组胺调控污损动物藤壶幼虫附着变态的作用研究

The marine biofouling and the widely applied toxic anti-fouling compounds brought serious damages to environments, economy, and human health. The developments of new anti-fouling technologies need accurate working mechanism urgently, since they should be no longer based on chemical toxicities. In the preliminary studies, the involvement of histamine in signaling transduction during the larvae settlement and metamorphosis of a typical biofouling species of barnacle was found. Based on this finding, this project intends to clarify the linkage of histamine and its receptors with the larval nervous system development and the larval capacities of signal perception or transmission, by the means of quantitative analysis and immunofluorescence labeling. By the exogenous enhancers and specific blockers of histamine signaling in barnacle larvae, tracking PKA and PKC expression, and following the changes of the second messagers including IP3, cAMP, cGMP, DAG, and free Ca2+, the downstream signaling pathways of histamine will be confirmed. Through the analysis of cell apoptosis and proliferation, mucus releasing, and Ca2+ absorption/accumulation, histamine regulatory mechanisms on barnacle larval metamorphosis will be investigated. Treated by the specific signal enhancers/blockers of neurotransmitters (including histamine) in sequence, the changes of signaling strength and the effects on barnacle larval settlement and metamorphosis will be demonstrated. The key sites sensitive to signaling interferences during larval settlement and metamorphosis will be located. In addition, the presence, signal strength, and the guidance to barnacle larvae of histamine in different biofilms will be studied to verify whether biofilm could guide larval settlement by the means of histamine. The results of histamine signaling pathways and regulatory mechanisms will provide new working mechanisions as targets for the non-toxic antifouling technologies.

海洋生物污损和现行毒性抗污损化合物均带来严重危害，非毒性抗污损技术的发展急需明确起效机制。前期关于典型污损动物藤壶幼虫附着变态的研究发现组胺参与该过程的信号传递。本项目在此基础上，通过定量分析与免疫荧光标记，阐明组胺及其受体与幼虫神经发育、信号感受与传导能力的关系；对组胺信号进行外源增强或专一性阻断，跟踪藤壶幼虫第二信使IP3、DAG、cAMP、cGMP、游离Ca2+等的变化，结合PKA、PKC表达量，确定组胺的信号途径；分析细胞凋亡与增生、黏胶释放、Ca2+吸收与富集等的变化，确定组胺调控幼虫附着变态的机制。针对包括组胺在内的各神经递质信号，逐一进行专一性阻断或增强，追溯下游信号强度和幼虫附着和变态的情况，寻找可干扰的敏感环节。通过分析不同生物膜中组胺的有无、信号强度、对藤壶幼虫的引导作用，验证生物膜可否通过组胺指引幼虫附着。发现的信号途径和调控机制可为非毒性抗污损技术提供新干扰靶标。

项目研究在前期关于典型污损动物藤壶幼虫附着变态的研究基础上，通过定性定量分析、免疫荧光标记、附着变态生物测试、转录组分析、以及蛋白表达水平、第二信使水平和酶活性水平分析，阐明了组胺及其受体与幼虫神经发育、信号感受与传导能力密切相关。利用组胺进行外源信号增强，利用抗组胺试剂进行专一性阻断，分析了藤壶幼虫各第二信使水平、PKA和PKC表达水平、差异基因表达、胞凋亡与增生、黏液蛋白等的变化与差异，推断出幼虫中枢神经系统的组胺受体是调控幼虫附着变态的重要位点；IP3极可能是藤壶幼虫附着变态体内信号传导的第二信使；组胺通过调节能量代谢蛋白AdipoR的表达，提升AMPK水平，从而控制幼虫脂质能量代谢，帮助幼虫尽快附着。另外，项目研究发现生物膜EPS单糖组成影响幼虫的附着，其附着效果和硅藻生物膜及硅藻培养液中的组胺和若干单糖信号有关，这些信号对藤壶幼虫的附着变态有促进和引导作用，是底栖硅藻生物膜对藤壶幼虫的引导作用的重要载体。