Download Biology 2015 – Evolution and Diversity

Biology 2015 – Evolution and Diversity
Lab 3: Protista, part I
During this week and next week’s lab you’ll be studying organisms that once would have been included in
the kingdom Protista. These are eukaryotic organisms, mostly single-celled, that are not plants, animals
or fungi. For convenience, these organisms are still widely referred to as protists. Those protists that are
heterotrophs are often referred to as protozoans. Those that are photosynthetic are usually referred to
as algae. None of these terms are phylogenetic clades – they are grades (e.g., herbivore is a grade that
unites organisms based on the consumption of plants, this does not indicate any taxonomic relatedness).
Protozoans are commonly found in aquatic, or at least very moist, habitats, and many protozoans are
internal symbionts or parasites of animals. The free-living forms include some fearsome predators, a
couple of which you will see today. On the basis of similarities in their motility, there were five traditionally
distinguishable groups of protozoans: sporozoans, amoebas, slime molds, ciliates and flagellates. Only
the sporozoans (non-motile parasites now referred to as Apicomplexans) and the ciliates are thought
to be monophyletic groups (clades). The protozoans with flagella result from convergent evolution and
are found in different, distinct Eukaryotic lineages. Amoeboid protozoans occur in at least two, and likely
three, groups of protists.
In today’s lab, we hope to provide you with organisms representing the following phyla: Protozoa, Ciliophora, Euglenophycota, Metamonada, Apicomplexa, Myxomycota. However, protists don’t travel well
in winter so, we generally order in backup organisms as well.
For most of these organisms, you should find that phase contrast microscopy with 10X and 40X objectives
will provide the best views. Be sure to describe, sketch and measure all the characteristics of the specimens you observe. As you observe them ask yourself the following questions: Do they move? In what
direction(s) do they move? What are their movements like? What internal structures are readily observed?
Do you observe any internal movement? Then use the answers to your questions to formulate good notes
for each organism. To get a good look at some of the faster organisms in today’s lab you’ll probably have
to slow them down. You should mount a small drop of the culture on a slide and then add in a drop of Detain, which is a viscous fluid used to slow down fast-moving organisms.
In a freshwater environment protozoans tend to take up water from their surroundings by osmosis. You’ve
heard about this in the lecture course. Unlike the bacteria and cyanobacteria you’ve already seen, the protozoans do not have a cell wall to ultimately limit the amount of water they may take up. Instead of relying
on a wall, they have intracellular structures called contractile vacuoles that collect excess water and expel
it from the cell. Contractile vacuoles should be visible in at least two of today’s organisms. Because they
don’t have a cell wall, many protozoans are able to engulf prey organisms whole, a process called phagotrophy. You may get to witness this process, or its results, today.
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Protozoa
Amoebas don’t have flagella or cilia, but are still able to move around by using their cytoskeleton to
change their cell shape and extend pseudopodia in a given direction, which is essentially the same process they use to surround and engulf food items.
Amoeba proteus
This is a fairly large amoeba. When you sample
the specimen jar, take a small drop from right
down at the bottom of the container. And avoid
shaking the container as you handle it. You
should then be able to find several amoebas
under your cover slip. You’ll probably also find a
variety of small, swimming organisms. They may
include several kinds of small flagellates, and
several kinds of bacteria.
Don’t expect the amoebas to move around a lot
when you first get them to the microscope - it
often takes them a little while to settle down.
But in a couple of minutes at least some of them
will start to extend their pseudopodia. Closely
examine them using phase contrast. You should Figure 1. Amoeba proteus engulfing prey.
be able to find their prominent single nucleus. You
should also notice the cytoplasmic streaming that accompanies the extension of the pseudopodia. You
may be able to find and observe the movements of the contractile vacuole(s). You may also notice some
small, living cell freshly trapped in a food vacuole.
Chaos carolinensis
This is the largest and best known species
of the genus Chaos. While most fall between
1 and 3 mm, some have been reported to
reach lengths of up to 5 mm. Members of
this genus closely resemble those in the
genus Amoeba in that they have a similar
morphology and move by producing pseudopodia that are rounded at the tip. One of
the ways they differ from Amoeba is in the
number of nuclei they posses. Members of
Amoeba have one nucleus while those within Chaos can have as many as a thousand.
It is this trait that led researchers to mistakenly move these into the genus Pelomyxa,
which is a genus of large, multinucleated
amoebae. Recent molecular studies have con- Figure 2. Chaos carolinensis
firmed that they are in fact more closely related
to Amoeba than to Pelomyxa. They are now placed in the genus Chaos, which is a sister group to Amoeba. Due to their large sizes, members of the genus Chaos are versatile heterotrophs that feed on bacteria,
other protists, and can even engulf small multicellular invertebrates.
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Ciliophora
Ciliates are probably the most structurally complex of the protozoans. They move through the coordinated
motion of cilia, which are short flagella distributed over the surface of their bodies. Some ciliates are almost completely covered with rows of many thousands of cilia. Others have fewer cilia distributed in very
characteristic patterns. Ciliates are now grouped with other Alveolates in the super-group Chromalveolata.
