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The reaction of the Pacific oyster, Crassostrea gigas, to homologous tissue implants
JOUHNAL
OF
INVERTEBRATE
PATHOLOGY
The Reaction
14, 293-300
( 1969)
of the Pacific Oyster, Crassostreu Homologous Tissue Implant& ‘, 3
DAVID College
M.
DESVOIGNE* of
AND
Fisheries, Unkersity Seattle, Warhington Received
March
1 Contribution No. 309 from the College of Fisheries, University of Washington. 3 This study was made possible by a project, No. 14-17-0001-1563 (Studies in Oyster Pathology) from the Bureau of Commercial Fisheries. 3 Part of a thesis in partial fulfillment of the requirements for a Master of Science degree in Fisheries, University of Washington, by the senior author. 4 Present address: Department of Civil Engineering, University of Washington, Seattle, Washington 98105. 293 0 1969
by Academic
Press, Inc.
of
K.
to
SPARKS
Washington,
98105 11, 1969
A study was made of the host reaction of intraspecific living tissue implants. A number host; but those that remained within the experiment. Although the implants appeared ultimately followed the typical pattern for wound and formed a union with the implants. host and became contiguous with it.
The inflammatory reaction to foreign substances among some of the invertebrate phyla is well-documented. Included in these investigations have been species of Coelenterata ( Prazdnikov and Mikhailova, 1962)) Annelida ( Cameron, 1932)) the Echinodermata (Bang and Lemma, 1962) Arthropoda (Cameron, 1934; Day, 1952; Schlumberger, 1952), and Mollusca (Bang, 1961; Feng, 1959; Jacquemain et al., 1947; Labbe, 1929, 1930; Mikhailova and Prazdnikov, 1961, 1952; Pauley, 1965; Pauley and Sparks, 1965, 1966; Stauber, 1950, 1961; Tripp, 1958, 1961, 1963). The reaction of the Pacific oyster, Crassostrea gigas, to a simple sterile surface wound without inter-
ALBERT
gigas,
the Pacific oyster, Crassostrea gigas, to of the implants were rejected by the host appeared normal throughout the to inhibit wound healing, the process the species. Leucocytes delineated the Finally, the implants fused with the
fering substances injected into the lesion, and with no secondary bacterial invasion, was studied by Pauley ( 1965), Pauley and Sparks (1965, 1967) and DesVoigne and Sparks ( 1968). Subsequent to the early work carried out by Drew and DeMorgan (lSlO), however, very few investigations dealt with the reaction of a host species to tissue implants. Studies conducted relatively recently on various phyla have examined this reaction (Bang and Bang, 1962; Bang and Lemma, 1962; Cooper, 1968). Triplett et al., (1958) noted that although homologous and autologous implants were retained on the skin of the sipunculoid worm, Dendrostomum xostericolum, for as long as 8 days, there was no evidence of healing and both types of grafts were eventually shed with no indication of selective affinities between the tissues. In experiments with asteroids, Ghiradella (1965) implanted either homologous or heterologous living tissues into two species. The results indicated that both species tend to tolerate tissue from their own species and eliminate extraspecific donor tissue.
