Kingdom Protista Sometimes called the “Junk drawer” Contains animal-like, plant-like and fungus-like organisms Plankton (zoo-/phyto-/myco)

CHARACTERISTICS OF PROTOZOANS Unicellular Eukaryotic Basically Ingestive Heterotrophs Lack cell walls but have definite shapes Most are motile Basically reproduce by asexual reproduction Aerobic but some can live in anaerobic conditions (ones living in digestive tracts)

Special structures: Macronucleus – controls metabolism Micronucleus - involved in conjugation Contractile vacuoles – maintains Meostasis Ingestion structures Anal pore – excretion of wastes Trichocysts – defense mechanism

HABITAT Majority of free-living Marine, terrestrial & freshwater. Some are parasites on algae to vertebrates Make up the zooplankton in marine ecosystems. Feed on phytoplankton Abundant in soil or on plants & animals Some live in guts of termites, roaches & ruminants (cows)

DIFFERENT PROTOZOANS Paramecium with trichocysts Paramecium Didinium Vorticella Stenor

PARAMECIUM Paramecium is a small unicellular organism. It is plentiful in freshwater ponds.

STRUCTURE

Position Of Protists In The Prokaryotic Kingdom

Classification Mastigophora – have one or more flagella Classified by method of locomotion Mastigophora – have one or more flagella - Have a flagella with a 9-2 microtubule arrangement - Flagella are polar & undulates, pushing protozoan in opposite direction - Longitudinal reproduction i.e. Peranema, Chilomonas

Ciliata (Ciliophora) - Have cilia. - Similar in structure to flagella but shorter and all over surface of organisms - Cilia usually arranged in rows & connected to each other - Cilia near oral cavity involved w/ food getting - Transverse fission, & sexual repro by conjugation Ie – Paramecium, Didinium, Blepharisma, Vorticella, Stentor

Sarcodina - Use Pseudopods for movement - Cytoplasmic streaming – amoeboid movement - Tips of pseudopods are less viscous so flow goes in that direction - Pseudopods for phagocytosis - Reproduce by binary fission i.e. Amoeba, Naegleria, Heliozoans, Radiolarians, Foraminifera

Sporozoa No method of motility All are parasites – use host for motility Reproduce by schizogamy (multiple fission) in host & sexual reproduction in a second host Ie. Plasmodium (malaria), Giardia, Toxoplasma, Trypanosoma, Trichomonas

Animal-Like Protists

PROTOZOAN PROTIST

EVOLUTION Evolved from the Archae approx. 1.5 billion years ago Polyphyletic group- protists arose by way of more than one ancestral group Represents separate evolutionary lineages Plant like b/c autotrophic (produce their own food) Animal-Like b/c they are heterotrophic (feed upon other organisms)

Figure 8.20

TYPES OF PROTOZOANS Zooflagellated Protozoans Flagellated Protozoans Phytoflagellated Protozoans

Zooflagellated Protozoa Lack chloroplast Heterotrophic Some members are important human parasites Species Trypanosoma brucei cause African sleeping sickness (Intermediate host- Tsetse flies )

Figure 8.8 (b)

Flagellated Protozoa Flagellates are the ancestors of ameoboid protozoan Phytoflagellated (photosynthesizing) Zooflagellated (particle feeding and parasitic)

pellicle contractile vacuole macronucleus cytoplasm: ectoplasm endoplasm micronucleus cilia oral groove anal pore gullet food vacuole

Phytoflagellated Protozoa Chlorophyll (oxygen for marine life) One or two flagella These protozoans are large portion of the marine food i.e dinoflagellates Two flagellates, chlorophyll, xanthophyll (bloom=red tides) and results in fill kills (Red sea, bible)

Figure 8.6

Other Phytoflagellated Protozoa Euglena Freshwater phytoflagellated protozoa Chloroplast has a pyrenoid (synthesizes and stores carbohydrates) Feed by absorption or are heterotrophic Stigma- photoreceptor at the base of the flagellum Haploid organisms and reproduce binary fission

