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Pathological changes in the nephrocytes of the shore crab, Carcinus maenas, following injection of bacteria
JOURNAL
OF
INVERTEBRATE
PATHOLOGY
38, 113- 121 (1981)
Pathological Changes in the Nephrocytes of the Shore Carcinus maenas, following Injection of Bacteria VALERIE
Crab,
J. SMITH' AND N. A. RATCLIFFE Received September 15, 1980
The gills of Curcinus maenas were examined by light and electron microscopy following injection ofeither sterile saline or the bacteriaBacillus cereus and Moraxella sp., to determine any role(s) for the nephrocytes in the host defense reactions. The results showed that although intact bacteria were not sequestered to the nephrocytes, these cells were active in the removal of large quantities of cell debris from the hemolymph. Much of this material was derived from the breakdown of the hemocytes in response to the presence of bacteria and it’s accumulation in the central vacuoles of the nephrocytes resulted in the degradation of these cells. It is proposed that while nephrocytes do not phagocytose intact bacteria, they augment the host defenses by clearing much of the hemocyte and associated bacterial debris from the gills, thus preventing blockage of the lamellar sinuses and subsequent impairment of respiration. KEY WORDS: Nephrocytes; phagocytosis; cell breakdown; host defense: bacteria: crab; Ctrrcimts
maenas.
tain large vacuolar cells, termed variously. nephrocytes (Drach, 1930; Wright, 1964; Previous studies of the host defense Ali, 1966; Foster and Howse, 1978); branreactions of the crab Carcinus maenas chial excretory cells (Cuenot, 1895); athroshowed that following injection of bacteria, cytes (Lison, 1942; Flemister and Flemisnumerous hemocyte clumps and entrapped ter, 1951) or podocytes (Strangways-Dixon bacteria accumulated in the gills and, to a lesser extent, the hepatopancreas and heart and Smith, 1970), which have been implicated in excretion (Parry, 1960; Strang(Smith and Ratcliffe, 1976, 1980a, b). We suggested that clump formation not only ways-Dixon and Smith, 1970), osmoregulation (Flemister and Flemister, 1951), and/ facilitates hemocyte:bacteria interaction or phagocytosis (Wright, 1964; Foster and but also prevents the spread of infection, Howse, 1978), these cells may be of sigand thus is probably an important componificance in the clearance of the gills. Connent of the cellular defenses. Also evident in the present investigation, in the gills in the early stages following in- sequently, histopathological changes in the nephrocytes oculation are large amounts of hemocyte of C. maenas following injection of bacteria debris and this, together with the cell are described, and the possible role(s) of clumps, often occludes the lamellar sinuses these cells in the disposal or elimination (Smith and Ratcliffe, 1980b). Many of the of degraded cellular and bacterial material cell clumps and much of the debris, howfrom the body is assessed. ever, are subsequently cleared from the gills and may either pass to other regions of MATERIALS AND METHODS the body or alternatively be eliminated from Animals, bacteria, and treatment of crabs the crab through the gills. were as described in Smith and Ratcliffe Since crustacean gills are known to con(1980a, b). Groups of four crabs were sampled at 1, 3, 12, or 24 hr after injection of 0.1 ’ Present address: University Marine Biological Station, Millport, Isle of Cumbrae KA28 OEG, Scot- ml of either heat-killed Bacillus cereus or Moraxelfa sp. (1 x lOVml), or, in the case land. INTRODUCTION
113 0022-201 l/81/0401 13-09$01.00/0 Copyright All rights
@ 1981 by Academic Press, Inc. of reproduction in any form reserved.
