activities, and respiratory burst and phagocytic activities of head-kidney macrophages) were measured in hybrid red tilapia that had been previously exposed to an Fno outbreak in a tilapia grow-out farm. The head-kidneys of these apparently healthy survivors, when checked by PCR were found to be Fno-positive with hemato-immunological parameters that were similar to fish without an a priori infection. The only exception was the percentage lymphocyte count in the peripheral blood, which was slightly, but significantly, lower in the Fno-infected fish, compared to those without the infection. When experimentally infected with S. agalactiae, the Fno-infected fish died more rapidly and at a significantly higher rate than fish without the infection. During the challenge, the hemato-immunological parameters of both groups of fish were very similar, although the Fno-infected fish, challanged with S. agalactiae expressed significantly higher plasma lysozyme and peroxidase activities, and their head kidney macrophages had significantly higher respiratory burst activity compared to non-Fno-infected fish challanged with S. agalactiae. The only two parameters for which Fno-infected fish showed significantly lower expressions than that of their non-infected counterparts were haematocrit and total red blood cell count. The cause of the rapidity and higher rates of mortality observed in the Fno-infected fish when challenged with S. agalactiae is unknown; but it may be due to a reduced erythropoiesis capability within the head-kidney because of the presence of Fno.

KatG plays an important role in Aeromonas hydrophila survival in fish macrophages and escape for further infection.

The success of the pathogenic bacteria is partly attributable to their ability to thwart host innate immune responses, which includes resisting the antimicrobial functions of macrophages. And reactive oxygen species (ROS) is one of the most effective antimicrobial components of macrophages to kill invading bacteria. Our previous studies found that Aeromonas hydrophila can survive in fish macrophages, which suggested that this bacterium might take fish macrophages as their shelters to resist drug killings and other immune damage. But how A. hydrophila survive in host macrophages remains unknown. Since KatG has been reported to have not only catalase activity but also peroxidase and peroxynitritase activity, the amino acid sequence and protein structure of KatG was analyzed in this study, the function of KatG in A. hydrophila survival in and escape from host macrophages was also carried out. The bioinformatics analysis displayed that KatG of A. hydrophila B11 showed >93% homologous to that of KatG in other Aeromonas. KatG of A. hydrophila was stable silenced by shRNA and RT-qPCR confirmed the expression of KatG in KatG-RNAi was significantly reduced. The survival rate of intracellular KatG-RNAi decreased by 80% compared to that of the wild type strain B11, while the intracellular ROS level of the macrophages that phagocytosed KatG-RNAi increased 65.9% when compared to that of the macrophages phagocytosed wild-type strain. The immune escape rate of A. hydrophila decreased by 85% when the expression of KatG was inhibited. These results indicated that (1) The amino acid sequence and protein structure of KatG of A. hydrophila is conserved; (2) KatG helped A. hydrophila to survive in fish macrophages by eliminating the harm of intracellular H2O2 and inhibiting intracellular ROS levels increased; (3) A small portion of intracellular A. hydrophila could escape from host macrophages for further infection, in this process KatG also played important role.

Dietary supplementation of probiotic Bacillus licheniformis Dahb1 improves growth performance, mucus and serum immune parameters, antioxidant enzyme activity as well as resistance against Aeromonas hydrophila in tilapia Oreochromis mossambicus.

The present study evaluated the dietary supplementation of probiotic Bacillus licheniformis Dahb1 on the growth performance, immune parameters and antioxidant enzymes activities in serum and mucus as well as resistance against Aeromonas hydrophila in Mozambique tilapia Oreochromis mossambicus. Fish (24 ±â€¯2.5 g) were fed separately with three diets, 1) commercial diet (control), 2) diet containing probiotic at 105 cfu g-1 (D1) and 3) diet containing probiotic at 107 cfu g-1 (D2) for 4 weeks. Growth performance in term of final weight (FW) specific growth rate (SGR) and feed conversion ratio (FCR), immune parameters of total protein (TP), alkaline phosphatase (ALP), myeloperoxidase (MPO), lysozyme (LYZ), reactive oxygen species (ROS), reactive nitrogen species (RNS) and antioxidant parameters of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in serum and mucus were evaluated after 2nd and 4th weeks. The FW, SGR, and FCR of fish fed with D1 and D2 significantly improved (p < 0.05). The activities of ALP, LYZ and MPO in the mucus were significantly higher (p < 0.05) in fish that fed D1 and D2. The TP, ROS, RNS, SOD and GPx in the serum were significantly higher (p < 0.05) in fish that fed D1 and D2. In addition, the challenge test showed that fish fed D1 and D2 enhanced significantly (p < 0.05) the resistance against A. hydrophila (1 × 107 cells ml-1). In conclusion, probiotic B. licheniformis Dahb1 can be applied in diet at 107 cfu g-1 to improve healthy status and resistance against A. hydrophila in tilapia farming.