FUNGUS LIKE PROTISTS Like fungi, they are heterotrophs, have cell walls, use spores to reproduce. Unlike fungi, they can move at some points in their life cycle. Three types: - Water Molds and Downy Mildews: Both types live in water or moist places, & look like fuzzy threads. They attack food crops (potatoes) - Slime Moulds: Live in moist soil and on decaying plants and trees. Some are with beautiful colors. They move using pseudopods. They eat bacteria and other microorganisms. Can combine, forming a multicellular mass & spores. Spores develop into a new generations of slime moulds

PLANT LIKE PROTISTS Called algae & are autotrophs (pigments & photosynthesis). * Some are unicellular, living unconnected from other algae cells. * Others form colonies together with a few cells specializing for reproduction, etc. (Most colony cells continue to carry out all normal functions.) * Some algae are multicellular like seaweed, where all cells are specialized.

Paramecium Movement The outer surface of the cell is covered with many hundreds of tiny hair-like structures called cilia. These act like microscopic oars to push through the water, enabling the organism to swim. If Paramecium comes across an obstacle, it stops, reverses the beating of the cilia, swims backwards, turns through an angle and moves forward again on a slightly different course. It moves so quickly that we have to add a thickening agent or quieting solution to the slide to slow it down to study it.

Paramecium Feeding Paramecium has a permanent feeding mechanism, consisting of an oral groove and a funnel-shaped gullet into which food is drawn by the combined action of cilia which cover the body and other cilia lining the oral groove and the gullet. As it moves through the water it rotates on its axis and small particles of debris and food are collected and swept into the gullet. They feed on small organisms such as bacteria, yeasts, algae and even other smaller protozoa.

Paramecium Excretion Food waste left in a food vacuole is excreted through the anal pore (the vacuole and pore fuse. Other wastes left over from cellular activity (metabolic waste) simply diffuse through the pellicle. Excess water and some metabolic wastes are excreted through the contractile vacuole.

ASEXUAL REPRODUCTION IN PROTOZOANS

Asexual Reproduction in Protozoa

Conjugation in Paramecium

Paramecium Reproduction In favourable conditions the cell divides in two by a process called binary fission (asexual reproduction). This forms two new cells, each of which rapidly grows any new structures required and increases in size. This whole process may take place two or three times a day if conditions were right.

Paramecium Reproduction This is a more complicated method called conjugation (sexual reproduction). It involves two cells coming together to exchange nuclear material. The two cells then separate and continue to reproduce by simple division. It is similar in some ways to sexual reproduction in more complex animals.

Reproduction in Ciliates Paramecium conjugating Transverse Binary fission

Symbiotic lifestyles Symbiosis Parasitism- a form of symbiosis- organism lives in or on other (Host)

Other kinds of symbiosis Don’t harm host Commensalisms- one member benefits Mutualism- both benefit

Some parasites have life cycles involving multiple hosts Definite host- harbors the sexual stages of the parasite Intermediate host- the offspring enter another host where they reproduce asexually, to complete lifecycle the final asexual stage must have access to a Definite host

Paramecium Grammers Growth: 0.05 mm avg. Respond: -react to chemicals – salt and vinegar -live in slightly acidic environments (stagnant H2O) -anterior end sensitive – move by trial and error

Paramecium continued… Adaptations: Cilia to help feed and escape Contractile vacuoles Trichocysts

Paramecium continued… Movement: by cilia in circular motion, move ~ 60 mm/hr Metabolism: -food pulled into oral groove by cilia -food vacuole forms at gullet -lysosome aids with digestion Feed mostly on bacteria, smaller protozoans and algae.

