Photosynthetic Dinoflagellates Research Articles

Heterologous DNA Uptake in Cultured Symbiodinium spp. Aided by Agrobacterium tumefaciens.

Plant-targeted pCB302 plasmids containing sequences encoding gfp fusions with a microtubule-binding domain; gfp with the fimbrin actin-binding domain 2; and gfp with AtRACK1C from Arabidopsis thaliana, all harbored in Agrobacterium tumefaciens, were used to assay heterologous expression on three different clades of the photosynthetic dinoflagellate, Symbiodinium. Accessibility to the resistant cell wall and through the plasma membrane of these dinoflagellates was gained after brief but vigorous shaking in the presence of glass beads and polyethylene glycol. A resistance gene to the herbicide Basta allowed appropriate selection of the cells expressing the hybrid proteins, which showed a characteristic green fluorescence, although they appeared to lose their photosynthetic pigments and did not further divide. Cell GFP expression frequency measured as green fluorescence emission yielded 839 per every 106 cells for Symbiodinium kawagutii, followed by 640 and 460 per every 106 cells for Symbiodinium microadriaticum and Symbiodinium sp. Mf11, respectively. Genomic PCR with specific primers amplified the AtRACK1C and gfp sequences after selection in all clades, thus revealing their presence in the cells. RT-PCR from RNA of S. kawagutii co-incubated with A. tumefaciens harboring each of the three vectors with their respective constructs, amplified products corresponding to the heterologous gfp sequence while no products were obtained from three distinct negative controls. The reported procedure shows that mild abrasion followed by co-incubation with A. tumefaciens harboring heterologous plasmids with CaMV35S and nos promoters can lead to expression of the encoded proteins into the Symbiodinium cells in culture. Despite the obvious drawbacks of the procedure, this is an important first step towards a stable transformation of Symbiodinium.

Microbiome structure of the fungid coral Ctenactis echinata aligns with environmental differences.

The significance of bacteria for eukaryotic functioning is increasingly recognized. Coral reef ecosystems critically rely on the relationship between coral hosts and their intracellular photosynthetic dinoflagellates, but the role of the associated bacteria remains largely theoretical. Here, we set out to relate coral‐associated bacterial communities of the fungid host species Ctenactis echinata to environmental settings (geographic location, substrate cover, summer/winter, nutrient and suspended matter concentrations) and coral host abundance. We show that bacterial diversity of C. echinata aligns with ecological differences between sites and that coral colonies sampled at the species’ preferred habitats are primarily structured by one bacterial taxon (genus Endozoicomonas) representing more than 60% of all bacteria. In contrast, host microbiomes from lower populated coral habitats are less structured and more diverse. Our study demonstrates that the content and structure of the coral microbiome aligns with environmental differences and denotes habitat adequacy. Availability of a range of coral host habitats might be important for the conservation of distinct microbiome structures and diversity.

Plastid genome-based phylogeny pinpointed the origin of the green-colored plastid in the dinoflagellate Lepidodinium chlorophorum.

Unlike many other photosynthetic dinoflagellates, whose plastids contain a characteristic carotenoid peridinin, members of the genus Lepidodinium are the only known dinoflagellate species possessing green alga-derived plastids. However, the precise origin of Lepidodinium plastids has hitherto remained uncertain. In this study, we completely sequenced the plastid genome of Lepidodinium chlorophorum NIES-1868. Our phylogenetic analyses of 52 plastid-encoded proteins unite L. chlorophorum exclusively with a pedinophyte, Pedinomonas minor, indicating that the green-colored plastids in Lepidodinium spp. were derived from an endosymbiotic pedinophyte or a green alga closely related to pedinophytes. Our genome comparison incorporating the origin of the Lepidodinium plastids strongly suggests that the endosymbiont plastid genome acquired by the ancestral Lepidodinium species has lost genes encoding proteins involved in metabolism and biosynthesis, protein/metabolite transport, and plastid division during the endosymbiosis. We further discuss the commonalities and idiosyncrasies in genome evolution between the L. chlorophorum plastid and other plastids acquired through endosymbiosis of eukaryotic photoautotrophs.

