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Components of the cellular defense and detoxification system of the common cuttlefish Sepia officinalis (Mollusca, Cephalopoda)
Tissue & Cell, 2002 34 (6) 390–396 © 2002 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0040-8166(02)00070-8, available online at http://www.idealibrary.com
Tissue&Cell
Components of the cellular defense and detoxification system of the common cuttlefish Sepia officinalis (Mollusca, Cephalopoda) Knut Beuerlein, Sandra L öhr, Bettina Westermann, Peter Ruth, Rudolf Schipp
Abstract. Endocytotic-active cells in the branchial heart complex of Sepia officinalis were studied by in situ injection of different types of xenobiotics and by in vitro perfusion of the organ complex with a bacterial suspension. The rhogocytes (ovoid cells) ingest particles of all tested sizes by endocytosis and phagocytosis. The hemocytes of the circulating blood and the adhesive hemocytes in the wall of the branchial heart incorporate all tested kinds of foreign materials, including bacterial cells due to phagocytosis achieved by the triangular mesenchymatic cells. The ultrastructural findings also give strong evidence that the triangular mesenchymatic cells are fixed hemocytes that have migrated into the branchial heart tissue. The ingestion and digestion of allogeneic substances and bacteria or their debris by rhogocytes and/or all (forms of) hemocytes suggests the involvement of these either fixed or mobile endocytotic-active cells in the defense and detoxification system of cephalopods. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords: branchial heart, rhogocytes, hemocytes, phagocytosis, endocytosis, bacterial infection
Introduction In coleoid cephalopods, the circulatory system includes a single systemic heart, contractile vessels and additional paired branchial hearts. Apart from their pulsatile activity in the venous part of the circulatory system, the branchial hearts and in particular their appendages fulfill a main function in the excretory system (Harrison & Martin, 1965; Potts, 1967; Schipp & von Boletzky, 1975; Schipp & Institut für Allgemeine und Spezielle Zoologie, Bereich Entwicklungsbiologie, Justus-Liebig-Universität, Giessen, Germany Received 10 April 2002 Revised 6 July 2002 Accepted 18 July 2002 Correspondence to: K. Beuerlein, Institut f ür Allgemeine und Spezielle Zoologie, Bereich Entwicklungsbiologie, Justus-Liebig-Universit ät, Stephanstrasse 24, D-35390 Giessen, Germany. Tel.: +49 641 99 35611; Fax: +49 641 99 35609; E-mail: [email protected]
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Hevert, 1981). Cytobiological studies point to the involvement of these organ complexes in hemocyanin metabolism of coleoid cephalopods (Beuerlein et al., 1998, 2000). Moreover, the branchial hearts probably serve as storage organs for heavy metals and transuranics (Nardi & Steinberg, 1974; Palumbo et al., 1977; Schipp & Hevert, 1978; Nakahara et al., 1979; Miramand & Guary, 1980, 1981; Guary et al., 1981; Miramand et al., 1991). The main constituent of the spongy branchial heart tissue—the ovoid cells—are considered as accumulation sites of foreign substances (Sundermann, 1980; Fiedler & Schipp, 1987; Beuerlein & Schipp, 1998). The endocytotic-active cells were compared with the pore cells in gastropods (renamed rhogocytes by Haszprunar, 1996, as originally proposed by Witmer & Martin, 1973 and Witmer, 1974, respectively) and the pericardial cells (nephrocytes) in arthropods (Sminia, 1981; Crossley, 1985). In contrast to the non-circulating rhogocytes (ovoid cells), phagocytotic-active hemocytes of the circulating blood and adhesive hemocytes
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in the wall of the branchial hearts are responsible for the elimination of bacteria from the hemolymph (Stuart, 1968; Bayne, 1973; Budelmann et al., 1997; Malham et al., 1997; Beuerlein & Schipp, 1998). These studies support our hypothesis that the rhogocytes together with the hemocytes form a component of a more comprehensive defense and detoxification system. In this regard, the localization of triangular shaped mesenchymatic cells integrated into the branchial heart tissue should be mentioned (Beuerlein & Schipp, 1998) as likely indicating the additional involvement of these cells in the defense and detoxification system of coleoid cephalopods. In order to classify the components involved in the cellular defense and detoxification system and to elucidate both origin and function of the triangular mesenchymatic cells within the branchial heart tissue, ultrastructural studies, tracer experiments with colloidal gold and fluorescent microspheres as well as a bacterial infection were carried out, especially to investigate their endocytotic activity and to characterize the cellular immune response of the coleoid cephalopod Sepia officinalis to allogeneic materials.
