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Morphological, structural and functional characteristics of the hemocytes of the oyster, Crassostrea ariakensis.

ABSTRACT Light and electron microscopical studies were carried out to characterize the hemocytes of the oyster, Crassostrea ariakensis. Three types of hemocytes were recognized: granulocytes, large hyalinocytes and small hyalinocytes. The large hyalinocytes and small hyalinocytes were agranular cells. The large hyalinocytes presented thin cytoplasm and high nucleus: cytoplasm ratios. The small hyalinocytes were the most homogeneous in shape and showed round or oval. They have the highest nucleus: cytoplasm ratios and contained very thin cytoplasm. The granulocytes showed abundant cytoplasm and low nucleus: cytoplasm ratios. At the ultrastructural level, the large hyalinocytes cytoplasm contained a variable number of mitochondria, Golgi complex and endoplasmic reticulum. They showed a total absence of cytoplasmic granules or a few small electron-lucid vesicles of different sizes. The granulocytes showed more polymorphic than the agranular cells, with numerous pseudopodia sprouting off their surface. The granulocytes showed similar organelles, but on the contrary they had abundant electron-dense particles or electron-lucent granules in the cell cytoplasm. The small hyalinocytes presented a total absence of cytoplasmic granules and few organelles and only one or two mitochondria were sometimes observed in the cytoplasm. Total hemocyte counts gave a mean ([+ or -]SE) concentration of 2.06 [+ or -] 0.20 x [10.sup.7] cells [mL.sup.-1] of hemolymph. Differential hemocyte counts identified the granulocytes as the predominant cell type, followed by the large hyalinocytes and the small hyalinocytes. The percentages of the three cell types in the hemocyte population of C. ariakensis were 68.4%, 21.6% and 9.0%, respectively. The quantitative study of phagocytosis showed that the granulocytes were phagocytic cells and the agranular cells showed a limited phagocytic ability. After the separation in Percoll density gradient, the granulocytes were separated from agranulocytes and pure granulocytes were obtained. However, the separation of different agranular cell types (large hyalinocytes and small hyalinocytes) was not achieved by Percoll or Ficoll gradient centrifugation. Our study provides a morphofunctional basis for the cellular defense mechanisms in C. ariakensis.

KEY WORDS: Crassostrea ariakensis, hemocytes, granulocytes, hyalinocytes, phagocytosis, separation

INTRODUCTION

Hemocytes of bivalve molluscs play an important and central role in the internal defense, and are known to be involved in other processes like wound and shell repair, nutrient digestion, transport and excretion (Cheng 1981). There have been many studies on the morphology, structure, function and classification of hemocytes in bivalves. The most important reviews of the various morphofunctional aspects of the hemocytes of the whole Mollusca phylum are those of Cheng (1981) and Hine (1999), who identified 2 fundamental hemocyte types in bivalve hemolymph: granulocytes and hyalinocytes (or agranulocytes). The presence of these two types was confirmed in Mya arenaria (Huffman & Tripp 1982), Mytilus edulis (Pipe 1990), Mytilus galloprovincialis (Cajaraville & Pal 1995, Carballal et al. 1997a, 1997b), Mercenaria mercenaria (Tripp 1992), Crassostrea virginica (Ford et al. 1994) and Ruditapes decussatus (Lopez et al. 1997a). However, the classification schedules were so varied that three, four or even more morphologically different populations have been proposed by authors for various bivalve species (Moore & Lowe 1977, Cheng & Downs 1988, Hine & Wesney 1994, Nakayama et al. 1997).

The oyster, Crassostrea ariakensis, is one of the most important commercial mollusk species in China, whose natural range is from the south China coast through Southeast Asia to the western coast of the Indian subcontinent. In China, the culture of C. ariakensis has a long history in the Pearl River Delta, Guangdong Province. In recent years, the mass mortality has occurred in cultivated oysters with a great loss. Some studies revealed that the oysters were infected by the pathogen, a Rickettsia-like organism (RLO) (Wu & Pan 2000, Sun & Wu 2004). However, no systematic studies have been carried out to investigate the morphology, structure, function and classification of hemocytes of C. ariakensis. A better understanding of the defense mechanisms in this bivalve species may lead to practical approaches to control RLOs and to avoid mass damage. Here we report the systematic morphological and structural characteristics of the hemocytes in the hemolymph of the oysters. Phagocytosis and separation of hemocytes by discontinuous density gradient centrifugation were also studied. Our study provides a morphofunctional basis for the cellular defense mechanisms in C. ariakensis.

