Environmentally-Driven Variation in the Physiology of a New Caledonian Reef Coral

Given the widespread threats to coral reefs, scientists have lost the opportunity to understand the basic biology of “pristine” corals whose physiologies have not been markedly perturbed by human activity. For instance, high temperature-induced bleaching has been occurring annually since 2014 in New Caledonia. Because most corals cannot withstand repeated years when bleaching occurs, an analysis was undertaken to showcase coral behavior in a period just before the onset of “annual severe bleaching” (ASB; November 2013) such that future generations might know how these corals functioned in their last bleaching-free year. Pocillopora damicornis colonies were sampled across a variety of environmental gradients, and a subset was sampled during both day and night to understand how their molecular biology changes upon cessation of dinoflagellate photosynthesis. Of the 13 environmental parameters tested, sampling time (i.e., light) most significantly affected coral molecular physiology, and expression levels of a number of both host and Symbiodiniaceae genes demonstrated significant diel variation; endosymbiont mRNA expression was more temporally variable than that of their anthozoan hosts. Furthermore, expression of all stress-targeted genes in both eukaryotic compartments of the holobiont was high, even in isolated, uninhabited, federally protected atolls of the country’s far northwest. Whether this degree of sub-cellular stress reflects cumulative climate change impacts or, instead, a stress-hardened phenotype, will be unveiled through assessing the fates of these corals in the wake of increasingly frequent marine heatwaves.

Seabird-Derived Nutrients Supply Modulates the Trophic Strategies of Mixotrophic Corals

The ability of corals to modulate their nutrition strategy in response to variable nutrient supply remains poorly understood, limiting our understanding of energy flow in coral reef ecosystems and thus our comprehension of their resilience to global changes. We used a naturally occurring nutrient gradient along the reef flat of two seabird-inhabited islets in the SW Pacific to characterize spatiotemporal fluctuations in coastal nutrient availability, and how it modulates the trophic response of the mixotrophic coral Pocillopora damicornis. The clear gradients in dissolved [NOx] and δ15N values of macroalgae and both P. damicornis tissues and symbionts observed along the reef flat during the dry and the rainy season revealed that seabird-derived-N is supplied year-round to the reef flat. Yet, nitrogen isotope values of macroalgae show that the seabirds’ effect on coral reefs varies with sites and seasons. Metrics derived from the SIBER framework revealed that coral nutrition seasonally favored autotrophy when exposed to higher seabird guano concentrations and at inshore stations, while heterotrophy dominated in corals less exposed to seabird-derived nutrient supply. P. Damicornis is therefore able to cope with large changes in nitrogen supply induced by seabird island communities by switching between autotrophy and heterotrophy. These results shed light on the flexibility of resource sharing within the coral-algae symbiosis and highlight the importance of seabird populations to the functioning of coral reef ecosystems.

Binding Pattern Reconstructions of FGF-FGFR Budding-Inducing Signaling in Reef-Building Corals

Reef-building corals play an important role in marine ecosystems. However, owing to climate change, ocean acidification, and predation by invasive crown-of-thorns starfish, these corals are declining. As marine animals comprise polyps, reproduction by asexual budding is pivotal in scleractinian coral growth. The fibroblast growth factor (FGF) signaling pathway is essential in coral budding morphogenesis. Here, we sequenced the full-length transcriptomes of four common and frequently dominant reef-building corals and screened out the budding-related FGF and FGFR genes. Thereafter, three-dimensional (3D) models of FGF and FGFR proteins as well as FGF-FGFR binding models were reconstructed. Based on our findings, the FGF8-FGFR3 binding models in Pocillopora damicornis, Montipora capricornis, and Acropora muricata are typical receptor tyrosine kinase-signaling pathways that are similar to the Kringelchen (FGFR) in hydra. However, in P. verrucosa, FGF8 is not the FGFR3 ligand, which is found in other hydrozoan animals, and its FGFR3 must be activated by other tyrosine kinase-type ligands. Overall, this study provides background on the potentially budding propagation signaling pathway activated by the applications of biological agents in reef-building coral culture that could aid in the future restoration of coral reefs.

