Kingdom Protozoa

Dr. Elizabeth A. Bergey and Dr. Eric G. Bright, University of Oklahoma

Modified, with permission, from Invertebrate Anatomy OnLine
copyright 2003 by Richard Fox (Lander University)

Introduction

Protozoa are animal-like protists; plant-like protists are called algae.  Protozoans include groups of organisms that have little in common and are not evolutionarily closely related.  Most protozoans are motile, most are unicellular (or form colonies of unicells), and are either heterotrophic or have heterotrophic ancestors.  Many, such as euglenoids and dinoflagellates, are derived from associations of two different organisms: protozoans and their photosynthetic endosymbionts.

Protozoa include about 92,000 described species.  They occur in all habitats, including marine, freshwater, and terrestrial (even in soil).  Many eat bacteria; others may consume algae, other protozoans or live symbiotically (including living in your gut!). Free-living protozoans are important in food webs. A few cause diseases (e.g., malaria). 

Most protozoans are enclosed by a skeletal structure known as the pellicle consisting of the plasma membrane and underlying cytoskeleton (with additional membranes, microtubules, microfilaments, or plates of cellulose or protein).  The pellicle maintains the shape of the cell. Sometimes there is a secreted, extracellular, non-living test (= shell, theca, lorica). 

The cytoplasm is typically divided into a thin outer ectoplasm and an inner endoplasm.  Protozoans often bear cilia or flagella.  Osmoregulation in freshwater protozoans is accomplished by contractile vacuoles that pump a hyposmotic urine from the cytoplasm back into the environment.  This counters the osmotic influx of water across the plasma membrane.  

The organisms included in this unit were chosen to represent the major protozoan taxa.  Whenever possible, species have been selected that are readily and inexpensively available alive.

NOTE: In looking at live cultures, please work with other nearby students. If you ‘show-off’ your specimens and they show you the ones they find, you will be able to see more taxa with less effort and time. Please share.

Euglena (a euglenoid flagellate)

Many euglenoids are green phototrophs; non-green ones are entirely heterotrophs. They have a long flagellum, plus a second, very short one. There are about 1000 described species. Most live in fresh water and are most common in nutrient-enriched still waters (e.g., farm ponds and drainage ditches). The cells are often spindle-shaped. Reproduction occurs by longitudinal division (= fission).  There is no known sexual reproduction. 

 Look at a wet mount of live Euglena. Note the characteristic fusiform shape. Observe their movement. Look for a long locomotory flagellum (= primary flagellum) at the anterior end.  There are actually two flagella but one, the accessory flagellum, is very short and can not be seen.  Does the flagellum pull or push the organism through the water?

Try to slow down the Euglena by removing some water from beneath the coverslip (touch a piece of tissue or blotting paper to the edge of the coverslip). Removing too much water may smash the cells!

The pellicle (which includes interlocking protein strips) holds the cell shape.  The primary flagellum emerges from an anterior pocket, the reservoir, which connects to the outside by a narrow canal.  The transparent reservoir is usually easy to see.  The second flagellum is rudimentary and does not emerge from the reservoir.  There is a small, red pigment eyespot, or stigma, located on one side of the reservoir.  It is associated with a swollen, light-sensitive region in the reservoir. (The stigma IS NOT an eye – it shades the light-sensitive region, allowing direction-sensing.)

The green color is from chlorophyll, which is located in chloroplasts. Some euglenoids lack chlorophyll. The carbohydrate reserve in euglenoids is paramylon, which is a glucose polymer.

If there is time, add a drop of iodine-potassium iodide to the edge of the coverslip; this will kill the cells.  The flagellum and other features should be more visible.  Look for the nucleus.   

Euglena  
  
Trypanosoma

Trypanosoma   (a kinetoplastid  flagellate)

Kinetoplastids are heterotrophs and most are parasitic. Unique to kinetoplastids is the kinetoplast, a large clump of DNA located at one end of an enormously long (compared to the length of the cell) mitochondrion.

Trypanosomes are small, parasitic kinetoplastids with one flagellum and an undulating membrane. They are blood parasites in vertebrate hosts, causing a number of diseases including sleeping sickness and Chagas’ disease.  The intermediate hosts are insects.  These diseases, and others, are caused by various members of the genera Leishmania and Trypanosoma.

Look at a slide of typanosomes from blood. The trypanosomes will be blue, if stained with Wright’s stain, and are considerably smaller than the red blood cells on the slide.  You may need to use the oil immersion lens to examine these small cells. Ask the TA to help.

