Parasitol Res (1991) 177:415-420 004432559100060C Parasitology

Research �9 Springer-Verlag 1991

Locomotion and attachment to the host of Myxinidocotyle and Acanthocotyle (Monogenea, Acanthocotylidae) G. Malmberg 1 and B. Fernholm 2

1 Swedish Museum of Natural History and Department of Zoology, University of Stockholm, S-106 91 Stockholm, Sweden 2 Research Department, Swedish Museum of Natural History, S-104 05 Stockholm, Sweden

Accepted February 19, 1991

Abstract. The locomotion and a t tachment of members of the genera Myxinidocotyle and Acanthocotyle (Mono- genoa, Acanthocotylidae) were studied and marks left by the posterior a t tachment organ on the hagfish and the rajid host skin were examined by scanning electron microscopy (SEM). The muscle-fibre arrangement in three types of pseudohaptors is described. The pseudo- haptor was found to have a suction function. Anatomi- cal data and a t tachment marks indicate that the hooks of the haptor have a strong a t tachment capacity, used in locomotion and for anchoring the pseudohaptor . We conclude that in larval stages as well as in adults, the haptor is the most impor tan t posterior locomotory or- gan, whereas the fully developed pseudohaptor is used for stat ionary attachment. The deep marks caused by both unarmed (Myxinidocotyle) and sclerite-armed (Acanthocotyle) pseudohaptora l ridges appeared to be harmless imprints, whereas the 16 haptoral hooks caused small wounds that may provide potential openings for secondary infections of the host skin.

body in front of the haptor (Kearn 1967; Malmberg and Fernholm 1989).

There are few descriptions of marks left by skin- dwelling monogeneans. Lester (1972) and Cone and Odense (1984) have described marks caused by the op- is thaptor of certain Gyrodactylus species. In this paper, for the first time, marks caused by the unique posterior a t tachment organ of acanthocotylid species are de- scribed.

Materials and methods

Living specimens

Specimens of Acanthocotyle lobianchi obtained from a specimen of Raja elavata (Rajidae) captured at Plymouth (UK) were studied in vivo in July 1974. Live specimens of Myxinidocotyle japonica Malmberg and Fernholm, 1989 on Eptatretus burgeri (Myxinidae) from Koajiro Bay, Japan, were studied in May 1973 at the Misaki Marine Biological Station, Tokyo University. Live specimens (post- larvae, juveniles and adults) of M. californica Malmberg and Fern- holm, 1989 on E. stoutii from La Jolla, California, were studied in Stockholm between May 23 and July 22, 1980.

Monogenean post-larvae move in a leech-like manner by means of an anterior and a posterior a t tachment or- gan. The posterior organ, the haptor , is armed with 10- 18 (marginal) hooks. In most of the monogenean fami- lies, the haptor develops into an opisthaptor, which in many families has marginal hooks and one or two pairs of anchors. In adult acanthocotylids, however, the poste- rior a t tachment organ consists o f a pseudohaptor and a small haptor with 16 hooks (marginal hooks) at its posterior end. This haptor is homologous with the hap- tor /opis thaptor described in other monogeneans (Price 1938), whereas the pseudohaptor originates as a speciali- zation of the peduncle, i.e, at the posterior end of the

Offprints requests to: G. Malmberg

Fixation of specimens for scanning electron microscopy

Detached specimens, besides specimens attached to skin fragments of their hosts, were fixed in a cold solution of 2.5% glutaraldehyde in 0A M sodium cacodylate buffer or in 5% formaldehyde, post- fixed for 1 h in a cold 1% solution of osmium tetroxide, dehydrated in ethanol or acetone, transferred to freon or acetone, critical-point dried, and coated with gold or gold/platinum for scanning electron microscopy (SEM). An entire (290 mm long) specimen of E. stoutii, captured near La Jolla, California, in 1980 and infested with M. californica, was anaesthetized in M.S. 222 (Cresent Research Chem- icals Inc.), kept for 90 rain in the vapour from a 2% osmium tetrox- ide solution and rinsed in distilled water. Fragments of skin were cut from the hagfish, rinsed again in distilled water, dehydrated and prepared for SEM as described above. A Cambridge Stereos- can S 4 and a Zeiss Novascan 30 were used for the SEM investiga- tions.

416 G. Malmberg and B. Fernholm: Acanthocotylid attachment to the host

Fixation of specimens for light microscopy

For light microscopy (bright-field, phase-contrast, interference- contrast microscopy) of the above three acanthocotylid species and the species Lophocotyle novaezeelandiea Malmberg and Fernholm, 1989, we used the same material (whole mounts; transverse, frontal and sagittal sections 3 or 4 gm thick) previously presented by Malmberg and Fernholm (1989).