Effectivity of oral recombinant DNA vaccine against Streptococcus agalactiae in Nile tilapia.

The effects of oral vaccination by two immune routes (gavage and mixed fodder administration) using a recombinant DNA vaccine SL7207-pVAX1-sip against Streptococcus agalactiae were assessed. SL7207-pVAX1-sip significantly enhanced various innate immune responses of tilapia, such as total serum protein, superoxide dismutase activity, lysozyme activity, complement C3 concentration in serum, serum antibacterial activity, and interleukin 1ß and tumor necrosis factor-α. Vaccinated fish had higher relative percent survival values (57% and 63% for gavage group and mix fodder administration, respectively) at 10 weeks after S. agalactiae infection. When administered mixed fodder, the DNA vaccine SL7207-pVAX1-sip against S. agalactiae may produce more effective protection. These findings can promote the application and development of DNA vaccines in aquaculture.

Metabolic Mechanism for l-Leucine-Induced Metabolome To Eliminate Streptococcus iniae.

Crucial metabolites that modulate hosts' metabolome to eliminate bacterial pathogens have been documented, but the metabolic mechanisms are largely unknown. The present study explores the metabolic mechanism for l-leucine-induced metabolome to eliminate Streptococcus iniae in tilapia. GC-MS-based metabolomics was used to investigate the tilapia liver metabolic profile in the presence of exogenous l-leucine. Thirty-seven metabolites of differential abundance were determined, and 11 metabolic pathways were enriched. Pattern recognition analysis identified serine and proline as crucial metabolites, which are the two metabolites identified in survived tilapias during S. iniae infection, suggesting that the two metabolites play crucial roles in l-leucine-induced elimination of the pathogen by the host. Exogenous l-serine reduces the mortality of tilapias infected by S. iniae, providing a robust proof supporting the conclusion. Furthermore, exogenous l-serine elevates expression of genes IL-1ß and IL-8 in tilapia spleen, but not TNFα, CXCR4 and Mx, suggesting that the metabolite promotes a phagocytosis role of macrophages, which is consistent with the finding that l-leucine promotes macrophages to kill both Gram-positive and Gram-negative bacterial pathogens. Therefore, the ability of phagocytosis enhanced by exogenous l-leucine is partly attributed to elevation of l-serine. These results demonstrate a metabolic mechanism by which exogenous l-leucine modulates tilapias' metabolome to enhance innate immunity and eliminate pathogens.

Oreochromis mossambicus diet supplementation with Psidium guajava leaf extracts enhance growth, immune, antioxidant response and resistance to Aeromonas hydrophila.

In this research, we focused on the efficacy of aqueous and ethanol leaf extracts of Psidium guajava L. (guava) based experimental diets on the growth, immune, antioxidant and disease resistance of tilapia, Oreochromis mossambicus following challenge with Aeromonas hydrophila. The experimental diets were prepared by mixing powdered (1, 5 and 10 mg/g) aqueous and ethanol extract of guava leaf with commercial diet. The growth (FW, FCR and SGR), non-specific cellular immune (myeloperoxidase activity, reactive oxygen activity and reactive nitrogen activity) humoral immune (complement activity, antiprotease, alkaline phosphatase activity and lysozyme activity) and antioxidant enzyme responses (SOD, GPX, and CAT) were examined after 30 days of post-feeding. A significant enhancement in the biochemical and immunological parameters of fish were observed fed with experimental diets compared to control. The dietary supplementation of P. guajava leaf extract powder for 30 days significantly reduced the mortality and increased the disease resistance of O. mossambicus following challenge with A. hydrophila at 50 µl (1 × 107 cells ml-1) compared to control after post-infection. The results suggest that the guava leaf extract could be used as a promising feed additive in aquaculture.

Thymol and Carvacrol Affect Hybrid Tilapia through the Combination of Direct Stimulation and an Intestinal Microbiota-Mediated Effect: Insights from a Germ-Free Zebrafish Model.

