The association of algicidal bacteria and raphidophyte blooms in South Carolina brackish detention ponds

Over the past 5 years, raphidophyte blooms have been frequently observed along the South Carolina coastal zone. During the 2002, 2003, and 2004 sampling seasons, we investigated temporal fluctuations of algicidal bacteria abundance against raphidophycean flagellates (Heterosigma akashiwo, Chattonella subsalsa, and Fibrocapsa japonica) using the microplate most probable number (MPN) method in three Kiawah Island brackish stormwater detention ponds (K1, K2, and K75). Local axenic isolates of H. akashiwo, C. subsalsa, and F. japonica were obtained and their susceptibility to algicidal bacteria tested. A total of 195 algicidal bacterial strains were isolated from raphidophyte blooms in the study ponds, and 6 of them were identified at the genus level, and the taxonomic specificity of their algicidal activity was tested against local (pond) and nonlocal isolates of raphidophytes (3 species, 10 total strains). In the ponds, a consistent association was found between raphidophyte bloom development and an increase in bacteria algicidal to the bloom species. In 12 of 15 cases, bloom decline followed the increase in algicidal bacteria to maximum abundances. Although variability was found in the taxonomic specificity of the algicidal bacteria effect (i.e. the number of raphidophyte species affected by a particular bacteria strain) and raphidophyte susceptibility (i.e. the number bacteria strains affecting a particular raphidophyte species), a toxic effect was always found when strains of a raphidophyte species were exposed to algicidal bacteria isolated from a bloom caused by that same species. The results suggest that algicidal bacteria may be an important limiting factor in raphidophyte bloom sustenance and can promote bloom decline in brackish lagoonal eutrophic estuaries.     

ANTIOXIDANT ENZYME RESPONSE AND REACTIVE OXYGEN SPECIES PRODUCTION IN MARINE RAPHIDOPHYTES1

Raphidophytes (class Raphidophyceae) produce high levels of reactive oxygen species (ROS), yet little is known regarding cellular scavenging mechanisms needed for protection against these radicals. Enzymatic activities of the antioxidants superoxide dismutase (SOD) and catalase (CAT) were measured in conjunction with the production of superoxide (O₂^??) and hydrogen peroxide (H₂O₂) in batch cultures of five different raphidophytes species during early exponential, late‐exponential, and stationary growth phases. The greatest concentrations of O₂^?? per cell were detected during exponential growth with reduced levels in stationary phases in raphidophytes Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara, Chattonella marina (Subrahman.) Y. Hara et Chihara, and Chattonella antiqua (Hada) Ono (strain 18). Decreasing trends from exponential to stationary phases for SOD activity and H₂O₂ per cell were observed in all species tested. Significant correlations between O₂^?? per cell and SOD activity per cell over growth phase were only observed in three raphidophytes (Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua strain 18), likely due to different cellular locations of externally released O₂^?? radicals and intracellular SOD enzymes measured in this study. CAT activity was greatest at early exponential phase for several raphidophytes, but correlations between H₂O₂ per cell and CAT activity per cell were only observed for Fibrocapsa japonica Toriumi et Takano, Chattonella antiqua (strain 18), and Chattonella subsalsa Biecheler. Our results suggest that SOD and CAT play important protective roles against ROS during exponential growth of several raphidophytes, while other antioxidant pathways may play a larger role for scavenging ROS during later growth.     

