Cell wall proteomic analysis of the cnidarian photosymbionts Breviolum minutum and Cladocopium goreaui.

The algal cell wall is an important cellular component that functions in defense, nutrient utilization, signaling, adhesion, and cell-cell recognition-processes important in the cnidarian-dinoflagellate symbiosis. The cell wall of symbiodiniacean dinoflagellates is not well characterized. Here, we present a method to isolate cell walls of Symbiodiniaceae and prepare cell-wall-enriched samples for proteomic analysis. Label-free liquid chromatography-electrospray ionization tandem mass spectrometry was used to explore the surface proteome of two Symbiodiniaceae species from the Great Barrier Reef: Breviolum minutum and Cladocopium goreaui. Transporters, hydrolases, translocases, and proteins involved in cell-adhesion and protein-protein interactions were identified, but the majority of cell wall proteins had no homologues in public databases. We propose roles for some of these proteins in the cnidarian-dinoflagellate symbiosis. This work provides the first proteomics investigation of cell wall proteins in the Symbiodiniaceae and represents a basis for future explorations of the roles of cell wall proteins in Symbiodiniaceae and other dinoflagellates.

The cell wall polysaccharides of a photosynthetic relative of apicomplexans, Chromera velia.

Chromerids are a group of alveolates, found in corals, that show peculiar morphological and genomic features. These organisms are evolutionary placed in-between symbiotic dinoflagellates and parasitic apicomplexans. There are two known species of chromerids: Chromera velia and Vitrella brassicaformis. Here, the biochemical composition of the C. velia cell wall was analyzed. Several polysaccharides adorn this structure, with glucose being the most abundant monosaccharide (approx. 80%) and predominantly 4-linked (approx. 60%), suggesting that the chromerids cell wall is mostly cellulosic. The presence of cellulose was cytochemically confirmed with calcofluor white staining of the algal cell. The remaining wall polysaccharides, assuming structures are similar to those of higher plants, are indicative of a mixture of galactans, xyloglucans, heteroxylans, and heteromannans. The present work provides, for the first time, insights into the outermost layers of the photosynthetic alveolate C. velia.

A candidate transporter allowing symbiotic dinoflagellates to feed their coral hosts.

The symbiotic partnership between corals and dinoflagellate algae is crucial to coral reefs. Corals provide their algal symbionts with shelter, carbon dioxide and nitrogen. In exchange, the symbiotic algae supply their animal hosts with fixed carbon in the form of glucose. But how glucose is transferred from the algal symbiont to the animal host is unknown. We reasoned that a transporter resident in the dinoflagellate cell membrane would facilitate outward transfer of glucose to the surrounding host animal tissue. We identified a candidate transporter in the cnidarian symbiont dinoflagellate Breviolum minutum that belongs to the ubiquitous family of facilitative sugar uniporters known as SWEETs (sugars will eventually be exported transporters). Previous gene expression analyses had shown that BmSWEET1 is upregulated when the algae are living symbiotically in a cnidarian host by comparison to the free-living state [1, 2]. We used immunofluorescence microscopy to localise BmSWEET1 in the dinoflagellate cell membrane. Substrate preference assays in a yeast surrogate transport system showed that BmSWEET1 transports glucose. Quantitative microscopy showed that symbiotic B. minutum cells have significantly more BmSWEET1 protein than free-living cells of the same strain, consistent with export during symbiosis but not during the free-living, planktonic phase. Thus, BmSWEET1 is in the right place, at the right time, and has the right substrate to be the transporter with which symbiotic dinoflagellate algae feed their animal hosts to power coral reefs.

Cell surface carbohydrates of symbiotic dinoflagellates and their role in the establishment of cnidarian-dinoflagellate symbiosis.

Symbiodiniaceae algae are often photosymbionts of reef-building corals. The establishment of their symbiosis resembles a microbial infection where eukaryotic pattern recognition receptors (e.g. lectins) are thought to recognize a specific range of taxon-specific microbial-associated molecular patterns (e.g. glycans). The present study used the sea anemone, Exaiptasia diaphana and three species of Symbiodiniaceae (the homologous Breviolum minutum, the heterologous-compatible Cladocopium goreaui and the heterologous-incompatible Fugacium kawagutii) to compare the surface glycomes of three symbionts and explore the role of glycan-lectin interactions in host-symbiont recognition and establishment of symbiosis. We identified the nucleotide sugars of the algal cells, then examined glycans on the cell wall of the three symbiont species with monosaccharide analysis, lectin array technology and fluorescence microscopy of the algal cell decorated with fluorescently tagged lectins. Armed with this inventory of possible glycan moieties, we then assayed the ability of the three Symbiodiniaceae to colonize aposymbiotic E. diaphana after modifying the surface of one of the two partners. The Symbiodiniaceae cell-surface glycome varies among algal species. Trypsin treatment of the alga changed the rate of B. minutum and C. goreaui uptake, suggesting that a protein-based moiety is an essential part of compatible symbiont recognition. Our data strongly support the importance of D-galactose (in particular ß-D-galactose) residues in the establishment of the cnidarian-dinoflagellate symbiosis, and we propose a potential involvement of L-fucose, D-xylose and D-galacturonic acid in the early steps of this mutualism.

Annual Reproductive Cycle and Unusual Embryogenesis of a Temperate Coral in the Mediterranean Sea.

The variety of reproductive processes and modes among coral species reflects their extraordinary regeneration ability. Scleractinians are an established example of clonal animals that can exhibit a mixed strategy of sexual and asexual reproduction to maintain their populations. This study provides the first description of the annual reproductive cycle and embryogenesis of the temperate species Caryophyllia inornata. Cytometric analyses were used to define the annual development of germ cells and embryogenesis. The species was gonochoric with three times more male polyps than female. Polyps were sexually mature from 6 to 8 mm length. Not only females, but also sexually inactive individuals (without germ cells) and males were found to brood their embryos. Spermaries required 12 months to reach maturity, while oogenesis seemed to occur more rapidly (5-6 months). Female polyps were found only during spring and summer. Furthermore, the rate of gamete development in both females and males increased significantly from March to May and fertilization was estimated to occur from April to July, when mature germ cells disappeared. Gametogenesis showed a strong seasonal influence, while embryos were found throughout the year in males and in sexually inactive individuals without a defined trend. This unusual embryogenesis suggests the possibility of agamic reproduction, which combined with sexual reproduction results in high fertility. This mechanism is uncommon and only four other scleractinians (Pocillopora damicornis, Tubastraea diaphana, T. coccinea and Oulastrea crispata) have been shown to generate their broods asexually. The precise nature of this process is still unknown.