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248 MEMOIRS OF THE QUEENSLAND MUSEUM REGULATORY MECHANISMS OF IMMUNE factor, NFkB, that are inhibited by Cyclosporin A, a CELLS IN SPONGES. Memoirs ofthe Queensland drugoftenusedmedicallytopreventrejectioninhuman Museum 44: 248, 1999;- Gray cells, large granular transplants. UsingBoydenChamberassays, theassays wanderingcellspresentthroughoutthetissuesofmany originally used to identify vertebrate immune systMem species of sponges, have been identified as cytokines, I have succeeded in establishing in iinmunocytes in two species ofsponges, Microciona proliferathatcontactwithforeigntissuestimulatesthe proliferaandCallyspongiadiffusa. Whenthetissuesof release ofcytokines activating the migration ofgray two differentsponge individuals areapposed, thegray cells toward the contacting tissue. Similarly, doses of cells accumulate attheboundary ofcontact atthe time CyclosporinAcommonly used to inhibittheactivation oftissuerejection. Ihavesuggestedthatthesecellsmay oMtvertebrateTcells,suppresseshistocompatibilityin be viewed as the most primordial examples of prolifera and allows the healing together oftissue evolutionary predecessors of the well-known from two individual sponges that would normally vertebrate lymphocytes. This comparison implies that undergo tissue rejection. These results provide further graycellssharefeaturesofvertebrate lymphocytesand evidence that the foundations ofthe cellular immune I have examined this idea with studies on two system of animals were already established in the prominent aspects ofactivation ofT and B cells. The spongesandthatstudyofgraycellswillprovideinsight primary signalling event upon activation of a into the course of evolution of animal immunity. O lymphocyte by recognition ofan appropriate immune Porifera, immunology, immunocytes, graycells. targetisthesynthesisandreleaseofcytokinesthatalert and coordinate theactivity ofotherlymphocytes in the Tom Humphreys (email: htom(a)Jiawaii,eduL Kewalo surroundingtissueandthroughoutthebody. Inaddition MarineLaboratory, UniversityofHawaii 41 AhuiStreet, theactivationoflymphocytes involves internal second Honolulu, HI96813. USA: IJune 1998. messenger pathways converging on the transcription NEGOMBATA MAGNIFICA-A MAGNIFICENT primarily archeocytes and choanocytes, (CHEMICAL) PET. Memoirs of (he Queensland membrane-limited inclusions which are perhaps Lat B Museum 44: 248. 1999:- Negombata magnifica secretoryand/orstorage vesicles. Theconcentration of {Latrunculia) is a Red Sea sponge known to produce Lat B in the sponge periphery correlates with the the toxin latrunculin (Lat). Since synthesis of this defensive role of the toxin, since encounters with compound is economically non-viable, we evaluated epibionts, predators and competitive neighbours take various ways of producing it, while determining its place through the surface layer. It may, therefore, be natural mechanism of production and ecological useful to isolatethesecells forculture. 2) Primarycell relevance.We examined the possibility of: I) cultures were established from adults and embryos. identifying the cells which produce and harbour Mechanical dissociation of inner parts (without the latrunculin;2)establishingcellcultures; 3) formingan external layer) proved to be superior (less underwatersponge'garden';and4)takingadvantageof contaminationandmorecelltypes)toothertechniques. die sponge's own reproduction and larval settlement. Primary cultures from embryos lasted significantly Early in the study itbecame evidentthat N. magnifica longer (up to 280 days) and cells survived a freezing might actually comprisetwoclosely related species of phase. Cell lines, however, have not yet been Negomhata, oneofthemanundescribed, newspecies. established. 3) Initial steps were taken toward The work reported here refers to the original N. establishing an in situ 'garden' ofN. magnifica from magnifica. 1)The location ofLat B, wasstudied using sponge fragments. Although growth rate of sponge specific rabbit anli-Lat B antibodies. Rabbits were fragments was superior to that ofnatural sponges in immunised with a conjugate of Lat B with Keyhole their vicinity, fragment survival overayear proved to Limpet Hemocyanin (KLH), and the antibodies were depend on sponge handling, water depth and affinitypurified overaLat B-Sepharosecolumn.Thick environmental conditions (currents, sedimentation and thin sections of the sponge were analysed by etc.). 4) Negomhata magnifica had a peak in sexual iminuno-histochemical and immuno-gold techniques reproduction during the summer. Sexually produced, using light and transmission electron microscopy, naturally released, larvae were settled on plates and respectively. Latrunculin B was prominently labelled theirgrowthanddevelopmentwerefollowedforupto4 in the sponge ectosome -endosome border, especially months. G Porifera, latrunculin, natural product, inthedensecell layerbeneaththecortex. Immuno-gold localization, antibodies, reproduction, immtino- localisation within the sponge revealed that Lat B histochem'tcal and immuno-gold techniques, cell resides in the sponge cells and not in its prokaryotic culture,Negombatamagnifica. symbionts.Thelabellingdensityofgoldparticlesinthe archeocytes and choanocytes was significantly higher Micha Bart (email: milan(dpost.tau.ac.il). Departmentof thanthatoftheotherspongecelltypes(specialcellsand Zoology TelAvivUnivcrsirw Tel'Aviv69978,Israel: 1June skeleton associated cells). The antibodies labelled 1998.