Intestinal Microbiota in Large Yellow Croaker,Larimichthys crocea,at Different Ages

The polymerase chain reaction–denaturing gradient gel electrophoresis (PCR‐DGGE) of 16S ribosomal RNA gene was used to investigate bacterial communities in the intestines of large yellow croaker at six different ages (12 d, 18 d, 26 d, 40 d, 3 mo, and 1 yr old) as well as within the corresponding feed and culture water. In addition, Illumina Miseq sequencing was utilized to compare intestinal microbiota between 12‐d‐old and 1‐yr‐old individuals. PCR‐DGGE results revealed that the culture water had the highest bacterial diversity, followed by the feed, while the intestines had the lowest diversity. The intestinal microbiota at six ages changed severely; however, the change did not follow any trend. The large yellow croaker intestines harbored specific bacterial communities that differed from those in both feed and water. Illumina Miseq sequencing results revealed that the diversity of intestinal bacteria in 12‐d‐old fish was higher than that in 1‐yr‐old fish, and the bacterial composition differed significantly between them. γ‐Proteobacteria and Pseudoalteromonas supplied the most abundant phylum and genus in the 12‐d‐old fish intestine. However, in the 1‐yr‐old fish intestine, Firmicutes and Clostridium were the most dominant, respectively. The study may contribute to a better understanding of gut microbiota and dynamics of the large yellow croaker and the relationship with their surrounding environment.     

小肽对大黄鱼仔鱼生长和小肠发育的影响及其在低温胁迫下的抗氧化应激效应

本试验分为生长试验和低温胁迫试验2部分。先分别投喂大黄鱼(Larimichthys crocea)仔鱼经不同浓度(0、0.5、1.0、2.0和3.0 mL/m 3)小肽营养强化后的轮虫和卤虫12 d,以探讨小肽对大黄鱼仔鱼生长和小肠发育的影响;再将大黄鱼仔鱼暴露在温度为12℃的水体中24 h,以探讨小肽对低温胁迫下大黄鱼仔鱼抗氧化能力和非特异性免疫力的影响。结果显示:不同浓度小肽均显著增加了轮虫和卤虫的必需氨基酸和总氨基酸含量(P<0.05),显著提高了大黄鱼仔鱼的体长及小肠绒毛高度、数量和组织面积(P<0.05)。小肽显著提高低温胁迫下大黄鱼仔鱼的谷胱甘肽(GSH)含量以及肝脏抗氧化酶[铜锌超氧化物歧化酶(Cu/Zn-SOD)、锰超氧化物歧化酶(Mn-SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶-1a(GPx-1 a)、谷胱甘肽过氧化物酶-1b(GPx-1 b)和谷胱甘肽还原酶(GR)]基因和非特异性免疫酶[c型溶菌酶(c-type LZM)、g型溶菌酶(g-type LZM)和碱性磷酸酶(AKP)]基因表达水平,显著降低活性氧(ROS)含量(P<0.05)。核转录因子NF-E2相关因子2(Nrf2)和核转录因子-κB(NF-κB)基因表达水平均与抗氧化酶基因和非特异性免疫酶基因表达水平呈显著正相关(P<0.05),并均与ROS含量呈显著负相关(P<0.05)。综上所述,小肽能够提高轮虫和卤虫的营养价值,从而改善大黄鱼仔鱼的生长和小肠发育;小肽通过诱导Nrf2和NF-κB的表达来增加抗氧化酶基因和非特异性免疫酶基因表达水平,从而缓解低温胁迫诱导的氧化损伤。     

Optimal dietary lipid level for large yellow croaker (Pseudosciaena crocea) larvae

A 30‐day feeding experiment was conducted in blue tanks (70 × 50 × 60 cm, water volume 180 L) to determine the effects of dietary lipid levels on the survival, growth and body composition of large yellow croaker (Pseudosciaena crocea) larvae (12 days after hatchery, with initial average weight 1.93 ± 0.11 mg). Five practical microdiets, containing 83 g kg^?1 (Diet 1), 126 g kg^?1 (Diet 2), 164 g kg^?1 (Diet 3), 204 g kg^?1 (Diet 4) and 248 g kg^?1 lipid (Diet 5), were formulated. Live feeds (Artemia sinicia nauplii and live copepods) were used as the control diet (Diet 6). Each diet was randomly assigned to triplicate groups of tanks, and each tank was stocked with 3500 larvae. During the experiment, water temperature was maintained at 23(±1) °C, pH 8.0 (±0.2) and salinity 25 (±2) g L^?1. The results showed that dietary lipid significantly influenced the survival and growth of large yellow croaker larvae. Survival increased with the increase of dietary lipid from 83 to 164 g kg^?1, and then decreased. The survival of larvae fed the diet with 83 g kg^?1 lipid (16.1%) was significantly lower than that of larvae fed other diets. However, the survival in larvae fed the diet with 16.4 g kg^?1 lipid was the highest compared with other artificial microdiets. Specific growth rate (SGR) significantly increased with increasing dietary lipid level from 83 to 164 g kg^?1 (P < 0.05), and then decreased. The SGR in larvae fed the diet with 164 g kg^?1 lipid (10.0% per day) was comparable with 204 g kg^?1 lipid (9.6% per day), but were significantly higher than other microdiets (P < 0.05). On the basis of survival and SGR, the optimum dietary lipid level was estimated to be 172 and 177 g kg^?1 of diet using second‐order polynomial regression analysis respectively.     

