多杀菌素抗性西花蓟马生殖适合度代价的调控机理研究

Insecticide resistance is a major hurdle in pest management. Fitness costs associated with insecticide resistance have been reported in a variety of insects, including spinosad resistant Frankliniella occidentalis. However, it remains unclear how pests regulate the trade-off between resistance and fitness costs. Reproduction and development is one of most hot fields in insect physiological research. Based on our previous research, three aspects will be conducted in this project: 1) Next-generation RNA sequence (RNA-Seq) will be performed to find and identify differentially expressed genes related to reproduction and development in spinosad-susceptible and -resistant near-isogenic lines of F. occidentalis. 2) The full-length of candidate genes, Vg, SgAbd-2, and Cp19 genes for instance, will be cloned by RT-PCR and RACE and allele sequence and expression profile will be compared between susceptible and resistant strains. The functions of each candidate gene will be ivestigated by RNA interference (RNAi) technique. 3) Transcription factors in juvenile hormone (Kr-h1), ecdysone (Br), and insulin-like peptide (FOXO) signaling pathways will be cloned and their expression profiles will be investigated as well. The functions and their effects of transcription factors on Vg synthesis will be conducted by RNAi methods. These results, on the one hand will clarify the regulation mechanisms underlying the reproduction trade-off associated with spinosad resistance in F. occidentalis, and to reveal the pathways regulating reproduction and development in insects of Thysanoptera. On the other hand, these results will be helpful to clarify the resistance development of F. occidentalis to spinosad, to explore candidate targets for designing novel insecticides, and to provide effective control strategies for insecticide resistant pest control.

抗药性是害虫防治中的主要障碍，与抗性相关的适合度代价在多种昆虫中存在（包括多杀菌素抗性西花蓟马），但抗性代价的调控机制尚不明确。本项目拟在前期研究的基础上，以多杀菌素敏感和抗性近等基因系西花蓟马为研究对象，开展三方面研究：利用RNA-Seq进行差异基因表达分析，筛选出与生殖发育相关的差异基因；采用RT-PCR结合RACE技术获得Vg、SgAbd-2、Cp19等差异基因的全序列，进行种群间序列和表达谱比对，采用RNAi技术研究候选基因的功能；研究保幼激素、蜕皮激素和胰岛素样多肽信号途径中的转录因子Kr-h1, Br和FOXO的表达谱、功能及RNAi后对Vg合成的影响等。研究结果一方面可以阐明多杀菌素抗性西花蓟马生殖适合度降低的调控机制，明确以西花蓟马为代表的缨翅目昆虫生殖和发育的调控途径；另一方面对明确西花蓟马对多杀菌素的抗性发展规律，挖掘害虫防治新途径，制订有效的抗性治理策略有重要意义。

抗药性已成为害虫防治中的主要障碍，大量的研究表明，与抗性相关的适合度代价在鞘翅目、半翅目、双翅目和鳞翅目昆虫中普遍存在，但这些普遍表型背后的内在机制和理论依据是,以及昆虫如何协调抗药性和生殖适合度之间的平衡尚未清楚。多杀霉素抗性西花蓟马的生物适合度降低54%，本项目以多杀霉素敏感和抗性近等基因系西花蓟马为研究对象，对两种品系西花蓟马的繁殖力、卵巢发育和保幼激素通路相关基因的作用进行了深入研究。.本研究首先建立了一种观察西花蓟马卵的方法—琼脂叶碟法，解决了长期以来无法准确观察蓟马的卵量（蓟马的卵产在表皮下面，肉眼无法看到）且无法进行后续生物学观察的局面。发现多杀霉素抗性西花蓟马的产卵量显著降低，卵巢管长度显著缩短，从解剖学上解释了抗性品系适合度降低的原因；不同日龄抗药性雌成虫中卵黄原蛋白基因(Vg)表达量降低，卵黄原蛋白生产量减少，而沉积量的减少，影响了抗性雌成虫卵巢的发育及其生殖力；发现抗性西花蓟马的保幼激素含量显著降低，可能调控了Vg的低表达而影响其生殖力；性种群的保幼激素甲氧基转移酶基因（JHAMT）和保幼激素酯酶基因（JHE）表达量显著降低，进一步的功能研究表明，JHE与JHAMT协同作用，调控JH的滴度平衡，从而抑制Vg基因的表达，进而影响西花蓟马的寿命和生殖力。研究结果，一方面可以阐明多杀霉素抗性西花蓟马生殖适合度降低的调控机制，明确以西花蓟马为代表的缨翅目昆虫生殖和发育的调控途径；另一方面可以明确西花蓟马对多杀霉素的抗性发展规律，对挖掘控制害虫的新靶标，制订有效的抗性治理策略有重要意义。