Diversity of bacteria in the marine sponge Aplysina fulva in Brazilian coastal waters

Abstract

Microorganisms can account for up to 60% of the fresh weight of marine sponges. Marine sponges have been hypothesized to serve as accumulation spots of particular microbial communities, but it is unknown to what extent these communities are directed by the organism or the site or occur randomly. To address this question, we assessed the composition of specific bacterial communities associated with Aplysina fulva, one of the prevalent sponge species inhabiting Brazilian waters. Specimens of A. fulva and surrounding seawater were collected in triplicate in shallow water at two sites, Caboclo Island and Tartaruga beach, Búzios, Brazil. Total community DNA was extracted from the samples using "direct" and "indirect" approaches. 16S rRNA-based PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analyses of the total bacterial community and of specific bacterial groups--Pseudomonas and Actinobacteria--revealed that the structure of these assemblages in A. fulva differed drastically from that observed in seawater. The DNA extraction methodology and sampling site were determinative for the composition of actinobacterial communities in A. fulva. However, no such effects could be gleaned from total bacterial and Pseudomonas PCR-DGGE profiles. Bacterial 16S rRNA gene clone libraries constructed from directly and indirectly extracted DNA did not differ significantly with respect to diversity and composition. Altogether, the libraries encompassed 15 bacterial phyla and the candidate division TM7. Clone sequences affiliated with the Cyanobacteria, Chloroflexi, Gamma- and Alphaproteobacteria, Actinobacteria, Bacteroidetes, and Acidobacteria were, in this order, most abundant. The bacterial communities associated with the A. fulva specimens were distinct and differed from those described in studies of sponge-associated microbiota performed with other sponge species.

FIG. 1.

TEM of A. fulva mesohyl. Several bacterial morphotypes (arrows), including membrane-bound nucleoid-containing bacteria (asterisks), can be observed. Microorganisms with an internal membrane system can also be observed (arrowheads). Scale bars, 0.5 μm.

FIG. 2.

PCR-DGGE 16S rRNA gene fingerprints of A. fulva and seawater DNA samples generated with “total-community” bacterial primers (a) and specific primer systems for Pseudomonas (c) and Actinobacteria (e). The arrows in panels a and e indicate bands that occur in the sponge profiles but not in water. Some of these bands, especially those in panel e, are characteristic of a specific DNA extraction strategy. The arrows in panel c show bands that were cloned and sequenced. Corresponding ordination biplots of PCR-DGGE fingerprints and qualitative environmental variables are shown in panels b, d, and f. Symbols: ⧫, A. fulva from Caboclo Island extracted directly (AfCD); ⋄, A. fulva from Caboclo Island extracted indirectly (AfCI); ▪, A. fulva from Tartaruga beach extracted directly (AfTD); □, A. fulva from Tartaruga beach extracted indirectly (AfTI); •, surrounding seawater from Caboclo Island (SWC); ○, surrounding seawater from Tartaruga beach (SWT). Shannon's indices of diversity are shown for each sample next to symbols. Symbol sizes correspond to their estimated diversity indices. Labels displayed on the diagram axes refer to the percentage variations of PCR-DGGE ribotypes; environment correlation accounted for the respective axis. The “star” symbols represent the centroid positions of the environmental variables in the diagram. Variables that significantly (P < 0.05) influence the bacterial community composition are indicated by an asterisk.

FIG. 3.

Evolutionary relationships of 145 gammaproteobacterial 16S rRNA sequences. Twenty-seven sequences retrieved from a dominant “Pseudomonas” band observed to be enriched in A. fulva DGGE profiles (Fig. 2c) were included in phylogenetic inference. The evolutionary history was inferred by using the neighbor-joining method (49). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed by using the Kimura two-parameter method (29) and are in the units of the number of base substitutions per site (note the scale bar). Bootstrap values (1,000 repetitions) greater than 50% are shown on three nodes. There were a total of 413 aligned nucleotide positions in the final data set. The 16S rRNA gene sequences of Burkholderia cepacia (T) ATCC 25416T (Betaproteobacteria) and Streptomyces thermocoprophilus (T) (Actinobacteria) served as outgroups.

FIG. 4.

Phylum distribution of bacterial 16S rRNA gene sequences based on the Classifier tool of the RDP, version 9.0. Bacterial sequences classified at the phylum level with a 70% CI (a) and unclassified sequences at 70% CI and reclassified at ≤69% CI (b) are shown.

FIG. 5.

Phylogenetic analysis of bacterial 16S rRNA genes retrieved from A. fulva. The trees shown were inferred and drawn as explained in the legend to Fig. 3. Solid circles next to tree branches correspond to bootstrap values higher than 50%. A. fulva-derived clones are indicated in boldface, with their respective tree leaves marked in red. Trees display one representative clone per OTU. A single OTU embraces, here, all of the clone sequences sharing at least 99% similarity. Red bars next to A. fulva clone labels indicate the number of clones belonging to the corresponding OTU. (a) Evolutionary relationships of 125 taxa representative of the Acidobacteria (light green) and the alpha (light blue), beta (blue), gamma (light purple), and delta (purple) classes of Proteobacteria. There were 468 nucleotide positions in the final data set. (b) Phylogeny of the Chloroflexi as delineated by 167 representative taxa. All previously described Chloroflexi subdivisions—as designated by Rappé and Giovanonni (45a) (clades 1 to 8) and Costello and Schmidt (8a) (clades C12, C05, H09, E05, G04, B12, and A07)—are shown and appear, mostly, as collapsed clades. Clades composed exclusively by uncultured (dark green) or containing cultured (light green) representatives are differentiated. Four novel Chloroflexi subdivisions represented solely by sponge-derived uncultured bacteria are highlighted (colored ranges on sequence labels). There were a total of 528 aligned nucleotide positions in the final data set.

FIG. 6.

16S rRNA-based phylogeny of sponge-associated Cyanobacteria. The tree was inferred and drawn as explained in the legend to Fig. 3. Labels in boldface represent A. fulva-derived Cyanobacteria sequences obtained in the present study. The accession numbers of the sequences obtained from the public databank are given in parentheses. There were a total of 612 aligned nucleotide positions in the final data set.