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Absence of alloimmune memory in the freshwater sponge Ephydatia fluviatilis
DEVELOPMENTAL AND COMPARATIVE
IMMUNOLOGY, Vol. 7, pp. 609-612, 0145-305X/83 $3°00 + .00 Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All rights reserved.
1983.
ABSENCE OF ALLOIMMUNE MEMORY IN THE FRESHWATER SPONGE EPHYOATIA FLUVIATILIS
Gys@le Van de Vyver Laboratoire de 8iologie animale et cellulaire Universit@ fibre de Bruxelles 50, av. F.D. Roosevelt, 1050 8ru×elles, Belgium
ABSTRACT.
Second-set allograft experiments were performed on the freshwater sponge Ephydatia fluviati~is in order to determine the eventual existence of a specific memory component. Graft rejection resides in an early collagen layer deposition at the interface of the foreign individuals. Based on the challenge time of collagen layering, cur results demonstrate that for Ephydatia fluviati~is, a second-set graft rejection gives neither accelerated nor intensified reactions. Consequently, a specific memory component fails in Ephydatia.
INTRODUCTION At present, the existence of an immunological memory-type response in sponges has been inadequately investigated and is still a point of controversy. 11, 2, 3) The experiments reported in this work were designed to determine more accurately, under standardized conditions, if an allogenic memory exists in the freshwater sponge Ephydatia f~uviatilis. This species has appeared to be particularly suitable to test this property because several definite strains are available and because freshwater sponges can be cultivated from gemmules under controlled conditions of medium, temperature, illumination and feeding. One of the best ways to test individuality and memory in higher organisms is to perform first-set and second-set grafts between individuals In freshwater sponges, however, individuality and memory can be demonstrated in a simpler way, allowing gemmuLes to hatch side by side. If the young sponges share the same individuality they will fuse. If they are different, they fail to fuse and a sharp layer of collagen is secreted between them. This technique is similar to grafting and non-merging is comparable to graft rejection, but it offers an advantage in the sense that it produces no physiological traumatism to the sponges. In order to compare the rejection behaviour of sponges, all at the same stage of development, the gemmules used to determine the first-set 6O9
610
ABSENCE OF MEMORY IN SPONGES
Vol. 7, No. 4
and the second-set rejection times, were placed simultaneously in the culture medium. However, the sponges that have to be checked for memory were kept isolated by glass capillaries of 20 sm in diameter, as ion Z as necessary. To do this, gemmules were placed in four parallel lines. The first and third lines, respectively, called al and a2 were composed of fifteen gemmules belonging to the strain a. ]he second and fourth lines, called BI and ~2 consisted of ~ gemmules [fig. 1},At the beginning of the experiments, a2 was isolated by a capillary from ~I and by another capillary from ~2. Rejection of foreign Ephydatia coming in contact involves a two step process. The first step consists of the secretion of a collagen barrier along the whole zone of contact. ]he second step occurring only if the available surface is large enough, results in the complete separation of the foreign sponges after a few days of contact. If a form of allogeneic immunological memory exist in Ephydatia, the time taken by sponges to produce the collagen barrier and to move apart must be shorter in the case of a second-set contact than in the case of a first-set contact.
RESULTS Production
of the collagen
ANO OISCUSSION
barrier
figs. Fig.
Fig.
I,
2
1. Non-merging front between aland 61, 115 h after the Remmules were placed in culture, a2and B2are still isolated by glass capillaries. 2. Non-merging between Bland a2and between m2and B2, 6 h after the removal of the capillaries [ > collagen barrier}.
In a set of fifteen identical experiments, ~ and B gemmules were aligned. Under our culture conditions, that is, in a mineral medium, at
Vol.
7, No. 4
ABSENCE OF MEMORY IN SPONGES
611
20°C, under constant i l h m i n a t i o n and without feeding, the collagen barrier between ~i and BI growing freely in contact was completely developed after an average time of 115 h with a standard deviation I o ] = 1.5, after the gemmules were placed in culture [£ig. I]. Other things being equal, this average time depends on the distance between the foreign gemmules that have been aligned. It is important to understand the experimental technique shown. In each experiment,the capillaries isolating ~2 from Bl and from B2 , were removed with the help of a razor blade, five hours after the complete formation of the collagen barrier between ~i and Bl This r e m o v a l brought ~2 simultaneously into contact with Bl which had a former contact with % a n d with B2 which is naive. After the removal of the capillaries, the average time required to produce the collagen barrier between Bl and e2 and between ~2 and B2 was essentially identical. In both situations, the average time was 5 h 44 min (fig. 2). The results indicate, that for sponges Kept under similar conditions, there is nothing to distinguish chronologically a first-set contact from a second-set contact.
