|关键词||寄生性甲藻 有害藻华 寄生感染 遗传多样性 体外培养|
1. 针对现有检测阿米巴藻感染细胞方法的不足，本论文将组织学及病理检查最常用的染色方法——苏木精-伊红染色技术用于阿米巴藻寄生感染的检测。通过鉴定阿米巴藻特征性的“蜂巢”状的多核体结构，本方法可以准确的检测阿米巴藻在宿主甲藻内的寄生感染。该方法简单易行，对操作技术和经验依赖较小，整个染色过程所需时间不超过3 h（最多20个样品/批次）。通过比例换算，苏木精-伊红染色方法可以比较准确的计算出阿米巴藻的实际感染率。总之，苏木精-伊红染色技术是一种可靠、便捷检测阿米巴藻感染细胞的方法，满足了大规模海洋生态调查的需求。
2. 长江口及其邻近海域2015年2月~2016年1月的10次调查期间，该海域浮游甲藻群落结构相对稳定，共检出浮游甲藻类39种，膝沟藻目在调查区域处于绝对优势，其次为裸甲藻目和多甲藻目。浮游甲藻年际平均丰度为6.62 × 105 cells/L，变动范围为9.06 × 103~6.10 × 106 cells/L。调查海域阿米巴藻宿主甲藻种类繁多，14种浮游甲藻被寄生感染。该海域阿米巴藻的宿主种类及感染率表现出明显的季节差异，夏、秋两季阿米巴藻宿主种类和感染率明显高于冬、春两季。6月为全年最高峰，11种甲藻被阿米巴藻感染，最高感染率为5.77 %。
3. 阿米巴藻在我国沿海主要海湾中广泛存在，分布较为复杂且存在明显的遗传多样性。9种浮游甲藻被寄生感染，感染率约为0.03~2.5 %。Amoebophrya sp. ex Gymnodinium instriatum和Amoebophrya sp. ex Karlodinium micrum在我国近岸水体出现频率最高，温州湾是含有阿米巴藻最丰富的海湾。我国阿米巴藻18S rDNA序列聚为三大支，不同地理种群间的样本呈现散在分布，没有形成明显的地理格局分布；所得样品涵盖13种阿米巴藻株系且不同株系间存在显著遗传差异，种间遗传距离在0.0252~1.5745之间，平均遗传距离为0.5672。
4. 通过建立阿米巴藻与其宿主甲藻红色赤潮藻的共培养体系，进行实验室感染实验，结果表明：Amoebophrya sp. ex Akashiwo sanguinea具有较强的感染力和较短的世代时间，能够显著抑制宿主甲藻的生长和增殖。孢子的感染率随着孢子-宿主比例的升高逐渐提高。孢子-宿主比为20：1时，孢子的感染率为91 ± 2.10 %；孢子-宿主比为40：1时，感染率逐渐趋于饱和。该阿米巴藻株系的整个世代时间约为58 ± 1.2 h，孢子的体外存活时间为78 h，且随着离体时间的延长，孢子的感染能力逐渐降低。Amoebophrya sp. ex A. sanguinea孢子不能感染其他甲藻类宿主，表明该株系具有较高的感染特异性。
|其他摘要||The parasitic dinoflagellate in the genus of Amoebophrya infects broad range hosts of planktonic dinoflagellates, ciliates and radiolarians in eutrophic and oligotrophic waters wordwidely. Its ecological significance was gradually recognized in recent years by researchers around the word. Lately, scattered evidences indicated that this type of parasitic dinoflagellates is widely distributed in the coastal waters of China; however, we have very limited understanding on the biological characteristics and ecological roles of Amoebophrya in the coastal waters of China due to the difficulty of establishing in vitro culture in laboratory and rare research conducted in field. Thus, this dissertation systematically explored the distribution and genetic diversity of Amoebophrya in the coastal waters of China through large-scale ecological survey. A typical strain of the parasites was isolated and cultured in laboratory, its infectivity and effects to host dinoflagellates were further investigated to assess the impacts of Amoebophrya infections during dinoflagellate blooms. This research will facilitate our understanding on the role of Amoebophrya during the succession of harmful algal blooms. The major findings were listed as following:|
1. The hematoxylin and eosin (H & E) staining assay was successfully adopted to detect Amoebophrya infection in planktonic dinoflagellates. The H & E staining assay provided reliable diagnosis of Amoebophrya infection by identifying the characteristic “beehive” of the multinucleate trophonts. The H & E staining assay is easy to use, that can be routinely performed within 3 h (up to 20 samples/batch). With suitable calculation, the modified H & E assay can be applied to assess the prevalence of Amoebophrya infection in planktonic dinoflagellates. Overall, the H & E staining assay offered reliable and convenient detection of Amoebophrya infection in planktonic dinoflagellates, meeting the demands of large-scale ecological surveys.
