Molecular cloning reveals co-occurring species behind red tide blooms of the harmful dinoflagellate Cochlodinium polykrikoides

Red tides caused by the marine dinoflagellate Cochlodinium polykrikoides Margalef pose significant environmental problems worldwide. Recently, the existence of severe blooms attributable to a single Cochlodinium Schütt species has been questioned by many researchers. Herein we investigated the dinoflagellate composition of harmful algal blooms (HABs) attributed to C. polykrikoides in Korean coastal waters at nine different stations (St.). The component species of Cochlodinium blooms were examined by using microscopic and gene-cloning methods. In the nine study areas, C. polykrikoides was the predominant species of HABs in St. 2, 4, 7, and St. 9. Based on the morphological identification, the bloom was initially thought to be caused only by C. polykrikoides; however, we detected additional bloom-forming dinoflagellates (Polykrikos schwartzii Bütschli and Polykrikos kofoidii Chatton), and diatoms (Pseudo-nitzschia americana (Hasle) Fryxell) along with C. polykrikoides. The parasitic dinoflagellates Amoebophrya Koeppen and Euduboscquella Coats, Bachvaroff & Delwiche were found to be co-located with Cochlodinium in our study, and for the first time, Cochlodinium fulvescens Iwataki, Kawami & Matsuoka was detected in Korea (west coast). These results suggest co-existence of multiple dinoflagellates in bloom populations of Cochlodinium and describe the composition of other dinoflagellate blooms (e.g., Polykrikos spp.) in Korean coastal regions. This co-occurrence may be considered during efforts to monitor and control HABs.     

Mapping the Distribution of Cysts from the Toxic Dinoflagellate Cochlodinium polykrikoides in Bloom-Prone Estuaries by a Novel Fluorescence In Situ Hybridization Assay

Cochlodinium polykrikoides is a cosmopolitan dinoflagellate that is notorious for causing fish-killing harmful algal blooms (HABs) across North America and Asia. While recent laboratory and ecosystem studies have definitively demonstrated that Cochlodinium forms resting cysts that may play a key role in the dynamics of its HABs, uncertainties regarding cyst morphology and detection have prohibited even a rudimentary understanding of the distribution of C. polykrikoides cysts in coastal ecosystems. Here, we report on the development of a fluorescence in situ hybridization (FISH) assay using oligonucleotide probes specific for the large subunit (LSU) ribosomal DNA (rDNA) of C. polykrikoides. The LSU rDNA-targeted FISH assay was used with epifluorescence microscopy and was iteratively refined to maximize the fluorescent reaction with C. polykrikoides and minimize cross-reactivity. The final LSU rDNA-targeted FISH assay was found to quantitatively recover cysts made by North American isolates of C. polykrikoides but not cysts formed by other common cyst-forming dinoflagellates. The method was then applied to identify and map C. polykrikoides cysts across bloom-prone estuaries. Annual cyst and vegetative cell surveys revealed that elevated densities of C. polykrikoides cysts (>100 cm⁻³) during the spring of a given year were spatially consistent with regions of dense blooms the prior summer. The identity of cysts in sediments was confirmed via independent amplification of C. polykrikoides rDNA. This study mapped C. polykrikoides cysts in a natural marine setting and indicates that the excystment of cysts formed by this harmful alga may play a key role in the development of HABs of this species.     

Field experiments on mitigation of harmful algal blooms using a Sophorolipid—Yellow clay mixture and effects on marine plankton

This study examined a new method of mitigating harmful algal blooms (HABs) by combining biosurfactant sophorolipid and yellow clay. To investigate the effects and practicability of this HAB mitigation method, field experiments were carried out during a Cochlodinium bloom near Miruk Island, South Korea, in August 2002. Field experiments examined the effects of sophorolipid and yellow clay on Cochlodinium bloom mitigation and on marine plankton such as bacteriaplankton, heterotrophic protists, and zooplankton. A mixture of 5 mg l⁻¹ sophorolipid and 1 g l⁻¹ yellow clay was sprayed directly on the sea surface and its effect was compared with that of 10 g l⁻¹ of yellow clay applied under similar conditions. The sophorolipid–yellow clay mixture more efficiently mitigated the Cochlodinium bloom (95% removal efficiency after 30 min) than yellow clay alone (79% after 30 min). Further, no variation in bacterial abundance occurred 30 min after spraying the sophorolipid–yellow clay mixture. After 30 min, heterotrophic protist abundance at the surface decreased 21 and 41%, respectively, following the sophorolipid–yellow clay mixture and yellow clay treatments. Zooplankton decreased by 38% 15 min after spraying the mixture and 67% 30 min after spraying the yellow clay. These results indicate that the mixture of sophorolipid and yellow clay had a less adverse effect on bacteriaplankton, heterotrophic protists, and zooplankton than the yellow clay, suggesting that the sophorolipid–yellow clay mixture can mitigate HABs efficiently with fewer negative effects on the pelagic ecosystem.     

