Targeted and non-targeted mass spectrometry to explore the chemical diversity of the genus Gambierdiscus in the Atlantic Ocean.

Dinoflagellates of the genus Gambierdiscus have been associated with ciguatera, the most common non-bacterial fish-related intoxication in the world. Many studies report the presence of potentially toxic Gambierdiscus species along the Atlantic coasts including G. australes, G. silvae and G. excentricus. Estimates of their toxicity, as determined by bio-assays, vary substantially, both between species and strains of the same species. Therefore, there is a need for additional knowledge on the metabolite production of Gambierdiscus species and their variation to better understand species differences. Using liquid chromatography coupled to mass spectrometry, toxin and metabolomic profiles of five species of Gambierdiscus found in the Atlantic Ocean were reported. In addition, a molecular network was constructed aiming at annotating the metabolomes. Results demonstrated that G. excentricus could be discriminated from the other species based solely on the presence of MTX4 and sulfo-gambierones and that the variation in toxin content for a single strain could be up to a factor of two due to different culture conditions between laboratories. While untargeted analyses highlighted a higher variability at the metabolome level, signal correction was applied and supervised multivariate statistics performed on the untargeted data set permitted the selection of 567 features potentially useful as biomarkers for the distinction of G. excentricus, G. caribaeus, G. carolinianus, G. silvae and G. belizeanus. Further studies will be required to validate the use of these biomarkers in discriminating Gambierdiscus species. The study also provided an overview about 17 compound classes present in Gambierdiscus, however, significant improvements in annotation are still required to reach a more comprehensive knowledge of Gambierdiscus' metabolome.

Using artificial substrates to quantify Gambierdiscus and other toxic benthic dinoflagellates for monitoring purposes.

Collecting methods generally used to determine cell abundances of toxic benthic dinoflagellates (BHAB) use cells dislodged from either macrophytes or artificial substrates. This article compares the advantages of the macrophyte and artificial substrate methods and discusses which method is more appropriate for use in monitoring programs that focus on toxic BHAB species identification and quantification. The concept of benthic dinoflagellate "preference" for specific macrophytes was also reviewed. Examination of data from 75 field studies showed macrophytes with higher surface area per unit biomass harbored higher concentrations of Gambierdiscus cells. There was no definitive evidence that cells were actively selecting one macrophyte over another. This observation supports the use of artificial substrates (AS) as a means of assessing cell abundances in complex habitats because cell counts are normalized to a standardized surface area, not macrophyte biomass. The artificial substrate method represents the most robust approach, currently available, for collecting toxic, benthic dinoflagellates for a cell-based early warning system.

Comparative Study on the Performance of Three Detection Methods for the Quantification of Pacific Ciguatoxins in French Polynesian Strains of Gambierdiscus polynesiensis.

Gambierdiscus and Fukuyoa dinoflagellates produce a suite of secondary metabolites, including ciguatoxins (CTXs), which bioaccumulate and are further biotransformed in fish and marine invertebrates, causing ciguatera poisoning when consumed by humans. This study is the first to compare the performance of the fluorescent receptor binding assay (fRBA), neuroblastoma cell-based assay (CBA-N2a), and liquid chromatography tandem mass spectrometry (LC-MS/MS) for the quantitative estimation of CTX contents in 30 samples, obtained from four French Polynesian strains of Gambierdiscus polynesiensis. fRBA was applied to Gambierdiscus matrix for the first time, and several parameters of the fRBA protocol were refined. Following liquid/liquid partitioning to separate CTXs from other algal compounds, the variability of CTX contents was estimated using these three methods in three independent experiments. All three assays were significantly correlated with each other, with the highest correlation coefficient (r2 = 0.841) found between fRBA and LC-MS/MS. The CBA-N2a was more sensitive than LC-MS/MS and fRBA, with all assays showing good repeatability. The combined use of fRBA and/or CBA-N2a for screening purposes and LC-MS/MS for confirmation purposes allows for efficient CTX evaluation in Gambierdiscus. These findings, which support future collaborative studies for the inter-laboratory validation of CTX detection methods, will help improve ciguatera risk assessment and management.

