Lipid profiling in chilled coral larvae.

Coral reefs are disappearing worldwide as a result of several harmful human activities. The establishment of cryobanks can secure a future for these ecosystems. To design effective cryopreservation protocols, basic proprieties such as chilling tolerance and lipid content must be assessed. In the present study, we investigated chilling sensitivity and the effect of chilling exposure on the lipid content and composition of larvae belonging to 2 common Indo-Pacific corals: Seriatopora caliendrum and Pocillopora verrucosa. The viability of coral larvae incubated with 0.5, 1, and 2 M ethylene glycol (EG), propylene glycol (PG), dimethyl sulfoxide (Me2SO), methanol, or glycerol and kept at 5 °C for different time periods was documented. In addition, we investigated the content of cholesterol, triacylglycerol (TAG), wax ester (WE), sterol ester (SE), lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, and several fatty acid (FA) classes in coral propagules incubated with 1 M PG or EG and kept at 5 °C for 6 h. Moreover, we examined seasonal changes in the aforementioned lipid classes in coral larvae. S. caliendrum incubated with 0.5 M PG or Me2SO and chilled for 2 h exhibited a viability rate of 11 ± 11%, whereas P. verrucosa exhibited a viability rate of 22 ± 14% after being chilled for 4 h. Furthermore, the results indicated that chilling exposure did not affect the content of any investigated lipid class in either species. The higher concentration of SE in P. verrucosa compared to S. caliendrum larvae may have contributed to the different cryotolerance displayed by the 2 larval species. A year-round lipid analysis of both coral larvae species revealed trends of homeoviscous adaptation and seasonal enhancement of lipid fluxes from symbionts to the host. During winter, the cholesterol/phospholipid ratio significantly increased, and P. verrucosa larvae exhibited an averagely decrease in FA chain lengths. During spring and summer, intracellular lipid content in the form of TAGs and WEs significantly increased in both species, and the average content of Symbiodiniaceae-derived FAs increased in P. verrucosa larvae. We concluded that the low cryotolerance displayed by S. caliendrum and P. verrucosa larvae is attributable to their chilling-sensitive membrane lipid profile and the high intracellular lipid content provided by their endosymbionts.

Supplementation of exogenous lipids via liposomes improves coral larvae settlement post-cryopreservation and nano-laser warming.

Coral reefs worldwide are receding because of detrimental human activities, and cryopreservation of coral larvae would ensure that their genetic biodiversity is not irremediably lost. In recent years, the vitrification and laser warming of coral propagules has demonstrated promising results. During cryopreservation, cellular membranes undergo substantial reconfigurations that may affect survival. Fat enrichment may alter the physical proprieties of cell membranes and improve resistance to low temperatures. Therefore, the aim of this study was to determine whether supplementation of exogenous lipids using liposomes would improve cryosurvival and further development of the vitrified and laser-warmed coral larvae of Seriatopora caliendrum and Pocillopora verrucosa. A vitrification solution (VS) composed of 2 M ethylene glycol (EG), 1 M propylene glycol (PG), 40% (w/v) Ficoll, and 10% gold nanoparticles (at a final concentration of 1.2 × 1018 particles/m3 and an optimised emission wavelength of 535 nm) was chosen. Coral larvae were subjected to vitrification with VS incorporating one of four lipid classes: phosphatidylcholine (PC), phosphatidylethanolamine (PE), erucic acid (EA), and linoleic acid (LA). Warming was achieved using a single laser pulse (300 V, 10 ms pulse width, 2 mm laser beam diameter). A significantly higher vitality rate was observed in S. caliendrum larvae subjected to vitrification and laser warming with EA-incorporated VS, and P. verrucosa larvae vitrified and laser warmed using PE-incorporated VS achieved a significantly higher settlement rate. Our study demonstrated that supplementation of exogenous lipids with liposomes enhances coral larvae cryotolerance and improves cryopreservation outcomes. Lipid enrichment may play a key role in cryobanking coral propagules, and in propagule development after thawing.

