Determination Of The Mitochondrial Genome Sequences And Phylogenetic Studies Of Several Anisakid Nematodes

Anisakid nematodes infect a wide range of invertebrates and vertebrates with a cosmopolitan distribution. Anisakiasis/anisakidosis caused by anisakid nematodes is an emerging infectious disease that humans are infected by eating raw or undercooked marine fish with third-stage larva（L3） that can be associated with mild to severe gastrointestinaldisease and allergic responses. At present, four genera of anisakids have been reported as pathogens of anisakiasis: Anisakis, Contracaecum, Pseudoterranova and Hysterothylacium. Accurate identification and differentiation of anisakid nematodes has important implications for the specific diagnosis, prevention and control of anisakiasis. Due to limitation of the morphological characters（easily influenced by environmental factors） and few gene sequences（lack of efficient genetic sites） in anisakid nematodes, over the last years, there has been considerable debate as to the classification and higher-level systematics of the family Anisakidae. Mitochondrial（mt） genome sequence provides useful genetic markers for investigating the taxonomy, population genetics and phylogenetics of parasites, but complete mt genomic data have been lacking for most anisakid nematodes. To date, complete mt genomes have been reported for only four species in the Anisakidae.In the first part, long PCR amplification method was used to amplify the complete mt DNA of P. azarasi, P. bulbosa, P. cattani, P. decipiens s.l, P. decipiens s.s and P. krabbei, which were 13,954 bp, 13,957 bp, 13,950 bp, 13,965 bp, 13,962 bp and 13,948 bp in size, respectively（Gen Bank accession numbers NC₀27163, KU558720, KU558721, KU558722, KU558723 and KU558724, respectively）. Each of the six genomes contains 36 genes, consisting of 12 genes for proteins, 2 genes for r RNA, and 22 genes for t RNA. As in most other nematodes, the atp8 gene is not present in any of the eight mt genomes sequenced, all mt genes are transcribed in the same direction. Comparative analyses of members of the Pseudoterranova decipiens complex. A comparison of the nucleotide sequences of each mt gene, and the amino acid sequences, conceptually translated from all mt protein-encoding genes among the six complex species. Across the entire mt genome, the sequence difference was 3.8⁹.4% among six P.decipiens complex species. The difference across both nucleotide and amino acid sequences of the 12 protein-coding was 3.8²5.8% and 1.7⁹.1% among six P. decipiens complex species, respectively. The 12 protein-coding genes have ATT, ATA or TTG as a start codon. All of the 12 protein-coding genes have TAA or TAG as a complete termination codon, except for cox1, cox2, cox3, nad5 and nad4 L which have an abbreviated stop codon（TA or T）. Phylogenetic analyses based on the 12 protein-coding genes by three different computational algorithms（Bayes, ML and MP）, all revealed consistent results that Pseudoterranova decipiens complex species consist of at least six not recognized sibling species, Pseudoterranova were more closely related to those of the Anisakis than they were to the Contracaecum.In the second part, long PCR amplification method was used to amplify the complete mt DNA of Contracaecum ogmorhini complex [ hosts Arctocephalus pusillus doriferus（Sample code COAPD）, Arctocephalus pusillus pusillus（Sample code COAPP） and Zalophus californianus（Sample code COZC） ], which were 14,012 bp, 14,019 bp and 14,010 bp in size, respectively（Gen Bank accession numbers KU558725, KU558726 and KU558727, respectively）. Each of the three genomes contains 36 genes, consisting of 12 genes for proteins, 2 genes for r RNA, and 22 genes for t RNA. Across the entire mt genome, the sequence difference was 0.6%（93 nucleotide substitutions） between the COAPD and COAPP, 1.7%（244 nucleotide substitutions） between the COAPP and COZC, and 1.7%（243 nucleotide substitutions） between the COAPD and COZC. The difference across both nucleotide and amino acid sequences of the 12 protein-coding was 0.6% and 0.9% between COAPD and COAPP; 1.8% and 1.1% between COAPP and COZC; and 1.8% and 1.0% between COAPD and COZC, respectively. Phylogenetic analyses was based on the 12 protein-coding genes by three different computational algorithms（Bayes, ML and MP）, and the results rejected previous proposal that Contracaecum ogmorhini represents a complex species.In the third part, the structural traits and evolution of Anisakid nematode mt genomes were examined by using comparative mt genomic and bioinformatics methods. In addition, we reconstructed the phylogeny of Ascaridomorpha, and assessed the utility of complete mt genome sequences as markers for phylogenetic analyses. The entire mt genomes of Anisakidae nematodes show negative AT-skew and positive GC-skew. The full mt genomes of Anisakidae nematodes did not show rearrangement and gene orders in Anisakidae nematodes were GA3. Phylogenetic analyses of the avaliable mt genome sequences strongly support the monophyly of the superfamilies Ascaridoidea（including three families Ascarididae, Toxocaridae and Anisakidae） and Heterakoidea（including two families Heterakidae and Ascaridiidae）.The present study determined seven mitochondrial genomes of anisakid nematodes, These results not only provided novel mt sequences for the studies of nematodes, but also have important implications for molecular taxonomy and population genetic structures of nematodes.