Biology of parasitic protozoa VII. Ciliophora, Opalinata (SAR) Andrea Bardůnek Valigurová andreav@sci.muni.cz Notice This presentation contains some material available on the web without the permission of the creator or the copyright owner. This presentation is to be used for educational purposes only. Educational purposes are defined as a communicating material for a particular course of instruction or for the administration of such course. Educational purposes do not cover the use of copyright material in PowerPoint for public lectures or other purposes. 5 supergroups = megagroups https://doi.org/10.1016/j.cub.2013.10.055 Ciliates https://doi.org/10.1016/j.cub.2013.10.055 Alveolataa https://doi.org/10.1016/j.cub.2013.10.055 Alveolata x Myzozoa Myzozoa Alveolata https://doi.org/10.3389/fmicb.2017.01594 Ciliophora • monophyletic taxon • quickly moving protists • files or rows of cilia on cell surface • cell surface covered with cortex • nuclear dualism - larger macronucleus (somatic, physiologically active, containing thousands of copies of genes) and smaller micronucleus (diploid, a germ nucleus whose meiotic products are exchanged during conjugation) • sexual reproduction = conjugation • asexual reproduction by binary fission (mostly transverse) • about 8,000 representatives (parasitic approx. 2,500) • mostly free-living in ponds, lakes, estuaries, saltmarshes, oceans • ectosymbionts on surface of animals (Trichodina on the gills and fish skin), cavity parasites and commensals (Balantidium in intestine, ciliates in rumen), tissue parasites (Ichthyophthirius multifiliis on fish skin) Impregnation of cytoskeletal structures with silver Ciliature • cilia typically arranged in longitudinal rows = kineties • exceptionally: i. lacking cilia ii. ciliated only in some phase of life cycle iii. cilia distributed on the cell surface irregularly • undulating membrane = longitudinal row of paired cilia • membranella = plate-like a cluster of cilia • cirrus = finger-like cluster of cilia Cortex • cell's own covering (pellicle) = unit membrane, subpellicular alveoli (Alveoata) • infraciliature = kinetosomes of cilia + associated microtubular and fibrillar formations: i. striated kinetodesmal fibrils ii. postciliary bands of microtubules iii. transverse bands of microtubules • basal microtubules courses beside the kinetosomes but is not directly connected to them Tetrahymena thermophila stained for tubulin (green), centrin (red) and DNA (blue) Generalised somatic cortex of a ciliate Cortex of a “typical” ciliate can be divided into somatic and oral regions. Somatic region (composed of a “skeletal” support opposed by the hydrostatic pressure within cell) functions in locomotion, sensing, attachment, and secretion of protective coverings, while oral region in sensing, acquiring, and ingesting nutrients. Nuclear dualism Macronucleus • excess of chromosomes = polyploidy (20 to more than 200n) • vegetative nucleus with an incomplete genome (e.g. only 5%) • 2,300 kb DNA = "gene sized fragments", equipped with telomeres) • number of nucleoli, active synthesis of RNA • forming from a micronuclear derivative after conjugation • unknown division of mechanism (amitosis?) Micronucleus • classic diploid nucleus • intranuclear spindle during division • amicronucleate clones incapable of conjugation Conjugation • sexual process - compatible mating strains meet and partly fuse (gamonts are morphologically indistinguishable) • cytoplasmic bridge forms between the gamonts (conjugants) • micronucleus undergoes meiotic division into 4 haploid micronuclei per cell  3 degenerate and 4th undergo mitosis and divides into stationary and migratory pronuclei • conjugants exchange migratory pronucleus and separate, old macronucleus degenerates • pronuclues merges with a stationary pronucleus  synkaryon which divides mitotically • new macronucleus forms from one new micronucleus • binary fission  identical daughter cells Conjugation (in Paramecium) Kinds of binary fission in ciliates Food intake • most ciliates are phagotrophs = absorption of food particles • combination of pinocytosis and osmosis in some ciliates • cell mouth = cytostome (cs) • vestibulum (v) at the bottom of the oral cavity (bc) or infundibulum (inf)  filter feeding more efficient • peristome (ps) • cytopharynx (cph) behind the mouth • cytopyge (cytoproct) to discharge the waste (undigested residues) • osmoregulation via pulsating vacuoles Variability in the oral structures of ciliates Postciliodesmatophorea • macronucleus does not divide and arises