МИНИСТЕРСТВО ЗДРАВООХРАНЕНИЯ РЕСПУБЛИКИ БЕЛАРУСЬ БЕЛОРУССКИЙ ГОСУДАРСТВЕННЫЙ МЕДИЦИНСКИЙ УНИВЕРСИТЕТ КАФЕДРА БИОЛОГИИ

В. Э. БУТВИЛОВСКИЙ, В. В. ГРИГОРОВИЧ, А. В. БУТВИЛОВСКИЙ

МЕДИЦИНСКАЯ БИОЛОГИЯ ДЛЯ ИНОСТРАННЫХ СТУДЕНТОВ, ОБУЧАЮЩИХСЯ ПО СПЕЦИАЛЬНОСТИ «ЛЕЧЕБНОЕ ДЕЛО»

MEDICAL BIOLOGY FOR INTERNATIONAL STUDENTS STUDYING IN THE SPECIALTY «GENERAL MEDICINE»

Учебно-методическое пособие

Минск БГМУ 2017 УДК 57(075.8)-054.6 ББК 28.70я73 Б93 Рекомендовано Научно-методическим советом университета в качестве учебно-методического пособия 17.05.2017 г., протокол № 9 А в т о р ы: доц. В. Э. Бутвиловский (темы 1–12); ассист. В. В. Григорович (темы 13–19); доц. А. В. Бутвиловский (темы 20–23, тесты) Р е ц е н з е н т ы: канд. биол. наук, доц. А. В. Колб; канд. мед. наук, доц. О. Н. Ринейская

Бутвиловский, В. Э. Б93 Медицинская биология для иностранных студентов, обучающихся по специальности «Лечебное дело» = Medical biology for international students studying in the specialty «General medicine» : учебно-методическое пособие / В. Э. Бутвиловский, В. В. Григоро- вич, А. В. Бутвиловский. – Минск : БГМУ, 2017. – 208 с. ISBN 978-985-567-741-4. Содержит теоретический материал 23 тем практических занятий по медицинской биологии и общей генетике, термины, открытые и закрытые тесты. Предназначено для иностранных студентов 1-го курса, обучающихся на английском языке. УДК 57(075.8)-054.6 ББК 28.70я73 ______Учебное издание

Бутвиловский Валерий Эдуардович Григорович Виктор Васильевич Бутвиловский Александр Валерьевич

МЕДИЦИНСКАЯ БИОЛОГИЯ ДЛЯ ИНОСТРАННЫХ СТУДЕНТОВ, ОБУЧАЮЩИХСЯ ПО СПЕЦИАЛЬНОСТИ «ЛЕЧЕБНОЕ ДЕЛО»

MEDICAL BIOLOGY FOR INTERNATIONAL STUDENTS STUDYING IN THE SPECIALTY «GENERAL MEDICINE» Учебно-методическое пособие На английском языке

Ответственная за выпуск Е. В. Чаплинская Переводчики В. В. Григорович, А. В. Бутвиловский Компьютерный набор В. Э. Бутвиловского Компьютерная верстка Н. М. Федорцовой

Подписано в печать 14.06.17. Формат 60 84/16. Бумага писчая «Снегурочка». Ризография. Гарнитура «Times». Усл. печ. л. 12,09. Уч.-изд. л. 12,13. Тираж 99 экз. Заказ 405.

Издатель и полиграфическое исполнение: учреждение образования «Белорусский государственный медицинский университет». Свидетельство о государственной регистрации издателя, изготовителя, распространителя печатных изданий № 1/187 от 18.02.2014. Ул. Ленинградская, 6, 220006, Минск. ISBN 978-985-567-741-4 © Бутвиловский В. Э., Григорович В. В., Бутвиловский А. В., 2017 © УО «Белорусский государственный медицинский университет», 2017 2 Topic 1. HUMAN IN THE SYSTEM OF NATURE. METHODS USED TO INVESTIGATE CELL

THE POSITION OF THE HUMAN IN THE ANIMAL WORLD SYSTEM The human as a species (table 1) refers to the phylum Chordates, subphylum Vertebrates, class Mammals, subclass Placentals, order Primates, suborder Anthropoids (narrow-nosed apes), family of Hominids (humans), genus Homo (man) and species Homo sapiens (wise man). Humans as biological and social beings. Humans have characters both of biological and social beings (table 1). The following signs are characteristic only of the species of Homo sapiens: straight walking, apparent thumb opposition, S-shaped spine, the brain volume of 1100–1700 cm3, prominence of the chin, abstract thinking, speech, producing tools, etc. The progress of humankind obeys social laws — laws of the society. The human life is impossible beyond the society. Social factors have played a great role in human development. Knowledge, skills and spiritual valuables are transferred in the society through training and education of young generations. Table 1 Similarity of humans and animals Taxons of animals Signs characteristics of human Phylum chordates Appearance of axial organs occurs in the embryonic period: a notochord, nerve tube, alimentary tube Subphylum The notochord transforms into the spine, the heart is at the abdominal vertebrates side. There are 2 pairs of extremities, 5 regions of the brain, jaws Class mammals 4-chambered heart, homoiothermy, well-developed cerebral cortex, mammary glands, presence of hair on skin coverings Subclass placentals Development of human fetus in the mother’s womb and its feeding through the placenta Order primates The thumb of the upper extremities is opposed to the others, nails on fingers, one pair of mammary glands, well-developed clavicles, teeth of three types and replacement of milk teeth by permanent ones, giving birth to one child in the majority of cases

THE SIGNIFICANCE OF BIOLOGY FOR MEDICINE Studying biology has great significance for training doctors. The important methods investigating etiology (cause) pathogenesis (development) of many human diseases and elaborating ways of their prevention and treatment are methods of biological modeling. Studying the biology of parasites is necessary for successful fighting against invasive diseases. World Health Organization suggests that about 90 % of all people are infected with helminthiases.

3 Studying the mechanisms of cell physiology is crucial for understanding the action mechanism of many drugs and elaborating new medicines. Genetic engineering (genetic designing of cells and organisms with definite characters) and biotechnologies (technological processes using living organisms) helped setting up the production of antibiotics, interferon, some hormones and enzymes, vitamins. Gene therapy is a promising trend in treatment of many hereditary disorders that could not be treated before. It is not possible without understanding of gene expression and work of human genome.

THE SUBJECT, TASKS AND METHODS OF CYTOLOGY Cytology (Latin cytos — cell, logos — science) is a science studying the structure, chemical composition and functions of cells, their multiplication, development and interaction in a multicellular organism. Tasks of cytology are: – research of the structure and function of cells and their components (membranes, organelles, inclusions and nuclei); – research of division of cells and possibilities of their adaptation to environmental changes; – research of interrelations between cells in a multicellular organism. Methods of cytology: 1. Microscopic methods are used to study morphology of cells and their components. Light microscopy uses visible light and a system of lenses to magnify images of small samples; electron microscopy uses a beam of accelerated electrons. The wavelength of an electron is thousand times shorter than that of visible light photons and electron Fig. 1. Mitochondrion under the electron microscope microscopes have a higher resolving power (figure 1). 2. Cytochemical (histochemical) methods help to investigate chemical composition or location of substances in cells (in tissue sections). They are based on staining the cell with dyes which color the substances of the nucleus and cytoplasm. 3. Biochemical methods are used for studying the chemical composition of cells, estimation of substance concentration in various tissues. They are based on a property of different biochemical compounds to absorb light waves of a definite length and allow to measure concentration of different compounds in the cell. 4. The method of differential centrifugation helps to study to the composition and properties of cell organelles: a tissue specimen is fragmentized to destroy

4 cell membranes and then placed into a centrifuge, where it is separated into several fractions. 5. The method of autoradiography (isotopic labeling) is used for studying dynamic of metabolic processes in cell components. It is based on introduction of radioactive isotopes into the cell. Molecules marked with radioactive isotopes (3H, 32P, 14C) participate in exchange reactions. Their location and accumulation, movement and excretion are determined by radiation registered with a photographic film (autoradiograph). 6. X-ray crystallography (X-ray structural analysis) is performed for studying the spatial structure and arrangement of molecules in the substance. This method is based on diffraction of X-rays passing through a crystal of substance. 7. Photo and video recording allows to monitor changes and processes of cell’s life such as division. 8. Microsurgery of cells is the method allowing removing and transplanting organelles of the cell. 9. Scanning electron microscopy form high-resolution images of specimen using a physical probe that scans its surfaces (figure 2). 10. Methods of cell culture are based on growing cells in artificial media. These cell cultures can be used Fig. 2. Image of pollen obtained by scanning to obtain hormones, enzymes, microscopy antibodies and another products.

MAGNIFYING DEVICES. BASIC STRUCTURE OF A LIGHT MICROSCOPE A microscope is intended for studying micro objects in a current of passing light. Light microscopes (figure 3) consist of mechanic, illuminating and optical parts. The mechanic part includes a base, stage, coarse adjustment knob (cremaliera), fine adjustment knob, arm and revolving nosepiece. The support of the microscope consists of an arm (draw-tube holder) and base. The arm carries: – revolving nosepiece — a rotating mechanism for changing objective lenses; – draw-tube — a hollow tube with an ocular lens; – a system of knobs for coarse and y adjustment of the microscope; – a stage for placing an investigation object; The illuminating part includes a lamp or mirror in simple microscopes and condenser.

5 The mirror of the microscope is double-sided and has flat and concave surfaces. The concave surface is used under natural illumination, while a flat one under intensive artificial illumination. The condenser is a lens system collecting light rays into a band. The diameter of the light band can be regulated with a small lever which changes the diaphragm lumen.

Fig. 3. Light microscope: 1 — ocular lens; 2 — draw-tube; 3 — arm (draw-tube holder); 4 — coarse adjustment knob (cremaliera); 5 — fine adjustment knob; 6 — base; 7 — mirror; 8 — condenser; 9 — stage; 10 — revolving nosepiece; 11 — objective lenses; 12 — lens collecting light; 13 — lamp, 14 — adapter;

The optical system consists of one ocular lens (or two in binocular microscopes) and several objective lenses with different magnifications. The ocular lens (oculus — eye) is a lens system directed towards the eye. Its magnification is indicated on the frame. A training microscope has spare ocular lenses with magnification 7×, 10× and 15×. The objective lens is located on the nosepiece at the lower end of the draw- tube. It is a lens system directed to the investigated object. Two kinds of objective lenses are commonly used in a training microscope: with low (8×) and high magnifications (40×). The total magnification of the microscope can be calculated by multiplying the magnifications of the objective and ocular lenses. For example, if the magnification of an objective lens is 40× and that of an ocular lens is 7×, then the total magnification of the microscope is equal to 280×. 6 DIRECTIONS FOR USE OF A MICROSCOPE 1. Put the microscope on a table (at the distance approximately equal to palm width from the edge of the table). Column should be directed towards you and the mirror towards the light source. 2. Turn the coarse adjustment knob to set the objective lens to the level 2–3 cm above the surface of the stage. 3. Turn and set the objective lens with low magnification (8×) towards the aperture of the stage. It should click when fixed properly. 4. Put the condenser to the middle position and open the diaphragm completely. 5. Look at the ocular lens and turn mirror surface to the light source for even illumination of the field of vision. 6. Put a micropreparation on the stage. Its side with the cover glass should be directed towards the objective lens. 7. Look at the stage, but not at the ocular lens, and lower the objective lens (turning the coarse adjustment knob) to the level 0.5 cm above the surface of the micropreparation. 8. Start looking at the ocular lens and turn coarse adjustment knob slowly until clear image of the object appears (the focal distance of the 8× objective lens is ~1 cm). 9. Study the object. Move the micropreparation manually. Note: If the object is too small and is not seen at low magnification, then adjust the microscope to the edge of the cover glass. Having obtained a clear image of the glass surface, move it and search for the object. Directions for work with a high-powered magnification (7 × 40): 1. Move the area of the micropreparation you need to see with high magnification to the center of the field of vision. 2. Turn and set the objective lens with high magnification (40×) instead of the current lens. It should click when fixed properly. 3. Put the condenser to the upper position to increase illumination. Look at the stage, but not at the ocular lens and carefully lower the objective lens (with coarse adjustment knob) until it touches the surface of the cover glass. 4. Looking at the ocular lens and slightly turn the coarse adjustment knob until object’s outlines appear (the focal distance of 40× objective is approximately 1–2 mm). 5. Use the fine adjustment knob for getting better image. 6. Study the needed area of the micropreparation. Terminating the work with the microscope: 1. Having finished studying the object, turn the coarse adjustment knob to raise the objective lens to the level 2–3 cm above the stage and take off the preparation from the stage.

7 2. Turn and set the objective lens with low magnification (8×) towards the aperture of the stage. It should click when fixed properly. 3. Lower the objective lens.

BASIC TERMS AND CONCEPTS Isotopic labeling (autoradiography) — technique based on tracking the passage of a substance labeled with an isotope. Life — functioning of open systems which decrease the internal entropy and are based on work of DNA and proteins. Cell — a membrane-bound structure which is the least structural and functional unit of living matter. X-ray crystallography — a technique used for determining the molecular structure of a crystal based on diffraction of X-rays. Microsurgery of cells — a technique allowing to remove and transplant organelles of the cell. Metabolism — complex of chemical transformations sustaining the life of an organism. Taxonomy of Homo sapiens — position of human being in biological classification: phylum Chordates, subphylum Vertebrates, class Mammals, subclass Placentals, order Primates, suborder Anthropoids, family of Hominids, genus Homo, species Homo sapiens. Cytology — science that deals with structure and functioning of cells.

Topic 2. BIOLOGY OF CELL. FLOW OF SUBSTANCES AND ENERGY IN THE CELL

MODERN CELL THEORY 1. Cell is the least structural, functional and genetic unit of all living things; it is an open self-regulating system and flows of substances, energy and information continuously pass through it. 2. Cells of all organisms have similar structure, chemical composition and processes of vital activity. 3. New cells are formed as result of the mother cell’s division. 4. Cells of a multicellular organism differentiate and form tissues for performing various functions.

DIFFERENTIATING SIGNS OF PRO- AND EUKARYOTIC CELLS (table 2). Any cell has membrane, cytoplasm and genetic apparatus (figure 4). However there are two wide groups of cells — pro- and eukaryotes, and they have numerous differences (figure 5). 8 CELL

Membrane Cytoplasm Nucleus

1. Supramembrane formations 1. Hyialoplasm 1. Nuclear membrane (glycocalyx) 2. Organelles 2. Nuclear sap 2. Plasma membrane 3. Inclusions 3. Chromatin 4. Nucleolus Fig. 4. Diagram of cell structure

C Fig. 5. Structure of prokaryotic and eukaryotic cells: a — prokaryotic cell; b, c — eukaryotic cells: 1 — nucleoid; 2 — plasma membrane; 3 — ribosomes; 4 — mesosome; 5 — cytoplasm; 6 — flagellum; 7 — cell wall; 8 — centrosome; 9 — mitochondria; 10 — rough ER; 11 — nucleolus; 12 — nucleus; 13 — Golgi complex; 14 — smooth ER 9 Table 2 Pro- and eukaryotic cells Prokaryotes Eukaryotes Mycoplasmas, bacteria, cyanobacteria Protozoans, plant and animal cells Sizes: 1–10 µm 10–100 µm There is no nucleus, but nucleoid There is a nucleus DNA is not bound with proteins-histones DNA is bound with proteins-histones There is no mitosis, no membrane-bound There is mitosis and membrane-bound organelles: their functions are performed by organelles mesosomes — ingrowths of the membrane

PLASMA MEMBRANE: STRUCTURE, PROPERTIES AND FUNCTIONS The basic membrane components are lipids. They compose from 20 to 80 % of its mass. They are phospholipids, lecithin and cholesterol. In 1943 H. Davson and J. Danielli proposed the first model of plasma membrane: two layers of lipid molecules are located between two layers of protein molecules. This model got the name sandwich model. Lipid molecules of plasma membrane — phospholipids — have two ends: the hydrophilic (water-soluble) head and the hydrophobic (water insoluble) tail. According to the sandwich model hydrophobic tails of those molecules are directed towards each other, hydrophilic ones — towards proteins (figure 6).

C Fig. 6. Models of plasma membrane: a — sandwich model; b, c — fluid mosaic model: 1 — solid protein layers; 2 — bilipid layer; 3 — hydrophilic heads of phospholipids; 4 — hydrophobic tails of phospholipids; 5 — glycolipid; 6 — glycoprotein; 7 — cholesterol; 8 — semi-integral protein; 9 — integral protein 10 In 1972 S. Singer and G. Nicolson proposed model that explains properties of the plasma membrane better: the fluid mosaic model. According to their concept lipid molecules form a «lipid sea» while protein molecules are embedded into that lipid bilayer. Protein molecules which penetrate both layers of lipid molecules are integral. Those protein molecules which are immersed into only one layer are semi-integral. Peripheral proteins lay on the surface of the membrane. The third component of a plasma membrane is glycoproteins and glycolipids forming glycocalyx on its surface — the receptor apparatus. Properties of the plasma membrane: – plasticity (it restores quickly after impairment and also stretches and constricts in cell movements); – semi-permeability (passes molecules selectively); – ability for self-locking (forms vesicles and vacuoles). Functions of the plasma membrane: – structural (membranes are components of all cell organelles except ribosomes and centrosomes); – barrier (protects the cell from external factors and sustains its composition); – metabolic (many enzymes are located on membranes); receptor (receives signals, recognizes substances). Types of transport of substances through the membrane: Passive transport runs down the concentration gradient (from the area of higher concentration to the area of lower concentration) without spending energy. Water and small molecules can pass into the cell by filtration, diffusion, through pores or dissolve in lipids of the membrane. Facilitated diffusion is a passive transport performed by carrier proteins (permeases). Amino acids, sugar, fatty acids cannot pass the membrane and are transported through it by means of such diffusion. Active transport requires energy because it proceeds against the concentration gradient (from the area of lower concentration to the area of higher concentration). Such transport requires enzymes, ATP molecules and special ion canals. An example of such transport is sodium-potassium pump. Endocytosis and exocytosis are associated with participation of the membrane in catching particles or molecules and transporting them into or from the cell. Plasma membrane surrounds a particle and forms a vesicle. Endocytosis of macromolecules or hard particles is phagocytosis, while transport of fluidsis pinocytosis. In case of exocytosis a vesicle with content joins the membrane of the cell and excrete the substances.

11 ORGANELLES OF THE ANABOLIC SYSTEM OF THE CELL The anabolic system performs reactions of constructive metabolism, or assimilation. Organelles are differentiated areas of the cytoplasm which have constant structure and perform specific functions. Ribosomes (figure 7) are spherical bodies (diameter is 15–35 nm) consisting of two subunits. The function of ribosomes is biosynthesis of proteins — translation. They are in hyaloplasm, on the external membrane of the nucleus, on membranes of rough ER. The large subunit of a ribosome contains three different molecules rRNA and 40 molecules of proteins, small subunit — one rRNA molecule and 33 protein molecules (figure 5). Ribosome subunits Fig. 7. Ribosome: are assembled in the nucleolus. The information 1 — small subunit; 2 — mRNA; about the rRNA structure is contained in 3 — tRNA, 4 — amino acids; 5 — nucleolar-organizer regions (NOR) which are large subunit; 6 — membrane of parts of DNA molecule corresponding to ER; 7 — protein secondary constrictions of satellite chromosomes. The subunits are made separately and unite into a ribosome only for translation. Endoplasmic reticulum (ER) is a system of canals located throughout all the cell (figure 8). It is connected with the perinuclear space of the nucleus and cavities of Golgi complex. Its wall is plasma membrane. ER canals perform compartmentalization of the cytoplasm (division into areas where various biochemical reactions take place). Fig. 8. Granular ER: The granular or rough ER 1 — canal; 2 — ribosomes; 3 — membrane (ribosomes are placed on its membranes) participate in biosynthesis of proteins, which are later transported to the Golgi complex. Carbohydrates and lipids are synthesized on membranes of the smooth ER (which does not contain ribosomes). It takes part in synthesis of steroid hormones, detoxication of toxic substances (in liver cells). Golgi complex consists of vesicles, tubules, sacs. Dictyosomes are basic elements of the complex. 12 Dictyosomes are piles of closed sacs composed of 10–15 plasma membranes that have widenings on the ends. These widenings form vesicles that separate and transform into lysosomes and vacuoles (figure 9). A number of these vesicles excrete secretes and metabolites from the cell. Functions of Golgi complex: – sorting and packing substances synthesized in ER; – synthesis of complex compounds (lipoproteins, glycoproteins); – assembling plasma membranes; – forming lysosomes, glyoxysomes and vacuoles; – taking part in substance secretion.

Fig. 9. Structure of Golgi complex

The catabolic system of the cell. The catabolic system performs energy exchange or dissimilation. Primary lysosomes are formed in Golgi complex. They are spheroidal bodies (0.2–2 µm in diameter) covered with a plasma membrane. They contain approximately 50 different hydrolytic enzymes. Secondary lysosomes (phagolysosomes) contain digested substances. Functions of lysosomes: – breaking up substances passed into the cell by phagocytosis; – destroying impaired structures and organelles of the cell. Peroxisomes are formed in ER. Their enzymes (oxidazes) oxidize amino acids with formation of peroxide (H2O2). Glyoxysomes are formed in Golgi complex, their enzymes transform fats into carbohydrates. Mitochondria have shapes of rods, filaments and granules. The sizes of mitochondria are from 0.5 to 7 µm (figure 10). Their number is not same in cells with different activity. Wall of mitochondrion consists of external and internal 13 membranes. Ingrowths of the internal membrane form cristae (singular — crista). There is the matrix containing enzyme systems of an aerobic stage of energy exchange and an autonomous system of protein biosynthesis (circular DNA, RNA and ribosomes) under the inner membrane. The interspace between the membranes of mitochondrion is perimitochondrial space. Functions of mitochondria: – ATP synthesis; – Synthesis of specific proteins and participating in synthesis of steroid hormones.

Fig. 10. Mitochondrion: 1 — ribosome; 2 — matrix; 3 — cristae; 4 — internal membrane; 5 — external membrane

ENERGY EXCHANGE IN THE CELL. ENZYME SYSTEMS OF MITOCHONDRIA Energy exchange is the sum of all enzymatic reactions which break down complex organic compounds and release energy used for ATP synthesis. The preparatory stage proceeds in the digestive system and in phagosomes of cells where breaking up of complex organic compounds into simpler ones occurs. Polysaccharides are split into monosaccharides, proteins into amino acids, fats into glycerol and fatty acids. All the released energy dissipates as warmth. The anaerobic (anoxic) stage or glycolysis occurs in the cytoplasm of cells. It is a chain of reaction catalyzed with ten enzymes. Glucose is broken down into 2 molecules of pyruvic acid and two ATP molecules are formed. The pyruvic acid passes into mitochondria for further transformations. Aerobic stage of energy exchange occurs in mitochondria. There are 3 basic enzyme systems in mitochondria: – enzymes of Krebs cycle (citric acid cycle) in the internal matrix; – enzymes of tissue respiration on the internal membrane; – enzymes of oxidative phosphorilation on ATP-somes (mushroom-shaped protein complexes of the inner membrane ). Pyruvic acid comes into the matrix of mitochondria and interacts with coenzyme A (CoA) to form acetyl CoA (an activated form of acetic acid). + During the citric acid cycle, CO2 and H are chipped off from acetyl CoA.

14 + CO2 is released from mitochondria, H and electrons pass through the enzyme system of tissue respiration. Protons accumulate on the external surface of the internal membrane and electrons on the internal one. Having reached a critical potential (200 mv), protons pass through canals of ATP-somes. Electrons give the energy for adding the residue of phosphoric acid to ADP (ATP synthesis) and join protons. Hydrogen atoms are formed; they interact with oxygen and form water molecules. All reactions of energy exchange in the cells of brain and skeletal muscles form 30 mol of ATP from 1 mol of glucose. Cells of cardiac muscle, liver, kidneys and other organs form 32 mol of ATP from 1 mol of glucose.

CONNECTION BETWEEN FLOWS OF SUBSTANCES AND ENERGY IN THE CELL Flows of substances and energy are closely related. Substances are source of energy in the cell and energy can be used for synthesis of other substances from organic and inorganic material. This integrity of substance and energy is shown in the figure 11.

Fig. 11. Integrity of energy and substance passing through the cell 15 BASIC TERMS AND CONCEPTS Glycocalyx — receptor apparatus on membranes of animal cells. Glycolysis — process of breaking down glucose without oxygen. Concentration gradient — the difference of substance concentrations. Enzymes of oxidative phosphorylation — are enzymes of mitochondria located on ATP-somes. Enzymes of tissue respiration — enzymes of mitochondria located in cristae. Enzymes of Krebs cycle — enzymes of mitochondria located in the matrix. Dictyosome — system of flat disc-like cisterns formeb by the membrane of the Golgi complex. Mesosomes — ingrowths of plasma membrane which perform a role of membrane organelles in prokaryotic cells. Nucleoid — a genetic apparatus of prokaryotes. Plasma membrane — bilipid layer with proteins and carbohydrates covering the cell. Peroxisomes — organelles, where oxidation of amino acids occurs and hydrogen peroxide is formed.

Topic 3. FLOW OF GENETIC INFORMATION IN THE CELL

STRUCTURE AND FUNCTIONS OF NUCLEUS Basic genetic information of the cell is in the nucleus. The nucleus (Latin — nucleus; Greek — karyon) was described in 1831 by R. Brown. The shape of the nucleus depends on the shape and functions of the cell. The covering of an interphase nucleus (karyolemma) consists of external and internal membranes. Perinuclear space is between those membranes (figure 12). There are openings in membranes (pores) with protein molecules forming pore complexes. When the cell is active, the majority of pores are open. The substance flow Fig. 12. Diagram of nucleus: passes through them from the cytoplasm into 1 — internal membrane; 2 — the nucleus and back. external membrane; 3 — pore; The number of pores in one nucleus reaches 4 — nucleoli; 5 — chromatin; 3–4 thousands. The external nuclear membrane is 6 — nuclear sap connected with canals of endoplasmic reticulum and carries ribosomes on the surface. Proteins of the internal nuclear membrane form a nuclear lamina. It sustains a constant shape of the nucleus and anchors the chromosomes (they are attached to it). 16 Nuclear sap or karyolymph, is colloid jelly-like solution that contains proteins, lipids, carbohydrates, RNA, nucleotides and enzymes. Nucleolus is a temporary component of the nucleus: it disappears in the beginning of cell division and restores in its end. Chemical composition of nucleolus: protein (~90 %), rRNA (~6 %), lipids, enzymes. Nucleoli form in the sites of secondary constrictions of satellite chromosomes. Their function is assembling ribosome subunits. Chromatin of the nucleus is chromosomes during the interphase. It contains DNA, proteins-histones and RNA in ratio 1:1.3:0.2. DNA together with proteins forms deoxiribonucleoprotein (DNP). DNP condenses to form chromosomes during mitosis. Functions of the nucleus: a) storage of hereditary information; b) taking part in division; c) regualtion of cell work.

TYPES OF CHROMOSOMES. STRUCTURE OF CHROMOSOMES. RULES OF CHROMOSOMES Chromosomes (Greek — chromo — color, soma — body) are condensed chromatin. The length of chromosomes is 0.2–5.0 µm, diameter — 0.2–2.0 µm. A metaphase chromosome consists of 2 chromatids linked in the area of a centromere (primary constriction). It divides the chromosome into 2 arms. Some chromosomes have secondary constrictions. The area they separate is a satellite, and such chromosomes are called satellite chromosomes. Terminal areas of chromosomes are telomeres (figure 13). Each chromatid includes one DNA molecule bound with proteins-histones. Chromosome areas with intense staining are areas of strong condensation (heterochromatin). Lighter areas are areas of weak condensation (euchromatin).

Fig. 13. Diagram of chromosome

17 Types of chromosomes according to the centromere position (figure 14): 1. Metacentric — the centromere is in the middle of the chromosome; arms are of approximately same length. 2. Submetacentric — the centromere is not in the center but not far from it; the arms are of different length. 3. Acrocentric — the centromere is situated far from the center; one arm is very short, and the other is very long.

Fig. 14. Types of chromosomes

Some cells of salivary glands of many insects (Drosophila) have giant polytene chromosomes containing multiply copied genetic material. There are four rules for chromosomes of all organisms: 1. The rule of the constant number of chromosomes. Every cell (except some specialized cells) have the certain and constant number of chromosomes typical for the species: human — 46, dog — 78, Drosophila — 8 and etc. 2. Parity of chromosomes. Every chromosome in a diploid set has a pair — a homologous chromosome with identical shape and size and allelic genes. 3. Individuality of chromosomes. Chromosomes of different pairs differ in shape, structure and size and contain different alleles. 4. Continuity of chromosomes. When genetic material is doubled, a chromosome originates from a chromosome. Function of chromosomes is storing, reproduction and transmission of genetic information, when cells and organisms multiply.

KARYOTYPE AND IDIOGRAM. CLASSIFICATION OF HUMAN CHROMOSOMES Karyotype (figure 15) is a diploid complement of chromosomes in a somatic cell which is characteristic of the species. Human karyotype includes 46 chromosomes. Pairs of chromosomes which are same in males and females and not responsible for sex determination are autosomes. There are 22 pairs of such chromosomes. The 23rd pair of chromosomes is different in males and females and determines the sex of the organism. These chromosomes are heterochromosomes or sex chromosomes. Men have Х and Y chromosomes, women — Х and Х. 18

Fig. 15. Human karyotype

An idiogram is a photograph or diagram of chromosomes arranged by their size. It is an ordered karyotype where chromosomes are shown as pairs. In 1960 year Denver System of Classification of Chromosomes was proposed. Human chromosomes are systematized according to their size, shape, position of the centromere, presence of satellites. It takes into consideration the centromere index (CI). It is the ratio of the length of the short chromosome’s arm to the total length of the chromosome. Denver Classification divides all the chromosomes into 7 groups (figure 16): 1. Group А: 1st–3rd pairs. Big metacentric and submetacentric chromosomes. CI is 38–49 %. 2. Group В: 4th and 5th pairs. Big metacentric chromosomes. CI is 24–30 %. 3. Group С: 6th–12th pairs and X-chromosome: submetacentric chromosomes of moderate size. CI is 27–35 %. 4. Group D: 13th–15th pairs. Acrocentric chromosomes. CI is about15 %. 5. Group Е: 16th–18th pairs. Quite small metacentric and submetacentric chromosomes. CI is 26–40 %. 6. Group F: 19th–20th pairs. Small submetacentric chromosomes. CI is 36–46 %. 7. Group G: 21st–22nd pairs and Y-chromosome. Small acrocentric chromosomes. CI is 13–33 %. The Paris Nomenclature of human chromosomes was elaborated in 1971. Chromosomes are processed with certain dyes which reveal dark and bright bands. These bands are named by the type of staining: Q-bands are stained by acrichine yperite; G-bands — Giemsa stain; R-bands are stained after heat denaturation and etc. The nomenclature indicates short arm of a chromosome as p and long arm as q. Each arm is divided into regions which are numbered from centromere to telomere. Bands in each region are numbered in the same direction. This allows indicating location of a certain gene. For example, esterase D is situated in the 4th band of the 1st region in the short arm of the 13th chromosome — 13p14. 19

Fig. 16. Denver classification of human chromosomes

MITOTIC AND CELL CYCLES. INTERPHASE. CAUSES OF MITOSIS Life cycle of cells is called cell cycle (figure 17).

Fig. 17. Cell cycle

Cell cycle or life cycle of the cell is a period from the appearance of the cell (from division) until its death or the end of next cell division. The life cycle of somatic cells includes growth and differentiation, performing specific functions, preparation for division and the division itself. The majority of cells 20 can undergo mitotic cycle. It includes a period of preparation for division (interphase) and the division itself (mitosis). The interphase includes three periods: G1 — pre-synthetic period (post- mitotic), S — synthetic period and G2 — post-synthetic period (pre-mitotic). The content of genetic material in the cell changes during the interphase. Keys used to denote it are: «n» is a set of chromosomes, «chr» is the number of chromatids in each chromosome, «c» is the number of DNA copies in each pair of chromosomes. Pre-synthetic period. During this period, the cell grows, performs its functions, accumulates RNA, proteins, DNA nucleotides, ATP. The period may last 12 hours or sometimes take several months. The content of genetic material is 2n1chr2c. During the synthetic period, replication of DNA molecules occurs: each chromatid adds one more identical to itself. The content of genetic material becomes 2n2chr4c. Centrioles duplicate. RNA, ATP and proteins-histones are synthesized. The cell continues performing its functions. The duration of the period is up to 8 hours. During the post-synthetic period energy of ATP accumulates; RNA, nuclear proteins and proteins-tubulins necessary for chromatin division spindle are actively synthesized. The content of genetic material does not change: 2n2chr4c. By the end of the period all synthetic processes become slower and the cytoplasm viscosity changes. Causes of mitosis: 1 1 1 1 – changing of the nuclear-cytoplasmic ratio from /6– /7 to /69– /89; – presence of «mitogenetic rays» which stimulate division of adjacent cells; – action of «wound hormones» released from impaired cells to stimulate division of unimpaired cells.

REGULATORS OF THE CELL CYCLE: CYCLINS AND CYCLIN-DEPENDENT KINASES There are checkpoints in the cell cycle. Their work is based on interaction of proteins cyclins (figure 18) and cyclin-dependent kinases which rule the proliferation. Cyclin-dependent kinases are constantly present in the cell, but their concentration changes at different stages of the cell cycle. They are not active and can be activated by cyclins (proteins). Concentration of the cyclins changes and corresponds to the stage of the cell cycle. There are cyclins А, B, D, and E which can bind the cyclin-dependent kinases when reach their critical concentration. Maximal concentration of the cyclin D is observed by the end of the presynthetic period and begins decreasing in S-period. The complex cyclin D-cyclin-dependent kinase runs DNA replication. Decrease of the cyclin D concentration comes with increase of cyclin E concentration which forms a complex cyclin E-cyclin-dependent kinase and

21 stimulated replication directly. The trigger which begins G2 period is activation by the cyclin A.

Fig. 18. Changes in the concentration level of cyclins during the cell cycle

The maximal concentration of the cyclin B is observed during metaphase. The complex cyclin B-cyclin-dependent kinase initiate fragmentation of the karyolemma, formation of the spindle apparatus and rearrangement of the cytoskeleton for the cytokinesis. Another factors having effect on the cell cycle are volume of substrate for growing, presence of free space, substances secreted by another cells.

COMPARISON OF MITOSIS AND MEIOSIS: CONTENT OF GENETIC MATERIAL DURING DIFFERENT STAGES OF DIVISION The basic way of cell division is mitosis. Mitosis has four basic stages: prophase, metaphase, anaphase and telophase (figure 19).

1 2 3 4 5 Fig. 19. Mitosis in a plant cell: 1 — interphase; 2 — prophase; 3 — metaphase; 4 — anaphase; 5 — telophase

The prophase starts with condensation of chromatin. Long chromatin fibers are shortened and thickened to form chromosomes. Centrioles move to cell poles and filaments of the division spindle are formed. The volume of the nucleus increases. Nucleoli and nuclear membrane disappear. The content of genetic material is 2n2chr4c. 22 During the metaphase, chromosomes are located at the equator of the cell forming metaphase plate. Microtubules of the division spindle are attached to the kinetochores at the centromere regions of chromosomes. It is clearly visible that each chromosome consists of two chromatids. The content of genetic material does not change — 2n2chr4c. During the anaphase, microtubules of the spindle apparatus provide separation of chromatids. These chromatids are now daughter chromosomes. They are pulled apart and move to the poles of the cell. The content of genetic information at each half of the cell is 2n1chr2c. During the telophase, formation of nuclei occurs: nuclear membranes are restored, chromosomes decondense and lose their clear outlines and nucleoli appear. Now, when genetic material is divided, cytokinesis (division of the cytoplasm) occurs — the final stage of the cell division. Two cells are formed with the content of genetic material 2n1chr2c. The significance of mitosis: – sustaining the constancy of the chromosome number, providing genetic succession in cellular populations; – equal distribution of chromosomes and genetic information between daughter cells. Meiosis is a variety of mitosis. Meiosis is division of somatic cells of gonads (gametocytes) which leads to formation of gametes. Meiosis consists of two divisions — meiosis I and meiosis II. Each division has four phases: prophase I and prophase II, metaphase I and metaphase II, anaphase I and anaphase II, telophase I and telophase II (figure 20). The prophase of meiosis I is the most complicated. It has 5 stages: 1. Leptotene: chromatin condenses forming thin chromatin filaments that start moving to each other with centromere regions; complement of genetic material is 2n2chr4c. 2. Zygotene: chromosomal synapsis starts. Homologous chromosomes stick to each other; complement of genetic information does not change — 2n2chr4c. 3. Pachytene: homologous chromosomes are tightly joined along the whole length; bivalents of chromosomes or tetrads of chromatids are formed; complement of genetic material is lnbiv4chr4c; by the end of the stage antagonizing forces start acting in the area of centromeres and crossing-over occurs (homologous recombination). 4. Diplotene: antagonizing forces act, but chromosomes are connected at the regions of their crossings which are called chiasmata; the complement of genetic material is same — lnbiv4chr4c; 5. Diakinesis: chromosomal condensation is finished, the nuclear membrane and nucleolus disappear; bivalents of chromosomes linked at their ends come into the cytoplasm and move to the center of the cell; microtubules of

23 the division spindle attach to kinetochores — protein structures in the regions of centromeres; Complement of genetic material is lnbiv4chr4c.

Fig. 20. The diagram of meiosis

During the metaphase of meiosis I, bivalents are located along the equator of the cell; chromosomes are clearly seen; complement of genetic material — lnbiv4chr4c. Anaphase I: bivalents separate into homologous chromosomes. Microtubules of the spindle apparatus pull the chromosomes to cell poles. Each chromosome is still composed of 2 chromatids. The complement of genetic material at each pole of the cell is ln2chr2c. During this phase the reduction (decrease) of the chromosome number occurs and this division is called reductonal. Diploid complement of chromosomes was reduced into haploid one. During the telophase of meiosis I, cytokinesis takes place and two- daughter haploid cells are formed — ln2chr2c. Decondensation of chromosomes does not occur. Interkinesis follows the meiosis I. It is a short interval between divisions of meiosis. DNA replication does not occur. Meiosis II starts after interkinesis. Meiosis II almost does not differ from mitosis. During prophase II (ln2chr2c), condensation of chromatin does not occur, and in anaphase II chromatids (but not chromosomes!) are pulled to the poles of the cell. Each daughter cell gets a complement of genetic information 1n1chr1c. 24 During meiosis one mother diploid cell forms 4 haploid cells (gametes). The significance of meiosis: it is a mechanism of gamete formation; it sustains the constancy of the number of chromosomes; provides combinative variation.

BASIC TERMS AND CONCEPTS Bivalents — two homologous chromosomes, connected to one another during the prophase of meiosis I. Karyolymph (nucleoplasm) — the viscous liquid within the nucleus. Cell cycle — a period from the appearance of the cell to its death or to the end of next division. Synapsis — connection of homologous chromosomes during prophase of meiosis I leading to formation of a bivalent. Meiosis — division of specialized somatic cells of gonads (gametocytes) resulting in formation of gametes. Mitotic cycle — period of cell’s life which includes preparation for division (interphase) and the division itself (mitosis). Telomeres — terminal parts of chromosome arms. Cemtromere index (CI) — length of the short chromosome arm divided by the entire length of the chromosome and expressed as percentage. Chiasmata — crossings of chromatids of homologous chromosomes observed during synapsis. Chromatin — a complex of DNA and histone proteins in the nucleus of the cell. Nuclear-cytoplasmic ratio — is a physiologically and morphologically regular ratio of the volume of the nucleus to the volume of the cytoplasm.

Topic 4. ARRANGEMENT OF HEREDITARY MATERIAL (PART 1)

LEVELS OF DNA CONDENSATION DNA is linked with histone and non-histone proteins and forms nuclear- protein fibers (DNP). Total length of such fibers in a human cell is close to 2 meters while length of a metaphase chromosome is approximately 150 µm. This is possible due to condensation of genetic material at four levels (figure 21). Nucleosomal level. A nucleosome is a globule containing per 2 histone molecules of each type: H2A, H2B, H3, H4. DNA wraps around such octamere almost 2 turns (146 pairs of nucleotides). The diameter of such nucleosomal thread is 10–13 nm. The DNA becomes 5–7 times shorter. This level is characteristic of the interphase.

25 Supernucleosomal level. The nucleosomes are connected by histone H1 and coil into «solenoid» diameter of which is approximately 25 nm. One turn of such spiral contains 6–10 nucleosomes. DNA shortens 6-fold more. The supernucleosomal level can be observed during interphase and in mitosis. Chromatid level. Supernucleosome spiral bends forms loops supported by DNA-binding (SAR) proteins, comprising basis of a chromatid. The diameter of the loops is 50 nm. DNP fiber is 10–20 shorten. Such condensation is typicalfor prophase of mitosis. Level of a metaphase chromosome. Chromatid fiber coils into spiral and forms regions of euchromatin (with lower condensation) and heterochromatin (dense, with higher condensation); genetic material shortens 20 times. The length of human chromosomes is 2–11 μm, diameter is 0.2–5.0 μm. Due to all these mechanisms DNA shortens 10 000 times. Fig. 21. Four package levels of DNP STRUCTURAL-FUNCTIONAL LEVELS OF GENETIC MATERIAL (GENE, CHROMOSOME, GENOME LEVELS) The structural unit at the gene level of genetic material is gene. As genes are separate units, they can be inherited independently of one another (3rd Mendel’s law) and independent changes (mutations which lead to changes of the characters) in the genes are possible. Genes of eukaryotes are in chromosomes — chromosome level of genetic material. The genes of each chromosome (pair of homologous chromosomes) comprise linkage groups and are inherited together. At this level, linkage and recombination (combining the parental genes during sexual reproduction due to random segregation of chromatids and crossing over) of genes occur. All the genes of the organism are functionally integrated and comprise a system — genotype (or genome). The same gene can have different effect in different genotypes. The genome level explains intra- and inter-allelic interactions of genes of a chromosome or even several chromosomes.

26 PROPERTIES OF GENES. PRIMARY FUNCTIONS OF GENES: AUTOSYNTHETIC (REPLICATION) AND HETEROSYNTHETIC (PROTEIN BIOSYNTHESIS) The gene is a part of a DNA molecule coding for a definite polypeptide. Genes are characterized by the following properties: 1. Specificity — each gene has its own unique sequence of nucleotides. 2. Integrity — being considered as a functional unit, the gene is indivisible (entire gene is unit of protein synthesis). 3. Discretion — genes have subunits such as a muton (subunit responsible for mutations), recon (responsible for recombination). Their minimal length is equal to a pair of nucleotides. 4. Stability — genes are relatively stable. The frequency of spontaneous mutations of a gene is approximately 10–5 per a generation of cells. 5. Lability — genes can to modify — mutate. 6. Pleiotropy — multiple gene action (one gene is responsible for several characters of the organism). 7. Expressivity is the degree of phenotypic manifestation of the gene. It depends on environmental factors and effect of other genes. 8. Penetrance is frequency of gene manifestation. It quotient of the number of individuals having the character divided by the number of individuals having the gene. Genes have 2 essential functions. Heterosynthetic function is protein biosynthesis. Autosynthetic function is replication of DNA. Replication of DNA (its duplication) occurs during the synthetic period of interphase. Replication is semi-conservative: strands of DNA are separated and serve as matrixes for newly created complementary strands; consequently, each new DNA consists of old and new (mother and daughter) strands. The enzyme which assembles new strand is DNA-polymerase. Replication begins at many points of DNA. A segment from beginning of one replication to the other one is replicon. Eukaryotic chromosomes have many replicons where replication occurs simultaneously while bacterial nucleoid is one replicon.. During replication, DNA double helix is uncoiled and separated by topoisomerase and helicase. This site is called replication fork (figure 22). DNA-polymerase can move along the matrix only in one direction: 3' → 5'. As DNA strands are antiparallel, polymerases should move in opposite directions. One DNA-polymerase (at leading strand) follows helicase and assembles new strand continuously while DNA-polymerase at the other one (lagging or template strand) moves backwards and synthesize short (150–200 nucleotides) fragments (Okazaki fragments). Bonds between them are formed by an enzyme DNA-ligase. The entire genome is replicated once a cell cycle.

27

Fig. 22. DNA replication

Genetic code and its properties. Protein biosynthesis. System of rules («language») by which genetic information is encoded as a sequence of nucleotides is a genetic code. A nucleotide triplet (3 nucleotides) coding for a certain amino acid is codon. Codon is the functional unit of genes. Properties of the genetic code are: – tripletness — one amino-acid is encoded by one triplet (three nucleotides); such triplet is called codon; – universality — certain codon codes for the same amino acid in all living organisms; – no overlapping — codons are read one by one and one nucleotide cannot be read twice as part of two adjacent triplets; – redundancy — several different triplets can code for the same amino acid (there are 64 triplets for 20 amino acids); – continuity — there are no disjunctive symbols between codons; – unidirectionality (absence of feedback) — enzymes reading the code can move along the matrix inly in one direction (from 3’to 5’end in DNA); – presence of codons-terminators which determine the end of protein biosynthesis. The correspondence of the nucleotide order in a DNA molecule to the order of amino acids in the polypeptide molecule is co-linearity. Protein biosynthesis is an enzymatic process, where nucleic acids play the main role. It begins from synthesis of mRNA in the nucleus of the cell. RNA- polymerase «reads» the nucleotides of the antisense strand of a gene and assembles complementary mRNA. This process is transcription. The mRNA is then transported through the pores of the nucleus to the cytoplasm where ribosomes are situated. 28 Another RNA participating in protein synthesis is tRNA which is carrier of amino acids. A tRNA molecule has 5 arms. One arm carries amino acid (amino acid arm). Each tRNA can bind only one type of amino acid. At the opposite side of tRNA is anticodon arm. The anticodon is complementary to an mRNA codon coding for the amino acid of this tRNA. Connection of amino acid to its tRNA is recognition. It occurs in the cytoplasm. This complex of amino acid bound to its tRNA is called aminoacyl tRNA, It is made by the enzyme aminoacyl tRNA synthetase with participation of ATP. The process occurring in ribosomes is translation: the information of nucleotide sequence is used to create amino acid sequence — a protein. The first step of this process is initiation: the mRNA, first aminoacyl tRNA, small and large ribosomal subunits unite (the aminoacyl tRNA is bound with the start-codon AUG of the mRNA). The large subunit of the ribosome has 2 active centers: aminoacyl and peptidyl sites (A- and P-sites). The A-site is empty at this stage while P-site contains the first aminoacyl tRNA. The second step of translation in which amino acids are connected with peptide bonds is elongation (figure 23). New aminoacyl tRNA comes to the A-site. If its anticodon matches to the mRNA codon, temporary hydrogen bonds are formed between them. Enzymes of the ribosome disconnect the first amino acid (of the first aminoacyl tRNA which is in the P-site) from its tRNA and connect to the amino acid of the second aminoacyl tRNA (which had just come to the A-site). The mRNA is pulled through the ribosome and the second aminoacyl tRNA carrying both amino acids pass from A- to P-site, the first tRNA without amino acid leaves the ribosome. And now the A-site is empty again while P-site contains aminoacyl tRNA.

Fig. 23. Elongation

Next aminoacyl tRNA comes to the A-site and these steps repeat until a stop-codon is reached. 29 The third step is termination. When a stop-codon (UAA, UGA or UAG) enter the ribosome termination proteins separate the polypeptide from the last tRNA, the tRNA from the P-site, ribosomal subunits from each other.

THE CENTRAL DOGMA OF MOLECULAR BIOLOGY In 1958 F. Krik formulated the central dogma of Molecular Biology. According to the dogma, the genetic information is transmitted from DNA to DNA (during replication), from DNA to mRNA (during transcription) and back (reverse transcription) from mRNA to protein (translation). Schematic view of the dogma: ↻ DNA ⇆ RNA → protein. Transmission of information from protein to nucleic acids is not known.

BASIC TERMS AND CONCEPTS Gene — a segment of DNA coding for a certain polypeptide (or RNA). Initiation — the first stage of translation. Revertase — the enzyme performing reverse transcription. Recon — unit of recombination equal to one nucleotide pair. Supernucleosome — the second level of DNA condensation. Stability of gene — ability of gene to maintain its structure. Termination — ending of the protein synthesis. Transcription — creation of mRNA on DNA matrix in the nucleus. Cistron — gene as a functional unit responsible for protein synthesis. Elongation — the second step of translation which begins from formation of the first peptide bond and finishes with connection of the last amino acid to ate polypeptide.

