中国科学院海洋研究所机构知识库

  平端深海偏顶蛤（Gigantidas platifrons曾用名：Bathymodiolus platifrons）是西北太平洋常见的深海大型化能营养生物，隶属于软体动物门、双壳纲、贻贝科、深海偏顶蛤亚科。在冲绳海槽的多处热液区，日本相模湾的冷泉区，以及我国南海冷泉区等多个还原性生境中均有其广泛分布，是这些热液/冷泉生态系统中优势的大型生物。平端深海偏顶蛤的最显著特点是以体内共生菌化能合成的有机物作为主要营养来源，因此其空间分布和数量变动都受制于热液、冷泉这些深海特殊的环境。由于深海调查采样较为困难，平端深海偏顶蛤的种群变动与补充机制尚缺乏系统认知。本文选取我国台西南福尔摩沙冷泉区作为研究区域，重点研究了该区域内平端深海偏顶蛤的繁殖生物学的相关问题，主要包括平端深海偏顶蛤的繁殖周期、性别比例、年龄组成、幼体的补充频率以及该密集种群的补充来源等；同时研究了平端深海偏顶蛤生活史中密不可分的内共生菌——甲烷氧化菌的获取途径，以及甲烷氧化菌在平端深海偏顶蛤稚贝生长过程中的分布转移模式；并且在非原位环境中对平端深海偏顶蛤进行人工繁殖尝试。以期为深入了解平端深海偏顶蛤的种群补充机制及环境适应策略提供基础理论和数据支撑。本研究获得的主要结果如下：

  台西南冷泉区的平端深海偏顶蛤绝大部分为雌雄异体，但存在极低频率的雌雄同体现象（＜0.76%）。平端深海偏顶蛤的繁殖是不连续的，存在周期变化，根据性腺指数（GI）的变化判断其每年下半年存在繁殖高峰。平端深海偏顶蛤的卵径和近海的贻贝科物种类似（约48.99–70.14μm），并且其稚贝的次生胚壳（ProdissoconchII，PII）远大于初生胚壳（Prodissoconch I，PI），因此推测其浮游幼体附着前需经历浮游营养型的发育过程。1年期回收的幼体捕获器中稚贝的壳长频率分布图及成体性腺发生周期数据表明其幼体补充频率为1周年内有1次补充高峰；群体简化基因组测序结果表明，平端深海偏顶蛤遗传结构世代间稳定，福尔摩沙冷泉区的群体具有稳定的种群结构，且主要以自我补充为主。

  平端深海偏顶蛤稚贝体内含有大量的内共生菌。通过稚贝的16s rDNA测序及稚贝宏基因测序结果表明，稚贝与成贝体内共生菌的类型相同，均为甲烷氧化菌（MOB），但是稚贝与成贝体内的共生菌空间分布模式及含菌细胞的状态存在差异。在平端深海偏顶蛤浮游幼体刚附着时或较小的稚贝体内，共生菌分布于稚贝全身器官组织的上皮细胞中，常见的有外套膜、鳃、足、肾脏等上皮细胞中。而在壳长约大于6mm的稚贝和成贝体内，共生菌的分布模式非常单一，均在鳃上皮的含菌细胞中分布。平端深海偏顶蛤性腺组织中不含有共生菌，因此在其繁殖发育过程中不会通过雌雄配子将共生菌传递给后代，每个世代均需通过从环境中重新获取甲烷氧化菌作为内共生菌，因此其共生菌的传递方式为水平传递。

  在对平端深海偏顶蛤的非原位长期蓄养过程中发现，鳃上皮含菌细胞中共生菌数量逐步减少，溶酶体含量降低。根据现有结果推测，由于宿主营养匮乏和胁迫响应条件下，溶酶体可能主动对含菌细胞中的甲烷氧化菌进行分解消化，过程中伴随着溶酶体含量先逐渐增多，甲烷氧化菌逐渐减少，当分解消化甲烷氧化菌之后，溶酶体随之在细胞内消失，使细胞呈现空泡化。这种能量缺乏条件下含菌细胞中溶酶体与共生菌的动态变化，是平端深海偏顶蛤在共生互作中的通过溶酶体主导消化和调控共生菌的直观体现。在循环水蓄养系统中对平端深海偏顶蛤的非原位蓄养时间长达388天，期间利用贝类繁殖的常规手段对其进行人工催产，遗憾的是无有活性的幼体产生，可能是由于环境差异巨大，贝体并没有处于最佳的生理状态导致。

    The genus Gigantidas belongs to one of the subfamilies, Bathymodiolinae, in the family Mytilidae of molluscan Bivalvia. The deep-sea mussel Gigantidas platifrons (previously named Bathymodiolus platifrons), widely distributed in both hydrothermal vents in the Okinawa Trough, and cold seeps in Sagami Bay of Japan and the Taixinan Formosa of the South China Sea (SCS), is one of the dominant macrofauna in these extreme ecosystems. The most significant characteristics of the deep-sea mussel G. platifrons is that they rely primarily on chemoautotrophic endosymbionts for nutrition and energy. Thus, the mussel’s spatial distribution and population variation are subject to the surrounding environment they habitat. Because of the difficulty in deep-sea survey sampling, there is no systematic understanding of the population dynamics and supplementation mechanism of the deep-sea mussel G. platifrons. In this paper, the reproductive biology of the deep-sea mussel G. platifrons from the Formosa cold seep area in southwest Taiwan was mainly studied. In details, the study contents included the reproductive characteristics, the frequency of population replenishment and the population genetic structures of G. platifrons, the acquisition ways of endosymbiotic methane-oxidizing bacteria (MOB) and the distribution and transfer model of symbiotic bacteria in the growth of G. platifrons. The main results are as follows:

    G. platifrons is a functionally dioecious species given the extremely low (0.76%) percentage of hermaphrodites found in the sampled population. The reproduction of G. platifrons is discontinuous with the maturity peak around the latter half of the year. Given the small oocyte size (48.99–70.14 μm) comparable to coastal mussels, and the shell length of the second Prodissoconch (PII) was much longer than the first Prodissoconch (PI), we proposed that G. platifrons developed via a free-living, planktotrophic larvae stage before its settlement. The replenishment of juvenile G. platifrons is once a year through one peak in the plot of juvenile shell length frequency distribution in one year. The supplementary source of the dense population was reflected by the 2b-RAD genome sequences of different shell length populations, and the results showed that the genetic structure of G. platifrons population is stable in the intergenerational and the mussel population mainly is self-replenishing in Formosa cold seep.

