In this paper, the growth requirements, fermentation pattern, and hydrolytic enzymatic activities of anaerobic ciliates collected from the hindgut of the African tropical millipede Archispirostreptus gigas are described. Single-cell molecular analysis showed that ciliates from the millipede hindgut could be assigned to the Nyctotherus velox and a new species named N. archispirostreptae n. sp. The ciliate N. velox can grow in vitro with unspecified prokaryotic populations and various plant polysaccharides (rice starch-RS, xylan, crystalline cellulose20-CC, carboxymethylcellulose-CMC, and inulin) or without polysaccharides (NoPOS) in complex reduced medium with soluble supplements (peptone, glucose, and vitamins). Specific catalytic activity (nkat/g of protein) of α amylase of 300, xylanase of 290, carboxymethylcellulase of 190, and inulinase of 170 was present in the crude protein extract of N. velox. The highest in vitro dry matter digestibility was observed in RS and inulin after 96 h of fermentation. The highest methane concentration was observed in xylan and inulin substrates. The highest short-chain fatty acid concentration was observed in RS, inulin, and xylan. In contrast, the highest ammonia concentration was observed in NoPOS, CMC, and CC. The results indicate that starch is the preferred substrate of the N. velox. Hydrolytic enzyme activities of N. velox showed that the ciliates contribute to the fermentation of plant polysaccharides in the gut of millipedes.

Institute of Animal Physiology Centre of Biosciences Slovak Academy of Sciences 040 00 Košice Slovakia

Institute of Hydrobiology Biology Centre AS CR 370 05 České Budějovice Czech Republic

Institute of Soil Biology and Biogeochemistry Biology Centre AS CR 370 05 České Budějovice Czech Republic

Zobrazit více v PubMed

Albaret J.L. Étude systématique et cytologique sur les ciliés hétérotriches endocommensaux (Systematic and cytological study on heterotrichous endocommensal ciliates) Mémoires Muséum Natl. D’histoire Nat. Ser. A Zool. 1975;89:1–114.

Gijzen H.J., Broers C.A., Barughare M., Stumm C.K. Methanogenic bacteria as endosymbionts of the ciliate Nyctotherus ovalis in the cockroach hindgut. Appl. Environ. Microbiol. 1991;57:1630–1634. doi: 10.1128/aem.57.6.1630-1634.1991. PubMed DOI PMC

Gijzen H.J., Barugahare M. Contribution of anaerobic protozoa and methanogens to hindgut metabolic activities of the American cockroach, Periplaneta americana. Appl. Environ. Microbiol. 1992;58:2565–2570. doi: 10.1128/aem.58.8.2565-2570.1992. PubMed DOI PMC

Gijzen H.J., van der Drift C., Barugahare M., Op den Camp H.J. Effect of Host Diet and Hindgut Microbial Composition on Cellulolytic Activity in the Hindgut of the American Cockroach, Periplaneta americana. Appl. Environ. Microbiol. 1994;60:1822–1826. doi: 10.1128/aem.60.6.1822-1826.1994. PubMed DOI PMC

De Graaf R.M., Ricard G., van Alen T.A., Duarte I., Dutilh B.E., Burgtorf C., Kuiper J.W.P., van der Staay G.W.M., Tielens A.G.M., Huynen M.A., et al. The Organellar Genome and Metabolic Potential of the Hydrogen-Producing Mitochondrion of Nyctotherus ovalis. Mol. Biol. Evol. 2011;28:2379–2391. doi: 10.1093/molbev/msr059. PubMed DOI PMC

Balch H.E. The Cultivation of Nyctotherus ovalis and Endamoeba blattae. Science. 1967;76:237. doi: 10.1126/science.76.1967.237.a. PubMed DOI

Nelson E.C. Cultivation of Nyctotherus cordiformis. J. Parasitol. 1943;29:292. doi: 10.2307/3272764. DOI

Lom J. Experiments with the cultivation of our three species of the genus Nyctotherus and of Balantidium entozoon and B. coli. Věst. Českoslov. Zool. Spol. 1956;20:16–61.

