Exercise 1 Protists: Observation and Classification of Specimens
EXERCISE 1 PROTISTS: OBSERVATION AND CLASSIFICATION OF SPECIMENS
Structure
1.1 Introduction Objectives 1.2 Materials Required 1.3 General Characters and Classification Super Group – Excavata Super Group – ‘SAR’ Clade Super Group – Unikonta 1.4 Observation of Slides of Protists Amoeba Euglena Paramecium Plasmodium 1.5 Terminal Questions 1.1 INTRODUCTION
In Unit 2 of Block 1 of Animal Diversity course you have already learnt that animal protists or the protozoans constitute a large assemblage of microscopic, unicellular organisms. These organisms exhibit all sorts of symmetry; show varied modes of nutrition and have diverse life histories. Some are autotrophic (chlorophyll bearing flagellates); others are heterotrophic including the saprozoic, phagotrophic or holozoic protozoa. They may be free living or mutualistic or commensals or parasites. They do not have tissues or organs, rather they have specialised organelles, and possess one or many nuclei.
In this exercise you will observe prepared slides of some representative protozoans. You will learn to classify these protozoans giving justification for placing them within their various groups and classes. You will also draw diagrams of the specimens displayed in the prepared slides as you observe them and compare them with figures given in this manual. Objectives
After performing this exercise you should be able to:
• identify the specimens belonging to the genera – Amoeba , Paramecium , Euglena , and Plasmodium and give their scientific and common names,
• draw labelled diagrams of the identified genera,
• classify the identified protozoans up to the level of class,
• list characters justifying their classification and mention special features, if any,
Animal Diversity: • mention the habitat and geographical distribution of the identified Laboratory genera, and
• mention the economic importance, if any, of the identified specimens. 1.2 MATERIALS REQUIRED
1. Compound microscope.
2. Prepared slides of Amoeba, Paramecium , Euglena , and Plasmodium .
3. Drawing sheets/note book, an HB pencil and eraser. 1.3 GENERAL CHARACTERERS AND CLASSIFICATION
Unicellular eukaryotes make up a group of all other eukaryotes that are not green plants, fungi, or animals. Unlike prokaryotes, eukaryotic cells have a membrane bound nucleus to enclose their genetic material and other membrane bound organelles such as mitochondria and Golgi apparatus or dictyosomes. These provide specific locations in the cell for its diverse functions, making the structural organization more complicated than the prokaryotic cell.
Unicellular eukaryotes are a very diverse group. The single celled eukaryotes or protists are considered to be the simplest eukaryotes but at the cellular level they are very complex as they perform all the functions in a single cell that multicellular organisms perform in various organs. Of these the animal like protists which were traditionally called protozoans have organelles and cytoskeleton that vary in structure, and they also use different modes of nutrition. Some are parasitic and others are predatory or mixotrophs (combining photosynthesis and heterotrophic nutrition). Some may spend their lifetime in one position and others may be constantly motile. Reproduction and life cycles are also highly varied. Genetic and morphological studies have shown that some protists are more closely related to plants, or fungi or animals than they are to each other; as a result Kingdom Protista in which all protozoans and plant like protists were placed has been abandoned. Though various lineages of protists are now recognized, the terms protists and their animal like representatives, that is, protozoans is still in use for convenience.