Note the elongate, narrow shape of the cell.  The cells are flattened and you are probably looking at a wide side.  You may occasionally see a cell that is turned on its edge to reveal its narrow margin.  The flagellum starts at the anterior end of the cell and is directed posteriorly so that it runs along the edge and then trails from the posterior end.  The flagellum is flattened and, together with the edge of the cell, forms a thin undulating membrane     

Peridinium and Ceratium  (amoured dinoflagellates)

Dinoflagellates (dino = whirling) are characterized by having two flagella that point different directions. They occur in both marine and freshwater habitats.  Ancestrally, all dinoflagellates were heterotrophs; but some have become autotrophs.  A few dinoflagellates produce toxins and are responsible for harmful algal blooms including “red” tides of various colors and paralytic shellfish poisoning.  Dinoflagellates may be endosymbiotic algae (e.g., the zoothanthallae of corals). A few marine species are bioluminescent. Species with thick cellulose plates in the pellicle are said to be armored; others are unarmoured. 

Peridinium

Ceratium and other armored dinoflagellates have a pellicle consisting of the cell membrane and cellulose armor plates.  The junctions between adjacent plates may be visible.  Can you see the outlines of the armor plates?  Peridinium looks similar to Ceratium, but lacks Ceratium’s long projections.

Dinoflagellates have two flagella:  the transverse flagellum encircles the cell and causes it to spin; the trailing (= longitudinal) flagellum trails behind the cell.  The flagella lie in grooves (sulcus and cingulum) in the pellicle, which are easily seen, though the groove with the trailing flagellum is not visible from all sides of the cell.

The green chlorophyll is often masked by carotinoids, which produce a reddish or brown color.

The debris on the bottom of the culture jar may include empty pellicles which appear as colorless ghosts, on which the armor plates, junctions, and grooves are easier to see.  Prepared slides sometimes contain some of these ghosts. 

Chilomonas  (a unicellular flagellate)

Chilamonas are colorless and found in organically rich freshwaters. Cells have a pocket in the anterior end, which is associated with the two flagella, and a narrowed posterior end.

Volvox (a colonial flagellate)

 Volvox colonies are hollow spheres with a large number of single cells imbedded in a gelatinous matrix. Volvox are photosynthetic and are generally considered algae. Cells have two flagella, which produce movement of the colony. Volvox belongs to a group of flagellated green algae that have a predetermined number of cells (2n number of cells, formed by successive divisions); although only Volvox forms such large spherical colonies. Smaller daughter colonies form within the hollow sphere and are released by rupture of the spherical colony. Daughter colonies form asexually from specialized cells that lack flagella. Volvox can also reproduce sexually.

Paramecium  (a ciliate)

Ciliates have two types of nuclei and a pellicle with associated cilia. They are heterotrophic, although a few harbor photosynthetic endosymbionts.  There 8000 known species of ciliates and many are symbionts, either commensals or parasites. A well defined permanent mouth, or cytostome, occupies a fixed location where food vacuoles are formed.  Cilia cover the cell in longitudinal rows and specialized cilia occur in the mouth region. In some ciliates, cilia can occur in clumps or be absent on parts of the cell.  Trichocysts can be discharged from the cell surface for defense or to assist in prey capture. Osmoregulation is by contractile vacuoles.

Paramecium is large, common, readily available, and easily maintained in the laboratory.  These are active, slipper-shaped ciliates. 

Prepare a wet mount. Warning: ciliates move much faster than flagellates.

Watch as it swims. The blunter end is anterior and the slightly pointed end is posterior.  Paramecium swims with the anterior end forward.  Paramecium swims in a helix because the ciliary beat is oblique to the long axis of the body.  Paramecium can reverse the direction of its ciliary beat and move backwards.  It can maneuver by locally regulating the ciliary beat.  Watch an individual and observe what it does when it hits an obstacle.

The oral groove is easy to see as the animal slowly rotates on its long axis.  Cilia in the groove move food particles (bacteria, small protozoa, organic particles) to the cytostome (cyto = cell, stome = mouth) at the posterior end of the groove, where food vacuoles form.  Vacuoles move in a circuitous route through the cell while digestion occurs.  The pH of the vacuole is initially highly acidic (pH = 3) then rises to about pH 5.  After digestion and absorption, the contents of the vacuole are discharged through a fixed anus.  

Paramecium

Paramecium has at least two contractile vacuoles, which are large and are in fixed positions. Watch a contractile vacuole for a minute and observe it fill and empty.  How long does it take to fill and empty?  What effect would increasing the osmolarity of the water have on the emptying rate?

The pellicle of Paramecium contains trichocysts, which can be discharged in defense.

Observe prepared slides of Paramecium in conjugation.  How can you quickly distinguish between Paramecium in fission and those in conjugation? 

