Coral larvae conservation: physiology and reproduction

Coral species throughout the world's oceans are facing severe environmental pressures. We are interested in conserving coral larvae by means of cryopreservation, but little is known about their cellular physiology or cryobiology. These experiments examined cryoprotectant toxicity, dry weight, water and cryoprotectant permeability using cold and radiolabeled glycerol, spontaneous ice nucleation temperatures, chilling sensitivity, and settlement of coral larvae. Our two test species of coral larvae, Pocillopora damicornis (lace coral), and Fungia scutaria (mushroom coral) demonstrated a wide tolerance to cryoprotectants. Computer-aided morphometry determined that F. scutaria larvae were smaller than P. damicornis larvae. The average dry weight for P. damicornis was 24.5%, while that for F. scutaria was 17%, yielding osmotically inactive volumes (V(b)) of 0.22 and 0.15, respectively. The larvae from both species demonstrated radiolabeled glycerol uptake over time, suggesting they were permeable to the glycerol. Parameter fitting of the F. scutaria larvae data yielded a water permeability 2 microm/min/atm and a cryoprotectant permeability = 2.3 x 10(-4) cm/min while modeling indicated that glycerol reached 90% of final concentration in the larvae within 25 min. The spontaneous ice nucleation temperature for F. scutaria larvae in filtered seawater was -37.8+/-1.4 degrees C. However, when F. scutaria larvae were chilled from room temperature to -11 degrees C at various rates, they exhibited 100% mortality. When instantly cooled from room temperature to test temperatures, they showed damage below 10 degrees C. These data suggest that they are sensitive to both the rate of chilling and the absolute temperature, and indicate that vitrification may be the only means to successfully cryopreserve these organisms. Without prior cryopreservation, both species of coral settled under laboratory conditions.     

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.     

Chilling sensitivity of carp (Cyprinus carpio) embryos at different developmental stages in the presence or absence of cryoprotectants: work in progress

The unsolved problem of cryopreservation of the yolk-rich teleost embryos may be related, in part, to their sensitivity to chilling and cryoprotective agents. The aim of this study was to gain data on the sensitivity of carp embryos to low temperatures at different developmental stages and on the possible protective and toxic effects of cryoprotectants. A total of 86,400 morulae, half-epiboly and heartbeat-stage embryos was selected and then placed in water or in 1 M methanol, dimethyl sulfoxide (Me2SO), glycerol or 0.1 M sucrose solution at 0, 4 or 24 degrees C for 5 min or 1 h. Following these treatments, the embryos were held in a 24 degrees C water bath until the evaluation of hatching rates. In every developmental stage a significant decrease of hatching rates following exposure to 4 or 0 degree C was detected. Sensitivity to chilling changed significantly with development (heartbeat < morula < half-epiboly). Half-epiboly stage embryos were less sensitive to a short period of exposure to cryoprotectants than morula and heartbeat stages. A 1-h exposure to cryoprotectants revealed a stage dependent sensitivity. Toxicity increased in the order of methanol < Me2SO < glycerol in morula and half-epiboly stages, and methanol < glycerol < Me2SO in the heartbeat stage. The results show morulae are partially protected against chilling in Me2SO and sucrose, half-epiboly in Me2SO, sucrose and methanol, and heartbeat-stage in methanol and glycerol. The results further suggest that carp embryos are sensitive to chilling and that toxicity and protective effects against chilling of cryoprotectants are stage-dependent. The finding on the low chilling sensitivity of heartbeat-stage embryos and the protective effect of certain cryoprotectants may be useful in designing cryopreservation protocols.     

