Effects of high temperatures on functional responses of haemocytes in the clam Chamelea gallina

The effects of high temperatures on the clam, Chamelea gallina, generally recognised as a low tolerant bivalve species, were studied by evaluating some functional responses of the haemocytes. The animals were kept for 7days at 20, 25 and 30 degrees C and total haemocyte count (THC), phagocytosis, lysozyme activity (in both haemocyte lysate and cell-free haemolymph), activity and expression of the antioxidant enzyme superoxide dismutase (SOD) (in both haemocyte lysate and cell-free haemolymph) were chosen as biomarkers of exposure to high temperatures. The survival-in-air test was also performed. During the experiment, the clams showed differing burrowing behaviour: the animals kept at 20 and 25 degrees C burrowed completely, whereas at 30 degrees C the clams progressively emerged from the sediment and then remained on the surface. The highest temperature significantly increased THC, whereas it decreased the phagocytic activity of haemocytes. The haemocyte size frequency distribution in clams kept at 30 degrees C showed that the cell population of about 8-10microm was markedly reduced compared to clams kept at 20 and 25 degrees C. In clams maintained at 25 degrees C, lysozyme activity was significantly increased in haemocyte lysate, whereas it was markedly decreased in cell-free haemolymph. Total SOD activity significantly decreased in haemocytes from clams held at 30 degrees C whereas it increased in cell-free haemolymph from clams held at 25 degrees C and 30 degrees C. A significant decrease in haemocyte Mn-SOD and Cu/Zn-SOD activities was found with increasing temperature. In cell-free haemolymph, the highest Mn-SOD activity was recorded at 30 degrees C, whereas the Cu/Zn-SOD activity showed no significant changes in clams maintained at different temperatures. SOD isoform expression exhibited different patterns in haemocyte lysate and cell-free haemolymph. The resistance to air exposure of clams kept at 30 degrees C was shown to decrease significantly, LT(50) values fell from 6days in clams kept at 20 degrees C and 25 degrees C to 4days in those kept at 30 degrees C.     

Differential binding of lectins to haemocytes of the mussel Mytilus edulis

Summary Pre- and post-embedding techniques were used to investigate the ultrastructural binding of a range of lectins to the haemocytes of the mussel Mytilus edulis. Direct and indirect labelling procedures were employed using colloidal gold and ferritin-labelled lectins, or biotinylated lectins followed by gold-labelled streptavidin. Cell surface receptors were present for lectins from Helix pomatia (HPA), Helix aspersa (HAA), Triticum vulgaris (WGA) and Tetragonolobus purpureas (TPA). Double labelling of haemocytes with HPA and WGA demonstrated binding sites for both lectins on the plasma membrane of the majority of haemocytes. Endocytosis of colloidal gold-labelled HPA was observed for unfixed haemocytes. Three classes of haemocyte were identified by use of morphological criteria: hyalinocytes; granulocytes containing small granules; and granulocytes containing large granules. Lectin binding showed the small granules of the granulocytes to be HPA-positive and the large granules of the granulocytes to be WGA-positive. The WGA-positive granules demonstrated a differential pattern of binding according to granule size. Binding sites for the lectin from Arachis hypogaea (PNA) were not demonstrated on the cell surface, but did show an affinity for the heterochromatin region of the nucleus in post-embedding protocols.     

Insight on cellular and humoral components of innate immunity in Squilla mantis (Crustacea, Stomatopoda)

