Cintia P. J. Rua

Taxonomic and Functional Microbial Signatures of the Endemic Marine Sponge Arenosclera brasiliensis

The endemic marine sponge Arenosclera brasiliensis (Porifera, Demospongiae, Haplosclerida) is a known source of secondary metabolites such as arenosclerins A-C. In the present study, we established the composition of the A. brasiliensis microbiome and the metabolic pathways associated with this community. We used 454 shotgun pyrosequencing to generate approximately 640,000 high-quality sponge-derived sequences (∼150 Mb). Clustering analysis including sponge, seawater and twenty-three other metagenomes derived from marine animal microbiomes shows that A. brasiliensis contains a specific microbiome. Fourteen bacterial phyla (including Proteobacteria, Cyanobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Cloroflexi) were consistently found in the A. brasiliensis metagenomes. The A. brasiliensis microbiome is enriched for Betaproteobacteria (e.g., Burkholderia) and Gammaproteobacteria (e.g., Pseudomonas and Alteromonas) compared with the surrounding planktonic microbial communities. Functional analysis based on Rapid Annotation using Subsystem Technology (RAST) indicated that the A. brasiliensis microbiome is enriched for sequences associated with membrane transport and one-carbon metabolism. In addition, there was an overrepresentation of sequences associated with aerobic and anaerobic metabolism as well as the synthesis and degradation of secondary metabolites. This study represents the first analysis of sponge-associated microbial communities via shotgun pyrosequencing, a strategy commonly applied in similar analyses in other marine invertebrate hosts, such as corals and algae. We demonstrate that A. brasiliensis has a unique microbiome that is distinct from that of the surrounding planktonic microbes and from other marine organisms, indicating a species-specific microbiome.

Diversity and antimicrobial potential of culturable heterotrophic bacteria associated with the endemic marine sponge Arenosclera brasiliensis

Marine sponges are the oldest Metazoa, very often presenting a complex microbial consortium. Such is the case of the marine sponge Arenosclera brasiliensis, endemic to Rio de Janeiro State, Brazil. In this investigation we characterized the diversity of some of the culturable heterotrophic bacteria living in association with A. brasiliensis and determined their antimicrobial activity. The genera Endozoicomonas (N = 32), Bacillus (N = 26), Shewanella (N = 17), Pseudovibrio (N = 12), and Ruegeria (N = 8) were dominant among the recovered isolates, corresponding to 97% of all isolates. Approximately one third of the isolates living in association with A. brasiliensis produced antibiotics that inhibited the growth of Bacillus subtilis, suggesting that bacteria associated with this sponge play a role in its health.

New polymorphic mitochondrial markers for sponge phylogeography

Phylogeography and population genetic studies in the Porifera have been limited by the lack of available polymorphic DNA markers. In this paper, we tested four new mitochondrial markers in nine demosponge species from a wide taxonomic range: partial sequences of the ATP synthase 6 (ATP6) and the cytochrome oxidase 2 (CO 2 ) genes and two spacers: one located between ATP6 and CO 2 and the other between the NADH dehydrogenase subunit 5 (ND 5 ) and the small subunit ribosomal RNA (rns) genes. The new markers presented levels of nucleotide diversity up to 2 . 4 times higher (π = 0.015 for CO 2 ) than those observed for the most commonly used mitochondrial marker in sponges, the cytochrome oxidase 1 gene (π = 0.006), making them suitable for alpha-level systematics, phylogeography and population genetics studies.

Polyketide Synthase Gene Diversity within the Microbiome of the Sponge Arenosclera brasiliensis, Endemic to the Southern Atlantic Ocean

ABSTRACT Microbes associated with marine sponges are considered important producers of bioactive, structurally unique polyketides. The synthesis of such secondary metabolites involves type I polyketide synthases (PKSs), which are enzymes that reach a maximum complexity degree in bacteria. The Haplosclerida sponge Arenosclera brasiliensis hosts a complex microbiota and is the source of arenosclerins, alkaloids with cytotoxic and antibacterial activity. In the present investigation, we performed high-throughput sequencing of the ketosynthase (KS) amplicon to investigate the diversity of PKS genes present in the metagenome of A. brasiliensis. Almost 4,000 ketosynthase reads were recovered, with about 90% annotated automatically as bacterial. A total of 235 bacterial KS contigs was rigorously assembled from this sequence pool and submitted to phylogenetic analysis. A great diversity of six type I PKS groups has been consistently detected in our phylogenetic reconstructions, including a novel and A. brasiliensis-exclusive group. Our study is the first to reveal the diversity of type I PKS genes in A. brasiliensis as well as the potential of its microbiome to serve as a source of new polyketides.

