Cent. Eur. J. Biol.• 5(5) • 2010 • 702-709 DOI: 10.2478/s11535-010-0040-2

Central European Journal of Biology

The surface topography of Eudiplozoon nipponicum () developmental stages parasitizing carp (Cyprinus carpio L.)

Research Article Iveta Hodová1,*, Iveta Matejusova2, Milan Gelnar1

1Department of Botany and Zoology, Faculty of Science, Masaryk University, 611 37 Brno, Czech Republic 2Marine Scotland, Marine Laboratory, AB11 9DB Aberdeen, United Kingdom

Received 26 October 2009; Accepted 23 April 2010

Abstract: Using scanning electron microscopy (SEM), the external morphology of all developmental stages (egg, oncomiracidium, diporpa, just fused juvenile and adult) of the parasite, Eudiplozoon nipponicum (Monogenea, Diplozoidae), from the gills of carp was studied. During the ontogeny, the tegument, tegumentary and sensory structures are subsequently developed. The tegument of free swimming oncomiracidium occurs in two types - the ciliated and non-ciliated with numerous uniciliated sensory structures. An attachment apparatus starts to form during the oncomiracidium stage. Further developmental stages are adapted to the environment of the gills. Tegumentary folds become more apparent later in development and assist to the parasite’s attachment. In connection with its reproductive strategy, the two morphological structures of diporpa (ventral sucker and dorsal papilla) appear to play important role. On the gills, two individuals need to meet and these structures mediate the fusion between two diporpae. The hindbody of adult parasite is highly modified for attachment. The haptor, folds and lobular extensions are most developed. The forebody is flexible and able to interact with host gill tissue via the mouth and associated mouth structures. The process of food intake of the parasite was discussed. Keywords: Scanning electron microscopy (SEM) • Eudiplozoon nipponicum • Monogenea • Surface topography © Versita Sp. z o.o.

1. Introduction its eyespots and surface cilia, and develops into the unpaired post-larval stage, diporpa, originally named Monogeneans belongs to the family Diplozoidae, and described by Dujardin [14]. In the diporpae which are blood-feeding parasites of mainly cyprinid stage, two structures important in the pairing process fish that parasitize their gills. Diplozoids are oviparous (the ventral sucker and the dorsal papilla) develop. platyhelminths with interesting life history and have been Diporpae are able to migrate across the gill in a subject of many biological, morphological, molecular caterpillar-like movement, using both mouth succers and phylogenetic studies [e.g. 1-11], but nevertheless and attachment apparatuses equipped with clamps and the present study is the first demonstration of the surface hooks. When a diporpa encounters the ventral sucker topography of all its developmental stages via scanning of another diporpa, it attaches to the dorsal papilla of electron microscopy. the second diporpa and both bodies twist [7]. The two Eudiplozoon nipponicum (Goto, 1891) originated individuals fuse permanently and are commonly seen in East Asia and was first recorded in Europe on a like this, in the shape of the letter X. The development farmed carp (Cyprinus carpio L.) in France in 1983 [12] of attachment the apparatus continues and a fully and rapidly spread to other European countries. This formed attachment apparatus consists of four pairs of parasite has a direct life cycle. It has an invasive larval clamps and one pair of central hooks [13]. stage, during which oncomiracidium hatches from its The SEM study of tegument and tegumentary egg in water and actively searches for a suitable host. structures was previously carried out on other One pair of attachment clamps and a pair of central monogenean (polyopisthocotylean) parasites such hooks are used by oncomiracidia to attach to the gill of as Diclidophora merlangi [15], Pseudothoracocotyla a host fish [13]. After attachment, the oncomiracidium indica [16], Bicotyle vellavoli [17], Heterapta chorinemi undergoes major morphological modifications. It loses [18], Pricea multae [19], Vallisia indica [20], Diplorchis

* E-mail: [email protected] 702 The surface topography of Eudiplozoon nipponicum (Monogenea) developmental stages parasitizing carp (Cyprinus carpio L.)