Materials and methods Animals Adult male (n = 7) and female (n = 6) S. officinalis caught on the Mediterranean cost near Banyuls sur Mer (France) were used. The animals were kept in tanks with circulating fresh seawater and fed regularly for several days. Prior to the tracer experiments and the dissections of the branchial hearts the animals were anaesthetized with 2% ethanol in seawater. The animals were considered as anaesthetized when the arms ceased to move and no postural reflex occurred when the animal was turned on its back. Histology and cytomorphology After opening of the mid-body region and the visceropericardial coelom, the branchial heart complexes were immediately removed. For light microscopy (LM) and transmission electron microscopy (TEM), tissue was prefixed in 3.8% glutardialdehyde in phosphate buffer (0.2 M; pH 7.4; 930 mOsmol), postfixed with 1% OsO4 for 2 h, dehydrated in a graded series of acetone and embedded in Durcupan (Fluka, Taufkirchen, Germany). Semi-thin sections were studied with a Photomicroscope II (Zeiss, Jena, Germany) and ultra-thin sections in the TEM (EM 9A, EM 10; Zeiss). Injection of colloidal gold The mid-body region was opened from the ventral side and an injection needle was pushed into the vena cephalica. Solutions of 1% colloidal gold (∅ 15 nm; British Biocell, Cardiff, UK) diluted in seawater (pH 7.4; 960 mOsmol; 0.17% glucose) were in situ injected into the branchial hearts for 1 h at 20 ◦ C. The controls were performed with the same seawater solution but without the tracer. During the experiments
the gills were rinsed with aerated seawater. For electron microscopy, tissue was prefixed with 3.5% glutardialdehyde in cacodylate buffer (0.1 M; pH 7.4; 1000 mOsmol) for 2 h at 20 ◦ C. Postfixation was done with 1% OsO4 for 1.5 h. Tissue samples were embedded in Durcupan (Fluka). Application of fluorescent microspheres After opening the viscero-pericardial coelom, suspensions of 1% carboxylate-modified fluorescent polystyrene microspheres (fluospheres; bead diameter: 20, 100, 200 and 500 nm, 1 and 2 m; Molecular Probes, Leiden, The Netherlands) in seawater (pH 7.4; 1000 mOsmol; 0.17% glucose) were in situ injected into the branchial hearts via the vena cava for 1 h at 20 ◦ C. During the experiments the gills were rinsed with aerated seawater. First, the heart beat amplitude flattened, but recovered to regular contractions 15 min after the beginning of the injections. The branchial heart complexes were dissected, tissue was fixed with 4% paraformaldehyde in seawater (24 h) and embedded in a low-melting paraffin (51–53 ◦ C). Roticlear (Roth, Karlsruhe, Germany) was used as decerating agent. Sections were hydrated in a graded series of ethyl alcohols. To mark the nuclei, the sections were counterstained with 0.1% bis-benzimide (Sigma, Taufkirchen, Germany) for 10 min, covered with Kaiser’s glycerol gelatine (Merck, Darmstadt, Germany) and investigated by fluorescence microscopy (FM) (Dialux fluorescence microscope equipped with a Ploemopak 2.3 illuminator; filter block D; excitation filter BP 355–425 nm; barrier filter LP 460 nm; Leitz, Wetzlar, Germany). Additionally, TEM observations were done as described above. Bacterial infection of the branchial hearts The branchial hearts of anaesthetized animals were immediately removed from the viscero-pericardial coelom and rinsed with seawater solution. The in vitro perfusions of the isolated organs were carried out as described by Schipp et al. (1986). After a 10 min perfusion, during which the organs showed regular rhythmical contractions, the seawater solution was replaced by a bacterial suspension. The rod-shaped bacteria (Pseudomonas sp., Schipp et al., 1991), derived from the pericardial coelom of an adult Nautilus pompilius (Philippinean Sea), had been cultivated for 2 days in a liquid culture (LB broth enrichment medium; pH 7.0). A sample of the liquid bacterial culture was centrifuged at 2200 g for 10 min at 4 ◦ C. The resulting pellet was resuspended and diluted 1:1 in sterile seawater to the final bacterial suspension. The branchial hearts ceased to beat 1–2 min after the beginning of a 15 min perfusion with that suspension, but recovered to regular contractions when rinsed with seawater for 10 min. Tissue samples were prefixed with 3.8% glutardialdehyde in phosphate buffer (0.2 M; pH 7.4; 930 mOsmol) for 2 h starting at 4 ◦ C and slowly increasing to 20 ◦ C, postfixed with 1% OsO4 for 1.5 h and embedded in Durcupan (Fluka).
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Fig. 1 Cytological analysis of the branchial heart tissue. (A) ×400. Scattered triangular mesenchymatic cells (arrows) are located among the predominant rhogocytes. LM, toluidine-blue staining. (B) ×4000. Single triangular mesenchymatic cell (arrow) integrated into a cluster of rhogocytes. TEM. (C) ×4000. Hemocytes of the circulating blood in the process of migrating into the branchial heart tissue; note their different phenotypes. TEM. (D) ×4000. Adhesive hemocyte typically surrounded by rhogocytes. TEM. bs, blood space; er, rough endoplasmic reticulum; ly, lysosome; mf, muscle fibre; mi, mitochondrium; nu, nucleus; rc, rhogocyte; va, vacuole.
