Anderson B. Mayfield

Lipid bodies in coral–dinoflagellate endosymbiosis: Proteomic and ultrastructural studies

Gastrodermal lipid bodies (LBs) are organelles involved in the regulation of the mutualistic endosymbiosis between reef‐building corals and their dinoflagellate endosymbionts (genus Symbiodinium). As their molecular composition remains poorly defined, we herein describe the first gastrodermal LB proteome and examine in situ morphology of LBs in order to provide insight into their structure and function. After tissue separation of the tentacles of the stony coral Euphyllia glabrescens, buoyant LBs of the gastroderm encompassing a variety of sizes (0.5–4 μm in diameter) were isolated after two cycles of subcellular fractionation via stepwise sucrose gradient ultracentrifugation and detergent washing. The purity of the isolated LBs was demonstrated by their high degree of lipid enrichment and as well as the absence of contaminating proteins of the host cell and Symbiodinium. LB‐associated proteins were then purified, subjected to SDS‐PAGE, and identified by MS using an LC‐nano‐ESI‐MS/MS. A total of 42 proteins were identified within eight functional groups, including metabolism, intracellular trafficking, the stress response/molecular modification and development. Ultrastructural analyses of LBs in situ showed that they exhibit defined morphological characteristics, including a high‐electron density resulting from a distinct lipid composition from that of the lipid droplets of mammalian cells. Coral LBs were also characterized by the presence of numerous electron‐transparent inclusions of unknown origin and composition. Both proteomic and ultrastructural observations seem to suggest that both Symbiodinium and host organelles, such as the ER, are involved in LB biogenesis.

The effects of a variable temperature regime on the physiology of the reef-building coral Seriatopora hystrix: results from a laboratory-based reciprocal transplant

SUMMARY To understand the effects of global climate change on reef-building corals, a thorough investigation of their physiological mechanisms of acclimatization is warranted. However, static temperature manipulations may underestimate the thermal complexity of the reefs in which many corals live. For instance, corals of Houbihu, Taiwan, experience changes in temperature of up to 10°C over the course of a day during spring-tide upwelling events. To better understand the phenotypic plasticity of these corals, a laboratory-based experiment was conducted whereby specimens of Seriatopora hystrix from an upwelling reef (Houbihu) and conspecifics from a non-upwelling reef (Houwan) were exposed to both a stable seawater temperature (26°C) regime and a regime characterized by a 6°C fluctuation (23–29°C) over a 12 h period for 7 days. A suite of physiological and molecular parameters was measured in samples of both treatments, as well as in experimental controls, to determine site of origin (SO) and temperature treatment (TT) responses. Only chlorophyll a (chl a) concentration and growth demonstrated the hypothesized trend of higher levels when exposed to a TT that mimicked SO conditions. In contrast, chl a, maximum dark-adapted quantum yield of photosystem II (Fv/Fm), and Symbiodinium ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL), photosystem I (psI, subunit III) and phosphoglycolate phosphatase (pgpase) mRNA expression demonstrated significant TT effects. Specifically, levels of these response variables were higher in samples exposed to a variable temperature regime, suggesting that S. hystrix may acclimate to fluctuating temperatures by increasing its capacity for photosynthesis.

Assessing the Impacts of Experimentally Elevated Temperature on the Biological Composition and Molecular Chaperone Gene Expression of a Reef Coral

Due to the potential for increasing ocean temperatures to detrimentally impact reef-building corals, there is an urgent need to better understand not only the coral thermal stress response, but also natural variation in their sub-cellular composition. To address this issue, while simultaneously developing a molecular platform for studying one of the most common Taiwanese reef corals, Seriatopora hystrix, 1,092 cDNA clones were sequenced and characterized. Subsequently, RNA, DNA and protein were extracted sequentially from colonies exposed to elevated (30°C) temperature for 48 hours. From the RNA phase, a heat shock protein-70 (hsp70)-like gene, deemed hsp/c, was identified in the coral host, and expression of this gene was measured with real-time quantitative PCR (qPCR) in both the host anthozoan and endosymbiotic dinoflagellates (genus Symbiodinium). While mRNA levels were not affected by temperature in either member, hsp/c expression was temporally variable in both and co-varied within biopsies. From the DNA phase, host and Symbiodinium hsp/c genome copy proportions (GCPs) were calculated to track changes in the biological composition of the holobiont during the experiment. While there was no temperature effect on either host or Symbiodinium GCP, both demonstrated significant temporal variation. Finally, total soluble protein was responsive to neither temperature nor exposure time, though the protein/DNA ratio varied significantly over time. Collectively, it appears that time, and not temperature, is a more important driver of the variation in these parameters, highlighting the need to consider natural variation in both gene expression and the molecular make-up of coral holobionts when conducting manipulative studies. This represents the first study to survey multiple macromolecules from both compartments of an endosymbiotic organism with methodologies that reflect their dual-compartmental nature, ideally generating a framework for assessing molecular-level changes within corals and other endosymbioses exposed to changes in their environment.

