Topography and ultrastructure of the tegument of Deropristis inflata Molin, 1859 (Digenea: Deropristidae), a parasite of the European eel Anguilla anguilla (Osteichthyes: Anguillidae)
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- Filippi, JJ., Quilichini, Y. & Marchand, B. Parasitol Res (2013) 112: 517. doi:10.1007/s00436-012-3162-9
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The tegumental ultrastructure of the intestine fluke Deropristis inflata was studied using scanning and transmission electron microscopy. The surface of the tegument was covered by transverse cytoplasmic ridges from which protrude numerous thorn-like spines showing crenelated tips on the posterior part. Spines were arranged in staggered rows. Cobblestone-like units of the tegument were observed on a semicircle-shaped formation over the oral sucker. A tegumental excrescence was observed in the dorsal anterior side of the fluke. Ultrastructural study revealed that the tegument of D. inflata had a typical syncytial organization with a distal cytoplasm lying over a basal matrix and cytons. Cytoplasmic bridges allowed transit of secretory vesicles and granules packed in gland cells. Two types of sensory structures were examined. Type 1 sensory receptor was a button-like uniciliated papilla mounted on a folded tegumental base and surrounded by cytoplasmic ridges. This receptor consisted of a nerve bulb and a cilium that extended from a centriole. Type 2 sensory receptor was a smooth bulb-like non-ciliated papilla. It was only recovered on the ventral sucker. This receptor consisted of a nerve bulb enclosing an ovoid electron-dense structure. For both receptors, the nerve bulbs contained numerous mitochondria, nerve fibers, and electron-lucent material. Particular distributions of the sensory receptors were observed with a concentration on the anterior third of the body around the oral and ventral suckers. Diagrams were made to help in understanding the nature of these structures.
According to Threadgold (1984), the tegument is considered to be the major host–parasite interface. It is involved in protection against host enzymes and immune systems, excretion processes, and absorption of nutrients (Burton 1966; Lumsden 1975; Threadgold 1984; Paperna and Dzikowski 2006). It may also be involved in ion regulation and osmoregulation (Threadgold 1984), which makes it a living, complex tissue (Roberts and Janovy 2000). According to Halton (2004), microscopy has for long dramatically expanded horizons in the field of helminthology by providing fundamentally important information on the structure and functional correlates of a number of key organ systems of helminths such as their body surface. Detailed studies on the ultrastructural characteristics of digeneans can provide useful information in the taxonomic study of the groups (Mata-López and Leon-Regagnon 2006). Moreover, Bakke and Lien (1978) and Southgate et al. (1986) considered the microtopographical features, such as papillae, spines and tubercles as taxonomically aspects of adult trematodes.
Among Digenea, studies on tegumental sensory structures mainly focused on cercaria or metacercaria stages (Fujino et al. 1979b; Matthews and Matthews 1988; Žd’árská 1992; Bogea and Caira 2001; Abdul-Salam and Sreelatha 2004; Poddubnaya et al. 2010) as they require orientation to stimuli such as light and gravity more than adults do, a condition related to the necessity of finding a host quickly (Roberts and Janovy 2000). However, some authors have emphasized the description of tegumental structures of adult digeneans (Žd’árská 1993; Ferrer et al. 1996; Ibraheem 2000; Filippi et al. 2010, 2012). Electron microscopy techniques such as scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are of a particular interest in the description of tegumental structures. Indeed, SEM can provide data on the external morphology and localization of these structures, while TEM allows ultrastructural investigations at a cellular level. Thus, the combination of both techniques provides a full range morphological description of the tegumental structures.
Among Digenea, the Deropristidae Cable and Hunninen 1942 is a small group of digeneans parasitic mainly in the spiral intestine of chondrosteans and, occasionally, the intestine of teleosts in euryhaline conditions in the eastern Nearctic and western Palaearctic region (Choudhury and Dick 1998; Jones et al. 2005). The genus Deropristis has been considered as belonging to families such as the Lepocreadiidae Odhner, 1905 (Cable and Hunninen 1942), or the Acanthocolpidae Lühe, 1906 (Yamaguti 1971). Most recent authors have recognized its belonging to the family Deropristidae (Choudhury and Dick 1998; Jones et al. 2005). Among the Deropristidae, Deropristis inflata is the only species that have been studied using SEM (Dezfuli et al. 1997; Kanev et al. 1999). However, the authors focused on the collar region of the fluke and its tegumentary spines. No TEM study has been made on the tegument of a deropristid.
