长牡蛎糖原磷酸化酶基因SNPs与生长性状和糖原含量的相关性分析

为研究糖原磷酸化酶基因多态性与长牡蛎(Crassostrea gigas)生长和糖原含量的相关性,本研究对长牡蛎糖原磷酸化酶基因(Cg-GPH)编码区单核苷酸多态性(SNPs)与来自5个家系322个长牡蛎个体的生长性状(包括壳高、壳长、壳宽、总体质量以及软体部质量)和糖原含量进行了关联分析.结果表明,在1940 bp的长牡蛎糖原磷酸化酶基因中共检测到82个SNPs位点,其中63个SNPs位点位于外显子区域,1个SNPs位点位于5′-UTR区,18个SNPs位点位于3′-UTR区,编码区SNPs平均密度为1/25 bp;5个SNPs位点与生长性状存在显著相关(P<0.05),未检测到与糖原含量相关SNPs.根据以上5个SNPs位点共构建得到6个SNP单倍型,其中,Cg-GPH基因的H6(CTGAT)单倍型在总体质量性状方面均显著高于其他5种单倍型(P<0.05),表明H6单倍型可能是对长牡蛎体质量增加最有利的单倍型.研究结果为今后长牡蛎生长性状的遗传改良提供了基础资料.     

不同育性长牡蛎性腺发育过程中糖原和脂质的组织化学研究

为探讨不同育性长牡蛎(Crassostrea gigas)性腺发育过程中主要供能物质的利用和变化差异,利用过碘酸雪夫氏染色(PAS染色)和油红O染色法对不同育性三倍体长牡蛎及二倍体长牡蛎性腺组织进行观察分析。结果显示,增殖期,二倍体和三倍体长牡蛎性腺结缔组织中均含有大量糖原和脂质。随着性腺发育,二倍体和可育型三倍体(3nα型)长牡蛎性腺结缔组织中糖原含量明显下降,到成熟期几乎检测不到糖原,说明性腺结缔组织中的糖原为配子发生提供能量。脂质主要存在于性腺结缔组织和卵母细胞的细胞质中,说明脂质是卵细胞发育的重要组成物质。雌性二倍体和3nα型长牡蛎性腺结缔组织中的脂类含量随着性腺发育并未发生明显下降,推测糖原可能转化为脂质以满足性腺发育的物质需求。脂质在二倍体雄性长牡蛎的性腺发育过程中出现明显的减少,说明脂类在雄性性腺发育中的主要功能可能是供能而不是结构组成。糖原和脂类作为长牡蛎性腺发育过程中重要的供能和组成物质,在不育型三倍体(3nβ型)长牡蛎性腺发育过程中含量没有发生明显的变化,这与3nβ型配子发生受阻密切相关。推测3nβ型长牡蛎由于配子发育受阻,性腺发育初期积累的糖原和脂质并未分配至生殖细胞,而留在结缔组织中,从而使其在繁殖季节仍然能够保持快速生长。研究结果为牡蛎生殖发育调控机制及育性控制育种提供重要信息。     

长牡蛎高糖原含量新品系正反交家系的生理代谢

为了探究糖原含量差异显著的长牡蛎(Crassostrea gigas)自交家系和正反交家系在生理代谢水平上的差异,本研究开展了在一定温度(20℃)和盐度(32)下自交组和正反交组的滤水率、耗氧率和排氨率实验。结果显示,滤水率方面,自交组AA最大,BB最小,二者差异显著(P0.05);正交组AB和反交组BA二者差异不显著且大小介于2个自交组之间,正反交组的杂种优势值为8.44%。耗氧率方面,自交组BB最大,正交组AB最小;自交组AA与BB差异显著(P0.05),正反交组杂种优势值为-43.87%;耗氧率与糖原含量呈负相关,相关系数为-0.884,达到极显著性水平(P0.01)。排氨率方面,各组之间的排氨率无显著差异,大小为0.04 mg NH_3-N/(g·h)。研究表明,糖原含量品系正反交家系的差异在生理代谢上主要表现为滤水率和耗氧率的差异。     

