【作者】 周媛；

【导师】 唐学玺；

【作者基本信息】 中国海洋大学 ， 生态学， 2010， 博士

【摘要】 浮游动物作为浮游植物和渔业生产的中间连接环节在海洋生态系统中扮演着重要的角色。海洋浮游动物对于赤微潮藻具有一定的潜在调控作用,浮游动物的摄食和数量变化将直接影响到浮游植物的种类、生长和数量,对有害赤潮的发生、发展过程有着重要的影响作用；反过来,有害赤潮微藻又会影响浮游动物的存活、生殖、生长和摄食等生理过程,从而对浮游动物的种群数量和发展产生不利作用。研究浮游植物和浮游动物的相互作用对揭示海洋生态系统的物质循环和能量流动过程有着重要意义。本文以海洋桡足类生物指状许水蚤(Schmackeria inopinus Burckhardt,1913)以及四种海洋赤潮微藻--塔玛亚历山大藻(Alexandrium tamarense)、东海原甲藻(Prorocentrum donghaiense Lu)、赤潮异弯藻(Heterosigma akashiwo)和中肋骨条藻(Skeletonema costatum)为实验生物,在实验生态条件下系统的研究了二者之间的相互作用,并对可能的作用机理进行了探讨,结果发现：1.指状许水蚤对四种赤潮微藻的摄食作用指状许水蚤对四种赤潮微藻：塔玛亚历山大藻、东海原甲藻、赤潮异弯藻和中肋骨条藻的摄食率呈现出一定的差异性：指状许水蚤对中肋骨条藻的摄食率相对较大,对塔玛亚历山大藻和东海原甲藻具有一定的摄食潜力,对赤潮异弯藻基本不摄食。按摄食率的高低排序依次为：中肋骨条藻>塔玛亚历山大藻>东海原甲藻>赤潮异弯藻。指状许水蚤雌体和雄体对四种赤潮微藻摄食率同样体现出一定差异性：雌体对塔玛亚历山大藻、东海原甲藻和中肋骨条藻的摄食率明显高于雄体,但二者对于赤潮异弯藻的摄食率差异不明显。2.指状许水蚤对四种赤潮微藻种群动态的影响指状许水蚤的加入能明显改变共培养体系中东海原甲藻、塔玛亚历山大藻和中肋骨条藻的种群增长规律：与对照组相比,三种微藻种群进入指数生长期和稳定期的时间延迟,种群最大细胞密度降低(P<0.05),这种影响随着指状许水蚤数量的增加而愈加明显；指状许水蚤对赤潮异弯藻种群动态没有明显的影响,与对照组相比,赤潮异弯藻种群密度变化较小(P>0.05),种群增长规律也基本类似。3.赤潮微藻对指状许水蚤生殖、生长和发育的影响四种赤潮微藻：赤潮异弯藻、东海原甲藻、塔玛亚历山大藻和中肋骨条藻均能够延迟指状许水蚤的抱卵时间和降低抱卵率,延长其卵囊发育所需要的时间和抱卵间隔(P<0.05)。与对照组相比,四种赤潮微藻对指状许水蚤的生长没有明显的影响(P>0.05),但是明显抑制了指状许水蚤发育的进程,延长了指状许水蚤从NⅡ期到成体所需的整个发育时间和每个发育阶段所需的时间(P<0.01)。四种赤潮微藻相比,赤潮异弯藻对指状许水蚤生殖和发育的抑制作用最明显,其次是东海原甲藻和塔玛亚历山大藻,中肋骨条藻相对不明显。赤潮异弯藻的四种组分：藻细胞破碎液、藻细胞过滤液、藻细胞再悬液和藻细胞液不但能够延迟指状许水蚤的抱卵时间,还能抑制其抱卵率,与对照组相比差异显著(P<0.05)；藻细胞过滤液、藻细胞再悬液和藻细胞液能显著延长指状许水蚤卵囊发育所需时间和抱卵间隔时间(P<0.05),但藻细胞破碎液的作用并不明显(P>0.05)。四种组分对指状许水蚤生长的影响作用不明显(P>0.05),但对指状许水蚤的发育影响显著(P<0.05)：一方面抑制了指状许水蚤发育的进程；另一方面延长了从一个时期发育到下一个时期所需的时间,其中藻细胞液对生殖和发育的抑制作用最明显,其次是藻细胞再悬液和藻细胞过滤液,藻细胞破碎液的影响相对较小。4.赤潮微藻对指状许水蚤种群数量的影响单养条件下,塔玛亚历山大藻和东海原甲藻能够抑制与其共培养的指状许水蚤的种群增长,与对照组相比种群数量增加缓慢,藻密度越高,抑制作用越明显,呈现出较明显的密度依赖性；中肋骨条藻对指状许水蚤种群数量的增长有一定的抑制作用,在2x104 cells mL-1--20x104 cells mL-1密度范围内,随着中肋骨条藻密度的增加,抑制作用减弱,当密度达到30x104 cells mL-1时,抑制作用明显增强。与上述三种微藻不同,在不同藻密度的赤潮异弯藻作用下,指状许水蚤种群数量不但没有增加,反而出现了降低的现象,并且随着藻密度的提高,种群数量降低越明显,显示赤潮异弯藻对指状许水蚤有较明显的致死效应。在塔玛亚历山大藻和东海原甲藻的混养体系中,二者起始密度比例的不同(8：5、1：1和5：8)并没有引起指状许水蚤种群数量的变化；随着培养时间的延长,三个起始密度比例下指状许水蚤种群数量均持续增加,但不同密度比例组之间种群数量的差异不显著(paired r-test, P> 0.05)。在中肋骨条藻和赤潮异弯藻的混养体系中,二者的起始接种密度比例对指状许水蚤实验种群数量的影响具有明显的差异,体系中中肋骨条藻所占的比例越高,对指状许水蚤种群的增长越有利。5.赤潮异弯藻对指状许水蚤抗氧化活性的影响赤潮异弯藻可使共培养体系中指状许水蚤的超氧化物歧化酶(SOD)、谷胱甘肽还原酶(GR)和谷胱甘肽过氧化物酶(Gpx)的活性降低,而且,随着藻密度的提高,其活性下降愈加明显,与对照组比较差异显著(P<0.05)；赤潮异弯藻还能够导致指状许水蚤的二种抗氧化因子类胡萝卜素(CAR)和谷胱甘肽(GSH)含量的下降(P<0.05),总抗氧化活性(Total antioxidant, TA)与对照组相比也有显著的降低(P<0.05)。同期对活性氧(ROS)的检测结果表明,赤潮异弯藻作用能导致指状许水蚤体内ROS含量的增高并贯穿于整个实验过程中；随着起始微藻密度的提高,指状许水蚤体内的ROS明显提高,并且膜脂过氧化程度(MDA含量)也有相应的升高,与对照组比较差异显著(P<0.05),说明在此条件下指状许水蚤体内产生了氧化胁迫(oxidative stress)。6.赤潮异弯藻对指状许水蚤生殖、发育和种群数量影响的机制分析综合分析本文的研究结果,我们推测赤潮异弯藻对指状许水蚤的生殖、发育和种群数量影响的机制可能为,在培养体系中,赤潮异弯藻分泌了某种有毒物质(他感物质),有毒物质一方面直接对蚤体产生毒害作用,另一方面,在有毒物质的胁迫作用下,指状许水蚤的抗氧化系统的活性下降,引起指状许水蚤ROS清除能力的降低,打破了其体内活性氧产生和清除之间的动态平衡,使得ROS在指状许水蚤体内大量积累；大量积累的ROS引起了膜脂过氧化作用加强,进一步对生物体的各项生理功能产生伤害。这种直接和间接伤害作用共同导致了指状许水蚤生理机能的异常,最终表现为生殖、发育和种群发展的抑制。更多还原

【Abstract】 Zooplankton plays an important role in marine ecosystems as a key loop between phytoplankton and fish. Marine zooplankton has a potentially regulatory effect on bloom-forming microalgae. Its feeding activity and population fluctuation directly affect the microalgal species, growth and population size, and then remarkably influence the occurrence and development of harmful algal blooms (HABs). One the other hand, bloom-forming microalgae will affect the zooplankton’s survival, reproduction, growth and feeding activity, and thus exert adverse effect on its population size and development. Therefore, a research on the interaction between phytoplankton and zooplankton is of important meaning to illuminate the processes of matter cycle and energy flow of marine ecosystem. In present study, the interactions among Schmackeria inopinus (Burckhardt,1913) and four species of bloom-forming microalgae (Alexandrium tamarense, Prorocentrum donghaiense, Heterosigma