Identification Of Major Tephritid Fruit Fly Pests Based On DNA Barcoding

Fruit flies of the family Tephritidae (Diptera:Tephritidae) are one of the important pests group of fruits and vegetables in tropical, subtropical and temperate areas all over the world, with a number of described species, a wide range of hosts and causes great damage. COI gene which lies in the respiratory chain of mitochondria encodes cytochrome oxidase subunit Ⅰ. COI gene has two important advantages over other molecular markers. Firstly, it is relatively conservative and easily amplified by universal primer. Secondly, the evolution of this gene is rapid enough to distinguish different species. DNA barcoding technology is able to identify species at DNA level successfully through sequencing and analyzing a fragment of about650bp which near the5’ end of the cytochrome c oxidase Ⅰ (COI) mitochondrial gene. In this research, the immatures (including egg, larva and pupa) and adult debris (including leg. wing, head, thorax and abdomen) of common tephritid fruit fly pests were used to develop a rapid identification technique for tephritid fruit flies based on DNA barcoding technology. In addition, Twenty one species which belong to genus Bactrocera and frequently intercepted at port of entry were identified using DNA barcoding technology and analysed of molecular phylogenetic relationships, which provided evidence at molecular level to understand the evolution of genus Bactrocera. The general aim of this study is to establish DNA barcoding identification system to achive remote detection and monitoring of tephritid fruit flies to prevent them from further spreading in China. The main results are as follows:1Establishment and application of DNA barcoding technology for identification of the immatures and adult debris of Bactrocera dorsalis (Hendel)(Diptera:Tephritid)Many species in the family Tephritidae are quarantine pests worldwide. Usually, identification of the tephritid fruit flies is mainly based on external morphological characteristics of adults. In this research, the immatures (including egg, larva and pupa) and adult debris (including leg, wing, head, thorax and abdomen) of Bactrocera dorsalis were used to develop a rapid identification technique for tephritid fruit flies based on DNA barcoding technology. The other four tephritid fruit fly species, i.e., B. correcta, B. cucurbitae, B. tau and B. minax, were used to verify the feasibility of the tephritid fruit fly identification technique developed. The results showed that the nucleotide sequence identity of the partial COI gene between the immatures or adult debris of B. dorsalis with the target gene from GenBank database is99.51%-99.84%, and that of other four tephritid fruit fly species with the target gene from GenBank database is100%,100%,99.81%-99.83%and100%, respectively. Neighbor-joining tree was established based on the analysis of COI gene sequences. The target species and the corresponding species in the database clustered in the same branches. All bootstrap values of the original divergence within a same species are100%. The intra-and inter-species genetic distances were calculated with MEGA version5.0software using the Kimera2-Parameter model. The intra-species genetic distances are0.0000-0.0041, with an average of0.0019. The interspecies genetic distances are0.0597-0.2363, with an average of0.1693. There was no overlap between intra-and inter-species genetic distances. The results indicated that the developed DNA barcoding identification techniques based on the partial COI gene can provide a rapid and accurate method for identification of immatures or adult debris of tephritid tephritid fruit fly species.2. Identification and Molecular phylogeny analysis of genus Bactrocera by DNA barcoding technologyTwenty one species belonged to eight subgenus of genus Bactrocera were identified using DNA barcoding technology. Sequenced the partial mitochondrial cytochrome c oxidase subunit I (COI) gene (about650bp) which used to identify species and analyze the relationship of the molecular phylogeny. The phylogenetic tree was established by three types of phylogenetic (NJ, MP, ML) method. The intra-and inter-species genetic distances were calculated with MEGA version5.0software using the Kimura2-parameter model. The clustering analysis of phylogenetic tree was consistent with the published morphological analyses in twenty one Bactrocera species. Each branch corresponded to one species and all bootstrap values were over95%. The intra-species genetic distances are0.0003to0.0068, with an average of0.0043. The inter-species genetic distances are0.0154to0.2395, with an average of0.1540. The genetic distance between species in these21species was35.8times higher than the corresponding values within species (0.1540vs.0.0043). There was no overlap between inter-and intra-species genetic distances. The results indicated that the DNA barcoding based on partial COI gene can provide rapid and accurate identification of Bactrocera species.Develop this technology will play a important role in tephritid fruit fly pest identifying and monitoring.3. Establishment of DNA barcoding recognition system for tephritid fruit fly pestsThe work was supported by the National Basic Research Program of China. The DNA barcode identification system for tephritid fruit fly pests (including Ceratitis capitata, Bactrocera dorsalis, B. oleae, B. minax etc), a significant part of the Database of Invasive Alien Species in China (DIASC) developed by Centre for Management of Invasive Alien Species of Ministry of Agriculture(CMIAS), was established by collecting the barcode information about economical tephritid fruit fly pests and applying ASPNET technology and C#programming language. The DNA barcode identification system contains about2405mtDNA COI sequences of185species of5genus of Tephritid which were obtained from NCBI, BOLD and DIASC. The system has three genus thirty six species tephritid fruit fly (1932samples), such as B. dorsalis (561samples), B. scutellata (296samples), B. tsuneonis (128samples). There were291sequences of29species of3genus fruit flies from DIASC,1696sequences of181species of5genus fruit flies from NCBI and417sequences of100species of5genus fruit flies from BOLD. The species in this study such as B. dorsalis, B. cucurbitae, B. tau, B. mianx which are from different geographic populations. In the database, related information for3genus31most common tephritid fruit fly species was available, including Chinese name, scientific name, synonym, common name, classification status, morphology (illustration with images), distribution both in home and abroad, host plants and damage, country of origin, pathway for introduction and spread, intended spreading areas, measures for control and management. The DNA barcoding database was applied preliminarily, molecular identification of tephritid fruit fly pests was performed successfully via mtDNA COI sequences from samples. The establishment of DNA barcoding recognition system for tephritid fruit flies will play a very important role in improving quarantine capability at ports and international trade in China.