On the control of HAB species using low biosurfactant concentrations

Biosurfactants have been suggested as a method to control harmful algal blooms (HABs), but warrant further and more in-depth investigation. Here we have investigated the algicidal effect of a biosurfactant produced by the bacterium Pseudomonas aeruginosa on five diverse marine and freshwater HAB species that have not been tested previously. These include Alexandrium minutum (Dinophycaee), Karenia brevis (Dinophyceae), Pseudonitzschia sp. (Bacillariophyceae), in marine ecosystems, and Gonyostomum semen (Raphidophyceae) and Microcystis aeruginosa (Cyanophyecae) in freshwater. We examined not only lethal but also sub-lethal effects of the biosurfactant. In addition, the effect of the biosurfactant on Daphnia was tested. Our conclusions were that very low biosurfactant concentrations (5 μg mL⁻¹) decreased both the photosynthesis efficiency and the cell viability and that higher concentrations (50 μg mL⁻¹) had lethal effects in four of the five HAB species tested. The low concentrations employed in this study and the diversity of HAB genera tested suggest that biosurfactants may be used to either control initial algal blooms without causing negative side effect to the ecosystem, or to provoke lethal effects when necessary.     

Recurrent vernal presence of the toxic Alexandrium tamarense/Alexandrium fundyense (Dinoflagellata) species complex in Narragansett Bay,USA

The vernal occurrence of toxic dinoflagellates in the Alexandrium tamarense/Alexandrium fundyense species complex in an enclosed embayment of Narragansett Bay (Wickford Cove, Rhode Island) was documented during 2005 and 2009–2012. This is the first report of regular appearance of the Alexandrium fundyense/Alexandrium tamarense species complex in Narragansett Bay. Thecal plate analysis of clonal isolates using SEM revealed cells morphologically consistent with both Alexandrium tamarense Lebour (Balech) and Alexandrium fundyense Balech. Additionally, molecular analyses confirmed that the partial sequences for 18S through the D1–D2 region of 28S were consistent with the identity of the two Alexandrium species. Toxin analyses revealed the presence of a suite of toxins (C1/2, B1 (GTX-5), STX, GTX-2/3. Neo, and GTX-1/4) in both Alexandrium tamarense (6.31 fmol cell⁻¹ STX equiv.) and Alexandrium fundyense (9.56 fmol cell⁻¹ STX equiv.) isolated from Wickford Cove; the toxicity of a Narragansett Bay Alexandrium peruvianum isolate (1.79 fmol cell⁻¹ STX equiv.) was also determined. Combined Alexandrium tamarense/Alexandrium fundyense abundance in Wickford Cove reached a peak abundance of 1280 cells L⁻¹ (May of 2010), with the combined abundance routinely exceeding levels leading to shellfishing closures in other systems. The toxic Alexandrium tamarense/Alexandrium fundyense species complex appears to be a regular component of the lower Narragansett Bay phytoplankton community, either newly emergent or previously overlooked by extant monitoring programs.     

Direct relationship between osmotic and ionic conforming behavior and tissue water regulatory capacity in echinoids

Echinoderms are considered marine osmoconforming invertebrates. However, many are intertidal or live next to estuaries, tolerating salinity changes and showing extracellular gradients to dilute seawater. Three species of echinoids – Lytechinus variegatus, which can occur next to estuarine areas, the rocky intertidal Echinometra lucunter, and the mostly subtidal Arbacia lixula – were submitted to a protocol of stepwise (rate of 2–3 psu/h) dilution, down to 15 psu, or concentration, up to 45 psu, of control seawater (35 psu). Coelomic fluid samples were obtained every hour. The seawater dilution experiment lasted 8 h, while the seawater concentration experiment lasted 6 h. Significant gradients (40–90% above value in 15 psu seawater) for osmolality, sodium, magnesium, and potassium were shown by L. variegatus and E. lucunter. A. lixula showed the smallest gradients, displaying the strongest conforming behavior. The esophagus of the three species was challenged in vitro with 20 and 50% osmotic shocks (hypo- and hyperosmotic). A. lixula, the most “conforming” species, showed the highest capacity to avoid swelling of its tissues upon the ? 50% hyposmotic shock, and was also the species less affected by salinity changes concerning the observation of spines and ambulacral feet movement in the whole-animal experiments. Thus, the most conforming species (A. lixula) displayed the highest capacity to regulate tissue water/volume, and was also the most euryhaline among the three studied species. In addition, tissues from all three species swelled much more than they shrank under osmotic shocks of same magnitude. This distinct trend to gain water, despite the capacity to hold some gradients upon seawater dilution, helps to explain why echinoderms cannot be fully estuarine, or ever enter fresh water.     