环境因子和免疫刺激对香港牡蛎免疫指标的影响

病害问题已严重威胁香港牡蛎(Crassostrea hongkongensis)养殖业的发展,寻找适用于香港牡蛎健康评价的生物标志物,对预防其病害具有重要的实用价值.该研究以盐度胁迫、氨氮胁迫、免疫刺激以及干露胁迫为实验条件,研究不同因素胁迫下香港牡蛎肝胰腺中免疫因子的变化情况,以期找出1～2个香港牡蛎对环境因子变化敏...     

升温与聚苯乙烯微塑料复合暴露对长牡蛎血细胞功能、免疫基因表达和能量代谢的影响

为阐明全球气候变暖和微塑料复合胁迫对长牡蛎(Crassostrea gigas)免疫应答、氧化应激和能量代谢的影响,本研究采用3个微塑料(microplastics, MPs)水平[无微塑料、小粒径聚苯乙烯微塑料(SPS-MPs,6μm)和大粒径聚苯乙烯微塑料(LPS-MPs,50～60μm)]和2个温度水平(20℃和25℃)对长牡蛎进行了为期21 d的单一和复合暴露,检测分析了各组长牡蛎血细胞功能[吞噬活性、活性氧(reactive oxygen species,ROS)含量]、糖原含量以及免疫相关基因表达的变化。研究结果表明,SPS-MPs暴露能增加长牡蛎血淋巴细胞中ROS含量,降低血细胞吞噬活性,揭示SPS-MPs毒性作用更强。升温与微塑料的协同作用增加了长牡蛎消化腺组织中的糖原含量。实时荧光定量PCR结果显示,升温与SPS-MPs复合暴露组长牡蛎消化腺组织通过上调热休克蛋白90 (heat shock protein90, HSP90)、核因子κB抑制蛋白(inhibitor of NF-κB, IκB)和p53基因表达量进行免疫应答;升温与微塑料的拮抗作用增加了鳃组织p53基...     

长牡蛎呼吸、排泄及钙化的日节律研究

通过室内实验与海区现场实验相结合的方法,研究了10℃、18℃及20℃下3种不同规格(壳高:S:2.5cm;M:5.5cm;B:6.8cm)的长牡蛎耗氧率、排氨率及钙化率的日变化。实验结果表明,长牡蛎的代谢有一定节律性,其中,呼吸表现为昼夜节律,在水温10℃时为夜高昼低,夜间的耗氧率比白天平均高0.07mg/ind.h;水温20℃时,室内实验的呼吸率无明显节律。而现场实验则表现为昼高夜低,白天比夜间高0.08mg/ind.h。排氨率与耗氧率变化不一致,白天和夜间分别有相近的变化趋势,可能是受到潮汐节律的影响;长牡蛎的钙化则表现出复杂的变化,不同时间段间有显著差异(P<0.05),但没有明显的节律性。在进行牡蛎生理实验时,要避免取短时间的生理指标计算其代谢水平,应选择不同时段进行重复实验。     