de novo from micronucleus, or when dividing, MT spindle are outside the nucleus, small group Intramacronucleata • macronucleus divides, MT spindle inside of dividing nucleus, most ciliates • oldest - based on somatic ciliature • later - oral ciliature: Kinetophragminophorea, Oligohymenophorea, Polyhymenophorea • recent - type of infraciliature, whether the macronucleus divides and whether during its division the MT spindles are inside or outside the macronucleus Classification of Ciliophora Intramacronucleata Trichodinidae • ecto commensals × ectoparasites • direct cycle • transmission by direct contact • indicator of poor water quality • pathogens of aquarium fish (infected fish often rubs against objects in the aquarium and gradually weakens) • spiral cilia around the cytostome = identification feature • binary fission • genera Trichodina, Trichodinella, Hemitrichodina, Paratrichodina, Tripartiella, Vauchomia,… genus Trichodina Trichodina heterodentata https://doi.org/10.1017/S0031182013001480 Prochilodus lineatus larvae highly parasitised by Trichodina heterodentata. A) Oedema (asterisk) in the secondary lamella, parasite (arrow). B) Discrete hyperplasia with focal fusion of secondary lamella (arrow), discrete mononuclear inflammatory infiltrate and oedema. C) Trichodinids (arrows) present inside and outside the operculum cavity (oc) showing no altered epithelium. D) Parasite (arrow), and pocket scale without alteration (arrow head). Abundant parasites (arrows) on the fish body surface. Abundant parasites on eyes (white arrow) with suction areas (arrowhead) (A) and on skin near the nostril with many suction areas (B); ulceration on dorsal fin with cocci attached (C, arrows) and numerous bacteria surrounding the ciliate (D, arrows). https://doi.org/10.1080/03079457.2016.1153798 Phallus. Trichodinids associated with acanthosis, superficial heterophilic and submucosal lymphoplasmacytic inflammation. Chilodonellidae genus Chilodonella • prominent cytopharynx with 3 circumoral kineties; ventral ciliary rows (kineties) forming 2 longitudinal bands close to the body margins • parasitising fish skin and gills - break down the host surface and feeding on the debris  emaciation, loss of condition, hypoxia, lethargy and even death; heavy infections - parasites cover almost the entire host surface Carp with heavy Chilodonella infection, displaying excess mucus Chilodonella hexasticha https://doi.org/10.1016/j.vetpar.2018.10.009 https://doi.org/10.1016/j.vetpar.2012.07.030 Chilodonella hexasticha Histopathological sections of Carassius auratus gills. Chilodonellids scattered over gill filaments (A-B, arrows) and located between or at the tips of lamellae (C-D, arrows), resulting in severe fusion of lamellae (B-C, yellow double arrow) and desquamation of epithelium cells (B, arrowhead). Necrosis of the epithelium between gill lamellae evident in some areas (D, asterisk). HE. Histopathology of the gills of Piaractus mesopotamicus parasitised by Chilodonella hexasticha. Epithelial hyperplasia (A-F), lamellar fusion (B-F), necrosis (C - arrows; F asterisk), cellular desquamation (D - asterisk), interstitial hemorrhage (E), and chilodonellids (D, F - arrows). HE. https://doi.org/10.1016/j.vetpar.2018.10.009 Chilodonella uncinata • cosmopolitan parasite of freshwater fish gills and skin • suspected facultative endoparasite of mosquito larva (potential biocontrol of medically important mosquito vectors ???) https://www.youtube.com/watch?v=TPrLu8-H_h4 https://www.youtube.com/watch?v=e-btCEUR-hYhttps://www.jstor.org/stable/24108543 https://actascientific.com/ASMI/pdf/ASMI-02-0435.pdf Ichthyophthiriidae Ichthyophthirius multifilis • only species within the genus • infecting most freshwater fish species • feeding stage = macroscopically visible trophonts (trophozoite resides inside host = endoparasite, visible as a white spot on the surface of the fish) (up to 1 mm in diameter) • using cytostome, trophont eats away the corridors in the skin, gill or fin epithelium of fish • encystation and rapid division (within cyst) to form tomites inside the cyst at the bottom • tomites transform into theronts with a perforatorium that are looking for a new fish host Ichthyophthirius multifilis https://www.youtube.com/watch?v=cOfPk3i0clw&t=3s Life cycle of Ichthyophthirius multifilis https://doi.org/10.1111/raq.12708 1) Theront invades host epidermis and grows into a trophont. 2) Trophont leaves host to become a protomont, which can swim freely. 3) Protomont secretes a cyst wall to become a tomont. Tomont undergoes binary fission, producing large numbers of tomites. 