PROTOZOA

A Solitary Flagellate: Euglena Phylum Sarcomastigophora

Subphylum Mastigophora

Euglena (figure 5.1) is a common green flagellate often found in the greenish surface scum of standing or slowly moving water. Euglena is an enigmatic organism with a curious mixture of plant and animal characteristics, and, therefore, sometimes is considered to represent a borderline case between the plant and animal kingdoms. Euglena is smaller than Amoeba and Paramecium and, therefore, the details of its internal structure are more difficult to observe.

· Prepare a wet mount from a culture of living Euglena and observe the locomotion of an active specimen under your compound microscope. The active swimming movements result from the beating of the long flagellum, which pushes the organism through the water. A second, shorter flagellum is present within the flagellar pocket, but does not aid in the swimming movements. At certain times Euglena also exhibits another type of wormlike locomotion during which waves of contraction pass along the body in a characteristic fashion. This type of locomotion is peculiar to Euglena and related organisms, and is appropriately termed euglenoid movement or metaboly. It appears to result in part from the elasticity of the thick outer covering of the body, the pellicle.

After the wet mount begins to dry out, temporarily immobilizing some of your specimens, study the anatomy of a stationary Euglena. You will also find it useful to supplement your observations with the study of a prepared microscope slide.

Identify the following structures under high power on your compound microscope: (1) pellicle, the thick outer covering of the body; (2) chloroplasts with green chlorophyll; (3) nucleus, exhibiting a large central endosome in stained preparations; (4) flagellar pocket; (5) contractile vacuole; (6) a red stigma, or eye spot; (7) the long anterior flagellum; and (8) paramylum grains, a type of starch that represents stored food materials.

Euglena is quite sensitive to light, and changing the light intensity tends to cause the Euglena to move away. Bright light tends to make this protozoon remain stationary.

· After you have completed your observations of the living specimen, add a drop of Lugol's solution (iodine and potassium iodide). This solution will kill the specimen and stain the flagellum to make it more readily visible.

As suggested by the presence of chloroplasts, the nutrition of Euglena is normally autotrophic; organic molecules (sugars) are synthesized from inorganic nutrients absorbed from the medium. Light from the sun provides the energy necessary for this process.

Biochemical tests have shown the paramylum granules to be a form of starch similar to that found in plants. Thus, both the presence of the chloroplasts and the storage of a plantlike form of starch indicate a close relationship of Euglena and its relatives to the plant kingdom.

· Some species of Euglena are also able to survive, grow, and reproduce in the dark with no visible evidence of chloroplasts, chlorophyll, or stored food materials.

How might such organisms obtain their food?

A Colonial (?) Flagellate: Volvox

Phylum Sarcomastigophora

Subphylum Mastigophora

Volvox (figure 5.2) is a common green alga that often occurs in great numbers in freshwater ponds and lakes. Although Volvox is photosynthetic and is sometimes considered to be an alga, it is often studied in zoology courses because it illustrates the organization of a simple colonial (or multicellular) organism well and because of its usefulness in illustrating one popular theory for the evolution of multicellular organisms from unicellular ancestors. It also demonstrates some basic similarities between plants and animals.

The spherical green Volvox are large enough to be seen swimming near the surface of a pond or in a laboratory culture even without a microscope.

Fig. 5.2 Volvox, asexual spheroid. (Courtesy of Carolina Biological Supply Company, Burlington, N. C.)

Fig. 5.3 Volvox cell structures, longitudinal section of cells at surface of spheroid.

Obtain some living Volvox from a culture and prepare a wet mount. Be sure to add a few bits of broken coverslip or sand grains to protect the spherical Volvox bodies from being crushed by the weight of the coverslip. Observe the spheroid shape of Volvox, its swimming movements, and its prominent green color.

The spherical Volvox bodies are usually called colonies in textbooks, but recent studies have suggested that they are more similar to multicellular individuals. The coordination of cells and cellular function within the spheroid body is much greater than in most other colonial algae and protozoons; thus, recent workers use the term spheroids for the Volvox body.

The hollow Volvox spheroids average about 0.5mm in diameter and have many small green cells embedded in their outer walls (see figure 5.3). Each of the tiny body cells of Volvox contains a nucleus, a contractile vacuole, a green chloroplast, and two whiplike flagella. Some or all of the cells (depending on the species) may also have a red stigma (light-sensitive spot). The flagella project outward from the surface, and their beating keeps the spheroids in a constant spinning motion. Although the somatic cells of Volvox are very small and difficult to observe except with special microscopic preparations, the adjacent cells in some species of Volvox are connected by thin cytoplasmic bridges or strands. Other species of Volvox, however, lack these intercellular connections.

