威海海域发现文昌鱼

文昌鱼(Branchiostoma belcheri)在我国南海、黄海和渤海的近岸均有发现，是我国二级保护动物，主要分布于厦门、青岛、烟台等地。2002年3月份在威海国际海水浴场进行鲸类资源调查时，在该海域首次发现了文昌鱼。     

北部湾的文昌鱼

文昌鱼是海洋稀有的原始脊索动物,它不但有很高的经济价值,而且是研究动物进化系统的一种珍贵材料。我国的文昌鱼,据以往报道,主要分布在福建厦门同安刘五店和山东青岛、烟台等地。1982年7月至1985年3月,我们在北部湾进行文昌鱼资源调查,采到的文昌鱼经鉴定为白氏文昌鱼(Branchiostoma belcheri Gray,即厦门文昌鱼,图版Ⅰ:1)。现将它的形态特征、生态习性和采捕方法叙述如下。     

文昌鱼鳃组织原代培养

采用组织块培养法培养文昌鱼(Branchiostoma belcheri japonicum)鳃细胞,使用L15,M199,Eargle'sMEM为基础培养基,加入一定量的NaCl及5%,10%,20%等不同体积分数的牛血清.经条件优化后得到最适合的培养条件为2L15+20%FBS+0.45%NaCl,在26～28℃条件下培养,组织块贴壁后12 h内就有至少4类上皮样细胞从中迁移出来,这些细胞在前3天迅速增殖,到第6天基本上达到平稳期,往后细胞开始凋亡,最长可以存活近两个星期.鳃细胞的原代培养为文昌鱼细胞体外培养的深入研究提供了实验依据.     

文昌鱼胚胎整装荧光原位杂交技术的建立

文昌鱼(Branchiostoma belcheri tsingtauense)是进化发育生物学研究的重要模式生物,文昌鱼胚胎整装荧光原位杂交(WFISH,whole-mounted fluorescent in situ hybridization)技术将有助于鉴定文昌鱼胚胎发育过程中具有重要调控作用的功能基因。报告了文昌鱼胚胎整装荧光原位杂交技术,用以快速灵敏分析特定基因在文昌鱼胚胎发育过程中的时空表达图式。用体外转录合成的地高辛标记文昌鱼FGFR基因的反义RNA探针,检测到FGFR在文昌鱼原肠胚中表达于发生内陷的中内胚层细胞中,而预定发育为外胚层的细胞不表达FGFR。     

文昌鱼精子发生过程中的超微结构研究

本文在超微结构水平上研究文昌鱼(Branchiostoma belcheri Gray)精子发生过程中细胞形态结构的变化和细胞器的演变规律。观察到精细胞核形态变化的特点、线粒体的演变及其原始结构形式。同时,也观察到精细胞变态过程中高尔基体的演变、顶体的形成及超微结构的特点和微管的重组。此外,我们还首次观察到尾丝外膜上有特殊的刺状结构。对以上观察结果,进行了讨论。     

厦门文昌鱼人工繁殖和幼虫发育及其变态的研究

用组织学和胚胎学方法对厦门国家级自然保护区文昌鱼的生殖季节、胚胎发育和幼虫培育进行详细研究,结果表明,自然保护区文昌鱼生殖季节可分为两个繁殖时期,从6月初开始至7月初为繁殖高峰期,8和9月为繁殖小产期.观察人工繁殖得出,文昌鱼(Branchiostoma belcheri)产卵时间在傍晚19:00前后,可以观察到雌、雄文昌鱼的产卵和排精行为:雄性文昌鱼先离开沙,并跃出水面快速游动和排精,接着雌鱼也跃出水面,所有的卵通过破裂的性腺壁进入到围鳃腔并通过围鳃孔到水中受精,受精率在98.5%以上,还观察了文昌鱼幼虫发育并变态为幼鱼(长度为1.05～13.5mm)的全过程.     

