微生物膜培养及其对大珠母贝幼虫附着的影响
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摘要
本研究以珊瑚石为培养载体,分析海水中无机氮与活性磷酸盐的变化、微生物膜的形态和底栖硅藻的种类,并比较附苗器上微生物膜培养时间对大珠母贝幼虫附着率的影响。结果显示:添加营养盐时可溶性无机氮和活性磷酸盐质量浓度均随时间逐渐下降,至16 d基本稳定,分别较初始值减少了99.11%和84.78%,而未添加营养盐时可溶性无机氮和活性磷酸盐质量浓度均无明显变化;添加营养盐的海水中培养了17 d的珊瑚石表面附着的硅藻包括菱形藻属、圆筛藻属、双眉藻属等,与球状细菌、胞外分泌物等共同形成微生物膜;未添加营养盐的海水中培养了17 d的珊瑚石表面附着的硅藻主要有异极藻属、胸隔藻属和曲壳藻属等,微生物膜结构相对疏松,珊瑚石孔洞间几乎没有微生物膜;在添加营养盐的微生物膜培养水体中处理4 d的附苗器48 h大珠母贝附苗率为25.78%,较对照组提升了9.87%,且显著高于处理2 d和3 d的附苗器附苗率。因此,自然海水中可以培育形成底栖硅藻和细菌微生物膜,促进大珠母贝幼虫附着。
In this study, several events, including the variation of inorganic nitrogen and active phosphate of seawater, the structure of biofilm and species of benthic diatom, were analyzed based on the culture carrier of corallite. Besides, settlement ratios of Pinctada maxima larvae were also compared among seedling apparatus disposed different times for biofilms culture. Results showed that concentrations of both the inorganic nitrogen and active phosphate decreased gradually until 16 d(with a decrease ratio of 99.11% and 84.78%, respectively) when nutrient was added, whereas no obvious change of concentrations were observed for control without nutrient added. Besides, biofilm consisted of spherical bacteria, extracellular secretion and diatom(such as Nitzschia, Synedra,Stephanodiscus, Amphora) on the surface of the corallites in 17 d for experiment groups of nutrient added seawater,while biofilm was loose and even scarce inside holes of corallite for control with different diatom from experiment groups, such as Gomphonema, Mastogloia, Achnanthes. The settlement ratio of Pinctada maxima larvae at 48 h was25.78% for seedling apparatus which was cultured to form biofilm for 4 d in nutrient adding seawater. It was not only obvious higher(9.87%) than that of control without biofilm culture but also higher than that of experiment groups which seedling apparatus was cultured 2 d or 3 d. Therefore, the biofilms constituted of diatom and bacteria might be developed from natural seawater, and the biofilms could induce settlement of Pinctada maxima larvae.
In this study, several events, including the variation of inorganic nitrogen and active phosphate of seawater, the structure of biofilm and species of benthic diatom, were analyzed based on the culture carrier of corallite. Besides, settlement ratios of Pinctada maxima larvae were also compared among seedling apparatus disposed different times for biofilms culture. Results showed that concentrations of both the inorganic nitrogen and active phosphate decreased gradually until 16 d(with a decrease ratio of 99.11% and 84.78%, respectively) when nutrient was added, whereas no obvious change of concentrations were observed for control without nutrient added. Besides, biofilm consisted of spherical bacteria, extracellular secretion and diatom(such as Nitzschia, Synedra,Stephanodiscus, Amphora) on the surface of the corallites in 17 d for experiment groups of nutrient added seawater,while biofilm was loose and even scarce inside holes of corallite for control with different diatom from experiment groups, such as Gomphonema, Mastogloia, Achnanthes. The settlement ratio of Pinctada maxima larvae at 48 h was25.78% for seedling apparatus which was cultured to form biofilm for 4 d in nutrient adding seawater. It was not only obvious higher(9.87%) than that of control without biofilm culture but also higher than that of experiment groups which seedling apparatus was cultured 2 d or 3 d. Therefore, the biofilms constituted of diatom and bacteria might be developed from natural seawater, and the biofilms could induce settlement of Pinctada maxima larvae.