Different strategies of energy storage in cultured and freshly isolated Symbiodinium sp.

The endosymbiotic relationship between cnidarians and Symbiodinium is critical for the survival of coral reefs. In this study, we developed a protocol to rapidly and freshly separate Symbiodinium from corals and sea anemones. Furthermore, we compared these freshly-isolated Symbiodinium with cultured Symbiodinium to investigate host and Symbiodinium interaction. Clade B Symbiodinium had higher starch content and lower lipid content than those of clades C and D in both freshly isolated and cultured forms. Clade C had the highest lipid content, particularly when associated with corals. Moreover, the coral-associated Symbiodinium had higher protein content than did cultured and sea anemone-associated Symbiodinium. Regarding fatty acid composition, cultured Symbiodinium and clades B, C, and D shared similar patterns, whereas sea anemone-associated Symbiodinium had a distinct pattern compared coral-associated Symbiodinium. Specifically, the levels of monounsaturated fatty acids were lower than those of the saturated fatty acids, and the level of polyunsaturated fatty acids (PUFAs) were the highest in all examined Symbiodinium. Furthermore, PUFAs levels were higher in coral-associated Symbiodinium than in cultured Symbiodinium. These results altogether indicated that different Symbiodinium clades used different energy storage strategies, which might be modified by hosts.

New Scalarane Sesterterpenoids from the Formosan Sponge Ircinia felix.

Five new scalarane sesterterpenoids, felixins A-E (1-5), were isolated from the Formosan sponge Ircinia felix. The structures of scalaranes 1-5 were elucidated on the basis of spectroscopic analysis. Cytotoxicity of scalaranes 1-5 against the proliferation of a limited panel of tumor cell lines was evaluated.

New Cytotoxic 24-Homoscalarane Sesterterpenoids from the Sponge Ircinia felix.

Two new 24-homoscalarane sesterterpenoids, felixins F (1) and G (2), were isolated from the sponge Ircinia felix. The structures of new homoscalaranes 1 and 2 were elucidated by extensive spectroscopic methods, particularly with one-dimensional (1D) and two-dimensional (2D) NMR, and, by comparison, the spectral data with those of known analogues. The cytotoxicity of 1 and 2 against the proliferation of a limited panel of tumor cell lines was evaluated and 1 was found to show cytotoxicity toward the leukemia K562, MOLT-4, and SUP-T1 cells (IC50 ≤ 5.0 µM).

Organic ultraviolet filter mixture promotes bleaching of reef corals upon the threat of elevated seawater temperature.

Global reef degradation is a critical environmental health issue that has triggered intensive research on ocean warming, but the implications of emerging contaminants in coral habitats are largely overlooked. Laboratory experiments assessing organic ultraviolet (UV) filter exposure have shown that these chemicals negatively affect coral health; their ubiquitous occurrence in association with ocean warming may pose great challenges to coral health. We investigated both short- (10-day) and long-term (60-day) single and co-exposures of coral nubbins to environmentally relevant organic UV filter mixtures (200 ng/L of 12 compounds) and elevated water temperatures (30 °C) to investigate their effects and potential mechanisms of action. The initial 10-day exposure of Seriatopora caliendrum resulted in bleaching only under co-exposure conditions (compounds + temperature). The 60-day mesocosm study entailed the same exposure settings with nubbins of three species (S. caliendrum, Pocillopora acuta and Montipora aequituberculata). Bleaching (37.5 %) and mortality (12.5 %) of S. caliendrum were observed under UV filter mixture exposure. In the co-exposure treatment, 100 % S. caliendrum and P. acuta bleached associating with 100 % and 50 % mortality, respectively, and significant increase of catalase activities in P. acuta and M. aequituberculata nubbins were found. Biochemical and molecular analyses indicated significant alteration of oxidative stress and metabolic enzymes. The results suggest that upon the adverse effects of thermal stress, organic UV filter mixture at environmental concentrations can cause bleaching in corals by inducing a significant oxidative stress and detoxification burden, suggesting that emerging contaminants may play a unique role in global reef degradation.

