Cultivation of Sponge-Associated Bacteria from Agelas sventres and Xestospongia muta Collected from Different Depths.

Sponge-associated bacteria have been mostly cultured from shallow water (≤30 m) sponges, whereas only few studies targeted specimens from below 30 m. This study assessed the cultivability of bacteria from two marine sponges Xestospongia muta and Agelas sventres collected from shallow (<30 m), upper mesophotic (30-60 m), and lower mesophotic (60-90 m) reefs. Sponge-associated bacteria were cultivated on six different media, and replicate plates were used to pick individual colonies or to recover the entire biomass. Prokaryotic community analysis was conducted using Illumina MiSeq sequencing of 16S rRNA gene amplicons. A total of 144 bacterial isolates were picked following a colony morphology coding scheme and subsequently identified by 16S rRNA gene sequence analysis. Sponge individuals at each depth-range harboured specific cultivable bacteria that were not retrieved from specimens collected at other depths. However, there were substantial differences in the number of colonies obtained for replicate sponges of the same species. In addition, source of inoculum and cultivation medium had more impact on the cultured prokaryotic community than sample collection depth. This suggests that the "plate count anomaly" is larger than differences in sponge-associated prokaryotic community composition related to depth.

Variations in Microbial Diversity and Metabolite Profiles of the Tropical Marine Sponge Xestospongia muta with Season and Depth.

Xestospongia muta is among the most emblematic sponge species inhabiting coral reefs of the Caribbean Sea. Besides being the largest sponge species growing in the Caribbean, it is also known to produce secondary metabolites. This study aimed to assess the effect of depth and season on the symbiotic bacterial dynamics and major metabolite profiles of specimens of X. muta thriving in a tropical marine biome (Portobelo Bay, Panamá), which allow us to determine whether variability patterns are similar to those reported for subtropical latitudes. The bacterial assemblages were characterized using Illumina deep-sequencing and metabolomic profiles using UHPLC-DAD-ELSD from five depths (ranging 9-28 m) across two seasons (spring and autumn). Diverse symbiotic communities, representing 24 phyla with a predominance of Proteobacteria and Chloroflexi, were found. Although several thousands of OTUs were determined, most of them belong to the rare biosphere and only 23 to a core community. There was a significant difference between the structure of the microbial communities in respect to season (autumn to spring), with a further significant difference between depths only in autumn. This was partially mirrored in the metabolome profile, where the overall metabolite composition did not differ between seasons, but a significant depth gradient was observed in autumn. At the phyla level, Cyanobacteria, Firmicutes, Actinobacteria, and Spirochaete showed a mild-moderate correlation with the metabolome profile. The metabolomic profiles were mainly characterized by known brominated polyunsaturated fatty acids. This work presents findings about the composition and dynamics of the microbial assemblages of X. muta expanding and confirming current knowledge about its remarkable diversity and geographic variability as observed in this tropical marine biome.

Prokaryotic communities of Indo-Pacific giant barrel sponges are more strongly influenced by geography than host phylogeny.

Sponges harbor complex communities of microorganisms that carry out essential roles for the functioning and survival of their hosts. In some cases, genetically related sponges from different geographic regions share microbes, while in other cases microbial communities are more similar in unrelated sponges collected from the same location. To better understand how geography and host phylogeny cause variation in the prokaryotic community of sponges, we compared the prokaryotic community of 44 giant barrel sponges (Xestospongia spp.). These sponges belonged to six reproductively isolated genetic groups from eight areas throughout the Indo-Pacific region. Using Illumina sequencing, we obtained 440 000 sequences of the 16S rRNA gene V3V4 variable region that were assigned to 3795 operational taxonomic units (OTUs). The prokaryotic community of giant barrel sponges was characterized by 71 core OTUs (i.e. OTUs present in each specimen) that represented 57.5% of the total number of sequences. The relative abundance of these core OTUs varied significantly among samples, and this variation was predominantly related to the geographic origin of the sample. These results show that in giant barrel sponges, the variation in the prokaryotic community is primarily associated with geography as opposed to phylogenetic relatedness.

