Findings on three endocommensal scuticociliates (Protista, Ciliophora) from freshwater mollusks, including their morphology and molecular phylogeny with descriptions of two new species

Abstract

Species of the ciliate genera Myxophyllum and Conchophthirus are found as endocommensals of terrestrial and freshwater mollusks, respectively. So far, there have been few studies of these genera and morphological data for most members are often incomplete. In the present work, two new species, Myxophyllum weishanense sp. nov. and Conchophthirus paracurtus sp. nov., and a known species, Conchophthirus lamellidens, were isolated from hosts in Lake Weishan Wetland, China. Taxonomic studies indicate that M. weishanense sp. nov. can be recognized mainly by the combination of about 60 somatic kineties on both ventral and dorsal sides and the presence of caudal cilia. Conchophthirus paracurtus sp. nov. differs from congeners in its body shape and size, having a glabrous area on the posterior right side, and having fewer somatic kineties. In addition, differences in their ITS2 (Internally Transcribed Spacer 2) secondary structures support the discrimination of the two new species from their highly similar congeners. An improved diagnosis for the poorly known species, C. lamellidens is also provided. Phylogenetic analyses reveal that members of the genus Myxophyllum belong to a fully supported clade that is sister to a large, poorly supported clade consisting of Hemispeiridae, Ancistridae, and several lineages of the nonmonophyletic Cyclidiidae. The Myxophyllum clade also includes Protophyra ovicola JQ956552, a possible misidentification. Sequences of the two new Conchophthirus species cluster with other congeners in a fully supported clade that is unrelated to either the ‘typical’ thigmotrichs or to pleuronematids, thus conflicting with the traditional classification, and may represent an orphan scuticociliate lineage.

Keywords

Ciliates / Endocommensal / ITS2 secondary structure / Mollusks / New taxa / Wetland

References

[1] Adl SM, Bass D, Lane CE, Lukeš J, Schoch CL, Smirnov A, Agatha S, Berney C, Brown MW, Burki F, Cárdenas P, Čepička I, Chistyakova L, del Campo J, Dunthorn M, Edvardsen B, Eglit Y, Guillou L, Hampl V, Heiss AA. Revisions to the classification, nomenclature, and diversity of eukaryotes. J Eukaryot Microbiol, 2019, 66: 4-119,

[2] Alfaro ME, Zoller S, Lutzoni F. Bayes or bootstrap? A simulation study comparing the performance of Bayesian Markov chain Monte Carlo sampling and bootstrapping in assessing phylogenetic confidence. Mol Biol Evol, 2003, 20: 255-266,

[3] Antipa GA. Structural differentiation in the somatic cortex of a ciliated protozoan, Conchophthirus curtus Engelmann, 1862. Protistologica, 1971, 4: 471-501

[4] Antipa GA. Hausmann K, Radek R. Cellular architecture, growth, morphogenesis, chemoattractants, and loose ends. Cilia and flagella, ciliates and flagellates, 2014 Stuttgart Schweizerbart 23-45

[5] Antipa GA, Hatzidimitriou G. Morphogenesis in Conchophthirus curtus: a study of the morphological events associated with binary fission. J Protozool, 1981, 28: 206-214,

[6] Antipa GA, Small EB. A redescription of Conchophthirus curtus Engelmann, 1862. J Protozool, 1971, 18: 491-501,

[7] Antipa GA, Small EB. The occurrence of thigmotrichous ciliated protozoa inhabiting the mantle cavity of unionid molluscs of Illinois. Trans Am Microsc Soc, 1971, 90: 463-472,

[8] Antipa GA, Dolan J, Lynn DH, Obolkina LA, Strüder-Kypke MC. Molecular phylogeny and evolutionary relationships between the ciliate genera Peniculistoma and Mytilophilus (Peniculistomatidae, Pleuronematida). J Eukaryot Microbiol, 2016, 63: 642-650,

[9] Antipa GA, Strüder-Kypke MC, Lynn DH. Molecular phylogeny, taxonomic relationships and North American distribution of Conchophthirus (Conchophthiridae, Scuticociliatia). Aquat Ecosyst Health Manag, 2020, 1: 58-68,

