Morphological and genetic characterization of bloom‐forming Raphidophyceae from Brazilian coast

Massive fish kills caused by bloom‐forming species of the Raphidophyceae occur in many marine coastal areas and often cause significant economic losses. The ultrastructure and phylogeny of marine raphidophytes from the Brazilian coast have not been fully analyzed. Here, we present the first combined morphological and genetic characterization of raphidophyte strains from the Brazilian coast. Ten strains of four raphidophyte species (Chattonella subsalsa, C. antiqua, Heterosigma akashiwo, and Fibrocapsa japonica) were characterized based on morphology (including ultrastructure) and LSU rDNA sequences. Chattonella subsalsa and C. antiqua formed two distinct genetic clades. We found that the cell size is the only phenotypic feature separating C. subsalsa and C. antiqua strains from Brazil, whereas traditional characteristics used for species separation in the genus Chattonella (i.e., tail size, chloroplast presence in the tail, ‘oboe‐shaped’ mucocysts, and presence of thylakoids in the pyrenoid matrix) were not sufficiently discriminative, due to their overlapping in the two taxa. The phylogenetic analysis indicated intra‐specific geographic differences among C. subsalsa sequences, with two subclades: one formed by isolates from Brazil, USA, and Iran, and another by a sequence from the Adriatic Sea (Italy). Fibrocapsa japonica also showed intra‐specific geographic differences, with a sequence from a Brazilian strain grouped with strains from Japan, Australia, and Germany, all of them distinct from the Italian isolates. This is the first combined morphological and phylogenetic analysis of raphidophytes from the South Atlantic. Our findings broaden knowledge of the biodiversity of this important bloom‐forming algal group.     

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     

Broad Salinity Tolerance as a Refuge from Predation in the Harmful Raphidophyte Alga Heterosigma akashiwo (Raphidophyceae)

The ability of harmful algal species to form dense, nearly monospecific blooms remains an ecological and evolutionary puzzle. We hypothesized that predation interacts with estuarine salinity gradients to promote blooms of Heterosigma akashiwo (Y. Hada) Y. Hada ex Y. Hara et M. Chihara, a cosmopolitan toxic raphidophyte. Specifically, H. akashiwo's broad salinity tolerance appears to provide a refuge from predation that enhances the net growth of H. akashiwo populations through several mechanisms. (1) Contrasting salinity tolerance of predators and prey. Estuarine H. akashiwo isolates from the west coast of North America grew rapidly at salinities as low as six, and distributed throughout experimental salinity gradients to salinities as low as three. In contrast, survival of most protistan predator species was restricted to salinities >15. (2) H. akashiwo physiological and behavioral plasticity. Acclimation to low salinity enhanced H. akashiwo's ability to accumulate and grow in low salinity waters. In addition, the presence of a ciliate predator altered H. akashiwo swimming behavior, promoting accumulation in low‐salinity surface layers inhospitable to the ciliate. (3) Negative effects of low salinity on predation processes. Ciliate predation rates decreased sharply at salinities <25 and, for one species, H. akashiwo toxicity increased at low salinities. Taken together, these behaviors and responses imply that blooms can readily initiate in low salinity waters where H. akashiwo would experience decreased predation pressure while maintaining near‐maximal growth rates. The salinity structure of a typical estuary would provide this HAB species a unique refuge from predation. Broad salinity tolerance in raphidophytes may have evolved in part as a response to selective pressures associated with predation.     

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.     

Using quantitative real-time PCR to study competition and community dynamics among Delaware Inland Bays harmful algae in field and laboratory studies