Diel rhythmicity of lipid-body formation in a coral-Symbiodinium endosymbiosis

The biogenesis of intracellular lipid bodies (LBs) is dependent upon the symbiotic status between host corals and their intracellular dinoflagellates (genus Symbiodinium), though aside from this observation, little is known about LB behavior and function in this globally important endosymbiosis. The present research aimed to understand how LB formation and density are regulated in the gastrodermal tissue layer of the reef-building coral Euphyllia glabrescens. After tissue fixation and labeling with osmium tetroxide, LB distribution and density were quantified by imaging analysis of serial cryo-sections, and a diel rhythmicity was observed; the onset of solar irradiation at sunrise initiated an increase in LB density and size, which peaked at sunset. Both LB density and size then decreased to basal levels at night. On a seasonal timescale, LB density was found to be significantly positively correlated with seasonal irradiation, with highest densities found in the summer and lowest in the fall. In terms of LB lipid composition, only the concentration of wax esters, and not triglycerides or sterols, exhibited diel variability. This suggests that the metabolism and accumulation of lipids in LBs is at least partially light dependent. Ultrastructural examinations revealed that the LB wax ester concentration correlated with the number of electron-transparent inclusion bodies. Finally, there was a directional redistribution of the LB population across the gastroderm over the diel cycle. Collectively, these data reveal that coral gastrodermal LBs vary in composition and intracellular location over diel cycles, features which may shed light on their function within this coral–dinoflagellate mutualism.

Rubisco Expression in the Dinoflagellate Symbiodinium sp. Is Influenced by Both Photoperiod and Endosymbiotic Lifestyle

Although the importance of anthozoan-dinoflagellate (genus Symbiodinium) endosymbioses in the establishment of coral reef ecosystems is evident, little is known about the molecular regulation of photosynthesis in the intra-gastrodermal symbiont communities, particularly with respect to the rate-limiting Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco). In this study, we analyzed rubisco mRNA (rbcL) and protein (RBCL) concentrations over the diel cycle in both cultured and endosymbiotic Symbiodinium samples. In the former, rbcL expression increased upon illumination and decreased during the dark, a pattern that was upheld under continual dark incubation. A different trend in rbcL expression was observed in endosymbiotic Symbiodinium residing within sea anemone (Aiptasia pulchella) tissues, in which illumination gradually led to decreased rbcL mRNA expression. Unexpectedly, RBCL protein expression did not vary over time within anemone tissues, and in neither cultured nor endosymbiotic samples was a correlation between gene and protein expression documented. It appears, then, that photoperiod, lifestyle, and posttranscriptional regulation are all important drivers of RBCL expression in this ecologically important dinoflagellate.

Fatty acid and phospholipid syntheses are prerequisites for the cell cycle of Symbiodinium and their endosymbiosis within sea anemones.

Lipids are a source of metabolic energy, as well as essential components of cellular membranes. Although they have been shown to be key players in the regulation of cell proliferation in various eukaryotes, including microalgae, their role in the cell cycle of cnidarian-dinoflagellate (genus Symbiodinium) endosymbioses remains to be elucidated. The present study examined the effects of a lipid synthesis inhibitor, cerulenin, on the cell cycle of both cultured Symbiodinium (clade B) and those engaged in an endosymbiotic association with the sea anemone Aiptasia pulchella. In the former, cerulenin exposure was found to inhibit free fatty acid (FFA) synthesis, as it does in other organisms. Additionally, while it also significantly inhibited the synthesis of phosphatidylethanolamine (PE), it did not affect the production of sterol ester (SE) or phosphatidylcholine (PC). Interestingly, cerulenin also significantly retarded cell division by arresting the cell cycles at the G0/G1 phase. Cerulenin-treated Symbiodinium were found to be taken up by anemone hosts at a significantly depressed quantity in comparison with control Symbiodinium. Furthermore, the uptake of cerulenin-treated Symbiodinium in host tentacles occurred much more slowly than in untreated controls. These results indicate that FFA and PE may play critical roles in the recognition, proliferation, and ultimately the success of endosymbiosis with anemones.

A Compartmental Comparison of Major Lipid Species in a Coral-Symbiodinium Endosymbiosis: Evidence that the Coral Host Regulates Lipogenesis of Its Cytosolic Lipid Bodies.