Natural product chemistry of gorgonian corals of the family Plexauridae distributed in the Indo-Pacific Ocean.

The structures, names, bioactivities and references of 105 natural products obtained from gorgonian corals belonging to the family Plexauridae with an Indo-Pacific distribution are described in this review. All compounds mentioned in this review were obtained from gorgonian corals belonging to the genera Astrogorgia, Bebryce, Echinomuricea, Euplexaura and Menella.

The characteristics of host lipid body biogenesis during coral-dinoflagellate endosymbiosis.

Intracellular lipid body (LB) biogenesis depends on the symbiosis between coral hosts and their Symbiodinaceae. Therefore, understanding the mechanism(s) behind LB biosynthesis in corals can portentially elucide the drivers of cellular regulation during endosymbiosis. This study assessed LB formation in the gastrodermal tissue layer of the hermatypic coral Euphyllia glabrescens. Diel rhythmicity in LB size and distribution was observed; solar irradiation onset at sunrise initiated an increase in LB formation, which continued throughout the day and peaked after sunset at 18:00. The LBs migrated from the area near the mesoglea to the gastrodermal cell border near the coelenteron. Micro-LB biogenesis occurred in the endoplasmic reticulum (ER) of the host gastrodermal cells. A transcriptomic analysis of genes related to lipogenesis indicated that binding immunoglobulin protein (BiP) plays a key role in metabolic signaling pathways. The diel rhythmicity of LB biogenesis was correlated with ER-localized BiP expression. BiP expression peaked during the period with the largest increase in LB formation, thereby indicating that the chaperoning reaction of abnormal protein folding inside the host ER is likely involved in LB biosynthesis. These findings suggest that the host ER, central to LB formation, potentially facilitates the regulation of endosymbiosis between coral hosts and Symbiodiniaceae.

Proteomic analysis of symbiosome membranes in Cnidaria-dinoflagellate endosymbiosis.

Symbiosomes are specific intracellular membrane-bound vacuoles containing microalgae in a mutualistic Cnidaria (host)-dinoflagellate (symbiont) association. The symbiosome membrane is originally derived from host plasma membranes during phagocytosis of the symbiont; however, its molecular components and functions are not clear. In order to investigate the protein components of the symbiosome membranes, homogenous symbiosomes were isolated from the sea anemone Aiptasia pulchella and their purities and membrane intactness examined by Western blot analysis for host contaminants and microscopic analysis using various fluorescent probes, respectively. Pure and intact symbiosomes were then subjected to biotinylation by a cell impermeant agent (Biotin-XX sulfosuccinimidyl ester) to label membrane surface proteins. The biotinylated proteins, both Triton X-100 soluble and insoluble fractions, were subjected to 2-D SDS-PAGE and identified by MS using an LC-nano-ESI-MS/MS. A total of 17 proteins were identified. Based on their different subcellular origins and functional categories, it indicates that symbiosome membranes serve as the interface for interaction between host and symbiont by fulfilling several crucial cellular functions such as those of membrane receptors/cell recognition, cytoskeletal remodeling, ATP synthesis/proton homeostasis, transporters, stress responses/chaperones, and anti-apoptosis. The results of proteomic analysis not only indicate the molecular identity of the symbiosome membrane, but also provide insight into the possible role of symbiosome membranes during the endosymbiotic association.

Norterpenoids and related peroxides from the formosan marine sponge Negombata corticata.

Six norterpenes including negombatoperoxides A and B (4 and 5), the inseparable epimers negombatoperoxides C and D (6 and 7), negombatodiol (8), and negombatolactone (9), in combination with three known compounds, (+)-nuapapuin B (1), (+)-nuapapuin B methyl ester (2), and (+)-aikupikoxide C (3), were isolated from the Formosan marine sponge Negombata corticata. In addition, 6,6-dimethylundecane-2,5,10-trione (10) was isolated for the first time from a natural source. Their structures, including relative configurations, were elucidated on the basis of interpretation of spectroscopic data and by the application of the empirical rule established by Capon and MacLeod. The absolute configurations of 8 and 9 were established by the application of Mosher's method and comparison of CD data with known lactones, respectively. Cytotoxicity of these isolates against human breast carcinoma, human liver carcinoma, and human lung carcinoma cell lines was evaluated.

