| 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 Mainland China, 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 mainland China, 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 (GSDICE) 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: ①Jiangling, 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 Mainland China. 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 Mainland China.In section two, Objective To explore morphologic variations and their spatial correlation among Oncomelania hupensis populationsfrom Mainland China. 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 Mainland China.
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