DESVOIGNE
294
AND SPARKS
days 15 through 28. It was hoped that the advanced stages of healing and implant This investigation was divided into two fusion would be completed by the end of time periods; the first experiment began in the experimental periods. At each sampling January, the second in April. Oysters for period, all oysters were checked for loss the first experiment were collected in De- of implant. In each experiment, 80 control cember to ensure that the phagocytosis ac- oysters were opened and wounded in the companying normal gonadal resorption same manner as the experimentals except was essentially complete and would not that no tissue was implanted. As soon as obscure the host reaction to the wound. each sample was randomly taken, it was The second experiment was initiated prior immediately shucked, fixed in Davidson’s to the onset of leucocytic infiltration of the fluid and . returned to the College of gonadal region in the resorption process. Fisheries; after 24 hr, the oysters were Thus, any tissue reaction would be a result placed in a 50% ethanol solution for of the wound and implant and would not storage. The specimens were dehydrated be part of the processes involved in the through a graded series of ethanol and normal gonadal cycle. zylene for 24 hr, embedded in Paraplast, The oysters were conditioned at water sectioned at approximately seven microns temperatures ranging from 7°C to 12°C and stained with either Harris hematoxylin for 7 days in the flowing sea water system and eosin or Mallory’s trichrome. at the U.S. Public Health Service Shellfish Sanitation Laboratory at Purdy, WashingRESULTS ton. This system, described by Kelly (1961), is sterile, therefore, secondary The wound-healing process has been bacterial invasion of the injured tissues described previously ( DesVoigne and was avoided. After conditioning, the oys- Sparks, 1968). Briefly, the gross appearters were removed from the system and a ance is one of dark discoloration of the one-half inch square hole was rasped in tissue and the production of a conchiolin the right valve of 80 oysters (in each ex- layer covering the window in the shell; periment). After the windows were cut, histologically, the leucocytes of the oyster two oysters were killed, and small portions invade the injured tissue, delineate the of the living mantle were placed in a slit wound margin, and fill the lesion. Interin the connective tissue near the palps. In nally, some of the hemocytes remove all the initial experiment, a No. 11 X-&o injured tissues while others differentiate knife blade was used for the incision. This into cells similar in appearance to normal blade proved to be too large resulting in connective tissue cells. On the surface, the the loss of several implants; therefore, in wound is closed, the hemocytes align and the second experiment, a No. 62s cataract elongate along the exterior and, ultimately, knife blade was used. The blades were form a pseudoepithelium which is indiswashed after each incision in 95% ethanol, tinguishable from the normal adjacent rinsed in heat-sterilized distilled water, epithelium. and allowed to air dry. Grossly, in the experimental group, the After wounding, the oysters were re- discoloration around the wound and the placed in the flowing sea water. Two oys- formation of the conchiolin layer around ters from each group were fixed at 8-hr the window in the valve occurred in a intervals for 7 days; at 24-hr intervals for similar manner and the sequence was days 8 through 14 and at 48-hr intervals for much the same as in the controls. METHODS
AND MATERIALS
REACTION
OF
OYSTJZR
In the initial experiment, loss of the implanted tissue began within 20 hr, possibly due to the size of the Iesion caused by the large scalpel. Specimens were judged to be losing the tissue if the implants were protruding from the lesion or were com-
FIG. 1. Healthy tissue of the oyster Cr~ssostrea response and the nomlal appearance of the implant. FIG. 2. Connective tissue implant-host interface cellular response. About 120 X.
TO
TLSSUE
295
IMPLANTS
pletely out of the lesion but in (:on (tact with the host. No tissue was lost afl :er 168 hr. Tissue loss in the second expe :x-innent was not initiated until 36 hr. The vi: ;ible process of implant loss did, howevc:r, last for 312 hr so that, although loss w as ini-
gigas 14 hr after implantation. About 30 X. 14 hr after insertion. Note
Note the
the lack complete
host ,k of
296
DES
VOIGNE
tially slower, overall the result was very nearly the same. Histologically, the healing process in this group followed the same pattern as the controls; however, in the specimens which retained the implant, the process was considerably slower. At 16 hr, the implant had elicited no host response; only a few leucocytes were infiltrating the region of the lesion or the implant. The implants appeared normal although they contained an increased amount of collagen. The Leydig cells of the implant were normal except at the excised end where they had pycnotic or karyolytic nuclei and fading cytoplasm (Fig. 1 and 2). The first ejected tissue was observed histologically at 40 hr. The implant contained an abnormally high number of leucocytes, possibly derived from the host before loss was completed. The host epithelium beneath the implant and adjacent to the site of implantation was either metaplastic, apparently due to the irritation of the rejecting tissue while in contact with the host, or was rapidly formed pseudoepithelium. By 80 hr, the lost implant had caused desquamation of the epithelium adjacent to the lesion (or production of pseudoepithelium was in its initial stages) and much of the host epithelium had been replaced by a band of fusiform hemocytes. After 144 hr, the host response increased infiltration into the slightly. Hemocytic lesion and implant area increased. Fusiform hemocytes aligned themselves along the interface between the host tissue and implant and, gradually, these cells formed a cross connection with the implant. At 160 hr, although an implant was partially lost, the fusiform cells of the host had partially migrated into the implant and incomplete fusion of the tissue occurred (Fig. 3). In one specimen, after 280 hr, an implant was visible in the palp region which had no epithelium and contained a heavy concen-