Novel algicidal evidence of a bacterium Bacillus sp. LP-10 killing Phaeocystis globosa,a harmful algal bloom causing species

While searching for effective bio-agents to control harmful algal blooms (HABs), the bacterial strain LP-10, which has strong algicidal activity against Phaeocystis globosa (Prymnesiophyceae), was isolated from surface seawater samples taken from the East China Sea. 16S rDNA sequence analysis and morphological characteristics revealed the strain LP-10 belonged to the genus Bacillus. The lytic effect of Bacillus sp. LP-10 against P. globosa was both concentration- and time-dependent. Algicidal activities of different growth stages of the bacterial culture varied significantly. The lytic effect of different parts of the bacterial cultures indicated that the algal cells were lysed by algicidal active compounds in the cell-free filtrate. Analysis of the properties of the active compounds showed that they had a molecular weight of less than 1000 Da and that the active compounds were stable between −80 and 121 °C. The algicidal range assay indicated that five other algal species were also suppressed by strain LP-10, including: Alexandrium catenella, A. tamarense, A. minutum, Prorocentrum micans and Asterionella japonica. Our results suggested that the algicidal bacterium Bacillus sp. LP-10 could be a potential bio-agent to control the blooms of harmful algal species.     

Combined physical,chemical and biological factors shape Alexandrium ostenfeldii blooms in The Netherlands

Harmful algal blooms (HABs) are globally expanding, compromising water quality worldwide. HAB dynamics are determined by a complex interplay of abiotic and biotic factors, and their emergence has often been linked to eutrophication, and more recently to climate change. The dinoflagellate Alexandrium is one of the most widespread HAB genera and its success is based on key functional traits like allelopathy, mixotrophy, cyst formation and nutrient retrieval migrations. Since 2012, dense Alexandrium ostenfeldii blooms (up to 4500 cells mL⁻¹) have recurred annually in a creek located in the southwest of the Netherlands, an area characterized by intense agriculture and aquaculture. We investigated how physical, chemical and biological factors influenced A. ostenfeldii bloom dynamics over three consecutive years (2013–2015). Overall, we found a decrease in the magnitude of the bloom over the years that could largely be linked to changing weather conditions during summer. More specifically, low salinities due to excessive rainfall and increased wind speed corresponded to a delayed A. ostenfeldii bloom with reduced population densities in 2015. Within each year, highest population densities generally corresponded to high temperatures, low DIN:DIP ratios and low grazer densities. Together, our results demonstrate an important role of nutrient availability, absence of grazing, and particularly of the physical environment on the magnitude and duration of A. ostenfeldii blooms. Our results suggest that predicted changes in the physical environment may enhance bloom development in future coastal waters and embayments.     

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 Pseudo-nitzschia 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.     

Killing potential protist predators as a survival strategy of the newly described dinoflagellate Alexandrium pohangense

Blooms caused by some species belonging to the dinoflagellate genus Alexandrium are known to cause large-scale mortality of fish. Thus, the dynamics of these species is important and of concern to scientists, officials, and people in the aquaculture industry. To understand the dynamics of such species, their growth and mortality due to predation need to be assessed. The newly described dinoflagellate Alexandrium pohangense is known to grow slowly, with a maximum autotrophic growth rate of 0.1 d⁻¹. Thus, it may not form bloom patches if its mortality due to predation is high. Therefore, to explore the mortality of A. pohangense due to predation, feeding on this species by the common heterotrophic dinoflagellates Gyrodinium dominans, Gyrodinium moestrupii, Luciella masanensis, Noctiluca scintillans, Oxyrrhis marina, Oblea rotunda, Polykrikos kofoidii, and Pfiesteria piscicida, as well as by the ciliate Tiarina fusus, was examined. None of these potential predators was able to feed on A. pohangense. In contrast, these potential predators were killed and their bodies were dissolved when incubated with A. pohangense cells or cell-free culture filtrates. The survival of G. moestrupii, O. marina, P. kofoidii, and T. fusus on incubation with 10 cells ml⁻¹ of A. pohangense was 20–60%, while that at the equivalent culture filtrates was 20–70%. With increasing A. pohangense cell-concentration (up to 1000 cells ml⁻¹ or equivalent culture filtrates), the survival rate of G. moestrupii, O. marina, P. kofoidii, and T. fusus rapidly decreased. The lethal concentration (LC₅₀) for G. moestrupii, O. marina, P. kofoidii, and T. fusus at the elapsed time of 24 h with A. pohangense cells (cultures of 11.4, 13.3, 1.6, and 3.3 cells ml⁻¹, respectively) was lower than that with A. pohangense filtrates (culture filtrates of 35.5, 30.6, 5.5, and 5.0 cells ml⁻¹, respectively). Furthermore, most of the ciliates and heterotrophic dinoflagellates in the water collected from the coast of Tongyoung, Korea, were killed when incubated with cultures of 1000 A. pohangense cells ml⁻¹ and equivalent culture filtrates. The relatively slow growing A. pohangense may form blooms by reducing mortality due to predation through killing potential protist predators.     

