Lab 3: Protozoans

1. Taxonomy for Lab 3

Phylum Euglenozoa ― Trypanosoma brucei, Trypanosoma cruzi, Leishmania

Phylum Ciliophora ― Paramecium, Stentor, Spirostomum, Vorticella

Phylum Apicomplexa ― Plasmodium

Phylum Amoebozoa ― Amoeba proteus, Entamoeba histolytica, Physarum

Phylum Parabasalia ― Trichonympha, Trichomonas vaginalis

Phylum Foraminifera ― foraminiferans

Phylum Radiolaria ― radiolarians

2. Introduction to the Protozoans

The organisms referred to as protozoans (“first animals”) constitute of diverse group of eukaryotic (mostly) unicellular organisms. In protozoans all life functions are carried out within the confines of a single cell. Although there are obviously no organs or tissues in protozoans, they are far from “simple” organisms as they are sometimes described. In fact, the cells of some species show the greatest complexity and internal organization of any organisms on Earth!

General protozoan characteristics include: small size, unicellular (but some species are colonial or have multicellular stages), body naked or covered by an exoskeleton (test) formed of silica or calcium carbonate. With over 64,000 living species, protozoans show a fantastic diversity of forms. Although they are found wherever life exists, protozoans always require moisture, which restricts them to a narrow range of environmental conditions in fresh water or marine habitats, the soil, decaying organic matter or inside the bodies of plants and animals. Many forms are ecologically important, forming essential links in food chains and decomposer systems.

About 10,000 species have close (symbiotic) relationships with animals or plants. These relationships may be mutualistic (both partners benefit), commensalistic (one benefits, while the other is neither helped nor harmed) or parasitic (the parasite benefits; the host is harmed). In fact, some of the most important diseases of humans and domestic animals are caused by parasitic protozoans!

3. Classification of the Protozoans

Although the protozoans used to be lumped into four groups based on their type of locomotion (i.e., whether they are propelled by flagella, cilia, pseudopodia or those forms that lack locomotor organelles), evidence from an analysis of the genes coding for the small subunit of ribosomal RNA as well as for several proteins has significantly changed (and continues to change) our concepts of the phylogenetic affinities and relationships not only of protozoan groups but of all eukaryotes and has forced a revision in protozoan classification. What follows, then, is an introduction to some of the currently recognized protozoan phyla as well as some of the more important clades and informal groupings of these organisms.

4. Phylum Euglenozoa

All members of this phylum move by flagella, whip-like projections composed of microtubules sheathed in an extension of the plasma membrane. Although some members of this phylum such as Euglena are autotrophic, a number of heterotrophic species cause serious diseases in humans and domestic animals. For example, Trypanosoma brucei causes African sleeping sickness in humans and a related disease in domestic animals. This disease, which is transmitted by the bite of a tsetse fly (Glossina spp.), causes death in about half of the infected individuals and permanent brain damage in many of those that survive.

Another dangerous euglenozoan parasite is Trypanosoma cruzi that causes Chagas’ disease, which affects some two to three million people in Central and South America, 45,000 of which die each year. Finally, several species of Leishmania that are transmitted by the bites of sand flies cause serious diseases in humans that may affect the liver or spleen or cause disfiguring lesions of the mucous membranes of the nose and throat and skin ulcers.

5. Phylum Ciliophora

 This large and diverse group includes some of the most complex protozoans known such as Paramecium, Stentor, Spirostomum and Vorticella. Locomotion is always by cilia, and all forms are multinucleate, having at least one macronucleus (responsible for metabolic and developmental functions of the cell) and one or more micronuclei that are involved in sexual reproduction). Most are holozoic but a few forms are parasitic and cause damage to their hosts, including humans. Several parasitic species can cause serious problems for aquarium fish and fish in farm enclosures.

In addition to a number of complex organelles, many ciliates have a sculptured, rigid outer covering called a pellicle. Embedded in the pellicle are the cilia plus a number of thread-like structures called trichocysts. Upon mechanical or chemical stimulation, these trichocysts can be discharged to produce long, sticky protein threads that remain attached to the organism. Although the function of these structures is probably defensive, it has been hard to demonstrate this.

6. Phylum Dinoflagellata

Included in this group are many species that form a large component of the marine phytoplankton, making them some of the most important producers in marine environments. Toxins produced by excessive profusions (blooms) of some of these marine species can lead to the so called red tides that poison fish or lodge in shellfish, making them poisonous to eat! Others such as Noctiluca produce light (bioluminescence). Other important dinoflagellates are the zooxanthellae that are mutualistic in reef-building orals and giant clams. Without their photosynthetic activity, coral reefs (and all that depend on them) would cease to exist!

