Morphological Characterization of the Hemocytes of the Clam,Ruditapes decussatus (Mollusca: Bivalvia)

CARMEN LOPEZ,* M. J. CARBALLAL,* CARLOS AZEVEDO,† AND ANTONIO VILLALBA**Centre of Marine Research. Ministry of Fisheries and Aquaculture of Galicia. P.O.B. 208, 36600 Vilagarcıa de Arousa, Spain;

and †Department of Cell Biology, Institute of Biomedical Sciences, University of Porto, P-4000 Porto, Portugal

Received June 3, 1996; accepted October 28, 1996

Hemocytes play an important role in internal de-fence in molluscs. The morphology of hemolymph cellswas studied for the first time in Ruditapes decussatus.Twomain types of hemocytes (hyalinocytes and granu-locytes) exist in R. decussatus. Three types of granulo-cytes were identified by light microscopy, in accor-dance with the presence of basophilic or acidophilicgranules or a mixture of both in the cytoplasm. Theexistence of hyalinocytes and granulocytes was con-firmed by electron microscopy. Some monoclonal anti-bodies (MABs) raised against hemocytes ofCrassostreagigas showed cross-reactivity with the total popula-tion of hemocytes of R. decussatus; however, noneof the MABs raised against hemocytes of Mytilusedulis showed cross-reactivity. The MABs assayed didnot allow us to distinguish hemocyte subpopulations.r 1997Academic Press

KEY WORDS: Ruditapes decussatus; hemocytes; hemo-lymph; immunological assays.

INTRODUCTION

In bivalve molluscs, blood cells or hemocytes play anessential role in internal defence. Bivalve hemocytesare known to be involved in other processes like woundand shell repair, nutrient digestion and transport, andexcretion (Cheng, 1981).Several authors have distinguished in bivalves two

hemocyte types (hyalinocytes and granulocytes), basedon morphological, cytochemical, and functional criteria(Cheng, 1975, 1981; Auffret, 1988; Suresh and Mohan-das, 1990; Pipe, 1990b; Kumazagua et al., 1991), exceptin Pectinidae, which have only the hyalinocyte type(Auffret, 1988). In addition, density gradient centrifuga-tion and immunological techniques have been used toseparate and identify hemocyte subpopulations (Chenget al., 1980; Yoshino and Granath, 1983; Bachere et al.,1988; Pipe, 1990a; Noel et al., 1994).Most of the studies on bivalve hemocytes have been

carried out in oysters and mussels, and only a few were

performed in species belonging to the Veneridae family:Mercenaria mercenaria, Ruditapes philippinarum, andSunetta scripta (Cheng, 1975; Foley and Cheng, 1974;Auffret, 1985; Cheney, 1971; Suresh and Mohandas,1990). Ruditapes decussatus is one of the most impor-tant commercial species of this family in Europe.Populations of this species are affected by two impor-tant epizootics caused by Perkinsus atlanticus (Aze-vedo, 1989) and Vibrio P1 (Paillard et al., 1994).Understanding the defence mechanisms of this bivalvespecies could contribute to finding ways to avoid epizo-otic damage.The aim of the present study was the morphological

characterization of the hemocyte types occurring in thehemolymph of the clam R. decussatus in order to makefurther studies in cellular defence mechanism researcheasier.

MATERIALS AND METHODS

Specimens

The clams, R. decussatus, used in this study werecollected from a cultured bed located in Carril (Ria deArosa, Galicia NW of Spain).

Hemolymph Collection

The hemolymph was taken from the posterior adduc-tor muscle of R. decussatus clams using a 23-gauge 3 1in. needle and syringe, through a hole made in the shellmargin. Depending on the assay, the hemolymph wascollected pure, diluted 1:3 in modified anti-aggregantAlsever solution (MAS) (20.80 g/liter glucose, 8.00g/liter sodium citrate, 3.36 g/liter EDTA, 22.50 g/litersodium chloride in distilled water) or in a fixativesolution.

Light Microscopy

Hemolymph smears and hemocyte cytospins wereused to study hemocyte morphology by light micros-copy.

JOURNAL OF INVERTEBRATE PATHOLOGY 69, 51–57 (1997)ARTICLE NO. IN964639

51 0022-2011/97 $25.00Copyright r 1997 by Academic Press

All rights of reproduction in any form reserved.

Smears. Smears were prepared as follows: one dropof pure hemolymph was added over one drop of filteredseawater (FSW) onto the glass slide. Hemocytes wereallowed to settle onto the glass surface for 15–30 min ina moist chamber at room temperature. Then, theattached cells were rinsed in FSW, fixed inmethanol for1 min, stained with rapid Hemacolor kit (Merck), airdried, and mounted.