The Delaware Inland Bays (DIB) have experienced harmful algal blooms of dinoflagellates and raphidophytes in recent years. We used quantitative polymerase chain reaction (QPCR) techniques to investigate the community dynamics of three DIB dinoflagellates (Karlodinium veneficum, Gyrodinium instriatum, and Prorocentrum minimum) and one raphidophyte (Heterosigma akashiwo) at a single site in the DIB (IR-32) in summer 2006 relative to salinity, temperature and nutrient concentrations. We also carried out complementary laboratory culture studies. New primers and probes were developed and validated for the 18S rRNA genes in the three dinoflagellates. K. veneficum, H. akashiwo, and G. instriatum were present in almost all samples throughout the summer of 2006. In contrast, P. minimum was undetectable in late June through September, when temperatures ranged from 20 to 30 °C (average 25.7 °C). Dissolved nutrients ranged from 0.1 to 2.8 μM PO₄³⁻ (median = 0.3 μM), 0.7–30.2 μM NO_x (median = 12.9 μM), and 0–19.4 μM NH₄⁺ (median = 0.7 μM). Dissolved N:P ratios covered a wide range from 2.6 to 177, with a median of 40. There was considerable variability in occurrence of the four species versus nutrients, but in general P. minimum and H. akashiwo were most abundant at higher (>40) N:P ratios and dissolved nitrogen concentrations, while K. veneficum and G. instriatum were most abundant at low dissolved N:P ratios (<20) and dissolved nitrogen concentrations < 10 μM. The semi-continuous laboratory competition experiment used mixed cultures of K. veneficum, P. minimum, and H. akashiwo grown at dissolved N:P ratios of 5, 16, and 25. At an N:P of 16 and 25 P. minimum was the dominant alga at the end of the experiment, even at a temperature that was much higher than that at which this alga was found to bloom in the field (27 °C). P. minimum and H. akashiwo had highest densities in the N:P of 25. K. veneficum grew equally well at all three N:P ratios, and was co-dominant at times at an N:P of 5. H. akashiwo had the lowest densities of the three algae in the laboratory experiment. Laboratory and field results showed both interesting similarities and significant differences in the influences of important environmental factors on competition between these harmful algal species, suggesting the need for more work to fully understand HAB dynamics in the DIB.     

The effects of salinity on the cellular permeability and cytotoxicity of Heterosigma akashiwo

A laboratory study using the fish‐killing raphidophyte Heterosigma akashiwo was conducted to examine its capability to grow at salinities below oceanic, and to test the perceived relationship between reduced salinities and increased cytotoxicity. A nonaxenic strain of H. akashiwo isolated from the U.S. Pacific Northwest was exposed to a combination of three salinity (32, 20, and 10) and five temperature (14.7°C, 18.4°C, 21.4°C, 24.4°C and 27.8°C) conditions. Our results demonstrate that cell permeability and cytotoxicity are strongly correlated in unialgal cultures of H. akashiwo, which both increased as salinity decreased from 32 to 10. Furthermore, over a broad median range of salinities (10 and 20), neither temperature nor specific growth rate was correlated with cytotoxicity. However, in cultures grown at the salinity of 32, both temperature and specific growth rate were inversely proportional to toxicity; this relationship was likely due to the effect of contamination by an unidentified species of Skeletonema in those cultures. The presence of Skeletonema sp. resulted in a cytotoxic response from H. akashiwo that was greater than the response caused by salinity alone. These laboratory results reveal the capability of H. akashiwo to become more toxic not only at reduced salinities but also in competition with another algal species. Changes in cell permeability in response to salinity may be an acclimation mechanism by which H. akashiwo is able to respond rapidly to different salinities. Furthermore, due to its strong positive correlation with cytotoxicity, cellular permeability is potentially associated with the ichthyotoxic pathway of this raphytophyte.     

The photobiology of Heterosigma akashiwo. Photoacclimation,diurnal periodicity,and its ability to rapidly exploit exposure to high light

