【作者】 周艺彪；

【导师】 姜庆五； 赵根明； 陈跃；

【作者基本信息】 复旦大学 ， 流行病与卫生统计学， 2006， 博士

【摘要】 日本血吸虫病是一种严重危害人类健康的人畜共患性疾病，在流行区严重影响居民的健康和当地经济的发展。湖北钉螺是日本血吸虫的唯一中间宿主，在血吸虫病流行中起着极其重要的作用。在我国大陆，钉螺主要分布于长江以南的12个省、市、自治区。至2003年全国共有钉螺面积3，786.8Km²。以往国内外对钉螺的形态学、地理分布、孳生环境、生理和生化特性等方面进入了较为深入的研究，显示湖北钉螺不仅在我国分布较广，而且在形态学、遗传学以及钉螺对血吸虫易感性等方面都存在着地域上的差异。国内外对钉螺遗传变异的研究中，以同工酶的研究较为多见，其次是对线粒体DNA和染色体核型等的研究，较少见对钉螺基因组遗传变异的研究。湖北钉螺的分类，多年来一直存在着争议，至今仍无定论，对钉螺种群内的遗传变异程度也存在着截然相反的结论。本次研究从钉螺形态形状和基因组DNA水平2个层次上对我国湖北钉螺的遗传变异进行系统的研究。第一部分第一节，目的探讨扩增片段长度多态性（AFLP）分子标记在钉螺遗传变异研究中应用的可能性，并进行实验条件优化和引物筛选。方法随机抽取云南大理和湖南君山阴性钉螺各1只，用异硫氰酸胍和Resin等抽取DNA，然后用64对引物对基因组DNA进行AFLP扩增，扩增产物用6％的变性聚丙烯酰胺凝胶电泳，荧光检测扩增产物。结果每对引物扩增的AFLP标记数在5—55之间，大理钉螺平均每对引物出现38.30（95％CI36.03～40.57）个标记，君山平均每对引物扩增出39.14（95％CI 36.71～41.57）个标记：每对引物扩增的多态性标记数和多态性频率分别在3～37个和28.6％～76.2％之间，分别平均为23.67（95％CI 22.12～25.22）和47.36％（95％CI 45.22％～49.50％）结论AFLP标记技术能应用于湖北钉螺的分类与遗传多样性的研究。第二节，目的探讨AFLP分子标记电泳图谱信息数量化数据的分析方法。方法随机抽取湖南君山阴性钉螺40只，进行AFLP扩增，用Glyko BandScan软件将钉螺AFLP电泳图谱信息数量化，使用不同的读带标准读带，得到相应数据集，然后对这些数据集进行遗传学统计分析与描述性总结。结果不同的标准所得到的遗传变异结果均有所差别，但随着读带标准值的增加，反映钉螺种群遗传多样性指标（如：Shannon’s信息指数）也增加，当其增加到一定水平时，又开始下降，而基因流和基因一致度则刚好相反。不同读带标准所得的遗传变异结果均呈明显的正态分布（P＞0.05）。以总灰度或以总灰度百分比划分读带标准，所得遗传变异结果的平均值均十分接近。将采集的钉螺中筛选出40只阴性钉螺，随机分为两组，平均基因一致度在总灰度百分比数据中为0.956，在总灰度数据中为0.958；两组间的平均遗传距离在总灰度百分比数据中为0.045，在总灰度数据中为0.043。结论将电泳图谱信息数量化，再以不同的读带标准去处理与分析数据的模式，是一种较为合理且准确的分析方法。第二部分第一节，目的探讨用AFLP分子标记研究湖北钉螺遗传变异的合理样本量与分子位点数。方法选取来自于湖南君山的钉螺为研究材料，用AFLP方法对钉螺基因组DNA进行扩增，然后分析钉螺样本量和分子位点数与遗传变异信息可靠性的关系。结果钉螺样本量和分子位点数与遗传多样性信息的可靠性之间存在明显的关系。当样本量低于7只时，AFLP，总位点数、多态位点数、多态位点频率、Nei’s基因多样性指数和Shannon’s信息指数变化很大，而当样本量超过30只时，这些指标值的变化趋于平稳。当AFLP分子位点数低于128时，多态位点频率、Nei’s基因多样性指数、Shannon’s信息指数以及这两个指数的标准差变化相当剧烈，当分子位点数超过338时，这些指标值的变化趋于稳定。结论在用AFLP分子标记技术研究湖北钉螺的遗传变异时，每个钉螺种群内的样本量最好不应低于30只，用于研究分析的分子位点数最好不低于338个。第二节，目的探讨湖北钉螺形态形状变异信息的可靠性与样本量的关系。方法从云南大理和湖南君山两地区随机抽取成年钉螺各60只，分别对钉螺壳形态数量性状进行测量，计算各数量性状指标的变异系数、组间变异百分比和多样性指数。结果当样本量低于30，所得到的遗传变异的结果极不可靠，随着样本量的增加遗传变异信息的可靠性也增加，当样本量超过55时，所得到的遗传变异的结果趋于稳定。结论。在湖北钉螺形态形状的遗传变异研究中，每个钉螺种群以分析不少于55个钉螺标本为好。第三部分第一节，目的探讨湖北钉螺种群内的遗传变异及其程度。方法采用扩增片段长度多态性（AFLP）分子标记技术对9省（云南、四川、广西、福建、湖南、湖北、江西、安徽、江苏）13个钉螺种群基因组DNA进行扩增，分析钉螺种群内的遗传变异。结果13个钉螺种群AFLP扩增片段数在403～472之间，江西星子钉螺种群内遗传多样性较高，多态位点频率、Nei’s基因多样性指数和Shannon’s信息指数分别为93.22％、0.345和0.510，而广西宜州钉螺种群内遗传多样性较低，以上3指标分别为55.80％、0.191和0.287；广西宜州钉螺种群内的相似性较大，相似系数（中位数）为0.904，而江苏丹徒钉螺种群内的相似性较低，相似系数（中位数）为0.748；13个钉螺种群内的遗传变异差异显著（P＜0.01），5个光壳钉螺种群内的相似系数普遍高于8个肋壳钉螺种群内的相似系数。结论我国大陆广泛分布的钉螺，种群内存在一定程度的遗传变异。不同地区钉螺种群内遗传变异程度不同，有的相差较大。第二节，目的探讨湖北钉螺种群内的形态形状变异及其程度。方法在中国大陆血吸虫病流行的7个省中，采集不同环境类型的21个钉螺种群，每个种群各测量60只湖北钉螺的11个形态性状。采用形态性状的变异系数、种群内个体间的欧氏距离与多样性指数、及主成分分析3种方法，分析不同湖北钉螺种群内形态性状的变异程度。结果在种群内个体间的欧氏距离和形态性状多样性方面，都以江西都昌的变异最大，平均欧氏距离为2.88，方差为2.33，极差为9.05，多样性指数为1.56，个体间平均距离以四川西昌湖北钉螺种群的最小，仅为1.37，而多样性指数以四川丹棱湖北钉螺种群的最小，为0.91。在不同光壳钉螺种群中，以云南大理钉螺个体间的平均距离最大，为1.90，但方差和极差以江苏宜兴的最大，分别为0.55和4.46。结论不同湖北钉螺种群内形态性状变异程度存在较大的差异，肋壳钉螺种群内的形态性状变异程度普遍高于光壳钉螺种群内的变异程度。第四部分第一节，目的探讨湖北钉螺种群间的遗传变异及其程度。方法采用AFLP分子标记技术对来自中国大陆10省的25个种群钉螺基因组DNA样品池进行扩增，分析钉螺各种群间的遗传变异并对钉螺种群进行聚类分析。结果25个钉螺种群间的相似系数GS_DICE在0.694～0.831之间，Nei无偏遗传一致性在0.635～0.799之间，遗传距离D在0.169～0.306之间，Nei无偏遗传距离在0.225～0.452之间，指名亚种（包括或不包括广西钉螺）钉螺种群间的遗传变异程度明显高于滇川亚种钉螺种群间的遗传变异程度（P＜0.01），光壳钉螺种群间的遗传变异程度明显高于肋壳钉螺种群间的遗传变异程度（P＜0.01）。25个钉螺种群被聚成3类，A类包括来自福建福清和广西宜州的光壳钉螺种群；B类包括来自四川西昌、普格、丹棱、蒲江、广汉和云南大理的光壳钉螺种群；C类则由其它来自长江中下游地区的17个钉螺种群组成。结论在我国分布的湖北钉螺已发生较大的遗传变异，基因组水平上的钉螺种群聚类结果和其地理分布基本一致。第二节，目的探讨湖北钉螺种群间的形态形状变异及其程度。方法采用数量分类法对27个钉螺种群的11个螺壳形态形状指标进行了聚类和主成分分析。结果27个钉螺种群中，以湖南华容钉螺种群与四川丹棱钉螺种群间的形态形状变异最大，其欧氏距离达10.29，而安徽贵池与枞阳钉螺种群间的形态形状变异最小，其欧氏距离仅为0.62。