Polyphasic characterization of water bloom forming Raphidiopsis species (cyanobacteria) from central China

Two strains of Raphidiopsis Fritsch et Rich were isolated from a fishpond in Wuhan city, China and rendered axenic, and characterized by a combination of morphological, physiological, biochemical and genetic methods. Morphologically the strains were identified as Raphidiopsis mediterranea Skuja (straight trichomes) and R. curvata Fritsch et Rich (coiled trichomes). These two strains demonstrated slight differences in optimal temperature range and GC content, while sharing some common characteristics including inability to grow hetertrophically, similar salinity tolerance (up to 0.78%) and an identical fatty acid composition. Cyanotoxins were not found in the strain of R. mediterranea, however, the strain of R. curvata contained both deoxycylindrospermopsin and cylindrospermopsin. Phylogenetic affiliations inferred from 16S rRNA gene sequences demonstrated that both Raphidiopsis strains clustered with Cylindrospermopsis, demonstrating their phylogenetic ties to Nostocaceae. (c) 2007 Elsevier B.V. All rights reserved.

Stable carbon isotope variations in surface bloom scum and subsurface seston among shallow eutrophic lakes

Carbon stable isotope analysis of surface bloom scum and subsurface seston samples was conducted in shallow eutrophic lakes in China during warm seasons from 2003 to 2004. delta C-13 values of bloom scum were always higher (averaged 5 parts per thousand) than those of seston in this study, and the possible reasons were attributed to (i) direct use of atmospheric CO2 at the air-water interface, (ii) decrease in C-13 fractionation due to higher carbon fixation, (iii) active CO2 transport, and/or (iv) HCO3 accumulation. Negative correlation between delta C-13(scum) - delta C-13(seston) and pH in the test lakes indicated that phytoplankton at the subsurface water column increased isotopic enrichment under the-carbon limitation along with the increase of pH, which might in turn decreased the differences in 313 C between the subsurface seston and the surface scums. Significant positive correlations of seston 8 13C with total concentrations of nitrogen and phosphorus in water column suggested that the increase in delta C-13 of seston with trophic state was depending on nutrient (N or P, or both) supply. Our study showed that delta C-13 of phytoplankton was indicative of carbon utilization, primary productivity, and nutrient supply among the eutrophic lakes. (C) 2007 Elsevier B.V All rights reserved.

Morphological and genetic study of Prorocentrum donghaiense Lu from the East China Sea, and comparison with some related Prorocentrum species

In this paper, the detailed morphology of Prorocentrum donghaiense Lu from both field samples and cultures was examined, and a taxonomic comparison was made between P donghaiense and some related Prorocentrum spp. using morphological and molecular data and other published information. There were distinct differences among these species in morphological characteristics that historically have been presented as conservative features. The discrepancies extended beyond that of individual variations within the same species due to environmental factors. Therefore, these morphological features may not be conservative but, rather, polymorphic depending on environmental conditions. Based on this analysis, we suggest that the high-biomass bloom-forming species in the East China Sea, previously reported as Prorocentrum dentatum Stein, is P donghaiense Lu. The species reported from the East China Sea and Japanese and Korean waters appear to be the same species. Molecular data also suggest that P. dentatum (CCMP1517) and P. donghaiense are genetically identical. Therefore, the geographic distribution of P. donghaiense may be much wider than expected. (C) 2004 Elsevier B.V. All rights reserved.

Experimental study on the impact of dinoflagellate Alexandrium species on populations of the rotifer Brachionus plicatilis