Results Histological and cytological findings The triangular mesenchymatic cells are diffusely dispersed throughout the whole wall of the branchial heart (Fig. 1A). Generally, they are completely surrounded by rhogocytes forming close cell aggregations similar to the adhesive hemocytes in the branchial heart (Fig. 1B). With the exception of the segmented nucleus, the triangular mesenchymatic cells exhibit similar size and ultrastructure as the adhesive hemocytes and/or the hemocytes of the circulating blood (Fig. 1C & D). Apart from a similar nucleo-plasmic ratio, these cells also correspond to hemocytes in the number and the size of vacuoles and lysosomes and in the perinuclear formation of the rough endoplasmic reticulum. With the
exception of the rhogocytes, all of them are able to develop extended pseudopod-like processes. Localization of injected colloidal gold After the in situ injection of colloidal gold the color of the branchial hearts immediately turned from white to dark red. At the electron-microscopic level, gold particles were mainly detected in the blood space but also in the adhesive hemocytes and the hemocytes of the circulating blood as well as in the rhogocytes of the branchial heart tissue, even though at lower levels. Gold particles were also found in the triangular mesenchymatic cells (Fig. 2A). Generally, the injected gold particles were located in the cytoplasm and more particularly in cell invaginations, endocytotic vesicles and lysosomes (Fig. 2B & C).
cellular defense and detoxification in cephalopods 393
in the central branchial heart tissue showed a distinct accumulation of fluospheres of all sizes. Yellowish-red fluorescences of fluospheres of all sizes were found in the circulating and in the adhesive hemocytes of the branchial heart (Fig. 3A). The triangular mesenchymatic cells in the branchial heart also revealed a strong ingestion of fluospheres of all sizes. By using electron microscopy, polystyrene beads (fluospheres) of all sizes were localized in endocytotic vesicles, vacuoles and lysosomes of all investigated cell types (Fig. 3B–D). Cytomorphology after bacterial infection The electron-microscopic analysis reveals that the triangular mesenchymatic cells are able to internalize whole bacterial cells by phagocytosis. The engulfed bacteria are enclosed by pseudopod-like processes or encapsulated in vacuoles and/or lysosomes showing different stages of lysis (Fig. 4). In contrast to the triangular mesenchymatic cells and to the adhesive and/or the circulating hemocytes that also have phagocytized bacterial cells, the rhogocytes of the branchial hearts exhibit no incorporation of whole bacteria.
Discussion Cytomorphological investigations The histological and cytological findings on structure and size of the triangular mesenchymatic cells presented in this study agree with the results of previous investigations on the general cytomorphology of hemocytes in cephalopods (Stuart, 1968; Bayne, 1973; Cowden & Curtis, 1981; Ford, 1992; Claes, 1996; Budelmann et al., 1997; Malham et al., 1997). In addition to a similar nucleo-plasmic ratio, the triangular mesenchymatic cells also correspond in number and size of lysosomes and vacuoles and in the perinuclear formation of the rough endoplasmic reticulum to the adhesive hemocytes in the gills and in the branchial heart complex (Schipp et al., 1979; Beuerlein & Schipp, 1998). These findings give strong evidence that the triangular mesenchymatic cells are also fixed hemocytes which have migrated into the branchial heart tissue much like the adhesive hemocytes; presumably, this observation relates to scarcely different phenotypes of the same cell form (Claes, 1996). Fig. 2 Detection of injected gold particles in the branchial heart tissue. (A) ×12 000. Triangular mesenchymatic cell filled with gold particles. TEM. (B) ×16 000. Gold particles (arrows) detected in cell invaginations and endocytotic vesicles of two rhogocytes bordering on each other. TEM. (C) ×20 000. Gold particles (arrows) located in transport vesicles of rhogocytes. ci, cell invagination; ly, lysosome; mi, mitochondrium; nu, nucleus; va, vacuole.
Detection of injected fluospheres Apart from green auto-fluorescence of the tissue and blue bis-benzimide-fluorescence of the nuclei, the injected fluospheres appeared as distinct yellowish-red spots. Rhogocytes
Uptake of colloidal gold The present electron-microscopic findings on the uptake of colloidal gold (∅ 15 nm) into the rhogocytes, the circulating and the adhesive hemocytes to which we count the triangular mesenchymatic cells are in agreement with the observations on the accumulation of gold particles (∅ <20 nm) in comparable cells, e.g. the pore cells (rhogocytes), the amoebocytes (hemocytes) and the reticulum cells (fixed phagocytes) in gastropods (Sminia et al., 1979; Sminia, 1981). In this context, it should be mentioned that the distinct cell surface structure of the rhogocytes, which is composed of extended cell invaginations covered by a glycocalyx, represents a barrier for
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Fig. 3 Localization of injected fluospheres in the branchial heart tissue. (A) ×500. Fluospheres are located in rhogocytes (arrows) and in adhesive hemocytes (arrowheads). FM, bis-benzimide staining. (B) ×15 500. Single triangular mesenchymatic cell exhibits incorporated fluospheres (arrows) of different sizes. TEM. (C) ×11 500. Rhogocyte in the process of phagocytizing polystyrene beads (arrows). TEM. (D) ×9000. Fluospheres engulfed by pseudopod-like processes of an adhesive hemocyte (arrowheads); note that adjacent rhogocytes have incorporated fluospheres (arrows). TEM. bs, blood space; ci, cell invagination; ly, lysosome; mf, muscle fibre; mi, mitochondrium; nu, nucleus; pp, pseudopod-like processes; rc, rhogocyte; va, vacuole.
