Direct identification of continuous second - order plus dead-time model

Describes a direct identification of continuous second - order plus dead-time model

아치사량 살충제에 반응하는 3종 해충의 유전자 동정 및 특성 분석

학위논문 (박사) -- 서울대학교 대학원 : 농업생명과학대학 농생명공학부, 2021. 2. Si Hyeock Lee.Since one of the major insecticide resistance mechanisms is the enhanced xenobiotic detoxification, characterization of these detoxification factors would facilitate the understanding how insects develop metabolic resistance to insecticides. As the expression of many detoxification gene is inducible by sublethal treatment of insecticides, analysis of the transcriptome profiles of insects treated with a sublethal dose of insecticide has been employed as a general method for identifying the major metabolic factors associated with insecticide tolerance and resistance. In this study, Plutella xylostella (diamondback moth, DBM), Frankliniella occidentalis (western flower thrips, WFT) and Drosophila melanogaster (common fruit fly, CFF) were selected as model insect species. These insects were treated with sublethal amounts of various insecticides, and their transcriptomes were analyzed and compared within and between species to common metabolic factors possibly associated with insecticide tolerance and resistance. In chapter I, third instar larvae of the P. xylostella were pretreated with sublethal concentrations (LC10) and then subsequently exposed to medium lethal concentrations (LC50) of chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb and spinosad via leaf dipping, their tolerance to insecticides was significantly enhanced. Transcriptome data determined that 125, 143, 182, 215 and 149 transcripts were overexpressed whereas 67, 45, 60, 60 and 38 transcripts were underexpressed following treatments with chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb and spinosad, respectively. When further characterized the differentially expressed genes (DEGs), the most notable over-transcribed genes were two cytochrome P450 genes (Cyp301a1 and Cyp9e2) and nine cuticular protein genes. On the contrary, several genes composing the mitochondrial energy generation system were under-transcribed in all treated larvae. These results showed at least in the case of P. xylostella, the common DEGs appeared to be involved in general chemical defense, regardless of the structures and modes of actions of these insecticides, at the initial stage of intoxication. In chapter II, pretreatment with sublethal concentrations (LC10) of chlorfenapyr, dinotefuran and spinosad, then subsequently treated with medium lethal concentrations (LC50) of the respective insecticide via residual contact vial plus water (RCVpW) method and the pretreatments enhanced the tolerance in F. occidentalis female adult significantly. Transcriptome analysis showed that 404, 386 and 756 genes were up-regulated, meanwhile 124, 107 and 169 genes were down-regulated following the treatment of chlorfenapyr, dinotefuran and spinosad, respectively. Among these, 199 transcripts were commonly up-regulated, whereas 31 transcripts were commonly down-regulated. Most up-regulated transcripts were categorized as basic biological processes, including proteolysis and lipid metabolism. Detoxification genes, such as one glutathione S transferase, three UDP-glucuronosyltransferases, four CYP450s, and one ABC transporter, were commonly up-regulated in all three insecticide-treated groups. RNA interference of five commonly overexpressed genes increased mortalities to all three insecticides, since these three tested insecticides have distinct structures and modes of action, the roles of commonly expressed