- Email: [email protected]
Light and electron microscopy of hemocyte of the hard clam, Meretrix lusoria (Röding)
Comp. Biochem.
Pergamon
Physiol. Vol. 108A. Nos 213, pp.219-286, 1994 Ekvier Science Ltd Printed in Great Britain 0300-9629/94 $7.00 + 0.00
Light and electron microscopy of hemocyte of the hard clam, Meretrix Zusoria (Ridding) Chiou-Ming
Wen, Guang-Hsiung
Kou and Shiu-Nan Chen
Department of Zoology, National Taiwan University, Taipei, Taiwan 106, ROC Hemocytes of the hard clam, Meretrix Zusoriu(R&hog), were studied using light and electron microscopy. On the basis of staining characteristics and morphological criteria, the hemocytes were class&xl into four major categories: hyalinocytes, larger eosinophilic granulocytes (LEGS), smaller eosinophilic granulocytes (SEGs) and fibrocytes. SEGs were the most numerous components of the hemocytes, while few hyalinocytes were observed under normal conditions in the clam. Morphological characteristics of the granulocytes differed from the granulocytes of marine bivalves that have been reported previously. Granules of LEGS, though, were similar to those of SEGs using light microscopy but were completely different with electron microscopy. Key words: Hemocyte; Hard clam; Meretrix lusoriu; Bivalve; Microscopy. Comp. Biochem. Physiol. lO$A, 279-286,
1994.
Introduction Hemocytes have been shown to play a significant role in the defense mechanisms of bivalves (see reviews by Cheng, 1967; Cheng and Rifkin, 1970; Feng et al., 1977; Feng, 1988; Fisher, 1986). Generally, they protect against foreign materials by an inflammatory process, aggregation, phagocytosis, hyperplasia or encapsulation. Furthermore, hemocytes participate in wound repair, digestion and detoxification processes of different xenobiotics (Bang, 1961; Cheng, 1981; Cheng and Cali, 1974; Feng, 1966; Moore and Lowe, 1977; Ruddell, 1971b; Sparks and Morado, 1988). In addition, hemocytes of some species have been shown to be capable of secreting cytotoxic molecules (Wittke and Renwrantz, 1985; Leippe and Renwrantz, 1985). As hemocytes are predominantly responsible for deCorrespondence to: Chiou-Ming Wen, Room 201, Institute of Fishery Biology, National Taiwan University, Taipei, Taiwan 106. Fax 886-23687122. Received 8 June 1993; accepted 23 July 1993. 279
fense against pathogens, a more thorough understanding of the morphology and functions of hemocytes will help us to better understand these defense mechanisms. Hemocytes have been clearly classified into two major types: agranulocytes and granulocytes, depending on the presence of cytoplasmic granulocytes (see review by Auffret, 1988). Granulocytes are further subdivided into eosinophilic, basophilic, small or large granulocytes, while agranulocytes are divided into hyalinocytes and fibrocytes, depending on the characteristics of the granules and cell size. However, there is diversity in bivalve hemocyte types even among the same family. Granulocytes in the Pacific oyster, Crassostrea gigus, for example, are subclassified into acidophilic granulocytes and basophilic granulocytes, whereas they are subclassified into basophilic granulocytes, eosinophilic granulocytes, and small granulocytes for the Eastern oyster, C. virginicu (Ruddell, 1971a, b, c; McCormick-Ray and Howard,
280
C.-M. Wen et al.
1991). As relatively few species have been investigated, hemocytes of more species need to be studied, and a suitable scheme established that could be applied to a variety of species. The purpose of this paper, therefore, is to describe the morphological characteristics of the hemocytes of Meretrix lusoriu. Such descriptions are obviously essential as a preface to studies concerned with their functions as related to internal defense. In addition, results of the present study will be helpful for the establishment of a bivalve hemocyte classification scheme.
Materials and Methods of the hard clam, Meretrix 3.5-4.5 cm), were obtained from a commercial source in Taipei and maintained in seawater (salinity of 15%) prior to use. Approximately 0.5 ml hemolymph was collected from the posterior adductor muscle sinus of each clam using a 2.5-ml syringe fitted with a 21-gauge hypodermic needle. Specimens
Zusoria Ridding (shell length
Fixed and stained smears
Cells examined as stained preparations consisted of 0.25 ml of freshly collected hemolymph placed on glass slides and permitted to settle for 10 min at room temperature (26 + 1C). The adhered cells were fixed with 10% seawater-buffered formalin, absolute methanol, or Carnoy’s fixative (acetic acid and methanol 1: 3) for 5 min. Subsequently, the attached cells fixed by formalin were rinsed twice in Sorenson’s buffer, pH 6.5, while the absolute methanol and Carnoy’s-fixed cells were rinsed with fresh absolute methanol. The slides were stained with 10% Giemsa’s, Wright’s, or Mayer’s hematoxylin and eosin stains (H & E), dehydrated, and mounted in Entellan@ (Merck). Electron microscopy preparations
Five millilitres of hemolymph were collected and centrifuged at 800 t-pm for 10 min (Hitachi 05PR-22). Cell pellets were subsequently pre-fixed with 2.5% seawater-glutaraldehyde and post-fixed with 0~0~ for 30 min, at 4°C. Samples were dehydrated in a graded series of ethanol
and absolute acetone, and embedded in Spurr’s resin. Ultrathin sections were cut with a LKB-2088 ultramicrotome equipped with a glass knife, mounted on Formvar@ grids, and stained with uranyl acetate and lead citrate, according to Frasca and Parks (1965). The sections were examined using an Hitachi H-6000 electron microscope at 75 kV.
