Parasitology Research

, Volume 116, Issue 11, pp 3065–3076 | Cite as

An investigation of the host-specificity of metacercariae of species of Apophallus (Digenea: Heterophyidae) in freshwater fishes using morphological, experimental and molecular methods

  • Diána Sándor
  • Kálmán Molnár
  • David I. Gibson
  • Csaba Székely
  • Gábor Majoros
  • Gábor CechEmail author
Original Paper


Metacercariae of species of the genus Apophallus Lühe, 1909, infecting the fins and skin of freshwater fishes, frequently cause black spot disease. Two species, Apophallus muehlingi (Jägerskiöld, 1899) and A. donicus (Skrjabin & Lindtrop, 1919), are known to occur in Hungarian fishes. It has generally been thought that metacercariae of A. muehlingi infect cyprinid fishes, whereas those of A. donicus develop in percids. As part of a morphological, experimental and molecular study, metacercariae were collected from 99 infected specimens of five cyprinid hosts (Abramis brama, Blicca bjoerkna, Chondrostoma nasus, Squalius cephalus, Scardinius erythrophthalmus) and 18 infected specimens of two percid hosts (Gymnocephalus cernua, Perca fluviatilis) in Hungarian natural waters (Lake Balaton, River Danube). Moreover, 1024 common carp (Cyprinus carpio) specimens collected from Hungarian fish ponds were investigated for Apophallus infection, but without positive results. For reliable species identification, experimental infections of chicks were carried in order to produce adult specimens from metacercariae collected from the fins and skin of the cyprinid and percid hosts. Within 8 days, adult specimens of both A. muehlingi and A. donicus developed in chicks infected with metacercariae from the cyprinid common bream (Abramis brama) and the white bream (Blicca bjoerkna) and the ruffe (Gymnocephalus cernua), a percid, respectively. The morphology of the collected metacercariae and adult individuals developed in the feeding experiments was characterised. A molecular analysis was extended to cercarial samples from the snail Lithoglyphus naticoides and to a single adult specimen of Apophallus from a fox. Sequences of 28 specimens were analysed using molecular methods (sequencing the internal transcribed spacer region and the cytochrome oxidase I subunit). Phylogenetic analysis was executed, and the Apophallus samples clustered into three distinct branches using both genes, A. muehlingi from cyprinids, A. donicus from percids and, a third, previously unknown, Apophallus clade, also from cyprinids.


Metacercariae Black spot disease Apophallus infections ITS region and COI 



This project has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement no. 634429 (ParaFishControl), the Hungarian Scientific Research Fund (OTKA PD 108813) and a Bolyai Scholarship (BO/00417/15/4).