For deeper insights into the function of crustacean haemocytes in immune responses, we studied the morphology and enzyme content of circulating cells of the mantis shrimp Squilla mantis from the North Adriatic Sea, together with their ability to phagocytose foreign cells. We also assayed the enzyme content and the agglutinating and haemolytic activities of cell-free haemolymph. Three haemocyte types, i.e., hyalinocytes, semigranulocytes and granulocytes, can be distinguished, according to cell and nuclear morphology and the presence of cytoplasmic granules. All of them share the same patterns of enzyme activities and are recognised by the same lectins. Spreading cells (hyalinocytes and semigranulocytes) can ingest foreign cells; granules of semigranular and granular cells have similar cytochemical properties. Injection of Micrococcus luteus into the heart sinus results in an increase in the frequency of hyaline cells and a decrease in the frequency of granulocytes. After 24 h from the injection, a decrease in the number of phagocytosing hyalinocytes, and a general decrease in the frequency of acid phosphatase-positive cells was reported. Our data match previous results and suggest the existence of a single differentiation pathway for Squilla haemocytes with the three haemocyte morphs as different stages of cell differentiation. Results also indicate that Squilla haemolymph performs immunosurveillance, through rapid changes in haemocyte distribution, increase of antimicrobial and antioxidant enzymes and secretion of lectins stimulating agglutination, phagocytosis and encapsulation.     

Characterisation of monoclonal antibodies to haemocyte types of scallop (Chlamys farreri)

Four monoclonal antibodies (MAbs) (1E7, 1F12, 2H5, 2C6) against haemocytes of scallop (Chlamys farreri) were produced by immunising Balb/C mice. Analysed by the indirect immunofluorescence assay test (IIFAT), immunocytochemical assay, flow cytometry (FCM) and Western-blotting, they showed specificity for more than one haemocyte type (hyalinocyte and granulocyte) and various haemocyte components of scallop. Using IIFAT to detect monolayers separated from gradient density centrifugation, the four MAbs were positive with haemocytes at different interfaces. The percentage of positive cells (percent reactivity, PR) that MAb 1E7 reacted with at the 20-30%, 30-40% and 40-50% interfaces were 43.50%, 41.25% and 60.00% respectively, PR of MAb 1F12 were 31.00%, 63.50% and 41.00%, MAb 2C6 were 11.00%, 51.00%, 77.00%, and MAb 2H5 were 20.25%, 34.75%, 38.25%. For the immunocytochemical assay, MAb 1E7 1F12 and 2H5 was positive with the cytoplasm of both hyalinocyte and granulocyte, 2C6 was positive with the membrane and cytoplasm of hyalinocyte and granulocyte. Analysed by FCM, the PR of the four MAbs (1E7, 1F12, 2H5, 2C6) with haemocytes were 54.23%, 38.56%, 56.4%, and 79.7% respectively; moreover, the PR with different haemocyte types was variable. The results of Western-blotting showed that MAb 1E7 recognised an antigen of molecular weight 200 kDa, MAb 2C6 an antigen of 60 kDa, however, MAb 1F12 reacted with antigens of 70 kDa, 60 kDa and 45 kDa. There was no protein band that MAb 2H5 detected. In conclusion, 2C6 seems to be a very promising MAb to identify and differentiate granulocytes, and the four MAbs will be used in further studies on cellular defence mechanism research.     

Haemocytes of the cockle Cerastoderma glaucum: morphological characterisation and involvement in immune responses

For the first time, morpho-functional characterisation of haemocytes from the cockle Cerastoderma glaucum was performed to identify circulating cell types and to study their involvement in immune responses. Haemocyte mean number was 5.5 (x 10(5)) cells/mL haemolymph. Two main haemocyte types were found in haemolymph: granulocytes (85%), about 10 microm in diameter and with evident cytoplasmic granules, and hyalinocytes (15%), 8 to 14 microm in diameter, with a few or no granules. Most of the cytoplasmic granules stained in vivo with Neutral Red, indicating that they were lysosomes. On the basis of haemocyte staining properties, granulocytes and hyalinocytes were further classified as basophils and acidophils. Acidophil hyalinocytes were the largest haemocyte type (about 14 microm in diameter) and had an eccentric nucleus and a large cytoplasmic vacuole. Both granulocytes and hyalinocytes (except acidophils) were able to phagocytise yeast cells, although the basal phagocytic index was very low (about 2%). It increased significantly (up to 26%) after pre-incubation of yeast in cell-free haemolymph, suggesting that haemolymph has opsonising properties. Haemocytes also produced superoxide anion. Moreover, both granulocytes and hyalinocytes (except acidophils) were positive for some important hydrolytic and oxidative enzymes. Lysozyme-like activity was recorded in both cell-free haemolymph and haemocyte lysate, although enzyme activity in cell lysate was significantly higher. Results indicate that haemocytes from C. glaucum are effective cells in immune responses.     