The lipid body (LB) formation in the host coral gastrodermal cell cytoplasm is a hallmark of the coral-Symbiodinium endosymbiosis, and such lipid-based entities are not found in endosymbiont-free cnidarian cells. Therefore, the elucidation of lipogenesis regulation in LBs and how it is related to the lipid metabolism of the host and endosymbiont could provide direct insight to understand the symbiosis mechanism. Herein, the lipid composition of host cells of the stony coral Euphyllia glabrescens, as well as that of their cytoplasmic LBs and in hospite Symbiodinium populations, was examined by high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS), and six major lipid species were identified: wax esters, sterol esters, triacylglycerols, cholesterols, free fatty acids, and phospholipids. Their concentrations differed significantly between host coral cells, LBs, and Symbiodinium, suggesting compartmental regulation. WE were only present in the host coral and were particularly highly concentrated in LBs. Amongst the four species of WE, the monoene R = C18:1/R = C16 was found to be LB-specific and was not present in the host gastrodermal cell cytoplasm. Furthermore, the acyl pool profiles of the individual LB lipid species were more similar, but not equal to, those of the host gastrodermal cells in which they were located, indicating partially autonomous lipid metabolism in these LBs. Nevertheless, given the overall similarity in the host gastrodermal cell and LB lipid profiles, these data suggest that a significant portion of the LB lipids may be of host coral origin. Finally, lipid profiles of the in hospite Symbiodinium populations were significantly distinct from those of the cultured Symbiodinium, potentially suggesting a host regulation effect that may be fundamental to lipid metabolism in endosymbiotic associations involving clade C Symbiodinium.

The Impacts of Ex Situ Transplantation on the Physiology of the Taiwanese Reef-Building Coral Seriatopora hystrix

We sought to determine whether the Indo-Pacific reef-building coral Seriatopora hystrix performs in a similar manner in the laboratory as it does in situ by measuring Symbiodinium density, chlorophyll a (chl-a) concentration, and the maximum quantum yield of photosystem II () at the time of field sampling (in situ), as well as after three weeks of acclimation and one week of experimentation (ex situ). Symbiodinium density was similar between corals of the two study sites, Houbihu (an upwelling reef) and Houwan (a nonupwelling reef), and also remained at similar levels ex situ as in situ. On the other hand, both areal and cell-specific chl-a concentrations approximately doubled ex situ relative to in situ, an increase that may be due to having employed a light regime that differed from that experienced by these corals on the reefs of southern Taiwan from which they were collected. As this change in Symbiodinium chl-a content was documented in corals of both sites, the experiment itself was not biased by this difference. Furthermore, increased by only 1% ex situ relative to in situ, indicating that the corals maintained a similar level of photosynthetic performance as displayed in situ even after one month in captivity.

Biomarker profiling in reef corals of Tonga’s Ha’apai and Vava’u archipelagos

Given the significant threats towards Earth’s coral reefs, there is an urgent need to document the current physiological condition of the resident organisms, particularly the reef-building scleractinians themselves. Unfortunately, most of the planet’s reefs are understudied, and some have yet to be seen. For instance, the Kingdom of Tonga possesses an extensive reef system, with thousands of hectares of unobserved reefs; little is known about their ecology, nor is there any information on the health of the resident corals. Given such knowledge deficiencies, 59 reefs across three Tongan archipelagos were surveyed herein, and pocilloporid corals were sampled from approximately half of these surveyed sites; 10 molecular-scale response variable were assessed in 88 of the sampled colonies, and 12 colonies were found to be outliers based on employment of a multivariate statistics-based aberrancy detection system. These outliers differed from the statistically normally behaving colonies in having not only higher RNA/DNA ratios but also elevated expression levels of three genes: 1) Symbiodinium zinc-induced facilitator-like 1-like, 2) host coral copper-zinc superoxide dismutase, and 3) host green fluorescent protein-like chromoprotein. Outliers were also characterized by significantly higher variation amongst the molecular response variables assessed, and the response variables that contributed most significantly to colonies being delineated as outliers differed between the two predominant reef coral species sampled, Pocillopora damicornis and P. acuta. These closely related species also displayed dissimilar temporal fluctuation patterns in their molecular physiologies, an observation that may have been driven by differences in their feeding strategies. Future works should attempt to determine whether corals displaying statistically aberrant molecular physiology, such as the 12 Tongan outliers identified herein, are indeed characterized by a diminished capacity for acclimating to the rapid changes in their abiotic milieu occurring as a result of global climate change.

Identifying corals displaying aberrant behavior in Fiji’s Lau Archipelago

Abstract Given the numerous threats against Earth’s coral reefs, there is an urgent need to develop means of assessing reef coral health on a proactive timescale. Molecular biomarkers may prove useful in this endeavor because their expression should theoretically undergo changes prior to visible signs of health decline, such as the breakdown of the coral-dinoflagellate (genus Symbiodinium) endosymbiosis. Herein 13 molecular- and physiological-scale biomarkers spanning both eukaryotic compartments of the anthozoan-Symbiodinium mutualism were assessed across 70 pocilloporid coral colonies sampled from reefs of Fiji’s easternmost province, Lau. Eleven colonies were identified as outliers upon employment of a detection method based partially on the Mahalanobis distance; these corals were hypothesized to have been displaying aberrant sub-cellular behavior with respect to their gene expression signatures, as they were characterized not only by lower Symbiodinium densities, but also by higher levels of expression of several stress-targeted genes. Although these findings could suggest that the sampled colonies were physiologically compromised at the time of sampling, further studies are warranted to state conclusively whether these 11 scleractinian coral colonies are more stress-prone than nearby conspecifics that demonstrated statistically normal phenotypes.