Macroalgal-associated dinoflagellates belonging to the genus Symbiodinium in Caribbean reefs

Coral-algal symbiosis has been a subject of great attention during the last two decades in response to global coral reef decline. However, the occurrence and dispersion of free-living dinoflagellates belonging to the genus Symbiodinium are less documented. Here ecological and molecular evidence is presented demonstrating the existence of demersal free-living Symbiodinium populations in Caribbean reefs and the possible role of the stoplight parrotfish (Sparisoma viride) as Symbiodinium spp. dispersers. Communities of free-living Symbiodinium were found within macroalgal beds consisting of Halimeda spp., Lobophora variegata, Amphiroa spp., Caulerpa spp. and Dictyota spp. Viable Symbiodinium spp. cells were isolated and cultured from macroalgal beds and S. viride feces. Further identification of Symbiodinium spp. type was determined by length variation in the Internal Transcribed Spacer 2 (ITS2, nuclear rDNA) and length variation in domain V of the chloroplast large subunit ribosomal DNA (cp23S-rDNA). Determination of free-living Symbiodinium and mechanisms of dispersal is important in understanding the life cycle of Symbiodinium spp.     

Thylakoid differentiation in endocyanelles

Cyanophora paradoxa Korshikov synchronized autotrophically in a light-dark regime of 14 h light and 10 h dark divides in the last two hours of the dark period. The division rate of the free-living blue-green alga, Synechococcus leopoliensis Raciborski, at identical culture conditions (24°C; 32 W m⁻²) is only slightly lowered in the light period. The comparison of thylakoid differentiation in the endocyanelles of Cyanophora paradoxa and in Synechococcus leopoliensis during the light-dark regime yields (1) the same ensemble of pigment-protein complexes in both organisms, (2) comparable syntheses of chlorophyll and phycobilins of Cyanophora paradoxa grown under 32 W m⁻² and of Synechococcus leopoliensis grown under light intensities below 9.2 W m⁻², and (3) identical photosynthetic oxygen evolution during the light period of the light-dark regime with minima at the beginning, in the middle (6th–7th h), and at the end of the light period. In both organisms this stage-specific oxygen evolution is inhibited by treatment with chloroamphenicol. Cycloheximide, however, causes no significant alterations. Results are discussed in view of the endosymbiotic theory.     

Immunolocalization and Western Blot Analysis of Nitrogenase in Oscillatoria limosa During a Light-dark Cycle

Nitrogenase of the non-heterocystous nitrogen-fixing cyanobacterium Oscillatoria limosa was subjected to western blot analysis and immunogold electron microscopy using antisera raised against dinitrogenase (MoFe-protein, Component I) and dinitrogenase reductase (Fe-protein, Component II). O. limosa was grown diazotrophically under an alternating light-dark cycle (16–8h light-dark). Although nitrogenase activity (acetylene reduction) was found predominantly during the dark phase, being absent during most of the light period, immunogold electron microscopy revealed label of both subunits of nitrogenase in samples taken throughout the light-dark cycle. It was also shown that the nitrogenase label was distributed homogeneously in the cell and that it was present in every cell of every trichome whether fixing nitrogen or not. On average, 34 (± 6) gold particles μm^?2 thin section were detected. Nitrate-grown cells did not contain nitrogenase label. Western blot analysis of the Fe-protein in samples taken during the light phase, revealed a single band with an apparent molecular weight of 37 kDa. At the end of the light period, and during the dark phase when high nitrogenase activities were observed, an additional band of 36 kDa was found. The anti-MoFe-protein antiserum revealed a single band of 56 kDa which was present throughout the light-dark cycle. Nitrate-grown cells were not recognized by either antiserum. It is concluded that nitrogenase enzyme is present in O. limosa throughout the light-dark cycle but that the Fe-protein is modified (inactive form) during the light period when nitrogenase activity is absent.     

Highly infectious symbiont dominates initial uptake in coral juveniles

The majority of reef-building corals acquire their obligate algal symbionts ( Symbiodinium ) from the environment. However, factors shaping the initial establishment of coral–algal symbioses, including parental effects, local environmental conditions and local availability of symbionts, are not well understood. This study monitored the uptake and maintenance of Symbiodinium in juveniles of two common corals, Acropora tenuis and Acropora millepora , that were reciprocally explanted between sites where adult colonies host different types of Symbiodinium . We found that coral juveniles were rapidly dominated by type D Symbiodinium , even though this type is not found in adult colonies (including the parental colonies) in four out of the five study populations. Furthermore, type D Symbiodinium was found in less than one-third of a wide range of coral species ( n  > 50) sampled at the two main study sites, suggesting that its dominance in the acroporid juveniles is not because it is the most abundant local endosymbiotic type. Moreover, dominance by type D was observed irrespective of the light intensity to which juveniles were exposed in a field study. In summary, despite its relatively low abundance in coral assemblages at the study sites and irrespective of the surrounding light environment, type D Symbiodinium is the main symbiont type initially acquired by juveniles of A. millepora and A. tenuis . We conclude that during early ontogeny in these corals, there are few barriers to the uptake of Symbiodinium types which differ from those found in parental colonies, resulting in dominance by a highly infectious and potentially opportunistic symbiont.     

