An ultrastructural study of the surface and attachment structures of Paradiplozoon homoion (Bychowsky & Nagibina, 1959) (Monogenea: Diplozoidae)
© The Author(s). 2017
Received: 7 March 2017
Accepted: 17 May 2017
Published: 25 May 2017
Species of Diplozoon Palombi, 1949 (Monogenea: Diplozoidae) are blood-feeding ectoparasites mainly parasitising the gills of cyprinid fishes. Although these parasites have been the subject of numerous taxonomic, phylogenetic and ecological studies, the ultrastructure of the surface and haptor attachment structures remains almost unknown. In this study, we used transmission electron microscopy to examine the ultrastructure of attachment clamps and neodermal surface of Paradiplozoon homoion (Bychowsky & Nagibina, 1959), family Diplozoidae Palombi, 1949, thereby broadening our knowledge of platyhelminth biology.
The hindbody surface of P. homoion is distinctly ridged, each ridge being supported by several muscle fibers and equipped with scales on the surface plasma membrane. Such structures have not been recorded previously in species of the family Diplozoidae. Comparisons of the surface structure of different body parts revealed slight differences in the thickness and number of organelles. Each of the clamps has a flattened bowl-like structure composed of sclerites, movable skeletal-like structures that are anchored by robust, radially oriented muscle bundles. The base of the posterior median plate sclerites is equipped with glandular cells possessing secretory vesicles.
This study brings detailed ultrastructural data for the surface and haptoral attachment clamps of P. homoion and provides new insights into the ultrastructure of Diplozoidae. Glandular cells at the base of the attachment clamps responsible for sclerite development in diplozoid species were observed for the first time. Our findings support the hypothesis that the structure of particular neodermal compartments is similar within the Platyhelminthes. On the other hand, the diplozoid glandular system and the mechanism of sclerite development clearly merits further attention.
KeywordsParadiplozoon homoion Ultrastructure Neodermis Tegument Attachment clamps
Species of Diplozoon Nordmann, 1832 are blood-feeding ectoparasites mainly parasitising the gills of cyprinid fishes, where they can cause mechanical damage to the gill filaments, initiating the development of secondary infections (bacterial, mycotic) and anemia [1, 2]. During the diplozoon life-cycle, two larvae (diporpa) pair and subsequently fuse permanently, producing the typical X-shaped diplozoon body arrangement, a characteristic unique to the Diplozoidae Palombi, 1949 . Diplozoon spp. have developed a number of adaptations for successful attachment to gills, including central hooks and clamps located on the posterior adhesive organ, the haptor. The neodermis (tegument) in these species is also associated with a number of surface structures that play an important role in the ectoparasitic life-style. The neodermis has been investigated at the ultrastructural level in other monogenean species, including Gyrodactylus sp. [4, 5], Entobdella soleae, Acanthocotyle elegans , Diclidophora merlangi , Rajonchocotyle emarginata, Plectanocotyle gurnardi , Diplectanum aequans , Allodiscocotyla diacanthi , and Metamicrocotyla macracantha . Studies on species within the Diplozoidae, however, are missing. In general, the external surface of monogeneans is a syncytium formed by neodermal (tegumental) cells. The distal cytoplasm is connected via cytoplasmic junctions with the proximal cytoplasm of cell bodies located in the subsurface parenchyma [7, 12–14].
An important part of the diplozoid monogenean body is the complex of posterior attachment structures on each opisthaptor, comprising a pair of central hooks and four pairs of clamps [15, 16]. These allow the parasite to attach to the gills using an extrinsic muscle/tendon system associated with a median J-shaped sclerite . The clamps comprise a posterior and anterior jaw, joined with a median plate by anterior and posterior joining sclerites [18, 19]. These clamps are morphologically distinct and can be used for species determination [19–22]. While the ultrastructure of heteronchoinean clamps has been investigated previously [23–28], ultrastructural data for diplozoids are presently missing. In general, the clamp wall surrounding the inner sclerites is formed of a specialized muscle complex with radially oriented muscle fibers . Chemical analysis has shown that heteronchoinean sclerites, including those in Diplozoon paradoxum, consist of scleroproteins [29–32].