BACKGROUND: Essential oils (EOs) are commonly used as animal feed additives. Information is lacking on the mechanisms driving the beneficial effects of EOs in animals, especially the role played by the intestinal microbiota of the host. OBJECTIVE: The purpose of this study was to clarify the relative contribution of direct effects of EOs on the physiology and immune system of tilapia and indirect effects mediated by the intestinal microbiota by using a germ-free zebrafish model. METHODS: Juvenile hybrid tilapia were fed a control diet or 1 of 4 treatment diets containing 60-800 mg Next Enhance 150 (NE) (an EO product containing equal levels of thymol and carvacrol)/kg for 6 wk. The key humoral and cellular innate immune parameters were evaluated after the feeding period. In another experiment, the gut microbiota of tilapia fed a control or an NE diet (200 mg/kg) for 2 wk were transferred to 3-d postfertilization (dpf) germ-free (GF) zebrafish, and the expression of genes involved in innate immunity and tight junctions was evaluated in zebrafish at 6 dpf. Lastly, NE was directly applied to 3-dpf GF zebrafish at 3 doses ranging from 0.2 to 20 mg/L, and the direct effect of NE on zebrafish was evaluated after 1 and 3 d. RESULTS: NE supplementation at 200 mg/kg enhanced phagocytosis activity of head kidney macrophages (×1.36) (P < 0.05) and plasma lysozyme activity (×1.69) of tilapia compared with the control (P < 0.001), indicating an immunostimulatory effect. Compared with those colonized with control microbiota, GF zebrafish colonized with NE microbiota showed attenuated induction of immune response marker genes serum amyloid a (Saa; ×0.62), interleukin 1ß (Il1ß; ×0.29), and interleukin 8 (Il8; ×0.62) (P < 0.05). NE treatment of GF zebrafish at 2 and 20 mg/L for 1 d upregulated the expression of Il1ß (×2.44) and Claudin1 (×1.38), respectively (P < 0.05), whereas at day 3 the expression of Occludin2 was higher (×3.30) in the 0.2-mg NE/L group compared with the GF control (P < 0.05). CONCLUSION: NE may affect the immunity of tilapia through a combination of factors, i.e., primarily through a direct effect on host tissue (immune-stimulating) but also an indirect effect mediated by microbial changes (immune-relieving).

Development and characterization of a cell line from tilapia head kidney with melanomacrophage characteristics.

A novel cell line THK, derived from the tilapia head kidney, was developed and characterized. The THK cell line comprised fibroblastoid cells that markedly proliferated in Leibovitz L-15 medium containing 2%-15% fetal bovine serum (FBS) at 20 °C-35 °C. Cell proliferation was dependent on the FBS concentration, and the optimal temperature for proliferation ranged between 25 °C and 30 °C. THK cells were characterized for the presence of phagocytic activity, acid phosphatase, alkaline phosphatase, α-naphthyl acetate esterase, lipofuscin, and tyrosinase. Transcripts of CD33, CD53, CD82, CD205, macrophage colony stimulating factor receptor, GATA2, and GATA3 that are specific for leucocytes or monocytes/macrophages or both were detected in the THK cells through PCR. However, THK cells lacked for CD83, a specific marker for dendritic cells. The results indicated that the fibroblastoid THK cells were melanomacrophage-related progenitors. PCR revealed that the THK cells exhibited the transcripts of toll-like receptor 1 (TLR1), TLR2, TLR3, and CD200, of which concern with immunity as well as the transcripts of vascular endothelial growth factor receptor 3, angiomotin, and angiopoietin-like protein 2 that associate with angiogenesis regulation and macrophage proliferation. THK cells were subcultured more than 90 times and can be useful for investigating the development and functioning of the teleostean innate immune system.

Immunostimulation by phospholipopeptide biosurfactant from Staphylococcus hominis in Oreochromis mossambicus.

The immunostimulatory effect of phospholipopeptide biosurfactant from Staphylococcus hominis (GenBank Accession No: KJ564272) was assessed with Oreochromis mossambicus. The non-specific (serum lysozyme activity, serum antiprotease activity, serum peroxidase activity and serum bactericidal activity), specific (bacterial agglutination assay) immune responses and disease resistance activity against Aeromonas hydrophila were examined. Fish were intraperitonially injected with water soluble secondary metabolite (biosurfactant) of S. hominis at a dose of 2 mg, 20 mg and 200 mg kg(-1) body weight. Commercial surfactant surfactin (sigma) at 20 mg kg(-1) was used as standard and saline as negative control. All the doses of water soluble biosurfactant tested, significantly enhanced the specific, nonspecific immunity and disease resistance from the day of post administration of phospholipopeptide biosurfactant till the tail of the experimental period. These results clearly indicated that the secondary metabolite isolated from S. hominis stimulates the immunity of finfish thereby could enhance aquaculture production.

