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HISTOCHEMICAL DISTRIBUTION OF THREE TYPES OF ENZYMES AND MUCOUS CELLS IN THE GILL, MANTLE AND HEPATOPANCREAS OF THE ARK SHELL SCAPHARCA SUBCRENATA.

INTRODUCTION

The bivalve mollusc has various enzymes and mucous cells distributing widely in the host tissues. They play an important role in the growth, development and immune defense of the host (Pipe 1990). Among them, alkaline phosphatase (ALP) can promote the metabolism of calcium and phosphorus in the body, maintain its proportion, and is related to the secretion of keratin and other proteins, consequently playing an important role in the formation of the shells (Xie et al. 2000). Nonspecific esterase (NSE) is involved in many functions including lipid metabolism, protein metabolism and signal transduction (Teng & Sun 2003). Peroxidase (POD) can promote cells adhesion and encapsulation (Ma & Mai 2003) as well catalyze the transformation of horseradish POD into quinone, which can kill the pathogenic bacteria and protect the host from invasion. Mucous cells can secrete mucopolysaccharides, glycoproteins and various hydrolytic enzymes (Harris et al. 1973), which promote digestion and absorption, and are also the first defense line of the immune mechanism of the host (Liu & Mai 2003).

In the mollusc, the gill is a filter-feeding and respiratory organ and this tissue also plays an important role in the defense of the body (Sun et al. 2017). The mantle is an important part of the body, with the primary function of being to enclose and protect the internal organs. It has the continuous and substantive secretory ability and also functions in respiration, waste disposal and sensory reception. The hepatopancreas is also called digestive gland, which is the main organ for digestion and absorption (Owen 1970).

Previous studies showed that the hydrolase and oxidase were distributed among the various tissues of the mollusc, such as the scallops Patinopecten yessoensis (Ran et al. 2007) and Chlamys farreri (Sun et al. 2002a). Mucous cells were commonly related to the hydrolytic enzymes, and their distributing patterns in the various tissues of some molluscs, such as C. farreri (Sun et al. 2002b), Meretix meretrix (Ren et al. 2003) and Argopectens irradias (Ren & Fu 2006), have been reported.

The ark shell Scapharca subcrenata (Lischke), which can survive in a broad temperature range, is widely distributed among the shallow coasts of China and Korea (Chen et al. 2006). It is a commercial bivalve with important economic value. In recent years, there have been some reports on the morphology, reproductive habits and immune mechanism of S. subcrenata (Liu et al. 2003, Xu et al. 2005, Park et al. 2011). The distributing patterns of the enzymes and mucous cells in the gill, mantle and hepatopancreas of S. subcrenata have still not been reported. Information regarding the distribution of the enzymes and mucous cells in the various tissues of S. subcrenata is essential for the elucidation of their absorption, metabolism, and immune defense mechanisms. The present study was aimed at evaluating the histochemical distribution of ALP, NSE, POD and various types of mucous cells in the gill, mantle and hepatopancreas of S. subcrenata.

MATERIALS AND METHODS

Animals and Sampling

Fifteen Scapharca subcrenata with a weight range of 11.6-19.9 g were purchased from a large aquatic wholesale market in Tianjin, China. Samples were immediately dissected, and the tissues, including the gill, mantle and hepatopancreas, were removed and placed into physiological saline; each tissue was divided into three equal portions. The first portion was fixed in Bouins solution (75 mL of a saturated solution of picric acid, 25 mL of formalin and 5 mL of glacial acetic acid) for 8 h, embedded in paraffin wax using an embedding machine (Thermo Microm EC 350, Thermo Fisher Scientific) and then subjected to Alcian blue (pH 2.5) periodic acid Schiff staining (AB-PAS). The second portion was fixed in cold (4[degrees]C) formol-calcium for 24 h and then stained for ALP and NSE, and the third portion was fixed in cold (4[degrees]C) 25% glutaric dialdehyde for 30 min, embedded in the optimum cutting temperature compound (OCT Compound; Sakura Tissue Tek) and then stained for POD.

Enzyme Histochemistry

The tissues, including the gill, mantle and hepatopancreas, were sectioned with a Thermo Microm HM 525 freezing microtome into 7 gm thick slices, and these samples were used for the ALP, NSE and POD staining. Three sections were used for the evaluation of enzyme activity in each tissue, and 10 fields of each tissue were randomly imaged and analyzed. The negative controls were incubated without substrates.

