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

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

Effects of a bacterial algicide,IRI-160AA,on dinoflagellates and the microbial community in microcosm experiments

Filtrates from the bacterium Shewanella sp. IRI-160 (termed IRI-160AA) have been shown to inhibit population growth and kill a variety of dinoflagellates grown in culture. Here we test the immediate efficacy of IRI-160AA in laboratory microcosms initiated from three natural dinoflagellate blooms (Prorocentrum minimum, Karlodinium veneficum and Gyrodinium instriatum). We measured target dinoflagellate abundance, total chlorophyll-a, photosystem II (PSII) photochemistry, and changes to the prokaryotic and eukaryotic community composition over 2–3 days of IRI-160AA incubation. Naked dinoflagellates were impacted more, while abundance of the thecate P. minimum was not affected. However, dinoflagellate growth inhibition was generally lower than that observed in uni-algal cultures, and took longer to occur. Eukaryotic community composition in IRI-160AA treated microcosms was significantly different from control incubations, and was driven predominantly by increases in heterotrophic protists (e.g. Euplotes sp. and Paraphysomonas sp.). Similarly, significant changes to the prokaryotic community structure were evident. Microcosms of G. instriatum with higher algicide concentrations indicated that algicidal activity was enhanced in a dose dependent manner. Furthermore, total ciliate abundance as well as a bactivorous chyrsophyte (Paraphysomonas sp.) increased in a dose dependent manner. Total diatom abundance increased at lower IRI-160AA concentrations, but increased less with increasing dose. Overall, the bio-activity of IRI-160AA on naturally occurring dinoflagellates in mixed natural microbial communities is encouraging from the applied perspective of using the active compound(s) in IRI-160AA as natural agent(s) to manage harmful dinoflagellate blooms.     

Investigation of the algicidal exudate produced by Shewanella sp. IRI-160 and its effect on dinoflagellates

The bacterium, Shewanella sp. IRI-160, was previously shown to have negative effects on the growth of dinoflagellates, while having no negative effects on other classes of phytoplankton tested (Hare et al., 2005). In this study, we investigated the mode of algicidal activity for Shewanella sp. IRI-160 and found that the bacterium secretes a bioactive compound. The optimum temperature for production of the algicidal compound by this bacterium was at 30 °C. Bacteria-free filtrate of medium containing the algicide (designated IRI-160AA) was stable at temperatures ranging from −80 °C to 121 °C, and could be stored at room temperature for at least three weeks with no loss in activity. Algicidal activity was eluted in the aqueous portion after C18 extraction, suggesting that the active compound is likely polar and water-soluble. The activity of IRI-160AA was examined on a broad range of dinoflagellates (Karlodinium veneficum, Karenia brevis, Gyrodinium instriatum, Cochlodinium polykrikoides, Heterocapsa triquetra, Prorocentrum minimum, Alexandrium tamarense and Oxyrrhis marina) and three species from other classes of algae as controls (Dunaliella tertiolecta, Rhodomonas sp. and Thalassiosira pseudonana). Algicidal activity was observed for each dinoflagellate and little to no negative effect was observed on chlorophyte and cryptophyte cultures, while a slight (non-significant) stimulatory effect was observed on the diatom culture exposed to the algicide. Finally, the effect of the algicide at different growth stages was investigated for K. veneficum and G. instriatum. IRI-160AA exhibited a significantly greater effect during logarithmic growth compared to stationary phase, suggesting a potential application of the algicide for prevention and control of harmful dinoflagellate blooms in the future.     

A bacterium that inhibits the growth of Pfiesteria piscicida and other dinoflagellates

Toxic dinoflagellate blooms have increased in estuaries of the east coast of the United States in recent years, and the discovery of Pfiesteria piscicida has brought renewed attention to the problem of harmful algal blooms (HAB) in general. Many bacteria and viruses have been isolated that have algicidal or algistatic effects on phytoplankton, including HAB species. Twenty-two bacterial isolates from the Delaware Inland Bays were screened for algicidal activity. One isolate (Shewanella IRI-160) had a growth-inhibiting effect on all three dinoflagellate species tested, including P. piscicida (potentially toxic zoospores), Prorocentrum minimum, and Gyrodinium uncatenum. This bacterium did not have a negative effect on the growth of any of the other four common estuarine non-dinoflagellate species tested, and in fact had a slight stimulatory effect on a diatom, a prasinophyte, a cryptophyte, and a raphidophyte. Shewanella IRI-160 is the first non-microzooplankton example of a microbe with the ability to control and inhibit the growth of P. piscicida, suggesting that bacteria in the natural environment could play a role in controlling the growth and abundance of P. piscicida and other dinoflagellates. Such bacteria could also potentially be used as management tools to prevent the proliferation of potentially harmful dinoflagellates in estuaries and coastal waters.     

