Journal of INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 96 (2007) 48–63 www.elsevier.com/locate/yjipa Sydney rock oyster (Saccostrea glomerata) hemocytes: Morphology and function Saleem Aladaileh, Sham V. Nair, Debra Birch, David A. Raftos * Department of Biological Sciences, Macquarie University, North Ryde, NSW 2109, Australia Received 28 November 2006; accepted 8 February 2007 Available online 1 March 2007 Abstract In this study, three major hemocyte types were identified in the Sydney rock oyster. They were characterized primarily by light and electron microscopy based on the presence or absence of granules and nucleus to cytoplasm ratios. Hemoblast-like cells were the smallest cell type 4.0 ± 0.4 lm and comprised 15 ± 3% of the hemocyte population. They had large nuclei and scanty basic cytoplasm. This cell type also had some endoplasmic reticuli and mitochondria. The second major type were hyalinocytes. Hyalinocytes represented 46 ± 6% of all hemocytes. They were large cells (7.1 ± 1.0 lm) that had low nucleus:cytoplasm ratios and agranular basic or acidic cytoplasm. Hyalinocytes had the ability to phagocytose yeast cells and formed the core of hemocyte aggregates associated with agglutination. Four discrete sub-populations of hyalinocytes were identified. The third major cell type were the granulocytes, comprising 38 ± 1% of the hemocyte population. These cells were large (9.3 ± 0.3 lm) and were characterized by cytoplasm containing many acidic or basic granules. Granulocytes were more phagocytic than hyalinocytes and they formed the inner layer of hemocytes during the encapsulation of fungal hyphae. Five discrete sub-populations of granulocytes were identified based on the types of granules in their cytoplasm. Flow cytometry showed that the hemocytes of rock oysters could be divided into between two and four major cell types based on their light scattering properties. The most common of the cell types identified by flow cytometry corresponded to hyalinocytes and granulocytes. Cytochemical assays showed that most enzymes associated with immunological activity were localized in granulocytes. Their granules contained acid phosphatase, peroxidase, phenoloxidase, superoxide and melanin. Hyalinocytes were positive only for acid phosphatase. All of these observations suggest that Sydney rock oysters have a broad variety of functionally specialized hemocytes, many of which are involved in host defense.  2007 Elsevier Inc. All rights reserved. Keywords: Transmission electron microscopy; Saccostrea glommerata; Hemocytes; Flow cytometry; Phenoloxidase; Acid phosphatase 1. Introduction The immune system has two main branches yielding innate or adaptive immunity. Current evidence suggests that invertebrates do not have adaptive immune systems, which are intrinsic to vertebrates. Hypervariable recognition molecules and pathogen-specific defense reactions have been reported recently in some invertebrate groups (Litman et al., 2005). However, the consensus is still that invertebrates lack both long term immunological memory * Corresponding author. Fax: +61 2 9850 8245. E-mail address: draftos@rna.bio.mq.edu.au (D.A. Raftos). 0022-2011/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2007.02.011 and the ability to produce high affinity antibodies directed against particular pathogens. Instead, they have well-developed innate immune systems that retain the ability to discriminate between self and non-self but lack precise antigen specificity (Jiravanichpaisal et al., 2006). The innate immune systems of invertebrates incorporate both humoral and cellular defenses. Humoral responses include effector proteins such as antimicrobial peptides phenoloxidase and lysozyme (Boulanger et al., 2006; Ovchinnikova et al., 2006). Cellular defenses include hemocyte- or coelomocyte-mediated responses like phagocytosis, nodule formation, encapsulation and the production of reactive oxygen intermediates (Pipe, 1992; Hegaret et al., 2003). In addition to their role in host defense, invertebrate S. Aladaileh et al. / Journal of Invertebrate Pathology 96 (2007) 48–63 hemocytes also perform a variety of other important physiological functions, including nutrient transport, digestion, wound healing, shell mineralization and excretion (Cheng, 1981; Franchini and Ottaviani, 2000; Mount et al., 2004). Hemocytes have been classified using a variety of approaches based on combinations of morphology, cytochemistry and function. They have also been characterized by flow cytometry (Xue et al., 2001; Allam et al., 2002a; Hegaret et al., 2003), density gradient centrifugation (Cheng et al., 1980; Bachere et al., 1988; Friebel and Renwrantz, 1995; Xue et al., 2000) and by using monoclonal antibodies that react with hemocyte surface proteins (Noel et al., 1994; Dyrynda et al., 1997; Xue and Renault, 2001; Jing and Wenbin, 2006). Hemocytes from bivalve mollusks have most often been divided into two types, hyalinocytes and granulocytes, both of which are phagocytic (Cheng and Foley, 1975; Auffret, 1988; Suresh and Mohandas, 1990; Wootton and Pipe, 2003). These two cell types have been found in many bivalve species including Anodonta cygnea, Dreissena polymorpha, Mytilus