Overview of Australian and New Zealand harmful algal species occurrences and their societal impacts in the period 1985 to 2018, including a compilation of historic records.

Similarities and differences between Australia and New Zealand in Harmful Algal species occurrences and Harmful Algal Events impacting on human society (HAEDAT) are reported and factors that explain their differences explored. Weekly monitoring of harmful phytoplankton and biotoxins commenced in Australia in 1986 and in New Zealand in 1993. Anecdotal historic HAB records in both countries are also catalogued. In Australia, unprecedented highly toxic Paralytic Shellfish Toxin (PST)-producing blooms of Alexandrium catenella have impacted the seafood industry along the 200 km east coast of Tasmania from 2012 to present. Toxic blooms in 1986-1993 by Gymnodinium catenatum in Tasmania were effectively mitigated by closing the affected area for shellfish farming, while a bloom by this same species in 2000 in New Zealand caused significant economic damage from restrictions on the movement of greenshell mussel spat. The biggest biotoxin event in New Zealand was an unexpected outbreak of Neurotoxic Shellfish Poisoning (NSP) in 1993 in Hauraki Gulf (putatively due to Karenia cf. mikimotoi) with 180 reported cases of human poisonings as well as reports of respiratory irritation north of Auckland. Strikingly, NSP never recurred in New Zealand since and no NSP events have ever been reported in Australia. In New Zealand, Paralytic Shellfish Poisoning (PSP) was the predominant seafood toxin syndrome, while in Australia Ciguatera Fish Poisoning (CFP) was the major reported seafood toxin syndrome, while no CFP has been recorded from consumption of New Zealand fish. In Australia, Diarrhetic Shellfish Poisoning (DSP) illnesses were recorded from two related outbreaks in 1997/98 following consumption of beach harvested clams (pipis) from a previously non-monitored area, whereas in New Zealand limited DSP illnesses are known. No human illnesses from Amnesic Shellfish Poisoning (ASP) have been reported in either Australia or New Zealand. Selected examples of HABs appearing and disappearing (NSP in New Zealand, Alexandrium catenella in Tasmania), species expanding their ranges (Noctiluca, Gambierdiscus), and reputed ballast water introductions (Gymnodinium catenatum) are discussed. Eutrophication has rarely been invoked as a cause except for confined estuaries and fish ponds and estuarine cyanobacterial blooms. No trend in the number of HAEDAT events from 1985 to 2018 was discernible.

Disentangling the environmental processes responsible for the world's largest farmed fish-killing harmful algal bloom: Chile, 2016.

The dictyochophyte microalga Pseudochattonella verruculosa was responsible for the largest farmed fish mortality ever recorded in the world, with losses for the Chilean salmon industry amounting to US$ 800 M in austral summer 2016. Super-scale climatic anomalies resulted in strong vertical water column stratification that stimulated development of a dynamic P. verruculosa thin layer (up to 38 µg chl a L-1) for several weeks in Reloncaví Sound. Hydrodynamic modeling (MIKE 3D) indicated that the Sound had extremely low flushing rates (between 121 and 200 days) in summer 2016. Reported algal cell densities of 7000-20,000 cells mL-1 generated respiratory distress in fish that was unlikely due to low dissolved oxygen (permanently >4 mg L-1). Histological examination of salmon showed that gills were the most affected organ with significant tissue damage and circulatory disorders. It is possible that some of this damage was due to a diatom bloom that preceded the Pseudochattonella event, thereby rendering the fish more susceptible to Pseudochattonella. No correlation between magnitude of fish mortality and algal cell abundance nor fish age was evident. Algal cultures revealed rapid growth rates and high cell densities (up to 600,000 cells mL-1), as well as highly complex life cycle stages that can be easily overlooked in monitoring programs. In cell-based bioassays, Chilean P. verruculosa was only toxic to the RTgill-W1 cell line following exposures to high cell densities of lysed cells (>100,000 cells mL-1). Fatty acid profiles of a cultured strain showed elevated concentrations of potentially ichthyotoxic, long-chain polyunsaturated fatty acids (PUFAs) (69.7% ± 1.8%)- stearidonic (SDA, 18:4ω3-28.9%), and docosahexaenoic acid (DHA, 22:6ω3-22.3%), suggesting that lipid peroxidation may help to explain the mortalities, though superoxide production by Pseudochattonella was low (< 0.21 ± 0.19 pmol O2- cell-1 h-1). It therefore remains unknown what the mechanisms of salmon mortality were during the Pseudochattonella bloom. Multiple mitigation strategies were used by salmon farmers during the event, with only delayed seeding of juvenile fish into the cages and towing of cages to sanctuary sites being effective. Airlift pumping, used effectively against other fish-killing HABs in the US and Canada was not effective, perhaps because it brought subsurface layers of Pseudochattonella to the surface, or and it also may have lysed the fragile cells, rendering them more lethal. The present study highlights knowledge gaps and inefficiency of contingency plans by the fish farming industry to overcome future fish-killing algal blooms under future climate change scenarios. The use of new technologies based on molecular methods for species detection, good farm practices by fish farms, and possible mitigation strategies are discussed.

