Introductory comments

Abstract

Associated microorganisms have been described in numerous marine sponges. Their metabolic activity, however, has not yet been investigated in situ. We quantified for the first time microbial processes in a living sponge. Sulfate reduction rates of up to 1200 nmol cm^?3d^?1 were measured in the cold-water bacteriosponge Geodia barretti . Oxygen profiles and chemical analysis of sponge tissue and canal water revealed steep oxygen gradients and a rapid turnover of oxygen and sulfide, dependent on the pumping activity of the sponge. Identification of the microbial community with fluorescently labelled oligonucleotide probes (FISH) indicates the presence of sulfate-reducing bacteria belonging to the Desulfoarculus/Desulfomonile/Syntrophus -cluster in the choanosome of this sponge. Analysis of lipid biomarkers indicates biomass transfer from associated sulfate-reducing bacteria or other anaerobic microbes to sponge cells. These results show the presence of an anoxic micro-ecosystem in the sponge G. barretti, and imply mutualistic interactions between sponge cells and anaerobic microbes. Understanding the importance of anaerobic processes within the sponge/microbe system may help to answer unsolved questions in sponge ecology and biotechnology.     

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.     

Metaproteogenomic analysis of a community of sponge symbionts

Sponges harbour complex communities of diverse microorganisms, which have been postulated to form intimate symbiotic relationships with their host. Here we unravel some of these interactions by characterising the functional features of the microbial community of the sponge Cymbastela concentrica through a combined metagenomic and metaproteomic approach. We discover the expression of specific transport functions for typical sponge metabolites (for example, halogenated aromatics, dipeptides), which indicates metabolic interactions between the community and the host. We also uncover the simultaneous performance of aerobic nitrification and anaerobic denitrification, which would aid to remove ammonium secreted by the sponge. Our analysis also highlights the requirement for the microbial community to respond to variable environmental conditions and hence express an array of stress protection proteins. Molecular interactions between symbionts and their host might also be mediated by a set of expressed eukaryotic-like proteins and cell–cell mediators. Finally, some sponge-associated bacteria (for example, a Phyllobacteriaceae phylotype) appear to undergo an evolutionary adaptation process to the sponge environment as evidenced by active mobile genetic elements. Our data clearly show that a combined metaproteogenomic approach can provide novel information on the activities, physiology and interactions of sponge-associated microbial communities.     

Deep sequencing reveals diversity and community structure of complex microbiota in five Mediterranean sponges

Marine sponges harbor dense microbial communities of exceptionally high diversity. Despite the complexity of sponge microbiota, microbial communities in different sponges seem to be remarkably similar. In this study, we used a subset of a previously established 454 amplicon pyrosequencing dataset (Schmitt and Taylor, unpublished data). Five Mediterranean sponges were chosen including the model sponge Aplysina aerophoba to determine the extent of uniformity by defining (i) the core microbial community, consisting of bacteria found in all sponges, (ii) the variable microbial community, consisting of bacteria found in 2–4 sponges, and (iii) the species-specific community, consisting of bacteria found in only one sponge. Using the enormous sequencing depth of pyrosequencing the diversity in each of the five sponges was extended to up to 15 different bacterial phyla per sponge with Proteobacteria and Chloroflexi being most diverse in each of the five sponges. Similarity comparison of bacteria on phylum and phylotype level revealed most similar communities in A. aerophoba and A. cavernicola and the most dissimilar community in Pseudocorticium jarrei. A surprising minimal core bacterial community was found when distribution of 97% operational taxonomic units (OTUs) was analyzed. Core, variable, and species-specific communities were comprised of 2, 26, and 72% of all OTUs, respectively. This indicates that each sponge contains a large set of unique bacteria and shares only few bacteria with other sponges. However, host species-specific bacteria are probably still closely related to each other explaining the observed similarity among bacterial communities in sponges.     

