Silicatein expression in Haliclona indistincta (Phylum Porifera, Order Haplosclerida) at different developmental stages.

Silicatein is the main protein responsible for the formation of spicules, tiny structures that constitute the silica skeleton of marine demosponges (Phylum Porifera). A unique innovation in Porifera that evolved from the cathepsin L family of proteins, it has been reported that two amino acids (S and H) are necessary to form the catalytic triad (SHN) to enable silica condensation. However, a diversity of silicatein sequence variants has since been reported with a variable pattern of presence/absence across sponge groups. Variants containing CHN or C/SQN at the active site appear more common in sponges from the Haplosclerida. Here, we report the expression levels of five silicatein variants through different developmental stages in the haplosclerid Haliclona indistincta. All five silicatein variants were expressed at low levels in the free-swimming larvae, which lack spicules and expression significantly increased at the two developmental phases in which spicules were visible. At these two phases, silicateins of CHN and C/SQN types were much more highly expressed than the SHN type indicating a possible ability of active sites with these alternative amino acids to condense silica and a more complex evolutionary story for spicule formation in marine demosponges than previously understood.

Antibiotic activity and microbial community of the temperate sponge, Haliclona sp.

AIMS: Sessile marine invertebrates engage in a diverse array of beneficial interactions with bacterial symbionts. One feature of some of these relationships is the presence of bioactive natural products that can defend the holobiont from predation, competition or disease. In this study, we investigated the antimicrobial activity and microbial community of a common temperate sponge from coastal North Carolina. METHODS AND RESULTS: The sponge was identified as a member of the genus Haliclona, a prolific source of bioactive natural products, based on its 18S rRNA gene sequence. The crude chemical extract and methanol partition had broad activity against the assayed Gram-negative and Gram-positive pathogenic bacteria. Further fractionation resulted in two groups of compounds with differing antimicrobial activity, primarily against Gram-positive test organisms. There was, however, notable activity against the Gram-negative marine pathogen, Vibrio parahaemolyticus. Microbial community analysis of the sponge and surrounding sea water via denaturing gradient gel electrophoresis (DGGE) indicates that it harbours a distinct group of bacterial associates. CONCLUSIONS: The common temperate sponge, Haliclona sp., is a source of multiple antimicrobial compounds and has some consistent microbial community members that may play a role in secondary metabolite production. SIGNIFICANCE AND IMPACT OF THE STUDY: These data suggest that common temperate sponges can be a source of bioactive chemical and microbial diversity. Further studies may reveal the importance of the microbial associates to the sponge and natural product biosynthesis.

Evaluation of differential protein expression in Haliclona aquarius and sponge-associated microorganisms under cadmium stress.

A comparative proteomic approach was used to assess differentially expressed proteins in marine sponges after 36 h of exposure to cadmium (Cd). After separation performed by 2-D polyacrylamide gel electrophoresis, 46 protein spots indicated differential expression, and 17 of these proteins were identified by electrospray ionization quadrupole time-of-flight mass spectrometry. From the proteins identified, 76% were attributed to sponge-associated microorganisms (fungi and bacteria), and 24% were attributed to Haliclona aquarius. Some of the proteins that were identified may be related to cell proliferation and differentiation or processes of oxidative stress repair and energy procurement. An integrated evaluation based on spot expression levels and the postulated functions of these proteins allowed a more accurate evaluation of the stress caused to the sponge holobiont system by cadmium exposure. This study could provide new insights into the use of a proteomic approach in the marine sponge to assess the effects of Cd pollution in a marine environment.

Isolation identification and biochemical characterization of a novel halo-tolerant lipase from the metagenome of the marine sponge Haliclona simulans.

