Archaeocytes from the spongeEphydatia fluviatilis were dissociated and then isolated on Ficoll density gradients. Their aggregation and reconstitution processes were studied by transmission electron microscopy to determine their capabilities for differentiation.Archaeocyte aggregates follow a well defined sequence of differentiation to generate the characteristic structures of a sponge. Pinacoderm is the first structure to be regenerated and appears progressively at the surface of the 12 h aggregates. Pinacocytes which have differentiated in archaeocyte aggregates are identical to native ones except that the nucleolus remains in most cells. The choanocytes appear only after 24 h by a two step process. First, small cells (choanoblasts) are formed from archaeocytes by mitosis. These cells then transform into fully differentiated choanocytes possessing collars and flagella. The early choanocyte chambers are small, irregular and randomly dispersed in the aggregates. Finally, collencytes and sclerocytes begin to appear just before the aggregates spread on the substrate.The differentiation of a suspension of pure archaeocytes is a unique model system to study sponge cell differentiation and has allowed us to demonstrate that archaeocytes isolated from developed sponges maintain the capacity to differentiate even though this capacity is not usually expressed.

The analysis and understanding of immune reactions in sponges are presently impaired by numerous discrepancies and inconsistencies between the results obtained for different species that make critical judgment difficult. In order to determine the possible influence of experimental procedures, parabiotic allografts were performed on Axinella verrucosa and Axinella damicornis. !lkree reactions were observed in allografts of Axinella verrucosa: acceptance, nonfusion, and chronic rejection. The acceptance reaction differed from the fusion of control autografts by a transient phase of dense cell accumulation between the parabionts. Nonfusion resulted in the virtual absence of reaction between the incompatible sponges. Chronic rejection was characterized by: 1) intensive cell migration and accumulation of collencytes in the zone of contact; 2) regular alignment of these cells on each side of that zone; 3) deposition of a collagen barrier that progressively joined but demarcated the parabionts. Some sponges presented nonfusion or chronic rejection depending on their graft partner. All allografts in Axinella damicornis displayed a chronic rejection reaction, which was essentially similar, but more intense and faster, than in Axinella verrucosa. However, in Axinella damicornis, the parabionts which were separated by the barrier became independent instead of joined. Reasons for and the implications of such a diversity of immune reactions in sponges are discussed.

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Allograft rejection in sponges has been repeatedly reported to be associated with cytotoxicity and tissue necrosis. The present work was undertaken in order to investigate the cellular aspects of antagonistic allograft rejection in Axinella polypordes, as part of an extended analysis of sponge immune reactions. Allografts were made by parabiosis of sponge branches, and their reaction was followed using light, transmission, and scanning electron microscopy. Rejection began paradoxically with the strong adherence of the parabionts. After three days, the zone of contact was characterized by extensive archaeocyte invasion, intensive phagocytosis, and cell lysis. Phagocytosis, achieved by the accumulated archaeocytes, involved healthy spherulous cells, and a substantial accumulation of cellular debris resulting from lysis of randomly dispersed single cells, mainly archaeocytes. The reaction lasted seven to nine days, after which cleavage of the reaction zone occurred, resulting in the separation of the parabionts. The reaction appeared to be limited to the former zone of contact, leaving a scar of denuded skeleton network. Our results clearly point out the role of archaeocytes in the rejection processes of Axinella polypoides; they suggest that cell lysis in antagonistic rejection of sponges depends on cytotoxic interactions between archaeocytes, since these cells appear to achieve both cytotoxicity, or killing activity, and phagocytosis. Some aspects of histoincompatibility in sponges and their analogies with those found in vertebrates are discussed.

A pure archeocyte fraction was isolated from the fresh-water spongeEphydatia fluviatilis by density gradient centrifugation of dissociated cell suspensions. The nature and purity of the fraction were confirmed by electron microscopy.