To investigate harmful effects of the dinoflagellate Alexandrium species on microzooplankton, the rotifer Brachionus plicatilis was chosen as an assay species, and tested with 10 strains of Alexandrium including one known non-PSP-producer (Alexandrium tamarense, AT-6). HPLC analysis confirmed the PSP-content of the various strains: Alexandrium lusitanicum, Alexandrium minutum and Alexandrium tamarense (ATHK, AT5-1, AT5-3, ATC102, ATC103) used in the experiment were PSP-producers. No PSP toxins were detected in the strains Alexandrium sp1, Alexandrium sp2. Exposing rotifer populations to the densities of 2000 cells ml(-1) of each of these 10 Alexandrium strains revealed that the (non-PSP) A. tarnarense (AT-6) and two other PSP-producing algae: A. lusitanicum, A. minutum, did not appear to adversely impact rotifer populations. Rotifers exposed to these three strains were able to maintain their population numbers, and in some cases, increase them. Although some increases in rotifer population growth following exposures to these three algal species were noted, the rate was less than for the non-exposed control rotifer groups. In contrast, the remaining seven algal strains (A. tamarense ATHK, AT5-1, AT5-3, ATC102, ATC103; also Alexandrium sp1 and Alexandrium sp2) all have adverse effects on the rotifers. Dosing rotifers with respective algal cell densities of 2000 cells ml-1 each, for Alexandrium spl, Alexandrium sp2, and A. tamarense strains ATHK and ATC103 showed mean lethal time (LT50) on rotifer populations of 21, 28, 29, and 36h, respectively. The remaining three species (A. tamarense strains AT5-1, AT5-3, ATC102) caused respective mean rotifer LT50S of 56, 56, and 71 h, compared to 160 h for the unexposed "starved control" rotifers. Experiments to determine ingestion rates for the rotifers, based on changes in their Chlorophyll a content, showed that the rotifers could feed on A. lusitanicum, A. minutum and A. tamarense strain AT-6, but could graze to little or no extent upon algal cells of the other seven strains. The effects on rotifers exposed to different cell densities, fractions, and growth phases of A. tamarense algal culture were respectively compared. It was found that only the whole algal cells had lethal effects, with strongest impact being shown by the early exponential growth phase of A. tamarense. The results indicate that some toxic mechanism(s), other than PSP and present in whole algal cells, might be responsible for the adverse effects on the exposed rotifers. (C) 2004 Elsevier B.V. All rights reserved.

A comparison of future increased CO2 and temperature effects on sympatric Heterosigma akashiwo and Prorocentrum minimum

Very little is known about how global anthropogenic changes will affect major harmful algal bloom groups. Shifts in the growth and physiology of HAB species like the raphidophyte Heterosigma akashiwo and the dinoflagellate Prorocentrum minimum due to rising CO2 and temperature could alter their relative abundance and environmental impacts in estuaries where both form blooms, such as the Delaware Inland Bays (DIB). We grew semi-continuous cultures of sympatric DIB isolates of these two species under four conditions: (1) 20 degrees C and 375 ppm CO2 (ambient control), (2)20 degrees C and 750 ppm CO2 (high CO2),(3) 24 degrees C and 375 ppm CO2 (high temperature), and (4) 24 degrees C and 750 ppm CO2 (combined). Elevated CO2 alone or in concert with temperature stimulated Heterosigma growth, but had no significant effect on Prorocentrum growth. P-Bmax (the maximum biomass-normalized light-saturated carbon fixation rate) in Heterosigma was increased only by simultaneous CO2 and temperature increases, whereas P-Bmax in Prorocentrum responded significantly to CO2 enrichment, with or without increased temperature. CO2 and temperature affected photosynthetic parameters alpha, Phi(max), E-k, and Delta F/F'(m) in both species. Increased temperature decreased and increased the Chl a content of Heterosigma and M Prorocentrum, respectively. CO2 availability and temperature had pronounced effects on cellular quotas of C and N in Heterosigma, but not in Prorocentrum. Ratios of C:P and N:P increased with elevated carbon dioxide in Heterosigma but not in Prorocentrum. These changes in cellular nutrient quotas and ratios imply that Heterosigma could be more vulnerable to N limitation but less vulnerable to P-limitation than Prorocentrum under future environmental conditions. In general, Heterosigma growth and physiology showed a much greater positive response to elevated CO2 and temperature compared to Prorocentrum, consistent with what is known about their respective carbon acquisition mechanisms. Hence, rising temperature and CO2 either alone or in combination with other limiting factors could significantly alter the relative dominance of these two co-existing HAB species over the next century. (c) 2007 Elsevier B.V. All rights reserved.

Ocean urea fertilization for carbon credits poses high ecological risks

The proposed plan for enrichment of the Sulu Sea, Philippines, a region of rich marine biodiversity, with thousands of tonnes of urea in order to stimulate algal blooms and sequester carbon is flawed for multiple reasons. Urea is preferentially used as a nitrogen source by some cyanobacteria and dinoflagellates, many of which are neutrally or positively buoyant. Biological pumps to the deep sea are classically leaky, and the inefficient burial of new biomass makes the estimation of a net loss of carbon from the atmosphere questionable at best. The potential for growth of toxic dinoflagellates is also high, as many grow well on urea and some even increase their toxicity when grown on urea. Many toxic dinoflagellates form cysts which can settle to the sediment and germinate in subsequent years, forming new blooms even without further fertilization. If large-scale blooms do occur, it is likely that they will contribute to hypoxia in the bottom waters upon decomposition. Lastly, urea production requires fossil fuel usage, further limiting the potential for net carbon sequestration. The environmental and economic impacts are potentially great and need to be rigorously assessed. (C) 2008 Elsevier Ltd. All rights reserved.