Using RDNA sequences to define dinoflagellate species.

Dinoflagellate species are traditionally defined using morphological characters, but molecular evidence accumulated over the past several decades indicates many morphologically-based descriptions are inaccurate. This recognition led to an increasing reliance on DNA sequence data, particularly rDNA gene segments, in defining species. The validity of this approach assumes the divergence in rDNA or other selected genes parallels speciation events. Another concern is whether single gene rDNA phylogenies by themselves are adequate for delineating species or if multigene phylogenies are required instead. Currently, few studies have directly assessed the relative utility of multigene versus rDNA-based phylogenies for distinguishing species. To address this, the current study examined D1-D3 and ITS/5.8S rDNA gene regions, a multi-gene phylogeny, and morphological characters in Gambierdiscus and other related dinoflagellate genera to determine if they produce congruent phylogenies and identify the same species. Data for the analyses were obtained from previous sequencing efforts and publicly available dinoflagellate transcriptomic libraries as well from the additional nine well-characterized Gambierdiscus species transcriptomic libraries generated in this study. The D1-D3 and ITS/5.8S phylogenies successfully identified the described Gambierdiscus and Alexandrium species. Additionally, the data showed that the D1-D3 and multigene phylogenies were equally capable of identifying the same species. The multigene phylogenies, however, showed different relationships among species and are likely to prove more accurate at determining phylogenetic relationships above the species level. These data indicated that D1-D3 and ITS/5.8S rDNA region phylogenies are generally successful for identifying species of Gambierdiscus, and likely those of other dinoflagellates. To assess how broadly general this finding is likely to be, rDNA molecular phylogenies from over 473 manuscripts representing 232 genera and 863 described species of dinoflagellates were reviewed. Results showed the D1-D3 rDNA and ITS phylogenies in combination are capable of identifying 97% of dinoflagellate species including all the species belonging to the genera Alexandrium, Ostreopsis and Gambierdiscus, although it should be noted that multi-gene phylogenies are preferred for inferring relationships among these species. A protocol is presented for determining when D1-D3, confirmed by ITS/5.8S rDNA sequence data, would take precedence over morphological features when describing new dinoflagellate species. This protocol addresses situations such as: a) when a new species is both morphologically and molecularly distinct from other known species; b) when a new species and closely related species are morphologically indistinguishable, but genetically distinct; and c) how to handle potentially cryptic species and cases where morphotypes are clearly distinct but have the same rDNA sequence. The protocol also addresses other molecular, morphological, and genetic approaches required to resolve species boundaries in the small minority of species where the D1-D3/ITS region phylogenies fail.

Quantifying Karenia brevis bloom severity and respiratory irritation impact along the shoreline of Southwest Florida.

Nearly all annual blooms of the toxic dinoflagellate Karenia brevis (K. brevis) pose a serious threat to coastal Southwest Florida. These blooms discolor water, kill fish and marine mammals, contaminate shellfish, cause mild to severe respiratory irritation, and discourage tourism and recreational activities, leading to significant health and economic impacts in affected communities. Despite these issues, we still lack standard measures suitable for assessing bloom severity or for evaluating the efficacy of modeling efforts simulating bloom initiation and intensity. In this study, historical cell count observations along the southwest Florida shoreline from 1953 to 2019 were used to develop monthly and annual bloom severity indices (BSI). Similarly, respiratory irritation observations routinely reported in Sarasota and Manatee Counties from 2006 to 2019 were used to construct a respiratory irritation index (RI). Both BSI and RI consider spatial extent and temporal evolution of the bloom, and can be updated routinely and used as objective criteria to aid future socioeconomic and scientific studies of K. brevis. These indices can also be used to help managers and decision makers both evaluate the risks along the coast during events and design systems to better respond to and mitigate bloom impacts. Before 1995, sampling was done largely in response to reports of discolored water, fish kills, or respiratory irritation. During this timeframe, lack of sampling during the fall, when blooms typically occur, generally coincided with periods of more frequent-than-usual offshore winds. Consequently, some blooms may have been undetected or under-sampled. As a result, the BSIs before 1995 were likely underestimated and cannot be viewed as accurately as those after 1995. Anomalies in the frequency of onshore wind can also largely account for the discrepancies between BSI and RI during the period from 2006 to 2019. These findings highlighted the importance of onshore wind anomalies when predicting respiratory irritation impacts along beaches.

