Morphological and functional characterization of the hemocytes of the scallop, Chlamys farreri.

ABSTRACT Light, transmission and scanning electron microscopical studies were carried out to characterize the hemocytes of the scallop Chlamys farreri (Jones & Preston). Five types of hemocytes were recognized: type 1 small hyalinocytes (2.44 [+ or -] 0.11 [micro]m, 45% to 50%), type 2 large hyalinocytes (4.83 [+ or -] 0.28 [micro]m, 15-20%), type 3 small granulocytes (4.07 [+ or -] 0.15 [micro]m, 15% to 20%), type 4 medium granulocytes (7.20 [+ or -] 0.26 [micro]m, 20-25%) and type 5 large granulocytes (13.87 [+ or -] 0.73 [micro]m, 3% to 5%). Granulocytes showed larger sizes and smaller N/C ratios than hyalinocytes. The mean hemocyte concentration was about (3.03 [+ or -] 0.11) x [10.sup.7] cells [mL.sup.-1]. Among hemocytes, 42.6% are granular cells and 57.4% are agranular cells. These gave a relatively systematic classification scheme for hemocytes of Chlamys farreri. Three kinds of granules were identified: high electron-dense granules, low electron-dense granules and medium electron-dense granules based on TEM studies. Both granulocytes and hyalinocytes showed phagocytic response to the two strains of bacteria, E. coli and Rickettsia-like organisms. The phagocytic ability of granulocyte was significantly higher (30% to 40%) than that of hyalinocyte (4.8% to 14%).

KEY WORDS: scallop, Chlamys farreri, hemocyte, hyalinocytes, granulocytes, morphology, phagocytosis 

INTRODUCTION

Internal defense in molluscan species utilizes an innate, non-lymphoid immune system, which consists of cellular and humoral components of the circulatory system that work together to eliminate potentially harmful microorganisms. Scallops, like all other bivalve molluscan, have an open circulatory system. Bivalve blood cells or hemocytes play an important 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. Foley & Cheng (1972) identified two hemocyte types in Crassostrea virginica hyalinocytes and granulocytes. Several other authors have reported this classification scheme in bivalves based on morphological, cytochemical and functional criteria (Cheng 1975, Cheng 1981, Suresh & Mohandas 1990, Pipe 1990, Kumazagua et al. 1991). It has been known that there are various types of hemocytes in bivalves, and there are some differences between different species.

Most of the studies on bivalve hemocytes have been carried out in oysters, mussels and clams. Only a few were performed in species belonging to the Pectinidae family: Patinopecten yessoensis (Nakamura et al. 1985), Pecten maximus (Le Gall et al. 1991), Chlamys farreri (Xing et al. 2002, Sun & Li 2003). Chlamys farreri is one of the most important commercial species in China because it is a traditional cultivated species in northern China, and its mass scale cultivation has been successful for more than 20 y along the coast of the Bohai Sea, the Yellow Sea and the adjacent waters in China. However, mass mortalities have occurred in the cultivated scallop with a great loss in recent years. Some studies revealed that populations of this species were affected by several pathogens: Rickettsia-like organisms (RLO), mycoplasma-like organisms (MLO) and virus-like particles (VLP) (Zhang & Wu 2003 [double dagger]). However, no systematic studies have been carried out to investigate the morphology, structure, function and classification of hemocytes of Chlamys farreri. A better understanding of the defense mechanisms in this bivalve species may lead to practical approaches to control RLOs or other diseases and to avoid mass damage. Here we report our systematic morphological and structural characterization of hemocytes in the hemolymph of the scallop. Our study provides a morphological basis for the cellular defense mechanisms in this organism.

MATERIALS AND METHODS

Hemolymph

About 200 scallops (shell length 3-7 cm) were collected from farms in Changdao, Shandong province, China. Approximately 0.2-0.3 mL of hemolymph was extracted from the posterior adductor muscle of each animal using a 25-gauge needle and added to an equal volume of either Baker's formol-calcium fixative (4% formaldehyde, 2% sodium chloride, 1% calcium acetate) or to 0.05 M Tris-HCl buffer (TBS, pH 7.6, containing 2% sodium chloride), or to an equal volume of EM fixative (2% formaldehyde, 2.5% glutaraldehyde, 2%NaCl, 2 mM calcium chloride in 0.2 M cacodylate buffer, pH 7.4), as appropriate. Hemolymph sample was pooled in several centrifugal tubes (10 mL). A minimum of 20 individuals was used for each hemocyte parameter investigated.