Is inorganic nutrient enrichment a driving force for the formation of red tides? A case study of the dinoflagellate Scrippsiella trochoidea in an embayment

Red tides (high biomass phytoplankton blooms) have frequently occurred in Hong Kong waters, but most red tides occurred in waters which are not very eutrophic. For example, Port Shelter, a semi-enclosed bay in the northeast of Hong Kong, is one of hot spots for red tides. Concentrations of ambient inorganic nutrients (e.g. N, P), are not high enough to form the high biomass of chlorophyll a (chl a) in a red tide when chl a is converted to its particulate organic nutrient (N) (which should equal the inorganic nutrient, N). When a red tide of the dinoflagellate Scrippsiella trochoidea occurred in the bay, we found that the red tide patch along the shore had a high cell density of 15,000 cells ml(-1), and high chl a (56 mu g l(-1)), and pH reached 8.6 at the surface (8.2 at the bottom), indicating active photosynthesis in situ. Ambient inorganic nutrients (NO3, PO4, SiO4, and NH4) were all low in the waters and deep waters surrounding the red tide patch, suggesting that the nutrients were not high enough to support the high chl a >50 mu g l(-1) in the red tide. Nutrient addition experiments showed that the addition of all of the inorganic nutrients to a non-red-tide water sample containing low concentrations of Scrippsiella trochoidea did not produce cell density of Scrippsiella trochoidea as high as in the red tide patch, suggesting that nutrients were not an initializing factor for this red tide. During the incubation of the red tide water sample without any nutrient addition, the phytoplankton biomass decreased gradually over 9 days. However, with a N addition, the phytoplankton biomass increased steadily until day 7, which suggested that nitrogen addition was able to sustain the high biomass of the red tide for a week with and without nutrients. In contrast, the red tide in the bay disappeared on the sampling day when the wind direction changed. These results indicated that initiation, maintenance and disappearance of the dinoflagellate Scrippsiella trochoidea red tide in the bay were not directly driven by changes in nutrients. Therefore, how nutrients are linked to the formation of red tides in coastal waters need to be further examined, particularly in relation to dissolved organic nutrients. (C) 2008 Elsevier B.V. All rights reserved.

Ecotoxicology of marine biotoxins in bivalve shellfish

A small proportion of harmful algae produce toxins which are harmful to human health. Strict monitoring programmes are in place within Ireland and the EU to effectively manage risk to human consumers of shellfish species that have accumulated marine biotoxins in their tissues. However, little is known about the impacts of HABs on shellfish health. This study used Solid Phase Adsorption and Toxin Tracking (SPATT) for the passive sampling of algal biotoxins at Lough Hyne Marine Nature Reserve in West Cork, Ireland. Spatial and temporal monitoring of the incidence of a wide range of lipophilic toxins was assessed over a 4-month period. Active sampling accumulated sufficient quantities of toxin for use in subsequent experimentation. In addition to commonly occurring Diarrhetic Shellfish Poisoning (DSP) toxins, Dinophysis toxin-1 and Pinnatoxin-G were both detected in the samples. This is the first identification of these latter two toxins in Irish waters. The effects of the DSP toxin okadaic acid (OA) were investigated on three shellfish species: Mytilus edulis, Ruditapes philippinarum and Crassostrea gigas. Histological examination of the gill, mantle and hepatopancreas tissues revealed varying intensity of damage depending both on the tissue type and the species involved. At the cellular level, flow cytometric analysis of the differential cell population distribution was assessed. No change in cell population distribution was observed in Mytilus edulis or Ruditapes philippinarum, however significant changes were observed in Crassostrea gigas granulocytes at the lower levels of toxin exposure. This indicated a chemically-induced response to OA. DNA fragmentation was measured in the haemolymph and hepatopancreas cells post OA-exposure in Mytilus edulis and Crassostrea gigas. A significant increase in DNA fragmentation was observed in both species over time, even at the lowest OA concentrations. DNA fragmentation could be due to genotoxicity of OA and/or to the induction of cell apoptosis.

