Remote,subsurface detection of the algal toxin domoic acid onboard the Environmental Sample Processor: Assay development and field trials

The ability to detect harmful algal bloom (HAB) species and their toxins in real- or near real-time is a critical need for researchers studying HAB/toxin dynamics, as well as for coastal resource managers charged with monitoring bloom populations in order to mitigate their wide ranging impacts. The Environmental Sample Processor (ESP), a robotic electromechanical/fluidic system, was developed for the autonomous, subsurface application of molecular diagnostic tests and has successfully detected several HAB species using DNA probe arrays during field deployments. Since toxin production and thus the potential for public health and ecosystem effects varies considerably in natural phytoplankton populations, the concurrent detection of HAB species and their toxins onboard the ESP is essential. We describe herein the development of methods for extracting the algal toxin domoic acid (DA) from Pseudo-nitzschia cells (extraction efficiency >90%) and testing of samples using a competitive ELISA onboard the ESP. The assay detection limit is in the low ng/mL range (in extract), which corresponds to low ng/L levels of DA in seawater for a 0.5 L sample volume acquired by the ESP. We also report the first in situ detection of both a HAB organism (i.e., Pseudo-nitzschia) and its toxin, domoic acid, via the sequential (within 2–3 h) conduct of species- and toxin-specific assays during ESP deployments in Monterey Bay, CA, USA. Efforts are now underway to further refine the assay and conduct additional calibration exercises with the aim of obtaining more reliable, accurate estimates of bloom toxicity and thus their potential impacts.     

Concurrent exposure of bottlenose dolphins (Tursiops truncatus) to multiple algal toxins in Sarasota Bay, Florida, USA

Sentinel species such as bottlenose dolphins (Tursiops truncatus) can be impacted by large-scale mortality events due to exposure to marine algal toxins. In the Sarasota Bay region (Gulf of Mexico, Florida, USA), the bottlenose dolphin population is frequently exposed to harmful algal blooms (HABs) of Karenia brevis and the neurotoxic brevetoxins (PbTx; BTX) produced by this dinoflagellate. Live dolphins sampled during capture-release health assessments performed in this region tested positive for two HAB toxins; brevetoxin and domoic acid (DA). Over a ten-year study period (2000–2009) we have determined that bottlenose dolphins are exposed to brevetoxin and/or DA on a nearly annual basis (i.e., DA: 2004, 2005, 2006, 2008, 2009; brevetoxin: 2000, 2004, 2005, 2008, 2009) with 36% of all animals testing positive for brevetoxin (n = 118) and 53% positive for DA (n = 83) with several individuals (14%) testing positive for both neurotoxins in at least one tissue/fluid. To date there have been no previously published reports of DA in southwestern Florida marine mammals, however the May 2008 health assessment coincided with a Pseudo-nitzschia pseudodelicatissima bloom that was the likely source of DA observed in seawater and live dolphin samples. Concurrently, both DA and brevetoxin were observed in common prey fish. Although no Pseudo-nitzschia bloom was identified the following year, DA was identified in seawater, fish, sediment, snails, and dolphins. DA concentrations in feces were positively correlated with hematologic parameters including an increase in total white blood cell (p = 0.001) and eosinophil (p<0.001) counts. Our findings demonstrate that dolphins within Sarasota Bay are commonly exposed to two algal toxins, and provide the impetus to further explore the potential long-term impacts on bottlenose dolphin health.     

Note on the occurrence of Pseudo-nitzschia australis and domoic acid in squid from Monterey Bay, CA (USA)

