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

Abstract

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. An average force of 6.1 ± 2.7 mN (about 246 times of the animals' 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 mM potassium chloride solution (58.26 ± 13.44 µm), followed by those treated in 20 mM 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 caused by the elasticity of the clamp material and that muscle activity is required for clamp opening.

Full text

3008
INTRODUCTION
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. paradoxum
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 animals’ 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−1potassium
chloride solution (58.26±13.44μm), followed by those treated in 20mmol l−1magnesium 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
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
(Monogenea), 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 (wlwong@zoologie.uni-kiel.de)
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3009Attachment force of Diplozoon paradoxum
is 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.
MATERIALS AND METHODS
Collection and preparation of monogeneans
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.
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
(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).
Measuring the clamp openings
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
Fig.1. Two Diplozoon paradoxum fuse together at the middle of their
bodies. (A)Light microscopic image showing a paired D. paradoxum
attached on a gill filament. (B)Scanning electron micrograph of a detached
paired D. paradoxum. c, cup-shaped structure; gf, gill filament; arrowheads
indicate the fusion ‘bridge’ between the two bodies of the monogeneans.
Scale bars, 500μm.
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3010
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−1KCl (Carl Roth), (2) 20mmoll−1MgCl2
(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−1KCl
solution, within 24h in the 20mmoll−1MgCl2solution, and after
4–6days in the filtered lake water. Experiments using 20mmoll−1
MgCl2solution and filtered lake water were conducted from March
2011 to June 2012, and those using 100mmoll−1KCl solution from
September to December 2012. The clamp openings of the immobilised
The Journal of Experimental Biology 216 (16)
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 postero-
lateral 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
Fig.2. (A)Experimental setup for pull-off force measurement (lateral view).
co, computer; ft, force transducer; gh, gill holder; gi, gill; ho, hook; is,
immobile stage; mc, micromanipulator control; mm, motorised
micromanipulator; mo, monogeneans; pe, Petri dish containing filtered lake
water; sc, sensor control. Not drawn to scale. (B)An example of a typical
force–time curve. (C)Light microscopic image showing the openings
(double-headed arrows) of clamps II and III.
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.
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3011Attachment force of Diplozoon paradoxum
Application Suite v3.8. The diameter of the secondary lamellae of
A. brama was 56.05±7.99μm (mean ± s.d.).
RESULTS
Observations on the movement of live clamps
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.
Scanning electron microscopy
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).
Attachment force of a D. paradoxum pair
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).
The width of clamp opening after various treatments
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
Body mass of a D. paradoxum pair (mg)
0
Pull-off force of a D. paradoxum pair (mN)
0
2
4
6
8
10
12
1234
r=0.692, r2=0.478
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.
Fig.4. A scatter plot with a linear regression of the pull-off force versus
body mass of a paired Diplozoon paradoxum showing a positive correlation
between these two variables (r=0.692, r2=0.478).
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3012
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−1KCl
had the widest clamp openings (58.26±13.44μm), followed by those
treated in 20mmoll−1MgCl2solution (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 pair-
wise 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).
DISCUSSION
Attachment performance of clamps
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
The Journal of Experimental Biology 216 (16)
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/Wratio of 73 (Betz, 2002), those of
the beetle Pachnoda marginata have a F/Wratio of 38 (Dai et al.,
2002), those of the mite Archegozetes longisetosus have a F/Wratio
of 1182 (Heethoff and Koerner, 2007) and those of the aphid
Megoura viciae have a F/Wratio of 17 (Lees and Hardie, 1988). The
claws of six species of Cancer crabs have F/Wratios that range from
60 to 388 (Taylor, 2000). The calculated average F/Wratio 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.
Muscular action during clamping: active or passive?
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
Positions of clamps
III III IV I II III IV I II III IV
Width opening of clamps (µm)
0
20
40
60
80
100
Monogeneans treated with KCl
Monogeneans treated with MgCl2
Natural dead monogeneans
aa
a
a
b
bbb
c
ccc
Fig.6. Box-and-whisker diagram showing the widths of
clamp opening of Diplozoon paradoxum treated in (1)
100mmoll−1KCl, (2) 20mmoll−1MgCl2and (3) filtered lake
water. The ends of boxes are defined as the 25th and 75th
percentiles, with a median line and error bars with the 10th
and 90th percentiles. The positions of clamps are defined
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
different clamps treated by the same solution. Boxes with
different letters indicate significant difference.
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3013Attachment force of Diplozoon paradoxum
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 MgCl2or 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.
MgCl2solution 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, MgCl2treatment 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 MgCl2solution
and those that died in lake water could be due to (1) the specific
concentration of MgCl2solution that was insufficient to cause a
total relaxation of the muscles or (2) the specific nature of the
response of monogenean muscles to the MgCl2solution.
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
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.
ACKNOWLEDGEMENTS
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.
AUTHOR CONTRIBUTIONS
W.-L.W. designed the study, collected the specimens, performed the experimental
studies, analysed the data and wrote the manuscript. S.N.G participated in the
design of the study, in interpretation of the results and in critical manuscript
revision. Both authors read and approved the final manuscript.
COMPETING INTERESTS
No competing interests declared.
FUNDING
This project was supported by the Alexander von Humboldt Foundation to W.L.W.
[3.2-MAY/1137309STP] and the Industrie and Handelskammer Schleswig-
Holstein to S.N.G. [Transfer Award IHK SH 2011].
B
Posterior
Anterior
Distal
Proximal
t
a
b
c
d
em
im
A
Posterior
Anterior
Distal
Proximal
Clamp opens
Clamp closes
Fig.7. Schematic diagrams illustrating the clamp sclerites and muscles
during the opening (A) and closing (B) of the clamp of Diplozoon
paradoxum. a, median J-shaped sclerite; b, anterior marginal sclerite; c and
d, posterior marginal sclerites supporting the periphery of the movable
posterior jaw; em, extrinsic muscles; im, intrinsic muscles; t, tendon. Black
arrowheads indicate that the median J-shaped sclerite is in a fixed position;
thick black arrows indicate the directions of the movable posterior jaws;
grey arrows indicate the actions of the extrinsic muscles. Diagrams are
modified from Owen (Owen, 1963a) (not drawn to scale).
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3014 The Journal of Experimental Biology 216 (16)
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