Digestive Gland Ultrastructure of the Tunicate, Halocynthia Roretzi (Ascidiacea: Pyuridae) in Relation to Function

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Journal of Coastal Life Medicine 2014; 2(12): 947-952 947

Journal of Coastal Life Medicine

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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. N: Nucleus. vesicles (Pv), multivesicular body (Mb) and intercellular junction (ij) in the apical cytoplasm of ciliated columnar epithelium. N: Nucleus; V: Vacuole; Ly: The non-ciliated columnar epithelium can be divided into Lysosome; Bm: Basal membrane; Gg: Glycogen granules; Mt: Mitochondria. two types according to the ultrastructural characteristics of the nucleus and the cytoplasm. The first type of the The epithelial layer of the blind ampulla portion is non-ciliated columnar epithelium has development of composed of columnar epithelia. However, these cells could microvilli on the free surface, and these microvilli were be divided into one type of ciliated columnar epithelium also covered with glycocalyx of high electron density. The and two types of non-ciliated columnar epithelium in nucleus was located in the basal area and had condensed accordance with the presence of cilia, electron density heterochromatin in a circular shape in the vicinity of the of cytoplasm, shape of the nucleus and ultrastructural karyomembrane (Figure 5A). The cytoplasm exhibited the characteristics of the cytoplasm (Figure 3B). development of oil droplets with a diameter of approximately μ The ciliated columnar epithelium displayed higher 1 m of low electron density, lysosomes, smooth endoplasmic electron density of the cytoplasm in comparison to the non- reticula and mitochondria (Figures 5B and 5C). ciliated columnar epithelium, with the development of cilia ( 3 ) Ld Mt and microvilli on the free surface Figure C . The cilia Mt “ ” Ly displayed a 9+2 microtubular structure in cross section sER (Figure 3D) and the surface of microvilli was covered with L glycocalyx of high electron density (Figure 3E). Numerous N d Ly N pinocytotic vesicles and multi-vesicular bodies were μ μ μ 1 m 1 m A 2 m B N C observed in the cytoplasm, in the vicinity of the free surface Figure 5. The first type of the non-ciliated columnar epithelium in the with scattered glycogen granules (Figure 3F). Multiple H. roretzi digestive gland of the tunicate, . G numbers of lysosomes and olgi complex were developed in A: Non-ciliated columnar epithelium with heterochromatin condensed the mid cytoplasm (Figure 4A), and condensed vesicles with nucleus (N) in the blind ampulla portion; B: Section showing the several high electron density existed in the boundaries of cisternae lipid droplets (Ld) and lysosome (Ly); C: Section showing the heterochromatin ( ) of the Golgi complex (Figure 4B). Glycogen granules of high condensed nucleus and well developed smooth endoplasmic reticula sER and mitochondria (Mt) in the cytoplasm. electron density were concentrated in the basal cytoplasm Yun Kyung Shin et al./Journal of Coastal Life Medicine 2014; 2(12): 947-952 950 H. The second type of the non-ciliated columnar epithelium longer[5,11]. As a result of this study, the digestive gland of roretzi also displayed microvilli development on the free surface could also be divided into the tubular and the blind like the first type. Although the nucleus is located in the ampulla portion. However, while the shape of the epithelium basal area, unlike the non-ciliated columnar epithelium in the tubular portion was cuboidal, three types of columnar of the first type, heterochromatin was scattered within the epithelia were confirmed in the blind ampulla portion, B. schlosseri nucleus (Figure 6A). Pinocytotic vesicles and multivesicular thereby displaying the difference between [10] D. grossularia body were observed in the cytoplasm in the vicinity of and [5]. free surface (Figure 6B). Lysosomes with fibrous substance Glycogen deposition, which is one of the digestion and multiple numbers of microsomes were observed in functions of the ascidian digestive gland, has been well- the mid cytoplasm (Figures 6C and 6D). Moreover, multiple researched in observation of the electronic microscopic B. schlosseri Styela clava D. numbers of Golgi complex, smooth endoplasmic reticula and structure of [10], [4] and grossularia lysosomes were distributed in the