Preliminary characterisation and extraction of anterior adhesive secretion in monogenean (platyhelminth) parasites

Secreted anterior adhesives, used for temporary attachment to epithelial surfaces of fishes (skin and gills) by some monogenean (platyhelminth) parasites have been partially characterised. Adhesive is composed of protein. Amino acid composition has been determined for seven monopisthocotylean monogeneans. Six of these belong to the Monocotylidae and one species, Entobdella soleae (van Beneden et Hesse, 1864) Johnston, 1929, is a member of the Capsalidae. Histochemistry shows that the adhesive does not contain polysaccharides, including acid mucins, or lipids. The adhesive before secretion and in its secreted form contains no dihydroxyphenylalanine (dopa). Secreted adhesive is highly insoluble, but has a soft consistency and is mechanically removable from glass surfaces. Generally there are high levels of glycine and alanine, low levels of tyrosine and methionine, and histidine is often absent. However, amino acid content varies between species, the biggest differences evident when the monocotylid monogeneans were compared with E. soleae. Monogenean adhesive shows similarity in amino acid profile with adhesives from starfish, limpets and barnacles. However, there are some differences in individual amino acids in the temporary adhesive secretions of, on the one hand, the monogeneans and, on the other hand, the starfish and limpets. These differences may reflect the fact that monogeneans, unlike starfish and barnacles, attach to living tissue (tissue adhesion). A method of extracting unsecreted adhesive was investigated for use in further characterisation studies on monogenean glues.

A comparison of the anterior adhesive system in the oncomiracidium and adult of the monogenean parasite Merizocotyle icopae (Monocotylidae)

The anterior adhesive system of the oncomiracidium and adult of Merizocotyle icopae (Monogenea: Monocotylidae) were compared. The oncomiracidium has one ventrally placed aperture on either side of the head near the anterior extremity. In the adult, there are three ventrally placed apertures on either side of the head region. Both systems have three types of electron-dense secretory bodies opening into each aperture. A rod-shaped secretion (S1) and a small electron dense ovoid secretion (S2) are common to larvae and adults. The third secretion type differs: in adults, it is a large, spherical (S3) type but in larvae, it is an ovoid (S4) body. S4 bodies do occur in adults, but appear to be secreted as a general body secretion. An additional anteromedian secretion (S5) is also present in the oncomiracidium, but is not secreted into the anterior apertures. Homology and function of secretions are discussed.

Host-specificity of monogenean (platyhelminth) parasites: a role for anterior adhesive areas?

Monogeneans (flatworms) are among the most host-specific of parasites in general and may be the most host-specific of all fish parasites. Specificity, in terms of a restricted spatial distribution within an environment, is not unique to parasites and is displayed by some fungi, insects, birds, symbionts and pelagic larvae of free-living marine invertebrates. The nature of cues, how habitats are recognised and how interactions between partners are mediated and maintained is of interest across these diverse associations. We review some experiments that demonstrate important factors that contribute to host-specificity at the level of infective stages (larvae of oviparous monogeneans; juveniles of viviparous gyrodactylids) and adult parasites. Recent research on immune responses by fish to monogenean infections is considered. We emphasise the critical importance of host epidermis to the Monogenea. Monogeneans live on host epidermis, they live in its products (e.g. mucus), monopisthocotyleans feed on it, some of its products are attractants and it may be an inhospitable surface because of its immunological activity. We focus attention on fish but reference is made to amphibian hosts. We develop the concept for a potential role in host-speciality by the anterior adhesive areas, either the specialised tegument and/or anterior secretions produced by monogeneans for temporary but firm attachment during locomotion on host epithelial surfaces. Initial contact between the anterior adhesive areas of infective stages and host epidermis may serve two important purposes. (1) Appropriate sense organs or receptors on the parasite interact with a specific chemical or chemicals or with surface structures on host epidermis. (2) A specific but instant recognition or reaction occurs between component(s) of host mucus and the adhesive(s) secreted by monogeneans. The chemical composition of fish skin is known to be species-specific and our preliminary analysis of the chemistry of some monogenean adhesives indicates they are novel proteins that display some differences between parasite families and species. (C) 2000 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Morphology, ultrastructure, and implied function of ciliated sensory structures on the developmental stages of Merizocotyle icopae (Monogenea : Monocotylidae)