引文
Bao W.Y.,Yang J.L.,Satuito C.G.,and Kitamura H.,2007,Larval metamorphosis of the mussel Mytilus galloprovincialis in response to Alteromonas sp.1:evidence for two chemical cues?Marine Biology,152(3):657-666
Cao S.,2013,Research on adhesion mechanism and antifouling technologies of a marine,Dissertation for Ph.D.,Tsinghua University,Supervisor:Wang J.D.,pp.2-3(曹杉,2013,海洋底栖硅藻附着机理及其防污技术研究,博士学位论文,清华大学,导师:汪家道,pp.2-3)
Cui Y.L.,Gu Z.F.,Zheng X.,and Wang A.M.,2015,Comparison of five biofilter media in ammonia-removing efficiency in the recirculation aquaculture system,Redai Shengwu Xuebao(Journal of Tropical Biology),6(3):235-241(郑兴,王爱民,2015,5种滤料在循环水养殖系统中去除氨氮效果的比较,热带生物学报,6(3):235-241)
Dobretsov S.V.,1999,Effects of macroalgae and biofilm on settlement of blue mussel(Mytilus edulis I.)larvae,Biofouling,14(2):153-165
Fu M.H.,Chen X.L.,and Yan G.H.,2015,Effect of light and different forms of nitrogen on the activity of glutamine synthetase in water hyacinth(Echhornia crassipes),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(3):635-639(傅明辉,陈肖丽,严国花,2015,光照和不同氮素对水葫芦谷酰胺合成酶活性的影响,基因组学与应用生物学报,34(3):635-639)
Ganesan A.M.,Alfaro A.C.,Brooks J.D.,and Higgins C.M.,2010,The role of bacterial biofilms and exudates on the set tlement of mussel(Perna canaliculus)larvae,Aquaculture,306(1-4):388-392
Gu Z.F.,Wang A.M.,Liang X.C.,Lin D.D.,Wang Y.,and Shi Y.H.,2012,A spat appurtenant device for Pteria penguin,China Patent,CN201010203279.5(顾志峰,王爱民,梁晓春,林冬冬,王嫣,石耀华,2012,一种企鹅珍珠贝稚贝附苗器,中国专利,CN201010203279.5)
Hadfield M.G.,and Paul V.J.,2001,Natural chemical cues for settlement and metamorphosis of marine-invertebrate larvae,Marine Chemical Ecology,pp.431-460
Hoagland K.D.,Rosowski J.R.,Gretz M.R.,and Roemer S.C.,1993,Diatom extracellular polymeric substances:function,fine structure,chemistry,and physiology,Journal of Phycology,29(5):537-566
Jiang Y.P.,and He M.X.,2009,Survey of the studies on Pinctada maxima(Jameson),Haiyang Kexue(Marine sciences),33(2):92-96(姜因萍,何毛贤,2009,大珠母贝的研究概况,海洋科学,33(2):92-96)
Jin D.X.,Cheng Z.D.,Lin J.M.,and Liu S.C.,eds.,1982,Marine benthic diatoms in China,China Ocean Press,Beijing,Chi na,pp.198(金德祥,程兆第,林均民,刘师成,编著,1982,中国海洋底栖硅藻类(上卷),海洋出版社,中国,北京,pp.93,162,175,198)
Krammer K.,and Lange-Bertalot H.,eds.,Liu W.,Zhu Y.S.,and Huang Y.Y.,trans.,Bacillariophyceae,Sun Yat-Sen Univer sity Press,Guangzhou,China,pp.35,107,138(克拉默,兰格-贝尔塔洛,编著,刘威,朱远生,黄迎艳,主译,2012,欧洲硅藻鉴定系统,中山大学出版社,中国,广州,pp.35,107,138)
Lai Z.N.,Shi C.B.,and Wu S.Q.,1998,Study on the concentration of nitrogen and phosphorus and the growth state of algae in fish ponds,Zhongshan Daxue Xuebao Luncong(Supplement to the Journal of Sun Yatsen University),(6):65-69(赖子尼,石存斌,吴淑勤,1998,池塘氮、磷浓度及藻相水色的研究,中山大学学报论丛,(6):65-69)
Lasa I.,2006,Towards the identification of the common features of bacterial biofilm development,International Microbiology,9(1):21-28
Sun J.C.,2009,A preliminary study on larval settlement and metamorphosisof the sea cucumber(Apostichopus japonicus)in response to benthic diatoms,Thesis for M.S.,Ocean University of China,Supervisor:Ma S.,and Bao W.Y.,pp.27(孙景春,2012,底栖硅藻诱导仿刺参(Apostichopus japonicus)幼体附着变态的初步研究,硕士学位论文,中国海洋大学,导师:马甡,包卫洋,pp.27)
