紫贻贝和厚壳贻贝杂交及F1代杂交优势初探

2005年-2007年对紫贻贝和厚壳贻贝种间进行了杂交生产性试验,通过亲贝强化培育、确认雌雄亲贝、改革育苗水体交换方法、投喂混合单细胞藻类、流水培养附着稚贝等技术,获得F₁代。结果表明,各试验组生长特性,正交F₁代﹥紫贻贝﹥厚壳贻贝﹥反交F₁代。其中正交F₁代获附着稚贝3.77亿粒,平均壳长为1.13 mm、平均壳高为0.78 mm,育苗成活率达35.57%。正交F₁除在孵化率上低于紫贻贝、厚壳贻贝外,在壳长、壳高、成活率等方面指标具有一定杂交优势,而反交F1代的杂交优势不明显。     

杂交贻贝同工酶表达的初步分析

运用聚丙烯酰胺凝胶电泳技术,对2种贻贝(厚壳贻贝、紫贻贝)稚贝及通过人工培育所得的二者的杂交后代稚贝同工酶表达状况进行分析.结果显示,所检测5种酶(SOD、MDH、ME、ATP、ADH)在上述3种贻贝中的表达极为相似,MDH在3种贻贝中除r迁移率相同的条带外,在紫贻贝中表达出迁移率较小、表达较弱的条带;SOD和ATP仅在厚壳贻贝中表达出比另外2种多1条迁移率最大的条带,ME则表现为紫贻贝少1条中间的条带,ADH在杂交贻贝和紫贻贝中表达完全相同,厚壳贻贝虽也表现为3条酶带,但迁移率明显不同.试验结果说明,厚壳贻贝和紫贻贝具有较近的亲缘关系,其杂交后代与二者均有一定的相似性,这与传统的分类方法所得的结论相一致.     

厚壳贻贝与贻贝遗传渐渗的分子生物学鉴定

本文运用ISSR标记对厚壳贻贝与贻贝遗传渐渗进行研究。结果表明,厚壳贻贝与贻贝的特异性标记在杂交贻贝中有所反应,进一步证实了在浙江嵊泗部分厚壳贻贝与贻贝发生了杂交和基因渐渗现象。所采集的杂交群体样本中,65%与厚壳贻贝的遗传距离近,可能是杂交后与厚壳贻贝回交的后代。目前由于过度采捕、环境改变以及引进外来种等影响,应该对厚壳贻贝的种质资源进行保护。     

厚壳贻贝的同化率及其生物沉积作用

2001年3～9月在青岛海区自然养殖状态下,利用沉积物收集器测定厚壳贻贝(Mytiluscrassitesta)的生物沉积及其对物质的输运,并采用灰分比例法计算厚壳贻贝的同化率。结果显示,厚壳贻贝的同化率分别为:小个体(壳长42～49mm)43 2%～59 9%、中等个体(壳长54～60mm)41 3%～56 1%、大个体(壳长65～74mm)47 6%～53 5%,平均值分别为51 6%、49 5%和52 5%。厚壳贻贝通过生物沉积作用加速海洋中颗粒物质的沉积,生物沉积率随个体的增大而增加,呈正相关关系,分别为:小个体[(42 3±4 4)～(77 9±10 8)]mg·ind-1·d-1,中等个体[(68 5±5 8)～(134 1±12 7)]mg·ind-1·d-1和大个体[(83 4±10 4)～(167 1±10 8)]mg·ind-1·d-1。海水温度和环境中饵料数量是影响厚壳贻贝的生物沉积的重要因子。     

厚壳贻贝人工繁殖技术的研究

厚壳贻贝(Mytilus coruscus)是我国贻贝的主要养殖品种,其苗种主要依靠天然苗种和半人工采苗获得。2006年浙江海洋学院于嵊泗县石柱育苗厂,首次突破了厚壳贻贝规模化全人工繁殖和稚贝海区中间培育技术。研究结果表明,厚壳贻贝亲贝通过室内强化培养,经人工催产可获得成熟受精卵,受精率可达95%。在水温16℃时,受精卵在受精后25min出现第一极体,受精后39h50min发育至直线绞合幼虫期,胚胎孵化率达92%,在水温15.8～21℃的条件下,经39d室内人工培育,获平均壳长0.694mm的附着稚贝1304.7×104ind;附着稚贝经102d海区中间培育,获平均壳长13.95mm的稚贝404.46×104ind,海区保苗成活率达31%。该研究结果为今后厚壳贻贝大规模苗种生产奠定了重要理论和技术基础。     

