Optimization of in vitro culture method for zooxanthellae associated with reef-building corals

Abstract:

[Objective] To develop an efficient method for isolating and cultivating zooxanthellae from reef-building corals and lay a foundation for the research on germplasm banking and physiological functions of zooxanthellae species associated with corals. [Methods] The zooxanthellae were isolated and enriched from reef-building coral tissues by micro-strainer filtration and density gradient centrifugation, and the cells were cultured in 96-well plates with the modified L1 medium. Single clones of zooxanthellae were obtained by single cell isolation, culture, and/or plate streaking. The species and phylogenetic relationship were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis in combination with internal transcribed spacer 2 (ITS2) and large subunit (LSU) sequences. [Results] Three zooxanthellae strains were isolated from Acropora pruinose of Weizhou Island, Galaxea fascicularis and Acropora tenuis of Xisha Islands and numbered AP21C1, GF21D1, and AT21A113, respectively. The three strains showed the ITS2 genotypes of C1, D1, and A113 and the sequences basically consistent with Cladocopium goreaui, Durusdinium trenchii, and Symbiodinium natans, respectively. All the three strains demonstrated self-spinning motion and adherence during the logarithmic growth phase, and strain AP21C1 was unable to grow on agar plates. [Conclusion] This study develops an efficient method for the in vitro culture of zooxanthellae associated with corals and provides a technical and theoretical basis for the research on the germplasm banking and physiological functions of zooxanthellae species associated with corals.