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. 2019 Dec 11;9(1):18851.
doi: 10.1038/s41598-019-55374-6.

First instance of settlement by cryopreserved coral larvae in symbiotic association with dinoflagellates

Luca Cirino  1   2 , Zhi-Hong Wen  1 , Kevin Hsieh  3 , Cheng-Liang Huang  4 , Qi Lun Leong  4 , Li-Hsueh Wang  2   5 , Chii-Shiarng Chen  1   2   5 , Jonathan Daly  6   7 , Sujune Tsai  8 , Chiahsin Lin  9   10
Affiliations

First instance of settlement by cryopreserved coral larvae in symbiotic association with dinoflagellates

Luca Cirino et al. Sci Rep. .

Abstract

Coral reefs are suffering on a global scale due to human impacts, thereby necessitating cryopreservation efforts. The objective of this study was to develop a suitable vitrification and laser warming protocol for larvae of the scleractinian coral Seriatopora caliendrum, which inherit their dinoflagellate algal symbionts vertically. Toxicity experiments were conducted with the cryoprotectants (CPAs) ethylene glycol (EG), propylene glycol (PG), dimethyl sulfoxide (DMSO), glycerol (GLY), and methanol (METH; listed in order from least to most toxic), and larvae were subjected to vitrification and laser warming using 2 M EG + 1 M PG and 2 M EG + 1 M DMSO. Vitrification and laser warming (300 V, 10 ms pulse width, 2 mm beam diameter) using a vitrification solution of 2 M EG + 1 M PG, 40% w/v Ficoll, and 10% v/v gold nanobars (GNB) at a final concentration of 1.2 × 1018 GNB/mL and a characteristic wavelength of 535 nm resulted in larvae with vitality and settlement percentages of 55 and 9%, respectively. This represents the first successful instance of cryopreservation of coral larvae that proceeded to settle upon warming, and suggests that the vitrification and ultra-fast laser warming approach may be applicable to other threatened marine species.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
A specimen of Seriatopora caliendrum sampled by SCUBA divers (a), the coral larvae hatchery (b), and details of the water flow throughout the hatchery (c).
Figure 2
Figure 2
A Seriatopora caliendrum larva used for vitrification and laser warming.
Figure 3
Figure 3
Gold nanobars (GNBs) used for laser warming. (a) The UV-vis-NIR spectrum of the GNB colloids; (b) Transmission electron microscopy (TEM) image of GNBs. Figure of High magnification (100 K) shows in the top-right corner.
Figure 4
Figure 4
Toxic effects (mean +/− SEM) of different cryoprotectants (CPA) on larval viability (top row of each panel) and settlement rate (bottom row of each panel) of Seriatopora caliendrum larvae: ethylene glycol (EG; a), propylene glycol (PG; 2), DMSO (c), glycerol (GLY; d), and methanol (METH; e). Each CPA was used at concentrations of 0.5, 1, 2, or 3 M for 10, 20, or 30 min. The bars with different numbers and letters on top, respectively, indicate significant differences (Tukey’s p < 0.05) in ATP content and settlement rate, respectively. Viability was referred by ATP content.
Figure 5
Figure 5
A Seriatopora caliendrum larva (a) that settled 12 hours after vitrification and warming using vitrification solution 1 (VS1). Integrity of vitrified and laser-warmed larvae (b; as an index; see methods), vitality (c; as a rate), and settlement (d; %) represented as registered (raw) and normalized data.
See this image and copyright information in PMC

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