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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 Granulocytes
White blood cells.

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granulocytes (granˑ·y
, large hyalinocytes and small hyalinocytes. The large hyalinocytes and small hyalinocytes were agranular cells. The large hyalinocytes presented thin cytoplasm cytoplasm: see protoplasm.
cytoplasm

Portion of a eukaryotic cell outside the nucleus. The cytoplasm contains all the organelles (see eukaryote).
 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 Golgi complex
n.
A complex of parallel, flattened saccules, vesicles, and vacuoles that lies adjacent to the nucleus of a cell and is concerned with the formation of secretions within the cell. Also called Golgi apparatus.
 and endoplasmic endoplasmic

pertaining to or arising from endoplasm.


endoplasmic ribosomes
small, cytoplasmic granules consisting of approximately 60% RNA and 40% protein.
 reticulum reticulum /re·tic·u·lum/ (re-tik´u-lum) pl. retic´ula   [L.]
1. a small network, especially a protoplasmic network in cells.

2. reticular tissue.
. They showed a total absence of cytoplasmic cytoplasmic

pertaining to or included in cytoplasm.


cytoplasmic inclusions
include secretory inclusions (enzymes, acids, proteins, mucosubstances), nutritive inclusions (glycogen, lipids), pigment granules (melanin, lipofuscin,
 granules Granules
Small packets of reactive chemicals stored within cells.

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 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 hemocyte /he·mo·cyte/ (he´mo-sit) blood cell.

he·mo·cyte
n.
A cellular component or formed element of the blood.
 counts gave a mean ([+ or -]SE) concentration of 2.06 [+ or -] 0.20 x [10.sup.7] cells [mL.sup.-1] of hemolymph hemolymph /he·mo·lymph/ (he´mo-limf?)
1. blood and lymph.

2. the bloodlike fluid of those invertebrates having open blood-vascular systems.


he·mo·lymph
n.
. 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 phagocytosis: see endocytosis.
Phagocytosis

A mechanism by which single cells of the animal kingdom, such as smaller protozoa, engulf and carry particles into the cytoplasm.
 showed that the granulocytes were phagocytic cells Phagocytic cells
A cell that ingests microorganisms and foreign particles.

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 and the agranular cells showed a limited phagocytic phag·o·cyt·ic
adj.
1. Of or relating to phagocytes.

2. Of, relating to, or characterized by phagocytosis.



phagocytic

emanating from or pertaining to phagocytes.
 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 Centrifugation

A mechanical method of separating immiscible liquids or solids from liquids by the application of centrifugal force. This force can be very great, and separations which proceed slowly by gravity can be speeded up enormously in centrifugal
. 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 bivalve, aquatic mollusk of the class Pelecypoda ("hatchet-foot") or Bivalvia, with a laterally compressed body and a shell consisting of two valves, or movable pieces, hinged by an elastic ligament.  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 phylum, in taxonomy: see classification.  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 mollusk: see Mollusca.
mollusk
 or mollusc

Any of some 75,000 species of soft-bodied invertebrate animals (phylum Mollusca), many of which are wholly or partly enclosed in a calcium carbonate shell secreted by the mantle, a soft
 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 The Pearl River Delta Region (PRD) in China occupies the low-lying areas alongside the Pearl River estuary where the Pearl river flows into the South China Sea. Since the "Open Door Policy" was adopted by the Communist Party of China in the late 1970s, the portion of the delta in , 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 RLO Reusable Learning Object
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) (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 Noun 1. adductor muscle - a muscle that draws a body part toward the median line
adductor

skeletal muscle, striated muscle - a muscle that is connected at either or both ends to a bone and so move parts of the skeleton; a muscle that is characterized by
 of each animal using a 25-gauge needle into an equal volume of either Baker formol-calcium fixative fixative /fix·a·tive/ (fik´sit-iv) an agent used in preserving a histological or pathological specimen so as to maintain the normal structure of its constituent elements.

fix·a·tive
adj.
 (4% formaldehyde, 2% sodium chloride sodium chloride, NaCl, common salt. Properties


Sodium chloride is readily soluble in water and insoluble or only slightly soluble in most other liquids. It forms small, transparent, colorless to white cubic crystals.
, 1% calcium acetate calcium acetate

Phos-Ex (UK), PhosLo, PhosLo Gelcap

Pharmacologic class: Mineral

Therapeutic class: Dietary supplement, electrolyte replacement agent

Pregnancy risk category C
) 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 glutaraldehyde /glu·ta·ral·de·hyde/ (gloo?tah-ral´de-hid) a disinfectant used in aqueous solution for sterilization of non-heat–resistant equipment; also used as a tissue fixative for light and electron microscopy. , 2% NACI NACI National Advisory Committee on Immunization (Canada)
NACI National Advisory Council on Innovation (South Africa)
NACI National Agency Check with Inquiries
, 2 mM calcium chloride calcium chloride, CaCl2, chemical compound that is crystalline, lumpy, or flaky, is usually white, and is very soluble in water. The anhydrous compound is hygroscopic; it rapidly absorbs water and is used to dry gases by passing them through it.  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 hemocytometer /he·mo·cy·tom·e·ter/ (-si-tom´e-ter) hemacytometer.

he·mo·cy·tom·e·ter
n.
An instrument for counting the blood cells in a measured volume of blood.
 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 “TEM” redirects here. For other uses, see TEM (disambiguation).

