An assessment of Pseudo-nitzschia population dynamics and domoic acid production in coastal Louisiana

Over 1200 samples were collected from Louisiana estuarine and coastal shelf waters between 1989 and 2002, and analyzed to examine the population dynamics of Pseudo-nitzschia and to assess the potential threat posed by domoic acid (DA), a potent neurotoxin produced by some members within this toxigenic diatom genus. Results demonstrated that three species in this region (Pseudo-nitzschia multiseries, P. pseudodelicatissima complex, P. delicatissima) produce DA, and that particulate toxin levels were highest (up to 3.05 μg L⁻¹) during the spring bloom, while cellular concentrations were highest in the winter/early spring when P. multiseries was most abundant (up to 30 pg cell⁻¹). These particulate toxin levels are comparable to those seen in other regions (e.g., United States west coast) where DA poisoning events have occurred in the past. Pseudo-nitzschia were most abundant under dissolved inorganic nitrogen-replete conditions coupled with lower silicate and/or phosphate concentrations, and in the early spring months when temperatures were cooler. Pseudo-nitzschia were occasionally well-represented in the phytoplankton assemblage (≥10⁶ cells L⁻¹ in 14% of samples, over 50% of total phytoplankton in 5% of samples), indicating that planktivores (e.g., Gulf menhaden, Brevoortia patronus) may have little choice but to consume Pseudo-nitzschia cells, thereby providing potential vectors for DA transfer to higher trophic levels. By comparison, eastern oysters (Crassostrea virginica) present in estuarine waters may be more exposed to this toxin when Pseudo-nitzschia cells are part of a mixed assemblage, reducing selective grazing by these bivalves. C. virginica may thus represent the most effective vector for DA exposure in humans.     

Rising temperatures may increase growth rates and microcystin production in tropical Microcystis species

Rising temperatures (1.4–6 °C) due to climate change have been predicted to increase cyanobacterial bloom occurrences in temperate water bodies; however, the impacts of warming on tropical cyanobacterial blooms are unknown. We examined the effects of four different temperatures on the growth rates and microcystin (MC) production of five tropical Microcystis isolates (M. ichthyoblabe (two strains), M. viridis, M. flos-aquae, and M. aeruginosa). The temperature treatments are based on current temperature range in Singapore's reservoirs (27 °C and 30 °C), as well as projected mean (33 °C) and maximum temperatures (36 °C) based on tropical climate change estimates of +6 °C in air temperature. Increasing temperatures did not significantly affect the maximum growth rates of most Microcystis strains. Higher growth rates were only observed in one M. ichthyoblabe strain at 33 °C and M. flos-aquae at 30 °C where both were isolated from the same reservoir. MC-RR and MC-LR were produced in varying amounts by all four species of Microcystis. Raised temperatures of 33 °C were found to boost total MC cell quota for three Microcystis strains although further increase to 36 °C led to a sharp decrease in total MC cell quota for all five Microcystis strains. Increasing temperature also led to higher MC-LR:MC-RR cell quota ratios in M. ichthyoblabe. Our study suggests that higher mean water temperatures resulting from climate change will generally not influence growth rates of Microcystis spp. in Singapore except for increases in M. ichthyoblabe strains. However, toxin cell quota may increase under moderate warming scenarios depending on the species.     

Estimating spatial variation in the abundance of potential microcystin-producing Microcystis spp. using real-time PCR during summer bloom in Lake Taihu