· Add a drop of 0.1 % methylene blue solution to a wet mount of Volvox and study under high power on your compound microscope. Can you observe any cytoplasmic bridges?

The green color of the spheroid individuals results from the presence of a chloroplast in each cell. What can you therefore infer about the nutrition of Volvox?

Within the spheroid you should be able to observe one or more large reproductive cells or gonidia. Reproduction in Volvox involves both sexual and asexual processes. In asexual development, embryos are formed from the gonidia.. Locate several gonidia within the interior of an asexual parent individual (see figure 5.4).

During asexual development, the gonidia undergo a series of cell divisions remarkably like those seen in the embryonic development of many animal species. See if you can locate several different developmental stages of gonidia in the living specimens provided.

The beginning of sexual reproduction can be recognized when the gonidia form male and/or female spheroids. Most species of Volvox have separate male and female individuals (i.e., are dioecious, "two houses"), but some species produce both eggs and sperm in the same individual and are thus monoecious ("one house"). Figure 5.4 also shows both male and female individuals.

The living cultures available for study in the laboratory are usually all asexual. You should study the demonstration chart to learn about the life cycle of Volvox.

Fig. 5.4 Volvox, male, female, and asexual spheroids. (Photograph by Barbara Grimes.)

Study the structure of Volvox using the living specimens provided in the laboratory. In a drop of water on a clean microscope slide, observe the swimming of Volvox spheroids first under low power, and then add a coverslip over the water drop and observe more details of the structure of the colony under higher magnification. Observe somatic cells, gonidia, eggs, sperm, and zygotes, using both the living specimens and the demonstration materials as necessary.

Evolution of Multicellularity

Volvox and several related green flagellates are often studied as models to illustrate one popular theory for the evolution of multicellular organisms. Most scientists believe that multicellular organisms arose from some unicellular form. The particular kind of unicellular organism is not known because this major evolutionary step took place more than 600 million years ago in the Precambrian Era. No well-preserved fossils have been found that actually document this transition from one to many cells, so biologists have searched among living plants and animals to seek models that might help their understanding of early evolution.

Volvox and several related green algae comprise the most popular model discussed by scientists. These related forms exhibit a graded series of solitary and colonial forms of increasing complexity and are called the Volvocine Series (figure 5.5). Among the important genera of algae comprising the series are: Chlamydomonas, Gonium, Pandorina, Eudorina, Pleodorina, and Volvox.

Other Mastigophora

Other important mastigophorans include the dinoflagellates, the symbiotic flagellates that inhabit the digestive tracts of termites and the wood roaches, some peculiar flagellates that may be related to sponges, and several important parasites of humans.

Dinoflagellates are found in both fresh and marine waters; many species form a characteristic outer covering, called a test, that is made of cellulose. Certain freshwater dinoflagellates may cause an unpleasant odor or taste in human water supplies. Gonyaulax and Gymnodinium are two marine dinoflagellates often associated with the red tides of coastal waters of North America, Europe, and Africa, that sometimes result in massive fish kills.

Fig. 5.6 Symbiotic flagellates from termite gut. (Courtesy of Carolina Biological Supply Company, Burlington, N.C.)

Some flagellates live as symbionts in the digestive tracts of wood roaches and termites (figure 5.6). Experiments have shown that the termites lack the digestive enzymes necessary to digest the cellulose in the wood they eat. The flagellates ingest splinters of wood and form food vacuoles around them. Later, digested products from the breakdown of the cellulose are released from the protozoa and provide nutrients for the termites. Termites from which the flagellates are experimentally removed soon die of starvation no matter how much wood they ingest. The flagellates benefit from the continuous supply of cellulose and from the suitable anaerobic environment of the host hindgut. Such a mutually beneficial symbiotic relationship is called mutualism.

Proterospongia is a colonial flagellate with species that closely resemble the flagellated collar cells, or choanocytes, characteristic of sponges (see chapter 6). Some biologists have suggested that the sponges may have evolved from some ancient protozoon similar to Protero

spongia.

Still other mastigophorans are parasites. Trypanosoma and Leishmania are two important genera that include several serious human parasites. Trypanosoma has a thin, undulating membrane connecting its long, whiplike flagellum with its body (see figures 5.7 and 5.8). Several species of Trypanosoma cause sleeping sickness and other diseases in humans. Leishmania includes species that cause severe diseases in tropical areas of Africa, Asia, and South America.