genes in tolerance were supported and further discussed. In chapter III, sublethal concentrations (LC10) of chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb, ivermectin and spinosad were introduced to D. melanogaster female adults, and subsequently treated these insecticides with medium lethal concentrations (LC50) via topical treatment. Similar with the previous cases, the tolerance to insecticides was enhanced significantly. Transcriptome analysis identified 123, 173, 75, 245, 368 and 145 over-transcribed genes, as well as 137, 108, 202, 83, 59 and 126 under-transcribed genes in chlorantraniliprole-, cypermethrin-, dinotefuran-, indoxacarb-, ivermectin- and spinosad-treatment, respectively. Among these DEGs, 26 and 30 genes were found commonly up- and down-regulated in all insecticide treated groups. The major part of commonly up-regulated genes are immune induced antibacterial peptides, such as attacin-A/C, diptericin A/B, drosocin and immune induced molecule 18, etc. On the other hand, many components of mitochondrial respiratory chain were commonly down-regulated in all treatments. Their roles in general chemical tolerance were discussed.현대 농업에서는 여러 종류의 해충방제를 위해 다양한 살충제가 도입되었으며, 이러한 화학 물질의 범지구적 사용으로 살충제 내성/저항성이 등장하였고 이는 시급하게 해결해야 될 문제이다. 따라서 살충제 스트레스에 의해 유도될 수 있는 유전자를 찾아내고 특징을 확인하기 위해 Plutella xylostella (배추좀나방, DBM), Frankliniella occidentalis (꽃노랑총채벌레, WFT) 및 Drosophila melanogaster (노랑 초파리, CFF)를 모델 시험곤충으로 선정하여 다양한 살충제들의 아치사량으로 처리한 후 전사체 데이터를 분석하였다. 1장에서는 잎침지법으로 P. xylostella 의 3령 유충에 chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb 및 Spinosad 약제를 아치사농도(LC10)로 전처리 한 후 반수치사농도 (LC50)에 노출 시켰을 때 살충제에 대한 내성이 크게 향상됨을 확인하였다. 전사체 데이터를 통해 chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb 및 spinosad 처리군에서 과발현된 전사체는 각각 125, 143, 182, 215 및 149개인 반면, 저발현된 전사체는 67, 45, 60, 60 및 38 개임을 확인하였다. 차별발현유전자(DEG) 중 가장 전사량 차이가 컸던 유전자는 두 개의 사이토크롬 P450 유전자(Cyp301a1 및 Cyp9e2)와 9 개의 표피단백질 유전자였다. 반대로, 미토콘드리아 에너지 생성 시스템을 구성하는 몇몇 유전자는 모든 처리군에서 적게 전사되었다. 이 결과는 P. xylostella의 경우, 대부분의 DEG가 살충제의 구조와 작용 기작에 관계없이 중독 초기 단계에서 일반적인 화학적 방어에 관여함을 시사한다. 2장에서는 잔류접촉법(RCVpW)으로 F. occidentalis 의 암컷 성충에 chlorfenapyr, dinotefuran 및 spinosad 약제의 아치사농도(LC10)를 전처리한 후 반수치사농도(LC50)에 노출시켜 살충제 내성이 크게 향상됨을 확인했다. 전사체 분석 결과 chlorfenapyr, dinotefuran 및 spinosad 처리시 404, 386, 756 개의 유전자의 발현량이 증가했고, 124, 107, 169개의 유전자의 발현량이 감소했다. 이 중 199개의 전사체는 세 가지 약제 처리 시 공통적으로 상향조절 되었으며, 31개가 하향조절 되었다. 대부분의 상향조절 된 전사체는 단백질 분해와 지질 대사와 같은 기본적인 생물학적 과정으로 분류되었다. 해독 유전자에 속하는 glutathione S transferase 1개, UDP- glucuronosyltransferases 3개, CYP450 4개, ABC transporter 1개는 세 살충제 처리군에서 공통적으로 상향조절되었다. 공통적으로 과발현 된 유전자 중 5개의 RNAi를 시행했을 때, 3개의 살충제 모두에서 사망률이 증가하였는데, 세 살충제의 구조와 작용 기작이 서로 다르기 때문에 공통과발현 유전자는 살충제 내성에 관여한다고 보았다. 3장에서는 D. melanogaster 암컷 성충을 국소처리법으로 chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb, ivermectin and spinosad 아치사농도 (LC10)에 노출시킨 후 반수치사농도(LC50)로 처리하였고, 이전 실험들과 마찬가지로 살충제 내성이 크게 증가하였다. 전사체분석을 통해 chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb, ivermectin and spinosad에서 각각123, 173, 75, 245, 368, 145개의 과발현된 유전자를 확인하였고, 137, 108, 202, 83, 59, 126개의 저발현된 유전자를 확인하였다. 이러한 DEG 중 26개와 30개의 유전자가 6개 살충제 모두에서 공통적으로 상향, 하향조절 되는 것으로 나타났으며, 공통적으로 상향조절된 유전자는 attacin-A/C, dptericin A/B, drosocin, immune induced molecule 18 등과 같은 항균성 펩타이드를 만드는 면역관련 유전자가 대부분이었다. 