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Results

Locomotion of Myxinidocotyle californica and Acanthocotyle Iobianchi specimens

As in A. lobianchi (see Kearn 1967), early post-larvae o f M . californica had no pseudohaptor (Fig. 2). Locomo- tion was achieved by the anterior a t tachment organ and the haptor. Small worms with a developed pseudohaptor and adults were found all over the surface of the host. They were highly mobile; when attached to the host skin by their posterior end, like specimens of A. lobian- chi, they could turn their bodies through almost 360 ~ and often undulated their body vigorously. When at- tempts were made to remove a specimen from the host skin, the parasite often tried to escape or pressed its extremely flat body against the host. The body and the posterior a t tachment organ of such specimens could be moved sideways several centimeters. Parasites readily at- tached their posterior a t tachment organ to the bo t tom of a plastic or glass petri dish. Even worms fixed (glutar- aldehyde) in this position could be moved over the bot- tom of the dish. We observed that A. lobianchi specimens could extrude and retract (Fig. 5) the haptor in relation to the pseudohaptor .

Muscle fibres in the peduncle, pseudohaptor and haptor

The muscle-fibre organization (Fig. 1) is basically the same in M. californica, M. japonica, Lophocotyle novae- zeelandica and A. lobianchi, although in the latter species it is especially prominent. Through the peduncle, bun- dles of muscle fibres run into the dorsal centre of the pseudohaptor , with some of them continuing to its ven- tral side. Two prominent bundles pass posteriorly into the haptor (Fig. 3). Other muscle-fibre bundles diverge and run radially to the margin of the pseudohaptor , forming a thin, dorsal pseudohaptoral muscle-fibre layer. There are dorso-ventral muscle fibres throughout the pseudohaptor ; a round its margin, however, especial- ly posteriorly and laterally, there is a region of closely spaced dorso-ventral muscle fibres (Fig. 1 a). In M. cali- fornica as well as M. japonica, small bundles of dorso- ventral muscle fibres run ventro-posteriorly to the trans- versely arranged ridges. On the ventral side of the pseu- dohap tor quite close to the pseudohaptoral margin, there is a thin, concentric band of muscle fibres (Fig. 1 b). In the haptor, most of the muscle fibres f rom the pedun- cle run to the 2 centrally situated hooks (Fig. 3); other fibres radiate and attach to the handles of the 14 periph- eral hooks.

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Fig. 1 a. A diagrammatic paratransversal section through the pseu- dohaptor of Myxinidoeotyle californica. Dorso-medially (dots), lon- gitudinal muscle fibres run from the peduncle, Under the dorsal tegument, broken lines indicate a thin layer of radial muscle fibres. From a dorso-medial area, muscle-fibre bundles run ventrally, cross and continue to the ventral side. In the lateral margins (con- tinuous lines), a region of closely spaced dorso-ventral muscle fibres can be seen. In the ventral peripher (dots), a thin layer of concentric muscle fibres occur. All over the pseudohaptor (vertical, continuous lines), dorso-ventral muscle fibres are visible. Fig. 1 b. Diagrammat- ic presentation of radial and circular muscle fibres in the pseudo- haptor of Myxinidocotyle; ventral view. On the ventral side, there is a thin layer of concentric muscle fibres inside the pseudohaptoral margin. On the dorsal side, a thin layer of radial muscle fibres are visible. The crossing dorso-ventral muscle fibres (Fig. 1 a) are partly shown

Marks left by the posterior attachment organ of acanthocotylids

In A. lobianchi the pseudohaptoral ridges are covered by tegument (Malmberg and Fernholm 1989; also see Fig. 5). Studies of the site of a t tachment of A. lobianchi specimens (fixed in situ on the host skin and experimen- tally detached) revealed a convex mark caused by the pseudohaptoral disk, a circular impression left by its margin and a radial pattern of deep impressions made

Fig. 2. Interference-contrast micrograph of a live, early post-larva of Myxinidocotyle californiea Malmberg and Fernholm J989, lack- ing pseudohaptoral anlages; ventral view. Scale bar=0.05 ram. Fig. 3. Horizontal section through the posterior attachment organ of M. japonica. Two bundles of muscle fibres (opposing arrows) run from the pseudohaptor into the haptor, where most fibres of each bundle attach to one of the two centrally situated marginal hooks (arrow). Scale bar=0.05 mm. Fig. 4. Scanning electron mi- crograph of the posterior attachment organ of M. japonica; side view showing the anteriorly directed pseudohaptoral ridges and the haptor (arrow). Scale bar = 0.04 mm. Fig. 5. Scanning electron micrograph of Aeanthoeotyle lobianchi; ventral view. The haptor (in retracted position) and the posterior-most part of the pseudo- haptor along with the peripheral part of three radial pseudohaptor- al ridges can be seen. Note the tegumental covering of the sclerites, Scale bar=0.01 ram. Fig. 6. Scanning electron micrograph of the posterior attachment organ of A. lobianchi; ventral view, haptor side to the right. Note the radial pattern of sclerite-armed ridges. Scale bar = 0.1 mm. Fig. 7. Scanning electron micrograph of the attachment mark left by the A. lobianchi pseudohaptor shown in Fig. 6; haptor side to the left (cf. Fig. 6). Note the circular imprint made by the border of the pseudohaptor and the radial pattern of imprints left by the pseudohaptoral sclerites. Scale bar = 0.1 mm