厦门文昌鱼遗传多样性研究

运用分子标记技术，对厦门现有文昌鱼(Branchiostoma balcheri Gray)海域(蟹口海域、南线海域、黄厝海域)文昌鱼种群进行遗传多样性研究。通过10个有效引物的RAPD反应，共扩增出238条带，其中多态性条带为121，占总扩增条带的50．84％。Nei指数法分析和UP(小俄统计分析表明：厦门3个海域文昌鱼可分为2个组。平均遗传距离为0．11。南线海域文昌鱼和黄厝海域文昌鱼的遗传距离为0．07，亲缘关系较近，聚类于同一组；蟹口海域文昌鱼与南线海域文昌鱼和黄厝海域文昌鱼的遗传距离分别为0．12和0．14，亲缘关系较远，单独为一组，结论 与3个海域的文昌鱼形态比较结果一致，出现遗传分化。Shannon遗传多样性指数统计结果表明：蟹口海域种群为0．616，南线海域种群为0．497，黄厝海域种群为0．391。厦门文昌鱼种群遗传多样性是各种群的遗传特性与该海域各种环境生态因子长期作用的结果，三元线性相关分析发现目前主要生态因素是底质和海水深度，3个海域中以蟹口海域的自然环境最为优越(其底质为中粗沙或粗中沙、水深8—15m)。根据以上研究结果，本文提出了厦门海域文昌鱼资源的保护建议。     

亚致死剂量铜对文昌鱼和玫瑰无须的组织病理学影响

采用半静水实验法测试研究了重金属Cu2 对文昌鱼(Branchiostoma belcheri)和玫瑰无须(Puntius conchonius)的急性致死毒性和亚急性毒性.实验结果表明,Cu2 对文昌鱼和玫瑰无须 24,48,72,96 h的半致死浓度分别为0.561,0.373,0.261,0.211 mg/L及0.976,0.783,0.562,0.473 mg/L,其安全质量浓度分别为0.021和0.047 mg/L.亚急性毒性实验中,文昌鱼和玫瑰无须 分别用浓度为20%和30%的 96 h LC50的Cu2 处理.不同器官的组织病理学观察结果表明,过量Cu2 对玫瑰无须 的鳃、肝脏、肾脏等器官产生毒害,对文昌鱼鳃和肝盲囊细胞也产生了严重的毒害,表现为细胞空泡化等明显的结构破坏.实验结果显示文昌鱼的肝盲囊和脊椎动物的肝脏一样,也是受过量铜显著毒害的主要靶器官器官.因此,文昌鱼的肝盲囊至少在经受过量Cu2 毒害方面与脊椎动物肝脏具有相似性,从生理功能方面为类文昌鱼祖先的盲囊(digestive caecum)是脊椎动物肝脏的前体这一假说提供了资料.     

青岛文昌鱼肌球蛋白重链基因片段的重组表达

将青岛文昌鱼(Branchiostoma belcheri tsingtauense)肌球蛋白重链基因片段亚克隆到pET-30a质粒,构建出重组表达载体并转化入大肠杆菌Escherichia coli BL21中.经SDS-PAGE和Western杂交检测表明,在IPTG诱导下含有重组载体的菌株可表达分子质量约30 ku的融合蛋白.诱导条件优化实验结果显示,该菌株经0.2 mmol/L IPTG诱导1 h就可大量表达此蛋白.可溶性实验确认该重组蛋白是可溶性蛋白.本研究为文昌鱼肌球蛋白重链特异性抗体的制备奠定了基础.     

