【作者】 黄园；

【导师】 刘建国；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2014， 博士

【摘要】 微藻在食品、营养和保健等领域具有重要应用价值，开发微藻资源可获得人类大量需求的高附加值生物活性物质，同时也是获得再生生物能源的理想途径。但随培养规模逐步扩大，敌害生物污染严重制约着微藻规模化培养过程，限制其生物资源开发。其中以浮游动物对藻类摄食危害最为严重，轻者影响微藻生长，重者则会在几天时间内将微藻食光，造成严重的经济损失。但目前在微藻工程化培养中尚缺乏有效的污染控制方法。本实验基于浮游动植物的基本生物学特征差异，在筛选轮虫高毒性植物源杀虫剂的基础上，获得具有增效作用的植物源杀虫剂二元复配，研究了植物源杀虫剂单剂和增效二元复配对微藻培养中轮虫污染的控制效果，探讨其使用经济性和实用性，并初步了解植物源杀虫剂对轮虫毒性的作用机理，以期为微藻工程化培养中敌害生物防治提供理论和技术指导。主要结果如下：1、通过测定4种植物源杀虫剂（苦皮藤素、印楝素、苦参碱和川楝素）对褶皱臂尾轮虫的急性毒性，筛选出3种对轮虫高急性毒性的植物源杀虫剂—苦皮藤素、苦参碱和川楝素，并得到其24h内的有效致死浓度（LC50），分别为0.175mg/L、0.061mg/L和2.132×10-3mg/L；亚致死浓度苦皮藤素（≥0.110mg/L）、苦参碱（≥0.050mg/L）和川楝素（≥0.191×10-3mg/L）对轮虫个体繁殖和种群累积具强慢性毒性，可显著降低轮虫个体净生殖力、内禀增长率和周限增长率，并缩短其时代时间；种群抑制效应具有明显的剂量依赖性和时间依赖性，药物浓度越高，作用时间越长，对轮虫种群繁殖的抑制作用越强。2、研究植物源杀虫剂单剂对海水微藻培养中轮虫污染的控制效果，结果表明：（1）全致死浓度（LC100）苦皮藤素（0.316mg/L）可在24h内快速杀灭微拟球藻中褶皱臂尾轮虫，同时对微拟球藻净光合放氧和呼吸速率均无显著影响，藻细胞保持正常生长分裂，但其色素含量有降低，微藻生物量可达到空白对照组84%；（2）相比苦皮藤素，川楝素对抑制褶皱臂尾轮虫种群繁殖慢性毒性更强，LC30~LC50（1.755~2.132×10-3mg/L）浓度川楝素在48~72h内有效降低轮虫种群密度，并导致存活轮虫产生明显的拒食反应，轮虫怀卵率和种群密度均维持在较低水平，同时对小球藻和微拟球藻光化学效率无明显抑制作用，藻细胞平均比生长速率与空白对照无显著差异。苦皮藤素和川楝素对环境非靶标生物低毒或无毒，同时具有易降解、残留量低、价格低廉等优势，是微藻培养中控制轮虫污染的有效候选药物。3、苦皮藤素、苦参碱和川楝素二元复配对褶皱臂尾轮虫的联合毒性同时受复配药物种类和复配比例影响，苦参碱和川楝素均以神经系统为主要作用靶标，其二元复配对轮虫联合毒性以相加作用为主；苦皮藤素/苦参碱和苦皮藤素/川楝素复配中，随苦参碱或川楝素含量增加，二元复配联合毒性基本呈增强趋势；复配比例达到1:9时，苦参碱或川楝素含量可充分抑制轮虫体内对苦皮藤素的解毒酶系统，从而导致苦皮藤素对轮虫毒性增强，二元复配表现为增效作用，提示可通过应用苦皮藤素/苦参碱（1:9）或苦皮藤素/川楝素（1:9）提高轮虫防治效率，减少药剂使用量，进而降低防治成本。4、基于低剂量多次添加的防治策略，连续四次添加0.006mg/L苦皮藤素/川楝素（1:9）增效混剂，可破坏轮虫消化系统、麻痹神经系统，引起轮虫生理紊乱并最终抑制轮虫能量吸收，从而在四天内逐渐降低轮虫种群密度（至1尾/ml），有效控制种群繁殖；期间增效混剂对小球藻和微拟球藻细胞实际光化学效率（φPSII）和光能利用效率（α）无抑制作用，细胞密度保持连续增长，其色素含量达到空白对照组70~90%。增效复配药剂控制轮虫污染可明显减少单剂使用量30~90%，降低对环境非靶标生物的潜在毒性，同时减少药剂经济成本29%以上，有效降低轮虫污染的防控成本。5、户外培养实验表明，两次添加0.003~0.006mg/L苦皮藤素/川楝素（1:9）增效复配在3天内有效抑制了钝顶螺旋藻中的萼花臂尾轮虫种群繁殖，对螺旋藻生物量、叶绿素和藻蓝蛋白含量无显著抑制作用；与目前生产中常用的碳酸氢铵方法相比较，0.003mg/L增效复配通过抑制轮虫繁殖可长期控制轮虫污染；碳酸氢铵处理组中由于氨挥发，培养后期轮虫种群将有部分恢复；碳酸氢铵和增效复配对螺旋藻藻丝形态均无明显影响；但碳酸氢铵作用短期内对螺旋藻生长抑制作用较强，增效药剂控制轮虫污染后螺旋藻生物量可达更高水平；增效复配可增加螺旋藻培养经济收入，在螺旋藻户外大规模培养控制轮虫污染中具有较高的实用性和经济可行性。6、以川楝素为例初步研究了植物源杀虫剂对轮虫毒性机理，表明LC30~LC70（1.755~2.59×10-3mg/L）浓度川楝素可改变轮虫体壁细胞膜的膜蛋白结构，引起细胞内外渗透压失衡，轮虫体壁出现明显褶皱；导致存活轮虫个体体长和体宽均明显缩小，体积迅速减小，并促使胃蛋白酶-底物中间体形成，但抑制酶-底物复合物进一步分解，从而降低胃蛋白酶酶促反应速度，同时抑制类胰蛋白酶-底物复合体形成，降低类胰蛋白酶酶促反应速度，从而引起轮虫明显的拒食反应，并随川楝素浓度升高，拒食反应越明显。更多还原

【Abstract】 Microalgae have great potential applications in varied fields such as the food, nutritionand health care. Microalgal resource development can produce large amounts ofhigh-additional-value substances that are in great demand now, and is also considered to bean ideal way for renewable energy production. However, the contaminations caused bybiological pollutants constrain the algal large-scale cultivation and seriously impede thedevelopment of microalgal resource. Microalgal growth is especially susceptible to thegrazing by zooplanktons, which can inhibit algal growth and reduce the algal concentrationto low levels within just a few days, resulting in heavy economic loss. For now, no effectiveapproaches are found to control the biological contaminations. Based on the biologicaldifferences between zooplankton and phytoplankton, this study screened the individualbotanical pesticide that had high toxicity on rotifers, and obtained the synergistic binarycombinations of the pesticides. The rotifer-control effects of individual botanical pesticideand the synergistic combinations, and their safety for algal growth were studied. Theeconomics, practical