Author Correction: The effects of aquarium culture on coral oocyte ultrastructure.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Cryopreservation of a Thermotolerant Lineage of the Coral Reef Dinoflagellate Symbiodinium.

Dinoflagellates of the genus Symbiodinium form symbiotic relationships with corals, other marine invertebrates, and protists; thus, they are considered as important species in coral reef ecosystems. If Symbiodinium could be successfully cryopreserved, the cell bank generated could prove to be a valuable resource for researchers interested in basic biological research of Symbiodinium-invertebrate symbioses. Herein, successful cryopreservation of clade D Symbiodinium was achieved using a two-step freezing protocol. Symbiodinium cells were exposed to cryoprotectants (CPAs) for 30 minutes before being vapor frozen for 20 minutes in liquid nitrogen (LN2); afterward, cells were immediately immersed in LN2 for 2 hours or 10 days. The initial experiment was conducted with the following CPAs at 1, 2, and 3 M concentrations: methanol, dimethyl sulfoxide, glycerol, ethylene glycol (EG), and propylene glycol (PG). It was found that infiltration with 2 M EG and PG yielded cells with the highest percentage viability. Upon thawing, culture of these Symbiodinium was carried out for 2 months in a growth chamber, and cells continued to grow and proliferate over this period. This represents successful cryopreservation of a dominant reef coral symbiont, a feat that will ideally aid in future research of this important lineage of dinoflagellate.

Comparative toxicities of four benzophenone ultraviolet filters to two life stages of two coral species.

The benzophenone (BP) organic ultraviolet (UV) filters have been measured in seawater at ng/L to µg/L levels, but more data on their effects in non-target marine organisms are needed. Corals can be exposed to BPs due to wastewater discharges and coastal recreational activities. In this study, toxicities and bioaccumulation of BP-1 (2,4-dihydroxybenzophenone), BP-3 (oxybenzone), BP-4 (sulisobenzone) and BP-8 (dioxybenzone) to larvae and adults of two coral species, Pocillopora damicornis and Seriatopora caliendrum, were assessed at concentrations ranging from 0.1-1000 µg/L. BP-1 and BP-8 exposure caused significant settlement failure, bleaching and mortality of S. caliendrum larvae [lowest observed effect concentration (LOEC): ≥10 µg/L] compared to the other BPs, while none of the tested compounds and concentrations affected P. damicornis larvae. Nubbins were more sensitive to BP-3, BP-1 and BP-8 than larvae. Overall, BP-1 and BP-8 were more toxic to the two tested species than BP-3 and BP-4, which matches the relative bioaccumulation potential of the four BPs (BP-8 > BP-1 ≈ BP-3 > BP-4). A conservative risk assessment using the effect concentrations derived from this study showed that BP-3, BP-1 and BP-8 pose high or medium risk to the health of corals in popular recreational areas of Taiwan and Hong Kong. Our study suggests that future ecotoxicological studies of corals should take their sensitivities, life stages and metabolic capacities into consideration.

First instance of settlement by cryopreserved coral larvae in symbiotic association with dinoflagellates.

Coral reefs are suffering on a global scale due to human impacts, thereby necessitating cryopreservation efforts. The objective of this study was to develop a suitable vitrification and laser warming protocol for larvae of the scleractinian coral Seriatopora caliendrum, which inherit their dinoflagellate algal symbionts vertically. Toxicity experiments were conducted with the cryoprotectants (CPAs) ethylene glycol (EG), propylene glycol (PG), dimethyl sulfoxide (DMSO), glycerol (GLY), and methanol (METH; listed in order from least to most toxic), and larvae were subjected to vitrification and laser warming using 2 M EG + 1 M PG and 2 M EG + 1 M DMSO. Vitrification and laser warming (300 V, 10 ms pulse width, 2 mm beam diameter) using a vitrification solution of 2 M EG + 1 M PG, 40% w/v Ficoll, and 10% v/v gold nanobars (GNB) at a final concentration of 1.2 × 1018 GNB/mL and a characteristic wavelength of 535 nm resulted in larvae with vitality and settlement percentages of 55 and 9%, respectively. This represents the first successful instance of cryopreservation of coral larvae that proceeded to settle upon warming, and suggests that the vitrification and ultra-fast laser warming approach may be applicable to other threatened marine species.

