Allogeneic cell interactions during graft rejection in Callyspongia diffusa (Porifera, Demospongia); a study with monoclonal antibodies

Many aspects of the cellular immune system in the marine sponge Callyspongia diffusa, have been defined by using artificially transplanted allogeneic tissues. Rejections show specificity of 'non-self' recognition, cytotoxic effector responses and short-term immunological memory. Histological investigations reveal a generalized mesohyl migration to the graft zone where archaeocytes line up at the allogeneic interface. Monoclonal antibodies (mAbs) raised to sponge cells have shown that little or no allogeneic cell mixing occurs at the graft interface and that certain mesohyl cell types do not appear to be directly involved in graft rejections. However, all mesohyl cell types are present in autograft fusion zones and in inflammatory responses to injury. The involvement of only some of the mesohyl cell types in graft rejections suggests specific interactions of an effector 'immunocyte'.     

Scanning electron microscope evidence for diatom uptake by two Antarctic sponges

Scanning electron microscopy (SEM) investigation of two Antarctic sponges, Phorbas glaberrima and Tedania charcoti, showed that the exopinacoderm effects a direct uptake of benthic diatoms which settle on the sponge surface. In P. glaberrima, planktonic diatoms were also observed penetrating through the inhalant system, the primary way of feeding in sponges. Benthic diatoms which accumulate in the mesohyl underneath the exopinacoderm help to strengthen the sponge cortex and may be an alimentary source during oligotrophic periods in the Antarctic environment.     

繁茂膜海绵原细胞富集细胞团培养过程中的细胞迁移规律

海绵是重要的生物活性物质来源, 近10年来, 从海绵中发现的具有生物活性的新化合物占海洋生物来源的30%以上, 并且大多具有显著的抗肿瘤, 抗艾滋病病毒的活性。但是, 由于海绵生物量不能满足这些活性物质进一步研究和商业化的需求, 目前仅有一种活性物质被成功的商业化, 这不仅是商业开发的损失, 也是提高人类生活质量活动的一种损失。为了解决海绵供给不足的问题, 人们进行了包括化学合成、海绵养殖以及海绵细胞培养在内的多种尝试,目前的研究结果表明, 海绵细胞离体培养技术是最有可能彻底解决海绵供给不足的途径之一。但是由于海绵自身的特殊性, 还没有人成功的建立起海绵细胞系以满足生产需要。人们发现, 海绵细胞的相互接触对于离体海绵细胞长期培养至关重要。经过多年的探索, 大连化物所海洋生物产品工程组建立了开发出了海绵原细胞富集细胞团培养技术, 通过对海绵组织内的原细胞进行富集来获得可长期培养的海绵细胞。海绵原细胞是海绵组织内的“干细胞”, 具有很强的分化、增殖潜力, 同时也是海绵组织内负责消化的主要细胞类型。为了探索海绵原细胞的增殖、分化规律, 本研究基于海绵原细胞富集细胞团培养体系, 构建了海绵细胞培养实时观测平台, 对繁茂膜海绵原细胞、领细胞、上皮细胞3类主要海绵细胞类型在海绵细胞团形成及生长的全过程进行观察, 了解不同类型细胞迁移规律的变化。通过对视频记录进行分析,发现离散的海绵细胞与细胞团内的海绵细胞具有截然相反的运动规律, 海绵细胞的运动具有很强的协同性。伴随原细胞在细胞团内不停息的迁移, 还观察到海绵细胞团内新生骨针的迁移以及细胞间进行颗粒物质的传递。这些信息的获得, 将有助于进一步了解不同细胞的功能与作用, 也有助于在此基础上探索海绵细胞的增殖、分化控制规律。     

The vacuolar cells of Oscarella lobularis (Porifera,Demospongiae): Ulatrastructural organization,origin, and function

This work is concerned with the ultrastructural organization and some histochemical features of the vacuolar cells of the demosponge Oscarella lobularis. Vacuolar cells are characterized by large clear vacuoles containing an aqueous fluid. They are commonly found in the mesohyl of this sponge and tend to constitute a sort of parenchyma in the choanosome. Mobile cells of the mesohyl appear to differetiate into vacuolar cells through the progressive formation of wide cytoplasmic lacunae. We have identified four types of cells showing progressive transformation toward the vacuolar cell type. Precursors (types 1–4) of the vacuolar cells probably derive from endopinacocytes, since they share several histochemical and ultrastructural characteristics with them. Our data support the notion that vacuolar cells are involved in the synthesis of collagen, act as a mechanical support of the sponge body, and are eventually extruded from the sponge through the canals of the aquiferous system.     