BACKGROUND: Lipases (EC 3.1.1.3) catalyze the hydrolysis of triacyl glycerol to glycerol and are involved in the synthesis of both short chain and long chain acylglycerols. They are widely used industrially in various applications, such as baking, laundry detergents and as biocatalysts in alternative energy strategies. Marine ecosystems are known to represent a large reservoir of biodiversity with respect to industrially useful enzymes. However the vast majority of microorganisms within these ecosystems are not readily culturable. Functional metagenomic based approaches provide a solution to this problem by facilitating the identification of novel enzymes such as the halo-tolerant lipase identified in this study from a marine sponge metagenome. RESULTS: A metagenomic library was constructed from the marine sponge Haliclona simulans in the pCC1fos vector, containing approximately 48,000 fosmid clones. High throughput plate screening on 1% tributyrin agar resulted in the identification of 58 positive lipase clones. Following sequence analysis of the 10 most highly active fosmid clones the pCC1fos53E1 clone was found to contain a putative lipase gene lpc53E1, encoded by 387 amino acids and with a predicted molecular mass of 41.87 kDa. Sequence analysis of the predicted amino acid sequence of Lpc53E1 revealed that it is a member of the group VIII family of lipases possessing the SXTK motif, related to type C ß-lactamases. Heterologous expression of lpc53E1 in E. coli and the subsequent biochemical characterization of the recombinant protein, showed an enzyme with the highest substrate specificity for long chain fatty acyl esters. Optimal activity was observed with p- nitrophenyl palmitate (C16) at 40°C, in the presence of 5 M NaCl at pH 7; while in addition the recombinant enzyme displayed activity across broad pH (3-12) and temperature (4 -60°C) ranges and high levels of stability in the presence of various solvents at NaCl concentrations as high as 5 M and at temperatures ranging from 10 to 80°C. A maximum lipase activity of 2,700 U/mg was observed with 10 mM p-nitrophenyl palmitate as substrate, in the presence of 5 mM Ca2+ and 5 M NaCl, and a reaction time of 15 min at pH 7 and 40°C; while KM and Vmax values were calculated to be 1.093 mM-1 and 50 µmol/min, respectively. CONCLUSION: We have isolated a novel halo tolerant lipase following a functional screen of a marine sponge fosmid metagenomic library. The activity and stability profile of the recombinant enzyme over a wide range of salinity, pH and temperature; and in the presence of organic solvent and metal ions suggests a utility for this enzyme in a variety of industrial applications.

First certain record of Demospongiae class (Porifera) alien species from the Mediterranean Sea.

In this paper, we identify some sponge specimens collected in the Faro Lake in Sicily, and belonging to Haliclona (Halicoclona) by using morphological analysis accompanied by molecular analysis through amplification of several molecular markers (18S and 28S rRNA, CO1 and ITS). The samples are identified as. H. (Halichoclona) vansoesti de Weerdt, de Kluijver & Gómez, 1999, a species native to the Caribbean, and therefore this is the first record of an alien species of the Demospongiae class (Porifera) from the Mediterranean Sea. This presence can be ascribed as results of global change (mainly global warming) that are affecting marine environment.

Localized production of defence chemicals by intracellular symbionts of Haliclona sponges.

Marine sponges often house small-molecule-producing symbionts extracellularly in their mesohyl, providing the host with a means of chemical defence against predation and microbial infection. Here, we report an intriguing case of chemically mediated symbiosis between the renieramycin-containing sponge Haliclona sp. and its herein discovered renieramycin-producing symbiont Candidatus Endohaliclona renieramycinifaciens. Remarkably, Ca. E. renieramycinifaciens has undergone extreme genome reduction where it has lost almost all necessary elements for free living while maintaining a complex, multi-copy plasmid-encoded biosynthetic gene cluster for renieramycin biosynthesis. In return, the sponge houses Ca. E. renieramycinifaciens in previously uncharacterized cellular reservoirs (chemobacteriocytes), where it can acquire nutrients from the host and avoid bacterial competition. This relationship is highly specific to a single clade of Haliclona sponges. Our study reveals intracellular symbionts as an understudied source for defence chemicals in the oldest-living metazoans and paves the way towards discovering similar systems in other marine sponges.

