A Semi-Automated Workflow for the Cryopreservation of Coral Sperm to Support Biobanking and Aquaculture.

Coral reefs are facing a crisis as the frequency of bleaching events caused by ocean warming increases, resulting in the death of corals on reefs around the world. The subsequent loss of genetic diversity and biodiversity can diminish the ability of coral to adapt to the changing climate, so efforts to preserve existing diversity are essential to maximize the resources available for reef restoration now and in the future. The most effective approach to secure genetics long-term is cryopreservation and biobanking, which permits the frozen storage of living samples at cryogenic temperatures in liquid nitrogen indefinitely. Cryopreservation of coral sperm has been possible since 2012, but the seasonal nature of coral reproduction means that biobanking activities are restricted to just a few nights per year when spawning occurs. Improving the efficiency of coral sperm processing and cryopreservation workflows is therefore essential to maximizing these limited biobanking opportunities. To this end, we set out to optimize cryopreservation processing pathways for coral sperm by building on existing technologies and creating a semi-automated approach to streamline the assessment, handling, and cryopreservation of coral sperm. The process, which combines computer-assisted sperm analysis, barcoded cryovials, and a series of linked auto-datasheets for simultaneous editing by multiple users, improves the efficiency of both sample processing and metadata management in the field. Through integration with cross-cutting research programs such as the Reef Restoration and Adaptation Program in Australia, cryopreservation can play a crucial role in large-scale reef restoration programs by facilitating the genetic management of aquaculture populations, supporting research to enhance thermal tolerance, and preventing the extinction of coral species. The described procedures will be utilized for coral cryopreservation and biobanking practitioners on reefs worldwide and will provide a model for the transition of cryopreservation technologies from research laboratories to large-scale applications.

Conduction-Dominated Cryomesh for Organism Vitrification.

Vitrification-based cryopreservation is a promising approach to achieving long-term storage of biological systems for maintaining biodiversity, healthcare, and sustainable food production. Using the "cryomesh" system achieves rapid cooling and rewarming of biomaterials, but further improvement in cooling rates is needed to increase biosystem viability and the ability to cryopreserve new biosystems. Improved cooling rates and viability are possible by enabling conductive cooling through cryomesh. Conduction-dominated cryomesh improves cooling rates from twofold to tenfold (i.e., 0.24 to 1.2 × 105  °C min-1 ) in a variety of biosystems. Higher thermal conductivity, smaller mesh wire diameter and pore size, and minimizing the nitrogen vapor barrier (e.g., vertical plunging in liquid nitrogen) are key parameters to achieving improved vitrification. Conduction-dominated cryomesh successfully vitrifies coral larvae, Drosophila embryos, and zebrafish embryos with improved outcomes. Not only a theoretical foundation for improved vitrification in µm to mm biosystems but also the capability to scale up for biorepositories and/or agricultural, aquaculture, or scientific use are demonstrated.

Solar radiation, temperature and the reproductive biology of the coral Lobactis scutaria in a changing climate.

Coral reefs worldwide are at risk due to climate change. Coral bleaching is becoming increasingly common and corals that survive bleaching events can suffer from temporary reproductive failure for several years. While water temperature is a key driver in causing coral bleaching, other environmental factors are involved, such as solar radiation. We investigated the individual and combined effects of temperature, photosynthetically active radiation (PAR), and ultraviolet radiation (UVR) on the spawning patterns and reproductive physiology of the Hawaiian mushroom coral Lobactis scutaria, using long-term experiments in aquaria. We examined effects on spawning timing, fertilisation success, and gamete physiology. Both warmer temperatures and filtering UVR altered the timing of spawning. Warmer temperatures caused a drop in fertilisation success. Warmer temperatures and higher PAR both negatively affected sperm and egg physiology. These results are concerning for the mushroom coral L. scutaria and similar reproductive data are urgently needed to predict future reproductive trends in other species. Nonetheless, thermal stress from global climate change will need to be adequately addressed to ensure the survival of reef-building corals in their natural environment throughout the next century and beyond. Until then, reproduction is likely to be increasingly impaired in a growing number of coral species.

