吳文哲臺灣大學：昆蟲學研究所許如君Hsu, Ju-ChunJu-ChunHsu2007-11-262018-06-292007-11-262018-06-292004http://ntur.lib.ntu.edu.tw//handle/246246/55046東方果實蠅(Bactrocera dorsalis (Hendel))是台灣最重要的果樹害蟲之一，目前有多種殺蟲劑防治其在田間的發生。以室內東方果實蠅品系分別用乃力松、三氯松、撲滅松、芬殺松、福木松、馬拉松、納乃得、賽滅寧、賽扶寧及芬化利等篩選十個品系30代後，證實東方果實蠅會對上述篩選的殺蟲劑產生抗藥性，其抗性程度從對乃力松的4.7倍到福木松的594倍。以DEF（S,S,S-tributylphosphorotrithioate）、DEM （diethyl maleate）及PBO（piperonyl butoxide）三種協力劑抑制昆蟲代謝解毒酵素，測試感性品系和十種抗性品系對殺蟲劑的感受性，據以推測代謝酵素對抗性的貢獻並解釋十種抗性品系對其它種殺蟲劑之間的交互抗性。乃力松、三氯松及馬拉松等三個抗性品系對這三個原始篩選的殺蟲劑在測試時有顯著的交互抗性，推測是因此三品系對其篩選藥劑之抗性皆具DEF協力作用所造成；賽扶寧、賽滅寧及芬化利之三抗性品系對這原始篩選的三個合成除蟲菊殺蟲劑也產生了顯著的交互抗性，究其原因則可能是因此三品系對其篩選藥劑的抗性皆具PBO協力作用所造成。在所測試的十種抗性品系中，對殺蟲劑的交互抗性現象不僅存在於同類別的殺蟲劑亦存在於不同類別的殺蟲劑上，尤以賽滅寧及芬化利抗性品系為烈，二者對其它殺蟲劑的抗性程度甚至大於對原篩選藥劑本身。相反地，六種有機磷及納乃得抗性品系對賽滅寧及芬化利則沒有交互抗性存在。進一步探討四種生化機制對馬拉松及撲滅松抗性的貢獻，只有麩胺基硫轉移酶（glutathione S-transferase）在馬拉松抗性品系和感性品系上無差異外，在抗性蟲上酯酶（esterase）及多功能氧化酶（mixed function oxidase）的量明顯高於感性蟲。另在標的酵素- 乙醯膽鹼酯酶（acetylcholinesterase (AChE)）在抗性蟲上對有機磷抑制劑則明顯較感性蟲不敏感。酯酶及氧化酶代謝的增加及乙醯膽鹼酯酶對抑制劑的不敏感性是東方果實蠅對馬拉松產生抗性的主要貢獻。在撲滅松抗性部分，則排除水解酶、麩胺基硫轉移酶及氧化酶等代謝酵素是對撲滅松產生抗性的原因，其抗性果實蠅頭部乙醯膽鹼酯酶對有機磷劑抑制的不敏感性為東方果實蠅對撲滅松產生抗性的主要機制。再藉由乙醯膽鹼酯酶基因的解序，得知感性果實蠅的乙醯膽鹼酯酶基因的胺基酸編碼區有2022個鹼基對，可轉譯成673個胺基酸。撲滅松的抗性蟲則在胺基酸序列上有三個點突變，分別為 I214V、G488S及Q643R，這些突變是造成抗性蟲對撲滅松產生不敏感性的主要原因。Oriental fruit flies, Bactrocera dorsalis (Hendel), were treated with ten insecticides, including six organophosphorus insecticides (naled, trichlorfon, fenitrothion, fenthion, formothion, and malathion), one carbamate (methomyl), and three pyrethroids (cyfluthrin, cypermethrin, and fenvalerate), by a topical application assay under laboratory conditions. Sub-parental lines of each generation of the oriental flies treated with the same insecticide were selected for 30 generations and were designated as x-r lines (x: insecticide; r: resistant). The parent colony was maintained as the susceptible colony. The line treated with naled exhibited the lowest increase in resistance (4.7-fold) while the line treated with formothion exhibited the highest increase in resistance (up to 594-fold) compared to the susceptible colony. Synergism bioassays were also carried out. Based on this, when oriental fruit flies were treated with S,S,S-tributyl phosphorotrithioate displayed a synergistic effect for naled, trichlorfon and malathion resistance, whereas the colonies treated piperonyl butoxide displayed a synergistic effect for pyrethroid resistance. All ten resistant lines also exhibited some cross resistance to other insecticides, not only to the same chemical class of insecticides but also to other classes. However, none of the organophosphorus-resistant or the methomyl-resistant lines exhibited cross-resistance to two of the pyrethroids (cypermethrin and fenvalerate). Overall, the laboratory resistance and cross-resistance data of the fruit flies treated with insecticides developed here should provide useful tools and information for designing an insecticide application strategy for controlling this fruit fly in the field. Extended study to explore the biochemical mechanism of resistance to the organophosphorus insecticides, malathion and fenitrothion, in the fly showed that the enzyme activity of glutathione S-transferase was not significantly different between the malathion-resistant line and susceptible colony. However, malathion-resistant line exhibited higher activity in esterase and mixed