Journal of Coastal Life Medicine 2014; 2(12): 947-952 947 Journal of Coastal Life Medicine journal homepage: www.jclmm.com Document heading doi: 10.12980/JCLM.2.2014JCLM-2014-0090 2014 by the Journal of Coastal Life Medicine. All rights reserved. 襃 Digestive gland ultrastructure of the tunicate, Halocynthia roretzi (Ascidiacea: Pyuridae) in relation to function 1 1 1 2 2 Yun Kyung Shin , Jung Jun Park , Jeong-In Myeong , Mi Ae Jeon , Jung Sick Lee * 1Aquaculture Management Division, Aquaculture Research Institute, NFRDI, Busan 619-902, Republic of Korea 2Department of Aqualife Medicine, Chonnam National University, Yeosu 550-749, Republic of Korea PEER REVIEW ABSTRACT Peer reviewer Objective: To examine the glycHalocynthiaogen storag eroretzi and lipid digestion, as well as detoxification of the Roberto Najle, Doctor in Animal Science, dMethods:igestive g land of the tunicate, . University Professor, Researcher. Tunicates used in this study were collected from tunicate aquafarm located in Hansan Tel: 054-249-4439850 BResults:ay on the southern coast of Korea. Light and electron microscopy was performed. Fax: 054-249-4439850 The digestive gland was divided into the tubular and blind ampulla portions. Fine E-mail: [email protected] vacuolar granules were present, and lipofuscin granules were confirmed in the cytoplasm of the Comments epithelia of the blind ampulla portion. Epithelial cells of the blind ampulla portion could be cConclusions:lassified into three types. This is a research paper where the The results indicate that these three types of cells are involved in glycogen storage, authors characterize the cellular level lipid digestion and detoxification. (light microscopy) and ultracellular (transmission electron microscopy) epithelial cellsH. o f roretzithe digestive gland of the tunicate, and compared with results obtained in other species of ascidians, finding differences in the types of epithelial cells. Also describes some of the features found in the gland such as the storage of glycogen and lipid KEYWORDS digestion and detoxification. Halocynthia roretzi Details on Page 880 Detoxification, Digestive gland epithelia, Glycogen, , Lipid 1. Introduction on the digestive system of tunicate, most studies are on the pyloric gland. In all tunicates, there is a network of digestive Class Ascidiacea is a group of primitive chordates belonging glands on the outer walls of the intestine. The digestive to subphylum Urochordata, with approximately 2 000 species gland of tunicate is the organ that performs osmoregulation, distributed worldwide. They mostly have marine habitats, and digestion and excretory function[3]. Researches on the are filter feeders that live in a sessile state either alone or as digestive functions of the gland of tunicate have concentrated [1] [3-6] part of a colony during mBoltenia,ost of th eHalocynthia,ir adult stage Herdmania,. A total of mostly on the glycogen metabolism and storage . 16 5 ( Microcomus, species of Pyura genera However, the digestive gland of the tunicate has been ) of family Pyuridae of class Ascidiacea determined to perform lipid digestion and detoxification have been reported in Korea[2]. functions similar to those of the liver in vertebrates or Although there are not many ultrastructural researches the analogous organ of the hepatopancreas of aquatic *Corresponding author: Dr. Jung Sick Lee, Department of Aqualife Medicine, Article history: Chonnam National University, Daehak-ro 50, Yeosu 550-749, Republic of Korea. Received 17 Jul 2014 Tel: +82-1089103172, +82-616597172 Received in revised form 20 Jul, 2nd revised form 24 Jul, 3rd revised form 31 Jul 2014 Fax: +82-616597179 Accepted 2 Aug 2014 E-mail: [email protected] Available online 8 Oct 2014 Foundation Project: Supported by National Fisheries Research and Development Institute (RP-2013-AQ-117) grant funded by the Korea government. Yun Kyung Shin et al./Journal of Coastal Life Medicine 2014; 2(12): 947-952 948 invertebrates, thereby necessitating research on this aspect[3,7]. the specimens were washed with 0.1 mol/L phosphate buffer This research employed ultrastructural study to examine the 4 times for 2 h and dehydrated with graded series of ethanol. 812 glycogen storage and lipHalocynthiaid digestion ,roretzi as we lH.l a roretzis detoxification Specimens were embedded in Epon , cut to ultrathin of the digestive gland of ( ). sections (70 nm in thickness) and placed on copper grids (200 mesh) in order to double-stain with uranyl acetate and lead TEM (LIBRA 120 2. Materials and methods citrate. Specimens were examined using a , Zeiss, Germany). 