厚壳贻贝稚贝附着机理研究

Settlement of marine invertebrate larvae is cued by physical, chemical and biological properties associated with surface and surface properties. In contrast to many marine invertebrate larvae, mussel settlement is not permanent, and plantigrades of many species can detach and resettle in an alternative substrate. Understanding the mechanism of plantigrade settlement, will facilitate the manipulation of surfaces to selectivity deter or promote settlement of targeted marine invertebrate. In the present study, the effects of physical (temperature, light density and surface property), chemical (biofilms) and biological (gregariousness) cues on plantigrade settlement of the mussel Mytilus coruscus will be investigated. In order to clarify the nature of settlement cues of the biofilms, the activity of potential cue was evaluated after treatments with fixative agents, heat, ethanol and antibiotics. The inducing activities of conditioned water will be also investigated. The extracellular polymeric substances from biofilms will be purified by Gel Permeation Chromatography, their molecular weights can be determined by SDS-PAGE and the structure and function of these chemicals will be analyzed by amino acid sequence and mass spectrometry analysis. In addition, composition differences of the substances from marine bacteria can be analyzed through SDS-PAGE and two-dimensional electrophoresis. The finding is important to clarify the settlement mechanism of the mussel M. coruscus, in the field of biofouling and antifouling researches and aquaculture industry, and subsequently develop more environmentally friendly antifouling technologies.

厚壳贻贝与许多其他海洋无脊椎动物不同，附着不是永久性的。厚壳贻贝稚贝具有切断足丝、移动位置和进行附着的能力，而它的附着机理尚未清楚。本研究探讨物理诱因（温度、光照、附着基质表面性质）、化学诱因（微生物被膜）和生物诱因（同种个体）等对厚壳贻贝稚贝附着行为影响，解析这些因子在调控稚贝附着的协同和耦合作用机制。深入开展化学诱因研究，筛查调控稚贝附着的细菌微生物被膜或硅藻微生物被膜；通过凝胶液相色谱法进行分离纯化微生物被膜的代谢产物，检测分离的化合物对稚贝附着的诱导活性；通过SDA-PAGE法及双向电泳法分析代谢产物组成的差异性和推算分子量；通过氨基酸测序、飞行质谱法等手段查清具有活性的化学信息物质的结构和功能，本研究结果将为今后新型海洋防污技术的研发提供崭新的研究视野。

针对厚壳贻贝稚贝附着机制尚不清楚的问题，本项目从物理诱因、化学诱因和生物诱因等方面开展了对厚壳贻贝稚贝附着行为影响研究。我们发现环境因子主要通过调控微生物被膜中细菌密度、被膜厚度以及细菌的聚集情况等生物学特征，最终影响了稚贝的附着。研究发现光照强度和稚贝密度单一因素均显著影响稚贝的群聚性；温度单一因子对稚贝的群聚性无显著影响，但其与光照强度的两者间交互影响稚贝群聚性，同样的结论适用于温度和稚贝密度的相互影响；温度、光照强度以及稚贝密度三者的交互作用共同对稚贝群聚性产生显著影响。附着基质的表面润湿性、表面颜色、微貌结构等表面性质与厚壳贻贝稚贝附着相互关系研究证明，表面性质对海洋微生物被膜的细菌密度、硅藻密度、干重和叶绿素a等生物量产生影响；进一步通过高通量测序技术发现表面性质的改变影响生物被膜的细菌群落和硅藻群落结构，从而导致稚贝附着行为的改变；特别是纳米银、二氧化钛和碳纳米管等基质具有很好抗附着效果。通过对源于自然微生物被膜以及野生厚壳贻贝肠道中的细菌生物被膜的作用进行了调查，发现均对稚贝附着具有显著诱导活性；被膜诱导活性与细菌来源无关，但与微生物被膜密度显著相关；通过对假交替单胞菌属细菌生物被膜形成特性和对厚壳贻贝稚贝附着关系进行了探讨，发现在生物被膜形成过程中菌株由于过量黑色素的合成，导致细菌突变，表型产生改变；通过全基因组重测序技术和基因敲除技术，发现hmgA基因调控黑色素的产生，并最终影响和调控厚壳贻贝稚贝的附着。我们研究结果对今后阐明不同诱因，特别是生物被膜调控厚壳贻贝附着的分子机制具有重要学术价值，同时对于新型海洋防污技术的发展具有潜在应用价值。

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