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动物对不同浮游植物和微型浮游动物猎物的就地消耗率。该方法将使用18S核糖体RNA基因作为标记来识别桡足类肠道中不同的猎物种类，并使用定量实时聚合酶链反应（q-PCR）来测量桡足类胃中每种猎物的数量。通过将这些肠道内容信息与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)开展实验室实验以确定桡足类动物的DNA消化率，(3)确定选定猎物物种的每个猎物生物的18S拷贝数，(4)在春末对缅因湾/乔治滩地区的Calanus finmarchicus进行取样，并使用群体特异性引物从肠道内容物中生成克隆库。从该克隆文库中开发的物种特异性引物将与qPCR一起用于定量猎物DNA。实验将在同一组动物中进行，以测量在野外选定猎物物种的DNA消化率。将计算摄入率，并与采用肠道色素法的摄入率进行比较。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。了解不断变化的环境条件对浮游动物的影响及其在控制浮游植物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（如薄层、冰缘和冰下）的食物环境具有很强的梯度，我们认为传统的瓶孵育技术不适合测量桡足类的原位摄食。正在开发的方法将允许直接就地估计不同猎物种类的消耗量，克服这些以前的限制。这项研究将导致研究方法的发展，这些方法将有许多应用于调查海洋环境中捕食者-猎物的原位相互作用，因为它的敏感性和测量环境中低浓度单个猎物物种的摄食率的能力。例子包括鱼苗、深海鱼类、拟鱼类和片脚类。这一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.