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Research Article Histological and Histochemical Studies of Digestive Iran. J. Ichthyol. (June 2020), 7(2): 125-135 Received: February 14, 2019 © 2020 Iranian Society of Ichthyology Accepted: March 14, 2020 P-ISSN: 2383-1561; E-ISSN: 2383-0964 http://www.ijichthyol.org Research Article Histological and histochemical studies of digestive system in the megrim, Lepidorhombus whiffiagonis (Teleostei: Scophthalmidae) Martina BEBIĆ1, Nives KEVIĆ1, Ivana RESTOVIĆ2, Mate ŠANTIĆ1, Ivana BOČINA*1 1Department of Biology, Faculty of Science, University of Split, Ruđera Boškovića 33, 2100 Split, Croatia. 2Faculty of Humanities and Social Sciences, University of Split, Poljička cesta 35, 21 000 Split, Croatia. *Email: [email protected] Abstract: The digestive system of Lepidorhombus whiffiagonis was studied using histological and histochemical techniques. The wall of oesophagus and stomach fundus consisted of four histological layers: mucosa, submucosa, muscularis and the outer layer. Mucosa, the innermost layer, usually consisted of three different layers, including epithelium, lamina propria and lamina muscularis mucosae. Submucosa of connective tissue with blood vessels and nerve fibres underlined the mucosa and usually protrudes into the mucosal folds except in the gallbladder where it is completely absent. The muscular layer of the digestive system consisted of smooth muscles arranged in longitudinal, circular or oblique layers. Striated fibres were found in the oesophageal wall. The outermost layer comes as tunica adventitia in upper parts and tunica serosa in lower parts of the digestive system. Hepatocytes were surrounded by a mass of fat-storing cells. Serous acini of pancreas were scattered down the length of the stomach. Alcian blue/PAS technique revealed that alkaline and neutral mucopolysaccharides were present in oesophagus while acidic mucopoly- saccharides was found in the stomach and intestines. Keywords: Histoarchitecture, Histochemistry, Pleuronectiformes, Adriatic Sea. Citation: Bebić, M.; Kević, N.; Restović, I.; Šantić, M. & Bočina, I. 2020. Histological and histochemical studies of digestive system in the Megrim, Lepidorhombus whiffiagonis (Teleostei: Scophthalmidae). Iranian Journal of Ichthyology 7(2): 125-135. Introduction commercial value, the megrim was an important The megrim, Lepidorhombus whiffiagonis object of several research studies concerning its age (Walbaum, 1792) (Pleuronectiformes) is a benthic and growth (Landa & Piñeiro 2000; Šantić 2013), flatfish, widespread in the eastern part of the Atlantic distribution and abundance (Sánchez et al. 1998), from Scandinavia and Iceland to the Western Sahara exploitation (Fahy & Fannon 1992), feeding habits and in the Mediterranean, mainly the central and (Šantić et al. 2009), population differentiation western part. It is also distributed through Adriatic (Danancher & Garcia-Vazquez 2009). The spatial Sea, except in the northernmost part, most numerous variations in life history characteristics of megrim in the Middle and Southern Adriatic, inhabiting were also considered, as well as differences in habitat sandy and sandy-moody bottoms, between 20 to 360 selection of male and female individuals (Gerritsen meters in depth, rarely deeper, mostly 250m (Jardas et al. 2010). 1996). Megrim is caught by bottom-travelling and it The megrim is a predatory carnivorous fish, is considered as palatable and economically whose diet is dominated by fish, cephalopods and important food species. France and Spain are the decapod crustaceans (Jardas 1996). Its prey also largest consumers of this species with most of the includes four large groups: Teleostei, Decapoda, megrim caught in British waters being exported to Mysidacea and Cephalopoda. Teleosts are the most these nations (FAO 2018). Due to its widespread and important prey of larger fishes, while Mysidacea 125 Iran. J. Ichthyol. (June 2020), 7(2): 125-135 constituted the main prey for smaller fishes. At the Nevertheless, the morphological studies could species level, the teleost ntonogadus megalo- help to better understand the relationship between kynodon and decaspods Processa canaliculate physiological and biochemical functions and constitute the most frequent prey (Šantić et al. 2009). molecular mechanisms (Kalhoro et al. 2018). Even in The digestive system of fish has shown a aquaculture, anatomical and histological remarkable diversity of morphological and functional characteristic of the fish digestive system could help features, particularly in connection with special to elucidate the development of pathological nutritional behaviour, body shape and sex (Kapoor et conditions, to improve nutritional formulations and al. 1975; Díaz et al. 2008). In fish, as in other thus promote the feed management (Kalhoro et al. vertebrates, the digestive