Monogenea), Gill Parasites from European Eel Anguilla Anguilla

DISEASES OF AQUATIC ORGANISMS Published June 9 Dis. aquat. Org. l

Ultrastructural evaluation of mebendazole action in Pseudodactylogyrus bini (Monogenea), gill parasites from European eel Anguilla anguilla

'Section of Fish Diseases, Department of Veterinary Microbiology, *Zoology Section. Department of Ecology and Molecular Biology, Royal Veterinary and Agricultural University, 13 Bulowsvej, DK-1870 Frederiksberg C, Denmark

ABSTRACT: The broad-spectrum anthelmintic mebendazole (MBZ) has den~onstrated its efficacy against a range of fish-parasitizing monogeneans. The action of this drug in the gill parasitic monogenean Pseudodactylogyrus bini has been elucidated by the use of transmission electron microscopy (TEM). Parasites were exposed in vitro to MBZ (1 and 10 mg 1-l) for up to 42 h whereafter they were fixed and prepared for TEM studies. It was demonstrated that the tegument and subtegumental layers in parasites exposed to 10 mg MBZ 1-' for 36 h were disorganized and partly destroyed, and that the drug interfered w.ith the organization of the flame-cell microtubule system. This is in accordance with the suggestion that tubulin and thereby the cytoskeleton is the main MBZ target, although other drug action mechanisms cannot be excluded.

KEY WORDS: Benzimidazoles . Mebendazole Monogenea Pseudodactylogyrus . Mode of action . Ultrastructure . Microtubules

INTRODUCTION transferred to a 96 well cell incubation plate (Nunc, Denmark) kept at 24 "C. Benzimidazoles have been extensively used as an- The parasites were incubated in groups of 8 in 1 or thelmintics in human medicine (Cook 1990) and in tra- 10 mg MBZ I-', in freshwater alone or in freshwater ditional livestock industries (Campbell 1990). Recently containing the solvent formic acid (10 p1 I-') (Table 1). this drug group has also been used against helminth The drug used was pure MBZ. Parasites were incu- infections in aquaculture systems (Goven & Amend bated for 36 h (10 mg MBZ 1-l) or 42 h (1 mg MBZ I-'). 1982, Szhkely & Molnar 1987, Buchmann & Bjerre- Half of the control worms were kept for 36 h and the gaard 1990a, b, Mellergaard 1990, Tojo et al. 1992). rest for 42 h. After the experiment all parasites were However, although the mode of action of benzimida- fixed in 2.5 % glutaraldehyde in 0.1 M cacodylate buf- zoles in parasites from homoiothermic animals has fer (pH 7.4) for 2 h. They were then postfixed in 1 %OS- been the subject of numerous investigations (Lacey mium tetroxide for 1 h at 4 "C and block stained for 2 h 1988) similar studies for fish parasites have not been at room temperature in 0.5 % aqueous uranyl acetate. published. The study elucidates the action of meben- Epon was used as embedding medium. The sections dazole (MBZ) in the gill monogeneans Pseudodactylo- were cut with a diamond knife on a Reichert Jung gyrus bini parasitizing the European eel Anguilla an- ultracut ultramicrotome, stained with uranyl acetate guilla. and lead citrate and examined with a Jeol 1200 Ex electron microscope at 60 kV. Controls (non-treated MATERIALS AND METHODS and formic-acid-exposed parasites) were handled like- wise. In order to reduce artefacts and secondary post- A total of 32 Pseudodactylogyrus bini were removed mortem changes of parasite structure, only parasites from the gills of l large eel (100 g body weight), rinsed showing some motility were used for further studies. in freshwater to remove mucus and cell debris and The exposure of parasites to MBZ for up to 42 h is

O Inter-Research 1994 5 6 Dis. aquat. Org 19: 55-60, 1994

Table 1. Pseudodacty1ogyrus bini. Number of parasites exposed to various media for 36 or 42 h at 24 "C. The number of dead parasites afterexposure in brackets. MBZ: mebendazole

Formlc acid Water l mg l ' IOmg I-' 10 p1 I-' 36/42 h 36/42 h

F! bini

relevant for study as this drug is slow-acting and at least 3 d (Buchmann & Bjerregaard 1990) or up to 4 to 6 d (Szekely & Molnar 1987) are needed for parasite eradication. In addition both concentrations used have shown high efficacy (Szekely & Molnar 1987).

