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The Journal of Experimental Biology 216, 3008-3014 © 2013. Published by The Company of Biologists Ltd doi:10.1242/jeb.076190

RESEARCH ARTICLE Attachment ability of a clamp-bearing fish parasite, Diplozoon paradoxum (), on gills of the common bream, Abramis brama

Wey-Lim Wong* and Stanislav N. Gorb Department of Functional Morphology and Biomechanics, Zoological Institute, Christian-Albrechts-Universität zu Kiel, Am Botanischen Garten 1-9, 24118 Kiel, Germany *Author for correspondence ([email protected])

SUMMARY Monogeneans, which are mainly fish ectoparasites, use various types of haptoral (posterior) attachment apparatus to secure their attachment onto their hosts. However, it remains unclear how strongly a monogenean can attach onto its host. In the present study, we aimed for the first time to (1) measure pull-off forces required to detach a pair of clamp-bearing monogeneans, Diplozoon paradoxum, from gills of Abramis brama and (2) determine the contribution of muscles to the clamp movements. A mean force of 6.1±2.7mN (~246 times the ’ weight) was required to dislodge a paired D. paradoxum vertically from the gills. There were significant differences (P<0.05, Tukey test) between the widths of clamp openings in D. paradoxum treated in three different solutions: the widest clamp openings were observed in the monogeneans treated in 100mmoll−1 potassium chloride solution (58.26±13.44μm), followed by those treated in 20mmol l−1 magnesium chloride solution (37.91±7.58μm), and finally those treated in filtered lake water (20.16±8.63μm). This suggests that the closing of the clamps is probably not due to the continuous contraction of extrinsic muscles but is caused by the elasticity of the clamp material and that muscle activity is required for clamp opening. Key words: biomechanics, monogenean, muscle, pull-off force, Platyhelminthes, resilin. Received 15 June 2012; Accepted 27 March 2013

INTRODUCTION attachment apparatus and associated muscular systems. Although Attachment is an essential feature of all parasitic organisms for their investigation of the haptoral attachment mechanism of monogeneans survival, and monogeneans, which are mainly ectoparasites, are no was conducted, to the best of our knowledge, as early as 1896 by exception. Monogenea is one of the largest classes within the phylum Cerfontaine (Cerfontaine, 1896), who examined the clamping Platyhelminthes and they usually possess anterior and posterior mechanism of Diclidophora denticulata on the gill of the fish Gadus attachment apparatus that are used for settlement, feeding, virens, no attempt has been made to measure the forces generated locomotion and transfer from host to host (Bychowsky, 1957; by the haptoral attachment systems. Additionally, it remains Yamaguti, 1963; Kearn, 1998). The anterior attachment apparatus unknown how muscles control the operation of the attachment of monogeneans (viz. head organs, bothria, pits or suckers) is usually apparatus. However, such information is important not only for a used for temporary attachment during their leech-like movement better understanding on the biology of monogeneans, but also for (Bychowsky, 1957; Kearn, 1999; Wong et al., 2006). The haptor the development of novel methods to control parasites in medicine or posterior attachment apparatus of monogeneans is more diverse and veterinary contexts. in its structure, usually used for a more secure and permanent Diplozoon paradoxum Nordmann 1832 (Platyhelminthes: attachment, and considered as the ‘hallmark’ for monogeneans. The Monogenea: Diplozoidea) is a gill parasite of freshwater fishes. haptors are generally equipped with various sclerotized armaments, This monogenean uses four pairs of clamps (four each on the left which include marginal hooks, anchors, suckers, clamps and and right side of the haptor) and a pair of relatively small hooks squamoid discs, and also adhesive secretions, or a combination of for posterior attachment at the host secondary gill lamellae these (Bychowsky, 1957; Yamaguti, 1963; Kearn, 1999; Wong et (Bychowsky and Nagibina, 1959; Owen, 1963a; Bovet, 1967). al., 2008). These unique haptoral attachment apparatus have The clamps are thought to provide the major role in the attachment encouraged many scientists to investigate how they operate of D. paradoxum, while the relatively small hooks most likely efficiently. Several studies have been undertaken to elucidate the function only during the initial stage of attachment (Owen, 1963a). attachment mechanism of the anchors (Llewellyn, 1960; Kearn, Each clamp of D. paradoxum possesses two jaws, hinged to each 1971), marginal hooks (Shinn et al., 2003; Arafa, 2011), clamps other, and each jaw is supported peripherally by marginal sclerites. (Cerfontaine, 1896; Llewellyn, 1956; Llewellyn, 1957; Llewellyn, The clamp is thought to close by an extrinsic muscle/tendon 1958; Llewellyn and Owen, 1960; Owen, 1963a; Bovet, 1967), system associated with a median J-shaped sclerite (Owen, 1963a). squamoid discs (Paling, 1966; Sánchez-García et al., 2011) and During their sexual maturation, two parasites fuse together haptoral secretions (Rand et al., 1986; Wong et al., 2008). However, permanently at the middle of their bodies forming a joint H-shaped conclusions about the functional principles of the attachment body (Fig.1) (Bychowsky and Nagibina, 1959; Bovet, 1967). The apparatus are mainly based on morphological investigations of the body connection or fusion ‘bridge’ between two D. paradoxum

