应用微卫星DNA标记探讨我国长江中下游地区湖北钉螺群体遗传结构

目的应用微卫星DNA分子标志分析中国长江中下游地区湖北钉螺群体的遗传结构。方法利用6对微卫星DNA引物,对采集自湖沼型血吸虫病流行区的湖南汉寿县、湖北阳新县、江西星子县、安徽当涂县和江苏扬州邗江区的湖北钉螺的P84、T5-13、T5-11、T4-22、T6-27和P82等6个位点进行荧光标记通用引物PCR扩增。每个采集点采集20～50个钉螺标本,共165个。统计分析各群体的等位基因数(Na)、近交系数(FIS)、期望杂合度(He)、观察杂合度(Ho)、多态信息含量(PIC)、群体间的遗传分化系数(FST)和遗传距离,根据遗传距离进行聚类分析,并进行分子变异方差分析(AMOVA)。结果 5个湖北钉螺群体在6个微卫星位点等位基因数范围为3～33,平均为15.833。群体的平均He和Ho范围分别为0.600～0.883和0.308～0.759,两者在湖北群体最高,在江苏群体最低。群体的FIS范围为0.143～0.539。成对群体间的FST范围为0.0006～0.0531,由于FST值较小,提示群体间未出现明显遗传分化。各群体的平均PIC为0.511～0.850,除江苏群体外,均为高度多态。分级AMOVA结算结果显示,变异主要存在于个体间,占总变异的95.2%。聚类分析结果显示,安徽群体与江苏群体首先聚为一支,然后依次同湖南、江西、湖北群体聚在一起。结论湖北钉螺多样性较为丰富;聚类分析结果符合湖北钉螺的地理分布格局;5个群体之间遗传差异较小,微卫星DNA的变异主要存在个体间。     

湖北钉螺数量性状研究Ⅲ种群间形态性状变异及其空间相关分析

目的 探讨湖北钉螺种群间形态性状变异及其空间相关性。方法 采集10个省的27个现场螺点的钉螺种群标本,分别测量螺壳的形态学指标及钉螺采集点之间的地理距离,分析钉 螺种群间形态性状变异及其与地理距离的相关性。结果20个指名亚种（包括1个广西钉螺种群）钉螺种群间欧氏距离的中位数为3. 24（95% CI:2. 88～3.81）;不包括广西钉螺种群时,中位数为 3.15 (95% CI: 2.86～3.76)。6个滇川亚种钉螺种群间欧氏距离的中位数为1.69 (95% CI：1.33～ 2. 27)。无论指名亚种是否包括广西钉螺种群,滇川亚种钉螺种群间的欧氏距离均明显低于指 名亚种种群(P<;0.05)。16个肋壳钉螺种群间欧氏距离的中位数为2. 86(95% CI：2.56～3, 23);11 个光壳钉螺种群间欧氏距离的中位数为2. 24（95% CI：2.00～2.43）,明显低于肋壳钉螺种群间的 欧氏距离(P<0.05)。指名亚种包括广西钉螺种群时γ为0. 265 5 (P<0. 01),不包括时γ为0．256 7(P<0.01),肋壳钉螺种群的γ为0．312 1(P<0．01)。结论 湖北钉螺指名亚种种群间的形态性状变异大于滇川亚种,肋壳钉螺种群间的形态性状变异大于光壳钉螺。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问结构。     