Results In preparations fixed with 10% buffered formalin, absolute methanol or Carnoy’s with Giemsa’s, fixative and stained Wright’s, or H & E stains, spread hemocytes verified the presence of two categories: those with granules and those without. Granulocytes were recognized as oblong, pleomorphic, or round cells with granules of varying density and intensity. Agranulocytes either completely lacked, or contained very few, granules. Depending on the fixative and/or stain employed, there were some variations in the staining characteristics of constituents of the cells. In comparison, cells fixed with 10% formalin resulted in better cytoplasmic staining, while those fixed with Carnoy’s resulted in more vivid contrast of their nuclei. Granular size, intensity and staining provided the basis for differentiating the granulocytes. Eosinophilic granulocytes contained pink, orange or purple granules surrounded by slight blue cytoplasm. Depending on the granular size, the eosinophilic granulocytes could be divided into large and small granular granulocytes (Fig. 1). The small eosinophilic granular granulocytes (SEGs) were the most numerous cell type, while the large eosinophilic granular granulocytes (LEGS) were the largest cell type but found in fewer numespecially hyalibers. Agranulocytes, nocytes, were rarely observed in smear preparations as compared with granulocytes. In addition, profiles of the LEGS generally showed that the granules filled up the endoplasm, pushing the nucleus to one side. Occasionally, LEGS with multinuclei and vacuoles were observed (Fig. 2). The agranulocytes could be classified as hyalinocytes and fibrocytes based on size and cytoplasmic characteristics, according to the classification scheme of Foley and
Hemocytes of the hard clam, Meretrix lusoriu
Fig. 1. Photomicrograph of stained hemocytes of Meretrix lusoria. Eosinophilic granulocytes. Hemocytes were fixed with 10% buffered formalin and stained with 50% Wright’s stain. Bar = 50 pm. Fig. 2. Photomicrograph of stained hemocytes of M. iusoria. Note the multinuclei LEGS. (Formalin fixed, Wright’s stained. Bar = 50 pm. Fig. 3. Photomicrograph of stained hemocytes of M. lusoria. Note the hyalinocyte (arrow) and fibrocyte (arrow head). Methanol fixed, Giemsa stained. Bar = 50pm. Fig. 4. Photo~~rog~ph of stained hemocytes of M. lusoria. Cell clumps. Note the pleomorphic nuclei of hemocytes. Carnoy’s fixed, Giemsa stained. Bar = 50 pm. Fig. 5. Photomicrograph of stained hemocytes of M. lusoria. Note the basophilic granulocyte. Carnoy’s fixed, Giemsa stain. Bar = 50 pm.
282
C.-M.
Wen et al.
Cheng (1974). The hyalinocytes were smaller (usually 4-5 pm), basophilic and contained a large, central nucleus (Fig. 3). They regularly represented spheres with few cytoplasmic processes. Fibrocytes were larger, with slight basophilic cytoplasm,
and in some cases, contained many vacuales (Fig. 3). Also, clumps of two to 10 adhered hemocytes were observed. The clumps formed immediately after the hemolymph was withdrawn. Cells would migrate from
Fig. 6. Electron micrograph of a Meretrix lusoria large eosinophilic granular granulocyte. Bar = 1 pm. GA: Golgi apparatus; LL: large lysosome-like vesicle; N: nucleus; M: mitochondrion; P: pseudopodium. Fig. 7. Electron micrograph of a smaller eosinophilic granular granulocyte with prominent Golgi apparatuses and microtubules. Bar = 1 pm. GA: Golgi apparatus; LV: lucid vesicle; SL: small lysosome-like vesicle. Fig. 8. Electron micrograph of portion of a small eosinophilic granular granulocyte containing smaller lysosome-like vesicle. Bar = 1 pm. C: centriole; GA: Golgi apparatus; LV: lucid vesicle; MT: microtubule; SL: small lysosome-like vesicle. Fig. 9. Electron micrograph of a portion of a small eosinophilic granular granulocyte. Note the fusion of vesicles (arrow). Bar = 1 pm. LV: lucid vesicle; SL: small lysosome-like vesicle.
Hemocytes of the hard clam,
Meretrix
lusoria
283
Fig. 10. Electron pseudopodia and G: Fig. 11. Electron
micrograph of two fibrocytes. The upper one shows several long projected the lower one shows glycogen-like granules in the cytoplasm. Bar = 1 pm. glycogen-like granules; rER: rough endoplasmic reticulum. micrograph of a fibrocyte, phagosomes (Pg) and fibrils (F) are shown. Bar = 1 pm. Fig. 12. Electron micrograph of a hyalinocyte. Bar = 1 pm. Fig. 13. Electron micrograph of a slightly granular hyalinocyte with larger nuclear/cytoplasm ratio. Bar = 1 pm.
the clumps after adhering to the slide. Usually, pleomorphicnuclear granulocytes and/or fibrocytes constituted the center of the clumps (Fig. 4). Basophilic granulocytes were occasionally observed (Fig. 5). Electron micrographs of the hemocytes of M. lusoria revealed the occurrence as shown under light microscopy. The most conspicuous type of organelles in granulocytes was the membrane-bound vesicles, which were the large or small eosinophilic granules present during light microscopy.