4) Tomites transform into theronts that break out from the cyst wall to find new host. c - cilia; cc, caudal cilium; cv, contractile vacuole; fv - food vacuole; ma - macronucleus; mi micronucleus; oa - oral apparatus. Detailed processes of a tomont's division. A) Tomonts. B) One-cell period. C) Two-cell period. D) Fourcell period. E) Eight-cell period. F) Sixteen-cell period. G) Multi-cell period. H) Theront. Symptoms of ichthyophthiriasis • trophonts, continuously rotating, are surrounded by host epidermal cells and leukocytes, producing a minute elevation of the skin (light-reflecting nodules) = white spot disease • infection challenges hosts’ osmoregulation and respiration + secondary bacterial and fungal infections Clinical signs: anorexia, hyperventilation, discoloration, abnormal behaviour (inactivity, isolation), resting on the bottom, flashing (rubbing and scratching against objects), balance disturbance, upside-down swimming near the surface A) Histology of gills with widespread lamellar epithelial hyperplasia with lamellar fusion (asterisks). B) Subepithelial ciliated protozoal cysts (arrows) contain a characteristic crescent-shaped macronucleus (asterisks) with abundant phagocytized erythrocytic and cellular debris. Pathology of ichthyophthiriasis Cryptocaryonidae Cryptocaryon irritans • only species within the genus • originally classified as Ichthyophthirius marinus • invading epithelium of fish skin, fins, gills and even the eyes • feeding on the body fluids, tissue debris and whole cells of its host • marine white spot disease or „marine ich (pronounced ick)“ • symptoms and life-cycle are generally similar to those of I. multifilis • most common agens of disease in marine aquaria https://www.youtube.com/watch?v=wfa25MKR0nY Life cycle of Cryptocaryon irritans Life stages of Cryptocaryon irritans A) 2 trophonts settling in a gill cavity B) early toront C) later tomont phase D) theronts https://doi.org/10.1111/raq.12594 Symptoms of Cryptocaryon irritans infection Infected fish hover just under the water surface (A) are lethargic and unresponsive, suddenly scratch their body on the bottom / wall of the tank or net cage, breathe more rapidly and lose their appetite. Clinical signs: pinhead-sized white nodules on the gills and body (B-D), mucus hyperproduction, skin discoloration, corneal cloudiness, ragged fins and pale gills  often fatal due to asphyxiation, osmotic imbalance and/or secondary bacterial infections https://doi.org/10.1111/raq.12594 https://doi.org/10.3390/ijms23020937 Cryptocaryon irritans trophonts in the gills of gilthead seabream. Macroscopical and binokulár representatives of healthy (control in A, C) and infected (B, D) gilthead seabream. Gill sections stained from control (E) and infected (F-H) gilthead seabream. Note the parasite adhering to the gills (F). Black arrows point to skin injuries during C. irritans infection. Grey arrows and black arrow heads point to C. irritans trophonts. White arrows point to the undifferentiated tissue between the secondary lamellae. Red arrows point to eosinophilic cells. Pathology of Cryptocaryon irritans infection Sessilida - Epistylididae genus Epistylis • urn-shaped peritrich ciliate • mature zooids or trophonts sedentary or sessile • ring of cilia on the distal end • rigid dichotomous stalk, attaches by a disk to host‘s hard surfaces (spines, scales, gill) • often branching to form a colony • skin, fins, and gills of fish • ectocommensals x ectoparasites = feeding primarily on bacteria and organic material, but eroding scales and hard spines of fins  irritation and inflammation of host epithelium at the attachment point  site for secondary infections of Aeromonas hydrophila • except for injury incurred by attachment, they are seldom harmful to the host unless there is massive infection Epistylis sp. A) in fresh-mounted slide under LM. B) Zooid in binary fission and detail of bacteria (arrow heads) attached on the peduncle of the ciliate. C) Oreochromis niloticus with fin erosion and loss of scale (arrowheads) associated with Epistylis colonisation. D) Pseudoplatystoma sp. showing total erosion of dorsal fin and Epistylis colonies on the dorsal fin and head (arrow heads). https://doi.org/10.1590/S1984-29612015013 Balantidiidae Balantidium coli • Neobalantidium coli, Balantioides coli • trophozoite (50 x 200 µm), cyst (50 x 60 µm) • colonising lumen of the large intestine and caecum • commensal, but under certain circumstances, acting as an opportunistic