Experimental infections were used to track the fate of the dorsal sensilla of Merizocotyle icopae (Monogenea: Monocotylidae) from nasal tissue of the shovelnose ray, Rhinobatos typus (Rhinobatidae). Scanning and transmission electron microscopy revealed that 3 types of uniciliate dorsal sensilla exist at different times in the development of the monogenean. Type 1 sensilla have little or no invagination where the cilium exits the distal end of the dendrite and possess a ring of epidermis surrounding the cilium distal to the invagination. Type 2 sensilla have a deep invagination where the cilium exits the dendrite. Type 3 sensilla can be distinguished from the other types by the shape of the dendrite. The larvae have predominantly Type I dorsal sensilla, most of which are lost approximately 24 h after infection and a few Type 2 sensilla, which are retained. Additional Type 2 sensilla (termed Adult Type 2 sensilla), which are slightly different morphologically from the Type 2 sensilla of the larvae, form in later stages of development. Numerous Type 3 sensilla are unique to the dorsal surface of adults. Loss of all Type I sensilla upon attachment to the host, R. typus, suggests that these may be chemo- or mechanoreceptors responsible for host location by the swimming infective larvae. Type 2 sensilla appear to be important in the larvae, juveniles, and adults whereas the modality mediated by Type 3 is specific to adults. (C) 2003 Wiley-Liss, Inc.

A revision of Benedenia Diesing, 1858 including a redescription of B-sciaenae (van Beneden, 1856) Odhner, 1905 and recognition of Menziesia Gibson, 1976 (Monogenea : Capsalidae)

Benedenia Diesing, 1858, a genus of capsalid (benedeniine) monogeneans, is redefined. The generic diagnosis is amended to include: the path of tendons in the haptor from extrinsic muscles in the body; presence and form of the marginal valve; a penis occupying a penis canal with weakly muscular wall; a weakly muscular accessory gland reservoir proximal to the penis and enclosed by a proximal extension of the wall of the penis canal; male and female genital apertures usually common, rarely separate; vagina with pore usually close to the common genital pore but may open in mid body between the germarium and the common genital pore, or anterior to the common genital pore. A conservative approach is adopted and the generic diagnosis is clarified and broadened to accommodate species that display some variation in reproductive anatomy, especially of the female system. We argue against potential alternative actions such as defining Benedenia strictly to contain species with separate male and female genital apertures and against recognition of a separate genus, Tareenia Hussey, 1986, for species with a vaginal pore anterior to the common genital pore. Under our conception, Benedenia comprises 21 species: B. sciaenae (van Beneden, 1856) Odhner, 1905 (type species); B. acanthopagri (Hussey, 1986) comb. nov.; B. anticavaginata Byrnes, 1986; B. bodiani Yamaguti, 1968; B. elongata (Yamaguti, 1968) Egorova, 1997; B. epinepheli (Yamaguti, 1937) Meserve, 1938; B. hawaiiensis Yamaguti, 1968; B. hendorffi(von Linstow, 1889) Odhner, 1905; B. hoshinai Ogawa, 1984; B. innobilitata Burhnheim Gomes and Varela, 1973: B. jaliscana Bravo-Hollis, 1952; B. lolo Yamaguti, 1968; B. lutjani Whittington and Kearn, 1993: B. monticellii (Parona and Perugia, 1895) Johnston, 1929; B. ovata (Goto, 1894) Johnston. 1929: B. pompatica Burhnheim, Gomes and Varela, 1973; B. rohdei Whittington, Kearn and Beverley-Burton, 1994; B. scari Yamaguti, 1968; B. sekii (Yamaguti, 1937) Meserve, 1938; B, seriolae (Yamaguti, 1934) Meserve, 1938; and B. synagris Yamaguti, 1953. The type species, B. sciaenae, is redescribed based on new material from Australia. No types for this taxon were designated and we have assigned a series of voucher specimens. Tareenia acanthopagri Hussey, 1986 becomes B. acanthopagri (Hussey, 1986) comb. nov. and T. anticavaginata (Byrnes, 1986) Egorova, 1997 and T. lutjani (Whittington and Kearn, 1993) Egorova, 1997 are returned to Benedenia as B. anticavaginata and B. lutjani Benedenia akaisaki Iwata, 1990 is considered a synonym of B. ovata and B. kintoki Iwata, 1990 is considered a synonym of B. elongata. Two species, B, madai Ishii and Sawada, 1938 and B. pagrosomi Ishii and Sawada, 1938, are considered species inquirendae. Based on the redefinition of Benedenia, the diagnosis for the Benedeniinae is amended. Tareenia is synonymized with Benedenia but Menziesia Gibson, 1976 is recognized and its generic diagnosis amended to include: anterior attachment organs tending to form a 'hooded' appearance; prominent anterior gland cells between the pharynx and the anterior margin of the body: long penis, tapering proximally, occupying a penis canal with weakly muscular wall: penis canal and penis describe a sigmoid; accessory gland reservoir dorsal and alongside, or posterior and lateral to, proximal end of the penis and enclosed by a proximal extension of the wall of the penis canal. Under this conception. Menziesia comprises: M. noblei (Menzies. 1946) Gibson, 1976 (type species); M. malaboni (Velasquez. 1982) comb. nov.: M. merinthe (Yamaguti, 1968) Gibson. 1976: M. ovalis (Yamaguti, 1968) Gibson, 1976: and M. sebastodis (Yamaguti, 1934) comb, nov. A key to valid species of Benedenia and Menziesia is provided and a list is presented of published records of undescribed or unattributed species of Benedenia. Some protocols are suggested for preparation of benedeniine material to enhance future taxonomic studies and comparisons. The host-specificity and geographic distribution of species in these revised genera are discussed. The composition of the Capsalidae is discussed and some difficulties in defining and distinguishing between its different subfamilies are considered.