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Scanning electron microscopy The fixed specimens were washed with 0.01moll–1 phosphate buffer, post-fixed in 1% aqueous osmium tetroxide for 1h at 4°C, washed with distilled water (10min × 3), dehydrated in a series of ascending concentrations of ethanol, critical point dried and mounted on aluminium stubs. The specimens were then sputter-coated (Leica EM SCD 500) with gold-palladium (15nm thickness) and examined in a scanning electron microscope (Hitachi S-4800, HISCO Europe, Krefeld, Germany) at 5kV.

Pull-off force measurement of clamp One of the main challenges of conducting such experimental studies is the difficulty in handling relatively small monogeneans. In the case of D. paradoxum, the fusion ‘bridge’ between the two fused monogeneans provides a perfect site to attach the force sensor for pull-off force measurements. The gill sections with attached D. paradoxum were processed prior to the experimental studies, because the monogeneans are usually attached at the inner hemibranchs of the gills. The gill filaments found above the fusion ‘bridge’ between the paired individuals were trimmed carefully to expose the attached monogeneans (Fig.1A). The trimmed gill section was then used in the experimental design illustrated in Fig.2A. First, the trimmed gill section was fixed in position using a steel rod that terminated with a fixed ring (~25mm in diameter). A smooth-ended Nirosta stainless steel hook (Thüringische Nadelfertigung Gerhard Ziggel, Wüllersleben, Germany) with a diameter of 300μm was used to hook the fusion ‘bridge’ between the paired monogeneans. The hook was then attached vertically to a 25g load cell force transducer Fig.1. Two Diplozoon paradoxum fuse together at the middle of their bodies. (A)Light microscopic image showing a paired D. paradoxum (World Precision Instruments, Sarasota, FL, USA) mounted on a attached on a gill filament. (B)Scanning electron micrograph of a detached motorised micromanipulator capable of a constant movement at paired D. paradoxum. c, cup-shaped structure; gf, gill filament; arrowheads various velocities (MS314, Märzhäuser, Wetzlar, Germany). To indicate the fusion ‘bridge’ between the two bodies of the monogeneans. avoid any damage to monogeneans during the experiment, the Scale bars, 500μm. micromanipulator was moved in a vertical direction at a constant velocity of 100μm–1. The movement of the hook during the pulling process was observed in a binocular to ensure that the fusion ‘bridge’ is flat in shape and has a length of approximately 0.37mm (Fig.1). between the two monogeneans was being hooked without any In the present study, we aimed to: (1) measure the force required obstacles in the vertical direction. After each experiment, the gills to detach a paired adult D. paradoxum from the gills of the and clamps were observed under the stereomicroscope to ensure freshwater bream, Abramis brama (Linnaeus 1758), and (2) that the detachment had occurred only between the clamps and the determine the contribution of muscle action to the closing or gills but not by tearing away from the gills. Force–time curves opening of the clamps. (Fig.2B) were recorded using Acqknowledge 3.7.0 software (Biopac Systems, Goleta, CA, USA) and the pull-off forces of the paired MATERIALS AND METHODS monogeneans were extracted from the recorded data. The pull-off Collection and preparation of monogeneans force (F) is here defined as the maximum force required to detach Live freshwater bream (Abramis brama), which were obtained from a paired D. paradoxum vertically from the fish gills (i.e. the ability the Wrohe Fischerei and the Fischzucht Reese, were caught from of a monogenean to remain attached onto the gills, when lifted up two lakes, Lake Westensee and Lake Selenter See, respectively, vertically from its fish host). To estimate the body mass of the located in Schleswig-Holstein, Germany. The fish were euthanised detached monogenean pair, the worms were blot-dried carefully and in the laboratory and the gills were examined for the occurrence of rapidly on a filter paper, and weighed using an analytical balance paired adult Diplozoon paradoxum under a stereomicroscope (Wild (Mettler Toledo, AG 204 DeltaRange, Greifensee, Switzerland) with M3Z, Leica Microsystems, Wetzlar, Germany). The monogeneans a sensitivity of 0.1mg. Linear regression analysis of the pull-off were identified based on the morphology of their sclerites and force versus body mass of monogenean pairs was performed using reproductive organs (Bychowsky and Nagibina, 1959; Bovet, 1967). SigmaStat software (Systat Software, San Jose, CA, USA). Ten living adult D. paradoxum were studied under a stereomicroscope (Leica M205A) to observe the movement of the Measuring the clamp openings clamps. Five gill sections with attached D. paradoxum were fixed Previous studies have shown that in general contract their in 2.5% glutaraldehyde (Carl Roth, Karlsruhe, Germany) (in muscles when treated with potassium chloride (KCl) solutions 0.01moll–1 phosphate buffer containing 3% sucrose at pH7.4) for (Fetterer et al., 1980; Moneypenny et al., 2001; Cobbett and Day, 6h at 4°C for scanning electron microscopy. A small section of a 2003), but relax them when treated with magnesium chloride (MgCl2) gill with attached living adult D. paradoxum (N=20) was excised solutions (Tyler, 1976; Shaw, 1979; Rees and Kearn, 1984; carefully and further used in the pull-off force measurements of Schürmann and Peter, 1998; Salvenmoser et al., 2010; Vizoso et al., clamps. 2010). The following three different solutions were used to investigate