Taxonomy,biogeography and DNA barcodes of Geodia species (Porifera,Demospongiae, Tetractinellida) in the Atlantic boreo‐arctic region

Geodia species north of 60°N in the Atlantic appeared in the literature for the first time when Bowerbank described Geodia barretti and G. macandrewii in 1858 from western Norway. Since then, a number of species have been based on material from various parts of the region: G. simplex, Isops phlegraei, I. pallida, I. sphaeroides, Synops pyriformis, G. parva, G. normani, G. atlantica, Sidonops mesotriaena (now called G. hentscheli), and G. simplicissima. In addition to these 12 nominal species, four species described from elsewhere are claimed to have been identified in material from the northeast Atlantic, namely G. nodastrella and G. cydonium (and its synonyms Cydonium muelleri and Geodia gigas). In this paper, we revise the boreo‐arctic Geodia species using morphological, molecular, and biogeographical data. We notably compare northwest and northeast Atlantic specimens. Biological data (reproduction, biochemistry, microbiology, epibionts) for each species are also reviewed. Our results show that there are six valid species of boreo‐arctic Atlantic Geodia while other names are synonyms or mis‐identifications. Geodia barretti, G. atlantica, G. macandrewii, and G. hentscheli are well established and widely distributed. The same goes for Geodia phlegraei, but this species shows a striking geographical and bathymetric variation, which led us to recognize two species, G. phlegraei and G. parva (here resurrected). Some Geodia are arctic species (G. hentscheli, G. parva), while others are typically boreal (G. atlantica, G. barretti, G. phlegraei, G. macandrewii). No morphological differences were found between specimens from the northeast and northwest Atlantic, except for G. parva. The Folmer cytochrome oxidase subunit I (COI) fragment is unique for every species and invariable over their whole distribution range, except for G. barretti which had two haplotypes. 18S is unique for four species but cannot discriminate G. phlegraei and G. parva. Two keys to the boreo‐arctic Geodia are included, one based on external morphology, the other based on spicule morphology. © 2013 The Linnean Society of London     

Cultivable Bacterial Community from South China Sea Sponge as Revealed by DGGE Fingerprinting and 16S rDNA Phylogenetic Analysis

The cultivable bacterial communities associated with four South China Sea sponges—Stelletta tenuis, Halichondria rugosa, Dysidea avara, and Craniella australiensis in mixed cultures—were investigated by microbial community DNA-based DGGE fingerprinting and 16S rDNA phylogenetic analysis. Diverse bacteria such as α-, γ-, δ-Proteobacteria, Bacteroidetes, and Firmicutes were cultured, some of which were previously uncultivable bacteria, potential novel strains with less than 95% similarity to their closest relatives and sponge symbionts growing only in the medium with the addition of sponge extract. According to 16S rDNA BLAST analysis, most of the bacteria were cultured from sponge for the first time, although similar phyla of bacteria have been previously recognized. The selective pressure of sponge extract on the cultured bacterial species was suggested, although the effect of sponge extract on bacterial community in high nutrient medium is not significant. Although α- and γ-Proteobacteria appeared to form the majority of the dominant cultivable bacterial communities of the four sponges, the composition of the cultivable bacterial community in the mixed culture was different, depending on the medium and sponge species. Greater bacterial diversity was observed in media C and CS for Stelletta tenuis, in media F and FS for Halichondria rugosa and Craniella australiensis. S. tenuis was found to have the highest cultivable bacterial diversity including α-, γ-, δ-Proteobacteria, Bacteroidetes, and Firmicutes, followed by sponge Dysidea avara without δ-Proteobacteria, sponge Halichondria rugosa with only α-, γ-Proteobacteria and Bacteroidetes, and sponge C. australiensis with only α-, γ-Proteobacteria and Firmicutes. Based on this study, by the strategy of mixed cultivation integrated with microbial community DNA-based DGGE fingerprinting and phylogenetic analysis, the cultivable bacterial community of sponge could be revealed effectively.     