 

7. Phylum Apicomplexa

This group contains endoparasitic protozoans, all of which possess (at least at certain developmental stages) a specialized combination of organelles called an apical complex, which contains structures that aid in penetrating the host. Although there are a number of apicomplexans that cause disease in humans and their animals, the most serious of these is malaria, which is caused in humans by four species of Plasmodium that are transmitted by the bite of the female Anopheles mosquito. There are over 600 million people in the world with the disease, and each year about 2 million people (mostly children) die from its effects directly and many other die indirectly.

8. Phylum Parabasalia

Parabasalids constitute another clade of flagellated protozoans that lack mitochondria. Although some parabasalids such as Trichonympha live as mutualists in the guts of termites and cockroaches where they (with the help of bacterial endosymbionts) produce enzymes that break down the wood (cellulose) in their host’s diet, others are human pathogens.

Trichomonas vaginalis is a sexually transmitted parabasalid protozoan that causes urogenital tract infections. Infection with T. vaginalis is one of the most common and curable sexually transmitted diseases with five million new infections reported each year in the United States alone and over 200 million worldwide! The parasite reproduces asexually through longitudinal fission, but unlike many other protozoans, the organism does not have a cyst stage as part of reproduction.

9. Phylum Amoebozoa

This group contains amebas and other protozoans that move by using their mobile extensions of the cytoplasm called pseudopodia. These pseudopodia come in a variety of sizes and shapes, the most common of which are rather large and blunt. Some species have thin, needle-like pseudopodia, while others have ones that form a netlike mesh around the organism. Nutrition in most forms is holozoic by engulfing prey (phagocytosis).

Amebas are naked protozoans often found in shallow, clear water. Although most amebas are free-living, feeding on small organisms with their pseudopodia, some forms are parasitic and can cause problems for humans. For example, Entamoeba histolytica is an important intestinal parasite of humans living in parts of the world with poor sanitary facilities. The parasite (which causes amebic dysentery), is contracted by drinking water contaminated with human waste or by eating raw vegetables washed with such water. Under the right conditions, the feeding stage can explosively reproduce, erode the intestinal wall and generate ulcers. In addition to causing diarrhea, E. histolytica can create problems outside of the digestive tract by invading the blood stream. Once in the blood stream it can migrate to the brain, liver and lungs – often with very serious outcomes.

10. The Slime Molds

Slime mold is a broad term describing fungus-like organisms that use spores to reproduce. Although slime molds were formerly classified as fungi, they are no longer considered part of this kingdom. Their common name refers to part of some of these organisms' life cycles where they can appear as gelatinous “slime”. Slime molds have been found all over the world and feed on microorganisms that live in any type of dead plant material. For this reason, these organisms are usually found in soil, lawns, and on the forest floor, commonly on deciduous logs. However, in tropical areas they are also common on inflorescences, fruits and in aerial situations (e.g., in the canopy of trees). In urban areas, they are found on mulch or even in the leaf mold in gutters. Most slime molds are smaller than a few centimeters, but some species may reach sizes of up to several square meters and masses of up to 30 grams, and many have striking colors such as yellow, brown and white.

11. Plasmodial Slime Molds

The true plasmodial slime molds exist in nature as a plasmodium, a multinucleate blob of protoplasm up to several centimeters in diameter, without cell walls and only a cell membrane to keep everything in. This “supercell” (a syncytium) is essentially a large ameba with thousands of individual nuclei that feeds by engulfing its food (mostly bacteria) with pseudopodia in a process called phagocytosis. Thus the slime mold ingests its food and then digests it.

When the plasmodium runs out of food, or environmental conditions become harsh, they often form elaborate (often beautiful) fruiting bodies made mostly from calcium carbonate and protein that produce spores that allow them to move to a new food source. These later germinate to form uninucleate amebas or flagellated swarm cells. These later fuse and then divide mitotically to form a plasmodium, completing the life cycle. One fascinating thing about plasmodial slime molds is that the millions of nuclei in a single plasmodium all divide at the same time. This makes slime molds ideal tools for scientists studying mitosis, the process of nuclear division.

Occasionally, during rainy periods, large plasmodia (up to a few meters in diameter) crawl out of the woods and into people's lawns and gardens. The plasmodium may be ugly to some, but it is not harmful. Slime molds cause very little damage. The plasmodium ingests bacteria, fungal spores, and maybe other smaller protozoa. Their ingestion of food is one reason slime molds are not considered to be fungi. Fungi produce enzymes exogenously (outside of their bodies) that break down organic matter into chemicals that are absorbed through their cell walls, not ingested.