RAPHIDOPHYCEAE [CHADEFAUD EX SILVA] SYSTEMATICS AND RAPID IDENTIFICATION: SEQUENCE ANALYSES AND REAL‐TIME PCR ASSAYS1

Species within the class Raphidophyceae were associated with fish kill events in Japanese, European, Canadian, and U.S. coastal waters. Fish mortality was attributable to gill damage with exposure to reactive oxygen species (peroxide, superoxide, and hydroxide radicals), neurotoxins, physical clogging, and hemolytic substances. Morphological identification of these organisms in environmental water samples is difficult, particularly when fixatives are used. Because of this difficulty and the continued global emergence of these species in coastal estuarine waters, we initiated the development and validation of a suite of real‐time polymerase chain reaction (PCR) assays. Sequencing was used to generate complete data sets for nuclear encoded small‐subunit ribosomal RNA (SSU rRNA; 18S); internal transcribed spacers 1 and 2, 5.8S; and plastid encoded SSU rRNA (16S) for confirmed raphidophyte cultures from various geographic locations. Sequences for several Chattonella species (C. antiqua, C. marina, C. ovata, C. subsalsa, and C. verruculosa), Heterosigma akashiwo, and Fibrocapsa japonica were generated and used to design rapid and specific PCR assays for several species including C. verruculosa Hara et Chihara, C. subsalsa Biecheler, the complex comprised of C. marina Hara et Chihara, C. antiqua Ono and C. ovata, H. akashiwo Ono, and F. japonica Toriumi et Takano using appropriate loci. With this comprehensive data set, we were also able to perform phylogenetic analyses to determine the relationship between these species.     

Assessment of Microzooplankton Grazing on <Emphasis Type="Italic">Heterosigma akashiwo</Emphasis> Using a Species- Specific Approach Combining Quantitative Real-Time PCR (QPCR) and Dilution Methods

Delaware’s Inland Bays (DIB) are subject to numerous mixed blooms of harmful raphidophytes each year, and Heterosigma akashiwo is one of the consistently occurring species. Often, Chattonella subsalsa, C. cf. verruculosa, and Fibrocapsa japonica co-occur with H. akashiwo, indicating a dynamic consortium of raphidophyte species. In this study, microzooplankton grazing pressure was assessed as a top–down control mechanism on H. akashiwo populations in mixed communities. Quantitative real-time polymerase chain reaction (QPCR) with species-specific primers and probes were used in conjunction with the dilution method to assess grazing pressure on H. akashiwo and other raphidophytes. As a comparison, we measured changes in chlorophyll a (chl a) to determine whole community growth and mortality caused by grazing. We detected grazing on H. akashiwo using QPCR in samples where chl a analyses indicated little or no grazing on the total phytoplankton community. Overall, specific microzooplankton grazing pressure on H. akashiwo ranged from 0.88 to 1.88 day⁻¹ at various sites. Experiments conducted on larger sympatric raphidophytes (C. subsalsa, C. cf. verruculosa and F. japonica) demonstrated no significant microzooplankton grazing on these species. Grazing pressure on H. akashiwo may provide a competitive advantage to other raphidophytes such as Chattonella spp. that are too large to be consumed at high rates by microzooplankton and help to shape the dynamics of this harmful algal bloom consortium. Our results show that QPCR can be used in conjunction with the dilution method for evaluation of microzooplankton grazing pressure on specific phytoplankton species within a mixed community. An erratum to this article can be found at     

Growth,pigment, and chromophoric dissolved organic matter responses of tropical Chattonella subsalsa (Raphidophyceae) to nitrogen enrichment

Blooms of the raphidophyte Chattonella subsalsa have been associated with massive fish‐kill events in several parts of the world. However, there have been few studies into physiological responses of tropical strains that could contribute to bloom outcomes. Such knowledge could provide insight into the C. subsalsa blooms recently documented within tropical coastal waters (e.g., 2010 and 2012 events in Singapore). Strains used in this study were isolated from the East Johor Straits (EJS), Singapore, an enclosed water channel frequently subjected to high levels of eutrophication. These cells were classified within the ‘global’ clade (and distinct from the ‘Adriatic Sea’ clade) based on morphology. The present study examined cellular responses to varying inputs of different forms of nitrogen (N), specifically nitrate, ammonium, and urea. Results from the study indicated that cells were unable to utilize urea as an N‐source, but grew well on a nitrate (V_max = 0.73 day^?1) and ammonium (V_max = 0.81 day^?1) supply. These growth rates were high compared to other strains from around the world, indicating that tropical C. subsalsa could exhibit elevated bloom potential within frequently eutrophic environments such as the EJS. Six pigments were detected in all cultures. These pigments were chlorophylls a and c; fucoxanthin; diadinoxanthin; violaxanthin; and β‐carotene. Chlorophyll‐a and fucoxanthin were the dominant pigments under both nitrate and ammonium regimes. Measurements of chromophoric dissolved organic matter generally increased both in molecular weight and in total content across the N‐concentration ranges. Such outcomes could have consequences for the chemical and optical conditions of the coastal environment.     