[10] Burlakova LE, Karatayev AY, Molloy DP. Field and laboratory studies of zebra mussel (Dreissena polymorpha) infection by the ciliate Conchophthirus acuminatus in the Republic of Belarus. J Invertebr Pathol, 1998, 71: 251-257,

[11] Burlakova LE, Padilla DK, Karatayev AY, Minchin D. Endosymbionts of Dreissena polymorpha in Ireland: evidence for the introduction of adult mussels. J Molluscan Stud, 2006, 72: 207-210,

[12] Chen D, Zhang G (2004) Fauna sinica, invertebrata, vol. 37. Mollusca: Gastropoda: Stylommatophora: Bradybaenidae. Science Press, Beijing

[13] Chuševė R, Mastitsky SE, Zaiko A. First report of endosymbionts in Dreissena polymorpha from the brackish Curonian Lagoon, SE Baltic Sea. Oceanologia, 2012, 54: 701-713,

[14] Conn DB, Simpson SE, Minchin D, Lucy FE. Occurrence of Conchophthirus acuminatus (Protista: Ciliophora) in Dreissena polymorpha (Mollusca: Bivalvia) along the River Shannon, Ireland. Biol Invasions, 2008, 10: 149-156,

[15] Corliss JO. Silver impregnation of ciliated protozoa by the Chatton–Lwoff technic. Stain Technol, 1953, 28: 97-100,

[16] Corliss JO. Raven PH, Williams T. Biodiversity, classification, and numbers of species of protists. Nature and human society: the quest for a sustainable world, 2000 Washington, DC National Academy Press 130-155

[17] Darty K, Denise A, Ponty Y. VARNA: interactive drawing and editing of the RNA secondary structure. Bioinformatics, 2009, 25: 1974-1975,

[18] de Puytorac PD, Groliere CA, Neto IDS. L'ultrastructure du cilié Myxophyllum steenstrupi (Stein, 1961) parasite de mollusques pulmonés et la famille des Thigmophryidae Chatton et Lwoff, 1923. Acta Protozool, 1992, 31: 221-231

[19] de Puytorac PD, Batisse A, Fleury G, Grain J, Laval-Peuto M, Silva-Neto I. Proposition d’une nouvelle classification du phylum des protozoaires Ciliophora Doflein, 1901. C R Acad Sci Ser III, 1994, 316: 716-720

[20] Deshmukh NZ, More BV, Jaid EL, Nikam SV. Study of ciliate commensals from the gills of freshwater bivalves (Molluscs: Bivalvia) of Jayakwadi Dam, Paithan, (M.S.), India. J Ecobiotechnol, 2011, 3: 9-12

[21] Dobrzańska J. Investigations on ciliates living in lamellibranchiates of small water bodies. Bull Acad Pol Sci, 1958, 6: 113-118

[22] Ehrenberg CG (1838) Die Infusionsthierchen als vollkommene Organismen. Ein Blick in Das Tiefere Organische Leben Der Natur. Leopold Voss, Leipzig

[23] Engelmann TW. Zur Naturgeschichte der Infusionstiere. Zeitschr f Wiss Zool Bd, 1862, 11: 347-393

[24] Fenchel T. Ciliates from Scandinavian molluscs. Ophelia, 1965, 2: 71-174,

[25] Fenchel T. On the ciliated Protozoa inhabiting the mantle cavity of lamellibranchs. Malacologia, 1966, 5: 35-36

[26] Fokin SI, Schweikert M. Bacterial endocytobionts within endosymbiotic ciliates in Dreissena polymorpha (Lamellibranchia: Mollusca). Eur J Protistol, 2003, 39: 311-318,

[27] Gao F, Katz LA, Song W. Insights into the phylogenetic and taxonomy of philasterid ciliates (Protozoa, Ciliophora, Scuticociliatia) based on analyses of multiple molecular markers. Mol Phylogenet Evol, 2012, 64: 308-317,

[28] Gao F, Katz LA, Song W. Multigene-based analyses on evolutionary phylogeny of two controversial ciliate orders: Pleuronematida and Loxocephalida (Protista, Ciliophora, Oligohymenophorea). Mol Phylogenet Evol, 2013, 68: 55-63,

