Parasitology Research

, Volume 118, Issue 12, pp 3349–3357 | Cite as

Morphological and molecular-genetic characterization of Chloromyxum trilineatum n. sp. (Myxosporea: Bivalvulida) in the gall bladder of pale chub (Zacco platypus) in Japan

  • Mariko Sekiya
  • Imron Rosyadi
  • Jinyong Zhang
  • Hiroshi SatoEmail author
Fish Parasitology - Original Paper


The genus Chloromyxum (Myxozoa: Myxosporea: Bivalvulida) is defined as having ridged or smooth bivalvular myxospores containing four polar capsules, with/without caudal filaments. Currently containing more than 140 nominal species, this genus is reasonably speciose with myxospores of unique but heterogeneous morphology. Recent phylogenetic studies have demonstrated its polyphyletic nature. During our myxosporean survey of freshwater fish, a new coelozoic myxosporean species, Chloromyxum trilineatum n. sp., was detected in the gall bladder of the pale chub, Zacco platypus (Cypriniformes: Cyprinidae), which originated from central Japan. Spores were subspherical, measuring 8.5–9.1 (8.8) μm in length, 7.6–8.2 (8.0) μm in width, and 6.8–7.8 (7.4) μm in thickness (n = 20). The valvular surface was smooth and three or four distinct ridges ran parallel to the suture line. Four almost equal polar capsules, 2.9–3.8 (3.3) μm in length and 1.6–2.4 (2.0) μm in width, assembled at the apical part of the spores. The partial nucleotide sequence of the 18S ribosomal RNA gene, 2014 bp in length, was closest to that of morphologically distinct Chloromyxum ellipticum, infecting the gall bladder of grass carp (Ctenopharyngodon idella) in China with 96.99% (1673/1725) identity and three insertion/deletion (indel) sites, followed by Chloromyxum legeri, infecting the gall bladder of common carp (Cyprinus carpio) in the Czech Republic with 89.97% (1803/2004) identity and 14 indel sites. Other myxosporean species, including Chloromyxum spp. from the gall bladder or urinary system of freshwater and marine fish, were phylogenetically distant from the present species.


Myxozoa Chloromyxum Zacco platypus Morphology SEM 18S rDNA Phylogeny 



We are indebted to Prof. Shuhei Tanaka, Faculty of Agriculture, Yamaguchi University, for his help with SEM. We also thank the staff of Gifu World Fresh Water Aquarium (Aqua Totto Gifu) for kindly providing the preserved fish materials for this study. Their daily health checks and dedicated care of the fish under their supervision enabled freshly dead samples to be promptly available for parasitological examination.

Funding information

This study was supported in part by Grant-in-Aid for Scientific Research 2017 and 2019 from the Towa Foundation for Food Science and Research (HS) and JSPS KAKENHI (Grant Number 18K05995). The collaborative research visit to Yamaguchi University in 2018 by the third author (JZ) was supported by the Chinese Academy of Science.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

436_2019_6500_MOESM1_ESM.xlsx (48 mb)
ESM 1 (XLSX 47.9 kb)