2.1. Specimens 3. Results Tunicates used in this study were collected from tunicate 3.1. Light microscopical structure H B K aqu° af’arm ’l’ocated i°n ’ ans’a’n ay on the southern coast of orea (34 46 36.8 N, 128 28 13.7 E). Morphological characteristics of the specimens were measured by using vernier calipers and The digestive gland of the tunicate could be classified into electronic scale. Twenty specimens used in this study were tubular and blind ampulla portions. The epithelial layer was adults with body heights of 10.2 cm (body heights: 9.4-10.8 cm) a single layer, with the tubular portion composed of cuboidal (Figure 1A). Specimens for histological analysis were produced epithelia, while the epithelial layer of blind ampulla portion by extracting the digestive gland following removal of the tunic was also composed of columnar epithelia (Figure 2A). A striated (Figure 1B). border was developed on the free surface of the epithelial layer. As the result of AB-PAS (pH 2.5) reaction, while the Bw striated border of the tubular portion displayed a blue color, the striated border of the blind ampulla portion displayed a Bs A s red color (Figure 2A). The cytoplasm of epithelial cells of the blind ampulla portion contained fine vacuo’les that displayed negative reaction upon H & E stain, Masson s trichrome stain, Bh Dg T AB-PAS (pH 2.5) reaction and AF-AB (pH 2.5) reaction (Figures 2 2 ) P A- D . In addition, lipofuscin granules that reacted to crimson color in the Long-Ziehl Neelsen stain were observed in the S cytoplasm of some of the cells (Figure 2E). A B Figure 1. The external morphology and morphometric characteristics (A) and H. roretzi specimen sampling area (B) of the tunicate, . V As: Atrial siphon; Bh: Body height; Bs: Buccal siphon; Bw: Body width; P: Process; S: Stolon; T: Tunic; Dg: Digestive gland. 2.2. Light microscopy B Specimen preparation for light microscopy was performed [8] according to the methodology of Drury’ and Wallington . V Specimens were fixed in aqueous Bouin s solution and were μ rinsed in running water and then dehydrated through a graded C 25 m ethanol series (70%-100%). The specimens were then embedded El (M C USA) in paμraplast c ormick, , and subsequently sectioned at 4 6 (RM2235 L G ) - m thickness using a microtome ’ , eica, ermany . 0 5% Specimens were stained’ with Mayer s hematoxylin and . V eosin (H & E), Masson s trichrome stain, alcian blue and μ (AB PAS 2 5) 25 m periodic acid-Schiff solution - , pH . , and aldehyde D fuchsin-alcian blue (AF-AB, pH 2.5) reaction. Long Ziehl- Ba Neelsen stain was performed to confirm the presence of Sb Lg lipofuscin granules. T 2.3. Transmission electron microscopy (TEM) Lg μ μ 25 m A E 25 m Figure 2. S Light microscopical feature of the digestive gland of the tunicate, pecimen preparation for electron microscopy was performed H. roretzi according to the methodology of Cormack[9]. Specimens . were fixed in 2.5% glutaraldehyde solution (pH 7.2, 0.1 mol/ A: Digestive gland consists of tubule (T) and blind ampulla (Ba) portion, AB- ° PAS (pH 2.5) reaction; B-D: Small vacuoles (V) in the cytoplasm of epithelium, L ) 2 4 4 C 0 1 ’ phosphate buffer for - h at and rinsed in . mol/ B: H & E stain, C: Masson s trichrome stain, D: AF-AB (pH 2.5) reaction; E: 1% L phosphate buffer, after which they were p° ost-fixed in Lipofuscin granules (Lg) in the cytoplasm of epithelium, Long Ziehl-Neelsen osmium tetroxide (OsO4) solution for 2 h at 4 C. After fixation, stain. El: Epithelial layer; Sb: Striated border. Yun Kyung Shin et al./Journal of Coastal Life Medicine 2014; 2(12): 947-952 949 3.2. Transmission electron microscopical structure of the ciliated columnar epithelium (Figure 4C). Moreover, well-developed mitochondria, microsomes and glycogen As the result of TEM observation, epithelial cells of the granules were scattered throughout the basal cytoplasm of tubular portion were cuboidal shape with the development the other same shaped cells. Membrane interdigitations were of microvilli on the free surface. The cytoplasm of these cells well-developed on the lateral cell membrane near the basal contained lysosomes and vacuoles of a wide range of sizes membrane of these cells, and the infoldings were formed in filled with fibrous substances (Figure 3A). portions of the basal membrane (Figure 4D). M Mv C s Cv N Gc Ly G Gg c V Ly μ 1 m 500 nm μ A N B 2 m μ A Bm B 2 m Gl Gg C Mv Gg Mt D 500 nm Pv 500 nm E Mi Mt Mb Bm 500 nm μ lj C D 2 m μ 1 m 500 nm Figure 4. C F Transmission electron micrographs of mid and basal cytoplasm in Figure 3. H. roretzi Transmission electron micrographs of the digestive gland epithelia the digestive gland epithelium of the tunicate, . H. roretzi of the tunicate, . A: Golgi complex (Gc) and lysosomes (Ly) in the mid cytoplasm; B: Golgi A: Epithelia in the tubular portion; B: Ciliated columnar epithelia in the blind complex with condensed vesicle (Cv) and microsomes (Ms) in the mid ampulla portion; C: Free surface of ciliated columnar epithelium in the blind ( ) “ ” cytoplasm; C: Accumulated glycogen granules Gg in the basal cytoplasm; D: ampulla portion; D: Cross section of cilia (C) showing 9+2 microtubular Mitochondria (Mt), membrane interdigitations (Mi) and basal membrane (Bm) ( ) ( ) structure; E: Microvilli Mv covered with glycocalyx Gl ; F: Pinocytotic infoldings.