The synthesis of mycosporine-like amino acids (MAAs) by cultured, symbiotic dinoflagellates

We tested the hypothesis that there is a relation between phylotypes (phylogenetic types, as determined by restriction fragment length polymorphism (RFLP) and partial sequence analysis of the small subunit ribosomal RNA gene (SSUrDNA)) and the synthesis of mycosporine-like amino acids (MAAs) by symbiotic dinoflagellates under the influence of ultraviolet radiation (UV-B/A) and photosynthetically active radiation (PAR). We exposed 27 isolates of symbiotic dinoflagellates simultaneously to UV-B/A and PAR, and subsequently determined the MAAs present in cell extracts and in the media. The algae used included 24 isolates of Symbiodinium spp. originating from jellyfishes, sea anemones, zoanthids, scleractinians, octocorals, and bivalves, and three others in the genera Gymnodinium, Gloeodinium and Amphidinium from a jellyfish, an hydrocoral and a flatworm, respectively. In this study, all of the phylotype A Symbiodinium spp. synthesized up to three identified MAAs. None of the 11 cultured phylotypes B and C Symbiodinium spp. synthesized MAAs. The three non-Symbiodinium symbionts also synthesized up to three MAAs. The results support a conclusion that phylotype A Symbiodinium spp. have a high predilection for the synthesis of MAAs, while phylotypes B and C do not. Synthesis of MAAs by symbiotic dinoflagellates in culture does not appear to relate directly to depths or to the UV exposure regimes from which the consortia were collected.     

SYMBIODINIUM (PYRRHOPHYTA) GENOME SIZES (DNA CONTENT) ARE SMALLEST AMONG DINOFLAGELLATES1

Using flow cytometric analysis of fluorescence, we measured the genome sizes of 18 cultured “free‐living” species and 29 Symbiodinium spp. isolates cultured from stony corals, gorgonians, anemones, jellyfish, and giant clams. Genome size directly correlated with cell size, as documented previously for most eukaryotic cell lines. Among the smallest of dinoflagellates, Symbiodinium spp. (6–15 μm) possessed the lowest DNA content that we measured (1.5–4.8 pg·cell^?1). Bloom‐forming or potentially harmful species in the genera Alexandrium, Karenia, Pfiesteria, and Prorocentrum possessed genomes approximately 2 to 50 times larger in size. A phylogenetic analysis indicated that genome/cell size has apparently increased and decreased repeatedly during the evolution of dinoflagellates. In contrast, genome sizes were relatively consistent across distantly and closely related Symbiodinium spp. This may be the product of intracellular host habitats imposing strong selective pressures that have restricted symbiont size.     

Uptake of phosphate by symbiotic and free-living dinoflagellates

Uptake of phosphate in the light by Amphidinium carterae, Amphidinium klebsii, cultured and symbiotic Gymnodinium microadriaticum conformed to Michaelis-Menten type saturation kinetics with all organisms showing similar K _m values, namely 0.005 to 0.016 M phosphorus. V _max values were 0.009–0.32 nmol phosphorus · 10⁵ cells^-1 · 10 min^-1. Phosphate uptake by all the dinoflagellates was greater in the dark than in the light. The metabolic inhibitor 3-(3,4-dichlorophenyl) 1,1-dimethylurea stimulated phosphate uptake in the light by A. carterae and A. klebsii, but inhibited uptake by cultured and symbiotic G. microadriaticum. Carbonylcyanide 3-chlorophenylhydrazone (CCCP) inhibited phosphate uptake by A. carterae and A. klebsii under both light and dark conditions. Uptake of phosphate by cultured and symbiotic G. microadriaticum in the light, but not in the dark, was inhibited by CCCP. Low concentrations of arsenate (5 g As · l^-1) stimulated phosphate by A. carterae and A. klebsii, but inhibited uptake by cultured and symbiotic G. microadriaticum. High concentrations of arsenate (100 g As · l^-1) did not affect uptake of phosphate by A. carterae and A. klebsii.     

