The Molecular Identification And Mitochondrial Genome Sequence Analysis Of Echinostoma Revolutum From Grus Japonensis

Grus japonensis is one of the rare birds on the verge of extinction in the world,which has been listed as one of China’s key protected birds.It mainly feeds on freshwater fish and shrimp,so it is easy to be infected with a variety of trematode parasites.Among them,the most common is Echinostoma revolutum.E.revolutum is one of the common parasites of poultry and some wild birds,and it can also infect human beings,belonging to polyxenous zoonotic parasite.Grus japonensis infected with the worm can behave symptoms such as loss of appetite,dysentery,weight loss and stunted growth and development,lead to death in severe cases.Presently,there are only morphological reports on E.revolutum,In this paper,the morphological and molecular biological identification of the worm was carried out,and the whole mitochondrial genome sequence was amplified by long PCR for sequence analysis.Firstly,the morphology of the worms stained with borax carmine was observed by microscopy.Primers were designed according to the ITS sequence of Echinostomatidae published on NCBI.The upstream primers ERF:5’-AGGACTTGGCTACGGCT-3’ and downstream primers ERR:5’-CGATAGAGTGACAGGTAACGGAACG-3’ were used to amplify the r DNA ITS sequence of the worm.The results showed that the morphological characteristics of this worm were consistent with those of E.revolutum,the amplified fragment was 710 bp,the highest homology with E.revolutum(GQ463130)in Gen Bank database(99%).The evolutionary tree constructed by NJ method showed that the worm was clustered with E.revolutum,which was identified as E.revolutum.Secondly,the whole mitochondrial genome sequence of E.revolutum amplified by long PCR was 15714 bp,which contained 36 genes(12 protein coding genes,22 t RNA genes and 2 r RNA genes)and one non-coding region(NCR),with a base content of A+T = 61.73%,codon usage prefers to A and T.The AT content of 12 protein-coding genes was highest in nad4 L gene(66.26%)and lowest in atp6 gene(59.73%).The main starting codons are GTG and TTG,and the terminating codons are TAA and TAG.The size of 22 t RNA genes ranged from 59 bp to 70 bp,and the secondary structure was typical clover structure and D-loop structure.The size of rrn L and rrn S genes was 1011 bp and 742 bp,respectively.Finally,the whole mitochondrial genome of E.revolutum was compared with that of E.miyagawai and E.hortense published by Gen Bank.The results showed that the mitochondrial genomes of three species of Echinostoma consisted of 36 genes and 1 noncoding region.The longest one was E.revolutum(15714 bp)and the shortest one was E.miyagawai(14416 bp).There were 38 bp overlapping regions between nad4 L and nad4 genes,long non-coding regions behind t RNA-Glu genes,AT preference for the base composition of all genes of three species of Echinostoma.Among 12 protein-coding genes atp6 was the highest(7.72)and cox1 was the lowest(0.33)at ka/ks value.Phylogenetic trees showed that E.revolutum and E.miyagawai clustered into one branch,into a branch of Echinostomatidae with Hypoderaeum conoideum.Schistosomatidae,Fasciolidae,Notocotylus,Paramphistomatidae,Paragonimidae,Opisthorchiidae,Heterophyidae and Clinostomatidae formed different branches,indicating that they belonged to different evolutionary groups.To sum up,the molecular identification and whole mitochondrial genome sequence analysis of E.revolutum in Grus japonensis was conducted in this paper,which not only enriched the mitochondrial gene database of Echinostoma,but also provided important information for comparative genomics research.It also provided scientific basis for elucidating the genetic diversity among Echinostomatidae trematodes.