高温胁迫对长牡蛎‘海大3号’品系存活及5项免疫指标的影响

夏季海水温度升高会对长牡蛎(Crassostrea gigas)体内生化反应产生重要影响,进而影响养殖牡蛎的存活率。本研究探讨了长牡蛎‘海大 3 号’在受到温度突升和温度渐升胁迫时的高温耐受性,以及在温度突升胁迫条件下过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、溶菌酶(LSZ)、总抗氧化能力(T-AOC)和丙二醛(MDA)5 种相关免疫指标在 72 h 内的变化。温度渐升实验中,实验海水的温度从 16℃逐渐升温(2℃/d)。温度突升实验中,将长牡蛎从暂养水温(16℃)分别直接转移至 18℃、22℃、26℃、30℃和 34℃。实验结果显示,温度渐升时长牡蛎‘海大 3 号’的最高存活温度(survival temperature maximum, STMax)为 33.63℃,最高临界温度(critical temperature maximum, CTMax)为 40.13℃,存活率为 50%时的温度(50% critical temperature maximum, 50%CTMax)为 36.67℃。在温度突升胁迫实验中, 72 h 的半数致死高温(median lethal temperature)72-h LT50为 30.13℃。各实验组内脏团中 5 项相关免疫指标随时间增加变化显著。在前 12 h, CAT、SOD 及 LSZ 活性和 T-AOC 显著升高,之后逐渐下降恢复到初始水平。MDA含量在 6 9 h 达到最大值,之后逐渐下降。这些结果表明高温胁迫会引起长牡蛎‘海大 3 号’体内的抗氧化免疫和溶菌酶活性发生显著变化,进而对牡蛎的存活产生重要影响。本研究检测的高温环境的耐受能力,将为长牡蛎新品系‘海大 3 号’的应用推广提供参考。     

温度和盐度急性胁迫对岩牡蛎存活及免疫指标的影响

为探究岩牡蛎(Crassostreanippona)对不同温度和盐度急性变化的适应性,本研究通过将岩牡蛎从暂养环境(27℃,盐度32)直接转移至5个温度梯度(15℃、19℃、23℃、31℃、35℃)和6个盐度梯度(16、20、24、28、36、40),研究了急性胁迫对岩牡蛎超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、碱性磷酸酶(AKP)、溶菌酶(LSZ)活性、丙二醛(MDA)含量及存活的影响。温度实验结果显示,15～31℃组岩牡蛎存活率均高于71.11%,35℃组存活率最低(34.44%),而15℃组免疫指标活性均受到明显抑制, MDA含量在144 h时达到最大值(46.93 nmol/mg prot);温度19℃、23℃、31℃组SOD、CAT、AKP和LSZ活性及MDA含量受应激显著上升后均逐渐恢复至对照水平。盐度实验结果表明, 40组盐度变化对岩牡蛎存活抑制明显,显著低于其他处理组(P0.05),适宜岩牡蛎存活的盐度变化为20～36组;盐度16组SOD、CAT、AKP和LSZ活性144 h时均显著低于对照组(P0.05), MDA含量与胁迫时间成正相关最终达到最大值(45.76 nmol/mg prot)。研究表明,温度和盐度突变均会对岩牡蛎5项免疫指标和存活率产生显著影响,岩牡蛎对温度19～31℃组和盐度20～36组突变具有较强的适应能力。     

长牡蛎夏季死亡与养殖环境及自身体质关系的初步研究

选取桑沟湾和爱连湾两个典型牡蛎养殖区为研究对象,调查了两个海区高温季节牡蛎的死亡情况,监测了不同养殖区的水温、盐度、pH和DO等环境指标,并测定了牡蛎体内超氧化物歧化酶(SOD)活性和生长指标的差异,初步探讨了桑沟湾养殖长牡蛎夏季高温死亡的原因。结果表明,实验期间(5～10月)桑沟湾崖头养殖海区(E122.498,N37.151)长牡蛎夏季累积死亡率为37%,最高死亡率出现在8月17日,高达51%,而爱连湾养殖海区(E122.582,N37.179)的累积死亡率仅为2.5%,最高死亡率出现在9月28日,为4.5%。单因子方差分析(ANOVA)表明死亡率与水温具有很强的正相关(R=0.804,P0.01),与溶氧呈明显负相关(R=-0.377,P0.01);死亡率与体重、软体湿重、性腺湿重等生长状况指标呈明显的负相关(R分别为-0.690、-0.498、-0.358),与SOD活性呈正相关(R=0.21,P0.05)。     