molecules and particles of something special size (Witmer, 1974; Sundermann, 1980; Beuerlein & Schipp, 1998). Taking into consideration that the slit-like openings of these cell invaginations are approximately 45 nm wide, the localization of the injected gold particles beyond this barrier suggests that 15 nm gold particles are able to penetrate the glycocalyx of the rhogocytes. The subsequent uptake of the gold particles into the rhogocytes, therefore, is probably due to obligatory endocytotic processes. Nevertheless, the incorporation of foreign materials by rhogocytes and all (forms of) hemocytes which are in contact with the hemolymph indicates that they are involved in the defense and detoxification system of S. officinalis (Beuerlein & Schipp, 1998), similar to the cellular defense system described for Loligo vulgaris (Meister, 1977; Sundermann, 1980) and Eledone cirrhosa (Malham & Runham, 1998).
Incorporation of fluospheres Because of their low non-specific binding, the chosen carboxylate-modified fluospheres appear to be suitable for applications involving phagocytosis but, in spite of the fact that the fluospheres were clearly distinguishable from the background fluorescence, it could not be ascertained by using fluorescence microscopy that the fluospheres were definitively located in the cell or whether they were only bound at the cell surface. However, the transmission electron microscopy provided more precise results. The most remarkable feature is that the rhogocytes phagocytize particles of large size, although their distinct cell surface structure strongly pleaded against this capacity. It seems that the rhogocytes of the branchial heart are not only responsible for the elimination of xenobiotics of small size like fluospheres (∅ 20 nm), gold particles (∅ 15 nm) and ferritin (∅ 10 nm;
cellular defense and detoxification in cephalopods 395
supported by previous investigations (Bayne, 1973; Witmer, 1974; Meister, 1977; Sundermann, 1980; Guary et al., 1981; Ford, 1992; Bayne, 1983; Budelmann et al., 1997; Malham et al., 1997; Beuerlein & Schipp, 1998; Malham & Runham, 1998), we propose that the triangular mesenchymatic cells are also adhesive hemocytes that, together with the circulating hemocytes, form the first line in the defense against allogeneic materials, being followed by the rhogocytes of the branchial heart tissue. Phagocytosis of bacteria In contrast to the above-mentioned phagocytosis of fluospheres, the rhogocytes of the branchial heart complex of S. officinalis revealed no uptake of whole allogeneic cells after bacterial infection. However, the increased endocytotic activity and plasmatic vacuolization as well as the apparent fusion of transport vesicles with lysosomes indicate that they are able to recognize, incorporate and catabolize allogeneic cellular material; e.g. bacterial toxins and/or cellular debris of bacteria (Beuerlein & Schipp, 1998). In the present study, phagocytosis of bacterial cells is demonstrated for the triangular mesenchymatic cells in the wall of the branchial heart. These cells reveal all stages of lysis of bacteria engulfed by lysosomes as do the adhesive and/or the circulating hemocytes. These findings agree with previous results on phagocytotic-active cells that are involved in the cellular defense in coleoid cephalopods (Stuart, 1968; Bayne, 1973; Budelmann et al., 1997; Malham et al., 1997; Beuerlein & Schipp, 1998) and other molluscs (Sminia, 1972, 1981; Bayne, 1983, Sminia & van der Knaap, 1987; Hansen et al., 1991; Cima et al., 2000; Fournier et al., 2001; Lacoste et al., 2001).
Conclusion
Fig. 4 Cytomorphological analysis of the branchial heart tissue after bacterial infection. ×13 500. Bacterial cells detected in a triangular mesenchymatic cell but not in the rhogocytes; note the different stages of lysis (I–III) of phagocytized bacteria within lysosomes and vacuoles. TEM. er, rough endoplasmic reticulum; ev, endocytotic vesicles; ly, lysosome; mi, mitochondrium; nu, nucleus; rc, rhogocyte; va, vacuole.
Beuerlein & Schipp, 1998) by selective endocytosis, but that they are also able to incorporate large foreign particles by probably receptor-mediated phagocytosis enabling them to inactivate allogeneic material by its catabolism and/or storage within the lysosomal compartment (Schipp & von Boletzky, 1975; Schipp & Hevert, 1978; Fiedler & Schipp, 1987; Beuerlein & Schipp, 1998). However, the higher rate of the uptake of fluospheres of all sizes by (all forms) of hemocytes points to the fact that above all else, these cells are responsible for the elimination of large foreign materials from the hemolymph. Confirmed by the present findings and
Based on the present findings, we conclude that the hemocytes of the circulating blood, the adhesive hemocytes (to which we count the triangular mesenchymatic cells) and the rhogocytes form the cellular defense and detoxification system in coleoid cephalopods.
ACKNOWLEDGEMENTS We would like to thank Brigitte Fronk and Anneliese Hudel for excellent help with the photographical evaluations and we also have benefited from discussions with Henrike Schmidtberg and Frank Berghaus. We are especially grateful to Sigurd von Boletzky who provided the technical facilities for work at the Laboratoire Arago in Banyuls sur Mer. This contribution was supported by grant Schi 99/10-1 of the DFG (German Research Foundation).