厚壳贻贝SOD和POD同工酶的组织特异性研究

采用聚丙烯酰胺垂直电泳对厚壳贻贝的鳃、性腺、外套膜、足、闭壳肌等5种组织的超氧化物歧化酶(SOD)和过氧化物酶(POD)进行了检测分析。SOD共检测出6条酶带,其中酶a在5种组织中都得到较强表达;酶b只在鳃中有表达,且活性高;酶c和d在鳃以外的4个组织中有表达,在足和外套膜中的表达活性低于闭壳肌和性腺;酶e在鳃、足、闭壳肌中有较强表达;酶f只在闭壳肌、性腺中有微弱表达。POD共检测到3条酶带;其中,酶a只在鳃中有表达;酶b在5种组织中都有表达;酶c只在足和性腺中有表达,该酶除在足中表达较弱外,在其它组织中都有较高的浓度。从检测结果来看,SOD和POD在厚壳贻贝体内的表达有一定的组织特异性,这种特异性不仅与各组织的不同生理分工相关,而且也与机体对自身基因表达进行调控相联系。     

厚壳贻贝苗期饵料培养技术

单细胞藻类的培养方法多种多样 ,本文所讨论的培养方法归为连续分级培养。即培养工艺为 :保种培养 ,在 30 0 0毫升三角烧瓶小规模接种→接种 ,转移到较大玻璃器皿内扩大规模培养→接种 ,转移到培养池内大规模培养→养成并输送至贻贝育苗池。1　保种培养种类作为厚壳贻贝苗种饵料的微藻主要是金藻、扁藻、褐指藻和小球藻。藻种一定要选择生长良好 ,生活力旺盛 ,色泽鲜艳 (硅藻类为黄褐色、绿藻类为鲜绿色 ) ,无沉淀及无明显附壁现象 ,镜检无其他混杂种类、浮游动物、原生动物之类污染的藻种。培养用的海水是经煮沸或过滤除菌法消毒后的海水。…     

厚壳贻贝抗菌肽Mytilin的初步鉴定

厚壳贻贝是我国具有重大经济价值的水产养殖贝类,对其抗菌肽的研究有助于人们了解厚壳贻贝的免疫机制。为了解厚壳贻贝血清中抗菌肽Mytilin的分子组成和特性,采用多维高效液相色谱对厚壳贻贝血清进行分离纯化,从中获得3种对革兰氏阳性菌以及阴性菌均有抑制作用的抗菌肽分子,序列分析表明3种抗菌肽具有较高的序列相似性,均属于贻贝抗菌肽Mytilin家族,分别命名为Mytilin-1,Mytilin-2和Mytilin-3。其中,Mytilin-1为34个氨基酸残基构成的多肽,其分子量为3885.17u,含8个半胱氨酸,形成4对二硫键。根据所测Mytilin-1的氨基酸序列设计特异性引物,通过菌落PCR方法筛选厚壳贻贝cDNA文库,获得Mytilin-1的cDNA基因并进行了序列分析。以上研究结果表明,作为贻贝的主要抗菌肽家族,Mytilin在厚壳贻贝血清中具有较高丰度,对其蛋白质序列以及基因序列的研究为深入了解厚壳贻贝Mytilin抗菌肽的分子多样性奠定了基础。     

厚壳贻贝3群体的形态差异与判别分析

运用多变量形态度量学分析方法,对浙江沿海嵊泗、舟山、台州3个地理群体厚壳贻贝的4个形态性状进行了形态差异研究。聚类分析和主成分分析结果表明,嵊泗群体和台州群体首先聚类,之后与舟山群体聚类。主成分分析中,2个主成分的贡献率分别为：主成分1为47．01％,2为25．07％,累积贡献率为72．08％。判别分析结果表明,3群体间形态差异显著（P〈0．01）。建立3群体的判别函数,判别准确率P。N80．00％。86．66％,P2为80．65％。85．71％。3群体的综合判别率为83．33％。     

