Sparganosis is a zoonotic parasitic disease caused by the spargana of Spirometra spp.,which is distributed worldwide and causes harm to the health of humans and animals.Up to now,over half of world’s sparganosis cases have been reported in China.The severity of sparganosis is depend on the organ which being invaded by Spirometra in the host.At present,biopsy is the best way to diagnose human sparganosis and determine the specific species within the genus Spirometra.Previous studies have indicated that S.erinaceieuropaei may be the only species of Spirometra and be responsible for sparganosis in China.The single gene or single region of the mitochondrial genome has been used frequently as the genetic marker to identifying Spirometra.Mitochondrial DNA(mt DNA)sequencing,a tool for population genetics investigation,has been helping researchers to clarify the genetic diversity and phylogenetic relationship of many tapeworms.The object of our study was the 7 S.erinaceieuropaei adult or spargana specimens which were collected from different hosts.The aims of the present study were:(i)to sequence and assemble mt genome of 7 S.erinaceieuropaei or spargana;(ii)to characterize the mt genome of 7 S.erinaceieuropaei or spargana;(iii)to identify the best genetic marker for identification of S.erinaceieuropaei;(iv)to verify the feasibility and suitability of the genetic marker.The mt genome of S.erinaceieuropaei was successfully amplified,sequencing and assembled by long-range PCR.Sequence analysis showed that the total length of the mt genome of S.erinaceieuropaei ranged from 13,640 bp to 13,680 bp,with A+T base content ranged from 66.14% to 66.48%.The nucleotide similarity of the 7 S.erinaceieuropaei mt genomes ranged from 97.40% to 99.90%,and the identity of nucleotide sequence and amino acid of 12 coding sequences(CDS)are 97.7% to 100.0%and 99.00% to 99.90%,respectively.The mt genome of 7 S.erinaceieuropaei isolates all contained 12 CDS,22 t RNA and 2 RNA,and all of which were arranged in the same sequential and direction.Among the 12 CDS,ATG and GTG are the two most common initiation codons;and the most common termination codon are TAA,TAG and incomplete termination codon T.Bayesian analysis indicated that S.erinaceieuropaei have different genotypes,but there was no significant association between genotypes and hosts,developmental stage and regions.Based on the mt DNA sequences sequenced in the present study and those available in the Gen Bank database,sliding window analysis indicated that nad1 gene could be the most appropriate genetic marker for identification and differentiation of S.erinaceieuropaei from other tapeworms.Thus,we amplified the nad1 gene of 7 S.erinaceieuropaei isolates using primers designed in this study,and constructed phylogenetic relationship based on nad1 gene sequences.The results revealed that the nucleotide identity of 7 isolates ranged from 97.90% to 100.00%,with the nucleotide diversity 0.01015 ± 0.00336 among 7 isolates.Phylogenetic relationship constructed based on nad1 gene sequences using neighbor-joining method showed clear discrimination between S.erinaceieuropaei and other Spirometra species.Thus,nad1 gene sequences could be the genetic marker for differentiation of S.erinaceieuropaei and diagnosis of sparganosis.In summary,the present study determined the mt genome sequences of 7 S.erinaceieuropaei isolates the first time,clarified the genetic and evolution relationship of S.erinaceieuropaei,and identified the best genetic marker for the identification of S.erinaceieuropaei,and have implications for better understanding the molecular identification,taxonomy,population genetics of S.erinaceieuropaei and sparganosis diagnosis. |