Molecular Characterization and Antibacterial Activity of a G-Type Lysozyme in Yellow Drum(Nibea albiflora)

Lysozyme(EC3.2.1.17)plays an important role in the immune response;as a nonspecific immune factor,it can resist causative agents.Lysozyme can be divided into c-type and g-type in fish.In a previous study,through genome-wide association analysis,the g-type lysozyme gene,which is named NaLyg in yellow drum(Nibea albiflora),was found to be a key candidate gene for disease resistance in response to Vibrio harveyi infection.The cDNA of NaLyg was 1025 bp,including four exons and three introns,and its open reading frame(ORF)had a full-length of 582 bp,encoding 193 amino acids.NaLyg was found to be conserved during evolution through bioinformatic analyses.The NaLyg protein possessed a sugar binding domain and three catalytic sites,including Glu71,Asp84 and Asp101.Quantitative qRT-PCR results confirmed that NaLyg gene mRNA was visibly increased after V.harveyi infection.The NaLyg protein purified by prokaryotic expression killed some gram-negative bacterial pathogens by inducing cell wall destruction,including V.harveyi,Aeromonas hydrophila and Edwardsiella tarda.Moreover,the NaLyg protein killed two gram-positive bacteria,Bacillus subtilis and Staphylococcus aureus.Taken together,the experimental results suggested that the NaLyg protein of N.albiflora played an important role in fighting bacterial infections.

Tilapia Head Glycolipid Alleviates Indomethacin-Induced Gastric Ulcer via Regulating Oxidative Stress and Inflammation Through COX/PGE2 Signaling Pathway in Adult Rats

The aim of this experiment was to investigate the ameliorative effect and molecular mechanism of tilapia head glycolipid(TH-GL)on indomethacin(IDM)-induced gastric ulcer in male Sprague Dawley(SD)rats.The gastric ulcer model was established by oral administration of 30mgkg^(-1) IDM after 7 days of TH-GL or omeprazole(OME)administration in rats.Then the macroscopic gastric injury symptoms,gastric mucosa protective factor cyclooxygenase 1(COX-1),cyclooxygenase 2(COX-2),prostaglandin E_(2)(PGE_(2)),the levels of oxidative stress,and inflammatory cytokine expression levels in the rats were analyzed.The experimental results showed that multiple ulcers appeared on the gastric surface of the rats in the model group.Compared to the model group,TH-GL significantly alleviated gastric ulcers and reduced the gastric damage index in rats.In addition,TH-GL significantly promoted the expression of constitutive enzyme COX-1 while inhibited the expression of inducible enzyme COX-2,and make PGE2 maintain at normal levels.TH-GL also inhibited oxidative stress and inflammatory responses,increased superoxide dismutase(SOD)activity and glutathione(GSH)content,decreased the level of malondialdehyde(MDA)and the content of pro-inflammatory factor.In conclusion,these results suggested that TH-GL could maintain the expression levels of COX-1 and PGE2 while inhibit the expression of COX-2 in the gastric of rat and then prevent IDM-induced gastric ulcer,which may be related to the regulation of oxidative stress and inflammatory response.Therefore,TH-GL might be a new option for the prevention of gastric diseases induced by IDM.

Effects of Salinity on Growth of Tilapia nilotica and Tilapia mossambica

Tilapia has wide-range tolerance to salinity, and most Tilapia species can survive in both fresh water and seawater. In this study, the survival rates, absolute growth rates and instantaneous growth rates of Tilapia nilotica and Tilapia mossambica in waters with four different salinities（0‰, 10‰, 20‰, 30‰） were investigated.The results showed that the growth of T. mossambica was trended to be increased with the increased salinity, and it is suitable to be farmed in seawater; however, the growth of T. nilotica was trended to be decreased with the increased salinity, and it is suitable to be farmed in fresh water.

