长牡蛎SNP标记的开发及多态性检测

长牡蛎是重要的经济养殖贝类，良种化、抗逆性状及快速生长个体的培育是长牡蛎养殖业得以持续发展的基础。目前飞速发展的分子标记辅助育种技术为优良品种的快速培育提供了理论基础和实践经验。本研究以长牡蛎为主要研究材料，探讨了长牡蛎SNP标记的筛选和多态性评价。 本研究利用已有长牡蛎EST库中的序列进行单核苷酸多态（SNP）标记开发。通过对长牡蛎（Crassostrea gigas）已有的EST序列数据库检索，经过序列聚类和拼接得到EST簇4548个，含有不少于4条EST序列的簇共1079个，经过进一步设置筛选条件，整理出可供利用的EST簇313个，得到候选SNP位点共计1140个。目前根据候选SNP位点共设计引物82组，通过片段长度差异等位基因特异性PCR（fragment length discrepant allele specific PCR，FLDAS-PCR）的分型方法，在一野生群体中进行检测和验证，结果共有17个SNP候选位点显示多态性，期望杂合度分布区间为0.088至0.506，观测杂合度分布区间为0.091至0.667；通过哈代-温伯格(HW) 平衡、连锁不平衡检验，结果显示除3个SNP位点的差异显著（P值<0.05）,不符合HW平衡之外，其他14个位点没有明显的连锁不平衡。对含有17个SNP的EST的共同序列进行BlastX分析，推测其功能并确定开放阅读框，从而预测17个SNP的性质。 本研究表明对于目前基因组学研究尚处在初级阶段的海洋生物物种，通过基于EST数据库的SNP开发是一条重要途径，可以有效弥补海洋生物基因组学滞后影响SNP标记开发的现状。

牡蛎的比较解剖学及系统分类和演化的研究

本文对我国己发表的牡蛎作了详细的比较解剖学研究。在强蛎亚科（Pycnodonteinae）发现了第三个心耳－－右上心耳和与其相连的第三条回心静脉－－后静脉，这在双壳类中实属首次报道。第一次提出了牡蛎循环系统的两种类型，附心脏型和无附心脏型。两者之间的主要区别是：前者无肌套血管，其外套血液主要来自环外套动脉和附心脏，而后者由于无附心脏和环外套动脉，其外套血液主要来自肌套血管；前者是通过出鳃静脉和外套静脉分别将鳃前部和后部的血液送回心耳，而后者的外套静脉与外入鳃血管不通，鳃中的血液只能通过出鳃静脉回到心耳。根据外套腔的形态特点，本文将中国的牡蛎分为三种类型六个组。第一种类型，左右侧都具侧水腔，包括一个组；第二种类型，仅右侧具有侧水腔，包括四个组；第三种类型；不具侧水腔，包括一个组。在比较解剖学研究的基础上，文章还讨论了消化系统，神经系统，循环系统等一些主要系统的演化过程，并推测了现生牡蛎各属间的演化关系。提出了一些新的分类依据，发现了一个具有重要演化意义的单行属种，爪蛎属（Talonostrea），猫爪牡蛎（T. talonata）。澄清了目前世界上在舌骨蛎属（Hyotissa）、拟舌骨蛎属（Parahyotissa）、小蛎属（Saccostrea）和巨蛎属（Crassostrea）分类中的混乱。解决了我国牡蛎分类中存在己久的疑难问题，将己发表的20种牡蛎重新鉴定为15种，即舌骨牡蛎（Hyotissa hyotis），复瓦牡蛎（Parahyotissa imbricata）。中华牡蛎（P. sinensis），鸡冠牡蛎（Lopha cristagalli）。薄片牡蛎（Dendostren folium），缘齿牡蛎（D. cre-nulifera），褶牡蛎（Alectryonella plicatula），猫爪牡蛎（T. talonata），长牡蛎（Crassostrea gigas），近江牡（C. rivularis），拟近江牡蛎（Crassostrea sp.），僧帽牡蛎（Saccostrea cucullata），棘刺牡蛎（S. echinata），鹅掌牡蛎（Planostrea pestigris）和密鳞牡蛎（Ostrea denselamellosa）。它们分别隶属于二个科，曲蛎科（Grypheidae）和牡蛎科（Ostreidae）；四个亚科，强蛎亚科（Pycnodonteinae）。冠蛎亚科（Pycnodonteinae），巨蛎亚科（Lopheinae）和牡蛎亚科（Crassostreinae）。

