Metrics of Coral Microfragment Viability

Abstract

Coral reefs are being degraded at unprecedented rates and decisive intervention actions are urgently needed to help them. One such intervention in aid of reefs is coral cryopreservation. Although the cryopreservation of coral sperm and larvae has been achieved, preservation of coral fragments including both its tissue and skeleton, has not. The goal of this paper was to understand and assess the physiological stressors that might underlie coral fragment cryopreservation and the long-term consequences of these physiological exposures to continued growth. Therefore, we assessed small fragments (~0.5 x0.5 mm2) from the Hawaiian coral, Porites compressa, examining: 1) the sensitivity of the fragments and their algal symbionts to chilling temperatures; 2) the sensitivity of the coral to complex cryoprotectants; 3) methods to safely remove the algal symbionts from the coral fragment for cryopreservation, given the two symbiotic partners may require different cryopreservation protocols; 4) continued growth over time of coral fragments once returned to running seawater after treatment exposures; and, 5) assessment of health and viability of microfragments after treatments examining the distribution of green fluorescent protein and fluorescent symbionts. Technological advances in cryo-technology promise to support successful coral fragment cryopreservation soon, and its success could help secure much of the genetic and biodiversity of reefs in the next decade.

Cryopreservation and revival of Hawaiian stony corals via isochoric vitrification

“…While existing techniques are adequate in many circumstances, they fall short when external stressors (including thermal and chemical stressors) may elicit visible physiological responses in the coral that do not necessarily equate to cell death, tissue damage, or other long term deleterious effects. Our lab established the utilities and limits of some of these methods for Porites compressa in previous work 33 , concluding ultimately that simple visual morphological evaluation, brightfield microscopic evaluation, and even confocal fluorescent evaluation provided limited use in establishing the relative health of stressed coral fragments for these experiments. In need of a more robust and quantitative method by which to evaluate the health of coral fragments, we used a microrespirometry technique to record oxygen consumption rates over time in order to differentiate between healthy and severely injured coral or dead coral 38 .…”

“…Due to differences in cryopreservation sensitivity between coral tissue and symbionts (Symbiodiniaceae require more time for cryoprotectant equilibration and faster freezing rates 34 ), corals were bleached according to the protocol of Lager et al 33 . After 3-5 days of menthol and light exposure to induce bleaching, microfragments for each genotype were visually inspected under a dissecting microscope for tissue integrity and overall coloration.…”

“…The microfragments are brought into the laboratory and then bleached with a combination of menthol and light, according to the protocol of Lager et al 33 (detailed also in the SI). This causes them to shed their algal symbionts, which in previous work and preliminary trials were found to interfere with the CPA-penetration / coral tissue dehydration process due to the differences in cryobiological parameters between the symbionts and the tissue 34,35 .…”