Petrosia ficiformis (Poiret, 1789): an excellent model for holobiont and biotechnological studies

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The aggregation of prokaryotic and eukaryotic cells has resulted in evolution of organisms with remarkable abilities to synthetize natural bioactive compounds of biotechnological relevance. Marine sponges such as Petrosia ficiformis are examples of this evolutionary strategy. The P. ficiformis microbiome, which produces a diversity of chemical compounds, plays a fundamental role in this sponge’s extraordinary adaptation to various ecological conditions. The microbial community of P. ficiformis seems representative of sponge microbiomes, but it has an unusual exclusively horizontal transmission. This uncommon feature, together with its wide environmental distribution, its ability to generate 3D cell cultures that host symbionts, and the availability of meta-omics and physiology information make this sponge an effective model to study the complexity of holobionts.     

Seasonal production of primmorphs from the marine sponge Petrosia ficiformis (Poiret, 1789) and new culturing approaches

The need to produce bioactive compounds from marine sponges leads several groups of research to the culture of primmorphs from different species, which are generally maintained in aquaria for long time before processing. Here we present a study where the importance of several parameters on primmorphs production from the symbiotic sponge Petrosia ficiformis has been evaluated: (i) the sterility of sea water, (ii) the maintenance in aquarium before processing, (iii) the seasonal cycle. Sterility of sea water does not improve primmorphs production in this species. The maintenance of sponges in aquaria before processing negatively affects cell cultures. Regarding seasonality, it is evident that both the number and the size of primmorphs can deeply change depending on the period of the year the sponge is collected. April and July are the months that lead to the highest number of primmorphs, May and June are the months that lead to their biggest sizes. Possible relationships of these results with the life cycle of P. ficiformis are discussed.     

Optimizing the formation of in vitro sponge primmorphs from the Chinese sponge Stylotella agminata (Ridley)

The establishment and optimization of in vitro primmorph formation from a Chinese sponge, Stylotella agminata (Ridley), collected from the South China Sea, were investigated. Our aims were to identify the key factors affecting primmorph formation in this species and to optimize the technique for developing an in vitro primmorph culture system. The size of dissociated cells from S. agminata is relatively small, in the range between 5 and 10 microm. Round-shaped primmorphs of less than 100 microm were formed 3 days after transferring the dissociated cells into seawater containing Ca(2+) and Mg(2+). The effect of various cell dissociation conditions, inoculum cell density, concentration of antibiotics, pH, and temperature was further investigated upon the formation of primmorphs. The time required for primmorph formation, primmorph size distribution, and the proliferating capability were microscopically documented. Healthy sponge S. agminata, inoculum cell density and culture temperature play a critical role for the successful formation of primmorphs and that the microbial contamination will have to be controlled.     

Cultivation of primmorphs from the marine sponge Suberites domuncula: morphogenetic potential of silicon and iron

Marine demosponges (phylum Porifera) are rich sources for potent bioactive compounds. With the establishment of the primmorph system from sponges, especially from Suberites domuncula, the technology to cultivate sponge cells in vitro improved considerably. This progress was possible after the elucidation that sponges are provided with characteristic metazoan cell adhesion receptors and extracellular matrix molecules which allow their cells a positioning in a complex organization pattern. This review summarizes recent data on the cultivation of sponges in aquaria and--with main emphasis--of primmorphs in vitro. It is outlined that silicon and Fe(+++) contribute substantially to the formation of larger primmorphs (size of 10 mm) as well as of a canal system in primmorphs; canals are probably required for an improved oxygen and food supply. We conclude that the primmorph system will facilitate a sustainable use of sponges in the production of bioactive compounds; it may furthermore allow new and hitherto not feasible insights into basic questions on the origin of Metazoa.     

The architecture of the canal systems of Petrosia ficiformis and Chondrosia reniformis studied by corrosion casts (Porifera,Demospongiae)

Summary The three-dimensional organization of the canal system in two sponge species, Petrosia ficiformis and Chondrosia reniformis, was studied using corrosion casts. Casts were made of live animals, in situ, and canal replicas were analzyed by scanning electron microscopy (SEM). In P. ficiformis the incurrent system consists of a superficial canal network giving rise to large radial canals, which ramify and anastomosize forming an internal web. Excurrent canals are arranged into modular ramified systems radiating from atrial cavities opening to the exterior. Main excurrent canals run at various depths within the sponge, even through the superficial incurrent network. Both incurrent and excurrent canal replicas show smooth, blind-ending capillaries. Some large incurrent canals merge with excurrent ones, thus bypassing choanocyte chambers. In C. reniformis there is a cortical collagen layer crossed by three-like incurrent canals, the twigs of which communicate with groups of inhalant pores. The stems of tree-like canals penetrate into the sponge medulla where they ramify and anastomosize to form a web. Main excurrent canals arise from large cloacal ducts leading to the oscular openings. They give rise to a sequence of branches intersecting the incurrent web. Both incurrent and excurrent canals have sharp, blind-ending capillaries. Morphometric data functions show that diameter scaling in canal branches is exponential in Petrosia and linear in Chondrosia. Structural differences and homologies between the two species are discussed.     

