Ultrastructure of the peritoneal exudate cells of seawater teleosts, seabream (Sparus aurata) and sea bass (Dicentrarchus labrax)

Transcription

1 Cell Tissue Res (1993) 273: Cell.Tissue Research 9 Springer-Verlag 1993 Ultrastructure of the peritoneal exudate cells of seawater teleosts, seabream (Sparus aurata) and sea bass (Dicentrarchus labrax) J. Meseguer, M.A. Esteban, J. Mufioz, A. L6pez-Ruiz Department of Cell Biology, Faculty of Biology, University of Murcia, E Murcia, Spain Received: 16 July 1992 / Accepted: 3 January 1993 Abstract. The peritoneal exudates of seabream and sea bass consist of granulocytes, lymphocytes and macrophages. These cells show conspicuous ultrastructural differences from the same cell-types of blood and head-kidney, which have not been reported previously. Peritoneal exudate granulocytes differ from their corresponding circulating or head-kidney forms in the following way: (a) they are larger in size, and (b) their abundant cytoplasmic granules have some new ultrastructural features, and a new granule population might also be present. Likewise, lymphocytes also show a noticeable difference; they contain a sparse population of small dense cytoplasmic granules. Monocytes, macrophages, and transitional forms between these two cell-types, are also found. The percentage of peritoneal exudate celltypes is different in seabream and sea bass. Macrophages in sea bass represent the most abundant peritoneal exudate cell-type. However, seabream shows lower percentages of macrophages than granulocytes. Key words: Peritoneum - Peritoneal exudate cell (PEC) Granulocytes - Lymphocytes - Macrophages - Sparus aurata, Dicentrarchus labrax (Teleostei) Introduction Fish peritoneal exudate cells (PECs) play important roles in both non-specific and specific immune mechanisms (Ellis 1982; Sakai 1984; Suzuki 1986; Olivier et al. 1992), and the peritoneal exudate (PE) has been used as a source of leucocytes for cell-mediated immunity studies (Bodammer 1986). However, there are no specific references to the fish PEC ultrastructure in the literature, and the sparse morphological data available, are derived from studies on phagocytosis (Finn and Nielson 1971; Ellis etal. 1976; MacArthur etal. 1984; Bodammer 1986). Correspondence to: J. Meseguer The morphofunctional features of the "histic form" of the mature piscine leucocyte are poorly understood since most electron-microscopic and functional studies have been based on leucocytes and related cell-types of blood and head-kidney (see Ellis 1977; Rowley etal. 1988). Likewise, there is limited quantitative data about the PE cell-types involved in teleost immune processes (Suzuki 1986). In this paper, we report the results of ultrastructural and quantitative studies of the PECs of two seawater teleosts, seabream (Sparus aurata L.) and sea bass (Dicentrarchus labrax L.). We compare the morphological features of the various cell-types with those of ontogenic and circulating forms of the same fish species, and with those of other fish. Materials and methods Ten immature male specimens (~ 450 g body wt. and 25 cm long) of the hermaphroditic protandrous teleost, seabream (Sparus aurata L.), and 10 male and female specimens of sea bass (Dicentrarehus labrax L.) (~350 g body wt. and cm long), were obtained from CULMAREX, S.A. (Murcia, Spain), and kept in running seawater aquaria at 17 ~ C and a natural photoperiod. They were fed daily with a commercial pellet diet. The specimens were anaesthetized with MS222 (Sandoz). Isolation of peritoneal exudate cells After disinfecting the fish ventral surface with 70% ethyl alcohol, the peritoneal cavity was injected with RPM[-1640 medium (Gibco) (10 ml/fish). The injected medium, with contained PECs, was then harvested after massaging the ventral surface of the fish for 10 min, and the thus obtained cell suspensions were centrifuged at 400 g for 10 min at 4~ and processed for transmission and scanning electron microscopy. Transmission electron microscopy PEC pellets were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, ph , for 1 h at 4 ~ C, postfixed in 1% OsO4 and