MATERIALS AND METHODS

Hemolymph Collection

The oysters, C. ariakensis (length: 6.0-9.8 cm; width: 4.5-6.6 cm; height: 9.0-14.0 cm) were collected from Hailing Bay in Yangxi County of Guangdong Province, China. Approximately 0.5-1 mL of hemolymph was extracted from the posterior adductor muscle of each animal using a 25-gauge needle into an equal volume of either Baker formol-calcium fixative (4% formaldehyde, 2% sodium chloride, 1% calcium acetate) or 0.05 M Tris-HCl buffer (TBS; pH 7.6, containing 2% sodium chloride), or into an equal volume of EM fixative (2% formaldehyde, 2.5% glutaraldehyde, 2% NACI, 2 mM calcium chloride in 0.2 M cacodylate buffer, pH 7.4), as appropriate. A minimum of 20 samples was used for each immune parameter investigated.

Light Microscopy Observation

To characterize the hemocytes, the staining technique with Hemacolor kit (Merck) on hemolymph smears was carried out to distinguish the hyalinocytes and granulocytes cells. According to the presence or absence of granules in the cytoplasm of the cells, differential hemocyte counts were carried out on Hemacolor smears, and the percentages of different cell types were calculated.

Total hemocyte counts were carried out with an improved Neubauer hemocytometer using Baker's fixed hemolymph samples. Mean cell parameters (cell diameters and nuclear diameter) were calculated by measuring each cell type on hemacolor stained smears using Motic images system.

Transmission Electron Microscopy

The suspension of hemolymph fixed with transmission electron microscopy (TEM) fixative solution was centrifuged (x750 g, 10 min). The pellets were washed in Pipes buffer with sucrose for 2 h at 4[degrees]C and post fixed in 1% osmium tetroxide in Pipes buffer for 75 min at 4[degrees]C. After being washed in Pipes buffer, the cells were embedded in 1.5% agar at 40[degrees]C and quickly centrifuged (x1,700 g, 5 min). Then, the pellets were dehydrated and embedded in Epon. Ultrathin sections, (50-70 nm), were stained with uranyl acetate and lead citrate and examined in a TEM JEOL 100CXII.

Scanning Electron Microscopy

Fresh hemolymph was fixed with glutaraldehyde at 2% (v/v) in Millonig 0.2 M; pH 7.3 buffer solution, washed in buffer, post-fixed with osmium tetraoxide at 1% (p/v) and placed in dehydration slides with ethanol and amilum acetate, followed by a critical point desiccation process with C[O.sub.2] and a platinum/palladium covering. Observation was done using a JEOL MODEL 1200 scanning electron microscopy (SEM).

Separation of Hemocytes by Discontinuous Density Gradient Centrifugation

Hemolymph (0.5-1.5 mL) was withdrawn from the posterior adductor muscle of each animal using a 25-gauge needle, then collected and diluted 1:1 in a modified antiaggregant Alsever solution (MAS) (20.8 g/l glucose; 8 g/l Na citrate; 3.36 g/l EDTA; 22.5 g/l NaCl in distilled water). The hemocytes were then collected by centrifugation at x640g (4[degrees]C, 10 min). The commercial Percoll (Sigma) solution was adjusted to 1,100 mOsm by adding NaCl to a final concentration of 0.41% (w/v) and the gradients (10%, 30%, 50% and 70% (v/v)) were prepared in MAS. Oyster hemocyte pellets were resuspended in MAS and layered onto the top of a Percoll gradient composed of 70%, 50%, 30% and 10% Percoll. After centrifugation at x640g (4[degrees]C, 15 min), the hemocytes present at each density interface were collected separately with a syringe. The hemocytes appearing at interface 30/ 10% of the gradient were further separated by centrifugation through a Ficoll (type 400, Sigma) density gradient containing 20%, 15%, 10% and 5% (w/v) Ficoll prepared in MAS. After centrifugation, the cells appearing at each density interface were collected separately. The density gradient centrifugation was carried out in 13-mL tubes. Each gradient layer of 2.5 mL of Percoll or Ficoll solution and 2 mL of hemocyte suspension were layered in each tube. Each separated hemocyte subpopulations with Percoll gradient or Ficoll gradient were recovered by centrifugation and washed once in MAS. With Each separated hemocyte subpopulations at the different interfaces were made smears, and rapid hemacolor coloration was used.