Over the past decade diatom blooms of domoic acid (DA)-producing Pseudo-nitzschia spp. have been responsible for numerous marine mammal and bird mortalities in Monterey Bay, CA. One possible toxin vector is the market squid, Loligo opalescens, a small pelagic mollusk that plays an important role in the near-shore food web of the California Current ecosystem as a favored vertebrate prey species. This study examined the trophic link between toxic Pseudo-nitzschia and L. opalescens using toxin and stomach content analyses of animals collected from Monterey Bay, CA in 2000. Receptor binding assay data (confirmed by tandem mass spectrometry), demonstrated the presence of DA in squid during a toxic Pseudo-nitzschia event, with P. australis frustules observed in stomach samples. Though DA levels were low (<0.5 μg DA g⁻¹ tissue) in L. opalescens during the study period, it is now clear that this potent neurotoxin can occur in squid and is likely delivered through its krill prey species, which are known to retain DA after feeding on toxic Pseudo-nitzschia. Our findings suggest that further study of the relationship between Pseudo-nitzschia blooms and DA contamination of squid is warranted to better evaluate the potential health risk to humans and wildlife associated with this major commercial seafood species and important prey item.     

Domoic acid exposure in pygmy and dwarf sperm whales (Kogia spp.) from southeastern and mid-Atlantic U.S. waters

The neurotoxin domoic acid (DA) was detected in urine and fecal samples recovered from pygmy sperm whales (Kogia breviceps) and dwarf sperm whales (Kogia sima) stranding along the U.S. Atlantic coast from 1997 to 2008. Of the 41 animals analyzed from Virginia, North Carolina, South Carolina and Florida, 24 (59%) tested positive for DA at concentrations of 0.4–1.8 ng/mL in urine and 12–13,566 ng/g in feces as determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Feces appeared to be the best indicator of DA exposure in Kogia spp., with 87% of all fecal samples analyzed testing positive for this toxin. Additional stranded animals (n = 40) representing 11 other cetacean species were recovered from the same region between 2006 and 2008 and analyzed by LC–MS/MS, however DA was not detected in any of these individuals. DA is produced naturally by diatoms in the genus Pseudo-nitzschia. Although blooms of DA-producing Pseudo-nitzschia have been associated with repeated large-scale marine mammal mortalities on the west coast of the U.S., there is no documented history of similar blooms on the southeast U.S. coast, and there were no observed Pseudo-nitzschia blooms in the region associated with any of these strandings. The feeding habits of Kogia spp. are poorly documented; thus, the vector(s) for DA exposure to these deep-diving species remains to be identified. Toxin accumulation in these pelagic whale species may be an indication of cryptic harmful algal bloom activity in offshore areas not currently being monitored. This study highlights the need for a better understanding of the role of toxigenic algae in marine mammal morbidity and mortality globally.     

A shift in the dominant toxin-producing algal species in central California alters phycotoxins in food webs

In California, the toxic algal species of primary concern are the dinoflagellate Alexandrium catenella and members of the pennate diatom genus Pseudo-nitzschia, both producers of potent neurotoxins that are capable of sickening and killing marine life and humans. During the summer of 2004 in Monterey Bay, we observed a change in the taxonomic structure of the phytoplankton community—the typically diatom-dominated community shifted to a red tide, dinoflagellate-dominated community. Here we use a 6-year time series (2000–2006) to show how the abundance of the dominant harmful algal bloom (HAB) species in the Bay up to that point, Pseudo-nitzschia, significantly declined during the dinoflagellate-dominated interval, while two genera of toxic dinoflagellates, Alexandrium and Dinophysis, became the predominant toxin producers. This change represents a shift from a genus of toxin producers that typically dominates the community during a toxic bloom, to HAB taxa that are generally only minor components of the community in a toxic event. This change in the local HAB species was also reflected in the toxins present in higher trophic levels. Despite the small contribution of A. catenella to the overall phytoplankton community, the increase in the presence of this species in Monterey Bay was associated with an increase in the presence of paralytic shellfish poisoning (PSP) toxins in sentinel shellfish and clupeoid fish. This report provides the first evidence that PSP toxins are present in California's pelagic food web, as PSP toxins were detected in both northern anchovies (Engraulis mordax) and Pacific sardines (Sardinops sagax). Another interesting observation from our data is the co-occurrence of DA and PSP toxins in both planktivorous fish and sentinel shellfish. We also provide evidence, based on the statewide biotoxin monitoring program, that this increase in the frequency and abundance of PSP events related to A. catenella occurred not just in Monterey Bay, but also in other coastal regions of California. Our results demonstrate that changes in the taxonomic structure of the phytoplankton community influences the nature of the algal toxins that move through local food webs and also emphasizes the importance of monitoring for the full suite of toxic algae, rather than just one genus or species.     