The aim of this study is to investigate the tegument of D. inflata using SEM and TEM, giving special consideration to the morphology and distribution of sensory receptors. To our knowledge, no electron microscope study using both techniques has been made on the tegument of a trematode of the Deropristidae family.
Materials and methods
Adult specimens of D. inflata were collected alive from the intestine of 20 eels (Anguilla anguilla) obtained from fishermen of the Urbino Pond, Corsica, France (42°03′N, 9°28′E) on November 2011. The fish were brought alive to the laboratory, anesthetized in 0.1 ml l−1 Eugenol (Merck Schuchardt OHG, Hohenbrunn, Germany), and dissected the day of the capture. During necropsy, helminths from the intestine were collected alive and prepared for SEM and TEM observations at the “Service d’Étude et de Recherche en Microscopie Électronique” of the University of Corsica Pascal Paoli.
A total of 35 adult flukes were fixed for at least 1 h in cold (4°C) 2.5 % glutaraldehyde in a 0.1 M sodium cacodylate buffer at pH 7.2. Specimens were then dehydrated in a graded acetone series and dried with the use of CO2 in an Emitech K850 critical point dryer (Quorum Technologies Ltd, Ashford, UK). Specimens were mounted on aluminium stubs with carbon double-sided adhesive discs. Specimens were then coated with gold/palladium in a Quorum Technologies SC7640 sputter coater (Quorum Technologies Ltd) and examined with the use of a Hitachi S-3400 N scanning electron microscope (Hitachi High-Technologies Corporation, Tokyo, Japan) at an acceleration voltage of 5 kV.
Transverse sections of ten adult flukes were made and fixed for at least 1 h in cold (4°C) 2.5 % glutaraldehyde in a 0.1 M sodium cacodylate buffer at pH 7.2, rinsed in a 0.1 M sodium cacodylate buffer at pH 7.2, postfixed in cold (4°C) 1 % osmium tetroxide in the same buffer for 1 h, dehydrated in ethanol and propylene oxide, embedded in Spurr (1969), and then polymerized at 60°C for 24 h. Ultrathin sections (60 nm in thickness) were obtained with the use of an RMC Boeckeler Power Tome PC ultramicrotome (Boeckeler Instruments, Inc., Tucson, AZ) placed on 300-mesh copper grids, and double stained with uranyl acetate and lead citrate according to Reynolds (1963). The sections were examined with the use of a Hitachi H-7650 transmission electron microscope (Hitachi High-Technologies Corporation) at an acceleration voltage of 80 kV.