Hemocyte Viability Evaluation

The viabilities of the fresh hemocytes collected from the oysters and the separated hemocyte subpopulations by Percoll discontinuous density gradient centrifugation were estimated by the 0.1% (w/v) Trypan blue test.

Phagocytosis of Zymosan

Zymosan (cell walls of Saccharomyces cerevisiae, zymosan A, Sigma) suspensions were prepared as described by Bachere et al. (1991). Zymosan particles at 40 mg 10 [mL.sup.-1] were suspended in sterile sea water (SSW) and boiled for 30 min, then washed twice and suspended in SSW before divided into aliquots and stored at -20[degrees]C. The aliquots were thawed and counted in a Mallassez cell immediately before use.

To study the phagocytosis of the external materials by oyster hemocytes, hemolymph from 20 oysters was extracted 1:3 in MAS and pooled. Eppendorf vials containing 1 x [10.sup.6] hemocytes (he) were prepared, then centrifuged (x200 g, 10 min, 4[degrees]C) to remove MAS. One milliliter of filtered seawater (FSW) containing 2.5% of MAS was then added to the vials. Phagocytosis assays were carried out by adding zymosan (zy) suspensions to the Eppendorf vials. The ratios of zy/he were 5:1. The assays were carried out at room temperature (20-23[degrees]C) for 60 min. Smears were performed at the end of phagocytosis assay. Hemocytes were fixed and stained with Hemacolor kit. The percentage of cells with phagocytosed particles was evaluated in 30 random selected microscope fields at a magnification of x1,000 in the slides.

To study the phagocytosis process with electron microscopy, hemolymph from five oysters was extracted in MAS (1:3) and pooled. Phagocytosis vials consisted of 1 x [10.sup.6] hemocytes, 5zy/he, and 80[micro]L of MAS and FSW to a final volume of 2 mL. These assays were carried out at room temperature (20[degrees]C to 23[degrees]C) for 60 min. After this time, cells were fixed (1 h, 4[degrees]C) by adding 2.5% glutaraldehyde to the phagocytosis vials. Hemocytes were then washed for 2 h, 4[degrees]C in 0.1 M PIPES buffer containing 7% sucrose (pH = 7.2), postfixed in 1% osmium tetroxide and embedded in agar and epon. Ultrathin sections (50-70 nm) were contrasted with uranyl acetate and lead citrate.

Statistical Analysis

The SPSS software was used for statistical analysis. Differences in all studied parameters were evaluated by 1-way ANOVA followed by Tukey test for comparisons. LSD test was used for multiple comparisons. Values of P < 0.05 were considered significant.

RESULTS

Light Microscopy Observation

On Hemacolor smears, 2 hemocyte types were distinguished by light microscopy: granulocytes and hyalinocytes (or agranulocytes) according to the presence or the absence of cytoplasmic granules. Hyalinocytes appeared to have 2 types: small and large hyalinocyte cells, according to the cell diameter in size (Fig. 1). The granulocytes were abundant and often appeared as either spherical cells (round hemocytes) or amoebocytes (spreading hemocytes) on the smears. The granulocytes contained numerous basophilic or refringent cytoplasmic granules and had small nuclei, which often showed the oval or eccentric shapes. On smears, endoplasm and ectoplasm of the cytoplasm could be clearly distinguished in the granulocytes. The ectoplasm presenting some thin pseudopodia showed a hyaline aspect whereas endoplasm was denser and contained cytoplasmic granules (Fig. 2). The large hyalinocytes were less abundant and often showed the round or oval shape. They often had the round and large nuclei in the center of the cells and reduced acidophilic cytoplasms, generally without granules or with few granules in the cytoplasms (Fig. 1). The small hyalinocytes were the least abundant on the smears. The cells and the nuclei often showed the round shape, and they showed large nuclei and reduced acidophilic and thin cytoplasms. Cytoplasmic granules were seldom observed in the small hyalinocytes (Fig. 1). In addition, multinucleate (2-4 nuclei) hemocytes with cytoplasmic granules were observed on these smears (Fig. 3). These multinucleate structures could be the result of the fusion of granulocytes.

