养殖近江牡蛎致病弧茵的分离与鉴定

从广东省疫区患病近江牡蛎Crassostrea ariakensis消化盲囊及血液中分离纯化出10株细菌,人工注射感染实验发现其中3株细菌注射组死亡率显著高于对照组。3株细菌均为革兰氏阴性,短杆状,能运动。常规生理生化和Biolog细菌鉴定系统测试结果表明,菌株A3G-2与溶藻弧菌Vibrio alginolyticus亲缘关系最近,菌株A2G-5B与解蛋白弧菌V．proteolyticus亲缘关系最近,菌株B1X-1与鲨鱼弧菌V．carchariae亲缘关系最近。为了进一步确定它们的分类学地位,分别测定了3株弧菌的16S rDNA序列,分析相关细菌相应序列的同源性,并构建系统进化树。结果表明,菌株A3G-2与溶藻弧菌亲缘关系最近,菌株A2G-5B与解蛋白弧菌亲缘关系最近,菌株B1X-1与鲨鱼弧菌亲缘关系最近。因此菌株A3G-2、A2G-5B和BIX-1分别被鉴定为溶藻弧菌、解蛋白弧菌和鲨鱼弧菌。组织病理观察发现患病牡蛎主要受损器官是消化盲囊,表现为腺泡管内壁吸收细胞崩解脱落,管腔扩大,进而管壁解体,消化盲囊出现大面积受损。     

重金属Cd2+胁迫对近江牡蛎超氧化物歧化酶活性的影响

探讨了将近江牡蛎抗氧化酶防御体系参数作为海洋重金属污染生物监测指标的可行性。在实验室条件下,研究重金属Cd2 胁迫对近江牡蛎Crassostrea rivularis鳃和消化腺组织超氧化物歧化酶(SOD)活性的诱导作用。结果表明,Cd2 对近江牡蛎消化腺组织SOD活性诱导的剂量效应呈抛物线型,而对鳃组织SOD活性诱导曲线呈非抛物线型,由于二者结构和功能的不同,前者的SOD活性高于后者;低浓度Cd2 (0.1mg.L-1)对近江牡蛎鳃组织SOD活性表现为显著诱导,中(0.5mg.L-1)、高(1.5mg.L-1)浓度Cd2 对近江牡蛎鳃组织SOD活性诱导呈先升后降的变化趋势;低、中浓度Cd2 对近江牡蛎消化腺组织SOD活性均表现为显著诱导,高浓度Cd2 对近江牡蛎消化腺组织SOD活性诱导呈先升后降的变化趋势。研究表明,对于水体中单一因子的Cd2 污染物而言,近江牡蛎消化腺组织中的SOD活性对其有一定的指示意义,但将其作为指标应用到实际养殖水体尚须进一步研究。     

湛江港近江牡蛎的生态研究

湛江港石门海区的近江牡蛎Crassostrea rivularis(Gould)养殖,出现苗种大批死亡、生产不稳定等现象。本文对该海区近江牡蛎的生活环境和个体生态进行了调查研究,并对它的采苗、养成、生物敌害的防除等问题作了初步探讨。     

溶藻弧菌内毒素免疫Balb/c小鼠方法的研究

采用热酚水法提取点带石斑鱼(Epinephelus malabaricus)病原溶藻弧菌VAL02菌株的内毒素脂多糖(lipopolysaccharide,LPS)。用提取的LPS按15、30、60μg·只-1和VAL02菌株颗粒抗原按1×108(不加佐剂)和1×108cell·只-1。(加佐剂)的方式免疫Balb/c小鼠,经1次基础免疫和6次加强免疫后,间接ELISA检测。结果表明颗粒抗原加佐剂免疫组效果最好。     

