SGER: Development of a method to measure feeding by copepods using qPCR

本项目将尝试开发一种新的方法，利用分子技术定量测量桡足类对不同浮游植物和微型浮游动物猎物物种的原位消耗率。该方法将使用18 S核糖体RNA基因作为标记，以确定不同的猎物物种的肠道中的桡足类，并使用定量实时聚合酶链反应（q-PCR），以测量的量的每种猎物的胃中的桡足类。通过将这些肠道内容物信息与DNA消化率相结合，研究人员将能够计算出这些不同猎物的摄食率。这项工作将分几个阶段进行：（1）开发和测试群体特异性引物，（2）进行实验室实验以确定桡足类的DNA消化速率，（3）确定选定猎物物种的每个猎物生物的18 S拷贝数，（4）在晚春期间从缅因州/乔治海岸地区的海湾采集芬兰哲水蚤样品，并使用以下组从肠道内容物产生克隆文库：特异引物从该克隆文库开发的物种特异性引物将与qPCR一起用于定量猎物DNA。将用同一组动物进行实验，以测量实地选定的猎物物种的DNA消化率。将计算摄食率，并与使用肠道色素法的摄食率进行比较。更广泛的影响：虽然研究的某些方面是高风险的，但潜在的回报将是可观的。了解浮游动物的环境条件变化的影响和它们在控制浮游植物的命运中的作用，需要了解它们的原位摄食率。由于海洋许多区域的食物环境存在很强的梯度，例如薄层、冰边缘和冰下区域，我们认为用于测量桡足类原位进食的传统瓶孵育技术是不合适的。正在开发的方法将允许直接在现场消费估计不同的猎物物种克服这些以前的局限性。这项研究将导致开发的方法，将有许多应用调查在海洋环境中的现场捕食者-猎物的相互作用，因为它的灵敏度和能力，以测量在环境中的低浓度存在的个别猎物物种的摄食率。例子包括鱼苗、深海鱼类、磷虾类和端足类。这些知识将导致对海洋食物网如何运作的新见解。该项目将涉及一名研究生的参与。

This project will attempt to development a new method using molecular techniques to quantitatively measure in situ consumption rates of copepods on different phytoplankton and microzooplankton prey species. The approach will be to use the 18S ribosomal RNA gene as a marker to identify different prey species in the guts of copepods and to use quantitative real-time polymerase chain reaction (q-PCR) to measure of the amount of each species of prey in the stomachs of the copepods. By combining this gut content information with DNA digestion rates the investigators will be able to calculate feeding rate on these different prey species. The work will proceed in several phases: (1) develop and test group-specific primers, (2) carry out laboratory experiments to determine DNA digestion rates by copepods, (3) determine the 18S copy number per prey organism for selected prey species, (4) sample Calanus finmarchicus from the Gulf of Maine/Georges Bank region during late spring and generate a clone library from the gut contents using the group-specific primers. Species-specific primers developed from this clone library will be used with qPCR to quantify the prey DNA. Experiments will be carried out with the same group of animals to measure DNA digestion rates for selected prey species in the field. Ingestion rates will be calculated and compared with rates using the gut pigment method. Broader Impacts: While some aspects of the research are high risk, the potential payoff will considerable. Understanding the effect of changing environmental conditions on zooplankton and their role in controlling the fate of phytoplankton requires knowledge of their in situ feeding rates. Because of the strong gradients in the food environment in many regions of the ocean e.g. thin layers, ice edge and under ice regions, we argue traditional bottle incubation techniques for measuring in situ feeding by copepods are not appropriate. The methods being developed will allow direct in situ consumption estimates of different prey species overcoming these previous limitations. This research will result in the development of methods that will have many applications for investigating in situ predator-prey interactions in the marine environment because of its sensitivity and ability to measure ingestion rate of individual prey species present at low concentrations in the environment. Examples include fish larvae, deep-sea fishes, euphausiids, and amphipods. This knowledge would lead to new insights as to how the oceanic food web is functioning. The project will involve participation of a Graduate Student.