Concentration and dispersal of a Pseudo-nitzschia bloom in Penn Cove, Washington, USA

A bloom of the pennate diatom Pseudo-nitzschia, several species of which are associated with the production of the potent excitotoxin domoic acid, was observed in a Puget Sound, Washington embayment in July and August of 1997. Penn Cove, which receives nutrients from the nearby Skagit River and abundant sunshine during summer months due to its location in the rain shadow of the Olympic Mountains, is the home of a commercial mussel farm which supplies shellfish to many coastal areas of the USA. Levels of domoic acid in mussels increased to 3 ppm on 6 and 10 July, corresponding to the observation of a brown algal bloom in Penn Cove. Four species of Pseudo-nitzschia (P. pungens, P. multiseries, P. australis, and P. pseudodelicatissima) were present in our samples from the cove, corresponding to levels of domoic acid in seawater ranging from 0.1-0.8 mirog l(-1) as measured by a receptor binding assay. The highest Pseudo-nitzschia concentration during the time of our sampling was 13 million cells per liter on 28 July. The bloom of Pseudo-nitzschia occurred after a period of strong discharge from the Skagit River and rain accompanied by elevated south and southeasterly winds. Stratification of the cove, providing optimal bloom conditions, was facilitated by weak winds, sunshine, and a freshwater lens at the mouth of the cove. The position of the Pseudo-nitzschia bloom was influenced by buoyancy fronts caused by exchange of water within the cove with that of Saratoga Passage. The decay of this bloom in Penn Cove was accompanied by decreasing nitrate levels at all measured depths. These and future observations aid in the development of a model for prediction of toxic bloom events in the shallow embayments of Puget Sound.     

大亚湾水域并基拟菱形藻的种类鉴定和产毒特征分析

为了丰富我国海域拟菱形藻(Pseudo-nitzschia Peragallo)的物种多样性, 并澄清其产毒特征, 研究从广东大亚湾海域分离并建立了一株拟菱形藻的单克隆培养株系MC298, 通过光学显微镜下的群体特征和透射电镜下的超微形态特征观察, 结合基于核糖体转录间隔区(Internal Transcribed Spacer, ITS)的分子系统学数据, 以及基于ITS2转录RNA的二级结构分析, 鉴定到我国拟菱形藻属的1个新记录种: 并基拟菱形藻P. decipiens Lundholm & Moestrup。研究对其形态学特征进行了较为详细地描述, 并与相似种进行了比较, 还对其ITS2-RNA的特有标志结构进行了阐述。同时, 利用高效液相色谱-质谱联用法(Liquid chromatography tandem mass spectrometry, LC-MS/MS)对该藻株的产毒特征进行了检测, 结果未检测到DA的存在。研究不仅丰富了我国拟菱形藻属的物种多样性, 也可为拟菱形藻的产毒特征研究提供基础数据。     

Feeding,egg production,and egg hatching success of the copepods Acartia tonsa and Temora longicornis on diets of the toxic diatom Pseudo-nitzschia multiseries and the non-toxic diatom Pseudo-nitzschia pungens