    Research on the distribution and transfer model of symbiotic bacteria showed that there was no symbiotic bacteria in gonad tissue of both male and female mussels, but symbiotic bacteria were found in majority organs of juvenile mussels, suggesting that G. platifrons should obtain symbiotic bacteria by horizontal transmission from environment rather than vertical transmission by gametes. Furthermore, the bacteriocytes were widely distributed in the epithelial cells of gill, mantle, kidney and foot tissues of the juvenile mussels, but limited in the gill epithelial cells of adult mussel. These results confirmed an ontogenetic shift in symbiont colonization from indiscriminate infection of a wide range of epithelial tissues in early life stages to spatially restricted colonization of gills in later developmental stages in G. platifrons. Further research on the translocation mechanism of symbiont with G. platifrons development is required.

    During the long-term on-board culture experiment of G. platifrons, the abundance of the symbionts was found to be gradually decreased along with the content of lysosomes. We proposed that the lysosomes functioned to digest symbionts and eliminate the senile or premortal symbionts actively, but when there were no symbiotic bacteria to digest and the lysosomes would gradually disappear. This conclusion is further supported by the suppressed lysosomes after the loss of the symbionts. Although the deep-sea mussel G. platifrons could survive for more than one year in our circulating water systems, we could not breed the larva of G. platifrons, presumably because the mussels were not in their best physiological state in the on-board culture system.

第1章  研究现状与进展... 1

1.1  深海... 1

1.2  深海中的还原性生态系统... 2

1.2.1  热液生态系统（Hydrothermal vent）... 2

1.2.2  冷泉生态系统（Cold seep）... 5

1.2.3  鲸落生态系统（Whale-fall）... 7

1.2.4  沉木生态系统（Wood-fall）... 8

1.3  共生体系中代表性物种的繁殖特点与种群结构的研究... 9

1.3.1  管状蠕虫的繁殖生物学与种群结构研究... 10

1.3.2  腹足纲生物种群结构研究... 12

1.3.3  巨蛤种群结构研究... 13

1.3.4  深海偏顶蛤亚科的种群结构研究... 15

1.4  深海偏顶蛤亚科代表性物种共生关系研究... 24

1.4.1  含有甲烷氧化菌的深海偏顶蛤... 25

1.4.2  含有硫氧化菌的深海偏顶蛤... 26

1.4.3  深海偏顶蛤与内共生菌的关系... 26

1.4.4  共生系统的进化... 27

1.5  研究内容和意义... 30

第2章  平端深海偏顶蛤的繁殖生物学分析... 33

2.1  前言... 33

2.2  材料与方法... 34

2.2.1  样品采集... 34

2.2.2  样品处理... 36

2.2.3  数据处理... 40

2.3  结果... 43

2.3.1  平端深海偏顶蛤配子发生与繁殖特点... 43

2.3.2  平端深海偏顶蛤种群补充频率的推测... 52

2.3.3  平端深海偏顶蛤种群遗传结构... 54

2.4  讨论... 59

2.4.1  平端深海偏顶蛤性腺解剖特征... 59

2.4.2  深海偏顶蛤亚科物种的繁殖特征... 59

2.4.3  平端深海偏顶蛤的种群补充特征... 63

2.5  小结... 64

第3章  平端深海偏顶蛤繁殖发育过程中共生菌分布转移研究    65

3.1  前言... 65

3.2  材料与方法... 65

3.2.1  样品采集... 65

3.2.2  样品保存... 67

3.2.3  组织学观察与荧光原位杂交（FISH）分析... 67

3.2.4  电镜样品处理与观察... 68

3.2.5  稚贝与成贝DNA提取及16S rDNA序列与宏基因组分析... 68

3.3  结果... 70

3.3.1  平端深海偏顶蛤稚贝与成贝体内共生菌鉴定与分析... 70

3.3.2  平端深海偏顶蛤体内共生菌分布... 73

3.4  讨论... 86

3.4.1  平端深海偏顶蛤共生菌分布与转移... 87

3.4.2  平端深海偏顶蛤共生菌的获得... 89

3.5  小结... 89

第4章  平端深海偏顶蛤人工培养及繁殖研究    91

4.1  前言... 91

4.2  材料与方法... 92

4.2.1  样品采集... 92

4.2.2  样品处理... 92

4.2.3  人工繁殖... 93

4.3  实验结果... 95

4.3.1  长期蓄养过程中鳃丝超微结构观察... 95

4.3.2  人工催产结果... 100

4.4  讨论... 101

4.4.1  共生菌在长期蓄养中的变化... 101

4.4.2  平端深海偏顶蛤人工催产失败原因分析... 103

4.4.3 长期蓄养系统的使用检测及平端深海偏顶蛤死亡分析... 104

4.5  小结... 105

第5章  总结与展望... 107

5.1  主要研究结论... 107

5.2  本文的创新点... 108

5.3  展望... 108

参考文献... 110

致  谢... 127

作者简历及攻读学位期间发表的学术论文与研究成果    129