对近年来发生在厦门海域的裸甲藻(Gymnodinuum)和原甲藻(Prorocentrum)赤潮的发生情况进行了监测分析,采用了采样、分离、单种培养、显微镜和扫描电镜观察r、DNA序列分析等系列监测、分离培养和赤潮生物鉴定技术,重点观察并确证了厦门海域存在的赤潮原因种为微小原甲藻(Prorocentrum minimum)、Takayama pulchellum、无纹环沟藻(Gyrodinium instriatum)。光学显微镜观察表明,赤潮发生海域存在着许多原甲藻和裸甲藻种类,但不能进一步确认到种。利用电子显微镜观察,可根据微小原甲藻体表规则的花纹等特征,根据Takayama pulchellum具有明显的特异性反S形顶沟等特征分别对它们进行有效地分类鉴定。分子分类学分析表明,T.pulchellum(株名为TPXM)28S rDNA D1-D2区序列长度为721 bp,与基因库中同种相似株的同源性大于99%;微小原甲藻(株名PMDH)的ITS和28S rDNA序列与基因库中同种序列的同源性高达99%;无纹环沟藻(株名GIXM)的ITS与基因库中登记的分离自中国深圳海域的4株同种藻的同源性也高达99%。用ITS序列和28S rDNA序列建立的系统进化树也能很好地显示微小原甲藻、Takayama pulchellum、无纹环沟藻之间以及它们与其他藻之间的亲缘关系。将上述结果结合文献记录和环境条件进行了分析,证实这3种赤潮种类Takayama pulchellum、无纹环沟藻、微小原甲藻是厦门海域较为常见的赤潮原因种。对上述检测和鉴定方法的系统应用也表明,这些方法可应用于对现场赤潮生物进行有效监测。Harmful algal blooms(HABs) is increasing globally,and HABs is one of the major disasters along the coast of Xiamen Harbour.Further investigations and researches on the main types of HABs and the algal species causing HAB in coast water of Xiamen Harbour should be made;the environmental monitoring networks and methods along the coast of Xiamen Harbour should be set up and consummated;science researches with local characteristics should be fully developed,and the exotic HAB species from the cabin seawater of foreign ships should also be studied.Aiming directly at rapid identification and regular monitoring of HABs species,this paper tried to develop essential methods of identificating and monitoring HABs species,and evaluated systemic methods for rapidly monitoring and classifying HABs species in morphology and molecular taxonomy.Meanwhile,series of studies were also undertaken to evaluate in the field and tried to find these important HABs causing species along the coast water of western Xiamen Harbour.The harmful algal bloom events caused by Prorocentrum and Gymnodinium species in coast water of western Xiamen Harbour were monitored during recent years.A series of detecting methods based on the analysis of environmental factors,sampling,cell isolation and unicellular culture of these HABs species,observation under microscope and scanning electron microscope(SEM),and sequence analysis of rDNA were used together to monitor and identify these harmful algae.The methods used for collecting samples and establishing specific HAB unicellular cultures were described in detail.Methods of SEM(scanning electron microscope) and ESEM(environmental scanning electron microscope) for Gymnodinium were developed and optimized.These important species that caused harmful algal bloom in Xiamen Harbour were observed and identified as Takayama pulchellum(strain name:TPXM),Gyrodinium instriatum(strain name:GIXM) and Prorocentrum minimum(strain name:PMXM).Algal morphology was observed under LM,and the results suggested that there were many Prorocentrum and Gymnodinium species in coast water of western Xiamen Harbour,and these species could not be identified as specific species under LM.Two strain of Prorocentrum and Gymnodinium were isolated during blooms in Xiamen Harbor and their morphology were observed under SEM.The results showed that there were regular patterns and other characteristics on Prorocentrum cells,which indicated that PMXM was Prorocentrum minimum,and cells of TPXM were broadly oval with a conspicuous and well-defined sigmoid apical groove present on the epitheca,and the apical groove was a characteristic reversed S-shape.These characteristics showed that TPXM was Takayama pulchellum.Molecular biological analyses of rDNA sequences of Prorocentrum and Gymnodinium showed that the D1-D2 region of partial LSU(large subunit) rDNA of T.pulchellum(TPXM) had 721bp and shared more than 99% similarity to the same species whose data was deposited in the GenBank,the ITS and D1-D2 region of partial LSU of P.minimum(PMXM) also shared more than 99% similarity to the same species in the GenBank,and ITS region of G.instriatum(GIXM) shared more than 99% similarity to the same species isolated from Shenzhen Bay in China and its sequences were deposited in the GenBank,too.The analysis of rDNA sequences and phylogenetic trees could be an appropriate taxonomic evidence,especially the length and whole sequences analysis of ITS and variable regions D1-D2 of 28S rDNA could be more reliable and could provide more exact taxonomic informations than other methods.The phylogenetic trees constructed from ITS and partial 28S rDNA could show the relationships of T.pulchellum(TPXM),G.instriatum(GIXM) and P.minimum(PMXM) and their correlative species,and could also separate these species from their disrelated species clearly.According to above results,the characteristics and causation of HABs in western Xiamen Harbour caused by the three species were discussed based on the analysis of related references and environmental conditions.All the results suggested that the three HABs causing species,such as T.pulchellum(TPXM),G.instriatum(GIXM),P.minimum (PMXM) were common HAB species in coast water of western Xiamen Harbour.The results of methodological application in field indicated that these methods and protocols might become useful,feasible and reliable tools to monitor in situ distribution of bloom-forming taxa,such as Gymnodinium and Prorocentrum in natural water.国家重点基础研究发展规划973项目(CEOHAB2001CB409704);; 厦门市创新科技基金项目(3502Z20041059);; 中国博士后基金项目(20060400854);; 农业部淡水鱼类种质资源与生物技术重点实验室开放课题(LFB20070611