栉孔扇贝♀和长牡蛎♂杂交受精及早期胚胎的细胞学研究

以栉孔扇贝Chlamys farreri为母本、长牡蛎Crassostrea gigas为父本进行人工杂交。采用Bouin氏液固定、石蜡切片和苏木精-伊红染色在光镜下对其受精和第1次卵裂过程中核相变化进行观察;运用压片法和热滴片法分别对4～8细胞期和担轮幼虫期胚胎进行染色体制片。结果表明,长牡蛎精子可以进入栉孔扇贝卵子,并激活卵子减数分裂使其释放第1极体（PB1）和第2极体（PB2）,能够形成雌、雄原核并融合为合子核,接着受精卵开始第1次卵裂;杂交胚胎发育过程中,囊胚期很长、胚胎畸形严重,只能发育到担轮幼虫期;杂种胚胎染色体数目变化范围较大,在20～85之间,染色体不能平均分配到两个子细胞中是杂种胚胎死亡的主要原因。杂交胚胎不能存活保证了异源精子诱导栉孔扇贝雌核发育的可靠性。     

夏季高温期三倍体和二倍体长牡蛎生理能量学及碳收支的比较研究

为解析夏季高温期三倍体与二倍体长牡蛎(Crassostrea gigas)摄食和代谢生理以及能量/碳分配策略的差异,于2022年8月,以三倍体和二倍体长牡蛎为研究对象,在山东荣成桑沟湾采用现场流水法测定滤水率、吸收效率、耗氧率、排氨率等摄食和代谢相关生理参数,并基于能量收支方程估算能量分配与碳分配情况。结果显示,三倍体长牡蛎的滤水率、同化效率均高于二倍体长牡蛎,但无显著差异(P>0.05);三倍体与二倍体长牡蛎的耗氧率、排氨率存在显著差异(P<0.05),三倍体长牡蛎的耗氧率显著低于二倍体长牡蛎(P<0.05),但排氨率显著高于二倍体长牡蛎(P<0.01)。能量收支与碳收支的结果显示,三倍体长牡蛎的摄食能/碳、同化能/碳均高于二倍体长牡蛎,但无显著差异(P>0.05);三倍体与二倍体长牡蛎的呼吸能/碳、排泄能/碳、生长余力存在显著差异(P<0.05),三倍体长牡蛎的呼吸能/碳显著低于二倍体长牡蛎(P<0.05),但排泄能/碳、生长余力显著高于二倍体长牡蛎(P<0.05)。三倍体和二倍体长牡蛎的氧氮比波动范围分别为7.91~14.11和59.81~94.19,因此,三倍体长牡蛎的主要供能物质为蛋白质,二倍体长牡蛎的主要供能物质为糖类和脂肪。研究结果为揭示夏季高温期长牡蛎倍性效应关联的能量分配方式差异提供了数据支撑。     

基于微卫星标记整合长牡蛎遗传图谱

为了提高长牡蛎遗传图谱上的微卫星标记密度,实验采用6个家系图谱间的共有微卫星标记作为锚定标记,构建了长牡蛎的整合图谱.该整合图谱共有161个微卫星标记,覆盖10个连锁群,图谱长度和平均间距分别为615.4和3.8 cM.各连锁群的标记数介于10 ～ 24个之间,连锁群长度为47.3～73.3 cM,是目前密度最高的长牡蛎微卫星图谱.不同作图家系连锁群上的标记分组保持一致,但标记顺序出现差异,可能与长牡蛎自然群体中存在大量的染色体重排现象有关.结果表明,该图谱可以为今后长牡蛎的遗传育种研究提供新的遗传工具.     

Sex-specific growth and condition of the Pacific oyster (Crassostrea gigas Thunberg)