成体文昌鱼实验室培养产卵、排精及其受精卵早期发育的观察

文昌鱼(Branchiostoma belcheri tsingtauense)属于脊索动物门(Chordata)头索动物亚门(Cephalochordata)文昌鱼科(Branchiostomidae),为国家二级保护动物。据认为(Carrol,1988),文昌鱼是和脊椎动物亲缘关系最近的现存无脊椎动物,在系统演化上,它介于无脊椎动物和脊椎动物之间,是研究脊椎动物起源的珍贵材料。此外,文昌鱼体内蛋白含量高达70%,并含有丰富的碘、铁和磷,脂肪含量低(Svetashev et al.,1994),在我国福建省和东南亚地区被视为美味佳肴。但是,近年来由于生存环境的改变和环境污染,文昌鱼种群在世界各海区都在减少(方永强,1987;Nishikawa,1992)。为保护这一具有重要学术意义和一定经济价值的濒危动物,开展全人工养殖的研究是十分必要和急需的。 杜琦等(1990)以及Wu等(1994)曾先后进行过文昌鱼人工培育的研究,取得了一些成果,目前,我们又开展了实验室培养文昌鱼的繁殖生物学研究。有关天然海区文昌鱼的产卵、排精及其受精卵胚胎发育已有大量描述(Wickstead,1975;金德祥,1964),但是,实验室培养文昌鱼的产卵、排精及其受精卵早期胚胎发育观察却未见报道。通过研究发现：(1)文昌鱼只有在雌、雄同时存在时才能产卵、排精,而雌、雄分开后都不产卵排精,说明文昌鱼产卵、排精很可能受性外激素的控制;(2)实验室培养的文昌鱼受精卵能正常发育,并形成正常幼虫。     

The interplay of sediment characteristics,depth, water temperature,and ocean currents shaping the biogeography of lancelets (Subphylum Cephalochordata) in the NW Pacific waters

Lancelets (or amphioxus) are often found within the soft bottom of shallow tropical and temperate seas. The present study is the first to provide a fine‐scale biogeography of five species of lancelets (Asymmetron lucayanum, Branchiostoma belcheri, Branchiostoma japonicum, Epigonichthys cultellus, and Epigonichthys maldivensis) in the NW Pacific and examine the effects of multiple environmental parameters. From multivariate analyses, the distribution and abundance patterns of lancelets were explained by a combination of factors comprising depth, temperature of the collecting month, mean temperature of the coldest month in the year, medium particle size and silt/clay ratio of the sediments. In addition, ocean currents also affect their distribution range. The major occurrence of A. lucayanum and E. maldivensis was associated with the warm Kuroshio current, but E. maldivensis exhibited higher tolerance to low salinity and low temperature, and preferred substrata of slightly larger grain size, a lower ratio of suspended sediments, and deeper water. The closely related B. belcheri and B. japonicum exhibited fine‐scale habitat differentiation, and B. japonicum was abundant along the southern coast of China, where the salinity is lower in winter because of the China Coastal Current and the substratum is composed of very coarse sand with suspended sediments. Branchiostoma belcheri occurred in low abundance and was only recorded in Northern Taiwan and the Taiwan Banks. Epigonichthys cultellus was found only at the Taiwan Banks, which had deep water, coarse sand, and a negligible amount of suspended sediments.     

底质细化对厦门潮下带文昌鱼栖息地大型底栖动物群落的影响

本文根据2014年至2017年在厦门文昌鱼保护区黄厝、南线十八线、鳄鱼屿和小嶝岛四处潮下带以及参照点白城潮下带获得的大型底栖动物和沉积物粒径数据,探讨底质细化对厦门潮下带文昌鱼栖息地大型底栖动物群落的影响。聚类分析表明个别黏土含量较高的取样站没有发现文昌鱼且大型底栖动物种数少。单因素方差分析表明,厦门潮下带部分大型底栖动物群落参数和优势种数量存在显著差异。底质粒径分析表明, 2017年9月,鳄鱼屿和白城潮下带黏土和粉砂含量高于黄厝、南线十八线和小嶝岛潮下带的黏土和粉砂含量。2016年夏季黄厝、南线十八线潮下带黏土和粉砂含量明显高于2014年夏季的黏土和粉砂含量。相关分析表明,5个大型底栖动物群落参数如物种数、栖息密度、生物量、多样性指数(H′)和丰度指数(d), 7个常见种的栖息密度和生物量与粉砂和黏土含量呈显著正相关,均匀度指数(J)和4种常见种的数量与粉砂和黏土含量无显著相关,白氏文昌鱼(Branchiostoma belcheri)的栖息密度和生物量与粉砂和黏土含量呈显著负相关。上述结果证实,黏土含量增加到15%和粉砂含量增加到60%有利于多数大型底栖动物栖息密度和生物量的增加,但导致白氏文昌鱼栖息密度和生物量的减少,而红角沙蚕(Ceratonereiserythraeenis)、滑指矶沙蚕(Eunice indica)、光滑倍棘蛇尾(Amphioplus laevis)受底质细化的影响较小。     

