The role of sponge-bacteria interactions: the sponge Aplysilla rosea challenged by its associated bacterium Streptomyces ACT-52A in a controlled aquarium system.

Sponge-associated bacteria play a critical role in sponge biology, metabolism and ecology, but how they interact with their host sponges and the role of these interactions are poorly understood. This study investigated the role of the interaction between the sponge Aplysilla rosea and its associated actinobacterium, Streptomyces ACT-52A, in modifying sponge microbial diversity, metabolite profile and bioactivity. A recently developed experimental approach that exposes sponges to bacteria of interest in a controlled aquarium system was improved by including the capture and analysis of secreted metabolites by the addition of an absorbent resin in the seawater. In a series of controlled aquaria, A. rosea was exposed to Streptomyces ACT-52A at 106 cfu/ml and monitored for up to 360 h. Shifts in microbial communities associated with the sponges occurred within 24 to 48 h after bacterial exposure and continued until 360 h, as revealed by TRFLP. The metabolite profiles of sponge tissues also changed substantially as the microbial community shifted. Control sponges (without added bacteria) and Streptomyces ACT-52A-exposed sponges released different metabolites into the seawater that was captured by the resin. The antibacterial activity of compounds collected from the seawater increased at 96 and 360 h of exposure for the treated sponges compared to the control group due to new compounds being produced and released. Increased antibacterial activity of metabolites from treated sponge tissue was observed only at 360 h, whereas that of control sponge tissue remained unchanged. The results demonstrate that the interaction between sponges and their associated bacteria plays an important role in regulating secondary metabolite production.

Global conservation status of sponges.

Sponges are important for maintaining ecosystem function and integrity of marine and freshwater benthic communities worldwide. Despite this, there has been no assessment of their current global conservation status. We assessed their status, accounting for the distribution of research effort; patterns of temporal variation in sponge populations and assemblages; the number of sponges on threatened species lists; and the impact of environmental pressures. Sponge research effort has been variable; marine sponges in the northeastern Atlantic and Mediterranean and freshwater sponges in Europe and North America have received the most attention. Although sponge abundance has increased in some locations since 1990, these were typically on coral reefs, in response to declines in other benthic organisms, and restricted to a few species. Few data were available on temporal trends in freshwater sponge abundance. Despite over 8500 described sponge species, only 20 are on threatened species lists, and all are marine species from the northeastern Atlantic and Mediterranean. Of the 202 studies identified, the effects of temperature, suspended sediment, substratum loss, and microbial pathogens have been studied the most intensively for marine sponges, although responses appear to be variable. There were 20 studies examining environmental impacts on freshwater sponges, and most of these were on temperature and heavy metal contamination. We found that most sponges do not appear to be threatened globally. However, little information is available for most species and more data are needed on the impacts of anthropogenic-related pressures. This is a critical information gap in understanding sponge conservation status.

Sponge-associated actinobacterial diversity: validation of the methods of actinobacterial DNA extraction and optimization of 16S rRNA gene amplification.

Experiments were designed to validate the two common DNA extraction protocols (CTAB-based method and DNeasy Blood & Tissue Kit) used to effectively recover actinobacterial DNA from sponge samples in order to study the sponge-associated actinobacterial diversity. This was done by artificially spiking sponge samples with actinobacteria (spores, mycelia and a combination of the two). Our results demonstrated that both DNA extraction methods were effective in obtaining DNA from the sponge samples as well as the sponge samples spiked with different amounts of actinobacteria. However, it was noted that in the presence of the sponge, the bacterial 16S rRNA gene could not be amplified unless the combined DNA template was diluted. To test the hypothesis that the extracted sponge DNA contained inhibitors, dilutions of the DNA extracts were tested for six sponge species representing five orders. The results suggested that the inhibitors were co-extracted with the sponge DNA, and a high dilution of this DNA was required for the successful PCR amplification for most of the samples. The optimized PCR conditions, including primer selection, PCR reaction system and program optimization, further improved the PCR performance. However, no single PCR condition was found to be suitable for the diverse sponge samples using various primer sets. These results highlight for the first time that the DNA extraction methods used are effective in obtaining actinobacterial DNA and that the presence of inhibitors in the sponge DNA requires high dilution coupled with fine tuning of the PCR conditions to achieve success in the study of sponge-associated actinobacterial diversity.

The Relationship Between Microbiomes and Selective Regimes in the Sponge Genus Ircinia.

Sponges are often densely populated by microbes that benefit their hosts through nutrition and bioactive secondary metabolites; however, sponges must simultaneously contend with the toxicity of microbes and thwart microbial overgrowth. Despite these fundamental tenets of sponge biology, the patterns of selection in the host sponges' genomes that underlie tolerance and control of their microbiomes are still poorly understood. To elucidate these patterns of selection, we performed a population genetic analysis on multiple species of Ircinia from Belize, Florida, and Panama using an F ST -outlier approach on transcriptome-annotated RADseq loci. As part of the analysis, we delimited species boundaries among seven growth forms of Ircinia. Our analyses identified balancing selection in immunity genes that have implications for the hosts' tolerance of high densities of microbes. Additionally, our results support the hypothesis that each of the seven growth forms constitutes a distinct Ircinia species that is characterized by a unique microbiome. These results illuminate the evolutionary pathways that promote stable associations between host sponges and their microbiomes, and that potentially facilitate ecological divergence among Ircinia species.

The Effects of Sampling and Storage Conditions on the Metabolite Profile of the Marine Sponge Geodia barretti.

