Molecular Phylogenetics Analysis Of Heartworms In China Based On COI, 12S RDNA And ITS-2 Genes

Comparing with high speed development of molecular biology,molecular systematization has been used in all areas of species classified as a branch,included in parasitic species taxonomy.Heartworm,as a filarial nematode,was placed in genus Dirofilaria under the family Onchocercidae based on morphological characters,which belongs to the Filarioidea superfamily(order Spirurida).Heartworm disease is a worldwide parasitic zoonosis,which mainly infected husbandry animal(dogs and cats) but could infect some wildlife,such as wolves,foxes,lions,ferrets,Raccoon dogs,Saki monkeys, Alopexs,Wolverines,tigers,bears,Cougars,Zalophuses,Neofelis nebulosas,harbor seals, castor fibers,otters,Raccoons,muskrats,Gorillas,chimpanzees and so on.To learn this disease has an important effect on developing of animal husbandry-raising and protecting human health.In this paper,mtDNA gene included cytochrome coxidase subunitⅠ(COI) and small subunit ribosomal RNA(12S rDNA),and ribosomal gene internal transcribed spacer 2 (ITS-2) as targets were used to analyse phylogentic relation about heartworm from red panda and dog with other filariae.Sequence alignments were used to analyse the difference of sequences by DNAStar and Clustal X program.Different evolutional approaches include minimal evolution(ME),neighbour-joiner(NJ) and maximum parsimony(MP), from distance matrix and character state,were used to construct the phylogenetic tree by MEGA 3.1 program.The result of sequences alignment and phylogenetic tree were used to evaluate the evolutional relation between Dirofilaria spp and other species included in the family Onchocercidae.The COI gene sequences alignment included 18 taxa(17 ingroup and 1 outgroup) and 652bp.The pairwise distances of different COI gene sequences were displayed by percent identity and divergence value.The percent identities were larger than 82.8%with all sequences,species Dirofilaria ailure and Drofilaria immitis had higher percent identity, which exceed 99.8%.The divergences value were 0～19.6 in all COI gene sequences,the value between the specie D.ailure and D.immitis was less than 0.2.The 12S rDNA gene sequences alignment included 16 taxa(15 ingroup and 1 outgroup) and 465bp.The percent identities were larger than 78.0%with all sequences,species D.ailure and D.immitis had higher percent identity,which exceed 98.4%.The divergence values were 0.2～26.1 in all 12S rDNA gene sequences,the value between the specie D.ailure and D.immitis was less than 1.6.The ITS-2 gene sequences aligenment included 7 taxa(6 ingroup and 1 outgroup) and 521bp.The percent identities were larger than 71.2,species D.ailure and D.immitis had higher percent identity,which was exceed 98.5%.The divergence value were 0.4～36.3 in all ITS-2 gene sequence.The value between the specie D.ailure and D.immitis was less than1.6.These different phylogenetic trees,included ME and MP trees based on COI gene sequences,ME and MP trees based on 12S rDNA gene sequences,NJ and MP trees based on ITS-2 gene sequences,showed a definitive conclusion,that D.ailure and D.immitis were put together as a depth branch tree with a high confidence level(99%or 100%). Meanwhile,the family Onchocercidae was assembled to three major groupings of species/genera,including Onchocerca spp.;Dirofilaria spp.;Brugia+Wuchereria.Our investigation results implied that D.ailure and D.immitis have a high homology, D.ailure was the synonym of D.immitis.Systematization based on morphological characters was not congruent with molecular characters in the family Onchocercidae.From a molecule phylogenetics visual angle,systematization should be evaluated again in the Filarioidea superfamily.