Physiological and Biochemical Performances of Menthol-Induced Aposymbiotic Corals

The unique mutualism between corals and their photosynthetic zooxanthellae (Symbiodinium spp.) is the driving force behind functional assemblages of coral reefs. However, the respective roles of hosts and Symbiodinium in this endosymbiotic association, particularly in response to environmental challenges (e.g., high sea surface temperatures), remain unsettled. One of the key obstacles is to produce and maintain aposymbiotic coral hosts for experimental purposes. In this study, a simple and gentle protocol to generate aposymbiotic coral hosts (Isopora palifera and Stylophora pistillata) was developed using repeated incubation in menthol/artificial seawater (ASW) medium under light and in ASW in darkness, which depleted more than 99% of Symbiodinium from the host within 4∼8 days. As indicated by the respiration rate, energy metabolism (by malate dehydrogenase activity), and nitrogen metabolism (by glutamate dehydrogenase activity and profiles of free amino acids), the physiological and biochemical performances of the menthol-induced aposymbiotic corals were comparable to their symbiotic counterparts without nutrient supplementation (e.g., for Stylophora) or with a nutrient supplement containing glycerol, vitamins, and a host mimic of free amino acid mixture (e.g., for Isopora). Differences in biochemical responses to menthol-induced bleaching between Stylophora and Isopora were attributed to the former digesting Symbiodinium rather than expelling the algae live as found in the latter species. Our studies showed that menthol could successfully bleach corals and provided aposymbiotic corals for further exploration of coral-alga symbioses.     

Endosymbiotic flexibility associates with environmental sensitivity in scleractinian corals

Flexibility in biological systems is seen as an important driver of macro-ecosystem function and stability. Spatially constrained endosymbiotic settings, however, are less studied, although environmental thresholds of symbiotic corals are linked to the function of their endosymbiotic dinoflagellate communities. Symbiotic flexibility is a hypothesized mechanism that corals may exploit to adapt to climate change. This study explores the flexibility of the coral–Symbiodinium symbiosis through quantification of Symbiodinium ITS2 sequence assemblages in a range of coral species and genera. Sequence assemblages are expressed as an index of flexibility incorporating phylogenetic divergence and relative abundance of Symbiodinium sequences recovered from the host. This comparative analysis reveals profound differences in the flexibility of corals for Symbiodinium, thereby classifying corals as generalists or specifists. Generalists such as Acropora and Pocillopora exhibit high intra- and inter-species flexibility in their Symbiodinium assemblages and are some of the most environmentally sensitive corals. Conversely, specifists such as massive Porites colonies exhibit low flexibility, harbour taxonomically narrow Symbiodinium assemblages, and are environmentally resistant corals. Collectively, these findings challenge the paradigm that symbiotic flexibility enhances holobiont resilience. This underscores the need for a deeper examination of the extent and duration of the functional benefits associated with endosymbiotic diversity and flexibility under environmental stress.     

Photosynthetic circadian rhythmicity patterns of Symbiodium,the coral endosymbiotic algae

Biological clocks are self-sustained endogenous timers that enable organisms (from cyanobacteria to humans) to anticipate daily environmental rhythms, and adjust their physiology and behaviour accordingly. Symbiotic corals play a central role in the creation of biologically rich ecosystems based on mutualistic symbioses between the invertebrate coral and dinoflagellate protists from the genus Symbiodinium. In this study, we experimentally establish that Symbiodinium photosynthesis, both as a free-living unicellular algae and as part of the symbiotic association with the coral Stylophora pistillata, is ‘wired’ to the circadian clock mechanism with a ‘free-run’ cycle close to 24 h. Associated photosynthetic pigments also showed rhythmicity under light/dark conditions and under constant light conditions, while the expression of the oxygen-evolving enhancer 1 gene (within photosystem II) coincided with photosynthetically evolved oxygen in Symbiodinium cultures. Thus, circadian regulation of the Symbiodinium photosynthesis is, however, complicated as being linked to the coral/host that have probably profound physiochemical influence on the intracellular environment. The temporal patterns of photosynthesis demonstrated here highlight the physiological complexity and interdependence of the algae circadian clock associated in this symbiosis and the plasticity of algae regulatory mechanisms downstream of the circadian clock.     