厚壳贻贝早期幼虫低温保存的影响研究

探讨低温(4 ℃)条件下的厚壳贻贝早期幼虫保存可能性,同时调查了不同培育密度对低温保存的影响。在正常条件下,继续培育低温保存后的幼虫,并调查其存活率和生长的变化。 结果表明,在低温保存后,早期幼虫存活率较高,超过95%;厚壳贻贝早期幼虫的壳长和壳高出现显著性的增长。不同培育组间,培育密度对厚壳贻贝早期幼虫的存活和生长影响不同,表明密度是幼虫低温保存的一个重要因素。在正常培育条件下,低温保存后的幼虫,3周后其存活率明显低于对照组,但仍超过50%,且其生长速度明显高于对照组。因此,低温培育是保存厚壳贻贝早期幼虫的有效方法,可用于今后贝类幼虫生物学实验和人工育苗技术的改善研究。     

微生物膜对厚壳贻贝稚贝附着的影响

为研究微生物膜在厚壳贻贝稚贝附着过程中的作用,通过海洋化学生态学和分子微生物学方法分析了微生物膜形成过程中其干重、附着细菌密度、底栖硅藻密度、叶绿素a含量等随日龄变化情况及其对厚壳贻贝稚贝附着的影响。同时,利用DGGE指纹图谱技术对不同日龄微生物膜中的细菌群落结构多样性进行了分析。结果发现,微生物膜的干重、附着细菌密度及底栖硅藻密度明显随着日龄的增加而增加,在28 d达到最高值,其干重、细菌和硅藻密度分别为0.87 mg/cm2、1.5×107/cm2、1.0×106/cm2,均与日龄显著相关。叶绿素a含量在14 d时达到最大,为2.2μg/cm2,随日龄的增加呈持续下降的趋势,相关性分析表明叶绿素a含量与日龄无直接关系。随着日龄的增加,微生物膜诱导的稚贝附着率逐渐增加,28 d时达到最高值,为76%。相关性分析显示,微生物膜的活性与干重、附着细菌密度及底栖硅藻密度显著相关,其相关性系数分别为0.717、0.711和0.754。然而,微生物膜的附着诱导活性与叶绿素a无直接相关性。细菌群落结构在厚壳贻贝稚贝附着过程中发挥了重要作用。     

Effect of temperature on survival,growth and development of Mytilus galloprovincialis larvae

Mussel aquaculture is widely prevalent worldwide, but generally relies on natural seed collection, which does not always meet the needs of the producers. Thus, development of mussel hatcheries is of economic interest in some parts of the world, such as Europe; it provides opportunities not only on annual reliability of seed but also on genetic improvements. To broaden knowledge on mussel larval physiology, we carried out temperature treatments (17, 20 and 24 °C) on Mytilus galloprovincialis larvae under laboratory conditions. The trials ended when 30% of the larval population was in the post‐larval stage. The temperature coefficient Q₁₀ indicated a strong relationship between temperature and increase in growth from 17 to 20 °C, but not between 20 and 24 °C. Exposure of M. galloprovincialis larvae to 17 °C resulted in poor growth, low survival and a delayed development and was considered to be inadequate for M. galloprovincialis larval culture. Rearing the larvae at 20 or 24 °C produced better growth, higher survival rates and faster metamorphosis as compared with 17 °C. The temperature region within 20 and 24 °C was suggested as adequate for the mussel M. galloprovincialis larval culture, and implications of these results on the development of commercial hatcheries were discussed.     

Influence of microalga lipid composition on the sexual maturation of Mytilus galloprovincialis: a hatchery study

The effect of microalga lipid composition on the reproduction of the mussel Mytilus galloprovincialis has been assessed to determine the best feeding strategy for producing large quantities of mussel seed. Three diets based on two microalgae Isochrysis galbana (clon T‐iso) and Chaetoceros gracilis were tested. Besides, hatchery groups were compared with mussels from natural populations. Lipid content and fatty acid profile of digestive gland and mantle of both sexes, eggs and microalgae were analysed and related to sexual maturation. Hatchery groups, specifically the one fed on T‐iso, showed better results in reproduction success, and these differences were reflected on tissue and egg lipid composition. Microalga fatty acid profile influenced tissues and sexes, and higher levels of 18:1n‐9, 22:6n‐3 (DHA) and 18:2n‐6 were detected in groups fed on T‐iso while higher level of 20:5n‐3 (EPA) and 16:1n‐7 in groups fed on C. gracilis. Evidence of synthesis capacity of EPA from 18:4n‐3 and DHA from EPA is detected comparing their levels and the mobilization between tissues. Egg fatty acid profile was influenced by the female diet, and differences among groups were detected and confirmed by PCAs.     

