History of Protistology
Antony van Leeuwenhoek (1623-1723) 1674-1716 first reported protists, recorded in letters to the Royal Society of London.
Huygens 1678 ciliates observed
Buonami 1691 Colpoda and euglenoids
Harris 1696 Euglena
Joblot, 1718 treatise on microscopic organisms, disproved abiogenesis using infusions
Trembly 1744 division in ciliates
Hill, 1752 Paramecium named
Baker, 1953 found Noctiluca
Rösel von Rosenhof, 1755 Various protists
Wrisberg, 1764 coined the term "Infusoria"
Ellis 1769 trichocysts of Paramecium with Geranium juice
Eichhorn, 1783 Heliozoan Actinospherium
O.F. Müller 1773, 1786 Monographs on protists, ciliate drawings still valid
Lamark 1816 named Folliculina
Gruithusen early 1800's cyclosis
Goldfuss 1817, coined term Protozoa
d'Orbigny 1826 study of Foraminiferida
Ehrenberg 1828-1838 major works on protists, many names still valid
Dujardin 1835-1841 monograph on protists, coined Rhizpoda
Purkinje 1840 coined the term Protoplasm
Siebold 1845 distinct definition of "protozoa"
Müller 1858 Radiolaria
Perty 1852 Ciliata
Cohn 1853 Flagellata
Claparède and Lachmann 1858 Suctoria
Haeckel 1862 Heliozoa; 1866 Protista
Diesing 1865 Mastigophora
Stein monographs 1854, 1859 on Ciliata and Mastigophora
Leidy 1879 first comprehensive work on protists of North America "Freshwater Rhizopods of North America"
Stokes 1888 "The Freshwater Infusoria of the United States"
Fossil remains from the begining of the Cambrian 570 mya
Calculated divergence of Kinetoplastids: 1BYA
rRna Sequences data shows polyphyletic origin of protists
phyla or kingdoms?
36+ phyla proposed with 100, extant described species
Descriptive research centered in europe/freshwater: cosmopolitanism
Many habitats and geographic regions not explored
protists not included in ecosystem level trophic dynamics until recently
Corliss, J.O. 1975. Three centuries of protozoology: a brief tribute to its founding father, A. van Leewenhoek of Delft. J. Protozool. 22:3-7.
Corliss, J.O. 1978-79. A salute to fifty-four great microscopists of the past: a pictoral footnote to the history of protozoology. Parts I & II. Trans. Amer. Micros. Soc. 97:419-458; 98:26-58.
Corliss, J.O. 1979. The Ciliated Protozoa: Characterization, Classification, and Guide tot he Literature, 2nd ed. Pergamon Press, Oxford & New York.
Corliss, J.O. 1986. Progress in protistology during the first decade following reeemergence of the field as a respectable interdisciplanry area in modern biological research. Prog. Protistol. 1:11-63.
Corliss, J.O. 1986. The 200th aniversary of "O.F.M., 1786": a tribute to the first comprehensive taxonomic treatment of the protozoa. J. Protozool. 33:475-478.
Dobell, C. 1932. Antony van Leewenhoek and his "Little Animals". Swets & Zietlinger, Amsterdam.
Montagnes, D.J.S., Taylor, F.J.R., & Lynn, D.H. 1990. Strombidium inclinatum n. sp. and a reassessment of Strombidium sulcatum Claparede and Lachmann (Ciliophora). J. Protozool. 37:318-323.
Carl Zeiss (1816-1888)
Sir George Airy
Ernst Abbe (1840-1905)
Otto Schott (1851-1935)
August Köhler (1866-1948)
Lens grinding and microscope building was trial and error until Enrst Abbe figured out the physics of image formation by the lenses of the microscope
Airy and Abbe: diffraction theory of image formation:
Image formation not explained by geometric optics, only by wave theory of light. A spot will produce an image of a bright central disk (the Airy disk) and concentric rings of diffracted light. Resolution is detemined by the ability to separate airy disks, limited to 0.13 Ám for optical systems using white light.
Microscope objective images the light source and
changes in the light source image due to the specimen
Amplitude = Brightness
Wavelength = Color
Objects in microscope transparent, seen only if contrast background in intensity or color.
Object will deviate waves: change amplitude and shift phase
Numerical apeture of lenses = N.A. =i sine
i = refractive index of white light (wavelength 5890 A)
(Red=2.41; Violet=2.47; Dispersion = 0.06)
Refractive index different for different wavelengths, dispersion.
Resolution = R = N.A./wavelength
Thus resolution is dependant on the wavelength of light and the N.A. of the objective.
Limit of resolution by light microscope = 0.24 Ám, max N.A. of 1.4
Dark field: Only waves deviated by the oject pass through.
Phase contrast: Object retarts waves 0.25 wavelength,
Bringing background, object deviated waves back into phase.
Differential Interference Contrast: using a prism to separate colors of deviated and undeviated waves.
Confocal Microscopy: Block out all deviated and undeviated waves above and below the plane of focus.
Epifluorescence Microscopy: Mercury Bulbs, observing objects below the limit of resolution by making them light sources
Scanning Laser Microscopy: Krypton-Argon laser, point scanned excitation.
Transmission Electron Microscopy: utilizing a smaller wavelength, magnetic lenses, degree of electron retardation by object.
Scanning Electron Microscopy: Reflected secondary electrons, surface imaging
Useful Magnification Range
Anonymous. Basics of the Optical Microscope. Olympus Corporation, N.Y.
Gray, P. 1964. Handbook of Basic Microtechnique, 3rd ed. McGraw-Hill Book Co., N.Y.
Haugland, R.P. Molecular Probes (catalog). Handbook of fluorescent probes and research chemicals. Molecular Probes, Inc., Eugene, OR.
Hayat, M.A. 1981. Fixation for Electron Microscopy. Academic Press, N.Y.
Kemp, P.F., Sherr, B.F., Sherr, E.B., & Cole, J.J, eds. 1993. Handbook of Methods in Aquatic Microbial Ecology. Lewis Publishers, Boca Raton, FL.
Köhler Illumination Centenary, 1984. Reprints by the Royal Microscopical Society, Seacourt Press Ltd., Oxford. (Available from Zeiss, Inc.).
Möldner, K. 1983. Preparation Techniques for Electron Microscopy. Carl Zeiss, Oberkochen, West Germany.
Möllring, F.K. 1981. Microscopy from the Beginning. Carl Zeiss, Oberkochen, West Germany.
Murphy, J.A. & Roomans, G.M., eds. 1984. Preparation of Biological Specimens for Scanning Electron Microscopy. Scanning Electron Microscopy, Inc., O'Hare, IL.
Smith, R.F. 1987. A tribute: "The four horsemen of microscopy". Functional Photography Sept/Oct.
Cytological Approach: function from structure
Biochemical Approach: function from in vitro and in vivo reaction products
Think Small- Think Unicellular Organisms
Molecular solutions that parallel Cellular solutions of metazoans.
Locomotion, feeding, digestion, osmoregulation
Characters of Living Cells:
1) complexity of structure
2) Change energy from one form to another: oxidation-reduction
3)Response to stimuli: plasticity and irritability
4) Homeostasis: maintain an inherently unstable system at some level of stability
6) Capacity to Reproduce
"the development, maintenance, modification and evolution of form" (Lynn, 1981)
Major components of cells:
2-3% complex carbohydrates
1.1% nucleic acids
0.4% small organic molecules
1.5% inorganic ions
Protein and water: 10% solution gel or sol?
Some proteins crystalize at 20%, 10% should be viscous
Water in cell is not all free, bound up by proteins and molecules,
affects what compounds are disolved and where
Ground Substance of Cytoplasm: Porter & Tucker (1981, Sci. Amer.)
high voltage TEM: microtrabicular lattice: cytoplasm not a haphazard solution, Organelles held in place by a cytoskeletal system
Centrioles Observed in light microsocpy as bright "asters"
1960's electron microscopy revealed tubular nature
Highly conserved: Histones only proteins to have undergone less evolutionary change (Dales, 1972. J. Cell Biol. 52:748-753)
Nagleria gruberi has two immunologically distinct tubulins sets: flagella and basal bodies tubulin unique
2 globular proteins, 55 Kda each: tubulin alpha and beta dimers has greater electrophoretic mobility due to charge difference, guanine binding site locations differ
Microtubules will self assemble given GTP and both dimers
Each molecule binds with GTP, one GTP hydrolyzed
alpha + beta+ 2 GTP = alpha-beta-GDP + GTP
GTP exchanges with medium, GDP tightly bound
Both GTP and GDP remain associated with tubulin
Availability of Ca++ controls polymerization
Forms tubules with 13 protofilaments 5 nm diameter,
Tublules 24 2 nm in diameter, core 15 nm, subunits have a pitch of 10-25 degrees
50% of tubulin remains as monomers within the cell, equilibrium process, tubulin monomers recycled Cruder cell preps polymerize better: MAPS: microtubule associated proteins
Role in assembly of microtubules into structures, stability
Mammalian brain:37degC required for polymerization
Dissociation at 4deg C, flagella & cilia tubules stable
Euplotes from Antarctica (-1.8 to -2 C) has gene sequence differences in dimer binding site, very different than temperate species (Miceli et al., 1994)
MicroTubule Organization Centers (MTOC's)
(Pickett-Heaps, 1975. Ann. N.Y. Acad. Sci. 253:352-361)
Centrioles, Centrosomes:electron dense regions, bright "asters" under light microscope from eminating microtubules
Tubular ribbons side by side (one to one) linkages: basal bodies, oral structures
Irregular hexigons: three or three pairs of linkages
Six linkages: dense hexagonal packing, cytopharyngeal baskets of ciliate Nassula
Centrioles & Basal Bodies: nine triplet microtubules, 400 nm long,
0.15-0.25 Ám dia, One complete tubule, two10 subunit tubules attached
Tetrahymena basal bodies have a single strand of RNA by AO, RNAse
Role in mitosis
Microfilaments 4-5 nm thick, cell shape, attached to membranes.
Singer and Nicholson 1972 Fluid Mosaic Model
Lipid bilayer with traversing proteins (unit membrane 80 A)
Glycocalyx: glycoproteins, carbohydrates, sensory, solute transport functions
Golgi (dictyosome in plants): smooth membranes stains with Osmium & silver salt, acid phosphatase concentrated , processing of ER produced proteins into glycoproteins; glycocalyx, extrusomes, scales, lysomes may originate
ER: less lipid than golgi membrane, attached ribosomes make secreted proteins, may be continuous with nuclear and mitochodrial membranes
Nuclear Membrane + one layer of ER = Nuclear envelope
Chloroplasts: Pigments, DNA, membrane layers
Mitochondria: laminar, tublular, pingpong paddle cristae, DNA
Lysosomes: hydrolytic (36 different) and acid phosphatase enzymes, single unit membrane; come from ER and golgi
Microbodies: Glyoxysomes & Peroxisomes
Glyoxysomes: Plant cells, conversion of lipid to carbohydrate, production of hydrogen peroxide: has catylase Glyoxylate Cycle
Peroxisomes: amino acid oxidases, production and removal of hydrogen peroxide.