DNA barcoding species in Alexandrium tamarense complex using ITS and proposing designation of five species

Alexandrium species can be very difficult to identify, with A. catenella, A. tamarense, and A. fundyense that compose “Alexandrium tamarense species complex” (Atama complex) as a distinct example. DNA barcoding is promising to offer a solution but remains to be established. In this study, we examined the utility of ITS in resolving the Atama species complex, by analyzing previously studied strains plus unstudied Chinese strains within the LSU- and SSU-rDNA based group/clade frameworks recently established. We further investigated the presence of intragenomic polymorphism and its implications in species delimitation. Similar to the previous SSU and LSU results, our ITS-based phylogenies divided the complex to five clusters, but with longer and evener branch lengths between the clusters. Based on the ITS region, the inter-cluster genetic distances (p = 0.134–0.216) were consistently and substantially greater than intra-cluster genetic distances (p = 0.000–0.066), with an average inter-cluster (species) distance (p = 0.167) 7.6-fold of the average intraspecific difference (p = 0.022), qualifying the approximately 510–520 bp ITS as a DNA barcode for Atama complex. We detected varying levels of intragenomic polymorphism in ITS but found that this did not impact the taxon-resolving power of this gene. With this DNA barcode, the new East and South China Sea strains and one Antarctic strain were placed in Clade IIC/Group IV, even though there were 7–10 polymorphic sites in their ITS, in contrast to none in SSU. Furthermore, our results suggest that the five clusters are recognizable as distinct species according to the phylogenetic species concept. Based on the phylogenetic placements of the type-locality strains of the existing three morphospecies and the dominant localities of other strains, we propose that Group I/Clade I be designated as A. fundyense, Group III/Clade IIB as A. tamarense, Group IV/Clade IIC as A. catenella, Group II/Clade IIA as A. mediterranis, and Group V/Clade IID as A. australis.     

Long-term decline of brown algal assemblages from southern Brazil under the influence of a nuclear power plant

We investigated long-term trends in brown macroalgal assemblages inhabiting shallow subtidal rocky bottoms under the influence of thermal effluent discharge from the Brazilian nuclear power plant (BNPP). Three operational periods based on the units of the BNPP were analysed: T0 = pre-operational, between the years 1980 and 1983; T1 = operational period of unit 1, between 1988 and 1999; and T2 = operational period of units 1 and 2, between 2005 and 2012. Using generalized linear mixed models (GLMMs), we found significant declines in the numbers of genera and species over time. More than half of the species of brown macroalgae disappeared during T2. In addition, the numbers of macroalgal genera and species were inversely related to the local surface seawater temperatures. Multivariate analyses revealed a clear separation between the years of T2 and those of T0, indicating long-term changes in the community assemblages. Among the most common species in the area, the frequencies of Canistrocarpus cervicornis, Dictyopteris delicatula, Hincksia mitchelliae, Sargassum spp. and Sphacelaria tribuloides decreased during T2, while Padina gymnospora maintained rather high yearly frequencies during T2 (>40%). Our data suggest that seawater temperatures consistently higher than 30 °C together with peaks higher than 35 °C may have triggered the decline in the brown algae on rocky bottoms under the influence of the BNPP discharge. These results from southern Brazil are consistent with studies from other locations that ascribe changes in seaweed diversity to increasing seawater temperatures, highlighting the sensitivity of brown macroalgae to thermal stress and demonstrating their effectiveness as an ecological indicator for monitoring the effects of nuclear power plant effluents on coastal environments.     