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Tissue&Cell
Components of the cellular defense and detoxification system of the common cuttlefish Sepia officinalis (Mollusca, Cephalopoda) Knut Beuerlein, Sandra L öhr, Bettina Westermann, Peter Ruth, Rudolf Schipp
Abstract. Endocytotic-active cells in the branchial heart complex of Sepia officinalis were studied by in situ injection of different types of xenobiotics and by in vitro perfusion of the organ complex with a bacterial suspension. The rhogocytes (ovoid cells) ingest particles of all tested sizes by endocytosis and phagocytosis. The hemocytes of the circulating blood and the adhesive hemocytes in the wall of the branchial heart incorporate all tested kinds of foreign materials, including bacterial cells due to phagocytosis achieved by the triangular mesenchymatic cells. The ultrastructural findings also give strong evidence that the triangular mesenchymatic cells are fixed hemocytes that have migrated into the branchial heart tissue. The ingestion and digestion of allogeneic substances and bacteria or their debris by rhogocytes and/or all (forms of) hemocytes suggests the involvement of these either fixed or mobile endocytotic-active cells in the defense and detoxification system of cephalopods. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords: branchial heart, rhogocytes, hemocytes, phagocytosis, endocytosis, bacterial infection
Introduction In coleoid cephalopods, the circulatory system includes a single systemic heart, contractile vessels and additional paired branchial hearts. Apart from their pulsatile activity in the venous part of the circulatory system, the branchial hearts and in particular their appendages fulfill a main function in the excretory system (Harrison & Martin, 1965; Potts, 1967; Schipp & von Boletzky, 1975; Schipp & Institut für Allgemeine und Spezielle Zoologie, Bereich Entwicklungsbiologie, Justus-Liebig-Universität, Giessen, Germany Received 10 April 2002 Revised 6 July 2002 Accepted 18 July 2002 Correspondence to: K. Beuerlein, Institut f ür Allgemeine und Spezielle Zoologie, Bereich Entwicklungsbiologie, Justus-Liebig-Universit ät, Stephanstrasse 24, D-35390 Giessen, Germany. Tel.: +49 641 99 35611; Fax: +49 641 99 35609; E-mail: [email protected]
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Hevert, 1981). Cytobiological studies point to the involvement of these organ complexes in hemocyanin metabolism of coleoid cephalopods (Beuerlein et al., 1998, 2000). Moreover, the branchial hearts probably serve as storage organs for heavy metals and transuranics (Nardi & Steinberg, 1974; Palumbo et al., 1977; Schipp & Hevert, 1978; Nakahara et al., 1979; Miramand & Guary, 1980, 1981; Guary et al., 1981; Miramand et al., 1991). The main constituent of the spongy branchial heart tissue—the ovoid cells—are considered as accumulation sites of foreign substances (Sundermann, 1980; Fiedler & Schipp, 1987; Beuerlein & Schipp, 1998). The endocytotic-active cells were compared with the pore cells in gastropods (renamed rhogocytes by Haszprunar, 1996, as originally proposed by Witmer & Martin, 1973 and Witmer, 1974, respectively) and the pericardial cells (nephrocytes) in arthropods (Sminia, 1981; Crossley, 1985). In contrast to the non-circulating rhogocytes (ovoid cells), phagocytotic-active hemocytes of the circulating blood and adhesive hemocytes
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in the wall of the branchial hearts are responsible for the elimination of bacteria from the hemolymph (Stuart, 1968; Bayne, 1973; Budelmann et al., 1997; Malham et al., 1997; Beuerlein & Schipp, 1998). These studies support our hypothesis that the rhogocytes together with the hemocytes form a component of a more comprehensive defense and detoxification system. In this regard, the localization of triangular shaped mesenchymatic cells integrated into the branchial heart tissue should be mentioned (Beuerlein & Schipp, 1998) as likely indicating the additional involvement of these cells in the defense and detoxification system of coleoid cephalopods. In order to classify the components involved in the cellular defense and detoxification system and to elucidate both origin and function of the triangular mesenchymatic cells within the branchial heart tissue, ultrastructural studies, tracer experiments with colloidal gold and fluorescent microspheres as well as a bacterial infection were carried out, especially to investigate their endocytotic activity and to characterize the cellular immune response of the coleoid cephalopod Sepia officinalis to allogeneic materials.
