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Studies on the internal defense mechanisms of sponges
JOURNAL
OF
INVERTEBRATE
12, 29-35
I’ATHOLOGY
Studies
on the Internal
III.
Cellular Reactions Heterologous
THOMAS
C.
CHESG, AND
(1968)
Defense
Mechanisms
of Sponges
in Terpios zeteki to Implanted Biological Materials l
HERBERT
W.
D.
MARSHALL
F.
YEE,
ERIK
KIFKIN,
KRA~IE~I
Cellular responses in Ter),io.c- z2frki to implnnted heteroio:Wu3 I~iolo:+d materials coxisting of human erythrocytes, trematode rediae and ccrcariao, and molluscau muscles, were examined at 6, 1 2, 24, and 48 hr portimpli~ntation. It was determined that erythrocytes were either phagocytized or encapsulated by sponge archaeocytes and were gradually eliminated from the mesoglea via the migration of host cells into cxcurrtd canals. The meroglea was essentially frc? of implanted erythrocytes by the 48th hr. Implants of rediae only evoked slight ercapsulation involving archneocytes and collencytes, with some of the latter forming syncytia. Freed cercariac were enveloped by a relatively thick syncytinl caps& of archaeocytes and collencytes. This capsule, however, clisintegrntetl by the 48th hr when the cercarial tissues clecomposed. Simrlltaneously, a lxjie numlx~ of nrchaeocytes including amorphous mnterials, most probably fragments of disintegrated inlpl~mtetl tkues, were found in the lumina of excurrent canals. Response to mll?clc m~:~lants consisted primarily of an irregular distribution of collencytes ant1 archaeocytes on the surfaces, hut with Thus, incomplete or complde encapsulation no evidence of true capsule formation. of heteroqafts has been demonstrated in sponges for the first time. Incision sites in sponges of the control series revealed ;I gratlual corxentration of parenchymal cells along the cut surfaces. No fusion of these surfaces wx obser\,c,tl.
particles experimentally injected into the mesoglea. Since it has been demonstrated that these two types of cells, primarily archaeocytes, are capable of phagocytizing foreign particles and eliminating them via migration between pinacocytes into excurrent canals, it appeared to be of interest to determine whether these cells are also capable of contributing to encapsulation and eventual destruction or elimination of heterologous biological materials which are too large to be phagocytized. To our knowledge, the occurrence of encapsulation as an internal defense mechanism has not been demonstrated among the Porifera.
In the two earlier papers of this series (Cheng et al.. 1968a, b), the morphology and quantitative distribution of the five types of free parenchymal cells found in the mesoglea of the marine demospongid poriferan Terpios zetpki have been reported as well as the role of archaeocytes and to some extent that of collencytes in the arrest and removal of India ink and carmine 1 This study was from the American by a grant (Contract Bureau of Commercial of the Interior.
supported in part by a grant Cancer Society and in part 14-17-007-662G) from the Fisheries, U. S. Department
29
30
CHENG,
MATERIALS
ASD
YEE,
RtFKIN,
I\/IETIIODS
Specimens of T. zeteki collected from pilings at Ala Wai Yacht Harbor in Honolulu, Hawaii, were maintained in the laboratory at 22°C in aerated filtered seawater with a salinity of 35 parts per thousand for 24 hr prior to use. The sponges were divided into four series, with eight in each. Members of the first series were each injected with 0.2 ml of washed human erythrocytes suspended in isotonic (0.85%) saline. A small incision, approximately 3 mm deep, was made on each member of the second and third series followed by the implantation of four or five third generation rediae of the trematode Philopl~tl~alnzus gralli into all the members of the second series and a small piece, approximately 2 mm:‘, of the foot musculature of the marine gastropod Littorina scuba into each of the members of the third series. Members of the fourth series served as controls. A similar incision was made in each of these but no foreign material was implanted. All of the sponges were returned to seawater after the introduction of the foreign materials. Two specimens of each of the four series were fixed in 10% seawater-formalin at each of four time intervals postimplantation, viz., 6, 12, 24, and 48 hr. After a fixation period of 8 hr, all of the sponges were dehydrated via a closely graded ethanol series, embedded in Tissuemat, and sectioned at 7 p. The sections were all stained with Delafield’s hematoxylin and counterstained with eosin. RESULTS
Six Hours
Postimplantation