Sexual resting cyst production by the dinoflagellate Akashiwo sanguinea: a potential mechanism contributing to the ubiquitous distribution of a harmful alga

The dinoflagellate Akashiwo sanguinea is a well known, cosmopolitan harmful microalga that frequently forms harmful algal blooms (HABs) in marine estuaries from temperate to tropical waters, and has posed a severe threat to fish, shellfish, and sea birds. Therefore, it is important to understand the ecology of this species, particularly the mechanisms regulating its ubiquitous geographic distribution and frequent recurrence of. To date, the mechanisms regulating distribution and recurrence of HABs by this species have been poorly understood. While resting cyst production can play a central role in the geographic expansion and initiation of HABs, studies of the life cycle of this alga, including cyst production, have been lacking. Here, we demonstrate that A. sanguinea produces sexual resting cysts homothallically. We present evidence for cell pairs in sexual mating, biflagellated planozygote formation, and cysts of different morphologies, and we describe time series for germination of cysts to germlings with two longitudinal flagella, along with studies of possible factors affecting cyst production. Phylogenetic analysis of large sub‐unit rDNA sequences revealed a monophyly of this species and thus possibly a recent common ancestor for all global populations. The discovery of resting cyst production by A. sanguinea suggests its frequent recurrence of blooms and global distribution may have been facilitated by the natural and anthropogenic transport of resting cysts.     

The toxic dinoflagellate Cochlodinium polykrikoides (Dinophyceae) produces resting cysts

While harmful algal blooms (HABs) caused by the toxic dinoflagellate Cochlodinium polykrikoides have been known to science for more than a century, the past two decades have witnessed an extraordinary expansion of these events across Asia, North America, and even Europe. Although the production of resting cysts and subsequent transport via ships’ ballast water or/and the transfer of shellfish stocks could facilitate this expansion, confirmative evidence for cyst production by C. polykrikoides is not available. Here, we provide visual confirmation of the production of resting cysts by C. polykrikoides in laboratory cultures isolated from North America. Evidence includes sexually mating cell pairs, planozygotes with two longitudinal flagella, formation of both pellicular (temporary) cysts and resting cysts, and a time series of the cyst germination process. Resting cyst germination occurred up to 1 month after cyst formation and 2–40% of resting cysts were successfully germinated in cultures maintained at 18–21 °C. Pellicular cysts with hyaline membranes were generally larger than resting cysts, displayed discernable cingulum and/or sulcus, and reverted to vegetative cells within 24 h to ∼1 week of formation. A putative armored stage of C. polykrikoides was not observed during any life cycle stage in this study. This definitive evidence of resting cyst production by C. polykrikoides provides a mechanism to account for the recurrence of annual blooms in given locales as well as the global expansion of C. polykrikoides blooms during the past two decades.     

Harmful algal blooms: causes,impacts and detection

Blooms of autotrophic algae and some heterotrophic protists are increasingly frequent in coastal waters around the world and are collectively grouped as harmful algal blooms (HABs). Blooms of these organisms are attributed to two primary factors: natural processes such as circulation, upwelling relaxation, and river flow; and, anthropogenic loadings leading to eutrophication. Unfortunately, the latter is commonly assumed to be the primary cause of all blooms, which is not the case in many instances. Moreover, although it is generally acknowledged that occurrences of these phenomena are increasing throughout the world's oceans, the reasons for this apparent increase remain debated and include not only eutrophication but increased observation efforts in coastal zones of the world. There is a rapidly advancing monitoring effort resulting from the perception of increased impacts from these HABs, manifested as expanding routine coastal monitoring programs, rapid development and deployment of new detection methods for individual species, toxins, and toxicities, and expansion of coastal modeling activities towards observational forecasts of bloom landfall and eventually bloom prediction. Together, these many efforts will provide resource managers with the tools needed to develop effective strategies for the management and mitigation of HABs and their frequently devastating impacts on the coastal environment.     