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Fig.3. Scanning electron micrographs of (A) the four pairs of clamps in the haptor of Diplozoon paradoxum and (B) the former site of clamp attachments (arrows) on the gill of Abramis brama. Roman numerals (I–IV) indicate the clamp numbers. Scale bars, 200μm.

monogeneans were observed using a stereomicroscope (Leica Fig.2. (A)Experimental setup for pull-off force measurement (lateral view). M205A). The posterior clamp-bearing region was excised carefully co, computer; ft, force transducer; gh, gill holder; gi, gill; ho, hook; is, immobile stage; mc, micromanipulator control; mm, motorised and orientated in such a way that the distal lateral side of the clamp micromanipulator; mo, monogeneans; pe, Petri dish containing filtered lake was facing vertically to the stereomicroscope. Images of the distal water; sc, sensor control. Not drawn to scale. (B)An example of a typical lateral side of the clamp were captured using the image-processing force–time curve. (C)Light microscopic image showing the openings software Leica Application Suite v3.8. The width of the clamp opening (double-headed arrows) of clamps II and III. was measured as shown in Fig.2C. It was defined as the distance between the two most distal inner points of the antero- and postero- the effects of different experimental condition on the opening of D. lateral sides of the clamp sclerites. The numbering of the clamps was paradoxum clamps. Different pairs of D. paradoxum were kept according to earlier studies (Bychowsky and Nagibina, 1959; Gläser −1 −1 separately in: (1) 100mmoll KCl (Carl Roth), (2) 20mmoll MgCl2 and Gläser, 1964), in which the most posterior pair of clamps is (Carl Roth) and (3) filtered lake water (control) at 4°C in the 24-well designated as I, followed by II, II and IV for the most anterior pair plates. In each experimental condition, 10 pairs of living detached D. of clamps (Fig.3A). An average of three width measurements was paradoxum were used. The width of the clamp openings was taken for each clamp opening. To compare the effect of two different measured after the monogeneans were immobilised or did not move physiological solutions (KCl and MgCl2) on the clamp openings, the when disturbed using a fine needle. The monogeneans were data were tested using the Kruskal–Wallis one-way ANOVA on ranks immobilised within 45min of incubation in the 100mmoll−1 KCl followed by all pair-wise multiple comparison procedures (Tukey test) −1 solution, within 24h in the 20mmoll MgCl2 solution, and after (SigmaStat), to evaluate the differences in the widths of clamp opening 4–6days in the filtered lake water. Experiments using 20mmoll−1 between monogeneans treated in different experimental conditions. MgCl2 solution and filtered lake water were conducted from March Images of the fresh secondary gill lamellae of A. brama were 2011 to June 2012, and those using 100mmoll−1 KCl solution from also captured using the Leica M205A stereomicroscope. The widths September to December 2012. The clamp openings of the immobilised of the secondary gill lamellae (N=50) were estimated with Leica

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shortening their bodies. The opening and closing actions of all the 12 r=0.692, r2=0.478 eight clamps in the haptors are independent of each other.