G. Malmberg and B. Fernholm: Acanthocotylid attachment to the host 417

418 G. Malmberg and B. Fernholm: Acanthocotylid attachment to the host

G. Malmberg and B. Fernholm: Acanthocotylid attachment to the host 419

by the sclerites in the pseudohaptoral ridges (Figs. 6, 7).

The transversely arranged pseudohaptoral ridges of Myxinidocotyte lack sclerites (Malmberg and Fernholm I989); however, we found that these ridges can cause imprints in the host tegument (Fig. 8) that are as distinct as those made by A. lobianchi. Marks left by a Myxinido- cotyle posterior attachment organ (Fig. 9) consisted of an impression made by the pseudohaptor and, posterior to that, a mark caused by the haptor. The mark left by the pseudohaptoral disk was convex and displayed a circular margin and deep impressions made by the transversely arranged pseudohaptoral ridges. The haptor mark consisted of a circular depression bordered by 14 perforated distentions, with 2 similarly perforated dis- tentions lying in the centre of the depression.

On the skin of an M. californica-infested hag fish spec- imen that had been entirely fixed in the vapour from a 2% osmium tetroxide solution, two types of attach- ment marks were found: those left by the pseudohaptor and the haptor (Figs. 8-10) and those caused by the hap- tor only (haptor mark; Fig. 12). In marks of the first type, the impressions left by the pseudohaptoral ridges were sometimes indistinct or even incomplete in number, whereas the mark made by the haptor was always dis- tinct (Fig. 11). Less distinct haptor marks (Fig. 13) con- sisted of 1 depression and ]6 perforations that showed few, if any, indistinct distentions.

In attachment marks of both types, the perforations made by the 16 haptoral hooks seemed to be actual wounds in the host skin. In contrast, marks left by the

Fig. 8. Scanning electron micrograph of an attachment mark left by Myxinidocotyle californica. Note the imprints caused by the pseudohaptoral margin and by the 8 + 8 pseudohaptoral ridges and the mark left by the haptor. Scale bar=0.05 ram. Fig. 9. Scanning electron micrograph of an attachment mark caused by the posterior attachment organ of a newly detached M. californica specimen_ Note the deep imprints left by the 8+8 pseudohaptoral ridges and by the pseudohaptoral margin (bottom) and the 14 peripheral and 2 central, perforated distentions made by the marginal hooks. Scale bar=0.05 mm. Fig. 10. Scanning electron micrograph of an M. californica specimen on the skin of its host; dorsal view. The posterior attachment organ has become unattached (anaesthetic effect); to the left of the organ, the haptor mark and a part of the pseudohaptor mark are visible. Scale bar=0.1 mm. Fig. 11. Scanning electron micrograph of an indistinct attachment mark left by the posterior attachment organ of an M. californica speci- men. Note the incomplete imprint made by the pseudohaptoral margin, the incomplete number of imprints caused by the pseudo- haptoral ridges and the distinct mark left by the haptor and its hooks. Scale bar = 0.05 mm. Fig. 12. Scanning electron micrograph of a distinct mark left by an M. californica haptor. A tegumental depression showing 14 peripherally and 2 centrally situated disten- tions can be seen. Each distention has a perforation (wound) caused by a haptoral hook; the two central distentions indicate that the two central hooks can work as an opposing pair. Cell borders in the host tegument are visible. Scale bar=0.025 mm. Fig. 13. Scanning electron micrograph of a less distinct mark left by an M. caliJbrnica haptor. A faint tegumental depression showing 14 peripheral and 2 central perforations (wounds) can be seen. As compared with the mark shown in Fig. 16, this one exhibits fewer and less distinct tegumental distentions. Cell borders in the host tegument are visible. Scale bar = 0.25 mm

pseudohaptoral ridges (M. californica, M. japonica, A. lobianchi) did not appear to be wounds, instead resem- bling deep, temporary impressions. On transverse sec- tions of an A. lobianchi pseudohaptor and its site of attachment, the host skin under the imprints made by sclerites (covered by the parasite's tegument) appeared to be intact.