Light Microscopy Observation

To characterize the hemocytes, three staining techniques, Giemsa, Wright and Hemacolor stain on hemolymph smears were used, from which hyalinocytes, granulocytes, acidophilic and basophilic cells were distinguished. The percentages of hyalinocytes and granulocytes were calculated depending on Hemacolor smears.

Total hemocyte counts were carried out with an improved Neubauer hemocytometer using Baker fixed hemolymph samples. Mean cell diameters were calculated by measuring 25 of each cell type in Hemacolor stained smears using Motic Images system. Statistical analysis was performed by using the SPSS software to determine whether there was a significant size difference between cell types. Values of P < 0.05 were considered significant.

Scanning Electron Microscopy Observation

Fresh hemolymph was fixed with glutaraldehyde at 2% (v/v) in Millonig 0.2 M, washed in buffer (pH 7.3 buffer solution), postfixed with osmium tetraoxide at 1% (v/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. Observations were made under a Jeol model 1200 scanning electron microscope.

Transmission Electron Microscopy Observation

The suspension of hemolymph fixed with EM fixative solution was centrifuged (750g, 10 min). The pellets were washed in Pipes buffer with sucrose for 2 h at 4[degrees]C and postfixed in 1% osmium tetraoxide 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 (1,700g, 5 min). Then, the pellets were dehydrated and embedded in Epon 812. Ultrathin sections (50-70 nm) were stained with uranyl acetate and lead citrate and observed under a JEOL 100CXII transmission electron microscope.

Phagocytosis of Bacteria

Two microorganisms E. coli and Rickettsia-like-organisms (RLO) were used in this study. The E. coli strain was obtained from the Laboratory of Immunology, College of Veterinary Medicine, China Agricultural University. RLO was suggested a putative pathogen to Chlamys farreri causing Rickettsia disease. RLO suspension was obtained by purification of homogenate of diseased scallop tissues. Before the phagocytosis assay, both microorganisms were suspended in filtered sea water (FSW) (~[10.sup.8]) and activated 1 h at room temperature (20[degrees]C to 23[degrees]C).

To study the phagocytosis of these bacteria by scallop hemocytes, hemolymph from 20 scallops was extracted 1:3 in modified antiaggregant Alsever solution (MAS) (20.8 g/liter glucose; 8 g/liter Na citrate; 3.36 g/liter EDTA; 22.5 g/liter NaCl in distilled water) and pooled. Phagocytosis vials contained 50 [micro]l MAS with 1 x [10.sup.6] hemocytes, and 50 [micro]l bacteria suspension. The experiments were performed at room temperature (20[degrees]C to 23[degrees]C) for 30 min in triplicate. Subsequently, smears were prepared and stained with Giemsa and Hemacolor. The percentage of phagocytic cells corresponding to each hemocyte type was estimated in 20 random selected microscope fields at x400.

RESULTS

Light Microscopy Observation

In Hemacolor smears two hemocyte categories, granulocytes and hyalinocytes, were distinguished by light microscopy observation (LMO) according to the presence or the absence of cytoplasmic granules. It was possible to distinguish acidophilic and basophilic granulocytes according to the staining affinities of their cytoplasmic granules. Acidophilic cytoplasmic granules stained red-brown or pink, and basophilic cytoplasmic granules stained blue. Hyalinocytes appeared as small and large cells. The small hyalinocytes had an average size of 2.44 [+ or -] 0.11 [micro]m and ranged from 1.28-3.56 [micro]m. The nucleus was 1.45 [+ or -] 0.38 [micro]m. The large hyalinocytes had an average size of 4.83 [+ or -] 0.28 [micro]m and ranged from 3.38-8.25 [micro]m. Multinucleate cells were usually observed in this type. Their cytoplasm was not stained and diaphanous, and no granules detected (Figs. 1, 2).

[FIGURES 1-2 OMITTED]

There were 3 types of granuloctytes: small cells, medium cells and large cells. Small granulocytes were 4.07 [+ or -] 0.15 [micro]m and ranged from 2.44 [micro]m to 5.3 [micro]m. The medium granulocytes were 7.20 [+ or -] 0.26 [micro]m and ranged from 5.44 [micro]m to 9.80 [micro]m. The large granulocytes were 13.87 [+ or -] 0.73 [micro]m and ranged from 10.32 [micro]m to 23.50 [micro]m.