An Amoeba: Amoeba proteus

Phylum Sarcomastigophora

Subphylum Sarcodina

Amoeba proteus (figure 5.9) is a protozoon found in ponds and streams. It often occurs on the undersides of plant leaves, and among diatoms and desmids. The transparent amoeba constantly changes shape by extending pseudopodia, footlike extensions of the cytoplasm, which serve for locomotion and in food capture. Amoeba proteus feeds on bacteria, small algae, and small protozoons.

In feeding, an advancing pseudopodium flows over one or more food organisms to trap the food in a waterfilled cup. The opening of the food cup then narrows until the food is completely enclosed in a food vacuole.

Demonstrations

1. Large flagella in other Mastigophora, such as Peranema.

2. Microscope slide of trypanosomes.

3. Microscope slide of symbiotic flagellates from digestive tract of termite or wood roach.

4. Microscope slide of dinoflagellates.

5. Microscope slide showing cell walls in Volvox.

6. Chart illustrating the life cycle of Volvox.

7. Microscope slides with related volvocine flagellates,

such as: Gonium, Pandorina, Eudorina, and Pleodorina.

white blood cell

Fig. 5.7 Trypanosoma, blood smear. (Courtesy of Carolina Biological Supply Company, Burlington, N.C.)

Fig. 5.8 Trypanosoma. Magnification 3,525X. (Scanning electron micrograph by Louis de Vos.)

Fig. 5.9 Amoeba proteus.

Prepare a wet mount to study living amoebae under your compound microscope.

Preparing a Wet Mount and a Hanging Drop. Obtain a clean microscope slide and add a drop of amoeba culture solution to the center of the slide. Take care to withdraw the drop of culture solution from the bottom of the culture dish or jar with a clean eyedropper or pipette. The amoebae are slightly heavier than the culture solution and are usually concentrated on the bottom of the vessel. Add a few bits of broken coverslip or grains of sand around the periphery of the drop to protect your specimens from being crushed, then carefully cover your preparation with a coverslip. This preparation is called a wet mount.

Another method of studying living Protozoa is to prepare a hanging drop. With this method you place a drop of the amoeba culture in a coverslip and invert the coverslip over the cavity of a depression slide. Both types of preparations can be observed for long periods of time if the outside edge of the coverslip is coated with petroleum jelly before it is placed in position on the microscope slide. Be sparing in your use of the petroleum jelly; avoid getting it into the culture droplet and on your microscope lens.

Study your preparation under the low power of the compound microscope to observe the general appearance of the amoeba. For best observation of a living amoeba, reduce the illumination to a minimum since living specimens are nearly transparent and almost invisible in bright light. Search your slide carefully for a specimen before discarding it or asking your instructor for a new preparation.

Locate an actively moving amoeba and note its constantly changing shape. The long, fingerlike projections are pseudopodia ("false feet"). Observe the lack of permanent orientation of the body of an amoeba; any portion may temporarily be anterior, posterior, right, or left.

With the aid of figure 5.9, identify and study the following structures found in the amoeba.

1. Endoplasm the inner granular region, which forms the bulk of the cytoplasm.

2. Ectoplasm the thin layer of clear cytoplasm which surrounds the endoplasm.

3. Cell membrane the outer membrane surrounding the amoeba. Sometimes also called the plasmalemma.

4. Plasmagel the stiff, jellylike, granular outer layer of colloidal endoplasm in the gel state.

5. Plasmasol the central mass of colloidal endoplasm in a fluid, or sol state. Note the streaming movements within the plasmasol.

6. Nucleus a transparent structure with no fixed position in the cell. It has the shape of a biconcave disc and often exhibits a folded or wrinkled appearance. Examine also the nucleus in a stained microscope slide of Amoeba proteus. Observe the darkly staining granular chromatin material within the nucleus.

7. Contractile vacuole a clear vacuole found in the endoplasm which collects excess water from the surrounding cytoplasm and body. Shortly after one contractile vacuole discharges its contents at the cell surface, a new contractile vacuole forms. Where are the new contractile vacuoles formed? Formerly, it was believed that the contractile vacuole also played an important role in the excretion of waste products from protein metabolism, but recent evidence has not supported this belief, and most specialists now agree that the contractile vacuole functions primarily in maintaining water balance in the cell (osmoregulation)discharges it outside the cell Fig. 5.10 Pelomyxa carolinensis. A large multinucleate amoeba. (Courtesy of Carolina Biological Supply Company, Burlington, N. C.)