Cytospins. Hemolymph was withdrawn diluted inMAS (1:3). The number of suspended hemocytes wasestimated with a Malassez hemocytometer. A volume ofthe suspension with 503 104 hemocytes was cytocentri-fuged (92g, 5 min, 4°C) using a Megafuge 1 or acentrifuge (Heraeus). Then, haemocytes were fixed andstained as described previously.

Hemocyte Measurements

Cell and nucleus diameters of hemocytes in cytospinswere measured using a microscope with a gradedocular. In addition, measurements were performed onsuspended hemocytes using image analysis equipment.In the latter case the hemolymph was withdrawndiluted 1:1 in MASwith 6% formaldehyde.After 15 minthe suspension was centrifuged (750g, 10 min, 4°C).Hemocytes were resuspended in 400 µl of MAS andstained with 100 µl of trypan blue. After 30 min a dropwas deposited onto a slide.

Electron Microscopy

Hemolymph was collected diluted 1:1 in a fixativesolution (5% glutaraldehyde in Pipes buffer 0.1 M, atpH 7.2, and 7% sucrose) and kept at 4°C for 1 h. Thesuspension was centrifuged (750g, 10 min). The pelletwas washed in Pipes buffer with sucrose for 2 h at 4°Cand postfixed in 1% osmium tetroxide in Pipes bufferfor 75 min at 4°C. After being washed in Pipes buffer,the cells were embedded in 1.5% agar at 40°C andquickly centrifuged (1700g, 5 min). Then, the pellet wasdehydrated and embedded in Epon. Ultrathin sections(50–70 nm) were stained with uranyl acetate and leadcitrate and examined in a transmission electronmicros-copy JEOL 100CXII.

Separation of Hemocytes by Discontinuous DensityGradient Centrifugation

Hemolymph was collected diluted 1:1 in MAS with6% formaldehyde. The gradients of Percoll were madewith isosmotic Percoll (20.8 g/liter glucose; 8 g/liter Nacitrate; 22.5 g/liter NaCl; 11 Percoll) diluted in MAS(10, 20, 30, 40, and 50%). Discontinuous density gradi-ents were made adding 2.5 ml of each concentrationinto a centrifuge tube. Into 2.5 ml of a suspensioncontaining 40 3 106 hemocytes in MAS was added intothe tube. After centrifugation (500g 30 min), the cells

concentrated at each density interface were collectedseparately with a syringe, diluted in MAS, and layeredonto a cushion of 10% sucrose–MAS in order to elimi-nate the Percoll. After a new centrifugation the pelletsof cells were resuspended in MAS. With the differentinterfaces were made cytospins, and a rapid hemacolor(Merck) coloration was used (Bachere et al., 1988).

Immunological Assays

Immunological assays were performed to test if clamhemocytes have epitopes for 7 monoclonal antibodies(MABs) raised against hemocytes of M. edulis and 18against Crassostrea gigas haemocytes. MABs (hybri-doma culture supernatants) used in our study wereprepared in the IFREMER laboratory (La Tremblade,France) (Morvan, 1991; Noel et al., 1994) in order tohave a tool to distinguish hemocyte subpopulations inthe respective species.Cytospins of clam hemocytes were used for indirect

immunofluorescence. Air-dried cytospins were fixed inacetone for 5 min, washed in distilled water, and airdried again. Cytospins were either immediately used orstocked at 220°C until used.Indirect immunofluorescence assays were performed

following the procedure described by Noel et al. (1994).The slides were overlaid with MABs and incubated 30min at room temperature in a moist chamber. Theslides were washed with immunofluorescence buffer(IF) (Diagnostics Pasteur) and then overlaid with fluo-rescein isothiocyanate-conjugated goat anti-mouse im-munoglobulin antiserum (Diagnostics Pasteur), dilutedin the same buffer containing 0.01% Evans blue. Theslides were incubated 30 min at room temperature in amoist chamber, washed, and mounted using glycerin-buffer fluid. MABs raised against Bonamia ostraea(Rogier et al., 1991) and cytospins incubated with bufferlacking MABs were used as negative controls.

Statistics

Differences in cell size, nucleus size, and nucleus/cytoplasm (N/C) ratio between hemocyte types werecompared byANOVA.ANewman–Keul’s range test wasused for multiple comparison. Tests were performedusing SOLO 2.0 (BMDPStatistical Software Inc., 1988).