These vesicles were scattered throughout the endoplasm. The membrane-bound specific vesicles of the granular hemocytes showed different morphological characteristics. Therefore, granulocytes were easily divided into two types, one with small vesicles (diameter ranging from 0.4-l .3 ,um) that contained fine electron-dense particles and the other with larger vesicles (diameter ranging from 1.O-2.9 pm) which were homogeneous (Figs 6-8). Also, the vesicles differed in their outline, the former being
284
C.-M. Wen et al.
irregular, while the latter was circular. In some cases, fusion of small vesicles was observed (Fig. 9). Besides the electrondense vesicles, electron-lucid vesicles were frequently seen in the cytoplasm of hemocytes. Granulocytes (as shown in Figs 6-8) revealed some other characteristics in electron microscopy. Smooth and rough endoplasmic reticulum were distributed throughout the cytoplasm and Golgi apparatuses were frequently observed. The nuclei of granulocytes, depending on the plane of section, were spherical, bean shaped or lobule. Microtubules associated with a centriole in the cytoplasm were observed at different times (Fig. 8). Fibrocytes showed morphologies similar to granulocytes except that there were few or no electron-dense vesicles in the cytoplasm. They usually had a number of filopodial projections at their peripheries. Glycogen-like granules and fibrils were present in some profiles (Figs 10 and 11). The hyalinocytes (as shown in Figs 12 and 13) were also generally small with a circular or slightly oval outline. Their nuclei usually filled the entire cell. Some of the cells contained visible Golgi apparatus, with a few electron-dense vesicles.
Discussion Although many studies on the hemocytes of bivalves exist, few species have been studied and there is, as yet, no widely accepted nomenclature for bivalve hemocyte types. In an attempt to classify hemocytes, Cheng (1981) presented a morphological scheme based on numbers of cytoplasmic granules, dividing the cells into two types: (1) hyalinocyte, a cell containing few or no granules; and (2) granulocyte, a cell containing granules ranging from very few to numerous. Fibrocytes were granulocytes, in some cases degranulated, meaning there were no granules in the cytoplasm (Cheng and Foley, 1975). The results reported herein are in agreement with these two types of hemocytes. Hyalinocytes (named lymphocyte-like cells by Feng et al., 1971; McCormick-Ray and Howard, 1991) are the most striking aspect in hemocyte morphology; however, their functions remain unclear. According
to a model established by Mix (1976), hyalinocytes are an original cell type of granulocytes which could differentiate into various types of granulocyte. Results of this study tend to support this idea, as intermediate cells of slightly granular hyalinocytes with larger and smaller nuclear/cytoplasm ratios have been found under electron microscopy. It seems that the differentiation of granulocytes is associated with Golgi apparatus because the Golgi apparatus is (1) rarely found in agranular hemocytes; (2) occasionally observed in slightly granular hyalinocytes; and (3) well developed in the two types of eosinophilic granulocytes. The Golgi apparatus is suggested to be the producer of numerous electron-lucid and electron-dense vesicles, so that those vesicles increase following the cells’ differentiation. Two types of eosinophilic granulocytes were observed in this investigation, and compared with those found in species discussed in previous studies. Granulocytes of A4. Zusoriu were different from those of Mercenariu mercenuriu (Cheng and Foley, 1975; Foley and Cheng, 1974), MytiZus edulis (Rasmussen et al., 1985), Crussostreu gigus (Ruddell, 1971a, c), C. oirginicu (Feng et al., 1971; Foley and Cheng, 1972), Pecten muximus, or Tapes philippinurum (Auffret, 1988) as compared using figures of light and electron microscopy. It should be noted, in the case of M. Zusoriu, the electron-dense vesicles of SEGs clearly include fine particles with an irregular outline, while those of LEGS contained homogeneous smeared materials with a circular edge. The functions and compositions of these two types of granules are unclear. Degranulation of bivalve hemocytes has been demonstrated by several investigators (Hirsch and Chon, 1960; Feng et al., 1977; Mohandas et al., 1985), and could be associated with the intracellular fusion of lysosomes with phagosomes, as well as with the extrusion of intact lysosomes from hemocytes into the extracellular medium (see review by Feng, 1988). Therefore, the granules (vesicles) of the two types of eosinophilic granulocytes are two different forms of lysosomes. However, the contents of these vesicles remain unclear. Centrioles, with ordered microtubules observed in this study, have been seen previously (Cheng and Foley,
Hemocytes of the hard clam, Meretrix lusoria
1975), but their significance remains to be understood. Multinucleated cells, as reported in C. gigas by Sparks and Pauley (1964) and in M. mercenaria by Foley and Cheng (1974), were also found in M. hsoria. Cheng and Galloway (1970), who found similar cells in the gastropod, Helisoma duryi normale, when challenged with allografts and xenografts, suggested they may only occur under pathological conditions. The number or cell size of multinucleated cells tend to increase following a period of in vitro culture (authors’ unpublished data). The roles of these multi- or binucleated cells in ho, however, are unclear. In conclusion, our results support the summary made by Cheng (1981) and Auffret (1988) in their critical reviews of the existing literature concerning the hematology of bivalves. The ultrastructure of the granulocytes of M. lusoria, however, is different from granulocytes of bivalves that have been studied previously. The Golgi apparatus found in granulocytes may play a significant role in hemocyte maturation. Functions and compositions of these two forms of eosinophilic granulocytes require further research. Acknowledgements-This study was supported by the National Science Council, Republic of China (Grants: NSC 80-0209-B002-02 and 81-0209-002-06).