QUANTITATIVE REAL‐TIME POLYMERASE CHAIN REACTION FOR COCHLODINIUM FULVESCENS (DINOPHYCEAE), A HARMFUL DINOFLAGELLATE FROM CALIFORNIA COASTAL WATERS1

Harmful blooms formed by species of the dinoflagellate Cochlodinium have caused massive fish kills and substantial economic losses in the Pacific Ocean. Recently, prominent blooms of Cochlodinium have occurred in central and southern California (2004–2008), and Cochlodinium cells are now routinely observed in microscopical analysis of algal assemblages from Californian coastal waters. The first documented economic loss due to a Cochlodinium bloom in California occurred in Monterey Bay and resulted in the mortality of commercially farmed abalone. Increasing occurrences of Cochlodinium blooms, the fact that these cells preserve poorly using standard techniques, and the difficulty of identifying preserved specimens using morphological criteria make Cochlodinium species prime candidates for the development of a quantitative real‐time polymerase chain reaction (qPCR) approach. The 18S rDNA gene sequenced from Cochlodinium cells obtained from California coastal waters, as well as GenBank sequences of Cochlodinium, were used to design and test a Molecular Beacon^® approach. The qPCR method developed in this study is species specific, sensitive for the detection of C. fulvescens that has given rise to the recent blooms in the eastern Pacific Ocean, and spans a dynamic abundance range of seven orders of magnitude. Initial application of the method to archived field samples collected during blooms in Monterey Bay revealed no statistically significant correlations between gene copy number and environmental parameters. However, the onset of Cochlodinium blooms in central California was consistent with previously reported findings of correlations to decreased surface temperature and increased inputs of nitrogenous nutrients.     

Eutrophication and harmful algal blooms: A scientific consensus

In January 2003, the US Environmental Protection Agency sponsored a “roundtable discussion” to develop a consensus on the relationship between eutrophication and harmful algal blooms (HABs), specifically targeting those relationships for which management actions may be appropriate. Academic, federal, and state agency representatives were in attendance. The following seven statements were unanimously adopted by attendees based on review and analysis of current as well as pertinent previous data:

• (1)Degraded water quality from increased nutrient pollution promotes the development and persistence of many HABs and is one of the reasons for their expansion in the U.S. and other nations;
• (2)The composition–not just the total quantity–of the nutrient pool impacts HABs;
• (3)High-biomass blooms must have exogenous nutrients to be sustained;
• (4)Both chronic and episodic nutrient delivery promote HAB development;
• (5)Recently developed tools and techniques are already improving the detection of some HABs, and emerging technologies are rapidly advancing toward operational status for the prediction of HABs and their toxins;
• (6)Experimental studies are critical to further the understanding about the role of nutrients in HABs expression, and will strengthen prediction and mitigation of HABs; and
• (7)Management of nutrient inputs to the watershed can lead to significant reduction in HABs.

Supporting evidence and pertinent examples for each consensus statement are provided herein.     

Growth,death, and photobiology of dinoflagellates (Dinophyceae) under bacterial-algicide control

Naturally occurring allelopathic compounds, specific to some phytoplankton, may be a good source of bio-control agents against microalgae responsible for harmful algal blooms (HABs). Global expansion of HABs has invigorated research into different approaches to control these algae, including the search for naturally derived algicidal compounds. Here, we investigated the effects of a filtrate from the algicidal marine bacterium Shewanella sp. IRI-160 on photochemical function of four cultured dinoflagellates, Karlodinium veneficum, Gyrodinium instriatum, Prorocentrum minimum, and Alexandrium tamarense. The filtrate (designated IRI-160AA) contains bioactive compound(s), which were recently shown to inhibit growth of several dinoflagellate species. Results of this study show that all dinoflagellates but P. minimum exhibited photosystem II (PSII) inhibition, loss of photosynthetic electron transport, and varying degrees of cellular mortality. Exposure assays over 24 h showed that PSII inhibition and loss of cell membrane integrity occurred simultaneously in G. instriatum, but not in K. veneficum, where PSII activity declined prior to losing outer-membrane integrity. In addition, PSII inhibition and population growth inhibition were dose-dependent in K. veneficum, with an average EC-50 of 7.9 % (v/v) IRI-160AA. Application of IRI-160AA induced significantly higher PSII inhibition and cell mortality in K. veneficum subjected to continuous darkness as compared to cells maintained with 12:12 h light/dark cycles, while no such dark effect was noted for G. instriatum. The marked differences in the rate and impact of this algicide suggest that multiple cellular targets and different cascades of cellular dysfunction occur across these dinoflagellates.     

