Studies On Resistance Of Diamondback Moth Plutella Xylostella (L.) To Tebufenozide And The Mechanisms

The diamondback moth, Plutella xylostella (L.), is one of the most destructive pests of cruciferae vegetable and has been reported to produce severious resistance to various convenient insecticides, including organophosphates, pyrethroids, Bacillus thuringiensis (Bt), acylurea insect growth regulators, abamectin, and DDT, etc.Tebufenozide developed by Rohm and Hass Co has been introduced for control of this pest nowadays. This insecticide binds to the ecdysone receptor in insect body, like the steroidal molting hormone, but initiates a premature incomplete molt and results in death of the larva. Tebufenozide appears to be lepidoperan-specific and has been used as an environmentally friendly insecticide. In order to make better use of this insecticide and keep it from developing resistance quickly in diamondback moth, studies have been carried out for implementation of effective resistance managements.1) Resistance selection and risk evaluationA susceptible strain of diamondback moth had been used to select for tebufenozide resistance by treatment of every two generations in laboratory. After selection for 17 times during 35 generations, a resistant strain was achieved with high resistance to tebufenozide (RR 93.8). During the selection, there were two peaks of resistance increase. The first peak appeared during the first 3 selection treatments, which made a resistance increase of 15.7 times. The second one occurred from the 8^th to 14^th selection treatments, and made the resistance increase from 17.7 times to 94.4 times. This result indicated that diamondback moth has the potential to develop high resistance to tebufenozide.2) Cross-resistance between tebufenozide and some common insecticidesThe toxicity of 11 insecticides on the susceptible strain and a laboratory-selected tebufenozide resistant strain of diamondback moth was tested to evaluate the cross-resistance between tebufenozide and other insecticides. The results showed that the selection with tebufenozide resulted in not only high level of resistance to tebufenozide (RR 93.8), but also obvious cross-resistance to abamectin (35.7), methoxyfenozide (29.1), JS118 (16.5), and deltamethrin (3.9). However, the selected resistant strain showed no obvious resistance to cypermethrin (1.3), fipronil (1.3), trichlorfon (1.1), chlorfenapyr (1.0), phoxim (0.9) and acephate (0.8). Thus, these insecticides were thought as non-cross-resistant insecticides, which could be used rotationally or mixed with tebufenozide to delay resistance development in this pest.3) Fitness costs for tebufenozide resistance in diamondback mothLife tables for experimental populations of the susceptible strain (S) and the tebufenozide-resistant strain (R) were constructed to evaluate the effects of tebufenozide resistance on the fitness of diamondback moth. The results showed that insects from both strains were similar in development duration, pupa weight, adult emergence rate and sex ratio. However, in the R strain, the survive rate of larvae and pupae, copulation rate of adults, effective fecundity of females and hatchability of eggs decreased significantly. As calculated with the S strain as standard, it was found that the R strain showed very low fitness with a value of only 0.35. This means that tebufenozide resistance costs much fitness of diamondback moth. Under the conditions free from tebufenozide, the proportion of the resistant individuals in populations will decrease and the sensitivity to tebufenozide will recover gradually. Thus, limited use of tebufenozide and alternate use it with some other insecticides without cross-resistance can be an effective method for the resistance management in this pest.4) Heredity of tebufenozide resistance in diamondback mothThe genetic characteristics of the tebufenozide resistance in diamondback moth, were analyzed by a serious of cross experiments with a laboratory susceptible strain and a selected resistant strain (RR 270). It was found that the resistance was partial biased in female heredity, with the LC50 540μg/ml for F1 and 2696.4μg/ml for rF1 progeny. Further analysis showed that the susceptible male and female were similar in sensitivity to tebufenozide, but resistant ones were much different. Thus, this bias had not relations with plasma heredity, but may be resulted from the possible resistance gene located on the female sex chromosome W. This was the second case for the sex linkage of insecticide resistance in insects.5) Mechanisms for resistance of diamondback moth to tebufenozideFor analysis of the resistance mechanisms, the synergism of PBO, TPP and DEM on tebufenozide in susceptible and resistant strain of diamondback moth was tested, and the activity of microsomal-O-demethylase (MFO), esterase(EST) and glutathione-S-transferase (GST) was also compared between these two strains. The results showed that PBO, TPP and DEM did not show any synergism on tebufenozide in the susceptible strain. But their synergism (5.56, 3.42 and 1.77, respectively) was significantly high in the resistant strain. The resistant strain also showed higher activity of MFO (2.42 folds), EST (2.10 folds) and GST (2.00 folds) than the susceptible strain. So, it was concluded that MFO, EST and GST were all involved in the resistance of diamondback moth to tebufenozide. Otherwise, the activity of chitinase and arylamidase in these two strains was also compared. It was found that the resistant strain had much lower activity of chitinase (0.26 fold), but higher activity of arylamidase (2.55 fold). However, the relationship between the activity of these two enzymes and tebufenozide resistance needs further studies to declear.6) Clonning of the genes correlated with tebufenozide resistanceWith RT-PCR technique, the cDNA fragments of 4 different genes, ecdysteroid receptor (EcR), ultraspiracle (USP), dopa decarboxylase (DDC) and larval cuticle protein (LCP), correlated with the tebufenozide resistance were cloned from diamondback moth. Of which, EcR was cloned to its full length by using RACE strategy. Sequence analysis indicated that the cloned cDNA fragments were USP 750bp, DDC 489bp and LCP 207bp, and the full length EcR is 2358bp with a ORF encoding 551 amino acides. These work funded the base for further work on molecular resistance mechanisms.7) Mutation in ecdysteroid receptor related to tebufenozide resistanceFull length genes of ecdysteroid receptor (EcR) from resistant and susceptible diamondback moth were cloned and sequenced. Alignment analysis demonstrated that some amino acid polymorphism existing in this gene, and one amino acid substitutes (P514S) in N terminal of 11 helix of the pocket of ligand binding was found to be resistance related. Comparision of the genotype composition (RR:RS:SS) between the cross F2 generation (3:5:2) and its survivers after tebufenozide treatment (8:1:0) proved that this mutation made diamondback moth more resistant. From the 3D model, it was also found that this mutation causes changes in the dimensional structure of EcR, and should be a resistance mutation.In this investigation, it was found that tebufenozide have risk of developing resistance in this pest, though it has a special mode of action. We should pay attention on the rational use of insecticides and management of resistance. Because the resistance at early developing stage depends on the enhancement of the activity of the detoxicating metabolism enzymes, as the results showed, we should consider of adding synergists to the insecticide formulations to increase the effect of tebufenozide on the resistant diamondback moth and delay the resistance development. Otherwise, the decrease in the fitness of resistant strain indicate that limited use of this insecticide and alternate use it with some other insecticides without cross-resistance can be also a effective method for the resistance management. Otherwise, the cloned genes and the identified mutation correlated with the tebufenozide resistance foundated the base for further study on molecular mechanism for the interaction between tebufenozide and EcR.