Bioluminescent Dinoflagellates Research Articles

Effects of salinity, pH and temperature on the re-establishment of bioluminescence and copper or SDS toxicity in the marine dinoflagellate Pyrocystis lunula using bioluminescence as an endpoint

Pyrocystis lunula is a unicellular, marine, photoautotrophic, bioluminescent dinoflagellate. This organism is used in the Lumitox ® bioassay with inhibition of bioluminescence re-establishment as the endpoint. Experiments determined if acute changes in pH, salinity, or temperature had an effect on the organisms’ ability to re-establish bioluminescence, or on the bioassay's potential to detect sodium dodecyl sulfate (SDS) and copper toxicity. The re-establishment of bioluminescence itself was not very sensitive to changes in pH within the pH 6–10 range, though reducing pH from 8 to levels below 6 decreased this capacity. Increasing the pH had little effect on Cu or SDS toxicity, but decreasing the pH below 7 virtually eliminated the toxicity of either compound in the bioassay. Lowering the salinity from 33 to 27‰ or less resulted in a substantial decrease in re-establishment of bioluminescence, while increasing the salinity to 43 or 48 ‰ resulted in a small decline. Salinity had little influence on the bioassay's quantification of Cu toxicity, while the data showed a weak negative relationship between SDS toxicity and salinity. Re-establishment of bioluminescence showed a direct dependence on temperature, but only at 10 °C did temperature have an obvious effect on the toxicity of Cu in this bioassay.

The Function of Circadian RNA‐Binding Proteins and Their cis‐Acting Elements in Microalgae

An endogenous clock regulates the temporal expression of genes/mRNAs that are involved in the circadian output pathway. In the bioluminescent dinoflagellate Gonyaulax polyedra circadian expression of the luciferin‐binding protein (LBP) is controlled at the translational level. Thereby, a clock‐controlled RNA‐binding protein, called circadian controlled translational regulator (CCTR), interacts specifically with an UG‐repeat, which is situated in the lbp 3′ UTR. Its binding activity correlates negatively with the amount of LBP during a circadian cycle. In the green alga Chlamydomonas reinhardtii, a clock‐controlled RNA‐binding protein (CHLAMY 1) was identified, which represents an analog of the CCTR from the phylogenetically diverse alga G. polyedra. CHLAMY 1 binds specifically to the 3′ UTRs of several mRNAs and recognizes them all via a common cis‐acting element, composed of at least seven UG‐repeats. The binding strength of CHLAMY 1 is strongest to mRNAs, whose products are key components of nitrogen metabolism resulting in arginine biosynthesis as well as of CO2 metabolism. Since temporal activities of processes involved in nitrogen metabolism have an opposite phase than CHLAMY 1 binding activity, the protein might repress the translation of the cognate mRNAs.

Hydromechanical stimulation of bioluminescent plankton

The response of the bioluminescent dinoflagellate Pyrocystis fusiformis was investigated for different hydraulic conditions ('hydromechanical stimulation'). Pipe flow and oscillating shear produced luminescence, whereas changes in hydrostatic pressure were not stimulating. More intense fluid motion led to higher intensity, mainly due to a higher probability of cell response. The organism was also able to emit light in a glucose-salt mixture. The experiments suggest that the cells are effectively stimulated if the flow conditions change in time.

Variations nycthémérales de la bioluminescence marine en Méditerranée et dans l’Atlantique nord-est

Bioluminescence measurements have been made using a bathyphotometer allowing the determination of stimulated light intensities down to 2 000 m depth, in the Mediterranean Sea on the Almeria–Oran front, during the winter 1997–1998, and in the northeastern Atlantic, on the Armorican continental shelf, during summers 1999 and 2000. Bioluminescence is weaker in the Mediterranean than in the Atlantic. In the epipelagic waters, day/night variations appear clearly, stimulated bioluminescence is higher at night than during the day. These diel variations can be explained by vertical migration of bioluminescent organisms and by photoinhibition of dinoflagellate bioluminescence. Fluorescence measurements made at the same time give information about potential bioluminescent sources, autotrophic and heterotrophic.

