饲料中添加L-肉碱对牙鲆幼鱼生长、生化组成及血液指标的影响

研究0、300、600、900、1 200、1 500 mg/kg 6个L-肉碱添加水平对牙鲆(Paralichthys olivaceus)幼鱼生长、生化组成和血液指标的影响。结果表明:添加量为1 200 mg/kg的处理组牙鲆幼鱼的增重率和特定生长率显著提高,饲料系数显著降低(P<0.05);随着饲料中L-肉碱含量的增加,肝指数呈现下降趋势,且在添加量为900~1 500 mg/kg时,各组较对照组差异显著(P<0.05);饲料中添加L-肉碱显著降低各实验组脏体比(P<0.05),而对各处理组间存活率和肥满度均无影响(P<0.05);肌肉和肝脏中的脂肪含量与L-肉碱添加量呈负相关,当添加量为1 200~1 500 mg/kg时,较对照组差异显著(P<0.05);肌肉中的蛋白含量与L-肉碱添加量呈正相关,但各组差异不显著(P<0.05);饲料中添加L-肉碱降低血清中的谷草转氨酶、胆固醇、甘油三酯和尿素氮含量,且添加量1 200 mg/kg组各指标含量均显著低于对照组(P<0.05);各处理组的血糖含量较对照组均有所增加,其中1 200 mg/kg组血糖含量较对照组提高41.6%(P<0.05);血清中总蛋白和低密度脂蛋白均随着L-肉碱添加量的增加而呈下降趋势,高密度脂蛋白却得以提升,但差异均不显著(P<0.05)。分析认为,在本实验条件下,饲料中添加L-肉碱可促进牙鲆幼鱼生长,且降脂效果显著。建议L-肉碱最适添加量为1 200 mg/kg。     

Histological Study on the Skin of Japanese Flounder Paralichthys olivaceus

Histological development of Japanese flounder Paralichthys olivaceus larval skin and ultrastructural difference of skin between reared normal and malpigmented Japanese flounder were studied with light microscopy (LM) and transmission electron microscopy (TEM). The results show that the skin develops slowly before the metamorphosis, while at the onset of metamorphosis, the skin develops quickly and becomes complete in structure till about 50d after being hatched. Ultrastructural observation on the normal and malpigmented skins shows that the iridophore and melanophore are adjacent to each other. Profile and structure of the two kinds of pigment cells are more complete in the skin of normal ocular side than in the skin of pigmented blind side. The ultrastructure of typical chloride cell was observed in the skin of Japanese flounder larvae for the first time.     

Ontogeny of the Lymphoid Organs of Japanese Flounder, Paralichthys olivaceus

Histological study on the ontogeny of the lymphoid organs, kidney, thymus and spleen of Japanese flounder, Paralichthys olivaceus, from hatching to 40d was carried out. The pronephric kidney duct appeared early in hatching although the primordial haemopoietic stem cells wereobserved within a week after hatching. The spleen was first seen after 8d of hatching. The thymus appeared after 15d, situated near the pronephric kidney. Small lymphoid cells appeared during the later phase of the post-larval stage in the sequence of thymus, kidney and spleen. During the 40d of observations, there were no distinct inner or outer zones in thymus and no red or white pulp in spleen. These results suggest that the nonspecific defense immune system plays a very important role in the early larval stage of Japanese flounder.     

牙鲆出血性败血病病原的初步研究

经病原菌的分离、纯化、人工感染和生理生化测定 ,认为变异微球菌为牙鲆出血性败血病的病原菌。菌体为圆形或椭圆形 ,单个、成对排列或成短链、四联及不规则的堆团。单个菌大小为 0 .5～ 1.8μm。无鞭毛 ,不运动 ,无牙孢 ,无荚膜。革兰氏阳性 ,不抗酸。好氧菌、葡萄糖 O- F试验为氧化型。测定的2 2种糖苷醇类中 ,仅葡萄糖、D-果糖、蔗糖、麦芽糖、甘油为好氧产酸 ,D-甘露糖弱产酸 ,且均不产气。氧化酶、过氧化氢酶阳性 ,硝酸盐还原、美兰还原为阳性。甲基红、V- P反应为阳性。能水解明胶、马尿酸钠 ,不能水解七叶苷、淀粉。不产生硫化氢、吲哚。不能利用柠檬酸盐、丙二酸盐。精氨酸双水解酶、赖氨酸脱羧酶、鸟氨酸脱羧酶、脲酶阴性 ,卵磷脂酶、凝固酶阳性。不溶血。先锋霉素、氯霉素、青霉素、红霉素为该病原菌的敏感药物     

The Toxic Mechanism of High Lethality of Herbicide Butachlor in Marine Flatfish Flounder,Paralichthys olivaceus