SMALL SUBUNIT rDNA SEQUENCE ANALYSIS OF SYMBIOTIC DINOFLAGELLATES FROM SEVEN SCLERACTINIAN CORALS IN A TAHITIAN LAGOON

The diversity of symbiotic dinoflagellates from reef-building corals collected in the lagoon of Tahiti (South Pacific ocean) was investigated by using a molecular approach. Populations of symbionts (strains or species) of 7 coral species ( Fungia scutaria , F. paumotensis Stutchbury, Pavona cactus Forskål, Leptastrea transversa Kluzinger, Pocillopora verrucosa Ellis and Solender, Montastrea curta Dana, and Acropora formosa Dana) were delimited by phylogenetic analysis of small subunit rDNA sequences. Coral P. verrucosa harbored 2 populations of symbiont SSU rDNA sequences that may correspond to two different Symbiodinium species. Corals F. scutaria and M. curta also seemed to contain two different Symbiodinium species. SSU rDNA dinoflagellate sequences from P. cactus , L. transversa , F. scutaria , F. paumotensis , and P. verrucosa were in the same phylogenetic cluster and showed low variability. For these distantly related coral species, dinoflagellate strains from the same species, rDNA paralogues from the same strain, or closely related Symbiodinium species could not be distinguished because monophyletic subgroups were not observed. SSU rDNA dinoflagellate sequences from A. formosa and M. curta were clearly different from the other Symbiodinium sequences and may represent specific species. This molecular approach highlighted a greater diversity of symbiotic dinoflagellates from corals in South Pacific ( Symbiodinium groups A, B, and C) than that observed in the rest of the Pacific ocean ( Symbiodinium group C). The diversity of symbiotic associations in a restricted area of the lagoon of Tahiti may reflect the complexity of interactions between species of Symbiodinium and corals.     

Assessing Symbiodinium diversity in scleractinian corals via next‐generation sequencing‐based genotyping of the ITS2 rDNA region

The persistence of coral reef ecosystems relies on the symbiotic relationship between scleractinian corals and intracellular, photosynthetic dinoflagellates in the genus Symbiodinium. Genetic evidence indicates that these symbionts are biologically diverse and exhibit discrete patterns of environmental and host distribution. This makes the assessment of Symbiodinium diversity critical to understanding the symbiosis ecology of corals. Here, we applied pyrosequencing to the elucidation of Symbiodinium diversity via analysis of the internal transcribed spacer 2 (ITS2) region, a multicopy genetic marker commonly used to analyse Symbiodinium diversity. Replicated data generated from isoclonal Symbiodinium cultures showed that all genomes contained numerous, yet mostly rare, ITS2 sequence variants. Pyrosequencing data were consistent with more traditional denaturing gradient gel electrophoresis (DGGE) approaches to the screening of ITS2 PCR amplifications, where the most common sequences appeared as the most intense bands. Further, we developed an operational taxonomic unit (OTU)‐based pipeline for Symbiodinium ITS2 diversity typing to provisionally resolve ecologically discrete entities from intragenomic variation. A genetic distance cut‐off of 0.03 collapsed intragenomic ITS2 variants of isoclonal cultures into single OTUs. When applied to the analysis of field‐collected coral samples, our analyses confirm that much of the commonly observed Symbiodinium ITS2 diversity can be attributed to intragenomic variation. We conclude that by analysing Symbiodinium populations in an OTU‐based framework, we can improve objectivity, comparability and simplicity when assessing ITS2 diversity in field‐based studies.     

Presence of Symbiodinium spp. in macroalgal microhabitats from the southern Great Barrier Reef

Coral reefs are highly dependent on the mutualistic symbiosis between reef-building corals and dinoflagellates from the genus Symbiodinium. These dinoflagellates spend part of their life cycle outside the coral host and in the majority of the cases have to re-infect corals each generation. While considerable insight has been gained about Symbiodinium in corals, little is known about the ecology and biology of Symbiodinium in other reef microhabitats. This study documents Symbiodinium associating with benthic macroalgae on the southern Great Barrier Reef, including some Symbiodinium that are genetically close to the symbiotic strains from reef-building corals. It is possible that some of these Symbiodinium were in hospite, associated to soritid foraminifera or ciliates; nevertheless, the presence of Symbiodinium C3 and C15 in macroalgal microhabitats may also suggest a potential link between communities of Symbiodinium associating with both coral hosts and macroalgae.     