Detection and quantification of cultured marine Alexandrium species by real-time PCR

The occurrence of harmful algal blooms (HABs) throughout the world has increased and poses a large threat to human health, fishery resources and tourism industries. The genus Alexandrium includes a number of toxic species associated with HABs. Therefore, it is very important to rapidly detect and monitor the harmful algae, such as Alexandrium genus. In this study, a standard curve of plasmid containing 18S rDNA-28S rDNA region from Alexandrium catenella was constructed and 5.8S rDNA sequence served as the primer of the real-time PCR. Cultured A. catenella, Alexandrium affine, Alexandrium lusitanicum and Alexandrium minutum samples were analyzed by real-time PCR using the same set of primers simultaneously. Using microscopy cells counts, 5.8S rDNA copies per cell and total DNA per cell were estimated. This assay method is promising for rapid detection of large number of Alexandrium samples.     

Identification of unique microbiomes associated with harmful algal blooms caused by Alexandrium fundyense and Dinophysis acuminata

Biotic interactions dominate plankton communities, yet the microbial consortia associated with harmful algal blooms (HABs) have not been well-described. Here, high-throughput amplicon sequencing of ribosomal genes was used to quantify the dynamics of bacterial (16S) and phytoplankton assemblages (18S) associated with blooms and cultures of two harmful algae, Alexandrium fundyense and Dinophysis acuminata. Experiments were performed to assess changes in natural bacterial and phytoplankton communities in response to the filtrate from cultures of these two harmful algae. Analysis of prokaryotic sequences from ecosystems, experiments, and cultures revealed statistically unique bacterial associations with each HAB. The dinoflagellate, Alexandrium, was strongly associated with multiple genera of Flavobacteria including Owenweeksia spp., Maribacter spp., and individuals within the NS5 marine group. While Flavobacteria also dominated Dinophysis-associated communities, the relative abundance of Alteromonadales bacteria strongly co-varied with Dinophysis abundances during blooms and Ulvibacter spp. (Flavobacteriales) and Arenicella spp. (Gammaproteobacteria) were associated with cells in culture. Eukaryotic sequencing facilitated the discovery of the endosymbiotic, parasitic dinoflagellate, Amoebophrya spp., that had not been regionally described but represented up to 17% of sequences during Alexandrium blooms. The presence of Alexandrium in field samples and in experiments significantly altered the relative abundances of bacterial and phytoplankton by both suppressing and promoting different taxa, while this effect was weaker in Dinophysis. Experiments specifically revealed a negative feedback loop during blooms whereby Alexandrium filtrate promoted the abundance of the parasite, Amoebophrya spp. Collectively, this study demonstrates that HABs formed by Alexandrium and Dinophysis harbor unique prokaryotic and eukaryotic microbiomes that are likely to, in turn, influence the dynamics of these HABs.     

Evidence for Production of Sexual Resting Cysts by the Toxic Dinoflagellate Karenia mikimotoi in Clonal Cultures and Marine Sediments

The toxic dinoflagellate Karenia mikimotoi has been well-known for causing large-scale and dense harmful algal blooms (HABs) in coastal waters worldwide and serious economic loss in aquaculture and fisheries and other adverse effects on marine ecosystems. Whether K. mikimotoi forms resting cysts has been a puzzling issue regarding to the mechanisms of bloom initiation and geographic expansion of this species. We provide morphological and molecular confirmation of sexually produced thin-walled resting cysts by K. mikimotoi based on observations of laboratory cultures and their direct detection in marine sediments. Light and scanning electron microscopy evidences for sexual reproduction include attraction and pairing of gametes, gamete fusion, formation of planozygote and thin-walled cyst, and the documentation of the thin-walled cyst germination processes. Evidence for cysts in marine sediments was in three aspects: positive PCR detection of cysts using species-specific primers in the DNA extracted from whole sediments; fluorescence in situ hybridization detection of cysts using FISH probes; and single-cell PCR sequencing for cysts positively labeled with FISH probes. The existence of sexually produced, thin-walled resting cysts by K. mikimotoi provides a possible mechanism accounting for the initiation of annually recurring blooms at certain regions and global expansion of the species during the past decades.     