Polyketide derivatives from the sponge associated fungus Aspergillus europaeus with antioxidant and NO inhibitory activities.

DPPH assay of the in-house marine-derived fungi uncovered that the EtOAc extract of the cultured fungus Aspergillus europaeus WZXY-SX-4-1, which was isolated from the marine sponge Xestospongia testudinaria, possesses radical scavenging activity. Chromatographic separation of the bioactive extract resulted in the isolation of 20 polyketide derivatives, including six new compounds namely eurobenzophenones A-C (1-3), euroxanthones A-B (4-5), and (+)1-O-demethylvariecolorquinones A (6). The structures of new compounds were determined on the basis of the analyses of spectroscopic data, including the Snatzke method for the configurational assignment. Benzophenones 3, 9 and 10 exhibited potent radical scavenging activity against DPPH. All polyketides were evaluated for the inhibitory effects toward the LPS induced nitric oxide (NO) production in mouse microglia BV2 cells and the NF-κB activation in human colon carcinoma cell line SW480. Compound 9 with the significant DPPH radical scavenging activity is corresponded to the potent inhibition against NF-κB in SW480 cells induced by LPS. Compounds 2, 4, 16-18 exerted remarked down-regulation of NF-κB in LPS-induced SW480 cells with weak inhibitory effects against NO production and the DPPH radical scavenging activity.

Nocardia xestospongiae sp. nov., isolated from a marine sponge in the Andaman Sea.

A marine sponge-derived actinomycete, strain ST01-07T, was isolated from Xestospongia sp. collected from the Andaman Sea. The strain was characterised taxonomically using a polyphasic approach. The strain contained meso-diaminopimelic acid in the peptidoglycan, whole-cell sugars were arabinose, galactose, glucose, mannose and ribose. Mycolic acids that co-migrated with those from Nocardia araoensis NBRC 100135T were observed in whole-cell extracts. MK-8(H4ω-cycl) was the predominant menaquinone. Major cellular fatty acids were C17 : 1ω8c, C16 : 0 and C17 : 0. The diagnostic phospholipids in the cell consisted of diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain ST01-07T belonged to the genus Nocardia and was most closely related to N. araoensis IFM 0575T (98.71 % 16S rRNA gene sequence similarity), Nocardia niwae W9241T (98.56 %), Nocardia beijingensis AS4.1521T (98.41 %) and Nocardia arthritidis IFM 10035T (98.36 %). In addition, low DNA-DNA relatedness values (13.6±0.1% to 40.1±0.6%) confirmed that strain ST01-07T represents a novel species of the genus Nocardia, for which the name Nocardia xestospongiae sp. nov., is proposed. The type strain is ST01-07T (=BCC 45622T=NBRC 109069T).

Peroxide Natural Products from Plakortis zyggompha and the Sponge Association Plakortis halichondrioides-Xestospongia deweerdtae: Antifungal Activity against Cryptococcus gattii.

Cryptococcus gattii is a human pathogen and causative agent of a pernicious, sometimes fatal, disseminated fungal disease. Investigation of antifungal extracts of the marine sponge association Plakortis halichondrioides-Xestospongia deweerdtae and the sponge Plakortis zyggompha from the Bahamas led to the discovery and isolation of 6-epi-7,8-dihydroplakortide K (1), plakortide AA (2), and three new plakinic acids, N-P (4-6; unstable 1,2-dioxolanes bearing benzyl-substituted conjugated dienes), along with known plakinic acids L, K, and M.5 Chiroptical comparisons and DFT calculations of (13)C NMR chemical shifts were used to assign the absolute stereostructure of 4. The stereospecific base-promoted rearrangement-saponification of 1 to 10 was briefly investigated and showed tight kinetic control and stereospecific formation of the new C-2 stereocenter with inversion at C-3. Plakinic acid M and plakortides 9 and 11 exhibited antifungal activity against C. gattii (MIC90 = 2.4 to 36 µM), but plakinic acids N-P were inactive under the same conditions.

Environmental drivers of microbial community shifts in the giant barrel sponge, Xestospongia muta, over a shallow to mesophotic depth gradient.

