BACKGROUND: Monogeneans, in general, show a range of unique adaptations to a parasitic lifestyle, making this group enormously diverse. Due to their unique biological properties, diplozoid monogeneans represent an attractive model group for various investigations on diverse biological interactions. However, despite numerous studies, there are still gaps in our knowledge of diplozoid biology and morphofunctional adaptations. RESULTS: In this study, we provide a comprehensive microscopic analysis of systems/structures involved in niche searching, sensing and self-protection against the host environment, and excretory/secretory processes in Eudiplozoon nipponicum. Freeze-etching enabled us to detect syncytium organisational features not visible by TEM alone, such as the presence of a membrane subjacent to the apical plasma membrane (separated by a dense protein layer) and a lack of basal plasma membrane. We located several types of secretory/excretory vesicles and bodies, including those attached to the superficial membranes of the tegument. Giant unicellular glands were seen accumulating predominantly in the apical forebody and hindbody haptor region. Muscle layer organisation differed from that generally described, with the outer circular and inner longitudinal muscles being basket-like interwoven by diagonal muscles with additional perpendicular muscles anchored to the tegument. Abundant muscles within the tegumentary ridges were detected, which presumably assist in fixing the parasite between the gill lamellae. Freeze-etching, alongside transmission electron and confocal microscopy with tubulin labelling, enabled visualisation of the protonephridia and nervous system, including the peripheral network and receptor innervation. Three types of receptor were identified: 1) uniciliated sensory endings with a subtle (or missing) tegumentary rim, 2) obviously raised uniciliated receptors with a prominent tegumentary rim (packed with massive innervation and muscles) and 3) non-ciliated papillae (restricted to the hindbody lateral region). CONCLUSIONS: This study points to specific morphofunctional adaptations that have evolved in diplozoid monogeneans to confront their fish host. We clearly demonstrate that the combination of different microscopic techniques is beneficial and can reveal hidden differences, even in much-studied model organisms such as E. nipponicum.

Department of Botany and Zoology Faculty of Science Masaryk University Kotlářská 2 611 37 Brno Czech Republic

Institute of Scientific Instruments of the Czech Academy of Sciences v v i Královopolská 147 612 64 Brno Czech Republic

See more in PubMed

Poulin R. Evolutionary ecology of parasites: from individuals to communities. London: Chapman and Hall; 1998.

Kearn GC. Parasitism and the platyhelminths. London: Chapman & Hall; 1998.

Woo PTK. Editor. Fish diseases and disorders: 1. Protozoan and metazoan infections. 2nd. Ed. Oxfordshire: CAB International; 2006.

Pečínková M, Matějusová I, Koubková B, Gelnar M. Investigation of Paradiplozoon homoion (Monogenea, Diplozoidae) life cycle under experimental conditions. Parasitol Int. 2007;56(3):179–183. doi: 10.1016/j.parint.2007.01.010. PubMed DOI

Zurawski TH, Mousley A, Maule AG, Gelnar M, Halton DW. Cytochemical studies of the neuromuscular systems of the diporpa and juvenile stages of Eudiplozoon nipponicum (Monogenea: Diplozoidae) Parasitology. 2003;126(4):349–357. doi: 10.1017/S0031182002002871. PubMed DOI

Valigurová A, Hodová I, Sonnek R, Koubková B, Gelnar M. Eudiplozoon nipponicum in focus: monogenean exhibiting a highly specialized adaptation for ectoparasitic lifestyle. Parasitol Res. 2011;108(2):383–394. doi: 10.1007/s00436-010-2077-6. PubMed DOI

Košková E, Matějusová I, Civáňová K, Koubková B. Ethanol-fixed material used for both classical and molecular identification purposes: Eudiplozoon nipponicum (Monogenea: Diplozoidae) as a case parasite species. Parasitol Res. 2010;107(4):909–914. doi: 10.1007/s00436-010-1949-0. PubMed DOI

Košková E, Špakulová M, Koubková B, Reblánová M, Orosová M. Comparative karyological analysis of four diplozoid species (Monogenea, Diplozoidae), gill parasites of cyprinid fishes. Parasitol Res. 2011;108(4):935–941. doi: 10.1007/s00436-010-2135-0. PubMed DOI