Mechanism of adhesion and detachment at the anterior end of Merizocotyle icopae (Monogenea : Monocotylidae) including ultrastructure of the anterior adhesive matrix

The anterior adhesive mechanism was studied for Merizocotyle icopae (Monogenea: Monocotylidae). Adult anterior apertures can open and close. In addition, duct endings terminating within the apertures are everted or retracted depending on the stage of attachment. Adhesive in adults is synthesized from all 3 secretory types (rod-shaped, small and large spheroidal bodies) found within anterior apertures. All exit together and undergo mixing to produce the adhesive matrix, a process that depletes duct contents. A greater number of ducts carrying rod-shaped bodies is depleted than ducts containing spheroidal bodies which changes the ratio of secretory types present on detachment. Detachment involves elongation of duct endings and secretion of additional matrix as the worm pulls away from the substrate. The change in secretory type ratio putatively modifies the properties of the secreted matrix enabling detachment. Only after detachment do ducts refill. During attachment, individual secretory bodies undergo morphological changes. The larval and adult adhesive matrix differs. Anterior adhesive in oncomiracidia does not show fibres with banding whereas banded fibres comprise a large part of adult adhesive. The data Suggest that this is the result of adult spheroidal secretions modifying the way in which the adult adhesive matrix forms.

Euzetia occultum n. g., n. sp (Euzetiinae n. subf.), a monocotylid monogenean from the gills of Rhinoptera neglecta (Rhinopteridae) from Moreton Bay, Queensland, Australia

Euzetia occultum n. g., n. sp. (Monogenea: Monocotylidae) is described from the gills of the Australian cownose ray Rhinoptera neglecta Ogilby collected in Moreton Bay, Queensland, Australia. Euzetia has one central and ten peripheral loculi, which is similar to species in Decacotyle Young, 1967. However Euzetia is distinguished from other genera in the family by the presence of an additional loculus on either side of the central loculus. Because Euzetia does not fit into any of the six existing subfamilies in the Monocotylidae Taschenberg, 1879, as currently recognised, we propose the Euzetiinae n. subf. to accommodate the new genus. Euzetia occultum is described and illustrated fully. This is the first published record of a monocotylid from a species of Rhinoptera Cuvier.