Topic 5. ARRANGEMENT OF HEREDITARY MATERIAL (PART 2)

CLASSIFICATION OF GENES (STRUCTURAL AND FUNCTIONAL, UNIQUE, REPEATED SEQUENCES, TRANSPOSONS) Classification of nucleotide sequences: 1. Unique sequences are solitary sequences in the genome. They are in structural genes and carry information about the structure of polypeptides. They compose approximately 56 % of the human genome. 2. Repeated sequences are nucleotide sequences repeated ten, hundred, million times play functional role: they are promoters, they regulate DNA replication, participate in crossing-over, participate in separation of exons and introns of transcriptons. 3. Transposons or jumping genes are mobile genetic elements able to move within the genome. 30 Classification of genes according to their function: 1. Structural genes carry information about structural proteins, enzymes, histones and nucleotide sequences in various types of RNA. 2. Functional genes such as genes-modulators and genes-regulators have effect on the work of structural genes. Genes-modulators are inhibitors, intensifiers, modifiers. They enhance, weaken or modify functioning of structural genes. Regulatory genes and operators rule the work of structural genes. According to the area of action: 1. Functioning in all cells such as genes coding enzymes of energy exchange. 2. Functioning in cells of one tissue. For example genes coding for myosin in muscle tissue. 3. Specific for only one type of cells. For example genes of hemoglobin in immature erythrocytes.

REGULATION OF TRANSCRIPTION IN PROKARYOTES (F. JACOB, J. MONOD) AND EUKARYOTES (G. P. GEORGIEV) Regulation of gene work in prokaryotes was described in 1961 by Andre Michel Lwoff, François Jacob and Jacques Lucien Monod. The transcription unit of prokaryotes is operon which has several structural genes ruled by a single operator (figure 24). DNA is shown as a straight line with functional segments: – promoter is a nucleotide sequence that RNA-polymerase recognizes and binds to; – operator is the place where the work of operon is switched off by binding with repressor protein; – structural genes (А, В, С) — code for proteins; – terminator of transcription initiate separation of RNA-polymerase from the DNA. The structural genes are not always active. Their work is ruled by a regulatory gene situated at a distance from the operon. This gene is active and codes for protein repressor. The repressor can bind the operator of the operon and inactivate it. The structural genes code for enzymes breaking certain substance (for example an enzyme lactase breaks sugar lactose). Therefore, such substance should induce production of these enzymes and activate operon. Such substance is inductor. Each operon has its own specific inductor (lactose is inductor of a lac-operon which codes for lactase). It is able to block the repressor and, consequently, release the operator. Then transcription occurs, and proteins encoded in the operon are created.

31 Operon

Regulatory Operator gene Promoter Structural genes Terminator

А В С

mRNA RNA-polymerase The operon is not active

Repressor Repressor binds the operator There is no inductor

Operon

Regulatory Operator gene Promoter Structural genes Terminator

А В С Transcription RNA- mRNA polymerase

Enzymes break Repressor Repressor is blocked the inductor with inductor The operon is active

Inductor Fig. 24. Activation and deactivation of an operon (in prokaryotes)

The created enzymes work until all the inductor is broken. As soon as nothing can block the repressor protein, it binds the operator again and switches the operon off. Therefore enzymes are not produced if they have nothing to process. Regulation of transcription in eukaryotes (G. P. Georgiev). In 1972 G. P. Georgiev proposed the scheme explaining the regulation of transcription in eukaryotes. The principle of its work is the same as this of the operon though its mechanisms are more complicated (figure 25). A transcription unit in eukaryotes is a transcripton. It consists of non- informative and an informative zones. The non-informative or regulatory zone includes promoter and several operators. The informative zone contains one structural gene consisting of exons (informative fragments) and introns (non- informative fragments). There is a terminator of transcription in the end of the structural gene. The work of a transcripton is ruled by several regulatory genes. They code for repressors which are able to block operators. Just as in the operon, the structural gene is transcribed if inductors get into the cell. Inductors can have complex structure (hormones). Due to inductors, operators 32 get rid of repressors and RNA-copy of the structural gene is created — pre mRNA. It contains nucleotide sequence of all the transcripton including informative and non-informative parts. Transcripton

Non-informative region Informative region

Regulatory Structural gene genes Promoter Operators Exons Introns

RNA- Terminator polymerase pre-mRNA

Repressors Procesing of pre-mRNA: Cutting out introns (loops) Splicing (connection) of exons into mRNA

mRNA

Enzyme

Inductors Fig. 25. Transcription in eukaryotes

Endo- and exonucleases of participate in processing of pre-mRNA. It includes destruction of non-informative parts cutting out introns and splicing of exons, adding poly(A)tail to 3’ end and cap to the 5’ end (figure 26).

Fig. 26. An mRNA of eukaryote

Exons code for different domains of proteins. They can be spliced in different order, so different mRNAs can be assembled and different proteins created from one gene. This is alternative splicing. After processing the mRNA is transported to cytoplasm where ribosomes produce protein it codes for. When the work of the protein results in splitting of the inductors, repressors block operators again and stop transcription. 33 CYTOPLASMIC INHERITANCE The basic genetic information of any organism is contained in the nucleus. However mitochondria and plastids contain genetic material (cytogenes) which can be inherited as well. Apart from them, cytoplasm of the cell can contain foreign DNA of viruses and bacteria. Criteria for revealing cytoplasmic inheritance: – inheritance by mother line — through the cytoplasm of an egg or ovum; – absence of expected segregation (according to Mendel’s laws) in descendants; – impossibility to reveal linkage groups; – different results of recurrent crossing (in case of nuclear inheritance they are identical). Mitochondrial inheritance was described by Boris Ephrussi in 1949. He discovered that about 1 % of yeast colonies are dwarf. They grow very slowly, because of mutation in mitochondrial cytogenes which resulted in absnce of respiratory enzymes. There are some human diseases that are caused by mutations of mitochondrial genes (mitochondrial cytopathy, incomplete closing of the backbone and membranes around the spinal cord — spina bifida, senile dementia, Leber disease (atrophy of an optic nerve), anencephaly (absence of the brain) and others. Plastid inheritance (K. Korrens, 1908). The plant Mirabilis jalapa (the four o'clock) has ―molted‖ leaves — with white spots. A mutation occurred in the DNA of plastids and cells lost the ability to synthesize chlorophyll. Plastids are distributed unevenly during the cell division. Some cells get normal plastids and have green leaves. The others get plastids without chlorophyll — leaves are white and the plant dies. The third case — a plant gets both kinds of plastids and have ―molted‖ leaves. Pseudocytoplasmic inheritance is associated with getting a viral or bacterial DNA into the cell. Some mice are predisposed to tumors of mammary glands. If normal little mice were fed by a cancer-predisposed female, these mice would have tumors of the mammary gland. And vice versa: if little mice born from cancer-predisposed female are fed by a healthy mouse, all the little mice would stay healthy. The causative agent of the tumor is a virus transmitted with milk.

BASIC TERMS AND CONCEPTS Operator — functional element of operon that the repressor attaches to. Inductor — substance that binds a certain protein-repressor. Intron — non-informative fragment of structural genes in eukaryotes. Operon — a transcription unit of prokaryotes. Promoter — a site of an operon recognized by RNA-polymerase. RNA processing — formation of mRNA including removal non- informative parts, splicing of exons, capping and polyadenylation. 34 Pseudocytoplasmic inheritance — inheritance of traits caused by foreign DNA present in the cell. Repressor — protein encoded by a regulatory gene for blocking the operator. Splicing — reactions that combine and bind fragments of pre-mRNA to form the mRNA. Transcripton — a transcription unit of eukaryotes. Transposon — mobile genetic elements able to move within the genome.. Exon — informative part of structural genes of eukaryotes.

Topic 6. GENETIC ENGINEERING

GENETIC ENGINEERING AS A SCIENCE Genetic engineering is the direct manipulation of an organism's genome with biotechnological techniques. It is also called genetic modification. Purpose of genetic engineering is designing genetic structures according to a plan. In another words it is creation of organisms with a new genetic program by translocation of genetic information from one organism to the other. Stages of genetic modification: I. Obtaining required genetic material II. Insertion of the obtained DNA into a vector DNA molecule. III. Incorporation of the recombinant DNA into a recipient cell. IV. Selection of cell clones which have functional required DNA.

OBTAINING GENETIC MATERIAL: TECHNIQUES. RESTRICTION ENDONUCLEASES 1. Artificial gene synthesis. If the gene is sequenced (its nucleotide sequence is decoded and known), then nucleotides can be assembled into a gene artificially. Short (8–16 nucleotides) single-strand oligonucleotide sequences are synthesized in a laboratory. They can be linked by ligases and treated with high temperature for the formation of double-strand DNA. Another technique allows many sequences to connect into a molecule with hydrogen bonds at their complementary regions (there must be properly selected sequences). The «gaps» between them are filled by polymerase chain reaction (figure 27). 2. Reverse transcription. A complementary DNA can be produced on mRNA matrix by an enzyme revertase. As eukaryotic mRNA has poly(A) tail on the 3’ end, primers can be easily selected — olygo dT sequences (figure 28). The second strand is then created. The obtained DNA is not functional as it has only informative region of the structural gene and has no regulatory region. The gene is able to work in a bacterial cell after adding a promoter. 3. Cleaving DNA from a genome with restriction endonucleases (restriction enzymes). 35

Fig. 27. Artificial gene synthesis

Fig. 28. Making a complementary DNA on an mRNA matrix

Restriction endonucleases (restriction enzymes, restrictases) are enzymes causing hydrolysis of DNA and formation of its fragments. Today more than 3000 restriction enzymes are known. They act on DNA of any organism if they have recognition sites. Such sites are usually palindromes consisting of 4–6 nucleotide pairs. Each acts only on its own recognition site. Restriction endonucleases can cut strands of DNA in different places or in the same place. In the first case single-stranded sticky ends are formed; in the second one blunt ends are formed (figure 29).

A B Fig. 29. Formation of sticky ends (A) and blunt ends (B) 36 Usage of restriction enzymes has a number of disadvantages: – it is not always possible to select restriction enzymes, which allow to cut out a proper DNA part with a required gene; – the DNA fragment may contain introns, then recombinant DNA will not be able to work in prokaryotic cells due to its disability for processing and splicing.

INSERTION OF DNA FRAGMENTS INTO A VECTOR MOLECULE. VECTORS Vector is a small autonomously replicated DNA molecule, which carries the foreign gene and provides its multiplication and/or expression. Requirements for vectors: – contain a replication origin (ori) for autonomous replication; – be inherited by a host cell; – be contained in a great number of copies in the cell; – have enough capacity that allows to clone big genes; – have convenient sites of restriction; – have markers for selection of cells where the vector works. The most common host for vector molecules is a bacterium E. сoli and most common vectors are plasmids. Plasmids are autonomously replicated circular DNA molecules that are normally present in bacterial cells (figure 30).

Fig. 30. Plasmid pBL-1 37 Phage vectors are bacteriophage genome units containing a recombinant DNA. Vectors for E. coli are constructed on the basis of phage λ and phage M13. Phage λ contains a double-strand DNA that consists of 48 500 nucleotide pairs. It is packed into the head as a linear molecule with sticky ends. After introduction into the cell, the sticky ends connect, the molecule closes into a circle with DNA-ligase. It is possible to clone fragments with the length 15 000 nucleotide pairs in vectors made on the basis of the phage λ. Cosmids are vectors made on the basis of plasmids and phage λ. A cosmid has cos-sites from phage λ on its ends. These sites are complementary single- strand regions 12 nucleotides length which work as sticky ends. Naturally these sites separate multiple copies of the phage genome in a long linear replicated DNA called concatamer. Cosmids have high capacity and allow to clone genes consisting of 33 000–39 000 nucleotide pairs. Phasmids or phagemids are plasmids having viral DNA that can develop both as a phage and a plasmid. The capacity of plasmids is comparable to that of phage vectors.

INCORPORATION OF THE RECOMBINANT DNA INTO A RECIPIENT CELL The following techniques based on the following phenomena can be used to incorporate the recombinant DNA into a cell: – conjugation — bacteria can transmit genetic material by means of direct intercellular contact. Genetic material is transmitted only in one direction; – transformation — direct uptake and incorporation of exogenous genetic material from medium through the cell membrane. The bacterial cell must be in a state of competence; – competence — state of a cell to take DNA from environment; – transduction — transmission of DNA by bacteriophages; – transfection — incorporation of the DNA by means of infection with phages λ, ψ X174 or Т4; – microinjections of DNA into animal cells; – usage of liposomes — vesicles of lipids with wall similar to plasma membrane.

TECHNIQUES USED IN GENETIC ENGINEERING AND BIOTECHNOLOGY Polymerase chain reaction (PCR) In 1980 Kary Banks Mullis elaborated a technique which allows amplifying DNA fragments in vitro (at the laboratory) by heat-resistant enzyme Taq polymerase. Apart from the enzyme, PCR requires nucleotides, primers and sample DNA. Taq polymerase is variety of DNA-polymerase extracted from a microorganism Thermus aquaticus and optimal temperature for its work is approximately 70 ºС. 38 Primers are short (20–30 nucleotides), single-strand DNA fragments complementary to 3'-ends of a matrix DNA. Due to the primers certain region of DNA can be chosen for amplification. PCR consists of 3 repeating steps (figure 31):

Fig. 31. Polymerase chain reaction

I. Denaturation: the mixture of reagents is heated up to 90 °C. During 15 seconds hydrogen bonds between DNA strands break and two single-strand molecules are formed. II. Annealing: the temperature is lowered to +50 °C. Primers connect to the complementary region of the DNA sample. This stage requires about 30 seconds. III. Elongation (or extension, polymerization) the reaction is heated again to 70 °C. At this temperature the Taq-polymerase assembles complementary strands moving from primers to the 5’ end of the matrix. This process takes 90 sec. As a result, the number of DNA increases by many times. During 20 cycles the number of DNA copies reaches to 106. At the present day PCR is performed automatically in a thermocycler. Southern blot is a technique elaborated in 1975 for detection of a specific DNA sequences in DNA samples (figure 32). A DNA is processed with restriction enzymes and placed on agar jelly in a chamber for electrophoresis. Electric field makes DNA fragments move from an anode to a cathode. Short fragments move further. After electrophoresis the DNA fragments separate: the longer the fragment the less the distance it had passed. DNA molecules of different are separated into fractions located some distance from each other. The fragments of the fractions, denaturate (strands separate) and is then carried to a nitrocellulose filter where they are fixated. This filter is placed into medium containing a DNA-probe — a short DNA fragment complementary to required nucleotide sequence. 39

Fig. 32. Southern blot hybridization

The probe is marked with radioactive isotopes. This is done for detecting the fractions which were bound with the probe. The filter is applied to a photographic film which acquires lighted spots due to the isotope. Revealing certain nucleotide sequences in a DNA sample allows detecting certain genes and making diagnosis of gene mutations.

DNA FINGERPRINTING (DNA PROFILING) There is minisatellite DNA — short (9–64 b. p.) variable tandem repeats. A tandem repeat is two or more identical adjacent nucleotide sequences. Human genome includes a plenty of various tandem repeats in different chromosomes forming a set of minisatellite DNA which is unique for every person. Analysis of these fragments based on southern blot in order to identify a person is called DNA fingerprinting.

Fig. 33. Example of DNA fingerprinting

Technique: DNA samples are extracted from cells and are cut by restriction enzymes into fragments of different length. Then southern blot is done. The fractions containing the minisatellites are detected by probes 40 complementary to 13 repeating nucleotides. The probe contains isotope and makes spots on the photographic film. These spots correspond to fractions containing the minisatellites. Such picture is unique for a person (figure 33).

BASIC TERMS AND CONCEPTS Autoradiogram — photographic film where spots corresponding to the marked DNA fractions are shown. Thermocycler — a machine performing PCR. Vector — a small autonomously replicated DNA molecule providing multiplication and/or work of an artificially inserted gene. DNA-probe — a radioactively marked short single-strand DNA sequence able to bind certain DNA site. Sticky ends — ends of DNA formed after cutting with restriction endonucleases which have complementary single-strand regions and able to join together. Liposomes — vesicles surrounded by one or several membranes of lipids. Plasmids — small autonomously replicated circular DNA molecules of bacterial genome. Polymerase chain reaction (PCR) — technique used to amplify (make multiple copies) DNA or its fragment in vitro. Primers — short (20–30 nucleotides) single-strand DNA fragments complementary to certain DNA site and serving as beginning for the new DNA strand during PCR. Recognition sites — usually short DNA fragments recognized processed by DNA restriction enzymes. Transfection — infection of cells with phages λ, ψ X174 and Т4. Blunt ends — ends of DNA formed after cutting with restriction endonucleases which have no complementary single-strand regions.

Topic 7. GENE INTERACTIONS. GENETIC LNKAGE. GENETICS OF SEX

INHERITANCE OF BLOOD GROUPS: SYSTEMS АВ0, MN AND RH Blood group in the ABO system is determined by a gene I which has alleles IO, IA and IB. The IA and IB code for antigens of erythrocytes while IO does not. If the genotype of a person is homozygous (IAIA; IBIB) or heterozygous (IAI0; IBI0) these alleles cause production of antigens А or В and determine blood groups A (or II) and B (or III). Therefore IA and IB are dominant alleles while IO is recessive one: it does not cause production of such antigens (group O or I). The genes IA and IB do not dominate over one another — both antigens А and В are produced if they are in the genotype IAIB (group AB or IV). 41 Multiple alleles are alleles which have more than two variations in a population: alleles of the gene I are I0, IA, IB. Blood group in the Rh system (Rhesus factor) is mostly determined by a gene D which causes presence of a protein rhesus factor in erythrocytes. Blood of such people (DD or Dd) is rhesus-positive (Rh+). If this protein is absent (genotype dd) then blood is rhesus-negative (Rh–). Blood group in the MN system is determined by two alleles — LN and LM. The LM is responsible for the antigen M in erythrocytes (blood group М) while the LN — for the antigen N (group N). Presence of both alleles in the genotype causes appearance of both antigens M and N (group MN).

NON-ALLELIC (INTER-ALLELIC) GENE INTERACTIONS Inter-allelic gene interaction is interaction of non-allelic genes having effect on the same trait or group of traits. Complementation is a gene interaction, when a gene complements the action of another non-allelic gene. For example, flowers of a plant sweat pea are colored due to dominant genes A and B. If one of them is absent in the genotype, flowers have no pigment and are white. Gene Genotype Color P: AAbb x aaBB A+B A-B- colored white white A+b A-bb white G Ab aB a+B aaB- white a+b aabb white F1 AaBb —100 % colored

9 3 3 1 F2 /16 A-B-; /16 A-bb; /16 aaB-; /16 aabb colored white white white Epistasis is a type of non-allelic interactions in which an allele of a gene (dominant or recessive) suppresses the effect of an allele of another gene. The gene-suppressor is called epistatic (or inhibitor), the suppressed one is called hypostatic. An example of epistasis is feather coloring in hens. Gene Genotype Color P: CCII x ccii C+I C-I- white white white (suppressed)

C+i C-ii colored G CI ci c+I ccI- white c+i ccii white F1 CcIi — 100 % white

9 3 3 1 F2 /16 C-I-; /16 C-ii; /16 ccI-; /16 ccii white colored white white

42 Coloring is determined by a gene С; a dominant allele of a gene I suppresses its effect. Example of recessive epistasis is Bombay blood group. A woman who had got an allele IB from mother had no antigens (group O). Further investigations revealed that the gene IB was suppressed by a rare recessive gene which caused epistatic effect in homozygous state. Polymeria is a non-allelic gene interaction of several gene pairs in which the number of their alleles defines development of a trait. This is inheritance of such quantitative characters as body mass, height, pigmentation of skin, arterial blood pressure. In most of cases the degree of character’s development depends on the number of dominant genes in the genotype: the more dominant genes person has, the more is the expressiveness of the character. This is cumulative polymeria. Development of most of qualitative characters depends on presence of the minimal number of dominant genes in the genotype. This is non-cumulative polymeria. The number of dominant genes does not change the degree of character’s development, but only its presence or absence (for example foot feathers of chicken). Such genes are denoted with the same letters but with different index numbers. For example genes coding for skin pigmentation of human are Р1Р1Р2Р2Р3Р3 for Negroid ethnicity, р1р1р2р2р3р3 for European ethnicity, Р1р1Р2р2Р3р3 for mulatto.

AUTOSOMAL AND GONOSOMAL LINKAGE GROUPS Linkage group is a set of genes situated in the same chromosome which are inherited together as one unit. The number of linkage groups is equal to haploid chromosome set of the organism (as homologous chromosomes represent the same genes). The 22 linkage groups of autosomes are celled autosomal linkage groups, those of sex chromosomes are gonosomal linkage groups.

CHROMOSOME THEORY OF INHERITANCE 1. Genes are situated in chromosomes in linear order in definite loci. Allelic genes are in the same loci of homologous chromosomes. 2. All the genes in a chromosome compose a linkage group and are inherited together. The number of linkage groups is equal to the number chromosome pairs of the species. 3. Crossing over can happen between homologous chromosomes and cause exchange of their alleles. 4. Probability of crossing over depends on the distance between genes in the chromosome. 1 % of its probability is equal to 1 centimorgan (cM, map unit, m.u.) — a unit of the distance between genes named after T. Morgan.

43 DETERMINATION OF SEX IN HUMAN AND ITS DISORDERS Anlagen of human external and internal sex organs develop by 4th week of embryogenesis under the influence of only one X-chromosome. Primary sex cells can be found during the 3rd week of embryogenesis in the ectoderm of the yolk sac. Differentiation of the anlagen into sex organs occurs since 4th till 12th weeks of intrauterine development and depends on the second sex chromosome: the X-chromosome causes transformation of sex cells into oogonia and female development of sex organs. Male development of sex organs occurs if the Y-chromosome is present in the genotype. The primary sex cells differentiate into spermatogonia, testes and male external sex organs develop. There are physical, intermediate and social-psychological determinants of sex. Physical determinants are: genetic sex (combination of sex chromosomes), gonad sex (gonads of the organism), hormonal sex (estrogens or androgens produced by gonads), gametic sex (gametes produced by the gonads). Influence of sex hormones defines morphological sex (male or female phenotype). Physical (morphophysiological) determinants are same or similar for human and most of animals. The intermediate determinant is civil sex (according to documents). Social-psychological determinants are: upbringing sex (different upbringing of boys and girls). It defines sexual identity and idea оf sexual role, and this predetermines choice of sexual partner. In most of cases it is a person of the opposite sex (heterosexuality), sometimes it is a person of the same sex (homosexuality). Transsexualism is a persistent discrepancy of sexual identity and true genetic and gonad sex and a wish to change sex. Transvestism is sexual perversion when the excitement and satisfaction are reached during putting on clothes of the opposite sex. An example of a disorder of sex determination is androgen insensitivity syndrome (AIS, Morris syndrome) when female traits or even female phenotype develop in persons having the sex chromosomes ХY (testicular feminization). In this case male sex hormones are produced in the body but there is lack of receptor providing sensitivity to the hormones in cells. This causes female development of the body.

X-CHROMOSOME’S SEX CHROMATIN. MARY F. LYON’S HYPOTHESIS OF X-CHROMOSOME INACTIVATION In 1949 Murray Barr and Ewart Bertram discovered a clump of heterochromatin in nuclei of cat’s neurons. It was present only in cells of female cats and absent in male animals. Later on, it was discovered that heterochromatin is an X-chromosome which was named Barr body (figure 34). 44 It can be connected with the membrane of the nucleus, stay freely in karyolymph or form an outgrowth of the nucleus (neutrophils). At the initial stage of embryogenesis, both X chromosomes function in all the cells of female embryo, i.e. the diversity of proteins there is higher than that of male embryos. This is one of the explanations why female embryos have higher vitality. In 1962 Mary F. Lyon suggested a hypothesis that one of the X-chromosomes is inactivated in the cells of female organism on 16th day of embryogenesis. This chromosome forms a clump of sex chromatin. This inactivation is random and some cells Fig. 34. Barr body in a neutrophil inactivate father’s X-chromosome while maternal one (Xm) remains active, other cells keep paternal (Xf) chromosome active. As alleles of these homologous pair are not identical, different variants of enzymes can be produced. They can have different thermal or pH optimum, different substrate affinity or repressor affinity. This enzyme diversity increases adaptability of female organism, especially in case of physical exertions and pathological states.

SEX CHROMOSOME DISORDERS In case of non-separation of chromatids during meiosis, abnormal gametes are formed (with two, more or no sex chromosomes). This leads to sex chromosome disorders. ♀ Х ХХ 0 ♂ Х ХХ ХХХ Х0 Y XY XXY Y0 XY XXY XXXY XY* 0 X0 XX* 00

XX and XY — normal male and female. ХХ* — normal female who got both X-chromosomes from mother. XY* — normal male who got both sex chromosomes from father. Y0, 00 — unviable genotypes. ХХХ — X trisomy. Karyotype is 47, ХХХ, phenotype is female. Incidence of the syndrome is 1 : 800–1 : 1000. Somatic cells contain two Barr bodies. Because of inactivation of X-chromosomes, symptoms are mild or absent and

45 their degree is variable. Height is tall, body constitution can have male traits. Intellect is usually lower than in healthy people. Functions of sex organs can be impaired. Can have healthy children, but they have higher risk of chromosome disorders. The more X-chromosomes are in the karyotype, the severer the symptoms. Х0 — Shereshevsky-Turner syndrome. Karyotype is 45, Х0, phenotype is female. The incidence is 1 : 2000–1 : 3000. Barr body is absent. If not treated, height of adult patients is 135–145 cm. Characteristic symptoms are short neck with skin fold following from the occiput to shoulders, low position of ears, low growth of hair at the occiput, changes in joints of fingers and toes; 15 % have malformations of the heart and kidneys. Ovaries and secondary sex characters are underdeveloped and these patients are sterile. Intellect is not impaired. Treatment is based on early hormonotherapy. ХХY, XXXY — Klinefelter syndrome. Karyotype is 47, XXY, 48, ХXXY, phenotype is male. Incidence is 1 : 400–1 : 500 among boys. Nuclei of somatic cells contain a Barr body (or two). Height is taller than average, body constitution is female. Gynecomastia is possible (development of breast tissue). Sick persons have less facial and body hair, underdeveloped testes, impaired spermatogenesis. Such patients are sterile, but sexual reflexes are preserved. Intellect is lowered, especially with increase of the number of X-chromosomes.

BASIC TERMS AND CONCEPTS Crossover gametes — gametes that contain chromatids that have undergone the crossing-over and exchanged certain alleles. Hemizygosity — a state when an allele is single in diploid chromosome set as it is contained in a non-homologous (differential) region of X or Y chromosome of a person with heterogametic sex. True hermaphroditism — a state of an organism associated with ability to produce both normal male and normal female gametes. Pseudohermaphroditism — a state of an organism associated with mismatch of primary and secondary sex characters. Complementation — non-allelic interaction in which a gene complements the action of another non-allelic gene and they both determine development of a character. Polymeria — non-allelic gene interaction of several gene pairs in which the number of their dominant alleles defines development or degree of a character. Recombinants — organisms who got crossover gametes. Klinefelter syndrome — a sex chromosome disorder caused by presence of an extra X-chromosome in a male karyotype. Androgen insensitivity syndrome — development of female phenotype in a person having the genotype ХY.

46 X trisomy — a sex chromosome disorder caused by presence of an extra X-chromosome in a female karyotype. Shereshevsky–Turner syndrome — a sex chromosome disorder caused by presence of only one X-chromosome in a karyotype. Physical determinants of sex — morphological and physiological sex determinants of an organism. Epistasis — type of non-allelic interactions in which an allele of a gene (dominant or recessive) suppresses the phenotypic effect of an allele of another gene.

Topic 8. VARIATION

PHENOTYPIC VARIATION. REACTION NORM Phenotypic variation (modificatory variation, phenotypic plasticity) is change of the phenotype not caused by change of the genotype, consequently it is non-hereditary. Such modifications are caused by environmental factors and can be predicted even for a group of individuals as they have same reaction to the same factors. Commonly modifications are useful and adaptive (for example, increased skin pigmentation under the action of ultraviolet). Limits of phenotypic variation are determined by norm of reaction. It depends on genotype which is inherited. If a trait varies insignificantly (fat content of milk), then its reaction norm is narrow. A trait which can change considerably (body mass) has wide reaction range.

GENOTYPIC VARIATION AND ITS TYPES (COMBINATIVE AND MUTATIONAL). COMPARISON OF MUTATIONS AND MODIFICATIONS Genotypic variation is represents changes of the phenotype caused by changes of the genotype. As genotype changes, such variation can be inherited by descendants. It includes a combinative and mutational variation. Combinative variation is associated with recombination of parental genes in leading to formation of new genotypes in their children without changes in the structure of their genes (for example a blue-eyed child in a family of heterozygous brown-eyed parents). Mechanisms providing combinative variation: 1. Crossing-over in the beginning of meiosis leads to recombination of parental genes. 2. Random distribution of chromosomes and chromatids to daughter cells during meiosis causes formation of gametes with different genotypes.

47 3. Random combination of these different gametes during fertilization provides different combinations of genotypes in children. Mutational variation or mutation is a sudden change of genetic material caused by environmental factors. It is inherited. Differences between mutations and modifications are shown in the figure 35. PROPERTIES

Modifications Mutations

They are not inherited They are inherited Have a group character Individual Are predictable Appear suddenly, unevenly Are not a matter for selection Matter for natural selection Have a reversible character Are constant Adaptive character of changes In general are harmful for the organism Fig. 35. Differentiation of mutations from modifications

MUTAGENIC FACTORS, THEIR CLASSIFICATION AND ACTION Mutagenic factors are factors causing mutations. Mutagenic factors are grouped into physical, chemical and biological. Physical mutagens are various kinds of radiation, temperature, humidity, etc. They cause structural impairments of genes and chromosomes, form free radicals interacting with DNA, destroy microtubules of spindle apparatus; dimers of adjacent pyrimidine bases in a DNA strand (T-T, T-C) and etc. Chemical mutagens are some medicines, formalin, sulfur mustard (yperite), colchicine, food preservation agents, etc. They cause desamination and alkylation of DNA molecule nucleotides; replacement of nitrogenous bases with their analogues (substances with similar structure); suppress synthesis of precursors of nucleic acids (nucleotides, ribose, deoxyribose). Biological mutagens are viruses, bacteria, metabolites of protozoans and helminthes. They cause impairments of DNA synthesis, impair separation of chromosomes and chromatids in the anaphase of meiosis and mitosis; waste products of parasites act as chemical mutagens, destroy chromosome telomeres and impair the process of crossing-over.

CLASSIFICATION OF MUTATIONS The process when mutations forms is called mutagenesis. According to etiological factors: 1. Spontaneous mutations — appear under the influence of natural factors (mutagens) without participation of human. 2. Induced mutations — result of directed effect of definite mutagenic factors. 48 According to mutated cells: 1. Gametic mutations — occur in sex cells and are transmitted by sexual reproduction. 2. Somatic mutations — occur in somatic cells, express in the individual itself and are inherited by asexual (vegetative) reproduction. According to the outcome for the organism: 1. Negative: lethal (incompatible with life) and semi-lethal (reducing vitality). 2. Neutral, affecting the vitality inconsiderably. 3. Positive, increasing the vitality. According to modification of the phenotype: 1. Morphological (small eyes, 6 fingers on the hand). 2. Biochemical (albinism, hemophilia). According to the level where mutation occurs: 1. Genome mutations. 2. Chromosome mutations. 3. Gene mutations.

GENE, CHROMOSOME AND GENOME MUTATIONS, THEIR CHARACTERISTICS, BIOLOGICAL AND MEDICAL SIGNIFICANCE Genome mutations are changes of the number of chromosomes caused by impairment of mitosis or meiosis. Haploidy is a single chromosome set: 1n. It is natural in drones (male bees). The vitality of such organisms is lower as all the recessive genes are expressed. Polyploidy is multiple chromosome set (3n, 4n, 5n). Polyploidy is used in plant cultivation as it increases fruitfulness. For human being, haploidy and polyploidy are lethal mutations. Heteroploidy (aneuploidy) is an abnormal number of chromosomes which is indivisible by a haploid one (2n ± 1, 2n ± 2 and so on). Trisomy is presence of 3 homologous chromosomes instead of normal two. An example is trisomy X. In that case X-chromosome is added to a pair of woman’s sex chromosomes and karyotype becomes (47, XXX). If such addition of X-chromosome happens in a male karyotype, Klinefelter syndrome develops (47, XXY). Monosomy is absence of one chromosome in a homologous pair. The example is Shereshevsky–Turner syndrome (45, X0). Nullisomy: absence of a chromosome pair. It is a lethal mutation for human. Chromosome mutations (or chromosome aberrations) are modifications of chromosome’s structure. There are interchromosomal or intrachromosomal mutations. Structural abnormalities within one chromosome or intrachromosomal mutations are inversions, deficiency and deletion (losses), duplications. Deletion

49 is loss of a middle region of a chromosome; deficiency — of an end of chromosome arm; duplication is doubling of a chromosome region; inversion is wrong order of genes’ arrangement in a chromosome when its part turns. If telomere regions of both chromosome arms are deleted, connection of the remaining ends into a ring may occur and form of ring chromosomes. Interchromosomal mutations are translocations. Translocations can be reciprocal when two chromosomes exchange their parts or non-reciprocal if part of a chromosome is relocated on another one. Robertsonian translocation is connection of two acrocentric chromosomes at the region of their centromeres. Losses and duplications have phenotypic manifestation because a set of genes changes. Inversions and translocations are not always revealed as they can be balanced — set of genes stays same. In such cases synapsis of homologous chromosomes becomes difficult and the distribution of genetic material between daughter cells is impaired. Gene mutations or transgenations are associated with changes of the gene structure and lead to metabolic diseases. Mutations of structural genes: 1. Reading frame shift is deletion or insertion of one or several nucleotide pairs of into a gene. 2. Transition is a mutation that substitutes a nucleotide with another nucleotide of the same type (purine with another purine or pyrimidine with another pyrimidine: A ↔ G, C ↔ T). Such mutation changes the codon where it happened and can lead to change of encoded amino acid. 3. Transversion is a mutation that substitutes a purine nucleotide with pyrimidine one or vice versa: pyrimidine nucleotide with purine (A ↔ C; G ↔ T). It also results in changes of codons and encoded amino acids. Such case when amino acid of a protein changes because of mutated nucleotide is called missense-mutations. If senseless stop-codons (UAA, UAG, UGA) determining the end of protein biosynthesis are formed then nonsense- mutations happened. Mutations of functional genes can have the following consequences: 1. The protein-repressor is modified and does not suit the operator. In this case structural genes are not switched off and work permanently. 2. The protein-repressor is tightly binds the operator and is not removed when inductor is delivered to the cell. Structural genes do not begin their work. 3. Impairment of alternation of repression and induction. In this case the product of a gene is synthesized without cause (inductor) and vice versa: it is not produced when inductor is in the cell. This happens if operator or regulatory gene mutate. In the majority of cases gene mutations are have phenotypic effect.

50 STABILITY AND REPAIR OF GENETIC MATERIAL, ANTIMUTAGENS Antimutagenesis is the impact on the cell or organism, which prevents or reduces the probability of mutations. Stability of genetic material is provided by anti-mutagenic mechanisms: 1. Natural barriers: diploid set of chromosomes, DNA is double helix, redundancy of the genetic code. 2. DNA repair: there is a natural mechanism repairing impaired DNA in the cell. In 1962 Claud Rupert described photoreactivation. He discovered that exposition of phages, bacteria and protozoans to ultraviolet rays depresses their vitality. Though if they are exposed to visible light after that, their vitality restores. Ultraviolet rays form dimers in DNA (connection of adjacent thymines in a DNA strand) and this disturbes gene expression. Visible light activated enzymes which can repair DNA from these dimers. More common type of DNA repair is excision repair (A. Herren). Four groups of enzymes take part in it: a) endonuclease recognizes an impaired DNA part and cuts the strand; b) exonuclease removes the impaired part of the strand; c) DNA polymerase synthesizes a fragment of the strand instead of a removed one due to the other strand; d) ligase links these DNA fragments. The impairment of DNA repair may result in such diseases as Xeroderma pigmentosum and Fankoni anemia. 3. Antimutagens. These are substances of various origins, small concentrations of which are able to stabilize a mutation process and decrease the number of both gene and chromosome mutation. They are biologically active substances — histamine and serotonin, antioxidants, sulphanilamide drugs, components of fresh vegetable juices, α-tocopherol.

BIOLOGICAL BASIS OF ONCOGENESIS Oncogenesis is a process of formation and development of tumors. 1. Mutation concept — the basis of oncogenesis is genomic or chromosomal mutations of somatic cells (G. de Freeze, 1901). 1. Virogenetic concept — viruses are causative agents of malignant tumors. Mutagens and oncogenesis stimulate the activity of viruses; their genome includes into the cell DNA and changes its properties (L. A. Zilber, 1946). 2. Epigenomic concept — the basis of transformation of a normal cell into a tumor are persistent impairments of the structure of functional genes (Yu. M. Olenov, 1967; A. Yu. Bronovitsky, 1972). 3. Oncogene concept. Cell DNA contains protooncogenes. These genes can be received from parents or introduced into the cell by viruses. The protooncogenes are activated by mutations or when a viral promoter gets 51 into the cell. Their active forms are oncogenes which make the cell give rise to a tumor (R. Hubner, 1969; G. I. Abelev, 1975).

BASIC TERMS AND CONCEPTS Genocopies — same phenotypic manifestation of different mutations. Deletions — intrachromosomal mutations associated with a loss of a middle part of the chromosome. Duplications — intrachromosomal mutations associated with doubling of a part of the chromosome. Isochromosomes — chromosomes originating from transverse division of chromosomes instead of longitudinal division of chromatids and consisting of two same arms. Inversion — intrachromosomal mutations characterized by gene arrangement order impairment occurs. Oncogenesis — a process of origination and development of tumor. Ring chromosome — chromosomes formed when its telomere regions are deleted and the remaining ends connect to each other. Modifications — changes of phenotype not caused by changes of the genotype. Reaction norm — range of phenotypic variation. Reading frame shift — a mutation of structural genes caused by insertion or deletion of nucleotides that move reading of nucleotides. Transgenations — genome mutations. Translocations — relocation of a chromosome region on another chromosome.

Topic 9. FUNDAMENTALS OF HUMAN GENETICS (PART 1)

MODERN TASKS OF HUMAN GENETICS Human genetics studies regularities of inheriting normal and pathologic characters, their modification under the influence of environment. Its section medical genetics studies mechanisms of hereditary pathology, elaborates techniques of diagnosis, treatment and prophylaxis of human’s hereditary disorders. This discipline is extremely important. At present day about 5000 hereditary disorders are describes, 2.5 % of newborns are affected, 40 % of neonatal mortality and permanent disability are caused by hereditary pathology. Tasks of human genetics are: – researching pathogenesis, clinical presentation, diagnosis, prophylaxis, pharmacological and another types of treatment (such as gene therapy) of hereditary disorders; 52 – early diagnosis of hereditary disorders by improvement of instant diagnosis tests and tests for prenatal diagnosis; – researching the mechanisms of genetic predisposition and innate resistance to multifactorial diseases; – studying genetic aspects of immunity, allergy, transplantology, oncogenesis, genetic engineering and etc.; – large-scale implementation of genetic counseling into medical service.

HUMAN AS AN OBJECT OF GENETIC INVESTIGATIONS Studying the human being has a number of difficulties such as: 1) hybridological method is inadmissible in human genetics; 2) complicated karyotype with many chromosomes and linkage groups; 3) late sexual maturity, low number of descendants leading to impossibility to analyze many genotypes and many generations; 4) high diversity of ecological and social conditions troubling analysis of gene manifestations. Though there are facts facilitating studying human: 1) high number of individuals; it is possible to analyze characters in numerous groups of people; 2) international cooperation of geneticists; 3) clinically, human is the most studied biological object; 4) elaboration especial methods to bridge over these difficulties.

CLASSIFICATION OF METHODS USED IN HUMAN GENETICS There are basic methods of human genetics, instant diagnostic tests and methods of prenataldiagnosis used to make possible diagnosis of a disorder before birth: Basic methods: – genealogical method; – twin study; – karyotyping (cytogenetic method); – boichrmical tests; – somatic cell hybridization; – methods of population genetics; – modeling; – methods of molecular genetics. Instant diagnosis tests: – barr body testing; – chemical and biochemical tests; – dermatoglyphic analysis; – microbiology tests.

53 Methods of prenatal diagnosis: – detection of embryo-specific proteins; – ultrasonography; – amniocentesis; – chorion biopsy; – fetoscopy.

GENEALOGICAL ANALYSIS Genealogical analysis was proposed by Francis Galton in 1883. It becomes a basis for genealogical method of human genetics. It relies on drawing and analysis of pedigree charts that illustrate inheritance of a certain character (disorder). The method allows to define: – whether the character is hereditary or not; – how it is inherited (type of inheritance); – zygosity of family members (homo- or heterozygotes); – gene penetrance (frequency of its manifestation); – genetic risk — probability of giving birth to children with the character. Legend keys used for drawing pedigree charts are shown in the figire 36. -female (the analyzed character is absent)

- male (the analyzed character is present)

- sex of the individual is not known to the proband

- male proband

- marriage (parents)

- children (siblings)

. - heterozygous carrier of the analyzed character

Fig. 36. Legend keys used for drawing pedigree charts

A person who is studied and from whom the pedigree is composed is called proband. It is indicated with arrow. Stages of genealogical analysis: – collection of data about proband’s relatives; – drawing a pedigree chart; – analysis of the chart and drawing conclusions. 54 Inheritance of characters can be of 5 types (table 3). Autosomal dominant inheritance: – sick children are born by only sick parents; – both men and women fall ill with equal probability; – sick persons are in every generation; – if one of the parents is dominant homozygote then the probability of the character for children is 100 %; if both parents are heterozygous — 75 %, if one parent is heterozygous and the other is recessive homozygote —50 %. Table 3 Types of inheritance Recessive gene Dominant gene Autosomal recessive Autosomal dominant Autosomal gene inheritance (a) inheritance (A) In the X- X-linked recessive X-linked dominant Gonosomal chromosome inheritance (Xa) inheritance (XA) gene In the Holandric inheritance (Y*) Y-chromosome

Autosomal recessive inheritance: – sick children can be born by healthy parents; – both men and women fall ill with equal probability; – sick persons are not in every generation; – if both parents are heterozygous then the probability of the character for their children is 25 %; if one parent is heterozygous and the other is a recessive homozygote — it is 50 %; if both parents are recessive homozygotes — 100 %. X-linked dominant inheritance has the same signs as autosomal-dominant one. The key feature is the fact that a man having the X-linked dominant character can transmit the gene only to daughters (sons get his Y-chromosome). X-linked recessive inheritance: – mostly men fall ill; – sick children can be born by healthy parents; – sick persons are not in every generation; – if parents are healthy then probability giving birth to a sick child is 50 % for boys, and 0 % for girls (i. e. 25 %). Holandric (Y-linked) inheritance: – only men can have the character; – such men are in all generations; – if a father is sick then all his sons are sick and vice versa.

TWIN STUDY. CRITERIA DETERMINING ZYGOSITY OF TWINS. HOLZINGER’S FROMULA In 1876 Francis Galton proposed the method of twin study. The method is used to estimate roles of heredity and environment in development of a character. 55 The frequency of giving birth to twins is 1 %. Twins can be monozygotic (MT) if they develop from the same zygote and have identical genotype. Dizygotic twins (DT) develop from different ova that develop and are fertilized at the same period. Sucgh twins have similar but not identical genotype (as siblings). Criteria of zygosity:MT always have same sex, blood group and fingerprints; in DT these factors can be different. The degree of twins’ similarity on a character is called concordance, the degree of their difference is discordance. In other words concordance is percent of twins who both have a character among all twins; discordance is the percentage of twins who are different in the character. Roles of heredity and environment for development of a character can be calculated by the Holzinger's formula:

Key: H — role of heredity; CMT — concordance for monozygotic twins; CDT — concordance for dizygotic twins. If H = 1.0, only heredity is responsible for development of the character; if H tends to 0, then mostly environment affects the character.

KARYOTYPING (CYTOGENETIC METHOD) Karyotyping is studying all chromosomes (karyotype) with microscope. Technique: lymphocytes or cells of bone marrow are obtained from a patient and grown on culture medium. Then mitosis is stimulated and stopped (it is necessary to do it at the moment of metaphase). The cells are treated with NaCl hypotonic solution and then chromosomes are stained. They are studied under microscope, photographs are taken. These photographs are used to comply an ideogram and analyze it. Fluorescent analysis is used for determining the karyotype and mapping chromosomes. The method reveals genome and chromosome mutations. There are keys to write karyotypes and mutations it has: «q» — long arm of a chromosome, «р» — short arm of a chromosome, «+» — excess of genetic material, «–» — loss of genetic material. For example the karyotype of a man sick with Down syndrome is 47, ХY, 21+.

CULTIVATION AND HYBRIDIZATION OF SOMATIC CELLS It is possible to clone somatic cells (fibroblasts) in artificial media. All the offspring of such cell would have the same genotype and this allows to study the influence the genotype and environment on characters at the level of cell. It is also possible to select cells with required characteristics. In this case selective media are used.

56 Cultivation of offer opportunities for somatic cell hybridization (figure 37). If Sendai virus inactivated with ultraviolet is added to a cell culture then frequency of cell hybridization rapidly increases. A culture containing different types of cells can form heterokaryotes — cells containing two different nuclei. Some of them are able to multiply by mitosis and form two single-nucleated cells — synkaryotes. Each synkaryote is a hybrid cell containing chromosomes of both parental fused cells.

Fig. 37. Somatic cell hybridization

Such hybridization allows to fuse cells of different species (human-mouse) and even of different phyla (human-mosquito). Synkaryotes are usually successfully obtained if cells taken from species of the same class are used. Hybrid cells of human and mouse have 43 pairs of chromosomes (23 and 20 from human and mouse). Later on, chromosomes of the cell which has longer terms of division are eliminated: human chromosomes are eliminated from human-mouse synkaryon.

BIOCHEMICAL GENETIC TESTS Biochemical genetic tests are used for revealing metabolic diseases by measuring activity of enzymes or the quantity of the reaction product that is catalyzed by an enzyme. Chromatography, fluorometry, radio immunological assay and other methods are used for revealing gene mutations causing metabolic diseases. For example, phenylketonuria (the impairment of phenylalanine (PhA) 57 exchange) can be detected by measuring concentration of phenylalanine in blood: in healthy people it is 1–2 mg %, in sick ones — 50–60 mg %. In different population one of 30–40 people is a heterozygous carrier of phenylketonuria-causing gene. Carriage can be revealed by loading tests: phenylalanine is injected to a person and its concentration in blood is recorded. If the concentration of PhA becomes normal slowly then a person is a carrier of phenylketonuria.

GENETIC ANALYSIS. THE HUMAN GENOME PROJECT Methods of genetic analysis allow detect a pathologic gene in genome. Stages of the methods: 1. DNA specimen is cut into short fragments by restriction endonucleases. 2. The fragments are separated into fractions of different length (and mass) by electrophoresis in an agar gel. 3. The DNA is amplified by PCR. 4. DNA strand are separated by heating as singe-strand molecule is required for hybridization with a probe. 5. The fragments of DNA are placed into the medium containing radioactive probe — single-strand DNA complementary to the gene need to be found. The probe binds the complementary DNA segment with the gene. 6. Presence of probes in the DNA fraction is detected by X-ray sensitive film. In 1990 an international project on making a genetic human map (Human Genome Project) was started. One of the tasks of the project was sequencing (decoding of nucleotide sequence) of human DNA. In 2000 the human genome was sequenced.

BASIC TERMS AND CONCEPTS Dizygotic twins — twins that developed from different zygotes at the same period. Monozygotic twins — twins that developed from same zygote. DNA hybridization — connection of DNA strands such as specimen and probe in case of their complementarity. Discordance — percentage of twins who are different in a character. Concordance — percentage of twins who both have the same character. DNA cloning — making unlimited number of DNA copies for further usage. Proband — a person from whom making a genealogy starts. Sequencing — decoding the nucleotide sequence of DNA. Synkaryote — a hybrid cell having chromosomes of both parental cells. Pedigree chart (genealogy) — a genealogic map illustrating the proband with relatives and allowing to analyze inheritance of a gene in the family.

58 Topic 10. FUNDAMENTALS OF HUMAN GENETICS (PART 2)

MATHEMATICAL AND BIOLOGICAL MODELING. VAVILOV’S LAW OF HOMOLOGOUS SERIES Biological modeling is study of congenital malformations and hereditary disorders in animal shaving similar pathology (hemophilia of dogs, diabetes mellitus of rats, etc.). This is based on the Vavilov’s Law of Homologous Series: closely related genera and species have similar series of hereditary variation. Certain variation types of a species allow to presume similar variation in another species or genera. Mathematical modeling is used by population genetics to estimate frequencies of genes and genotypes for populations living in different conditions.

METHOD OF POPULATION STATISTIC. THE CONCEPT OF POPULATION. PANMICTIC AND NON-PANMICTIC POPULATIONS Method of population statistic of human genetics is based on Hardy– Weinberg principle and allows to calculate frequencies of genes and genotypes in a population. It belongs to diagnostics as can be used to estimate genetic risk of hereditary disorders for different countries and populations. Population is a group of individuals of same species having one whole genetic pool, capable of free crossing, inhabiting same territory for a long time and relatively isolated from other populations. There are ecological and genetic characteristics of populations. Main ecological characteristics are the number of individuals, the area of habitat, density of population, distribution of individuals on the area, age and gender composition, birth rate and death rate. The sum of genes in a population is its genetic pool. All the pools of populations form the genetic pool of the species. The individuals in the population have different genotypes (АА, Аа, аа), i. e. they are genetically polymorphous unlike pure lines consisting of homozygotes (all have the same genotype: АА or аа). A population is panmictic if crossing of its individuals is random and choice of partner is not limited. If such limitation are present then the population is not panmictic. Most of natural populations are not panmictic as a number of factors (weaker males, distance between individuals and so on) restrict choice of partners for coupling.