Blooms of Pseudo-nitzschia and domoic acid in the San Pedro Channel and Los Angeles harbor areas of the Southern California Bight, 2003–2004

Abundances of Pseudo-nitzschia spp. and concentrations of particulate domoic acid (DA) were determined in the Southern California Bight (SCB) along the coasts of Los Angeles and Orange Counties during spring and summer of 2003 and 2004. At least 1500 km² were affected by a toxic event in May/June of 2003 when some of the highest particulate DA concentrations reported for US coastal waters were measured inside the Los Angeles harbor (12.7 μg DA L⁻¹). Particulate DA levels were an order of magnitude lower in spring of 2004 (February and March), but DA concentrations per cell at several sampling stations during 2004 exceeded previously reported maxima for natural populations of Pseudo-nitzschia (mean = 24 pg DA cell⁻¹, range = 0–117 pg DA cell⁻¹). Pseudo-nitzschia australis dominated the Pseudo-nitzschia assemblage in spring 2004. Overall, DA-poisoning was implicated in >1400 mammal stranding incidents within the SCB during 2003 and 2004. Ancillary physical and chemical data obtained during our regional surveys in 2004 revealed that Pseudo-nitzschia abundances, particulate DA and cellular DA concentrations were inversely correlated with concentrations of silicic acid, nitrogen and phosphate, and to specific nutrient ratios. Particulate DA was detected in sediment traps deployed at 550 and 800 m depth during spring of 2004 (0.29–7.6 μg DA (g sediment dry weight)⁻¹). The highest DA concentration in the traps was measured within 1 week of dramatic decreases in the abundances of Pseudo-nitzschia in surface waters. To our knowledge these are the deepest sediment trap collections from which DA has been detected. Sinking of the spring Pseudo-nitzschia bloom may constitute a potentially important link between DA production in surface waters and benthic communities in the coastal ocean near Los Angeles. Our study indicates that toxic blooms of Pseudo-nitzschia are a recurring phenomenon along one of the most densely populated coastal stretches of the SCB and that the severity and magnitude of these events can be comparable to or greater than these events in other geographical regions affected by domoic acid.     

Detection of the toxin domoic acid from clam extracts using a portable surface plasmon resonance biosensor

The amnesic shellfish poison domoic acid is produced by marine algae of the genus Pseudo-nitzschia. We have developed a portable surface plasmon resonance (SPR) biosensor system for the detection of domoic acid. Because of concerns with domoic acid contamination of shellfish, there is a need for rapid field quantification of toxin levels in both shellfish and seawater. Antibodies were raised against domoic acid and affinity purified. These antibodies were used to develop competition- and displacement-based assays using a portable six-channel SPR system developed in our laboratories. Standard curves for detection of domoic acid in phosphate buffered saline and in diluted clam extracts analyzed by the competition-based SPR assay demonstrated a limit of detection of 3 ppb (10 nM) and a quantifiable range from 4 to 60 ppb (13–200 nM). Comparison of analyses for domoic acid levels in Pacific razor clams, Siliqua patula, containing moderate to high levels of domoic acid by the standard HPLC analysis protocol and the SPR-based assay gave an excellent correlation. This same technology should also function for detection of domoic acid in concentrated algal extracts or high dissolved levels in seawater.     