Potential metabolic strategies of widely distributed holobionts in the oceanic archipelago of St Peter and St Paul (Brazil)

Sponges are one of the most complex symbiotic communities and while the taxonomic composition of associated microbes has been determined, the biggest challenge now is to uncover their functional role in symbiosis. We investigated the microbiota of two widely distributed sponge species, regarding both their taxonomic composition and their functional roles. Samples of Didiscus oxeata and Scopalina ruetzleri were collected in the oceanic archipelago of St Peter and St Paul and analysed through metagenomics. Sequences generated by 454 pyrosequencing and Ion Torrent were taxonomically and functionally annotated on the MG-RAST server using the GenBank and SEED databases, respectively. Both communities exhibit equivalence in core functions, interestingly played by the most abundant taxa in each community. Conversely, the microbial communities differ in composition, taxonomic diversity and potential metabolic strategies. Functional annotation indirectly suggests differences in preferential pathways of carbon, nitrogen and sulphur metabolisms, which may indicate different metabolic strategies.

Rapid isolation of culturable microalgae from a tropical shallow lake system

AbstractMicroalgae diversity is constantly being studied and explored for biotechnological uses. The shallow lake system of Lençois Maranhenses (SLLM) is a unique coastal ecosystem in northeast Brazil found interspersed in a field of sand dunes. Organisms in these tropical lakes are constantly exposed to high temperatures and solar irradiance. Yet, little is known about the diversity of culturable microalgae in this aquatic ecosystem. This study reports the use of flow cytometry with fluorescence-activated cell sorting (FACS) to isolate single microalgae cells/coenobia from five lakes in SLLM, accessing the efficiency of this isolation technique with two types of culture media. To retrieve the highest diversity of culturable microalgae, planktonic, benthic, and epiphytic samples were collected and processed by FACS. The diversity of microalgae in natural lake communities was described by morphology-based taxonomy. Isolates of the most abundant phylum established in cultures (Chlorophyta) were characterized by gene sequencing (18S rDNA). A total of 3072 microalgal cells/coenobia were sorted into 96-well plates. From these, 945 wells presented algal growth (31% success rate). Based on morphological diversity and adaptability to culture conditions, a set of 171 strains were selected to be incorporated in a culture collection. Microalgae present in the lakes belonged to six phyla, with four of them represented in the cultured strains. Our sampling strategy coupled with FACS isolation retrieved a fairly large number and diversity of microalgal strains with minimum isolation effort from a unique coastal environment. The monoclonal cultures established in this study offer new opportunities for basic and applied research on microalgae biotechnology. Graphical abstractᅟ

Microbial and Functional Biodiversity Patterns in Sponges that Accumulate Bromopyrrole Alkaloids Suggest Horizontal Gene Transfer of Halogenase Genes

Marine sponge holobionts harbor complex microbial communities whose members may be the true producers of secondary metabolites accumulated by sponges. Bromopyrrole alkaloids constitute a typical class of secondary metabolites isolated from sponges that very often display biological activities. Bromine incorporation into secondary metabolites can be catalyzed by either halogenases or haloperoxidases. The diversity of the metagenomes of sponge holobiont species containing bromopyrrole alkaloids (Agelas spp. and Tedania brasiliensis) as well as holobionts devoid of bromopyrrole alkaloids spanning in a vast biogeographic region (approx. Seven thousand km) was studied. The origin and specificity of the detected halogenases was also investigated. The holobionts Agelas spp. and T. brasiliensis did not share microbial halogenases, suggesting a species-specific pattern. Bacteria of diverse phylogenetic origins encoding halogenase genes were found to be more abundant in bromopyrrole-containing sponges. The sponge holobionts (e.g., Agelas spp.) with the greatest number of sequences related to clustered, interspaced, short, palindromic repeats (CRISPRs) exhibited the fewest phage halogenases, suggesting a possible mechanism of protection from phage infection by the sponge host. This study highlights the potential of phages to transport halogenases horizontally across host sponges, particularly in more permissive holobiont hosts, such as Tedania spp.

Environmental conditions affect activity and associated microorganisms of marine sponges

Changes in environmental conditions can influence sponges and their holobionts. The present study investigated the effect of upwelling and anthropogenic pollution on the bioactivity of marine sponges, microbial communities and functional genes, and composition of their chemical compounds. The species Dysidea etheria, Darwinella sp., Hymeniacidon heliophila and Tedania ignis were collected from areas with distinct influence of upwelling and low anthropogenic impact and from areas without influence of upwelling but affected by sewage and the port. In most cases, the same sponge species collected from areas with distinct environmental conditions had a different chemical composition, antifouling activity, composition and diversity of associated microorganisms. Antimicrobial, quorum sensing inhibitory and anti-larval activities of sponge extracts were more pronounced in the area without upwelling showing higher level of anthropogenic pollution. This study suggests that upwelling and anthropogenic pollution affect the chemical activity and holobiome composition of sponges.