Symbiodinium clade C dominates zooxanthellate corals (Scleractinia) in the temperate region of Japan

Endosymbiotic algae of the genus Symbiodinium have been divided into nine clades (A-I) following genetic classification; some clades are known to have physiological properties that enable the coral hosts to adapt to different environmental conditions. To understand the relationships of coral-alga symbioses, we focused on Symbiodinium diversity in zooxanthellate corals living under the severe environmental conditions of the temperate region (30°-35°N) of Japan. We investigated Symbiodinium clades in 346 colonies belonging to 58 coral species from six locations. We then selected three coral species-Acropora hyacinthus, Acropora japonica, and Cyphastrea chalcidicum-to investigate whether Symbiodinium clades changed during winter or summer over the course of year (May 2009-Apr 2010) in Tanabe Bay, Japan. Three Symbiodinium clades (C, D, and F) were detected in corals in the temperate region. Notably, 56 coral species contained Symbiodinium clade C. Oulastrea crispata predominantly contained clade D, but traces of clade C were also detected in all samples. The temperate-specific species Alveopora japonica contained clades C and F simultaneously. Seasonal change of symbiont clades did not occur in the three coral species during the investigation period where SSTs range on 12.5-29.2°C. However, we found Acropora (2 spp.) and Cyphastrea (1 sp.) contained different subcladal types of clade C. These results reveal that most coral species harbored Symbiodinium clade C stably throughout the year, suggesting that Symbiodinium clade C shows low-temperature tolerance, and that two hypothetical possibilities; genetic differences of subcladal types generating physiological differences or wide physiological flexibility in the clade C.     

Recognizing diversity in coral symbiotic dinoflagellate communities

A detailed understanding of how diversity in endosymbiotic dinoflagellate communities maps onto the physiological range of coral hosts is critical to predicting how coral reef ecosystems will respond to climate change. Species-level taxonomy of the dinoflagellate genus Symbiodinium has been predominantly examined using the internal transcribed spacer (ITS) region of the nuclear ribosomal array (rDNA ITS2) and downstream screening for dominant types using denaturing gradient gel electrophoresis (DGGE). Here, ITS2 diversity in the communities of Symbiodinium harboured by two Hawaiian coral species was explored using direct sequencing of clone libraries. We resolved sixfold to eightfold greater diversity per coral species than previously reported, the majority of which corresponds to a novel and distinct phylogenetic lineage. We evaluated how these sequences migrate in DGGE and demonstrate that this method does not effectively resolve this diversity. We conclude that the Porites spp. examined here harbour diverse assemblages of novel Symbiodinium types and that cloning and sequencing is an effective methodological approach for resolving the complexity of endosymbiotic dinoflagellate communities harboured by reef corals.     

LIPID SYNTHESIS AND FATTY ACID COMPOSITION IN NANNOCHLOROPSIS SP. (EUSTIGMATOPHYCEAE) GROWN IN A LIGHT-DARK CYCLE1

Chemical composition and lipid biosynthesis were studied in the marine eustigmatophyte Nannochloropsis sp. Grown under a 12:12 h light-dark regime. Cellular division occurred in the dark and was associated with a reduction in cell volume. The cellular content of chlorophyll a and carotenoids increased during the light period and decreased during the dark period. Other cellular components, such as proteins, carbohydrates and lipids, followed a similar pattern. Nannochloropsis sp. Incorporated acetate, mostly into lipids during the light period, whereas a low rate of acetate incorporation was observed during the dark period, mostly into nonlipid compounds. Neutral lipids such as triacylglycerol were synthesized and accumulated in the light and showed a rapid turnover in the dark. Polar structural lipids such as monogalactosyl diacylglycerol were synthesized during the light period and hardly turned over during the dark period. Changes in lipid content were associated with variations in cellular fatty acid composition. The light period was characterized by an increase in the percentage of C16:0 and 16:1 fatty acids associated with triacylglycerols. However, in the dark period, as triglycerides were consumed for cellular maintenance, the relative distribution of the C20:5 fatty acid associated with the galactolipids increased.     

