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.

Cryopreservation of the collector urchin embryo, Tripneustes gratilla.

The collector urchin, Tripneustes gratilla, is an ecologically important member of the grazing community of Hawai'i's coral reefs. Beyond its ability to maintain balance between native seaweeds and corals, T. gratilla has also been used as a food source and a biocontrol agent against alien invasive algae species. Due to overexploitation, habitat degradation, and other stressors, their populations face local extirpation. However, artificial reproductive techniques, such as cryopreservation, could provide more consistent seedstock throughout the year to supplement aquaculture efforts. Although the sperm and larvae of temperate urchins have been successfully cryopreserved, tropical urchins living on coral reefs have not. Here, we investigated the urchin embryos' tolerance to various cryoprotectants and cooling rates to develop a cryopreservation protocol for T. gratilla. We found that using 1 M Me2SO with a cooling rate of 9.7 °C/min on gastrula stage embryos produced the best results with survival rates of up to 85.5% and up to 50.8% maturation to the 4-arm echinopluteus stage, assessed three days after thawing. Continued research could see cryopreservation added to the repertoire of artificial reproductive techniques for T. gratilla, thereby assisting in the preservation of this ecologically important urchin, all while augmenting aquaculture efforts that contribute to coral reef restoration.

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.

Cryopreservation and revival of Hawaiian stony corals using isochoric vitrification.

Corals are under siege by both local and global threats, creating a worldwide reef crisis. Cryopreservation is an important intervention measure and a vital component of the modern coral conservation toolkit, but preservation techniques are currently limited to sensitive reproductive materials that can only be obtained a few nights per year during spawning. Here, we report the successful cryopreservation and revival of cm-scale coral fragments via mL-scale isochoric vitrification. We demonstrate coral viability at 24 h post-thaw using a calibrated oxygen-uptake respirometry technique, and further show that the method can be applied in a passive, electronics-free configuration. Finally, we detail a complete prototype coral cryopreservation pipeline, which provides a platform for essential next steps in modulating post-thaw stress and initiating long-term growth. These findings pave the way towards an approach that can be rapidly deployed around the world to secure the biological genetic diversity of our vanishing coral reefs.

Tank fouling community enhances coral microfragment growth.

Anthropogenic stressors threaten reefs worldwide and natural in situ coral reproduction may be inadequate to meet this challenge. Land-based culture can provide increased coral growth, especially with microfragments. We tested whether culture methods using different algal fouling communities could improve the growth and health metrics of microfragments of the Hawaiian coral, Porites compressa. Culture method fouling communities were: (1) similar to a reef environment (Mini Reef); (2) clean tanks managed to promote crustose coralline algae (Clean Start); and (3) tanks curated beforehand with poorly-competing algae (Green Film) assessed in winter and summer months. The Green Film method during the winter produced the fastest microfragment mean growth at 28 days until the first row of new polyps developed, and also the highest tank and plate metric health scores. Time efficient, standardized methods for land-based culture designed to maximize growth and production of coral fragments will contribute considerably to the success of large-scale restoration efforts.

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.

Unified methods in collecting, preserving, and archiving coral bleaching and restoration specimens to increase sample utility and interdisciplinary collaboration.

Coral reefs are declining worldwide primarily because of bleaching and subsequent mortality resulting from thermal stress. Currently, extensive efforts to engage in more holistic research and restoration endeavors have considerably expanded the techniques applied to examine coral samples. Despite such advances, coral bleaching and restoration studies are often conducted within a specific disciplinary focus, where specimens are collected, preserved, and archived in ways that are not always conducive to further downstream analyses by specialists in other disciplines. This approach may prevent the full utilization of unexpended specimens, leading to siloed research, duplicative efforts, unnecessary loss of additional corals to research endeavors, and overall increased costs. A recent US National Science Foundation-sponsored workshop set out to consolidate our collective knowledge across the disciplines of Omics, Physiology, and Microscopy and Imaging regarding the methods used for coral sample collection, preservation, and archiving. Here, we highlight knowledge gaps and propose some simple steps for collecting, preserving, and archiving coral-bleaching specimens that can increase the impact of individual coral bleaching and restoration studies, as well as foster additional analyses and future discoveries through collaboration. Rapid freezing of samples in liquid nitrogen or placing at -80 °C to -20 °C is optimal for most Omics and Physiology studies with a few exceptions; however, freezing samples removes the potential for many Microscopy and Imaging-based analyses due to the alteration of tissue integrity during freezing. For Microscopy and Imaging, samples are best stored in aldehydes. The use of sterile gloves and receptacles during collection supports the downstream analysis of host-associated bacterial and viral communities which are particularly germane to disease and restoration efforts. Across all disciplines, the use of aseptic techniques during collection, preservation, and archiving maximizes the research potential of coral specimens and allows for the greatest number of possible downstream analyses.

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.

Challenges of sperm cryopreservation in transferring heat adaptation of corals across ocean basins.

