2.2　DNA Extraction and PCR Amplification

The host insect mitochondrion was isolated using a mitochondrion extraction kit(Solarbio，Beijing)according to the manufacturer's recommendation.Total mt DNA was extracted by a modified CTAB method(Kang et al.，2011)and sequenced in two ways.In one method，we sequenced the mt genome by paired-end 150 sequencing with an insert size of 400bp using Illumina HiSeq Platform.In the other method，11 overlapping fragments(F1 to F11)totally covered the entire mt genome by long PCR using PrimeSTARⒸGXL DNA Polymerase(Takara，Dalian).The sequences of primer pairs 1～9 are from Cao and coworkers(Cao et al.，2012)，while primer pairs 10(F：5＇-CAAAATAAYCCTTTARTCAGGCAC-3＇；R：5＇-CGCAACTGCTGGCACA AAAT-3＇)and 11(F：5＇-ATGAAAAATGAATCGGTTGTAAAT-3＇，R：5＇ATTTA GCAAGATTTATTACTCGTT 3＇)were designed using Primer Premier 5 based on the obtained sequence results.All primers were synthesized by Sangon Biotechnology Co.Ltd.(Shanghai，China).(https://www.daowen.com)

The fragments were amplified in a volume with 200μM of each d NTPs，0.2μM of each primers，1.25 U/50μL of DNA polymerase and 20～100 ng of DNA.The reaction was performed as follows：an initial denaturation at 95℃for 4min，followed by 30 cycles of denaturation at 98℃for 10 s，annealing at 56℃for 15 s，extension at 68℃for 2min，and then a final extension at 68℃for 5min.All products were checked using agarose gels and sequenced directly in two directions at Sangon Biotechnology Co.Ltd.(Shanghai，China).