海湾扇贝超氧化物歧化酶家族基因结构、表达和多态性分析

海湾扇贝Argopecten irradian Lamarck于1982年从美国引种到中国，由于具有较快的生长速度和很高的经济效益，海湾扇贝成为中国最主要的养殖贝类之一。近年来海湾扇贝养殖遇到了死亡率高等问题，深入开展海湾扇贝功能基因的研究，尤其是免疫相关基因及其机制研究并在此基础上寻找扇贝疾病防治的有效方法对海湾扇贝的健康养殖十分重要。 对于贝类免疫系统来说，其血细胞在先天性免疫防御中起着重要的作用。当受到外界病原侵染时，贝类血细胞的一个重要免疫反应就是吞噬作用。在吞噬病原过程中，受到病原侵染的贝类还会产生其他多种免疫反应，这些免疫反应将消耗大量的能量（ATP），产能的呼吸链会加速运转，由此也会引发与呼吸链相耦联的活性氧（ROS）的大量产生。这些活性氧具有极强的反应特性，能破坏病原微生物的结构和功能分子，实现对入侵病原的杀灭。利用活性氧对被吞噬的病原进行杀灭，这是吞噬作用消除病原抵御侵染的重要机制。但由于活性氧分子反应的非特异性，它们也会破坏宿主机体细胞内的功能蛋白分子、不饱和脂肪酸分子和核酸等，对细胞造成严重的伤害，进而导致机体生理机能的损伤和免疫系统的破坏。所以，及时消除病原感染机体内过量产生的ROS，维持相关细胞的正常代谢，对提高机体抵抗力和免疫力具有重要的作用。O2-是生物体内产生的第一种活性氧分子，其他的活性氧分子也是由它衍生而来，消除过量O2-是消除过量活性氧危害的第一步也是关键一步。生物体内，超氧化物歧化酶（SOD）是催化O2-发生歧化反应，消除O2-的关键酶。 首先，本文通过RACE方法获得了海湾扇贝SOD家族全部三种基因的cDNA全长并对其进行了序列的生物信息学分析，海湾扇贝AiCuZnSOD全长cDNA为1047个碱基，其中开放阅读框为459个碱基，编码152个氨基酸，与栉孔扇贝Chlamys farreri的CuZnSOD相似度为77.5%，与长牡蛎Crassostrea gigas的相似度为75%，与人的相似度为74.7%。AiMnSOD全长cDNA为1207个碱基，其中开放阅读框为678个碱基，编码226个氨基酸，序列比对结果发现AiMnSOD的氨基酸序列与虾夷扇贝Mizuhopecten yessoensis和皱纹盘鲍Haliotis discus hannai的相似度分别为85%和78.4%，与哺乳动物相似度也在68%~72%之间。AiECSOD全长cDNA为893个碱基，其中开放阅读框为657个碱基，编码218个氨基酸。AiECSOD与其它物种ECSOD相似度比较低。与线虫Brugia pahangi的相似度为27.9%，与疟蚊Anopheles gambiae的相似度为31.4%，与斑马鱼Danio rerio的相似度为27.8%，与人的相似度也只有28.6%，与同是贝类的长牡蛎ECSOD也只有28.1%的相似性。主要原因是AiECSOD的信号肽和肝磷脂结合区域在各物种中无同源性。 其次，采用qRT-PCR（quantitative real time PCR）方法分析三种SOD基因在不同组织中的表达情况，结果表明三种SOD基因的组织表达有所差异。AiCuZnSOD基因在鳃中表达水平最高，其次是血细胞和性腺，在外套膜、闭壳肌和肝胰脏表达水平较低。AiMnSOD基因在鳃中表达水平最高，其次是外套膜，在血细胞、性腺，而在肝胰脏和闭壳肌表达较弱。