The giant barrel sponge, Xestospongia muta, is a high microbial abundance sponge found on Caribbean coral reefs along shallow to mesophotic depth gradients where multiple abiotic factors change with depth. Sponges were collected along a depth gradient at Little Cayman (LC) and Lee Stocking Island (LSI), and the microbiome of these samples was analysed using 16S rRNA amplicon sequencing. Statistically significant shifts in community structure and dissimilarity (∼ 40%) were detected from 10 to 90 m in LC sponges, but a similar shift was not identified in sponges from 10 to 60 m at LSI (only 17% dissimilar). Additionally, inorganic nutrient levels steadily increased with depth at LSI but not at LC. Based on bulk stable isotopic variability, sponges collected from LC were generally more enriched in (15) N and less enriched in (13) C as depth increased, suggesting a transition from dependency on photoautotrophy to heterotrophy as depth increased. Patterns of stable isotopic enrichment were largely invariant at LSI, which is also reflected in the more stable microbial community along the depth gradient. It appears that environmental factors that change with depth may contribute to differences in X. muta microbial assemblages, demonstrating the importance of contemporaneous environmental sampling in studies of the microbiome of sponges.

Composition and Predictive Functional Analysis of Bacterial Communities in Seawater, Sediment and Sponges in the Spermonde Archipelago, Indonesia.

In this study, we used a 16S rRNA gene barcoded pyrosequencing approach to sample bacterial communities from six biotopes, namely, seawater, sediment and four sponge species (Stylissa carteri, Stylissa massa, Xestospongia testudinaria and Hyrtios erectus) inhabiting coral reefs of the Spermonde Archipelago, South Sulawesi, Indonesia. Samples were collected along a pronounced onshore to offshore environmental gradient. Our goals were to (1) compare higher taxon abundance among biotopes, (2) test to what extent variation in bacterial composition can be explained by the biotope versus environment, (3) identify dominant (>300 sequences) bacterial operational taxonomic units (OTUs) and their closest known relatives and (4) assign putative functions to the sponge bacterial communities using a recently developed predictive metagenomic approach. We observed marked differences in bacterial composition and the relative abundance of the most abundant phyla, classes and orders among sponge species, seawater and sediment. Although all biotopes housed compositionally distinct bacterial communities, there were three prominent clusters. These included (1) both Stylissa species and seawater, (2) X. testudinaria and H. erectus and (3) sediment. Bacterial communities sampled from the same biotope, but different environments (based on proximity to the coast) were much more similar than bacterial communities from different biotopes in the same environment. The biotope thus appears to be a much more important structuring force than the surrounding environment. There were concomitant differences in the predicted counts of KEGG orthologs (KOs) suggesting that bacterial communities housed in different sponge species, sediment and seawater perform distinct functions. In particular, the bacterial communities of both Stylissa species were predicted to be enriched for KOs related to chemotaxis, nitrification and denitrification whereas bacterial communities in X. testudinaria and H. erectus were predicted to be enriched for KOs related to the toxin-antitoxin (TA) system, nutrient starvation and heavy metal export.

Bacterial community composition and predicted functional ecology of sponges, sediment and seawater from the thousand islands reef complex, West Java, Indonesia.

In the present study, we assessed the composition of Bacteria in four biotopes namely sediment, seawater and two sponge species (Stylissa massa and Xestospongia testudinaria) at four different reef sites in a coral reef ecosystem in West Java, Indonesia. In addition to this, we used a predictive metagenomic approach to estimate to what extent nitrogen metabolic pathways differed among bacterial communities from different biotopes. We observed marked differences in bacterial composition of the most abundant bacterial phyla, classes and orders among sponge species, water and sediment. Proteobacteria were by far the most abundant phylum in terms of both sequences and Operational Taxonomic Units (OTUs). Predicted counts for genes associated with the nitrogen metabolism suggested that several genes involved in the nitrogen cycle were enriched in sponge samples, including nosZ, nifD, nirK, norB and nrfA genes. Our data show that a combined barcoded pyrosequencing and predictive metagenomic approach can provide novel insights into the potential ecological functions of the microbial communities. Not only is this approach useful for our understanding of the vast microbial diversity found in sponges but also to understand the potential response of microbial communities to environmental change.