First published record of the pathogenic monogenean parasite Neobenenenia melleni (Capsalidae) from Australia

The monogenean Neobenedenia melleni (Mac- Callum, 1927) Yamaguti 1963 is a well-known and virulent pathogen in culture conditions recorded from the skin of many teleost fish species worldwide. Until now, N. melleni has not been reported from wild or cultured fish in Australian waters. This study documents a recent outbreak of N. melleni that occurred on Lates calcarifer (barramundi) cultivated in sea cages in Hinchinbrook Channel between Hinchinbrook Island and mainland Queensland, Australia, which resulted in the loss of 200 000 fish (50 tonnes). The origin of this outbreak is unclear because N. melleni has not been recorded from any wild host species in Australia and strict quarantine regulations exclude the possibility of its introduction on imported fish. We propose that N. melleni occurs naturally on wild populations of some teleost species in Australian waters and that the few surveys of wild fish conducted along the eastcoast have failed to report this species. The possibility that uncharacteristically low water temperatures led to the outbreak is discussed.

The effect of the cleaner fish Labroides dimidiatus on the capsalid monogenean Benedenia lolo parasite of the labrid fish Hemigymnus melapterus

The cleaner fish Labroides dimidiatus affected the pigmented monogenean parasite Benedenia lolo on the fish Hemigymnus melapterus (Labridae) held in aquaria. The effect of cleaner fish varied with the size class of fish; only small fish [a posteriori size class < 11-5 cm standard length (L-S)] exposed to cleaner fish had fewer monogeneans compared with fish not exposed to cleaner fish. The abundance of monogeneans on large fish (a posteriori size class > 11-5 cut L-S) was not affected by cleaner fish. The size-frequency distributions of monogeneans on both size-classes of H. melapterus were affected by cleaner fish. Fish exposed to cleaner fish had fewer large (> 3 mm) and more small (< 1 mm) monogeneans than fish not exposed to cleaner fish, suggesting cleaner fish selectively removed larger monogeneans. This difference was more pronounced on large fish. In the absence of cleaner fish, small fish had almost as many monogeneans as large fish; they also had more small monogeneans than the large fish, suggesting small fish were more vulnerable to infection by monogeneans than larger fish. This suggests that the cleaner fish L. dimidiatus has the potential to control benedeniine monogeneans on captive fish and highlights the importance of taking into account fish size in studies of the effect of cleaner fish on ectoparasites. (C) 2002 The Fisheries Society of the British Isles. Published by Elsevier Science Ltd. All rights reserved.

Simultaneous fixation using glutaraldehyde and osmium tetroxide or potassium ferricyanide-reduced osmium for the preservation of monogenean flatworms: An assessment for Merizocotyle icopae

Simultaneous fixation was investigated for a marine organism: the monogenean flatworm ectoparasite Merizocotyle icopae. Four protocols for primary fixation were compared: 3% glutaraldehyde alone in OAM cacodylate buffer for a minimum of 2 hours; 1% glutaraldehyde in combination with 1% osmium tetroxide, both in 0.1M cacodylate buffer, until tissues darkened (5-20 minutes); 1% glutaraldehyde in OAM cacodylate buffer in combination with 0.5% potassium ferricyanide-reduced osmium until tissues darkened (5-20 minutes); 1% glutaraldehyde in combination with 1% osmium tetroxide, both in 0.1M cacodylate buffer, for 30 minutes. The study confirms that the standard method for transmission electron microscopic fixation (first listed protocol) routinely applied to platyhelminths is optimal for ultrastructural preservation, but some simultaneous fixation methods (second and third listed protocols) are acceptable when rapid immobilization is needed. Scanning electron microscopic preparations may be improved using simultaneous primary fixation. (C) 2004 Wilcy-Liss, Inc.