General organization of the tegument
Scanning electron microscopy
Transmission electron microscopy
Morphological characteristics of the sensory receptors of Deropristis inflata as seen in SEM
Button-like ciliated structure
Smooth bulb-like structure
General organization of the tegument
The tegumental organization of D. inflata is similar to the general tegumental structure described for digeneans (Morris and Threadgold 1968; Bennett and Threadgold 1975; Threadgold 1984; Roberts and Janovy 2000). As for the Monogenea and the Cestoidea, digenean parasites possess a distal continuous and anucleate peripheral layer making up a “sunken” epidermis, also known as a distal cytoplasm. Cytons (cell bodies containing the nuclei) connected to the distal cytoplasm via cytoplasmic bridges (or internuncial processes) lie beneath a superficial layer of circular and longitudinal muscles (Hockley and McLaren 1973; Bennett and Threadgold 1975; Threadgold 1984; Roberts and Janovy 2000). An extracellular matrix (ECM) composed of a basal matrix and an interstitial matrix is occurring through this syncytium, between the cytons and the distal cytoplasm (Conn 1993). Tegumental folds, such as cytoplasmic ridges and cobblestone-like zones, could participate in nutrition by amplifying the free surface area (Paperna and Dzikowski 2006). Cytoplasmic ridges of the tegument have also been observed in SEM micrographs of other digeneans, such as Bucephalus anguillae (Filippi et al. 2010), Fasciola hepatica (Bennett 1975a), Lecithochirum musculus (Filippi et al. 2012), and Schistosoma mansoni (Hockley 1973). The contraction of longitudinal muscles is thought to impact on the presence of these ridges (Williams and McKenzie 1995). Cobblestone-like areas of the tegument have also been described for other digeneans, such as B. anguillae (Filippi et al. 2010), L. musculus (Filippi et al. 2012), Cyclocoelum mutabile (Tajrine et al. 1999), Heterophyopsis continua (Hong et al. 1991), Leucochloridiomorpha constantiae (Font and Wittrock 1980), and Metagonimus yokogawai (Lee et al. 1984). Such mechanisms increasing the surface area of the body would be beneficial in large, stout worms, which have a much lower surface/volume ratio than smaller species (Dias et al. 2003).
The glycocalyx observed on the outer membrane of D. inflata is thought to provide permeability control, ionic regulation, protection against host enzymes, and antibody/antigen reactions (Burton 1966; Lumsden 1975; Paperna and Dzikowski 2006).
Secretory bodies are usually present in the syncytium of digeneans (Threadgold 1984; Roberts and Janovy 2000). These secretory vesicles are involved in the surface membrane turnover which becomes a protective device for the parasite (Threadgold 1984). Secretory vesicles have been recorded in other adult digeneans such as B. anguillae (Filippi et al. 2010), F. hepatica (Bennett and Threadgold 1975), Prosorhynchoides arcuatus (Cohen et al. 1996), Zoogonoides viviparus (Køie 1971) and Zygocotyle lunata (Irwin et al. 1991). Secretory vesicles probably contribute to the formation and maintenance of the glycocalyx (Bogitsh 1968; Shannon and Bogitsh 1971). Others authors have suggested that they are involved in maintenance of the outer membrane or in immunoprotection (Lumsden 1975; Hanna 1980a, b). Secretory vesicles pass from cytons to the distal cytoplasm with the use of cytoplasmic bridges such as for B. anguillae (Filippi et al. 2010) and S. mansoni (Hockley and McLaren 1973).
Large secretory granules enclosed by microtubules in gland cells are common in the D. inflata tegument. Similar structures were also recorded in the tegument of other trematodes (Halton and Dermott 1967; Halton and Lyness 1971; Swarup and Jain 1984; Filippi et al. 2010) and other flatworms (Jones and Beveridge 1998). According to Whittington and Cribb (2001), similar secretions are may be involved in adhesion to the host mucosa.
The basal matrix of the tegument of D. inflata was observed infolded into the interstitial matrix. Generally, such structures are considered to be characteristic of epithelia specializing in active transport (Halton 1972). It is accepted that many of the cells of the flatworms are connected physically to the ECM through intercellular junctions (Conn 1993). Thus, basal and interstitial matrices make part of the general structure described for the tegument of digeneans (Threadgold 1984; Conn 1993; Roberts and Janovy 2000). ECM, such as basal or interstitial matrices, are thought to be involved in skeletal support, nutrient storage, motility, transport, oxygen storage, and may be a source of undifferentiated cells for regeneration. It is also accepted that it can be a modified tissue for morphogenesis and that structural interactions with other tissues can occur (Conn 1993). Cytoplasmic bridges occurring through the ECM and connecting the cytons to the distal cytoplasm were observed in the integument of D. inflata as for the general tegumental structure described for digeneans (Hockley and McLaren 1973; Bennett and Threadgold 1975; Threadgold 1984; Roberts and Janovy 2000).
In the tegument of D. inflata, as for other digeneans, cytons of the syncytium occur in the nucleated region of the tegument below the ECM (Morris and Threadgold 1968; Bennett and Threadgold 1975; Lumsden 1975; Threadgold 1984; Roberts and Janovy 2000; Paperna and Dzikowski 2006). Thus, the cytons can avoid any adverse influence of the host, thereby permitting regional differentiation and specialization (Halton and Johnston 1982).