[29] Gao F, Warren A, Zhang Q, Gong J, Miao M, Sun P, Xu D, Huang J, Yi Z, Song W. The all-data-based evolutionary hypothesis of ciliated protists with a revised classification of the phylum Ciliophora (Eukaryota, Alveolata). Sci Rep, 2016, 6: 24874,

[30] Ghosh E. Studies on Infusoria. Rec Indian Mus, 1918, 15: 129-134

[31] Grizzle JM, Brunner CJ (2007) Assessment of current information available for detection, sampling, necropsy, and diagnosis of diseased mussels. Alabama Department of Conservation and Natural Resources Wildlife and Freshwater Fisheries Division, Montgomery, pp 21–24

[32] Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser, 1999, 41: 95-98

[33] He J, Zhuang Z. . The freshwater bivalves of China, 2013 Harxheim ConchBooks

[34] Hillis DM, Bull JJ. An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Syst Biol, 1993, 42: 182-192,

[35] Hu X, Lin X, Song W. . Ciliate atlas: species found in South China Sea, 2019 Beijing Science Press

[36] International Commission on Zoological Nomenclature (ICZN). . International Code of Zoological Nomenclature, 1999 London International Trust for Zoological Nomenclature,

[37] Irwin NAT, Lynn DH. Molecular phylogeny of mobilid and sessilid ciliates symbiotic in eastern Pacific limpets (Mollusca: Patellogastropoda). J Eukaryot Microbiol, 2015, 62: 543-552,

[38] Jerome CA, Lynn DH, Simon EM. Description of Tetrahymena empidokyrea n. sp., a new species in the Tetrahymena pyriformis sibling species complex (Ciliophora, Oligohymenophorea), and an assessment of its phylogenetic position using small-subunit rRNA sequences. Can J Zool, 1996, 74: 1898-1906,

[39] Kahl A. Urtiere oder Protozoa I: Wimpertiere oder Ciliata (Infusoria) 2. Holotricha Tierwelt Dtl, 1931, 21: 181-398

[40] Karatayev AY, Burlakova LE. What we know and don’t know about the invasive zebra (Dreissena polymorpha) and quagga (Dreissena rostriformis bugensis) mussels. Hydrobiologia, 2022,

[41] Karatayev AY, Burlakova LE, Molloy DP, Volkova LK. Endosymbionts of Dreissena polymorpha (Pallas) in Belarus. Internat Rev Hydrobiol, 2000, 85: 539-555,

[42] Karatayev AY, Burlakova LE, Molloy DP. Seasonal dynamics of Conchophthirus acuminatus (Ciliophora, Conchophthiridae) infection in Dreissena polymorpha and D. bugensis (Bivalvia, Dreissenidae). Eur J Protistol, 2000, 36: 397-404,

[43] Karatayev AY, Burlakova LE, Molloy DP, Mastitsky SE. Dreissena polymorpha and Conchophthirus acuminatus: what can we learn from host-commensal relationships. J Shellfish Res, 2007, 26: 1152-1160,

[44] Katoh K, Rozewicki J, Yamada KD. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform, 2019, 20: 1160-1166,

[45] Kazubski SL. New hosts for Myxophyllum steenstrupi (Stein) and Trichodinopsis paradoxa Claparede et Lachmann in Caucasus. USSR and Greece J Protozool, 1964, 11: 33

[46] Kazubski SL. Buccal apparatus of Myxophyllum steenstrupi (Stein, 1861) Raabe, 1934 (Thigmotricha). Acta Protozool, 1973, 12: 169-172

[47] Kidder GW. Studies on the ciliates from fresh water mussels. I. The structure and neuromotor system of Conchophthirius anodontae Stein, C. curtus Engl., and C. magna sp. nov. Biol Bull, 1934, 66: 69-90,

[48] Laruelle F, Molloy DP, Fokin SI, Ovcharenko MA. Histological analysis of mantle-cavity ciliates in Dreissena polymorpha: their location, symbiotic relationship, and distinguishing morphological characteristics. J Shellfish Res, 1999, 18: 251-257