The main aim of this study was to undertake the first detailed ultramicroscopic analysis of the neodermal surface and attachment structures of Paradiplozoon homoion Bychowsky & Nagibina, 1959, a generalist parasite infecting a range of cyprinid fish species, including roach Rutillus rutilus (L.), bleak Alburnus alburnus (L.) and gudgeon Gobio gobio Fleming [33, 34]. The species is an oviparous monogenean parasite with a relatively high prevalence in European populations of wild and farmed fish. Our paper forms part of a complex study focusing on diplozoid morphology and ultrastructure and phylogenetic and functional indicators for their specialized life strategy.
Sample origin and collection
Adult P. homoion were collected from the gills of bleak caught in the littoral zone of the Mušov lowland reservoir (Czech Republic; 48°53′12″N, 16°34′37″E) in 2013. The fish were transported live in oxygenated water to the parasitological laboratory at the Faculty of Science, Masaryk University, where they underwent a standard parasitological autopsy  modified for the detection of diplozoid species, i.e. only the gills were examined. After euthanasia, the gills were extracted and checked for the presence of all diplozoid ontogenetic stages under an Olympus SZX 7 zoom stereo microscope. Live worms were determined based on their morphology examined under an Olympus BX50 light microscope equipped with Nomarski differential interference contrast and an Olympus Stream Motion digital image analysis system v. 1.9.2. The parasites were then processed and fixed for further light and transmission electron microscopy analysis.
Transmission electron microscopy
Parasites intended for transmission electron microscopy (TEM) were washed three times in freshwater to remove any remaining mucus. Live specimens were fixed directly in 2% osmium tetroxide for 1 h and dehydrated through an ascending acetone series. The dehydrated samples were immediately embedded in Spurr resin . In order to obtain a better orientation in basic diplozoon morphology, ten longitudinal and ten transversal semi-thin sections (0.5 μm) of whole worm bodies were cut (from the anterior extremity of body through the pharynx to the posterior extremity) using a Leica EM UC6i ultramicrotome. Sections were stained with toluidine blue for 30 s at 80 °C to contrast the acidic components (sulfates, carboxylates and phosphate radicals) of the different tissues . Selected ultrathin sections were contrasted with uranyl acetate lead by citrate and examined using a JEOL JEM-1010 TEM operating at 60 kV. Images were taken for further analysis using Megaview II software (ResAlta Research Technologies).
General morphological characterization
The musculature lying proximally to the basal lamina is well developed and formed by several layers of longitudinal and circular muscle fibers, with the circular muscle fibers placed proximally to the basal lamina (Fig. 3b). All muscle fibers are attached to the internal side of the basal lamina by hemidesmosomes (Figs. 1c; 5d, f). The space between the muscle fibers is filled with interstitial material and the fibers themselves are bound by sarcolemma.
Each sclerite is closely bound by the basal lamina of the surrounding muscle complex (Fig. 6b). Between the body of the sclerites and the surrounding basal lamina there is a space filled with closely packed fibrous content known as the outer layer (Fig. 6b). The sclerite body is composed of a moderately electron-dense material containing fibrils, which are irregularly organized and of different lengths; many of them are branched, forming randomly orientated three-dimensional fibrous structures (Fig. 6a, b). Large cavities with closely packed fibrils, mitochondria and numerous small electron-dense granules inside the lumen are clearly visible in longitudinal sections through the sclerites (Fig. 6a). Nerve fibers were located relatively close to the terminal and basal parts of sclerites (Figs. 5a, b, d and 6c, d). Very small (0.1 μm in diameter) electron-dense, membrane-bound vesicles were detected adhering to the external surface of the sclerites (Fig. 5c). Some vesicles were also located in the parenchyma space and inside sclerites on the internal surface of the cavities.
The base of the posterior median plate sclerites is equipped with glandular cells possessing secretory vesicles (Fig. 5e). The same secretory vesicles are located in the surface external syncytial layer covering the medial plate sclerite.