The single cell of a ciliate may contain a “mouth” (an oral groove that is specialized for engulfing prey,
often bacteria or smaller protozoans and a cytostome), digestive vacuoles that break down the prey, and
an anal pore region where waste is excreted. Ciliates often have complex contractile vacuoles that collect
and expel the excess water they take up by osmosis. They may have two types of nuclei. A single macronucleus may contain many copies of the genome, and controls the ongoing activities of the organism. One
or more diploid micronuclei may be involved in exchange of genetic material during conjugation between
two cells.
Paramecium multimicronucleatum
You should first mount Paramecia in a drop of
water with plenty of Detain. These cells are very
fast. This species has one macronucleus and
potentially many micronuclei. Look for food vacuoles, and for the contractile vacuoles (there are
generally two), and for the oral groove. Watch
as the contractile vacuoles fill and empty - how
does the appearance of the vacuoles change
during this cycle? Try mounting some Paramecia in water- no Detain -and see if you can
calculate its relative speed. How fast would a
human have to run to be competitive in proporFigure 3. P. multimicronucleatum.
tion to body size? Note that Paramecium is roughly
foot-shaped.
Blepharisma
Blepharisma are found in fresh and salt water. Regardless
of size, most species are easily identified by a red or pinkish color caused by granules of the pigment Blepharismin,
a photosensitive pigment found in granules just under
the plasma membrane of the cell. They are photo-phobic
and will seek out darker areas as light intensity increases.
Blepharisma feed on bacteria, algae, rotifers, other ciliates
and smaller members of the same species.
Figure 4. Blepharisma.
All species of Blepharisma are uniformly ciliated, with the cilia arranged in longitudinal rows. Between
neighboring rows of cilia there is a row of pink or red pigment. Sometimes the pigmentation may be quite
pale or absent altogether. Blepharisma tapers posteriorly, often with a large contractile vacuole located
at the posterior-most point of the cell. Each possesses Macronucleus which can take a variety of forms
depending on species and phase of life. For this reason I am not sure what they look like this week. Look
for a structure that is rod-shaped, ovoid, spherical, or monolithiform.
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Stentor
Stentor is a trumpet-shaped genus of filter-feeding ciliated
protists. They are named after the Greek herald Stentor
and are among the biggest known extant unicellular organisms, reaching lengths of up to two millimeters.
There is a ring of prominent cilia around the anterior end
that can sweep in food and aid in swimming. Stentor uses
a contractile vacuole to deal with water that enters it by osmosis. Each cell has one (often elongated) macronucleus
and several micronuclei.
Figure 5. Stentor.
Vorticella
Vorticella is a ciliate that typically remains attached to a
substrate. It uses its cilia to create a ‘vortex’ that pulls in
prey organisms it then engulfs. Each cell has a separate
stalk anchored onto the substrate, which contains a contractile fibril. When stimulated, the fibril shortens, causing the stalk to coil. Vorticella are ubiquitous, they live in
freshwater ponds and streams, and anywhere protists
are plentiful. We will see these again and again over the
semester because they are a common organism in many
different samples we order from the two biological supply
companies we use.
Figure 6. Vorticella.
Didinium
Didinium is a genus of unicellular carnivorous protists
Their diet consists largely of Paramecium, although they
will also attack and consume other ciliates.
Didinium nasutum is a voracious predator. It uses specialized structures called toxicysts to ensnare and paralyze its prey. Once captured, the prey is engulfed through
it’s expandable cytostome. While Didinium nasutum is
sometimes described as feeding exclusively upon Paramecia, it has been shown that it will readily devour other
ciliate species. Moreover, strains of Didinium raised on a
specific species will actually show a preference for that
species and a diminished ability to kill and ingest ParaFigure 7. Didinium nasutum eating Paramecium.
mecia.
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Flagelate Protozoans
These protozoans move using flagella. Eukaryotic flagella are very different in structure and function from
the prokaryotic flagella that provided motility for the bacteria we observed two weeks ago. Eukaryotic
flagella are sometimes referred to as “undulipodia” because of the way they function. Flagellates include
both free-living organisms and parasitic or symbiotic organisms. We hope to have examples of both kinds
for today’s lab
Euglenophycota
Peranema
This is a genus of free-living flagellate protists
found in freshwater lakes, ponds and at the bottom
of stagnant pools rich in decaying organic material.
They are spindle shaped and pointed at the anterior end. They possess a pellicle that wraps around
the cell in a helical fashion. This gives them the
ability to deform/contort their body as they move.
This type of movement is also seen in Euglena, a
photoautotrophic alga we will see next week.
Although they are morphologically similar to and
taxonomically related to Euglena, they do not
contain chloroplasts so they must capture live prey.
Using a rigid feeding structure called the “rod-orFigure 8. Peranema.
gan” they consume yeast, bacteria, and other protists. Peranema also lack both a stigma (“eye spot”)
and it’s accompanying photo receptor (the paraflagellar body) that are present in Euglenids. Although
they lack a localized photo receptor, they do possess the light-sensitive protein rhodopsin and are therefore able to respond to changes in light.