AND
SPARKS
tration of collagen. There was only light hemocytic infiltration into the area. At the innermost portion of the implant, the fusiform hemocytes ran partiallv along the host-implant interface and -incompletely fused the two tissues. An implant which had fused completely with the host tissue in the palp region was observed at 448 hr. The tissue appeared healthy and all portions of the implant were intact (Fig. 4). The two epithelia, or pseudoepithelia, fused and the connective tissues were also continuous (Fig. 5 and 6). There were numerous, apparently normal, round hcmocytes present in the connective tissues of both the implant and the host but the fusiform cells typical of healing were not present. DISCUSSION
Homologous tissues experimentally implanted in vertebrate hosts are commonly rejected. Antibody production of the vertebrates is refined to the extent that homografts are distinguishable from autologous tissues. Rejection or encapsulation and subsequent lysis usually follows such implants. However, if antibodies can be climinated, homologous tissue and organ implantation may be accomplished with a slight degree of success, as recent organ transplants have indicated. Tripp (1961) investigated the reaction of the gastropod Australorbis glubratus to tissue implants of formalin-fixed A. glabratus tissue and homologous an d heterologous living tissues. The formalinfixed heterologous tissue was encapsulated within 24 hr by fibroblasts migrating from nearby connective tissue. Implantation of fresh homologous tissue elicited only a transient hemocytic infiltration, probably in response to the lesion; there was no fibroblastic response. Canzonier (1963; 1967, personal communication) reported 50% rejection of
REACTION
OF
OYSTER
norma ~1 and diseased implants of homologous tissues in Cmssostrea uirginica. In those oysters which retained the implants, howe\ ler, there was no indication of rejection clr overt host response once fusion to
FIG. 3.
Partially rejected implant ) 160 hr. About 120 X. FIG. 4. Mantle implant fused in tissues. After 448 hr. About 30 X.
with
incomplete
TO
TISSUE
297
IMPLANTS
the host had occurred. The blood sP aces joined with those of the host and the author assumed cross circulation was act tornplished. In our experimental oysters, whc ere loss
fusion
of
the
connective
tissue.
(Hosi t on
the
right.
the
palps.
Note
the
apparent
continuity
of the
host
and
im] slant
298
5. The epithelium FIG. 6. The FIG.
palp
DES
fused and fused
boundary the newly connective
VOIGNE
AND
SPARKS
between the host and implant. Note the difference in staining of the formed pseudo-epithelium of the implant. After 448 hr. About 120 X. tissue of the host (right) and implant. After 448 hr. About 120 X.
of the implant was common (but considerably less than 50%), the rejection of the tissues was probably not a result of antibody production since antibodies (gamma globulins) are not known to occur in invertebrates; more likely it was due to the nature of the implant. The tissue used in
the experiments was normal living mantle tip which is commonly surrounded by shell liquor only. As the tissues were implanted, they were surrounded by the host tissues. The nature of the experiment was such that, instead of attempting to elicit fusion of epidermal layers at the surface, the tis-
REACTION
OF
OYSTER
sues were forced into the connective tissue of the visceral hump of the oysters in the hope that the normal healing process would seal the implant in the host. The wounds did not close as rapidly as the implants were lost. Loss of the implanted tissues was probably due to a combination of two factors: (1) contraction and movement of the host in the normal activity and (2) the cilia and muscle fibers of the implant pulling or pushing the implant free of the surrounding host tissues, i.e., the implants rejecting the host instead of the reverse. ACRXOWLEDGMENTS The authorsare gratefully indebted for the aid of the following people: Dr. Max Katz for reviewing the manuscript; Mr. William Beck for the use of the U.S. Public Health Service facilities at Purdy, Washington; Mr. Ted Ericksen for aid in sampling; Mr. Eugene J. Yamashita for providing the experimental oysters; and Mrs. Lieselotte Schwartz for processing the specimens.
REFEREXCES BANG, F. B.
1961. Reaction to injury in the oyster (Crassostrea virginica). Biol. Bull., 121, 57-68. BANG, F. B., AND BANG, B. G. 1962. Studies on sipunculoid blood; immunologic properties of coelomic fluid and morphology of “urn cells.” Cab. Biol. Mar., 3, 363-374. BANG, F. B., AND LEMMA, A. 1962. Bacterial infection and reaction to injury in some echinoderms. J. insect Puthol., 4, 401-414. CAHN, A. H. 1949. Pearl culture in Japan. USFWS Fish Leaflet 357, 91 pp. CA~ZEHON, G. R. 1932. Inflammation in earthworm. J. Pathol. Bacterial., 35, 933-972. CAI\IERON, G. R. 1934. Inflammation in the caterpillars of Lepidoptera. J. Pathol. Bacteriol., 38, 441466. CANZONIER, W. J. 1963. Histological observations on the response of oysters to tissue implants. PTOC. Natl. Shellfih. Assoc., 54, 1. COOPER, E. L. 1968. Transplantation immunity in annelids. I. Rejection of xenografts exchanged between Lumbricus terre.rtris and Eisenia foetidu. Transplantation, 6, 322-337.