CHARACTERISTIC OF HUMAN POPULATIONS. TYPES OF MARRIAGES. GENETIC PROCESSES OCCURRING IN LARGE POPULATIONS. HARDY–WEINBERG PRINCIPLE There are big and small populations. A big population includes more than 4000 individuals. Demes and isolates are small populations. 59 The number of individuals in demes is 1500–4000, marriages within that groups comprise 80–90 %, inflow of genes from another groups is no more than 1–2 %. Isolates are populations of less than 1500 individuals, intragroup marriages exceed 90 %, inflow of genes from another groups is less than 1 %. There is inbreeding (consanguineous marriages) in demes and isolates. As relatives have higher probability to carry the same recessive pathological gene, consanguineous marriages have higher probability of recessive homozygous children. This homozygotization leads to reduced biological fitness (inbreeding depression) due to high probability homozygosity on recessive deleterious genes. Outbreeding is breeding of unrelated individuals. It maintains high level of heterozygosity in populations and lowers the frequency of hereditary pathology in the population. Human populations are described with demographic characteristics such as the number of individuals, birth rate, death rate, age and gender composition. Effect of natural selection in human populations is low and isolation is broken. A very numerous population is close to an ideal population — a population with unlimited number of individuals, complete isolation, complete panmixia, absence of mutations and natural selection, so all genes stay in the population. Hardy–Weinberg principle: gene and genotype frequencies in an ideal population will remain constant from generation to generation. Large populations have genetic polymorphism (АА, Аа, аа) and panmixia. In this case 9 combinations of marriages are possible: 1. АА × АА → АА. 2. АА × Аа → АА + Аа. А А а 3. АА × аа → Аа. А а а 4. Аа × АА → АА + Аа. АА 1 4 7 5. Аа × Аа → АА + 2Аа + аа. Аа 2 5 8 6. Аа × аа → Аа + аа. аа 3 6 9 7. аа × АА → Аа. 8. аа × Аа → Аа + аа. 9. аа × аа → аа. Sum: 4АА + 8Аа + 4аа or АА + 2Аа + аа. Frequencies of genes are denoted as: А — р, а — q. Frequencies of genotypes: АА — р2, 2Аа — 2рq, аа — q2. Therefore, mathematical writing of the Hardy– Weinberg principle is: р + q = 1 for genes and p2 + 2pq + q2 = 1 for genotypes.

FACTORS IMPAIRING THE EQUILIBRIUM OF GENES AND GENOTYPES IN POPULATIONS (MUTATIONS, NATURAL SELECTION, POPULATION WAVES, ISOLATION, MIGRATIONS, GENETIC DRIFT) AND THEIR CHARACTERISTIC Formation of mutations is random and nondirectional. It maintains heterozygosity on the popuation. Mutations can be neutral, positive or negative

60 or the organism. The neutral mutations can become positive or negative if environmental factors change. The frequency of spontaneous mutation of a gene is 10–5–10–7 for one generation. Dominant mutations manifest in the first generation and become matter for natural selection. Recessive mutations manifest only in recessive homozygotes, for this reason they accumulate in the population and only then manifest and become matter for natural selection. Population waves are periodical fluctuations in the number of individuals associated with environmental factors. The waves change genetic composition of populations as they eliminate weakest individuals. Isolation leads to limitation of free crossing. It separates the population into groups and change frequencies of genotypes. There are various types of isolation: 1. Geographic (mountains, rivers). 2. Biological: genetic (sterility of hybrids); ecological and ethological (lowering the probability to meet a partner); morphological and physiological (incapability for crossing caused by morphological mismatch of sex organs). A factor which can increase heterozygosity is migration. Immigration brings new alleles to the population while emigration changes ratio of alleles by carrying them away from the population. Another factor typical for small populations is genetic drift — accidental fluctuations of gene frequencies. It can result in homozygotization of a population. It can be demonstrated in population of self-fertilizing plants. For example composition of population is 1АА + 2Аа + 1аа (heterozygotes are 50 % of population). In F2 their number will decrease to 25 %, in F3 — to 12.5 % and so on. Lethal genes in the population cause extinction. Evolution in small populations is barely possible as there is no genetic diversity. The most important evolutionary factor is natural selection. It eliminates unsuccessful gene combinations and saves the most efficient ones. It also changes gene frequencies in populations. There are 3 types of natural selection: stabilizing, directional and disruptive.

GENETIC LOAD AND ITS NATURE Sum of recessive mutations that decrease biological fitness of a population is genetic load or genetic burden. Part of the genetic load has no phenotypic manifestation and is transmitted from generation to generation (heterozygous carriage). In addition, new mutations occur in every generation. As they are caused by environmental factors, genetic load correlated with the degree of environmental pollution (it is about 5 %).

61 METHODS OF PRENATAL DIAGNOSIS OF HEREDITARY DISORDERS AND MALFORMATIONS Direct methods of prenatal diagnosis (diagnosis before birth) are based on examination of fetus; indirect methods are tests of a pregnant woman (obstetric- gynecological, genealogical, biochemical tests). Important diagnostic marker is α-fetoprotein (AFP). It is an embryo- specific protein produced by fetal cells and placenta which then passes into the mother’s blood. Low concentration of α-fetoprotein at the 13–15th weeks of embryonic development is typical for some trisomies, fetal growth retardation, threatened miscarriage and fetal death. High concentration of AFP can be associated with, plural pregnancy, nerve tube defects, congenital nephrosis and other malformations. Ultrasonography is direct non-invasive methods (non-invasive = without tissues injury). It is a diagnostic imaging technique based on the application of ultrasound. It allows to obtain image of the fetus and embryonic membranes. All pregnant women are tested by the method because it is safe and can be repeated. Ultrasonography reveals vitality of the fetus, twin pregnancy and severe development defects of the skeleton, brain and spinal cord. Direct invasive methods are diagnostic procedures on the fetus with tissue injury. They are associated with some risk and performed only for indications such as: – diagnosed hereditary disease in the family; – mother’s age over 37 years; – carriage of X-linked recessive disorder by mother; – cases of spontaneous abortions at early stages of pregnancy, stillbirths, children with multiple congenital anomalies and chromosome pathology; – heterozygosity of both parents with an autosomal-recessive disorder. Direct invasive methods are: 1. Chorion biopsy is taking chorion cilia through the uterine cervical canal for cytogenetic and biochemical investigations and DNA analysis. It is performed within 8th–13th weeks of gestation under control of ultrasonography. The method reveals gene, chromosome and genome mutations. 2. Amniocentesis is performed within 15–17th weeks. It is puncture of the amniotic sac through the abdominal wall under control of ultrasonography in order to take 15–20 ml of amniotic fluid with fetal cells. Complications in this method arise in 1 % of cases.

INSTANT DIAGNOSIS TESTS Instant diagnosis tests are methods of fast preliminary diagnosis of hereditary disorders. These methods should be economical, safe and diagnostically accurate; they must require small volume of easily accessible (blood, urine) material. 62 Guthrie microbiological test (neonatal heel prick). It is used for diagnosis of phenylketonuria. This disorder is associated with increased concentration of phenylalanine in blood. Technique: a drop of newborn’s blood is taken on a blotting paper. The paper is placed on agar medium containing anti-metabolite of phenylalanine and bacterial culture. The anti-metabolite inhibits bacterial growth, but if the blood contains a lot of phenylalanine bacterial colonies appear on the medium. Sex chromatin test. Buccal (cheek) epithelial cells or leukocytes are taken for the test. The X-chromatin is stained with acetoorseine; Y-chromatin — with acrichine yperite. Smear is studied under the microscope. The method is used for making diagnosis of chromosomal sex and sex chromosome disorders. Biochemical and chemical methods are used for fast preliminary diagnosis of hereditary metabolic diseases. For example phenylketonuria can be diagnosed by addition 10 % FeCl3 solution to urine — the reaction becomes dark blue-green. Dermatoglyphic analysis studies skin patterns of the fingers, palms and feet. They often have some changes in case of malformations. Dermatoglyphic patterns are very individual and do not change during life. There are patterns of three types on finger tips: an arch (A), a loop (L) and a whorl (W). There are tri-radii in interfinger spaces: a, b, c and d. Near the bracelet crease is a palm tri-radius t. Connnection of tri-radii a, d, t, with lines shows a main palm angle. Normally it is not more than 57 °. The combination of radial loops on 4–5th fingers, palm angle of 60–86° and single transverse palmar crease (fusion of an oblique and transverse line) are typical for those who have congenital malformations.

BASIC TERMS AND CONCEPTS Amniocentesis — method of prenatal diagnosis based on sampling amniotic fluid with fetal cells for further tests. α-fetoprotein — protein contained in the amniotic fluid and blood serum of a pregnant woman. Demes — are human populations where the number of individuals is 1500–4000. Genetic drift — incidental fluctuations of genes’ frequencies in small populations. Incest marriage — marriage between family members or close relatives (such as brother and sister, parent and child) forbidden in most of cultures by law and religion. Panmixia — absence of limitations for free choosing in a population. Population — group of individuals of same species having one whole genetic pool, capable of free crossing, inhabiting same territory for a long time and relatively isolated from other populations.

63 Guthrie test — microbiological tests for diagnosis of phenylketonuria in newborns. Ultrasonography — diagnostic method using ultrasound for visualisation of fetus and embryonic membranes. Chorion biopsy — method of prenatal diagnosis based on sampling chorion cilia for further tests.

Topic 11. HUMAN GENETIC AND CHROMOSOME DISORDERS

GENE MUTATIONS AS A CAUSE OF METABOLIC DISEASES Gene mutations are revealed phenotypically in the human as hereditary metabolic diseases — enzymopathies. About 3000 such disorders are described. Their frequency in human populations is from 2 to 4 %. Gene disorders may have the following causes: 1) mutations of structural genes that lead to qualitative changes of proteins, formation of mutated proteins (for example, mutant forms of hemoglobin); 2) mutations of functional genes cause quantitative changes — the concentration of normal protein in the cell decreases. Substances which accumulate in case of abnormal enzyme activity may cause toxic effect or impairments of cell structure and function.

CHARACTERISTIC OF GENE DISORDERS OF HUMAN Gene disorders are classified according to the type of metabolic impairment. Impairment of amino acid exchange. Phenylketonuria is autosomal-recessive disorder with incidence frequency 1 : 10 000. The enzyme phenylalanine hydroxylase is impaired. Phenylalanine is not transformed into tyrosine but transformed into phenylpyruvic acid which is for nervous system. Symptoms: «mice» smell, progressing mental retardation, increased excitability and tone of muscles, hyperreflexia, tremor, epileptic seizures, low pigmentation of the skin. Diagnosis: Guthrie test, an instant diagnostic test with FeCl3, biochemical methods (detection of high concentration of phenylpyruvic acid in urine and phenylalanine in the blood). Treatment: diet-therapy (food without surplus contents of phenylalanine) from the first weeks of life till 7–10 years and longer. Albinism is caused by impairments in the enzyme tyrosinase which participates in formation of the pigment melanin. Incidence frequency is 1 : 5000–1 : 25 000. The inheritance is autosomal-recessive.

64 Symptoms: depigmentation of the skin, hair, eyes, photophobia, decreased acuty of vision, increased sensitivity to ultraviolet rays. Diagnosis — clinical examination. Treatment is not elaborated. Impairment of carbohydrate exchange. Galactosemia autosomal-recessive disorder. The incidence frequency is 1 : 100 000. The disease is caused by insufficiency of the enzyme, galactose-1- phosphate uridylyltransferase, which participates in metabolism of galactose. Symptoms: hepatomegaly, jaundice, vomiting, diarrhea, arrest of psychical and motor development, cataract. Diagnosis: decreased content of glucose is in blood, the content of protein and galactose in urine is increased. Treatment: diet therapy without lactose from. Impairment of lipid exchange. Hyperlipoproteinemia is caused by the impairment of lipid exchange in blood plasma (fatty acids, triglycerides, cholesterol) due to defects of enzymes or cell receptors. Incidence frequency of the disorder is 1 : 500. The inheritance is autosomal-dominant. Symptoms: increased concentration of cholesterol results leads to atherosclerosis, ischemic heart disease, early myocardial infarctions (33–45 years). Diagnosis: measuring lipoproteins in the blood serum. Impairment of purine exchange. Lesch–Nyhan syndrome. Incidence frequency is 1 : 300 000. The inheritance is X-linked recessive. The disease is caused by insufficiency of the enzyme that catalyzes attachment of purine bases to nucleotides. They are broken down into uric acid. Symptoms: muscle hypertone, intellectual disability, propensity of a child to self-injuries, urinary calculi, deposits of the uric acid in joints. Diagnosis: measuring the concentration of uric acid in blood. Impairment of mineral exchange. Wilson–Konovalov disease. Incidence frequency is 2 : 100 000; the inheritance is autosomal-recessive. The cause of the disease is insufficiency of an enzyme that impairs the synthesis of ceruloplasmin which performs copper transport. Concentration of copper in blood increases and it accumulates in the brain and liver. The disease is usually diagnosed at school age. Symptoms: hepatomegaly, jaundice, vomiting, cirrhosis of the liver, impairment of intellect, tremor, impairment of swallowing, muscle hypertone. Diagnosis: measuring the concentration of ceruloplasmin in blood serum. Impairment of blood coagulation. Hemophilia A. Incidence frequency is 1 : 6500 of newborn boys. The inheritance is X-linked recessive. Cause of the disease is decreased activity of coagulation factor VIII (anti-hemophilic globulin A). The disease is revealed in 2–3 years of life, sometimes after birth (bleeding from the umbilical cord and

65 intracutaneous hemorrhages). Symptoms: hemorrhages, a hematome type of bleeding, hemarthroses (hemorrhages into a knee, elbow, mortis joint), joint stiffness, blood in urine. Diagnosis: determination of coagulation factor VIII of the blood. Treatment: injection of coagulation factor and exchange transfusion. Impairments of the hemoglobin structure (hemoglobinopathies). Sickle-cell anemia. In case of this disorder the glutamic acid in position 6 of the hemoglobin β-chain is replaced with valine. In homozygotes, erythrocytes are sickle-shaped. Chronic hypoxia and anemia, hemolysis and breakdown of erythrocytes occur. Lethal outcomes are possible. Heterozygous carriers of a HbS gene are healthy under normal conditions. Diagnosis of gene disorders is made by biochemical methods and methods of DNA hybridization.

CHROMOSOME AND GENOME MUTATIONS AS A CAUSE OF HUMAN CHROMOSOME DISORDERS Chromosome disorders result from of chromosome and genome mutations. Their frequency is 0.24–0.4 %. About 90 % of chromosome disorders are trisomies of autosomes. All the cases of polyploidy, haploidy, monosomies (except an X-monosomy) and trisomies of large chromosomes are lethal for human. Diagnosis of chromosome disorders is made by means of karyotyping. The most frequent mutations are trisomies of the 13th, 18th and 21st pairs of chromosomes.

CHARACTERISTICS OF HUMAN CHROMOSOME DISORDERS Patau syndrome (47, XX, 13+; 47, XY, 13+). The frequency is 1:6000. The disorder can be caused by trisomy or Robertsonian translocation. Minimal symptoms: microcephaly, polydactyly, short neck, narrow eye slits, low nasal bridge, cleft lip and palate, microphthalmia, deformed auricles. Children are born with low body weight (less than 2500 g); 80 % of such newborns have heart defects, 65 % — abnormalities of the brain, 60 % — abnormalities of kidneys, 50 % — defects of digestive organs. About 95 % of them die by 1 year. Edwards syndrome (47, XX, 18+; 47, XY, 18+) has incidence frequency 1:7000. For women older 45 years the risk to give birth to a sick child is 0.7 %. The syndrome can be caused by trisomy, rarely by mosaic forms or translocation. Minimal symptoms: low weight at birth (on an average 2100 g), abnormalities of the skull (step-like depression of frontal bones near the fontanel, small lower jaw and mouth, eye slits are narrow and short), deformed auricles, a rocker bottom foot, defects of the heart and large vessels. Prognosis — 60 % of the children die by the age of three months. Down syndrome (47, XX, 21+; 47, XY, 21+) is the most frequent chromosome disorder (1:750). Such children are more often born by mothers 66 of 41–46 years as the probability to give birth to a sick child for them to 4.1 %. The syndrome can be caused by trisomy, translocation or mosaicism. Minimal symptoms: intellectual disability, flat face, short neck, epicanthus, upslanting palpebral fissures, thick lips, thick tongue protruding from the mouth, defects of the cardiovascular system and digestive organs. Life span is about 36 years. Cri-du-chat (cat’s cry) syndrome (46, XX, 5p- or46, XY, 5p-) results from deletion of the short arm of the 5th chromosome. The incidence frequency is 1:45 000. Minimal symptoms: specific cry resembling cat meowing, physical underdevelopment, intellectual disability, microcephaly, moon face, broad nose, short neck, strabismus, low-set ears, abnormalities of occlusion, muscular hypotony. Life span is reduced: only 14 % of the patients live over 10 years.

BASIC TERMS AND CONCEPTS Hemophilia — disease associated with impairment of blood coagulation. Microphthalmia — malformation associated with reduced sizes of the eye-ball. Microcephaly — malformation associated with reduced size of the brain. Monosomy — mutation in which only one homologous chromosome is present in the karyotype instead of a pair. Syndactylia — malformation associated in which phalanges of adjacent fingers fuse together. Trisomy — mutation associated with presence of three homologous chromosome instea of two. Enzymopathy — hereditary metabolic disorder caused by impairments in synthesis and functions of enzymes. Chromosome disorders — complexes of congenital defects caused by the impairment of the structure and number of chromosomes. Ceruloplasmin — the protein providing copper transport in the organism. Epicanthus — skin fold of the upper eyelid covering the inner corner of the eye.

Topic 12. GENETIC COUNSELING

THE AIM AND TASKS OF GENETIC COUNSELING Genetic counseling is advice of individuals with established or potential genetic problems of consequences and nature of the disorder and risks to future offspring. The aim of genetic counseling is the estimation of the genetic risk in the examined families and their counseling.

67 Tasks of genetic counseling: – advice of families and patients having hereditary pathology; – prenatal diagnosis of malformations and hereditary diseases; – assistance to doctors of various specialties in making diagnosis; – making a territorial register of families and patients with hereditary and congenital pathology and their follow-up monitoring; – popularization of medical-genetic knowledge among the population.

STAGES OF MAKING GENETIC PROGNOSIS AND THEIR CHARACTERISTICS 1. Determination of genetic risk. Genetic risk is a probability of appearing a hereditary pathology in a family. There are: low risk — up to 5 %, mild risk — to 10 % medium risk — to 20 % and a high risk — over 20 %. Depending on severity of medical and social consequences of the pathology, the mild, medium and high risks are indications for termination of pregnancy (medical abortion). 2. Estimating the severity of medical and social consequences of the disorder. The degree of genetic risk does not always correspond to a severity of a disease. For example, polydactylia (the risk of inheritance is 50 %) can be easily eliminated by surgery, while phenylketonuria (the risk is 25 % if parents are carriers) is a severe disease and is hardly cured. Social and medical severity of phenylketonuria for the patient and his family are severe. 3. Prenatal diagnostics. The decision concerning the termination of pregnancy is made by the spouses. A doctor may only inform them.

INDICATIONS FOR DIRECTION OF A FAMILY TO GENETIC COUNSELING: – similar hereditary pathology in some members of the family; – sterility and a miscarriage in the first pregnancy; – intellectual disability and arrested physical development of a child; – the 1st child which has malformations; – primary amenorrhea (absence of menstruations) and underdevelopment of secondary sex characters; – contact of spouses with mutagenic factors; – consanguinity of the spouses.

TREATMENT PRINCIPLES OF HUMAN HEREDITARY DISORDERS At the present time, the following approaches are used for treatment of hereditary diseases and diseases with genetic predisposition. 1. Symptomatic treatment. Particular symptoms of all hereditary diseases are treated with medicines: antiinflammatories for inflammatory processes, pain killers for pains, sedatives for states of excitation. Surgical treatment is often used in case of congenital defects such as stenosis and atresia of vessels, polydactylia, heart defects, defects of the skull. 68 2. Pathogenic treatment (for metabolic diseases): – exchange correction — diet therapy (phenylketonuria, galactosemia); – metabolic inhibition — suppression of synthesis of the product which is not excreted from the organism (uric acid in the Lesch-Nyhan syndrome); – substitution therapy — injection of the product that is not produced in the organism (growth hormone in case of dwarfism, insulin in diabetes mellitus). 3. Etiotropic treatment — elimination of the cause of the disease. The most perspective method is gene therapy which to replaces mutated genes by methods of genetic engineering. Gene therapy: 1. Antisense oligonucleotides ‒ short nucleotide sequences complementary to fragments of mRNA or DNA. Linking with target (promoter or mRNA) they block synthesis of a pathologic protein. 2. Ribozymes — polyribonucleotides having enzyme (ribonuclease) activity. Specific nucleotide activity of ribozymes allows to use the nucleotides complementary to mRNA of viruses to destroy them. 3. Implanting genes into the DNA of somatic cells for treating tumors ‒ own tumor cells of patients with genes coding for the tumor necrosis factor or interleukins activating lymphocytes and macrophages are injected).

BASIC TERMS AND CONCEPTS Mild genetic risk — the probability of hereditary pathology in children up to 10 %. Medium genetic risk — the probability of hereditary pathology in children up to 20 %. High genetic risk — the probability of hereditary pathology in children over 20 %. Diet therapy — treatment by a diet. Metabolic inhibition — suppression of synthesis of the product which is not excreted from the organism. Gene therapy — treatment using methods of genetic engineering. Substitution therapy — injection metabolites which are not produced in the organism because of a pathology. Pathogenic therapy — therapy that interrupts the development of the disorder but not eliminates its cause. Symptomatic therapy — elimination of symptoms of a disorder without effect on its cause and mechanism of its development. Etiotropic therapy — treatment that eliminates of the cause of the disease.

69 Topic 13. REPRODUCTION OF LIVING MATTER

REPRODUCTION AS ESSENTIAL PROPERTY OF LIVING MATTER Reproduction is a universal property of living matter which provides multiplication of organisms and is based on transmission of genetic information from generation to generation. Basis of reproduction at the molecular level is DNA replication, at the subcellular level — duplication of some organelles, at the cellular level — cell division. The division of cells is basis for reproduction of living matter.

TYPES OF REPRODUCTION Reproduction can be sexual or asexual. Characteristics of asexual reproduction (figure 38) are: – one individual participates in reproduction; – origin of genetic material is somatic cell; – genotypes of daughter individuals are same as parental one; – the number of individuals increases quickly; – optimal for living in constant environmental conditions. Asexual reproduction

Vegetative (by body parts) Spore formation (by spores)

in unicellular in multicellular organisms organisms

in plants in animals Fig. 38. Asexual reproduction

Asexual reproduction of unicellular organisms: a) binary fission — longitudinal (Euglena), transverse (Paramecium caudatum); b) schizogony — multiple division: nucleus divide into several parts, then cytoplasm divides (malaria parasite); c) budding — a bud forms on the mother cell; it grows and ultimately separates from the mother individual (yeast). Asexual reproduction of multicellular organisms: a) plants — vegetative reproduction (by vegetative organ: roots, stems, leafs); b) animals: – budding (hydra); – fragmentation division of the body into several parts (annelids); – polyembryony — division of a zygote or embryo into parts which form separate organisms (flukes). 70 Sporogenesis: special organs (sporogonia) form spores that give rise to a new organism (water plants, mushrooms, mosses, lycopodia, horsetails, ferns). Characteristics of sexual reproduction (figure 39): – two individuals take part in reproduction (except self-fertilization); – parental sex cells are a source of genetic information; – genotypes of descendants differ from the parental ones due to combinative variation; – provides the adaptability of organisms to changing environmental conditions. Sexual reproduction

With fertilization Without fertilization (gametic copulation) (partenogenesis)

androgenesis gynogenesis Fig. 39. Sexual reproduction

GAMETOGENESIS (OOGENESIS AND SPERMATOGENESIS) Species that have sexual reproduction can be dioecious (with separate males and females) or hermaphrodites (have both male and female reproductive organs. As hermaphrodites have both male and female gonads, they form both spermatozoa and ova. Such hermaphroditism occurs in flatworms and ringworms. It is a true hermaphroditism. In case of pseudohermaphroditism, an individual has sex organs and secondary sex characters of both sexes, but gonads of only one sex (male or female). The human may have false hermaphroditism as result of disorders. Dioecious species have only female or only male gonads. Males and females are characterized by the characters of sexual dimorphism: differences in body sizes, coloration, structure, voice specificities, behavior and other characters. The characters of sexual dimorphism in the human are: peculiarities of the musculoskeletal system, distribution of subcutaneous adipose tissue, degree of hair covering, voice quality, peculiarities of behavior, etc. Gametogenesis is a process of gamete formation: diploid somatic cells transform into haploid sex cells. Formation of spermatozoa is spermatogenesis; formation of ova is oogenesis (figure 40). Peculiarities of human gametogenesis: 1. Mitotic division of oogonies is finished before the birth of the organism. Mitosis of spermatogonies starts with puberty. 2. The growth period of oogenesis is very considerable. 3. In oogenesis, the 1st meiotic division stops at diakinesis until puberty is reached. The 2nd division of meiosis stops at the metaphase and resumes after fertilization.

71 4. Unlike in spermatogenesis, period of transformation is absent in oogenesis. 5. A newborn girl has about 30 000 oocytes in the ovaries; only 300–600 of them reach their maturity (about 13 cells a year). 6. A male organism produces up to 500 billion spermatozoa during the period of sexual life.

Genetic Cells names Spermatogenesis Ovogenesis Cells names Periods information

2n2chr4c Spermatogonia Ovogonia Proliferation (mitosis)

2n2chr4c Primary Primary spermatocytes ovocytes Growth

1n2chr2c Secondary Secondary Maturation spermatocytes ovocytes (meiosis) 1n1chr1c 1n1chr1c Spermatides Transformation 1n1chr1c Spermatozoa Ovum

Fig. 40. Gametogenesis

INSEMINATION AND ITS TYPES. FERTILIZATION AND ITS STAGES The processes providing contact of female and male gametes are called insemination. The insemination is followed by fertilization — fusion of gametes into a zygote. Insemination of water animals occurs in water: gametes are excreted and fuse there (consequently external fertilization occurs). Insemination of terrestrial animals requires delivery of male gametes into reproductive tracts of females. Fertilization of such animals is internal. Contact of gametes is provided by: – opposite charges of gametes; – movement of spermatozoa and contraction of wall of female reproductive tracts; – ovum secretes gynogamones and spermatozoa have positive chemotaxis for them. External stage of fertilization is entrance of a spermatozoon into the ovum. During the contact with the ovum, secretion of acrosome containing hyaluronidase is excreted. This enzyme dissolves the membrane of the ovum, an acrosomal process stretch from the acrosome; it penetrates membranes of the ovum and fuses with its membrane. A receptive spot is formed in this area of the ovum. It encloses the head and centriole of the spermatozoon and takes them

72 into the ovum’s cytoplasm. In case of monospermy the ovum can be fertilized by only one spermatozoon (mammals). Fertilization membrane is formed on the surfase of the ovum and another spermatozoa cannot pass through it. In case of polyspermy several spermatozoa enter the ovum (insects, fishes, birds). Internal stage of fertilization is associated with karyogamy — fusion of haploid pronuclei (nuclei of fused gametes in the ovum’s cytoplasm) into a diploid nucleus. The male pronucleus (nucleus of the spermatozoon) enlarges to the sizes of a female pronucleus (nucleus of the ovum), turns through 180° and moves (together with the centrosome at its front end) to the female pronucleus. They unite to form diploid chromosome set. There is a phenomenon of parthenogenesis. It is development of an organism from an unfertilized ovum. A natural partenogenesis occurs in lower cancroids, bees, butterflies, rock lizards. Nuclei of somatic cells in such individuals can be haploid. Diploid chromosome set can be restored by fusion of the ovum’s nucleus with the nucleus of a directing body.

BIOLOGICAL PECULIARITIES OF HUMAN REPRODUCTION 1. The human is not only biological but a social being. 2. The ability for reproduction is acquired with puberty. Its signs are first periods in girls (on an average from 12–15 years) and pollutions in boys (from 13–16 years). 3. Reproductive period in women lasts till 40–45 years, in men — till old age (gamete production by the testes occurs during the whole life). 4. During one intercourse about 200 million of spermatozoa are released with the semen fluid. 5. Since puberty female organism produces one secondary oocyte a moon month. 6. Fertilization occurs in upper parts of a fallopian tube, usually within 12 hours after ovulation. 7. Spermatozoa are able for fertilization during 1–2 days after getting into the female reproductive tract. 8. Human reproduction, unlike that of animals, is not seasonal. It depends on a number of social-economic factors. 9. The human can regulate birthrate.

BASIC TERMS AND CONCEPTS Acrosome — modified Golgi complex of a spermatozoon providing its entrance into the ovum. Anisogamy — form of sexual process in which gamets that fuse together are morphologically different.

73 Gynogenesis — type of sexual reproduction in which male and female pronuclei do not contact and only the female nucleus is used for the development of a zygote. Copulation — sexual process in which genetic information of two unicellular organisms fuse. Oogenesis — process of development and maturation of ova. Insemination — processes providing contact of gametes. Sexual process — exchange of genetic information between two cells or fusion of the genetic information of two cells which does not increase the number of individuals. Pronucleus — nucleus of ova or spermatozoon before their fusion. Synkaryon — nucleus of a zygote. Spermatogenesis — process of spermatozoa development.

Topic 14. FUNDAMENTALS OF ONTOGENESIS

ONTOGENESIS, ITS TYPES AND PERIODS Ontogenesis is individual development of an organism from formation of a zygote to death. Division of ontogenesis into periods (figure 41). Periods of ontogenesis

progenesis prenatal ontogenesis postnatal ontogenesis Fig. 41. Periods of ontogenesis

Progenesis (prezygotic period) is a period of formation and maturation of those parental gametes that formed a zygote. Prenatal ontogenesis starts at the moment of a zygote formation and ends with birth of a new organism or when it leaves the egg. Postnatal ontogenesis period that begins at the moment of birth or hatching from an egg and ends with death. There are several types of organism development (table 4). Table 4 Types of development Direct development Indirect development (with metamorphosis) (without metamorphosis) Laying eggs with a lot of Stages of incomplete metamorphosis: yolk (birds) egg – larva – mature individual (intestinal helminthes) Intrauterine development Stages of complete metamorphosis: (mammals) egg – larva – pupa – mature individual (butterflies, dipterans)

74 Human prenatal development includes: – Germinative or initial period is the 1st week after fertilization, when cleavage of a zygote takes place; – Embryonic period — the 2nd–3rd weeks after fertilization when a blastula and gastrula are formed; formation of germ layers and anlagen of axial organs takes place; – Prefetal period — the 4th–8th weeks, when formation of organ systems and placenta takes place; – Fetal period — since the 9th week the embryo is called fetus; growth of the fetus and formation of organs and organ systems take place. Periods of postnatal ontogenesis are 1. Neonatal period (1–10 days) is a complex period when reconfiguration of the whole organism occurs in order to adapt to new existence conditions. 2. Infancy, or breastfeeding period (11 days – 12 months). A child is feed with mother’s milk. The baby grows rapidly. 3. Early childhood (1–3 years). The child learns to walk and speak, gets acquainted with the world around. 4. The 1st period of childhood (4–6 years). The child is interested in everything and tries to understand everything, get hang of basic game skills. 5. The 2nd period of childhood (7–11 years in girls, 7–12 years in boys). The growth slows, intensive development of the muscular system occurs. In this period children go to school. 6. Puberty, or adolescence (12–15 years in girls, 13–16 years in boys) Sexual maturation starts and growth speed intensity increases. 7. Youths (16–20 years in girls, 17–21 years in young men) Sexual maturation, growth and physical development have completed. 8. 1st period of middle age (21–35 years in women, 22–35 years in men) an optimal period for childbirth; mastering professional skills. 9. 2nd period of middle age (36–55 years in women, 36–60 years in men) is a period of the most active professional activity. The first signs of ageing appear after 35 years). 10. Advanced age (56–75 years in women, 61–75 years in men). The processes of aging are going on; this is the age of retirement. 11. Senile age (76–90 years) Senile changes are marked; some people still can work creatively at this age. 12. Longevity (over 90 years).

CHARACTERISTIC OF PROGENESIS Progenesis is a prezygotic maturation of a female gamete that is a basis for a zygote. It starts in the embryonic period of the mother’s organism and is finished when a sperm delivers its genetic material to the cell. That is why the older is the woman, the longer is this period. Usually its length coincides

75 with the mother’s age. Progenesis of a spermatozoon lasts about 70 days. The quality of gametes and possible mutations of their genes have a considerable effect on health of future children.

STAGES OF EMBRYOGENESIS (CLEAVAGE, GASTRULATION, HYSTO- AND ORGANOGENESIS). PROVISIONAL ORGANS OF CHORDATES. PECULIARITIES OF EMBRYONIC DEVELOPMENT OF HUMAN Zygote is a unicellular development stage of a multicellular organism; it is formed after fusion of male and female gametes. The cleavage type of a zygote depends on the type of the ovum. The type of the ovum depends on amount and distribution of yolk (nutrients) it contains. Daughter cells of the zygote that are formed during the cleavage are blastomeres. Some animals have the development stage when the cleaving zygote resembles a raspberry — morula. Eventually blastula forms. It is embryo with layer of blastomeres and cavity inside. The layer of cells is called blastoderm; the cavity of the blastula is blastocoel. The stage of blastula is followed by gastrullation — formation of gastrula. Gastrula is an embryo having two (or three) layers of cells called germ layers. They give rise to all tissues and organs. There are different types of gastrulation (figure 42).

Fig. 42. Types of gastrulation

1. Invagination. The vegetative pole of the blastula is drawn inside to the inner side of the animal pole. A bilaminar (double-layer) embryo is formed. The external layer is ectoderm, the internal one is endoderm. The cavity of the gastrula is called a gastrocoel (primary intestine). The entrance to this intestine is a primary mouth or blastopore. Its edges form upper and lower lips of the blastopore. In the secondary-mouthed 76 (echinodermata, chordates) it transforms into anal opening and the mouth is formed at an opposite end of the embryo. 2. Ingression (immigration) — movement of some cells into the cavity for formation of the second germ layer (endoderm). This way is characteristic of coelenterate. 3. Epiboly is typical for telolecithal ova having much yolk on the vegetative pole. Cells of the animal pole divide faster than cells of the vegetative pole, which form an endoderm. 4. Delamination. All cells of a layer divide parallelly to its surface and form two germ layers: ectoderm and endoderm. Single type of gastrulation is seldom present and has some peculiarities, humans gastrulation is «mixed» — several types are combined simultaneously. Peculiarities of embryonic development of human. Gastrulation of human and mammals has two stages. The first stage is delamination taking place at the period of 7th–14th days. Cells of embryoblast divide into two layers — epiblast (gives rise to ectoderm, neural plate and notochord) and hypoblast (gives rise to endoderm). At the same period provisional organs form (amnion, chorion, yolk sac). During the first stage of gastrulation, implantation occurs: embryo adheres to the wall of the uterus (7th–9th days). The second stage of gastrulation takes place since 4th till 21st days by means of ingression. The ingesting cells form the third germ layer mesoderm, anlagen of axial organs (neural tube and notochord) and a provisional organ allantois. Mesoderm is not unique for human: all animals except sponges and coelenterates have three germ layers. The most common approaches to from anlage of mesoderm are teloblastic and enterocoelous gastrulation. In case of teloblastic gastrulation large cells teloblasts are formed on the two sides of the blastopore. They start dividing; small cells settle between the ectoderm and endoderm and form mesoderm. Such gastrulation is characteristic of invertebrates. The enterocoelous gastrulation is typical for chordates. Ingrowths (coelomic sacs) form on the two sides of the primary intestine. They separate from the primary intestine, grow between the ectoderm and endoderm and give rise to mesoderm. After the formation of germinal layers hystogenesis (formation of tissues) and organogenesis (formation of organs) occur, anlagen of axial organs are formed. Ectoderm gives rise to integument, central nervous system, proximal and diatal regions of the alimentary tube. Endoderm gives rise to notochord, middle of alimentary tube, liver, pancreas and respiratory system.

77 Mesoderm gives rise to connective and muscle tissues, skeleton and skeletal muscles, dermis, dentine, genitourinary system, smooth muscles, heart and blood vessels, blood and lymphatic system. Provisional (temporary) organs of embryo and fetus are: Amnion is a sac with fluid which forms aquatic environment for the embryo and fetus, protects it from drying out and injury. Chorion is the external covering contacting with the shell of an egg or mother’s tissues. It provides exchange with environment and participates in formation of placenta. Yolk sac participates in feeding of the embryo and is the first hematopoietic (blood-creating) organ. Allantois is an outgrowth of the posterior region of the gut. It is a reservoir for urea and uric acid. It participates in formation of placenta in mammals as well.

REALIZATION OF GENETIC INFORMATION DURING PRENATAL ONTOGENESIS. MECHANISMS OF EMBRYOGENESIS AND MORPHOGENESIS Realization of genetic information during prenatal ontogenesis. Genetic information of DNA is realized through mRNA causing creation of enzymes or another types of proteins; these proteins define presence or absence of various traits. Gene expression depends on other genes. They may have effect on activity of a gene and consequently on a trait. The gene can have effect on expression of other genes. Realization of gene action also depends on environmental factors that may change structure of DNA, mRNA, proteins and change phenotypic manifestations of the gene (figure 43). Other genes

Gene mRNA protein-enzyme biochemical character (DNA) reaction

Other characters

Environmental factors

Fig. 43. Realization of genetic information during prenatal development

Mechanisms of embryogenesis: 1. Differential activity of genes during the embryonic development various groups of genes are activated and deactivated in certain order. 2. Determination. This means that cells «choose» specific way of development and can transform only into certain types of cells. At the initial stages of embryogenesis blastomeres are totipotent (they can give rise to a whole

78 organism). Their development depends on external inductors and adjacent cells. At later stages of embryogenesis, the cells become determined and can develop only according to a plan. 3. Differentiation is the process of biochemical, functional and morphological specialization of cells when relatively uniform formations become more and more different. Stages of differentiation: – dependent differentiation (till the stage of early gastrula) cells are totipotent and their development depends on adjacent cells; independent differentiation (at the stage of late gastrula) cells are determined and can transform into only certain types of cells. Genetic basis of differentiation. Genetic differentiation is associated with unique property ovum — heterogeneity of the cytoplasm. It means that regions of the cytoplasm have different complements of chemical substances and have different potencies (figure 44). Chemically heterogeneous cytoplasm of the ovum (increases after fertilization) ↓ Chemically heterogeneous cytoplasm in blastomeres ↓ Different blastomeres contain different inductors ↓ Different inductors activate different transcriptons ↓ Different enzymes are synthesized to catalyze different biochemical reactions ↓ Cells produce different tissue-specific proteins ↓ Cells become different ↓ Different types of cells form different tissues ↓ Different tissues form different organs Fig. 44. Stages of differentiation

4. Morphogenesis is a process when new structures appear and change in course of embryonic development. Mechanisms of morphogenesis: 1. Embryonic induction is influence of a group of embryonic cells on adjacent cells (G. Shpeman, G. Mangold). The primary inductor (an upper lip of the blastopore) determines creation of nerve tube from ectoderm on the dorsal side of the embryo, then creation of the notochord bellow the tube is caused and then formation of the alimentary tube.

79 2. Morphogenetic fields (A. G. Gurvich) are fields formed by groups of cells which can respond to certain localized biochemical signals and develop into certain anatomical structures. 3. Gradient of physiological activity (Ch. Child) — intensity metabolism in the anterior region (head) of the embryo is higher than that in the caudal one. 4. Positional information of the cell — due to intercellular interactions, every cell «estimates its own position» in the anlage of an organ and then differentiates according to this position.

CRITICAL PERIODS OF THE ONTOGENESIS. TERATOGENS Periods of the maximal sensitivity of the embryo or fetus to environmental factors are called critical periods. The human has 3 main critical periods in embryogenesis: 1) implantation of an embryo in the mucous membrane of the uterus (6th–7th day after fertilization); 2) placentation — beginning of the placenta formation (14th–15th day after fertilization); 3) delivery — at this period reconfiguration of all organ systems occurs (39th–40th weeks). In critical periods organism undergoes crucial functional changes and readjusts to new conditions of existence. The impairment of the course of embryogenesis caused by environmental factors is called teratogenesis (Greek teras — monster). Factors causing teratogenesis are teratogens. They are medicines (antibiotics, quinine, chloride, anti-depressants, etc.), alcohol, nicotine, waste products of parasites, ionizing radiation. Causes and development mechanisms of malformations are studied by teratology. Incidence frequency of malformations in human populations is 1–2 %. Variants of congenital development defects: – aplasia — organ is not laid down; – hypoplasia — underdevelopment of a tissue or organ; – hypotrophy — degeneration of an organ or tissue caused by loss of cells; – hypertrophy — increase in volume of a tissue or organ produced entirely by enlargement of existing cells; – heterotopy — normal tissue is misplaced; – atresia — the absence or closure of a normal body orifice or tubular passage; – stenosis — constriction or narrowing of a duct or passage. There are 3 critical periods in the postnatal human ontogenesis: 1. Neonatal period (the first days after birth) — reconfiguration of all organ systems for a new environment.

80 2. Puberty period (12–16 years) — a hormonal readjustment, formation of secondary sex characters. 3. Period of sexual involution (about 50 years in women, 60–70 years in men) — reproductive function fades functional depression of gonads and endocrine glands occur.

GROWTH. GROWTH TYPES OF HUMAN TISSUES AND ORGANS. ACCELERATION Growth the process providing enlargement of sizes and mass of the body. The growth can be unlimited and limited. Unlimited growth lasts all the life (crawfishes, fish and reptiles) while limited one stops at the certain age (insects, birds, mammals) (figure 45). Regulation of growth

Somatotropin (hypophysis); Environmental factors: light, food, vitamins (А, В, thyroxin (thyroid); sex hormones D), microelements, social and economic factors Fig. 45. Regulation of tissue growth

Speed of body growth is not uniform. Periods of the most intensive body growth are the first year of life: body length increases by 25 cm. In the 2nd year it increases approximately by 10–11 cm, in the 3rd by 8 cm, from 4 to 7 years by 5–7 cm per year, during 2nd period of childhood it increases by 4–5 cm per year. The second period of intensive growth is puberty. During this period the growth speed increases to 7–8 cm a year and then slows down and is 1–2 cm per year till the 20–25 years. Speed of growth is not same for different tissues and body parts. Basic growth types for tissues and organs are: – General type of growth. The whole body, muscles, skeleton, respiratory organs, liver grow intensively during the 1st year of life puberty; – Lymphoid type of growth. The thymus, lymph nodes and the lymphoid tissue of the intestine, spleen, tonsils reach their maximal size by 11–12 years and then their volume decrease; – Cerebral type of growth. The brain, spinal cord, eyes, head develop earlier than other parts of the body and reach sizes characteristic of adults by 10–12 years; – Reproductive type of growth. Organs of the reproductive system grow rapidly during puberty. Somatotropic hormone (growth hormone) is produced by hypophysis. Its intensive production occurs since birth till 13–16 years. Hypofunction of hypophysis causes, pituitary dwarfism, hyperfunction causes giantism and human height can surpass 2m. Production of hormone in adult age causes acromegalia — enlargement of bones of palms, feet and face.

81 Thyroxin enhances energy exchange in the body. Hypofunction of thyroid gland causes delayed growth, delayed puberty, impairment of body proportions, mental disturbance. Sex hormones have effect on metabolic processes as well. Environmental factors also have considerable effect on growth. Normal growth of a child requires balanced meal with vitamins and microelements. Synthesis of vitamin D is influenced by sunlight. In recent decades, acceleration of physical and mental development of children and adolescents is observed. It is marked even at the stage of intrauterine development — body length of newborns increased 0.5–1.0 cm, body mass — 50–100 g, the terms of teeth cutting out changed. The human height has increased on an average by 8 cm over the recent 100 years. The numerous factors were supposed to cause acceleration: mixed marriages (increase the heterozygosity), better food, urbanization, increased background radiation, a number of social factors and even changes in the Earth magnet field. Human age: 1. Biological age — correspondence of body functional capacities to certain age or the age person looks. 2. Chronological age — the number of years a person has lived or passport age. Criteria for determination of a biological age: – skeletal maturity: ossification of cartilaginous regions and growth zones; – teeth maturity: appearance of milk teeth and their replacement with permanent ones; – time when secondary sex characters appear and their development degree.

HUMAN CONSTITUTION AND HABITUS Constitution of human are genetically conditioned peculiarities of human morphology, physiology and behavior. In 1927 M. V. Chernorutsky proposed the classification including three types of constitution: Ectomorphic type (asthenics): a narrow chest, low position of the diaphragm, elongated lungs, short intestine provides lower absorption, thin bones and long extremities, thin layer of subcutaneous fat. Statistically, asthenics are characterized by high excitability. They more often have neuroses, hypotonia, ulcers, tuberculosis. Mesomorphic type (sthenics): balanced constitution, moderate development of the subcutaneous fat tissue. They are usually people of action; more often have neuralgias, atherosclerosis and diseases of the upper airways. Endomorphic type (hypersthenics): a broad chest, voluminous stomach and long intestine, considerable fat tissue. The amounts of cholesterol, uric acid, erythrocytes and hemoglobin in the blood are higher than in other constitution

82 types. Assimilation processes predominate. Hyperstenics have tendencies to obesity, diabetes mellitus, hyper-tension, diseases of kidneys and gallbladder. Habitus includes peculiarities of morphology, physiology and behavior in a definite period. Habitus shows overall condition of a person and his health at a given moment. It includes: peculiarities of the constitution, pose, bearing, gait, skin color, facial expression, concordance of a biological and chronological age.

AGEING. BASIC THEORIES OF AGEING Ageing is a common biological regularity characteristic of all living organisms. Old age is a final stage of ontogenesis. The science about ageing and old age is called Gerontology. It studies regularities of ageing of various organ systems and tissues. Geriatrics is a science about diseases of old people. It studies peculiarities of their development, course, treatment and prophylaxis. There are more than 300 hypotheses of ageing. The most common of them are. 1. Energetic theory (M. Rubner, 1908): the organism of each species has a definite energetic fund. It is being spent during the life and then the organism dies. 2. Intoxication theory (I. Mechnikov, 1903): self-poisoning of the organism due to accumulation of products of nitrogenous exchange and putrefaction in the intestine. 3. Theory associated with the connective tissue (A. Bogomolets, 1922): the connective tissue is a nutrition regulator of cells and tissues; its changes impair the inter-tissue interactions and result in ageing. 4. Overstrain of the central nervous system (I. Pavlov, 1912. G. Celie, 1936): stress and long nervous strain cause ageing. 5. Changes of colloidal properties of the cell cytoplasm (V. Ruzhichka, M. Marinesku, 1922): cytoplasm modifies and does not retain water properly, hydrophilic colloids transform into hydrophobic ones, colloidal particles become bigger and their biological properties change. 6. Predetermined number of cells mitoses (A. Heiflick, 1965): different species have different numbers of possible cell divisions: human fibroblasts of embryos can form about 50 generations (those of mice and hen has about 15 generations). 7. Genetic theory is associated with accumulation of mutations, decreasing of its intensity and impairment of DNA transcription, translation and repair; impairment of self-renewal of proteins. Social factors: living conditions, lifestyle and various diseases considerably impact ageing. Ageing and the life span depend also on the ecological situation. The science that studies a healthy lifestyle and conditions increasing human life span is called Valeology.

83 The theoretically possible human age is 150–200 years; the maximum registered one is 115–120 years. An average life span of men in Belarus is 62–70 years, that of women — 72–79 years.

CLINICAL AND BIOLOGICAL DEATH. REANIMATION. EUTHANASIA Ageing of the organism is terminated by death. Death provides alternation of generations. Causes of death can be different. Physiological death, or natural death, occurs due to ageing. Pathological death, or untimely death, is the result of a disease or an accident. Clinical death occurs as a result of termination of vital functions (heart or respiration failure), but processes of substances exchange in the cells and organs are retained. Biological death is termination of processes of self-renewal in cells and tissues, impairment of chemical processes, autolysis and decay of cells. In the most sensitive cells of the cerebral cortex, necrotic changes are revealed already in 5–6 minutes after clinical death. Prolongation of the period of clinical death is possible by using general hypothermia of the organism that slows down metabolic processes and increases the resistance to anoxia. Reanimation is complex of actions performed to return a person to life from the state of a clinical death (when vital organs are not impaired) within 5–6 minutes while cells of the brain are still alive. Reanimation methods are used in medicine in any threatening conditions. Euthanasia is a medical assistance to pass from life for a terminally ill patient at his will or request of his relatives. Euthanasia is allowed by law only in some countries.