Significantly enhanced thermal stability of HMX by phase-transition lysozyme coating

A new robust bio-inspired route by using lysozyme aqueous solution for surface modification on 1,3,5,7-tetranitro-1,3,5,7-tetrazocane(HMX)was described in this paper.HMX crystals were coated by in situ phase transition of lysozyme(PTL)molecules.The HMX decorated by PTL was characterized by SEM,XRD,FTIR and XPS,demonstrating a dense core-shell coating layer.The coverage of lysozyme on HMX crystal was calculated by the ratio of sulfur content.The surface coverage increased from 60.5% to 93.5% when the content of PTL was changed from 0.5 wt% to 2.0 wt%,indicating efficient coating.The thermal stability of HMX was investigated by in situ XRD and DSC.The thermal phase transition temperature of HMX(β to δ phase)was delayed by 42℃ with 2.0 wt% PTL coating,which prevented HMX from thermal damage and sensitivity by the effect of PTL coating.After heating at 215℃,large cracks appeared in the naked HMX crystal,while the PTL coated HMX still maintained intact,with the impact energy of HMX dropped dramatically from 5 J to 2 J.However,the impact energy of HMX with 1.0 wt% and 2.0 wt% coating content(HMX@PTL-1.0 and HMX@PTL-2.0)was unchanged(5 J).Present results potentially enable large-scale fabrication of polymorphic energetic materials with outstanding thermal stability by novel lysozyme coating.

RAPD-SCAR Markers for Genetically Improved NEW GIFT Nile Tilapia (Oreochromis niloticus niloticus L.) and Their Application in Strain Identification

The NEW GIFT Nile tilapia （Oreochromis niloticus niloticus L.） is a nationally certificated new strain selected over 14 years and 9 generations from the base strain of GIFT Nile tilapia, introduced in 1994. This new variety has been extended in most of areas of China. The management of genetically improved strains, including the genetic markers for identification is needed urgently. RAPD analysis was conducted and their conversion to SCAR markers was developed. From NEW GIFT Nile tilapia, two strain-specific RAPD bands, S304624 bp and S36568 bp were identified. The strain-specific RAPD bands were gel-purified, cloned, and sequenced. Locus-specific primers were then designed to amplify the strain-specific bands. PCR amplification was conducted to test the variations in allele frequencies of two converted SCAR markers among the NEW GIFT Nile tilapia and its base strains, as well as 7 additional farmed strains worldwide. The frequency of SCAR marker Ⅰ （553 bp） was 85.7% in NEW GIFT Nile tilapia, but 16.7% in the base strain. The frequency of SCAR marker Ⅱ （558 bp） was 91.4% in NEW GIFT Nile tilapia, but 0% 70% in the 7 other strains. In order to confirm the utility of these two markers, an examination was conducted for a wild population from Egypt, resulted the frequency of SCAR Ⅰ and Ⅱ was 10% and 70%, respectively, much lower than that of New GIFT strain. The increase in allele frequency of these two SCAR markers suggests that these markers might be genetically linked to the quantitative trait loci （QTL） underlining the performance traits by long term selection, and indicate the bright potential of SCAR marker technology for tracking generations during selection progress and for distinguishing among genetically improved strain and other strains.

RAPD Analysis of Micropterus salmoides and Tilapia nilotica

The intra-population phylogenetic relationship between Micropterus salmoides and Tilapia nilotica was analyzed by random amplified polymorphic DNA （ RAPD） technique. The results showed that the similar coefficient of bands between M. salmoides and T. nilotica was 0.236, and the genetic distance was 0. 764, which indicated that M. salmoides had a distant relationship with T. nilotica.

Changing Rule of Physical and Chemical Indicators of Sediment in Tilapia Aquaculture Ponds

To provide basic information for studying balancing mechanism of nutrition environment in aquaculture ponds, sediment samples were collected periodically during the breeding process of tilapia in ponds. Then various types of nutrient content of the samples were detected and the changing rule was summarized. The results showed that dry weight of the sediment and content of TP, TN and TOC generally presented a trend of first increasing and then decreasing. The main production period of each nutrient was from July to September and the production percentage was close to 80% of the total production. The content of NH4^＋-N increased gradually and reached a peak in early August, then maintained at the peak level. NO2^--N content of the sediment in each pond was basically the same in the earlier aquaculture stage and decreased in middle stage with a big difference, then rose again in the later stage.