DNA divergency and speciation inSymbiodinium (Dinophyceae)

Relationship among symbiotic dinoflagellates of the genusSymbiodinium derived from seven different host species has been studied by means of DNA/DNA hybridization. DNA homologies range from about 70 to 30%D. Highest homology is regarded as subspecific level. Based on the characteristics of DNA and phenotypes,Symbiodinium microadriaticum subsp.microadriaticum is separated fromSymbiodinium microadriaticum subsp.condylactis. Lowest homology occurs at the methodical background, and is similar to that obtained with DNA of algae belonging to different classes. The data are in excellent agreement with DNA base composition, karyotypes, and morpohological as well as biochemical markers, emphasizing speciation among these gymnodinioid zooxanthellae.Dedicated to DrRobert K. Trench, Professor of Biology and Geology, University of California at Santa Barbara, whose work initiated recognition of speciation in zooxanthellae.     

Phylogenetic incongruence between dinoflagellate endosymbionts (Symbiodinium) and their host foraminifera (Sorites): small-subunit ribosomal RNA gene sequence evidence

Phototrophic dinoflagellate zooxanthellae commonly occur as endosymbionts in many planktic and certain benthic foraminifera (soritids). Many taxonomic issues and specific identities of foraminiferal dinoflagellates are not yet resolved. To assess taxonomic affinities among other dinoflagellates, we have determined the complete nucleotide sequence of the small-subunit rRNA coding region from Symbiodinium sp., an endosymbiotic dinoflagellate of the larger foraminifer Sorites orbiculus. The poly merase chain reaction was adopted for the in vitro amplification of ribosomal DNA, utilizing primers complementary to conserved regions. PCR-amplified DNA was directly sequenced and the sequence was aligned to all complete 18S-rDNA dinoflagellate sequences currently available through GenBank. Apicomplexan, ciliate, chromistacean, and rhodophycean sequences were added to infer across-kingdom phylogenetic relationships. Phylogenetic analysis of aligned nucleotide sequences produced a single most parsimonious tree (generated by the branch and bound method of PAUP). The inferred phylogeny indicates that the dinoflagellate extracted from the foraminifer Sorites orbiculus is a sister taxon to the symbiont present in the larger foraminifera Marginopora kudakajimaensis, but only distantly related to the dinoflagellate isolated from the soritid Amphisorus hemprichii. The sequence heterogeneity demonstrates a high degree of genetic diversity among Symbiodinium-like zooxanthellae and re-emphasizes that they are a variety of distinct entities.The inferred molecular phylogenetic relationships among symbiotic dinoflagellates are not congruent with the foraminiferal phylogeny based on cladistic methodology. The lack of correlation between the evolutionary history of dinoflagellate symbionts and their foraminiferal hosts argues against co-evolution. This lack of co-evolution implies that flexible recombinations among hosts and symbionts are evolutionarily favorable over permanently associated lineages, at least in these benthic foraminifera.     

Population genetic data of a model symbiotic cnidarian system reveal remarkable symbiotic specificity and vectored introductions across ocean basins