Comparison of antigenicity among haemocytes of seven bivalve species by monoclonal antibodies against haemocytes of scallop (Chlamys farreri)

In this paper, haemocyte antigenicity of seven bivalve species (scallop (Chlamys farreri), bay scallop (Argoecten irradians), oyster (Crassostrea talienwhanensis), asiatic hard clam (Meretrix meretrix), monila clam (Ruditapes philipinarum), purplish washington clam (Saxidomus purpuratus) and horny ark (Scapharca subcrenta)) were analysed using monoclonal antibodies (MAbs) 1E7, 1F12, 2C6 and 2H5 against haemocytes of C. farreri, employed methods of immuno-dotblotting (IDB), indirect immunofluorescence assay (IIFA) and western-blotting (WB). The four MAbs react with haemocytes of seven bivalve species. As the results for both IDB and IIFA, MAb 1E7 was positive with haemocytes of R. philipinarum, MAb 1F12 with haemocytes of A. irradians, M. meretrix, R. philipinarum and S. purpuratus; MAb 2C6 with haemocytes of the other five bivalve species except for S. purpuratus. MAb 2H5 was negative with haemocytes of the other six bivalve species in IDB, but was positive with haemocytes of R. philipinarum and S. purpuratus in IIFA. Further experiments by WB showed MAb 1F12 was able to recognise the protein of A. irradians haemocyte at molecular weights of 156 and 80 kDa, haemocytes of M. meretris, R. philipinarum, S. purpuratus, at 60, 30, 58 kDa, respectively. MAb 2C6 recognised haemocyte M. meretris proteins at 50 and 37 kDa, A. irradians, C. talienwhanensis, R. philipinarum, S. subcrenta at 40, 38, 38, 45 kDa, respectively. There were no protein bands reacting with MAb 1E7 and MAb 2H5. The results indicate antigenic similarities exist among haemocytes of the seven bivalve species.     

Characterization and immunocytochemical localization of actin and fibronectin in haemocytes of the musselMytilus galloprovincialis

Summary Cell-extracellular matrix interactions are recognized to be important for human leucocyte functions, including chemotaxis and phagocytosis. These activities depend on a reorganization of the microfilament actin (F-actin) promoted by fibronectin, one of the major components of extracellular matrices. Although invertebrate haemocytes are, in many aspects, similar to the human granulocyte-monocyte-macrophage cell lineage, actin and fibronectin have not been well studied in these cells. Consequently, the characterization and structural organization of actin and fibronectin in mussel (Mytilus galloprovincialis) haemocytes was investigated using Western blotting analysis, indirect immunofluorescence and immunoelectron microscopy. Actin was immunocharacterized by an anti-total actin monoclonal antibody. Fibronectin was immunocharacterized by an autologous polyclonal antiserum directed against the protein of mussel haemolymph. Actin was mainly localized along the peripheral cytoplasm of the haemocyte. The distribution of the F-actin microfilaments was assayed with Rhodamine-labelled phalloidin. F-actin was associated mainly with stress-fibres of spreading haemocytes and with microspikes at the adhesion sites. The labelling by the anti-fibronectin antiserum of the haemocyte rough endoplasmic reticulum vesiles, revealed by immunoelectron microscopy, suggests that these cells are involved in fibronectin biosynthesis. Gold particles were also present along the outer surfaces of the cell plasma membrane and its protrusions. Mussel fibronectin was localized immunohistochemically at the adhesion sites and in the extracellular matrix fibrils. The relationships between fibronectin and the actin cystoskeleton inMytilus galloprovincialis haemocytes are discussed.     