114
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of control crabs, with sterile Carcinus saline (Smith and Ratcliffe, 1978). Small pieces of gill were excised and processed for electron microscopy, and the thick and thin sections cut from the peripheral, dorsal region of the lamellae, were stained, examined, and photographed as described previously (Smith and Ratcliffe, 1980b). RESULTS
In C. maenas the nephrocytes are concentrated in the peripheral, dorsal region of the gill lamellae (Ali, 1966). Light microscopy observations of the control, salineinjected crabs, showed they were large (ca. 20-50 pm in diameter), spherical or ovoid cells, characteristically containing a large, central vacuole with several smaller, peripheral vacuoles scattered throughout the cytoplasm (Fig. 1). At the ultrastructural level, the nephrocytes from the control animals were surrounded by a thin basal lamina (Fig. 2) which was usually intact. Adjacent to the basal lamina, the plasma membrane was frequently infolded to form many small tubules and coated vesicles (Fig. 3). The central and peripheral vacuoles were separated from the ground substance of the cytoplasm by single bounding membranes (Figs. 2, 3) and these vacuoles were either electron transparent or enclosed electronopaque, flocculent material (Figs. 2, 3). In addition, the central vacuole sometimes contained a small but variable number of myelin configurations and/or crystalline inclusions (Fig. 2). The chemical nature of these crystals is unknown but they may represent excretory products (Ali, 1966). Examination of the nephrocytes from the crabs injected 1 hr previously with bacteria showed a marked increase in the activity of the cytoplasm, with large areas of the plasma membrane infolded and budding off numerous coated vesicles and tubules (Fig. 4, see also Fig. 11). Also evident, at this time, were a variable number of small (ca. 0.5 x 1.0-0.7 x 1.5 pm), electron-dense bodies enclosed by the peripheral vacuoles
RATCLIFFE
(Figs. 4, 5). These inclusions closely resembled in structure, size, and electron density the granules of the refractile hemocytes (Smith and Ratcliffe, 1980b) and clearly lacked the characteristic features of bacteria. At 1 hr incubation, these structures were confined to the peripheral vacuoles. By 3 hr, however, not only were there greater numbers of these inclusions present, but a few were also contained within the central vacuole (Fig. 6). The granules appeared to be transported across the nephrocyte cytoplasm, to the central vacuole into which they were released by disintegration of the cytoplasm (Fig. 7). Actual discharge of the granules, some of which had a flocculent and degraded appearance at this time (Figs. 6, 7), was not observed. Again, the nephrocyte cytoplasm appeared very active in vesicle formation, and although variable in extent, many nephrocytes now showed general signs of degeneration, with disruption of portions of the bounding membrane of the central vacuole and fragmentation of the adjacent cytoplasm (Fig. 6). By 12 hr incubation, many more myelin configurations were present in the central vacuole (Fig. 8) and most of the granules in the peripheral vacuoles showed some degree of breakdown (Figs. 9, 10). Coated vesicles were also observed fusing with the peripheral vacuoles (Fig. 11) and may have been involved in granule breakdown by, as yet, some undetermined process. The cytoplasmic breakdown, seen previously in the 3 hr experimental crabs, although again variable in extent, was also generally observed at 12 hr (Fig. 8), but was even more pronounced by 24 hr (Fig. 12). Often by this latter time, the nephrocytes were almost completely degraded, leaving only a thin rim of highly eroded cytoplasm surrounding a huge central vacuole (Fig. 12) which frequently contained a variable number of myelin configurations, cell remnants and/or crystalline inclusions (Fig. 12). The ultimate fate of this material is unknown. Not all the nephrocytes, seen after
NEPHROCYTES
OF C. maenn~
FIGS. l-3. Gill lamellae, 1 hr after injection of sterile saline. FIG. 1. Nephrocytes (Ne) in the connective tissue matrix of a gill lamella. Note the large central vacuoles (CV) and smaller, peripheral vacuoles (PV) in the nephrocyte cytoplasm. Araldite section, x239 (scale bar = 100 wm). FIG. 2. Nephrocyte, showing the thin basal lamina (BL), cytoplasm (Cy), and eccentric nucleus(N). Note the large, central vacuole (CV) containing myelin configurations (m) and crystalline inclusions (CT). Some of the smaller peripheral vacuoles (PV) enclose electron-opaque material (unlabeled arrowheads). x4725 (scale bar = 5 wm). FIG. 3. Electron micrograph showing the formation of coated vesicles (cov) and tubules (t) by infolding of the plasma membrane (unlabeled arrow heads). Two peripheral vacuoles (PV) are present. one containing electron-opaque material. Basal lamina (BL) and the bounding membranes (bm) of the peripheral (PV) and central (CV) vacuoles are also evident. x22,500 (scale bar = 0.5 pm).
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FIG. 4. Part of a nephrocyte, 1 hr after injection of Mm-axe/la sp., showing peripheral vacuoles (PV) containing small electron-dense bodies (E). Large numbers of coated vesicles and tubules (unlabeled arrow heads) are present in thqcytoplasm (Cy). The central vacuole (CV) contains dense, flocculent material and myelin configurations (m). Nucleus (N). x5625 (scale bar = 5 pm). FIG. 5. Electron-dense body (E) within one of the peripheral vacuoles of a nephrocyte 1 hr after injection of Moraxda sp. This structure closely resembles a refractile hemocyte granule. x29,700 (scale bar = 0.5 pm).