Growth and competition of several harmful dinoflagellates under different nutrient and light conditions

Three near-shore type harmful dinoflagellates, Prorocentrum minimum, Prorocentrum donghaiense and Karlodinium veneficum, and one off-shore dinoflagellate, Karenia brevis, were grown in laboratory monocultures and mixed batch cultures. The dinoflagellate cultures were grown on treatments of two ambient nitrogen (N):phosphorus (P) ratios; two N substrates (nitrate and urea) and two light intensities. The microalgae Rhodomonas and Synechococcus were also added in separate treatments to the mixed culture treatments as potential food sources. All tested species grew well on both N substrates. In mixed culture, P. minimum outgrew K. veneficum, and P. donghaiense outgrew K. brevis in most treatments reaching higher growth rates and higher biomass. However, when a third algae, Rhodomonas, was added, the growth of P. minimum was inhibited relative to that of K. veneficum. In contrast, when grown with K. brevis, the growth rate of P. donghaiense was not significantly affected by the addition of potential prey. K. brevis had a longer growth phase, and kept growing after P. donghaiense reached stationary phase, suggesting better adaptation of K. brevis to low inorganic nutrient conditions. The growth of K. brevis was also significantly limited in the low light treatment. K. veneficum overgrew P. minimum in the presence of Rhodomonas, a potential nutrient source. The growth rates of both K. brevis and P. donghaiense were reduced with the presence of Synechococcus. In addition to nutrient competition, mixotrophy and allelopathy were likely mechanisms in determining the dominant species.     

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.     

Ecological niche partitioning of the invasive dinoflagellate Prorocentrum minimum and its native congeners in the Baltic Sea

This study analyses three decades of the peculiar bloom-formation history of the potentially toxic invasive planktonic dinoflagellates Prorocentrum minimum (Pavillard) Schiller in the SW Baltic Sea. We tested a research hypothesis that the unexpectedly long delay (nearly two decades) in population development of P. minimum prior to its first bloom was caused by competition with one or several closely related native dinoflagellate species due to ecological niche partitioning which hampered the spread and bloom-forming potential of the invader. We applied the ecological niche concept to a large, long-term phytoplankton database and analysed the invasion history and population dynamics of P. minimum in the SW Baltic Sea coastal waters using the data on phytoplankton composition, abundance and biomass. The ecological niche dimensions of P. minimum and its congener P. balticum were identified as the optimum environmental conditions for the species during the bloom events based on water temperature, salinity, pH, concentration of nutrients (PO₄³⁻; total phosphorus, TP; total nitrogen, TN; SiO₄⁴⁻), TN/TP-ratio and habitat type. The data on spatial distribution and ecological niche dimensions of P. minimum have contributed to the development of the “protistan species maximum concept”. High microplankton diversity at critical salinities in the Baltic Sea may be considered as a possible reason for the significant niche overlap and strong competitive interactions among congeners leading to prolonged delay in population growth of P. minimum preceding its first bloom in the highly variable brackishwater environment.     

In situ identification and localization of bacteria associated with Gyrodinium instriatum (Gymnodiniales,Dinophyceae) by electron and confocal microscopy

The presence of intracellular bacteria in the dinoflagellate Gyrodinium instriatum Freudenthal & Lee has previously been described but the bacterial flora associated with this species has not been characterized. In this study, new results of transmission electron microscopy (TEM) and in situ hybridization using several bacterial group-specific oligonucleotide probes are presented. The long-term association of endocytoplasmic and endonuclear bacteria with G. instriatum has been confirmed. All endonuclear and most of the endocytoplasmic bacteria labelled were identified as belonging to the betaproteobacteria. Large clusters of Cytophaga-Flavobacterium-Bacteroides (CFB) were labelled and observed in the cytoplasm of the dinoflagellate cells, but were absent from the nucleus. Gammaproteobacteria were only observed outside the dinoflagellates. No alphaproteobacteria were detected either free-living or intracellular. Empirical observation of intracellular CFB reflected a degradation process of moribund dinoflagellate cells, whereas the systematic colonization of dinoflagellate nucleoplasm by betaproteobacteria suggested a true symbiotic relationship. Natural colonization may have occurred, perpetuated by vertical transmission of intracellular bacteria to the dinoflagellate daughter cells, via a pool of bacteria sequestered within the nucleus. Dividing bacteria were observed in the nucleus and equilibrium may be maintained by release of endonuclear bacteria to the cytoplasm through nuclear envelope constrictions.     