微粒饲料替代生物饵料对大黄鱼稚鱼生长、存活和消化酶活力的影响

以初始体重为(1.93±0.11) mg的大黄鱼稚鱼(12日龄)为实验对象,以微粒饲料(micro-diet,MD)分别替代0%、25%、50%、75%和100%生物饵料(live prey,LP),探讨微粒饲料替代生物饵料对大黄鱼稚鱼生长、存活、体成分和消化酶活力的影响.30 d的摄食生长实验表明:微粒饲料替代生物饵料显著影响大黄鱼稚鱼的生长、存活、体成分和消化酶活力.当微粒饲料替代50%和75%生物饵料时,两组间的特定生长率(SGR)差异不显著(P＞0.05),但均显著高于100%替代水平(P＜0.05);同时,75%替代水平SGR显著高于0%和25%替代水平(P＜0.05).存活率在各处理组间的差异关系与SGR的变化趋势类似.鱼体粗蛋白含量随替代水平的升高有下降的趋势,其中50%、75%和100%替代水平鱼体粗蛋白含量显著低于0%和25%替代水平(P＜0.05),而鱼体粗脂肪含量变化趋势与之相反.当微粒饲料替代100%生物饵料时,其胰段和肠段淀粉酶活力显著高于其余各处理组(P＜0.05),而其余各处理组之间淀粉酶活力差异均不显著;微粒饲料替代生物饵料对各处理组蛋白酶活力无显著影响.由此可以看出,大黄鱼苗种生产中,在12日龄以后使用优质微粒饲料替代50%～75%的生物饵料是可行的.     

大黄鱼C型凝集素受体Clec4e的分子鉴定及凝集特性

C型凝集素受体（C-type lectin receptor，CTLR）是可以特异性结合糖类病原体相关分子模式（pathogen-associated molecular patterns，PAMPs）的模式识别受体（pattern recognition receptors，PPRs），在先天性免疫中发挥着重要作用。为了揭示硬骨鱼CTLR的生物学功能，本研究以从大黄鱼（Larimichthys crocea）转录组数据库中筛选出的一个CTLR基因—C型凝集素结构域家族4成员E基因（C-type lectin domain family 4 member E gene，Clec4e）为研究对象，研究其分子特征、表达分布和凝集特性。结果显示，LcClec4e cDNA全长1546 bp，开放阅读框（open reading frame，ORF）771 bp，编码254个氨基酸。LcClec4e的N端有一个跨膜区，无信号肽，C端含有一个糖识别结构域（carbohydrate recognition domain，CRD），其中含有糖结合位点EPN和WFD以及6个可形成二硫键的保守半胱氨酸。系统发育分析表明，LcClec4e与多种鲈形目鱼类Clec4e具有较近的亲缘关系。荧光定量PCR结果显示，LcClec4e在所检测的10种组织中呈组成型分布，且在肝脏中表达量最高；LcClec4e在来源于大黄鱼头肾组织的原代巨噬细胞、淋巴细胞和粒细胞中均有表达，且在巨噬细胞中表达量最高；经灭活溶藻弧菌刺激后，LcClec4e在3种免疫细胞中的表达均极显著上调。原核表达的重组LcClec4e胞外段（recombinant LcClec4e-extracellular domain，rLcClec4e-ex）具有Ca2+依赖性的凝集活性，可凝集小鼠、家兔的红细胞，以及嗜水气单胞菌、变形假单胞菌、溶藻弧菌和坎氏弧菌等4种水产常见的革兰氏阴性菌。D-葡萄糖、D-果糖、D-甘露糖、D-麦芽糖、α-乳糖和脂多糖均可抑制rLcClec4e-ex对大黄鱼重要病原菌变形假单胞菌的凝集作用，说明LcClec4e可能与变形假单胞菌表面的糖类物质结合。这些研究结果提示，LcClec4e可能作为一种PPR，通过结合病原菌表面的糖类PAMPs来识别病原，参与大黄鱼抗细菌感染的免疫防御。     