无论指名亚种包括还是不包括广西钉螺，滇川亚种钉螺种群间欧氏距离均明显低于指名亚种种群间的（P＜0.05）：11个光壳钉螺种群间的欧氏距离明显低于16个肋壳钉螺种群间的欧氏距离（P＜0.05）。主成分分析将27个钉螺种群分为三组，第一组包括江陵、贵池、枞阳、丹徒、铜陵1、江宁的钉螺。第二组包括南昌、华容、都昌、汉川、阳新、蔡甸、南县、星子、君山、江山的钉螺。第三组包括宜兴、西昌、广汉、蒲江、丹棱、大理、福清、石门、铜陵2、宜州、普格的钉螺。结论指名亚种钉螺种群间的形态形状变异大于滇川亚种的，肋壳钉螺种群间的形态形状变异大于光壳钉螺的。主成分分析的分类结果与环境类型基本一致。第五部分第一节，目的探讨湖北钉螺的空间遗传结构。方法分析25个钉螺种群间的遗传距离与地理距离的相关性。结果25个钉螺种群间的遗传距离D和Nei无偏遗传距离，都与其地理距离存在明显的正相关性（P＜0.001），相关系数分别为0.5234和0.5622；湖北钉螺指名亚种种群间的遗传距离与地理距离也存在正相关（P＜0.001），遗传距离D的相关系数为0.5276，Nei无偏遗传距离的为0.5770；无论是肋壳钉螺还是光壳钥一螺，钉螺种群间的遗传距离都与地理距离存在正相关(（P＜0.001），肋壳钉螺种群间的遗传距离D和Nei无偏遗传距离与地理距离的相关系数分别为0.3612和0.3916，光壳钉螺的相关系数分别为0.7535和0.7500。结论在我国大陆广泛分布的湖北钉螺种群间具有明显的空间遗传结构。第二节目的探讨湖北钉螺种群间的形态形状变异的空间相关性。方法分析27个钉螺种群间形态形状变异及其与地理距离的相关性。结果27个钉螺种群间形态形状变异与其地理距离存在明显的正相关（P＜0.001），相关系数r为0.2791；这种显著的相关性在指名亚种和肋壳钉螺种群中仍然存在，但在滇川亚种和光壳钉螺种群中却未发现（（P＞0.05），指名亚种包括广西钉螺种群时相关系数为0.2655（P＜0.01）），不包括时相关系数为0.2567（P＜0.01），肋壳钉螺种群的相关系数r为0.3121（P＜0.001）。结论在我国大陆分布的肋壳钉螺种群间的表型变异具有明显的空间结构。更多还原

【Abstract】 Schistosomiasis japonicum is a major zoonose threatening human health, and impacts severely the development of economy in endemic areas. Oncomelania hupensis is only an intermediate host of Schistosomiasis japonicum, and takes a very important role of transmitting schistosomiasis, In China’s mainland, snail is mainly distributed along the Yangtze River and down to the basin of the Yangtze River including 404 counties/cities in 12 province level in China. Up to 2003 year, intermediate host snails were found on a total surface area of 3786.8 km2. In the past, there were a lot of reports on morphology, geographical distribution, breeding environment, physiology, biochemistry and so on, and the results show that differences between O. hupensis populations include shell size and structure, genetics breeding environment, isoenzyme, gene, growth rates, potential for infection by S. japonicum and so forth. In the studies of snail genetic variation, the reports on isoenzyme were the most, and the next were mitochondrial DNA and chromosome et al. However, the variations of snail genome DNA were studied in a very few reports. The taxonomy of Oncomelania has been confused over the years in part because of one character （shell sculpture） and two characterstates （Shell smooth/shell ribbed）, and there is also a discrepancy about the extent of genetic diversities within populations of Oncomelania hupensis. In our study, the genetic diversity of Oncomelania hupensis was studied in morphologic and genome DNA levels.In part oneIn section one, Objective The application probability of AFLP （Amplified fragment length polymorphism） was explored in the study of genetic diversity of Ocomelania hupensis. Methods A negative snail was token at random from Dali, Yunnan province and Junshan, Hunan province respectively, and DNA was drawn with Guanidiniumn thiocyanate and Resin et al. Genomic DNA was amplified selectively by the AFLP technique with 64 pairs of primers, and the polymorphism of PCR products was examined by SDS—PAGE. Results The number of AFLP makers was between 5 and 55 for per a pair of primer. The average number of a pair of primers was 38.30（95%CI 36.03～40.57） for the snail from Yunnan province, and 39.14 （95%CI 36.71～41.57） for the one from Hunan province. For a pair of primers, the number of amplified polymorphic markers was between 3 and 37, and the