Gas Chromatography-Mass Spectrometry-Based Targeted Metabolomics of Hard Coral Samples.

Gas chromatography-mass spectrometry (GC-MS)-based approaches have proven to be powerful for elucidating the metabolic basis of the cnidarian-dinoflagellate symbiosis and how coral responds to stress (i.e., during temperature-induced bleaching). Steady-state metabolite profiling of the coral holobiont, which comprises the cnidarian host and its associated microbes (Symbiodiniaceae and other protists, bacteria, archaea, fungi, and viruses), has been successfully applied under ambient and stress conditions to characterize the holistic metabolic status of the coral. However, to answer questions surrounding the symbiotic interactions, it is necessary to analyze the metabolite profiles of the coral host and its algal symbionts independently, which can only be achieved by physical separation and isolation of the tissues, followed by independent extraction and analysis. While the application of metabolomics is relatively new to the coral field, the sustained efforts of research groups have resulted in the development of robust methods for analyzing metabolites in corals, including the separation of the coral host tissue and algal symbionts. This paper presents a step-by-step guide for holobiont separation and the extraction of metabolites for GC-MS analysis, including key optimization steps for consideration. We demonstrate how, once analyzed independently, the combined metabolite profile of the two fractions (coral and Symbiodiniaceae) is similar to the profile of the whole (holobiont), but by separating the tissues, we can also obtain key information about the metabolism of and interactions between the two partners that cannot be obtained from the whole alone.

Inter-partner specificity limits the acquisition of thermotolerant symbionts in a model cnidarian-dinoflagellate symbiosis.

The ability of corals and other cnidarians to survive climate change depends partly on the composition of their endosymbiont communities. The dinoflagellate family Symbiodiniaceae is genetically and physiologically diverse, and one proposed mechanism for cnidarians to acclimate to rising temperatures is to acquire more thermally tolerant symbionts. However, cnidarian-dinoflagellate associations vary in their degree of specificity, which may limit their capacity to alter symbiont communities. Here, we inoculated symbiont-free polyps of the sea anemone Exaiptasia pallida (commonly referred to as 'Aiptasia'), a model system for the cnidarian-dinoflagellate symbiosis, with simultaneous or sequential mixtures of thermally tolerant and thermally sensitive species of Symbiodiniaceae. We then monitored symbiont success (relative proportional abundance) at normal and elevated temperatures across two to four weeks. All anemones showed signs of bleaching at high temperature. During simultaneous inoculations, the native, thermally sensitive Breviolum minutum colonized polyps most successfully regardless of temperature when paired against the non-native but more thermally tolerant Symbiodinium microadriaticum or Durusdinium trenchii. Furthermore, anemones initially colonized with B. minutum and subsequently exposed to S. microadriaticum failed to acquire the new symbiont. These results highlight how partner specificity may place strong limitations on the ability of certain cnidarians to acquire more thermally tolerant symbionts, and hence their adaptive potential under climate change.

The physiological response of the deep-sea coral Solenosmilia variabilis to ocean acidification.

Several forms of calcifying scleractinian corals provide important habitat complexity in the deep-sea and are consistently associated with a high biodiversity of fish and other invertebrates. How these corals may respond to the future predicted environmental conditions of ocean acidification is poorly understood, but any detrimental effects on these marine calcifiers will have wider impacts on the ecosystem. Colonies of Solenosmilia variabilis, a protected deep-sea coral commonly occurring throughout the New Zealand region, were collected during a cruise in March 2014 from the Louisville Seamount Chain. Over a 12-month period, samples were maintained in temperature controlled (∼3.5 °C) continuous flow-through tanks at a seawater pH that reflects the region's current conditions (7.88) and an end-of-century scenario (7.65). Impacts on coral growth and the intensity of colour saturation (as a proxy for the coenenchyme tissue that covers the coral exoskeleton and links the coral polyps) were measured bimonthly. In addition, respiration rate was measured after a mid-term (six months) and long-term (12 months) exposure period. Growth rates were highly variable, ranging from 0.53 to 3.068 mm year-1 and showed no detectable difference between the treatment and control colonies. Respiration rates also varied independently of pH and ranged from 0.065 to 1.756 µmol O2 g protein-1 h-1. A significant change in colour was observed in the treatment group over time, indicating a loss of coenenchyme. This loss was greatest after 10 months at 5.28% and could indicate a reallocation of energy with physiological processes (e.g.  growth and respiration) being maintained at the expense of coenenchyme production. This research illustrates important first steps to assessing and understanding the sensitivity of deep-sea corals to ocean acidification.

The scavenger receptor repertoire in six cnidarian species and its putative role in cnidarian-dinoflagellate symbiosis.