nigromaculatus [21], Gotocotyla secunda, G. bivaginalis protuberances (Figure 1A,B,C,D). The egg is ovoid, [22], Bifurcohaptor lucknowensis [23], Diplozoon with a single long filament opposite to the operculum lucknowensis [24], Allodiscocotyla diacanthi [25], (Figure 1A,B,C,D). The filament is convoluted and exits Polystoma integerrimum [26] or Paranaella luquei [27]. uterus opening behind the egg (Figure 1A,B). The line The family Diplozoidae is very unique among between the operculum and the rest of the egg is very polyopisthocotyleans. Aims of the present study were, thin and difficult to observe in a closed egg (Figure 1C). on the basis of the model parasite E. nipponicum, The shape of operculum is shown on Figure 1D. to investigate surface topography and tegumentary changes during the entire ontogenetic development, 3.2 Oncomiracidium to clarify the active role of the tegument and accessory The shape of the is fusiform (Figure 2A,B). structures during attachment, mate-seeking and Groups of ciliated cells enable movement of the following fusion. The SEM technique is used first time oncomiracidium. Ciliated cells are distributed in four to examine all stages of E. nipponicum and allows us to zones – one anterolateral, two medial and one posterior hypothesize on possible routes of food intake. (Figure 2A,B). The posterior end of oncomiracidium forms a conical projection (Figure 2A,B). A mouth opening is present in the anterior part of 2. Experimental Procedures ventral side (Figure 2B,C). A single pair of clamps and pair of hooks (not visible in SEM) are used for Live specimens of several stages (except for the attachment of the oncomiracidium (Figure 2B,D). oncomiracidia) of Eudiplozoon nipponicum (Goto, 1891) The tegument in the non-ciliated parts of were collected from the gills of experimentally oncomiracidium is formed by irregularly arranged into infected carp (1-3 years old) (Cyprinus carpio) bred low-rise surface ridges and shallow pits (Figure 2F). in the experimental pond of the Research Institute of Aquaculture and Hydrobiology in Vodňany, South Bohemia, Czech Republic. Eggs laid by mature diplozoids into the water were collected and cultivated in embryo dishes at room temperature (25°C), with water being changed daily. Oncomiracidia (n=13) hatched in 7 days after collection of eggs. The parasites used for the SEM analysis were washed in tap water several times to remove the remains of their mucus and fixed in hot 4% formaldehyde or in 4% glutaraldehyde at 4oC for

24 h, followed by postfixation in 1% OsO4 for 1 h. The specimens of oncomiracidia were fixed in 4% formaldehyde at room temperature. The material was subsequently dehydrated through a graded ethanol series, dried in a Pelco CPD II or CPD 030 critical point

drying apparatus (Bal-tec) using liquid CO2, mounted on aluminium stubs with double sided adhesive tape or discs, coated with gold in Polaron E5100 or SCD 040 sputter coating unit (Balzers) and examined in a JEOL 6300 or VEGA scanning electron microscope operating at 15-20 kV.

3. Results 3.1 Egg The egg exits the body of adult parasites via the Figure 1. Eudiplozoon nipponicum – egg, SEM micrographs. uterus opening (Figure 1A,B). The uterus opening (A) egg, apical view; (B) egg, lateral view; (C) detail of egg, has a triangularly-shaped flap (Figure 1A,B). lateral view; (D) opened egg; egg (eg); triangular-like flap (t); convoluted filament (fi); line between operculum and The surface of egg is smooth, without any rest of the egg (arrows); Scale – bars: A–D=100 µm.