Chemotaxonomic survey of sterols and fatty acids in six marine raphidophyte algae

Fatty acid and sterol profiles allowed for clear discrimination betweentheraphidophyte genera Chattonella,Heterosigma, Fibrocapsa andOlisthodiscus, but exhibited little differentiation forindividual Chattonella species(C.marina, C. antiqua and C.subsalsa). Sterol and fatty acid profiles do not support theseparation of Chattonella antiqua and C.marina as distinct species. Ecophenotypic variations in lipidprofiles were also observed between Chattonella strainsfromdifferent geographic locations. Sterol signatures which may be useful aschemotaxonomic markers were: the absence of C₂₇ sterols (cholesteroland 24-dihydrozymosterol) in Heterosigma akashiwo; thepresence of isofucosterol in Chattonella; and theoccurrence of brassicasterol, poriferasterol and fucosterol inOlisthodiscus luteus. High levels of eicosapentaenoic acid(EPA; 17-27% of fatty acids) were present in all raphidophyte species. Lipidcomposition correlated more closely to recent molecular classification ofraphidophytes than carotenoid pigments.     

Issue Information

Bloom‐forming Raphidophyceae from the Brazilian coast. Chattonella subsalsa (left), C. antiqua (center), and Heterosigma akashiwo (right) were photographed at the same view. See Branco et al. in this issue. The link to the article here

     

DESCRIPTION OF VIRIDILOBUS MARINUS (GEN. ET SP. NOV.), A NEW RAPHIDOPHYTE FROM DELAWARE'S INLAND BAYS

Delaware's Inland Bays (DIB), USA, are subject to blooms of potentially harmful raphidophytes, including Heterosigma akashiwo. In 2004, a dense bloom was observed in a low salinity tributary of the DIB. Light microscopy initially suggested that the species was H. akashiwo; however, the cells were smaller than anticipated. 18S rDNA sequences of isolated cultures differed substantially from all raphidophyte sequences in GenBank. Phylogenetic analysis placed it approximately equidistant from Chattonella and Heterosigma with only ~96% sequence homology with either group. Here, we describe this marine raphidophyte as a novel genus and species, Viridilobus marinus (gen. et sp. nov.). We also compared this species with H. akashiwo, because both species are superficially similar with respect to morphology and their ecological niches overlap. V. marinus cells are ovoid to spherical (11.4 × 9.4 μm), and the average number of chloroplasts (4 per cell) is lower than in H. akashiwo (15 per cell). Pigment analysis of V. marinus revealed the presence of fucoxanthin, violaxanthin, and zeaxanthin, which are characteristic of marine raphidophytes within the family Chattonellaceae of the Raphidophyceae. TEM and confocal microscopy, however, revealed diagnostic microscopic and ultrastructural characteristics that distinguish it from other raphidophytes. Chloroplasts were in close association with the nucleus and thylakoids were arranged either parallel or perpendicular to the cell surface. Putative mucocysts were identified, but trichocysts were not observed. These features, along with DNA sequence data, distinguish this species from all other raphidophyte genera within the family Chattonellaceae of the Raphidophyceae.     

Pigments and fatty acids of marine raphidophytes: A chemotaxonomic re-evaluation

The patterns of occurrence of photosynthetic pigments and fatty acids among seven available species (11 strains) of marine raphidophytes were determined and used as chemotaxonomic markers. All currently recognized genera of marine raphidophytes were included for analysis: that is, Chattonella, Fibrocapsa, Heterosigma, Olisthodiscus and Haramonas. The characteristic pigment composition was shown to be chlorophyll a, chlorophylls c₁ and/or c₂, fucoxanthin as the major carot-enoid, β,β-carotene and any or all of zeaxanthin, violaxanthin and an auroxanthin-like pigment as the minor carotenoids. The carotenoid composition of all marine raphidophyte genera investigated was virtually the same, except in Fibrocapsa and Haramonas, which differed due to the occurrence of fucoxanthinol and 19′-butanoyloxyfucoxanthin, respectively. These fucoxanthin derivatives, in addition to fucoxanthin, have potential chemotaxonomic use for differentiating the two species. In all 11 strains, 15 fatty acids (saturated, mono-unsaturated and polyunsaturated) were determined. Significant taxonomic distinctions between genera were reflected by their fatty acid profiles. A rapid key for the differentiation of genera, in addition to morphological features, may be the absence of the 18:4 fatty acid in Olisthodiscus; presence of 18:5 in Heterosigma; the presence of fucoxanthinol in Fibrocapsa and presence of 19′-butanoyloxyfucoxanthin in Haramonas.     

