Associated bacterial flora, growth, and toxicity of cultured benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus toxicus.

The growth, toxicity, and associated bacterial flora of 10 clonal cultures of the toxic benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus toxicus isolated from the coastal waters of southwest Puerto Rico have been examined. Clonal cultures of O. lenticularis grew more rapidly and at broader temperature ranges than those of G. toxicus. All five Ostreopsis clones were toxic, while only one of the five Gambierdiscus clones was poisonous. The degree of toxicity among poisonous clones was highly variable. The number of associated bacterial genera and their frequency of occurrence were quite variable among clones of both dinoflagellate genera. Bacterial isolates represented six genera (Nocardia, Pseudomonas, Vibrio, Aeromonas, Flavobacterium, and Moraxella) in addition to coryneform bacteria. Extracts of dinoflagellate-associated bacteria grown in pure culture were not toxic. Gambierdiscus clones were characterized by the frequent presence of Pseudomonas spp. (four of five clones) and the absence of coryneforms. In O. lenticularis, only one of five clones showed the presence of Pseudomonas spp., and Moraxella sp. was absent altogether. Detailed analyses of toxicity and associated microflora in a selected Ostreopsis clone, repeatedly cultivated (four times) over a period of 160 days, showed that peak cell toxicities developed in the late static and early negative culture growth phases. Peak Ostreopsis cell toxicities in the stationary phase of culture growth were correlated with significant increases in the percent total bacteria directly associated with these cells. Changes in the quantity of bacteria directly associated with microalgal cell surfaces and extracellular matrices during culture growth may be related to variability and degree of toxicity in these laboratory-cultured benthic dinoflagellates.     

Associated bacterial flora, growth, and toxicity of cultured benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus toxicus

The growth, toxicity, and associated bacterial flora of 10 clonal cultures of the toxic benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus toxicus isolated from the coastal waters of southwest Puerto Rico have been examined. Clonal cultures of O. lenticularis grew more rapidly and at broader temperature ranges than those of G. toxicus. All five Ostreopsis clones were toxic, while only one of the five Gambierdiscus clones was poisonous. The degree of toxicity among poisonous clones was highly variable. The number of associated bacterial genera and their frequency of occurrence were quite variable among clones of both dinoflagellate genera. Bacterial isolates represented six genera (Nocardia, Pseudomonas, Vibrio, Aeromonas, Flavobacterium, and Moraxella) in addition to coryneform bacteria. Extracts of dinoflagellate-associated bacteria grown in pure culture were not toxic. Gambierdiscus clones were characterized by the frequent presence of Pseudomonas spp. (four of five clones) and the absence of coryneforms. In O. lenticularis, only one of five clones showed the presence of Pseudomonas spp., and Moraxella sp. was absent altogether. Detailed analyses of toxicity and associated microflora in a selected Ostreopsis clone, repeatedly cultivated (four times) over a period of 160 days, showed that peak cell toxicities developed in the late static and early negative culture growth phases. Peak Ostreopsis cell toxicities in the stationary phase of culture growth were correlated with significant increases in the percent total bacteria directly associated with these cells. Changes in the quantity of bacteria directly associated with microalgal cell surfaces and extracellular matrices during culture growth may be related to variability and degree of toxicity in these laboratory-cultured benthic dinoflagellates.     

The effect of elevated temperature on the toxicity of the laboratory cultured dinoflagellate Ostreopsis lenticularis (Dinophyceae)

Ostreopsis lenticularis Fukuyo 1981, is the major benthic dinoflagellate vector implicated in ciguatera fish poisoning in finfish on the southwest coast of Puerto Rico. Clonal laboratory cultures of O. lenticularis (clone 301) exposed to elevated temperatures (30-31 degrees C) for 33 and 54 days showed significant increases in the quantity of extractable toxin they produced as compared to their toxicities versus cells grown at temperatures of 25-26 degrees C. O lenticularis samples collected directly from the field following exposure to elevated temperatures for comparable periods of time also showed significant increases in extractable toxin. The increased toxicity of both field sampled and laboratory grown O. lenticularis exposed to elevated temperatures may result from the effects of elevated temperatures on their metabolism and/or the bacterial symbionts found associated with these microalgae. The number of bacteria associated with cultured O. lenticularis exposed to elevated temperatures was significantly reduced. Increased toxin recovery from O. lenticularis exposed to elevated temperatures may have resulted from the direct effect of temperature on toxin production and/or the reduction of Ostreopsis associated bacterial flora that consume toxin in the process of their growth. This reduction in the quantity of associated bacterial flora in temperature treated cultures may result in increased toxin recovery from O. lenticularis due to a reduction in the consumption of toxin by these symbiont bacteria.     