Transient transformation of cultured photosynthetic dinoflagellates (Symbiodinium spp.) with plant-targeted vectors

Reproducible and reliable genetic transformation methods are a key tool for understanding the physiology and cell biology of Symbiodinium. Nevertheless, transformation methods previously applied to cells such as microalgae, including those utilizing glass beads, have not been tested on these microorganisms. Here, we report a simple, transient transformation method, which allowed plasmid incorporation into three distinct clades of cultured Symbiodinium cells with the plant-targeted plasmid pCB302 harboring sequences encoding a fusion of green fluorescent protein (gfp) with RACK1C from Arabidopsis thaliana (AtRACK1C). Accessibility of the plasmid to the resistant cell wall and through the plasma membrane of the dinoflagellates was achieved through vigorous shaking in the presence of glass beads and polyethylene glycol. A resistance gene to the herbicide Basta allowed appropriate selection in the photosynthetic cells. The transformation frequency per every 106 cells was 107 ± 7 transformants for Symbiodinium kawagutii, 74 ± 8 for Symbiodinium microadriaticum ssp. microadriaticum, and 65 ± 5 for Symbiodinium sp. Mf11. Moreover, Symbiodinium pulchrorum cultures were successfully transformed with a different vector (pCAMBIA-FABD2-gfp) under the same conditions, further validating our procedure. The observation of green fluorescent emission from the cell cytoplasm in all performed transformations indicated that the procedure allowed the heterologous plasmids to enter and undergo expression in the Symbiodinium cells. The success of this transient transformation method opens interesting possibilities for functional genomics studies in Symbiodinium spp.

Pellucidodinium psammophilum gen. & sp. nov. and Nusuttodinium desymbiontum sp. nov. (Dinophyceae), two novel heterotrophs closely related to kleptochloroplastidic dinoflagellates

:Nusuttodinium spp. use ingested cryptomonad chloroplasts as kleptochloroplasts but form a sister group to Spiniferodinium spp., which are typical photosynthetic dinoflagellates. Our survey of the diversity of Nusuttodinium-related dinoflagellates resulted in the discovery of two colourless dinoflagellates, referred to as DAI (dinoflagellate collected from Aininkappu) and DIS (dinoflagellate collected from Ishikari). Cells of DAI possess a right-handed cingulum, an apical groove connecting with the sulcal extension at a right angle and nuclear chambers. Cells of DIS have a projection on their epicone encircled by a counterclockwise-directed apical groove and lack nuclear chambers. Cells of DAI digest cryptomonads directly, and DIS never ingests cryptomonads, making both species nonkleptochloroplastidic. Phylogenetic analyses showed that DIS is a member of the Nusuttodinium clade; whereas, DAI is a sister of the clade, and the photosynthetic genus Spiniferodinium is positioned at the base of these dinoflagellates. Based on a unique combination of features, including a unique shape to the apical groove, a right-handed cingulum, nuclear chambers and lack of a chloroplast, we describe DAI as Pellucidodinium psammophilum gen. & sp. nov. Based on a projection on the epicone, a counterclockwise-directed apical groove and the lack of nuclear chambers, we describe DIS as Nusuttodinium desymbiontum sp. nov. despite its inability to display kleptochloroplastidy. The phylogenetic position of P. psammophilum, together with its shared presence of nuclear chambers with Spiniferodinium, implies that it represents an evolutionary intermediate prior to the acquisition of kleptochloroplastidy; N. desymbiontum appears to have lost this competence secondarily.

Trehalose is a chemical attractant in the establishment of coral symbiosis.