Erytlarocytes. In specimens fixed at this time interval, in addition to finding a large number of free erythrocytes, a number of archaeocytes free within the mesoglea were found to have phagocytized whole erythrocytes (Fig. 1, A, B; Fig. 2, A). Two additional mechanisms were also employed by
AND
KRAMER
sponge archaeocytes for the arrest of human erythrocytes. One involved two or more, commonly three, archaeocytes encapsulating a single erythrocyte. The cytoplasm of these sponge cells had become fused SO that the enveloping capsule consisted of a syncytium containing several nuclei (Fig. 1, C). The other mechanism involved the formation of a similar syncytium by three or more archaeocytes surrounding two or more erythrocytes (Fig. 1, D). Although chemical evidence is yet unavailable, the appearance of several phagocytized erythrocytes suggested that intracellular degradation had occurred. In these cells, the characteristic homogeneous pinkish orange appearance of the cytoplasm was altered to a much lighter and clumped appearance (Fig. 1, B). In addition to finding erythrocytes either phagocytized or encapsulated by sponge archaeocytes within the mesoglea, a few archaeocytes, each enclosing a single erythrocyte, were observed in the process of passing through the stratum of pinacocytes lining excurrent canals and being deposited in the lumina of the canals (Fig. 2, B). Rediae. A number of archaeocytes and collencytes were found adhering to the external surfaces of rediae implanted in sponges examined at the 6th hr ( Fig. 2, C ) . The sponge cells did not form a continuous tunica but were unevenly and irregularly distributed with some adjacent collencyter forming syncytia. It is of interest to note that among the sections examined, the walls of two cercariae-bearing rediae had ruptured. releasing fully developed cercariae. A relatively thick layer of sponge cells, both archaeocytes and collencytes, was observed enveloping each freed cercaria (Fig. 2, D ) . Many of the collencytes had fused. Muscle. A few archaeocytes and collencytes, primarily the former, were found adhering to the surfaces of the implanted molluscan muscles.
INTERNAL
DEFENSE
MECHANISMS
OF
SPONGES.
III.
en
FIG. 1. Single erythrocytes within single phagocytic archaeocytes and free erythrocytes. B, Single erythrocyte within phagocytic erythrocyte showing clnmped appearance of cytoplasm of erythrocyte suggesting degradation. C, Single erythrocytes encapsulated by two or more fused archaeocytes, phagoD, Two or more erythrocytes encytic archaeocyte enclosing single erythrocyte, and free erythrocyte. (All drawings made from cells observed in sections.) capsulated by two or more fused archaeocytes. e, erythrocyte; en, single erythrocyte encapsulated by two or more sponge archaeocytes; men, two or more erythrocytes encapsulated by two or more sponge archaeocytes; ph, single erythrocyte phagocytizetl by sin,qle archaeocyte.
Controls.
The cut surfaces of sponges the control series did not reveal any noticeable aggregation of cells.
comprising
Ttoelce Hours Postinzplantation Erythrocytes. The number of free erythrocytes in the mesoglea of T. xeteki was conspicuously reduced in specimens examined at this time interval. The majority were either phagocytized or encapsulated in the same manner as in 6-hr specimens. Those phagocytized were frequently observed in the process of being carried to the lumina of excurrent canals within Again, the appearance of archaeocytes. many of the phagocytized erythrocytes suggested intracellular degradation. Although some encapsulated erythrocytes were found along the borders of excurrent canals, th(a cwilddcd in the mesomajorit!, rc~maincd gka. Recline. Irregular clumps of archaeocytes and collencytes were found adhering to
the surfaces of implanted rediae as in 6-hr specimens. Again, a few fully developed cercariae which had been released from ruptured rediae were observed. It is of interest to note that the sponge cells which had formed a packed layer surrounding cercariae in 6-hr specimens had coalesced, forming a relatively thick syncytial capsule around freed cercariae in 12-hr specimens (Fig. 2, E). Mzcscle. The number of host cells adhering to the surfaces of implanted muscles had increased in 12-hr specimens (Fig. 2, F). These, however, retained their integrity rather than form a syncytial tunica. A few had migrated beneath the muscle surfaces, between the myofibers.
Controls.
There was a slight increase in the number of free cells. primarily collencytes and archaeocytes, along the cut edges in the control animals (Fig. 3, A).