Levels of organization (Lynn 1981): Structural Conservatism
Level of Organization
G1: diploid vegetative
S: centriole replication at begining, DNA replication throughout
Porter, K.R. & Tucker, J.B. 1981. The ground substance of the living cell. Scientific American.
Lynn, D.H. 1981. The organization and evolution of microtubular organelles in ciliated protozoa. Biol. Rev. 56:243-292.
Giese, A.C. 1979. Cell Physiology. Saunders College Publishing, Philadelphia, PA.
Dustin, P. 1978. Microtubules. Springer-Verlag. Berlin.
Dales, S. 1972. Concerning the universality of a microtubule antigen in animal cells. J. Cell Biol. 52:748-753.
Pickett-Heaps, J.D. 1969. The evolution of the mitotic apparatus: an attempt at comparative cytology in dividing plant cells. Cytobios 1:257-280
Avery, S.V., D. Lloyd, and J.L. Harwood. 1994. Changes in membrane fatty acid composition and D12-desaturase activity during growth of Acanthamoeba castellanii in batch culture. J. Euk. Microbiol. 41: 396-
Jacobson, K., E.D. Sheets, and R. Simson. 1995. Revisiting the fluid mosaic model of membranes. Science 268: 1441-1442.
Ramesha, C.S. and G.A. Jr. Thompson. 1983. Cold stress induces in situ phospholipid molecular species changes in cell surface membranes. Biochimica et Biophysica Acta 731: 251-260.
Ramesha, C.S. and G.A. Jr. Thompson. 1984. The mechanism of membrane response to chilling. J. Biol. Chem. 259: 8706-8712
In the protists "one finds the extreme adaptations of cell structure and organization, and by the study of some of the more bizarre forms we may gain some insight into the possibilities and also the limits of eukaryotic cell organization and specialization" Berger & Taylor, 1981.
One theory suggests there was a time when RNA served both genetic and catalytic functions (Joyce, 1991). Catalytic functions of RNA known, information content inherent.
DNA-protein life thought to have begun 4 bya
Between 4.2 and 4.0 bya environmental conditions became favorable for evolution of life, chiefly due to the fall off in level of meteor impacts
Organic soup formation is well understood, organic complexes and amino acids detected in meteorites
Jump from organic soup to self replicating life remains a mystery.
Fossils reveal life on earth 3.5 to 3.6 bya: stromatolites and microfossils
Stromatolites:phototrophic photoautotrophic, mucus sheaths = cyanobacteria 3,556 +/- 0.032 bya
First Eukaryote fossils in rock
possible at 2.1 bya
more probable at 1.5 bya
for certain at 1.2 bya
600 mya invertebrate-vertebrate split
530 mya Cambrian Explosion
400 mya Fish
300 mya Birds
Inheretance of structure above the level of molecules independant of genetics
ciliate asexual reproduction: sex vs. reproduction segmentation of growing cylinders
Omins forma ex DNA: all form from DNA
Two different perspectives:
1) continuity of structural oganization: inheritance of phenotypic differences
2) indispensibility of preexisting structure for genesis of new structure:
physical rules that constrain and determine biological organization
Differences in number of ciliary rows is inherited in clones
Clones from a mixed population of 8s and 9s do not develop mixtures of 8s and 9s unitil many generations later
Figure 4.1: stability ranges for phenotypic variation: structural inheritance bounded by genetics:
Variability within stable region inherited for long periods,
extremes tend to fall back into stability region
Autonomy of ciliary rows:
inverted ciliary rows inherited for 800-1500 fissions
structure "...determined by the molecular geography within the unit territory and not by any other outside influence, either molecular or cellular"
Monophyletic: Single origin within a phyla
Paraphyletic: Single origin and not all descendants
Polyphyletic: Multiple origins within a phyla
Neutral mutations in sequence analysis
Protists are not "relicts" of the evolutionary process from prokaryotes to metazoa and metaphyta, they are evolutionary endproducts in their own right with comon ancestry with metazoa
Problems with protists:
1) extreme diversity
2) tremedous convergence of characters
3) symbiotic events
4) they have left very little fossil records
5) biochemcial data is lacking for many groups
Naegleria-like cysts in Cretaceous amber (Waggoner, 1993)
Lack of structural features and fossil record prevented evolutionary analysis of prokaryotes and hampered analysis of protists
Fossil record for those forms that create tests and loricae
Oceanic oozes >30% biogenic particles CaCO3 and SiO2,
mm's /year, kms deep
Two ways to examine phyologeny of Protists:
Structural/Morphologic: Lynn (1981) Structural conservatism: "The conservation of structure through time is inversely related to the level of biological organization."
Biochemical/Molecular: rRNA, other genes, conserved proteins (enzymes)
Sequence homologies of conserved genes and proteins compared
Theories of eukaryotic origin:
archebacterial theory: based on 16s rRNA (Woese, 1987)
Methanogens, Halobacteria, Thermoacidophiles:
no muramic acid walls
actin and myosin-like molecules
DNA associated with a histone-like basic protein
Prolific symbionts in protists
Shared 11 amino acid sequence in highly conserved EF-1 alpha
Involved in protein synthesis (Hoffman, 1992; Rivera & Lake, 1992) other prokaryotes have a four amino acid sequence in this region EF-tu
Autogenous vs Serial Endosymbiosis
Specialization and development of cellular compartments: membrane invaginations, mesosomes
Serial Endosymbiosis Theory (Taylor, 1974)
Lynn Sagin /Margulis
Origins with Portier (1918), Wallin (1927), Schanderl (1948)
co-metabolism, evolving atmosphere - O2 toxicity
requires translocation of genes from symbiont to host.
Predatory bacteria (Guerrero et al., 1986):
epibiotic attachment: Vampirococcus on Chromatium
penetration to periplasmic space: Bdellovibrio
Penetration of the cytoplasm: Daptobacter
Chloroplasts and mitochondria (Gray 1988) have:
prokaryotic -like DNA: more closely related to prokaryotes than eukayotes
sensitive to antibiotics
Arise only from themselves: If cured Euglena plastids do not spontaneously reform
Symbioses ( in the broadest sense) abound: Legionella, Chlorella
cyanobacteria (a only), prochloron (a & b chlorophyl), no brown analog
Plastids with two memebranes: prokaryotic endosymbionts
plastids bearing more that two memebranes: endosymbionts were Eukaryotic
three membranes: euglenoids & dinoflagellates: Dinos with Peridinin have unique form of RiBisCO
previously only found in some Proteobacteria encoded in dino nuclear DNA: unique evolutionary history
four membranes: chlorophyll c containing organisms other than dinoflagellates
Cryptomonad with "nucleomorph": double membrane bound with RNA & DNA (Douglas et al, 1991; Penny & O'Kelly, 1991)
nucleomorph, chloroplast and cytoplasm, eukaryotic ribosomes enclosed by chloroplast ER
when first described 25 years ago recognized that it might be endosymbiontic eukaryote
Nuclear rRNA close to fungi, Acanthamoeba, and green plants
Nucleomorph rRNA associated with red algae.
Ribulose biphosphate carboxylase enzyme DNA agrees with this placement
Crytpto chloroplast and red algae chloroplasts in a different clade than cyanobacteria, cyanelles, & green plant chloroplasts
Blue-Green dinoflagllate Gymnodinium acidotum contains a cryptomonad.
Ciliate Mesodinium rubrum has cryptomond (Wilcox & Wedemayer, 1985)
both symbionts lack periplast, ejectosomes, & flagella
Transformation stages of symbionts to chloroplasts
Amphidinium wigrense contains blue-green chloroplasts: three membranes, lack of any other non-dinoflagellate organelles, but chloroplast structure is typical of cryptophytes
further degeneration of symbiont
Dinos Peridinium balticum, Kryptoperidinium foliaceum contain Chrysophyceans
Mitochondria: Paracoccus, Rhodopseudomonas like bacteria
many cases of obligate bacterial symbioses in mitochondria free protists
Protist groups separated very early, with the most deeply divergent organisms belonging to orders of flagellates = ancient origin for 9+2 organelle, fungi, plants that lack flagella or centrioles are derived and developed other types of mtoc for mitotic spindle formation
Ciliates diverge late with Dinos, close to metazoan-metaphyte radiation
nuclear peculiarites probably derived: in ciliates nuclear dualism and two of the stop codons that are elsewhere universal code for glutamine, dinos lack histones.
Protists represent lineages from the transitions from prokaryote to eukaryote and unicellualr to multicellular organisms
as much or more distance between some flagellates and ciliates as between ciliates and metazoa
Trichomonas, a quadraflagellated mitochondria-less flagellate, diverges very early, suggesting a long period of eukaryotic life prior to the aquisition of mitochondria
Hydrogenosomes derived from mitochondria
Mitochnodrial genes found in the nuclues-gene transfer
16s rRNA (Hinkle & Sogin, 1993)
Radiation in eukaryotic lineages: rougly co-occurring divergences of plant, animal, Straemopiles and aveolate (dinoflagellate, ciliate, apicomplexan) Crown Eukaryotes
Incomplete understanding of the 16s-like rRNA clock speed makes dating difficult, but estimates of one billion years ago (Sogin, 1991) for eukaryotic radiation.
First eukaryotes were likely flagellates: all organisms at base of tree are flagellates, amoeboid forms convergent, arose numerous times Hinkle & Sogin (1993).
Classifications of Protists
Natural groupings based on presumed evolution:
Cladagrams common ancestry
Artificial but convienient
Natural Ecological functional
Sapropic system in Europe
Haeckel, 1866 three kingdoms:Plantae, Protista, Animalia
Whittaker: 5 kingdoms: Monera, Protista, Fungi, Plantae, Animalia
Levine et al., 1980 Subkingdom Protozoa in kingdom Protista
seven Phyla: Sacromastigophora
Empires: Bacteria, Eukaryota
Eukaryota Kingdoms: Plantae, Animalia, Fungi, Chromista, Protozoa, Archezoa;
same six kingdoms, 34 Phyla, 83 classes
eliminates many inter-level classfications
Baroin, A. et al., 1988. Patial phylogeny of the unicellular eukaryotes based on rapid sequencing of a portion of 28s ribosomal RNA. PNAS 85:3474-3478.