8. Food vacuoles vacuoles containing bits of ingested food and the digestive enzymes that act to break down these food materials into soluble materials that can be utilized by the amoeba. How are the food vacuoles formed? How are the undigested contents of a food vacuole disposed of after digestion has taken place?

Amoeboid Movement. Amoeba moves about by extending pseudopodia into which some of the innermost cell contents flow. Various kinds of amoebae form pseudopodia of different size and form. These pseudopodia are important in feeding, support, and locomotion. The mechanism of amoeboid movement has been studied by many scientists because of its intriguing nature and because similar movements occur in many other kinds of cells, including human leucocytes. Also, scientists now believe that amoeboid movement may be closely related to the phenomenon of cytoplasmic streaming, which occurs in virtually all kinds of living cells.

The movement of an amoeba is accomplished by the forward flow of the relatively liquid plasmasol from the center of the amoeba toward and into an expanding pseudopodium. Around the periphery of the pseudopodium, the

plasmasol changes into a stiff plasmagel. Thus, the plasmasol moves the pseudopodium forward, and the plasmagel serves to fix it in position.

Recent biochemical and biophysical studies have demonstrated that the mechanism of amoeboid movement is similar to that in muscle contraction. Contractile proteins similar to the actin and myosin found in vertebrate muscles are present in the cytoplasm of an amoeba. Amoeboid movement results from folding, unfolding, polymerization, and depolymerization of these proteins.

∎ Study the locomotion of an Amoeba on your microscope slide and also the pattern of its internal protoplasmic movements. In an active specimen, locate and carefully follow the movement of some granules in the plasmagel at the temporary posterior end. Observe how the plasmagel of the endoplasm changes into plasmasol, which flows forward and then changes into the gel state again, just back of the tip of the forming pseudopodium.

Reproduction. The reproduction of Amoeba proteus occurs only through the asexual process of binary fission. The nucleus and cytoplasm of a parent cell divide to form two daughter cells approximately equal in size. Thus, each of the daughter cells is genetically identical to the parent cell-excluding the rare occurrence of a mutation in one of the daughter cells.

Other Sarcodina

Many members of this group of Protozoa are more specialized than Amoeba. Pelomyxa carolinensis is a large multinucleate amoeba often studied in zoology classes (figure 5.10). Numerous species of amoebae live in shellsaperture
Fig. 5.11 Test of a freshwater amoeba. (Scanning electron micrograph by F. W. Harrison.)

Fig. 5.12 Actinosphagerium. (Courtesy of Carolina Biological Supply Company, Burlington, N.C.)

or tests which they secrete, or which they form from sand grains or other materials (figure 5.11). Difflugia and Arcella are two common testate amoebae found in freshwater ponds and streams. Other species of amoebae are parasites or symbionts in the digestive tracts of various animals. Entamoeba histolytica, an important parasite of humans, is the cause of amoebic dysentery, a disease often spread by drinking water or by eating raw vegetables contaminated by human wastes in parts of the world with poor sanitary facilities.

Some freshwater members of the Subphylum Sarcodina have many long, thin pseudopodia supported by axial rods of microtubules. Actinosphaerium (figure 5.12)

is a common freshwater example of a group called heliozoans because of the resemblance to the sun and its rays of sunlight.

Members of three marine classes of this subphylum, called radiolarians, form skeletons or tests of silicon and/ or strontium compounds and exhibit many beautiful shapes (figure 5.13). The radiolarians are among the oldest known protozoa, and their tests are abundant in marine sediments in many parts of the world.

The foraminiferans (figure 5.14), representing another class of sarcodines, are an ancient and important group of marine sarcodines which form tests of calcium carbonate or other materials. The shells of foraminiferans Fig. 5.13 Radiolarian tests. (Courtesy of Carolina Biological Supply Company, Burlington, N.C.)

Fig. 5.14 Foraminiferan tests. (Photograph by Barbara Grimes.)

accumulate on the sea bottom and contribute to the formation of chalk and limestone. England's White Cliffs of Dover are made up largely of foraminiferan tests, as is much of the Bedford limestone found in Indiana and Illinois, and some of the limestone that was used to build the Egyptian pyramids. The distribution of certain species of foraminifera is also very important to petroleum geologists as an indication of ancient environmental conditions that may have been favorable for the formation of petroleum and thus as important clues to the location of petroleum pools.

∎ Observe several of these other types of Sarcodina among the demonstrations.

Demonstrations

1. Models and charts of Amoeba proteus.

2. Culture of Amoeba under a stereomicroscope.

3. Microscopic slides with testate and parasitic representatives of the Subphylum Sarcodina, such as Arcella, Difflugia, Entamoeba histolytica, Actinosphaerium (a heliozoan), and Globigerina (a foraminiferan).