AiECSOD基因在血细胞中表达水平最高，其次是肝胰脏，在鳃、闭壳肌表达水平较低，而性腺和外套膜没有检测到。同时，采用qRT-PCR对鳗弧菌Vibrio angullarum感染后海湾扇贝血细胞中三种SOD基因mRNA表达变化进行了检测。AiCuZnSOD表达量在各个时间段没有显著差异(P > 0.05)。AiMnSOD的表达量在1.5 h时略有下降，在3 h时达到最高表达量，是空白组（0h）的3倍(P < 0.01)，从6 h到24 h表达量逐渐下降，24 h时表达量是空白组的1.6倍，24 h到48 h又稍有升高。AiECSOD的表达量在1.5 h时有所下降，是空白组的0.3倍（P < 0.05)，随后逐渐升高，在12 h时达到最高表达量，是空白组（0h）的4.5倍（P < 0.01），从24 h到48 h表达量逐渐下降并恢复到空白组的水平。在对照组，各个时间点没有显著差异（P > 0.05）。在鳗弧菌感染后，海湾扇贝三种SOD的表达并不一致，且差异比较显著。AiCuZnSOD被认为是构成性表达基因，其受外界刺激的影响最小，AiMnSOD和AiECSOD受刺激后表达上调比较明显。 第三，采用Genome-walking的方法得到了海湾扇贝三种SOD基因的基因组全长和近端启动子序列并对其进行了相关分析。AiCuZnSOD的基因组序列全长为4279bp，包含有4个外显子和3个内含子。AiMnSOD的基因组序列全长为10692bp，包含有4个外显子和3个内含子。AiECSOD的基因组序列全长为5276bp，包含有5个外显子和4个内含子。三种基因外显子和内含子的结合处序列遵循-AT/GT-原则。我们把海湾扇贝SOD家族的三个基因的近端启动子进行了比较分析。发现三种SOD在靠近起始密码子的位置都有Oct-1结合位点。三种SOD共有的转录位点有：Oct-1、C/EBPalp、Oct2.1、Sp-1和GATA-1。AiCuZnSOD和AiMnSOD共有的转录位点有：ICSBP、Ftz、TATA-box、C/EBPbeta和Antp。AiCuZnSOD和AiECSOD共有的转录位点有：AP-1和NFκB。AiMnSOD和AiECSOD共有的转录位点有：GR和ER。AiCuZnSOD独有的位点有：SRF、YY-1和NF-1。AiMnSOD独有的位点有：HNF-1、Hb、MEB、NF-muE1、Pit-1a和Eve。AiECSOD独有的位点有：CREB、RATA-alph、Kruppel-like和AP-3。 此外，通过构建原核表达载体，本研究对AiCuZnSOD和AiECSOD基因进行了体外重组表达，并对纯化的重组蛋白进行了酶活分析。酶活分析表明，重组AiCuZnSOD蛋白有较高的酶活和稳定性。 最后，我们对海湾扇贝三种SOD基因的部分区域，包括启动子、编码区，部分内含子区域进行了SNP检测，并对SOD基因部分SNP位点多态性和鳗弧菌敏感性进行了相关分析。三种SOD基因中，我们共发现了59个SNP位点，其中AiECSOD的SNP位点最多，特别是在启动子区，AiCuZnSOD和AiMnSOD多态性较低。其中AiCuZnSOD启动子区的-1739 T-C 位点的基因型和等位基因，AiECSOD启动子区的-498 A-T和-267 G-A等位基因频率，AiECSOD的第一个外显子38 Thr-Lys的多态性在敏感和抗菌群体中存在显著差异（P < 0.05）。