system is a tube of variable 2018). diameter which is longitudinally divided into the In view of the paucity of knowledge on the oesophagus, stomach, intestines, and rectum (Treer et histophysiology of the digestive system of al. 1995). The tongue and the teeth in the oral cavity L. whiffiagonis in relation to their food and feeding are also included as associated organs in the digestive habits, the aim of the present study was to elucidate system, as well as the extramural digestive organs: the histological structure of the digestive system as liver, gallbladder and pancreas. The salivary glands well as the histochemical nature of the mucins are generally missing in the oral cavity of fish (Treer secreted by the epithelial mucous cells in the gut of et al. 1995). From the cranial end of the oesophagus this fish. to the caudal end of the rectum, the wall of the alimentary canal consists of four distinctive layers. Materials and Methods Starting from the lumen, these layers are: mucosa, A total of five adult L. whiffiagonis individuals of submucosa, muscularis externa and serosa or average length 20.9cm and average weight 73,574 g adventitia (Kierszenbaum & Tres 2012). Although were sampled to study the histology and the general plan of digestive system histology in histochemical characterization of the alimentary fishes is very similar to those of other vertebrates, tract. The specimens were collected in the southern however, it shows noticeable diversity in its part of the Adriatic Sea, in May 2017. Immediately morphology and function related to both taxonomy after collecting the parts of digestive tube and different feeding habits (Al Abdulhadi 2005). (oesophagus, stomach, intestines, liver, pancreas and Mucosal epithelium of the alimentary tube in fishes gallbladder) were fixed in 10% formalin. is usually provided with mucous cells which show Histological study: After two days of fixation, the different histochemical features. These features tissues samples for histological study (oesophagus, depend on different content of muccopolysaccharides stomach, intestines, liver, pancreas and gallbladder) (MPS) secreted by these cells which is in correlation were dehydrated in an ascending series of ethanol, with species, age, regions of the alimentary tract and starting from 70% ethanol. Tissue samples were then feeding habits of the fish (Park & Kim 2001, cleared with xylene and embedded in paraffin. Sarasquete et al. 2001; Cao & Wang 2009). In most Tissues sections were cut transversally using Microm fishes, the intestinal mucous cells contain acidic MPS microtome at 6µm and mounted on glass slides. The (Cataldi et al. 1987; Al-Abdulhadi, 2005; Bočina et sections were deparaffinised with xylene and stained al. 2016). It seems that the quality and quantity of the with haematoxylin-eosin staining to present the main mucus from the intestinal goblet cells could also be morphological features of the mentioned organs of correlated to environmental pollution (Ferrando et al. the megrim’s digestive system. The sections were 2006). observed using an Axioskop Zeiss light microscope 126 Bebić et al.- Histological and histochemical studies of digestive system in the megrim Fig.1. Anatomy of the megrim’s digestive system. (A) s, stomach; l, liver, (B) o, oesophagus, (C) o, oesophagus; s, stomach; l, liver. and (D) s, stomach; l, liver; i, intestines; gb, gallbladder. and photos were taken under Sony DSC-S75 digital could be distinguished in the oesophageal wall of camera (Sheehan & Hrapchak 1980). megrim: mucosa (tunica mucosa), submucosa (tunica Histochemical study: Alcian blue/ PAS staining was submucosa), muscular layer (tunica muscularis) and used to establish the histochemical nature of mucins outer layer (tunica adventitia). The mucosa is secreted by the epithelial mucous cells in megrim’s innermost, thin layer, which formed high folds alimentary canal. The sections were deparaffinised deeply protruding into the oesophageal lumen (Fig. first and then were brought to distilled water through 2A). It is lined with a simple columnar epithelium downgraded ethanol series and oxidized in 1% containing microvilli (Fig. 2B). The epithelial cells aqueous periodic acid solution for 10-15 minutes at were high with centrally positioned nuclei. The room temperature. After washing in distilled water, numerous, big, bubble-shaped goblet cells were the sections were immersed in Schiff’s reagent for 45 scattered among columnar epithelial cells (Fig. 2A min and washed again in water. The sections were and 2B). When treated with PAS/AB, apical parts of then immersed in 1% AB in 3% acetic acid (pH 2.5) epithelial cells stained purple, indicating the presence for 45 min. Then the slides were finally washed in of alkaline and neutral mucopolysaccharide (Fig. running tap water,
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