Resting cysts,and effects of temperature and salinity on the growth of vegetative cells of the potentially harmful species Alexandrium insuetum Balech (Dinophyceae)

The potentially harmful species Alexandrium insuetum established by the incubation of resting cysts isolated from sediment trap samples collected at Jinhae-Masan Bay, Korea was characterized by morphological and phylogenetic analysis. The effects of temperature and salinity on the growth of A. insuetum were also investigated. The resting cysts are characterized by a spherical shape, a small size (20–25 μm) and the presence of either three or four red accumulation bodies. The similarity of morphological features of the resting cysts to those of other species of the minutum group (consisting of Alexandrium minutum and A. tamutum) indicates that the morphological features of resting cysts might improve the accuracy of the grouping of Alexandrium species. A. insuetum germinated from the resting cysts is morphologically consistent with vegetative cells reported from Korean and Japanese coastal areas, and has an partial large subunit (LSU) rDNA sequence identical to that from Japanese strains. The growth of A. insuetum was observed between salinity 20 and 35, with increasing temperature; however at 25 °C, A. insuetum could grow even at the salinity of 15. The highest growth rate (0.60 d⁻¹) was observed at 25 °C and the salinity of 25, which is higher than the previously reported growth rate of A. tamarense, which is responsible for outbreaks of paralytic shellfish poisoining and blooms in Jinhae-Masan Bay. These results suggest that the proliferation of A. insuetum in Jinhae-Masan Bay is likely to be highest during the summer.     

Toxic strains of the Alexandrium ostenfeldii complex in southern South America (Beagle Channel,Argentina)

During phytoplankton monitoring in the Beagle Channel (≈54°52′ S, 67°32′ W) a previously undetected Alexandrium species was observed in coincidence with mouse bioassay toxicity. Detailed thecal plates analysis using epifluorescence and scanning electron microscopy revealed the presence of the Alexandrium ostenfeldii species complex, showing a mixture of the diagnostic features usually used to discriminate between the morphospecies A. ostenfeldii and A. peruvianum. Cells of the A. ostenfeldii complex were commonly observed during spring after the main annual diatom bloom, when temperatures and salinities were respectively around 7.5–10 °C and 30–30.5 psu, and nutrients showed a seasonal decrease. Toxin analysis by liquid chromatography–mass spectrometry revealed the production of 13-desmethyl spirolide C and 20-methyl spirolide G in cell cultures. The cellular contain of spirolides during exponential phase growth was 0.5906 ± 0.0032 and 0.1577 ± 0.0023 pg cell⁻¹ for 13-desMe-C and 20-Me-G, respectively. A third unknown compound, with a structure resembling that of spirolides was also detected in culture. Moreover, an additional compound with a similar m/z (692) than that of 13-desMe-C but presenting a higher retention time (Rt = 40.5 min) was found in high proportions in mussel samples. PSP toxins were present at low concentration in mussels but were not detected in cultures. These results extend the world-wide distribution of toxic strains of the A. ostenfeldii complex to the Beagle Channel (southern South America), where toxic events have been traditionally linked to the presence of Alexandrium catenella. This is the first confirmed occurrence of spirolides in mussels and plankton from Argentina, which highlights the importance of monitoring these toxins and their producing organisms to protect public health and improve the management of shellfish resources.     

Determination of seed viability of eight wild Saudi Arabian species by germination and X-ray tests

Our purpose was to evaluate the usefulness of the germination vs. the X-ray test in determining the initial viability of seeds of eight wild species (Salvia spinosa, Salvia aegyptiaca, Ochradenus baccatus, Ochradenus arabicus, Suaeda aegyptiaca, Suaeda vermiculata, Prosopisfarcta and Panicumturgidum) from Saudi Arabia. Several days were required to determine viability of all eight species via germination tests, while immediate results on filled/viable seeds were obtained with the X-ray test. Seeds of all the species, except Sa.aegyptiaca, showed high viability in both the germination (98–70% at 25/15 °C, 93–66% at 35/25 °C) and X-ray (100–75%) test. Furthermore, there was general agreement between the germination (10% at 25/15 °C and 8% at 35/25 °C) and X-ray (5%) tests that seed viability of Sa.aegyptiaca was very low, and X-ray analysis revealed that this was due to poor embryo development. Seeds of P.farcta have physical dormancy, which was broken by scarification in concentrated sulfuric acid (10 min), and they exhibited high viability in both the germination (98% at 25/15 °C and 93% at 35/25 °C) and X-ray (98%) test. Most of the nongerminated seeds of the eight species except those of Sa.aegyptiaca were alive as judged by the tetrazolium test (TZ). Thus, for the eight species examined, the X-ray test was a good and rapid predictor of seed viability.     

