条斑紫菜叶状体细胞的发育与分化

条斑紫菜壳孢子经室内10～80d培养后长成的叶状体分别被酶解成单个细胞，后者在液体培养基中发育成正常叶状体、畸形叶状体、细胞团、性细胞囊等10种不同类型的再生体，它们的形态与结构、细胞排列与大小，放散单孢子难易程度及最终发育结果等均不同。来自日龄不同的种藻的细胞，其再生体类型的数目和各类再生体的百分数不同。当种藻日龄为10～30d时，在它们的单离细胞再生体中出现了极少的细胞团，正常叶状体和具假根畸形叶状体的百分数随着种藻日龄的增加，急剧减少，但不具假根．的畸形叶状体百分数却随着种藻日龄的增加而增加。当种藻日龄大于40d以后，它们的单离细胞就几乎不能直接再生成正常叶状体。随着种藻日龄的增加，细胞再生体中不具假根的畸形叶状体百分数也显著减少，而细胞团的百分数急剧上升，精子囊和果胞子囊的数量也逐渐上升。来自同一藻体不同部位的细胞，在它们的再生体中，随着细胞所处藻体部位的上移，正常叶状体和畸形叶状体的百分数急剧减少，而细胞团百分数却不断增加。上述结果说明，在离体条件下，条斑紫菜叶状体的单离细胞发育成不同类型的再生体，是由于它们在种藻里处于不同分化阶段的缘故；同时，初步得出在条斑紫菜叶状体的发育过程中，从壳孢子细胞分化成性原细胞的过程可分为8个不同的分化阶段。     

黄海绿潮浒苔(Enteromorpha prolifera)生活史的初步研究

实验室条件下对2008年6月黄海中南部海域暴发的绿潮浒苔(Enteromorpha prolifera)样品进行培养,观察记录绿潮浒苔生活史不同世代的生长发育情况.结果显示,成熟藻体释放出的雌、雄配子结合后固着,随后发育形成新个体;刚释放出的孢子具有聚集生长的趋势,随后发育形成具有假根和叶状体分化的新个体;在生长衰败期,部分藻体体细胞发生明显变化,细胞膨胀后发生分裂,发育形成新个体并能在死去的藻体上附着生长;培养的叶状体片段两端迅速愈合,并显示生长极性,两端分别形成叶状体和假根.后两种单性生殖方式目前尚未见公开报道.     

礁膜原生质体的分离及培养

为了开发礁膜人工育苗的新技术,以4％果胶酶和2％纤维素酶的甘露醇溶液,分离酶解经无菌处理的礁膜叶状体,在40mmol·L~(-1)CaCl_2,0.7mol·L~(-1)甘露醇,pH5.5,温度28℃,摇床速度50r·min~(-1)旋转振荡3h的条件下,1.0g湿重礁膜可获原生质体约2×10~6个。原生质体分离后依次用比重为1.030、1.026、1.022的消毒海水(添加N、P、Fe~(2 )、IAA、KB、Vit C)培养,2d后已形成明显的再生细胞壁,3～4d后细胞开始分裂,8d后形成由4～8个细胞组成的细胞团,以后原生质有不同的发育形态,较常见的是形成由一共同胶质膜包被的类似亲本的叶状体组织块,另一种是原生质体形成孢子囊,成熟后,可产生许多游动孢子,再由游动孢子发育成正常形态的膜状配子体,第三种发育形态为偏离亲本正常形态的管状体。     

鼠尾藻有性繁殖和幼孢子体发育的形态学观察

野外跟踪观测烟台芦洋湾地区鼠尾藻的成熟繁殖状况,采集临近成熟的鼠尾藻至实验室培养,跟踪观测其卵子的排放、卵子和受精卵的发育、假根的生长以及幼孢子体的前期发育。试验结果表明,本地区鼠尾藻繁殖期集中在6—8月,鼠尾藻的卵子发育属8核1卵型,卵子受精后约20 h开始脱离生殖托,受精卵先连续进行2次的横分裂,而后再由一端的1个细胞发育成假根,另外2个发育成体细胞。幼孢子体在约2 mm时开始形成分枝突起。     