长牡蛎可存活四倍体的诱发机制和异源三倍体的诱发

采用乙酸地衣红染色技术（Acetic orcein staining technique）较系统地研究了长牡蛎 Crassostrea gigas (Thunberg)三倍体产生的卵子在受精且第一极体释放受抑制后的减数分裂过及染色体的分离行为以阐明可存活四倍体的产生体的机制。用浓度为0.5 mg/L的细胞松驰素B （CB）处理受卵以抑制其第一极体的释放。在观察到个别受精卵出现第一极体时开始CB处理，持续至对照组中50％的受精卵出现的第一极体。对处理组和对照组的受精卵从受精后隔5分钟取样一次，用卡诺氏液（Carnoy's fixative, 冰醋酸和甲醇按1：3的体积比充分混合）固家样品。采用0.5%的乙酸－地衣红染料进行受精卵的染色，而后压片观察受精卵染色体行为。长牡蛎三倍体产生的卵子，中期I同源染色体构型呈现单价体（Univalents）,二价体（Bivalents）、三价体（Trivalents）以及大于三价体的多价体（Multivalents）混合出更的特征。在第一极体释放受抑制的受精卵的第二次减数分裂过程中，可确认四种染色体分离类型：三极分离（Tripolar segregation） (54.5%)、联合二极分离（United bipolar segregation） (12%)、独立二级分离（Incomplete united bipolar segregation）(4%)。其余卵子的染色体分离行为（23％）不规律，呈现不同程度的紊乱，但总体看来介于上述四种分离类型之间。此外，某些特定的独立二级也可能是四位体形成的最主要的细胞遗传学体制。此外，某些特定的独立二极分离也可能产生四倍体。轻细胞体驰素B 处理的受精卵的减数分裂过程具有显著的不同步性，表现在三个方面：第一，在两个重复组之间，即两个雌体之间，存在第二次减数分裂的时间进程的不同步性；第二，同一个雌体产生的卵子之间的发育速度不同步性，表现为不同的卵子进入第一次减数分裂的时间不同；第三，同一卵子内的染色体之间，其行为有时存在的不同步性。另外，探讨了中心类在支配第二减数分裂时各种染色体分离行为的可能机制。以长牡蛎二倍体与近江牡蛎二倍体的染交作为对照，探讨了能长牡蛎四倍体与近江牡蛎二倍体杂并诱导异源三倍体的可行性。长牡蛎Crassostrea gigas (Thunberg)四倍体和二倍体与近江牡蛎Crassostrea rivularis (Gould)二倍体的杂交以及相应的对照组共进行了三批重复实验，杂交实验采用高密度的精子。研究结果表明，自交组平均受精率依次为94％（GG）、77％ （RR）,88% （G／GG）和85％ （GG／G）。双方差分析（ANOVA）表明，各自交组之间受精率没有显著差异（F＝3.118, P=0.132）。在杂交组，直接授精后180分钟，尚未观察到受精迹象，因而无法估计受精率。授精后48小时的孵化率各组之间差异很大，并经双方差分析（ANOVA）表明存在显著性差异，（F＝3.188, P=0.018）。其中GGR和RGG组的孵化率相近似，产生的幼虫数量明显少于对照组。在四种类型的杂交实验中，二倍体C. gigas (雌体) ＊ 二倍体 C. rivularis (雌体)（ＧＲ）早最成功的。虽基ＧＲ组幼虫的生长率低于对照组，但其存活率接近于对照组。长牡蛎四倍体与近江牡蛎二倍体杂交组（GR），在授精后两天的孵化率较低，但幼虫的生长状况与对照组接近。另外两个杂交组，即近江牡蛎二倍体与长牡蛎四倍体（RGG），二倍体近江牡蛎江与二倍体长牡蛎（RG），授精后两天的孵化率很低，幼虫生长得缓慢。三个重复组的GR杂交组和一个重复组的GGR杂交组获得稚贝。聚合酶链式反应／限制性酶切片段长度的多态性（PCR／PFLP）检支分析结果证实这些稚贝均是杂交种；流式细胞术分析结果证明GGR获得的稚贝是三倍体，从而证明获得了长牡蛎与近江牡蛎的异源三倍体。有迹象表明三倍体与二倍体杂交种之间（GGR对GR）存在生长上的差异。首先，GGR的眼点幼虫大约比GR组早出现5－7天即仅次于对照组GG，G／GG，和GG／G；第二，尽管仅获得少量GGR幼贝，这些幼贝在授精后90天的大小显著大于GR组的个体。在RGG和RG组中，幼虫没能存活到眼点幼点阶段。细胞学检查结果表明，杂交组的绝大多数卵子发育停滞在第一次减数分裂中期（Metaphase I）,这一过程至少持续到授精后180分钟。仅有2％的GGR 组的卵子在授精后180分钟进入第一次减数分裂后期）（Anaphase I）. 而在此时期，GR，RGG和RG组的卵子中，仍只观察到10第二价体（Bivalents）.

