小菜蛾對二醯胺類殺蟲劑抗藥性之生化與分子解析

Biochemical and molecular genetic analyses of the resistance against diamide insecticides in the diamondback moth Plutella xylostella

"小菜蛾（Plutella xylostella）是世界性十字花科作物的重要害蟲，而二醯胺類（diamide）是防治小菜蛾的最新型殺蟲劑。本研究初步發現小菜蛾剋安勃抗性品系（chlorantraniliprole resistant strain; CTPR-R strain）對剋安勃與氟大滅的抗藥性程度分別為實驗室內飼養之剋安勃感性品系（chlorantraniliprole susceptible strain; CTPR-S strain）的4048.9與208.5倍，因此接著從生化、分子等不同方面，分析CTPR-R品系對剋安勃及氟大滅產生抗藥性之因子，與對此兩種殺蟲劑於抗藥性程度差異的可能成因。首先，使用能專一性抑制昆蟲體內解毒酵素之協力劑（synergist）針對CTPR-R品系進行協力效果（synergistic effect）分析，藉此確認解毒酵素是否為CTPR-R品系代謝殺蟲劑之因子。結果顯示協力精（piperonyl butoxide. PBO）這種協力劑不論對剋安勃或氟大滅均有協力效果（協力比Synergistic Ratio,SR>2），顯示由此協力劑所抑制之解毒酵素，即細胞色素P450單氧化酶（cytochrome P450 monooxygenase）可能參與CTPR-R品系對二醯胺類之代謝抗藥性（metabolic resistance）；而協力劑triphenyl phosphate（TPP）則僅對剋安勃具有協力效果（SR>2），顯示TTP所抑制之酯酶（esterase）可能參與CTPR-R品系對剋安勃之代謝抗藥性；協力試驗顯示PBO對細胞色素P450單氧化酶及酯酶都有協力作用，由此推論剋安勃的抗性和這二種代謝酵素有關。另一方面，二醯胺類殺蟲劑作用目標之魚尼丁受器基因（Ryanodine receptor, RyP）的表現量，於小菜蛾接觸不同濃度之氟大滅後，均會隨著時間進展而逐漸提高，且接觸25ppm於72小時之處理組，其基因表現量顯著高於對照組，顯示此基因在CTPR-R品系中可能參與對氟大滅的抗藥性。在作用點抗性方面，過去已被證實與小菜蛾對二醯胺類殺蟲劑產生抗藥性相關之G4946E點突變，在本次研究中也證實其存在於CTPR-R品系中。研究結果顯示該品系在不接觸二醯胺類殺蟲劑時，其感性同型合子（homozygotes of sensitive allele；SS）、突變異型合子（heterozygous；SR）與突變同型合子（Homozygotes of resistant allele；RR）頻率分別為30%、25%與45%。然而在剋安勃處理後之存活個體中，有86.7%具有G4946E點突變，但仍有13.3%之突變個體能夠存活於剋安勃逆境下，顯示此突變並非CTPR-R品系對剋安勃產生抗藥性之主因；相較之下，以不同濃度氟大滅處理後存活之個體中100%為G4946E突變的同結合型，顯示此突變應為CTPR-R品系對抗氟大滅之重要因子。本研究顯示，CTPR-R品系對剋安勃及氟大滅在解毒酵素參與之種類及點突變的反應均不相同，而是否還有其他抗性因子參與其中，則還需進一步之研究進行驗證。"

"Plutella xylostella is an important pest of cruciferous crops worldwide. Diamide is the newest insecticide for the control of diamondback moth. However, this study showed that the resistance ratio of chlorantraniliprole resistance strain (CTPR-R strain) to chlorantraniliprole and flubendiamide were 4048.9 and 208.5 folds compared with chlorantraniliprole susceptible strain (CTPR-S strain). Therefore, this report explore why resistance mechanism of the two species of diamide will differ in biochemical, and molecular levels in CTPR-R strain. First, we determine whether the detoxification enzymes were the metabolic factor of insecticides resistance through mixed the diamides and synergists, which can specifically inhibit detoxification enzymes. The results showed that the synergist PBO had a synergistic effect by combined with chlorantraniliprole and flubendiamide respectively (SR>2), indicating that cytochrome P450 monooxygenase might be involved the resistant to both diamides in CTPR-R strain. On the other hand, the synergist TPP has only synergistic effect by combined with chlorantraniliprole in CTPR-R strain (SR>2), suggested the esterase may involved in resistance to diamide in CTPR-R strain. On the other hand, gene expression of ryanodine receptor (RyP) which is target site of diamides, was also analyzed. The result showed no significant difference in the RyP expression level of the larvae between CTPR-S and CTPR-R strain. However, the expression of RyP was significantly increased after exposure to flubendiamide, indicating that RyP may be involved in the resistance to flubendiamide in CTPR-R strain. In addition, the mutation of G4946E, which is thought to be related to the resistance of chlorantraniliprole, is also found in the CTPR-R strain. The frequency of different genotypes of G4946E mutation were 30%, 25% and 45% from homozygotes of sensitive allele (S/ S), heterozygous (S/R) and homozygotes of resistant allele (R/R), respectively. Afterwards analysis of the G4946E mutation frequency of the surviving samples against the diamines. The results showed that although this mutation is associated with the CTPR-R strain against to chlorantraniliprole, but not the main cause of resistance to chlorantraniliprole. In contrast, 100% of the G4946E mutants were found to be one of the major causes of the CTPR-R strain against to flubendiamide. To sum up, the G4946E mutation and the detoxification enzymes are involved in the different levels of chlorantraniliprole and flubendiamide resistance in CTPR-R strain. Whether there are other resistance factors involved, still need further research to support, and hope that through the results of this study."