basal cytoplasm. These [5]. The common ultrastructural characteristics lysosomes contained characteristically homogeneous core of these gland epithelia included the development of cilia granules of high electron density (Figure 6E). and microvilli on the free surface, and the development of pinocytotic vesicles, lysosomes, endoplasmic reticula, Mv mitochondria and glycogen granules in the cytoplasm. In this study, the ultrastructural characteristics that were confirmed B. schlosseri Styela clava D. grossularia Pv Ms in the , and were confirmed in the ciliated columnar epithelium of the H. roretzi Mt digestive glands of . Accordingly, these cells Mb were determined to participate in glycogen deposition. In N N addition, the development of mitochondria and infoldings of

μ the basal membrane in the ciliated columnar epithelium of μ 2 m H. roretzi A 2 m B C 500 nm are determined to be structural characteristics that relate to the active transport of glycogen. Ly Mugnaini and Harboe reported that the digestive glands Ly Gc of ascidian have functional similarity to the liver of vertebrates[7]. Burighel and Cloney asserted that there is a Cg need to pursue further research on this aspect[3]. sER Liver cells of vertebrates synthesize triglycerides, μ 2 m μ D E 1 m cholesterol and phospholipids, and store them in the Figure 6. The second type of the non-ciliated columnar epithelium in the form of large lipid droplets in the cytoplasm. Such H. roretzi. digestive gland of the tunicate, ultrastructural characteristics of lipid synthesis are also A: Two non-ciliated columnar epithelia with heterochromatin scattered nucleus; B: Microvilli (Mv), pinocytotic vesicles (Pv) and multivesicular body displayed as numerous smooth endoplasmic reticula, (Mb) in the apical cytoplasm; C: Numerous microsomes (Ms) in the cytoplasm; mitochondria and large lipid droplets of low electronic ( ) D: Lysosome Ly of low electron density with fibrous substance; E: Golgi density[12]. Lipid storage and metabolic functions were complex (Gc), smooth endoplasmic reticula (sER) and several lysosomes with (C ) M M N N also reported in the digestive gland of marine mollusks. core granules g of high electron density. t: itochondria; : ucleus. Viviparus The digestive gland epithelium (digestive cell) of ater performs hexosaminidase activity, and displays an 4. Discussion ultrastructure which participates in lipid storage and metabolism, with the development of apical microvilli, The digestive glands of ascidian are structurally composed pinocytotic vesicles, rough endoplasmic reticula, Golgi of tubules and blind ampullae[3]. The microscopic structure complex, lysosomes, mitochondria and oil droplets[13]. of the digestive glands of ascidian has been described in the In addition, lipid digestion was confirmed in the Botryllus schlosseri B. schlosseri ( ). The epithelium of these cytochemistry and ultrastructure of the digestive gland Aplysia punctata glands is composed of a single cell type. The epithelium is epithelium of [14]. In the digestive glands Mytilus galloprovincialis cuboidal and has a few long cilia and numerous microvilli of , digestive cells (digestive gland Dendrodoa on the free surface[10]. The digestive glands of epithelium) which perform lipid digestive functions are grossularia D. grossularia ( ) are also composed of tubules distinguished. The digestive cells contain granules that β and blind ampullae, and, although the shapes of epithelia display positive reaction in N-acetyl- -hexosaminidase. are all columnar, the epithelia of the ampulla portion are Moreover, the digestive cell was confirmed to participate Yun Kyung Shin et al./Journal of Coastal Life Medicine 2014; 2(12): 947-952 951 H. roretzi in lipid metabolism through observation of Golgi complex, Three types of cells of the digestive gland of lysosomes, residual body and lipid inclusions under the are presumed to be involved in the glycogen storage, lipid electron microscope[15]. In this study, the presence of cells digestion and detoxification. The main functions of the three among the epithelial cells containing vacuoles that display types of cells in the digestive glands will differ according to ’ negative reaction in H & E stain, Masson s trichrome cell type, although all the types of cells perform all of the stain, AB-PAS (pH 2.5) reaction and AF-AB (pH 2.5) reaction three aforementioned functions