Coccolith volume of the Southern Ocean coccolithophore Emiliania huxleyi as a possible indicator for palaeo-cell volume.

Coccolithophores are a key functional phytoplankton group and produce minute calcite plates (coccoliths) in the sunlit layer of the pelagic ocean. Coccoliths significantly contribute to the sediment record since the Triassic and their geometry have been subject to palaeoceanographic and biological studies to retrieve information on past environmental conditions. Here, we present a comprehensive analysis of coccolith, coccosphere and cell volume data of the Southern Ocean Emiliania huxleyi ecotype A, subject to gradients of temperature, irradiance, carbonate chemistry and macronutrient limitation. All tested environmental drivers significantly affect coccosphere, coccolith and cell volume with driver-specific sensitivities. However, a highly significant correlation emerged between cell and coccolith volume with Vcoccolith  = 0.012 ± 0.001 * Vcell  + 0.234 ± 0.066 (n = 23, r2  = .85, p < .0001, σest  = 0.127), indicating a primary control of coccolith volume by physiological modulated changes in cell volume. We discuss the possible application of fossil coccolith volume as an indicator for cell volume/size and growth rate and, additionally, illustrate that macronutrient limitation of phosphorus and nitrogen has the predominant influence on coccolith volume in respect to other environmental drivers. Our results provide a solid basis for the application of coccolith volume and geometry as a palaeo-proxy and shed light on the underlying physiological reasons, offering a valuable tool to investigate the fossil record of the coccolithophore E. huxleyi.

Perceived global increase in algal blooms is attributable to intensified monitoring and emerging bloom impacts.

Global trends in the occurrence, toxicity and risk posed by harmful algal blooms to natural systems, human health and coastal economies are poorly constrained, but are widely thought to be increasing due to climate change and nutrient pollution. Here, we conduct a statistical analysis on a global dataset extracted from the Harmful Algae Event Database and Ocean Biodiversity Information System for the period 1985-2018 to investigate temporal trends in the frequency and distribution of marine harmful algal blooms. We find no uniform global trend in the number of harmful algal events and their distribution over time, once data were adjusted for regional variations in monitoring effort. Varying and contrasting regional trends were driven by differences in bloom species, type and emergent impacts. Our findings suggest that intensified monitoring efforts associated with increased aquaculture production are responsible for the perceived increase in harmful algae events and that there is no empirical support for broad statements regarding increasing global trends. Instead, trends need to be considered regionally and at the species level.

Detection of Paralytic Shellfish Toxins in Southern Rock Lobster Jasus edwardsii Using the Qualitative Neogen™ Lateral Flow Immunoassay: Single-Laboratory Validation.

