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The fine structure of a parasitic ciliate Terebrospira during ingestion of the exoskeleton of a shrimp Palaemonetes
TISSUE L CELL 1976 8 (4) 573-582 Published by Longman Group Ltd. Printed in Great Britain
PHYLLIS
CLARKE
THE FINE STRUCTURE CILIATE TEREBROSPIRA OF THE EXOSKELETON
BRADBURY
and VIBHA
GOYAL
OF A PARASITIC DURING INGESTION OF A SHRIMP
PALAEMONETES
ABSTRACT. The ciliated protozoan, Terebrospira chattoni, invades the exoskeleton of the shrimp, Palaemoneres pugio, eating out long galleries parallel to the surface of the exoskeleton. Solubilization of the exoskeleton occurs around an area of the elaborately infolded surface membrane at the anterior of the organism. Dissolved products of the digestion of the exoskeleton are taken into the body by the formation of coated vesicles at pores in the membrane. The surface membrane that is taken in by pinocytosis is apparently recycled by the introduction into the membrane of organelles implicated in mambrane recycling in other ciliates. Acid phosphatase can be demonstrated on the surface membrane as well as in the endocuticle around the organism.
stage of Terebrispira confirms the lack of a cytostome or other oral structures and the absence of organelles for boring and triturating. The solid exoskeleton must be solubilized by extracellular enzymes. Terebrospira has been examined by electron microscopy to learn how it feeds, and experiments were begun to demonstrate enzymatic activity at the advancing face of the gallery.
Intmduction THE crustacean exoskeleton is a rich store of carbohydrates, protein, and lipid, that is impervious to the digestive enzymes of most organisms. In the animal kingdom only a few ciliated protozoa, belonging to the order Apostomatida, are able to feed solely on the exoskeleton or its degradation products. One of these, T’erebrospira chattoni, forms a division cyst on the surface of the brackish water shrimp, Palaemonetes pugio, and divides into two to eight daughters. (Bradbury et al., 1974). Each daughter cell dissolves a hole in the floor of the parent cyst and abandons the cyst to eat long galleries in the shrimp’s endocuticle. The galleries radiate from the empty parent cyst like the points of a star (Fig. 1) and in heavy infections the shrimp exoskeleton resembles lace or lattice-work with galleries running in all directions through the endocuticle. Each gallery contains a daughter ciliate closely appressed to the advancing face of the gallery. Electron microscopy of this feeding Department of Zoology, North Carolina University, Raleigh, North Carolina 27607. Revised 29 August 1976. Received 30 July 1976. 37
State
Materials and Methods Palaemonetes pugio (Holthuis) was collected
by dip net from South Creek at the Pamlico Marine Station in Aurora, N.C. Shrimp heavily infected with Terebrospira could be easily identified at low magnifications by the star-shaped patterns formed in the exoskeleton by the galleries. Shrimp were bisected at the junction of the carapace and the abdomen; infected fragments of exoskeleton were pulled off with forceps and immersed in 2.8% glutaraldehyde in 0.1 M cacodylate (pH 7.3). Adhering shreds of flesh were picked away by fine forceps, and the cleaned fragments were placed in fresh fixative for 1 h at room temperature, washed in buffered 6% sucrose, postfixed for 2 hr in 2% 0~04 in 0.1 M Na cacodylate, dehydrated in ethanol 573
BRADBURY
and embedded in Maraglas. Thin sections were cut with a diamond knife mounted on an LKB Ultrotome 8800 and picked up on grids covered with carbon-stabilized Parlodion films. The sections were stained with uranyl acetate and lead citrate and examined and photographed in a Siemens 1A electron microscope operating at 80 kV. To demonstrate acid phosphatase (Wise and Flickinger, 1970), fragments of infected exoskeleton were fixed for 3 hr at room temperature in 2% glutaraldehyde in O-l M Na cacodylate (pH 7.3) and then washed 6 x in 7 % buffered sucrose. The fragments were incubated for 20 min at room temperature in the following medium: 5.0 ml 1.25 % (w/v) Na-,%glycerophosphate, 5.0 ml 0.2 Trismaleate buffer (pH S.O), IO.0 ml 0.2% (w/v) Pb(NO&, and 5.0 ml distilled water. After incubation the fragments were washed 3 x in distilled water, once in 1 % acetic acid, and once again in distilled water. As a control, fragments of the same exoskeleton were incubated under the same con-
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ditions except that the medium did not contain the Na-8-glycerophosphate. Sections from the experimentally treated material were left unstained. Results When observed on living shrimp, Terebrospiru in its gallery is about 14-2 times longer than t is broad (60 x 45 p) but sections show that it is dorsoventrally flattened and only a few micra thick. The course of its gallery follows three to five laminae of the endocuticle, deviating neither upward nor downward. The ciliate always occupies the advancing face of the gallery, being snugly enclosed by exoskeleton on all sides except the rear where the lumen of the gallery extends back, sometimes as much as 1 mm to the empty parent cyst. Electron microscopy reveals the unsuspected presence of a very thin cyst wall around the feeding organism, thinner than any previously reported in ciliates (Fig. 2).
