Bioluminescent Dinoflagellates Research Articles

Comparison of the Qwiklite™ algal bioluminescence test with marine algal growth rate inhibition bioassays

Although marine algal bioassays based on growth rate inhibition over 72-96 h have been widely used to assess the toxicity of contaminants in waters and sediments, changes in pH over the test duration can lead to changes in contaminant speciation and consequently an under- or over-estimation of toxicity. In addition, high cell densities are used in order to obtain a detectable response, further reducing the tests' environmental relevance in marine waters. There is a need for rapid acute tests with ecologically relevant test endpoints that may be used as surrogates for longer-term chronic tests. This study compares the sensitivity and reproducibility of a rapid marine dinoflagellate (Pyrocystis lunula) bioluminescence test (QwikLite) with standard algal growth rate bioassays (Nitzschia closterium and Entomoneis c.f. punctulata) using ammonia and several antifouling agents (tributyltin [TBT], copper, and diuron) as reference toxicants. QwikLite was of similar sensitivity to ammonia as standard algal growth rate tests, but was less sensitive to copper, diuron and TBT, with 24-h EC50 values of 10 +/- 1.1 mg N/L, 0.128 +/- 0.021 mg Cu/L, 19 +/- 13 mg diuron/L, and 0.226 +/- 0.028 mg TBT/L. Inter-test precision using different batches of P. lunula was generally acceptable. On the basis of NOEC values, QwikLite was more sensitive to copper and ammonia at 25 degrees C than at 21 degrees C. QwikLite shows promise as a rapid, inexpensive screening test for acute toxicity of contaminants in marine environments.

OLIGONUCLEOTIDE PRIMERS FOR THE DETECTION OF BIOLUMINESCENT DINOFLAGELLATES REVEAL NOVEL LUCIFERASE SEQUENCES AND INFORMATION ON THE MOLECULAR EVOLUTION OF THIS GENE(1).

Bioluminescence is reported in members of 18 dinoflagellate genera. Species of dinoflagellates are known to have different bioluminescent signatures, making it difficult to assess the presence of particular species in the water column using optical tools, particularly when bioluminescent populations are in nonbloom conditions. A "universal" oligonucleotide primer set, along with species and genus-specific primers specific to the luciferase gene were developed for the detection of bioluminescent dinoflagellates. These primers amplified luciferase sequences from bioluminescent dinoflagellate cultures and from environmental samples containing bioluminescent dinoflagellate populations. Novel luciferase sequences were obtained for strains of Alexandrium cf. catenella (Whedon et Kof.) Balech and Alexandrium fundyense Balech, and also from a strain of Gonyaulax spinifera (Clap. et Whitting) Diesing, which produces bioluminescence undetectable to the naked eye. The phylogeny of partial luciferase sequences revealed five significant clades of the dinoflagellate luciferase gene, suggesting divergence among some species and providing clues on their molecular evolution. We propose that the primers developed in this study will allow further detection of low-light-emitting bioluminescent dinoflagellate species and will have applications as robust indicators of dinoflagellate bioluminescence in natural water samples.

Fundamental Study for Measuring Spatial Distributions of Impact Pressure by Bioluminescence

Fundamental optical responses of bioluminescent dinoflagellates, Pyrocystis lunura under impacting water pressure were measured in the imaging experiment. Effects of biological parameters, such as fatigue andbiorhythm, to the responses were also examined. A good correlation of the bioluminescent intensity and the dynamic pressure was obtained on the basis of our proposing statistical image preprocessing, indicating a possibility to develop new bio-measurement tool for acquiring spatial distributions of hydro-dynamic pressure.

The use of bioluminescent dinoflagellates as an environmental risk assessment tool

A novel toxicity method to determine sublethal and lethal effects of manmade contaminants on the bioluminescence output from marine dinoflagellates has been developed and tested over the course of 16 years. The toxicity system, QwikLite™, was developed for the sole purpose of evaluating the potential toxicity of various materials used in bay sediments, storm water discharges, industrial discharges from Naval facilities, and antifoulant paints. Bioluminescence inhibition was observed in the following dinoflagellates: Lingulodinium polyedrum (formerly known as Gonyaulax polyedra), Ceratocorys horrida, Pyrocystis noctiluca, Pyrocystis lunula, Pyrocystis fusiformis, and Pyrophacus steinii. Cultured cells were exposed to various concentrations of contaminants from hours through 10 days. Further application with bioluminescent dinoflagellates in a variety of toxicity testing schemes have shown that these species can be used as a screening assay organism in lieu of the more costly, labor intensive bioassays presently in use.

