PHOTOINHIBITION OF MECHANICALLY STIMULABLE BIOLUMINESCENCE IN THE AUTOTROPHIC DINOFLAGELLATE CERATIUM FUSUS (PYRROPHYTA)1

Ceratium fusus (Ehrenb.) Dujardin was exposed to light of different wavelengths and photon flux densities (PFDs) to examine their effects on mechanically stimulable bioluminescence (MSL). Photoinhibition of MSL was proportional to the logarithm of PFD. Exposure to I μmol photons·m^?2s^?1 of broadband blue light (ca. 400–500 nm) produced near-complete photoinhibition (≥90% reduction in MSL) with a threshold at ca. 0.01 μmol photons·m^?2·s^?1. The threshold of photoinhibition was ca. an order of magnitude greater for both broadband green (ca. 500–580 nm) and red light (ca. 660–700 nm). Exposure to narrow spectral bands (ca. 10 nm half bandwidth) from 400 and 700 nm at a PFD of 0.1 μmol photons·m^?2·s^?1 produced a maximal response of photoinhibition in the blue wavelengths (peak ca. 490 nm). A photoinhibition response (≥ 10%) in the green (ca. 500–540 nm) and red wavelengths (ca. 680 nm) occurred only at higher PFDs (1 and 10 μmol photons·m^?2·s^?1). The spectral response is similar to that reported for Gonyaulax polyedra Stein and Pyrocystis lunula Schütt and unlike that of Alexandrium tamarense (Lebour) Balech et Tangen. The dinoflagellate's own bioluminescence is two orders of magnitude too low to result in self-photoinhibition. The quantitative relationships developed in the laboratory predict photoinhibition of bioluminescence in populations of C. fusus in the North Atlantic Ocean.     

EFFECTS OF LIGHT INTENSITY ON DIVISION RATE,STIMULABLE BIOLUMINESCENCE AND CELL SIZE OF THE OCEANIC DINOFLAGELLATES DISSODINIUM LUNULA,PYROCYSTIS FUSIFORMIS AND P. NOCTILUCA12

Preadapted cultures were grown in a 12:12 LD cycle at a series of light intensities under cool-white, fluorescent lamps. Pyrocystis fusiformis Murray maintained high division rates at low light intensities at the expense of cell size. In contrast, Dissodinium lunula (Schuett) Taylor had relatively lower division rates at low light intensities with little concomitant decrease in size. The response of P. noctiluca Murray was intermediate between these two species. For all three, cell numbers did not increase above an intensity of 5–10 μEin·m^?2·sec^?1 and division rate was saturated at ca. 30, 60, and 60μEin·m^?2·sec^?1 for P. fusiformis, P. noctiluca, and D. lunula, respectively. The capacity for stimulable bioluminescence was saturated at light intensities of 0.15 μEin·m^?2·day in short-term (2-day) experiments. In cultures of P. fusiformis and P. noctiluca, maintained for at least one month at lower intensities than needed to saturate division rate, a decrease in the capacity for stimulable bioluminescence was accompanied by a reduction in cell size. Our results suggest that cell size and bioluminescent capacity may prove to be a potentially useful indication of the history of exposure of natural populations of Pyrocystis spp. to ambient intensities.     

PHOTOINHIBITION OF MECHANICALLY STIMULABLE BIOLUMINESCENCE IN THE HETEROTROPHIC DINOFLAGELLATE PROTOPERIDINIUM DEPRESSUM (PYRROPHYTA)1