A Ciliate: Paramecium caudatum Phylum Ciliophora (Ciliata)

Paramecium (figure 5.15) is a large, common, ciliated protozoon often found in water containing bacteria and decaying organic matter. There are several species of Paramecium that differ in various details of structure and that range in length from about 120-300 microns. The laboratory directions provided here are based upon Paramecium caudatum, a species frequently used for laboratory study and experimentation, but they will also apply, with minor modification (such as body size and number of micronuclei), to the study of other species of Paramecium.

∎ Obtain a drop of Paramecium culture in a clean pipette and place it on a clean microscope slide with a similar-sized drop of methyl cellulose solution (or other similar agent) to slow movement. Methyl cellulose is a viscous material and serves mechanically

I Fig. 5.15 Paramecium caudatum.

Fig. 5.16 Paramecium, nigrosin stain to illustrate sculpturing of bilayered pel icle. (Photograph by Barbara Grimes.)

to slow the swimming of the fast-moving Paramecium. Add a coverslip and observe your preparation under low power with your compound microscope. Note the form, color, and behavior of the animals in your preparation. Observe the slipper-shaped body with an oral groove beginning at the anterior end and running diagonally across the anterior portion of the animal. At the posterior end of this groove is the cytostome, or "cell mouth," through which food particles are passed as a result of the action of the specialized oral cilia lining the oral groove.

Paramecium is much more complex in its structure than Amoeba. Select a large, immobile, or slowly moving specimen, and with the aid of figure 5.15, identify and study the following structures.

1. Cilia the numerous cylindrical protoplasmic extensions that cover the surface of the Paramecium and that function in locomotion and in food gathering.

2. Pellicle the thick outer covering of the body through which the cilia project. The pellicle has a complex structure, but its details are difficult to observe without special techniques. Figure 5.16 shows some of the surface depressions in the bilayered pellicle.

3. Trichocysts tiny, rodlike structures embedded in the cortical (outer) cytoplasm beneath the pellicle. When properly stimulated, the trichocysts discharge their contents and form long threads. There is some evidence that the trichocysts may serve as a defense against predators, and they also serve to anchor the animal during feeding. In other types of ciliated protozoa, trichocysts have been found to have still other functions. Observe the microscopic demonstration of discharged trichocysts.

4. Macronucleus the large nucleus located near the center of the cell. Since it is transparent in a living animal, the structure of the macronucleus is best studied in a stained preparation. Experiments have demonstrated that the macronucleus controls most metabolic functions of the cell.

5. Micronucleus a smaller nucleus located close to and lying partly within a depression on the oral side of the macronucleus. The micronucleus is involved primarily in the reproductive and hereditary functions of the animal. This presence of two distinct types of nuclei is called nuclear dimorphism and is a condition found only in the Phylum Ciliophora. Paramecium caudatum has only a single micronucleus, but some other species of Paramecium have two or more micronuclei. As with the macronucleus, the structure of the micronucleus is best studied in a prepared microscope slide.

6. Contractile vacuoles two clear, slowly pulsating vesicles located near each end of the body. Each contractile vacuole is surrounded by several radiating canals (not often seen in ordinary student preparations) which collect water from the surrounding cytoplasm. Observe the behavior of the contractile vacuoles. Are they fixed in position? Do they contract alternately or simultaneously? The function of the contractile vacuoles in Paramecium is the same as in Amoeba, the collection and discharge of excess water from the cell. Freshwater protozoa often have contractile vacuoles; marine protozoa generally lack them. How would you explain this difference?

7. Cytostome (cell mouth) a permanent opening near the posterior end of the oral groove through which food is passed.

8. Cytopharynx a short tube extending from the cytostome posteriorly and downward into the cytoplasm where food vacuoles are formed.

9. Food vacuoles vacuoles located within the cytoplasm where they are carried by the streaming movements of the cytoplasm. Undigested materials are discharged through the cytopyge, or anal pore, located posterior to the oral groove.

Feeding

Paramecium is a filter-feeding organism and normally feeds on bacteria and yeast cells collected by a specialized food-collecting apparatus. An oral groove extends diagonally back along the body to a funnel-shaped cytopharynx. Food is swept along the oral groove by the action of specialized cilia lining the groove, is passed through the circular cytostome ("cell mouth") at the opening of the cytopharynx, and is passed through the cytopharynx into a newly forming food vacuole.

