The Species Identification And Mitochondrial Genome Sequence Analysis Of Dipetalonema Gracile From Squirrel Monkey

Dipetalonema gracile is a common parasite in squirrel monkeys.Its infection can lead to host's malnutrition and progressive wasting,its severe infection can lead to host's death,thus posing a serious threat to the health and safety of the squirrel monkey population.At present,there are only a few morphological descriptions about D.gracile at home and abroad,and there were no reports on molecular identification and mitochondrial genome.This study aimed to identify suspected D.gracile worms in a dead squirrel monkey from a zoo in Guangzhou by the morphological and molecular biological means,in order to diagnosis the pathogen.Moreover,its complete mitochondrial genome was amplified by PCR method and subjected to sequence analysis,so as to provide a scientific basis for further study of molecular identification and molecular diagnosis of Dipetalonema nematodes.1.Species identification of Dipetalonema sp.from squirrel monkey.Microscopic examination was used to observe the morphology of the adult worms collected in the dead squirrel monkeys from a zoo in Guangzhou.Its morphological characteristics were consistent with those of Dipetalonema nematodes.According to the published sequence of cox1 in the conserved region of Dipetalonema,primers CX1?F?:5'-GAC CAGGAAGTAGTTGAA-3'and CX1?R?:5'-CAGCCTCACTAATAATACCA-3'were designed to perform PCR amplification and sequence analysis of the corresponding fragments.The results showed that the size of the amplified fragment was 632 bp,and its homology with cox1 sequence of D.gracile?KP760182?was the highest?98.9%?.The phylogenetic tree showed that the parasite was located in the same branch as D.gracile.Therefore,it was identified as D.gracile.2.Sequence analysis of D.gracile complete mitochondrial genome.The mitochondrial genome sequences of D.gracile were amplified by ordinary PCR and long-range PCR.The full-length of D.gracile complete mitochondrial genome was13584 bp,which contained 22 tRNA genes,12 encoded protein genes and 2 rRNA genes,and their nucleotide composition included A?16.89%?,G?20.19%?,T?56.22%?and C?6.70%?,among which A+T=73.12%.The 12 protein-coding genes used TTG and ATT as start codons and TAG and TAA as stop codons.Among the 22 tRNA genes,only trnS1^AGN and trnS2^UCN lacked the DHU loops,showing the D-loop structure,and the other 20 tRNAs lacked the T?C loops,showing the TV-loop structure.The rrn L?986 bp?and rrnS?678 bp?genes are single-stranded and conserved in secondary structures.Using Angiostrongylia as an outgroup,the phylogenetic tree was constructed by the ML method and the MP method based on the cox1 gene of 12 species of nematodes from 4 different genus of Spirulina.Results showed that the D.gracile was located in Dipetalonema belonging to Onchocercidae of Spirurida.The Dipetalonema with Brugia,Setaria and Dirofilaria are individually divided into separate evolutionary groups.In summary,the D.gracile collected from dead squirrel monkey from a zoo in Guangzhou was accurately identified by morphological and molecular biology methods.Its complete mitochondrial genome sequence was obtained and analyzed for the first time.This study provides molecular identification information of the parasite,enriches the mitochondrial gene database of Dipetalonema,and lay the foundation for the future research on the classification,identification and diagnosis of Dipetalonema at the molecular level.