2 302 Table 1. Quantitative and cell surface features of seabream and sea bass peritoneal exudate cells Seabream Sea bass Cell Cell size Cell surface Cell percentage (Ixm) percentage (.g_+ SD) ()[_+ SD) (X_+ SD) Cell size (~m) (~_+ SD) Cell surface Granulocyte I 22.6_ _+1.71 Nearly smooth 5.2_+1.19 Granulocyte II 37.4 _ _ Rugged with short 28.1 _ protrussions Lymphocyte 15.2_ _+0.52 Smooth with short and 10.4_ thin cell processes Macrophage 24.8 _ _ Rugged with weak 57.4_ depressions and finger-like processes 7.4 _ Short cell processes Occasional short thick processes Smooth with short and thin cell processes 7.13 _ 1.62 Very irregular embedded in Epon. Sections were obtained with a Reichert-Jung ultramicrotome. Semithin sections were stained with toluidine blue and ultrathin sections were stained with uranyl acetate and lead citrate, then examined with a Zeiss EM 10C electron microscope. Scanning electron microscopy PECs were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, ph , at room temperature, and postfixed in 1% OsO4. The samples were then dehydrated in acetone, critical-point dried, sputter-coated with gold and studied with a Jeol JSM T-300 scanning electron microscope. Quantification procedure The total number of PECs harvested per fish, estimated in a Neubauer haemocytometer (Absher 1973), varied from 9 x 106 to 12 x 106 for seabream, and from 12 x 106 to 16 x 106 for sea bass. The cell percentage was evaluated by counting 5000 PECs after their characterization on toluidine-blue-stained semithin sections which were examined with a Leitz Orthoplan photomicroscope. The celltypes were categorized according to the presence or absence of cytoplasmic granules. The non-granular cells (lymphocytes and macrophages) were distinguished by their different size, and the granular cells (granulocytes I and II) by their tinctorial features. The PEC size was established by estimating the cell mean volume (see Gundersen et al. 1988). Results The PE of seabream and sea bass displayed 2 types of granulocytes (G I and G II), lymphocytes and macrophages (Table 1). Granulocytes The seabream G I cells had an eccentric euchromatinic nucleus containing some peripheral heterochromatin (Fig. 1). Some mitochondria, numerous free ribosomes, some cisternae of rough endoplasmic reticulum and a Golgi apparatus were present in their cytoplasm. The most notable cytoplasmic feature was the presence of 2 granule populations differentiated by their electron- density (Fig. 1 a, b). Both of them showed a progressive vacuolation process which appears to start with some small rounded vacuoles inside the granules and culminates with the formation of a large, central or eccentric, light vacuole. Some of the granules fused to form large irregular bodies. The G II cell-type (Fig. 2) appeared as light or dark cells with a round or oval eccentric nucleus with a small rim of heterochromatin, accompanied by small, sparse heterochromatin blocks, particularly evident in the dark cells. Three granule types were present in the cytoplasm. The first had a homogeneous, medium electron-dense content (Fig. 2a); the second contained 1 to 4 dense cores surrounded by low density material (Fig. 2b), and occasionally was elliptical and contained a crystalloid (Fig. 2c); and the third granuletype, had an eccentric electron-dense core surrounded by a light ring (Fig. 2d). Besides the granules, a few mitochondria, some flat cisternae of rough endoplasmic reticulum, free ribosomes, light vesicles and occasional lipidic droplets were also present. By scanning electron microscopy (Fig. 3), both granulocyte types of seabream Fig. 1. Granulocytes I and II of seabream peritoneal exudate. N nucleus; Ga Golgi area. x Bar: 6.66 gm Figs. 1 a-b, Detail of the cytoplasmic granules of a seabream peritoneal exudate granulocyte I. a x b x Bars: 1.6 gm Fig. 2. Light and dark granulocytes II of seabream peritoneal exudate. x Bar: 3.63 gm Figs. 2 a-d. Detail of the cytoplasmic granules of a seabream peritoneal exudate granulocyte II. a First granule type. x b Second granule type. x e Detail of an elliptical granule of the second type containing a crystalloid, x d Third granule type. x Bars: 0.66 gm Fig. 3. Scanning electron micrograph of the granulocyte I and II of seabream peritoneal exudate, x Bar: 4.44 [xm Fig, 4. Granulocyte I of sea bass peritoneal exudate showing first granule-type (GI) and second granule-type (G2) granules Inset: Detail of third granule-type granule Bars: 1.29 gm (4); 0.64 gm (inset) Fig. 5. Granulocyte II of sea bass peritoneal exudate. Arrows: First granule type. Arrowheads: Third granule type. x Inset: Second granule type. x Bars: 1.66 gm (5); 0.5 gm (inset) >

