Determination Of Mitochondrial Genome Sequences And Phylogenetic Studies Of Gnathostoma Nematodes

Gnathostoma nematodes are food-borne zoonotic parasites,which can cause serious public health problems.Gnathostomiasis cases are distributed worldwide,mostly in tropical and subtropical regions.Through consuming raw fish,frogs,snakes and uncooked chicken,the patients get infected with Gnathostoma spp..Gnathostoma nematodes invade the body’s gastrointestinal tract,destroy the skin and the central nervous system,which can lead to subarachnoid hemorrhage and eosinophilia,and even meningitis,causing fatal permanent neurological damage.At present,six Gnathostoma species have been reported to cause human gnathostomiasis: G.spinigerum,G.doloresi,G.nipponicum,G.malaysiae,G.hispidum and G.binucleatum.Accurate identification and differentiation of Gnathostoma nematodes has important implications for the specific diagnosis,treatment and control of gnathostomiasis.Because of the stability,conservatism,less recombination and high rate of evolution of mitochondrial(mt)genome sequence,it has been an important molecular marker for the studies of parasite classification,genetic variation and phylogenetic reconstruction.However,there are limited studies on the mitochondrial genomes of the Gnathostoma nematodes,and only one Gnathostoma mitochondrial genome sequence was reported.In this study,the complete mt sequences of four Gnathostoma nematodes from different countries were amplified by long-range PCR and determined by subsequent primer walking.The sizes of each complete mt genome were 13,809 bp for G.doloresi China isolate;13,812 bp for G.doloresi Japan isolate;14,093 bp for G.nipponicum;and 14,391 bp for Gnathostoma sp.Korea isolate.The GenBank accession numbers were KU975390,KX231806,KX826911 and KX826912,respectively.All of the four mt genome sequences contained a total of 36 genes including 12 protein coding genes(without apt8 gene),2 rRNA genes and 22 tRNA genes,a total of 36 genes.The gene orders and transcription directions are the same as that of G.spinigerum reported before.The sequence diversity across the complete mt genomes varied from14.4% to 18.2% among G.doloresi of China and Japan isolates,G.nipponicum,Gnathostoma sp.and G.spinigerum.The 12 protein-coding genes always use TTG,ATA,ATG and ATT as the initiation codons;and use TAG and TAA as the stop codons;but some of them also used TA and T as incomplete stop codons.In this study,phylogenetic analyses based on concatenated amino acid sequences of the 12 protein-coding genes of the four kinds of Gnathostoma nematodes and the other 20 nematodes by Bayesian inference(BI)was carried out,and the results showed that G.doloresi is more closely related to G.spinigerum than to other Gnathostoma spp.;G.nipponicum is more closely related to Gnathostoma sp.than to G.spinigerum;Gnathostoma sp.is a distinct species of Gnathostoma based on its mt genome sequence,which may represent a new Gnathostoma species.The phylogenetic tree reconstructed in this study showed the phylogenetic relationships between Gnathostoma spp.and other 20 nematode species.In conclusion,this study firstly determined the complete mt genome sequences of four Gnathostoma nematodes.These mt sequence data not only provide valuable resources for the studies of molecular epidemiology,phylogeny and population genetics of Gnathostoma nematodes,but also provide novel genetic markers for the development of diagnostic and detection methods for Gnathostoma spp,which will have important implications for the better prevention and control of human gnathostomiasis.