Chen L. Züchtungsversuche an parasitischen Protozoen von Periplaneta orientalis. Zeitschrift. Parasitenkd. 1933;6:207–219. doi: 10.1007/BF02122064. DOI

Byzov B.A. Intestinal Microbiota of Millipedes. In: König H., Varma A., editors. Intestinal Microorganisms of Termites and Other Invertebrates. Springer; Berlin/Heidelberg, Germany: 2006. pp. 89–114. DOI

Šustr V., Tajovský K., Semanová S., Chroňáková A., Šimek M. The giant African millipede, Archispirostreptus gigas (Diplopoda: Spirostreptida), a model species for ecophysiological studies. Acta Soc. Zool. Bohem. 2013;77:145–158.

Nardi J.B., Bee C.M., Taylor S.J. Compartmentalization of microbial communities that inhabit the hindguts of millipedes. Arthropod Struct. Dev. 2016;45:462–474. doi: 10.1016/j.asd.2016.08.007. PubMed DOI

Taylor E.C. Role of Aerobic Microbial Populations in Cellulose Digestion by Desert Millipedes. Appl. Environ. Microbiol. 1982;44:281–291. doi: 10.1128/aem.44.2.281-291.1982. PubMed DOI PMC

Šustr V., Semanová S., Rost-Roszkowska M.M., Tajovský K., Sosinka A., Kaszuba F. Enzymatic activities in the digestive tract of spirostreptid and spirobolid millipedes (Diplopoda: Spirostreptida and Spirobolida) Comp. Biochem. Physiol. Part B Biochem. Mol. Biol. 2020;241:110388. doi: 10.1016/j.cbpb.2019.110388. PubMed DOI

Regensbogenova M., Kisidayova S., Michalowski T., Javorsky P., Moon-Van Der Staay S.Y., Moon-Van Der Staay G.W.M.M., Hackstein J.H.P., Mcewan N.R., Jouany J.-P., Newbold J.C., et al. Rapid identification of rumen protozoa by restriction analysis of amplified 18S rRNA gene. Acta Protozool. 2004;43:219–224.

Katoh K., Standley D.M. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol. Biol. Evol. 2013;30:772–780. doi: 10.1093/molbev/mst010. PubMed DOI PMC

Hall T.A. BioEdit: A User-Friendly Biological Sequence Alignment Editor and Analysis Program for Windows 95/98/NT. Information Retrieval Ltd.; London, UK: 1999. pp. 95–98. (Nucleic Acids Symposium Series).

Ronquist F., Teslenko M., Van Der Mark P., Ayres D.L., Darling A., Höhna S., Larget B., Liu L., Suchard M.A., Huelsenbeck J.P. MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 2012;61:539–542. doi: 10.1093/sysbio/sys029. PubMed DOI PMC

Guindon S., Dufayard J.F., Lefort V., Anisimova M., Hordijk W., Gascuel O. New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0. Syst. Biol. 2010;59:307–321. doi: 10.1093/sysbio/syq010. PubMed DOI

Swofford D.L. PAUP*: Phylogenetic Analysis Using Parsimony and Other Methods, Version 4. Sinauer Associates; Sunderland, MA, USA: 2003.

Foissner W. An update of “basic light and scanning electron microscopic methods for taxonomic studies of ciliated protozoa”. Int. J. Syst. Evol. Microbiol. 2014;64:271–292. doi: 10.1099/ijs.0.057893-0. PubMed DOI

Foissner W. Basic light and scanning electron microscopic methods for taxonomic studies of ciliated protozoa. Eur. J. Protistol. 1991;27:313–330. doi: 10.1016/S0932-4739(11)80248-8. PubMed DOI

Doddema H.J., Vogels G.D. Improved identification of methanogenic bacteria by fluorescence microscopy. Appl. Environ. Microbiol. 1978;36:752–754. doi: 10.1128/aem.36.5.752-754.1978. PubMed DOI PMC

Williams A.G., Coleman G.S. The Rumen Protozoa. Springer; New York, NY, USA: 1992. DOI

