Henry G. Trapido-Rosenthal

Molecular Quantification of Symbiotic Dinoflagellate Algae of the Genus Symbiodinium

The dinoflagellate microalga Symbiodinium is the dominant algal symbiont in corals and related marine animals. To explore the incidence of mixed infections, methods employing real-time quantitative polymerase chain reaction (QPCR) and fluorescence in situ hybridization (FISH) were developed. In experiments focusing on Symbiodinium clades A and B, QPCR and FISH results were well correlated and generally more precise and sensitive than those from the endpoint PCR–restriction fragment length polymorphism analysis (PCR-RFLP) traditionally used for this application, thus increasing the detected incidence of mixed infections. For example, the prevalence of mixed infections in the sea anemone Condylactis gigantea was 40% by PCR-RFLP and 80%–90% by QPCR and FISH. However, the use of QPCR and FISH was limited by inter-host variation in the rRNA gene copy number per Symbiodinium cell, precluding any single conversion factor between QPCR signal and Symbiodinium cell number; and one FISH probe that gave excellent hybridization efficiency with cultured Symbiodinium yielded variable results with Symbiodinium from symbioses. After controlling for these caveats, QPCR studies revealed that field-collected hosts previously described as universally unialgal bore up to 1.6% of the alternative clade. Further research is required to establish the contribution that algal cells at low density in symbiosis and external to the symbiosis make to the minor clade.

Glutamine synthetase is a glial-specific marker in the olfactory regions of the lobster (Panulirus argus) nervous system.

Glutamine synthetase (GS) has been qualified as a very specific marker of astroglial‐type neuroglia in vertebrate neural tissues. In this paper we have begun to examine the possibility that glial localization of GS could be a ubiquitous characteristic of complex nervous systems. To this end we have used immunohistochemistry to localize GS‐like immunoreactivity in the olfactory regions of the complex nervous system of the arthropod, the spiny lobster Panulirus argus. We describe a novel method for affinity isolation of antibodies from crude serum. Using this approach we purified GS‐specific antibodies to chick retina GS and used these to analyze the lobster brain and the primary olfactory organ. Western blots showed that the lobster brain contains an immunoreactive peptide with nearly the same molecular mass as that of chick retina GS. Northern blot analyses of mRNA and enzymatic activity assays also confirm that the lobster brain produces GS. Immunohistochemical staining of sectioned lobster olfactory lobes and sensory sensilla showed strong reactivity in specific cells. Comparison of the GS immunostaining pattern with that for FMRFamide, a well characterized marker of neurons in invertebrate neural tissues, it became clear that GS is indeed glial‐specific in lobster neural tissues as it is in vertebrates. These results suggest that the compartmentalization of GS in non‐neuronal cells is either an early step in neural evolution or is an obligate and fundamental characteristic of complex neural systems composed of both neurons and neuroglia. GLIA 20:275–283, 1997.

Importance of Time and Place: Patterns in Abundance of Symbiodinium Clades A and B in the Tropical Sea Anemone Condylactis gigantea

The capacity of some corals and other cnidarians to form symbioses with multiple algae (Symbiodinium) is a candidate route by which these symbioses tolerate variable environmental conditions. On Bermuda, the coral reef dwelling anemone Condylactis gigantea bears Symbiodinium of clades A and B. At thermally variable inshore and nearshore sites, clade A predominates (as sole symbiont or in mixed infection with clade B), whereas animals at offshore sites with more uniform temperatures bear only clade B or mixed infections. Individual animals at one nearshore site monitored over a year by sampling tentacles showed increased prevalence of clade A in March-November, when sea waters were warm (average 26 °C), and increased clade B in November-March when cool waters prevailed (average 18.5 °C). In laboratory analyses of excised tentacles, the symbiosis with clade B, but not clade A, bleached at elevated temperature (32 °C), suggesting that thermal tolerance may contribute to the higher prevalence of clade A at inshore/nearshore sites and in the summer. The temporal changes in the algal complement were not accompanied by bleaching, and Symbiodinium density fluctuated in hosts with stable Symbiodinium composition but not in hosts with variable composition. This suggests that changes in the relative abundance of Symbiodinium clades do not require bleaching and may even protect the symbiosis from large fluctuations in algal density.