NEPHROCYTES
24 hr incubation, however, showed this degeneration, and a few intact cells were still evident. Bacteria were not detected associated with the nephrocytes of the experimental crabs at any of the incubation times. Occasionally, in the saline-injected controls, a few electron-dense bodies were seen in the peripheral and central vacuoles but they were much less numerous than in the experimental crabs. Some nephrocyte breakdown was also seen but again this was much less frequent and drastic than in the bacteria-injected animals. Saline injection, by itself, also had little effect on the nephrocytes since the structure of these cells in the control crabs was similar to that of untreated animals, previously described by Ali (1966). DISCUSSION Relatively few detailed studies of crustacean nephrocytes have been made (Wright, 1964; Ali, 1966; Strangways-Dixon and Smith, 1970), and their precise function is uncertain. Similar cells, termed pericardial cells, also occur in insects (Hollande, 1922; Bowers, 1964; Wigglesworth, 1970; Crossley, 1972) and are considered to be equivalent to the reticuloendothelial system of vertebrates (Wigglesworth, 1970), selectively taking up certain protein and other molecules for breakdown, as well as manufacturing substances, such as lysozyme, in response to injected bacteria (Crossley, 1972). Suggestions have been made for the physiological role of crustacean nephrocytes. Drach (1930), Lison (1942), and Ah (1966) reported that injected dyes and parti-
OF
C. maenas
117
cle suspensions were sequestered to the nephrocyte vacuoles, and proposed an excretory function for these cells. Further evidence for an excretory/detoxifying activity is provided by their close structural resemblance to the podocytes of vertebrate kidney (Strangways-Dixon and Smith, 1970). An alternative theory suggesting that the nephrocytes participate in chloride ion regulation (Flemister and Flemister, 195 1) was not confirmed by a subsequent detailed histological investigation (Flemister, 1959). More recently, a possible phagocytic role has also been ascribed to crustacean nephrocytes (Wright, 1964; Foster and Howse, 1978), although convincing experimental evidence for this hypothesis is lacking. In the present study, neither intact nor degraded bacteria were found associated with the nephrocytes of either the control or the experimental crabs. Johnson (1976) similarly failed to observe bacteria in the nephrocytes or Callinectes sapidus infected with a Gram-negative bacterium, so that in crustaceans, these cells do not appear to play a direct role in the removal of microbial invaders from the blood. In C. maenas, however, much larger quantities of cell debris were seen in the nephrocytes of the bacteria-injected crabs than in the saline-injected controls, indicating that these cells may, in some way, be indirectly involved in the host defense reactions. Much of the cell debris, present in the peripheral vacuoles at 1 and 3 hr postinjection, appeared to be of refractile cell origin and these cells have previously been shown to be unstable in the presence of bacteria or other non-self materials (Smith and Ratcliffe, 1978, 198Ob), although it is also possible that many of the myelin
FIG. 6. Nephrocyte, 3 hr after injection of Bacillus cereus. Several electron-dense bodies (E) are present in the central vacuole (CV) and some appear flocculent and degraded (unlabeled arrowheads). Part of the bounding membrane of the central vacuole is now broken down and the cytoplasm is disintegrating (D) (compare with Fig. 4). x7200 (scale bar = 2 wm). FIG. 7. Central region of a nephrocyte, 3 hr after injection of Moraxella sp. A peripheral vacuole (PV) is about to discharge an electron-dense body (E) into the central vacuole (CV). The bounding membrane is disrupted (arrowheads). x 16,200 (scale bar = 0.5 pm).
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FIG. 8. Nephrocyte, 12 hr after injection of Moraxella sp. Note the large number of myelin configurations (m) in the central vacuole (CV) and numerous coated vesicles and tubules (unlabeled arrowheads) in the active looking cytoplasm (Cy). x6300 (scale bar = 5 pm). FIG. 9. Part of a nephrocyte, 12 hr after injection of Eacillus cereus, showing two peripheral vacuoles (PV) enclosing electron-dense bodies (E) of varying degrees of breakdown. Basal lamina (BL); central vacuole (CV). x 13,500 (scale bar = 1 pm).
NEPHROCYTES
OF
C. mrrenas
119
FIG. 12. Nephrocyte, 24 hr after injection of Moraxella sp., showing the extreme erosion of the cytoplasm (Cy). Note the large crystalline inclusion (cr) in the central vacuole (CV) and the electrondense bodies (E) contained within the degenerating cytoplasm. x 3250 (scale bar = 10 pm).
configurations, seen after 12 and 24 hr of incubation, may have represented other totally degraded cells and/or organelles. Furthermore, the accumulation of material in the nephrocytes reaches a maximum between 3 and 12 hr postinoculation, a time coinciding with the clearance of many of
the degraded hemocytes and large hemocyte:bacteria complexes (= cell clumps) sequestered to the gills in response to the injection of bacteria (Smith and Ratcliffe, 1980b). Thus, the nephrocytes probably play an important role in the host defenses by removing much of the cell debris which
FIG. 10. As in Fig. 9, a peripheral vacuole (PV) containing some flocculent electron-dense material (E) and a crystalline inclusion (cr). Central vacuole (CV): coated vesicles (unlabeled arrowheads). x 10,800 (scale bar = 1 km). FIG. 11. Part of a nephrocyte, 12 hr after injection of Badus cereus. showing fusion of a coated vesicle (cov) with a peripheral vacuole (PV). Note the formation of the coated vesicles (unlabeled arrowheads) from the infolded plasma membrane adjacent to the basal lamina (BL). x45.900 (scale bar = 0.5 pm).