function oxidase than the susceptible colony did. The target enzyme, acetylcholinesterase (AChE), from the resistant line was less sensitive to the inhibition of organophosphorus inhibitors than that from the susceptible colony. These results suggested that elevated hydrolytic and oxidative metabolic enzymes in conjunction with an altered AChE with poorer catalytic efficiency might contribute to the resistance of this fly to malathion. To fenitrothion, the resistant line exhibited reduced AChE activity compared to that in susceptible colony, while the activities of the other enzymes did not significantly differ between these two fruit fly colonies. The resistant line also exhibited at least a 10-fold reduced sensitivity to a series of AChE inhibitors compared to that of susceptible colony. To investigate the molecular basis of this fenitrothion resistance, cDNAs from the gene encoding AChE were characterized from individuals representing both the resistant lines and susceptible colony. Three point mutations, I214V, G488S and Q643R, resulting in nonsynonymous changes in the amino acid sequence of this gene were detected in the resistant flies. These changes appear to correspond to key catalytic sites affecting the function of AChE.TABLE OF CONTENTS ACKNOWLEDGMENTS i ABSTRACT OF THE DISSERTATION iii CHINESE ABSTRACT vi LIST OF TABLES xii LIST OF ILLUSTRATIONS xiv CHAPTER 1. INTRODUCTION 1 Insecticide Resistance 1 Insecticide Resistance Mechanism 3 The Biology of Bactrocera dorsalis 6 History of Chemical Control of B. dorsalis in Taiwan 8 CHAPTER 2. RESISTANCE AND SYNERGISTIC EFFECTS OF INSECTICIDES IN BACTROCERA DORSALIS (DIPTERA: TEPHRITIDAE) IN TAIWAN 11 Abstract 11 Introduction 12 Materials and Methods 14 Insects 14 Chemicals 15 Resistant Lines and Bioasssays 16 Synergism Bioassays 18 Cross-resistance Bioassays 18 Results 19 Susceptible Colony 19 Establishment of Resistant Lines 19 Synergism Bioassays 20 Cross-resistance Bioassays 21 Discussion 22 CHAPTER 3. BIOCHEMICAL MECHANISMS OF MALATHION RESISTANCE IN ORIENTAL FRUIT FLIES (BACTROCERA DORSALIS) 32 Abstract 32 Introduction 33 Materials and Methods 34 Chemicals 34 Laboratory Colonies 34 Enzyme-activity Assays 36 Esterases 36 Glutathione S-transferases 37 Mixed Function Oxidases 37 AChE Activity and Insensitivity 38 Enzyme Kinetic Studies 39 Results 40 Discussion 41 CHAPTER 4. ASSOCIATION OF POINT MUTATIONS IN THE ACE GENE WITH FENITROTHION RESISTANCE IN THE ORIENTAL FRUIT FLY 52 Abstract 52 Introduction 53 Materials and Methods 56 Oriental Fruit Flies 56 Enzyme-activity Assays 57 Esterases 57 Glutathione S-transferases 58 Mixed Function Oxidases 58 AChE Activity and Insensitivity 59 Cloning and Sequencing of AChE Gene of Susceptible Flies 60 Cloning and Sequencing of AChE Gene of Resistant Flies 62 AChE Inhibition Assay and Sequencing the cDNA 62 Results 63 Enzyme-activity Assays 63 cDNA and Deduced Amino Acid Sequences of Susceptible AChE 64 Comparison of AChE Genes from Fenitrothion-susceptible and -resistant Flies 64 AChE Inhibition Assay plus Sequencing the cDNA 65 Discussion 65 CHAPTER 5. CONCLUSION 77 REFERENCES 80 APPENDIX607089 bytesapplication/pdfen-US抗藥性乙醯膽鹼酯酶生化機制基因東方果實蠅insecticide resistancebiochemical mechanismAceBactrocera dorsalisthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/55046/1/ntu-93-D88632001-1.pdf