32
CHENG,
YEE,
RIFKIN,
AND
KAAI\IER
or implanted tissues stainetl FIG. 2. All photomicrographs are of sections of either ?‘cr)lios zdeki within phagocytic archaeocytes in mc~pl~a ( 6 hr po-twith hematoxylin and eosin. A. Erythrocytes implantation). B. Erythrocyte-laden archaeocytes migrating into escurrent canal of sponge ( 6 hr postimplantation). C. Irregulnr aggregates of archarocytes and collencytes on surface of impl:mted redia ( 6 hr postimplantation). D. Relatively imiform layer of nrcharocytrs and collrncytrs adherir~ to surface of cercaria (6 hr postimplantation). E. Formation of syrtcytinl tunic of hoct c:~lls cllrroundinv cercaria ( I2 hr po?timplantation ) F. Irregular aggregatcas of archacocytcs and collcncytcs on surfarc ) of implanted molluscan muscle ( L q hr postimplautntion v\c’, v\current vand: m11, 11111~c.le; pc’, pll:i~:oarcharocytc~s and collencytes; cer, cercaria; ac, sponge cytized erythrocytes: rw, redial wall: s, syncytial capsnk of ho\t ccb.
INTERNAL
DEFENSE
MECHANISMS
OF
SPONGES.
III.
33
FIG. 3. All photomicrograph; are of sections of either Terpios Zrteki or iniulnnted materials stained with hematoxylin and eosin. A. Slight concentration of parenchymal cells along cut edges in control specimen ( 12 hr po;tincision) . B. Freed erythrocytes in excurrent canal (24 hr postimplantation). C. Section of cercaria showing presence of syncytial capsule of host cells. Notice that the implanted tissue is commencing to disintegrate (24 hr postimplantation). D. Section of disintegrating cercaria showing disappearance of syccytial capsule of host cells and infiltration of foreign tissue by archaeocytes (48 hr postimplantation). E. Relatively heavy concentration of parenchymal cells along cut edges in control specimen (49 hr postincision). a, archaeocytes; cat, concentration of archaeocytes and collencytes; dc, decomposing cercaria; e. human erythrocytes; exe, excurrent canal; in, incision; s. syncytial capsule of host cells.
34
CHENG,
Twenty-four
Hours
YEE,
RIF’KIN,
Postimplantation
Erythrocytes. Practically no free erythrocytes occurred in the mesoglea of sponges examined at this time interval. Those found were encapsulated, either singly or in small groups, by fused archaeocytes. On the other hand, large numbers of erythrocytes occurred in the lumina of excurrent canals (Fig. 3, B). Most of these had ruptured out of archaeocytes and were found as clumps in the canals. Rediae. It was evident by the 24th hr postimplantation that the rediae had died and their tissues were disintegrating. Despite the changes associated with disintegration, no true encapsulation of redial tissues occurred although archaeocytes and collencytes were observed not only adhering to the redial surfaces as at previous time intervals but a few were also found infiltrating the decomposing redial tissues. Again, cercariae which had escaped from ruptured rediae were found in sponges examined at .this time interval. These cercariae were also disintegrating. The syncytial encapsulating layer of host cells was still evident surrounding each decomposing cercaria (Fig. 3, C). Muscle. The cellular reactions of the hosts observed at this time interval were comparable to those found at the 6th and 12th hr although critical examination revealed the fusing of the cytoplasm of adjacent collencytes. These, however, did not form a continuous capsule. Controls. The edges of the incision made in control sponges were no different from those observed at 12 hr. Forty-eight
Hours
Postimplantation
Erythrocytes. Very few erythrocytes, free, encapsulated, or phagocytized, were found in the mesoglea of sponges examined at this time interval. A large number of encapsulated and phagocytized erythro-
AND
KRAMER
cytes, however, were found along the borders and in the lumina of excurrent canals. As was the case in 24-hr specimens, many of the erythrocytes found in the canals had ruptured out of phagocytic archaeocytes. Rediae. By the 48th hr postimplantation, both rediae and freed cercariae were at advanced stages of disintegration. In fact, their tissues had decomposed to such a state that their integrities were barely distinguishable. In the case of both disintegrating rediae and cercariae, sponge cells, primarily archaeocytes, had infiltrated the foreign tissues. Furthermore, in the case of cercariae, the encapsulating syncytial layer of host cells was no longer visible (Fig. 3, D). It is of interest to note that large numbers of archaeocytes enclosing amorphous materials were found in the lumina of excurrent canals. These may represent phagocytic cells which had removed disintegrating cells and cell fractions of rediae and cercariae. Muscle. Muscle implants examined at this time interval were also decomposing. The individual myofibers were no longer discrete and were poorly stained. Host cells, both archaeocytes and collencytes, were found not only forming an uneven layer covering the surface, but were also found infiltrating throughout the decomposing myofibers. Controls. The two surfaces resulting from the incision were not fused. There were heavy concentrations of free cells, primarily collencytes, but also some archaeocytes, along the wounded surfaces (Fig. 3, E). Many of the collencytes had fused to form a netlike reticulum. The appearance and arrangement of these cells suggested that fusion of the two opposing surfaces would not follow but that the exposed layers would become new surfaces lined with cells originating as collencytes.
