Studies On Genetic Variations In Ribosomal ITS And Mitochondrial DNA Sequences Of Two Kalicephalus Nematodes

Kalicephalus spp.are parasitic nematodes of the family Diaphanocephalidae with worldwide distribution.Kalicephalus spp.mainly infect snakes,causing inappetence,hypopepsia,emaciation and metabolic disorders,causing serious economic losses to snake breeding industry.The frequently-used method for identification of nematodes mainly depends on morphological features,which,however,eould not distinguish adults,eggs and larvae with similar morphological characteristics.Sequences of the intermal transcribed spacers(ITS)of ribosomal DNA(rDNA),together with mitochondrial DNA(mtDNA)have been frequently used as genetic markers for molecular systematics and phylogenetics studies of parasites.However,studies on mitochondrial genomes of Kalicephalus spp.were very limited.The only available research preliminarily reported the partial coxl(cytochrome c oxidase subunit I)sequence from one undetermined Kalicephalus species,but the information of complete mitochondrial genome sequences of Kalicephalus spp.is still unclear.Therefore,it is necessary to carry out mitochondrial genome analysis of Kalicephalus spp.,which is helpful for studying the genetic evolution,molecular classification of Kalicephalus spp.and anti-Kalicephalus spp.drugs.In this study,extraction of the genomic DNA,PCR amplification of ITS rDNA target genes,sequencing and analysis of the positive amplification product were carried out on 21 samples of Kalicephalus spp.from Langli Town,Changsha County,Changsha City,Hunan Province.Results showed that the length of the target gene obtained by PCR amplification was 695 bp,including ITS-1 rDNA,5.8S rDNA and ITS-2 rDNA.After sequence alignment analysis and comparing,there were 20 K.indicus samples and one K.branchycephlus specimens identified.The total A+T content of K.indicus ITS(ITS-l rDNA and ITS-2 rDNA)was 52.64%?53.83%,and the total A+T content of K branchycephlus ITS was 53.25%.In the 20 K.indicus specimens,the similarity of K.indicus was 98.64%?100%for ITS sequences,99.18.%?100%in ITS-1 and 97.29%?100%in ITS-2,but 5.8S rDNA was highest conserved,with the similarity being 100%.The interspecific differences between K.indicus and K.branchycephlus were 13.3%-14.8%in ITS,9.6%-11.6%in ITS-1 and 22.9%-26.3%in ITS-2,indicating that the intraspecific differences of ITS rDNA sequences were conserved than the interspecific differences among Kalicephalus spp..Therefore,the ITS sequences could be used as a genetic marker molecule for the classification and identification of the genus Kalicephalus.PCR amplification,sequencing and analysis were carried out on four genes in the mitochondrial genome of K.indicus and K.branchycephlus,including coxl:mitochondrial small subunit 12S of ribosomal RNA,12S;ATP synthase subunit 6,atp6 and nicotinamide adenine dinucleotide dehydrogenase subunit 4,nad4.The sequences of coxl,12S,apt6 and nad4 for seven K.indicus were orderly spliced,and then Bayesian Inference(BI)was used to study the phylogenetic developmental relationship.Results showed that the lengths of the cox1,12S,atp6 and nad4 sequences obtained by PCR amplification were 400 bp,600 bp,500 bp and 400 bp,respectively.Sequence analysis showed that gene variability of coxl for K.indicus was 0%-4.23%,0-3.57%in 12S,0.67%-8.67%in atp6 and 0%-11.01%in nad4,respectively.The interspecific difference of K.branchycephlus were 11.1%-12%in coxl;10.6%-11.8%in 12S;0.9%-8.9%in atp6 and 0-13.2%in nad4,respectively.The results of genetic phylogenetic tree analysis showed that the seven K.indicus were placed in the same branch and were closest to the genetic distance of Ancylostoma ceylanicum.The K.indicus and K.branchycephlus mitochondrial genomes were amplified by PCR,and their complete mitochondrial genome sequences were determined by subsequent primer walking,and their sequences were analyzed by phylogenetic tree.The sequencing results showed that the sizes of mitochondrial genomes were 13,588 bp for K.indicus and 13,572 bp for K.branchycephlus.Their mt genome sequences contain 12 protein-encoding genes(PCGs),22 tRNA genes and 2 rRNA genes,but lacking atpS gene.Their mt genome of gene arrangements and transcription directions were identical to A.ceylanicum in the Ancylastomatidae family.Phylogenetic analysis based on 12 PCGs demonstrated that the representatives of Diaphanocephalidae family K.indicus and K.branchycephlus were clustered together in a single branch,with the Ancylastomatidae represented by A.ceylanicum as the closely related family.In summary,this study determined the complete mt genome sequences of K.indicus and K.branchycephlus in the Diaphanocephalidae family and studied their sequence polymorphisms of four mt genes(coxl,12S,atp6 and nadA)for the first time.The results of this study not only enrich the resources of the mt genome database,but also provide novel genetic markers for further exploring molecular taxonomy and population genetics of parasites in reptiles.