【作者】 周立明；

【导师】 唐学玺；

【作者基本信息】 中国海洋大学 ， 生态学， 2008， 博士

【摘要】 日益严重的臭氧层侵蚀与破坏是当今最引入注目的全球性环境变化之一。臭氧层的减薄使得到达地面的紫外线，尤其是对生物具严重损伤作用的紫外线B波段（UV-B）的辐射增强，从而引起生物从生态系统、群落、种群、个体、细胞以及分子各个层次水平上发生变化。进而对全球产生明显的生态学和生物学效应。同时，随着沿海水域富营养化的加剧，赤潮频繁爆发已成为世界范围内的海洋生态灾害。自上世纪90年代以来，我国赤潮的发生呈现出“大范围、高频率、多种类、快节奏”的特点，并有连年增加的态势，这对原本已经非常严峻的近岸水域生态环境和沿岸水产业的可持续发展造成了极大的威胁。采用切实有效的赤潮防治措施成为赤潮研究的重要内容之一。由于赤潮防治的物理方法和化学方法具有自身无法克服的带来二次污染的缺点，生物防治法以其经济和环保的巨大优势成为理想的赤潮防治途径。其中利用大型海藻与赤潮微藻间的相互作用来预防或控制赤潮是目前致力于发展的崭新研究方向。本文研究小珊瑚藻（Corallina pilulifera）对三种赤潮微藻一赤潮异弯藻（Heterosigma akashiwo）、亚历山大藻（Alexandrium tamarense）和中肋骨条藻（Skeletonema costatum）的克生作用及其对UV-B辐射增强的相应变化，一方面为发展和建立利用大型海藻治理赤潮的生物防治法提供科学依据，另一方面对阐明UV-B辐射增强对海洋生态系统的影响有重要意义。主要实验结果如下：1．小珊瑚藻和3种赤潮微藻对UV-B辐射增强的敏感性比较研究UV-B辐射处理对赤潮异弯藻的生长始终表现出抑制效应，而低剂量的UV-B辐射处理对亚历山大藻和中肋骨条藻的生长有刺激作用，高剂量的UV-B辐射处理对亚历山大藻和中肋骨条藻的生长仍然显示出抑制作用。UV-B辐射处理对赤潮异弯藻、亚历山大藻和中肋骨条藻的96 h半数有效抑制剂量分别为1．63、2．34和2．58 J m^-2。因此3者对UV-B辐射处理的敏感性依次是赤潮异弯藻＞亚历山大藻＞中肋骨条藻。与3种赤潮微藻相比，小珊瑚藻对UV-B辐射表现出较强的抗性。在实验设置的剂量范围内，没有检测出UV-B辐射对小珊瑚藻生长的半数有效抑制剂量EC₅₀(96h·EC₅₀＞3．0J m^-2)。2．小珊瑚藻对3种赤潮微藻克生效应的研究通过排除营养盐、光照和pH等因素的影响，利用共培养系统研究了小珊瑚藻的新鲜组织、干粉末、水溶性组织抽提液及培养水过滤液对3种赤潮微藻--赤潮异弯藻、亚历山大藻及中肋骨条藻生长的作用。实验结果表明，小珊瑚藻的新鲜组织和干粉末对3种赤潮微藻的生长均有强烈的抑制作用，而且在较高的浓度下能够完全杀死赤潮异弯藻细胞和中肋骨条藻细胞，证明了小珊瑚藻对3种赤潮微藻具有克生效应。小珊瑚藻水溶性抽提液对3种赤潮微藻的生长同样表现出抑制作用，且抑制作用随浓度的升高而加强，表明小珊瑚藻组织中具有抑制赤潮微藻生长的克生物质，这进一步证明小珊瑚藻对3种赤潮微藻的克生效应主要是通过克生物质的来完成的。小珊瑚藻培养水过滤液在一次性和半连续性添加方式下，对3种赤潮微藻的生长均没有明显抑制作用，表明小珊瑚藻新鲜组织不能够或非常微量的向环境中释放克生物质，证明小珊瑚藻对赤潮微藻的克生作用方式是细胞间直接接触。小珊瑚藻新鲜组织对赤潮异弯藻、中肋骨条藻和亚历山大藻的克生效应由强到弱依次为：中肋骨条藻＞赤潮异弯藻＞亚历山大藻。因此3种有害赤潮微藻对小珊瑚藻的克生效应的敏感性表现出一定的差异性。这可能与3者的细胞外层结构不同有关。3．小珊瑚藻对赤潮异弯藻的克生效应对UV-B辐射增强的响应研究小珊瑚藻的新鲜组织和干粉末对赤潮异弯藻的克生效应对UV-B辐射处理都作出了响应性变化。高剂量(3．0 Jm^-2)UV-B辐射处理使小珊瑚藻新鲜组织对赤潮异弯藻的克生效应明显减弱，低剂量(0．9 J m^-2)的UV-B辐射处理使小珊瑚藻新鲜组织和干粉末对赤潮异弯藻的克生效应明显增强。小珊瑚藻水溶性抽提液对赤潮异弯藻的克生效应对UV-B辐射处理同样作出了响应性变化。高剂量(3．0 J m^-2)UV-B辐射处理使小珊瑚藻水溶性抽提液对赤潮异弯藻的克生效应明显减弱，低剂量(0．9 J m^-2)的UV-B辐射处理使小珊瑚藻水溶性抽提液对赤潮异弯藻的克生效应明显增强。这表明小珊瑚藻组织中的克生物质的克生活性或合成量在UV-B辐射处理下发生了显著变化，即高剂量(3．0 J m^-2)UV-B辐射处理使小珊瑚藻组织中的克生物质的克生活性或合成量显著降低，而低剂量(0．9 J m^-2)的UV-B辐射处理使小珊瑚藻组织中的克生物质的克生活性或合成量显著升高。无论是在一次性还是在半连续添加方式下，UV-B辐射处理都没有引起小珊瑚藻培养水过滤液对赤潮异弯藻生长作用的变化（p＞0．05），表明UV-B辐射处理没有改变小珊瑚藻赤潮异弯藻克生作用的方式，即仍然主要通过细胞间的直接接触方式完成的。4．小珊瑚藻组织提取物对赤潮异弯藻的生长抑制作用研究小珊瑚藻组织甲醇提取物在5种提取物中具有最高的对赤潮异弯藻生长抑制活性。在相对较高浓度的甲醇提取物作用下，赤潮异弯藻呈现出完全死亡的现象，小珊瑚藻组织甲醇提取物对赤潮异弯藻的EC₅₀值为0．004 ppt。较高浓度的小珊瑚藻组织蒸馏水提取物对赤潮异弯藻的生长具有显著的抑制作用，但是在最高浓度下也未能使其完全死亡。另外3种有机溶剂（丙酮、乙醚和氯仿）的提取物对赤潮异弯藻的生长无明显的影响。证明小珊瑚藻组织中含有对赤潮异弯藻生长有抑制作用的克生物质，且这些克生物质具有相对较高的极性。对小珊瑚藻的甲醇提取物进行了液液萃取，然后将其分离为石油醚相、乙酸乙酯相、正丁醇相和蒸馏水相。并用这4相对赤潮异弯藻进行克生物质的生物活性检测，结果表明，石油醚相对赤潮异弯藻具有较高的克生活性，其有效浓度为20 mg L^-1；乙酸乙酯相次之，其有效浓度为35 mg L^-1。而正丁醇相和蒸馏水相对赤潮异弯藻没有明显的杀藻作用。5．小珊瑚藻组织提取物对赤潮异弯藻的生长抑制作用对UV-B辐射增强的响应研究与未经UV-B辐射处理的小珊瑚藻蒸馏水提取物相比，低剂量UV-B辐射处理组(0．9 J m^-2)的小珊瑚藻组织蒸馏水提取物对赤潮异弯藻的生长有显著（p＜0．05）抑制作用；而高剂量UV-B辐射处理组(3．0 J m^-2)的小珊瑚藻组织蒸馏水提取物对赤潮异弯藻的生长有显著（p＜0．05）促进作用。说明低剂量和高剂量的UV-B辐射处理组都改变了小珊瑚藻蒸馏水提取液对赤潮异弯藻的生长的抑制效应，分别使抑制效果加强和减弱。与未经UV-B辐射处理的小珊瑚藻甲醇提取物相比，低剂量(0．9 J m^-2)和高剂量(3．0 J m^-2)UV-B辐射处理组的小珊瑚藻组织甲醇提取物对赤潮异弯藻的生长都有显著（p＜0．05）促进作用，并且高剂量处理组的促进作用明显（p＜0．05）大于低剂量处理组。表明低剂量和高剂量的UV-B辐射处理都能引起小珊瑚藻甲醇提取液对赤潮异弯藻的生长的抑制效应的改变，使抑制效果明显减弱。无论是高剂量(3．0 J m^-2)的UV-B辐射处理还是低剂量(0．9 J m^-2)的UV-B辐射处理都没有引起小珊瑚藻组织丙酮、乙醚和氯仿抽提物对赤潮异弯藻的生长效应的变化。UV-B辐射处理(0．9和03．0 J m^-2)能够改变小珊瑚藻甲醇提取物石油醚相和乙酸乙酯相对赤潮异弯藻的克生活性，使其对赤潮异弯藻生长的抑制率明显降低（p＜0．05）。正丁醇相和蒸馏水相对赤潮异弯藻的作用没有对UV-B辐射处理作出响应变化（p＞0．05）。更多还原