Some additional ciliates

Didinium (a ciliate)

This ciliate is a voracious predator, which may feed on other protozoans, including Paramecium. The mouth is located on a protruding anterior cone and the species moves using two bands of cilia. Fiber-like organelles in the cone are extruded and impale the prey.

Vorticella (a peritrich ciliate)

Peritrichs are ciliates with well-developed oral cilia and reduced body cilia. Adult peritrichs are attached to the substrate by a stalk, which may be contractile.  Some may swim free in the water and be mistaken for rotifers.  Some are solitary and some are colonial, with many individuals sharing a branching stalk.

Vorticella is a solitary species with a contractile stalk and can usually be found attached to vegetation, rocks, docks, or other firm substrates in freshwater.

Make a wet mount and examine Vorticella.  Note the stalk that is attached to the bell-shaped cell body.  

Vorticella lacks cilia on the cell body. Oral cilia on the disk opposite the stalk are specialized for feeding, producing currents that carry food, chiefly bacteria, into the oral cavity on the disk. These oral cilia are also used for locomotion in peritrichs.

Tap the stage of the microscope to induce contraction of the stalk.  Note the rapid contraction and the helical shape of the stalk after contraction.  Recovery and re-extension is much slower.  Compare the length of the contracted stalk with that of the fully extended stalk.  Oral cilia are not evident in contracted individuals.

Stentor  (a ciliate)

Stentor is a large contractile ciliate with well developed body and oral cilia.  The common blue stentor, Stentor coeruleus, often appears on floating docks, or vegetation in freshwater ponds, lakes, or reservoirs. It can be as large as 1-2 mm when fully extended.

Stentor attaches to substrates but can release and be free-swimming. When attached, they are trumpet-shaped (Stentor was a mythological Greek herald who yelled louder than 50 ordinary men) or cylindrical.  Stentor is often oval when swimming. 

Observe a stationary attached individual.  The apical end flares out to form a large disc encircled by a ring of ciliary membranelles.  At one side of the ring, the membranelles spiral tightly downward to form an oral cavity leading to the cytostome. 

The macronucleus of most species, including S. coeruleus, resembles a string of beads.  The micronucleus is not usually visible. The contractile vacuole is elongate.

Amoeba  (amoeboid Protozoa, Amebas, naked amebas)

The “ameboid protozoans”, which all have pseudopods, are not a monophyletic group.  Ameboid protozoans include marine, terrestrial, and freshwater species; a few are parasites.  Their cells have one to many nuclei and many species have internal or external skeletons.  “Ameboid protozoa” includes amebas, Foraminiferea, and Actinopoda.           

Ameba cytoplasm is divided into a thin layer of stiff, clear ectoplasm covering the remaining more fluid and granular endoplasm. Ameba pseudopods may be thick or slender. Although amebas have no internal skeleton, a secreted, external test, to which particles may be added, is present in testate species. Naked amebas have no test. Contractile vacuoles are present in freshwater species but lacking in marine amebas. 

Amebas occur in most aquatic or moist habitats including soil and in symbiosis with other organisms.  A few species, such as Entamoeba histolytica - the causative agent of amoebic dysentery - are pathogens. 

Amoeba proteus is a large and easily obtained naked ameba.  Remove a drop of debris from the bottom of the Amoeba culture jar and place it on a slide.  Scatter a few grains of fine sand in the drop and add a coverslip.  The sand protects the amebas from being crushed by the coverslip.  Close the iris diaphragm partly to improve contrast and make a systematic search for AmoebaBe sure you are focused on the surface of the slide and that you do not have too much light. On your first attempt you may confuse inanimate debris with amebas.  The debris in commercial cultures is usually brown whereas amoebae are gray but both appear to be shapeless.  If close observation reveals very slow movement, you probably have an ameba.

The pseudopodia of Amoeba are thick and blunt and may appear at any point on the surface of the cell.  The ectoplasm at the tip of each pseudopodium is a little thicker than it is elsewhere and forms a hyaline cap.  The remainder of the cytoplasm is the more fluid, granular endoplasm.  Most of the cytoplasm is the granular endoplasm.  Observe the formation of pseudopodia.
Look for the nucleus near the center of the cell.  It is large and shaped like a thick disk.

A large, hyaline, spherical contractile vacuole is located in the trailing end of the cell.  The contractile vacuole is hard to see if it has just emptied or is on the other side of the cell.

The endoplasm contains numerous, mostly spherical, food vacuoles of various sizes.  These are formed by phagocytosis of the small unicells that are the food of Amoeba.  Food vacuoles are darker than the contractile vacuole and are usually opaque.  Numerous other inclusions, inorganic crystals and oil droplets, give the endoplasm its granular appearance.

