Molecular Identification And Phylogeny Of Acaroid Mites

Objiective: Based on mitochondrial COI gene,ribosomal ITS sequence and PCR-RFLP technique,a molecular identification method for acaroid mites was established.At the same time,we constructed and discussed the phylogenetic relationship of acaroid mites in common storage based on COI gene and ITS sequence.Methods: Acarid mites were isolated from flour and herbs in some provinces and cities of China.On the basis of morphological identification,genomic DNA of a single mite was extracted.Mitochondrial COI gene and ribosomal ITS sequence of acarid mites were obtained by PCR amplification,cloning and sequencing.The sequence of mitochondrial COI gene and ribosomal ITS of acarid mites were retrieved by comparing and identifying the corresponding sequences in NCBI database.The sequence analysis was carried out by DNA Star and MEGA6.0 software,and the phylogenetic tree was constructed by Neighbor-Joining(NJ)method.At the same time,restriction enzyme was used to carry out restriction enzyme digestion to ribosomal ITS sequence.The agarose gel electrophoresis of 2% concentration was used to detect the results of enzyme digestion,and a rapid identification method for stored product mites was established.Results:Acarus siro,Aleuroglyphus ovatus,Tyrophagus putrescentiae,Lepidoglyphus destructor,Chortoglyphus arcuatus,Gohier fusca and Dermatophagoides farinae were collected,isolated and identified in some areas of Anhui Province.Seven mitochondrial COI gene sequences were obtained and 27 known mitochondrial COIsequences were retrieved from NCBI database.Sequences of the same location and size were intercepted and analyzed together.The length of the sequences was 377 bp.No deletion or insertion of bases was found in all sequences.The A+T base content of COI gene of 17 acarid mites was 63.4%,which was consistent with AT preference of mitochondrial gene of Acari.A total of 220 conservative loci,156 mutation loci and130 simple information loci were detected.The results of molecular and morphological identification of mitochondrial COI gene were consistent.The genetic distance between species was 0.055~0.309,and within species was 0.000~0.015.The genetic distance between species was much larger than that within species.In the phylogenetic tree of NJ,different individuals of the same species are clustered under the same branch,which is consistent with the species identified by morphology.At the same time,17 acaroid mites can be divided into 5 branches,which is basically consistent with the results of traditional morphological taxonomy.Seven species of Acarus siro,Suidasia nesbitti,Tyrophagus putrescentiae,Lepidoglyphus destructor,Chortoglyphus arcuatus,Gohieria fusca and Dermatophagoides farinae were isolated and identified by collecting storage samples from some provinces and municipalities in China.A total of 241 ribosomal ITS sequences were obtained.Their length ranged from 906 bp to 1405 bp,including 57 bp 18S rRNA,complete ITS1,5.8S rRNA and ITS2 sequences,and 43 bp 28 S rRNA.Among them,the ITS sequence of Acaroid Coarsefoot was 1405 bp and that of Dermatophagoides farinae was 906 bp.The A + T base content of ribosomal ITS sequence was 57.19%.249 conservative sites,1167 mutation sites and 1117 simple information sites were detected.The results of molecular identification and morphological identification of ribosomal ITS sequences were consistent.The genetic distances of seven acaroid mites ranged from 0.2007~0.7009,within species from0.0079~0.0322,and within species from the same geographical population from0.0025~0.0373.Fifty ribosomal ITS sequences of 10 acaroid mites were retrieved from NCBI database for phylogenetic analysis.In the phylogenetic tree of NJ,the ITS sequence of acaroid mites and the database of the same species were clustered intoone branch,and the clustering results were consistent with the morphological identification results,and the acaroid mites of different geographical populations dispersed within each branch of NJ tree to supplement the results of phylogenetic development.At the same time,13 acaroid mites could be divided into 4 branches,which was basically consistent with the results of traditional morphological taxonomy.Identification of Acaroid Mites by ITS-PCR:The amplified ITS products of 7acaroid mites showed different RFLP electrophoresis patterns after Taq I,Hpa II,Rsa I,Hinf I digestion.The electrophoretic bands produced by the four enzymes were identical with the clonal sequences obtained.The bands produced by different acaroid mites were specific.Conclusion:1 Mitochondrial COI gene,ribosomal ITS sequence and PCR-RFLP technology can accurately identify acaroid mites,which can be used as a powerful complement to morphological identification methods.2 Mitochondrial COI gene is suitable for molecular phylogenetic studies of lower order elements within acaroid mites,and ribosomal ITS sequence is suitable for molecular phylogenetic studies among geographical populations within acaroid mites.