edulis, Scrobicularia plana, Ruditapes philippinarum, Mercenaria mercenaria, Meretrix lusoria and Crassostrea gigas (Giamberini et al., 1996; Pipe et al., 1997; Allam et al., 2002a; Soares-da-Silva et al., 2002; Wootton and Pipe, 2003; Chang et al., 2005). In many of these species, granulocytes have been further subclassified as eosinophilic, basophilic and neutrophilic (McCormickRay and Howard, 1991; Lopez et al., 1997c). Granulocytes have the ability to phagocytose microbial pathogens and they contain a mixture of hydrolytic enzymes that contribute to intracellular killing. They are often more active phagocytes than hyalinocytes. Hyalinocytes are smaller than granulocytes. Some contain a few granules and are usually more morphologically heterogeneous than granulocytes. Hyalinocytes have been divided into two types, small hyalinocytes, with large nuclei and scanty cytoplasm, and large hyalinocytes with low nucleus:cytoplasm ratios (Cheng, 1981). In the present study, we characterized hemocytes of the Sydney rock oyster, Saccostrea glomerata, using light and electron microscopy, cytochemistry and flow cytometry. We also investigated their role in phagocytosis and encapsulation. S. glomerata is one of the most important invertebrate aquaculture species in Australia. Unlike some other commercially exploited oysters, Sydney rock oysters suffer from a variety of infectious diseases and parasitic infestations. These include Winter Mortality Syndrome and QX disease, as well as mudworm and flatworm infestation. Our interest in the hemocytes of S. glomerata arises from their potential roles in immune responses against these infectious agents, and particularly the role that environmental stress plays in disease susceptibility via its effects on hemocytes. A number of environmental factors are known to affect the invertebrate defense systems that are associated with hemocyte function (Cheng and Chen, 2000). Temperature, salinity and chemical 49 pollutants alter the activities of hemocytes in several species of molluscs (Fisher et al., 1987; Cheng et al., 2004a,b). It is believed that the effects of these factors on hemocytes might increase the susceptibility of oysters to infection. In order to understand the nature of this interaction between the environmental factors and the Sydney rock oyster immune system, there is an urgent need to characterize the hemocytes of this species. The results of the current study will be used in further investigations to find links between disease susceptibility and environmental stress. 2. Materials and methods 2.1. Hemolymph preparation Sydney rock oysters were purchased from the Sydney Fish Markets (Sydney, Australia). The animals were maintained in aerated aquaria (20 L) at 25 C. Hemocytes were harvested by shucking the oysters and withdrawing the hemolymph directly from the heart near the adductor muscle using 22-gauge needles fitted to 5-ml syringes. Hemolymph was mixed immediately with an equal volume of marine anticoagulant (MAC1; 0.1 M glucose, 15 mM trisodium citrate, 13 mM citric acid, 10 mM EDTA, 0.45 M NaCl, pH 7.0). 2.2. Live cell analysis The analysis of live hemocytes was carried out by placing a 30 ll of whole hemolymph on acid alcohol cleaned microscope slides and allowing the cells to adhere for 10 min. The slides were then covered with a glass cover slip and viewed at high magnification with an Olympus BH-2 microscope equipped for epi-fluorescence and differential interference contrast (DIC). The percentage of the total hemocyte population comprised by the different cell sub-populations was calculated from differential cell counts of hemolymph from five different oysters. The data are presented as mean percentages ± SEM. The aggregation of hemocytes, their mobility and degranulation were monitored by time-lapse photomicroscopy (one image per 5 s). The images were analyzed by using ImagJ 1.37c (Wayne Rasband, National Institutes of Health, USA). 2.3. Cytochemical procedures for light microscopy Immediately after withdrawal from oysters, 30 ll whole hemolymph were loaded onto acid alcohol cleaned micro1 Abbreviations used: LPS, lipopolysaccharide; FSW, sterile filtered sea water; 4HA, hydroquinine monomethyl ether; MBTH, 3-methyl-2-benzothiazolinone hydrazone; MAC, marine anti-coagulant, EDTA, ethylenediaminetetraacetic acid; NBT, nitroblue tetrazolium; PIPES, piperazine1,4-bis(2-ethanesulfonic acid). 50 S. Aladaileh et al. / Journal of Invertebrate Pathology 96 (2007) 48–63 scope slides. Cells were allowed to adhere to the slides for 20 min before being stained according to the following cytochemical methods. Unless stated otherwise, all reagents and buffers were from Sigma–Aldrich (Castle Hill, NSW). 2.3.1. Giemsa/May–grünwald stain After adhesion to slides, hemocytes were fixed for 20 min in formaldehyde (4% in filtered seawater, FSW) and then immersed in May–Grünwald stain for 6 min. Slides were then transferred to Giemsa stain for 30 min before being washed in phosphate buffer saline (PBS) and air dried. Slides were mounted in Ultramount No. 7 (Fronine Laboratory Supplies) and observed by light microscopy. 