polymorphic ratewas between 28.6% and 76.2%, the average, 23.67 （95%CI 22.12～25.22） and 47.36%（95%CI 45.22%～49.50%）,respectively. Conclusion The AFLP technique will break a new path for classifying Ocomelania hupensis and studying the genetic diversity of Ocomelania hupensis.In section two, Objective To explore an analysis method for AFLP electrophoresis Pattern quantitative data. Methods Forty snail individuals from Junshan, Hunan Province were selected for AFLP analysis. AFLP electrophoresis Pattern were first transformed into quantitative data by Glyko BandScan software, and the bands were read according to different standards of reading band, then the corresponding data sets were acquired. These data sets were analyzed by genetic statistics respectively to get an inference set, and the analysis of this inference set were performed to arrive at summary description. Results The results of genetic variation from different standards of reading band were different. With the increase of the standard value of reading band, the indices indicating the genetic polymorphism of Ocomelania hupensis population （e.g. Shannon’s information index） also increased. When the standard value arrived at a certain level, the values of these indices began to fall. Compared with the above indices, there were the opposite change for gene flow and genetic identity. The distributions of inference results form different standards of reading band were all significant normal distribution. The mean values of results of genetic variation according to total gray were very close to the ones according to proportion of total gray. The average genetic identity between the "subpopulations" was 0. 956 according to proportion of total gray or 0.958 according to total gray; the average genetic distance between the "subpopulations" was 0.045 according to proportion of total gray or 0.043 according to total gray. Conclusion The paradigm, in which the electrophoresis Pattern were transformed into quantitative data and then the data were dealt with and analyzed according to the different standard of reading band, was a kind of more reasonable and accuracy analysis method.In part twoIn section one, Objective To explore the reasonable sample size and the reasonable number of molecular marker loci in the study of AFLP being used to analyze the genetic diversity of Ocomelania Hupensis. Methods the ribbed-shelled snails coming from Junshan. Hunan province, were selected to analyze the relationship of the number of AFLP molecular marker loci and sample size with the reliability of information on genetic variation forOcomelania Hupensis by AFLP method. Results There were a obvious relationship of the number of AFLP molecular marker loci and sample size with the reliability of information on genetic variation for Ocomelania Hupensis. When sample size was less than 7 individuals, the total number of AFLP loci, the number of polymorphic loci, Nei’s gene diversity and Shannon’s information index changed greatly. But when sample size was more than 30 individuals, the values of these indices tended towards stabilization. When the number of AFLP loci was less than 128, the frequency of polymorphic loci, Nei’s gene diversity, Shannon’s information index and the standard deviation of these two indices changed greatly. But when the number of loci was more