Assiut Vet. Med. J. Vol. 60 No. 141 April 2014 LIGHT AND SCANNING ELECTRON MICROSCOPY ON MONOGENEAN PARASITE FROM RED SEA FISH NEMIPTERUS JAPONICUS (TRIEADIN BRAENS) IN EGYPT SAMAR W. TADROS*; AMAL K. EL-GAYAR** and GHADA M. MOHAYEMEN*** * Animal Health Research Institute, Port Said branch, Parasitology Unit. ** Department of Parasitology, Faculty of Veterinary Medicine, Suez Canal University *** Animal Health Research Institute, Fish Disease Department, Fish Parasites Unit. Email: [email protected] ABSTRACT During a survey from November 2012 till October 2013 on a marine fish’s species Received at: 26/2/2014 Nemipterus japonicas, a microcotylid monogenean species Lutianicola haifonensis was recorded for the first time from 42 out of 80 (52.5%) examined Accepted: 3/4/2014 fish’s species in Red Sea. Nemipterus japonicas were found to be recorded as a new host for both the monogenea and crustacean parasite species. A copepod crustacean, Hatschekia gracilis also recorded for the first time from 16 out of 80 (20%) of N. japonicas fishes. A brief description for Lutianicola haifonensis and Hatschekia gracilis was presented. Scanning electron microscopy (SEM) for L. haifonensis is studied. Key words: Monogenea- marine fish- electron microsope- Nemipterus japonicus (Trieadin braens), Microcotylid, Hatschekia INTRODUCTION diagnostic and taxonomic characters (Ramasamy et al., 1995; Ramasamy and Brennean 2000; Marine fisheries are of lesser production to Egypt Bayoumy et al., 2007 and Yoon et al., 2013). than its major freshwater fisheries and aquaculture industries, and represent only about 17 % of total fish Nevertheless, different species of Monogenea may production in Egypt. However, of the marine present ultrastructural variations of the tegument, fisheries, approximately 55 % of the total marine fish such as presence of microvilli and secretory pores and catched comes from the Red Sea and Gulf of Suez a wide variation in morphological aspect of surface (FAOLex, 2004). Most studies of monogenean modifications and sensory structures (Smyth and parasites in Egypt were carried out on freshwater Halton 1983). Therefore, it is important to investigate fishes (Khidr, 1997; El-Naggar et al., 2001; El- the tegument of each individual species in order to Naggar & El-Abassy, 2003). Little attention has paid elucidate questions on maintenance and survival of to parasites of marine fish, especially monogenetic the parasite on their microhabitat. In Microcotylidae, trematodes except that carried by Paperna the tegument of Atriaster sp. and Microcotyle sp. has (1972a,b,c); Bayoumy, (2003), Bayoumy et al. (2007) been studied by transmission electron microscopy and Bayoumy and Abd El-Monem (2011). During a (TEM) (Justine and Bonami 1993). The gills are a recent assessment of Red sea fish species by the favorite site for the attachment of several parasitic animal Health Research Institute (Port-Said branch), a copepods. They damage the gills by feeding on the samples of Nemipterus japonicas (Trieadin braens) delicate tissue of the gill lamellae or on the blood was found to be infected with a microcotylid circulating within the lamellae, leading to a loss of monogenean parasite, whose morphology is respiratory surface area (Pillai, 1985; Lester and consistent with a Microcotyle omanae, which was Hayward, 2006; Purivirojkul and Areechon, 2008). firstly described from the gill of Cheimerius nufar Lutianicola haifonensis species is described for the from Arabian Sea (Machkewskyi et al., 2013). The first time from Nemipterus japonicus (Trieadin Microcoylidae is the largest family in monogenean braens) in the Al Ghardaqa, Red Sea, Egypt. The subclass Oligonchoinea Bychowsky, 1937. At the copepod family Hatschekiidae Kabata, 1979 consists present some 150 species belonging to more than 40 of eight genera known as gill parasites of marine genera were counted within this family. However, the teleost fishes (Boxshall and Halsey 2004). Of these, specific composition and status of some genera Hatschekia Poche, 1902 is the largest genus of the remain obscure up to now (Mamaev, 1986). family, containing 97 valid species found on the gills of nearly 140 actinopterigian fish species (Uyeno and Scanning electron microscopy provide resolution at Nagasawa 2010). high magnification, which has been exploited in helminthology to elucidate and describe the MATERIALS and METHODS specialized organs of attachment, the position of oral 80 specimens of Nemipterus japonicus, (Trieadin and genital apertures considered as additional braens), 30–34 cm in total length, and weight 300- 54 Assiut Vet. Med. J. Vol. 60 No. 141 April 2014 500 gm were caught in the Red Sea off al- Ghardaqah level of the germarium. No terminal anchors present. (27°15′28″N 33°48′42″E) Red sea Governorate, from Paired muscular, unarmed, septate buccal organs (Fig. November 2012 till October 2013 and identified Ι, 2). Oesophagus simple without diverticula. using Randall (1995) and Abd-essalaam (1995). The Oesophagus 134 µm (100–175 µm) long. Intestinal fish specimens were transported to the Laboratory of caeca terminates post-testicular in the narrow animal Health Research institute (Port-Said branch) peduncle leading to the haptor. Genital atrium in an ice box to be examined. The gills removed in muscular, unarmed 85 µm (75-90 µm) wide, 84 µm small Petri dishes containing 0. 