Lake Taihu, which is the third largest freshwater lakes in China, is a hypertrophic shallow lake in eastern China that has experienced lake-wide cyanobacterial blooms annually during the last few decades. In this study, quantitative real-time PCR assays targeting on phycocyanin intergenic spacer (PC-IGS) and a microcystin synthetase gene mcyD were established, respectively. Water samples collected from eight sampling sites (including Zhushan Bay (N5), Meiliang Bay (N2), Gonghu Bay (N4), West lake areas (W2 and W4), south-middle lake areas (S2, S4 and S5)) in August of 2009 and 2010 were analyzed using real time PCR for the distribution and abundance of toxic and total Microcystis populations. The results showed that Microcystis exists as a mixed population of potential toxic and non-toxic genotypes, and there was significant spatial changes in the abundance of potential toxic Microcystis on the basis of quantification by quantitative real-time PCR analysis: the abundance of toxic Microcystis population in 2009 and 2010 varied from 4.08 × 10⁴ to 8.28 × 10⁶ copies mL^?1, from 4.45 × 10⁵ to 5.22 × 10⁷ copies mL^?1, respectively. Meanwhile the ratio of the mcyD subpopulation to the total Microcystis varied considerably, from 5.7% to 41.1% in 2009 and from 10.3% to 65.8% in 2010 in all sampling sites, and the value is high in Zhushan Bay and Meiliang Bay with the high level of eutrophication. Correlation analysis showed the abundance of toxic and total Microcystis being strongly related (P < 0.01). However, there is different effects of environmental factors on the abundance of toxic and non-toxic Microcystis populations. The abundance of toxic and total Microcystis populations were positively correlated with chlorophyll-a (Chl-a) concentration (P < 0.01) suggesting that Microcystis is dominated genera of cyanobacterial bloom in Lake Taihu. It was also found that the abundance of toxic Microcystis and the proportion of toxic subpopulation to the total Microcystis were positively correlated with total phosphorus and orthophosphate concentrations (P < 0.01), whereas there was no significant correlation with total nitrogen and nitrate concentration (P > 0.05). All data suggest that phosphorus concentration is a critical factor for determining the abundance of toxic Microcystis population.     

Estimating microcystin levels at recreational sites in western Lake Erie and Ohio

Cyanobacterial harmful algal blooms (cyanoHABs) and associated toxins, such as microcystin, are a major global water-quality issue. Water-resource managers need tools to quickly predict when and where toxin-producing cyanoHABs will occur. This could be done by using site-specific models that estimate the potential for elevated toxin concentrations that cause public health concerns. With this study, samples were collected at three Ohio lakes to identify environmental and water-quality factors to develop linear-regression models to estimate microcystin levels. Measures of the algal community (phycocyanin, cyanobacterial biovolume, and cyanobacterial gene concentrations) and pH were most strongly correlated with microcystin concentrations. Cyanobacterial genes were quantified for general cyanobacteria, general Microcystis and Dolichospermum, and for microcystin synthetase (mcyE) for Microcystis, Dolichospermum, and Planktothrix. For phycocyanin, the relations were different between sites and were different between hand-held measurements on-site and nearby continuous monitor measurements for the same site. Continuous measurements of parameters such as phycocyanin, pH, and temperature over multiple days showed the highest correlations to microcystin concentrations. The development of models with high R² values (0.81–0.90), sensitivities (92%), and specificities (100%) for estimating microcystin concentrations above or below the Ohio Recreational Public Health Advisory level of 6 μg L⁻¹ was demonstrated for one site; these statistics may change as more data are collected in subsequent years. This study showed that models could be developed for estimates of exceeding a microcystin threshold concentration at a recreational freshwater lake site, with potential to expand their use to provide relevant public health information to water resource managers and the public for both recreational and drinking waters.     

Uptake of paralytic shellfish poisoning and spirolide toxins by paddle crabs (Ovalipes catharus) via a bivalve vector

The uptake of paralytic shellfish poisoning (PSP) toxins and spirolides by the paddle crab (Ovalipes catharus) was investigated in two laboratory feeding trials using Greenshell? mussels (Perna canaliculus), which had been fed toxic strains of either Alexandrium catenella or A. ostenfeldii, as a vector. Toxin uptake by crabs occurred in both feeding trials and was limited to the visceral tissue; no toxins were detected in the body meat or the gills. The first trial utilized a strain of A. catenella that had high total PSP toxin content, 442.3 ± 91.6 fmol/cell, that was dominated by low toxicity N-sulfocarbamoyl toxins resulting in a low cellular toxicity, 5.5 ± 1.6 pg STXequiv./cell. In this trial, toxin accumulation in the crabs was highly variable and ranged from 3.8 to 221.5 μg STXequiv./100 g, with 3/4 of the crabs exceeding the regulatory limit of 80 μg STXequiv./100 g. Eight days after feeding on toxic mussels the crabs still retained high levels of toxin suggesting that depuration rates in this species may be slow. In the second feeding trial, the A. ostenfeldii strain fed to mussels produced low levels of both PSP toxins (52.0 ± 19.5 fmol/cell; 1.4 ± 0.3 pg STXequiv./cell) and spirolides (1.8 pg/cell) and, as a result, the concentration transferred to crabs via the mussels was very low-PSP toxins ranged from 2.5 to 6.8 μg STXequiv./100 g and spirolides from 6 to 7 μg/kg. The results of our study demonstrate that paddle crabs are capable of acquiring both PSP toxins and spirolides and suggest that this may occur in the wild during a toxic shellfish event. It also highlights the need to remove the viscera before consumption.     