Traditionally, the protozoans have been classified as flagellates, amoebae, sporozoans and ciliates. However, over the past thirty years molecular phylogenetic studies have led to extensive modifications of the traditional classification schemes and the most dramatic changes have occurred in the protist groups. The recent system of classification places all unicellular eukaryotes in four major supergroups namely Excavata, SAR clade, Archaeplastida and Unikonta. We advise you to reread the Section on classification in Unit 1of the Animal Diversity course and refer to Figure 1.1 before you take up this exercise. We will only refer to the classifications of those protists (that were put under the former Kingdom Protozoa) which you will observe during this exercise. 6
1.3.1 Super Group – Excavata Protists: Observation and Classification of All members of Excavata have similar cytoskeletal features. Some of the Specimens unicellular species that form this group have a distinguishing morphological feature that is an ‘excavated’ feeding groove found on one side of the cell. They include photosynthetic, parasitic, heterotrophic and symbiotic taxa. Mitochondria may be reduced or modified in some groups and have flagella in some that differ from other organisms. The main protozoans of this super- group are placed in three phyla as given below one of which is Phylum Euglenozoa that is of interest to us:
Super Group Excavata
Phylum
Diplomonada Parabasala Euglenozoa Subphylum
Kinetoplastida Euglenida
Class Euglenoidea Phylum Euglenozoa: This phylum includes predatory heterotrophs, photosynthetic autotrophs, mixotrophs and parasites. Members of this phylum have a series of longitudinal microtubules that lie below the cell membrane and help it to stiffen into a pellicle.The main distinguishing feature is the presence of a rod with either a spiral or crystalline structure inside each of their flagella. The two best studied groups of euglenozoans are. Subphylum Kinetoplasta and Subphylum Euglenida.
Subphylum Kinetoplasta: Members of this subphylum have a single large mitochondrion with a mass of DNA associated with the kinetosome or basal body of the flagellum forming a unique organelle kinetoplast. .
Class Trypanosomatidea: This class includes member which have one or two flagella arising out of a pocket typically with paraxial rod. All are parasitic; examples, Trypanosoma gambiense that infects humans causing sleeping sickness, Trypanosoma cruzi that causes an important disease i.e. Chagas’s disease. Leishmania that causes kala azar or visceral leishmaniasis which is widely distributed in eastern India.
Subphylum Euglenida: Members have pellicular microtubules that stiffen pellicle.
Class Euglenoidea: Members of this class have a pocket at one end from which arise two flagella, one is small and the other is large. Mostly photosynthetic but some euglinids are mixotrophs. They have chloroplasts and these chloroplasts are surrounded by a double membrane and perform photosynthesis when sunlight is available and become heterotrophic in the absence of sunlight, absorbing organic nutrients from the environment. Many 7
Animal Diversity: other species ingest prey by phagocytosis. The most frequently studied Laboratory euglenid in the laboratory is Euglena. A red eyespot or stigma is present that
functions to orient the organism towards light. 1.3.2 Super Group – ‘SAR’ Clade
The SAR clade has been proposed recently (Adl et al. (2012) ) based on the whole genome DNA sequence analysis which has revealed that the three major groups of protists Stramenopiles, Alveolata and Rhizaria can be placed together in a super group. This is a highly diverse collection of protists. Of the three major groups, Stramenopiles include the photosynthetic diatoms and golden and brown algae (these will be dealt in the course on Plant Diversity) and the protozoan members are placed in Alveolata and Rhizaria.
Super Group SAR-Clade Group
Stramenopiles Alveolata Rhizaria
Phylum Phylum
Ciliophora Dinoflagellata Apicomplexa Cercozoa Foraminifera Radiolaria
Class
Gregarinea Coccidia
Group Alveolata (unranked): They have membrane enclosed sacs or alveoli under the plasma membrane. They include both photosynthetic and heterotrophic protists.
Phylum Ciliophora: Ciliates are named because of the cilia that cover them. A structural system of fibers along with the kinetosomes (basal bodies of the cilia) forms the infraciliature just beneath the pellicle. They are present in fresh water and marine habitats as well in moist soils. They are free living, parasitic and symbiotic. The distinguishing feature is the presence of two types of nuclei, one large macronucleus and tiny micronuclei. Macronucleus is responsible for the functioning of the organism and the micronucleus is diploid and involved only in reproduction. They have a special type of reproduction – conjugation during which two cells line up and micronuclei are exchanged via a cytoplasmic bridge between the two cells. Example is Paramecium .