than 338, the values of these indices tended towards stabilization. Conclusion When the genetic diversity of Ocomelania Hupensis were analyzed by AFLP method, the sample size coming from each snail population should be not less than 30 individuals, and the number of molecular loci analyzed should not less than 338.In section two, Objective To explore the relationship between the reliability of information of morphologic diversity and sample size in morphologic characteristics of Oncomelania hupensis. Method Sixty adult snails （smooth shell） from Dali, Yunnan Province （smooth shells） and sixty adult snails （ribbed shell） from Junshan, Hunan Province （ribbed shells）, were sampled at random, and the morphologic characteristics of the snails were measured, and then the coefficient of variation, proportion of variation between group and Shannon’s information index were computed respectively. Results When the sample size was less than 30. the result of morphologic diversity gained from the study was not very reliable, and with the increase of sample size the reliability of the information of morphologic diversity was also augmented. When the sample size was more than 55. the result of morphologic diversity was reliable. Conclusion In order to get reliable information of morphologic variation in the study of morphologic characteristics of Oncomelania hupensis, at least 55 individuals should be investigated for each population.In part threeIn section one Objective To explore the degree of genetic diversity within populations of Oncomelania hupensis. Methods AFLP method was used to amplify the genomic DNA of thirteen snail populations from nine provinces （i.e. Yunnan, Sichuan, Guangxi, Fujian, Hunan, Hubei, Jiangxi, Anhui and Jiangsu） and the genetic diversities within snail populations were analyzed. Results The number of AFLP fragments amplified ranged from 403 to 472 for thirteen Oncomelania populations. Among thirteen snail populations, the genetic diversitywithin the population from Xingzi county, Jiangxi province was most, and the percentage of polymorphic loci, Nei’s genetic diversity and Shannon’s information index were 93.22%, 0.345 and 0.510 respectively, while these indices for the snail population from Yizhou city, Guangxi province were lowest, they were 55.80%, 0.191 and 0.287 respectively. The similarity between the ingroup-individuals from Yizhou city, Guangxi province was most, and the average coefficient of similarity was 0.904, and that from Dantu county, Jiangsu province was lowest, and was 0.748. The genetic diversities within snail populations were different significantly for the thirteen snail populations （P<0.01）. The coefficients of similarity within snail population of five smooth-shell snail populations were all higher than that of eight ribbed shell snail populations. Conclusion There was the certain extent of genetic variation within snail populations from China’s mainland, further more, the degree of genetic variation within snail populations from different areas was different significantly.In section two, Objective To explore the degree of morphologic diversity within populations of Oncomelania hupensis. Methods 21 Oncomelania hupensis populations was collected form the different environment types of seven provinces with schistosomiasis japonicum in mainland of China. Sixty