单一性别日本血吸虫尾蚴的获得

Isolation of single-sex cercaria of Schistosoma japonicum

阐明人工制备阳性钉螺释放日本血吸虫尾蚴性别类型的规律和特点,为需要获取单一性别尾蚴的研究提供理论依据。通过毛蚴对钉螺"一对一"(1只毛蚴感染1只钉螺)和"多对一"(约10只毛蚴感染1只钉螺)感染方式分别制备阳性钉螺。分别用单只钉螺释放的尾蚴感染单只小鼠, 3周后收集小鼠体内的日本血吸虫童虫,通过显微镜观察鉴定童虫的性别,提取童虫DNA,用RRPS、 M5A、 MPA、 2AAA、 MF1、 J5N等6对微卫星引物进行PCR扩增,扫板分析短串联重复(STR)位点的多态性,比较每一个钉螺所释放全部尾蚴所发育成的童虫基因型差异。镜检结果表明,"一对一"感染的4只小鼠所收集的血吸虫童虫, 1只为全雌虫, 3只为全雄虫;"多对一"感染的43只小鼠所收集的血吸虫童虫, 19只为全雌虫(44.2%), 7只为全雄虫(16.3%), 17只为雌雄虫共存(39.5%)。各虫体DNA样品, PCR产物琼脂糖电泳的结果和STR扫板分析结果基本一致,但是PCR产物直接电泳分析结果不能精确显示扩增对象的多态性。STR扫板分析结果显示,"一对一"感染的虫体,STR的峰型相似,提示基因型相似,但是同一钉螺来源的不同虫体间仍存在细微差异;"多对一"感染的虫体,STR样品峰型有较大差异,提示样品具有不同的基因型,其中单一钉螺释放的所有尾蚴发育成的童虫有如下特点:雌雄共存,但可分为雌多雄少、雌少雄多和雌雄对等3种情况;全雄虫,基因型相同或不同;全雌虫,基因型相同或不同。总之,毛蚴"一对一"感染钉螺,可以获得基因型一致的、单一性别的尾蚴;混合感染的"多对一"感染方式也可得到单一性别尾蚴,但单性尾蚴基因型不一致。

To obtain the single-sex cercaria for relevant research of schistosomiasis a single miracidium or multiple miracidia were used to infect one snail(one-to-one or multiple-to-one models). The released cercaria from each infected snail were used to infect a mouse. Immature adult worms or schistosomula were collected from each infected mouse 3 weeks after infection and the sex of the worms was identified under microscope. The genotype variation was determined by PCR with 6 short tandem repeats(STRs) primers(RRPS, M5 A, MPA, 2 AAA, MF1 and J5 N). Among 4 mice infected with 'one-to-one' cercaria, single-sex worms were obtained in all mice, one mouse with female and three mice with male worms. However, among 43 mice infected with cercaria from 'multiple-to-one' snails, 19 contained female worms(44.2%), 7 contained male worms(16.3%) and 17 contained mixed-sex worms(39.5%). STR analysis with 6 pairs of microsatellite primers, better than electrophoresis analysis of PCR products,showed the nearly identical genotype for worms collected from 'one-to-one' groups and different genotypes for worms collected from 'multiple-to-one' groups. Minor genetic variation was detected between single-sex worms collected from 'one-to-one' groups possibly due to the mutation(s) occurred during self-reproduction and development in snail. Worms from 'multiple-to-one' groups showed significant genetic polymorphism in terms of different STR patterns, even for the single-sex worms compared to those collected from 'one-to-one' groups. In conclusion, single-sex cercaria can be obtained by infecting a snail with single miracidium( 'one-to-one' model) with nearly identical genotype. Even though the single-sex cercaria are possibly obtained by the multiple-to-one infection model, the genotype is mixed and complicated.