In 1987, there was an episode of shellfish poisoning in Canada with human fatalities caused by the diatom Pseudo-nitzschia multiseries, which produced the toxin domoic acid. In order to examine whether domoic acid in this diatom serves as a grazing deterrent for copepods, we compared feeding rates, egg production rates, egg hatching success and mortality of the calanoid copepods Acartia tonsa and Temora longicornis feeding on unialgal diets of the toxic diatom P. multiseries and the similarly-sized non-toxic diatom Pseudo-nitzschia pungens. Copepods were collected in summers of 1994, 1995 and 1996 from Shediac Bay, New Brunswick, Canada, near Prince Edward Island, the site of the 1987 episode of domoic acid shellfish poisoning. Rates of ingestion of the toxic versus the non-toxic diatom by A. tonsa and T. longicornis were similar, with only one significantly different pair of values obtained in 1994, for which A. tonsa had a higher mean rate of ingestion of the toxic than the non-toxic diatom. Thus, domoic acid did not appear to retard grazing. Analyses of copepods with high performance liquid chromatography (HPLC) revealed that copepods accumulated domoic acid when feeding on P. multiseries. Egg production rates of copepods when feeding on P. multiseries and P. pungens were very low, ranging from 0 to 2.79 eggs female^–1 d^–1. There did not appear to be differential egg production or egg hatching success on diets of the toxic and non-toxic diatoms. Mortality of females on the toxic diet was low, ranging from 0 to 20%, with a mean of 13%, and there was no apparent difference between mortality of copepods feeding on toxic versus non-toxic diatoms. Egg hatching success on both diets, although based on few eggs, ranged between 22% and 76%, with a mean percentage hatching of 45%. Diets of the non-toxic diatom plus natural seawater assemblages supplemented with dissolved domoic acid, revealed similar rates and percentages when compared to previous experiments. In summary, none of the variables measured indicated adverse effects on copepods feeding on the toxic compared to the non-toxic diatom.     

PSEUDO-NITZSCHIA SPECIES (BACILLARIOPHYCEAE) IN LOUISIANA COASTAL WATERS: MOLECULAR PROBE FIELD TRIALS, GENETIC VARIABILITY, AND DOMOIC ACID ANALYSES

An 18-month field survey of the Pseudo-nitzschia population present in Louisiana coastal waters was conducted comparing species abundance estimates by novel fluorescent molecular probes (16S large subunit rDNA oligonucleotide sequences) with traditional electron and differential-interference light microscopy. While the probe and microscopic analyses agreed on the presence or absence of four common Pseudo-nitzschia species ( P. multiseries (Hasle) Hasle, P. pseudodelicatissima (Hasle) Hasle, P. delicatissima (P.T. Cleve) Heiden, and P. pungens (Grunow) Hasle in 66% of the samples analyzed, the probes gave conflicting results with the microscopic methods in the remaining 34% of the samples. The majority of the discrepancies appear to be because of genetic variation within the Pseudo-nitzschia population, especially in P. pseudodelicatissima, indicating that the Monterey Bay Pseudo-nitzschia spp. may not be appropriate reference strains for distinguishing Louisiana Pseudo-nitzschia spp. Additionally, P. pseudodelicatissima has been associated with domoic acid (DA) activity in three field samples, at levels up to 22 times higher than the highest value given inother published reports of DA production by this species. The contemporaneous existence of multiple strains of P. pseudodelicatissima (toxic and non-toxic) presents new challenges to the study of the ecophysiology and population dynamics of this bloom-forming species.     

Pseudo-nitzschia simulans sp. nov. (Bacillariophyceae), the first domoic acid producer from Chinese waters

The genus Pseudo-nitzschia has attracted attention because of production of the toxin, domoic acid (DA), causing Amnesic Shellfish Poisoning (ASP). Pseudo-nitzschia blooms occur frequently in Chinese coastal waters, and DA has been detected in several marine organisms, but so far no Pseudo-nitzschia strains from Chinese waters have been shown to produce DA. In this study, monoclonal Pseudo-nitzschia strains were established from Chinese coastal waters and examined using light microscopy, electron microscopy and molecular markers. Five strains, sharing distinct morphological and molecular features differentiating them from other Pseudo-nitzschia species, represent a new species, Pseudo-nitzschia simulans sp. nov. Morphologically, the taxon belongs to the P. pseudodelicatissima group, cells possessing a central nodule and each stria comprising one row of poroids. The new species is characterized by the poroid structure, which typically comprises two sectors, each sector located near opposite margins of the poroid. The production of DA was examined by liquid chromatography tandem mass spectrometry (LC–MS/MS) analyses of cells in stationary growth phase. Domoic acid was detected in one of the five strains, with concentrations around 1.05–1.54 fg cell⁻¹. This is the first toxigenic diatom species reported from Chinese waters.     

