Fluorescence localization of epidermal cathepsins L and B in the Japanese eel

Histochemical localization of proteolytic activities in the dorsal epidermis of Japanese eel was demonstrated by fluorescent microscopy utilizing 4-methoxy-2-naphthylamide (4M$\beta$NA) derivatives as substrates and 5-nitrosalicylaldehyde as a trapping agent. Carbobenzoxy-L-phenylalanyl-L-arginyl-4M$\beta$NA (Cbz-Phe-Arg-4M$\beta$NA) and Cbz-Arg-Arg-4M$\beta$NA were used for direct detection of cathepsins L and B activities, respectively, in fresh frozen sections and unfixed cells of the eel epidermis. The fluorescing areas, where Cbz-Phe-Arg-4M$\beta$NA was hydrolyzed by cathepsin L, were shown in mucus secretory cells and club cells and broadly around skin surface. The fluorescing areas due to Cbz-Arg-Arg-4M$\beta$NA hydrolysis by cathepsin B were localized similarly in these tissues. The fluorescing intensity for both catheptic activities in mucus secretory cells was higher than that in club cells, where small fluorescing granules were distributed. These results indicate that eel cathepsins L and B are stored in epidermal secretory cells at different levels and probably serve as defense factors before or after secretion by these cells. Abbreviations: Cbz – carbobenzoxy; 4M$\beta$NA – 4-methoxy-2-naphthylamide; NSA – 5-nitrosalicylaldehyde.     

养殖刺参保苗期重大疾病“腐皮综合征”病原及其感染源分析

2003～2005年冬、春季,山东省和辽宁省众多养殖区域的保苗期刺参(Apostichopus japonicus)暴发了较为严重的传染性疾病-"腐皮综合征".该病波及面广,传染性强,死亡率常高达90%以上.发病症状主要表现为厌食、摇头、肿嘴、排脏、身体萎缩、口部溃烂乃至体表大面积溃疡.本研究对症状较为典型的3家海参保苗期的发病幼参进行了详细的病原学分析,从所有病参的病灶组织分离得到了1种占有绝对优势的菌株,经人工感染实验证明它对健康刺参具有较强的致病性,且感染病参的症状与自然发病海参的症状相同.通过形态学、生理生化和16S rDNA分子生物学方法对该菌进行的分类鉴定表明,导致保苗期刺参"腐皮综合征"的致病菌是假交替单胞菌(Pseudoalteromonas nigrifaciens).同时也对3家养殖场刺参保苗养殖系统进行了分析研究.结果表明,水源中细菌含量不高,为(0.8×102)～(1.2×102)cells/mL,其特征与病灶处优势菌不同;而饵料中细菌含量最高可达3.2×106 cells/mL.饵料、池水和池底污物的优势菌与病灶处优势菌基本一致,说明饵料可能是病原菌的主要来源.本研究首次揭示了"腐皮综合征"导致保苗期刺参大规模死亡的原因及其致病原,对刺参健康养殖和病害防治具有重要的指导意义.     

三角帆蚌HcTyr基因内壳色性状相关SNP筛选及图谱定位

为研究三角帆蚌HcTyr基因与内壳色性状的相关性,本实验根据已分离的三角帆蚌HcTyr基因进行引物设计和片段扩增,并用144只三角帆蚌对其多态性进行筛选和验证,卡方检验分析了SNP位点和三角帆蚌内壳色相关性,并对HcTyr基因进行了图谱定位。结果显示,在HcTyr基因上筛选出8个SNP位点,其中有7个SNP位点与三角帆蚌内壳色L、a及d E呈显著相关性,用这7个SNP位点做单倍型构建和分析,发现Ⅱ、Ⅲ及Ⅳ这3种单倍型在白色群体中出现的频率极显著高于在紫色群体中出现的频率,而Ⅴ和Ⅶ这2两种单倍型在紫色群体中出现的频率极显著高于白色群体。进一步在商业养殖群体中验证发现,Ⅳ和Ⅶ这2种单倍型可分别作为白色和紫色群体的优势单倍型。研究表明,三角帆蚌HcTyr基因可作为内壳色相关的候选基因,其部分SNP位点可用于三角帆蚌分子辅助育种。另外本实验还将HcTyr基因定位在三角帆蚌LG16连锁群上,这为进一步解析该基因调控珍珠颜色形成的分子机制奠定了基础。     