Total hemocyte counts gave a mean concentration of (3.03 [+ or -] 0.11) x [10.sup.7] cells [mL.sup.-1] of hemolymph. Differential hemocyte counts, which separated the cells by the presence or absence of granules, identified a hemocyte population containing 42.6% granular cells and 57.4% agranular cells (Table 1).

Table 2 shows the ranges and mean values of the cell and nucleus size, and the N/C ratio measured in Hemacolor smears. Granulocyte types were larger in size and smaller in N/C ratios than hyalinocytes. Significant differences were found in hemocyte diameter among five types of scallop hemocytes, which were determined by SPSS software (df = 4, F = 138.224, P < 0.001).

Scanning Electron Microscopy Observation

Four hemocyte shapes could be observed according to size and shape using SEM.

(1) Round cell: These cells were small, about 1-3 [micro]m, round-shaped with no pseudopodia and with a smooth surface (Fig. 3). They occupied a higher percentage of about 55% in the hemocyte population.

(2) Pear cell: These cells were relatively small, about 1.5-3.0 x 3.5-5.0 [micro]m. They usually were pear-shaped with no pseudopodia and with a smooth surface (Fig. 3). They occupied a percentage of about 20% in the hemocyte population.

(3) Irregular polyhedral cell: These cells were large, about 6-10 [micro]m, and usually appeared to have irregular polyhedral shapes. The surfaces of these cells were usually corrugated, with surface secretion particles and several pseudopodia (Fig. 3). They occupied a percentage of about 24% in the hemocyte population.

(4) Large cell: These cells were large, about 10-25 [micro]m, and usually appeared to have irregular polyhedral shapes and honeycomb-like surface structure. The surfaces of these cells were usually corrugated with spongy projections, more surface secretion particles and several pseudopodia (Fig. 4). They comprised only about 1-2% in the hemocyte population.

[FIGURES 3-4 OMITTED]

Transmission Electron Microscopy Observation

Two hemocyte categories, namely granulocytes and hyalinocytes could be determined under TEM.

The hyalinocytes showed thin cytoplasm with no granules, and almost no cell organelles could be seen in this cell cytoplasm. The cell and the nucleus were mostly spherical (Fig. 5,6). Transmission electron microscopy observation (TEMO) permitted us to confirm the occurrence of many small hyalinocytes (~2 [micro]m) (Fig. 7). Generally, hyalinocytes were smaller in size and larger in N/C ratio than that of granulocytes as previously described using LM.

[FIGURES 5-7 OMITTED]

The granulocytes showed a denser cytoplasm, which contained granules with different sizes and different electron density (Fig. 8, 9, 10, 11, 12). Three kinds of granules could be distinguished from electron micrographs (Fig. 8-12). Electron-dense granules were about 0.1-0.6 [micro]m in size, either with or without a surrounding membrane. Low electron-dense granules were about 0.1-0.5 [micro]m in size, and usually surrounded by a membrane. Medium electrondense granules were about 0.2-1.0 [micro]m in size, and usually surrounded by a membrane. The cell and nuclear morphology of granulocytes showed more diversity than that of the hyalinocytes. Most of the granulocytes were polymorphic and have long pseudopodia (Fig. 8, 9). The nucleus appeared polymorphic including round, kidney, and bell shapes with hilobate nuclei, trilobate nuclei and tetralobate nuclei (Fig. 12). The cytoplasm contained a variable number of mitochondria, Golgi complex, endoplasmic reticulum, and abundant ribosome granules (Fig. 8-13).

[FIGURES 8-13 OMITTED]

Phagocytosis of Bacteria

LMO revealed both granulocytes and hyalinocytes phagocytosed the two strains of bacteria, E. coli and RLO incubated in suspension. But not all cells were shown to be actively phagocytic. Table 3 showed the results of scallop hemocyte phagocytosis of E. coli and RLO. Percentages of phagocytic cell for all hemocytes were 25.4% for E. coli, and 21.7% for RLO, respectively. The percentages of phagocytic granulocytes were 40.1% for E. coli, and 30.3% for RLO, respectively, and phagocytic hyalinocytes were 14.5% for E. coli, and 4.8% for RLO, respectively. Data indicated the phagocytic ability of granulocytes was significantly higher than that of hyalinocytes.