BACKGROUND: Paralytic shellfish toxins (PST) are a significant problem for the Tasmanian shellfish and Southern Rock Lobster (Jasus edwardsii) industries, and the introduction of a rapid screening test in the monitoring program could save time and money. OBJECTIVE: The aim was to perform a single-laboratory validation of the Neogen rapid test for PST in the hepatopancreas of Southern Rock Lobster. METHODS: The AOAC INTERNATIONAL guidelines for the validation of qualitative binary chemistry methods were followed. Three different PST profiles (mixtures) were used, of which two were commonly found in naturally contaminated lobster hepatopancreas (high in gonyautoxin 2&3 and saxitoxin), and the third toxin profile was observed in a few select animals (high in gonyautoxin 1&4). RESULTS: The Neogen test consistently returned negative results for non-target toxins (selectivity). The probability of detection (POD) of PST in the lobster hepatopancreas using the Neogen test increased with increasing PST concentrations. POD values of 1.0 were obtained at ≥0.57 mg STX-diHCl eq/kg in mixtures 1 and 2, and 0.95 and 1.0 for mixture 3 at 0.79 and 1.21 mg STX-diHCl eq/kg, respectively, with a fitted POD of 0.98 for 0.80 mg STX-diHCl eq/kg. The performance of the Neogen test when using four different production lots (ruggedness) showed no significant differences. CONCLUSIONS: The results of the validation study were satisfactory and the Neogen test is being trialed within the Tasmanian PST monitoring program of Southern Rock Lobster. HIGHLIGHTS: The Neogen rapid kit was successfully validated for the detection of PST in Southern Rock Lobster hepatopancreas.

An optimized method for the extraction of ancient eukaryote DNA from marine sediments.

Marine sedimentary ancient DNA (sedaDNA) provides a powerful means to reconstruct marine palaeo-communities across the food web. However, currently there are few optimized sedaDNA extraction protocols available to maximize the yield of small DNA fragments typical of ancient DNA (aDNA) across a broad diversity of eukaryotes. We compared seven combinations of sedaDNA extraction treatments and sequencing library preparations using marine sediments collected at a water depth of 104 m off Maria Island, Tasmania, in 2018. These seven methods contrasted frozen versus refrigerated sediment, bead-beating induced cell lysis versus ethylenediaminetetraacetic acid (EDTA) incubation, DNA binding in silica spin columns versus in silica-solution, diluted versus undiluted DNA in shotgun library preparations to test potential inhibition issues during amplification steps, and size-selection of low molecular-weight (LMW) DNA to increase the extraction efficiency of sedaDNA. Maximum efficiency was obtained from frozen sediments subjected to a combination of EDTA incubation and bead-beating, DNA binding in silica-solution, and undiluted DNA in shotgun libraries, across 45 marine eukaryotic taxa. We present an optimized extraction protocol integrating these steps, with an optional post-library LMW size-selection step to retain DNA fragments of ≤500 base pairs. We also describe a stringent bioinformatic filtering approach for metagenomic data and provide a comprehensive list of contaminants as a reference for future sedaDNA studies. The new extraction and data-processing protocol should improve quantitative paleo-monitoring of eukaryotes from marine sediments, as well as other studies relying on the detection of highly fragmented and degraded eukaryote DNA in sediments.

Environmental drivers of unprecedented Alexandrium catenella dinoflagellate blooms off eastern Tasmania, 2012-2018.

Blooms of the highly toxic dinoflagellate Alexandrium catenella (previously referred to as tamarense group 1) were first detected off eastern Tasmania in 2012 and have since been responsible for incidences of human paralytic shellfish poisoning and extended closures (up to 25 weeks) of mussel, oyster, scallop, abalone and rock lobster industries (up to 150 mg/kg PST in mussels). Investigation of meteorological and oceanographic influences indicate that the annually recurrent winter-spring blooms (June-Oct) occur within a narrow water temperature window (10-15 °C) under two distinct sets of conditions: (1) following high rainfall and land run-off, under relatively light winds; and (2) following periods of anomalously low air temperatures and associated cooling of shallow coastal waters, again under relatively light winds. The common driver of blooms appears to be the development of stratification in coastal waters, via salinity and/or temperature gradients. We propose a framework for evaluating the risk of Alexandrium with the aim of developing a forecasting capability, and compare these environmental conditions with historic data to understand the recent advent of these blooms.

Fish gill damage by harmful microalgae newly explored by microelectrode ion flux estimation techniques.

