Cultivation of explants of the marine sponge Crambe crambe in closed systems

Explants of the sponge Crambe crambe were cultured in natural seawater, with or without marine microalga (Phaeodactylum tricornutum) in discontinuous flow through systems and in continuous flow-through systems (DFTHS and CFTHS, respectively). Growth was measured as the increase in underwater weight. In the experiment carried out in the CFTHS, the explants average underwater weight increased by up to 1380% of the initial weight in 22-45 days. Growth in DFTHS was much slower producing a gain of up to an average value of 322% of the initial weight in 100-210 days. Growth kinetics varied considerably for different explants. Explants grew fastest in the first 10-days of subculture. The sponges grew better in CFTHS compared with the DFTHS. The high growth rates observed in CFTHS suggest that this technique is a promising method for culturing C. crambe in closed systems.     

Growth of the sponge Pseudosuberites (aff.) andrewsi in a closed system

Explants of the Indo-Pacific sponge Pseudosuberites aff. andrewsi were fed with the microalgae Chlorella sorokiniana and Rhodomonas sp. It was microscopically observed that these algae were ingested and digested by the sponge cells, suggesting that they were consumed by the sponges. The algae were further used for two growth experiments with five explants of P. aff. andrewsi and four explants of P. andrewsi. Growth was measured as the increase in projected body area. The explants showed considerable growth (up to 730% in 54 days for P. aff. andrewsi and up to 680% in 22 days for P. andrewsi), which is much higher than previously reported growth rates for sponges. Growth started after a stationary phase of 5–20 days in which the projected body area did not increase. The growth of P. aff. andrewsi appeared to be linear and was inhibited at the end of the experiment. Two explants of P. andrewsi showed exponential growth instead of linear growth. Hence, no general statements about the growth kinetics of these sponges can be made at this time. However, the high growth rates found in this study suggest a promising future for cultivation of sponges in closed systems.     

A bioreaction–diffusion model for growth of marine sponge explants in bioreactors

Marine sponges are sources of high-value bioactives. Engineering aspects of in vitro culture of sponges from cuttings (explants) are poorly understood. This work develops a diffusion-controlled growth model for sponge explants. The model assumes that the explant growth is controlled by diffusive transport of at least some nutrients from the surrounding medium into the explant that generally has a poorly developed aquiferous system for internal irrigation during early stages of growth. Growth is assumed to obey Monod-type kinetics. The model is shown to satisfactorily explain the measured growth behavior of the marine sponge Crambe crambe in two different growth media. In addition, the model is generally consistent with published data for growth of explants of the sponges Disidea avara and Hemimycale columella. The model predicted that nutrient concentration profiles for nutrients, such as dissolved oxygen within the explant, are consistent with data published by independent researchers. In view of the proposed model’s ability to explain available data for growth of several species of sponge explants, diffusive transport does play a controlling role in explant growth at least until a fully developed aquiferous system has become established. According to the model and experimental observations, the instantaneous growth rate depends on the size of the explant and all those factors that influence the diffusion of critical nutrients within the explant. Growth follows a hyperbolic profile that is consistent with the Monod kinetics.     

Progress towards a controlled culture of the marine sponge Pseudosuberites andrewsi in a bioreactor

Explants of the tropical sponge Pseudosuberites andrewsi were fed with the marine diatom Phaeodactylum tricornotum. The food was supplied either as intact algae or as a filtered crude extract. Growth (measured as an increase in underwater weight) was found in both experiments. The explants fed with intact algae increased to an average underwater weight of 255% of the initial weight in 45-60 days. The explants fed with crude extract increased to an average of 200% of the initial weight in 30 days. These results show that it is possible to grow a sponge using a single microorganism species as a food source. In addition, it was demonstrated that sponges are also capable of growing on non-particulate food. Therefore, this study is an important step forward towards the development of controlled, in vivo sponge cultures.     