Application of terminal restriction fragment length polymorphism (T-RFLP) analysis to monitor effect of biocontrol agents on rhizosphere microbial community of hot pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

Microbial communities in hot pepper (Capsicum annuum L.) cultivation fields under different cultivation methods were investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis. Rhizosphere soil and leaf samples were collected from control, conventional and nature-friendly cultivation fields between May and July, 2009. Two Bacillus subtilis strains were applied to nature-friendly cultivation fields as biocontrol agents during the sampling period. Relative abundances of bacteria and plant pathogenic fungi related T-RFs were also measured to monitor the effect of biocontrol agents on potential plant pathogenic fungi. In the principal component analysis (PCA) based on T-RFLP profiles, the microbial communities from rhizosphere soil samples in July, including bacteria and fungi, showed distinct difference between nature-friendly cultivation fields and other cultivation fields. However, there was no correlation between cultivation methods and leaf microbial communities at any sampling period. Changes in the abundance of bacteria related T-RF in the rhizosphere of nature-friendly cultivation fields were observed clearly two months after application of biocontrol agent, while the abundance of plant pathogenic fungi related T-RFs significantly decreased.     

Monitoring of accelerated naphthalene-biodegradation in a bioaugmented soil slurry

The effect of microbial inoculation on the mineralization of naphthalene in a bioslurry treatment was evaluated in soil slurry microcosms. Inoculation by Pseudomonas putida G7 carrying the naphthalene dioxygenase (nahA) gene resulted in rapid mineralization of naphthalene, whereas indigenous microorganisms in the PAH-contaminated soil required a 28 h adaptation period before significant mineralization occurred. The number of nahA-like gene copies increased in both the inoculated and non-inoculated soil as mineralization proceeded, indicating selection towards naphthalene dioxygenase producing bacteria in the microbial community. In addition, 16S rRNA analysis by denaturing gradient gel electrophoresis (DGGE) analysis showed that significant selection occurred in the microbial community as a result of biodegradation. However, the indigenous soil bacteria were not able to compete with the P. putida G7 inoculum adapted to naphthalene biodegradation, even though the soil microbial community slightly suppressed naphthalene mineralization by P. putida G7.     

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.     

Comparison of the Bacterial Communities of Wild and Captive Sponge Clathria prolifera from the Chesapeake Bay

The red-beard sponge Clathria prolifera, which is widely distributed in the USA, has been widely used as a model system in cell biology and has been proposed as a suitable teaching tool on biology and environmental sciences. We undertook the first detailed microbiological study of this sponge on samples collected from the Chesapeake Bay. A combination of culture-based studies, denaturing gradient gel electrophoresis, and bacterial community characterization based on 16S rRNA gene sequencing revealed that C. prolifera contains a diverse assemblage of bacteria that is different from that in the surrounding water. C. prolifera individuals were successfully maintained in a flow-through or recirculation aquaculture system for over 6 months and shifts in the bacterial assemblages of sponges in aquaculture compared with wild sponges were examined. The proteobacteria, bacteroidetes, actinobacteria, and cyanobacteria represented over 90% of the species diversity present in the total bacterial community of the wild C. prolifera. Actinobacteria, cyanobacteria, and spirochetes were not represented in clones obtained from C. prolifera maintained in the aquaculture system although these three groups comprised ca. 20% of the clones from wild C. prolifera, showing a significant effect of aquaculture on the bacterial community composition. This is the first systematic characterization of the bacterial community from a sponge found in the Chesapeake Bay. Changes in sponge bacterial composition were observed in sponges maintained in aquaculture and demonstrate the importance of monitoring microbial communities when cultivating sponges in aquaculture systems.     

