3种驴寄生圆线科线虫体表结构观察及其遗传进化研究

Observation of body surface structure and genetic evolution study on three species of donkey-parasitic nematodes of the family Strongylidae

为了探究寄生于驴消化道的3种圆线科线虫的体表结构及其分子生物学分类,试验对采自我国新疆地区驴(Equus asinus)肠道的槽形盅口线虫(Cyathostomum alveatum)、蒙氏盅口线虫(Cyathostomum montgomeryi)和双冠双冠线虫(Cylicodontophorus bicoronatus)标本使用扫描电镜观察并测量,同时对3种线虫的ITS基因进行PCR扩增,阳性产物测序后进行同源性分析并构建系统发育树。结果表明:槽形盅口线虫外叶冠细长,约为21枚,雄虫交合伞较短,生殖锥背唇有指状附属物,腹唇有乳突状突起,交合刺呈线状,远端有钩;蒙氏盅口线虫外叶冠细长,蜷曲向外,共21枚,雄虫交合伞较长,生殖锥背唇有1对囊状附属物,每个附属物上有1个乳突状凸起,交合刺呈线状,远端有钩;双冠双冠线虫口领与体部相接处只有1个较细的环沟,外叶冠约为30枚,雄虫生殖锥发育良好,腹唇呈圆锥形且有1个丘状突,背唇上有两个对称的刺状附属物,交合刺粗壮。测序得到的ITS基因序列与NCBI中其他双冠双冠线虫ITS基因序列的相似性为99.88%~100%,与盅口亚科其他种类线虫ITS基因序列的相似性为86.46%~96.45%;槽形盅口线虫和蒙氏盅口线虫ITS基因序列与盅口亚科其他种类线虫ITS基因序列的相似性分别为85.38%~96.42%和85.38%~96.11%。槽形盅口线虫和蒙氏盅口线虫与盅口属、杯冠属和冠环属线虫聚为一支,双冠双冠线虫与双冠属线虫聚为一支。说明使用扫描电镜可以对3种圆线科线虫体表结构进行精确观察,基于ITS基因的系统发育分析结果与形态学分类结果一致。

In order to explore the body surface structure and molecular biological classification of three species of the nematodes of the family Strongylidae parasitizing the digestive tract of donkeys,in this experiment,scanning electron microscope was used to observe and measure the specimens of Cyathostomum alveatum,Cyathostomum montgomeryi and Cylicodontophorus bicoronatus from the intestines of Equus asinu from Xinjiang region,China.At the same time,the ITS gene of three nematodes was amplified by PCR,and the homology analysis was carried out after the sequencing of positive products and the phylogenetic tree was constructed.The results showed that the outer leaf crown of Cyathostomum alveatum was slender,which amounted to about 21.The male copulatory bursa was short,with a finger-like appendage on the dorsal lip of the genital cone and a papillomatoid protrusion on the ventral lip;the copulatory spines were linear and hooked at the distal end.The outer leaf crown of Cyathostomum montgomeryi was slender,curled outward,with a total number of 21.The male copulatory bursa was long,with a pair of sac-like appendages on the dorsal lip of the genital cone,one mastoid protrusion on each appendage;the copulatory spines were linear and hooked at the distal end.There was only one thin annular groove at the junction of the oral collar and body of Cylicodontophorus bicoronatus,and the outer leaf crown amounted to about 30;the male reproductive cone was well developed,and the ventral lip was conical and had a mound-like process;on the dorsal lip,there were two symmetrical spiny appendages with thick intersecting spines.The nucleotide similarity between the sequenced ITS gene sequence and other Cylicodontophorus bicoronatus ITS gene sequences in NCBI was 99.88%-100%.The similarity with the ITS gene sequences of other species of nematodes in the subfamily Cyathostominae was 86.46%-96.45%.The similarities between the ITS gene sequences of Cyathostomum alveatum and Cyathostomum montgomeryi and the ITS gene sequences of other species of the nematodes in the subfamily Cyathostominae were 85.38%-96.42%and 85.38%-96.11%,respectively.Cyathostomum alveatum and Cyathostomum montgomeryi were clustered into one branch with Cyathostomum,Cylicostephanus,Coronocyclus nematodes,and Cylicodontophorus bicoronatus and Cylicodontophorus nematodes were clustered into one branch.The results suggested that the body surface structure of three Strongylidae nematodes was accurately observed by scanning electron microscopy,and the phylogenetic analysis results based on ITS gene were consistent with the morphological classification results.