Predator/Prey Interaction between <Emphasis Type="Italic">Pfiesteria piscicida</Emphasis> and <Emphasis Type="Italic">Rhodomonas</Emphasis> Mediated by a Marine Alpha Proteobacterium

The dinoflagellate Pfiesteria piscicida coexists with bacteria in aquatic environments and as such, may interact with them at the physiological level. This study was designed to investigate the influence of bacteria, present in a clonal culture of Pfiesteria piscicida, on the predator/prey relationship of this dinoflagellate with the alga Rhodomonas. A series of replenishment experiments with bacteria isolated from P. piscicida clonal culture and the bacteria-free P. piscicida derived from the same culture were carried out. In the presence of bacteria, the number of P. piscicida increased significantly when incubated with alga Rhodomonas. This enhanced growth was almost entirely due to the increased consumption rate of Rhodomonas by P. piscicida since in bacteria-free (axenic) cultures Rhodomonas were consumed at significantly reduced rates relative to cultures with bacteria. Subsequent replenishment experiments with individual bacterial isolates showed that a single isolate was responsible for the increased predation rate of P. piscicida. The presence or absence of this specific bacterium determined the outcome of the interaction between P. piscicida and Rhodomonas. Partial sequence analysis of the 16S rDNA of this isolate indicated that it was a novel marine alpha proteobacterium with sequence similarities to a Roseobacter sp. and a bacterium recently isolated from a toxic dinoflagellate Alexandrium sp.     

THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM (DINOPHYCEAE) REQUIRES MARINE BACTERIA FOR GROWTH1

Interactions with the bacterial community are increasingly considered to have a significant influence on marine phytoplankton populations. Here we used a simplified dinoflagellate‐bacterium experimental culture model to conclusively demonstrate that the toxic dinoflagellate Gymnodinium catenatum H. W. Graham requires growth‐stimulatory marine bacteria for postgermination survival and growth, from the point of resting cyst germination through to vegetative growth at bloom concentrations (10³ cells · mL^?1). Cysts of G. catenatum were germinated and grown in unibacterial coculture with antibiotic‐resistant or antibiotic‐sensitive Marinobacter sp. DG879 or Brachybacterium sp., and with mixtures of these two bacteria. Addition of antibiotics to cultures grown with antibiotic‐sensitive strains of bacteria resulted in death of the dinoflagellate culture, whereas cultures grown with antibiotic‐resistant bacteria survived antibiotic addition and continued to grow beyond the 21 d experiment. Removal of either bacterial type from mixed‐bacterial dinoflagellate cultures (using an antibiotic) resulted in cessation of dinoflagellate growth until bacterial concentration recovered to preaddition concentrations, suggesting that the bacterial growth factors are used for dinoflagellate growth or are labile. Examination of published reports of axenic dinoflagellate culture indicate that a requirement for bacteria is not universal among dinoflagellates, but rather that species may vary in their relative reliance on, and relationship with, the bacterial community. The experimental model approach described here solves a number of inherent and logical problems plaguing studies of algal‐bacterium interactions and provides a flexible and tractable tool that can be extended to examine bacterial interactions with other phytoplankton species.     

Effect of associated bacteria on the growth and toxicity of Alexandrium catenella

Saprophytic bacteria in cultures of the marine dinoflagellate Alexandrium catenella were removed to assess their effect on growth and paralytic shellfish poisoning toxin production of this dinoflagellate. The actual axenic status was demonstrated by the lack of observable bacteria both immediately after treatment and following extended incubation in the absence of antibiotics. Bacteria were measured by counting CFU and also by epifluorescence microscopy and PCR amplification of bacterial 16S-23S spacer ribosomal DNA to detect noncultivable bacteria. Removal of bacteria did not have any effect on the growth of the dinoflagellate except for the inhibition of A. catenella disintegration after reaching the stationary phase. Toxicity was determined in dinoflagellate cell extracts by different methods: high-performance liquid chromatography (HPLC); an electrophysiological test called the Electrotest, which measures the inhibition of saxitoxin-sensitive Na(+) channels expressed in a cell line; and a mouse bioassay, which measures the toxic effect on the whole mammal neuromuscular system. A lower toxicity of the dinoflagellates in axenic culture was observed by these three methods, though the difference was significant only by the mouse bioassay and HPLC methods. Altogether the results indicate that axenic cultures of A. catenella are able to produce toxin, though the total toxicity is probably diminished to about one-fifth of that in nonaxenic cultures.     