Health and Ecological Impacts of Harmful Algal Blooms: Risk Assessment Needs

The symposium session, Indicators for Effects and Predictions of Harmful Algal Blooms, explored the current state of indicators used to assess the human health and ecological risks caused by harmful algal blooms, and highlighted future needs and impediments that must be overcome in order to provide a complete risk assessment of their impacts. Six recognized human poisoning syndromes resulting from algal toxins (paralytic, neurotoxic, amnesic, diarrhetic shellfish poisonings, ciguatera fish poisoning, and putative estuary associated syndrome) impact human health through consumption of contaminated seafood, direct contact with bloom water, or inhalation of aerosolized toxin. Thorough health risk assessment for the variety of algal toxins is hampered to varying degrees because either the toxin has not been identified or indicators for exposure and effects remain poorly defined. Predicting the occurrence and determining the impacts of harmful algal blooms in coastal ecosystems are the two major ecological risk assessment needs. In the former case, the hazard is the suite of conditions that trigger bloom initiation, magnify bloom intensity or support bloom longevity, whereas in the latter case, the hazard is the algal toxin. In both cases, indicators (of triggering mechanisms, exposure, and effects) are better defined for some HAB species and toxins than others, but are by no means complete.     

我国沿海拟菱形藻属的2新记录种及其产毒特征分析

为澄清我国沿海拟菱形藻属(Pseudo-nitzschia)的物种多样性,并确认中国海域拟菱形藻属是否具有产生多莫酸(Domoicacid)的能力,采用毛细管显微操作技术从我国沿海水体中分离、纯化拟菱形藻细胞,建立了单克隆培养株系,并基于核糖体转录间隔区ITS1-5.8S-ITS2 序列构建了分子系统树。结果表明,结合在光学显微镜和透射电镜下观察的形态学特征和分子系统发育分析数据,鉴定到我国拟菱形藻属的2新记录种:银河拟菱形藻(P. galaxiae Lundholm & Moestrup)和微孔拟菱形藻(P. micropora Priisholm, Moestrup & Lundholm),对其形态学特征进行了详细描述,并与相似种类进行了比较研究。利用高效液相色谱(HPLC)技术对多莫酸特征进行了检测,结果表明培养株系并不产生多莫酸。这些为我国拟菱形藻属物种多样性和产毒特征研究提供了基础数据。     

First record of the dynamics of domoic acid producing Pseudo-nitzschia spp. in Indonesian waters as a function of environmental variability

Within the past few decades, harmful algal blooms (HABs) have occurred frequently in Indonesian waters, resulting in environmental degradation, economic loss and human health problems. So far, HAB related studies mainly addressed ecological traits and species distribution, yet toxin measurements were virtually absent for Indonesian waters. The aim of the present study was to explore variability of the potentially toxic marine diatom genus Pseudo-nitzschia, as well as its neurotoxin domoic acid as a function of environmental conditions in Ambon Bay, eastern Indonesia. Weekly phytoplankton samples, oceanographic (CTD, nutrients) and meteorological (precipitation, wind) parameters were analyzed at 5 stations in the bay during the dry and wet seasons of 2018. Liquid chromatography – tandem mass spectrometry (LC–MS/MS) was used to detect particulate DA (pDA). Vegetative cells of Pseudo-nitzschia spp. and pDA were found in 98.6% and 51.4% of the samples, respectively. pDA levels were low, yet detected throughout the campaign, implying that Ambon Bay might potentially be subject to amnesic shellfish poisoning. The highest levels of both Pseudo-nitzschia spp. cell abundance and pDA were found in the wet season, showing a strong positive correlation between both parameters, compared to the dry season, (r = 0.87 and r = 0.66 (p < 0.01), respectively). Statistical analyses revealed that temperature and mixed layer depth positively correlated with Pseudo-nitzschia spp. and pDA during the dry season, while ammonium showed positive correlations in both seasons. This study represents the first successful investigation of the presence and variability of Pseudo-nitzschia spp. and its neurotoxin DA in Indonesian waters.     