  1. Abdel-Baki AAS (2007) Chloromyxum alii sp. n. (Myxozoa: Myxosporea) infecting the gallbladder of African butter catfish Schilbe mystus (Linnaeus 1758) from the River Nile, Egypt: light and scanning electron microscopy. Acta Protozool 46:263–267Google Scholar
  2. Anisimova M, Gascuel O (2006) Approximate likelihood-ratio test for branches: a fast, accurate, and powerful alternative. Syst Biol 55:539–552PubMedGoogle Scholar
  3. Auerbach M (1908) Bemerkungen über Myxosporidien heimischer Süsswasserfischen. Zool Anz 32:456–465Google Scholar
  4. Azevedo C, Casal G, Garcia P, Matos P, Teles-Grilo L, Matos E (2009) Ultrastructural and phylogenetic data of Chloromyxum riorajum sp. nov. (Myxozoa), a parasite of the stingray Rioraja agassizii in Southern Brazil. Dis Aquat Org 85:41–51PubMedGoogle Scholar
  5. Bahri S, Andree KB, Hedrick RP (2003) Morphological and phylogenetic studies of marine Myxobolus spp. from mullet in Ichkeul Lake, Tunisia. J Eukaryot Microbiol 50:463–470PubMedGoogle Scholar
  6. Bartošová P, Fiala I (2011) Molecular evidence for the existence of cryptic species assemblages of several myxosporeans (Myxozoa). Parasitol Res 108:573–583PubMedGoogle Scholar
  7. Cantatore DMP, Irigoitia MM, Holzer AS, Bartošová-Sojková P, Pecková H, Fiala I, Timi JT (2018) The description of two new species of Chloromyxum from skates in the Argentine Sea reveals that a limited geographic host distribution causes phylogenetic lineage separation of myxozoans in Chondrichthyes. Parasite 25:47PubMedPubMedCentralGoogle Scholar
  8. Casal G, Garcia P, Matos P, Monteiro E, Matos E, Azevedo C (2009) Fine structure of Chloromyxum menticirrhi n. sp. (Myxozoa) infecting the urinary bladder of the marine teleost Menticirrhus americanus (Sciaenidae) in Southern Brazil. Eur J Protistol 45:139–146PubMedGoogle Scholar
  9. Chandran A, Zacharia PU, Sathianandan TV, Shamal P, Binesh CP, Kaur P, Sanil NK (2018) Morphological and molecular characterization of Chloromyxum argusi n. sp. (Myxosporea) infecting the urinary bladder of Scatophagus argus Linnaeus 1766 (Scatophagidae) from the southwest coast of India. Parasitol Res 117:3145–3156PubMedGoogle Scholar
  10. Chernova TN (1969) New species of Myxosporidae–Chloromyxum colchicus Chernova sp. n.- from the gall bladder of the roach. Bull Acad Sci Georgian SSR 53:731–732 (in Russian)Google Scholar
  11. Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, Chevenet F, Dufayard JF, Guindon S, Lefort V, Lescot M, Claverie J-M, Gascuel O (2008) robust phylogenetic analysis for the non-specialist. Nucleic Acids Res 36:465–469Google Scholar
  12. Eiras JC, Lu YS, Gibson DI, Fiala I, Saraiva A, Cruz C, Santos MJ (2012) Synopsis of the species of Chloromyxum Mingazinni, 1890 (Myxozoa: Myxosporea: Chloromyxidae). Syst Parasitol 83:203–225PubMedGoogle Scholar
  13. Ferguson JA, Atkinson SD, Whipps CM, Kent ML (2008) Molecular and morphological analysis of Myxobolus spp. of salmonid fishes with the description of a new Myxobolus species. J Parasitol 94:1322–1334PubMedGoogle Scholar
  14. Fiala I (2006) The phylogeny of Myxosporea (Myxozoa) based on small subunit ribosomal RNA gene analysis. Int J Parasitol 36:1521–1534PubMedGoogle Scholar
  15. Fiala I, Dyková I (2004) The phylogeny of marine and freshwater species of the genus Chloromyxum Mingazzini, 1890 (Myxosporea, Bivalvulida) based on small subunit ribosomal RNA gene sequences. Folia Parasitol 51:211–214PubMedGoogle Scholar
  16. Fiala I, Bartošová-Sojková P, Whipps CM (2015) Classification and phylogenetics of Myxozoa. In: Okamura B, Gruhl A, Bartholomew JL (eds) Myxozoan evolution, ecology and development. Springer International Publishing, Cham, pp 85–110Google Scholar
  17. Fujita T (1913) On a new species of Chloromyxum from the gall bladder of the carp. Annot Zool Jpn 8:257–259Google Scholar
  18. Fujita T (1923) Studies on Myxosporidia of Japan. J Coll Agri Hokkaido Imp Univ 10:191–248Google Scholar
  19. Fujita T (1925) On two new species of Chloromyxum found in freshwater fishes in Sapporo. Annot Zool Jpn 10:278–280Google Scholar
  20. Fujita T (1927) Studies on Myxosporidia of Japan. 5. On Myxosporidia in fishes of Lake Biwa. J Coll Agri Hokkaido Imp Univ 16:229–247Google Scholar
