The fall armyworm, Spodoptera frugiperda, has developed extremely high levels of resistance to chlorantraniliprole and other classes of insecticides in the field. As microRNAs (miRNAs) play important roles in various biological processes through gene regulation. we examined the miRNA profile of S. frugiperda in response to Chlorantraniliprole, Indoxacarb and Thiamethoxam. Transcriptome analysis showed significant changes in the abundance of some miRNAs after treatment of S. frugiperda larvae with LC20 concentrations of three insecticides. A total of 197 miRNAs were systematically identified from S. frugiperda, and 16, 9, 2 miRNAs were differentially expressed after treatments of three insecticides. Importantly, three miRNAs were significantly downregulated and three were upregulated by RT-qPCR after treatment the LC50 of three insecticides with S. frugiperda larvae. Microinjection of agomirs of these six miRNAs into S. frugiperda larvae resulted in significant changes in mortality rates when exposed to three insecticides. Additionally, we also screened potential target genes for some of differentially expressed miRNAs, which may play important roles in insecticide resistance development. These findings provide valuable insights into the molecular mechanisms of insecticide resistance and underscore the potential of miRNAs as targets for the development of novel pest control strategies in S. frugiperda.

The Ryanodine Receptor in wild type Drosophila melanogaster has an amino acid substitution which is known to cause Chlorantraniliprole resistance in Pluteall xylostella. Even though we have reported that two Chlorantraniliprole resistant Drosophila strains have elevated total esterase activities, our report does not fully explain the significantly increased resistant ratios in two Chlorantraniliprole resistant Drosophila strains. Thus, we further analyzed alteration of reactive oxygen species and mitochondria activities in two Chlorantraniliprole resistant Drosophila strains. Our result suggested that Chlorantraniliprole resistance development in Drosophila requires alteration of various signal transduction pathways.

Chlorantraniliprole에 대한 저농도 및 고농도 저항성 계통을 이용하여 다른 약제들에 대한 교차 저항성 구명 연구를수행하였다. Chlorantraniliprole과는 다른 작용 기전을 가지는 작물보호제들에 대한 저항성 지수를 계산하는 연구를수행하였다. 그리고, 저농도 및 고농도 저항성 게통에서 발현이 차이가 나는 Glutathione-S-Transferase를 알아내기위하여 발현 양을 정량 분석 하였다. 본 연구의 결과는 향후 저항성 회피 기전의 개발에 활용될 수 있을 것으로사료된다.

The diamondback moth (DBM), Plutella xylostella, is a globally distributed and important economic pest. Chemical control is the primary approach to regulate populations of this pest. Chlorantraniliprole is the first commercial insecticide that belongs to the new chemical class of diamide insecticides. In this study, the resistant strain was observed 1578-fold resistance to chlorantraniliprole. Point mutation (G4946E) in ryanodine receptor (RyR) showed a high frequency. Enzyme assays indicated that glutathione S-transferase (GST) activity in the resistant strain was 2.4 times higher compared with the susceptible strain, whereas no difference was seen for P450 and esterase. In addition, the expression of two GSTs genes was up-regulated. These findings pave the way for the complete understanding of the mechanisms of diamide insecticides resistance in insects.

Ryanodine receptors (RyRs) regulate the contractions of insect muscles by altering intracellular Ca2+ concentration and are the targets of chlorantraniliprole. Here we established two resistant strains of Drosophila melanogaster, which were treated with low or high concentrations of chlorantraniliprole, and their resistance levels were determined on the basis of contact and ingestion toxicities. Compared with the wild type, the two resistant strains did not show any significant differences in contact toxicity. However, they showed significantly increased resistance ratios in ingestion toxicity than that by the wild type. The resistant strains had altered expression levels of RyRs and more enhanced Acetylcholinesterase and Glutathione-S-transferase activities than that by the non-selected strain. These results suggested that the resistance development of chlorantraniliprole in the two strains might be mediated by the activation of detoxification pathways in D. melanogaster.

The diamondback moth, Plutella xylostella, is known worldwide as a pest that affects cruciferous plants. We surveyed insecticide susceptibility in chlorantraniliprole-resistant strain of P. xylostella to 13 insecticides (3 diamaids, 2 synthetic pyrethroids, 2 spinosyns, 1 organophosphorus, 1 carbamate, 1 marcrolide, and 3 others) to examine resistance ratios. The levels of insecticide resistance were extremely high, especially to chlorantraniliprole and flubendiamide. Enzyme assays indicated that glutathione S-transferase (GST) activity in the resistant strain was 2.4 times higher compared with the susceptible strain, whereas no difference was seen for P450 and esterase. Its F1 progeny resulting from reciprocal crosses with a susceptible strain revealed no maternal effects and a diamide susceptible phenotype, suggesting an autosomally almost recessive mode of inheritance.

Chlorantraniliprole은 diamide계통의 나비목 유충 방제제로서 기존 살충제들과는 다르게 Ryanodine Receptor (RyR)에 작용하여 Ca2+ 분비에 이상을 유도하여 치사시킨다. 하지만 반복 사용으로 일부 나비목 해충에서 RyR 유전자의 돌연변이에 의한 저항성 계통이 보고되었다. 본 연구에서는 Chlorantraniliprole 저항성 발달 기작을 초파리를 모델 곤충으로 선정하여 수행하였다. EMS 처리로 비특이적 돌연변이를 유도시킨 후, Chlorantraniliprole을 낮은 농도 (5ppm)와 높은 농도 (30ppm)에서 도태를 시작하여 2종류의 저/고도태압 저항성 계통을 확보하였다. 섭식독성 수준을 비교한 결과, 고도태압군의 저항성 수준이 대조군에 비해 약 8배 정도 높게 나타났다. 또한 RyR의 발현량과 생화학적 해독 기전의 차이를 구명한 결과, 2개의 저항성 군집은 대조군보다 전반적으로 높은 생화학적인 해독 기전의 활성화를 보여주었다. 향후 연구는 저항성 기전을 밝혀냄으로써 저항성 회피기작을 알아내는데 도움을 줄 것이다. (본 연구는 농촌진흥청 연구과제 (PJ010821032016)의 지원을 받았음)

노랑초파리(Drosophila melanogaster)는 가장 연구가 많이 되어져 있는 모델동물로서 다양한 연구에 활용이 되어져 왔다. 하지만, 작물보호제에 대한 약제 저항성 모델로는 활용이 되어지지 않고 있다. 우리는 노랑초파리의 수컷에 Ethylene methane sulfonate를 처리하여 전체염색체에 돌연변이를 유도 한 후에 Chlorantraniliprole을 처리하여 약제에 대하여 저항성을 가지는 초파리 계통을 제작, 확립하였다. 저항성 계통에 대한 전체발현 유전체 분석과 micro-RNA분석을 수행하여 발현이 변화된 유전자를 밝혀내고 돌연변이 유전자를 발굴하는 연구를 수행하고 있다. 이러한 연구는 저항성 관련 기전을 밝혀내는 것과 동시에 내성에 관련된 기전을 밝혀내는데도 커다란 도움을 줄 수 있을 것이다. (본 연구는 농촌진흥청 협동연구사업 “초파리를 이용한 교차저항성 발달 모델 및 기작 구명(PJ01082103)”으로 수행되었음).