Strongylophorine-8, a pro-electrophilic compound from the marine sponge Petrosia (Strongylophora) corticata, provides neuroprotection through Nrf2/ARE pathway

Green plant-origin electrophilic compounds are a newly-recognized class of neuroprotective compounds that provide neuroprotection through activation of the Nrf2/ARE pathway. Electrophilic hydroquinones are of particular interest due to their ability to become electrophilic quinones upon auto-oxidation. Although marine organisms frequently produce a variety of electrophilic compounds, the detailed mechanisms of action of these compounds remain unknown. Here, we focused on the neuroprotective effects of strongylophorine-8 (STR8), a para-hydroquinone-type pro-electrophilic compound from the sponge Petrosia (Strongylophora) corticata. STR8 activated the Nrf2/ARE pathway, induced phase 2 enzymes, and increased glutathione, thus protecting neuronal cells from oxidative stress. Microarray analysis indicated that STR8 induced a large number of phase 2 genes, the regulation of which is controlled by the Nrf2/ARE pathway. STR8 is the first example of a neuroprotective pro-electrophilic compound from marine organisms.     

Sterols of the marine sponge Petrosia weinbergi: implications for the absolute configurations of the antiviral orthoesterols and weinbersterols

The marine sponge Petrosia weinbergi was found to contain isofucosterol and clionasterol as its major sterols. The rare cyclopropyl sterol (24S,28S)-24,28-methylenestigmast-5-en-3beta-ol, previously detected as only 0.07% of the total sterols of a pelagophytic alga Pulvinaria sp., made up 6.6% of the total sterols. These sterols are believed to be the biosynthetic precursors of the antiviral orthoesterols and weinbersterols found in the same sponge. Based on the side chains of the isolated sterols, the absolute configurations of the antiviral steroid side chains are assigned to be (24R,28S)- for orthoesterol B, (24R)- for orthoesterol C, and (24S,28S)- for weinbersterols A and B.     

Branching out: a remarkable new branching syllid (Annelida) living in a Petrosia sponge (Porifera: Demospongiae)

We describe the morphology and biology of a previously unknown form of branching annelid, Ramisyllis multicaudata gen. et sp. nov. , an endosymbiont of shallow‐water marine sponges (Petrosia sp., Demospongiae) in northern Australia. It belongs to the polychaete family Syllidae, as does Syllis ramosa McIntosh, 1879, the only other named branching annelid, which was collected from deep‐water hexactinellid sponges during the 1875 Challenger expedition. It differs from S. ramosa in parapodial and chaetal morphology. Ramisyllis multicaudata gen. et sp. nov. has segments of several types, including specialized posterior segments on the emergent portions of the worm, and simplified elongate segments that bridge larger cavities in the sponge interior. Aside from the obvious branching form, the new annelid is similar to Parahaplosyllis, differing from it in lacking pharyngeal armature and in the details of the parapodial chaetae and dorsal cirri. Molecular evidence from 16S and 18S rDNA supports a sister‐group relationship with Parahaplosyllis, with both being sister to Trypanosyllis and Eurysyllis. The phylogenetic position of R. multicaudata gen. et sp. nov. indicates that branching has evolved independently in Ramisyllis gen. nov. and Syllis. This is supported by differences in the branching process between the two taxa: in S. ramosa branching is initiated by segment addition at the parapodium, whereas in R. multicaudata gen. et sp. nov. segments are added from a region between parapodia. A model for branching in R. multicaudata gen. et sp. nov. is proposed and possible developmental processes underlying branching in Annelida, and body symmetry comparisons with other invertebrates, are also discussed. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 164 , 481–497.     

Primary cultures from the marine sponge Xestospongia muta (Petrosiidae,Haplosclerida)

In the context of the investigations on the origin and in vitro production of bioactive compounds, primary cultures were developed from ectosomal and choanosomal cell suspensions from the sponge Xestospongia muta. Dissociated cells aggregated and reorganized into a striking reticulated network of cells, typical for X. muta. Moreover, in some cultures an isotropic reticulation of small spicules, very similar to that found in the ectosome of adult sponges, was observed. Phytohaemagglutinin promoted aggregation and the reorganization of the cells. HPLC analyses revealed that straight-chain acetylenic compounds were recovered from short-term cultures and that they were synthesized during culture. Heterotrophic bacteria were assumed to be involved in the process. Together our results established that X. muta would be an excellent experimental model to study, in laboratory conditions, the differentiation of the skeleton and the in vitro biosynthesis of straight-chain acetylenic compounds.     

