Resistance Monitoring For Neonicotinoids Insecticide In Q-biotype Bemisia Tabaci From Eastern China And Cloning Of The Target Gene

The whitefly, Bemisia tabaci Gennadius (Homoptera:Aleyrodidae), is a worldwide agricultural pest. The two most devastating biotypes are referred as "B" and "Q". The pesticide resistance of Q-biotype stain is higher than the B-biotype. And, the Q-biotype of B. tabaci has developed resistance to a wide range of insecticides including organophosphates, pyrethroids, insect growth regulators, and especially neonicotinoids. In this paper, the resistance levels of eight Q-biotype B. tabaci field populations collected from eastern China to neonicotinoids (imidacloprid, nitenpyram, thiamethoxam and dinotefuran) and sulfoxaflor were detected. The full-length cDNA of Q-biotype B. tabaci nicotinic acetylcholine receptor (nAChR) (31 subunit gene was cloned by RT-PCR, and resistance-associated mutation were detected in Q-biotype B. tabaci field populations. In addition, the flanking sequences of insecticide resistance-associated cytochrome P450 gene CYP4C64 of Q-biotype B.tabaci were cloned by genome walking, and polymorphisms between B-biotype and Q-biotype B. tabaci were compared and analyzed.The resistance levels of Q-bitype B. tabaci in eight field populations including Yangzhou, Jiangyin, Dongtai, Hexian, Hefei, Shanghai, Hangzhou and Nangchang to neonicotinoids were determined by leaf dipping method. The results showed that, compared with the reference strain, the eight field populations had developed low to moderate levels of resistance to imidacloprid and nitenpyram with the resistance ratio ranging between 4.07-21.75 fold and 3.37-16.14 fold, respectively. While Yangzhou population exhibited high resistance (RF 40.38) to thiamethoxam, all other populations only developed low levels of resistance to thiamethoxam with the resistance ratio ranging between 3.50-8.58 fold. However, all eight field populations were relatively susceptible to both dinotefuran and sulfoxaflor with the highest sensitivity observed in Nanchang population (RF 0.5 and 0.41, respectively), which suggested that there is no cross-resistance among dinotefuran, sulfoxaflor and imidacloprid, nitenpyram and thiamethoxam.The 1597 bp full-length cDNA of Q-biotype B. tabaci nAChR β1 subunit gene was cloned by RT-PCR, which showed 84.8% amino acid identity with that of M. persicae. Sequence analysis of nAChR β1 subunit gene in eight field populations of Q-biotype B.tabaci revealed 23 nucleotide polymorphisms, which lead to 19 amino acids replacement. While R87 corresponding to T81R mutation in Loop D of nAChR β1 subunit in M. persicae was not mutated in all populations, a 45 bp fragment deletion was found in Jiangyin population, which encodes 15 amino acids including R78.The 51 and 3’ flanking sequence of insecticide resistance-associated cytochrome P450 gene CYP4C64 of Q-biotype B. tabaci was cloned by genome walking. Multiple transcription factor binding sites were predicted in the 5’ flanking sequence of CYP4C64 gene by CONSITE program, including 51 GATA-1,24 Ahr-ARNT,7 CREB,2 NRF-2 and 33 Broad-complex-4. Sequence comparison of the 5’ flanking region of Q-biotype and B-biotype CYP4C64 gene revealed one transposon insertion in Q-biotype B. tabaci, which contains several transcription factor binding sites, including 3 Ahr-ARNT,7 Broad-complex-4 and one NRF-2.