Satellite discrimination of Karenia mikimotoi and Phaeocystis harmful algal blooms in European coastal waters: Merged classification of ocean colour data

The detection of dense harmful algal blooms (HABs) by satellite remote sensing is usually based on analysis of chlorophyll-a as a proxy. However, this approach does not provide information about the potential harm of bloom, nor can it identify the dominant species. The developed HAB risk classification method employs a fully automatic data-driven approach to identify key characteristics of water leaving radiances and derived quantities, and to classify pixels into “harmful”, “non-harmful” and “no bloom” categories using Linear Discriminant Analysis (LDA). Discrimination accuracy is increased through the use of spectral ratios of water leaving radiances, absorption and backscattering. To reduce the false alarm rate the data that cannot be reliably classified are automatically labelled as “unknown”. This method can be trained on different HAB species or extended to new sensors and then applied to generate independent HAB risk maps; these can be fused with other sensors to fill gaps or improve spatial or temporal resolution. The HAB discrimination technique has obtained accurate results on MODIS and MERIS data, correctly identifying 89% of Phaeocystis globosa HABs in the southern North Sea and 88% of Karenia mikimotoi blooms in the Western English Channel. A linear transformation of the ocean colour discriminants is used to estimate harmful cell counts, demonstrating greater accuracy than if based on chlorophyll-a; this will facilitate its integration into a HAB early warning system operating in the southern North Sea.     

Inhibitory effects of Chinese traditional herbs and herb-modified clays on the growth of harmful algae,Phaeocystis globosa and Prorocentrum donghaiense

In order to develop an improved method of mitigating harmful algal blooms (HABs), we assessed the inhibitory effects of five Chinese traditional herbs, Andrographis paniculata (Burm.f.) Nees, Galla chinensis, Punica granatum L., Cortex phellodendri chinensis and Radix scutellariae, as well as clay modified with herb extract on the growth of two harmful algae, Phaeocystis globosa and Prorocentrum donghaiense. The results showed that the five Chinese herbs had varying effects on the target microalgae and inhibitory rates ranged from −35% to 100% at concentration of 0.3 g dry wt./L during 96 h treatments. Among the five herbs, G. chinensis exhibited the strongest inhibitory effect, almost 100% on P. globosa and P. donghaiense. The growth of P. globosa and P. donghaiense was completely inhibited by G. chinensis extracts (0.3 g dry wt./L) during all growth phases, and lag phase cultures were more sensitive than exponential phase and stationary phase cultures. The highest inhibitory rate (100% inhibition) on P. globosa was observed at lag phase, followed by exponential phase (73.1% inhibition), and stationary phase (57% inhibition). The highest inhibitory rate of 100% on P. donghaiense was also found at lag phase, 40.3% at exponential phase, and 23.4% at stationary phase. Furthermore, modified clay with G. chinensis extract significantly enhanced the inhibitory impact. Modified clay (0.3 g L⁻¹) produced 95% of growth inhibition for both algae species at 24 h, and maintained the inhibition thereafter. Our study demonstrated that G. chinensis and clays modified with its extract significantly inhibited the growth of harmful species, therefore may provide ideas and another option for control of harmful algal blooms.     

The central role of selenium in the biochemistry and ecology of the harmful pelagophyte,Aureococcus anophagefferens

