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Cryopreservation of the gorgonian endosymbiont Symbiodinium.

ABSTRACT: The study focused on finding a suitable cryoprotectant (CPA) and an optimum freezing protocol for the cryopreservation of the endosymbiotic dinoflagellates (Symbiodinium, clade G) of Junceella fragilis wherein the success of experiments is crucial to both scientific and ecology studies. A two-step freezing technique was developed. The viability of the thawed dinoflagellates was assayed using the adenosine triphosphate (ATP) bioassay for the first time and was further confirmed through the culturing of dinoflagellates in vitro. The results suggested that 30 min was the most suitable holding time for the dinoflagellates, and the samples produced highest viability when suspended at 5 cm from the surface of LN2. Results also showed that 1 M methanol with 0.4 M sucrose was the most effective CPA, yielding the highest viability (56.93%). Although cell densities of both cryopreserved and control group suffered an initial decline of culture, the cell densities were maintained throughout the remaining duration. In the present study, the cryopreservation of clade G endosymbiont algae was studied for the first time and the method described here could be applied for future studies on symbiotic algae cryopreservation.

SUBMITTER: Chong G

PROVIDER: S-EPMC4709583 | biostudies-other | 2016 Jan

REPOSITORIES: biostudies-other

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Project description:Ribosomal DNA sequence data abounds from numerous studies on the dinoflagellate endosymbionts of corals, and yet the multi-copy nature and intragenomic variability of rRNA genes and spacers confound interpretations of symbiont diversity and ecology. Making consistent sense of extensive sequence variation in a meaningful ecological and evolutionary context would benefit from the application of additional genetic markers. Sequences of the non-coding region of the plastid psbA minicircle (psbA(ncr)) were used to independently examine symbiont genotypic and species diversity found within and between colonies of Hawaiian reef corals in the genus Montipora. A single psbA(ncr) haplotype was recovered in most samples through direct sequencing (~80-90%) and members of the same internal transcribed spacer region 2 (ITS2) type were phylogenetically differentiated from other ITS2 types by substantial psbA(ncr) sequence divergence. The repeated sequencing of bacterially-cloned fragments of psbA(ncr) from samples and clonal cultures often recovered a single numerically common haplotype accompanied by rare, highly-similar, sequence variants. When sequence artifacts of cloning and intragenomic variation are factored out, these data indicate that most colonies harbored one dominant Symbiodinium genotype. The cloning and sequencing of ITS2 DNA amplified from these same samples recovered numerically abundant variants (that are diagnostic of distinct Symbiodinium lineages), but also generated a large amount of sequences comprising PCR/cloning artifacts combined with ancestral and/or rare variants that, if incorporated into phylogenetic reconstructions, confound how small sequence differences are interpreted. Finally, psbA(ncr) sequence data from a broad sampling of Symbiodinium diversity obtained from various corals throughout the Indo-Pacific were concordant with ITS lineage membership (defined by denaturing gradient gel electrophoresis screening), yet exhibited substantially greater sequence divergence and revealed strong phylogeographic structure corresponding to major biogeographic provinces. The detailed genetic resolution provided by psbA(ncr) data brings further clarity to the ecology, evolution, and systematics of symbiotic dinoflagellates.

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Cryopreservation of the gorgonian endosymbiont Symbiodinium.

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