Paralytic shellfish poisoning toxins in butter clams (Saxidomus gigantea) from the Kodiak Archipelago, Alaska.

Consumption of toxic butter clams (Saxidomus gigantea) is the most frequent cause of paralytic shellfish poisoning (PSP) in Alaskan coastal communities. This study examines seasonal variation in total paralytic shellfish toxin concentrations and congener distribution in tissues of butter clams collected in three communities in the Kodiak Islands, Alaska: the City of Kodiak, Ouzinkie and Old Harbor. In response to questions from local harvesters, the efficacy of removing particular clam tissues on total toxin levels was also assessed. Butter clam samples were collected ∼monthly during 2015-2020 in each community to monitor shellfish toxin levels. Results were combined with clam monitoring data collected previously (2013-2015) to document the seasonal distribution of saxitoxin (STX) and its congeners (neosaxitoxin, gonyautoxin) in clam tissues. Seasonally, paralytic shellfish toxin levels in butter clams were highest in summer, declined in winter, but often remained above regulatory limits throughout the year in the three Kodiak communities. Butter clams collected from Ouzinkie (2013-2020) averaged 165 ± 87 µg STX equivalents (Eq.) 100 g - 1, compared to Kodiak 73 ± 54 µg STX Eq. 100 g - 1 and Old Harbor 143 ± 103 µg STX Eq. 100 g - 1. STX accounted for 59-71% of the total toxin concentration in clams at Ouzinkie, Kodiak, and Old Harbor, while neosaxitoxin (neoSTX) accounted for 12-18%. Gonyautoxins (GTXs) represented 31-60% of the total toxin concentration during the seasonal Alexandrium catenella bloom in June-July, with lower percentages in other months. The fraction of total toxin varied among clam tissues: the siphon tip (2-29%), the neck (3-56%), the gut (3-65%) and the body (6-85%). Removal of the siphon tip reduced total toxin content substantially in some samples but had little effect in others. Saxitoxin congeners varied greatly and somewhat unpredictably among clam tissues, and the results indicate removal of specific tissues was not an effective strategy for reducing paralytic shellfish toxin levels in butter clams for safe consumption.

Sulfo-Gambierones, Two New Analogs of Gambierone Produced by Gambierdiscus excentricus.

Ciguatera poisoning is caused by the ingestion of fish or shellfish contaminated with ciguatoxins produced by dinoflagellate species belonging to the genera Gambierdiscus and Fukuyoa. Unlike in the Pacific region, the species producing ciguatoxins in the Atlantic Ocean have yet to be definitely identified, though some ciguatoxins responsible for ciguatera have been reported from fish. Previous studies investigating the ciguatoxin-like toxicity of Atlantic Gambierdiscus species using Neuro2a cell-based assay identified G. excentricus as a potential toxin producer. To more rigorously characterize the toxin profile produced by this species, a purified extract from 124 million cells was prepared and partial characterization by high-resolution mass spectrometry was performed. The analysis revealed two new analogs of the polyether gambierone: sulfo-gambierone and dihydro-sulfo-gambierone. Algal ciguatoxins were not identified. The very low ciguatoxin-like toxicity of the two new analogs obtained by the Neuro2a cell-based assay suggests they are not responsible for the relatively high toxicity previously observed when using fractionated G. excentricus extracts, and are unlikely the cause of ciguatera in the region. These compounds, however, can be useful as biomarkers of the presence of G. excentricus due to their sensitive detection by mass spectrometry.

Cyanobacterial bloom phenology in Saginaw Bay from MODIS and a comparative look with western Lake Erie.

