LAB # 1. THE PROTOZOANS
1. Overview
Almost everything about Protozoan phylogeny and biology is controversial. No two
biologists can agree whether this group should be alligned with the plants, the animals or
even the fungi. We won’t go into the details of this controversy in the lab and we will
adopt the view of Pechenik who simply refers to all organisms that are NOT plants,
animals or fungi as Protozoans. They are truly a fascinating group, inhabiting all
environments and displaying immense variety, especially in terms of their mechanisms to
obtain food and to reproduce. As you look at the various species we have available,
always remember that you are observing a single cell. Many of the processes that we
have come to know in multicellular animals, occur within this cell. This cell must also
avoid predators, compete, feed and avoid its own parasites and diseases. Thus, all the
‘problems and constraints’ that we will talk about over the term, are solved within this
single cell.
The field of Protozoology is vast and we can only scratch the surface of this fascinating
group. The main purpose of this lab is to introduce you to the diversity of selected
Protozoans and to compare selected living material with preserved specimens. We will
focus first on the free-living Protozoans and then shift to examples of parasitic types.
This lab will tax your skills as a microscopist. Have patience and ask me for assistance if
required. First, we will focus on the Ciliates, the Sarcodinids (Ameobas), the nonparasitic Flagellates and some other ‘misfits’. Concentrate on the living material, as we
won’t have a chance to see much of it again this term. Make careful, labeled drawings
where appropriate. Concentrate next on your slide-box material, and make careful
comparisons to the appropriate figures in your text.
Non-parasitic Protozoans
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1. Essential components of the lab
review set-up, cleaning and use of compound and dissecting microscopes
observe the locomotion, feeding behaviour and functional morphology of cultures of
ciliates (especially Paramecium), amoebas, and perhaps others
observe samples of locally-collected pond water for diversity of commensal and freeliving ciliates (especially Stentor, Vorticella and perhaps Volvox)
2. Classification
We will follow the convention used by Pechenik who considers the Protozoa as
comprising at least 16 separate phyla. We won’t be concerned with the taxonomy but
you will need to be able to recognize selected major groups (e.g. ciliates, sarcodinids,
dinoflagellates, choanoflagellates, radiolarians).
Phylum Ciliophora
Paramecium, Vorticella, Stentor
Phylum Rhizopoda (the ameba-like organisms)
Amoeba, foraminiferans, radiolarians
Phylum Euglenozoa
Euglena, Volvox
Phylum Dinozoa (the dinoflagellates)
Phylum Choanozoa (the choanoflagellates)
3. Protozoan ultrastructure and functional ecology
Phylum Ciliophora
We will have available free-living forms of a variety of ciliates. They are among the
most structurally complex of the protozoans. Review your knowledge of Paramecium
from Biol 1020 and from figures in your text. You should recognize unique features such
as cilia, double nuclei, complex modes of reproduction, cytostome and at least one fixed
contractile vacuole. Other structures such as toxicysts (eject toxins to subdue prey),
haptocysts (prey capture) and trichocysts (probably defense) add to the complexity.
The double nucleus distinguishes ciliates from other protists. The smaller of the two
deals with reproduction; the larger with regulation of normal metabolism. Cilia usually
covers most of the cell. Reproduction is typically by binary fission, with the plane of
division along the long axis of the body.
Place a drop of the Paramecium culture on a slide and observe for locomotion and
functional morphology. Focus on mechanisms of locomotion and feeding. Note the
forward, spiral movement (the anterior end is narrower than the posterior). Locate the
oblique depression on the side called the oral groove, that then leads to the buccal cavity.
Note the longitudinally-arranged cilia covering the entire body and lining the oral groove.
Food trapped in the oral groove travels through the buccal cavity to the cytostome, where
a food vacuole is formed. The
outermost membrane is called the pellicle, which provides structure and support. A
contractile vacuole with radiating canals occurs at each end. These are in fixed
positions and open to the pellicle. Compare these features with stained specimens from
your slide box and by comparison with the figures in your text. The nuclei should be
more obvious in the stained specimens.