BIOLUMINESCENCE IN THE MARINE DINOFLAGELLATE PYROCYSTIS LUNULA

Marine dinoflagellates are the main contributors to a phenomenon commonly known as “phosphorescence of the sea”. Several investigators using different dinoflagellate species have shown that dinoflagellate bioluminescence is regulated by a circadian rhythm. Diverse bioluminescent systems exist in different organisms; however, all systems utilize an enzyme luciferase and the substrates luciferins, which show inter‐species cross‐reactivity. In marine dinoflagellates, luciferase and luciferin are localized in specific organelles, termed scintillons, which are usually located in close proximity to the vacuole during their active state. Bioluminescence is stimulated by mechanical stress or by acidification of the medium. Nicolas and co‐workers (1987, J. Cell Biol. 105: 723‐735) suggested that bioluminescence is triggered by an acidification of the scintillon caused by events that depolarize the tonoplast (proton trigger model). However, little is known about the signal transduction cascade that relays the mechanical stimulus perceived at the plasma membrane to the tonoplast. This study investigates the response of the bioluminescent system of Pyrocystis lunula to various parameters such as age of culture, light quality and quantity, disturbance (handling), sensitivity to chemicals, etc. to determine, if the bioluminescent response of P. lunula suitable for measuring environmental toxicants. The assay system used measures bioluminescence in response to mechanical stimuli or acidification allowing us to determine, if the response varies amongst the two different types of stimulation under different experimental conditions. The use of cell‐permeant inhibitors for certain signaling pathways in this assay may help to biochemically characterize events involved in signal transduction from the plasma membrane to the scintillon.

Luminescent response of the red tide dinoflagellate Lingulodinium polyedrum to laminar and turbulent flow

While it is universally accepted that plankton continually experience a dynamic fluid environment, their sensitivity to the features of the surrounding flow field at the relevant length and time scales of the organism is poorly characterized. The present study uses bioluminescence as a tool to understand how the red tide dinoflagellate Lingulodinium polyedrum (= Gonyaulax polyedra) responds to well‐characterized hydrodynamic forces present in fully developed laminar and turbulent pipe flow. The response of L. polyedrum to hydrodynamic stimulation was best characterized by wall shear stress; at similar values of wall shear stress, the response was similar for laminar and turbulent flows. The response threshold occurred in laminar flow at a wall shear stress of approximately 0.3 N m−2. At these low flow rates, video analysis of the velocity of flash trajectories revealed that responding cells were positioned only near the pipe wall, where local shear stress levels were equal to or greater than threshold. For cell concentrations ranging over four orders of magnitude, threshold values of wall shear stress were restricted to a narrow range, consistent with an antipredation function for dinoflagellate bioluminescence. For laminar flows with above‐threshold wall shear stress values ≤= 1 N m−2, mean bioluminescence increased with wall shear stress according to a power (log–log) relationship, with the slope of the power function dependent on cell concentration. The increase in bio‐luminescence within this range was due primarily to an increasing population response rate and, to a lesser extent, an increase in maximum flash intensity per cell and the increased flux of organisms with higher flow rates. For wall shear stress levels > 1 N m−2, the maximum intensity per cell remained approximately constant with increasing wall shear stress, even as the flow transitioned from laminar to turbulent, and the smallest turbulent length scales became less than the average cell size.

Dolphins glow with the flow

The dynamics of flow over the surface of animals — ranging from insects in flight to swimming fish — have been examined using optical methods and markers such as dye, air bubbles and smoke. At the same time, amateur and professional seafarers (and biologists) have considered themselves extremely lucky if they have seen the firework displays produced by dolphins swimming in waters that contain high levels of bioluminescent plankton (Fig. 1). Now, physicist Jim Rohr, biologist Michael Latz and their colleagues have ingeniously combined the two approaches, and they report their findings in the Journal of Experimental Biology1. The authors analysed the light output that is induced from natural populations of bioluminescent dinoflagellates by free-swimming dolphins, and they have used the results to interpret some of the characteristics of flow over these animals.