Rapid quantification of mcyB copy numbers on dry chemistry PCR chips and predictability of microcystin concentrations in freshwater environments

Microcystin-producing cyanobacteria cause serious water quality problems worldwide, which has led to growing pressure for more intensive monitoring. Molecular biology methods that are based on identification and enumeration of biosynthetic genes, such as quantitative PCR, show promise in this respect. To be practical in a wide range of settings, these methods need to be usable also by laboratory personnel who do not have previous experience in PCR setup. Here we present a real-time quantitative mcyB dry chemistry PCR assay capable of identifying the three globally most common microcystin-producing cyanobacterial genera, Anabaena, Microcystis and Planktothrix. It minimizes the amount of liquid handling and avoids direct contact with the PCR reagents at the time of analysis. Large quantities of virtually identical chips can be manufactured, improving the comparability of results. Using the dry chemistry PCR chips, freshwater environmental samples from Finnish and Estonian lakes, rivers and reservoirs were analyzed for mcyB. The chip format was found to be highly suitable for water sample analysis due to its ease-of-use, good sensitivity and amplification efficiency. Significant positive correlation (Spearman's rank correlation, ρ > 0.66, P < 0.001) was observed between combined mcyB copy numbers from Microcystis, Anabaena, Planktothrix and total microcystin concentrations, regardless of the method used to measure the toxins (ELISA or LC–MS). Positive correlations were observed also for single lakes.     

Interaction between toxic dinoflagellate Alexandrium catenella exposure and disease associated with herpesvirus OsHV-1 μVar in Pacific oyster spat Crassostrea gigas

Blooms of toxic dinoflagellates can co-occur with mass mortality events associated with herpesvirus OsHV-1 μVar infection that have been decimating Pacific oyster Crassostrea gigas spat and juveniles every summer since 2008 in France. This study investigated the possible effect of a harmful dinoflagellate, Alexandrium catenella, a producer of Paralytic Shellfish Toxins (PSTs), upon the oyster spat–herpesvirus interaction. Oyster spat from a hatchery were challenged by cohabitation with oysters contaminated in the field with OsHV-1 μVar and possibly other pathogens. Simultaneously, the oysters were exposed to cultured A. catenella. Infection with OsHV-1 μVar and PST accumulation were measured after 4 days of experimental exposure.Exposure to Alexandrium catenella modified the host–pathogen interaction by reducing prevalence of OsHV-1 μVar infection. In addition, oysters challenged with OsHV-1 μVar and possibly other pathogens from the environment accumulated smaller amounts of PSTs than unchallenged oysters. Three possible mechanisms are suggested by these results: (i) possible direct interactions between A. catenella and herpesvirus (or associated pathogens) could reduce viral transmission and algal availability for oyster consumption; (ii) oyster feeding behavior or digestive functions may have been altered, thus decreasing both uptake of viral particles and consumption or digestion of toxic algae and consequent toxin accumulation; (iii) immuno-activation by A. catenella could enhance defense efficiency against OsHV-1 μVar infection. These findings suggest further research on relationships between OsHV-1 μVar and toxic dinoflagellates and their combined effects upon disease transmission and proliferation processes, as well as on oyster physiological and immunological involvement in this complex, tripartite interaction.     