The copepod Calanus finmarchicus: A potential vector for trophic transfer of the marine algal biotoxin, domoic acid

The marine algal biotoxin, domoic acid (DA), is produced by certain members of the diatom genus Pseudo-nitzschia. This neurotoxin has been responsible for several mass mortality events involving marine birds and mammals. In all cases, the toxin was transferred from its algal producers through marine food webs by one or more intermediate vectors. The ability of some copepod taxa to serve as vectors for DA has been demonstrated; however, the role played in DA trophic transfer by Calanus finmarchicus, which often dominates N. Atlantic zooplankton assemblages and is a primary dietary component of the highly endangered N. Atlantic right whale (Eubalaena glacialis), has been uncertain. In the present study, we examined the ability of C. finmarchicus to consume DA-producing algae and retain the toxin. Results of grazing and toxin accumulation/depuration experiments showed that C. finmarchicus consumed DA-producing Pseudo-nitzschia multiseries regardless of the presence or absence of morphologically similar, but non-toxic, P. pungens, across initial cell concentrations ranging from 1000-4000 cells mL^− 1. Furthermore, C. finmarchicus did not appear to preferentially consume or avoid either Pseudo-nitzschia species tested. After ingestion of P. multiseries, copepods accumulated DA and retained it for up to 48 h post-removal of the toxin source. These findings provide evidence for the potential of C. finmarchicus to facilitate DA trophic transfer in marine food webs where toxic Pseudo-nitzschia is present.     

Pseudo-nitzschia physiological ecology,phylogeny, toxicity,monitoring and impacts on ecosystem health

Over the last decade, our understanding of the environmental controls on Pseudo-nitzschia blooms and domoic acid (DA) production has matured. Pseudo-nitzschia have been found along most of the world's coastlines, while the impacts of its toxin, DA, are most persistent and detrimental in upwelling systems. However, Pseudo-nitzschia and DA have recently been detected in the open ocean's high-nitrate, low-chlorophyll regions, in addition to fjords, gulfs and bays, showing their presence in diverse environments. The toxin has been measured in zooplankton, shellfish, crustaceans, echinoderms, worms, marine mammals and birds, as well as in sediments, demonstrating its stable transfer through the marine food web and abiotically to the benthos. The linkage of DA production to nitrogenous nutrient physiology, trace metal acquisition, and even salinity, suggests that the control of toxin production is complex and likely influenced by a suite of environmental factors that may be unique to a particular region. Advances in our knowledge of Pseudo-nitzschia sexual reproduction, also in field populations, illustrate its importance in bloom dynamics and toxicity. The combination of careful taxonomy and powerful new molecular methods now allow for the complete characterization of Pseudo-nitzschia populations and how they respond to environmental changes. Here we summarize research that represents our increased knowledge over the last decade of Pseudo-nitzschia and its production of DA, including changes in worldwide range, phylogeny, physiology, ecology, monitoring and public health impacts.     

Recent domoic acid closures of shellfish harvest areas in Washington State inland waterways

Several species of the toxigenic diatom Pseudo-nitzschia, together with low concentrations of domoic acid (DA) in shellfish have been observed in Puget Sound, Washington State, since 1991. However, for the first time in September 2003, high-density blooms of Pseudo-nitzschia forced the closure of recreational, commercial, and tribal subsistence shellfish harvesting in Puget Sound. Here we report on the environmental conditions associated with shellfish closures in two inland waterways of Washington State during the Fall 2005. In Sequim Bay, shellfish harvest losses occurred on September 12 following the measurement of elevated macronutrient levels on September 2, and a bloom of P. pseudodelicatissima (up to 13 million cells/L) on September 9. Ambient NH₄ concentrations >12 μM (measured on September 2) were likely due to anthropogenic sources, ostensibly from sewage inputs to Sequim Bay. The closure of a Penn Cove commercial shellfish farm on October 16 was caused by a bloom of P. australis that followed a period of sustained precipitation, elevated Skagit River flow, and persistent southeasterly winds. The relative importance of a number of environmental factors, including temperature, stratification caused by rivers, and nutrient inputs, whether natural or anthropogenic, must be carefully studied in order to better understand the recent appearance of massive blooms of toxigenic Pseudo-nitzschia in the inland waterways of Washington State.     

