東方果實蠅對納乃得之抗性機制探討

Methomyl-Resistant Mechanisms of the Oriental Fruit Fly, Bactrocera dorsalis (Diptera: Tephritidae)

黃凱揚(Kai-Yang Huang)

指導教授 ： 許如君

國立臺灣大學/生物資源暨農學院/昆蟲學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.01317

摘要

東方果實蠅 (Bactrocera dorsalis) 為相當嚴重的農業害蟲，納乃得 (methomyl)於1995年在台灣被推薦使用於防治東方果實蠅，但2002年在田間已發現東方果實蠅對納乃得產生數十倍抗藥性。在本論文研究當中，我們利用實驗室建立之東方果實蠅納乃得抗性品系 (具140倍抗性) 以及感性品系進行協力劑實驗、代謝酵素活性及乙醯膽鹼酯酶 (acetylcholinesterase, AChE) 活性及ace基因 (為轉譯AChE之基因) 序列等實驗以探討可能的抗性機制。另加入曾深入研究乙醯膽鹼酯酶之撲滅松 (fenitrothion) 抗性品系 (具538倍抗性) 以做對照比較。以協力劑探討納乃得的代謝機制，結果顯示協力劑PBO (piperonyl butoxide) 及TPP (triphenyl phosphate) 的協力效果顯著 (PBO, 4.04倍；TPP, 4.82倍)，另協力劑DEM (diethyl maleate) 對納乃得抗性品系之協力效果比感性品系高約1.7倍；在代謝酵素活性實驗當中，也同樣發現納乃得抗性品系在代謝酵素酯酶 (受質：α-及β-naphthyl acetate) (1.5倍)、麩胺基硫轉移酶 (受質：CDNB及DCNB) (1.5至2倍) 及多功能氧化酶 (受質：7-ethoxycoumarin) (4倍) 等活性上均較感性品系來的高。推測多功能氧化酶 (mixed function oxidases)、酯酶 (esterases) 及麩胺基硫轉移酶 (glutathione S-transferases) 可能和東方果實蠅之納乃得抗性的產生有所關聯。在乙醯膽鹼酯酶的活性、乙醯膽鹼酯酶不敏感性實驗 (抑制劑: 納乃得及氧基巴拉松 (paraoxon)) 以及ace基因的表現實驗當中，並沒有找到感性品系和納乃得抗性品系之間的差異。另在乙醯膽鹼酯酶抑制活性實驗當中，僅有撲滅松抗性品系對氧基巴拉松的抑制較不敏感。雖然在納乃得抗性品系ace基因定序實驗中，發現乙醯膽鹼酯酶後轉譯修飾區域上有一點突變 (T659A) 位置，但此點突變也同樣發生在感性品系當中，感性品系之點突變基因頻度較納乃得抗性品系低 (感性品系：TT = 0.27；TA = 0.55；AA = 0.18。納乃得抗性品系：TA = 0.16；AA = 0.84)。T659A點突變會稍微增加後轉譯修飾前之乙醯膽鹼酯酶序列疏水性C端的疏水性；不過並沒有從納乃得抗、感性品系間或是不同的T659A基因型間，找到對於乙醯膽鹼酯酶的活性以及乙醯膽鹼酯酶受納乃得抑制能力之間的關聯性。目前研究發現東方果實蠅對於納乃得的抗性機制可能主要是在代謝機制上；而且東方果實蠅對於納乃得之標的抗性機制，和果實蠅屬 (Bactrocera spp.) 中曾深入研究探討之有機磷類標的抗性機制不同。

關鍵字

東方果實蠅 ； 納乃得 ； 協力劑 ； 代謝酵素 ； 乙醯膽鹼酯酶點突變

並列摘要

Oriental fruit flies (Bactrocera dorsalis) are notorious agricultural pests, methomyl has been used to manage B. dorsalis in Taiwan since 1995. Unfortunately, several times of methomyl resistance has been observed in B. dorsalis from field investigation in 2002. In this study, the synergism bioassay, metabolic enzyme activity, acetylcholinesterase (AChE) activity and the ace gene (which can encode AChE protein) molecular assays were used to explore the possible methomyl resistant mechanisms in the methomyl-resistant strain (Meth-R, 140-fold resistance ratio) of B. dorsalis. A well-documented fenitrothion-resistant strain (Fenit-R, 538-fold resistance ratio) was used to compare with the Meth-R and susceptible strains on AChE assays. Synergism tests indicated synergistic ratios toward methomyl at 4.04-fold with piperonyl butoxide (PBO) and 4.82-fold with triphenyl phosphate (TPP) on Meth-R, and there was a 1.7-fold diethyl maleate (DEM) synergistic ratio on Meth-R as compared to the results of the susceptible strain. Enzyme activity results supported the theory that Meth-R had greater activity, close to 1.5-fold in ESTs (by α- or β-naphthyl acetate as the substrates), 1.5- to 2-fold in GSTs (by CDNB or DCNB as the substrates), and 4-fold in MFOs (by 7-ethoxycoumarin as the substrates) over the susceptible strain, which suggests that mixed function oxidases (MFOs), esterases (ESTs), and glutathione S-transferases (GSTs) are involved in methomyl resistance. No significant difference was found on the AChE activity, AChE inhibition (by using methomyl and paraoxon as inhibitors), and quantitative PCR (qPCR) of ace gene between the Meth-R and susceptible strains. The AChE of the Fenit-R strain was found to be insensitive to the paraoxon inhibitor. Although a point mutation (T659A) located at the post-translational modification region was discovered on the ace gene of the Meth-R; T659A also occurred in the susceptible strain, but with lower gene frequency (susceptible strain: homozygous genotype of T = 0.27, heterozygous = 0.55, homozygous genotype of A = 0.18; Meth-R strain: TA = 0.16; AA = 0.84). T659A might slightly influence the hydrophobicity of AChE precursor protein due to an increase of the hydrophobic results on the C-terminal region. However, there was no correlation between the Meth-R and susceptible strains or among different genotypes of T659A on the results of AChE activity or AChE inhibition assays. As a result, our study indicates that the possible methomyl resistance is the metabolic mechanism in B. dorsalis. The mechanism in methomyl resistance is different from the well-known altered target-site mechanism in organophosphate resistance in Bactrocera spp.

並列關鍵字

Bactrocera dorsalis ； methomyl ； synergists ； metabolic enzyme ； acetylcholinesterase ； point mutation

參考文獻

Chuang, Y. Y., and R. F. Hou. 2005. Field tests and evaluation of effectiveness using the longer efficiency trap for the oriental fruit fly, Bactrocera dorsalis. Research Bulletin, KDARES. 16: 50-59. (in Chinese)

Liu, T. D., C. J. Huang, and H. S. Lin. 1996. Evaluation of guava fruits on the control of oriental fruit fly (Bactrocera dorsalis (Hendel)). Bull. Taichung Dist. Agri. Improv. Sta. 50: 61-71. (in Chinese)

Ahmed, S., and R. M. Wilkins. 2002. Studies on some enzymes involved in insecticide resistance in fenitrothion-resistant and -susceptible strains of Musca domestica L. (Dipt, Muscidae). J. Appl. Entomol. 126: 510-516.

Amichot, M., S. Tares, A. Brun-Barale, L. Arthaud, J. M. Bride, and J. B. Berge. 2004. Point mutations associated with insecticide resistance in the Drosophila cytochrome P450 Cyp6a2 enable DDT metabolism. Eur. J. Biochem. 271: 1250-1257.

Armstrong, R. N. 1997. Structure, catalytic mechanism, and evolution of the glutathione transferases. Chem. Res. Toxicol. 10: 2-18.