Molecular identification of four species of trematode larvae in freshwater snails from Qiqihar area
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.
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