Photoinhibition of mechanically stimulable bioluminescence (MSL) in the heterotrophic dinoflagellate Protoperidinium depressum Bailey was investigated using samples collected from the Massachusetts and southern Texas coasts. The times for both photoinhibition of MSL (ca. 10 min) and dark recovery from photoinhibition of MSL (ca. 45 min) in this species were similar to those reported for autotrophic dinoflagellates. The degree of photoinhibition of MSL was a linear function of the logarithm of photon flux density (PFD). The threshold PFDs for the photoinhibition of MSL were 0.02, 0.6, and 21 μmol photons · m^?2· s^?1 for broad-band blue, green, and red light, respectively. These PFDs are lower than those required for photoinhibition of MSL by the autotrophic dinoflagellates Pyrocystis lunula and Ceratium fusus. We speculate that photosynthetic pigments in autotrophic dinoflagellates shield the photoreceptor that causes photoinhibition of MSL, thus lowering the sensitivity of these dinoflagellates to light. When field-collected P. depressum were kept in the laboratory without growth for a week, photoinhibition of MSL's sensitivity to light increased progressively along with 1) a decrease in its bioluminescence capacity (BCAP), 2) a decrease in the ratio of MSL to BCAP (MSL/BCAP), and 3) a decrease in the orange pigmentation (probably carotenoid) of the dinoflagellate. The action spectrum for photoinhibition of MSL in P. depressum was characterized primarily with a broad peak in the blue extending into the green. We suggest that carotenoid was not a photoreceptor for the photoinhibition of MSL in P. depressum because the peak of the action spectrum was too broad and extended too far into the green part of the spectrum, and because the orange pigment present decreased as photoinhibition of MSL became more sensitive to light.     

SPECIES OF OCEANIC DINOFLAGELLATES IN THE GENERA DISSODINIUM AND PYROCYSTIS: INTERCLONAL AND INTERSPECIFIC COMPARISONS OF THE COLOR AND PHOTON YIELD OF BIOLUMINESCENCE1

We have examined aspects of the bioluminescence of 5 clones of Dissodinium, 1 clone of Pyrocystis acuta, 4 clones of Pyrocystis fusiformis, and 5 clones of Pyrocystis noctiluca. All clones produced the same color bioluminescence with an intensity peak near 474 nm. The in vivo emission spectra of these clones agreed with those previously determined, for 4 other species of marine dinoflagellates. The amount of light emitted by the dinoflagellates in scotophase when mechanically stimulated to exhaustion was determined for most of the clones. The largest species, P. noctiluca and P. fusiformis, emitted 37–89 × 10⁹ photons cell^?1 and 23–62 × 10⁹ photons cell^?1, respectively, about a thousand, times as much light as Gonyaulax species. Pyrocystis acuta emitted 3–6 × 10⁹ photons cell^?1. Three of the 5 clones of Dissodinium were bioluminescent. The range for 3 clones was 5–13 × 10⁹ photons cell^?1. All 5 clones of Dissodinium are morphologically distinct. Both the clones of Dissodinium and Pyrocystis produced much higher numbers of photons per cell nitrogen (ca. 7–50 times) than Gonyaulax polyedra or Pyrodinium bahamense. The data suggested that enzyme turnover occurred in the reactions producing light during mechanical stimulation of Dissodinium and Pyrocystis species.     

The chemical mimicking of the mechanical stimulation, photoinhibition, and recovery from photoinhibition of bioluminescence in marine dinoflagellate, Gonyaulax polyedra

Mechanically stimulable bioluminescence and photoinhibition of sensitivity to mechanical stimulation in the marine dinoflagellate Gonyaulax polyedra can be mimicked by a number of cations, proportional to the logarithm of their external concentrations. The data are consistent with mechanical stimulability as a membrane depolarization resulting in an increase in H+ ions at bioluminescence sites and with photoinhibition as a hyperpolarization of the cell membrane.     