  21. Fujita T (1936) Notes on some Myxosporidia in freshwater fishes. Zool Mag (Tokyo) 48:595–601Google Scholar
  22. Gleeson RJ, Adlard RD (2012) Phylogenetic relationships amongst Chloromyxum Mingazzini, 1890 (Myxozoa: Myxosporea), and the description of six novel species from Australian elasmobranchs. Parasitol Int 61:267–274PubMedGoogle Scholar
  23. Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704PubMedGoogle Scholar
  24. Hallett SL, Diamant A (2001) Ultrastructure and small-subunit ribosomal DNA sequence of Henneguya lesteri n. sp. (Myxosporea), a parasite of sand whiting Sillago analis (Sillaginidae) from the coast of Queensland, Australia. Dis Aquat Org 46:197–212PubMedGoogle Scholar
  25. Hallett SL, Atkinson SD, Holt RA, Banner CR, Bartholomew JL (2006) A new myxozoan from feral goldfish (Carassius auratus). J Parasitol 92:357–366PubMedGoogle Scholar
  26. Holzer AS, Sommerville C, Wootten R (2006) Molecular identity, phylogeny and life cycle of Chloromyxum schurovi Shul’man & Ieshko 2003. Parasitol Res 99:90–96PubMedGoogle Scholar
  27. Jirků M, Bartošová P, Kodádková A, Mutschmann F (2011) Another chloromyxid lineage: molecular phylogeney and redescription of Chloromyxum careni from the Asian horned frog Megophrys nasuta. J Eukaryot Microbiol 58:50–59PubMedGoogle Scholar
  28. Kato E, Kasai A, Tomochi H, Li YC, Sato H (2017) Four Myxobolus spp. (Myxosporea: Bivalvulida) from the gill lamellae of common carp (Cyprinus carpio) and Japanese silver crucian carp (Carassius langsdorfii) in the western part of Japan, with the description of three new species (M. tanakai n. sp., M. paratoyamai n. sp., and M. ginbuna n. sp.). Parasitol Res 116:2427–2441PubMedGoogle Scholar
  29. Kopečná J, Jirku M, Oborník M, Tokarev YS, Lukes J, Modrý D (2006) Phylogenetic analysis of coccidian parasites from invertebrates: search for missing links. Protist 157:173–183PubMedGoogle Scholar
  30. Kudo RR (1916) Contributions to the study of parasitic protozoa. III. Notes on some Myxosporidia found in some fresh-water fishes of Japan, with the description of three new species. J Parasitol 3:3–9Google Scholar
  31. Kudo RR (1920) Studies on the Myxosporidia: a synopsis of genera and species of Myxosporidia. Illinois Biological Monographs 5:1–265Google Scholar
  32. Li Y-C, Sato H, Kamata Y, Ohnishi T, Sugita-Konishi Y (2012) Tree novel myxobolid species of genera Henneguya and Myxobolus (Myxosporea: Bivalvulida) from marine fish in Japan. Parasitol Res 111:819–826PubMedGoogle Scholar
  33. Li Y-C, Sato H, Tanaka S, Ohnishi T, Kamata Y, Sugita-Konishi Y (2013) Characterization of the ribosomal RNA gene of Kudoa neothunni (Myxosporea: Multivalvulida) in tunas (Thunnus spp.) and Kudoa scomberi n. sp. in a chub mackerel (Scomber japonicus). Parasitol Res 112:1991–2003PubMedGoogle Scholar
  34. Liu Y, Gu ZM, Luo YL (2010) Some additional data to the occurrence, morphology and validity of Myxobolus turpisrotundus Zhang, 2009 (Myxozoa: Myxosporea). Parasitol Res 107:67–73PubMedGoogle Scholar
  35. Liu Y, Whipps CM, Gu ZM, Huang MJ, He C, Yang HL, Molnár K (2013) Myxobolus musseliusae (Myxozoa: Myxobolidae) from the gills of common carp Cyprinus carpio and revision of Myxobolus dispar recorded in China. Parasitol Res 112:289–296PubMedGoogle Scholar
  36. Liu XH, Yuan S, Zhao YL, Fang P, Chen H, Zhang JY (2016) Morphological and molecular characterization of Myxobolus sheyangensis n. sp. (Myxosporea: Myxobolidae) with intralamellar sporulation in allogynogenetic gibel carp, Carassius auratus gibelio (Bloch) in China. Parasitol Res 115:3567–3574PubMedGoogle Scholar
  37. Liu XH, Voronin VN, Dudin AS, Morozova DA, Zhang JY (2017) Morphological and molecular characterization of a new cyprinid gall bladder-infecting Chloromyxum species, Chloromyxum peleci sp. n. (Myxozoa: Chloromyxidae), from Pelecus cultratus (L.) in Russia. Parasitol Res 116:2239–2248PubMedGoogle Scholar
  38. Lom J, Arthur JR (1989) A guideline for the preparation of species descriptions in Myxosporea. J Fish Dis 12:151–156Google Scholar
  39. Lom J, Dyková I (1993) Scanning electron microscopic revision of common species of the genus Chloromyxum (Myxozoa: Myxosporea) infecting European freshwater fishes. Folia Parasitol 40:161–174Google Scholar