Isolation and identification of gastrointestinal microbiota from the short-nosed fruit bat Cynopterus brachyotis brachyotis

Studies on the microbial ecology of gut microbiota in bats are limited and such information is necessary in determining the ecological significance of these hosts. Short-nosed fruit bats (Cynopterus brachyotis brachyotis) are good candidates for microbiota studies given their close association with humans in urban areas. Thus, this study explores the gut microbiota of this species from Peninsular Malaysia by means of biochemical tests and 16S rRNA gene sequences analysis. The estimation of viable bacteria present in the stomach and intestine of C. b. brachyotis ranged from 3.06 × 10¹⁰ to 1.36 × 10¹⁵ CFU/ml for stomach fluid and 1.92 × 10¹⁰ to 6.10 × 10¹⁵ CFU/ml for intestinal fluid. A total of 34 isolates from the stomach and intestine of seven C. b. brachyotis were retrieved. A total of 16 species of bacteria from eight genera (Bacillus, Enterobacter, Enterococcus, Escherichia, Klebsiella, Pantoea, Pseudomonas and Serratia) were identified, Enterobacteriaceae being the most prevalent, contributing 12 out of 16 species isolated. Most isolates from the Family Enterobacteriaceae have been reported as pathogens to humans and wildlife. With the possibility of human wildlife transmission, the findings of this study focus on the importance of bats as reservoirs of potential bacterial pathogens.     

Mixotrophy in the phototrophic dinoflagellate Takayama helix (family Kareniaceae): Predator of diverse toxic and harmful dinoflagellates

Takayama spp. are phototrophic dinoflagellates belonging to the family Kareniaceae and have caused fish kills in several countries. Understanding their trophic mode and interactions with co-occurring phytoplankton species are critical steps in comprehending their ecological roles in marine ecosystems, bloom dynamics, and dinoflagellate evolution. To investigate the trophic mode and interactions of Takayama spp., the ability of Takayama helix to feed on diverse algal species was examined, and the mechanisms of prey ingestion were determined. Furthermore, growth and ingestion rates of T. helix feeding on the dinoflagellates Alexandrium lusitanicum and Alexandrium tamarense, which are two optimal prey items, were determined as a function of prey concentration. T. helix ingested large dinoflagellates ≥15 μm in size, except for the dinoflagellates Karenia mikimotoi, Akashiwo sanguinea, and Prorocentrum micans (i.e., it fed on Alexandrium minutum, A. lusitanicum, A. tamarense, A. pacificum, A. insuetum, Cochlodinium polykrikoides, Coolia canariensis, Coolia malayensis, Gambierdiscus caribaeus, Gymnodinium aureolum, Gymnodinium catenatum, Gymnodinium instriatum, Heterocapsa triquetra, Lingulodinium polyedrum, and Scrippsiella trochoidea). All these edible prey items are dinoflagellates that have diverse eco-physiology such as toxic and non-toxic, single and chain forming, and planktonic and benthic forms. However, T. helix did not feed on small flagellates and dinoflagellates <13 μm in size (i.e., the prymnesiophyte Isochrysis galbana; the cryptophytes Teleaulax sp., Storeatula major, and Rhodomonas salina; the raphidophyte Heterosigma akashiwo; the dinoflagellates Heterocapsa rotundata, Amphidinium carterae, Prorocentrum minimum; or the small diatom Skeletonema costatum). T. helix ingested Heterocapsa triquetra by direct engulfment, but sucked materials from the rest of the edible prey species through the intercingular region of the sulcus. With increasing mean prey concentration, the specific growth rates of T. helix on A. lusitanicum and A. tamarense increased continuously before saturating at prey concentrations of 336–620 ng C mL⁻¹. The maximum specific growth rates (mixotrophic growth) of T. helix on A. lusitanicum and A. tamarense were 0.272 and 0.268 d⁻¹, respectively, at 20 °C under a 14:10 h light/dark cycle of 20 μE m⁻² s⁻¹ illumination, while its growth rates (phototrophic growth) under the same light conditions without added prey were 0.152 and 0.094 d⁻¹, respectively. The maximum ingestion rates of T. helix on A. lusitanicum and A. tamarense were 1.23 and 0.48 ng C predator⁻¹d⁻¹, respectively. The results of the present study suggest that T. helix is a mixotrophic dinoflagellate that is able to feed on a diverse range of toxic species and, thus, its mixotrophic ability should be considered when studying red tide dynamics, food webs, and dinoflagellate evolution.     