Saginaw Bay and western Lake Erie basin (WLEB) are eutrophic catchments in the Laurentian Great Lakes that experience annual, summer-time cyanobacterial blooms. Both basins share many features including similar size, shallow depths, and equivalent-sized watersheds. They are geographically close and both basins derive a preponderance of their nutrient supply from a single river. Despite these similarities, the bloom phenology in each basin is quite different. The blooms in Saginaw Bay occur at the same time and place and at the same moderate severity level each year. The WLEB, in contrast, exhibits far greater interannual variability in the timing, location, and severity of the bloom than Saginaw Bay, consistent with greater and more variable phosphorus inputs. Saginaw Bay has bloom biomass that corresponds to relatively mild blooms in WLEB, and also has equivalent phosphorus loads. This result suggests that if inputs of P into the WLEB were reduced to similarly sized loads as Saginaw Bay the most severe blooms would be abated. Above 500 t P input, which occur in WLEB, blooms increase non-linearly indicating any reduction in P-input at the highest inputs levels currently occurring in the WLEB, would yield disproportionately large reductions in cyanobacterial bloom intensity. As the maximum phosphorus loads in Saginaw Bay lie just below this inflection point, shifts in the Saginaw Bay watershed toward greater agriculture uses and less wetlands may substantially increase the risk of more intense cyanobacterial blooms than presently occur.

Investigation of Algal Toxins in a Multispecies Seabird Die-Off in the Bering and Chukchi Seas.

Between 2014 and 2017, widespread seabird mortality events were documented annually in the Bering and Chukchi seas, concurrent with dramatic reductions of sea ice, warmer than average ocean temperatures, and rapid shifts in marine ecosystems. Among other changes in the marine environment, harmful algal blooms (HABs) that produce the neurotoxins saxitoxin (STX) and domoic acid (DA) have been identified as a growing concern in this region. Although STX and DA have been documented in Alaska (US) for decades, current projections suggest that the incidence of HABs is likely to increase with climate warming and may pose a threat to marine birds and other wildlife. In 2017, a multispecies die-off consisting of primarily Northern Fulmars (Fulmarus glacialis) and Short-tailed Shearwaters (Ardenna tenuirostris) occurred in the Bering and Chukchi seas. To evaluate whether algal toxins may have contributed to bird mortality, we tested carcasses collected from multiple locations in western and northern Alaska for STX and DA. We did not detect DA in any samples, but STX was present in 60% of all individuals tested and in 88% of Northern Fulmars. Toxin concentrations in Northern Fulmars were within the range of those reported from other STX-induced bird die-offs, suggesting that STX may have contributed to mortalities. However, direct neurotoxic action by STX could not be confirmed and starvation appeared to be the proximate cause of death among birds examined in this study.

Effects of substratum and depth on benthic harmful dinoflagellate assemblages.

Microhabitats influence the distribution and abundance of benthic harmful dinoflagellate (BHAB) species. Currently, much of the information on the relationships between BHABs and microhabitat preferences is based on non-quantitative anecdotal observations, many of which are contradictory. The goal of this study was to better quantify BHAB and microhabitat relationships using a statistically rigorous approach. Between April 2016 to May 2017, a total of 243 artificial substrate samplers were deployed at five locations in the Perhentian Islands, Malaysia while simultaneous photo-quadrat surveys were performed to characterize the benthic substrates present at each sampling site. The screen samplers were retrieved 24 h later and the abundances of five BHAB genera, Gambierdiscus, Ostreopsis, Coolia, Amphidinium, and Prorocentrum were determined. Substrate data were then analyzed using a Bray-Curtis dissimilarity matrix to statistically identify distinct microhabitat types. Although BHABs were associated with a variety of biotic and abiotic substrates, the results of this study demonstrated differing degrees of microhabitat preference. Analysis of the survey results using canonical correspondence analysis explained 70.5% (horizontal first axis) and 21.6% (vertical second axis) of the constrained variation in the distribution of various genera among microhabitat types. Prorocentrum and Coolia appear to have the greatest range being broadly distributed among a wide variety of microhabitats. Amphidinium was always found in low abundances and was widely distributed among microhabitats dominated by hard coral, turf algae, sand and silt, and fleshy algae and reached the highest abundances there. Gambierdiscus and Ostreopsis had more restricted distributions. Gambierdiscus were found preferentially associated with turf algae, hard coral and, to a lesser extent, fleshy macroalgae microhabitats. Ostreopsis, almost always more abundant than Gambierdiscus, preferred the same microhabitats as Gambierdiscus and were found in microbial mats as well. With similar habitat preferences Ostreopsis may serve as an indicator organism for the presence of Gambierdiscus. This study provides insight into how BHAB-specific microhabitat preferences can affect toxicity risks.