Alkaline Phosphatase Activity

To evaluate the ALP activity, the sections were incubated in a mixture containing 5% nitrotetrazolium blue chloride (Sangon) and 5% 5-bromo-4-chloro-3-indolyl phosphate (Sangon) in 10 mL ALP buffer (100 mM sodium chloride, 5 mM magnesium chloride, 100 mM Tris--HCI, pH 9.5) for 20 min at room temperature. After incubation, the sections were washed with distilled water, dehydrated in alcohol, subjected to dimethylbenzene hyalinization and then mounted in balsam.

Nonspecific Esterase Activity

The NSE activity was evaluated using the naphthyl AS acetate method (Pearse 1972). The sections were incubated in a solution containing 0.1 mL of 1% naphthyl AS acetate, 10 mL phosphate buffer (0.05 M, pH 7) and 30 mg fast red TR at 37[degrees]C for 30 min. The sections were then stained with 2% methyl green, washed with distilled water and then mounted in glycerogelatin.

Peroxidase Activity

The sections were stained for POD with the diaminobenzidine (DAB, Sigma) method. Sections were incubated in a solution containing 10 mg DAB-4HC1 dissolved in 5 mL distilled water and 5 mL phosphate buffer (0.2 M, pH 7) at room temperature for 20 min. Approximately 100 [micro]L of H2O2 were then added to the solution, and the sections were incubated at room temperature for 30 min. The sections were then stained with methylene blue, washed with distilled water, dehydrated with alcohol, subjected to dimethyl benzene hyalinization and then mounted in balsam.

Alcian Blue (pH 2.5) Periodic Acid Schiff Histochemistry

Samples were sectioned with a Thermo Microm HM 355S microtome into 7.tm thick slices. The sections were stained with an AB pH 2.5/PAS staining kit (Solarbio), dehydrated with alcohol, subjected to dimethyl benzene hyalinization and then mounted in balsam. Four sections of each tissue were prepared and used in the mucous cell evaluation, and 10 fields of each section were randomly imaged and analyzed.

Photomicrography and Statistical Analyses

The sections were examined and photographed with a Leica DM 4000B microscope. Enzyme activities were analyzed quantitatively by using the ImageJ (http://www.imagej.net) to determine the mean optical density (MOD). A higher MOD value indicated a stronger enzymatic reaction. The number of the different types of mucous cells were counted under 400 X magnification. The MOD value and number of mucous cells were expressed as the means [+ or -] SD. The data were analyzed using SPSS 16.0 (IBM). Differences in the activities of the three enzymes, and the number of the four types of mucous cells in the different tissues, including the gill, mantle and hepatopancreas (factor), were assessed using one-way ANOVA with Duncan test. A P value of <0.05 was required for significance.

RESULTS

Enzyme Histochemistry

The distribution and staining intensity of ALP, NSE and POD in the gill, mantle and hepatopancreas of Scapharca subcrenata were shown in Table 1. Enzyme activity was determined by observing photomicrographs and quantitatively analyzing the optical density.

A positive ALP histochemical reaction was indicated by blue-purple staining (Fig. 1A--D). According to the dyeing results, the ALP activity was observed primarily in the epithelial cells (EP) of the gill filament and mantle (Fig. 1A, C), whereas limited staining was observed in the connective tissue (CT) of the gill arch and mantle (Fig. lB, C). The closer to the gill axis the gill filament was, the higher ALP activity it had. In the hepatopancreas, a strong positive staining for ALP was located among the basal lamina of the glandular duct and digestive cells. Whereas, the positive distribution in the columnar EP of the duct and CT was sparse (Fig. 1 D). The ALP activities were analyzed quantitatively by using the ImageJ to determine the MOD. The analysis indicated that the ALP activity in the mantle was higher than that in the hepatopancreas, which was higher than that in the gill (P < 0.05, Table 1).

A positive NSE histochemical reaction was indicated by purple-red staining. The NSE activity was located mostly in the EP of the gill, mantle and hepatopancreas (Fig. 2A--D). In the gill, the NSE activity was observed mostly in the EP of the gill filament and sparsely in the CT and interlamellae junction of the gill filament (Fig. 2A). In the hepatopancreas, the NSE activity was distributed mainly among the columnar EP of ducts and the glandular duct epithelium, and sparsely among the CT (Fig. 2B). In the mantle, the lateral epithelium, medial epithelium and CT showed purple-red staining (Fig. 2C, D). Additionally, the staining intensity of the medial epithelial CT was higher than that observed in the lateral epithelial CT. Quantitative analysis indicated that the NSE activity in the hepatopancreas was significantly higher than that in the gill (P < 0.05). In addition, the NSE activity between the hepatopancreas and mantle varied but was not statistically significant (P > 0.05, Table 1).