In Vitro CuIture of the Tropical Sponge <Emphasis Type="Italic">Axinella corrugata</Emphasis> (Demospongiae): Effect of Food Cell Concentration on Growth,Clearance Rate,and Biosynthesis of Stevensine

In vitro culture is one possible method for supplying sponge metabolites for pharmaceutical applications, but appropriate feeding regimens that maximize both growth and metabolite biosynthesis are largely unknown. According to the natural concentration (NC) of cells 1 to 50 µm in size that are available to wild Axinella corrugata, we fed explants a multispecific diet of bacteria, microalgae, and yeast at 4 different concentrations: 1NC, 3NC, 5NC, and 5+1NC (the last consisted of 5 NC of bacteria and 1 NC of microalgae and yeast). Explants fed a 3NC diet had the best culture response, growing on average from 8.5 g to 10.3 g in 8 weeks, and showing a 110% increase in concentration (milligrams per gram of dry weight) of the antitumor compound stevensine. Stevensine production in 3NC explants, representing the total milligrams of metabolite per explant, increased by 157% over the study. Explants fed at 1NC had relatively stable weights, indicating that the diet met metabolic costs only. Explants fed at the two highest concentrations lost weight after 4 weeks, possibly because long-term high cell concentration blocked their aquiferous system, reducing their ability to feed efficiently. Stevensine production in explants fed the 1NC, 5NC, or 5+1NC diets were similar, and varied little from the initial amount. A separate experiment showed that the clearance rate for A. corrugata is similar between the examined food types and cell concentrations over 5 hours, averaging 766 ml h^–1 g DW^–1.Overall, this study demonstrates that relatively small changes in food abundance can greatly affect both sponge growth and metabolite biosynthesis. The good growth and increased production of the target metabolite stevensine for A. corrugata explants fed a 3NC diet suggests that in vitro culture is a viable method of supplying some sponge metabolites.     

Cultivation of Marine Sponges

There is increasing interest in biotechnological production of marine sponge biomass owing to the discovery of many commercially important secondary metabolites in this group of animals. In this article, different approaches to producing sponge biomass are reviewed, and several factors that possibly influence culture success are evaluated. In situ sponge aquacultures, based on old methods for producing commercial bath sponges, are still the easiest and least expensive way to obtain sponge biomass in bulk. However, success of cultivation with this method strongly depends on the unpredictable and often suboptimal natural environment. Hence, a better-defined production system would be desirable. Some progress has been made with culturing sponges in semicontrolled systems, but these still use unfiltered natural seawater. Cultivation of sponges under completely controlled conditions has remained a problem. When designing an in vitro cultivation method, it is important to determine both qualitatively and quantitatively the nutritional demands of the species that is to be cultured. An adequate supply of food seems to be the key to successful sponge culture. Recently, some progress has been made with sponge cell cultures. The advantage of cell cultures is that they are completely controlled and can easily be manipulated for optimal production of the target metabolites. However, this technique is still in its infancy: a continuous cell line has yet to be established. Axenic cultures of sponge aggregates (primmorphs) may provide an alternative to cell culture. Some sponge metabolites are, in fact, produced by endosymbiotic bacteria or algae that live in the sponge tissue. Only a few of these endosymbionts have been cultivated so far. The biotechnology for the production of sponge metabolites needs further development. Research efforts should be continued to enable commercial exploitation of this valuable natural resource in the near future. Received November 5, 1998; accepted June 20, 1999.     

Oxygen dynamics in choanosomal sponge explants

Oxygen microprofiles were measured over the boundary layer and into the tissue of 10-day-old cultivated tissue fragments (explants of 2-4 cm³) from the choanosome of the cold-water sponge Geodia barretti with oxygen-sensitive Clark-type microelectrodes. At this time of cultivation, the surface tissue and the aquiferous system of the explants is regenerating, which makes oxygen and nutrient supply by pumping activity impossible. Oxygen profiles showed a parabolic shape, indicating oxygen flux over a diffusive boundary layer and into the tissue. Oxygen was always depleted only 1 mm below the sponge surface, leaving the major part of the explants anoxic. Diffusive oxygen flux into the explant was calculated from three oxygen profiles using Fick's first law of diffusion and revealed 9 μmol O₂ cm^-3 day^-1, which is in the lower range of in situ oxygen consumption of whole sponges. The ability of G. barretti to handle continuous tissue anoxia enables choanosomal explants to survive the critical first weeks of cultivation without a functional aquiferous system, when oxygen is supplied to the sponge explant by molecular diffusion over its surface.     

Do associated microbial abundances impact marine demosponge pumping rates and tissue densities?