不同植物激素对条斑紫菜体细胞生长发育的影响

用添加不同浓度植物激素(IBA和6一BA)的PES培养液对条斑紫菜体细胞进行培养，观察其对条斑紫菜体细胞生长发育的影响。结果表明：酶解的条斑紫菜近基部体细胞离体培养可发育成细胞团和叶状体，低浓度的IBA有利于体细胞向叶状体方向发育，而低浓度的6一BA有利于体细胞发育成细胞团。     

坛紫菜体细胞单克隆叶状体途径及海上养殖

用海螺酶酶解坛紫菜叶状体制备游离的营养细胞,在显做镜下挑选出单个细胞放入96孔板,在20℃,1500～2000 lx,12D:12L条件下进行隔离培养。一部分细胞直接发育形成了叶状体:一种途径是单个细胞通过典型的两级分裂发育成叶状体,也有一些单细胞先形成叶片状的细胞团,然后在细胞团一端形成假根;另一种途径是部分单细胞先形成“愈伤组织”,经过一段时间培养后放散出类似“孢子”的细胞,其中部分“孢子”发育成叶状体。由单细胞克隆培养获得的叶状体经组织培养形成纯合丝状体,这种纯系丝状体扩大培养后转入传统的育苗途径下海养殖。纯系在苗网附着率、叶片质量和性成熟时间等经济性状方面比未经选育的普通种有优势。     

铜藻的受精卵发生与幼孢子体发育

通过室内培养, 观察了铜藻受精卵的发生与幼孢子体发育, 研究了幼孢子体的适宜培养条件, 为全人工育苗奠定理论基础。结果表明, 铜藻雌性生殖托的基部和中部首先集中排卵, 卵子粘附于生殖托表面完成受精和早期发生。刚释放的卵子具有8个核, 受精后, 8个核迅速融合成1个大核, 开始细胞分裂。前二次的细胞分裂均为横裂, 在萌发体的一端产生一个很小的“假根原细胞”, 后者最终发育成假根, 萌发体的其它细胞发育成苗体。受精后约48 h, 受精卵发育成具有假根芽的幼孢子体, 开始脱落附着; 培养15 d, 发育成具有2个叶片、体长超过3 mm的幼孢子体。在幼苗的早期培育阶段, 较高的温度和长光照时有利于幼苗的生长和叶片增加, 适宜培养条件为温度21~24 ℃, 光密度40 μmol photons/(m2?s), 光周期14 L∶10 D。     

坛紫菜雌雄叶状体的细胞分化比较

以室内培养20～90 d的坛紫菜雌雄叶状体为研究材料，用酶解法分别获取单离细胞进行再生培养。在雌雄叶状体的体细胞再生体中，都出现9种不同发育类型。再生体发育类型的数目和比例与种藻日龄密切相关等结果，证实了离体培养的单离细胞发育成不同形态的再生体是基于其离体前处于不同分化时期所致；由壳孢子分化成性母细胞大致可划分成8个不同阶段。雌雄叶状体的细胞分化途经大致相同，但也有一定差异，雌性叶状体的细胞最终分化成雌性性母细胞，并产生大量的丝状体；而雄性叶状体的细胞最终分化成雄性性母细胞，绝大部分生成精子，但极少数产生丝状体。在雌雄叶状体的细胞再生体中，均产生 “类单孢子”并长成正常叶状体。雄性叶状体成熟较雌性早，与其细胞分化速度较快有关。成熟期不同的雌性品系观察结果表明，叶状体成熟越早、生长期越短，其体细胞分化速度也越快。     

长心卡帕藻四分孢子发育成配子体的形态建成观察

2007年11月到2009年4月在海南黎安，以养殖多年的红褐色长心卡帕藻为材料，跟踪观察了四分孢子形成、释放、萌发以及配子体苗形态建成过程。结果表明，（1）该长心卡帕藻被确定为四分孢子体，四分孢子形成和释放主要发生在夏季和秋季（2008年4月到2008年10月）。（2）人工诱导可促使四分孢子萌发并形成胚苗，胚苗色泽出现明显分化，有红褐色、黄绿色、深绿色、黄绿—红嵌合等不同类型。（3）四分孢子萌发率达到87.1%±7.2%，培养皿内胚苗平均日生长率为（6.3±1.1） %/d，在四分孢子萌发10 d左右假根出现并于培养30 d左右消失。（4）胚苗经过约5个月实验室培养发育成的配子体苗达到了下海挂养程度大小，在海上培养初期配子体苗日生长率大多在10 %/d以上，最高可达21.2 %/d，但随着藻体长大生长速率逐渐下降。经过4个多月海上挂养栽培，获得了藻枝形态、粗细、疏密程度和生长速率等差异明显的多种配子体，部分配子体活力和抗逆性比其母本有显著增加。     