Algal toxins in Alaskan seabirds: Evaluating the role of saxitoxin and domoic acid in a large-scale die-off of Common Murres.

Elevated seawater temperatures are linked to the development of harmful algal blooms (HABs), which pose a growing threat to marine birds and other wildlife. During late 2015 and early 2016, a massive die-off of Common Murres (Uria aalge; hereafter, murres) was observed in the Gulf of Alaska coincident with a strong marine heat wave. Previous studies have documented illness and death among seabirds resulting from exposure to the HAB neurotoxins saxitoxin (STX) and domoic acid (DA). Given the unusual mortality event, corresponding warm water anomalies, and recent detection of STX and DA throughout coastal Alaskan waters, HABs were identified as a possible factor of concern. To evaluate whether algal toxins may have contributed to murre deaths, we tested for STX and DA in a suite of tissues obtained from beach-cast murre carcasses associated with the die-off as well as from apparently healthy murres and Black-legged Kittiwakes (Rissa tridactyla; hereafter, kittiwakes) sampled in the preceding and following summers. We also tested forage fish and marine invertebrates collected in the Gulf of Alaska in 2015-2017 to evaluate potential sources of HAB toxin exposure for seabirds. Saxitoxin was present in multiple tissue types of both die-off (36.4 %) and healthy (41.7 %) murres and healthy kittiwakes (54.2 %). Among birds, we detected the highest concentrations of STX in liver tissues (range 1.4-10.8 µg 100 g-1) of die-off murres. Saxitoxin was relatively common in forage fish (20.3 %) and invertebrates (53.8 %). No established toxicity limits currently exist for seabirds, but concentrations of STX in birds and forage fish in our study were lower than values reported from most other bird die-offs in which STX intoxication was causally linked. We detected low concentrations of DA in a single bird sample and in 33.3 % of invertebrates and 4.0 % of forage fish samples. Although these results do not support the hypothesis that acute exposure to STX or DA was a primary factor in the 2015-2016 mortality event, additional information about the sensitivity of murres to these toxins is needed before we can discount their potential role in the die-off. The widespread occurrence of STX in seabirds, forage fish, and invertebrates in the Gulf of Alaska indicates that algal toxins should be considered in future assessments of seabird health, especially given the potential for greater occurrence of HABs in the future.

An extraordinary Karenia mikimotoi "beer tide" in Kachemak Bay Alaska.

In autumn of 2013 an immense dinoflagellate bloom developed in Kachemak Bay, AK, USA. Much of the Bay was discolored a dark amber color and raised public concerns as small scale fish kills were reported in a few locations. Light microscopy revealed a monospecific bloom of gymnodinoid dinoflagellates that were previously unknown from the Bay. Gene sequencing of SSU rDNA from cells collected from the bloom confirmed the causative species to be Karenia mikimotoi. This represents the first report of a K. mikimotoi bloom in Alaska. After the bloom organism was confirmed, a K. mikimotoi species-specific qPCR assay was developed and used to assess K. mikimotoi abundances in DNA extracted from phytoplankton samples from Kachemak Bay and Lower Cook Inlet (LCI) obtained over a six-year period. The K. mikimotoi abundances were compared with corresponding time series of environmental variables (water temperature, salinity, water column stability, nutrients, precipitation and wind speed) to assess the factors contributing to the development of the bloom. The results showed early bloom development occurred in August when snow melt reduced salinities and increased water column stability during a period of calm winds. Peak bloom concentrations occurred in late September (107 cell eq. L-1) even as water temperatures were decreasing. The bloom gradually declined over the winter but persisted until April of 2014. Karenia mikimotoi cells were not detected two years prior or three years following the bloom, suggesting cells were introduced to Kachemak Bay at a time when conditions allowed K. mikimotoi to thrive.