Microsatellite loci for assessing genetic diversity, dispersal and clonality of coral symbionts in 'stress-tolerant' clade D Symbiodinium

Reef corals harbouring clade D Symbiodinium spp. (endosymbiotic dinoflagellates) appear more tolerant of environmental stress. As sea surface temperatures rise, symbioses involving Symbiodinium D may increase in prevalence. For this reason, eight polymorphic microsatellite loci were developed for clade D Symbiodinium. From the analysis of 132 samples originating from cnidarian hosts in the Atlantic, Pacific and Indian Oceans, 4 to 35 alleles were found at each haploid locus and diversity indices ranged from 0.35 to 0.97. Population genetic analyses of these symbionts should reveal how environmental perturbations affect genetic diversity, geographical distributions, and possible host-range expansions to new coral species.     

Effect of Si-status on diel variation of intracellular free amino acids in Thalassiosira weissflogii under low-light intensity

Twenty-one intracellular free amino acids were analysed during a 12-12 h light-dark cycle, on duplicate axenic cultures of Thalassiosira weissflogii (clone Actin, Provasoli-Guillard CCMP) under either Si-sufficient or Si-starved conditions. Total concentrations ranged between 40 and 165 fmol/cell. Total level as well as individual levels of amino acids decreased during the dark period, and GLN/GLU ratio was lower during the dark period. All these results were correlated with the light-dark carbon metabolism of the algae and related to the protein synthesis at night. The Si-starved cultures showed a lower total level of FAA compare to the Si-sufficient cultures, especially in the light period. Silica status of the cells affected more the metabolites of the dark respiration than the photorespiratory metabolites SER and GLY. Si deprivation induced higher range of ALA and VAL, and a decrease of the TCA metabolites GLU & ASP. Additionally, the relative percentage of ASP increased under Si starvation, at the expense of GLU, and this shift was emphasized in the dark period.     

Influence of the quantity and quality of light on photosynthetic periodicity in coral endosymbiotic algae

Symbiotic corals, which are benthic organisms intimately linked with their environment, have evolved many ways to deal with fluctuations in the local marine environment. One possible coping mechanism is the endogenous circadian clock, which is characterized as free running, maintaining a ～24 h periodicity of circuits under constant stimuli or in the absence of external cues. The quantity and quality of light were found to be the most influential factors governing the endogenous clock for plants and algae. Unicellular dinoflagellate algae are among the best examples of organisms that exhibit circadian clocks using light as the dominant signal. This study is the first to examine the effects of light intensity and quality on the rhythmicity of photosynthesis in the symbiotic dinoflagellate Symbiodinium sp., both as a free-living organism and in symbiosis with the coral Stylophora pistillata. Oxygen production measurements in Symbiodinium cultures exhibited rhythmicity with a periodicity of approximately 24 h under constant high light (LL), whereas under medium and low light, the cycle time increased. Exposing Symbiodinium cultures and corals to spectral light revealed different effects of blue and red light on the photosynthetic rhythm, specifically shortening or increasing the cycle time respectively. These findings suggest that the photosynthetic rhythm is entrained by different light cues, which are wired to an endogenous circadian clock. Furthermore, we provide evidence that mRNA expression was higher under blue light for two potential cryptochrome genes and higher under red light for a phytochrome gene isolated from Symbiodinium. These results offer the first evidence of the impact of the intensity and quality of light on the photosynthetic rhythm in algal cells living freely or as part of a symbiotic association. Our results indicate the presence of a circadian oscillator in Symbiodinium governing the photosynthetic apparatus through a light-induced signaling pathway that has yet to be described.     

Studies on the biochemistry and fine structure of silica shell formation in diatoms

Summary Cell division in Navicula pelliculosa (Bréb.) Hilse, strain 668 was synchronized with an alternating regime of 5 h light and 7 h dark. Cell volume and dry weight increased only during the light period. DNA synthesis, which began during the third h of light, was followed sequentially by mitosis, cytokinesis, silicic acid uptake, cell wall formation, and cell separation. Silicification and a small amount of net synthesis of DNA, RNA and protein occurred during the dark at the expense of carbohydrate reserves accumulated during the light period. Cells kept in continuous light, after synchronization with the light-dark regime, remained synchronized through a second division cycle; the sequence of morphological events was the same as that in the light-dark division cycle, but the biosynthesis of macromolecular components changed from a stepwise to a linear pattern. The silicon-starvation synchrony was improved by depriving light-dark synchronized cells of silicic acid at the beginning of their division cycle, then resupplying silicic acid to cells blocked at wall formation.Abbreviation L light - D dark Portions based on a thesis submitted by W.M.D. to the University of California, San Diego in partial fulfillment of the requirements for the PH.D degree     