TO
TISSUE
IMPLANTS
GUSHING, J.
299
1957. Tissue transplantation in Pecten irradians. Biol. Bull., 113, 327. DAY, hf. F. 1952. Wound healing in the gut of the cockroach Periplaneta. Atlstralian J. Sci. Res., B5, 282-289. DESVOIGNE, D. &I., AND SPARKS, A. K. 1968. The process of wound healing in the Pacific oysters, Crassostrea gigas (Thunberg). J. Invertebrate Pathol., 12, 53-65. DREW, G. H., AX;D DEMORGAN, W. 1910. The origin and formation of fibrous tissue produced as a reaction to injury in Pectin maximus, as a tyl’e of Lamellibranchiata. Quart. I. Micros. SC?., 55, 595-610. FENG, S. Y. 1959. Defense mechanisms of the oyster. Bull. New Jersey Acad. Sci., 4, 17. GHIRADELLA, HELER‘ T. 1965. The reaction of two starfishes, Putiria mini&a and Asterias forbesi to foreign tissue in the coelom. Biol. Bull., 128, 77-89. JACQUEIXIAIS, R., JULLIAN, A., AND NOEL, R. 1947. Sur l’action de certains corps cancerigenes chez les cephalopodes. Compt. Rend Acad. Sci., 225, 441-443. LABBE, A. 1929, Reactions experimentales des mollusques a l’introduction de stylets de celloidine. Compt. Rend. Sot. Biol., 100, 116-168. LABBE, A. 1930. Reaction du conjonetif au goudron chez un mollusque Doris tubercrtluta Cuvier, Compt. Rend. Sot. Biol., 103, 20-22. MIKHAILOVA, I. G., AND PRAZDNIKOV, E. V. 1961. Two questions on the morphological reactivity of mantle tissues in Mytilus edulis L. Tr. Murmansk. Morskogo Biol. Inst., 3, 125-130. ( In Russian. ) MIKHAILOVA, I. G., ASD PRAZDNIKOV, E. V. 1962. Inflammatory reactions in the Barents Sea sea mussel ( Mytilrts edulis L. ). Tr. Murmansk. Mor.Tkogo Biol. blrt., 4, 208-220. (In Russian. ) PAULEY, G. B. 1965. Observations on the acute inflammatory reaction and the wound repair process in the Pacific oyster, Crassostrea gigas (Thunberg). Master of Science Thesis, Univ. of Wash., 65 pp. PAULEY, G. B., AND SPARICS, A. K. 1965. Preliminary observations on the acute inflammatory reaction in the Pacific oyster, Crassostrea gigas (Thunberg). J. Invertebrate Pathol., 7, 248-256. PAULEY, G. B., AND SPARKS, A. K. 1966. The acute inflammatory reaction in two different tissues of the Pacific oyster, Crmsostrea gigas. J. Fish. Res. Bd. Canada, 23, 19131921.
300
DESVOIGSE
AND
G. B., AND SPARKS, A. Ii. 1967. Observations on experimental wound repair in the adductor muscle and the Leydig cells of the oyster Crassostrea gigas. J. Invertebrate Pathol., 9, 298309.
PAULEY,
PHAZDNIKOV, E. V., AKU MIKHAILOVA, I. G. 1962. A note on the problem of the character of the early inflammatory reaction in some coelenterates ( Staurophora mertensii Brandt, 1935, Aurelia aurita L., Beroe crtcumis Fabr.) Tr. Murmansk. Morskogo Biol. Inst., 4, 22228. ( In Russian.) H. G. 1952. A comparative study of the reactions to injury: The cellular response to methylcholanthrene and to talc in the body cavity of the cockroach (Periplaneta americana). Arch. Pathol., 51, 98-113.
SCHLUMBERGER,
STAUBER,
L.
A.
1950.