INTERNAL ~KUSSION
AND
DEFENSE
MECHANISMS
COSCLUSIONS
It is evident from our observations that the sponge Terpios zeteki is capable of encapsulating experimentally introduced heterologous biological materials (heterografts) although the efficiency of this mechanism varies, depending on the nature of the implanted material. For example, it has been noted that human erythrocytes are completely encapsulated in addition to being phagocytized and cercariae freed from ruptured rediae are completely encapsulated. On the other hand, although sponge archaeocytes and collencytes do adhere to the outer surfaces of implanted rediae and molluscan muscles, the formation of a complete capsule does not occur. The complete encapsulation of cercariae by a relatively thick syncytium is of interest. It is recalled that Thakur and Cheng (1968) have demonstrated that Philopl~thalnw gralli cercariae commence to secrete cytogenous materials from three distinct types of subsurfacial gland cells prior to their escape from rediae. This material is uniformly deposited on the body surface of each cercaria. It is thus possible that the more spectacular and efficient encapsulation of cercariae, as compared with cellular reactions towards rediae or muscles, represents the response of sponge archaeocytes and collencytes to the proteinaceous cystogenous material as is the case in molluscs harboring trematode metacercariae (Cheng et al., 1966a, b). It is interesting that human erythrocytes, which average 7.74 p in diameter, can become phagocytized by archaeocytes which are smaller, averaging 6 by 5 p (Cheng et al., 1968a). This indicates that phagocytic archaeocytes can become hypertrophied to accommodate foreign cells of slightly greater dimensions. The fact that both phagocytized and encapsulated erythrocytes are all essentially eliminated from the parenchyma of T. zeteki by the 48th hr via the migration of
OF
SPONGES,
III.
35
erythrocyte-laden or enveloping archaeocytes across lining pinacocytes into excurrent canals indicates that not only arc archaeocytes enclosing foreign bodies capable of migration, but small encapsulation complexes, involving several archaeocytes surrounding a foreign body, are also capable although with less rapidity. Finally, the aggregation of sponge cells around implanted rediae, cercariae, and muscles should not be totally attributed to attraction by the heterografts since examination of our controls has revealed that there is some migration of cells to the wounded surfaces as related to would healing. However, the fact that sponge cells do become adhered to the surfaces of the heterografts and form a complete capsule in the case of cercariae indicates that the cellular response can be attributed, at least in part, to the presence of the implanted materials. REFERENCES
T. C., SIIUSTER, C. N., JR., AND ANDERSON, A. H. 1966a. A comparative study of the susceptibility and response of eight species of marine pelecypods to the trematode Himasthlu quissetensis. Truns. Am. Microscop. Sot., 85, 284-295. CHENG, T. C., SHUSTER, C. N., JR., AND ANDERSON, A. H. 1966b. Effects of plasma and tissue extracts of marine pelecypods on the cercaria of Himasthla qniwetcnsis. Exptl. Purusitol., 19, 9-14. CHENG, T. C., YEE, H. \V. F., ANI) RIFKIN, E. 1968a. Studies on the internal defense mechanisms of sponges. I. The cell types occurring in the mesoglea of Terpios z&&i (de Laubenfels ) ( Porifera: Demospongiae ) Pacific Sci., m press. CIIENG, T. C., RIFKIN, E., AND YEE, H. W. F. 1968b. Studies on the internal defense mechanisms of sponges. II. Phagocytosis and elimination of India ink and carmine particles by certain parenchymal cells of Terpios ~eteki. J. Invertebrate Pathol., 10, 302-309. THAKUR, A. S., AND CHEN~, T. C. 1968. The formation, structure, and histochemistry of the metacercarial cyst of Philophthalmu gralli Mathis and Lwer.,I ParusitoZoct/. .“_ iu _ press.