Cloning and primary immunological study of TGF-ß1 and its receptors TßR I /TßR II in tilapia(Oreochromis niloticus).

The transforming growth factor ß (TGF-ß) superfamily plays critical roles in tumor suppression, cell proliferation and differentiation, tissue morphogenesis, lineage determination, cell migration and apoptosis. Recently, TGF-ß1, one important member of TGF-ß superfamily, is suggested as an immune regulator in the teleost. In this study, we cloned the cDNAs of TGF-ß1 and its receptors, TßR I and TßR II (including three isoforms) from tilapia (Genbank accession numbers: KP754231- KP754235). A tissue distribution profile analysis indicated that TGF-ß1 was highly expressed in the head kidney, gill, spleen, kidney and PBLs (peripheral blood leukocytes); TßR I only showed considerable expression in the liver; and TßR II-2 was highly expressed in the kidney, gill, liver, head kidney and heart. We determined that the mRNA expressions of TGF-ß and TßR I /TßR II-2 were significantly increased in tilapia head kidney and spleen leukocytes by the stimulation of Lipopolysaccharide (LPS) or Poly I: C. We also examined their expressions in the spleen and head kidney of tilapia after IP injection of streptococcus agalactiae. The results showed that the mRNA expressions of these three genes all increased in the head kidney as early as 6 h post infection, and in the spleen 3 d post infection. In addition, the protein level of TGF-ß1 was also up-regulated in the head kidney and the spleen after infection. Taken together, our data indicate that the TGF-ß1-TßR I /TßR II-2 system functions potentially in tilapia immune system.

Effect of UV-B radiation on the antibody response of fish - implication on high altitude fish culture.

Literally, all living forms are either directly or indirectly dependent upon sun for energy. Radiation from sun is differentiated into several components of a spectrum based on the wavelength. Ultraviolet (UV) radiation may be one of the infamous radiations emitted by the sun. Ozone depletion is another critical factor by which UV induced ill-effects are intensified. Though there are numerous studies on effects of UV radiation on terrestrial organisms, its effect on freshwater and aquaculture ecosystems has been largely neglected. Here, we report that enhanced UV irradiation may suppress the primary and secondary antibody responses to a soluble protein antigen in fish. Fishes exposed for longer periods (80min) were particularly very sensitive to infection, as shown by our sensitivity index.

Molecular cloning, functional identification and expressional analyses of FasL in Tilapia, Oreochromis niloticus.

FasL is the most extensively studied apoptosis ligand. In 2000, tilapia FasL was identified using anti-human FasL monoclonal antibody by Evans's research group. Recently, a tilapia FasL-like protein of smaller molecule weight was predicted in Genbank (XM_003445156.2). Based on several clues drawn from previous studies, we cast doubt on the authenticity of the formerly identified tilapia FasL. Conversely, using reverse transcription polymerase chain reaction (RT-PCR), the existence of the predicted FasL-like was verified at the mRNA level (The Genbank accession number of the FasL mRNA sequence we cloned is KM008610). Through multiple alignments, this FasL-like protein was found to be highly similar to the FasL of the Japanese flounder. Moreover, we artificially expressed the functional region of the predicted protein and later confirmed its apoptosis-inducing activity using a methyl thiazolyl tetrazolium (MTT) assay, Annexin-V/Propidium iodide (PI) double staining, and DNA fragment detection. Supported by these evidences, we suggest that the predicted protein is the authentic tilapia FasL. To advance this research further, tilapia FasL mRNA and its protein across different tissues were quantified. High expression levels were identified in the tilapia immune system and sites where active cell turnover conservatively occurs. In this regard, FasL may assume an active role in the immune system and cell homeostasis maintenance in tilapia, similar to that shown in other species. In addition, because the distribution pattern of FasL mRNA did not synchronize with that of the protein, post-transcriptional expression regulation is suggested. Such regulation may be dominated by potential adenylate- and uridylate-rich elements (AREs) featuring AUUUA repeats found in the 3' untranslated region (UTR) of tilapia FasL mRNA.

Immunological enhancement action of endotoxin-free tilapia heat shock protein 70 against Streptococcus iniae.

