Weber Donald C, Lundgren Jonathan G
USDA Agricultural Research Service, Invasive Insect Biocontrol and Behavior Laboratory, BARC-West 11 1A, Beltsville, MD 20705 USA.
J Insect Sci. 2009;9:41. doi: 10.1673/031.009.4101.
Using quantitative PCR that amplified a prey-specific mtDNA 214 bp amplicon from the COI mitochondrial gene of the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), prey eggs of known age and number were fed to larvae of the generalist predator lady beetle Coleomegilla maculata (De Geer) (Coleoptera: Coccinellidae), to elucidate the effects of time and diet since consumption, number of prey eggs, and methods for sample fixation and preservation, on the quantity of target DNA detected. Signal was strongly attenuated directly after cessation of feeding, even when predators were immediately frozen at -20 degrees C. However, the quantity of target detected was significantly related to the number of eggs consumed and the time elapsed since eating. Decrease in detected prey DNA was consistent with a negative exponential model. The target DNA sequence disappeared from starved predators (quantitative half-life estimate of 59 min) more slowly than those fed potato aphids after consuming the target prey eggs (half-life estimate 16 min), whereas those fed C. maculata eggs as a chaser were intermediate in the rate at which they degraded the target prey DNA sequence. Fixative protocols are of critical importance in proper use of the qPCR technique. Among seven methods tested, storing the predator immediately in 70% ethanol prechilled to -20 degrees C yielded the highest amount of target sequence, 22.8% of that recovered directly from a single intact prey egg. Samples frozen without solvent at -80 degrees C and -20 degrees C yielded only 6.0% and 2.3% of the target DNA respectively, and room temperature ethanol and ethylene glycol-based antifreeze averaged below 1% recovery of target DNA. Nevertheless, target prey was detected in more than 80% of antifreeze-stored predators. Predators killed and held at room temperature for 4 h or 5 days yielded no target prey DNA in 18 of 20 cases. These results emphasize both the value and the complexities of application of the qPCR technique to field predation studies.
利用定量PCR扩增来自科罗拉多马铃薯甲虫（Leptinotarsa decemlineata (Say)，鞘翅目：叶甲科）线粒体细胞色素氧化酶亚基I（COI）基因的一段214 bp的猎物特异性线粒体DNA扩增子，将已知年龄和数量的猎物卵投喂给多食性捕食者黄斑盘瓢虫（Coleomegilla maculata (De Geer)，鞘翅目：瓢虫科）的幼虫，以阐明自捕食后时间和食物、猎物卵数量以及样本固定和保存方法对检测到的目标DNA数量的影响。即使捕食者立即在-20℃冷冻，停止取食后信号也会迅速衰减。然而，检测到的目标数量与消耗的卵数以及取食后的时间显著相关。检测到的猎物DNA减少符合负指数模型。饥饿捕食者（定量半衰期估计为59分钟）中目标DNA序列消失的速度比在消耗目标猎物卵后投喂马铃薯蚜的捕食者（半衰期估计为16分钟）慢，而投喂黄斑盘瓢虫卵作为后续食物的捕食者降解目标猎物DNA序列的速度则介于两者之间。固定方案对于qPCR技术的正确使用至关重要。在测试的七种方法中，将捕食者立即储存在预冷至-20℃的70%乙醇中可获得最高量的目标序列，为直接从单个完整猎物卵中回收量的22.8%。在-80℃和-20℃无溶剂冷冻的样本分别仅产生目标DNA的6.0%和2.3%，室温乙醇和基于乙二醇的防冻液平均目标DNA回收率低于1%。然而，在超过80%的用防冻液储存的捕食者中检测到了目标猎物。在20个案例中有18个案例显示，在室温下杀死并放置4小时或5天的捕食者未检测到目标猎物DNA。这些结果强调了qPCR技术在野外捕食研究应用中的价值和复杂性。
