Sequence Variability In Mitochondrial DNA Regions And Analyses Of The Entire Mitochondrial Genome Sequences Of Gasterophilus Intestinalis From Two Regions

Horse gastrointestinal myiasis is a chronic wasting disease caused by larvae of Gasterophilus spp. parasitising in the digestive tract of the equids. This disease is distributesd all over the world and is mainly popular in the northeast, northwest and Inner Mongolia in China. There are four Gasterophilus species, namely G. intestinalis, G. haemorrhoidalis, G. pecorum and G. veterinus, and the infection rate of G. intestinalis is highest among them. Horse gastrointestinal myiasis mainly damages the host’s gastrointestinal tract, causing ulcers, abscess, erosion, perforation, peritonitis and other damage, which can cause severe anemia, weight loss, immune system collapse, even cause the horse death, resulting in considerable economic losses to the horse breeding.The recent reports about gasterophilus equina were mainly focused on traditional parasitic studies, such as morphology, life history, epidemiological investigation and diagnosis etc.Studies on the molecular aspects werelimited. Therefore, the first part of the study examined the nucleotide variation among G. intestinalis larvae isolates collected from Heilongjiang Provienceand Xinjiang Uygur Autonomous Region in partial mitochondrial(mt)regions.The complete cox1, nad5 and rrn L gene of the mitochondrial genome were amplified by PCR, separately from individual G. intestinalis larvae. Sequence alignment and polymorphism analysis were performed by using molecular biology software, which showed that the intra-specific sequence variations within G. intestinalis larvae were 0.1-3.1% for cox1, 0.4-3.4% for nad5 and 0.1-1.5% for rrn L.nad5 gene had the largest intra-specific variation, and rrn L gene had the smallest intra-specific variation. The inter-specific sequence variation in cox1 gene among G. intestinalis larvaes, G. nasilis larvaes and G.pecorum larvaes were 11.6-17.6% and 17.5-18.5% respectively. Phylogenetic tree was constructed by Bayesian inference method, which indicated that all the G. intestinalis isolates and other G. intestinalis larvaesgathered together and formed into a single branch, but all samples from the same region did not form a branch conclusivley. The phylogenetic relationship of the samples from Heilongjiang and Xinjiang wasgenetically closer than to samples in other countries.The second part of this study determined the complete mitochondrial genome sequences for G. intestinalis larvae from Heilongjiang Provience(HLJ) and Xinjiang Uygur Autonomous Region(XJ), and constructed phylogenetic tree based on protein coding genes of the mitochondrial genome, and further discussed about the phylogenetic relationships with other dipteran flies. The results showed that the length of the complete mt genome of G. intestinalis was 15,687 bp(HLJ) and 15,660 bp(XJ), which contained 37 genes, namely 13 genes for proteins, 22 genes for t RNA, 2 genes for r RNA and a D-loop region. The content of A+T(70.2% and 70.3%) was significantly higher than that of G+C, and this was also common in most of the spiders and insects.With concatenated amino acid sequences of 13 protein-coding genes as genetic markers, the results of phylogenetic re-construction by Bayesian inference(BI), maximum likelihood(ML), and Maximum parsimony(MP) three methods were quite similar. The results showed that phylogenetic tree formed two big branches, namely Gasterophilidae, Oestridae and Tachinidae formed a branch and other families formed another branch.In the first branch, Heilongjiang and Xinjiang samples formed a small branch, and phylogenetic relationship between G. intestinalis(HLJ and XJ) and Hypoderma lineatum was closer than to the others. The results of this study were consistent with the previous results, which indicated that the whole mt genome sequence was a good genetic marker for the phylogenetic study of parasites.The present study examined sequence variation among G. intestinalis larvae isolates collected from Heilongjiang Provience and Xinjiang Uygur Autonomous Region, determined the complete mt genome sequences of G.intestinalis larvae for the first time. These data providedfoundation for further studying the population genetics and molecular evolution of G. intestinalis.