An automated modular open-technology device to measure and adjust concentration of aquatic sperm samples for cryopreservation.

Repositories for aquatic germplasm are essential for safeguarding valuable genetic diversity for species relevant to aquaculture, biomedical research, and conservation. Development of aquatic germplasm repositories is impeded by a lack of standardization within laboratories and across the research community. Protocols for cryopreservation are often developed ad hoc and without close attention to variables, such as cell concentration, that strongly affect the success and reproducibility of cryopreservation. The wide dissemination and use of specialized tools and devices as open hardware can improve processing reliability and save costs. The goal of the present work was to develop and prototype a modular and open-technology approach to help to standardize the cell concentration of germplasm samples prior to cryopreservation. The specific objectives were to: 1) design and fabricate prototypes of the automated concentration measurement and adjustment system (CMAS), incorporating custom peristaltic pumps and optical evaluation modules, and 2) evaluate the performance of the CMAS with biological samples. Linear regression models were obtained for estimation of aquatic sperm concentration >108 cells/mL and for algae concentration > (3 × 105) cells/mL. Algae were diluted with extender medium by an automated process, resulting in a dilution precision of ±12.6% and ±6.7% in two trials, attaining means of 89% and 71% of the target cell concentration. The development of the CMAS as open technology can provide opportunities for community-level standardization in cryopreservation of aquatic germplasm and can invite new users, makers, and developers into the open-technology community. This will increase the reach and capabilities of much-needed aquatic germplasm repositories.

Contrasting reproductive strategies of two Hawaiian Montipora corals.

Sessile invertebrates often engage in synchronized spawning events to increase likelihood of fertilization. Although coral reefs are well studied, the reproductive behavior of most species and the relative influence of various environmental cues that drive reproduction are not well understood. We conducted a comparative examination of the reproduction of the well-studied Hawaiian coral Montipora capitata and the relatively unknown reproduction of its congener, Montipora flabellata. Both are simultaneous hermaphroditic broadcast spawners that release egg-sperm bundles with external fertilization. Montipora capitata had a distinct reproductive pattern that resulted in coordinated gamete maturation and the synchronized release of thousands of egg-sperm bundles across two spawning pulses tightly coupled to consecutive new moon phases in June and July. Montipora flabellata exhibited a four month reproductive season with spawning that was four-fold less synchronous than M. capitata; its spawning was aperiodic with little linkage to moon phase, a broadly distributed release of only dozens or hundreds of bundles over multiple nights, and a spawning period that ranged from late June through September. The reproductive strategy of M. flabellata might prove detrimental under climate change if increased frequency and severity of bleaching events leave it sparsely populated and local stressors continue to degrade its habitat.

Low-Cost Resin 3-D Printing for Rapid Prototyping of Microdevices: Opportunities for Supporting Aquatic Germplasm Repositories.

Germplasm repositories can benefit sustainable aquaculture by supporting genetic improvement, assisted reproduction, and management of valuable genetic resources. Lack of reliable quality management tools has impeded repository development in the past several decades. Microfabricated open-hardware devices have emerged as a new approach to assist repository development by providing standardized quality assessment capabilities to enable routine quality control. However, prototyping of microfabricated devices (microdevices) traditionally relies on photolithography techniques that are costly, time intensive, and accessible only through specialized engineering laboratories. Although resin 3-D printing has been introduced into the microfabrication domain, existing publications focus on customized or high-cost (>thousands of USD) printers. The goal of this report was to identify and call attention to the emerging opportunities to support innovation in microfabrication by use of low-cost (

Assisted gene flow using cryopreserved sperm in critically endangered coral.