The evolution of marine demosponges has led to two basic life strategies: one involving close associations with large and diverse communities of microorganisms, termed high microbial abundance (HMA) species, and one that is essentially devoid of associated microorganisms, termed low microbial abundance (LMA) species. This dichotomy has previously been suggested to correlate with morphological differences, with HMA species having a denser mesohyl and a more complex aquiferous systems composed of longer and narrower water canals that should necessitate slower seawater filtration rates. We measured mesohyl density for a variety of HMA and LMA sponges in the Florida Keys, and seawater pumping rates for a select group of these sponges using an in situ dye technique. HMA sponges were substantially denser than LMA species, and had per unit volume pumping rates 52–94% slower than the LMA sponges. These density and pumping rate differences suggest that evolutionary differences between HMA and LMA species may have resulted in profound morphological and physiological differences between the two groups. The LMA sponge body plan moves large quantities of water through their porous tissues allowing them to rapidly acquire the small particulate organic matter (POM) that supplies the majority of their nutritional needs. In contrast, the HMA sponge body plan is suited to host large and tightly packed communities of microorganisms and has an aquiferous system that increases contact time between seawater and the sponge/microbial consortium that feeds on POM, dissolved organic matter and the raw inorganic materials for chemolithotrophic sponge symbionts. The two evolutionary patterns represent different, but equally successful patterns and illustrate how associated microorganisms can potentially have substantial effects on host evolution. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Distinct histomorphology for growth arrest and digitate outgrowth in cultivated Haliclona sp. (Porifera: Demospongiae)

The use of sponges in biotechnological processes is limited by the supply problem, and sponge biomass production is becoming a current topic of research. The distinction between characteristics for growth and growth arrest is also important for environmental monitoring. In this study, we analyze the morphology of the digitate outgrowths from the sponge Haliclona sp . The sponge Haliclona sp . was successfully cultivated for 14 months in a closed system. The morphological characterization of growth arrest was performed after submitting explants to starvation‐stress for approximately 2 weeks, to correlate morphology with growth and growth arrest. The digitate outgrowth showed three distinct regions: mature (MR), transition (TR) and immature (IR). Our data suggest a growth developmental program, with collagen fascicles guiding axial growth in IR, followed by progressive development of choanocyte chambers and large aquiferous systems at the more mature proximal region (choanosome). The intercalation of choanocyte chambers and small aquiferous systems inside collagen fascicles previously originated at the IR region can be responsible for thickening expansion and conversion of the collagen fascicles into columnar choanosome in MR. The growth arrest after starvation‐stress assay showed morphological changes in the IR corroborating collagen in the extreme tip of the digitate outgrowth as an important role in guiding of axial growth of Haliclona sp . The identification of distinct morphologies for growth and growth arrest suggest a growth developmental program, and these data could be useful for further investigations addressing sponge biomass gain and environmental monitoring.     

Sustained growth of explants from Mediterranean sponge Crambe crambe cultured in vitro with enriched RPMI 1640

Marine sponges are potential sources of many unique metabolites, including cytotoxic and anticancer compounds. Natural sponge populations are insufficient or inaccessible for producing commercial quantities of metabolites of interest. It is commonly accepted that tissue (fragments, explants, and primmorphs) and in vitro cell cultivation show great potential. However, there is little knowledge of the nutritional requirements of marine sponges to carry out efficient and sustained in vitro culture and progress has been slow. In marine invertebrate fila many unsuccessful attempts have been made with in vitro cultures using typical commercial animal cell media based on sources of dissolved organic carbon (DOC) (e.g., DMEM, RPMI, M199, L-15, etc.). One of the reasons for this failure is the use of hardly identifiable growth promoters, based on terrestrial animal sera. An alternative is the use of extracts from marine animals, since they may contain nutrients necessary for growth. In this work we have cultivated in vitro explants of the encrusting marine sponge Crambe crambe. It is one of the most abundant sponges on the Mediterranean coastline and also possesses an array of potentially active metabolites (crambines and crambescidins). Initially a new approach was developed in order to show consumption of DOC by explants. Thus, different initial DOC concentrations (300, 400, 700 and 1200 mg DOC L(-1)) were assayed. Consumption was evident in all four assays and was more marked in the first 6 h. The DOC assimilation data were adjusted to an empirical model widely used for uptake kinetics of organic dissolved compounds in marine invertebrates. Second, a protocol was established to cultivate explants in vitro. Different medium formulations based on RPMI 1640 commercial medium enriched with amino acids and inorganic salts to emulate seawater salinity were assayed. The enrichment of this medium with an Octopus aqueous extract in the proportions of 10% and 20% (v/v) resulted in an evident sustained long-term growth of C. crambe explants. This growth enhancement produced high metabolic activity in the explants, as is confirmed by the high ammonium and lactate content in the medium a few days after its renewal and by the consumption of glucose. The lactate accumulation increased with the size and age of explants. Prior to these experiments, we successfully developed a robust new alternative method, based on digital image treatment, for accurate determination of the explant apparent volume as growth measure.     