Climate change and harmful benthic microalgae.

Sea surface temperatures in the world's oceans are projected to warm by 0.4-1.4 °C by mid twenty-first century causing many tropical and sub-tropical harmful dinoflagellate genera like Gambierdiscus, Fukuyoa and Ostreopsis (benthic harmful algal bloom species, BHABs) to exhibit higher growth rates over much of their current geographic range, resulting in higher population densities. The primary exception to this trend will be in the tropics where temperatures exceed species-specific upper thermal tolerances (30-31 °C) beyond which growth slows significantly. As surface waters warm, migration to deeper habitats is expected to provide refuge. Range extensions of several degrees of latitude also are anticipated, but only where species-specific habitat requirements can be met (e.g., temperature, suitable substrate, low turbulence, light, salinity, pH). The current understanding of habitat requirements that determine species distributions are reviewed to provide fuller understanding of how individual species will respond to climate change from the present to 2055 while addressing the paucity of information on environmental factors controlling small-scale distribution in localized habitats. Based on the available information, we hypothesized how complex environmental interactions can influence abundance and potential range extensions of BHAB species in different biogeographic regions and identify sentinel sites appropriate for long-term monitoring programs to detect range extensions and reduce human health risks.

Ciguatoxin Occurrence in Food-Web Components of a Cuban Coral Reef Ecosystem: Risk-Assessment Implications.

In Cuba, ciguatera poisoning associated with fish consumption is the most commonly occurring non-bacterial seafood-borne illness. Risk management through fish market regulation has existed in Cuba for decades and consists of bans on selected species above a certain weight; however, the actual occurrence of ciguatoxins (CTXs) in seafood has never been verified. From this food safety risk management perspective, a study site locally known to be at risk for ciguatera was selected. Analysis of the epiphytic dinoflagellate community identified the microalga Gambierdiscus. Gambierdiscus species included six of the seven species known to be present in Cuba (G. caribaeus, G. belizeanus, G. carpenteri, G. carolinianus, G. silvae, and F. ruetzleri). CTX-like activity in invertebrates, herbivorous and carnivorous fishes were analyzed with a radioligand receptor-binding assay and, for selected samples, with the N2A cell cytotoxicity assay. CTX activity was found in 80% of the organisms sampled, with toxin values ranging from 2 to 8 ng CTX3C equivalents g-1 tissue. Data analysis further confirmed CTXs trophic magnification. This study constitutes the first finding of CTX-like activity in marine organisms in Cuba and in herbivorous fish in the Caribbean. Elucidating the structure-activity relationship and toxicology of CTX from the Caribbean is needed before conclusions may be drawn about risk exposure in Cuba and the wider Caribbean.

Improved Accuracy of Saxitoxin Measurement Using an Optimized Enzyme-Linked Immunosorbent Assay.

Paralytic shellfish poisoning (PSP) is precipitated by a family of toxins produced by harmful algae, which are consumed by filter-feeding and commercially popular shellfish. The toxins, including saxitoxin, neosaxitoxin, and gonyautoxins, accumulate in shellfish and cause intoxication when consumed by humans and animals. Symptoms can range from minor neurological dysfunction to respiratory distress and death. There are over 40 different chemical congeners of saxitoxin and its analogs, many of which are toxic and many of which have low toxicity or are non-toxic. This makes accurate toxicity assessment difficult and complicates decisions regarding whether or not shellfish are safe to consume. In this study, we describe a new antibody-based bioassay that is able to detect toxic congeners (saxitoxin, neosaxitoxin, and gonyautoxins) with little cross-reactivity with the low or non-toxic congeners (decarbamoylated or di-sulfated forms). The anti-saxitoxin antibody used in this assay detects saxitoxin and neosaxitoxin, the two most toxic congers equally well, but not the relatively highly toxic gonyautoxins. By incorporating an incubation step with L-cysteine, it is possible to convert a majority of the gonyautoxins present to saxitoxin and neosaxitoxin, which are readily detected. The assay is, therefore, capable of detecting the most toxic PSP congeners found in commercially relevant shellfish. The assay was validated against samples whose toxicity was determined using standard HPLC methods and yielded a strong linear agreement between the methods, with R2 values of 0.94-0.96. As ELISAs are rapid, inexpensive, and easy-to-use, this new commercially available PSP ELISA represents an advance in technology allowing better safety management of the seafood supply and the ability to screen large numbers of samples that can occur when monitoring is increased substantially in response to toxic bloom events.