Kišidayová S., Váradyová Z., Zeleňák I., Siroka P. Methanogenesis in rumen ciliate cultures of Entodinium caudatum and Epidinium ecaudatum after long-term cultivation in a chemically defined medium. Folia Microbiol. 2000;45:269–274. doi: 10.1007/BF02908958. PubMed DOI

Hino T., Kametaka M., Kandatsu M. The cultivation of rumen oligotrich protozoa. III. White clover factors which stimulate the growth of Entodinia. J. Gen. Appl. Microbiol. 1973;19:397–413. doi: 10.2323/jgam.19.397. DOI

Coleman G.S. Rumen entodiniomorphid protozoa. In: Taylor A.E.R., Baker J.R., editors. Methods of Cultivating Parasites In Vitro. Academic Press Inc.; London, UK: 1978. pp. 39–54.

Horáková K. Mikroskopické metódy (Microscopic methods) In: Betina V., Barátová H., Fargašová A., Frank V., Horáková K., Šturdík E., editors. Mikrobiologické Laboratórne Metódy (Microbiological Laboratory Methods) Alfa; Bratislava, Slovakia: 1988. pp. 173–206.

Grishagin I.V. Automatic cell counting with ImageJ. Anal. Biochem. 2015;473:63–65. doi: 10.1016/j.ab.2014.12.007. PubMed DOI

Abramoff M.D., Magalhães P.J., Ram S.J. Image processing with ImageJ. Biophotonics Int. 2004;11:36–42.

Kišidayová S., Váradyová Z. Effect of insulin on in vitro fermentation activity of microrganism community of rumen ciliate culture. Cell Biol. Int. 2005;29:147–152. doi: 10.1016/j.cellbi.2004.10.002. PubMed DOI

Cottyn B.G., Boucque C.V. Rapid method for the gas-chromatographic determination of volatile fatty acids in rumen fluid. J. Agric. Food Chem. 1968;16:105–107. doi: 10.1021/jf60155a002. DOI

Chaney A.L., Marbach E.P. Modified Reagents for Determination of Urea and Ammonia. Clin. Chem. 1962;8:130–132. doi: 10.1093/clinchem/8.2.130. PubMed DOI

Mellenberger R.W., Satter L.D., Millett M.A., Baker A.J. An in vitro Technique for Estimating Digestibility of Treated and Untreated Wood. J. Anim. Sci. 1970;30:1005–1011. doi: 10.2527/jas1970.3061005x. DOI

Nevel C.J., Demeyer D.I. Stoichiometry of carbohydrate fermentation and microbial growth efficiency in a continous culture of mixed rumen bacteria. Eur. J. Appl. Microbiol. Biotechnol. 1979;7:111–120. doi: 10.1007/BF00505016. DOI

Ungerfeld E.M. Shifts in metabolic hydrogen sinks in the methanogenesis-inhibited ruminal fermentation: A meta-analysis. Front. Microbiol. 2015;6:37. doi: 10.3389/fmicb.2015.00037. PubMed DOI PMC

Čársky J. Príprava presných, tlmivých a izotonických roztokovo (Preparation of precise, buffered and isotonic solutions) In: Ferenčík M., Škárka B., editors. Biochemické Laboratórne Metódy (Biochemical Laboratory Methods) ALFA-VTEL; Bratislava, Slovakia: 1981. pp. 39–71.

Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976;72:248–254. doi: 10.1016/0003-2697(76)90527-3. PubMed DOI

Miller G.L. Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Anal. Chem. 1959;31:426–428. doi: 10.1021/ac60147a030. DOI

Bailey M.J., Biely P., Poutanen K. Interlaboratory testing of methods for assay of xylanase activity. J. Biotechnol. 1992;23:257–270. doi: 10.1016/0168-1656(92)90074-J. DOI

Béra-Maillet C., Devillard E., Cezette M., Jouany J.-P., Forano E. Xylanases and carboxymethylcellulases of the rumen protozoa Polyplastron multivesiculatum, Eudiplodinium maggii, and Entodinium sp. FEMS Microbiol. Lett. 2005;244:149–156. doi: 10.1016/j.femsle.2005.01.035. PubMed DOI