pathogen  invasive, attacking the intestinal wall, rarely extraintestinal infections • primary reservoir: pig (usually without symptoms) • humans can also be reservoirs, and other potential animal hosts include rodents, dogs, nonhuman primates and birds • in older literature, human balantidiosis also known as "butchers' disease" = referring to its frequent occurrence in butchers who often come into contact with the contents of the pig's colon Ma Ma cytostome cilia Mi Mi contractile vacuole Life cycle of Balantidium coli Morphology of B. coli life stages A) Trophozoite stained with Lugol’s iodine. Note the macronucleus (N) and vacuoles (V). B) HE stained histological section of pig intestine showing 3 trophozoites; cytostome (C), macronucleus (N). C) Cysts from gorillan stained with Lugol’s iodine. Macronucleus (N) is visible in some cysts. D-F) Cysts stained with Lugol’s iodine; macronucleus (N), vacuoles (V) varying from 0-6 or more.trofozoite cyst • excystation in small intestine • often asymptomatic • mild chronic form: diarrhoea, constipation, abdominal pain (many years) • acute form: dysentery (blood and mucus in stool) • extraintestinal infection is rare but potentially serious, typically secondary to intestinal infection • rupture of fulminant colonic ulcers, rarely intestinal perforation  peritonitis and liver abscesses – can be fatal • invasion of urogenital tract due to contamination from anal region or through fistulae caused by severe infection Human balantidiasis Virulence factor: hyaluronidase helping B. coli to penetrate intestinal mucosa Pathology of human balantidiasis Balantidium coli trophozoites among necrotic debris and acute inflammatory cells found in colonic ulcers. Urinary balantidiasis Skin condition after treatment with metronidazole and apremilast. Skin condition of the infected patient before the systemic treatment for psoriasis and balantidiasis was started. B. coli was also incidentally found in the urine of a female after being in contact with infected pig. This patient had numerous comorbidities  immunocompromise suspected key factor in urinary balantidiasis DOI: 10.4172/2472-1212.1000177 Accidental finding of B. coli in urine of patient with acute renal failure. Microscopic examination showing few red cells and ovoid to oblong ciliated parasites. https://www.jcdr.net/article_fulltext.asp?id=4343 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8957295 https://doi.org/10.1016/j.actatropica.2021.106069 https://doi.org/10.1590/S1984-29612019056https://doi.org/10.4142/jvs.2006.7.2.207 • B. coli is frequently associated with enteric diseases in man and nonhuman primates, with rare disease manifestations of swine or other mammalians B. coli typhlocolitis in horses A) grossly characterised by diffusely reddened mucosa of the cecum and ventral large colon. B) Mucosa of large colon and cecum present superficial necrosis associated with multiple trophozoites of B. coli. HE. Pathology of animal balantidiasis https://doi.org/10.1016/j.vetpar.2008.08.013 Colon of Finnish horse with balantidiasis. A) Severe haemorrhage and destruction of mucosa with congestion and oedema at the submucosa. B) Ciliated protozoan invading the colonic surface and CD) are embedded within the colonic mucosa. HE. A B B. coli in duct of gastric lymph node of Barbary sheep Trophozoite of B. coli in submucosa of the abomasum of Barbary sheep. 5 supergroups = megagroups Proteromonadea • 1-2 pairs of anisokont cilia; uninucleate endobionts in intestinal tract of amphibians, reptiles and mammals Proteromonas lacertae • model species in intestine of lizards, 2 flagella Karotomorpha bufonis • intestine of toads, 4 flagella Opalinea Blastocystis Opalinata • formerly Slopalinida • multiciliated – cilia (flagella) without tubular hairs evenly spaced cortical ridges underlain by microtubules, ranging from singlets to ribbons • cyst forming; many osmotrophic in vertebrate guts Opalinea • unusual protists with large cells, previously placed in Ciliophora • name derived from iridescent appearance when light reflects on their delicately folded surface • flagella originating from an anterior morphogenetic centre = falx, organised in oblique longitudinal rows or files • microtubular ribbons supporting longitudinal pellicular ridges between ciliary rows • 2 to hundreds monomorphic nuclei • intestinal endobionts of amphibians and some fish • life cycle complex - sexual process induced by host hormones and linked to its life cycle • genera Cepedea, Opalina, Protoopalina, Protozelleriella, Zelleriella 1) Cysts are excreted by adult frog and ingested by a tadpole. 