Food sources of three bivalves living in two habitats of Jiaozhou Bay (Qingdao, China): Indicated by lipid biomarkers and stable isotope analysis

Food Sources of three filter-feeding bivalves from two habitats (intertidal oyster Crassostrea gigas, mussel Mytilus galloprovincialis. and subtidal cultured scallop Chlamys farreri) of Jiaozhou Bay (Qingdao,China) were determined by fatty acid and stable isotope in analysis. Cultured scallop was characterized by significant diatom markets such as 16:1/16:0 close to 1 and high ratio of 20:5(n - 3)/22:6(n - 3), hence we assume that the scallop mainly feeds on diatoms. Fatty acid biomarkers specific to bacteria and terrestrial materials were also found in considerable amounts in scallop tissue, which suggested that there were Substantial bacterial and terrestrial input into the food of the species. Intertidal oyster and mussel, however, exhibited significant flagellate marker. 22:6(n - 3). and lower level of diatom markers. which indicated that flagellates are also part of intertidal bivalves' Planktonic food Sources: meanwhile, high level of Chlorophyta fatty acid marker, Sigma 18:2(n - 6) + 18:3(n - 3), suggested that Ulva pertusa (Chlorophyta) seaweed bed supplied important food sources to intertidal bivalves. Additionally, result of stable isotope analysis showed that phytoplankton contributed 86.2 to 89.0% to intertidal bivalves' carbon budget; macroalga U. pertusa origin source had a contribution of MIX, to 11.0%, which indicated its role Lis in important supplemental food source to intertidal bivalves. From this study. it is concluded that the dietary difference of three bivalves probably relates to the different potential food sources in the scallop farm and intertidal zone in Jiaozhou Bay.

Chromosomal rearrangement in Pectinidae revealed by rRNA loci and implications for bivalve evolution

Karyotype and chromosomal localization of major (18-5.8-28S) and minor (5S) ribosomal RNA genes were studied in two species of Pectinidae, zhikong (Chlamys farreri) and bay (Argopecten irradians irradians) scallops. using fluorescence in situ hybridization (FISH). C. farreri had a haploid number of 19 with a karyotype of 3m + 4sm + 7sm-st + 4st + 1st-t, and A. i. irradians had a haploid number of 16 with a karyotype of 5st + 11t. In C. farreri, the major and minor rRNA genes had one locus each and were mapped to the same chromosome-Chromosome 5. In A. i. irradians, the major rRNA genes had two loci, located on Chromosomes 4 and 8, and the 5S rRNA gene was found at a third chromosome-Chromosome 10. Results of this and other studies indicate that karyotype of A. i. irradians (n = 16, 21 arms) is secondary and derived from an ancestral karyotype similar to that of C. farreri (n = 19, 38 arms) through considerable chromosomal loss and rearrangements. The ability to tolerate significant chromosomal loss suggests that the modal karyotype of Pectinidae and possibly other bivalves with a haploid number of 19 is likely tetraploid; i.e., at least one genome duplication has occurred during the evolution of Bivalvia.