akashiwo and Skeletonema costatum) were investigated under controlled laboratory conditions, and the possible effective mechanism was discussed. Results showed that:1. Feeding activities of S. inopinus on four species of bloom-forming microalgaeFeeding rates of S. inopinus differed significantly when fed on A tamarense, P. donghaiense, H. akashiwo and S. costatum in co-culture. S. inopinus was preferred to S. costatum, while presented a certain feeding rates on A. tamarense and P. donghaiense, but almost not fed on H. akashiwo. The feeding selectivity was:S. costatum> A. tamarense> P. donghaiense> H. akashiwo.Feeding rates of male and female S. inopinus were different: The feeding rates of female ones were higher than those of male when fed on A. tamarense, P. donghaiense and S. costatum, but no difference was observed between male and female when fed on H. akashiwo.2. Influences of S. inopinus on population dynamics of bloom-forming microalgae in co-cultureS. inopinus showed significantly influences on population dynamics of P. donghaiense, A. tamarense and S. costatum in co-cultures. The addition of S. inopinus not only regulated the population growth, but also decreased the microalgal population density (P< 0.05):The time at which microalgal population achieving exponential growth phase and platform phase delayed, and the maximum cell density obviously decreased (P< 0.05) in the co-culture as compared with that in the control growing in the mono-culture. The changes became more remarkable with the increaseed amount of S. inopinus. In contrast, S. inopinus had no obvious effect on the growth and cell density of H. akashiwo in the co-culture and almost no significance was observed as compared with the control group (P> 0.05). This result further demonstrated that S. inopinus almost didn’t feed on H. akashiwo.3. Influences of bloom-forming microalgae on reproduction, growth and development of S. inopinus in co-cultureAll the four bloom-forming microalgae (A. tamarense, P. donghaiense, H. akashiwo and S. costatum) delayed the egg-carrying time, decrease the egg mass rate, and prolonged the duration of egg mass development and the interval of egg carrying of S. inopinus as compared to the control (P< 0.05). However, the tested microalgae had no obvious influence on the growth of S. inopinus (P> 0.05) while significantly restrained the development of S. inopinus and prolonged the duration from N II stage to adult and the duration time of each larval stage as compared to the control which cultured in normal conditions (P< 0.01). Among the microalgae, H. akashiwo showed the strongest inhibitory effect on reproduction and development of S. inopinus followed by P. donghaiense and A. tamarense. S. costatum showed little inhibition as compared to the other three.Four components of H. akashiwo (water extracts, culture medium filtrate, re-suspended cells and microalgal culture medium) not only delayed the egg-carrying time, but also led to restrain of egg carrying (P< 0.05). The cell-free water extracts had no obvious effects on prolonging the duration time of egg mass development and the interval of reproduction (P> 0.05); however, the culture medium filtrate, re-suspended cells and algal medium significantly prolonged the duration time of egg mass development and the interval of reproduction as compared to the control (P< 0.05). These four components had little effect on the growth of S. inopinus (P> 0.05), but significantly affected the development of S. inopinus (P< 0.05). On one hand, the four components presented inhibitory effects on developmental process of S. inopinus, and prolonged duration time of each developmental stage, among which microalgal culture medium showed the strongest inhibitory effects