Preserving underwater forests: Developing a protocol for crayweed cryopreservationNovel Approaches

Catalina Musrri Fuenzalida¹, Georgina Wood^2,3, Ezequiel M. Marzinelli^1,4, Justine K. O’Brien^5,6, Adriana Vergés⁵, Rachel Venhuizen⁵, Jonathan Daly^5,6

¹The University of Sydney, Australia - ²Flinders University, Australia - ³University of Western Australia, Australia - ⁴Sydney Institute of Marine Science, Australia - ⁵UNSW Sydney, Australia - ⁶Taronga Conservation Society Australia, Australia

Underwater forests formed by large brown seaweeds are declining in many temperate reefs globally. Such declines are resulting in lower genetic diversity, which can impact the resilience of species to changing environmental conditions. To conserve genetic diversity and secure these important coastal habitats, new preservation strategies are needed. Cryopreservation is a tool that can be used to store genetic material and has been used to obtain genetic insurance against environmental stress. Here, we aimed to develop a cryopreservation protocol for gametes and germlings of the fucoid seaweed Phyllospora comosa (“crayweed”), a species highly vulnerable to thermal and other stressors in the context of environmental change. We tested the cytotoxicity of commonly used cryoprotectant agents (CPAs) in seaweed cryopreservation (DMSO, ethylene glycol, glycerol, methanol and propylene glycol). Non-lethal cytotoxic effects of CPAs on germlings and sperm were observed, including a reduction in length (germlings), motility, and density (sperm). Freezing trials resulted in post-thaw sperm survival. Best results were observed with 15% DMSO and a freezing rate of 35-45°C/min, displaying 23% post-thaw motility and 0.6 x 106 sperm/mL, compared to 10.3 x 106 sperm/mL in the initial sample (no CPA). Germlings could not be revived after thawing, despite testing several freezing rates, CPA concentrations (5-30%), equilibration times (10-60 min) and added sugars. Our results indicate that crayweed sperm can be cryopreserved, giving us the chance to store diverse genotypes that could be bred with fresh eggs; however, further work will be required to improve the efficiency of cryopreservation protocols. Our findings provide guidelines for future trials with P. comosa and other fucoid species to incorporate cryopreservation techniques into the management and conservation of marine forests to mitigate the effects of climate change.