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

Attachment is an essential feature of all parasitic organisms for their survival, and monogeneans, which are mainly ectoparasites, are no exception. Monogenea is one of the largest classes within the phylum Platyhelminthes and they usually possess anterior and posterior attachment apparatus that are used for settlement, feeding, locomotion and transfer from host to host (Bychowsky, 1957; Yamaguti, 1963; Kearn, 1998). The anterior attachment apparatus of monogeneans (viz. head organs, bothria, pits or suckers) is usually used for temporary attachment during their leech-like movement (Bychowsky, 1957; Kearn, 1999; Wong et al., 2006). The haptor or posterior attachment apparatus of monogeneans is more diverse in its structure, usually used for a more secure and permanent attachment, and considered as the ‘hallmark’ for monogeneans. The haptors are generally equipped with various sclerotized armaments, which include marginal hooks, anchors, suckers, clamps and squamoid discs, and also adhesive secretions, or a combination of these (Bychowsky, 1957; Yamaguti, 1963; Kearn, 1999; Wong et al., 2008). These unique haptoral attachment apparatus have encouraged many scientists to investigate how they operate efficiently. Several studies have been undertaken to elucidate the attachment mechanism of the anchors (Llewellyn, 1960; Kearn, 1971), marginal hooks (Shinn et al., 2003; Arafa, 2011), clamps (Cerfontaine, 1896; Llewellyn, 1956; Llewellyn, 1957; Llewellyn, 1958; Llewellyn and Owen, 1960; Owen, 1963a; Bovet, 1967), squamoid discs (Paling, 1966; Sánchez-García et al., 2011) and haptoral secretions (Rand et al., 1986; Wong et al., 2008). However, conclusions about the functional principles of the attachment apparatus are mainly based on morphological investigations of the attachment apparatus and associated muscular systems. Although investigation of the haptoral attachment mechanism of monogeneans was conducted, to the best of our knowledge, as early as 1896 by Cerfontaine (Cerfontaine, 1896), who examined the clamping mechanism of Diclidophora denticulata on the gill of the fish Gadus virens, no attempt has been made to measure the forces generated by the haptoral attachment systems. Additionally, it remains unknown how muscles control the operation of the attachment apparatus. However, such information is important not only for a better understanding on the biology of monogeneans, but also for the development of novel methods to control parasites in medicine and veterinary contexts.

Diplozoon paradoxum Nordmann 1832 (Platyhelminthes: Monogenea: Diplozoidea) is a gill parasite of freshwater fishes. This monogenean uses four pairs of clamps (four each on the left and right side of the haptor) and a pair of relatively small hooks for posterior attachment at the host secondary gill lamellae (Bychowsky and Nagibina, 1959; Owen, 1963a; Bovet, 1967). The clamps are thought to provide the major role in the attachment of D. paradoxum, while the relatively small hooks most likely function only during the initial stage of attachment (Owen, 1963a). Each clamp of D. paradoxum possesses two jaws, hinged to each other, and each jaw is supported peripherally by marginal sclerites. The clamp is thought to close by an extrinsic muscle/tendon system associated with a median J-shaped sclerite (Owen, 1963a). During their sexual maturation, two parasites fuse together permanently at the middle of their bodies forming a joint H-shaped body (Fig. 1) (Bychowsky and Nagibina, 1959; Bovet, 1967). The body connection or fusion ‘bridge’ between two D. paradoxumis flat in shape and has a length of approximately 0.37 mm (Fig. 1). In the present study, we aimed to: (1) measure the force required to detach a paired adult D. paradoxum from the gills of the freshwater bream, Abramis brama (Linnaeus 1758), and (2) determine the contribution of muscle action to the closing or opening of the clamps.