[FIGURES 1-3 OMITTED]

Hemocyte Count and Measurements

Total hemocyte counts gave a mean ([+ or -]SE) concentration of (2.06 [+ or -] 0.20) x[10.sup.7] cells [mL.sup.-1] of hemolymph. A total of 1,016 hemocytes of eight oysters were counted for this study. Differential type hemocytes counts showed that the mean percentage compositions ([+ or -]SE) of granulocytes, large hyalinocytes and small hyalinocytes were 68.4 [+ or -] 1.55, 21.6 [+ or -] 1.21 and 9.0 [+ or -] 0.74 respectively. Table 1 showed the percentage of three cell types in the hemocyte population of C. ariakensis.

Table 2 showed the ranges and mean values ([+ or -]SE) of the cell and nucleus sizes and the nucleus/cytoplasmic (N/C) ratios measured on Hemacolor stained smears. Granulocyte types showed larger sizes and smaller N/C ratios than hyalinocytes. Granulocytes were about 6.8 [+ or -] 0.15 [micro]m and ranged from 3.2-9.5 [micro]m. Their nuclei were about 2.08 [- or +] 0.05 [micro]m and ranged from 1.29-4.23 [micro]m. The large hyalinocytes are homogenous in size but smaller than granulocytes, with about 3.8 [+ or -] 0.08 [micro]m in diameter ranging from 2.35-6.35 [micro]m. Their nuclei were about 2.20 [+ or -] 0.05 [micro]m and ranged from 1.2-3.07 [micro]m. The small hyalinocytes had an average size of 2.05 [micro] 0.04 [micro]m, ranging from 1.32-2.78 [micro]m. Their nuclei diameter was 1.20 [+ or -] 0.03 [micro]m, and ranged from 0.78-1.75 [micro]m. Their N/C ratios were the highest, but different among cells.

The results of the ANOVA comparison indicated a significant difference for the cell size, N/C ratio and nucleus size. The multiple comparisons with the LSD indicated a significant difference between all hemocyte types for the cell size. However, in the case of the N/C ratio, multiple comparisons indicated no significant difference between the large and small hyalinocytes, and in the case of the nuclear diameter, multiple comparisons indicated no significant difference between the large hyalinocytes and granulocyte types (Table 2).

Electron Microscopy

Electron microscopy permitted us to confirm the occurrence of three hemocyte types: granulocytes, large and small hyalinocytes in the hemolymph of C. ariakensis. The large hyalinocytes often showed round or oval shapes, and presented smooth surface. The large hyalinocytes presented the high N/C ratios and thin cytoplasms, which contained a variable number of mitochondria, Golgi complex and endoplasmic reticulum (Fig. 4-6). They showed a total absence of cytoplasmic granules or a few small electron-lucid vesicles of different sizes, some of them probably originating in the Golgi complex or the smooth endoplasmic reticulum (Fig. 4). The nucleus of the hyalinocyte appeared round or oval and often was in a central position of the cell. Some large hyalinocyte nuclei had abundant euchromatin, and some showed abundant heterochromatin in the central and the peripheral positions. The nuclei of some hyalinocytes were surrounded only by small cytoplasmic rim (Fig. 5).

[FIGURES 4-6 OMITTED]

The small hyalinocytes were the most homogeneous in shape and showed round or oval. They had the highest N/C ratios and contained very thin cytoplasm. They showed a total absence of cytoplasmic granules. The organelles such as Golgi complex and endoplasmic reticulum were not observed in the cytoplasm; however, one or two mitochondria were sometimes observed in the cytoplasm (Fig. 6, 7). The nucleus often appeared oval and held most position of the cell. The nucleus sometimes showed abundant heterochromatin in the central and the peripheral positions (Fig. 6).

[FIGURE 7 OMITTED]

The granulocytes showed more polymorphic than the hyalinocytes, and were oval or eccentric in shape. The most prominent features of the granulocytes were the numerous pseudopodia sprouting off their surfaces (Fig. 8, 9, 10), which were suggested related with the phagocytic ability of the granulocytes. Some pseudopodia of the granulocytes were slim and long, however some were thick and short (Fig. 8). Some pseudopodia complected together (Fig. 10). Engulfed vacuoles and residual bodies were also observed in the granulocytes. The granulocytes showed abundant cytoplasm and low N/C ratios. They presented similar organelles but on the contrary had abundant electron-dense particles or electron-lucent granules in the cell cytoplasm. The electron dense granules were spherical, often different in size and measured 0.2~0.4 [micro]m in diameter. The electron-dense granules were composed of a homogenous electron-dense matrix (Fig. 8). The electron-lucent granules were round, with an electron-lucent core, surrounded by an electron-dense membrane unit and often very different in size and measured 0.2~0.6 [micro]m in diameter (Fig. 9, 10). Some electron-lucent granules were rough along the peripheral; some were very smooth along the peripheral. Their nuclei appeared polymorphic such as round shape, kidney shape and bell shape (Figs. 8, 9). They often were in one end of the cells or in eccentric positions in the cell cytoplasms.