Transmission electron microscopy (TEM) is an imaging technique whereby a beam of electrons is transmitted through a specimen, then an image is formed, magnified and directed to appear either
 

The suspension of hemolymph fixed with transmission electron microscopy (TEM TEM

1. transmission electron microscope.

2. triethylenemelamine.

3. transmissible encephalopathy of mink.
) 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 de·hy·drate  
v. de·hy·drat·ed, de·hy·drat·ing, de·hy·drates

v.tr.
1. To remove water from; make anhydrous.

2. To preserve by removing water from (vegetables, for example).
 and embedded in Epon. Ultrathin sections, (50-70 nm), were stained with uranyl acetate and lead citrate citrate /cit·rate/ (sit´rat) a salt of citric acid.

citrate phosphate dextrose  (CPD) anticoagulant citrate phosphate dextrose solution.
 and examined in a TEM JEOL JEOL Japan Electron Optics Laboratory  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 osmium (ŏz`mēəm), metallic chemical element; symbol Os; at. no. 76; at. wt. 190.2; m.p. 3,045±30°C;; b.p. 5,027±100°C;; sp. gr. 22.57 at 20°C;; valence usually +0 to +8.  tetraoxide at 1% (p/v) and placed in dehydration slides with ethanol and amilum acetate, followed by a critical point desiccation des·ic·ca·tion
n.
The process of being desiccated.



desic·ca
 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 EDTA: see chelating agents. ; 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 try·pan blue
n.
An acid dye used for staining of the reticuloendothelial system, the kidney tubules, and cells in tissue culture.



trypan blue

a supravital stain and a stain for amyloid.
 test.

Phagocytosis of Zymosan zy·mo·san  
n.
An insoluble carbohydrate from the cell wall of yeast, used especially in the immunoassay of properdin.



[zymos(is) + -an2.]
 

Zymosan (cell walls of Saccharomyces Saccharomyces: see yeast.  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 SSW
abbr.
south-southwest

Noun 1. SSW - the compass point midway between south and southwest
sou'-sou'-west, south southwest
) 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 milliliter /mil·li·li·ter/ (mL) (-le?ter) one thousandth (10-3) of a liter.

mil·li·li·ter
n. Abbr.
 of filtered seawater (FSW FSW Friction Stir Welding
FSW Flight Software
FSW Full Spectrum Warrior (video game)
FSW Family Support Worker
FSW Female Sex Worker
FSW Fox Sports World (cable TV channel) 
) 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 A statistical package from SPSS, Inc., Chicago (www.spss.com) that runs on PCs, most mainframes and minis and is used extensively in marketing research. It provides over 50 statistical processes, including regression analysis, correlation and analysis of variance.  software was used for statistical analysis. Differences in all studied parameters were evaluated by 1-way ANOVA anova

see analysis of variance.

ANOVA Analysis of variance, see there
 followed by Tukey test for comparisons. LSD LSD or lysergic acid diethylamide (lī'sûr`jĭk, dī'ĕth`ələmĭd, dī'ĕthəlăm`ĭd), alkaloid synthesized from lysergic acid, which is found in the fungus ergot (  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 basophilic /ba·so·phil·ic/ (-fil´ik)
1. pertaining to basophils.

2. staining readily with basic dyes.


basophilic

staining readily with basic dyes.
 or refringent re·frin·gent
adj.
Of, relating to, or producing refraction; refractive.
 cytoplasmic granules and had small nuclei, which often showed the oval or eccentric shapes. On smears, endoplasm endoplasm /en·do·plasm/ (en´do-plazm?) the central portion of the cytoplasm of a cell.endoplas´mic

en·do·plasm
n.
 and ectoplasm ectoplasm

an old-fashioned term which referred to a peripheral band of gel-like cytoplasm, free of organelles, found in free and motile cells.
 of the cytoplasm could be clearly distinguished in the granulocytes. The ectoplasm presenting some thin pseudopodia showed a hyaline hyaline /hy·a·line/ (hi´ah-lin) glassy and translucent.

hy·a·line
adj.
Resembling glass, as in translucence or transparency; glassy.

n.
1.
 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 acidophilic /ac·i·do·phil·ic/ (as?i-do-fil´ik)
1. easily stained with acid dyes.