10 Scanning electron microscopy In the posterior region of D. paradoxum, four pairs of widely opened 8 clamps were observed (Fig.3A). The first clamp (clamp I) of each row of clamps is usually smaller than the other three clamps 6 (Fig.3A). Often, monogeneans were dislodged spontaneously from D. paradoxum pair (mN) the fish gills during the dehydration process. Microscopic study of 4 the gill section with a former attachment site, left by a D. paradoxum, revealed that each clamp is able to grasp one or two secondary gill 2 lamellae (Fig.3B). A cup-shaped structure was observed in the region just anterior to the four pairs of clamps (Fig.1B). 0 Pull-off force of a 0 1 234 Attachment force of a D. paradoxum pair Body mass of a D. paradoxum pair (mg) Observations under the stereomicroscope showed that there was no rupture of gill tissues left in the clamps after each pull-off Fig.4. A scatter plot with a linear regression of the pull-off force versus experiment. Results from a total of 20 pull-off force measurements body mass of a paired Diplozoon paradoxum showing a positive correlation showed that the pull-off force for a D. paradoxum pair is 6.1±2.7mN between these two variables (r=0.692, r2=0.478). (mean ± s.d.), ranging from 1.4 to 10.8mN. The paired D. paradoxum has a mean body mass of 2.5±0.8mg ranging from 1.0 Application Suite v3.8. The diameter of the secondary lamellae of to 3.6mg. A positive correlation (correlation coefficient, r=0.692; A. brama was 56.05±7.99μm (mean ± s.d.). coefficient of determination, r2=0.478) was observed between the pull-off forces and the body mass of paired D. paradoxum (Fig.4). RESULTS Observations on the movement of live clamps The width of clamp opening after various treatments Examination of live detached adult D. paradoxum showed that their Diplozoon paradoxum can shorten, twist and elongate its body in clamps are usually closed and directed ventrally. Occasionally, the filtered lake water (Fig.5A–C). When D. paradoxum were monogeneans can open some of their clamps while elongating or immobilised in the KCl solution, the monogeneans contracted and

Fig.5. Light microscopic images of Diplozoon paradoxum. (A–C) Video image sequences showing D. paradoxum shortens (A), twists (B) and elongates (C) its body in filtered lake water. (D)The monogenean elongates its body when treated with MgCl2 solution. (E)The monogenean shortens its body when treated with KCl solution. (F)Magnified image of the selected area in E showing that the clamps are opened. (G)Magnified image of the selected area in D showing that the clamps are closed. Scale bars, (A–E) 1mm, (F,G) 100μm.

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100 Fig.6. Box-and-whisker diagram showing the widths of a Monogeneans treated with KCl a clamp opening of Diplozoon paradoxum treated in (1) Monogeneans treated with MgCl2 −1 −1 a a Natural dead monogeneans 100mmoll KCl, (2) 20mmoll MgCl2 and (3) filtered lake water. The ends of boxes are defined as the 25th and 75th 80 percentiles, with a median line and error bars with the 10th and 90th percentiles. The positions of clamps are defined

µ m) as I, II, III and IV. Statistical differences (P<0.05, Tukey test) are found among three experimental conditions. There were no significant differences (P>0.05, Tukey test) among the 60 b b b different clamps treated by the same solution. Boxes with b different letters indicate significant difference. c 40 c c c Width opening of clamps ( 20