∎ Prepare a wet mount with a drop of Paramecium culture to study the feeding process. Add a small amount of congo red stained yeast with the tip of a toothpick or clean dissecting needle. Try to pick up the smallest amount of yeast possible on the toothpick; too much yeast will cloud your preparation and obscure the Paramecium.

With this preparation, you can study the movement of the food particles, the formation of food vacuoles, and the subsequent movement of the food vacuoles within the cytoplasm. After the food vacuoles are formed, digestive enzymes are released into them and chemical digestion of the food particles begins. Note the color change in the vacuoles as the enzymes work. The color change is due to a change of pH in the vacuoles. Where do the digestive enzymes come from? Why don't they digest the other materials in the cell such as mitochondria and ribosomes? The diffusible products of digestion are released into the cytoplasm, and the undigestible remains are discharged at a specific site on the surface of the animal. This site is the cytopyge, or cell anus.

Cilia and Flagella

Most of the surface of Paramecium is covered by thin, hairlike projections called cilia (singular: cilium). Cilia are extensions of the cortical (outer) cytoplasm of the cell and play important roles in feeding and locomotion.

A great deal has been learned in recent years about the structure and function of cilia. These studies have revealed that cilia are closely related to the flagella (singular: flagellum) found on the surface of other kinds of protozoa. The structural differences between cilia and flagella are minor. When the projections are short and numerous, they are called cilia. When they are long and few, they are flagella. Cilia generally exhibit a relatively simple back and forth movement. The movements of flagella are often more complex and may involve a series of helical waves propagated along the flagellum.

Both cilia and flagella have a common basic structure. A cross section reveals an outer membrane enclosing a circle of nine pairs of microtubules and two single microtubules in the center of the cilium or flagellum. This basic pattern is found in all cilia and flagella, not only among the protozoa but also on the gills of molluscs, the ciliated epithelium lining the trachea of vertebrates, and the tail of spermatozoa.

Recent biochemical studies have also demonstrated that the movements of cilia and flagella involve contractile proteins similar to those found in striated muscle. This is another important illustration of the basic similarity of all living organisms.

Reproduction

Paramecium reproduces by a simple type of asexual reproduction in which the parent divides into two equal daughter cells. This type of asexual reproduction is termed transverse fission and is found in many kinds of protozoa. Living specimens are occasionally seen in the process of fission, but the details of fission are best studied in a stained microscope slide. Obtain a prepared slide of Paramecium in fission and observe the nuclei. During fis sion, the micronucleus first divides by mitosis, and the macronucleus later divides by amitosis. No visible chro mosomes are formed in the macronucleus; the macronu cleus simply constricts, and the two portions separate. Macronuclear division is followed by cytoplasmic division (cytokinesis). The process of fission may be completed rapidly, and under optimal conditions, Paramecium can reproduce asexually two or more times per day.

∎ Draw the representative stages of fission in Paramecium as indicated on figure 5.17.

Unlike Amoeba, Paramecium can also reproduce sexually. The specialized type of sexual process exhibited by Paramecium is called conjugation (figure 5.18). During this process, two individuals come together, adhere by their oral surfaces, undergo a complex series of changes in both the macronuclei and the micronuclei, exchange a single pair of micronuclei (one from each cell), separate, and resume asexual reproduction. Following the exchange of micronuclei in each Paramecium, the newly introduced micronucleus fuses with another (nonmigrating) micronucleus. Thus, there is an exchange of hereditary material and a subsequent fusion of hereditary material from the two parents, a situation analogous to that of ordinary sexual reproduction studied earlier. Fig. 5.18 Paramecium in conjugation. (Courtesy of Carolina Biological Supply Company, Burlington, N.C.)

∎ Examine a prepared slide or a demonstration of Paramecium in conjugation.

Fig. 5.20 Didinium, a carnivorous ciliate ingesting a Paramecium. (Courtesy of Carolina Biological Supply Company, Burlington, N.C.)

Fig. 5.19 Stentor. (Courtesy of Carolina Biological Supply Company, Burlington, N. C.)

Other Ciliata

The Phylum Ciliophora (Ciliata) is a large and diverse group of protozoa. One important form is Stentor (figure 5.19), a large, trumpet-shaped ciliate which is common in lakes, ponds, and streams, and which has been used in many experimental studies. Stentor has a spiral array of complex ciliary organelles leading to its cytostome and a beaded macronucleus. Didinium (figure 5.20) is a barrelshaped predaceous ciliate with a voracious appetite. It feeds on other ciliates including Paramecium. A hungry Didinium can eat a Paramecium every two hours.