[49] Laruelle F, Molloy DP, Roitman VA. Histological analysis of trematodes in Dreissena polymorpha: their location, pathogenicity, and distinguishing morphological characteristics. J Parasitol, 2002, 88: 856-863,

[52] Li T, Pan X, Lu B, Miao M, Liu MJ (2021) Taxonomy and molecular phylogeny of a new freshwater ciliate Frontonia apoacuminata sp. nov. (Protista, Ciliophora, Oligohymenophorea) from Qingdao, P.R. China. Int J Syst Evol Microbiol 71:005071

[50] Li T, Liu M, Warren A, Al-Farraj SA, Yi Z, Sheng Y. Morphology and SSU rRNA gene-based phylogeny of three peniculid ciliates (Ciliophora, Oligohymenophorea) from China, including a new Frontonia species. Eur J Protistol, 2022, 85: 125910,

[51] Li R, Zhuang W, Feng X, Hines HN, Hu X. First redescription and molecular phylogeny of Trimyema claviforme Kahl, 1933 with the description of a Chinese population of Plagiopyla nasuta Stein, 1860 (Ciliophora, Plagiopylea). Eur J Protistol, 2023, 90: 126003,

[53] Liu MJ, Liu YJ, Zhang TT, Lu BR, Gao F, Gu J, Al-Farraj SA, Hu XZ, Song WB. Integrative studies on the taxonomy and molecular phylogeny of four new Pleuronema species (Protozoa, Ciliophora, Scuticociliatia). Mar Life Sci Technol, 2022, 4: 179-200,

[54] Lu BR, Hu XZ, Warren A, Song WB, Yan Y. From oral structure to molecular evidence: new insights into the evolutionary phylogeny of the ciliate order Sessilida (Protista, Ciliophora), with the establishment of two new families and new contributions to the poorly studied family Vaginicolidae. Sci China Life Sci, 2023, 66: 1535-1553,

[55] Lynn DH. . The ciliated protozoa: characterization, classification, and guide to the literature, 2008 3 Dordrecht Springer

[56] Lynn DH, Doerder FP, Gillis PL, Prosser PS. Tetrahymena glochidiophila n. sp., a new species of Tetrahymena (Ciliophora) that causes mortality to glochidia larvae of freshwater mussels (Bivalvia). Dis Aquat Org, 2018, 127: 125-136,

[57] Lynn DH, Small EB (2002) Phylum Ciliophora. In: Lee JJ, Leedale GF, Bradbury P (eds) An illustrated guide to the Protozoa, organisms traditionally referred to as Protozoa, or newly discovered groups, 2nd edn. Society of Protozoologists, Lawrence

[58] Mastitsky SE. Infection of Dreissena polymorpha (Bivalvia: Dreissenidae) with Conchophthirus acuminatus (Ciliophora: Conchophthiridae) in lakes of different trophy. Bioinv Rec, 2012, 1: 161-169,

[59] Mastitsky SE, Lucy F, Gagarin VG. First report of endosymbionts in Dreissena polymorpha from Sweden. Aquat Invasions, 2008, 1: 83-86,

[60] Mayén-Estrada R, Dias RJP, Ramírez-Ballesteros M, Rossi M, Reyes-Santos M, Durán-Ramírez CA, Cruz-Jiménez G (2021) Ciliates as symbionts. In: Pereira L, Gonçalves AMM (eds) Plankton communities. IntechOpen, London

[61] Mermod G. Recherches sur la faune infusorienne des tourbieres et des eaux voisines de Sainte-Croix (Jura Vaudois). Rev Suisse Zool, 1914, 22: 31-114,

[62] Miao M, Warren A, Song W, Wang S, Shang H, Chen Z. Analysis of the internal transcribed spacer 2 (ITS2) region of scuticociliates and related taxa (Ciliophora, Oligohymenophorea) to infer their evolution and phylogeny. Protist, 2008, 159: 519-533,