The toxic mechanism of herbicide butachlor to induce extremely high lethality in marine flatfish flounder,Paralichthys Olivaceus,was analyzed by histopathological examination,antioxidant enzymes activities and ATP content assay.Histopathological examination of gill,liver and kidney of exposed fishes showed that gill was a target organ of butachlor.The butachlor seriously impaired the respiration of gills by a series of lesions such as edema,lifting and detachment of lamellar epithelium,breakdown of pillar cells,and blood congestion.The dysfunction of gill respiration caused suffocation to the exposed flounder with extremely high acute lethality.Antioxidant enzyme activity assay of the in vitro cultured flounder gill(FG) cells exposed to butachlor indicated that butachlor markedly inhibited the antioxidant enzyme activities of Superoxide dismutase(SOD),catalase(CAT) and glutathione peroxidase(GPX).Furthermore,along with the decline of antioxidant enzyme activities,ATP content in the exposed FG cells decreased,too.This infers that the oxidative stress induced by butachlor can inhibit the production of cellular ATP.Similar decrease of ATP content was also observed in the exposed flounder gill tissues.Taken together,as in FG cells,butachlor possibly induced a short supply of ATP in pillar cells by inhibiting the antioxidant enzyme activities and then affecting the contractibility of the pillar cells,which in turn resulted in the blood congestion and suffocation of exposed flounder.     

Establishment and Characterization of Four Long-Term Cultures of Neural Stem/Progenitor Cell Lines from the Japanese Flounder Paralichthys olivaceus

Neurogenesis is an important progress wherein the neural stem cells(NSCs) differentiate into functional neurons under conductive conditions. Neurogenesis occurs continuously in different areas of the central neural system in adult teleosts compared with adult mammals. Therefore, NSC cell lines must be established to offer a valuable in vitro system for studies on neurogenesis and other related functions. In this study, four cell lines designated as PoB1, PoB2, PoBf and PoBh were established from the brain of the Japanese flounder Paralichthys olivaceus. The cell lines were sub-cultured over 150 times and still grew well in DMEM/F12 medium at 24℃. PoB1, PoB2, PoBf and PoBh were identified as neural stem/progenitor cell lines on the basis of the mRNA expression of nestin and/or aldh111 or slc6 a4 and the formation of neurospheres. The cells transfected with the pEGFP-C1 plasmid showed fluorescent signals with distinct reagent dependencies. The established cell lines from the brain of P. olivaceus offer a valuable system in vitro for the study of neurogenesis, fish neural regulation and endocrinology-related functions.     

Effects of Calanoid Copepod Schmackeriapoplesia as a Live Food on the Growth,Survival and Fatty Acid Composition of Larvae and Juveniles of Japanese Flounder,Paralichthys olivaceus

Zooplankton constitutes a major part of the diet for fish larvae in the marine food web, and it is generally believed that copepods can meet the nutritional requirements of fish larvae. In this study, calanoid copepod Schmackeria poplesia, rotifer Brachionus plicatilis and anostraca crustacean Artemia sp. were analyzed for fatty acid contents, and were used as live food for cul- turing larval Japanese flounder, Paralichthys olivaceus. The total content of three types of HUFAs （DHA, EPA and ARA） in S. po- plesia was significantly higher than that in the other two live foods （P〈O.O1）. Three live organisms were used for raising larvae and juveniles of Paralichthys olivaceus respectively for 15 and 10 d. Then the growth, survival and fatty acid composition of the larvae and juveniles were investigated. The results showed that the larvae and juveniles fed with copepods （S. poplesia） had significantly higher growth rate than those fed with the other two organisms （P〈0.01）. The survival of the flounder larvae fed with copepods was significantly higher than that of the others （P〈0.01）, and the survival of the juvenile fish fed with copepods was higher than that fed with Artemia （P〈0.05）. The contents of three types of HUFAs （DHA, EPA and ARA） and the ratio of DHA/EPA in larval and juve- nile flounder P. olivaceus were analyzed. The results showed that the contents of DHA, EPA and ARA in the larvae and juveniles fed with S. poplesia were higher than those fed with a mixed diet orArtemia only, and the ratio of EPA/ARA in larvae and juveniles of P. olivaceus fed with S. poplesia was lower than that in the case of feeding with a mixed diet or Artemia only. The present data showed that copepod is the best choice for feeding the larvae and juveniles of fish considering its effects on the survival, growth and nutrition composition of the fish.     

限食水平与饥饿时间对卵形鲳鲹幼鱼补偿生长的影响

限食水平与饥饿时间对卵形鲳鲹幼鱼生长的影响实验共分7组,其中3个限食处理组日投喂量分别为对照组的20%、40%和60%,限食12 d后饱食投喂28 d;3个饥饿处理组饥饿时间分别为4、8、12 d,分别饱食投喂36、32和28 d;对照组饱食投喂40 d。结果表明:短期限食都能发生完全补偿生长效应,补偿生长效果随饥饿时间延长而减弱,饥饿12 d发生部分补偿生长;各组的摄食率和饲料转化率都有所提高,饥饿程度较大组补偿生长效应主要是通过提高摄食率来实现;在限食或饥饿期间,鱼体肌肉中的水分和粗灰分含量均有所升高,粗蛋白和粗脂肪含量呈现不同程度下降。     