The Aiptasia–Symbiodinium symbiosis is a promising model for experimental studies of cnidarian–dinoflagellate associations, yet relatively little is known regarding the genetic diversity of either symbiotic partner. To address this, we collected Aiptasia from 16 localities throughout the world and examined the genetic diversity of both anemones and their endosymbionts. Based on newly developed SCAR markers, Aiptasia consisted of two genetically distinct populations: one Aiptasia lineage from Florida and a second network of Aiptasia genotypes found at other localities. These populations did not conform to the distributions of described Aiptasia species, suggesting that taxonomic re‐evaluation is needed in the light of molecular genetics. Associations with Symbiodinium further demonstrated the distinctions among Aiptasia populations. According to 18S RFLP, ITS2‐DGGE and microsatellite flanker region sequencing, Florida anemones engaged in diverse symbioses predominantly with members of Symbiodinium Clades A and B, but also C, whereas anemones from elsewhere harboured only S. minutum within Clade B. Symbiodinium minutum apparently does not form a stable symbiosis with other hosts, which implies a highly specific symbiosis. Fine‐scale differences among S. minutum populations were quantified using six microsatellite loci. Populations of S. minutum had low genotypic diversity and high clonality (R = 0.14). Furthermore, minimal population structure was observed among regions and ocean basins, due to allele and genotype sharing. The lack of genetic structure and low genotypic diversity suggest recent vectoring of Aiptasia and S. minutum across localities. This first ever molecular‐genetic study of a globally distributed cnidarian and its Symbiodinium assemblages reveals host–symbiont specificity and widely distributed populations in an important model system.     

Sterols of Amphidinium species in the Operculatum Clade: Predominance of cholesterol instead of Δ8(14) sterols previously considered Amphidinium-specific biomarkers

The dinoflagellates Amphidinium carterae and Amphidinium corpulentum have been previously characterized as having Δ⁸⁽¹⁴⁾-nuclear unsaturated 4α-methyl-5α-cholest-8(14)-en-3β-ol (C_28:1) and 4α-methyl-5α-ergosta-8(14),24(28)-dien-3β-ol (amphisterol; C_29:2) as predominant sterols, where they comprise approximately 80% of the total sterol composition. These two sterols have hence been considered as possible major sterol biomarkers for the genus. Here, we have examined the sterols of four recently identified species of Amphidinium (Amphidinium fijiense, Amphidinium magnum, Amphidinium theodori, and Amphidinium tomasii) that are closely related to Amphidinium operculatum as part of what is termed the Operculatum Clade to show that each species has its sterol composition dominated by the common dinoflagellate sterol cholesterol (cholest-5-en-3β-ol; C_27:1), which is found in many other dinoflagellate genera, rather than Δ⁸⁽¹⁴⁾ sterols. While the Δ⁸⁽¹⁴⁾ sterols 4α-methyl-5α-cholest-8(14)-en-3β-ol and 4α,23,24-trimethyl-5α-cholest-8(14),22E-dien-3β-ol (C_30:2) were present as minor sterols along with another common dinoflagellate sterol, 4α,23,24-trimethyl-5α-cholest-22E-en-3β-ol (dinosterol; C_30:1), in some of these four species, amphisterol was not conclusively observed. From a chemotaxonomic perspective, while this does reinforce the genus Amphidinium's ability to produce Δ⁸⁽¹⁴⁾ sterols, albeit here as minor sterols, these results demonstrate that caution should be used when considering Δ⁸⁽¹⁴⁾ sterols, especially amphisterol, as Amphidinium-specific biomarkers within these species where cholesterol is the predominant sterol.     

Morphology of the symbiosis between Corculum cardissa (Mollusca: Bivalvia) and Symbiodinium corculorum (Dinophyceae).

Light and transmission electron microscopy of tissues of the symbiotic clam Corculum cardissa (L) showed that a symbiotic dinoflagellate, Symbiodinium corculorum (Trench), is found predominantly in the mantle and the gills. The data suggest that in C. cardissa the algae are located in a zooxanthellal tubular system that is associated with the hemocoel and is similar to that seen in tridacnine ("giant") clams. The algae occur within the lumen of the tertiary tubules and are thus separated from the hemolymph by a tissue that is one cell layer thick. Under a light microscope the tertiary tubules appear as rows of symbionts originating from the digestive diverticulum, presumably branching from the primary tubules that are also seen in symbiotic tridacnine clams. This morphological arrangement is discussed with regard to the ontogeny and the evolution of the tubular system within symbiotic bivalves.     