Live freshwater bream (Abramis brama), which were obtained from the Wrohe Fischerei and the Fischzucht Reese, were caught from two lakes, Lake Westensee and Lake Selenter See, respectively, located in Schleswig-Holstein, Germany. The fish were euthanised in the laboratory and the gills were examined for the occurrence of paired adult Diplozoon paradoxum under a stereomicroscope (Wild M3Z, Leica Microsystems, Wetzlar, Germany). The monogeneans were identified based on the morphology of their sclerites and reproductive organs (Bychowsky and Nagibina, 1959; Bovet, 1967). Ten living adult D. paradoxum were studied under a stereomicroscope (Leica M205A) to observe the movement of the clamps. Five gill sections with attached D. paradoxum were fixed in 2.5% glutaraldehyde (Carl Roth, Karlsruhe, Germany) (in 0.01 mol l⁻¹ phosphate buffer containing 3% sucrose at pH 7.4) for 6 h at 4°C for scanning electron microscopy. A small section of a gill with attached living adult D. paradoxum (N=20) was excised carefully and further used in the pull-off force measurements of clamps.

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.

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 (World Precision Instruments, Sarasota, FL, USA) mounted on a motorised micromanipulator capable of a constant movement at various velocities (MS314, Märzhäuser, Wetzlar, Germany). To avoid any damage to monogeneans during the experiment, the 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’ between the two monogeneans was being hooked without any obstacles in the vertical direction. After each experiment, the gills and clamps were observed under the stereomicroscope to ensure that the detachment had occurred only between the clamps and the gills but not by tearing away from the gills. Force–time curves (Fig. 2B) were recorded using Acqknowledge 3.7.0 software (Biopac Systems, Goleta, CA, USA) and the pull-off forces of the paired monogeneans were extracted from the recorded data. The pull-off force (F) is here defined as the maximum force required to detach a paired D. paradoxum vertically from the fish gills (i.e. the ability of a monogenean to remain attached onto the gills, when lifted up vertically from its fish host). To estimate the body mass of the detached monogenean pair, the worms were blot-dried carefully and rapidly on a filter paper, and weighed using an analytical balance (Mettler Toledo, AG 204 DeltaRange, Greifensee, Switzerland) with a sensitivity of 0.1 mg. Linear regression analysis of the pull-off force versus body mass of monogenean pairs was performed using SigmaStat software (Systat Software, San Jose, CA, USA).

Previous studies have shown that flatworms in general contract their muscles when treated with potassium chloride (KCl) solutions (Fetterer et al., 1980; Moneypenny et al., 2001; Cobbett and Day, 2003), but relax them when treated with magnesium chloride (MgCl₂) solutions (Tyler, 1976; Shaw, 1979; Rees and Kearn, 1984; Schürmann and Peter, 1998; Salvenmoser et al., 2010; Vizoso et al., 2010). The following three different solutions were used to investigate the effects of different experimental condition on the opening of D. paradoxum clamps. Different pairs of D. paradoxum were kept separately in: (1) 100 mmol l⁻¹ KCl (Carl Roth), (2) 20 mmol l⁻¹ MgCl₂ (Carl Roth) and (3) filtered lake water (control) at 4°C in the 24-well plates. In each experimental condition, 10 pairs of living detached D. paradoxum were used. The width of the clamp openings was measured after the monogeneans were immobilised or did not move when disturbed using a fine needle. The monogeneans were immobilised within 45 min of incubation in the 100 mmol l⁻¹ KCl solution, within 24 h in the 20 mmol l⁻¹ MgCl₂ solution, and after 4–6 days in the filtered lake water. Experiments using 20 mmol l⁻¹ MgCl₂ solution and filtered lake water were conducted from March 2011 to June 2012, and those using 100 mmol l⁻¹ KCl solution from September to December 2012. The clamp openings of the immobilised monogeneans were observed using a stereomicroscope (Leica M205A). The posterior clamp-bearing region was excised carefully and orientated in such a way that the distal lateral side of the clamp was facing vertically to the stereomicroscope. Images of the distal lateral side of the clamp were captured using the image-processing software Leica Application Suite v3.8. The width of the clamp opening was measured as shown in Fig. 2C. It was defined as the distance between the two most distal inner points of the antero- and posterolateral sides of the clamp sclerites. The numbering of the clamps was according to earlier studies (Bychowsky and Nagibina, 1959; Gläser and Gläser, 1964), in which the most posterior pair of clamps is designated as I, followed by II, II and IV for the most anterior pair of clamps (Fig. 3A). An average of three width measurements was taken for each clamp opening. To compare the effect of two different physiological solutions (KCl and MgCl₂) on the clamp openings, the data were tested using the Kruskal–Wallis one-way ANOVA on ranks followed by all pair-wise multiple comparison procedures (Tukey test) (SigmaStat), to evaluate the differences in the widths of clamp opening between monogeneans treated in different experimental conditions.

