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Home , Amoeba, Amoeba (genus), Amoeba proteus, Blepharisma, Chaos (genus), Ciliate

2015 – Evolution and Diversity Lab 2: Protista, part I

During this week and next week’s lab you’ll be studying that once would have been included in the Protista. These are eukaryotic organisms, mostly single-celled, that are not , or fungi. For convenience, these organisms are still widely referred to as . Those protists that are are often referred to as protozoans. Those that are photosynthetic are usually referred to as . 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, , slime molds, and . 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: , Cil- iophora, Euglenophycota, Metamonada, , . 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 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 . 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 . You heard about this in lecture. Unlike the and you’ve already seen, the protozoans do not have a wall to ultimately limit the amount of water they may take up. Instead of relying on a wall, they have intracellular structures called contractile 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 , many protozoans are able to engulf prey organisms whole, a process called phagotrophy. You may get to witness this process, or its results, today.

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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 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 . 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 - 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. engulfing prey. be able to find their prominent single nucleus. You should also notice the that accompanies the extension of the pseudopodia. You may be able to find and observe the movements of the contractile (s).You may also notice some small, living cell freshly trapped in a .

Chaos carolinenis This is the largest and best known of the . 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 , 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 in the super-group .

The single cell of a may contain a “mouth” (an oral groove that is specialized for engulfing prey, often bacteria or smaller protozoans and a ), 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 , 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 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 is roughly foot-shaped.

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, , 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 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 . 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 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 .

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 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. When stimulated, the fibril shortens, causing the stalk to coil. Vorticella sp. are ubiquitous, they live in freshwater ponds and streams, and anywhere protists are plentiful. We will see these again and again over the semester because they are a common organism in many different samples we order from the two biological supply companies we use.

Figure 6. Vorticella sp.

Didinium nasutum is a genus of unicellular carnivorous protists Their diet consists largely of Paramecium, although they will also attack and consume other ciliates.

Didinium nasutum is a voracious predator. It uses specialized structures called toxicysts to ensnare and paralyze its prey. Once captured, the prey is engulfed through it’s expandable cytostome. While D. nasutum is sometimes described as feeding exclusively upon Paramecia, it has been shown that it will readily devour other ciliate species. Moreover, strains of Didinium raised on a specific species will actually show a prefer- ence for that species and a diminished ability to kill and ingest Paramecia. Figure 7. Didinium nasutum.

5 Flagelate Protozoans Unlike the ciliates, this group of protozoans move using flagella. Eukaryotic flagella are very different in structure and function from the prokaryotic flagella that provided motility for the bacteria we observed two weeks ago. Eukaryotic flagella are sometimes referred to as “undulipodia” because of the way they func- tion. Flagellates include both free-living organisms and symbiotic organisms. We hope to have examples of both kinds for today’s lab

Euglenophycota

Peranema sp. This is a genus of free-living protists found in freshwater lakes, ponds and at the bottom of stagnant pools rich in decaying organic material. They are spindle shaped and pointed at the anterior end. They possess a pellicle that wraps around the cell in a helical fashion. This gives them the ability to deform/contort their body as they move. This type of movement is also seen in , a photoautotrophic alga we will see next week.

Although they are morphologically similar to and taxonomically related to Euglena, they do not contain so they must capture live prey. Using a rigid feeding structure called the “rod-organ” they consume yeast, bacteria, and Figure 8. . sp other protists. Peranema also lack both a stigma (“eye spot”) and it’s accompanying photo receptor (the paraflagellar body) that are present in . Although they lack a localized photo receptor, they do possess the light-sensitive protein rhodopsin and are therefore able to respond to changes in light.

Metamonada

Trichonympha campanula campanula is one of several genera of flagellate protozoans that live as symbionts in the guts of . T. campanula is a in the super-group . It ingests particles of wood harvested by its host . And then symbiotic bacteria within the protozoan digest the wood. There are two ‘levels’ of in this system!

T. campanula is a fairly large, short, stout flagellate that tapers steeply toward its anterior end. You will find very numerous flagella extending from near the anterior end along all sides of the organism, past its posterior end. It’s almost as though it has long ‘hair’ on its ‘head’. If you examine T. campanula carefully, Figure 9. Trichonympha campanula.