盐度对近江牡蛎幼虫生长及存活的影响

采用室内培育手段,研究了盐度对近江牡蛎(Crassostrea rivularis)幼虫生长及存活的影响。将人工授精孵育的面盘幼虫分别置于盐度依次为16,25,30的砂滤海水中培育至眼点幼虫。每隔一天检测各盐度组幼虫的壳长、壳高和存活数量。结果表明,16盐度组的幼虫生长最快,25盐度组次之,30盐度组最慢。从受精后第14天开始,16盐度组幼虫的生长显著快于30盐度组(P<0.05)。在整个浮游幼虫阶段,高盐度组幼虫的存活率低于低盐度组,其中30盐度组的存活率最低。16和25盐度组在幼虫出现眼点之前均出现大量死亡现象,而30盐度组并没有出现大量死亡现象。上述结果说明,近江牡蛎幼虫经过高盐驯化,对高盐环境的抗性具有一定的提高。     

近江牡蛎鳃细胞的原代培养

采用改良的DMEM(HG)培养基,建立了近江牡蛎(Crassostrea hongkongensis)鳃细胞体外培养技术,包括鳃组织块培养法和胰酶消化培养法。结果表明,组织块培养法接种6 h后,细胞开始从组织块中迁出,细胞形态较小,呈圆形、椭圆形或多边形,直径3～6μm。培养至3 d时,细胞在组织块周围形成生长晕。培养至6 d时可进行细胞传代,本次实验细胞已传至第6代。胰酶消化法接种约2 h后,细胞逐渐贴壁,从形态上主要分为两类,一类为小型细胞,形态为圆形、椭圆形或多边形,直径3～6μm,数量多,增殖速度较快;另一类为大型细胞,形态为圆形、椭圆形或多边形,直径10～20μm,部分细胞内部含有颗粒,数量较少,增殖速度较慢。培养至2 d时可进行细胞传代,传代培养物中的优势细胞皆为小型细胞。本次实验细胞已传至第6代。     

微塑料对近江牡蛎免疫力的影响

为探究微塑料对牡蛎免疫力的影响,该研究对近江牡蛎(平均壳长:6.6cm)采用壳内推注的方式注入不同浓度的聚苯乙烯微塑料(0.05、0.5、5 mg·L^–1,以无菌生理盐水为稀释液),分别胁迫24 h、48 h和72 h后,检测分析牡蛎鳃组织的免疫指标(碱性磷酸酶(AKP)、超氧化物歧化酶(SOD)、丙二醛(MDA)、过氧化氢酶(CAT)、一氧化氮合成酶(NOS)和乙酰胆碱酯酶(AChE))的变化。结果显示,在胁迫24 h后,微塑料浓度对AKP的活力有显著影响(P<0.05)。SOD、AKP和结构型一氧化氮合成酶的活性呈现明显的先升后降的趋势。在胁迫48 h后,微塑料浓度上升导致A-chE活力显著下降(P<0.05), CAT、SOD和AKP呈现先升后降的趋势。在胁迫72 h后,微塑料浓度的升高, AKP和AChE的活力呈现先降后升的趋势,而SOD、CAT和诱导型一氧化氮合成酶的活力与之呈现相反的趋势,显示先升后降的趋势。随时间和浓度的增长, MDA处于一直上升的趋势。以上结果表明,微塑料可使牡蛎产生免疫应激反应,且胁迫时间越长,牡蛎免疫应激反应越剧烈。     

广西钦州湾近江牡蛎体有机氯农药和多氯联苯的残留水平与分布

对广西钦州湾近江牡蛎(Crassostrea rivularis)体有机氯农药(HCHs和DDTs)和多氯联苯(PCBs)的残留水平进行监测与分析。结果表明:近江牡蛎体中HCHs、DDTs和PCBs的质量比分别为ND～0.23μg/kg、0.01～0.21μg/kg和ND～2.3μg/kg;检出率分别为45%、100%和95%,3种污染物含量符合国家《海洋生物质量》第一类标准和无公害水产品质量要求。相对国内其他海区而言,钦州湾近江牡蛎受HCHs、DDTs和PCBs污染程度处于低污染水平。     

