Virginia L. Carter

Oxidative Stress in Zebrafish (Danio rerio) Sperm

Laboratories around the world have produced tens of thousands of mutant and transgenic zebrafish lines. As with mice, maintaining all of these valuable zebrafish genotypes is expensive, risky, and beyond the capacity of even the largest stock centers. Because reducing oxidative stress has become an important aspect of reducing the variability in mouse sperm cryopreservation, we examined whether antioxidants might improve cryopreservation of zebrafish sperm. Four experiments were conducted in this study. First, we used the xanthine-xanthine oxidase (X-XO) system to generate reactive oxygen species (ROS). The X-XO system was capable of producing a stress reaction in zebrafish sperm reducing its sperm motility in a concentration dependent manner (P<0.05). Second, we examined X-XO and the impact of antioxidants on sperm viability, ROS and motility. Catalase (CAT) mitigated stress and maintained viability and sperm motility (P>0.05), whereas superoxide dismutase (SOD) and vitamin E did not (P<0.05). Third, we evaluated ROS in zebrafish spermatozoa during cryopreservation and its effect on viability and motility. Methanol (8%) reduced viability and sperm motility (P<0.05), but the addition of CAT mitigated these effects (P>0.05), producing a mean 2.0 to 2.9-fold increase in post-thaw motility. Fourth, we examined the effect of additional cryoprotectants and CAT on fresh sperm motility. Cryoprotectants, 8% methanol and 10% dimethylacetamide (DMA), reduced the motility over the control value (P<0.5), whereas 10% dimethylformamide (DMF) with or without CAT did not (P>0.05). Zebrafish sperm protocols should be modified to improve the reliability of the cryopreservation process, perhaps using a different cryoprotectant. Regardless, the simple addition of CAT to present-day procedures will significantly improve this process, assuring increased and less variable fertilization success and allowing resource managers to dependably plan how many straws are needed to safely cryopreserve a genetic line.

Preserving and Using Germplasm and Dissociated Embryonic Cells for Conserving Caribbean and Pacific Coral

Coral reefs are experiencing unprecedented degradation due to human activities, and protecting specific reef habitats may not stop this decline, because the most serious threats are global (i.e., climate change), not local. However, ex situ preservation practices can provide safeguards for coral reef conservation. Specifically, modern advances in cryobiology and genome banking could secure existing species and genetic diversity until genotypes can be introduced into rehabilitated habitats. We assessed the feasibility of recovering viable sperm and embryonic cells post-thaw from two coral species, Acropora palmata and Fungia scutaria that have diffferent evolutionary histories, ecological niches and reproductive strategies. In vitro fertilization (IVF) of conspecific eggs using fresh (control) spermatozoa revealed high levels of fertilization (>90% in A. palmata; >84% in F. scutaria; P>0.05) that were unaffected by tested sperm concentrations. A solution of 10% dimethyl sulfoxide (DMSO) at cooling rates of 20 to 30°C/min most successfully cryopreserved both A. palmata and F. scutaria spermatozoa and allowed producing developing larvae in vitro. IVF success under these conditions was 65% in A. palmata and 53% in F. scutaria on particular nights; however, on subsequent nights, the same process resulted in little or no IVF success. Thus, the window for optimal freezing of high quality spermatozoa was short (∼5 h for one night each spawning cycle). Additionally, cryopreserved F. scutaria embryonic cells had∼50% post-thaw viability as measured by intact membranes. Thus, despite some differences between species, coral spermatozoa and embryonic cells are viable after low temperature (-196°C) storage, preservation and thawing. Based on these results, we have begun systematically banking coral spermatozoa and embryonic cells on a large-scale as a support approach for preserving existing bio- and genetic diversity found in reef systems.

Betaines and Dimethylsulfoniopropionate as Major Osmolytes in Cnidaria with Endosymbiotic Dinoflagellates