Growth dynamics of non-toxic Pseudo-nitzschia delicatissima and toxic P. seriata (Bacillariophyceae) under simulated spring and summer photoperiods

Marine planktonic diatoms of the genus Pseudo-nitzschia Peragallo have been responsible for amnesic shellfish poisoning (ASP) events worldwide through the production of the neurotoxin domoic acid (DA). The appearance and toxicity of Pseudo-nitzschia species is variable throughout the year and potentially linked to changes in environmental parameters; many ASP events occur in relatively high latitudes where day length is particularly variable with season. In UK waters, shellfish monitoring has prevented any impact on human health but has led to long-term closures of fisheries, with severe economic consequences. Laboratory experiments on two Pseudo-nitzschia species typically found in Scottish West Coast waters during spring (short photoperiod (SP)) and summer (long photoperiod (LP)) conditions were conducted to determine the influence of photoperiod on their growth and toxicity. Results indicated that non-toxic P. delicatissima (Cleve) Heiden achieved a greater cell density under SP (9-h light:15-h dark (L:D) cycle). For toxin-producing P. seriata (Cleve) H. Peragallo, a LP (18-h L:6-h D cycle) resulted in an enhanced growth rate, cell yield and total toxin production, but it decreased the toxin production per cell. A better understanding of the response of Pseudo-nitzschia species to photoperiod and other foreseeable environmental variables may help predict the appearance of toxic strains.     

Host-specific adaptation governs the interaction of the marine diatom,Pseudo-nitzschia and their microbiota

The association of phytoplankton with bacteria is ubiquitous in nature and the bacteria that associate with different phytoplankton species are very diverse. The influence of these bacteria in the physiology and ecology of the host and the evolutionary forces that shape the relationship are still not understood. In this study, we used the Pseudo-nitzschia–microbiota association to determine (1) if algal species with distinct domoic acid (DA) production are selection factors that structures the bacterial community, (2) if host-specificity and co-adaptation govern the association, (3) the functional roles of isolated member of microbiota on diatom–hosts fitness and (4) the influence of microbiota in changing the phenotype of the diatom hosts with regards to toxin production. Analysis of the pyrosequencing-derived 16S rDNA data suggests that the three tested species of Pseudo-nitzschia, which vary in toxin production, have phylogenetically distinct bacterial communities, and toxic Pseudo-nitzschia have lower microbial diversity than non-toxic Pseudo-nitzschia. Transplant experiments showed that isolated members of the microbiota are mutualistic to their native hosts but some are commensal or parasitic to foreign hosts, hinting at co-evolution between partners. Moreover, Pseudo-nitzschia host can gain protection from algalytic bacteria by maintaining association with its microbiota. Pseudo-nitzschia also exhibit different phenotypic expression with regards to DA production, and this depends on the bacterial species with which the host associates. Hence, the influences of the microbiota on diatom host physiology should be considered when studying the biology and ecology of marine diatoms.     

Toxigenic algae and associated phycotoxins in two coastal embayments in the Ebro Delta (NW Mediterranean)

Harmful Algal Bloom (HAB) surveillance is complicated by high diversity of species and associated phycotoxins. Such species-level information on taxonomic affiliations and on cell abundance and toxin content is, however, crucial for effective monitoring, especially of aquaculture and fisheries areas. The aim addressed in this study was to determine putative HAB taxa and related phycotoxins in plankton from aquaculture sites in the Ebro Delta, NW Mediterranean. The comparative geographical distribution of potentially harmful plankton taxa was established by weekly field sampling throughout the water column during late spring–early summer over two years at key stations in Alfacs and Fangar embayments within the Ebro Delta. Core results included not only confirmed identification of HAB taxa that are common for the time period and geographical area, but also provided evidence of potentially new taxa. At least 25 HAB taxa were identified to species level, and an additional six genera were confirmed, by morphological criteria under light microscopy and/or by molecular genetics approaches involving qPCR and next generation DNA pyrosequencing. In particular, new insights were gained by the inclusion of molecular techniques, which focused attention on the HAB genera Alexandrium, Karlodinium, and Pseudo-nitzschia. Noteworthy is the discovery of Azadinium sp., a potentially new HAB species for this area, and Gymnodinium catenatum or Gymnodinium impudicum by means of light microscopy. In addition, significant amounts of the neurotoxin domoic acid (DA) were found for the first time in phytoplankton samples in the Ebro Delta. While the presence of the known DA-producing diatom genus Pseudo-nitzschia was confirmed in corresponding samples, the maximal toxin concentration did not coincide with highest cell abundances of the genus and the responsible species could not be identified. Combined findings of microscopic and molecular detection approaches underline the need for a synoptic strategy for HAB monitoring, which integrates the respective advantages and compensates for limitations of individual methods.     