Growth-promoting effects of a bacterium on raphidophytes and other phytoplankton

On 29 April 2003, a Heterosigma akashiwo bloom (9.5 × 10⁴ cells mL⁻¹) associated with a fish kill (>10⁴ dead fishes estimated from aerial surveys) was observed offshore of Bulls Bay, McLellanville, South Carolina, USA. To assess a potential cause of this bloom event, we investigated the bacterial diversity and algal/bacterial interactions in the bloom microbial community. Thirty-five bacterial strains were isolated and screened for algicidal or algal growth-promoting activities. One strain (BBB25) had significant growth-promoting effects on all 7 algal species tested: three raphidophytes (Heterosigma akashiwo, Chattonella subsalsa, Fibrocapsa japonica), two diatoms (Chaetoceros neogracile, Nitzschia sp.), a cryptophyte (Cryptomonas sp.), and a chlorophyte, Ankistrodesmus sp. This strain (BBB25) is a Gram-positive, rod-shaped spore-forming bacterium. Partial 16S rDNA gene sequence and morphological characters indicated that BBB25 is related closely to the genus Bacillus. The general nature of the algal response indicates that the growth-promoting effects of BBB25 are not specific to H. akashiwo, and suggests potentially widespread effects. Since the presence or relative abundance of the other algal species was not assessed during the bloom initiation period, the selective stimulatory effect on H. akashiwo bloom formation in Bulls Bay is unknown. These results demonstrate, however, the potential for bacterial species to play a regulatory role in bloom formation.     

Phylogeography of the freshwater raphidophyte Gonyostomum semen confirms a recent expansion in northern Europe by a single haplotype

Gonyostmum semen is a freshwater raphidophyte that has increased in occurrence and abundance in several countries in northern Europe since the 1980s. More recently, the species has expanded rapidly also in north‐eastern Europe, and it is frequently referred to as invasive. To better understand the species history, we have explored the phylogeography of G. semen using strains from northern Europe, United States, and Japan. Three regions of the ribosomal RNA gene (small subunit [SSU], internal transcribed spacer [ITS] and large subunit [LSU]) and one mitochondrial DNA marker (cox1) were analyzed. The SSU and partial LSU sequences were identical in all strains, confirming that they belong to the same species. The ITS region differentiated the American from the other strains, but showed high intra‐strain variability. In contrast, the mitochondrial marker cox1 showed distinct differences between the European, American, and Japanese strains. Interestingly, only one cox1 haplotype was detected in European strains. The overall low diversity and weak geographic structure within northern European strains supported the hypothesis of a recent invasion of new lakes by G. semen. Our data also show that the invasive northern European lineage is genetically distinct from the lineages from the other continents. Finally, we concluded that the mitochondrial cox1 was the most useful marker in determining large‐scale biogeographic patterns in this species.     

Haemolytic activity within the species Fibrocapsa japonica (Raphidophyceae)

Strong haemolytic activity was observed for extracts of 15 Fibrocapsa japonica strains collected from different global regions. The EC₅₀ values ranged between 0.4 × 10⁴ and 1.9 × 10⁴ F. japonica cells ml⁻¹. The relationship between the haemolytic activity observed in the cell extracts and the amount and nature of polyunsaturated fatty acids (PUFAs) was investigated. Between 7% and 89% of the haemolytic activity could be attributed to the amount and toxicity of the PUFAs C18:4n3 (OTA), C20:5n3 (EPA), and C20:4n6 (AA). However, the higher the haemolytic activity of a strain the more it appeared to be caused by haemolytic compounds other than the PUFA's. The EC₅₀ of the extracts with the highest haemolytic activity strongly depended on light intensity and was in line with observations made on Chattonella marina.In the cell extracts of the strains studied brevetoxins were always below detection limits. For strains where 20–35% of the haemolytic activity was not caused by PUFAs, the concentration of these compounds was close to LC₅₀ values reported for fish. Overall the haemolytic activity of the F. japonica strains was in the range of known harmful microalgae. Therefore, PUFAs in combination with potent light dependent haemolytic compounds could be a major cause of ichthyotoxicity observed during F. japonica blooms.     