Amoeba
Arcella

Arcella  (ameboid Protozoa, amebas, testate amebas)

The shelled, or testate, amebas are protected by an external organic test (shell) which they secrete and sometimes augment with additional siliceous secretions or debris.  Arcella vulgaris (vulgar = common) is a shelled freshwater ameba that inhabits vegetation and the bottom of quiet freshwater habitats.

Make a wet mount of material from the bottom of the Arcella culture jar, using sand grains to support the coverslip.   

Arcella has a transparent, brownish hemispherical test.  Slender, sometimes branched pseudopodia extend through the opening of the test.  They are not always evident - and are extended and retracted, and change shape rather quickly for an ameba.      

Entamoeba  (an ameba)

Entamoeba are parasites, commensals, or symbionts of animals. They are generally found in the digestive tract and are spread by fecal contamination.  Entamoeba hystolytica lives in the large intestine of humans and causes amebic dysentery; there are several other entamoebas that also occur in humans and are not pathogenic. (Amoebic dysentery is characterized by diarrhea = runny stools and dysentery = bloody stools; the blood comes from breaks in the intestinal lining).

foraminiferan (an ‘amoeboid protozoan’)

Foraminifereans, or “forams”, are found primarily in marine habitats where they may be either benthic or planktonic.  Forams construct a test of calcium carbonate, organic material, or debris.  The surface of the shell is often sculptured with numerous small pits, spines, bumps, teeth, or little peaks.  The tests of most species are composed of many chambers, called locules, and are said to be multilocular.  As the protozoan grows, locules of increasing size are added, so that the oldest locule is the smallest. The single cell occupies all the locules.

The shell has a large aperture (= opening) from which cytoplasm protrudes. The exterior of the shell has a thin covering of cytoplasm with numerous long, narrow, often branching hair-like ‘reticulopodia’ (reticulo=net). In some, including Globigerina (illustrated), the test has tiny pores through which cytoplasm may extend.  Reticulopods are the characteristic pseudopodia of Foraminiferea.

Living forams are not usually available for classroom studies and prepared slides of the empty tests of calcareous species are used instead.  Globigerina is a common planktonic genus and is used as an example.

Find a foram (such as Globigerina) on your slide.  Note the spiral progression of spherical locules.  The largest locule is the youngest and bears the aperture, which is visible in properly oriented specimens.  Carefully use high power to focus on the surface of a locule and note the surface sculpture, if any.  Globigerina tests may have minute, sharp peaks with pits at their tips.

Look at the shells of other species of forams, if any are present on your slide.  Note the many shapes and arrangements of locules.  Look for features, such as the aperture, pits, surface sculpture, locules, and secondary apertures, that distinguish taxa. 

Globigerina

Actinopods (‘ameboid protozoans’; radiolarians and helizoans)

Actinopoda includes taxa of spherical protozoa with axopodia (thin pseudopods with a core of stiff microtubles). They occur in both marine and freshwater habitats.

Radiolarians are marine and have axopodia radiating from the cell.  An inner organic test and an outer mineral (generally siliceous) skeleton are both present. Most are planktonic and most are spherical although the skeleton often is not. Siliceous spines may be present.

Radiolarians, like forams, are usually examined as empty skeletons.  Although they are beautiful, they provide little insight into the morphology of intact radiolarian cells.

Note the diversity on the slide.  Observe the conspicuous pores in the skeletons through which the microtubular cores of the axopodia pass. 

two radiolarians (skeletons)

Actinosphaerium (actinopods, heliozoans)

Mount Actinosphaerium using a few grains of fine sand to support the coverslip.  Actinosphaerium is spherical.  The outer part of the cell has fluid-filled vacuoles, which give it a frothy appearance and may reduce density, helping maintain suspension in the water column.

Numerous thin, conspicuous, needlelike axopodia radiate from the surface of the sphere.  Each axopodium has a core of microtubules and a thin covering of cytoplasm.  The length of the axopodia is changeable; in fact, these organisms can move using a rolling locomotion accomplished by shortening the axopodia on the leading margin.

Actinosphaerium feeds on a variety of small algae, diatoms, protozoa, and small metazoans such as rotifers.  Upon blundering into one of the axopodia, the prey sticks and becomes immobile, apparently paralyzed.  The axopodium bends or shortens so the food can be phagocytized.  The prey, and the resulting food vacuoles, may be quite large.

Actinosphaerium

© Copyright by Elizabeth Bergey and Eric Bright 2016

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