Stoodley P.,Sauer K.,Davies D.G.,and Costerton J.W.,2002,Biofilms as complex differentiated communities,Annual Review of Microbiology,56(3):187-209
Su Z.X.,Huang L.M.,Yan Y.,and Li H.X.,2007,The effect of different substrates on pearl oyster Pinctada martensii(Dunker)larvae settlement,Aquaculture,271(1-4):377-383
Wang R.C.,and Wang Z.P.,eds.,1998,Marine shellfish culture,Qingdao Ocean University Press,Qingdao,China,pp.149-202(王如才,王昭萍,编著,1998,海水贝类养殖学,青岛海洋大学出版社,中国,青岛,pp.149-202)
Wieczorek S.K.,and Todd C.D.,1998,Inhibition and facilitation of settlement of epifaunal marine larvae by microbial biofilm cues,Biofouling,12(1-3):81-118
Xie Y.K.,Lin B.P.,Hu Y.P.,and Xu Z.J.,eds.,1985,Shellfish and pearls of Pinctada maxima,China Ocean Press,Beijing,China,pp.46-47(谢玉坎,林碧萍,胡亚平,许志坚,编著,1985,大珠母贝及其养殖珍珠,海洋出版社,中国,北京,pp.46-47)
Xiong D.Q.,Du X.M.,Tang W.H.,Huang M.X.,Gu Q.B.,Guo L.J.,and Li F.S.,2005,Nitrogen distribution in the water of the Haihe river mainstream and estuary seawater and its relationship with water dissolved oxygen levels,Research of Environmental Sciences,18(3):1-8
Xu Z.J.,and Li K.K.,1980,A preliminary study on the rearing of artificially fertilized larvae of Pinctada maxima(Jameson),Shuichan Xuebao(Journal of Fisheriesof China),4(3):295-301(许志坚,李孔开,1980,大珠母贝人工育苗培育的初步研究,水产学报,4(3):295-301)
Zhao B.,Zhang S.,and Qian P.Y.,2003,Larval settlement of the silver-or goldlip pearl oyster Pinctada maxima(Jameson)in response to natural biofilms and chemical cues,Aquaculture,220(1):883-901
Cao S.,2013,Research on adhesion mechanism and antifouling technologies of a marine,Dissertation for Ph.D.,Tsinghua University,Supervisor:Wang J.D.,pp.2-3(曹杉,2013,海洋底栖硅藻附着机理及其防污技术研究,博士学位论文,清华大学,导师:汪家道,pp.2-3)
Cui Y.L.,Gu Z.F.,Zheng X.,and Wang A.M.,2015,Comparison of five biofilter media in ammonia-removing efficiency in the recirculation aquaculture system,Redai Shengwu Xuebao(Journal of Tropical Biology),6(3):235-241(郑兴,王爱民,2015,5种滤料在循环水养殖系统中去除氨氮效果的比较,热带生物学报,6(3):235-241)
Dobretsov S.V.,1999,Effects of macroalgae and biofilm on settlement of blue mussel(Mytilus edulis I.)larvae,Biofouling,14(2):153-165
Fu M.H.,Chen X.L.,and Yan G.H.,2015,Effect of light and different forms of nitrogen on the activity of glutamine synthetase in water hyacinth(Echhornia crassipes),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(3):635-639(傅明辉,陈肖丽,严国花,2015,光照和不同氮素对水葫芦谷酰胺合成酶活性的影响,基因组学与应用生物学报,34(3):635-639)
Ganesan A.M.,Alfaro A.C.,Brooks J.D.,and Higgins C.M.,2010,The role of bacterial biofilms and exudates on the set tlement of mussel(Perna canaliculus)larvae,Aquaculture,306(1-4):388-392
Gu Z.F.,Wang A.M.,Liang X.C.,Lin D.D.,Wang Y.,and Shi Y.H.,2012,A spat appurtenant device for Pteria penguin,China Patent,CN201010203279.5(顾志峰,王爱民,梁晓春,林冬冬,王嫣,石耀华,2012,一种企鹅珍珠贝稚贝附苗器,中国专利,CN201010203279.5)
Hadfield M.G.,and Paul V.J.,2001,Natural chemical cues for settlement and metamorphosis of marine-invertebrate larvae,Marine Chemical Ecology,pp.431-460