Most marine invertebrates and algae are osmoconformers whose cells accumulate organic osmolytes that provide half or more of cellular osmotic pressure. These solutes are primarily free amino acids and glycine betaine in most invertebrates and small carbohydrates and dimethylsulfoniopropionate (DMSP) in many algae. Corals with endosymbiotic dinoflagellates (Symbiodinium spp.) have been reported to obtain from the symbionts potential organic osmolytes such as glycerol, amino acids, and DMSP. However, corals and their endosymbionts have not been fully analyzed for osmolytes. We quantified small carbohydrates, free amino acids, methylamines, and DMSP in tissues of the corals Fungia scutaria, Pocillopora damicornis, Pocillopora meandrina, Montipora capitata, Porites compressa, and Porites lobata (all with symbionts) plus Tubastrea aurea (asymbiotic) from Kaneohe Bay, Oahu (Hawaii). Glycine betaine, at 33–69 mmol/kg wet mass, was found to constitute 90% or more of the measured organic solutes in all except the Porites species. Those were dominated by proline betaine and dimethyltaurine. DMSP was found at 0.5–3 mmol/kg in all species with endosymbionts. Freshly isolated Symbiodinium from Fungia, P. damicornis, and P. compressa were also analyzed. DMSP and glycine betaine dominated in the first two; Porites endosymbionts had DMSP, proline betaine, and dimethyltaurine. In all specimens, glycerol and glucose were detected by high‐performance liquid chromatography only at 0–1 mmol/kg wet mass. An enzymatic assay for glycerol plus glycerol 3‐phosphate and dihydroxyacetone phosphate yielded 1–10 mmol/kg. Cassiopeia andromeda (upside‐down jelly; Scyphozoan) and Aiptasia puchella (solitary anemone; Anthozoan) were also analyzed; both have endosymbiotic dinoflagellates. In both, glycine betaine, taurine, and DMSP were the dominant osmolytes. In summary, methylated osmolytes dominate in many Cnidaria; in those with algal symbionts, host and symbiont have similar methylated amino acids, as do congeners. However, little glycerol was present as an osmolyte and was probably metabolized before it could accumulate.

Physiology and cryosensitivity of coral endosymbiotic algae (Symbiodinium).

Coral throughout the world are under threat. To save coral via cryopreservation methods, the Symbiodinium algae that live within many coral cells must also be considered. Coral juvenile must often take up these important cells from their surrounding water and when adult coral bleach, they lose their endosymbiotic algae and will die if they are not regained. The focus of this paper was to understand some of the cryo-physiology of the endosymbiotic algae, Symbiodinium, living within three species of Hawaiian coral, Fungia scutaria, Porites compressa and Pocillopora damicornis in Kaneohe Bay, Hawaii. Although cryopreservation of algae is common, the successful cryopreservation of these important coral endosymbionts is not common, and these species are often maintained in live serial cultures within stock centers worldwide. Freshly-extracted Symbiodinium were exposed to cryobiologically appropriate physiological stresses and their viability assessed with a Pulse Amplitude Fluorometer. Stresses included sensitivity to chilling temperatures, osmotic stress, and toxic effects of various concentrations and types of cryoprotectants (i.e., dimethyl sulfoxide, propylene glycol, glycerol and methanol). To determine the water and cryoprotectant permeabilities of Symbiodinium, uptake of radio-labeled glycerol and heavy water (D(2)O) were measured. The three different Symbiodinium subtypes studied demonstrated remarkable similarities in their morphology, sensitivity to cryoprotectants and permeability characteristics; however, they differed greatly in their sensitivity to hypo- and hyposmotic challenges and sensitivity to chilling, suggesting that standard slow freezing cryopreservation may not work well for all Symbiodinium. An appendix describes our H(2)O:D(2)O water exchange experiments and compares the diffusionally determined permeability with the two parameter model osmotic permeability.

First frozen repository for the Great Barrier Reef coral created

To build new tools for the continued protection and propagation of coral from the Great Barrier Reef (GBR), an international group of coral and cryopreservation scientists known as the Reef Recovery Initiative joined forces during the November 2011 mass-spawning event. The outcome was the creation of the first frozen bank for Australian coral from two important GBR reef-building species, Acropora tenuis and Acropora millepora. Approximately 190 frozen samples each with billions of cells were placed into long-term storage. Sperm cells were successfully cryopreserved, and after thawing, samples were used to fertilize eggs, resulting in functioning larvae. Additionally, developing larvae were dissociated, and these pluripotent cells were cryopreserved and viable after thawing. Now, we are in a unique position to move our work from the laboratory to the reefs to develop collaborative, practical conservation management tools to help secure Australias coral biodiversity.

Analysis of Internal Osmolality in Developing Coral Larvae, Fungia scutaria

Coral species throughout the world are facing severe local and global environmental pressures. Because of the pressing conservation need, we are studying the reproduction, physiology, and cryobiology of coral larvae with the future goal of cryopreserving and maintaining these organisms in a genome resource bank. Effective cryopreservation involves several steps, including the loading and unloading of cells with cryoprotectant and the avoidance of osmotic shock. In this study, during the time course of coral larvae development of the mushroom coral Fungia scutaria, we examined several physiologic factors, including internal osmolality, percent osmotically active water, formation of mucus cells, and intracellular organic osmolytes. The osmotically inactive components of the cell, Vb, declined 33% during development from the oocyte to day 5. In contrast, measurements of the internal osmolality of coral larvae indicated that the internal osmolality was increasing from day 1 to day 5, probably as a result of the development of mucus cells that bind ions. Because of this, we conclude that coral larvae are osmoconformers with an internal osmolality of about 1,000 mOsm. Glycine betaine, comprising more than 90% of the organic osmolytes, was found to be the major organic osmolyte in the larvae. Glycerol was found in only small quantities in larvae that had been infected with zooxanthellae, suggesting that this solute did not play a significant role in the osmotic balance of this larval coral. We were interested in changes in cellular characteristics and osmolytes that might suggest solutes to test as cryoprotectants in order to assist in the successful cryopreservation of the larvae. More importantly, these data begin to reveal the basic physiological events that underlie the move from autonomous living to symbiosis.