湖北省庙河地区钉螺细胞色素C氧化酶1基因差异的研究

目的　比较湖北省庙河沿岸地区钉螺CO1基因序列的差异 ,探讨光壳螺与肋壳螺差异的原因。方法　在该地区选 7个点 (上游 4个点 ,下游 3个点 )采集钉螺 ,用CTAB法提取钉螺基因组DNA ,PCR方法扩增CO1基因 ,纯化后测序 ,运用ESEE软件排序并比较变异位点 ,观察各点钉螺的CO1基因单倍体型 ,运用PHYLIP软件计算遗传距离 ,绘制基因进化树。结果　获得CO1基因大小为 638bp ,上游和下游累积变异位点数分别为 2 9和 46,两者差异具有显著性 ;各采集点内钉螺按变异位点多少可分为两组 ;各采集点间存在有相同的基因单倍体型 ;上游和下游螺群之间的遗传距离为 0 0 2 2 1± 0 0 10 5 ;在FITCH绘制的基因进化树上 ,上游和下游地区钉螺交错分布在同一亚种不同的两个分支中。结论　在不同的生态环境下 ,下游地区钉螺CO1基因的变异强度比上游大 ;庙河各采集点螺群间存在一定的基因流动 ;庙河地区螺群属湖北钉螺湖北亚种 (O h hupensis) ,可能存在着两种不同进化速率的螺群。     

我国湖北钉螺各地域株外形数据库的建立及其分类的初步应用

目的建立湖北钉螺外形地域分布数据库并探讨其应用价值，为钉螺分类的进一步研究奠定基础。方法利用1950年代及1998年以后收集的钉螺标本，测量其螺层、壳高、壳宽、次螺层体长及外唇；观察螺壳表面纵肋有无及粗细程度，螺顶为粉红色及螺顶磨损情况，并按照地区以县为单位归类，建立外形地域分布数据库。对该数据库进行钉螺外形特征分类，并进行部分区域的钉螺50年前后外形对比；检索与姚氏钉螺相关具红色螺顶特征的钉螺分布区域；检索与湖北大口钉螺相关具螺顶磨损特征的钉螺分布区域。结果建立了湖北钉螺外形地域分布数据库和50年前后外形对比数据库；具螺顶磨损特征的钉螺除湖北大口外尚有其他2个分布区域；螺顶泛红的湖北钉螺除广西宾阳及其周边的两个县（市）外尚有其他9个分布区域。结论根据钉螺纵肋形态可分为4类：光壳、细肋、肋壳、粗肋；钉螺外形在50年内未发生明显变异。大口钉螺螺顶磨损现象罕见。螺顶泛红是广西宾阳、武鸣、忻城三地钉螺极重要的特征，但并非惟该地区所拥有。     

湖北钉螺数量性状研究Ⅰ.遗传变异信息的可靠性与样本量的关系

目的　探讨湖北钉螺数量性状遗传变异信息的可靠性与样本量的关系。方法　从云南大理和湖南岳阳两地区随机抽取钉螺各6 0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(CV)、组间变异百分比和多样性指数。结果　CV以外唇缘厚度变异最大,肋壳钉螺达到了2 0 .5 7% ,光壳钉螺为14 .14 % ,其次是次螺层长度,分别12 .98%和11.4 9% ;肋壳钉螺有近四分之三的性状指标CV大于光壳钉螺的,且平均CV9.35 %也大于光壳钉螺的7.83% ;肋壳钉螺的平均多样性指数为1.5 4 1,高于光壳钉螺的1.2 2 0 ;当样本量<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>5 5时,所得到的遗传变异的结果趋于稳定。结论　在湖北钉螺形态数量性状的遗传变异研究中,每个钉螺种群以分析不少于5 5个钉螺标本为好。     

中国大陆钉螺种群遗传学研究 Ⅰ.种群遗传变异

用采自34个现场螺点的钉螺进行水平淀粉凝胶电泳,并进行13种酶系统染色.结果17个等位基因中,7个为多态基因位点.种群间遗传距离的变化范围0.03—0.27.UPGMA、Fitch-Mar-golish最小方差法、最大相似性聚类分析图显示,聚类的组别与形态学或/和地理分布相一致.各分析指标提示,肋壳钉螺种群显著地不同于光亮钉螺种群,两组间的遗传距离为0.15.     