Coral reefs have evolved with a crucial symbiosis between photosynthetic dinoflagellates (genus Symbiodinium) and their cnidarian hosts (Scleractinians). Most coral larvae take up Symbiodinium from their environment; however, the earliest steps in this process have been elusive. Here we demonstrate that the disaccharide trehalose may be an important signal from the symbiont to potential larval hosts. Symbiodinium freshly isolated from Fungia scutaria corals constantly released trehalose (but not sucrose, maltose or glucose) into seawater, and released glycerol only in the presence of coral tissue. Spawning Fungia adults increased symbiont number in their immediate area by excreting pellets of Symbiodinium, and when these naturally discharged Symbiodinium were cultured, they also released trehalose. In Y-maze experiments, coral larvae demonstrated chemoattractant and feeding behaviors only towards a chamber with trehalose or glycerol. Concomitantly, coral larvae and adult tissue, but not symbionts, had significant trehalase enzymatic activities, suggesting the capacity to utilize trehalose. Trehalase activity was developmentally regulated in F. scutaria larvae, rising as the time for symbiont uptake occurs. Consistent with the enzymatic assays, gene finding demonstrated the presence of a trehalase enzyme in the genome of a related coral, Acropora digitifera, and a likely trehalase in the transcriptome of F. scutaria. Taken together, these data suggest that adult F. scutaria seed the reef with Symbiodinium during spawning and the exuded Symbiodinium release trehalose into the environment, which acts as a chemoattractant for F. scutaria larvae and as an initiator of feeding behavior- the first stages toward establishing the coral-Symbiodinium relationship. Because trehalose is a fixed carbon compound, this cue would accurately demonstrate to the cnidarian larvae the photosynthetic ability of the potential symbiont in the ambient environment. To our knowledge, this is the first report of a chemical cue attracting the motile coral larvae to the symbiont.

Menthol-induced bleaching rapidly and effectively provides experimental aposymbiotic sea anemones (Aiptasia sp.) for symbiosis investigations.

Experimental manipulation of the symbiosis between cnidarians and photosynthetic dinoflagellates (Symbiodinium spp.) is crucial to advancing the understanding of the cellular mechanisms involved in host-symbiont interactions, and overall coral reef ecology. The anemone Aiptasia sp. is a model for cnidarian-dinoflagellate symbiosis, and notably it can be rendered aposymbiotic (i.e. dinoflagellate-free) and re-infected with a range of Symbiodinium types. Various methods exist for generating aposymbiotic hosts; however, they can be hugely time consuming and not wholly effective. Here, we optimise a method using menthol for production of aposymbiotic Aiptasia. The menthol treatment produced aposymbiotic hosts within just 4weeks (97-100% symbiont loss), and the condition was maintained long after treatment when anemones were held under a standard light:dark cycle. The ability of Aiptasia to form a stable symbiosis appeared to be unaffected by menthol exposure, as demonstrated by successful re-establishment of the symbiosis when anemones were experimentally re-infected. Furthermore, there was no significant impact on photosynthetic or respiratory performance of re-infected anemones.

Symbiont type influences trophic plasticity of a model cnidarian-dinoflagellate symbiosis.

The association between cnidarians and photosynthetic dinoflagellates within the genus Symbiodinium is a prevalent relationship in tropical and subtropical marine environments. Although the diversity of Symbiodinium provides a possible axis for niche diversification, increased functional range and resilience to physical stressors such as elevated temperature, how such diversity relates to the physiological balance between autotrophy and heterotrophy of the host animal remains unknown. Here, we experimentally show interspecific and intraspecific variability of photosynthetic carbon fixation and subsequent translocation by Symbiodinium to the model cnidarian host Aiptasia pallida. By using a clonal anemone line harboring different species of Symbiodinium, we determined that symbiont identity influences trophic plasticity through its density, capacity to fix carbon, quantity of translocated carbon and ultimately the host's capacity to ingest and digest prey. Symbiont carbon translocation and host prey ingestion were positively correlated across symbiont combinations that consisted of different isoclonal lines of Symbiodinium minutum, while a combination with type D4-5 Symbiodinium displayed lower carbon translocation, and prey capture and digestion more similar to Aiptasia lacking symbionts. The absence of a shift toward greater heterotrophy when carbon translocation is low suggests that the metabolic demand of feeding and digestion may overwhelm nutritional stores when photosynthesis is reduced, and amends the possible role of animal feeding in resistance to or recovery from the effects of climate change in more obligate symbioses such as reef-building corals.