2.3.2. Acid phosphatase Acid phosphatase was detected in hemocytes using a Lymphocyte Enzyme Kit. The slides were fixed in citrateacetone formaldehyde for 30 s and then washed in deionized water. The slides were then transferred to Coplin jars containing a working solution with 1 ml sodium nitrate, 1 ml Fast Grant GBC Base solution, 38 ml prewarmed deionized water, 5 ml acetate solution and 5 ml naphthol AS-BI phosphate. Slides were incubated in this working solution for 1 h at 37 C before being removed, rinsed in deionized water for 2 min and allowed to air dry for 15 min. They were then counterstained for 2 min in methylene blue. The slides were rinsed in deionized water and mounted for inspection by light microscopy. Acid phosphatase activity was detected as red to violet staining in the cytoplasm (Hose et al., 1990). 2.3.3. Peroxidase Slides were fixed in formaldehyde (4% in FSW) for 10 min, washed in phosphate buffer and transferred to a Coplin jar containing 5 mg 3,3 0 -diaminobenzidine tetrahydrochloride (DAB) in 10 ml Tris–HCl and 100 ll H2O2. Slides were incubated at 37 C for 2 h and then washed in PBS. DAB peroxidase development was detected as yellow/brown granules in the cytoplasm (Cima et al., 2001). 2.3.4. Phenoloxidase Thirty microliters of phenoloxidase stain was applied to live hemocytes on slides and allowed to stand for 20 min. The stain contained 0.02 mg ml 1 lipopolysaccharide (LPS), 5 mM hydroquinine monomethyl ether (4HA, Fluka, Switzerland) and 5 mM 3-methyl-2-benzothiazolinone hydrazone (MBTH) in FSW. The slides were then mounted and examined by light microscopy. Phenoloxidase staining appeared as a red coloration in hemocytes (Cima et al., 2001). 2.3.5. Superoxide stain Intracellular superoxide anions (O2 ) were detected using slides that had been coated with nitroblue tetrazolium (NBT). Thirty microliters of NBT (1 mg ml 1 in FSW) were loaded onto acid alcohol washed slides and allowed to adhere for 1 h at 37 C. Hemocytes (1 · 106 ml 1) were pipetted onto NBT coated slides and incubated for 30 min at room temperature. NBT was reduced in the presence of O2 to form blue granules of formazan inside hemocytes (Song and Hsieh, 1994). 2.3.6. Melanin stain After adhesion to slides, hemocytes were rinsed and the slides were transferred to Coplin jars containing freshly prepared 10% ammoniacal silver solution (Fontana-Masson silver solution; (Sheehan and Hrapchak, 1980). The solution was prepared by adding concentrated ammonium hydroxide to 20 ml 10% silver nitrate drop by drop until the solution turned dark and then cleared. The slides were incubated in a water bath at 60 C for 20 min before being washed gently in water and fixed with 5% sodium thiosulfate for 2 min. Finally, the slides were washed with distilled water and mounted. Melanin was detected as a brown precipitate. 2.4. Phagocytosis Congo red stained yeast, Saccharomyces cerevisiae (Sigma–Aldrich), were used as target cells for phagocytosis. Five milligrams yeast was suspended in 5 ml FSW and 5 ml of filtered Congo red (Sigma–Aldrich; 0.8% in FSW). The suspension was autoclaved at 120 C for 15 min. It was then washed two times and centrifuged at 1300g for 5 min. The pellets were resuspended in 10 ml FSW and stored at 4 C. To measure phagocytic activity, 30 ll whole hemolymph adjusted with FSW to 1 · 106 cells ml 1 were placed on glass coverslips and the cells were allowed to adhere to the coverslips for 30 min at room temperature (25 C) in a moist chamber. The adherent cells were then washed five times with FSW, overlaid with 100 ll of Congo red stained yeast (7 · 105 ml 1) and incubated for 30 min at room temperature. Non-phagocytosed yeast cells were removed by dipping each coverslip in FSW 10 times. The slides were then viewed at high magnification (1000·) with an Olympus BH-2 microscope equipped for epi-fluorescence and differential interference contrast (DIC). Phagocytic activity was determined as the percentage of hemocytes that had ingested at least one yeast cell by counting a minimum of 200 granulocytes and 200 hyalinocytes on each coverslip. 2.5. Encapsulation Encapsulation assays were carried out by placing 30 ll of whole hemolymph onto coverslips coated with fungal hyphae. Forty microliters of fungal hyphae (Cordyceps bassiana) in suspension (1 · 105) were added to coverslips and incubated at room temperature (25 C) for 25 min. The hyphae were kindly supplied by Chris Turnball (Macquarie University). After 30 min, coverslips were mounted and S. Aladaileh et al. / Journal of Invertebrate Pathology 96 (2007) 48–63 assessed for capsule formation. Some coverslips were stained for melanin formation by using Fontana-Masson silver solution as described above. 51 K550). Hemocytes were viewed using a JEOL 6480 LA scanning electron microscope. 3. Results 2.6. Flow cytometry One milliliter hemolymph was collected and placed directly into a flow cytometry tube containing 1 ml 4% paraformaldehyde. Hemocytes were then analyzed using a FACSCalibur flow cytometer (Becton–Dickinson, North Ryde, NSW). For each hemocyte sample, the flow rate was adjusted to maintain the total events below 250 s 1. The forward scatter (FSC) detector was set to E01 sensitivity, 52V, and the side scatter (SSC) detector was set to 350 V. Ten thousand events were counted and sorted for each sample. Data were analyzed using WinMDI (J. Trotter, Salk Institute for Biological Studies, La Jolla, CA, USA). 