individuals were sampled randomly for each snail population and 11 morphological traits were measured for each snail. The coefficient of variation of morphological characters, Euclidean distances between snail individual within population, Morphological polymorphism and Principal components analysis were conducted to analyze the variation of morphological trait within snail population. Results Among 21 snail populations, the morphological variation of the snail population from Duchang, Jiangxi province was the most, and the median, variance and range of its Euclidean distance and its Shannon-Winer index were 2.88, 2.33, 9.05 and 1.56 respectively. The median of Euclidean distance of the snail population from Xichang, Sichuan province was the least （1.37）, but the Shannon-Winer index of the population from Danling, Sichuan province was the least（0.91）. Among smooth-shell populations, the median of Euclidean distance of the snail population from Dali, Yunnan province was the most （1.90）, but the variance and range of Euclidean distance of the snail population from Yixing, Jiangsu province were the most （0.55 and 4.46 respectively）. Conclusion There was the different degree of morphological variation within different snail populations, and the degree of morphological variation within ribbed-shell snail populations was higher than that of within smoothed-shell snail populations.In part fourIn section one Objective. To explore the genetic diversity among populations of Oncomelania hupensis. Methods AFLP method was used to amplify the genomic DNA pool of twenty five snail populations from ten provinces, and the genetic diversities among these snail populations were analyzed. Results The coefficient of similarity (GS_DICE) among twenty five snail populations ranged from 0.694 to 0.831, Nei’s unbiased genetic identity from 0.635 to 0.799, Genetic distance D from 0.169 to 0.306, and Nei’s unbiased genetic distance from 0.225 to 0.452.The genetic variation among populations of O.h.hupensis （including or not including a snail population from Guangxi Province） was higher than that of O. h. robertsoni populations （P<0.01）. The genetic variation among smooth-shell snail populations was higher than that of ribbed shell snail populations （P<0.01）. Twenty five snail populations were divided into three groups: group A included smooth-shell snail from Fuqing of Fujian province and Yizhou of Guangxi province; group B consisted of smooth-shell snail Dali of Yunnan province and Xichang, Puge,Danleng, Pujiang,Guanghan of Sichuan; group C was composed of other seventeen snail populations from the Yangtze River drainage below the Three Gorges. Conclusion There was big genetic variation among these populations of Oncomelania hupensis. The clustering result of snail populations in genomic level was consistent basically with geographical distribution.In section two, Objective To explore the morphologic variations among snail populations. Method 27 snail populations were used and 11 shell morphological traits were measured for quantitative taxonomic analysis. The Euclidean distances between different snail populations were computed for cluster analysis, and a correlation matrix was computed among the variables for the principal components analysis. Results Among 27 snail populations, The Euclidean distance between snail populations