Additions to the Genus Phyllodesmium, with a Phylogenetic Analysis and its Implications to the Evolution of Symbiosis

The facelinid genus Phyllodesmium (Ehrenberg, 1831) consists of approximately 24 described species that prey upon soft-bodied corals. At least five additional species have yet to be described, making it an interesting genus for testing phylogenetic hypotheses. The genus is extremely morphologically diverse, with many species adapting specifically to a specific host coral. One of the most interesting adaptations found in this genus is the widespread participation in a symbiotic relationship with photosynthetic dinoflagellates in the genus Symbiodinium. Two new species, Phyllodesmium acanthorhinum n. sp. and Phyllodesmium undulatum n. sp., from the Philippine Islands and Japan are described, and a morphological phylogeny is created to include the two new species, as well as three undescribed species. An examination of the Phyllodesmium phylogeny suggests that species with digestive gland branching and zooxanthellae are more derived. Confidence and robustness in this analysis are lacking, however, and further studies using molecular data could add confidence to this conclusion.

Selective feeding and foreign plastid retention in an Antarctic dinoflagellate.

The peridinin-containing plastid found in most photosynthetic dinoflagellates is thought to have been replaced in a few lineages by plastids of chlorophyte, diatom, or haptophyte origin. Other distinct lineages of phagotrophic dinoflagellates retain functional plastids obtained from algal prey for different durations and with varying source species specificity. 18S rRNA gene sequence analyses have placed a novel gymnodinoid dinoflagellate isolated from the Ross Sea (RSD) in the Kareniaceae, a family of dinoflagellates with permanent plastids of haptophyte origin. In contrast to other species in this family, the RSD contains kleptoplastids sequestered from its prey, Phaeocystis antarctica. Culture experiments were employed to determine whether the RSD fed selectively on P.antarctica when offered in combination with another polar haptophyte or cryptophyte species, and whether the RSD, isolated from its prey and starved, would take up plastids from P.antarctica or from other polar haptophyte or cryptophyte species. Evidence was obtained for selective feeding on P.antarctica, plastid uptake from P.antarctica, and increased RSD growth in the presence of P.antarctica. The presence of a peduncle-like structure in the RSD suggests that kleptoplasts are obtained by myzocytosis. RSD cells incubated without P.antarctica were capable of survival for at least 29.5months. This remarkable longevity of the RSD's kleptoplasts and its species specificity for prey and plastid source is consistent with its prolonged co-evolution with P.antarctica. It may also reflect the presence of a plastid protein import mechanism and genes transferred to the dinokaryon from a lost permanent haptophyte plastid.

Genus Gambierdiscus in the Canary Islands (NE Atlantic Ocean) with Description of Gambierdiscus silvae sp. nov., a New Potentially Toxic Epiphytic Benthic Dinoflagellate

Species of the dinoflagellate genus Gambierdiscus are the cause of Ciguatera Fish Poisoning, common in tropical areas. Nevertheless, until recently this syndrome was not reported in the NE Atlantic Ocean. A new photosynthetic dinoflagellate species, Gambierdiscus silvae sp. nov. is described based on samples taken from tide pools on rocky shores of the Canary Islands (NE Atlantic Ocean). Its morphology was studied by light and scanning electron microscopy. The new species is anterioposteriorly compressed, lenticular in shape with an epitheca slightly higher than the hypotheca. It is round in apical view and has a thick smooth theca with many scattered pores. Plate formula is Po, 4', 0a, 6″, 6c, 6s?, 5'″, 0p, 2″″. Plate 2' is hatchet-shaped and Plate 2″″ is very wide and the largest of the hypotheca. Phylogenies inferred from the large subunit nuclear rRNA showed that three G. silvae strains clustered in a well supported sister clade to G. polynesiensis, distinct from the other species. G. australes was observed for the first time in the Atlantic, together with G. excentricus already reported from these islands. This work increases the number of Gambierdiscus species described and shows their unexpected diversity in the Canary Islands.

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 SymbiodiniumITS2 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.

A genomic approach to coral-dinoflagellate symbiosis: studies of Acropora digitifera and Symbiodinium minutum.

