Studies On ITS Sequence And The Entire Mitochondrial Genome Sequence Of Haemaphysalis Flava And Haemaphysalis Longicornis

Ticks are obligate hematophagous ectoparasites carrying a large number of pathogens,direct and indirect harm to people and animals.People have been classifying ticks according to their morphological and structural characteristics,but this method is very difficult to identify ticks which similar morphological characteristics,leading to the error in classification results.In the recent years,the sequences of internal transcribed spacers(ITS)and some mitochondrial DNA(mtDNA)genes have widely been used as good genetic markers for population structure,molecular markers,biological evolution,phylogeny,species differentiation and evolutionary genomics.The aim of the present study is to provide the reference for ticks classification and to provide useful information for the studies on mitochondrial genome and molecular phylogeny of ticks by PCR amplify the entire sequence of ITS rDNA and mtDNA of Haemaphysalis flava(H.flava)and Haemaphysalis longicornis(H.longicornis).The complete ITS(ITS-1,ITS-2)region sequences of H.flava(n=20)and H.longicornis(n=45)were 1302 bp,1160 bp,1582 bp and 1610 bp in length,respectively.The result of our study showed that the intra-specific and inter-specific differences of the ITS(ITS-1,ITS-2)regions within H.flava were 0%-1.2%,0%-1.6%,50.66%-52.77% and 37.0%-52.0%,respectively;The inter-specific and inter-specific differences of the ITS(ITS-1,ITS-2)regions within H.longicornis were 0%-2%,0%-2.2%,35.1%-55.2% and 37.0%-52.0%,respectively.The results of our study suggested that the ITS rDNA as a good gene marker for tick identification and classification because the ITS rDNA sequence is more conservative and the difference of inter-specific among different species.The complete mt genome sequence of H.flava and H.longicornis was 14689 bp,14693 bp in length,respectively.Both mt genomes include 37 genes(13 protein-coding genes,22 transfer RNA genes,two ribosomal RNA genes).Among 13 protein-coding genes of H.flava and H.longicornis,the longest gene is nad5,which was 1656 bp,1658 bp in length,respectively;The shortest gene is atp8,which was 162 bp,154 bp in size,respectively.The ML analysis showed that the H.flava and H.longicornis are located on the same branch,consistent with the morphological classification.The result of the gene variation in three genes of H.flava(cox1,nad1,nad4)showed that the intra-specific and inter-specific differences of H.flava were 0-0.4%(for cox1),0-0.4%(for nad1),0-0.3%(for nad4),2.5-14.3%(for cox1),13.6-24.8%(for nad1),14.8-19%(for nad4),respectively.The result of the gene variation in three genes of H.longicornis(cox1,16 S,nad5)showed that the intra-specific variations of H.longicornis were 0-2.84%(for cox1),0-3.0%(for 16S),0-6.7%(for nad5),respectively,and their inter-specific variations were 13.8-15.34%(for cox1),14.73-15.66%(for 16S),13.3-18.11%(for nad5),respectively.The ML analyzed showed that H.flava from two provinces didn't appear regional differentiation,but H.longicornis from three provinces have appeared regional differentiation.