DISCUSSION

Two categories of hemocyte, granulocytes and hyalinocytes, were distinguished based on the presence or absence of cytoplasmic granules, respectively. Acidophilic and basophilic granules, could be distinguished according to the staining affinities of their cytoplasmic granules. Acidophilic cytoplasmic granules stained red-brown or pink, whereas basophilic cytoplasmic granules stained blue. These results conformed to those of other bivalves reported by many authors (Cheng 1975, Cheng 1981, Suresh & Mohandas 1990, Kumazagua et al. 1991). This study showed that Chlamys farreri hemocytes could be classified 5 types: small hyalinocytes (type 1), large hyalinocytes (type 2), small granulocytes (type 3), medium granulocytes (type 4), and large granulocytes (type 5) according to cell size, granular features, and some structural characteristics.

Total hemocyte number for Chlamys farreri had showing high variability (Xing et al. 2002, Sun & Li 2003). It is possible because the density of hemolymph may vary with scallop age, physical status and the methods used. Differential hemocyte counts, which identified hyalinocyte as the predominant cell type (G/H: 42.6%/ 57.4%), were not in agreement with the results (G/H: 66%/34%) from Xing et al. (2002). The multinucleate cells were observed in granulocytes and hyalinocytes. These results agreed with Wootton et al. (2003) for Scrobicularia plana. The type 1 small hyalinocytes corresponded to lymphocyte-like cells by Sun & Li (2003) on Chlamysfarreri, depending on their structure and size. But the percentage of type 1 cell (45% to 50%) was much higher than that of lymphocyte-like cells (5% to 10%) by Sun & Li (2003). We have noticed small hyalinocyte easily lost from Giemsa stained smear slides.

Four hemocyte shapes observed under SEM did not wholly conform to the five types of hemocyte characterized under LM. In SEM, special emphasis is placed on surface structure. However, round cells might correspond to the type 1 small hyalinocytes identified under LM; Pear cell might correspond to type 2 hyalinocytes and type 3 small granulocytes identified with LM; Irregular polyhedral cell might correspond to type 4 medium granulocytes and partly type 2 large hyalinocytes identified with LM; Large cell might correspond to type 5 large granulocytes identified with LM.

The ultrastructure of the hemocytes revealed that granulocytes contained three kinds of granules. The peculiarity of granules should be determined by their origin or function, not by their size. It was hypothesized that the electron-dense (type 1) granules were phagocytosed foreign materials (microorganisms, or other granules) or internal waste materials (organelles or fragments); the low electron-dense granules (type 2) primary lysosomes, probably originating in the Golgi complex or endoplasmic reticulum; and the medium electron-dense granules, secondary lysosomes, formed by fusion of type 1 and type 2 granules or matured by type 1 granules, which absorbed some hydrolytic enzymes. Phagocytic hemocytes require considerable amount of energy in the process of phagocyting particles, secretion of hydrolytic enzymes, fusion and decomposition of granules and discharge of wastes. Therefore, phagocytic hemocytes often contain numerous mitochondria, endoplasmic reticulum and a great quantity of glycogen (Fig. 8-13).

Two strains of bacteria E. coli and RLO were used and each was phagocytosed by the blood cells. The quantitative study of phagocytosis showed that there are functional differences between Chlamysfarreri hemocyte categories in this study. The percentage of phagocytic cell in granulocyte (about 40%) was significantly higher than that in hyalinocyte (14.5%). It was suggested that the granulocytes might be phagocytic cells. On the other hand, the hyalinocytes showed a limited phagocytic ability. Similar results were reported in most bivalves (Foley & Cheng 1975, Hine & Wesney 1994, Russell-Pinto et al. 1994). The role of bivalve hyalinocytes is unknown. It is possible that they develop other functions different from phagocytosis such as nutrition or coagulation process.

ACKNOWLEDGMENTS

This study was supported by NSFC (NO. 30170741), Key Science Program (KSCX2-SW-302-8) of Chinese Academy of Sciences.

LITERATURE CITED

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Cheng, T. C. 1981. Bivalves. "Invertebrate Blood Cells" N. A. Ratcliffe & A. F. Rowley, editors. London: Academic Press. pp. 233-300.

Foley, D. A. & T. C. Cheng. 1972. Interaction of mollusks and foreign substances: The morphology and behavior of hemolymph cells of the American oyster, Crassostrea virginica, in vitro. J. Invertebr. Pathol. 19:383-394.

Foley, D. A. & T. C. Cheng. 1975. A quantitative study of phagocytosis by hemolymph cells of the pelecypods Crassostrea virginica and Mercenaria mercenaria J. Invertebr. Pathol. 25:189-197.