Distribution of Karlodinium veneficum in the coastal region of Xiangshan Bay in the East China Sea,as detected by a real-time quantitative PCR assay of ribosomal ITS sequence

Athecate dinoflagellate Karlodinium veneficum is a universal toxic species possessing karlotoxins recognized especially as ichthyotoxic as well as cytotoxic and hemolytic. Blooms of K. veneficum, both single-species or accompanied with other species, occurred more frequently worldwide in recent years, including the coastal region of China. Normally, K. veneficum present in relatively low abundance in phytoplankton communities in estuary regions. Being small and difficult to identify with light microscopy, it has been ignored for a long time till its blooming and toxins being confirmed. How it presents in background level and what is its relationship with critical geological and hydrological environment factors are basically not clear. In this study, the paper reports the application of a real-time quantitative PCR (qPCR) method to investigate the abundance and distribution of K. veneficum in the coastal waters of Xiangshan Bay in the East China Sea (ECS), a typical bay area of harmful algae blooms and heavily affected by anthropogenic activities. The real-time qPCR assay came out being an efficient method at detecting even low cell densities of K. veneficum of different genotypes. A total of 38 field samples of surface (0.5 m) and bottom water (9–100 m in depth) were analyzed and 12 samples were found positive for K. veneficum. At least 3 genotypes of K. veneficum present in this region. Temperatures in sites of K. veneficum positive ranged from 21.7 to 23.4 °C, and salinity levels were between 21.1 and 26.3. The K. veneficum distributed quite extensively in the waters of Xiangshan Bay, cell abundance varied from a low of 4 cells/L to a maximum of 170 cells/L. Most of the samples containing K. veneficum were collected from bottom water in different sites. At three of the 19 sampling sites, K. veneficum was detected in both surface and bottom water samples. Especially at sampling site near Beilun port, where the water is typically muddy with low transparency, relative high cell numbers of K. veneficum were found in both surface and bottom waters. Mixotrophy and vertical migration of K. veneficum could be important eco-physiological factors to consider in terms of understanding these distribution characteristics. The ideal conditions for K. veneficum growth and aggregation in this area still needs further study.     

Genetic relations among basil taxa (Ocimum L.) based on molecular markers, nuclear DNA content, and chromosome number

Twenty-eight basil accessions including six Ocimum species and six botanical varieties or cultivars of O. basilicum were studied using molecular markers, nuclear DNA content, and chromosome counting. This is the first study reporting the nuclear DNA content in the genus Ocimum. The results supported the existence of more infrageneric groups within the genus. The section Ocimum was further divided into two separate clades. The first clade contained the accessions belonging to different botanical varieties and cultivars of O. basilicum as well as O. minimum, indicating that the separate species rank of O. minimum was not justified. The second clade, comprising O. americanum, O. africanum, and two O. basilicum var. purpurascens accessions, could represent a set of allopolyploid species sharing some common parental genomes. O. tenuiflorum was the most divergent species according to genetic distance; it had the smallest genome size, organized in small chromosomes, and the lowest chromosome number. Chromosome data obtained in our research could indicate that the basic chromosome number for species belonging to section Ocimum is x = 12. This suggestion implies that species belonging to O. basilicum clade are tetraploids, while species belonging to O. americanum clade are hexaploids. It seems that the basic chromosome number for O. gratissimum could be x = 10 and for O. tenuiflorum x = 9. The differences in genome size and chromosome number among Ocimum species indicate that evolution of their genomes was accompanied by both sequence deletion/amplification and chromosome rearrangements and polyploidization.     