Purification and in vitro cultivation of archaeocytes (stem cells) of the marine sponge <Emphasis Type="Italic">Hymeniacidon perleve</Emphasis> (Demospongiae)

Marine sponges (Porifera) are the best source of marine bioactive metabolites for drug discovery and development, although the sustainable production of most sponge-derived metabolites remains a difficult task. In vitro cultivation of sponge cells in bioreactors has been proposed as a promising technology. However, no continuous cell line has as yet been developed. Archaeocytes are considered to be toti/multipotent stem cells in sponges and, when purified, may allow the development of continuous sponge cell lines. As a prerequisite, we have developed a novel four-step protocol for the purification of archaeocytes from a marine sponge, Hymeniacidon perleve: (1) differential centrifugation to separate large sponge cells including archaeocytes; (2) selective agglomeration in low-Ca²⁺/Mg²⁺ artificial seawater in which living archaeocytes form small loose aggregates with some pinacocytes and collencytes; (3) differential adherence to remove anchorage-dependent pinacocytes, collencytes and other mesohyl cells; (4) Ficoll-Vrografin density gradient centrifugation to purify archaeocytes. The final purity of archaeocytes is greater than 80%. The proliferation potential of the archaeocytes has been demonstrated by high levels of BrdU incorporation, PCNA expression and telomerase activity. In 4-day primary cultures, the purified archaeocytes show a 2.5-fold increase in total cell number. This study opens an important avenue towards developing sponge cell cultures for the commercial exploitation of sponge-derived drugs. The authors are grateful for the financial support of the Chinese Academy of Sciences under the “100 Talent Project”, the “Innovation Fund” from the Dalian Institute of Chemical Physics, the “Hi-Tech Research and Development Program of China” (2001AA620404), and the European Commission (project: Silicon Biotechnology).     

Localization of Two Brominated Metabolites,Aerothionin and Homoaerothionin,in Spherulous Cells of the Marine Sponge Aplysina fistularis (=Verongia thiona)

Energy dispersive X-ray microanalysis was used to localize the two brominated natural products (aerothinonin and homoaerothionin) in the tissues of a marine demosponge, Aplysina fistularis. Virtually all of these compounds were localized within the spherules of the spherulous cells in the mesohyl. This is the first localization of any secondary metabolite at the cellular or sub-cellular level in any marine invertebrate. In Aplysina fistularis, as in other species of the same genus studied by Vacelet, the spherulous cells are concentrated just beneath the exopinacoderm and just beneath the endopinacoderm of the excurrent canals. Moreover, there is electron microscopic evidence for degeneration of some spherulous cells throughout the mesohyl. Presumably, this degeneration can release some aerothionin and homoaerothionin, which are known to have antibiotic properties. After release from the spherulous cells, these brominated natural products could function (1) within the mesohyl to exclude some types of bacteria or to aggregate ingested bacteria and/or (2) within the boundary layer of the surrounding seawater for defense or offense, as considered in the discussion section.     

Cyclosporin A suspends transplantation reactions in the marine sponge Microciona prolifera

Sponges are the simplest extant animals but nevertheless possess self-nonself recognition that rivals the specificity of the vertebrate MHC. We have used dissociated cell assays and grafting techniques to study tissue acceptance and rejection in the marine sponge Microciona prolifera. Our data show that allogeneic, but not isogeneic, cell contacts trigger cell death and an increased expression of cell adhesion and apoptosis markers in cells that accumulate in graft interfaces. Experiments investigating the possible existence of immune memory in sponges indicate that faster second set reactions are nonspecific. Among the different cellular types, gray cells have been proposed to be the sponge immunocytes. Fluorescence confocal microscopy results from intact live grafts show the migration of autofluorescent gray cells toward graft contact zones and the inhibition of gray cell movements in the presence of nontoxic concentrations of cyclosporin A. These results suggest that cell motility is an important factor involved in sponge self/nonself recognition. Communication between gray cells in grafted tissues does not require cell contact and is carried by an extracellular diffusible marker. The finding that a commonly used immunosuppressor in human transplantation such as cyclosporin A blocks tissue rejection in marine sponges indicates that the cellular mechanisms for regulating this process in vertebrates might have appeared at the very start of metazoan evolution.     

