齐齐哈尔地区淡水螺体内4种吸虫幼虫的分子鉴定

doi:10.12140/j.issn.1000-7423.2024.03.012

摘要/Abstract

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（2 771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。

关键词: 淡水螺, 吸虫, 幼虫, ITS2序列, 种类鉴定

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 section 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 isolated. 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 Express 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 amurensis (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 larvae 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 consistency 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, suggesting that larva c was a trematode of the genus Echinochasmus of the family Echinostomatidae. The larvae d ITS2 sequence 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 Echinostomatidae, and Asymphylodora of the Monorchiidae, potentially endangering the health of fish, poultry and mammals.

Key words: Freshwater snail, Trematodes, Cercaria, ITS2 sequence, Species identification

中图分类号:

R383.2

李健科, 张静, 刘柳, 刘谦豪, 张浩. 齐齐哈尔地区淡水螺体内4种吸虫幼虫的分子鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(3): 360-366.

LI Jianke, ZHANG Jing, LIU Liu, LIU Qianhao, ZHANG Hao. Molecular identification of four species of trematode larvae in freshwater snails from Qiqihar area[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2024, 42(3): 360-366.

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	幼虫a（n = 5） Larva a (n = 5)	幼虫b（n = 5） Larva b (n = 5)	幼虫c（n = 5） Larva c (n = 5)	幼虫d（n = 5） Larva d (n = 5)
体部大小/mm Body size/mm	0.607 × 0.072	1.118 × 0.576	0.544 × 0.374	1.387 × 0.368
尾部大小/mm Tail size/mm	0.789 × 0.104	0.876 × 0.153	0.990 × 0.216	-
口吸盘大小/mm Oral sucker size/mm	0.062 × 0.063	0.158 × 0.106	0.191 × 0.089	0.212 × 0.179
腹吸盘大小/mm Ventral sucker size/mm	-	0.155 × 0.152	0.083 × 0.089	0.245 × 0.229
口腹吸盘距离/mm Distence between oral sucker and ventral sucker/mm	-	0.612	0.228	0.453
特征性结构 Characteristic structure	眼点2个 2 ocellis	头冠处可见一排小棘 A row of small spines is visible at the crown of the head	尾部宽大，末端有透明囊状物 Tail broad, terminating in a transparent vesicle	睾丸、卵巢各一 One testicle, one oarium