Inorganic carbon acquisition in potentially toxic and non-toxic diatoms: the effect of pH-induced changes in seawater carbonate chemistry

The effects of pH-induced changes in seawater carbonate chemistry on inorganic carbon (C(i)) acquisition and domoic acid (DA) production were studied in two potentially toxic diatom species, Pseudo-nitzschia multiseries and Nitzschia navis-varingica, and the non-toxic Stellarima stellaris. In vivo activities of carbonic anhydrase (CA), photosynthetic O(2) evolution and CO(2) and HCO(3)(-) uptake rates were measured by membrane inlet MS in cells acclimated to low (7.9) and high pH (8.4 or 8.9). Species-specific differences in the mode of carbon acquisition were found. While extracellular carbonic anhydrase (eCA) activities increased with pH in P. multiseries and S. stellaris, N. navis-varingica exhibited low eCA activities independent of pH. Half-saturation concentrations (K(1/2)) for photosynthetic O(2) evolution, which were highest in S. stellaris and lowest in P. multiseries, generally decreased with increasing pH. In terms of carbon source, all species took up both CO(2) and HCO(3)(-). K(1/2) values for inorganic carbon uptake decreased with increasing pH in two species, while in N. navis-varingica apparent affinities did not change. While the contribution of HCO(3)(-) to net fixation was more than 85% in S. stellaris, it was about 55% in P. multiseries and only approximately 30% in N. navis-varingica. The intracellular content of DA increased in P. multiseries and N. navis-varingica with increasing pH. Based on our data, we propose a novel role for eCA acting as C(i)-recycling mechanism. With regard to pH-dependence of growth, the 'HCO(3)(-) user' S. stellaris was as sensitive as the 'CO(2) user' N. navis-varingica. The suggested relationship between DA and carbon acquisition/C(i) limitation could not be confirmed.     

Quantifying rates of evolutionary adaptation in response to ocean acidification

The global acidification of the earth's oceans is predicted to impact biodiversity via physiological effects impacting growth, survival, reproduction, and immunology, leading to changes in species abundances and global distributions. However, the degree to which these changes will play out critically depends on the evolutionary rate at which populations will respond to natural selection imposed by ocean acidification, which remains largely unquantified. Here we measure the potential for an evolutionary response to ocean acidification in larval development rate in two coastal invertebrates using a full-factorial breeding design. We show that the sea urchin species Strongylocentrotus franciscanus has vastly greater levels of phenotypic and genetic variation for larval size in future CO(2) conditions compared to the mussel species Mytilus trossulus. Using these measures we demonstrate that S. franciscanus may have faster evolutionary responses within 50 years of the onset of predicted year-2100 CO(2) conditions despite having lower population turnover rates. Our comparisons suggest that information on genetic variation, phenotypic variation, and key demographic parameters, may lend valuable insight into relative evolutionary potentials across a large number of species.     

Understanding the blob bloom: Warming increases toxicity and abundance of the harmful bloom diatom Pseudo-nitzschia in California coastal waters

The toxic diatom genus Pseudo-nitzschia produces environmentally damaging harmful algal blooms (HABs) along the U.S. west coast and elsewhere, and a recent ocean warming event coincided with toxic blooms of record extent. This study examined the effects of temperature on growth, domoic acid toxin production, and competitive dominance of two Pseudo-nitzschia species from Southern California. Growth rates of cultured P. australis were maximal at 23 °C (∼0.8 d⁻¹), similar to the maximum temperature recorded during the 2014–2015 warming anomaly, and decreased to ∼0.1 d⁻¹ by 30 °C. In contrast, cellular domoic acid concentrations only became detectable at 23 °C, and increased to maximum levels at 30 °C. In two incubation experiments using natural Southern California phytoplankton communities, warming also increased the relative abundance of another potentially toxic local species, P. delicatissima. These results suggest that both the toxicity and the competitive success of particular Pseudo-nitzschia spp. can be positively correlated with temperature, and therefore there is a need to determine whether harmful blooms of this diatom genus may be increasingly prevalent in a warmer future coastal ocean.     