The trace element selenium (Se) is required for the biosynthesis of selenocysteine (Sec), the 21st amino acid in the genetic code, but its role in the ecology of harmful algal blooms (HABs) is unknown. Here, we examined the role of Se in the biology and ecology of the harmful pelagophyte, Aureococcus anophagefferens, through cell culture, genomic analyses, and ecosystem studies. This organism has the largest and the most diverse selenoproteome identified to date that consists of at least 59 selenoproteins, including known eukaryotic selenoproteins, selenoproteins previously only detected in bacteria, and novel selenoproteins. The A. anophagefferens selenoproteome was dominated by the thioredoxin fold proteins and oxidoreductase functions were assigned to the majority of detected selenoproteins. Insertion of Sec in these proteins was supported by a unique Sec insertion sequence. Se was required for the growth of A. anophagefferens as cultures grew maximally at nanomolar Se concentrations. In a coastal ecosystem, dissolved Se concentrations were elevated before and after A. anophagefferens blooms, but were reduced by >95% during the peak of blooms to 0.05 nℳ. Consistent with this pattern, enrichment of seawater with selenite before and after a bloom did not affect the growth of A. anophagefferens, but enrichment during the peak of the bloom significantly increased population growth rates. These findings demonstrate that Se inventories, which can be anthropogenically enriched, can support proliferation of HABs, such as A. anophagefferens through its synthesis of a large arsenal of Se-dependent oxidoreductases that fine-tune cellular redox homeostasis.     

Spatial, seasonal and species variations of harmful algal blooms in the South Yellow Sea and East China Sea

The occurrences of harmful algal blooms (HABs), in terms of frequency and area in the Chinese coastal waters, have been increasing since 1980s and caused considerable economic losses. In the present study, we have analyzed spatial and seasonal characteristics of HAB events in the southern Yellow Sea and East China Sea along Chinese coast from 1933 to 2004. With a total 435 HAB records, the most frequent HAB occurrence area (FHA) is off the Yangtze River mouth and another two FHA areas are located south of the Yangtze River estuary along about isobaths of 30–60 m coastal water in the East China Sea. The time of HAB occurrence shifted during our study period: from autumn (August–October) before 1980s to July–August in 1980s, during May–July in 1990s, and May–June for the period of 2000–2004. Causative species were found to be different: Noctiluca scintillans and Skeletonema costatum were dominant causative species prior to 2000; and Prorocentrum donghaiense Lu was dominant from 2000 to 2004 and also caused large blooms in May. Trichodesmium sp. caused many HABs in autumn (August–October) prior to 1980s with only one HAB between 1980 and 2004. The changes of the dominant HAB species may have affected the timings of HAB occurrence, as well as the increasing HAB-affected areas in recent years.     

Intraspecific facilitation by allelochemical mediated grazing protection within a toxigenic dinoflagellate population

Dinoflagellates are a major cause of harmful algal blooms (HABs), with consequences for coastal marine ecosystem functioning and services. Alexandrium fundyense (previously Alexandrium tamarense) is one of the most abundant and widespread toxigenic species in the temperate Northern and Southern Hemisphere and produces paralytic shellfish poisoning toxins as well as lytic allelochemical substances. These bioactive compounds may support the success of A. fundyense and its ability to form blooms. Here we investigate the impact of grazing on monoclonal and mixed set-ups of highly (Alex2) and moderately (Alex4) allelochemically active A. fundyense strains and a non-allelochemically active conspecific (Alex5) by the heterotrophic dinoflagellate Polykrikos kofoidii. While Alex4 and particularly Alex5 were strongly grazed by P. kofoidii when offered alone, both strains grew well in the mixed assemblages (Alex4 + Alex5 and Alex2 + Alex5). Hence, the allelochemical active strains facilitated growth of the non-active strain by protecting the population as a whole against grazing. Based on our results, we argue that facilitation among clonal lineages within a species may partly explain the high genotypic and phenotypic diversity of Alexandrium populations. Populations of Alexandrium may comprise multiple cooperative traits that act in concert with intraspecific facilitation, and hence promote the success of this notorious HAB species.     

Tracking losses of brevetoxins on exposure to phytoplankton competitors: Ecological impacts

The frequent occurrence of devastating blooms of the harmful dinoflagellate Karenia brevis in the Gulf of Mexico has motivated research into bloom dynamics and potential mitigation strategies. The use of competing phytoplankton to lower waterborne concentrations of the most abundant and toxic brevetoxins produced during these blooms has been proposed. However the ecological impacts of using such biocontrol agents have not been addressed. This study investigated the impact on marine invertebrates of lowered brevetoxin concentrations due to the presence of competing phytoplankton. Even at low brevetoxin concentrations, the presence of the common diatom Skeletonema grethae ameliorated harmful toxic effects of brevetoxins upon the brine shrimp, Artemia salina, and reduced the incidence of negative physiological and morphological responses of the sea anemone Aiptasia pallida. In addition, brevetoxin biotransformation products formed by competing phytoplankton appear to be non-toxic or do not trigger the same physiological responses as brevetoxins in the model organisms used. These findings may impact the interpretation of ecotoxicological data gathered during bloom events, since the presence of phytoplankton competitors in Karenia blooms is likely to reduce the harmful effects seen on many marine invertebrates.     