Phylum Apicomplexa : All the species are parasitic and specialised for living and reproducing in animal tissue; the infectious stage is called sporozoite. The sporozoites have a system of organelles at one end known as apical complex that allows the apicomplexan parasite to penetrate the host cell membrane. Although apicomplexans are not photosynthetic, they retain a Noctiluca has the same compound as modified plastid which suggests their algal origin. Most apicomplexans have fireflies elaborate lifecycles involving one or two hosts. that makes it glow Class Gregarinea: Mature gamonts (individuals that produce gametes) are and when millio 8 of these protists float they form spectacular glowing tides.
large, extracellular parasites of digestive tract or body cavity of invertebrates; Protists: Observation and Life cycle with one host. Example , Monocystis and Gregarina . Classification of Specimens Class Coccidea : Mature gamonts are small, and typically intracellular. Life cycle with two hosts. The best known example, Plasmodium that causes malaria, lives both in the mosquitoes and humans. Another important disease causing apicomplexan Toxoplasma gondii is a parasite of cats and can infect humans. 1.3.3 Super Group – Unikonta
Unikonta includes protists that are closely related to fungi and animals. These protists form two large groups as given in the flow chart below:
Super Group Unikonta Group
Ameobozoa Opisthokonta Class
Myxogastrida Dictyostelida Tubulina Archamoeba 1. Group Amoebozoa (Unranked): It includes many Amoeba species that have lobe shaped or tube shaped pseudopodia. Amoebozoans include the slime moulds, tubulinids and entamoebas.
a) Class Myxogastrida: This class includes the plasmodial slime moulds that grow to form a mass called the plasmodium which is not multicellular but multinucleated. It feeds by pseudopodia and when it lacks nutrients it stops growing but gives out fruiting bodies which functions in sexual reproduction. b) Class Dictyostelida: This group includes the cellular slime moulds. The feeding stages are individually functioning cells but when nutrients are depleted the cells come together and form the slug like aggregate which functions as a unit, but the cells are still separated by their individual cell membranes. Ultimately the sluglike aggregate forms asexual fruiting bodies. c) Class Tubulinea: This class make a large and varied group of amoebas that have lobe or tube shaped pseudopodia. These protists are widely present in freshwater, marine environments and also in soil. Most are heterotrophs that prey on bacteria and other protists. The most well known example is Amoeba proteus . d) Class Archamoebea: Most amoebozoans are free living, however, individuals of genus Entamoeba are parasitic on vertebrates and some invertebrates. They are unusual in having flagellated stages in their life cycle and cysts with several nuclei.
2. Group Opisthokonta: It is a diverse group of eukaryotes that includes the animals and fungi as well as protists that are more closely related to fungi and animals than to other protists. 9
Animal Diversity: Laboratory 1.4 OBSERVATION OF SLIDES OF PROTISTS
We had said in the earlier section that a protozoan is not as simple an organism as one would think it to be. It is so designed that even though it is a single cell, it still functions as a complete organism.
The slide that you will get for observation is a permanently, stained whole mount. While observing it you should sketch it as accurately as you can. You will note that as you sketch the specimen under observation you are more likely to look for details you would not see otherwise, and if you put those details in the right places in the sketch and in the right dimensions, you will recognise the specimen when you see it again.
Some times you may not be able to see all the details in a single specimen but by observing several specimens and drawing a composite picture from your observation you will be able to recognise the specimen in any given slide. Colour coding also helps. You can devise a colour coding system for your self, for example blue for the nucleus, green for the chloroplast, etc. 1.4.1 Amoeba
Place a slide of Amoeba (common species is A. proteus ) under the microscope and focus it under low power (5X ×5X), and observe it carefully. Amoeba looks like an irregular colourless drop of gelatinous protoplasm.
Now focus it under high power (10X ×10X) and note the following features:
i) The body is covered by a thin elastic, semi-permeable membrane called plasmalemma.
ii) Under the plasmalemma, the protoplasm is clearly distinguished into an outer clear ectoplasm and an inner granular fluid-like endoplasm .
iii) Try to identify the anterior and the posterior ends of Amoeba . The anterior end is characterised by a thick layer of ectoplasm (the hyaline cap ), while the posterior end is modified in the form of a tail like region the uroid (The anterior end of Amoeba is the one in which direction a pseudopodium is given out for progression).
iv) You will see some blunt, finger-like projections. These are the pseudopodia and are the extensions of the protoplasm. Pseudopodia help the Amoeba to move in the medium and also in capturing food.