Monoclonal antibodies specific to haemocytes of black tiger prawn Penaeus monodon

Monoclonal antibodies specific to haemocytes of Penaeus monodon were generated from a mouse immunized with a mixture of SDS-treated and formalin-fixed haemocytes. Hybridoma clones were selected by immunohistochemistry against fixed haemocytes, heart, lymphoid organ, and haemopoietic tissue, and Western blot against haemocyte extract and haemolymph. Sixteen monoclonal antibodies specific to haemocytes were obtained and could be divided into six groups according to their binding capacities to various haemocyte proteins in Western blot analyses, 102, 43, approximately 20, 61, 175 and approximately 230 kDa, and their differences in recognition of haemocyte sub-populations. The first group of antibodies strongly recognized a small subset of semi-granulocytes (SG) and hyalinocytes (H) but occasionally stained lightly a very small population of granulocytes (G). The antibodies also bound to a group of cells in haemopoietic tissue as well as cells located at the inner layers of the tubules in the lymphoid organ but not in the spheroid. The second group of antibodies strongly bound to a large sub-population of G and SG with coarse granules but did not bind to most of the H. This group of antibodies also cross-reacted with cells in the outer layer of the tubules in the lymphoid organ. The third group of antibodies recognized all G and only a small portion of SG. The fourth, fifth and sixth groups bound to sub-populations of G, SG and H in similar proportions. None of the antibodies showed any cross-reactivity to other components in haemolymph. The common antigens recognized by the first and the second groups of antibodies in the haemopoietic tissue and the lymphoid organ may reflect relationships among these organs in the development of the sub-populations of G and SG. Haemopoietic tissue may be the site for haemocyte production and the lymphoid organ may be the site for further differentiation of at least two different lines of haemocytes.     

Haemocyte reactions in WSSV immersion infected Penaeus monodon

White spot syndrome virus (WSSV) has been a major cause of shrimp mortality in aquaculture worldwide in the past decades. In this study, WSSV infection (by immersion) and behaviour recruitment of haemocytes is investigated in gills and midgut, using an antiserum against the viral protein VP28 and a monoclonal antibody recognising haemocytes (WSH8) in a double immunohistochemical staining and in addition transmission electron microscopy was applied. More WSH 8(+) haemocytes were detected at 48 and 72 h post-infection in the gills of infected shrimp compared to uninfected animals. Haemocytes in the gills and midgut were not associated with VP28-immunoreactivity. In the gills many other cells showed virus replication in their nuclei, while infected nuclei in the gut cells were rare. Nevertheless, the epithelial cells in the midgut showed a clear uptake of VP28 and accumulation in supranuclear vacuoles (SNV) at 8h post-infection. However, epithelial nuclei were never VP28-immunoreactive and electron microscopy study suggests degradation of viral-like particles in the SNV. In contrast to the gills, the midgut connective tissue shows a clear increase in degranulation of haemocytes, resulting in the appearance of WSH8-immunoreactive thread-like material at 48 and 72 h post-infection. These results indicate recruitment of haemocytes upon immersion infection in the gills and degranulation of haemocytes in less infected organs, like the midgut.     

Electron microscope study of haemolymph cells of Metrioptera roeselii (Orthoptera,Tettigoniidae)

On the basis of patterns of haemocyte ultrastructure and functions at preimago and imago stages of Metrioptera roeselii, secretory cells of granulocyte type were recognized in the haemolymph. The development of granulocytes was traced starting from their formation up to cell death and destruction. The haemocytes develop as "dark" and "light" cells, differing in their functional activity, although their ultrastructure is similar. In the cytoplasm of granulocytes, granules of both mitochondrial and nuclear origin were detected. Simultaneously two processes occur in the cells--the accumulation and discharge of granules.     