Molecular confirmation of Planktothrix rubescens as the cause of intense,microcystin—Synthesizing cyanobacterial bloom in Lake Ziros,Greece

Cyanobacterial blooms occur increasingly often and raise ecological concerns worldwide. In Mediterranean freshwater ecosystems algal blooms are commonly attributed to Microcystis, Anabaena, and Aphanizomenon genera while Planktothrix is the most common bloom forming cyanobacterium in deep Northern and prealpine European oligotrophic to mesotrophic lakes. In the framework of an undertaken study of cyanobacterial species in lakes of Northwestern Greece we investigated the cyanobacterial diversity in Lake Ziros throughout a 15-month period (January 2006–March 2007) by using molecular methods. Surprisingly, a severe cyanobacterial bloom occurred during the study period, which upon microscopic examination and detailed molecular characterization found to be caused by Planktothrix rubescens species. The appearance of P. rubescens from November 2006 coincided with poor cyanobacterial diversity and resulted in a thick epilimnetic bloom in March 2007 (3.1 × 10⁸ cells/l and microcystin concentration 199 μg/l). Genotype composition of the total cyanobacterial community of the lake was analyzed by using denaturing gradient gel electrophoresis (DGGE) profiling of the intergenic transcribed spacer region of the rnn operon (rRNA-ITS). A P. rubescens strain closely related to Kpr strain from Lake Klinckenberg, The Netherlands, was found to dominate. The importance of this observation is expanded by the fact that microcystin concentrations recorded in Lake Ziros were the highest measured ever in Greek aquatic ecosystems examined so far and also found amongst the highest recorded worldwide.     

A validated UPLC–MS/MS method for the surveillance of ten aquatic biotoxins in European brackish and freshwater systems

Over the past few decades, there has been an increased frequency and duration of cyanobacterial Harmful Algal Blooms (HABs) in freshwater systems globally. These can produce secondary metabolites called cyanotoxins, many of which are hepatotoxins, raising concerns about repeated exposure through ingestion of contaminated drinking water or food or through recreational activities such as bathing/swimming. An ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) multi-toxin method has been developed and validated for freshwater cyanotoxins; microcystins-LR, -YR, -RR, -LA, -LY and -LF, nodularin, cylindrospermopsin, anatoxin-a and the marine diatom toxin domoic acid. Separation was achieved in around 9 min and dual SPE was incorporated providing detection limits of between 0.3 and 5.6 ng/L of original sample. Intra- and inter-day precision analysis showed relative standard deviations (RSD) of 1.2–9.6% and 1.3–12.0% respectively. The method was applied to the analysis of aquatic samples (n = 206) from six European countries. The main class detected were the hepatotoxins; microcystin-YR (n = 22), cylindrospermopsin (n = 25), microcystin-RR (n = 17), microcystin-LR (n = 12), microcystin-LY (n = 1), microcystin-LF (n = 1) and nodularin (n = 5). For microcystins, the levels detected ranged from 0.001 to 1.51 μg/L, with two samples showing combined levels above the guideline set by the WHO of 1 μg/L for microcystin-LR. Several samples presented with multiple toxins indicating the potential for synergistic effects and possibly enhanced toxicity. This is the first published pan European survey of freshwater bodies for multiple biotoxins, including two identified for the first time; cylindrospermopsin in Ireland and nodularin in Germany, presenting further incentives for improved monitoring and development of strategies to mitigate human exposure.     

The efficiency of combined coagulant and ballast to remove harmful cyanobacterial blooms in a tropical shallow system

We tested the hypothesis that a combination of coagulant and ballast could be efficient for removal of positively buoyant harmful cyanobacteria in shallow tropical waterbodies, and will not promote the release of cyanotoxins. This laboratory study examined the efficacy of coagulants [polyaluminium chloride (PAC) and chitosan (made of shrimp shells)] alone, and combined with ballast (lanthanum modified bentonite, red soil or gravel) to remove the natural populations of cyanobacteria collected from a shallow eutrophic urban reservoir with alternating blooms of Cylindrospermopsis and Microcystis. PAC combined with ballast was effective in settling blooms dominated by Microcystis or Cylindrospermopsis. Contrary to our expectation, chitosan combined with ballast was only effective in settling Cylindrospermopsis-dominated blooms at low pH, whereas at pH ≥ 8 no effective flocculation and settling could be evoked. Chitosan also had a detrimental effect on Cylindrospermopsis causing the release of saxitoxins. In contrast, no detrimental effect on Microcystis was observed and all coagulant-ballast treatments were effective in not only settling the Microcystis dominated bloom, but also lowering dissolved microcystin concentrations. Our data show that the best procedure for biomass reduction also depends on the dominant species.     