Characterization of a toxic Pseudo-nitzschia spp. bloom in the Northern Gulf of Mexico associated with domoic acid accumulation in fish

A toxic bloom of Pseudo-nitzschia spp. was observed in the Alabama coastal waters of the northern Gulf of Mexico (NGOM) in June 2009 that resulted in the accumulation of domoic acid (DA) in fish. The bloom initiated following a large storm event that likely caused increased groundwater discharge 16–20 days prior to peak densities. Eleven sites, located in littoral shoreline waters and inshore embayments spanning the entire Alabama NGOM coastline, were sampled during peak densities to assess Pseudo-nitzschia species composition and toxicity, and associated water-quality parameters. Small fish (0.27–11.9 g body weight) were collected at six of these sites for analysis of DA content. High Pseudo-nitzschia spp. densities (8.27 × 10⁴–5.05 × 10⁶ cell l⁻¹) were detected at eight sites located in the littoral shoreline and particulate DA was detected at six of these littoral sites (48.0–540 pg ml⁻¹). The bloom consisted primarily (>90%) of Pseudo-nitzschia subfraudulenta, a species previously characterized as forming only a minor component of Pseudo-nitzschia assemblages and not known to produce DA. Pseudo-nitzschia spp. were at low densities or not detected at the inshore sites and DA was detected at these sites. Pseudo-nitzschia spp. density varied along an estuarine gradient, with greater densities occurring in the most saline, clear, and nutrient-poor waters. Cell density was strongly and negatively correlated with silicate (Si) concentrations and the ratios of silicate to dissolved inorganic nitrogen and phosphate (Si:DIN and Si:PO₄). Cell toxin quota was negatively correlated with phosphate, and strongly and positively correlated with the ratio of total nitrogen to total phosphorus (TN:TP). These relationships are consistent with previous observations that indicate Pseudo-nitzschia spp. density and toxicity are likely to be greater in high salinity, high irradiance, and nutrient-poor waters. DA was detected in 128 of 131 (98%) of the fish collected, which included seven primary and secondary consumer species. This is the first demonstration of trophic transfer of DA in this region of the NGOM, indicating that toxic blooms of Pseudo-nitzschia spp. in Alabama coastal waters have the potential to transfer DA to recreationally and commercially important fish species.     

Monitoring Karenia brevis blooms in the Gulf of Mexico using satellite ocean color imagery and other data

Harmful algal blooms (HABs) of Karenia brevis are a recurrent problem in the Gulf of Mexico, with nearly annual occurrences on the Florida southwest coast, and fewer occurrences on the northwest Florida and Texas coasts. Beginning in 1999, the National Oceanic and Atmospheric Administration has issued the Gulf of Mexico HAB Bulletins to support state monitoring and management efforts. These bulletins involve analysis of satellite imagery with field and meteorological station data. The effort involves several components or models: (a) monitoring the movement of an algal bloom that has previously been identified as a HAB (type 1 forecast); (b) detecting new blooms as HAB or non-HAB (type 2); (c) predicting the movement of an identified HAB (type 3); (d) predicting conditions favorable for a HAB to occur where blooms have not yet been observed (type 4). The types 1 and 2 involve methods of bloom detection requiring routine remote sensing, especially satellite ocean color imagery and in situ data. Prediction (types 3 and 4) builds on the monitoring capability by using interpretative and numerical modeling. Successful forecasts cover more than 1000 km of coast and require routine input of remotely sensed and in situ data.The data sources used in this effort include ocean color imagery from the Sea-Viewing Wide Field-of-View Sensor/OrbView-2 satellite and processed using coastal-specific algorithms, wind data from coastal and offshore buoys, field observations of bloom location and intensity provided by state agencies, and forecasts from the National Weather Service. The HAB Bulletins began in coordination with the state of Florida in autumn of 1999 and included K. brevis bloom monitoring (type 1), with limited advisories on transport (type 3) and the detection of blooms in new areas (type 2). In autumn 2000, we improved both the transport forecasts and detection capabilities and began prediction of conditions favorable for bloom development (type 4). The HAB Bulletins have had several successes. The state of Florida was advised of the potential for a bloom to occur at the end of September 2000 (type 4), and the state was alerted to the position of blooms in January 2000 and October 2001 in areas that had not been previously sampled (type 3). These successful communications of HAB activity allowed Florida agencies responsible for shellfish management and public health to respond to a rapidly developing event in a timely, efficient manner.     