In order to study the possibility of gaining commercial benefit from culturing an excess of one sex of the Pacific oyster (Crassostrea gigas), comparative data on the growth rate and condition of male and female oysters are reported. Historically, measurement of sex‐specific growth rate in oysters has been overlooked or confounded by protandric sex. The recent conclusion that the sex ratio of Pacific oysters is predominantly under genetic rather than environmental control introduces the possibility of manipulating sex ratio for commercial gain if they exhibit asynchronous sex‐specific growth rates. Pacific oysters were cultured intertidally in Smoky Bay, south Australia. The observations, made over the 7‐month gametogenic cycle from August to February to ensure no sex reversal, were of growth rates of male and female oysters and ambient chlorophyll a concentrations. Mean shell growth of female oysters was significantly faster than that of males (4.5 ± 3.3 compared with 3.8 ± 3.2 μm day^?1 mm^?1 total length). Sex‐specific asymmetries in length and weight were generally significant and increased in magnitude during the 7‐month study period, suggesting potential commercial benefits from increasing the proportion of cultured female oysters. The fastest increase in the sex‐specific disparity in growth and condition came after the October chlorophyll a peak, suggesting that females utilize blooms more efficiently than males. Our results compare favourably with methods currently used to increase oyster growth (e.g. triploidy can provide growth gains of 13–51%).     

福建漳浦太平洋牡蛎微卫星等位基因比较

本文采用Ucdcg153、157、202微卫星分子标记对福建漳浦从广东饶平引人的太平洋牡蛎育苗亲贝进行等位基因比较.两组样品(Cg♀和cg♂)携带的等位基因在微卫星重复区间及其附近存在高度相似的变异特征.这些特征区间分别是:Ucdcg153 23～40位碱基区间,Ucdcg157 73～100位碱基区间,Ucd-cg2...     

长牡蛎壳金选育群体性腺发育与营养成分的周年变化

为了评估长牡蛎(Crassostrea gigas)壳金选育群体的繁殖周期及营养价值,自2013年11月至2014年10月,对山东省刘公岛海区长牡蛎第三代壳金选育群体的性腺发育、营养成分(总蛋白、总脂肪、糖原)季节变化及其与环境因子的关系进行了研究,并比较了与普通养殖群体营养成分的差异。结果表明,长牡蛎壳金选育群体雌雄比例接近1︰1,性腺同步发育;配子发生开始于2月,全年只有1个产卵季节(6―8月),7月为两性配子排放高峰期。配子成熟及排放期间,水温处于较高水平,食物充足,可为性腺发育提供能量,也有利于幼体的存活和生长。卵母细胞直径从配子发生开始逐渐增大,并在产卵前达到最大值(36.88μm),产卵后则降低。营养成分分析表明,脂肪含量在性腺–内脏团中随配子发育积累储存,产卵后显著降低;糖原在性腺发育到4月时开始下降,为配子的发育提供能量;蛋白质在配子发育过程中大量合成,与繁殖活动存在密切联系。长牡蛎壳金选育群体外套膜的总蛋白含量显著高于普通养殖群体(P0.05),糖原和总脂肪含量显著低于普通养殖群体(P0.05),其他组织的一般营养成分未出现显著差异。研究表明,长牡蛎在壳金群体选育过程中营养成分已表现出分化,这为长牡蛎壳色选育提供了重要参考依据。     

Reproduction, condition and mortality of the Pacific oyster Crassostrea gigas (Thunberg) in Sonora, México

Starting in 1997, mortality outbreaks in cultured oysters Crassostrea gigas have been reported in northwestern México. Previous studies have shown that massive die‐offs result from multi‐factor processes related to reproduction of the species. We studied the reproductive cycle and the condition index of cultured oysters in the coastal lagoon of El Soldado, Sonora, as well as the relationship of the life cycle with environmental parameters. We used oocyte diameter to determine reproductive stages and the condition index to describe the physiological state of oysters. Additionally, the temperature, salinity, seston and chlorophyll a were recorded at the study site. The results showed that C. gigas began accelerated reproductive activity in March under the influence of high temperature and increased concentrations of food. No spawning events were recorded and gametes were reabsorbed within the gonad in September and October. The results showed a period of nutrient storage during autumn–winter and another period of gamete production in spring–summer. A mortality event occurred at the end of winter, tied to significant increases in temperature and availability of food and in accelerated reproductive activity and high condition index. These conditions were very similar to those reported in other countries during summer die‐offs of C. gigas.     

