齐齐哈尔地区淡水螺体内4种吸虫幼虫的分子鉴定
Molecular identification of four species of trematode larvae in freshwater snails from Qiqihar area
李健科 1张静 1刘柳 1刘谦豪 1张浩1
作者信息
- 1. 齐齐哈尔医学院,黑龙江齐齐哈尔,161000
- 折叠
摘要
目的 对分离自齐齐哈尔市嫩江流域淡水螺体内的4种吸虫幼虫进行分子生物学鉴定.方法 2023年3-7月在齐齐哈尔市嫩江浏园段水域内采集淡水螺,分类鉴定后压碎螺壳,显微镜下观察内脏团,分离螺体内的吸虫幼虫.分别提取不同吸虫幼虫总DNA,PCR扩增吸虫幼虫内转录间隔区2(ITS2),对扩增产物进行测序,利用Contig Express软件拼接后在NCBI网站进行序列比对.用MEGA 11.0软件以邻接法构建系统进化树并计算遗传距离.结果 共采集到7种淡水螺(2771只),其中阳性螺3种,分别为黑龙江短沟蜷(282只)、中国圆田螺(709只)和绘环棱螺(142只).共检出4种吸虫幼虫,每只淡水螺只寄生1种吸虫幼虫,分别为寄生于黑龙江短沟蜷和绘环棱螺体内的幼虫a(阳性率25.23％,107/424),寄生于中国圆田螺体内的幼虫b(阳性率2.82％,20/709),寄生于黑龙江短沟蜷体内的幼虫c(阳性率0.70％,2/282)和寄生于中国圆田螺体内的幼虫d(阳性率0.56％,4/709).幼虫a～d的ITS2目的序列扩增长度分别约为523、701、960和554 bp.基因测序及序列比对结果显示,幼虫a ITS2序列与Notocotylus ephemera(GenBank:OP720890.1)序列一致性最高,为98.49％;与N.ephemera和Notocotylidae sp.的遗传距离最近,均为0.014,推测幼虫a为背孔科吸虫.幼虫b ITS2序列与卷棘口吸虫(GenBank:GQ463130.1)序列一致性最高,为94.56％;与卷棘口吸虫的遗传距离最近,为0.085,推测幼虫b 为棘口科棘口属吸虫.幼虫 c ITS2 序列与 Echinochasmus suifunensis(GenBank:MT447049.1)序列一致性最高,为99.82％;与E.milvi的遗传距离最近,低于0.001,推测幼虫c为棘口科棘隙属吸虫.幼虫d ITS2序列与马尔科维奇侧殖吸虫(GenBank:OP106430.1)序列一致性最高,为92.36％;与Asymphylodora parasquamosa的遗传距离最近,为0.090,推测幼虫d为单睾科侧殖属吸虫.结论 齐齐哈尔嫩江流域内淡水螺体内可能寄生有背孔科吸虫、棘口科棘口属和棘隙属吸虫、单睾科侧殖属吸虫,提示可能危害鱼类、家禽及哺乳动物健康.
Abstract
Objective Molecular identification of four species of trematode larvae isolated from freshwater snails in the Nenjiang River Basin in Qiqihar City.Methods Freshwater snails were collected from the Liuyuan sec-tion of the Nenjiang River in Qiqihar City from March to July 2023.After classification and identification,the shell was crushed,the visceral mass was observed under a microscope,and the trematode larvae in the snails were iso-lated.The total DNA of different trematode larvae was extracted,and the internal transcribed spacer region 2(ITS2)of trematode larvae was amplified by PCR,and the amplified products were sequenced.After splicing using Contig Ex-press software,the sequence consistency was compared on the NCBI website.The phylogenetic tree was using the neighbor-joining method,and the genetic distance was calculated using MEGA 11.0 software.Results A total of 7 species of freshwater snails(2 771 snails)were collected,of which 3 species were positive,including Koreoleptoxis amu-rensis(282 snails),Cipangopaludina chinensis(709 snails)and Bellamya limnophila(142 snails).A total of 4 species of trematode larvae were detected,and each freshwater snail only parasitized 1 species of trematode larvae,which were lar-vae a(positive rate 25.23％,107/424)parasitized in K.amurensis and B.limnophila,larvae b(positive rate 2.82％,20/709)parasitized in C.chinensis,larvae c(positive rate 0.70％,2/282)parasitized in K.amurensis and larvae d(positive rate 0.56％,4/709)parasitized in C.chinensis.The amplified lengths of ITS2 target sequences of larvae a-d were about 523 bp,701 bp,960 bp and 554 bp,respectively.The results of gene sequencing and sequence alignment showed that the larvae a ITS2 sequence had the highest identity with Notocotylus ephemera(GenBank:OP720890.1)sequence,which was 98.49％.It had the closest genetic distance with N.ephemera and Notocotylidae sp.,both of which were 0.014.It was speculated that larva a was a trematode of the Notocotylidae.The larvae b ITS2 sequence had the highest consis-tency with the sequence of Echinostoma revolutum(GenBank:GQ463130.1),which was 94.56％.The genetic distance with E.revolutum is the closest,which is 0.085.It is speculated that larva b is a trematode of the genus Echinostoma in the family Echinostomatidae.The larvae c ITS2 sequence had the highest consistency with Echinochasmus suifunensis(GenBank:MT447049.1)sequence(99.82％).The genetic distance with E.milvi was the closest,less than 0.001,suggest-ing that larva c was a trematode of the genus Echinochasmus of the family Echinostomatidae.The larvae d ITS2 se-quence had the highest consistency with the sequence of Asymphylodora markewitschi(GenBank:OP106430.1),which was 92.36％.The genetic distance with A.parasquamosa is the closest,which is 0.090,suggesting that the larva d was a trematode of the genus Asymphylodora of the family Monorchiidae.Conclusion Freshwater snails in the Nenjiang River Basin of Qiqihar may harbour trematodes of the Notocotylidae,Echinostoma and Echinochasmus of the Echinosto-matidae,and Asymphylodora of the Monorchiidae,potentially endangering the health of fish,poultry and mammals.
关键词
淡水螺/吸虫/幼虫/ITS2序列/种类鉴定Key words
Freshwater snail/Trematodes/Cercaria/ITS2 sequence/Species identification引用本文复制引用
基金项目
齐齐哈尔医学院科研项目(QYS2019Z-06)
校企合作横向课题()
出版年
2024
NETL
NSTL
万方数据