“Symbio-Cryobank”: Toward the Development of a Cryogenic Archive for the Coral Reef Dinoflagellate Symbiont Symbiodiniaceae

Introduction

Applied Symbiodiniaceae cryopreservation has progressed in recent years in response to environmental changes threatening the integrity of reefs worldwide. With the development of new cryotechniques, success has been achieved in Symbiodiniaceae freezing.^1–4 Two-step freezing improves the survival of Symbiodiniaceae. ³ The advantages of the two-step freezing protocol are that (1) more Symbiodiniaceae can be cryopreserved through this method than through vitrification (cryotop-related carriers), (2) the ease of setup and operation, (3) mobility, and (4) low cost.

Results and Discussion

Successful protocols utilized for Symbiodiniaceae cryopreservation are listed in Table 1. Symbiodiniaceae were cryopreserved through various methods and required different cryoprotective agents (CPAs) and concentrations, equilibria, and holding times. Seriatopora caliendrum larvae with Cladocopium were successfully cryopreserved through vitrification and nanogold-laser warming. ⁵ This was the first study on the cryopreservation of coral larvae containing Symbiodiniaceae. Other studies recorded two-step freezing as the optimum cryopreservation protocol (Table 1).^1,3 The results from the assay by Santiago-Vázquez et al. ⁴ suggested that the two-step freezing is more effective than a three-step protocol regarding the viability of Symbiodiniaceae (Table 1). During the three-step protocol, the slower rate of cooling likely led to excessive cell dehydration, which reduced the efficiency of freezing. For all CPAs tested on Symbiodiniaceae, 2 M concentration was the upper limit, which suggested that they had a protective effect when used at lower concentrations, except Breviolum with CPAs of 20% methonol (MeOH) and 20% EtOH may be disadvantageous if an unsuitable or chemically toxic CPA is used for a prolonged equilibration (Table 1). A concentration of 1 M of CPAs is generally preferred for Fugacium (Table 1). The equilibration period of 30 minutes was sufficient for CPAs to permeate Symbiodiniaceae; however, this period was unsuitable for ethylene glycol (EG) and propylene glycol (PG) in Symbiodinium and Fugacium because they are less permeable than the other CPAs (Table 1).

Table 1 also presents the Symbiodiniaceae species that were collected and deposited in the symbio-cryobank in the National Museum of Marine Biology and Aquarium (NMMBA), Taiwan, after two-step freezing (patented device no.: M394447). The Symbiodiniaceae species were preserved in 0.25 mL straws (1–2 × 10⁶/mL) in liquid nitrogen, and coding was used to identify the cryopreserved samples. The sample placed from the surface of LN2 (7 cm) had the final temperature at −112.9°C, whereas the sample placed from the surface of LN2 (5 cm) had the final temperature at −123.9°C (Table 1). ¹ Symbiodiniaceae species placed in the cryobank were extracted from corals collected in Nanwan, Taiwan (GPS coordinates: 21°56′N, 120°44′E) at 10 m depth, and were transferred to the NMMBA husbandry center with a permit issued by the Kenting National Park Office. Freshly isolated Cladocopium was extracted from the fragments of S. caliendrum. Cladocopium and coral host cells were isolated using a density gradient technique involving sucrose. Freshly isolated Gerakladium was extracted from the epidermis layer of Junceella fragilis. Free-living Symbiodiniaceae species were collected from a single cell culture at 25°C under a 12-h light–dark cycle. The viability of Symbiodiniaceae was validated by an ATP bioassay with a Lumat 9507 luminometer (Berthold Technologies, Bad Wildbad, Germany), followed by cell culture. After two-step freezing, Symbiodiniaceae species were long-term cryopreserved by being plunged into liquid nitrogen, with coding for their identification. Six Symbiodiniaceae species were cryopreserved in a total of 634 straws.

EG is an effective CPA for Symbiodiniaceae. EG and PG were the only two CPAs that provided excellent cryoprotection for Durusdinium ³ and Fugacium (herein; Table 1). EG was effective for Symbiodinium, free-living Cladocopium, and Fugacium (herein). In various studies, they exhibited protective effects only when combined with other CPAs. ⁶ Symbiodinium, Breviolium, Cladocopium, Fugacium, and Gerakladium were successfully cryopreserved using MeOH through two-step freezing (Table 1) [herein, Refs.^1,4], and freshly isolated Cladocopium was successfully cryopreserved using a vitrification solution comprising MeOH and dimethyl sulfoxide. ² MeOH was less toxic to Breviolum when equilibrated 10 minutes at 20% (Table 1). Nevertheless, the high penetrative ability may be disadvantageous if an unsuitable or chemically toxic CPA is used for a prolonged period. ⁴ For Cladocopium, glycerol (Gly) was conducted because it is the CPA with the highest level of cryoprotection, which was consistent to the results of Chong et al. ¹ for Gerakladium, in which 1 M Gly for a 30-minute equilibrium period produced 26% post-thaw viability. In addition, Durusdinium exhibited >50% post-thaw viability with Gly. ³ Gerakladium was also cryopreserved with Gly using a controlled slow-cooling protocol (Table 1). ⁷ Cladocopium may produce intracellular amounts of Gly that may protect cells and increase the total concentration of all solutes in the system to reduce the amount of ice formed. To verify this hypothesis, additional studies are necessary.

Cladocopium under a two-living status (free-living and freshly isolated) was successfully cryopreserved through two-step freezing (Table 1). Free-living Cladocopium was cryopreserved with MeOH, EG, and Gly (herein). However, MeOH and EG were not effective for Cladocopium isolated from S. caliendrum, which suggested that MeOH combined with EG has more toxic effects on freshly isolated Cladocopium and that Gly has higher viability. However, MeOH is effective for other freshly isolated Symbiodiniaceae (Gerakladium and Breviolium), and EG and PG show cryoprotection only for free-living Symbiodiniaceae (Table 1). Comparison between freshly isolated and free-living cultured cells revealed several differences: After culturing in the medium, they became smaller and their cell walls became less thick, and their chloroplast content and number of lipid droplets decreased. ⁸ Fuentes-Grünewald et al. ⁹ stated that high amounts of lipids in marine dinoflagellates cause problems in cryopreservation. Because free-living Symbiodiniaceae species have thin cell walls and fewer lipid droplets, free-living Symbiodiniaceae species may be suitable for freezing and as CPAs can be more effective when permeable. This explains why EG and PG viability was effective only for free-living Symbiodiniaceae species.

Cryobanks of coral symbionts reflect a new and major type of preservation that can be used instantly. Taiwan's first Symbiodiniaceae cryobank was developed in the NMMBA. We created a symbiocryobank (1) to protect viable specimens of each high-priority species, (2) provide biomaterials to scientists for basic and applied research, (3) allow a portion of the bank to retain frozen but alive specimens for hundreds of years in liquid nitrogen, if necessary, allowing threats to be mitigated and habitats to be restored, (4) thaw samples from the repository to “seed” new gene diversity into damaged and bleached reefs, and (5) provide the flexible and thermally tolerant Symbiodiniaceae species, which is key to the successful formation of a robust and comprehensive cryobank for the world's Symbiodiniaceae species. This approach may provide a crucial tool for selective breeding experiments and the development of thermally resilient strains of coral.

Authors' Contributions

C.L. and S.T. conceived the experiment, S.D.G. and L.H.W. conducted the experiment, P.J.M. and S.T. analyzed the results, and S.D.G. and C.L. wrote the article.