Antimicrobial activity of marine sponge Clathria indica (Dendy, 1889)

Sponges are sessile filter feeders that have developed efficient defense mechanisms against foreign invaders such as viruses, bacteria or eukaryotic organisms. Antimicrobial peptides are known as major components of the innate immune defense system in marine invertebrates. The aim of the present work was to study the antimicrobial properties of the Indian sponge Clathria indica with special reference to the identification of antimicrobial peptides. Crude methanolic extract and its chloroform, n-butanol and aqueous fractions were tested against 16 human pathogens which include eleven bacteria with four of them being multidrug resistant and five pathogenic fungi. All fractions showed effective antibacterial activity against common and multidrug-resistant Salmonella typhi and antifungal activity against C. albicans and C. neoformans. However, they were ineffective against Escherichia coli, Pseudomonas aeruginosa, Streptococcus pyogenes and Staphylococcus aureus. Chloroform fraction being the most potent among the fractions tested on chemical investigation was indicative of the presence of peptides as evidenced by ninhydrin positive spots on TLC and presence of peptide bonds by NMR. Its ESI-MS showed presence of several peptides in the range of m/z 850 to 980. Structure of three peptides has been tentatively assigned by ESI-MS/MS or tandem mass analysis, on the basis of the amino acid sequence established. The results clearly show that the sponge C. indica represent an interesting source of marine invertebrates-derived antimicrobial peptides in the development of new strategies to treat various infectious diseases.     

Pyrosequencing reveals sponge specific bacterial communities in marine sponges of Red Sea,Saudi Arabia

Bacterial communities of marine sponges are believed to be an important partner for host survival but remain poorly studied. Sponges show difference in richness and abundance of microbial population inhabiting them. Three marine sponges belonging to the species of Pione vastifica, Siphonochalina siphonella and Suberea mollis were collected from Red sea in Jeddah and were investigated using high throughput sequencing. Highly diverse communities containing 105 OTUs were identified in S. mollis host. Only 61 and 43 OTUs were found in P. vastifica and S. siphonella respectively. We identified 10 different bacterial phyla and 31 genera using 27,356 sequences. Most of the OTUs belong to phylum Proteobacteria (29%–99%) comprising of Gammaproteobacteria, Alphaproteobacteria, and Deltaproteobacteria where later two were only detected in HMA sponge, S. mollis. A number of 16S rRNA sequences (25%) were not identified to phylum level and may be novel taxa. Richness of bacterial community and Shannon, Simpson diversity revealed that sponge S. mollis harbors high diversity compared to other two LMA sponges. Dominance of Proteobacteria in sponges may indicate an ecological significance of this phylum in the Red sea sponges. These differences in bacterial composition may be due to difference in location site or host responses to environmental conditions. To the best of our knowledge, the microbial communities of these sponges have never been studied before and this is first attempt to unravel bacterial diversity using PCR-based 454-pyrosequencing method.     

Biosilica formation in spicules of the sponge Suberites domuncula: synchronous expression of a gene cluster

The formation of spicules is a complicated morphogenetic process in sponges (phylum Porifera). The primmorph system was used to demonstrate that in the demosponge Suberites domuncula the synthesis of the siliceous spicules starts intracellularly and is dependent on the concentration of silicic acid. To understand spicule formation, a cluster of genes was isolated. In the center of this cluster is the silicatein gene, which codes for the enzyme that synthesizes spicules. This gene is flanked by an ankyrin repeat gene at one side and by a tumor necrosis factor receptor-associated factor and a protein kinase gene at the other side. All genes are strongly expressed in primmorphs and intact animals after exposure to silicic acid, and this expression is restricted to those areas where the spicule formation starts or where spicules are maintained in the animals. Our observations suggest that in S. domuncula a coordinated expression of physically linked genes is essential for the synthesis of the major skeletal elements.     

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.     

Induction of apoptosis by the marine sponge (Mycale) metabolites, mycalamide A and pateamine

The marine sponge metabolites mycalamide A (myca-lamide) and pateamine are extremely cytotoxic. While mycalamide has been shown to inhibit protein synthesis, the mechanism by which these compounds induce cell death is unknown. Using DNA laddering, Annexin-V staining, and morphological analysis, we demonstrate that both metabolites induce apoptosis in several different cell lines. Furthermore, both mycalamide and pateamine were more potent inducers of apoptosis in the 32D myeloid cell line after transformation with either the ras or bcr-abl oncogenes. This increased sensitivity was also observed in response to the protein synthesis inhibitors cycloheximide and puromycin, and cytosine--D-arabinofurano-side (Ara-C), an inducer of DNA damage. We propose, therefore, that in 32D cells where Ras signalling has been altered either by constitutive expression of oncogenic ras or by Bcr/abl-mediated perturbation of upstream signalling events, increased susceptibility to apoptosis by a range of stimuli is conferred.     