Protein kinase A affects Galleria mellonella (Insecta: Lepidoptera) larval haemocyte non-self responses

We used the protein kinase A (PKA) specific activator Sp-8-Br-cAMPS and type I inhibitor Rp-8-Br-cAMPS alone and in combination to define the role of PKA in the non-self responses of larval Galleria mellonella haemocytes in vitro and in vivo. Active PKA depressed haemocyte responses whereas PKA inhibition enhanced activities, including bacterial phagocytosis, the number of haemocytes with adherent bacteria, bacterial-induced haemocytic protein release and haemocyte adhesion to slides in vitro, as well as in vivo bacterial removal from the haemolymph. Non-attached haemocytes had more PKA activity than attached haemocytes; therefore, active PKA limited haemocyte response to foreign materials. We found that (i) PKA inhibitor alone induced non-self responses, including haemocyte protein discharge and lowered haemocyte counts in vivo, and induced nodulation; (ii) the enzyme activator produced effects opposite to those of the inhibitor; and (iii) together, the modulators offset each others' effects and influenced haemocyte lysate PKA activity. These findings establish PKA as a mediator of haemocytic non-self responses.     

The roles of haemocytes and the lymphoid organ in the clearance of injected Vibrio bacteria in Penaeus monodon shrimp

In order to study the reaction of Penaeus monodon haemocytes, live Vibrio anguillarum bacteria were injected and the shrimp were periodically sampled. Immuno-double staining analysis with specific antisera against the haemocyte granules and bacteria showed that large numbers of haemocytes encapsulated the bacteria at the site of injection. A rapid decrease of live circulating bacteria was detected in the haemolymph. Bacterial clearance in the haemolymph was induced by humoral factors, as observed by agglutinated bacteria, and followed by uptake in different places in the body. Bacteria mainly accumulated in the lymphoid organ (LO), where they, or their degradation products, could be detected for at least 7 days after injection. The LO consists of folded tubules with a central haemal lumen and a wall, layered with cells. The haemolymph, including the antigens, seemed to migrate from the central tubular lumen through the wall, where the bacteria are arrested and their degradation is started. Electron microscopy of the LO revealed the presence of many phagocytic cells that morphologically resemble small-granular haemocytes. It is proposed that haemocytes settle in the tubule walls before they phagocytose. Immunostaining suggests that many of the haemocytes degranulate in the LO, producing a layer of fibrous material in the outer tubule wall. These findings might contribute to the reduced haemocyte concentration in the haemolymph of diseased animals or following injection of foreign material. It is proposed that the LO is a filter for virtually all foreign material encountered in the haemolymph. Observations from the present study are similar to clearance mechanisms in the hepatic haemolymph vessel in most decapod crustaceans that do not possess a LO. The experimental shrimp appeared to contain many LO spheroids, where bacterial antigens were finally observed as well. It is proposed that the spheroids have a degradation function for both bacterial and viral material, and that their presence is primarily related to the history of the infectious burden of the shrimp.     

Preliminary study on haemocyte response to white spot syndrome virus infection in black tiger shrimp Penaeus monodon

White spot syndrome virus (WSSV) has been a major cause of shrimp mortality in aquaculture in the past decade. In contrast to extensive studies on the morphology and genome structure of the virus, little work has been done on the defence reaction of the host after WSSV infection. Therefore, we examined the haemocyte response to experimental WSSV infection in the black tiger shrimp Penaeus monodon. Haemolymph sampling and histology showed a significant decline in free, circulating haemocytes after WSSV infection. A combination of in situ hybridisation with a specific DNA probe for WSSV and immuno-histochemistry with a specific antibody against haemocyte granules in tissue sections indicated that haemocytes left the circulation and migrated to tissues where many virus-infected cells were present. However, no subsequent haemocyte response to the virus-infected cells was detected. The number of granular cells decreased in the haematopoietic tissue of infected shrimp. In addition, a fibrous-like immuno-reactive layer appears in the outer stromal matrix of tubule walls in the lymphoid organ of infected shrimp. The role of haemocytes in shrimp defence after viral infection is discussed.     