基于微卫星DNA标记的山东微山湖区放养钉螺遗传结构分析

目的分析在山东微山湖地区长期放养的湖北钉螺(Oncomelania hupensis)群体与其来源地江苏省长江江滩钉螺群体之间的基因差异。方法收集江苏省扬州市江都区、仪征市和丹阳市、南京市六合区的长江钉螺以及山东省济宁市微山湖独山岛的钉螺。微山湖区放养的钉螺来自于扬州市江都区,放养时间为10年(至2014年)。选择A18、C22、T4-33、T6-17等4对微卫星位点对钉螺群体进行PCR扩增,分析各钉螺群体的等位基因数(Na)、期望杂合度(He)、观察杂合度(Ho)和群体间遗传差异平均数等指标,并进行分子变异方差分析(AMOVA),用非加权组平均法(UPGMA)和邻接法(NJ)构建系统进化树描述群体遗传分化及聚类情况。使用Mantel检验探讨遗传距离与地理距离的相关性。结果本研究收集5个钉螺群体共103只钉螺。江都、仪征、丹阳、六合、微山湖群体的Na分别为7.50、12.50、10.00、11.50和12.75;Ho分别为0.16、0.27、0.17、0.30和0.22;He分别为0.81、0.91、0.84、0.90和0.92。微山湖群体的3个遗传多样性指标均在较高水平,提示有较好的遗传多样性,但与其他群体差异无统计学意义(P0.05)。微山湖与江都钉螺群体间遗传差异平均数最小(0.79),丹阳群体与其他群体之间差异较大,为0.87~0.97。AMOVA分析的遗传变异主要来源于钉螺个体间(占92.50%)。NJ和UPGMA系统进化树显示,江都和微山湖的钉螺群体聚为一支,仪征和六合的钉螺群体聚为一支,丹阳钉螺群体与其他4个群体聚为一支。Mantel检验显示遗传距离与地理距离没有相关性。结论江苏省长江钉螺群体与微山湖钉螺群体基因多样性均较高,微山湖地区放养的钉螺与来源地的长江钉螺群体尚未出现明显遗传分化。     

钉螺在巢湖生存繁殖的现场研究

目的了解钉螺能否在巢湖生存繁殖.方法选择巢湖湖区的马尾河口和巢湖闸两处为实验区,巢湖东岸高林山区为对照区,投放无为县江外滩的肋壳钉螺和巢湖东岸高林山区的光壳钉螺,观察钉螺在放养不同时间后的生存繁殖情况.结果1年后,实验区和对照区均有相当数量存活的肋壳钉螺和光壳钉螺成螺;研究期间在实验区和对照区均检获了一定数量的光壳钉螺和肋壳钉螺幼螺;产卵期间实验区和对照区肋壳钉螺存活率差异无显著性(P＞0.05),而光壳钉螺存活率差异具有显著性(P＜0.05);产卵期间无论是光壳钉螺还是肋壳钉螺,各实验区雌螺卵巢中所含螺卵数与同一月份对照区同类雌螺卵巢中所含螺卵数的差异均无显著性(P均＞0.05).结论肋壳钉螺、光壳钉螺均能够在巢湖生存繁殖,但光壳钉螺在巢湖的生存状况不及其在高林山区的生存状况理想.     

湖北钉螺与中华圆田螺体内酚氧化酶活力比较

目的 比较湖北钉螺及中华圆田螺体内酚氧化酶活力差异。方法 以螺软体组织制备粗制酶液,以邻苯二酚为底物,测定肋壳钉螺、光壳钉螺和中华圆田螺体内酚氧化酶活力。结果 光壳钉螺、肋壳钉螺、中华圆田螺体内酚氧化酶活力值分别为（25.72 ± 2.27）、（14.56 ± 1.24） U/mL和（13.72 ± 1.06） U/mL,光壳钉螺体内酚氧化酶活力值高于肋壳钉螺（q = 21.46,P < 0.05）及中华圆田螺（q = 12.00,P < 0.05）,中华圆田螺与肋壳钉螺体内酚氧化酶活力值差异无统计学意义（q = 1.62,P > 0.05）。结论 湖北钉螺及中华圆田螺体内酚氧化酶活力存在差异,可能与螺的生活环境有关。     