The influence of salinity in the domoic acid effect on estuarine phytoplankton communities

Toxic species of the diatom genus Pseudo-nitzschia, observed worldwide from coastal waters to the open ocean, produce the neurotoxin domoic acid (DA). DA is an important environmental and economic hazard due to shellfish contamination with subsequent effects on higher trophic levels. Previous research has demonstrated that, among other environmental factors, salinity influences the abundance and toxicity of Pseudo-nitzschia. In this study, the environmental factors driving the growth of Pseudo-nitzschia and the production of dissolved DA (dDA) in North Inlet estuary were examined. The effect of salinity on the growth inhibition of phytoplankton induced by the initial presence as well as by an addition of dDA was also assessed. Initially, the diatom abundance was negatively correlated with the abundance of Pseudo-nitzschia and with the concentration of dDA. With the addition of a concentrated solution of dDA, the percent inhibition of cryptophytes and diatoms was significantly correlated with salinity and suggested a higher sensitivity to dDA at extreme salinities. These results emphasize the importance of salinity in assessing the properties of DA and potentially of other phycotoxins on phytoplankton.     

Species of the planktonic diatom genus Pseudo-nitzschia of the Pacific coasts of Mexico

Species of the diatom genus Pseudo-nitzschia are common in the marine phytoplankton world-wide. Some species of this genus have been proved to be source of domoic acid (DA), a powerful toxin causing Amnesic Shellfish Poisoning (ASP) in humans and probably mass mortality in sea birds and mammals. Net plankton samples obtained during several cruises and seasons from the Pacific coasts of Mexico: western coasts of Baja California, Gulf of California, coasts of the tropical Pacific of Mexico (including the Gulf of Tehuantepec), were analyzed to study the species of the diatom genus Pseudo-nitzschia. Four species ( P. australis, a presumed toxic species, P. fraudulenta, P. lineola, P. pungens) and one former species of the genus, Nitzschia americana were recorded and studied by light and electron microscopy. The most common species was P. pungens, widely distributed along the Pacific coasts of Mexico. All other species appeared occasionally and in low relative abundances. The probable misidentification of P. australis as P. seriata is discussed, as well as the presence of another potentially toxic species, P. delicatissima, in the Gulf of California. No case of toxicity (ASP) has been fully documented and therefore related to toxic Pseudo-nitzschia species in the Gulf of California. This revised version was published online in July 2006 with corrections to the Cover Date.     

Novel symptomatology and changing epidemiology of domoic acid toxicosis in California sea lions (Zalophus californianus): an increasing risk to marine mammal health

Harmful algal blooms are increasing worldwide, including those of Pseudo-nitzschia spp. producing domoic acid off the California coast. This neurotoxin was first shown to cause mortality of marine mammals in 1998. A decade of monitoring California sea lion (Zalophus californianus) health since then has indicated that changes in the symptomatology and epidemiology of domoic acid toxicosis in this species are associated with the increase in toxigenic blooms. Two separate clinical syndromes now exist: acute domoic acid toxicosis as has been previously documented, and a second novel neurological syndrome characterized by epilepsy described here associated with chronic consequences of previous sub-lethal exposure to the toxin. This study indicates that domoic acid causes chronic damage to California sea lions and that these health effects are increasing.     

Ocean acidification-induced food quality deterioration constrains trophic transfer