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would otherwise occlude the lamellar sinuses of the gills and impair respiration. The mechanism by which particulate material enters the nephrocytes was not ascertained in the present investigation. Drach (1930) proposed that dyes entered by diffusion, but it is unlikely that the refractile cell granules could penetrate the nephrocyte wall in this way. Possibly, entry may occur by infolding or intucking of the plasma membrane to form the peripheral vacuoles. The mode of degradation of the sequestered material inside the peripheral and central vacuoles is also unknown, although the process may be aided by substances released from the coated vesicles which were frequently seen fusing with the nephrocyte vacuoles. A similar phenomenon has been reported for the pericardial cells of C. erythrocephala, where transfer of materials from the coated vesicles to the vacuoles has been demonstrated using horseradish peroxidase label (Crossley, 1972). Finallv. _ , the reason for the breakdown of the nephrocytes in the bacteria-injected crabs over the 24-hr experimental period is unclear, but it may be a result of the enhanced activity of these cells so that ageing and death are accelerated. This process is thus an indirect histopathological consequence of infection. Ultimately, the degraded nephrocytes and enclosed material may be discharged from the crab at the next moult as suggested by Drach (1930) and Wright (1964). In conclusion, crustacean nephrocytes probably augment the host defense reactions by removing large quantities of cell debris, which may include degraded microorganisms, from the gills. They thus maintain respiratory activity and may also produce enzymes capable of degrading the enclosed material. Further investigations are necessary to elucidate the exact nature of the sequestered material, and of any vaculolar/vesicular secretions, as well as the mechanism(s) by which elimination of debris is finally achieved.
ACKNOWLEDGMENTS We would like to thank Dr. B. L. Bayne of the Institute for Marine Environmental Research, Plymouth, England, for helpful advice and criticism throughout much of this work. We are also grateful to Professor E. W. Knight-Jones for facilities provided and to the Natural Environment Research Council for a studentship to V. J. S., and finally to the Royal Society for financial support.
REFERENCES ALI, M. 1966. “The Histology of the Gills of C’arcinus maenas (L.) and Other Decapod Crustacea.” Ph.D. thesis, University of Newcastle-upon-Tyne. BOWERS, B. 1964. Coated vesicles in the pericardiaf cells of the aphid (Myzus persicae Sulz). Protoplasma, 59, 351-367. CROSSLEY,A. C. 1972. The ultrastructure and function of pericardial cells and other nephrocytes in an insect: Calliphora erythrocephala. Tissue Cell, 4, 529-560. CLJI?NOT, L. 1895. etudes physiologiques sur les crustacts dbcapodes. Arch. Biol., 13, 245-303. DRACH, P. 1930. etude sur le systtme branchial des crustaces decapodes. Arch. Anat. Microsc. Morphol. Exp., 26, 83- 133. FLEMISTER, L. J., AND FLEMISTER, S. C. 1951. Chloride ion regulation and oxygen consumption in the crab, Ocypode albicans (Bosq). Biol. Bull., 101, 259-273. FLEMISTER, S. C. 1959. Histophysiology of gill and kidney of the crab, Ocypode albicans. Biol. BUN.. 116, 37-48. FOSTER, C. A., AND HOWSE, H. D. 1978. A morphological study on gills of the brown shrimp Penaeus aztecus. Tissue Cell., 10, 77-92. HOLLANDE, A. C. 1922. La cellule ptricardial des insectes: Cytologie, histochimie, rBle physiologique. Arch. Anat. Microsc.. 18, 85-307. JOHNSON, P. T. 1976. Bacterial infection in the blue crab, Callinectes sapidas: Course of infection and histopathology. J. Invertebr. Pathol., 28, 25-36. LISON, L. 1942. Recherches sur I’histophysiologie cornparke de I’excrCtion chez les arthropodes. Mem. Acad. Roy. Med. Belg.. 19, l- 107. PARRY, G. 1960. Excretion. In “The Physiology of Crustacea” (T. H. Waterman, ed.), Vol. I, pp. 341-366. Academic Press, New York/London. SMITH, V. J., AND RATCLIFFE, N. A. 1976. Defensive reactions of the shore crab, Carcinus maenas, to foreign particles. In “Proceedings 1st Colloquium on Invertebrate Pathology” (T. A. Angus, P. Faulkner, and A. Rosenfield, eds.), pp. 312-313. Queens University, Kingston, Ontario. SMITH, V. J., AND RATCLIFFE, N. A. 1978. Host defence reactions of the shore crab. Carcinus maenas (L.) in vitro. J. Mar. Biol. Assoc. U.K., 58, 367- 376.
NEPHROCYTES
SMITH. V.J., AND RATCLIFFE,N. A. 198Oa. Hostdefence reactions of the shore crab, Carcinus mnenas CL.). clearance and distribution of injected test particles. J. Mar. Bid. Assoc. U.K., 60, 89- 102. SWTH, V. J., AND RATCLIFFE, N. A. 198Ob. Cellular defense reactions of the shore crab, Carcinus nwenas, in viw hemocytic and histopathological responses to injected bacteria. J. Intvrtrbr. Pathd. 35, 65-74.
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STRANCWAYS-DIXON,J..ANDSMITH. D. S. 1970.The fine structure of gill podocytes in Panlrlirus ureas (Crustacea). Tissue Cell. 2, 61 l-624. WIGGLESWORTH. V. B. 1970. The pericardial cells of insects: Analogue of the reticuloendothelial system. J. Reti~ul~~endorhrl. SIC-., 7, 208-216. WRIGHT. K. A. 1964. The fine structure of the nephrocytes of the gills of two marine decapods. J. l/lfru.sfruc’t. Rcs.. 10, 1- 13.