[FIGURES 8-10 OMITTED]

Scanning Electron Microscopy Observation

Observation under SEM confirmed the surface structures of the hemocytes. According to the size and the surface structure, 3 hemocyte types could be identified: (1) large cells that were usually round and showed no pseudopodia with relatively smooth surfaces, however, sometimes they showed some tiny refractive inclusions or spherule (Fig. 11); (2) small round cells that were the smallest, they were usually round and showed no pseudopodia with relatively smooth surfaces (Fig. 12); (3) irregular cells that often were irregular in shape and presented abundant tenuous and long pseudopodia they usually appeared irregular twist, poly-angle shapes and honeycomb-like surface structure; the surfaces of these cells were usually with corrugation, spongy projections and surface secretion particles (Fig. 13).

[FIGURES 11-13 OMITTED]

Separation of Hemocytes

After centrifugation in Percoll gradients, the total hemocyte population was separated into three cell fractions. Fraction present at the interface 10/30% contained only agranular hemocytes including the large and small hyalinocytes. Fraction collected from the interface 50/70% was composed of pure granulocytes. Fraction appearing at the interface 30/50% was a mixture of all hemocyte types.

However, the large and small hyalinocytes could not be separated with Percoll gradients or Ficoll gradient. By using Ficoll gradient, both fractions at the interface of 10/15% and 5/10% consisted of the large and small hyalinocytes simultaneously; the fraction at the interface of 15/20% contained cells whose composition was variable in different experiments.

Hemocyte Viability After Separation

The dead cells appeared blue staining with the Trypan blue, and the live cells did not stain with the Trypan blue (Fig. 14). The viability of the separated hemocytes collected from fractions at the interface 10/30% and 30/50% of Percoll gradients were compared with the nonseparated hemocytes. For the total hemocyte population before separation, the mean cell viability was 86.4%. After separation, the corresponding values for fractions at the interface 10/30% and 30/50% of Percoll gradients were 81.8, 82.8%, respectively. No significant difference (t-test) was detected between the viabilities of the total hemocyte population before separation and any of the two separated cell fractions.

[FIGURE 14 OMITTED]

Phagocytosis

In vitro phagocytosis assays showed that among the hemocyte types, the granulocytes showed an important phagocytic activity whereas the hyalinocytes did not. Light microscopy revealed that several zymosans were located inside the hemocytes after 60 min, and a few particles were encircled by pseudopodia. The number of zymosan particles phagocytosed per hemocyte was variable, and 6.24 [+ or -] 0.74%, 20.61 [+ or -] 1.19% and 29.07 [+ or -] 1.08% of hemocytes phagocytosed, one, two or three particles, respectively, whereas 39.08 [+ or -] 1.69% of hemocytes phagocytosed four or more particles. Electron microscopy confirmed the intracellular location of zymosan. Particles were found inside phagosomes (Fig. 15), some of the particles partially degraded. Hemocytes containing zymosan had many vacuoles that included degraded products.

[FIGURE 15 OMITTED]