Paramecium continued… Excretion: -Contractile vacuole removes excess H20. -C02 across pellicle by diffusion -anal pore removes waste. Reproduction: Asexual - cell division Sexual – conjugation (exchange of micronucleus DNA)

CLADOGRAM OF PROTOZOA RELATIONSHIPS

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Endosymbiosis and Cytoplasmic Inheritance in Paramecium

This Topic Will Focus On The Following : Altenburg paper (1948) Plasmagene hypothesis Kappa body symbiosis Current understanding of Kappa bodies (Preer 1974) Other cytoplasmic inheritance in Paramecium (Meyer 2002) Paramecium biology Cell biology Life cycle

Altenburg paper (1948) investigates the evidence that Kappa bodies are a symbiont Kappa bodies are elements within Paramecium that cause them to be killers Killer Paramecium kill other Paramecium in the immediate environment Kappa particles, thought to be plasmagenes by Sonneborn, but Altenburg suggest they may be symbionts

The plasmagene theory suggested kappa bodies were genes within the cytoplasm Plasmagenes defined as self-replicating structure capable of producing traits that exist in the cytoplasm and are independent of chromosomal genes. The trait that Kappa bodies produce is the killing factor Kappa bodies are inherited through the cytoplasm and not through chromosomes Sonneborn wrote in 1976, “It was awful of me to be so attached to a pet idea. That was an ordeal between my mind and my heart and it took a while for the mind to win and the heart to accept. Impersonal scientific objectivity is a goal to be sought by hard self-discipline; we are not born with it.”

Altenburg’s evidence that Kappa bodies are symbionts is strongly supported by evidence Preer (1948) showed Kappa is large enough to see under a light microscope 38o C kills Kappa but not Paramecium Division of Kappa and Paramecium is independent of each other Paramecium with symbiont There is an upper limit of the number of Kappa in Paramecium More likely a symbiont than a parasite

Preer (1974) reviewed the overwhelming evidence that Kappa bodies are symbionts Kappa contains DNA, RNA, protein, and lipids in proportions expected in bacteria Kappa contains electron transport system with cytochromes similar to bacteria and not eukaryotes Electron micrograph of symbionts Cytochrome Glycolysis & Kreb Cycle Electron microscopy clearly showed that Kappa is prokaryotic Electron micrograph of flagellated Kappa

Current information has shown why Kappa induces killing and the different types of bacteria symbiosis Kappa bodies kill other Paramecium by releasing toxins into the environment The presence of the symbiont makes the host resistant to the toxin Kappa bodies are transmitted by the cytoplasm during asexual division Many other types of symbionts found gamma sigma lambda alpha Kappa is the most common delta pi omega mu

The discovery of bacterial symbionts within Paramecium allows for their taxonomic classification Kappa, mu, gamma, and nu are in genera Caedobacter Alpha bodies are in the genera Cytophaga Lambda and sigma are in genera Lyticum Delta bodies are in genera Tectobacter

Differences have been found between Kappa bodies in the same host Some Kappa bodies contain refractile ( R ) bodies R body is a type of inclusion body When genes from one organism are within another organism and are transcribed, a inactive protein may form Magnified image of coiled R body (2)

Kappa bodies may contain ‘R’ bodies and it affects their reproductive capability Non bright Kappa bodies do not contain R bodies but can reproduce Bright Kappa bodies do contain R bodies but cannot reproduce Dividing symbiont Non bright produce other non bright, but occasionally a non bright turns into a bright Toxicity associated only with Brights

There is still unsolved questions regarding Kappa body symbiosis What benefit does Paramecium get from the symbiosis? How does the presence of a Kappa body induce resistance to the toxin? Resistance can be overcome with large toxin dose The presence of Kappa with or without R bodies induces resistance to the toxin

Other types of cytoplasmic inheritance discovered in Paramecium and other ciliates is: Genome-wide DNA rearrangements Mating type Serotypes

Paramecium has a complex cellular biology Eukaryotic Ciliates contain at least 2 nuclei Germ-line micronucleus (MIC) Somatic macronucleus (MAC) MAC is generated from the MIC Extensive genome rearrangements occur in the MAC Diagram of Paramecium

The two nuclei make the life cycle of Paramecium more complicated than other eukaryotes MIC goes through meiosis and the haploid MIC goes through mitosis Result is 4 haploid MIC, but 2 are degraded Paramecium exchange 1 haploid MIC MIC fuse and form diploid MIC and duplicate via mitosis Old MAC degrades and duplicated MIC is processed into new MAC In asexual reproduction, the MIC goes through mitosis and the MAC goes through amitosis