日本鳗鲡腐皮病病原菌的分离及鉴定

从患腐皮病的日本鳗鲡(Anguilla japonica)体表溃烂处分离到1株病原菌(322A),对其进行了人工感染实验和生理生化分析,测定了该菌株的16S rRNA基因序列和促旋酶(gyrase)B亚单位gyrB基因序列,并分别构建系统发育树。结果显示:感染实验证实菌株322A具有致病性;生理生化分析鉴定该菌株属于气单胞菌属(Aero-monassp.);16S rRNA基因分析显示,该菌株与气单胞菌属细菌的同源性均在99%~100%,构建的系统树显示,菌株322A与嗜水气单胞菌(A.hydrophila(FJ462702))亲缘关系最近;gyrB基因分析表明,该菌株与A.hydrophila种内序列的相似性为96%~98%,种间序列的相似性为94%~95%,构建的系统树结果显示,该菌株与A.hydrophila(FJ608553、FJ608552、AF208259)聚为一个分支。综合上述实验结果,菌株322A可鉴定为嗜水气单胞菌(A.hydrophila)。     

Quality characteristics in raw and smoked fillets of Atlantic salmon, Salmo salar, fed high-energy diets

Atlantic salmon, Salmo salar , were fed four levels of dietary fat (256, 308, 346 and 389 g kg⁻¹ of diet) for 138 days, with the purpose of studying quality characteristics in raw and smoked fish fillets. Dietary fat levels up to 346 g kg⁻¹ resulted in increased fat content of the raw fillets. The dietary fat levels had a less systematic effect on perceived fatness of the smoked fillets and caused a trend towards better odour and flavour. The fat content of raw fillets was significantly ( P ≤ 0.05) correlated to lower smoke odour, greater rancid flavour, fatness, and a yellower hue of the smoked fillets. Astaxanthin levels of the raw fillets varied between 6 and 11 mg kg⁻¹ of fillet and were significantly ( P ≤ 0.05) correlated with greater intensity of smoke odour, lower off-odour, and less whiteness, greater colour intensity and a redder hue of the smoked fish. It is proposed that salmon fillets can be graded according to weight, fat content and colour values prior to smoking to obtain more standardized quality characteristics of the final product after processing.     

Characterization and Film-Forming Properties of Gelatins from Whitemouth Croaker (Micropogonias furnieri) Skin and Bones

The Whitemouth croaker (Micropogonias furnieri), due to its abundance, is considered one of the most traditional and important species fished in the coastal waters and estuaries of South America. The aim of this work was to produce biopolymeric films of gelatins obtained from skin and bones of Whitemouth croaker. The fish gelatins were characterized through the yield, chemical composition, physiochemical, and rheological properties. The films were characterized by tensile strength (TS), elongation at break (EAB), water vapor permeability (WVP), thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FT-IR), and optical properties. The values of protein yield, hydroxyproline content, gel strength, melting temperature, gelling temperature, and viscosity of the skin gelatin were higher than the values of the bone gelatin. The films obtained of skin gelatin showed all properties and thermal analysis superior in relation to the films of bone gelatin; this can be justified by the best physicochemical and rheological characteristics of the skin gelatin.     

Natural bixin as a potential carotenoid for enhancing pigmentation and colour in goldfish (Carassius auratus)

Discovering natural carotenoids for colour enhancement and health benefits of fish is important to develop new feed formulations. We have purified natural bixin from achiote seeds and evaluated the effect of colour enhancing and pigmentation in goldfish. Varying levels of bixin‐based diets were prepared with 420 g kg^?1 of crude protein and 120 g kg^?1 of lipid content. Our results clearly showed that bixin (0.05, 0.10, 0.20 and 0.60 g kg^?1) based diets significantly (P < 0.05) enhanced the skin and fin colour at 30 and 60 days compared to diet without bixin. Interestingly, diet which contains 0.20 g kg^?1 bixin and commercial feed (with astaxanthin) had similar effect on carotenoid deposition in skin. Moreover, total carotenoid deposition in fin was higher than in skin of all bixin‐containing diets. However, 0.60 g kg^?1 bixin‐containing diet had lower specific growth rate (1.01 ± 0.01) and higher feed conversion ratio (2.05 ± 0.19) compared to the control group. The present results demonstrate that achiote bixin can be successfully used as an alternative natural carotenoid source against synthetic astaxanthin in fish feed. Our data indicate that 0.20 g kg^?1 is a suitable dietary level of bixin to ensure strong pigmentation, acceptable growth and feed utilization in goldfish.     