General morphological characterization
Comparison with previous studies concerning platyhelminth surface structures [38–40] confirms that P. homoion share analogous morphological characteristics with other Platyhelminthes. While there have been numerous previous studies focused on the morphology and ultrastructure of monogenean surface and attachment structures using TEM [10, 11, 23, 24, 26, 27, 41], none have included species from the family Diplozoidae. Highly developed transverse tegumentary annular ridges were identified on the hindbody of Eudiplozoon nipponicum using scanning electron microscopy . The same ridges were later recognized on histological sections and termed “tegumental folds” . Our findings on P. homoion suggest that such ridges are not solely of outer neodermal (tegumental) syncytium origin, the ridges comprising well-developed musculature, the surface of which is covered with an outer syncytial layer. The outer surface layer of the hindbody ridges is equipped with several electron-dense scales on the external plasmatic membrane. Epidermal scales were also recorded on the posterior surface of the monogenean D. aequans . These scales, which were composed of moderately electron-dense material, were located within the tegumental external syncytial layer cytoplasm, beneath the outer membrane. Relatively large and apparently hard scale-like neodermal structures have been reported as ‘spines’ or ‘scales’ in the monogenean Pseudorhabdosynochus spp. (Diplectanidea) , but these were not examined at an ultrastructural level. While the function of the scales on P. homoion is still not fully understood, their presence could suggest a protective role.
The primary surface structure of P. homoion is very similar to that of other monogenean species, e.g. D. merlangi ; R. emarginata, P. gurnardi ; Pricea multae ; Atriaster sp. ; Tetraonchoides sp. ; M. macracantha ; Dictyocotyle coeliaca . In each of these, the outer syncytial layer is connected to the nucleated cell body via cytoplasmic bridges.
Different granular and vesicular bodies have been observed in the syncytial layer cytoplasm in all previously studied monogeneans [12, 26, 45, 48, 49]. These bodies are produced by Golgi stacks in the inner nucleated regions and exported to the outer syncytial layer. This corresponds well with our TEM results for P. homoion, whereby the vesicles were present in both inner bodies and the outer layer. Unlike Gotocotyla bivaginalis , for which four vesicle types were documented in the outer cytoplasm layer, we observed only three types in P. homoion.
Previous studies have noted differences in the surface ultrastructure of two heteronchoinean monogeneans (R. emarginata and P. gurnardi) . Uniquely among monogeneans, for example, R. emarginata possess a series of shallow pore-like infoldings at their free edge, and lack microvilli. The surface of the diclidophorid P. gurnardi, on the other hand, is similar to that of other monogeneans. The surface structure of P. homoion differs from both of these species in that no shallow pore-like infoldings or microvilli were recorded anywhere on the surface (except around the mouth cavity). Finally, while the apical membranes of Atriaster sp.  have some undulations and microvilli, similar to E. soleae or A. elegans , they are lacking in both R. emarginata  and P. homoion.
While the P. homoion neodermis possesses the same morphological properties in different body regions, slight differences could be discerned in the width of the outer syncytial layer and the amount of individual organelles. The outer syncytial layer on the middle forebody and cross-body region of P. homoion, for example, contains a higher proportion of V1 vesicles, in contrast to other body parts where V2 and V3 vesicles tend to predominate. Although the meaning of the vesicles has not yet been clarified in Diplozoon spp., these may have a storage function. The neodermal organelles and the role of the neodermis (tegument) in nutrient uptake by parasitic platyhelminths have previously been discussed by Dalton et al. . In P. homoion, the outer syncytial layer around the apical mouth cavity, with its microvilli, ranging between 3 and 7 μm in total length. The different measurements recorded could be related to the parasite’s feeding technique, the mouth cavity having to be very flexible while sucking fish blood. Although the microvilli on the surface neodermis of the mouth have been discussed previously , their true function is still far from being understood, though it is likely to be related either to disruption of host tissue or uptake of host blood.
The clamps of P. homoion are covered with a very thin outer syncytial layer, which correlates well with results obtained for other monogenean species, such as G. bivaginalis and C. leptogaster [26, 27]. This reduction in external syncytial layer thickness probably reflects an adaptation related to the parasite’s life strategy, in that the clamps must be able to grab hold of tissue between the host’s gill lamellae in a very limited space.
While morphology and sclerite composition has frequently been studied in relation to species identification, morphological abnormalities, and taxonomic characterization of species of the family Diplozoidae [22, 51–53], there have been no ultrastructural studies on the clamps. Our findings on P. homoion correlate with observations on the clamps of other heteronchoinean monogeneans in that the clamp frame is usually formed of several electron-dense sclerites bound by radial muscle fibers, the surface is covered with a very thin outer syncytial layer, and nerve fibers are located close to the terminal and basal parts of the sclerites [26–28]. Association of nerve plexuses with the clamps has also been observed in E. nipponicum (a species closely related to P. homoion) using immunostaining and microscopic observation .
Early studies suggested that Paradiplozoon sp. sclerites were composed of chitin ; however, later work showed that the sclerites of D. paradoxum from the same family consisted of a resilin-like protein . More recently, studies on the chemical composition of monogenean clamps have shown that they are formed of scleroproteins, probably stabilized by tyrosyl residues in dityrosine [29, 56].
Studies on the development and ultrastructure of Gastrocotyle trachuri clamp walls have detected numerous differentiating cells associated with the region around the epidermal infoldings and developing clamp [24, 57]. These cells produce cytoplasmic extensions that form a syncytial region around the epidermal infolding near the region of clamp formation. The same cells also produce groups of electron-lucent membrane-bound vesicles that pass along the extensions towards the region of clamp formation. In a previous study describing the mechanism of sclerite development , the authors noted formation of short hollow tubules at the start of the process, followed by an increasing number of tubules and development of areas of dense material within the developing sclerites, after which the sclerites became uniformly electron-dense prior to forming the clamps. We recorded electron-dense material migrating through the parenchyma to the sclerite surface, an observation supported by previous observations on G. trachuri , in which electron-lucent membrane-bound vesicles were observed moving along the differentiating cell extensions towards the region of clamp formation.
Here, we document for the first time, glandular cells containing secretory bodies at the base of the posterior median plate sclerite. Numerous gland cells have been observed opening ventrally into the ducts around the haptor suckers in Polystomoides spp. . Cyton-containing secretory bodies of different sizes and shapes have been described between numerous membranous folds near the marginal hooklets in Macrogyrodactylus congolensis , although it is not thought that the glandular secretions play any role in attachment to the host’s skin by the marginal hooklets. The function of the haptoral gland cells at the base of the median plate sclerite, observed for the first time in our study, is still not fully understood and merits further attention. However, as three types of secretory products related to attachment have been observed in the anterior adhesive areas of two capsalid Benedenia monogeneans (B. rohdei and B. lutjani) , they could be associated with the process of attachment to the fish gills.
Our work represents a first approach for examination the surface and attachments clamp ultrastructure in a species of the family Diplozoidae using TEM. Ultrastructural details were characterized for a range of tegumental compartments, including scales located on the hindbody, the outer surface layer, and the cytoplasmic bridge connecting the inner nucleated cell bodies and the outer syncytial layer. Our findings for P. homoion contradict previous information that the surface ridges are purely of neodermal origin; rather, the ridges show well-developed musculature and a neodermal outer layer localized externally to the basal lamina that covers the ridge’s surface. This study provides a unique comparison of the neodermis from different body parts for species from the family Diplozoidae. We were able to produce unique photo documentation of electron-dense material (responsible for sclerite development) migrating through the parenchyma to the sclerite surface. Similarly, we also documented the occurrence of glandular cells at the base of attachment clamps for the first time in a diplozoid species. The mechanisms involved in sclerite and glandular cell development merit further attention.
The authors would like to thank the Faculty of Science, Masaryk University Brno. A big thank you goes to Professor Iva Dyková for her help with TEM observations, her helpful comments and kind approach. We acknowledge the Laboratory of EM, Biology Centre of CAS, institution supported by the MEYS CR (LM 2015062 Czech - BioImaging). Many thanks go to Gabriela Vágnerová and Eliška Šrámová for their help with fish dissections and to Dr. Kevin Roche for English correction of this text.
The study, VK, BK and MG were supported by Czech Science Foundation Grant No. GAP506/12/1258. MK and JI were supported by the Czech Science Foundation (GBP505/12/G112-ECIP). VK and JI also received financial support via the Grant Agency of Zasaryk University, Category A, No. MUNI/A/1325/2015.
Availability of data and materials
The data supporting the conclusions of this article are included within the article.
VK, BK and MG conceived and designed the study. MG provided scientific background in the field of monogenean research. VK, BK, MK and JI provided the dissections. VK conducted the TEM analysis. JI designed the tables. MK revised the manuscript critically for important intellectual content. VK analyzed data and wrote the paper. All authors contributed to improve the manuscript and read and approved the final version.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Kawatsu H. Studies on the anemia of fish – IX. Hypochromic microcytic anemia of crucian carp caused by infestation with a trematode, Diplozoon nipponicum. Bull Japan Soc Sci Fish. 1978;44:1315–9.View ArticleGoogle Scholar
- Buchmann K, Bresciani J. Monogenea (Phylum Platyhelminthes). Fish Dis Disorders. 2006;1:297–344.Google Scholar
- Khotenovsky IA. Life cycle of several monogeneans of the genus Diplozoon. Parazitol Sb. 1977;27:35–43. (In Russian).Google Scholar
- Lyons KM. Fine structure of the outer epidermis of the viviparous monogenean Gyrodactylus sp. from the skin of Gasterosteus aculeatus. J Parasitol. 1970;56:1110–7.View ArticleGoogle Scholar
- Kritsky DC, Kruidenier FJ. Fine structure and development of the body wall in the monogenean, Gyrodactylus eucaliae Ikezaki and Hoffman, 1957. Proc Helminthol Soc Wash. 1976;43:47–58.Google Scholar
- Lyons KM. The fine structure and function of the adult epidermis of two skin parasitic monogeneans, Entobdella soleae and Acanthocotyle elegans. Parasitology. 1970;60:39–52.View ArticlePubMedGoogle Scholar
- Morris GP, Halton DW. Electron microscope studies of Diclidophora merlangi (Monogenea: Polyopisthocotylea). II. Ultrastructure of the tegument. J Parasitol. 1971;57:49–61.View ArticleGoogle Scholar
- Lyons KM. Ultrastructural observations on the epidermis of the polyopisthocotylinean monogeneans Rajonchocotyle emarginata and Plectanocotyle gurnardi. Z Parasitenkd. 1972;40:87–100.View ArticlePubMedGoogle Scholar
- Shaw MK. The ultrastructure of the epidermis of Diplectanum aequans (Monogenea). Parasitology. 1980;80:9–21.View ArticleGoogle Scholar
- 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;25:43–54.View ArticlePubMedGoogle Scholar
- Cohen SC, Kohn A, Baptista-Farias MFD. Ultrastructure of the tegument of Metamicrocotyla macracantha (Alexander, 1954) Koratha, 1955 (Monogenea, Microcotylidae). Braz J Biol. 2004;64:27–31.View ArticlePubMedGoogle Scholar
- Smyth JD, Halton DW. The physiology of trematodes. Cambridge: Cambridge University Press; 1983.Google Scholar
- Tyler S, Hooge M. Comparative morphology of the body wall in flatworms (Platyhelminthes). Can J Zool. 2004;82:194–210.View ArticleGoogle Scholar
- Konstanzová V, Koubková B, Kašný M, Ilgová J, Dzika E, Gelnar M. Ultrastructure of the digestive tract of Paradiplozoon homoion (Monogenea). Parasitol Res. 2015;114:1485–94.View ArticlePubMedGoogle Scholar
- Llewellyn J. The adhesive mechanisms of monogenetic trematodes: the attachment of Plectanocotyle gurnardi (v. Ben. & Hesse) to the gills of Trigla. J Mar Biol Assoc UK. 1956;35:507–14.View ArticleGoogle Scholar
- Llewellyn J, Owen IL. The attachment of the monogenean Discocotyle sagittata Leuckart to the gills of Salmo trutta L. Parasitology. 1960;50:51–60.View ArticlePubMedGoogle Scholar
- Owen IL. The attachment of the monogenean Diplozoon paradoxum to the gills of Rutilus rutilus LI Micro-habitat and adhesive attitude. Parasitology. 1963;53:455–61.View ArticleGoogle Scholar
- Khotenovsky IA. Fauna of the USSR. Monogenea. Suborder Octomacrinae Khotenovsky. Leningrad: Nauka; 1985. (In Russian).Google Scholar
- Pečínková M, Vøllestad LA, Koubková B, Gelnar M. Asymmetries in the attachment apparatus of a gill parasite. J Zool. 2007;272:406–14.View ArticleGoogle Scholar
- Bychowsky BE. Monogenetic trematodes, their classification and phylogeny. Moscow - Leningrad: Academy of Sciences USSR; 1957. (In Russian).Google Scholar
- Bychowsky BE, Nagibina LF. On the systematics of the genus Diplozoon Nordmann (Monogenoidea). Zool Zh. 1959;38:362–77. (In Russian).Google Scholar
- Šebelová Š, Kuperman B, Gelnar M. Abnormalities of the attachment clamps of representatives of the family Diplozoidae. J Helminthol. 2002;76:249–68.View ArticlePubMedGoogle Scholar
- Shaw MK. The ultrastructure of the clamp sclerites in Gastrocotyle trachuri and other clamp-bearing monogeneans. Z Parasitenkd. 1979;59:43–51.View ArticleGoogle Scholar
- Shaw MK. The ultrastructure of the clamp wall of the monogenean gill parasite Gastrocotyle trachuri. Z Parasitenkd. 1979;58:243–58.View ArticleGoogle Scholar
- Ramasamy P, Hanna REB, Threadgold T. The surface topography and ultrastructure of the tegument and haptor of Pricea multae (Monogenea). Int J Parasitol. 1986;16:581–9.View ArticleGoogle Scholar
- Ramasamy P, Bhuvaneswari R. The ultrastructure of the tegument and clamp attachment organ of Gotocotyla bivaginalis (Monogenea, Polyopisthocotylea). Int J Parasitol. 1993;23:213–20.View ArticlePubMedGoogle Scholar
- Poddubnaya LG, Hemmingsen W, Gibson DI. Clamp ultrastructure of the basal monogenean Chimaericola leptogaster (Leuckart, 1830) (Polyopisthocotylea: Chimaericolidae). Parasitol Res. 2014;113:4023–32.View ArticlePubMedGoogle Scholar
- 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;115:2285–97.View ArticlePubMedGoogle Scholar
- Lyons KM. The chemical nature and evolutionary significance of monogenean attachment sclerites. Parasitology. 1966;56:63–101.View ArticlePubMedGoogle Scholar
- Kayton RJ. Histochemical and X-ray elemental analysis of the sclerites of Gyrodactylus spp. (Platyhelminthes: Monogenoidea) from the Utah chub, Gila atraria (Girard). J Parasitol. 1983;69:862–5.View ArticleGoogle Scholar
- Shinn AP, Gibson DI, Sommerville C. A study of the composition of the sclerites of Gyrodactylus Nordmann, 1832 (Monogenea) using X-ray elemental analysis. Int J Parasitol. 1985;25:797–805.View ArticleGoogle Scholar
- Wong WL, Michels J, Gorb SN. Resilin-like protein in the clamp sclerites of the gill monogenean Diplozoon paradoxum Nordmann, 1832. Parasitology. 2013;140:95–8.View ArticlePubMedGoogle Scholar
- Matejusová I, Koubková B, Gelnar M, Cunningham CO. Paradiplozoon homoion Bychowsky & Nagibina, 1959 versus P. gracile Reichenbach-Klinke, 1961 (Monogenea): two species or phenotypic plasticity? Syst Parasitol. 2002;53:39–47.View ArticlePubMedGoogle Scholar
- 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;108:935–41.View ArticlePubMedGoogle Scholar
- Ergens R, Lom J. Causative agents of parasitic diseases of fish. Prague: Academia; 1970.Google Scholar
- Spurr AR. A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultra Res. 1969;26:31–43.View ArticleGoogle Scholar
- Trump BF, Smuckler EA, Benditt EP. A method for staining epoxy sections for light microscopy. J Ultra Res. 1961;5:343–8.View ArticleGoogle Scholar
- Morseth DJ. The fine structure of the tegument of adult Echinococcus granulosus, Taenia hydatigena, and Taenia pisiformis. J Parasitol. 1966;52:1074–85.View ArticlePubMedGoogle Scholar
- Morris GP, Threadgold LT. Ultrastructure of the tegument of adult Schistosoma mansoni. J Parasitol. 1968;54:15–27.View ArticlePubMedGoogle Scholar
- Filippi JJ, Quilichini Y, Foata J, Marchand B. Topography and ultrastructure of the tegument of Lecithochirium musculus (Digenea: Hemiuridae), a parasite of the European eel Anguilla anguilla (Osteichthyes: Anguillidae). J Morphol. 2012;273:361–70.View ArticlePubMedGoogle Scholar
- Santos CP, Lanfredi RM. Ultrastructure and cytochemistry of the tegument of Atriaster heterodus (Platyhelminthes: Monogenea). Mem Inst Oswaldo Cruz. 2000;95:899–904.View ArticlePubMedGoogle Scholar
