Bioluminescent Dinoflagellates Research Articles

Intensity differences in bioluminescent dinoflagellates impact foraging efficiency in a nocturnal predator

Bioluminescence in dinoflagellates is thought to function as a alarm, alerting visual predators to the presence of dinoflagellate grazers. However, many bioluminescent dinoflagellates, particularly those associated with harmful algal blooms (HABs), have a much lower bioluminescence capacity that seems less well-adapted for attracting the attention of distant secondary predators. The present study was motivated by a question regarding the impact of extreme differences in bioluminescence potential among dinoflagellates, particularly those with the capacity to form HABs. This study examined the function of bioluminescence in the bright emitter, Pyrocystis noctiluca (Murray, 1876), compared to the much dimmer HAB species, Lingulodinium polyedrum F. Stein (Stein 1883). The foraging efficiency of the nocturnal teleost, Apagon maculatus (Poey, 1860), was determined at a range of cell concentrations with both dinoflagellate species. At low cell concentrations of P. noctiluca, both the foraging efficiency and the orientation distance of the fish to the prey increased, indicating that bioluminescence functions as a burglar alarm. However, neither fish foraging efficiency nor orientation distance increased in the presence of luminescent L. polyedrum at low cell concentrations. At higher concentrations, the bioluminescence of L. polyedrum improved the foraging efficiency of the fish, but the orientation distance to the prey was no greater than with non-luminescent cells, indicating that at low cell concentrations, bioluminescence does not function as a burglar alarm in L. polyedrum. The role of bioluminescence as a possible aposematic signal in L. polyedrum is discussed, along with the implications for the role of bioluminescence in HAB dynamics.

Molecular detection of bioluminescent dinoflagellates in surface waters of the Patagonian shelf during early austral summer 2008.

We investigated the distribution of bioluminescent dinoflagellates in the Patagonian Shelf region using “universal” PCR primers for the dinoflagellate luciferase gene. Luciferase gene sequences and single cell PCR tests, in conjunction with taxonomic identification by microscopy, allowed us to identify and quantify bioluminescent dinoflagellates. We compared these data to coincidental discrete optical measurements of stimulable bioluminescence intensity. Molecular detection of the luciferase gene showed that bioluminescent dinoflagellates were widespread across the majority of the Patagonian Shelf region. Their presence was comparatively underestimated by optical bioluminescence measurements, whose magnitude was affected by interspecific differences in bioluminescence intensity and by the presence of other bioluminescent organisms. Molecular and microscopy data showed that the complex hydrography of the area played an important role in determining the distribution and composition of dinoflagellate populations. Dinoflagellates were absent south of the Falkland Islands where the cold, nutrient-rich, and well-mixed waters of the Falklands Current favoured diatoms instead. Diverse populations of dinoflagellates were present in the warmer, more stratified waters of the Patagonian Shelf and Falklands Current as it warmed northwards. Here, the dinoflagellate population composition could be related to distinct water masses. Our results provide new insight into the prevalence of bioluminescent dinoflagellates in Patagonian Shelf waters and demonstrate that a molecular approach to the detection of bioluminescent dinoflagellates in natural waters is a promising tool for ecological studies of these organisms.

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.

Pharmacological investigation of the bioluminescence signaling pathway of the dinoflagellate Lingulodinium polyedrum: evidence for the role of stretch‐activated ion channels