DISCUSSION

Morphological criteria were generally used to characterize hemocytes in bivalves, however, the existing nomenclature of bivalve hemocytes is inconsistent, being dependent on the observer and the technique used (Feng et al. 1971, Ruddell 1971a, Ruddell 1971b, Foley & Cheng 1972, Cheng 1975, Cheng 1981, Moore & Lowe 1977, Hawkins & Howse 1982, Rasmussen et al. 1985, Chang et al. 2005). Cheng (1981) presented a morphological scheme based on numbers of cytoplasmic granules, dividing cells into 2 types: granulocytes, cells containing granules that ranged from very few to numerous; and agranulocytes, cells containing few or no granules. In this study, we identified 2 main hemocyte types in C. ariakensis, with both light and electron microscopy observation: granulocytes and agranulocytes (large hyalinocytes and small hyalinocytes). The granulocytes were characterized by the abundant content of granules, presenting a noncentral small nucleus, spreading with pseudopodia related with the phagocytic abilities. According to the cell size, agranulocytes appeared to have two types: small hyalinocytes and large hyalinocytes. The large hyalinocytes were morphologically characterized by the relatively large and central nuclei surrounded by the small volume of cytoplasm and by few or no cytoplasmic granules. The small hyalinocytes have the least cell diameter and large and central nuclei surrounded by the small volume of cytoplasm with the absence of cytoplasmic granules. Many authors suggested that the hyalinocytes were non-different cells; however, hyalinocytes were classed into 2 types, small and large hyalinocytes, by Xue et al. (2000), which supported our classification. Some studies suggested that it was possible to distinguish acidophilic and basophilic granulocytes according to the staining affinities of the cytoplasmic granules of the granulocytes (Cheng 1975, Cheng 1981, Suresh & Mohandas 1990, Nakayama et al. 1997, Lopez et al. 1997b, Wootton & Pipe 2003, Zhang et al. 2005). However, Xue et al. (2000) found the granulocytes of Ostrea edulis contained only numerous basophilic or refringent cytoplasmic granules, in agreement with our study on the staining characteristic of C. ariakensis granulocytes.

In addition, the multinucleate granulocytes were observed on smears. These cells have been observed in other mollusks (Sparks & Pauley 1964, Cheng 1981, Anderson 1987, Wootton & Pipe 2003) with light microscopy. They are considered to be the result of a fusion of granulocytes in some pathological conditions such as postmortem changes or rejection of grafts. However, the origin and progress of their forming are not clear.

The ultrastructure of the hemocytes in this study revealed that granulocytes mainly contained two types of granules: electrondense particles and electron-lucent granules. They were different in size of both types of granules. Furthermore, the granules of different size often existed inside most granulocytes simultaneously. Therefore, granulocytes were not differentiated in this study. Some investigators distinguished the granulocytes in other bivalve with only small or large granules and classed the granulocytes into large and small granulocytes. They considered that the granulocytes with small or large granules were immature or mature granulocytes, respectively (Klebanoff & Clark 1978, Rasmussen et al. 1985 Pipe 1990). However, we agree with the option of Zhang et al. (2005) that the peculiarity of granules should be determined by their origin or function, not by their size and did not believe that granulocytes with different type of granules were in different development phases, though their origin and functions were not wholly clear.

The ultrastructural study of C. ariakensis hemocytes showed that some large hyalinocytes contained nuclei with abundant euchromatin and others contained nuclei with large clumps of heterochromatin. Carballal et al. (1997c) also found this phenomenon, but they suggested that both types of hyalinocytes could belong to a different cell line, one for hyalinocytes and another for granulocytes, or hyalinocytes containing abundant euchromatin might give rise to hyalinocytes with more heterochromatin and granulocytes. However, we consider that maybe the both types of large hyalinocytes are in different development phases of the hemocytes, the large hyalinocytes containing nuclei with abundant euchromatin can give rise to hyalinocytes with more heterochromatin that are overripe. The most granulocytes containing nuclei with abundant euchromatin probably are in their bloom stage and present more phagocytic competence. Maybe some large hyalinocytes containing nuclei with abundant euchromatin also result from the overripe granulocytes after fulfilling their phagocytic functions because the nuclear diameters were not significantly different between the large hyalinocytes and the granulocytes.

There are several theories on bivalve hemocytes renewal and maturation. Moore and Eble (1977) suggested that different hemocytes are maturing stages within a single cell line. Cheng (1981) proposed an ontogenetic model with two cell lines, one for hyalinocytes and another for granulocytes, each originating from a different prohemocyte. Auffret (1988) also suggested this last hypothesis for O. edulis and C. gigas hemocytes. However, in our study the morphological variability found by light and electron microscopy in the oyster hemocytes do not allow us to confirm any of the previous hypotheses.

Total hemocyte counts showed high variability in the number of circulation hemocytes because the density of hemolymph might vary with different species, age and physical status. Because spreading ability was different between hyalinocytes and granulocytes, results of cell measurements were different according to the method used. Lopez et al. (1997a, 1997b) suggested that fixing hemocytes in suspension before measuring was a better method. In our study, differential hemocyte counts after fixing identified granulocytes as the predominant cell type (68.4%), followed by the large hyalinocytes (21.6%) and the small hyalinocytes (9.0%). In the study of other mollusks, similar results were reported. The granulocytes corresponded to roughly 75% and 66% of the total population of hemocytes and the hyalinocytes corresponded to the remaining 25% and 34% in Scrobicularia plana (Wootton & Pipe 2003) and Argopecten irradians (Xing et al. 2002) respectively. However, some authors reported the hyalinocytes were the predominant cell type (i.e., granulocytes/hyalinocytes were 44.7%/ 55.3% and 37.3%/62.7%) in A. irradians (Zhang et al. 2005) and C. virginica (H6garet et al. 2003) respectively.