Microbial symbionts of the Australian Great Barrier Reef sponge, Candidaspongia flabellata

Microbial symbionts of the newly described rare, biochemically active Dictyoceratid sponge, Candidaspongia flabellata (Very White Fan) found in the Australian Great Barrier Reef, are being studied in detail. The chemistry of this sponge species is distinctive, and includes a previously undescribed compound, fanolide as well as homosesterterpene and bishomoscalarane secondary metabolites (Bergquist et al., 1999). Current research is focused on assessing the diversity of the microbial community associated with this sponge. The entire culturable community of this sponge has been studied in detail. A total of 228 bacteria, 25 fungi, 3 actinomycetes and 9 cyanobacteria were isolated from 10 individuals of this sponge. Eight eubacteria (designated AB001–AB008), along with seven cyanobacteria were consistently found associated with C. flabellata and absent from the surrounding water column, suggesting that these bacteria have a specific association with the sponge. Partial 16S ribosomal RNA gene sequencing of these isolates was done for phylogenetic characterisation. Electron microscopy was also used to confirm the presence of many morphotypes of bacteria and indicated spatial arrangements of particular morphotypes.     

Antiangiogenic, Antimicrobial, and Cytotoxic Potential of Sponge-Associated Bacteria

The bacteria associated with marine invertebrates are a rich source of bioactive metabolites. In the present study bacteria associated with the sponge Suberites domuncula and its primmorphs (3-dimensional aggregates containing proliferating cells) were isolated and cultured. These bacteria were extracted, and the extracts were assayed for antiangiogenic, hemolytic, antimicrobial, and cytotoxic activities. Our studies revealed that extract obtained from the bacterium (PB2) isolated from sponge primmorphs is a potent angiogenesis inhibitor. In the chick chorio-allantoic membrane (CAM) assay, it showed 50% activity at 5 μg ml⁻¹ and 100% activity at 10 and 20 μg ml⁻¹ concentrations. Extracts obtained from 5 bacterial strains isolated from sponge and its primmorphs showed hemolytic activity. The sponge-associated bacteria belonging to the α subdivision of Proteobacteria and the primmorph-associated bacterium identified as a possible novel Pseudomonas sp. displayed remarkable antimicrobial activity. It is important to note that these bacterial extracts were strongly active against multidrug-resistant clinical strains such as Staphylococcus aureus and Staphylococcus epidermidis, isolated from hospital patients. The bacterial extracts having antimicrobial activity also showed cytotoxicity against HeLa and PC12 cells. In summary, this investigation explores the importance of sponge-associated bacteria as a valuable resource for the discovery of novel bioactive molecules.     

Cambrian Series 3 lithistid sponge–microbial reefs in Shandong Province,North China: reef development after the disappearance of archaeocyaths

The Cambrian Series 3 Zhangxia Formation in Shandong Province, North China, includes small‐scale lithistid sponge–microbial reefs. The lithistid sponges grew on oolitic and bioclastic sediments, which were stabilized by microbial activities. The relative abundances of microbial components (e.g. calcimicrobe Epiphyton and stromatolites) vary among the reefs. However, the microbial components commonly encrusted or bound the lithistid sponges, formed remarkable encrustations on the surfaces of the sponges. Epiphyton especially grew upward and downward. The lithistid sponges thus provided substrates for the attachment and development of microbes, and the microbes played essential roles as consolidators, by encrusting reef‐building sponges. Additionally, the lithistid sponges were prone to degradation via microbial activities and diagenetic processes, and were thus preserved as micritic bodies, showing faint spicular networks or abundant spicules. Such low preservation potential within the reef environment obscured the presence of the sponges and their widespread contribution as reef‐building organisms during the Cambrian. During the prolonged interval after the demise of archaeocyaths, purely microbial reefs, such as stromatolites and thrombolites have been considered to be the principal reef builders, in association with rare lithistid sponge–microbial associations. However, recent findings, including those from Shandong Province and Korea, suggest that the lithistid sponge‐bearing reefs were more extensive during the Epoch 3 to the Furongian than previously thought. These lithistid sponge–microbial reefs were precursors of the sponge–microbial reefs that dominated worldwide in the Early Ordovician.     