The
fate
of
India
ink
SPARKS
injected intracardially into the oyster, Ostrea virgin& Gmelin. Biol. Bull., 98, 2,27-241. STAUBER, L. A. 1961. Immunity in invertebrates, with special reference to the oyster. Proc. Natl. Shell. Assoc., 50, 7-20. TRIPLETT, E. L., GUSHING, J. E., AW DURALL, G. L. 1958. Observations on some immune reactions of the sipunculoid worm Dendroatomun~ zo!tericolum. Am. Nat., 92, 287-293. TRIPP, M. R. 1958. Studies on the defense mechanism of the oyster, Crassostrea oirginica. J. Parasitol., 44, 35-36. 1961. The fate of foreign maTRIPP, M. R. terials experimentally introduced into the snail Australorbis glabratus. J. Parasitol., 47, 745-751. 1963. Cellular responses of mob TRIPP, M. R. lusks. Ann. N.Y. Acad. Sci.. 113, 467474.
OF
INVERTEBRATE
PATHOLOGY
The Reaction
14, 293-300
( 1969)
of the Pacific Oyster, Crassostreu Homologous Tissue Implant& ‘, 3
DAVID College
M.
DESVOIGNE* of
AND
Fisheries, Unkersity Seattle, Warhington Received
March
1 Contribution No. 309 from the College of Fisheries, University of Washington. 3 This study was made possible by a project, No. 14-17-0001-1563 (Studies in Oyster Pathology) from the Bureau of Commercial Fisheries. 3 Part of a thesis in partial fulfillment of the requirements for a Master of Science degree in Fisheries, University of Washington, by the senior author. 4 Present address: Department of Civil Engineering, University of Washington, Seattle, Washington 98105. 293 0 1969
by Academic
Press, Inc.
of
K.
to
SPARKS
Washington,
98105 11, 1969
A study was made of the host reaction of intraspecific living tissue implants. A number host; but those that remained within the experiment. Although the implants appeared ultimately followed the typical pattern for wound and formed a union with the implants. host and became contiguous with it.
The inflammatory reaction to foreign substances among some of the invertebrate phyla is well-documented. Included in these investigations have been species of Coelenterata ( Prazdnikov and Mikhailova, 1962)) Annelida ( Cameron, 1932)) the Echinodermata (Bang and Lemma, 1962) Arthropoda (Cameron, 1934; Day, 1952; Schlumberger, 1952), and Mollusca (Bang, 1961; Feng, 1959; Jacquemain et al., 1947; Labbe, 1929, 1930; Mikhailova and Prazdnikov, 1961, 1952; Pauley, 1965; Pauley and Sparks, 1965, 1966; Stauber, 1950, 1961; Tripp, 1958, 1961, 1963). The reaction of the Pacific oyster, Crassostrea gigas, to a simple sterile surface wound without inter-
ALBERT
gigas,
the Pacific oyster, Crassostrea gigas, to of the implants were rejected by the host appeared normal throughout the to inhibit wound healing, the process the species. Leucocytes delineated the Finally, the implants fused with the
fering substances injected into the lesion, and with no secondary bacterial invasion, was studied by Pauley ( 1965), Pauley and Sparks (1965, 1967) and DesVoigne and Sparks ( 1968). Subsequent to the early work carried out by Drew and DeMorgan (lSlO), however, very few investigations dealt with the reaction of a host species to tissue implants. Studies conducted relatively recently on various phyla have examined this reaction (Bang and Bang, 1962; Bang and Lemma, 1962; Cooper, 1968). Triplett et al., (1958) noted that although homologous and autologous implants were retained on the skin of the sipunculoid worm, Dendrostomum xostericolum, for as long as 8 days, there was no evidence of healing and both types of grafts were eventually shed with no indication of selective affinities between the tissues. In experiments with asteroids, Ghiradella (1965) implanted either homologous or heterologous living tissues into two species. The results indicated that both species tend to tolerate tissue from their own species and eliminate extraspecific donor tissue.