0 III III IV I II III IV I II III IV Positions of clamps shortened their bodies (Fig.5E). However, the monogeneans relaxed compared with those available for some other biological structures, and elongated their bodies when they were immobilized in MgCl2 which function like a clamp (viz. claws of insects and crustaceans). solution (Fig.5D). The monogeneans treated in 100mmoll−1 KCl Previous studies reported that on rough surfaces the claws of the beetle had the widest clamp openings (58.26±13.44μm), followed by those Stenus cicindeloides have a F/W ratio of 73 (Betz, 2002), those of −1 treated in 20mmoll MgCl2 solution (37.91±7.58μm) and those the beetle Pachnoda marginata have a F/W ratio of 38 (Dai et al., treated in filtered lake water (20.16±8.63μm). Results from the 2002), those of the mite Archegozetes longisetosus have a F/W ratio Kruskal–Wallis one-way ANOVA by ranks test followed by all pair- of 1182 (Heethoff and Koerner, 2007) and those of the aphid wise multiple comparison procedures (Tukey test) showed that there Megoura viciae have a F/W ratio of 17 (Lees and Hardie, 1988). The were significant differences (P<0.05) between the widths of clamp claws of six species of Cancer crabs have F/W ratios that range from openings (clamp positioned I, II, III, IV) in monogeneans treated 60 to 388 (Taylor, 2000). The calculated average F/W ratio for D. with three different solutions, but no significant differences between paradoxum is ~246 and generally higher than the abovementioned the widths of the four clamp openings treated by the same solution ratios in other invertebrates, except for A. longisetosus, Cancer (Fig.6). branneri and C. oregonensis. However, the comparison is not conclusive, as there are differences in the morphology of attachment DISCUSSION structures and in the attachment mechanisms of the clamps and the Attachment performance of clamps claws. We also assume that the most important comparison in the Adult individuals of D. paradoxum use four pairs of clamps to case of D. paradoxum has to be made with the drag forces acting on establish their posterior attachment onto the gill filaments of A. its body due to the water flow in the gills of the fish. Unfortunately, brama. To detach a paired D. paradoxum vertically from the fish such data are not available in the literature. gills, an average force of 6.1±2.7mN was required. During the experiment, we assumed that there was (1) no change in the material Muscular action during clamping: active or passive? properties of the gill filaments after the gills are excised, (2) no Previous studies have suggested that the clamps of D. paradoxum injury or other physiological effect on the fusion ‘bridge’ of the are operated by an ‘extrinsic muscle–tendon–fair-lead–hinged-jaw’ paired D. paradoxum caused by the vertical pulling of the system (see Fig.7) in which the closing of the clamp hinge jaws is experimental hook, (3) no capillary force contributed by the water caused by the contraction of extrinsic muscles associated with the meniscus formed on the edges of the fine experimental hook and clamps (Owen, 1963a; Bovet, 1967). These extrinsic muscles (4) a negligible effect, if any, due to the attachment force contributed originate anteriorly from the dorsal and the ventral longitudinal by the relatively small posterior anchors. However, if there were muscles of the body wall. If the abovementioned hypothesis is minimal contributions from both the capillary force and the correct, the extrinsic muscles have to be contracted constantly in attachment force of the posterior hooks, we suggest that the values order to enable the clamps to grasp securely the fish gill lamellae. obtained in the present study were slightly overestimated. Although some muscles present in the monogeneans are presumed The clamp of D. paradoxum consists of a framework of sclerites to be able to perform continuous contraction (Halton et al., 1998; that forms a fixed anterior jaw and a hinged posterior jaw (Owen, Kearn, 1966), this would hinder or stop the probing or searching 1963a). Careful examination of the literature indicated that there is movement of the monogeneans. Similar problems of such continuous no information on the pull-off forces for either D. paradoxum or other contraction of extrinsic muscles, associated with the haptoral monogeneans or other parasitic animals possessing clamp structures attachment apparatus in monogeneans, have also been noted in other similar to those of D. paradoxum. To obtain an impression of the studies (Llewellyn, 1960; Kearn, 1966; Halton et al., 1998). In attachment performance of the clamps in relationship to the ’s addition, if one end of the extrinsic muscles were not ‘fixed’ or body weight, a ratio of the pull-off force to the weight (F/W) of a attached to a stiff support, the contraction of the extrinsic muscles, paired D. paradoxum was calculated. The obtained results were associated with the clamps, would only cause the retraction of the