CIIENG,
OF
INVERTEBRATE
12, 29-35
I’ATHOLOGY
Studies
on the Internal
III.
Cellular Reactions Heterologous
THOMAS
C.
CHESG, AND
(1968)
Defense
Mechanisms
of Sponges
in Terpios zeteki to Implanted Biological Materials l
HERBERT
W.
D.
MARSHALL
F.
YEE,
ERIK
KIFKIN,
KRA~IE~I
Cellular responses in Ter),io.c- z2frki to implnnted heteroio:Wu3 I~iolo:+d materials coxisting of human erythrocytes, trematode rediae and ccrcariao, and molluscau muscles, were examined at 6, 1 2, 24, and 48 hr portimpli~ntation. It was determined that erythrocytes were either phagocytized or encapsulated by sponge archaeocytes and were gradually eliminated from the mesoglea via the migration of host cells into cxcurrtd canals. The meroglea was essentially frc? of implanted erythrocytes by the 48th hr. Implants of rediae only evoked slight ercapsulation involving archneocytes and collencytes, with some of the latter forming syncytia. Freed cercariac were enveloped by a relatively thick syncytinl caps& of archaeocytes and collencytes. This capsule, however, clisintegrntetl by the 48th hr when the cercarial tissues clecomposed. Simrlltaneously, a lxjie numlx~ of nrchaeocytes including amorphous mnterials, most probably fragments of disintegrated inlpl~mtetl tkues, were found in the lumina of excurrent canals. Response to mll?clc m~:~lants consisted primarily of an irregular distribution of collencytes ant1 archaeocytes on the surfaces, hut with Thus, incomplete or complde encapsulation no evidence of true capsule formation. of heteroqafts has been demonstrated in sponges for the first time. Incision sites in sponges of the control series revealed ;I gratlual corxentration of parenchymal cells along the cut surfaces. No fusion of these surfaces wx obser\,c,tl.
particles experimentally injected into the mesoglea. Since it has been demonstrated that these two types of cells, primarily archaeocytes, are capable of phagocytizing foreign particles and eliminating them via migration between pinacocytes into excurrent canals, it appeared to be of interest to determine whether these cells are also capable of contributing to encapsulation and eventual destruction or elimination of heterologous biological materials which are too large to be phagocytized. To our knowledge, the occurrence of encapsulation as an internal defense mechanism has not been demonstrated among the Porifera.
In the two earlier papers of this series (Cheng et al.. 1968a, b), the morphology and quantitative distribution of the five types of free parenchymal cells found in the mesoglea of the marine demospongid poriferan Terpios zetpki have been reported as well as the role of archaeocytes and to some extent that of collencytes in the arrest and removal of India ink and carmine 1 This study was from the American by a grant (Contract Bureau of Commercial of the Interior.
supported in part by a grant Cancer Society and in part 14-17-007-662G) from the Fisheries, U. S. Department
29
30
CHENG,
MATERIALS
ASD
YEE,
RtFKIN,
I\/IETIIODS
Specimens of T. zeteki collected from pilings at Ala Wai Yacht Harbor in Honolulu, Hawaii, were maintained in the laboratory at 22°C in aerated filtered seawater with a salinity of 35 parts per thousand for 24 hr prior to use. The sponges were divided into four series, with eight in each. Members of the first series were each injected with 0.2 ml of washed human erythrocytes suspended in isotonic (0.85%) saline. A small incision, approximately 3 mm deep, was made on each member of the second and third series followed by the implantation of four or five third generation rediae of the trematode Philopl~tl~alnzus gralli into all the members of the second series and a small piece, approximately 2 mm:‘, of the foot musculature of the marine gastropod Littorina scuba into each of the members of the third series. Members of the fourth series served as controls. A similar incision was made in each of these but no foreign material was implanted. All of the sponges were returned to seawater after the introduction of the foreign materials. Two specimens of each of the four series were fixed in 10% seawater-formalin at each of four time intervals postimplantation, viz., 6, 12, 24, and 48 hr. After a fixation period of 8 hr, all of the sponges were dehydrated via a closely graded ethanol series, embedded in Tissuemat, and sectioned at 7 p. The sections were all stained with Delafield’s hematoxylin and counterstained with eosin. RESULTS
Six Hours
Postimplantation