MONO‐ AND DIGALACTOSYLDIACYLGLYCEROL COMPOSITION OF RAPHIDOPHYTES (RAPHIDOPHYCEAE): A MODERN INTERPRETATION USING POSITIVE‐ION ELECTROSPRAY/MASS SPECTROMETRY/MASS SPECTROMETRY1

Raphidophyte algae (Raphidophyceae) can be divided according to pigment composition and plastid ancestry into two categories, brown‐ and green‐pigmented taxa. We sought to examine if there are any biochemical differences in plastid lipid composition between the two groups. To this end, the composition and positional distribution of fatty acids of the chloroplast lipids, mono‐ and digalactosyldiacylglycerol (MGDG and DGDG, respectively), were examined using positive‐ion electrospray/mass spectrometry (ESI/MS) and electrospray/mass spectrometry/mass spectrometry (ESI/MS/MS). Brown‐pigmented strains from the genera Chattonella, Fibrocapsa, and Heterosigma primarily consisted of 20:5/18:4 (sn‐1/sn‐2) MGDG and 20:5/18:4 DGDG, while isolates of the green‐pigmented raphidophyte Gonyostomum semen (Ehrenb.) Diesing contained these as well as 18:3/18:4 MGDG and DGDG, thus underscoring its green algal plastid lineage. Although previously unseen without the regiochemical information provided by ESI/MS/MS, Chattonella subsalsa Biecheler possessed 20:5/18:3 DGDG as a major form, a potential biosynthetic intermediate in the production of 20:5/18:4 DGDG. These results provide a modern interpretation of the fatty acid regiochemistry of MGDG and DGDG.     

Life cycle,physiology, ecology and red tide occurrences of the fish-killing raphidophyte Chattonella

The marine fish-killing raphidophytes of the genus Chattonella currently consist of five species, i.e. C. antiqua, C. marina, C. minima, C. ovata and C. subsalasa. The distribution of Chattonella species was confirmed in tropical, subtropical and temperate regions in the world accompanying mass mortalities of fishes in nature and in aquaculture. The fish-killing mechanisms are still unclear, but suffocation is the ultimate cause of fish death. Increasing evidence is pointing towards the generation of reactive oxygen species (ROS, e.g. superoxide), which are responsible for the gill tissue injury and mucus production that leads to death of fishes. A taxonomic revision was proposed based on morphology and genetic diversity that Chattonella antiqua and Chattonella ovata should be varieties of Chattonella marina possessing nomenclatural priority. Optimum temperatures for growth are 25 °C for C. antiqua and C. marina, 25–30 °C for C. ovata and 20–30 °C for Chattonella subsalsa. Adequate ranges of salinity for growth were about 20–30 for Chattonella species. Chattonella cells generally divide once a day. Laboratory culture experiments with artificial synthetic medium demonstrated that C. antiqua, C. marina and C. ovata used only Fe chelated with EDTA for growth, although tested diatoms and dinoflagellates used rather many kinds of chelated Fe. A suitable concentration of humic acid supplied with iron also had enhancing effects on the growth of C. antiqua. Diel vertical migration was observed in Chattonella, and the cells reached 7.5 m deep at night in the case of C. antiqua demonstrated by a mesocosm experiment in the Seto Inland Sea. Chattonella species have diplontic life history and have haploid cyst stage in their life cycle. Encystment was observed through formation of pre-encystment small cells after the depletion of nitrogen, and the small cells sink to the sea bottom to complete cyst formation by attachment to the solid surface such as diatom frustules and sand grains. Newly formed cysts are in the state of spontaneous dormancy and they need cold temperature period of four months or longer for maturation (acquisition of germination ability). Cysts germinate in early summer and resultant vegetative cells play an important role as seed populations in blooming in the summer season. However, relatively small part of cyst populations actually germinate from bottom sediments, and success of red tide formation is dependent on the growth in water columns. Since red tides of Chattonella were observed when diatoms were scarce in seawater, diatoms appear to have a key for the predominance of Chattonella in water columns. Diatom resting stages in sediments need light for germination/rejuvenation, whereas Chattonella cysts can germinate even in the dark, implying the selective germination of Chattonella cysts at the sea bottom under calm oceanographic conditions which contribute to bloom formation of Chattonella. As a mechanism of red tide occurrences of Chattonella in coastal sea, “diatom resting hypothesis” was presented. Biological control using diatoms is proposed through the germination/rejuvenation of resting stages suspending from bottom sediments to euphotic layer by sediment perturbation with submarine tractors or fishing trawling gears. Since diatoms have much higher growth rates, and newly joined diatom vegetative cells grow faster and prevent occurrence of Chattonella red tides as a result. As another prevention strategy for Chattonella red tides, algicidal bacteria inhabiting in seaweed beds and seagrass beds are presented. Co-culture of fish and seaweeds in aquaculture areas, and the developments of seaweed- and seagrass-beds would be practical and ultimately environment-friendly strategies for the prevention of harmful red tides of Chattonella by virtue of natural algicidal bacteria supplied from seaweeds and leaves of seagrass.     