河蚌结缔组织细胞发生及发育观察

对河蚌外套膜组织学观察发现,外套膜结缔组织发源于细胞聚合体。聚合体细胞发育形成结缔组织细胞及组织构造。外套膜边缘“类聚合体”细胞增殖和发育形成边缘结缔组织。聚合体细胞发育形成不同形状细胞(成纤维细胞和颗粒细胞)。两者不断变化:成纤维细胞积累颗粒最终发育成球状颗粒细胞。球状细胞解体释放颗粒后恢复分泌纤维质状态。纤维物质重新组合排列构成贝体组织骨架,颗粒物质参与贝壳形成。     

紫菜遗传育种研究进展

对中国紫菜(Pyropia/Porphyra)种质资源进行了详细的介绍,从育种技术、育种目标和育种成果3个方面综述了紫菜育种领域的研究进展。育种技术以选择育种、杂交育种和诱变育种为主,分子遗传育种在探索之中。育种目标在优质、高产的基础上,结合气候变化、不同海区环境,培育耐高温、耐低盐等抗逆性强的品种。目前,中国已培育出紫菜新品种6个,其中坛紫菜4个,条斑紫菜2个。最后,作者提出中国紫菜产业发展面临的一些问题,期望良种选育与产业发展相结合,为科研工作者进一步开展紫菜育种研究提供参考。     

5.8S rDNA-ITS区片段的序列分析在坛紫菜种质鉴定中的应用

为探讨DNA序列标记技术在坛紫菜种质鉴定中的应用，对10个野生坛紫菜种质材料的5.8S rDNA-ITS区进行PCR扩增和序列分析，结果发现扩增的片段长度在1 208~1 219 bp之间，可以分为ITS1区，5.8S区和ITS2区3个部分，其中5.8S区片段的长度完全一致，均为160 bp；ITS1区和ITS2区片段的长度也非常接近，只有几个碱基的差异。多重序列比对发现10个种质材料的ITS区（包括ITS1和ITS2）序列都存在一定差异，序列同源性在95.82％~99.73％之间，而5.8S区序列则完全一致，但与其它种紫菜的5.8S区序列有很大差异，序列同源性在79.7％~95.0％之间。由此认为5.8S rDNA-ITS区这种高度保守区和高变区交替排列的形式可以成为坛紫菜种质鉴定及系统进化分析的强有力工具。     

A review of the environmental effects and alternative production strategies of marine aquaculture in Chile

In Chile, fish, mussel and seaweed cultivation has expanded significantly over the last decade. This review considers the accumulated knowledge on the environmental effects of aquaculture in Chilean coastal areas, analyses the capacity of the industry to treat its waste and also gives some insight into new culture technologies and strategies that are currently under research and discussion in Chile. Data relating to the environmental impact of aquaculture in Chile are scarce and much is subject to severe methodological restrictions with regard to sampling design. Results related to the environmental effects show that seaweed cultivation can have an impact on sedimentation processes, increase of invertebrate assemblages and algal epiphytic abundances. It has also been ascertained that mollusc farming causes biodeposition, faunal changes and possible effects related to the introduction of new species, as well as pathogens and other unforeseen species. It has been affirmed that fish cultivation, in particular that of salmon species, also has an environmental impact related to organic sedimentation and changes in the fauna. However, these results indicate that, in general, the current dynamics of bays and fjords seem to be an important factor for the environmental sustainability of the salmon culture areas. Salmon cultivation has also been associated with phytoplankton blooms, but this point was not supported by a monitoring programme in southern Chile. Furthermore, there is concern related to new pathogen introduction and therapeutical applications to the fish cultures, and further research is required in this field. Regulations to protect the environment from the consequences of aquaculture activities have been adopted during the last couple of years. The main regulations are provided by international market standards. Nevertheless, these regulations can only be effective if other human activities, such as urban discharge, intensive agriculture fertilisation and pesticide utilisation, are taken into consideration, in an integrated perspective. On the other hand, the Chilean salmon farming industry in particular, would be in a position to cover the costs involved in the treatment of waste waters, if feeding management were improved in the future. Finally, active research is currently being undertaken into new cultivation strategies, such as the use of integrated cultivation and the recycling of nutrient-rich waters, which should permit the diversification of this economic activity in Chile, while minimising the environmental impact.     