Tn5 MUTAGENESIS OF PSEUDOMONAS STUTZERI SF/PS, A BACTERIUM ASSOCIATED WITH ALEXANDRIUM LUSITANICUM (DINOPHYCEAE) AND PARALYTIC SHELLFISH POISONING

It is becoming increasingly clear that bacteria can play an important role in the toxin and population dynamics of harmful algal bloom (HAB) events. In this paper, we document protocols and strategies that can be used to identify bacterial genes involved in either the production of toxic compounds and/or the establishment and maintenance of relationships between bacteria and algae. The protocols we tested involved transposon mutagenesis and complementation with broad-host-range plasmids. We tested six bacterial strains thought to be involved, either directly or indirectly, in the production of toxins associated with paralytic shellfish poisoning (PSP). Five strains were resistant to transformation under the growth conditions used. However, a single strain, Pseudomonas stutzeri SF/PS, was readily transformed when grown under appropriate conditions. This bacterium has been shown to accumulate PSP toxins and to increase toxin production when added to axenic cultures of a toxic dinoflagellate, Alexandrium lusitanicum . We conclude that a transposon mutagenesis strategy can be used to identify genes involved in HAB events.     

Bacterial community associated with Pfiesteria-like dinoflagellate cultures

Dinoflagellates (Eukaryota; Alveolata; Dinophyceae) are single-cell eukaryotic microorganisms implicated in many toxic outbreaks in the marine and estuarine environment. Co-existing with dinoflagellate communities are bacterial assemblages that undergo changes in species composition, compete for nutrients and produce bioactive compounds, including toxins. As part of an investigation to understand the role of the bacteria in dinoflagellate physiology and toxigenesis, we have characterized the bacterial community associated with laboratory cultures of four ' Pfiesteria -like' dinoflagellates isolated from 1997 fish killing events in Chesapeake Bay. A polymerase chain reaction with oligonucleotide primers specific to prokaryotic 16S rDNA gene sequences was used to characterize the total bacterial population, including culturable and non-culturable species, as well as possible endosymbiotic bacteria. The results indicate a diverse group of over 30 bacteria species co-existing in the dinoflagellate cultures. The broad phylogenetic types of dinoflagellate-associated bacteria were generally similar, although not identical, to those bacterial types found in association with other harmful algal species. Dinoflagellates were made axenic, and the culturable bacteria were added back to determine the contribution of the bacteria to dinoflagellate growth. Confocal scanning laser fluorescence microscopy with 16S rDNA probes was used to demonstrate a physical association of a subset of the bacteria and the dinoflagellate cells. These data point to a key component in the bacterial community being species in the marine alpha-proteobacteria group, most closely associated with the α-3 or SAR83 cluster.     

A procedure to estimate okadaic acid in whole dinoflagellate cells using immunological techniques

A single procedure to detect and estimate okadaic acid in isolated whole cells was developed based on immunofluorescence and microscope photometry. This procedure allows the study of variations in okadaic acid concentration per cell although it is no substitute for HPLC procedures. Cells from mid-log exponential and stationary phase from two different clonal cultures of the okadaic-acid-producing dinoflagellate Prorocentrum lima (PI 5V and PI 7V) were analyzed. The results showed that: (1) cells from saturated phase cultures contain more okadaic acid than those from exponentially-growing mid-log phase; (2) genetic differences exist in okadaic acid production between the clones used; (3) okadaic acid is synthesized continuously during the whole cell cycle.     

