Light and Scanning Electron Microscope Studies Of

Proc. Helminthol. Soc. Wash. 55(2), 1988, pp. 224-228 Light and Scanning Electron Microscope Studies of Fundulotrema prolongis (Monogenea: Gyrodactylidea) Parasitizing Fundulus diaphanus (Cyprinodontidae) in Nova Scotia, Canada, with an Emended Diagnosis of Fundulotrema DAVID K. CONE' AND PAUL H. ODENSE2 1 Department of Biology, Saint Mary's University, Halifax, Nova Scotia, Canada B3H 3C3 and 2 National Research Council of Canada, Regional Laboratory, Nova Scotia, Canada B3H 3Z1 ABSTRACT: The haptor and peduncle of Fundulotrema prolongis (Hargis, 1955) parasitizing the body surface of Fundulus diaphanus in Nova Scotia is examined with light and scanning electron microscopy. The study shows that this attachment organ is almost rectangular in ventral view, with marginal hooks I-III in 2 anterolateral groups set away from the posteriorly located hooks IV-VIII. The so-called peduncular bar is an apparently static, V-shaped sclerite positioned with its apex directed anteroventrally beyond the level of the surrounding tissue. The bar is covered by a smooth spineless tegument and may have its origin within this outer layer. In unextended animals the lateral regions of the bar can rest on the well-developed hamulus roots and may serve as a stabilizer or physical stop for the hamuli. The bar is not a supplementary organ of attachment and thus is functionally different from the prehensile squamodisc of diplectanid monogeneans. The study provides an emended diagnosis for the genus Fundulotrema. KEY WORDS: SEM, Monogenea, Fundulotrema prolongis, Fundulus diaphanus, Gyrodactylidea, killifish. Scanning electron microscopy (SEM) has been follows the system proposed by Llewellyn (1963) be- used successfully to study the haptor of species cause of its consistency in an evolutionary context. of Gyrodactylus Nordmann, 1832 (Monogenea: Gyrodactylidea) (Hawkes, 1977; Ergens, 1983; Results Kayton, 1983;LinnenbachandHausmann, 1983; Malmberg, 1983; Cone and Odense, 1984; Cone The haptor of F. prolongis is almost rectan- and Cusack, 1988). In the present study we use gular in shape and constitutes lh of the total body the technique along with light microscopy for length in relaxed specimens (Fig. 1). studying Fundulotrema prolongis (Hargis, 1955) SEM enabled the relative positions of the scler- Kritsky and Thatcher, 1977, the type species of otized components, seen in flattened, glycerine- a closely related but little-studied genus. mounted preparations (Fig. 2), to be visualized in 3 dimensions (Fig. 3). The pair of hamuli are splayed laterally with the bare blades of each Materials and Methods directed anteriorly and parallel to the ventral surface of the body. Two distinct muscular sheaths Samples of banded killifish (Fundulus diaphanus), 3-5 cm long and lightly infected with F. prolongis, were enclose the well-developed anteroventrally di- collected in June 1986 from Porters Lake, Nova Scotia rected hamulus roots (Fig. 3). Full outlines of the (44°45'N, 63°18'W). Several hundred fish were held in ventral bar and associated anterolateral process- a 200-liter aquarium supplied with 15°C dechlorinated es and posteriorly directed membrane are visible tapwater. The fish were fed tropical fish food ad libi- tum. After 2 mo the intensities of F. prolongis increased beneath a taut tegument surface (Fig. 3). The 16 in some cases to 500 worms per fish. Infected fish were marginal hooks are peripheral on the haptor with frozen by immersion in liquid Freon 22 and freeze- ventromedially directed sickle blades; pairs I-III dried overnight. Dried parasites were mounted ventral are in 2 anterolateral groups; pairs IV-VIII form side up on SEM stubs prior to sputter coating with gold an evenly spaced posterior group (Figs. 1-3, 5). and examination with a JEOL 35 scanning electron microscope. Infected fish were also embedded in Para- An apparently static peduncular bar, covered plast for histological study; sections were stained in by smooth tegument, is present immediately an- hematoxylin and eosin. Live parasites were fixed in 5% terior to the haptor (Figs. 2, 3). Histological sec- formalin and mounted unstained in glycerine jelly for tions reveal that it is V-shaped in cross section light microscope studies. Live specimens were studied in wet mounts. Identification of the parasites was con- (Fig. 4), and, like the ventral bar, is intensely firmed by examination of type material (USNM Helm. eosinophilic. It appears to lie within the ventral Coll. No. 49331). Numbering of the marginal hooks tegument, protruding anteroventrally beyond the 224 Copyright © 2011, The Helminthological Society of Washington 225 surrounding tissue. In nonextended specimens the lateral regions of the bar are positioned immediately above or directly on the underlying hamulus roots. Fundulotrema prolongis attaches to the host by means of the blades of the marginal hooks that impale epithelial cells on the skin surface. The 3 pairs of hooks grouped anteriorly maintain an anterior position when adhered to host tissue (Fig. 5). The skin beneath the haptor is