BASIC TERMS AND CONCEPTS Acceleration — speeding-up of physical and mental development of new generations of children and adolescents. Valeology — a science that studies a healthy lifestyle and conditions for increasing the life span. Biological age — the number of years a person looks. Chronological age — age confirmed by documents. Critical periods — periods of prenatal ontogenesis when the embryo or fetus is particularly sensitive to environmental factors. Human habitus — peculiarities of morphology, physiology, behavior in a definite time moment. Human constitution — genetically conditioned peculiarities of human morphology, physiology and behavior Morphogenetic fields — are fields formed by groups of cells which can respond to certain localized biochemical signals and develop into certain anatomical structures. 84 Ontogenesis — individual development of an organism from the moment of zygote formation till death. Progenesis — period of formation and maturation of those parental gametes that formed a zygote. Geriatrics — science that studies diseases of old people, peculiarities of their development, course, treatment and prophylaxis. Gerontology — science about aging and old age Reanimation — complex of actions performed to return a person to life from the state of a clinical death.

Topic 15. EVOLUTION OF ORGAN SYSTEMS

CONNECTION OF THE ONTOGENESIS AND PHYLOGENESIS, BIOGENETIC LAW, A. N. SEWERTZOFF’S THEORY ABOUT PHYLEMBRYOGENESES Ontogenesis is individual development of an organism or all the development processes of an individual from the moment of zygote formation to death. This development proceeds due to expression of genetic information received from parents. Environmental conditions have considerable effect on this expression and development of characters. Phylogenesis is the evolution history of a species. Ontogenesis and phylogenesis are closely connected. Knowledge of phylogenesis explains ontogenesis and mechanism of malformations that can develop during prenatal ontogenesis. Correlation of ontogenesis and phylogenesis was shown by Karl Ernst von Baer in 1828 when he formulated the following laws: Law of embryonic similarity — the embryo of a higher form (i. e. higher animal) never resembles any other form, but only its embryo. Law of successive appearance of characters — the more general characters of a large group appear earlier in the embryo than the more special characters. Law of embryonic divergence — every embryo of a given animal form, instead of passing through the other forms, rather becomes separated from them. This laws state that early stages of embryogenesis of vertebrates (such as fishes, birds, mammals) are very similar. In course of time they get differentiated and acquire traits of their classes, then those of their orders and etc. In 1866 Ernst Haeckel formulated the biogenetic law: ontogenesis is a short and fast repeat of phylogenesis; though not adult ancestral stages repeat, but traits of their embryos. Ch. Darvin confirmed the correlation between onto- and phylogenesis and developed the theory of recapitulations. Recapitulation is a repeat of ancestral characters in embryos. For example lying down and development of

85 the respiratory system in a mammal embryo undergoes the stages when gill slits, then gills and only then the lungs are formed. A. N. Severtsev elaborated the theory of phylembryogeneses. This theory explains relations between ontogenesis and phylogenesis. Phylembryogenesis is an embryonic reconstruction that is preserved in adults and has adaptive nature. There are 3 types of phylembryogeneses: 1) archallaxis is an early deviation from ancestral developmental pattern that occurs simultaneously with formation of the organ anlage (an example is development of a hair coat in mammals). Mutated genes get involved in morphogenesis at its initial stages and make the new course for development of the organ (recapitulations are absent); 2) deviation development that begins in accordance with ancestral pattern and deviate in the middle of the course (an example is development of scales in reptiles). Initially morphogenesis proceeds according to ancestral patterns (partial recapitulation) but later on mutated genes activate and make new course for the organ’s development. 3) anaboly development that follows ancestral pattern up to its last stage and then new stages are added (a two-chambered heart into a four-chamber heart). At first all stages of the organ development recapitulate, and then mutated genes activate to form new character. In cases of some malformations the body acquires characteristics of another orders or classes of chordates. They appear due to ontophylogenetic mechanisms such as recapitulations and parallelisms. Recapitulations occur as a result of incomplete anaboly or its absence. Examples of such disorders are three- chambered heart, preservation of embryonic vessels, two aortal arches, arrested development of kidneys, duplication of ureters. Parallelism is independent development of similar characters in closely related species during their evolution (human and animals that have similar origin). An example of parallelism in human is polymastia (abnormal number of nipples).

EVOLUTION OF THE NERVOUS SYSTEM IN CHORDATES The nervous system originates from ectoderm and forms as a nerve tube. Basic directions of evolution: 1. Differentiation of the nerve tube into the brain and the spinal cord. 2. Evolution of the brain: a) transformation of 3 brain vesicles into 5 brain vesicles and therefore 5 brain regions; b) appearance of the cerebral cortex and enlargement of its surface due to its sulci (grooves) and gyri (folds); c) transformation of the ichthyopsidian brain into sauropsidian one and ultimately into mammalian brain. 3. Differentiation of the peripheral nervous system.

86 The CNS of lancelet is nerve tube. Its anterior part is dilated and has olfactory pit. Photosensitive cells (Hesse organs) are located throughout the whole length of the tube. The brain of mammals consists of 5 regions. It undergoes same stages during its formation. At first the nerve tube is formed and 3 brain vesicles appear in its anteior end: forebrain (prosencephalon), midbrain (mesencephalon) and hindbrain (rhombencephalon). Then the forebrain and hindbrain divide to form 5 brain vesicles which transform into a certain brain region: cerebrum (telencephalon), interbrain (diencephalon), midbrain (mesencephalon), pons and cerebellum (metencephalon) and medulla oblongata (myelencephalon). There are cavities in the bran (cerebral ventricles) that are followed by the spinal canal in the spinal cord. The part of the brain located above the ventricles is called roof and the part below is the floor of the brain. The brain of fishes is small. The cerebrum is not divided into hemispheres. The roof is epithelial; the floor of the brain consists of striate bodies. Olfactory lobes are small. The interbrain consists of thalamus and hypothalamus. The midbrain is large as it is the integrating center of the CNS (ichthyopsidian type of the brain). A flexure appears in the area of the midbrain. The cerebellum is developed well. There are 10 pairs of cranial nerves (figure 46).

5 2 6 2 5 6 1 9 9 1

8 3 7 4 3 7 B А 8 4 2 5 6 2 5 6 9 9 1

1

7 3 D 8 4 C 4 7 8 3 Fig. 46. The brain of vertebrates (longitudinal section): A — bony fish, B — amphibian, C — reptile, D — mammal: 1 — forebrain; 2 — epiphysis; 3 — hypophysis; 4 — interbrain; 5 — midbrain; 6 — cerebellum; 7 — medulla oblongata; 8 — striated bodies; 9 — roof

In amphibians: 1) volume of the forebrain increases; 2) cerebrum divides into 2 hemispheres; 3) nervous tissue appears in the brain roof; 4) striated bodies are well developed. Olfactory lobes are separated from the hemispheres. The interbrain consists of thalamus and hypothalamus. The midbrain is large and 87 still serves as the integrating center. The cerebellum is poorly developed. The medulla oblongata is developed same as in fish. There are 10 pairs of cranial nerves. In reptiles cerebrum is the largest brain region. Large olfactory lobes are differentiated, parietal lobes are separated. Hemispheres of the brain have primordial cortex on their lateral surfaces. The structure of the cortex is primitive (3 layers of cells) — archipallium. The striated bodies of the forebrain serve as the integrating center. Such type of the brain is called sauropsidian (striatal). The size of the midbrain is lower than in amphibians (it is no longer the integrating center of the brain). The cerebellum is considerably larger than that of amphibians. The medulla oblongata forms a sharp flexure in the vertical plane. There are 12 pairs of cranial nerves. In mammals the forebrain reaches maximal development due to the secondary cortex (neopallium). In lower mammals the surface of the cortex is smooth, in higher mammals it has sulci and gyri. The secondary cortex is an integrating center (mammalian type of the brain). The forebrain covers the interbrain. The size of the midbrain decrease. This region consists of quadrigemina (2 superior colliculi are subcortical centers of vision, 2 inferior colliculi are subcortical centers of hearing). The cerebellum is considerably larger. It is differentiated into two hemispheres with the vermis in the middle. The brain has 12 cranial nerves. There are 3 flexures of the brain: 1) cephalic flexure at the level of the midbrain, 2) cervical flexure in the region where the medulla oblongata passes into the spinal cord, 3) pontine flexure in the area of the hindbrain.

EVOLUTION OF THE CIRCULATORY SYSTEM OF CHORDATES The circulatory system originates from mesoderm. Basic directions of evolution: 1. Appearance and differentiation of the heart (change of two-chambered heart into the four-chambered one). 2. Appearance of the 2nd (pulmonary) circulation and a complete separation of venous and arterial blood. 3. Transformation of branchial arteries (arterial arches) and differentiation of vessels following from the heart. Lancelet has one circulation. The abdominal aorta carrying venous blood forms afferent branchial arteries (their number corresponds to the number of branchial arches — up to 150 pairs), where it gets enriched with oxygen. Through efferent branchial arteries blood flows to left and right branches of dorsal aorta. Anterior parts of the branches proceed into carotid arteries and carry blood to the anterior region of the body; posterior parts of the branches join together and form the dorsal aorta that divides into multiple arteries carrying blood to all organs (figure 47).

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Fig. 47. The circulatory system of the Lancelet: 1 — abdominal aorta; 2 — afferent branchial arteries; 3 — efferent branchial arteries; 4 — branches of a dorsal aorta; 5 — carotid arteries; 6 — dorsal aorta; 7 — intestinal artery; 8 — subintestinal vein; 9 — liver portal vein; 10 — hepatic vein; 11 — right posterior cardial vein; 12 — right anterior cardial vein; 13 — left Cuvier’s duct

After gas exchange the venous blood accumulates in paired anterior and posterior cardial veins located symmetrically. The anterior and posterior cardial veins join together into the Cuvier’s ducts. They empty into the abdominal aorta. Portal system is formed near the hepatic cecum. Blood from there passes through the hepatic vein into the abdominal aorta. Fishes have one circulation. The heart is located beneath the mandible and consists of two chambers (atrium and ventricle) filled with venous blood. A venous sinus borders upon the atrium; an arterial cone follows by the ventricle and passes into the abdominal aorta. Anlages of 5th–7th pairs of branchial arteries are formed during embryogenesis but then the 1st, 2nd and 7th are reduced, and only the 3rd–6th pairs continue functioning. Due to appearance of lungs, 2nd circulation develops in amphibians. The heart consists of two atria and one ventricle. A venous sinus borders upon the right atrium, an arterial cone follows by the ventricle (figure 48). The atria open into the ventricle with one aperture. Venous and arterial blood come from the right and left atria. Blood in the right part of the ventricle is venous, mixed in the center and arterial in the left part. The blood is distributed into 3 pairs of vessels through the arterial cone: venous blood goes to the skin and lungs through the pulmocutaneous arteries; mixed blood goes to all organs through aortal arches; arterial blood goes to the brain through carotid arteries. Anlages of 6th–7th pairs of branchial arteries are formed in embryo- genesis and then the 1st, 2nd, 5th and 7th are reduced. The 3rd one transforms into carotid arteries, the 4th one form arches of the aorta, the 6th — pulmocutaneous arteries (figure 49). In reptiles the heart consists of 3 chambers, an incomplete septum appears in the ventricle. The pulmonary artery springs from the right part of the ventricle, it carries venous blood to the lungs; from the left part springs the right arch of the aorta that carries arterial blood to the brain and forelimbs. The left arch of the aorta springs from the center of the ventricle, it carries mixed blood. Behind 89 the heart 2 arches of the aorta fuse into one vessel and carry mixed blood to all organs. Anlages of 6 pairs of branchial arteries are formed. They transform into the same vessels as in amphibians (the 5th pair — into pulmonary arteries).

7 6 А B 4 5 4 2 2 1 3 3 1

6 8 7 6 5 D 5 C 8 7 2 10 9 1 2 1 3 4 3 4 Fig. 48. Heart evolution of vertebrates: A — fish: 1 — venous sinus; 2 — atrium; 3 — ventricle; 4 — bulb of aorta; B — amphibian: 1 — right atrium; 2 — left atrium; 3 — ventricle; 4 — arterial cone; 5 — left pulmocutaneous artery; 6 — right arch of the aorta; 7 — carotid arteries; C — reptiles: 1 — right atrium; 2 — left atrium; 3 — ventricle; 4 — interventricular septum; 5 — right pulmonary artery; 6 — right arch of the aorta; 7 — left arch of the aorta; 8 — left Botallo duct; 9 — pulmonary veins; 10 — vena cava; D — mammal: 1 — right atrium; 2 — left atrium; 3 — right ventricle; 4 — left ventricle; 5 — left pulmonary artery; 6 — left arch of the aorta; 7 — pulmonary veins; 8 — vena cava

Fig. 49. Development of arterial arches in vertebrate animals: A — anlage in a vertebrate, B — fish, C — anura amphibian, D — reptile, E — mammal: 1–6 — arterial (branchial) arches; 7 — abdominal aorta; 8 — dorsal aorta; 9 — carotid arteries; 10 — right arch of aorta; 11 — left arch of aorta; 12 — pulmonary arteries; 13 — carotid duct; 14 — Botallo duct

90 In mammals the heart is completely divides into the left and right halves to ultimately separate arterial and venous blood. The right heart contains venous blood while the left one is filled with arterial blood. The pulmonary circulation starts from the right ventricle with pulmonary arteries and terminates in the left atrium with pulmonary veins. The systemic circulation starts from the left ventricle with a left arch of the aorta and ends in the right atrium with vena cava. Anlages of 6 pairs of branchial arteries are formed in embryogenesis, then in the 1st and 2nd pairs are reduced; the 3rd pair forms carotid arteries; the right one of 4th pair is reduced while the left one forms an arch of the aorta; the 5th pair is reduced; the 6th pair transforms into pulmonary arteries.

EVOLUTION OF THE RESPIRATORY SYSTEM OF CHORDATES The respiratory system has an endodermal origin. Basic directions of evolution of the respiratory system: 1. Transformation of interbranchial septa of lancelets into the gill apparatus of fishes. 2. Enlargement of the respiratory surface due to gill filaments; formation of gill capillaries. 3. Transformation of the gill apparatus into terrestrial respiratory organs (lungs). 4. Development and differentiation of respiratory tract, formation of a bronchial tree. 5. Enlargement of the respiratory surface of the lungs; formation of the chest and appearance of the diaphragm. A lancelet has 100–150 pairs of interbranchial septa piercing the pharynx and gas exchange takes place in their vessels. These are afferent branchial artery and efferent branchial artery. There are no branchial capillaries. Fishes have branchiae (gills) in the anterior part of the pharynx. Gas exchange takes place in the capillaries of gill filaments. Crossopterygians acquired organs able to breath with air — paired outgrowth of the pharyngeal wall at the abdominal side. They are anlagen of lungs of terrestrial vertebrates. Anura amphibians have a laryngotracheal chamber, in caudate amphibians it separates into the larynx and trachea (figure 50); arytenoid cartilages and vocal folds appear in the pharynx. Anurans have septa in the lungs. The lungs of caudates are presented by two thin-walled sacs without septa. Ventilation of the lungs is low and skin participates in respiration. In reptiles the respiratory surface of the lungs is increased by honeycomb shaped structures faveoli with blood vessels. There are extrapulmonary bronchi; cricoid cartilage appears in the pharynx, cartilaginous rings appear in the trachea. There is a chest. Ribs are movably connected to the spine and breastbone, there are intercostal muscles.

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7 4 8 3 5 6 1 2 E C А 8

6 9 4 5

B D F

10 Fig. 50. Evolution of the lungs in vertebrates: A — pharynx and a swimming bladder (lungs) of the crossopterygian fishes, B — pharynx and lungs of amphibians, C — caudate amphibian, D — anuran amphibian, E — reptile, F — mammal: 1 — pharynx; 2 — unpaired chamber connecting the swimming bladder with the pharynx; 3 — sacs of the swimming bladder; 4 — laryngotracheal chamber; 5 — pulmonary sacs; 6 — intrapulmonary septa; 7 — trachea; 8 — bronchus; 9 —branches of bronchi; 10 — alveoli

In mammals appear the nasal cavity, nasopharynx. Thyroid cartilage appear in the larynx. Bronchial tree is formed. Bronchioles and alveoli considerably increase the respiratory surface (the number of alveoli is up to 500 million). The chest is separated from the abdominal cavity by the diaphragm and takes part in respiration.

EVOLUTION OF THE DIGESTIVE SYSTEM OF CHORDATES The digestive system originate from the endoderm, its beginning and ending regions regions develop from the ectoderm. Basic directions of evolution: 1. Differentiation of the alimentary tube into regions. 2. Appearance of digestive glands. 3. Appearance of teeth and their differentiation. 4. Enlargement of the absorption surface due to the elongation of the intestine and appearance of villi. Lancelet’s digestive system is presented by a straight tube that is differentiated into a pharynx and intestine. The pharynx has gill slits. The alimentary tube forms a hepatic cecum. Fishes have jaws with homogenous teeth (homodontous animals). There are an esophagus, stomach, small and large intestines. The liver is well developed; there is a gallbladder. The pancreas is differentiated poorly. Amphibians have an oropharyngeal cavity with homogenous teeth, esophagus, small and large intestine, liver, pancreas. A muscular tongue and salivary glands appear. There are no enzymes in saliva. Amphibians have a duodenum and rectum. The intestine ends with a cloaca. 92 Reptiles has an oral cavity that is separated from the pharynx, differentiation of teeth begins (fangs), walls of the stomach are thick. There is a primordial cecum, the intestine becomes longer and ends with a cloaca. Mammals are heterodonts (have incisors, canines and molars); lips appeared. The saliva contains enzymes. The intestine is differentiated into a small and large intestine, the caecum is well developed and has an appendix. The rectum ends with an anal opening. The mucous membrane of the intestine has a great number of folds, the small intestine has villi.

EVOLUTION OF THE EXCRETORY SYSTEM OF CHORDATES The excretory system originates from mesoderm. It is is represented with nephridia in lancelets, and by kidneys in vertebrates. Basic directions of evolution: 1. Substitution of nephridia (lancelet) with kidneys (vertebrates). 2. Transformation of a pronephros (head kidney) into a mesonephros (mesonephric kidney) and ultimatly metanephros (pelvic kidney) by increasing the number of nephrons and convergence of the nephrons and blood capillaries, elongation of nephron tubules. The lancelet has 100–150 pairs of nephridia. They are short tubules that have one end open into a coelom, and the other one — into a peribranchial cavity. A glomerule of capillaries is situated in the coelom wall near tubules. In course of evolution, vertebrates successively change 3 generations of kidneys: pronephros, mesonephros, metanephros. A nephron is a basic structural and functional unit of an excretory organ. The pronephros (in larvae of fishes and amphibians) has 6–12 nephrons. The nephron consists of a funnel (nephrostome) and a short tubule. Nephrostomes open into the coelom and tubules into the ureter of the kidney. The glomerulus is located in the coelom wall near nephrostomes (figure 51).

Fig. 51. Evolution of the nephron: A — pronephros, B — mesonephros, C — metanephros: 1 — nephrostome; 2 — tubule of the nephron; 3 — ureter; 4 — glomerulus; 5 — coelom; 6 — capsule of the nephron Dissimilation products pass from the blood into the coelom, then through the nephrostome into the tubule, and then into the ureter of the pronephros (pronephric duct). The ureter opens into the cloaca. 93 The mesonephros (mature fishes and amphibians) contain approximately 100 nephrons. Some glomeruli have an outgrowth of tubule wall in shape of two-walled capsule. Nephrostomes are preserved. Dissimilation products are removed from the blood in two ways: from the nephrostome into the tubule or from the glomerulus into the tubule. During further development of the urinary system, the pronephric duct splits longitudinally into Muller duct and Wolffian duct. In males of lower vertebrates the Muller duct atrophies but in females it is transformed into an oviduct. The Wolffian canal transforms into a ureter in females or it functions as both the ureter and seminal duct in males. Amniotes (higher vertebrates) have metanephros. It contains about 1 million nephrons. There is no nephrostome, the wall of the tubule completely envelopes the glomerulus (renal corpuscle consisting of a Shumlyansky– Bowman capsule and glomerulus, then the tubule is differentiated into a descending part, the Henle loop and an ascending part. Removal of dissimilation products from the blood occurs directly into a tubule. Filtration of blood plasma occurs in the glomerulus while tubules perform reabsorption of water, amino acids and glucose from primary urine. The dilation of the distal part of the ureter forms a urinary bladder. The phylogenetical relation of the excretory and genital systems: – the gonads of Vertebrates are germinated as paired folds on ventral parts of the primary kidney; – canaliculi of the prokidney and its ureter form a funnel and an oviduct in females; a ureter of the primary kidney serves as a semen duct in males (figure 52).

I IV V VI II III Fig. 52. Development of the excretory and genital systems in vertebrates: I — neutral embryonic state in a lower vertebrate; II — female lower vertebrate; III — male higher vertebrate; IV — neutral embryonic state of a higher vertebrate; V — female higher vertebrate; VI — male higher vertebrate: 1 — pronephros; 2 — mesonephros; 3 — metanephros; 4 — pronephric canal; 5 — Muller duct serving as oviduct in females; 6 — Wolffian duct serving as semen duct in males; 7 — uterus; 8 — ureter; 9 — bladder; 10 — cloaca; 11 — gonad; 12 — anus 94 ONTOPHYLOGENETIC ETIOLOGY OF MALFORMATIONS IN THE NERVOUS, CARDIOVASCULAR, RESPIRATORY, DIGESTIVE AND UROGENITAL SYSTEMS IN THE HUMAN Ontophylogenetic etiology of brain malformations (causes are recapitulations): undifferentiation of hemispheres, incomplete separation of hemispheres of the telencephalon (prosencephalia); ichthyopsidian or sauropsidian types of the brain. Ontophylogenetic etiology of cardiovascular malformations: ventricular septal defect, open Botallo duct, underdevelopment of aortopulmonary septum (incomplete separation of the arterial trunk into an aorta and a pulmonary trunk), transposition of the great vessels, preservation of both aortal arches, etc. Ontophylogenetic etiology of malformations of the respiratory system: underdevelopment of the pharynx or lungs, cystic lung hypoplasia, abnormal branching of bronchi, hypoplasia of the diaphragm, etc. Ontophyloigenetic etiology of malformations of the digestive system: cervical fistulae (rupture gill pouch), homodontous teeth, additional lobes of the liver and pancreas, shortening of the intestine. Ontophylogenetic etiology of urogenital malformations: a pelvic position of kidneys, preservation of a mesonephros, doubling of the ureter, bicornuate uterus, duplex uterus and vagina (parallelism).

BASIC TERMS AND CONCEPTS Anaboly — development that follows ancestral pattern up to its last stage and then new stages are added. Archallaxis — early deviation from ancestral developmental pattern that occurs simultaneously with formation of the organ anlage. Arterial cone — pulsating muscular tube that starts from the ventricle and divides into a pulmocutaneous arteries, carotid arteries and aortal arches. Botallo duct — duct connecting the aorta with pulmonary arteries and carrying arterial blood from the systemic circulation into the pulmonary one. Venous sinus — site where cava veins join the heart. Mesonephros — excretory organ of fishes and amphibians. Metanephros — kidney of terrestrial animals. Metanephric duct — ureter of pelvic kidney. Sauropsidian brain — brain where the integrating center is striate bodies. Ichthyopsidian brain — brain where the integrating center is midbrain. Parallelism — independent development of similar characters in closely related species in course of their evolution. Recapitulation — appearance of ancestral characters in embryos during their ontogenesis. Phylembryogenesis — embryonic reconstruction that is preserved in adults and have adaptive nature. 95 Topic 16. INTRODUCTION TO PARASYTOLOGY

ORIGIN AND AGE OF PARASITISM. CRITERIA OF PARASITISM According to the study of Yevgeny Pavlovsky, «parasites are animals that live at the expense of individuals of other species; they are closely associated with these species biologically and ecologically during long or short period of their life cycle». Criteria of parasitism: 1) spacial relations with the host; 2) feeding at the expense of the host; 3) pathogenic action on the host. The host of a parasite is an organism that provides the parasite with inhabitation and food and is harmed by it. A specific habitation is characteristic of the parasite. Primary habitation is the host’s organism. It actively reacts to the presence of a parasite. The secondary habitation is external environment. The host is a link between the parasite and the environment. Parasitism is a most common form of symbiosis: all viruses, many bacteria, some kinds of fungi and higher plants are parasites. Parasites are 10 000 species of protozoans, 7000 species of arthropods, 20 000 species of helminthes. Some classes includes only parasites — Sporozoa, Flukes and Tapeworms. Diseases caused by various parasites have different names: those caused by viruses and bacteria are called infections (flue, hepatitis, tuberculosis, etc.); by protozoans and helminthes are invasions (ascariasis, teniasis, enterobiasis, etc.); diseases caused by arthropods (ticks, insects) are infestations (pediculosis, myiasis, scabies, etc.). There are various biological interactions between species in the nature: Competition is interaction of organisms that require same existence conditions or resources. Predation — interactions of organisms of different species in which one predator organism kills the other one — prey — and uses it for feeding. Antibiosis (Greek anti — against, bios — life) — interactions of organisms of different species in which metabolites of one of them suppress the development of other one. These substances have a chemical nature. An example is production of antibiotics by mildew fungi, secretion of phytoncides (Greek phyton — plant, caedo — kill) by some higher plants (pine, cedar, onion, garlic). Antibiotics and phytoncides are used in medicine for treating various diseases.

96 Symbiosis is any form of interactions between different species. The term was introduced into biology by de Barry in 1879 (Greek sym — near, bios — life). The following forms of symbiosis are distinguished: – synoikia or hosting (Greek syn — together, oikos — house) —one species uses the other one as habitation without causing any harm or benefit (cancroid sea acorns on a mollusks’ shells); – commensalism (French commensal — co-eater) — permanent or temporary co-habitation of individuals of different species in which one of them eats food remains or excretion products of the other one without any harm (shark and sticking fish); – mutualism (French mutuus — mutually beneficial) — mutually beneficial co-habitation of organisms of different species; – parasitism (para — near; sitos — feeding) is antagonistic sybiosis. The most common form of symbiosis is one variety of interspecies relations. Age of parasitism. Theoretically, parasites presumably could appeared together with protists as parasitic bacteria were found in the amoebae. Multicellular parasites existed in the paleozoic era: ichnolites of the stems of sea lilies (Echinodermata) had gall-like growths caused by nematodes. Origin of parasitism: 1. Predator → ectoparasite. Medicinal leeches are temporary ectoparasites for the human; the leech can be predators for small animals as it sucks out a great amount of blood and the animal dies. 2. Free-living organism → attached mode of life → ectoparasitism. Free-living cirripedia may pass to an attached mode of life. They attach to underwater parts of wooden buildings or bottoms of ships. They pass to ectoparasitism if they attach to living objects —shells of mollusks or fish bodies. 3. Commensalism → ectoparasitism. Commensalism → endoparasitism. If a commensal settles on body coverings of the animal, it may become an ectoparasite. It becomes an endoparasite when gets inside the organism (in body cavities connected with the environment). Entamoeba coli is an endocommensal in the human organism. Transit through the digestive tract → endoparasitism (larvae of a domestic fly).

CLASSIFICATION OF PARASITES AND THEIR HOSTS Classification of parasites: 1. According to relation with the host: – obligate parasites — parasitism is the only possible way of living for such species (Ascaris, lice); – facultative parasites are free living organisms that can get into a living organism and behave as parasites (larvae of the domestic fly);

97 – hyperparasites or superparasites are parasites of parasites (bacteria in parasitizing protozoans). 2. According to location in the host: – ectoparasites inhabit body coverings of the host (lice, fleas); – endoparasites live inside the host’s organism: a) intracellular parasites (toxoplasma); b) cavity parasites (Ascaris); c) tissue parasites (liver fluke); d) intradermal parasites (itch mite). 3. According to duration of the relation with the host: – permanent parasite — all life cycle proceeds in the host (Ascaris); – temporary parasite — some stages of their life cycle require development in the host: larval parasitism (larvae of a botfly); imago parasitism — parasitism of sexually mature individuals (mosquitoes, fleas). Classification of hosts: 1. According the parasite’s life stage: a) principal (definitive) host — a host where a parasite maturate and reproduces sexually (human for Taenia solium); b) intermediate host — a host where a parasite lives for period and reproduces asexually (human for malaria parasite); c) supplementary or accessory host — additional intermediate host (fish for a cat liver fluke). d) reservoir host — in this host invasive stage of the parasite accumulates (predatory fish for larvae of Diphyllobothrium latum). 2. According to conditions of parasite's development: a) obligate (or natural) host provides optimal conditions for parasite’s development and there is biocenotic contact (natural ways of invasion) — the human for the Ascaris lumbricoides; b) optional (or permissive, accidental) host there are biocenotic contact, but no normal biochemical conditions for the parasite’s development (the human for the Ascaris suum — affects pigs); c) potential host can provide normal biochemical conditions for the development of the parasite, but there are no biocenotic contact — no ways for invasion (Guiney pig for trichinella).

THE PARASITE-HOST SYSTEM Parasitism is an ecological phenomenon. Ecological Parasitology studies interrelations of parasites and their populations with each other, with the host’s organism and the environment. The parasite-host system includes one host individual and a parasite (or an entire group of parasites) of the same species.

98 Conditions necessary for the formation of this system: a) contact between the parasite and the host; b) the host must provide proper conditions for the development of the parasite; c) the parasite must resist host’s protective reactions. Evolution of the system tends to improve its stability, reach equilibrium, diminish antagonism between the parasite and the host. Lessen of the antagonism is achieved due to co-adaptation: – in the parasite — morphologic and biologic adaptations; – in the host — complication of defense mechanisms. Directions of evolution are also different (co-evolution): – in the parasite — complication of adaptation mechanisms to the host; – in the host — improving all defensive reactions (to destroy the parasite).

TRANSMISSION ROTES OF PARASITES 1) alimentary — orally with food and water (eggs of helminthes, cysts of protists); 2) respiratory (droplet) — through the respiratory tract (cysts of some Amoebae, some viruses and bacteria); 3) vertical (transplacental) — from mother to fetus (toxoplasma, malaria parasite); 4) iatrogenic — due to medical procedures, for example transfusion of infected blood or usage of unsterile surgical tools (trypanosomes, malaria parasite); 5) indirect and direct contact — contact with a sick one through household goods (itch mite) or with his body surface; 6) vector-borne — carriage of a pathogen by an arthropod (trypanosomes, malaria parasite); 7) sexual — in sexual contacts (Trichomonas vaginalis).

ADAPTATIONS TO PARASITISM Parasites are highly specialized organisms, maximally adapted to their inhabitation and way of living. Morphological and physiological adaptations of parasites: a) progressive adaptations: – enlargement of the body (up to 20 m in tapeworms); – high development of the reproductive system; – hermaphroditism; – fixation organs (adhesive discs of Giardia lamblia, suckers of flukes, bothria or hooks of tape worms, claws of lice, etc); – integument that protects the parasite from host’s defense; – molecular mimicry — similarity of proteins of the parasite and the host; 99 – excretion of anti-enzymes. b) regressive adaptations: – simplification of sense organs — endoparasites have only tactile and chemical sense organ; – simplification of the organ systems — absence of alimentary tract in tape worms. Biological adaptations are associated with structural peculiarities of the reproductive system, reproduction and life cycles of parasites: a) high fertility (Taenia solium excretes 100 thousand eggs with every mature segment, an ascaris — 250 thousand eggs per day); b) diversity of asexual reproduction (schizogony in malaria parasite, polyembryony in flukes); c) migrations within the host's organism (larvae of Taenia solium and Ascaris lumbricoides); d) complex life cycles with alternation of hosts. The results of interactions of the parasite and the host on the level of organism may be different: death of the parasite, death of the host and carriage of the parasite.

PATHOGENIC ACTION AND SPECIFICITY OF PARASITES Pathogenicity is the ability to cause a disease. It depends on: – genotype of the parasite, its species; – host’s age (children and old people are more susceptible to invasion); – diet regimen (improper diet weakens the organism and contributes to increasing the number of parasites in the organism and their sizes, reduces the terms of their development); – dose and degree of invasion (the more eggs or larva get to the host’s organism, the severer is the course of the disease); – resistance of the host; – presence of other parasites and diseases. Specificity of the parasite is degree of a historically formed adaptation to certain hosts. Its types are: a) hostal specificity: monohostal parasites have one species of the host (Ascaris lumbricoides), polyhostal parasites have hosts of several species (trichinella); b) topical specificity (a site of parasitizing): Ascaris lumbricoides live in intestine, head louse — on the hairy region of the head and etc.; c) age specificity: enterobiasis in more common for children; d) seasonal specificity: outbreaks of amebic dysentery are more typical for the end of spring and summer).

100 Pathogenic action of parasites: 1. Mechanic: parasites harm tissues by their body mass (ball of Ascaris lumbricoides in the intestine, a cyst of echinococcus in the brain), by fixation organs (injury of the intestinal mucous membrane by suckers), impairment of skin, etc. This action is revealed due to a pain syndrome. 2. Toxicoallergic action is produced by metabolites of parasites that are antigens; histolysins and decay products of dead parasites. Manifestations of this action: skin eruptions, dermatitis, eosinophilia, allergic reactions. 3. Absorption of nutrients and vitamins results in avitaminosis (mainly A and C), loss of weight, exhaustion. 4. Impairment of the metabolic process reduces host's resistance and increases sensitivity to pathogens of other diseases. 5. Biologically active substances of some parasites have immune- depressive effect on the host. 6. Some parasites stimulate oncogenesis: schistosomes may cause cancer of the bladder and rectum. 7. Parasites produce an unfavorable effect on the course of pregnancy and development of a fetus (malaria parasite, toxoplasma, cat liver fluke, etc.).

RESPONSE OF THE HOST TO PARASITIC INVASION The basis of all reactions is the host’s immune response. Allergy is a kind of immune reactivity. The first reaction to a parasite is an attempt to kill it with enzymes, then — to neutralize factors of its «aggression» by proteases, inhibitors of enzymes. Reactions at cellular level show as hypertrophy and modification of the shape of affected cells (erythrocytes in malaria). At tissue level: isolation of the parasite from a healthy tissue (formation of a capsule in trichinellosis, formation of pseudocysts in toxoplasmas). At organism level: humoral reactions (production of anti-bodies) and various forms of immunity: complete — relative, active — passive, inborn — acquired.

BIOLOGICAL BASIS OF PROPHYLAXIS OF PARASITIC DISEASES K. I. Skriabin elaborated biological basis of prophylaxis to control parasitic diseases. It is a complex of prophylactic measures based on detailed studying of the pathogen’s biology, migration ways, life cycle, biology of intermediate hosts. It is possible to interrupt any link of the parasite life cycle. The final practical aim of Parasitology is protection of the human, animals and plants from parasitic action and elimination of parasitic diseases.

101 BASIC TERMS AND CONCEPTS Invasions — diseases caused by protozoans and helminthes. Infections — diseases caused by viruses and bacteria. Hyperparasitism — relations between parasites of different species when one parasite parasitize the other parasite. Molecular mimicry — similarity of parasite’s antigens to host’s antigens. Parasitocenosis — all the parasites in the organism of a host. Parasite — organism which is biologically adapted for life at the expense of another organism. Pathogenicity — capability of the parasite to cause a disease. Symbiosis — and kind of persistent interactions of organisms of different species. Specificity of the parasite — historically formed adaptation degree of the parasite to its host. Invasive stage — that life stage of the parasite which gets to the organism of the host to continue the lie cycle.

Topic 17. PHYLUM SARCOMASTIGOPHORA, CLASSES SARCODINA, ZOOMASTIGOTA

GENERAL CHARACTERISTIC OF THE KINGDOM PROTISTA Inhabitance: water reservoirs, damp soil, plants, animals and humans. Over 10 000 of 65 000 species are parasites. A cell of protozoans performs functions of the whole organism. The cell caovering consists of plasma membrane, elastic membrane pellicle or a denser cuticle. The shape is constant (Zoomasticota and Infusoria) or changeable (Sarcodina). The sizes are from 3 to 150 μm. There are 2 layers in the cytoplasm: ectoplasm which is the external layer and endoplasm — the internal one. There are organelles of typical for all cells (mitochondria, ER, ribosomes, Golgi complex, etc.) and those characteristic of only such protists (contractile and digestive vacuoles, cilia, flagella, etc.). Organelles of movement are pseudopodia, flagella and cilia. The majority of protozoans are heterotrophs. They receive substances by endocytosis, active transport, osmotically or through the cell mouth (cytostome)., Food vacuoles are formed and lysosomes with enzymes join them. Digested substances are transported to the cytoplasm, and undigested remains are removed from the cell through any region of plasma membrane or through anal pore. Protozoans have contractile vacuoles performing osmoregulation and excretion of dissimilation products, they also stimulate gas exchange.

102 Cells of protozoans contain one or several nuclei. Reproduction is asexual: binary fission or schizogony. There is a sexual process (conjugation or copulation). In unfavorable conditions protozoans transform into cysts. When cysts get in favorable conditions, excystation occurs when protist forms the vegetative form (trophozoite). Irritability of protists is taxis. Classification: phylum Sarcomastigophora (classes Sarcodina and Zoomastigota), phylum Apicomplexa (class Sporozoa) and phylum Infusoria (class Ciliata).

PARASITIC SARCODINAE (PHYLUM SARCOMASTIGOPHORA, CLASS SARCODINA) About 10 000 species of Sarcodina are the most primitive representatives of Sarcomastigophora. The cell covering consists of plasma membrane, pellicle is absent and the body shape is changeable. The cell contains one nucleus. Organelles of movement are pseudopodia. In unfavorable conditions cysts are formed. Organelles of parasitic Sarcodinae are poorly developed. Feeding occurs by endocytosis (bacteria, organic substances, enteric cells, erythrocytes). Entamoeba histolytica (Dysenteric amoeba) is a pathogen of amoebiasis (amoebic dysentery). The disease is common everywhere, more often in countries with warm climate. Morphological peculiarities: two stages: trophozoite (vegetative stage) and cyst. Cysts (8–16 μm in size) contain 4 nuclei (figure 53).

Fig. 53. Morphology of trophozoites and cysts of E. histolytica and E. coli: A — E. histolytica, B — E. coli: 1, 4 — diagrams of trophozoites; 2, 5, 6 — trophozoites (7×40); 3 — f. magna with swallowed erythrocytes (7×40); 7, 8 — cysts (7×40)

Trophozoites of Entamoeba histolytica may exist as 3 forms: a minor vegetative (lat. forma minuta), major vegetative (lat. forma magna) and tissue form. Forma minuta (12–20 μm in diameter) are capable of moving and feed on

103 bacteria. Forma magna (30–40 μm) excretes proteolytic enzymes and is able to engulf erythrocytes. Tissue trophozoites (20–25 μm) can move fast. Forma minuta is not pathogenic, forma magna and tissue form are pathogenic. Life cycle: Amoebiasis is transmitted by the fecal-oral route. Infection occurs by ingestion of cysts. Transmitting factors are contaminated vegetables, fruit and water. Mechanic vector of cysts are flies and cockroaches. Four trophozoites (forma minuta) come out from the swallowed cyst in the intestine. They can exist there for a long time (feed, multiply) and transform into cysts (cyst carrier state) (figure 54).

Fig. 54. Life cycle of Entamoeba histolytica

When the host’s organism is weakened (by infections, using spicy food, fasting, hyperthermia, etc.) forma minuta transforms into forma magna that harms the mucous membrane of the large intestine. In the intestinal wall it transforms into tissue form. It is often carried into the liver, brain and other organs by blood. In remission, pathogenic trophozoites in the intestinal lumen transform into forma minuta and cysts. Pathogenic action: 1. Mechanic (destruction of mucous membrane of the large intestine cause bleeding ulcers with diameter from several millimeters to 2–2.5 cm). 2. Toxicoallergic (poisoning by waste products). 3. Feeding at the expense of the host’s organism and impairment of metabolic processes (engulfment of erythrocytes, absorption of vitamins and impairment of fluid-and-electrolyte balance). Symptoms: bloody diarrhea up to 10 times a day and more, pains in the abdomen in area of the large intestine (mostly in the right hypochondrium). Intoxication may be in various degrees. Complication of amebiasis: amoebic abscess in the liver or lungs, purulent peritonitis, inflammatory processes of the skin in the perineal area. Laboratory diagnostics: microscopy of feces smears and the content of ulcers in order to reveal pathogenic trophozoites. It is possible to reveal cysts during remissions and cyst carriage.

104 Personal prophylaxis: observing hygien rules (washing hands, washing vegetables and fruits with hot water, protection of food from flies and cockroaches). Social prophylaxis: revealing and treating sick people; control over sanitary condition of reservoirs, food manufacturer, shops and markets; prophylactic examination of workers of food manufacturer; elimination of flies and cockroaches; personal and social health education.

PARASITIC FLAGELLATES (PHYLUM SARCOMASTIGOPHORA, CLASS ZOOMASTIGOTA) There are 8 000 species of Sarcomastigophora. Many representatives are parasites of animals and human. They have a constant body shape (due to pellicle), one nucleus. Locomotion is provided by flagella and undulating membrane (outgrowth of cytoplasm). Parasitic species are heterotrophs, their feeding is osmotic. They multiply by a longitudinal binary fission. Some secies are capable of sexual process copulation. Leishmania. Leishmaniasis is a natural-focal disease. Visceral leishmaniasis is common in the area of the Mediterranean Sea, Middle and South Asia, Africa and South America. Cutaneous leishmaniasis is revealed in South Europe, North and West Africa, Near East, Central and South Asia. The focus of mucocutaneous leishmaniasis is in South and Central America. Morphological peculiarities: leishmania has 2 stages: promastigote (has flagellum, size is 10–20 μm) and amastigote (nonflagellate round or oval cell, 3–5 μm). Pathogens of leishmaniasis are morphologically similar but have biochemical and antigenic differences (figure 55).

Fig. 55. Morphology of leshmania and its vector: A — diagram; B — flagellate form (7×40); C — aflagellate form in a macrophage (7×40); D — vector mosquito 105 Life cycle: biological vectors are sand flies of the genus Phlebotomus. They contain promastigotes (figure 56).

Fig. 56. Life cycle of leishmania

Infection occurs in mosquito bites (vector-borne route). In the human organism Leishmaniae lose their flagella, transform into amastigotes, begin intracellular parasitizing and intensively multiply. Natural reservoirs of L. donovani are coyotes, dogs, rodents; those of L. tropica are rodents, those of L. braziliens are rodents, primates and slothes. L. donovani and L. infantum cause visceral leishmaniasis (Dumdum fever, black fever, kala-azar). Pathogenic action: 1. Mechanic (destruction of hepatic cells, lymphatic nodes, red bone marrow). 2. Toxicoallergic (poisoning by waste products). Incubation period lasts from several weeks to 6–8 months. L. donovani and L. infantum are called visceral leishmaniasis (black disease, dumdum fever, kala-azar, infantile leishmaniasis). Clinical manifestations: Intermittent fever, weakness, headache, inanition, rash, enlargement of the liver and spleen, anemia. Children fall ill more often. After leishmaniasis they acquire long-lived immunity. Laboratory diagnostics: revealing leishmania in puncture samples of red bone marrow (of breastbone) or lymphatic nodes. L. tropica major and L. tropica minor cause cutaneous leishmaniasis (oriental sore).

106 Pathogenic action: 1. Mechanic (destruction of cutaneous cells). 2. Toxicoallergic (poisoning by waste products). Clinical manifestations. Small erythematic tubercles appear on skin 2–6 weeks after bite. Later on, an ulcer with elevated edges forms (leishmanioma). All the course of ulcer formation till healing over may last 3–4 months to 2 years. An unpleasant-looking scar stays on the affected area. Laboratory diagnostics: finding leishmaniae in smears taken from the ulcers. L. brasiliensis, L. mexicana and L. peruviana cause mucocutaneous leishmaniasis. Pathogenic action: 1. Mechanic (destruction of the skin, mucous membranes, cartilages). 2. Toxicoallergic (poisoning by waste products). Incubation period lasts 2–3 weeks to 1–3 months. Clinical manifestations: Ulcers gradually destroying all soft tissues. Overgrowing of the tissues of the nose, lips, pharynx, larynx. The disease is hardly treated and it often leads to death. Laboratory diagnostics: finding leishmaniae in smears taken from the ulcers. Prophylaxis: protection from sandfly bites (repellents, insect nets) and vaccination, revealing and treating sick people, elimination of sandflies and animals-reservoirs of the disease, health education. Genus Trypanosoma. Trypanosoma brucei gambiense (West Africa) and Trypanosoma brucei rhodesiense (East Africa) are pathogens of sleeping sickness, or African trypanosomiasis. Trypanosoma cruzi (South America) is a pathogen of Chagas disease, or American trypanosomaisis (figure 57). Both diseases are natural-focal and vector-borne. Life cycle of trypanosomes incudes several stages: trypomastigote is an elongated cell with long flagellum and undulating membrane. It affects vertebrate hosts and is an invasion stage for them; epimastigote is similar to trypomastigote, but its flagellum is shorter and the undulating membrane is smaller. It exists only in the organism of the vector and can transform into trypomastigote; amastigote is an immobile intracellular parasite. It affects vertebrate hosts and can transform into trypomastigote. The cell of a trypanosoma is curved, length is 13–40 μm. The flagellum extends along the edge of the undulating membrane. Feeding is osmotic, multiplication occurs by binary fission. Life cycle: Tsetse flies (g. Glossina) are biological vectors. When the fly takes a blood meal on a sick person, trypomastigotes are taken into its stomach (figure 58).

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Fig. 57. Morphologhical peculiarities of pathogens of trypanosomaisis: A — diagram: 1 — erythrocytes; 2 — flagellum; 3 — nucleus; 4 — undulating membrane; B — T. cruzi (7×40); C — T. brucei (7×40); D — Triatoma infestans; E — Glossina palpalis

Fig. 58. Life cycle of pathogens of African trypanosomiasis

They transform into epimastigotes, multiply and accumulate in salivary glands (this lasts 20 days). Infection of a host occurs through bite by the infected fly (vector-borne rote). Human also can become infected by blood transfusion, unsterile surgical armaments, by transplacental rote. 108 The life cycle proceeds in the organism of the human or reservoir hosts (pigs for T. brucei gambiense, antelopes and cattle for T. brucei rhodesiense). At first trypomastigotes settle subcutaneous adipose tissue, then lymphatic system. They multiply and in 20–25 days pass to blood that carries them to all organs. Trypanosomes are more commonly found in cerebrospinal fluid, they affect the brain and spinal cord. Pathogenic action: 1. Mechanic (destruction of cells and tissues of affected organs). 2. Toxicoallergic (poisoning with waste products). Incubation period is 1–3 weeks to 2 and more years. Clinical manifestations. A trypanosome chancre appears at the site of bite, there is enlargement of lymphatic nodes on the posterior surface of the neck, rise of temperature, weakness, attenuation. Later on, symptoms suggesting affection of CNS appear: sleepiness, dementia, sopor and coma. In gambian form of the disease develops into progressing encephalitis with typical sleepiness (sleeping sickness). The outcome is lethal if the disease is not treated. Laboratory diagnostics: finding trypanosomes in peripheral blood smears, puncture samples of lymphatic nodes, cerebrospinal fluid; immunoassay (detection of anti-bodies in the blood serum). Prophylaxis: prevention of tsetse fly's bites, treatment of sick people and carriers, personal and social health education. Trypanosoma cruzi. Morphological peculiarities are similar to that of T. brucei. Life cycle: Trypanosoma cruzi affects human and animals (armadillos, ant- bears, etc.) that are natural reservoirs the disease. Biological vectors are kissing bugs of the g. Triatoma. While biting a sick person or animals, the bug ingests trypomastigotes. In the intestine of the bug they transform into epimastigotes, multiply, transform into trypomastigotes and are excreted with feces. Infection of another person (vector-borne rote) occurs when the feces of the bug get to the injured skin (bites, scratchings). Infection also may occur by transfusion of blood and transplacentally. In the human organism trypomastigotes transform into amastigotes and multiply. In 1–2 weeks amastigotes transform into trypomastigotes inside the injured cells and enter the bloodstream. They circulate within the organism and invade new cells (cardiac and skeletal muscles, nervous system, etc.) where the cycle repeats. Pathogenic action: 1. Mechanic (destruction of cells and tissues, edema). 2. Toxicoallergic (poisoning by waste products). Incubation period lasts 4–14 days. Clinical manifestations: at the site of the bite where trypanosomes pass thes kin appears inflammatory nodule with hyperemia and edema (chagoma).

109 In 1–2 weeks (when parasites enter the blood) appears fever, headache, face edema, pains in the area of the heart and signs of heart failure. Complications: meningoencephalitis, impairment of the vegetative nervous system, heart, liver, kidneys and other organs. Mortality rate is up to 14 %. Laboratory diagnostics: detection of trypanosomes in peripheral blood smears, cerebrospinal fluid, puncture samples of lymphatic nodes, spinal cord; immunoassay (revealing anti-bodies in the blood serum). Prophylaxis: revealing and treating sick people, elimination of vectors and protection from their bites (repellents, etc.), health education. Genus Giardia. Lamblia intestinealis is a pathogen of lambliasis (giardiasis). Lamblia can affect only human. The disease is spread everywhere. Morphological peculiarities: A pear-like cell (10–18 μm) with 4 pairs of flagella, 2 supporting axes (axostyles) dividing the body into two symmetrical halves. Each half has per 1 nucleus and an adhesive disc (figure 59). Cysts have an oval shape.