三峡库区上、下游湖北钉螺基因组DNAITS2遗传多态性研究

目的分析三峡库区上、下游湖北钉螺核糖体DNA第2转录间隔区（rDNA-ITS2）基因的遗传变异。方法采集三峡库区上游四川、云南及下游安徽、湖北4省8地（市）钉螺,提取其基因组DNA,PCR特异性扩增rDNA ITS2基因并测序,ClustalX（1.81）软件进行多序列比对,MEGA（3.1）软件Kimura2-parameter法计算遗传距离,非加权组平均法（UPGMA）和最小进化法（ME）构建系统发生树。结果上游与下游不同地域株钉螺间ITS2基因差异约为11%,下游地区的肋壳与光壳钉螺ITS2基因差异约为3.8%,上游螺群间的遗传距离为0.001～0.013,下游螺群间的遗传距离为0.004～0.017,上游与下游螺群间的遗传距离在0.033～0.041之间。2种方法构建的系统发生树其拓扑结构一致,分为2大支,上游的云南和四川地理株形成一支系,下游的湖北、安徽地理株形成另一支系,既有肋壳钉螺又有光壳钉螺。结论上、下游地理株湖北钉螺间ITS2基因遗传差异较显著,但下游肋壳和光壳钉螺ITS2基因遗传变异小,表明地域距离和生态环境对湖北钉螺的遗传变异影响较大,不能以钉螺纵肋表型的有无作为钉螺种株的判断标准。     

云南省钉螺螺壳形态学性状指标测量

研究云南省钉螺螺壳形态学变异,为钉螺遗传进化研究和控制提供依据。方法　在云南省现有钉螺分布区,综合考虑钉螺密度、地理位置、海拔、水系、环境类型等多个因素后,选取3个州（市）10个血吸虫病流行县（市、区）12个行政村作为钉螺采集点。2021年12月至2022年1月,在每个采集点采集约200只钉螺,随机选取其中30只成年钉螺（螺旋数6～7个）,对钉螺的11个螺壳形态学性状指标进行测量,并进行聚类分析和主成分分析。结果　12个钉螺样本群中,壳高最长的钉螺样本采自洱源县永乐村,为7.33 mm;最短的样本采自古城区东元村,为4.68 mm。壳顶角度最大的样本采自弥渡县蔡庄村,为59.47°;角度最小的样本采自洱源县前甸村,为41.40°;12个钉螺样本群的螺壳形态学性状平均变异系数为9.075%,其中以外唇缘厚度变异系数最大,达29.809%。12个钉螺样本群间平均欧式距离为2.26,其中以洱源县前甸村和大理市五星村两地钉螺样本群间欧式距离最小（0.26）,以弥渡县蔡庄村和楚雄县苍岭村两地样本群间欧式距离最大（8.17）。聚类分析和主成分分析均将12个钉螺样本群划分为3类：弥渡县蔡庄村钉螺样本群,楚雄县苍岭村钉螺样本群,洱源县前甸村、大理市五星村、永胜县杨伍村、祥云县小桥村、洱源县永乐村、大理市小岑村、南涧县安定村、古城区东元村、鹤庆县莲义村、巍山县甸中村钉螺样本群;3类样本群差异主要体现在与壳顶角度和外唇缘厚度相关的主成分2上。结论　随采集点纬度逐渐减小,云南省12个钉螺样本群螺壳形态指标欧式距离及螺壳形态变异程度逐渐增加,壳顶角度可作为反映螺层成长过程的指标之一。     