Effects of Temperature and Salinity on Coral Bleaching in Laboratory

Coral bleaching occurs when cell density or the concentration of photosynthetic pigments of the endosymbionts, zooxanthellae are decreased. This incident may possibly be caused by some environmental stresses, especially under conditions of elevated temperature, decrease in water salinity, or a combination of these factors. To determine the role of temperature and salinity on zooxanthellae and coral bleaching this study was conducted in aquariums under laboratory conditions on cauliflower coral Pocillopora damicornis. The samples were collected from three sites around Samaesan Island, Chonburi, Thailand. Three sets of experiments were conducted at three levels of temperature: room temperature 27 (control), 30, and 33 oC respectively. At each temperature level, three levels of salinities; 10, 20 and 30 (control) psu were tested as well. Coral bleaching percentage and zooxanthellae density in the water column were observed every 6 hours during the period of 72 hours. The results showed that when coral exposed to the highest temperature (33 oC) under the lowest salinity (10 psu), 50-90% bleaching was found and higher symbiont densities in the water column were detected. These results suggested that the combination of the high temperature and low salinity had synergistic effects on coral bleaching and zooxanthellae.

Differential Affinities of a Pocillopora damicornis Galectin to Five Genera of Symbiodiniaceae at Different Temperatures

The symbiosis of coral-Symbiodiniaceae is the quintessential basis of the coral reef ecosystem, and its breakdown results in coral bleaching, one of the most severe ecological catastrophes in the ocean. Critical to the establishment of the symbiosis is the host’s specific recognition of the symbionts through the binding of the coral host’s pattern recognition receptors (PRRs) to the symbiont cell surface’s glycoconjugates. However, the molecular basis for this recognition process is poorly understood. The present study investigated the binding affinities of the coral galectin PdGLT-1 to different symbiodiniacean species under different temperatures. At 25°C, the PdGLT-1 recombinant protein (rPdGLT-1) exhibited different binding affinities to different symbiodiniacean species from five genera, with a significantly higher binding affinity (p < 0.05) to Fugacium kawagutii (2.6-fold) and Cladocopium goreaui (1.9-fold) than Symbiodinium microadriaticum. The binding topology of rPdGLT-1 differed among the five symbiodiniacean species; for S. microadriaticum, Breviolum minutum, and Durusdinium trenchii, the binding was on some specific sites on the cell surface, whereas for C. goreaui and F. kawagutii, the binding signals were detected over the whole cell surface. Interestingly, PdGLT-1 binding induced agglutination of F. kawagutii cells but not of C. goreaui, explaining why C. goreaui was the most dominant symbiodiniacean symbionts in corals. Moreover, the affinity of rPdGLT-1 to Symbiodiniaceae was affected by temperature, and the highest binding affinities were observed at 30, 20, 30, 35, and 30°C for S. microadriaticum, B. minutum, C. goreaui, D. trenchii, and F. kawagutii, respectively. The optimal binding temperatures were consistent with the current understanding that D. trenchii was the most thermal resistant among these species. These results suggest that the binding affinity of the PRR PdGLT-1 may determine the specificity of host-symbiont pairing and explain why Cladocopium is the dominant symbionts of coral P. damicornis at normal temperature, and corals with Durusdinium symbionts may survive better at high temperature.

The Meta-Organism Response of the Environmental Generalist Pocillopora damicornis Exposed to Differential Accumulation of Heat Stress

Coral bleaching events in the marine environment are now occurring globally, and the frequency and severity of these events are increasing. Critically, these events can cause the symbiosis between Symbiodiniaceae and their coral hosts to break down, but how the microbial community within the coral responds to bleaching is still equivocal. We investigated the impact of thermal stress exposure on the meta-organism responses of the generalist scleractinian coral species Pocillopora damicornis. Using mesocosms to recreate warming scenarios previously observed at Heron Island, we show that P. damicornis symbiont densities and photophysiological parameters declined at a similar rate under thermal stress regardless of the length of pre-bleaching thermal stress, defined here as temperatures above the monthly maximum mean (MMM) for Heron Island but below the local bleaching threshold (MMM + 2°C). However, we find that the P. damicornis microbiome remains stable over time regardless of the degree of thermal stress and the accumulation of pre-bleaching thermal stress. Our study therefore suggests that while P. damicornis is physiologically impacted by bleaching temperatures, the microbial community identified through 16S rRNA sequencing remains unchanged at the ASV level throughout bleaching. Understanding the capacity of a generalist species to withstand bleaching events is imperative to characterizing what coral species will exist on coral reefs following disturbances, as it has been suggested that the success of environmental generalist species may simplify community structure and lead to changes in biodiversity following environmental disturbance.

Intra-genomic variation in symbiotic dinoflagellates: Recent divergence or natural hybridization?