on reproduction and development, re-suspended cells and culture medium filtrate had stronger one than that of extracts from disrupted cells.4. Influences of bloom-forming microalgae on the population quantity of S. inopinusUnder controlled laboratory conditions, A. tamarense and P. donghaiense inhibited the growth of S. inopinus resulting in a decreased growth rate and the inhibitory effect, and the inhibition increased accordingly with the initial microalgal density increasing. The population quantity of S. inopinus decreased significantly with the increase of H. akashiwo density in the co-culture, and the decrease was density-dependent, inferring that H. akashiwo present a lethal effect on S. inopinus in the co-culture. S. costatum had inhibitory effect on population growth of S. inopinus. The inhibitory effect decreased with the increase when cell density of S. costatum was set at the range from 2×104 cells ml-1 to 20x10 cells ml-1, but the inhibition was obviously increased when the density was set at 30x104 cells ml-1.In microalgal co-culture systems, the population fluctuation of S. inopinus did not depend on the density ratio of A. tamarense to P. donghaiense. Along with culture time, the population quantity of S. inopinus constantly increased at three density ratios (8:5,1:1 and 5:8), but no obvious difference of population density was observed among the three density ratios (P> 0.05). Under co-culture systems of H. akashiwo and S. costatum, the population fluctuation of S. inopinus remarkably depended on the microalgal density ratio, and the growth rate of population quantity of S. inopinus increased with the increase in the ratio of S. costatum/ H. akashiwo.5. Influence of H. akashiwo on the antioxidant system of S. inopinusH. akashiwo decreased the activities of superoxide dismutase (SOD), glutathione reductase (GR) and glutathione peroxidase (Gpx), the enzymatic activities decreased with the increase of microalgae density (P< 0.05). Likewise, H. akashiwo decreased the contents of two antioxidant metabolites (CAR and GSH) (P< 0.05), and the total antioxidant activity was greatly inhibited as compared to the control (P< 0.05).The effect of H. akashiwo led to a significant accumulation of reactive oxygen species (ROS), and the generation of ROS was observed throughout the whole culture process. ROS content elevated with the increase in microalgal density, and was significantly higher than that in control groups (P< 0.05). simutaneous analysis on the malonaldehyde (MDA) showed that S. inopinus were subjected to a serious peroxidative stress of membrane lipid.6. The possible mechanism of H. akashiwo on reproduction, development and population dynamics of S. inopinusWhen considered the results as a whole, we conjectured the possible mechanism of H. akashiwo on reproduction, development and population quantity of S. inopinus. In co-culture system, H. akashiwo secreted toxic substance (allelopathy) which caused direct injuries on S. inopinus. On the other hand, the activities of antioxidant enzymes and the antioxidant metabolites contents decreased when exposed to these toxic substances, resulting in a decrease in total antioxidant activity. Furthermore, the decease led to the over-production of ROS, and the overwhelming ROS could break the ROS homeostasis in S. inopinus and initiate the membrane lipid peroxidation which could be characterized by the increase of MDA. It decreased the antioxidant system activities thereafter, which further aggravated the oxidative stress. ROS-mediated oxidative stress might be the possible mechanisms that damage the growth and reproduction of S. inopinus in the co-culture, and the allelochemicals secreted by the bloom-forming microalga was the original reason.更多还原