Circadian rhythmicity of tryptophan hydroxylase in the marine dinoflagellate gonyaulax polyedra stein and the role of tryptophan hydroxylation in bioluminescence

In the bioluminescent dinoflagellate Gonyaulax polyedra, tryptophan hydroxylase activity exhibits a circadian rhythm of high amplitude, which persists in constant light. The maximum is found in the middle of photophase. Melatonin or exposure to a decreased temperature (15°C), a treatment leading to melatonin accumulation, suppresses tryptophan hydroxylase activity. 5-Hydroxytryptophan, but not tryptophan, stimulates bioluminescence. The dosage dependence of this effect of 5-hydroxytryptophan is similar to that of its metabolite serotonin. These actions seem to be due to the further conversion to a much more potent metabolite, 5-methoxytryptamine. Inhibition of tryptophan hydroxylase by p-chlorophenylalanine, p-fluorophenylalanine or 5-fluoro-tryptophan decreases light emission. This inhibition can be overcome by addition of various indolic metabolites originating from the tryptophan hydroxylation pathway, in particular, 5-hydroxytryptophan, serotonin, melatonin and 5-methoxytryptamine.

Cephalopod Predation Facilitated by Dinoflagellate Luminescence.

Predation by nocturnal cephalopods on nonluminous prey was examined in the presence of dinoflagellate bioluminescence. Sepia officinalis Linnaeus and Euprymna scolopes Berry were tested for predation efficiency in darkness illuminated by the luminescent dinoflagellate Pyrocystis fusiformis Murry. Prey were mysids, Holmesimysis sculpta (Tattersall); grass shrimp, Palaemonetes pugio Holthuis; and mosquito fish, Gambusia affinis Baird and Girard. Tests were conducted in aquaria containing 0-20 cells ml-1 of P. fusiformis. Predation increased as numbers of luminescent dinoflagellates increased. Controls were predation tests in the presence of P. fusiformis during nonluminescent photophase or in the absence of dinoflagellates. Movements of squid and prey readily stimulated luminescence. Behavior and correlated luminescence in infrared-illuminated aquaria were recorded by image-intensified and infrared video cameras. Sepia strikes on prey were common under luminescent conditions--85% occurred in less than 10 min; but strikes in darkness were rare. E. scolopes attacked more frequently than Sepia, and almost 90% obtained prey under luminescent conditions. This study demonstrates the ability of squid to use dinoflagellate bioluminescence to locate and capture nonluminous prey. The burglar alarm theory of the adaptive significance of dinoflagellate bioluminescence is supported.

AUTOMATED MECHANICAL STIMULATION AND MEASUREMENT OF BIOLUMINESCENCE IN MARINE DINOFLAGELLATES

AbstractA new integrated system for reproducible, automated mechanical stimulation and measurement of bioluminescence (BL) in multiple samples of marine dinoflagellate cell suspensions is described. The system was designed to allow the application of standardized experimental routines to parallel test vials for the purpose of toxicity testing. A sample tray delivered test vials to the position of mechanical stimulation and BL measurement. Mechanical stimulation of BL was applied as sharp rotation‐onset of the test vial about its vertical axis. Thus, any direct chemical or physical perturbation of the cell suspension was avoided. A silicon photovoltaic cell measured the emitted light. Stimulation, measurement and recording of BL were integrated and controlled by specially developed software, which runs on a personal computer in the graphic environment of MS‐Windows. Precise scheduling, flexible programming and identical repetition of experimental routines are possible in practice. For Gonyaulax polyedra, details of BL, as stimulated and measured with the new system, are presented and discussed. We conclude that the system exhibits specific features that offer wide potential of application in several fields of research on dinoflagellate BL, particularly for toxicity testing.

On the primary functions of melatonin in evolution: mediation of photoperiodic signals in a unicell, photooxidation, and scavenging of free radicals.