Zurawski TH, Mousley A, Mair GR, Brennan GP, Maule AG, Gelnar M, Halton DW. Immunomicroscopical observations on the nervous system of adult Eudiplozoon nipponicum (Monogenea: Diplozoidae) Int J Parasitol. 2001;31(8):783–792. doi: 10.1016/S0020-7519(01)00192-8. PubMed DOI

Zurawski TH, Mair GR, Maule AG, Gelnar M, Halton DW. Microscopical evaluation of neural connectivity between paired stages of Eudiplozoon nipponicum (Monogenea: Diplozoidae). J Parasitol. 2003;89(1):198–200. 10.1645/0022-3395(2003)089[0198:meoncb]2.0.co;2. PubMed

Hodová I, Matějusová I, Gelnar M. The surface topography of Eudiplozoon nipponicum (Monogenea) developmental stages parasitizing carp (Cyprinus carpio L.) Cent Eur J Biol. 2010;5:702–709.

Konstanzová V, Koubková B, Kašný M, Ilgová J, Dzika E, Gelnar M. Excretory system of representatives from family Diplozoidae (Monogenea) Parasitol Res. 2016;115(4):1493–1500. doi: 10.1007/s00436-015-4882-4. PubMed DOI

Konstanzová V, Koubková B, Kašný M, Ilgová J, Dzika E, Gelnar M. Ultrastructure of the digestive tract of Paradiplozoon homoion (Monogenea) Parasitol Res. 2015;114(4):1485–1494. doi: 10.1007/s00436-015-4331-4. PubMed DOI

Konstanzová V, Koubková B, Kašný M, Ilgová J, Dzika E, Gelnar M. An ultrastructural study of the surface and attachment structures of Paradiplozoon homoion (Bychowsky & Nagibina, 1959) (Monogenea: Diplozoidae) Parasit Vectors. 2017;10(1):261. doi: 10.1186/s13071-017-2203-8. PubMed DOI PMC

Hodová I, Sonnek R, Gelnar M, Valigurová A. Architecture of Paradiplozoon homoion: a diplozoid monogenean exhibiting highly-developed equipment for ectoparasitism. PLoS One. 2018;13(2):e0192285. doi: 10.1371/journal.pone.0192285. PubMed DOI PMC

Threadgold LT. Parasitic platyhelminthes. In: Bereiter-Hahn J, Matoltsy AG, Richards KS, editors. Biology of the tegument. Berlin: Springer-Verlag; 1984. pp. 132–212.

Smyth JD, Halton DW. The physiology of trematodes. 1. Cambridge, UK: Cambridge University Press; 1984.

Ramasamy P, Brennan GP, Halton DW. Ultrastructure of the surface structures of Allodiscocotyla diacanthi (Polyopisthocotylea: Monogenea) from the gills of the marine teleost fish, Scomberoides tol. Int J Parasitol. 1995;25(1):43–54. doi: 10.1016/0020-7519(94)E0039-P. PubMed DOI

Mehlhorn H, editor. Parasitology in focus: facts and trends. Berlin Heidelberg: Springer-Verlag; 1988.

Ramasamy P, Hanna REB, Threadgold LT. The surface topography and ultrastructure of the tegument and haptor of Pricea multae (Monogenea) Int J Parasitol. 1986;16(6):581–589. doi: 10.1016/0020-7519(86)90024-X. DOI

Conder GA, Marchiondo AA, Williams JF, Andersen FL. Freeze-etch characterization of the teguments of three metacestodes: Echinococcus granulosus, Taenia crassiceps, and Taenia taeniaeformis. J Parasitol. 1983;69(3):539–548. doi: 10.2307/3281368. PubMed DOI

Ramasamy P, Brennan G. Ultrastructure of the surface structures and haptor of Empleurosoma pyriforme (Ancyrocephalinae; Monopisthocotylea: Monogenea) from the gills of the teleost fish Therapon jarbua. Parasitol Res. 2000;86(2):129–139. doi: 10.1007/s004360050022. PubMed DOI

