Biology 2015 – Evolution and Diversity Lab 2: Protista, part I During this week and next week’s lab you’ll be studying organisms that once would have been included in the kingdom Protista. These are eukaryotic organisms, mostly single-celled, that are not plants, animals or fungi. For convenience, these organisms are still widely referred to as protists. Those protists that are heterotrophs are often referred to as protozoans. Those that are photosynthetic are usually referred to as algae. None of these terms are phylogenetic clades – they are grades (e.g., herbivore is a grade that unites organisms based on the consumption of plants, this does not indicate any taxonomic relatedness). Protozoans are commonly found in aquatic, or at least very moist, habitats, and many protozoans are internal symbionts or parasites of animals. The free-living forms include some fearsome predators, a couple of which you will see today. On the basis of similarities in their motility, there were five traditionally distinguishable groups of protozoans: sporozoans, amoebas, slime molds, ciliates and flagellates. Only the sporozoans (non-motile parasites now referred to as Apicomplexans) and the ciliates are thought to be monophyletic groups (clades). The protozoans with flagella result from convergent evolution and are found in different, distinct Eukaryotic lineages. Amoeboid protozoans occur in at least two, and likely three, groups of protists. In today’s lab, we hope to provide you with organisms representing the following phyla: Amoebozoa, Cil- iophora, Euglenophycota, Metamonada, Apicomplexa, Mycetozoa. However, protists don’t travel well in winter so, we generally order in backup organisms as well. For most of these organisms, you should find that phase contrast microscopy with 10X and 40X objectives will provide the best views. Be sure to describe, sketch and measure all the characteristics of the spec- imens you observe. As you observe them ask yourself the following questions: Do they move? In what direction(s) do they move? What are their movements like? What internal structures are readily observed? Do you observe any internal movement? Then use the answers to your questions to formulate good notes for each organism. To get a good look at some of the faster organisms in today’s lab you’ll probably have to slow them down. You should mount a small drop of the culture on a slide and then add in a drop of De- tain, which is a viscous fluid used to slow down fast-moving organisms. In a freshwater environment protozoans tend to take up water from their surroundings by osmosis. You heard about this in lecture. Unlike the bacteria and cyanobacteria you’ve already seen, the protozoans do not have a cell wall to ultimately limit the amount of water they may take up. Instead of relying on a wall, they have intracellular structures called contractile vacuoles that collect excess water and expel it from the cell. Contractile vacuoles should be visible in at least two of today’s organisms. Because they don’t have a cell wall, many protozoans are able to engulf prey organisms whole, a process called phagotrophy. You may get to witness this process, or its results, today. 1 Protozoa Amoebas don’t have flagella or cilia, but are still able to move around by using their cytoskeleton to change their cell shape and extend pseudopodia in a given direction, which is essentially the same pro- cess they use to surround and engulf food items. Amoeba proteus This is a fairly large amoeba. When you sample the specimen jar, take a small drop from right down at the bottom of the container. And avoid shaking the container as you handle it. You should then be able to find several amoebas under your cover slip. You’ll probably also find a variety of small, swimming organisms. They may include several kinds of small flagellates, and several kinds of bacteria. Don’t expect the amoebas to move around a lot when you first get them to the microscope - it often takes them a little while to settle down. But in a couple of minutes at least some of them will start to extend their pseudopodia. Closely examine them using phase contrast. You should Figure 1. Amoeba proteus engulfing prey. be able to find their prominent single nucleus. You should also notice the cytoplasmic streaming that accompanies the extension of the pseudopodia. You may be able to find and observe the movements of the contractile vacuole(s).You may also notice some small, living cell freshly trapped in a food vacuole. Chaos carolinenis This is the largest and best known species of the genus Chaos. While most fall between 1 and 3 mm, some have been reported to reach lengths of up to 5 mm. Members of this genus closely resemble those in the genus Amoeba in that they have a similar morphology and move by producing