  1. Bykhovskaya-Pavlovskaya IE (1962) Class Trematoda Rudolphi, 1808. In: Bykhowski BE (ed) [keys to of the parasites of freshwater fishes of the USSR.] Akademii Nauk SSSR, Moscow-Leningrad, Russia, pp 428-520 (in Russian)Google Scholar
  2. Bykhovskaya-Pavlovskaya IE, Kulakovskaya AP (1987) Class Trematoda Rudolphi, 1808. In: Bauer ON (ed) [key to determination the parasites of freshwater fishes of the USSR.] Vol. 3. Nauka, Leningrad, pp 77-198 (in Russian)Google Scholar
  3. Cameron TWM (1937) Studies on the heterophyid trematode Apophallus venustus (Ransom, 1920) in Canada. Part II. Life history and bionomics. Can J Res 15:38–51CrossRefGoogle Scholar
  4. Cameron TWM (1945) Fish-carried parasites in Canada: (1) parasites carried by fresh-water fish. Can J Comp Med Vet S 9:245–254Google Scholar
  5. Castresana J (2000) Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 17:540–552CrossRefPubMedGoogle Scholar
  6. Cech G, Molnár K, Székely C (2017) Molecular biological studies of adult and metacercarial stages of Petasiger exaeretus Dietz, 1909 (Digenea: Echinostomatidae). Acta Vet Hung 65:198–207CrossRefPubMedGoogle Scholar
  7. Chernogorenko MI (1977) Trematode fauna of mollusks in the Kremenchug Reservoir. Hydrobiol J 13:87–94Google Scholar
  8. Cichy A, Faltýnková A, Zbikowska E (2011) Cercariae (Trematoda, Digenea) in European freshwater snails—a checklist of records from over one hundred years. Folia Malacol 19(3):165–189. CrossRefGoogle Scholar
  9. Dönges J (1964) Der Lebenszyklus von Posthodiplostomum cuticola (v. Nordmann 1832) Dubois 1936 (Trematoda, Diplostomatidae). Z Parasitenk 24:169–248Google Scholar
  10. Dönges J (1967) Parasitär induzierte Melaninbildung in Fischen. Z Parasitenk 29:310–312PubMedGoogle Scholar
  11. Ferguson JA, Schreck CB, Chitwood R, Kent ML (2010) Persistence of infection by metacercariae of Apophallus sp., Neascus sp., and Nanophyetus salmincola plus two myxozoans (Myxobolus insidiosus and Myxobolus fryeri) in coho salmon Oncorhynchus kisutch. J Parasitol 96:340–347. CrossRefPubMedGoogle Scholar
  12. Ferguson JA, Locke SA, Font WF, Steinauer ML, Marcogliese DJ, Cojocaru CD, Kent ML (2012) Apophallus microsoma n. sp. from chicks infected with metacercariae from coho salmon (Oncorhynchus kisutch) and review of the taxonomy and pathology of the genus Apophallus (Heterophyidae). J Parasitol 98:1122–1132. CrossRefPubMedGoogle Scholar
  13. Fried B (1994) Metacercarial excysment of trematodes. Adv Parasit 33:128Google Scholar
  14. Galazzo DE, Dayanandan S, Marcogliese DJ, McLaughlin JD (2002) Molecular systematics of some North American species of Diplostomum (Digenea) based on rDNA-sequence data and comparisons with European congeners. Can J Zool 80:2207–2217. CrossRefGoogle Scholar
  15. Hoffman GL (1958) Experimental studies on the cercaria and metacercaria of a strigeoid trematode, Posthodiplostomum minimum. Exp Parasitol 7:23–50CrossRefPubMedGoogle Scholar
  16. Ivanov VM, Semenova NN (2004) Life cycle of the trematode Rossicotrema donicum (Opisthorchiida, Heterophyidae) in the Volga River delta. Zool Zh 83:1206–1215 (in Russian)Google Scholar
  17. Izvekova GI, Tyutin AV (2011) Occurrence of partenites in mollusks and the influence that metacercaria of Apophallus muehlingi (Jagerskiold, 1898) and Posthodiplostomum cuticola (Nordmann, 1832) has on some biochemical parameters in fish. Inland Water Biol 4(3):367–372. CrossRefGoogle Scholar
  18. Jousson O, Bartoli P, Pawlowski J (1999) Molecular identification of developmental stages in Opecoelidae (Digenea). Int J Parasitol 29:1853–1858CrossRefPubMedGoogle Scholar
