New Species-Specific Primers for Molecular Diagnosis of Bactrocera minax and Bactrocera tsuneonis (Diptera: Tephritidae) in China Based on DNA Barcodes.

Linyu Zheng,Jia Wan,Fan Jiang,Lijun Liu,Yue Zhang,Wenzhao Hu,Yujia Qin,Dijin Guo,Wenzhao Yang,Zihua Zhao,Yiying Zeng,Zhaochun Jiang,Zhihong Li,Jiafeng Zhang

doi:10.3390/insects10120447

Abstract

Tephritidae fruit flies (Diptera: Tephritidae) are regarded as important damage-causing species due to their ability to cause great economic losses in fruit and vegetable crops. Bactrocera minax and Bactrocera tsuneonis are two sibling species of the subgenus Tetradacus of Bactrocera that are distributed across a limited area of China, but have caused serious impacts. They share similar morphological characteristics. These characteristics can only be observed in the female adult individuals. The differences between them cannot be observed in preimaginal stages. Thus, it is difficult to distinguish them in preimaginal stages morphologically. In this study, we used molecular diagnostic methods based on cytochrome c oxidase subunit I and species-specific markers to identify these two species and improve upon the false-positive results of previous species-detection primers. DNA barcode sequences were obtained from 900 individuals of B. minax and 63 individuals of B. tsuneonis. Based on these 658 bp DNA barcode sequences of the cytochrome c oxidase subunit I gene, we successfully designed the species-specific primers for B. minax and B. tsuneonis. The size of the B. minax specific fragment was 422 bp and the size of the B. tsuneonis specific fragment was 456 bp. A series of PCR trials ensured the specificity of these two pairs of primers. Sensitivity assay results demonstrated that the detection limit for the DNA template concentration was 0.1~1 ng/μL for these two species. In this study, we established a more reliable, rapid, and low-cost molecular identification method for all life stages of B. minax and B. tsuneonis. Species-specific PCR can be applied in plant quarantine, monitoring and control of B. minax and B. tsuneonis.

Highlights

Tephritidae, consisting of the true fruit files, is one of the largest families in Diptera
Bactrocera minax (Chinese citrus fly) and Bactrocera tsuneonis (Japanese orange fly) are two sibling species of subgenus Tetradacus of Bactrocera that are native to the East Asia region
According to many studies on B. minax and B. tsuneonis, we found that the host range of the two species is different

Summary

Introduction

Tephritidae, consisting of the true fruit files, is one of the largest families in Diptera. Tephritidae includes over 5000 species classified into 500 genera [1,2], and is distributed worldwide except in Antarctica [1]. The members of the genus Bactrocera of Tephritidae, distributed primarily in tropical Asia, Australia and South Pacific regions, are regarded as damage-causing species due to their devastation of fruit and vegetable crops [1]. Bactrocera minax (Chinese citrus fly) and Bactrocera tsuneonis (Japanese orange fly) are two sibling species of subgenus Tetradacus of Bactrocera that are native to the East Asia region. There are two theories on the origin of B. minax.

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Results