Assisted gene flow (AGF) is a conservation intervention to accelerate species adaptation to climate change by importing genetic diversity into at-risk populations. Corals exemplify both the need for AGF and its technical challenges; corals have declined in abundance, suffered pervasive reproductive failures, and struggled to adapt to climate change, yet mature corals cannot be easily moved for breeding, and coral gametes lose viability within hours. Here, we report the successful demonstration of AGF in corals using cryopreserved sperm that was frozen for 2 to 10 y. We fertilized Acropora palmata eggs from the western Caribbean (Curaçao) with cryopreserved sperm from genetically distinct populations in the eastern and central Caribbean (Florida and Puerto Rico, respectively). We then confirmed interpopulation parentage in the Curaçao-Florida offspring using 19,696 single-nucleotide polymorphism markers. Thus, we provide evidence of reproductive compatibility of a Caribbean coral across a recognized barrier to gene flow. The 6-mo survival of AGF offspring was 42%, the highest ever achieved in this species, yielding the largest wildlife population ever raised from cryopreserved material. By breeding a critically endangered coral across its range without moving adults, we show that AGF using cryopreservation is a viable conservation tool to increase genetic diversity in threatened marine populations.

Reproductive plasticity of Hawaiian Montipora corals following thermal stress.

Ocean warming, fueled by climate change, is the primary cause of coral bleaching events which are predicted to increase in frequency. Bleaching is generally damaging to coral reproduction, can be exacerbated by concomitant stressors like ultraviolet radiation (UVR), and can have lasting impacts to successful reproduction and potential adaptation. We compared morphological and physiological reproductive metrics (e.g., sperm motility, mitochondrial membrane integrity, egg volume, gametes per bundle, and fertilization and settlement success) of two Hawaiian Montipora corals after consecutive bleaching events in 2014 and 2015. Between the species, sperm motility and mitochondrial membrane potential had the most disparate results. Percent sperm motility in M. capitata, which declined to ~ 40% during bleaching from a normal range of 70-90%, was still less than 50% motile in 2017 and 2018 and had not fully recovered in 2019 (63% motile). By contrast, percent sperm motility in Montipora spp. was 86% and 74% in 2018 and 2019, respectively. This reduction in motility was correlated with damage to mitochondria in M. capitata but not Montipora spp. A major difference between these species is the physiological foundation of their UVR protection, and we hypothesize that UVR protective mechanisms inherent in Montipora spp. mitigate this reproductive damage.

Freezing on the beach: A robust coral sperm cryopreservation design.

Cryopreservation of coral sperm requires reliable, travel-ready, inexpensive hardware. To this end, we developed and tested a robust, second-generation, conduction-based cryovial cooling rack assembled from 3D-printed and commercially available parts. Cooling rates from -10 to -80 °C were found to be repeatable at -22.9 ± 1.9 (rate ± SD) °C/min for 1-mL samples and -35.4 ± 3.3 °C/min for 0.5-mL samples. This represents an improvement on the variability of cooling rates in an older design, which was found to be -31.8 ± 7.1 °C/min for 1-mL samples. Design files and a manual were produced to encourage widespread use and the development of derivative designs.

Assessing coral sperm motility.

The declining reproductive viability of corals threatens their ability to adapt to changing ocean conditions. It is vital that we monitor this viability quantitatively and comparatively. Computer-assisted sperm analysis (CASA) systems offer in-depth analysis used regularly for domestic and wildlife species, but not yet for coral. This study proposes quality control procedures and CASA settings that are effective for coral sperm analysis. To resolve disparities between CASA measurements and evaluations by eye, two negative effects on motility had to be resolved, slide adhesion (procedural) and sperm dilution (biological). We showed that the addition of bovine serum albumin, or caffeine, or both to fresh sperm reduced adhesion in the CASA cassettes, improved motility and motile sperm concentration (P < 0.0001), yet these additions did not affect measurements of total sperm concentration. Diluting coral sperm reduced sperm motility (P = 0.039), especially from heat-stressed corals. We found CASA concentration counts comparable to haemocytometer and flow cytometer measures (P = 0.54). We also found that motile sperm per egg is a useful predictor of fertilisation success, using cryopreserved sperm. Standard measurements of coral reproductive characteristics inform our understanding of the impacts of climate change on reef populations; this study provides a benchmark to begin this comparative work.