Harmful algal blooms (HAB) are responsible for massive mortalities of wild and aquacultured fish due to noticeable gill damage, but the precise fish-killing mechanisms remain poorly understood. A non-invasive microelectrode ion flux estimation (MIFE) technique was successfully applied to assess changes in membrane-transport processes in a model fish gill cell line exposed to harmful microplankton. Net Ca2+, H+, K+ ion fluxes in the rainbow trout cell line RTgill-W1 were monitored before and after addition of lysed cells of this Paralytic Shellfish Toxins (PST) producer along with purified endocellular dinoflagellate PST. It was demonstrated that PST alone do not play a role in fish gill damage during A. catenella outbreaks as previously thought, but that other ichthyotoxic metabolites from lysed algal cells (i.e. lipid peroxidation products or other unknown metabolites) result in net K+ efflux from fish gill cells and thereby gill cell death.

Detection of Paralytic Shellfish Toxins in Mussels and Oysters Using the Qualitative Neogen Lateral-Flow Immunoassay: An Interlaboratory Study.

Paralytic shellfish toxins (PSTs) in bivalve molluscs represent a public health risk and are controlled via compliance with a regulatory limit of 0.8 mg saxitoxin (STX)⋅2HCl equivalents per kilogram of shellfish meat (eq/kg). Shellfish industries would benefit from the use of rapid immunological screening tests for PSTs to be used for regulation, but to date none have been fully validated. An interlaboratory study involving 16 laboratories was performed to determine the suitability of the Neogen test to detect PSTs in mussels and oysters. Participants performed the standard protocol recommended by the manufacturer and a modified protocol with a conversion step to improve detection of gonyautoxin 1&4. The statistical analysis showed that the protocols had good homogeneity across all laboratories, with satisfactory repeatability, laboratory, and reproducibility variation near the regulatory level. The mean probability of detection (POD) at 0.8 mg STX⋅2HCl eq/kg using the standard protocol in mussels and oysters was 0.966 and 0.997, respectively, and 0.968 and 0.966 using the modified protocol. The estimated LOD in mussels was 0.316 mg STX⋅2HCl eq/kg with the standard and 0.682 mg STX⋅2HCl eq/kg with the modified protocol, and 0.710 and 0.734 mg STX⋅2HCl eq/kg for oysters, respectively. The Neogen test may be acceptable for regulatory purposes for oysters in accordance with European Commission directives in which the standard protocol provides, at the regulatory level, a probability of a negative response of 0.033 on 95% of occasions. Its use for mussels is less consistent at the regulatory level due to the wide prediction interval around the POD.

Single-Laboratory Validation of the Neogen Qualitative Lateral Flow Immunoassay for the Detection of Paralytic Shellfish Toxins in Mussels and Oysters.

Detection of paralytic shellfish toxins (PSTs) in bivalve shellfish by analytical methods is complicated and costly, requiring specific expertise and equipment. Following extensive blooms of Alexandrium tamarense Group 1 in Tasmania, Australia, an investigation was made into commercially available screening test kits suitable for use with the toxin profiles found in affected bivalves. The qualitative Neogen rapid test kit, with a modified protocol to convert gonyautoxins GTX1&4 and GTX2&3 into neosaxitoxin and saxitoxin (STX), respectively, with higher cross-reactivities, was the best fit-for-purpose. This validation study of the test kit and the modified protocol was undertaken following AOAC INTERNATIONAL guidelines for the validation of qualitative binary chemistry methods. The validation used four different PST profiles representing natural profiles found in Australia and in Europe: two in a mussel matrix and two in an oyster matrix. The test kit was shown to have appropriate selectivity of the toxin analogs commonly found in bivalve shellfish. The matrix and probability of detection (POD) study showed that the rapid test kit used with the modified protocol was able to consistently detect PST at the bivalve regulatory level of 0.8 mg STX⋅2HCl eq/kg, with a POD estimated via the binomial logistic regression of 1.0 at 0.8 mg STX⋅2HCl eq/kg in all tested profiles in both matrixes. The POD at 0.4 mg STX⋅2HCl eq/kg was 0.75 and 0.46 for the two toxin profiles in an oyster matrix and 0.96 and 1.0 for the two toxin profiles in a mussel matrix. No significant differences in the PODs of the PSTs at the regulatory level were found between production lots of the test kits. The results suggest the method is suitable to undergo a collaborative validation study.

Independence of nutrient limitation and carbon dioxide impacts on the Southern Ocean coccolithophore Emiliania huxleyi.