Problems of Coloniality,Modularity, and Individuality in Sponges and Special Features of Their Morphogeneses During Growth and Asexual Reproduction

A comparative analysis of the organization of sponges has been carried out to clarify problems of their coloniality, individuality, and modularity. The morphological, physiological, morphogenetic, and immunological aspects of the problem have been analyzed. The followers of the hypothesis of colonial organization of sponges interpret the process of “new zooid” formation as an “incomplete asexual reproduction.” A comparative analysis of morphogeneses in sponges during growth processes and asexual reproduction has clearly shown them to be different. A rearrangement (remodeling) of structures accompanied by disorganization and reorganization of tissues in neighboring elements of aquiferous system is the basis of growth. Migration of polypotent and secretory cells into the core of bud development is the major mechanism of budding. The formation of new aquiferous units (aquiferous modules) does not represent an “incomplete asexual reproduction.” Thus, the terms “colony” and “zooid” cannot be applied to the sponges. A morphologically separate sponge, irrespective of its level of organization (ascon, sycon, or leucon) and the number of oscula (aquiferous modules) should be considered as an individual.     

The marine sponge Ianthella basta can recover from stress-induced tissue regression

Sponges often exhibit tissue regression in response to stressful conditions. This study investigated whether handling stress invoked tissue regression in Ianthella basta and assessed whether sponges could recover from this regressed tissue state. Six necrotic specimens and 12 healthy explants were collected at Orpheus Is. Australia and transported to aquarium facilities. Sponges were photographed daily and an integrated density (ID) measurement was used to quantify tissue regression. Histological samples were taken from sponge explants to compare cellular organization. Bacterial communities of regressed and recovered tissue were compared using Denaturing Gradient Gel Electrophoresis (DGGE). After 12 h both necrotic and healthy sponges displayed substantial tissue regression. However, within 72 h all sponges recovered to their original condition. The ID of the sponge tissue doubled, confirming tissue recovery in I. basta. Sponges affected by tissue regression had significantly fewer choanocyte chambers and more densely packed granulated cells than recovered sponges. DGGE revealed the same microbial symbionts in both regressed and recovered sponges. Handling stress associated with collection and transportation is sufficient to invoke tissue regression in this species, but sponges can rapidly recover. This study contributes to our understanding of how sponges respond to environmental pressures, influencing population resilience and persistence.     

In vitro sponge fragment culture of Chondrosia reniformis (Nardo, 1847)

In vitro cultivation systems for sponges (Porifera) have to be developed to produce compounds of value in biotechnological processes. Organotypic culture attempts, which maintain or mimic the natural tissue structure, are promising ways towards a biotechnology of sponges. We used the Mediterranean species Chondrosia reniformis for sponge fragment in vitro cultivation. The species is common throughout the Mediterranean, easy to keep in aquariums and shows good recovery and regeneration after fragmentation. The regeneration process of the 50-80 mm(3) fragments lasted for several days and resulted in a rounded or ovoid body shape. The aquiferous system was reduced. Cells performed proliferation during the first weeks as we could demonstrate by 5-bromo-2'-deoxy-uridine (BrdU) incorporation. No proliferation could be demonstrated after a culture period of 3 months, but silicate uptake. Cellular density decreased with cultivation length, but collagen production increased. Fragments have been kept in culture up to 19 months. C. reniformis can be used as a model system to develop feeding strategies and evaluate the biotechnological potential of sponge fragment in vitro cultivation.     

