Molecular Identification And Population Genetic Structure Of The Genus Anisakis From Marine Fishes In The East China Sea

The present study includes two parts. In the part I, molecular identification of926samples of Anisakis larvae from10marine fishes from the East China Sea was made usingPCR-RFLP coupled sequence analysis of nuclear ribosomal DNA. In the part II, populationgenetic structure of Anisakis pegreffii from10marine fishes from the East China Sea wasanalyzed based on the part of the mitochondrial cytochrome coxidase subunit1gene(pcox1)using PCR-DGGE coupled sequence analysis.1. Molecular identification of Anisakis larvae from the East China SeaThree electrophoresis patterns were detected by digestion of nuclear ribosomal DNAwith restriction endonuclease HinfI, the first type is coincidence with that of Anisakis pegreffii(903samples), the second type is coincidence with that of Anisakis typica(2samples) and thethird type is coincidence with that of a recombinant genotype (21samples). Typical sampleswere selected from the three types by digestion with restriction endonucleas HhaⅠand TaqⅠ,which lead to the same conclusion. ITS-1and ITS-2for the selected samples (5samples of A.pegreffii,1samples of A. typica and all samples of recombinant genotypes) were sequenced.5samples of A. pegreffii have same sequences of ITS-1and ITS-2which coincided with that ofreported in GenBank previously. The A. typica also has same sequences of ITS-1and ITS-2which coincided with that of reported in GenBank previously. While two different sequencesof ITS-1were found among the recombinant genotypes, one sequence representingrecombinant genotype I (Rec1) has C/T and C/T polymorphism at alignment positions280and296respectively, which is coincidence with that of recombinant genotype reported byAbollo et al.(2003) and Shi (2010); the other sequence representing recombinant genotype II(Rec2) has only one C/T polymorphism at alignment position296, which is coincidence withthat of recombinant genotype reported by Shi (2010). MtDNA SSU sequencing of two typesof recombinant genotypes indicated sequence of Rec1and Rec2is as same as that of A.pegreffii. The result supported the recombinant genotypes may be retention of ancestralpolymorphism because no specimens of Anisakis simplex s.s were collected from the EastChina Sea. 2. Population genetic structure analysis of A.pegreffii from marine fishes in the EastChina SeaTen populations（As、Tr、Ps、Lo、Ze、Sa、Pn、Tra、Sc、Br）of903samples of A.pegreffii from the East China Sea were divided based the hosts. Ten haplotypes (hap1-hap10)were detected among them using DGGE coupled with sequencing. MtDNA cox1of allsamples have the same length (384bp) with14multiloci including3transversions and11transitions. Nucleotide acid was similar component and A+T is much higer than G+C.AMOVA analysis showed the variations of intra-populations significantly bigger than thevariations of inter-populations. Pairwise Fst values and P examination of Fst between tenpopulations of Anisakis pegreffii in the East China Sea showed Fst values between Ps and Saor Tra were0.32308and0.25824respectively, and P examination were0.06306and0.01802respectively; Fst values between Ps and Lo、Ze, between Br and Ze、Sa、Tra have a range from0.15759～0.23430, and P examination is significant(P<0.05), showing the geneticdifferentiation is relatively high among these populations. However, Fst values range from0～0.15for most of populations, indicating the genetic differentiation is relatively low; Someother Fst values with negative showed no genetic differentiation among these populations.The phylogentic tree of haplotypes based on the sequences of cox1showed10haplotypesdivided two branch, one including Hap1、Hap4、Hap8、Hap9、Hap10, the other includingHap2、Hap3、Hap5、Hap6、Hap7.Hap1and Hap2were located in the two different branch andwere shared by all populations. Therefor, A. pegreffii from the East China Sea have no hostspecificity.