Erytlarocytes. In specimens fixed at this time interval, in addition to finding a large number of free erythrocytes, a number of archaeocytes free within the mesoglea were found to have phagocytized whole erythrocytes (Fig. 1, A, B; Fig. 2, A). Two additional mechanisms were also employed by
AND
KRAMER
sponge archaeocytes for the arrest of human erythrocytes. One involved two or more, commonly three, archaeocytes encapsulating a single erythrocyte. The cytoplasm of these sponge cells had become fused SO that the enveloping capsule consisted of a syncytium containing several nuclei (Fig. 1, C). The other mechanism involved the formation of a similar syncytium by three or more archaeocytes surrounding two or more erythrocytes (Fig. 1, D). Although chemical evidence is yet unavailable, the appearance of several phagocytized erythrocytes suggested that intracellular degradation had occurred. In these cells, the characteristic homogeneous pinkish orange appearance of the cytoplasm was altered to a much lighter and clumped appearance (Fig. 1, B). In addition to finding erythrocytes either phagocytized or encapsulated by sponge archaeocytes within the mesoglea, a few archaeocytes, each enclosing a single erythrocyte, were observed in the process of passing through the stratum of pinacocytes lining excurrent canals and being deposited in the lumina of the canals (Fig. 2, B). Rediae. A number of archaeocytes and collencytes were found adhering to the external surfaces of rediae implanted in sponges examined at the 6th hr ( Fig. 2, C ) . The sponge cells did not form a continuous tunica but were unevenly and irregularly distributed with some adjacent collencyter forming syncytia. It is of interest to note that among the sections examined, the walls of two cercariae-bearing rediae had ruptured. releasing fully developed cercariae. A relatively thick layer of sponge cells, both archaeocytes and collencytes, was observed enveloping each freed cercaria (Fig. 2, D ) . Many of the collencytes had fused. Muscle. A few archaeocytes and collencytes, primarily the former, were found adhering to the surfaces of the implanted molluscan muscles.
INTERNAL
DEFENSE
MECHANISMS
OF
SPONGES.
III.
en
FIG. 1. Single erythrocytes within single phagocytic archaeocytes and free erythrocytes. B, Single erythrocyte within phagocytic erythrocyte showing clnmped appearance of cytoplasm of erythrocyte suggesting degradation. C, Single erythrocytes encapsulated by two or more fused archaeocytes, phagoD, Two or more erythrocytes encytic archaeocyte enclosing single erythrocyte, and free erythrocyte. (All drawings made from cells observed in sections.) capsulated by two or more fused archaeocytes. e, erythrocyte; en, single erythrocyte encapsulated by two or more sponge archaeocytes; men, two or more erythrocytes encapsulated by two or more sponge archaeocytes; ph, single erythrocyte phagocytizetl by sin,qle archaeocyte.
Controls.
The cut surfaces of sponges the control series did not reveal any noticeable aggregation of cells.
comprising
Ttoelce Hours Postinzplantation Erythrocytes. The number of free erythrocytes in the mesoglea of T. xeteki was conspicuously reduced in specimens examined at this time interval. The majority were either phagocytized or encapsulated in the same manner as in 6-hr specimens. Those phagocytized were frequently observed in the process of being carried to the lumina of excurrent canals within Again, the appearance of archaeocytes. many of the phagocytized erythrocytes suggested intracellular degradation. Although some encapsulated erythrocytes were found along the borders of excurrent canals, th(a cwilddcd in the mesomajorit!, rc~maincd gka. Recline. Irregular clumps of archaeocytes and collencytes were found adhering to
the surfaces of implanted rediae as in 6-hr specimens. Again, a few fully developed cercariae which had been released from ruptured rediae were observed. It is of interest to note that the sponge cells which had formed a packed layer surrounding cercariae in 6-hr specimens had coalesced, forming a relatively thick syncytial capsule around freed cercariae in 12-hr specimens (Fig. 2, E). Mzcscle. The number of host cells adhering to the surfaces of implanted muscles had increased in 12-hr specimens (Fig. 2, F). These, however, retained their integrity rather than form a syncytial tunica. A few had migrated beneath the muscle surfaces, between the myofibers.
Controls.
There was a slight increase in the number of free cells. primarily collencytes and archaeocytes, along the cut edges in the control animals (Fig. 3, A).