References Auffret M. (1988) Bivalve hemocytes morphology. Am. Fish. Sot. Sp. Publ. 18, 167-177.
Bang F. B. (1961) Reaction in injury in the oyster (Crassostrea virginica). Biol. Bull. 121, 57-68.
Cheng T. C. (1967) Marine molluscs as hosts for symbioses: with a review of known parasites of commercially important species. Ado. Mar. Biol. 5, 1424.
T. C. (1981) Bivalves. In Invertebrate Blood Cells (Edited by Ratcliffe N. A. and Rowley
Cheng
A. F.), Vol. 1, pp. 233-330. Academic Press, New York. Cheng T. C. and Cali A. (1974) An electron microscope study of the fate of bacteria phagocytosed by granulocytes of Crassostrea virginica. Contemp. Topics in Immunobiol. 4, 25-35.
Cheng T. C. and Foley D. A. (1975) Hemolymph cells of the bivalve mollusc Mercenaria mercenaria: an electron microscopical study. J. Invert. Pathol. 26, 341-351.
Cheng T. C. and Rifkin (1970) Cellular reactions in marine molluscs in response to helminth parasitism. Am. Fish. Sot. Sp. Publ. 5, 443-496.
285
Cheng T. C., Rodrick G. E., Foley D. A. and Koehler S. A. (1975) Release of lysozyme from hemolymph cells of Mercenaria mercenaria during phagocytosis. J. Invert. Pathol. 25, 161-165. Feng S. Y. (1966) Experimental bacteria1 infections in the oyster, Crassostrea virginica. J. Invert. Pathol. 8,505511.
Feng S. Y. (1988) Cellular defense mechanisms of oysters and mussels. Am. Fish. Sot. Sp. Publ. 18, 153-168. Feng S. Y., Feng J. S., Burke C. N. and Khairallah L. H. (1971) Light and electron microscopy of the leucocytes of Crassostrea uirginica (Mollusca: Pelecypoda). 2. Zellforsch. 120, 222-245. Feng S. Y., Feng J. S. and Yamasu T. (1977) Roles of Mytilus coruscus and Crassostrea gigas blood cells in defense and nutrition. In Comparative Pathobiology (Edited by Bulla L. A. and Cheng T. C.), Vol. 3, pp. 31-67. Plenum Press, New York. Fisher W. S. (1986) Structure and function of oyster hemocytes. In Immunity in Invertebrates (Edited by Brehelin M.), pp. 25-35. Springer, Berlin. Foley D. A. and Cheng T. C. (1972) Interaction of molluscs and foreign substances: the morphology and behavior of hemolymph cells of oyster, Crassostrea virginica, in vitro. J. Invert. Pathol. 19, 383-394.
Foley D. A. and Cheng T. C. (1974) Morphology, hematological parameters, and behavior of hemolymph cells of the quahaug clam, Mercenaria mercenaria. Biol. Bull. 146, 341-356.
Frasca J. M. and Parks V. E. (1965) A routine technique for double staining ultrathin sections using uranyl and lead salts. J. Cell Biol. 25, 157. Hirsch J. C. and Chon Z. A. (1960) Degranulation of polymorphonuclear leucocytes following phagocytosis of microorganism. J. exp. Med. 112, 1005-1014.
Leippe M. and Renwrantz L. (1985) On the capability of bivalve and gastropod hemocytes to secrete cytotoxic molecules. J. Invert. Pathol. 46, 209-210. McCormick-Ray M. G. and Howard T. (1991) Morphology and mobility of oyster hemocytes: evidence for seasonal variation. J. Invert. Pathol. 58, 219-230.
Mix M. C. (1976) A general model for leucocyte cell renewal in bivalve mollusks. Mar. Fish. Rev. 38, 3741.
Mohandas A., Cheng T. C. and Cheng J. B. (1985) Mechanism of lysosomal enzyme release from Mercenaria mercenaria granulocytes: a scanning electron microscope study. J. Invert. Pathol. 46, 189-197.
Moore M. N. and Lowe D. M. (1977) The cytology and cytochemistry of the hemocytes of Mytilus edulis and their responses to experimentally injected carbon particles. J. Invert. Pathol. 29, 18-30.
Ramssussen L. P. D., Hage E. and Karlog 0. (1985) An electron microscope study of the circulating leucocytes of the marine mussel, Mytilus edulis. J. Invert. Pathol. 45, 158-167. Ruddell C. L. (1971a) Elucidation of the nature and function of the granular oyster amoebocytes through histochemical studies of normal and traumatized oyster tissue. Histochemie 26, 98-112.
286
C.-M. Wen et al.
Ruddell C. L. (197lb) The fine structure of oyster agranular amoebocytes from regenerating mantle wounds in the Pacific oyster, Crassostrea gigas. J. Invert. Pathol. 18, 260-268. Ruddell C. L. (1971~) The fine structure of the granular amoebocytes of the Pacific oyster, Crassostrea gigas. J. Invert. Pathol. 18, 269-275. Sparks A. K. and Morado J. F. (1988) Inflammation
and wound repair in bivalve molluscs. Am. Fish. Sot. Sp. Publ. 18, 139-152. Sparks A. K. and Pauley G. B. (1964) Studies of the normal postmortem changes in the oyster, Crassostrea gigas (Thunberg) J. Invert. Pathol. 6, 78-101. Wittke M. and Renwratz L. (1985) On the capability of bivalve and gastropod hemocytes to secrete cytotoxic molecules. J. Invert. Pathol. 46, 209-210.