仿生态条件下星洲红鱼(Singapore red tilapia)和奥尼罗非鱼(Oreochromis niloticus)生长和肌肉营养特性的比较研究

为比较星洲红鱼(Singapore red tilapia)和奥尼罗非鱼(Oreochromis niloticus)的生长和肌肉营养特性,实验利用两种鱼的不同体色将其混养在同一池塘中,在相同的环境条件和养殖管理下研究其生长特性,并分析肌肉营养成分。结果表明:两种罗非鱼生长在前60 d无差异,60 d后星洲红鱼的体长、体质量日增长和特定生长率均大于奥尼罗非鱼,且差异显著(P<0.05)。据此得到了星洲红鱼和奥尼罗非鱼的体长(L)、体质量(W)生长与养殖天数(d)的回归方程。星洲红鱼含肉率高于奥尼罗非鱼9.65%(P<0.05),肌肉水分高于奥尼罗非鱼,蛋白质、粗脂肪和灰分含量均低于奥尼罗非鱼,其中脂肪含量差异显著(P<0.05),其他不显著。两种罗非鱼肌肉的氨基酸组成全面,氨基酸组成均符合FAO/WHO的理想模式,除精氨酸含量有差异外,其他氨基酸含量稍有差异但不显著。星洲红鱼和奥尼罗非鱼肌肉EAA/TAA、EAA/NEAA、DAA/TAA和EAAI分别为41.04%和40.95%,70.00%和69.34%,40.71%和39.08%,67.46%和67.08%,差异不显著。星洲红鱼、奥尼罗非鱼肌肉∑SFA和∑UFA分别为28.03%和28.60%,63.87%和61.38%,各脂肪酸含量除C14:0和C18:2外,其他含量差异不显著。研究表明,星洲红鱼和奥尼罗非鱼生长快、营养丰富、食用价值高,是优质淡水鱼类养殖品种,其中星洲红鱼含肉率高,生长快,养殖效益明显。

Induction and expression of T4 lysozyme gene in Pichia pastoris

Aim To induce and express the T4 lysozyme in Pichia pastoris and test the antibacterial activity of the protein. Methods T4 lysozyme gene was inserted into expression vector pPIC9K of Pichia pastoris with the fusion at N terminal. The recombinant plasmid was digested by Sal I and then introduced into prepared GS115 competent cells by electroporation. Positive clone and multiple inserts were screened. The secreted proteins in the supernatants were tested. In the agar holes diffusion assay, our expressed protein showed significant antibacterial circles. Results T4 lysozyme protein inhibited the growth of staphylococcus aureus and streptococcus Pneumoniae. There was no difference in the bactericidal activity and the amount of protein expression between the single and multiple copies. The antibacterial activity of expressed protein remained the same during the heat stability test. Conclusion T4 lysozyme was successfully induced and expressed in Pichia pastoris. There is no relationship between copy number and expression. T4 lysozyme protein is heat stable.

Impacts of Climate Change on Economic Performance of Tilapia Pond Operations in Minalin, Pampanga: A Case of Intensive Large-Scale Pond Culture

Pampanga is the top tilapia-producing province in Philippines. One of its municipalities, Minalin, is considered as one of its major centers of production. This study aims to determine the impacts of climate change hazards on the economic performance of tilapia-intensive pond culture in Minalin, Pampanga considering the threats to the industry, livelihoods, and food security. Results revealed that the economic performance of tilapia pond operations using semi-intensive large-scale culture was affected by climate change, as reflected in the reduction of yield among tilapia pond operators. This makes intensive tilapia pond farming in Minalin still a viable venture but will now require more than a year to pay for investment (variable and fixed) costs if under climate change risks. The study recommends strengthening capacity building related to climate-smart tilapia farming technologies for pond operators and to implement appropriate technologies to address climate change risks.