A DNA hybridization assay to identify toxic dinoflagellates in coastal waters: detection of Karenia brevis in the Rookery Bay National Estuarine Research Reserve

A DNA hybridization assay was developed in microtiter plate format to detect the presence of toxic dinoflagellates in coastal waters. Simultaneous detection of multiple species was demonstrated using Karenia brevis, Karenia mikimotoi, and Amphidinium carterae. Molecular probes were designed to detect both K. brevis and K. mikimotoi and to distinguish between these two closely related species. The assay was used to detect K. brevis in coastal waters collected from the Rookery Bay National Estuarine Research Reserve. Assay results were verified by species-specific PCR and sequence analysis. The presence/absence of K. brevis was consistent with microscopic observation. Assay sensitivity was sufficient to detect K. brevis in amounts defined by a regional monitoring program as “present” (≤1000 cells/L). The assay yielded quick colorimetric results, used a single hybridization temperature, and conserved the amount of genomic DNA utilized by employing one set of PCR primers. The microplate assay provides a useful tool to quickly screen large sample sets for multiple target organisms.     

cDNA cloning and phylogenetic and expression analyses of actin in symbiotic dinoflagellates (Symbiodinium spp.)

Three cDNAs encoding actins were identified in two culturable strains (clades A and F) of the symbiotic dinoflagellates Symbiodinium spp. In a molecular phylogenetic analysis these actin sequences formed a monophyletic group with known dinoflagellate actins, remote from Syact-p that had been isolated from a clade A Symbiodinium strain (HG39). One of the newly identified actin sequences (SyAct-F1) was the most closely related to partial actin cDNA sequences (named AGfact-p and AFcact-p) isolated from adult colonies of two reef corals (Galaxea fascicularis and Favites chinensis) that were inhabited by Symbiodinium spp., suggesting the possibility that the latter two were from the symbionts. Partial AFcact-p sequences could be amplified by PCR using genomic DNA prepared from a symbiotic adult colony of F. chinensis as the template, but not from planula larvae in which zooxanthellae could not be detected, also arguing for the origin of AFcact-p in the symbiont. An expression analysis showed that the levels of the SyAct-A1 mRNA were comparable in symbiotic and non-symbiotic states, and also in motile and non-motile phases in a cultured condition, suggesting its usefulness as a constitutively expressed control gene in expression analysis of Symbiodinium mRNAs.     

PHYLOGENETIC POSITION OF SYMBIODINIUM (DINOPHYCEAE) ISOLATES FROM TRIDACNIDS (BIVALVIA), CARDIIDS (BIVALVIA), A SPONGE (PORIFERA), A SOFT CORAL (ANTHOZOA), AND A FREE-LIVING STRAIN

The genus Symbiodinium is the commonly observed symbiotic dinoflagellate (zooxanthellae) that forms mutual associations with various marine invertebrates. Numerous studies have revealed that the genus is comprised of a group of diverse taxa, and information on the phylogenetic relationships among the genus’ members is increasing. In this study, small subunit (SSU) ribosomal RNA (ssrRNA) gene sequences were determined for 15 more Symbiodinium strains from 12 relatively unstudied host taxa (Indo-Pacific tridacnids, cardiids, sponge, and soft coral), 1 hitherto unreported free-living Symbiodinium strain, and 4 other Symbiodinium strains from four other host taxa (Indo-Pacific zoanthid, foraminifer, jellyfish, and mid-Pacific hard coral). Their respective phylogenetic positions were inferred, and strains that are either closely related to or distinct from previously reported Symbiodinium taxa were revealed. The cultured Symbiodinium strains isolated from individuals of six species of tridacnids and three species of cardiids all had identical ssrRNA gene sequences, are closely related to S. microadriaticum Freudenthal, and are indistinguishable from the RFLP Type A strain previously reported. However, the ssrRNA gene sequences of clam symbionts that were obtained via gene cloning were different from those of the cultured isolates and represent strains that are close to the RFLP Type C strains. The Symbiodinium-like dinoflagellate from the Indo-Pacific sponge Haliclona koremella De Laubenfels is distinct from any of the Symbiodinium taxa studied and may be similar to the symbiont previously isolated from the stony coral Montipora patula Quelch. The isolates from the soft coral Sarcophyton glaucum Quoy et Gaimard and from the zoanthid Zoanthus sp. are both very closely related to S. pilosum Trench et Blank. The free-living Symbiodinium isolate is very closely related to the symbiont isolated from the Indo-Pacific foraminifer Amphisorus hemprichii Ehrenberg, which in turn is distinct from the Red Sea strain isolated from a similar host. Theisolate from Cassiopeia sp. is different from S. microadriaticum F., the type species harbored by Cassiopeia xamachana Bigelow, and is instead very closely related to S. pulchrorum Trench isolated from a sea anemone. The symbiont from the stony coral M. verrucosa Lamarck is a sister taxon to the symbionts isolated from the foraminifera Marginopora kudakajimensis Gudmundsson and Sorites orbiculus Forskål. These data suggest that polymorphic symbioses extend from cnidarians to some bivalve, foraminifer, and jellyfish host species.     