Pergamon
Physiol. Vol. 108A. Nos 213, pp.219-286, 1994 Ekvier Science Ltd Printed in Great Britain 0300-9629/94 $7.00 + 0.00
Light and electron microscopy of hemocyte of the hard clam, Meretrix Zusoria (Ridding) Chiou-Ming
Wen, Guang-Hsiung
Kou and Shiu-Nan Chen
Department of Zoology, National Taiwan University, Taipei, Taiwan 106, ROC Hemocytes of the hard clam, Meretrix Zusoriu(R&hog), were studied using light and electron microscopy. On the basis of staining characteristics and morphological criteria, the hemocytes were class&xl into four major categories: hyalinocytes, larger eosinophilic granulocytes (LEGS), smaller eosinophilic granulocytes (SEGs) and fibrocytes. SEGs were the most numerous components of the hemocytes, while few hyalinocytes were observed under normal conditions in the clam. Morphological characteristics of the granulocytes differed from the granulocytes of marine bivalves that have been reported previously. Granules of LEGS, though, were similar to those of SEGs using light microscopy but were completely different with electron microscopy. Key words: Hemocyte; Hard clam; Meretrix lusoriu; Bivalve; Microscopy. Comp. Biochem. Physiol. lO$A, 279-286,
1994.
Introduction Hemocytes have been shown to play a significant role in the defense mechanisms of bivalves (see reviews by Cheng, 1967; Cheng and Rifkin, 1970; Feng et al., 1977; Feng, 1988; Fisher, 1986). Generally, they protect against foreign materials by an inflammatory process, aggregation, phagocytosis, hyperplasia or encapsulation. Furthermore, hemocytes participate in wound repair, digestion and detoxification processes of different xenobiotics (Bang, 1961; Cheng, 1981; Cheng and Cali, 1974; Feng, 1966; Moore and Lowe, 1977; Ruddell, 1971b; Sparks and Morado, 1988). In addition, hemocytes of some species have been shown to be capable of secreting cytotoxic molecules (Wittke and Renwrantz, 1985; Leippe and Renwrantz, 1985). As hemocytes are predominantly responsible for deCorrespondence to: Chiou-Ming Wen, Room 201, Institute of Fishery Biology, National Taiwan University, Taipei, Taiwan 106. Fax 886-23687122. Received 8 June 1993; accepted 23 July 1993. 279
fense against pathogens, a more thorough understanding of the morphology and functions of hemocytes will help us to better understand these defense mechanisms. Hemocytes have been clearly classified into two major types: agranulocytes and granulocytes, depending on the presence of cytoplasmic granulocytes (see review by Auffret, 1988). Granulocytes are further subdivided into eosinophilic, basophilic, small or large granulocytes, while agranulocytes are divided into hyalinocytes and fibrocytes, depending on the characteristics of the granules and cell size. However, there is diversity in bivalve hemocyte types even among the same family. Granulocytes in the Pacific oyster, Crassostrea gigus, for example, are subclassified into acidophilic granulocytes and basophilic granulocytes, whereas they are subclassified into basophilic granulocytes, eosinophilic granulocytes, and small granulocytes for the Eastern oyster, C. virginicu (Ruddell, 1971a, b, c; McCormick-Ray and Howard,
280
C.-M. Wen et al.
1991). As relatively few species have been investigated, hemocytes of more species need to be studied, and a suitable scheme established that could be applied to a variety of species. The purpose of this paper, therefore, is to describe the morphological characteristics of the hemocytes of Meretrix lusoriu. Such descriptions are obviously essential as a preface to studies concerned with their functions as related to internal defense. In addition, results of the present study will be helpful for the establishment of a bivalve hemocyte classification scheme.
Materials and Methods of the hard clam, Meretrix 3.5-4.5 cm), were obtained from a commercial source in Taipei and maintained in seawater (salinity of 15%) prior to use. Approximately 0.5 ml hemolymph was collected from the posterior adductor muscle sinus of each clam using a 2.5-ml syringe fitted with a 21-gauge hypodermic needle. Specimens
Zusoria Ridding (shell length
Fixed and stained smears
Cells examined as stained preparations consisted of 0.25 ml of freshly collected hemolymph placed on glass slides and permitted to settle for 10 min at room temperature (26 + 1C). The adhered cells were fixed with 10% seawater-buffered formalin, absolute methanol, or Carnoy’s fixative (acetic acid and methanol 1: 3) for 5 min. Subsequently, the attached cells fixed by formalin were rinsed twice in Sorenson’s buffer, pH 6.5, while the absolute methanol and Carnoy’s-fixed cells were rinsed with fresh absolute methanol. The slides were stained with 10% Giemsa’s, Wright’s, or Mayer’s hematoxylin and eosin stains (H & E), dehydrated, and mounted in Entellan@ (Merck). Electron microscopy preparations
Five millilitres of hemolymph were collected and centrifuged at 800 t-pm for 10 min (Hitachi 05PR-22). Cell pellets were subsequently pre-fixed with 2.5% seawater-glutaraldehyde and post-fixed with 0~0~ for 30 min, at 4°C. Samples were dehydrated in a graded series of ethanol
and absolute acetone, and embedded in Spurr’s resin. Ultrathin sections were cut with a LKB-2088 ultramicrotome equipped with a glass knife, mounted on Formvar@ grids, and stained with uranyl acetate and lead citrate, according to Frasca and Parks (1965). The sections were examined using an Hitachi H-6000 electron microscope at 75 kV.