Successful cryopreservation of coral larvae using vitrification and laser warming.

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.

Effects of toxic compounds in Montipora capitata on exogenous and endogenous zooxanthellae performance and fertilization success.

Studies have identified chemicals within the stony coral genus Montipora that have significant biological activities. For example, Montiporic acids A and B and other compounds have been isolated from the adult tissue and eggs of Montipora spp. and have displayed antimicrobial activity and cytotoxicity in cultured cells. The ecological role of these toxic compounds is currently unclear. This study examines the role these toxins play in reproduction. Toxins were found in the eggs and larvae of the coral Montipora capitata. Releasing these toxins by crushing both the eggs and larvae resulted in irreversible inhibition of photosynthesis in endogenous and exogenous zooxanthellae within minutes. Moreover, these toxins were stable, as frozen storage of eggs and larvae did not affect toxicity. Photosynthetic competency of Porites compressa zooxanthellae treated with either frozen or fresh, crushed eggs was inhibited similarly (P > 0.05, ANCOVA). Addition of toxic eggs plugs to live P. compressa fragments caused complete tissue necrosis under the exposed area on the fragments within 1 week. Small volumes of M. capitata crushed eggs added to sperm suspensions reduced in vitro fertilization success by killing the sperm. After 30 min, untreated sperm maintained 90 ± 1.9% SEM motility while those treated with crushed eggs were rendered immotile, 4 ± 1.4% SEM. Flow cytometry indicated membrane disruption of the immotile sperm. Fertilization success using untreated sperm was 79 ± 4% SEM, whereas the success rate dropped significantly after exposure to the crushed eggs, 1.3 ± 0% SEM. Unlike the eggs and the larvae, M. capitata sperm did not reduce the photosynthetic competency of P. compressa zooxanthellae, suggesting the sperm was nontoxic. The identity of the toxins, cellular mechanism of action, advantage of the toxins for M. capitata and their role on the reef are still unknown.

Trehalose is a chemical attractant in the establishment of coral symbiosis.

Coral reefs have evolved with a crucial symbiosis between photosynthetic dinoflagellates (genus Symbiodinium) and their cnidarian hosts (Scleractinians). Most coral larvae take up Symbiodinium from their environment; however, the earliest steps in this process have been elusive. Here we demonstrate that the disaccharide trehalose may be an important signal from the symbiont to potential larval hosts. Symbiodinium freshly isolated from Fungia scutaria corals constantly released trehalose (but not sucrose, maltose or glucose) into seawater, and released glycerol only in the presence of coral tissue. Spawning Fungia adults increased symbiont number in their immediate area by excreting pellets of Symbiodinium, and when these naturally discharged Symbiodinium were cultured, they also released trehalose. In Y-maze experiments, coral larvae demonstrated chemoattractant and feeding behaviors only towards a chamber with trehalose or glycerol. Concomitantly, coral larvae and adult tissue, but not symbionts, had significant trehalase enzymatic activities, suggesting the capacity to utilize trehalose. Trehalase activity was developmentally regulated in F. scutaria larvae, rising as the time for symbiont uptake occurs. Consistent with the enzymatic assays, gene finding demonstrated the presence of a trehalase enzyme in the genome of a related coral, Acropora digitifera, and a likely trehalase in the transcriptome of F. scutaria. Taken together, these data suggest that adult F. scutaria seed the reef with Symbiodinium during spawning and the exuded Symbiodinium release trehalose into the environment, which acts as a chemoattractant for F. scutaria larvae and as an initiator of feeding behavior- the first stages toward establishing the coral-Symbiodinium relationship. Because trehalose is a fixed carbon compound, this cue would accurately demonstrate to the cnidarian larvae the photosynthetic ability of the potential symbiont in the ambient environment. To our knowledge, this is the first report of a chemical cue attracting the motile coral larvae to the symbiont.