Kováčiková M, Simdyanov TG, Diakin A, Valigurová A. Structures related to attachment and motility in the marine eugregarine Cephaloidophora cf. communis (Apicomplexa) Eur J Protistol. 2017;59:1–13. doi: 10.1016/j.ejop.2017.02.006. PubMed DOI

Ilgová J, Kavanová L, Matiašková K, Salát J, Kašný M. Effect of cysteine peptidase inhibitor of Eudiplozoon nipponicum (Monogenea) on cytokine expression of macrophages in vitro. Mol Biochem Parasitol. 2020;235:111248. doi: 10.1016/j.molbiopara.2019.111248. PubMed DOI

Goto S. On Diplozoon nipponicum, n.sp. J Coll Sci Imp Univ Tokyo. 1891;4:151–192.

El-Naggar M, Kearn G. Glands associated with the anterior adhesive areas and body margins in the skin-parasitic monogenean Entobdella soleae. Int J Parasitol. 1983;13(1):67–81. doi: 10.1016/S0020-7519(83)80067-8. DOI

Rees JA, Kearn GC. The anterior adhesive apparatus and an associated compound sense organ in the skin-parasitic monogenean Acanthocotyle Iobianchi. Z Parasitenk. 1984;70(5):609–625. doi: 10.1007/BF00926591. DOI

El-Naggar MM, Arafa SZ, El-Abbassy SA, Kearn GC, Cable J. Ultrastructure of the anterior adhesive apparatus of the gill parasite Macrogyrodactylus clarii and skin parasite M. congolensis (Monogenea; Gyrodactylidae) from the catfish Clarias gariepinus. Parasitol Int. 2019;71:151–159. doi: 10.1016/j.parint.2019.03.005. PubMed DOI

Wong W-L, Gorb SN. Attachment ability of a clamp-bearing fish parasite, Diplozoon paradoxum (Monogenea), on gills of the common bream, Abramis brama. J Exp Biol. 2013;216(Pt 16):3008–3014. doi: 10.1242/jeb.076190. PubMed DOI

Wong W-L, Brennan GP, Halton DW, Maule AG, Lim L-HS. Secretory products of the haptoral reservoirs and peduncular glands in two species of Bravohollisia (Monogenea: Ancyrocephalidae) Invertebr Biol. 2008;127(2):139–152. doi: 10.1111/j.1744-7410.2007.00118.x. DOI

El-Naggar M, Kearn GC. Haptor glands in the gill-parasitic, ancyrocephaline monogenean Cichlidogyrus halli typicus and the report of a possible prokaryotic symbiont. Int J Parasitol. 1989;19(4):401–408. doi: 10.1016/0020-7519(89)90096-9. DOI

Kearn GC. The survival of monogenean (platyhelminth) parasites on fish skin. Parasitology. 1999;119:57–88. doi: 10.1017/S003118200008464X. PubMed DOI

Roudnický P, Potěšil D, Zdráhal Z, Gelnar M, Kašný M. Laser capture microdissection in combination with mass spectrometry: approach to characterization of tissue-specific proteomes of Eudiplozoon nipponicum (Monogenea, Polyopisthocotylea) PLoS One. 2020;15(6):e0231681. doi: 10.1371/journal.pone.0231681. PubMed DOI PMC

Valverde-Islas LE, Arrangoiz E, Vega E, Robert L, Villanueva R, Reynoso-Ducoing O, Willms K, Zepeda-Rodriguez A, Fortoul TI, Ambrosio JR. Visualization and 3D reconstruction of flame cells of Taenia solium (Cestoda) PLoS One. 2011;6(3):e14754. doi: 10.1371/journal.pone.0014754. PubMed DOI PMC

Rohde K, Watson NA, Roubal FR. Ultrastructure of the protonephridial system of Anoplodiscus cirrusspiralis (Monogenea Monopisthocotylea) Int J Parasitol. 1992;22(4):443–457. doi: 10.1016/0020-7519(92)90145-B. PubMed DOI

Poddubnaya LG, Xylander WER, Gibson DI. Ultrastructural characteristics of the protonephridial terminal organ and associated ducts of adult specimens of the Aspidogastrea, Digenea and Monogenea, with comments on the relationships between these groups. Syst Parasitol. 2012;82(2):89–104. doi: 10.1007/s11230-012-9359-6. PubMed DOI