Future oceanic conditions induced by anthropogenic greenhouse gas emissions include warming, acidification and reduced nutrient supply due to increased stratification. Some parts of the Southern Ocean are expected to show rapid changes, especially for carbonate mineral saturation. Here we compare the physiological response of the model coccolithophore Emiliania huxleyi (strain EHSO 5.14, originating from 50oS, 149oE) with pH/CO2 gradients (mimicking ocean acidification ranging from 1 to 4 × current pCO2 levels) under nutrient-limited (nitrogen and phosphorus) and -replete conditions. Both nutrient limitations decreased per cell photosynthesis (particulate organic carbon (POC) production) and calcification (particulate inorganic carbon (PIC) production) rates for all pCO2 levels, with more than 50% reductions under nitrogen limitation. These impacts, however, became indistinguishable from nutrient-replete conditions when normalized to cell volume. Calcification decreased three-fold and linearly with increasing pCO2 under all nutrient conditions, and was accompanied by a smaller ~30% nonlinear reduction in POC production, manifested mainly above 3 × current pCO2. Our results suggest that normalization to cell volume allows the major impacts of nutrient limitation (changed cell sizes and reduced PIC and POC production rates) to be treated independently of the major impacts of increasing pCO2 and, additionally, stresses the importance of including cell volume measurements to the toolbox of standard physiological analysis of coccolithophores in field and laboratory studies.

Comparative performance of four immunological test kits for the detection of Paralytic Shellfish Toxins in Tasmanian shellfish.

Blooms of the toxic dinoflagellate Alexandrium tamarense (Group 1) seriously impacted the Tasmanian shellfish industry during 2012 and 2015, necessitating product recalls and intensive paralytic shellfish toxin (PST) product testing. The performance of four commercial PST test kits, Abraxis™, Europroxima™, Scotia™ and Neogen™, was compared with the official AOAC LC-FLD method for contaminated mussels and oysters. Abraxis and Europroxima kits underestimated PST in 35-100% of samples when using standard protocols but quantification improved when concentrated extracts were further diluted (underestimation ≤18%). The Scotia kit (cut off 0.2-0.7 mg STX-diHCl eq/kg) delivered 0% false negatives, but 27% false positives. Neogen produced 5% false negatives and 13% false positives when the cut off was altered to 0.5-0.6 mg STX-diHCl eq/kg, the introduction of a conversion step eliminated false negatives. Based on their sensitivity, ease of use and performance, the Neogen kit proved the most suitable kit for use with Tasmanian mussels and oysters. Once formally validated for regulatory purposes, the Neogen kit could provide shellfish growers with a rapid tool for harvesting decisions at the farm gate. Effective rapid screening preventing compliant samples undergoing testing using the more expensive and time consuming LC-FLD method will result in significant savings in analytical costs.

Role of resting cysts in Chilean Alexandrium catenella dinoflagellate blooms revisited.

The detection of sparse Alexandrium catenella-resting cysts in sediments of southern Chilean fjords has cast doubts on their importance in the recurrence of massive toxic dinoflagellate blooms in the region. The role of resting cysts and the existence of different regional Chilean populations was studied by culturing and genetic approaches to define: (1) cyst production; (2) dormancy period; (3) excystment success; (4) offspring viability and (5) strain mating compatibility. This study newly revealed a short cyst dormancy (minimum 69 days), the role of key abiotic factors (in decreasing order salinity, irradiance, temperature and nutrients) controlling cyst germination (max. 60%) and germling growth rates (up to 0.36-0.52div.day-1). Amplified fragment length polymorphism (AFLP) characterization showed significant differences in genetic distances (GD) among A. catenella populations that were primarily determined by the geographical origin of isolates and most likely driven by oceanographic dispersal barriers. A complex heterothallic mating system pointed to variable reproductive compatibility (RCs) among Chilean strains that was high among northern (Los Lagos/North Aysén) and southern populations (Magallanes), but limited among the genetically differentiated central (South Aysén) populations. Field cyst surveys after a massive 2009 bloom event revealed the existence of exceptional high cyst densities in particular areas of the fjords (max. 14.627cystscm-3), which contrast with low cyst concentrations (<221.3cystscm-3) detected by previous oceanographic campaigns. In conclusion, the present study suggests that A. catenella resting cysts play a more important role in the success of this species in Chilean fjords than previously thought. Results from in vitro experiments suggest that pelagic-benthic processes can maintain year-round low vegetative cell concentrations in the water column, but also can explain the detection of high cysts aggregations after the 2009-bloom event. Regional drivers that lead to massive outbreaks, however, are still unknown but potential scenarios are discussed.