applicability, and insecticidal mechanism of botanical pesticides onexterminating rotifers were also discussed. This study is aimed to provide theoretical andtechnical directions to the control of biological pollutants in microalgal mass cultivation. Themain results were as follows:1. By determining the acute toxicity of four botanical pesticides (celangulin,azadirachtin, matrine and toosendanin) on the rotifer B. plicatilis, three botanical pesticides,celangulin, matrine and toosendanin, that were highly acute toxic to rotifers were screened,with24h LC50values of0.175mg/L,0.061mg/L and2.132×10-3mg/L, respectively.Sublethal concentrations of celangulin (≥0.110mg/L), matrine (≥0.050mg/L) andtoosendanin (≥0.191×10-3mg/L) showed highly chronic toxicity toward the rotiferreproduction and population growth, with significant decreases of the net reproduction,intrinsic rate and finite rate of increase, and shortened generation time. The inhibitory effects of celangulin, matrine and toosendanin on population growth of rotifers were significantlyconcentration-dependent and time-dependent. The longer the time or higher theconcentration the rotifers were exposed to, the stronger the population growth of rotifers wasinhibited.2. The rotifer-control effects of individual botanical pesticide and its safety in algalcultivation were evaluated. Results demonstrated that,(1) the LC100(0.316mg/L) ofcelangulin exterminated the B. plicatilis rotifers effectively in the Nannochloropsis gaditanacultures in24hours. The celangulin treatment had no significant toxicity to the netphotosynthetic and respiratory rates of algal cells. Though partial decreases of chlorophyllcontent were observed, the N. gaditana kept growing during the cultivation period, with theharvest dry biomass reaching84%of the control;(2) Compared with the celangulin,toosendanin showed stronger chronic toxicity on inhibiting the population growth of rotifers.The LC30~LC50(1.755~2.132×10-3mg/L) of toosendanin decreased the rotifer density in48~72h, with the obvious antifeeding responses of survivals in the Chlorella sp. and N.oceanica cultures. Thus, both the population density and egg ratio of survival rotifers kept inlow levels. The toosendanin treatments had no negative influences on the photosyntheticefficiency of algal cells. There was no significant difference on the average specific growthrate between the algae treated by toosendanin and the control. Celangulin and toosendaninshowed none or low toxicity to non-target organisms in aquatic environments. Together withtheir advantages of rapid degradation, low residue and low commercial price, celangulin andtoosendanin are considered to be good potential botanical pesticides for controlling rotifersin microalgal mass cultivation.3. Interaction toxicity of binary combinations between celangulin, matrine andtoosendanin were dependent on the pesticides themselves and their ratios in combinations.Mixtures of matrine/toosendanin mainly produced addition owing to their similar modes ofaction aiming at the nervous system. As the content of matrine or toosendanin incombinations increased, there was an increased trend in the binary toxicity of either celangulin/matrine or celangulin/toosendanin combinations. Combinations of celangulinmixed with matrine or toosendanin at the1:9ratio exhibited synergism, which