The shape and distribution of tegumental spines are related to the size or shape of worms and their migration pattern (Lee et al. 1985, 1987). D. inflata spines are thought to be an intermediate form between the tegumentary spines of acanthostome parasites of fish and the tegumentary spines of echinostome parasites of birds (Dezfuli et al. 1997). Dezfuli et al. (1992) also observed a decrease in spine density in the ventral part of the small portion between the two suckers, while the lateral spines of this region are bigger and stockier than any other spine observed on the entire body surface. However, in their previous study, Dezfuli et al. (1992) observed two rings of circumferential spines around the ventral sucker when we only found one. The spines they observed were also slightly shorter (6 μm) than those observed in the present study (8 μm). Circumoral rings of spines are widely spread and typical for parasites of fish (Yamaguti 1971). However, Jones et al. (2005) mentioned the absence of circumoral rings of spines in their key to the determination of the genus Deropristis while other authors recovered such structures (Cable and Hunninen 1942; Dezfuli et al. 1992, 1997). Spines with a crenelated tip were recovered over the posterior extremity of the body of D. inflata. Similar spines were also recovered on the body surface of Karyakartia egyptensis (Abdou 2008). Differences in spine structures and their distribution over the body may involve a significant difference in host–parasite relationships (Chai et al. 2000). Køie (1977) suggested that large pointed spines may be involved in feeding, with spines acting as abrasive structures for host tissue. In contrast, Senft et al. (1961) and Lumsden (1975) opined that spines aided in attachment to the host tissue. Some authors also argued that spines could operate in locomotion (Bennett 1975b; Ursone and Fried 1995). TEM micrographs of D. inflata revealed increased opacity at the base of the spines. These are thought to be involved in spine motility (Bennett and Threadgold 1975; Abbas and Cain 1987; Ferrer et al. 2001). Dezfuli et al. (1992) showed that the presence of spines on the body surface of D. inflata increase the damage to intestinal folds of the eel. Thus, damages to the mucosal structure can affect the absorptive efficiency of the eel’s alimentary canal making D. inflata responsible for a physiological dysfunction of the A. anguilla digestive tract. No spines were recovered on the ventral sucker of D. inflata. According to Bakke (1976) and Bennett (1975b), the absence of spines may be less irritating for the host mucosa. The decrease in spine density on the non-feeding sucker (the oral sucker encircled the mouth here) may be related to a specialization of the area which facilitates attachment to the host tissue without causing damage (Pandey and Tewari 1984).
The tegument of D. inflata bears numerous sensory papillae scattered over the body. Sensory receptors are common and varied among digeneans. These structures have been studied in species such as B. anguillae (Filippi et al. 2010), Clonorchis sinensis (Fujino et al. 1979a), Cryptocotyle lingua (Køie 1977), F. hepatica (Bennett 1975b), Gorgoderina vitelliloba (Hoole and Mitchell 1981), H. continua (Hong et al. 1991), and L. musculus (Filippi et al. 2012). According to Halton (2004), these structures represent the terminations of fine nerve processes that extend from the peripheral nervous system to the base of the body surface layer.