Dinoflagellate bioluminescence serves as a whole-cell reporter of mechanical stress, which activates a signaling pathway that appears to involve the opening of voltage-sensitive ion channels and release of calcium from intracellular stores. However, little else is known about the initial signaling events that facilitate the transduction of mechanical stimuli. In the present study using the red tide dinoflagellate Lingulodinium polyedrum (Stein) Dodge, two forms of dinoflagellate bioluminescence, mechanically stimulated and spontaneous flashes, were used as reporter systems to pharmacological treatments that targeted various predicted signaling events at the plasma membrane level of the signaling pathway. Pretreatment with 200μM Gadolinium III (Gd(3+) ), a nonspecific blocker of stretch-activated and some voltage-gated ion channels, resulted in strong inhibition of both forms of bioluminescence. Pretreatment with 50μM nifedipine, an inhibitor of L-type voltage-gated Ca(2+) channels that inhibits mechanically stimulated bioluminescence, did not inhibit spontaneous bioluminescence. Treatment with 1mM benzyl alcohol, a membrane fluidizer, was very effective in stimulating bioluminescence. Benzyl alcohol-stimulated bioluminescence was inhibited by Gd(3+) but not by nifedipine, suggesting that its role is through stretch activation via a change in plasma membrane fluidity. These results are consistent with the presence of stretch-activated and voltage-gated ion channels in the bioluminescence mechanotransduction signaling pathway, with spontaneous flashing associated with a stretch-activated component at the plasma membrane.

Selectivity of the Voltage Gated Proton Channel HV1

The voltage gated proton channel, HV1, triggers the flash in bioluminescent dinoflagellates, aids calcification by coccolithophores, and in humans, regulates pH in the respiratory tract, sperm capacitation, histamine secretion, the phagocyte respiratory burst, and B lymphocyte signaling. On the down-side, HV1 exacerbates breast cancer metastasis and brain damage from ischemic stroke. All of these functions require extreme proton selectivity, because [H+] is 106 lower than that of other ions. We studied proton selectivity in HV1 from humans and Karlodinium, a dinoflagellate, which share only 15% sequence identity. A crucial aspartate in the S1 transmembrane domain of each (Asp112 in hHV1, Asp51 in kHV1) is essential for proton selectivity; neutralizing mutations result in anion permeability or abolish conduction (Musset et al, 2011, Nature 480:273-277; Smith et al, 2011, Proc. Natl. Acad. Sci.U.S.A. 108:18162-18168). That the two proteins exhibit the same phenomenology despite limited sequence identity indicates that the selectivity mechanism is highly conserved. A combination of approaches, including accessibility studies of the three Arginine residues in the S4 helix, provides a picture of the open human HV1 channel, in which Arg205 (R1) is accessible to the external solution, Arg208 (R2) forms a salt bridge with Asp112, and Arg211 (R3) is accessible to the internal solution. Further studies of the molecular requirements for proton permeation and selectivity are underway.Support: NSF MCB-0943362, NIH R01-GM087507.

Hv1 Structure and Function: Insights from Evolutionary Information

The voltage gated proton channel, HV1, is the most selective ion channel known, and plays an important role in innate immunity, human sperm maturation, tumorigenesis, and dinoflagellate bioluminescence, to name a few examples. When the gene was identified in 2006, HV1 was found to be homologous to the voltage sensor domains of classic voltage gated cation channels; these homologous domains do not conduct ions. Phylogenetic analysis of VSDs showed that HV1, voltage sensing phosphatases, and c15orf27 are three twigs that occupy a branch distinct from other voltage gated ion channels, and that this branch is part of the larger group comprising voltage gated sodium and calcium channels, which split from potassium channels at least a billion years ago. We used a combination of sequence and phylogenetic analysis, structural modeling, and electrophysiological experimentation to identify the proton selectivity filter of HV1, an important milestone in elucidating the functional components of the protein. We proposed a “signature sequence” that defines all presently known HV1, and used it to identify the first dinoflagellate HV1, despite only 15% identity with hHV1. We combined molecular dynamics and sampling methods with other approaches, to help us choose and experimentally validate a refined model that we are using to help us discover the determinants of proton specific conduction.