Results In preparations fixed with 10% buffered formalin, absolute methanol or Carnoy’s with Giemsa’s, fixative and stained Wright’s, or H & E stains, spread hemocytes verified the presence of two categories: those with granules and those without. Granulocytes were recognized as oblong, pleomorphic, or round cells with granules of varying density and intensity. Agranulocytes either completely lacked, or contained very few, granules. Depending on the fixative and/or stain employed, there were some variations in the staining characteristics of constituents of the cells. In comparison, cells fixed with 10% formalin resulted in better cytoplasmic staining, while those fixed with Carnoy’s resulted in more vivid contrast of their nuclei. Granular size, intensity and staining provided the basis for differentiating the granulocytes. Eosinophilic granulocytes contained pink, orange or purple granules surrounded by slight blue cytoplasm. Depending on the granular size, the eosinophilic granulocytes could be divided into large and small granular granulocytes (Fig. 1). The small eosinophilic granular granulocytes (SEGs) were the most numerous cell type, while the large eosinophilic granular granulocytes (LEGS) were the largest cell type but found in fewer numespecially hyalibers. Agranulocytes, nocytes, were rarely observed in smear preparations as compared with granulocytes. In addition, profiles of the LEGS generally showed that the granules filled up the endoplasm, pushing the nucleus to one side. Occasionally, LEGS with multinuclei and vacuoles were observed (Fig. 2). The agranulocytes could be classified as hyalinocytes and fibrocytes based on size and cytoplasmic characteristics, according to the classification scheme of Foley and
Hemocytes of the hard clam, Meretrix lusoriu
Fig. 1. Photomicrograph of stained hemocytes of Meretrix lusoria. Eosinophilic granulocytes. Hemocytes were fixed with 10% buffered formalin and stained with 50% Wright’s stain. Bar = 50 pm. Fig. 2. Photomicrograph of stained hemocytes of M. iusoria. Note the multinuclei LEGS. (Formalin fixed, Wright’s stained. Bar = 50 pm. Fig. 3. Photomicrograph of stained hemocytes of M. lusoria. Note the hyalinocyte (arrow) and fibrocyte (arrow head). Methanol fixed, Giemsa stained. Bar = 50pm. Fig. 4. Photo~~rog~ph of stained hemocytes of M. lusoria. Cell clumps. Note the pleomorphic nuclei of hemocytes. Carnoy’s fixed, Giemsa stained. Bar = 50 pm. Fig. 5. Photomicrograph of stained hemocytes of M. lusoria. Note the basophilic granulocyte. Carnoy’s fixed, Giemsa stain. Bar = 50 pm.
282
C.-M.
Wen et al.
Cheng (1974). The hyalinocytes were smaller (usually 4-5 pm), basophilic and contained a large, central nucleus (Fig. 3). They regularly represented spheres with few cytoplasmic processes. Fibrocytes were larger, with slight basophilic cytoplasm,
and in some cases, contained many vacuales (Fig. 3). Also, clumps of two to 10 adhered hemocytes were observed. The clumps formed immediately after the hemolymph was withdrawn. Cells would migrate from
Fig. 6. Electron micrograph of a Meretrix lusoria large eosinophilic granular granulocyte. Bar = 1 pm. GA: Golgi apparatus; LL: large lysosome-like vesicle; N: nucleus; M: mitochondrion; P: pseudopodium. Fig. 7. Electron micrograph of a smaller eosinophilic granular granulocyte with prominent Golgi apparatuses and microtubules. Bar = 1 pm. GA: Golgi apparatus; LV: lucid vesicle; SL: small lysosome-like vesicle. Fig. 8. Electron micrograph of portion of a small eosinophilic granular granulocyte containing smaller lysosome-like vesicle. Bar = 1 pm. C: centriole; GA: Golgi apparatus; LV: lucid vesicle; MT: microtubule; SL: small lysosome-like vesicle. Fig. 9. Electron micrograph of a portion of a small eosinophilic granular granulocyte. Note the fusion of vesicles (arrow). Bar = 1 pm. LV: lucid vesicle; SL: small lysosome-like vesicle.
Hemocytes of the hard clam,
Meretrix
lusoria
283
Fig. 10. Electron pseudopodia and G: Fig. 11. Electron
micrograph of two fibrocytes. The upper one shows several long projected the lower one shows glycogen-like granules in the cytoplasm. Bar = 1 pm. glycogen-like granules; rER: rough endoplasmic reticulum. micrograph of a fibrocyte, phagosomes (Pg) and fibrils (F) are shown. Bar = 1 pm. Fig. 12. Electron micrograph of a hyalinocyte. Bar = 1 pm. Fig. 13. Electron micrograph of a slightly granular hyalinocyte with larger nuclear/cytoplasm ratio. Bar = 1 pm.
the clumps after adhering to the slide. Usually, pleomorphicnuclear granulocytes and/or fibrocytes constituted the center of the clumps (Fig. 4). Basophilic granulocytes were occasionally observed (Fig. 5). Electron micrographs of the hemocytes of M. lusoria revealed the occurrence as shown under light microscopy. The most conspicuous type of organelles in granulocytes was the membrane-bound vesicles, which were the large or small eosinophilic granules present during light microscopy.