RESULTS

All parasites exposed to pure freshwater or freshwater containing solvent (formic acid) for both 36 and id' 42 h survived the exposure time. Some of the treated 1::; parasites survived the MBZ exposure (Table 1). l. ,: :

TEM examination findings of non-treated worms

The non-treated parasites showed a well-defined structure (Figs. 1 & 2). The outer tegument with a thickness of up to 2.2 pm (2200 nm) is constructed of an outer membrane and an inner membrane connected with a basal lamina with finger-like invaginations. Fig. 1. Pseudodaclylogyrus bmj. Tegument of an untreated Under the basal lamina, circular and longitudinal mus- specimen. OM, outer membrane; V. vacuole; BL: basal lamina cle layers are found, under which a syncytium with with inner membrane;CM: circular muscle, LM: longitudinal nuclei, numerous larger mitochondria with well-devel- muscle; MI: mitochondria. Scale bar = 0.5 pm (500nm) oped cristae and Golgi bodies are located. Smaller mitochondria are found in the outer tegument where membrane stacks, a large numbers of vesicles with a The formic acid treated parasites did not show aber- granular content and a few electron lucent vesicles are rations from the untreated control group. also located. A few vacuolar sacs bounded by a double membrane were detected in the tegument. Numerous bacteria associated with the outer membrane were TEM examination of MBZ-treated worms frequently observed. The intact flame bulb of an untreated Pseudo- Specimens of Pseudodactylogyrus bini which were dactylogyrus bini is composed of a terminal cell con- exposed to MBZ (l mg l-') for 42 h showed slight aber- taining a large nucleus and surrounded by large mito- rations from the normal structure of the tegument and chondria with well-developed cristae. Internal ribs the described flame cell composition. Only few blebs extend apically and connect wlth th.e capillary at their were observed in the treated tegument and in the external ribs. The flame consists of more than 20 cilia flame cell the connection between the capillary and extending into the collecting duct. the internal ribs of the terminal cell became loosened The control sections comprised both highly con- (Fig. 3). Some of the moribund specimens of P bin1 ex- tracted worms with extended invaginations of the posed to 10 mg MBZ I-' for 36 h showed a total disinte- basal lamina and the outer tegument in addition to re- gration and sloughing of the tegument in some places, laxed specimens with smooth basal lamina and outer leaving only the basal lamina enclosing cytoplasmic tegumental membrane. residues, interstitial fibers and deteriorating muscle Buchrnann & Bresciani: Effect of lnebendazole on Pseudodactylogyrus Oini

Fig. 2. Pseudodactylog~~rusbini. Tegument of an untreated specimen. MS: Membrane stack; F: finger-like invaginations of basal lamina and inner membrane. Other symbols as in Fig. 1. Scale bar = 0.25 pm (250 nm) layers (Fig. 4).One specimen exhibited partial destruc- However, the present study did not establish a direct tion of the microtubule arrangement in the flame cell causality between MBZ treatment and tegumental and disintegration of the terminal cell (Fig. 5). damage as these injuries at least theoretically could be explained as secondary. Thus, Bresciani (1973) demonstrated that the tegument of Polystoma integerrimum DISCUSSION in old specimens was empty and that the teguments often were damaged during fixation. Rohde (1975) As was previously demonstrated in the genera even found that some areas of the body wall of Pseudodactylogyrus(Schmahl & Mehlhorn 1988) and Polystomoidessp. were without tegument. Dactylogyrus (El-Naggar & Kearn 1983), we often The protonephridial system of monogeneans has found the intact tegument of Pseudodactylogyrus to been extensively studied by a number of authors have a monogenean structure consisting of the outer (Rohde 1973,1993, Rohde et al. 1989a, b, 1992) and the anucleate layer under which the basal lamina and flame bulbs of Pseudodactylogyrusbini conform with muscle layers are located (see Fried & Haseeb 1991). descriptions given for other monogeneans. The flame In some moribund parasites exposed to 10 mg cells and the protonephridial capillary showed a well- MBZ I-', part of the tegument was sloughed off. defined structure in the intact parasite. In this study However, a low concentration (1 mg 1-l) for 42 h did adverse effects on the flame cells were seen associated not elicit severe changes in the tegument as only few with mebendazole exposure, which interfered with the blebs were observed in the outer anucleate layer. The organization of microtubule associated structures. The impact of the drug on the tegument could play an slightest effect was seen on the connection between important part in the parasiticidal effect of MBZ. the terminal cell and the capillary, which was found Dis. aquat. Org 19 55-60, 1994