Fig. 59. Morphology of Lamblia intestinalis: A — diagram of trophozoite; B — trophozoites (7×40); C — binary division; D — cyst

Life cycle. Giardia has vegetative stage (trophozoite) and a cyst. Infecting occurs by alimentary rote, when cysts are ingested with unwashed vegetables, fruit or water. Excystation occurs in the duodenum. Location of trophozoites is the upper part of the small intestine and bile ducts. Pathogenic action. 1. Mechanic (irritation of the duodenal mucosa, impairment of parietal digestion and absorption of nutritions). 2. Toxicoallergic (poisoning by waste products). 110 3. Feeding at the expense of the host’s organism and impairment of metabolic processes (absorption of nutrients and vitamins). Clinical manifestations: general uneasiness, bad appetite, nausea, aches in the epigastric region and right hypochondrium, unstable stool (diarrhea, constipation). Lambliasis aggravates the course of other diseases of the digestive system. Laboratory diagnostics: revealing vegetative forms (trophozoites) in feces or duodenal content. Prophylaxis: observing rules of personal hygiene, revealing and treating sick people, personal and social health education. Trichomonas vaginalis is a pathogen of trichomoniasis. The disease is common everywhere. Morphological peculiarities (figure 60): the cell is oval, has 5 flagella. An axostyle is in the middle of the body, it form sharpened long spike at the end of the cell. Size is up to 30 μm. One flagellum extends along the undulating membrane. There is a nucleus and digestive vacuoles in the cytoplasm.

Fig. 60. Morphology of a Trichomonas: A — diagram; B — microphotograph

Life cycle: infection occurs in by means of sexual contacts with infected people or usage of unsterile gynecological tools. Trichomonas affects genitourinary tracts; it does not form cysts. Pathogenic action. 1. Mechanic (destruction of the urinary mucous membranes). 2. Toxicoallergic (poisoning by waste products). Clinical manifestations. In acute form: itching, burning sensation in genitourinary tracts, local inflammation, profuse greenish discharge with unpleasant smell. Laboratory diagnostics: revealing trophozoites in direct smears from genitourinary tracts. Prophylaxis: revealing and treating sick people, avoiding accidental sexual relationships, sterility of gynecological tools, personal and social health education.

111 BASIC TERMS AND CONCEPTS Amoebiasis — disease caused by Entamoeba histolytica. Chagas disease — disease caused by Trypanosoma cruzi. Visceral leischmaniasis — disease caused by Leischmania donovani and Leischmania infantum. Cutaneous leischmaniasis — disease caused by Leischmania tropica major and Leischmania tropica minor. Lambliasis — disease caused by Lamblia intestinalis. Pellicle — elastic membrane covering a cell of a protozoan. Taxis — oriented movement of a motile organism in response to an external stimulus. Sleeping sickness — disease caused by Trypanosoma brucei. Trichomonaisis — disease caused by Trichomonas vaginalis. Undulating membrane — locomotor organelle of some protists consisting of fin-like fold of plasma membrane with flagellum.

Topic 18. PHYLUM INFUSORIA, CLASS CILIATA. PHYLUM APICOMPLEXA, CLASS SPOROZOA

CHARACTERISTICS OF THE CLASSES CLIATA AND SPOROZOA Ciliates are the most developed protists. Their cells are covered with pellicle, its shape is constant and its size is 30 to 1000 micrometers. Locomotor organs are cilia. Between the cilia are trichocysts — organelles used for protection and attack. Food of ciliates is organic particles, bacteria and other small unicellular organisms. Ciliates have deepening peristome near the cell mouth cytostome and cytopharynx where food vacuoles are formed. Undigested remains are eliminated through the anal pore. Two contractile vacuoles maintain osmotic pressure and participate in removal of liquid dissimilation products. There are macronucleus (the vegetative nucleus) and micronucleus (participates in conjugation). Reproduction is asexual (binary fission). Exchange of genetic information occurs by means of sexual process conjugation. If environmental conditions become unfavorable they form cysts. All the species of the class Sporozoa are parasites. They do not have locomotor organelles, digestive and contractile vacuoles. The life cycles of sporozoans are complicated and require alternation of hosts where sexual and asexual reproduction occur.

BALANTIDIUM COLI Balantidium coli is a human parasite of the class Ciliata. It causes balantidiasis (balantidial dysentery). The disease is common everywhere. 112 Morphological peculiarities (figure 61): oval cell 30–150 × 40–70 μm. There is a peristome at the frontal end, which passes into a cytostome and a funnel-like cytopharynx. At the posterior side is anal pore (cytoproct). The macronucleus has shape of bean or rod. There are 2 contractile vacuoles. Balantidium can form cysts.

A B C D Fig. 61. Morphology of Balantidium coli: A — diagram; B — trophozoite (7×40); C — parasites in tissues (7×40); D — cyst (7×40)

Life cycle: trophozoites colonize the large intestine (cecum). Infection occurs alimentary: Cysts (invasive stage) are swallowed with contaminated vegetables, fruits, while drinking water. Workers of pig farms are affected more commonly because pigs are the source of invasion. Cysts form trophozoites in the alimentary tract of the host. Pathogenic action: 1. Mechanic (impairment of the intestinal mucous membrane and formation of deep ulcers). 2. Toxicoallergic (poisoning by waste products). 3. Feeding at the expense of the host’s organism (food particles, sometimes erythrocytes and leukocytes are found in the cytoplasm). Clinical manifestations: diarrhea with blood, pains in the abdomen, vomiting, malaise, weakness, headache. Complications: perforation of ulcers and liver abscesses. Laboratory diagnostics: detection of trophozoites in feces. Prophylaxis: observing rules of personal hygiene, revealing and treating sick people; protection of the environment from contamination by feces of pigs and sick people, personal and social health education.

LIFE CYCLE OF MALARIA PATHOGEN. TYPES OF MALARIA PARASITES, THEIR MORPHOLOGICAL CHARACTERISTICS IN A THIN BLOOD SMEAR Pathogens of human malaria (figure 62) refer to order Haemosporidia, genus Plasmodium. There are of 4 types of plasmodia: Plasmodium vivax causes tertian malaria, Plasmodium ovale causes ovale malaria, Plasmodium malaria causes quartan malaria, Plasmodium falciparum causes malignant tertian malaria.

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Fig. 62. Morphology of Plasmodia: A — ring stage of Pl. falciparum; B — amoeboid stage of Pl. vivax; C — schizont of Pl. ovale; D — band stage of Pl. malaria; E — morula of Pl. vivax; F — morula of Pl. ovale; G — gametocyte of Pl. vivax; H — gametocyte of Pl. falciparum

Malaria is common mostly in countries with a subtropic and tropic climate. Life cycle. The human is an intermediate host for a malaria parasite while female mosquitoes are principal hosts (figure 63).

Fig. 63. Life cycle of malaria pathogens The disease is transmitted by female mosquitoes of g. Anopheles. While biteing human, the mosquito injects sporozoites of the malaria parasite together 114 with saliva. Blood carries sporozoites to the cells of the liver, spleen and endothelium of blood capillaries, where they transform into liver-stage schizonts. The schizonts grow and in 5–16 days divide by schizogony into numerous liver- stage merozoites. All these stages form the cycle of exoerythrocytic schizogony (liver schizogony) which corresponds to the incubation period. The merozoites destroy hepatic cells, enter the blood and settle in erythrocytes to form trophozoites. This is the start of erythrocytic schizogony. Each merzoite that got into an erythrocyte is now called an erythrocytic schizont. During maturation it undergoes ring stage and amoeboid stage. Then the nucleus divide many times (into 6–24), cytoplasm arranges around these nuclei. This stage is called morula. The cells formed as a result of erythrocytic schizogony are erythrocytic merozoites. The membrane of erythrocyte is broken and merozoites with their metabolites are released into blood (merulation). At this moment attack of malaria starts. Some merozoites invade new erythrocytes to repeat the cycle of erythrocytic schizogony (it may repeat many times). Other merozoites get into erythrocytes to transform into gamonts (micro- and macrogametocytes). Their further development (gametogony) may occur only in the mosquito. If the sick prson is biten on this stage, microgametocytes and macrogametocytes get into the stomach of a female mosquito and transform into micro- and macrogamets. They fuse and form a zygote capable of moving (ookinete). It implants into the wall of the stomach getting to its outer surface. There the ookinete covers with a shell and transforms into a sporocyst (sporogony).The membrane of a mature oocyst breaks, sporozoites get into the body cavity of the mosquito. They are carried to all organs by hemolymph and accumulate predominantly in salivary glands.

WAYS OF INFECTING HUMAN WITH MALARIA. PATHOGENIC ACTION OF THE PARASITE. SYMPTOMS AND DIAGNOSIS OF MALARIA. BIOLOGICAL BASICS OF MALARIA PROPHYLAXIS As noted above, infection of human occurs through a bite of a female Anopheles mosquito that injects sporozoites into the blood with saliva (vector- bone rote of transmission). Infection is also possible in blood transfusion and transplacentally. In this case an invasive stage for the human is an erythrocytic schizont, and such malaria is called schizontic malaria. Pathogenic action: 1. Mechanic (destruction of erythrocytes and hepatocytes). 2. Toxicoallergic (poisoning by waste products). 3. Feeding at the expense of the host (on hemoglobin) and impairment of metabolic processes. Clinical manifestations: intermittent fever attacks. An attack lasts 6–12 hours and includes a cold stage, hot stage and sweating stage. The attack starts with sensation of cold and shivering lasting 0.5 to 2–3 hours. Then temperature

115 rapidly increase up to 40–41 °C. Patients have severe fever and intoxication symptoms. In 6–8 hours (or later for malaria caused by P. falciparum) the temperature drops to 35–36 °C and a profuse sweating starts, intoxication decreases, patients feel better. In a tertian malaria these attacks repeat every 48 hours; for quartan malaria this period is 72 hours. It is explained by the duration of erythrocytic schizogony: for Plasmodium vivax, Plasmodium ovale and Plasmodium falciparum it is 48 hours, for Plasmodium malaria — 72 hours. Enlargement of the liver and spleen is observed (here affected erythrocytes are destroyed). The disease is accompanied by anemia. Malignant tertian malaria has a more severe course and results in lethal outcomes. Basic causes of complications (malaria coma, acute renal failure, etc.): erythrocytes of all ages are affected; a great number of blood merozoites; erythrocyte schizogony occurs not in large blood vessels, as in other types of plasmodia, but in capillaries of organs (such as brain). Laboratory diagnostics: finding parasites in the blood (thick blood smear). It is necessary to take blood during the attack or immediately after it. To determine the specious of the malaria parasite the following signs are used: 1. Plasmodium vivax has noticeable amoeboid stage. 2. Erythrocytes affected by Plasmodium ovale are enlarged and have an distorted shape with fringed edges. 3. The gamont of Plasmodium falciparum has semi-lunar shape. 4. Schizont of Plasmodium malaria has stage of band. Methods of immunoassay (determination of anti-bodies in blood) are also significant for diagnosis of malaria. Personal prophylaxis: prevention of mosquitoes bites (using repellents) and chemical prophylaxis. Social prophylaxis: revealing and treating sick people and carriers, personal and social health education, eliminnation of mosquitoes of g. Anopheles. Fighting mosquitoes includes the following directions: 1. Protection from bites — wearing covering-up clothes, using repellents, nets on windows; zooprophylaxis — making biologic barriers (cattle-breeding farms) between places of mosquitoes’ reproduction and dwelling houses, etc.). 2. Elimination of adult mosquitoes — dispersion of insecticides in places of wintering of mosquitoes (basements, garrets, cattle yards). 3. Elimination of larvae: a) drainage of small water reservoirs having no economic significance; b) using toxic chemicals; c) shading water reservoirs with trees; d) drainage of mashes, deepening of reservoirs, straightening of river-beds; e) dispersion of mineral oils over the surface of water reservoirs (they block spiracles of larvae) f) raising gambusia fish.

116 TOXOPLASMA Toxoplasma gondii belongs to the class Sporozoa, order Coccidia. It is a pathogen of toxoplasmosis. The disease is common everywhere, 30 % of people on the Earth are infected. Morphological peculiarities (figure 64): a trophozoite has a semilunar shape, sizes of 4–7 × 2–4 μm.

Fig. 64. Morphology of T. gondii: A — diagram; B — trophozoite (7×40); C — oocyst (7×40)

One of its ends is sharpened, the other is rounded. The body is covered with 2 membranes. The nucleus is large. There is a conoid on the sharpened end; it serves for attachment of the parasite to a host’s cell. Life cycle: principal hosts are representatives of the Felidae species (cats, lynx, etc.) (figure 65).

Fig. 65. Life cycle of T. gondii 117 Intermediate hosts are all mammals, birds and reptiles. Invasion sources: 1) cats, excreting oocysts with sporozoites into the environment; 2) wild and domestic animals, birds and humans excreting tissue cysts with trophozoites in saliva, nose mucus, sperm, feces and milk; 3) meat of domestic animals and wild animals and birds. Rotes of transmission: 1) alimentary — with contaminated food of animal origin (meat, milk and eggs); 2) contact — in contact with cats (contamination with oocysts), through the damaged skin during processing skins of affected animals; 3) vertical. Pathogenic action: 1. Mechanic (impairment of cells, hemorrhages in serous membranes, necrotic foci in the liver, spleen, brain). 2. Toxicoallergic (poisoning by waste products). Clinical manifestations. Acquired toxoplasmosis has no symptoms. In people with weakened immunity the disease has symptoms of chronic intoxication: long-duration rise of temperature to 37.3–37.5 °C, weakness, apathy, poor appetite, headache, worsening of memory, etc., lymphatic nodes are enlarged (cervical, occipital, inguinal). Congenital toxoplasmosis. If infection occurs during the first months of pregnancy, miscarriages or still-birth may be observed. When infection occurs at a later term of pregnancy, the development of the brain of a fetus can be impaired (hydrocephaly), meningoencephylitis develops, sometimes inflammation of ocular membranes, jaundice, enlargement of the liver and spleen. Laboratory diagnostics: Immunoassay (revealing anti-bodies in the blood of sick people). Sometimes parasites are found in blood smears, puncture samples of lymphatic nodes and cerebrospinal fluid. Personal prophylaxis: observing rules of hygiene after contacts with cats, proper cooking oof meat, boiling milk, observing rules of working with animal carcasses. Social prophylaxis prevention of contamination of the environment and water sources with animal feces, personal and social health education. Timely examination of pregnant women is necessary for prophylaxis of congenital toxoplasmosis.

PNEUMOCYSTIS CARINII Pneumocystis carinii is a sporozoan that causes pneumocystosis. The disease is common everywhere, about 10 % of word population are infected. Morphology: there are 2 main forms of the parasite: small (1–5 micrometers) mononucleated trophozoites and sporocysts, containing 2 to 8 sporozoites.

118 Rote of transmission: droplet. Pathogenic action: 1. Mechanic (injure of alveoli). 2. Toxicoallergic (intoxication caused by metabolites of the parasite). Symptoms. Only children and people with low immunity develop the symptoms of the disease. In children, pneumocystosis occur at 4–6th months of life: appetite lowers, body mass do not increase, pallor and cyanosis of nasolabial triangle maydevelop; there are semicough, subfebrile temperature, rales in lungs, labored breathing, barking cough. Laboratory diagnosis: X-ray reveals focal lung lesions of different size and intensity. Sometimes the parasites can be found in sputum. Prophylaxis: medical examinations of high-risk groups, health education.

BASIC TERMS AND CONCEPTS Balantidiasis — disease caused by Balantidium coli. Schizontic malaria — malaria occuring when the invasive stage is an erythrocyte schizont. Merozoite — vegetative stage in the life cycle of sporozoans. Merulation — coming out of mature merozoites from erythrocytes into the blood plasma. Oocyst — life stage of malaria parasite situated on the external surface of the mosquito's stomach abd it containing sporozoites. Pseudocyst — cyst in tissues that is formed as a result of accumulation of trophopzoits of the toxoplasma in a cell. Gametogony — development of plasmodium’s gametes in the body of the intermediate host. Hypnozoites — sporozoites of plasmodia that affectliver cells and continue their development only after some dormant period of time. Pneumocystosis — disease caused by Pneumocystis carinii. Congenital toxoplasmosis — disease caused by Toxoplasma gondii which affects fetustransplacentally. Schizogony — asexual reproduction of sporozoans in which mother cell divides into multiple daughter cells. True cyst — cyst formed as a result of copulation of gametes.

119 Topic 19. PHYLUM PLATHELMINTHES, CLASS TREMATODA

GENERAL CHARACTERISTIC AND CLASSIFICATION OF THE PHYLUM The number of species: 15 000. Mode of life: free living (turbellarians) and parasites (flukes, tapeworms). Characteristic features of the phylum: 1) 3 germ layers; 2) bilateral symmetry of the body; 3) elongated and flattened body; 4) dermo-muscular wall of the body; 5) absence of the body cavity; 6) organ systems: digestive, excretory, nervous and reproductive. The dermo-muscular body wall consists of epithelium (tegument) and 3 layers of smooth muscles (circular, longitudinal and diagonal) beneath. Digestive system: 2 regions: foregut (mouth, pharynx) and a blind-ended midgut. Tapeworms have no digestive system. The excretory organs are pronephridia. The nervous system: circumpharyngeal nerve ring, suprapharyngeal and subpharyngeal ganglions and nerve chords. The lateral cords are most developed. There are tactile organs and organs of chemical senses. Interspace between organs is filled with the connective tissue. The majority of species are hermaphrodites. The phylum includes 3 classes: eddy worms (Turbellaria), flukes (Trematoda) and tapeworms (Cestoidea).

THE FEATURES OF FLUKES OF ADAPTABILITY TO PARASITISM. PECULIARITIES OF LIFE CYCLES IN FLUKES The body of flukes is leaf-shaped from 2 to 80 mm long. Fixation organs are located on the abdominal side (oral and ventral suckers). The tegument protects the parasite from digestion in the host’s organism. The majority of flukes are hermaphrodites. Male reproductive system: testes, vasa defferentia, ejaculatory duct, cirrus. Female reproductive system: singke ovary, uterus, vitelline gland, ootype, Mehlis gland. Flukes have complex life cycles and produce thousands or tens of thousands of eggs per day. Asexual reproduction of fluke’s larvae is called polyembryony. Principal hosts are vertebrate animals and human, intermediate hosts are fresh-water snails (1st intermediate host), fish, crustaceans, crabs (2nd intermediate host). A sexually mature life stage of suckers is called marita. It lays eggs in the organism of the principal host (figure 66). The egg should be in water to produce miracidium. The miracidium swims and finds intermediate host (snail) where it undergoes stage of sporocyst during which a generation of redia develops. Each redia form a generation of cercariae. They leave the mollusk’s body and freely swim in water. A dormant stage of cercaria situated on water plants is adolescaria. The majority of fluke species have the 2nd intermediate host (fishes, crawfishes, crabs). Cercaria permeate its body by a sharp stilet and transforms into metacercaria. 120 Therefore, several invasive stages are possible for principal host (human): metacercaria, adolescercaria or cercaria. Diseases caused by flukes are called trematodoses.

External environment Principal host (Fresh-water reservoir) 1st intermediate (vertebrates) host (mollusks) Sporocyst Mature stage (marita) Egg Miracidium Redia parthenitae

Cercaria

Cercaria Shistosom es P.westermani Adolescaria O.felineus F.hepatica

Metacercaria 2nd intermediate host (fishes, cancroids, crabs)

Fig. 66. Diagram of the flukes’ life cycle

COMMON LIVER FLUKE Fasciola hepatica is a biohelminth, pathogen of fasciolasis. The disease is common everywhere. Morphological peculiarities: leaf-like shape of the body, size 3–5 cm in length. Has 2 suckers (oral and ventral). Canals of the gut greatly branch. There is a uterus behind the abdominal sucker and branching ovarium beneath it. Viteline glands are in the body sides, testes are in the middle part of the body (figure 67). Life cycle: principal hosts are herbivorous animals, sometimes human. An intermediate hosts are mollusks (Limnea truncatula). Stages of the life cycle: marita – egg – miracidium – sporocyct – redia – cercaria – adolescaria. The human becomes infected while drinking water from stagnant reservoirs or eating plants watered with that water and vegetables washed with it (swallowing adolescaria). In the intestine the membrane of adolescaria is dissolved, parasites permeate into the liver through the portal vein or through the intestinal wall into the abdomen, and then into the liver.

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Fig. 67. Morphological peculiarities of F. hepatica: A —of the parasite; B — reproductive system; C — digestive system; D — photograph; E — egg (7×40)

Pathogenic action: 1. Mechanic (destruction of hepatic cells and obstruction of bile ducts). Cirrhosis of the liver can develop in case of intensive invasion. 2. Toxicoallergic (poisoning by waste products). 3. Feeding at the expense of the host and impairment of normal metabolism (absorption of nutrients and vitamins). Clinical manifestations: ache in the right hypochondrium (where the liver is situated), nausea, vomiting, jaundice of sclerae, indigestion, weakness, headache, skin itching, rash and fever. The liver is enlarged, dense and painful when palpated. Complications: inflammation of bile ducts, liver abscess, jaundice. Laboratory diagnostics: finding eggs in feces or duodenal content. Eggs are large (135 × 80 μm), oval, yellowish-brown, have lid on one of the poles. Sometimes transit eggs are revealed in healthy people after eating liver of animals sick with fasciolasis. Another effective method is immunoassay. Prophylaxis: not to use water from reservoirs for drinking and watering vegetable gardens; to wash vegetables thoroughly; to reveal and treat sick persons; personal and social health education; sanitation of animals; prevention of contaminating water reservoirs with feces of sick animals and people.

СAT LIVER FLUKE Opisthorchis felineus is a biohelminth, pathogen of opisthorchiasis (opistorchosis). The disease is common in Siberia along the bads of large rivers. Some foci can appear in Belarus and other countries (figure 68). Morphological peculiarities: the body length is 10 mm. There are a uterus in its middle, a rounded ovarium and bean-shaped seminal receptacle behind it. There are 2 rosette-shaped testes in the hind part of the body, and S-shaped

122 canal of the excretory system between them. The canals of midgut do not branch. Viteline glands are situated on both sides of the body.

Fig. 68. Morphological peculiarities of O. felineus: A — diagram; B — photograph (×20); C, D — egg (7×40)

Life cycle: principal hosts are human, cats, dogs and other fish-eating animals. The 1st intermediate host is freshwater mollusks (Bithynia leachi), the 2nd intermediate host is freshwater fish. Life cycle stages: marita – egg – miracidium – sporocyst – redia – cercaria – metacercaria. Infection of a human occurs while eating undercooked fish which contains a metacercaria. Marita are located in the liver and pancreas of a principal host. Pathogenic action: 1. Mechanic (injury of the walls of bile ducts by suckers and their obstruction; damaging the liver and pancreas). 2. Toxicoallergic (poisoning by waste products). 3. Feeding at the expense of the host and impairment of metabolic processes. 4. Mutagenic (primary liver cancer is more often in case of the disease). Clinical manifestations: pains in the right hypochondrium, loss of appetite, nausea, vomiting, indigestion, weakness, headache. The liver is enlarged. Laboratory diagnostics: revealing eggs of the parasite in feces or duodenal content. Eggs are 26–30 × 10–15 μm in size, of yellowish-brown color, oval, there is a lid on one pole. Revealing antibodies in the blood serum (immunoassay). Prophylaxis: proper boiling, frying and salting fish (observing the rules of salting), revealing and treating sick people; prevention of contaminating water reservoirs with feces of sick animals and people; personal and social health education.

123 ORIENTAL LUNG FLUKE Paragonimus westermani is a biohelminth, pathogen of paragonimiasis. The disease is common in the South-Eastern Asia and South Asia, Central Africa and South America. Morphological peculiarities: egg-shaped slightly flattened body; the length is 7.5–12 mm (figure 69).

Fig. 69. Morphology of P. westermani: A — diagram of marita; B — view of marita (×20); C — parasites in the tissue of the lung; D — egg (7×40)

There is a lobular ovarium on one side from an abdominal sucker and the uterus on the other side. Viteline glands are located in lateral parts of the body. Backward from the uterus and ovarium there are 2 lobe-shaped testes. Life cycle: principal hosts are human, dogs, cats, pigs and other mammals. The 1st intermediate host is freshwater mollusks of the genus Melania, the 2nd intermediate host is crawfish and crabs. Stages of the life cycle: marita – egg – miracidium – sporocyst – redia – cercaria – metacercaria. Infection of the human occurs while eating crawfishes and crabs having metacercaria. In the gastro-intestinal tract of the host, parasites get rid of their membranes, permeate into the abdomen through the intestinal wall, and then into the pleura and lungs through the diaphragm. Fluke’s maritas are situated in small bronchi, where cavities are formed around them; these cavities are filled with parasite’s metabloites and products of tissue decay. Eggs are excreted into the environment with phlegm or feces. Pathogenic action: 1. Mechanic (injury of the intestinal wall, diaphragm, pleura and lungs). 2. Toxicoallergic (poisoning by waste products). 3. Feeding at the expense of the host’s organism and impairment of metabolic processes. Clinical manifestations: pains in the chest, breathlessness, cough with purulent sputum and sometimes with blood, high temperature, headache. Complications: pulmonary heart disease, brain abscesses, meningoencephalitis.

124 Laboratory diagnostics: finding eggs in the sputum or feces. Eggs are large (up to 100 μm), oval, of yellowing color, with a lid and a thick membrane. Prophylaxis: not to eat improperly cooked crawfishes and crabs; prevention of contaminating water reservoirs with feces of sick animals and people; personal and social health education.

BLOOD FLUKES Schistosomes are common in the countries with a tropical and subtropical climate. Several types of schistosomes can affect human: S. haematobium is a pathogen of genitourinary schistosomiasis (bilharzia); S. Mansoni is a pathogen of gastrointestinal schistosomiasis; S. japonica is a pathogen of oriental schistosomiasis (Katayama fever). It is variety of gastrointestinal schistosomiasis with severe affections of the intestine, liver, and sometimes CNS. Morphological peculiarities: dioecious (have separate sexes). Male’s body is short and broad (10–15 mm), female’s body is up to 20 mm (figure 70). Females are situated in the gynecophoral canals on the abdominal side of male flukes. Males have a developed abdominal sucker, which ensures a reliable fixation to the walls of blood vessels.

Fig. 70. Morphology of schistosomes: A — diagram of the maritas; B — egg of S. haematobium (7×40); C — egg of S. Mansoni (7×40); D — egg of S. japonicum (7×40); E — maritas (×20); F — schistosomule (7×40); G — egg of S. Mansoni in the wall of the intestine (7×40)

Life cycle: principal hosts are human and various mammals, intermediate hosts are fresh-water mollusks. Stages of life cycle: marita – egg – miracidium –

125 primary sporocyst – secondary sporocyst – cercaria. Maritas are located in veins of the abdominal cavity and urogenital system. Females lay eggs in the vascular lumen of the walls of urinary bladder or intestine. Eggs have sharp spike, which helps them to get into the lumen of the organ. Then they should get to water, larvae develop in the mollusk’s body. Cercaria leaves the mollusk and penetrates human skin during bathing, working in water, drinking water from reservoirs. Clothes do not protect the body from cercariae. In the organism cercariae migrate through lymphatic and blood vessels into the right atrium, right ventricle and then to the lungs, then to veins of mesentery, intestine, genitourinary system. Pathogenic action: 1. Mechanic (injury of the genitourinary organs and intestine by eggs). 2. Toxicoallergic (poisoning by waste products). 3. Feeding at the expense of the host’s organism and impairment of metabolic processes (absorption of nutrients, vitamins, blood cells). 4. Mutagenic (induce cancer of the urinary bladder, tract and intestine). Clinical manifestations: dermatitis and itching at the site of cercaria invasion. During the migration of young schistosomes there are cough with mucus and spitting of blood, symptoms of bronchial asthma with general malaise, headache, weakness and loss of appetite. Symptoms of genitourinary schistosomiasis are disuria (impairment of micturition) hematuria (excretion of blood in urine), painful micturition. Characteristic signs of gastrointestinal schistosomiasis are pains in the abdomen, irregular bowel movements, blood and mucus in feces, diarrhea, oedema of lower extremities and the abdomen. Laboratory diagnostics: finding eggs of S. Mansoni and S. japonicum in feces and bioptates of the intestinal mucous membrane; eggs of S. haematobium in urine and bioptates of the bladder mucous membrane. Immunoassay. Prophylaxis: not to bathe, wash in water of reservoirs which may contain cercaria, not to drink it, not to use it for domestic needs; revealing and treating sick people, prevention of contaminating water reservoirs with feces and urine; personal and social health education.

TECHNIQUES OF LABORATORY DIAGNOSIS OF TREMATODOSES Most of helminthes are situated in intestine or organs that are connected with it by means of ducts. For this reason, most of techniques are based on looking for the parasites in fecies. There are macroscopic and microscopic methods that are used to detect parasites and their parts (helminthoscopy) or their eggs (helminthoovoscopy). Performing such tests requires strict adherence to Hygiene rules, sterilize inoculation loops and glass sticks in flame, sterilizing laboratory dish, glasses and tools, cleanliness of the place of work.

126 BIOLOGICAL BASICS OF PROPHYLAXIS OF TREMATODOSIS It is a complex of measures that are based on studying biology of the pathogen, its migration ways, life stages, biology of intermediate hosts. That gives possibility to interrupt some link of the parasite life cycle.

BASIC TERMS AND CONCEPTS Cercarial dermatitis — syndrome caused by the cercariae of certain species of schistosomes whose normal hosts are birds and mammals other than humans. Dermo-muscular body wall — a body wall of flatworms that consists of tegument and 3 layers of smooth muscles. Marita — stage of sexually mature worm in the life cycle of flukes. Metacercaria — invasive stage for a principal host in the life cycle of flukes. Miracidium — the 1st larval stage in the life cycle of flukes. Redia — larval stage of flukes in which parasitize the 1st intermediate host. Sporocyst — larval stage of flukes that develops in the organism of the 1st intermediate host from a miracidium. Tegument — external layer of a dermo-muscular body wall of flukes. Cercaria — mobile larva of the fluke that is leaves the mollusk’s organism and gets to water. Ootype — part of the female reproductive system of flukes where eggs are fertilized, surrounded with yolk and covers with a shell. Polyembryony — asexual reproduction of living matter consisting in development of more than one embryo from a single zygote. Cirrus — copulatory orgnan of flukes which is modified appendage of the male reproductive duct.

Topic 20. PHYLUM PLATHELMINTHES, CLASS CESTOIDEA

CHARACTERISTIC OF THE CLASS TAPEWORMS, THEIR ADAPTAION TO PARASITISM There are 1800 species of endoparasites. Their bodies are flattened in a dorsal-ventral direction and look like tapes. Length is from 1 mm to 10–18 m. A head of tapeworms is called scolex. It is equiped with fixation organs such as suckers, rostellum with hooks, bothria (sucking grooves); the next body regions are a neck and strobila (body). Strobila consists of segments (proglottids). Mature proglottids detach from the strobila and come outside. An external layer of the dermo-muscular wall (tegument) has hair-like growths (microthriches) for absorbtion of nutrients in the host’s intestine. The digestive, circulatory and respiratory systems are absent. The excretory organs are protonephridia. 127 The nervous system and sense organs are poorly developed. Cestodes are hermaphrodites. In young proglottids, male reproductive organs develops first (near the neck), then female organs do. Therefore proglottids in the middle of the strobila are hermaphroditic. Mature segments (at the end of the body) have only uterus filled with eggs. Uterus of taeniae is closed, that of Diphyllobothria is open.

PECULIARITIES OF LIFE CYCLES OF TAENIA AND DIPHYLLOBOTHRIA Types of measle. An egg of taenia contains spherical 6-hooked larva called oncosphere. Having get to the intestine of an intermediate host, the oncosphere comes out from egg, permeates into blood vessels using hooks. Blood carries it to tissues of various organs and transformed in to a measle (figure 71). LIFE CYCLE OF LIFE CYCLE OF DIPHYLLOBOTHRIUM Principal host TAENIA (development occurs in water) Sexually mature stage (development occurs aland)

Egg Mature proglottids Coracidium Egg Procercoid (contains an oncosphere) 1st intermediate host Oncosphere (Little crawfish, Cyclopes) (in the intestines) Intermediate Measle host Plerocercoid (measle) (in tissues) (vertebrate animals) 2nd intermediate host (Fish) TAENIA SOLIUM DIPHYLLOBOTHRIUM LATUM TAENIARHYNCHUS SAGINATUS ECHINOCOCCUS GRANULOSUS Fig. 71. Life cycles of cestodes

A cysticercus is a vesicle-like measle filled with fluid. Inside the measle there is one screwed scolex. The coenurus is a measle with several screwed scloexes. A cysticercoid has a widening with a screwed scolex and a tail-like appendage. An echinococcus is a mother cyst with many daughter cyst inside; each of them contains a scolex. A plerocercoid is a worm-shaped larva with two bothria. Measles transform into an adult individual in the intestine of principal hosts. By the action of digestive juices the scolex screws outside, attaches to the intestinal wall and proglottids start growing from the neck. Diseases caused by cestodes are cestodoses.

128 TAENIA SOLIUM AND TAENIARHYNCHUS SAGINATUS Taeniarhynchus saginatus (beef tapeworm) is a biohelminth, a pathogen of taeniarhynchosis. The disease is common everywhere. Morphological peculiarities: the length of a mature parasite is 4–10 m. There are 4 suckers on the scolex. Hermaphroditic progottids have a bilobed ovaries and viteline glands behind them; there are vesicle-like testes in lateral parts of a proglottid. Mature segments contains uterus with 17–35 branches and up to 175 000 eggs inside (figure 72). Mature segments may crawl out of the anus and move along the human body and clothes.

Fig. 72. Morphology of Taeniarhynchus saginatus: A–D — diagrams, E–H — microphotographs: A, E — scolexes, B, F — hermaphroditic progloditts, C, G — mature proglottids, D, H — eggs: 1 — testes; 2, 3 — semen ducts; 4 — cirrus; 5 — genital atrium; 6 — vagina; 7 — ovarium; 8 — viteline gland; 9 — ootype; 10, 14 — uterus; 11, 12 — canals of excretory system; 13 — suckers; 15 — radial striation

Life cycle: a principle host is a human, an intermediate one is cattle that get infected while swallowing eggs of tenia with grass. The human gets infected while eating undercooked beef with measles (cysticerci). The life span of taenia in the human body is up to 25 years. Pathogenic action: 1. Mechanic (by irritation of the intestinal mucous membrane by suckers). 2. Toxicoallergic (poisoning by waste products). 3. Feeding ate the expen the host’s organism and impairment of metabolic processes. Clinical manifestations: itching around the anus, ache in the abdomen, unstable stool, weakness, loss of appetite, loss of weight. Laboratory diagnostics: revealing proglottids or eggs in feces. Eggs are round, have a thick striated shell and six-hooked oncosphere inside. 129 Prophylaxis. Personal: not to eat untested beef. Social: making a veterinary evaluation of cattle corpses, revealing and treating sick people, prevention of contaminating pastures with feces, sanitary improvements of settlements (closed toilets in rural areas), personal and social health education. Taenia solium (pork tapeworm) is a biohelminth. Its sexually mature life tage causes teniasis in the human and its larvae cause cysticercosis. Morphological peculiarities: the length of the tapeworm is 2–3 m, the scolex is equipped with 4 suckers and a rostellum with 2 rows of hooks (figure 73).

Fig. 73. Morphology peculiarities of Taenia solium: A–D — diagrams, E–H — microphotographs: A, E — scolexes, B, F — hermaphroditic proglottids, C, G — mature proglottids, D, H — eggs: 1 — hooks; 2 — suckers; 3 — testes; 4 — semen duct; 5 — genital atrium; 6 — vagina; 7 — ovarium; 8 — ootype; 9 — viteline glan; 10 — excretory canals; 11, 13 — uterus; 12 — additional lobe of the ovary; 14 — radial striation

A hermaphroditic proglottid contains a thrilobed ovarium. A mature proglottid contains a uterus with 7–12 branches. Mature segments are immobile. Life cycle: a principal host is human; an intermediate ones are domestic pigs or wild boars, sometimes human. Getting infected by teniasis occurs while eating undercooked pork with cysticerci. In the intestine a scolex screws out from the cysticercus by action of digestive juices. It attaches to the intestinal wall and begin forming proglottids. In 2–3 months a helminth reaches its sexual maturity. The life span of a tenia is several years. Pathogenic action is similar to that of Taeniarhynchus saginatus. Clinical manifestations: pains in the abdomen, nausea, vomiting, indigestion, headache, dizziness. Laboratory diagnostics: revealing proglottids or eggs in feces. Eggs of Taeniarhynchus saginatus and Taenia solium look same. 130 Prophylaxis. Personal prophylaxis consists in not eating untested pork. Social examination of pig and boar corpses, revealing and treating sick people, prevention of contaminating pastures with feces, sanitary improvements of settlements (closed toilets in rural areas), personal and social health education. Cysticercosis. The pathogen of cysticercosis is cysticercus of a Taenia solium. The human gets infected with cysticercosis: 1) because of noncompliance with rules of personal hygiene and swallowing eggs which can be on hands and food; 2) in autoinvasion: if a person is sick with teniasis, proglottids may get into the stomach during vomiting, then oncospheres are released from eggs, get to various organs (subcutaneous fat tissue, muscles, eyes, brain) and transform into measles; 3) in treating teniasis with drugs that dissolve proglottids. Pathogenic action: 1. Mechanic (pressure on tissues). 2. Toxicoallergic (poisoning by waste products). Clinical manifestations depend on intensity of invasion and location of cysticerci. Their presence in CNS may cause headaches, convulsions, paralysis of extremities and may end up with death. Intraocular cysticercosis may cause a tolal loss of vision. Laboratory diagnostics: immunoassay. Personal prophylaxis is observing rules of hygiene, social measures are health education, revealing and treating sick people.

HYMENOLEPIS NANA Hymenolepis nana (dwarf tapeworm) is a contact helminth, a pathogen of hymenolepidosis. Preschool children fall ill more often. Morphological peculiarities: the length of the dwarf tapeworm is 1–5 cm, its body cosists of about 200 proglottids; the scolex has 4 suckers and a rostellum with a double circlet of hooks. The uterus is closed, but a thin wall of proglottids is easily destroyed and eggs get into the intestinal lumen (figure 74). Life cycle: the human and is both principal and intermediate host. Invasion occurs due to not obeying rules of personal hygiene and swallowing eggs of the tapeworm. Oncospheres come out from eggs in the small intestine. They implant into villi of the intestinal mucous membrane and transform into cysticercoids. Measles destroy villi, get into the intestinal lumen, attach to the mucous membrane and in 2 weeks rich sexual maturity. The life span of the parasite is 1–2 months. The development of oncospheres is possible without changing the hosts due to autoreinvasion. Pathogenic action: 1. Mechanic (destruction of villi of a small intestine, irritation of the mucous membrane by fixation organs of the parasite).

131 2. Toxicoallergic (poisoning by waste products). 3. Feeding at the expense of the host’s organism and impairment of metabolic processes.

Fig. 74. Morphology of Hymenolepis nana: A — diagram of the life cycle in the small intestine: 1 — oncosphere; 2 — cysticercoid; 3 — scolex; B — mature tapeworm (×20); C — scolex (7×8); D — mature proglottids (7×8); E — egg (7×40)

Clinical manifestations: ache in the abdomen, loss of appetite, nausea, indigestion, general weakness, irritability. In case of intensive invasions vomiting, dizziness, seizures, fainting are possible. Children are arrested in mental and physical development. Laboratory diagnostics: finding eggs in feces. Eggs are round and have two membranes and filaments between them; oncosphese has the shape of a lemon. Personal prophylaxis is observing hygiene rules. Measures of social prophylaxis are: 1) inculcation of hygien skills in children; 2) revealing, isolating and treating sick people; 3) thorough moist mopping of children’s rooms and sanitary treatment of toys; 4) personal and social health education.

ECHINOCOCCUS GRANULOSUS Echinococcus granulosus is a biohelminth, a pathogen of echinococcosis. Morphological peculiarities: the length is 3–5 mm. The scolex has suckers and a rosellum with 2 circlets of hooks. The strobila consists of 3–4 proglottids. The second to the last proglottid is hermaphroditic, the last one is mature. The uterus is branched and closed (figure 75).

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Fig. 75. Morphology of Echinococcus granulosus: A1 — a rostellum with two circlets of hooks; 2 — sucker; 3 — neck; 4 — hermaphroditic proglottid; 5 — mature proglottid; 6 — uterus; A — diagrams; B — a microphotograph (7×8)

Life cycle: principal hosts are carnivorous animals (dogs, wolfs, coyotes), intermediate hosts are human, herbivorous and omnivorous animals (large and small cattle, pigs, camels, deer, etc.). Invasion of principal hosts occurs in eating organs of affected animals. Measles in the intestine form a great number of sexually mature worms. Mature proglottids of the echinococcus are capable of crawling from the anus. They crawl on the animal coat and disseminate eggs. Having got to the grass, eggs and proglottids, can be swallowed by intermediate hosts. In the intestine, oncospheres come out from eggs, get into the bloodstream and are carried to various organs (liver, lungs), where they transform into measles (hydatid cysts). The human gets infected from sick dogs while neglecting rules of personal hygiene. It is possible to get infected from sheeps and other animals if their wool carries eggs that have got there from grass or soil. In the human echinococcus affects the liver, lungs, brain, muscles and bones. Pathogenic action: 1. Mechanic (pressure on tissues and destruction of affected organs). 2. Toxicoallergic (poisoning by waste products). Clinical manifestations: skin itching and rash, pain and pressure in the right hypochondrium. Patient may suffer from pains in the chest, cough, breathlessness, sometimes hemoptysis. The hydatid cyst may burst into a bronchus, abdominal and thoracic cavity or become purulent. These complications may result in a lethal outcome.

133 Laboratory diagnostics: is based on an X-ray examination and immunoassay (revealing antibodies in the blood serum). Measures of personal prophylaxis are observing hygiene rules, washing hands after contact with dogs, sheeps since their wool can contain eggs of echinococcus. Social prophylaxis consists in dehelminthization of service dogs, never feed them with organs of animals that can be affected with echinococcus; trapping stray dogs, personal and social health education.

DYPHYLLOBOTHRIUM LATUM Dyphyllobothrium latum is a biohelminth, pathogen of diphyllobothriasis. Morphological peculiarities: the body length is 10–18 m. There are 2 sucking grooves (bothria) on the scolex. The width of proglottids is significantly more than their length (figure 76). Mature proglottids contain an open rosette-shaped uterus.

Fig. 76. Morphology of Diphyllobothrium latum: A–E — diagrams; F–I — microphotographs; A — scolex, B, F — transverse section of the scolex, C, G — hermaphrodite proglottid, D, H — mature proglottid, E, I — egg: 1 — bothria; 2 — uterus; 3 — lid of the egg

Life cycle: principal hosts are human and fish-eating mammals (cats, dogs, polar foxes, bears). The 1st intermediate hosts are small crustaceans (cyclops, daphnia), the 2nd one is fish. Predator fishes can be reservoir hosts. Eggs are excreted from the organism of a principal host with feces. They should get into water where in 3–5 weeks they excrete a larva called coracidium. The coracidium is swallowed by the 1st intermediate host and transforms into a procercoid in its intestine. When a fish swallows the affected crustacean, the procercoid transforms into a plerocercoid in its muscles and reproductive organs. Principal hosts get 134 infected while eating fish or caviar with plerocercoids. The life span of Diphyllobothrium latum in the human organism is up to 25 years. The location of the parasite is a small intestine. Pathogenic action: 1. Mechanic (injures a mucous membrane of the intestine by bothria). 2. Toxicoallergic (poisoning by waste products). 3. Feeding at the expense of the host’s organism and impairment of metabolic processes (selectively absorbs vitamin B12, which results in the development of malignant anemia). Clinical manifestations: weakness, nausea, ache in the abdomen, meteorism, subfebrile temperature. Signs of anemia appear: general weakness, sleepiness, dizziness, and dyspeptic events. Bright-red spots and fissures appear on the tongue, atrophy of nipples occurs. The skin is pale, slightly yellowish; the liver and spleen are enlarged. Laboratory diagnostics: finding eggs in feces. Eggs are oval, there is a lid on one pole, and a prominence on the other one. Personal prophylaxis exclusion of raw, half-raw, improperly cooked fish and caviar from racion. Social prophylaxis is prevention of reservoirs from contamination with feces, revealing and treating sick people, personal and social health education.

TECHNIQUES USED FOR LABORATORY DIAGNOSIS OF CESTODOSES Visual examination of feces. Small portions of feces are mixed with water in a flat bath or a Petrie dish. They are examined in a good illumination against a dark background with a magnifying glass if needed. This allows to reveal helminthes, their scolexes, parts of a strobila, proglottids. Sedimentation techniques: if the specific weight of eggs is greater than the specific weight of the fluid, then eggs concentrate in the sediment, which is studied under the microscope. It is used for revealing trematodes’ eggs. Krasilnikov technique. Under the action of detergents of washing materials helminthes’ eggs are concentrated in the sediment. The technique allows revealing eggs of all helminthes excreted with feces. Direct smear. A small part of feces is brought by a stick on the preparation glass into a drop of the 50 % of water-glycerin solution and rubbed untill an even smear is obtained, then it is examined under the microscope. Cellophane thick smear (Kato technique). Eggs of helminthes are revealed in a thick smear of feces with glycerin that stained with malachite green dye. The method reveals eggs of ascaris, whipworms, diphyllobothria, trematodes, tenia. Floatation techniques: if the specific weight of eggs is less than the specific weight of the fluid, then eggs float to the surface of the fluid and are taken to the film which is studied under the microscope. It is used for revealing eggs of ancylostoma, whipworm and dwarf tapeworm.

135 Fulleborn technique. Saturated solution of NaCl is used. Eggs of nematodes, dwarf tapeworm and Diphyllobothrium latum are revealed. Kalantaryan technique. Excrenments are mixed up with a saturated solution of NaNO3 in ratio 1 : 20. Eggs of the majority of helminthes quickly float up and are revealed in a scum. Oncospheres of teniae and eggs of trematodes do not float up.

BIOLOGICAL BASIS OF CESTODOSES PROPHYLAXIS It is a complex of preventive measures that are based on studying the biology of pathogens, ways of their migration, their life stages and biology of intermediate hosts. This gives a allows to interrupt the parasite’s life cycle and prevent its further development.

BASIC TERMS AND CONCEPTS Biohelminthes — helminthes that requires an intermediate host for development of their larvae. Bothria — fixation organs of Diphyllobothrium latum. Contact helminthes — helminthes whose eggs are transmitted during contact of a healthy person with a sick one or through domestic objects. Plerocercoid — measle of a Diphyllobothrium latum. Prglottid — segment of tapeworm’s body. Scolex — head of a tapeworm. Strobila — body of tapeworms consisting of segments. Cisticercus — measle of Taeniarhynchus saginatus and Taenia solium. Cysticercoid — measle of Hymenolepis nana. Echinococcus — tapeworm, pathogen of echinococcosis.

Topic 21. PHYLUM NEMATHELMINTHES, CLASS NEMATODA

GENERAL CHARACTERISTIC OF THE PHYLUM NEMATHELMINTHES AND THE CLASS NEMATODA Over 15 000 species inhabit water, soil, decaying organic matter; many of them have adapted to a parasitic mode of life. Characteristic features of the phylum: 1) three germ layers; 2) bilateral symmetry of the body; 3) cylindrical or spindle-like shape of the body; 4) presence of a dermo-muscular body wall and the primary body cavity; 5) presence of nervous, digestive, excretory and reproductive systems; 6) they are dioecious (have separate sexes); 7) hindgut and the anus have appeared. The phylum includes 5 classes. The class Nematoda has a medical significance. Class Nematoda. The body is spindle-like, its length can be from 136 1 mm to 1.5 m. Cross-section of the body is round. The body is covered with dermo-muscular wall, consisting of a cuticle, hypodermis and a layer of smooth muscles. The body cavity is primary (pseudocoelom). It contains internal organs. The digestive system consists of 3 regions: foregut, midgut and hindgut. The excretory organs are 1–2 cutaneous glands. Removal of wastes is also performed by phagocytes. The nervous system consists of a suprapharyngeal and subpharyngeal ganglions, circumpharyngeal nerve ring and longitudinal nerve chords. Nematodes have tactile and chemical sense organs. Nematodes are dioecious and have noticeable sexual dimorphism: males are smaller than females and their posterior end spirally curved. The reproductive system is tubular. In females it starts with long paired ovaries. Each ovarium widen to form an oviduct, the oviducts are followed by uteri which join together to form vagina. The reproductive system of males consists of an unpaired testis, vas defferens, ejaculatory duct that opens into the hindgut. Some species are viviparous. The majority of nematodes are geohelminthes. Diseases caused by ring worms are called nematodoses.