SPONTANEOUS AND STIMULATED BIOLUMINESCENCE OF THE DINOFLAGELLATE CERATOCORYS HORRZDA (PERIDINIALES)1

This is the first report of spontaneous bioluminescence in the autotrophic dinoflagellate Ceratocorys horrida von Stein. Bioluminescence was measured, using an automated data acquisition system, in a strain of cultured cells isolated from the Sargasso Sea. Ceratocorys horrida is only the second dinoflagellate species to exhibit rhythmicity in the rate of spontaneous flashing, flash quantum flux (intensity), and level of spontaneous glowing. The rate of spontaneous flashing was maximal during hours 2–4 of the dark phase [i.e. circadian time (CT)16–18 for a 14:10 h LD cycle (LD14:10)], with approximately 2% of the population flashing-min^?1, a rate approximately one order of magnitude greater than that of the dinoflagellate Gonyaulax polyedra. Flash quantum flux was also maximal during this period. Spontaneous flashes were 134 ms in duration with a maximum flux (intensity) of 3.1×10⁹ quanta-s^?1. Light emission presumably originated from blue fluorescent microsources distributed in the cell periphery and not from the spines. Values of both spontaneous flash rate and maximum flux were independent of cell concentration. Isolated cells also produced spontaneous flashes. Spontaneous glowing was dim except for a peak of 6.4× 10⁴quanta-s^?1 cell^?1, which occurred at CT22.9 for LD14:10 and at CT22.8 for LD12:12. The total integrated emission of spontaneous flashing and glowing during the dark phase was 4×10⁹ quantacell^?1, equivalent to the total stimulable luminescence. The rhythms for C. horrida flash and glow behavior were similar to those of Gonyaulax polyedra, although flash rate and quantum flux were greater. Spontaneous bioluminescence in C. horrida may be a circadian rhythm because it persisted for at least three cycles in constant dark conditions. This is also the first detailed study of the stimulated bioluminescence of C. horrida, which also displayed a diurnal rhythm. Cultures exhibited >200 times more mechanically stimulated bioluminescence during the dark phase than during the light phase. Mechanical stimulation during the dark phase resulted in 6.7 flashes. cell^?1; flashes were brighter and longer in duration than spontaneous flashes. Cruise-collected cells exhibited variability in quantum flux with few differences in flash kinetics. The role of dinoflagellate spontaneous bioluminescence in the dynamics of near-surface oceanic communities is unknown, but it may be an important source of natural in situ bioluminescence.     

The mechanical triggering of bioluminescence in marine dinoflagellates: chemical basis

In both photosynthetic (Pyrodinium bahamense, Gonyaulax polyedra, Pyrocystis Iunula, P. noctiluca, P. fusiformis) and nonphotosynthetic (Noctiluca miliaris) bioluminescent dinoflagellates chemical stimulation can by-pass mechanical stimulation. The effective ions are Ca⁺⁺, K⁺, NH₄⁺ and H⁺. Other chemicals found effective are those implicated in Ca⁺⁺ transport or binding. There are interspecies differences in degrees of mechanical and chemical stimulability. Photoinhibition of mechanical stimulability is the result of two effects, the first a reduction in total bioluminescence potential and the second a decrease in mechanical stimulability resulting experimentally in a decreased rate of light emission. This latter effect can be reversed with Ca⁺⁺ ions. Chemicals which bind Ca⁺⁺ or displace Ca⁺⁺ can mimic the effects of photoinhibition. The chemical inhibition of mechanical stimulability is also reversed by Ca⁺⁺ ions. A scheme is proposed which is consistent for all species examined.     

Stimulable and Spontaneous Bioluminescence in the Marine Dinoflagellates, Pyrodinium bahamense, Gonyaulax polyedra, and Pyrocystis lunula

P. bahamense, G. polyedra, and P. lunula exhibit interspecies differences in stimulable and spontaneous bioluminescence. For each species the total number of photons that can be emitted upon mechanical stimulation is a constant, regardless of the time during scotophase at which stimulation occurs. Ratios of stimulable bioluminescence per organism during scotophase and photophase are as high as 950:1 for laboratory cultures and have been observed as high as 4000: 1 for natural populations of P. bahamense. Spontaneous emission in darkness shows flashing as well as low-level continuous emission. Natural populations of P. bahamense, placed in darkness during natural photophase, exhibit a dual character to their stimulable bioluminescence. Mechanical stimulation techniques are described for rapid and reproducible stimulation of bioluminescence.     

OCCURRENCE AND ABUNDANCE OF GREEN-FLUORESCING DINOFLAGELLATES IN SURFACE WATERS OF THE NORTHWEST ATLANTIC AND NORTHEAST PACIFIC OCEANS1

We have cultured green fluorescing heterotrophic dinoflagellates whose continuous green fluorescence is due to an unidentified compound, probably a flavin, that excites with blue (～460 nm) light and emits green (～535 nm) light. No evidence of bioluminescence was found, but we note that compounds with similar fluorescence characteristics have been associated with bioluminescence in other taxa. These cells, all naked gymnodinoids, are widespread and abundant in the Northwest Atlantic and Northeast Pacific Oceans (10³–10⁵ L^?1). They comprise 4–100% of the total heterotrophic dinoflagellate component which, in turn, is usually equivalent magnitude to the phototrophic naked dinoflagellate component of the phytoplankton community.     