RESULTS

Light Microscopy

Two hemocyte types were distinguished by lightmicroscopy: granulocytes and hyalinocytes, accordingto the presence or the absence of cytoplasmic granules,respectively.In smears, endoplasm and ectoplasm could be clearly

distinguished in granulocytes, but not in hyalinocytes.Cytoplasmic granuleswere present in granulocyte endo-

52 LOPEZ ET AL.

plasm, whereas ectoplasm showed a hyaline aspect(Fig. 1). Granulocytes showed greater ability to producepseudopods. In addition, multinucleate (2–14 nuclei)hemocytes with cytoplasmic granules were abundant inthese monolayers (Fig. 2). These multinucleate struc-tures could be the result of the fusion of granulocytes.In hemocyte cytospins (Fig. 3), the number of hyalino-

cytes was higher than that in smears, thus suggesting alimited ability to adhere to a glass surface of thishemocyte type. Granulocytes showed fewer pseudopodsthan in smears.It was possible to distinguish acidophilic, basophilic,

andmixed granulocytes according to the staining affini-ties of their cytoplasmic granules, in both smears andcytospins.

Electron Microscopy

Electron microscopy permitted us to confirm theoccurrence of two hemocyte types (granulocytes and

hyalinocytes) in the hemolymph of R. decussatus(Fig. 4).The hyalinocytes showed a total absence of cytoplas-

mic granules. The nucleus appeared in a central or aneccentric position. The nucleus showed abundant het-erochromatin in the central and the peripheral posi-tions. The cytoplasm contained a variable number ofmitochondria, Golgi complex, endoplasmic reticulum,and small electron-lucid vesicles of different sizes, someof them probably originating in the Golgi complex orthe smooth endoplasmic reticulum.The granulocytes showed similar organelles but on

the contrary had abundant electron-dense cytoplasmicparticles surrounded by a membrane unit, that is,cytoplasmic granules with diameters between 0.2 and1.1 µm.

Hemocyte Measurements

Table 1 shows the ranges and mean values (6 SE) ofthe cell and nucleus size and the N/C ratio measured in

TABLE 1Mean Values 6 Standard Error and Ranges of Cell and

Nuclear Diameters and Nuclear/Cytoplasmic Ratio (N/C) ofHemocytes of R. decussatus

Hyalinocytes(N 5 98)

Basophilic Gr.(N 5 35)

Acidophilic Gr.(N 5 166)

Cell diameter 11.44 6 0.26 µm 12.24 6 0.35 µm 13.42 6 0.16 µm(6–20 µm) (9–20 µm) (10–20 µm)

Nucleardiameter 7.34 6 0.14 µm 7.46 6 0.23 µm 7.65 6 0.10 µm

(5–12 µm) (6–13 µm) (5–14 µm)N/C 0.70 6 0.01 0.63 6 0.02 0.50 6 0.01

(0.4–1) (0.4–0.9) (0.4–0.9)

Note.Measurements were made on cytospins.N, sample size.

TABLE 2Results of ANOVA Made for Comparison of Different Cell

Dimensions (Cell and Nuclear Diameter, (N/C) betweenHemocyte Types (Hyalinocytes and Granulocytes) of R.decussatus

Source DF Sum-Square F-ratio Prob. . F

Variable cell diameterA (Type) 2 306.2991 24.49 0.0000Error 296 1851.219Total 298 2157.518

Variable nucleardiameter

A (Type) 2 2.7508 0.58 0.5632Error 296 707.7576Total 298 710.5084

Variable nuclear/cyto-plasm ratio

A (Type) 2 0.937844 30.65 0.0000Error 296 4.528109Total 298 5.465953

Note.Measurements were made on cytospins.

TABLE 3Mean Values 6 Standard Error and Ranges of Cell andNuclear Diameter and N/C of Hemocytes of R. decussatus

Hyalinocytes(N 5 117)

Granulocytes(N 5 121)

Cell diameter 10.89 6 0.16 µm 8.88 6 0.16 µm7.61–15.69 µm 5.17–15.69 µm

Nuclear diameter 4.67 6 0.08 µm 3.88 6 0.08 µm2.38–7.69 µm 2.18–7.13 µm

N/C 0.43 6 0.01 0.44 6 0.010.24–0.75 0.27–0.68

Note.Measurements were made on hemocytes fixed in suspension.N, sample size.

TABLE 4Results of ANOVA Made for Comparison of Different Cell

Dimensions (Cell and Nuclear Diameter, (N/C)) betweenHemocyte Types (Hyalinocytes and Granulocytes) of R.decussatus

Source DF Sum-Square F-Ratio Prob. . F

Variable cell diameterA (Type) 1 239.8728 76.62 0.000Error 236 738.8348Total 237 978.7076

Variable nucleardiameter

A (Type) 1 37.49272 44.67 0.000Error 236 198.0948Total 237 235.5875

Variable nuclear/cyto-plasm ratio

A (Type) 1 9.467E-03 1.30 0.2549Error 236 1.723647Total 237 1.733115

Note.Measurements were made on hemocytes fixed on suspension.

