Species Identification of Puparium Samples of Common Sarcosaphagous Flies Based on Molecular Marker Analysis

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Species Identification of Puparium Samples of Common Sarcosaphagous Flies Based on Molecular Marker Analysis

LI Xue-bo1，NING Shu-hua2，ZHANG Gui-qin3，YU Hong-li3，WANG Qing-shan4，Li Hong-wei4 *   

  1. 1 Shandong Provincial Key Laboratory of Evidence Identification in Colleges and Universities， Shandong University of Political Science and Law，Jinan 250014，China；2 Jiangbei Branch of Chongqing Public Security Bureau，Chongqing 400020，China；3 Institute of Forensic Science，Shijiazhuang Public Security Bureau，Shijiazhuang 050021，China；4 Institute of Forensic Science， Chongqing Public Security Bureau，Chongqing 400021，China
  • Online:2015-02-28 Published:2015-05-04

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Abstract:

Objective  To investigate the application value of empty puparia in species identification of common sarcosaphagous flies.  Methods  Fifty-five samples of adult flies and their empty puparia were collected. All the samples were identified as 2 families， 6 genera and 8 species by morphological characteristics. The samples were divided into 3 groups according to their time period between eclosion and our analyses： less than 2 years（n=23）， 2-5 years（n=20）， and more than 5 years（n=12）. The mtDNA of each sample was extracted by CTAB method. The purity and concentration of DNA were tested. PCR products were amplified using two sets of primers. Two sequences of COⅠ gene（sequenceⅠ： 498 bp， sequenceⅡ： 841 bp） from each sample were compared to the sequences in GenBank using BLAST for species identification.  Results  The mtDNA was extracted successfully from all the samples. DNA concentration of adult chest muscle preserved less than or equal to 5 years and empty puparia preserved less than 2 years ranged from 1.0 to 3.0 μg/μl， and the value of A260/A280 ranged from 1.6 to 1.8. The purity and concentration was lower than 1.6 and 1.0 μg/μl， when the adult chest muscle and empty puparia preserved more than 5 years and 2 years， respectively. DNA concentration of the samples significantly decreased with the prolonged preservation time（P＜0.01）. Two sequences of COⅠ gene was amplified in adult chest muscle and empty puparia which preserved less than 2 years. The success rates of amplification decreased with the prolonged preservation time， especially for the sequence Ⅱ（P＜0.01）. The morphological identification  of 8 species did not match exactly with the results based on the COI gene， correct species identification occurred in 6 and 7 species out of 8 based on the two sequences， respectively， and their Max ident value exceeded 97%.  Conclusion  Empty puparium samples can be used to extract mtDNA and identify species.

Key words: Sarcosaphagous fly, Puparium, Species identification