The dynamics of toxic Microcystis strains and microcystin production in two hypertrofic South African reservoirs

The South African impoundments of Hartbeespoort and Roodeplaat experience excessive blooms of Microcystis species each year. Microcystins, produced primarily by strains of cyanobacteria belonging to the genera Microcystis, Anabaena and Planktothrix, are harmful cyanobacterial hepatotoxins. These bloom-forming cyanobacteria form toxic and non-toxic strains that co-occur and are visually indistinguishable, but can be identified and quantified molecularly. We described the relationships between microcystin production and the genotypic composition of the Microcystis community involved together with environmental conditions in both the Roodeplaat and Hartbeespoort reservoirs using quantitative real time PCR. DNA copy number of the Microcystis-specific 16S rRNA and toxin biosynthesis genes, mcyE and mcyB, were measured. Planktothrix spp. occurred in both reservoirs during autumn, but no toxin-producing species was present as measured with mcyE specific primers, whereas both toxic and non-toxic strains of Microcystis were recorded in both reservoirs, with Microcystis spp. dominating in the summer months. Water-surface temperature correlated strongly with microcystin concentration, mcyE and mcyB copy number. Microcystin production was associated by temperatures higher than 23 °C. This suggests that should current environmental trends persist with surface water temperatures continuing to rise and more and more nutrients continued to be loaded into fresh water systems toxic Microcystis may outgrow non-toxic Microcystis and synthesise even more microcystins.     

Potential human health risk assessment of cylindrospermopsin accumulation and depuration in lettuce and arugula

The cyanobacterial toxin cylindrospermopsin (CYN) has become a globally important secondary metabolite due to the negative effect it has on human and animal health. As a means of evaluating the risk of human exposure to CYN, the bioaccumulation and depuration of the toxin in lettuce (Lactuca sativa L.) and arugula (Eruca sativa Mill.) were investigated, after irrigation with contaminated water. The vegetables were irrigated for 7 days with CYN (3, 5 and 10 μg/L) contaminated water (bioaccumulation phase), and subsequently, irrigated for 7 days with uncontaminated distilled water (depuration phase). In general, the bioaccumulation of CYN in both vegetables decreased with increasing exposure concentration. Bioconcentration factor (BCF) of CYN increased with the progression of the experiment at 3.0 μg/L CYN, while the reverse occurred at 5 and 10 μg/L CYN. In arugula, BCF increased at all CYN exposure concentrations throughout the study. The depuration of CYN decreased with increasing exposure concentration but was highest in the plants of both species with the highest bioaccumulation of CYN. Specifically, in plants previously irrigated with water contaminated with 3, 5 and 10 μg/L CYN, the depuration of the toxin was 60.68, 27.67 and 18.52% for lettuce, and 47, 46.21 and 27.67% for arugula, respectively. Human health risks assessment revealed that the consumption of approximately 10 to 40 g of vegetables per meal will expose children and adults to 1.00-6.00 ng CYN/kg body mass for lettuce and 2.22-7.70 ng CYN/kg body mass for arugula. The irrigation of lettuce and arugula with contaminated water containing low CYN concentrations constitutes a potential human exposure route.     

Cyanotoxin diversity and food web bioaccumulation in a reservoir with decreasing phosphorus concentrations and perennial cyanobacterial blooms

In a shallow multifunction dam reservoir, perennial water blooms formed by several toxin-producing cyanobacteria (Anabaena spp., Aphanizomenon spp., Planktothrix agardhii and Microcystis spp.) were observed. Over a seven-year period, concomitantly with a gradual decrease in phosphate and total phosphorus concentrations in the water and an increase in the DIN to DIP ratio, a reduced biomass of cyanobacteria was noted. Simultaneously, a twofold increase in cyanobacterial species richness was found. The concentration of intracellular anatoxin-a was positively correlated with the total cyanobacterial biomass, but the concentration of intracellular microcystins was significantly negatively correlated with the level of phosphorus in the water. Therefore, in a period with a very low (2.3–3.6) DIN:DIP ratio, intracellular ANTX prevailed in the reservoir, while in the following years (at DIN:DIP = 23–36) much higher MC levels were noted. The highest total concentrations (22.2 μg L⁻¹) of intracellular MCs (MC-LF > -LY > -LR > -LA = -LW) and ANTX (14.4 μg L⁻¹) were found in 2010. In the following year, eight MC iso-forms were detected (MC-LF > -LY > -LA > -LR > -LW > -WR > -YR > -RR). The number of MC variants was positively correlated with the increased contribution of Anabaena planctonica/A. affinis and Microcystis spp. to cyanobacteria biomass. The indigenous bentho-pelagic fish Abramis brama L. accumulated in their tissues relatively high amounts of both ANTX (e.g. 6.2–18.4 μg g⁻¹ FW of liver) and different variants of MCs (up to 4.4 μg g⁻¹ FW of liver). Cyanotoxin tissue contents decreased in the following order: gills > liver > muscles. These observed strong changes in the species structure of cyanobacteria assemblages, even at their considerably smaller biomass, appeared to be an undesirable phenomenon due to the predominance of the efficient MC and ANTX producers, such as Anabaena spp., which is easily digested by fish. The variability of the profile of cyanobacterial blooms that depends on nutrient fluctuations and may account for the diverse toxin accumulation and tissue distribution in freshwater ichthyofauna is noteworthy, especially in water bodies used for fishery.     