The stem cell concept in sponges (Porifera): Metazoan traits

Sponges are considered the oldest living animal group and provide important insights into the earliest evolutionary processes in the Metazoa. This paper reviews the evidence that sponge stem cells have essential roles in cellular specialization, embryogenesis and Bauplan formation. Data indicate that sponge archaeocytes not only represent germ cells but also totipotent stem cells. Marker genes have been identified which are expressed in totipotent stem cells and gemmule cells. Furthermore, genes are described for the three main cell lineages in sponge, which share a common origin from archaeocytes and result in the differentiation of skeletal, epithelial, and contractile cells.     

Dense populations of Archaea associated with the demosponge Tentorium semisuberites Schmidt, 1870 from Arctic deep-waters

The associated microbial community in the mesohyl of the Arctic deep-water sponge Tentorium semisuberites Schmidt, 1870 (Hadromerida, Demospongiae) is dominated by Archaea. This is the result of an integral approach applying analyses of microbial lipid biomarkers as well as microscopic investigations using differential fluorescence in situ hybridisation with universal probes and counterstaining with 4′,6′-diamidino-2-phenylindole (DAPI) on sponge sections based on samples collected in the Greenland Sea in 2001, 2002 and 2005. The distribution of isoprenoidal C₄₀ hydrocarbons of the biphytane series suggests that affiliates of both major archaeal kingdoms, the Crenarchaeota and the Euryarchaeota, are present in the choanosome of T. semisuberites. Positive signals using the oligonucleotide probe ARCH915 indicate high numbers of Archaea in the mesohyl of this sponge. Based on optical estimations 70–90% of all microbial DAPI signals accounted for archaeal cells. Archaea in these high proportions have never been described in an Arctic deep-sea hadromerid sponge, nor in any other demosponge species. Similar observations in specimens collected over a time scale of 4 years suggest permanent sponge-Archaea associations.     

The significance of syncytial tissues for the position of the hexactinellida in the metazoa

Hexactinellid sponges are metazoans in which the major tissuecomponent is a multinucleated syncytium. The preferred deepwaterhabitat of these sponges makes collection of hexactinellidsin good condition difficult, and has hindered extensive examinationof their body plan. Nonetheless, over the last three decadesa number of studies have explored their ecology, histology andphysiology. It has been shown that hexactinellids are extremelylong-lived animals. Their cytoplasm consists of a giant, multinucleatedtissue, the trabecular syncytium, which is connected via openand plugged cytoplasmic bridges to cells such as archaeocytes,choanoblasts, and cells with spherical inclusions. Because allof the sponge is cytoplasmically interconnected, electricalsignals can propagate through the animal. The effector responseis arrest of the feeding current. The perforate plugged junctionapparently allows tissues to specialize in different ways whilemaintaining limited cytoplasmic continuity. Larvae of hexactinellidsponges are already largely syncytial. Although it is not knownwhen the first syncytial tissues are formed or when perforateplugged junctions first appear during embryogenesis, evidencethat embryos are cellular until gastrulation suggests that hexactinellidsponges may have evolved from cellular sponges and that syncytialtissues are not a primitive trait of the Metazoa.     

Cellular location of (2R, 3R, 7Z)-2-aminotetradec-7-ene-1, 3-diol, a potent antimicrobial metabolite produced by the Caribbean sponge Haliclona vansoesti

The Caribbean sponge Haliclona vansoesti has been found to contain large amounts of a new sphingosine derivative, (2R, 3R, 7Z)-2-aminotetradec-7-ene-1, 3-diol (compound 1). To determine the localization of this compound within the organism, cell distribution and quantitative determination of the aminodiol content of cell fractions obtained by differential centrifugation have been performed. Results show that choanocytes and archaeocytes are the major sponge cell types and that H. vansoesti harbour small photosynthetic symbionts (cyanobacteria) and few heterotrophic bacteria. Reverse-phase HPLC analyses of the cell fractions reveal that the aminodiol 1 is not associated with the prokaryotic endobionts but with the sponge cells, in particular the archaeocytes. This is clearly established by the positive significant correlation existing between the numbers of archaeocytes and the amounts of aminodiol 1. The mean aminodiol concentration is estimated to be 2 microg/10(5) archaeocytes. The aminodiol 1 is also found in substantial amounts in primary cell cultures, so that cell culture can be envisaged as an option for its production. Sponge cell suspensions display potent antibacterial and antiyeast activities, in correlation with their aminodiol content, indicating that this compound is at least in part responsible for these activities in the sponge. The release of the aminodiol I into the external medium suggests that this substance may be involved in the defence mechanisms of the sponge.     