内脏团插核术刺激对三角帆蚌血细胞的影响

为了探讨三角帆蚌(Hyriopsis cumingii Lea)血细胞的类型及内脏团插核手术刺激对血细胞形态结构和数量的影响,研究利用相差显微镜、光学显微镜、透射电子显微镜和流式细胞仪对三角帆蚌血细胞进行了形态学研究。流式细胞术光散射图谱显示血细胞被分两类,一类为颗粒度高的大细胞,另外一类为颗粒度低的小细胞;相差显微镜观察显示,血细胞可分为胞体暗、折光性差和胞体明亮、折光性强的两类;Giemsa和H.E染色显示细胞分为胞质染色不均一、胞内颗粒明显和胞质染色均一、胞内颗粒不明显的两类;透射电镜超薄切片观察显示,颗粒明显的细胞胞质内线粒体、高尔基体等细胞器较丰富,颗粒不明显的细胞胞质内细胞器较少;负染结果表明血细胞主要分为表面不光滑、突起明显和细胞表面光滑、突起较不明显的两类。综合上述实验结果可见,三角帆蚌血细胞分为颗粒明显的细胞和颗粒不明显的透明细胞两大类。内脏团插核术刺激后,血细胞的形态和比例均发生显著变化。血细胞形态更多样,伪足状突起更明显,细胞内囊泡状物质增多,血细胞密度显著增高(PP<0.01)。研究表明,作为三角帆蚌免疫系统重要组成部分的血细胞,在插核手术后,其类型、形态结构和数量均产生明显变化,这是机体对外界刺激产生的免疫防御反应,其中颗粒细胞担负着主要的免疫功能。       

Cresolase, catecholase and laccase activities in haemocytes of the red swamp crayfish

Phenoloxidase activity in crayfish haemocyte lysates and extracts of haemocyte membranes were studied using native PAGE and SDS-PAGE gels and staining for cresolase, catecholase and laccase activities. The activation of the proenzyme, prophenoloxidase to phenoloxidase, in native PAGE was demonstrated following exposure to SDS. By staining samples separated in SDS-PAGE followed by renaturation, a high molecular mass phenoloxidase activity was identified in both the soluble and membrane fractions of haemocyte preparations. The membrane-associated activity appeared at only relatively high molecular mass (> 300 kDa), and could easily be eluted from membranes using detergents or NaCl. Further, this membrane-associated activity has a catecholase activity but not the cresolase activity seen in the soluble preparations. In addition, several other phenoloxidase enzymes were identified with different relative mobilities (250, 80, 72 and 10 kDa). Crayfish haemocytes also contained laccase activity, thought to be restricted to cuticle sclerotisation in the integument. Laccase activity in haemocytes might aid in the formation of capsule used to contain pathogens.     

Possible involvement of cockroach haemocytes in the storage and synthesis of cuticle proteins

The injection of UL¹⁴C-leucine into newly ecdysed immature cockroaches resulted in the labelling of both haemocyte and serum proteins. Serum proteins were purified by gel filtration, concentrated and reinjected into other freshly ecdysed animals. After incubations of one hour, radioactivity was detected in serum, haemocyte, and cuticle proteins. Similar experiments using labelled soluble blood cell proteins also produced radioactivity in the serum, cells and cuticle. The possible rôle of haemocytes in cuticle protein synthesis is denoted and its significance in regard to cuticular tanning is discussed.     

Heavy metals affect the circulating haemocyte number in the shrimp Palaemon elegans