32
CHENG,
YEE,
RIFKIN,
AND
KAAI\IER
or implanted tissues stainetl FIG. 2. All photomicrographs are of sections of either ?‘cr)lios zdeki within phagocytic archaeocytes in mc~pl~a ( 6 hr po-twith hematoxylin and eosin. A. Erythrocytes implantation). B. Erythrocyte-laden archaeocytes migrating into escurrent canal of sponge ( 6 hr postimplantation). C. Irregulnr aggregates of archarocytes and collencytes on surface of impl:mted redia ( 6 hr postimplantation). D. Relatively imiform layer of nrcharocytrs and collrncytrs adherir~ to surface of cercaria (6 hr postimplantation). E. Formation of syrtcytinl tunic of hoct c:~lls cllrroundinv cercaria ( I2 hr po?timplantation ) F. Irregular aggregatcas of archacocytcs and collcncytcs on surfarc ) of implanted molluscan muscle ( L q hr postimplautntion v\c’, v\current vand: m11, 11111~c.le; pc’, pll:i~:oarcharocytc~s and collencytes; cer, cercaria; ac, sponge cytized erythrocytes: rw, redial wall: s, syncytial capsnk of ho\t ccb.
INTERNAL
DEFENSE
MECHANISMS
OF
SPONGES.
III.
33
FIG. 3. All photomicrograph; are of sections of either Terpios Zrteki or iniulnnted materials stained with hematoxylin and eosin. A. Slight concentration of parenchymal cells along cut edges in control specimen ( 12 hr po;tincision) . B. Freed erythrocytes in excurrent canal (24 hr postimplantation). C. Section of cercaria showing presence of syncytial capsule of host cells. Notice that the implanted tissue is commencing to disintegrate (24 hr postimplantation). D. Section of disintegrating cercaria showing disappearance of syccytial capsule of host cells and infiltration of foreign tissue by archaeocytes (48 hr postimplantation). E. Relatively heavy concentration of parenchymal cells along cut edges in control specimen (49 hr postincision). a, archaeocytes; cat, concentration of archaeocytes and collencytes; dc, decomposing cercaria; e. human erythrocytes; exe, excurrent canal; in, incision; s. syncytial capsule of host cells.
34
CHENG,
Twenty-four
Hours
YEE,
RIF’KIN,
Postimplantation
Erythrocytes. Practically no free erythrocytes occurred in the mesoglea of sponges examined at this time interval. Those found were encapsulated, either singly or in small groups, by fused archaeocytes. On the other hand, large numbers of erythrocytes occurred in the lumina of excurrent canals (Fig. 3, B). Most of these had ruptured out of archaeocytes and were found as clumps in the canals. Rediae. It was evident by the 24th hr postimplantation that the rediae had died and their tissues were disintegrating. Despite the changes associated with disintegration, no true encapsulation of redial tissues occurred although archaeocytes and collencytes were observed not only adhering to the redial surfaces as at previous time intervals but a few were also found infiltrating the decomposing redial tissues. Again, cercariae which had escaped from ruptured rediae were found in sponges examined at .this time interval. These cercariae were also disintegrating. The syncytial encapsulating layer of host cells was still evident surrounding each decomposing cercaria (Fig. 3, C). Muscle. The cellular reactions of the hosts observed at this time interval were comparable to those found at the 6th and 12th hr although critical examination revealed the fusing of the cytoplasm of adjacent collencytes. These, however, did not form a continuous capsule. Controls. The edges of the incision made in control sponges were no different from those observed at 12 hr. Forty-eight
Hours
Postimplantation
Erythrocytes. Very few erythrocytes, free, encapsulated, or phagocytized, were found in the mesoglea of sponges examined at this time interval. A large number of encapsulated and phagocytized erythro-
AND
KRAMER
cytes, however, were found along the borders and in the lumina of excurrent canals. As was the case in 24-hr specimens, many of the erythrocytes found in the canals had ruptured out of phagocytic archaeocytes. Rediae. By the 48th hr postimplantation, both rediae and freed cercariae were at advanced stages of disintegration. In fact, their tissues had decomposed to such a state that their integrities were barely distinguishable. In the case of both disintegrating rediae and cercariae, sponge cells, primarily archaeocytes, had infiltrated the foreign tissues. Furthermore, in the case of cercariae, the encapsulating syncytial layer of host cells was no longer visible (Fig. 3, D). It is of interest to note that large numbers of archaeocytes enclosing amorphous materials were found in the lumina of excurrent canals. These may represent phagocytic cells which had removed disintegrating cells and cell fractions of rediae and cercariae. Muscle. Muscle implants examined at this time interval were also decomposing. The individual myofibers were no longer discrete and were poorly stained. Host cells, both archaeocytes and collencytes, were found not only forming an uneven layer covering the surface, but were also found infiltrating throughout the decomposing myofibers. Controls. The two surfaces resulting from the incision were not fused. There were heavy concentrations of free cells, primarily collencytes, but also some archaeocytes, along the wounded surfaces (Fig. 3, E). Many of the collencytes had fused to form a netlike reticulum. The appearance and arrangement of these cells suggested that fusion of the two opposing surfaces would not follow but that the exposed layers would become new surfaces lined with cells originating as collencytes.