Classification of common oysters from North China

Oysters are commonly found on rocky shores along China's northern coast, although there is considerable confusion as to what species they are. To determine the taxonomic status of these oysters, we collected specimens from nine locations north of the Yangtze River and conducted genetic identification using DNA sequences. Fragments from three genes, mitochondrial 165 rRNA, mitochondria! cytochrome oxidase I (COI), and nuclear 285 rRNA, were sequenced in six oysters from each of the nine sites. Phylogenetic analysis of all three gene fragments clearly demonstrated that the small oysters commonly found on intertidal rocks in north China are Crassostrea gigas (Thunberg, 1793), not C. plicatula (the zhe oyster) as widely assumed. Their small size and irregular shell characteristics are reflections of the stressful intertidal environment they live in and not reliable characters for classification. Our study confirms that the oysters from Weifang, referred to as Jinjiang oysters or C. rivularis (Gould, 1861), are C. ariakensis (Wakiya, 1929). We found no evidence for the existence of C. talienwhanensis (Crosse, 1862) and other Crassostrea species in north China. Our study highlights the need for reclassifying oysters of China with molecular data.

Ecotoxicology of marine biotoxins in bivalve shellfish

A small proportion of harmful algae produce toxins which are harmful to human health. Strict monitoring programmes are in place within Ireland and the EU to effectively manage risk to human consumers of shellfish species that have accumulated marine biotoxins in their tissues. However, little is known about the impacts of HABs on shellfish health. This study used Solid Phase Adsorption and Toxin Tracking (SPATT) for the passive sampling of algal biotoxins at Lough Hyne Marine Nature Reserve in West Cork, Ireland. Spatial and temporal monitoring of the incidence of a wide range of lipophilic toxins was assessed over a 4-month period. Active sampling accumulated sufficient quantities of toxin for use in subsequent experimentation. In addition to commonly occurring Diarrhetic Shellfish Poisoning (DSP) toxins, Dinophysis toxin-1 and Pinnatoxin-G were both detected in the samples. This is the first identification of these latter two toxins in Irish waters. The effects of the DSP toxin okadaic acid (OA) were investigated on three shellfish species: Mytilus edulis, Ruditapes philippinarum and Crassostrea gigas. Histological examination of the gill, mantle and hepatopancreas tissues revealed varying intensity of damage depending both on the tissue type and the species involved. At the cellular level, flow cytometric analysis of the differential cell population distribution was assessed. No change in cell population distribution was observed in Mytilus edulis or Ruditapes philippinarum, however significant changes were observed in Crassostrea gigas granulocytes at the lower levels of toxin exposure. This indicated a chemically-induced response to OA. DNA fragmentation was measured in the haemolymph and hepatopancreas cells post OA-exposure in Mytilus edulis and Crassostrea gigas. A significant increase in DNA fragmentation was observed in both species over time, even at the lowest OA concentrations. DNA fragmentation could be due to genotoxicity of OA and/or to the induction of cell apoptosis.

Assessing the sensitivity of blue mussel beds to pressures associated with human activities.