A positive POD histochemical reaction was indicated by dark brown staining (Fig. 3A--C). In the gill, the POD activity occurred in the CT of the gill filaments and showed no obvious distribution in the EP (Fig. 3A). In the mantle, the POD activity was located mostly in the EP and sparsely in the CT of the lateral epithelium (Fig. 3B). In the hepatopancreas, the positive reaction was observed only in the EP of the glandular duct (Fig. 3C). Quantitative analysis indicated that the POD activity in the mantle was significantly higher than that in the hepatopancreas (P < 0.05). Additionally, the POD activity in the mantle and gill varied but was not statistically significant (P> 0.05, Table 1).

Aldan Blue (pH 2.5) Periodic Acid Schiff Histochemistry

The distribution of various types of mucous cells was assessed by photomicrography and statistical analysis. Four types of mucous cells were detected in the gill, mantle and hepatopancreas of Scapharca subcrenata. Type I cells (PAS+AB-) were stained purple-red, indicating that these were PAS activity-positive and AB activity-negative, thereby revealing the presence of glycogen, neutral mucins and glycoproteins. In the gill, the mucous cells identified as type I cells were observed among the EP of the gill filament (Fig. 4A). In the mantle, type I cells were distributed among the medial epithelium (Fig. 4B, C). In the hepatopancreas, type I cells were not detected. Type II cells (PAS--AB+) were stained pale blue, indicating that these were AB activity-positive and PAS activity-negative, thereby revealing the presence of acid mucins. The type II cells were distributed sparsely in the CT of the mantle (Fig. 4C) and mostly among the ciliated columnar cells in the digestive ducts of the hepatopancreas (Fig. 4D). Type III cells (nPAS+AB+) were stained purple-red and strongly positive for PAS, thereby revealing the presence of abundant neutral mucins (Sun et al. 2002b). The type III cells were distributed mostly in the gill arch (Fig. 4E) and the glandular duct of the hepatopancreas (Fig. 4F), whereas they were distributed sparsely in the gill filament (Fig. 4A) and among the EP of the mantle (Fig. 4B, C). Type IV cells (aPAS+AB+) were stained blue-purple and strongly positive for AB, thereby revealing the presence of abundant acid mucins (Ren & Fu 2006). In the gill, type IV cells were observed in the gill arch (Fig. 4E) and among the EP of gill filament (Fig. 4A). In the mantle, type IV cells were distributed among the medial epithelium in its forefront (Fig. 4C). In the hepatopancreas, type IV cells were distributed sparsely in the digestive duct (Fig. 4F). The four types of mucous cells in the gill, mantle and hepatopancreas of S. subcrenata were summarized in Table 2. The gill showed the highest number of type III cells, more type IV cells than type I cells. The mantle showed the highest number of type IV cells, more type I cells than type II cells and the lowest number of type III cells. The hepatopancreas showed the highest number of type II cells, more type IV cells than type III cells. The number of type I cells in the mantle was higher than that in the gill (P < 0.05). The number of type II cells in the hepatopancreas was higher than that in the mantle (P < 0.05). The number of type III cells and type IV cells in the gill and mantle were higher than those in the hepatopancreas (P < 0.05). The number of type IV cells did not significantly differ between the gill and mantle (P > 0.05).

DISCUSSION

The ALP, NSE and POD activities and the mucous cells were distributed ubiquitously throughout the gill, mantle and hepatopancreas of Scapharca subcrenata, but different tissues exhibited various distribution of the detected enzyme activities and mucous cells.

Alkaline phosphatase is an important regulatory enzyme distributed widely among animals, which can catalyze the hydrolysis and transfer various phosphate groups (Posen 1967). In the mollusc, the transmembrane transport functions of ALP can participate in the transport of nutrients (Blasco et al. 1993). In the present study, the finding of the abundant distribution of ALP among the EP of gill filaments and mantle was similar to that observed in Chlamys farreri (Sun et al. 2002a). Because the mantle and gill are feeding organs of the mollusc, the food particles are mainly filtered through the ciliary movement on the gills and the mantle membrane. This distribution feature of ALP might be related to their functions associated with the absorption and transport of nutrients. And the finding of higher ALP activity in the gill filament closer to the gill axis might be related with the fact of the accumulation of food at this location. In addition, the mantle secretes, repairs, and maintains the shells of those molluscs that have shells. The epithelium of the mantle can concentrate the calcium from the environment via water and food, adds it to the extrapallial space where the shell forms. In a previous study on Hyriopsis cumingii, ALP promoted the calcareous deposition in the mantle (Shi et al. 2008). Because there was the most abundant ALP in the mantle of Scapharca subcrenata, it was speculated that the ALP was commonly involved in the process of the calcium metabolism. The hepatopancreas provided the main function of food digestion and nutrient absorption. It was previously reported that ALP was involved in the process of the transferring food to the blood cavities between the glandular ducts of the hepatopancreas (Cui et al. 2005). The finding of higher ALP activity at the base of the glandular duct of the hepatopancreas in this study was similar to that observed in Mytilus edulis (Cui et al. 1999) and Mactra antiquata (Xu et al. 2007). And this indicated that nutrients were absorbed at this location with the involving of ALP activities.