<p>The perpetuity of coral reefs will ultimately depend on the ability of corals to adapt to changing conditions. Inter-specific hybridization can provide the raw genetic material necessary for adaptation, and stimulate macro-evolutionary leaps during periods of environmental upheaval. Though well-documented in corals, hybridization has yet to be identified in their dinoflagellate symbionts (genus Symbiodinium), despite growing evidence of sexual reproduction in this genus. The integral roles that these symbiotic algae play in coral productivity, reef accretion and ‘coral bleaching’ emphasize the need to better understand their short-term evolutionary potential. In this thesis, I develop new molecular and statistical methodology, and combine lab- and field-based analysis to explore the potential for hybridization between divergent Symbiodinium taxa. To screen for putative Symbiodinium hybrids, intra-genomic variation was examined within individual symbionts isolated from the reef-building coral Pocillopora damicornis at Lord Howe Island (Australia). A nested quantitative PCR (qPCR) assay was developed to quantify polymorphic internal transcribed spacer 2 (ITS2) sequences within the genome of each symbiont cell. Three genetically distinct Symbiodinium populations were detected co-existing within the symbiont consortium of P. damicornis. Mixed populations of ‘pure’ Symbiodinium types C100 and C109 coexisted with a population of cells hosting co-dominant C100 and C109 ITS2 repeats. Genetically heterogeneous Symbiodinium cells were more common than homogeneous symbionts in four of the six colonies analysed, with a maximum proportional abundance of 89%. Morphological, functional and ecological attributes of heterogeneous Symbiodinium cells were characterized to assess their candidacy as putative hybrids. The proportional abundance of genetically heterogeneous symbionts was spatially and temporally conserved within colonies, indicating a lack of competition between Symbiodinium populations. However, this abundance ratio varied considerably between colonies separated by metres to tens of metres, and to a greater extent between sites isolated by hundreds to thousands of metres. The local thermal maximum emerged as a significant predictor of the proportional abundance of genetically heterogeneous Symbiodinium cells, suggesting that the distribution of these ‘putative hybrids’ is influenced by a reduced affinity for thermal stress. Genetically heterogeneous Symbiodinium cells were around 50% larger (by volume) than homogeneous cells, occupied tissue of the coral host at reduced densities, and showed relatively poor light-harvesting efficiency. Colonies hosting a higher proportion of these symbionts suffered a reduction in overall photosynthetic performance (maximum gross photosynthesis normalised to respiration; P:R) at the ambient temperature of 25 °C. This disparity was maintained when the temperature was elevated to simulate the maximum experienced within the LHI lagoon (29 °C). Under these stressful conditions, colonies dominated by putative Symbiodinium hybrids were only marginally capable of net oxygen production. The influence of putative Symbiodinium hybrids on the growth and survival of P. damicornis was tested by reciprocally transplanting coral colonies between reef sites featuring distinct temperature regimes. Neither calcification nor mortality was influenced by the proportional abundance of genetically heterogeneous cells in the symbiont consortium. This uncoupling of symbiont performance and host fitness may be explained by stochastic events such as predation and disease, which substantially increase variation in growth and mortality in field experiments. Alternatively, it may represent some unknown benefit associated with hosting hybrid symbionts, belying their relatively poor photosynthetic performance, and explaining the widespread abundance of these heterogeneous Symbiodinium cells on the Lord Howe Island reef. Our inability to maintain many clade C Symbiodinium types in culture prevents direct observations of hybridization between C100 and C109. Unequivocal evidence of this phenomenon will therefore likely remain elusive until high-resolution, single-copy nuclear markers can be developed, since the incomplete displacement of ancestral polymorphisms can leave a similar genomic signature to that of hybridization. However, this study serves to provide an initial proof-of-principle for hybridization between divergent Symbiodinium taxa. In doing so, it highlights the need to better understand the evolutionary processes underpinning coral- and symbiont-adaptation in a changing climate.</p>

Living on the Edge: Protective Mechanisms Underlying Thermal Tolerance in High Latitude Symbiodinium spp.