OF
INVERTEBRATE
PATHOLOGY
38, 113- 121 (1981)
Pathological Changes in the Nephrocytes of the Shore Carcinus maenas, following Injection of Bacteria VALERIE
Crab,
J. SMITH' AND N. A. RATCLIFFE Received September 15, 1980
The gills of Curcinus maenas were examined by light and electron microscopy following injection ofeither sterile saline or the bacteriaBacillus cereus and Moraxella sp., to determine any role(s) for the nephrocytes in the host defense reactions. The results showed that although intact bacteria were not sequestered to the nephrocytes, these cells were active in the removal of large quantities of cell debris from the hemolymph. Much of this material was derived from the breakdown of the hemocytes in response to the presence of bacteria and it’s accumulation in the central vacuoles of the nephrocytes resulted in the degradation of these cells. It is proposed that while nephrocytes do not phagocytose intact bacteria, they augment the host defenses by clearing much of the hemocyte and associated bacterial debris from the gills, thus preventing blockage of the lamellar sinuses and subsequent impairment of respiration. KEY WORDS: Nephrocytes; phagocytosis; cell breakdown; host defense: bacteria: crab; Ctrrcimts
maenas.
tain large vacuolar cells, termed variously. nephrocytes (Drach, 1930; Wright, 1964; Previous studies of the host defense Ali, 1966; Foster and Howse, 1978); branreactions of the crab Carcinus maenas chial excretory cells (Cuenot, 1895); athroshowed that following injection of bacteria, cytes (Lison, 1942; Flemister and Flemisnumerous hemocyte clumps and entrapped ter, 1951) or podocytes (Strangways-Dixon bacteria accumulated in the gills and, to a lesser extent, the hepatopancreas and heart and Smith, 1970), which have been implicated in excretion (Parry, 1960; Strang(Smith and Ratcliffe, 1976, 1980a, b). We suggested that clump formation not only ways-Dixon and Smith, 1970), osmoregulation (Flemister and Flemister, 1951), and/ facilitates hemocyte:bacteria interaction or phagocytosis (Wright, 1964; Foster and but also prevents the spread of infection, Howse, 1978), these cells may be of sigand thus is probably an important componificance in the clearance of the gills. Connent of the cellular defenses. Also evident in the present investigation, in the gills in the early stages following in- sequently, histopathological changes in the nephrocytes oculation are large amounts of hemocyte of C. maenas following injection of bacteria debris and this, together with the cell are described, and the possible role(s) of clumps, often occludes the lamellar sinuses these cells in the disposal or elimination (Smith and Ratcliffe, 1980b). Many of the of degraded cellular and bacterial material cell clumps and much of the debris, howfrom the body is assessed. ever, are subsequently cleared from the gills and may either pass to other regions of MATERIALS AND METHODS the body or alternatively be eliminated from Animals, bacteria, and treatment of crabs the crab through the gills. were as described in Smith and Ratcliffe Since crustacean gills are known to con(1980a, b). Groups of four crabs were sampled at 1, 3, 12, or 24 hr after injection of 0.1 ’ Present address: University Marine Biological Station, Millport, Isle of Cumbrae KA28 OEG, Scot- ml of either heat-killed Bacillus cereus or Moraxelfa sp. (1 x lOVml), or, in the case land. INTRODUCTION
113 0022-201 l/81/0401 13-09$01.00/0 Copyright All rights
@ 1981 by Academic Press, Inc. of reproduction in any form reserved.
114
SMITH
AND
of control crabs, with sterile Carcinus saline (Smith and Ratcliffe, 1978). Small pieces of gill were excised and processed for electron microscopy, and the thick and thin sections cut from the peripheral, dorsal region of the lamellae, were stained, examined, and photographed as described previously (Smith and Ratcliffe, 1980b). RESULTS
In C. maenas the nephrocytes are concentrated in the peripheral, dorsal region of the gill lamellae (Ali, 1966). Light microscopy observations of the control, salineinjected crabs, showed they were large (ca. 20-50 pm in diameter), spherical or ovoid cells, characteristically containing a large, central vacuole with several smaller, peripheral vacuoles scattered throughout the cytoplasm (Fig. 1). At the ultrastructural level, the nephrocytes from the control animals were surrounded by a thin basal lamina (Fig. 2) which was usually intact. Adjacent to the basal lamina, the plasma membrane was frequently infolded to form many small tubules and coated vesicles (Fig. 3). The central and