12. Cellular Slime Molds

In contrast to the plasmodial slime molds, cellular slime molds, or social amebas, spend most of their lives as individual unicellular organisms, and as long as there is enough food (usually bacteria) the amebas thrive. However, when food runs out, they send out chemical signals to surrounding amebas, which then stream toward a central point, forming a slug like multicellular pseudoplasmodium (“false” plasmodium), which can then migrate like a single organism. When conditions are right, the pseudoplasmodium stops migrating and forms a multicellular fruiting body. Some of the cells become spores that disseminate, while the rest form stalk cells whose only function is to raise the spores up into the air to be more easily caught in air currents.

13. Phylum Foraminifera

Lab-3 01

This slide shows two exoskeletons, or tests, from a group of marine protozoans called foraminiferans. The shells of these ancient protozoans, which are composed of calcium carbonate, accumulate on sea bottoms and contribute over time to the formation of chalk and limestone. It is largely the bodies of these foraminiferans that have formed England's White Cliffs of Dover and the limestone used to build the Egyptian pyramids. 

14. Phylum Radiolaria

Lab-3 02

This slide contains a number of exoskeletons, or tests, of marine protozoans called radiolarians. These beautiful tests, which are abundant in marine sediments in many parts of the world, are composed of principally of silica. 

15. Amoeba proteus w.m.

Lab-3 03

  1. Nucleus
  2. Contractile vacuole
  3. Food vacuole
  4. Pseudopodium

This slide shows several stained specimens of Amoeba proteus (Proteus was a Greek god that could assume various forms). These relatively large protozoans use mobile extensions of the cytoplasm called pseudopodia for movement and food capture. Ingested food is surrounded by a food vacuole and digested by enzymes. Clear areas called contractile vacuoles collect excess water from the surrounding cytoplasm and discharge it to the outside of the body. Also note the darkly stained nuclei, which contain granular chromatin and control the activities of these unicellular organisms. 

Photographs of living amoebas

Lab-3 04

This phase contrast microscope image shows a specimen of a living ameba. Note the large contractile vacuole on the left-hand side of the organism. This organelle is used to collect and expel excess water that enters the ameba by osmosis.  

Lab-3 05

This phase contrast microscope image shows a living ameba. Note the many food vacuoles forming within this "well-fed" individual as well as the mobile extensions of the body called pseudopodia.  

Lab-3 06

This phase contrast microscope image shows another living ameba using its pseudopodia (upper right hand corner) to surround a prey item. Once inside, the food will enter food vacuoles to be digested.  

16. Paramecium caudatum w.m.

Lab-3 7

  1. Macronucleus
  2. Micronucleus
  3. Contractile vacuoles

This is a slide of the large and complex ciliate Paramecium caudatum, which is often found in water containing bacteria and decaying organic matter. Note the large, kidney-shaped macronucleus that controls most of the metabolic functions of the organism. Located close to and often within a depression on the macronucleus is the much smaller micronucleus, which is involved in reproduction. As in other freshwater protozoans, contractile vacuoles are used to remove excess water that is constantly entering the organism by osmosis.

Photographs of Living Paramecia

Lab-3 08

This phase contrast microscope image shows two live specimens of Paramecium caudatum. Note the large contractile vacuole at the anterior end of the organism on the right (pointed to by the red arrow). This organelle is used to collect and expel excess water that enters by osmosis. Also note the oral groove on the surface of the organism. This depression leads to a permanent cell mouth called a cytostome through which food particles enter the protozoan.  

Lab-3 09

 

  1. Food vacuole
  2. Oral groove
  3. Micronucleus
  4. Macronucleus
  5. Contractile vacuoles

This phase contrast microscope image shows a magnified view of a specimen of Paramecium caudatum. Note the large macronucleus and smaller micronucleus. The two fixed contractile vacuoles shown are filled with fluid soon to be expelled. Note the radial canals of this organelle that collect the fluid from cytoplasm. A food vacuole can also be seen in this specimen. 

 

Lab-3 10

 

  1. Macronucleus
  2. Contractile vacuoles
  3. Food vacuoles

This phase contrast microscope image shows a highly magnified view of a another specimen of Paramecium caudatum. Note the large macronucleus, food vacuoles and two fixed contractile vacuoles. The radial canals that collect water from the cytoplasm and deliver it to the vacuole are easily seen in this specimen.

