Molecular Identification And Genetic Variation Study Of Ascaridia Ne Matodes From Macaw(Ara Chloroptera)

Ascaridia nematodes are the most common parasitic nematodes of parrots,causing serious harm to the host.They parasitize in small intestines of the order Psittaciformes birds,with many species and wide distribution.Parrots infected with Ascaridia sp.behave depressed,inappetence,diarrhea,anemia,emaciation and even death with serious infections,which may result in great economic losses to the parrot breeding.Therefore,the accurate identification of Ascaridia species from parrots,as well as studies on the genetic variation and phylogenetic relationship,have an important significance for prevention and control of ascariasis in parrots.In the present study,a total of 6 roundworms from macaw in Guangzhou zoo were identified molecularly.At the same time,the mitochondrial genes named cytochrome c oxidase subunit 1(cox1),cytochrome c oxidase subunit 2(cox2),NADH dehydrogenase subunit 1(nad1),N ADH dehydrogenase subunit 2(nad2)and NADH dehydrogenase subunit 4(nad4)were sequenced to study the genetic variation of Ascaridia nematodes from parrots.Firstly,after the roundworms from macaw were identified morphologically,and their internal transcribed spacer ribosomal DNA(ITS rDN A)sequences were amplified by using primers NC5 and NC2,cloned and sequenced.Then the homology of their sequences with those of related Ascaridae nematodes and phylogenetic analysis were conducted by the DNAStar softwares.The result showed that the length o f all amplicons were 1015 bp,which had the highest similarity(93.8%)with the sequences of Ascaridia columbae(KF147909)deposited in GenBank,the similarity with other roundworms of Ascaridata were less than 68.6%.Phylogenetic analysis based on ITS seq uence displayed that all isolates from macaw were clustered in one clade,and they were more closely related to Ascaridia columbae than other members of Ascaridada.Results indicate that those roundworms from the macaw belong to Ascaridia species.Secondly,partial mitochondrial genes cox1,cox2,nad1,nad2 and nad4 of Ascaridia nematodes isolated from the macaw were amplified by PCR method and sequenced for genetic variation analysis.The results showed that the lengths of pcox1,pcox2,pnad1,pnad2 and pnad4 were 441 bp,341 bp,512 bp,591 bp and 430 bp,respectively.The content of A+T of pcox1,pcox2,pnad1,pnad2 and pnad4 gene were 64.38%~65.14%,58.82%~59.86%,65.96%~66.60%,70.99%~71.71% and 63.96%~64.72%,respectively.There were 7,4,9,12 and 9 variable sites in the pcox1,pcox2,pnad1,pnad2 and pnad4 gene sequences,the intraspecific sequence variations within Ascaridia nematodes from parrots were 0.0%~1.6% for pcox1 gene,0.0%~1.0% for pcox2 gene,0.2%~1.7% for pnad1 gene,0.5%~1.6% for pnad2 gene and 0.0%~1.8% for pnad4 gene,the interspecific sequence differences among members of the Ascaridida were 11.9%~18.6%,11.3%~19.0%,18.0%~31.4%,22.1%~46.7% and 19.2%~45.9% for pcox1,pcox2,pnad1,pnad2 and pnad4,respectively.There were 5,4,6,6 and 5 haplotypes in the pcox1,pcox2,pnad1,pnad2 and pnad4 genes,the nucleotide diversities were 0.00746±0.00171,0.00461±0.00154,0.00837±0.00175,0.00793±0.00182 and 0.00914±0.00211,and the average genetic distances were 0.008,0.005,0.009,0.008 and 0.009 respectively.Phylogenetic trees were constructed with Maximum parasimony(MP),Neighbor-joining(NJ)and Maximum Likelihood(ML)methods based on these five genes were the same.All Ascaridia nematodes from parrots were in one clade,they were more closely related to Ascaridia columbae than any other members of Ascaridada.The result indicated that there is no significant variation in pcox1,pcox2,pnad1,pnad2 and pnad4 sequences within Ascaridia nematodes in parrots,while interspecies difference is obvious.The five mt DNA fragments can be served as genetic marker for interspecific genetic variation studies.In conclusion,this study identified the roundworms from macaw by molecular biology means,and examined the population genetic variation of Ascaridia nematodes from parrots based on mitochondrial genes(cox1,cox2,nad1,nad2,nad4).These may provide useful molecular information for molecular dentification and population genetics of ascarid and the prevention and control of ascariasis.