Investigation of the genotoxic potential of the marine biotoxins okadaic acid and azaspiracids

The present study investigated the genotoxic potential of the marine biotoxins okadaic acid (OA) and azaspiracids (AZAs). Harmful algae blooms (HABs) are an increasing global problem with implications for the ecosystem, economy and human health. Most data available on human intoxication are based on acute toxicity. To date, limited data has been published on possible long term effects, carcinogenicity and genotoxicity. To investigate genotoxicity in the present study, DNA fragmentation was detected using the COMET assay. In contrast to most other available studies, two further endpoints were included. The Trypan Blue Exclusion assay was used to provide information on possible cytotoxicity and assess the right concentration range. Flow cytometer analysis was included to detect the possible involvement of apoptotic processes. In house background data for all endpoints were established using positive controls. Three different cell lines, Jurkat T cells, CaCo-2 cells and HepG-2 cells, representing the main target organs, were exposed to OA and AZA1-3 at different concentrations and exposure times. Data obtained from the COMET assay showed an increase in DNA fragmentation for all phycotoxins, indicating a modest genotoxic effect. However, the data obtained from the Trypan Blue Exclusion assay showed a clear reduction in cell viability and cell number, indicating the involvement of cytotoxic and/or apoptotic processes. This is supported by data obtained by flow cytometer analysis. All phycotoxins investigated showed signs of early/late apoptosis. Therefore, the combined observations made in the present study indicate that OA and AZA1-3 are not genotoxic per se. Apoptotic processes appear to make a major contribution to the observed DNA fragmentation. The information obtained in this study stresses the importance of inclusion of additional endpoints and appropriate positive controls in genotoxicity studies. Furthermore, these data can assist in future considerations on risk assessment, especially regarding repeated exposure and exposure at sub-clinical doses.

Validation of the detection of Alexandrium species using specific RNA probes tested in a microarray format: Calibration of signal using variability of RNA content with environmental conditions.

The dinoflagellate genus Alexandrium contains several toxin producing species and strains, which can cause major economic losses to the shell fish industry. It is therefore important to be able to detect these toxin producers and also distinguish toxic strains from some of the morphologically identical non-toxic strains. To facilitate this DNA probes to be used in a microarray format were designed in silico or developed from existing published probes. These probes targeted either the 18S or 28S ribosomal ribonucleic acid (rRNA) gene in Alexandrium tamarense Group I, Group III and Group IV, Alexandrium ostenfeldii and Alexandrium minutum. Three strains of A. tamarense Group I, A. tamarense Group III, A. minutum and two strains of A. ostenfeldii were grown at optimal conditions and transferred into new environmental conditions changing either the light intensity, salinity, temperature or nutrient concentrations, to check if any of these environmental conditions induced changes in the cellular ribonucleic acid (RNA) concentration or growth rate. The aim of this experiment was the calibration of several species-specific probes for the quantification of the toxic Alexandrium strains. Growth rates were highly variable but only elevated or lowered salinity significantly lowered growth rate for A. tamarense Group I and Group III; differences in RNA content were not significant for the majority of the treatments. Only light intensity seemed to affect significantly the RNA content in A. tamarense Group I and Group III, but this was still within the same range as for the other treatments meaning that a back calibration from RNA to cell numbers was possible. The designed probes allow the production of quantitative information for Alexandrium species for the microarray chip.

Validation of the detection of Pseudo-nitzschia spp. using specific RNA probes tested in a microarray format: Calibration of signal based on variability of RNA content with environmental conditions.

Harmful algal blooms (HAB) occur worldwide and cause health problems and economic damage to fisheries and tourism. Monitoring for toxic algae is therefore essential but is based primarily on light microscopy, which is time consuming and can be limited by insufficient morphological characters such that more time is needed to examine critical features with electron microscopy. Monitoring with molecular tools is done in only a few places world-wide. EU FP7 MIDTAL (Microarray Detection of Toxic Algae) used SSU and LSU rRNA genes as targets on microarrays to identify toxic species. In order to comply with current monitoring requirements to report cell numbers as the relevant threshold measurement to trigger closure of fisheries, it was necessary to calibrate our microarray to convert the hybridisation signal obtained to cell numbers. Calibration curves for two species of Pseudo-nitzschia for use with the MIDTAL microarray are presented to obtain cell numbers following hybridisation. It complements work presented by Barra et al. (2012b. Environ. Sci. Pollut. Res. doi: 10.1007/s11356-012-1330-1v) for two other Pseudo-nitzschia spp., Dittami and Edvardsen (2012a. J. Phycol. 48, 1050) for Pseudochatonella, Blanco et al. (2013. Harmful Algae 24, 80) for Heterosigma, McCoy et al. (2013. FEMS. doi: 10.1111/1574-6941.12277) for Prymnesium spp., Karlodinium veneficum, and cf. Chatonella spp. and Taylor et al. (2014. Harmful Algae, in press) for Alexandrium.

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 Pseudonitzschia 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. Published by Elsevier B.V.