Britten, R.J. 1986. Rates of DNA sequence evolution differ between taxonomic groups. Science 231:1393-1398
Douglas et al., 1991. Crytomonad algae are evolutionary chimeras of two phylogentically distinct unicellular eukaryotes. Nature 350:148-151.
Gray, M.W. 1988. Organelle origins and ribosomal RNA. Biochem. Cell Biol. 66:325-348.
Guerrero, R. et al. 1986. Predatory prokaryotes: predation and primary consumption evolved in bacteria. PNAS 83:2138-2142.
Gutell, R.R. et al., 1994. Lessons from an evolving rRNA: 16s and 23s rRNA structures from a comparative perspective. Microbiol. Reviews 58:10-26.
Hinkle, G. & Sogin, M.L. 1993. The evolution of the Vahlkampfiidae as deduced from 16s-like ribosomal RNA anlaysis. J. Euk. Microbiol. 40:599-603.
Hoffman, M. 1992. Reserchers find an organism they can really relate to. Science 257:32.
Joyce, G.F. 1991. The rise and fall of the RNA world. The New Biologist 3:399-407.
Margulis, L. 1981. Symbiosis in cell evolution. W.H. Freeman & Co., San Francisco.
Penny, D. & O'Kelly, C.J. 1991. Seeds of a universal tree. Nature 350:106-107
Rivera, M.C. & Lake, J.A. 1992. Evidence that eukaryotes and eocyte prokaryotes are immediate relatives. Science 257:74-76.
Waggoner, B.M. 1993. Naeglaria-like cysts in cretaceous amber from central Kansas. L. Euk. Biol. 40:97-100.
Wilcox, L.W. & Wedemayer, G.J. 1985. Dinoflagellate with blue-green chloroplasts derived from an endosymbiotic eukaryote. Science 227:192-194.
Woese, C.R. 1994. There must be a prokaryote somewhere: Microbiology's search for itself. Microbiol. Reviews 58:1-9.
Kingdom Archezoa (maybe should be in Kingdom Protozoa)
Widespread anaerobic/aerotolerant free living in organic rich habitats
Lack of golgi, mitochondria, peroxisomes
Uniflagellate amoebae ( Phylum Achamoebae)
Pelobiontea: single flagellum associated with nucleus cap of microtubules from base of basal bodies and covers part of nucleus; thin connections to nuc membrane flagella poorly motile
Pelomyxa, Mastigna, Mastigamoebae, Phreatamoebae, Mastigella
Tetraflagellate non-amoeboid (Phylum Metamonada) well developed cytopharynx and cytostome
Trepomonadea, Retortamonadea, Oxymonadea
Trepomonadea: Diplomonadida, Enteromonadida
Doublet organisms: 2 nucleii and 2 flagellar complexes, two cytostomes
occurs as single and doublet organism insertion on nuc. mem.
4 flagella per mastigont system, 3 anterior, one recurrent
Subnuclear Fiber: anterior to nucleus
Infranuclear Fiber: crosses under nucleus and descend along trailing flagellum of opposite side
Direct Cytostomal Fiber: descends posteriorly, surrounds cytopharynx
reduction or elimination of cytostome in more "advanced" forms
trailing flagella axonemes in these forms pass through cytoplasm to anterior
specialized adhesive organelles
mitosis semi-open with intranuclear spindle
nuclei divide synchonously
Free-living genera: Trepomonas, Hexamita
lateral insertion of mastigont systems
Cresant shaped nuclei
Grooved cytostomal pockets
one free flagellum, three descend through cytostomal groove
Supranuclear splays under plasma membrane
2 pairs basal bodies connected by striated fiber, basal bodies at right angles
heterodynamic flagella, one or 3 anterior, one trailing descending through a
Supranuclear fiber anterior to nucleus
Direct Cytostomal fiber posteriorly surrounds cytopharynx
Lacks infranuclear fiber
tube-like cytostome channel to the outside
cytostopharynx extends posteriorly to form funnel shaped depression, lips
Trailing flagellum has flattened vane like ridges.
cytostome and cytopharynx are stengthened by microtubules
No golgi, few membrane organelles except food vacuoles
mitotic spindle is intranuclear, nuclear membrane remains intact.
resistant cysts for transmission
Two free living genera:
Retortamonas: two flagella, salt marsh, cytostopharyns groove/pockets
Chilomastix: four flagella
may be related to retorts
2 pairs of basal bodies connected by thick fiber
ribbon of microtubules arises from this fiber and descends to posterior of cell joined by other ribbons to form a compound structure: the axostyle
Axostyle may bend actively, contractile, main form of locomotion
Anterior of cell supported by sheet of microtubules: pelta arises from one basal body
no golgi, no cytostome
posterior site of phagocytosis, pinocytosis known
mitosis closed, intranuclear spindle
Not resolved whether branch is before or after Euglenozoa
Trichomonadea: difference between two species as great as vertebrate classes
endozoic, have parabasal bodies: golgi + striated fiber
cytoskeleton of microtubules and striated fibers, parabasal fibers, costa, attractophores,
parabasal fibers: arise from anterior most basal body, descend either side of the nucleus, support two golgi
Costa: underlies and supports undulating membrane, thick striated fiber
Attractophores (rhizoplasts) extend from a basal body to the nuclear envelope
lack mitochondria, contain basilliform hydrogenosomes: two membranes, contain DNA
lack cytostome, posterior phagocytosis and pinocytosis
closed mitosis with external spindle, kinetochores inserted in nuclear membrane
Ditrichomonas hornigbergii: free living anaerobe trichomonad. Suggests features are not specializations of parasitic/commensal life Farmer (1993)
Pseudotrichomonas: free living
kinetoplast:polymerized AT rich DNA in single mitochondrion.
20% of nuclear DNA, largest chunk of extranuclear DNA known
polymerized small circles and some larger circles more typical of mitochondrial DNA
single chunk near basal bodies or several dispersed
divides before nucleus
1 or 2 flagella from a pocket or pit, paraflagellar rod
single mitochondrion extends length of cell, basal bodies associated with mitochondrion not nuclear membrane
microtubule supported cytopharynx
singular nucleus, prominent nucleolus, persists through mitosis
golgi in region of flagelar pocket but not attached
CV if present empties into flagellar pocket
rRNA suggest close relatives of Euglenoidea:
heterodynamic flagellaw/ paraxial rods
flagellar pocket w/cv
microtubule supported pharynx
Orders Bodonida & Trypanosomatida
small 3-15 um bacterivores and plant pathogens
oval or elongate with two heterodynamic flagella arise from an anterior flagellar pocket, no expanded flagellar pocket reservoir
flagella smooth, or with non-tubular mastigonemes, sometimes in tufts
paracrystalline paraflagellar rod in each flagellum adjacent to axoneme
Longitudinal microtubules under plasma membrane
Some forms have a well developed cytostome opening near anterior of cell
supporting microtubules originating adjacent to basal bodies
contractile vacuole empties into flagellar pocket
single mitochondria with kinetoplast and ping-pong paddle cristae
mitosis closed with internal spindle, binary longitudinal fission
parasites of blood and tissues of plant and animals.
single flagellum, two basal bodies
epimastigote, trypanomastigote stages free swimming
flagellum connected to cell body with undulated membrane
amastigote flagellum reduced and non-emergent
nutrition aquired by phagoccytosis at base of expanded flagellar pocket
transmission by sucking insects, complex life histories
African sleeping sickness, Chaga's disease, Leishmaniasis
Phylum Choanozoa: class Choanoflagellatea
single anterior flagellum, collar of slender cytoplasmic tentacles form a collar or basket
no mastigonemes, may have vane like projections
cytoskeleton of subsurface microtubules extend from basal bodies
no rhizoplast or stirated fibers
flattened cristae possible affinity to chrysomonads
secreted stalks common, loricae of organic or thin silica rods
Cortico-Flagellates: Katablepharis, Colponema
Ciliate like flagellates
tubular cristae, cortical aveoli, rigid tubular mastigonemes
endozooic flagellates in amphibians
flattened, taper posterior
multiple flagellar apparati arranged in kineties, some short, do not extend length of cell
two striated fibers directed to the left of basal bodies (kd to the right in ciliates)
mts only arise from the basal bodies of the falx, extend between kineties in cytoplasmic ridges
pinocytosis between ridges
numerous diploid nucleii
cell divides longitudinally
thought to be remant of primitve ciliate ancestor
small <20 um resemble karyorelecteans
free living, marine sediments, bacteria, diatoms, flagellates
homokaryotic, multinucleate, longitudinal division in cysts,
each product binucleate
Infraciliature unique, resembles some flagellates
no aveoli, pc or t mts, kds, parasomal sacs
Brugerolle, G. 1993. Evolution and diversty of amitochondrial zooflagellates. J. Euk. Microbiol. 40:616-618.
Dodge, J.D. 1979. The phytoflagellates: fine structure and phylogeny. In: Levandowsky, M. & Hutner, S.H. 1979. Biochemistry and Physiology of Protozoa. Vol 1:7-58
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"lacking useful phenotypic characters for comparative analysis, amoebae are a classic example of a highly paraphyletic group of protists tradditionally united in taxonomic schemes universally recognized as artificial."
Hinkle & Sogin, 1993
Growing sentiment that taxon Sarcodina is not valid
Formation of pseudopods shared with leukocytes, dinoflagellates,
many cellular forms
Granuloreticulose: anastomosing networks
Actionopods: rigidly cross linked microtubules
ectoplasm, hyaloplasm: clear,
in actinopoda, reticular network surrounding cell
Limax (tongue shaped), mono or polypodial, finger shaped etc
Most amoebae small 5-50 microns with one or few lobopodia or filopodia
Most information from Amoeba proteus and Chaos carolinenesis
three types of nuclei:
1) Spherical to discoidal, sometimes dented or folded, nucleoli (endosomes) scattered throughout or near the nuclear membrane: Amoeba proteus
2) Spherical with central nucleolus: Nagleria, Entamoeba
3) Spherical with one or more eccentric nucleoli: Polychaos, Iodamoeba
Presence of nucleus required for locomotion: enucleate half cells round up, move again after reintroduction of nucleus.