温度对全缘马尾藻幼孢子体生长和生理组分的影响

为探究全缘马尾藻(Sargassum intrgerrimum)幼孢子体对温度的适应性,在照度为10 000 lx,光照周期为12L:12D,温度分别为11.0、16.0、21.0、26.0、31.0、36.0℃的条件下,研究不同温度对全缘马尾藻幼孢子体生长、光合色素含量、SOD活力和CAT活力的影响。结果表明,幼孢子体在16.0~26.0℃的温度范围内生长明显,差异有统计学意义(P﹤0.05),温度为21.0℃时生长最快;温度为11.0℃时生长缓慢,温度为31.0和36.0℃时幼孢子体的生长因温度过高有溃烂现象。在本实验条件下,全缘马尾藻幼孢子体的适宜温度范围为16.0~26.0℃,最适温度为21.0℃。     

Genome-Wide Identification of heat shock protein 10/60 Genes in Japanese Flounder(Paralichthys olivaceus) and Their Regulated Expression After Bacterial Infection

Heat shock proteins 10/60(hsp10/60) are a family of conserved ubiquitously expressed heat shock proteins which are produced by cells in response to exposure to stressful conditions. Besides the chaperone and housekeeping functions, they are also known to be involved in immune response during bacterial infection. In this study, we identified and annotated 10 hsp10/60 genes through bioinformatic analysis in Japanese flounder(Paralichthys olivaceus). Among them one member of hsp10(hspe) family and nine members of hsp60(hspd) family were identified. Phylogenetic and selection pressure analysis showed that the hsp10/60 genes were evolutionarily constrained and their function was conserved. Besides, hsp10/60 genes were involved in different embryonic and larval stages and acted as the sentinel role in an unchallenged organism. In addition, we also observed the expression patterns of hsp10/60 genes after Edwardsiella tarda infection, for the first time in Japanese flounder. Eight out of 10 genes were differentially expressed after bacterial challenges, the significantly regulated expressions of flounder hsp10/60 genes after bacterial infections suggested their involvement in immune response in flounder. Our results provide valuable information for clarifying the evolutionary relationship, and early insights of the immune functions of hsp10/60 genes in Japanese flounder.     

Weighted Correlation Network Analysis(WGCNA) of Japanese Flounder(Paralichthys olivaceus) Embryo Transcriptome Provides Crucial Gene Sets for Understanding Haploid Syndrome and Rescue by Diploidization

Artificial gynogenesis is of great research value in fish genetics and breeding technology. However, existing studies did not explain the mechanism of some interesting phenomena. Severe developmental defects in gynogenetic haploids can lead to death during hatching. After diploidization of chromosomes, gynogenetic diploids may dispense from the remarkable malformation and restore the viability, although the development time is longer and the survival rate is lower compared with normal diploids. The aim of this study was to reveal key mechanism in haploid syndrome of Japanese flounder, a commercially important marine teleost in East Asia. We measured genome-scale gene expression of flounder haploid, gynogenetic diploid and normal diploid embryos using RNA-Seq, constructed a module-centric co-expression network based on weighted correlation network analysis(WGCNA) and analyzed the biological functions of correlated modules. Module gene content analysis revealed that the formation of gynogenetic haploids was closely related to the abnormality of plasma proteins, and the up-regulation of p53 signaling pathway might rescue gynogenetic embryos from haploid syndrome via regulating cell cycle arrest, apoptosis and DNA repair. Moreover, normal diploid has more robust nervous system. This work provides novel insights into molecular mechanisms in haploid syndrome and the rescue process by gynogenetic diploidization.     

Optimization of the purification methods for recovery of recombinant growth hormone from Paralichthys olivaceus

This study aimed to optimize the purification of recombinant growth hormone from Paralichthys olivaceus. Recombinant flounder growth hormone (r-fGH) was expressed by Escherichia coli in form of inclusion body or as soluble protein under different inducing conditions. The inclusion body was renatured using two recovery methods, i.e., dilution and dialysis. Thereafter, the refolded protein was purified by Glutathione Sepharase 4B affinity chromatography and r-fGH was obtained by cleavage of thrombin. For soluble products, r-fGH was directly purified from the lysates by Glutathione Sepharase 4B affinity chromatography. ELISA-receptor assay demonstrated that despite its low receptor binding activity, the r-fGH purified from refolded inclusion body had a higher yield (2.605 mgL-1) than that from soluble protein (1.964 mgL-1). Of the tested recovery methods, addition of renaturing buffer (pH 8.5) into denatured inclusion body yielded the best recovery rate (17.9%). This work provided an optimized purification method for high recovery of r-fGH, thus contributing to the application of r-fGH to aquaculture.     

不同饵料对方斑东风螺幼螺生长的影响

在盐度29.5~30,p H 7.6~7.8、水温29~29.5℃条件下,投喂凡纳滨对虾、小黄鱼、太平洋牡蛎3种饵料30 d,研究不同饵料对方斑东风螺幼螺生长的影响。研究表明,投喂凡纳滨对虾的方斑东风螺幼螺生长最快,壳宽(9.41±0.72)mm,壳高(15.08±1.18)mm,体质量(0.61±0.13)g,特定生长率5.153%·d-1,均优于投喂小黄鱼和太平洋牡蛎处理组。