INTERNAL ~KUSSION
AND
DEFENSE
MECHANISMS
COSCLUSIONS
It is evident from our observations that the sponge Terpios zeteki is capable of encapsulating experimentally introduced heterologous biological materials (heterografts) although the efficiency of this mechanism varies, depending on the nature of the implanted material. For example, it has been noted that human erythrocytes are completely encapsulated in addition to being phagocytized and cercariae freed from ruptured rediae are completely encapsulated. On the other hand, although sponge archaeocytes and collencytes do adhere to the outer surfaces of implanted rediae and molluscan muscles, the formation of a complete capsule does not occur. The complete encapsulation of cercariae by a relatively thick syncytium is of interest. It is recalled that Thakur and Cheng (1968) have demonstrated that Philopl~thalnw gralli cercariae commence to secrete cytogenous materials from three distinct types of subsurfacial gland cells prior to their escape from rediae. This material is uniformly deposited on the body surface of each cercaria. It is thus possible that the more spectacular and efficient encapsulation of cercariae, as compared with cellular reactions towards rediae or muscles, represents the response of sponge archaeocytes and collencytes to the proteinaceous cystogenous material as is the case in molluscs harboring trematode metacercariae (Cheng et al., 1966a, b). It is interesting that human erythrocytes, which average 7.74 p in diameter, can become phagocytized by archaeocytes which are smaller, averaging 6 by 5 p (Cheng et al., 1968a). This indicates that phagocytic archaeocytes can become hypertrophied to accommodate foreign cells of slightly greater dimensions. The fact that both phagocytized and encapsulated erythrocytes are all essentially eliminated from the parenchyma of T. zeteki by the 48th hr via the migration of
OF
SPONGES,
III.
35
erythrocyte-laden or enveloping archaeocytes across lining pinacocytes into excurrent canals indicates that not only arc archaeocytes enclosing foreign bodies capable of migration, but small encapsulation complexes, involving several archaeocytes surrounding a foreign body, are also capable although with less rapidity. Finally, the aggregation of sponge cells around implanted rediae, cercariae, and muscles should not be totally attributed to attraction by the heterografts since examination of our controls has revealed that there is some migration of cells to the wounded surfaces as related to would healing. However, the fact that sponge cells do become adhered to the surfaces of the heterografts and form a complete capsule in the case of cercariae indicates that the cellular response can be attributed, at least in part, to the presence of the implanted materials. REFERENCES
T. C., SIIUSTER, C. N., JR., AND ANDERSON, A. H. 1966a. A comparative study of the susceptibility and response of eight species of marine pelecypods to the trematode Himasthlu quissetensis. Truns. Am. Microscop. Sot., 85, 284-295. CHENG, T. C., SHUSTER, C. N., JR., AND ANDERSON, A. H. 1966b. Effects of plasma and tissue extracts of marine pelecypods on the cercaria of Himasthla qniwetcnsis. Exptl. Purusitol., 19, 9-14. CHENG, T. C., YEE, H. \V. F., ANI) RIFKIN, E. 1968a. Studies on the internal defense mechanisms of sponges. I. The cell types occurring in the mesoglea of Terpios z&&i (de Laubenfels ) ( Porifera: Demospongiae ) Pacific Sci., m press. CIIENG, T. C., RIFKIN, E., AND YEE, H. W. F. 1968b. Studies on the internal defense mechanisms of sponges. II. Phagocytosis and elimination of India ink and carmine particles by certain parenchymal cells of Terpios ~eteki. J. Invertebrate Pathol., 10, 302-309. THAKUR, A. S., AND CHEN~, T. C. 1968. The formation, structure, and histochemistry of the metacercarial cyst of Philophthalmu gralli Mathis and Lwer.,I ParusitoZoct/. .“_ iu _ press.
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