Far more intimate knowledge of scleractinian coral biology is essential in order to understand how diverse coral-symbiont endosymbioses have been established. In particular, molecular and cellular mechanisms enabling the establishment and maintenance of obligate endosymbiosis with photosynthetic dinoflagellates require further clarification. By extension, such understanding may also shed light upon environmental conditions that promote the collapse of this mutualism. Genomic data undergird studies of all symbiotic processes. Here we review recent genomic data derived from the scleractinian coral, Acropora digitifera, and the endosymbiotic dinoflagellate, Symbiodinium minutum. We discuss Acropora genes involved in calcification, embryonic development, innate immunity, apoptosis, autophagy, UV resistance, fluorescence, photoreceptors, circadian clocks, etc. We also detail gene loss in amino acid metabolism that may explain at least part of the Acropora stress-response. Characteristic features of the Symbiodinium genome are also reviewed, focusing on the expansion of certain gene families, the molecular basis for permanently condensed chromatin, unique spliceosomal splicing, and unusual gene arrangement. Salient features of the Symbiodinium plastid and mitochondrial genomes are also illuminated. Although many questions regarding these interdependent genomes remain, we summarize information necessary for future studies of coral-dinoflagellate endosymbiosis.

PSI Mehler reaction is the main alternative photosynthetic electron pathway in Symbiodinium sp., symbiotic dinoflagellates of cnidarians.

Photosynthetic organisms have developed various photoprotective mechanisms to cope with exposure to high light intensities. In photosynthetic dinoflagellates that live in symbiosis with cnidarians, the nature and relative amplitude of these regulatory mechanisms are a matter of debate. In our study, the amplitude of photosynthetic alternative electron flows (AEF) to oxygen (chlororespiration, Mehler reaction), the mitochondrial respiration and the Photosystem I (PSI) cyclic electron flow were investigated in strains belonging to three clades (A1, B1 and F1) of Symbiodinium. Cultured Symbiodinium strains were maintained under identical environmental conditions, and measurements of oxygen evolution, fluorescence emission and absorption changes at specific wavelengths were used to evaluate PSI and PSII electron transfer rates (ETR). A light- and O2 -dependent ETR was observed in all strains. This electron transfer chain involves PSII and PSI and is insensitive to inhibitors of mitochondrial activity and carbon fixation. We demonstrate that in all strains, the Mehler reaction responsible for photoreduction of oxygen by the PSI under high light, is the main AEF at the onset and at the steady state of photosynthesis. This sustained photosynthetic AEF under high light intensities acts as a photoprotective mechanism and leads to an increase of the ATP/NADPH ratio.

Ultrastructure of trichocysts in Hematodinium spp. infecting Atlantic snow crab, Chionoecetes opilio

Trichocyst morphology and development were explored using transmission electron microscopy in Hematodinium spp. isolated directly from Atlantic snow crab (Chionoecetes opilio) hemolymph and from in vitro cultures. Appearance of trichocysts defines the initiation of a morphological transition in the parasites life cycle from vegetative stage to the transmission stage. Trichocysts within sporonts were found in distinct clusters near the nucleus in close apposition to the Golgi. As cells transitioned to more mature dinospores however, trichocysts were found randomly distributed throughout the cytoplasm. Clusters contained both primordial and maturing trichocysts at various stages indicating an asynchronous development. The random distribution of mature trichocysts suggests deployment to the cell membrane for future extrusion. Mature trichocysts of Hematodinium spp. appeared structurally similar to trichocysts from photosynthetic dinoflagellates. Hematodinium spp. trichocysts differed by the presence of peripheral tubules associated with novel cuboidal appendages in the apical region rather than a network of central electron dense fibres as found in photosynthetic dinoflagellates. Additionally, the trichocyst membrane of Hematodinium spp. was in close apposition to the square crystalline core.Trichocyst expulsion was not observed during our study which along with features of development and maturation within Hematodinium life stages should provide insight into proposed roles in host attachment or defense that could further our understanding of the mechanisms of pathogenesis and transmission of the parasite.