ASCARIS LUMBRICOIDES Ascaris lumbricoides is a geohelminth, pathogen of ascariasis. The disease is antroponosis. It is common everywhere exept arctic areas, deserts and semi- deserts. Morphological peculiarities: the length of a female is up to 40 cm, that of a male — 25 cm. The body is cylindrical, sharpened at the ends. There are are cuticular lips on the anterior end of the body (figure 77).

Fig. 77. Morphology of Ascaris lumbricoides: A — sexually mature helminthes (photograph); B — a transverse section (7×8); C — a fragment of the transverse section in the area of the uterus (7×40): 1 — the uterus filled with eggs; 2 — midgut; 3, 4 — ovarium; 5 — muscular fibers; 6 — cuticle; 7 — cylinder of hypodermis; D, E — fertilized eggs with a larva (7×40); F — unfertilized egg (7×40) 137 Life cycle: a sexually mature ascaris is located in a small intestine. A fertilized female lays up to 240 000 eggs per day, they are excreted into the environment with feces. Eggs develop in soil in proper temperature (20–25 °C), humidity and oxygen. This takes 21–24 days. Such eggs get into the human organism with unwashed vegetables, fruit and water. In the small intestine larvae come out of eggs, perforate its wall, get into blood vessels and migrate. Blood carries them through the liver, right atrium, right ventricle, pulmonary trunk and alveolar capillaries. Through the capillary walls larvae get into alveoli, ascend to bronchioles, bronchi, trachea and get into the pharynx to be swallowed. In 2.5–3 months they transform into sexually mature worms in a small intestine. Larval migration lasts about 2 weeks. The life span of mature ascaris is about 1 year. Larvae of another ascaris species (ascaris of pigs, dogs, etc.) may also migrate in the human organism but cannot complete the life cycle. The syndrome they cause is called Larva migrans. Pathogenic action of larvae of ascaris: 1. Toxicoallergic (poisoning by waste products). 2. Mechanic (injury of the liver, rupture of capillaries, injury of alveoli). 3. Feeding at the expense of the host’s organism and impairment of metabolic processes (absorption of nutrients and vitamins). 4. Mutagenic. Clinical manifestations of larval migration stage of ascariasis: persistent spastic cough especially at night, skin rash and itching, weakness, fever, headache, perspiration, oedema of lids and face. Clinical manifestations of intestinal stage of ascariasis: pains in the abdomen, nausea, vomiting, diarrhea, worsening of appetite, weakness, irritancy, worsening of memory, loss of weight. Complications of intestinal ascariasis: obstructive jaundice, purulent pancreatitis, purulent cholangitis, appendicitis, peritonitis, spastic and mechanic intestinal obstruction. Sometimes ascarides are found in frontal sinuses, cranial cavity, middle ear and ovaries. Personal prophylaxis involves observing rules of hygiene, washing vegetables, fruits and berries with hot water. It is necessary to protect food from flies and cockroaches as they are mechanic vectors for eggs of ascaris. Social prophylaxis is revealing and treating sick people, protection of the environment from contamination with ascaris eggs, health education.

TRICHOCEPHALUS TRICHIURUS Trichocephalus trichiurus (human whipworm) is a geohelminth, pathogen of trichocephaliasis. The disease is common everywhere. Morphological peculiarities: the length of a female is up to 5 cm, males are a shorter. The anterior region of the body is thin and filament-like snd

138 contains only esophagous. The posterior one is thicker, it contains all other organs (figure 78).

Fig. 78. Morphology of Trichocephalus trichiurus: A, B, E — diagrams; C, D, F — microphotographs; A, С — mature females, B, D — males, E, F — eggs: 1 — an anterior end of the body; 2 — «plug» on the pole

Life cycle. A fertilized female lays up to 60 000 eggs a day; they are excreted to the environment with feces. The development of eggs occurs in soil. In optimal conditions (temperature 25–30 °C, high humidity, presence of oxygen), an invasion larva maturates in 25–30 days. The human gets infected while eating vegetables, fruit and water contaminated with parasite’s eggs. In the intestine larvae come out of eggs and in 1–1.5 months become sexually mature. Migration of larvae does not occur. The whipworm's life span in the body is more than 5 years. Parasites are located in the upper region of the large intestine (mainly in the caecum). Pathogenic action: 1. Mechanic (injury of the mucous membrane of the intestine). 2. Toxicoallergic (poisoning by waste products). 3. Feeding at the expense of the host’s organism and impairment of metabolic processes (they perforate intestinal mucous membrane by an anterior end and feed on blood). 4. Mutagenic. Clinical manifestations: ache along the large intestine, irregular stool, meteorism, poor appetite, nausea, vomiting, weakness, headache. Complications: anemia, appendicitis and convulsive attacks. Laboratory diagnostics: finding eggs in feces. Eggs have a lemon shape and «plugs» on the poles. Prophylaxis: the same as in ascariasis. 139 ENTEROBIUS VERMICULARIS Enterobius vermicularis (seatworm or pinworm) a contact helminth, a pathogen of enterobiasis. The disease is common everywhere. Morphological peculiarities: the length of a female is about 10 mm, that of a male is 2–5 mm (figure 79). There are vesicles (cuticular swellings) at the anterior part of the body. Posterior part of the esophagus has a bulb — a ball-like dilation that takes part in fixation of the parasite to intestinal walls.

Fig. 79. Morphology of Enterobius vermicularis: A–C — diagrams; D–G — microphotographs; A, D, F — female, B, G — male: 1 — vesicle; 2 — esophagus; 3 — bulb; 4 — genital opening; 5 — uterus; 6 — anus; C, E — egg

Life cycle. Pinworms settle in the terminal region of the small intestine and in the beginning of the large intestine. After fertilization females crawl out of the anus, lay eggs on the skin of the perineum and excrete irritating fluid that causes itching. In proper conditions (temperature is 34–36 °C, humidity 70–90 %, oxygen), the eggs maturate in 4–6 hours. Sick people scratch itching skin and eggs get under nails. Later on they can be brought into the mouth or household goods. If the eggs are swallowed and get to the intestine of the host, larvae come out of eggs and in 2 weeks reach sexual maturity. The life span of a seatworm is about a month. Pre-school and junior school children fall ill more often. Pathogenic action: 1. Mechanic (injury of the intestinal mucous membrane). 2. Toxicoallergic (poisoning by waste products). 3. Feeding at the xpence of the host’s organism and impairment of metabolic processes. 140 Clinical manifestations: itching and a burning sensation around the anus. Itching troubles patients day and night, becomes unbearable, spreads to the perineum, sex organs and abdomen. The well-being and sleep of patients become worse, there appears irritancy, diarrhea with mucus, nausea, vomiting, borborygmus and aerocolia, academic progress of children worsens. Laboratory diagnostics: finding eggs by an adhesive tape test. Eggs are colorless, asymmetric, one side is flattened. Personal prophylaxis: observing personal hygiene, clean hands and bed- linen. Social prophylaxis inculcation of hygien skills in children, examination of the staff of child-care establishments, isolation and treatment of sick people, regular wet cleaning of rooms, sanitary treatment of toys, health education of parents and educators of pre-school establishments.

TRICHINELLA SPIRALIS Trichinella spiralis is a biohelminth, a pathogen of trichinellosis. Morphological peculiarities: females have sizes of 3–4 mm, males — 1.5–2.0 mm. Female reproductive tract is unpaired. Larvae are coiled like a spiral and encapsulated with connective tissue (figure 80).

Fig. 80. Morphology of Trichinella spiralis: A — diagram of a sexually mature worm, B — diagram of an encapsulated larva, C — an encapsulated larva (7×8): 1 — esophagus; 2 — uterus; 3 — ovarium; 4 — testis; 5 — muscle fiber; 6 — larva; 7 — capsule; D — male (7×40); E — decapsulated larvae (7×8)

Life cycle. Trichinella parasitizes carnivorous and omnivorous animals (pigs, wild boars, cats, dogs, mice, rats, bears, etc.). One and the same organism is a principal host at first (sexually mature forms are in the intestine) and then

141 an intermediate host (larvae are in muscles). Getting infected occurs while eating meat contaminated with larvae (pork, meat of wild boars, bears, etc.). In the small intestine capsules of larvae are digested, larvae transform in sexually mature worms. After fertilization females implant into the mucous membrane of the small intestine and give birth to new larvae. The larvae are carried within the organism with blood flow of and lymph and settle in the skeletal muscles. The diaphragm, intercostal and mastication muscles are affected more frequently. Larvae get into muscles and coil into spirals. Each larva covers with a capsule which calcifies in a year. Larvae preserve their vitality in the capsule up to 20–25 years. To continue the life cycle and transform into sexually mature forms, larvae must get into the intestine of another host. The human is a biological end for the cycle. Pathogenic action: 1. Toxicoallergic (poisoning of the organism by waste products of metabolism and decaying). 2. Mechanic (injury of intestinal walls and muscles). 3. Feeding at the expense of the host’s organism and impairment of metabolic processes. 4. Mutagenic. Clinical manifestations: ache in the abdomen, nausea, vomiting, diarrhea. Allergic rash and ache in muscles (ocular, masticatory muscles and muscles of calves, waist and shoulder girdle) appear, the temperature rises to 40–41 °C, edema of lids and face. Complications: myocarditis, pneumonia, meningoencephalitis, polyneuritis, thromboembolia, etc. Diagnosis: clinical presentation of the disease (edema of the lids and the face, muscular pains), background (eating untested meat of pigs, wild boars). Laboratory examinations: clinical blood analysis (eosinophilia), imunoassay, microscopy of bioptates of gastrocnemius and deltoid muscles. Personal prophylaxis: exclusion of untested meat from the diet (heat processing of meat does not kill larvae). Social prophylaxis consists in elimination of rodents (reservoirs of the pathogen), veterinary checks of meat, zoohygienic keeping of pigs (not allowing them to eat rats), deratization and health education.

STRONGYLOIDES STERCORALIS Strongyloides stercoralis (treadworm) is a geohelminth, pathogen of strongyloidiasis. The disease is common in the South-East Asia, East and South Africa and South America. Morphological peculiarities: colorless thread-like nematodes 1–3 mm in size. Life cycle: parasite settles the duodenum, bile and pancreatic ducts. After fertilization females lay eggs and males die. Rhabditiform (non-infectious)

142 larvae come out of eggs, which are excreted into the environment with feces. The further development of rhabditiform larvae occurs in soil in two ways: 1) if the environmental conditions are unfavorable, they turn into filariform (invasive) larvae that able to penetrate the host’s skin and migrate as ancylostoma’s larvae do; 2) if the conditions are favorable, the rhabditiform larvae transform into free living males and females. After fertilization free living females lay eggs. New formed rhabditiform larvae can transform into sexually mature free-living worms or in filariform larvae. Development also may proceed in the organism of one host: rhabditiform larvae undergo several moltings and transform into filariform ones in the intestine, they migrate and maturate. Migrating larvae may maturate already in the lungs. Pathogenic action: 1. Mechanic (rupture of capillaries and alveoli by larvae, injury of the mucous membrane of a small intestine). 2. Toxicoallergic (poisoning by waste products). 3. Feeding at the expense of the host’s organism (content of the intestine) and impairment of metabolic processes. Clinical manifestations: skin inflammation, weakness, irritancy, headache, skin itching, symptoms of bronchitis, pneumonia. Then appear signs of enteritis, gastroenteritis. Complications: perforation of the intestine with peritonitis, pancreatitis. Laboratory diagnostics: finding rabditform larvae in fresh feces, sometimes in duodenal content, sputum, vomited matter. A high eosinophilia reaching 70–80 % is noted. Personal prophylaxis: observing rules of hygiene. It is recommended not to walk barefoot or lie on the ground in foci of anсylostomiasis. Social prophylaxis: revealing and treating sick people, building sanitary facilities in settlements (water supply, sewage systems), personal and social health education.

TOXOCARA CANIS Toxocara canis (dog roundworm) is a geohelminth causing toxocariasis. Morphology: long body has the shape of cylinder with sharp ends. The length of females is 6.5‒ 10 cm, that of males is 4‒ 6 cm. Mouth is equipped with three cuticular lips. Life span is 4–6 maonths. Life cycle: mature individuals live only in the stomach and intestine of dogs and not common for human. A female toxocara lays about 200 000 eggs a day, theses eggs are eliminated with feces and require development in soil. The size of such eggs is 65–75 micrometers, color is brown (bright or dark), shape is round. Development of eggs in soil requires proper temperature and humidity and lasts 5–30 days, maturated eggs can survive for several months or even years. Human becomes infected when swallows eggs of toxocarae with

143 food or water which are polluted with dog feces. Larvae hatch from the eggs and migrate through blood vessels to various organs and tissues where they cover with capsules and cause the larval form of the disease. Pathogenic action: 1. Toxucoallergic. 2. Mechanic (injures of the liver, capillaries, alveoli). Symptoms. Children of 1‒ 4 years old fall ill more often. The course of the disease is characterized by clear allergic symptoms (inching rush) fever, enlargement of the liver and spleen, bronchial pneumonia with attacks of cough and breathlessness, puffiness of the face, formation of granulomae containing larvae of toxcarae in various organs. Toxocarae may affect eyes and can cause abscess, keratitis, larvae can migrate in the vitreous body. Laboratory diagnosis: detection of larvae in sputum, immunoassay, X-ray, eosinophilia in blood. Prophylaxis: general sanitary practice for prevention of environmental pollution from dog feces, their treatment, creation of dog run areas, obeying Hygiene rules.

DIAGNOSTIC TECHNIQUES USED FOR DIAGNOSIS OF NEMATODOSES Adhesive tape technique is used for diagnosis of enterobiasis. A sticky side of piece of transparrent tape 4–5 cm long is applied to perineum folds near the anus and is taken off. It is stuck to the preparation glass and studied under the microscope. The examination should be performed in morning. Diagnosis of tissue helminthoses. To diagnose tissue helminthes (thrichinelliasis, cysticercosis and etc.) immunotechnique are used: Immunoprecipitation (IP), complement-fixation test (CFT), indirect hemagglutination test and others. Muscular biopsy technique for diagnosis of trichinellosis: a specimen of the gastrocnemius or deltoid muscles is taken. Coiled larvae of thrichinelles in capsules are well-seen under the microscope. Muscle digesting technique: artificial gastric juice added to finely cut muscle specimen and placed into the termostat in 37 °C for 12–16 hours. Then the sediment is put on the preparation glass with a dropper and examined under the microscope. Trichinella larvae are revealed to be free of capsules.

BIOLOGICAL BASICS OF PROPHYLAXIS OF NEMATODOSES It is a complex of preventive measures that are based on studying the biology of pathogens, ways of their migration, their life stages and biology of intermediate hosts. This allows to interrupt the parasite’s life cycle and prevent its further development.

144 BASIC TERMS AND CONCEPTS Migration stage of ascariasis — period of ascariasis associated with migration larvae of ascaris. Bulb — dilation of the esophagus in some nematodes. Vesicule — swelling of a cuticle around the oral opening of a seatworm. Geohelminthes — helminthes that require development in soil for their larvae or eggs. Dehelmithization — complex of measures taken to eliminate helminthes in the human organism. Hypodermis — epithelial tissue of roundworms.. Capsule of a trichinella — covering of trichinella's larvae formed by connective tissue of the host. Migration — movement of a larval stage of ringworms in the host’s organism in order to continue their life cycle. Nematodoses — a group of diseases caused by ringworms. Muscle biopsy — diagnostic technique allowing to find larvae of trichinella in muscle specimens under the microscope. Adhesive tape test — diagnostic technique consisting in applying a piece of adhesive tape to perianal area with its further microscopy for diagnosis of enterobiosis. Immunoassay — diagnostic methods that detect antibodies to a certain pathogen in blood plasma of a patient. Surgical implications of ascariasis — complications of ascariasis caused by abnormal location of ascaris. Eutely — phenomenon in which an organism has fixed number of somatic cells.

Topic 22. PHYLUM ARTHROPODA, CLASS ARACHNIDA. POISONOUS AND VENOMOUS ORGANISMS

GENERAL CHARACTERISTIC AND TAXONOMY OF THE PHYLUM ARTHROPODA The number of species is over 1.5 million. Characteristic features: 1) development of organ systems from 3 germ layers; 2) bilateral symmetry of the body; 3) heteronomous segmentation; 4) body consists of 2 regions (cephalothorax and abdomen) or 3 regions (head, thorax and abdomen); 5) segmentated extremities; chitinized cuticle (exoskeleton); 7) appearance of striated muscles and separation of muscular groups; 8) mixed body cavity (mixocoel); 9) developed circulatory, respiratory, digestive, excretory, reproductive and nervous systems.

145 The digestive system consists of 3 regions: foregut, midgut and hindgut. It begins with a mouth opening having mouthparts and ends with an anal opening. There are digestive glands such as salivary glands and liver in the middle region. Excretory organs are modified metanephridia (green and coxal glands) or Malpighian tubules. Respiratory organs are: gills in aquatic artropods, book lungs and trachea in terrestrial ones. The circulatory system is open. The heart is located on the dorsal side of the body. The nervous system includes a large cerebral ganglion, performing the function of the brain, a circumpharyngeal nerve ring and ventral nerve chord. Threre are sensory organs of sight, smell, tactile sense, taste, hearing and equilibrium. Arthropods are dioecious (have separate sexes). Males and females are differed in size and colour, such distinction is sexual dimorphism. Development is direct or indirect (with metamorphosis). Classes: Crustacea, Arachnida and Insecta.

GENERAL CHARACTERISTIC AND CLASSIFICATION OF THE CLASS ARACHNIDA The number of species is about 40 000. They adapted for living on land. They have 2 regions of the body: the cephalothorax and abdomen. The body is covered with a cuticle saturated with chitin, the hypodermis is located beneath it. Spiders have silk glands and venomous glands. 6 pairs of appendages are located on the cephalothorax. The first 2 pairs (chelicerae and pedipalps) are used to grasp and to fragmentize food. The rest 4 pairs are walking legs. The digestive system is adapted for feeding semi-fluid food. Excretory organs of arachnids are coxal glands and Malpighian tubules. Respiratory organs are book lungs and trachea. The circulatory system is open. There is a tube-shaped heart with ostia (3–7 pairs) that acts as non-return valves allowing blood to enter the heart and prevent it from leaving, 2 short aortae (anterior and posterior) and lateral arteries branching from heart. The hemolymph contains hemocyanin. The nervous system consists of a cerebral ganglion that performs functions of the brain, ventral nerve chord and nerves. Sensory organs are simple eyes, organs of smell and chemical teste. Arachnids are dioecious. Sexual dimorphism is marked. Reproduction is sexual, development is direct or indirect (with metamorphosis). Orders: scorpions (Scorpiones), spiders (Araneae), ticks (Acarina).

ACARI Acari (or Acarina) is a group of arachnids that contains ticks and mites. Classification: phylum Arthropoda, class Arachnida, order Acarina, families: Ixodidae, Tyroglyphidae, Sarcoptidae and Demodicidae. 146 a. Family Ixodidae. Representatives: Ixodes ricinus or a castor bean tick, Ixodes persulcatus or the taiga tick, Dermacentor pictus, Dermacentor marginatus, Hyalomma anatolicum. Morphology: the body sizes are from 5 to 25 mm. They inhabit forests and steppe. The body is dorso-ventrally flattened and has no regions. There are 1 pair of eyes and 4 pairs of walking legs. The first 2 pairs of appendages are transformed into mouthparts of a piercing-sucking type. Mouthparts of ticks are sometimes called capitulum. It is located terminally on the anterior end of the body and can be seen from the dorsal side. There is a chitin «shield» (scutum) covering the whole dorsal side in a male and only the frontal part of the back in a female. This provides greater elasticity of the female’s abdomen during blood sucking. Ixodes ticks have dark-brown scutum; Dermacentor ticks have scutum with marble appearance (figure 81).

♂ ♀ Dermacentor pictus Ixodes ricinus Fig. 81. Ticks of the family Ixodidae

Life cycle: ticks go through four life stages: egg, six-legged larva, eight- legged nymph, and adult (imago). After hatching from the eggs, ticks must feed on blood at every stage to survive. Blood meal lasts up to several days. The ticks can starve up to 3 years. Their bites are painless, as saliva contains anesthetics. The female lays about 17 000 eggs in soil, in bark of dead trees. Medical significance: they are biological vectors of the tick-borne encephalitis. The encephalitis virus (family Flaviviridae) affects salivary glands and gonads of ticks. Encephalitis is transmitted by the bite of ick (from tick to human) and transovarially (from tick to its larva). Reservoirs of the encephalitis virus are birds and rodents. Ixodes ticks also transmit viral hemorrhagic fevers, brucellosis, typhus, plague and tularemia. Ticks of the genus Dermacentor transmit a virus of Scotland encephalitis. b. Family Tyroglyphidae. Representative: Tyroglyphus farinae (flour mite). Morphology: the body of a slightly-yellow color, sizes are 0.4–0.7 mm, has no eyes.

147 Tyroglyphus ticks may inhabit granary (flour, groats, corn, cheese, etc.), spoil grain and seeds with their excretions (figure 82).

A B C Fig. 82. Ticks: A — Tyroglyphus farinae; B — Sarcoptes scabiei; C — Dеmodex folliculorum

Medical significance: eating contaminated food may cause diarrhea, catarrhal symptoms of the gastrointestinal tract. During harvesting ticks may get into respiratory tract and cause asthmatic symptoms or bite a human and cause grain itch. c. Family Sarcoptidae. Representative: Sarcoptes scabiei (itch mite). Morphology: the mites have wide, oval, slightly-yellow colored body, covered with bristles. The body sizes are from 0.3–0.4 mm, legs are conical- shaped and shortened, eyes are absent. They breathe with the whole body surface. Life cycle: Sarcoptes scabiei is permanent parasite of the human and animals that burrows into skin and causes scabies. Fertilized female burrows into the stratum corneum of the skin per 2 mm a day and lays about 50 eggs in the burrow. Males do not burrow the skin. The mites feed on the host’s tissues. The development from an egg to an imago takes about 1–2 weeks. Adult mites live up to 2 months. Medical significance: the most common symptom is severe itchiness becoming worse at night. These symptoms can be present across most of the body or just certain areas such as the wrists, between fingers, or along the waistline. Scabies is spread during a direct skin contact with a sick person or their clothes. Secondary infection may get in scratches and cause suppuration. Prophylaxis of scabies: following basic hygiene rules in comminicating with animals and sick people; maintaining the purity of the body; revealing and treating sick persons; sanitary inspection of hostels and bathhouses. d. Family Demodicidae. Dеmodex folliculorum (figure 82). 148 Morphology: the worm-shaped body has the length about 0.4 mm. The cticule is thin and transparent cuticle. Legs are very short and have per a couple of claws. Life cycle: the mites affect sebaceous glands and hair follicles on the face, neck, shoulders (head of such mite is directed to the depth of the gland). They are often found in healthy people. In people predisposed to allergy, the mites may intensively multiply and obturate ducts of sebaceous glands. Medical significcance: cause demodicosis. Sumptoms are acne of pinc color with pus. Infection occurs by means of direct contact with a sick person. Diagnosis is based on microscopy of the content of sebaceous glands or hair bulb which reveals imago, larvae, numphs or eggs of the parasites.

POISONOUS FUNGI: CLASSIFICATION, CHARACTERISTICS OF MYCOTOXINS, FIRST AID AND PROPHYLAXIS OF POISONONGS There are two morphological groups of fungi: micro- and macromycetes. The micromycetes are microscopic fungi. They are the most frequent agents of severe food intoxication caused by fungi (aspergillus, penicillium, fusarium, ergot). Macroscopic fungi are macromycetes. They usually cause intoxication when eaten by mistake (amanita, gyromitra). According to edibility, macromycetes (commonly known as mushrooms) are: edible, edible after proper cooking (blewit, sharp agaric), unpalatable, and poisonous (death cap amanita, fly amanita, lorchel). Characteristics of the poisons. The poison of death cup amanita contains bicyclic polypeptides, amanitins and phalloidins. Fly amanita contains muscarine (para-sympathotropic agent) and hallucinogens (bufotenin, muscasone and others). Lorchel (gyromitrin which is similar to toxins of death cup amanita). Symptoms of the poisoning: vomit, abdominal pains, hypersalivation and hyperhidrosis, lachrymation, breathlessness, miosis. In cases of severe poisonngs — diarrhea, low artherial pressure, heart beat disorder, convulsions, collapse, coma. Death may occur as result of toxic hepatitis and acute heart failure. First aid: gastric lavage with suspending of activated carbon, 1 % potassium permanent, usage of saline laxatives.

POISONOUS PLANTS: CLASSIFICATION, CHARACTERISTICS OF PHYTOTOXINS, FIRST AID AND PROPHYLAXIS OF POISONONGS There are plants which produce and accumulate poisons which can cause toxication and even death of animals or human in various types of contacts. By now more than 10 000 species of poisonous plants are described. They are lily of the valley, blister buttercup, marsh tea and others. There are extremely poisonous plants such as devil's trumpets, black henbane, belladonna, Zwerg- Holunder and others. Some plants can be poisonous only when grow in certain conditions. 149 Characteristics of the poisons: plant poisons contain different groups of chemical compounds such as alkaloids, saponins, essential oils, glycosides, flavonoids, tannins, resinous substances, carboxylic acids, cyanic compounds and others. Toxicity of plant poisons is associated with a number of factors. Cardiac glycosides are not destroyed for a long time, are excreted through kidneys and affect them. Alkaloids have toxic effect on liver. Many glycosides are hydrolyzed and then broken into hydrocyanic acid which causes adverse effects. Symptoms: The most clinically important syndromes in case of acute poisonings are: psy-choneurological, respiratory, cardiovascular, gastrointestinal, hepatic, renal. First aid in case of poisonings with phytotoxins. 1. Removal of toxins from the organism: gastric lavage, vomiting, removal of the intestinal content, usage of adsorbents such as activated carbon. 2. Antidotes: substance with opposite effect on the organism can be used for some toxins. 3. Detoxication: artificial diuresis, replacement transfusion, dialysis, hemosorption. 4. Relief of symptoms: antishock therapy, normalization of work of respiratory, cardiovascular, central and peripheral nervous systems.

CLASSIFICATION OF TOXIC ANIMALS According to the presence of specific apparatus for injecting venom into the victim («armed»), animals are divided into venomous and poisonous. Primarily-toxic animals have special glands to produce toxine or specific toxic metabolites. As a rule, toxixity of these animals is a character of the species (jellyfish, scorpions, snakes, fishes). Secondarily-toxic animals accumulate exogenous toxines from the environment. Such animals are poisonous if they are eaten by other organisms (fish adsorb industrial toxins from water). The primarily-toxic animals are divided according to the ways of producing and using their toxines. Actively-venomous animals have a specialized apparatus («armed») such as thread cells on tentacles of jellyfishes, a stinger in hymenoptera and fangs in snakes. Venom is injected into the body of the victim parenterally (avoiding the digestive tract). Actively-poisonous animals have no such apparatus («unarmed»). Secretions of their glands are poisonous in case of direct contact with integuments of the victim (skin glands of amphibians, anal glands of insects). Passively-poisonous animals (fishes, caudate amphibians, mollusks) can have toxic metabolites that are accumulated in various organs and tissues. They are dangerous only when eaten by a victim.

150 PHYSIOLOGICAL CHARACTERISTIC OF TOXINS OF INVERTEBRATES (JELLYFISH, ARACHNIDS, HYMENOPTERANS), THEIR EFFECT ON THE HUMAN BODY; THE FIRST AID AND PROPHYLAXIS OF BITES AND POISONINGS Characteristic of animal toxines. Animal toxines (zootoxins) are biologically active substances that actively interact with biological structures of the body. Zootoxins are diverse by their chemical structure (alkaloids, histamine, various enzymes and their inhibitors). According to the physiological effect on living systems, zootoxins are subdivided into: 1) neurotoxins affecting predominantly the nervous system; 2) cytotoxins causing damage of cells and tissues; 3) hemorrhagins impairing normal permeability of blood vessels; 4) hemolysins destroying erythrocytes. A clinical presentation of toxication of human depends on composition of the toxine, the site of affection, the season of the year and the time of the day as well as a overall condition of the person. Coelenterates (orange-striped jellyfish and physalia) refers to actively- venomous animals. Thread cells excrete a neurotropic venom that blocks synapses. Clinical presentation. In sites of sting by tentacles of the orange-striped jellyfish appears sharp pain, erythema, rash. Symptoms temperature rise, rapid decrease of muscle tone, pains in extremities and lumber area, impairment of consciousness, hallucinations, delirium, respiratory and cardiac affection, in severe cases — death. First aid. It is required to remove parts of tentacles and striking threads from the skin, treat the affected sites with alcohol or solution of soda. Prophylaxis. Not to bathe in the thicket of water plants and in places of jellyfish gatherings. Phylum Arthropoda, class Arachnida, order Scorpions (yellow, Italian, black). They are actively-venomous, have venomous glands located in the last segment of the abdomen. They excrete neurotropic venom that blocks neuromuscular synapses. Clinical presentation. At the site of a bite appears a severe pain, edema, erythema, vesicles. Symptoms: headache, weakness, impairment of consciousness, and respiration, tachycardia in children. Lethal outcomes are possible. First aid. Sucking off the venom, applying cold to the site of a bite, taking pain-killers. Injection of specific antiserum. Prophylaxis. Protection from bites: examination of dwellings, bedding, clothes, shoes. Order Arachnida. Spiders are actively-venomous. Ducts of their venomous glands open on chelicerae.

151 Karakurt has neurotropic venom that blocks neuromuscular synapses. Clinical presentation. At a bite site appears pain, numbness of extremities. Symptoms: pain quickly spreading throughout the body, headaches, breathlessness, heartbeat, bronchial spasms, vomiting and impairment of consciousness. Lethal outcomes are possible. First aid. Sucking off the venom, slight of the bite site, injection of an antikarakurt serum can be used. Prophylaxis. Prevention from getting caracurts to the places of human lodging for the night. Tarantulas’s venom contains cytotoxins and hemorrhagins and impair permeability of capillary walls. Clinical presentation. At a bite site appear pain, reddening, edema, skin necrosis. Symptoms: malaise, sleepiness, chills, pulse acceleration, perspiration. First aid. To treat the site with disinfectants, ensure rest, abundant drinking, pain-killers for the patient. Prophylaxis: protection from bites. Class Insecta, order Hymenoptera (bees, wasps). These insects are actively-venomous, have toxic glands and a sting at the end of the abdomen. The venom has a neurotropic and cytotoxic action and is a strong allergen. Clinical presentation. After a bite — pain, edema, erythema. Possible symptoms: allergic reactions.

PHYSIOLOGICAL CHARACTERISTIC OF TOXINS OF VERTEBRATE ANIMALS (FISHES, AMPHIBIANS, REPTILES), THEIR EFFECT ON THE HUMAN; THE FIRST AID AND PROPHYLACXIS OF BITES AND POISONING Toxic fishes are divided into 2 groups: 1. Venomous species having toxic glands; the secretion of these glands is injected into the wound made by fin rays, teeth or thorns of branchial covers. Representatives: sting ray, sea dragons, ruffs and perches, moray eels, devilfish, firefish. They are spread predominantly in tropic latitudes of the Pacific and Atlantic Oceans. Pathogenic action and clinical presentation. Toxins pass into the organism through a wound on the skin. At the moment of a prick victim feels pain that quickly spreads to the whole extremity. Then appear fear, breathlessness, heart pain, vomiting and sometimes loss of consciousness. Inflammation, sometimes ulcers and tissue necrosis develop at the bite site. A severe poisoning ends with death within a day. Treatment: sucking off the venom from the wound, applying a rope, symptomatic treatment. Prophylaxis includes putting on special clothes if deal with the fishes. 2. Fishes that are poisonous when eaten (moray eels, thons, perciformes, pufferfish). When these fishes are used as food, poisoning develops in 20–30 minutes. There appears numbness of the tongue and fingers, nausea, vomiting,

152 breathlessness, respiratory and speech affection. The treatment is symptomatic. As prophylaxis, the mentioned fishes should be excluded from the diet. Amphibians. There are some toxic substances in the skin of some amphibians. The most virulent poison is produced by African tree-frogs and tree-toads. Toxine of the Columbian cocoa frog (the length of 2–3 cm, the weight is a bit more than 1 g) is 50 times stronger than a tetanus toxin. Other toxic amphibians are not dangerous for the human (they have no mechanism for injecting the toxin into tissues). When their poison gets on the skin or mucous membranes, erythema and inflammation are observed. These symptoms are relieved by washing with water. It is necessary to take care lest amphibians’ poison gets to the eyes. Class Reptila. Families elapids and sea serpents (king cobra and Indian cobra, long-glanded coral snakes, sea kraits). These are primarily-toxic actively- venomous animals. They have toxic immobile fangs with canals for the venom on the anterior part of the maxilla. Pathogenic action and clinical presentation. The venom contains neurotoxins, cytotoxins, hemolysins. At a bite site develops pain, edema, inflammation. Symptoms: excitation and then depression of CNS; swallowing, speech and breathing are impaired. Lethal outcomes are possible. Family Viperidae (blunt-nosed viper, phoorsa, Orsini's viper, copperhead snake, rattlesnakes). They are primarily-toxic actively-venomous animals. They have toxic glands and fangs with canals. Pathogenic action and clinical presentation. The venom contains neurotoxins, cytotoxins, hemolysins, they stimulate blood coagulation. At a bite site develops pain, edema, tissue necrosis. Symptoms: weakness, nausea, dizziness, impairment of blood coagulation. Lethal outcomes are possible. First aid. The bite site should be treated with an antiseptic and a compressing bandage should be applied. The patient should be transported in a lying position. Injection of snakes’ antitoxins should be done. Prophylaxis: in places of snakes’ inhabitance one should not touch them and wear high boots. BASIC TERMS AND CONCEPTS Anthroponoses — diseases in which causing agents are transmitted from a human to human. Actively-venomous animals — have venomous glands and a specialized apparatus for injection of their venom. Vector-borne diseases — diseases pathogens of which are transmitted by blood-sucking arthropods. Secondary-toxic animals — animals that accumulate exogenous poisons and are toxic when eaten. Passively-poisonous animals — animals that have toxic metabolites accumulated in various organs and tissues. 153 Primarily-toxic animals — animals having special glands for production of toxic secretion or some toxic metabolites. Chelicerae — the first pair of spider appendages used to inject venom into victims body. Pedipalps — the second pair of spider appendages which function as mouthparts. Poisonous animals — animals that can cause adverse effects on human organism when eaten or contacted by means of their toxins. Phytotoxins — toxins which are produced by plants.

Topic 23. PHYLUM ARTHROPODA, CLASS INSECTA

GENERAL CHARACTERISTIC AND TAXONOMY OF CLASS INSECTA The number of species is over 1 million. There are 3 body regions: the head, thorax and abdomen. There are one pair of antennae (sense organs), mouthparts and compound eyes on the head. The thorax consists of three segments. It carries six segmented legs (per one pair for each segment). There are 1 or 2 pairs of wings on the 2nd and 3rd segments on the dorsal side of the thorax. The abdomen consists of 6–12 segments. The body is covered with chitin, beneath is the hypodermis containing odoriferous glands, wax glands, prothoracic glands and other. The muscular system is differentiated and specialized. The digestive system consists of a foregut, midgut and hindgut. Mouthparts include two mandibles, two maxillae, upper and lower lips and a tongue (hypopharynx). Types of mouthparts depend of the way of feeding: chewing (bugs), piercing-sucking (mosquitoes, fleas), licking (flies), sucking (butterflies). Excretory organs are Malpighian tubules and a corpus adiposum (fat body). The respiratory system of insects is represented by trachea. The circulatory system is poorly developed. A tube-like heart with aorta is located on the dorsal side. The blood of insects is called hemolymph. It transports nutrients and dissimilation products. The nervous system consists of suprapharyngeal ganglion (the brain) having 3 regions — anterior, middle and posterior. Ventral nerve cord has a tendency to confluence of ganglions. Tactile organs are sensitive hairs around the body. Olfactory organs are located on palps and antennae, on mandibles. Taste receptors are located on mouthparts and leg segments. Eyes are simple or compound. Insects are dioecious, sexual dimorphism is marked. Their reproduction is sexual. Development is direct or indirect (complete or incomplete metamorphosis). The following criteria are used for the division into classes: type of mouthparts, presence and the number of wings, type of development (table 5).

154 Table 5 Orders of insects Order Metamorphosis Structure of wings Mouthparts Hemiptera Incomplete 2 pairs: fore wings are Piercing-sucking (true bugs) partially membranous, hind wings are membranous Blattoidea (cockroaches Incomplete 2 pairs: fore wings are leathery, Chewing and termites) hind wings are membranous Phthiraptera (lice) Incomplete Absent Piercing-sucking Siphonaptera (fleas) Complete Absent Piercing-sucking Diptera (true flies, Complete 2 pairs: forewings are Piercing- mosquitoes) membraneous; the back wings sucking, licking are reduced and transformed into halteres

Medical significance: they are vectors or pathogens of diseases (ectoparasites) and toxic animals.

SUCKING LICE (ORDER ANOPLURA) Taxonomy: trere are two genera in order Anoplura: genus Pediculus and Phthirus. Genus Pediculus is represented by one species. Pediculus humanus includes 2 subspecies — the head louse and the body louse which freely cross and give fertile offspring, but they have some morphological and biological differences. Head louse (Pediculus humanus capitis). Morphology: the length of a male is about 2–3 mm, female — 3–4 mm. The posterior end of male’s body is rounded, the female body is forked. Mouthparts are piercing-sucking (figure 83). Life cycle: lice live in the hairy area of the head. They feed on human blood 2–3 times a day, may starve for several days. The life cycle of the body louse consists of three stages: egg (nit), nymph, and adult. Nits are attached to hair with a sticky secretion. During the whole life (about 38 days) a female lays about 300 eggs. A larva comes from an egg and in several days transforms into imago (a mature form). Body louse (Pediculus humanus humanus). Morphology: body louse has larger body sizes than the head louse (to 4.7 mm), carvings along the body edge are not so deep and pigmentation is slightly marked. Life cycle: body lice live on underwear and clothes, but feed on the skin. Nits are sticked to the clothes or to the hair on human body. The life span is up to 48 days, the development lasts not less than 16 days. By the end of its life female can have about 4000 offspring.

155

Fig. 83. Representatives of the order Anoplura: A — Pediculus humanus capitis (scheme); B, C, D — Pediculus humanus capitis (7×8); F — Pediculus humanus humanus (scheme); G, H — nits; I, J — Pediculus humanus humanus (7×8); E — Phthirus pubis (scheme); K — Phthirus pubis (7×8)

Medical significance: lice of genus Pediculus cause pediculosis (or Vagabond’s disease). During blood meal, lice inject saliva into the wound. This causes itching. Pediculosis is characterized by pigmentation and scraching on skin. Lice are biological vectors of epidemic typhus (caused by bacterium Rickettsia prowazekii) and a louse-borne relapsing fever (caused by bacterium Borrelia recurrentis). Infection of epidemic typhus occurs by a specific contamination — rubbing louse intestine content into wounds or into scratches on the skin, and by contamination in rubbing lice feces into the skin during scratching. Human can also get louse-borne relapsing fever by a specific contamination — smashing louse and rubbing its hemolymph into the skin during scratching. Pubic louse (Phthirus pubis) is an ectoparasite of humans; feeds on blood. Morphology: sizes up to 1.5 mm. The body is short, almost round. Life cycle: parasitizes usually in the human pubic hair but can also live in other body areas covered with coarse hair, such as armpits, eye-lashes, beard. The female lays about 50 eggs during its life. The life cycle from an egg to a mature form lasts 22–27 days. Medical significance: pubic lice cause phthiriasis (severe itching usually in the pubic-hair area). Human can get phthiriasis by sexual contacts, rarely — through underwear and clothes. Protective measures against lice: extermination them in the environment, on the human body and on clothes.

156 FLEAS (ORDER APHANIPTERA) Morphology: fleas are wingless insects their body is flattened from the sides, covered with chitin and bristles, mouthparts adapted to blood feeding. There are short feelers and a pair of simple eyes on the head. The last pair of legs is long and well adapted for jumping. Fleas have complete metamorphosis lasting about 19 days. Fleas lay eggs in slits of the floor, in garbage. There are four life cycle stages of egg, larva, pupa, and imago. Larvae have a worm-like shape without limbs. In some time a larva pupates. Adult fleas must feed on blood before they become capable of reproduction. Larvae feed on organic leftovers. The life span of fleas lasts over 1 year. Representatives: human flea (Pulex irritans) and oriental rat flea (Ceratophyllus fasciatus and Xenopsylla cheopis) (figure 84).

Fig. 84. Morphology of fleas: A — Pulex irritans (scheme); B — Pulex irritans (7×8); C — jigger-infested extremity; D — Sarcopsylla penetrans (7×8) Medical significance: fleas are temporary ectoparasites (bites cause itching, dermatitis). Fleas are also biological vectors of bacteria causing plague and tularemia. Natural reservoirs of plague are rodents, such as rats, gophers and marmots. The human gets plague infectection during the contacts with a sick animals (skinning) or with a sick person (by airborne route) and transmissively (vector-borne rote). Infection occurs during blood-sucking (inoculation) or by contamination: when the plague bacteria with fleas feces get into injured skin during scratching. Fleas of genus Xenopsylla are biological vectors of tularemia and murine typhus (pathogen bacteria of genus Rickettsia), they are also intermediate hosts of animal tapeworms. Jigger flea (Sarcopsylla penetrans). It is common in countries of South America and Africa, lives in sand, in dry grass and in cabines. 157 Morphology: jigger flea is 1 mm long, has a yellow-grey color. Life cycle: fertilized females attack the human, burrow the skin between toes or get under the nails. They feed on blood and lymph, enlarge to the sizes of a pea and develop thousands of eggs. A tumor-like tissue growth around such a flea is marked. Mature eggs are excreted into the environment, females die and cause decomposition of injured tissues. Medical significance: jigger fleas are parasites of the human and mammals (dogs, pigs and rodents), they cause sarcopsyllosis. The formed wounds get inflamed and are very painful; often a secondary infection follows. Complications of sarcopsyllosis are tetanus and gangrene. Protective measures against fleas: keeping a rooms clean, elimination of slits in the floor and walls, fighting against rodents (deratization), using insecticides and repellents. It is not recommended to walk barefoot in the sand in Africa and South America.

COCKROACHES (ORDER BLATTOIDEA) Morphology: large insects, the body length reaches 3 cm. The body is flattened in a dorsal-ventral direction. They have 2 pairs of wings: fore wings are leathery, protective and hind wings are thin, membranous. In females the wings are reduced. Cockroaches have chewing mouthparts (figure 85).

Fig. 85. Morphology of cockroaches: A — Blattella germanica; B — Blatta orientalis; C — Periplaneta americana; D — mouthparts of a oriental cockroach: 1 — upper lip; 2 — upper jaw; 3 — lower jaw; 4 — palps; 5 — lower lip 158 Life cycle: the development occurs with an incomplete metamorphosis lasts for several months. Females lay eggs in cocoons, which they carry with them about 14–15 days. They are active at night, at daytime they hide in floor slits. They are met in human habitations, at food fabrics and public food services, in shops and canteens. Obligatory conditions for their life in human houses are the presence of fluid, a definite temperature and amount of food. They feed foods, human excretions and various wastes. Representatives: the oriental cockroach (Blatta orientalis), the German cockroach (Blattella germanica) and the American cockroach (Periplaneta americana). Medical significance: cockroaches are mechanic vectors of infectious and invasive diseases. Protective measures against cockroaches: insecticides are used to kill them. It is also necessary to clean the rooms, not to leave leftovers on the table, to fix slits in a floor and walls.

BUGS (ORDER HETEROPTERA) Bed bug (Cimex lectularius) is dark-brown bug that can reach to 8 mm in lenght (males are smaller than females), with reduced wings (figure 86).

Fig. 86. Morphology of bugs: A — Cimex lectularius; B — Triatoma infestans

It has a specific smell from odorous glands. The body is flattened in a dorsal-ventral direction. The abdomen shape is changeable and depends on blood saturation. Life cycle: bed bugs live in human habitations near or inside beds or other sleep areas. At daytime and under artificial illumination bugs hide in slits of the floor, under wallcovering, in furniture slits, behind the curtains. At night they go out of their shelter, attack the human and feed on blood. Females lay 159 eggs in slits of the floor, in books, on clothes. In 2–3 weeks (depending on the temperature) larvae come out of eggs and also feed on blood. Larvae moult several times and transform into imagos. Mature bugs and larvae may starve for several months. Medical significance: the saliva of the bed bug is poisonous, its bites are painful. Kissing bug (Triatoma infestans). Morphology: the body of kissing bug is oval, flattened in a dorsal-ventral direction and has large sizes (1.5–3.5 cm), also it has well developed wings. Life cycle: kissing bugs inhabit rodent holes and human habitations. They attack sleeping people at night and insert their proboscis into the skin of the neck, face, more often around lips and feed blood. After feeding the bug turns around and defecates into the bite wound. Medical significance: kissing bugs are temporal ectoparasites and biological vectors of Trypanosoma cruzi which causes Chagas disease — a natural-focal disease common in South America. In some people, saliva of bugs causes severe allergic reaction. Protective measures against bugs: insecticides are used to kill bugs. Elimination of rodents living near human, as they are feeders of bugs. It is also necessary to clean the rooms and to fix slits in a floor, walls and furniture.

MOSQUITOES (FAMILY CULICIDAE). GENERA CULEX, ANOPHELES AND AEDES Morphology: mature mosquitoes have a slender stretched body and small sizes. There are large compound eyes, palps and mouthparts on the head. Females have piercing-sucking mouthparts and feed on blood. Males have sucking mouthparts. They feed flower nectar. Segmented palps are on the sides of the mouthparts. A pair of translucent wings is attached to the thorax. The abdomen has 10 segments, the last two of them are modified into sexual appendices (figure 87). Life cycle: mosquitoes go through four stages in their life cycles: egg, larva, chrysalid, and adult or imago. A new generation of mosquitoes undergoes a period of physiological maturation lasting about 4 days. During this time they stay near water reservoirs and feed on nectar. In twilight males form a swarm, females fly into it, fertilization occurs, and then females must obligatorily feed on blood to provide development of eggs. During blood digestion maturation of eggs occurs (gonotrophic cycle). When the eggs maturate, females get to a water reservoir and lay about 350–450 eggs on its surface. Larvae come out of eggs. A minimal term of their development is 15 days in an optimal temperature (25 °C). Fertilized females (Anopheles, Culex) and eggs (Aedes) pass the winter. When autumn colds come males fertilize females and die. Eggs. Aedes mosquitoes lay eggs by one into temporary reservoirs: in puddles or tree hollows. Eggs are oval-shaped without air floats. Culex

160 mosquitoes lay eggs on the surface of stagnant water stucked in a form of a raft. Eggs are V-shaped without air floats. Anopheles mosquitoes lay eggs in stagnant or slowly running clean water. Eggs have floats with air on their sides and swim separately.

Fig. 87. Morphology of mosquitoes: A — eggs of Culex; B — larva of Culex; C — chrysalid of Culex; D — head of a male Culex; E — head of a female Culex; F — eggs of Anopheles; G — larva of Anopheles; H — chrysalid of Anopheles; I — head of a male Anopheles; J — head of a female Anopheles

Larvae. Larvae of Culex and Aedes mosquitoes have a respiratory trumpet in the shape of narrow tube on penultimate segment. Such larvae form an angle with the water surface. Larvae of Anopheles mosquitoes have no respiratory siphon and are located parallel with water surface. Chrysalides are comma-shaped. They breathe through a pair of respiratory trumpets on the dorsal side of cephalothorax. It means they float on water surface. Chrysalides of Culex and Aedes have straight tube-shaped respiratory trumpets. Chrysalides of Anopheles have funnel-shaped respiratory trumpets. Mature forms (imagos) differ by their body position, wing pattern and the structure of mouthparts. The abdomen of Culex and Aedes mosquitoes is located parallel with the surface where they sit. Posterior end of Anopheles mosquitoes abdomen is elevated. There are dark spots on the wings of some Anopheles mosquitoes, on the wings of Culex and Aedes mosquitoes they are absent. Antennae of males are hairy, antennae of females are hairy much less. Palps of Anopheles females are equal in length to the proboscis, palps of 1 1 Culex and Aedes females comprise /3– /4 of the proboscis length.

161 Palps of Anopheles males are equal in length to the proboscis and have club-shaped thickenings, palps of Culex and Aedes are usually longer than the proboscis and have no thickenings. Medical significance: mosquitoes are temporary ectoparasites. Anopheles mosquitoes are biological vectors and principal hosts of malaria parasites, biological vectors and intermediate hosts of wuchereria bancrofti and brugia malayi. Aedes mosquitoes are biological vectors of Japanese encephalitis, yellow fever, dengue fever, lymphocytic choriomeningitis, anthrax, tularemia, Wuchereria bancrofti and Brugia malayi. Culex mosquitoes are biological vectors of Japanese encephalitis, tularemia and brugia malayi.

FLIES (MUSCIDAE FAMILY) AND SUBFAMILY PHLEBOTOMIDAE House fly (Musca domestica) is common everywhere. Adult flies have grey or black slightly hairy body and can reach up to 7.5 mm in length (figure 88).

