Genome sequence of PSonyx, a singleton bacteriophage infecting Corynebacterium glutamicum.

PSonyx is a newly isolated phage that infects Corynebacterium glutamicum. This siphovirus was isolated from a French pond in the south of Paris by students from Paris-Saclay University. Its 80,277-bp singleton genome carries 136 protein-coding genes and 5 tRNAs.

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.

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.

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.

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.

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.

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.

Cryopreservation of Fish Spermatogonial Cells: The Future of Natural History Collections.

As global biodiversity declines, the value of biological collections increases. Cryopreserved diploid spermatogonial cells meet two goals: to yield high-quality molecular sequence data; and to regenerate new individuals, hence potentially countering species extinction. Cryopreserved spermatogonial cells that allow for such mitigative measures are not currently in natural history museum collections because there are no standard protocols to collect them. Vertebrate specimens, especially fishes, are traditionally formalin-fixed and alcohol-preserved which makes them ideal for morphological studies and as museum vouchers, but inadequate for molecular sequence data. Molecular studies of fishes routinely use tissues preserved in ethanol; yet tissues preserved in this way may yield degraded sequences over time. As an alternative to tissue fixation methods, we assessed and compared previously published cryopreservation methods by gating and counting fish testicular cells with flow cytometry to identify presumptive spermatogonia A-type cells. Here we describe a protocol to cryopreserve tissues that yields a high percentage of viable spermatogonial cells from the testes of Asterropteryx semipunctata, a marine goby. Material cryopreserved using this protocol represents the first frozen and post-thaw viable spermatogonial cells of fishes archived in a natural history museum to provide better quality material for re-derivation of species and DNA preservation and analysis.

Effectiveness of coral relocation as a mitigation strategy in Kane'ohe Bay, Hawai'i.

Coral reef restoration and management techniques are in ever-increasing demand due to the global decline of coral reefs in the last several decades. Coral relocation has been established as an appropriate restoration technique in select cases, particularly where corals are scheduled for destruction. However, continued long-term monitoring of recovery of transplanted corals is seldom sustained. Removal of coral from a navigation channel and relocation to a similar nearby dredged site occurred in 2005. Coral recovery at the donor site and changes in fish populations at the receiving site were tracked periodically over the following decade. Coral regrowth at the donor site was rapid until a recent bleaching event reduced coral cover by more than half. The transplant of mature colonies increased spatial complexity at the receiving site, immediately increasing fish biomass, abundance, and species that was maintained throughout subsequent surveys. Our research indicates that unlike the majority of historical accounts of coral relocation in the Pacific, corals transplanted into wave-protected areas with similar conditions as the original site can have high survival rates. Data on long-term monitoring of coral transplants in diverse environments is central in developing management and mitigation strategies.

Effects of terrigenous sediment on settlement and survival of the reef coral Pocillopora damicornis.

Survival and settlement of Pocillopora damicornis larvae on hard surfaces covered with fine (<63 µm) terrigenous red clay was measured in laboratory Petri dishes. The dishes were prepared with sediment films of various thicknesses covering the bottoms. Coral larvae were incubated in the dishes for two weeks and the percent that settled on the bottom was determined. There was a statistically significant relationship between the amount of sediment and coral recruitment on the bottom, with no recruitment on surfaces having a sediment cover above 0.9 mg cm(-2). Experimental conditions for the delicate coral larvae were favorable in these experiments. Total survival over the two week settlement tests expressed as the sum of coral recruits and live larvae at the end of the experiment did not show a significant decline, so the major impact of the sediment was on successful settlement rather than on mortality. Larval substrate selection behavior was the primary factor in the observed result.

Response of reef corals on a fringing reef flat to elevated suspended-sediment concentrations: Moloka'i, Hawai'i.

A long-term (10 month exposure) experiment on effects of suspended sediment on the mortality, growth, and recruitment of the reef corals Montipora capitata and Porites compressa was conducted on the shallow reef flat off south Moloka'i, Hawai'i. Corals were grown on wire platforms with attached coral recruitment tiles along a suspended solid concentration (SSC) gradient that ranged from 37 mg l(-1) (inshore) to 3 mg l(-1) (offshore). Natural coral reef development on the reef flat is limited to areas with SSCs less than 10 mg l(-1) as previously suggested in the scientific literature. However, the experimental corals held at much higher levels of turbidity showed surprisingly good survivorship and growth. High SSCs encountered on the reef flat reduced coral recruitment by one to three orders of magnitude compared to other sites throughout Hawai'i. There was a significant correlation between the biomass of macroalgae attached to the wire growth platforms at the end of the experiment and percentage of the corals showing mortality. We conclude that lack of suitable hard substrate, macroalgal competition, and blockage of recruitment on available substratum are major factors accounting for the low natural coral coverage in areas of high turbidity. The direct impact of high turbidity on growth and mortality is of lesser importance.