Shear-Stress Dependence of Dinoflagellate Bioluminescence

Fluid flow stimulates bioluminescence in dinoflagellates. However, many aspects of the cellular mechanotransduction are incompletely known. The objective of our study was to formally test the hypothesis that flow-stimulated dinoflagellate bioluminescence is dependent on shear stress, signifying that organisms are responding to the applied fluid force. The dinoflagellate Lingulodinium polyedrum was exposed to steady shear using simple Couette flow in which fluid viscosity was manipulated to alter shear stress. At a constant shear rate, a higher shear stress due to increased viscosity increased both bioluminescence intensity and decay rate, supporting our hypothesis that bioluminescence is shear-stress dependent. Although the flow response of non-marine attached cells is known to be mediated through shear stress, our results indicate that suspended cells such as dinoflagellates also sense and respond to shear stress. Shear-stress dependence of flow-stimulated bioluminescence in dinoflagellates is consistent with mechanical stimulation due to direct predator handling in the context of predator-prey interactions.

Evidence for the role of G-proteins in flow stimulation of dinoflagellate bioluminescence

Luminescent dinoflagellates respond to flow by the production of light. The primary mechanotransduction event is unknown, although downstream events include a calcium flux in the cytoplasm, a self-propagating action potential across the vacuole membrane, and a proton flux into the cytoplasm that activates the luminescent chemistry. Given the role of GTP-binding (G) proteins in the mechanotransduction of flow by nonmarine cells and the presence of G-proteins in dinoflagellates, it was hypothesized that flow-stimulated dinoflagellate bioluminescence involves mechanotransduction by G-proteins. In the present study, osmotic swelling of cells of the dinoflagellate Lingulodinium polyedrum was used as a drug delivery system to introduce GDPbetaS, an inhibitor of G-protein activation. Osmotically swollen cells produced higher levels of flow-stimulated bioluminescence at a lower threshold of shear stress, indicating they were more flow sensitive. GDPbetaS inhibited flow-stimulated bioluminescence in osmotically swollen cells and in cells that were restored to the isosmotic condition following hypoosmotic treatment with GDPbetaS. These results provide evidence that G-proteins are involved in the mechanotransduction of flow in dinoflagellates and suggest that G-protein involvement in mechanotransduction may be a fundamental evolutionary adaptation.

Variability in the bioluminescence response of the dinoflagellate Pyrocystis lunula

Light emission in dinoflagellates is induced by water motions. But although it is known that mechanical stimulations of these organisms trigger the bioluminescent response, the exact mechanism that involves some cell membrane excitations by fluid motions is not yet fully understood and is still controversial. We show in this experimental study that the accelerated shear flow, created by abrupt rotations of one or both co-axial cylinders of a Couette shearing chamber excites the light emission from cultured dinoflagellates Pyrocystis lunula. Following our first results published earlier that state that pure laminar shear does not excite the main bioluminescent response in dinoflagellates, our present experiments show that both shear and acceleration in the flow are needed to trigger the bioluminescent response. Besides, the probability to stimulate this bioluminescent response under acceleration and shear is deduced from the response curves. This response follows a Gaussian distribution that traduces a heterogeneity in individual cell thresholds for the stimulation of bioluminescence in a dinoflagellate population. All these results will have a repercussion in the possible applications of dinoflagellate bioluminescence in flow visualizations and measurements. Moreover, this study opens a new way in studying mechanically-induced stimulus thresholds at the cell level.

Two different domains of the luciferase gene in the heterotrophic dinoflagellate Noctiluca scintillans occur as two separate genes in photosynthetic species

Noctiluca scintillans , a heterotrophic unarmored unicellular bioluminescent dinoflagellate, occurs widely in the oceans, often as a bloom. Molecular phylogenetic analysis based on 18S ribosomal DNA sequences consistently has placed this species on the basal branch of dinoflagellates. Here, we report that the structural organization of its luciferase gene is strikingly different from that of the seven luminous species previously characterized, all of which are photosynthetic. The Noctiluca gene codes for a polypeptide that consists of two distinct but contiguous domains. One, which is located in the N-terminal portion, is shorter than but similar in sequence to the individual domains of the three-domain luciferases found in all other luminous dinoflagellates studied. The other, situated in the C-terminal part, has sequence similarity to the luciferin-binding protein of the luminous dinoflagellate Lingulodinium polyedrum , encoded there by a separate gene. Western analysis shows that the native protein has the same size (≈100 kDa) as the heterologously expressed polypeptide, indicating that it is not a polyprotein. Thus, sequences found in two proteins in the L. polyedrum bioluminescence system are present in a single polypeptide in Noctiluca .