Non-specific esterase, a major hydrolase, is found primarily in the endoplasmic reticulum, mitochondria and lysosome as well as in other organelles (Ran et al. 2007). The NSE is mainly involved in the bioprocess of biotransformation, protein and lipid metabolism (Teng & Sun 2003). In addition, Laureau et al. (2003) suggested that the NSE in the mollusc was also involved in the denfence function. They found that the percentage of NSE positive cells among blood cells were significantly decreased after infection with Bonamia ostreae, indicating that NSE was involved in the degradation process after phagocytosis of blood cell to fight against invading pathogens. In this study, the NSE activity of Scapharca subcrenata mainly existed on the epidermis of the organs that were in close contact with the external environment, such as the gill filament and mantle. This finding was similar to the results in Chlamys farreri (Sun et al. 2003). Because the organ epidermis was prone to encounter various pathogens from the external environment and consequently involved in the defense mechanism against pathogen infection, it was speculated that NSE in the gill and mantle of S. subcrenata probably participated in the immune defense mechanism. The finding of the strongest NSE activity in the digestive glands also matched the fact that it was the site for food digestion and fat storage in the mollusc (Franchini & Ottaviani 1992, Walker 1970), and functioned in the process of the digestion of esters and lipids.

Peroxidase can be discharged into the phagosomes in the process of phagocytosis and is a part of microbicidal system. Previous results showed that it was an important enzyme for killing invading microorganisms in the mollusc (Wootton & Pipe 2003). In the present study, the finding of the POD distributing among the EP of the gill filaments and mantle in Scapharca subcrenata was similar to that observed in Chlamys farreri (Liang et al. 2007, Wang et al. 2004). The gill and mantle of the mollusc were more likely to be subjected to opportunistic pathogens exiting in the seawater. It was an important physiological function for the epidermis of these organs to secrete some antimicrobial substances to prevented the invasion of the pathogens (Wang et al. 2004). The extensive and high activities of POD in the gill and mantle of S. subcrenata indicated that POD was involved in the defense mechanism of the gill and mantle against these opportunistic pathogens. The reason why POD activity was weaker in the gill EP than that in the CT still needs to be further studied.

The AB-PAS histochemical results revealed various distribution of the four types of mucous cells (Type I, II, III, IV) in the gill, mantle and hepatopancreas of Scapharca subcrenata.

The gill of Scapharca subcrenata is a filter organ (Sun et al. 2017). It was previously reported that in bivalves, filter feeding mainly relied on the mucus and cilia in the gills (Ward et al. 1993) and the mucous cells in the gill could secrete mucous substances to wrap up food particles (Deng & Li 2002). In this study, the findings of the gill of S. subcrenata containing a large number of mucous cells, mainly type III and IV mucous cells, were similar to the results observed in Chlamys farreri (Sun et al. 2002b). The type III cells secreted abundant neutral glycoconjugates, which facilitated the absorption and transport of macromolecules through the cell membranes. Whereas, acid glycoconjugates commonly secreted by the type IV cells contributed to the lubrication of the food. It was speculated that the type III and IV mucous cells in the gill of the mollusc were commonly involved in the mechanism of nutrition transportation.

It was reported that the more chance to contact with water or the more complex functions the organ had, the more types and number of the mucous cells it had (Singh & Mittal 1990). The four types of mucous cells were found, as well the most mucous cells were detected in the mantle of Scapharca subcrenata. This finding was similar to the results in the mantle of Chlamys farreri (Sun et al. 2002b), Meretix meretrix (Ren et al. 2003) and Argopectens irradias (Ren & Fu 2006). More types and amounts of the mucous cells in the mantle were probably due to the fact that it contacted directly with sea water and easily affected by water flow. In addition, Among the four types of mucous cell in the mantle of S. subcrenata, the type IV, which occupied the highest abundance, primarily secreted the acidic glycoconjugates, including sulfur-acidic mucus and salivary acidic mucus. These substances were considered to be involved in the absorption and transport of calcium and the formation of calcium carbonate (Sun & Shi 2000). It was speculated that the type IV mucous cells played an important role in the formation of shells.

