黑龙江省扶余市麦穗鱼舌状绦虫裂头蚴的分子鉴定和系统发育分析

Molecular Identification and Phylogenetic Analysis of Ligula intestinalis Pleroceroid in Pseudorasbora parva from Fuyu City,Heilongjiang Province

为鉴定从黑龙江省扶余市麦穗鱼腹腔内收集的绦虫裂头蚴的种类,本试验采用PCR方法分别对其核糖体内转录间隔区(ITS)和线粒体细胞色素C氧化酶亚基Ⅰ(COⅠ)基因进行扩增和序列测定。运用DNASTAR软件的MegAlign程序分析核苷酸同源性,采用邻接法构建基于ITS和COⅠ基因序列的系统进化树,以此分析分离虫株种内与种间的系统发育关系。结果显示,各分离虫株的ITS1和ITS2基因与舌状绦虫(AY121751)的核苷酸序列同源性最高,分别为98.8%~100%和99.6%~99.7%;各分离虫株的COⅠ基因与肠舌状绦虫(EU241273)的核苷酸序列同源性最高,为91.2%~94.7%。基于ITS序列构建的系统进化树显示,舌状绦虫和双线绦虫的ITS序列具有高度的保守性,ITS区域可能不适宜作为区分舌状绦虫属内部种类的可靠分子标记。基于COⅠ基因构建的系统进化树显示,分离虫株与土耳其、俄罗斯和欧洲的肠舌状绦虫分离株进化关系最近。根据序列同源性和COⅠ基因进化树结果,初步将本试验所分离虫株鉴定为肠舌状绦虫。结果表明,COⅠ基因更适合于舌状绦虫属种类的鉴别。本试验为舌状绦虫的分子流行病学调查提供了参考。

To identify the species of plerocercoids collected from the abdominal cavity of Pseudorasbora parva in Fuyu,Heilongjiang Province,PCR was used to amplify and sequence the ribosomal internal transcribed spacer(ITS)regions and mitochondrial cytochrome C oxidase subunit I(COⅠ)genes.Nucleotide homology was analyzed using the MegAlign program in the DNASTAR software,and phylogenetic trees based on ITS and COⅠgene sequences were constructed using the Neighbor-Joining method to analyze the phylogenetic relationships within and between species.The results showed that the ITS1 and ITS2 genes of the isolated plerocercoids had the highest nucleotide sequence homology with Ligula intestinalis(AY121751),at 98.8%-100%and 99.6%-99.7%,respectively.The COⅠgene of the isolated plerocercoids had the highest nucleotide sequence homology with Ligula intestinalis(EU241273),at 91.2%-94.7%.The phylogenetic tree constructed based on ITS sequences shows that the ITS sequences of Ligula and Digramma tapeworms are highly conserved,indicating that the ITS region may not be a reliable molecular marker for distinguishing species within the genus Ligula.The phylogenetic tree based on the COⅠgene indicated that the isolated plerocercoids were most closely related to Ligula intestinalis isolates from Turkey,Russia,and Europe.Based on sequence homology and the COⅠgene phylogenetic tree,the isolated plerocercoids were preliminarily identified as Ligula intestinalis.The results indicate that the COⅠgene is more suitable for identifying species of the genus Ligula.This study provides a reference for the molecular epidemiological investigation of Ligula intestinalis.