These vesicles were scattered throughout the endoplasm. The membrane-bound specific vesicles of the granular hemocytes showed different morphological characteristics. Therefore, granulocytes were easily divided into two types, one with small vesicles (diameter ranging from 0.4-l .3 ,um) that contained fine electron-dense particles and the other with larger vesicles (diameter ranging from 1.O-2.9 pm) which were homogeneous (Figs 6-8). Also, the vesicles differed in their outline, the former being
284
C.-M. Wen et al.
irregular, while the latter was circular. In some cases, fusion of small vesicles was observed (Fig. 9). Besides the electrondense vesicles, electron-lucid vesicles were frequently seen in the cytoplasm of hemocytes. Granulocytes (as shown in Figs 6-8) revealed some other characteristics in electron microscopy. Smooth and rough endoplasmic reticulum were distributed throughout the cytoplasm and Golgi apparatuses were frequently observed. The nuclei of granulocytes, depending on the plane of section, were spherical, bean shaped or lobule. Microtubules associated with a centriole in the cytoplasm were observed at different times (Fig. 8). Fibrocytes showed morphologies similar to granulocytes except that there were few or no electron-dense vesicles in the cytoplasm. They usually had a number of filopodial projections at their peripheries. Glycogen-like granules and fibrils were present in some profiles (Figs 10 and 11). The hyalinocytes (as shown in Figs 12 and 13) were also generally small with a circular or slightly oval outline. Their nuclei usually filled the entire cell. Some of the cells contained visible Golgi apparatus, with a few electron-dense vesicles.
Discussion Although many studies on the hemocytes of bivalves exist, few species have been studied and there is, as yet, no widely accepted nomenclature for bivalve hemocyte types. In an attempt to classify hemocytes, Cheng (1981) presented a morphological scheme based on numbers of cytoplasmic granules, dividing the cells into two types: (1) hyalinocyte, a cell containing few or no granules; and (2) granulocyte, a cell containing granules ranging from very few to numerous. Fibrocytes were granulocytes, in some cases degranulated, meaning there were no granules in the cytoplasm (Cheng and Foley, 1975). The results reported herein are in agreement with these two types of hemocytes. Hyalinocytes (named lymphocyte-like cells by Feng et al., 1971; McCormick-Ray and Howard, 1991) are the most striking aspect in hemocyte morphology; however, their functions remain unclear. According
to a model established by Mix (1976), hyalinocytes are an original cell type of granulocytes which could differentiate into various types of granulocyte. Results of this study tend to support this idea, as intermediate cells of slightly granular hyalinocytes with larger and smaller nuclear/cytoplasm ratios have been found under electron microscopy. It seems that the differentiation of granulocytes is associated with Golgi apparatus because the Golgi apparatus is (1) rarely found in agranular hemocytes; (2) occasionally observed in slightly granular hyalinocytes; and (3) well developed in the two types of eosinophilic granulocytes. The Golgi apparatus is suggested to be the producer of numerous electron-lucid and electron-dense vesicles, so that those vesicles increase following the cells’ differentiation. Two types of eosinophilic granulocytes were observed in this investigation, and compared with those found in species discussed in previous studies. Granulocytes of A4. Zusoriu were different from those of Mercenariu mercenuriu (Cheng and Foley, 1975; Foley and Cheng, 1974), MytiZus edulis (Rasmussen et al., 1985), Crussostreu gigus (Ruddell, 1971a, c), C. oirginicu (Feng et al., 1971; Foley and Cheng, 1972), Pecten muximus, or Tapes philippinurum (Auffret, 1988) as compared using figures of light and electron microscopy. It should be noted, in the case of M. Zusoriu, the electron-dense vesicles of SEGs clearly include fine particles with an irregular outline, while those of LEGS contained homogeneous smeared materials with a circular edge. The functions and compositions of these two types of granules are unclear. Degranulation of bivalve hemocytes has been demonstrated by several investigators (Hirsch and Chon, 1960; Feng et al., 1977; Mohandas et al., 1985), and could be associated with the intracellular fusion of lysosomes with phagosomes, as well as with the extrusion of intact lysosomes from hemocytes into the extracellular medium (see review by Feng, 1988). Therefore, the granules (vesicles) of the two types of eosinophilic granulocytes are two different forms of lysosomes. However, the contents of these vesicles remain unclear. Centrioles, with ordered microtubules observed in this study, have been seen previously (Cheng and Foley,
Hemocytes of the hard clam, Meretrix lusoria
1975), but their significance remains to be understood. Multinucleated cells, as reported in C. gigas by Sparks and Pauley (1964) and in M. mercenaria by Foley and Cheng (1974), were also found in M. hsoria. Cheng and Galloway (1970), who found similar cells in the gastropod, Helisoma duryi normale, when challenged with allografts and xenografts, suggested they may only occur under pathological conditions. The number or cell size of multinucleated cells tend to increase following a period of in vitro culture (authors’ unpublished data). The roles of these multi- or binucleated cells in ho, however, are unclear. In conclusion, our results support the summary made by Cheng (1981) and Auffret (1988) in their critical reviews of the existing literature concerning the hematology of bivalves. The ultrastructure of the granulocytes of M. lusoria, however, is different from granulocytes of bivalves that have been studied previously. The Golgi apparatus found in granulocytes may play a significant role in hemocyte maturation. Functions and compositions of these two forms of eosinophilic granulocytes require further research. Acknowledgements-This study was supported by the National Science Council, Republic of China (Grants: NSC 80-0209-B002-02 and 81-0209-002-06).