3 303

4 304

5 305 PE could be clearly differentiated because of their respective sizes and surface features (Table 1). In sea bass, the G I cells had an eccentric, slightly indented or bi-lobed nucleus containing some coarse peripheral heterochromatin blocks (Fig. 4). Three cytoplasmic granule populations were observed. The first consisted of oval or round granules containing a homogeneous, granular or fibrous, medium electron-dense material; the second population consisted of round granules containing granular, medium electron-dense, material; and the third, consisted of scattered, small, round dense granules with a light halo (Inset). Flattened cisternae of rough endoplasmic reticulum, free ribosomes, mitochondria and clusters of fl-glycogen were also present. G II cells had a rounded or elongated, eccentric nucleus with peripheral heterochromatin and a small nucleolus (Fig. 5). Three cytoplasmic granule types were present. The first was fusiform or three-cornered in shape and filled by a homogeneous, medium electron-dense, granular material (Fig. 5); the second contained a granular, medium electron-dense material (Inset); and the third, contained a light peripheral halo surrounding a dense core (Fig. 5). The size and surface features of sea bass granulocytes are summarized in Table 1. Lymphocytes Lymphocytes of both seabream and sea bass were rounded, with short cell processes, and they possessed a large nucleus with an eccentric nucleolus (Fig. 6). In the cytoplasm, there were numerous free ribosomes, 3-4 large mitochondria, a few fiat cisternae of rough endoplasmic reticulum, and an occasional small Golgi apparatus, with the notable presence of a few round to oval dense granules. By scanning electron microscopy (Fig. 7) they were seen as rounded cells with very short, thin, randomly distributed processes, which corresponded to those observed by transmission electron microscopy. Fig. 6. Lymphocyte of the sea-bass peritoneal exudate showing a few round dense granules, x Bar: 1.37 pm Fig. 7. Scanning electron micrograph of a peritoneal exudate lymphocyte (L) next to a granuloeyte II. (I/). x Bar: 3.5 gm Fig. 8. Sea-bass peritoneal exudate monocyte with a prominent kidney-shaped, eccentric nucleus. RER Rough endoplasmic reticulum, Mt mitochondria, "L lysosome, PV pinocytotic vesicles. x Bar: 1.6 p,m Fig. 8a. Three monocytes in close apposition, polarized towards and surrounding a damaged cell (Dc). x Bar: 2.66 gm Figs. 9 a-c. Peritoneal exudate macrophages of seabream and sea bass. a Macrophage showing a lobed nucleus (N) and a large lysosome (Ly). x b Macrophage with a light vesicle, x c Macrophage with a very large vesicle. The cytoplasm form a peripheral ring containing the nucleus Bars: 2.0 gm (a), 2.0 gm (b), 2.1 pm (e) Fig. 10. Scanning electron micrograph of peritoneal exudate macrophages, x Bar: 3.63 gm Macrophages Monocytes and macrophages were observed in the PE of both species. Monocytes (Fig. 8) had a prominent kidney-shaped, eccentric nucleus, and profiles of rough endoplasmic reticulum, free ribosomes, large mitochondria, a Golgi apparatus, lysosomes and some peripheral pinocytic vesicles, in their cytoplasm. Two or more monocytes were frequently observed in close apposition, polarized towards and surrounding other cells. These latter cells had the ultrastructural features of damaged cells (cytoplasmic vacuolation, organelle swelling and membrane disruptions) (Fig. 8 a). Cells with ultrastructural features intermediate between those of monocytes and macrophages, were also observed. Macrophages (Fig. 9 a-c) had a lobed or irregularly outlined nucleus with a nucleolus, and their cytoplasm had an heterogeneous appearance owing to the presence of