Algicidal effects of yellow clay and the thiazolidinedione derivative TD49 on the fish-killing dinoflagellate Cochlodinium polykrikoides in microcosm experiments

In order to evaluate the potential to control the fish-killing dinoflagellate Cochlodinium polykrikoides, we compared the algicidal effects of the thiazolidinedione derivative TD49 with those of yellow clay in 10-L microcosms. The responses of higher trophic level marine organisms and microbial loop communities to the algicide were also evaluated. In the yellow clay treatments, the concentration of C. polykrikoides was slightly reduced at day 1 of the experiment but remained higher than that of the control, suggesting that the reduction ratio of C. polykrikoides was <20 %. In the 0.8-μM TD49 treatment, the abundance of C. polykrikoides declined by 98 % 1 day following the addition of the algicide. The algicide did not affect nontarget algae including Chaetoceros spp., Skeletonema spp., Cylindrotheca spp., and other species. In all microcosms, bacterial abundance increased abruptly after day 1, then declined over the next 2 days as a result of predation by heterotrophic nanoflagellates and the small protozoan Uronema sp. Predation by the large protozoan species Euplotes sp. on Uronema sp. gradually increased with increasing incubation time in the TD49 treatment. Zooplankton were particularly affected by the environmental changes that occurred in the microcosms following collapse of the C. polykrikoides populations. Striped beak perch were not affected by the yellow clay treatments and concentrations of TD49?C. polykrikoides, whereas the algicide TD49 is effective in controlling the harmful alga. The results imply that the algicide has positive effects on natural microbial communities and is not toxic to nonharmful algae and higher trophic level marine organisms.     

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.     

Concentrations of dimethylsulphoniopropionate and activities of dimethylsulphide-producing enzymes in batch cultures of nine dinoflagellate species

Dinoflagellates are recognised as one of the major phytoplankton groups that produce dimethylsulphoniopropionate (DMSP), the precursor of the marine trace gas dimethylsulphide (DMS) which has climate-cooling potential. To improve the prospects for including dinoflagellates in global climate models that include DMSP-related processes, we increased the data base for this group by measuring DMSP, DMS-producing enzyme activity (DPEA), carbon, nitrogen and Chl a in nine clonal dinoflagellate cultures (1 heterotrophic and 8 phototrophic strains). Growth rates ranged from 0.11 to 1.92?day^?1 with the highest value being for the heterotroph Crypthecodinium cohnii. Overall, we observed two orders of magnitude variability in DMSP content (11–364?mM) and detected DPEA in five of the nine strains (0.61–59.73?fmol?cell^?1?h^?1). Cell volume varied between 454 and 18,439?μm³ and whilst C and N content were proportional to the cell volume, DMSP content was not. The first DMSP measurements for a dinoflagellate from Antarctic waters and a species with diatom-like plastids are included. Lower DMSP concentrations were found in three small athecate species and a dinoflagellate with haptophyte-like plastids. The highest concentrations and production rates tended to be in globally distributed dinoflagellates and the heterotroph. Photosynthetic species that are distributed in temperate to tropical waters showed low DMSP concentrations and production rates and the polar representative showed moderate concentration and a low production rate. Estuarine species had the lowest concentrations and production rates. These data should help refine the inclusion of dinoflagellates as a functional group in future global climate models.     

Marine microalgae attack and feed on metazoans

Free-living microalgae from the dinoflagellate genus Karlodinium are known to form massive blooms in eutrophic coastal waters worldwide and are often associated with fish kills. Natural bloom populations, recently shown to consist of the two mixotrophic and toxic species Karlodinium armiger and Karlodinium veneficum have caused fast paralysis and mortality of finfish and copepods in the laboratory, and have been associated with reduced metazooplankton biomass in-situ. Here we show that a strain of K. armiger (K-0688) immobilises the common marine copepod Acartia tonsa in a density-dependent manner and collectively ingests the grazer to promote its own growth rate. In contrast, four strains of K. veneficum did not attack or affect the motility and survival of the copepods. Copepod immobilisation by the K. armiger strain was fast (within 15 min) and caused by attacks of swarming cells, likely through the transfer and action of a highly potent but uncharacterised neurotoxin. The copepods grazed and reproduced on a diet of K. armiger at densities below 1000, cells ml⁻¹, but above 3500 cells ml⁻¹ the mixotrophic dinoflagellates immobilised, fed on and killed the copepods. Switching the trophic role of the microalgae from prey to predator of copepods couples population growth to reduced grazing pressure, promoting the persistence of blooms at high densities. K. armiger also fed on three other metazoan organisms offered, suggesting that active predation by mixotrophic dinoflagellates may be directly involved in causing mortalities at several trophic levels in the marine food web.     