Symbiophagy and biomineralization in the “living fossil” Astrosclera willeyana

Representatives of all major metazoan lineages form biominerals. The molecular mechanisms that underlie this widespread and evolutionarily ancient ability are gradually being revealed for some lineages. However, until a wider range of metazoan biomineralization strategies are understood, the true diversity, and therefore the evolutionary origins of this process, will remain unknown. We have previously shown that the coralline demosponge, Astrosclera willeyana, in some way employs its endobiotic bacterial community to form its highly calcified skeleton. Here, using in situ hybridization and immunohistochemistry, we show that an ortholog of ATG8 (most likely a GABARAPL2/GATE-16 ortholog) is expressed in cells that construct the individual skeletal elements of the sponge. In TEM sections sponge cells can be observed to contain extensive populations of bacteria, and frequently possesses double-membrane structures which we interpret to be autophagosomes. In combination with our previous work, these findings support the hypothesis that the host sponge actively degrades a proportion of its bacterial community using an autophagy pathway, and uses the prokaryotic organic remains as a framework upon which calcification of the sponge skeleton is initiated.     

Functional convergence of microbes associated with temperate marine sponges

Most marine sponges establish a persistent association with a wide array of phylogenetically and physiologically diverse microbes. To date, the role of these symbiotic microbial communities in the metabolism and nutrient cycles of the sponge‐microbe consortium remains largely unknown. We identified and quantified the microbial communities associated with three common Mediterranean sponge species, Dysidea avara, Agelas oroides and Chondrosia reniformis (Demospongiae) that cohabitate coralligenous community. For each sponge we quantified the uptake and release of dissolved organic carbon (DOC) and nitrogen (DON), inorganic nitrogen and phosphate. Low microbial abundance and no evidence for DOC uptake or nitrification were found for D. avara. In contrast A. oroides and C. reniformis showed high microbial abundance (30% and 70% of their tissue occupied by microbes respectively) and both species exhibited high nitrification and high DOC and NH₄⁺ uptake. Surprisingly, these unique metabolic pathways were mediated in each sponge species by a different, and host specific, microbial community. The functional convergence of microbial consortia found in these two sympatric sponge species, suggest that these metabolic processes may be of special relevance to the success of the holobiont.     

Recipient Environment More Important than Community Composition in Determining the Success of an Experimental Sponge Transplant

Human activities have inflicted profound damage upon many ecosystems, and ecologists are now seeking effective means of restoring ecosystems to their natural state. Industrial ports and harbors are highly modified and often depauperate in native fauna. They are typically characterized by poor water quality and modified community composition, both of which may hinder attempts to reintroduce native species. Here, we conducted a field experiment to separate the effects of the recipient environment and community composition on the success of endemic sponge explants in Port Kembla Harbor, NSW, Australia. A reciprocal transplant was conducted between communities originating from six sites that varied in water quality and community composition, enabling us to assess the relative factors simultaneously. A colony of the endemic sponge Tedania anhelans was then inserted into the center of each community, and we quantified the survival, growth, and metal bioaccumulation of sponges over three months. Endemic sponges consistently performed better against resident assemblages when water quality was good. Sponges transplanted to cleaner sites had over double the survivorship and approximately three times the surface area of sponges transplanted to disturbed sites. These patterns were independent of community composition. Bioaccumulation of metals in sponges was correlated with survival; however, other factors such as turbidity may be required to explain sponge mortality at some sites. This study adds to evidence that remediation of the physical and chemical environments may be a prerequisite for biological remediation and demonstrates the value of experimental transplants in assessing restoration potential.     

Characterization of putative circular plasmids in sponge‐associated bacterial communities using a selective multiply‐primed rolling circle amplification