Images of the fresh secondary gill lamellae of A. brama were also captured using the Leica M205A stereomicroscope. The widths of the secondary gill lamellae (N=50) were estimated with Leica Application Suite v3.8. The diameter of the secondary lamellae of A. brama was 56.05±7.99 μm (mean ± s.d.).

Examination of live detached adult D. paradoxum showed that their clamps are usually closed and directed ventrally. Occasionally, the monogeneans can open some of their clamps while elongating or shortening their bodies. The opening and closing actions of all the eight clamps in the haptors are independent of each other.

In the posterior region of D. paradoxum, four pairs of widely opened clamps were observed (Fig. 3A). The first clamp (clamp I) of each row of clamps is usually smaller than the other three clamps (Fig. 3A). Often, monogeneans were dislodged spontaneously from the fish gills during the dehydration process. Microscopic study of 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 lamellae (Fig. 3B). A cup-shaped structure was observed in the region just anterior to the four pairs of clamps (Fig. 1B).

Observations under the stereomicroscope showed that there was no rupture of gill tissues left in the clamps after each pull-off experiment. Results from a total of 20 pull-off force measurements showed that the pull-off force for a D. paradoxum pair is 6.1±2.7 mN (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 to 3.6 mg. A positive correlation (correlation coefficient, r=0.692; coefficient of determination, r²=0.478) was observed between the pull-off forces and the body mass of paired D. paradoxum (Fig. 4).

Diplozoon paradoxum can shorten, twist and elongate its body in filtered lake water (Fig. 5A–C). When D. paradoxum were immobilised in the KCl solution, the monogeneans contracted and shortened their bodies (Fig. 5E). However, the monogeneans relaxed and elongated their bodies when they were immobilized in MgCl₂ solution (Fig. 5D). The monogeneans treated in 100 mmol l⁻¹ KCl had the widest clamp openings (58.26±13.44 μm), followed by those treated in 20 mmol l⁻¹ MgCl₂ solution (37.91±7.58 μm) and those treated in filtered lake water (20.16±8.63 μm). Results from the Kruskal–Wallis one-way ANOVA by ranks test followed by all pairwise multiple comparison procedures (Tukey test) showed that there were significant differences (P<0.05) between the widths of clamp openings (clamp positioned I, II, III, IV) in monogeneans treated with three different solutions, but no significant differences between the widths of the four clamp openings treated by the same solution (Fig. 6).

Adult individuals of D. paradoxum use four pairs of clamps to establish their posterior attachment onto the gill filaments of A. brama. To detach a paired D. paradoxum vertically from the fish 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 properties of the gill filaments after the gills are excised, (2) no injury or other physiological effect on the fusion ‘bridge’ of the paired D. paradoxum caused by the vertical pulling of the experimental hook, (3) no capillary force contributed by the water meniscus formed on the edges of the fine experimental hook and (4) a negligible effect, if any, due to the attachment force contributed by the relatively small posterior anchors. However, if there were minimal contributions from both the capillary force and the attachment force of the posterior hooks, we suggest that the values obtained in the present study were slightly overestimated.

The clamp of D. paradoxum consists of a framework of sclerites that forms a fixed anterior jaw and a hinged posterior jaw (Owen, 1963a). Careful examination of the literature indicated that there is no information on the pull-off forces for either D. paradoxum or other monogeneans or other parasitic animals possessing clamp structures similar to those of D. paradoxum. To obtain an impression of the attachment performance of the clamps in relationship to the animal's body weight, a ratio of the pull-off force to the weight (F/W) of a paired D. paradoxum was calculated. The obtained results were compared with those available for some other biological structures, which function like a clamp (viz. claws of insects and crustaceans). Previous studies reported that on rough surfaces the claws of the beetle Stenus cicindeloides have a F/W ratio of 73 (Betz, 2002), those of the beetle Pachnoda marginata have a F/W ratio of 38 (Dai et al., 2002), those of the mite Archegozetes longisetosus have a F/W ratio of 1182 (Heethoff and Koerner, 2007) and those of the aphid Megoura viciae have a F/W ratio of 17 (Lees and Hardie, 1988). The claws of six species of Cancer crabs have F/W ratios that range from 60 to 388 (Taylor, 2000). The calculated average F/W ratio for D. paradoxum is ~246 and generally higher than the abovementioned ratios in other invertebrates, except for A. longisetosus, Cancer branneri and C. oregonensis. However, the comparison is not conclusive, as there are differences in the morphology of attachment structures and in the attachment mechanisms of the clamps and the claws. We also assume that the most important comparison in the case of D. paradoxum has to be made with the drag forces acting on its body due to the water flow in the gills of the fish. Unfortunately, such data are not available in the literature.

