Transcriptome-based identification and characterization of genes commonly responding to five different insecticides in the diamondback moth, Plutella xylostella

https://doi.org/10.1016/j.pestbp.2017.11.007Get rights and content

Highlights

  • DBM larvae treated with insecticide sublethal doses exhibited enhanced tolerance.

  • Transcriptomes of larvae treated with insecticide sublethal doses were analyzed.

  • Genes commonly responding to all insecticide treatments were further characterized.

  • Cyp450 and cuticle protein genes were commonly up-regulated in all treated larvae.

  • Mitochondrial energy generation genes were down-regulated in all treatments.

Abstract

When the 3rd instar larvae of the diamondback moth (DBM), Plutella xylostella, were pretreated with sublethal doses (LC10) and then subsequently exposed to lethal doses (LC50) of chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb and spinosad via leaf dipping, their tolerance to insecticides was significantly enhanced. To identify genes that commonly respond to the treatment of different insecticides and are responsible for the tolerance enhancement, transcriptomic profiles of larvae treated with sublethal doses of the five insecticides were compared with that of untreated control. A total of 117,181 transcripts with a mean length of 662 bp were generated by de novo assembly, of which 35,329 transcripts were annotated. Among them, 125, 143, 182, 215 and 149 transcripts were determined to be up-regulated whereas 67, 45, 60, 60 and 38 genes were down-regulated following treatments with chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb and spinosad, respectively. Gene ontology (GO) analysis of differentially expressed genes (DEGs) revealed little differences in their GO profiles between treatments with different insecticides except for spinosad. Finally, the DEGs commonly responding to all insecticides were selected for further characterization, and some of their over-transcription levels were confirmed by quantitative PCR. The most notable examples of commonly responding over-transcribed genes were two cytochrome P450 genes (Cyp301a1 and Cyp9e2) and nine cuticular protein genes. In contrast, several genes composing the mitochondrial energy generation system were significantly down-regulated in all treated larvae. Considering the distinct structure and mode of action of the five insecticides tested, the differentially expressed genes identified in this study appear to be involved in general chemical defense at the initial stage of intoxication. Their possible roles in the tolerance/resistance development were discussed.

Introduction

Diamondback moth (DBM), Plutella xylostella (L.), which originated from the Mediterranean region, is one of the most devastating agricultural pests. It feeds on foliar tissue of cruciferous crops, including cabbage, broccoli, cauliflower, etc. The economic loss caused by DBM is estimated as US $ 4–5 billion worldwide annually [1], [2]. Many insecticides have been used to control DBM in the past decades. As a result of the overuse of insecticides and its high adaptability, DBM has developed resistance to most commercial insecticides, including organophosphates, carbamates and pyrethroids [3]. To overcome the resistance problem, various other groups of insecticides, including neonicotinoids, macrocyclic lactones, diamides, etc., have been introduced to control DBM [4], [5], [6]. Although some cases of DBM resistance to these newly introduced insecticides have been reported, resistance is not yet widespread and underscores the critical need for proactive resistance management strategies for them. Since one of the major mechanisms for insecticide resistance is enhanced xenobiotic detoxification, including proteins that can metabolize or efflux xenobiotics [7], characterization of these detoxification factors would facilitate the understanding how insects develop metabolic resistance to insecticides.

Metabolic resistance to insecticides can be developed when pests acquire heritable traits that result in either constitutive/inducible over-expression or functional alteration of protein products involved in metabolism [8]. Since the expression of many detoxification gene is inducible by sublethal treatment of insecticides, analysis of transcript profiles, either in a small scale of using a subset of detoxification genes or in a full scale of using entire transcriptome, has been employed as a general method for identifying the major metabolic factors associated with insecticide resistance [8], [9], [10]. In the transcriptional profiling of body louse detoxification genes [cytochrome P450 (Cyp450) and ABC transporters (ABCT)], three Cyp450 and one ABCT genes, which are also known to be involved in insecticide metabolism in other organisms, were significantly overexpressed upon induction by a brief, sublethal exposure to ivermectin that resulted in tolerance [8]. Heterologous expression of CYP6CJ or ABCC4 resulted in the oxidative metabolism or ATP-dependent efflux of ivermectin, respectively [11]. In the case of DBM larvae induced by sublethal doses of cypermentrin, eight of 11 Cyp450 genes tested were over-transcribed in a cypermethrin-resistant strain whereas only a single Cyp450 gene was induced in a susceptible strain, suggesting that the selective Cyp450 induction by cypermethrin is also a metabolic resistance mechanism [12].

In this study, significant enhancements of tolerance to insecticides were verified in the DBM larvae individually pretreated with sublethal doses of five different insecticides (chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb and spinosad). To identify genes that commonly respond to different insecticides, thereby being putatively responsible for tolerance, the transcriptomes of the treated larvae were compared with that of untreated larvae, and a systematic analysis of metabolic factors that are induced by different groups of insecticides was carried out. In addition, by comparing deferentially expressed genes between insecticides, gene groups that commonly responded to different groups of insecticide were identified in order to determine generalist detoxification (defense) factors associated with tolerance. Identification of these insecticide-induced genes would contribute to our understanding on the xenobiotic detoxification factors in DBM and their role in developing tolerance and perhaps eventually resistance.

Section snippets

Diamondback moth stocks and rearing conditions

The insecticide-susceptible strain of DBM was obtained from Rural Development Administration, South Korea. They were reared in plastic cages (40 cm × 35 cm × 30 cm) using Chinese cabbage plants (Brassica napus) at 24 °C, 60% humidity and a 16-h light:8-h dark cycle.

Determination of insecticide sublethal doses and tolerance bioassay

Technical grade insecticides (> 93.9% purity) of chlorantraniliprole {5-bromo-N-[4-chloro-2-methyl-6-(methylcarbamoyl)phenyl]-2-(3-chloropyridin-2-yl)pyrazole-3-carboxamide}, dinotefuran [2-methyl-1-nitro-3-(oxolan-3-ylmethyl)guanidine] and

Determination of insecticide sublethal doses

The LC10 values at 24 h post-treatment were determined to be 0.32, 0.40, 16.1, 0.37 and 0.04 ppm for chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb and spinosad, respectively (Table 1). All p values were > 0.05 for the chi-square, suggesting that the bioassay data well fit the probit model. After treating the sublethal concentrations of chlorantraniliprole, cypermethrin, indoxacarb and spinosad, treated DBM larvae were collected for RNA extraction at 10 h post-treatment as these

Tolerance induction

Pretreatment of sublethal doses of insecticides induced the tolerance enhancement in DBM larvae although its level by indoxacarb pretreatment was not significant (p = 0.065) (Fig. 1). A similar case of tolerance induction was also previously reported in body lice that were briefly exposed to a sublethal dose of ivermectin [8]. In current study, the tolerance enhancement was observed in all examined cases regardless of insecticide type, suggesting that the tolerance induction by sublethal

Conclusions

Comparative transcriptome analysis followed by separate treatment of five insecticides enabled the identification of commonly responding genes to the sublethal challenge, thereby being involved in tolerance enhancement. The notable examples of over-transcribed genes include two Cyp450 genes and nine cuticular protein genes. Interestingly, many genes involved in the mitochondrial energy generation were down-regulated in all treated groups. Considering the physiological functions of these genes,

Acknowledgements

This work was supported by grant PJ010821022017 from Rural Development Administration (RDA), Korea. Yue Gao and Kyungmun Kim were supported in part by Brain Korea 21 program.

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