2) After hatching the young gamont migrates to cloaca. 3-4) Formation of micro- and macrogametes (meiosis). 5) Fusion of the heterogametes. 6) Encystation of the zygote and excretion via faeces. 7-8) After oral uptake of a cyst by another tadpole the trophozoite grows up in the cloaca (up to 0.5 mm). 8.1-8.2) Inside tadpole the small trophozoite may start division, leading to formation and excretion of cysts (1) giving rise to new trophozoites (2-8) after ingestion by another tadpole. 9) After tadpole metamorphosis completion, trophozoites (agamonts, trophonts) grow up and form up to 2,000 nuclei. 10) During frog nonbreeding season, trophozoites multiply by binary fission (either longitudinal or oblique-transverse). 11-12) During breeding season frog hormones induce rapid trophozoites' divisions without compensatory nuclear divisions and growth. Thus the parasites (precystic forms) become successively smaller. These stages, finally having 2–12 nuclei, encyst (1), are set free with the faeces of the host, and become infectious for tadpoles. CI - cilia, CW - cyst wall, N - nucleus Life cycle of Opalina ranarum Opalinea • Opalina (flattened, 1a) and Cepedea (cylindrical; 1b, 6a) are multinucleate genera • Zelleriella (flattened; 1c) and Protoopalina (cylindrical; 1d, 6b) have 2 nuclei Opalina ranarum • model organism https://www.youtube.com/watch?v=U6thaFVYhAM&t=2s Cepedea longa https://doi.org/10.1051/parasite/2017006 A) Cepedea trophonts; B) Opalina (left) and Cepedea (right) trophonts; C) putative protrophont of Cepedea; D) Cepedea trophont white arrow - direction of movement of metachronal waves, yellow arrow - nuclei white arrow - nuclei, red arrow - basal bodies with flagella arranged in oblique rows, black arrow - falx (3-4 basal bodies visible in one row = Cepedea) α-tubulin F-aktin nuclei • α-tubulin immunolabeling: strong signal in the flagella and area of the pellicle between individual rows of flagella • longitudinal folds between the rows of flagella are supported by bands of microtubules • rows of flagella supported on the left side by a strip of microfilaments (F-actin) α-tubulin F-aktin nuclei 5 supergroups = megagroups genus Blastocystis • single genus belonging to extremely diverse (genetically and phenotypically) group of Stramenopiles • no flagella, usually observed in the form of spherical cells with huge vacuoles • quite common in the intestines of many vertebrates (including humans) and invertebrates • classic form found in human stools is the cyst with 6-40 μm in diameter • potentially associated with human intestinal pathology (usually referend to as B. hominis), in some cases also with skin problems such as urticaria – but clinical significance of Blastocystis is unclear • often connected with irritable bowel syndrome and other gastrointestinal symptoms – not clear whether Blastocystis can cause these disorders or is just more efficient in colonisation of the altered environment of unhealthy intestine • more complicated by the fact that Blastocystis genetically very variable (some subtypes are more pathogenic than other ones) - based on analysis of ribosomal genes 17 subtypes (subtypes arguably represent separate species) were identified  9 in humans • detected in both symptomatic and asymptomatic persons • no effective therapeutic strategy Blastocystis spp. Blastocystis life cycle Blastocystis life cycle A) Vacuolar forms of Blastocystis with large centrally placed vacuole that shows extensive variation in size (arrows). B) Granular forms with distinct granules filling the central body. C) Amoeboid form with characteristic pseudopodia. D) Cyst forms. Note the smaller size and the characteristic refractile cyst wall surrounded by loose irregular outer coat. Morphological forms of Blastocystis https://doi.org/10.1016/j.pt.2013.08.006 Possible interactions and explanations for Blastocystis carriage and symptomatic status of the host https://doi.org/10.1186/s13071-022-05418-0 https://doi.org/10.1186/s13071-022-05418-0 Thank you for your attention ☺ Lectures ✓ Introduction: BPP 2022 I ✓ Euglenozoa (Excavata): BPP 2022 II ✓ Fornicata / Preaxostyla / Parabasala (Excavata): BPP 2022 III ✓ Apicomplexa I (SAR): BPP 2022 IV ✓ Apicomplexa II (SAR): BPP 2022 V ✓ Amoebae (Excavata, Amoebozoa): BPP 2022 VI ✓ Ciliophora, Opalinata (SAR): BPP 2022 VII  Pneumocystis (Opisthokonta, Fungi): BPP 2022 VIII • Microsporidia (Opisthokonta, Fungi): BPP 2022 IX • Myxozoa (Opisthokonta, Animalia): BPP 2022 X