9% NACL to get rid (75–100 µm) long at its base; projects away from the excess mucus. For light microscopy, the isolated body as a small cone. Two large, paired, muscular, parasites were fixed in 5% formal saline and washed dorsal, vaginae, unarmed. Large, fusiform, with distilled water to remove excess fixative. For operculated eggs (275-310) µm long, 85-108 µm staining acetic acid alum carmine was used according wide (present in 4 specimens). Very long, tangled, to (Carleton, 1967) for 10-30 minutes. Dehydration apical filament ~1060 µm long, which tapers towards was maintained by passing in ascending grads of its extremity, the terminus of which is not thickened; ethyl alcohol, cleared in clove oil and mounted in posterior filament 110 µm long (Fig. Ι, 3). Testes Canada balsam. All dimensions are given in numerous, post-ovarian 42 µm (45–55) in number, micrometers as the mean, with the range and number situated in post-ovarian intercaecal field, not of measurements in parentheses. extending into the haptoral penduncle. Conspicuous Y-shaped vitelline duct, irregular, well defined, dark- Parasites specimens for SEM 10 parasitic fish specimens were used in this study. brownish in coloration, co-extensive with intestinal Worms were collected and prepared for SEM as caecae, extending from the genital atrium to mid-way follows. Living specimens were kept for 30 minutes along the haptoral peduncle (Fig. Ι, 4), branches 191 in the refrigerator before fixation in 4% aqueous µm (185–200 µm) long, the posterior piece 242 µm gluteraldehyde solution at 4°C for 48 hrs. (Halton, (230–255 µm) long, leads into the genito-intestinal 1979). Then the specimens washed thoroughly with canal. Haptor symmetrical, with numerous clamps cacodylate buffer and post fixed for 4 hrs. With arranged in two equal rows of approximate equal size aqueous osmium tetroxide (OsO4), dehydrated containing 98-135 of clamps arranged in two equal through acetone, dried in Polaron Equipmement, rows (Fig. Ι, 5a). Clamps, of approximate similar E300 critical point drying apparatus using liquid shape but size slightly dissimilar; clamps develop in a posterior to anterior direction (Fig. Ι, 5b). CO2, mounted on aluminum stub with double phase sticker. The specimens then coated with gold- Scanning electron microscopy: palladium in an E5000 sputter coating unit (Polaron The body of Lutianicola haifonensis is elongated, Equipment) coating unit and examined in a Joel SEM with a posterior haptor (Fig. Π, 6). The buccal cavity T330 scan-electron microscope operating at 20 Kev. and the opening of the genitalatrium lie in the anterior For copepods collection: part of the ventral surface (Fig Π, 7). The tegument of All gill arches from both sides were removed one by the body presents regular intervals, forming folds in one by cutting them at their extremities and examined the ventral and dorsal surfaces (Fig. Π, 7). It presents immediately in seawater. Parasites were collected microvillous-like tegumental projections, covering under a binocular microscope. both ridges and folds (Fig. Π, 8). Isolated, papillae were observed irregularly distributed all over the Copepods were removed with fine forceps or with the body surface (Fig. Π, 8). The tegument around the help of a fine needle, or brush and immediately fixed opening of genital atrium is folded, and contains in 70% ethanol. Copepod identification was based on microvillous-like projections and non-ciliated sensory morphological features according to Yamaguti papillae (Fig. Π, 8). (1963); Kabata (1979); Pillai (1985). The haptor is separated from the body proper at the RESULTS posterior region. It is triangular and composed of two symmetrical rows of clamps, formed by two 1- Lutianicola haifonensis opposable hinged jaws and covered by a thin Class: Monogenea Carus, 1863 tegument. The internal margins of the clamps are Family: Microcotylidae Taschenberg, 1879 serrated and, depending on state of contraction, may Subfamily Microcotylinae Monticelli, 1892 be opened or closed (Fig. Π, 9). The haptoral Site: Gill of 50 fish specimens out of 80 (62%) tegument is similar to the rest of the body, differing Host: Nemipterus japonicus (Trieadin braens) only in the fact that the microvillous- like projections Locality: al- Ghardaqah (27°15′28″N 33°48′42″E), are absent. Red sea Governorate 2- Copepods Light microscopy Hatschekia gracilis Yamaguti, 1954 Body; lanceolate, elongate (Fig Ι,1) body Habitat and locality. Gill of Nemipterus japonicus length,4,400-5,900 µm, haptor length, (2750–7375 (Trieadin braens) , Red Sea µm); 585 µm (275–775 µm) in maximum width at the Material. Five mature females. 55 Assiut Vet. Med. J. Vol. 60 No. 141 April 2014 Body slender, 1.9-2.1 mm long (Fig. ΠΙ, 10, 11). shaped (Fig.