(a) (b)
10 Fig. 1.1: Amoeba proteus . a) light micrograph (160X), b) diagrammatic view.
v) You can also see the single, clearly stained nucleus, a large single Protists: Observation and contractile vacuole and large number of food vacuoles in the endoplasm. Classification of Compare your slide with Figure 1.1. Specimens
Habit and Habitat
Amoeba is commonly found in the mud, in fresh water ponds and ditches, and slow running streams. It is abundantly found in the water with lots of decaying vegetation and bacteria.
Geographical Distribution: All over the world.
Classification and its Justification
Domain Eukaryota Membrane bound nucleus and organelles multiple chromosomes complexed with histones.
Super-Group Unikonta Unicellular eukaryotes closely related to animals and fungi.
Group Amoebozoa Have lobe shaped or tube shaped locomotory organelles called pseudopodia.
Class Tubulinea Tubular/finger shaped pseudopodia. Freshwater heterotrophs.
Genus Amoeba 1.4.2 Euglena
Take the prepared slide of Euglena and focus it first under low power and next under the high power of the microscope. Observe the following characteristics: i) Euglena is an oval spindle shaped organism with a blunt anterior end and a pointed posterior end. ii) Body is externally covered with a pellicle. iii) Underneath the pellicle the cytoplasm is clearly differentiated into an outer ectoplasm and an inner endoplasm.
(a) (b)
Fig. 1. 2: a) Photomicrograph showing Eug ena (200 x), b) Diagrammatic view. 11
Animal Diversity: iv) Anterior end of the body bears an opening cytostome, which continues Laboratory internally as a tubular cytopharynx . The cytopharynx leads into a large
spherical reservoir. v) From the base of the reservoir a single whip-like flagellum arises. vi) A single large spherical nucleus is located towards the posterior region of the body. vii) Cytoplasm also contains chlorophyll which is contained in bodies known as chloroplasts . viii) You can also see a brightly coloured spot – stigma, closely adhered to the cytopharynx (hence the name Euglena , which means Eu = true + glena = eye ball) . ix) Compare your slide with Figure 1.2.
Habit and Habitat
Euglena is a solitary protozoan found in fresh water ponds, ditches, lakes and slow running streams with a lot of vegetation. It is abundantly found in those ponds, which contain decaying nitrogenous organic matter such as animal faeces, leaves, etc.
Geographical Distribution: Abundant in warm waters throughout the world.
Classification and its Justification
Domain Eukaryota Membrane bound nucleus and organelles multiple chromosomes complexed with histones. .
Super Group Excavata Unicellular eukaryotes having similar cytosketetal features. Some with excavated feeding groove on one side of cell.
Phylum Euglenozoa Have a series of longitudinal microtubules under the cell membrane; rod like structure inside their flagella. Predatory, heterotrophs, photosynthetic autotrophs, mixotrophs and parasites.
Sub-phylum Euglenida Have peculiar microtubules with stiffen pellicle.
Class Euglenoidea Have pocket at one end from which arise two flagella;one small and the other large. Mostly photosynthetic but some are mixotrophs.
Genus Euglena 1.4.3 Paramecium Place a prepared slide of Paramecium under the microscope and focus it 12 under low power (5X × 5X) and observe the following:
i) It has a cigar-shaped or slipper-shaped body, hence commonly called Protists: Observation and slipper animalcule (=little animal). Classification of Specimens ii) Now focus it under high power (10X × 40X): The entire body is covered with pellicle, which shows rows of tiny depressions.
iii) Look carefully at these depressions. From each such depression arises a cilium .
iv) These cilia cover the entire body and are uniform in size except at the posterior end of the body where they are larger and form a caudal tuft . These cilia help the Paramecium in locomotion.
v) The anterior end is bluntly rounded and the posterior end is slightly pointed.
vi) One side of the organism has a depression, which leads into an oral groove. This groove ends in the mouth or cytostome.