Stable Symbiodinium composition in the sea fan Gorgonia ventalina during temperature and disease stress

Like most Caribbean octocorals, Gorgonia ventalina, the common sea fan, harbors endosymbiotic dinoflagellates belonging to the genus Symbiodinium. When stressed, the host can lose these algal symbionts, a phenomenon termed "bleaching." Many cnidarians host multiple types of algal symbionts within the genus Symbiodinium, and certain types of algae may be more tolerant of stress than others. We examined the effects of temperature, temperature-induced bleaching, and infection by Aspergillus sydowii, a fungal pathogen, on Symbiodinium types harbored by the sea fan Gorgonia ventalina in the Florida Keys. Symbiont type, identified on the basis of variation in small subunit nuclear ribosomal genes or large subunit chloroplast ribosomal genes, did not vary with temperature treatment or infection status. Although allelic variation based on one microsatellite locus was found among samples and reef site, it did not consistently correlate with temperature, treatment, or disease status, suggesting that the symbiont-host relationship is stable. An aberrant PCR product was found in samples collected at one site and could be used to differentiate Symbiodinium populations among sites in the Florida Keys.     

A connection between colony biomass and death in Caribbean reef-building corals

Increased sea-surface temperatures linked to warming climate threaten coral reef ecosystems globally. To better understand how corals and their endosymbiotic dinoflagellates (Symbiodinium spp.) respond to environmental change, tissue biomass and Symbiodinium density of seven coral species were measured on various reefs approximately every four months for up to thirteen years in the Upper Florida Keys, United States (1994-2007), eleven years in the Exuma Cays, Bahamas (1995-2006), and four years in Puerto Morelos, Mexico (2003-2007). For six out of seven coral species, tissue biomass correlated with Symbiodinium density. Within a particular coral species, tissue biomasses and Symbiodinium densities varied regionally according to the following trends: Mexico≥Florida Keys≥Bahamas. Average tissue biomasses and symbiont cell densities were generally higher in shallow habitats (1-4 m) compared to deeper-dwelling conspecifics (12-15 m). Most colonies that were sampled displayed seasonal fluctuations in biomass and endosymbiont density related to annual temperature variations. During the bleaching episodes of 1998 and 2005, five out of seven species that were exposed to unusually high temperatures exhibited significant decreases in symbiotic algae that, in certain cases, preceded further decreases in tissue biomass. Following bleaching, Montastraea spp. colonies with low relative biomass levels died, whereas colonies with higher biomass levels survived. Bleaching- or disease-associated mortality was also observed in Acropora cervicornis colonies; compared to A. palmata, all A. cervicornis colonies experienced low biomass values. Such patterns suggest that Montastraea spp. and possibly other coral species with relatively low biomass experience increased susceptibility to death following bleaching or other stressors than do conspecifics with higher tissue biomass levels.     

Light-limited growth of two strains of the marine diatom Phaeodactylum tricornutum Bohlin: Chemical composition,carbon partitioning and the diel periodicity of physiological processes

Two isolates of the marine pennate diatom Phaeodactylum tricornutum Bohlin were grown in semi-continuous, nutrient-sufficient culture at varying irradiances on a 12-h light, 12-h dark illumination cycle. The reponse of the isolates to varying degrees of light limitation differed with respect to all of the compositional parameters measured, including growth rates, elemental composition, chlorophyll content, and the partitioning of cellular carbon into four biochemical classes: proteins, lipids, polysaccharides, and low-molecular weight intermediates. The isolates also differed with respect to the relative contributions of light-period and dark-period uptake to the total uptake of ammonium and phosphate ions, although in all cases uptake took place at a reduced rate in the dark. They did not differ with respect to the diel periodicity of cell division, chlorophyll synthesis, and biochemical synthesis. Slightly more cell division took place during the dark period than during the light period. The specific rate of chlorophyll synthesis in the light period, when expressed as a function of irradiance, saturated rapidly; the rate was nearly constant for all irradiances > 100 βE · m^?2 · s^?1. Chlorophyll synthesis in the dark was positively correlated with irradiance over the entire range of irradiances, except where photoinhibition was involved. Protein was synthesized in both the light and dark periods, but at a reduced rate in the dark. Polysaccharides were synthesized during the light period and consumed during the dark period. Lipids and low molecular weight intermediates were synthesized during the light period, but showed little net change during the dark period.     