Profile of J. Woodland Hastings.

J. Woodland Hastings, elected to the National Academy of Sciences in 2003, reports in his Inaugural Article in this issue of PNAS on the structure of the luciferase gene in Noctiluca scintillans, one of the largest and most primitive bioluminescent dinoflagellates (1). Hastings' work shows that Noctiluca's luciferase possesses domains for both catalysis and substrate binding, whereas in some previously described dinoflagellate species, these domains occur as separate proteins.

Real‐time monitoring of nutrient concentrations and red‐tide outbreaks in the southern sea of Korea

In order to determine the physical and chemical factors controlling the outbreak of red tides, we monitored nutrients and other environmental parameters using a novel real‐time monitoring buoy system during the summer of 2003 in the southern sea of Korea, where red‐tide outbreaks occur every year. The real‐time monitoring data on bioluminescence, which may indicate the presence of bioluminescent dinoflagellate species, showed a sudden increase under the lowest concentrations of dissolved inorganic nutrients. Our monitoring system for the first time provides real‐time variations in nutrients and associated outbreaks of dinoflagellate red tides. This result supports the previous hypothesis by others that the outbreak of dinoflagellate red tides is associated with the limited growth of diatoms under depleted DIP or DIN conditions. We suggest that this real‐time monitoring system can be utilized as a powerful tool for studying and predicting harmful dinoflagellate red tides in the coastal ocean.

A quantitative model for flow-induced bioluminescence in dinoflagellates

A model is presented for the flash response of bioluminescent dinoflagellates stimulated by fluid shear. The model is based on the idea that the response of an individual cell to stimulation is inherently probabilistic, and can be modeled as a Poisson process over short time scales. A new cell parameter, the cell anxiety, is introduced to parameterize the probability of flashing. The statistical model is incorporated into a description of fully developed fluid flow in pipes and a cylindrical Couette chamber, and found to compare favorably with previously published data from experiments.

Bioluminescent response of the dinoflagellate Lingulodinium polyedrum to developing flow: Tuning of sensitivity and the role of desensitization in controlling a defensive behavior of a planktonic cell

Dinoflagellate bioluminescence is believed to serve a defensive function, decreasing grazing at night. Previous characterization of bioluminescence stimulated by fully developed flows might have underestimated the true sensitivity of bioluminescence by not observing the initial response. Also, it has been suggested that bioluminescence may be more sensitive to time-varying flow than to constant flow conditions. We used developing laminar Couette flow to characterize the sensitivity of the initial bioluminescent response of the dinoflagellate Lingulodinium polyedrum in time-varying flow. Both the absolute sensitivity (threshold) and dynamic sensitivity were consistent with that determined previously in fully developed flows, although there were differences between different cultured isolates of the same species and between those isolates and cells harvested from a unialgal bloom of the same species. When the rate of increase of shear was varied while keeping the maximum shear level similar, the threshold was independent of the rate of increase of shear. Surprisingly, the integrated bioluminescence was strongly dependent on the rate of increase of shear. The mechanism behind the preferential response to rapidly increasing shear was determined to be desensitization. Desensitization may influence which naturally occurring flows strongly stimulate bioluminescence either by allowing cells to avoid producing a primary response in certain slowly changing flows or, more generally, to avoid the cost of repeated stimulation when entrained in environmental flows containing above-threshold shears.

Dinoflagellate bioluminescence in response to mechanical stimuli in water flows

Abstract. Bioluminescence of plankton organisms induced by water movements has long been observed and is still under investigations because of its great complexity. In particular, the exact mechanism occurring at the level of the cell has not been yet fully understood. This work is devoted to the study of the bioluminescence of the dinoflagellates plankton species Pyrocystis noctiluca in response to mechanical stimuli generated by water flows. Several experiments were performed with different types of flows in a Couette shearing apparatus. All of them converge to the conclusion that stationary homogeneous laminar shear does not trigger massive bioluminescence, but that acceleration and shear are both necessary to stimulate together an intense bioluminescence response. The distribution of the experimental bioluminescence thresholds is finally calculated from the light emission response for the Pyrocystis noctiluca species.

