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.

Biogeography of two species of Symbiodinium (Freudenthal) inhabiting the intertidal sea anemone Anthopleura elegantissima (Brandt).

We have analyzed the genetic profiles of dinoflagellate populations obtained from the Pacific coast sea anemone Anthopleura elegantissima (Brandt) at collection sites from Washington to California. Genetic differences within the symbiont populations of California anemones have been uncovered by restriction length polymorphism (RFLP) analysis of the small subunit (SSU) and large subunit (LSU) ribosomal RNA genes, and by denaturing gradient gel electrophoresis (DGGE) of the internal transcribed spacer region 2 (ITS 2). The existence of two Symbiodinium species is substantiated by sequence analysis of the variable regions V1, V2, and V3 of the SSUrDNA, which also establishes their phylogenetic relatedness to other members of the genus Symbiodinium. Anemones from Washington and Oregon harbor a single dinoflagellate species, for which we propose the name S. muscatinei sp. nov. At these northern locations, S. muscatinei either exists alone or co-occurs with the Chlorella-like green algal symbiont. Our results indicate that S. muscatinei co-occurs with a second dinoflagellate, S. californium, in mixed populations in central and southern California. We suggest that the geographic distribution of these dinoflagellates is related to the temperature cline created by latitude.

Photosynthetic response to elevated temperature in the symbiotic dinoflagellate Symbiodinium microadriaticum in culture.

Elevated temperature (28-34 degrees C) has been hypothesized as the primary cause of the loss of algal endosymbionts in coral reef-associated invertebrates, a phenomenon observed on a world-wide scale over the last decade. In past studies of this "bleaching" phenomenon, there has been an underlying assumption that temperature adversely affects the animal hosts, the algae thereby being relegated to a more passive role. Because photosynthesis is a sensitive indicator of thermal stress in plants and has a central role in the nutrition of symbiotic invertebrates, we have tested the hypothesis that elevated temperature adversely affects photosynthesis in the symbiotic dinoflagellate Symbiodinium microadriaticum. The results, based on analyses of light-mediated O2 evolution and in vivo fluorescence, indicate that photosynthesis is impaired at temperatures above 30 degrees C and ceases completely at 34-36 degrees C. These observations are discussed in the context of possible mechanisms that may function in the disassociation of algal-invertebrate symbioses in response to elevated temperature.

Isolation and sequence analysis of the small subunit ribosomal RNA gene from the euryhaline yeast Debaryomyces hansenii.

The small subunit ribosomal RNA gene (SSU rDNA) from the euryhaline yeast Debaryomyces hansenii has been isolated and sequenced. After appropriate alignment of this sequence with SSU rDNA sequences from 30 other taxa, phylogenetic reconstruction using distance matrix and maximum parsimony methods indicates that D. hansenii is most closely affiliated with Candida albicans, and occurs in the cluster of the yeasts Saccharomyces cerevisiae, Torulaspora delbruekii, Candida glabrata, and Kluyveromyces lactis. It appears that the capacity to tolerate high salt is independent of phylogenetic affiliations based on SSU rDNA analyses.

The nucleotide sequence of the small subunit ribosomal RNA gene from Symbiodinium pilosum, a symbiotic dinoflagellate.

The complete sequence of the small subunit ribosomal RNA (SSU rRNA) gene was determined for the symbiotic dinoflagellate Symbiodinium pilosum. This sequence was compared with sequences from two other dinoflagellates (Prorocentrum micans and Crypthecodinium cohnii), five Apicomplexa, five Ciliata, five other eukaryotes and one archaebacterium. The corresponding structurally conserved regions of the molecule were used to determine which portions of the sequences could be unambiguously aligned. Phylogenetic relationships were inferred from an analysis of distance matrices, where pair-wise distances were determined using a maximum likelihood model for transition and transversion ratios, and from maximum parsimony analysis, with bootstrap resampling. By either analytical approach, the dinoflagellates appear distantly related to prokaryotes, and are most closely related to two of the Apicomplexa, Sarcocystis muris and Theileria annulata. Among the dinoflagellates, C. cohnii was found to be more closely affiliated with the Apicomplexa than either P. micans or S. pilosum.

The biogenesis of the cyanellae of Cyanophora paradoxa. I. Polypeptide composition of the cyanellae.