Diurnal variations of dinoflagellate bioluminescence within the open-ocean north-east Atlantic

In regions where dinoflagellates dominate bioluminescent emissions, diurnal variations in bioluminescence potential (BPOT) can be influenced by both exogenous and endogenous factors. In summer 2009, measurements were made in the north-east Atlantic to examine the diurnal variations in BPOT in natural dinoflagellate communities and determine the influence of circadian regulation and light exposure. The maximum night BPOT was >23 times greater than the daytime levels for the same populations. Photosynthetic species were responsible for 55–75% of measured BPOT based on calculated light budgets. Under continual darkness, diurnal variability of BPOT was retained over a 48-h period, demonstrating a degree of circadian control. Results suggest that both photosynthetic and heterotrophic dinoflagellates exhibit circadian regulation of their bioluminescent capacity and light strongly influences the diurnal variation of BPOT. Circadian rhythms were photo-entrained to the phase of the natural photoperiod and light further inhibited daytime bioluminescence. Maximum night BPOT was significantly correlated with the previous day integrated photosynthetically active radiation (PAR) suggesting photo-enhancement within natural populations. A 21% decrease in integrated PAR was associated with a 26–29% decrease in maximum night BPOT for constant populations. Maximum night BPOT was damped by up to 73% when organisms were kept in constant darkness. Findings further quantify diurnal variations in BPOT in natural dinoflagellate populations and their relation to a number of taxonomic, cellular and environmental factors. Results emphasize the importance of considering the recent light history of bioluminescent communities when analysing or predicting in situ BPOT.

Late-Holocene paleoenvironmental history of bioluminescent Laguna Grande, Puerto Rico

Coastal lagoons are valuable natural resources and ecotourism hotspots that are at great risk due to human development and global climate change. In order to protect these resources it is important that we fully understand the paleoenvironmental history of these unique ecosystems and their sensitivity to environmental change. Here we present a multi-proxy (pollen, charcoal, grain-size, dinoflagellate cyst, stable isotope geochemistry, and foraminifera) 3600calyr B.P. record of paleoenvironmental change for Laguna Grande, Puerto Rico, which is a particularly unique coastal lagoon as it contains high abundances of the bioluminescent dinoflagellate Pyrodinium bahamense. Sedimentary evidence indicates that the Laguna Grande basin began accumulating sediment ~3600calyr B.P. coincident with regional sea level rise, but the modern lagoon itself is geologically quite young and did not become fully enclosed until ~575calyr B.P. due to longshore depositional processes and mangrove swamp development. Foraminifera assemblages suggest a potential breach of a protective barrier ~1550calyr B.P. perhaps as a result of a tropical storm or tsunami and is coincident with an increase in fire activity. Only two high-energy depositional units (tempestites) could be identified in the lagoon sediments over the last 575calyr B.P. indicating minimal impact by tropical storms or tsunamis on the lagoon since isolation from the open ocean. Fossil dinoflagellate cyst concentrations and influxes do not approach modern levels until ~200calyr B.P. and are currently at the highest levels in the history of the lagoon, indicating the lagoon may have become bioluminescent only recently, perhaps as a result of anthropogenic nutrient additions. Sedimentation rates support this hypothesis indicating a recent tripling of sedimentation rates when compared to millennial-scale sedimentation rates. This study highlights the utility of paleolimnological data in the management and preservation of at-risk coastal lagoon ecosystems.

Abstracts of the 17th International Symposium on Bioluminescence and Chemiluminescence ‐ (ISBC 2012)

WARNING : The light-emitting molecular structures responsible for the chemiluminescence and fluorescence phenomena are not necessarily the same!

Swimming helps phytoplankton avoid being carried by currents

Observations of an upwelling event in Monterey Bay, California, show that non‐bioluminescent phytoplankton from the northern part of the bay are carried with the southward flowing currents along the entrance to the bay, but many bioluminescent dinoflagellates avoid such advection. It has been suggested that the vertical swimming ability of bioluminescent dinoflagellates to descend to deeper layers helped them avoid being carried away by currents. Shulman et al. tested that hypothesis using a tracer model in which the dinoflagellates population is modeled as a concentration, and vertical swimming speeds are introduced into the model. They show that a swimming speed of 20 meters per day, which is in the middle of the observed swimming velocity ranges for observed species of dinoflagellates, leads to only 40% of the population being advected with the current in comparison to the case without vertical swimming. This is in agreement with the observed ratio of mean bioluminescence intensity at the entrance to the bay to the observed mean intensity in the northern part of the bay. Thus, the model confirms that dinoflagellates with moderate vertical swimming speed could avoid being carried away by currents. (Journal of Geophysical Research‐Oceans, doi:10.1029/ 2011JC007480, 2012)