Melatonin is widely abundant in many eukaryotic taxa, including various animal phyla, angiosperms, and unicells. In the bioluminescent dinoflagellate Gonyaulax polyedra, melatonin is produced in concentrations sometimes exceeding those found in the pineal gland, exhibits a circadian rhythm with a pronounced nocturnal maximum, and mimics the short-day response of asexual encystment. Even more efficient as a cyst inducer is 5-methoxyptryptamine (5MT), which is also periodically formed in Gonyaulax. In this unicell, the photoperiodic signal-transduction pathway presumably involves melatonin formation, its deacetylation to 5MT, 5MT-dependent transfer of protons from an acidic vacuole, and cytoplasmic acidification. According to this concept, we observe that cyst formation can be induced by various monoamine oxidase inhibitors and protonophores, that 5MT dramatically stimulates H(+)-dependent bioluminescence and leads to a decrease of cytoplasmic pH, as shown by measurements of dicyanohydroquinone fluorescence. Cellular components from Gonyaulax catalyze the photooxidation of melatonin. Its property of being easily destroyed by light in the presence of cellular catalysts may have been the reason that many organisms have developed mechanisms utilizing this indoleamine as a mediator of darkness. Photooxidative reactions of melatonin, as studied with crude Gonyaulax extracts and, more in detail, with protoporphyrin IX as a catalyst, lead to the formation of N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) as one of the main products. Photochemical mechanisms involve interactions with a photooxidant cation radical leading to the formation of a melatonyl cation radical, which subsequently combines with a superoxide anion.(ABSTRACT TRUNCATED AT 250 WORDS)

Bioluminescence in Dinoflagellates: A Test of the Burgular Alarm Hypothesis

Bioluminescence in dinoflagellates, unicellular aquatic organisms, has attracted considerable attention, primarily due to the striking nature of this phenomenon-during blooms, disturbances in the water (e.g., breaking waves, the wakes of boats, etc.) can be intensely phosphorescent. Despite a number of hypotheses regarding the function of bioluminescence in dinoflagellates, it is not clear why dinoflagellates bioluminesce (see Morin [1983] for a review). Dinoflagellates are stimulated to bioluminesce by a deformation of their cell membrane generated by shear forces (Hamman and Seliger 1972). These shear forces are often generated by strong stirring of water, such as breaking waves, or the rapid swimming of fish or invertebrates (Sweeney 1987). Sweeney (1987) noted that the light emitted from dinoflagellates is blue-green in color, with the maximum emission being at 474-476 nm. These wavelengths have a low extinction coefficient in water, allowing the light to be visible over relatively long distances. For this reason many researchers have assumed that bioluminescence serves some communication function. Schantz (1971) suggested that bioluminescence is a form of aposematic coloration, warning potential grazers of noxious substances contained by the prey. Indeed, many of the species of dinoflagellate that bioluminesce also contain toxins. There are also many nontoxic bioluminescent dinoflagellates, perhaps sug-

Epipelagic planktonic bioluminescence in the marginal ice zone of the Greenland Sea

Epipelagic planktonic bioluminescence in the marginal ice zone of the Fram Strait was investigated during April and May 1989. Vertical profiles of bioluminescence potential were measured using a HIDEX (High Input Defined EXcitation) design bathyphotometer. Mesozooplankton samples were collected with oblique tows using a 153 μm mesh net. The amount of bioluminescence produced by mechanical stimulation of individual organisms was measured using an integrating sphere and photon-counting system. These measures of bioluminescence potential along with estimated abundances of bioluminescent organisms allow an independent estimated of integrated epipelagic bioluminescence potential. The zooplankton community structure was relatively simple in this region and the number of bioluminescent species correspondingly small. The majority of epipelagic bioluminescence was produced by copepods (Metridia spp.), larvaceans (Oikopleura spp.), euphausiids (Thysanoeassa spp.) and ostracods (Conchoecia spp.), although the relative contribution of these organisms to the overall bioluminescence potential varied considerably with time and location over the 2 mo period. Bioluminescent dinoflagellates were rare and did not contribute significantly to epipelagic bioluminescence. Integrated bioluminescence potential in the water column was significantly correlated with zooplankton biomass, but not with any other measured environmental parameter. No enhancement of chlorophyll, zooplankton biomass or bioluminescence was observed in the marginal ice zone compared to the adjacent open waters of the Fram Strait during this spring cruise.