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A passive action of elastic material of the clamp (Ramalingam, 1973; Proximal Wong et al., 2013) in concert with relaxed intrinsic muscles, the Clamp opens monogeneans are able to maintain their attachments securely with minimum expenditure of energy. In addition, this enables the monogeneans to use muscles for functions other than attachment. The a em diameter of the secondary gill lamellae of A. brama, which are b ~1.5–2.0 times larger than the average width of the clamp openings of D. paradoxum that either were treated with MgCl or died Anterior c 2 naturally, provides an appropriate geometry for the clamps to grasp d during attachment. It remains unclear, however, whether the clamping forces created by the passive action of deformed clamp material and relaxed intrinsic muscles are sufficient to maintain their haptoral im attachment. An earlier study suggested that a suction under pressure may be produced by the intrinsic muscles of the clamp wall (Bovet, t 1967), but this statement needs further investigation. MgCl solution has been widely used to relax muscles in many Posterior 2 invertebrates including bivalves and flatworms (Tyler, 1976; Shaw, Distal 1979; Culloty and Mulcahy, 1992; Butt et al., 2008; Salvenmoser et al., 2010). In bivalves, MgCl treatment leads to the relaxation B 2 Proximal of the abductor muscle holding the shells closed. The effect of MgCl2 Clamp closes solution on the muscular systems of monogeneans has not been investigated. The statistically significant difference of the width of the clamp opening between D. paradoxum treated in MgCl2 solution and those that died in lake water could be due to (1) the specific concentration of MgCl2 solution that was insufficient to cause a Anterior total relaxation of the muscles or (2) the specific nature of the response of monogenean muscles to the MgCl2 solution.

Conclusions and outlook This is the first experimental study demonstrating the pull-off force of a clamp-bearing fish parasite, D. paradoxum. An adult paired D. paradoxum is able to maintain its attachment onto the fish gills under up to 6.1±2.7mN of external force before it can be dislodged. Clamps Posterior of D. paradoxum are able to grasp onto the fish gill lamellae without Distal continuous contraction of extrinsic muscles, and this contradicts the hypothesis proposed earlier for D. paradoxum (Owen, 1963a; Bovet, 1967). The closing of the clamps is most likely due to the Fig.7. Schematic diagrams illustrating the clamp sclerites and muscles passive action of the resilin-like material of the clamp sclerites during the opening (A) and closing (B) of the clamp of Diplozoon (Wong et al., 2013). Subsequently, the expenditure of energy is paradoxum. a, median J-shaped sclerite; b, anterior marginal sclerite; c and minimised during the life-long attachments of the monogeneans onto d, posterior marginal sclerites supporting the periphery of the movable their fish hosts. In future, detailed studies of the functional posterior jaw; em, extrinsic muscles; im, intrinsic muscles; t, tendon. Black arrowheads indicate that the median J-shaped sclerite is in a fixed position; morphology and electrophysiological experiments on the muscle thick black arrows indicate the directions of the movable posterior jaws; system associated with the clamps should be performed in order to grey arrows indicate the actions of the extrinsic muscles. Diagrams are provide further confirmation of the passive functional mechanism modified from Owen (Owen, 1963a) (not drawn to scale). of D. paradoxum clamps.

ACKNOWLEDGEMENTS monogenean body instead of closing its clamps. Our experimental We thank the ‘Landesamt für Landwirtschaft, Umwelt und ländliche Räume’, study indicates that D. paradoxum close their clamps when the Schleswig-Holstein, Germany provided the permit for collecting fish. Special corresponding muscles are in a relaxed state and, vice versa, muscle thanks to Dr Alexander Kovalev for his technical advice. action opens the clamp (Fig.5F,G, Fig.6, Fig.7). This result resolves some abovementioned contradictions and additionally provides a AUTHOR CONTRIBUTIONS W.-L.W. designed the study, collected the specimens, performed the experimental plausible explanation that the haptoral attachment system of D. studies, analysed the data and wrote the manuscript. S.N.G participated in the paradoxum does not consume much energy in its long-term attached design of the study, in interpretation of the results and in critical manuscript condition. As the adult paired D. paradoxum are believed not likely revision. Both authors read and approved the final manuscript. to change their positions on the gills (Owen, 1963b), energy is only required for a short period of detachment. COMPETING INTERESTS The facts that D. paradoxum can (1) either open or close their No competing interests declared. clamps when their bodies are elongated or shortened and (2) open and close their clamps independently strongly suggest that the closing FUNDING This project was supported by the Alexander von Humboldt Foundation to W.L.W. and opening of each clamp is also probably effected by their intrinsic [3.2-MAY/1137309STP] and the Industrie and Handelskammer Schleswig- muscles. By assuming that the closing of the clamps is caused by the Holstein to S.N.G. [Transfer Award IHK SH 2011].

