中国沿海三种重要有害藻华甲藻的生活史研究

Resting cyst has been proven to be one of the most important facilities used by dinoflagellates to form harmful algal blooms (HABs) and expand their geographic distribution. A handful of dinoflagellates that form HABs most widely and thus are most intensively investigated are unexceptionally resting cyst producers and have been proven to expand their geographic distribution via resting cysts. In coastal waters of China, the events and scales of HABs caused by the dinoflagellates Karenia mikimotoi and Prorocentrum donghaiense have increaesd considerably since 1990s, which caused catastrophic losses in fishery and aquaculture. The HABs caused by these two species have been predictable annual events in some coastal areas and have exhibited trends of geographic expansion. Another well-known, worldwide distributed, and toxic dinoflagellate, Karlodinium veneficum, has been recently reported forming toxic blooms in China and presents in many estuaries along the coastline of China. Yet, it is still an open question regarding whether or not the recurrences and geographic expansion of HABs caused by these species are attributable to production of resting cysts in their life histories. Our recent preliminary results, however, have indicated these species are most likely cyst producers. Therefore, here we propose a systematic investigation into their life histories (or life cycles) using multiple clonal cultures each of the abovementioned dinoflagellates already available in the laboratory with the aim of clarifying whether or not they do produce cysts in laboratory cultures and in the field. The approaches proposed will include induction of cyst production with multiple stimulating measures, germination of cysts if/when available, and detection of cysts in sediment samples taken from areas where these species recurrently bloom，via a combined approach of morphological identification and molecular detectiona (FISH and qPCR). We believe that accomplishment of the proposed project will lead to an appreciably deeper understanding about the mechanisms of the initiation, recurrence, and geographic expansion of HABs caused by the abovementioned dinoflagellates, and provide a solid basis for the development of models for forecasting and measures for preventing HABs in the future.

休眠孢囊已被证明在甲藻藻华的形成和地理扩散中起着非常关键的作用。世界上分布最广因而研究最多的几种有毒藻华种都在其生活史中形成且通过休眠孢囊在地理上扩散。由甲藻米氏凯伦藻和东海原甲藻引起的有害藻华过去十几年以来在我国沿海发生频率和规模不断增大且有扩散趋势，给沿海经济造成巨量损失。世界广布种微小卡罗藻的有毒藻华最近也在我国出现且南北各海域都已发现其分布。这些藻华在同海域连年爆发且分布范围扩大是否与其特殊生活史如产生休眠孢囊有关目前还远不清楚。我们的初步证据表明它们可能是产孢囊的种类。基于此，本项目将利用已有上述三种藻类纯培养首先在实验室诱导孢囊的形成和萌发，以探明其生活史过程和特征，同时运用形态学与分子探针相结合的手段检测藻华爆发典型海域的沉积物中是否存在它们的孢囊。项目的实施对认识上述几种重要藻华的形成、年际复发、地理扩散机制具有重要的理论意义，也为后续藻华预测与防控研究提供科学依据。

休眠孢囊已被证明在甲藻藻华的形成和地理扩散中起着非常关键的作用。我国自上个世纪九十年代起由甲藻尤其是米氏凯伦藻和东海原甲藻引起的赤潮发生频率和规模不断增大且有扩散趋势，给沿海经济造成巨量损失。世界广布种剧毒卡尔藻形成的赤潮最近也在我国出现且南北各海域都发现其分布。目前，这三种甲藻是否在它们的生活史中产生休眠孢囊以及休眠孢囊是否与它们引发的藻华在某一地区连年爆发且分布范围扩大有关尚不清楚。为此，本项目通过建立实验室纯培养，研究了米氏凯伦藻、东海原甲藻和剧毒卡尔藻的有性生殖过程、孢囊形成过程的细胞形态学变化、孢囊的萌发过程观察，还研究了温度、营养盐、颗粒添加物等环境因子对孢囊形成的影响。在课题执行期间，通过搭载基金委共享航次及自行组织的近海采样，获得了百余份中国近海沉积物样品，优化和建立了野外孢囊检测的FISH技术、单孢囊PCR检测技术和甲藻拷贝数、倍性的确定等；利用FISH、显微观察和单孢囊PCR结合的技术在野外沉积物中检测了米氏凯伦藻、东海原甲藻和剧毒卡尔藻休眠孢囊。最后，开展了中国近海沉积物中米氏凯伦藻、东海原甲藻和剧毒卡尔藻孢囊分布的拓展性研究。通过研究得出以下主要结论：（1）米氏凯伦藻、东海原甲藻和剧毒卡尔藻均具有有性生活史（有性交配细胞对、双纵鞭毛合子等证据），且能形成有性休眠孢囊；（2）营养限制能明显促进米氏凯伦藻和东海原甲藻休眠孢囊的形成，一定的温度（20°Ϲ）条件适于米氏凯伦藻孢囊的萌发；（3）FISH技术和单孢囊PCR技术证明在中国近海沉积物中存在这三种甲藻的休眠孢囊，其形态学特征与室内纯培养中发现的孢囊一致；（4）通过沉积物的孢囊浓缩和定量PCR检测，在我国渤海、黄海、东海和南海均检测到了米氏凯伦藻和剧毒卡尔藻孢囊较广泛的分布，但仅从东海检测到了东海原甲藻孢囊。这一结果与这三种甲藻在中国海域爆发藻华的历史纪录相一致。米氏凯伦藻、东海原甲藻和剧毒卡尔藻休眠孢囊的发现和在沉积物中的广泛存在的证明，为这三种藻华的种源、形成、年际复发、地理扩散提供了更深入的认识和机理性解释，也为后续基于孢囊分布和丰度的藻华预测预警及防控研究提供科学依据。