Comparative Study on the Antioxidant Activity of Peptides from Pearl Oyster(Pinctada martensii) Mantle Type V Collagen and Tilapia(Oreochromis niloticus) Scale Type Ⅰ Collagen

In this study, Pearl oyster mantle type V collagen(POMC) and tilapia scale type I collagen(TSC) were extracted and hydrolyzed by various proteases in order to obtain peptides. The antioxidant activity of the peptides was investigated by DPPH, hydroxyl radical scavenging experiments and a dynamic digestion model in vitro. The results show that there are significant differences in amino acid composition between POMC and TSC. The collagen peptides obtained from pearl oyster mantle(POMCP) by treating with alkaline protease exhibited higher antioxidant activity than that from tilapia scale(TSCP) treated with papaya protease, and both of them showed greater DPPH and hydroxyl radical scavenging activity than other peptides. After being separated via Sephadex G-25 chromatography, the M1 fraction isolated from POMCP, and the S1 fraction from TSCP with which both had higher molecular weights showed the strongest antioxidant activity than other fractions, and the M1 fraction exhibited stronger antioxidant activity than the S1 fraction in scavenging free-radicals and protecting cells from the oxidation damage. Furthermore, after treating the dynamic digestion system model in vitro, the DPPH and hydroxyl radical scavenging activity of the M1 fraction increased slightly. These results suggest that POMCP exhibits stronger antioxidant activity than TSCP, which means that PMOP may be a good candidate to be a potential natural antioxidant in the food-processing industry.

Intramammary expression and therapeutic effect of a human lysozyme-expressing vector for treating bovine mastitis

To develop a gene therapy strategy for treating bovine mastitis, a new mammary-specific vector containing human lysozyme (hLYZ) cDNA and kanamycin resistance gene was constructed for intramammary expression and clinical studies. After one time acupuncture or intracisternal infusion of healthy cows with 400 μg of the p215C3LYZ vector, over 2.0 μg/ml of rhLYZ could be detected by enzymatic assay for about 3 weeks in the milk samples. Western blotting showed that rhLYZ secreted into milk samples from the vector-injected cows had molecular weight similar to that of the natural hLYZ in human colostrums. Twenty days after the primary injection, the quarters were re-injected with the same vector by quarter acupuncture and even higher concentrations of rhLYZ could be detected. Indirect competitive ELISA of milk samples showed that the vector injection did not induce detectable humoral immune response against hLYZ. Clinical studies showed that twice acupuncture of quarters with the p215C3LYZ vector had overt therapeutic effect on clinical and subclinical mastitis previously treated with antibiotics, including disappearance of clinical symptoms and relatively high microbiological cure rates. These data provide a solid rationale for using the vector to develop gene therapy for treating bovine mastitis.

Behavioral response of tilapia (Oreochromis niloticus) to acute ammonia stress monitored by computer vision

The behavioral responses of a tilapia (Oreochromis niloticus) school to low (0.13 mg/L), moderate (0.79 mg/L) and high (2.65 mg/L) levels of unionized ammonia (UIA) concentration were monitored using a computer vision system. The swimming activity and geometrical parameters such as location of the gravity center and distribution of the fish school were calculated continuously. These behavioral parameters of tilapia school responded sensitively to moderate and high UIA concen-tration. Under high UIA concentration the fish activity showed a significant increase (P<0.05), exhibiting an avoidance reaction to high ammonia condition, and then decreased gradually. Under moderate and high UIA concentration the school’s vertical location had significantly large fluctuation (P<0.05) with the school moving up to the water surface then down to the bottom of the aquarium alternately and tending to crowd together. After several hours’ exposure to high UIA level, the school finally stayed at the aquarium bottom. These observations indicate that alterations in fish behavior under acute stress can provide important in-formation useful in predicting the stress.

尼罗罗非鲫(Tilapia nilotia)仔鱼前期器官发育与分化的组织学观察

本文报道了尼罗罗非鲫发育到仔鱼前期器官发育与分化的研究结果。其中,消化管、肝脏、胰脏、胆囊、鳔、眼和鳃等的发育已达到最后阶段的造型,分化程度较高。胸腺、甲状腺、肾脏仍属器官原基,分化水平较低。口中孵化的方式对眼的分化有一定影响。直到仔鱼前期末,消化管和消化腺没有实质性变化。但胃,肠内出现许多大的卵黄颗粒。在这一阶段,性腺增大十分明显,且被毛细血管及血岛将组织分成许多小叶。性腺的迅速发育与卵黄快速吸收和利用可能有一定的对应关系。