Miller G.L., Blum R., Glennon W.E., Burton A.L. Measurement of carboxymethylcellulase activity. Anal. Biochem. 1960;1:127–132. doi: 10.1016/0003-2697(60)90004-X. DOI

Small E.B., Marszalek D.S., Antipa G.A. A Survey of Ciliate Surface Patterns and Organelles as Revealed with Scanning Electron Microscopy. Trans. Am. Microsc. Soc. 1971;90:283. doi: 10.2307/3225188. DOI

Imai S., Katsuno M., Tsunoda K. Scanning Electron Microscopy of Rumen Ciliates in Cattle. Zool. Mag. 1977;86:194–207.

Wang Q., Zhao X. A three-dimensional phase diagram of growth-induced surface instabilities. Sci. Rep. 2015;5:8887. doi: 10.1038/srep08887. PubMed DOI PMC

Lynn D.H. The implications of recent descriptions of kinetid structure to the systematics of the ciliated protists. Protoplasma. 1991;164:123–142. doi: 10.1007/BF01320819. DOI

De Beaurepaire Aragão B. Noticia sobre o Nyctotherus cordiformis Stein. Mem. Inst. Oswaldo Cruz. 1912;4:125–129. doi: 10.1590/S0074-02761912000100009. DOI

Pinto C. Anatomia e biologia dos Nyctotherus do batrachios do Brasil. Bol. Biol. 1926;3:45–48.

Nelson E.C. Alcohol–preserved tissue–cultivation medium: Methods of preparation and use and results obtained in the cultivation of Nyctotherus cordiformis. Am. J. Epidemiol. 1943;38:185–192. doi: 10.1093/oxfordjournals.aje.a118880. DOI

Lucas C.L.T. A Study of Excystation in Nyctotherus ovalis: With Notes on Other Intestinal Protozoa of the Cockroach. J. Parasitol. 1928;14:161. doi: 10.2307/3271996. DOI

Kudo R.R. Studies on Nyctotherus ovalis Leidy, with special reference to its nuclear structure. Arch. Protistenkd. 1936;87:10–42.

Suzuki J., Kobayashi S., Yoshida N., Azuma Y., Kobayashi-Ogata N., Kartikasari D.P., Yanagawa Y., Iwata S. Phylogenetic position of Nyctotherus teleacus isolated from a tortoise (Astrochelys radiata) and its electron microscopic features. J. Vet. Med. Sci. 2020;82:699–703. doi: 10.1292/jvms.20-0004. PubMed DOI PMC

Hoyte H.M.D. The protozoa occurring in the hind-gut of cockroaches. I. Responses to changes in environment. Parasitology. 1961;51:415–436. doi: 10.1017/S0031182000070700. DOI

Lom J. Polysacharidové reservy nálevníků rodu Balantidium a Nyctotherus. Českoslov. Biol. 1955;4:394–409.

Dutta G.P. The Cytoplasmic Inclusions of Nyctotherus macropharyngeus: Histochemical Studies. J. Cell Sci. 1958;s3-99:517–521. doi: 10.1242/jcs.s3-99.48.517. DOI

Hoyte H.M.D. The protozoa occurring in the hind-gut of cockroaches. II. Morphology of Nyctotherus ovalis. Parasitology. 1961;51:437–463. doi: 10.1017/S0031182000070712. DOI

Nakai Y., Imai S. Cytochemical Identification of Reserve Polysaccharides in Rumen Ciliates by Microspectrophotometry. Jpn. J. Parasitol. 1989;38:216–220.