Discussion

Marks left by the haptor

On the skin of an entirely fixed hagfish, some marks were left by Myxinidocotyle californica haptors only (Fig. 12); they may have been made by larvae or post- larvae that lacked a functional pseudohaptor. Other marks were caused by a pseudohaptor and a haptor (Figs. 8-10) and could have been made by juveniles with a fully developed pseudohaptor or by adults. The hap- toral marks left by worms of different sizes exhibited about the same diameter, because the haptor reaches its full size as early as during post-larval development (Malmberg and Fernholm 1989).

The presence of two large bundles of muscle fibres running to the two central hooks (all four acanthocotylid species) indicates that the latter have an attachment ca- pacity different from that of an individual peripheral hook. The observation that the central hooks can func- tion as an opposing, gaffing pair (Malmberg and Fern- holm 1989; also see Fig. 12) indicates the same. A capac- ity to extrude the haptor (Acanthocotyle lobianchi) may facilitate the attachment of this organ.

The haptor marks left by early post-larvae were as distinct as those left by specimens with a fully developed pseudohaptor. This suggests that the haptor is an active attachment organ in adult acanthocotylids as well. It probably remains the posterior locomotory organ. If this is the case, a moving adult acanthocotylid will leave haptoral marks only.

The function of the posterior attachment organ in adult acanthocotylids

Live specimens of M. caIifornica could be moved side- ways over the host skin; the posterior end of the parasite attached readily to glass and plastic surfaces and, even after fixation, the pseudohaptor could be moved over the substrate. These observations, as well as the arrange- ment of the pseudohaptoral muscle fibres, indicate that the pseudohaptor has a suction capacity. Most likely, contraction of the different fibre bundles increases the firmness of the thin disk and the concavity of the pseu- dohaptor 's ventral side. Such contractions may also cause firmness at the margin of the pseudohaptoral border and diminish the diameter of the pseudohaptor. Finally, the strong muscle fibres running from the pe- duncle centrally into the pseudohaptor and continuing to its ventral side may elevate the central area of the disk and increase the convexity of the pseudohaptor.

420 G. Malmberg and B. Fernholm: Acanthocotylid attachment to the host

In a functional position, the pseudohaptoral ridges certainly give the pseudohaptor an increased attachment stability. However, transversely arranged ridges (Myxini- docotyle) obviously could not prevent a lateral displace- ment. Presumably, a radial arrangement (Lophocotyle and Acanthocotyle) of the ridges diminishes the risk of displacement; sclerite-armed, radial pseudohaptoral ridges (Acanthocotyle) may lower this risk even more.

Certain dorso-ventral muscle fibres were attached just posterior to the pseudohaptoral ridges in Myxinidoco- tyle. Possibly, the contraction of these fibres changes the anterior orientation of the ridges (Fig. 4) to a more vertical position when a worm begins moving, thus facili- tating the detachment of the ridges.

The suction of the pseudohaptor most likely depends on proper contact between the margin of the suction disk and the host skin and on a firm attachment of the posterior end of the disk; the latter condition seems to be attained by means of the small haptor. Posterior to the imprint left by the pseudohaptor of M. californica, a distinct mark caused by each of the 16 haptoral hooks was always visible (Figs. 8-10). Proper contact to the host skin may be attained by contraction of the promi- nent muscle fibres running from the posterior end of the body, through the peduncle, and on to the haptor and its hooks.

the pseudohaptoral imprintings, indicating that the Myxinidocotyle ridges cause no wounds (Fig. 11, right side of the mark). Judging by our studies of transverse sections of A. Iobianchi, not even sclerite-armed radial ridges cause wounds in the host tegument; the impressed host skin under the sclerites appeared to be intact.

Thus, only the haptoral hooks of the acanthocotylid posterior attachment organ seem to damage the host skin. In a moderate parasite infection, such small wounds may easily heal. If the infection is severe, how- ever, the posterior attachment organ of acanthocotylids may increase the risk of secondary infections via the small wounds caused by haptors.

Acknowledgements. The following persons have been most helpful in acquiring material for this study: H. Kobayashi, Japan; J. More- land and I. Mannering, New Zealand; N. Holland and R. McCon- naughey, California; and J. Llewellyn, United Kingdom. Working areas were kindly provided by the University of California, San Diego, and by the Marine Biological Laboratory, Plymouth, Eng- land. Y. Lilliemarck, Department of Zoology, Stockholm, sec- tioned the material. Financial support was given by the Japan Soci- ety for the Promotion of Science, the Danish Natural Science Re- search Council, the Swedish Natural Science Research Council and the Foundation Olle Engkvist Byggm/istare.

References

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On the skin of E. stoutii we observed distinct (Fig. 12) as well as less distinct haptor marks (Fig. 13) consisting of small wounds made by the 16 hooks. The absence of distentions and indistinctness of perforations caused by haptoral hooks may be a result of different degrees of healing of the host tegument.

Indistinct or incomplete impressions left by the pseu- dohaptoral ridges may represent steps in a receding of

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