Morphology, toxicity and molecular characterization of Gambierdiscus spp. towards risk assessment of ciguatera in south central Cuba.

Ciguatera poisoning is caused by the consumption of reef fish or shellfish that have accumulated ciguatoxins, neurotoxins produced by benthic dinoflagellates of the genera Gambierdiscus or Fukuyoa. Although ciguatera constitutes the primary cause of seafood intoxication in Cuba, very little information is available on the occurrence of ciguatoxins in the marine food web and the causative benthic dinoflagellate species. This study conducted on the south-central coast of Cuba reports the occurrence of Gambierdiscus and Fukuyoa genera and the associated benthic genera Ostreopsis and Prorocentrum. Gambierdiscus/Fukuyoa cells were present at low to moderate abundances depending on the site and month of sampling. This genus was notably higher on Dictyotaceae than on other macrophytes. PCR analysis of field-collected samples revealed the presence of six different Gambierdiscus and one Fukuyoa species, including G. caribaeus, G. carolinianus, G. carpenteri, G. belizeanus, F. ruetzleri, G. silvae, and Gambierdiscus sp. ribotype 2. Only Gambierdiscus excentricus was absent from the eight Gambierdiscus/Fukuyoa species known in the wider Caribbean region. Eleven clonal cultures were established and confirmed by PCR and SEM as being either G. carolinianus or G. caribaeus. Toxin production in each isolate was assessed by a radioligand receptor binding assay and found to be below the assay quantification limit. These novel findings augment the knowledge of the ciguatoxin-source dinoflagellates that are present in Cuba, however further studies are needed to better understand the correlation between their abundance, species-specific toxin production in the environment, and the risk for fish contamination, in order to develop better informed ciguatera risk management strategies.

HABscope: A tool for use by citizen scientists to facilitate early warning of respiratory irritation caused by toxic blooms of Karenia brevis.

Blooms of the toxic microalga Karenia brevis occur seasonally in Florida, Texas and other portions of the Gulf of Mexico. Brevetoxins produced during Karenia blooms can cause neurotoxic shellfish poisoning in humans, massive fish kills, and the death of marine mammals and birds. Brevetoxin-containing aerosols are an additional problem, having a severe impact on beachgoers, triggering coughing, eye and throat irritation in healthy individuals, and more serious respiratory distress in those with asthma or other breathing disorders. The blooms and associated aerosol impacts are patchy in nature, often affecting one beach but having no impact on an adjacent beach. To provide timely information to visitors about which beaches are low-risk, we developed HABscope; a low cost (~$400) microscope system that can be used in the field by citizen scientists with cell phones to enumerate K. brevis cell concentrations in the water along each beach. The HABscope system operates by capturing short videos of collected water samples and uploading them to a central server for rapid enumeration of K. brevis cells using calibrated recognition software. The HABscope has a detection threshold of about 100,000 cells, which is the point when respiratory risk becomes evident. Higher concentrations are reliably estimated up to 10 million cells L-1. When deployed by volunteer citizen scientists, the HABscope consistently distinguished low, medium, and high concentrations of cells in the water. The volunteers were able to collect data on most days during a severe bloom. This indicates that the HABscope can provide an effective capability to significantly increase the sampling coverage during Karenia brevis blooms.

Species-specific PCR assays for Gambierdiscus excentricus and Gambierdiscus silvae (Gonyaulacales, Dinophyceae).