Symbiodinium clades A and D differentially predispose Acropora cytherea to disease and Vibrio spp. colonization

Coral disease outbreaks have increased over the last three decades, but their causal agents remain mostly unclear (e.g., bacteria, viruses, fungi, protists). This study details a 14‐month‐long survey of coral colonies in which observations of the development of disease was observed in nearly half of the sampled colonies. A bimonthly qPCR method was used to quantitatively and qualitatively evaluate Symbiodinium assemblages of tagged colonies, and to detect the presence of Vibrio spp. Firstly, our data showed that predisposition to disease development in general, and, more specifically, infection by Vibrio spp. in Acropora cytherea depended on which clades of Symbiodinium were harbored. In both cases, harboring clade D rather than A was beneficial to the coral host. Secondly, the detection of Vibrio spp. in only colonies that developed disease strongly suggests opportunistic traits of the bacteria. Finally, even if sporadic cases of switching and probably shuffling were observed, this long‐term survey does not suggest specific‐clade recruitment in response to stressors. Altogether, our results demonstrate that the fitness of the coral holobiont depends on its initial consortium of Symbiodinium, which is distinct among colonies, rather than a temporary adaptation achieved through acquiring different Symbiodinium clades.     

Functional significance of dinitrogen fixation in sustaining coral productivity under oligotrophic conditions

Functional traits define species by their ecological role in the ecosystem. Animals themselves are host–microbe ecosystems (holobionts), and the application of ecophysiological approaches can help to understand their functioning. In hard coral holobionts, communities of dinitrogen (N₂)-fixing prokaryotes (diazotrophs) may contribute a functional trait by providing bioavailable nitrogen (N) that could sustain coral productivity under oligotrophic conditions. This study quantified N₂ fixation by diazotrophs associated with four genera of hermatypic corals on a northern Red Sea fringing reef exposed to high seasonality. We found N₂ fixation activity to be 5- to 10-fold higher in summer, when inorganic nutrient concentrations were lowest and water temperature and light availability highest. Concurrently, coral gross primary productivity remained stable despite lower Symbiodinium densities and tissue chlorophyll a contents. In contrast, chlorophyll a content per Symbiodinium cell increased from spring to summer, suggesting that algal cells overcame limitation of N, an essential element for chlorophyll synthesis. In fact, N₂ fixation was positively correlated with coral productivity in summer, when its contribution was estimated to meet 11% of the Symbiodinium N requirements. These results provide evidence of an important functional role of diazotrophs in sustaining coral productivity when alternative external N sources are scarce.     

Transmission Mode Predicts Specificity and Interaction Patterns in Coral-Symbiodinium Networks

Most reef-building corals in the order Scleractinia depend on endosymbiotic algae in the genus Symbiodinium for energy and survival. Significant levels of taxonomic diversity in both partners result in numerous possible combinations of coral-Symbiodinium associations with unique functional characteristics. We created and analyzed the first coral-Symbiodinium networks utilizing a global dataset of interaction records from coral reefs in the tropical Indo-Pacific and Atlantic Oceans for 1991 to 2010. Our meta-analysis reveals that the majority of coral species and Symbiodinium types are specialists, but failed to detect any one-to-one obligate relationships. Symbiont specificity is correlated with a host’s transmission mode, with horizontally transmitting corals being more likely to interact with generalist symbionts. Globally, Symbiodinium types tend to interact with only vertically or horizontally transmitting corals, and only a few generalist types are found with both. Our results demonstrate a strong correlation between symbiont specificity, symbiont transmission mode, and community partitioning. The structure and dynamics of these network interactions underlie the fundamental biological partnership that determines the condition and resilience of coral reef ecosystems.     

Chronic parrotfish grazing impedes coral recovery after bleaching

Coral bleaching, in which corals become visibly pale and typically lose their endosymbiotic zooxanthellae (Symbiodinium spp.), increasingly threatens coral reefs worldwide. While the proximal environmental triggers of bleaching are reasonably well understood, considerably less is known concerning physiological and ecological factors that might exacerbate coral bleaching or delay recovery. We report a bleaching event in Belize during September 2004 in which Montastraea spp. corals that had been previously grazed by corallivorous parrotfishes showed a persistent reduction in symbiont density compared to intact colonies. Additionally, grazed corals exhibited greater diversity in the genetic composition of their symbiont communities, changing from uniform ITS2 type C7 Symbiodinium prior to bleaching to mixed assemblages of Symbiodinium types post-bleaching. These results suggest that chronic predation may exacerbate the influence of environmental stressors and, by altering the coral-zooxanthellae symbiosis, such abiotic-biotic interactions may contribute to spatial variation in bleaching processes.     