The significant differences of cell size and N/C ratios existed in either hyalinocytes or granulocytes. Granulocyte types showed larger sizes and smaller N/C ratios than hyalinocytes. These fundamental features are common to many bivalve species (e.g., M. mercenaria; Foley & Cheng 1974, C. virginica; Feng 1965, C. edule; Russell-Pinto et al. 1994, M. edulis; Rasmussen et al. 1985, Friebel & Renwrantz 1995, M. lusoria; Wen et al. 1994 and A. irradians; Zhang et al. 2005).

In SEM, special emphasis had been placed on the surface structure. The observation under SEM confirmed the pseudopodia observed under TEM. Moreover, the irregular cells, large round cells and small round cells under SEM might correspond to the granulocytes, large hyalinocytes and small hyalinocytes observed under TEM.

The quantitative study of phagocytosis showed that there were functional differences between hemocyte types of C. ariakensis. The granulocytes were phagocytic cells. On the contrary, the hyalinocytes showed a limited phagocytic ability. Similar results were reported in M. edulis (Moore & Lowe 1977), Tapes semidecussatus (Montes et al. 1995), Tridacna crocea (Nakayama et al. 1997), C. virginica (Foley & Cheng 1975, Renwrantz et al. 1979), M. mercenaria (Foley & Cheng 1975), Mytilus californianus (Bayne et al. 1979), O. edulis and C. gigas (Mourton et al. 1992), Cerastoderma edule (Russell-Pinto et al. 1994), Tiostrea chilensis (Hine & Wesney 1994), M. galloprovincialis (Carballal et al. 1997c) and A. irradians (Zhang et al. 2005), but these reports were in contrast with those previously reported by Tripp (1992) on M. mercenaria, Lopez et al. (1997b) on R. decussatus and Cima et al. (2000) on Tapes philippinarum. In the latter species, the authors considered that the agranular hemocytes and granulocytes were both active phagocytes.

Phagocytic hemocytes require considerable amounts of energy in the process from phagocytosis of particles, secretion of hydrolysis enzymes, fusion and decomposition of granules to discharge of wastes. Therefore, phagocytic hemocytes often contained abundant numerous mitochondria, Golgi complex, endoplasmic reticulum, and a great quantity of glycogen. Moreover, abundant granules and many spreading pseudopodia of the phagocytic hemocytes were related to the phagocytic competence.

The role of bivalve hyalinocytes with no phagocytosis is unknown. It is possible that they develop other functions different from phagocytosis because in Cerastoderma edule they are involved in rosette formation with sheep erythrocytes (Russell-Pinto et al. 1994) and in other invertebrates, such as the crustaceans, they participate in the coagulation process (Hose et al. 1990).

Density gradient centrifugation is one of the most used techniques for separating molluscan hemocytes into subpopulations. Different materials have been applied as gradient substrates. Cheng et al. (1980) separated fixed hemocytes of C. virginic into several subpopulations by sucrose density gradient centrifugation. Clearly, sucrose is not suitable for separation of live cells, in particular, when the separation aims at studies about cell function, because sucrose solutions cannot keep the same osmolarity at different concentrations. However, Percoll, a colloidal suspension of polyvinylpyrolidone coated silica particles, has been extensively used for separation of living cells. Some investigators have applied it in separating hemocytes of C. gigas (Bachere et al. 1988), C. virginica (Cheng & Downs 1988), Lymnaea stagnalis (Adema et al. 1994), M. edulis (Friebel & Renwrantz 1995) and R. decussatus (Lopez et al. 1997b) and O. edulis (Xue et al. 2000). The results obtained in this study indicated that it was also applicable to the separation of C. ariakensis hemocytes. After the separation in this type of density gradient, the granulocytes were separated from agranulocytes and pure granulocytes were obtained. However, the separation of different agranular cell types was not achieved by Percoll gradient centrifugation. The agranulocytes had the low percentage of population of hemocytes and were difficult in adhering on the smears, so the observed agranulocytes were fewer than the granulocytes after Percoll gradient centrifugation. In addition, the granulocytes were very stained because the Percoll was not wholly washed off.