Subcellular fractionation of Cu exposed oysters,Crassostrea virginica,and Cu accumulation from a biologically incorporated Cu rich oyster diet in Fundulus heteroclitus in fresh and sea water

In order to examine the effect of salinity on Cu accumulation from a naturally incorporated diet, oysters (Crassostrea virginica) were exposed in sea water for 96 days to four waterborne [Cu]: 2.9 ± 0.7 (control), 4.3 ± 0.6, 5.4 ± 0.5, and 10.7 ± 1.0 µg L^? 1. After 96 days, the control whole body [Cu] increased from 2.1 ± 0.5 to 9.1 ± 1.1 µg g^? 1 w.w. and the highest [Cu] was 163.4 ± 27.1 µg g^? 1 w.w. in the oysters. Despite large differences in tissue [Cu], there was no effect on the fraction of trophically available metal in the oyster suggesting that trophic transfer will correlate well with tissue [Cu]. The control and highest [Cu] oysters became diet for killifish (Fundulus heteroclitus) in fresh and seawater for 40 days. The two diets contained 84.7 ± 5.1 and 850.5 ± 8.8 µg Cu g^? 1 d.w. Fish were fed a combined diet of oyster and a pellet supplement (20.5 ± 1.0 µg Cu g^? 1 d.w.) both at 5% body mass day^? 1. In killifish, Cu increased ~ 7% in gills and 100% in intestines after 6 weeks of exposure to the high Cu diet. No other tissues accumulated Cu above control levels. An 11-fold difference free Cu²⁺ concentrations was predicted in intestinal fluid between fresh and sea water, but there was no corresponding effect of salinity on intestinal Cu accumulation suggesting that Cu is not accumulated as the free ion.     

Marine snow formation by the toxin-producing diatom,Pseudo-nitzschia australis

The formation of marine snow (MS) by the toxic diatom Pseudo-nitschia australis was simulated using a roller table experiment. Concentrations of particulate and dissolved domoic acid (pDA and dDA) differed significantly among exponential phase and MS formation under simulated near surface conditions (16 °C/12:12-dark:light cycle) and also differed compared to subsequent particle decomposition at 4 °C in the dark, mimicking conditions in deeper waters. Particulate DA was first detected at the onset of exponential growth, reached maximum levels associated with MS aggregates (1.21 ± 0.24 ng mL⁻¹) and declined at an average loss rate of ∼1.2% pDA day⁻¹ during particle decomposition. Dissolved DA concentrations increased throughout the experiment and reached a maximum of ∼20 ng mL⁻¹ at final sampling on day 88. The succession by P. australis from active growth to aggregation resulted in increasing MS toxicity and based on DA loading of particles and known in situ sinking speeds, a significant amount of toxin could have easily reached the deeper ocean or seafloor. MS formation was further associated with significant dDA accumulation at a ratio of pDA: dDA: cumulative dDA of approximately 1:10:100. Overall, this study confirms that MS functions as a major vector for toxin flux to depth, that Pseudo-nitzschia-derived aggregates should be considered ‘toxic snow’ for MS-associated organisms, and that effects of MS toxicity on interactions with aggregate-associated microbes and zooplankton consumers warrant further consideration.     

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.     