A comparison of anterior adhesive areas and secretions in Troglocephalus rhinobatidis and Neoheterocotyle rhinobatidis (Monogenea : Monocotylidae) from the gills of the shovelnose ray, Rhinobatos typus (Rhinobatidae)

This study continues the collection of data on the anterior adhesive areas and secretions of monopisthocotylean monogenean (flatworm) parasites and begins an investigation of their phylogenetic usefulness. Here, two species of parasitic worms from an elasmobranch, Troglocephalus rhinobatidis (Monocotylidae: Dasybatotreminae) and Neoheterocotyle rhinobatidis (Monocotylidae: Heterocotylinae), are compared and contrasted. It has been suggested in recent literature that these two taxa are more closely related than is currently recognised. Our data support this view. Both species have multiple apertures on the ventral anterior margin through which adhesive is secreted. Two types of secretion exit from multiple adjacent duct endings terminating in each aperture: rod-shaped (S1) and spherical-shaped (S2) bodies. S1 bodies of both species show nano-banding of similar size and are membrane bound. Ultrastructure of the glands, ducts, duct endings and secreted adhesive is similar for both species, but aperture shape differs. Away from the adhesive areas, tegumental inclusions are found to differ between the two species and another, apparently non-adhesive, secretion is found in N. rhinobatidis.

Platyhelminth phylogenetics - a key to understanding parasitism?

The comparative method, the inference of biological processes from phylogenetic patterns, is founded on the reliability of the phylogenetic tree. In attempting to apply the comparative method to the understanding of the evolution of parasitism in the phylum Platyhelminthes, we have highlighted several points we consider to be of value along with many problems. We discuss four of these topics. Firstly, we view the group at a phylum level, in particular discussing the importance of establishing the sister taxon to the obligate parasite group, the Neodermata, for addressing such questions as the monophyly, parasitism or the endo or ectoparasitic nature of the early parasites. The variety of non-congruent phylogenetic trees presented so far, utilising either or both morphological and molecular data, gives rise to the suggestion that any evolutionary scenarios presented at this stage be treated as interesting hypotheses rather than well-supported theories. Our second point of discussion is the conflict between morphological and molecular estimates of monogenean evolution. The Monogenea presents several well-established morphological autapomorphies, such that morphology consistently estimates the group as monophyletic, whereas molecular sequence analyses indicate paraphyly, with different genes giving different topologies. We discuss the problem of reconciling gene and species trees. Thirdly, we use recent phylogenetic results on the tapeworms to interpret the evolution of strobilation, proglottization, segmentation and scolex structure. In relation to the latter, the results presented indicate that the higher cestodes are diphyletic, with one branch difossate and the other tetrafossate. Finally, we use a SSU rDNA phylogenetic tree of the Trematoda as a basis for the discussion of an aspect of the digenean life-cycle, namely the nature of the first intermediate host. Frequent episodes of host-switching, between gastropod and bivalve hosts or even into annelids, are indicated.

Experimental susceptibility of some reef fish species to Benedenia lutjani (Monogenea : Capsalidae), a parasite of Lutjanus carponotatus (Pisces : Lutjanidae)

The susceptibility of species of lutjanid, lethrinid and serranid fish to infection by either larval or post-larval (juvenile and adult) specimens of the capsalid monogenean Benedenia lutjani Whittington and Kearn (1993) was examined experimentally. Four species of lutjanids became infected when exposed to larvae of B. lutjani, but three species of lethrinids and four species of serranids were not susceptible to larvae under the same conditions. Variability in the intensity of infection by larvae occurred within and between lutjanid species. Few post-larval specimens of B. lutjani transferred between individuals of the specific host Lutjanus carponotatus (Richardson 1842) in 60-l aquaria and none transferred between specimens of L. carponotatus in a 7,500-l concrete tank. These results indicate that transfer of post-larval B. lutjani between individuals of the specific host is unlikely to occur in the wild. Other lutjanid species did not become infected when exposed to specimens of L. carponotatus infected heavily by post-larval B. lutjani, but two lethrinid species were susceptible to infection under the same conditions. These data indicate that different factors may mediate host-specificity for larval and post-larval B. lutjani.