Silica spicules and axial filaments of the marine sponge Stelletta grubii (Porifera,Demospongiae)

Summary In all cases an organic axial filament within the silica spicules of Stelletta grubii forms the core of the major axes of the glass. In the small, star-shaped silica spicules (asters) the filament is shown for the first time to be radial with an enlarged center; in the large four-rayed spicules (triaenes) it is four-rayed; and in the large single-rayed spicules (oxeas) the filament is single-rayed. In situ, the filament is not dissolved by boiling nitric acid and thus is apparently protected by encasement within the glass which can also be stratified. The small silica asters are formed by single cells which resemble the so-called spherulous cells of other sponges. The very large size of triaenes and oxeas suggests that they may possibly be formed by more than one cell. The diameter of the filament in the much smaller asters is much narrower than the filament in the larger spicules, indicating a possible relationship between filament diameter and spicule diameter. While the axial filament in larger spicules frequently has a triangular cross-section it can also be hexaognal. Some aster filaments also retain a close to hexagonal cross-section. Filaments freed from large spicules by hydrofluoric acid display a complex morphology; possibly there is an internal silicified core. Some reported aspects of filament morphology are, however, probably artefacts of desilicification with hydrofluoric acid. Offprint requests to: T.L. Simpson, Department of Biology, University of Hartford, West Harford, Connecticut 06117, USA (Permanent affiliation)     

Antifouling activity exhibited by secondary metabolites of the marine sponge,Haliclona exigua (Kirkpatrick)

Bioassay guided purification of the acetone extract of the marine sponge, Haliclona exigua, (Gulf of Mannar, India) yielded a fraction rich in bis-1-oxaquinolizidine alkaloids, active against seven strains of fouling bacteria as well as cyprids of the cosmopolitan barnacle, Balanus amphitrite. The major alkaloids in the mixture have been tentatively identified as nor-araguspongine C (33.76%), araguspongine C (6.49%), dihydroxy araguspongine (36.36%), methyl and dimethyl derivatives of the latter (12.98 and 10.38%, respectively) from HRMS studies. The lower EC₅₀ (6.6 μg/ml as against the US Navy standard of 25 μg/ml for NPAs) and low toxicity (LC₅₀ 18 μg/ml as compared to 0.00001 μg/ml for TBT) values, coupled with its favourable therapeutic ratio (2.7 as against the requirement of >1) makes these compounds ideal NPAs in environmentally compatible antifouling coatings.     

Polymorphic microsatellite loci isolated from the marine sponge Scopalina lophyropoda (Demospongiae: Halichondrida)

A total of seven microsatellites out of 88 isolated from a genomic library enriched for (CA)_n and (GA)_n repeats were characterized in the Mediterranean marine sponge Scopalina lophyropoda. The microsatellite motifs were large (34.81 ± 13.9 bp) and imperfect. The seven microsatellite loci were screened in 30 individuals collected from Blanes, northwestern Mediterranean. All of them were polymorphic (allele numbers and observed heterozygosities ranged from 3 to 6 and from 0.16 to 0.76, respectively). No significant linkage disequilibrium between pairs of loci and no departure from Hardy–Weinberg equilibrium were found. These markers are therefore promising for studies of the population structure of the species.     

Biochemical and biophysical characterization of collagens of marine sponge, Ircinia fusca (Porifera: Demospongiae: Irciniidae)

Collagens were isolated and partially characterized from the marine demosponge, Ircinia fusca from Gulf of Mannar (GoM), India, with an aim to develop potentially applicable collagens from unused and under-used resources. The yield of insoluble, salt soluble and acid soluble forms of collagens was 31.71 ± 1.59, 20.69 ± 1.03, and 17.38 ± 0.87 mg/g dry weight, respectively. Trichrome staining, Scanning & Transmission Electron microscopic (SEM & TEM) studies confirmed the presence of collagen in the isolated, terminally globular irciniid filaments. The partially purified (gel filtration chromatography), non-fibrillar collagens appeared as basement type collagenous sheets under light microscopy whereas the purified fibrillar collagens appeared as fibrils with a repeated band periodicity of 67 nm under Atomic Force Microscope (AFM). The non-fibrillar and fibrillar collagens were seen to have affinity for anti-collagen type IV and type I antibodies raised against human collagens, respectively. The macromolecules, i.e., total protein, carbohydrate and lipid contents within the tissues were also quantified. The present information on the three characteristic irciniid collagens (filamentous, fibrillar and non-fibrillar) could assist the future attempts to unravel the therapeutically important, safer collagens from marine sponges for their use in pharmaceutical and cosmeceutical industries.