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On the dorsal and ventral surfaces, there are numerous 3.4 Paired stages (juveniles and pre-adult stages) uniciliated sensory structures (Figure 2C,F). Figure 3F illustrates the first paired stage so-called “just married” – a connection is formed between the dorsal 3.3 Diporpa papilla of one individual and ventral sucker of the other Two structures typical for diporpae were observed. individual (Figure 3G shows the detail). The tegument The dorsal knob-like papilla (Figure 3A,B) develop on the is similar to the tegument of diporpae. After pairing, dorsal surface of the medial region, and on the ventral the concretion of the tegument and inner structures surface the ventral sucker is formed (Figure 3C,D). continues in the region of fusion. In the central region of The ventral sucker and dorsal knob-like papilla are involved the hindbody, lobular extensions typical for the specimen in a first contact between diporpae when pairing occurs. E. nipponicum are gradually formed. The attachment The mouth is subterminal (Figure 3C). The tegument is apparatus is subsequently developed into the final covered by discontinuous ridges and fine surface folds form, four pairs of clamps and one pair of central hooks and pits (Figure 3A,B,C,D). Single uniciliated sensory (compare Figure 3F and 5E). endings, basally supported by a collar of tegument, were observed around the mouth and on the body. The 3.5 Adult uniciliated sensory endings without tegumentary collars The body of the adult parasite, composed of two were abundant on the entire forebody (Figure 3E). individuals, has a typical X-shape (Figure 5A).

Figure 2. Eudiplozoon nipponicum – oncomiracidium, SEM Figure 3. Eudiplozoon nipponicum – diporpa and juvenile stage, micrographs. (A) oncomiracidium, dorsal view; SEM micrographs. (A) diporpa, dorsal view; (B) detail of (B) oncomiracidium, lateroventral view; (C) detail of mouth dorsal papilla; (C) diporpa, ventral view; (D) detail of ventral opening; (D) detail of clamp; (E) detail of zone with partly sucker; (E) detail of tegument; (F) paired juvenile stage; (G) lacked cilia; (F) detail of non-ciliated part of tegument; detail of region of fusion; dorsal papilla (dp); ventral sucker anterolateral zone with ciliated cells (alz); medial zone (vs); mouth opening (mo); clamps (cl); secretory products (mz); posterior zone (pz); mouth opening (mo); clamp (arrows); uniciliated sensory ending with collar (arrowhead); (cl); uniciliated sensory structure (arrow); Scale – bars: A, uniciliated sensory ending without collar (star); Scale – bars: B=50 µm; F=20 µm; C, E=10 µm; D=5 µm. A, C, F=200 µm; G = 50 µm; B, D=10 µm; E=5 µm.

704 The surface topography of Eudiplozoon nipponicum (Monogenea) developmental stages parasitizing carp (Cyprinus carpio L.)

3.5.1 Forebody 3.5.2 Region of fusion The mouth is large, subterminal and situated on the The transverse ridges and folds of two fused adults are ventral side of each of two forebodies. Uniciliated continuous (Figure 5F). The region of fusion is relatively sensory structures may occur at the opening of the mouth wide (Figure 5G) compared to the juvenile stage. The (Figure 4C,D). A pair of the rounded buccal suckers in uterus openings of both individuals are situated on the the buccal cavity is shown in Figure 4D. The pharynx is ventral surface in the region of fusion (Figure 5F). circular with an opening in the middle (Figure 4E). The surface of the buccal cavity is covered by 3.5.3 Hindbody short dense microvilli-like prejections (Figure 4C,D,E). Transverse tegumentary annular ridges on the parasite The tegument of the forebody is highly folded, forming hindbody are highly developed, especially in the central annular transverse ridges (Figure 4A,B). These ridges region where they form prominent, fleshy, flange-like are discontinuous at the lateral margins. Configuration structures (Figure 5B,C,D). The lobed enlargement of the parallel lower folds is superimposed on these (Figure 5C) in the middle of hindbody is a typical feature major ridges (Figure 4B). The ridges and folds are more of E. nipponicum. obvious in this stage than in diporpae stage. There Each haptor is symmetrical, with two rows of the four are many ciliated structures on the dorsal and ventral pincer-like clamps (Figure 5A,B,E). There is a row of surfaces of the forebody which may have a sensory papilla-like dome-shaped non-ciliated structures running function. The entire surface of the forebody, including longitudinally along each of the lateral sides of the its ridges and folds, is covered by shallow pits and hindbody, from anterior of the flanges and almost reaching depressions, that lack microvilli. the haptor (i.e. along the peduncle) (Figure 5B,C).