Diversity of the Luciferin Binding Protein Gene in Bioluminescent Dinoflagellates – Insights from a New Gene in Noctiluca scintillans and Sequences from Gonyaulacoid Genera

Dinoflagellate bioluminescence systems operate with or without a luciferin binding protein, representing two distinct modes of light production. However, the distribution, diversity, and evolution of the luciferin binding protein gene within bioluminescent dinoflagellates are not well known. We used PCR to detect and partially sequence this gene from the heterotrophic dinoflagellate Noctiluca scintillans and a group of ecologically important gonyaulacoid species. We report an additional luciferin binding protein gene in N. scintillans which is not attached to luciferase, further to its typical combined bioluminescence gene. This supports the hypothesis that a profound re‐organization of the bioluminescence system has taken place in this organism. We also show that the luciferin binding protein gene is present in the genera Ceratocorys, Gonyaulax, and Protoceratium, and is prevalent in bioluminescent species of Alexandrium. Therefore, this gene is an integral component of the standard molecular bioluminescence machinery in dinoflagellates. Nucleotide sequences showed high within‐strain variation among gene copies, revealing a highly diverse gene family comprising multiple gene types in some organisms. Phylogenetic analyses showed that, in some species, the evolution of the luciferin binding protein gene was different from the organism's general phylogenies, highlighting the complex evolutionary history of dinoflagellate bioluminescence systems.     

Dinoflagellate bioluminescence: a comparative study of invitro components.

In vitro bioluminescence components of the dinoflagellates Gonyaulax polyedra, G. tamarensis, Dissodinium lunual, and Pyrocystis noctiluca were studied. The luciferases and luciferins of the four species cross-react in all combinations. All of these species possess high-molecular weight luciferases (200,000-400,000 daltons) with similar pH activity profiles. The active single chains of luciferases from the Gonyaulax species have a MW of 130,000 while those from P. noctiluca and D. lunula have a MW of 60,000. Extractable luciferase activity varies with time of day in the two Gonyaulax species, but not in the other two. A luciferin binding protein (LBP) can easily be extracted from the two Gonyaulax species (MW approximately 120,000 daltons), but none could be detected in extracts of either D. lunula or P. noctiluca. Scintillons are extractable from all four species, but they vary in density and the degree to which activity can be increased by added luciferin. Although the biochemistry of bioluminescence in these dinoflagellates is generally similar, the observations that D. lunula and P. noctiluca apparently lack LBP and have luciferases with low MW single chains require further clarification.     

In situ measurements of bioluminescence response of Gonyaulax spinifera to various mechanical stimuli

A conspicuous bioluminescence during nighttime was reported in an aquaculture farm in the Cochin estuary due to Gonyaulax spinifera bloom on March 20, 2020. In situ measurements on bioluminescence was carried out during nighttime to quantify the response of G. spinifera to various mechanical stimuli. The bioluminescence intensity (BI) was measured using Glowtracka, an advanced single channel sensor, attached to a Conductivity–Temperature–Depth Profiler. In steady environment, without any external stimuli, the bioluminescence generated due to the movement of fishes and shrimps in the water column was not detected by the sensor. However, stimuli such as a hand splash, oar and swimming movements, and a mixer could generate measurable bioluminescence responses. An abundance of?~?2.7?×?10⁶ cells L^?1 of G. spinifera with exceptionally high chlorophyll a of 25 mg m^?3 was recorded. The BI in response to hand splash was recorded as high as 1.6?×?10¹¹ photons cm^?2 s^?1. Similarly, BI of?~?1–6?×?10¹⁰ photons cm^?2 s^?1 with a cumulative bioluminescence of?~?2.51?×?10¹² photons cm^?2 (for 35 s) was recorded when there is a mixer with a constant force of 494 N/800 rpm min^?1. The response of G. spinifera was spontaneous with no time lapse between application of stimuli and the bioluminescence response. Interestingly, in natural environment, application of stimulus for longer time periods (10 min) does not lower the bioluminescence intensity due to the replenishment of water thrusted in by the mixer from surrounding areas. We also demonstrated that the bioluminescence intensity decreases with increase in distance from the source of stimuli (mixer) (av. 1.84?×?10¹⁰ photons cm^?2 s^?1 at 0.2 m to av. 0.05?×?10¹⁰ photons cm^?2 s^?1 at 1 m). The BI was highest in the periphery of the turbulent wake generated by the stimuli (av. 3.1?×?10¹⁰ photons cm^?2 s^?1) compared to the center (av. 1.8?×?10¹⁰ photons cm^?2 s^?1). When the stimuli was applied vertically down, the BI decreased from 0.2 m (0.3?×?10¹⁰ photons cm^?2 s^?1) to 0.5 m (0.10?×?10¹⁰ photons cm^?2 s^?1). Our study demonstrates that the BI of G. spinifera increases with increase in mechanical stimuli and decreases with increase in distance from the stimuli.