Fig. 1. Fragment of exoskeleton from a uropod of Palaemonetes pugio showing the star-shaped patterns formed by the exodus of daughter cells from the reproductive cysts of Terebrospira chattoni. The compartmented reproductive cyst can be observed at the center of each star. The galleries form the rays of the stars and the dark body at the distal end of each ray is the ciliate. x 24. Fig. 2. Section through the cyst wall, cell membrane, and peripheral cytoplasm in the postero-lateral region of Terebrospira. The cyst wall shows a faint periodicity (arrows). Fibrous strands extend from the cyst wall to the exoskeleton (E) and to the cell membrane. At the cell membrane a membrane organelle (MO) is rejoining the membrane at a pore (P). Another membrane organelle is free in the cytoplasm. x 40,000. Fig, 3. Longitudinal section through the anterior end of Terebrospira. The conspicuous hummocks formed by infoldings of the surface membrane enclose an ellipse of smooth endoplasmic reticulum (SER). Coated vesicles (CV) are visible at the pores (P) and also free in the cytoplasm. x 31,000. Fig. 4. Longitudinal section illustrating surface of Terebrospira.
the relative simplicity of the postero-lateral
Fig. 5. Section tangential to part of the surface of the anterior region of Terebrospira. Coagulum (C) of degraded endocuticie (E) surrounds the cyst wall (W) which shows subunits with electron-lucent centers. x 31,000. Inset: x 60,000. Fig. 6. Coated vesicles (arrows) joining a vacuole with an irregular profile, perhaps a food vacuole. Other coated vesicles and similar vesicles without coats are free in the cytoplasm. x 55,000. Fig. 7. Section through the periphery of the anterior end of the organism where apostome organelles (MO) are rejoining the cell membrane at the pores (P). A coated vesicle (CV) appears to be forming simultaneously at the pore to the right. x 55,000.
PARASITIC
CILIATE
571
TEREBROSPIRA
In transverse section the wall shows a faint periodicity (Figs. 2, 4) but sections cut tangential to the ciliate’s surface indicate that it is formed of close-packed short rods with electron-lucent centers and fuzzy dense walls (Fig. 5). In the few sections in which the cyst wall is not close to the cell membrane, the fibrous strands of a conspicuous cell coat are visible, extending from the cell membrane to the cyst wall, and similar fibrous strands extend from the wall to the dense coagulum lining the gallery (Fig. 2). Typically, ciliates have rather elaborate pellicles, but Terebrospiru’s surface is bounded only by a single unit membrane. At the advancing end, the anterior pole, the membrane is raised in an irregular pattern of elliptical or rectangular hummocks, greatly increasing the area of the membrane adjacent to the dissolving exoskeleton (Figs. 3, 5). The surface of the posterior half of the body is smoother with only random flattened invaginations (Fig. 4). A single subpellicular cisterna of smooth endoplasmic reticulum encloses the entire body, maintaining a constant position in relation to the cell membrane. The complexity of Terebrospiru’s surface visible in horizontal or transverse section is due to the rectangle or ellipse of SER enclosed within a hummock (Figs. 3, 5). The cell membrane and its underlying SER are interrupted by pores at which coated vesicles are formed (Figs. 3, 5, 7). (In ciliates, pellicular pores are specialized areas or invaginations of the surface where the cell membrane is presumably different.) At the organism’s anterior pole the pores occur close to one another, between adjacent hummocks; on the more flattened posterior surface they are spaced far apart. In some sections through the anterior pole of Terebrospira almost every pore ends in a coated vesicle, but coated vesicles apparently can form at any pore that is next to the exoskeleton. Coated vesicles can be observed at some pores along the sides of the organism, but the number of coated vesicles in the peripheral cytoplasm diminishes markedly toward the posterior of the ciliate. Apparently vesicles lose their coats soon after they leave the periphery of the organism, because a coated vesicle is rarely observed at a distance from the cell membrane. Other vesicles of the same size and
general appearance, but without coats may occur more centrally. A few sections seem to show fusion of the coated vesicles (and smooth-walled vesicles of the same size) with irregularly shaped bodies that are probably food vacuoles (Fig. 6). Some of these vacuoles appear almost empty except for dense fibrous material adhering to the inner surface of the membrane. Others may contain markedly greater amounts of the fibrous material. These vacuoles are always small, less than 0.5 p in their largest dimension. However, they are identical in visible structure to vacuoles more centrally located that may be several micra in diameter. Because of the irregular outlines of the large and small vacuoles, it has been impossible to determine if the larger vacuoles are formed by the fusion of smaller vacuoles. Also in the peripheral cytoplasm beneath and usually parallel to the proximal surface of the SER are unit-membrane-bounded lamellar structures with fuzzy interiors. These resemble organelles (membrane organelles) in other ciliates that are implicated in the recycling of membrane for food vacuoles (Allen, 1974, 1976; Bradbury, 1973, 1974; Howell and Paulin, 1976; Kloetzel, 1974; McKanna, 1973). Occasionally a section will show one of these lamellae in direct continuity with a pore (Figs. 2, 7), supporting the hypothesis that they are stored membranes that replenish the surface membrane, thereby replacing the membrane that is continuously being drawn into the interior during the formation and inward migration of the coated vesicles. By light microscopy the galleries in the endocuticle are smooth-walled tunnels with no melanization or differentiation of their walls. Electron microscopy adds little to this description except that the exoskeleton surrounding the lumen of the gallery is markedly denser and more coarsely fibrous. Dense fibrous ‘clots’ may appear between the cyst wall and the endocuticle, and in the rare instances when a space can be distinguished between the cyst wall and the exoskeleton the space is traversed by fine filaments (Figs. 2, 4). Acid phosphatase
Extracellular deposits of lead reaction product appear strewn through the laminae of the shrimp’s cuticle around and in front of the
*
Fig. IO. Lead deposits x 24,000. Fig. I I. x 36,000.
on the membranes
of vacuoles
lhat may be food vacuoles.