6 you will likely see a brownish color within the organism near its posterior end. That color is from the wood particles the protozoan has ingested. You’ll probably notice that T. campanula is very active as it moves under your cover slip, and that it seems to twist and turn and pulsate vigorously as it moves. It turns out that there are bacteria attached to the of T. campanula that are now believed to contribute in some way to this characteristic movement.

You will likely see additional flagellate symbionts from the guts of these termites. Two that are fairly com- mon are Pyrsonympha major and Streblomastix strix. These organisms are , a group you may hear about in the lecture course.

Pyrsonympha major This has a more slender appearance than Trichonympha and it appears to spiral at its broad end. Its flagella are located at its anterior end but they don’t extend away from the body of the cell but rather extend toward the posterior end and produce ‘ridges’ on the surface of the cell that have the appearance of ruffles that are very evident as the organism moves.

Figure 10. Pyrsonympha major.

Streblomastix strix Another symbiont, Streblomastix strix, will be very abun- dant in your slides. It is a very slender, needle-like organ- ism with spiral ridges that appear to extend the length of the cell.

Figure 11. Streblomastix strix.

7 Apicomplexa

The Apicomplexans are a large group of parasitic protists. They may have single hosts or may have alter- nate hosts, as in the case of , the parasite. They may have fairly complex life cycles with stages and processes described by unique sets of terms. Fortunately, you won’t have to learn any Apicomplexan life cycles. Male gametes may have flagella, and Gregarina trophozoites (a stage in the life cycle) may exhibit an apparently unique form of gliding motility.

Gregarina Gregarina are a genus of intestinal parasites found in ar- thropods. Mealworm (Tenebrio molitor) guts may contain two or more species, the most common being Gregarina polymorpha. The Gregarina trophozoites never seem to be numerous in our specimens, but we generally find enough for everyone to see them. And we often also find them in termite guts as well, so we’ll hopefully have two chances to find them in this week’s lab.

You may want to try your hand at preparing a slide of fresh guts. Use a razor blade to cut off the head and the next adjacent segment from your Mealworm, along with the last one or two segments of the abdomen. Use forceps to slowly and gently pull out the digestive tract, and remove any adhering . You should then open up the gut a bit, by puncturing it with a dissecting Figure 12. Gregarina. needle, tearing it up with the forceps, or dicing it up with a razor blade. Place the gut in a drop of saline solution on a microscope slide, and gently press a cover slip down to cover your specimen. The same general approach will work for finding the symbionts of termites. Why do you need to use saline solution?

Mycetozoa Slime molds come in two forms: cellular slime molds and plasmodial slime molds. They are now both placed as amoebozoans in the super-group Unikonta, and they are both pretty strange kinds of organ- isms. As the name Mycetozoa suggests, it was once thought that these organisms were allied with fungi.

Cellular slime molds can exist as single-celled feeding amoeba, aggregates of amoebae that can migrate as slug-like colonies, stalked fruiting bodies, resistant spores, and large diploid .

Plasmodial slime molds are also quite strange. The feeding form is an amoeba-like mass of , called a plasmodium that can become very large and may contain many thousands of diploid nuclei. It in moist places, feeding on bacteria and directly absorbing nutrients from the substrate. When food becomes in short supply, the plasmodium may produce stalked sporangia that produce haploid spores following . The spores germinate to produce either single-celled haploid amoebae or flagellated “swarm” cells. When two of these cells fuse, a diploid amoeba is formed that may in turn develop into a new plasmodium. Under unfavorable environmental conditions, as when the environment becomes too dry, the plasmodium may turn into a sclerotium. This is a dry, dessication-resistant structure that may sur- vive for months. When conditions improve, the sclerotium can resume growth as a new plasmodium.

8 polycephalum is a plasmodial , and we have living cultures for you to observe. In these cultures you should be able to see the characteristic fan-like growth pattern of this bright yellow organism, and the ‘veins’ or ‘tubes’ of cytoplasm that make up the plasmodium.

You should also observe the example of a sclerotium that is available next to the dis- secting microscope. This is the resistant structure that allows the organism to survive dry or cold periods, to resume active plasmo- dial growth when conditions improve.

Figure 13. Physarum polycephalum growing on a log.

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