Periodic and seasonal exposure to high light is a common occurrence for many near‐shore and estuarine phytoplankton. Rapid acclimatization to shifts in light may provide an axis by which some species of phytoplankton can outcompete other microalgae. Patterns of photoacclimation and photosynthetic capacity in the raphidophyte Heterosigma akashiwo (Hada) Hada ex Hara et Chihara isolated from the mid‐Atlantic of the United States were followed in continuous cultures at low‐ and high‐light intensities, followed by reciprocal shifts to the opposite light level. The maximum quantum yield (F_v/F_m) as well as the photosynthetic cross‐section (σ_PSII) of photosystem II was higher in high‐light cultures compared to low‐light cultures. Significant diurnal variability in photochemistry and photoprotection was noted at both light levels, and high‐light‐acclimated cultures displayed greater variability in photoprotective pathways. When shifted from low to high light, there was only a slight and temporary decline in maximum quantum yield, while cell specific growth more than doubled within 24 h. Rapid acclimation to high light was facilitated by short‐term photoprotection (nonphotochemical quenching), reduced PSII reaction center connectivity, and electron transport. Short‐term increases in de‐epoxidated xanthophyll pigments contributed to nonphotochemical protection, but lagged behind initial increases in nonphotochemical quenching and were not the primary pathway of photoprotection in this alga. By 48 h, photochemistry of cultures shifted from low to high light resembled long‐term high‐light‐acclimated cultures. This isolate of H. akashiwo appears well poised to exploit rapid shifts in light by using unique cellular adjustments in light harvesting and photochemistry.     

Pigment composition and photoacclimation as keys to the ecological success of Gonyostomum semen (Raphidophyceae,Stramenopiles)

Aquatic habitats are usually structured by light attenuation with depth resulting in different microalgal communities, each one adapted to a certain light regime by their specific pigment composition. Several taxa contain pigments restricted to one phylogenetic group, making them useful as marker pigments in phytoplankton community studies. The nuisance and invasive freshwater microalga Gonyostomum semen (Raphidophyceae) is mainly found in brown water lakes with sharp vertical gradients in light intensity and color. However, its pigment composition and potential photoadaptations have not been comprehensively studied. We analyzed the photopigment composition of 12 genetically different strains of G. semen by high performance liquid chromatography after acclimation to different light conditions. We confirmed the pigments chl a, chl c1c2, diadinoxanthin, trans‐neoxanthin, cis‐neoxanthin, α and β carotene, which have already been reported for G. semen. In addition, we identified, for the first time, the pigments violaxan‐thin, zeaxanthin, and alloxanthin in this species. Alloxanthin has never been observed in raphidophytes before, suggesting differences in evolutionary plastid acquisition between freshwater lineages and the well‐described marine species. The amount of total chl a per cell generally decreased with increasing light intensity. In contrast, the increasing ratios of the prominent pigments diadinoxanthin and alloxanthin per chl a with light intensity suggest photoprotective functions. In addition, we found significant variation in cell‐specific pigment concentration among strains, grouped by lake of origin, which might correspond to genetic differences between strains and populations.     

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.     

Community-Level and Species-Specific Associations between Phytoplankton and Particle-Associated Vibrio Species in Delaware's Inland Bays

Vibrio species are an abundant and diverse group of bacteria that form associations with phytoplankton. Correlations between Vibrio and phytoplankton abundance have been noted, suggesting that growth is enhanced during algal blooms or that association with phytoplankton provides a refuge from predation. Here, we investigated relationships between particle-associated Vibrio spp. and phytoplankton in Delaware''s inland bays (DIB). The relative abundances of particle-associated Vibrio spp. and algal classes that form blooms in DIB (dinoflagellates, diatoms, and raphidophytes) were determined using quantitative PCR. The results demonstrated a significant correlation between particle-associated Vibrio abundance and phytoplankton, with higher correlations to diatoms and raphidophytes than to dinoflagellates. Species-specific associations were examined during a mixed bloom of Heterosigma akashiwo and Fibrocapsa japonica (Raphidophyceae) and indicated a significant positive correlation for particle-associated Vibrio abundance with H. akashiwo but a negative correlation with F. japonica. Changes in Vibrio assemblages during the bloom were evaluated using automated ribosomal intergenic spacer analysis (ARISA), which revealed significant differences between each size fraction but no significant change in Vibrio assemblages over the course of the bloom. Microzooplankton grazing experiments showed that losses of particle-associated Vibrio spp. may be offset by increased growth in the Vibrio population. Moreover, analysis of Vibrio assemblages by ARISA also indicated an increase in the relative abundance for specific members of the Vibrio community despite higher grazing pressure on the particle-associated population as a whole. The results of this investigation demonstrate links between phytoplankton and Vibrio that may lead to predictions of potential health risks and inform future management practices in this region.     