山东微山湖区子代钉螺壳形变异的研究

目的 目的 探讨钉螺被人为迁移至南水北调山东受水区后其子代钉螺壳形变化特点。方法 方法 取微山湖区独山岛饲 养的钉螺, 以亲代钉螺107只为对照组, 同时测量子1代螺115只、 子2代螺107只, 收集螺体壳长 （L）、 壳宽 （W）、 壳口宽 （AW）、 壳口长 （AL）、 唇脊厚度 （LIT） 等数据, 并计数体螺旋纵肋数。结果 结果 子代钉螺LIT明显减少, 并随子代数增加呈递减 状态, 三代螺两两比较, LIT差异有统计学意义 （P < 0.01）; 同时, 子代螺L和AL降低, W和AW增加, 壳口长与宽之积增加, 体螺旋纵肋数增加。结论 结论 微山湖子代钉螺的螺体变小、 螺壳变薄, 提示微山湖区非完全适宜钉螺孳生, 但是种群可以生 存多年。     

南通市“有螺无病”地区不同条件下血吸虫毛蚴感染钉螺的研究

目的探索南通市有钉螺无血吸虫病流行的原因,验证既往研究结论,为该类地区血吸虫病监测提供科学依据。方法采用控制现场试验方法,现场观察人工输入血吸虫毛蚴感染钉螺情况,同时实验室比较观察"有螺无病"和"有螺有病"区土壤对血吸虫毛蚴感染钉螺的影响。结果除如东县新店镇汤园村(有螺无病)现场光壳螺组外,实验钉螺在"有螺无病"和"有螺有病"现场控制环境均可以被血吸虫毛蚴感染。各组光壳钉螺感染率低于肋壳钉螺,但无统计学意义;各组光壳钉螺死亡率高于肋壳钉螺,差异有统计学意义(χ~2新店=135.118,χ~2双甸=122.836,χ~2白蒲=154.436,χ~2丁堰=138.288,χ~2对照=151.923,P均0.01)。"有螺无病"和"有螺有病"区土壤试验组钉螺感染率差异无统计学意义(χ~2如皋=0.071,χ~2如东=0.216,P均0.05),各组与无土实验对照组差异亦无统计学意义(χ~2=7.148,P0.05)。结论南通地区"有螺无病"区在输入足够数量血吸虫病传染源后有可能形成血吸虫病的传播,有必要开展血吸虫病和钉螺监测工作。     

用RAPD技术对湖北大口等3地的钉螺遗传差异初探

目的对3个不同地点的湖北钉螺的遗传差异进行研究。方法采用随机引物扩增多态性DNA(RAPD)技术,对湖北省三个不同地域的钉螺进行PCR扩增,根据DNA扩增产物的琼脂糖凝胶电泳结果,计算湖北钉螺之间的遗传距离,绘制系统进化树,并结合形态学观察,生态环境调查,综合分析得出结论。结果不同地点湖北钉螺标本PCR扩增产物均呈多态性,钟祥市的大口与冷水距离很近,但两地的钉螺遗传距离大于远安县与钟祥市钉螺的遗传距离。大口林场的钉螺壳顶磨损明显。结论生态环境对遗传变异的影响较大,地理距离与遗传距离不成正比。     

Allozyme-based genetic variation within an unstable "population" of Chinese Oncomelania hupensis (Gastropoda: Rissoacea: Pomatiopsidae)]