Pezizomycotina dominates the fungal communities of South China Sea sponges Theonella swinhoei and Xestospongia testudinaria.

Compared with the knowledge of sponge-associated bacterial diversity and ecological roles, the fungal diversity and ecological roles of sponges remain largely unknown. In this study, the fungal diversity and protein synthesis potential in two South China Sea sponges Theonella swinhoei and Xestospongia testudinaria were investigated by rRNA vs. rRNA gene analysis. EF4/fung5 was chosen after a series of PCR tests to target fungal 18S rRNA and 18S rRNA gene. Altogether, 283 high-quality sequences were obtained, which resulted in 26 Operational taxonomic units (OTUs) that were assigned to Ascomycota, Basidiomycota, and Blastocladiomycota. At subphylum level, 77.3% of sponge-derived sequences were affiliated with Pezizomycotina. The fungal compositions of T. swinhoei and X. testudinaria were different from that of ambient seawater. The predominant OTU shared between two sponges was rare in seawater, whereas the most abundant OTUs in seawater were not found in sponges. Additionally, the major OTUs of sponge cDNA datasets were shared in two sponges. The fungal diversity illustrated by sponge cDNA datasets correlated well with that derived from sponge DNA datasets, indicating that the major members of sponge-associated fungi had protein synthesis potential. This study highlighted the diversity of Pezizomycotina in marine sponge-fungi symbioses and the necessity of investigating ecological roles of sponge-associated fungi.

Symbiotic prokaryotic communities from different populations of the giant barrel sponge, Xestospongia muta.

The prokaryotic community composition of the ecologically dominant sponge, Xestospongia muta, and the variability of this community across in different populations of sponges from the Caribbean and Bahamas were quantified using 454 pyrosequencing of the 16S rRNA gene. The symbiotic prokaryotic communities of X. muta were significantly different than the surrounding bacterioplankton communities while an analysis of similarity (ANOSIM) of the sponge prokaryotic symbionts from three geographically distant sites showed that both symbiont and bacterioplankton populations were significantly different between locations. Comparisons of individual sponges based on the UniFrac P-test also revealed significant differences in community composition between individual sponges. The sponges harbored a variety of phylum level operational taxonomic units (OTUs) common to many sponges, including Cyanobacteria, Poribacteria, Acidobacteria, Chloroflexi, and Gemmatimonadetes, but four additional symbiotic phyla, previously not reported for this sponge, were observed. Additionally, a diverse archaeal community was also recovered from X. muta including sequences representing the phyla Euryarchaeota and Thaumarchaeota. These results have important ecological implications for the understanding of host-microbe associations, and provide a foundation for future studies addressing the functional roles these symbiotic prokaryotes have in the biology of the host sponge and the nutrient biogeochemistry of coral reefs.

Phylogenetically diverse ureC genes and their expression suggest the urea utilization by bacterial symbionts in marine sponge Xestospongia testudinaria.

Urea is one of the dominant organic nitrogenous compounds in the oligotrophic oceans. Compared to the knowledge of nitrogen transformation of nitrogen fixation, ammonia oxidization, nitrate and nitrite reduction mediated by sponge-associated microbes, our knowledge of urea utilization in sponges and the phylogenetic diversity of sponge-associated microbes with urea utilization potential is very limited. In this study, Marinobacter litoralis isolated from the marine sponge Xestospongia testudinaria and the slurry of X. testudinaria were found to have urease activity. Subsequently, phylogenetically diverse bacterial ureC genes were detected in the total genomic DNA and RNA of sponge X. testudinaria, i.e., 19 operative taxonomic units (OTUs) in genomic DNA library and 8 OTUs in cDNA library at 90% stringency. Particularly, 6 OTUs were common to both the genomic DNA library and the cDNA library, which suggested that some ureC genes were expressed in this sponge. BLAST and phylogenetic analysis showed that most of the ureC sequences were similar with the urease alpha subunit of members from Proteobacteria, which were the predominant component in sponge X. testudinaria, and the remaining ureC sequences were related to those from Magnetococcus, Cyanobacteria, and Actinobacteria. This study is the first assessment of the role of sponge bacterial symbionts in the regenerated utilization of urea by the detection of transcriptional activity of ureC gene, as well as the phylogenetic diversity of ureC gene of sponge bacterial symbionts. The results suggested the urea utilization by bacterial symbionts in marine sponge X. testudinaria, extending our understanding of nitrogen cycling mediated by sponge-associated microbiota.