Flg 3 Pseudodactylogyrusbin1 Flame bulb after treatment with rnebenddzole (MBZ)I1 rng 1-', 42 h). T.terminal cell; N: nucleus: C cllia, Ca: capillary; IR Internal nb. ER external rib; MI, m~tochondna Scale bar = 0.25 pm (250 nrn) partly separated after 42 h in MBZ (1 mg 1 l). The tylus bullatarudis. This is partly in accordance with our moribund parasites (36 h In l0 mg I-') showed a results. disintegration of flame bulbs and rn~crotubulesIn the Stud~eson the effects of mebendazole on the ultra- flame. structure of Ascans suum (Borgers & De Nollin 1975) It could be suggested that the Pseudodactylogyrus and of other microtubule inhibitors on Fasciola hepat- bin1 protonephridial system, containing essential ml- ica (Stitt & Fairweather 1992, 1993) also disclosed that crotubule structures, IS affected by mebendazole, organelles became disorganized due to treatment with whereby the osmoregulatory ability of the paraslte de- these substances. However, although the microtubule tenorates and the parasite eventually is killed. A pos- system IS considered as the main target for benzimida- sible direct effect of MBZ on cytoplasmic microtubules zoles (Lacey 1988) this drug group has been shown to in the tegument which would elicit blebbing and affect a range of enzymatic reactions in helminths sloughing of the tegument m addition to disorganiza- (Kohler & Bachrnann 1978, Sarwal et al. 1989) as well, tion of muscle layers could assist this kllhng. Schmahl and ~tcannot be excluded that a number of other action (1993) Indicated that MBZ 1s able to interfere with the mechan~smsare lnvolved in the described effect on the tegument, muscle and nerve structures of Gyrodac- monogenean. uru 001 = 1eq aleJS -eppuosollu~x .auIelj aql 6u1urloj ell13 aql Jo uollsas aslansuell :3 .zgyy lalje q1nq auIe[j 'S '61~'(mu 00~)urrl z.0 = 1eq aless -salq~j[eg!ls -1alu1 :JI .lalie[ alssnur 1~u!pnl!6uo[ '~aAe[apsnur 1elnsl1s :w3'euyurer pseq :lg .luaurJea11 01 anp juaurn6al jo 6u1q6nols lalje uau~sadspunq!lom ,m .(yg~ ',-Ifjur 01 '~gp~)aIozepuaqam yl!~luawlea.11 JaJje rurq sn.rXBolX~~epopnasd-S v P .s61~ 60 Dis. aquat. Org. 19: 55-60, 1994