Soft coral Sarcophyton (Cnidaria: Anthozoa: Octocorallia) species diversity and chemotypes

Research on the soft coral genus Sarcophyton extends over a wide range of fields, including marine natural products and the isolation of a number of cembranoid diterpenes. However, it is still unknown how soft corals produce this diverse array of metabolites, and the relationship between soft coral diversity and cembranoid diterpene production is not clear. In order to understand this relationship, we examined Sarcophyton specimens from Okinawa, Japan, by utilizing three methods: morphological examination of sclerites, chemotype identification, and phylogenetic examination of both Sarcophyton (utilizing mitochondrial protein-coding genes MutS homolog: msh1) and their endosymbiotic Symbiodinium spp. (utilizing nuclear internal transcribed spacer of ribosomal DNA: ITS- rDNA). Chemotypes, molecular phylogenetic clades, and sclerites of Sarcophyton trocheliophorum specimens formed a clear and distinct group, but the relationships between chemotypes, molecular phylogenetic clade types and sclerites of the most common species, Sarcophyton glaucum, was not clear. S. glaucum was divided into four clades. A characteristic chemotype was observed within one phylogenetic clade of S. glaucum. Identities of symbiotic algae Symbiodinium spp. had no apparent relation to chemotypes of Sarcophyton spp. This study demonstrates that the complex results observed for S. glaucum are due to the incomplete and complex taxonomy of this species group. Our novel method of identification should help contribute to classification and taxonomic reassessment of this diverse soft coral genus.     

Symbiodinium Photosynthesis in Caribbean Octocorals

Symbioses with the dinoflagellate Symbiodinium form the foundation of tropical coral reef communities. Symbiodinium photosynthesis fuels the growth of an array of marine invertebrates, including cnidarians such as scleractinian corals and octocorals (e.g., gorgonian and soft corals). Studies examining the symbioses between Caribbean gorgonian corals and Symbiodinium are sparse, even though gorgonian corals blanket the landscape of Caribbean coral reefs. The objective of this study was to compare photosynthetic characteristics of Symbiodinium in four common Caribbean gorgonian species: Pterogorgia anceps, Eunicea tourneforti, Pseudoplexaura porosa, and Pseudoplexaura wagenaari. Symbiodinium associated with these four species exhibited differences in Symbiodinium density, chlorophyll a per cell, light absorption by chlorophyll a, and rates of photosynthetic oxygen production. The two Pseudoplexaura species had higher Symbiodinium densities and chlorophyll a per Symbiodinium cell but lower chlorophyll a specific absorption compared to P. anceps and E. tourneforti. Consequently, P. porosa and P. wagenaari had the highest average photosynthetic rates per cm² but the lowest average photosynthetic rates per Symbiodinium cell or chlorophyll a. With the exception of Symbiodinium from E. tourneforti, isolated Symbiodinium did not photosynthesize at the same rate as Symbiodinium in hospite. Differences in Symbiodinium photosynthetic performance could not be attributed to Symbiodinium type. All P. anceps (n = 9) and P. wagenaari (n = 6) colonies, in addition to one E. tourneforti and three P. porosa colonies, associated with Symbiodinium type B1. The B1 Symbiodinium from these four gorgonian species did not cluster with lineages of B1 Symbiodinium from scleractinian corals. The remaining eight E. tourneforti colonies harbored Symbiodinium type B1L, while six P. porosa colonies harbored type B1i. Understanding the symbioses between gorgonian corals and Symbiodinium will aid in deciphering why gorgonian corals dominate many Caribbean reefs.     

Diel Pattern of Photosynthate Partitioning in Phytoplankton Population in an Oligomesotrophic Lake

The aim of this study was to measure the short-term changes in inorganic carbon allocation into various macromolecular compounds (proteins, polysaccharides and lipids) throughout a diurnal cycle in the oligomesotrophic Lake Pavin (Massif Central of France) at the depths of 5, 15 and 30 m. Biochemical fractionation was done by consecutive differential extractions in order to separate proteins, polysaccharides, lipids and low molecular weight compounds (LMW) by virtue of their relative solubilities in different extraction solvents. Over the entire diurnal cycle inorganic carbon was preferentially incorporated into proteins (M = 30%), then into polysaccharides (M = 28%), LMWs (M = 27%) and lipids (M = 15%). However, at 5 m, diurnal variations were reflected by the high percentage of the inorganic carbon incorporated into polysaccharides during periods of high light intensity and decreased at dawn and dusk. The reverse pattern was observed for the allocation of inorganic carbon to proteins.     

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.