peripheral vacuoles were separated from the ground substance of the cytoplasm by single bounding membranes (Figs. 2, 3) and these vacuoles were either electron transparent or enclosed electronopaque, flocculent material (Figs. 2, 3). In addition, the central vacuole sometimes contained a small but variable number of myelin configurations and/or crystalline inclusions (Fig. 2). The chemical nature of these crystals is unknown but they may represent excretory products (Ali, 1966). Examination of the nephrocytes from the crabs injected 1 hr previously with bacteria showed a marked increase in the activity of the cytoplasm, with large areas of the plasma membrane infolded and budding off numerous coated vesicles and tubules (Fig. 4, see also Fig. 11). Also evident, at this time, were a variable number of small (ca. 0.5 x 1.0-0.7 x 1.5 pm), electron-dense bodies enclosed by the peripheral vacuoles
RATCLIFFE
(Figs. 4, 5). These inclusions closely resembled in structure, size, and electron density the granules of the refractile hemocytes (Smith and Ratcliffe, 1980b) and clearly lacked the characteristic features of bacteria. At 1 hr incubation, these structures were confined to the peripheral vacuoles. By 3 hr, however, not only were there greater numbers of these inclusions present, but a few were also contained within the central vacuole (Fig. 6). The granules appeared to be transported across the nephrocyte cytoplasm, to the central vacuole into which they were released by disintegration of the cytoplasm (Fig. 7). Actual discharge of the granules, some of which had a flocculent and degraded appearance at this time (Figs. 6, 7), was not observed. Again, the nephrocyte cytoplasm appeared very active in vesicle formation, and although variable in extent, many nephrocytes now showed general signs of degeneration, with disruption of portions of the bounding membrane of the central vacuole and fragmentation of the adjacent cytoplasm (Fig. 6). By 12 hr incubation, many more myelin configurations were present in the central vacuole (Fig. 8) and most of the granules in the peripheral vacuoles showed some degree of breakdown (Figs. 9, 10). Coated vesicles were also observed fusing with the peripheral vacuoles (Fig. 11) and may have been involved in granule breakdown by, as yet, some undetermined process. The cytoplasmic breakdown, seen previously in the 3 hr experimental crabs, although again variable in extent, was also generally observed at 12 hr (Fig. 8), but was even more pronounced by 24 hr (Fig. 12). Often by this latter time, the nephrocytes were almost completely degraded, leaving only a thin rim of highly eroded cytoplasm surrounding a huge central vacuole (Fig. 12) which frequently contained a variable number of myelin configurations, cell remnants and/or crystalline inclusions (Fig. 12). The ultimate fate of this material is unknown. Not all the nephrocytes, seen after
NEPHROCYTES
OF C. maenn~
FIGS. l-3. Gill lamellae, 1 hr after injection of sterile saline. FIG. 1. Nephrocytes (Ne) in the connective tissue matrix of a gill lamella. Note the large central vacuoles (CV) and smaller, peripheral vacuoles (PV) in the nephrocyte cytoplasm. Araldite section, x239 (scale bar = 100 wm). FIG. 2. Nephrocyte, showing the thin basal lamina (BL), cytoplasm (Cy), and eccentric nucleus(N). Note the large, central vacuole (CV) containing myelin configurations (m) and crystalline inclusions (CT). Some of the smaller peripheral vacuoles (PV) enclose electron-opaque material (unlabeled arrowheads). x4725 (scale bar = 5 wm). FIG. 3. Electron micrograph showing the formation of coated vesicles (cov) and tubules (t) by infolding of the plasma membrane (unlabeled arrow heads). Two peripheral vacuoles (PV) are present. one containing electron-opaque material. Basal lamina (BL) and the bounding membranes (bm) of the peripheral (PV) and central (CV) vacuoles are also evident. x22,500 (scale bar = 0.5 pm).
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FIG. 4. Part of a nephrocyte, 1 hr after injection of Mm-axe/la sp., showing peripheral vacuoles (PV) containing small electron-dense bodies (E). Large numbers of coated vesicles and tubules (unlabeled arrow heads) are present in thqcytoplasm (Cy). The central vacuole (CV) contains dense, flocculent material and myelin configurations (m). Nucleus (N). x5625 (scale bar = 5 pm). FIG. 5. Electron-dense body (E) within one of the peripheral vacuoles of a nephrocyte 1 hr after injection of Moraxda sp. This structure closely resembles a refractile hemocyte granule. x29,700 (scale bar = 0.5 pm).