大黄鱼弧菌病综合防治对策的初步研究

在5月下旬用哈维氏弧菌(Vibrio harveyi)灭活菌苗对大黄鱼(Pseudosciaena crocea)幼鱼进行浸泡免疫，7月至8月高温季节投喂添加防病药物或者uharveyi灭活菌苗的配合饲料，进行了综合防病试验。试验结果表明，uharveyi灭活菌苗浸泡免疫接种可以有效地预防uharveyi对受免大黄鱼的感染，但是，受免鱼对副溶血弧菌(V.parahaemolyticus)的感染则没有显著的保护；高温季节投喂添加药物的配合饲料能够有效地降低大黄鱼的死亡率，而将药物添加在鱼糜中后的投喂方式则不能获得明显防病效果；对受免鱼口服灭活菌苗后在56d内仍然可以观察到加强免疫效果。     

大黄鱼体表溃烂症病原菌的鉴定

从网箱养殖的患体表溃烂症的大黄鱼心、肝、肾、脾等组织分离到8株细菌，大黄鱼人工感染试验表明分离菌株XS01为致病菌。对该菌株进行形态、生理生化试验鉴定为费氏弧菌(Vibrio fucheri)。药敏试验结果表明，该菌株对磺胺甲基异恶唑等13种药物敏感，对氨苄青霉素等8种药物不敏感。进行了该菌株对温度和盐度的耐受性试验，认为该菌有较广的温度和盐度适应范围。     

肠道消化吸收相关因子对大黄鱼生长速度的影响

为了分析大黄鱼Pseudosciaena croce在人工养殖过程中,常常出现同一批次鱼苗在相同养殖条件下个体差异明显、部分鱼生长速度缓慢的原因,试验选取福州市连江县某鱼场同一网箱内同一批次投放的鱼苗,根据个体大小分组,体质量为60~100 g的为生长正常组,体质量为10~40 g的为生长缓慢组,在相同养殖条件下饲喂300 d,试验结束时,采用消化酶活性检测、荧光定量PCR和石蜡组织切片3种方法,分别测定大黄鱼肠道的消化酶活性和菌群数量,观察肠道的组织形态学变化并进行对比分析。结果表明:两组鱼的各种消化酶活性均无显著性差异(P0.05);在生长缓慢组鱼肠道内,酵母菌和弧菌等兼性厌氧菌数量高于生长正常组,且两组之间酵母菌数量有显著性差异(P0.05);生长正常组鱼肠道绒毛密集、修长且完整,而生长缓慢组鱼肠道绒毛稀疏,有大量损伤,前肠上皮内淋巴细胞数量显著高于生长正常组(P0.05)。研究表明,大黄鱼鱼群生长速度与肠道菌群及肠绒毛状态有关,而与肠道消化酶活性无关。     

福建三沙湾养殖大黄鱼肠道菌群研究

为研究大黄鱼（Pseudosciaena crocea）肠道细菌的群落结构,提取大黄鱼肠道内容物和肠壁样品总DNA,构建细菌16SrDNA克隆文库．分别从两个文库中随机挑选阳性克隆子测序,序列同源性分析和系统进化分析结果表明：在大黄鱼肠道中存在梭杆菌纲（Fusobacteria）细菌、拟杆菌纲细菌（Bacteroidetes）和γ-变形细菌纲（γ-Proteobacteria）细菌．肠壁样品细菌多样性高于肠道内容物样品．梭杆菌属细菌在肠道内容物样品中占据优势地位,而肠壁样品则以拟杆菌属细菌为最优势细菌类群,其次是梭杆菌属细菌。两个样品中均只检测到少量γ-变形细菌纲细菌．     

饵料类型对岱衢族大黄鱼鱼体和肌肉营养成分、体色、肉色的影响

岱衢族大黄鱼为我国优质大黄鱼养殖群体,养殖中所投喂的饵料往往影响着鱼体和肌肉营养成分、体色和肉色,这些均为衡量岱衢族大黄鱼品质的重要指标.本试验旨在通过测定岱衢族大黄鱼鱼体和肌肉营养成分、体色和肉色指标,研究投喂鲜杂鱼和配合饲料对岱衢族大黄鱼品质的影响.试验设置2组,分别投喂鲜杂鱼和配合饲料22个月,采样后所测鱼体和肌肉营养成分指标包括干物质、粗蛋白质、粗脂肪、粗灰分和氨基酸含量,体色和肉色指标包括L、a、b值.结果表明:与投喂鲜杂鱼相比,投喂配合饲料显著降低了岱衢族大黄鱼全鱼的粗脂肪含量(P＜0.05),但显著提高了肌肉的粗脂肪、亮氨酸(Leu)、赖氨酸(Lys)、谷氨酸(Glu)、总必需氨基酸、总呈味氨基酸和总氨基酸含量(P＜0.05);饵料类型对岱衢族大黄鱼的体色和肉色无显著影响(P＞0.05),但岱衢族大黄鱼体表不同部位颜色存在显著差异(P＜0.05),与背部相比,腹部的颜色较亮,且偏红黄.由此得出,相较于鲜杂鱼,采用配合饲料养成的岱衢族大黄鱼肌肉具有更高的营养价值.