Based on polypeptide separation, protein purification and immunoblotting techniques using heterologous antibodies, we have been able to identify several photosynthetically important polypeptide components of the cyanellae of Cyanophora paradoxa. Cytochrome c-552 and ferredoxin have been purified to electrophoretic homogeneity and exhibit apparent molecular masses of 10.5 and 9.0 kDa, respectively. Cytochrome c-552 has an isoelectric point of pH 4.2 +/- 0.1. Plastocyanin was immunologically and spectrally undetectable even in cells grown in the presence of Cu2+. Polypeptides for cytochromes f, b-6 and c-552 have been located in electrophoretically resolved thylakoid samples by using the TMBZ-staining procedure. Intact phycobilisomes have been purified and characterized with respect to polypeptide composition and absorption and emission spectra. Photosystems I and II have been isolated and characterized with respect to their photochemical activities, spectral characteristics and polypeptide composition. Photochemically active PS I complexes fluoresce maximally at 720 nm at 77 K and comprise five polypeptide subunits resolved under denaturing conditions with apparent molecular masses of 66, 21, 18, 14 and 11 kDa. PS II core complexes mediate light-dependent 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-sensitive electron transfer between 1,5-diphenylcarbazide (DPC) and 2,6-dichlorophenolindophenol (DPIP) at rates of 140-200 mumol h-1 mg-1 chlorophyll. These complexes exhibit absorption maxima at 436 and 673 nm and show fluorescence emission maxima at 685 and 695 nm at 77 K. Rubisco was separated by two-dimensional electrophoresis and immunologically characterized.

The biogenesis of the cyanellae of Cyanophora paradoxa. II. Pulse-labelling of cyanellar polypeptides in the presence of transcriptional and translational inhibitors.

Cycloheximide and chloroamphenicol, specific inhibitors of protein translation in the cytoplasmic and cyanellar compartments, respectively, of Cyanophora paradoxa, have been employed in 30 min pulse-labelling experiments by using [NaH-14C]O3 to label total cell proteins in vivo. Cyanellae purified from host cell lysates were separated into soluble and thylakoid fractions and analysed by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) to determine the distribution of radioactivity in the cyanellar polypeptides. Analysis of the autoradiograms of electrophoretically resolved proteins of the cyanellae indicates that about 70% of the total number of cyanellar proteins visualized in the controls are synthesized on cytoplasmic ribosomes. The majority (81%) of the soluble cyanellar proteins appear to be cytoplasmically synthesized. In contrast, the majority (70%) of the thylakoid proteins are synthesized within the cyanellae. The observations also suggest that the polypeptides synthesized within the cyanellae include species that are the most abundant and rapidly turned over. A number of the polypeptides previously identified have now been characterized with regard to their sites of synthesis. In addition, we report on labelling experiments involving rifampicin, a specific inhibitor of cyanellar transcription, which indicate that different mRNAs within the cyanellae have markedly different stabilities.

The biogenesis of the cyanellae of Cyanophora paradoxa. III. In vitro synthesis of cyanellar polypeptides using separated cytoplasmic and cyanellar RNA.

RNA from Cyanophora paradoxa was separated into cytoplasmic and cyanellar fractions by using a combination of subcellular fractionation and oligo-dT chromatography. In vitro translation of the separated cytoplasmic and cyanellar RNAs in a rabbit reticulocyte lysate system in the presence of [35S]methionine resulted in the incorporation of radiolabel into electrophoretically distinct sets of polypeptides. Monospecific and polyspecific antibodies that react with cyanellar polypeptides were used to probe the in vitro translation products by indirect immunoprecipitation by using Staphylococcus protein A conjugated to Sepharose beads. The results indicate that linker polypeptide L1 of the phycobilisome, the gamma subunit of coupling factor CF1, and subunit II of PS I are synthesized in the cytoplasm as precursor molecules that are 5-8 kDa larger than their mature sizes. Antibodies directed against the psbA gene product (the D1 protein) precipitated a polypeptide found in the translation products of the cyanellar RNA-directed reactions, which is about 1.5 kDa larger than the mature protein.

Speciation and symbiotic dinoflagellates.

Morphometric analyses based on three-dimensional reconstruction of the nuclei of four different strains of the symbiotic dinoflagellate Symbiodinium microadriaticum, the algae that inhabit corals, giant clams, and other marine invertebrates, revealed marked differences in chromosome numbers and chromosome volumes. The differences are not consistent with different ploidy states within the same species, but can most easily be interpreted as indicating different species.

Persistence and coexistence of a nonsymbiotic coral in open reef environments.

In the Republic of Belau (western Caroline Islands), the nonsymbiotic stony coral Tubastraea micrantha (Ehrenberg) occurs commonly on open reef habitats that are dominated by symbiotic corals. This observation calls into question the often-held view that nonsymbiotic corals are restricted to cryptic environments because of their inability to compete successfully for space with rapidly growing symbiotic corals. In this study, we investigated aspects of the biology of Tubastraea that might account for its coexistence and persistence on the reef. Our results indicate that despite low calcification rates, linear skeletal extension for Tubastraea is surprisingly rapid for a nonsymbiotic species and comparable to those of many symbiotic corals. This factor, coupled with Tubastraea's resistance to direct competitive dominance (i.e., by aggressive interactions) and avoidance of predation, promotes its coexistence with symbiotic species. The relative importance of these biological interactions in different coral communities leads to predictions corroborated by observed patterns of local distribution and abundance.