17. Paramecium undergoing fission w.m.

Lab-3 11

This slide shows a single Paramecium that is dividing in the process of asexual reproduction called binary fission. During this process, the micronuclei first divide mitotically and then redistribute themselves throughout the cytoplasm, after which the macronucleus elongates amitotically into two halves. On the specimen shown, this division of the macronucleus into two distinct halves has been completed. 

18. Paramecium conjugation w.m.

Lab-3 12

The blue arrows point to a pair of conjugants

This slide shows a number of stained specimens of Paramecium engaged in various stages of a type of sexual reproduction called conjugation. During this process, two individuals of different mating types come together and form a cytoplasmic bridge between them. This is followed by a complex set of divisions and degenerations of the macronuclei and micronuclei that ultimately results in an exchange in genetic material between the conjugants analogous to the sexual reproduction seen in multicellular organisms. 

19. Stentor w.m.

Lab-3 13

The red arrows point to the macronuclei

This slide shows two stained specimens of the large, trumpet-shaped ciliate Stentor, a common inhabitant of freshwater lakes, ponds and streams. Although Stentor can use its cilia to actively move through the water column in search of food, it is often found attached by a long stalk to submerged sticks, stones and vegetation where it uses an array of complex ciliary organelles to draw food particles into its mouth (cytostome). Note the long, beaded macronuclei whose great size most likely reflect the special problems of controlling such a large cell. 

Photograph of a Living Stentor

Lab-3 14

This phase contrast microscope image shows a living ciliate called Stentor. Note the long, trumpet-shaped body of this exceptionally large protozoan as well as the beaded macronucleus that carries control to all parts of this long and large cell.

20. Vorticella w.m.

Lab-3 15

This slide shows numerous stained specimens of the ciliate Vorticella attached to a small piece of debris by long contractile stalks. Cilia around the mouth create water currents that draw small food particles into the organism.

Photograph of living Vorticella

Lab-3 16

This phase contrast microscope image shows a living Vorticella. Note the long stalk by which this ciliate is attached to the substrate (a piece of pond debris). Although this stalk can reach a length of 3,000 microns, it can be retracted in a fraction of a second when the organism is disturbed (see next photo in the series).

21. Spirostomum w.m.

Lab-3 18

This slide shows three stained specimens of an exceptionally large ciliate called Spirostomum. This spiral-shaped protozoan can reach a length of 3 mm and has a highly contractile body. Like Stentor, it also has a long, beaded-macronucleus. 

22. Termite gut flagellates

Lab-3 19

Living in the digestive tracts of most termites (and some cockroaches) are mutualistic parabasalids of the genus Trichonympha that help their hosts digest cellulose and other structural components of wood. Surprisingly, the protozoans themselves lack the ability to produce cellulases and must depend on a population of endosymbiotic bacteria to produce these enzymes. In exchange for this service, the protozoans and their endosymbionts benefit from a continuous supply of energy-rich cellulose and from the suitable environment of the host's gut.

Interestingly, although Trichonympha has a large number of typical eukaryotic flagella that surround most of the organism, it also harbors a population of motile spirochaete bacteria that cling to sites on the protozoan lacking flagella. At present, researchers are unsure as to the role these ectosymbiotes play in the protozoan’s ecology.

Photographs of living Termite Gut Flagellates

Lab-3 20

This phase contrast microscope image shows the large protozoan Trichonympha that inhabits the gut of primitive termites. Other smaller zooflagellates as well as bacterial species can also be seen on the slide. 

Lab-3 21

This phase contrast microscope image shows a more magnified view of the large zooflagellate Trichonympha that inhabits the gut of primitive termites. 

23. Paramecium pellicle

Lab-3 22

This slide shows two specimens of Paramecium that have been treated with a special stain that highlights a structure called the pellicle, a semi-rigid outer covering that provides support for the cilia which project through it. On the slide, these structures appear to be composed of numerous ridges and grooves.

24. Trypanosoma brucei

Lab-3 23

 This slide shows a blood smear containing the flagellate Trypanosoma brucei that causes African sleeping sickness in humans. Although there are two subspecies of the parasite that cause slightly different forms of the disease, both are transmitted by the bite of the tsetse fly (Glossina). Numerous purple-stained trypanosomes (pointed to by the blue arrows) can be seen among the lightly stained, circular erythrocytes (red blood cells). A large, darkly stained lymphocyte (white blood cell) can also be seen on the slide.