Nitric Oxide and Cnidarian-Dinoflagellate Symbioses: Pieces of a Puzzle1

Abstract The presence of nitric oxide synthase (NOS) activity is demonstrated in the tropical marine cnidarian Aiptasia pallida and in its symbiotic dinoflagellate algae, Symbiodinium bermudense. Enzyme activity was assayed by measuring the conversion of arginine to citrulline. Biochemical characterization of NOS from Aiptasia was characterized with respect to cellular localization, substrate and cofactor requirements, inhibitors, and kinetics. In response to acute temperature shock, anemones retracted their tentacles. Animals subjected to such stress had lower NOS activities than did controls. Treatment with NOS inhibitors caused tentacular retraction, while treatment with the NOS substrate L-arginine inhibited this response to stress, as did treatment with NO donors. These results provide a preliminary biochemical characterization of, and suggest a functional significance for, NOS activity in anthozoan-algal symbiotic assemblages.

Characterisation of nitric oxide synthase activity in the tropical sea anemone Aiptasia pallida

The presence of nitric oxide synthase (EC 1.14.23 NOS) activity is demonstrated in the tropical marine cnidarian Aiptasia pallida (Verrill). Enzyme activity was assayed by measuring the conversion of [3H]arginine to [3H]citrulline. Optimal NOS activity was found to require NADPH. Activity was inhibited by the competitive NOS inhibitor NG-methyl-L-arginine (L-NMA), but not the arginase inhibitors L-valine and L-ornithine. NOS activity was predominantly cytosolic, and was characterised by a Km for arginine of 19.05 microM and a Vmax of 2.96 pmol/min per microgram protein. Histochemical localisation of NOS activity using NADPH diaphorase staining showed the enzyme to be predominantly present in the epidermal cells and at the extremities of the mesoglea. These results provide a preliminary biochemical characterisation and histochemical localisation of NOS activity in A. pallida, an ecologically important sentinel species in tropical marine ecosystems.

The Efflux of Amino Acids from the Olfactory Organ of the Spiny Lobster: Biochemical Measurements and Physiological Effects

The amino acids taurine and glycine are odorants that activate specific chemosensory cells in the olfactory sensilla (aesthetascs) of the spiny lobster, Panulirus argus. We show that the aesthetascs themselves contain large intracellular concentrations of taurine (≈2 mM) and glycine (≈ 85 mM); these concentrations are more than 10,000-fold greater than the response thresholds of the chemosensory cells. A net efflux of at least five amino acids occurs when the olfactory organ is immersed in amino acid-free seawater. With taurine and glycine, efflux continues until an apparent equilibrium is reached between the sensilla and the external medium; for taurine the equilibrium with seawater occurs at ≈12 to 28 nM, and for glycine at ≈100 to 500 nM. Aesthetascs may achieve these equilibria within 300 ms. Hence, even during the brief interval between consecutive flicks of the antennule, olfactory receptors are exposed to a background of odorants escaping from intracellular stores. Electrophysiological studies show that both the spontaneous and evoked activities of taurine-sensitive chemosensory cells are markedly affected by a taurine background simulating that measured in the efflux studies. Uptake systems may participate in establishing the equilibria between sensilla and seawater since (1) the net efflux of amino acids increases in sodium-free seawater; and (2) guanidinoethane sulfonate, a competitor for taurine uptake, selectively increases net taurine efflux. Effluxes from an olfactory organ may contribute noise to the chemosensory process; alternatively, background substances could contribute functionally by affecting membrane proteins.

Development of nitric oxide and nitric oxide synthase as ecotoxicological biomarkers in the tropical marine environment

Abstract The enzyme nitric oxide synthase (NOS) catalyses reactions that result in the generation of nitric oxide (NO), a multipotent signal molecule. We have begun to characterise the activity of NOS found in the host tissue of the symbiotic tropical/sub-tropical anemone Aiptasia pallida , and have monitored changes in the activity of this enzyme under different environmental conditions. Our characterisation studies indicate that the NOS activity from Aiptasia is similar to activities from other organisms in terms of cellular localisation, cofactor requirements, and inhibition profiles. Our environmental experiments indicate that: (1) the incubation of whole animals at temperatures representative of the highest and lowest extremes to which the Bermuda population would be exposed results in an NOS activity reduction relative to animals maintained at the control temperature of 25 °C; and (2) treatment with copper produces variable changes in NOS activity relative to untreated controls.