Amoebae proteus nucleus: trilaminar porous membrane with honeycomb support matrix internal
nuclei can be removed and inserted into other amoebae with high rate of survival
Surface membrane covered with plasmalemma: glycocalyx integrated with membrane, fibrous protrusions, scales, spines, frothy vesicles
Mitochondria have tubular cristae
Plasmalemma (sarcolemma): protection, adhesion, sensory reception, chemical detection, alterations of chemical balances
Filaments: anionic, sensitive to positively charged substances: blockage with nuetral red or alcian blue prevents pinocytosis, proteases destroy ability of Chaos to recognize prey
filaments may be piezo-electric: mechnoreceptors
Some crystals (unknown) and lipids storage
Triuet crystals (urea) accumulate unitl divsion-presumable used as nitrogen source for nucleic acid production
Contractile vacuole in freshwater amoebae, rarely in marine
Golgi site of plasmellemma formation, scale and test plates
Testate amoebae (both filo and lobopodia)
1) proteinaceous, 2) agglutinate, 3) silicious secreted, 4) calcareous secreted
older shells accumulate minerals and become darker
Some amoebae (Euglypha & Paulinella ovalis make scales, store them near nucelus until cell divsion, scales donated entirely to daughter cell, adults do not replace lost scales Ions that inhibit silica deposition (germanium) block scale formation. Selective ingestion of siliceous particles also donated to daughter cell. If daughter separated from parent after transfer of nucleus and scales, assembly of test will proceed: genetic mechanisms seem to determine shell formation: used as a taxonmic criterion
Cytoskeleton: actin fibers network. Attached to the inner lamina of cell membrane
myosin links to actin Ca++ dependant contraction
Cysts may be mucilagenous, cellulosic, proteinaceaous or all three.
Giant amoebae Chaos carolinense emits an anesthetizing substance for prey ciliates
some amoebae secret enzymes to extracellularly digest bacteria
1) metamitosis-nuclear membrane disintegrates, amphiastral spindle forms, membrane reconstructed during telophase
2) mesomitosis- nuclear membrane persists through part of mitosis, spindle has neither astral poles or polar caps
3) promitosis- dome shaped half cups of divided endosome form poles of spindle, envelope persists
Immune response: nuclear transplants from an amoeba that has not recovered from a cytoplasmic bacterial infection to an enucleated amoeba that has developed resitance cannot prevent reinfection.
Pelamyxa palustris (vanBruggen et al., 1988): Archezoa: primitively lack mitochondria, golgi, mitotic spindle, mtoc, any 9+2 arrangement.
Methanogenic symbionts (and others).
Entamoebae: lacking mitochondria, peroxisomes, hydrogenosomes.
Small if any golgi, parasitic, some glycogen storage
Slime Molds: thought to be closely related to soil amoebae, aggregation and fruiting body stages with spores (Hartmanella + Protostelids)
Protostelids:amoeboflagellates; Acrasids: lobose, eruptive pseudopods
Myxogastrea: acellular slime molds, Physarium
Dictyostelea: cellular slime molds: Dictyostelium
Nagleria: amoeboflagellates, cells change from amoeboid form with no obvious polarity to elongate flagellated form by de novo synthesis of basal bodies
Exclusively marine Most benthic, some planktonicgranulorecticulopods
1)vegetative or somatic, irregular, eliptical, amoeboid protrusions, many peripheral nucleoli, fibrous inner surface to membrane, many pores
2) generative smaller, fewer pores, few central nucleolei
microtubules in pseudopods, branching tubules into reticular networks
cross bridges react with antibiodies to atpase dynein molecules of cilia
Goltz & Hauser, 1994
Shell is "external" but covered by cytoplasm
fibrous plasmellema. May include bubble vesicles for floatation, sticky fibrous coat on reticulopods
Peroxisomes and lysosomes, mitochondria occur in the reticulopods.
Each filopd retains its identity in bidirectional cytoplasm (granular, rheoplasm) motion despite fusion in bundles of reticular networks (anastomoses): constant motion of the cytoplasm through out the reticulonetwork and cell body in shell
each filopod contains coiled core of microtubultes and microfilaments.
microtubules and bidirectional flow establish tension in reticulopods
Shell distinctive most calcium carbonate , some agglutinated
Digestion initiated along reticular network, with fusion of lysosomes (gastriole), completed in cell body
prey opn unicellualars or meiofauna
Life cycle: 1) Agamont: does not produce gametes, develops from zygote
2) Gamont: develops from agamont, meiotic divsions, gametes are amoeboid or flagellated, shellless
Actinopods axiopodia stiff with rigid cross-linked microtubules
also possess reticulopods
few with mineral skeletons, few with ecotplasm, mostly freshwater
nucleus can be deformed by axopods
Nucleus has fibrous layer on outside of membrane, nucleoli around periphery, some multinucleate,
axoneme contains two whorls of microtubules of variable length, tapers microtubules oringinate at the surface of the nucleus. Cell memebranes extends out and along each axopod with extrusomes and mitochondria
surface covered by sticky mucus
some cysts known
axopods range from few microtubules to extremely complex from MTOC's, give radiate shape.
MTOC's can be
1) plaques on nucleus surface,
2) plaques on an interior dense layer of cytoplasm,
3) single central centroplast, ,
axopods can provide locomotory function
bidirectional flow of cytoplasms along axonemes, saltatory movement controlled by localized changes in the ion balance across the cytolasmic membrane
may "row" or "walk" on axonemes
trap prey by adhesion to axopods, retratction draws prey to cell body, large lobopod extends from cell body to form gastriole
binary fission, some swarmers, cell fusion known
nuclei with central endosome, nucleolus has eccentric clear globule
cytoplasm clearly delineated from outer vesicular cytopplasm by organic membranous boundary caled central capsule, enclosed by complex "endoskeleton" of strontium sulfate (Acantharea) or opaline silicates (Polycystinea, solid spines & Phaeodarea, hollow spines, organics)
central capsule surrounds nucleus and axoplast, pores through capsule connect ecto and endoplasm. axoplast (MTOC) near nucleus, microtubules radiate out through fusules into axopods ectoplasm organized into a mass of reticulopods.
mucus or gelatinous coat
some cysts known
cytoskeletons simple to elaborate
in Acantharea, spines anchored in an ectoplasmic cortex, myophrisks (cups) elastic and contractile
cytoplasmic movement continual bidirectional along axopods and throughout ectoplasm
Many with algal symbionts
digestion similar to forams with gastriole forming on reticulopods
multiple mitotic fission with biflagellate swarmers in acantaria
polycystinia swarmers from endomitoses and and fragmentation of the nucleus, swarmers may have crystal of strontium sulfate, some binary fission
Phasodaria binary or multiple fission
Hinkle, G. & Sogin, M.L. 1993. J. Euk. Microbiol. 40:599-603.
Ability to transform from amoebae to flagellates, One mode usually predominates: Nagleria mostly amoeba;
Tetramitus mostly flagellate
eruptive motility, closed mitosis with dividing nucleolus, cyst
Name from genus Vahlkampfia (only member not to form flagellates)
Only other amoeba-flagellate is myxomycota (plasmodial-acellular
By 16s rRNA, Monophyletic group that branches early
Nagleria splits early
Acanthamoeba diverge within the "grand radiation of eukaryotes"
Entamoeba emerges much later, related, but not significantly clustered
Patterson, 1983. Nuclearia moebiusi J. Proto 30:301-307
Filose pseudopodia without mts, homogeneous cytoplasm in Pseudopods
Mitochondria with flattened cristae
Lacks extrusomes, MTOC's, cytoplasmic mts
mts inside nucleus only during mitosis
reorganization of nuc envelope late in division
Pattern of cellular organization unique from other amoebae
ill-defined genus due to small number of characters
Classification by pseudopod type: Class Filosea, includes testaceans
naked filosea in two families Vampyrellidae and Nucleariidae based on feeding behavior
Only other group with flattened cristae is centrohelid heliozoa
Nuclearia lacks extrusomes, cytoplasmic mts, mtoc and surface structures of that group
Caan, 1986. Nuclearia delicatula J. Proto 33:392-396.
ingestion of Phormidium, pentration of Spirogyra,
but not at cross wall junction
finely pointed and filose pseudopodia can branch but do not anastomose
fine pointed pseudopods in diretion of motion , hyaline zone of cytoplasm
filopods not from hyaline cytoplasm
mesomitotic nuclear division, intact nuclear membrane
contractile vacuole pattern unique, small spherical vesicles enlarge around CV prior to CV filling.
Chavez et al, 1986. Phreatamoeba balamuthi n.g., n.sp. J. Proto. 33:397-404
Anaerobic or microaerophilic amoeboflagellate but different from others by eruptive limax locomotion, polymorphism, multinucleate
flattened locomotive form with broad anterior hyaline zone like mayorellid Paramoebidae, Conodina, but flagellate and multinucleate
Multinucleate forms in Pelobiontida
other amoeboflagellates in Schizopyrenida which locomote by hemispherical hyaline eruptions, nucleolus and nuclear membrane persist during division.
P.balamuthi nucleoulus breaks down
Flagellate stage lacks rostrum, collar, and cytostome of other amoeboflagellates
Like Naeglaria gruberi and Adelphamoeba cannot reproduce as flagellate
Cone like microtubular structure associated with flagellar basal bodies suggests an affinity to the Eumycetozoa: intermeadiate between "true" amoebae
van Bruggen et al., 1988 Methanogens in Pelamyxa palustris. J. Proto 35:20-23.
P. palustris lacks golgi, mitochondria, mitotic apparatus, mtoc, 9+2
Patterson 1985. J. Protozool. 32:241-246. Pompholyxophrys punicea
Spherical bodied amoebae with fine radiating pseudopods considered a heliozoan
Layer of siliceous spheres "pearles"
rare organism not culturable
no extrusomes, cytoplasmic microtubules, or microtubular axonemes as found in heliozoa
acentric nucleus, mitochondria, nuclear dictyosomes superficial layer of homogeneous cytoplasm continuous with filopods
feeding by ingestion
Shares these characters with the Nucleariid amoebae
Golz & Hauser, 1994.
reticulopod networks contain highly ordered regions of microtubules and less structured arrays.
Structural MAPs occur in the loosely arranged tubule regions
Ordered arrays connected by ciliary dynein-like proteins
reacts with ciliary dynein ATPase antibody-motor proteins for microtubules
Anderson, O.R. 1983. Radiolaria. Springer-Verlag, Berlin. 355 pp.
Anderson, O.R. 1996. The physiological ecology of planktonic sarcodines with applications to paleoecology: patterns in space and time. J. Euk. Microbiol. 43:261-274.
Caan, 1986. Nuclearia delicatula J. Proto 33:392-396.
Caron, D.A., A.F. Michaels, N.R. Swanberg and F.A. Howse. 1995. Primary productivity of symbiont-bearing planktonic sarcodines (Acantharia, Radiolaria, Foraminifera) in surface waters near Bermuda. J. Plankton Res. 17: 103-129.