Cellular events in the reestablishment of a symbiosis between a marine dinoflagellate and a coelenterate.

Summary. Within 24 h after the initial phagocytotic uptake of freshly isolated (from host tissue) symbiotic algae (Symbiodinium mieroadriaticum) by the endodermal cells of the polyp (scyphistoma) stage of the jellyfish Cassiopeia xamachana,the algal population was observed to decline despite evidence of algal cell division. Analyses of the frequency of phago-lysosome fusion as an indicator of possible attempts of the host to digest the algae indicated that, althoughphago-lysosome fusion did occur, the low frequency of occurrence is inconsistent with the interpretation that the animals digested the algae. Animal cell lysosomes were located predominantly at the apices of the endodermal cells,and the symbiotic algae were transported toward the bases of the endodermal cells.Within 3 days after initial infection, most endodermal cells with algae ceased to be phagocytotically active (with respect to the uptake of carmine particles). Many of these endodermal cells soon migrated into the mesoglea to become what are traditionally referred to as "amoebocytes".Within amoebocytes the algae proliferated. The onset of strobilation by the scyphistomae was directly correlated with the increase in the algal population within these amoebocytes.

Endocytosis of the symbiotic dinoflagellate Symbiodinium microadriaticum Freudenthal by endodermal cells of the scyphistomae of Cassiopeia xamachana and resistance of the algae to host digestion.

The ingestion and fate of four types of particles by endodermal cells of the scyphistomae of Cassiopeia xamachana were investigated by scanning and transmission electron microscopy. Ferritin was endocytosed pinocytotically by invagination of the plasmalemma. These small pinocytotic vesicles fuse with other similar vesicles to form larger ferritin-containing vacuoles, which eventually fuse with lysosomes. Such secondary lysosomes exhibit acid phosphatase activity. The co-occurrence of acid phosphatase activity and ferritin in secondary lysosomes achieved maximum frequency within 2 h of uptake of ferritin and was evident for at least 4 h following uptake. Artemia particles, live freshly isolated symbiotic algae (Symbiodinium microadriaticum), and heat-killed S. microadriaticum are phagocytosed by endodermal cells. Ferritin-labelled lysosomes fused with food vacuoles containing particles of Artemia. Vacuoles containing heat-killed S. microadriaticum also showed evidence of phago-lysosome fusion. S. microadriaticum in situ (i.e. in host cells) after 3 days exposure to the photosynthetic inhibitor, 3-(3-4-dichlorophenyl)-1,1-dimethylurea, appeared degenerate, and were found in loose-fitting host vacuoles, many in mid and apical portions of the host cell. More than 70% of these vacuoles with moribund algae contained the ferritin label, indicating that lysosome fusion had occurred. In contrast, live S. microadriaticum in control animals were almost always found at the base of the host cell in individual tight-fitting vacuoles with no evidence of lysosome fusion. Live S. microadriaticum apparently escape host digestion by prohibiting the fusion of lysosomes with the vacuole in which they reside. Vacuoles containing defunct algal symbionts, in contrast, were subject to lysosomal attack.

Selectivity in phagocytosis and persistence of symbiotic algae in the scyphistoma stage of the jellyfish Cassiopeia xamachana.

We have investigated whether interactions between cell-surface macromolecules play a role in cellular recognition leading to specificity in the establishment of intracellular symbiosis between dinoflagellates and the polyp (scyphistoma) stage of the jellyfish Cassiopeia xamachana. All strains of the symbiotic dinoflagellate Symbiodinium microadriaticum were phagocytosed by the endodermal cells of the scyphistomae when presented to them as cells freshly isolated from their respective hosts. The rates of phagocytosis of such cells were high, and were directly correlated with the presence of a membrane, thought to be the host cell vacuolar membrane that surrounds the freshly isolated algae. Cultured algae lack this membrane. All cultured algae, even those that proliferate in host tissues, were phagocytosed at very low or undetectable rates. Freshly isolated algae treated with reagents that removed the host membrane were phagocytosed at low rates. The endodermal cells of the scyphistomae of the non-symbiotic medusa Aurelia aurita also phagocytosed freshly isolated algae, but did not phagocytose cultured algae. Phagocytosis of algae and carmine particles was found to be a competitive process in scyphistomae of C. xamachana. No correlation was observed between the surface electrical charge on algae and their phagocytosis by host endodermal cells. Neither was there any correlation between phagocytosis and persistence. We conclude that the specificity in symbioses between marine invertebrates and dinoflagellates appears to be regulated by processes that occur after potential algal symbionts are phagocytosed.