Abundance and Bioactivity of Cultured Sponge-Associated Bacteria from the Mediterranean Sea

In this study, the search for new antibiotics was combined with quantitative ecological studies. The cultured fraction of the associated bacterial communities from ten different Mediterranean sponge species was investigated. To obtain quantitative and qualitative data of sponge-associated bacterial communities and to expand the cultured diversity, different media were used. The largest morphological diversity and highest yield of isolates was obtained by using oligotrophic media, which consisted of natural habitat seawater amended with (1% additional carbon sources. The dominant bacterial morphotypes were determined and bacterial isolates were tested for antimicrobial activity and identified using 16S rDNA sequencing. The sponge-associated most abundant morphotypes were all affiliated to the Alphaproteobacteria and showed antimicrobial activity against at least one of the tested strains. In contrast, the ambient seawater was dominated by Gammaproteobacteria. One single alphaproteobacterium, which was related to Pseudovibrio denitrificans, was shown to dominate the cultured community of at least six of the sponges. This designated MBIC3368-like alphaproteobacterium has been isolated from sponges before and seems to be restricted to associations with members of the phylum Porifera. It displays a weak and unstable antimicrobial activity, which gets easily lost during cultivation. However, this bioactive bacterium was present in the sponges by up to 10⁶ cells per gram wet-weight sponge tissue and dominated the cultured fraction with up to 74%. The association of this alphaproteobacterium with sponges is probably evolutionary young and facultative and possibly involves biologically active secondary metabolites. Besides a demonstrated vertical transfer, additional horizontal transfer between the sponges is assumed. Members of the genus Bacillus displaying antimicrobial activity were found regularly, too. However, actinomycetes, which are known for their production of bioactive substances, were present in very low abundance.     

Farming Sponges to Supply Bioactive Metabolites and Bath Sponges: A Review

Sponges have been experimentally farmed for over 100 years, with early attempts done in the sea to supply “bath sponges”. During the last 20 years, sponges have also been experimentally cultured both in the sea and in tanks on land for their biologically active metabolites, some of which have pharmaceutical potential. Sea-based farming studies have focused on developing good farming structures and identifying the optimal environmental conditions that promote production of bath sponges or bioactive metabolites. The ideal farming structure will vary between species and regions, but will generally involve threading sponges on rope or placing them inside mesh. For land-based sponge culture, most research has focused on determining the feeding requirements that promote growth. Many sea- and land-based studies have shown that sponges grow quickly, often doubling in size every few months. Other favorable results and interesting developments include partially harvesting farmed sponges to increase biomass yields, seeding sexually reproduced larvae on farming structures, using sponge farms as large biofilters to control microbial populations, and manipulating culture conditions to promote metabolite biosynthesis. Even though some results are promising, land-based culture needs further research and is not likely to be commercially feasible in the near future. Sea-based culture still holds great promise, with several small-scale farming operations producing bath sponges or metabolites. The greatest potential for commercial bath sponge culture is probably for underdeveloped coastal communities, where it can provide an alternative and environmentally friendly source of income.     

Bacterial Community Dynamics in the Marine Sponge <Emphasis Type="Italic">Rhopaloeides odorabile</Emphasis> Under In Situ and Ex Situ Cultivation

Cultivation of sponges is being explored to supply biomaterial for the pharmaceutical and cosmetics industries. This study assesses the impact of various cultivation methods on the microbial community within the sponge Rhopaloeides odorabile during: (1) in situ cultivation under natural environmental conditions, (2) ex situ cultivation in small flow-through aquaria and (3) ex situ cultivation in large mesocosm systems. Principal components analysis of denaturing gradient gel electrophoresis profiles indicated a stable microbial community in sponges cultured in situ (grown in the wild) and in sponges cultured ex situ in small flow-through aquaria over 12 weeks. In contrast, a shift in the microbial community was detected in sponges cultivated ex situ in large mesocosm aquaria for 12 months. This shift included (1) a loss of some stable microbial inhabitants, including members of the Poribacteria, Chloroflexi and Acidobacteria and (2) the addition of new microbes not detected in the wild sponges. Many of these acquired bacteria had highest similarity to known sponge-associated microbes, indicating that the sponge may be capable of actively selecting its microbial community. Alternatively, long-term ex situ cultivation may cause a shift in the dominant microbes that facilitates the growth of the more rare species. The microbial community composition varied between sponges cultivated in mesocosm aquaria with different nutrient concentrations and seawater chemistry, suggesting that these variables play a role in structuring the sponge-associated microbes. The high growth and symbiont stability in R. odorabile cultured in situ confirm that this is the preferred method of aquaculture for this species at this time.     