     

Bioluminescence of Marine Dinoflagellates : I. An underwater photometer for day and night measurements

Portable light-baffled underwater photometers have been designed for the measurement of dinoflagellate bioluminescence by day and night. Maximal light emission is obtained by mechanical stimulation in a defined volume. The pump which stimulates the dinoflagellates also constantly replenishes the sample volume so that continuous measurements are possible. Evidence for both diurnal variation and vertical migration is presented. Using luminous bacteria for calibration a single dinoflagellate has been found to emit of the order of 10¹⁰ light quanta per flash. The technique suggests that large scale mapping of bioluminescence is feasible.     

Chemistry,clones, and circadian control of the dinoflagellate bioluminescent system. The Marlene DeLuca memorial lecture

Bioluminescence in the dinoflagellate Gonyaulax polyedra occurs as brief bright flashes, originating from many (～400) small (～0.5 μm) cytoplasmic organelles which protrude into the acidic vacuole, and are thus surrounded by the tonoplast. Biochemically, the substrate is unusual; it is an open chain tetrapyrrole, highly unstable to air but protected in the cell at pH? 8 by virtue of a luciferin binding protein (LBP). This molecule is a dimer of 72 kDa subunits which, upon a decrease in pH, releases luciferin to react with oxygen in the luciferase (～140 kDa) catalysed luminescent reaction. cDNAs for both luciferase and LBP have been isolated and cloned, and the identity of LBP was confirmed by hybrid selection and in vitro translation of the message. The tenfold circadian (day to night) change in the amount of LBP, which parallels the in vivo rhythm of luminescence, is due to de novo synthesis and subsequent degradation of the protein each day. The LBP mRNA levels, as determined by in vitro translations and by Northern hybridizations, do not vary over the daily cycle, indicating that circadian control of bioluminescence in this species is mediated at the level of translation.     

EFFECTS OF SMALL‐SCALE TURBULENCE ON NET GROWTH RATE AND SIZE OF TEN SPECIES OF MARINE DINOFLAGELLATES1

Field observations and results from previous laboratory studies on the effects of turbulence on dinoflagellates have led to a paradigm in phytoplankton ecology that dinoflagellate growth is negatively affected by turbulence. To test the paradigm, 10 species of autotrophic dinoflagellates were exposed to quantified three‐dimensional turbulence generated by vertically oscillating cylindrical rods in 20‐L rectangular culture tanks. Turbulence was quantified in the tanks (as the turbulent energy dissipation rate, ε ) using an acoustic Doppler velocimeter. Dinoflagellates were exposed to two turbulence treatments: high turbulence ( ε ～ 10 ^{? 4} m²·s ^{? 3}), low turbulence ( ε ～ 10 ^{? 8} m²·s ^{? 3}), and an unstirred control. In accord with the paradigm, Ceratium fusus (Ehrenberg) Dujardin had lower net growth rates in high turbulence, whereas Pyrocystis noctiluca Murray ex Haeckel and Ceratium tripos (O. F. Müller) Nitzsch did not increase their numbers in high turbulence. However, Alexandrium tamarense (Lebour) Balech, Pyrocystis fusiformis Wyville‐Thomson ex Murray, Alexandrium catenella (Whedon and Kofoid) Balech, and a Gyrodinium sp. Kofoid and Swezy were apparently unaffected by turbulence and had the same net growth rates across all turbulence treatments. Contradicting the paradigm, Lingulodinium polyedrum (Stein) Dodge (= Gonyaulax polyedra), Gymnodinium catenatum Graham, and Alexandrium fundyense Balech had increased net growth rates in high turbulence treatments. Cross‐sectional area (CSA) varied little across turbulence treatments for 8 of 10 dinoflagellate species tested, CSA in C. fusus increased when net growth rate decreased in high turbulence, and, conversely, CSA decreased in L. polyedrum when net growth rate increased in high turbulence.     