Assessment of EvaGreen-based quantitative real-time PCR assay for enumeration of the microalgae Heterosigma and Chattonella (Raphidophyceae)

Heterosigma akashiwo and Chattonella species (Raphidophyceae) are difficult to detect and quantify in environmental samples because of their pleomorphic and fragile cell nature. In this study, we developed a quantitative real-time polymerase chain reaction (qRT-PCR) assay for the enumeration of these algal taxa using a new DNA-binding dye, EvaGreen. Species-specific qRT PCR primers to H. akashiwo, Chattonella antiqua, Chattonella marina, Chattonella ovata, and Chattonella subsalsa were designed to target the ITS2 rRNA gene intergenic region. Primer specificities were tested via BLAST searches. In addition, specificity was verified using empirical tests, including competitive PCR. The qRT PCR assay analyzing C _t value and the log of cell number showed a significant linear relationship (r ²?≥?0.997). When light microscopy was used to monitor the population dynamics of targeted Raphidophyceae from Lake Shihwa, H. akashiwo was detected in ten samples and no Chattonella spp. were detected (70 samples collected from May, 2007 to January, 2008). In contrast, when the qRT-PCR assay was used, H. akashiwo was detected in 41 samples. C. antiqua, C. marina, and C. ovata were detected in eight samples. Most of the samples analyzed using qRT-PCR assays showed higher algal numbers than did those assayed via microscopy, suggesting that the enumeration of Raphidophyceae via classic microscopic methods most likely underestimates true algal concentration.     

Comparative studies on the fish-killing activities of Chattonella marina isolated in 1985 and Chattonella antiqua isolated in 2010, and their possible toxic factors

Chattonella antiqua isolated in 2010 showed extremely more potent fish-killing activities against red sea bream, Japanese horse mackerel, and blue damselfish than those of Chattonella marina isolated in 1985. Chemiluminescence and electron spin resonance (ESR) analyses suggested greater reactive oxygen species (ROS)-producing activity of C. antiqua than that of C. marina. Sodium benzoate, a hydroxyl radical scavenger, significantly suppressed the fish-killing activity of C. antiqua on blue damselfish. The chlorophyll level in the gill tissue of blue damselfish exposed to flagellate cells increased along with the exposure time, and the cell count of gill-associated C. antiqua estimated with chlorophyll level was higher than that of C. marina. These results suggest that the ROS-producing activity and affinity of Chattonella cells to the gill surface may be important factors influencing the fish-killing activity of Chattonella species.     