Sponge epizoism in the Caribbean and the discovery of new Plakortis and Haliclona species, and polymorphism of Xestospongia deweerdtae (Porifera).

The new discovery by Vicente et al. (2014) of specialized epizoic symbioses between sponges of the genera Plakortis and Xestospongia revealed the obligate interaction of two new Plakortis spp. associating with Xestospongia deweerdtae and a new Xestospongia sp. In this study we formally describe the two new Plakortis spp. as Plakortis deweerdtaephila sp. nov. (previously reported as Plakortis sp. 1), Plakortis symbiotica sp. nov. (previously reported as Plakortis sp. 2) and describe the new Xestospongia sp. epibiont as Haliclona (Halichoclona) plakophila sp. nov.  Plakortis deweerdtaephila associates only with X. deweerdtae, and has very small to large straight diods (24.2-233.7 µm long) and triods (26.4-102.6 µm long) that form large ectosomal circular meshes (114-329 µm diameter). P. symbiotica associates with both X. deweerdtae and H. plakophila, has larger curved diods (71.9-141.8 µm long) and triods (20.4-70.6 µm long) that form smaller ectosomal circular meshes (43-121 µm diameter) than P. deweerdtaephila. Phylogenetic analysis of cox1 and cob gene fragments revealed a strongly supported clade that grouped both Plakortis spp. nov. distantly from any other known Plakortis spp. H. plakophila is described as a thin encrusting veneer of tissue with occasional papillae, so far only found associated with P. symbiotica in La Parguera, Puerto Rico. Phylogenetic analysis of 18S rRNA and cox1 gene fragments place it distantly from any known clade of Haplosclerida. We found a new associated morphotype of X. deweerdtae from Bocas del Toro Panama, which completely overgrew P. deweerdtaephila. In addition, free-living morphotypes from Panama produce larger S-shaped and round bracket shaped strongyles never before observed for this species, leading us to redescribe X. deweerdtae. All X. deweerdtae morphotypes shared >99% sequence homology of cox1, 18S rRNA and 28S rRNA genes with the holotype of X. deweerdtae. This study highlights the highly variable morphological characters of X. deweerdtae influenced by lifestyle and environmental factors. This is also the first time that an obligate symbiosis with a heterospecific sponge is used as a key taxonomic character.

A new species of Haliclona (Demospongiae: Haplosclerida: Chalinidae) from southeastern Brazil and the first record of Haliclona vansoesti from the Brazilian coast.

In this paper we describe two species of the cosmopolitan sponge genus Haliclona from Rio de Janeiro State, SE Brazil, one of which is new to science and the other a new record to Brazil. Haliclona (Rhizoniera) fugidia sp. nov. is brownish-pink, salmon or cream, thickly encrusting, without oscular tubes and tangential ectosomal reticulation. Choanosomal skeleton is a mostly unispicular ladder-like reticulation of oxeas, very organized near the sponge surface and denser and more disorganized in the interior of the sponge. Haliclona (Halichoclona) vansoesti de Weerdt et al., 1999 was originally described from the Caribbean. It has a very loose connection between ectosome and choanosome, a whitish translucent ectosome combined with a purplish choanosome, a cavernous structure and a friable or crispy consistency. The conspecificity of SE Brazilian and Caribbean populations of H. (Halich.) vansoesti was verified through phylogenetic analysis of small subunit 18S rRNA (18S) and mitochondrial cytochrome oxidase subunit I (COI) gene sequences. A maximum likelihood phylogenetic tree constructed with 18S sequences indicates that specimens of H. (Halich.) vansoesti from Rio de Janeiro were phylogenetically closer to the same species from the Caribbean than to other species of Haliclona. Although not available for H. (Halich.) vansoesti from the Caribbean, COI sequences of our specimens were also quite distinct from those of other Haliclona species. Molecular identification based on DNA sequences is a useful complement to traditional morphology-based taxonomy, especially in highly plastic sponges such as Haliclona spp. and other haplosclerids.