Spatial and seasonal changes in the species composition of armored dinoflagellates in the Southwestern Atlantic Ocean

Summary To compare the spatial and temporal (seasonal) distribution of dinoflagellates, vertical net hauls were taken along similar cruise tracks in the Scotia Sea, Weddell Sea and across the Polar Front Zone in the austral spring and the austral fall. Sixty-three species of armored dinoflagellates were identified and enumerated. Chisquare and hierarchical cluster analyses were performed to define spatial and seasonal patterns in genera and species assemblages. The dominant genera were Protoperidinium, Dinophysis and Ceratium. The Polar Front Zone was an important biogeographical barrier with Blepharocysta, Gonyaulax, Heteroschisma, Oxytoxum and Podolampas occurring mainly north of the Front. Species found primarily in the austral spring were Ceratium fusus, Ceratium lineatum, Dinophysis antarctica, Dinophysis simplex, Gonyaulax digitale, Protoperidinium pyriforme and Protoperidinium variegatum. Austral fall species included Dinophysis tuberculata and Protoperidinium elegantissum. Distribution of armored dinoflagellates in the Southwestern Atlantic Ocean is influenced at the generic level by spatial considerations, particular with relation to the Polar Front Zone, whereas species composition can be effected by both region and season.     

Bioluminescence of colonial radiolaria in the western Sargasso Sea

Colonial radiolaria (Protozoa: Spumellarida) were a conspicuous feature in surface waters of the Sargasso Sea during the April (1985) Biowatt cruise. The abundance of colonies at the sea surface at one station was estimated to be 23 colonies · m⁻².Bioluminescence by colonial radiolaria, representing at least six taxa, was readily evoked by mechanical stimuli and measured by fast spectroscopy and photon-counting techniques. Light emission was deep blue in color (peak emissions between 443 and 456 nm) and spectral distributions were broad (average half bandwidth of 80 nm). Single flashes were 1–2 s in duration at ≈23 °C, with species-dependent kinetics which were not attributed to differences in colony morphology, since colonies similar in appearance could belong to different species (even families) and display different flash kinetics. Although the presence of dinoflagellate symbionts was confirmed by the presence of dinoflagellate marker pigments in the colonies, luminescence in the radiolaria examined most likely did not originate from symbiotic dinoflagellates because of (1) differences in the emission spectra, (2) unresponsiveness to low pH stimulation, (3) differences in flash kinetics and photon emission of light emission, and (4) lack of light inhibition.The quantal content of single flashes averaged 1 × 10⁹ photons flash⁻¹, and colonies were capable of prolonged light emission. The mean value of bioluminescence potential based on measurements of total mechanically stimulated bioluminescence was 1.2 × 10¹¹ photons · colony⁻¹. It is estimated that colonial radiolaria are capable of producing ≈2.8 × 10¹² photons · m⁻² of sea surface. However, this represented only 0.5% of in situ measured bioluminescence potential.     

CIRCADIAN REGULATION OF BIOLUMINESCENCE IN THE DINOFLAGELLATE PYROCYSTIS LUNULA1

In the unicellular algae Pyrocystis lunula Schütt and Gonyaulax polyedra Stein, bioluminescence and its circadian regulation are similar in several respects, but there are also several important differences. As in G. polyedra, P. lunula emits light both as bright flashes and as a low intensity glow. At 20° C, the individual flashes are considerably brighter than in G. polyedra, and their durations are typically less than 500 ms. Both species show a circadian rhythm in the frequency of spontaneous flashes, which peaks in the night-phase under light–dark cycles and continues in both continuous light and dark. However, compared to G. polyedra, the circadian system in P. lunula is more sensitive to light: 10 min exposures (500 μmol · m^–2· s^–1 white light) can shift the phase of the rhythm by more than 8 h, and rhythmicity is completely suppressed at an irradiance above 20 μmol · m^–2· s^–1, where the G. polyedra rhythym persists for weeks. Like G. polyedra, period length increases with increasing irradiance of continuous red light but decreases with increasing intensity of continuous blue light. The glow in P. lunula differs markedly from that in G. polyedra in that it occurs at about the same intensity at all times during the circadian cycle; thus, it is not under circadian control but may fluctuate 5–10-fold in intensity within a time frame of seconds. This suggests that the glow may differ in its physiological basis in the two organisms. The results also indicate that the circadian regulation of luciferase activity differs in the two species. In G. polyedra, the organelle responsible for bioluminescence and luciferase is lost and then reformed on a daily basis; in P. lunula, the luciferase is conserved and localized elsewhere during the nonbioluminescent phase of the cycle.     