Chavez et al, 1986. Phreatamoeba balamuthi n.g., n.sp. J. Proto. 33:397-404
Espanosa-Cantellano, M. et al., 1998. Entamoeba dispar: ultrastructure, surface properties and cytopathic effect. J. Euk. Microbiol. 45:265-272.
Goltz, R. & Hauser, M. 1994. Europ. J. Protistol. 30:221-226.
Hülsman, N. 1993. Lateromyxa gallica N.G., N.Sp. (Vampyrellidae): a filopodial amoeboid protist with a novel life cycle and conspicuous ultrastructural characters. J. Euk. Microbiol. 40:141-149.
Patterson, 1983. Nuclearia moebiusi J. Protozool. 30:301-307
Patterson 1985. J. Protozool. 32:241-246. Pompholyxophrys punicea
Röpstorf et al., 1994. Comparative fine structureal investigations of interphase and mitotic nuclei of Vampyrellid filose amoebae J. Euk. Microbiol. 41:18-30.
Sawyer, T.K. et al. 1998. Learamoeba waccamawensis, N.G., N.Sp. (Heterologosea: Vahlkampfiidae), a new temperature=tolerant cyst-forming soil amoeba. J. Euk. Microbiol. 45:260-264.
Stothard, D.R., et al., 1998. The evolutionary history of the Genus Acanthamoeba and the identification of eight new 18S rRNA sequence types. J. Euk. Microbiol. 45:45-54.
van Bruggen et al., 1988 Methanogens in Pelamyxa palustris. J. Proto 35:20-23.
Weekers, P.H.H. et al., 1993. Effects of grazing by the free-living soil amoebae Acanthamoeba castellanii, Acanthamoeba polyphaga, and Hartmanella vermiformis on various bacteria. Appl. Environ. Microbiol. 59:2317-2319.
Yamaoka, I et al., 1984. Scale formation in an amoeba, Cochliopodium sp. J. Protozool. 31:267-272.
"Indeed, amoung the protists, ciliates have taken subcellular specialization to its limit."
Lynn & Corliss, (1991)
Most complex cellular archetecture of all known eukaryotes
Archetecture of ciliature and infraciliature, oral apparatus
used to distinguish groups:
tendancy for decreased polymerization:
less but more specialized oral structures
historically, patterns of ciliation on somatic and oral surfaces used.
Cell division complex: most binary but many variations
dikaryotic: macronucluei, micronuclei
amicronucleate forms known
endomitosis, intact membrane, internal microtubules in some
external in others: heterotrichs and Karyorelecteans
macs and mics divide, except Karyorelecteans,
in which macs can only come from mics
Hammerschmidt et al., 1996: Karyorelectians not relects
Karyorelects and heterotrichs sister group with all other cilia
Two subphyla proposed:
Postciliodesmatophora Gerrassimova and Seravin 1976:
Mac division evolved with external mac spindle
Intramacronucleata subphylum nov.: internal mac spindle
Mac divsion evolved with internal spindle
most phagotrophic: cytostome-cytopharynx, oral kinetids, cilia, cytoproct-cytopyge
contractile vacuole-nephridial plasm
extrusomes: mucocysts, toxicysts, fibrous trichocysts
divsion binary or multiple, some complex in suctoria and chonotrichs
somatic: locomotion, attachment, protection, sensing
oral: capture and ingestion of nutrients
infraciliature:basal bodies (kinetosomes) and associated fibrils
Kinetid: Kinetodesmal fiber KD, Transverse and Postciliary microtubular ribbons
mts and fibers link adjacent kinetids into kineties
somatic dikinetids with anterior or both ciliated
well developed overlapping pc ribbons
conspicuous right or left handedness to oral structures
Oral polykinetids traverse anterior and extend into an oral cavity.
Fused somatic cilia in some = cirri
some loricate, sessile forms
left serial polykinetids may encircle anterior end
external microtubles in mac mitosis in some
may be primitve branch from ciliate radiation
some loricate forms Folliculina, Stentor
Class Amophorida (position uncertain)
Anaerobic Metopidae, Caenomorphidae
Formerly thought to be "primitive" ciliates.
long, vermiform, thigmotactic, many with one barren surface
extremely contractile, marine interstitial, one genus freahwater
pc ribbons overlap
macs contain aprox 2x mic DNA but composition unknown
Macs do not divide
Generally lack corical aveloi
Protocruzia single large chromosome condenses in mac,
may be all chromosomes end to end,
cytophoran oral structure, kinetid and karyo type
Often found in marine anerobic, microaerophilic and sufurous conditions
Kentrophorous, Tracheloraphis: only known pseudopodial feeding
Loxodes Anaerobic, has Mullerian vesicles
Little specialization of cilia surrounding the mouth
somatic kinetid of one kinetosome + 2 transverse ribbons
one lateral into cortex
mt nematadesmata extend into cytoplasm from bases of dikinetids that surround the cytostome: rhabdos
subclass Haptoria carnivores, protistovores: toxicysts
Didinium, Lacrymaria, autotrophic mesodinium
endosymbionts of vertebrates
Balantidium only human ciliate parasite
Oral apparatus at or near apical part of cell
Relatively simple oral ciliature, may be simple polykinetids = brosse
many have litostome like toxicysts
monokinetids on somatic surface, transverse mt ribbons oriented radially
Prorodon, Coleps, Metacystis
conical or bell shaped body
somatic cilia poorly developed
Oral polykinetids surround anterior end, used for locomotion and feeding
Tintinnids, Strobilidiids, Strombidiids
Subclasses Stichotrichia and Hypotrichia
somatic cilias as rows or scattered polykinetids Cirri
Benthic, cirri used as walking appendages
Class Colpodea: radiation mirrors ciliate radiation
oral morphology diverse, resemble many other groups:
prostomes, oligohymenophoreans, nassophoreans, heterotrichs
reticulate silverline system
somatic dikinetid kinetid distinct, with transverse mt ribbon extending from posterior of dikinetids toward posterior of ciliate forming a desmos
Oral ciliature in right and left fields
terrestrial, freshwater, many edaphic
division cysts, slime mold ciliate
Oral nematodesmata, can be quite elaborate
mono & dikinetid with tranverse ribbon tangential,
well developed KD, may have kd desmos
feed on filamentous cyanobacteria
prominant radially arranged mt ribbons that form the cytopharynx.
somatic monokinetids transverse mts reduced or absent,
distinctive lateral KD
somatic kineties underlain by subkinetal mts
Phyllophryngia: free living, commensal and parasitic forms
Chonotrichia: ectocommensals on crustaceans
Suctoria: only larval forms ciliated
"adults" also have kinetosomes-unciliated
tentacles with haptocysts sessile free floating some with stalks
most suck out contents of prey, one penetrates
Reproduction by budding "Birthing"
most speciose of the ciliates
usually three oral polykinetids to left or anterior of cytostome,
ODK membrane (Paraoral, Undulating) on right
somatic monokinetids, some dikinetids,
radially directed transverses,
anterior only in dikinetids directed tangential
buccal cavity with PO + peniculi and quadrulus
Frontonia, Paramecium, Lembadion
mono and dikinetids, dikinetids sometimes restricted to posterior half
Paraoral dikinetid in three segments, third is stomatogenic
chondriome in some species
somatic monokinetids, right most post oral kinetid stomatogenic
Oral ciliature UM + AZM
Astomatia: mouthless symbionts in anelids and amphibians
may have holdfast appendage
Peritrichia: prominant oral ciliature encircles anterior end
somatic cilia reduced: telotroch band on larvae
stalked sessile bacterivores vorticella, some loricate, colonial,
strongly contractile stalks, Vorticella, Zoothamnium, Opercularia
ectosymbionts with complex life cycles
tied to physiology of marine crustacean hosts
Baroin-Tourancheau, A. et al., 1992. A broad molecular phylogeny of ciliates: identification of major evolutionary trends and radiations within the phylum. PNAS 89:9764-9768.
Corliss, J.O 1979. The Ciliated Protozoa. Characterization, classification, and guide to the literature. 2nd Ed. Pergamon Press, New York. 455 pp.
Hammerschmidt, B., M. Schlegel, D.L. Lynn, D.D. Leipe, M.L. Sogin, & I.B. Raikov. 1996. Insights intothe evolution of nuclear dualism in the ciliates revealed by phylogenetic analysis of rRNA sequences. J. Euk. Microbiol. 43:225-230.
Kurtz & Tiedtke 1993. J. Euk. Microbiol. 40:10-13.
Lynn, D.H. 1996. My journey in ciliate systematics. J. Euk. Microbiol. 43:253-260.
Small, E.B. & Lynn, D.H. 1981. A new macrosystem for the phylum Ciliophora Doflein, 1901. BioSystems 14:387-401.
Small, E.B. & Lynn, D.H. 1985. Phylum Ciliophora Doflein, 1901. Pp 393-575 In: Lee, J.J., S.H. Hutner, & E.C. Bovee, eds., An Illustrated Guide to the Protozoa. Society of Protozoologists and Allen Press, Lawrence, KA.
Any in or on another: mutual to parasitic
Establishment of Symbionts:
1. Breakage & loss of phagosomal membrane: free in cytoplasm
2. Loss or inactivation of Lysosome receptors
3. Non-digestion after fusion of lysosome
1 & 2 common
Symbiont: codes for all structural and functional molecules needed
Organelle: some sequence (genes) transferrred to host,
transport of products required
Mitochondria: 4 destinations: OM, IM, Periplasmic Space, Matrix
Chloroplasts: 5 destinations: thylakoid
multiple membrane layers in some protists: higher complexity
Review by Lee et al., 1985
synchronous division common
predominantly ciliates and amoebae
Fresh water :Chlorophyceae mostly more common than cyanobacteria: Cyanelles
Marine:Chlorophysceae, Rhodophyceae, Dinophyceae, Bacillariophyceae
Chlorella in Paramecium
EM observations of Chlorella: indistigusihable from free living form
Physiological differences in carbon metabolism and release of photosynthate
Few substances released: carbohydrates:fructose, glucose, xylose, maltose,
some amino acids
Released carbon can be 85% of photosynthetically fixed carbon
Release is pH dependant, energy consuming process
Released carbohydrates not detected within cells in high amounts
higher specific activity of ribulose-1,5-biphosphate carboxylase
All symbionts examined are in perialgal vesicles, not free in the cytoplasm
divided symbionts each enclosed separately
divide synnchronously with host
Different from food vacuoles:
Vacuoles: only one alga per vacuole
small space 0.05 um between alga and membrane
do not circulate
do not fuse with lysosomes
no acid phospahtase activity
exogenously supplied symbiont cells ingested and digested
cell surface properties and ability to translocate sugar important to infectivity
In darkness, algae grow heterotrophically and maltose secretion is reduced
growth rate of symbionts slow relative to free living ones: maltose release
lytic enzymes of host may be lower when alga population high
ammonia and glutamine provided by host
higher symbiont loads at higher light levelsresult in higher ingestion rates of particulate food: nitrogen demand on host.