Study of selective feeding in the marine cladoceran Penilia avirostris by HPLC pigment analysis

Food selection by the marine cladoceran Penilia avirostris was studied in the field by HPLC analysis of phytoplankton marker pigments and in the laboratory by microscopic measurement of cell removal. Comparison between pigment composition in natural phytoplankton and in P. avirostris showed that P. avirostris preferred diatoms, cryptophytes and chlorophytes, and ignored prymnesiophytes and dinoflagellates. Peridinin, the marker pigment for dinoflagellates was found in P. avirostris only when the dinoflagellate populations were dominated by Prorocentrum. Pigment degradation rates ranged from 13.73% for alloxanthin to 36.62% for chlorophyll a. Clearance rates measured in the laboratory provided further evidence of strong preference for diatoms and cryptophytes, and avoidance of dinoflagellates. Microscopic counts suggested that P. avirostris was feeding on prymnesiophytes, although ingestion of prymnesiophytes could not be confirmed by pigment analysis.     

Roles of mixotrophy in blooms of different dinoflagellates: Implications from the growth experiment

Studies over the last two decades suggested that mixotrophy could be an important adaptive strategy for some bloom-forming dinoflagellates. In the coastal waters adjacent to the Changjiang River estuary in the East China Sea, recurrent blooms of dinoflagellates Prorocentrum donghaiense, Karenia mikimotoi and Alexandrium catenella started to appear from the beginning of the 21 century, but roles of mixotrophy in the formation of dinoflagellate blooms were not well understood. In the current study, mixotrophy-based growth of four selected bloom-causative dinoflagellate species, i.e. K. mikimotoi, A. catenella, P. donghaiense and Prorocentrum micans, were studied. Dinoflagellates were co-cultured with different prey organisms, including bacterium Marinobacter sp., microalgae Isochrysis galbana and Hemiselmis virescens, under a variant of nutrient conditions. It was found that growth of dinoflagellate K. mikimotoi was significantly promoted with the presence of prey organisms. Growth of P. donghaiense and P. micans was only slightly improved. For A. catenella, the addition of prey organisms has no effects on the growth, while both of the two prey microalgae I. galbana and H. virescens were killed, probably by allelochemicals released from A. catenella. There was no apparent relationship between nutrient conditions and the mixotrophy-based growth of the tested dinoflagellates. Based on the results of the growth experiment, it is implicated that mixotrophy may play different roles in the growth and bloom of the four dinoflagellate species. It can be an important competitive strategy for K. mikimotoi. For the two Prorocentrum species and A. catenella, however, the role of mixotrophy is much limited. They may depend more on other competitive strategies, such as phototrophy-based growth and allelopathic effect, to prevail in the phytoplankton community and form blooms.     

Discrimination of marine algal taxonomic groups using delayed fluorescence spectroscopy

We present a method for in situ monitoring of phytoplankton composition changes in a marine environment. The method is based on delayed fluorescence excitation spectra analyzed with CHEMTAX software, which is generally used for determination of phytoplankton communities with HPLC pigment data. Delayed fluorescence (DF) is a photosynthetic parameter that can only be measured in living cells. Algal DF excitation spectra are group-specific, based on their composition of photosynthetic pigments.DF excitation spectra of 14 marine algal species from different families were measured with a delayed fluorescence spectrometer. Mixtures were prepared from northern Adriatic algal species representing six taxonomic groups: dinoflagellates (Prorocentrum minimum), diatoms (Skeletonema costatum), cyanobacteria (Synechococcus sp.), prasinophytes (Micromonas sp.), cryptophytes (Teleaulax sp.), and prymnesiophytes (Isochrysis galbana). The DF excitation spectra (DFS) and HPLC pigment compositions of the mixtures were analyzed with CHEMTAX software. The prediction power of DFS–CHEMTAX method was comparable to HPLC–CHEMTAX.     

Perkinsosis in the Manila clam Ruditapes philippinarum affects responses to the harmful-alga, Prorocentrum minimum