was attributedto the interference of matrine or toosendanin with the detoxification enzymes of celangulin.The synergistic celangulin/matrine (1:9) or celangulin/toosendanin (1:9) treatment will leadto increased insecticidal efficacy, reduced dosage of biocides, thereby reducing the cost ofexterminating rotifers.4. Based on the management strategy of frequent low-dose treatments, four dailytreatments of0.006mg/L celangulin/toosendanin (CA/TSN)(1:9) combination reduced thepopulation density of rotifers to approximately one rotifers/ml in four days. Thelow-concentration treatments do not directly cause rotifer death, but rather causephysiological disorders by damaging the digestive system, paralyzing the neuromuscularsystem and finally inhibiting energy uptake of the rotifers. Four additions of0.006mg/LCA/TSN treatment had no inhibitory effects on the effective quantum yield (φPSII) andphotosynthetic efficiency (α) of Chlorella sp. and N. oceanica. The microalgae treated by thecombination kept growing, with the pigment content reaching70~90%of the control.Application of the synergistic CA/TSN combination leads to an obvious dosage reduction by30~90%of celangulin and toosendanin for rotifer elimination. As a result, the potentialtoxic impacts for non-target organisms were decreased. The cost for rotifer exterminationwould also be reduced by29%.5. The outdoor experiments showed that two additions of0.003~0.006mg/L of thecelangulin/toosendanin (CA/TSN)(1:9) combination effectively inhibited rotiferreproduction within3days, and showed no toxicity toward the algal biomass, including thechlorophyll and phycocyanin levels of S. platensis. A comparison between the CA/TSNcombination and NH4HCO3treatments indicated that0.003mg/L CA/TSN showed a lastingeffect on controlling rotifers by sterilizing the rotifers. In the NH4HCO3treatment, therotifers still reproduced at a low level, partially owing to the dissipation of thetreatment-generated ammonia. Neither the NH4HCO3nor the CA/TSN treatment changed algal filament morphology; however, the NH4HCO3treatment initially inhibited algal growthmore seriously. S. platensis treated by CA/TSN reached a higher biomass concentration inthe same time frame. Given its effectiveness, together with the projected increase in farmearnings, the synergistic CA/TSN combination showed excellent potential and economicfeasibility for rotifer extermination in outdoor mass cultures of S. platensis.6. The insecticidal mechanism of toosendanin for rotifers was preliminarily studied.Results suggested that LC30~LC70(1.755~2.59×10-3mg/L) of toosendanin seriouslywrinkled the body wall of rotifers by isomerizing the membrane proteins in the epidermalcells, thereby resulting in the imbalance of osmotic pressures inside and outside of cells.Toosendanin decreased the body length and width, and shrunk the body size of survivalrotifers significantly. For the digestive enzymes, toosendanin decreased the rate ofpepsase-catalyzed reaction by improving the binding but inhibiting the further decomposingof pepsase and substrates. In contrast, toosendanin decreased the rate of tryptase-catalyzedreaction by reducing the affinity of tryptase with substrates. The inhibition of toosendanin onpepsase and tryptase activities resulted in the obvious antifeeding responses of rotifers,which was dosage-dependent.更多还原