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REFERENCES Llewellyn, J. (1960). Amphibdellid (monogenean) parasites of electric rays Arafa, S. (2011). Ultrastructure of musculature of the marginal hooklets of (Torpedinidae). J. Mar. Biol. Assoc. U. K. 39, 561-589. Macrogyrodactylus congolensis, a monogenean skin parasite from the catfish Clarias Llewellyn, J. and Owen, I. L. (1960). The attachment of the monogenean Discocotyle gariepinus. Acta Parasitol. 56, 122-130. sagittata Leuckart to the gills of Salmo trutta L. Parasitology 50, 51-60. Betz, O. (2002). Performance and adaptive value of tarsal morphology in rove beetles Moneypenny, C. G., Kreshchenko, N., Moffett, C. L., Halton, D. W., Day, T. A. and of the genus Stenus (Coleoptera, Staphylinidae). J. Exp. Biol. 205, 1097-1113. Maule, A. G. (2001). Physiological effects of FMRFamide-related peptides and Bovet, J. (1967). Contribution à la morphologie et à la biologie de Diplozoon classical transmitters on dispersed muscle fibres of the turbellarian, Procerodes paradoxum von Nordmann 1832. Bull. Soc. Neuchatel. Sci. Nat. 90, 63-159. littoralis. Parasitology 122, 447-455. Butt, D., O’Connor, S. J., Kuchel, R., O’Connor, W. A. and Raftos, D. A. (2008). Owen, I. L. (1963a). The attachment of the monogenean Diplozoon paradoxum to the Effects of the muscle relaxant, magnesium chloride, on the Sydney rock oyster gills of Rutilus rutilus L. II. Structure and mechanism of the adhesive apparatus. (Saccostrea glomerata). Aquaculture 275, 342-346. Parasitology 53, 463-468. Bychowsky, B. E. (1957). Monogenetic Trematodes, Their Systematics and Owen, I. L. (1963b). The attachment of the monogenean Diplozoon paradoxum to the Phylogeny. Moscow/Leningrad: Academy of Sciences of the U.S.S.R. [in Russian]. gills of Rutilus rutilus L. I. Micro-habitat and adhesive attitude. Parasitology 53, 455- (English translation by W. J. Hargis and P. C. Oustinoff, 1961). Washington DC: 461. American Institute of Biological Sciences. Paling, J. E. (1966). The attachment of the monogenean Diplectanum aequans Bychowsky, B. E. and Nagibina, L. F. (1959). Systematics of the genus Diplozoon (Wagener) Diesing to the gills of Morone labrax L. Parasitology 56, 493-503. Nordmann (Monogenoidea). Zool. Zhurnal. 38, 362-377. Ramalingam, K. (1973). Chemical nature of monogenean sclerites. I. Stabilization of Cerfontaine, P. (1896). Contribution à l’étude des Octocotylides. I–III. Arch. Biol. Paris clamp-protein by formation of dityrosine. Parasitology 66, 1-7. 14, 497-560. Rand, T. G., Wiles, M. and Odense, P. (1986). Attachment of Dermophthirius Cobbett, P. and Day, T. A. (2003). Functional voltage-gated Ca2+ channels in muscle carcharhini (Monogenea: Microbothriidae) to the Galapagos shark Carcharhinus fibers of the platyhelminth Dugesia tigrina. Comp. Biochem. Physiol. 134A, 593-605. galapagensis. Trans. Am. Microsc. Soc. 105, 158-169. Culloty, S. C. and Mulcahy, M. F. (1992). An evaluation of anaesthetics for Ostrea Rees, J. A. and Kearn, G. C. (1984). The anterior adhesive apparatus and an edulis (L.). Aquaculture 107, 249-252. associated compound sense organ in the skin-parasitic monogenean Acanthocotyle Dai, Z., Gorb, S. N. and Schwarz, U. (2002). Roughness-dependent friction force of lobianchi. Parasitol. Res. 70, 609-625. the tarsal claw system in the beetle Pachnoda marginata (Coleoptera, Salvenmoser, W., Egger, B., Achatz, J. G., Ladurner, P. and Hess, M. W. (2010). Scarabaeidae). J. Exp. Biol. 205, 2479-2488. Electron microscopy of flatworms: Standard and cryo-preparation methods. In Fetterer, R. H., Pax, R. A. and Bennett, J. L. (1980). Praziquantel, potassium and Methods in Cell Biology (ed. T. Müller-Reichert), pp. 307-330, London: Academic 2,4-dinitrophenol: analysis of their action on the musculature of Schistosoma Press. mansoni. Eur. J. Pharmacol. 