芳樟醇、桉叶油素取食胁迫下香樟齿喙象(鞘翅目:象甲科)幼虫的生长发育及抗性基因的转录表达

Growth,Development and the Resistance Gene Transcriptional Expression of Pagiophloeus tsushimanus(Coleoptera:Curculionidae)Larvae after Feeding on Linalool and Eucalyptol

【目的】探究香樟齿喙象与香樟互作中的寄主选择和适应机制,明确香樟齿喙象幼虫对芳樟醇、桉叶油素胁迫生境下的生长发育适应性及其解毒代谢方式,以阐明香樟齿喙象专性为害香樟的潜在机制。【方法】采用人工饲料混药法,测定香樟齿喙象初孵幼虫取食不同质量分数芳樟醇和桉叶油素3天后的死亡率,明确香樟齿喙象幼虫对2种萜类物质的敏感性。分别设定10.0、19.7 mg·g^(-1)的芳樟醇及5.0、15.2 mg·g^(-1)的桉叶油素进行长期取食处理,测定香樟齿喙象幼虫阶段的存活曲线、发育历期、体重等生长发育指标,明确香樟齿喙象幼虫对2种萜类物质的生物学响应。利用qRT-PCR技术,测定香樟齿喙象4龄幼虫取食芳樟醇和桉叶油素后的解毒酶基因(细胞色素P450酶、羧酸酯酶、UDP-葡萄糖醛酸转移酶、谷胱甘肽-S-转移酶和ABC转运蛋白)和表皮蛋白基因的相对表达量变化。【结果】芳樟醇和桉叶油素在2种处理质量分数条件长期胁迫下,对香樟齿喙象幼虫的生长发育产生显著的抑制作用,并且抑制作用随质量分数增加而增强,即发育历期延长、体重降低以及死亡率提高。此外,还表现出幼虫在蜕皮期前后出现高频死亡的特征。相较于桉叶油素,香樟齿喙象幼虫对芳樟醇表现出更强的耐受性。qRT-PCR结果显示,芳樟醇胁迫能够显著地诱导香樟齿喙象幼虫体内细胞色素P450酶和表皮蛋白的基因表达,而桉叶油素取食胁迫下仅有1条UDP-葡萄糖醛酸转移酶的基因表达量显著上调。【结论】香樟齿喙象幼虫对2种寄主植物次生物质的取食胁迫产生不同生理适应性。同时,其幼虫的解毒酶相关基因和表皮蛋白基因对2种萜类物质胁迫的表达响应模式存在差异性。香樟齿喙象幼虫能够有目的地调控不同生理适应机制,以克服不同的寄主次生物质防御,以实现寄主定殖和种群暴发。

【Objective】Pagiophloeus tsushimanus(Coleoptera:Curculionidae)is a serious wood borer on Cinnamomum camphora,which has rapidly outbroken in Shanghai.Linalool and eucalyptol are important terpene metabolites in C.camphora,and also constitute the chemical defense system in C.camphora.This study aims to explore the host selection and adaptation mechanism in the interaction between P.tsushimanus and C.camphora and clarify the development adaptability and detoxification mechanism that P.tsushimanus copes with the linalool and eucalyptol,so as to analyze the potential mechanism that P.tsushimanus obligates to C.camphora.【Method】The newly hatched P.tsushimanus larvae were fed with linalool and eucalyptol for 3 days using residual film method and the sensitivity of P.tsushimanus larvae to the two terpenoids was determined.Linalool(10.0 mg·g^(–1) and 19.7 mg·g^(–1))and eucalyptol(5.0 mg·g^(–1) and 15.2 mg·g^(–1))were set for the long term feeding treatment,to measure the survival curve,developmental duration,body weight and other growth and development indexes of P.tsushimanus larvae in order to evaluate the phenotypic response of P.tsushimanus to the two terpenoids.The qRT PCR technique was used to determine the relative expressions of detoxification enzyme genes(CYP450,carboxylesterase,UDP-glucuronosyltransferase,Glutathione Stransferase and ATP binding cassette transporter)and insect cuticle protein genes in the 4th instar larvae of P.tsushimanus.【Result】Linalool and eucalyptol significantly inhibited growth and development of P.tsushimanus larvae under the long-term feeding of two mass fraction of the terpenoids and this inhibitory effect was correlated with the dose,that is,prolonged the developmental duration,reduced the weight and showed high mortality.Moreover,the larvae also exhibited high-frequency mortality before and after the molting period.Compared with eucalyptol,P.tsushimanus larvae showed stronger tolerance to linalool.The qRT-PCR results revealed that the expression of four cytochrome P450 genes were significantly up-regulated,four cuticle protein genes were up-regulated in P.tsushimanus treated with linalool and one UGT gene was significantly up-regulated in P.tsushimanus larvae treated with eucalyptol.【Conclusion】The larvae of P.tsushimanus show different physiological adaptability to the two secondary metabolites.Meanwhile,the expression patterns of detoxification enzyme genes and cuticle protein genes in P.tsushimanus larvae are different under two secondary metabolites.The result also suggests that P.tsushimanus larvae could detoxify different plant secondary metabolites through different mechanism so as to achieve host colonization and population outbreak.