Hoagland K.D.,Rosowski J.R.,Gretz M.R.,and Roemer S.C.,1993,Diatom extracellular polymeric substances:function,fine structure,chemistry,and physiology,Journal of Phycology,29(5):537-566
Jiang Y.P.,and He M.X.,2009,Survey of the studies on Pinctada maxima(Jameson),Haiyang Kexue(Marine sciences),33(2):92-96(姜因萍,何毛贤,2009,大珠母贝的研究概况,海洋科学,33(2):92-96)
Jin D.X.,Cheng Z.D.,Lin J.M.,and Liu S.C.,eds.,1982,Marine benthic diatoms in China,China Ocean Press,Beijing,Chi na,pp.198(金德祥,程兆第,林均民,刘师成,编著,1982,中国海洋底栖硅藻类(上卷),海洋出版社,中国,北京,pp.93,162,175,198)
Krammer K.,and Lange-Bertalot H.,eds.,Liu W.,Zhu Y.S.,and Huang Y.Y.,trans.,Bacillariophyceae,Sun Yat-Sen Univer sity Press,Guangzhou,China,pp.35,107,138(克拉默,兰格-贝尔塔洛,编著,刘威,朱远生,黄迎艳,主译,2012,欧洲硅藻鉴定系统,中山大学出版社,中国,广州,pp.35,107,138)
Lai Z.N.,Shi C.B.,and Wu S.Q.,1998,Study on the concentration of nitrogen and phosphorus and the growth state of algae in fish ponds,Zhongshan Daxue Xuebao Luncong(Supplement to the Journal of Sun Yatsen University),(6):65-69(赖子尼,石存斌,吴淑勤,1998,池塘氮、磷浓度及藻相水色的研究,中山大学学报论丛,(6):65-69)
Lasa I.,2006,Towards the identification of the common features of bacterial biofilm development,International Microbiology,9(1):21-28
Sun J.C.,2009,A preliminary study on larval settlement and metamorphosisof the sea cucumber(Apostichopus japonicus)in response to benthic diatoms,Thesis for M.S.,Ocean University of China,Supervisor:Ma S.,and Bao W.Y.,pp.27(孙景春,2012,底栖硅藻诱导仿刺参(Apostichopus japonicus)幼体附着变态的初步研究,硕士学位论文,中国海洋大学,导师:马甡,包卫洋,pp.27)
Stoodley P.,Sauer K.,Davies D.G.,and Costerton J.W.,2002,Biofilms as complex differentiated communities,Annual Review of Microbiology,56(3):187-209
Su Z.X.,Huang L.M.,Yan Y.,and Li H.X.,2007,The effect of different substrates on pearl oyster Pinctada martensii(Dunker)larvae settlement,Aquaculture,271(1-4):377-383
Wang R.C.,and Wang Z.P.,eds.,1998,Marine shellfish culture,Qingdao Ocean University Press,Qingdao,China,pp.149-202(王如才,王昭萍,编著,1998,海水贝类养殖学,青岛海洋大学出版社,中国,青岛,pp.149-202)
Wieczorek S.K.,and Todd C.D.,1998,Inhibition and facilitation of settlement of epifaunal marine larvae by microbial biofilm cues,Biofouling,12(1-3):81-118
Xie Y.K.,Lin B.P.,Hu Y.P.,and Xu Z.J.,eds.,1985,Shellfish and pearls of Pinctada maxima,China Ocean Press,Beijing,China,pp.46-47(谢玉坎,林碧萍,胡亚平,许志坚,编著,1985,大珠母贝及其养殖珍珠,海洋出版社,中国,北京,pp.46-47)
Xiong D.Q.,Du X.M.,Tang W.H.,Huang M.X.,Gu Q.B.,Guo L.J.,and Li F.S.,2005,Nitrogen distribution in the water of the Haihe river mainstream and estuary seawater and its relationship with water dissolved oxygen levels,Research of Environmental Sciences,18(3):1-8
Xu Z.J.,and Li K.K.,1980,A preliminary study on the rearing of artificially fertilized larvae of Pinctada maxima(Jameson),Shuichan Xuebao(Journal of Fisheriesof China),4(3):295-301(许志坚,李孔开,1980,大珠母贝人工育苗培育的初步研究,水产学报,4(3):295-301)
Zhao B.,Zhang S.,and Qian P.Y.,2003,Larval settlement of the silver-or goldlip pearl oyster Pinctada maxima(Jameson)in response to natural biofilms and chemical cues,Aquaculture,220(1):883-901