Many cnidarians engage in a mutualism with endosymbiotic photosynthetic dinoflagellates that forms the basis of the coral reef ecosystem. Interpartner interaction and regulation includes involvement of the host innate immune system. Basal metazoans, including cnidarians have diverse and complex innate immune repertoires that are just beginning to be described. Scavenger receptors (SR) are a diverse superfamily of innate immunity genes that recognize a broad array of microbial ligands and participate in phagocytosis of invading microbes. The superfamily includes subclades named SR-A through SR-I that are categorized based on the arrangement of sequence domains including the scavenger receptor cysteine rich (SRCR), the C-type lectin (CTLD) and the CD36 domains. Previous functional and gene expression studies on cnidarian-dinoflagellate symbiosis have implicated SR-like proteins in interpartner communication and regulation. In this study, we characterized the SR repertoire from a combination of genomic and transcriptomic resources from six cnidarian species in the Class Anthozoa. We combined these bioinformatic analyses with functional experiments using the SR inhibitor fucoidan to explore a role for SRs in cnidarian symbiosis and immunity. Bioinformatic searches revealed a large diversity of SR-like genes that resembled SR-As, SR-Bs, SR-Es and SR-Is. SRCRs, CTLDs and CD36 domains were identified in multiple sequences in combinations that were highly homologous to vertebrate SRs as well as in proteins with novel domain combinations. Phylogenetic analyses of CD36 domains of the SR-B-like sequences from a diversity of metazoans grouped cnidarian with bilaterian sequences separate from other basal metazoans. All cnidarian sequences grouped together with moderate support in a subclade separately from bilaterian sequences. Functional experiments were carried out on the sea anemone Aiptasia pallida that engages in a symbiosis with Symbiodinium minutum (clade B1). Experimental blocking of the SR ligand binding site with the inhibitor fucoidan reduced the ability of S. minutum to colonize A. pallida suggesting that host SRs play a role in host-symbiont recognition. In addition, incubation of symbiotic anemones with fucoidan elicited an immune response, indicating that host SRs function in immune modulation that results in host tolerance of the symbionts.

THE EFFECTS OF ULTRAVIOLET-B RADIATION ON ANTARCTIC SEA-ICE ALGAE(1).

The impacts of ultraviolet-B radiation (UVB) on polar sea-ice algal communities have not yet been demonstrated. We assess the impacts of UV on these communities using both laboratory experiments on algal isolates and by modification of the in situ spectral distribution of the under-ice irradiance. In the latter experiment, filters were attached to the upper surface of the ice so that the algae were exposed in situ to treatments of ambient levels of PAR and UV radiation, ambient radiation minus UVB, and ambient radiation minus all UV. After 16 d, significant increases in chl a and cell numbers were recorded for all treatments, but there were no significant differences among the different treatments. Bottom-ice algae exposed in vitro were considerably less tolerant to UVB than those in situ, but this tolerance improved when algae were retained within a solid block of ice. In addition, algae extracted from brine channels in the upper meter of sea ice and exposed to PAR and UVB in the laboratory were much more tolerant of high UVB doses than were any bottom-ice isolates. This finding indicates that brine algae may be better adapted to high PAR and UVB than are bottom-ice algae. The data indicate that the impact of increased levels of UVB resulting from springtime ozone depletion on Antarctic bottom-ice communities is likely to be minimal. These algae are likely protected by strong UVB attenuation by the overlying ice and snow, by other inorganic and organic substances in the ice matrix, and by algal cells closer to the surface.

Growth anomalies on the coral genera Acropora and Porites are strongly associated with host density and human population size across the Indo-Pacific.

Growth anomalies (GAs) are common, tumor-like diseases that can cause significant morbidity and decreased fecundity in the major Indo-Pacific reef-building coral genera, Acropora and Porites. GAs are unusually tractable for testing hypotheses about drivers of coral disease because of their pan-Pacific distributions, relatively high occurrence, and unambiguous ease of identification. We modeled multiple disease-environment associations that may underlie the prevalence of Acropora growth anomalies (AGA) (n = 304 surveys) and Porites growth anomalies (PGA) (n = 602 surveys) from across the Indo-Pacific. Nine predictor variables were modeled, including coral host abundance, human population size, and sea surface temperature and ultra-violet radiation anomalies. Prevalence of both AGAs and PGAs were strongly host density-dependent. PGAs additionally showed strong positive associations with human population size. Although this association has been widely posited, this is one of the first broad-scale studies unambiguously linking a coral disease with human population size. These results emphasize that individual coral diseases can show relatively distinct patterns of association with environmental predictors, even in similar diseases (growth anomalies) found on different host genera (Acropora vs. Porites). As human densities and environmental degradation increase globally, the prevalence of coral diseases like PGAs could increase accordingly, halted only perhaps by declines in host density below thresholds required for disease establishment.

Gross and microscopic morphology of lesions in Cnidaria from Palmyra Atoll, Central Pacific.

We conducted gross and microscopic characterizations of lesions in Cnidaria from Palmyra Atoll, Central Pacific. We found growth anomalies (GA) to be the most commonly encountered lesion. Cases of discoloration and tissue loss were rare. GAs had a focal or multi-focal distribution and were predominantly nodular, exophytic, and umbonate. In scleractinians, the majority of GAs manifested as hyperplasia of the basal body wall (52% of cases), with an associated absence or reduction of polyp structure (mesenteries and filaments, actinopharynx and tentacles), and depletion of zooxanthellae in the gastrodermis of the upper body wall. In the soft corals Sinularia sp. and Lobophytum sp., GAs exclusively manifested as prominent hyperplasia of the coenenchyme with an increased density of solenia. In contrast to scleractinians, soft coral GAs displayed an inflammatory and necrotizing component with marked edema of the mesoglea, accompanied by infiltrates of variably-sized granular amoebocytes. Fungi, algae, sponges, and Crustacea were present in some scleractinian GAs, but absent in soft coral GAs. Fragmentation of tissues was a common finding in Acropora acuminata and Montipora cf. dilatata colonies with tissue loss, although no obvious causative agents were seen. Discoloration in the zoanthid, Palythoa tuberculosa, was found to be the result of necrosis, while in Lobophytum sp. discoloration was the result of zooxanthellar depletion (bleaching). Soft corals with discoloration or tissue loss showed a marked inflammatory response, however no obvious causative organisms were seen. Lesions that appeared similar at the gross level were revealed to be distinct by microscopy, emphasizing the importance of histopathology.