黄河口与长江口近江牡蛎的比较转录组分析

为进一步开发近江牡蛎Crassostreaariakensis的基因资源,探究黄河口与长江口近江牡蛎的遗传差异,采用Illumina HiSeq 4000测序平台对东营垦利和南通海门的近江牡蛎进行高通量转录组测序。对测序数据进行拼接后分别得到83680条和71269条unigene,并对unigene进行了基因功能注释和正选择基因的富集分析。结果表明,同源基因经筛选得到正选择基因259个,GO (Gene Ontology)功能分析中共涉及到967个相关功能类别,其中有关到生物学过程的分类条目占比最多,为590项。总体上看,与细胞器、代谢过程、结合以及催化活性相关的类别占主导地位。KEGG( Kyoto Encyclopedia of Genes and Genomes)富集分析表明,共涉及48个代谢通路,其中与核糖体、细胞凋亡相关的通路占比最大。上述结果不仅加深了对黄河口以及长江口近江牡蛎基因表达水平差异的认识,同时也为今后进行近江牡蛎各类遗传学分析和一些关键基因的克隆以及具体功能分析提供了基础数据。     

近江牡蛎与太平洋牡蛎的同工酶鉴别

福建省罗源湾于上世纪80年代初先后引进来自日本、台湾和澳大利亚的太平洋牡蛎．该品种引进后不久就开始出现养殖品质退化的现象；对此，有人怀疑是与本地近江牡蛎杂交所致。本文通过同工酶分析，确认罗源湾太平洋牡蛎被测样品中不舍与近江牡蛎杂交的个体．苹果酸脱氢酶电泳可作为鉴定相关种群的技术手段．但是，本项调查结果也显示罗源湾太平洋牡蛎很可能不是纯系品种，培育纯化品系、加强种质管理对振兴福建牡蛎产业具有重要意义。     

厦门浔江湾牡蛎养殖区弧菌研究

利用ＭＰＮ法及ＡＰＩ２０Ｅ系统对厦门浔江湾牡蛎养殖区的特牡蛎体、水体及底质三类榈进行弧菌分析。结果表明，三类榈的弧菌密度随季节呈春，夏〉秋，冬趋而变化。牡蛎体弧菌密度与水样、底质样弧菌密度之间的相关数值分别为０．８１４３和０．５９５５，二者都高于０．３２５，因此呈显著相关，而且前两者相关关系更密切。     

Accumulation and cytosolic partitioning of metals in the American oyster Crassostrea virginica

The ability of the oyster to accumulate trace metals, particularly the transition metals (Cd, Cu, Zn, and Hg), from food and water has been well documented. There is little information, however, on how these metals interact to affect whole animal retention, partitioning, and binding to metallothionein (Mt). In this study oysters were exposed to Cd and Cu, both alone and in combination, and significant effects have been demonstrated on subcellular partitioning. These studies showed that Cu can displace Cd from Mt, but the Cd is not lost from the animal. Two metal-binding protein peaks have been separated by gel chromatography (<10 K and 24 K daltons). The lower molecular weight protein peak has been characterized as Mt, consisting of two isoproteins, but the higher molecular weight protein peak appears to be an aggregation of metal-binding proteins, some of which may be Mt or a Mt dimer. In environments that are contaminated with trace metals, Mt binds primarily Hg, Cu, and/or Cd and Zn, but in noncontaminated areas Mt binds primarily Zn on one of the two isoMts.     

人工诱导香港巨牡蛎雌核发育单倍体的研究

研究利用紫外线诱导香港巨牡蛎Crassostrea hongkongensis雌核发育的条件.结果表明,在强度为1820μW · (cm2·s)-1的紫外线(254nm)下分别照射精子0,5,10,15,20,25,30,35,40,45,50,55,60,70,80和90s后,照射30s的实验组能够保持较高的受精率(61.6%),该组的D形幼虫发生率为0,出现"Hertwig效应".研究表明,在强度为1 820 μW·(cm2·s)-1的紫外线下照射精子30s是获得香港巨牡蛎雌核发育单倍体的适宜条件.研究还表明受精率和D形幼虫发生率随照射时间的增加而下降,遗传失活的精子与正常卵子受精后其胚胎发育至D形幼虫前期停止.经紫外线照射的精子授精后所产生的单倍体胚胎发育速度慢于正常受精所产生的二倍体胚胎,各照射组均出现非整倍体.     