[63] Molloy DP, Giamberini L, Burlakova LE, Karatayev AY, Cryan JR, Trajanovski SL, Trajanovska SP (2010) Investigation of the endosymbionts of Dreissena stankovici with morphological and molecular confirmation of host species. In: van der Velde G, Rajagopal S, Bij de Vaate A (eds) The zebra mussel in Europe. Backhuys Publishers, Leiden, pp 227–238

[64] Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum likelihood phylogenies. Mol Biol Evol, 2015, 32: 268-274,

[65] Peck RK. Cortical ultrastructure of the scuticociliates Dexiotricha media and Dexiotricha colpidiopsis (Hymenostomata). J Protozool, 1977, 24: 122-134,

[66] Penn JH. Studies on ciliates from mollusks of Iowa. Proc Iowa Acad Sci, 1958, 65: 517-534

[67] Poláková K, Čepička I, Bourland WA. Phylogenetic position of three well-known ciliates from the controversial order Loxocephalida Jankowski, 1980 (Scuticociliatia, Oligohymenophorea) and Urozona buetschlii (Schewiakoff, 1889) with improved morphological descriptions. Protist, 2021, 172: 125833,

[68] Prosser RS, Lynn DH, Salerno J, Bennett J, Gillis PL. The facultatively parasitic ciliated protozoan, Tetrahymena glochidiophila (Lynn, 2018), causes a reduction in viability of freshwater mussel glochidia. J Invertebr Pathol, 2018, 157: 25-31,

[69] Qu Z, Groben R, Marteinsson V, Agatha S, Filker S, Stoeck T. Redescription of Dexiotricha colpidiopsis (Kahl, 1926) Jankowski, 1964 (Ciliophora, Oligohymenophorea) from a hot spring in Iceland with identification key for Dexiotricha species. Acta Protozool, 2019, 57: 95-106

[70] Raabe Z. Untersuchungen an einigen Arten des Genus Conchophthirus Stein. Bull Acad Pol Sci Lettr Sér B II, 1933, 1932: 295-310

[71] Raabe Z. Weitere Untersuchungen an einigen Arten des Genus Conchophthirus Stein. Mém Acad Pol Sci Lettr Sér B Sci Nat, 1934, 1934: 221-235

[72] Raabe Z. Recherches sur les ciliés thigmotriches (Thigmotricha C.-Lw.). V. Ciliés thigmotriches du lac Balaton (Hongrie). Annls Univ M Curie-Sklod Sec C Biol Sci, 1950, 5: 197-215

[73] Raabe Z. The parasitic ciliates of Dreissensia polymorpha and other Bivalvia in the Ohrid Lake. Acta Protozool, 1966, 4: 1-14

[74] Raabe Z. Ordo Thigmotricha (Ciliata-Holotricha). I. Pars generalis. Acta Protozool, 1967, 5: 1-36

[75] Raabe Z. Ordo Thigmotricha (Ciliata-Holotricha). II. Familia hemispeiridae. Acta Protozool, 1970, 7: 117-180

[76] Raabe Z. Ordo Thigmotricha (Ciliata-Holotricha). IV. Familia Thigmophryidae. Acta Protozool, 1971, 9: 121-170

[77] Raabe Z. Ordo Thigmotricha (Ciliata–Holotricha). V. Familiae Hysterocinetidae et Protoanoplophryidae. Acta Protozool, 1972, 10: 115-184

[78] Rataj M, Vďačný P. Dawn of astome ciliates in light of morphology and time-calibrated phylogeny of Haptophrya planariarum, an obligate endosymbiont of freshwater turbellarians. Eur J Protistol, 2018, 64: 54-71,

[79] Rataj M, Vďačný P. Putative ITS2 secondary structure model and multi-gene phylogenies of tetrahymenids (Ciliophora, Hymenostomatia) parasitizing planarians and crayfish worms. Zool Scr, 2022, 51: 246-265,

[80] Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol, 2012, 61: 539-542,

[81] Seibel PN, Müller T, Dandekar T, Schultz J, Wolf M. 4SALE—a tool for synchronous RNA sequence and secondary structure alignment and editing. BMC Bioinformatics, 2006, 7: 498,