The hepatopancreas of Scapharca subcrenata is the site of digestion and absorption in the body (Xu et al. 2005). The ABPAS histochemical results showed that the glandular duct contained a large number of type II mucous cells, which were similar to the observation in Meretrix meretrix (Yang et al. 2005) and Argopecten irradias (Wang et al. 2003). Type II mucous cells mainly secreted the acid glycoconjugates, which had the functions of binding food and lubricating the digestive tract. Moreover, these substances maintained the digestive tract a certain acidic environment in which a majority of digestive enzymes had high activities. Therefore, it was speculated that the type II mucous cells played a role in promoting food digestion and absorption in the hepatopancreas.

In this study, the histochemical distribution of ALP, NSE and POD, as well as the types of mucous cells in the gill, mantle and hepatopancreas of Scapharca subcrenata ware evaluated for the first time. These results will provide the basis for further studies on the absorption mechanisms and immune defensive functions of the gill, mantle and hepatopancreas of S. subcrenata.

ACKNOWLEDGMENTS

This study was partially supported by the Innovation Team of Tianjin Fisheries Research System (ITTFRS2017009), the Modern Agroindustry Technology Research System (CARS-48), the Scientific Program of Tianjin City (16JCQNJC09500), the Major Project for Tianjin Seed Technology (15ZXZYNC00050).

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QING MAO, (1[dagger]) ZHUORAN HAN, (1,2[dagger]) YINGLAN LI, (1) JINGFENG SUN, (1*) YONGJUN GUO, (1) XUELIANG SUN, (1) LIMEI CHEN (1) AND KEZHI XING (1)

(1) Tianjin Key Laboratory of Aqua-Ecology and Aquaculture, College of Fisheries, Tianjin Agricultural University, 22 Jinjing Road, Xiqing District, Tianjin 300384, China; (2) Key Laboratory of Ecology and Environment Science of Higher Education Institutes, Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, College of Life Science, South China Normal University, 55 Zhongshan Avenue West, Tianhe District, Guangzhou 510631, China

(*) Corresponding author. E-mail: sun jf@ l63.com

([dagger]) These authors contributed equally to this work.

DOI: 10.2983/035.038.0209
TABLE 1.
Distribution of three enzymes in the gill, mantle and hepatopancreas of
Scapharca subcrenata, as measured by MOD.

                            MOD mean [+ or -] SD
Distribution              ALP                       NSE

Gill            0.307 [+ or -] 0.018 (a)  0.287 [+ or -] 0.05 (a)
Mantle          0.390 [+ or -] 0.011 (b)  0.301 [+ or -] 0.007 (b)
Hepatopancreas  0.376 [+ or -] 0.027 (c)  0.305 [+ or -] 0.005 (b)

                  MOD mean [+ or -] SD
Distribution             POD

Gill            0.486 [+ or -] 0.032 (a)
Mantle          0.498 [+ or -] 0.033 (a)
Hepatopancreas  0.315 [+ or -] 0.031 (b)

Different lower-case superscript letters within the same column
indicated significant differences among the gill, mantle and
hepatopancreas of S. subcrenata (P < 0.05).

TABLE 2.
Distribution of mucous cells in the gill, mantle and hepatopancreas of
Scapharca subcrenata.

                       Number of mucous cells (mean [+ or -] SD)
Distribution              Type I                    Type II

Gill             8.40 [+ or -] 7.33 (a,A)    0 (a,A)
Mantle          22.71 [+ or -] 14.15 (a,B)  19.93 [+ or -] 13.75 (a,B)
Hepatopancreas   0 (a,A)                    60.93 [+ or -] 19.58 (b,C)

                       Number of mucous cells (mean [+ or -] SD)
Distribution             Type III                  Type IV

Gill            67.63 [+ or -] 12.38 (b,A)  55.47 [+ or -] 17.76 (bc,A)
Mantle          18.58 [+ or -] 15.37 (a,B)  53.40 [+ or -] 12.73 (b,A)
Hepatopancreas   8.93 [+ or -] 10.29 (a,C)   9.33 [+ or -] 10.10 (a,B)

Different upper-case superscript letters within the same column
indicated significant differences among the different tissues of S.
subcrenata, and different lower-case superscript letters within the
same row indicated significant differences among the different types of
mucous cells within the same tissue of S. subcrenata (P < 0.05).
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Author:Mao, Qing; Han, Zhuoran; Li, Yinglan; Sun, Jingfeng; Guo, Yongjun; Sun, Xueliang; Chen, Limei; Xing,
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:9CHIN
Date:Aug 1, 2019
Words:4923
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