Phylum Protozoa (PROTOZOANS)
The term Protozoa embraces the vast assemblage of living organisms, some free-living, some parasitic, that appear to organise their whole lives as single cells. Estimates of the total number of species are difficult to make because the definition of the term species is particularly unclear in the Protozoa. It is safe to say that over 80,000 species have been described and that there is more than three times this number as yet undescribed. Although a few are visible to the unaided eye, being up to 5 mm. long, the vast majority require a good microscope to reveal them and their structure as they are only from 2μ to 8μ long. Whether they are animal or plant, whether this or that group is truly protozoan, whether they form a subkingdom, superphylum or phylum are all matters that have produced diverse opinions. But the one generally accepted criterion for the inclusion of a particular organism in the phylum is that all functions are confined to a unicellular structure. It is therefore valuable to consider cell construction and function before reviewing the more varied and complex aspects of micro-organisms. Without knowledge of basic structure, it is impossible to begin to make sense of the enormous variety of this versatile and cosmopolitan group. Historically protozoology is relatively recent. Protozoans were first seen in 1674 by Van Leeuwen-hock using a simple microscope. Their small size has meant that knowledge about them has been linked with the slow development of the microscope. Improved techniques and instruments have brought with them a wider understanding of the form, physiology and behaviour of these tiny creatures.
Examples of PROTOZOANS
Prymnesium ( x 2,175) is a marine species that live by photosynthesis. The chloroplasts, extending from the base of the flagella, are pigmented and trap the light energy necessary for building up the living cell. This species sometimes grows huge populations and when it forms the diet of fishes is poisonous.
Ceratium is one of the dinoflagellates which are mostly marine, though freshwater forms occur. The spines are stiff and two flagella are used in locomotion. One lies in the equatorial groove seen in the photograph while the other is in a shallow groove running from the equatorial groove.
The structure on a small scale Certain functions are essential to life in its highest and lowest forms. In man, for example, the essentials are movement in search of food and away from harmful agents; awareness of the environment through the senses and integration of responses; capture and processing of food; respiration through the lungs; controlling the water content and soluble substances of the body by means of the urinary system; and reproduction by means of specialised organs. In man, these six functions demand specialised structures or organ systems in which each organ consists of many minutes, and usually specialised cells. In other words, the cell is the lowest structural denominator of life. In higher forms, some cells are so specialised that they cannot exist or multiply outside the organism of which they are part.
Basic cell plan In its basic form the plan of most cells, including most protozoans, is that shown in the diagram of a hypothetical cell below. It will be seen at once that the traditional definition of a cell as a single nucleus surrounded by cytoplasm is now no more than a convenient simplification. Even Amoeba, one of the simplest known forms of animal life, cannot be reduced to quite such fundamentals. With the recent advent of the electron microscope the cell membrane, the mitochondria, the Golgi apparatus and other structures have been elevated from the area of hypothesis to that of common and certain knowledge. The existence of other structures (ribosomes, nuclear pores and the endoplasmic reticulum) was not even suspected by earlier cytologists using light microscopes.
The cell membrane of Protozoa
The cell membrane is composed of two asymmetrical layers, each one formed by a complex of fatty and protein substances known as lipo-protein. The molecules are composed of fat at one end and protein at the other. In a membrane, they are arranged in such a way that the fatty ends are together and the two protein ends face outwards. The distance between the protein layers is between 8 p and 12 p. This typical structure is known as a unit membrane and forms not only the covering of all animal cells but also their projecting organelles and some internal structures.
Mitochondria of Protozoa
Mitochondria of cells other than those of protozoans are ovoid structures with internal anatomy consisting of cristae or layers of double unit membranes crossing transversely. It is these cristae that characterise mitochondria, and are particularly important, as protozoan mitochondria are seldom ovoid, but generally elongated to various extents. In trypanosomes, mitochondria change in size and function during the life-history. In one phase, the bloodstream form, mitochondria are very reduced and poor in cristae, by which characteristic such forms are distinguished. The function of mitochondria is to release energy in usable form to the cell in the process called cellular respiration.
Chloroplasts in Protozoa
Chloroplasts are structures somewhat resembling mitochondria but which contain colour pigments. They give most plants their characteristic green colour. In some flagellated protozoans chloroplasts are green, but more typically they are yellowish-brown. These organelles serve to absorb energy from sunlight and convert light energy into chemical energy of sugar or starch. This is a process requiring dissolved carbon dioxide and water and is generally known as photosynthesis.