Recharging the Phylogenetic Analysis of Voltage Sensor Domains

Sequence and phylogenetic analysis of voltage-gated ion channels has famously resulted in the discovery of novel ion channels in previously unexplored phyla, and has provided insight into the molecular underpinnings of ion channel function. Because of their crucial role in human health, most current evolutionary analysis has quite reasonably centered on ‘traditional’ pore-forming channels (e.g., Yu, et al, 2005, Pharmacol. Rev., 57:387-395, among many others). Earlier phylogenetic analyses of voltage sensor domain (VSD) modules (e.g., Komanovics et al, 2002, FASEB J., 16:1623-1629, among others) were undertaken before the discovery and characterization of the genes for voltage sensitive phosphatases (VSP) and voltage gated proton channels (HV1), which contain VSDs but do not contain traditional ion pores. We recently reported two new discoveries: in one line of inquiry we found and characterized a dinoflagellate HV1, supporting the prediction of its existence in bioluminescent dinoflagellates by Fogel and Hastings in 1972 (PNAS, 3:690-693); and in the other we uncovered the universal selectivity filter of HV1's. Both of these studies were informed by large-scale sequence and phylogenetic analyses that focused on homologs of VSDs themselves, separate from their N- or C-terminal appendages. Here we present a full sequence and phylogenetic analysis that updates earlier work and reveals that VSDs may have taken a different evolutionary path from associated ion channel pore domains, which has mechanistic and physiological implications.

Can vertical migrations of dinoflagellates explain observed bioluminescence patterns during an upwelling event in Monterey Bay, California?

Extensive AUVs surveys showed that during the development of upwelling, bioluminescent dinoflagellates from the northern part of the Monterey Bay, California (called the upwelling shadow area), were able to avoid advection by southward flowing currents along the entrance to the Bay, while non‐bioluminescent phytoplankton were advected by currents. It is known that vertical swimming of dinoflagellates to deeper layers helps them avoid losses due to advection. In the present paper, we investigate if modeling dinoflagellates' vertical swimming can explain the observed dinoflagellates' ability to avoid advection during the upwelling development. The dynamics of a dinoflagellate population is modeled with the tracer model with introduced vertical swimming velocity. Three swimming behaviors are considered: sinking, swimming to the target depth and diel vertical migration. Velocities in all swimming cases are considered in the ranges of documented velocities for the observed dinoflagellates species during the upwelling development in the Monterey Bay. Our modeling confirmed that observed bioluminescent dinoflagellates' avoidance of advection during the upwelling development can be explained by their vertical swimming ability. In the case of swimming with 20 m/day (which is half of observed maximum swimming velocity), around 40% of dinoflagellates population from the northern part of the Bay were advected along the entrance to the Bay in comparison to the case without swimming. This is in agreement with the ratio of around 45% of observed mean bioluminescence intensity at the entrance to the Bay to the observed mean intensity in the northern part of the Bay. This mechanism also helps explain the general persistence of dinoflagellates in this part of the coastline.

Bioluminescence of Scintillons Isolated from Noctiluca Miliaris is Inhibited by Divalent Metal Cations, Suggesting Proton Channel Involvement