Continuous cell movements rearrange anatomical structures in intact sponges.

Time-lapse cinemicrography was used to record the active movements of cells in living intact sponges. Each of the three main cell types (pinacocytes, mesohyl cells, and choanocytes) continuously moved and rearranged themselves so that the internal anatomy of the sponge was continuously remodeled. The shape and appearance of the sponges anatomical structures often changed substantially within a few hours. The most motile were the mesohyl cells, with many moving as fast as one cell-length per minute (15 microns/min). Mesohyl cell locomotion was often accompanied by displacements of spicules, canals, and choanocyte chambers; the patterns of these displacements suggested that the mesohyl cells were providing the motive forces for these rearrangements. The locomotion of the pinacocytes varied according to position: those along the outer sponge margins were most active, whereas those in other parts of the surface moved relatively little. Choanocytes were never observed to undergo independent locomotion but were always found grouped together in choanocyte chambers. These choanocyte chambers interacted with pinacocytes and mesohyl cells to form excurrent canals, which continuously moved, fused with, and branched from one another. These observations suggest that the experimental phenomenon of sponge cell-reaggregation and reconstitution, discovered by H. V. Wilson, represents an extreme version of morphogenetic processes that normally go on continuously within intact sponges. The results from the present study also suggest that these cellular rearrangements are controlled by active cell movements and behavioral responses that include but are not limited to selective cell adhesion.     

Phagocytic efficiency and cytotoxic responses of Indian freshwater sponge (Eunapius carteri) cells isolated by density gradient centrifugation and flow cytometry: a morphofunctional analysis

The freshwater sponge Eunapius carteri (Porifera: Demospongiae: Spongillidae), a resident of Indian freshwater ecosystems, has pharmaceutical and ecological potential, but there is inadequate information on its cellular spectrum and cell-mediated immune responses. Microscopical analysis revealed the existence of eight distinct cellular variants, i.e. blast-like cells, choanocytes, small amoebocytes, granular cells, pinacocytes, large amoebocytes, archaeocytes and sclerocytes. The cells were isolated by density gradient centrifugation and flow cytometry and used for a morphofunctional analysis. We investigated the phagocytic efficiency of E. carteri cells under the challenge of yeast particles in vitro and spectrophotometrically quantified the generation of cytotoxic molecules (superoxide anions and nitric oxide) in different isolated cellular fractions. The two cell separating technologies did not yield any significant differences in the major findings on morphology, phagocytic response and generation of superoxide anions and nitric oxide. Archaeocytes, granular cells and large amoebocytes were identified as chief phagocytes with a high phagocytic potential as recorded by light microscopy. Archaeocytes were the principal generators of superoxide anions, whereas nitric oxide was recorded in the fractions rich in archaeocytes and large amoebocytes. The present investigation thus provides useful information regarding cellular variation, cytotoxic status and innate phagocytic response of the cells of E. carteri, a common but less studied sponge of India.     

Dynamic structure of the mesohyl in the sponge Chondrosia reniformis (Porifera, Demospongiae)

The common demosponge Chondrosia reniformis possesses the capacity to undergo an unusual creep process which results in the formation of long outgrowths from the parent body. These shape changes, which have been interpreted as adaptive strategies related to environmental factors, asexual reproduction or localised locomotor phenomena, are due mainly to the structural and mechanical adaptability of the collagenous mesohyl. This contribution describes the morphological correlates of mesohyl plasticisation in C. reniformis. The microscopic anatomy of the mesohyl was examined when it was in different physiological conditions: (1) standard ”resting” condition, (2) ”stiffened” condition and (3) dynamic ”creep” condition. In this last case four representative regions of the sponge body were analysed: the parent region, the elongation region, the transition region and the propagule region. The results show that the histological modification of the sponge mesohyl during plasticisation is limited and localised. The most significant structural changes involve mainly cytological features of specific cellular components characterised by granule inclusions (i.e. the spherulous cells) and the arrangement and density of the collagenous extracellular framework, though the integrity of the collagen fibrils themselves is not affected. Morphological and functional aspects of mesohyl plasticisation invite comparison with the mutable collagenous tissue of echinoderms. Possible functional analogies between these two tissues are hypothesised. Accepted: 29 June 2001     