Sublethal effects of the toxic alga Heterosigma akashiwo on the southeastern oyster (Crassostrea virginica)

Over the last three years, several blooms of Heterosigma akashiwo (Raphidophyceae) were documented in South Carolina (SC) brackish waters, including areas containing extensive oyster (Crassostrea virginica) beds. This study examined the sublethal effects of H. akashiwo on C. virginica, based on cellular biomarker responses after exposure to laboratory cultures of H. akashiwo isolated from SC waters, and to water collected from two SC H. akashiwo blooms. Exposure to laboratory cultures or blooms of H. akashiwo significantly increased oyster hepatopancreas lysosomal destabilization rates, but had little effect on gill p-glycoprotein (p-gp) expression. Lysosomal destabilization in oysters continued to increase even after a 7-day recovery period in clean seawater, suggesting that H. akashiwo toxin or other cellular byproducts continued to damage the hepatopancreas. These results suggest that even short-term exposures of oysters to high cell densities of H. akashiwo could have long-term adverse physiological effects, and imply that oyster health may be compromised in areas where repetitive H. akashiwo blooms occur.     

Structural and ultrastructure changes show an increase in amoeboid forms in Heterosigma akashiwo (Raphidophyceae), during recovery after nutrient depletion

Heterosigma akashiwo shows remarkable ultrastructural changes during the recovery from a late stationary phase (“aged” culture) induced by nutrient depletion. H. akashiwo cells showed different morphological types in “aged” cultures, with an increase in irregular cells and cell fragments. The irregular cells mostly corresponded to an amoeboid shape of the cell. Many of these cells showed chloroplasts with a homogeneous matrix of medium electron density lacking most thylakoids and condensed nucleus, probably as a result of cyst/resting cells germination. In other cells, we observed nuclear blebbing without chromatin condensation and changes in mitochondrion ultrastructure. Some vegetative cells in active phase (“young” culture) were connected to each other, apparently phagocytizing cytoplasmic fragments and intact chloroplasts in the medium. An explanation for the phenomenon may reside in the need of acquiring organic material after nutrient reduction for a faster recovery. On the basis of our observations, we conclude that some ultrastructural features, normally used to distinguish between different species and strains of Raphidophyceae, may be related to different physiological states and should be used with caution for systematic purposes.     

Ability of marine eukaryotic red tide microalgae to utilize insoluble iron

Iron is an essential trace metal and a limiting factor for microalgal growth, but bioavailable dissolved iron concentrations in seawater are low. Microalgal blooms have frequently occurred in coastal areas under such iron limitation accompanied by mass mortalities of fish and bivalves. Their massive growth despite physiological iron-deficiency has long been an enigma, because most of them cannot grow in chemically defined artificial media. We developed a feasible artificial medium for the culture of many species of red tide microalgae modified for investigation of iron utilization. Here, we report on the ability of marine eukaryotic red tide microalgae to utilize insoluble iron. Some microalgal species could utilize particulate FePO₄ and FeS for growth. Particulate FePO₄ was available for the growth of the raphidophyte Heterosigma akashiwo, the dinoflagellate Heterocapsa triquetra and the diatom Ditylum brightwellii. The dinoflagellates Heterocapsa circularisquama and Karenia mikimotoi, and the cryptophyte Rhodomonas ovalis utilized both particulate FePO₄ and particulate FeS for growth. In contrast, particulate FeO(OH) and Fe₂O₃ did not support the growth of any of the red tide microalgae examined. Except for Chattonella species (Raphidophyceae), the growth of red tide microalgae were confirmed in the medium with very easily soluble FeCl₃ added. The order of bioavailability of tested iron-source species were Fe–EDTA > FeCl₃ > FePO₄ > FeS > FeO(OH), Fe₂O₃ for most of microalgae examined, although for H. circularisquama the utilization of FeCl₃ was higher than that of Fe–EDTA. The results suggest that red tide microalgae show different patterns of specific strategies for the utilization of various iron sources. The occurrence of red tides in coastal areas may depend on the combination of microalgal species and insoluble iron species present.     