  40. Lom J, Dyková I (2006) Myxozoan genera: definition and notes on taxonomy, life-cycle terminology and pathogenic species. Folia Parasitol 53:1–36PubMedGoogle Scholar
  41. Matsukane Y, Sato H, Tanaka S, Kamata Y, Sugita-Konishi Y (2010) Kudoa septempunctata n. sp. (Myxosporea: Multivalvulida) from an aquacultured olive flounder (Paralichthys olivaceus) imported from Korea. Parasitol Res 107:865–872PubMedGoogle Scholar
  42. Mitchell LG, Listebarger JK, Bailey WC (1980) Epizootiology and histopathology of Chloromyxum trijugum (Myxosporea: Myxosporida) in centrarchid fishes from Iowa. J Wildl Dis 16:233–236PubMedGoogle Scholar
  43. Mutschmann F (1999) A new myxozoan, Chloromyxum careni sp. n. (Myxosporea: Chloromyxidae) from the kidney of Megophrys nasuta Schlegel, 1858 (Anura: Pelobatidae) from Indonesia. Acta Protozool 38:83–86Google Scholar
  44. Reda ASA (2010) Chloromyxum aegypticus n. sp. (Myxozoa: Chloromyxidae) infecting the testicular tissue of the Egyptian toad, Bufo regularis (Amphibia: Bufonidae), and its pathogenicity. Parasitol Res 107:1299–1306PubMedGoogle Scholar
  45. Rocha S, Casal G, Al-Quraishy S, Azevedo C (2013) Morphological and molecular characterization of a new myxozoan species (Myxosporea) infecting the gall bladder of Raja clavata (Chondrichthyes), from the Portuguese Atlantic coast. J Parasitol 99:307–317PubMedGoogle Scholar
  46. Rocha S, Casal G, Al-Quraishy S, Azevedo C (2014) Morphological and ultrastructural redescription of Chloromyxum leydigi Mingazzini, 1890 (Myxozoa: Myxosporea), type species of the genus, infecting the gall bladder of the marine cartilaginous fish Torpedo marmorata Risso (Chondrichthyes: Torpedinidae), from the Portuguese Atlantic coast. Folia Parasitol 61:1–10PubMedGoogle Scholar
  47. Salim KY, Desser SS (2000) Descriptions and phylogenetic systematics of Myxobolus spp. from cyprinids in Algonquin Park, Ontario. J Eukaryot Microbiol 47:309–318PubMedGoogle Scholar
  48. Sanders JL, Jaramillo AG, Ashford JE, Feist SW, Lafferty KD, Kent ML (2015) Two myxozoans from the urinary tract of topsmelt, Atherinops affinis. J Parasitol 10:577–586Google Scholar
  49. Sarkar NK (2007) Two new myxosporean species of Chloromyxum Mingazzini 1890 (Myxosporea: Chloromyxidae) from the gall bladder of two freshwater fishes. Uttar Pradesh J Zool 27:233–237Google Scholar
  50. Shul’man SS (1966) Myxosporidia of the USSR. In: Kothekar VS (ed) Izdatel’stvo Nauka AN SSSR, Moscow-Leningrad (in Russian; English version in 1988). Amerind Publishing Co., New Delhi 631 ppGoogle Scholar
  51. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680PubMedPubMedCentralGoogle Scholar
  52. Urawa S, Hiroi O (1986) Two species of Chloromyxum (Myxozoa: Myxosporea) in chum salmon, Oncorhynchus keta (Walbaum): taxonomy and life cycle. Sci Rep Hokkaido Fish Hatchery 40:11–20 (in Japanese with English summary)Google Scholar
  53. Zhang JY, Wang JG, Li AH, Gong XN (2010) Infection of Myxobolus turpisrotundus sp. n. in allogynogenetic gibel carp, Carassius auratus gibelio (Bloch), with revision of Myxobolus rotundus (s. l.) Nemaczek reported from C. auratus auratus (L.). J Fish Dis 33:625–638PubMedGoogle Scholar
  54. Zhang JY, Zhao YL, Batueva MD, Luo D, Xing ZF, Zhang QQ, Liu XH (2017) Redescription of Chloromyxum ellipticum Li & Nie, 1973 (Myxosporea: Chloromyxidae) infecting the gall bladder of gras carp Ctenopharyngodon idellus Valenciennes, 1844, supplemented by morphlogical and molecular characteristics. Parasitol Res 116:1479–1486PubMedGoogle Scholar
  55. Zhao YJ, Li NN, Tang FH, Dong JL (2013) Remarks on the validity of Myxobolus ampullicapsulatus and Myxobolus honghuensis (Myxozoa: Myxosporea) based on SSU rDNA sequences. Parasitol Res 112:3817–3823PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Laboratory of Parasitology, Joint Faculty of Veterinary MedicineYamaguchi UniversityYamaguchiJapan
  2. 2.Fish Diseases Laboratory, State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of HydrobiologyChinese Academy of SciencesWuhanChina
  3. 3.Faculty of Veterinary MedicineAirlangga UniversitySurabayaIndonesia

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