Your slide box also contains an example of Paramecium conjugation. We will cover the
details of this unique form of ‘sexual’ reproduction in class. Examine the specimen under
the microscope and refer to fig. 3.17 in your text for comparison.
Vorticella is common in our freshwater ponds. It is a solitary genus but often gregarious,
attached to virtually any substrate. Take a sample of pond vegetation (or a snail shell)
and observe under the scope. Note the inverted bell-shaped body and stalk. The stalk can
retract and extend because of a large fiber that is composed of contractile filaments. Use
fig. 3.19 in Pechenik to assist you in interpreting functional morphology.
Stentor is a large, trumpet-shaped ciliate, often with delicate internal pigmentation (Fig.
3.19). The anterior end is broadened. The macronucleus is slender and elongate and
extends longitudinally. Micronuclei are present but hard to see. There should also be a
rootlike holdfast at the tapered posterior end.
Phylum Rhizopoda
These Protozoans (commonly known as Amebas) should also be familiar to you from
Biol 1020. Recall their use of pseudopodia, protoplasmic extensions of the motile cell,
as their primary means of locomotion and feeding. The body can be naked (as in
Amoeba) or with a test (shell) (e.g. Foramineferans). Almost all species are free-living
and reproduction is by binary fission. They live in almost every conceivable habitat and
are especially common in the soil.
Amoeba proteus is a heterotrophic ameba. To view this species under the scope, try
putting cracked pieces of a coverslip around the specimen before covering it (i.e. raise the
coverslip off the surface of the slide). Take a sample from the bottom of the dish and
find it under low power. Observe movement of the organism. This species has large
pseudopodia used in food capture and locomotion. Refer to the figure 3.27 and to stained
specimens to help find some of the important organelles. Try to observe a contractile
vacuole discharging its liquid waste. You may also find food vacuoles.
As you observe Amoeba movement, consider the mechanisms responsible. Based solely
on your observations of pseudopod action, try to articulate this mechanism to one of your
classmates. How does it compare to the mechanisms used by ciliates, flagellates,
radiolarians and dinoflagellates?
Phylum Euglenozoa (flagellated protozoans)
These are the chlorophyll-containing, flagellated protists. Euglena should be familiar to
you. Members of this phylum have a cell wall, cellulose and chloroplasts. Use your slide
box and Fig. 3.30 in Pechenik to observe the functional morphology of Euglena (try to
find chloroplasts, flagellum, eyespots, cytostome and pharynx). In addition to the basic
functional morphology of this species, also note that in addition to obtaining nutrients via
photosynthesis, Euglena can also absorb nutrients directly across the body wall (but it
can’t capture its own prey).
Volvox is a bizarre, colonial Euglenid. This is an extremely important group in
phylogenetic terms because the more advanced species show division of the colony into
specific groups of cells with specific functions (i.e. this is the first time we come across
‘division of labor’). There are usually two flagella per cell. All are green with distinct
cellulose cell walls. They live mostly in fresh water and both asexual and sexual
reproduction is common. In Volvox, there is differentiation between somatic and
reproductive cells within each colony. These large colonies also move with a particular
pole consistently directed forward. The cells of the ‘anterior end’ bear well–developed
eyespots and have significantly larger flagella than the trailing cells.
Phylum Dinozoa (the dinoflagellates)
You might know the dinoflagellates for their ability to cause bioluminescence (the
biochemical production of light) and also for their ability to cause pathogenic ‘red tides’.
In class, we will talk about the recently-discovered Pfeisteria, arguably one of the most
advanced protozoans. They are common in all aquatic habitats, especially in marine ones.
Most are free-living. Many are odd-shaped indeed! There are two flagella used for
locomotion and most species obtain their nutrition via photosynthesis. Most species have
a distinctive ‘armor’ around the cell made of cellulose, thus providing nutrition, structure
and protection. Possibly their most important ecological role is as intracellular symbionts
in many marine invertebrates (we’ll see these live in two weeks in the tentacles of sea
anenomes). These forms are known as zooxanthellae and they are known to play critical
roles in the formation of coral reefs.