Figure 4. Eudiplozoon nipponicum – adult, SEM micrographs. Figure 5. Eudiplozoon nipponicum – adult, SEM micrographs. (A) forebody of adult; (B) detail of tegument; (C) detail of (A) overall view of adult; (B) hindbody of adult; (C) hindbody, mouth; (D) detail of opened mouth; (E) detail of everted detail of enlargement and row of non-ciliate papillae; (D) mouth; microvilli (mv); buccal sucker (bs); pharynx detail of tegument with annular disconnected ridges; (E) (ph); uniciliated sensory structure (arrow); Scale – bars: detail of haptor with four pair of clamps; (F) region of fusion, A=100 µm; B – E=10 µm. lateral view; (G) region of fusion, caudal view; transverse tegumentary annular ridge (tr); uterus opening (uo); lobed enlargement (arrows); non ciliate papillae (arrowheads); Scale - bars: A=1 mm; B-G=100 µm.

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4. Discussion the loss of cilia from ciliary cells and delayed hatching of oncomiracidia. Other authors suggested the loss This paper describes surface structures of all of cilia is triggered by manipulation in experimental developmental stages of E. nipponicum using SEM, environments [37] or during oncomiracidia death [34]. describing some of the structures, such as the When an oncomiracidium of E. nipponicum triangularly-shaped tegumental flap of uterus opening, attaches to the gills of its host, it undergoes changes for the first time. The shape of the egg and a single long in its morphology to become a diporpa. It also loses filament are similar to the egg ofZeuxapta seriolae, and its pigmented eyes, develops an intestine and two organism previously studied using SEM [28]. However, new structures – a small muscular sucker on the E. nipponicum eggs differ from Z. seriolae in shape of ventral surface of its forebody and knob-like papilla their opercular tip: conical in E. nipponicum but rounded on the dorsal surface. The surface of the dorsal knob- in Z. seriolae. The egg surface of E. nipponicum is like papilla is covered by small globular formations smooth, unlike the rounded protuberances observed (Figure 3B). We propose that these formations might in Z. seriolae. The position of filament on the opposite be products of secretory glands described in Zurawski pole than the operculum is specific for E. nipponicum et al. [7]. These authors noted that the dorsal papilla of among other diplozoids, but is similar to Z. seriolae and E. nipponicum diporpa failed to give a positive reaction Heteraxinoides xanthophilis [29]. with phaloidin, therefore it was concluded that this The oncomiracidium´s ciliated cells are arranged structure was not muscular. This finding corresponds to in anterolateral, medial and posterior zones, serving to the work of Khotenovsky [13] who described this dorsal facilitate its movement while searching for a host and papilla as “a tegumental plug” associated with group of might serve as mechanoreceptors or proprioreceptors. gland cells located in the parenchyma. Khotenovsky Their presence and distribution was first observed by [13] suggested that the secretory products of these Kamegai [30] and Khotenovsky [13]. Oncomiracidium gland cells assist tegument lysis during diporpae tegument in the non-ciliated parts with irregularly pairing. This corresponds with our observation of the arranged surface folds and shallow pits is similar to justpaired juvenile stage where tegument in the place of tegument of Rajonchocotyle emarginata studied by attachment is interconnected (Figure 3F,G). Whittington [31]. However, there are differences in The large subterminal mouth is located on the arrangement of the uniciliated sensillae and distribution ventral forebody of E. nipponicum adults. The buccal of zones with locomotive ciliated cells compared with cavity is covered by microvilli-like projections to increase E. nipponicum. The position of the uniciliated sensillae its surface area for attachment and for absorption of E. nipponicum was first described by Lambert and during suction. The sequence of the three pictures Denis [32], frequently being located on the dorsal and (Figure 4C,D,E) represents their mode of feeding in ventral tegument and surrounding the buccal suckers. accordance with our current hypothesis. The mouth is Their function as rheoreceptors or mechanoreceptors progressively opened and the buccal suckers are forced was suggested as oncomiracidium require active further out, exposing the pharynx. The buccal suckers movement in water current during location of its host. attach to the surface of the secondary gill lamellae. It is generally believed that mobile stages of the Pharynx is pulled back by muscles causing suction parasites lose their ciliary cells after the parasite which ruptures the gill lamellae. The buccal cavity is attaches [33]. Later on, Kearn [34] described filled with blood. Alternatively, the gland secretions the process of ciliary loss during attachment of participate in digestion and the rupture of the lamellae. oncomiracidia of Entobdella soleae after contact with Blood is then probably pumped back into the intestine by its host and Tinsley [35] referred to loss of the intact the pharynx. The mechanisms of the pharynx eversion ciliary cells from the body surface of E. soleae. In and protruding is very similar to the mechanism of intake one case, we noted that E. nipponicum oncomiracidia of eroded epidermal tissue as described on Entobdella lost their locomotory cilia (Figure 2E), however, the soleae by Kearn [38]. The contribution of these glands ciliary cells remained in the place. In this case, the in the digestion process is an aim of our future research. exact shape and position of each individual ciliary cell When the tegument of an E. nipponicum adult was observable. The analogous structures similar to individual is finally formed, connecting both parasitic ciliary cells of E. nipponicum were observed by du individuals, the forebody is covered by transverse Preez and Kok [36] on surface of Polystoma australis ridges that are well developed compared either to oncomiracidia which failed to attach to its host. A diporpa or juvenile stage. The ridged body surface is detailed study is needed to fully understand process formed by shallow pit-like depressions. The analogous of cilia loss. There is a possible relationship between transverse tegumental ridges were described in