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.

Cryobiology of coral fragments.

Around the world, coral reefs are dying due to human influences, and saving habitat alone may not stop this destruction. This investigation focused on the biological processes that will provide the first steps in understanding the cryobiology of whole coral fragments. Coral fragments are a partnership of coral tissue and endosymbiotic algae, Symbiodinium sp., commonly called zooxanthellae. These data reflected their separate sensitivities to chilling and a cryoprotectant (dimethyl sulfoxide) for the coral Pocillopora damicornis, as measured by tissue loss and Pulse Amplitude Modulated fluorometry 3weeks post-treatment. Five cryoprotectant treatments maintained the viability of the coral tissue and zooxanthellae at control values (1M dimethyl sulfoxide at 1.0, 1.5 and 2.0h exposures, and 1.5M dimethyl sulfoxide at 1.0 and 1.5h exposures, P>0.05, ANOVA), whereas 2M concentrations did not (P<0.05, ANOVA). A seasonal response to chilling was observed in the coral tissue, but not in the zooxanthellae. During the winter when the fragments were chilled, the coral tissue remained relatively intact (∼25% loss) post-treatment, but the zooxanthellae numbers in the tissue declined after 5min of chilling (P<0.05, ANOVA). However, in the late spring, coral tissue (∼75% loss) and zooxanthellae numbers declined in response to chilling alone (P<0.05, ANOVA). When a cryoprotectant (1M dimethyl sulfoxide) was used in concert with chilling it protected the coral against tissue loss after 45min of cryoprotectant exposure (P>0.05, ANOVA), but it did not protect against the loss of zooxanthellae (P<0.05, ANOVA). The zooxanthellae are the most sensitive element in the coral fragment complex and future cryopreservation protocols must be guided by their greater sensitivity.

Seasonal Preservation Success of the Marine Dinoflagellate Coral Symbiont, Symbiodinium sp.

Coral reefs are some of the most diverse and productive ecosystems on the planet, but are threatened by global and local stressors, mandating the need for incorporating ex situ conservation practices. One approach that is highly protective is the development of genome resource banks that preserve the species and its genetic diversity. A critical component of the reef are the endosymbiotic algae, Symbiodinium sp., living within most coral that transfer energy-rich sugars to their hosts. Although Symbiodinium are maintained alive in culture collections around the world, the cryopreservation of these algae to prevent loss and genetic drift is not well-defined. This study examined the quantum yield physiology and freezing protocols that resulted in survival of Symbiodinium at 24 h post-thawing. Only the ultra-rapid procedure called vitrification resulted in success whereas conventional slow freezing protocols did not. We determined that success also depended on using a thin film of agar with embedded Symbiodinium on Cryotops, a process that yielded a post-thaw viability of >50% in extracted and vitrified Symbiodinium from Fungia scutaria, Pocillopora damicornis and Porites compressa. Additionally, there also was a seasonal influence on vitrification success as the best post-thaw survival of F. scutaria occurred in winter and spring compared to summer and fall (P < 0.05). These findings lay the foundation for developing a viable genome resource bank for the world’s Symbiodinium that, in turn, will not only protect this critical element of coral functionality but serve as a resource for understanding the complexities of symbiosis, support selective breeding experiments to develop more thermally resilient strains of coral, and provide a ‘gold-standard’ genomics collection, allowing for full genomic sequencing of unique Symbiodinium strains.

Potential bleaching effects on coral reproduction

Bleaching profoundly impacts coral reproduction, often for years after an event. However, detailed reproductive characteristics of coral after bleaching have not been broadly described, especially as they relate to cryopreservation. Therefore, in the present study we measured several reproductive characteristics in coral in Kaneohe Bay, Hawaii, for two species, namely Fungia scutaria and Montipora capitata, during the bleaching period of 2014 and 2015. We examined spawning periods, egg morphometry, sperm concentration, fresh and cryopreserved sperm motility exposed to different concentrations of dimethyl sulfoxide, time of first cleavage, larval survival with fresh and cryopreserved spermatozoa, infection success and settlement success. Many of these reproductive parameters were reduced in 2015, especially sperm motility. Once the reduced-motility spermatozoa from 2015 post-bleach were cryopreserved, there was a steep decline in post-thaw viability and this would prevent any substantive further use of these samples in reproduction for conservation benefit. Worldwide, as bleaching events become more frequent, the ability to bank and conserve coral ex situ may be significantly reduced. Thus, it is imperative that while genetic diversity is still high in these populations, intensive efforts are made to bank coral species during non-bleaching periods.