53MORPHOLOGICAL CHARACTERIZATION OF CLAM HEMOCYTES

54 LOPEZ ET AL.

cytospins. Granulocyte types showed larger sizes andsmaller N/C ratios than hyalinocytes. The results of theANOVA comparison (Table 2) indicated a significantdifference for cell size and N/C ratio and the differencewas not significant for nucleus size. The multiplecomparison with the Newman/Keul’s test indicated asignificant difference between all hemocyte types forthe cell size. However, in the case of the N/C ratio,Newman/Keul’s test indicated a significant differenceonly between granulocytes and hyalinocytes but notbetween granulocyte types.Table 3 shows the range and mean values (6 SE) of

the cell and nucleus size and N/C ratio measured onhemocytes fixed in suspension. In this case hyalino-cytes showed larger cell size and nucleus size thangranulocytes but the N/C ratio was similar. The resultsof ANOVA comparison (Table 4) showed a significantdifference for cell and nucleus size and were nonsignifi-cant for the N/C ratio.

Hemocyte Separation

With the discontinuous Percoll density gradient cen-trifugation it was not possible to separate hemocytetypes. Most of the hemocytes were concentrated at theinterfaces 20–30% and 30–40% (Table 5).

Immunological Assays

Only 5 MABs of a total of 18 tested MABs raisedagainst C. gigas hemocytes showed cross-reactivitywith R. decussatus hemocytes. 100% of the hemocytesin cytospins cross-reacted with these 5 MABs (Table 6).Therefore, those MABs did not allow us to distinguishamong hemocyte types. On the contrary, none of the 7

MABs against M. edulis hemocytes showed cross-reactivity with R. decussatus hemocytes.

DISCUSSION

Two morphological hemocyte types occur in R. decus-satus hemolymph, granulocytes, and hyalinocytes, thatcan be distinguished according to the presence or theabsence of granules inside the cytoplasm. Other au-thors have described the occurrence of these hemocytetypes in various marine bivalves, with the exception ofPectinidae, which lack the granulocyte type (Auffret,1988).Earlier studies on clam hemocytes of the Veneroida

order indicated the only occurrence of granulocytes(Cuenot, 1891, in Cheng, 1981; Zaks, 1955, in Cheng,

FIG. 1. Photomicrograph of granulocytes in smears. en, endoplasm; ec, ectoplasm; ps, pseudopods; gr, cytoplasmic granules; n, nucleus(31150). FIG. 2. Photomicrograph of a multinucleate granulocytes in smears. This multinucleate cell could be the result of the fusion ofgranulocytes. n, nucleus (31150). FIG. 3. Photomicrograph from a hemocyte cytospin showing granulocytes (Gr) with cytoplasmic granules(gr) and nucleus (n) and hyalinocytes (Hy) with hyaline cytoplasm and nucleus (n). (31325). FIG. 4. Electronmicrograph showing thinsections of a granulocyte (Gr) and hyalinocyte (Hy). N, nucleus; gr, electron-dense particles; mi, mitochondria; g, Golgi complex; ps,pseudopods; v, electron-lucid vesicles. (35800).

TABLE 5Percentage of Cells Corresponding to Each Hemocyte Type

Occurring in Every Interface after Centrifugation in aDiscontinuous Gradient of Percoll

Interface

Granulocytes

%Hyalinocytes %Acidolphic %Basophilic%Acidophilic/basophilic

10/20 23.16 6 2.16 61.00 6 2.00 14.50 6 1.17 1.33 6 1.3420/30 23.45 6 0.91 60.66 6 1.20 12.44 6 0.99 3.45 6 1.1330/40 23.33 6 1.39 60.89 6 1.44 12.89 6 1.55 2.89 6 0.4840/50 21.00 6 3.33 62.50 6 0.83 14.50 6 4.50 2.00 6 2.00

TABLE 6Reactivity (Percentage of Reacting Hemocytes and

Reaction Intensity) of R. decussatus Hemocytes with MABsagainst C. gigas and M. edulis Hemocytes, in FluorescentImmunoassays

MABs Reactivity

C. gigasHCG2 100% (11)HCG4 0%HCG5 100% (11)HCG9 0%HCG10 0%HCG12 0%HCG13 0%HCG14 0%HCG15 100% (11)HCG16 0%HCG18 0%HCG19 0%HCG21 96% (1)HCG22 0%HCG23 100% (11)HCG26 0%HCG27 0%HCG29 0%

M. edulis18C5 0%20D3 0%9F10 0%11A1 0%13B9 0%18E10 0%6E2 0%

Note. (1) light, (11) moderate, (111) strong.