Relationships of Dissolved Oxygen with Chlorophyll-a and Phytoplankton Composition in Tilapia Ponds

This study investigated the relationships among the parameters of dissolved oxygen, chlorophyll-a and phytoplankton composition in tilapia ponds. Each pond (a total of 18 ponds) was sampled once in the dry, winter season between January and March and again early in the rainy season between May and June. The data were analyzed by examining correlations among parameters as affected by season, altitude and culture system. Observations were made at sites located in 5 selected provinces of northern Thailand: Chiangrai, Chiangmai, Phayao, Lampang and Nakornsawan. Mean elevation of these areas range from 25 to 582 meters above sea level (masl) and were categorized into low (400 masl) elevation sites. Ponds were 0.8 - 2.0 m deep, 0.16 - 0.64 ha in area and could be further categorized into high and low input systems.Mean air temperature in winter ranged between 16.5°C - 35.8°C while mean water temperature ranged between 25.5°C - 27.1&#176;C. In rainy season, air temperature ranged between 22.0°C - 37.3°C and water temperature ranged between 29.4°C - 31.8°C. The amount of chlorophyll-a in both seasons were comparable (p > 0.05), but chlorophyll-a in high input system was significantly higher (p 0.05) than in low input ponds. Only weak correlation was found between chlorophyll-a, DOmax and DOmin. Multifactor-ANOVA was used to analyze the difference of total bacteria and filamentous cyanobacteria in ponds based upon elevation, culture systems and season. Result shows that there is a significant interaction observed between elevation, culture system and season (p 0.05). Species diversity and composition of phytoplankton in fish ponds in 2 seasons revealed the presence of 90 genera of phytoplankton under all 7 divisions. Divisions Chlorophyta and Cyanophyta had the most number of genera identified in both seasons with Pediastrum spp., and Scendesmus spp., and Anabaena spp. as dominant genera/genus, respectively.

Application of Lactic Acid Bacteria(LAB) in Freshness Keeping of Tilapia Fillets as Sashimi

Aquatic products are extremely perishable food commodities. Developing methods to keep the freshness of fish represents a major task of the fishery processing industry. Application of Lactic Acid Bacteria(LAB) as food preservative is a novel approach. In the present study, the possibility of using lactic acid bacteria in freshness keeping of tilapia fillets as sashimi was examined. Fish fillets were dipped in Lactobacillus plantarum 1.19(obtained from China General Microbiological Culture Collection Center) suspension as LAB-treated group. Changes in K-value, APC, sensory properties and microbial flora were analyzed. Results showed that LAB treatment slowed the increase of K-value and APC in the earlier storage, and caused a smooth decrease in sensory score. Gram-negative bacteria dominated during refrigerated storage, with Pseudomonas and Aeromonas being relatively abundant. Lactobacillus plantarum 1.19 had no obvious inhibitory effect against these Gram-negatives. However, Lactobacillus plantarum 1.19 changed the composition of Gram-positive bacteria. No Micrococcus were detected and the proportion of Staphylococcus decreased in the spoiled LAB-treated samples. The period that tilapia fillets could be used as sashimi material extended from 24 h to 48 h after LAB treatment. The potential of using LAB in sashimi processing was confirmed.

罗非鱼(Tilapia nilotica)简达气单胞菌病的病原分离鉴定及药敏试验

从患病罗非鱼(Tilapia nilotica)体内分离得到细菌NL05,通过回归感染试验确定NL05为致病菌,并测出NL05对罗非鱼的半致死量(LD50)为1×103 CFU/g。结合细菌形态学特征、生理生化指标和16S r RNA基因同源分析,鉴定NL05为简达气单胞菌(Aeromonas jandaei)。形态学观察发现,NL05为革兰氏阴性、短杆状;生理生化试验中麦芽糖、甘露醇、葡萄糖、水杨素、硫化氢等13种指标为阳性,蔗糖、阿拉伯糖、木糖、肌醇、卫矛醇等10种指标为阴性。药敏试验显示,NL05对奥复星、丙氟哌酸、丁胺卡那霉素、多粘菌素B、氟哌酸、利福平、洁霉素等13种抗生素敏感,对阿奇霉素、菌必治、卡那霉素、链霉素、美满霉素5种抗生素中介,对氨苄青霉素、阿莫西林、恩诺沙星、复方新诺明、甲氧苄啶等13种抗生素耐药。