Our present understanding of ocean acidification (OA) impacts on marine organisms caused by rapidly rising atmospheric carbon dioxide (CO(2)) concentration is almost entirely limited to single species responses. OA consequences for food web interactions are, however, still unknown. Indirect OA effects can be expected for consumers by changing the nutritional quality of their prey. We used a laboratory experiment to test potential OA effects on algal fatty acid (FA) composition and resulting copepod growth. We show that elevated CO(2) significantly changed the FA concentration and composition of the diatom Thalassiosira pseudonana, which constrained growth and reproduction of the copepod Acartia tonsa. A significant decline in both total FAs (28.1 to 17.4 fg cell(-1)) and the ratio of long-chain polyunsaturated to saturated fatty acids (PUFA:SFA) of food algae cultured under elevated (750 μatm) compared to present day (380 μatm) pCO(2) was directly translated to copepods. The proportion of total essential FAs declined almost tenfold in copepods and the contribution of saturated fatty acids (SFAs) tripled at high CO(2). This rapid and reversible CO(2)-dependent shift in FA concentration and composition caused a decrease in both copepod somatic growth and egg production from 34 to 5 eggs female(-1) day(-1). Because the diatom-copepod link supports some of the most productive ecosystems in the world, our study demonstrates that OA can have far-reaching consequences for ocean food webs by changing the nutritional quality of essential macromolecules in primary producers that cascade up the food web.     

伪柔弱拟菱形藻复合群的形态分类学研究

于2007年夏季(8月)在广东省大亚湾海域观察到伪柔弱拟菱形藻复合群的6个种类:花形拟菱形藻(P.caciantha Lundholm,Moestrup Hasle)、靓纹拟菱形藻(P.calliantha Lundholm,Moestrup Hasle)、尖细拟菱形藻[P.cuspidata(Hasle)Lundholm,Moestrup Hasle]、曼氏拟菱形藻(P.mannii Amato Montresor)、伪柔弱拟菱形藻[P.pseudodelicatissima(Hasle)Lundholm,Hasle Moestrup]和中华拟菱形藻(P.sinica QiWang),其中花形拟菱形藻、靓纹拟菱形藻和曼氏拟菱形藻为我国新记录种类。对该复合群种类的形态学特征、生活习性和生态分布进行了观察和描述,并对其种类之间的形态学特征进行了比较研究。     

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。     

Environmental stress and domoic acid production by Pseudo-nitzschia: a physiological perspective

Production of domoic acid (DA) by the pennate diatom Pseudo-nitzschia multiseries is associated with physiological stress caused by silicate (Si) and/or phosphate (P) limitation. Such limitation may promote DA synthesis by (1) reducing primary metabolic activity, thus making available necessary precursors, high energy compounds, and cofactors, and (2) favoring the expression of genes involved in the biosynthesis of this toxin. In the case of Si and P-limitation, DNA synthesis and the progression through the cell division cycle are slowed, perhaps prolonging or arresting the cells in the stage of the division cycle which is most conducive to DA production. However, N-limitation results in an insufficient pool of cellular free N, which restricts synthesis of this nitrogenous toxin. A continuous supply of photophosphorylated high-energy intermediates (e.g., ATP and NADPH) is necessary for DA synthesis. In order to better understand the mechanism(s) of DA production, more studies are needed to elucidate: (1) the details of the biosynthetic pathway, (2) the regulation of enzymes involved in the pathway, (3) the relation between DA synthesis and the cell division cycle, (4) the cellular compartmentalization of DA biosynthesis, and (5) other environmental factors that may trigger DA production. Finally, these studies should be extended to include toxigenic Pseudo-nitzschia species other than P. multiseries, to confirm the commonality of these mechanisms.     

Coastal acidification impacts on shell mineral structure of bivalve mollusks

Ocean acidification is occurring globally through increasing CO₂ absorption into the oceans creating particular concern for calcifying species. In addition to ocean acidification, near shore marine habitats are exposed to the deleterious effects of runoff from acid sulfate soils which also decreases environmental pH. This coastal acidification is being exacerbated by climate change‐driven sea‐level rise and catchment‐driven flooding. In response to reduction in habitat pH by ocean and coastal acidification, mollusks are predicted to produce thinner shells of lower structural integrity and reduced mechanical properties threatening mollusk aquaculture. Here, we present the first study to examine oyster biomineralization under acid sulfate soil acidification in a region where growth of commercial bivalve species has declined in recent decades. Examination of the crystallography of the shells of the Sydney rock oyster, Saccostrea glomerata, by electron back scatter diffraction analyses revealed that the signal of environmental acidification is evident in the structure of the biomineral. Saccostrea glomerata, shows phenotypic plasticity, as evident in the disruption of crystallographic control over biomineralization in populations living in coastal acidification sites. Our results indicate that reduced sizes of these oysters for commercial sale may be due to the limited capacity of oysters to biomineralize under acidification conditions. As the impact of this catchment source acidification will continue to be exacerbated by climate change with likely effects on coastal aquaculture in many places across the globe, management strategies will be required to maintain the sustainable culture of these key resources.     