from Guichi and Zongyang in Anhui province was the least （0.62） and between snail populations from Danleng, Sichuan province and Huarong, Hunan province was the most （10.29）. Whether including a snail population from Guangxi Province or not, the median among O.h.robertsoni was less than that among O.h.hupensis （P<0.05）. The median among ribbed-shell populations was bigger than that among smooth-shell populations （P<0.05）. The result of the principle component analysis showed that three species-groups could be divided: ① Jiangling, Guichi, Zongyang, Dantu, Tongling 1, Jiangning; ② Nanchang, Huarong, Duchang, Hanchuan, Yangxin, Caidian, Nanxian, Xingzi, Junshan, Jiangshan; ③ Yixing, Xichang, Guanghan, Pujiang, Danleng, Dali, Fuqing, Shimen, Tongling 2, Yizhou, Puge. Conclusion Morphologic variation among O.h.hupensis was bigger than that among O.h.robertsoni, andMorphologic variation among ribbed-shell populations was bigger than that among smooth-shell populations. The clustering result of snail populations in morphologic level was consistent basically with environmental type.In part fiveIn section one, Objective To explore the spatial genetic structure of Oncomelania hupensis from China’s mainland. Methods The correlations between genetic variations and geographical distances were analyzed. Results There was significant positive correlation between genetic and geographical distance for twenty five snail populations （P<0.001）, and the correlation coefficient was 0.5234 for genetic distance D, was 0.5622 for Nei’s unbias genetic distance; The correlation between genetic and geographical distance was significant among snail populations of O. h. hupensis （P<0.001）, and the correlation coefficient was 0.5276 for genetic distance D, was 0.5770 for Nei’s unbias genetic distance; Whether ribbed-shelled or smooth-shelled snail, there were significant positive correlations between genetic and geographical distances （P<0.001）. For ribbed-shelled snail, the correlation coefficient was 0.3612 for genetic distance D, was 0.3916 for Nei’s unbias genetic distance, and for smooth-shelled snail, the correlation coefficient was 0.7535 for genetic distance D, 0.7500 for Nei’s unbias genetic distance. Conclusion There was significant spatial genetic structure among snail populations of Oncomelania hupensis widespread distributed in China’s mainland.In section two, Objective To explore morphologic variations and their spatial correlation among Oncomelania hupensis populationsfrom China’s mainland. Methods twenty seven snail populations from ten provinces were collected, and the correlations between morphologic variations and geographical distances were analyzed. Results There was a significant correlation between morphologic variation and geographic distance among 27 snail populations （P<0.001） and the correlation coefficient was 0.2791; The significant correlations also were found among O.h.hupensis and ribbed-shell populations, but not found among O.h.robertsoni and smooth-shell populations （P>0.05）. The correlation coefficient was 0.2655 for O.h.hupensis including a snail population from Guangxi province, 0.2567 not including it; was 0.3121 for ribbed-shell population. Conclusion There was significant spatial structure among ribbed-shell snail populations of Oncomelania hupensis distributed in China’s mainland.更多还原