  19. Kent ML, Watral VG, Whipps CM, Cunningham ME, Criscione CD, Heidel JR, Curtis LR, Spitsbergen J, Markle DF (2004) A digenean metacercaria (Apophallus sp.) and a myxozoan (Myxobolus sp.) associated with vertebral deformities in cyprinid fishes from the Willamette River, Oregon. J Aquat Anim Health 16:116–129CrossRefGoogle Scholar
  20. Locke SA, McLaughlin JD, Marcogliese DJ (2010) DNA barcodes show cryptic diversity and a potential physiological basis for host specificity among Diplostomoidea (Platyhelminthes: Digenea) parasitizing freshwater fishes in the St. Lawrence River, Canada. Mol Ecol 19:2813–2827. CrossRefPubMedGoogle Scholar
  21. Lyster LL (1940) Apophallus imperator sp. nov., a heterophyid encysted in trout, with a contribution to its life history. Can J Res 18:106–121CrossRefGoogle Scholar
  22. Malek EA (1980) Snail-transmitted parasitic diseases. CRC Press, Boca Raton 658 ppGoogle Scholar
  23. Mastitsky SE (2007) First report of parasites in Lithoglyphus naticoides (Gastropoda: Hydrobiidae) from Lake Lukomskoe (Belarus). Aquat Inv 2:149–151CrossRefGoogle Scholar
  24. Miller MJ (1941) The life history of Apophallus brevis Ransom, 1920. J Parasitol 27(suppl):12Google Scholar
  25. Miller MJ (1942) Black spot disease of speckled trout. Rev Can Biol 1:464–471Google Scholar
  26. Milne I, Wright F, Rowe G, Marshal DF, Husmeier D, McGuire G (2004) TOPALi: software for automatic identification of recombinant sequences within DNA multiple alignments. Bioinformatics 20:1806–1807. CrossRefPubMedGoogle Scholar
  27. Mödlinger G (1934) Beiträge zur Biologie von Apophallus donicus. (Adatok az Apophallus donicus biológiájához). Arb I Abt Ungar Biol Forsch-Inst 7:60–65 (in Hungarian)Google Scholar
  28. Molnár K (1963) Black spot disease in Danube fishes. [Fekete pettyes betegség a dunai halakon]. Halászat 9:174 (in Hungarian)Google Scholar
  29. Molnár K, Székely Cs, Csaba Gy, Láng M, Majoros G (2001) Results of veterinary-pathological research of Lake Balaton fishes (Balatoni halak kórtani kutatásának állategészségügyi eredményei). In: Results of Balaton research in 2000 [A Balaton kutatásának 2000. évi eredményei.] Budapest: Magyar Tudományos Akadémia, pp 158–166 (in Hungarian)Google Scholar
  30. Moravec F (2001) Checklist of the metazoan parasites of fishes of the Czech Republic and the Slovak Republic, 1873–2000. Academia, Prague 168 ppGoogle Scholar
  31. Morozov FN (1952) Superfamily Heterophyoidea Faust, 1929. In: Skrjabin KI (ed) [trematodes of animals and men]. Osnovy Trematologii 6: 153–601. (in Russian)Google Scholar
  32. Niemi D, Macy R (1974) The life cycle and infectivity to man of Apophallus donicus (Skrjabin and Lindtop, 1919) (Trematoda: Heterophyidae) in Oregon. Proc Helm Soc Wash 41:223–229Google Scholar
  33. Odening K (1970) Der Entwicklungszyklus von Apophallus muehlingi (Trematoda: Opisthorchiida: Heterophyidae) in Berlin. Z Parasitenk 33:194–210. CrossRefPubMedGoogle Scholar
  34. Odening K (1973) Der Lebenszyklus des Trematoden Apophallus donicus in Berlin im Vergleich zu A. muehlingi. Biol Zentralbl 92:455–494Google Scholar
  35. Paperna I (1995) Digenea (phylum Platyhelminthes). In: Woo PTK (ed) Fish diseases and disorders: protozoan and metazoan infections. CAB International, Wallingford, pp 329–389Google Scholar
  36. Pike AW, Burt MDB (1983) The tissue response of yellow perch, Perca flavescens Mitchill to infections with the metacercarial cyst of Apophallus brevis Ransom, 1920. Parasitology 87:393–404CrossRefGoogle Scholar
  37. Quist MC, Bower MR, Hubert WA (2007) Infection by a black spot-causing species of Uvulifer and associated opercular alterations in fishes from a high-desert stream in Wyoming. Dis Aquat Org 78:129–136. CrossRefPubMedGoogle Scholar