Ficoll has very high viscosity, and Ficoll density gradient had been used to separate the large and small hyalinocytes of the O. edulis (Xue et al. 2000). In this study, we also applied the technique of centrifugal elutriation, however, the large hyalinocytes and small hyalinocytes could not be separated in this study. In O. edulis, the large hyalinocytes could be separated from the small hyalinocytes, but the separation was not complete. However, the density gradient centrifugation with successive Percoll and Ficoll solutions is a practical technique for molluscan hemocyte separation, although it has different efficiency with the different molluscan hemocytes.

ACKNOWLEDGMENTS

The authors thank the team of Marine and Fishery Bureau of Yangxi County, especially Mr. M.Y. Qiu, Mr. X.B. Zhang and Mr. C.G. Cheng for their assistance with sampling the oysters. This study was supported by NSFC (30170741 and 30371107), Scientific Program of Zhejiang Province (2004C23041) and Key Science Program (KSCX2-SW-302-8) of Chinese Academy of Sciences.

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JINGFENG SUN, (1,3) XINZHONG WU (1,2) * AND WEIZHU ZHANG (1)

(1) The South China Sea Institute of Oceanology, the Chinese Academy of Sciences, Guangzhou 510301, China; (2) College of Animal Sciences, Zhejiang University, Hangzhou 310029, China; (3) College of Life Science, South China Normal University, Guangzhou 510631, China

* Corresponding author. E-mail: wuxz@zju.edu.cn
TABLE 1.

The percentage of 3 cell types (granulocytes, large hyalinocytes and
small hyalinocytes) in the hemocyte population of Crassostrea
ariakensis analyzed by the SPSS software.

 Cell Sum
 Type of cell Mean (a) [+ or -] SEM N (1016)

Granulocytes 86.7500 [+ or -] 4.45112 8 694
Large Hyalinocytes 28.8750 [+ or -] 2.40117 8 231
Small Hyalinocytes 11.375 [+ or -] 1.05115 8 91

 Percentage
 Type of cell [+ or -] SEM

Granulocytes 68.4125 [+ or -] 1.54796
Large Hyalinocytes 22.5958 [+ or -] 1.20902
Small Hyalinocytes 8.9918 [+ or -] 73910

A total of 1,016 Haemocytes were distinguished and counted on
Hemacolor's stained smears.

(a) Mean of cells on 8 smears. N, the observed smear size

TABLE 2.

Mean values ([micro]m) [+ or -] Standard Error and ranges of cell and
nuclear diameters and nuclear/cytoplasmic (N/C) ratio of hemocytes of
Crassostrea ariakensis.

 Hemocyte type N Cell diameter [+ or -] sem

Granulocyte 107 6.7937 [+ or -] .14657 (a)
Ranges 3.2857-9.5
Large Hyalinocytes 92 3.7637 [+ or -] .07966 (b)
Ranges 2.35-6.35
Small Hyalinocytes 68 2.0490 [+ or -] .04052 (c)
Ranges 1.32-2.78

 Hemocyte type Nuclear diameter [+ or -] sem

Granulocyte 2.0816 [+ or -] .05408 (a)
Ranges 1.29-4.23
Large Hyalinocytes 2.1959 [+ or -] .04491 (a)
Ranges 1.2-3.07
Small Hyalinocytes 1.2013 [+ or -] .03312 (b)
Ranges 0.78-1.75

 N/C
 Hemocyte type ratio [+ or -] sem

Granulocyte .3144 [+ or -] .01061 (a)
Ranges 0.171291-0.79520
Large Hyalinocytes .5924 [+ or -] .01020 (b)
Ranges 0.321809-0.822148
Small Hyalinocytes .5988 [+ or -] .01809 (b)
Ranges 0.337662-0.890052

Measurements were made on Hemacolor's stained smears. N, sample size.

The multiple comparisons were made by LSD. Different letters (a, b, c)
at the same column showed significant difference, and the same letters
(a, b, c) at the same column showed no significant difference between
the mean values. Values of P < 0.05 were considered significant.
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Author:Zhang, Weizhu
Publication:Journal of Shellfish Research
Geographic Code:1USA
Date:Apr 1, 2006
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