Spirostomum (figure 5.21) is a long, wormlike ciliate that has contractile fibrils that seem to function in a way similar to striated muscle fibrils. Tetrahymena (figure 5.22) is a small, ovoid ciliate that has been used in many experimental studies of biochemistry and genetics.

Vorticella (figure 5.23) is a sessile form with a long, contractile stalk which attaches to submerged stones, shells, plants, animals, and other objects. Some relatives of Vorticella, such as Carchesium and Zoothamnion, form stalked colonies.

Podophyra is a suctorian, a specialized group of sessile ciliates that have suctorial tentacles in their mature stages and are predators of other ciliates. Cilia are found only on juvenile stages of the suctorians.

1. Models and charts of Paramecium.

2. Stained slide showing pellicle of Paramecium.

3. Stained slide to show discharged trichocysts.

4. Stained slides with representative members of the

Class Ciliata, such as Stentor, Euplotes, Tetrahymena, Vorticella, Didinium, Blepharisma, Trichodina, and Podophyra.

Phylum Apicomplexa

Members of this group were formerly included among the Sporozoa, but recent investigations have indicated that they should be considered a separate phylum. All members of this phylum are parasitic on other organisms.

Many species in this group parasitize invertebrate animals such as earthworms, crabs, and oysters, but the most important species are parasites of vertebrates, including humans. Among the most important sporozoans are Plasmodium and Eimeria. Several species of Plasmodium cause various forms of malaria in humans and other animals.

Demonstrations

Chapter 5

Fig. 5.21 Spirostomum, a ciliate with contractile fibers and a flexible pellicle that exhibits a peculiar, wormlike locomotion. (Courtesy of Carolina Biological Supply Company, Burlington, N. C.)

Eimeria is a genus of sporozoan parasites that causes coccidiosis in birds, rabbits, and other animals. This dis cytostome ease has great economic impact on the poultry industry. Eimeria, like Plasmodium, has a complex life cycle that includes both sexual and asexual forms. Study the demonstrations illustrating the life history and importance of Eimeria and coccidiosis.

The Malaria Parasite: Plasmodium

More than 50 species of Plasmodium have been described. All are parasites of vertebrate animals, including amphibians, reptiles, birds, and mammals. Four species cause human malaria, P. vivax, P. ovale, P. malariae, and P. falciparum. The life cycles of these species are all similar.

Malaria, one of the most serious and debilitating of human diseases, has had an important role in history from the fall of the Roman Empire to the war in Vietnam. Although modern medicine has made some progress in eliminating malaria, the disease is yet to be conquered. It is most prevalent in tropical and semitropical areas, and costs millions of lives and trillions of dollars annually. Fig. 5.22 Tetrahymena. (Scanning electron micrograph by Jolanta Nunley.)

Protozoa

Fig. 5.23 Vorticella. A sessile ciliate with along contractile stalk for attachment and a ring of cilia surrounding the mouth. (Courtesy of Carolina Biological Supply Company, Burlington, N.C.)

Life Cycle of Plasmodium

The life cycle of Plasmodium (see figure 5.24) is complex like those of other apicomplexa and includes several generations with both sexual and asexual reproduction. The life cycle can best be understood by starting with the zygote in the gut of a mosquito, one of the two hosts necessary for the completion of the life cycle.

The zygote becomes motile and passes through the lining and wall of the stomach or midgut of the mosquito and is now called an ookinete. The ookinete then rounds up and encysts on the outside of the gut wall and is called an oocyst. After several days of growth, the oocyst divides internally to form several hundred sporozoites. The sporozoites escape by rupturing the external wall of the oocyst and migrate through the hemocoel to the salivary glands of the mosquito.

When a mosquito bites a human, the sporozoites and the mosquito's salivary secretions are injected into this host. Sporozoites that find their way into the human bloodstream are eventually carried to the liver, where the sporozoites enter host cells. Inside the host liver cells, the sporozoites transform into amoeboid multinuclear schizonts and feed upon the contents of the host liver cells. The schizonts reproduce asexually to form many merozoites (the next stage in the life cycle), escape from the liver cells, and, in some cases, invade other liver cells to repeat the process.

Merozoites escaping into the bloodstream penetrate erythrocytes to initiate the erythrocytic phase of the life cycle. Parasitic stages in the liver prior to entry into the erythrocytes are termed the exoerythrocytic phase.

