OBJECTIVE: To investigate the sequences of internal transcribed spacer 2 (ITS2) and cyclooxygenase 1 (COX1) genes of Paragonimus metacercariae in freshwater crabs in Henan Province, identify the species of Paragonimus and evaluate its genetic relationships with Paragonimus isolates from other provinces in China.
METHODS: Freshwater crabs were collected from 8 survey sites in Zhengzhou, Luoyang, Pingdingshan, Nanyang and Jiyuan cities of Henan Province from 2016 to 2021, and Paragonimus metacercariae were detected in freshwater crabs. Genomic DNA was extracted from Paragonimus metacercariae, and the ITS2 and COX1 genes were amplified using PCR assay, followed by sequencing of PCR amplification products. The gene sequences were spliced and aligned using the software DNASTAR, and aligned with the sequences of Paragonimus genes in the GenBank. Phylogenetic trees were created using the MEGA6 software with the Neighbor-Joining method based on ITS2 and COX1 gene sequences, with Fasciola hepatica as the outgroup.
RESULTS: The detection rates of Paragonimus metacercariae were 6.83% (11/161), 50.82% (31/61), 18.52% (5/26), 8.76% (12/137), 14.29% (9/63), 17.76% (19/105), 18.50% (32/173) and 42.71% (41/96) in freshwater crabs from 8 survey sites in Zhengzhou, Luoyang, Pingdingshan, Nanyang and Jiyuan cities of Henan Province, with a mean detection rate of 19.46% (160/822), and a mean infection intensity of 0.57 metacercariae/g. The amplified ITS2 and COX1 gene fragments of Paragonimus were approximately 500 bp and 450 bp in lengths, respectively. The ITS2 gene sequences of Paragonimus metacercariae from 8 survey sites of Henan Province showed the highest homology (99.8% to 100.0%) with the gene sequence of P. skrjabini (GenBank accession number: MW960209.1), and phylogenetic analysis showed that the Paragonimus in this study was clustered into the same clade with P. skrjabini from Sichuan Province (GenBank accession number: AY618747.1), Guangxi Zhuang Autonomous Region (GenBank accession number: AY618729.1) and Hubei Province (GenBank accession number: AY618751.1), and P. miyazaki from Fujian Province (GenBank accession number: AY618741.1) and Japan (GenBank accession number: AB713405.1). The COX1 gene sequences of Paragonimus metacercariae from 8 survey sites of Henan Province showed the highest homology (90.0% to 100.0%) with the gene sequence of P. skrjabini (GenBank accession number: AY618798.1), and phylogenetic analysis showed that the Paragonimus in this study was clustered into the same clade with all P. skrjabini and clustered into the same sub-clade with P. skrjabini from Hubei Province (GenBank accession numbers: AY618782.1 and AY618764.1).
CONCLUSIONS: Paragonimus species from freshwater crabs in Henan Province were all characterized as P. skrjabini, and the ITS2 and COX1 gene sequences had the highest homology to those of P. skrjabini from Hubei Province. The results provide insights into study of Paragonimus in Henan Province and China.
[摘要] 目的 分析河南省溪蟹体内并殖吸虫囊蚴内转录间隔区 2 (internal transcribed spacer 2, ITS2) 和环氧化酶 1 (cyclooxygenase 1, COX1) 基因序列, 鉴定并殖吸虫虫种, 并分析其与国内其他省份并殖吸虫间的遗传学关系。方法 2016—2021 年, 从河南省郑州市、洛阳市、平顶山市、南阳市及济源市等 5 个市 8 个调查点采集溪蟹, 检出溪蟹中并殖吸虫囊蚴, 计算 溪蟹囊蚴感染率; 提取囊蚴 DNA 进行 PCR 扩增并测序。使用 DNASTAR 软件拼接测序结果、进行基因序列比对, 同时与 GenBank 数据库中并殖吸虫基因序列进行 BLAST 比对。以肝片吸虫为外群, 使用 MEGA 6.0 软件, 采用邻接法构建基于 ITS2、COX1 基因序列的系统进化树。结果 河南省郑州市、洛阳市、平顶山市、南阳市及济源市等 5 个市 8 个调查点溪蟹 并殖吸虫囊蚴感染率分别为 6.83% (11/161)、50.82% (31/61)、18.52% (5/26)、8.76% (12/137)、14.29% (9/63)、17.76% (19/105)、18.50% (32/173) 和 42.71% (41/96), 平均阳性率为 19.46% (160/822), 平均感染度为 0.57 个/g。PCR 扩增结果显 示, 并殖吸虫ITS2、COX1 基因片段长度分别约为 500、450 bp。河南省 8 个调查点溪蟹并殖吸虫囊蚴ITS2 基因序列与斯 氏并殖吸虫 (GenBank登录号: MW960209.1) 同源性最高, 为 99.8%~100.0%; 基因进化树中与四川省 (GenBank登录号: AY618747.1)、广西壮族自治区 (GenBank登录号: AY618729.1)、湖北省斯氏并殖吸虫 (GenBank 登录号: AY618751.1) 以 及福建 (GenBank 登录号: AY618741.1) 和日本宫崎并殖吸虫 (GenBank 登录号: AB713405.1) 聚为 1 个大分支。COX1 基因 序列与斯氏并殖吸虫 (GenBank 登录号: AY618798.1) 同源性最高, 为 90.0%~100.0%; 在进化树中与所有斯氏并殖吸虫 聚为 1 个大分支, 且与湖北省斯氏并殖吸虫 (GenBank 登录号: AY618782.1、AY618764.1) 聚为 1 个小支。结论 河南省 溪蟹体内并殖吸虫虫种均为斯氏并殖吸虫, 且与湖北省斯氏并殖吸虫基因序列同源性最高。本研究结果可望为河南省 以及全国并殖吸虫研究提供参考。.