单一性别日本血吸虫尾蚴的获得
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摘要
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.
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.
引文
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[14]毛守白.血吸虫生物学与血吸虫病的防治[M].北京:人民卫生出版社,1990:101-102.
[15]何毅勋,倪传华,刘和香,等.不同数量及时龄的日本血吸虫毛蚴感染钉螺的研究[J].寄生虫与医学昆虫学报,1994(1):22-26.
[16]Minchella DJ,Sollenberger KM,Pereira De Souza C.Distribution of schistosome genetic diversity within molluscan intermediate hosts[J].Parasitology,1995,111(2):217-220.
[17]Sire C,Langand J,Barral V,et al.Parasite(Schistosomamansoni)and host(Biomphalaria glabrata)genetic diversity:population structure in a fragmented landscape[J].Parasitology,2001,122(Pt 5):545-554.
[18]Eppert A,Lewis FA,Grzywacz C,et al.Distribution of schistosome infections in molluscan hosts at different levels of parasite prevalence[J].J Parasitol,2002,88(2):232-236.
[2]WHO.PCT databank[DB/OL].(2014-04-10)[2018-08-02].http://www.who.int/neglected_diseases/preventive_chemotherapy/sch/en/.
[3]张燕,董惠芬,蒋明森,等.血吸虫分子生物学检测技术研究进展[J].中国血吸虫病防治杂志,2017,29(6):798-801.
[4]张利娟,徐志敏,戴思敏,等.2017年全国血吸虫病疫情通报[J].中国血吸虫病防治杂志,2018,30(5):481-488.
[5]许正元,刘惠生,沈明学,等.不同数量血吸虫毛蚴感染钉螺后感染螺逸蚴的计量研究[J].中国血吸虫病防治杂志,1989,1(4):4-8.
[6]杨佩才,张洪英,谢朝勇,等.日本血吸虫单性感染虫体与复性感染虫体的比较[J].中国血吸虫病防治杂志,2016,28(6):683-686.
[7]Sun J,Li C,Wang S.The up-regulation of ribosomal proteins further regulates protein expression profile in female Schistosoma japonicum after pairing[J].PLoS One,2015,10(6):e0129626.
[8]Sun J,Wang S,Li C,et al.Novel expression profiles of microRNAs suggest that specific miRNAs regulate gene expression for the sexual maturation of female Schistosoma japonicum after pairing[J].Parasit Vectors,2014,7:177.
[9]Sun J,Wang SW,Li C,et al.Transcriptome profilings of female Schistosoma japonicum reveal significant differential expression of genes after pairing[J].Parasitol Res,2014,113(3):881-892.
[10]张聪,万伦,李博,等.毛蚴人工感染湖北钉螺的对比观察[J].公共卫生与预防医学,2014,25(4):96-97.
[11]钱宝珍,Shrivastava J,Webster JP,等.中国大陆日本血吸虫微卫星DNA的初步研究[J].中国血吸虫病防治杂志,2004,16(4):241-245.
[12]Shi HP,Lu DB,Shen L,et al.Single-or mixed-sex Schistosoma japonicum infections of intermediate host snails in hilly areas of Anhui,China[J].Parasitol Res,2014,113(2):717-721.
[13]Yin M,Hu W,Mo X,et al.Multiple near-identical genotypes of Schistosoma japonicum can occur in snails and have implications for population-genetic analyses[J].Int J Parasitol,2008,38(14):1681-1691.
[14]毛守白.血吸虫生物学与血吸虫病的防治[M].北京:人民卫生出版社,1990:101-102.
[15]何毅勋,倪传华,刘和香,等.不同数量及时龄的日本血吸虫毛蚴感染钉螺的研究[J].寄生虫与医学昆虫学报,1994(1):22-26.
[16]Minchella DJ,Sollenberger KM,Pereira De Souza C.Distribution of schistosome genetic diversity within molluscan intermediate hosts[J].Parasitology,1995,111(2):217-220.
[17]Sire C,Langand J,Barral V,et al.Parasite(Schistosomamansoni)and host(Biomphalaria glabrata)genetic diversity:population structure in a fragmented landscape[J].Parasitology,2001,122(Pt 5):545-554.
[18]Eppert A,Lewis FA,Grzywacz C,et al.Distribution of schistosome infections in molluscan hosts at different levels of parasite prevalence[J].J Parasitol,2002,88(2):232-236.