  38. Rodnick KJ, St.-Hilaire S, Battiprolu PK, Seiler SM, Kent ML, Powell MS, Ebersole JL (2008) Habitat selection influences sex distribution, morphology, tissue biochemistry, and parasite load of juvenile coho salmon in the West Fork Smith River, Oregon. Trans Am Fish Soc 137:1571–1590CrossRefGoogle Scholar
  39. Sewell RBS (1922) Cercariae indicae Indian. J Med Res 10:1–370Google Scholar
  40. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis, version 6.0. Mol Biol Evol 30:2725–2729. CrossRefPubMedPubMedCentralGoogle Scholar
  41. Taylor LH, Hall BK, Cone DK (1993) Experimental infection of yellow perch (Perca flavescens) with Apophallus brevis (Digenea, Heterophyidae): parasite invasion, encystment, and ossicle development. Can J Zool 71:1886–1894. CrossRefGoogle Scholar
  42. Taylor LH, Hall BK, Miyake T, Cone DK (1994) Ectopic ossicles associated with metacercariae of Apophallus brevis (Trematoda) in yellow perch, Perca flavescens (Teleostei): development and identification of bone and chondroid bone. Anat Embryol 190:29–46. CrossRefPubMedGoogle Scholar
  43. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680CrossRefPubMedPubMedCentralGoogle Scholar
  44. Tkach VV, Pawlowski J, Sharpilo VP (2000) Molecular and morphological differentiation between species of the Plagiorchis vespertilionis group (Digenea, Plagiorchiidae) occurring in European bats, with a redescription of P. vespertilionis (Müller, 1780). Syst Parasitol 47:9–22. CrossRefPubMedGoogle Scholar
  45. Tobler M, Schlupp I (2008) Influence of black spot disease on shoaling behaviour in female western mosquitofish, Gambusia affinis (Poeciliidae, Teleostei). Environ Biol Fish 81:29–34CrossRefGoogle Scholar
  46. Tyutin AV, Izvekova GI (2013) Infection of mollusks and fish by the trematode Apophallus muehlingi (Jagerskiold, 1898) and its interrelations with intermediate hosts. Inland Water Biol 6:52–56. CrossRefGoogle Scholar
  47. Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JAM (2007) Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 35:W71–W74. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Van Steenkiste N, Locke SA, Castelin M, Marcogliese DJ, Abbott CL (2015) New primers for DNA barcoding of digeneans and cestodes (Platyhelminthes). Mol Ecol Resour 15:945–952. CrossRefPubMedGoogle Scholar
  49. Villeneuve DL, Curtis LR, Jenkins JJ, Warner KE, Tilton FA, Kent ML, Watral VG, Cunningham ME, Markle DF, Sethajintanin D, Krissanakriangkrai O, Johnson ER, Grove R, Anderson KA (2005) Environmental stresses and skeletal deformities in fish from the Willamette River, Oregon. USA. Environ Sci Technol 39:3495–3506. CrossRefPubMedGoogle Scholar
  50. Vojtek J (1989) The present situation of the research into the stages of development of trematodes in Czechoslovakia. Scri Fac Sci Nat Univ Purkyn Brun 19:339–352Google Scholar
  51. Wierzbicka J, Wierzbicki K (1973) Metacercariae of the genus Apophallus Lühe, 1909 (Trematoda: Heterophyidae) in Western Pomerania of Poland. Acta Ichthyol Piscat 3:75–89CrossRefGoogle Scholar
  52. Yamaguti S (1971) Synopsis of digenetic trematodes of vertebrates. Vols. I and II. Keigaku Publishing Company, Tokyo 1074 ppGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Institute for Veterinary Medical Research, Centre for Agricultural ResearchHungarian Academy of SciencesBudapestHungary
  2. 2.Department of Life SciencesNatural History MuseumLondonUK
  3. 3.Department of Parasitology and ZoologyUniversity of Veterinary MedicineBudapestHungary

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