lysosomes and vesicles. Some macrophages had a large light vesicle containing homogeneous material and osmiophilic peripheral condensations (Fig. 9b). This vesicle was occasionally very large and the cytoplasm formed a peripheral ring containing the nucleus (Fig. 9 c). By scanning electron microscopy (Fig. 10), the macrophages had a rugged cell surface with weak depressions randomly distributed and finger-like or irregularly outlined processes. Discussion The present results show that PECs isolated from seabream and sea bass, consist of granulocytes, lymphocytes and macrophages, which have ultrastructural features comparable to those described for blood and headkidney cells of the same species (Zuasti and Ferrer 1988; Meseguer et al. 1990, 1991; L6pez-Ruiz et al. 1992). However, important differences have been noted, which indicate that these cells in circulating blood may enter into tissues and act as mature cells. Unlike well-known vertebrate PE cell-types, the few morphological data available on fish PECs have been mainly reported after intraperitoneal injection of foreign materials during phagocytosis assays (Finn and Nielson 1971; Ellis et al. 1976; MacArthur et al. 1984; Bodammer 1986; Suzuki 1986). In the present study, however, the ultrastructural data have been obtained from the study of cells flushed out in suspension using a culture medium. The number and viability of fish leucocytes have previously been calculated and tested as a prerequisite to phagocytosis assays (Braun-Nesje etal. 1982; Parish et al. 1985; Fletcher and White 1987; Weeks et al. 1987; Graham etal. 1988; Saggers and Gould 1989; Sakai et al. 1989), but the percentage of each cell-type involved in this process has not yet been established. Suzuki (1986) has pointed out that the relative number of PE monocyte/macrophages decreases gradually, whereas granulocytes increase with time after injection of foreign materials. The percentage of PECs in untreated seabream is different from that of sea bass. It seems likely that the G I and G II granulocytes, noted in the present study, correspond to neutrophils/

6 306 heterophils and acidophils, respectively, that have been characterized in the blood and head-kidney of the same species (Meseguer et al. 1990; L6pez-Ruiz et al. 1992). However, the ultrastructure of these cells differs from that of the corresponding circulating or head-kidney forms in the following way: (a) they are larger in size and (b) their abundant cytoplasmic granules show some new ultrastructural features, and a new granule population might be present. More specifically, seabream PE contains a few neutrophilic granulocytes similar to those observed in peripheral blood (Ldpez-Ruiz et al. 1992), although most consist of large cells whose granules contain material of different electron-density in progressive vacuolation. In sea bass G Is, 3 granule-types can be observed as in the heterophils of head-kidney (Meseguer et al. 1990), which can be compared with the 3 granuletypes of mammalian neutrophilic granulocytes (Brederoo et al. 1983, 1986). The G IIs of seabream PE, have more than one dense core, and occasionally an elliptical crystalloid, within their cytoplasmic granules, granuletypes that do not occur in head-kidney (Zuasti and Ferrer 1988) or peripheral blood cells (L6pez-Ruiz et al. 1992). The most significant differentiating feature of sea bass PE G IIs, however, is a new cytoplasmic granule population consisting of abundant fusiform or three-cornered granules. These granules are similar to those present in mature PE acidophils from striped bass (Morone saxatilis) (Bodammer 1986). The above suggests that definitive maturation of fish acidophils might take place after they leave peripheral blood. It has been pointed out that fish have fewer circulating granulocytes than higher vertebrates, probably because of the abundance of other leucocyte types (Rowley et al. 1988). Mature heterophils are the most frequent granular leucocyte in teleosts (Ellis 1977; Meseguer et al. 1990), although mature acidophilic granulocytes are the most numerous in other piscine groups (Bielek 1981; Cenini 1984). Variations in the percentages of