Progress in Understanding Algal Bloom-Mediated Fish Kills: The Role of Superoxide Radicals, Phycotoxins and Fatty Acids.

Quantification of the role of reactive oxygen species, phycotoxins and fatty acids in fish toxicity by harmful marine microalgae remains inconclusive. An in vitro fish gill (from rainbow trout Oncorhynchus mykiss) assay was used to simultaneously assess the effect in superoxide dismutase, catalase and lactate dehydrogenase enzymatic activities caused by seven species of ichthyotoxic microalgae (Chattonella marina, Fibrocapsa japonica, Heterosigma akashiwo, Karenia mikimotoi, Alexandrium catenella, Karlodinium veneficum, Prymnesium parvum). Quantification of superoxide production by these algae was also performed. The effect of purified phycotoxins and crude extracts was compared, and the effect of fatty acids is discussed. The raphidophyte Chattonella was the most ichthyotoxic (gill cell viability down to 35%) and also the major producer of superoxide radicals (14 pmol cell-1 hr-1) especially after cell lysis. The raphidophyte Heterosigma and dinoflagellate Alexandrium were the least toxic and had low superoxide production, except when A. catenella was lysed (5.6 pmol cell-1 hr-1). Catalase showed no changes in activity in all the treatments. Superoxide dismutase (SOD) and lactate dehydrogenase exhibited significant activity increases of ≤23% and 51.2% TCC (total cellular content), respectively, after exposure to C. marina, but SOD showed insignificant changes with remaining algal species. A strong relationship between gill cell viability and superoxide production or superoxide dismutase was not observed. Purified brevetoxins PbTx-2 and -3 (from Karenia brevis, LC50 of 22.1 versus 35.2 µg mL-1) and karlotoxin KmTx-2 (from Karlodinium; LC50 = 380 ng mL-1) could almost entirely account for the fish killing activity by those two dinoflagellates. However, the paralytic shellfish toxins (PST) GTX1&4, C1&C2, and STX did not account for Alexandrium ichthyotoxicity. Only aqueous extracts of Alexandrium were cytotoxic (≤65% decrease of viability), whereas crude methanol and acetone extracts of Chattonella, Fibrocapsa, Heterosigma, Karlodinium and Prymnesium decreased cell viability down to 0%. These and our previous findings involving the role of fatty acids confirm that superoxide radicals are only partially involved in ichthyotoxicity and point to a highly variable contribution by other compounds such as lipid peroxidation products (e.g. aldehydes).

Strain variability in fatty acid composition of Chattonella marina (Raphidophyceae) and its relation to differing ichthyotoxicity toward rainbow trout gill cells.

Lipid profiles of three strains (Mexico, Australia, Japan) of Chattonella marina (Subrahmanyan) Hara et Chihara were studied under defined growth (phosphate, light, and growth phase) and harvest (intact and ruptured cells) conditions. Triacylglycerol levels were always <2%, sterols <7%, free fatty acids varied between 2 and 33%, and polar lipids were the most abundant lipid class (>51% of total lipids). The major fatty acids in C. marina were palmitic (16:0), eicosapentaenoic (EPA, 20:5ω3), octadecatetraenoic (18:4ω3), myristic (14:0), and palmitoleic (16:1ω7c) acids. Higher levels of EPA were found in ruptured cells (21.4-29.4%) compared to intact cells (8.5-25.3%). In general, Japanese N-118 C. marina was the highest producer of EPA (14.3-29.4%), and Mexican CMCV-1 the lowest producer (7.9-27.1%). Algal cultures, free fatty acids from C. marina, and the two aldehydes 2E,4E-decadienal and 2E,4E-heptadienal (suspected fatty acid-derived products) were tested against the rainbow trout fish gill cell line RTgill-W1. The configuration of fatty acids plays an important role in ichthyotoxicity. Free fatty acid fractions, obtained by base saponification of total lipids from C. marina showed a potent toxicity toward gill cells (median lethal concentration, LC50 (at 1 h) of 0.44 µg · mL(-1) in light conditions, with a complete loss of viability at >3.2 µg · mL(-1) ). Live cultures of Mexican C. marina were less toxic than Japanese and Australian strains. This difference could be related to differing EPA content, superoxide anion production, and cell fragility. The aldehydes 2E,4E-decadienal and 2E,4E-heptadienal also showed high impact on gill cell viability, with LC50 (at 1 h) of 0.34 and 0.36 µg · mL(-1) , respectively. Superoxide anion production was highest in Australian strain CMPL01, followed by Japanese N-118 and Mexican CMCV-1 strains. Ruptured cells showed higher production of superoxide anion compared to intact cells (e.g., 19 vs. 9.5 pmol · cell(-1)  · hr(-1) for CMPL01, respectively). Our results indicate that C. marina is more ichthyotoxic after cell disruption and when switching from dark to light conditions, possibly associated with a higher production of superoxide anion and EPA, which may be quickly oxidized to produce more toxic derivates, such as aldehydes.