Vertical transmission of cyanobacterial symbionts in the marine sponge Chondrilla australiensis (Demospongiae)

The cyanobacterial symbionts of the marine sponge Chondrilla australiensis (Demospongiae) were examined using fluorescent microscopy and Transmission Electron Microscopy. Unicellular cyanobacteria with ultrastructure resembling Aphanocapsa feldmannii occur in the cortex and bacterial symbionts are located throughout the mesohyl. In C. australiensis, the developing eggs are distributed throughout the mesohyl and are surrounded by nurse cells attached to them by thin filaments. The nurse cells form cytoplasmic bridges with the eggs, apparently releasing their contents into the egg cytoplasm. The presence of cyanobacterial and bacterial symbionts inside developing eggs and nurse cells in 25% of female Chondrilla australiensiswas established using Transmission Electron Microscopy, suggesting that these symbionts are sometimes passed on to the next generation of sponges via the eggs.     

Specific and nonspecific infiltration of sponge matrix allografts by specifically sensitized cytotoxic lymphocytes

Urethane sponges coated with allogeneic or syngeneic cells were implanted subcutaneously into mice and the cytotoxicity of infiltrating host cells was assessed in vitro. First-set allogeneic sponges attracted a population of lymphocytes enriched in cytotoxic T cells directed against the alloantigens in the sponge. If two sponges bearing cells of different H-2 specificity were grafted simultaneously to a single recipient, specifically sensitized cytotoxic cells (SSCL) were found in both sponges directed against both sets of alloantigens, although specific infiltration predominated. If a syngeneic and allogeneic sponge were transplanted, SSCL were found in both the syngeneic sponge and allogeneic sponge. These data are interpreted to suggest that chemotactic substances are elaborated at graft sites which can attract circulating SSCL into sites of inflammation and that those released at the specific site are more attractive for SSCL than are those elaborated at sites of nonspecific rejection or healing. In recipients who had previously been sensitized to alloantigens, second-set grafts were rapidly infiltrated by SSCL directed against the sensitizing antigen. First-set indifferent allografts in sensitized recipients were infiltrated by SSCL directed against the previous alloantigens as well as SSCL directed against its own alloantigens. Syngeneic grafts were not infiltrated by SSCL in presensitized recipients. These data suggest that any alloantigenic stimulus can induce the mobilization from lymphoid depots of preformed SSCL directed against another set of antigens; syngeneic grafts cannot. Once mobilized, however, circulating SSCL can respond to specific and nonspecific chemotactic factors elaborated by either healing or rejecting grafts.     

Embryogenesis in the glass sponge Oopsacas minuta: Formation of syncytia by fusion of blastomeres

Sponges (Porifera) are unusual animals whose body plans makeinterpreting phylogenetic relationships within the group andwith other basal metazoan taxa a difficult task. Although molecularapproaches have offered new insights, some questions requirea morphological approach using detailed ultrastructural or lightmicroscopical studies of developing embryos and larvae. Glasssponges (Hexactinellida) have perhaps the most unusual bodyplan within the Metazoa because the majority of the tissue ofthe adult consists of a single giant multinucleated syncytiumthat forms the inner and outer layers of the sponge and is joinedby cytoplasmic bridges to uninucleate cellular regions. Herewe have used serial section transmission and high-resolutionscanning electron microscopy to examine when syncytia firstform in the cave-dwelling glass sponge Oopsacas minuta. We confirmthat in O. minuta blastomeres are separate until the 32-cellstage; cleavage is equal but asynchronous until a hollow blastulais formed. The sixth division yields a collection of variouslysized micromeres at the surface of the embryo and large yolk-and lipid-filled macromeres lining the blastocoel. Syncytiathen form by the fusion of micromeres to form cytoplasmic bridgeswith each other and the fusion of macromeres to form the futuremultinucleated trabecular tissue of the larva and adult sponge.The multinucleated trabecular tissue envelops and forms cytoplasmicbridges with all uninucleate cells, covering the developinglarva with a continuous syncytial epithelium. Differentiationof tissues occurs very early during embryogenesis with the separationof uninucleate and multinucleate lineages, but all cells andsyncytia are joined by cytoplasmic bridges such that there iscytoplasmic continuity throughout the entire larva. Althoughglass sponges begin life as a cellular embryo, the unusual mechanismof syncytia formation at such an early stage in developmentdistinguishes this group of animals from their closest multicellularrelatives, the Demospongiae. Most important, however, thesedata lend support to the hypothesis that the original metazoanswere cellular, not syncytial.     