NEPHROCYTES
24 hr incubation, however, showed this degeneration, and a few intact cells were still evident. Bacteria were not detected associated with the nephrocytes of the experimental crabs at any of the incubation times. Occasionally, in the saline-injected controls, a few electron-dense bodies were seen in the peripheral and central vacuoles but they were much less numerous than in the experimental crabs. Some nephrocyte breakdown was also seen but again this was much less frequent and drastic than in the bacteria-injected animals. Saline injection, by itself, also had little effect on the nephrocytes since the structure of these cells in the control crabs was similar to that of untreated animals, previously described by Ali (1966). DISCUSSION Relatively few detailed studies of crustacean nephrocytes have been made (Wright, 1964; Ali, 1966; Strangways-Dixon and Smith, 1970), and their precise function is uncertain. Similar cells, termed pericardial cells, also occur in insects (Hollande, 1922; Bowers, 1964; Wigglesworth, 1970; Crossley, 1972) and are considered to be equivalent to the reticuloendothelial system of vertebrates (Wigglesworth, 1970), selectively taking up certain protein and other molecules for breakdown, as well as manufacturing substances, such as lysozyme, in response to injected bacteria (Crossley, 1972). Suggestions have been made for the physiological role of crustacean nephrocytes. Drach (1930), Lison (1942), and Ah (1966) reported that injected dyes and parti-
OF
C. maenas
117
cle suspensions were sequestered to the nephrocyte vacuoles, and proposed an excretory function for these cells. Further evidence for an excretory/detoxifying activity is provided by their close structural resemblance to the podocytes of vertebrate kidney (Strangways-Dixon and Smith, 1970). An alternative theory suggesting that the nephrocytes participate in chloride ion regulation (Flemister and Flemister, 195 1) was not confirmed by a subsequent detailed histological investigation (Flemister, 1959). More recently, a possible phagocytic role has also been ascribed to crustacean nephrocytes (Wright, 1964; Foster and Howse, 1978), although convincing experimental evidence for this hypothesis is lacking. In the present study, neither intact nor degraded bacteria were found associated with the nephrocytes of either the control or the experimental crabs. Johnson (1976) similarly failed to observe bacteria in the nephrocytes or Callinectes sapidus infected with a Gram-negative bacterium, so that in crustaceans, these cells do not appear to play a direct role in the removal of microbial invaders from the blood. In C. maenas, however, much larger quantities of cell debris were seen in the nephrocytes of the bacteria-injected crabs than in the saline-injected controls, indicating that these cells may, in some way, be indirectly involved in the host defense reactions. Much of the cell debris, present in the peripheral vacuoles at 1 and 3 hr postinjection, appeared to be of refractile cell origin and these cells have previously been shown to be unstable in the presence of bacteria or other non-self materials (Smith and Ratcliffe, 1978, 198Ob), although it is also possible that many of the myelin
FIG. 6. Nephrocyte, 3 hr after injection of Bacillus cereus. Several electron-dense bodies (E) are present in the central vacuole (CV) and some appear flocculent and degraded (unlabeled arrowheads). Part of the bounding membrane of the central vacuole is now broken down and the cytoplasm is disintegrating (D) (compare with Fig. 4). x7200 (scale bar = 2 wm). FIG. 7. Central region of a nephrocyte, 3 hr after injection of Moraxella sp. A peripheral vacuole (PV) is about to discharge an electron-dense body (E) into the central vacuole (CV). The bounding membrane is disrupted (arrowheads). x 16,200 (scale bar = 0.5 pm).
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FIG. 8. Nephrocyte, 12 hr after injection of Moraxella sp. Note the large number of myelin configurations (m) in the central vacuole (CV) and numerous coated vesicles and tubules (unlabeled arrowheads) in the active looking cytoplasm (Cy). x6300 (scale bar = 5 pm). FIG. 9. Part of a nephrocyte, 12 hr after injection of Eacillus cereus, showing two peripheral vacuoles (PV) enclosing electron-dense bodies (E) of varying degrees of breakdown. Basal lamina (BL); central vacuole (CV). x 13,500 (scale bar = 1 pm).
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FIG. 12. Nephrocyte, 24 hr after injection of Moraxella sp., showing the extreme erosion of the cytoplasm (Cy). Note the large crystalline inclusion (cr) in the central vacuole (CV) and the electrondense bodies (E) contained within the degenerating cytoplasm. x 3250 (scale bar = 10 pm).
configurations, seen after 12 and 24 hr of incubation, may have represented other totally degraded cells and/or organelles. Furthermore, the accumulation of material in the nephrocytes reaches a maximum between 3 and 12 hr postinoculation, a time coinciding with the clearance of many of
the degraded hemocytes and large hemocyte:bacteria complexes (= cell clumps) sequestered to the gills in response to the injection of bacteria (Smith and Ratcliffe, 1980b). Thus, the nephrocytes probably play an important role in the host defenses by removing much of the cell debris which
FIG. 10. As in Fig. 9, a peripheral vacuole (PV) containing some flocculent electron-dense material (E) and a crystalline inclusion (cr). Central vacuole (CV): coated vesicles (unlabeled arrowheads). x 10,800 (scale bar = 1 km). FIG. 11. Part of a nephrocyte, 12 hr after injection of Badus cereus. showing fusion of a coated vesicle (cov) with a peripheral vacuole (PV). Note the formation of the coated vesicles (unlabeled arrowheads) from the infolded plasma membrane adjacent to the basal lamina (BL). x45.900 (scale bar = 0.5 pm).