55MORPHOLOGICAL CHARACTERIZATION OF CLAM HEMOCYTES

1981), but Auffret (1985) noted the presence of thegranulocytes and hyalinocytes in R. phillipinarum.Foley and Cheng (1974) reported the presence of threehemocyte types inM. mercenaria (hyalinocytes, granu-locytes, and fibrocytes), but Cheng and Foley (1975),after an ultrastructural study, considered the fibrocytesto be granulocytes which have degranulated.In R. decussatus hemocyte types some morphological

characteristics showed variability depending on thetechnique used. Cheney (1971) made the same observa-tion.The small capacity of adhesion showed byR. decussa-

tus hyalinocytes agrees with the observations on M.mercenaria indicated by Foley and Cheng (1974).The multinucleate cells observed in cytospins were

similar to these reported by Auffret (1985). However,multinucleated cells found in the smears in this studyevoked these found by Sparks and Pauley (1964), Foleyand Cheng (1974), Anderson (1987), and Cheng (1981).These authors considered that these cells could arisefrom the fusion of granulocytes. These cells were foundin postmortem oysters (Sparks and Pauley, 1964) andassociated with allografts and xenografts (Cheng andGalloway, 1970).The results of the ultrastructural study ofR. decussa-

tus hemocytes agree with the results of Cheng andFoley (1975) andAuffret (1985) inM.mercenaria andR.philippinarum, respectively. Different authors distin-guished in other bivalves granulocytes with only smallor large granules that were considered to be immatureor mature granulocytes, respectively (Klebanoff andClark, 1978; Rasmussen et al., 1985; Pipe, 1990b).In our study, euchromatin was found in the nuclei of

both hemocyte types, as indicated byAuffret (1985).Since spreading ability is different between hyalino-

cytes and granulocytes, results of cell measurementswere different according to the method used. Hyalino-cytes appeared larger than granulocytes when they hadbeen fixed in suspension. This is in agreement withSuresh and Mohandas’s (1990) observations on theclams Sunetta scripta and V. cyprinoides var. cochinen-sis, who used the same method for measurements. Onthe contrary, when hyalinocytes were cytocentrifugedand then measured, granulocytes appeared larger, asstated by Auffret (1985) for R. philippinarum hemo-cytes. It seems that fixing hemocytes in suspensionbefore measuring is a better method. There are othertechniques for measuring cells, such as flow cytometryand the use of a Coulter counter (Ford et al., 1994).Several authors suggested different hemopoietic paths

in bivalves (Mix, 1976; Moore and Eble, 1977; Balouetand Poder, 1979; Cheng, 1981). Cheng (1981) saidabout the presence of basophilic and acidophilic gran-ules inside granulocytes that the basophilic granulescould be immature granules which mature and becomeacidophilic. The simultaneous occurrence of both kinds

of granules inside some hemocytes in R. decussatuscould support this hypothesis.The immunological assays did not permit identifica-

tion of hemocyte subpopulations in this study, sincenone of the MABs was specific for any hemocyte type.For this purpose it would be necessary to produceMABs against clam hemocytes. MABs have been usedfor identifying hemocyte subpopulations in vertebrates(Takeya et al., 1989; Flo et al., 1991; Leven andRodriguez, 1991; Naume et al., 1991) and molluscs(Yoshino and Granath, 1983; Dikkeboom et al., 1985;Noel et al., 1994).According to immunological results it would be pos-

sible to hypothesize a major phylogenetic proximitybetween clams and oysters opposite to mussels. Sincenone of the MABs rised against mussel hemocytesreacted with clam hemocytes, whereas 5 of the MABsrised against oyster hemocytes cross-reacted with clamhemocytes.

ACKNOWLEDGMENTS

We thank the team of Eric Mialhe (IFREMER, La Tremblade,France) for supplying the monoclonal antibodies and advising us inimmunological techniques and Jose Molares and Jose Fuentes fortheir collaboration with image analysis and statistics. The clams forthis study were supplied by Manuel Franco. This study was partiallysupported by a scholarship of CAIXA DE GALICIA in 1991 forCarmen Lopez during a stay in the laboratory of IFREMER (LaTremblade, France).

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57MORPHOLOGICAL CHARACTERIZATION OF CLAM HEMOCYTES