5. Questions for discussion
1. You’ve seen various ciliates and are hopefully impressed with their ability to move.
Most have bodies completely covered by cilia. In order to chase prey, avoid predators
and generally assess their environments, they must somehow coordinate the beating of
countless numbers of cilia. Without a complex nervous system, how do they do this ?
2. Paramecium and Amoeba are small, slow and full of protein, making them highly
attractive to predators. What options to they have to avoid predators ? How has
Vorticella and the dinoflagellates attempted to solve the problem ?
3. What ecological roles do Amoebas play in the soil ? Ciliates in fresh water ?
PART II: THE PARASITIC PROTOZOANS
1. Overview
Parasitism has evolved independently within almost every group of protozoans. Some
groups (e.g. The sporozoans) are exclusively parasitic. You should not find it surprising
that parasitism has evolved so often in these small, single-celled organisms. In this part
of the lab, we will only be covering 2 examples (3 if we have time) of this immense
diversity. There is no great logic in the 2 I have selected for study. Very simply, one is
found locally in earthworms and represents a precursor to the evolution of a most
important human parasite, Plasmodium. It is also among the largest protozoans known.
The second is a species that is also a local problem, and one that many parasitologists
consider will be one of our most important pathogens in the next century. As you go
through these examples, pay attention to the morphological differences between them, the
diversity of life-cycles, and their adaptations for parasitism. You will see that there are
direct and indirect modes of transmission, sexual and asexual multiplicative phases, and
various levels of pathology for the host.
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2. Essential features of the lab
observe the life-cycle stages of Monocystis from local earthworms
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understand the similarities and differences in life-histories between Monocystis,
Plasmodium and Cryptosporidium and Giardia.
3. Classification
Phylum Sporozoa
Monocystis sp.
Plasmodium spp.
Cryptosporidium
Phylum Diplomonida
Giardia lamblia
4. Life-cycle of Monocystis in earthworms
The Phylum Sporozoa is entirely parasitic and contains some of the most pathogenic
parasites of humans. It also contains species that have incredibly complex life-cycles,
often involving more than one host. The phylum is named after the presence of an
‘apical complex’ that is typically used to penetrate host cells. The only other generalized
characteristic is that all of the approximately 5,000 species typically alternate between
sexual and asexual phases and often have complex life cycles. There are two major
groups within the Sporozoa: The gregarines (e.g. Monocystis), and the coccidians (e.g.
Plasmodium and Cryptosporidium).
Monocystis lumbrici is an Apicomplexan parasite that lives in the seminal vesicles of
terrestrial earthworms. The worm becomes infected when it ingests a spore containing
several sporozoites. These hatch in the gizzard, where the released sporozoites penetrate
the intestinal wall, enter the dorsal blood vessel, and then make their way to one of the
host’s 5 or so ‘hearts’. From there they penetrate the seminal vesicle, where they enter the
sperm-forming cells in the wall. At this point they ingest and destroy the developing
spermocytes. Then they move into the lumen of the vesicle where they become mature
trophozoites. After a period of feeding, two of these will come together, flatten against
each other, and secrete a common cyst around each other. This is the gametocyst, usually
containing 2 gamonts. Each now undergoes extensive division of their nuclei, pinches off
a small portion of cell cytoplasm, which together then bud off to become the gametes.
The fusion of a pair of gametes forms a zygote, each ultimately becoming a spore. Three
cell divisions later forms 8 sporozoites. Thus, each gametocyst now contains many
oocysts. New hosts become infected by ingesting gametocysts, or more commonly, by
ingesting individual oocysts. Thus, meiosis is zygotic. Only the zygote is diploid, and
reductional division in sporogony returns the sporozoites to the haploid condition.
Proceedure:
Dissect the anterior end of a freshly anesthetized worm. Remove the seminal vesicles and
place in a drop of water. Take small pieces of seminal vesicle, squash under a cover slip
and look for the different stages of Monocystis (see figures). Make drawings of each
stage and construct an annotated life-cycle. Record as many different stages of infection
as you can.