Toxicological effects of two organic ultraviolet filters and a related commercial sunscreen product in adult corals.

Corals are exposed to organic ultraviolet (UV) filters and other personal care product (PCP) ingredients in the environment, but the toxicities of organic UV filters and their related PCP to corals are not well understood. In this study, 7-day exposures were conducted to evaluate the toxicities and bioaccumulation of two organic UV filters, ethylhexylmethoxy-cinnamate (EHMC; octinoxate) and octocrylene (OC) (single- and combined-chemical tests), and diluted sunscreen wash-off water containing both active ingredients to the adult life stage of two hard coral species, Seriatopora caliendrum and Pocillopora damicornis. In the single-chemical tests, death (33.3%) and bleaching (83.3%) were only observed in the 1000 µg/L EHMC treatment of S. caliendrum. In the sunscreen product exposures, 5% sunscreen water (containing 422.34 ±â€¯37.34 µg/L of EHMC and 33.50 ±â€¯7.60 µg/L of OC at Day 0) caused high mortality in S. caliendrum (66.7-83.3%) and P. damicornis (33.3-50%), and tissue concentrations were up to 10 times greater than in the single-chemical exposures; co-exposure to EHMC and OC at similar levels to those in the sunscreen product resulted in bioaccumulation similar to the single-chemical tests. These results confirm the bioaccumulation potential of EHMC and OC and show that other ingredients in sunscreen products may increase the bioavailability of active ingredients to corals and exacerbate the toxicity of sunscreen products. Future studies on the toxicities of PCPs to aquatic organisms should not only focus on the toxicities of active ingredients.

The effects of aquarium culture on coral oocyte ultrastructure.

As the world's oceans are currently threatened by anthropogenic pollution and climate change, coral breeding has become an important conservation method, since it can limit marine organisms' exposure to sub-optimal environment conditions. However, the aquarium environment is inherently different from the ocean, and this could manifest in physiological changes in the reared organisms, particularly with respect to their reproduction. Therefore, the aim of this study was to observe and compare the ultrastructure of the oocytes from wild Oxypora lacera and Echinopora gemmacea with the oocytes from cultured corals using transmission electron microscope. The oocytes from Wild O. lacera and E. gemmacea were larger than cultured ones, though their microvillus layers were significantly thiner. Internally, lipid granule areas and yolk material density in the oocytes of wild O. lacera and E. gemmacea were ~25% lower than in their cultured counterparts. Food availability and the presence and availability of symbiotic dinoflagellates (genus Symbiodinium) may have played a role in driving these lipid-based differences, in particular, as cultured corals had limited potential for heterotrophic feeding. These data will aid in future coral husbandry efforts.

Generation of clade- and symbiont-specific antibodies to characterize marker molecules during Cnidaria-Symbiodinium endosymbiosis.

The endosymbiosis between cnidarians and dinoflagellates is responsible for the formation of coral reefs. Changes in molecules have been identified during the process of cnidaria-Symbiodinium endosymbiosis. However, the complexity of the molecular interaction has prevented the establishment of a mechanistic explanation of cellular regulation in this mutualistic symbiosis. To date, no marker molecules have been identified to specifically represent the symbiotic status. Because the endosymbiotic association occurs in the symbiotic gastrodermal cells (SGCs), whole cells of isolated SGCs were used as an antigen to generate monoclonal antibodies (mAb) to screen possible molecular candidates of symbiotic markers. The results showed that one of the generated monoclonal antibodies, 2-6F, specifically recognized clade C symbiotic Symbiodinium but not its free-living counterpart or other Symbiodinium clades. The expression levels of 2-6F mAb-recognized proteins are highly correlated with the symbiotic status, and these proteins were characterized as N-linked glycoproteins via treatment with peptide N-glycosidase F. Furthermore, their glycan moieties were markedly different from those of free-living Symbiodinium, potentially suggesting host regulation of post-translational modification. Consequently, the 2-6F mAb can be used to detect the symbiotic state of corals and investigate the complex molecular interactions in cnidaria-Symbiodinium endosymbiosis.