Crystal structure of a pH-regulated luciferase catalyzing the bioluminescent oxidation of an open tetrapyrrole

The luciferase of Lingulodinium polyedrum, a marine bioluminescent dinoflagellate, consists of three similar but not identical domains in a single polypeptide. Each encodes an active luciferase that catalyzes the oxidation of a chlorophyll-derived open tetrapyrrole (dinoflagellate luciferin) to produce blue light. These domains share no sequence similarity with any other in the GenBank database and no structural or motif similarity with any other luciferase. We report here the 1.8-A crystal structure of the third domain, D3, at pH 8, and a mechanism for its activity regulation by pH. D3 consists of two major structural elements: a beta-barrel pocket putatively for substrate binding and catalysis and a regulatory three-helix bundle. N-terminal histidine residues previously shown to regulate activity by pH are at the interface of the helices in the bundle. Molecular dynamics calculations indicate that, in response to changes in pH, these histidines could trigger a large molecular motion of the bundle, thereby exposing the active site to the substrate.

Dinoflagellates: a remarkable evolutionary experiment

In this paper, we focus on dinoflagellate ecology, toxin production, fossil record, and a molecular phylogenetic analysis of hosts and plastids. Of ecological interest are the swimming and feeding behavior, bioluminescence, and symbioses of dinoflagellates with corals. The many varieties of dinoflagellate toxins, their biological effects, and current knowledge of their origin are discussed. Knowledge of dinoflagellate evolution is aided by a rich fossil record that can be used to document their emergence and diversification. However, recent biogeochemical studies indicate that dinoflagellates may be much older than previously believed. A remarkable feature of dinoflagellates is their unique genome structure and gene regulation. The nuclear genomes of these algae are of enormous size, lack nucleosomes, and have permanently condensed chromosomes. This chapter reviews the current knowledge of gene regulation and transcription in dinoflagellates with regard to the unique aspects of the nuclear genome. Previous work shows the plastid genome of typical dinoflagellates to have been reduced to single-gene minicircles that encode only a small number of proteins. Recent studies have demonstrated that the majority of the plastid genome has been transferred to the nucleus, which makes the dinoflagellates the only eukaryotes to encode the majority of typical plastid genes in the nucleus. The evolution of the dinoflagellate plastid and the implications of these results for understanding organellar genome evolution are discussed.

Bioluminescence response of four species of dinoflagellates to fully developed pipe flow

Dinoflagellate bioluminescence provides a nearly instantaneous index of flow sensitivity. This study compared flow sensitivity in four species of morphologically diverse luminescent dinoflagellates (Ceratium fusus, Ceratocorys horrida, Lingulodinium polyedrum and Pyrocystis fusiformis) using fully developed laminar and turbulent pipe flow. Bioluminescence response thresholds always occurred in laminar flows with wall shear stress levels that, depending on species, ranged from 0.02 to 0.3 N m � 2 . With few exceptions, such as breaking waves and wave-forced bottom shears in shallow nearshore areas, these threshold shear stress levels are several orders of magnitude larger than typical oceanic ambient flows. For laminar flows above threshold, species also differed in the proportion of organisms responding and the minimum shear stress level where individual flashes reached their highest intensity. Following transition to turbulent flow, there was never a dramatic increase in bioluminescence, even when energetic turbulent length scales were similar to the cell size. On the basis of their bioluminescence response in laminar flow, these species were ranked in order of decreasing sensitivity as C. horrida > P. fusiformis > C. fusus > L. polyedrum. This ranking, though not conclusive, is consistent with increased flow sensitivity due to increasing size and the presence of spines. With the exception of a small fraction of the C. horrida population that is sensitive enough to flash within the feeding current of a predator, the present study suggests that flashes only occur with predator contact. Nevertheless, flow sensitivity may serve as an index of the response to mechanical agitation during predator contact/handling. Flow sensitivity may be constrained to maximize the response to predator contact/handling while minimizing stimulation by background oceanic flows to avoid depleting luminescent reserves.

Bioluminescence of the dinoflagellate Pyrocystis noctiluca induced by laminar and turbulent Couette flow