Harmful algal bloom (HAB) in the East Sea identified by the Geostationary Ocean Color Imager (GOCI)

Harmful Cochlodinium polykrikoides blooms have frequently appeared and caused fatal harm to aquaculture in Korean coastal waters since 1995. We investigated the applicability of GOCI, the world's first Geostationary Ocean Color Imager, in monitoring the distribution and temporal movement of a harmful algal bloom (HAB) that was discovered in the East Sea near the Korean peninsula in August 2013. We identified the existence of C. polykrikoides at a maximum cell abundance of over 6000 cells/mL and a chlorophyll a concentration of over 400 mg/m³. In areas of C. polykrikoides blooms, GOCI remote sensing reflectance (R_rs) spectra demonstrated the typical radiometric features of a HAB, and from the diurnal variations using GOCI-derived chlorophyll concentration images, we were able to identify the vertical migration of the red tide species. We also found that the formation and propagation of the HAB had relations with cold water mass in the coastal region. GOCI can be effectively applied to the monitoring of short-term and long-term movements of red tides.     

Physical processes leading to the development of an anomalously large Cochlodinium polykrikoides bloom in the East sea/Japan sea

An anomalously large Harmful Algal Bloom (HAB) was observed in the southwest coast of the East/Japan Sea (hereafter the East Sea) during the summer of 2013. During this time period, the presence of Cochlodinium polykrikoides (C. polykrikoides) was detected by the Geostationary Ocean Color Imager (GOCI) and validated by in-situ observations. GOCI observations have been available since 2011, thus allowingto examine various stages of the physical condition of the developing C. polykrikoides bloom, thereby other multi-satellite and buoy measurements obtained between 2011 and 2013. Research results indicate that this HAB is related to four processes: the transport of C. polykrikoides from the south coast of Korea to the HAB area; a relatively high insolation; continuous coastal upwelling; and a favorable Sea Surface Temperature (SST) for C.polykrikoide growth. In examination of the main transport mechanisms, geostrophic current measurements were used to estimate the flow trajectories, showing water from the south coast to the HAB area off the southeast coast of Korea. Result shows that ninety percent of the water from the south coast reached the HAB area in 2013. Furthermore, to examine the insolation mechanism, the Photosynthetically available radiation (PAR) value was derived from the Moderate Resolution Imaging Spectoradiometer (MODIS), showing that PAR values were relatively high in the HAB area during HAB period (47 Ein m⁻¹ day⁻¹). Moreover, Upwelling age (UA) was calculated in order to investigate the strength of coastal upwelling events, which were found to support relatively high UA values during the HAB period. The mean UA value during the HAB period was 1.01, higher than those in 2011 and 2012 which were 0.61 and 0.76, respectively. Finally, SST in the HAB area was also analyzed to examine which conditions were most favorable for HAB growth. Therefore, the results of this study suggest that the four mechanisms can explain the relative contributions of the anomalously HAB development observed off the southeast coast of Korea.     

Toxic mucus traps: A novel mechanism that mediates prey uptake in the mixotrophic dinoflagellate Alexandrium pseudogonyaulax

The functional role of harmful substances (i.e. toxins) produced by marine planktonic algae is still, in many cases, unknown. This study describes a novel mechanism by which the phototrophic dinoflagellate Alexandrium pseudogonyaulax secretes a toxic mucus trap where prey items are caught and immobilized prior to ingestion. Prey cells remain entrapped and immobile in the mucus trap, but most stay intact, readily available as whole-cell prey. It is shown that food uptake by A. pseudogonyaulax increases its growth rate considerably even in nutrient-replete, high-light conditions. The increase in growth rate was more enhanced in light-limited treatments and A. pseudogonyaulax grew significantly faster when fed Heterocapsa rotundata, than when fed Teleaulax acuta under both light conditions. For comparison, strains of Alexandrium catenella and Alexandrium minutum were studied for their mixotrophic capabilities. None of these strains were mixotrophic under the conditions provided. In addition, the toxic effects on various protistan targets of these Alexandrium strains as well as Alexandrium tamarense and Alexandrium ostenfeldii were compared to that of A. pseudogonyaulax. A. tamarense and A. catenella did immobilize and lyse target cells through substances leaked directly into the water, differing from all the strains of A. pseudogonyaulax studied. Results show that the toxic effect of A. pseudogonyaulax is non-specific causing nearly 100% immobilization of a variety of protistan targets at relatively low cell concentrations (500 cells ml⁻¹ of donor cell). A critical donor cell density was not required as only one A. pseudogonyaulax cell was able to cause immobilization of target cells. For the first time, the connection between excreted toxins and phagotrophy is evident in an Alexandrium species and this particular strategy has the potential to severely impact competing phytoplankton communities.     