2.7. Transmission electron microscopy (TEM) Hemocytes were fixed for 10 min at 4 C in 4% paraformaldehyde, 2.5% glutaraldehyde and 0.3 M sucrose in 0.1 M PIPES buffer (Sigma–Aldrich, pH 7.2). The cells were then centrifuged at 400g for 10 min and fresh fixative was carefully added for a further 2 h at room temperature. The cells were washed 3 · 10 min in PIPES with 0.3 M sucrose and then post-fixed with 1% osmium tetroxide (OsO4) in 0.1 M PIPES for 1 h at 4 C. After post-fixation, the pellets were washed in the same buffer, the hemocytes were embedded in melted 2% agar and then centrifuged at 2000g for 5 min. The pellets were dehydrated through a graded ethanol series and embedded in L.R. White resin. Ultrathin sections were cut using a Reichert Ultracut-S ultramicrotome and mounted onto Pioloform coated, copper grids. Sections were stained with saturated aqueous uranyl acetate for 35 min and Reynold’s lead citrate for 4 min. Sections were examined with a Philips CM 10 transmission electron microscope. 2.8. Scanning electron microscopy (SEM) Coverslips (12 mm · 12 mm) were cleaned with acid alcohol (1% HCl in 70% ethanol) and coated with 0.1% ethylene imine polymer solution (PEI). Thirty microliters of whole hemolymph containing 1 · 106 cells ml 1 were added to the coverslips and hemocytes were allowed to adhere for 25 min at room temperature in a moist chamber. The adherent cells were fixed in 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M PIPES buffer (pH 7.2 with 0.3 M sucrose) for 2 h. They were then washed in buffer, post-fixed in 1% osmium tetroxide in 0.1 M PIPES buffer (pH 7.2 with 0.3 M sucrose), dehydrated in a graded series of ethanol and transferred to a critical point dryer (Emitech K850). The coverslips were mounted on aluminum stubs and coated with gold using a sputter coater (Emitech 3.1. Light microscopy Three major hemocyte types were identified by light microscopy based on nucleus:cytoplasm ratio and the presence or absence of cytoplasmic granules. The three hemocyte types were hemoblast-like cells, hyalinocytes and granulocytes. Hemoblast-like cells were the smallest cells observed in the hemolymph. They had a spherical shape with a high nucleus:cytoplasm ratio (Fig. 1A). Hemoblast-like cells comprised 15 ± 3% (n = 5 ± SEM) of the total hemocyte population and were 4.0 ± 0.4 lm in diameter. These cells were not motile and they required about 50 min to adhere to glass slides. Hyalinocytes (Fig. 1B) represented 46 ± 6% of the total hemocyte population. They varied in size within a range of 5–9 lm, having an average diameter of 7.1 ± 1.0 lm. Hyalinocytes had the capacity for amoeboid movement and they formed filopodia. They had round or oval nuclei and adhered to glass slides within 15–20 min. Hyalinocytes were also characterized by a cytoplasm containing no, or a few granules. Their cytoplasm were packed with vacuoles of varying sizes. Some hyalinocytes maintained a round shape when attached to glass slides and spread only slightly, forming some small filopodia. Hyalinocytes played a central role in hemocyte aggregation. Fig. 1G shows an aggregation of hemocytes with hyalinocytes in its core. By monitoring the aggregation process by time-lapse imaging, we found that hyalinocytes initially formed small aggregates and that these aggregates continued to grow in size after they form. Initially the aggregates contain few granulocytes. However, granulocytes start to move toward the aggregates after their initial formation and start to degranulate. We also found that the hyalinocytes detected in aggregates were not simply degranulated granulocytes. The degranulation process occured in two stages. First, granulocytes formed vacuoles that enclosed their granules. Then, the granules began moving within the vacuoles in a manner analogous to Brownian motion, before disappearing from within the cell within few minutes. The process continued until all the granules had disappeared from granulocytes (Fig. 1H). Granulocytes were the third type of hemocytes identified in S. glommerata (Fig. 1C and D). These cells comprised 38 ± 1% of the total hemocyte population. They varied in size from 5–9 lm with an average diameter of 9.3 ± 0.3 lm. Granulocytes adhered to glass slides within 15–20 min and they were highly ameboid after they adhered. They also had the ability to form many long filopodia, and were characterized by eccentric oval nuclei and numerous cytoplasmic granules. Their granules varied in size and were concentrated in the endoplasmic area. The 52 S. Aladaileh et al. / Journal of Invertebrate Pathology 96 (2007) 48–63 Fig. 1. Differential interference contrast micrograph of live hemocytes from S. glommerata. (A) Hemoblast-like cell, (B) hyalinocyte, (C) large granulocyte, (D) small granulocyte, (E and F) large granulocytes under UV light, (G) a hemocyte aggregate showing hyalinocytes in its core, (H) degranulated granulocyte. Scale bar = 5 lm. ectoplasm did not contain granules. Strong auto-fluorescence was observed in granulocytes under ultraviolet light (Fig. 1F). The intensity of auto-fluorescence was variable, with some granulocytes showing a bright yellow/blue coloration and others a much lighter yellow auto-fluorescence (Fig. 1E). After staining with Giemsa/May–Grünwald stain, hemoblast-like cells were light basophilic in appearance (Fig. 2A). Hyalinocytes could be subdivided after Giemsa/May–Grünwald staining into small hyalinocytes with a basophilic cytoplasm (Fig. 2B) and large hyalinocytes with acidophilic cytoplasm (Fig. 2C). The cytoplasm of the large hyalinocytes was packed with small vacuoles. Granulocytes could also be subdivided after staining into three types; basophilic, acidophilic and an intermediate form. The basophilic granulocytes were small with large granules (Fig. 2D), whereas the acidophilic form were large with small granules (Fig. 2E). The intermediate population contained a mixture of acidic and basic granules (Fig. 2F). 3.2. Cytochemistry Acid phosphatase activity was detected in 13 ± 7% of granulocytes and 3 ± 7% of hyalinocytes. Activity was seen as a brown granular staining in the granulocytes (Fig. 3A), and as a red to light brown coloration in the cytoplasm in hyalinocytes (Fig. 3B). Peroxidase activity was localized as dark brown deposits in granulocytes only (Fig. 3C). It was S. Aladaileh et al. / Journal of Invertebrate Pathology 96 (2007) 48–63 53 Fig. 2. Light micrographs of fixed hemocytes of S. glommerata stained with Giemsa/May–Grünwald stain. (A) Hemoblast-like cell, (B) basophilic hyalinocyte, (C) acidophilic hyalinocyte, (D) basophilic granulocyte, (E) acidophilic granulocyte, (F) granulocyte with a combination of basophilic and acidophilic granules. Scale bar = 5 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) detected in 22 ± 7% of granulocytes. Phenoloxidase activity was observed as pink to red granular deposits in 84 ± 9% of granulocytes. These deposits yielded variable intensities of color (Fig. 3D and E). There was no phenoloxidase activity in hyalinocytes. Similarly, melanin production was observed as brown deposits in granulocytes only (Fig. 3F). Superoxide activity was also observed only in granulocytes (94 ± 4%), where it was detected as violet or dark blue deposits in the granules (Fig. 3G). ulocytes was formed around the target hyphae (Fig. 4D and E). Some granulocytes started to degranulate even without a direct contact with hypha. Within 40 min, all the granulocytes contributing to encapsulation had degranulated. The hyalinocytes had only minor participation in the encapsulation process. Melanization occurred inside the capsules formed by granulocytes (Fig. 4F) and also in solution surrounding the capsule (Fig. 4G). 3.4. Flow cytometry 3.3. Phagocytosis and encapsulation Both granulocytes and hyalinocytes were capable of ingesting yeast cells (Fig. 4A and B). Granulocytes were more efficient phagocytes than hyalinocytes. Fig. 4I shows that 48 ± 2% of granulocytes ingested yeast compared to 36 ± 3% of hyalinocytes (p < 0.05). Individual granulocytes could phagocytose up to 30 yeast cells (Fig. 4C). Granulocytes lacked the ability to extend long pseudopodia during phagocytosis. Instead they engulfed foreign particles by cell surface invagination or by forming very short pseudopodia. Hyalinocytes had the ability to extend long pseudopodia toward foreign particles and engulf them into phagosomes (Fig. 4H). Granulocytes could also encapsulate fungal hyphae. During fungal encapsulation experiments, a layer of gran- Fig. 5A and B shows flow cytometric light scatter plots of hemocytes from two different oysters. In total, five oysters were analyzed in this way (data not shown). Variability among individuals was high, but four recognizable populations could be identified (Fig. 5A). Population R1 had high forward and 90 light angle scatter, consistence with large size and high granularity. R2 represented a population of cells with medium size (intermediate forward scatter) and moderate granularity (intermediate 90 light scatter). R3 had high forward scatter and low 90 light scatter, indicative of a population of large agranular cells. R4 represented a population with small size and high granularity (low forward and high 90 light scatter). Some oysters had only two populations, R1 and R2 (Fig. 5B), whilst others had all four. Microscopic observation of sorted hemocyte showed that 54 S. Aladaileh et al. / Journal of Invertebrate Pathology 96 (2007) 48–63 Fig. 3. Light and differential interference contrast micrographs of S. glommerata hemocytes showing cytoplasmic localization of different enzymes or products. (A and B) Acid phosphatase, (C) peroxidase, (D and E) phenoloxidase, (F) melanin, (G) superoxide. Scale bar = 5 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this paper.) R1 contained granulocytes (Fig. 5C) and R2 incorporated hyalinocytes (Fig. 5D). R3 and R4 could not be efficiently sorted because of the low cell numbers in each region. 3.5. SEM The three main hemocyte types (hemoblast-like cells, hyalinocytes and granulocytes) were also identified by SEM. Hemoblast-like cells were characterized under SEM by their small size and round shape (Fig. 6A). They extended some short filopodia and the cell surface was undulated. Hyalinocytes were characterized by their smooth surface and long filopodia (Fig. 6B), whereas granulocytes tended to have roughened surface membranes consistent with granule exocytosis or the presence of granules immediately beneath the plasmalemma (Fig. 6C). 