Reef-building corals live very close to their upper thermal limits and their persistence is imperiled by a rapidly warming climate. Human interventions may be used to increase the thermal limits of sensitive corals by cross-breeding with heat-adapted populations. However, the scope of breeding interventions is constrained by regional variation in the annual reproductive cycle of corals. Here we use cryopreservation technology to overcome this barrier and cross-breed conspecific coral populations across ocean basins for the first time. During regional spawning events, sperm samples were cryopreserved from populations of the widespread Indo-Pacific coral, Platygyra daedalea, from the southern Persian Gulf (maximum daily sea surface temperature of 36 °C), the Oman Sea (33 °C), and the central Great Barrier Reef (30 °C). These sperm samples were thawed during a later spawning event to test their ability to fertilize freshly spawned eggs of P. daedalea colonies from the central Great Barrier Reef. Average fertilization success for the Persian Gulf (9%) and Oman Sea (6%) sperm were 1.4-2.5 times lower than those for the native cryopreserved sperm from Great Barrier Reef (13-15%), potentially due to lower sperm quality of the Middle Eastern sperm and/or reproductive incompatibility between these distant populations. Overall, fertilization success with cryopreserved sperm was low compared with fresh sperm (>80%), likely due to the low motility of thawed sperm (≤5%, reduced from 50% to >90% in fresh sperm). To evaluate whether cross-bred offspring had enhanced thermal tolerance, the survival of larvae sired by Persian Gulf cryopreserved sperm, Great Barrier Reef cryopreserved sperm, and Great Barrier Reef fresh sperm was monitored for six days at ambient (27 °C) and elevated (33 °C) temperature. Against expectations of thermal tolerance enhancement, survival of larvae sired by Persian Gulf cryopreserved sperm was 2.6 times lower than larvae sired by Great Barrier Reef fresh sperm at 33 °C (27% versus 71%), but did not differ at 27 °C (77% versus 84%). This lack of enhanced thermal tolerance was unlikely due to outbreeding depression as survival was equally poor in larvae sired by Great Barrier Reef cryopreserved sperm. Rather, follow-up tests showed that cryoprotectant exposure during fertilization (0.1% DMSO) has a negative effect on the survival of P. daedalea larvae which is exacerbated at elevated temperature. Collectively, our findings highlight challenges of breeding corals for enhanced thermal tolerance using cryopreserved sperm, which may be overcome by methodological advances in the collection and preservation of high-quality motile sperm and minimizing the exposure time of eggs to cryoprotectants.

Growth and survival among Hawaiian corals outplanted from tanks to an ocean nursery are driven by individual genotype and species differences rather than preconditioning to thermal stress.

The drastic decline in coral coverage has stimulated an interest in reef restoration, and various iterations of coral nurseries have been used to augment restoration strategies. Here we examine the growth of two species of Hawaiian Montipora that were maintained in mesocosms under either ambient or warmed annual bleaching conditions for two consecutive years prior to outplanting to determine whether preconditioning aided coral restoration efforts. Using coral trees to create a nearby ocean nursery, we examined whether: (1) previous ex situ mesocosm growth would mirror in situ coral tree nursery growth; and (2) thermal ex situ stress-hardening would predict future success during natural warming events in situ for corals moved from tanks to trees. For Montipora capitata, we found that variation in growth was explained primarily by genotype; growth rates in the mesocosms were similar to those in situ, irrespective of preconditioning. Variation in M. flabellata growth, however, was explained by both genotype and culture method such that an individual M. flabellata colony that grew well in the tanks did not necessarily perform as well on the coral trees. For both species, previous exposure to elevated temperatures in the mesocosms provided no benefit to either growth or survival during a warming event in the coral tree nursery compared to those grown in ambient temperatures. Overall, M. capitata performed better in the tree nursery with higher net growth, lower mortality, and was subject to less predation than M. flabellata. Our results show little benefit of the additional cost and time of stress-hardening these corals prior to outplanting because it is unlikely to aid resilience to future warming events. These results also suggest that selecting corals for restoration based on long-term genotype growth performance may be more effective for optimal outcomes but should be weighed against other factors, such as coral morphology, in situ nursery method, location, and other characteristics.

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.

Towards a method for cryopreservation of mosquito vectors of human pathogens.