Separation of foreign sponges After the formation of the barrier between ~l and B1 ; Bl and ~2 , and between ~2 and B2, the close contact lasted for several days. After that period, however, a31 of the sponges began to move apart. Two separation stages were checked for memory : the very beginning, defined by the appearance of a fissure between the foreign sponges; and the stage following complete separation. In order to dispose of the problem of a separation reference time, we set up 15 series of controls by pairing ~ and B sponges that were able to move apart in any direction. The results obtained with the control series appear to be important. Indeed, they clearly demonstrate that for sponges of the same age, grown under controlled conditions and able to move freely apart, the time required for separation is completely random. In these experiments, the onset of separation varied from 77 - 270 h~ the average was 152 h with a o of 55. The time required for complete separation ranged from 148-437 h; the average was 260 h with o of 83. Moreover, the separation times recorded for the experimental series indicate that the time is dependent on the space available for migration. Two sponges, one having a large migration space and the other only a small space, separated more slowly than pairs in which both members were able to migrate freely. They separate more quickly, however, than sponges that were "sandwiched" between others. Thus. the time required for separation for ~2 and the naive B2 cannot be used as a test for immunological memory, since 81 and B2 are not under similar conditions as far as the space available for migration is concerned Ifig. 2). Theoretically, it would be possible to prepare a set of experiments in which naive and sensitized sponges would be under similar migration conditions. Nevertheless, the
612
ABSENCE OF MEMORY IN SPONGES
Vol. 7, No.
random nature of the separation time, as indicated by the results of the control series, brings us to a realization thab the separation of foreign sponges is definitely not a suitable test for memory. On the contrary, the chronological regularity with which the collagen barrier is produced, provides a good criterion for determining whether an alloimmune memory exists. The results o f our experiments indicate that in the freshwater sponge cultivated and tested under standardized conditions, no form of immunological memory can be detected since the rejection processes correlated with a second-set contact do not differ from those correlated with a first-set contact.
EpAydat¢a
We propose the hypothesis that the rejection phenomenon in sponges is primarily a form of cellular immunity shared by all cell types [4) and that no particular immunotype cell exists, The absence of such a cell type would explain why there is no alloimmunological memory in sponges.
REFERENCES I.
HILDEMANN, W.H., JOHNSTON, I.S., and JOKIEL, P.L. Immunocomoetence in the lowest metazoan ohylum : transplantation immunity in sponges. Science 204, 420, 1978.
2.
EVANS, C.O., KERR, J., and CURTIS A.S.O. Graft rejection and immune memory in marine sponges. In Phylogeny of Immunolozical Memory. M.J. MANNING {Ed.] Elsevier, 1980, p. 27.
3.
VAN DE VYVER, G. Second-set allograft rejection in two sponges species and the problem of an alloimmune memory. In Phylogeny of Immunological Memory. M.J. MANNING lEd.] Elsevier, 1980, p. 15.
4.
DE SUTTER, O., and VAN DE VYVER, G. Isolation and recognition properties of some definite sponge cell types. Oev. Comp. Immunol. 3, 389, 1979.
IMMUNOLOGY, Vol. 7, pp. 609-612, 0145-305X/83 $3°00 + .00 Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All rights reserved.
1983.
ABSENCE OF ALLOIMMUNE MEMORY IN THE FRESHWATER SPONGE EPHYOATIA FLUVIATILIS
Gys@le Van de Vyver Laboratoire de 8iologie animale et cellulaire Universit@ fibre de Bruxelles 50, av. F.D. Roosevelt, 1050 8ru×elles, Belgium
ABSTRACT.
Second-set allograft experiments were performed on the freshwater sponge Ephydatia fluviati~is in order to determine the eventual existence of a specific memory component. Graft rejection resides in an early collagen layer deposition at the interface of the foreign individuals. Based on the challenge time of collagen layering, cur results demonstrate that for Ephydatia fluviati~is, a second-set graft rejection gives neither accelerated nor intensified reactions. Consequently, a specific memory component fails in Ephydatia.
INTRODUCTION At present, the existence of an immunological memory-type response in sponges has been inadequately investigated and is still a point of controversy. 11, 2, 3) The experiments reported in this work were designed to determine more accurately, under standardized conditions, if an allogenic memory exists in the freshwater sponge Ephydatia f~uviatilis. This species has appeared to be particularly suitable to test this property because several definite strains are available and because freshwater sponges can be cultivated from gemmules under controlled conditions of medium, temperature, illumination and feeding. One of the best ways to test individuality and memory in higher organisms is to perform first-set and second-set grafts between individuals In freshwater sponges, however, individuality and memory can be demonstrated in a simpler way, allowing gemmuLes to hatch side by side. If the young sponges share the same individuality they will fuse. If they are different, they fail to fuse and a sharp layer of collagen is secreted between them. This technique is similar to grafting and non-merging is comparable to graft rejection, but it offers an advantage in the sense that it produces no physiological traumatism to the sponges. In order to compare the rejection behaviour of sponges, all at the same stage of development, the gemmules used to determine the first-set 6O9
610
ABSENCE OF MEMORY IN SPONGES
Vol. 7, No. 4
and the second-set rejection times, were placed simultaneously in the culture medium. However, the sponges that have to be checked for memory were kept isolated by glass capillaries of 20 sm in diameter, as ion Z as necessary. To do this, gemmules were placed in four parallel lines. The first and third lines, respectively, called al and a2 were composed of fifteen gemmules belonging to the strain a. ]he second and fourth lines, called BI and ~2 consisted of ~ gemmules [fig. 1},At the beginning of the experiments, a2 was isolated by a capillary from ~I and by another capillary from ~2. Rejection of foreign Ephydatia coming in contact involves a two step process. The first step consists of the secretion of a collagen barrier along the whole zone of contact. ]he second step occurring only if the available surface is large enough, results in the complete separation of the foreign sponges after a few days of contact. If a form of allogeneic immunological memory exist in Ephydatia, the time taken by sponges to produce the collagen barrier and to move apart must be shorter in the case of a second-set contact than in the case of a first-set contact.