DOMOIC ACID PRODUCTION IN NITZSCHIA SP. (BACILLARIOPHYCEAE) ISOLATED FROM A SHRIMP-CULTURE POND IN DO SON, VIETNAM

Domoic acid (DA), a neuroexcitatory amino acid, was detected in batch culture of the newly recognized species Nitzschia navis-varingica Lundholm et Moestrup . The production of DA by this diatom was confirmed by electrospray ionization mass spectrometry. The diatom was collected from a shrimp-culture pond in Do Son, Vietnam. The production of DA (1.7 pg·cell ^{− 1}) is within the levels reported for Pseudo-nitzschia multiseries (Hasle) Hasle. The DA production started during the late exponential growth phase and reached a maximum during the early stationary growth phase. Maximum DA levels in the axenic culture decreased to about half that of the nonaxenic culture (0.9 pg·cell ^{− 1} vs. 1.7 pg·cell ^{− 1}), suggesting that DA production by the new species is influenced by bacteria.     

Ocean acidification and the loss of phenolic substances in marine plants

Rising atmospheric CO(2) often triggers the production of plant phenolics, including many that serve as herbivore deterrents, digestion reducers, antimicrobials, or ultraviolet sunscreens. Such responses are predicted by popular models of plant defense, especially resource availability models which link carbon availability to phenolic biosynthesis. CO(2) availability is also increasing in the oceans, where anthropogenic emissions cause ocean acidification, decreasing seawater pH and shifting the carbonate system towards further CO(2) enrichment. Such conditions tend to increase seagrass productivity but may also increase rates of grazing on these marine plants. Here we show that high CO(2) / low pH conditions of OA decrease, rather than increase, concentrations of phenolic protective substances in seagrasses and eurysaline marine plants. We observed a loss of simple and polymeric phenolics in the seagrass Cymodocea nodosa near a volcanic CO(2) vent on the Island of Vulcano, Italy, where pH values decreased from 8.1 to 7.3 and pCO(2) concentrations increased ten-fold. We observed similar responses in two estuarine species, Ruppia maritima and Potamogeton perfoliatus, in in situ Free-Ocean-Carbon-Enrichment experiments conducted in tributaries of the Chesapeake Bay, USA. These responses are strikingly different than those exhibited by terrestrial plants. The loss of phenolic substances may explain the higher-than-usual rates of grazing observed near undersea CO(2) vents and suggests that ocean acidification may alter coastal carbon fluxes by affecting rates of decomposition, grazing, and disease. Our observations temper recent predictions that seagrasses would necessarily be "winners" in a high CO(2) world.     

Comparison of two domoic acid-producing diatoms: a review

In the past five years, awareness of domoic acid has increased from localized problems in Canada to outbreaks along both North American coasts. The phycotoxin domoic acid causes Amnesic Shellfish Poisoning (ASP) in humans and can be fatal. The known species of phytoplankton responsible for production of domoic acid include some pennate diatom species of the genus Nitzschia, sensu latu, which form stepped chains typical of the Pseudonitzschia. These diatoms are widely distributed, but their life histories and population dynamics are poorly understood. This review addresses histories of occurrences, morphology, geographical distributions, seasonal patterns, growth requirements, domoic acid production, and trophic interactions, with emphasis on a comparison of Pseudonitzschia pungens f. multiseries (Hasle) Hasle and Pseudonitzschia australis Frenguelli. Through continued research it will become possible to provide guidelines for regulatory agencies that protect both the consumer and the seafood industry.