Geodia barretti is a deep-sea marine sponge common in the north Atlantic and waters outside of Norway and Sweden. The sampling and subsequent treatment as well as storage of sponges for metabolomics analyses can be performed in different ways, the most commonly used being freezing (directly upon collection or later) or by storage in solvent, commonly ethanol, followed by freeze-drying. In this study we therefore investigated different sampling protocols and their effects on the detected metabolite profiles in liquid chromatography-mass spectrometry (LC-MS) using an untargeted metabolomics approach. Sponges (G. barretti) were collected outside the Swedish west coast and pieces from three sponge specimens were either flash frozen in liquid nitrogen, frozen later after the collection cruise, stored in ethanol or stored in methanol. The storage solvents as well as the actual sponge pieces were analyzed, all samples were analyzed with hydrophilic interaction liquid chromatography as well as reversed phase liquid chromatography with high resolution mass spectrometry using full-scan in positive and negative ionization mode. The data were evaluated using multivariate data analysis. The highest metabolite intensities were found in the frozen samples (flash frozen and frozen after sampling cruise) as well as in the storage solvents (methanol and ethanol). Metabolites extracted from the sponge pieces that had been stored in solvent were found in very low intensity, since the majority of metabolites were extracted to the solvents to a high degree. The exception being larger peptides and some lipids. The lowest variation between replicates were found in the flash frozen samples. In conclusion, the preferred method for sampling of sponges for metabolomics was found to be immediate freezing in liquid nitrogen. However, freezing the sponge samples after some time proved to be a reliable method as well, albeit with higher variation between the replicates. The study highlights the importance of saving ethanol extracts after preservation of specimens for biology studies; these valuable extracts could be further used in studies of natural products, chemosystematics or metabolomics.

Genomic Insights Into the Lifestyles of Thaumarchaeota Inside Sponges.

Sponges are among the oldest metazoans and their success is partly due to their abundant and diverse microbial symbionts. They are one of the few animals that have Thaumarchaeota symbionts. Here we compare genomes of 11 Thaumarchaeota sponge symbionts, including three new genomes, to free-living ones. Like their free-living counterparts, sponge-associated Thaumarchaeota can oxidize ammonia, fix carbon, and produce several vitamins. Adaptions to life inside the sponge host include enrichment in transposases, toxin-antitoxin systems and restriction modifications systems, enrichments previously reported also from bacterial sponge symbionts. Most thaumarchaeal sponge symbionts lost the ability to synthesize rhamnose, which likely alters their cell surface and allows them to evade digestion by the host. All but one archaeal sponge symbiont encoded a high-affinity, branched-chain amino acid transporter system that was absent from the analyzed free-living thaumarchaeota suggesting a mixotrophic lifestyle for the sponge symbionts. Most of the other unique features found in sponge-associated Thaumarchaeota, were limited to only a few specific symbionts. These features included the presence of exopolyphosphatases and a glycine cleavage system found in the novel genomes. Thaumarchaeota have thus likely highly specific interactions with their sponge host, which is supported by the limited number of host sponge species to which each of these symbionts is restricted.

Spatial Cell Disparity in the Colonial Choanoflagellate Salpingoeca rosetta.

Choanoflagellates are the closest unicellular relatives of animals (Metazoa). These tiny protists display complex life histories that include sessile as well as different pelagic stages. Some choanoflagellates have the ability to form colonies as well. Up until recently, these colonies have been described to consist of mostly identical cells showing no spatial cell differentiation, which supported the traditional view that spatial cell differentiation, leading to the co-existence of specific cell types in animals, evolved after the split of the last common ancestor of the Choanoflagellata and Metazoa. The recent discovery of single cells in colonies of the choanoflagellate Salpingoeca rosetta that exhibit unique cell morphologies challenges this traditional view. We have now reanalyzed TEM serial sections, aiming to determine the degree of similarity of S. rosetta cells within a rosette colony. We investigated cell morphologies and nuclear, mitochondrial, and food vacuole volumes of 40 individual cells from four different S. rosetta rosette colonies and compared our findings to sponge choanocytes. Our analysis shows that cells in a choanoflagellate colony differ from each other in respect to cell morphology and content ratios of nuclei, mitochondria, and food vacuoles. Furthermore, cell disparity within S. rosetta colonies is slightly higher compared to cell disparity within sponge choanocytes. Moreover, we discovered the presence of plasma membrane contacts between colonial cells in addition to already described intercellular bridges and filo-/pseudopodial contacts. Our findings indicate that the last common ancestor of Choanoflagellata and Metazoa might have possessed plasma membrane contacts and spatial cell disparity during colonial life history stages.

Untapped sponge microbiomes: structure specificity at host order and family levels.

Sponges are complex holobionts in which the structure of the microbiome has seldom been characterized above the host species level. The hypothesis tested in this study is that the structure of the sponge microbiomes is specific to the host at the order and family levels. This was done by using 33 sponge species belonging to 19 families representing five orders. A combination of three primer sets covering the V1-V8 regions of the 16S rRNA gene provided a more comprehensive coverage of the microbiomes. Both the diversity and structure of sponge microbiomes were demonstrated to be highly specific to the host phylogeny at the order and family levels. There are always dominant operational taxonomic units (OTUs) (relative abundance >1%) shared between microbial communities of sponges within the same family or order, but these shared OTUs showed high levels of dissimilarity between different sponge families and orders. The unique OTUs for a particular sponge family or order could be regarded as their 'signature identity'. 70%-87% of these unique OTUs (class level) are unaffiliated and represent a vast resource of untapped microbiota. This study contributes to a deeper understanding on the concept of host-specificity of sponge microbiomes and highlights a hidden reservoir of sponge-associated microbial resources.