Planktonic bioluminescence in the pack ice and the marginal ice zone of the Beaufort Sea

An icebreaker cruise into the Beaufort Sea in the fall of 1986 provided a unique opportunity for studying planktonic bioluminescence in ice fields and in the marginal ice zone. Bathyphotometer casts (bioluminescence intensity, seawater temperature, beam attenuation coefficient, and salinity) and biological collections were made to a depth of 100 m. A light budget, which describes the planktonic species responsible for the measured bioluminescence, and a dinoflagellate species budget were constructed from the mean light output from luminescent plankton and plankton counts. The vertical distribution of bioluminescence among the ice stations was similar. The maximum intensities were 2 to 8×106 photons s-1 cm-3 in the upper 50 m of the sea-ice interface. The marginal ice zone station (MIZ) exhibited a maximum intensity of 2 to 3×108 photons s-1 cm-3 between 5 and 30 m depth. At Ice Station 2, Metridia longa and their nauplii contributed approximately 80% of stimulable bioluminescence in the upper 10 m but, overall, Protoperidinium spp. dinoflagellates contributed most of the light to a depth of 100 m. In the MIZ, Protoperidinium spp. dinoflagellates contributed ∼90% of the light within the upper 10 m, decreasing to 43% of the contributed light at a depth of 40 m. Below 40 m, dinoflagellate bioluminescence decreased to a few percent of the total to a depth of 90 m. Metridia spp. copepods contributed more than 50% of the light at depths from 40 to 90 m. Ostracods, larvaceans, and euphausiid furcilia contributed <1% of all bioluminescence at all depths sampled. Correlation analyses between measured bioluminescence (photons s-1 cm-3), the number of bioluminescent dinoflagellates and the light budget for the MIZ indicated highly significant associations: r=0.919, p=0.001, and r=0.912, p<0.001, respectively (Student's two-tailed t-tests). Bioluminescence was negatively correlated with seawater salinity at all stations (p=0.001). Maximum bioluminescence was measured in the less saline surface waters at all stations.

An encounter model to predict natural planktonic bioluminescence

Natural bioluminescence results from the emissions of bioluminescent organisms in the absence of man‐made disturbance. Most epipelagic and mesopelagic bioluminescence is caused by planktonic organisms. Limited attempts to measure natural bioluminescence in situ with surface deployed and moored instruments have recorded much higher levels of natural bioluminescence than have been observed from submersibles. We present a model predicting the frequency of interactions among planktonic organisms leading to natural bioluminescent emissions. The results suggest that natural bioluminescent flashes caused by interactions between zooplankters may be rare events in the Sargasso Sea, with our model predicting a maximum of nearly 600 flashes h−1 m−3 at night in surface waters of the northern Sargasso Sea where there was an unusually high concentration of euphausiids and small bioluminescent dinoflagellates, but &lt;1 flash h−1 m−3 in the southern Sargasso Sea. In both locations most of the natural bioluminescence was predicted to be caused by euphausiids and copepods of the genus Pleuromamma and natural bioluminescence generally decreased with depth. The more intense displays of natural bioluminescence are probably the result of the bioluminescence of numerous smaller dinoflagellates common in coastal waters and may also be the result of disturbances caused by interactions of larger organisms with bioluminescent plankters.

Correlations of planktonic bioluminescence with other oceanographic parameters from a Norwegian fjord

In August 1985 and 1986, a senes of bathyphotometric measurements were made In icefree waters of Vestfjord, Norway, to quantify stimulable in situ bioluminescence. In 1986, we tested and identified the causative bioluminescent plankton. In all profiles, peak or maximum bioluminescence intensity was always within a zone of 15 to 30m below sea surface, with marked decreases below 50m. Maximum biolurninescence intensity from all profiles for both years ranged from 3 X 10' to 2 X 10' photons S-' cc-' of turbulently-flowing seawater. Testing some of the plankton on board ship revealed that the brightest flashes were produced by the copepods Metridia longa and M lucens and the ostracod Conchoecia sp. Dinoflagellates of the genus Protopendnium emitted light at an intermediate level. The total number of dinoflagellates ranged from 1300 to 3700 cells I-' at water depths of 10 to 30m, and sharply decreased at 80 to lOOm (2 to 11 cells I-'). It was estimated that dinoflagellates accounted for 96 % of the measured light from the surface to a depth of loon?. Several species of the luminous d~noflagellate Ceratium were found. However, their contribution to the overall light budget never exceeded 25 %. P. curtipescontributed at least 50 % of the measured light field at 30m and above. In general, luminous zooplankton (larval copepods and euphausiids) contributed less than 4 % of the light produced above 100 m. The number of bioluminescent dinoflagellates correlated with bioluminescent intensity at the 0.05 significance level. Correlation analysis of beam attenuation coefficient w t h biolun7inescent intensity yielded high r values resulting in an overall level of significance of less than 0.01, and, therefore, may be a useful indicator for bioluminescence.