RESULTS Production
of the collagen
ANO OISCUSSION
barrier
figs. Fig.
Fig.
I,
2
1. Non-merging front between aland 61, 115 h after the Remmules were placed in culture, a2and B2are still isolated by glass capillaries. 2. Non-merging between Bland a2and between m2and B2, 6 h after the removal of the capillaries [ > collagen barrier}.
In a set of fifteen identical experiments, ~ and B gemmules were aligned. Under our culture conditions, that is, in a mineral medium, at
Vol.
7, No. 4
ABSENCE OF MEMORY IN SPONGES
611
20°C, under constant i l h m i n a t i o n and without feeding, the collagen barrier between ~i and BI growing freely in contact was completely developed after an average time of 115 h with a standard deviation I o ] = 1.5, after the gemmules were placed in culture [£ig. I]. Other things being equal, this average time depends on the distance between the foreign gemmules that have been aligned. It is important to understand the experimental technique shown. In each experiment,the capillaries isolating ~2 from Bl and from B2 , were removed with the help of a razor blade, five hours after the complete formation of the collagen barrier between ~i and Bl This r e m o v a l brought ~2 simultaneously into contact with Bl which had a former contact with % a n d with B2 which is naive. After the removal of the capillaries, the average time required to produce the collagen barrier between Bl and e2 and between ~2 and B2 was essentially identical. In both situations, the average time was 5 h 44 min (fig. 2). The results indicate, that for sponges Kept under similar conditions, there is nothing to distinguish chronologically a first-set contact from a second-set contact.
Separation of foreign sponges After the formation of the barrier between ~l and B1 ; Bl and ~2 , and between ~2 and B2, the close contact lasted for several days. After that period, however, a31 of the sponges began to move apart. Two separation stages were checked for memory : the very beginning, defined by the appearance of a fissure between the foreign sponges; and the stage following complete separation. In order to dispose of the problem of a separation reference time, we set up 15 series of controls by pairing ~ and B sponges that were able to move apart in any direction. The results obtained with the control series appear to be important. Indeed, they clearly demonstrate that for sponges of the same age, grown under controlled conditions and able to move freely apart, the time required for separation is completely random. In these experiments, the onset of separation varied from 77 - 270 h~ the average was 152 h with a o of 55. The time required for complete separation ranged from 148-437 h; the average was 260 h with o of 83. Moreover, the separation times recorded for the experimental series indicate that the time is dependent on the space available for migration. Two sponges, one having a large migration space and the other only a small space, separated more slowly than pairs in which both members were able to migrate freely. They separate more quickly, however, than sponges that were "sandwiched" between others. Thus. the time required for separation for ~2 and the naive B2 cannot be used as a test for immunological memory, since 81 and B2 are not under similar conditions as far as the space available for migration is concerned Ifig. 2). Theoretically, it would be possible to prepare a set of experiments in which naive and sensitized sponges would be under similar migration conditions. Nevertheless, the
612
ABSENCE OF MEMORY IN SPONGES
Vol. 7, No.
random nature of the separation time, as indicated by the results of the control series, brings us to a realization thab the separation of foreign sponges is definitely not a suitable test for memory. On the contrary, the chronological regularity with which the collagen barrier is produced, provides a good criterion for determining whether an alloimmune memory exists. The results o f our experiments indicate that in the freshwater sponge cultivated and tested under standardized conditions, no form of immunological memory can be detected since the rejection processes correlated with a second-set contact do not differ from those correlated with a first-set contact.
EpAydat¢a
We propose the hypothesis that the rejection phenomenon in sponges is primarily a form of cellular immunity shared by all cell types [4) and that no particular immunotype cell exists, The absence of such a cell type would explain why there is no alloimmunological memory in sponges.
REFERENCES I.
HILDEMANN, W.H., JOHNSTON, I.S., and JOKIEL, P.L. Immunocomoetence in the lowest metazoan ohylum : transplantation immunity in sponges. Science 204, 420, 1978.
2.
EVANS, C.O., KERR, J., and CURTIS A.S.O. Graft rejection and immune memory in marine sponges. In Phylogeny of Immunolozical Memory. M.J. MANNING {Ed.] Elsevier, 1980, p. 27.
3.
VAN DE VYVER, G. Second-set allograft rejection in two sponges species and the problem of an alloimmune memory. In Phylogeny of Immunological Memory. M.J. MANNING lEd.] Elsevier, 1980, p. 15.
4.
DE SUTTER, O., and VAN DE VYVER, G. Isolation and recognition properties of some definite sponge cell types. Oev. Comp. Immunol. 3, 389, 1979.