Growth rate is temperature‐dependent,but the proportion of resource allocation between the growth and gonads is not,in early adult stage of the lancelet branchiostoma japonicum

The pattern of resource allocation into multiple life‐history traits can vary with the environment; for example, high temperatures generally facilitate growth. Changes in investment in growth due to temperature may alter the allocation to other traits that are in trade‐off with that. Here, we investigated the trade‐off between growth and reproduction and whether the proportion of allocation to these varies with increasing seawater temperature in the lancelet Branchiostoma japonicum. We reared the lancelets of early adult stage under three different temperature treatments (control, + 1°C and + 2°C treatments) for 427 days in the laboratory. Although the total body length at initiation did not differ between treatments, the length was influenced by the interaction between elapsed days and temperature treatments, suggesting that growth rate was temperature‐dependent; the lancelets under higher temperature conditions showed a more rapid growth rate than did those under the control conditions. The frequency of individuals with gonads did not differ between treatments throughout their reproductive season. The monthly growth increments were also not significantly affected by the interaction between the presence/absence of gonads and temperature treatments, although the presence of gonads caused the growth increments to decrease. In addition, the effect of temperature treatments was significant. These results suggest that changes in temperature did not alter the pattern of allocation between growth and reproduction that were in a trade‐off, whereas high temperature would solely result in an increase in resource intake, at least in the early adult stage of this species.     

青岛文昌鱼（Branchiostomabelcheri tsingtauense）染色体G类带型研究

以青岛文昌鱼的晚期囊胚和早期原肠胚的胚胎细胞为实验材料,利用低渗处理、空气干燥法制备染色体玻片标本。在此基础上,为进一步探索显示青岛文昌鱼染色体G带的新方法,采用片龄、蛋白酶K和胰蛋白酶显带技术对青岛文昌鱼的染色体进行了初步研究,显示了青岛文昌鱼染色体的G类带型,绘制了青岛文昌鱼染色体的G类带型模式图,并对三种G类带型...     

First observation of Karlodinium veneficum from the East China Sea and the coastal waters of Germany

Harmful algal blooms often occurred in the East China Sea (ECS) and the German coastal waters of the North Sea but Karlodinium veneficum had not been taxonomically reported. Two strains of Karlodinium (LAMB090611 and LAMB010601) were isolated from the two areas in 2009. The morphological characteristics and molecular phylogeny of two strains are compared on the basis of observation of a light microscope, a scanning electron microscope (SEM), a laser scanning micro-scope (LSM) and an internal transcribed spacer (ITS) sequence data. The mean cell length of strain LAMB090611 is (14.2 ± 1.8) μm (range 11.1-18.7 μm) and mean width is (10.8 ± 1.5) μm (range 8.2- 14.7 μm). The mean cell length of strain LAMB010601 is (15.1 ± 1.2) μm (range 12.7-17.9 μm) and the mean width is (11.4 ± 1.1) μm (range 9.1-14.7 μm), respectively. The two strains are similar in morphological characteristics, including a straight apical groove, distinct ventral pore, sulcal extension, cingulum displacement, two or four irregular shaped chloroplasts within the cell and almost equal sized epicone and hypocone. The large and round nucleus is located at the center or at the hypocone of the cell. The sequence length of the ECS strain LAMB090611 and the German strain LAMB010601 is 640 and 646 bp, respectively. The GC content is 49%. The nucleotide similarity of the two strains is 98.1%. The sequence divergence is 0.003. Both strains are confirmed as Karlo-dinium veneficum (D. Ballantine) J. Larsen and this is the first taxonomic report from China and Germany coastal waters. The population dynamics of this toxic species in the ECS and German coastal waters needs to be investigated in the near future.     