海绵生物活性物质及海绵细胞离体培养

介绍了来自海绵的生物活性物质种类、分布及其潜在的应用价值。讨论了其作为抗癌、抗病毒、抗细菌等药用的生物活性物质及其相关的海绵种属 ;强调海绵生物活性物质的商业化和临床应用所面临的“供给短缺问题”。作为解决这一问题的途径之一 ,海绵细胞离体培养是最有前景的技术。讨论了海绵细胞离体培养技术的研究现状 ,存在的问题及未来的发展趋势。对我国海域的海绵生物活性物质的研究开发现状进行总结 ,强调海绵研究对开发具有我国自主知识产权的新药、新化合物的必要性及重要性 ,并提出进行研发的可能优先领域     

Explant organ culture of hamster alimentary tract epithelium

Adult Syrian Golden hamster alimentary tract maintained as explants in organ culture was studied using the model system for hamster pancreas described by Resau et al. (1983a). Explants of esophagus, stomach, duodenum and colon were maintained in organ culture on Gelfoam® sponge rafts in a high-oxygen atmosphere with serum-supplemented CMRL-1066 medium. All of the tissues were observed to show evidence of sublethal acute cell injury during the first several days of culture. Subsequently, the epithelial tissues recovered from this injury, repopulated the denuded areas of the explants and replicated within the sponge matrix. Explants were maintained in a differentiated state for 30+ days and sampled for morphology to examine the process of cell injury, repair, differentiation and replication which occurs in mucosal epithelia. The percentage of basement membrane covered by epithelia in the explants from various tissues was compared to the level of LDH in the media to reveal the relationship between viability determined by biochemical and by morphological methods. Restitution of the mucosal surface occurred in all of the explants. We conclude that adequate populations of replicating cells are maintained within the epithelium of the hamster alimentary tract tissues in vitro so that restitution can occur through migration and subsequent differentiation of the epithelial cells within the mucosa of the explants.Abbreviations 4F-1G 4% formaldehyde 1% glutaraldehyde fixative - LDH lactate dehydrogenase - OsO₄ osmium tetroxide - PAS/PLH periodic acid, periodic acid Shiff lead hematoxylin stain     

Monitoring Microbial Community Composition by Fluorescence In Situ Hybridization During Cultivation of the Marine Cold-Water Sponge Geodia barretti

To determine the stability and specificity of microbes associated with the marine cold-water sponge Geodia barretti during cultivation, we compared the microbial community of freshly retrieved specimens to that of cultivated explants by fluorescence in situ hybridization (FISH). G. barretti hosts a specific homogeneous microbial community in its mesohyl, which is maintained during a cultivation period of 8 months. In 10-day-old explants, bright colonies of unusually large bacterial cells, located predominantly at canal walls, were observed in addition to the common bacteria. Bacteria of the aberrant type included both lineages present in whole sponges and foreign ones, notably numerous genera of sulfate-reducing bacteria. We assume that these represent infectious bacteria that eluded the innate immune system of the sponge. Explants that resist these microbial attacks during the critical phase of cultivation eliminate infectious bacteria. The intrinsic microbial community of G. barretti is not affected by these infections and remains persistent over a cultivation period of at least several months.     

Wnt signaling and induction in the sponge aquiferous system: evidence for an ancient origin of the organizer

The importance of polarity-the possession of a primary body axis-is evident in the functional features of animals, such as feeding, and therefore must have arisen simultaneously with the evolution of multicellular animal body plans. Sponges are thought to represent the most ancient extant lineage of multicellular animals and whereas adult sponges do not possess obvious polarity, they are useful study organisms in which to examine the origin and evolution of body polarity. We tested the effect of pharmacological agents known to disrupt the polarity of a wide variety of animals on sponge organization during development. Lithium chloride and alsterpaullone, which mimic canonical Wnt signaling in other animals, caused formation of ectopic oscula and disrupted the ability of the sponge to feed. Transplanted oscula were able to attach to and induce canal reorganization in host sponges suggesting that the osulum has inductive capabilities. This work suggests that canonical Wnt signaling is responsible for setting up the aquiferous system, which acts as an organizing center polarizing the sponge.