The characterization of the cyanobacterial passengers of open ocean marine eukaryotic hosts: The abundance, ultrastructure, and phylogenetic diversity

Cyanobacterial symbionts formed associations with a Radiolarian, Dictyocoryne sp., a Tintinnid, Codonella sp. and several genera of Dinophysoids: Amphisolenia, Citharistes, Dinophysis, Histioneis, and Ornithocercus. A Reverse Transcriptase Polymerase Chain Reaction was developed and used with cyanobacteria specific primers for 16S rRNA to reveal the cyanobiont's phylogeny, diversity, and specificity on individual hosts. The function of the cyanobacteria partners was interpreted by comparing the cyanobiont sequences to known organisms. Individuals were isolated for an Immunocytochemistry assay coupled with Electron Microscopy.; Each individual collected was closely associated with a mixed assemblage of 1--3 cyanobacterial and/or 1--2 bacterial morphotypes. The symbionts of Dictyocoryne sp. had similar size, cell shape, and peripheral thylakoids with intense phycoerythrin label to Prochlorococcus sp. Similarly, the 16S rRNA sequence from Dictyocoryne sp. symbionts showed 99% similarity to Prochlorococcus sp. isolates; Prochlorococcus has not previously been reported living symbiotically. Cyanobionts of Codonella sp. and Ornithocercus sp. were most similar in size and thylakoid distribution to Synechococcus sp., as was most of the 16S rRNA symbiont phylotypes. In one Histioneis sp. host, a few larger cyanobionts labeled with nitrogenase resided amongst many smaller non-nitrogenase labeled cyanobionts and bacteria. The presence of nitrogenase has not been previously shown in the cyanobionts of Dinophysoids. Clusters of bacteria were also observed in Dictyocoryne, Ornithocercus and Codonella. Mixed populations appeared to be the norm, and supported a 3-part reclassification of these symbioses.; Phylogenetic analyses of the symbiont sequences supported two distinct lineages, one clade contained the majority of sequences (n = 42, 84%) with high similarity (96--100%) to photosynthetic free-living cyanobacteria and the other clade had a few novel phylotypes (n = 8, 16%) dispersed with other free-living photosynthetic diazotrophs and plastid genes. One symbiont type was present in several genetically diverse hosts, while others were specific to one host genus. Mixed assemblages of symbiont phylotypes were found in 12 individual hosts. Several cyanobiont sequences had low sequence identity (81--86%) to the database; some clustered with known nitrogen-fixing taxa, Cyanothece sp. It appeared that symbioses between cyanobacteria are more abundant than previously assumed, and the discovery of several unique phylotypes suggested that other cyanobacterial lineages are yet undescribed.