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

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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.