64, 31-38. Sánchez-García, N., Padrós, F., Raga, J. A. and Montero, F. E. (2011). Comparative Gläser, H. J. and Gläser, B. (1964). Zur Taxonomie der Gattung Diplozoon study of the three attachment mechanisms of diplectanid monogeneans. Aquaculture Nordmann, 1832. Parasitol. Res. 25, 164-192. 318, 290-299. Halton, D. W., Maule, A. G., Mair, G. R. and Shaw, C. (1998). Monogenean Schürmann, W. and Peter, R. (1998). Inhibition of regeneration in the planarian neuromusculature: some structural and functional correlates. Int. J. Parasitol. 28, Dugesia polychroa (Schmidt) by treatment with magnesium chloride: a morphological 1609-1623. study of wound closure. Hydrobiologia 383, 111-116. Heethoff, M. and Koerner, L. (2007). Small but powerful: the oribatid mite Shaw, M. K. (1979). The ultrastructure of the clamp wall of the monogenean gill Archegozetes longisetosus Aoki (Acari, Oribatida) produces disproportionately high parasite Gastrocotyle trachuri. Parasitol. Res. 58, 243-258. forces. J. Exp. Biol. 210, 3036-3042. Shinn, A. P., Bron, J. E., Sommerville, C. and Gibson, D. I. (2003). Comments on Kearn, G. C. (1966). The adhesive mechanism of the monogenean parasite Tetraonchus monenteron from the gills of the pike (Esox lucius). Parasitology 56, the mechanism of attachment in species of the monogenean genus Gyrodactylus. 505-510. Invertebr. Biol. 122, 1-11. Kearn, G. C. (1971). The attachment of the ancyrocephalid monogenean Haliotrema Taylor, G. M. (2000). Maximum force production: why are crabs so strong? Proc. Biol. balisticus to the gills of the trigger fish, Balistes capriscus (=carolinensis). Sci. 267, 1475-1480. Parasitology 63, 157-162. Tyler, S. (1976). Comparative ultrastructure of adhesive system in the Turbellaria. Kearn, G. C. (1998). Parasitism and the Platyhelminths. New York, NY: Chapman and Zoomorphologie 84, 1-76. Hall. Vizoso, D. B., Rieger, G. and Schärer, L. (2010). Goings-on inside a worm: functional Kearn, G. C. (1999). The survival of monogenean (platyhelminth) parasites on fish hypotheses derived from sexual conflict thinking. Biol. J. Linn. Soc. Lond. 99, 370- skin. Parasitology 119 Suppl., S57-S88. 383. Lees, A. D. and Hardie, J. (1988). The organs of adhesion in the aphid Megoura Wong, W. L., Brennan, G. P., Halton, D. W. and Lim, L. H. S. (2006). Fine structure viciae. J. Exp. Biol. 136, 209-228. of the anterior adhesive apparatus (head organs) of Bravohollisia gussevi Lim, 1995 Llewellyn, J. (1956). The adhesive mechanisms of monogenetic trematodes: the (Monogenea: Ancyrocephalidae). Parasitology 132, 427-438. attachment of Plectanocotyle gurnardi (v. Ben. and Hesse) to the gills of Trigla. J. Wong, W. L., Brennan, G. P., Halton, D. W., Maule, A. G. and Lim, L. H. S. (2008). Mar. Biol. Assoc. UK 35, 507-514. Secretory products of the haptoral reservoirs and peduncular glands in two species Llewellyn, J. (1957). The mechanism of the attachment of Kuhnia scombri (Kuhn, of Bravohollisia (Monogenea: Ancyrocephalidae). Invertebr. Biol. 127, 139-152. 1829) (Trematoda: Monogenea) to the gills of its host Scomber scombrus L. Wong, W. L., Michels, J. and Gorb, S. N. (2013). Resilin-like protein in the clamp including a note on the of the parasite. Parasitology 47, 30-39. sclerites of the gill monogenean Diplozoon paradoxum Nordmann, 1832. Llewellyn, J. (1958). The adhesive mechanisms of monogenetic trematodes: the Parasitology 140, 95-98. attachment of species of the Diclidophoridae to the gills of gadoid fishes. J. Mar. Yamaguti, S. (1963). Systema Helminthum. IV. Monogenea and Aspidocotylea. New Biol. Assoc. UK 37, 67-79. York, NY: John Wiley and Sons.

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