various cell-types in fish PE seem to be related to the functional activity of these cells (Suzuki 1986). Our results demonstrate a wide range of variation in PE granular leucocyte percentages between seabream and sea bass. The G IIs are more numerous than GI cells, paralleling the abundance of acidophils in seabream peripheral blood (L6- pez-ruiz et al. 1992). The presence and role of fish acidophils, however, are poorly understood (Ellis 1977; Rowley et al. 1988). The percentage level of G IIs in both seabream and sea bass PE, is similar to that of tilapia (Oreochromis niloticus) and carp (Cyprinus carpio) macrophages, shortly after an injection ofzymosan or paraffin (Suzuki 1986). Using morphological criteria, fish lymphoid cells are subdivided into lymphoblasts (large lymphocytes), lymphocytes (small lymphocytes) and plasma cells (Romestand and Trilles 1984; Rowley etal. 1988; Meseguer et al. 1991). Our results show that small lymphocytes are the only type of lymphoid cell found in PE of seabream and sea bass, and ultrastructurally, these cells are similar to those present in the blood and head-kidney of several teleost species (Weinreb 1963; Ferguson 1976; Temmink and Bayne 1987; Bielek 1988). Only one no- ticeable difference could be established between PE lymphocytes and those in the head-kidney and blood of seabream and sea bass (Zuasti and Ferrer 1989; Meseguer et al ; L6pez-Ruiz et al. 1992), namely, the presence of a few small dense cytoplasmic granules. Granular lymphocytes have previously been reported in some fish (Cannon et al. 1980; Savage 1983; Temkin and McMillan 1986; Bielek 1988; Dogger and Harris 1989), their dense granules seeming to correspond to the azurophilic granules observed by light microscopy (Cannon et al. 1980). Fish lymphocytes play a central role in humoral immunity (Rowley et al. 1988), but the absence of plasma cells in the PE of seabream, sea bass and other fish (Sakai 1984; Bodammer 1986; Suzuki 1986), as well as the low percentage of PE lymphocytes, suggest that fish PE is not particularly involved in antibody production. Fish macrophages have been described as both haematogenous and histogenous forms (Jakowska 1956; Lester and Daniels 1976). On the other hand, fish monocytes or monocyte-macrophages have been found in most lymphomieloid tissues (Ellis 1976; Bielek 1980; Braun-Nesje et al ; Meseguer et al. 1991), and form part of the PE (MacArthur et al. 1984; Bodammer 1986). Fish monocytes and macrophages are probably components of the same cell lineage, and therefore have similar properties and functions (Rowley et al. 1988). Suzuki (1986) has suggested that most peritoneal macrophages are derived from circulating monocytes since few free PE monocytes can be observed without first injecting the fish with foreign material. However, in the present study, substantial numbers of free monocytes, as well as transitional forms between monocytes and macrophages, have been observed. The ultrastructure of monocytes and macrophages from seabream and sea bass PE is very similar to that of comparable cells present in blood and head-kidney of these species (Zuasti and Ferrer 1989; Meseguer et al. 1991; L6pez-Ruiz et al. 1992) and, that of other fish (Ferguson 1976; Bielek 1980; Page and Rowley 1983; Cenini 1984). According to some authors, fish peritoneal macrophages differ from blood monocytes of the same species, in their size, irregular nuclear outline, and in the presence of a greater abundance of lysosomes and vesicles (Bodammer 1986; Suzuki 1986). Sea-bass macrophages represent the most abundant PEC type, as in other fish (Sakai 1984; Suzuki 1986). Seabream, however, has fewer macrophages than acidophils (G II). A high level of phagocytotic activity, however, has been reported for acidophils from various fish species (Bodammer 1986; Parish etal. 1986; Suzuki 1986; Temmink and Bayne 1987; Dogger and Harris 1989). Acknowledgements. This work was supported in part by grants from the Ministry of Education and Science, the "Instituto de Fomento de la Regi6n de Murcia" and the "Consejeria de Cultura, Educaci6n y Turismo de la Comunidad Aut6noma de la Regi6n de Murcia" (PCT 91/46), Spain.

