一種羧酸酯酶對小菜蛾脫芬瑞抗藥性的影響

Abstract

小菜蛾 (Plutella xylostella) 是現今被認為對十字花科植物最具威脅性的害蟲，對許多不同機制的殺蟲劑都已經產生抗藥性，本論文針對田間常用的四種殺蟲劑 (剋安勃、因滅汀、脫芬瑞、氟大滅) 進行抗性篩選，之後搭配先前實驗室的小菜蛾次世代定序 (Next Generation Sequencing) 資料，分別挑出上述四個殺蟲劑抗性品系與當時第一子代相比 FPKM (Fragments Per Kilobase of transcript per Million mapped reads) 值有 3 倍以上差異，並從每個抗性品系中挑選出 4 個差異表現最高，且屬於細胞色素 P450 單氧化酶 (Cytochrome P450 monooxygenase)、穀胱甘肽-S-轉移酶 (Glutathione S-transferase) 或羧酸酯酶 (Carboxylesterases) 三種常見殺蟲劑代謝酵素類群之基因做為候選基因，結果發現一種羧酸酯酶的表現量在脫芬瑞抗性品系 F5 中有明顯增加，由協力試驗 (Synergism test) 證實羧酸酯酶類群基因被抑制後會影響小菜蛾對脫芬瑞的敏感性，最後再以 dsRNA 干擾此羧酸酯酶基因表現，72 小時後與控制組相比表現量顯著降低了 4.9 倍，並在 RNA 干擾後餵食脫芬瑞，72 小時與控制組相比，干擾此羧酸酯酶表現之小菜蛾於診斷劑量處理死亡率由 4.1% 增加至 43.8%；而以脫芬瑞對此品系 LC50 (Lethal Concentration 50%) 劑量處理死亡率由 46.9% 增加至 85.4%，證實此羧酸酯酶基因確實與小菜蛾對脫芬瑞抗性相關。

The diamondback moth (Plutella xylostella) is currently considered the biggest threat to cruciferous plants, as it has developed resistance to many insecticides. This study utilizes insectides (chlorantraniliprole, emamectin benzoate, tolfenpyrad, and flubendiamide) commonly used in the field to select resistant moths. Using Next Generation Sequencing (NGS) data, we compared four resistant strains with our current F1 strain. Four metabolic enzyme genes (cytochrome P450 monooxygenase, glutathione S-transferase, carboxylesterases) with more than 3-fold difference in FPKM values were chosen as candidate gene, and the four insecticides were used for selection. Expression levels of candidate genes in the fifth generation of these strains were tested using quantitative real-time PCR (qPCR) and expression levels of one carboxylesterase was shown to be heightened in tolfenpyrad-resistant strains. Esterase inhibitor TPP (triphenyl phosphate) given during synergism tests was shown to significantly increase tolfenpyrad susceptibility in tolfenpyrad-resistant strains, thereby proving that esterase inhibitors can increase tolfenpyrad susceptibility. Finally, dsRNA interference of carboxylesterase expression showed a 4.9-fold decrease in expression levels after 72 hours in comparison with the control group. The test group was given tolfenpyrad after RNA interference and a post-72h comparison with the control group showed that mortality rates increased from 4.1% to 43.8% under the diagnostic dose and LC50 mortality rates of tolfenpyrad increased from 46.9% to 85.4%, proving that reduction of expression levels in this carboxylesterase also decreased diamondback moth resistance to tolfenpyrad.