Brust H., Orzechowski S., Fettke J. Starch and Glycogen Analyses: Methods and Techniques. Biomolecules. 2020;10:1020. doi: 10.3390/biom10071020. PubMed DOI PMC

Wakita M., Hoshino S. Physicochemical properties of a reserve polysaccharide from sheep rumen ciliates genus Entodinium. Comp. Biochem. Physiol. Part B Comp. Biochem. 1980;65:571–574. doi: 10.1016/0305-0491(80)90315-6. DOI

Wani A.A., Singh P., Shah M.A., Schweiggert-Weisz U., Gul K., Wani I.A. Rice Starch Diversity: Effects on Structural, Morphological, Thermal, and Physicochemical Properties-A Review. Compr. Rev. Food Sci. Food Saf. 2012;11:417–436. doi: 10.1111/j.1541-4337.2012.00193.x. DOI

Zhao L., Pan T., Guo D., Wei C. A simple and rapid method for preparing the whole section of starchy seed to investigate the morphology and distribution of starch in different regions of seed. Plant Methods. 2018;14:16. doi: 10.1186/s13007-018-0283-x. PubMed DOI PMC

Horváthová T., Šustr V., Chroňáková A., Semanová S., Lang K., Dietrich C., Hubáček T., Ardestani M.M., Lara A.C., Brune A., et al. Methanogenesis in the Digestive Tracts of the Tropical Millipedes Archispirostreptus gigas (Diplopoda, Spirostreptidae) and Epibolus pulchripes (Diplopoda, Pachybolidae) Appl. Environ. Microbiol. 2021;87:e0061421. doi: 10.1128/AEM.00614-21. PubMed DOI PMC

Ellis J.E., Williams A.G., Lloyd D. Oxygen consumption by ruminal microorganisms: Protozoal and bacterial contributions. Appl. Environ. Microbiol. 1989;55:2583–2587. doi: 10.1128/aem.55.10.2583-2587.1989. PubMed DOI PMC

Ellis J.E., Mcintyre P.S., Saleh M., Williams A.G., Lloyd D. The influence of ruminal concentrations of O2 and CO2 on fermentative metabolism of the rumen entodiniomorphid ciliate Eudiplodinium maggii. Curr. Microbiol. 1991;23:245–251. doi: 10.1007/BF02092025. DOI

Park T., Yu Z. Aerobic cultivation of anaerobic rumen protozoa, Entodinium caudatum and Epidinium caudatum. J. Microbiol. Methods. 2018;152:186–193. doi: 10.1016/j.mimet.2018.08.006. PubMed DOI

Fievez V., Piattoni F., Mbanzamihigo L., Demeyer D. Reductive Acetogenesis in the Hindgut and Attempts to its Induction in the Rumen—A Review. J. Appl. Anim. Res. 1999;16:1–22. doi: 10.1080/09712119.1999.9706258. DOI

Laho T., Váradyová Z., Mihaliková K., Kišidayová S. Fermentation Capacity of Fecal Microbial Inocula of Przewalski Horse, Kulan, and Chapman Zebra and Polysaccharide Hydrolytic Activities of Fecal Microbial Constituents (Ciliates and Bacteria) of Kulan and Chapman Zebra. J. Equine Vet. Sci. 2013;33:143–149. doi: 10.1016/j.jevs.2012.05.064. DOI

Kišidayová S., Váradyová Z., Pristaš P., Piknová M., Nigutová K., Petrželková K.J., Profousová I., Schovancová K., Kamler J., Modrý D. Effects of high- and low-fiber diets on fecal fermentation and fecal microbial populations of captive chimpanzees. Am. J. Primatol. 2009;71:548–557. doi: 10.1002/ajp.20687. PubMed DOI

Cieslak A., Váradyová Z., Kišidayová S., Szumacher-Strabel M. The effects of linoleic acid on the fermentation parameters, population density, and fatty-acid profile of two rumen ciliate cultures, Entodinium caudatum and Diploplastron affine. Acta Protozool. 2009;48:51–61.

