Sequence analysis of ribosomal ITS2 gene and mitochondrial CO1 gene of Paragonimus metacercariae from freshwater crabs in Henan, Anhui, Fujian and Zhejiang provinces, China

doi:10.12140/j.issn.1000-7423.2020.01.013

Abstract

Abstract: Objective To analyze the sequences of ribosomal second internal transcribed spacer（ITS2） and mitochondrial cytochrome c oxidase subunit Ⅰ (CO1) genes of Paragonimus metacercariae in 5 areas from Henan, Anhui, Fujian and Zhejiang Provinces of China, and identify the species of Paragonimus metacercariae. Methods From October 2018 to September 2019, freshwater crabs were collected from five survey sites, Lantian Town in Xiuning County of Anhui Province, Yongjia County in Zhejiang Province, Shaoyuan Town in Jiyuan City of Henan Province, as well as Zhenghe County and Zhangzhou City in Fujian Province. Genomic DNA was extracted from Paragonimus metacercariae from the crabs, and the ribosomal ITS2 and mitochondrial CO1 genes were amplified by PCR and sequenced. The amplified sequences were spliced and aligned by DNA Star software along with Blast alignment with the Paragonimus genes in GenBank^TM. Based on the ITS2 and CO1 gene sequences, a phylogenetic tree was constructed with the neighbor-joining method using Fasciola hepatica as the outgroup. Results The metacercaria positive rates found in the crabs from the five survey sites in Lantian Town, Xiuning County (Anhui Province), Yongjia County (Zhejiang Province), Shaoyuan Town, Jiyuan City (Henan Province), Zhenghe County and Zhangzhou City (Fujian Province) were 82% (49/60), 41% (29/70), 55% (41/74), 65% (41/63), and 45% (32/71), respectively. The lengths of amplified ITS2 and CO1 gene sequences were approximately 500 bp and 450 bp, respectively. The ITS2 and CO1 gene sequences of Paragonimus metacercariae from Lantian Town of Xuning County(Anhui Province) and Yongjia County (Zhejiang Province) showed the highest similarity with those of P. westermani (KC417492.1 and AF219379.2), being 99%-100% and 96%-99%, respectively, and both clustered in the same branch with P. westermani. The ITS2 and CO1 genes of Paragonimus metacercariae from Shaoyuan Town of Jiyuan City(Henan Province) showed the highest similarity with those of P. skrjabini (KX129924.1 and MK568551.1), at 98%-100% and 95%-99%, respectively, both being clustered in the same branch with P. skrjabini. The ITS2 gene of Paragonimus metacercariae from Zhenghe County(Fujian Province) showed the highest similarity (98%-100%) with that of P. skrjabini (KX129924.1), forming a clade with P. skrjabini and P. miyazaki (the distance between the two species was not obvious). The CO1 gene from Zhenghe County showed the highest similarity (91%-94%) with that of P. miyazaki (AY618823.1 and AY618834.1), formed a clade with P. miyazaki but was distant from the clade of P. skrjabini. The ITS2 gene of Paragonimus metacercariae from Zhangzhou City (Fujian Province) showed the highest similarity (100%) with the gene of P. westermani (KC417492.1), and the CO1 gene sequence showed the highest similarity (97%-99%) with that of P. cenococopiosus (AF159595.1), being clustered in the with P. cenococopiosus branch. Conclusion The CO1 gene sequence amplified from the metacercariae of 5 areas in 4 Provinces was highly homologous to those of P. westermani, P. skrjabini, P. miyazaki and P. cenococopiosus. The CO1 gene of Paragonimus may be used as a potential molecular marker for species differentiation.

Key words: Paragonimus, Metacercariae, Freshwater crabs, Ribosomal second internal transcribed spacer, Mitochondrial cytochrome c oxidase subunitⅠ

CLC Number:

R383.23

HU Kun-min, CHEN Shao-hong, AI Lin, ZHENG Bin. Sequence analysis of ribosomal ITS2 gene and mitochondrial CO1 gene of Paragonimus metacercariae from freshwater crabs in Henan, Anhui, Fujian and Zhejiang provinces, China[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020, 38(1): 87-94.

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