The Joint Nature Conservation Committee (JNCC) commissioned this project to generate an improved understanding of the sensitivities of blue mussel (Mytilus edulis) beds, found in UK waters, to pressures associated with human activities in the marine environment. The work will provide an evidence base that will facilitate and support management advice for Marine Protected Areas, development of UK marine monitoring and assessment, and conservation advice to offshore marine industries. Blue mussel beds are identified as a Habitat of Principle Importance (HPI) under the Natural Environment and Rural Communities (NERC) Act 2006, as a Priority Marine Feature (PMF) under the Marine (Scotland) Act 2010, and included on the OSPAR (Annex V) list of threatened and declining species and habitats. The purpose of this project was to produce sensitivity assessments for the blue mussel biotopes included within the HPI, PMF and OSPAR habitat definitions, and clearly document the supporting evidence behind the assessments and any differences between them. A total of 20 pressures falling in five categories - biological, hydrological, physical damage, physical loss, and pollution and other chemical changes - were assessed in this report. The review examined seven blue mussel bed biotopes found on littoral sediment and sublittoral rock and sediment. The assessments were based on the sensitivity of M. edulis rather than associated species, as M. edulis was considered the most important characteristic species in blue mussel beds. To develop each sensitivity assessment, the resistance and resilience of the key elements are assessed against the pressure benchmark using the available evidence gathered in this review. The benchmarks were designed to provide a ‘standard’ level of pressure against which to assess sensitivity. Blue mussel beds were highly sensitive to a few human activities: • introduction or spread of non-indigenous species (NIS); • habitat structure changes - removal of substratum (extraction); and • physical loss (to land or freshwater habitat). Physical loss of habitat and removal of substratum are particularly damaging pressures, while the sensitivity of blue mussel beds to non-indigenous species depended on the species assessed. Crepidula fornicata and Crassostrea gigas both had the potential to outcompete and replace mussel beds, so resulted in a high sensitivity assessment. Mytilus spp. populations are considered to have a strong ability to recover from environmental disturbance. A good annual recruitment may allow a bed to recovery rapidly, though this cannot always be expected due to the sporadic nature of M. edulis recruitment. Therefore, blue mussel beds were considered to have a 'Medium' resilience (recovery within 2-10 years). As a result, even where the removal or loss of proportion of a mussel bed was expected due to a pressure, a sensitivity of 'Medium' was reported. Hence, most of the sensitivities reported were 'Medium'. It was noted, however, that the recovery rates of blue mussel beds were reported to be anywhere between two years to several decades. In addition, M. edulis is considered very tolerant of a range of physical and chemical conditions. As a result, blue mussel beds were considered to be 'Not sensitive' to changes in temperature, salinity, de-oxygenation, nutrient and organic enrichment, and substratum type, at the benchmark level of pressure. The report found that no distinct differences in overall sensitivity exist between the HPI, PMF and OSPAR definitions. Individual biotopes do however have different sensitivities to pressures, and the OSPAR definition only includes blue mussel beds on sediment. These differences were determined by the position of the habitat on the shore and the sediment type. For example, the infralittoral rock biotope (A3.361) was unlikely to be exposed to pressures that affect sediments. However in the case of increased water flow, mixed sediment biotopes were considered more stable and ‘Not sensitive’ (at the benchmark level) while the remaining biotopes were likely to be affected.

Using a clearly documented, evidence-based approach to create sensitivity assessments allows the assessment basis and any subsequent decision making or management plans to be readily communicated, transparent and justifiable. The assessments can be replicated and updated where new evidence becomes available ensuring the longevity of the sensitivity assessment tool. For every pressure where sensitivity was previously assessed as a range of scores in MB0102, the assessments made by the evidence review have supported one of the MB0102 assessments. The evidence review has reduced the uncertainty around assessments previously undertaken in the MB0102 project (Tillin et al., 2010) by assigning a single sensitivity score to the pressures as opposed to a range. Finally, as blue mussel bed habitats also contribute to ecosystem function and the delivery of ecosystem services, understanding the sensitivity of these biotopes may also support assessment and management in regard to these. Whatever objective measures are applied to data to assess sensitivity, the final sensitivity assessment is indicative. The evidence, the benchmarks, the confidence in the assessments and the limitations of the process, require a sense-check by experienced marine ecologists before the outcome is used in management decisions.