5. Life-cycle of Plasmodium spp.
Plasmodium falciparum
This intracellular parasite is the most dangerous of the 4 species that cause malaria in
humans. This species will be the focus of our discussions in lecture. It is the most
common and debilitating of human parasitic diseases. Over 2 million people each year
die from the disease. Moreover, the disease has played a major role in shaping human
history and civilizations. It is impossible for you to understand this disease without a
thorough understanding of its life cycle. Please spend time before lab going over the lifecycle diagram in your text (Fig. 3.35) and the web site.
Your slides only show a fraction of the various stages of the malaria life-cycle. One slide
shows gametocytes inside host red-blood cells as deeper-staining structures (often
crescent or bean-shaped). Using the oil immersion lens you should see that
microgametocytes have a large nucleus and irregularly distributed granules.
Macrogametocytes have a small compact nucleus with a dark red nucleolus. Further
development of these forms only continues inside a mosquito’s stomach. Sometimes, the
gametocyte-infected cells have been distorted to such an extent that it ruptures during the
fixing process.
You also have a slide that shows trophozoites. They can be distinguished by the large
food vacuole surrounded by a thin layer of cytoplasm and including a peripheral nucleus.
This gives the characteristic ‘ring’ that forms the well-known ‘ring-stage’. Again, the
infected RBC may appear distended or abnormal in shape. Use the demonstration slides
of the various life-cycle stages and life-cycle diagrams to help you understand the
biology of this important parasite.
6. Life-cycle of Cryptosporidium parvum
This waterborn parasite, together with Giardia, represents one of two major waterborn
parasites of humans. It is generally a non-lethal disease but does cause severe diarrhea,
vomiting, weight loss and cramping. People with normal immune systems expel the
parasites in about 2 weeks, and are then immune to further infection. However, it is
usually lethal in patients with compromised immune systems (especially the elderly and
HIV-infected hosts). It is a major concern in the US, and now in Canada.
The main concern is that cysts are highly resistant to standard disinfectants such as
chlorine and chlorine dioxide. One instance in Milwaukee in 1994 led to an estimated
370,000 illnesses (virtually the entire city); there was an outbreak in 1995 in Kelowna
and in Medicine Hat in 2001. The Lethbridge area is considered one of the most likely
areas in Canada for an outbreak of Cryptosporidiosis, due mostly to the large numbers of
cattle. In the US, the main method of control is the conversion of water-treatment plants
to use ozone rather than chlorine as a disinfectant. Costs run into the billions of dollars.
This parasite is a recent problem, so it is still hard to buy specimens. We will sketch the
generalized life cycle of this parasite in class.
7. Giardia
Your slides of Giardia lamblia contain the feeding form (trophozoite) and cysts of this,
the most common intestinal parasite of humans. They are not easy to find on your slides;
you will have to scan the slide at 40X to find the cysts. It is the causative agent of
giardiasis or “beaver fever”. This is the disease most associated with wilderness campers
and the parasite can be responsible for severe diarrhea. The parasite also matures in many
other hosts (including beavers) which is a factor in the epidemiology of this disease. See
also the SEM pictures from the website.
Giardia has four pairs of flagella arising from a central pair of rod-like structures called
axostyles (difficult to see as the flagella are usually lost during staining). Under oil
immersion, you should be able to see two nuclei and a central pair of median bodies. The
trophozoites are cup-shaped and the surface of the ventral side is concave and thickened
to form a large adhesive disc (used for attachment). Locate a cyst at 40X (this will try
your patience as a microscopist!) and advance to oil immersion. Note the thick cyst wall,
enclosed flagella, nuclei and median bodies (Fig. 3.30).
8. Questions for discussion
1. Other water-born diseases (such as Giardia) are killed by disinfectants such as
chlorine. Cryptosporidium on the other hand has a notoriously thick cyst wall, which
chlorine cannot penetrate. Why would Cryptosporidium evolve such a thick wall, while
other water-born parasites do not ?
2. What are the major differences in the life-cycles of the gregarines, Plasmodium and
Cryptosporidium ? What is the advantage of incorporating a vector into the life-cycle ?
3. What factors are leading to the worldwide resurgence in falciparum malaria ?