Viral Impacts on Total Abundance and Clonal Composition of the Harmful Bloom-Forming Phytoplankton Heterosigma akashiwo

Recent observations that viruses are very abundant and biologically active components in marine ecosystems suggest that they probably influence various biogeochemical and ecological processes. In this study, the population dynamics of the harmful bloom-forming phytoplankton Heterosigma akashiwo (Raphidophyceae) and the infectious H. akashiwo viruses (HaV) were monitored in Hiroshima Bay, Japan, from May to July 1998. Concurrently, a number of H. akashiwo and HaV clones were isolated, and their virus susceptibilities and host ranges were determined through laboratory cross-reactivity tests. A sudden decrease in cell density of H. akashiwo was accompanied by a drastic increase in the abundance of HaV, suggesting that viruses contributed greatly to the disintegration of the H. akashiwo bloom as mortality agents. Despite the large quantity of infectious HaV, however, a significant proportion of H. akashiwo cells survived after the bloom disintegration. The viral susceptibility of H. akashiwo isolates demonstrated that the majority of these surviving cells were resistant to most of the HaV clones, whereas resistant cells were a minor component during the bloom period. Moreover, these resistant cells were displaced by susceptible cells, presumably due to viral infection. These results demonstrated that the properties of dominant cells within the H. akashiwo population change during the period when a bloom is terminated by viral infection, suggesting that viruses also play an important role in determining the clonal composition and maintaining the clonal diversity of H. akashiwo populations. Therefore, our data indicate that viral infection influences the total abundance and the clonal composition of one host algal species, suggesting that viruses are an important component in quantitatively and qualitatively controlling phytoplankton populations in natural marine environments.     

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.     

Lethal effects of ichthyotoxic raphidophytes,Chattonella marina,C. antiqua,and Heterosigma akashiwo,on post-embryonic stages of the Japanese pearl oyster,Pinctada fucata martensii

The inimical effects of the ichthyotoxic harmful algal bloom (HAB)-forming raphidophytes Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua on the early-life stages of the Japanese pearl oyster Pinctada fucata martensii were studied. Fertilized eggs and developing embryos were not affected following exposure to the harmful raphidophytes; however, all three algal species severely affected trochophores and D-larvae, early-stage D-larvae, and late-stage pre-settling larvae. Exposure to C. marina (5 × 10² cells ml⁻¹), C. antiqua (10³ cells ml⁻¹), and H. akashiwo (5 × 10³ cells ml⁻¹) resulted in decreased success of metamorphosis to the trochophore stage. A complete inhibition of trochophore metamorphosis was observed following exposure to C. antiqua at 5 × 10³ cells ml⁻¹ and C. marina at 8 × 10³ cells ml⁻¹. In all experiments, more than 80% of newly formed trochophores were anomalous, and in the case of exposure to H. akashiwo at 10⁵ cells ml⁻¹ more than 70% of D-larvae were anomalous. The activity rates of D-larvae (1-day-old) were significantly reduced following exposure to C. antiqua (8 × 10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (10⁴ cells ml⁻¹, 24 h). The activity rates of pre-settling larvae (21-day-old) were also significantly reduced following exposure to C. antiqua (10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (5 × 10⁴ cells ml⁻¹, 24 h). Significant mortalities of both larval stages were induced by all three raphidophytes, with higher mortality rates registered for pre-settling larvae than D-larvae, especially following exposure to C. marina (5 × 10²–8 × 10³ cells ml⁻¹, 48–86 h) and C. antiqua (10³–8 × 10³ cells ml⁻¹, 72–86 h). Contact between raphidophyte cells and newly metamorphosed trochophores and D-larvae, 1-day-old D-larvae, and 21-day-old larvae resulted in microscopic changes in the raphidophytes, and then, in the motile early-life stages of pearl oysters. Upon contact and physical disturbance of their cells by larval cilia, H. akashiwo, C. marina and C. antiqua became immotile and shed their glycocalyx. The trochophores and larvae were observed trapped in a conglomerate of glycocalyx and mucus, most probably a mixture of larval mucous and raphidophyte tricosyts and mucocytes. All motile stages of pearl oyster larvae showed a typical escape behavior translating into increased swimming in an effort to release themselves from the sticky mucous traps. The larvae subsequently became exhausted, entrapped in more heavy mucous, lost their larval cilia, sank, become immotile, and died. Although other toxic mediators could have been involved, the results of the present study indicate that all three raphidophytes were harmful only for motile stages of pearl oysters, and that the physical disturbance of their cells upon contact with the ciliary structures of pearl oyster larvae initiated the harmful mechanism. The present study is the first report of lethal effects of harmful Chattonella spp. towards larvae of a bivalve mollusc. Blooms of H. akashiwo, C. antiqua and C. marina occur in all major cultivation areas of P. fucata martensii during the developmental period of their larvae. Therefore, exposure of the motile early-life stages of Japanese pearl oysters could adversely affect their population recruitment. In addition, the present study shows that further research with early-life development of pearl oysters and other bivalves could contribute to improving the understanding of the controversial harmful mechanisms of raphidophytes in marine organisms.     