Fig. 88. Morphology of the family Mucsidae: A — Stomoxys calcitrans; B — Musca domestica; C — Wohlfahrtia magnifica; D — Glossina palpalis; E — claws and sticky pads on the legs, F — mouthparts of the house fly: 1 — sticky pad; 2 — claw; 3 — lower lip; 4 — oral opening

There are claws and sticky pads on the legs, due to them flies can move on any surfaces. Flies have licking mouthparts. The saliva contains mucolytic enzymes for digestion of organic substances which flies lick after. They feed on food particles and decaying organic leftovers. 162 Life cycle: if the temperature is not lower than 17–18 °C females lay up to 150 eggs in 4–8 days after crossing on decaying organic matter such as garbage, carrion or human feces. If the tempereature is about 35–45 °C larvae come out of eggs in 1 day, they pupate in soil in 1–2 weeks if the temperature is not higher than 25 °C. A new generation of flies appears in a month. Their life span is about 1 month. Medical significance: flies are mechanic vectors of enteric infections (cholera, typhoid fever, paratyphus, dysentery), tuberculosis, diphtheria, helminthes eggs and protozoans’ cysts. There are more than 6 million bacteria on the fly’s body, and up to 28 million bacteria in the intestine. Protective measures against house flies. Insecticides, adhesive tapes, baits with poisons are used as protection form from house flies, they are also can be eliminated mechanicly. Stable fly (Stomoxys calcitrans). Adult flies can reach 5–6 mm in length. The body is grey with dark stripes on the thorax and spots on the abdomen. The adults of both sexes feed blood of warm-blooded animals during the daytime. With the help of its proboscis stable flies scrape off skin epidermis and lick off blood. The saliva contains toxic substances causing a severe itch. Bites are painful. The population of flies reaches its maximum in August- September. The female lives about 20 days. Stable flies are mechanic vectors of anthrax. Protective measures against stable flies are the same as against the house flies. Tsetse fly (Glossina palpalis) is large dark brown biting fly that inhabit much of midcontinental Africa. It lives near human habitations along river banks and lakes. They have large sizes (up to 13 mm), the proboscis is strongly chitinized, protrudes forward. Females are viviparous, they lay only one larva into the soil surface. The larva develops in pupa into the soil, than in 3–4 weeks an imago comes out. During the whole life (3–6 months) females lay about 6–12 larvae. Tsetse flies feed on blood of animals and humans, they are biological vector of African trypanosomiasis. Protective measures against tsetse fly: cutting down bushes and trees along river banks near human habitations and along roads. Insecticides are used against mature flies. Spotted flesh fly (Wohlfahrtia magnifica) is common in countries with a moderate and hot climate. The body is light-grey, the adults are about 9–13 mm in length and there are 3 dark longitudinal stripes on the thorax. Mature flies feed nectar. Females lay about 120–150 larvae in human open cavities (nasal cavity, in eyes, in ears), in the wounds and ulcers of the human or animalbody, sometimes in human open cavities during a sleep in the open air. Larvae live in ears, nasal cavity, frontal sinuses and eyes. Larvae destroy tissues and reach bones. Parasitizing larvae cause myiasis. Myiasis may complicate by

163 necrosis. In 5–7 days larvae fall out in the soil and pupate. Preventive measures are defence from flies attacks. Sand flies (subfamily Phlebotomidae) inhabit regions with a warm climate, close to human habitations. Besides they live in caves and holes of rodents. Their sizes are about 1.5–3.5 mm, the body is brown-grey or slightly yellow. The head is small. Mouthparts are piercing-sucking. Legs are long and thin. The body and wings are slightly hairy. They lay eggs in shadowed places: rodents’ holes, caves, in birds’ nests, in garbage. Males feed on flower nectar, females feed on blood (in twilight and at night). Their bites are painful and cause itching and scratching. Mosquitoes are biological vectors of leishmaniasis and pappataci fever. Transovarial transmission occurs in sand flies.

BASIC TERMS AND CONCEPTS Gonotrophic cycle — maturation of eggs in female insects of the Diptera order during digestion of blood. Zooprophylaxis — use of biological barriers (cattle farms) between hatching places of insects and human habitations. Inocculation — infection of the host that occurs during blood sucking through a proboscis. Insecticides — substances used to kill insects. Contamination — infection that occurs by rubbing vector’s feces into the skin during scratching. Mechanic vector — vector in which pathogens do not multiply and do not develop, but only transmitted to the host. Myiasis — disease caused by larvae of flies and bot-flies. Repellent — substance which discourages insects. Pediculosis — disease caused by lice of genus Pediculus. Phthiriasis — disease caused by a pubic louse.

164 GAP-FILLING TESTS

THE ROLE OF BIOLOGY IN MEDICAL EDUCATION. METHODS USED TO INVESTIGATE CELLS 1. Structure of cells is studied by … microscopy. 2. Chemical composition of cells and location of various substances can be assessed by … 3. Smallest structural components of cells can be studied by the … microscopy. 4. Chemical composition of cells and chemical reactions occurring there are studied with … 5. The method which allows to separate different components of cells is … 6. Homo sapiens belongs to the subclass … 7. Homo sapiens belongs to the family …

BIOLOGY OF THE CELL. THE FLOW OF SUBSTANCE AND ENERGY IN THE CELL 8. The division of cytoplasm of the cell by membranes is called … 9. The receptor apparatus located on the outer surface of a plasma membrane is called … 10. ER (endoplasmic reticulum) and … form the transport system of the cell. 11. The diameter of cytoskeleton microfilaments is … nm. 12. Peroxisomes are made in … 13. The large subunit of ribosomes contains 40–50 molecules of proteins and … molecules of r-RNA … 14. The destruction of cell organelles by its own lysosomes is called … 15. Integral proteins of membranes forming pores and providing their permeability are called … 16. The efficiency of the anaerobic stage of energy exchange is … %.

FLOW OF GENETIC INFORMATION IN THE CELL 17. Nuclear lamina mostly consists of … 18. There is a … in the area of primary constriction which connects with microtubules of the spindle apparatus. 19. The region of secondary constriction of satellite chromosomes is called … 20. Complement of genetic material in the cell during diplotene is … 21. During diplotene chromosomes of bivalents are connected only in the crossing regions called … 22. There are … on the equator of the cell during metaphase of the second meiotic division. 23. Complement of genetic material in the cell during the metaphase II is … 165 ARRANGEMENT OF HEREDITARY MATERIAL 24. A DNA segment bound with a protein octamer for condensation is … 25. DNA becomes … times shorter at the first level of condensation. 26. DNA becomes 10–20 times shorter at the … level of condensation. 27. As result of condensation at all levels, DNA becomes … times shorter. 28. Autosynthetic function of gene is its … 29. DNA-polymerase moves along the matrix strand from its … end to … end. 30. Process of detection and binding an amino acid by proper tRNA is … 31. There is mRNA triplet … in the P-site of the ribosome during initiation. 32. The process which begins at the moment when first peptide bond is formed and finishes at the moment when the last amino acid is connected to a polypeptide is called … 33. Some antibiotics are … of protein biosynthesis. 34. Regulatory genes code for proteins … 35. For transcription of structural genes, operators should get rid from proteins … 36. Assembling several variants of mRNA from the same exons is called … 37. A region in a transcripton determining the end of transcription is … 38. A substance which can be broken by enzymes encoded in the operon and causing its activation is … 39. Leber disease is caused by mutation of …

GENETIC ENGINEERING 40. Enzymes capable of cutting DNA in certain sites and form sticky ends are called … 41. Synthesis of genes by on mRNA matrix is based on a process which is called … 42. Vectors used in genetic engineering are bacterial plasmids, phage genomes, phasmids and … 43. The restriction enzyme Eco R I forms … ends in DNA. 44. Hybrid vectors capable of developing both as a phage and as a plasmid are called … 45. The plasmids containing cos-sites (sticky ends) of phage λ DNA are called … 46. Size of the DNA fragments which can be cloned in cosmids is about … thousand nucleotide pairs … 47. The basic vector for the animal genes cloning is the genome of the virus … 48. The restriction enzyme Hind II forms … when cuts both DNA strands in same points.

166 GENE INTERACTIONS. GENETIC LINKAGE. GENETICS OF SEX 49. Bombay blood group is an example of non-allelic interaction called … 50. Cross of diheterozygotes causes phenotypic segregation ratio 15:1 in case of inter-allelic gene interaction which is called … 51. Cross of diheterozygotes causes phenotypic segregation ratio 9:7 in case of inter-allelic gene interaction which is called. 52. A phenomenon which breaks genetic linkage is called … 53. One centimorgan is unit of the distance between genes equal one percent of … 54. In case of genetic linkage, the maximal percentage of crossing over is … %. 55. Such phenotypic characters of a female as low position of ears, skin fold on the neck are characteristic of … syndrome. 56. Men having female body constitution, gynecomastia and impairment of spermatogenesis is example of a person sick with … syndrome. 57. Civil sex is … determinant of sex. 58. Persistent discrepancy of sexual identity and true genetic and gonad sex and a wish to change sex is called …

VARIATION 59. A phenomenon when non-hereditary variation has the same phenotypic manifestation as the hereditary one is called … 60. Enzymes capable of cutting out the damaged part of DNA strand during repair are … 61. Transgenation when one purine base is replaced with another purine base is called … 62. … of chromosome telomeres and connection of remaining ends leads to formation of ring chromosomes. 63. Mutation of … genes leads to the impairment of alternation of repression and expression of genes. 64. Non-separation of chromosomes during mitosis or meiosis causes … mutations. 65. Aneuploidy when only one chromosome of a pair is present in the karyotype is called … 66. Genome mutation when somatic cells have single chromosome set is called … 67. Disease caused by the infringement of DNA repair and characterized by insufficiency of red bone marrow function resulting in deficit of blood cells and hyperpigmentation is called …

167 FUNDAMENTALS OF HUMAN GENETICS 68. If parents are heterozygous (complete dominance, autosomal dominant inheritance, gene penetrance is 25 %), then the probability of giving birth to a sick baby is … %. 69. If a mother is heterozygous and a father is healthy (X-linked dominant inheritance, gene penetrance is 40 %), then the probability of giving birth to a sick baby is … %. 70. The type of inheritance when the father transmits his character to all daughters, but neither to sons is called … 71. A hybrid somatic cell containing nuclei of two different cells … 72. Method of human genetic that allows to reveal the role of heredity and environment in development of a character is called … 73. Percentage of twins who are different in a certain character is called … 74. The method of human genetics that allows to reveal genome and chromosome mutations is called … 75. Heterozygous carriers of pathologic genes can be revealed by biochemical … tests. 76. Chorion biopsy is performed within … weeks of pregnancy. 77. Changes of genetic structure of a population can be predicted by a methods of … 78. Level of α-fetoprotein in the blood of a pregnant woman … in case of Down syndrome of the fetus. 79. Each pregnant woman compulsory undergoes … — a direct non- invasive method of prenatal diagnostics. 80. Mother’s age of over 37 years, spontaneous abortions and stillbirth in the anamnesis, children with congenital malformations are indications for carrying out … methods of prenatal diagnostics. 81. Sex chromatin Y is determined by staining the cells of buccal epithelium by … 82. Normally the main palmar angle is not more than … 83. Human populations with the number not exceeding 1500 people where intragroup marriages surpass 90 % are called … 84. Genetic load has no phenotypic manifestation when … of a pathological gene is observed. 85. Consanguineous marriages lead to … depression as relatives have higher probability to carry the same pathological gene.

HUMAN GENETIC AND CHROMOSOME DISORDERS 86. Increased concentration of copper in blood in case of Wilson– Konovalov disease is caused by mutation of the gene responsible for synthesis of the protein …

168 87. Sickle-cell anemia is caused by the mutation leading to replacement of glutamic acid with … in 6th position of the β-chain. 88. The … syndrome is associated with increased level of uric acid and its salts n the organism caused by deficit of the enzyme catalyzing addition of purine bases to nucleotides. 89. Hereditary deficiency of the enzyme tyrosinase causes … 90. Deficit of ceruloplasmin is the cause of the … 91. Genetic diseases caused by the impairment of lipid exchange in the blood plasma due to defects of enzymes or cells' receptors are called … 92. Mutations associated with changes of chromosome number or impairment of their structure cause … disorders. 93. … syndrome results from trisomy on the 18th pair of autosomes.

GENETIC COUNSELLING 94. Substitution therapy is an example of the … treatment of hereditary disorders. 95. Dietotherapy is an example of the … treatment of hereditary disorders. 96. Prescribing anesthetics is an example of the … treatment of hereditary disorders. 97. Surgical removal of the 6th finger is an example of the … treatment of hereditary disorders. 98. Gene therapy is an example of the … treatment of hereditary disorders.

REPRODUCTION OF LIVING MATTER 99. Exchange of genetic information between the individuals of the same species is … 100. Fusion of pronuclei during fertilization is called … 101. Sexual reproduction without fertilization is called … 102. A phenomenon when an organism develops on the genetic basis of only male gametes is called … 103. During period of proliferation, cells divide by … 104. During the period of maturation, cells divide by … 105. A phenomenon of asexual reproduction of an embryo is called … 106. Gamones contributing to spermatozoon’s fixation on the ovum’s membrane are called … 107. Spermatozoa possess the ability of fertilization within …

FUNDAMENTALS OF ONTOGENESIS 108. Mitotic division of the zygote into blastomeres that occurs at early stages of prenatal ontogenesis is called … 109. The period of human prenatal development since the beginning of the 4th and till the end of the 8th weeks is called … 169 110. Type of gastrulation in case of which cells of the blastoderm migrate to the blastocoel and multiply to form the second germ layer is called … 111. Organisms blastopore of which transforms into anus and the mouth is formed at the opposite side of the body during embryogenesis are called … 112. The primary cause of cell differentiation during embryogenesis is … of the ovum’s cytoplasm. 113. Influence of one group of cells on another one by means of specific substances is called … 114. Gradual change of intensity of metabolic activity at the ends of the embryo or fetus is the example of the … of physiological activity. 115. The growth type of thymus and spleen is … 116. The hormone of hypophysis … plays the main role in regulation of human growth. 117. One of the main caused of acceleration is increasing … of young generations due to mixed marriages. 118. People of … constitutional type are predisposed to neuroses, ulcerous disease, tuberculosis. 119. The state of an organism characterized by cardiac and respiratory arrest, loss of consciousness but not critical impairments of cell metabolism is called … death. 120. Medical assistance to pass from life for a terminally ill patient according to his will or request of his relatives is called …

EVOLUTION OF ORGAN SYSTEMS 121. Recapitulations are completely absent in a phylembryogenesises that is called … 122. Ontophylogenetic causes of congenital defects are recapitulations and … 123. Brain type with striate bodies performing function of integrating center is called … 124. The first chordates that have duodenum and rectum are … 125. In reptiles the sixth pair of branchial arteries transforms into … 126. In mammals the third pair of branchial arteries transforms into … 127. Lancelet’s excretory organs are … 128. The only chordates that has functioning pronephros in adults is … 129. Mesonephros consists of approximately … nephrons.

INTRODUCTION TO PARASITOLOGY 130. Free-living organisms which can become parasites if they get to the organism of other species are called … 131. Hosts providing optimal biochemical conditions for the parasite and have biocoenotic contact with it are called … 170 132. Hosts providing biochemical conditions for the parasite but don’t have biocoenotic contact with it are called … 133. Hosts characterized by the presence of biocoenotic contacts with parasites but absence of biochemical conditions for their development are called … 134. Route of transmission of parasites with water and foodstuffs is called … 135. Route of transmission of parasites through mucous membranes of respiratory pipes is called … 136. Route of transmission of parasites with household goods is called … 137. Route of transmission of parasites with infected donor blood is called …

PHYLUM SARCOMASTIGOPHORA, CLASSES SARCODINA, ZOOMASTIGOTA 138. Vegetative form of protozoans is called … 139. Lesion of mucous membrane of the large intestine leading to formation of bleeding ulcers 2.5 cm diameter is a pathogenic effect of … 140. Vector of African trypanosomiasis is … 141. Life cycle of Trypanosoma … includes flagellated and non-flagellated stages. 142. Site of the skin 10–15 cm in diameter with hyperemia and edema, caused by invasion of Trypanosoma cruzi is called … 143. Stage of Leishmania donovani's life cycle which affects the vector is called … 144. Trichomonas vaginalis has … flagella.

PHYLUM INFUSORIA, CLASS CILIATA PHYLUM APICOMPLEXA, CLASS SPOROZOA 145. Pathogen of malignant tertian malaria is Pl. … 146. Pathogen of quartan malaria is Pl. … 147. Stage of malaria parasite that is invasive for intermediate host is called … 148. Final stage of malaria parasite in the human organism is called … 149. Stage of band is characteristic of Pl. … 150. Semilunar shape of gamonts is characteristic of Pl. … 151. Specific organelle on sharp end of toxoplasma providing the parasite fixation to the host’s cell is called … 152. Principal hosts of toxoplasma are representatives of the family … 153. Life stages of toxoplasma that are invasive for the principal host are … 154. Life stages of toxoplasma that are invasive for the intermediate host are …

171 PHYLUM PLATHELMINTHES, CLASS TREMATODA 155. Metacercaria, adolescaria and cercaria of flukes are … for the principal host. 156. Dormant invasive stage in the life cycle of liver fluke is called … 157. Fluke which has 2 rosette-like testes and S-like canal of the excretory system between them is called … 158. Life cycle of the cat liver fluke: marita egg miracidium sporocyst redia … metacercaria. 159. Egg-shaped fluke with an abdominal sucker in the middle of the body is called … 160. Larva of Paragonimus westermani which is an invasive stage for the principal host is called … 161. Special fissure of a male schistosome where female schistosome is situated is called … 162. Life cycle of schistosomes includes stages: egg miracidium primary sporocyst … cercaria. 163. Larva of schistosomes which is invasive for the principal host is called …

PHYLUM PLATHELMINTHES, CLASS CESTOIDEA 164. A measle of a cestode which is a big maternal cyst with smaller daughter cysts where scolexes develop is called … 165. The contact helminth of the class tapeworms is … 166. Hermaphroditic progottids of Taeniarhynchus saginatus have an ovarium consisting of … lobes. 167. Mature proglottid of Taeniarhynchus saginatus have … branches of the uterus. 168. Ovary of pork tapeworm has … lobes. 169. Mature proglottid of Taenia solium have … branches of the uterus. 170. Measle of a Hymenolepis nana is called … 171. Strobila of a Hyminolepis nana consists of approximately … proglottids. 172. Human is a … host for Echinococcus. 173. Life cycle of Diphylobotrium latum includes the following stages: egg … procercoid plerocercoid adult individual.

PHYLUM NEMATHELMINTHES, CLASS NEMATODA 174. Body wall of roundworms is covered with … 175. There are … situated along the canals of the excretory system in nematodes. 176. The number of muscle layers in the body wall of ascaris is … 177. Life span of mature Ascaris in the human body is about … 172 178. Nematode which has thin thread-like anterior end of the body is called … 179. Whipworm inhabits the … of the host. 180. Whipworm feds on … 181. Life span of toxocara is … 182. A parasite which has vesicle on the anterior end of the body and bulb in the gut is … 183. Nematode with life cycle including parasitic and free living stages is called … 184. Techniques that allow diagnostic of tissue helminthiasis are biopsy, muscle digestion, thick-blood film, and … PHYLUM ARTHROPODA, CLASS ARACHNIDA. POISONOUS AND VENOMOUS ORGANISMS 185. Ticks of … family have eyes. 186. Ixodes ricinus is a biological vector of … 187. Dermacentor pictus belongs to the family … 188. Hyalomma anatolicum is a biological vector of … 189. According to physiological effect on the body zootoxins are divided into neurotoxins, cytotoxins, hemorrhagins and … 190. Physalia’s stinging organs are … 191. Toxin of a scorpion belongs to … 192. Toxin of a karakurt belongs to … 193. Toxin of Colombian cocoa frog is … times stronger than tetanus toxin. 194. Toxins of a Brazilian spider belong to cytotoxins and … 195. Toxins of hymenopterans belong to cytotoxins and … 196. Viper snakes are primarily-toxic … animals. PHYLUM ARTHROPODA, CLASS INSECTA 197. House fly is a … vector of infectious and invasive diseases. 198. Stomoxys calcitrans is a mechanic vector of … 199. Glossina palpalis is a biological vector of … 200. Mosquitoes of the genus lay eggs in stagnant or slowly running clean water. 201. Drainage of small water pond and spraying incecticides on their surface are done for elimination … of mosquitoes. 202. Raising mosquito fishes (Gambusia) in water ponds is example of … method of mosquito elimination. 203. Fleas are vectors of tularemia and … 204. Jigger flea causes … 205. Latin name of the order fleas is … 206. Eggs of lice are called … 207. The pathogen of louse-borne relapsing fever is … 173 MULTICHOICE TESTS

THE ROLE OF BIOLOGY IN MEDICAL EDUCATION. METHODS USED TO INVESTIGATE CELLS 1. Main tasks of cytology are: 1 — studying the transmission of genetic information, 2 — studying the structure of tissues, 3 — studying the structure and functions of the nucleus, 4 — studying the cell divisions, 5 — studying the functions of plasma membrane and organelles: a) 1, 2, 3, 4, 5; b) 1, 3, 4, 5, c) 3, 4, 5, d) 2, 3, e) 3, 4. 2. Methods of cytology are: a) light and electron microscopy, cytogenetic karyotyping; b) isotopic labeling and differential centrifugation; c) cytogenetic karyotyping and cell microsyrgery; d) genealogical and cytochemical; e) X-ray crystallography and twin study. 3. Certain components of the cell can be extracted by: a) light and electron microscopy; b) hyctochemical and biochemical methods; c) genealogical and hybridological methods; d) differential centrifugation; e) X-ray crystallography. 4. Characters of the species Homo sapiens: a) high development of the brain; b) thought, consciousness, straight walking; c) har coat and nails; d) differentiated teeth and straight walking; e) apparent thumb opposition. 5. As a biological being, numan has: a) heredity and variability; b) social life; c) struggling for existance; d) metabolism, thought and consciousness; e) speech. 6. As a social being, numan has: a) heredity and variability, thought; b) speech and social working; c) metabolism, growth, development, ability to perform work; d) growth, development, ability to perform work; e) social mode of life and thought.

BIOLOGY OF THE CELL. THE FLOW OF SUBSTANCE AND ENERGY IN THE CELL 7. Plasma membrane contains: a) bilayer of carbohydrates; b) bilayer of lipids; c) two layers of proteins covering the surface of the membrane; d) semi-integral proteins; e) integral proteins. 8. Properties of plasma membrane are: a) plasticity; b) impermeability and fluidity; c) semi-permeability; d) elasticity; e) self-locking. 9. Energy is not required for: a) diffusion; b) facilitated diffusion; c) phagocytosis and pinocytosis; d) endocytosis and diffusion; e) pinocytosis and osmosis. 10. Transport of substances into the cell that requires ATP energy is: a) transport of ions into the cell down the concentration gradient; b) phagocytosis; c) pinocytosis and diffusion; d) osmosis and endocytosis; e) transport of substances into the cell against the concentration gradient. 174 11. Energy is required for such transport as: a) phagocytosis and diffusion; b) facilitated diffusion and osmosis; c) osmosis and pinocytosis; d) endocytosis; e) active transport. 12. Organelles of the cell’s anabolic system are: a) mitochondria and rough endoplasmic reticulum; b) ribosomes and Golgi complex; c) endoplasmic reticulum; d) lysosomes and peroxisomes; e) glyoxysomes, ribosomes and lysosomes. 13. Organelles of the cell catabolic system are: a) mitochondria; b) ribosomes, glyoxysomes and endoplasmic reticulum; c) endoplasmic reticulum and mitochondria; d) Golgi complex and peroxisomes; e) peroxisomes and lysosomes. 14. Ribosomes are located: a) on membranes of endoplasmic reticulum and in hyaloplasm; b) in hyaloplasm and karyoplasm; c) on internal nuclear membrane and in chloroplasts; d) on external nuclear membrane and in the mitochondria; e) in mitochondrial matrix and lysosomes. 15. Functions of the ER are: a) synthesis of proteins; b) DNA synthesis and compartmentalization; c) synthesis of fats and carbohydrates; d) transport of substances and compartmentalization; e) formation of peroxisomes and RNA synthesis. 16. Parts of the Golgi complex: a) vesicles and cisternae; b) canals, criaste and stroma; c) granae, stroma and vesicles; d) subunits, criatae and vacuoles; e) cristae, matrix and canals. 17. Functions of Golgi complex are: a) sorting, packing and secretion of substances; b) formation of lysosomes and complex organic compounds; c) synthesis of ATP, proteins and glyoxysomes; d) synthesis of cell membranes; e) protein synthesis and substance secretion. 18. Primary lysosomes: a) are small spherical organelles, size up to 2 µm; b) are rod-shaped organelles having one membrane; c) are small spherical organelles, have two membranes, size up to 2 µm; d) have ribosomes in their matrix; e) have up to 40 hydrolytic enzymes in their matrix. 19. Functions of secondary lysosomes (phagosomes): a) splitting of proteins and polysaccharides; b) synthesis of proteins and polysaccharides; c) heterophagy; d) ATP synthesis and autophagy; e) destruction of larval organs in animals having indirect development. 20. Functions of peroxisomes: a) splitting of proteins and polysaccharides; b) oxidation of amino acids with production of Н2О2; c) synthesis of polysaccharides and fats; d) heterophagy and oxidation of amino acids with production of Н2О2; e) destruction of larval organs in animals having indirect development and autophagy. 21. Components of mitochondria: a) external and internal membranes, thylakoids; b) circular DNA, ribosomes and cristae; c) thylakoids and ATP- somes; d) cristae, cisternae and vesicles; e) matrix and thylacoids.

175 22. Functions of mitochondria are: a) synthesis of specific proteins; b) splitting of proteins into amino acids; c) synthesis of ATP; d) synthesis of AMP (adenylic acid); e) splitting of organic substances into Н2О and СО2. 23. The first stage of energy exchange proceeds in: a) digestive tract; b) mitochondria; c) digestive tract and ER; d) cytoplasm and mitochondria; e) nucleus and cytoplasm. 24. Anaerobic stage of energy exchange occurs in: a) intestine; b) cytoplasm and mitochondria; c) cytoplasm and endoplasmic reticulum; d) cytoplasm; e) Golgi complex and cell nucleus.

FLOW OF GENETIC INFORMATION IN THE CELL 25. Idiogram is: a) non-systematized image of karyotype; b) systematized image of karyotype; c) order of genes in a chromosome; d) order of nucleotides in a gene; e) scheme or photograph of chromosomes arranged by their size. 26. Processes occurring in the cell during the pre-synthetic period of interphase are: a) synthesis of RNA, various proteins and enzymes; b) synthesis of DNA, RNA, proteins and ATP; c) growth of the cell and АTP synthesis; d) accumulation of DNA nucleotides, synthesis of tubulins for the spindle apparatus; e) synthesis of DNA, RNA and tubulins for the spindle apparatus. 27. Processes occurring in the cell during the synthetic period of interphase are: a) doubling of plastids and mitochondria; b) synthesis of DNA; c) synthesis of ATP and proteins; d) accumulation of DNA nucleotides, synthesis of mRNA and proteins; e) synthesis of tubulins for the spindle apparatus and DNA. 28. Processes occurring in the cell during the post-synthetic period of interphase are: a) synthesis of DNA and enzymes; b) synthesis of DNA, rRNA, growth of the cell; c) synthesis of ATP, accumulation of DNA nucleotides; e) synthesis of tubulins for the spindle apparatus. 29. Complement of genetic material during the pre-synthetic period: a) 1n1chr1с; b) 1n2chr2с; c) 2n1chr2с; d) 2n2chr4с; e) 1nbiv4chr4с. 30. Complement of genetic material by the end of the synthetic period: a) 1n1chr1с; b) 1n2chr2с; c) 2n1chr2с; d) 2n2chr4с; e) 1n4chr4с. 31. Causes of mitosis are: a) increase of nuclear-cytoplasmic ratio; b) decrease of nuclear-cytoplasmic ratio; c) replication of DNA and «wound hormones»; d) «wound hormones» and mitogenetic rays; e) damage of karyolemma. 32. Complement of genetic material during the telophase: a) 1n1chr1с; b) 1n2chr2с; c) 2n1chr2с; d) 2n2chr4с; e) 1n4chr4с. 33. Cells that can divide by mitosis: a) somatic cells; b) gametes; c) gametogonia; d) prokaryotic cells; e) cells without nucleus.

176 34. Meiosis is a division of: a) somatic and prokaryotic cells; b) gametes and embryonic cells; c) gametocytes; d) stem cells; e) tumor cells. 35. The order of stages in the prophase of meiosis I: a) diakinesis, diplotene, pachytene, zygotene, leptotene; b) leptotene, diakinesis, diplotene, pachytene, zygotene; c) leptotene, zygotene, diakinesis, diplotene, pachytene; d) leptotene, zygotene, pachytene, diplotene, diakinesis; e) diplotene, pachytene, zygotene, leptotene, diakinesis. 36. Processes occurring in the cell during the metaphase of meiosis I: a) centrioles move to the poles of the cell; b) decondensation of chromatin; c) bivalents are in the equator of the cell; d) synapsis; e) crossing-over. 37. Complement of genetic material during the prophase of meiosis I: a) 1n1chr1с; b) 1n2chr2с; c) 2n1chr2с; d) 2n2chr4с; e) 1nbiv2chr2с.

ARRANGEMENT OF HEREDITARY MATERIAL (PART 1) 38. Structural-functional levels of eukaryotic genetic material: a) gene and genome levels; b) chromosome, cellular, genome levels; c) genome and subcellular levels; d) cellular, organism, gene levels; e) organism and population levels. 39. Consequences resulting from arrangement of genetic material at the gene level: a) genetic linkage; b) independent inheritance of genes; c) mutations of genes; d) crossing-over and interactions of genes; e) intraallelic interactions of genes and genetic linkage. 40. Consequences resulting from arrangement of genetic material at the chromosome level: a) genetic linkage; b) independent inheritance of genes; c) mutations of genes and interactions of genes; d) crossing-over; e) chromosome mutations. 41. Consequences resulting from arrangement of genetic material at the genome level: a) genetic linkage and crossing-over; b) independent inheritance of genes and chromosome mutations; c) mutations of genes and crossing-over; d) genome mutations; e) interactions of genes. 42. Properties of genes: a) stability and lability; b) integrity and pleiotropy; c) integrity, specificity and unambiguity; d) discretion and non-specificity; e) specificity, tripletness and universality. 43. Specificity is the gene property to: a) mutate; b) determine synthesis of the certain polypeptide; c) be responsible for development of several characters; d) vary the degree of its phenotypic manifestation; e) have different frequency of phenotypic manifestations. 44. Pleiotropy is the gene property to: a) mutate; b) determine synthesis of the certain polypeptide; c) be responsible for development of several characters; d) vary the degree of its phenotypic manifestation; e) have different frequency of phenotypic manifestations.

177 45. Lability is the gene property to: a) mutate; b) determine synthesis of the certain polypeptide; c) be responsible for development of several characters; d) vary the degree of its phenotypic manifestation; e) have different frequency of phenotypic manifestations. 46. Expressivity is the gene property to: a) mutate; b) determine synthesis of the certain polypeptide; c) be responsible for development of several characters; d) vary the degree of its phenotypic manifestation; e) have different frequency of phenotypic manifestations. 47. Penetrance is the gene property to: a) mutate; b) determine synthesis of the certain polypeptide; c) be responsible for development of several characters; d) vary the degree of its phenotypic manifestation; e) have different frequency of phenotypic manifestations. 48. The least structural unit of a gene is: a) nitrogenous base; b) pair of complementary nucleotides; c) codon; d) one nucleotide; e) triplet of nucleotides. 49. The least functional unit of a gene is: a) one nucleotide; b) pair of complementary nucleotides; c) codon; d) transcripton; e) triplet of nucleotides. 50. Heterosynthetic function of a gene is: a) transcription and replication; b) translation and transcription; c) DNA replication and reparation; d) transformation and translation; e) only translation.

ARRANGEMENT OF HEREDITARY MATERIAL (PART 2) 51. The roles of structural genes: a) сode for repressor protein; b) code for enzymes; c) code for histones; d) code for various types of RNA; e) code for various RNA and repressor. 52. The role of functional genes: a) code for repressor protein; b) code for enzymes; c) code for histones; d) code for products regulating the work of structural genes; e) code for ribosomal RNA. 53. The role of operator a) codes for repressor protein; b) codes for enzymes; c) participates in switching the work of structural genes on and off; d) codes for mRNA; e) regulates activity of functional genes. 54. Classification of genes: a) structural, modifiers and repressors; b) introns, exons, inhibitors; c) functional and structural; d) corepressors and operators; e) regulators and intensifiers. 55. Parts of transcripton are: a) exons and several operators; b) operators and regulatory genes; c) structural genes and promoter; d) promoter terminator and repressor; e) initiator and regulators. 56. Information about the structure of a polypeptide is encoded by: a) terminators; b) operators; c) introns; d) exons; e) promoter. 57. Repeated sequences participate in: a) regulation of DNA replication; b) formation of operators and exons; c) formation of introns and participation in crossing-over; d) formation of exons and terminators; e) formation of promoters and initiators. 178 58. Functions of introns: a) regulate translation and replication of DNA; b) separate exons; c) participate in crossing-over and regulation of translation; d) contain spare information providing variability; e) regulate translation. 59. Criteria of cytoplasmic heredity are: a) segregation of characters occurs in accordance with Mendel’s laws; b) segregation of characters does not correspond to Mendel’s laws; c) it is possible to reveal linkage groups; d) inheritance goes on mother’s line; it is not possible to reveal linkage groups; e) identical results of recurrent crossings. 60. Features of human mitochondrial genome are: a) circular DNA contains 16 500 pairs of nucleotides; b) circular DNA contains 500 pairs of nucleotides and includes r-RNA genes; c) both strands are transcribed, contains gene of cytochrome b; d) one strand is transcribed; includes r-RNA genes; e) contains information about 22 t-RNA, circular DNA contains 160 pairs of nucleotides.

GENETIC ENGINEERING 61. Purposes of genetic engineering are: a) designing of genetic structures according to a plan; b) decoding the nucleotide sequences of DNA; c) creation of organisms with the new genetic program; d) revealing linkage groups and sequencing of genes; e) construction of a chromosome genetic map. 62. Main stages of genetic engineering are: a) obtaining genetic material; b) making genetic maps of chromosomes; c) decoding the nucleotide sequence of human DNA and assembling recombinant DNA; d) selection of the transformed cells; e) incorporation of a recombinant DNA into the host cell. 63. Genes for cloning in a vector can be obtained: a) by artificial gene synthesis; b) synthesis on a protein matrix; c) by reverse transcription; d) by making a map of a chromosome; e) cleaving from the genome with restriction endonucleases. 64. Recombinant DNA can be made by insertion of genes into: a) proteins; b) plasmids; c) viral genome; d) lipid molecule; e) phage genome. 65. Enzymes used in genetic engineering: a) DNA-polymerases; b) lipases and restriction enzymes; c) revertases and restriction enzymes; d) restriction enzymes and amylases; e) ligases. 66. Achievements of genetic engineering: a) strains of E. coli producing inulin; b) strains of E. coli producing somatotropin; c) plants acquiring atmospheric nitrogen; d) microorganisms producing petrol from food proteins; e) antiviral serums. 67. The directions for further development of genetic engineering: a) transfer of genetic information in eukaryotes by means of sexual reproduction; b) inducing mutations by chemical mutagens; c) sequencing of human genome; d) substitution of mutated genes with normal ones; e) insertion of artificial genes into human genome. 179 GENE INTERACTIONS. GENETIC LINKAGE. GENETICS OF SEX 68. Complete linkage is observed: a) in female Drosophila and male silkworm; b) if different allelic pairs are situated in different chromosomes; c) if crossing over occurs; d) if crossing over does not occur; e) in male Drosophila and female silkworm. 69. Characteristics of complementation: a) mutual influence of different genes situated in adjacent loci of the same chromosome; b) two dominant alleles of different genes are required for development of a trait; c) two recessive alleles of different genes are required for development of a trait; d) dominant or recessive allele of one gene suppresses effect of dominant or recessive allele of another gene; e) alleles of different genes have effect on degree of character’s development. 70. Characteristics of epistasis: a) mutual influence of different genes situated in adjacent loci of the same chromosome; b) two dominant alleles of different genes are required for development of a trait; c) two recessive alleles of different genes are required for development of a trait; d) dominant or recessive allele of one gene suppresses effect of dominant or recessive allele of another gene; e) one gene has effect on development of several traits. 71. Incomplete genetic linkage is observed: a) if different allelic pairs are situated in the same chromosome; b) if different allelic pairs are situated in different chromosomes; c) if crossing over occurs; d) if crossing over does not occur; e) in male Drosophila and female silkworm. 72. Period when anlagen of sex organs differentiate into male or female sex organs: a) 1st–4th weeks; b) 4th–6th weeks; c) 4th–8th weeks; d) 4th–12th weeks; e) 10th-16th weeks. 73. Characteristics of polymeria: a) mutual influence of different genes situated in adjacent loci of the same chromosome; b) two dominant alleles of different genes are required for development of a trait; c) two recessive alleles of different genes are required for development of a trait; d) one gene has effect on several characters; e) alleles of different genes have effect on degree of character’s development. 74. Inter-allelic gene interactions: a) hemizygosity and recessive epistasis; b) epistasis and cumulative polymeria; c) co-dominance and polymeria; d) complementation and pleiotropy; e) superdominance and recessiveness. 75. Anlagen of genitalia are formed: a) by 1st week of embryogenesis; b) by 2nd week of embryogenesis; c) by 3rd week of embryogenesis; d) by 4th week of embryogenesis; e) by 5th week of embryogenesis. 76. By 4th week laying down anlagen of genitalia is determined by: a) autosomes; b) one X-chromosome; c) both X-chromosomes; d) Y-chromosome; e) both X- and Y- chromosomes. 77. If the second sex chromosome is absent in the genotype, then: a) gonads differentiate; b) gonads do not differentiate; c) normal tissue of gonads 180 is substituted with connective tissue; d) gonads partially atrophy; e) gonads completely atrophy. 78. Transvestism is a phenomenon when: a) physical determinants of sex are impaired, person choses sexual partner of the same sex; b) psychological determinants of sex are impaired, person choses sexual partner of the same sex; c) gametic and hormonal sexes are impaired; d) genetic sex is not impaired, person wishes to wear clothes of the opposite sex; e) genetic and gametic sexes are impaired, person is sterile. 79. Karyotype in case of Klinefelter syndrome: a) 47,ХХY; b) 45,X0; c) 47,XXX; d) 46,XY; e) 46,XY,9p+. 80. Karyotype in case of Shereshevsky-turner syndrome: a) 46,ХY,5p–; b) 45,X0; c) 47,XXY; d) 47,XX,21+; e) 46,XX,9p+. 81. Karyotype in case of X-trisomy: a) 46,ХY,5p–; b) 45,X0; c) 47,XXX; d) 47,XX,21+; e) 47,XXX, 5p-. 82. Karyotype in case of Androgen insensitivity syndrome: a) 46,ХY,5p–; b) 45,X0; c) 47,XXY; d) 47,XX,21+; e) 46,XY. 83. Barr body is: a) inactivated X-chromosome; b) inactivated Y-chromosome; c) active X-chromosome; d) active X-chromosome; e) inactivated X-or Y-chromosomes.

VARIATION 84. Properties of modifications: a) they are adaptive; b) they are inherited; c) they are not inherited; d) they are matter for natural selection; e) they are matter for artificial selection. 85. Physical mutagens cause: a) formation of thymine dimers; b) deamination and alkylation of nucleotides; c) replacement of bases with their analogs; d) breakage of microtubules in the spindle apparatus; e) embedding of foreign DNA in the DNA of a host cell. 86. Chemical mutagens cause: a) formation of thymine dimers; b) deamination and alkylation of nucleotides; c) replacement of bases with their analogs; d) breakage of microtubules in the spindle apparatus; e) embedding of foreign DNA in the DNA of a host cell. 87. Biological mutagens cause: a) structural defects of genes and chromosomes; b) polyploidy; c) formation of thymine dimers; d) haploidy; e) embedding of foreign DNA in the DNA of a host cell. 88. According to their causes, mutations are: a) somatic and genome; b) spontaneous and phylogenetic; c) gametic and chromosomal; d) induced and ecological; e) spontaneous and induced. 89. Characteristics of somatic mutations: a) occur in gametes; b) occur in somatic cells; c) have phenotypic manifestation in the individual who got them; d) are transmitted to descendants during sexual reproduction; e) are transmitted to descendants during asexual reproduction. 181 90. Characteristics of gametic mutations are: a) occur in sex cells; b) occur in somatic cells; c) have phenotypic manifestation in the individual who got them; d) are transmitted to descendants during sexual reproduction; e) are transmitted to descendants during asexual reproduction. 91. Mutations of functional genes cause: a) transpositions; b) impaired alternation of recognition and termination; c) impaired alternation of initiation and elongation; d) impaired alternation of induction and repression; e) transitions. 92. According to the level where they occur, mutations are: a) somatic and lethal; b) gene and genome; c) gametic and chromosome; d) gene and chromosome; e) chromosome and induced. 93. Genome mutations are caused by: a) non-separation of chromosomes and chromatids during mitosis and meiosis; b) impairment of crossing over; c) endomitosis; d) structural impairments of chromosomes; e) destruction of spindle apparatus. 94. Polyploidy is: a) increased abnormal number of chromosomes indivisible by 1n; b) increased abnormal number of chromosomes divisible by 1n; c) decreased abnormal number of chromosomes indivisible by 1n; d) decreased abnormal number of chromosomes divisible by 2n; e) haploid set of chromosomes. 95. Haploidy is: a) positive mutation; b) nullsomy; c) monosomy; d) absence of one chromosome; e) haploid set of chromosomes. 96. Types of mutations in structural genes: a) transductions; b) transpositions; c) translocations; d) reading frame shift; e) transitions. 97. Stability of genetic material is not provided by: a) haploid chromosome set; b) diploid chromosome set; c) double helix of DNA; d) redundancy of the genetic code; e) DNA repair. 98. Excision repair of a DNA occurs in the following order: 1) synthesis of a new fragment of DNA strand; 2) ligation of the synthesized strand with the rest of the repairing DNA; 3) recognition the damaged DNA strand; 4) cutting out the damaged fragment of DNA strand; 5) replication of the DNA: a) 1–5–2–3; b) 5–1–3–2; c) 3–4–5–2; d) 3–4–2–1; e) 3–4–1–2. 99. According to the oncogene concept, the basis of oncogeneis is: a) protooncogenes received from parents or introduced into the genome of the cell by viruses; b) chromosome mutations of somatic cells; c) presence of protooncogenes in somatic cells of an organism; d) genome mutations of somatic cells; e) incorporations of viral DNA in the genome of somatic cells.

FUNDAMENTALS OF HUMAN GENETICS (PART 1) 100. Studying the human being has a number of difficulties such as: a) simple karyotype; b) early sexual maturation; c) low number of children; d) high number of children; e) possibility to conduct experiments.

182 101. Modern tasks of human genetics are: a) early diagnosis of hereditary disorders by improvement of instant diagnosis tests and tests for prenatal diagnosis; b) elaboration of gene therapy on the basis of biotechnological techniques and genetic engineering; c) use of hybridological method; d) large- scale implementation of genetic counseling into medical service; e) study of primary and secondary sexual characters. 102. Methods used in human genetics are: a) basic and experimental methods; b) methods of prenatal diagnosis and crossing; c) instant diagnosis tests and basic methods; d) genetic and paleontological tests; e) sociological and comparative anatomical. 103. Stages of genealogical analysis are: a) collection of data about proband’s relatives; b) calculation of gene frequency in the population; c) drawing genetic map of chromosome; d) estimation of environment’s role in development of the character; e) analysis of the pedigree chart. 104. Criteria of twins’ zygosity are: a) mode of dress and blood group; b) sex and blood groups in Rh and MN systems; c) color of eyes and mood; d) height and body temperature; e) fingerprints. 105. Technique of karyotyping: 1) processing with hypotonic solution of NaCl; 2) staining; 3) arrest of mitosis at the stage of metaphase by colchicine; 4) cultivation of cells on artificial media; 5) induction of mitosis. a) 1-5-3-4-2; b) 4-5-3-1-2; c) 4-1-5-3-2; d) 5-3-4-1-2; e) 4-5-1-3-2. 106. Holzinger's formula is used to calculate: a) gene frequency in the population; b) role of heredity for development of a character; c) role of environment for development of a character; d) probability of inheritance; e) genetic risk. 107. Biochemical genetic tests study: a) complete blood count; b) activity of enzymes; c) activity of digestion; d) composition of primary urine; e) structure of enzymes in a crystal. 108. Loading tests are used to to reveal: a) heterozygotes carrying recessive pathological gene; b) chromosome mutations; c) genome mutations; d) gene mutations; e) inheritance type. 109. Methods of genetic analysis are based on: a) mathematical expression of Hardy–Weinberg principle; b) extraction of DNA fragments and their analysis; c) drawing and analysis of pedigree charts; d) studying activity of enzyme systems; e) studying a karyotype under the microscope. 110. Methods of genetic analysis are used to: a) obtain certain genes and their parts for analysis; b) reveal genome mutations; c) detect certain nucleotide sequences; d) reveal chromosome mutations; e) reveal type of inheritance. 111. Cultivation of somatic cells: a) is based on uses Hardy–Weinberg principle; b) is based on extraction of DNA fragments and their sequencing; c) allows to obtain clones of a single cell; d) allows to select cells with certain characters; e) is based on karyotyping.

183 112. Somatic cell hybridization is used to: a) obtain synkaryotes of various cells; b) extract genes and their fragments for further sequencing; c) obtain clones of a single cell; d) select cells with certain characters; e) study karyotype with microscopy.

FUNDAMENTALS OF HUMAN GENETICS (PART 2) 113. Demographic characteristics of human populations: a) the number of individuals and genetic composition; b) birth and death rates; c) panmixia and density; d) isolation and migration; e) age and gender composition. 114. Characteristics of ideal populations: a) unlimited number of individuals; b) low number of individuals; c) complete panmixia; d) absence of mutations; e) presence of mutations. 115. In mathematical expression of the Hardy–Weinberg principle, a «p» denotes the frequency of: a) dominant gene; b) recessive gene; c) dominant homozygotes; d) recessive homozygotes; e) heterozygotes. 116. In mathematical expression of the Hardy–Weinberg principle, a «p2» denotes the frequency of: a) dominant gene; b) recessive gene; c) dominant homozygotes; d) recessive homozygotes; e) heterozygotes. 117. In mathematical expression of the Hardy–Weinberg principle, a «q» denotes the frequency of: a) dominant gene; b) recessive gene; c) dominant homozygotes; d) recessive homozygotes; e) heterozygotes. 118. In mathematical expression of the Hardy–Weinberg principle, a «q2» denotes the frequency of: a) dominant gene; b) recessive gene; c) dominant homozygotes; d) recessive homozygotes; e) heterozygotes. 119. In mathematical expression of the Hardy–Weinberg principle, a «2pq» denotes the frequency of: a) dominant gene; b) recessive gene; c) dominant homozygotes; d) recessive homozygotes; e) heterozygotes. 120. Processes occurring in small populations: a) Hardy–Weinberg principle works; b) birth and death rates change; c) frequencies of genes and genotypes change; d) age and gender composition change; e) the number of individuals change. 121. A genetic pool is: a) sum of individual’s genes; b) all the genes of individuals in a population; c) all the genes of individuals of the same species; d) all the genes of family members; e) all the genes of all individuals. 122. Types of marriages in human populations: a) same-sex marriages; b) unequal marriages; c) outmarriages; d) interracial marriages; e) incest marriages. 123. Microbiological tests allow to: a) create genetic maps of human chromosomes; b) determine the number of X-chromosomes; c) determine the number of Y-chromosomes; d) reveal some chromosome mutations; e) reveal some metabolic defects. 124. Dermatoglyphic analysis allow to: a) study pathogenesis of skin diseases; b) elaborate prophylactic measures of skin diseases; c) determine 184 the causes of skin diseases; d) reveal possible hereditary origin of disease; e) diagnose metabolic defects. 125. Indirect methods of prenatal diagnostics are: a) α-fetoprotein test; b) ultrasonography; c) choroin biopsy; d) amniocentesis; e) fetoscopy. 126. Direct noninvasive methods of prenatal diagnostics are: a) alpha- fetoprotein test; b) ultrasonography; c) chorion biopsy; d) amniocentesis; e) fetoscopy. 127. Optimal terms for carrying out direct noninvasive methods of prenatal diagnostics are: a) 6–8 weeks; b) 8–10 weeks; c) 12–20 weeks; d) 23–30 weeks; e) 30–35 weeks. 128. Genetic load is: a) positive mutations saturating a population; b) all the mutations reducing adaptability in a population; c) neutral mutations saturating a population; d) negative mutations saturating a population; e) absence of mutations in populations.