The ultrastructural localization of luciferase in three bioluminescent dinoflagellates, two species of Pyrocystis, and Noctiluca, using anti-luciferase and immunogold labelling.

In order to discover the intracellular location of luciferase in dinoflagellates, sections from a number of species were treated with a polyclonal anti-luciferase and the bound antibody was visualized at the electron-microscope level by indirect immunogold labelling. In two species of Pyrocystis and in Noctiluca, as in Gonyaulax, antibody became bound to dense vesicles, which correspond in size and position to light-emitting bodies detected in previous work. These vesicles resemble microsomes, are bounded by a single membrane and sometimes project into the vacuole. Unexpectedly, the trichocysts of Gonyaulax and Noctiluca and the related mucocysts of Pyrocystis also bound the antibody. This cross-reaction seems quite independent of bioluminescence, since the trichocysts of the non-luminous Cachonina also reacted positively. The possibility is discussed that a protein, different from luciferase but having some antigenic similarity, is present in trichocysts and related organelles.

Dinoflagellates

Dinoflagellates are single-celled organisms of the Division Pyrrhophyta. Most people, although not familiar with dinoflagellates, are familiar with their effects. Bioluminescent dinoflagellates cause a sparkling of the sea at night as the waves break, and certain dinoflagellates may produce blooms called “red tides,” which poison marine life or which cause toxins to accumulate in shellfish, poisoning those who eat them. The Old Testament (Exodus 7:20–21) and the writings of the ancient Greeks refer to red water and the killing of fish. The Red Sea may have gotten its name from dinoflagellate blooms.

BEHAVIORAL RESPONSES OF OCEANIC ZOOPLANKTON TO SIMULATED BIOLUMINESCENCE

A defensive function often has been suggested for the bioluminescence of dinoflagellates and copepods, but there is only limited experimental evidence. Using closed circuit television equipment and infrared illumination we have recorded the behavioral responses of planktonic copepods, ostracods, polychaetes, chaetognaths, and euphausiids to simulated bioluminescent flashes. The swimming patterns of these organisms were then quantified using a video-computer system for motion analysis (the Bugwatcher). The photophobic response exhibited by certain copepod species in response to simulated dinoflagellate flashes, as well as the lack of response by several potential predators on copepods to their simulated bioluminescence, provide new insight into the roles of bioluminescence in plankton ecology. Com parison of the responses of the non-bioluminescent copepod Calanus finmarchicus and the bioluminescent copepod Metridia longa to simulated copepod biolumines cence show that Metridia is much more responsive than Calanus. This suggests that bioluminescence in Metridia may be recognized as a warning signal by conspecifics in addition to serving as a defense against predation.

Behavioral responses of the coastal copepod Acartia hudsonica (Pinhey) to simulated dinoflagellate bioluminescence

Light pulses were used to mimic dinoflagellate bioluminescence and test its effects on the swimming behavior of Acartia hudsonica (Pinhey). The horizontal swimming patterns of the copepod were tracked and described using a video-computer system. Single flashes of light of 60 ms duration, with a wavelength of peak emission of 475 nm and an intensity of 2 μE · m −2 · s −1 caused a “startle” response consisting of a short burst of high speed swimming. A series of these flashes repeated every 5 s resulted in higher average swimming speed, more swimming speed bursts, and straighter paths. These behavioral changes are similar to those previously found for A. hudsonica in the presence of bioluminescent dinoflagellates. The effects of altering the intensity, duration, and color of the simulated dinoflagellate flash were also tested. Our results support the hypothesis that dinoflagellate bioluminescence is a highly evolved adaptation for repelling nocturnal grazers.