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.     

Phylogenetic relationships of the Raphidophyceae and Xanthophyceae as inferred from nucleotide sequences of the 18S ribosomal RNA gene

Some earlier studies suggested an evolutionary relationship between the Raphidophyceae (chloromonads) and Xanthophyceae (yellow-green algae), whereas other studies suggested relationships with different algal classes or the öomycete fungi. To evaluate the relationships, we determined the complete nucleotide sequences of the 18S ribosomal RNA gene from the raphidophytes Vacuolaria virescens, Chattonella subsalsa, and Heterosigma carterae, and the xanthophytes Vaucheria bursata, Botrydium stoloniferum, Botrydiopsis intercedens, and Xanthonema debile. The results showed that the Xanthophyceae were most closely related to the Phaeophyceae. A cladistic analysis of combined data sets (nucleotide sequences, ultrastructure, and pigments) suggested the Raphidophyceae are the sister taxon to the Phaeophyceae-Xanthophyceae clade, but the bootstrap value was low (40%). The raphidophyte genera were united with high (100%) bootstrap values, supporting a hypothesis based upon ultrastructural features that marine and freshwater raphidophytes form a monophyletic group. We examined the relationship between Vaucheria, a siphoneous xanthophyte alga, and the öomycetes, and we confirmed that Vaucheria is a member of the class Xanthophyceae. Partial nucleotide sequences of the 18S rRNA gene from eight xanthophytes (including Bumillariopsis filiformis, Heterococcus caespitiosus, and Mischococcus sphaerocephalus) produce a phylogeny that is not congruent with the current morphology-based classification scheme.     

A quantitative real‐time PCR assay for identification and enumeration of the occasionally co‐occurring ichthyotoxic Pseudochattonella farcimen and P. verruculosa (Dictyochophyceae) and analysis of variation in gene copy numbers during the growth phase of single and mixed cultures

The ichthyotoxic genus Pseudochattonella forms recurrent extensive blooms in coastal waters in Japan, New Zealand and Northern Europe. It comprises of two morphologically similar species, P. verruculosa and P. farcimen, which complicates visual species identification and enumeration of live and fixed material. Primers designed previously could not quantitatively distinguish species in mixed assemblages. To address this issue we developed two primer sets: one revealed itself to be genus specific for Pseudochattonella and the other species‐specific for P. verruculosa. By subtracting cell estimates for P. verruculosa from combined results we could calculate cell numbers for P. farcimen. This approach has overcome the challenges posed by the very limited sequence availability and low gene variability between the two species. The qPCR assay was extensively tested for specificity, efficiency and sensitivity over an entire growth cycle in both single and mixed assemblages. Comparison of cell abundance estimates obtained by qPCR assay and microscopy showed no statistically significant difference until stationary and death phases. The assay was also tested on environmental samples collected during a small Pseudochattonella bloom in Denmark in March–April 2015. It was impossible to distinguish P. farcimen and P. verruculosa by light microscopy but qPCR showed both species were present. The two methods provided nearly identical cell numbers but the assay provided discrimination and enumeration of both species.     

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.