7 307 References Absher M (1973) Hemocytometer counting. In: Kruse PK, Patterson MJ (eds) Tissue culture: methods and applications. Academic Press, New York Bielek E (1980) Electron microscopical studies of blood cells in teleosts. IV. Monocytes and macrophages. Zool Jb Anat Bd 103: Bielek E (1981) Developmental stages and localization of peroxidatic activity in the leucocytes of three teleost species (Cyprinus carpio L.; Tinca tinca L.; Salmo gairdneri Richardson). Cell Tissue Res 220: Bielek E (1988) Ultrastructural analysis of leucocyte interaction with tumour targets in a teleost, Cyprinus carpio L. Dev Comp Immunol 12: Bodammer JE (1986) Ultrastructural observations on peritoneal exudate cells from the striped bass. Vet Immunol Immunopathol 12:12~140 Braun-Nesje R, Bertheussen K, Kaplan K, Seljelid R (1981) Salmonid macrophages : separation, "in vitro" culture and characterization. J Fish Dis 4: Braun-Nesje R, Kaplan G, Seljelid R (1982) Rainbow trout macrophages "in vitro": morphology and phagocytic activity. Dev Comp Immunol 6: Brederoo P, Van der Meulen J, Mommaas-Kienhuis AM (1983) Development of the granule population in neutrophil granulocytes from human bone marrow. Cell Tissue Res 228: Brederoo P, Van der Meulen J, Daems WTh (1986) Ultrastructural localization of peroxidase activity in developing neutrophil granulocytes from human bone marrow. Histochemistry 84: Cannon MS, Mollenhauer HH, Eurell TE, Lewis DH, Cannon AM, Tompkins CH (1980) An ultrastructural study of the leukocytes of the channel catfish, Ietaluruspunctatus. J Morphol 164: 1-23 Cenini P (1984) The ultrastructure of leucocytes in carp (Cyprinus carpio). J Zool 204: Doggett TA, Harris JE (1989) Ultrastructure of the peripheral blood Ieucocytes of Oreochromis mossambicus. J Fish Biol 33 : Ellis AE (1976) Leucocytes and related cells in the plaice Pleuronectes platessa. J Fish Biol 8 : Ellis AE (1977) The leucocytes of fish: A review. J Fish Biol 11: Ellis AE (1982) Differences between the immune mechanisms of fish and higher vertebrates. In: Roberts RJ (ed) Microbial Diseases of Fish. Academic Press, London, pp 1-29 Ellis AE, Munroe ALS, Roberts RJ (1976) Defence mechanisms in fish. 1. A study of the phagocytic system and the fate of intraperitoneally injected particulate material in the plaice (Pleuronectes platessa L.). J Fish Biol 8 : Ferguson HW (1976) The ultrastructure of plaice (Pleuronectes platessa) leucocytes. J Fish Biol 8: Finn JP, Nielson NO (1971) The inflammatory response of rainbow trout. J Fish Biol 3 : Fletcher TC, White A (1987) Metabolic and immunological effects of endotoxin in the plaice, Pleuronectes platessa L. J Fish Biol 31:81-85 Graham S, Jeffries AH, Secombes CJ (1988) A novel assay to detect macrophage bactericidal activity in fish. Factors influencing the killing of Aeromonas salmonicida. J Fish Dis 11: Gundersen HJG, Bendtsen TF, Korbo L, Marcussen N, Moler A, Nielsen K, Nyengaard JR, pakkenberg B, Sorensen FB, Vesterby A, West MJ (1988) Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. Acta Pathol Microbiol Immunol Scand 96: Jakowska S (1956) Morphologie et nomenclature des cellules du sang des t616ost6ens. Rev Hematol 11: Lester RJG, Daniels BA (1976) The esosinophilic cell of the white sucker, Catostomus comrnersoni. J