706 The surface topography of Eudiplozoon nipponicum (Monogenea) developmental stages parasitizing carp (Cyprinus carpio L.)

Allodiscocotyla diacanthi [25], Pseudothoracocotyla for E. nipponicum. The annular ridges and extensions indica [16], Pricea multae [19] and other monogeneans. on hindbody are important for attachment and secure The single uniciliated sensory endings with a collar of position of the parasite among the secondary gill tegument are most frequent around the mouth opening lamellae analogous to a zip fastener. and occur also on the forebody. Presumably, this type The non-ciliate dome-shaped papillae positioned of sensory structure has a significant function during in a row, running longitudinally along lateral side of the feeding process. These receptors are more frequent hindbody may have a function in the movement of the on forebody tegument of diporpae, compared to adults. parasite on the surface of the gill. Similar papillae were It is presumably related to signalling during mate described in Entobdella soleae [39]. selection. This type of sensillae was described in many In conclusion, this study illustrates several novel monogeneans, for example in Diclidophora merlangi sensory structures of E. nipponicum which were not [15]. The second type of uniciliated sensory endings previously described. Uniciliated receptors may function without the tegumental collar occurs on the surface of as tangoreceptors or rheoreceptors and may be involved the forebody and is also more common in diporpae than in orientation of the parasite body in relation to a direction in adults. The nonciliated papilla-like sensory endings of water current. The non-ciliated receptors may be occurring on the forebody were described in Gotocotyla mechanoreceptors and assist body orientation during secunda [22], Vallisia indica [20] and other monogenean feeding. The receptors on the hindbody may respond to a species. Microvilli are common structures on the surface need of movement of the haptor and its individual clamps. of the most monogeneans, however, they are absent on the body surface of E. nipponicum. The lack of microvilli was also reported in Pseudothoracocotyla indica [18]. Acknowledgements Because diplozoon is formed from two individuals, the reproductive organs are doubled and interconnected. This study was supported by the Czech Science Two uteri terminate on the ventral surfaces by two Foundation, grant No. GA 524/07/1610, Research openings in the region of fusion located cross-wise. Plan of Masaryk University No. MSM 0021622416 and The tegument of the hindbody in adults differs Ichthyoparasitology Research Centre of the Ministry between diporpae and juveniles. The transverse annular of Education, Youth and Sports of the Czech Republic ridges are highly developed and in the middle part of the No. LC 522. We thank Carey Cunningham for English body form prominent, fleshy extensions characteristic correction and helpful comments.

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