Linking microscopy-based identities to molecular identities for problem- or problematic protozoa.

地球上大多数复杂生命的主要群体不是动物、真菌、植物或藻类，而是各种自由生活的原生动物。这些单细胞捕食者具有重要的生态意义，因为它们往往是细菌和单细胞藻类(构成海洋生态系统的基础)最重要的消费者。有些病毒还会导致动物患病(见下文)。这项申请是为了支持关于被忽视的原生动物的生物多样性、分布和/或进化的三个不同项目。这些项目都涉及将DNA数据与基于显微镜的鉴定联系起来。 项目1：捕食性(吞噬)真甲藻是沉积物中重要的原生动物，具有进化意义，但由于培养物很少，研究一直很困难。例如，在所描述的100个物种中，有5%的DNA数据。此外，它们拥有快速进化的基因序列，这使得它们很难通过环境测序(用于确定栖息地中存在哪些微生物的主要工具)来检测。我们最近开发了从未培养的、单个的咽突细胞中获得DNA序列的方法，我们已经通过显微镜鉴定了这些细胞。我们将把这一点与新技术相结合，为单个真核生物生成多基因图谱(条形码)。我们将从大西洋、加拿大西海岸和韩国的海洋和淡水沉积物中提取约200个细胞。我们将使用这个数据集来估计包括所有主要亚型的食虫科动物的详细进化史(这在以前是不可能的)，并记录捕食性食虫性食虫科动物‘物种’内的遗传多样性。它还将被用于优化基于DNA的调查方法，以更好地检测自然样品中实际存在的真甲藻的生物多样性。 项目2：我们之前的研究表明，极端盐度的栖息地(盐分是海水的5倍)蕴藏着许多该环境所特有的原生动物。然而，几乎所有这类研究都使用了来自人造或短期栖息地(例如太阳能盐箱或小水池)的材料。我们将调查大型永久大盐湖的原生动物，检查水和浅层沉积物样本。这项调查将使用光学显微镜、单细胞分离和培养，以及环境基因序列调查。这将测试已经从人工/短暂系统培养的生物体是否很好地代表了从非常咸的系统中培养出来的和不能培养的多样性。我们还将能够与地中海海底奇怪的“深度高盐度缺氧盆地”推断的物种清单进行比较，以确定它们实际上有多不寻常。 项目3：海带森林是非常高产的栖息地，是鱼类的重要苗圃。海胆的放牧将新斯科舍省大西洋海岸的大片海藻森林变成了恰如其分的“荒地”。海胆种群水平由一种致病原生动物入侵帕罗米巴控制，这种原生动物是一种兼性寄生虫(即它也可以在宿主之外生活和生长)。这种疾病非常依赖温度，在夏末发作，但不知道原生动物来自哪里，特别是它是如何越冬的。我们将开发敏感的基于DNA的方法来检测帕拉莫巴，并使用这些方法来尝试在不同的环境水柱和沉积物样本中检测它。我们还将探索是否存在不同的原生动物遗传菌株，在不同的地点发现，或者在对海胆的致命性方面不同。需要这些数据来准确预测气候变化对大西洋地区海藻森林的未来范围和影响(以及它们所提供的生态系统服务)。

Most of the major groups of complex life on Earth are not animals, fungi, plants or algae, but are instead varieties of free-living protozoa. These single celled predators are ecologically important because are often the most important consumers of bacteria and single-celled algae (which form the base of the ocean ecosystem). Some can also cause disease in animals (see below). This application is to support three distinct projects on the biodiversity, distribution and/or evolution of neglected protozoa. The projects all involve connecting DNA data to microscopy-based identification. Project 1: Predatory (phagotrophic) euglenids are important protozoa in sediments, and are of evolutionary interest, but have been difficult to study because there are very few cultures. For example, there are DNA data from <5% of the 100s of described species. Also, they have rapidly evolving gene sequences, which makes them difficult to detect by environmental sequencing (the major tool used to determine which microorganisms are present in a habitat). We recently developed methods for obtaining DNA sequences from uncultured, single euglenid cells that we have identified by microscopy. We will combine this with new technologies to generate multi-gene profiles (’barcodes’) for single euglenids. We will profile about 200 cells from marine and freshwater sediments in Atlantic and West Coast Canada, and Korea. We will use this dataset to estimate a detailed evolutionary history for euglenids that includes all major subtypes (this has been impossible before), and to document the genetic diversity within ‘species’ of predatory euglenids. It will also be used to optimize DNA-based survey methods to better detect the biodiversity of euglenids that is actually present in natural samples. Project 2: Our prior studies show that extremely salty habitats (with >5 times more salt than seawater) harbor many protozoa that are unique to this environment. However, almost all such studies used material from artificial or short-lived habitats (e.g. solar salterns or tiny pools). We will survey the protozoa from the large permanent Great Salt Lake, examining water and shallow sediment samples. This survey will use light microscopy, single cell isolations, and culturing, along with an environmental gene-sequence survey. This will test whether the collection of organisms already cultured from artificial/ephemeral systems represents well the culturable and unculturable diversity from very salty systems in general. We will also be able to compare to species inventories inferred for the strange ‘deep hypersaline anoxic basins’ on the Mediterranean Sea floor, to determine how unusual they actually are. Project 3: Kelp forests are very productive habitats that are important nurseries for fish. Grazing by sea urchins turns large areas of kelp forest on the Atlantic Coast of Nova Scotia into aptly-named ‘barrens’ . The sea urchin population levels are controlled by a disease-causing protozoan, Paramoeba invadens, which is a facultative parasite (i.e. it can also live and grow outside the host). The disease is very temperature-dependent and strikes in late summer, but it is not known where the protozoan comes from, especially how it over-winters. We will develop sensitive DNA-based methods to detect the Paramoeba, and use these to try to detect it in different environmental water column and sediment samples over time. We will also explore whether there are different genetic strains of the protozoan, found in different locations, or that differ in lethality to sea urchins. This data is needed for making accurate predictions of its future extent and influence on kelp forests in the Atlantic region (and thus the ecosystem services they provide) under climate change.