<p>The association between symbiotic dinoflagellates (Symbiodinium spp.) and corals extends to subtropical and temperate regions, where sea surface temperatures (SSTs) are generally lower than in the tropics and can vary substantially over the course of the year due to seasonal changes. These high latitude coral-dinoflagellate symbioses might be better able to withstand thermal variability and might be particularly well equipped to cope with lower SSTs compared to their tropical relatives. The aim of this thesis was to analyze the cellular mechanisms that underlie heat and/or cold tolerance in a range of reef-building corals (Acropora yongei, Acropora solitariensis, Isopora palifera, Pocillopora damicornis, Porites heronensis and Stylophora sp.), as well as the symbiotic sea anemone Entacmaea quadricolor. In particular, the study focussed on protective mechanisms in their dinoflagellate symbionts as a potential determinant of thermal sensitivity (i.e. bleaching) or resistance of the intact symbiosis. High latitude reef-building corals were analyzed at the world’s southernmost coral reef at Lord Howe Island, while E. quadricolor was sampled at the subtropical coral community at North Solitary Island; both sites are located in New South Wales, Australia. The specific objectives were to assess the roles of: (1) xanthophyll deepoxidation; (2) thylakoid fatty acid composition; (3) Symbiodinium superoxide dismutase (SOD) and ascorbate peroxidase (APX) activity; and (4) D1 repair on the photophysiology, bleaching susceptibility and survivorship of a range of high-latitude coral-Symbiodinium associations from Lord Howe Island when exposed to elevated or decreased temperature. Furthermore, I aimed to: (5) characterise Symbiodinium diversity in the anemone E. quadricolor on the west coast of Australia; and (6) measure the dynamics of Symbiodinium ITS2 populations and SOD activity in two E. quadricolor phenotypes (green and pink colour phenotypes) in response to elevated temperature. I showed that thermal responses in high latitude corals and their dinoflagellate symbionts are highly variable, depending on host species (or phenotype) and Symbiodinium genotype, and that the activation of protective mechanisms in Symbiodinium was not necessarily correlated with sub-lethal bleaching susceptibility or survivorship of their coral hosts. More specifically: (1) In response to short-term heat stress and cold stress, xanthophyll de-epoxidation increased in some but not all bleaching susceptible (e.g. P. damicornis) and bleaching tolerant (P. heronensis) corals; (2) overall unsaturated thylakoid fatty acids increased in symbionts of a bleaching tolerant coral association, yet was not correlated with PSII photochemical efficiency; and (3) SOD and APX activity remained unchanged in the majority of Symbiodinium types regardless of bleaching susceptibility of the coral host, but decreased in bleaching susceptible Pocillopora damicornis when exposed to short-term heat stress. Elevated temperatures resulted in enhanced D1 turnover in two warm-water bleaching susceptible Symbiodinium-host combinations; however a direct link between increased dependence on D1 turnover and bleaching susceptibility was not demonstrated. From the results obtained it seems unlikely that the specific cellular adaptations in Symbiodinium alone determine the tolerance of Lord Howe corals to thermal variations. In contrast, the results highlight the significance of the particular host-symbiont combination and it appears that the host is important in determining, at least in part, the thermal response of the coral. Additionally, this study revealed a high diversity of Symbiodinium ITS2 (internal transcribed spacer 2) types in E. quadricolor from five locations on the west coast of Australia. E. quadricolor predominantly associated with six types of clade C (four of which were novel) and most anemones harboured multiple types simultaneously. At North Solitary Island, anemones simultaneously harboured Symbiodinium C25 and C3.25 (a novel variant of C3). Experimentally, I showed that anemones shuffled the relative proportions of C25 and C3.25 in response to elevated temperature, but not in both anemone colour phenotypes analyzed. Furthermore, baseline photobiological characteristics were distinct in the two different anemone colour morphs but were not correlated with the ratio of Symbiodinium C25 to C3.25, suggesting that host mechanisms such as pigmentation were involved in regulating light utilization by the symbionts. My hypothesis that symbiont shuffling was related to SOD activity, as such that those symbionts with enhanced SOD activity and increased capability to scavenge superoxide anion would increase in relative abundance in response to short-term heat stress, could not be proved. In summary, this thesis provides detailed information on some key cellular mechanisms that could underpin thermal sensitivity and resistance in high latitude Symbiodinium, and most importantly highlights the significance of the host-symbiont combination in determining the response to thermal stress. The various mechanistic findings described here further our understanding of the coral bleaching process in general and particularly give insight into physiological and cellular responses to coldwater stress in reef-building corals at high-latitude sites. The results of this thesis indicate that in light of ongoing climate change, as episodes of cold-water and warm-water anomalies will become more frequent, branching corals such as Acropora yongei or Pocillopora damicornis and their symbionts will experience physiological stress more frequently than massive species such as Porites heronensis. This might have profound impacts on the long-term stability and species composition of high latitude coral reefs.</p>