Localization of nitric oxide synthase in the developing gonads of amphioxus Branchiostoma belcheri tsingtauense

The localization of nitric oxide synthases （NOS） is dealed with in the developing gonads of amphioxus Branchiostoma belcheri tsingtauense. It was found by NADPH-diaphorase staining that （1） NOS activity was present in the nuclear membranes of germinal vesicles during the entire period ofoocyte development; （2） NOS was localized in both the nuclear membranes and the perinuclear region of cytoplasm in the vitellogenetic oocytes; （3） NOS was relocated in the cortical layer in the mature egg; （4） NOS activity was present in spermatocytes, but not in the spermatogonia in the middle of October; （5） NOS was detected in both spermatozoa and spermatids as well as spermatocytes during the breeding season. This is the first report on the distribution pattern of NOS in the developing gonads in protochordates. These results suggest a role for NOS in the functioning of the nuclear membranes and yolk synthesis during oogenesis and in cell division and differentiation during spermatogenesis.     

Morphological and molecular evidence supports the occurrence of a single species of Zebrias zebrinus along the coastal waters of China

The so-called zebra sole includes a group of small flatfishes characterized by transverse band pairs on the ocular side and distributed throughout shallow waters along the coast of the Indo-West Pacific Ocean. Sev-eral species of the zebra sole have been recorded from the coastal waters of China. Morphological analysis of 1 107 specimens of the zebra sole from 15 successive localities along the China’s coast demonstrated that no significant variations among these localities were found on the basis of meristic counts and morphometric characters. Phylogenetic analysis based onCOI gene sequences of 14 individuals and D-loop of 22 indi-viduals from eight localities showed that they were indistinguishable among these localities. Therefore, both morphological and molecular evidence supported the occurrence of a single species of the zebra sole along the China’s coast. The available name for this species isZebriaszebrinus （Temminck and Schlegel, 1846） in-stead ofZ.zebra （Bloch, 1787）.Zebrias fasciatus（Basilewsky, 1855） andSolea ommatura （Richardson, 1846） are considered here as two synonyms ofZ. zebrinus.     

青岛文昌鱼的生活史──年龄、生长和死亡研究

于１９８９年８月－１９９０年７月对青岛文昌鱼的生活史进行研究。结果表明：（１）青岛文昌鱼种群由４个齿组组成，它至少生产４ａ以上，甚至可达６ａ左右。（２）在Ⅰ、Ⅱ齿时生长较快，平均年生长１３－１５ｍｍ，Ⅲ龄后生长速度减慢，即使同一年龄组的文晶鱼，在不同季节其生长速度也不同。（３）在冬春采集的文昌鱼中Ⅲ龄组的峰值较小。（４）繁殖季节过后，老龄文昌鱼因衰老而死亡。     

中国沿海不同地区泥蚶(Tegillarca granosa)的遗传多样性分析

以采自海南海口、广西防城港、广西北海、广东湛江、福建漳州、山东荣成、山东威海7个地理群体62个泥蚶个体为材料,获取592bp线粒体COI基因片段序列,并进行遗传多样性及分化分析。多态性遗传参数统计显示:62个个体共检测出103个多态性位点,定义了26个单倍型;总群体单倍型多样性指数为0.834,核苷酸多样性指数为0.01665,平均核苷酸差异数为9.85772。7个群体均显示出较丰富的遗传多样性,群体内遗传距离及群体内遗传多样性参数显示中国沿海泥蚶遗传多样性由北向南呈升高趋势,而群体内遗传分化系数也呈现升高的趋势。基于26个单倍型COI序列构建的NJ树和UPGMA树以及基于群体间遗传距离构建的UPGMA树显示,荣成群体和威海群体亲缘关系较近,聚成一小支,而后与漳州群体相聚,然而南方类群并没有聚为独立的一支。