Molecular Characterization of Sub-cuticular Bacteria in New Zealand Echinoderms

Many echinoderms contain sub-cuticular bacteria (SCB), symbionts which reside in the lumen between the epidermal cells and the outer cuticle of the host. The relationship is very common, with ~60% of all echinoderms studied so far containing SCB. Currently, little is known about the function of the symbiosis, although it has been hypothesized that SCB may aid in host nutrition or antimicrobial defense. Whatever their function, the large numbers of SCB observed in many echinoderms (10 (to the power of 8) - 10 (to the power of 9) SCB g-1 AFDW host tissue) suggest that they may be important to the host. Factors contributing to the lack of knowledge about the echinoderm-SCB symbiosis include the difficulty associated with cultivating symbiotic bacteria, and the lack of studies identifying the SCB by molecular means. In this study, molecular techniques were employed to characterize the SCB of several common New Zealand echinoderms. The specific objectives of the study were to identify the SCB through sequencing of a region of the bacterial 16S rRNA gene, identify and locate SCB in situ through the use of fluorescence in situ hybridization (FISH), and cultivate SCB obtained from those echinoderms which were found to contain them. Phylogenetic analysis of 16S rRNA sequences obtained from echinoderm-associated bacteria resulted in the identification of four putative species of SCB. All four bacteria were isolated from samples of Stichopus mollis (class Holothuroidea), and two of the four were also found in samples of Patiriella sp (class Asteroidea). The first putative SCB belongs to the order Rhizobiales (a-proteobacteria), and is closely related to the SCB previously isolated from the brittle star Ophiactis balli. The second species belongs to the order Chromatiales (y-proteobacteria). Putative SCB species 3 falls within the Roseobacter clade (a-proteobacteria). The phylogenetic placement of the final putative SCB is more ambiguous, as this bacterium falls among members of the a- and y- subdivisions of the phylum Proteobacteria. The nearest relatives of this final bacterium are in the orders Rickettsiales and Thiotrichales. Results of FISH assays show that Patiriella sp. and S. mollis contain SCB, while a third species, Astrostole scabra (class Asteroidea) does not. The SCB community composition was found to vary between Patiriella sp. and S. mollis. In both species, the majority of the SCB present were found to belong to the a-subdivision of the phylum Proteobacteria (>80% in both species). However, in S. mollis, ~20% of the SCB community consists of bacteria belonging to the y-subdivision of the phylum Proteobacteria, whereas bacteria belonging to this subdivision were never observed in Patiriella sp. Cultivation experiments were carried out using a range of culture media, however results were inconclusive. Ten species of proteobacteria were successfully cultivated, three of which were obtained only from Patiriella sp. and S. mollis samples and were considered possible candidates for SCB. However, phylogenetic analysis of these three bacteria revealed that closely-related bacteria are predominantly free-living species. While the possibility remains that these three bacteria are in fact SCB, it seems more likely that they represent seawater or echinoderm surface-associated bacteria. This study contributes to the body of knowledge of the echinoderm-SCB symbiosis by identifying several potential SCB in Patiriella sp. and S. mollis, and is the first to identify SCB in situ through the use of FISH. An obvious goal in studies of the echinoderm-SCB symbiosis is to determine the function of the relationship. Potential functions of the symbiosis, based on the results obtained here, are discussed herein.