Successful cryopreservation of coral larvae using vitrification and laser warming

Abstract

Climate change has increased the incidence of coral bleaching events, resulting in the loss of ecosystem function and biodiversity on reefs around the world. As reef degradation accelerates, the need for innovative restoration tools has become acute. Despite past successes with ultra-low temperature storage of coral sperm to conserve genetic diversity, cryopreservation of larvae has remained elusive due to their large volume, membrane complexity, and sensitivity to chilling injury. Here we show for the first time that coral larvae can survive cryopreservation and resume swimming after warming. Vitrification in a 3.5 M cryoprotectant solution (10% v/v propylene glycol, 5% v/v dimethyl sulfoxide, and 1 M trehalose in phosphate buffered saline) followed by warming at a rate of approximately 4,500,000 °C/min with an infrared laser resulted in up to 43% survival of Fungia scutaria larvae on day 2 post-fertilization. Surviving larvae swam and continued to develop for at least 12 hours after laser-warming. This technology will enable biobanking of coral larvae to secure biodiversity, and, if managed in a high-throughput manner where millions of larvae in a species are frozen at one time, could become an invaluable research and conservation tool to help restore and diversify wild reef habitats.

Figure 1

Phalloidin Staining: Wholemount phalloidin fluorescent microscopic images of developing F. scutaria larvae stained for actin with AlexaFluor 488-conjugated phalloidin. This stain highlights the growth and development of the gastrovascular cavity (GVC; white arrows) and internal structure. Bar = 40 µm. (A) Day 1, the GVC was elongated and flat. There was a clear delineation between the endoderm and ectoderm but it appeared to be incomplete in the apical region. The plasmalemmas of cells lining the developing gastrovascular cavity were brightly stained. (B) Day 2, the actinopharynx began to open and widen and the mesenteries began to develop in the GVC. Additionally, there was more complete development of the endoderm and mesoglea. (C) Day 3 showed a well-developed actinopharynx and mouth and more complex GVC. (D) Day 4, the mesoglea began to thicken and complexity of the GVC continued to increase. (E) Day 5 presented a continued widening of muscular actinopharynx and development of mesenteries.

Figure 2

Changes in larval volume in response to the permeating cryoprotectants dimethyl sulfoxide (DMSO; 10% v/v in FSW; brown) and propylene glycol (PG; 10% v/v in FSW; yellow), and the non-permeating cryoprotectant trehalose (0.75 M in 50% FSW, green), compared to control larval volume in filtered seawater (blue), over a three-minute period. A reduction in larval volume was observed within 1 minute of exposure to permeating and non-permeating cryoprotectants on all four days. Increasing larval complexity affected the permeability of DMSO and PG from Day 2 to Day 5. Permeation of DMSO and PG caused larval volume to return to the control volume within the 3-minute analysis period on Days 2 and 3, but larval volume only returned to 85–90% of control volume on Day 4 and 75–80% on Day 5. Volumes are expressed as a proportion of the mean larval volume in FSW for a given day, and each data point is a mean of n ≥ 10 measurements.

Figure 3

Toxicity experiments measure the proportion of larvae exposed to two different cryoprotectant solutions (cryoprotectant solution 1: 7.5% (v/v) DMSO, 7.5% (v/v) PG, and 1 M trehalose in PBS, dark columns; cryoprotectant solution 2: 5% (v/v) DMSO, 10% (v/v) PG, and 1 M trehalose in PBS, light columns) that resumed swimming at two hours post-rehydration. Solution 2 demonstrated a better than 80% survival on all developmental days, whereas Solution 1 was slightly more damaging to larvae, especially on Day 3. No significant differences were found among any of the cryoprotectant solutions or days (P > 0.05; Kruskal-Wallis test with Dunn’s multiple-comparisons test). Data are mean ± SEM of n = 3 technical replicates. Each replicate contained 20–40 larvae from a pooled sample of larvae from 10 females.

Figure 4

Dehydration of larvae in response to complex cryoprotectant solutions reduced internal ice crystal formation during vitrification and even distribution of the gold nanorod particles permitted reproducible melting of 1-µL vitrified droplets, resulting in live F. scutaria larvae post-warming. (A) Changes in larval volume in response to cryopreservation solution 2 (5% v/v DMSO, 10% v/v PG, and 1 M trehalose in PBS) in larvae from Day 2 and Day 3 post-fertilization. Dehydration was more effective on Day 2 than on Day 3, which may account for the higher rate of larval recovery on that day. Volumes are expressed as a proportion of the mean larval volume in FSW for a given day, and each data point is a mean of n ≥ 10 measurements. (B) Confocal images of a Day 3 larva showing fluorescence from GFP (left) and distribution of DyLight-coated gold nanorod particles surrounding the larva in cryoprotectant (right). Gold nanorod particles were evenly distributed in the cryoprotectant solution and formed a dense field that completely surrounded the larvae. (C) Total number of Day 2 and Day 3 larvae that were vitrified and laser warmed (black), compared to the number remaining after rehydration (white), and the number that resumed swimming (grey). The number of larvae that survived rehydration and resumed swimming was reduced on Day 3 compared to Day 2 but this was not significant. Data are mean ± SEM. Columns with the same letter are not significantly different (P > 0.05, Kruskal-Wallis test with Dunn’s multiple-comparisons test).