Two dense bodies (Ly) that may be lysosomes
Fig. 12. Scattered lead granules treated with Na-/3-glycerophosphate.
near the cell membrane.
in food vacuoles in an organism that was not Section stained only with uranyl acetate. x 32,000.
Fig. 8. Acid phosphatase reaction products at the cell membrane of the hummocks at the anterior end of Terebrospira. Copious amounts of extracellular reaction product are visible in the exoskeleton at the right. x 19,000. Fig. 9. (EP) is at lower left not in the of ‘empty’
Cross-section through the posterior region of Terebrospira. The epicuticle the upper right corner and shreds of shrimp hypodermis are visible at the corner. Extracellular lead deposits can be observed next to the ciliate but exoskeleton near the hypodermis. Lead is also deposited on the membranes vacuoles that may be food vacuoles. x 11,500.
BRADBURY
ciliate (Figs. 8, 9), but never in uninfected parts of the cuticle and never in the control. No reaction product is ever seen on the cyst wall or in the space between the wall and the organism. Some sections include cells of the shrimp hypodermis, and there is no reaction product in the endocuticle adjacent to them (Fig. 9). Intracellular sites of deposition of lead in Terebrospira are in small dense bodies that are probably lysosomes (Fig. 1l), and around the periphery of vacuoles tentatively identified as food vacuoles (Figs. 9, 10) formed by an accumulation and fusion of coated vesicles. Conspicuous lead deposits may occur on restricted areas of the cell membrane, most commonly on the hummocks at the anterior end of the organism (Fig. 8), but some organisms have few surface-membrane-associated deposits and little reaction product within the cell. This failure to react strongly may be at least partly due to the problems of treating with reagents an organism that is embedded in exoskeleton. The pieces of exoskeleton incubated without substrate showed no deposition of reaction product outside the cell or on the cell membrane, but there was a deposition of fine granules inside the periphery of the putative food vacuoles (Fig. 12). Discussion Although Terebrospira belongs to a subphylum of protozoa in which complex rather tough pellicles are the rule, this ciliate is covered only by a simple unit membrane whose only modification is the pores. The surface of the membrane is covered by a conspicuous fibrous cell coat like the surface of free-living amoebae or the cells of absorptive epithelia of vertebrates. It is, however, remarkable that this fibrous surface is separated from the substrate that the ciliate acts upon by a cyst wall, a non-living structure secreted by the organism itself. Few ciliate cysts have been carefully studied, but Terebrospiru’s cyst wall is the thinnest of any yet described. Encystation is for protection against an unfavourable environment or, more rarely, for reproduction. Other ciliates do not feed while encysted. Terebrospira is unique in that it actively feeds and grows while enclosed in a cyst, The cyst wall material is obviously not impermeable
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to complex substances. There must be a twoway traffic through it, enzymes to degrade the endocuticle passing outward and the soluble products of degradation passing into the cell membrane. Visibly the cyst wall resembles somewhat the basal lamina of the mid-gut epithelium of the mosquito, another site of active exchange (Terzakis, 1967), but its function may be more like that of the peritrophic membrane of insects, protection of a delicate absorbing surface. Fibrous cell coats are involved in the recognition and binding of the molecular inducers of pinocytosis (Chapman-Andresen, 1972). In ciliates the only conspicuous fibrous coat is on the single unit membrane covering the cytostome, a gap in the complex pellicle at which food vacuoles are formed. The fibrous coat on the cytostomal membrane may recognize molecules that stimulate food vacuole formation. Terebrospira has no cytostome, but the membrane over its entire surface resembles (except for the pores) the cytostomal membranes of other ciliates. In many Apostomatida, the exuvial fluid of their crustacean hosts is the only inducer that stimulates the formation of food vacuoles (Bradbury, 1974). The surface of Terebrospira may be responding to degradation products similar to exuvial fluid but produced by its own enzymes rather than the enzymes of its host. Although most ciliates have a row of pellicular pores (parasomal sacs) along each ciliary meridian, Terebrospirn’s simple indentations in the surface membrane occur in much greater numbers and have no obvious relationship to its few straight rows of stubby cilia. Pores seem to be regularly spaced relative to one another all over the surface of the ciliate. At the anterior of the organism the hummocks between the pores bring the pores closer to one another. The hummocks may serve not only to increase membrane area, but to bring a greater number of pores into the region of active dissolution of the exoskeleton. The interpretation that the coated vesicles form rather than discharge at the cell membrane is supported by the fact that no structure in the interior of the cell seems to form them. Coated vesicles are organelles specialized for protein transport within the cell (e.g. transport of enzymes from the Golgi (Bruni and Porter.