Metamonada
Trichonympha
Trichonympha is one of several genera of flagellate protozoans that live as symbionts in the guts
of termites. Trichonympha is a Parabasalid in the
super-group Excavata. It ingests particles of wood
harvested by its host termite. And then symbiotic
bacteria within the protozoan digest the wood. There
are two ‘levels’ of symbiosis in this system!
Trichonympha is a fairly large, short, stout flagellate
that tapers steeply toward its anterior end. You will
find very numerous flagella extending from near the
anterior end along all sides of the organism, past its
posterior end. It’s almost as though it has long ‘hair’
on its ‘head’. If you examine Trichonympha carefully,
you will likely see a brownish color within the organFigure 9. Trichonympha.
ism near its posterior end. That color is from the wood
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particles the protozoan has ingested. You’ll probably notice that Trichonympha is very active as it moves
under your cover slip, and that it seems to twist and turn and pulsate vigorously as it moves. It turns out
that there are bacteria attached to Trichonympha’s cell membrane that are now believed to contribute in
some way to this characteristic movement.
You will likely see other flagellate symbionts from the guts of these termites. Two that are fairly common
are Pyrsonympha and Streblomastix. These organisms are Oxymonads, a group you may hear about in
the lecture course.
Pyrsonympha
This endosymbiont has a more slender appearance than
Trichonympha. Its flagella also extend from its anterior
end, but they don’t extend away from the body of the
organism until they reach the posterior end. Instead, they
produce ‘ridges’ at the surface of the cell that have the
appearance of ruffles that are very evident as the organism moves.
Streblomastix
Another symbiont, Streblomastix, will be very abundant in
your slides. It is a very slender, needle-like organism with
spiral ridges that appear to extend the length of the cell.
Figure 10. Pyrsonympha.
Figure 11. Streblomastix.
Apicomplexa
The Apicomplexans are a large group of parasitic protists. They may have single hosts or may have alternate
hosts, as in the case of Plasmodium, the malaria parasite. They may have fairly complex life cycles with stages
and processes described by unique sets of terms. Fortunately, you won’t have to learn any Apicomplexan life
cycles. Male gametes may have flagella, and Gregarina
trophozoites (a stage in the life cycle) may exhibit an
apparently unique form of gliding motility.
Gregarina
Gregarina are a genus of intestinal parasites found in arthropods. Mealworm (Tenebrio molitor) guts may contain
two or more species, the most common being Gregarina
polymorpha. The Gregarina trophozoites never seem
to be numerous in our specimens, but we generally find
Figure 12. Gregarina.
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enough for everyone to see them. And we often also find them in termite guts as well, so we’ll hopefully
have two chances to find them in this week’s lab.
You may want to try your hand at preparing a slide of fresh guts. Use a razor blade to cut off the head and
the next adjacent segment from your Mealworm, along with the last one or two segments of the abdomen.
Use forceps to slowly and gently pull out the digestive tract, and remove any adhering tissue. You should
then open up the gut a bit, by puncturing it with a dissecting needle, tearing it up with the forceps, or
dicing it up with a razor blade. Place the gut in a drop of saline solution on a microscope slide, and gently press a cover slip down to cover your specimen. The same general approach will work for finding the
symbionts of termites. Why do you need to use saline solution?
Myxomycota
Slime molds come in two forms: cellular slime molds and plasmodial slime molds. They are now both
placed as amoebozoans in the super-group Unikonta, and they are both pretty strange kinds of organisms. As the name Mycetozoa suggests, it was once thought that these organisms were allied with fungi.
Cellular slime molds can exist as single-celled feeding amoeba, aggregates of amoebae that can migrate
as slug-like colonies, stalked fruiting bodies, resistant spores, and large diploid zygotes.
Plasmodial slime molds are also quite strange. The feeding form is an amoeba-like mass of cytoplasm,
called a plasmodium that can become very large and may contain many thousands of diploid nuclei. It
lives in moist places, feeding on bacteria and directly absorbing nutrients from the substrate. When food
becomes in short supply, the plasmodium may produce stalked sporangia that produce haploid spores
following meiosis. The spores germinate to produce either single-celled haploid amoebae or flagellated
“swarm” cells. When two of these cells fuse, a diploid amoeba is formed that may in turn develop into a
new plasmodium. Under unfavorable environmental conditions, as when the environment becomes too
dry, the plasmodium may turn into a sclerotium. This is a dry, dessication-resistant structure that may survive for months. When conditions improve, the sclerotium can resume growth as a new plasmodium.
Physarum polycephalum
Physarum polycephalum is a plasmodial
slime mold, and we have living cultures for
you to observe. In these cultures you should
be able to see the characteristic fan-like
growth pattern of this bright yellow organism,
and the ‘veins’ or ‘tubes’ of cytoplasm that
make up the plasmodium.
You should also observe the example of a
sclerotium that is available next to the dissecting microscope. This is the resistant
structure that allows the organism to survive
dry or cold periods, to resume active plasmodial growth when conditions improve.
Figure 13. Physarum polycephalum growing on a log.
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