Statistical analysis

KT₅₀ (time to cause 50% knockdown) was calculated using the Bliss' probit method [21]. Chi-square tests were used for the comparison of subspecies composition of Aaa and Aaf between the collection in 2013 and 2014, and the comparison of insecticide susceptibility between the two subspecies. For the two phylogenetic datasets, a total of four phylogenetic analyses were conducted: 1) maximum parsimony analysis (MP), 2) maximum likelihood analysis (ML), and 3) neighbor joining analysis (NJ) were all conducted using the 1st data, and 4) MP was also conducted using the 2nd data. Based on the model selection program of MEGA, the Tamura 3-parameter model was the evolution model used. The first three analyses were conducted by MEGA, whereas the MP tree for the 2nd dataset was constructed using PHYLIP3.69 (http://evolution.genetics.washington.edu/phylip.html). For the all constructed trees, Bootstrap replication was operated for 1,000 times to calculate how strongly the branches were supported.