[82] Small EB, Lynn DH. Lee JJ, Hutner SH, Bovee EC. Phylum Ciliophora, Doflein, 1901. An illustrated guide to the Protozoa, 1985 Lawrence Society of Protozoologists 393-575

[83] Song W, Zhao Y, Xu K, Hu X, Gong J. . Pathogenic Protozoa in mariculture, 2003 Beijing Science

[84] Song W, Warren A, Hu X. . Free-living ciliates in the Bohai and Yellow Seas, 2009 Beijing Science Press

[85] Souidenne D, Furuya H. Gestal C, Pascual S, Guerra Á, Fiorito G, Vieites J. Protist (ciliates) and related diseases. Handbook of pathogens and diseases in cephalopods, 2019 Cham Springer 153-158,

[86] Stein F. Über ein neues parasitisches Infusionsthier (Ptychostomum paludinarum) aus dem Darmkanal von Paludinen und über die mit demselben zunächst verwandten Infusionsformen. Sber K Böhm Ges Wiss, 1861, 1861: 85-90

[87] Sweeney BA, Hoksza D, Nawrocki EP, Ribas CE, Madeira F, Cannone JJ, Gutell R, Maddala A, Meade CD, Williams LD, Petrov AS, Chan PP, Lowe TM, Finn RD, Petrov AI. R2DT is a framework for predicting and visualising RNA secondary structure using templates. Nat Commun, 2021, 12: 3494,

[88] Trifinopoulos J, Nguyen LT, von Haeseler A, Minh BQ. W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucl Acids Res, 2016, 44: W232-W235,

[89] Uyemura M. Über drei in der Süsswasser-Muscheln (Anodonta laura v. Martens) lebende Ciliaten (Conchophthirus). Sci Rep Tokyo Bunrika Daigaku Sect B, 1935, 2: 89-100

[90] Van As JG, Basson L. Barker GM. Ciliophoran (Ciliophora) parasites of terrestrial gastropods. Natural enemies of terrestrial molluscs, 2004 Wallingford CABI Publishing 559-578,

[91] Vd’ačný P, Rajter L’. Rapid radiation, gradual extinction and parallel evolution challenge generic classification of spathidiid ciliates (Protista, Ciliophora). Zool Scr, 2015, 45: 200-223

[92] Wang Z, Chi Y, Li T, Song W, Wang Y, Wu T, Zhang G, Liu Y, Ma H, Song W, Al-Rasheid KAS, Warren A, Lu B. Biodiversity of freshwater ciliates (Protista, Ciliophora) in the Lake Weishan Wetland, China: the state of the art. Mar Life Sci Technol, 2022, 4: 429-451,

[93] Wilbert N. Eine verbesserte Technik der Protargolimprägnation für Ciliaten. Mikrokosmos, 1975, 64: 171-179

[94] Zhang T, Vďačný P. Re-discovery and novel contributions to morphology and multigene phylogeny of Myxophyllum steenstrupi (Ciliophora: Pleuronematida), an obligate symbiont of terrestrial pulmonates. Zool J Linn Soc, 2021, 192: 1-23,

[95] Zhang T, Vďačný P. Multiple independent losses of cell mouth in phylogenetically distant endosymbiotic lineages of Oligohymenophorean ciliates: a lesson from Clausilocola. Mol Phylogenet Evol, 2022, 166: 107310,

[96] Zhang T, Vďačný P. Morphological and molecular characterization of the ciliate parasite Tetrahymena rostrata infecting the renal organ of the dusky slug (Arion fuscus). Can J Zool, 2023, 101: 32-45,

[97] Zhang T, Vďačný P. Re-discovery and novel contributions to morphology and multigene phylogeny of Protospirella mazurica (Raabe, 1968) Aescht, 2001 (Ciliophora: Pleuronematida), an obligate symbiont of the river nerite Theodoxus fluviatilis Linnaeus, 1758 (Mollusca: Gastropoda). Eur J Protistol, 2023, 88: 125956,

[98] Zhang T, Vďačný P. Deciphering phylogenetic relationships of and delimiting species boundaries within the controversial ciliate genus Conchophthirus using an integrative approach. Mol Phylogenet Evol, 2024, 190: 107931,