Golgi apparatus in Protozoa
Golgi apparatus and lysosomes are structures about which there is still some controversy. Typically the Golgi body has been shown to consist of flattened saccules arranged like a pile of paper bags. Recent electron micrographs strongly indicate that small bodies called lysosomes develop at the tips of the saccules and are freed into the cytoplasm. They are characterised by their typical content of enzymes, which perform a catalytic function in breaking down many biological substances in acid conditions. Lysosomes seem to function as the start of some digestive processes and also in protecting cells against noxious substances.
Cilia and flagella in Protozoa
Cilia and flagella are organelles of locomotion and have essentially the same basic structure. The unit is the flagellum and is composed of eleven hollow fibres inside a cylinder of 200-300mg in diameter. Two fibres lie together in the centre with nine outer fibres arranged in a ring around them. At the base of a flagellum, where it is inserted into the cell body, the centre fibres disappear, leaving only the ring of nine which may become fused. The fused region is known variously as the basal body or basal granule.
Flagella vary in length from 61.1 to 250 p and are found singly or only a few attached to each cell. Cilia, on the other hand, occur typically in rows, when many hundreds may more or less cover the cell, and arc seldom longer than 30p. in a few ciliates, however, which are described in some detail on p. 28, the cilia are much reduced in number or fused together to form paddle-like membranclles or tufted cirri. Thus it is difficult to generalise and it may well be that the distinction between cilia and flagella is artificial. When cilia occur in large numbers they beat in a co-ordinated way, controlled by additional structures running along, and some between, the rows of cilia immediately below the cell membrane.
Protozoan nuclei are so similar that it is best to describe first the structures of a mammalian nucleus and then draw contrasts with the protozoans. These structures are chromosomes, centrioles and spindle (when the nucleus is dividing), nucleolus, and nuclear membrane with its associated pores.
The chromosomes usually are seen clearly only during cell division, when they appear as sausage-shaped elements. The number in each cell is usually constant within a species but can be as low as four or as high as several hundred. It has been demonstrated that the chromosomes contain deoxyribonucleic acid (DNA), the substance which in all animal cells constitutes the encoded genetic information. Between divisions chromosomes are diffuse in the nucleus and not usually visible.
Centrioles and spindle in Protozoa
The centrioles and spindle, like the chromosomes, become visible only at division. The centrioles are short cylinders and arc frequently regarded as the same type of structure as the basal granule of a flagellum. From them arise numerous hollow fibres to which the chromosomes are attached. In a dividing nucleus there are two diametrically op-posed centrioles (see diagram p. 17). The fibres from each centriole approach each other from the poles.
Nucleolus of Protozoa
The nucleolus is a body within the nucleus. Characteristically it appears between divisions. It contains ribonucleic acid. Between divisions the nucleus can be seen to be made of a mesh of fibres or diffuse granules and one or more distinct bodies called karyosornes or endosomes.
Types of Protozoan locomotion
There are three types of protozoan locomotion.
- Amoeboid locomotion
- Flagellate locomotion
- Ciliate locomotion
(1) Protozoan Amoeboid locomotion
Amoeboid locomotion, illustrated here by Amoebas proteus, in which a pseudopodium is pushed out and the nucleus moves into it. Other pseudopodia are produced at the same time and the nucleus subsequently moves into one of these.
(2) Protozoan Flagellate locomotion
Flagellate locomotion presented here in schematised form, is by means of one or more flagella, used oar-fashion, except that the flagellum is flexible on the recovery stroke. This is the method used by the majority of flagellates, but others twist the flagellum so that a vortex is created in the water and the animal is sucked into it. (
Ciliate locomotion is produced by rows of short filaments or cilia that cover the surface of the animal and are interconnected by a network of subpellicular fibrils. The cilia behave rather like flagella, except that the strokes are co-ordinated by the subpellicular fibrils to beat in waves, and this is known as metachronal rhythm.
Hartmanella (x 1,220) is an amoeba with interesting pseudopodia. The tips show fine rhizopodia extending from the broader pseudopodia, and food vacuoles are numerous in the cytoplasm. The nucleus containing the nucleolus is clearly seen in this living specimen.
Nuclear membrane of Protozoa
The nuclear membrane is a typical unit mem-brane surrounding the nucleus and separating it from the surrounding cytoplasm. It was once thought that the nuclear membrane acted as a barrier against the flow of materials across it in either direction. Recent electron micrographs have shown that the nuclear membrane is pierced by numerous pores 8-20mµ in diameter. This fact makes its supposed role ‘as a barrier rather less than certain.