Materials and methods Animals Adult male (n = 7) and female (n = 6) S. officinalis caught on the Mediterranean cost near Banyuls sur Mer (France) were used. The animals were kept in tanks with circulating fresh seawater and fed regularly for several days. Prior to the tracer experiments and the dissections of the branchial hearts the animals were anaesthetized with 2% ethanol in seawater. The animals were considered as anaesthetized when the arms ceased to move and no postural reflex occurred when the animal was turned on its back. Histology and cytomorphology After opening of the mid-body region and the visceropericardial coelom, the branchial heart complexes were immediately removed. For light microscopy (LM) and transmission electron microscopy (TEM), tissue was prefixed in 3.8% glutardialdehyde in phosphate buffer (0.2 M; pH 7.4; 930 mOsmol), postfixed with 1% OsO4 for 2 h, dehydrated in a graded series of acetone and embedded in Durcupan (Fluka, Taufkirchen, Germany). Semi-thin sections were studied with a Photomicroscope II (Zeiss, Jena, Germany) and ultra-thin sections in the TEM (EM 9A, EM 10; Zeiss). Injection of colloidal gold The mid-body region was opened from the ventral side and an injection needle was pushed into the vena cephalica. Solutions of 1% colloidal gold (∅ 15 nm; British Biocell, Cardiff, UK) diluted in seawater (pH 7.4; 960 mOsmol; 0.17% glucose) were in situ injected into the branchial hearts for 1 h at 20 ◦ C. The controls were performed with the same seawater solution but without the tracer. During the experiments
the gills were rinsed with aerated seawater. For electron microscopy, tissue was prefixed with 3.5% glutardialdehyde in cacodylate buffer (0.1 M; pH 7.4; 1000 mOsmol) for 2 h at 20 ◦ C. Postfixation was done with 1% OsO4 for 1.5 h. Tissue samples were embedded in Durcupan (Fluka). Application of fluorescent microspheres After opening the viscero-pericardial coelom, suspensions of 1% carboxylate-modified fluorescent polystyrene microspheres (fluospheres; bead diameter: 20, 100, 200 and 500 nm, 1 and 2 m; Molecular Probes, Leiden, The Netherlands) in seawater (pH 7.4; 1000 mOsmol; 0.17% glucose) were in situ injected into the branchial hearts via the vena cava for 1 h at 20 ◦ C. During the experiments the gills were rinsed with aerated seawater. First, the heart beat amplitude flattened, but recovered to regular contractions 15 min after the beginning of the injections. The branchial heart complexes were dissected, tissue was fixed with 4% paraformaldehyde in seawater (24 h) and embedded in a low-melting paraffin (51–53 ◦ C). Roticlear (Roth, Karlsruhe, Germany) was used as decerating agent. Sections were hydrated in a graded series of ethyl alcohols. To mark the nuclei, the sections were counterstained with 0.1% bis-benzimide (Sigma, Taufkirchen, Germany) for 10 min, covered with Kaiser’s glycerol gelatine (Merck, Darmstadt, Germany) and investigated by fluorescence microscopy (FM) (Dialux fluorescence microscope equipped with a Ploemopak 2.3 illuminator; filter block D; excitation filter BP 355–425 nm; barrier filter LP 460 nm; Leitz, Wetzlar, Germany). Additionally, TEM observations were done as described above. Bacterial infection of the branchial hearts The branchial hearts of anaesthetized animals were immediately removed from the viscero-pericardial coelom and rinsed with seawater solution. The in vitro perfusions of the isolated organs were carried out as described by Schipp et al. (1986). After a 10 min perfusion, during which the organs showed regular rhythmical contractions, the seawater solution was replaced by a bacterial suspension. The rod-shaped bacteria (Pseudomonas sp., Schipp et al., 1991), derived from the pericardial coelom of an adult Nautilus pompilius (Philippinean Sea), had been cultivated for 2 days in a liquid culture (LB broth enrichment medium; pH 7.0). A sample of the liquid bacterial culture was centrifuged at 2200 g for 10 min at 4 ◦ C. The resulting pellet was resuspended and diluted 1:1 in sterile seawater to the final bacterial suspension. The branchial hearts ceased to beat 1–2 min after the beginning of a 15 min perfusion with that suspension, but recovered to regular contractions when rinsed with seawater for 10 min. Tissue samples were prefixed with 3.8% glutardialdehyde in phosphate buffer (0.2 M; pH 7.4; 930 mOsmol) for 2 h starting at 4 ◦ C and slowly increasing to 20 ◦ C, postfixed with 1% OsO4 for 1.5 h and embedded in Durcupan (Fluka).
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Fig. 1 Cytological analysis of the branchial heart tissue. (A) ×400. Scattered triangular mesenchymatic cells (arrows) are located among the predominant rhogocytes. LM, toluidine-blue staining. (B) ×4000. Single triangular mesenchymatic cell (arrow) integrated into a cluster of rhogocytes. TEM. (C) ×4000. Hemocytes of the circulating blood in the process of migrating into the branchial heart tissue; note their different phenotypes. TEM. (D) ×4000. Adhesive hemocyte typically surrounded by rhogocytes. TEM. bs, blood space; er, rough endoplasmic reticulum; ly, lysosome; mf, muscle fibre; mi, mitochondrium; nu, nucleus; rc, rhogocyte; va, vacuole.