2. growing best on acid media.
 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 mul·ti·nu·cle·ate or mul·ti·nu·cle·at·ed
adj.
Having two or more nuclei.



multinuclear, multinucleate

cells having more than one nucleus.
 (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 N/C No Charge
N/C No Change
N/C No Comment
N/C new condition
N/C Numerical Control
N/C No Connect (electronics)
N/C Normally Closed Contact
N/C Newton Per Coulomb
N/C Number of Users Per Cell Density
) ratios measured on Hemacolor stained smears. Granulocyte granulocyte /gran·u·lo·cyte/ (gran´u-lo-sit?) granular leukocyte.granulocyt´ic

band-form granulocyte  band cell.


gran·u·lo·cyte
n.
 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 homogenous - homogeneous  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 euchromatin /eu·chro·ma·tin/ (u-kro´mah-tin) that state of chromatin in which it stains lightly, is genetically active, and is considered to be partially or fully uncoiled.

eu·chro·ma·tin
n.
, and some showed abundant heterochromatin heterochromatin /het·ero·chro·ma·tin/ (-kro´mah-tin) that state of chromatin in which it is dark-staining, genetically inactive, and tightly coiled.

het·er·o·chro·ma·tin
n.
 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 com·plect·ed  
adj. Informal
Marked by or having a particular facial complexion. Often used in combination: "A white-haired and ruddy-complected priest stood on the deck of one of the trawlers" 
 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 refractive

capacity to refract light.


refractive error
a difference between the focal length of the cornea and lens, and the length of the eye, resulting in myopia or hyperopia.
 inclusions or spherule spher·ule  
n.
A miniature sphere; a globule.



[Late Latin sphaerula, diminutive of Latin sphaera, ball; see sphere.
 (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 cor·ru·ga·tion  
n.
1.
a. The act or process of corrugating.

b. The state of being corrugated.

2. A groove or ridge on a corrugated surface.

Noun 1.
, spongy spongy /spon·gy/ (spun´je) of a spongelike appearance or texture.

spong·y
adj.
Resembling a sponge in appearance, elasticity, or porosity.
 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 in vitro /in vi·tro/ (in ve´tro) [L.] within a glass; observable in a test tube; in an artificial environment.

in vi·tro
adj.
In an artificial environment outside a living organism.
 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 en·cir·cle  
tr.v. en·cir·cled, en·cir·cling, en·cir·cles
1. To form a circle around; surround. See Synonyms at surround.

2. To move or go around completely; make a circuit of.
 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 postmortem changes,
n.pl changes that occur after death.
 or rejection of grafts. However, the origin and progress of their forming are not clear.

The ultrastructure ultrastructure /ul·tra·struc·ture/ (-struk?chur) the structure beyond the resolution power of the light microscope, i.e., visible only under the ultramicroscope and electron microscope.  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 o·ver·ripe  
adj.
1. Too ripe.

2. Marked by decay or decline.



over·ripe
. 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 on·to·ge·net·ic
adj.
Of or relating to ontogeny.
 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 hydrolysis (hīdrŏl`ĭsĭs), chemical reaction of a compound with water, usually resulting in the formation of one or more new compounds.  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 glycogen (glī`kəjən), starchlike polysaccharide (see carbohydrate) that is found in the liver and muscles of humans and the higher animals and in the cells of the lower animals. . 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 Rosette

D’Albert’s pliable, versatile, talented, acknowledged bedmate. [Fr. Lit.: Mademoiselle de Maupin. Magill I, 542–543]

See : Courtesanship



(language) Rosette - A concurrent object-oriented language from MCC.
 formation with sheep erythrocytes Erythrocytes
Red blood cells.

Mentioned in: Bartonellosis

erythrocytes (ē·rithˑ·rō·sīts),
n.pl red blood cells.
 (Russell-Pinto et al. 1994) and in other invertebrates, such as the crustaceans, they participate in the coagulation coagulation (kōăg'ylā`shən), the collecting into a mass of minute particles of a solid dispersed throughout a liquid (a sol), usually followed by the precipitation or  process (Hose et al. 1990).

Density gradient centrifugation is one of the most used techniques for separating molluscan mol·lus·can also mol·lus·kan  
adj.
Of or relating to the mollusks.

n.
A mollusk.
 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 osmolarity /os·mo·lar·i·ty/ (oz?mo-lar´i-te) the concentration of a solution in terms of osmoles of solutes per liter of solution.

os·mo·lar·i·ty
n.
 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 elutriation /elu·tri·a·tion/ (e-loo?tre-a´shun) purification of a substance by dissolving it in a solvent and pouring off the solution, thus separating it from the undissolved foreign material. , 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 NSFC National Small Flows Clearinghouse
NSFC National Natural Science Foundation of China
NSFC National Society of Film Critics
NSFC National Science Foundation of China
NSFC North Shore Fencers Club (Long Island, New York) 
 (30170741 and 30371107), Scientific Program of Zhejiang Province (2004C23041) and Key Science Program (KSCX2-SW-302-8) of Chinese Academy of Sciences The Chinese Academy of Sciences (CAS) (Simplified Chinese: 中国科学院; Pinyin: Zhōngguó Kēxuéyuàn), formerly known as Academia Sinica .

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Adj. 1. auld - a Scottish word; "auld lang syne"
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Description

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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
Words:6892
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