Dataset Information

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.

SUBMITTER: Daly J

PROVIDER: S-EPMC6200823 | biostudies-other | 2018 Oct

REPOSITORIES: biostudies-other

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Successful cryopreservation of coral larvae using vitrification and laser warming.

Daly Jonathan J Zuchowicz Nikolas N Nuñez Lendo C Isabel CI Khosla Kanav K Lager Claire C Henley E Michael EM Bischof John J Kleinhans F W FW Lin Chiahsin C Peters Esther C EC Hagedorn Mary M

Scientific reports 20181024 1

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 ...[more]

PMID: 30356142

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Project description:BackgroundOrchids are under threat from human activities and climate change, with populations limited to small geographic hotspots. This makes them ideal candidates for ex situ conservation. Orchid seeds are desiccation tolerant, but often have poor longevity in seed banks and cryopreservation of orchid protocorms is complex and expensive. Therefore, simple methods for large-scale storage programs are essential to store orchid seeds of different life forms. Seeds of five species representing epiphytic, lithophytic and terrestrial orchids from the Central Highlands of Madagascar were studied to find a simple and effective system of cryopreservation. The use of a vitrification solution prior to cryopreservation to improve survival was investigated, as well as the use of symbiotic and asymbiotic germination media to maximise germination after cryopreservation. Using the filter paper packet method, dried seeds were stored in vapour phase above liquid nitrogen and recovered after thawing with both symbiotic and asymbiotic media.ResultsThe study revealed that cryoprotection is not essential for the species in this study, which represented a range of lifeforms. Vitrification generally led to a decrease in germination post cryopreservation. The use of a symbiotic germination medium post cryopreservation was found to be successful in the species in which it was tested. However, the use of an asymbiotic medium was successful for all the species in this study.ConclusionsVitrification was not essential for the species in this study as the orchid seeds were already ultralow temperature and desiccation tolerant. However, further studies using more species are required to validate this approach. This may be an ecophysiological or genetic trait of these species. Therefore, this form of dry seed cryopreservation could form part of ex situ orchid seed conservation using a standard method. The methods developed here will store greater genetic diversity compared to what can be achieved with protocorms and are suitable for both asymbiotic and symbiotic recovery after cryopreservation. This will help reduce the time and cost of ex situ conservation, and help develop universal protocols for large genera, compared to custom protocols required for protocorm cryopreservation.

Project description:Reef-building corals form nutritional symbioses with endosymbiotic dinoflagellates (Symbiodiniaceae), a relationship that facilitates the ecological success of coral reefs. These symbionts are mostly acquired anew each generation from the environment during early life stages ("horizontal transmission"). Symbiodiniaceae species exhibit trait variation that directly impacts the health and performance of the coral host under ocean warming. Here, we test the capacity for larvae of a horizontally transmitting coral, Acropora tenuis, to establish symbioses with Symbiodiniaceae species in four genera that have varying thermal thresholds (the common symbiont genera, Cladocopium and Durusdinium, and the less common Fugacium and Gerakladium). Over a 2-week period in January 2018, a series of both no-choice and four-way choice experiments were conducted at three temperatures (27, 30, and 31°C). Symbiont acquisition success and cell proliferation were measured in individual larvae. Larvae successfully acquired and maintained symbionts of all four genera in no-choice experiments, and >80% of larvae were infected with at least three genera when offered a four-way choice. Unexpectedly, Gerakladium symbionts increased in dominance over time, and at high temperatures outcompeted Durusdinium, which is regarded as thermally tolerant. Although Fugacium displayed the highest thermal tolerance in culture and reached similar cell densities to the other three symbionts at 31°C, it remained a background symbiont in choice experiments, suggesting host preference for other symbiont species. Larval survivorship at 1 week was highest in larvae associated with Gerakladium and Fugacium symbionts at 27 and 30°C, however at 31°C, mortality was similar for all treatments. We hypothesize that symbionts that are currently rare in corals (e.g., Gerakladium) may become more common and widespread in early life stages under climate warming. Uptake of such symbionts may function as a survival strategy in the wild, and has implications for reef restoration practices that use sexually produced coral stock.

Project description:Sperm cryopreservation by vitrification is a promising approach for small-bodied animals such as zebrafish (Danio rerio). However, most vitrification tools adopted in aquatic research were initially designed for applications other than sperm (such as human embryo freezing) and, thus, pose challenges for adoption to sperm vitrification. Three-dimensional (3D) printing combined with open hardware sharing is an emerging strategy to address challenges in the development of cryopreservation tools. The goal of this study was to develop a 3D printed Vitrification Device for Cryo-Vials (VDCV) that can be integrated with the existing vial storage systems. The VDCV combined the vitrification and handling components to achieve functions of sample handling, vitrification, storage, and identification. The vitrification component featured a base, a stem, and a loop. A total of 36 configurations with various loop lengths (8, 10, and 12 mm); loop widths (2.0, 2.5, 3.0, and 3.5 mm); and support structures (open, transverse, and axial) of the VDCD prototypes were designed, fabricated, and tested. Device handling orientations (horizontal and vertical holding angles prior to and during freezing) were also investigated. Computer simulations estimated that the cooling rate of the samples ranged from 0.6-1.5 × 105 °C/min in all the configurations. Prior to freezing, loops with axial supports produced a minimum of 92% film retention. The overall trends of full vitrification occurrence were observed: horizontal plunging > vertical plunging, and axial support > transverse support and open loop. A loop length of 8 mm had the highest overall vitrification occurrence (86-100%). No significant differences (p = 0.6584) were shown in a volume capacity (5.7-6.0 μL) among the three supporting configurations. A single unit of VDCV can provide loading efficiencies of about 6 × 107 sperm/vial, pooling of samples from 3-6 males/vial, and fertilization for 1800 eggs/vial. The VDCV are low-cost (<$0.5 material cost per unit) and can be customized, standardized, securely labeled, and efficiently stored. The prototypes can be accessed by user communities through open-fabrication file sharing and fabricated with consumer-level 3D printers, thus facilitating community-level standardization.

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Successful cryopreservation of coral larvae using vitrification and laser warming.

Publications

Successful cryopreservation of coral larvae using vitrification and laser warming.

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