HUMAN GENETIC AND CHROMOSOME DISORDERS 129. Symptoms of phenylketonuria are: a) mice smell, intellect is not impaired; b) increased excitability and tone of muscles, intellectual disability; c) low excitability and tone of muscles, low pigmentation of skin; d) convulsive attacks, hemorrhages in joints; e) high concentration of phenylalanine hydroxylase in blood. 130. Symptoms of albinism are: a) hyposensitivity to ultra-violet rays; b) milky-white skin color; c) hair depigmentation; d) hair pigmentation; e) decreased acuity of vision. 131. Symptoms of galactosemia are: a) jaundice of newborns; b) vomiting, diarrhea, hepatomegaly and splenomegaly; c) depigmentation of skin and hair; d) propensity to self-damages; e) intellectual disability. 132. Symptoms Wilson–Konovalov disease are: a) increased concentration of copper in blood; b) increased concentration of iron in blood; c) accumulation of copper in the liver and brain leading to their degeneration; d) accumulation of iron in the liver and brain leading to their degeneration; e) impairment of functions of liver and central nervous system. 133. Symptoms of hemophilia A are: a) time of blood coagulation is 5–6 minutes; b) nasal bleedings and paralysis of legs; c) plural hematomas; d) hemorrhages in large joints and intellect decrease; e) blood in urine and high arterial pressure. 134. The karyotype for Patau syndrome is: a) 47, XXY; b) 47, XX, 18+; c) 47, XXX; d) 48, XYY; e) 47, XY, 13+. 135. Symptoms of Edward syndrome are: a) macrocephaly; b) congenital heart defects; c) big lower jaw and oral opening; d) throat underdevelopment; e) rocker bottom foot.

185 136. The karyotype for Down syndrome is: a) 45, XX, 21-; b) 47, XY, 13+; c) 47, XX, 21+; d) 47, XY, 21+; e) 46, XX, 5q-. 137. The karyotype for cri du chat syndrome: a) 45, XX, 5-; b) 46, XY, 5p-; c) 47, XX, 18+; d) 47, ХY,5+; e) 46, XX, 5p-.

GENETIC COUNSELLING 138. The main aims of genetic counseling are: a) estimation of the genetic risk in the examined family; b) to decrease the frequency of all diseases; c) to decrease the frequency of genetic diseases; d) to decrease the frequency of congenital malformations; e) to increase the birthrate. 139. High genetic risk is: a) up to 5 %; b) 5–10 %; c) 10–20 %; d) 20–30 %; e) about 50 %. 140. Indications for direction of a family to genetic counseling are: a) presence of similar hereditary disorders in several family members; b) arrested physical development of a child; c) infection in the family; d) parasitic disease in the family; e) divorce of spouses. 141. Examples of symptomatic treatment of hereditary disorders are: a) pain killers for inflammatory processes; b) antibiotics for pain syndrome; c) sedatives for excitement; d) excluding substance that is not metabolized in the organism from the diet; e) surgical correction of congenital defects. 142. Hereditary disorders corrected by special diets are: a) Down syndrome; b) phenylketonuria; c) hemophilia; d) galactosemia; e) Edwards syndrome. 143. Examples of pathogenic treatment of hereditary disorders are: a) pain killers for a pain syndrome; b) metabolic inhibition; c) gene therapy; d) excluding substance that is not metabolized in the organism from the diet; e) restriction of non-metabolic substance in the diet. 144. Examples of etiological treatment of hereditary disorders are: a) metabolic inhibition; b) antibiotics; c) substitution therapy; d) excluding substance that is not metabolized in the organism from the diet; e) gene therapy. 145. Metabolic inhibition includes: a) restriction of substance receipt with food; b) elimination of the substrate of pathological reaction from the organism; c) compensation of not synthesized product; d) suppression of pathological substrate’s synthesis; e) protection of an organ against receipt of catabolic products.

REPRODUCTION OF LIVING MATTER 146. Characteristics of asexual reproduction is: a) two individuals participate in reproduction; b) only one individual participates in reproduction; c) the genotype of daughter individuals differ from parental ones; d) genotype of daughter individuals are identical to parental ones; e) the number of daughter individuals increases slowly.

186 147. Characteristics of sexual reproduction is: a) usually two individuals participate in reproduction; b) only one individual participates in reproduction; c) genotypes of daughter individual differs from parental ones; d) genotypes of daughter individuals are identical to parental ones; e) the number of daughter individuals increases quickly. 148. Asexual reproduction of animals: a) vegetative reproduction; b) conjugation; c) copulation; d) polyembryony; e) fragmentation. 149. Movement forward of spermatozoa in the female reproductive tracts is provided by: a) mobility of spermatozoa; b) ovum’s immobility; c) contraction of muscles of female reproductive tract; d) excretion of gynogamones; e) contraction of abdominal muscles. 150. Fertilization is: a) fusion of an ovum with a sperm; b) movement of gametes to one another; c) movement of spermatozoa through female reproductive tract; d) process when the ovum leaves an ovary; e) sexual process. 151. Phases of fertilization: a) destruction of ova with hyaluronidase; b) distal interaction of gametes; c) contact interaction of gametes; d) entrance of the sperm’s head into the ovum; e) cleavage of the ova. 152. Peculiarities of human reproduction: a) reproductive period in women lasts till old age; b) men are capable for reproduction since the puberty up to 50 years; c) since puberty female organism produces one secondary oocyte a moon month; d) the older is the man, the longer is the time between the divisions of meiosis; e) spermatozoa are produced periodically

FUNDAMENTALS OF ONTOGENESIS 153. The cleavage type of a zygote depends on: a) sizes of the ovum; b) shape of the ovum; c) volume of yolk; d) distribution of yolk in the cytoplasm; e) potentialities of ovum's cytoplasm. 154. Primary causes of cells differentiation during embryogenesis are: a) chemical homogeneity of the ovum’s cytoplasm; b) chemical heterogeneity of the ovum's cytoplasm; c) chemical homogeneity of spermatozoon's cytoplasm; d) chemical heterogeneity of spermatozoon’s cytoplasm; e) different potencials of animal and vegetative poles of the ovum. 155. The main mechanisms of cell differentiation are: a) block of different transcriptons at certain stages of development; b) turning on all genes at the certain stages of development; c) block of all genes at the certain stages of development; d) unblocking of different transcriptons at the certain stages of development; e) block of one gene at the certain stages of development. 156. Action of genes during the ontogenesis: a) DNA → enzyme → mRNA → biochemical reaction → character; b) DNA → mRNA → enzyme → biochemical reaction → character; c) other genes have effect on a character; d) other genes do not have effect on the character; e) environmental factors do not have effect on the character. 187 157. At the early stages of embryogenesis (before early stage of gastrula) cells: a) are totipotent; b) are determined; c) can activate most of their transcriptons; d) can activate only some transcriptons; e) almost all the transcriptons are blocked. 158. At the stage of late gastrula cells: a) are totipotent; b) are determined; c) can activate most of their transcriptons; d) can activate only some transcriptons; e) almost all the transcriptons are blocked. 159. Characteristics of totipotent cells are: a) their development is preprogrammed; b) their development is not preprogrammed; c) each of them can give rise to any type of cells; d) each of them can give rise to only one certain type of cells; e) the majority of transcriptons are blocked. 160. Characteristics of determined cells are: a) their development is finally preprogrammed; b) their development is not preprogrammed c) each of them can give rise to any type of cells; d) each of them can give rise to only one certain type of cells; e) the majority of genes can join the work. 161. Characteristics of general growth of organs and tissues: a) intensive growth since birth and till 10–12 years; b) uniform growth during the whole period of growing; c) intensive growth during the first year of life and puberty; d) tissue grows intensively till 11–12 years and then gradually decrease to the volume characteristic of adults; e) rapid growth during puberty. 162. Causes of critical periods of embryogenesis are: a) changes in conditions of embryo existence and feeding; b) transition from one development period to another one; c) appearance of new inductors; d) active dedifferentiation of cells; e) poor nutrition of the pregnant woman. 163. Cause of ageing according to genetic hypothesis is: a) changed colloidal properties of cytoplasm; b) decreased production of sexual hormones; c) impaired DNA repair and inability for replication; d) impaired adaptation and regulation of the body; e) genetically programmed number of cell mitoses. 164. Cause of ageing according to intoxication hypothesis is: a) changed colloidal properties of cytoplasm; b) decreased production of sexual hormones; c) accumulation of waste products in the large intestine and their adsorption to the blood; d) impaired adaptation and regulation of the body; e) accumulation of mutations.

EVOLUTION OF ORGAN SYSTEMS 165. Examples of chordates: a) lancelets and cephalopods; b) reptiles and amphibians; c) mammals and cyclostomes; d) bony fishes and arthropods; e) nematodes and birds. 166. Basic evolution directions of nervous system of chordates are: 1) differentiation of the nerve tube into the brain and spinal cord; 2) transformation of the mammalian brain into sauropsidian; 3) transformation of the ichthyopsidian brain into sauropsidian; 188 4) transformation of the sauropsidian brain into ichthyopsidian; 5) transformation of the sauropsidian brain into mammalian; 6) development of the peripheral NS: a) 1, 2, 4, 6; b) 1, 3, 5, 6; c) 1, 2, 5, 6; d) 6; e) 1. 167. Ontophylogenetically conditioned malformations of the nervous system are: a) complete separation of cerebral hemispheres; b) non- differentiation of the cerebral hemispheres; c) absence of gyri in the cerebral cortex; d) anencephaly; e) olygophernia. 168. Ontophylogenetically conditioned malformations of the respiratory system are: a) underdevelopment of laryngeal cartilages and improper branching of bronchi; b) sac-like and «honeycomb» lungs; c) book lungs and tracheae; d) bronchial pneumonia and tuberculosis; e) compensating emphysema and atriopore. 169. Basic evolution directions of circulatory system of chordates are: 1) transformation of 2-chambered heart into 3-chambered one; 2) transformation of 3-chambered heart into 4-chambered one; 3) decreasing the number branchial arches; 4) increasing the number of branchial arches; 5) transformation of 3-chambered heart into 2-chambered one; 6) appearance of pulmonary circulation and complete separation of the arterial and a venous blood: a) 1, 2, 4, 5; b) 1, 2, 4, 6; c) 2, 3, 4, 6; d) 1, 2, 3, 6; e) 1, 2, 5, 6. 170. Mixed blood in present in hearts of: a) lancelets and amphibians; b) reptiles and amphibians; c) mammals and fishes; d) fishes and amphibians; e) cyclostomes and reptiles. 171. Basic evolution directions of digestive system of chordates are: 1) differentiation of the alimentary tract into regions; 2) appearance of digestive glands; 3) appearance of teeth and their differentiation; 4) appearance of posterior region of the intestine; 5) appearance of the oral cavity; 6) enlargement of the absorption surface due to the elongation of the intestine and appearance of villi: a) 1, 2, 4, 5; b) 1, 2, 5, 6; c) 1, 2, 3, 6; d) 1, 2, 3, 5; e) 1, 3, 4, 6. 172. Ontophylogenetically conditioned malformations of the digestive system are: a) appendix and additional lobes of the liver; b) esophageal cervical fistulae and homodont teeth; c) wisdom teeth; d) cloaca and wisdom teeth; d) homodont teeth and appendix; e) additional lobes of pancreas and cloaca. 173. Basic evolution directions of urinary system of chordates are: 1) transformation of a mesonephros into metanephros; 2) transformation of a mesonephros into a pronephros; 3) transformation of a metanephros into a pronephros; 4) transformation of a metanephros into a mesonephros; 5) transformation of a pronephric duct into a mesonephrogenic duct 6) transformation of the mesonephrogenic duct to a metanephric duct: a)1, 3, 6; b) 1, 5, 6; c) 1, 4, 5; d) 1, 4, 5; e) 2, 5, 6.

189 174. Ontophylogenetically conditioned malformations of urinary system are: a) preservation of the pronephric duct and presence of only one kidney; b) presence of only one kidney and preservation of the mesonephric duct; c) presence of three kidneyans and preservation of the mesonephric duct; d) presence of mesonephric kidneys and preservation of mesonephric duct; e) preservation of mesonephric duct and duplication of ureters. 175. In female amniotes Muller duct: a) reduces; b) performs only the function of ureter; c) performs the function of a gonad; d) performs the function of oviduct e) performs functions of ureter and oviduct. 176. In male amniotes Wolffian canal: a) reduces; b) performs only the function of ureter; c) performs functions of ureter and seminal duct; d) performs only the function of seminal duct; e) performs only the function of gonad.

INTRODUCTION TO PARASITOLOGY 177. Types of biological interactions: a) competition and predation; b) symbiosis and parabiosis; c) parabiosis; d) symbiosis and antibiosis; e) anabiosis. 178. Competition is a biological interaction in which: a) one species hunts the other one; b) one species produces substances to suppress vital processes of the other one; c) two species require the same conditions or resources; d) any kind of interactions between two organisms; e) both species receive mutual benefit. 179. Antibiosis is a biological interaction in which: a) one species hunts the other one; b) one species produces substances to suppress vital processes of the other one; c) two species require the same conditions or resources; d) any kind of interactions between two organisms; e) both species receive mutual benefit. 180. Commensalism is a biological interaction in which: a) both species receive mutual benefit; b) one species uses the other one only as habitation without causing harm or benefit; c) one species uses the other one only as habitation and origin of food without causing harm or benefit; d) one species uses the other one only as habitation and harms it; e) none of the species have benefit. 181. Mutualism is a biological interaction in which: a) both species receive mutual benefit; b) one species uses the other one only as habitation without causing harm or benefit; c) one species uses the other one only as habitation and origin of food without causing harm or benefit; d) one species uses the other one only as habitation and harms it; e) none of the species have benefit. 182. Criteria of parasitism are: a) relation with a host; b) absence of contacts between species; c) feeding at the expense of the host and causing harm; 190 d) one species uses the other one only as habitation without causing harm or benefit; e) production of substances that are required for the host survival. 183. Conditions for formation of a parasite-host system: a) contact between parasite and the host; b) parasite should cause death of a host; c) parasites and hosts not always need to contact; d) host must provide optimal conditions for the parasite; e) parasite should not resist the protective reactions of the host. 184. Types of symbiosis: a) mutualism and synoikia; b) anabiosis a and parasitism; c) competition and anabiosis; d) predation and cannibalism; e) commensalism and parasitism. 185. Examples progressive morphological and physiological adaptations of parasites: a) presence of attachment organs and specialization of integument; b) simplification of the nervous system and sense organs; c) molecular mimicry and secretion of antienzymes; d) absence of the digestive tract in intestinal parasites; e) high fertility and complex life cycles. 186. Examples of biological adaptations of parasites: a) presence of attachment organs and anti-enzymes; b) simplification of the nervous system and sense organs; c) various forms of asexual reproduction and high fertility; d) complex life cycles, alternation of hosts and migration of larvae within the host; e) immunosuppressive action. 187. Pathogenic actions of parasites are: a) mechanic injury of tissues, toxicoallergic; b) supplying the host with vitamins; c) supplying the host with nutrients; d) absorption of nutrients and vitamins from the host; e) weakening the organism and increasing probability of secondary infection. 188. Pathogenicity of a parasite does not depend on: a) host’s genotype and environmental factors; b) parasite’s genotype; c) host’s age and diet; d) body height and sex of the host; e) presence of other parasites in the host. 189. Protective reactions of the host’s organism occur at levels: a) subcellular and cellular; b) cellular and organism; c) population and tissue; d) cellular and tissue; e) population-specific. 190. Adaptation of parasites at the population level: a) presence of cysts and active search for hosts; b) simplification of nervous system and absence of alimentary system in tapeworms; c) molecular mimicry and anti-enzymes; d) involvement of intermediate and reservoir hosts into the life cycle; e) synchronization of parasite's life cycle and hosts behavior.

PHYLUM SARCOMASTIGOPHORA, CLASSES SARCODINA, ZOOMASTIGOTA 191. Sequence of stages of dysenteric ameba life cycle is: a) forma minuta → forma magna → tissue form → cyst → forma magna; b) forma magna → forma minuta → tissue form → cyst → forma magna; c) cyst → forma minuta → forma magna → tissue form → forma magna; d) cyst → forma minuta → forma magna → tissue form → forma minuta → cyst; e) tissue form → forma magna → forma minuta → cyst. 191 192. Laboratory diagnostics of American trypanosomiasis is based on: a) finding trypanosomes in feces and duodenal content; b) immunoassay; c) finding trypanosomes in blood smears; d) finding trypanosomes in liquor and in puncture samples of lymph nodes; e) finding trypanosomes in skin specimens. 193. Symptoms of visceral leishmaniasis are: a) fever, asthenia, headache; b) bloody diarrhea; c) anemia and cachexia; d) enlargement of liver and the spleen; e) ache along the small intestine. 194. Reservoir hosts of African trypanosomiasis are: a) sick people and monkeys; b) cattle and antelopes; c) dogs and wolves; d) opossums and armadillos; e) pigs. 195. Symptoms of African trypanosomiasis are: a) drowsiness, fever, an cachexia; b) bloody diarrhea; c) lesion of the heart muscle; d) liver and spleen enlargement; e) trypanosomic chancre on the skin, enlargement of occipital lymph nodes. 196. Symptoms of mucocutaneous leishmaniasis are: a) only lesion of skin; b) lesion of skin, mucosa and cartilages; c) lesion of internal organs; d) secondary infection; e) impairment of vision and hearing. 197. Symptoms of lambliasis are: a) bad appetite and nausea; b) headache and drowsiness; c) pains in epigastrium and in dextral subcostal area; d) pains in left subcostal area; e) unstable stool.

PHYLUM INFUSORIA, CLASS CILIATA. PHYLUM APICOMPLEXA, CLASS SPOROZOA 198. Exoerythrocytic cycle of malaria parasites is: a) sporozoites → erythrocytic schizont → liver-stage schizonts → liver-stage merozoites; b) sporozoites → liver-stage schizonts → erythrocytic schizonts → liver-stage merozoites; c) sporozoites → liver-stage schizonts → liver-stage merozoites; d) erythrocytic schizonts → sporozoites → gametocytes; e) sporozoites → erythrocytic schizonts → liver-stage schizonts → gametocytes. 199. Cycle of erythrocytic schizogony of malaria parasites is: a) ring- stage schizont → аmoeboid-stage schizont → gametocyte → morula → erythrocytic merozoite; b) morula → erythrocytic merozoite → gametocyte → ring-stage schizont → аmoeboid-stage schizont; c) amoeboid-stage schizont → ring-stage schizont → morula → gametocytes → erythrocytic merozoite; d) ring- stage schizont → amoeboid-stage schizont → morula → erythrocytic merozoite → gametocyte; e) gamentocyte → morula → ring-shaped schizont → amoeboid schizont → erythrocytic merozoite. 200. Sequence of malaria parasite's gametogony: a) ооcyst → gametocyte → macro- and microgametes → zygote → ооkinete b) gametocytes → macro- and microgametes → zygote → ookinete; c) macro-and microgametes → ookinete → zygote → gametocytes; d) macro- and microgametes → zygote →

192 ооkinete → gametocytes; e) gametocytes → zygote → ооkinete → macro- and microgametes. 201. Laboratory diagnosis of toxoplasmosis is based on: a) detection of trophozoites in feces and duodenal content; b) immunoassay; c) detection of trophozoites in urine; d) detection of trophozoites in striated muscles; e) detection of trophozoites in liquor and biopsy specimens of lymph nodes. 202. Sequence of symptoms in attack of malaria is: a) sweating stage → cold stage → hot stage; b) cold stage → sweating stage → hot stage; c) hot stage → cold stage → sweating stage; d) cold stage → hot stage → sweating stage; e) hot stage → sweating stage → cold stage.

PHYLUM PLATHELMINTHES, CLASS TREMATODA 203. The female reproductive system of flukes includes: a) testes, ovaries and uterus; b) ovaries, viteline glands and cirrus; c) ovaries, uterus, viteline glands and seminal receptacle; d) ovaries, seminal ducts and uterus; e) ootype, cirrus and viteline glands. 204. The first intermediate hosts of flukes are: a) human and monkeys; b) cattle; c) cats and dogs; d) snails, e) fishes, shrimps and crabs. 205. The second intermediate hosts of flukes are: a) sometimes absent; b) cattle; c) wild boars house and pigs; d) mollusks; e) fishes, shrimps and crabs. 206. Laboratory diagnostics of fascioliasis: a) finding eggs in the phlegm and urine; b) finding eggs in duodenal contents and feces; c) immunoassay; d) radionuclide diagnostics of the liver and pancreas; e) finding maritas in feces and duodenal content. 207. Techniques used for laboratory diagnostics of opistorchosis: a) Fulleborn and Kalantaryan techniques; b) Gorachev technique; c) Schulman technique; d) direct smear and thick-blood film; e) adhesive tape technique. 208. Laboratory diagnostics of a paragonimiasis: a) finding eggs in feces and urine; b) finding eggs in feces and sputum; c) finding larvae in feces and sputum; d) finding maritas in the lung and liver; e) immunoassay and radionuclide diagnostics of the lungs. 209. What is affected in case of urinogenital schistosomiasis? a) mesentery veins and the wall of the small intestine; b) veins of the uterus and the upper third of the vagina; c) veins of the urinary bladder and prostate; d) veins of the large intestine; e) veins of the lungs. 210. What is affected in case of Мansоn schistosomiasis? a) veins of the mesentery and the intestine; b) veins of the uterus and the vagina; c) veins of the bladder; d) the system of the portal vein and the liver; e) the brain.

193 PHYLUM PLATHELMINTHES, CLASS CESTOIDEA 211. Correct sequence of broad tapeworm’s life cycle is: a) egg → coracidium → procercoid → oncosphere → plerocercoid; b) egg → oncosphere → measle; c) egg → coracidium → procercoid → plerocercoid; d) cercarium → coracidium → procercoid → measle; e) procercoid → меtacercaria → plerocercoid. 212. Invasion of human with teniasis occurs during: a) personal hygiene breaches; b) contacts with sick persons; c) eating undercooked beef; d) eating undercooked pork; e) eating undercooked fish, shrimps and crabs. 213. Invasion of a person with cysticercosis occurs during: a) swallowing eggs of pork tapeworm; b) eating undercooked pork and beef; c) eating undercooked shrimps and crabs; d) contact with domestic pigs; e) аutoinvasion in teniasis. 214. Pathogenic action of Taenia solium: is: a) lesion of the brain and a spinal cord; b) toxicoallergic; c) irritation of mucosa of the large intestine; d) irritation of mucosa of the small intestine; e) absorption of nutrients from the host's digestive tract. 215. Symptoms of Taeniarchynchus invasion are: a) blood-containing diarrhoea; b) fever and ache in the abdomen; c) ache in the abdomen, nausea, vomiting; d) laboured breathing, ache in the thorax; e) enlargement of liver and spleen. 216. Life stage of echinococcus that is invasive for human is: a) egg; b) oncosphere; c) plerocercoid; d) cysticercoid; e) cysticercus. 217. Human becomes affected with broad tapeworm: a) by means of contact with a sick person; b) by means of contact with a carnivorous anmals; c) when eats undercooked beef and pork; d) by vector-borne rote; e) when eats undercooked fish.

PHYLUM NEMATHELMINTHES, CLASS NEMATODA 218. Morphology of ascaris: a) segmented body 1–5 cm length; b) spindle-shaped long body 25–40 cm length; c) white color; d) white-pink body color; e) tape-like body up to 3 meters length. 219. Human becomes affected with ascaris: a) when swallows eggs of ascaris while breaching rules of Hygiene; b) when larvae actively invade the skin; c) by means of contact with a sick person; d) when eats undercooked beef; e) by vector-borne rote. 220. Migration way of ascaris larvae in the body is: a) intestine → right heart → lungs → blood vessels → liver → bronchi → trachea → pharynx → intestine; b) intestine → liver → bronchi → right heart → lungs → blood vessels → trachea → pharynx → intestine; c) liver → bronchi → right heart → lungs → blood vessels → trachea → pharynx → intestine; d) intestine → blood

194 vessels → liver → right heart → lungs → bronchi → trachea → pharynx → intestine; e) intestine → blood vessels → right heart → lungs → liver → bronchi → trachea → pharynx → intestine. 221. Symptoms of migration ascariasis are: a) intestinal obstruction; b) fever and an asthmatic bronchitis; c) non-constant eosinophilic infiltrations in lungs; d) occlusion of choledoch duct; e) appendicitis. 222. Symptoms of intestinal ascariasis are: a) cough with bloody sputum; b) ache in the abdomen; c) fever and rush; d) bad appetite, nausea, vomit; e) non-constant eosinophilic infiltrations in lungs and pneumonia. 223. Morphophysiological features of whipworm are: a) length of females is 5 cm, has vesicle on the anterior end of a body; b) length of female is 3–5 cm, has bulb and buccal capsule with teeth; c) length of a female is 3–5 cm, anterior end of the body is thread-like while the posterior one is thicker; d) has cuticular lips, feeds on the intestinal content; e) feeds on blood. 224. Surgical implications of аscariasis are: a) obstructive jaundice and obstruction of the intestine; b) affection of an eyeball by an adult worm; c) perforation of the intestinal wall; d) pneumonia and bronchitis; e) pancreatitis and appendicitis. 225. Symptoms of enterobiosis: a) distribution of sleep and defective memory; b) impairment of vision; c) aches in the area of the small intestine and in right hypochondriac area; d) cough; e) itch in the peritoneal area. 226. Laboratory diagnosis of enterobiosis is based on: a) immunoassay; b) detection of larvae in blood and striated muscles; c) detection of eggs and mature parasites on the peritoneal area; d) detection of eggs and mature parasites in feces; e) detection of larvae and eggs on the peritoneal area. 227. Symptoms of trichinellosis are: a) brain lesion; b) gastrointestinal disorders; c) rising of temperature and eosinophilla; d) oedema of eyelids and face, pains in muscles; e) enlargement of liver and spleen. 228. Laboratory diagnosis of trichinellosis is based on: a) detection of eggs in saliva and feces; b) detection of larvae in blood and lymph; c) immunoassay; d) detection of larvae in striated muscles; e) detection of mature parasites in striated muscles. 229. Peculiarities of the life cycle of the Strongyloides: a) larvae may develop without being in the invironment; b) in soil rhabditiform larvae transform in filariform larvae if environmental conditions are favorable; c) in soil rhabditiform larvae transform in mature worms if environmental conditions are unfavorable; d) larvae penetrate skin of the host and migrate; e) larvae are unable to migrate in the host. 230. Rotes of infection with Strongyloidiasis: a) droplet; b) alimentary — eating the polluted vegetables, fruits and drinking water from reservoirs; c) direct contact with a sick person; d) larvae invade the body through the skin; e) transplacentally.

195 PHYLUM ARTHROPODA, CLASS ARACHNIDA. VENOMOUS AND POISONOUS ANIMALS 231. Features of ticks and mites: a) bodies have no regions, respiratory organs are tracheae, heart is at the dorsal side; b) bodies have no regions, respiratory organs are gills; c) bodies consist of cephalothorax and abdomen, circulatory system is open; d) segmented bodies, heart is at the dorsal side, circulatory system is open; e) circulatory system is closed, heart is at the ventral side. 232. Biological vector of disease is an organism: a) where the pathogen undergoes definite development stages obligatory for the parasite; b) where the pathogen doesn’t undergo definite development stages, obligatory for the parasite; c) carryng pathogens on body surface or on mouthparts; d) where the pathogen desn’t undergo definite development stages, facultative for the parasite; e) where the pathogen passes through the intestinal tract without reproduction. 233. Features of Ixodidae family are: a) habitation is forests and steppe; b) habitation is caves, holes of rodents, abandoned buildings; c) blood meal lasts up to several days; d) can starve up to 12–15 years; e) females lay 50–200 eggs. 234. Scabies is spread: a) by vector-bone route; b) during a direct skin contact with a sick person; c) by eating of uncooked fish; d) by bedclothes of sick persons; e) by drinking water from the open sources. 235. Prophylaxis of scabies is: a) revealing and treating sick persons; b) elimination of vectors; c) maintaining the purity of the body; d) washing vegetables and fruits before eating; e) sanitary inspection of hostels, bathhouses and heath education. 236. Ticks belong to the class: a) Trematoda; b) Cestoda; c) Nematoda; d) Arachnida; e) Insecta. 237. Symptoms of toxication with cobra venom: a) sharp pain, inflammation of lymphatic vessels; b) inflammation of lymphatic vessels, a necrosis of tissues; c) sharp pain, necrosis of tissues; d) excitation and then depression of CNS, necrosis of tissues; e) excitation and then depression of CNS, impairment of respiration are observed. 238. Symptoms of toxication with Viper snakes venom: a) sharp pain and impairment of blood clotting; b) extremities numbness and hemorrhagic edema; c) hemorrhagic edema; d) numbness of extremities and impairment of respiration; e) impairment of blood clotting and respiration. 239. Clinical presentation of poisoning with fly amanita: a) vomiting, diarrhea; b) laboured breathing; c) elevation of temperature, tachycardia; d) excitation, euphoria, e) hallucinations and convulsions. 240. Clinical presentation of poisoning with Papaver somniferum: a) vomiting, dizziness; b) allergic reactions, arterial blood hypotension;

196 c) hallucinations, respiratory depression up to failure; d) death caused by cardiac arrest, e) retention of urine and bowel movement. 241. Clinical presentation of poisoning with cannabis: a) bloody diarrhea: b) vinose state, verbal and motor excitement, hallucinations; c) bradycardia, arterial hypotension, diarrhea; d) psychological functional disturbances leading to disintegration of personality, e) merriment passing into sleep with dreams. 242. First aid in a toxication with snake venom is: a) to suck the venom away and to treat the place of biting with disinfectants; b) to scorch the place of biting and to put the victim in a shade; c) to scorch and to treat the place of a biting with disinfectants; d) to transport a victim in lying position; e) to apply a hard bandage to a place of a biting and to transport the victim in any position.

PHYLUM ARTHROPODA, CLASS INSECTA 243. Morphological features of cockroaches: a) flattened body, length reaches 3 cm; b) narrowed body, length reaches 3 cm; c) chewing mouthparts, the length of the body is up to 8 cm; d) chewing mouthparts, the length of the body is up to 3 cm; e) flattened body, piercing and sucking mouthparts. 244. Medical significance of cockroaches: a) mechanic vectors transmitting eggs of helminthes and cysts of protists; b) biological vectors transmitting pathogens of tularemia and tuberculosis; c) biological vectors transmitting pathogens of malaria and filariases; d) may gnaw epidermis around lips; e) feed on blood, bites are painful. 245. Morphology of head lice: a) length of the body is 1–4 mm, wings are absent; b) length of the body is 1–4 mm, there is one pair of wings; c) chewing mouthparts; d) length of the body is 2–4 mm, wings are absent; e) mouthparts are piercing and sucking. 246. Medical significance of fleas: a) mechanic vectors transmitting pathogens of tuberculosis and dysentery; b) biological vectors transmitting cysts of protists and eggs of helminthes; c) biological vectors transmitting pathogens of plague and tularemia; d) bites are painful, may cause dermatitis; e) mechanic vectors transmitting pathogens of tularemia. 247. Features of the life cycle of lice belonging to the genus Pediculus: a) lay eggs in dry dust and on food products; b) stick nits to hair; c) development is direct; d) development is indirect with incomplete metamorphosis; e) duration of the life cycle is 2–3 months. 248. Medical significance of pubic lice: a) mechanic vectors transmitting eggs of helminthes and cysts of protists; b) biological vectors transmitting pathogens of the louse-borne relapsing fever; c) biological vectors transmitting pathogens of epidemic typhus; d) cause pediculosis; e) cause phthiriasis. 249. Morphology of pubic louse: a) bodies are short and wide in comparison with head lice, body length is up to 10 mm; b) bodies are short and 197 wide in comparison with head lice, body length is up to 1.5 cm; c) bodies are long, the length is up to 5 mm; d) piercing and sucking mouthparts; e) chewing mouthparts. 250. Morphology of house fly: a) the body is about 7.5 mm in length, licking mouthparts; b) the body is about 7.5 mm in length, piercing-licking mouthparts; c) body is covered with short hair, one pair of wings; d) piercing- licking mouthparts, body is covered with short hair; e) chewing mouthparts, two pairs of wings. 251. Medical significance of a house flies: a) biological vectors transmitting bacteria, cysts of protists and eggs of helminthes; b) mechanic vectors transmitting bacteria, cysts of protists and eggs of helminthes; c) biological vectors transmitting pathogens of plague and Japanese encephalitis; d) larvae may cause myiasis; e) biological vectors transmitting pathogen of African trypanosomiasis. 252. Medical significance of stable flies: a) mechanic vectors transmitting cysts of protists and eggs of helminthes; b) mechanic vectors transmitting pathogen of anthrax; c) biological vectors transmitting pathogen of anthrax; d) larvae may cause myiasis; e) bites are painful. 253. Morphology of pre-imago stages of Anopheles mosquitoes: a) eggs have no air floats, larvae have respiratory siphon; b) eggs have air floats, larvae have siphon; c) larvae have no siphon, chrysalides have a funnel-like respiratory trumpet; d) eggs have air floats, chrysalides have tube-shaped respiratory trumpets; e) eggs have air floats, chrysalides have funnel-like respiratory trumpet. 254. Morphology of mature mosquitoes of the genus Anopheles: a) antennae of females are hairy, palps and proboscis are equal in length; b) antennae of females are not hairy, palps and proboscis are equal in length; c) antennae of males are hairy and palps are shorter than the proboscis; d) antennae of males are hairy and palps have club-like thickenings at ends; e) antennae of males are hairy and palps have no club-shaped thickenings. 255. Medical significance of mosquitoes of the genus Anopheles: a) mechanic vectors transmitting cysts of protists and eggs of helminthes; b) biological vectors transmitting pathogens of tularemia and plague; c) biological vectors and principal hosts of malaria parasites; d) biological vectors transmitting pathogen of onchocercosis; e) biological vectors and intermediate hosts of Wuchereria bancrofti. 256. Medical significance of mosquitoes of the genus Aedes: a) biological vectors transmitting pathogens of tularemia and the Japanese encephalitis; b) biological vectors transmitting cysts of protists and eggs of helminthes; c) biological vectors transmitting pathogens of the plague and tuberculosis; d) biological vectors and principal hosts of malaria parasites; e) biological vectors transmitting Brugia malayi.

198 257. Medical significance of mosquitoes of the genus Culex: a) mechanic vectors transmitting pathogens of tularemia and Japanese encephalitis; b) biological vectors transmitting cysts of protists and eggs of helminthes; c) biological vectors transmitting pathogen of malaria; d) biological vectors of Wuchereria bancrofti and Brugia malayi; e) biological vectors of Brugia malayi. 258. Medical significance of pubic louse: a) mechanic vectors transmitting cysts of protists and eggs of helminthes; b) biological vectors transmitting pathogen of louse-borne relapsing fever; c) biological vectors transmitting pathogen of epidemic typhus; d) cause pediculosis; e) cause phthiriasis. 259. Measures to eliminate larvae of mosquitoes: a) fumigation, b) usage of repellents, c) raising mosquito fishes (Gambusia) in water ponds, d) usage of incecticides, e) maintaining purity of houses. 260. Measures to eliminate imago of mosquitoes: a) fumigation, b) usage of repellents, c) raising mosquito fishes (Gambusia) in water ponds, d) usage of incecticides, e) maintaining purity of houses.

199 ANSWERS TO THE GAP-FILLING TESTS

The role of Biology in medical education. Methods used to investigate cells 1. Light. 5. Differential centrifugation. 2. Isotope labelling. 6. Placentals. 3. Electron. 7. Hominids. 4. Cytochemical.

Biology of the cell. Flow of substance and energy in the cell 8. Compartmentalization. 13. 3. 9. Glycocalyx. 14. Autophagy. 10. Golgi complex. 15. Porin. 11. 6–8. 16. 40 %. 12. ER.

Flow of genetic information in the cell 17. Lamins. 21. Chiasmata. 18. Kinetochore. 22. Bivalents. 19. Nucleolar organizer. 23. 1n 2chr 2c. 20. 1nbiv 4chr 4c.

Arrangement of hereditary material 24. Nucleosome. 32. Elongation. 25. 5–7. 33. Inhibitors. 26. Chromatid. 34. Repressors. 27. 10 000. 35. Repressors 28. Replication. 36. Alternative splicing. 29. 3/–5/. 37. Terminator . 30. Recognition. 38. Inductor. 31. AUG. 39. Functional genes.

Genetic engineering 40. Restriction endonucleases. 45. Cosmids. 41. Reverse transcription. 46. 33–39. 42. Cosmids. 47. SV40. 43. Sticky. 48. Blunt. 44. Phasmids.

200 Gene interactions. Genetic linkage. Genetics of sex 49. Recessive epistasis. 54. 50. 50. Dominant epistasis. 55. Shereshevsky–Turner. 51. Complementation. 56. Klinefelter. 52. Crossing-over. 57. Intermediate. 53. Crossing-over. 58. Transsexualism.

Variation 59. Phenocopy. 64. Genome. 60. Exonucleases. 65. Monosomy. 61. Transition. 66. Haploidy. 62. Delition. 67. Fanconi anemia. 63. Functional.

Fundamentals of human genetics 68. 18.75 %. 77. Population statistic. 69. 20 %. 78. Decrease. 70. X-linked dominant. 79. Ultrasonography. 71. Synkaryote. 80. Direct invasive. 72. Twin study. 81. Acrichine yperite. 73. Discordance. 82. 57. 74. Karyotyping. 83. Isolates. 75. Genetic. 84. Heterozygosity. 76. 8th–13th. 85. Inbreeding.

Human genetic and chromosome disorders 86. Ceruloplasmin. 90. Wilson–Konovalov disease. 87. Valine. 91. Hyperlipoproteinemia. 88. Lesch-Nyhan syndrome. 92. Chromosome. 89. Albinism. 93. Edwards.

Genetic counselling 94. Pathogenetic. 96. Symptomatic. 98. Etiotropic. 95. Pathogenetic. 97. Symptomatic.

Reproduction of living matter 99. Sexual process. 104. Meiosis. 100. Syncaryogamy. 105. Polyembryony. 110. Partenogenesis. 106. Fertilizins. 102. Androgenesis. 107. 1–2 days. 103. Mitosis. 201 Fundamentals of ontogenesis 108. Cleavage. 115. Lymphoid. 109. Prefetal. 116. Somatotropin. 110. Ingression. 117. Heterozygosity. 111. Deuterostomes. 118. Ectomorphic. 112. Chemical heterogeneity. 119. Clinical. 113. Embryonic induction. 120. Euthanasia. 114. Gradient.

Evolution of organ systems 121. Archalaxes. 126. Carotid arteries. 122. Parallelisms. 127. Nephridia. 123. Sauropsidan. 128. Cyclostomes. 124. Amphibians. 129. 100. 125. Pulmonary arteries.

Introduction to parasitology 130. Facultative parasites. 134. Alimentary. 131. Obligate. 135. Respiratory 132. Potential. 136. Indirect contact. 133. Optional. 137. Iatrogenic .

Phylum Sarcomastigophora, classes Sarcodina, Zoomastigota 138. Trophozoite. 141. Cruzi. 139. Entaboeba histolytica 142. Chagoma. and Balantidium coli. 143. Promastigote. 140. Tsetse fly. 144. 5.

Phylum Infusoria, class Ciliata. Phylum Apicomplexa, class Sporozoa 145. Falciparum. 150. Falciparum. 146. Malaria. 151. Conoid. 147. Sporozoite. 152. Cats. 148. Micro- and macrogametocyte. 153. Sporozoite, trophozoite. 149. Malaria. 154. Sporozoite, trophozoite.

Phylum Plathelminthes, class Trematoda 155. Invasive stages . 160. Metacercaria. 156. Adolescaria. 161. Gynecophoral canal. 157. Cat liver fluke. 162. Secondary sporocyst. 158. Cercaria. 163. Cercaria. 159. Lung fluke.

202 Phylum Plathelminthes, class Cestoidea 164. Hydatid cyst. 169. 7–12. 165. Dwarf tapeworm. 170. Cysticercoid. 166. 2. 171. 200. 167. 17–35. 172. Intermediate. 168. 3. 173. Coracidium.

Phylum Nemathelminthes, class Nematoda 174. Cuticule. 180. Blood. 175. Phagocytes. 181. 4–6 months. 176. One. 182. Enterobius vermicularis. 177. One year. 183. Treadworm. 178. Whipworm. 184. Immunoassay. 179. Large.

Phylum Arthropoda, class Arachnida. Poisonous and venomous organisms 185. Ixodidae. 191. Neurotoxin. 186. Tick-borne encephalitis. 192. Neurotoxin. 187. Ixodidae. 193. 50. 188. Hemorrhagic fever. 194. Hemorrhagins. 189. Hemolyzins. 195. Neurotoxins. 190. Thread cells. 196. Actively-venomous.

Phylum Arthropoda, class Insecta 197. Mechanic. 203. Plague. 198. Anthrax. 204. Sarcopsilliasis. 199. African tripanosomiasis. 205. Anoplura. 200. Anopheles. 206. Nits. 201. Larvae. 207. Borrelia recurrentis. 202. Biological.

203 ANSWERS TO THE MULTICHOICE TESTS

The role of Biology in medical education. Methods used to study cells 1. c. 2. b. 3. d. 4. a, b. 5. a, c. 6. b, e.

Biology of the cell. Flow of substance and energy in the cell 7. b, d, e. 8. a, c, e. 9. a, b. 10. b, e. 11. d, e. 12. b, c. 13. a, e. 14. a, d. 15. c, d. 16. a. 17. a, b, d. 18. a, e. 19. a, c. 20. b. 21. b. 22. a, c. 23. a. 24. d.

Flow of genetic information in the cell 25. b, e. 26. a, c. 27. b, c. 28. e. 29. c. 30. d. 31. b, d. 32. c. 33. a, c. 34. c. 35. d. 36. c. 37. e.

Arrangement of hereditary material (Part 1) 38. a. 39. b, c. 40. a, d, e. 41. d, e. 42. a, b. 43. b. 44. c. 45. a. 46. e. 47. d. 48. b. 49. c. 50. e.

Arrangement of hereditary material (Part 2) 51. b, c, d. 52. a, d. 53. c . 54. c, e. 55. a, c. 56. d. 57. a, c, e. 58. b, d. 59. b, d. 60. a, c.

Genetic engineering 61. a, c. 62. a, d, e. 63. a, c, e. 64. b, c, e. 65. a, c, e. 66. b, c, e. 67. d, e.

Gene interactions. Genetic linkage. Genetics of sex 68. d, e. 69. b. 70. d. 71. a, c. 72. d. 73. e. 74. b, d. 75. d. 76. b. 77. b, c. 78. d . 79. a. 80. b . 81. c. 82. e. 83. a.

Variation 84. a. 85. a, d. 86. b, c. 87. a, e. 88. e. 89. b, c, e. 90. a, d. 91. d. 92. b, d. 93. a. 94. b. 95. e. 96. d, e. 97. a. 98. e. 99. a, c.

204 Fundamentals of human genetics (Part 1) 100. c. 101. a, b, d. 102. c. 103. a, e. 104. b, e. 105. b. 106. b. 107. b. 108. a 109. b. 110. a, c. 111. d. 112. a.

Fundamentals of human genetics (Part 2) 113. b, e. 114. a, c, d. 115. a. 116. c. 117. b. 118.d. 119. e. 120. c. 121. b. 122. d, e. 123. e. 124. d. 125. a. 126. b. 127. c. 128. b, d.

Human genetic and chromosome disorders 129. b. 130. b, c, e. 131. a, b, e. 132. a, c, e. 133. c. 134. e. 135. b, e. 136. c, d. 137. b, e.

Genetic counselling 138. a, c, d. 139. d, e. 140. a. 141. c, e. 142. b, d. 143. b, d, e. 144. e. 145. b, d.

Reproduction of living matter 146. b, d. 147. a, c. 148. d, e. 149. a, c, d. 150. a. 151. d. 152. c.

Fundamentals of ontogenesis 153. c, d. 154. b. 155. a, d. 156. b, c. 157. a, c. 158. b, d. 159. b, c. 160. a, d. 161. c. 162. a, b, c. 163. c. 164. c.

Evolution of organ systems 165. b, d. 166. b. 167. b, c, d. 168. a, b. 169. d. 170. b. 171. c. 172. b, e. 173. b. 174. d. 175. d. 176. d.

Introduction to parasitology 177. a, d. 178. c. 179. b. 180. c. 181. a. 182. a, c. 183. a, d, e. 184. a, e. 185. a, c. 186. c, d. 187. a, d, c. 188. c, d. 189. b, d. 190. a, d, e.

205 Phylum Sarcomastigophora, classes Sarcodina, Zoomastigota 191. d. 192. b, c, d. 193. a, c, d. 194. b, e. 195. a, d, e. 196. b, d. 197. a, c, e.

Phylum Infusoria, class Ciliata. Phylum Apicomplexa, class Sporozoa 198. c. 199 d. 200. b. 201. b, e. 202. d.

Phylum Plathelminthes, class Trematoda 203. c. 204. d. 205. a, e. 206. b, c. 207. b. 208. b, e. 209. b, c. 210. a, d.

Phylum Plathelminthes, class Cestoidea 211. c. 212. d. 213. a, e. 214. b, d, e. 215. c. 216. a. 217 e.

Phylum Nemathelminthes, class Nematoda 218. b, d. 219. a. 220. d. 221. b, c. 222. b, d. 223. c, e. 224. a, c, e. 225. a, e. 226. a, c. 227. b, c, d. 228. c, d. 229. a, d. 230. b, d.

Phylum Arthropoda, class Arachnida. Poisonous and venomous organisms 231. a. 232. a. 233. a, c. 234. b, d. 235. a, c, e. 236 d. 237. a, e. 238. a, c. 239. a, c, e. 240. a, c, e. 241. b, d, e. 242. a, d.

Phylum Arthropoda, class Insecta 243. a, d. 244. a, d. 245. d, e. 246. c, d. 247. b, d. 248. e. 249. b, d. 250. a, c. 251. b, d. 252. b, e. 253. c, e. 254. b, d. 255. c, e. 256. a, e. 257. e. 258. b, c, e. 259. c. 260. a, d.

206 LITERATURE

1. Bekish, O.-Y. L. Medical biology : textbook for student of higher educational establishments / O.-Y. L. Bekish. Vitebsk : VSMU, 2003. 346 p. 2. Medical biology for international students 1st year : lecture course / V. E. Butvilovsky [et al.]. Minsk : BSMU, 2017. 141 p. 3. Медицинская биология для иностранных студентов = Medical biology for international students : учеб.-метод. пособие / В. Э. Бутвиловский [и др.]. Минск : БГМУ, 2016. 224 с. 4. Медицинская биология и общая генетика : терминологический словарь для иностранных студентов / В. Э. Бутвиловский [и др.]. Минск : БГМУ, 2007. 55 с. 5. Медицинская биология и общая генетика : тесты / В. Э. Бутвиловский [и др.]. Минск : БГМУ, 2006. 228 с. 6. Медицинская биология и общая генетика : сборник задач / В. Э. Бутвиловский [и др.]. 2-е изд. Минск : БГМУ, 2010. 264 с. 7. Медицинская биология и общая генетика : учеб. / Р. Г. Заяц [и др.]. 2-е изд., испр.: Минск : Выш. школа, 2012. 496 с. 8. Частная паразитология : учеб.-метод. пособие. / В. Э. Бутвиловский [и др.]. Минск : БГМУ, 2007. 107 с.

207 CONTENTS

Topic 1. Human in the system of nature. Methods of studying cells ...... 3 Topic 2. Biology of the cell. The flow of substance in the cell. The flow of energy in the cell ...... 8 Topic 3. Temporal organization of the cell ...... 16 Topic 4. Arrangement of hereditary material (I) ...... 25 Topic 5. Arrangement of hereditary material (II) ...... 30 Topic 6. Genetic engineering ...... 35 Topic 7. Interaction of genes. Genetic linkage and genetics of sex ...... 41 Topic 8. Variation ...... 47 Topic 9. Fundamentals of human genetics (I) ...... 52 Topic 10. Fundamentals of human genetics (II) ...... 59 Topic 11. Human genetic and chromosome disorders ...... 64 Topic 12. Genetic counselling ...... 67 Topic 13. Reproduction of organisms ...... 70 Topic 14. Fundamentals of ontogenesis ...... 74 Topic 15. Evolution of organ systems ...... 85 Topic 16. Introduction to parasitology ...... 96 Topic 17. Phylum Sarcomastigophora, classes Sarcodina, Zoomastigota ...... 102 Topic 18. Phylum Infusoria, class Ciliata. Ph. Apicomplexa, cl. Sporozoa ...... 112 Topic 19. Phylum Plathelminthes, class Trematoda ...... 120 Topic 20. Phylum Plathelminthes, class Cestoidea ...... 127 Topic 21. Phylum Nemathelminthes, class Nematoda ...... 136 Topic 22. Phylum Arthropoda, class Arachnida. Poisonous and venomous organisms ...... 145 Topic 23. Phylum Arthropoda, class Insecta ...... 154 Gap-filling tests ...... 165 Multichoice tests ...... 174 Answers to the gap-filling tests ...... 200 Answers to the multichoice tests ...... 204 Literature ...... 207

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