Analysis Of Complete Mitochondrial Genome Of Two Hookworms From Dogs And Cats

Ancylostoma ceylanicum and A.tubaeforme may inhabit the small intestine of canids,felids and humans,and may cause dyspepsia,abdominal pain and diarrhea,as well as iron deficiency anemia and even death under heavy infection,which can pose a potential risk to public health.As eukaryotic cell organelle,the mitochondrion is a place for aerobic respiration and energy production,and plays a significant role in cell metabolism and homeostasis.There are many unique characteristics on gene composition,arrangement,heredity and variation,as well as codon usage of mitochondrial(mt)genome of parasites.So,the objectives of the present study were to amplify the complete mt genome sequence of A.ceylanicum and A.tubaeforme and to analyse their sequence characteristics.The first part of this study is to identify morphologically and molecularly A.ceylanicum from dog and A.tubaeforme from cat.Their morphological characterics were consistent with A.ceylanicum and A.tubaeforme,respectively.The primer AF(5?-GACTGCGGACTGCTGTAT-3?)and its complementary primer AR(5?-AAGTTCAGCGGGTAGTCA-3?)were used to amplify the ITS1+ sequences of A.ceylanicum and A.tubaeforme.Their amplified ITS1+ fragments were 404 bp in length and BLAST analysis indicated highest(100% and 99%)similarity with A.ceylanicums from China(GenBank accession number: KF279132)and A.tubaeforme from America(GenBank Accession number: JQ812691).The phylogenetic tree based on NJ method showed that A.ceylanicum from dog in this study was clustered into a group with A.ceylanicum and A.tubaeforme from cat was clustered into a group with A.tubaeforme.The second part of this study is to amplify complete mt genome sequence of A.ceylanicum by ordinary or long PCR technique.The results showed that the complete mt genome sequence of A.ceylanicum from dog was 13,660 bp in length,including12 protein-coding genes,2 rRNA genes and 22 tRNA genes and 3 non-coding regions(AT-rich region,SNCR and LNCR).The base composition of A.ceylanicum mt genome was biased toward A and T and the total AT content was 78.35%.Twelve protein-coding genes used ATT and TTG as start codon,and TAA,TAG,TA and T as stop codon and the lowest content of AT was cox1 gene(71.39%),and the highest content was and2 gene(83.57%).The length of 22 tRNA genes ranged in 54 ~ 63 bp,and its predicted secondary structures were D-loop and V-loop.The length of two ribosomal RNA(rrnL and rrnS)was 960 and 694 bp,respectively.The third part of this study is to amplify complete mt genome sequence of A.tubaeforme by ordinary or long PCR technique.The results showed that the complete mt genome of A.tubaeforme from cat was 13,730 bp in length,including 12protein-coding genes,22 transfer RNA genes,two ribosomal RNA genes,two non-coding regions and an AT rich region.The nucleotide content of A and T was77.93%,which was biased toward A and T.Twelve protein-coding genes used ATT,TTG and GTG as start codon,and TAA,TAG,TA and T as stop codon and the lowest content of AT was cox1 gene(70.9%),and the highest content was and2 gene(81.6%).The length of 22 tRNA genes ranged in 52 ~ 62 bp,and its predicted secondary structures were D-loop and V-loop.The length of two ribosomal RNA(rrnL and rrnS)was 958 and 697 bp,respectively.The four part of this study is to analyze complete mitochondrial genome of four hookworms including A.ceylanicum and A.tubaeforme in this study and A.duodenale and A.caninum from GenBank.The results showed that the mt genome organization of these four hookworms was composed of 36 genes and 3 non-coding regions.Among them the mtDNA of A.ceylanicum was minimum and A.tubaeforme was the longest in length.The longest gene of A.ceylanicum and A.duodenale was nad5(1579 bp),and the shortest gene was nad4L(234 bp)while the longest gene of A.tubaeforme and A.caninum was cox1(1578 bp).In addition,the lowest content of AT was cox1 gene,and the highest content was and2 gene,except for A.caninum whichthe highest content of AT was nad6 gene.The minimum Ka/Ks was cox1 gene and the maximum Ka/Ks was nad2 gene.A.ceylanicum used TTG and ATT as initiation codons of nad5 and nad6 genes but other Ancylostoma species uesd ATT as initiation codons of nad5 and uesd GTG or ATG for nad6 gene.Although 22 tRNA genes of these Ancylostoma species were different in length,the anticodon codons were the same.The phylogenetic tree based on the 12 protein-coding genes and cox1 and nad2 genes showed that in the Ancylostomatidae lineage,Necator nematodes had closer relationship with Ancylostoma nematodes.In the lineage of Ancylostoma,A.ceylanicum occurred on a branch external to the other three Ancylostoma species,and there are differences on phylogenetic relationship among A.tubaeforme,A.duodenale and A.caninum.A.tubaeforme and A.caninum clustered into a group based on the12 protein-coding genes and cox1 gene.However,A.tubaeforme and A.duodenale clustered into a group based on nad2 gene.Accordingly,the analysis of complete mitochondrial genome of two hookworms from dogs and cats can not only enhance the mt genome database of Ancylostomatidae nematodes which can provide important information for the comparative genomics studies,but also provide new clue for further phylogenetic studies among Ancylostomatidae nematodes by revisiting the phylogeny of Ancylostomidae nematodes.