3.6. TEM The same three major hemocyte types (hemoblast-like cells, hyalinocytes and granulocytes) were evident under TEM. Hemoblast-like cells were again found to be the smallest of the hemocytes (Fig. 7A). They had large nuclei with a single nucleolus and a high nucleus:cytoplasm ratio. The cytoplasm contained many rough endoplasmic reticuli and mitochondria. Hyalinocytes were highly variable in size and the content of their cytoplasm. We identified four subtypes (Types I– IV) by TEM according to size and ultrastructure. Type I hyalinocytes were small cells characterized by a central nucleus with relatively low nucleus:cytoplasm ratios (Fig. 7B). The cytoplasm of this subtype contained mito- S. Aladaileh et al. / Journal of Invertebrate Pathology 96 (2007) 48–63 55 Fig. 4. Differential interference contrast micrographs of S. glommerata hemocytes showing: (A) a granulocyte with engulfed yeast cell (red) (B) a hyalinocyte with engulfed yeast cell (red), (C) a granulocyte under UV light with more than one internalized yeast cell (red), (D) fungal hypha under UV excitation encapsulated by granulocytes. Granulocytes can be identified by their fluorescent granules (E) the same encapsulated fungal hypha under differential interference contrast microscopy, (F) fungal hypha melanized by an adherent granulocyte (brown coloration), (G) fungal hypha melanized (brown coloration) without any direct contact between the hypha and oyster hemocytes, (H) hyalinocyte with a long filopodia surrounding a yeast cell (arrow), (I) the percentage of hemocytes that had ingested one or more yeast (% phagocytic cells, n = 10, bars = SEM). Scale bar = 5 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this paper.) 56 S. Aladaileh et al. / Journal of Invertebrate Pathology 96 (2007) 48–63 Fig. 5. Flow cytometric scatter plots (forward scatter, FSC, vs. 90 light angle scatter, SSC) for hemocytes from two oysters. (A) Scatter plot of hemocytes from one of the oysters showing four hemocyte populations designated R1, R2, R3 and R4. (B) Scatter plot from a second oyster showing only two populations, R1 and R2. (D and C) Differential interference contrast micrographs of cells sorted from population R1 (D, granulocyte) and R2 (C, hyalinocyte). Scale bar = 5 lm. chondria, endoplasmic reticuli and some vacuoles. Type I hyalinocytes also had the ability to form some filopodia. The second subtype (Type II) were larger hyalinocytes (Fig. 7C), identified by their large nuclei with many chromatin clumps, a distinct nuclear cleft, small amounts of endoplasmic reticulum, numerous Golgi complexes and mitochondria. A few granules and some vacuoles were also present in the cytoplasm of Type II hylanocytes. Fig. 7D shows that Type III hyalinocytes were large cells that had a medium-sized eccentric nuclei. These hyalinocytes also had numerous vesicles, mitochondria, well developed Golgi complexes and endoplasmic reticulae. Type IV hyalinocytes were characterized by a large central nucleus with clumps of chromatin (Fig. 7E). These hyalinocytes had a few electron lucent granules, well developed Golgi complexes and endoplasmic reticulae. They were distinguished from other hyalinocytes by the distinctive arrangement of mitochondria located near the nucleus (Fig. 7F). Granulocytes were classified into five subtypes (Types – V) according to size and granularity. Type I granulocytes were small cells with large nuclei giving high nucleus:cytoplasm ratios (Fig. 8A). They had some electron dense granules, mitochondria and endoplasmic reticuli. These granulocytes also had the ability to form filopodia. Type II granulocytes (Fig. 8B) were medium-sized cells with one type of granule in their cytoplasm and a nucleus that had a light core surrounded by dense chromatin. The granules of Type II granulocytes were membrane bound with two distinct halves, one with electron dense material and the other with electron lucent material (Fig. 9A). Type II granulocytes had short filopodia and their granules tended to be secreted during cytological preparation. Type III granulocytes (Fig. 8C) had large central nuclei with many chromatin clumps. This cell type could be identified by the presence of some homogeneously electron dense granules and some membrane bound granules that had electron dense cores enclosed by a less dense region (Fig. 9B). They also had large granules that contained finely granular material with patches of lighter material (Fig. 9C), as well as some mitochondria. Type III granulocytes also formed filopodia. Type IV granulocytes were large cells with many membrane bound granules (Fig. 8D). These cells tend to be slightly larger than Type II and III granulocytes and they had small eccentric nuclei. The granules of Type IV granulocytes were elliptical with two or three distinct components including finely granular material and electron dense material (Fig. 9D and E). This cell type also had S. Aladaileh et al. / Journal