| The whitefly Bemisia tabaci (Gennadius) is one of the most devastating pest insects and occurs in subtropical and tropical agriculture as well as in greenhouse production systems. B. tabaci has been recorded from more than 600 plant specices. Over the last three decades, this pest has caused heavy losses of crop yield by direct feeding damage, excreting honey dew that favours sooty mould production and transmitting plant viruses. B. tabaci comprises a complex population with many biotypes, of which the B-biotype and Q-biotype are the most widespread biotypes. Both B-biotype and Q-biotype B. tabaci have invaded into China, and the Q-biotype is supplanting the B-biotype which used to be ubiquitous in China.Imidacloprid, the first commercial neonicotinoid insecticide, was introduced in 1991. As a neurotoxin, imidacloprid acts on the nicotinic acetylcholine receptor (nAChR) of insects and is selectively toxic to insects relative to mammals. Imidacloprid is used to control sucking and biting pest insects including ricehoppers, aphids, thrips, whiteflies, termites, turf insects, soil insects and some beetles. Control of B. tabaci is heavily dependent on chemical insecticides, and this pest has developed resistance to a wide range of insecticides including carbamates, organophosphates, pyrethroids, insect growth regulators and neonicotinoids. The Q-biotype B. tabaci from Spain has evolved resistance to imidacloprid since 1998.In the present study, the biotype and resistance status of B. tabaci collected from southeastern China was investigated. Inheritance mode, cross resistance spectrum, and biochemical mechanisms of imidacloprid resistance were studied in a resistant B-biotype B.tabaci strain. Combined transcriptome sequencing and real-time PCR,2 P450 genes associated with imidacloprid resistance were identified in both B-biotype and Q-biotype B. tabaci. These results will establish the technical basis of whitefly resistance management strategy in China.1. Biotype and insecticide resistance status of the whitefly B. tabaci from ChinaTwo different biotypes of B. tabaci (B-biotype and Q-biotype) were detected in south-eastern China, and the samples collected from geographical regions showed a prevalence of the Q-biotype and the coexistence of B-and Q-biotypes in some regions. Moderate to high levels of resistance to two neonicotinoids were established in both biotypes (28-1900-fold to imidacloprid,29-1200-fold to thiamethoxam. Medium to high levels of resistance to alpha-cypermethrin (22-610-fold) were also detected in both biotypes. Four out of 12 populations had low to medium levels of resistance to fipronil (10-25-fold). Four out of 12 populations showed low levels of resistance to spinosad (5.7-6.4-fold). All populations tested were susceptible to abamectin. We can get the conclusion that the Q-biotype B. tabaci is supplanting the B-biotype which used to be ubiquitous in China. Field populations of both B and Q-biotypes of B. tabaci have developed high levels of resistance to imidacloprid and thiamethoxam. Abamectin is the most effective insecticide against adult B. tabaci from all populations.2. Competition between the B- and Q-biotypes of B. tabaci and its relevance to insecticide resistanceThe insecticide resistance of specimens of a mixed population of the B- and Q-biotypes of B. tabaci collected in the city of Hangzhou, Zhejiang Province was monitored for several generations. When the population was reared on cotton without insecticides for 10 generations, the proportion of B-biotype to Q-biotype individuals in the F0 population was similar; however the B-biotype completely replaced the Q-biotype in the F10 population. Resistance to imidacloprid, abamectin and fipronil in the F10 population was significantly reduced compared with the F0 population, but resistance to a-cypermethrin was similar between the F0 and F10 populations. These results indicate that the B-biotype is competitively superior to the Q-biotype in the absence of insecticide selection pressure under laboratory conditions. However, the Q-biotype is competitively superior in field conditions where neonicotinoids are widely used, presumably because it has greater potential to develop resistance to neonicotinoids than the B-biotype.3. Cross-resistance and mode of inheritance in imidacloprid-resistant B-biotype B. tabaciThe NJ-Imi strain of B-biotype B. tabaci was selected from the NJ strain with imidacloprid for 30 generations. The NJ-Imi strain exhibited 490-fold resistance to imidacloprid, high levels of cross-resistance to three other neonicotinoids, low levels of cross-resistance to monosultap, cartap and spinosad, but no cross-resistance to abamectin and cypermethrin. Imidacloprid resistance in the NJ-Imi strain was autosomal and semi-dominant.4. Biochemical mechanism of imidacloprid resistance in B-biotype B. tabaciThe biochemical mechanisms of imidacloprid resistance in B-biotype B. tabaci were studied by synergism test, detoxifing enzyme activity. The synergistic effects of PBO, DEF and DEM on imidacloprid in strain NJ were compared with strain NJ-Imi at both G15 (15th generaion of selection) and G30 (30th generation of selection). The oxidase inhibitor PBO showed 2.5-and 2.1-fold synergism with imidacloprid in the NJ-Imi strain at G15 and G30 respectively, but no synergism of imidacloprid efficacy in strain NJ. P450 monooxygenase activity in strain NJ-Imi (elevated 2.5-fold) was significantly higher than in strain NJ. The glutathione depleter DEM did not show appreciable synergism in combination with imidacloprid in either the NJ or the NJ-Imi strain. The glutathione S-transferase activity towards CDNB was not significantly different between strains.The esterase inhibitor DEF synergised imidacloprid both in strain NJ (2.7-fold) and in strain NJ-Imi G15 (2.5-fold), but not in strain NJ-Imi G30. Esterase activity usingα-naphthyl acetate as substrate was significantly (0.6-fold) lower in strain NJ-Imi than in strain NJ. The results indicate oxidative degradation at least to some extent in the resistant strain.5. Data analysis of the transcriptome sequencing of B-biotype B. tabaci adultsA whitefly (B-biotype) transcriptome was de novo assembled using a short read sequencing technology (Solexa). Over 37 million short reads were produced and more than 70 thousand unique sequences (mean size 619 bp) were assembled. Scaffold gene annotation, gene ontology and COG function classification were presented based on the different databases of NCBI. All these scaffold genes were further mapped to pathways. We analyzed the novel insecticide resistance-related genes including P450s, ESTs, GSTs and nAChRs from our database. Our study will provide an important basis for investigating resistance mechanisms of B. tabaci.6. Genome wide screen of imidacloprid resistance related P450 genes in B. tabaci.A total of 141 unique sequences annotated as cytochrome P450 genes (partial or full length cDNA, from～100bp to over 2000bp) were identified from the transcriptome database, and 35 new P450 genes were named based on the database of NCBI. Real-time quantitative PCR was used to analyse the relative expression of the 37 P450 genes between resistant and susceptible strains. Of these, two P450 genes (CYP6CX4 and CYP6CM1) were upregulated in two resistant strains compared with a susceptible SUD-S strain (from 10 to 35 fold). Another 3 P450 genes were also upregulated in two strains (CYP6CX2, CYP6CX3, and CYP6DZ, from 1.9 to 6.7 fold). The expression levels of five P450 genes (CYP4C64, CYP4G69, CYP6DW2, CYP6DW3, and CYP6CX5) were similar among three strains (different biotypes). An evidently elevation of expression level of CYP6CX4 and CYP-6CM1 were observed in survivors after exposure to a high dose of imidacloprid compared with untreated adults, which confirmed that overexpression of these two P450 genes are involved in imidacloprid resistance in B. tabaci. |
PDF Full Text Request