Impacts of climate change on non-native species

Anthropogenic changes to climate and extreme weather events have already led to the introduction of non-native species (NNS) to the North Atlantic. Regional climate models predict that there will be a continuation of the current trend of warming throughout the 21st century providing enhanced opportunities for NNS at each stage of the invasion process. Increasing evidence is now available to show that climate change has led to the northwards range expansion of a number of NNS in the UK and Ireland, such as the Asian club tunicate Styela clava and the Pacific oyster Crassostrea gigas. Providing definitive evidence though of the direct linkage between climate change and the spread of the majority of NNS is extremely challenging, due to other confounding factors, such as anthropogenic activity. Localised patterns of water movement and food supply may also be complicating the overall pattern of northwards range expansion, by preventing the expansion of some NNS, such as the slipper limpet Crepidula fornicata and the Chilean oyster Ostrea chilensis, from a particular region. A greater understanding of the other aspects of climate change and increased atmospheric CO2, such as increased rainfall, heat waves, frequency of storm events, and ocean acidification may aid in increasing the confidence that scientists have in predicting the long term influence of climate change on the introduction, spread and establishment of NNS.

Polyopes lancifolius (Halymeniales, Rhodophyta), a new component of the Japanese marine flora introduced to Europe

Polyopes lancifolius (Harvey) S. Kawaguchi & H.W. Wang has been recorded for the first time in Europe, during the summer or 2008. A small population was discovered in the Gulf of Morbihan (northeast Atlantic, France). This is the first observation of P. lancifolius outside its native range. Vegetative and reproductive morphological features are compared with previous descriptions. rbcL sequences show no divergence from Japanese populations. Imports of Pacific oysters Crassostrea gigas (Thunberg 1793) are likely to be responsible for its accidental introduction into the Gulf of Morbihan, either directly from northwest Pacific regions or indirectly (secondary dispersal) by transfers from another European oyster farming site. The history of previous algal introductions from Japan suggests that if it becomes successfully established at Morbihan, the species is likely to spread to other European coastal areas.

Phylogeographical analyses of shellfish viruses: inferring a geographical origin for ostreid herpesviruses OsHV-1 (Malacoherpesviridae)

Mortality episodes have regularly been affecting the shellfish industry throughout its history. Some of these mortalities, especially in the oyster industry, have been attributed to herpesviruses. Purification of viral particles and molecular characterization have led to the development of routine monitoring, as well as improved taxonomic classification. Ostreid herpesviruses (Malacoherpesviridae), mostly affecting Pacific oysters Crassostrea gigas, have been sporadically recorded in the French oyster industry since the early 1990s (OsHV-1 'reference'). From 2008, a new variant of ostreid herpesvirus (OsHV-1 mu Var) has emerged and seriously impacted oyster production in France and other European countries. Consequently, the presence of ostreid herpesviruses has been monitored in different oyster producing areas around the world. The present study compiles molecular data that are available from survey efforts and takes a biogeographical approach, in order to infer an origin for ostreid herpesviruses. The highest genotype diversity was found in East Asia, despite a lower survey effort in that area than in Europe. Genotype network analyses show that both populations of ostreid herpesviruses present in Europe (OsHV-1 'reference' and OsHV-1 mu Var) are closely related to genotypes recorded in Asia. Moreover, ostreid herpesviruses have been detected in wild and symptom-free populations of various Asian native Crassostrea species. In the rest of the world, ostreid herpesvirus genotypes were recorded from cultivated C. gigas, and mostly associated with mortality episodes. Results of this study are therefore highly suggestive of an Asian origin for these viruses, which can be pathogenic under farming conditions. It also highlights the risks of European stock improvements, by means of overseas shellfish imports.

Evolution of the extracellular matrix in deuterostomes and the influence of calcitropic hormones

Tese de dout., Biologia (Biologia Molecular), Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2010

Development and standardization of a protocol for sperm cryopreservation of two important commercial oyster species

Dissertação de mestrado, Aquacultura e Pescas, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Crescimento e qualidade microbiológica das ostras produzidas em cultivos multitróficos versus monocultivos

Dissertação de mestrado, Aquacultura e Pescas (Aquacultura), Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015