DESVOIGNE
294
AND SPARKS
days 15 through 28. It was hoped that the advanced stages of healing and implant This investigation was divided into two fusion would be completed by the end of time periods; the first experiment began in the experimental periods. At each sampling January, the second in April. Oysters for period, all oysters were checked for loss the first experiment were collected in De- of implant. In each experiment, 80 control cember to ensure that the phagocytosis ac- oysters were opened and wounded in the companying normal gonadal resorption same manner as the experimentals except was essentially complete and would not that no tissue was implanted. As soon as obscure the host reaction to the wound. each sample was randomly taken, it was The second experiment was initiated prior immediately shucked, fixed in Davidson’s to the onset of leucocytic infiltration of the fluid and . returned to the College of gonadal region in the resorption process. Fisheries; after 24 hr, the oysters were Thus, any tissue reaction would be a result placed in a 50% ethanol solution for of the wound and implant and would not storage. The specimens were dehydrated be part of the processes involved in the through a graded series of ethanol and normal gonadal cycle. zylene for 24 hr, embedded in Paraplast, The oysters were conditioned at water sectioned at approximately seven microns temperatures ranging from 7°C to 12°C and stained with either Harris hematoxylin for 7 days in the flowing sea water system and eosin or Mallory’s trichrome. at the U.S. Public Health Service Shellfish Sanitation Laboratory at Purdy, WashingRESULTS ton. This system, described by Kelly (1961), is sterile, therefore, secondary The wound-healing process has been bacterial invasion of the injured tissues described previously ( DesVoigne and was avoided. After conditioning, the oys- Sparks, 1968). Briefly, the gross appearters were removed from the system and a ance is one of dark discoloration of the one-half inch square hole was rasped in tissue and the production of a conchiolin the right valve of 80 oysters (in each ex- layer covering the window in the shell; periment). After the windows were cut, histologically, the leucocytes of the oyster two oysters were killed, and small portions invade the injured tissue, delineate the of the living mantle were placed in a slit wound margin, and fill the lesion. Interin the connective tissue near the palps. In nally, some of the hemocytes remove all the initial experiment, a No. 11 X-&o injured tissues while others differentiate knife blade was used for the incision. This into cells similar in appearance to normal blade proved to be too large resulting in connective tissue cells. On the surface, the the loss of several implants; therefore, in wound is closed, the hemocytes align and the second experiment, a No. 62s cataract elongate along the exterior and, ultimately, knife blade was used. The blades were form a pseudoepithelium which is indiswashed after each incision in 95% ethanol, tinguishable from the normal adjacent rinsed in heat-sterilized distilled water, epithelium. and allowed to air dry. Grossly, in the experimental group, the After wounding, the oysters were re- discoloration around the wound and the placed in the flowing sea water. Two oys- formation of the conchiolin layer around ters from each group were fixed at 8-hr the window in the valve occurred in a intervals for 7 days; at 24-hr intervals for similar manner and the sequence was days 8 through 14 and at 48-hr intervals for much the same as in the controls. METHODS
AND MATERIALS
REACTION
OF
OYSTJZR
In the initial experiment, loss of the implanted tissue began within 20 hr, possibly due to the size of the Iesion caused by the large scalpel. Specimens were judged to be losing the tissue if the implants were protruding from the lesion or were com-
FIG. 1. Healthy tissue of the oyster Cr~ssostrea response and the nomlal appearance of the implant. FIG. 2. Connective tissue implant-host interface cellular response. About 120 X.
TO
TLSSUE
295
IMPLANTS
pletely out of the lesion but in (:on (tact with the host. No tissue was lost afl :er 168 hr. Tissue loss in the second expe :x-innent was not initiated until 36 hr. The vi: ;ible process of implant loss did, howevc:r, last for 312 hr so that, although loss w as ini-
gigas 14 hr after implantation. About 30 X. 14 hr after insertion. Note
Note the
the lack complete
host ,k of
296
DES
VOIGNE
tially slower, overall the result was very nearly the same. Histologically, the healing process in this group followed the same pattern as the controls; however, in the specimens which retained the implant, the process was considerably slower. At 16 hr, the implant had elicited no host response; only a few leucocytes were infiltrating the region of the lesion or the implant. The implants appeared normal although they contained an increased amount of collagen. The Leydig cells of the implant were normal except at the excised end where they had pycnotic or karyolytic nuclei and fading cytoplasm (Fig. 1 and 2). The first ejected tissue was observed histologically at 40 hr. The implant contained an abnormally high number of leucocytes, possibly derived from the host before loss was completed. The host epithelium beneath the implant and adjacent to the site of