Coral lipid bodies as the relay center interconnecting diel-dependent lipidomic changes in different cellular compartments.

Lipid bodies (LBs) in the coral gastrodermal tissues are key organelles in the regulation of endosymbiosis and exhibit a diel rhythmicity. Using the scleractinian Euphyllia glabrescens collected across the diel cycle, we observed temporally dynamic lipid profiles in three cellular compartments: host coral gastrodermal cells, LBs, and in hospite Symbiodinium. Particularly, the lipidome varied over time, demonstrating the temporally variable nature of the coral-Symbiodinium endosymbiosis. The lipidome-scale data highlight the dynamic, light-driven metabolism of such associations and reveal that LBs are not only lipid storage organelles but also act as a relay center in metabolic trafficking. Furthermore, lipogenesis in LBs is significantly regulated by coral hosts and the lipid metabolites within holobionts featured predominantly triacylglycerols, sterol esters, and free fatty acids. Given these findings through a time-varied lipidome status, the present study provided valuable insights likely to be crucial to understand the cellular biology of the coral-Symbiodinium endosymbiosis.

Influence of temperature and gonadal steroids on the ontogenetic expression of brain serotonin 1A and 1D receptors during the critical period of sexual differentiation in tilapia, Oreochromis mossambicus.

cDNA sequences of serotonin (5-hydroxytryptamine, 5-HT) 1A and 1D receptors were cloned from the tilapia, Oreochromis mossambicus, brain. The influence of both gonadal steroids and temperature on the ontogenetic expression of brain 5-HT1A and 5-HT1D receptors from days 5 to 15 post-hatch, a critical period of sexual differentiation, was investigated using quantitative real-time reverse transcription-polymerase chain reaction. Neither estrogen nor methyltestosterone had an effect on the ontogenetic expression of 5-HT1A or 5-HT1D receptors. Between days 5 and 10 post-hatch, a critical period for low-temperature-induced feminization, we found no significant difference in the ontogenetic expression of 5-HT1A between exposure to low and elevated temperature. A similar result was found for 5-HT1D. Between days 10 and 15 post-hatch, a critical period for elevated-temperature-induced masculinization, the ontogenetic expression of neither brain 5-HT1A nor 5-HT1D was altered by exposure to elevated temperature. These results suggest that neither brain 5-HT1A nor 5-HT1D plays a critical role in either gonadal steroid- or temperature-induced sexual differentiation.

Cryopreservation of the gorgonian endosymbiont Symbiodinium.

The study focused on finding a suitable cryoprotectant (CPA) and an optimum freezing protocol for the cryopreservation of the endosymbiotic dinoflagellates (Symbiodinium, clade G) of Junceella fragilis wherein the success of experiments is crucial to both scientific and ecology studies. A two-step freezing technique was developed. The viability of the thawed dinoflagellates was assayed using the adenosine triphosphate (ATP) bioassay for the first time and was further confirmed through the culturing of dinoflagellates in vitro. The results suggested that 30 min was the most suitable holding time for the dinoflagellates, and the samples produced highest viability when suspended at 5 cm from the surface of LN2. Results also showed that 1 M methanol with 0.4 M sucrose was the most effective CPA, yielding the highest viability (56.93%). Although cell densities of both cryopreserved and control group suffered an initial decline of culture, the cell densities were maintained throughout the remaining duration. In the present study, the cryopreservation of clade G endosymbiont algae was studied for the first time and the method described here could be applied for future studies on symbiotic algae cryopreservation.