Ecological meta-analysis of bloom-forming planktonic Cyanobacteria in Argentina

The aim of the present research was to summarize the main reasons that explain the distribution of harmful blooms of cyanobacteria in Argentina. It is a large territory with climates ranging from humid tropical to cold temperate. We performed a meta-analysis of the published data and information in technical reports published from 1945 to 2015, and included additional data from personal non-published studies. A total of 122 water bodies affected by planktonic cyanobacterial blooms were recorded and geo-referenced. The analysis showed that blooms, defined as events exceeding 5000 cells/mL, occurred in different types of water bodies, including shallow lakes, rivers, streams, reservoirs, estuaries and storage facilities. Maximum bloom abundance and species and ecological strategies (dispersive, scum-forming, nitrogen fixer) responsible for each event were related to the geographic and climatologic characteristics and type and origin of water bodies. The Puna and the Andean Patagonia eco-regions were mostly free of blooms. The most impaired aquatic systems were shallow lakes and reservoirs (46.7 and 24.6%, respectively). Deep lakes had no reports of blooms and rivers were mainly affected at the regulated reaches, with intensities generally decreasing downstream the dams. Besides, 74.3% of the blooms reported in Argentina exceeded WHO Alert Level 2 for drinking and bathing waters (100,000 cells/mL). Thirty-nine species, identified by Komárek’s polyphasic approach to taxonomy, were responsible for the blooms. Microcystis aeruginosa, Dolichospermum spiroides, Dolichospermum circinale, Raphidiopsis mediterranea and Cylindrospermopsis raciborskii were frequently found participating in either mixed or single species blooms. The species distribution was associated with the eco-region and aquatic system typologies and affected by seasonality and climatological and geographic variables. The eco-strategies of cyanobacterial species showed stronger associations with the qualitative and quantitative indicators used in the meta-analysis, and appeared as useful tools for management measures.     

In vitro interactions between several species of harmful algae and haemocytes of bivalve molluscs

Harmful algal blooms (HABs) can have both lethal and sublethal impacts on shellfish. To understand the possible roles of haemocytes in bivalve immune responses to HABs and how the algae are affected by these cells (haemocytes), in vitro tests between cultured harmful algal species and haemocytes of the northern quahog (= hard clam) Mercenaria mercenaria, the soft-shell clam Mya arenaria, the eastern and Pacific oysters Crassostrea virginica and Crassostrea gigas and the Manila clam Ruditapes philippinarum were carried out. Within their respective ranges of distribution, these shellfish species can experience blooms of several HAB species, including Prorocentrum minimum, Heterosigma akashiwo, Alexandrium fundyense, Alexandrium minutum and Karenia spp.; thus, these algal species were chosen for testing. Possible differences in haemocyte variables attributable to harmful algae and also effects of haemolymph and haemocytes on the algae themselves were measured. Using microscopic and flow cytometric observations, changes were measured in haemocytes, including cell morphology, mortality, phagocytosis, adhesion and reactive oxygen species (ROS) production, as well as changes in the physiology and the characteristics of the algal cells, including mortality, size, internal complexity and chlorophyll fluorescence. These experiments suggest different effects of the several species of harmful algae upon bivalve haemocytes. Some harmful algae act as immunostimulants, whereas others are immunosuppressive. P. minimum appears to activate haemocytes, but the other harmful algal species tested seem to cause a suppression of immune functions, generally consisting of decreases in phagocytosis, production of ROS and cell adhesion and besides cause an increase in the percentage of dead haemocytes, which could be attributable to the action of chemical toxins. Microalgal cells exposed to shellfish haemolymph generally showed evidence of algal degradation, e.g. loss of chlorophyll fluorescence and modification of cell shape. Thus, in vitro tests allow a better understanding of the role of the haemocytes and the haemolymph in the defence mechanisms protecting molluscan shellfish from harmful algal cells and could also be further developed to estimate the effects of HABs on bivalve molluscs in vivo.