rather than having functions were confirmed upon light microscopic examination of the which are different from one other. H. roretzi epithelium of digestive gland of . In addition, as the results of TEM observation, development of microvilli on Conflict of interest statement the free surface, and development of smooth endoplasmic reticula, mitochondria and oil droplets in the cytoplasm of non-ciliated columnar epithelia were observed. Such We declare that we have no conflict of interest. ultrastructural characteristics are similar to those of the liver cells of vertebrates and digestive gland cells of Acknowledgements marine mollusks which display lipid digestion. Detoxification is another important function of liver cells in vertebrates. The detoxification function in various types This work was supported by National Fisheries Research of cells, including liver cells, is explained by means of the and Development Institute (RP-2013-AQ-117) grant funded activation of smooth endoplasmic reticula, mitochondria by the Korea government. and lysosome, and ultrastructural characteristics that relate to the accumulation of lipofuscin granules[16-18]. Comments The ultrastructural characteristics of lysosomes that relate to the detoxification functions in the digestive glands of Background marine mollusks are very similar to those of vertebrates. Lysosomes also convert foreign substances infused into Research on the digestive system of tunicates is mostly the organism from outside, including marine pollutants, directed to the pyloric gland and those related to the into heterophagosomes, by dissolving them with hydrolytic digestive gland of tunicates have focused primarily on enzymes. Macromolecular lysosomes that remain in non- metabolism and glycogen storage. The hepatopancreas or disintegrated state with incomplete dissolution at this time digestive gland in aquatic invertebrates meets some similar are referred to as the residual body or lipofuscin. Pipe functions to the liver in vertebrates such as lipid digestion and Moore[19] explained the effect of phenanthrene in the and play an important role in detoxification of chemicals Littorina littorea digestive cells of periwinkle, through and drugs. This paper examines the storage of glycogen and the ultrastructural changes of lysosomes and distribution lipid digestion and detoxification of the digestive gland of β H. roretzi. of -glucuronidase, which is a lysosomal enzyme. The the tunicate, effect of stress arising from heavy metals or organic Research frontiers compounds was also explained through the lysosomal and Perna lipid alterations and lipofuscin in green mussel, This paper distinguishes morphologically and structurally viridis Mytilus galloprovincialis Scrobicularia plana [20], [21], different portions of the digestive gland (tubular and Tapes semidecussatus Gomphina veneriformis and [1] and [22]. blind the ampulla) and by light microscopy and electron In this study, cells among the epithelial cells which microscopy found that the three types of epithelial cells of contain lipofuscin granules (stained crimson as the result blind the ampulla portion are involved in storing glycogen, of Long Ziehl-Neelsen stain) were confirmed by light lipid digestion and detoxification. Good prepared for light H. roretzi microscopic examination of the digestive gland of . and electron microscopy. In addition, as the result of TEM observation, lysosomes Related reports with homogeneous core granules of high electron density and smooth endoplasmic reticula were characteristically Although ascidian glands are structurally composed of developed in the cytoplasm of the non-ciliated columnar tubules and blind blisters. Other authors have reported that Botryllus schlosseri Dendrodoa epithelium. Such ultrastructural characteristics were similar epithelial cells in differ from grossular to the characteristics of liver cells of vertebrates and the . This difference in the types of epithelial cells also digestive gland epithelium of marine mollusks which display has been found in this work, since three types of columnar detoxification function. epithelia were confirmed in blind the ampulla portion. Yun Kyung Shin et al./Journal of Coastal Life Medicine 2014; 2(12): 947-952 952 Innovations and breakthroughs Tissue developing hydranths of a hydromedusan coelenterate. 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