References Auffret M. (1988) Bivalve hemocytes morphology. Am. Fish. Sot. Sp. Publ. 18, 167-177.
Bang F. B. (1961) Reaction in injury in the oyster (Crassostrea virginica). Biol. Bull. 121, 57-68.
Cheng T. C. (1967) Marine molluscs as hosts for symbioses: with a review of known parasites of commercially important species. Ado. Mar. Biol. 5, 1424.
T. C. (1981) Bivalves. In Invertebrate Blood Cells (Edited by Ratcliffe N. A. and Rowley
Cheng
A. F.), Vol. 1, pp. 233-330. Academic Press, New York. Cheng T. C. and Cali A. (1974) An electron microscope study of the fate of bacteria phagocytosed by granulocytes of Crassostrea virginica. Contemp. Topics in Immunobiol. 4, 25-35.
Cheng T. C. and Foley D. A. (1975) Hemolymph cells of the bivalve mollusc Mercenaria mercenaria: an electron microscopical study. J. Invert. Pathol. 26, 341-351.
Cheng T. C. and Rifkin (1970) Cellular reactions in marine molluscs in response to helminth parasitism. Am. Fish. Sot. Sp. Publ. 5, 443-496.
285
Cheng T. C., Rodrick G. E., Foley D. A. and Koehler S. A. (1975) Release of lysozyme from hemolymph cells of Mercenaria mercenaria during phagocytosis. J. Invert. Pathol. 25, 161-165. Feng S. Y. (1966) Experimental bacteria1 infections in the oyster, Crassostrea virginica. J. Invert. Pathol. 8,505511.
Feng S. Y. (1988) Cellular defense mechanisms of oysters and mussels. Am. Fish. Sot. Sp. Publ. 18, 153-168. Feng S. Y., Feng J. S., Burke C. N. and Khairallah L. H. (1971) Light and electron microscopy of the leucocytes of Crassostrea uirginica (Mollusca: Pelecypoda). 2. Zellforsch. 120, 222-245. Feng S. Y., Feng J. S. and Yamasu T. (1977) Roles of Mytilus coruscus and Crassostrea gigas blood cells in defense and nutrition. In Comparative Pathobiology (Edited by Bulla L. A. and Cheng T. C.), Vol. 3, pp. 31-67. Plenum Press, New York. Fisher W. S. (1986) Structure and function of oyster hemocytes. In Immunity in Invertebrates (Edited by Brehelin M.), pp. 25-35. Springer, Berlin. Foley D. A. and Cheng T. C. (1972) Interaction of molluscs and foreign substances: the morphology and behavior of hemolymph cells of oyster, Crassostrea virginica, in vitro. J. Invert. Pathol. 19, 383-394.
Foley D. A. and Cheng T. C. (1974) Morphology, hematological parameters, and behavior of hemolymph cells of the quahaug clam, Mercenaria mercenaria. Biol. Bull. 146, 341-356.
Frasca J. M. and Parks V. E. (1965) A routine technique for double staining ultrathin sections using uranyl and lead salts. J. Cell Biol. 25, 157. Hirsch J. C. and Chon Z. A. (1960) Degranulation of polymorphonuclear leucocytes following phagocytosis of microorganism. J. exp. Med. 112, 1005-1014.
Leippe M. and Renwrantz L. (1985) On the capability of bivalve and gastropod hemocytes to secrete cytotoxic molecules. J. Invert. Pathol. 46, 209-210. McCormick-Ray M. G. and Howard T. (1991) Morphology and mobility of oyster hemocytes: evidence for seasonal variation. J. Invert. Pathol. 58, 219-230.
Mix M. C. (1976) A general model for leucocyte cell renewal in bivalve mollusks. Mar. Fish. Rev. 38, 3741.
Mohandas A., Cheng T. C. and Cheng J. B. (1985) Mechanism of lysosomal enzyme release from Mercenaria mercenaria granulocytes: a scanning electron microscope study. J. Invert. Pathol. 46, 189-197.
Moore M. N. and Lowe D. M. (1977) The cytology and cytochemistry of the hemocytes of Mytilus edulis and their responses to experimentally injected carbon particles. J. Invert. Pathol. 29, 18-30.
Ramssussen L. P. D., Hage E. and Karlog 0. (1985) An electron microscope study of the circulating leucocytes of the marine mussel, Mytilus edulis. J. Invert. Pathol. 45, 158-167. Ruddell C. L. (1971a) Elucidation of the nature and function of the granular oyster amoebocytes through histochemical studies of normal and traumatized oyster tissue. Histochemie 26, 98-112.
286
C.-M. Wen et al.
Ruddell C. L. (197lb) The fine structure of oyster agranular amoebocytes from regenerating mantle wounds in the Pacific oyster, Crassostrea gigas. J. Invert. Pathol. 18, 260-268. Ruddell C. L. (1971~) The fine structure of the granular amoebocytes of the Pacific oyster, Crassostrea gigas. J. Invert. Pathol. 18, 269-275. Sparks A. K. and Morado J. F. (1988) Inflammation
and wound repair in bivalve molluscs. Am. Fish. Sot. Sp. Publ. 18, 139-152. Sparks A. K. and Pauley G. B. (1964) Studies of the normal postmortem changes in the oyster, Crassostrea gigas (Thunberg) J. Invert. Pathol. 6, 78-101. Wittke M. and Renwratz L. (1985) On the capability of bivalve and gastropod hemocytes to secrete cytotoxic molecules. J. Invert. Pathol. 46, 209-210.