com- pressed and occasionally pierced by the hamulus blades. Histological sections revealed that the peduncular bar does not pierce or touch the skin surface during normal attachment. Discussion The haptor of F. prolongis is extended anteriorly to accommodate the unusually long hamulus roots and thus takes on an almost rectangular outline in ventral view. This development appears to have left marginal hooks I-III in 2 anterolateral groups set away from the posterior ones. The haptor has a distinct peduncular bar. This sclerite has been described as an anteriorly directed skirt with sclerotized points (Hargis, 1955; Beverley-Burton, 1984) and as a transverse peduncular bar with small oblong pits (Williams and Rogers, 1971). What is seen with light microscopy as a skirt is actually the outline of the V-shaped bar protruding anteroventrally beyond the surrounding tissue. The bar does not have spines but is pitted at its apex. In unextended specimens the bar is positioned in such a way that its lateral regions could exert passive pressure on the roots of the hamuli. In this position it might stabilize these sclerites while allowing greater freedom of movement for the Figure 1. Scanning electron photomicrograph of forebody. It might serve as a physical stop pro- Fundulotrema prolongis from the skin surface of Fun- tecting the soft peduncular tissue from potential dtiIns diaphanus. Ventral view. Scale bar is 100 /tm. damage of any dorsally directed action of the well-developed hamulus roots. As a stop it might also facilitate removal of the hamulus blades from ded in the tegument, thus supporting a possible host tissue when required. The bar does not pen- tegumental origin (Kearn, 1968). Although ul- etrate adjacent host skin and does not supple- trastructural studies are needed to confirm the ment permanent attachment to the host. It there- relationship of the peduncular bar with the ven- fore differs functionally from the diplectanid tral tegument, our light microscopical study sug- squamodisc with which it might be confused (see gest that it lies within the tegument. If shown to Paling, 1966; Oliver, 1976). be so, the bar may represent a sclerotized tegu- In cross section (Fig. 4), however, the bar re- mental fold. sembles the V-shaped spines present on the squa- Fundulotrema prolongis attaches to the host modisc of Diplectanum aequans (Wagener, 1857). in a manner similar to Gyrodactylus avalonia Shaw (1981) showed that these spines are embed- Hanek and Threlfall, 1969, and Gyrodactylus co- Copyright © 2011, The Helminthological Society of Washington •-^.«%- •« "~ — ^•^^ —^ w— *£ »i~-*^^ - ^" ^. j** i* : •' » ^ ^Br>.Y"HR Figures 2-4. Photomicrographs of Fundulotrema prolongis. 2. Light photomicrograph of the haptor of F. prolongis showing the various sclerite components: peduncular bar (PB), hamuli (H), and ventral bar and membrane (VB). The marginal hooks are peripheral on the haptor, with 2 anterolateral groups set apart from the others. Ventral view. Glycerine-cleared specimen. Scale bar is 20 pm. 3. Scanning electron photomicrograph of the haptor of F. prolongis, showing the sclerites described in Figure 2 covered with a taut smooth tegument. Ventral view. Scale bar is 10 ^im. 4. Longitudinal section through an attached F. prolongis showing the relative position of the peduncular bar (PB) and 1 of the hamulus roots (HR). Note the bar is V-shaped in cross section. 20/im. 226 Copyright © 2011, The Helminthological Society of Washington 227 lemanensis Mizelle and Kritsky, 1967. All 3 species use the blade tips of the marginal hooks to pierce epithelial cells on the host surface and the hamulus blades for supplementary attachment (Cone and Odense, 1984; Cone and Cu- sack, 1988). The genus Fundulotrema was established by Kritsky and Thatcher (1977) to accommodate species of Gyrodactylinae Monticelli, 1892, pos- sessing a peduncular bar and a haptor with a ventral pair of hamuli and 2 support bars and 16 evenly spaced marginal hooks. The present study reveals that the marginal hooks in fact have an uneven distribution. Examination of type specimens of Fundulotrema foxi (Rawson, 1973), Fundulotrema megacanthus (Wellborn and Rog- ers, 1967), Fundulotrema stableri (Hathaway and Herlevich, 1973), and Fundulotrema tremato- clithrus (Rogers, 1967) confirms that marginal hooks I-III are grouped anteriorly in all known members of the genus. The above diagnostic correction does not jeop- ardize the taxonomic validity of Fundulotrema but simply ties the genus closer to Polyclithrum Rogers, 1967, and Swingleus Rogers, 1969. Swingleus has 2 bilateral winglike support plates, a peduncular bar, and the first 3 pairs of the 16 marginal hooks grouped anteriorly (Rogers, 1969). Polyclithrum has numerous ventral sup- Figure 5. Dorsal view of the parasite attached in port bars, no peduncular bar, and the first 4 pairs situ. Note that the 2 anterolateral groups of marginal hooks (arrows) retain their anterior position in attached of the 16 marginal hooks grouped anteriorly parasites. Scale bar is 20 ^im. (Rogers, 1967).

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