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would otherwise occlude the lamellar sinuses of the gills and impair respiration. The mechanism by which particulate material enters the nephrocytes was not ascertained in the present investigation. Drach (1930) proposed that dyes entered by diffusion, but it is unlikely that the refractile cell granules could penetrate the nephrocyte wall in this way. Possibly, entry may occur by infolding or intucking of the plasma membrane to form the peripheral vacuoles. The mode of degradation of the sequestered material inside the peripheral and central vacuoles is also unknown, although the process may be aided by substances released from the coated vesicles which were frequently seen fusing with the nephrocyte vacuoles. A similar phenomenon has been reported for the pericardial cells of C. erythrocephala, where transfer of materials from the coated vesicles to the vacuoles has been demonstrated using horseradish peroxidase label (Crossley, 1972). Finallv. _ , the reason for the breakdown of the nephrocytes in the bacteria-injected crabs over the 24-hr experimental period is unclear, but it may be a result of the enhanced activity of these cells so that ageing and death are accelerated. This process is thus an indirect histopathological consequence of infection. Ultimately, the degraded nephrocytes and enclosed material may be discharged from the crab at the next moult as suggested by Drach (1930) and Wright (1964). In conclusion, crustacean nephrocytes probably augment the host defense reactions by removing large quantities of cell debris, which may include degraded microorganisms, from the gills. They thus maintain respiratory activity and may also produce enzymes capable of degrading the enclosed material. Further investigations are necessary to elucidate the exact nature of the sequestered material, and of any vaculolar/vesicular secretions, as well as the mechanism(s) by which elimination of debris is finally achieved.
ACKNOWLEDGMENTS We would like to thank Dr. B. L. Bayne of the Institute for Marine Environmental Research, Plymouth, England, for helpful advice and criticism throughout much of this work. We are also grateful to Professor E. W. Knight-Jones for facilities provided and to the Natural Environment Research Council for a studentship to V. J. S., and finally to the Royal Society for financial support.
REFERENCES ALI, M. 1966. “The Histology of the Gills of C’arcinus maenas (L.) and Other Decapod Crustacea.” Ph.D. thesis, University of Newcastle-upon-Tyne. BOWERS, B. 1964. Coated vesicles in the pericardiaf cells of the aphid (Myzus persicae Sulz). Protoplasma, 59, 351-367. CROSSLEY,A. C. 1972. The ultrastructure and function of pericardial cells and other nephrocytes in an insect: Calliphora erythrocephala. Tissue Cell, 4, 529-560. CLJI?NOT, L. 1895. etudes physiologiques sur les crustacts dbcapodes. Arch. Biol., 13, 245-303. DRACH, P. 1930. etude sur le systtme branchial des crustaces decapodes. Arch. Anat. Microsc. Morphol. Exp., 26, 83- 133. FLEMISTER, L. J., AND FLEMISTER, S. C. 1951. Chloride ion regulation and oxygen consumption in the crab, Ocypode albicans (Bosq). Biol. Bull., 101, 259-273. FLEMISTER, S. C. 1959. Histophysiology of gill and kidney of the crab, Ocypode albicans. Biol. BUN.. 116, 37-48. FOSTER, C. A., AND HOWSE, H. D. 1978. A morphological study on gills of the brown shrimp Penaeus aztecus. Tissue Cell., 10, 77-92. HOLLANDE, A. C. 1922. La cellule ptricardial des insectes: Cytologie, histochimie, rBle physiologique. Arch. Anat. Microsc.. 18, 85-307. JOHNSON, P. T. 1976. Bacterial infection in the blue crab, Callinectes sapidas: Course of infection and histopathology. J. Invertebr. Pathol., 28, 25-36. LISON, L. 1942. Recherches sur I’histophysiologie cornparke de I’excrCtion chez les arthropodes. Mem. Acad. Roy. Med. Belg.. 19, l- 107. PARRY, G. 1960. Excretion. In “The Physiology of Crustacea” (T. H. Waterman, ed.), Vol. I, pp. 341-366. Academic Press, New York/London. SMITH, V. J., AND RATCLIFFE, N. A. 1976. Defensive reactions of the shore crab, Carcinus maenas, to foreign particles. In “Proceedings 1st Colloquium on Invertebrate Pathology” (T. A. Angus, P. Faulkner, and A. Rosenfield, eds.), pp. 312-313. Queens University, Kingston, Ontario. SMITH, V. J., AND RATCLIFFE, N. A. 1978. Host defence reactions of the shore crab. Carcinus maenas (L.) in vitro. J. Mar. Biol. Assoc. U.K., 58, 367- 376.
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SMITH. V.J., AND RATCLIFFE,N. A. 198Oa. Hostdefence reactions of the shore crab, Carcinus mnenas CL.). clearance and distribution of injected test particles. J. Mar. Bid. Assoc. U.K., 60, 89- 102. SWTH, V. J., AND RATCLIFFE, N. A. 198Ob. Cellular defense reactions of the shore crab, Carcinus nwenas, in viw hemocytic and histopathological responses to injected bacteria. J. Intvrtrbr. Pathd. 35, 65-74.
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STRANCWAYS-DIXON,J..ANDSMITH. D. S. 1970.The fine structure of gill podocytes in Panlrlirus ureas (Crustacea). Tissue Cell. 2, 61 l-624. WIGGLESWORTH. V. B. 1970. The pericardial cells of insects: Analogue of the reticuloendothelial system. J. Reti~ul~~endorhrl. SIC-., 7, 208-216. WRIGHT. K. A. 1964. The fine structure of the nephrocytes of the gills of two marine decapods. J. l/lfru.sfruc’t. Rcs.. 10, 1- 13.