Interactive effects of elevated temperature and CO2 levels on metabolism and oxidative stress in two common marine bivalves (Crassostrea virginica and Mercenaria mercenaria)

Marine bivalves such as the hard shell clams Mercenaria mercenaria and eastern oysters Crassostrea virginica are affected by multiple stressors, including fluctuations in temperature and CO₂ levels in estuaries, and these stresses are expected to be exacerbated by ongoing global climate change. Hypercapnia (elevated CO₂ levels) and temperature stress can affect survival, growth and development of marine bivalves, but the cellular mechanisms of these effects are not yet fully understood. In this study, we investigated whether oxidative stress is implicated in cellular responses to elevated temperature and CO₂ levels in marine bivalves. We measured the whole-organism standard metabolic rate (SMR), total antioxidant capacity (TAOC), and levels of oxidative stress biomarkers in the muscle tissues of clams and oysters exposed to different temperatures (22 and 27 °C) and CO₂ levels (the present day conditions of ~ 400 ppm CO₂ and 800 ppm CO₂ predicted by a consensus business-as-usual IPCC emission scenario for the year 2100). SMR was significantly higher and the antioxidant capacity was lower in oysters than in clams. Aerobic metabolism was largely temperature-independent in these two species in the studied temperature range (22–27 °C). However, the combined exposure to elevated temperature and hypercapnia led to elevated SMR in clams indicating elevated costs of basal maintenance. No persistent oxidative stress signal (measured by the levels of protein carbonyls, and protein conjugates with malondialdehyde and 4-hydroxynonenal) was observed during the long-term exposure to moderate warming (+ 5 °C) and hypercapnia (~ 800 ppm CO₂). This indicates that long-term exposure to moderately elevated CO₂ and temperature minimally affects the cellular redox status in these bivalve species and that the earlier observed negative physiological effects of elevated CO₂ and temperature must be explained by other cellular mechanisms.     

Lethal effects of ichthyotoxic raphidophytes,Chattonella marina,C. antiqua,and Heterosigma akashiwo,on post-embryonic stages of the Japanese pearl oyster,Pinctada fucata martensii

The inimical effects of the ichthyotoxic harmful algal bloom (HAB)-forming raphidophytes Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua on the early-life stages of the Japanese pearl oyster Pinctada fucata martensii were studied. Fertilized eggs and developing embryos were not affected following exposure to the harmful raphidophytes; however, all three algal species severely affected trochophores and D-larvae, early-stage D-larvae, and late-stage pre-settling larvae. Exposure to C. marina (5 × 10² cells ml⁻¹), C. antiqua (10³ cells ml⁻¹), and H. akashiwo (5 × 10³ cells ml⁻¹) resulted in decreased success of metamorphosis to the trochophore stage. A complete inhibition of trochophore metamorphosis was observed following exposure to C. antiqua at 5 × 10³ cells ml⁻¹ and C. marina at 8 × 10³ cells ml⁻¹. In all experiments, more than 80% of newly formed trochophores were anomalous, and in the case of exposure to H. akashiwo at 10⁵ cells ml⁻¹ more than 70% of D-larvae were anomalous. The activity rates of D-larvae (1-day-old) were significantly reduced following exposure to C. antiqua (8 × 10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (10⁴ cells ml⁻¹, 24 h). The activity rates of pre-settling larvae (21-day-old) were also significantly reduced following exposure to C. antiqua (10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (5 × 10⁴ cells ml⁻¹, 24 h). Significant mortalities of both larval stages were induced by all three raphidophytes, with higher mortality rates registered for pre-settling larvae than D-larvae, especially following exposure to C. marina (5 × 10²–8 × 10³ cells ml⁻¹, 48–86 h) and C. antiqua (10³–8 × 10³ cells ml⁻¹, 72–86 h). Contact between raphidophyte cells and newly metamorphosed trochophores and D-larvae, 1-day-old D-larvae, and 21-day-old larvae resulted in microscopic changes in the raphidophytes, and then, in the motile early-life stages of pearl oysters. Upon contact and physical disturbance of their cells by larval cilia, H. akashiwo, C. marina and C. antiqua became immotile and shed their glycocalyx. The trochophores and larvae were observed trapped in a conglomerate of glycocalyx and mucus, most probably a mixture of larval mucous and raphidophyte tricosyts and mucocytes. All motile stages of pearl oyster larvae showed a typical escape behavior translating into increased swimming in an effort to release themselves from the sticky mucous traps. The larvae subsequently became exhausted, entrapped in more heavy mucous, lost their larval cilia, sank, become immotile, and died. Although other toxic mediators could have been involved, the results of the present study indicate that all three raphidophytes were harmful only for motile stages of pearl oysters, and that the physical disturbance of their cells upon contact with the ciliary structures of pearl oyster larvae initiated the harmful mechanism. The present study is the first report of lethal effects of harmful Chattonella spp. towards larvae of a bivalve mollusc. Blooms of H. akashiwo, C. antiqua and C. marina occur in all major cultivation areas of P. fucata martensii during the developmental period of their larvae. Therefore, exposure of the motile early-life stages of Japanese pearl oysters could adversely affect their population recruitment. In addition, the present study shows that further research with early-life development of pearl oysters and other bivalves could contribute to improving the understanding of the controversial harmful mechanisms of raphidophytes in marine organisms.     