The foregoing is a brief account of the generalised nuclear structure. The variations found in different protozoans inevitably strain this generalisation as they strain most others. Some protozoans appear not to have chromosomes; others appear to divide without either centrioles or spindle; some appear to have no nucleolus; some (typically of the class Ciliata) have two nuclei, and others (the Try-panosomatidac) have a fragment of DNA associated with the flagellum, and so on.
Diversity has two main explanations. First, the protozoans are an ancient group of organisms and have undergone extensive evolutionary diversification; second, some of the diversity may be only apparent either because the descriptions of the many species have been made by different authors using different techniques at different times, or because many of the structures are very close to the limit of resolution of the light microscope and are therefore difficult or impossible to see clearly.
The electron microscope has aided greatly the study of the smaller protozoan components both nuclear and extra-nuclear. In some cases a unity of structure has been demonstrated (unit membrane, the fibrillar structure of cilia and flagella). In other cases a considerable diversity has been shown (e.g. variety of cytoplasmic inclusions).
Symmetry of Protozoa
Applied to other animals the term means the spatial arrangement of parts according to some geometrical design. Thus most animals including man have bilateral symmetry, otherwise called mirror-image or two-fold symmetry. Here, the animal body has mirror-image right and left halves. Adult echinoderms such as starfishes with five-fold radial symmetry (the symmetry of cylinders and wheels), break the more usual pattern.
Among protozoans, the protean nature of the body form of some (amoebas) dictates that they be considered asymmetrical. Others, with a more constant body form, maybe imperfectly bilateral (and this group includes the majority, especially hypotrichous ciliates), radial (choanoflagellates), or even spherical (Heliozoa and Radiolaria).
Energy production in Protozoa
Energy is needed for all life processes. Animals obtain it by enzymatically breaking down complex molecules containing much energy into smaller molecules with less total energy. The difference between the two energy levels is available to the organism if it is capable of using it. The most frequent method of breakdown is cellular respiration, summarised in the overall equation: sugar + oxygen —,carbon + water + energy dioxide
C6H1206 + 6CO2 → 6CO2 +6H2O + energy
Actinopod is an example of protozoa
Actinopod (x 615). Here the food-gathering filopodia are seen fully extended. Most of the length of filopodium is stiffened by a spiral structure recently revealed by electron microscopy. Cytoplasm flows along the filopodia carrying attached food particles.
Respiration in Protozoa
The oxygen required for this aerobic respiration is obtained directly or indirectly from the air by respiration. In those protozoans which require oxygen, it is acquired by aerobic respiration, dissolved in the environmental medium (freshwater, seawater, host’s blood), and enters the cell by simple diffusion through the cell membrane. No special respiratory organs are known in the protozoan orders.
Anaerobic respiration in Protozoa
Anaerobic protozoans (e.g. entodiniomorphs) do not require oxygen to produce energy. These are typically the parasitic species that live in a nutrient-rich but oxygen-poor environment of the host, whose metabolism deals with the waste product, lactic acid. The conversion of sugars to these substances extracts only about one-fifth of the total energy that would be available if the complete process took place. Therefore, to obtain a given amount of energy, anaerobic organisms utilise much more sugar than aerobic species.
Aerobic respiration in Protozoa
Aerobic respiration may be considered as a three-stage process: glycolysis or splitting of simple sugar molecules into two 3-carbon molecules; the tricarboxylic acid cycle, in which the 3-carbon molecules are progressively broken down in the mitochondria to liberate carbon dioxide and some energy; and oxygen transport, the major energy source, in which oxygen is brought into combination ultimately with hydrogen.
The energy so produced is used to make a common cell fuel, adenosine triphosphate or ATP. This molecule gives up energy to whatever synthetic system needs it by the liberation of one phosphate group and becomes adenosine diphosphate or ADP.
Homeostasis and osmoregulation in Protozoa
A cell, whether protozoan or metazoan, has numerous substances within it which are essential for its proper function. Some of these constantly are being broken down and utilised and must therefore be replaced. Also, metabolic activity produces waste products that have to be eliminated if the cell is not to be self-poisoned. The functioning of cells is such that the internal constituents, both of cytoplasm and nucleus, tend to remain constant, except for necessary changes in growth and cell division. The general condition of keeping each cell in a state of constant composition is called homeostasis.
One feature of biological membranes is that they are selectively permeable; water and some small ions and molecules can flow more easily through them than larger molecules, although size is not the only regulating factor. This is the basis for the phenomenon of osmosis. In freshwater habitats it is mostly water molecules that bombard the outside of the cell, while the inside is bombarded by water containing dissolved salts, sugars and proteins.