Fogel and Hastings (1972, P.N.A.S. USA 69:690-693) first hypothesized the existence of voltage gated proton channels in bioluminescent dinoflagellates, where they were postulated to trigger the flash. The flash originates in discrete organelles termed scintillons (Nicolas et al., 1987 J. Cell Biol. 105: 723-735.) that contain luciferase and its substrate, luciferin. Luciferase is activated, and luciferin is released from a binding protein, at low pH. Proton channels in the membranes of the scintillon were postulated to conduct H+ from the acidic central flotation vacuole into the scintillon, activating luciferase and releasing luciferin, causing a bioluminescent flash. We have recently cloned a voltage gated proton channel (kHV1) from a non-bioluminescent dinoflagellate, Karlodinium veneficum (Smith et al., 2011 P.N.A.S.in-press). In heterologous expression systems, this channel exhibits a negative Vthreshold so that inward current is activated, consistent with the proposed role of allowing proton flux from the vacuole into the scintillon. The H+ current of heterologously expressed kHV1 is inhibited by Zn2+, but at much higher concentrations than in mammalian channels. We have developed a method to isolate functional scintillons from the bioluminescent species Noctiluca miliaris through gentle homogenization and sucrose density gradient centrifugation. The density of the scintillon fraction was found to be 1.213 g/cm3. Reducing the pH of the scintillon suspension to below pH 6 elicits a luminescent flash. Addition of divalent metals to the suspension diminishes the intensity of the flash. Zn2+, Ni2+, Cu2+, Co2+, and Cd2+ at millimolar concentrations abolished the luminescence from isolated scintillons. The sensitivity of scintillon luminescence to divalent metals is consistent with the idea that proton selective channels in the scintillon membrane mediate the activation of the flash.

Voltage-gated proton channel in a dinoflagellate

Fogel and Hastings first hypothesized the existence of voltage-gated proton channels in 1972 in bioluminescent dinoflagellates, where they were thought to trigger the flash by activating luciferase. Proton channel genes were subsequently identified in human, mouse, and Ciona intestinalis, but their existence in dinoflagellates remained unconfirmed. We identified a candidate proton channel gene from a Karlodinium veneficum cDNA library based on homology with known proton channel genes. K. veneficum is a predatory, nonbioluminescent dinoflagellate that produces toxins responsible for fish kills worldwide. Patch clamp studies on the heterologously expressed gene confirm that it codes for a genuine voltage-gated proton channel, kH(V)1: it is proton-specific and activated by depolarization, its g(H)-V relationship shifts with changes in external or internal pH, and mutation of the selectivity filter (which we identify as Asp(51)) results in loss of proton-specific conduction. Indirect evidence suggests that kH(V)1 is monomeric, unlike other proton channels. Furthermore, kH(V)1 differs from all known proton channels in activating well negative to the Nernst potential for protons, E(H). This unique voltage dependence makes the dinoflagellate proton channel ideally suited to mediate the proton influx postulated to trigger bioluminescence. In contrast to vertebrate proton channels, whose main function is acid extrusion, we propose that proton channels in dinoflagellates have fundamentally different functions of signaling and excitability.

Identification of a voltage‐gated proton channel gene in Karlodinium veneficum

Karlodinium veneficum, an ichthyotoxic dinoflagellate, is responsible for massive fish kills through the release of toxins (karlotoxins). The mechanism of release of the karlotoxin has not been elucidated. In bioluminescent dinoflagellates such as Noctiluca milaris and Lingulodinium polyhedra, voltage-gated proton channels serve as an unusual type of ion channel exhibiting several unique properties. It is known that voltage-gated proton channels form a low resistance pathway across the cell membrane that allows for the passage of particular ionic species in a controlled manner. Therefore, it was hypothesized that a voltage-gated proton channel is present in K. veneficum and that permits the release of the karlotoxins. Identification of the gene suspected to code for this channel in the Karlodinium genome was accomplished using an EST library database created at University of Maryland Biotechnology Institute. The 5′ end of the suspected proton channel gene in K. veneficum strain 1609 was identified and sequenced revealing the splice leader sequence at the 5′ end, four transmembrane domains and similarity with the EST identified in strain 2778. The 3′ end of the gene was not successfully sequenced. This gene will be ligated into a vector for expression which will permit for the study of its role in the release of karlotoxins. Supported by NIH 5T34GM008411

Bioluminescence Imaging Measurements of oscillatory shear flows

In this paper, flashing responses of bioluminescent dinoflagellates in oscillatory turbulent boundary layer flows over rippled bed are studied with the aim with future development of imaging technique to aquire planar distributions of turbulence in a shear layer. Foundamental statistical properties of the bioluminescence involved in strong turbulence caused by separation of the boundary layer from the ripple crest are discussed, and it is found that there is a correlation of time variation of the mean flash intensity with the turbulent energy.