Effects of light and prey availability on Arctic freshwater protist communities examined by high-throughput DNA and RNA sequencing

Protists in high-latitude lakes are constrained by cold temperatures, low inorganic nutrient supply and low light availability for much of the year due to ice cover and polar darkness. The lengthening ice-free periods in these freshwater ecosystems due to a warming climate results in increased light availability, but the overall impacts on phytoplankton and other protists are unknown. We experimentally investigated protist community responses to changes in light and prey availability in a dilution series in Ward Hunt Lake (latitude 83°05'N), in the Canadian High Arctic. The communities at the end of the experiment were characterized using high-throughput pyrosequencing of the V4 region of the 18S rRNA gene as a measure of taxonomic presence, and of 18S rRNA (from RNA converted to cDNA) as a taxon-specific indicator of community response. At the end of the experiment under low irradiance, cDNA reads were dominated by photosynthetic dinoflagellate genera, except at the greatest dilution where Cercozoa were most abundant. In contrast, the cDNA reads in the high light treatments were dominated by chrysophytes. Given the known trophic differences among dinoflagellates, cercozoans and chrysophytes, this apparent environmental selection implies that the rise in underwater irradiance associated with increasing ice-free conditions may affect microbial food web structure and function in polar lakes.

Excitation relaxation dynamics and energy transfer in fucoxanthin–chlorophyll a/c-protein complexes, probed by time-resolved fluorescence

In algae, light-harvesting complexes contain specific chlorophylls (Chls) and keto-carotenoids; Chl a, Chl c, and fucoxanthin (Fx) in diatoms and brown algae; Chl a, Chl c, and peridinin in photosynthetic dinoflagellates; and Chl a, Chl b, and siphonaxanthin in green algae. The Fx–Chl a/c-protein (FCP) complex from the diatom Chaetoceros gracilis contains Chl c1, Chl c2, and the keto-carotenoid, Fx, as antenna pigments, in addition to Chl a. In the present study, we investigated energy transfer in the FCP complex associated with photosystem II (FCPII) of C. gracilis. For these investigations, we analyzed time-resolved fluorescence spectra, fluorescence rise and decay curves, and time-resolved fluorescence anisotropy data. Chl a exhibited different energy forms with fluorescence peaks ranging from 677nm to 688nm. Fx transferred excitation energy to lower-energy Chl a with a time constant of 300fs. Chl c transferred excitation energy to Chl a with time constants of 500–600fs (intra-complex transfer), 600–700fs (intra-complex transfer), and 4–6ps (inter-complex transfer). The latter process made a greater contribution to total Chl c-to-Chl a transfer in intact cells of C. gracilis than in the isolated FCPII complexes. The lower-energy Chl a received excitation energy from Fx and transferred the energy to higher-energy Chl a. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.

Biomarker discovery and biotherapeutics applications of photosynthetic light-harvesting and bioluminescence light-emitting chromophore-protein complexes in stem cell biology and regenerative medicine.