Photoaccumulation of cells, not observed for free symbionts or symbiont free hosts
ciliary reversals controled by symbiont population requires >50 chlorella
Mesodinium rubrum red ciliate with Cryptomonad symbionts, mouthless
planktonic cilaites, benthic forams, freshwater Heliozoa
can be 40% of ciliate asssemblage in marine waters, mixotrophic
isolated chloroplasts quickly die, but functional in ciliate cytoplsm up to 2 weeks
chloroplasts do not divide
algal cells digested but not chloroplasts, free in cytoplasm
chloroplasts from several algal species can be held, some ciliates more restricted
may contribute 30-40 % of ciliate's carbon requirements
some obligate mixotrophs, chloroplasts are not digested during starvation
planktonic cilaites, benthis forams, freshwater Heliozoa
Zooxanthellae Gymnodinium dinos
Flagellate Leptomonas escapes food vacuole and penetrates host nucleus
Leptomonas found in Paramecium and Euplotes
epibionts common on all types of free living flagellates, ciliates and amoebae: Microbial Seascapes
Metachonal waves of attached spirochetes on Mixotricha
specialized attachment structures provided by the flagellates
Sulfate reducers common
Cytoplasmic, Macronuclear, micronuclear
Legionella: normal host is free living bacterivorus protists will not grow in tap water, filtrates of tetrahymena culture, or lysed cells of Tetrahymena,
Kappa or "killer" particles, lambda, mu, gamma
gram negative bacteria in Paramecium aurelia
lambda provides essential folic acid
Kappa: : Caedibacter
N particles (non-brights) some fraction of which become Brights with R-body
ability to kill paramecia is due to R-body or ribbon that pierces food vacuoles
stimulated by low pH
viruses associated with extruded R-body
viruses induce formation of R-bodies in N particles
R-body production dependant on a plasmid
R-bodies similar to ejectosomes in Cryptomonads
similar structures seen in free living bacteria (Proteus sp.)
Epibiotic bacteria on Euplotidium
external cells in matched depressions, honey comb like array around the cell
two types: with and without ejectable ribbon
ejectable filament up to 20 x length of cell
stains with DAPI on tip: Viruses?
found in many protists: may contribute to host metabolism by providing required compounds or vitamins
common in termit flagellates: Caryococcus
Holospora in Paramecium: uncultivatable but by PCR amplification
back testing with fluorescent probes
found to be related to subclass of the proteobacteria
closest relative is Rickettsia
short form: reproductive stage
long form: infective stage
Jeon 1992. Amoeba proteus strain with obligate bacterial symbiont
gram negative rod shaped bacteria in symbiosomes
three proteins and LPS from bacteria invovled in stability of the relationship.
29kD protiein in host cytoplasm
96 KD protein and LPS in symbiosome membrane Antigenic portion of LPS exposed on cytoplasmic face of symbiosome:prevention of lysosome fusion
Actin produced by the host accumulated by bacteria
Protein spectrin (220-225 kD) associated with symbiosome membranes
Methanogen associations common in rumen
and free living anaerobic protozoa:
utilizing H2 that inhibits protist metabolism
F420 coenzyme autofluorescence
Hydrogensomes: conversion of pyruvate to acetate and hydrogen
marker enzymes: ferridoxin oxidoreductase and hydrogenase
tolerant of oxygen at physiological levels
possible secondary development of mitochondria in some protists:
primitively anaerobic: Archezoa:Trichomonads, Diplomonads, microsporidia
in anaerobic Cyclidium hydrogenosme resembles mitochondriome of Uronmea
has methogens, and unidentified eubacterium in three part consortium
Embly and Finlay: anaerobiasis arose three times independnatly in ciliates
16s rRNA analysis of cilaites and symbionts
Amann, R., Springer, N., Ludwig, W., Görtz, H-D., and Scleifer, K-H. 1991. Identification in situ and phylogeny of uncultured bacterial endosymbionts. Nature 351:161-164.
Ball, G.H. 1969. Organisms living on and in protozoa. In Chen, T.-T., ed., Research in Protozoology Vol 3:565-718.
Embly, T.M. and Finlay, B.J. 1994. The use of small subunit rRNA sequences to unravel the relationships between anaerobic ciliates and their methanogen endosymbionts. Microbiology 140:225-235.
Esteban, G., Guhl, B.E., Clarke, K.J., Embly, T.M., and Finlay, B.J. 1993. Cyclidium porcatum n. sp.: a free-living anaerobic scuticociliate containing a stable complex of hydrogenosmes, eubacteria, and archeobacteria. Eur. J. Protistol. 29:262-270.
Fields, B.S., Shotts, E.B. Jr., Feeley, J.C., Gorman, G.W., Martin, W.T. 1984. Proliferation of Legionella pneumophila as an intracellular parasite of the ciliated protozoan Tetrahymena pyriformis. Appl. Environ. Microbiol. 47:467-471.
Fenchel, T and Finlay, B.J. 1991. The biology of free living anaerobic ciliates. Europ. J. Protistol. 26:201-215.
Fenchel, T. and Ramsing, N.B. 1992. Identification of sulphate-reducing ectosymbiotic bacteria from anaerobic cilaites using 16s rRNA binding oligonucleotide probes. Arch. Microbiol. 158:394-397.
Finlay, B.J. and Fenchel, T. 1989. Hydrogenosomes in some anaerobic protozoa resemble mitochondria. FEMS Microbiology Letters 65:311-314.
Fokin, S. and Görtz, H-D. 1993. Caedibacter macronucleorum sp. nov., a bacterium inhabiting the maconucleus of Paramecium duboscqui. Arch. Protistenkd. 143: 319-324.
Görtz, H-D. 1983. Endonuclear symbionts in ciliates. Int. Rev. Cytol. suppl 14:145-176.
Jeon, K.W. 1992. Macromolecules involved in the amoeba-bacteria symbiosis. J. Protozool. 39:199-204.
Lee, J.J., Soldo, A.T., Reisser, W., Lee, M.J., Jeon, K.W., and Görtz, H-D. 1985. The extent of algal and bacterial endosymbioses in protozoa. J. Protozool. 32:391-403.
Lindholm, T. Mesodinium rubrum- a unique photosynthetic ciliate. Adv. Aquat. Microbiol. 3:1-48.
Reisser, W. 1986. Endosymbiotic associations of freshwater protozoa and algae. Prog. Protistol. 1:195-214.
Stoecker, D.K. M.W. Silver, A.E. Michaels, and L.H. Davis. 1988. Obligate mixotrophy in Laboea strobila, a cilaite which retains chloroplasts. Marine Biology 99:415-423.
"Animal behavior is largely governed by biological electricity"
Saimi et al., 1988. TIBS 13:304-309.
Four "known" motility systems
microtubule+ dynein + ATP
actin + myosin + ATP
Cilia and flagella
0.2 m diameter
cilia 10's m long, flagella 100's m
cilia oar like motion
flagella more complex patterns
membrane controls motion
all eukaryotic cilia and flagella depend upon the same basic mechanism
motility, food gathering, behavioral response, cell recognition
9+2 axoneme surronded by membrane=specialized compartment of cytoplasm
doublet axonemal microtubules:
complete A subfiber, partial B subfiber c subfiber only in basal body
during self assembly, A fibers assemble above basal body, b sub fiber and other components assemble on A fibers
9 doublets linked into a ring by inner and outer dynein arms, radial spokes, interdoublet circumferential links.
several hundred known proteins in cilia, only a fraction have been identified
Cartwheel proteins of the basal body have been isolated by self assembly Gavin et al., 1994
Dynein: 9 polypeptides including 2 ATPases per arm
arms extend from A fiber to adjacent b fiber
radial spokes composed of 17 polypeptides
extend from A fiber to towards central complex (two microtubules and associated proteins)
Modeled as a cape, main body, and spherical head
Dynein activity must be regulated temporally and spatially to generate bends
Radial spokes invovled in regulating mt sliding:
Chlamydomonas mutants lacking radial spokes Smith & Sale 1992.
paralized or impaired motility
isolated axonemes can be induced to slide apart in telescopic fashion:
sliding disintigration assay.
sliding velocities diminished in axonemes missing radial spokes
restored by addition of isolated radial spokes from wild type flagella
radial spokes apparently interact with inner dynein arm
Switch Point hypothesis for control of doublet sliding:
one half on during power stroke, other half on during recovery.
ATP and Mg2+maintained at 100
Ca2+ at 10-7 M or less
Ciliary motion can change as
1) orientation reponse: direction of the power stroke changes counter clockwise in proportion to membrane depolarization: Ca concentration in cilia
2) Frequency respnonse: change in beat frequency in response to membrane hyperpolariztion
Chemically skinned cells ecxtracted with triton X-100
mixture of ATP, Mg swims forward
increasing addition of Ca results in counterclockwise shift in orientation
Hyperpolarization: adding K results in clockwise shift and beat frequency increase
1) initiation of active sliding at the basal region
2) propagation of active sliding along the doublet pairs tot he tip
3) determination of the patterns of cross-bridging activity
Orientation mechanism: changing the patterns of cross bridging
Ca does not appear to affect beat frequency, only bridging patterns that affect orientation and indirectly affect beat frequency
Ca2+ gradient 104:1 in, +116 mV
K+ gradient 40:1 out, -93 mV
net electrical potential -33 mV
energy expended in maintainingelectrical potentials
two major membrane conductances (up to 22 ion channels have been identified):
calcium and potassium
Calcium "calcium channel" excitation of membrane, rapid activation of cilary motor response. Only depolarization activates Ca channels
Potassium three types of channels with different acitvations on directly Ca activated
K channels are antagonists of Ca channels, maintain homeostasis
voltage dependant Ca channels on cilia only
depolarization of membrane by a few millivolts opens ciliary Ca channels
Ca influx triggers K channels for K outflow
In Bursaridium action potetnials are not graded, but all or nothing responses that are apparently spontaneous. Berg & Sand 1994
as eukaryotic cells exhibit irritabilty: response to external stimuli
Jennings 1906: the avoidance reaction
1. concentration threshold for response
2. responses to stimuli mediated by avoidance reaction: accumulation or dispersal
3. cells collect in areas of optimal stimulation by treating lesser stimuli as repellants:
attraction and repulsion are relative
an attractant can be a repellant relative to a stronger atractant
a repellant can be an attractant relative to a stronger repellant.