The developing Plasmodium inside the erythrocytes exhibit a characteristic morphology, as seen in Giemsastained microscope preparations, and are recognized by their red nucleus and blue ring-shaped cytoplasm (see figure 5.25). This characteristic morphology is very helpful in the laboratory diagnosis of malaria. The merozoites in the erythrocytes undergo further asexual reproduction within the erythrocytes. Later, the merozoites rupture the wall of the erythrocytes, escape into the blood, and enter most erythrocytes. This multiplication process may be repeated several times, so that an enormous number of parasites are produced within the host. The rupture of the erythrocytes by the merozoites also releases accumulated toxic wastes from the parasites and causes the symptomatic chills and fever commonly associated with human malaria.

Some of the merozoites develop into sexual forms instead of repeating the asexual merogony. These sexual forms develop within the erythrocytes and become microgametocytes (male) and macrogametocytes (female) (figure 5.26). These stages are the progenitors of the male and female gametes, and represent the start of the sexual portion of the life cycle.

If a mosquito bites an infected host and ingests infected erythrocytes, the gametocytes pass into the mosquito's stomach, where they mature into microgametes and

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Fig. 5.24 Life cycle of Plasmodium.

macrogametes. The microgametocytes develop flagella like outgrowths, which break free, become motile, and fertilize macrogametes. The fertilized macrogametes, or zygotes, then invade the gut wall of the mosquito to start uninfected red the cycle.

∎ Study the microscope slides with blood smears prepared with blood from humans infected with Plasmodium vivax or a similar species. Identify as many stages in the life cycle of Plasmodium as you can from your own slides and the demonstration slides. Observe the changes in morphology of the parasite during its development in the human erythrocytes. With the aid of figure 5.24, try to relate the portion of the Plasmodium life cycle in human erythrocytes to the other parts of the life cycle completed in the mosquito and in the exoerythrocytic stages in the human. Why do you think Plasmodium requires two hosts to complete its life cycle? What special adaptations for life as a parasite can you identify in Plasmodium? List several of these adaptations for parasitism in table 5.1. blood cell

Fig. 5.25 Plasmodium. Ring stage inside human erythrocyte. (Courtesy of Carolina Biological Supply Company, Burlington, N.C.)

Key Terms

Cilia cylindrical cytoplasmic extensions from the surface of certain protozoa and of some metazoan cells. Serve in locomotion and feeding of ciliated protozoa .uninfected Basically similar to flagella, but shorter and more nu blood cellmerous. All have the universal internal 9 + 2 pattern of microtubules. Singular: cilium.

Coccidiosis disease of birds, rabbits, and other animals caused by members of the protozoon genus Eimeria.

Macrogametocyte

Conjugation a specialized type of mating, nuclear exchange, and nuclear reorganization characteristic of ciliates. A form of sexual reproduction.

Contractile vacuole an organelle found in many freshwater protozoons that serves in osmoregulation (water balance).

Cytostome the "cell mouth" found in many protozoa, including ciliates, some flagellates, and some apicomplexa. In ciliates, the cytostome is often surrounded by specialized ciliary feeding organelles.

Flagella cylindrical cytoplasmic extensions from the surface of certain protozoa and some metazoan cells. Function in locomotion and feeding of mastigophorans. Similar to cilia but longer and usually fewer per cell. Singular: flagellum.

Gonidia specialized reproductive cells in Volvox. Singular: gonidium.

Macronucleus the large metabolic nucleus typical of ciliates. Often has a characteristic shape. Divides amitotically by pinching in two. Contains many duplicated sets of genes (polyploid).

Malaria disease of humans and other animals. Caused by members of the protozoon genus Plasmodium, which invade the blood and other tissues of the hosts.

Micronucleus the small reproductive nucleus in ciliates. Some ciliates have more than one micronucleus. Usually divides by ordinary mitosis.

Nuclear dimorphism having two distinct types of nuclei in the same cell. Characteristic of the ciliates (e.g., with macronucleus and micronucleus).

Pseudopodia the protoplasmic extensions, "false feet," of the Sarcodina used for locomotion and in feeding. Various types of sarcodines have pseudopodia specialized for certain purposes or modes of life. Singular: pseudopodium.

Demonstrations

1. Microscope slides with selected stages in the life cycle Stigma a light-sensitive spot found in certain flagellated

of Plasmodium. protozoa, such as Euglena and Volvox. 2. Chart illustrating the life cycle of Plasmodium.

3. Microscope slide of Eimeria.

4. Chart illustrating the life cycle of Eimeria.

Fig. 5.26 Plasmodium. Developing macrogametocyte in human erythrocyte. (Courtesy of Carolina Biological Supply Company, Burlington, N. C.)