The immunological effects of heat shock proteins (HSPs) had been found in humans and mice, but scarce data of endotoxin-free Hsp70 were reported in tilapia. In the current study, we reported that tHsp70 alone and antigen-tHsp70 compound increased the proliferations of lymphocytes and macrophages, significantly increased the NO release and phagocytotic ability of macrophages (p<0.05), and enhanced the levels of immune-related genes in lymphocytes and macrophages in a dose- and/or time-dependent manner. On the other hand, tHsp70 not only helped to reduce the proliferation inhibitions induced by the ECP treatment, but also assisted antigens to enhance the vaccine-induced protection against Streptococcus iniae (p<0.05). We described, for the first time, a critical role of endotoxin-free tHsp70 on activation of tilapia lymphocytes and macrophages post S. iniae exposure and its up-regulation effects on vaccine-induced protection. Our research highlights the immunological enhancement action of Hsp70 in teleost immunity.

A novel GH secretagogue, A233, exhibits enhanced growth activity and innate immune system stimulation in teleosts fish.

In teleosts fish, secretion of GH is regulated by several hypothalamic factors that are influenced by the physiological state of the animal. There is an interaction between immune and endocrine systems through hormones and cytokines. GH in fish is involved in many physiological processes that are not overtly growth related, such as saltwater osmoregulation, antifreeze synthesis, and the regulation of sexual maturation and immune functions. This study was conducted to characterize a decapeptide compound A233 (GKFDLSPEHQ) designed by molecular modeling to evaluate its function as a GH secretagogue (GHS). In pituitary cell culture, the peptide A233 induces GH secretion and it is also able to increase superoxide production in tilapia head-kidney leukocyte cultures. This effect is blocked by preincubation with the GHS receptor antagonist [d-Lys(3)]-GHRP6. Immunoneutralization of GH by addition of anti-tilapia GH monoclonal antibody blocked the stimulatory effect of A233 on superoxide production. These experiments propose a GH-mediated mechanism for the action of A233. The in vivo biological action of the decapeptide was also demonstrated for growth stimulation in goldfish and tilapia larvae (P<0.001). Superoxide dismutase levels, antiprotease activity, and lectin titer were enhanced in tilapia larvae treated with this novel molecule. The decapeptide A233 designed by molecular modeling is able to function as a GHS in teleosts and enhance parameters of the innate immune system in the fish larvae.

Transgenic expression of tilapia hepcidin 1-5 and shrimp chelonianin in zebrafish and their resistance to bacterial pathogens.

Recently, tilapia hepcidin (TH)1-5 was characterized, and its antimicrobial functions against several pathogens were reported. The antimicrobial functions of another shrimp antimicrobial peptide (AMP), chelonianin, were also characterized using a recombinant chelonianin protein (rcf) that was expressed by a stably transfected Chinese hamster ovary (CHO) cell line against pathogen infections in fish. The function of the overexpression of both AMPs in zebrafish muscles was not examined in previous studies. Herein, we investigated the antimicrobial functions of TH1-5 and chelonianin against Vibrio vulnificus (204) and Streptococcus agalactiae (SA48) in transgenic TH1-5 zebrafish and transgenic chelonianin zebrafish. The presence of TH1-5 and chelonianin enhanced the inhibitory ability in transgenic AMP zebrafish against the two different bacterial infections. The bacterial number of either V. vulnificus (204) or S. agalactiae (SA48) had decreased at 96 h after injection into transgenic AMP zebrafish muscle compared to non-transgenic zebrafish muscle. Additionally, immune-related gene expressions analyzed by real-time PCR studies showed the modulation of several genes including interleukin (IL)-10, IL-22, IL-26, MyD88, Toll-like receptor (TLR)-1, TLR-3, TLR-4, nuclear factor (NF)-κB, tumor necrosis factor (TNF)-α, and lysozyme, and significant differences were found between transgenic AMP zebrafish and wild-type zebrafish injected with PBS at 1-24 h. These results suggest that several immune-related gene expressions were induced in transgenic TH1-5 and chelonianin zebrafish which effectively inhibited bacterial growth. The survival rate dropped to 86.6% in transgenic chelonianin zebrafish after 28 days of infection compared of the 50% survival rate in transgenic TH1-5 zebrafish after 28 days of infection. Overall, these results indicate that TH1-5 and chelonianin possess the potential to be novel candidate genes for aquaculture applications to treat fish diseases.