There is therefore a net gain of water into the cell without any corresponding loss of the larger molecules. If this osmosis of water continued unchecked the cell would distend and finally bunt. Normally this does not happen because energy is spent in the cell membrane keeping the water out, though it is not clear by what mechanism this occurs.
However, in many freshwater species some water gets past this barrier and is removed from the cell through the expenditure of energy by mem-branes which form the pulsating vesicle called the tractile vacuole. This process permits the simultan-eous excretion of waste products. Cell membranes, by using energy, are able to regulate the rate of flow of many kinds of ions and molecules into and from cells.
The mechanisms involved are mostly not understood but are called active transport. The general principle demonstrated is that cells can conserve and in some cases concentrate necessary materials within themselves. The em-phasis lies on the properties of the cell membrane and on the healthy cell being able to keep this organelle in good repair to function properly.
In the Protozoa, this is as varied as the main body forms, amoeboid, flagellate or ciliate.
Descriptions of mechanical processes which often happen at considerable speed cannot replace direct observation or, especially, slow motion cinematographs. Amoeboid movement, a very slow form of progress, has been closely investigated recently and several theories have been proposed. As seen from above, amoebas move by a smooth and continuous process of pushing out a pseudopodium (`false foot’). The nucleus (one of the few reference points) moves slowly into it. In the meantime, newer pseudopodia have been formed and movement is then in the direction of one of them. A newly formed pseudopodium has a clear margin, known as the ectoplasm, and an inner granular endoplasm.
The granular bodies have been shown by the electron microscope to be numerous vacuoles, mitochondria and lysosomes. The earlier sol-gel hypothesis supposed that the ectoplasm at the tip of a growing pseudopodium was thinner and weaker than that surrounding the rest of the organism, especially at the rear. And so the jelly-like ectoplasm (gel) tended to contract, forcing the more fluid endoplasm (sol) forwards and thereby extending the weakest part, the new pseudopodium. A more recent theory has been put forward proposing that the ectoplasm is made up of parallel protein molecules which strengthen it. In the ectoplasm of a forming pseudopodium, a folding and regimentation of these molecules occur near the tip. The result is that endoplasm flows into the tip to occupy the newly formed space.
Still, more recently amoebas have been viewed from the side instead of from above. Seen from this angle, they seem to move on small strut-like projections which hold the main pseudopodia clear of the substratum. This important matter is still under active study. Another type of locomotion related to amoeboid movement is found in the Actinopoda, which have fine stiff filaments projecting from the main cell body. Cytoplasm streams along these filaments, carrying with it bits of matter stuck to the surface. The organic bits serve as food when they reach the main cell body.
Those pseudopodia in contact with the substratum slowly ease the animal along. A third type, flagellate movement, is invariably by means of one or more flagella. The exact method of obtaining the relatively high speeds achieved by such small .creatures (often 250μ per second or about a yard an hour) depends on the species.
The movement of the flagellum is by locally-formed waves which can pass in either direction along the length of the organelle. The waves act on the surrounding water to push or pull the animal forward. Most flagellates use the pulling method of propulsion and the flagellum moves first. Some species move by twisting the forward-projecting flagellum back on itself and then by whirling the tip to create a vortex into which the rest of the animal is sucked. The variations on the method are many. Recent slow-motion tine studies have shown that the flagella of trypanosomatids are subject to waves of contraction passing from the tip to the base. Since flagella can be observed to stop and start, the control for starting a wave presumably originates in the cell and some signal must travel along the flagellum from base to tip to trigger a contraction.
Euglenoid movement is an alternation of the shape of the body of Euglena and other similar flagellates apparently determined by the subpellicular fibrils. It is well developed in Euglena and is used to move the whole body. In others it appears to be less involved in locomotion although its exact function is not known. Yet another method of locomotion is found in the ciliates, which sweep themselves along by using the cilia with which most are more or less covered. Each cilium beats in a characteristic way. In the power stroke the filament is stiff and extended while in the recovery stroke it is bent and offers least resistance to the water. The cilia lie in rows interconnected by a complex network of subpellicular fibrils. The result is that the whole ciliated surface of the animal can beat in a coordinated way.