Shear effects on suspended marine sponge cells

Fractions of viable cells, apoptotic and irreversibly damaged cells, dead whole cells and cell fragments were measured by flow cytometry during the production of freely suspended primary cells from explants of the marine sponge Axinella damicornis. The explants were disintegrated using the well-known Müller protocol [W.E.G. Müller, M. Wiens, R. Batel, R. Steffen, R. Borojevic, M.R. Custodio, Establishment of a primary cell culture from a sponge: primmorphs from Suberites domuncula, Mar. Ecol. Progr. Ser. 178 (1999) 205–219]. Supplementation of the standard Ca²⁺- and Mg²⁺-free artificial seawater of the Müller protocol, with the shear protectant Pluronic F68 (0.1%, w/v) greatly reduced the cell damage and enhanced the recovery of viable cells at each of the four stages of the protocol. Agitation of cells on an orbital shaker at 75 rpm essentially killed all the viable cells within 2.5 h, but no loss of viability occurred at a higher agitation speed of 100 rpm for up to 6 h when the cells were supplemented with Pluronic F68. This time-dependent loss in viability could be significantly reduced by processing at 3 °C instead of the normal 17 °C. A four-step mechanistic model was shown to describe the kinetics of cell death and fragmentation within ±10% of the measured values. The damage to cells was modeled as a web of first-order processes that did not depend on cell–cell interactions. The forces in the agitated fluid killed the viable cells by impact, which was not accompanied by cell rupture (i.e. the cell was left dead, but intact).     

Effects of nutrient-limiting supply ratios on toxin content of Karenia brevis grown in continuous culture

Toxins produced as secondary metabolites can play important roles in phytoplankton communities and contribute to the ecological success of harmful algal bloom (HAB) taxa. Toxin composition and content in phytoplankton are affected by a suite of environmental factors, including nutrient availability. Changes in nutrient availability can increase or decrease toxin content and alter toxin composition, depending on toxin stoichiometry and the mechanisms by which nutrient limitation affects toxin production. The studies that have assessed the effects of nutrient availability on brevetoxin content of the HAB species Karenia brevis have reported contradictory results, although there is growing support that nutrient limitation increases brevetoxin content. In this study, we assessed the effects of decreased nitrogen (N) and phosphorus (P) availability on brevetoxin content and composition of K. brevis grown in chemostats at steady state by altering the nutrient supply ratios of incoming media from the Redfield Ratio. Overall, brevetoxin content was greatest in cultures grown at the lowest rate, regardless of the nutrient supply ratio (i.e., under both Redfield and N-limiting supply ratios). Compared to cultures grown at 0.2 d⁻¹, cultures grown at 0.1 d⁻¹ exhibited 5-fold increases in intracellular toxin content. In contrast, at constant growth rates, N-limiting supply ratios decreased intracellular brevetoxin content by approximately one-third, although this result was significant only in cultures growing at the fastest rate of 0.23 d⁻¹. P-limiting supply ratios had no effect on brevetoxin content or composition. In addition, when cultures grown at rates of 0.2 d⁻¹ were supplied with balanced/Redfield N:P supply ratios, but different absolute nutrient concentrations, toxin content was greater under greater nutrient concentrations. These findings suggest that when growth rate is not nutrient limited, there is a positive relationship between nutrient availability and brevetoxin content. This work contributes to previous studies by demonstrating strong growth rates effects on brevetoxin content and that growth rate and nutrient availability can independently or together affect toxin content of K. brevis. Moreover, our work underscores the value of the chemostat as a tool to elucidate the mechanisms by which nutrient availability and growth rate affect toxin production and content of HAB species.