미토콘드리아 호흡계를 구성하는 유전자들이 공통적으로 하향조절되었으며, 이러한 유전자들이 일반적으로 내성에 어떻게 관여하는지에 대해 기술하였다.GENERAL INTRODUCTION 1 CHAPTER I Transcriptomic identification and characterization of genes responding to sublethal concentrations of five different insecticides in the diamondback moth, Plutella xylostella 12 ABSTRACT 12 1. INTRODUCTION 14 2. MATERIAL AND METHODS 17 2.1 Diamondback moth stocks and rearing conditions 17 2.2 Determination of insecticide sublethal concentrations and tolerance bioassay 17 2.3 Insecticide treatment and total RNA extraction 19 2.4 Library construction and sequencing 19 2.5 Sequence processing, de novo assembly and annotation 20 2.6 Differentially expressed gene (DEG) analysis 21 2.7 Quantitative real-time PCR (qPCR) 22 3. RESULTS 24 3.1 Determination of insecticide sublethal concentrations 24 3.2 Determination of tolerance following sublethal pretreatment of insecticides 26 3.3 De novo assembly of transcriptome data 28 3.4 DEGs following insecticide treatment 33 3.5 Confirmation of DEG profiles by qPCR 39 4. DISCUSSION 41 4.1 Tolerance induction 41 4.2 GO profiles of DEGs 41 4.3 Commonly over-transcribed genes following the treatment of sublethal doses of insecticides 42 4.4 Commonly under-transcribed genes following the treatment of sublethal doses of insecticides 47 5. CONCLUSIONS 49 CHAPTER II Transcriptomic identification and characterization of genes responding to sublethal concentrations of three different insecticides in the western flower thrips, Frankliniella occidentalis 50 ABSTRACT 50 1. INTRODUCTION 52 2. MATERIAL AND METHODS 57 2.1 Insect strains and rearing 57 2.2 Insecticide treatment using the residual contact vial plus water (RCVpW) bioassay method, determination of sublethal doses, and tolerance bioassay 58 2.3 Insecticide treatment and total RNA extraction 59 2.4 Library construction and sequencing 60 2.5 Sequence processing and annotation 61 2.6 Reference-based differentially expressed gene (DEG) analysis 61 2.7 Quantitative real-time PCR (qPCR) 62 2.8 dsRNA synthesis 65 2.9 Ingestion RNAi and insecticide bioassay 68 3. RESULTS 69 3.1 Increased tolerance following sublethal pretreatment with insecticides 69 3.2 Transcriptome data analysis 72 3.3 DEGs following insecticide treatment 76 3.4 Validation of DEG profiles by qPCR 94 3.5 Effects of target gene RNAi on the insecticide toxicity 96 4. DISCUSSION 99 4.1 Tolerance induction 99 4.2 GO profiles of DEGs 100 4.3 Comparison of DEG profiles between F. occidentalis and P. xylostella 100 4.4 Commonly over-transcribed genes following treatment with sublethal concentrations of insecticides 102 4.5 Mortality increase in the thrips with representative target genes knocked down 106 5. CONCLUSION 107 CHAPTER III Transcriptomic identification and characterization of genes responding to sublethal concentrations of six different insecticides in the common fruit fly, Drosophila melanogaster 108 ABSTRACT 108 1. INTRODUCTION 110 2. MATERIAL AND METHODS 114 2.1 Insect strains and rearing 114 2.2 Determination of sublethal concentrations and tolerance bioassay 114 2.3 Insecticide treatment and total RNA extraction 115 2.4 Library construction and sequencing 116 2.5 Sequence processing and annotation 117 2.6 Reference-based differentially expressed gene (DEG) analysis 117 2.7 Quantitative real-time PCR (qPCR) 118 3. RESULTS 122 3.1 Determination of insecticide sublethal concentrations and tolerance induction 122 3.2 Transcriptome data analysis 125 3.3 DEGs following insecticide treatment 127 3.4 Validation of DEG profiles by qPCR 135 4. DISCUSSION 142 4.1 Tolerance induction 142 4.2 GO profiles of DEGs 142 4.3 Commonly over-transcribed genes following treatment with sublethal concentrations of insecticides 143 4.4 Commonly under-transcribed genes following treatment with sublethal concentrations of insecticides 148 4.5 Comparison of DEG profiles among D. melanogaster, F. occidentalis and P. xylostella 151 5. CONCLUSION 156 GENERAL DISCUSSION 158 REFERENCE 166 ABSTRACT IN KOREAN 181 ACKNOWLEDGEMENT 184Docto