We have since the 1970's embarked on the development of biologically derived fluorophore-protein complexes that will find applications in the communicable and non-communicable disease etiology processes and their cures. We have since then become largely successful in these endeavors along with interspersed contributions also from investigators who have generally restricted to working in confined disciplines. Their encompassment with our works as this investigator has traversed his definitely chosen and not merely a circumstantial, coincidental, or accidental step-wise multi-disciplinary scientific path from biophysics to regenerative medicine spanning these lines of investigations for last four decades have finally yielded the much necessitated disease related applied biological interventions for human benefits. Taking a cue from our early investigations and findings on which we call attention to the identification and characterization of the use of the primary light-emitting lumazine precursor of riboflavin which is 6,7-dimethyl-8-ribityl lumazine-protein complex which we had derived from the bioluminescence bacterium (Photobacterium phosphoreum) wherein it functions as a naturally occurring fluorescence light emitter (LumP). These in vivo phenomena have been a precursor to the subsequent developments in vitro. This in vivo to in vitro investigation path of ours has been also comprised among others of binding of photosynthetic light-harvesting marine dinoflagellate algae (Glenodinium sp.) derived peridinin-chlorophyll a-protein (PerCP) complex-labeled monoclonal antibodies useful in the development of flow cytometry. These fluorescence labeled antibodies bound antigens which include those of communicable infectious diseases (HIV/AIDS - env-gp160, gag-p24), non-communicable but also potential hereditary and malignant disorders (Cancer/Tumor Markers - Melan-A/Mart-1 of melanoma), normal immune response cells (Human/Mouse/species cellular MHC/TCR/CD45/CD33/CD56/CD19/CD41), and of types of stem cells (CD34/CD38/c-Mpl/Oct4/Neuropilin-1/SOX17). Such antigens have been analyzed by us and other investigators by fluorescence-activated cell sorting (FACS - cell surface and intracellular binding), confocal fluorescence microscopy, or/and immunohistochemistry, to determine qualitative and quantitative antigen expression levels and their mechanistic implications. We have followed stem cell differentiation patterns and signaling mechanisms through marker antigen-antibody binding wherein the antibodies are labeled with covalently linked fluorophore-protein complexes or fluorescence emitting chromophores. These complexes among others also have included PerCP. We are also now in the process of developing flow cytometry applications of additional visible light emitting chromophore-protein complexes through industrial collaborations. The United States Navy has long been known for interest in the estimation of changes in illumination intensity in and under the oceans to track movements of enemy ships and other naval vessels.

Cold-Induced Cysts of the Photosynthetic DinoflagellateLingulodinium polyedrumHave an Arrested Circadian Bioluminescence Rhythm and Lower Levels of Protein Phosphorylation

Dinoflagellates are microscopic, eukaryotic, and primarily marine plankton. Temporary cyst formation is a well-known physiological response of dinoflagellate cells to environmental stresses. However, the molecular underpinnings of cold-induced cyst physiology have never been described. Cultures of the photosynthetic dinoflagellate Lingulodinium polyedrum readily form temporary cysts when placed at low (8°C±1°C) temperature and excyst to form normal motile cells following a return to normal temperature (18°C±1°C). The normal circadian bioluminescence rhythm and the expected changes in Luciferin Binding Protein abundance were arrested in L. polyedrum cysts. Furthermore, after excystment, the bioluminescence rhythm initiates at a time corresponding to zeitgeber 12, independent of the time when the cells encysted. Phosphoprotein staining after two-dimensional polyacrylamide gel electrophoresis, as well as column-based phosphoprotein enrichment followed by liquid chromatography tandem mass spectrometry, showed cyst proteins are hypophosphorylated when compared with those from motile cells, with the most marked decreases found for predicted Casein Kinase2 target sites. In contrast to the phosphoproteome, the cyst proteome is not markedly different from motile cells, as assessed by two-dimensional polyacrylamide gel electrophoresis. In addition to changes in the phosphoproteome, RNA sequencing revealed that cysts show a significant decrease in the levels of 132 RNAs. Of the 42 RNAs that were identified by sequence analysis, 21 correspond to plastid-encoded gene products and 11 to nuclear-encoded cell wall/plasma membrane components. Our data are consistent with a model in which the highly reduced metabolism in cysts is achieved primarily by alterations in the phosphoproteome. The stalling of the circadian rhythm suggests temporary cysts may provide an interesting model to address the circadian system of dinoflagellates.

CPPB-aE is discovered from photosynthetic benthic dinoflagellates.

Although chlorophyll degradation pathways in higher plants have been well studied, little is known about the mechanisms of chlorophyll degradation in microalgae. In this article, we report the occurrence of a chlorophyll a derivative that has never been discovered in photosynthetic organisms. This chlorophyll derivative emits no fluorescence and has a peculiar absorbance peak at 425, 451, 625, and 685nm. From these features, it was identified as 13(2) ,17(3) -cyclopheophorbide a enol (cPPB-aE), reported as a degradation product of chlorophyll a derived from prey algal cells in heterotrophic protists. We discovered cPPB-aE in six benthic photosynthetic dinoflagellates that are phylogenetically separated into four clades based on SSU rDNA molecular phylogeny. This is the first report of this chlorophyll derivative in photosynthetic organisms and we suggest that the derivative is used to quench excess light energy.