1965) or protein
uptake
by the
PARASITIC
CILIATE
TEREBROSPIRA
cell (Roth and Porter, 1964)). Studies of insect molting provide numerous examples of coated vesicles forming at the surface of hypodermal cells and bringing in complex substances (Delachambre, 1970; Locke, 1969a, b). Pre-ecdysial arthropods reabsorb the endocuticle that is digested by exuvial fluid. A recent study shows that peroxidase, injected through the old cuticle into the exuvial space, is taken up in coated vesicles by the hypodermal cells of an insect (Delachambre, 1970). The coated vesicles lose their coats and eventually join larger bodies containing acid phosphatase and resembling the putative food vacuoles in Terebrospira. The process of endocuticular resorption by hypodermal cells would be similar to our interpretation of the path of nutrients in Terebrospira in which coated vesicles form at the pores and carry dissolved exoskeleton into the cytoplasm for further digestion. Membrane organelles also are more common in the anterior of the organism. In many sections membrane organelles may be observed in direct continuity with the surface, apparently opening up to expose their fibrous interiors. Structures resembling membrane organelles have recently been observed at sites where food vacuole formation or collapse is occurring (Allen, 1974; Allen and Wolf, 1974; Bradbury, 1973, 1974; Howell and Paulin, 1976; Kloetzel, 1974; McKanna, 1973). Other protozoan and metazoan cells form similar organelles for membrane storage under circumstances where surface area is rapidly decreasing (e.g. retraction of pseudopods (Bardele, 1972) collapse of the urinary bladder (Porter et al., 1967). In ciliates related to Terebrospira, membrane organelles form directly from condensing food vacuoles (Bradbury, 1973). Probably they form the same way in Terebrospira. Perhaps the irregular outline of the supposed food vacuoles may be explained by a condensation of the food material, but as yet no unequivocal intermediate stages in the formation of food vacuoles from coated vesicles have been observed. Like the coated vesicles, membrane organelles occur only at the periphery of the organism. The amounts rejoining the surface at a particular site must be regulated because
581
silver-impregnated whole amounts of the ciliate show that its meridional ciliary rows maintain a constant relationship to one another (Bradbury, et al., 1974). If large amounts of membrane rejoined the surface at random or in a single region, the rows of cilia would zig-zag or be displaced relative to one another. In experiments to demonstrate acid phosphatase by Gomori reactions, the enzyme appears in its expected locations in the lysosomes and the food vacuoles. The appearance of reaction product outside the cell and on the cell membrane was unusual but not without precedent. Some metazoan cells, dendritic reticulum cells in the human tonsil (Stephen and Blumke, 1971) epithelia of the mouse kidney (Sasaki and Fishman, 1973), primate uterine cells (Smith, 1969), and the digestive gland of the slug (Bowen and Davis, 1971), may have acid phosphatase associated with their cell membranes, but its function there is usually unclear. D’Day has demonstrated extracellular release of lysosomal enzymes including acid phosphatase, from amoebae of a cellular slime mold (O’Day, 1973). He suggests that this release is a common event and may be due to defecation of food vacuoles or autophagic vacuoles, or it may be for the purpose of digesting large molecules in the medium. Since Terebrospira is digesting a tunnel through a solid food source, the exoskeleton, the discovery of extracellular enzyme and enzyme bound to the cell membrane was not unexpected. In all probability, other enzymes (chitinases, proteases) for the digestion of this complex substrate will be found in the same location. Terebrospira promises to be a rewarding subject for work on the cytochemical digestion of the crustacean exoskeleton. Acknowledgements
This work was partially sponsored by Office of Sea Grant, N.O.A.A., U.S. Dept. of Commerce, under Grant No. 04-3-158-40, and the State of North Carolina, Department of Administration. The U.S. Government is authorized to produce and distribute reprints for governmental purposes notwithstanding any copyright that may appear hereon.
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References
ALLEN, R. D. 1974. Food vacuole membrane growth with microtubule-associated membrane transport in Paramecium. J. Cell Biol., 63, 904-922. ALLEN, R. D. 1976. Freeze fracture evidence for intramembrane changes accompanying membrane recycling in Paremecium. Cytobiologie, 12, 254-273. ALLEN, R. D. and WOLF, R. W. 1974. The cytoproct of Paramecium caudatum: structure and function, microtubules and fate of food vacuole membranes. J. Cell Sci., 14, 61 l-631. BARDELE, C. F. 1972. Cell cycle, morphogenesis, and ultrastructure in the pseudoheliozoan Clathrulina elegans. Z. Zellforsch. mikrosk. Anat., 130, 219-242. BOWEN, I. D. and DAVIS, P. 1971. The fine structural distribution of acid phosphatase in the digestive gland of Arion hortensis (Fer.) Protoplasma, 73, 73-81. BRADBURY,P. C. 1973. The fine structure of the cytostome of the apostomatous ciliate, Hyalophysa chaftoni. J. Protozool., 20,405-414. BRADBURY, P. C. 1974. Stored membranes associated with feeding in apostome trophonts with different diets. Protistologica, 10, 533-542. BRADBURY, P. C., CLAMP, J. C. and LYON, J. T., III. 1974. Terebrospira chattoni, sp.n., a parasite of the shrimp Palaemonetes pugio Holthuis. J. Protorool., 21, 678-686. BRUNI, C. and PORTER, K. R. 1965. The fine structure of the parenchymal cell of the normal rat liver. Am. J. Path., 46, 691-755. CHAPMAN-ANDRESEN,C. 1972. Membrane activity in freshwater amoebae. J. Protozool., 19, 225-231. DELACHAMBRE,J. 1970. Etudes sur l’epicuticule des insectes. II. Modifications de l’epiderme au tours de la secretion de l’epicuticule imaginale chez Tenebrio molitor L. Z. Zellforsch. mikrosk. Anat., 112, 97-l 19. HOWELL, E. F. and PAULIN, J. J. 1976. Membrane-bounded disks in Stentor coeruleus. J. Protozool., 23, 1lA. KLOETZEL, J. A. 1974. Feeding in ciliated protozoa. I. Pharyngeal disks in Euplotes: a source of membrane for food vacuole formation? J. Cell