New bisabolane sesquiterpenoids from a marine-derived fungus Aspergillus sp. isolated from the sponge Xestospongia testudinaria.

Three new phenolic bisabolane sesquiterpenoid dimers, disydonols A-C (1-3), and one known compound (S)-(+)-sydonol (4) were isolated from the fermentation broth of a marine-derived fungus Aspergillus sp., which was isolated from the sponge Xestospongia testudinaria collected from the South China Sea. Their structures were elucidated on the basis of comprehensive spectral analysis including 1D and 2D NMR spectra and HR-ESI-MS. These compounds were evaluated for cytotoxic activity against HepG-2 and Caski human tumour cell lines. Among them, compounds 1 and 3 exhibited cytotoxicity against the two cell lines.

Marinobacter xestospongiae sp. nov., isolated from the marine sponge Xestospongia testudinaria collected from the Red Sea.

A Gram-negative, catalase- and oxidase-positive, non-sporulating, rod-shaped and slightly halophilic bacterial strain, designated UST090418-1611(T), was isolated from the marine sponge Xestospongia testudinaria collected from the Red Sea coast of Saudi Arabia. Phylogenetic trees based on the 16S rRNA gene sequence placed strain UST090418-1611(T) in the family Alteromonadaceae with the closest relationship to the genus Marinobacter. The 16S rRNA gene sequence similarity between the strain and the type strains of recognized Marinobacter species ranged from 92.9 to 98.3%. Although strain UST090418-1611(T) shared high 16S rRNA gene sequence similarity with Marinobacter mobilis CN46(T), M. zhejiangensis CN74(T) and M. sediminum R65(T) (98.3, 97.4 and 97.3%, respectively), the relatedness of the strain to these three strains in DNA-DNA hybridization was only 58, 56 and 33%, respectively, supporting the novelty of the strain. In contrast to most strains in the genus Marinobacter, strain UST090418-1611(T) tolerated only 6% (w/v) NaCl, and optimal growth occurred at 2.0% (w/v) NaCl, pH 7.0-8.0 and 28-36 °C. The predominant cellular fatty acids were C(12:0) 3-OH, C(16:0), C(12:0) and summed feature 3 (C(16:1)ω6c and/or C(16:1)ω7c). The genomic DNA G+C content was 57.1 mol%. Based on the physiological, phylogenetic and chemotaxonomic characteristics presented in this study, we suggest that the strain represents a novel species in the genus Marinobacter, for which the name Marinobacter xestospongiae sp. nov. is proposed, with UST090418-1611(T) ( = JCM 17469(T)  = NRRL B-59512(T)) as the type strain.

Sponge-associated bacteria are strictly maintained in two closely related but geographically distant sponge hosts.

The giant barrel sponges Xestospongia muta and Xestospongia testudinaria are ubiquitous in tropical reefs of the Atlantic and Pacific Oceans, respectively. They are key species in their respective environments and are hosts to diverse assemblages of bacteria. These two closely related sponges from different oceans provide a unique opportunity to examine the evolution of sponge-associated bacterial communities. Mitochondrial cytochrome oxidase subunit I gene sequences from X. muta and X. testudinaria showed little divergence between the two species. A detailed analysis of the bacterial communities associated with these sponges, comprising over 900 full-length 16S rRNA gene sequences, revealed remarkable similarity in the bacterial communities of the two species. Both sponge-associated communities include sequences found only in the two Xestospongia species, as well as sequences found also in other sponge species and are dominated by three bacterial groups, Chloroflexi, Acidobacteria, and Actinobacteria. While these groups consistently dominate the bacterial communities revealed by 16S rRNA gene-based analysis of sponge-associated bacteria, the depth of sequencing undertaken in this study revealed clades of bacteria specifically associated with each of the two Xestospongia species, and also with the genus Xestospongia, that have not been found associated with other sponge species or other ecosystems. This study, comparing the bacterial communities associated with closely related but geographically distant sponge hosts, gives new insight into the intimate relationships between marine sponges and some of their bacterial symbionts.