Seasonal variations in the immunological and physiological parameters of the Pacific oyster Crassostrea gigas cultured in Bahía de Macapule (Sinaloa,Mexico)

Triploid Crassostrea gigas were cultured during 13 months in Nestier‐type oyster trays. The impact of environmental parameters on the physiological and immunological parameters was evaluated. Temperature, salinity and seston were recorded monthly. Seventeen oysters were sampled monthly for immunological and condition index (CI) analyses. Samples were obtained as a haemolymph lysate supernatant (HLS). Protein content was determined using the Bradford method. The activity of hydrolytic enzymes was determined using the API ZYM kit and the lysoplate assay. Seston showed different patterns throughout the cycle. Condition index showed a positive correlation with the protein content of HLS. Protein showed a negative correlation with temperature. Eleven hydrolytic enzymes were detected in samples and higher enzymatic activity corresponded to leucine arylamidase and esterase. Leucine arylamidase and lysozyme activity showed a positive correlation with temperature. Oyster mortality was 28% in our modules and 70% in the oyster farm. Oysters showed low values of CI and haemolymph protein content in summer–autumn when mortalities were observed in the culture system. This finding suggests that these stressed oysters may have insufficient energy to invest in their immune system. It appears that oyster mortality in the culture system resulted from a combination of animal overcrowding, high temperature and low salinity.     

干露胁迫对长牡蛎基因组DNA甲基化的影响

为探讨干露胁迫对海产贝类基因组DNA甲基化的影响,应用荧光标记甲基化敏感扩增多态性(fluorescencelabeled methylation sensitive amplified polymorphism,F-MSAP)技术,比较了不同干露条件下(0 d、0.5 d、1 d、3 d、5 d、7 d、9 d和11 d)长牡蛎(Crassostrea gigas)基因组DNA甲基化的变化。结果表明,对照组(干露处理0 d)闭壳肌与鳃组织的总体甲基化水平分别为29.76%和29.82%;干露处理0.5 d、1 d、3 d、5 d、7 d、9 d和11 d的长牡蛎全基因组甲基化水平呈现先增高后降低的趋势,其中,闭壳肌组织的总体甲基化水平分别为36.59%、38.86%、43.02%、39.30%、51.13%、46.79%和35.06%,鳃组织总体甲基化水平分别为39.39%、42.13%、39.36%、43.54%、56.19%、38.57%和28.99%;干露处理7 d的长牡蛎甲基化水平明显高于其他时期(P0.05),11 d时甲基化水平基本恢复至初始状态。甲基化变异模式分析发现,闭壳肌与鳃组织DNA甲基化变异位点存在差异,甲基化升高位点变化程度较大(P0.05)。以上结果表明,长牡蛎通过改变DNA甲基化模式来应答干露胁迫,发生了不同程度的甲基化与去甲基化反应,DNA甲基化与长牡蛎的抗逆性状密切相关。     

Seasonal variation in the condition index of Pacific oyster postlarvae (Crassostrea gigas) in a land‐based nursery in Sonora,Mexico

This study examined the seasonal variation in the condition index (CI) of Crassostrea gigas postlarvae (<5 mm) that were cultivated at a commercial hatchery. Oysters were sampled weekly at the nursery using seawater from a lagoon for the grow‐out that precedes commercialization. Temperature, salinity, seston, chlorophyll a, oxygen and pH were recorded at each sampling and water samples were taken to identify phytoplankton groups and their abundance. High levels of primary productivity, chlorophyll a and seston were detected during summer, but the highest CI occurred in winter. During winter, elevated phytoplankton biomass was composed by diatoms and phytoflagellates, which served as the main food source and promoted weight gain in this season. Variations in salinity, oxygen and pH were not related to differences in the CI. However, it appears that the wide temperature variation affected functions, such as feeding activity, apparently enhancing ingestion during winter (mean 16.5±1.4 °C) and reducing ingestion during summer (mean 31±1.5 °C). Winter production resulted in postlarvae with a homogeneous size range and a high CI, indicating that winter is more favourable to start cultivation. The CI represents a practical means to determine the physiological state of postlarvae before transfer to cultivation sites.