COMPARISON OF ENDOSYMBIOTIC AND FREE‐LIVING SYMBIODINIUM (DINOPHYCEAE) DIVERSITY IN A HAWAIIAN REEF ENVIRONMENT1

Many scleractinian corals must acquire their endosymbiotic dinoflagellates (genus Symbiodinium) anew each generation from environmental pools, and exchange between endosymbiotic and environmental pools of Symbiodinium (reef waters and sediments) has been proposed as a mechanism for optimizing coral physiology in the face of environmental change. Our understanding of the diversity of Symbiodinium spp. in environmental pools is poor by comparison to that engaged in endosymbiosis, which reflects the challenges of visualizing the genus against the backdrop of the complex and diverse micro‐eukaryotic communities found free‐living in the environment. Here, the molecular diversity of Symbiodinium living in the waters and sediments of a reef near Coconut Island, O‘ahu, Hawai‘i, sampled at four hourly intervals over a period of 5 d was characterized using a Symbiodinium‐specific hypervariable region of the chloroplast 23S. A comparison of Symbiodinium spp. diversity recovered from environmental samples with the endosymbiotic diversity in coral species that dominate the adjacent reef revealed limited overlap between these communities. These data suggest that the potential for infection, exchange, and/or repopulation of corals with Symbiodinium derived from the environment is limited at this location, a finding that is perhaps consistent with the high proportion of coral species in this geographic region that transmit endosymbionts from generation to generation.     

Osmotic tolerance and membrane permeability characteristics of rhesus monkey (Macaca mulatta) spermatozoa

Biophysical characteristics of the plasma membrane, such as osmotic sensitivity and water and cryoprotectant permeability are important determinants of the function of spermatozoa after cryopreservation. A series of experiments was conducted with rhesus macaque spermatozoa at 23 degrees C to determine their: (1) cell volume and osmotically inactive fraction of the cell volume; (2) permeability coefficients for water and the cryoprotectants dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol; (3) tolerance to anisosmotic conditions; and (4) motility after a one step addition and removal of the four cryoprotectants. An electronic particle counter and computer aided semen analysis were used to determine the cell volume and permeability coefficients, and motility, respectively. Rhesus spermatozoa isosmotic cell volume was 27.7+/-3.0 microm3 (mean+/-SEM) with an osmotically inactive cell fraction of 51%. Hydraulic conductivity in the presence of dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol was 1.09+/-0.30, 0.912+/-0.27, 1.53+/-0.53, and 1.94+/-0.47 microm/min/atm, respectively. Cryoprotectant permeability was 1.39+/-0.31, 2.21+/-0.32, 3.38+/-0.63, and 6.07+/-1.1 (x10(-3)cm/min), respectively. Rhesus sperm tolerated all hyposmotic exposures. However, greater than 70% motility loss was observed after exposure to solutions of 600 mOsm and higher. A one step addition and removal of all four cryoprotectants did not cause significant motility loss. These data suggest that rhesus sperm are tolerant to hyposmotic conditions, and ethylene glycol may be the most appropriate cryoprotectant for rhesus sperm cryopreservation, as it has the highest permeability coefficient of the tested cryoprotectants.     

The effect of chilling and cryoprotectants on hard coral (Echinopora spp.) oocytes during short-term low temperature preservation

Understanding chilling sensitivity and chilling injury of coral oocytes, in the presence and absence of a cryoprotectant, is important in developing cryopreservation protocols, as well as for short-term storage and transport (e.g., for species conservation). The objective of this study was to investigate the chilling sensitivity of hard coral (Echinopora spp.) oocytes and the effectiveness of methanol (as a cryoprotectant) in protecting these oocytes during short-term, low temperature preservation. Oocytes were exposed to 0.5, 1, or 2 m methanol at 5, 0, or −5 °C for 1, 2, 4, 6, 8, 16, or 32 h, and their quality determined based on adenosine triphosphate (ATP) content. Methanol at 0.5 m was the most effective means to reduce chilling-induced reduction in ATP concentrations. Coral oocytes can be stored at room temperature for 4 h in filtered nature seawater with no detrimental effect on oocyte quality; however, in the present study, oocyte survival was extended for 8 h by addition of methanol in low concentrations (0.5 or 1 m) at low temperatures (5 and 0 °C). These findings should enhance conservation efforts and facilitate low-temperature transport of endangered and threatened coral species.     