尼罗罗非鱼表皮和鳃黏膜共生菌结构特征及其与鱼体健康状况相关关系

黏膜及其表面的共生菌群是鱼类抵御外界不利环境的第一道屏障。为探索养殖罗非鱼表皮和鳃黏膜共生菌菌群的结构特征是否与其健康状况间存在相关关系,本研究运用高通量测序技术,以无乳链球菌腹腔注射攻毒48 h后存活和濒死尼罗罗非鱼为检测对象,检测攻毒前后罗非鱼表皮和鳃黏膜共生菌群落结构差异。结果显示,健康尼罗罗非鱼表皮和鳃黏膜共生菌均存在优势菌群,主要为特吕珀菌属、硫杆菌属、弓形杆菌属、海单胞菌属和弧菌属。人工感染无乳链球菌后存活尼罗罗非鱼表皮和鳃黏膜共生菌菌群与感染前无显著差异;与存活组相比,濒死尼罗罗非鱼的表皮和鳃黏膜共生菌菌群多样性下降,其中弓形杆菌属、假交替单胞菌属、海单胞菌属、假单胞菌属和弧菌属等含量显著下降,链球菌属含量占总菌群的55.30%±1.24%,表明养殖尼罗罗非鱼表皮和鳃黏膜共生菌群落结构可能与其健康状况相关。     

Effect of salinity on growth and energy budget of red and green colour variant sea cucumber Apostichopus japonicus (Selenca)

Sea cucumber, Apostichopus japonicus (Selenca), tolerates salinity fluctuations inhabiting intertide zone. This study deals with growth, food intake, food conversion and the bioenergetic responses of the red variant (wet weight of 2.60 ± 0.11g) and green variant (wet weight of 2.56 ± 0.08 g) A. japonicus to different salinities of 22, 26, 30, 34, and 38 psu at 16.5 ± 0.5°C. The results showed that salinity had a significant effect on specific growth rate (SGR) of both green and red variants A. japonicus (P < 0.05). Both colour variants of sea cucumber had highest SGR at 30 psu, and then decreased when salinity below or above this point. Maximum SGR (the green 1.07 ± 0.08% day^?1, the red 1.14 ± 0.09% day^?1 respectively) is related with maximum food intake (FI) and highest food conversion efficiency (FCE) (P < 0.05) occurring at 30 psu. Only under 22 psu, the green variant grew faster than the red variant (P < 0.05), and under other four salinity treatments there was no significant difference between SGR of two colour variant holothruians (P > 0.05). Values of adaptable salinity scope for green and red variants sea cucumber survival are 18.5~39 psu and 20.9~38.6 psu respectively. The average energy budget formula of sea cucumber at 30 psu was: 100C = 6G +42F +3U+49R (C, energy ingested; G, energy for growth; F, energy loss as faeces; U, energy used for ammonia excretion; R, energy loss for respiration). The sea cucumber had maximum energy ingested (C) and highest proportion of energy for growth (G) at 30 psu, and then decreased when salinity is above or below this salinity. Both red and green variants of A. japonicus deposited for growth were very low, and the energy loss in faeces and energy for respiration accounted for the majority of assimilation energy. The result clearly showed that the optimum condition for farming green and red variants A. japonicus, both with respect growth and energy allocation, is the salinity scope of 26 ~ 30 psu.     

Epidermal proteases of the Japanese eel

A fluorescent-sensitive assay was used to demonstrate the protease activity in the dorsal skin of Japanese eel (Anguilla japonica). Two distinct extracts were separately prepared from skin mucus and epidermal cell layers, with no mutual contamination. The epidermal extract was sensitive to various substrates, whereas there was no, or only marginal, susceptibility to the same substrates for the mucous extract. Optimum hydrolysis pHs of the epidermal extract was variable and below pH 7.0, and the optimum hydrolysis temperatures were between 40 and 50 °C. In addition, Tos-Phe-Ch₂Cl, chymostatin, CdCl₂, CuCl₂, HgCl₂ and ZnCl₂ inhibited protease activities to different extents. Several other reagents specifically affected the protease activities, and their induced effects were useful for the identification of epidermal proteases. The findings indicate that a proteolytic factor, exhibiting various enzymological specificities, is retained within epidermal cell layers of Japanese eel. This factor is composed of 4 distinct proteases, such as cathepsins L and B-like proteases, a serine protease and an aminopeptidase.