Mosquito-borne diseases are responsible for millions of human deaths every year, posing a massive burden on global public health. Mosquitoes transmit a variety of bacteria, parasites and viruses. Mosquito control efforts such as insecticide spraying can reduce mosquito populations, but they must be sustained in order to have long term impacts, can result in the evolution of insecticide resistance, are costly, and can have adverse human and environmental effects. Technological advances have allowed genetic manipulation of mosquitoes, including generation of those that are still susceptible to insecticides, which has greatly increased the number of mosquito strains and lines available to the scientific research community. This generates an associated challenge, because rearing and maintaining unique mosquito lines requires time, money and facilities, and long-term maintenance can lead to adaptation to specific laboratory conditions, resulting in mosquito lines that are distinct from their wild-type counterparts. Additionally, continuous rearing of transgenic lines can lead to loss of genetic markers, genes and/or phenotypes. Cryopreservation of valuable mosquito lines could help circumvent these limitations and allow researchers to reduce the cost of rearing multiple lines simultaneously, maintain low passage number transgenic mosquitoes, and bank lines not currently being used. Additionally, mosquito cryopreservation could allow researchers to access the same mosquito lines, limiting the impact of unique laboratory or field conditions. Successful cryopreservation of mosquitoes would expand the field of mosquito research and could ultimately lead to advances that would reduce the burden of mosquito-borne diseases, possibly through rear-and-release strategies to overcome mosquito insecticide resistance. Cryopreservation techniques have been developed for some insect groups, including but not limited to fruit flies, silkworms and other moth species, and honeybees. Recent advances within the cryopreservation field, along with success with other insects suggest that cryopreservation of mosquitoes may be a feasible method for preserving valuable scientific and public health resources. In this review, we will provide an overview of basic mosquito biology, the current state of and advances within insect cryopreservation, and a proposed approach toward cryopreservation of Anopheles stephensi mosquitoes.

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.

Cryopreservation and Laser Nanowarming of Zebrafish Embryos Followed by Hatching and Spawning.

This study shows for the first time the ability to rewarm cryopreserved zebrafish embryos that grow into adult fish capable of breeding normally. The protocol employs a single injection of cryoprotective agents (CPAs) and gold nanorods (GNRs) into the yolk and immersion in a precooling bath to dehydrate the perivitelline space. Then embryos are encapsulated within CPA and GNR droplets, plunged into liquid nitrogen, cryogenically stabilized, and rewarmed by a laser pulse. Postlaser nanowarming, embryos (n = 282) exhibit intact structure by 1 h (40%), continued development after 3 h (22%), movement after 24 h (11%), hatching after 48 h (9%), and swimming after Day 5 (3%). Finally, from fish that survives till Day 5, two larvae are grown to adulthood and spawned, yielding survival comparable to an unfrozen control. Future efforts will focus on improving the survival to adulthood and developing methods to cryopreserve large numbers of embryos for research, aquaculture, and biodiversity preservation.

Rapid counting and spectral sorting of live coral larvae using large-particle flow cytometry.

Research with coral embryos and larvae often requires laborious manual counting and sorting of individual specimens, usually via microscopy. Because many coral species spawn only once per year during a narrow temporal window, sample processing is a time-limiting step for research on the early life-history stages of corals. Flow cytometry, an automated technique for measuring and sorting particles, cells, and cell-clusters, is a potential solution to this bottleneck. Yet most flow cytometers do not accommodate live organisms of the size of most coral embryos (> 250 µm), and sample processing is often destructive. Here we tested the ability of a large-particle flow cytometer with a gentle pneumatic sorting mechanism to process and spectrally sort live and preserved Montipora capitata coral embryos and larvae. Average survival rates of mechanically-sorted larvae were over 90% and were comparable to those achieved by careful hand-sorting. Preserved eggs and embryos remained intact throughout the sorting process and were successfully sorted based on real-time size and fluorescence detection. In-line bright-field microscopy images were captured for each sample object as it passed through the flow-cell, enabling the identification of early-stage embryos (2-cell to morula stage). Samples were counted and sorted at an average rate of 4 s larva-1 and as high as 0.2 s larva-1 for high-density samples. Results presented here suggest that large-particle flow cytometry has the potential to significantly increase efficiency and accuracy of data collection and sample processing during time-limited coral spawning events, facilitating larger-scale and higher-replication studies with an expanded number of species.

The impact of short-term exposure to near shore stressors on the early life stages of the reef building coral Montipora capitata.

Successful reproduction and survival are crucial to the continuation and resilience of corals globally. As reef waters warm due to climate change, episodic largescale tropical storms are becoming more frequent, drastically altering the near shore water quality for short periods of time. Therefore, it is critical that we understand the effects warming waters, fresh water input, and run-off have on sexual reproduction of coral. To better understand the effects of these near shore stressors on Hawaiian coral, laboratory experiments were conducted at the Institute of Marine Biology to determine the independent effects of suspended sediment concentrations (100 mg l-1 and 200 mg l-1), lowered salinity (28‰), and elevated temperature (31 °C) on the successful fertilization, larval survival, and settlement of the scleractinian coral Montipora capitata. In the present study, early developmental stages of coral were exposed to one of three near shore stressors for a period of 24 h and the immediate (fertilization) and latent effects (larval survival and settlement) were observed and measured. Fertilization success and settlement were not affected by any of the treatments; however, larval survival was negatively affected by all of the treatments by 50% or greater (p > 0.05). These data show that early life stages of M. capitata may be impacted by near shore stressors associated with warming and more frequent storm events.