基于分子生物技术的蜘蛛摄食生态学分析及控虫效能评价

The rapid progress of molecular biotechnology of DNA barcoding and FQ-PCR provide us with valuable opportunities to evaluate generalist predators' insect-controlling efficiency with quantity accurately in the field. In line with the communities structure of insect pests and wolf spiders(Lycosidae) in farmland and utilize DNA barcoding and FQ-PCR techniques, we study predation of predators with quality accurately and evaluate the wolf spider' insect-controlling efficiency to pests with quantity accurately in the field. This study is significance for ecological pest management, utilizing predatorial natural enemies resource. In this research, the contents of the wolf spider' digestive tract were used to develop a rapid identification technique based on DNA barcoding technology. Sequenced the partial mitochondrial cytochrome-c oxidase subunit I (COI) gene which used to identify species and analyze the relationship of the molecular phylogeny. The general aim of this study is to establish DNA barcoding identification system of insect pests in farmland. FQ-PCR detection primers and probe were designed according to insect pests -specific fragment. A standard curve was developed by using target DNA. The numbers of DNA copies of different development stages of insect pests were detected; meanwhile, the relationships between quantitative detection ability of predation and environmental factors were established respectively. The numbers of target pest predation can be evaluated by the target DNA copies in the contents of the wolf spider' digestive tract. The establishment of qualitative and quantitative evaluation system on the predatory ability to insect pests provided plentiful knowledge to feeding ecology. Furthermore, the techniques were also important in protection and using natural enemies, achieve the sustainable development of agriculture.

基于申请者对狼蛛形成优势类群机制及其与猎物间信息联系机制的研究结果，为全面客观评价狼蛛亚群落控虫效能，本项目以DNA条形码技术和实时荧光PCR技术对狼蛛控虫效能进行定性定量分析。定性分析：在探明水、旱两类农田（稻田、菜地）系统中狼蛛和目标猎物群落结构及动态的基础上，结合COI分子标记和DNA测序技术等生物信息学方法，以狼蛛吸胃内容物为材料，进一步认证狼蛛的捕食食谱，以构建狼蛛摄食猎物DNA条形码数据库。定量分析以多拷贝线粒体DNA为基础，以害虫特异性片段设计实时荧光定量PCR检测引物和ALLGlo探针，测定目标害虫靶标DNA拷贝数，比较狼蛛吸胃内害虫靶标DNA拷贝数，结合害虫在狼蛛体内衰变率进行校正，换算出狼蛛捕食害虫量，由此构建拟合狼蛛数量、阳性比、吸胃内目标害虫的含量及衰变率等因素的控虫数模和评价指标体系。研究结果可为保蛛控虫提供新的理论依据，对减少农药用量保护生态环境有重要意义。

本研究严格按照项目计划书执行，以拟环纹豹蛛及其猎物为研究对象，应用DNA条形码、终点PCR、实时荧光PCR等技术，并比原计划新增基因二代测序、数字PCR技术，重点从微观上探讨蜘蛛在不同地理位置、不同生境及不同时期内的猎物食谱差异及其对猎物的消化速率、耐饥饿机理与控虫效能进行了一系列研究，获得如下主要结果：.1.基于分子生物学技术蜘蛛食性分析，结果表明不同纬度对蜘蛛的食谱差异无明显影响，其食谱丰度的趋势为：中低海拔＞高海拔，作物生长期＞收割期，生境多样（湿地）＞生境单一（草甸）。DNA条形码结合二代测序结果发现，蜘蛛对鞘翅目和双翅目的捕食效率（52%和28%）明显高于其他目的昆虫，该结果与以往报道的“蜘蛛嗜好飞虱、叶蝉类昆虫”完全不同。.2.利用PCR技术对蜘蛛消化道内猎物DNA残留量进行分析，以判断蜘蛛对果蝇、飞虱、叶蝉和蚜虫的消化速率。结果表明，猎物DNA浓度在蜘蛛头胸部的消化道内无明显变化，进入腹部后DNA浓度呈逐渐下降趋势。该结果验证了蜘蛛头胸部仅有储存作用，主要消化过程在腹部完成。消化速率与进食量、环境温度等因素相关：进食量与消化速率成正比，最适消化温度为25℃~30℃，环境温度过高或过低对消化速率均有负面影响。.3.分析了不同环境因素对蜘蛛捕食猎物效率的影响。单因素实验结果表明极差R最大的是环境温度，其次是光照颜色，最后是光照强度。正交实验以判断环境因素对蜘蛛捕食效率的影响，发现最有利的环境因素组合为光照强度15 LX，光照颜色绿色，环境温度26℃。这对制定保蛛治虫的生态调控措施提供了科学依据。.4. 自然状态下蜘蛛控虫效果的测定。在大田调查的基础上，并利用DNA条形码技术确定不同生境的稻田主要农业害虫的种类，其中以褐飞虱、灰飞虱、稻纵卷叶螟、管长麦蚜、黑尾叶蝉数量较多。再通过数字PCR技术定量检测蜘蛛的控虫效果，计算结果表明，农田生态系统中，每头蜘蛛平均能捕食褐飞虱2.6头、灰飞虱2.1头、稻纵卷叶螟0.2头、麦长管蚜1.0头、黑尾叶蝉0.2头。控虫效能与生境类型、猎物密度与人类活动频率（施药等）相关。.利用上述研究结果构建了拟合蜘蛛数量、阳性比、吸胃内目标害虫的含量及衰变率等因素的控虫数模和评价指标体系。研究结果可为在农田生态系统中探求发挥天敌控害作用的新方法新手段提供科学依据；为保蛛控虫提供新的理论依据，对减少农药用量保护生态环境有重要意义。