Van Houten: attaction saturatable and specific
Klinokinesis: modulation of turning or tumbling frequency
Orthokinesis: modualtion of speed
Most attractants for Paramecium found in the fermentation products of its food.
Didinium attracted to bacterial products
Specific chemical receptors have been identified for folate, cAMP
Amino acids induce hyperpolarizations in the nM range
work with Tetrahymena has shown that not all amino acids are attractants
Flagellate and amoebae responses to bacteria are variable: speciies dependant
Ca influx and binding to calmodulin associated with cAMP and cGMP formation. can be overridden by cAMP that accumulates as cells starve
anterior : depolarization, ciliary reversal
posterior: hyperpolarization speed up
posterior receptor channels may be selective to cations: aallowing only K+ flux
mechanoreceptors on soma membrane, not cilia membranes
cilia may physically transmit mechano stimuli to soma
peak at 520 nm photosynthesis peak at 440 and 680,
not inhibited by photosynthesis blockers
440 induces hyperpolarization (blue)
520 (green) and 680 (dark red) depolarization
photodispersal in white light and accumulation in dark: mechanism not understood
low reynolds numbers: ration of viscous to inertial forces
shear gradients from non-slip zone near surface to no-effect zone away from surface
the extent of influence of a cilium on the surrounding water limited by its proximity ot cell surface: net transport of water by the motion of a cilium
metachonal waves a function of mechanical hydraulics, not electrophysiology
Cytoplasm partioned into two distinct regions: viscous hyaline ectoplasm and less viscous ganular endolasm
layer of microfilaments associated with boundary and plasma membrane
one filament 5-8 nm in diameter-actin continuously distributed over cell surface
thick filaments myosin containing in region of uroid
filaments in intermeadiate and uroid regions randomly oriented
filaments in frontal region paralell to plasma membrane.
Two major theories of amoeboid motility:
1) ectoplasmic tube contraction model
2) frontal contraction model
ectoplasmic "toothpaste" tube model: pressure exerted all over cell, especially at uroid, released at poit of pseudopodial advancement
suction on uroid does not stope pseudopodial formation
laser ablation of uriod does not stop pseuodopodial advancement.
frontal contraction model:flow produced by conversion of endoplasm into a gel-like state at frontal zone, producing a pull on the endoplasm.
modified thoery may be more accurate:
pressure exerted all over cortex, separation of filaments from membrane at frontal zone allows membrane expansion, granuloplasm filtered through the mesh of filaments, filaments depolimerize, allowing endoplasm to advance
reformation of membrane attached filaments from monomers in endoplasm
Filopodia cytoplasmic streaming and extension mt dependant
membrane markers dmeonstrate that membrane flows tractor tread like,
attachment to substratum can occur: adhesive "micropodia" leave "footprints"
Motility in coccidian Toxoplasma and other apicomplexans
Mondragon et al., 1994
twirling, gliding motility, rotation of the cell around a fixed posterior
inhibited by cytochalasins: drugs that interfere with actin filament function.
insensitive to colchicine mt drugs
mild proteolysis permeabolizes cells, inducing response
Berg, T.O. & Sand, O. 1994. Spontaneous all-or-nothing action potentials in the ciliate Bursaridium difficile. J. EuK. Microbiol. 41:13-17.
Dunlap, K. 1977. Localizaiton of calcium channels in Paramecium caudatum. J. Physiol. 271:119-133.
Eckert, R. 1972. Bioelectric control of ciliary activity. Science 176:473-481.
Gavin, R.H., Duffus, W.A., & Contard, P.C. 1989. Charateristics of basal body cartwheel reassembly. J. Protozool. 36:391-397
Levandowsky, M & D.C.R. Hauser. 1978. Chemosensory responses of swimming algae and protozoa. Int. Rev. Cytol. 53:145-210.
Machemer, H. 1988. Electrophysiology. Chapt 13. in Gortz, H.-D. Paramecium. Springer Verlag.
Machemer, H.D. 1988. Motor control of cilia. Chapt 4. in Gortz, H.-D. Paramecium. Springer Verlag.
Mondragon, R., Meza, I., & Frixione, E. 1994. Divalent cation and ATP dependant motility of Toxoplasma gondii tachyzoites after mild treatment with trypsin. J. Euk. Microbiol. 41:330-337.
Naitoh, Y. & Ekert, R. 1969. Ionic mechanisms controlling behavioral responses of Paramecium to mechanical stimulation. Science 164:963-965.
Ogura, A. & Takahashi, K. Artificial deciliation causes loss of calcium-dependant responses in Paramecium. Nature 264:170-172.
Satir, P. 1974. How cilia move. Scientific American 231:44-52.
Smith, E.F. & Sale, W.S. 1992. Regulation of dynein-driven microtubule sliding by the radial spokes in flagella. Science 257:1557-1559.
Van Houten, J. 1979. Membrane potential changes during chemokinesis in Paramecium. Science 204:1100-1103.
Van Houten, J & Preston, R.R. 1988. Chemokinesis. Chapt. 18. in Gortz, H.-D. Paramecium. Springer Verlag.
Symposium on the Structure and Function of Cilia and Flagella. J. Protozool. 31:7-40
Feeding and Growth
binding and channel formation
Induction by binding to acidic groups of the mucopolysaccharides of the glycocalyx
Group one: very labile inorganic salts at neutral pH
Group 2: Reversible bound proteins at pH giving positive charge
Group 3: irreversible bound Alcian blue dyes-binding to surface coat
Food vacuole formation: binding of membrane dicoidal vesicles to ribbed wall to feed growing vacuole, Ca+ dependant actin polymerization
cycling of food vacuoles: selective digestion-cell wall materials of bacteria not digested, etc.
Stimulation of Phagocytosis
Common stimuli for phagocytosis found for amoebae, ciliates, and mammalian cells: conserved underlying biochemistry.
Binding to membrane triggering actin polymerization and food vacuole formation
lipids found to be effective Bailey et al., 1987..
lectins: Pseudomonas lectins stimulate phagocytosis in Tetrahymena
Bacteria fed Tetrahymena express a glycoconjugate not seen in axenic cells, derived in mucocysts
Mucous coat on amoebae capable of concentrating proteins and inorganic cations 10-50 x over the medium
Mechanical stimulation found to be necessary if chemcial receptors saturated
Changes in the structure of water at surfaces: ion displacement
peptide hormones and neuroppetides have been detected in bacteria and protists
biogenic amines: catecholamines, serotonin in:
Tetrahymena, Crithidia, Entamoeba
In Tetrahymena- cell divsion, glucose metabolism, regeneration of cilia can be regulated with biogenic amines
Histamine, serotonin, and epinephrine stimulate phagocytosis
andrenergic receptor antagonists block these responses
Conserved receptor, chmosensory transduction system:
receptor:guanine nucleotide binding protein adenylate cyclase
(GTPase, G- protein) generation of cAMP as a messenger
Particle feeding: sieving
optimal particle sizes: size selection demonstrated:
clearance rates and ingestion
filtration models discount selective capture or chemical recognition
however, not all bacteria support growth of protists: Later
1) preingestion recognition and selective feeding
2) selective digestion of randomly ingested particles
Also filtration models suggest water flow
between cilia of ODK, Collar tentacles of Choanoflagellates.
Chemical responses: gradients of particles and substrates
Life History Attributes:
amoebae communication cAMP & small oligopeptide: Glorin
For Human cells % of ATP:
Protein turnover 34.7%
Ca2+ dependant processes 27.8%
RNA synthesis 8.5%
DNA sysnthesis 7.6%
Gross vs net assimilation effciency
For protists: growth and reproduction continuous phenomena
growth to sexual maturity then energy expendture in reproduction
reproduction still as single cells!
Respiration: µ Mb, µ for metazoans 8x protists, b 0.75
Banse, 1982 MEPS 9:281-297
Crawford et al., 1994.
14C labelling method
high energetic cost of motility in amoebae: 56% of total metabolic activity
cytochalsin inhibited actin polymerization
Fenchel and Finlay estimated 1%, probably too low
10% for fast moving ciliates: may be more effcient than amoebae
Respiration rate may vary 50-60% for protists depending on nutritional state
respiration rate slows rapidly with starvation.
Chemical communication in amoebae
Small molecular weight molecules, taxa specific act as chemical messengers for "social amoebae".cAMP and Glorin a oligopeptide
toxins, physical impediments
Lambornella clarki: free-living cilaite feeds on bacteria, mosquito larvae prey on l. clarki. Water soluble compound from mosquito larvae induces divsion of L. clarki to form parasitic cells that encyst on larvae, penetration of the cuticle occurs and ciliates eat mosquito larvae from inside. May eliminate mosquito larvae and reappear as free living cells
two species of Euplotes spine/"wing" development: Water soluble factors (polypetides) produced by a number of predators (ameobae, Lembadion, Stenostomum). Protein sysnthesis necesary for change but not cell divison. Cost of 15% of generation time, presence of a predator defense that is inducable indicates an increased survivorship with defense, but cost of maintaining defense when not needed.
plantlike characteristic s and animla like characteristcs , as well as unique metabolic capabilities are found mixed throughout the protists
some photorophic and symbiont carrying forms have very few requirements, synthesizing all or most organics form inorganic materials
some parasitc forms are dependant on hosts for preformed organics
some dependance on preformed organic nitorgen is seen
amino acids, fatty acids, vitamins required by many protists
Ammonium preferred,but many phytofalgellates can use nitrate
Nitrate reductase (nitrate to nitrrite) also present in ciliate Loxodes
most protist can ssimilate amino acids, and nucleotides, mostly from particulate food
urea is not produced
some sterols required for growth
Tetrahymena and other ciliates do not produce any typical sterols but produce Tetrahymenol, a pentacyclic triterpenoid
Tetrahymena can accumulate other sterols from medium and incorporate them in membranes, added chloesterol blocks tetrahymenol production
gamma linoleic acid found in Chryomonads, but not prymnesiomonads:
Suuports separation of these groups
Acanthanamoeba has amoung its major fatty acids 20 carbon polyunsaturates like higher animals, but also can biosynthesize long chain fatty acids de novo like plants
Lipid compositions are known to change with physiological state, temperature, and pressure adaptations
Rumen and termit gut protists have cellulase (symbionts?)
Acanthamoeba also has a cellulase: but may be for cellulosic cyst walls rather than digestion of celluose as a substrate
Collagenolytic activity found in some pathogenic amoebae, but not non-pathogenic forms
Bailey, G.B., Day, D.B., Nokkaew, C., & Harper, C.C. 1987. Stimulation by target cell membrane lipid of actin polymerization and phagocytosis by Entamoeba histolytica. Infect. Immun. 55:1848-1853
Bolivar, I., Guiard-Maffia, J. 1986. Expression of surface coat glycoconjugates by bacteria-fed Tetrahymena. J. Protozool. 33:335-340.