Thin layers and species-specific characterization of the phytoplankton community in Monterey Bay, California, USA

During the summers of 2005 and 2006, experiments designed to understand the properties of densely concentrated, thin layers of plankton and the processes governing their dynamics were conducted in Monterey Bay, California, USA. Our goal was to elucidate the role that species-specific properties of phytoplankton play in thin layer dynamics. Using adaptive sampling, we collected water samples from inside and outside bio-optical features of the water column. Characterization of the phytoplankton was compiled from live and preserved samples, and analyzed within a framework of physical, optical, chemical and acoustical data. In both years, Monterey Bay was home to an extraordinarily diverse assemblage of phytoplankton and other protists. Bioluminescent dinoflagellates, and Harmful Algal Bloom (HAB) taxa were common. In 2005, community assemblages were widespread, thus advection of water through the experimental mooring array did not result in floristic changes. In 2006 phytoplankton were very patchy in horizontal distribution, and advection of water through the array was at times accompanied by dramatic shifts in community composition. Individual taxa often exhibited disparate patterns of vertical distribution, with some found throughout the water column, whereas others were restricted to narrow depth intervals. Thin layers were observed in both years. In 2005, the dinoflagellate Akashiwo sanguinea formed intense thin layers near the pycnocline at night, and migrated to near surface waters at dawn. In 2006, layer composition was more complex, and related to the water mass present at the time of sampling. Optically detected thin layers of phytoplankton can be studied from the perspective of the impact their high biomass has on both ecological processes, and ocean optics. But thin layers can also be studied from the species-specific perspective of each organism, its role within the thin layer habitat, and the impact that life within a thin layer has on its life history and ecology. Several low-abundance taxa appeared to be restricted to narrow depth intervals in the water column, and constitute species-specific thin layers with the potential to have a large ecological impact even if their biomass is too low to dominate an optically defined thin layer. Concentration into thin layers may also facilitate obligatory relationships between taxa, such as the hypothesized interrelationships between cryptomonads, Myrionecta rubra, and Dinophysis spp., all of which were observed in this system. Complexity of vertical structure in Monterey Bay rivals that already demonstrated in topographically constrained, stratified systems, and presents challenges to our theoretical framework of phytoplankton ecology.

Shipboard testing of the efficacy of SeaKleen® as a ballast water treatment to eliminate non‐indigenous species aboard a working tanker in Pacific waters

Trials were conducted aboard the tanker Seabulk Mariner to test a natural product, SeaKleen®, as a biocide controlling non‐indigenous populations of plankton and bacteria in ballast water. SeaKleen® was dosed into matched ballast tanks at two different concentrations, 0.8 mg L−1 active ingredient (a.i.) and 1.6 mg L−1 a.i. during ballasting off the Oregon coast during a three‐day passage to Prince William Sound, Alaska. Live organism counts from treated ballast water were compared with those from untreated (control tank) water collected from the same source location. Shipboard chemical analyses were made to verify dose and quantify chemical degradation and residuals following dilution. Results indicated that both SeaKleen® doses resulted in complete zooplankton and phytoplankton mortality and that the higher dose (1.6 mg L−1 a.i.) caused a two‐log removal of culturable bacteria over a 92 h grow‐out period. Spectrophotometry confirmed initial dosing to within 5% of nominal values. Shipboard bioassays were conducted using larval fish (Cyprinodon variegatus), brine shrimp (Artemia salina) and the bioluminescent dinoflagellate Pyrocystis lunula. Exposure of the test organisms to water drawn from treated ballast tanks 48 h after SeaKleen® was added to the tanks resulted in 100% mortalities in Cyprinodon and Pyrocystis at both doses. Corresponding mortalities for Artemia larvae were 100% and 60% for high and low SeaKleen® doses, respectively. Toxicity testing of treated water, subjected to varying dilutions, indicated that residual toxicity to even the most sensitive organisms would be eliminated once the discharge had dispersed beyond 100 feet from the vessel.