25. Trypanosoma cruzi

Lab-3 26

Trypanosoma cruzi is a parasitic protozoan that causes the potentially fatal Chagas’ disease. Transmission occurs through the bites of the assassin or “kissing” bug (Triatoma) when feces containing an infective stage of the parasite are deposited on the skin surface. Because the bite can cause pain and itching, the feces often get scratched into the wound or may be picked up by the hand and transferred to the eye, where they enter through the mucus membrane. Transmission can also occur through contaminated blood transfusions.

Chagas’ disease presents one of the highest disease burdens in Latin America. Approximately 16-18 million people are currently infected, 50,000 of which die each year. There are currently no good drugs available to treat disease, so elimination efforts primarily involve vector control and blood screening to prevent new infections. 

26. Trichomonas vaginalis

Lab-3 27

 Trichomonas vaginalis is a small anaerobic, parabasalid protozoan that moves with the aid of four whip-like flagella that protrude from its front end. It also has a fifth flagellum extending rearward from an undulating membrane that allows the parasite to attach to and tear the urethra or vaginal walls, causing inflammation that aids in speeding and intensifying infection. The adults (called trophozoites) then live in the urinary or reproductive tracts, until they are passed onto their next human host though unprotected sex.

27. Leishmania donovani

Lab-3 25

Leishmania is another trypanosome that infects humans. Like Trypanosoma brucei, the parasite requires two hosts to complete its life cycle: a mammal and an insect. Leishmania causes two forms of disease: cutaneous leishmaniasis and visceral leishmaniasis. The former typically results in cutaneous lesions that are often self-limiting. The latter is much more serious, often resulting in the destruction of the phagocytic cells of the immune system that can lead to secondary infection and eventual death of the human host. 

28. Plasmodium blood smear

Lab-3 24

This slide shows a blood smear taken from an individual infected with malaria, which is caused by the apicomplexan parasite Plasmodium. Although most of the red blood cells in the smear appear normal, notice the cell infected with an intracellular feeding stage of the parasite called a trophozoite (1). After feeding on the red blood cell’s hemoglobin, the parasite undergoes a form of asexual reproduction called schizogony (multiple fission), which results in the production of a number of nuclei seen in the red blood cell (2) above and to the left of the trophozoite. After cytokinesis is completed, the cell will rupture and release newly formed daughter cells called merozoites. It is the synchronous destruction of many erythrocytes and the release of their contents that produce the alternating bouts of fever and chills characteristic of this debilitating disease.

29. Amoeba model

Lab-3 28

This image shows a model of a relatively large protozoan called Amoeba. Amebas use mobile extensions of the cytoplasm called pseudopodia (4) for movement and food capture. Protozoans that form pseudopodia have two type of cytoplasm, an outer, more viscous portion called the ectoplasm and an inner, more fluid portion called the endoplasm. When a pseudopodium begins to form, a clear space at the leading edge of the pseudopodium called the hyaline cap (5) appears. After this occurs, endoplasm begins to flow into this space, causing the pseudopodium to be pushed forward through the medium. In addition to their locomotor role, pseudopodia can be used to engulf prey in a process known as phagocytosis. Once ingested, food enters food vacuoles (3) where it is digested by enzymes released from lysosomes. Clear areas called contractile vacuoles (2) collect excess water that enters by osmosis from the surrounding cytoplasm and discharge it to the outside of the body. Also note the darkly stained nucleus (1), which controls the activities of this unicellular organism. 

30. Paramecium model

Lab-3 29

This image shows a model of the large, complex ciliate protozoan known as Paramecium. These unicellular organisms are often found in water containing bacteria and decaying organic matter. Note the large, kidney-shaped macronucleus (1) that controls most of the metabolic functions of the organism. Located close to (and often within a depression on the macronucleus) is the much smaller micronucleus (2), which is involved in reproduction. As in other freshwater protozoans, contractile vacuoles (4) are used to remove excess water that enters the organism by osmosis. In addition to these organelles, note the ciliated oral groove (5) that directs food to a permanent opening called the cytostome, or cell mouth (6). Once inside the cell, the food is surrounded by food vacuoles (3) and is digested by enzymes released by lysosomes. Some species also maintain a permanent opening to outside called a cytoproct ("cell anus"). Located beneath the plasma membrane is a stiff but flexible structure called the pellicle that provides support for the protozoan, enabling it to maintain its shape. Embedded within this pellicle are the cilia that project through it as well as numerous thread-like structures called trichocysts (7). Upon mechanical or chemical stimulation, these trichocysts can be discharged (as shown on the model) to produce long, sticky protein threads that remain attached to the organism. It is believed that these structures can be used for defense.