AMPHIDINIUM REVISITED. II. RESOLVING SPECIES BOUNDARIES IN THE AMPHIDINIUM OPERCULATUM SPECIES COMPLEX (DINOPHYCEAE), INCLUDING THE DESCRIPTIONS OF AMPHIDINIUM TRULLA SP. NOV. AND AMPHIDINIUM GIBBOSUM. COMB. NOV.1

Amphidinium operculatum Claparède et Lachmann, the type species of the dinoflagellate genus Amphidinium, has long had an uncertain identity. It has been considered to be either difficult to distinguish from other similar species or a morphologically variable species itself. This has led to the hypothesis that A. operculatum represents a “species complex.” Recently, the problem of distinguishing A. operculatum from similar species has become particularly acute, because several morphologically similar species have been found to produce bioactive compounds of potential interest to the pharmaceutical industry. In this study, we cultured and examined existing cultures of several species of Amphidinium, most of which have been previously identified as A. operculatum or as species considered by some to be synonyms or varieties of A. operculatum. Thirty strains were examined using comparative LM, SEM, and partial large subunit (LSU) rDNA sequence data. Through morphological and molecular phylogenetic analyses, six distinct species were identified, including Amphidinium trulla sp. nov. and Amphidinium gibbosum comb. nov. Amphidinium operculatum was redescribed based on four cultures. Genetic variability within the examined Amphidinium species varied greatly. There was little difference among strains in partial LSU rDNA for most species, but strains of A. carterae and A. massartii Biencheler differed by as much as 4%. In both A. carterae and A. massartii, three distinct genotypes based on partial LSU rDNA were found, but no morphological differences among strains could be observed using LM or SEM. In the case of A. carterae, no biogeographically related molecular differences were found.     

Differential antioxidant response between two Symbiodinium species from contrasting environments

High sea surface temperature accompanied by high levels of solar irradiance is responsible for the disruption of the symbiosis between cnidarians and their symbiotic dinoflagellates from the genus Symbiodinium. This phenomenon, known as coral bleaching, is one of the major threats affecting coral reefs around the world. Because an important molecular trigger to bleaching appears related to the production of reactive oxygen species (ROS), it is critical to understand the function of the antioxidant network of Symbiodinium species. In this study we investigated the response of two Symbiodinium species, from contrasting environments, to a chemically induced oxidative stress. ROS produced during this oxidative burst reduced photosynthesis by 30 to 50% and significantly decreased the activity of superoxide dismutase. Lipid peroxidation levels and carotenoid concentrations, especially diatoxanthin, confirm that these molecules act as antioxidants and contribute to the stabilization of membrane lipids. The comparative analysis between the two Symbiodinium species allowed us to highlight that Symbiodinium sp. clade A temperate was more tolerant to oxidative stress than the tropical S. kawagutii clade F. These differences are very likely a consequence of adaptation to their natural environment, with the temperate species experiencing conditions of temperature and irradiance much more variable and extreme.     

Cover

Loss (bleaching) of symbiotic dinoflagellates (genus Symbiodinium) from the sea anemone Aiptasia pallida caused by elevated temperatures or disruption of symbiont photosynthesis can be restored through exposure to axenic Symbiodinium cultures (top part of figure; Symbiodinium appear red due to chlorophyll autofluorescence under blue light). [Vol. 49, No. 3, pp.447–458]