OBJECTIVE: To answer the following questions: 1. For Oncomelania snails collected two years apart from the same locality, has there been genetic divergence? 2. How much experimental error has there been in studying subsets of these populations? 3. As this is an unstable population, what has the net effect been on Hardy-Weinberg equilibrium (HWe)? METHODS: Allozymes were studied using horizontal starch gel electrophoresis. Data collected from numbers of experiments were compiled. Data from each collection were divided into two equal subsets based on chronology of the experiments. Thirty-four loci were studied using 72 to 180 snails per subset. RESULTS: The mean number of alleles per locus ranged from 1.5-1.9. With each consecutive subset, the % polymorphic loci dropped from 38.2 to 17.6. The mean heterozygosity was very low: 0.033 to 0.049 and not significantly different from Hardy-Weinberg expectations. Ten loci and 11 alleles exclusive to the first group were eliminated from the overall study reducing the number of polymorphic loci from 19 to 10. There were significant departures from HWe at five loci having a substantial number of individuals for each allele. Nei's and Wright's D were 0.003 +/- 0.001 and 0.054 +/- 0.006 respectively. CONCLUSION: 1. There were significant errors seen primarily in the results scored in the earliest experiments. 2. These earlier errors involving scoring difficult to resolve loci, and interpretation of rare alleles that were not found in later experiment had no significant effect on overall genetic distance. 3. The use of Wright's D for closely related populations is explained. Results with Nei's D indicated no significant difference among the four subunits; Wright's D yielded significant difference between the collections made two years apart, attributed to the annual flooding of the Yangtze River mixing snails from different localities. 4. Major polymorphic loci were not in Hwe as predicted using the unstable population model. 5. One must study 25 or more individuals to find relatively rare alleles and study population genetics.     

长江下游3省湖北钉螺不同地理种群的群体遗传结构研究

目的 目的 对长江下游江西、 安徽和江苏3省湖北钉螺的不同地理种群进行群体遗传结构分析, 以探讨rDNA?ITS分 子标记在钉螺扩散路径监测中的应用价值。 方法 方法 采集3省9个种群的钉螺样本, 提取基因组DNA, PCR特异性扩增rD? NA?ITS基因并克隆测序, 统计分析群体间的遗传分化系数 （Fst ）、 遗传距离和种群遗传多样性等参数, 利用单倍型构建家系 网络图。结果 结果 9个种群共获得有效序列93条, 检测到78个单倍型, 平均单倍型多样性、 核苷酸多样性分别为0.988和 0.012 88, 表明钉螺种群的遗传多样性水平较高; 其遗传变异主要来自群体内, 种群间遗传距离为0.001 6～0.002 3, 显示钉 螺种群间遗传分化程度较高; 家系网络图显示所有单倍型虽可形成3个主要的家系分支, 但各地理种群在家系网络图中无 明显分化。结论 结论 长江下游江西、 安徽和江苏3省沿长江分布的湖北钉螺群体遗传多样性主要存在个体间, 群体间未形成 明显的遗传分化。rDNA?ITS分子标记在钉螺扩散路径监测中的应用价值值得进一步探讨。     

Oncomelania hupensis (Gastropoda: rissooidea) in eastern China: molecular phylogeny, population structure, and ecology

The rissooidean snail genus Oncomelania is of medical interest as various taxa are hosts for the human blood fluke Schistosoma and the lung fluke Paragonimus; because of close co-evolved host-parasite-relationships, snail diversity may reflect parasite diversity. There is a considerable amount of confusion regarding the identity of smooth- and ribbed-shelled populations of Oncomelania hupensis in eastern China. We therefore studied the genetic variation, population structure, phylogenetic relationships and ecology of five smooth- and five ribbed-shelled populations in Hubei, Hunan, Anhui, Zhejiang, and Jiangsu provinces. Based on sequencing data of a fragment of the mitochondrial gene for cytochrome oxidase I from 80 individuals, we found little genetic variability within the ingroup-individuals studied here (average pi=0.01922). Moreover, within the ingroup, smooth-shelled individuals cluster together with ribbed-shelled individuals. We therefore consider all smooth- and ribbed-shelled populations of Oncomelania throughout the lower Yangtze River basin to belong to the subspecies O. hupensis hupensis. Our data indicate that ribbing in O. h. hupensis is associated with the annual floods of the Yangtze River. The greatest haplotype (d(H)) and nucleotide diversities (pi) are found in aggregates of ribbed-shelled snails along areas of the Yangtze River drainage subject to flooding. In areas not affected by flooding, the shells are smooth and genetic diversity decreases significantly.     