implantation was either metaplastic, apparently due to the irritation of the rejecting tissue while in contact with the host, or was rapidly formed pseudoepithelium. By 80 hr, the lost implant had caused desquamation of the epithelium adjacent to the lesion (or production of pseudoepithelium was in its initial stages) and much of the host epithelium had been replaced by a band of fusiform hemocytes. After 144 hr, the host response increased infiltration into the slightly. Hemocytic lesion and implant area increased. Fusiform hemocytes aligned themselves along the interface between the host tissue and implant and, gradually, these cells formed a cross connection with the implant. At 160 hr, although an implant was partially lost, the fusiform cells of the host had partially migrated into the implant and incomplete fusion of the tissue occurred (Fig. 3). In one specimen, after 280 hr, an implant was visible in the palp region which had no epithelium and contained a heavy concen-
AND
SPARKS
tration of collagen. There was only light hemocytic infiltration into the area. At the innermost portion of the implant, the fusiform hemocytes ran partiallv along the host-implant interface and -incompletely fused the two tissues. An implant which had fused completely with the host tissue in the palp region was observed at 448 hr. The tissue appeared healthy and all portions of the implant were intact (Fig. 4). The two epithelia, or pseudoepithelia, fused and the connective tissues were also continuous (Fig. 5 and 6). There were numerous, apparently normal, round hcmocytes present in the connective tissues of both the implant and the host but the fusiform cells typical of healing were not present. DISCUSSION
Homologous tissues experimentally implanted in vertebrate hosts are commonly rejected. Antibody production of the vertebrates is refined to the extent that homografts are distinguishable from autologous tissues. Rejection or encapsulation and subsequent lysis usually follows such implants. However, if antibodies can be climinated, homologous tissue and organ implantation may be accomplished with a slight degree of success, as recent organ transplants have indicated. Tripp (1961) investigated the reaction of the gastropod Australorbis glubratus to tissue implants of formalin-fixed A. glabratus tissue and homologous an d heterologous living tissues. The formalinfixed heterologous tissue was encapsulated within 24 hr by fibroblasts migrating from nearby connective tissue. Implantation of fresh homologous tissue elicited only a transient hemocytic infiltration, probably in response to the lesion; there was no fibroblastic response. Canzonier (1963; 1967, personal communication) reported 50% rejection of
REACTION
OF
OYSTER
norma ~1 and diseased implants of homologous tissues in Cmssostrea uirginica. In those oysters which retained the implants, howe\ ler, there was no indication of rejection clr overt host response once fusion to
FIG. 3.
Partially rejected implant ) 160 hr. About 120 X. FIG. 4. Mantle implant fused in tissues. After 448 hr. About 30 X.
with
incomplete
TO
TISSUE
297
IMPLANTS
the host had occurred. The blood sP aces joined with those of the host and the author assumed cross circulation was act tornplished. In our experimental oysters, whc ere loss
fusion
of
the
connective
tissue.
(Hosi t on
the
right.
the
palps.
Note
the
apparent
continuity
of the
host
and
im] slant
298
5. The epithelium FIG. 6. The FIG.
palp
DES
fused and fused
boundary the newly connective
VOIGNE
AND
SPARKS
between the host and implant. Note the difference in staining of the formed pseudo-epithelium of the implant. After 448 hr. About 120 X. tissue of the host (right) and implant. After 448 hr. About 120 X.
of the implant was common (but considerably less than 50%), the rejection of the tissues was probably not a result of antibody production since antibodies (gamma globulins) are not known to occur in invertebrates; more likely it was due to the nature of the implant. The tissue used in
the experiments was normal living mantle tip which is commonly surrounded by shell liquor only. As the tissues were implanted, they were surrounded by the host tissues. The nature of the experiment was such that, instead of attempting to elicit fusion of epidermal layers at the surface, the tis-
REACTION
OF
OYSTER
sues were forced into the connective tissue of the visceral hump of the oysters in the hope that the normal healing process would seal the implant in the host. The wounds did not close as rapidly as the implants were lost. Loss of the implanted tissues was probably due to a combination of two factors: (1) contraction and movement of the host in the normal activity and (2) the cilia and muscle fibers of the implant pulling or pushing the implant free of the surrounding host tissues, i.e., the implants rejecting the host instead of the reverse. ACRXOWLEDGMENTS The authorsare gratefully indebted for the aid of the following people: Dr. Max Katz for reviewing the manuscript; Mr. William Beck for the use of the U.S. Public Health Service facilities at Purdy, Washington; Mr. Ted Ericksen for aid in sampling; Mr. Eugene J. Yamashita for providing the experimental oysters; and Mrs. Lieselotte Schwartz for processing the specimens.
REFEREXCES BANG, F. B.
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