Sci., 15, 379401. LOCKE, M. 1969a. The structure of an epidermal cell during the development of the protein epicuticle and the uptake of molting fluid in an insect. J. Morph., 127, 7-40. LOCKE, M. 1969b. The localization of a peroxidase associated with hard cuticle formation in an insect, Calgodes ethlius Stoll, Lepidoptera, Hesperiidae. Tissue and CelI, 1, 555-574. MCKANNA, J. A. 1973. Cyclic membrane flow in the ingestive-digestive system of peritrich protozoans. I. Vesicular fusion at the cytopharynx. J. Cell Sci., 13, 663-675. O’DAY, D. H. 1973. Intracellular localization and extracellular release of certain lyosomal enzyme activities from amoebae of the cellular slime mould Polysphondylium pallidurn. Cytobios., 7, 223-232. PORTER, K. R., KENYON, K. and BADENHAUSEN,S. 1967. Specializations of the unit membrane. Protoplasma, 63, 262-274. ROTH, T. F. and PORTER, K. R. 1964. Yolk protein uptake in the oocyte of the mosquito Aedes aegypti L. J. Cell Biol., 20, 3 13-332. SASAKI, M. and FISHMAN, W. 1973. Dual ultrastructural localization of acid phosphatase in mouse kidney tubule cells. J. Histochem. Cytochem., 21, 653-660. SMITH, R. E. 1969. Phosphohydrolases in cell organelles: electron microscopy. Arm. N. Y. Acad. Sci., 166, 525-563. STEPHEN, R. and BLUMKE, S. 1971. Electronmicroskopischer Nachweis der saueren Phosphatase in Keimzentren menschlicher Tonsillen. Z. ZeIlforsch. mikrosk. Anat., 115, 114-136. TERZAKIS, J. A. 1967. Substructure in an epithelial basal lamina (basement-membrane). J. Cell Biol., 35, 273-278. WISE, G. E. and FLICKINGER, C. J. 1970. Cytochemical staining of the Golgi apparatus in Amoeba proteus. J. Cell Biol., 46, 620-626.
PHYLLIS
CLARKE
THE FINE STRUCTURE CILIATE TEREBROSPIRA OF THE EXOSKELETON
BRADBURY
and VIBHA
GOYAL
OF A PARASITIC DURING INGESTION OF A SHRIMP
PALAEMONETES
ABSTRACT. The ciliated protozoan, Terebrospira chattoni, invades the exoskeleton of the shrimp, Palaemoneres pugio, eating out long galleries parallel to the surface of the exoskeleton. Solubilization of the exoskeleton occurs around an area of the elaborately infolded surface membrane at the anterior of the organism. Dissolved products of the digestion of the exoskeleton are taken into the body by the formation of coated vesicles at pores in the membrane. The surface membrane that is taken in by pinocytosis is apparently recycled by the introduction into the membrane of organelles implicated in mambrane recycling in other ciliates. Acid phosphatase can be demonstrated on the surface membrane as well as in the endocuticle around the organism.
stage of Terebrispira confirms the lack of a cytostome or other oral structures and the absence of organelles for boring and triturating. The solid exoskeleton must be solubilized by extracellular enzymes. Terebrospira has been examined by electron microscopy to learn how it feeds, and experiments were begun to demonstrate enzymatic activity at the advancing face of the gallery.
Intmduction THE crustacean exoskeleton is a rich store of carbohydrates, protein, and lipid, that is impervious to the digestive enzymes of most organisms. In the animal kingdom only a few ciliated protozoa, belonging to the order Apostomatida, are able to feed solely on the exoskeleton or its degradation products. One of these, T’erebrospira chattoni, forms a division cyst on the surface of the brackish water shrimp, Palaemonetes pugio, and divides into two to eight daughters. (Bradbury et al., 1974). Each daughter cell dissolves a hole in the floor of the parent cyst and abandons the cyst to eat long galleries in the shrimp’s endocuticle. The galleries radiate from the empty parent cyst like the points of a star (Fig. 1) and in heavy infections the shrimp exoskeleton resembles lace or lattice-work with galleries running in all directions through the endocuticle. Each gallery contains a daughter ciliate closely appressed to the advancing face of the gallery. Electron microscopy of this feeding Department of Zoology, North Carolina University, Raleigh, North Carolina 27607. Revised 29 August 1976. Received 30 July 1976. 37
State
Materials and Methods Palaemonetes pugio (Holthuis) was collected
by dip net from South Creek at the Pamlico Marine Station in Aurora, N.C. Shrimp heavily infected with Terebrospira could be easily identified at low magnifications by the star-shaped patterns formed in the exoskeleton by the galleries. Shrimp were bisected at the junction of the carapace and the abdomen; infected fragments of exoskeleton were pulled off with forceps and immersed in 2.8% glutaraldehyde in 0.1 M cacodylate (pH 7.3). Adhering shreds of flesh were picked away by fine forceps, and the cleaned fragments were placed in fresh fixative for 1 h at room temperature, washed in buffered 6% sucrose, postfixed for 2 hr in 2% 0~04 in 0.1 M Na cacodylate, dehydrated in ethanol 573
BRADBURY
and embedded in Maraglas. Thin sections were cut with a diamond knife mounted on an LKB Ultrotome 8800 and picked up on grids covered with carbon-stabilized Parlodion films. The sections were stained with uranyl acetate and lead citrate and examined and photographed in a Siemens 1A electron microscope operating at 80 kV. To demonstrate acid phosphatase (Wise and Flickinger, 1970), fragments of infected exoskeleton were fixed for 3 hr at room temperature in 2% glutaraldehyde in O-l M Na cacodylate (pH 7.3) and then washed 6 x in 7 % buffered sucrose. The fragments were incubated for 20 min at room temperature in the following medium: 5.0 ml 1.25 % (w/v) Na-,%glycerophosphate, 5.0 ml 0.2 Trismaleate buffer (pH S.O), IO.0 ml 0.2% (w/v) Pb(NO&, and 5.0 ml distilled water. After incubation the fragments were washed 3 x in distilled water, once in 1 % acetic acid, and once again in distilled water. As a control, fragments of the same exoskeleton were incubated under the same con-