The term meta-chronal rhythm is used to describe the beating, as the cilia do not all exhibit the power stroke simultaneously but in an orderly sequence. This sends waves of power and recovery strokes alternately down the body. The varied and advanced methods of locomotion found especially in the Spirotrkha are produced partly by the loss of some rows of cilia and partly by fusion of others into cirri and membranellae. Many organisms in this group are able to ‘run’ over the surface of water plants with astonishing speed. The cirri serve as stronger leg-like units than single cilia, and although more widely separated from each other still beat in a coordinated way.
Euglena is an example of protozoa
Euglena(x 850). Binary, fission in this living cell is taking place by longitudinal splitting. In this way not only is the genetic material contained in the nucleus halved between each cell, but also the cytoplasmic particles, chloroplasts, food reserves, and mitochondria are equally divided.
Euglena ( x 800). The same cells have nearly completed division. When complete flagella are developed the separated cells swim away.
Vorticella is an example of protozoa
Vorticella has its main body on the end of a contractile stalk shown here shortened by forming a spiral. Feeding is achieved by ingesting particles wafted in the current of water created by the crown of cilia that can be seen around the top of the body.
Individual cells of this euglenoid species form stalks of mucilage. While they are in this pseudo-colonial phase there are no large flagella, but the cells can separate, grow a flagellum and swim around like others. flagellates. Euglena ( x 850). Binary. fission in this living cell is taking place by longitudinal splitting. In this way not only is the genetic material contained in the nucleus halved between each cell, but also the cytoplasmic particles, chloroplasts, food reserves, and mitochondria are equally divided.
Euglena ( x 800). The same cells have nearly completed division. When complete flagella are developed the separated cells swim away.
Nutrition of Protozoans
In the Protozoa this is so varied that it serves to emphasise their claim as founders of the animal kingdom. Some, like Euglena, are green and are very close to plants, some like Didinium ingest large particles or the whole bodies of other protozoans, some ‘drink’ in nutrients by forming small pockets in the cell membrane, some filter off particles in the surrounding water by setting up wide-ranging currents, and there are some in which the feeding mechanisms are still obscure. The commonest methods of obtaining food are undoubtedly filter feeding, phagocytosis—both phagotrophic feeding methods—and saprozoic feeding. Phagotrophy is the general term applied to feeding on particulate matter. If the particles are in suspension then the method usually employed is filter feeding.
As practiced by Vortieella, a sessile ciliate, a current of water is produced by the beating of the cilia around the crown of the body, so causing a vortex that draws particles into the mouth. Particles may also be taken when they are on the surface of water plants or in among other materials such as soil or intestinal contents.
The process is a modification of amoeboid movement and consists in forming a hollow conical pseudopod with the particle in the depression. The pseudopod rejoins with itself on the other side of the particle, which is now enclosed within the protozoan. This process is known as phagocytosis.
Saprozoic feeding was earlier thought to be diffusion of soluble food through the cell membrane. Two things have changed this concept considerably. First, the distinction between a solution and a suspension is no longer clear cut. For example, protein molecules which appear to form a stable solution under normal conditions can be thrown down to the bottom of the vessel by ultra-centrifugation. The distinction, then, is one of convenience: small particles (e.g. proteins) cannot be seen when in solution; larger particles can be seen and the mixture is then called a suspension.
The second point is that more careful observation of cells revealed pinocytosis (cell drinking), a process in which the cell membrane, instead of absorbing material in solution, forms very deep cones, smaller and more tube-like than in phago-cytosis, into which the solution is drawn. The tube closes behind it and so cuts off pinocytosis vacuole. Pinocytosis may be considered, rather crudely, as a modification of phagocytosis or vice versa. The mechanisms are similar but are far removed from
Mitosis in Phylum Protozoa
Mitosis is the usual process during which a cell divides into two. At first the nucleus is resting (A), then the chromosomes appear as threads (B) which shorten and coil into spirals. At this point the centrioles or poles begin to move apart, and a spindle forms between them. The chromosomes lie across the spindle (C), attached to it by their spindle attachments. By now the nuclear membrane has dissolved (D), and the duplicates of each chromosome separate and move towards the poles (E) and the spindle itself elongates, pushing the groups of chromosomes further apart. The chromosomes uncoil (F), elongate and disappear, and a new nuclear membrane forms to give two distinct new cells.
This class is characterized by non-motile spores, each containing two or more polar filaments, small sacs with curled spring-like structures within. They are all parasitic and small, with a host range that includes both invertebrates and vertebrates. The infections caused are mostly mild or occur in animals which are not important in human economy. An exception is filosema (order Microsporidia) of silk-worms and honey bees, where a debilitating and often fatal disease results from infection.
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