Release and degradation of amnesic shellfish poison from decaying Pseudo-nitzschia multiseries in presence of bacteria and organic matter

Domoic acid (DA), the neurotoxin produced by diatoms such as Pseudo-nitzschia multiseries is water-soluble and can bioaccumulate, causing mass death of birds and marine mammals worldwide. Humans eating contaminated shellfish most commonly suffer from memory loss but mortalities have been recorded. The fate of particulate and dissolved DA released from the cells or added as standards was studied when incubated with different bacterial abundances, copepod faecal pellets, mussel pseudo-faeces and bottom sediment. Strains of P. multiseries from Canada and Brazil were grown in non-axenic continuous monocultures with different nutrient conditions, or in a follow-up mesocosm experiment. Incubation lasted up to 75 days in the dark under quiescent conditions after the cells had been killed. Release of DA from decaying cells did not depend on bacterial abundance when the bacterial source was cultures of P. multiseries, and the dissolved toxin was stable with bacteria from P. multiseries cultures (at least 20 days with 1× or 4× bacterial concentration), or with a naturally occurring density of bacteria from surface waters of a known P. multiseries bloom area (35 days). However, four-fold concentration of the natural bacterial consortium from the bloom site reduced the onset of DA degradation to 16 days. Thus, this study suggests that when testing toxin degradation by bacteria, it is important to use bacterial consortia from known bloom areas of Pseudo-nitzschia. Copepod faecal pellets did not affect DA degradation, whereas the presence of mussel pseudo-faeces and bottom sediment rapidly removed most of the toxin. We believe that the rapid removal of DA in the two latter treatments was due to higher bacterial abundance and the presence of enzymes from the mussels and/or associated bacteria that are important for the degradation process. The mechanisms underlying the observed effects on DA degradation with mussel pseudo-faeces and sediment require further research, but suggest interesting possibilities as a potential future mitigation technique.     

Development of a high-resolution molecular marker for tracking Pseudo-nitzschia pungens genetic diversity through comparative analysis of mitochondrial genomes

The harmful algal bloom (HAB) species Pseudo-nitzschia pungens is widely distributed in almost all continents. Accumulating evidence suggests that P. pungens has high genetic diversity and many strains can produce the toxin domoic acid (DA) that harms animals and humans. Nevertheless, different P. pungens strains cannot be distinguished using morphological features or using common molecular markers including 18S rDNA, 28S rDNA, ITS, cox1, and rbcL. As such, high-resolution molecular markers need to be developed to resolve P. pungens genetic diversity, facilitating accurate tracking of toxic P. pungens strains. We hypothesized that molecular markers with high resolution and high specificity can be designed through identifying regions with high genomic variations in the mitochondrial genome. Here, we describe the development of a new molecular marker Pseudo-nitzschia pungens mitochondrial 1 (ppmt1) with high resolution and high specificity through comparative analysis of mitochondrial genomes of nine P. pungens strains isolated from coastal regions of China. In conclusion, we have developed ppmt1 as a high-resolution and high-specificity molecular marker for tracking strains and genetic diversity of the HAB species P. pungens.