Previous studies have suggested that the clamps of D. paradoxum are operated by an ‘extrinsic muscle–tendon–fair-lead–hinged-jaw’ system (see Fig. 7) in which the closing of the clamp hinge jaws is caused by the contraction of extrinsic muscles associated with the clamps (Owen, 1963a; Bovet, 1967). These extrinsic muscles originate anteriorly from the dorsal and the ventral longitudinal muscles of the body wall. If the abovementioned hypothesis is correct, the extrinsic muscles have to be contracted constantly in order to enable the clamps to grasp securely the fish gill lamellae. Although some muscles present in the monogeneans are presumed to be able to perform continuous contraction (Halton et al., 1998; Kearn, 1966), this would hinder or stop the probing or searching movement of the monogeneans. Similar problems of such continuous contraction of extrinsic muscles, associated with the haptoral attachment apparatus in monogeneans, have also been noted in other studies (Llewellyn, 1960; Kearn, 1966; Halton et al., 1998). In addition, if one end of the extrinsic muscles were not ‘fixed’ or attached to a stiff support, the contraction of the extrinsic muscles, associated with the clamps, would only cause the retraction of the monogenean body instead of closing its clamps. Our experimental study indicates that D. paradoxum close their clamps when the corresponding muscles are in a relaxed state and, vice versa, muscle action opens the clamp (Fig. 5F,G, Fig. 6, Fig. 7). This result resolves some abovementioned contradictions and additionally provides a plausible explanation that the haptoral attachment system of D. paradoxum does not consume much energy in its long-term attached condition. As the adult paired D. paradoxum are believed not likely to change their positions on the gills (Owen, 1963b), energy is only required for a short period of detachment.

The facts that D. paradoxum can (1) either open or close their clamps when their bodies are elongated or shortened and (2) open and close their clamps independently strongly suggest that the closing and opening of each clamp is also probably effected by their intrinsic muscles. By assuming that the closing of the clamps is caused by the passive action of elastic material of the clamp (Ramalingam, 1973; Wong et al., 2013) in concert with relaxed intrinsic muscles, the 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 diameter of the secondary gill lamellae of A. brama, which are ~1.5–2.0 times larger than the average width of the clamp openings of D. paradoxum that either were treated with MgCl₂ or died naturally, provides an appropriate geometry for the clamps to grasp 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 attachment. An earlier study suggested that a suction under pressure may be produced by the intrinsic muscles of the clamp wall (Bovet, 1967), but this statement needs further investigation.

MgCl₂ solution has been widely used to relax muscles in many invertebrates including bivalves and flatworms (Tyler, 1976; Shaw, 1979; Culloty and Mulcahy, 1992; Butt et al., 2008; Salvenmoser et al., 2010). In bivalves, MgCl₂ treatment leads to the relaxation of the abductor muscle holding the shells closed. The effect of MgCl₂ 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 total relaxation of the muscles or (2) the specific nature of the response of monogenean muscles to the MgCl₂ solution.

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 of D. paradoxum are able to grasp onto the fish gill lamellae without 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 passive action of the resilin-like material of the clamp sclerites (Wong et al., 2013). Subsequently, the expenditure of energy is minimised during the life-long attachments of the monogeneans onto their fish hosts. In future, detailed studies of the functional morphology and electrophysiological experiments on the muscle system associated with the clamps should be performed in order to provide further confirmation of the passive functional mechanism of D. paradoxum clamps.

We thank the ‘Landesamt für Landwirtschaft, Umwelt und ländliche Räume’, Schleswig-Holstein, Germany provided the permit for collecting fish. Special thanks to Dr Alexander Kovalev for his technical advice.