Reproductive cycle and gonadial changes in the New Zealand rock oyster Crassostrea glomerata

The annual reproductive cycle and the cyclical changes in the gonad of the New Zealand rock oyster, Crassostrea glomerata (Gould, 1850), during the breeding period of 1970–71 and 1971–72 have been described. The gonad passes through an intermediate phase, after a post‐spawning period, when sex is indeterminate. Gametogenesis begins in July and August, but follicles ripen mostly during the spring months of October and November. Maximum development is seen in November, and nearly all oysters are in spawning condition in December and January. Major spawning takes place in January or February depending upon water temperature, and spawning continues until the end of March. Larvae may be found in the‐ plankton until late in the season, to the end of April or even May. Following spawning, gonadial regression sets in, with leucocyte infiltration and phagocytosis of residual gonial cells. A greater percentage of oysters more than 1 y old are females. The majority of oysters of age 1 y and below are males.     

Esterification of vertebrate-like steroids in the eastern oyster (Crassostrea virginica)

The esterification of two model vertebrate steroid hormones - estradiol (E2) and dehidroepiandrosterone (DHEA) - was studied in the oyster Crassostrea virginica. The activity of acyl-CoA:steroid acyltransferase was characterized in microsomal fractions isolated from oyster digestive glands. The apparent Km and Vmax values changed with the fatty acid acyl-CoA used (C20:4, C18:2, C18:1, C16:1, C18:0 or C16:0), and were in the range of 9-17 microM, and 35-74 pmol/min/mg protein for E2, and in the range of 45-120 microM, and 30-182 pmol/min/mg protein for DHEA. Kinetic parameters were also assessed in gonadal tissue. The enzyme saturated at similar concentrations, although conjugation rates were lower than in digestive gland. Preliminary data shows that tributyltin (TBT) in the low microM range (1-50) strongly inhibits E2 and DHEA esterification, the esterification of E2 being more sensitive to inhibition than that of DHEA. Overall, results indicate that apolar conjugation occurs in oysters, in both digestive gland and gonads, at a very similar rate to mammals, suggesting that this is a well conserved conjugation pathway during evolution. Esterification, together with other mechanisms, can modulate endogenous steroid levels in C. virginica, and might be a target for endocrine disrupters, such as TBT.     

不同规格近江牡蛎的排氨率和耗氧率分析

采用实验生态学方法研究了不同规格近江牡蛎的呼吸和排泄生理生态学参数。结果显示,近江牡蛎耗氧率R_O随软体干重(W )的增加而减小, 体重对近江牡蛎耗氧率(R_O)的影响达极显著水平,R_O = 1.8232W^-0.7600, (R²= 0.8313 , P <0.01); 近江牡蛎的排氨率(R_N)和排磷率(R_P)随软体干重的增加而降低,呈负相关关系;而氧氮比O/N和氮磷比N/P在各组间未达显著水平(P >0.05) , R_N = 0.0930W^-0.6582 (R² = 0.7444, P <0.01)、R_P = 0.0126W^-0.8874 (R² = 0.9224,P <0.01);各组不同规格近(大小)江牡蛎的O/N值较高,而N/P值则随规格增大而呈上升趋势,但差异也不显著(P >0.05)。这些研究结果对牡蛎养殖密度和环境关系分析及牡蛎健康养殖具有重要参考价值。     

Expression and activity of a multixenobiotic resistance system in the Pacific oyster Crassostrea gigas

The multidrug resistance (MDR) mechanism corresponds to a defence system relying on the expression of high molecular membrane proteins that can actively lower the intracellular concentration of a wide variety of toxins, thus maintaining them below their toxic level. Using RT-PCR, expression levels of a gene belonging to the class I of mammalian mdr genes, has been assessed in different developmental stages of the oyster Crassostrea gigas. While no expression was found in the oocyte or the trocophore stage, a rise of mRNA content was observed from the veliger stage to the juvenile stage, thus indicating the induction of the system as the animal is developing in the environment. The incubation of gill fragments in the dye rhodamine B and subsequent measurements of intracellular fluorescence using a microplate reader indicates that the system can effectively decrease the accumulation of the test compound in a competitive manner with known inhibitors or environmental contaminants as observed in vertebrate cells. The oyster MXR system is thus becoming active in adult oyster and could be of importance in environmentally contaminated areas.