Fish Res Bd Can 33: L6pez-Ruiz A, Esteban MA, Meseguer J (1992) Blood cells of the gilthead seabream (Sparus aurata L.). Light and electron microscopic studies. Anat Rec 234: MacArthur JI, Fletcher TC, Pirie BJS, Davidson RJL, Thomson AW (1984) Peritoneal inflammatory cells in plaice, Pleuronectes platessa L. : effects of stress and endotoxin. J Fish Bio125 : Meseguer J, Esteban MA, Garcia-Ayala A, L6pez-Ruiz A, Agulleiro B (1990) Granulopoiesis in the head-kidney of the sea bass (Dicentrarchus labrax L.): an ultrastructural study. Arch Histol Cytol 53: Meseguer J, Esteban MA, Agulleiro B (1991) Stromal cells, macrophages and lymphoid cells in the head-kidney of sea bass (Dicentrarehus labrax L.). An ultrastructural study. Arch Histol Cytol 54: Olivier G, Moore AR, Fildes J (1992) Toxicity of Aeromonas salmonicida cells to Atlantic salmon Salmo salar peritoneal macrophages. Dev Comp Immunol 16:49-61 Page M, Rowley AF (1983) A cytochemical, light and electron microscopical study of the leucocytes of the adult river lamprey, Lampetrafluviatilis (L. Gray). J Fish Biol 22: Parish N, Wrathmell A, Harris JE (1985) Phagocytic cells in the dogfish (Scyliorhinus canicula L.). In: Manning M J, Tatner MF (eds) Fish immunology. Academic Press, London, pp Parish N, Wrathmell A, Hart S, Harris JE (1986) The leucocytes of the elasmobranch Scyliorhinus canicula L. A morphological study. J Fish Biol 28: Romestand B, Trilles JP (1984) Nomenclature and cytologie descriptive des 616ments figur6s du sang et des organes h6mopoi6- tiques du bar (Dicentrarchus labrax). Rec Med Vet 160: Rowley AF, Hunt TC, Page M, Mainwaring G (1988) Vertebrate blood cells. In: Rowley AF, Ratcliffe NA (eds) Fish. Cambridge University Press, Cambridge, pp Saggers BA, Gould ML (1989) The attachment of micro-organisms to macrophages isolated from the tilapia Oreochromis spilurus Gunther. J Fish Biol 35: Sakai DK (1984) Opsonization by fish antibody and complement in the immune phagocytosis by peritoneal exudate cells isolated from salmonid fishes. J Fish Dis 7:29-38 Sakai M, Ogasawara K, Atsuta S, Kobayashi M (1989) Comparative sensitivity of carp, Cyprinus carpio L. and rainbow trout, Salmo gairdneri Richardson, to Renibacterium salmoninarum. J Fish Dis 12: Savage AG (1983) The ultrastructure of the blood cells of the pike Esox lucius L. J Morphol 178: Suzuki K (1986) Morphological and phagocytic characteristics of peritoneal exudate cells in tilapia, Oreochromis niloticus (Trewavas), and carp, Cyprinus carpio L. J Fish Biol 29:34%364 Temkin RJ McMillan DB (1986) Gut-associated lymphoid tissue (GALT) of the goldfish, Carassius auratus. J Morphol 190:9-26 Temmink JHM, Bayne CJ (1987) Ultrastructura! characterization of leucocytes in the pronephros of carp (Cyprinus carpio L.). Dev Comp Immunol 11 : 125! 37 Weeks BA, Keisler AS, Myrvik QN, Warinner JE (1987) Differential uptake of neutral red by macrophages from three species of estuarine fish. Dev Comp Immunol 11 : Weinreb EL (1963) Studies on the fine structure of teleost blood cells. I. Peripheral blood. Anat Rec 147: Zuasti A, Ferret C (1988) Granulopoiesis in the head kidney of Sparus auratus. Arch Histol Cytol 51: Zuasti A, Ferret C (1989) Haemopoiesis in the head kidney of Sparus auratus. Arch Histol Cytol 52: