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The fine structure of the granular amebocytes of the Pacific oyster, Crassostrea gigas
JOURNAL
OF
INVERTEBRATE
The
PBTHOLOGY
Fine
18,
Structure
of the
269-275
(1971)
of the
Pacific
Oyster,
Granular
Amebocytes
Crassostrea
gigas’
CRAIG L. R~DDELL Department
of Pathology,
School
of Medicine,
Tlniversily Received
of California
April
at Davis,
Davis,
California
96616
6, 1971
Oyster granular amebocytes were characterized by electron microscopy to be of two types. Acidophiles contained relatively small, spherical, amorphous, osmiophilic cytoplasmic granules with a maximum dimension of 0.4-0.56 p. Basophiles contained large, cytoplasmic granules, approximately 0.7-1.2 p in diameter. The granules resembled hollow, spherical structures in which an outer membrane and a layer of closely associated granular material enclosed an electron-transparent interior.
INTRODUCTION
The work presented below is the second in a series of four papers directed t)oward the elucidation of the nature and funct,ion of oyster amebocytes (also see Ruddell, 1971) through hist,ochemical and ultrastJructural st,udies of normal and traumatized oyster &sues. In t)his report) is presented t,he fine st,ruct’ure of the granulocytes in t,he Pacific oyst,er, Crassostrea gigas. A brief review of the biology of oyster granular amebocytes based on my \vork with the Pacific oyst,er, Crassostrea gigas, (Ruddell, 1969, 1971) will also be presented herein. A Brief Review of the Biology Grarrular Amebocytes
of the Oyster
On t,he basis of a number of histological and histochemical staining reactions (Ruddell, 1969, 1971), tsvo t,ypes of oyster granular amebocytes, an acidophilic and basophilic granular amebocyte, Ivere recognized in plast)ic and paraffin sect,ions.Wit,11 bhe light microscope, the granular amebocyte-t,ypes were charact,erized by a cartwheel nucleus reminiscent of patterns displayed by mam1 This work was supported in part by USPHS contract 5 to 1 ESOD038-02; Health Sciences Advancement Award Number RR06138; Epidermal Papillomas in Pleuronectid Fishes, NIH CA 11618; and Tumor Biology Training Grant, NIH CA
05245. 269
malian plasma cells. Nucleoli in granular amebocytes were small or absent. Nuclei measured 3-5 p in diameter and whole cells, 5-S P in diameter. These observations were in accord with accounts of t,he morphology of granular cells given by Takamuki (1934) and Galt,soff (1964). The acidophilic granular cells, or acidophiles, contained small (< 0.5 p in diameter) cytoplasmic granules which were acidophilic and copper-positive and which reacted Mith diazotized p-nitroaniline to yield a red pigment (Ruddell, 1971). The basophiles contained large, basophilic cytoplasmic granules, approximately 1 p in diameter, which were zinc positive (Ruddell, 1971). The granular amebocytes were never observed undergoing mitosis; replication was confined t,o the agranular amebocytes, which were often observed dividing (Ruddell, 1969). In wounds, “immature” basophiles were commonly observed. These cells contained very small, basophilic granules and seemedto have differentiated from an agranular amebocyte precursor alt)hough this point, remains t)o be confirmed. In cross sections of t,he oyster mantle (Ruddell, 1969), the basophiles were found t,o out,number the acidophiles by at, least 3 : 1. The granular amebocytes were very common in the oyster mantle, and as many as 56 SOof
270
RUDDELL
all nuclei in a given field in cross sections of oyster mantle were granular amebocyt’es. The granulocytes were almost never observed phagocytosing particulate material (Ruddell, 1969). Carmine, carbon part,icles, and bact,eria inject,ed in seawater suspensions into oyster mantles were phagocytosed exclusively by the agranular amebocytes. The granular amebocytes responded to trauma in a singular and complex fashion (Ruddell, 1969). Chemical and physical injury to the mantle of an oyster initially resulted in (1) the release of copper and matrix from acidophilic granular amebocytes and (2) the swelling and possible rupture of basophilic granular amebocytes. In the later phase of the oyster’s response to trauma, copper and matrix released from t’he acidophilic granular amebocytes percolated throughout the traumatized area; copper was bound by all cells in the traumatized area but appeared to be selectively absorbed by the granules of the basophilic granular amebocytes, w-hereupon the granules of these amebocytes become “stabilized.” Copperstabilized basophiles were insensitive to mild traumatic stimuli and were never found in the swollen state typical of basophiles found in newly traumatized areas. The copperladen basophiles could also be induced t’o migrate through the epithelia of oysters, whereupon t’hey collected in such vast numbers on the external surfaces of oysters so as to color portions of the oyster green. Bang (1961) noted that oyster granular amebocytes formed clots in vitro and that bacteria were immobilized by these clots. He speculat,ed that the clots may have rendered bacteria more susceptible to phagocytosis. Bang’s observation suggested that one or both of the oyst,er granular amebocytes may function in vitro to immobilize, and perhaps destroy, bacteria and other pat’hogens. This assumption is not unwarranted since copper, one of the substances released by granular amebocytes during trauma, has long been known as a potent ant’imet’abolite (see Bowen, 1966; Albert, 1968; Ruddell, 1969).
My own observations and those of Pauley and Sparks (1965) and DesVoigne and Sparks (1968) indicated t’hat greening in oysters was associated with trauma. It is a very real possibility that the green oyster may be the reflection of a massive response to trauma. Boyce and Herdman (1897) foreshadowed this conclusion. These authors examined green American oysters (Crassostrea virginica) which had been relaid in England and observed that the green areas on t’he oyster’s surface were characterized by large numbers of copper-bearing, granular, ameboid leukocytes. It was hypot,hesized that the green patches were associated with a disease process: “There are evidently several kinds of greenness in oysters and whereas some may be due to normal and healthy processes, others must be regarded as abnormal or diseased conditions. It is the latter, in our experience, that contains t,he copper.” METHODS
AND
MATERIALS
The Pacific oysters, Crassostrea gigas, employed in this study were taken from the oyster beds of the Western Oyster Company, Purdy, Washington, and the Johnson Oyster Company, Point Reyes, Inverness, California. Small portions of tissue were excised from the auricles, from regenerating mantle wounds (72,144, and 240 hr after wounding), and from “green” mantles. Tissues were fixed according to the following scheme: 2% acrolein in seawater, 2-3 min, followed by 4% glutaraldehyde in seawater, 3-5 hr, cacodylate buffer, overnight Tissues were dehydrated and embedded in Epon 812 according to Luft’s (1961) procedure. Thin sections of the Purdy oysters were cut on a Sorvall Porter-Blum MT-I ultramicrotome with glass knives and stained with lead cit,rate (Reynolds, 1963). The sect,ions were examined with a Model 2A RCA electron microscope. Tissues from oysters collected at Inverness were cut on a Sorvall Porter-Blum MT-2 ultramicrotome with a diamond knife, stained with alcoholic uranyl acetate and
FINE STRUCTURE
OF PACIFIC
lead cit,rat,e, and examined with a Model 801 A.E.I. electron microscope. RESULTS
Recognitiotl of Gradar Electron Microscope
Amebocytes in the
Identification of the acidophilic granular amebocytes in the electron microscope was made on the basis of the following criteria: (1) size-1 looked for small amebocyte-type cells filled with small granules; and (2) a simple hist’ochemical react’ion. It! was found
FIG.
1. Electron
micrograph
of an oyster acidophile
271
OYSTER AMEBOCYTES
that by hydrolyzing thick (1 p) Epon sections with 0.2% solutions of NaOCl buffered to pH 8.0 and subsequently treating t,hesesections with 0.1% I- in acid solution, the small, osmium-blackened granules of a small cell with a cartwheel nuclear chromatin pattern were rendered intensely acidophilic. It seemedvery likely that t,hesesamesmall cells with osmiophilic granules Tvere acidophiles. Once having identified the acidophiles in thick sections, it was an easy matter to find t,he same cells in ultrathin sections of the same mat’erial.
from a 240-hr-old
regenerating
wound.
X20,000.
272
RUDDELL
Identification of basophiles in ultrathin sections was predicated on the observat’ion that, under the proper circumstances, basephiles could be induced to migrate across epithelial borders (Ruddell, 1969, 1971). Ultrathin sections of epithelial borders known to contain large numbers of migrating basophiles were compared with epithelial borders from control oysters cont’aining very few basophiles. The basophiles then could be easily identified in ultrat.hin sections of any oyster tissue.
FIG.
2. Electron
micrograph
of an oyster
General
Morphology
Except that the granular amebocytes contained granules of differing appearance, the ultrast.ruct.ural morphology of the trio types of granular amebocytes was quit)e similar. Their nuclei were enclosed in a double membrane occasionally perforat,ed with pores. As observed with the light, microscope, chromatin was arranged in small, evenly dispersed clumps, and recognizable nucleoli were generally absent. The amebocytes lacked large,
basophile
from
the auricle.
X15,400.
FINE
STRUCTURE
OF
PACIFIC
organized arrays of endoplasmic reticulum and Golgi apparatus. Mitochondria were rather small, being 0.3-0.8 cc in length. Flattened or tubular mitochondria were often observed. Small, membrane-bound vesicles, 0.07-0.2 P in diameter, were abundant in the cytoplasm of granular amebocytes. Glycogen particles and rosettes were scattered t8hroughout the cytoplasm of the granular amebocytes. Cellular and nuclear dimensions of the granular amebocytes were found to be within the size range as determined under the light microscope (see Introduction). The Acidophilic (Acidophile) Acidophiles brane-bound,
Granular
Amebocyte
were characterized by memosmiophilic cytoplasmic gran-
OYSTER
273
AMEBOCYTES
ules, circular or ovoid in cross section, with a maximum dimension of 0.44.56 p (Fig. 1). The matrix of the granules was composed of an amorphous, finely granular material. The Basophilic (Basophile)
Granular
Amebocyte
Basophiles contained large granules 0.71.2 CLin diameter; the granules were round or oval in cross section and at low magnification resembled hollow, ball-like structures in which an outer membrane and a layer of closely associated granular material enclosed an electron transparent interior (Fig. 2). The membrane-associat’ed granular material was usually distributed around t,he interior of the granule in an asymmetric fashion. The amount of granular material associated with granule membranes was variable,
FIG. 3. Electron micrograph of two basophile granules ture of t,he membrane-associated granular matrix. Two coarse granular network of one of the granules. X75,000.
clearly indicating “intragranular”
the variability in the texgranules can be seen in the
274
FIG.
HIJDDELL
4.
generating
Electron micrograph weunds. X75,oBO.
of a basophile
granule typioal of basophiles from green mantles and re-
even among granules of the same diameter within the samecell. The substructure of the granular layer was also variable, and the appearance of the gram&r matrix ranged from an amorphous, finely granular material to a coarse, granular-fibrillar network (Fig. 3). Small, membrane-bound, “intergranular” granules, 320-350 8 in diameter, commonly were found associated with this coarse network. The distinct and consistent variation ob-
served in granular substructure may indicate t.hat basophile granules are functionally polarized. Basophiles from tissues removed from green portions of oysters or from healing wounds were often markedly different from controls in that, the mitochondria were often swollen and cell membranes disarrayed, the granuIes were slightly swoIlen, distorted, and often contained glycogen, granular material, thin fibrils of lesst,han 80 H diameter derived
FINE
STRUCTURE
OF
PACIFIC
from the membrane-associated granular layer, and the presence of “intragranular” granules (Fig. 4). DISCUSSION
The observations on the fine structure of oyster granular ameboeytes present’ed contributed little 00 the understanding of the way in which these unique cells responded to trauma. It became apparent that examination of excised portions of oyster tissues was not the best “ay to observe oyster granular amebocytes in operation, as it were, since it was difficult to identify, with any certainty, acidophiles releasing copper and associated granular matrix or basophiles swelling. In order to do this, it seemed probable that experiment,al designs predicated on time-sequence studies of oyster amebocytes maintained in vitro, in which granular amebocytes could be induced to undergo their characteristic response to trauma, would have to be considered. It was interesting to note that basophiles from green portions of the oyster mantle often gave the appearance of being disarrayed. As basophiles from green portions of oysters were found to bind large amounts of copper (Ruddell, 1969, 1971), the disarrayed appearance of basophiles from green areas may have been the morphological manifestaCon of a cell poisoned with copper.
I wish to thank Miss Grete Nilson for expert technical assistance and Mr. Bob Munn for help in the use of the electron microscope and for reviewing this manuscript. Appreciat,ion is gratefully extended to Dr. S. R. Wellings and Dr. A. K. Sparks for their support of this study and also to Dr. J. Luft, Miss P. Phelps, Mr. Davies-Williams,
OYSTER
AMEBOCYTES
and the personnel of the Western pany, Purdy, Washington, and the ter Company, Inverness, California, valuable assistance.
275 Oyster ComJohnson Oysfor their in-
REFERENCES ALBERT, A. 1968. “Selective T0xicit.y.” Methuen, London. BANG, F. B. 1961. Reaction to injury in the oyster (Crassostrea virginica). Biol. Bdl., 121, 5748. BOWEN, H. J. 196G. “Trace Elements in Biochemistry.” Academic Press, New York. BOYCE, R., AND HERDMAN, W. A. 1897. On a green leucocytosis in oysters associated with the presence of copper in the leucocytes. Proc. Roy. Sot. Lond., 62, 30-38. DESVOIGNE, D. M., AND SPARKS, -4. K. 1968. The process of wound healing in the Pacific oyster, Crassostrea gigas. J. Invertebr. Pathol., 12, 53-65. GALTSOFF, P. S. 1964. The American oyster, Crassostrea virginica. Gmelin Fish. Bull., Fish Wddlife Serv., 64, 1480. LUFT, J. H. 1961. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. cytoz., 9, 409-514. PAULEY, G. B., AND SPARKS, A. K. 1965. Preliminary observations on the acute inflammatory reaction in the Pacific oyster, Crassostrea gigas, (Thunberg). J. Znvertebr. Pathol., 7, 248-257. REYNOLDS, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in elect.ron microscopy. J. Cell. Biol., 17,208-213. RUDDELL, C. L. 1959. A cytological and histochemical study of wound repair in the Pacific oyster, Crassostrea gigas. Ph.D. Thesis, University of Washington, Seattle. RTJDDELL, C. L. 1971. Elucidation of the nature and function of the granular oyster amebocytes through histochemical studies of normal and traumatized oyster tissues. Histochemie, 26, 95-112. TAKATSUKI, S. 1934. On the nature and function of the amebocytes of Ostrea edztlis. Quart. J. Microsc. Sci., 76, 379436.
OF
INVERTEBRATE
The
PBTHOLOGY
Fine
18,
Structure
of the
269-275
(1971)
of the
Pacific
Oyster,
Granular
Amebocytes
Crassostrea
gigas’
CRAIG L. R~DDELL Department
of Pathology,
School
of Medicine,
Tlniversily Received
of California
April
at Davis,
Davis,
California
96616
6, 1971
Oyster granular amebocytes were characterized by electron microscopy to be of two types. Acidophiles contained relatively small, spherical, amorphous, osmiophilic cytoplasmic granules with a maximum dimension of 0.4-0.56 p. Basophiles contained large, cytoplasmic granules, approximately 0.7-1.2 p in diameter. The granules resembled hollow, spherical structures in which an outer membrane and a layer of closely associated granular material enclosed an electron-transparent interior.
INTRODUCTION
The work presented below is the second in a series of four papers directed t)oward the elucidation of the nature and funct,ion of oyster amebocytes (also see Ruddell, 1971) through hist,ochemical and ultrastJructural st,udies of normal and traumatized oyster &sues. In t)his report) is presented t,he fine st,ruct’ure of the granulocytes in t,he Pacific oyst,er, Crassostrea gigas. A brief review of the biology of oyster granular amebocytes based on my \vork with the Pacific oyst,er, Crassostrea gigas, (Ruddell, 1969, 1971) will also be presented herein. A Brief Review of the Biology Grarrular Amebocytes
of the Oyster
On t,he basis of a number of histological and histochemical staining reactions (Ruddell, 1969, 1971), tsvo t,ypes of oyster granular amebocytes, an acidophilic and basophilic granular amebocyte, Ivere recognized in plast)ic and paraffin sect,ions.Wit,11 bhe light microscope, the granular amebocyte-t,ypes were charact,erized by a cartwheel nucleus reminiscent of patterns displayed by mam1 This work was supported in part by USPHS contract 5 to 1 ESOD038-02; Health Sciences Advancement Award Number RR06138; Epidermal Papillomas in Pleuronectid Fishes, NIH CA 11618; and Tumor Biology Training Grant, NIH CA
05245. 269
malian plasma cells. Nucleoli in granular amebocytes were small or absent. Nuclei measured 3-5 p in diameter and whole cells, 5-S P in diameter. These observations were in accord with accounts of t,he morphology of granular cells given by Takamuki (1934) and Galt,soff (1964). The acidophilic granular cells, or acidophiles, contained small (< 0.5 p in diameter) cytoplasmic granules which were acidophilic and copper-positive and which reacted Mith diazotized p-nitroaniline to yield a red pigment (Ruddell, 1971). The basophiles contained large, basophilic cytoplasmic granules, approximately 1 p in diameter, which were zinc positive (Ruddell, 1971). The granular amebocytes were never observed undergoing mitosis; replication was confined t,o the agranular amebocytes, which were often observed dividing (Ruddell, 1969). In wounds, “immature” basophiles were commonly observed. These cells contained very small, basophilic granules and seemedto have differentiated from an agranular amebocyte precursor alt)hough this point, remains t)o be confirmed. In cross sections of t,he oyster mantle (Ruddell, 1969), the basophiles were found t,o out,number the acidophiles by at, least 3 : 1. The granular amebocytes were very common in the oyster mantle, and as many as 56 SOof
270
RUDDELL
all nuclei in a given field in cross sections of oyster mantle were granular amebocyt’es. The granulocytes were almost never observed phagocytosing particulate material (Ruddell, 1969). Carmine, carbon part,icles, and bact,eria inject,ed in seawater suspensions into oyster mantles were phagocytosed exclusively by the agranular amebocytes. The granular amebocytes responded to trauma in a singular and complex fashion (Ruddell, 1969). Chemical and physical injury to the mantle of an oyster initially resulted in (1) the release of copper and matrix from acidophilic granular amebocytes and (2) the swelling and possible rupture of basophilic granular amebocytes. In the later phase of the oyster’s response to trauma, copper and matrix released from t’he acidophilic granular amebocytes percolated throughout the traumatized area; copper was bound by all cells in the traumatized area but appeared to be selectively absorbed by the granules of the basophilic granular amebocytes, w-hereupon the granules of these amebocytes become “stabilized.” Copperstabilized basophiles were insensitive to mild traumatic stimuli and were never found in the swollen state typical of basophiles found in newly traumatized areas. The copperladen basophiles could also be induced t’o migrate through the epithelia of oysters, whereupon t’hey collected in such vast numbers on the external surfaces of oysters so as to color portions of the oyster green. Bang (1961) noted that oyster granular amebocytes formed clots in vitro and that bacteria were immobilized by these clots. He speculat,ed that the clots may have rendered bacteria more susceptible to phagocytosis. Bang’s observation suggested that one or both of the oyst,er granular amebocytes may function in vitro to immobilize, and perhaps destroy, bacteria and other pat’hogens. This assumption is not unwarranted since copper, one of the substances released by granular amebocytes during trauma, has long been known as a potent ant’imet’abolite (see Bowen, 1966; Albert, 1968; Ruddell, 1969).
My own observations and those of Pauley and Sparks (1965) and DesVoigne and Sparks (1968) indicated t’hat greening in oysters was associated with trauma. It is a very real possibility that the green oyster may be the reflection of a massive response to trauma. Boyce and Herdman (1897) foreshadowed this conclusion. These authors examined green American oysters (Crassostrea virginica) which had been relaid in England and observed that the green areas on t’he oyster’s surface were characterized by large numbers of copper-bearing, granular, ameboid leukocytes. It was hypot,hesized that the green patches were associated with a disease process: “There are evidently several kinds of greenness in oysters and whereas some may be due to normal and healthy processes, others must be regarded as abnormal or diseased conditions. It is the latter, in our experience, that contains t,he copper.” METHODS
AND
MATERIALS
The Pacific oysters, Crassostrea gigas, employed in this study were taken from the oyster beds of the Western Oyster Company, Purdy, Washington, and the Johnson Oyster Company, Point Reyes, Inverness, California. Small portions of tissue were excised from the auricles, from regenerating mantle wounds (72,144, and 240 hr after wounding), and from “green” mantles. Tissues were fixed according to the following scheme: 2% acrolein in seawater, 2-3 min, followed by 4% glutaraldehyde in seawater, 3-5 hr, cacodylate buffer, overnight Tissues were dehydrated and embedded in Epon 812 according to Luft’s (1961) procedure. Thin sections of the Purdy oysters were cut on a Sorvall Porter-Blum MT-I ultramicrotome with glass knives and stained with lead cit,rate (Reynolds, 1963). The sect,ions were examined with a Model 2A RCA electron microscope. Tissues from oysters collected at Inverness were cut on a Sorvall Porter-Blum MT-2 ultramicrotome with a diamond knife, stained with alcoholic uranyl acetate and
FINE STRUCTURE
OF PACIFIC
lead cit,rat,e, and examined with a Model 801 A.E.I. electron microscope. RESULTS
Recognitiotl of Gradar Electron Microscope
Amebocytes in the
Identification of the acidophilic granular amebocytes in the electron microscope was made on the basis of the following criteria: (1) size-1 looked for small amebocyte-type cells filled with small granules; and (2) a simple hist’ochemical react’ion. It! was found
FIG.
1. Electron
micrograph
of an oyster acidophile
271
OYSTER AMEBOCYTES
that by hydrolyzing thick (1 p) Epon sections with 0.2% solutions of NaOCl buffered to pH 8.0 and subsequently treating t,hesesections with 0.1% I- in acid solution, the small, osmium-blackened granules of a small cell with a cartwheel nuclear chromatin pattern were rendered intensely acidophilic. It seemedvery likely that t,hesesamesmall cells with osmiophilic granules Tvere acidophiles. Once having identified the acidophiles in thick sections, it was an easy matter to find t,he same cells in ultrathin sections of the same mat’erial.
from a 240-hr-old
regenerating
wound.
X20,000.
272
RUDDELL
Identification of basophiles in ultrathin sections was predicated on the observat’ion that, under the proper circumstances, basephiles could be induced to migrate across epithelial borders (Ruddell, 1969, 1971). Ultrathin sections of epithelial borders known to contain large numbers of migrating basophiles were compared with epithelial borders from control oysters cont’aining very few basophiles. The basophiles then could be easily identified in ultrat.hin sections of any oyster tissue.
FIG.
2. Electron
micrograph
of an oyster
General
Morphology
Except that the granular amebocytes contained granules of differing appearance, the ultrast.ruct.ural morphology of the trio types of granular amebocytes was quit)e similar. Their nuclei were enclosed in a double membrane occasionally perforat,ed with pores. As observed with the light, microscope, chromatin was arranged in small, evenly dispersed clumps, and recognizable nucleoli were generally absent. The amebocytes lacked large,
basophile
from
the auricle.
X15,400.
FINE
STRUCTURE
OF
PACIFIC
organized arrays of endoplasmic reticulum and Golgi apparatus. Mitochondria were rather small, being 0.3-0.8 cc in length. Flattened or tubular mitochondria were often observed. Small, membrane-bound vesicles, 0.07-0.2 P in diameter, were abundant in the cytoplasm of granular amebocytes. Glycogen particles and rosettes were scattered t8hroughout the cytoplasm of the granular amebocytes. Cellular and nuclear dimensions of the granular amebocytes were found to be within the size range as determined under the light microscope (see Introduction). The Acidophilic (Acidophile) Acidophiles brane-bound,
Granular
Amebocyte
were characterized by memosmiophilic cytoplasmic gran-
OYSTER
273
AMEBOCYTES
ules, circular or ovoid in cross section, with a maximum dimension of 0.44.56 p (Fig. 1). The matrix of the granules was composed of an amorphous, finely granular material. The Basophilic (Basophile)
Granular
Amebocyte
Basophiles contained large granules 0.71.2 CLin diameter; the granules were round or oval in cross section and at low magnification resembled hollow, ball-like structures in which an outer membrane and a layer of closely associated granular material enclosed an electron transparent interior (Fig. 2). The membrane-associat’ed granular material was usually distributed around t,he interior of the granule in an asymmetric fashion. The amount of granular material associated with granule membranes was variable,
FIG. 3. Electron micrograph of two basophile granules ture of t,he membrane-associated granular matrix. Two coarse granular network of one of the granules. X75,000.
clearly indicating “intragranular”
the variability in the texgranules can be seen in the
274
FIG.
HIJDDELL
4.
generating
Electron micrograph weunds. X75,oBO.
of a basophile
granule typioal of basophiles from green mantles and re-
even among granules of the same diameter within the samecell. The substructure of the granular layer was also variable, and the appearance of the gram&r matrix ranged from an amorphous, finely granular material to a coarse, granular-fibrillar network (Fig. 3). Small, membrane-bound, “intergranular” granules, 320-350 8 in diameter, commonly were found associated with this coarse network. The distinct and consistent variation ob-
served in granular substructure may indicate t.hat basophile granules are functionally polarized. Basophiles from tissues removed from green portions of oysters or from healing wounds were often markedly different from controls in that, the mitochondria were often swollen and cell membranes disarrayed, the granuIes were slightly swoIlen, distorted, and often contained glycogen, granular material, thin fibrils of lesst,han 80 H diameter derived
FINE
STRUCTURE
OF
PACIFIC
from the membrane-associated granular layer, and the presence of “intragranular” granules (Fig. 4). DISCUSSION
The observations on the fine structure of oyster granular ameboeytes present’ed contributed little 00 the understanding of the way in which these unique cells responded to trauma. It became apparent that examination of excised portions of oyster tissues was not the best “ay to observe oyster granular amebocytes in operation, as it were, since it was difficult to identify, with any certainty, acidophiles releasing copper and associated granular matrix or basophiles swelling. In order to do this, it seemed probable that experiment,al designs predicated on time-sequence studies of oyster amebocytes maintained in vitro, in which granular amebocytes could be induced to undergo their characteristic response to trauma, would have to be considered. It was interesting to note that basophiles from green portions of the oyster mantle often gave the appearance of being disarrayed. As basophiles from green portions of oysters were found to bind large amounts of copper (Ruddell, 1969, 1971), the disarrayed appearance of basophiles from green areas may have been the morphological manifestaCon of a cell poisoned with copper.
I wish to thank Miss Grete Nilson for expert technical assistance and Mr. Bob Munn for help in the use of the electron microscope and for reviewing this manuscript. Appreciat,ion is gratefully extended to Dr. S. R. Wellings and Dr. A. K. Sparks for their support of this study and also to Dr. J. Luft, Miss P. Phelps, Mr. Davies-Williams,
OYSTER
AMEBOCYTES
and the personnel of the Western pany, Purdy, Washington, and the ter Company, Inverness, California, valuable assistance.
275 Oyster ComJohnson Oysfor their in-
REFERENCES ALBERT, A. 1968. “Selective T0xicit.y.” Methuen, London. BANG, F. B. 1961. Reaction to injury in the oyster (Crassostrea virginica). Biol. Bdl., 121, 5748. BOWEN, H. J. 196G. “Trace Elements in Biochemistry.” Academic Press, New York. BOYCE, R., AND HERDMAN, W. A. 1897. On a green leucocytosis in oysters associated with the presence of copper in the leucocytes. Proc. Roy. Sot. Lond., 62, 30-38. DESVOIGNE, D. M., AND SPARKS, -4. K. 1968. The process of wound healing in the Pacific oyster, Crassostrea gigas. J. Invertebr. Pathol., 12, 53-65. GALTSOFF, P. S. 1964. The American oyster, Crassostrea virginica. Gmelin Fish. Bull., Fish Wddlife Serv., 64, 1480. LUFT, J. H. 1961. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. cytoz., 9, 409-514. PAULEY, G. B., AND SPARKS, A. K. 1965. Preliminary observations on the acute inflammatory reaction in the Pacific oyster, Crassostrea gigas, (Thunberg). J. Znvertebr. Pathol., 7, 248-257. REYNOLDS, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in elect.ron microscopy. J. Cell. Biol., 17,208-213. RUDDELL, C. L. 1959. A cytological and histochemical study of wound repair in the Pacific oyster, Crassostrea gigas. Ph.D. Thesis, University of Washington, Seattle. RTJDDELL, C. L. 1971. Elucidation of the nature and function of the granular oyster amebocytes through histochemical studies of normal and traumatized oyster tissues. Histochemie, 26, 95-112. TAKATSUKI, S. 1934. On the nature and function of the amebocytes of Ostrea edztlis. Quart. J. Microsc. Sci., 76, 379436.