Results Histological and cytological findings The triangular mesenchymatic cells are diffusely dispersed throughout the whole wall of the branchial heart (Fig. 1A). Generally, they are completely surrounded by rhogocytes forming close cell aggregations similar to the adhesive hemocytes in the branchial heart (Fig. 1B). With the exception of the segmented nucleus, the triangular mesenchymatic cells exhibit similar size and ultrastructure as the adhesive hemocytes and/or the hemocytes of the circulating blood (Fig. 1C & D). Apart from a similar nucleo-plasmic ratio, these cells also correspond to hemocytes in the number and the size of vacuoles and lysosomes and in the perinuclear formation of the rough endoplasmic reticulum. With the
exception of the rhogocytes, all of them are able to develop extended pseudopod-like processes. Localization of injected colloidal gold After the in situ injection of colloidal gold the color of the branchial hearts immediately turned from white to dark red. At the electron-microscopic level, gold particles were mainly detected in the blood space but also in the adhesive hemocytes and the hemocytes of the circulating blood as well as in the rhogocytes of the branchial heart tissue, even though at lower levels. Gold particles were also found in the triangular mesenchymatic cells (Fig. 2A). Generally, the injected gold particles were located in the cytoplasm and more particularly in cell invaginations, endocytotic vesicles and lysosomes (Fig. 2B & C).
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in the central branchial heart tissue showed a distinct accumulation of fluospheres of all sizes. Yellowish-red fluorescences of fluospheres of all sizes were found in the circulating and in the adhesive hemocytes of the branchial heart (Fig. 3A). The triangular mesenchymatic cells in the branchial heart also revealed a strong ingestion of fluospheres of all sizes. By using electron microscopy, polystyrene beads (fluospheres) of all sizes were localized in endocytotic vesicles, vacuoles and lysosomes of all investigated cell types (Fig. 3B–D). Cytomorphology after bacterial infection The electron-microscopic analysis reveals that the triangular mesenchymatic cells are able to internalize whole bacterial cells by phagocytosis. The engulfed bacteria are enclosed by pseudopod-like processes or encapsulated in vacuoles and/or lysosomes showing different stages of lysis (Fig. 4). In contrast to the triangular mesenchymatic cells and to the adhesive and/or the circulating hemocytes that also have phagocytized bacterial cells, the rhogocytes of the branchial hearts exhibit no incorporation of whole bacteria.
Discussion Cytomorphological investigations The histological and cytological findings on structure and size of the triangular mesenchymatic cells presented in this study agree with the results of previous investigations on the general cytomorphology of hemocytes in cephalopods (Stuart, 1968; Bayne, 1973; Cowden & Curtis, 1981; Ford, 1992; Claes, 1996; Budelmann et al., 1997; Malham et al., 1997). In addition to a similar nucleo-plasmic ratio, the triangular mesenchymatic cells also correspond in number and size of lysosomes and vacuoles and in the perinuclear formation of the rough endoplasmic reticulum to the adhesive hemocytes in the gills and in the branchial heart complex (Schipp et al., 1979; Beuerlein & Schipp, 1998). These findings give strong evidence that the triangular mesenchymatic cells are also fixed hemocytes which have migrated into the branchial heart tissue much like the adhesive hemocytes; presumably, this observation relates to scarcely different phenotypes of the same cell form (Claes, 1996). Fig. 2 Detection of injected gold particles in the branchial heart tissue. (A) ×12 000. Triangular mesenchymatic cell filled with gold particles. TEM. (B) ×16 000. Gold particles (arrows) detected in cell invaginations and endocytotic vesicles of two rhogocytes bordering on each other. TEM. (C) ×20 000. Gold particles (arrows) located in transport vesicles of rhogocytes. ci, cell invagination; ly, lysosome; mi, mitochondrium; nu, nucleus; va, vacuole.
Detection of injected fluospheres Apart from green auto-fluorescence of the tissue and blue bis-benzimide-fluorescence of the nuclei, the injected fluospheres appeared as distinct yellowish-red spots. Rhogocytes
Uptake of colloidal gold The present electron-microscopic findings on the uptake of colloidal gold (∅ 15 nm) into the rhogocytes, the circulating and the adhesive hemocytes to which we count the triangular mesenchymatic cells are in agreement with the observations on the accumulation of gold particles (∅ <20 nm) in comparable cells, e.g. the pore cells (rhogocytes), the amoebocytes (hemocytes) and the reticulum cells (fixed phagocytes) in gastropods (Sminia et al., 1979; Sminia, 1981). In this context, it should be mentioned that the distinct cell surface structure of the rhogocytes, which is composed of extended cell invaginations covered by a glycocalyx, represents a barrier for
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Fig. 3 Localization of injected fluospheres in the branchial heart tissue. (A) ×500. Fluospheres are located in rhogocytes (arrows) and in adhesive hemocytes (arrowheads). FM, bis-benzimide staining. (B) ×15 500. Single triangular mesenchymatic cell exhibits incorporated fluospheres (arrows) of different sizes. TEM. (C) ×11 500. Rhogocyte in the process of phagocytizing polystyrene beads (arrows). TEM. (D) ×9000. Fluospheres engulfed by pseudopod-like processes of an adhesive hemocyte (arrowheads); note that adjacent rhogocytes have incorporated fluospheres (arrows). TEM. bs, blood space; ci, cell invagination; ly, lysosome; mf, muscle fibre; mi, mitochondrium; nu, nucleus; pp, pseudopod-like processes; rc, rhogocyte; va, vacuole.