【Abstract】 Increasingly serious erosion and destruction of ozonosphere is one of the mostconspicuous phenomena of global changes nowadays.Ozone depleting promoted the doseof UV,especially UV-B,radiation increasing,which could produce serious damage effectto biology.Enhanced UV-B radiation can induce various changes of organism at the levelsof ecosystem,community,population,individuals,cell and molecular and further presentobvious ecological and biological effects.At the same time,with intensifications of coastal water eutrophication,severe outbreaksof harmful algal blooms （HABs） had become marine ecological disasters in the world.Since 1990s,red tides presented the features of“large range,high frequency,multiplespecies”in China,and has an increasing trend.This caused greatly threaten to coastalaquaculture and ecological environment.Using feasibly and effective measures to controlHABs were one of important content about red tides study.Biological method controllingred tides were an ideal approach because of its economic and no pollution,whereas thephysical and chemical methods about controlling HABs had second pollution that theycan’t overcome by their self.Using interaction between macroalgae and red tidesmicroalgae were considered to be a new way to prevent and control HABs.The present studies deal with the allelopathic effects of macroalgae Corallina piluliferaon three red tide microalgae （Heterosigama akashiwo,Alexandrium tamarense andSkeletonema costatum）,and their responses to enhanced UV-B radiation.The mainobjectives of these studies were to provide evidence for the feasibility of using manymacroalgae to control the growth of red tide microalgae,and present the importantfunction about the effect of enhanced UV-B radiation on marine ecosystem.Results showthat: 1 Sensitivity comparative studies on the response of C.pilulifera and three redtides microalgae to enhanced UV-B radiationThe growth of H.akashiwo was inhibited after UV-B radiation treatment,while UV-Bradiation produced stimulatory effect to A.tamarense and S.costatum at lower dose,andstill exhibited inhibitition effect to these two red tides microalgae at high dose of UV-Bradiation.The median effective dose (ED₅₀) of UV-B radiation to H.akashiwo,A.tamarense and S.costatum were 1.63,2.34 and 2.58 J m^-2 respectively.Thus the sensitiveorder of three species microalgae to UV-B radiation was H.akashiwo＞A.tamarense＞S.costatum.Compared with these red tide microalgae,C.pilulifera presented a strongresistance to UV-B radiation,and the EC₅₀ of UV-B radiation to C.pilulifera was notshowed in the range of experimental doses (96h·EC₅₀＞3.0 J m^-2)2 Studies on the allelopathic effects of C pilulifera on three species of red tidemicroalgaeExperiments were conducted under the same laboratory environmental conditions,including the nutrient,pH,light conditions and effects of bacteria in order to investigate theallelopathic effects of fresh tissue,dry powder,aqueous extracts and culture mediumfiltrate of C pilulifera on the growth of microalgae （H.akashiwo,A.tamarense and S.costatum） were evaluated using coexistence culture systems.The results of coexistenceassay showed that the growth of the three microalgae was strongly inhibited by using freshtissues and dry powder of C pilulifera,and H.akashiwo and S.costatum could be killedcompletely at higher concentration.It indicated that there are allelopathic effects of Cpilulifera on three microalgae.Aqueous extracts of C pilulifera also inhibited the growth of three microalgae,andinhibitory effects were enhanced with increasing concentration.This indicated that thereare allelochemicals in C.pilulifera,which could inhibit the growth of red tides microalgae,and further proved that the allelopathic effects of C.pilulifera on three microalgae were carried out mainly through allelochemicals.The effects of C.pilulifera culture medium filtrate exhibited no apparent inhibitoryeffect under initial filtrate addition and semi-continuous addition,which suggested that thefresh tissue of C.pilulifera can not release or little quantifies of allelochemicals toenvironment.This proved that the way of allelopathic effects of C.pilulifera on threemicroalgae were conducted by direct contacting.Allelopathic effects of the fresh tissue of C.pilulifera on three microalgae from high tolow were:S.costatum＞H.akashiwo＞A.tamarense.The results showed that there weresensitivity differences to some extent in allelopathic effects of C.pilulifera on threemicroalgae.Perhaps it was related to the structure of amphithecia of the species red tidesmicroalgae.3 Response of allelopathic effects of C.pilulifera on H.akashiwo to enhancedUV-B radiationThe allelopathic effects of flesh tissue and dry powder of C.pilulifera on H.akashiwowere changed after UV-B radiation stressing.High dose of UV-B radiation treatment (3.0 Jm^-2) significantly decreased the allelopathic effects of fresh tissue on H.akashiwo,whilelow dose of UV-B radiation treatment (0.9 J m^-2) obviously enhanced the allelopathiceffects of fresh tissue and dry powder of C.pilulifera on H.akashiwo.The allelopathic effects of aqueous extracts of C.pilulifera on H.akashiwo were alsoaltered under UV-B radiation treatment.High dose of UV-B radiation treatment (3.0 J m^-2)significantly decreased the allelopathic effects of aqueous extracts on H.akashiwo,whilelow dose of UV-B radiation treatment (0.9 J m^-2) obviously enhanced the allelopathiceffects of aqueous extracts of C.pilulifera on H.akashiwo.This showed that UV-Bradiation treatment changed allelopathic effects or compound quantity of allelochemicalsfrom tissue of C.pilulifera.Namely,high dose of UV-B radiation treatment (3.0 J m^-2)significantly decreased the allelopathic effects or compound quantity of allelochemicals,and low dose of UV-B radiation treatment (0.9 J m^-2) obviously enhanced the allelopathic effects or compound quantity of allelochemicals that from tissue of C pilulifera.The allelopathic effects of C.pilulifera culture medium filtrate were not changed underinitial filtrate addition and semi-continuous addition after UV-B radiation treatment（p＞0.05）.It showed that UV-B radiation did not alter the way of allelopathic effects of C.pilulifera on H.akashiwo,and direct contact among cell-cell was still a main approach toconduct the allelopathic effects.4 Study on growth inhibition effects of H.akashiwo by the extracts of C.piluliferaMethanol extracts of C pilulifera showed the highest growth inhibitory activity on H.akashiwo,and they killed all the cells of H.akashiwo at relatively higher concentrations.The EC₅₀ of methanol extracts of C.pilulifera to H.akashiwo was 0.004 ppt.Growth of H.akashiwo was significantly inhibited by the distilled water extracts of C.pilulifera at relatively higher concentrations,but cells of H.akashiwo were not killedcompletely even at the highest concentration.The other three organic solvent extracts （acetone,aether and chloroform） of C.pilulifera had no apparent effects on H.akashiwo,which suggested that there areallelochemicals in C.pilulifera which could inhibit the growth of H.akashiwo,and thegrowth inhibitory substances from the extracts of C.pilulifera had relatively highpolarities.The methanol extracts of C.pilulifera were partitioned to petroleum ether phase,ethylacetate phase,butanol phase and distilled phase by liquid-liquid fractionation.Thebioassays of the activity of every fraction which were carried out on H.akashiwo,indicatedthat petroleum ether phase and ethyl acetate phase had stronger allelopathic effect on H.akashiwo and its effective concentration was 20 mg L^-1;ethyl acetate phase took the secondplace,and its effective concentration was 35 mg L^-1;butanol phase and distilled waterphase had not obvious algicidal effect on H.akashiwo. 5 Response of the extracts of C.pilulifera inhibited growth of H.akashiwo toenhanced UV-B radiation.Compared with distilled extracts of C.pilulifera,which was untreated by UV-B radiation,the distilled extracts of C.pilulifera presented significant inhibitive effects on H.akashiwoat the group of low dose (0.9 J m^-2) of UV-B radiation treatment （p＜0.05）;while thedistilled extracts of C.pilulifera promoted the growth of H.akashiwo obviously （p＜0.05）at the group of high dose (3.0 J m^-2) of UV-B radiation treatment.This indicated that boththe groups of low and high dose changed inhibitive effects of the distilled extracts of C.pilulifera on H.akashiwo,and the inhibitive effects were enhanced and weakenedrespectively.Compared with methanol extracts of C.pilulifera,which was untreated by UV-Bradiation,the methanol extracts of C.pilulifera presented significant accelerate effects onH.akashiwo both in the groups of low dose (0.9 J m^-2) and high dose (3.0 J m^-2) of UV-Bradiation treatment （p＜0.05）,and promoting effects in the groups of high dose wasstronger significantly than that in the groups of low dose.This indicated that inhibitiveeffects of the methanol extracts of C.pilulifera on H.akashiwo could be changed both inthe groups of low dose and high dose of UV-B radiation treatment,and the inhibitiveeffects were weakened.Extracts of acetone,aether and chloroform from C.pilulifera had not changed thegrowth of H.akashiwo after treatment by UV-B radiation either in high dose group (0.9 Jm^-2) or low dose group (3.0 J m^-2).Allelopathic activity of petroleum ether phase and ethyl acetate phase from themethanol extracts of C.pilulifera could be altered by UV-B radiation treatment,theinhibitive effects of C.pilulifera on H.akashiwo were significantly decreased （p＜0.05）.The effects of butanol phase and distilled phase from the methanol extracts of C.piluliferaon H.akashiwo were not changed after UV-B radiation treatment.更多还原