The Mitochondrial Genome Of Sinergasilus Polycolpus (Copepoda, Ergasilidae) Parasitizing The Gills Of Hypophthalmichthys Molitrix And Aristichthys Nobilis

The subclass Copepod,belonging to the phylum Arthropoda,the subphylum Crustacea,the Class Maxillopoda,comprises more than 11500 nominal species placed into 200 families and 1650 genera,but the researches about the relationships among orders of the subclass Copepod are based on morphological characteristics with limited amendments by molecular biological techniques chonsidering partial nuclear DNA,however,this is difficult in species identification.The present study aims to determine the nucleotide sequence of mitochondrial geneme of Sinergasilus polycolpus,a poicilostomatoid copepod,parasitizing the freshwater fish Hypophthalmichthys molitrix and Aristichthys nobilis.Sinergasilus polycolpus,belonging to the subclass copepod,the order Poecilostomatoida Thorell,the family Ergasilidae,is the mainly parasite on the filum branchiaie of Hypophthalmichthys molitrix and Aristichthys nobilis.In this study,The segments of Cyt b and COⅠwas amplified with the degenerate primers which were designed on the basis of the homologous sequences,and then the special primers were designed from the above determined segments to amplified the region between Cyt b and COⅠby using Long-PCR.Two segments with respectively 7k and 6k length were gotten.Finally a Sinergasilus polycolpus mt DNA segment was gotten with 12917 bp in length,and with A of 35.0%,T of 35.8%,C of 14.0%and G of 15.2%in base composition,from which the value of GC-Skew,and AT-Skew was calculated as 0.041and -0.011,after splicing,and has been deposited in GenBank database with accession number of EU 621723.On the basis of homologous comparison and structure characterization of the sequence,the total of 35 genes were characterized, including 13protein coding genes,21 tRNA and 1 12s rRNA genes.However,the segment between ND4-Atp6,in which 16s rRNA,tRNA^Arg and tRNA^Thr genes,and control region,may be located,could not be amplified.The 13 protein-coding genes begin respectively with ATA(COⅠ,COⅡ,ND 1,ND 2,ND 3,ND 5 and ND 6),ATG(ND 4L,COⅢand ATP 6),ATC(Cyt b) and ATT(ATP 8 and ND 4),as initiation codon,and had respectively TAA(COⅡ,ATP 6,ATP 8,Cyt b,ND 1,ND 2,ND 3,ND 4,ND 4L and ND 5),TAG(ND 6),TA(COⅠ) and T(COⅢ) as termination codon,however,the latter two being incomplete termination codons.All of 21 tRNA genes with 54-63 bp in length can be folded to the typical clover leaf structure,However,other than 3 tRNA genes withou TψC arms the length of TψC arms of these tRNA genes varied from 1bp to 4 bp.In addition,it is singular among animal that there were three tRNA^Leu genes in the mt genomes. Among 35 genes,4 protein-coding genes(COⅠ,ND 5,Cyt b and ND 4)and 7 tRNA genes(tRNA^Leu(CUN),tRNA^Met,tRNA^Pro,tRNA^Asp,tRNA^Glu,tRNA^Tyr and tRNA^His)were encoded on the N strand,the remainders were on the J strand.There were 12 overlap between genes,with the total of 159 bp in length,however,the significance of the overlap between relative genes in the Sinergasilus polycolpus mt genomes is unclear.The mitochondrial gene order in Sinergasilus polycolpus differs significantly from the mtDNA gene order of the archaic arthropod Limulus polyphemus,which is normally taken as the reference.Although it appears that such large-scale gene rearrangement have occurred in the ancestral species of Sinergasilus polycolpus none of the proposed mechanisms parsimoniously account for this eccentric gene arrangement.Therefore,to elucidate the mechnisms of the large-scale gene rearrangement in the copepod species it is necessary to further study the mt genomes of more species of copepods and,of the related groups.A molecular phylogenetic hypothesis for the subphylum Crustacea was generated, involving 21 specieses,based on amino-acid sequences of the mitochondrial genomic protein-coding genes.NJ,ME and MP analyses of this data set produced the topologies using Nematoda(Trichinella spiralis) as the outgroup.Our molecular data overwhelmingly supports that:compared with other two Harpacticoidas(Tigriopus japonicus and Tigriopus californicus),the Sinergasilus polycolpus(Poecilostomatoida Thorell) is closed to Lepeophtheirus salmonis(Siphonostomatoida Thorell); Malacostraca is monophyletic;the relationship of Vargula hilgendorfii(Ostracoda) is unambiguous,but the genetic divergence implies it is more closed to malacostraca; the branchiura and pentastomida are regard as sister group;all phylogenetic trees support that Maxillopoda is paraphyletic.