Rohde K, Watson N. Ultrastructure of the flame bulbs and protonephridial capillaries of Prorhynchus (Lecithoepitheliata, Prorhynchidae, Turbellaria) Zool Scr. 1991;20(2):99–106. doi: 10.1111/j.1463-6409.1991.tb00277.x. DOI

Rohde K, Justine J-L, Watson N. Ultrastructure of the flame bulbs of the Monopisthocotylean Monogenea Loimosina wilsoni (Loimoidae) and Calceostoma herculanea (Calceostomatidae) Ann Parasitol Hum Comp. 1989;64(6):433–442. doi: 10.1051/parasite/1989646433. DOI

Kearn GC. Some aspects of the biology of monogenean (platyhelminth) parasites of marine and freshwater fishes. Oceanography. 2014;2:117.

Mair GR, Maule AG, Shaw C, Halton DW. Muscling in on parasitic flatworms. Parasitol Today. 1998;14(2):73–76. doi: 10.1016/S0169-4758(97)01182-4. PubMed DOI

Halton DW, Maule AG, Mair GR, Shaw C. Monogenean neuromusculature: some structural and functional correlates. Int J Parasitol. 1998;28(10):1609–1623. doi: 10.1016/S0020-7519(98)00063-0. PubMed DOI

Poddubnaya LG, Hemmingsen W, Gibson DI. Clamp ultrastructure of the basal monogenean Chimaericola leptogaster (Leuckart, 1830) (Polyopisthocotylea: Chimaericolidae) Parasitol Res. 2014;113(11):4023–4032. doi: 10.1007/s00436-014-4070-y. PubMed DOI

Poddubnaya LG, Hemmingsen W, Gibson DI. Ultrastructural observations of the attachment organs of the monogenean Rajonchocotyle emarginata (Olsson, 1876) (Polyopisthocotylea: Hexabothriidae), a gill parasite of rays. Parasitol Res. 2016;115(6):2285–2297. doi: 10.1007/s00436-016-4973-x. PubMed DOI

Specian RD, Lumsden RD, Ubelaker JE, Allison VF. A unicellular endocrine gland in cestodes. J Parasitol. 1979;65(4):569–578. doi: 10.2307/3280324. PubMed DOI

Tyler S, Hooge M. Comparative morphology of the body wall in flatworms (Platyhelminthes) Can J Zool. 2004;82(2):194–210. doi: 10.1139/z03-222. DOI

Grano-Maldonado MI. Ultrastructure of the external sensory apparatus of Gyrodactylus gasterostei Gläser, 1974. Microsc Res Tech. 2014;77(9):740–747. doi: 10.1002/jemt.22396. PubMed DOI

Rohde K, Watson NA. Ultrastructure of the buccal complex of Polylabroides australis (Monogenea, Polyopisthocotylea, Microcotylidae) Int J Parasitol. 1995;25(3):307–318. doi: 10.1016/0020-7519(94)00083-Z. PubMed DOI

Lyons KM. The epidermis and sense organs of the Monogenea and some related groups. Adv Parasitol. 1973;11:193–232. doi: 10.1016/S0065-308X(08)60187-6. PubMed DOI

Lyons KM. Compound sensilla in monogenean skin parasites. Parasitology. 1969;59(3):625–636. doi: 10.1017/S0031182000031164. DOI

Ergens R, Lom J. Původci parasitárních nemocí ryb. 1. Praha: Academia; 1970.

Khotenovsky IA. Fauna of the USSR. Monogenea. Suborder Octomacrinae Khotenowsky. Nauka, Leningrad (in Russian) 1985.

Humason GL. Animal tissue techniques. 2. San Francisco and London: W.H. Freeman and Company; 1967.

Snow R. Alcoholic hydrochloric acid-carmine as a stain for chromosomes in squash preparations. Stain Technol. 1963;38(1):9–13. doi: 10.3109/10520296309061161. PubMed DOI

Valigurová A, Vaškovicová N, Musilová N, Schrével J. The enigma of eugregarine epicytic folds: where gliding motility originates? Front Zool. 2013;10(1):57. doi: 10.1186/1742-9994-10-57. PubMed DOI PMC