Molecular Mechanism Of Chlorantraniliprole Resistance In Plutella Xylostella(L.)

The diamondback moth (DBM), Plutella xylostella (L.), is the most widely distributed major pest of crucifers. The annual global costs caused by the DBM have increased from approximately＄1billion in the1990s to＄4-5billion in recent years. However, due to the intensive application of insecticides and the biological characters (such as genetic plasticity, high fecundity and fast cycle) of the DBM, this pest has developed resistance to92compounds and become the most resistant pest insect in the world.Chlorantraniliprole, a wildly applied diamide insecticide, owns a completely novel mode of action. While high levels of resistance to this insecticide in DBM were reported in Philippines, Thailand, Brazil and China after2-3years of extensive and repetitive application, while its mechanisms of resistance kept unknown. To reveal the molecular mechanisms of resistance to the chlorantraniliprole in DBM, several experiments were conducted, and the results were briefly described as follows:Chlorantraniliprole showed dramatic sublethal effects on DBM. When4th instar DBM larvae were exposed to LC25of chlorantraniliprole on a cabbage (Brassica oleracea var. capitata L.) leaf for96h, increased developmental time for4th instar larval to pupa period(4.27days vs.3.34days in the control), lower pupal weight (3.58mg vs.4.17mg in the control) and decreased adult fecundity (by42%) were observed. F1generation underwent transgenerational effects, i.e. higher developmental time from egg to pre-pupae and lower egg hatching rate. Demographic growth parameters, such as the net reproductive rate (Ro), the intrinsic rate of increase (rm), and the finite rate of increase (λ) were significantly lower for the LC25chlorantraniliprole treated group than for the untreated control.A full-length cDNA sequence of RyR was cloned from DBM through RT-PCR and rapid amplification of cDNA ends (RACE). The gene (named PxRyRl) is15,753bp long, with an open reading frame of15,354bp, encoding a predicted RyR of5117amino acids. An alternative splicing of the PxRyRl was also cloned and named PxRyR2. The PxRyRl shares77-93%identity with other insect RyRs. Quantitative real-time PCR analysis showed that the PxRyR was expressed at a high level in second-instar larvae and adults, at a low level in prepupae and pupae and abundantly in the body wall muscle and head (respectively6.00and3.12times of the expression in the gut). Western blot analysis with anti-RyR antibodies showed that the RyR was mainly present in the body wall muscle and head, but barely present in the haemocyte and gut.In the four field chlorantraniliprole resistant populations collected in Guangdong province, we identified a point mutation (G4946E) in PxRyR. Most importantly, the frequency of the G4946E mutation is significantly correlated to the chlorantraniliprole resistance ratios in P. xylostella (R2=0.82,.P=0.0003). Ligand binding assays showed that the binding affinities of the PxRyR to chlorantraniliprole in three field resistant populations were2.41-,2.54-and2.60-times lower than that in the susceptible one. These results showed that the point mutation G4946E confered high chlorantraniliprole resistance in field DBM populations from Guangdong province.Later, a field population was collected from Tonghai,Yunnan province, which showed2128-fold resistance to chlorantraniliprole. When examined the frequency of G4946E, only20%individuals were found mutant at position4946as heterozygous genotype. Then the full length of RyR was screened for other possible mutations, and three novel mutations, including a glutamic acid to valine substitution (E1338D), a glutamine to leucine substitution (Q4594L) and an isoleucine to methionine substitution (I4790M) were identified in highly conserved regions, and each mutation showed a high frequency of86.7%. More importantly, the co-existence of the three new mutations together with the previously reported G4946E were all involved in the chlorantraniliprole resistance through different combinations.After definiting the role of four mutationsin in resistance to chlorantraniliprole, an allele-specific PCR (AS-PCR) based method was established for the diagnosis of the three mutations (E1338D, Q4594L and G4946E), which could efficiently detect these mutations with a98%accuracy.Besides the four point muations, overexpression of PxRyR was also involved in chorantraniliprole resistance in DBM. qRT-PCR showed that the expression levels of PxRyR in several high resistant populations were2.28-4.14folds higher than that of the susceptible one. When the expression of PxRyR was successfully suppressed in both resistant and susceptible populations by injection of corresponding dsRNA,there showed no difference in mortality. However, when both resistant and susceptible populations were treated with LC25of chlorantraniliprole after dsRNA injection, the resistant population showed a significantly higher mortality than the susceptible one.