Acknowledgements. Thls work was supported by the Dan~sh Rohde, K. (1973). Ultrastructure of the protonephridial Veterinary and Agricultural Research Council. The authors system of Polystomoides malay~ Rohde and P. renschli are indebted to Mrs B. Weng Jergensen for her skillful techni- Rohde (Monogenea: Polystomatidae). Int. J. Parasitol. 3: cal assistance. 329-333 Rohde, K. (1975). Fine structure of the Monogenea, especially LITERATURE CITED P~lyst~rn~ldeSWard. Adv. Parasitol. 13: 1-33 Rohde, K. (1993). Ultrastructure of protonephridia in the Borgers, M., De Nollin, S. (1975). Ultrastructural changes in monogenea. Implications for the phylogeny of the group. Ascaris suum intestine after mebendazole treatment in Bull. Fr. Peche Piscicult. 328: 115-1 19 vivo. J. Parasitol. 61: 110-122 Rohde, K.,Justine, J.-L.,Watson, N. (1989a). Ultrastructure of Bresciani, J. (1973). The ultrastructure of the integument of the flame bulbs of the monopistocotylean monogenea the monogenean Polystoma integerrimurn (Frolich 1791). Loimosina wilsoni (Loimoidae) and Calceostoma hercu- Yearbook for the Royal Veterinary and Agricultural Uni- lanea (Calceostomatidae). Ann. Parasitol. Hum. Comp. 64: versity, Copenhagen, p. 14-27 433-442 Buchmann, K., Bjerregaard, J. (1990a). Comparative effica- Rohde, K.,Watson, N., Roubal, F. (1989b). Ultrastructure of cies of commerciallv available benzimidazoles asainst the protonephridial system of Dactylogyrus sp. and an Pseudodactylogyrusinfestations in eels. Dis. aquat. Org. 9: unidentified Ancyrocephalinae (Monogenea: Dactylogyri- 117-120 dae). Int. J. Parasitol. 19: 859-864 Buchmann, K.,Bjerregaard. J. (1990b). Mebendazol~ trpat- Rnhrlr?, K,Wiltson, N. .A., Rnnhz!, F R. (1992). 'l!!?as!?nct?lre ment of pseudodactylogyrosis in an intenswe eel-culture of the protonephridial system of Anoplodiscus cirrusspir- system. Aquaculture 86: 139-153 alis (Monogena: Monopistocotylea). Int. J Parasitol. 22: Campbell, W. C. (1990). Benzimidazoles: veterinary uses. 443-457 Parasitology Today 6: 130-133 Sarwcll, R., Sanyai, S. N., Khera, S. (1989). In vitro effect of Cook, G. C. (1990). Use of benzim~dazole chemotherapy in benzimidazole drugs on the enzymes of lipid metabolism human helminthiasis: indications and efficacy. Parasi- in Trichuris globulosa. J. Parasitol. 75: 808-810 tology Today 6: 133-136 Schmahl, G. (1993). Up-to-date chemotherapy against mono- El-Naggar, M. M,, Kearn, G. C. (1983). The tegument of the genea: a review. Bull. Fr. P&che Piscicult. 328: 74-81 monogenean gill parasites Dactylogyrus amphibothrium. Schmahl, G., Mehlhorn, H. (1988). Treatment of fish parasites Int. J. Pa~dsiloi.13: 579-592 4. Effects ot sym.triazinone (toltrazuril) on monogenea. Fried, B., Haseeb, M. A. (1991). Platyhelminthes: Aspido- Parasitol. Res. 75: 132-143 gastrea, Monogenea and Digenea. In: Harrison, F. W., Stitt. A. W.. Fairweather, I. (1992). Spermatogenesis in Bogitsh, B.J. (eds.) Microscopic anatomy of invertebrates. Fasciola hepatica: an ultrastructural comparison of the 3: 141-209 effects of the anthelmintic, triclabendazole (,,Fasinex") Goven, B. A., Amend, D. F. (1982). Mebendazole/trichlorfon and the microtubule inhibitor, tubulozole. Inv. Repr. Dev. combination: a new anthelmintic for removing rnonoge- 22: 139-150 netic trematodes from fish. J. Fish Biol. 20: 373-378 Stitt. A. W., Fairweather. I. (1993). Fasciola hepatica: the Kohler, P., Bachmann, R. (1978). The effects of the antipara- effect of the microtubule inhibitors, colchicine and tubulo- sitic drugs levamisole, thiabendazole, praziquantel and zole-C on the ultrastructure of the adult fluke. Para- chloroquine of mitochondrial electron transport in muscle sitology 107: 297-309 tissue from Ascaris suurn. Molec. Pharmacol. 14: 155-163 Szekely, CS.,Molnar, K. (1987). Mebendazole is an efficacious Lacey, E. (1988). The role of the cytoskeletal protein, tubulln, drug against pseudodactylogyrosis in the European eel In the mode of action and mechanism of drug resistance to (Anguilla anguilla). J. appl. Ichthyol. 3: 183-186 benzimidazoles. Int. J. Parasitol. 18: 885-936 Tojo, J., Santamarina, M. T., Ubeira. F. M., Estevez, J., Mellergaard. S. (1990). Mebendazole treatment against Sanmartin. M. L. (1992). Anthelmintic activity of benzi- Pseudodactylogyrus infections in eel (Anguilla anguilla). midazoles against Gyrodactylus sp. infecting rainbow Aquacu.lture 91: 15-2 1 trout Oncorhynchus rnykiss. Dis. aquat. Org. 12 185-189

Responsible Subject Editor: W. Korting, Hannover, Manuscript first received: October 12, 1993 Germany Revised version accepted: January 6, 1994