Bcl-2 transduction protects human endothelial cell synthetic microvessel grafts from allogeneic T cells in vivo

T cell interactions with vascular endothelial cells (EC) are of central importance for immune surveillance of microbes and for pathological processes such as atherosclerosis, allograft rejection, and vasculitis. Animal (especially rodent) models incompletely predict human immune responses, in particular with regard to the immunological functions of EC, and in vitro models may not accurately reflect in vivo findings. In this study, we describe the development of an immunodeficient SCID/bg murine model combining a transplanted human synthetic microvascular bed with adoptive transfer of human T lymphocytes allogeneic to the cells of the graft that more fully recapitulates T cell responses in natural tissues. Using this model, we demonstrate that transduced Bcl-2 protein in the engrafted EC effectively prevents injury even as it enhances T cell graft infiltration and replication.     

The reaction of the sponge Chondrosia reniformis to mechanical stimulation is mediated by the outer epithelium and the release of stiffening factor(s)

Although sponges are still often considered to be simple, inactive animals, both larvae and adults of different species show clear coordination phenomena triggered by extrinsic and intrinsic stimuli. Chondrosia reniformis, a common Mediterranean demosponge, lacks both endogenous siliceous spicules and reinforcing spongin fibers and has a very conspicuous collagenous mesohyl. Although this species can stiffen its body in response to mechanical stimulation when handled, almost no quantitative data are available in the literature on this phenomenon. The present work was intended to quantify the dynamic response to mechanical stimulation both of intact animals and isolated tissue samples in order to evaluate: (i) the magnitude of stiffening; (ii) the relationship between the amount of stimulation and the magnitude of the stiffening response; (iii) the ability of the whole body to react to localized stimulation; (iv) the possible occurrence of a conduction mechanism and the role of the exopinacoderm (outer epithelium). Data on mesohyl tensility obtained with mechanical tests confirmed the difference between stimulated and non-stimulated isolated tissue samples, showing a significant relationship between ectosome stiffness and the amount of mechanical stimulation. Our experiments revealed a significant difference in tensility between undisturbed and maximally stiffened sponges and evidence of signal transmission that requires a continuous exopinacoderm. We also provide further evidence for the presence of a chemical factor that alters the interaction between collagen fibrils, thereby changing the mechanical properties of the mesohyl.     

Cellular origin of chlorinated diketopiperazines in the dictyoceratid sponge Dysidea herbacea (Keller)

The tropical marine sponge Dysidea herbacea (Keller) contains the filamentous unicellular cyanobacterium Oscillatoria spongeliae (Schulze) Hauck as an endosymbiont, plus numerous bacteria, both intracellular and extracellular. Archaeocytes and choanocytes are the major sponge cell types present. Density gradient centrifugation of glutaraldehyde-fixed cells with Percoll as the support medium has been used to separate the cyanobacterial symbiont from the sponge cells on the basis of their differing densities. The protocol also has the advantage of separating broken from intact cells of O. spongeliae. The lighter cell preparations contain archaeocytes and choanocytes together with damaged cyanobacterial cells, whereas heavier cell preparations contain intact cyanobacterial cells, with less than 1% contamination by sponge cells. Gas chromatography/mass spectrometry analysis has revealed that the terpene spirodysin is concentrated in preparations containing archaeocytes and choanocytes, whereas nuclear magnetic resonance analysis of the symbiont cell preparations has shown that they usually contain the chlorinated diketopiperazines, dihydrodysamide C and didechlorodihydrodysamide C, which are the characteristic metabolites of the sponge/symbiont association. However, one symbiont preparation, partitioned by a second Percoll gradient, has been found to be devoid of chlorinated diketopiperazines. The capability to synthesize secondary metabolites may depend on the physiological state of the symbiont; alternatively, there may be two closely related cyanobacterial strains within the sponge tissue.