The two most toxic Gambierdiscus species identified from the Caribbean are G. excentricus and G. silvae. These species are the primary causes of ciguatera fish poisoning and likely contribute disproportionately to the toxicity of marine food webs. While Gambierdiscus species are difficult to distinguish using light or scanning electron microscopy, reliable species-specific molecular identification methods have been developed and used successfully to identify a number of other Gambierdiscus species. Corresponding species-specific assays are not yet available for G. excentricus and G. silvae, which imposes limitations on species identification and related ecological studies. The following note describes species-specific polymerase chain reaction assays for G. excentricus and G. silvae that can be used for these purposes.

Habitat complexity affects benthic harmful dinoflagellate assemblages in the fringing reef of Rawa Island, Malaysia.

Few studies have investigated the effect of fine-scale habitat differences on the dynamics of benthic harmful dinoflagellate assemblages. To determine how these microhabitat differences affect the distribution and abundance of the major benthic harmful dinoflagellate genera in a tropical coral reef ecosystem, a field study was undertaken between April-September 2015 and January 2016 on the shallow reef flat of the fringing reef of Rawa Island, Terengganu, Malaysia. Sampling of benthic dinoflagellates was carried out using an artificial substrate sampling method (fiberglass screens). Benthic microhabitats surrounding the sampling screens were characterized simultaneously from photographs of a 0.25-m2 quadrat based on categories of bottom substrate types. Five taxonomic groups of benthic dinoflagellates, Ostreopsis, Gambierdiscus, Prorocentrum, Amphidinium, and Coolia were identified, and cells were enumerated using a light microscope. The results showed Gambierdiscus was less abundant than other genera throughout the study period, with maximum abundance of 1.2 × 103 cells 100 cm-2. While most taxa were present on reefs with high coral cover, higher cell abundances were observed in reefs with high turf algal cover and coral rubble, with the exception of Ostreopsis, where the abundance reached a maximum of 3.4 × 104 cells 100 cm-2 in habitats with high coral cover. Microhabitat heterogeneity was identified as a key factor governing the benthic harmful dinoflagellate assemblages and may account for much of the observed variability in dominant taxa. This finding has significant implications for the role of variability in the benthic harmful algal bloom (BHAB) outbreaks and the potential in identifying BHAB-related toxin transfer pathways and the key vectors in the food webs.

Environmental factors influencing the distribution and abundance of Alexandrium catenella in Kachemak bay and lower cook inlet, Alaska.

Despite the long history of paralytic shellfish poisoning (PSP) events in Alaska, little is known about the seasonal distribution and abundance of the causative organism, Alexandrium, or the environmental factors that govern toxic bloom development. To address this issue, a five year study (2012-2017) was undertaken in Kachemak Bay and lower Cook Inlet Alaska to determine how the occurrence of Alexandrium catenella, the dominant PSP-causing Alexandrium species, was influenced by temperature, salinity, nutrient concentrations, and other environmental factors. Cell concentrations from 572 surface water samples were estimated using quantitative PCR. Monthly sampling revealed a seasonal pattern of A. catenella bloom development that was positively correlated with water temperature. Prevailing salinity conditions did not significantly affect abundance, nor was nutrient limitation a direct factor. Elevated cell concentrations were detected in 35 samples from Kachemak Bay (100-3050 cell eq. L-1) while a maximum abundance of 67 cell eq. L-1 was detected in samples from lower Cook Inlet sites. Monitoring data showed average water temperatures in Kachemak Bay increased by ∼2 °C over the course of the study and were accompanied by an increase in Alexandrium abundance. Based on these findings, 7-8 °C appears to represent a temperature threshold for significant bloom development in Kachemak Bay, with the greatest risk of shellfish toxicity occurring when temperatures exceed 10-12 °C. The role of temperature is further supported by time series data from the Alaska Coastal Current (station GAK1), which showed that summertime shellfish toxicity events in Kachemak Bay generally followed periods of anomalously high winter water temperatures. These data indicate monitoring changes in water temperatures may be used as an early warning signal for subsequent development of shellfish toxicity in Kachemak Bay.