Plasmid transfers among bacterial populations can directly influence the ecological adaptation of these populations and their interactions with host species and environment. In this study, we developed a selective multiply‐primed rolling circle amplification (smRCA) approach to enrich and characterize circular plasmid DNA from sponge microbial symbionts via high‐throughput sequencing (HTS). DNA (plasmid and total community DNA) obtained from sponge (Cinachyrella sp.) samples and a bacterial symbiont (Vibrio sp. CyArs1) isolated from the same sponge species (carrying unknown plasmids) were used to develop and validate our methodology. The smRCA was performed during 16 hr with 141 plasmid‐specific primers covering all known circular plasmid groups. The amplified products were purified and subjected to a reamplification with random hexamer primers (2 hr) and then sequenced using Illumina MiSeq. The developed method resulted in the successful amplification and characterization of the sponge plasmidome and allowed us to detect plasmids associated with the bacterial symbiont Vibrio sp. CyArs1 in the sponge host. In addition to this, a large number of small (<2 kbp) and cryptic plasmids were also amplified in sponge samples. Functional analysis identified proteins involved in the control of plasmid partitioning, maintenance and replication. However, most plasmids contained unknown genes, which could potentially serve as a resource of unknown genetic information and novel replication systems. Overall, our results indicate that the smRCA‐HTS approach developed here was able to selectively enrich and characterize plasmids from bacterial isolates and sponge host microbial communities, including plasmids larger than 20 kbp.     

Morphological,Bacterial, and Secondary Metabolite Changes of <Emphasis Type="Italic">Aplysina aerophoba</Emphasis> upon Long-Term Maintenance Under Artificial Conditions

The aim of this study was to analyze successional changes in the bacterial community over a period of 6 months of cultivation of Aplysina aerophoba sponges under different artificial cultivation conditions by use of denaturing gradient gel electrophoresis (DGGE). The cultivation conditions varied concerning the water temperature (20 ± 2 °C and 25 ± 2 °C) of the aquaria, additional illumination of one aquarium, and feeding of the sponges. Amplicons from DGGE separation of dominant colonizing or variably appearing bacteria were sequenced and aligned for taxonomical identification. In addition, secondary metabolites typically found in A. aerophoba were analyzed to investigate changes in the natural product profile during cultivation. The cultivation of sponges under any given condition did not lead to a depletion of their bacterial community in the course of the experiment. On the contrary, the distinctive set of associated bacteria was maintained in spite of a dramatic loss of biomass and morphological degradation during the cultivation period. Generally, all sequences obtained from the DGGE gels were related to bacteria of five phyla: Actinobacteria, Cyanobacteria, α-Proteobacteria, γ-Proteobacteria, and Chloroflexi. Despite the overall stability of the bacterial community in A. aerophoba, an unambiguous variability was detected for the Cyanobacteria “A. aerophoba clone TK09”. This variability was ascribed to the predominant light conditions. The analysis of the metabolic pattern revealed that the concentration of a class of characteristic-brominated compounds typically found in A. aerophoba, like aeroplysinin-1, aerophobin-1, aerophobin-2, and isofistularin-3, increased over the 6 months of cultivation.     

Culturable heterotrophic bacteria from the marine sponge <Emphasis Type="Italic">Dendrilla</Emphasis> <Emphasis Type="Italic">nigra</Emphasis>: isolation and phylogenetic diversity of actinobacteria

Culturable heterotrophic bacterial composition of marine sponge Dendrilla nigra was analysed using different enrichments. Five media compositions including without enrichment (control), enriched with sponge extract, with growth regulator (antibiotics), with autoinducers, and complete enrichment containing sponge extract, antibiotics, and autoinducers were developed. DNA hybridization assay was performed to explore host specific bacteria and ecotypes of culturable sponge-associated bacteria. Enrichment with selective inducers (AHLs and sponge extract) and regulators (antibiotics) considerably enhanced the cultivation potential of sponge-associated bacteria. It was found that Marinobacter (MSI032), Micromonospora (MSI033), Streptomyces (MSI051), and Pseudomonas (MSI057) were sponge-associated obligate symbionts. The present findings envisaged that “Micromonospora–Saccharomonospora–Streptomyces” group was the major culturable actinobacteria in the marine sponge D. nigra. The DNA hybridization assay was a reliable method for the analysis of culturable bacterial community in marine sponges. Based on the culturable community structure, the sponge-associated bacteria can be grouped (ecotypes) as general symbionts, specific symbionts, habitat flora, and antagonists.