Progress in understanding harmful algal blooms: paradigm shifts and new technologies for research, monitoring, and management.

The public health, tourism, fisheries, and ecosystem impacts from harmful algal blooms (HABs) have all increased over the past few decades. This has led to heightened scientific and regulatory attention, and the development of many new technologies and approaches for research and management. This, in turn, is leading to significant paradigm shifts with regard to, e.g., our interpretation of the phytoplankton species concept (strain variation), the dogma of their apparent cosmopolitanism, the role of bacteria and zooplankton grazing in HABs, and our approaches to investigating the ecological and genetic basis for the production of toxins and allelochemicals. Increasingly, eutrophication and climate change are viewed and managed as multifactorial environmental stressors that will further challenge managers of coastal resources and those responsible for protecting human health. Here we review HAB science with an eye toward new concepts and approaches, emphasizing, where possible, the unexpected yet promising new directions that research has taken in this diverse field.

PHOTOSYNTHETIC PIGMENT AND GENETIC DIFFERENCES BETWEEN TWO SOUTHERN OCEAN MORPHOTYPES OF EMILIANIA HUXLEYI (HAPTOPHYTA)1.

The widespread coccolithophorid Emiliania huxleyi (Lohmann) W. W. Hay et H. Mohler plays a pivotal role in the carbon pump and is known to exhibit significant morphological, genetic, and physiological diversity. In this study, we compared photosynthetic pigments and morphology of triplicate strains of Southern Ocean types A and B/C. The two morphotypes differed in width of coccolith distal shield elements (0.11-0.24 µm, type A; 0.06-0.12 µm, type B/C) and morphology of distal shield central area (grill of curved rods in type A; thin plain plate in type B/C) and showed differences in carotenoid composition. The mean 19'-hexanoyloxyfucoxanthin (Hex):chl a ratio in type B/C was >1, whereas the type A ratio was <1. The Hex:fucoxanthin (fuc) ratio for type B/C was 11 times greater than that for type A, and the proportion of fuc in type A was 6 times higher than that in type B/C. The fuc derivative 4-keto-19'-hexanoyloxyfucoxanthin (4-keto-hex) was present in type A but undetected in B/C. DNA sequencing of tufA distinguished morphotypes A, B/C (indistinguishable from B), and R, while little variation was observed within morphotypes. Thirty single nucleotide polymorphisms were identified in the 710 bp tufA sequence, of which 10 alleles were unique to B/C and B morphotypes, seven alleles were unique to type A, and six alleles were unique to type R. We propose that the morphologically, physiologically, and genetically distinct Southern Ocean type B/C sensu Young et al. (2003) be classified as E. huxleyi var. aurorae var. nov. S. S. Cook et Hallegr.

Succession and physiological health of freshwater microalgal fouling in a Tasmanian hydropower canal.