Ivan M., Neill L., Forster R., Alimon R., Rode L.M., Entz T. Effects of Isotricha, Dasytricha, Entodinium, and Total Fauna on Ruminal Fermentation and Duodenal Flow in Wethers Fed Different Diets. J. Dairy Sci. 2000;83:776–787. doi: 10.3168/jds.S0022-0302(00)74940-X. PubMed DOI

Boxma B., de Graaf R.M., van der Staay G.W.M., van Alen T.A., Ricard G., Gabaldón T., Van Hoek A.H.A.M., Der Staay S.Y.M.-V., Koopman W.J.H., Van Hellemond J.J., et al. An anaerobic mitochondrion that produces hydrogen. Nature. 2005;434:74–79. doi: 10.1038/nature03343. PubMed DOI

Šustr V., Chroňáková A., Semanová S., Tajovský K., Šimek M. Methane production and methanogenic Archaea in the digestive tracts of millipedes (Diplopoda) PLoS ONE. 2014;9:e102659. doi: 10.1371/journal.pone.0102659. PubMed DOI PMC

Finlay B.J., Esteban G., Clarke K.J., Williams A.G., Embley T.M., Hirt R.P. Some rumen ciliates have endosymbiotic methanogens. FEMS Microbiol. Lett. 1994;117:157–161. doi: 10.1111/j.1574-6968.1994.tb06758.x. PubMed DOI

Lewis W.H., Sendra K.M., Embley T.M., Esteban G.F. Morphology and Phylogeny of a New Species of Anaerobic Ciliate, Trimyema finlayi n. sp., with Endosymbiotic Methanogens. Front. Microbiol. 2018;9:140. doi: 10.3389/fmicb.2018.00140. PubMed DOI PMC

Vogels G.D., Hoppe W.F., Stumm C.K. Association of methanogenic bacteria with rumen ciliates. Appl. Environ. Microbiol. 1980;40:608–612. doi: 10.1128/aem.40.3.608-612.1980. PubMed DOI PMC

Tóthová T., Piknová M., Kišidayová S., Javorský P., Pristaš P. Distinctive archaebacterial species associated with anaerobic rumen protozoan Entodinium caudatum. Folia Microbiol. 2008;53:259–262. doi: 10.1007/s12223-008-0039-5. PubMed DOI

Stumm C.K., Zwart K.B. Symbiosis of protozoa with hydrogen-utilizing methanogens. Microbiol. Sci. 1986;3:100–105. PubMed

Goosen N.K., Horemans A.M.C., Hillebrand S.J.W., Stumm C.K., Vogels G.D. Cultivation of the sapropelic ciliate Plagiopyla nasuta Stein and isolation of the endosymbiont Methanobacterium formicicum. Arch. Microbiol. 1988;150:165–170. doi: 10.1007/BF00425157. DOI

Regensbogenova M., Michalowski T., Newbold C., McEwan N., Javorsky P., Kišidayová S., Hackstein J., Pristaš P. A re-appraisal of the diversity of the methanogens associated with the rumen ciliates. FEMS Microbiol. Lett. 2004;238:307–313. doi: 10.1111/j.1574-6968.2004.tb09771.x. PubMed DOI

Nunez F.S., Crawford C.S. Digestive enzymes of the desert millipede Orthoporus ornatus (Girard) (Diplopoda: Spirostreptidae) Comp. Biochem. Physiol. Part A Physiol. 1976;55:141–145. doi: 10.1016/0300-9629(76)90082-7. PubMed DOI

Profousová I., Mihaliková K., Laho T., Váradyová Z., Petrželková K.J., Modrý D., Kišidayová S. The ciliate, Troglodytella abrassarti, contributes to polysaccharide hydrolytic activities in the chimpanzee colon. Folia Microbiol. 2011;56:339–343. doi: 10.1007/s12223-011-0053-x. PubMed DOI

Kišidayová S., Pristaš P., Zimovčáková M., Blanár Wencelová M., Homol’ová L., Mihaliková K., Čobanová K., Grešáková Ľ., Váradyová Z. The effects of high dose of two manganese supplements (organic and inorganic) on the rumen microbial ecosystem. PLoS ONE. 2018;13:e0191158. doi: 10.1371/journal.pone.0191158. PubMed DOI PMC

González G., Murphy C.M., Belén J. Direct and Indirect Effects of Millipedes on the Decay of Litter of Varying Lignin Content. In: Sudarshana P., Nageswara-Rao M., Soneji J.R., editors. Tropical Forests. InTech; London, UK: 2012. pp. 37–50. DOI

Demeyer D.I., De Graeve K. Differences in Stoichiometry Between Rumen and Hindgut Fermentation. Adv. Anim. Physiol. Anim. Nutr. 1991;64:50–61.