The excitation of bioluminescence by different flow regimes generated within a Couette chamber was examined using the dinoflagellates Pyrocystis noctiluca. Cultured cells of P. noctiluca were gently transferred into a cylindrical Couette chamber in a dark room. In initial experiments, the velocity of the outer Couette cylinder was then gradually increased. The bioluminescence emissions in response to stationary-laminar and turbulent flows were quantified using a photomultiplier tube. Video images were also recorded in order to identify the location of bioluminescence emissions within the Couette chamber. Reflective flake flow visualizations were used to correlate these locations to the flow regimes in those parts of the chamber. These experiments clearly demonstrated that the strongest bioluminescence emissions were only triggered by the onset of turbulence at high rotation speeds. Below the turbulence threshold, much lower bioluminescence emissions were detected and appeared to be in response to a nonhomogeneity in the stationary-laminar flow (end cap effects and Ekman cells). In a second set of experiments, the excitation of bioluminescence in response to acceleration was studied by abrupt starts of the rotating Couette cylinder. These experiments also triggered massive bioluminescence emissions. We conclude that pure laminar-stationary, homogenous shear flow excites very little bioluminescence in P. noctiluca. The bulk of bioluminescence emissions primarily occurred under nonhomogenous or nonstationary flow conditions, where the cells experience velocity changes as they move through the flow. These findings are discussed in relation to the theory that bioluminescence in dinoflagellates is an antipredation mechanism.

Bioluminescence imaging of wave‐induced turbulence

The ability to measure turbulent processes on small spatial and temporal scales is a long standing problem in physical oceanography. Here we explore a novel means of measuring fluid shear stress using the cell flashing behavior of bioluminescent dinoflagellates. To illustrate this technique, we present estimates of the heterogeneous, time‐varying shear stress inside a breaking wave crest. These results have implications for a better understanding of upper ocean wave physics, air‐sea gas transfer, and the biology of planktonic near‐surface organisms as well as providing a new quantitative fluid visualization tool.

Coastal bioluminescent marine snow: fine structure of bioluminescence distribution

To determine if bioluminescent organisms were differentially concentrated within marine snow relative to ambient water, individual aggregates were photographed in situ and collected by hand using SCUBA, and mechanically stimulable bioluminescence (BL) was measured with an integrating sphere photomultiplier system. These are among the first measurements of BL on marine snow, and are the first quantitative estimates of BL marine snow over the water column in a coastal environment (East Sound, WA). BL in marine snow and ambient water varied significantly over short time scales (1–11 days) and vertical spatial scales (1–5 m resolution). BL was enriched up to 180 times within aggregates as compared to an equal volume of surrounding seawater. However, the contribution of BL associated with marine snow to total BL in the water column varied between <1%, due to the relatively low total volume occupied by marine snow in the water column, and 78%, when the majority of total BL was associated with marine snow. Even though BL marine snow was relatively rare at times, these aggregates produced BL many times brighter than individual dinoflagellates. Although BL on aggregates was correlated with depth, the brightest aggregate BL was not consistently found at the thermocline, a region where marine snow concentrations are frequently high. Aggregate mass was found to be a significant predictor of aggregate BL for detritus-based marine snow. The heterotrophic dinoflagellate, Protoperidinium leonis, made the largest contribution to both BL in aggregates and to the overall water column. Concentrations of the bioluminescent dinoflagellates Protoperidinium spp. and Noctiluca scintillans were significantly correlated with BL per aggregate as a function of depth, date, and type of marine snow. Because BL has been shown to deter herbivore grazing on individual BL dinoflagellates, BL-enriched marine snow may likewise, avoid consumption and play an important role in coastal carbon cycling and food web structure.

Antioxidant properties of the melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK): scavenging of free radicals and prevention of protein destruction

In numerous experimental systems, the neurohormone melatonin has been shown to protect against oxidative stress, an effect which appears to be the result of a combination of different actions. In this study, we have investigated the possible contribution to radical scavenging by substituted kynuramines formed from melatonin via pyrrole ring cleavage. N1-Acetyl-5-methoxykynuramine (AMK), a metabolite deriving from melatonin by mechanisms involving free radicals, exhibits potent antioxidant properties exceeding those of its direct precursor N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and its analog N1-acetylkynuramine (AK). Scavenging of hydroxyl radicals was demonstrated by competition with ABTS in a Fenton reaction system at pH 5 and by competition with DMSO in a hemin-catalyzed H2O2 system at pH 8. Under catalysis by hemin, oxidation of AMK was accompanied by the emission of chemiluminescence. AMK was a potent reductant of ABTS cation radicals, but, in the absence of catalysts, a poor scavenger of superoxide anions. In accordance with the latter observation, AMK was fairly stable in a pH 8 H2O2 system devoid of hemin. Contrary to AFMK, AMK was easily oxidized in a reaction mixture generating carbonate radicals. In an oxidative protein destruction assay based on peroxyl radical formation, AMK proved to be highly protective. No prooxidant properties of AMK were detected in a sensitive biological test system based on light emission by the bioluminescent dinoflagellate Lingulodinium polyedrum. AMK may contribute to the antioxidant properties of the indolic precursor melatonin.