of Invertebrate Pathology 96 (2007) 48–63 57 Fig. 6. Scanning electron micrographs of S. glommerata hemocytes. (A) Hemoblast-like cell, (B) hyalinocyte, (C) granulocyte. some homogeneously electron dense granules (Fig. 9F), endoplasmic reticuli and mitochondria, and they could form filopodia. Type V granulocytes were also large cells. They had small central nuclei and large cytoplasms containing many granules (Fig. 8E) with a distinctive electron lucent core surrounded by a membrane (Fig. 9G). The area between the electron lucent core and the outer membrane contained granular material. Type V granulocytes also had some granules with two kidney shaped inclusions (Fig. 9H). Each inclusion had a clear center surrounded by granular material. These cells had some mitochondria in their cytoplasm and they could form long filopodia. They were approximately the same size as Type IV granulocytes. 4. Discussion Hemocyte classification in invertebrates is often a controversial issue. Controversy is usually created because there is no universal procedure to classify hemocytes. In bivalves, hemocytes have been classified in many species, such as Ruditapes philippinarum, M. lusoria, C. gigas, Chlamys farreri, Tridacna crocea, D. polymorpha and Mytilus galloprovincialis (Giamberini et al., 1996; Carballal et al., 1997b; Nakayama et al., 1997; Allam et al., 2002b; Chang et al., 2005; Jing and Wenbin, 2005). Our interest in the hemocytes of S. glommerata comes from our work on the immune system of oysters. Previous studies have indicated that hemocytes play an important role in resistance and susceptibility to diseases that are currently impacting the Sydney rock oyster aquaculture industry (Peters and Raftos, 2003; Newton et al., 2004; Bezemer et al., 2006). As a result, our study is intentionally biased toward classifying hemocytes on the basis of factors that are often related to host defense, including the types of granules and enzymes found within cells. We have identified three distinct hemocyte types in S. glommerata; hemoblast-like cells, granulocytes and hyalinocytes. The classification in this study is similar to the 58 S. Aladaileh et al. / Journal of Invertebrate Pathology 96 (2007) 48–63 Fig. 7. Transmission electron micrographs of S. glommerata hemoblast-like cell and hyalinocytes. (A) Hemoblast-like cell, (B) Type I hyalinocyte, (C) Type II hyalinocyte with large nucleus and clear nuclear cleft, (D) Type III hyalinocyte with large number of vesicles, (E and F) Type IV hyalinocyte with central nucleus and mitochondria. N, nucleus; M, mitochondria; ER, endoplasmic reticulum; Nu, nucleolus; V, vacuole; GR, granule; G, Golgi; NC, nuclear cleft; F, filopodia. results of previous work on bivalves, such as C. gigas, Perna perna, M. lusoria (Barracco et al., 1999; Chang et al., 2005). Four distinct cell populations were identified based on their flow cytometric light scattering properties. S. Aladaileh et al. / Journal of Invertebrate Pathology 96 (2007) 48–63 59 Fig. 8. Transmission electron micrographs of S. glommerata granulocytes. (A) Type I granulocytes. (B) Type II granulocyte, (C) Type III granulocyte, (D) Type IV granulocyte, (E) Type V granulocyte. N, nucleus; M, mitochondria; ER, endoplasmic reticulum; V, vacuole; GR, granule; G, Golgi; F, filopodia. Similar data had been obtained in many bivalves species (Xue et al., 2001; Goedken and De Guise, 2004). In S. glomerata, flow cytometric population, R1, contained large granulocytes and R2 represented medium-sized hyalinocytes with moderate internal complexity. Unfortunately, R3 and R4 could not by sorted using flow cytometry. From 60 S. Aladaileh et al. / Journal of Invertebrate Pathology 96 (2007) 48–63 Fig. 9. Transmission electron micrographs of different types of granules (A–H) found in granulocytes of S. glommerata. Scale bar = 0.2 lm. their locations on the light scatter plots, R3 and R4 are likely to represent large hyalinocytes and small granulocytes, respectively. Populations equivalent to R4 have been found in other bivalves, such as Crassostrea virginica (Ford et al., 1994; Ashton-Alcox and Ford, 1998; Allam et al., 2002a). In S. glommerata both granulocytes and hyalinocytes contribute to phagocytosis, but granulocytes seem to be the most avid phagocytes. This agrees with studies of other bivalve species, such as Ruditapes decussata and P. perna (Lopez et al., 1997a; Barracco et al., 1999). The role of granulocytes in phagocytosis may be matched by their capacity for intracellular killing. We have shown that granulocytes contain high levels of acid phosphatase and phenoloxidase enzymatic activities, as well as the ability to form superoxides and peroxides. These features have been found in many other bivalves, like R. decussates, P. perna, C. farreri and M. galloprovincialis (Santarem et al., 1994; Carballal et al., 1997b; Lopez et al., 1997b; Barracco et al., 1999; Xing et al., 2002). In these species, the same intracellular enzymatic activities are associated with the ability of hemocytes to kill pathogens after phagocytosis. For example, in M. edulis and M. galloprovincialis the production of superoxide anions and hydrogen peroxide have been directly associated with