Effect of Associated Bacteria on the Growth and Toxicity of Alexandrium catenella

Saprophytic bacteria in cultures of the marine dinoflagellate Alexandrium catenella were removed to assess their effect on growth and paralytic shellfish poisoning toxin production of this dinoflagellate. The actual axenic status was demonstrated by the lack of observable bacteria both immediately after treatment and following extended incubation in the absence of antibiotics. Bacteria were measured by counting CFU and also by epifluorescence microscopy and PCR amplification of bacterial 16S-23S spacer ribosomal DNA to detect noncultivable bacteria. Removal of bacteria did not have any effect on the growth of the dinoflagellate except for the inhibition of A. catenella disintegration after reaching the stationary phase. Toxicity was determined in dinoflagellate cell extracts by different methods: high-performance liquid chromatography (HPLC); an electrophysiological test called the Electrotest, which measures the inhibition of saxitoxin-sensitive Na⁺ channels expressed in a cell line; and a mouse bioassay, which measures the toxic effect on the whole mammal neuromuscular system. A lower toxicity of the dinoflagellates in axenic culture was observed by these three methods, though the difference was significant only by the mouse bioassay and HPLC methods. Altogether the results indicate that axenic cultures of A. catenella are able to produce toxin, though the total toxicity is probably diminished to about one-fifth of that in nonaxenic cultures.     

Isolation and characterization of a marine algicidal bacterium against the toxic dinoflagellate Alexandrium tamarense

Interactions between bacteria and harmful algal bloom (HAB) species have been acknowledged as an important factor regulating both the population dynamics and toxin production of these algae. A marine bacterium SP48 with algicidal activity to the toxic dinoflagellate, Alexandrium tamarense, was isolated from the Donghai Sea area, China. Genetic identification was achieved by polymerase chain reaction amplification and sequence analysis of 16S rDNA. Sequence analysis showed that the most probable affiliation of SP48 was to the γ-proteobacteria subclass and the genus Pseudoalteromonas. Bacterial isolate SP48 showed algicidal activity through an indirect attack. Additional organic nutrients but not algal-derived DOM was necessary for the synthesis of unidentified algicidal compounds but β-glucosidase was not responsible for the algicidal activity. The algicidal compounds produced by bacterium SP48 were heat tolerant, unstable in acidic condition and could be easily synthesized regardless of variation in temperature, salinity or initial pH for bacterial growth. This is the first report of a bacterium algicidal to the toxic dinoflagellate A. tamarense and the findings increase our knowledge of bacterial–algal interactions and the role of bacteria during the population dynamics of HABs.     

Polynucleobacter bacteria in the brackish-water species Euplotes harpa (Ciliata Hypotrichia)

We have found a Polynucleobacter bacterium in the cytoplasm of Euplotes harpa, a species living in a brackish-water habitat, with a cirral pattern not corresponding to that of the freshwater Euplotes species known to harbor this type of bacteria. The symbiont has been found in three strains of the species, obtained by clonal cultures from ciliates collected in different geographic regions. The 16S rRNA gene sequence of this bacterium identifies it as a member of the beta-proteobacterial genus Polynucleobacter. This sequence shares a high similarity value (98.4-98.5%) with P. necessarius, the type species of the genus, and is associated with 16S rRNA gene sequences of environmental clones and bacterial strains included in the Polynucleobacter cluster (>95%). An oligonucleotide probe was designed to corroborate the assignment of the retrieved sequence to the symbiont and to detect similar bacteria rapidly. Antibiotic experiments showed that the elimination of the bacteria stops the reproductive cycle in E. harpa, as has been shown for the freshwater Euplotes species.     

Dinoflagellate community analysis of a fish kill using denaturing gradient gel electrophoresis

A molecular method using the polymerase chain reaction (PCR) amplification of small subunit gene sequences (18S rDNA) and denaturing gradient gel electrophoresis (DGGE) was used to determine both the population complexity and species identification of organisms in harmful algal blooms. Eighteen laboratory cultures of dinoflagellates, including Akashiwo, Gymnodinium, Heterocapsa, Karenia, Karlodinium, Pfiesteria, and Pfiesteria-like species were analyzed using dinoflagellate-specific oligonucleotide primers and DGGE. The method is sensitive and able to determine the number of species in a sample, as well as the taxonomic identity of each species, and is particularly useful in detecting differences between species of the same genus, as well as differences between morphologically similar species. Using this method, each of eight Pfiesteria-like species was verified as being clonal isolates of Pfiesteria piscicida. The sensitivity of dinoflagellate DGGE is approximately 1000 cells/ml, which is 100-fold less sensitive than real-time PCR. However, the advantage of DGGE lies in its ability to analyze dinoflagellate community structure without needing to know what is there, while real-time PCR provides much higher sensitivity and detection levels, if probes exist for the species of interest, attributes that complement DGGE analysis. In a blinded test, dinoflagellate DGGE was used to analyze two environmental fish kill samples whose species composition had been previously determined by other analyses. DGGE correctly identified the dominant species in these samples as Karlodinium micrum and Heterocapsa rotundata, proving the efficacy of this method on environmental samples. Toxin analysis of a clonal isolate obtained from the fish kill samples confirmed the presence of KmTx2, corroborating the earlier genetic identification of toxic K. micrum in the fish kill water sample.     

