Classification And Phylogenetic Analysis Of Cyathostomum And Coronocyclus

Infections of horses with parasites not only involve millions of nematodes, but also involve a wide range of species. The nematodes of the Strongylidae are of major veterinary importance, especially the Strongylinae subfamily and Cyathostominae subfamily. They usually cause significant diseases in equids,and brings animal husbandry a great economic loss. Although using anthelmintic drugs can readily control and prevent strongylids, this method has led to the increase of anthelmintic resistance in equine strongylids and high prevalence rate. The population of parasites in host has changed because of widespread use of drugs. Therefore, putting forward a new integrated control method is particularly important. But there are limitations in the understanding of fundamental biological and phylogenetic aspects of these parasites, owing to the limitations in specific identification and diagnosis by using traditional, morphological approaches. This study adopts the method of combining the classical taxonomy and molecular classification to analyze the classification and phylogenetic relationship of genus Cyathostomum and Coronocyclus. The author first observes and describes the body surface structures of three species of the genus Coronocyclus with scanning electron microscope. Then, determines and analyzes ITS and partial cox1 sequences of two genuses.This study obtains scanning electron micrographs of Coronocyclus(C. coronatus, C. labiatus, C. labratus), which collected from Henan Province, China. The results show that three kinds of Coronocyclus nematodes surface morphology characters are significantly different. There are some differences in the number of external leaf-crown, the shape of the appendages of genital cone in male, the shape of posterior end in female. These results compared with previous descriptions under optical microscopes are basically the same, and much clearer and more accurate than the observed under optical microscopes. The present research provides new evidence for taxonomy and identification of Cyathostomum and Coronocyclus by means of comparing the scanning electron micrographs of the two genuses.This study obtains ten ITS sequences from genome of the two genuses. The length of ITS sequences ranged from 748 to 843 bp, and the length of 5.8S representing six species is 153 bp. The length of ITS2 sequences representing C. coronatus is about 90 bp shorter than from other species. The 5.8S sequences had no variation among the six species; the ITS1+ITS2 sequences had 154 variable sites(including gap), 64 parsimony informative sites. The G+C content of ITS1 district ranged from 47% to 48.6%, and of ITS2 ranged from 37.7%-41.1%.The differences of ITS2(0-31.9%) are greater than ITS1(0-4.9%). Within species variation of 0-1.9% was detected in ITS1, 0-0.9% in ITS2. Sequence differences among six species ranged from 1.1% to 4.9% in ITS1, from 1.2% to 31.9% in ITS2. The sequence differences among species was greater than the within species sequence variation. Partial mitochondrial cytochrome oxidase c subunit I sequence(pcox1) for the six species have been investigated in this study. All pcox1 sequences obtained were 393 bp. The A+T composition for the pcox1 gene ranged from 65.9% to 68.9%, reflecting AT base bias obviously. The level of intraspecific sequence variation for pcox1 was low(0.5-2.5%) for six species, and sequence differences among species was 6.6-15.8%. NJ and MP trees constructed respectively based on ITS1+ITS2 and pcox1 shared a very similar branching pattern. Each dendrogram divided the species into two clades. One clade included C. pateratum and C. catinatum, the other clade included C. tetracanthum and Coronocyclus. In each analysis, there were two species “pairs” that always clustered together(C. pateratum with C. catinatum; C. labiatus with C. labratus). And C. tetracanthum always clustered with Coronocyclus. In the phylogenetic tree based on ITS1+ITS2 data, C. tetracanthum first clustered with C. labiatus and C. labratus, while in the phylogenetic tree based on pcox1 data, C. tetracanthum first clustered with C. coronatus. Each molecular data and dendrogram are not entirely congruent with the current morphological classification, it spurports the closer relationship between C. tetracanthum and Coronocyclus.