The pathology of sponge orange band disease affecting the Caribbean barrel sponge Xestospongia muta.

The aim of this study was to examine sponge orange band (SOB) disease affecting the prominent Caribbean sponge Xestospongia muta. Scanning and transmission electron microscopy revealed that SOB is accompanied by the massive destruction of the pinacoderm. Chlorophyll a content and the main secondary metabolites, tetrahydrofurans, characteristic of X. muta, were significantly lower in bleached than in healthy tissues. Denaturing gradient gel electrophoresis using cyanobacteria-specific 16S rRNA gene primers revealed a distinct shift from the Synechococcus/Prochlorococcus clade of sponge symbionts towards several clades of unspecific cyanobacteria, including lineages associated with coral disease (i.e. Leptolyngbya sp.). Underwater infection experiments were conducted by transplanting bleached cores into healthy individuals, but revealed no signs of SOB development. This study provided no evidence for the involvement of a specific microbial pathogen as an etiologic agent of disease; hence, the cause of SOB disease in X. muta remains unidentified.

Effects of sponge bleaching on ammonia-oxidizing Archaea: distribution and relative expression of ammonia monooxygenase genes associated with the barrel sponge Xestospongia muta.

Sponge-mediated nitrification is an important process in the nitrogen cycle, however, nothing is known about how nitrification and symbiotic Archaea may be affected by sponge disease and bleaching events. The giant barrel sponge Xestospongia muta is a prominent species on Caribbean reefs that contains cyanobacterial symbionts, the loss of which results in two types of bleaching: cyclic, a recoverable condition; and fatal, a condition associated with the disease-like sponge orange band (SOB) syndrome and sponge death. Terminal restriction fragment length polymorphism (TRFLP) analyses, clone libraries, and relative mRNA quantification of ammonia monooxygenase genes (amoA) were performed using a RNA transcript-based approach to characterize the active ammonia-oxidizing Archaea (AOA) community present in bleached, non-bleached, and SOB tissues of cyclically and fatally bleached sponges. We found that non-bleached and cyclically bleached tissues of X. muta harbored a unique Crenarchaeota community closely related to those reported for other sponges. In contrast, bleached tissue from the most degraded sponge contained a Crenarchaeota community that was more similar to those found in sediment and sand. Although there were no significant differences in amoA expression among the different tissues, amoA expression was higher in the most deteriorated tissues. Results suggest that a shift in the Crenarchaeota community precedes an increase in amoA gene expression in fatally bleached sponges, while cyclic bleaching did not alter the AOA community structure and its amoA gene expression.

Novel actinobacteria from marine sponges.

Actinobacteria exclusively within the sub-class Acidimicrobidae were shown by 16S rDNA community analysis to be major components of the bacterial community associated with two sponge species in the genus Xestospongia. Four groups of Actinobacteria were identified in Xestospongia spp., with three of these four groups being found in both Xestospongia muta from Key Largo, Florida and Xestospongia testudinaria from Manado, Indonesia. This suggests that these groups are true symbionts in these sponges and may play a common role in both the Pacific and Atlantic sponge species. The fourth group was found only in X. testudinaria and was a novel assemblage distantly related to any previously sequenced actinobacterial clones. The only actinobacteria that were obtained in initial culturing attempts were Gordonia, Micrococcus and Brachybacterium spp., none of which were represented in the clone libraries. The closest cultured actinobacteria to all the Acidimicrobidae clones from Xestospongia spp. are 'Microthrix parvicella' and Acidimicrobium spp. Xestospongia spp. can now be targeted as source material from which to culture novel Acidimicrobidae to investigate their potential as producers of bioactive compounds. Isolation of sponge-associated Acidimicrobidae will also make it possible to elucidate their role as sponge symbionts.