Bacterial communities and toxin profiles of Ostreopsis (Dinophyceae) from the Pacific island of Okinawa,Japan

Variations in toxicity of the benthic dinoflagellate Ostreopsis Schmidt 1901 have been attributed to specific molecular clades, biogeography of isolated strains, and the associated bacterial community. Here, we attempted to better understand the biodiversity and the basic biology influencing toxin production of Ostreopsis. Nine clonal cultures were established from Okinawa, Japan, and identified using phylogenetic analysis of the ITS-5.8S rRNA and 28S rRNA genes. Morphological analysis suggests that the apical pore complex L/W ratio could be a feature for differentiating Ostreopsis sp. 2 from the O. ovata species complex. We analyzed the toxicity and bacterial communities using liquid chromatography-mass spectrometry, and PCR-free metagenomic sequencing. Ovatoxin was detected in three of the seven strains of O. cf. ovata extracts, highlighting intraspecies variation in toxin production. Additionally, two new potential analogs of ovatoxin-a and ostreocin-A were identified. Commonly associated bacteria clades of Ostreopsis were identified from the established cultures. While some of these bacteria groups may be common to Ostreopsis (Rhodobacterales, Flavobacteria-Sphingobacteria, and Enterobacterales), it was not clear from our analysis if any one or more of these plays a role in toxin biosynthesis. Further examination of biosynthetic pathways in metagenomic data and additional experiments isolating specific bacteria from Ostreopsis would aid these efforts.     

16S rRNA Targeted Probes for the Identification of Bacterial Strains Isolated from Cultures of the Toxic Dinoflagellate Alexandrium tamarense

Abstract Bacteria have been implicated in the production of paralytic shellfish poison (PSP) toxins, which are normally associated with bloom-forming algal species, specifically toxic dinoflagellate algae. To clarify the role that these bacteria may play in the production of PSP toxins, it is desirable to identify and localize the bacteria associated with the dinoflagellates. 16S rRNA-targeted probes offer the possibility for both, and thus, probes have been made to putatively toxigenic bacteria isolated from the PSP-related dinoflagellate Alexandrium tamarense and tested for their specificity in dot blot and in situ hybridization experiments. Received: 5 August 1999; Accepted: 19 January 2000; Online Publication: 5 May 2000;     

Identification and characterization of potentially algal-lytic marine bacteria strongly associated with the toxic dinoflagellate Alexandrium catenella

The toxic dinoflagellate Alexandrium catenella isolated from fjords in Southern Chile produces several analogues of saxitoxin and has been associated with outbreaks of paralytic shellfish poisoning. Three bacterial strains, which remained in close association with this dinoflagellate in culture, were isolated by inoculating the dinoflagellate onto marine agar. The phenotypically different cultivable bacterial colonies were purified. Their genetic identification was done by polymerase chain reaction amplification of the 16S rRNA genes. Partial sequence analysis suggested that the most probable affiliations were to two bacterial phyla: Proteobacteria and the Cytophaga group. The molecular identification was complemented by morphological data and biochemical profiling. The three bacterial species, when grown separately from phytoplankton cells in high-nutrient media, released algal-lytic compounds together with aminopeptidase, lipase, glucosaminidase, and alkaline phosphatase. When the same bacteria, free of organic nutrients, were added back to the algal culture they displayed no detrimental effects on the dinoflagellate cells and recovered their symbiotic characteristics. This observation is consistent with phylogenetic analysis that reveals that these bacteria correspond to species distinct from other bacterial strains previously classified as algicidal bacteria. Thus, bacterial-derived lytic activities are expressed only in the presence of high-nutrient culture media and it is likely that in situ environmental conditions may modulate their expression.     