phagocytosis (Pipe, 1992; Noel et al., 1993; Carballal et al., 1997b; Arumugam et al., 2000). The presence of acid phosphatase indicates that some granules might function as lysosomes (Granath and Yoshino, 1984) and could participate in intracellular digestion of proteins, carbohydrates and lipids as a part of the phagosomal system (Cheng, 1983). The role of granulocytes in the S. glommerata immune system may not be limited to phagocytosis. Evidence is provided in this study that granulocytes are also the predominant cell types involved in encapsulation reactions. Encapsulation is often used by invertebrates including molluscs to sequester pathogens that are too big to be phagoctosed by individual hemocytes (Cheng, 1981; Field et al., 2004). In unionid mussels, the encapsulation of the Aspidogaster conchicola (Trematoda: Aspidogastrea) is S. Aladaileh et al. / Journal of Invertebrate Pathology 96 (2007) 48–63 achieved by fibrocytes (Huehner and Etges, 1981). Cheng (1981) demonstrated that these fibrocytes are granulocytes that are morphologically altered in the latter stages of encapsulation process. In other invertebrates, such as the cockroach, Blattella germanica, and the moth, Manduca sexta, encapsulation is also achieved by granulocytes (Han and Gupta, 1989; Ling and Yu, 2006). The contribution of S. glommerata granulocytes to encapsulation was shown here using fungal hyphae. Granulocytes aggregated around hyphae and altered their morphology during encapsulation. Capsule formation was accompanied by melanization, probably via the phenoloxidase cascade that is characteristic of granulocytes (Ling and Yu, 2005). Melanization was apparent both within and outside granulocytes, indicating that phenoloxidase activity and subsequent melanization may be both extracellular and intracellular killing mechanisms. In many invertebrates, such as M. sexta, the activation of prophenoloxidase initiates melanization (Ling and Yu, 2005), and both intracellular and extracellular activities have been reported for phenoloxidase in bivalve species including R. philippinarum, Chamelea gallina and Tapes decussatus (Munoz et al., 2006). Even though they were not as avidly phagocytic as granulocytes, hyalinocytes were shown in the current study to have a central role in hemocyte aggregation processes. This function has been demonstrated in many bivalves, like T. crocea, where hyalinocytes were located in the core of hemocyte aggregations associated with wound healing (Nakayama et al., 1997). Unlike granulocytes and hyalinocytes, hemoblast-like cells did not contribute to defensive responses like phagocytosis or encapsulation, and they lacked the common intracellular enzyme systems associated with host defense. The presence of only a few cytoplasmic organelles and their low enzymatic activities suggests that hemoblast-like cells are immature hemocytes (Hine, 1999; Cima et al., 2001). This leaves open the possibility that these cells act as stem cells from which the other two major cell classes are derived. In many invertebrates, multiple hemocyte types appear to be derived by the differentiation of stem cells that have a very similar morphology to the hemoblast-like cells present in S. glommerata (Johansson et al., 2000; Meister, 2004). In the clam, Tapes philippinarum, and the colonial ascidian, Botryllus schlosseri, hemoblast-like cells are recognized by anti-CD34 antibodies (Cima et al., 2000; Ballarin and Cima, 2005). CD34 is a transmembrane glycoprotein which acts as a surface marker for very early hematopoietic stem cells in mammals (Satterthwaite et al., 1992). Its expression on invertebrate cells suggests that they may have a similar role. Despite these observations we provide no evidence that can resolve cell lineage expansion in S. glommerata, or the precise relationships between hemocyte types. It is also clear that there is heterogeneity within both granulocytes and hyalinocytes. Five distinct sub-types of granulocytes were identified and four different sub-types of hyalinocytes. 61 These different sub-types may represent different stages on continuous cell differentiation pathways, or they may simply result from subtle changes in the differentiation states of individual hemocytes. The differences between granulocytes were reflected by the number and types of granules found in the cytoplasm. The diversity of granules that we have identified is a common feature in many bivalve species, such as M. galloprovincialis and C. virginica (Feng et al., 1971; Carballal et al., 1997a). In the current study, granulocytes were subdivided into basophilic, acidophilic and mixed forms based on the affinity of the granules for Giemsa/May–Grünwald stain. Similar distinctions have been found in many bivalves, like C. gigas, M. edulis and R. decussates (Lopez et al., 1997c; Pipe et al., 1997; Chang et al., 2005). We subdivided granulocytes into five subclasses, these were small granulocytes (Type I) with high nucleus:cytoplasm ratios and some granules and medium size cells including Type II granulocytes that had a single type of granule and Type III cells with large nuclei and more than one type of granules. 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