Preservation of manipulated yeast diet

Manipulated yeast diet can be usedfor seed production of aquacultural organisms.Various methods for preserving the yeast dietduring the periods of circulation in marketwere tested, and the preservation of the yeastdiet by freeze-drying was the best. With thispreservation method, the manipulated yeastswere maintained fairly well (up to 71%) whenstored for three weeks under refrigeratedcondition (4 °C), while more than 80% ofthe yeast protoplasts disappeared in 5 days incontrol. Preservation temperature had an effecton retaining the yeast diet, but adding 3.5%NaCl concentration in the culture medium at thetime of yeast cultivation did not.     

An improved enzyme preparation for rapid mass production of protoplasts as seed stock for aquaculture of macrophytic marine green algae

Preparation of protoplasts and their subsequent applications for both basic and applied research of marine macroalgae remains largely under developed due to lack of development of reliable methods with consistent yields of viable protoplasts. An improved enzyme preparation with a single commercial enzyme, e.g. 2% Cellulase Onozuka R-10 in 1% NaCl solution, was developed to produce protoplasts rapidly from different green algal genera of Ulva, Enteromorpha and Monostroma. The simple dissolution of enzyme powder in 1% NaCl resulted in exclusion of 2% Macerozyme R-10 from the mixture consisting of 2% Cellulase Onozuka R-10 with 3% NaCl earlier reported as superior for the same algae. Optimal conditions for the isolation of maximum yields of viable protoplasts were found to be with 2% Cellulase Onozuka R-10 incubated at 20 °C for 2 h in 1% NaCl solution with 0.8 M mannitol adjusted to pH 6.0. The protoplast yield with optimized enzyme mixture was as high as 102.8 × 10⁶ cells g^− 1 f. wt for M. oxyspermum while it was in the range of 74.4–88.6 × 10⁶ cells g^− 1 f. wt thallus for seven species of Ulva, and 82.5–95.4 × 10⁶ cells g^− 1 f. wt for three species of Enteromorpha. The regeneration rate of protoplasts isolated using this method ranged from 89 to 92% with normal morphogenesis. The seeding of nylon threads with isolated protoplasts of M. oxyspermum was successful and after 3–4 weeks the entire frame with nylon threads became thick green in color with tiny germlings in laboratory culture. Thus, the method described in the present study allow for rapid mass production of viable protoplasts that could be potentially used as a source for seed material for mariculture and for other applied phycological research.     

Preservation of manipulated yeast diet

Manipulated yeast diet can be usedfor seed production of aquacultural organisms.Various methods for preserving the yeast dietduring the periods of circulation in marketwere tested, and the preservation of the yeastdiet by freeze-drying was the best. With thispreservation method, the manipulated yeastswere maintained fairly well (up to 71%) whenstored for three weeks under refrigeratedcondition (4 °C), while more than 80% ofthe yeast protoplasts disappeared in 5 days incontrol. Preservation temperature had an effecton retaining the yeast diet, but adding 3.5%NaCl concentration in the culture medium at thetime of yeast cultivation did not.     

糙海参胚胎和幼体发育的形态观察

为提高糙海参育苗技术,研究描述了糙海参从受精卵发育到稚参的形态变化,在显微镜下测定了受精卵、胚胎和幼体的大小,确定了糙海参胚胎发育的过程。结果表明,通过利用阴干、流水刺激法对成熟亲参进行人工催产,得到大量的受精卵,其受精率为90%以上。糙海参胚胎和幼体发育可分为受精卵、卵裂期、囊胚期、旋转囊胚期、原肠期、初耳状幼体、中耳状幼体、大耳状幼体、樽形幼体和稚参等阶段;在平均水温29℃,平均盐度34条件下,糙海参受精卵经3 h发育形成囊胚,4 h进入旋转囊胚期,5 h进入原肠期,19 h完成胚胎发育变态为耳状幼体;并经过7 d的生长与发育进入樽形幼体;第15天变态为稚参。观察发现,多精入卵现象则会导致胚胎发育不正常,最终使胚胎发育停止并死亡。在糙海参幼体发育过程中,大耳状幼体期幼虫臂的大小及其球状体的形成均可作为判断其幼体发育健康状况的重要指标:幼虫臂越大,球状体出现率越高,其幼虫的变态率和成活率也越高。对比发现,位于糙海参尾部的突出结构——尾突,为仿刺参和新西兰刺参所没有,这一结构差异同时导致了其骨片位置也不同;并且,糙海参胚胎和幼体与其他种类的海参在发育时间上存在一定差异。     