Determination of 17<i>α</i>-Methyltestosterone in Freshwater Samples of Tilapia Farming by High Performance Liquid Chromatography

17α-methyltestosterone is used to induce the sex reversal of Tilapia sp. to obtain cultures mono-sex to an economically viable. This practice may lead to environmental contamination and problems in human health. Therefore methods need to be developed to detect residues of 17α-methyltestosterone in aqueous matrices. A simple high-performance liquid chromatographic method using ultraviolet detection (245 nm) and testosterone as internal standard has been developed for the monitoring 17α-methyltestosterone in freshwater samples of tilapia aquaculture. The method described involves limited sample preparation as it includes a filtration followed by a single solid-phase extraction step using C18 cartridge. Validation data indicated that the HPLC-UV method for 17α-methyltestosterone determination in the concentration range of 50 - 2000 μg/L provided good linearity, sensitivity, accuracy and precision. Method performance was efficiently applied to monitoring the freshwater samples of fish ponds and the surrounding aquatic channels.

Dietary L-arginine supplementation reduces lipid accretion by regulating fatty acid metabolism in Nile tilapia(Oreochromis niloticus)

Background: Excessive white fat accumulation in humans and other animals is associated with the development of multiple metabolic diseases. It is unknown whether dietary L-arginine supplementation reduces lipid deposition in high fat diet-fed Nile tilapia(Oreochromis niloticus).Results: In the present study, we found that dietary supplementation with 1% or 2% arginine decreased the deposition and concentration of fats in the liver;the concentrations of triglycerides, low-density lipoprotein, total cholesterol, and high-density lipoprotein in the serum;and the diameter of adipocytes in intraperitoneal adipose tissue. Compared with the un-supplementation control group, the hepatic activities of alanine aminotransferase,aspartate aminotransferase, and lactate dehydrogenase, and hepatic concentration of malondialdehyde were reduced but these for catalase and superoxide dismutase were enhanced by dietary supplementation with 2% arginine. Arginine supplementation reduced the total amounts of monounsaturated fatty acids, while increasing the total amounts of n-3 and n-6 polyunsaturated fatty acids in the liver. These effects of arginine were associated with reductions in mRNA levels for genes related to lipogenesis(sterol regulatory element-binding protein-1, acetyl-CoA carboxylase α, stearoyl-CoA desaturase, and fatty acid synthase) but increases in mRNA levels for genes involved in fatty acid β-oxidation(carnitine palmitoyltransferase 1α and peroxisome proliferator-activated receptor α). In addition, hepatic mRNA levels for Δ4 fatty acyl desaturase 2 and elongase 5 of very long-chain fatty acids were enhanced by arginine supplementation.Conclusion: These results revealed that dietary L-arginine supplementation to tilapia reduced high fat diet-induced fat deposition and fatty acid composition in the liver by regulating the expression of genes for lipid metabolism.

罗非鱼(Tilapia)鱼皮提取明胶的工艺优化

以罗非鱼(Tilapia)鱼皮为原料,分别用碱法、酸法、酶法进行前处理,对3种方法的提取效果进行比较,筛选出最佳水解方法为酶法。优化了酶法处理后的漂白、熬胶工艺,获得鱼皮提取明胶的最佳工艺。结果显示,前处理采用0.2%的混合酶,调p H值至3.5,于40℃水浴中酶水解鱼皮1 h,用6%高锰酸钾溶液进行氧化漂白3 h,再用1.5%草酸溶液进行还原漂白45 min。控制p H值为5?6,分3次熬胶,每次分别将水浴加热升温至60℃、65℃、70℃,各加热2 h,过滤后将3次胶液合并,在真空度不低于500 mm Hg,温度不超过60℃的情况下浓缩至20%左右。然后在60℃的鼓风干燥箱干燥至明胶含水量14%以下,经粉碎机粉碎,即可得成品明胶。该工艺产品得率19.93%(湿基)、粘度13.1 m Pa·s、凝胶强度1034.3 g/cm2。