Bonner, J.T., 1983. Chemical Signals of Social Amoebae. Scientific American 248:114-120.
Crawford, D.W., Rogerson, A., Laybourn-Parry, J. 1994. Respiration of the marine amoeba Trichosphaerium sieboldi determined by 14C labelling and Cartesian diver methods. Mar. Ecol. Prog. Ser. 112:135-142.
Dive, D. 1973. La nutrition holozoique des protozoaires cilies. Ses consequences dans l'epuration naturelle et artificielle. L'Annee Biologique XII:343-380.
Fenchel, T. 1986. Protozoan filter feeding. Prog. Protistol. 1:65:114.
Fenchel, T. & Finlay, B.J. 1983. Repiration rates in heterotrophic, free-living protozoa. Microb. Ecol. 9:99-122.
Gilboa-Gardinaer, N & Sharabi, Y. 1980. Increase in growth rate and phaocytic acitivty of Tetrahymena induced by Pseudomonas lectins. J. Protozool. 27:209-211.
Kusch, J. 1993. Induction of defensive morphological changes in ciliates. Oecologia 94:571-575.
Levitzki, A. 1988. From epinephrine to cyclic AMP. Science 241:800-806
Washburn, J.O., Gross, M.E., Mercer, D.R., & Anderson, J.R. 1988. Predator-induced trophic shift of a free-living cilaite: parasitism of mosquito larvae by their prey. Science 240:1193-1195
Cells studied: Paramecium, Tetrahymena, Stylonichia, Oxytricha, Euplotes
Nuclear dualism: germ line, somatic
Mating cells exchange haploid nuclei, develope new macronuclei
numbers of nuclei vary with species, multinuclei genetically identicle
mitosis intranuclear, individual chromosomes not identifiable
forms with multi macs fuse macs prior to mitosis
Karyorelecteans: no amitosis, mac derived at each divsion
switching mics and macs, phenotype determined by genes in mac
small amount of rna sythesis found by autorads in mics, but only during brief DNA sysnthesis period
genetic silence does not jibe with experimental removal of mics: lower asexual reproductive rates, no vegetative growth, cell death, although some forms survive well in amicronuc state
few mic specific genes eliminated in formation of mac, may escape deletion
some DNA sequences have been found in amicronucleate macs that are normally only found in mics
retention of essential vegetative genes in mic ensures survival of sexually competent cells
Mic structure: no nucleoli, uniform dense packed chromatin
Mac: chromatin bodies dispersed in nucleoplasm, multiple nucleoli, numerous nuc mem pores
Histones reflect other evolutionary data reflectin not only the ancient split from eukaryotic line but also extensive divergence witin the cilaites
in mics: genes in single copy: even rRNA gene, unlike other eukaryotes
Tetrahymena: DNA content of MAC 7.5-13.2 e9 bp in stable cultures, may vary more in different physiological conditions. Mac is a subset of sequences in mic
aprox. 46 x haploid mic (Paramecium: aprox. 800x): but not strictly polyploid. Mic 80% unique sequences, 20% repititous and eliminated (hypotrichs up to 98% repititious and deleted)
Aprox. 6000 cut/splice sites where sequence deletion occurs
deletions 10o bp to 10kbp, distribited thoughout 5 chromosomes
some deletions are interstitial tandem repeats of telomere sequence
may be as inverted repeating pairs defineing ends of transposable units excised and destroyed
functions of deleted sequences not known
mic DNA cut into permananet sub-chromosomal fragments
mic chromosomes average 44,000kbp
fragmented into 200 molecules 100 to over 1500 kbp at average of 57 copies/molecule
Telomeric repeates added to ends of molecules by Telomerase: provide stability and replication
rRNA 21 kbp molecule formed from excision of single rRNA gene, replicated and spliced into a palindrome telomeric sequences added both 17 and 25 s RNA encoded
Palindrome relpicated 9,000 molecules of 18,000 copies of the gene, aprox 300x other genes
telomeric sequences 20-70 tandem copies
transcription from center outward
Paramecium rRNA in molecules of various sizes with one to several genes arranged head to tail
Three Global changes identified with mac formation:
1) repititous sequences and some unique sequences deleted and destryed
2) 5 chromosomes fragmented in specific and repaetable pattern into aprox 200 molecules
3) each molecule replicated to an average of 57 copies
mac formation phenomena more extreme and slightly different
Mac to Mic DNA ratios: Euplotes crassus 27:1, 200:1 e. eurytomus
in Oxytricha and Stylonichia, 96 and 98% of mic DNA eliminated
mic DNA in Oxytricha is 70% unique sequences and 30% repeats
most of the unique sequences eliminated in mac formation, small subset of mic genes used in mac formation yet alre all that is necessary for vegetative growth
each sequence in mac aproximately 1900 copies in Oxytricha, 7500 copies in Stylonichia
high rates of transcription necesary for large, complex cells
rRNA gene in 1000,000 copies per mac
average of 950 for other molecules
Telomeres havesingle stranded overhangs of 16 to 20 bases, may interact to form circles or end to end associations: 4 stacked layers of G quartets hydrogen binding not known if this occurs in vivo
protein associations prevent double stranded exonuclease activity.
Replication of genomes
Mac & Mic replication separately regulated
Mics replicate rapidly, perhaps multiple replication origins, many deleted with mac formation macs may only have one or less per gene sized molecule
Macs: replication bands- wave of replication of milions of DNA molecules
pulse labeling: indicates dan in a gel state: labels a small band.
Prior to amitosis, dna sols, mac rounds up (multiples fuse) pulse labeled DNA mixes
nucleoii do not label
at least three replication band specific proteins that migrate with it
selective replication may also occur to maintain constant copy number of gene in mac
Tetrahymena DNA A+T rich, 76%, codoing regions only 56% spacer regions >76%
Hypotrichs: 75% for leaders, 69% for trailers, 52% for coding regions
Evolutionary drifting of non-coding sequences toward A+T richness is widespread amoung eukaryotes
Genetic code unique to ciliates
three universal stop codons in other eukaryotes: TAA, TAG, TGA have evolved different usage in ciliates, mostly glutamine coding, may be restricted to some genes
reflected in tRNAs
Tetrahymena: three glutamine tRNAs: two that reconize "stop" codons may have evolved from convential glutamine tRNA
Euplotes: TAA, and TAG used a s astop codon, TGA encodes cysteine
unusual condon useage may limit viruses: no viruses have ever been found in ciliates
other Codon usage is biased relative to other eukaryote usage
Two actin genes cloned from Oxytricha both code for 375 amino acid actin but are only 63% similarbut very different from actin conserved through yeasts & mammals
sequences are scrambled in micronuclueus relative to their final position in macronucleus 9 MDS separated by IES
three of six cloned genes from oxytricha have been scrambled in mic.
Amoebae: rRNA genes amplified as extrachromosomal circular molecules in Entamoeba histolytica
may be common feature of unicellular eukaryotes to have amplified genes as extrachromosomal elements
Genome undergoes frequent rearrangement permitting cells to avoid immune response by spontaneously changing surface coat.
Genome contains over 1000 genes encoding variant surface glycoproteins (VSG)
usually only one expressed at a time with aprox 107 copies of the protein on the surface, 5% of total protein, large for surface protein
Expression site associated genes (ESAG) also transcribed with VSG, 14 to 25 isogenes, glycoproteins, 0.01% of total protein, function unknown
Rearrangements by maneuvering VSG genes into expression sites for transcription
Several expression sites may exist, but only one active
Infection progresses in waves, 2 week intervals
sequential clone growth
From the N- terminal, 1st 25-30 amino acids different
successive peaks in a single rabbit resulted in 100 different VSGs
spontaneous switching at a rate of 10-6 to 10-7 per division, occurs in defined media, not a response to immune attack : unique genetic mechanism
protist chromosome number cannot be determined by staining because they do not condense sufficiently during mitosis to be visualized.
In Insect 1x, bloodstream 2 x
mixture of trypanosomes in insect phase may result in 1.5 x cells, twice the number of minichromosomes
pulse field electrophoresis: four size classes of chromosomes, some quite small for eukaryotes
VSG gene on a 80 kb minichromosome
100 minichromosomes 50-100 kb
5-7 200 to 700 kb; several 2000 kb
Some do not enter gel: very large or unusual structure
Chromosome size and number may vary within clones:
may fragment and fuse
minichromosomes less prominant in species that do not undergo antigenic variation
DNA probes hybridize to all chromosome sizes, several hundred to 1000 genes may exist
mRNA's contain 1600 bases, may be 4% OF genome
transpositional unit 2500 to 3500 bp, up to 9% of genome involved
transcriptionally regulated: VSG cDNA hybridizes only to RNA from tryp expressing that VSG and not to other Tryp RNA
Duplicative Transposition ELC formation
In some cases an extra copy of the transcribed gene is present:expression telomere linked copy more suseptable to DNAse cleavage
Switch to another VSG gene results in loss of the previous ELC
Telomere linked genes do not have to be duplicated for expression
All expression sites are next to telomeres: those genes internal must be copied and translocated to a telomere-linked expression site.
Transferred region replaces the previous segement in the expression site.
Most expresision sites located on large chromosomes
VSG genes may occur within sequences of 2 to 10 isogene families
Four possible acitivation events
1) ELC formation
2) teleomere exchange
3) teleomere conversion
4) switched activation without DNA transposition
Composite ELC's found with contributed elements from previosly active gene and others
Transcription in Tryps
Spliced leader sequence of 39 nucleotides at 5' end of all tryp mRNA, also found in non-variable kineto's
spliced leaders come from different locations than transcribed genes: Discontinuous Transcription
1) transport across nuc membrane
2)stability of mRNA
4) cytoplasmic compartmentalization
spliced leaders also found in nematod Caenorhabditis elegans, vaccinia virus
May be multiple promoter sites, some transposed, some upstream of transposed DNA
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Rizzo, P.J. 1991. The enigma of the Dinoflagellate chromosome. J. Protozool. 38:1246-252.
Donelson, J.E. 1989. DNA rearrangements and antigenic variation in African trypanosomes. Chapter 35 In: Berg, D.E. & Howe, M.M., eds. Mobile DNA. ASM publications, Washington, DC. pp763-781.
Seiwert, S.D. & Stuart, K. 1994. RNA editing: transfer of genetic information from gRNA to precursor mRNA in Vitro. Science 266:114-117.
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