Hine, P. M. & B. Wesney. 1994. Interaction of phagocytosed Bonamia sp. (Haplosporidia) with hemocytes of oysters Tiostrea chilensis. Dis. Aquat. Org. 20:219-229.

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Nakamura, M., K. Mori, S. Inooka & T. Nomara. 1985. In vitro production of hydrogen peroxide by the amoebocytes of the scallop, Patinopecten yessoensis. Dev. Comp. Immunol. 9:407-417.

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Xing, Jing, W. B. Zhan & L. Zhou. 2002. Endoenzymes associated with hemocyte types in the scallop (Chlamys farreri). Fish Shellfish Immunol. 13:271-278.

Zhang, W. Z. & X. Z. Wu. 2003. Advances in studies on pathogens and causes of mortalities of cultivated scallop in China (in Chinese)[C]. In: Chinese SCI & TECH Association, Green paper on Prevention and Treatment of Pest, 166-170.

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

(1) 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) Biology Department, Chuzhou University, Chuzhou, 23900, China

* Corresponding author. E-mail: [e-mail].

TABLE 1.
The densities of hemocyte and percentages of granulocyte and
hyalinocyte.

Hemolymph density             Granulocyte

(3.03 [+ or -] 0.11) x
  [10.sup.7] cell/ml     (42.6 [+ or -] 10.77)%

                              Hyalinocyte         Sample No.

(3.03 [+ or -] 0.11) x
  [10.sup.7] cell/ml     (57.4 [+ or -] 25.51)%       20

Note: (mean [+ or -] se).

TABLE 2.
Characteristics of different hemocyte types in Chlamys farreri

    Hemocyte type          Cell diameter       Nuclear diameter
                            ([micro]m)           ([micro]m)
Hyalinocytes
  Type 1 small cell      2.44 [+ or -] 0.11   1.45 [+ or -] 0.38
  Ranges                 1.28-3.56            1.03-2.25
  Type 2 large cell      4.83 [+ or -] 0.28   2.08 [+ or -] 0.51
  Ranges                 3.38-8.25            1.8-4.75
Granulocytes
  Type III small cell    4.07 [+ or -] 0.15   1.41 [+ or -] 0.32
  Ranges                 2.44-5.3             1.13-1.50
  Type IV middle cell    7.20 [+ or -] 0.26   2.09 [+ or -] 0.47
  Ranges                 5.44-9.80            1.5-3.56
  Type V large cell     13.87 [+ or -] 0.73   4.12 [+ or -] 0.39
  Ranges                10.32-23.50           2.5-6.25

    Hemocyte type             N/C ratio         Percentages   Sample
                                                               No.

Hyalinocytes            0.59 [+ or -] 1.94        45%-50%       25
  Type 1 small cell     0.29-0.93                   --
  Ranges                0.43 [+ or -] 0.34-       15%-20%       25
  Type 2 large cell     0.34-0.62                   --
  Ranges
Granulocytes            0.34 [+ or -] 0.47        15%-20%
  Type III small cell   0.25-0.46                   --
  Ranges                0.29 [+ or -] 0.68        20%-25%       25
  Type IV middle cell   0.16-0.55                   --
  Ranges                0.29 [+ or -] 0.47        3%-5.5%       25
  Type V large cell     0.22-0.34                   --
  Ranges

Note: Hemolymph collected from YaMei Scallop Farm, Changdao County,
Shandong province, China, Sept., 2003. Experiments were carried out
in Laboratory of Immunology, College of Veterinary Medicine, China
Agricultural University, Beijing, Sept., 2003. Distinguished and
counted on Hemocolor's stained smears.

TABLE 3.
Activities of phagocytosis of differential hemocyte in Chlamys
farreri on E. coli and RLOs (mean [+ or -] se).

Categories    Phagocytic rate on E. coli   Phagocytic rate on RLO

Hemocyte         (25.4 [+ or -] 4.0)%       (21.7 [+ or -] 9.7)%
Granulocyte      (40.1 [+ or -] 7.1)%       (30.3 [+ or -] 6.9)%
Hyalinocyte      (14.5 [+ or -] 3.3)%        (4.8 [+ or -] 7.3)%

Note: Hemolymph collected from YaMei Scallop Farm, Changdao County,
Shandong province, China, Sept., 2003. Experiments were carried out
in Laboratory of Immunology, College of Veterinary Medicine, China
Agricultural University, Beijing, Sept., 2003. Counted of Giemsa's
stained smears. Data statistics were made by SPSS software.