Bioluminescent response of individual dinoflagellate cells to hydrodynamic stress measured with millisecond resolution in a microfluidic device

Dinoflagellate bioluminescence serves as a model system for examining mechanosensing by suspended motile unicellular organisms. The response latency, i.e. the delay time between the mechanical stimulus and luminescent response, provides information about the mechanotransduction and signaling process, and must be accurately known for dinoflagellate bioluminescence to be used as a flow visualization tool. This study used a novel microfluidic device to measure the response latency of a large number of individual dinoflagellates with a resolution of a few milliseconds. Suspended cells of several dinoflagellate species approximately 35 microm in diameter were directed through a 200 microm deep channel to a barrier with a 15 microm clearance impassable to the cells. Bioluminescence was stimulated when cells encountered the barrier and experienced an abrupt increase in hydrodynamic drag, and was imaged using high numerical aperture optics and a high-speed low-light video system. The average response latency for Lingulodinium polyedrum strain HJ was 15 ms (N>300 cells) at the three highest flow rates tested, with a minimum latency of 12 ms. Cells produced multiple flashes with an interval as short as 5 ms between individual flashes, suggesting that repeat stimulation involved a subset of the entire intracellular signaling pathway. The mean response latency for the dinoflagellates Pyrodinium bahamense, Alexandrium monilatum and older and newer isolates of L. polyedrum ranged from 15 to 22 ms, similar to the latencies previously determined for larger dinoflagellates with different morphologies, possibly reflecting optimization of dinoflagellate bioluminescence as a rapid anti-predation behavior.

Bloom forming Alexandrium ostenfeldii (Dinophyceae) in shallow waters of the Åland Archipelago, Northern Baltic Sea

In the past years, late summer blooms of the bioluminescent dinoflagellate Alexandrium ostenfeldii have become a recurrent phenomenon in coastal waters of the central and Northern Baltic Sea. This paper reports exceptionally high cell concentrations (10 5 to 10 6 cells L −1) of the species found during bioluminescent blooms in 2003 and 2004 in a shallow embayment of the Åland archipelago at the SW coast of Finland. Clonal cultures were established for morphological, molecular, toxicological and ecophysiological investigations to characterize the Finnish populations and compare them to other global A. ostenfeldii isolates. The Finnish isolates exhibited typical morphological features of A. ostenfeldii such as large size, a prominent ventral pore and an orthogonally bent first apical plate. However, unambiguous differentiation from closely related Alexandrium peruvianum was difficult due to considerable variation of sulcal anterior plate shapes. The Finnish strains were genetically distinct from other isolates of the species, but phylogenetic analyses revealed a close relationship to isolates from southern England and an A. peruvianum morphotype from the Spanish Mediterranean. Together these isolates formed a distinct clade which was separated from a clade containing other Northern European, North American and New Zealand populations. Toxin analyses confirmed the presence of the PSP toxins GTX2, GTX3 and STX in both Finnish isolates with GTX3 being the dominant toxin. Total relative PSP toxin contents were moderate, ranging from approximately 6 to 15 fmol cell −1 at local salinities of 5 and 10 psu, respectively. Spirolides were not detected. Salinity tolerance experiments showed that the Finnish isolates were well adapted to grow at the low salinities of the Baltic Sea. With a salinity range of approximately 6 to 20–25 psu, Baltic populations are physiologically distinct from their marine relatives. Vigorous production of different cyst types in the cultures suggest that cysts may play a crucial role in the survival and retainment of A. ostenfeldii populations in the Baltic Sea.