应用同工酶技术研究我国湖北钉螺(腹足纲:麂眼螺超科:圆口螺科)不稳定螺群体内的遗传变异

[Objective] To answer the following questions:① For Oncomelania snails collected two years apart from the same locality,has there been genetic divergence?②How much experimental error has there been in studying subsets of these populations? ③As this is an unstable population,what has the net effect been on Hardy-Weinberg equilibrium(Hwe)?[Methods] Allozymes were studied using horizontal starch gel electrophoresis.Data collected from numbers of experiments were conapiled.Data from each collection were divided into two equal subsets based on chronology of the experiments.Thirty-four loci were studied using 72 to 180 snails per subset.[Results] The mean number of alleles per locus ranged frcra 1.5～1.9.With each consecutive subset,the 96 polymorphic loci dropped from 38.2 to 17.6.The mean heterozygosity was very low:0.033 to 0.049 and not significantly different from Hardy-Weinberg expectations.Ten loci and 11 alleles exclusive to the first group were eliminated from the overall study reducing the number of polymorphic loci from 19 to 10.There were significant departures from Hwe at five loci having a substantial number of individuals for each allele.Nei's and Wright's D were 0.003±0.001 and 0.054±0.006 respectively.[Conclusion] ①There were significant errors seen primarily in the results scored' in the earliest experiments.②These earlier errors involving scoring difficult to resolve loci,and interpretation of rare alleles that were not found in later experiment had no significant effect on overall genetic distance.③The use of Wright's D for closely related populations is explained.Results with Nei's D indicated no significant difference among the four subunits; Wright's D yielded significant difference between the collections made two years apart,attributed to the annual flooding of the Yangtze River mixing snails from different localities.④ Major polymorphic loci were not in Hwe as predicted using the unstable population model.⑤One must study 25 or more individuals to find relatively rate alleles and study population genetics.     

ALLOZYME-BASED GENETIC VARIATION WITHIN AN UNSTABLE “POPULATION” OF CHINESE ONCOMELANIA HUPENSIS (GASTROPODA: RISSOACEA: POMATIOPSIDAE)

[Objective] To answer the following questions:① For Oncomelania snails collected two years apart from the same locality,has there been genetic divergence?②How much experimental error has there been in studying subsets of these populations? ③As this is an unstable population,what has the net effect been on Hardy-Weinberg equilibrium(Hwe)?[Methods] Allozymes were studied using horizontal starch gel electrophoresis.Data collected from numbers of experiments were conapiled.Data from each collection were divided into two equal subsets based on chronology of the experiments.Thirty-four loci were studied using 72 to 180 snails per subset.[Results] The mean number of alleles per locus ranged frcra 1.5～1.9.With each consecutive subset,the 96 polymorphic loci dropped from 38.2 to 17.6.The mean heterozygosity was very low:0.033 to 0.049 and not significantly different from Hardy-Weinberg expectations.Ten loci and 11 alleles exclusive to the first group were eliminated from the overall study reducing the number of polymorphic loci from 19 to 10.There were significant departures from Hwe at five loci having a substantial number of individuals for each allele.Nei's and Wright's D were 0.003±0.001 and 0.054±0.006 respectively.[Conclusion] ①There were significant errors seen primarily in the results scored' in the earliest experiments.②These earlier errors involving scoring difficult to resolve loci,and interpretation of rare alleles that were not found in later experiment had no significant effect on overall genetic distance.③The use of Wright's D for closely related populations is explained.Results with Nei's D indicated no significant difference among the four subunits; Wright's D yielded significant difference between the collections made two years apart,attributed to the annual flooding of the Yangtze River mixing snails from different localities.④ Major polymorphic loci were not in Hwe as predicted using the unstable population model.⑤One must study 25 or more individuals to find relatively rate alleles and study population genetics.