一例海豚源典型异尖线虫形态观察和分子鉴定

Morphological Observation and Molecular Characterization of Anisakis Derived from Dolphin

旨在鉴定由海豚粪便样本中所分离所得线虫种类。首先对所分离虫体进行形态观察,然后提取虫体基因组DNA,并对样本28S rRNA D2-D3基因和ITS基因片段进行PCR扩增,以进行分子生物学鉴定。所得序列经BLAST比对并用邻接法构建系统进化树对所得序列进行分析。结果显示:虫体长120~140 mm,整体呈圆柱形,两端尖细,外观呈乳白色半透明,镜下观察其内部偏黄。可见食道前端细,向后逐渐膨大。PCR结果显示,28S rRNA D2-D3基因与ITS基因扩增长度分别为797和840 bp。BLAST比对结果显示:28S rRNA D2-D3基因(GenBank登录号OR345165)与已报道典型异尖线虫(Anisakis typical, GenBank登录号HF911524.1)的序列一致性为95.36%;ITS基因(GenBank登录号OR345164)与典型异尖线虫(GenBank登录号MF668919.1)一致性为97.11%,其中,ITS-1区域与典型异尖线虫(GenBank登录号MF668919.1)一致性为91.90%,ITS-2区域与典型异尖线虫(GenBank登录号JN968938.1)一致性为94.97%,5.8S rRNA区域与典型异尖线虫(GenBank登录号OQ244221.1)一致性为100.00%。基于ITS基因和28S rRNA D2-D3基因的系统发育分析结果显示,此次研究分离所得寄生虫样本与典型异尖线虫遗传距离最近,且同一分支上。本次分离所得寄生虫样本经过分子鉴定为典型异尖线虫。同源性分析结果显示,其与典型异尖线虫一致性均大于95%;同时,在28S rRNA D2-D3基因序列和ITS基因序列的系统发育分析方面,均与已报道的典型异尖线虫遗传距离最近。

In order to identify the species of nematodes isolated from dolphin fecal samples,the morphology of the isolated nematodes is first identified.The parasite′s body length was observed to be 120-140 mm long,with a cylindrical shape and slender ends.Its appearance was milky white and translucent,and its internal structure was observed to be yellowish under the microscope.It can be seen that the front end of the esophagus is thin and gradually expands backwards.We extracted the genomic DNA of the isolated worm and amplified the 28S rRNA D2-D3 gene and ITS gene fragments by PCR for molecular identification.The PCR results showed that the amplification products of the 28S rRNA D2-D3 gene and ITS gene were 797 and 840 bp,respectively.BLAST alignment results showed that the sequence similarity between the 28S rRNA D2-D3 gene(GenBank accession number OR345165)and the Anisakis typical(GenBank accession number HF911524.1)was 95.36%.The sequence similarity between the ITS gene(GenBank accession number OR345164)and the Anisakis typical(GenBank accession number MF668919.1)was 97.11%,with a 91.90%sequence identity between the ITS-1 region and the Anisakis typical(GenBank accession number MF668919.1).There is a 94.97%sequence identity between the ITS-2 region and the Anisakis typical(GenBank accession number JN968938.1)and a 100.00%sequence identity between the 5.8S rRNA region and the Anisakis typical(GenBank accession number OQ244221.1).Phylogenetic analyses based on the ITS gene and the 28S rRNA D2-D3 gene showed that the parasite samples isolated in this study were genetically closest to the Anisakis typical,and on the same branch.In summary,the isolated parasite samples obtained from this study should be the larvae of Anisakis typical based on microscopic examination and molecular analysis.