Freshwater microalgal biofouling in hydropower canals in Tarraleah, Tasmania, is dominated by a single diatom species, Gomphonema tarraleahae. The microfouling community is under investigation with the aim of reducing its impact on electricity generation. Species succession was investigated using removable glass slides. Fouled slides were examined microscopically and for chlorophyll a biomass. Chl a biomass increased steeply after 8 weeks (0.09-0.87 mg m(-2)), but increased much earlier on slides surrounded by a biofouled inoculum. Succession began with low profile diatoms such as Tabellaria flocculosa, progressing to stalked diatoms such as Gomphonema spp. and Cymbella aspera. Few chlorophytes and no filamentous algae were present. Pulse amplitude modulated fluorometry was used to measure the physiological health of fouling on the canal wall. Maximum quantum yield (F(v)/F(m)) measurements were consistently <0.18, indicating that the fouling mat consisted of dead or dying algae. The succession and physiological health of cells in the fouling community has broad implications for mitigation techniques used.

SURVEY FOR KARLOTOXIN PRODUCTION IN 15 SPECIES OF GYMNODINIOID DINOFLAGELLATES (KARENIACEAE, DINOPHYTA)(1).

Toxin analysis of 15 species of Kareniaceae revealed the presence of karlotoxin, KmTx 2, in only a single species (Karlodinium veneficum) but with variable activity in strains from the Swan (Km(Swan) Tx 2-1, 2.1 pg · cell(-1) ; and Km(Swan) Tx 2-2, 0.53 pg · cell(-1) ), Huon (Km(Huon) Tx 2, 0.86 pg · cell(-1) ), and Derwent rivers (<0.001 pg · cell(-1) ) in Australia. A newly isolated Southern Ocean species, Karlodinium conicum, contained a novel poorly hemolytic karlotoxin analogue (Kmconicum Tx, 2.8 pg · cell(-1) ). The hemolytic potency (HD50%) of the Australian karlotoxins were as follows: Km(Swan) Tx 2-1 (65.9 ± 4.8 ng) and Km(Swan) Tx 2-2 (63.4 ± 3.7 ng), Km(Huon) Tx 2 (343 ± 4.9 ng), and Kmconicum Tx (>4,000 ng). Species from the closely related genera Takayama (T. helix, T. tasmanica, T. tuberculata), Karenia (K. asterichroma, K. brevis, K. mikimotoi, K. papilionacea, K. umbella), and Karlodinium (Ka. australe, Ka. antarcticum, Ka. ballantinum, Ka. corrugatum, Ka. decipiens) were all consistently negative for karlotoxin production. Brevetoxin (PbTx) was only detected in K. brevis, and hemolytic activity was only observed in Ka. veneficum strains.

NOVEL UNARMORED DINOFLAGELLATES FROM THE TOXIGENIC FAMILY KARENIACEAE (GYMNODINIALES): FIVE NEW SPECIES OF KARLODINIUM AND ONE NEW TAKAYAMA FROM THE AUSTRALIAN SECTOR OF THE SOUTHERN OCEAN(1).

Six new species of unarmored dinoflagellates in the family Kareniaceae were isolated from the Australian sector of the Southern Ocean in March 2006: Takayama tuberculata de Salas sp. nov, Karlodinium antarcticum de Salas sp. nov., Karl. ballantinum de Salas sp. nov., Karl. conicum de Salas sp. nov., Karl. corrugatum de Salas sp. nov., and Karl. decipiens de Salas et Laza-Martínez sp. nov. These new taxa were characterized using light and electron microscopy and sequencing of the LSU rDNA and are well supported based either on their morphology or molecular phylogeny. Takayama tuberculata, isolated just north of the polar front (55°-57° S), is genetically close to T. tasmanica, but smaller, with a significantly reduced number of amphiesmal vesicles. Medium-sized Karl. antarcticum, also isolated from near the polar front, is characterized by its long ovoid cell outline and very long apical groove. The small Karl. ballantinum has a very short apical groove. The large Karl. conicum has a distinct conical epicone and spherical posterior nucleus. The small Karl. corrugatum, from just south of the polar front, has distinctive parallel striations on the epicone surface and a distinctively shaped and placed ventral pore. The large and widespread Karl. decipiens, distributed through Southern Ocean waters from the polar front to Tasmanian coastal waters, and coastal Spain, has a helicoidal chloroplast arrangement and a large central nucleus. This study represents the first description of species in the potentially ichthyotoxic family Kareniaceae recorded from the Southern Ocean.