Similar sensory receptors structures of some other digeneans as seen in TEM
Žd’árská and Nebesářová (2004)
Filippi et al. (2010)
Hoole and Mitchell (1981)
Fujino et al. (1979a)
Among Digenea, intra-tegumental (or non-ciliated) sensory structures have been observed using SEM or TEM in adults of Brachylaimus aequans (Žd’árská et al. 1990; Žd’árská 1993), C. sinensis (Fujino et al. 1979a), Echinostoma paraensei (Maldonado et al. 2001), F. hepatica (Bennett 1975b), Gorgoderina attenuata and Gorgoderina bilobata (Mata-López and Leon-Regagnon 2006), G. vitelliloba (Hoole and Mitchell 1981), Hasstilesia tricolor (Crites and Jilek 1981), Heterophyes nocens (Chai et al. 1992), H. continua (Hong et al. 1991), Himasthla alincia (Han et al. 2003), K. egyptensis (Abdou 2008), L. musculus (Filippi et al. 2012), Leucochloridium sp. (Bakke 1976), Metagonimus miyatai (Chai et al. 1998), Metagonimus takahashii (Chai et al. 2000), Nicolla skrajbini (Moravec 2009), Paragonimus westermani (Choi and Yoo 1985), Philophthalmus megalurus (Edwards et al. 1977), Pygidiopsis summa (Chai et al. 2002), and Transversotrema licinum (Abdul-Salam and Sreelatha 1992). Similar receptors have also been recovered in aspidogastreans (Halton and Lyness 1971; Ip and Desser 1984; Rohde and Watson 1990; Gao et al. 2003) and paramphistome digeneans (Dunn et al. 1987; Brennan et al. 1991; Irwin et al. 1991; Mattison et al. 1994). Sensory receptors with the same external morphology of the type 2 observed here had also been recovered in other trematodes such as G. attenuata (Nadakavukaren and Nollen 1975), L. musculus (Filippi et al. 2012), Phyllodistomum umblae (Bakke and Bailey 1987), Stephanostomum egypticum (Abdou and Ashour 2000), and T. licinum (Abdul-Salam and Sreelatha 1992). Hoole and Mitchell (1981) recovered domed papillae on the ventral surface of G. vitelliloba. These papillae are very similar in morphology and ultrastructure to the type 2 sensory receptor of D. inflata as they have the same electron-dense structure contained in a nerve bulb filled with mitochondria. A similar electron-dense structure was also observed on the ventral sucker of newly excysted juveniles of C. sinensis by Fujino et al. (1979a). Moreover, light microscopical observations on the aspidogastrean Multicotyle purvisi have revealed the presence of a centrally situated mass inside the bulbous extension of the types E and I papillae (Rohde 1966). This central mass, which stained intensely with urea-silver nitrate, may be comparable to the electron-dense structure recovered within the type 2 sensory receptor of D. inflata. The absence of connection to the exterior environment observed for the type 2 sensory receptor probably restrains its function as chemoreceptor. It may be specialized to respond to mechanical stimuli (Ip and Desser 1984). According to Žd’árská and Nebesářová (2003), the lack of connection to the surface indicates that non-ciliate receptors are possibly mechanoreceptors, serving to respond to compression. Several authors suggest that domed or bulb-like papillae may function as contact receptors (Bakke 1976; Fujino et al. 1979a; Hoole and Mitchell 1981). This type of sensory receptor, mostly distributed on or around the oral or ventral suckers of digeneans, is thought to be helpful to the fluke in locating surface for attachment (Abdou 2001). This position brings it into contact with the plug of host tissue formed by the fluke at the attachment site (Hoole and Mitchell 1981; Ashour et al. 1994; Ashour 1995). As for C. metoecus (Žd’árská and Nebesářová 2003), type 2 sensory receptors of D. inflata are close to the receptors of adult aspidogastreans.
Cytoplasmic ridges were observed surrounding both type 1 and type 2 sensory receptors. These ridges, or infoldings, indicate that sensory receptors may be subject to muscular movement and have a function in contact communications, as does the regional aggregations of these receptors around the oral sucker (Bakke 1976). Some authors indicated site-specificity in the distribution of sensory receptors (Bogea and Caira 2001; Abdul-Salam and Sreelatha 2004; Mata-López and Leon-Regagnon 2006). Most of the receptors observed on the D. inflata tegument were concentrated in the anterior third of the fluke, which contains oral and ventral suckers. Thus, the distribution of the sensory receptors relates well to the behavior of the worm because the parasite tends to attach to the host via the ventral sucker and probes the near environment with the oral one (surrounding the mouth).
We are grateful to Mr. Jean-Louis Guaitella and Mr. Louis Tarallo, fishermen of the Urbino pond, for providing us the eels. The experiments comply with the current laws of the country in which they were performed.