海水酸化胁迫对紫贻贝能量分配的影响

2016年5～6月在山东桑沟湾楮岛海区,采用野外围隔和现场流水相结合的方法,以紫贻贝(Mytilus galloprovincialis)为研究对象,以pH为8.0作为对照组,探讨了酸化胁迫(p H=7.7)对其能量分配的影响。结果显示,短期(10d)海水酸化胁迫下,紫贻贝的滤水率、同化效率、氧氮比显著下降(P0.05),排氨率极显著增加(P0.01),耗氧率无显著差异(P0.05);中期(30d)海水酸化胁迫下,紫贻贝的滤水率和氧氮比显著下降(P0.05),而同化效率、耗氧率、排氨率显著升高(P0.05)。能量收支的结果显示,短期(10 d)酸化胁迫下,紫贻贝的摄食能和吸收能显著降低(P0.05),呼吸能无显著差异(P0.05),排泄能显著增加(P0.05),生长余力极显著降低(P0.01);中期(30d)海水酸化胁迫下,摄食能降低(P0.05),吸收能、呼吸能、排泄能、生长余力显著升高(P0.05)。氧氮比的结果显示,在海水酸化胁迫下,氧氮比的波动范围为14.28～20.46,贝类体内的供能物质由脂肪和碳水化合物逐渐向蛋白质过渡。研究结果为揭示紫贻贝应对海水酸化胁迫的生理响应提供了基础数据。     

Effects of larval cryopreservation on subsequent development of the blue mussels,Mytilus galloprovincialis Lamarck

In this study, the effects of three commonly used chemicals, dimethyl sulphoxide (DMSO), ethylene glycol (EG), propylene glycol (PG) and their combinations with trehalose, were evaluated on the cryopreservation of D‐larvae of the blue mussel Mytilus galloprovincialis. The larvae were harvested 30 h post‐fertilization at 21 °C and cryopreserved using a standard protocol in 5%, 10% or 15% of DSMO, EG and PG either as single chemical solutions or in combination with 0.2 M trehalose. Among these cryoprotectants, 5% DMSO resulted in the highest post‐thaw survival rate of 55.3±7.8%, although it did not significantly differ from those with 10% and 15% EG. The addition of 0.2 M trehalose did not improve the post‐thaw larval survival rates in all the combinations assessed. The cryo‐effects on subsequent development were evaluated using the D‐larvae frozen with 5% DMSO. The results showed that cryopreservation affected both larval survival and growth in this species. The relative daily mortality rate was significantly higher in treated than control groups over the period from 3 h post‐thaw to day 11 post‐fertilization. On day 6 post‐fertilization, the average larval length in the treated group was significantly smaller than that in the control. From day 11 post‐fertilization, and onwards, differences in these two traits were not significant between treated and control groups. On day 21 post‐fertilization, about 80% of the larvae in both treated and control groups developed eyes and the normalized survival rate in the treated group was 12.5%.     

Comparison of the physiological energetics between Mytilus chilensis,Mytilus galloprovincialis and their hybrids,under laboratory conditions

Laboratory‐produced juvenile individuals of the species Mytilus chilensis, M. galloprovincialis and their hybrids were subjected to physiological measurements under an experimental diet of Isochrysis galbana (30 × 10⁶ cells L^?1), 13°C temperature and a salinity of 30 psu. Pure species individuals showed a higher clearance rate (CR). Mytilus chilensis had a CR of 1.13 L h^?1, while M. galloprovincialis registered only 0.78 L h^?1. Also, pure taxa registered higher values (above 70%) of absorption efficiency when compared with hybrid individuals. Ammonia excretion in M. chilensis and M. galloprovincialis was 1.5% and 0.4%, respectively, while hybrids registered significantly lower values. Under these experimental conditions, M. chilensis registered the highest scope for growth (P < 0.05), compared with M. galloprovincialis and their hybrids. However, the net growth efficiency index (K ₂) in hybridization type I (♀Mg × ♂Mc) was higher (P < 0.05) than other experimental groups. The invasive mytilid M. galloprovincialis showed values that are very similar to those obtained with the hybridization I group (♀Mg × ♂Mc). Finally, we discuss that water temperature is an important factor in the biogeographic separation of both species and the potential effects that the settlement of the invasive species may have for Chilean mussel production.