坛紫菜遗传连锁图谱的构建

以野生型坛紫菜纯系(♀)和红色型坛紫菜纯系(♂)作为杂交亲本,结合四分子分析法及单个体细胞克隆的丝状体途径,创建了由157个株系组成的坛紫菜DH作图群体,并用经过筛选的24对SRAP引物和16对SSR引物对父母本及作图群体各株系进行双标记分析,获得了224个多态性标记,其中157个标记符合孟德尔分离规律。根据标记间的连锁规律,首次构建了坛紫菜的分子遗传连锁图谱,所构建的遗传图谱由包含124个标记(含SRAP标记104个,SSR标记20个)的5个连锁群组成,总长度为879.2cM,平均标记间隔为7.09cM,各个连锁群长度为134.2～213.6cM,包含标记18～31个。最后采用3种不同方法计算得到坛紫菜的估计基因组长度平均为955.3cM,由此得到坛紫菜遗传连锁图谱的基因组覆盖率为92.0%。     

近江蛏精子超微形态结构观察及与缢蛏精子的比较

利用扫描电镜和透射电镜分别观察了近江蛏和缢蛏成熟精子的超微形态结构。发现近江蛏精子与缢蛏精子在超微形态结构上差异很大。近江蛏精子为典型的原生型,成熟精子由头部、中段和尾部组成,头部包括顶体和细胞核。近江蛏精子与缢蛏精子顶体均为保龄球状,但近江蛏精子顶体全长比缢蛏短。近江蛏精子细胞核略扁圆形,外缘具8～9个圆弧形凸起；缢蛏精子细胞核则呈圆球状。近江蛏精子中段由线粒体和中心粒复合体构成,线粒体一般5个,个别4～6个；缢蛏线粒体5个或6个。近江蛏精子尾部为细长的鞭毛,轴丝为“9 2”结构,与缢蛏的相同。从近江蛏与缢蛏的精子形态差异看,两者应属于种间差异。     

The introduction of the European bitterling (Rhodeus amarus) to west and central Europe

The European bitterling is considered to be a native species over much of its present range in Europe. A dramatic decline in its abundance from 1960 to 1980 in west and central Europe, attributed to aquatic pollution, led to the establishment of stringent national and international regulations for protection of the species. Here, we review the evidence that until AD 1100 the bitterling was restricted to the Ponto‐Caspian and Aegean regions (south‐eastern Europe and adjacent regions of Asia Minor) and only expanded into its present range during the 19th century. The earliest records of bitterling in west and central Europe are from regions where carp cultivation was common and the bitterling appears to have spread into this region in association with the gradual expansion of carp cultivation. After an initial period of expansion, between approximately 1150 and 1560 in regions with carp cultivation, the species virtually disappeared from Europe during the coldest period of the Little Ice Age. Bitterling reappeared at the end of the 18th century, initially in historical centres of carp cultivation, and was again abundant and widespread in Europe by around 1850. Its reappearance appears to have been through expansion of refuge populations as well as new invasions. The decline in abundance of bitterling during the period 1960–80 in west and central Europe appears not only to have been caused by pollution, as is generally believed, but may also be correlated with low spring temperatures. From approximately 1980 onwards the European bitterling once again expanded its distribution in many parts of Europe, particularly in eastern Europe. This recent expansion may be due to a combination of factors, including a rise in ambient temperature coupled with an increase in anthropogenic dispersal and changes to aquatic habitats favourable to bitterling. Thus, the bitterling, which is legally protected in Europe at a national and international level as an endangered indigenous species, is actually an invasive species and a parasite of freshwater mussels. Its current expansion in distribution could pose a hazard to freshwater mussel populations in regions where they are already threatened.