Sardar P., Šustr V., Chroňáková A., Lorenc F. Metatranscriptomic holobiont analysis of carbohydrate-active enzymes in the millipede Telodeinopus aoutii (Diplopoda, Spirostreptida) Front. Ecol. Evol. 2022;10:873. doi: 10.3389/fevo.2022.931986. DOI

Hogan M.E., Slaytor M., O’Brien R.W. Transport of volatile fatty acids across the hindgut of the cockroach Panesthia cribrata Saussure and the termite, Mastotermes darwiniensis Froggatt. J. Insect Physiol. 1985;31:587–591. doi: 10.1016/0022-1910(85)90116-7. DOI

Leidy J. Some Observations on Nematoidea Imperfecta, and Descriptions of Three Parasitic Infusoriae. Trans. Am. Philos. Soc. 1853;10:241–246. doi: 10.2307/1005276. DOI

Leidy J. Nyctotherus, a new genus of Polygastrica, allied to Plescoma. Proc. Acad. Nat. Sci. USA. 1849;4:233.

Savin M.B. Master’s Thesis. University of Pennsylvania; Philadelphia, PA, USA: 1931. Nyctotherus velox and Endolimax sp. with Remarks on Other Prozozoan Parasites Found in the Millipede, Spirobolus marginatus.

van Hoek A.H., van Alen T.A., Sprakel V.S., Hackstein J.H., Vogels G.D. Evolution of anaerobic ciliates from the gastrointestinal tract: Phylogenetic analysis of the ribosomal repeat from Nyctotherus ovalis and its relatives. Mol. Biol. Evol. 1998;15:1195–1206. doi: 10.1093/oxfordjournals.molbev.a026027. PubMed DOI

Lynn D.H., Wright A.-D.G. Biodiversity and Molecular Phylogeny of Australian Clevelandella Species (Class Armophorea, Order Clevelandellida, Family Clevelandellidae), Intestinal Endosymbiotic Ciliates in the Wood-Feeding Roach Panesthia cribrata Saussure, 1864. J. Eukaryot. Microbiol. 2013;60:335–341. doi: 10.1111/jeu.12037. PubMed DOI

Li M., Hu G., Li C., Zhao W., Zou H., Li W., Wu S.-G., Wang G.-T., Ponce-Gordo F. Morphological and molecular characterization of a new ciliate Nyctotheroides grimi n. sp. (Armophorea, Clevelandellida) from Chinese frogs. Acta Trop. 2020;208:105531. doi: 10.1016/j.actatropica.2020.105531. PubMed DOI

Pecina L., Vďačný P. Two New Endozoic Ciliates, Clevelandella lynni sp. n. and Nyctotherus galerus sp. n., Isolated from the Hindgut of the Wood-feeding Cockroach Panesthia angustipennis (Illiger, 1801) J. Eukaryot. Microbiol. 2020;67:436–449. doi: 10.1111/jeu.12793. PubMed DOI

Paiva T.D.S., Borges B.D.N., Silva-Neto I.D.D. Phylogenetic study of Class Armophorea (Alveolata, Ciliophora) based on 18S-rDNA data. Genet. Mol. Biol. 2013;36:571–585. doi: 10.1590/S1415-47572013000400017. PubMed DOI PMC

Pecina L., Vďačný P. Morphological versus molecular delimitation of ciliate species: A case study of the family Clevelandellidae (Protista, Ciliophora, Armophorea) Eur. J. Taxon. 2020;697:1–46. doi: 10.5852/ejt.2020.697. DOI

Affa’a F.M., Hickey D.A., Strüder-Kypke M., Lynn D.H. Phylogenetic position of species in the genera Anoplophrya, Plagiotoma, and Nyctotheroides (Phylum ciliophora), endosymbiotic ciliates of annelids and anurans. J. Eukaryot. Microbiol. 2004;51:301–306. doi: 10.1111/j.1550-7408.2004.tb00570.x. PubMed DOI