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

该项目将尝试开发一种新的方法，利用分子技术来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的不同猎物物种，并使用定量实时聚合酶链式反应(Q-PCR)来测量每种猎物在桡足类胃中的数量。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对这些不同猎物物种的摄食率。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，以确定桡足类对DNA的消化速度，(3)确定选定猎物物种的每个被捕食生物体的18S拷贝数，(4)在晚春期间从缅因湾/乔治斯浅滩地区采集Calanus finmarchicus样本，并使用群体特异性引物根据肠道内容建立克隆文库。根据该克隆文库开发的物种特异性引物将与qPCR一起用于定量检测猎物DNA。实验将在同一组动物身上进行，以测量选定的猎物物种在野外的DNA消化率。摄取率将被计算出来，并与使用肠道色素方法的摄取率进行比较。更广泛的影响：虽然研究的某些方面风险很高，但潜在的回报将是可观的。要了解环境条件变化对浮游动物的影响以及它们在控制浮游植物命运中的作用，需要了解它们的就地摄食率。由于在海洋的许多区域，如薄层、冰缘和冰下区域，食物环境的梯度很强，我们认为传统的瓶子培养技术用于测量桡足类的就地摄食是不合适的。正在开发的方法将能够直接就地估计不同的猎物物种的消费量，克服了以前的这些限制。这项研究将导致开发可用于现场调查海洋环境中捕食者-猎物相互作用的方法，因为它具有测量环境中低浓度个体猎物物种摄取率的敏感性和能力。例子包括鱼类幼体、深海鱼类、真甲鱼和两足类。这一知识将导致对海洋食物网如何运作的新见解。该项目将包括一名研究生的参与。

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.