pseu- dopodia that are rounded at the tip. One of the ways they differ from Amoeba is in the number of nuclei they posses. Members of Amoeba have one nucleus while those with- in Chaos can have as many as a thousand. It is this trait that led researchers to mistak- enly move these into the genus Pelomyxa, which is a genus of large, multinucleated amoebae. Recent molecular studies have con- Figure 2. Chaos carolinensis firmed that they are in fact more closely related to Amoeba than to Pelomyxa. They are now placed in the genus Chaos, which is a sister group to Amoe- ba. Due to their large sizes, members of the genus Chaos are versatile heterotrophs that feed on bacteria, other protists, and can even engulf small multicellular organisams. 2 Ciliophora Ciliates are probably the most structurally complex of the protozoans. They move through the coordinated motion of cilia, which are short flagella distributed over the surface of their bodies. Some ciliates are al- most completely covered with rows of many thousands of cilia. Others have fewer cilia distributed in very characteristic patterns. Ciliates are now grouped with other Alveolates in the super-group Chromalveolata. The single cell of a ciliate may contain a “mouth” (an oral groove that is specialized for engulfing prey, often bacteria or smaller protozoans and a cytostome), digestive vacuoles that break down the prey, and an anal pore region where waste is excreted. Ciliates often have complex contractile vacuoles that collect and expel the excess water they take up by osmosis. They may have two types of nuclei. A single macro- nucleus may contain many copies of the genome, and controls the ongoing activities of the organism. One or more diploid micronuclei may be involved in exchange of genetic material during conjugation between two cells. Paramecium multimicronucleatum You should first mount Paramecia in a drop of water with plenty of Detain. These cells are very fast. This species has one macronucleus and potentially many micronuclei. Look for food vac- uoles, and for the contractile vacuoles (there are generally two), and for the oral groove. Watch as the contractile vacuoles fill and empty - how does the appearance of the vacuoles change during this cycle? Try mounting some Para- mecia in water- no Detain -and see if you can calculate its relative speed. How fast would a human have to run to be competitive in propor- Figure 3. P. multimicronucleatum. tion to body size? Note that Paramecium is roughly foot-shaped. Blepharisma Blepharisma are found in fresh and salt water. Regardless of size, most species are easily identified by a red or pink- ish color caused by granules of the pigment Blepharismin, a photosensitive pigment found in granules just under the plasma membrane of the cell. They are photo-phobic and will seek out darker areas as light intensity increases. Blepharisma feed on bacteria, algae, rotifers, other ciliates and smaller members of the same species. Figure 4. Blepharisma sp. All species of Blepharisma are uniformly ciliated, with the cilia arranged in longitudinal rows. Between neighboring rows of cilia there is a row of pink or red pigment. Sometimes the pigmentation may be quite pale or absent altogether. Blepharisma tapers posteriorly, often with a large contractile vacuole located at the posterior-most point of the cell. Each possesses a Macronucleus which can take a variety of forms depending on species and phase of life. For this reason I am not sure what they look like this week. Look for a structure that is rod-shaped, ovoid, spherical, or monolithiform. 3 Stentor coeruleus Stentor is a trumpet-shaped genus of filter-feeding ciliated pro- tists. This genus is named after the mythical Greek warrior who posessed an unusually loud voice (note the horn shape in figure 5d) and are among the biggest known extant unicellular organ- isms, reaching lengths of up to two millimeters. The species we will be examining is Stentor coeruleus. There is a ring of prominent cilia near the oral pouch that can sweeps in food while the rest of the cilia aid in swimming. Like other protozoans Stentor uses a contractile vacuole to deal with excessive water that enters via osmosis. Each cell has one elon- gated macronucleus and several micronuclei. Stentor takes a few Figure 5a. Stentor coeruleus. different shapes depending on what it is doing (see figures 5b-5d below). Figure 5b. Shape of S. coeruleus at rest. Figure 5c. Shape of S. coeruleus moving. Figure 5d. Shape of S. coeruleus feeding. 4 Vorticella sp. Vorticella is a ciliate that typically remains attached to a substrate. It uses its cilia to create a ‘vortex’ that pulls in prey organisms it then engulfs. Each cell has a separate stalk anchored onto the substrate, which contains a con- tractile fibril.