Genome-wide rearrangements of the MAC genome consists of deletion of DNA sequences and chromosome amplification The developing new MAC loses 10 - 95% of the genome depending on the ciliate MAC chromosomes are amplified to a high ploidy level Deletion occurs after an initial amplification of the MIC genome but before the ploidy level is reached

The deletion of DNA is located at specific sequences called internal excised sequences (IES) IES are located in coding and non-coding regions of the MIC genome These sequences are not present in the MAC genome At some point in MAC development, the IES sequences are deleted

The mating type of Paramecium shows maternal inheritance Conjugation of P. caudatum by Yanagi Paramecium has 2 mating types - O and E Both are not determined by genetic differences as they are both produced in homozygous wild-type strains Mating type is the same through asexual reproduction but can change after sexual conjugation and MAC formation After conjugation O cells mostly produce other O cells and E cells produce other E cells

Paramecium mating types do not follow the Mendelian segregation of alleles Mendelian segregation of allelic pairs Maternal inheritance of mating types

Mating types O and E depends on different states of MAC genome Transferring E maternal MAC into O cell causes the progeny to become E Transferring O MAC does not change E cells O is the default mating type E cell O cell E cell Produces Insert E MAC

This differential state of MAC is dependent on the presence of IES in the MAC The mutation mTFE causes O cells to become E This mutation affects the excision of an IES on the G gene The G gene is a surface antigen and the failure of excision causes a nonfunctional protein to be translated Functional - type O excision Mutational retention Nonfunctional - type E MIC G gene MAC G gene

Microinjection studies have shown that the presence of an IES sequence in the MAC inhibits the excision of its homologous IES in the MIC O cells contain G gene in the MAC without its IES (IES-) E cells contain the G gene in the MAC with its IES (IES+) Injecting a plasmid of IES+ G gene into O cell’s MAC created the retention of the IES in the MAC of daughter cells Injection of IES- plasmid did not induce excision The presence of IES in the MAC causes the retention of the IES in subsequent generations after sexual conjugation

Microinjection of IES+ plasmid retains the IES in the MAC genome after autogamy

Meyer (2002) asked, “How can a sequence introduced in one nucleus affect the excision of the homologous sequence in another nucleus?” Two models developed Model 1: Sequence-specific protein factors are required for the excision of the IES in the developing MAC The problem with this model is the large number of protein factors needed, about 50,000 Model 2: Sequence specificity is achieved by homologous nucleic acid (most likely RNA) that is transported from the maternal MAC to the developing MAC

Mochizuki (2004) explained the Scanning Model, a synthesis of Meyer’s model 1 and 2 Entire MIC genome is transcribed bi-directionally and forms dsRNA dsRNA is cut up into smaller RNA called scn RNA Scn RNA move to the old MAC and any matching homologous sequences are degraded Scn RNA that were not degraded move to the developing MAC These scn RNAs target homologous sequences which are deleted in an RNA i-like mechanism

Polymorphic lifestyles Have different forms during their life cycle May form cysts (vegetative cells) when adverse conditions exist. Cysts are not heat and chemically resistant.

SUMMARY Paramecium has many instances of cytoplasmic and maternal inheritance Paramecium Kappa bodies are bacterial symbionts that produce a killing factor and they are inherited through the cytoplasm IES excision and retention in the MAC is maternally inherited by the genome present in the MAC Electron micrograph of Kappa

Ecological importance Members of the food chain – Primary or Secondary consumers Consume soil bacteria & algae (1 paramecium can ingest 5 million bacteria/day Involved in sewage disposal by metabolizing nutrients present to carbon dioxide & water

HARMS Cause disease in host organisms Malaria – Plasmodium via mosquito Toxoplasmosis – Toxoplasma African Sleeping Sickness – Trypanosoma via tsetse fly Chagas – Toxoplasma Vaginitis – Trichomonas Giardiasis – Giardia 150 million people/year in world contract Malaria & 1.5 mill/year die of it.

FACTS ABOUT PARAMECIUM “One scientist calculated that if all the progeny of a single Paramecium survived, assuming a division rate of once a day, then after 113 days, the mass of paramecia would equal the volume of the Earth! “