2. During the investigation period (from February 2015 to January 2016, 10 cruises), planktonic dinoflagellate community structure was relatively stable in the Changjiang estuary and adjacent sea and totally 39 dinoflagellate taxa were identified. The species richness of Gonyaulacales ranked first, followed by Gymnodiniales and Peridiniales. The average yearly abundance of planktonic dinoflagellates was 6.62 × 105 cells/L, ranged from 9.06 × 103 to 6.10 × 106 cells/L. Fourteen free-living marine dinoflagellates served as hosts for Amoebophrya in the investigated area. The host dinoflagellate species and the prevalence of Amoebophrya infection had obvious seasonal variation, of which summer and fall was obviously higher than spring and winter. The primary peak occurred in June, when eleven species of dinoflagellates were infected, with prevalence up to 5.77 %.
3. Amoebophrya was widely spread in coastal waters of China, which showed a complex distribution pattern and high genetic diversity. Nine species of dinoflagellates were found to be infected by Amoebophrya spp., with prevalence varied between 0.03 and 2.5 %. Amoebophrya sp. ex Gymnodinium instriatum and Amoebophrya sp. ex Karlodinium micrum were commonly observed in the coastal waters of China and Wenzhou Bay had the most Amoebophrya species. All Amoebophrya spp. 18S rDNA gene sequences formed into three major clades. However, sequences of the same area did not form a single cluster, indicating that Amoebophrya populations had not form a pattern of obvious geographical distribution. Thirteen species of Amoebophrya spp. were identified in the water column, of which the Kimura-2-parameter genetic distance ranged between 0.0252 and 1.5745, with a mean of 0.5672, indicating high genetic diversity among Amoebophrya spp..
4. An Akashiwo sanguinea-Amoebophrya sp. in vitro culture was established in laboratory, the biological interactions of Amoebophrya sp. were explored. Amoebophrya sp. ex A. sanguinea was aggressive and short in duration, which can induce mortality to its hosts, preventing its normal growth and proliferation. The prevalence of parasitic infection increased sharply with increasing dinospore: host ratios. The prevalence of parasitic infection was 91 ± 2.10 % when the dinospore: host cell was 20:1, becoming saturated at the dinospore: host ratio of 40: 1. The life span of Amoebophrya sp. ex. A. sanguinea was approximately 58 ± 1.2 hours. The dinospores can survive up to 78 hours in environment waters, but gradually lose their infectivity. Amoebophrya sp. ex. A. sanguinea cannot infect other dinoflagellate species, suggesting that the parasite has a high preference for specific host, with a single parasitic clade preferentially infecting a single dinoflagellate host species.
In conclusion, this dissertation presented the first comprehensive study on Amoebophrya spp. infections in planktonic dinoflagellates in the coastal waters of China and explored its primary ecological roles through series of field surveys and laboratory studies. The major finding of this dissertation will contribute to better understanding on the impact of parasitism by Amoebophrya to its host dinoflagellate populations as well as the ecological significance of the parasitic dinoflagellate in coastal environments of China.
|陈田田. 阿米巴藻（Amoebophrya）在我国近海的分布、多样性及寄生作用初步研究[D]. 北京. 中国科学院大学,2017.|
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