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ditions except that the medium did not contain the Na-8-glycerophosphate. Sections from the experimentally treated material were left unstained. Results When observed on living shrimp, Terebrospiru in its gallery is about 14-2 times longer than t is broad (60 x 45 p) but sections show that it is dorsoventrally flattened and only a few micra thick. The course of its gallery follows three to five laminae of the endocuticle, deviating neither upward nor downward. The ciliate always occupies the advancing face of the gallery, being snugly enclosed by exoskeleton on all sides except the rear where the lumen of the gallery extends back, sometimes as much as 1 mm to the empty parent cyst. Electron microscopy reveals the unsuspected presence of a very thin cyst wall around the feeding organism, thinner than any previously reported in ciliates (Fig. 2).
Fig. 1. Fragment of exoskeleton from a uropod of Palaemonetes pugio showing the star-shaped patterns formed by the exodus of daughter cells from the reproductive cysts of Terebrospira chattoni. The compartmented reproductive cyst can be observed at the center of each star. The galleries form the rays of the stars and the dark body at the distal end of each ray is the ciliate. x 24. Fig. 2. Section through the cyst wall, cell membrane, and peripheral cytoplasm in the postero-lateral region of Terebrospira. The cyst wall shows a faint periodicity (arrows). Fibrous strands extend from the cyst wall to the exoskeleton (E) and to the cell membrane. At the cell membrane a membrane organelle (MO) is rejoining the membrane at a pore (P). Another membrane organelle is free in the cytoplasm. x 40,000. Fig, 3. Longitudinal section through the anterior end of Terebrospira. The conspicuous hummocks formed by infoldings of the surface membrane enclose an ellipse of smooth endoplasmic reticulum (SER). Coated vesicles (CV) are visible at the pores (P) and also free in the cytoplasm. x 31,000. Fig. 4. Longitudinal section illustrating surface of Terebrospira.
the relative simplicity of the postero-lateral
Fig. 5. Section tangential to part of the surface of the anterior region of Terebrospira. Coagulum (C) of degraded endocuticie (E) surrounds the cyst wall (W) which shows subunits with electron-lucent centers. x 31,000. Inset: x 60,000. Fig. 6. Coated vesicles (arrows) joining a vacuole with an irregular profile, perhaps a food vacuole. Other coated vesicles and similar vesicles without coats are free in the cytoplasm. x 55,000. Fig. 7. Section through the periphery of the anterior end of the organism where apostome organelles (MO) are rejoining the cell membrane at the pores (P). A coated vesicle (CV) appears to be forming simultaneously at the pore to the right. x 55,000.
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571
TEREBROSPIRA
In transverse section the wall shows a faint periodicity (Figs. 2, 4) but sections cut tangential to the ciliate’s surface indicate that it is formed of close-packed short rods with electron-lucent centers and fuzzy dense walls (Fig. 5). In the few sections in which the cyst wall is not close to the cell membrane, the fibrous strands of a conspicuous cell coat are visible, extending from the cell membrane to the cyst wall, and similar fibrous strands extend from the wall to the dense coagulum lining the gallery (Fig. 2). Typically, ciliates have rather elaborate pellicles, but Terebrospiru’s surface is bounded only by a single unit membrane. At the advancing end, the anterior pole, the membrane is raised in an irregular pattern of elliptical or rectangular hummocks, greatly increasing the area of the membrane adjacent to the dissolving exoskeleton (Figs. 3, 5). The surface of the posterior half of the body is smoother with only random flattened invaginations (Fig. 4). A single subpellicular cisterna of smooth endoplasmic reticulum encloses the entire body, maintaining a constant position in relation to the cell membrane. The complexity of Terebrospiru’s surface visible in horizontal or transverse section is due to the rectangle or ellipse of SER enclosed within a hummock (Figs. 3, 5). The cell membrane and its underlying SER are interrupted by pores at which coated vesicles are formed (Figs. 3, 5, 7). (In ciliates, pellicular pores are specialized areas or invaginations of the surface where the cell membrane is presumably different.) At the organism’s anterior pole the pores occur close to one another, between adjacent hummocks; on the more flattened posterior surface they are spaced far apart. In some sections through the anterior pole of Terebrospira almost every pore ends in a coated vesicle, but coated vesicles apparently can form at any pore that is next to the exoskeleton. Coated vesicles can be observed at some pores along the sides of the organism, but the number of coated vesicles in the peripheral cytoplasm diminishes markedly toward the posterior of the ciliate. Apparently vesicles lose their coats soon after they leave the periphery of the organism, because a coated vesicle is rarely observed at a distance from the cell membrane. Other vesicles of the same size and
general appearance, but without coats may occur more centrally. A few sections seem to show fusion of the coated vesicles (and smooth-walled vesicles of the same size) with irregularly shaped bodies that are probably food vacuoles (Fig. 6). Some of these vacuoles appear almost empty except for dense fibrous material adhering to the inner surface of the membrane. Others may contain markedly greater amounts of the fibrous material. These vacuoles are always small, less than 0.5 p in their largest dimension. However, they are identical in visible structure to vacuoles more centrally located that may be several micra in diameter. Because of the irregular outlines of the large and small vacuoles, it has been impossible to determine if the larger vacuoles are formed by the fusion of smaller vacuoles. Also in the peripheral cytoplasm beneath and usually parallel to the proximal surface of the SER are unit-membrane-bounded lamellar structures with fuzzy interiors. These resemble organelles (membrane organelles) in other ciliates that are implicated in the recycling of membrane for food vacuoles (Allen, 1974, 1976; Bradbury, 1973, 1974; Howell and Paulin, 1976; Kloetzel, 1974; McKanna, 1973). Occasionally a section will show one of these lamellae in direct continuity with a pore (Figs. 2, 7), supporting the hypothesis that they are stored membranes that replenish the surface membrane, thereby replacing the membrane that is continuously being drawn into the interior during the formation and inward migration of the coated vesicles. By light microscopy the galleries in the endocuticle are smooth-walled tunnels with no melanization or differentiation of their walls. Electron microscopy adds little to this description except that the exoskeleton surrounding the lumen of the gallery is markedly denser and more coarsely fibrous. Dense fibrous ‘clots’ may appear between the cyst wall and the endocuticle, and in the rare instances when a space can be distinguished between the cyst wall and the exoskeleton the space is traversed by fine filaments (Figs. 2, 4). Acid phosphatase