Vibrio Zinc-Metalloprotease Causes Photoinactivation of Coral Endosymbionts and Coral Tissue Lesions

Background

Coral diseases are emerging as a serious threat to coral reefs worldwide. Of nine coral infectious diseases, whose pathogens have been characterized, six are caused by agents from the family Vibrionacae, raising questions as to their origin and role in coral disease aetiology.

Methodology/Principal Findings

Here we report on a Vibrio zinc-metalloprotease causing rapid photoinactivation of susceptible Symbiodinium endosymbionts followed by lesions in coral tissue. Symbiodinium photosystem II inactivation was diagnosed by an imaging pulse amplitude modulation fluorometer in two bioassays, performed by exposing Symbiodinium cells and coral juveniles to non-inhibited and EDTA-inhibited supernatants derived from coral white syndrome pathogens.

Conclusion/Significance

These findings demonstrate a common virulence factor from four phylogenetically related coral pathogens, suggesting that zinc-metalloproteases may play an important role in Vibrio pathogenicity in scleractinian corals.     

The promiscuous larvae: flexibility in the establishment of symbiosis in corals

Coral reefs thrive in part because of the symbiotic partnership between corals and Symbiodinium. While this partnership is one of the keys to the success of coral reef ecosystems, surprisingly little is known about many aspects of coral symbiosis, in particular the establishment and development of symbiosis in host species that acquire symbionts anew in each generation. More specifically, the point at which symbiosis is established (i.e., larva vs. juvenile) remains uncertain, as does the source of free-living Symbiodinium in the environment. In addition, the capacity of host and symbiont to form novel combinations is unknown. To explore patterns of initial association between host and symbiont, larvae of two species of Acropora were exposed to sediment collected from three locations on the Great Barrier Reef. A high proportion of larvae established symbiosis shortly after contact with sediments, and Acropora larvae were promiscuous, taking up multiple types of Symbiodinium. The Symbiodinium types acquired from the sediments reflected the symbiont assemblage within a wide range of cnidarian hosts at each of the three sites, suggesting potential regional differences in the free-living Symbiodinium assemblage. Coral larvae clearly have the capacity to take up Symbiodinium prior to settlement, and sediment is a likely source. Promiscuous larvae allow species to associate with Symbiodinium appropriate for potentially novel environments that may be experienced following dispersal.     

Evidence of photosymbiosis in Palaeozoic tabulate corals

Coral reefs form the most diverse of all marine ecosystems on the Earth. Corals are among their main components and owe their bioconstructing abilities to a symbiosis with algae (Symbiodinium). The coral–algae symbiosis had been traced back to the Triassic (ca 240 Ma). Modern reef-building corals (Scleractinia) appeared after the Permian–Triassic crisis; in the Palaeozoic, some of the main reef constructors were extinct tabulate corals. The calcium carbonate secreted by extant photosymbiotic corals bears characteristic isotope (C and O) signatures. The analysis of tabulate corals belonging to four orders (Favositida, Heliolitida, Syringoporida and Auloporida) from Silurian to Permian strata of Europe and Africa shows these characteristic carbon and oxygen stable isotope signatures. The δ¹⁸O to δ¹³C ratios in recent photosymbiotic scleractinians are very similar to those of Palaeozoic tabulates, thus providing strong evidence of such symbioses as early as the Middle Silurian (ca 430 Ma). Corals in Palaeozoic reefs used the same cellular mechanisms for carbonate secretion as recent reefs, and thus contributed to reef formation.     

Physiological responses to short-term sediment exposure in adults of the Caribbean coral Montastraea cavernosa and adults and recruits of Porites astreoides

Coral Reefs - Sedimentation is a common anthropogenic stressor known to reduce coral growth, reproduction, and the photosynthetic capacity of their endosymbiotic algae. This study assessed the...