(a) (b)
Fig. 1.3: Paramecium bursaria , a) photomicrograph (430 x), b) diagrammatic representation.
vii) Now observe the protoplasm, which is clearly divisible into an outer ectoplasm and an inner fluid-like endoplasm . You can see series of rod like trichocysts under the pellicle embedded in the endoplasm.
viii) The endoplasm contains two nuclei – one large kidney-shaped macronucleus and the other small dot-like micronucleus .
ix) You can also see two contractile vacuoles – one towards the anterior end and the other towards the posterior end.
x) Apart from these structures, many spherical food vacuoles can be seen in the endoplasm.
xi) Compare your stide with Fig. 1.3.
Habit and Habitat
Paramecium is found in fresh water ponds and ditches rich in dead and decaying vegetation.
Geographical Distribution : World wide. 13
Animal Diversity: Classification and its Justification Laboratory Domain Eukaryota Membrane bound nucleus and organelles, multiple chromosomes complexed with histones.
Super Group ‘SAR’Clade Unicellular eukaryotes Includes three groups i.e. Stramenopila, Alveolata and Rizaria.
Group Alveolata Share a system of sacs called alveoli under their plasma membrane.
Phylum Ciliophora Cilia cover the body, presence of two types of nuclei: one large macro-nucleus and another tiny micro-nucleus.
Genus Paramecium
Species caudatum/ bursaria 1.4.4 Plasmodium
Place a slide of Plasmodium (a malarial parasite of human) under microscope and focus it gently and nicely, so that you can see this apicomplexan . The parasites spread through their host as tiny infectious cells called sporozoites. Apicomplexans are so named because one end (the apex) of the sporozoite cell contains a complex of organelles specialised for penetrating host cells and tissues.
Most apicomplexans have intricate life cycles with both sexual and asexual stages.
i) Plasmodium is an intracellular parasite of human and other vertebrates and causes malaria.
ii) The life history of Plasmodium (Fig. 1.4) is completed in two hosts, viz partly in a definite host, the man and partly in an intermediate host, the female Anopheles mosquito.
iii) When an infected female Anopheles bites a human, sporozites are introduced in the blood from where they reach in linear cells through blood stream and multiply to form merozoites.
iv) After a few cycles in the liver the merozoites enter the red blood corpuscles (RBCs) and feed on its contents.
v) After having undergone 2-8 schizogonous changes in the main blood stream, the merozoites assume different shapes called gametocytes.
vi) Development of gametocytes cannot proceed further in blood of human, therefore, they wait for female Anopheles to bite and feed on the blood.
vii) When gametocytes reach through blood of human to Anopheles , they
14 undergo sporogony for further development.
viii) There are 4 species of Plasmodium causing different types of fever. Protists: Observation and Classification of i. P. vivax causes benign/tertian fever. Specimens ii. P. falciparum causes malignant tertian fever . iii. P. malariae causes quartan fever . iv. P. ovale causes mild tertian fever .
(a) (b)
Fig. 1.4: a) Sporozoite of Plasmodium, (b) Life cycle of Plasmodium.
Habit and Habitat
Plasmodium is found as an intracellular parasite in blood of hhumanuman s and other vertebrates.
Geographical Distribution: It is widely distributed in t ropical and temperate countries of the world like India, Srilanka, Bangladesh, Nepal and Pakistan.
Classification and its Justification
Domain Eukaryota Membrane bound nucleus and organelles multiple chromosomes complexed with histones.
Super Group SAR Clade Includes 3 groups ofunicellular eukaryotes, Stramenopila, Alveolata and Rizaria. Group Alveolata Share a system of sacs called alveoli under their plasma membrane . Phylum Apicomplexa Parasitic, sporozoites have a set of organ elles (apical complex) with the anterior end specialised for penetrating the host cell and tissue. Most with one or two hosts in their life cycle .
Class Coccidea Mature gamonts small; s pores or oocyst present which contains infective sporozoite. Two hosts in life cycle.
Genus Plasmodium 15
Animal Diversity: Laboratory 1.5 TERMINAL QUESTIONS
1. How do you distinguish ectoplasm from endoplasm?
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2. What are the functions of contractile vacuole, cytoplasm, pseudopodia and food vacuoles respectively?
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3. At what level of organisation are the protists placed?
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