Mortality in the initial ontogeny of Paralabrax maculatofasciatus (Actinopterygii, Perciformes, Serranidae) caused by Chattonella spp. (Raphidophyceae)

Fish are particularly sensitive to metabolites produced by Raphidophyte species and these have caused intensive fish kills in several countries. However, the effects on embryos of marine fish are unknown but could probably provoke an important impact on new stock recruitment and hence on fisheries. We evaluated the toxic effects of Chattonella spp. strains from the Gulf of California on three development stages of spotted sand bass (Paralabrax maculatofasciatus): embryo in segmentation stage (ES), embryo (EM), and eleutheroembryo (EL). Embryos (ES) were exposed to different cell concentrations of Chattonella subsalsa, Chattonella marina, Prorocentrum micans, and f/2 medium as control. Also, one set of embryos was tested with cell-free media for C. subsalsa cultures. Incubation lasted until embryos reached apterolarva phase. The ES was the most sensitive stage reaching 98% mortality with C. subsalsa, followed by cell-free media of C. subsalsa cultures, with mortalities close to 90%, whereas EM and EL phases presented mortalities below 60%. This work demonstrates that larval stages of P. maculatofasciatus are highly sensitive to short time exposure to all Chattonella spp. strains tested, that direct physical contact with cells is not required to cause mortality, and that the toxic effect is more pronounced when embryos hatch.     

Bottom-up controls on a mixed-species HAB assemblage: A comparison of sympatric Chattonella subsalsa and Heterosigma akashiwo (Raphidophyceae) isolates from the Delaware Inland Bays, USA

Recent novel mixed blooms of several species of toxic raphidophytes have caused fish kills and raised health concerns in the highly eutrophic Inland Bays of Delaware, USA. The factors that control their growth and dominance are not clear, including how these multi-species HAB events can persist without competitive exclusion occurring. We compared and contrasted the relative environmental niches of sympatric Chattonella subsalsa and Heterosigma akashiwo isolates from the bays using classic Monod-type experiments. C. subsalsa grew over a temperature range from 10 to 30 °C and a salinity range of 5–30 psu, with optimal growth occurring from 20 to 30 °C and 15 to 25 psu. H. akashiwo had similar upper temperature and salinity tolerances but also lower limits, with growth occurring from 4 to 30 °C and 5 to 30 psu and optimal growth between 16 and 30 °C and 10 and 30 psu. These culture results were confirmed by field observations of bloom occurrences in the Inland Bays. Maximum nutrient-saturated growth rates (μ_max) for C. subsalsa were 0.6 d⁻¹ and half-saturation concentrations for growth (K_s) were 9 μM for nitrate, 1.5 μM for ammonium, and 0.8 μM for phosphate. μ_max of H. akashiwo (0.7 d⁻¹) was slightly higher than C. subsalsa, but K_s values were nearly an order of magnitude lower at 0.3 μM for nitrate, 0.3 μM for ammonium, and 0.2 μM for phosphate. H. akashiwo is able to grow on urea but C. subsalsa cannot, while both can use glutamic acid. Cell yield experiments at environmentally relevant levels suggested an apparent preference by C. subsalsa for ammonium as a nitrogen source, while H. akashiwo produced more biomass on nitrate. Light intensity affected both species similarly, with the same growth responses for each over a range from 100 to 600 μmol photons m⁻² s⁻¹. Factors not examined here may allow C. subsalsa to persist during multi-species blooms in the bays, despite being competitively inferior to H. akashiwo under most conditions of nutrient availability, temperature, and salinity.     

Mixotrophy in red tide algae raphidophytes

Marine raphidophytes are common red tide organisms that are distributed worldwide. They are known to be harmful to other plankton and fish and have often caused large-scale fish mortality in many countries. Thus, the population dynamics of raphidophytes is a critical concern for scientists, the aquaculture industry, and government officers from many countries. Raphidophyte growth and mortality should be investigated to understand bloom dynamics. Raphidophytes were thought to be exclusively autotrophic organisms. However, several recent studies have revealed that raphidophytes are able to feed on heterotrophic and autotrophic bacteria, i.e. raphidophytes are mixotrophic algae. Further, high-resolution video microscopy has revealed the mechanism by which raphidophytes feed on bacteria, which involves capturing prey cells in the mucus excreted by mucocysts and engulfing the cells through mucocysts. These discoveries may influence the conventional view on both raphidophyte bloom dynamics and plankton energy flow and carbon cycling. In the present study, I review prey, feeding mechanisms, and ingestion rates of mixotrophic marine raphidophytes. In addition, I examine the ecological significance of raphidophyte mixotrophy.