圆线虫的分类及系统发育学研究

Equids (including horse, donkey, mule and zebra) can harbour a range of parasites in the large intestines, most importantly the strongyloid nematodes (family Strongylidae, subfamilies Strongylinae and Cyathostominae) which have substantial community richness.A severe internal parasite burden can directly affect the health of hosts and even death. Compared with the studies of the world,the investigations concentrated mainly on morphological classification in China, and did not consider molecular biological techniques.However,difficulties arise in identifying some species by morphology,leading to controversies over the status of certain taxa based on morphological characters,for example similar species or cryptic species, different developmental stages, in particular eggs and larvae.There has been controversy regarding the taxonomic and evolutionary relationships of equine strongyles based on morphological characters.From an applied perspective,there are serious limitations in the diagnosis of infections using currently available coproscopic methods. Also, there are significant problems linked to the excessive and uncontrolled use of anthelmintics, which has led to a substantial increase in the prevalence of cyathostomins and anthelmintic resistance and in the intensity of infection of this group. For these reasons, it is important to develop accurate diagnostic approaches.Thus,the program is the first study about molecular identification and phylogeny of strongyloid nematodes based on rDNA-ITS and mtDNA COⅠgene sequences in China.The main concents are as follows:①Strongyloid nematodes are measured, redescribed and photographed through observation under light microscopy and scanning electron microscopy (SEM). SEM observations of strongyles are made at first time in China and the results will provide new characters for the identification and taxonomy.② The interspecific differences and intraspecific differences of rDNA-ITS and mtDNA COI gene sequences from strongyloid nematodes are analyzed.③ Combining with the sequences of GenBank,the homology is examined by means of Blast software and molecular phylogenetic trees will be reconstructed by Neighbor-Joining (NJ), Maxmium parsmiony (MP) methods using PAUP software. Meanwhile,the origin and evolutionary relationship of strongyloid nematodes are analyzed and have a discussion through combining molecular data with morphological data.The findings of the present study have laid a foundation for classification and molecular diagnosis of strongyloid nematodes.

圆线虫主要寄生于马属动物大肠内，可引起宿主消瘦、贫血甚至死亡。目前，我国对圆线虫的研究仍然停滞在传统的形态学分类方面，关于分子鉴定和系统发育方面的报道仍是空白。而依据形态学特征建立的分类系统和进化关系也一直存在着争议。因此，本研究计划先从经典分类入手，首次将光镜和扫描电镜相结合对圆线虫进行重新鉴定和描述；然后，基于核糖体DNA-ITS序列和线粒体COⅠ序列对圆线虫进行分子鉴定，比较遗传物质的多样性及差异性；最后，结合GenBank中已记录种类和本研究测定种类的特定基因序列资料构建圆线虫系统发育拓扑结构，探讨圆线虫的起源、进化以及亲缘关系。本研究不仅可以为马属动物圆线虫病的诊断和防治提供分子技术手段，同时为解决形态分类与分子分类之间存在的一些争议问题提供参考依据。

圆线虫是马属动物线虫病的主要病原体，寄生部位是大肠。圆线虫大量寄生时，动物往往会出现精神萎靡，四肢无力，食欲不振，被毛粗乱无光泽，极度贫血等症状。因此，为了有效诊断和控制圆线虫疾病，国内外学者分别从多个方面对圆线虫进行了研究。本项目主要调查了河南省圆线虫的寄生情况，在前期工作中，已报道22个种，项目实施过程中又发现13种，目前共采集到35个种类，其中7种为河南省新纪录。研究成果如下：（1）在光学显微镜下对新增加的13种圆线虫进行了观察、形态测量和拍照，并详细描述了种类特征；（2）对9种圆线虫进行了扫描电镜观察、拍照和特征描述。通过扫描电镜能够很清楚地观察到线虫外部结构的特点，弥补了光学显微镜的不足，可以为该类线虫的分类鉴定提供新的依据。（3）通过分析和比对圆线虫的rDNA-ITS和mtDNA COІ 序列，结果发现，圆线虫的种内差异较小，而种间差异较大，进一步证明，两种目的基因均为鉴定线虫种类的理想标记；（4）通过构建圆线虫的系统发育树，对一些种属的分类地位进行了重新界定。本研究为解决圆线虫在分类学上存在的争议问题提供了有利的参考依据。