Morphology and toxicology of Prorocentrum arabianum sp. nov., (dinophyceae) a toxic planktonic dinoflagellate from the Gulf of Oman, Arabian Sea

A new species of planktonic dinoflagellate, Prorocentrum arabianum isolated from the Gulf of Oman, is described using both scanning electron microscopy (SEM) and light microscopy. This clonal isolate has the following morphological characteristics: (1) cell shape is asymmetric; (2) thecal surface is rugose, covered with small poroids; (3) periflagellar area is unornamented, and (4) intercalary band is horizontally striated. Analysis of P. arabianum confirms the production of one cytotoxic compound and one ichthyotoxic compound. P. arabianum is the second known toxic planktonic Prorocentroid dinoflagellate.     

IDENTIFICATION OF BACTERIA ASSOCIATED WITH DINOFLAGELLATES (DINOPHYCEAE) ALEXANDRIUM SPP. USING TYRAMIDE SIGNAL AMPLIFICATION–FLUORESCENT IN SITU HYBRIDIZATION AND CONFOCAL MICROSCOPY1

In the marine environment, phytoplankton and bacterioplankton can be physically associated. Such association has recently been hypothesized to be involved in the toxicity of the dinoflagellate genus Alexandrium. However, the methods, which have been used so far to identify, localize, and quantify bacteria associated with phytoplankton, are either destructive, time consuming, or lack precision. In the present study we combined tyramide signal amplification–fluorescent in situ hybridization (TSA‐FISH) with confocal microscopy to determine the physical association of dinoflagellate cells with bacteria. Dinoflagellate attached microflora was successfully identified with TSA‐FISH, whereas FISH using monolabeled probes failed to detect bacteria, because of the dinoflagellate autofluorescence. Bacteria attached to entire dinoflagellates were further localized and distinguished from those attached to empty theca, by using calcofluor and DAPI, two fluorochromes that stain dinoflagellate theca and DNA, respectively. The contribution of specific bacterial taxa of attached microflora was assessed by double hybridization. Endocytoplasmic and endonuclear bacteria were successfully identified in the nonthecate dinoflagellate Gyrodinium instriatum. In contrast, intracellular bacteria were not observed in either toxic or nontoxic strains of Alexandrium spp. Finally, the method was successfully tested on natural phytoplankton assemblages, suggesting that this combination of techniques could prove a useful tool for the simultaneous identification, localization, and quantification of bacteria physically associated with dinoflagellates and more generally with phytoplankton.     

EVIDENCE FOR ASEXUAL RESTING CYSTS IN THE LIFE CYCLE OF THE MARINE PERIDINOID DINOFLAGELLATE,SCRIPPSIELLA HANGOEI1

Scrippsiella hangoei (Schiller) Larsen is a peridinoid dinoflagellate that grows during winter and spring in the Baltic Sea. In culture this species formed round, smooth cysts when strains were mixed, indicating heterothallic sexuality and hypnozygote production. However, cysts of the same morphology were also formed in clonal strains exposed to slightly elevated temperature. To better understand the role of cysts in the life cycle of S. hangoei, cyst formation and dormancy were examined in culture experiments and the cellular DNA content of flagellate cells and cysts was compared in clonal and mixed strains using flow cytometry. S. hangoei exhibited a high rate of cyst formation in culture. Cysts produced in both clonal and mixed strain cultures were thick‐walled and underwent a dormancy period of 4 months before germinating. The S. hangoei flagellate cell population DNA distributions consisted of 1C, intermediate, and 2C DNA, indicative of respective eukaryotic cell cycle phases G1, S, and G2M. The majority (>95%) of cysts had a measured DNA content equivalent to the lower 1C DNA value, indicating a haploid nuclear phase and an asexual mode of cyst formation. A small percentage (<5%) of cysts produced in the mixed strain culture had 2C DNA, and thus could have been diploid zygotes. These findings represent the first measurements of dinoflagellate resting cyst DNA content, and provide the first quantitative evidence for dinoflagellate asexual resting cysts. Asexual resting cysts may be a more common feature of dinoflagellate life cycles than previously thought.     

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.