Environmental contamination by heavy metals produced by either anthropogenic or natural activities represents a threat to many species of aquatic animals worldwide. This study investigates the effect of short-term (96 h) exposure to dissolved heavy metals on the number of circulating haemocytes in the shrimp, Palaemon elegans (Rathke). Changes in haemocyte counts were determined in relation to time of exposure and with heavy metal concentration, relating the results to toxicity. It was found that immersion in artificial seawater containing Hg, Cd, Cu, Cr, Zn or Pb caused a decrease in the haemocyte count during the first 8 h exposure, although the haemocyte number returned to the initial (time 0) levels over the following 16 h immersion. In each case, the decrease in circulating haemocyte count induced by these metals was significantly different from the controls. The greatest decrease in haemocyte numbers (haemocytopenia) was induced by Pb, followed, in descending order, by Zn, Hg, Cr, Cu and Cd. The lethal level of haemocytopenia for the shrimps, defined as the number of haemocytes ml remaining in moribund animals (i.e. threshold of mortality) was found to be significantly lower than the levels tolerated by surviving shrimps (i.e. the limit of survival). The percentage of haemocytes remaining in the circulation at the threshold of mortality as a function of the number at time 0 was 56.6 +/- 8.8%. By contrast, the equivalent value for the threshold of survival was 63.7 +/- 12.4%. Importantly, the percentage decrease in haemocyte counts tolerated by P. elegans appears to vary with the metal. Animals treated with Pb or Zn survived with a lower number of circulating haemocytes than animals exposed to the other heavy metals.     

First cytochemical study of haemocytes from the crab Carcinus aestuarii (Crustacea,Decapoda)

For the first time, a morphological study of haemocytes from the crab Carcinus aestuarii was carried out by means of light microscopy and differing cytochemical assays. Analysis of haemocyte size frequency distribution (performed by means of a Coulter Counter) revealed the presence of two distinct haemocyte fractions in C. aestuarii haemolymph, depending on cell size. The first fraction was of about 3–5 µm in diameter and 30–50 fL in volume, the second was of about 6–12 µm in diameter and over 200 fL in volume. Mean cell diameter and volume were 8.20±1.7 µm and 272.30±143.5 fL, respectively. Haemocytes observed under light microscope were distinguished in three cell types: granulocytes (28%; 11.94±1.43 µm in diameter) with evident cytoplasmic granules, semigranulocytes (27%; 12.38±1.76 µm in diameter) with less granules than granulocytes, and hyalinocytes (44%; 7.88±1.6 µm in diameter) without granules. In addition, a peculiar cell type was occasionally found (about 1%): it was 25–30 µm in diameter and had a great vacuole and a peripheral cytoplasm with granules. Granulocyte and semigranulocyte granules stained in vivo with Neutral Red, indicating that they were lysosomes. Giemsa’s dye confirmed that granulocytes and semigranulocytes were larger than hyalinocytes. Pappenheim’s panoptical staining and Ehrlich’s triacid mixture allowed to distinguish granule-containing cells (including semigranulocytes) in acidophils (64%), basophils (35%) and neutrophils (1%). Hyalinocytes showed always a basophilic cytoplasm. Haemocytes were positive to the PAS reaction for carbohydrates, even if cytoplasm carbohydrate distribution varied among cell types. Lastly, lipids were found on cell membrane and in cytoplasm of all haemocyte types in the form of black spots produced after Sudan Black B staining. The morphological characterisation of C. aestuarii haemocytes by light microscopy was necessary before performing both ultrastructural and functional studies of circulating cells.Key words: Carcinus aestuarii, crab, haemocytes, light microscopy, cytochemical assays, morphological characterisation.     

Development of a monoclonal antibody specific to granulocytes and its application for variation of granulocytes in scallop Chlamys farreri after acute viral necrobiotic virus (AVNV) infection

A monoclonal antibody (MAb 6H7) specific to granulocytes of scallop Chlamys farreri was produced by immunising mice with separated granulocytes as an antigen. Characterised using a flow cytometric immunofluorescence assay, MAb 6H7 reacted to granulocytes by 87.1% of total positive haemocytes. At the ultrastructural level, MAb 6H7 demonstrated epitope in cytoplasmic granules of granulocytes. Western blotting analysis indicated that a peptide of 155 kDa was recognised by MAb 6H7. It was therefore used to investigate granulocyte variation in C. farreri after acute viral necrobiotic virus (AVNV) infection using an enzyme-linked immunosorbent assay. The result illustrated that granulocytes varied greatly by AVNV infection, and their amount significantly increased on day 1 post-injection, then decreased on days 2, 3 and 4, thereafter, rebounded and approached to a second peak on day 6, finally went down gradually to the control level on day 8.