POTENTIAL IMPORTANCE OF BENTHIC CYSTS OF GONYAULAX TAMARENSIS AND G. EXCAVATA IN INITIATING TOXIC DINOFLAGELLATE BLOOMS1,2,3

Thick-walled, nonmotile cysts (termed hypnocysts) of two dinoflagellates were isolated from estuarine sediments in Cape Cod, Massachusetts, and germinated to produce their respective motile, thecate stages. Hypnocysts from Orleans district were identified as Gonyaulax excuvata (Braarud) Balech sensu Loeblich & Loeblich. Visually identical hypnocysts from Falmouth district were provisionally identified as Gonyaulax tamarensis Lebour. Both species were toxic. A geographic survey in September detected hypnocysts in only the sediments of locations where toxic blooms developed the preceding and following Spring. Laboratory incubation (16 C) of hypnocysts from sediment samples stored in the dark (5 C) for 6 mo initiated excystment by the temperature increase, with no appreciable effect from light regime, nutrient, or chelator concentrations. Motility of excysted germlings was optimum in highly chelated medium and in the presence of light. We conclude that hypnocysts of both tasa are important in seeding recurrent annual blooms, synchronizing early bloom development with vernal warning of seawater and increasing the geographic range of the species. We suggest that many red tides in New England and eastern Canadian waters are initiated through the displacement of motile estuarine populations into nearshore area by tidal advection and surface runoff, although the potential existence and importance of offshore cyst reservoirs cannot be discounted. Evidence is presented that hypnocysts are probable sexual zygotes whereas the thin-walled cysts readily formed in laboratory cultures (pellicle cyst) are asexual. Pellicle cysts are of limited durability, do not overwinter in nature, and therefore do not play a significant role in initiating toxic blooms.     

THE ROLE OF CALCIUM IN FLOW‐STIMULATED BIOLUMINESCENCE OF THE RED TIDE DINOFLAGELLATE LINGULODINIUM POLYEDRUM

Many marine planktonic dinoflagellates emit flashes of light in response to either laminar or turbulent flows as well as direct mechanical stimulation. The production of a flash of light is known to be mediated by a proton‐mediated action potential across the vacuolar membrane; the mechanotransduction process initiating this action potential is unknown. Here we report on an investigation into the role of Ca⁺² in the mechanotransduction process regulating bioluminescence in the red tide dinoflagellate Lingulodinium polyedrum. Calcium ionophores and low concentrations of the membrane‐disrupting agent digitonin stimulated bioluminescence only when calcium was present in the media or added with the agent, indicating that the flash‐triggering vacuolar action potential is specifically stimulated by a calcium influx. A variety of known calcium channel blockers or antagonists inhibited mechanically stimulated bioluminescence but did not affect cellular bioluminescent capacity. In many cases the inhibitory affect occurred after only a brief exposure. In addition, gadolinium (Gd⁺³), a blocker of many stretch‐activated ion channels, caused potent inhibition of mechanically stimulated bioluminescence. The order of potency of the transition metals tested was La⁺³ > Gd⁺³ > Co⁺² > Mn⁺² > Ni⁺², similar to their potency as blockers of known calcium channels. Experiments with a quantified shear flow demonstrated that flow‐stimulated bioluminescence depended on the level of extracellular calcium. Future work will elucidate the signaling pathway involving calcium‐mediated flow‐stimulated mechanotransduction. Our goal is to use bioluminescence as a proxy for the initial cellular mechanotransduction events triggered by fluid flow.