The Symbiotic Cyanobacteria in Mediterranean Sponges

Globally, sponges are known to harbour various symbiotic cyanobacteria or algae; although cyanobactiera are more prevalent. These organisms have a mutually beneficial relationship that can boost the metabolism and increase the supply of nutrients to a host, whilst the symbiont receives protection from grazers and reduced UV exposure, and also receives a source of nitrogen from the host. Some work suggests that cyanobacteria in particular are able to provide defensive secondary metabolites and can increase the adaptability and ecological function of sponges. The genetic diversity of these sponge-associated cyanobacteria consists of multiple linneages such as Synechoccus, Synechocystis, Oscillataria, Lyngbya and Cyanobacterium. The most common and widespread symbiont is Candidatus Synechococcus spongiarum, a single-celled cyanobacertium that exsists in the peripheral region of the sponge. This particular symbiont can be found globally from tropical to temperate regions and makes up 85% of sponge-photosymbiont associations. Recent molecular evidence has found hidden diversity within the populations of S. sporangium. The cyanobacterial symbionts Synechocystis and Prochloron have also been described from sponges however, genetic characterization of these photosymbionts has only been reported from 5 locations and the ecological importance of these species is unknown. This study examins the diversity and activity of cyanobacterial symbionts in the Mediterranean sponge species Ircinia fasciculate and Ircinia variabilis. I. fasciculate inhabits high-irradiance zones whilst I. variabilis inhabits low irradiance, shaded zones. This study compares the genetic diversity, morphology and chlorophyll a (chl a) content of the cyanobacterial symbionts in these two temperate sponges.

Six individuals of each sponge species were collected from two neighbouring sites. Tissues samples were preserved in enthanol and stored in -20°C for genetic analysis. Three individuals of each species were assessed for chl a by determining the various absorbance of supernatant aliquots and using equations from Parsons et al (1984). The ultrastructure of the cyanobacteria species was determined by using transmission electron microscopy (TEM). Tissue from the different sponger species was fixed and incubated overnight before being embedded in a Spurr resin. This was then sliced into ultra-thin sections. Only cells that exhibited a clear center and thylakoids were digitally analysed and measured. Three individuals of each sponge species were prepared for metagenomic analysis following Qiagen® protocol. DNA extracts were used in PCR amplification using the 3’ end of the 16s region, the entire 16s-23s ITS region and the 5’ end of the 23S region. A low annealing temperature was used to reduce PCR bias. The PCR products were purified and individual clones were screened. These were subject to BLAST searches. Phylogentic reconstructions were then built using the different recovered rRNA gene fragments. S. spongiarum was reconstructed using 16S-23S rRNA ITS sequence whilst Synechocystis was constructed using only 16S rRNA sequences.

Chl a in I. fasciculate averaged around 248.1±27.8 μg/g whilst I. variabilis averaged 131.0±15.1 μg/g. These differences were shown to be significant with I. fasciculate being nearly twice the level of I. variabilis. The dominant symbiont cell seen under TEM represented the cell Candidatus S. spongiarum. These cells were seen to be actively reproducing in the mesohyl layer of the sponges. They also appeared to be interacting with the hosts’ archeocyte cells. The S. spongiarum was occasionally seen to be engulfed by the host cell although there was evidence of consumption. These symbionts were seen to be significantly larger in I. variabilis than in I. fasciculate. I. fasciculate also exhibited a significantly higher abundance of glycogen granules and a second morphotype of S. spongiarum that was three times larger than the average S. spongiarum cell. Synechocystis was not seen to be reproducing and there was no obvious interaction between host and symbiont cells. The clone libraries revealed two different cyanobacterial symbionts in I. fasciculata and I. variabilis. 85% of genetic sequencing in I. fasciculate and 100% in I. variabilis was found to correspond to Candidatus S. spongiarum. The remaining percentage was found to be Synechocystis. The ITS markers from Synechocystis were found to be shorter than S. spongiarum. Additionally a novel clade, named clade ‘M’, of S. spongiarum was identified in both sponges whilst a novel clade was also discovered for Synechocystis in I. fasciculata.

Although the sponge species exhibited the similar species of cyanobacteria, the symbionts were composed of different chl a pigments and storage products that correlated to the irradiance of the environment from which the host was recovered. Additionally the symbionts in I. fasciculata were engrained with glycogen granules that indicated the transfer of surplus carbon stores to the host. The irradiance conditions were thought to play a role in dictating the activity of sponge-associated cyanobacteria. Both hosts were dominated by the novel ‘M’ S. spongiarum clade but Synechocystis was only observed in I. fasciculata, this is different to the Caribbean Ircinia spp hosts that do not contain Synechocystis and hold a different clade of S. spongiarum however, the different ITS markers (3’ 16S, 16S-23S and 5’ 23S) used produced different results on this matter. Yet past work on this topic has also eluded to distinct clades per region and the use of different ITS markers may further shed light on the hidden cryptic diversity of these cyanobacterial symbionts. The different cell sizes suggest morphological plasticity in response to ambient irradiance levels and the various irradiance gradients in these flexible organisms suggests an environmental gradient rather than an expulsion/compositional shift as in coral/zooxanthellae symbiosis. Furthermore, the dependence of the host upon the symbiont was not significant, despite high abundance of S. spongiarum. This is in contrast to most other sponge-cyanobacteria symbiosis. It was suggested that this may be due to the active regulation of cyanobacteria to avoid predation on cyanobacteria-rich sponges, and to reduce the oxidative stress produced by the symbiont.

Erwin P.M & López-Legentil S. 2012. Ultrastructure, Molecular Phylogenetics and Chlorophyll a Content of Novel Cyanobacterial Symbionts in Temperate Sponges, Microb. Ecol. 64: 771-783.