| The carmine spider mite Tetranychus cinnabarinus is one of the most serious plant pests causing yield losses of crops. Owing to high reproductive potential and short life cycle, mites can easily develop acaricide resistance. As a novel acaricide, cyflumetofen was registered in China by the year of 2013. Cyflumetofen shows excellent efficacy for the control of spider mites and it was reported that cyflumetofen was safe to non-targeted organisms. In this study, a resistance strain of T. cinnabarinus against cyflumetofen was selected and its resistance mechanism was investigated. The main results were as follows:1. The resistance was selected for 64 generations, and the LC50 value increased from 2.19 mg/L to 42.99mg/L, resistance folds (Rf) were 19.63 and 54.42 when compared to SS and CyS strains respectively.2. Synergism experiments showed that three synergists (PBO、DEM and DEF) had different degrees of effects on CyR43 strain. The synergism ratios (SR) were 2.15,3.77 and 1.76 respectively, and the SR of three mixed synergists was 3.62. Besides, enzyme activity test showed that the activities of MFOs、GSTs and CarEs were significantly increased in CyR43 when compared to CyS, and the ratios of enzymes activity (CyR43/CyS) were 1.85,7.20 and 1.95-folds respectively. The results consistently showed that DEM (inhibitor of GSTs) can increase the sensitivity of mites to cyflumetofen and the activity of GSTs increased markedly in resistance strain, which demonstrated that GSTs was the key factor conferring cyflumetofen resistance.3. As one potential target of cyflumetofen, the activities of mitochondrial complex Ⅱ (SQR) were detected in CyR49 (Rf=31.19), CyR54 (Rf=43.38) and CyR64 (Rf=54.42). The results showed that the SQR activities decreased to 89%、81% and 58% when compared to CyS. The 50% inhibition concentration (IC50) of SQR activities by cyflumetofen were 23.141±0.173nmol/L and 63.207±1.900nmol/L in CyS and CyR64 respectively, the insensitive ratio of CyR64 (RIC50/S IC50) was 2.73 when compared to CyS. The reduction of the activity and sensibility of SQR might contribute to resistance against cyflumetofen.4. Five genes of SQR in T. cinnabarinus were cloned based on the genomes of T. urticae, which were named as SDHA, SDHB, SDHC, SDHD and SDH5 respectively (GenBank accession numbers were KP686429, KP686430, KP686431, KP686432 and KP686433 respectively). Mutations linked with resistance were not found in CyR64. The expression character of these genes were detected via the comparative quantitative real-time PCR (qPCR). The results showed that the expression levels of these genes were highest in the three-day-old adult female mites, furthermore, the mRNA expression of SDHA, SDHB and SDH5 were always significantly down regulated in the CyR, which might result in the depressed activity and sensibility of SQR in CyR. The strikingly decreased expression of three key genes prompted us to give a conjecture that the down-regulation of SDHA, SDHB and SDH5 genes probably change the proportion of five subunits and thereby modulate the shapes of protein complex, which might decrease the toxicological effect of cyflumetofen and change its inhibitory efficacy.5. The ATP contents in the CyS and CyR64 strains of T. cinnabarinus were measured by HPLC. The results showed that the ATP content decreased in CyR64 strain compared with CyS strain, which might be caused by the reduced activity and expression of SQR in CyR. The ATP content significantly decreased in CyS strain after the pretreatment of cyflumetofen, whereas the ATP content in CyR.64 did not change significantly. According to the results, it suggested that the CyR had stronger toleration to cyflumetofen than CyS as the increased activities of detoxifying enzymes and the depressed sensibility of SQR in CyR. |
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