- Hodová I, Matejusova I, Gelnar M. The surface topography of Eudiplozoon nipponicum (Monogenea) developmental stages parasitizing carp (Cyprinus carpio L.). Cent Eur J Biol. 2010;5:702–9.Google Scholar
- 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;108:383–94.View ArticlePubMedGoogle Scholar
- Santos CP, Buchmann K, Gibson DI. Pseudorhabdosynochus spp. (Monogenea: Diplectanidae) from the gills of Epinephelus spp. in Brazilian waters. Syst Parasitol. 2000;45:145–53.View ArticlePubMedGoogle Scholar
- Justine JL. Ultrastructure of spermiogenesis, spermatozoa and the tegument in Atriaster sp. (Platyhelminthes, Monogenea, Polyopisthocotylea, Microcotylidae). Zool Scr. 1992;21:231–8.View ArticleGoogle Scholar
- Justine JL, Mattei X, Euzet L. Ultrastructure of Tetraonchoides (Platyhelminthes, Monogenea): tegument, tegumentary receptors, oocytes and mineralous corpuscles. Ann Sci Nat Zool Biol Anim. 1994;15:151–62.Google Scholar
- Poddubnaya LG, Hemmingsen W, Gibson DI. Surface ultrastructural characteristics of Dictyocotyle coeliaca Nybelin, 1941 (Monopisthocotylea: Monocotylidae), an endoparasitic monogenean of rays. Parasitol Res. 2016;115:965–73.View ArticlePubMedGoogle Scholar
- El-Naggar MM, Kearn GC. The tegument of the monogenean gill parasites Dactylogyrus amphibothrium and D. hemiamphibothrium. Int J Parasitol. 1983;13:579–92.View ArticleGoogle Scholar
- Ramasamy P, Brennan GP. 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;86:129–39.View ArticlePubMedGoogle Scholar
- Dalton JP, Skelly P, Halton DW. Role of the tegument and gut in nutrient uptake by parasitic platyhelminths. Can J Zool. 2004;82:211–32.View ArticleGoogle Scholar
- Civáňová K, Koyun M, Koubková B. The molecular and morphometrical description of a new diplozoid species from the gills of the Garra rufa (Heckel, 1843) (Cyprinidae) from Turkey - including a commentary on taxonomic division of Diplozoidae. Parasitol Res. 2013;112:3053–62.Google Scholar
- Avenant-Oldewage A, Le Roux LE, Mashego SN, van Vuuren BJ. Paradiplozoon ichthyoxanthon n. sp. (Monogenea: Diplozoidae) from Labeobarbus aeneus (Cyprinidae) in the Vaal River, South Africa. J Helminthol. 2014;88:166–72.View ArticlePubMedGoogle Scholar
- Pečínková M, Matejusova I, Koubková B, Gelnar M. Classification and occurrence of abnormally developed Paradiplozoon homoion (Monogenea, Diplozoinae) parasitising gudgeon Gobio gobio. Dis Aquat Org. 2005;64:63–8.View ArticlePubMedGoogle Scholar
- 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;31:783–92.View ArticlePubMedGoogle Scholar
- Milne SJ, Avenant-Oldewage A. The fluorescent detection of Paradiplozoon sp. (Monogenea: Diplozoidae) attachment clamps’ sclerites and integumental proteins. Onderstepoort J Vet Res. 2006;73:149–52.View ArticlePubMedGoogle Scholar
- Ramalingam K. Chemical nature of monogenean sclerites. I. Stabilization of clamp-protein by formation of dityrosine. Parasitology. 1973;66:1–7.View ArticlePubMedGoogle Scholar
- Shaw MK. The development of the clamp attachment organs of the monogenean Gastrocotyle trachuri. Z Parasitenkd. 1979;59:277–94.View ArticleGoogle Scholar
- Rohde K. Fine structure of the Monogenea, especially Polystomoides Ward. Adv Parasitol. 1975;13:1–33.View ArticlePubMedGoogle Scholar
- Arafa S. Ultrastructure of musculature of the marginal hooklets of Macrogyrodactylus congolensis, a monogenean skin parasite from the catfish Clarias gariepinus. Acta Parasit. 2011;56:122–30.View ArticleGoogle Scholar
- Whittington ID, Cribb BW. Morphology and ultrastructure of the anterior adhesive areas of the capsalid monogenean parasites Benedenia rohdei from the gills and B. lutjani from the pelvic fins of Lutjanus carponotatus (Pisces: Lutjanidae). Parasitol Res. 1999;85:399–408.View ArticlePubMedGoogle Scholar