Characterization of multiple isolates from an Alexandrium ostenfeldii bloom in The Netherlands

Alexandrium ostenfeldii is an emerging harmful algal bloom species forming a global threat to coastal marine ecosystems, with consequences for fisheries and shellfish production. The Oosterschelde estuary is a shallow, macrotidal and mesotrophic estuary in the southwest of The Netherlands with large stocks of mussels, oysters, and cockles. These shellfish stocks were threatened by a recent A. ostenfeldii bloom in the Ouwerkerkse Kreek, which is a brackish water creek discharging water into the Oosterschelde. Little is yet known about the characteristics of the A. ostenfeldii population in this creek. We therefore isolated 20 clones during an A. ostenfeldii bloom in 2013, and characterized these clones on their growth and toxin profile in their exponential growth phase. The cyclic imines were identified by comparison of A. ostenfeldii extracts with the retention time and CID spectra of standard solutions, or with published CID spectra. We furthermore assessed the allelochemical potency and phylogeny of a selection of 10–12 clones. Morphology and molecular phylogeny showed that all clones belong to Group 1 of A. ostenfeldii. All clones showed comparable growth rates of on average 0.22 ± 0.03 d⁻¹. During exponential growth, they all produced a unique combination of paralytic shellfish poisoning toxins, spirolides and gymnodimines, of which particularly the latter showed a high intra-specific variability, with a 25-fold difference between clones with the lowest and highest cell quota. Furthermore, the selected 12 clones showed high allelopathic potencies with EC₅₀ values based on lysis assays against the cryptophyte Rhodomonas salina between 212 and 525 A. ostenfeldii cells mL⁻¹. Lytic activities were lower for cell extracts, indicating an important extracellular role of these compounds. A high intra-specific variability may add to the success of genotypically diverse A. ostenfeldii blooms, and make populations resilient to changes in environmental and climatic conditions.     

Environmental factors controlling colony formation in blooms of the cyanobacteria Microcystis spp. in Lake Taihu,China

Nitrogen (N) and phosphorus (P) over-enrichment has accelerated eutrophication and promoted cyanobacterial blooms worldwide. The colonial bloom-forming cyanobacterial genus Microcystis is covered by sheaths which can protect cells from zooplankton grazing, viral or bacterial attack and other potential negative environmental factors. This provides a competitive advantage over other phytoplankton species. However, the mechanism of Microcystis colony formation is not clear. Here we report the influence of N, P and pH on Microcystis growth and colony formation in field simulation experiments in Lake Taihu (China). N addition to lake water maintained Microcystis colony size, promoted growth of total phytoplankton, and increased Microcystis proportion as part of total phytoplankton biomass. Increases in P did not promote growth but led to smaller colonies, and had no significant impact on the proportion of Microcystis in the community. N and P addition together promoted phytoplankton growth much more than only adding N. TN and TP concentrations lower than about TN 7.75–13.95 mg L⁻¹ and TP 0.41–0.74 mg L⁻¹ mainly promoted the growth of large Microcystis colonies, but higher concentrations than this promoted the formation of single cells. There was a strong inverse relationship between pH and colony size in the N&P treatments suggesting CO₂ limitation may have induced colonies to become smaller. It appears that Microcystis colony formation is an adaptation to provide the organisms adverse conditions such as nutrient deficiencies or CO₂ limitation induced by increased pH level associated with rapidly proliferating blooms.