The dinoflagellate Prorocentrum minimum is increasingly recognized as a harmful algal bloom (HAB) species that affects filter-feeding shellfish. An experiment was done to investigate possible interactions between parasitic diseases and exposure to P. minimum in Manila clams, Ruditapes philippinarum. Manila clams, with variable levels of infection with Perkinsus olseni, were exposed for three or six days to the benign phytoplankton species Chaetoceros neogracile or a mixed diet of C. neogracile and P. minimum. After three or six days of exposure, clams were assessed individually for condition index, parasite status, and plasma and hemocyte parameters (morphological and functional) using flow-cytometry. Histological evaluation was also performed on individual clams to assess prevalence and intensity of parasitic infection, as well as other pathological conditions.Prorocentrum minimum caused several changes in Manila clams, especially after six days of exposure, such as decreased hemocyte phagocytosis and size and clam condition index. Pathological conditions observed in Manila clams exposed to P. minimum were hemocyte infiltration in the intestine and gonad follicles, myopathy, and necrosis of the intestine epithelial cells. The parasite P. olseni alone had no significant effect on Manila clams, nor did it modulate the hemocyte variables in clams exposed to P. minimum; however, the parasite did affect the pathological status of Manila clams exposed to the P. minimum culture, by causing atrophy and degeneration of residual ova in the gonadal follicles and hyaline degeneration of the muscle fibers, indicating synergistic effects of both stressors on the host over a short period of time. Additionally, an in vitro experiment also demonstrated detrimental effects of P. minimum and exudates upon P. olseni cells, thus suggesting HAB antagonistic suppression of transmission and proliferation of the parasite in the natural environment over a longer period of time. The results of this experiment demonstrate the complexity of interactions between host, parasite, and HAB.     

Rapid and sensitive detection method for Karlodinium veneficum by recombinase polymerase amplification coupled with lateral flow dipstick

The dinoflagellate Karlodinium veneficum that is usually present at relatively low cell abundances is a globally-distributed harmful algal bloom-forming species, which negatively affects marine ecosystems, fisheries, and human health. Hence, an efficient detection platform for the rapid and sensitive identification of K. veneficum is highly demanded. In this study, a method referred to as recombinase polymerase amplification coupled with lateral flow dipstick (RPA-LFD) was developed for the rapid detection of K. veneficum. The primers for RPA and the detection probe for LFD were designed to specially target the internal transcribed spacer of K. veneficum by molecular cloning and multiple alignments of the related sequences. The developed RPA can gain an approximately 300 bp specific band from K. veneficum. Successful amplification for RPA could be achieved at a temperature range of 35 °C–45 °C. RPA for 30 min could produce enough products that could generate clearly visible electrophoresis bands and were adequate for subsequent LFD analysis. The RPA products can be visually detected by the naked eyes through an LFD after an automatic chromatography for 5 min at room temperature. The developed RPA-LFD was exclusively specific for K. veneficum and displayed no cross-reactivity with other algal species that are commonly distributed along the Chinese coast. In addition, the lowest detection limit of RPA-LFD was 10 ng μL⁻¹ of genomic DNA and 0.1 cell mL⁻¹, which was 100-fold sensitive than conventional PCR. In conclusion, the developed RPA-LFD assay in this study can be used as a rapid and sensitive method to monitor K. veneficum in the future.     

Phosphate Stress in Cultures and Field Populations of the Dinoflagellate Prorocentrum minimum Detected by a Single-Cell Alkaline Phosphatase Assay

Alkaline phosphatase activity is a common marker of phosphate stress in many phytoplankton, but it has been difficult to attribute alkaline phosphatase activity to specific organisms or groups of phytoplankton in the field with traditional biochemical procedures. A new alkaline phosphatase substrate, ELF-97 (enzyme-labeled fluorescence), shows promise in this regard. When a phosphate group is cleaved from the ELF-97 reagent, the remaining molecule precipitates near the site of enzyme activity, thus fluorescently tagging cells with alkaline phosphatase activity. We characterized ELF-97 labeling in axenic cultures of a common dinoflagellate, Prorocentrum minimum, in order to understand ELF-97 labeling dynamics when phosphate nutrition varies. Enzyme activity, as detected by ELF-97 labeling, appears to be induced in late-log- or early-stationary-phase cultures if cells are grown in low-phosphate media and is lost when phosphate-stressed cells are refed with phosphate. ELF-97 appears to label an inducible intracellular alkaline phosphatase in P. minimum based on confocal microscopy studies. This may limit the use of this reagent to organisms that lack high levels of constitutive intracellular phosphatases. After laboratory cultures were characterized, ELF-97 was used to assay field populations of P. minimum in Narragansett Bay during two 1-week periods, and 12 to 100% of the P. minimum cells were labeled. The level of cell labeling was reduced by 3 days of incubation with added inorganic phosphate. Our results indicate that ELF-97 is an excellent new tool for monitoring phytoplankton phosphate stress in the environment when the data are supported by appropriate laboratory studies.