molecules and particles of something special size (Witmer, 1974; Sundermann, 1980; Beuerlein & Schipp, 1998). Taking into consideration that the slit-like openings of these cell invaginations are approximately 45 nm wide, the localization of the injected gold particles beyond this barrier suggests that 15 nm gold particles are able to penetrate the glycocalyx of the rhogocytes. The subsequent uptake of the gold particles into the rhogocytes, therefore, is probably due to obligatory endocytotic processes. Nevertheless, the incorporation of foreign materials by rhogocytes and all (forms of) hemocytes which are in contact with the hemolymph indicates that they are involved in the defense and detoxification system of S. officinalis (Beuerlein & Schipp, 1998), similar to the cellular defense system described for Loligo vulgaris (Meister, 1977; Sundermann, 1980) and Eledone cirrhosa (Malham & Runham, 1998).
Incorporation of fluospheres Because of their low non-specific binding, the chosen carboxylate-modified fluospheres appear to be suitable for applications involving phagocytosis but, in spite of the fact that the fluospheres were clearly distinguishable from the background fluorescence, it could not be ascertained by using fluorescence microscopy that the fluospheres were definitively located in the cell or whether they were only bound at the cell surface. However, the transmission electron microscopy provided more precise results. The most remarkable feature is that the rhogocytes phagocytize particles of large size, although their distinct cell surface structure strongly pleaded against this capacity. It seems that the rhogocytes of the branchial heart are not only responsible for the elimination of xenobiotics of small size like fluospheres (∅ 20 nm), gold particles (∅ 15 nm) and ferritin (∅ 10 nm;
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supported by previous investigations (Bayne, 1973; Witmer, 1974; Meister, 1977; Sundermann, 1980; Guary et al., 1981; Ford, 1992; Bayne, 1983; Budelmann et al., 1997; Malham et al., 1997; Beuerlein & Schipp, 1998; Malham & Runham, 1998), we propose that the triangular mesenchymatic cells are also adhesive hemocytes that, together with the circulating hemocytes, form the first line in the defense against allogeneic materials, being followed by the rhogocytes of the branchial heart tissue. Phagocytosis of bacteria In contrast to the above-mentioned phagocytosis of fluospheres, the rhogocytes of the branchial heart complex of S. officinalis revealed no uptake of whole allogeneic cells after bacterial infection. However, the increased endocytotic activity and plasmatic vacuolization as well as the apparent fusion of transport vesicles with lysosomes indicate that they are able to recognize, incorporate and catabolize allogeneic cellular material; e.g. bacterial toxins and/or cellular debris of bacteria (Beuerlein & Schipp, 1998). In the present study, phagocytosis of bacterial cells is demonstrated for the triangular mesenchymatic cells in the wall of the branchial heart. These cells reveal all stages of lysis of bacteria engulfed by lysosomes as do the adhesive and/or the circulating hemocytes. These findings agree with previous results on phagocytotic-active cells that are involved in the cellular defense in coleoid cephalopods (Stuart, 1968; Bayne, 1973; Budelmann et al., 1997; Malham et al., 1997; Beuerlein & Schipp, 1998) and other molluscs (Sminia, 1972, 1981; Bayne, 1983, Sminia & van der Knaap, 1987; Hansen et al., 1991; Cima et al., 2000; Fournier et al., 2001; Lacoste et al., 2001).
Conclusion
Fig. 4 Cytomorphological analysis of the branchial heart tissue after bacterial infection. ×13 500. Bacterial cells detected in a triangular mesenchymatic cell but not in the rhogocytes; note the different stages of lysis (I–III) of phagocytized bacteria within lysosomes and vacuoles. TEM. er, rough endoplasmic reticulum; ev, endocytotic vesicles; ly, lysosome; mi, mitochondrium; nu, nucleus; rc, rhogocyte; va, vacuole.
Beuerlein & Schipp, 1998) by selective endocytosis, but that they are also able to incorporate large foreign particles by probably receptor-mediated phagocytosis enabling them to inactivate allogeneic material by its catabolism and/or storage within the lysosomal compartment (Schipp & von Boletzky, 1975; Schipp & Hevert, 1978; Fiedler & Schipp, 1987; Beuerlein & Schipp, 1998). However, the higher rate of the uptake of fluospheres of all sizes by (all forms) of hemocytes points to the fact that above all else, these cells are responsible for the elimination of large foreign materials from the hemolymph. Confirmed by the present findings and
Based on the present findings, we conclude that the hemocytes of the circulating blood, the adhesive hemocytes (to which we count the triangular mesenchymatic cells) and the rhogocytes form the cellular defense and detoxification system in coleoid cephalopods.
ACKNOWLEDGEMENTS We would like to thank Brigitte Fronk and Anneliese Hudel for excellent help with the photographical evaluations and we also have benefited from discussions with Henrike Schmidtberg and Frank Berghaus. We are especially grateful to Sigurd von Boletzky who provided the technical facilities for work at the Laboratoire Arago in Banyuls sur Mer. This contribution was supported by grant Schi 99/10-1 of the DFG (German Research Foundation).
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