Extracellular deposits of lead reaction product appear strewn through the laminae of the shrimp’s cuticle around and in front of the
*
Fig. IO. Lead deposits x 24,000. Fig. I I. x 36,000.
on the membranes
of vacuoles
lhat may be food vacuoles.
Two dense bodies (Ly) that may be lysosomes
Fig. 12. Scattered lead granules treated with Na-/3-glycerophosphate.
near the cell membrane.
in food vacuoles in an organism that was not Section stained only with uranyl acetate. x 32,000.
Fig. 8. Acid phosphatase reaction products at the cell membrane of the hummocks at the anterior end of Terebrospira. Copious amounts of extracellular reaction product are visible in the exoskeleton at the right. x 19,000. Fig. 9. (EP) is at lower left not in the of ‘empty’
Cross-section through the posterior region of Terebrospira. The epicuticle the upper right corner and shreds of shrimp hypodermis are visible at the corner. Extracellular lead deposits can be observed next to the ciliate but exoskeleton near the hypodermis. Lead is also deposited on the membranes vacuoles that may be food vacuoles. x 11,500.
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ciliate (Figs. 8, 9), but never in uninfected parts of the cuticle and never in the control. No reaction product is ever seen on the cyst wall or in the space between the wall and the organism. Some sections include cells of the shrimp hypodermis, and there is no reaction product in the endocuticle adjacent to them (Fig. 9). Intracellular sites of deposition of lead in Terebrospira are in small dense bodies that are probably lysosomes (Fig. 1l), and around the periphery of vacuoles tentatively identified as food vacuoles (Figs. 9, 10) formed by an accumulation and fusion of coated vesicles. Conspicuous lead deposits may occur on restricted areas of the cell membrane, most commonly on the hummocks at the anterior end of the organism (Fig. 8), but some organisms have few surface-membrane-associated deposits and little reaction product within the cell. This failure to react strongly may be at least partly due to the problems of treating with reagents an organism that is embedded in exoskeleton. The pieces of exoskeleton incubated without substrate showed no deposition of reaction product outside the cell or on the cell membrane, but there was a deposition of fine granules inside the periphery of the putative food vacuoles (Fig. 12). Discussion Although Terebrospira belongs to a subphylum of protozoa in which complex rather tough pellicles are the rule, this ciliate is covered only by a simple unit membrane whose only modification is the pores. The surface of the membrane is covered by a conspicuous fibrous cell coat like the surface of free-living amoebae or the cells of absorptive epithelia of vertebrates. It is, however, remarkable that this fibrous surface is separated from the substrate that the ciliate acts upon by a cyst wall, a non-living structure secreted by the organism itself. Few ciliate cysts have been carefully studied, but Terebrospiru’s cyst wall is the thinnest of any yet described. Encystation is for protection against an unfavourable environment or, more rarely, for reproduction. Other ciliates do not feed while encysted. Terebrospira is unique in that it actively feeds and grows while enclosed in a cyst, The cyst wall material is obviously not impermeable
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to complex substances. There must be a twoway traffic through it, enzymes to degrade the endocuticle passing outward and the soluble products of degradation passing into the cell membrane. Visibly the cyst wall resembles somewhat the basal lamina of the mid-gut epithelium of the mosquito, another site of active exchange (Terzakis, 1967), but its function may be more like that of the peritrophic membrane of insects, protection of a delicate absorbing surface. Fibrous cell coats are involved in the recognition and binding of the molecular inducers of pinocytosis (Chapman-Andresen, 1972). In ciliates the only conspicuous fibrous coat is on the single unit membrane covering the cytostome, a gap in the complex pellicle at which food vacuoles are formed. The fibrous coat on the cytostomal membrane may recognize molecules that stimulate food vacuole formation. Terebrospira has no cytostome, but the membrane over its entire surface resembles (except for the pores) the cytostomal membranes of other ciliates. In many Apostomatida, the exuvial fluid of their crustacean hosts is the only inducer that stimulates the formation of food vacuoles (Bradbury, 1974). The surface of Terebrospira may be responding to degradation products similar to exuvial fluid but produced by its own enzymes rather than the enzymes of its host. Although most ciliates have a row of pellicular pores (parasomal sacs) along each ciliary meridian, Terebrospirn’s simple indentations in the surface membrane occur in much greater numbers and have no obvious relationship to its few straight rows of stubby cilia. Pores seem to be regularly spaced relative to one another all over the surface of the ciliate. At the anterior of the organism the hummocks between the pores bring the pores closer to one another. The hummocks may serve not only to increase membrane area, but to bring a greater number of pores into the region of active dissolution of the exoskeleton. The interpretation that the coated vesicles form rather than discharge at the cell membrane is supported by the fact that no structure in the interior of the cell seems to form them. Coated vesicles are organelles specialized for protein transport within the cell (e.g. transport of enzymes from the Golgi (Bruni and Porter.
1965) or protein
uptake
by the
PARASITIC
CILIATE
TEREBROSPIRA
cell (Roth and Porter, 1964)). Studies of insect molting provide numerous examples of coated vesicles forming at the surface of hypodermal cells and bringing in complex substances (Delachambre, 1970; Locke, 1969a, b). Pre-ecdysial arthropods reabsorb the endocuticle that is digested by exuvial fluid. A recent study shows that peroxidase, injected through the old cuticle into the exuvial space, is taken up in coated vesicles by the hypodermal cells of an insect (Delachambre, 1970). The coated vesicles lose their coats and eventually join larger bodies containing acid phosphatase and resembling the putative food vacuoles in Terebrospira. The process of endocuticular resorption by hypodermal cells would be similar to our interpretation of the path of nutrients in Terebrospira in which coated vesicles form at the pores and carry dissolved exoskeleton into the cytoplasm for further digestion. Membrane organelles also are more common in the anterior of the organism. In many sections membrane organelles may be observed in direct continuity with the surface, apparently opening up to expose their fibrous interiors. Structures resembling membrane organelles have recently been observed at sites where food vacuole formation or collapse is occurring (Allen, 1974; Allen and Wolf, 1974; Bradbury, 1973, 1974; Howell and Paulin, 1976; Kloetzel, 1974; McKanna, 1973). Other protozoan and metazoan cells form similar organelles for membrane storage under circumstances where surface area is rapidly decreasing (e.g. retraction of pseudopods (Bardele, 1972) collapse of the urinary bladder (Porter et al., 1967). In ciliates related to Terebrospira, membrane organelles form directly from condensing food vacuoles (Bradbury, 1973). Probably they form the same way in Terebrospira. Perhaps the irregular outline of the supposed food vacuoles may be explained by a condensation of the food material, but as yet no unequivocal intermediate stages in the formation of food vacuoles from coated vesicles have been observed. Like the coated vesicles, membrane organelles occur only at the periphery of the organism. The amounts rejoining the surface at a particular site must be regulated because
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silver-impregnated whole amounts of the ciliate show that its meridional ciliary rows maintain a constant relationship to one another (Bradbury, et al., 1974). If large amounts of membrane rejoined the surface at random or in a single region, the rows of cilia would zig-zag or be displaced relative to one another. In experiments to demonstrate acid phosphatase by Gomori reactions, the enzyme appears in its expected locations in the lysosomes and the food vacuoles. The appearance of reaction product outside the cell and on the cell membrane was unusual but not without precedent. Some metazoan cells, dendritic reticulum cells in the human tonsil (Stephen and Blumke, 1971) epithelia of the mouse kidney (Sasaki and Fishman, 1973), primate uterine cells (Smith, 1969), and the digestive gland of the slug (Bowen and Davis, 1971), may have acid phosphatase associated with their cell membranes, but its function there is usually unclear. D’Day has demonstrated extracellular release of lysosomal enzymes including acid phosphatase, from amoebae of a cellular slime mold (O’Day, 1973). He suggests that this release is a common event and may be due to defecation of food vacuoles or autophagic vacuoles, or it may be for the purpose of digesting large molecules in the medium. Since Terebrospira is digesting a tunnel through a solid food source, the exoskeleton, the discovery of extracellular enzyme and enzyme bound to the cell membrane was not unexpected. In all probability, other enzymes (chitinases, proteases) for the digestion of this complex substrate will be found in the same location. Terebrospira promises to be a rewarding subject for work on the cytochemical digestion of the crustacean exoskeleton. Acknowledgements
This work was partially sponsored by Office of Sea Grant, N.O.A.A., U.S. Dept. of Commerce, under Grant No. 04-3-158-40, and the State of North Carolina, Department of Administration. The U.S. Government is authorized to produce and distribute reprints for governmental purposes notwithstanding any copyright that may appear hereon.
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References
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