David M. Kulis

Biogeography of toxic dinoflagellates in the genusAlexandrium from the northeastern United States and Canada

Twenty-eight strains of toxic dinoflagellates in the genusAlexandrium from the northeastern United States and Canada were characterized on the basis of morphology, bioluminescence capacity, mating compatibility, and toxin composition. The distributions of these characters were evaluated in the context of regional patterns of paralytic shellfish poisoning (PSP) and coastal hydrography. Two morphospecies were identified-A. tamarense Lebour andA. fundyense Balech. The two are interspersed geographically though there are areas, such as the Gulf of Maine, where apparently onlyA. fundyense occurs. Southern waters (Cape Cod, Connecticut, and Long Island) have especially diverse populations. The two species are sexually compatible. Virtually all northern isolates are bioluminescent, whereas southern isolates include bioluminescent and non-bioluminescent strains. Cluster analyses, based on high performance liquid chromatography (HPLC) determinations of the suite of toxins produced by each isolate, revealed two and perhaps three distinct groups. One is comprised almost exclusively of northern strains, and the other of southern strains. A Cape Cod cluster may be separable from the southern group. These analyses explain a previously reported north-to-south trend of decreasing toxicity, as the northern isolates produce greater proportions of the more potent toxins than do southern forms. The overall perspective is that the biogeography of toxicAlexandrium spp. in the study region is not that of a single, widespread, homogeneous population, but rather is comprised of several sub-populations, each with its own physiological characteristics and history. Two scenarios are considered with respect to this regional biogeography. The first invokes recent and continuing dispersal of isolates to the south from a center of origin in the north, followed by recombination and strong selection. The second holds that the northern and southern populations diverged from a common ancestor (vicariance), but now represent localized populations with little mixing of genotypes. Neither hypothesis can be completely refuted by the data presented here, though the weight of the evidence favors the latter. The correct scenario may be a combination of both, with recent and continuous speading occuring within the Gulf of Maine and perphaps the Gulf of St. Laerence, but with endemic localized populations persisting without genetic exchange in most southern locations. These data also indicate that although morphological criteria separate toxicAlexandrim isolates from the study region into two morphospecies, these assignments do not coincide with clusterings based on toxin composition or allozyme electrophoresis, and they are further violated by mating results. A revision of taxon designations to the varietal level could be justified.

Dynamics and physiology of saxitoxin production by the dinoflagellatesAlexandrium spp.

Toxin content (fmol cell−1) and a suite of elemental and macromolecular variables were measured in batch cultures of the dinoflagellatesAlexandrium fundyense, A. tamarense andAlexandrium sp. from the southern New England region, USA. A different perspective was provided by semicontinuous cultures which revealed sustained, steady-state physiological adaptations by cells to N and P limitation. Two types of variability were investigated. In batch culture, changes in nutrient availability with time caused growth stage variability in toxin content, which often peaked in mid-exponential growth. A second type of variability that could be superimposed on growth stage differences is best exemplified by the high toxin content of cells grown at suboptimal temperatures. Calculations of the net rate of toxin production (Rtox; fmol cell−1 d−1) for these different culture treatments and modes made it possible to separate the dynamics of toxin production from cell division. Over a wide range of growth rates, cells produced toxin at rates approximating those needed to replace “losses” to daughter cells during division. The exception to this direct proportionality was with P limitation, which was associated with a dramatic increase in the rate of toxin production as cells stopped dividing due to nutrient limitation in batch culture. Growth stage variability in batch culture thus reflects small imbalances (generally within a factor of two) between the specific rates of toxin production and cell division. N limitation and CO2 depletion both affect pathways involved in toxin synthesis before those needed for cell division; P limitation does the opposite. The patterns of toxin accumulation were the same as for major cellular metabolites or elemental pools. The highest rates of toxin production appear to result from an increased availability of arginine (Arg) within the cell, due to either a lack of competition for this amino acid from pathways involved in cell division or to increased de novo synthesis. There were no significant changes in toxin content with either acclimated growth at elevated salinity, or with short term increases or decreases of salinity. These results demonstrate that toxin production is a complex process which, under some conditions, is closely coupled to growth rate; under other conditions, these processes are completely uncoupled. Explanations for the observed variability probably relate to pool sizes of important metabolites and to the differential response of key biochemical reactions to these pool sizes and to environmental conditions.

SEXUALITY AND CYST FORMATION IN THE DINOFLAGELLATE GONYAULAX TAMARENSIS: CYST YIELD IN BATCH CULTURES1

Encystment of the toxic dinoflagellate Gonyaulax tamarensis Lebour (var. excavata) was monitored in batch cultures exposed to a variety of nutritional and environmental treatments. Limitation by nitrogen (as ammonium or nitrate) or phosphorus (as phosphate) resulted in cyst formation. When the initial concentration of limiting nutrient was varied, total cyst yield (mL−1) was directly proportional to the cell yield at all but the highest nutrient concentrations (where encystment was minimal). Encystment efficiency was relatively constant (0.1–0.2 cysts · cell−1) over a 5‐fold range of cell densities, indicating that 20 to 40% of the vegetative populations successfully encysted. Cyst formation was negligible in nutrient‐replete medium, even with a significant reduction in growth rate due to non‐optimal light, temperature, or to high batch culture cell densities. Low light levels did decrease cyst yield once encystment was initiated by nutrient limitation, but this was probably linked to smaller motile cell yield and not to a specific inhibition of encystment. In contrast, encystment was more sensitive to temperature than was growth rate: optimal cyst production occurred over a relatively narrow temperature range and no cysts were formed at [Page missing]

Toxin composition variations in one isolate of the dinoflagellate Alexandrium fundyense

A commonly accepted paradigm in the study of saxitoxin-producing dinoflagellates is that the total concentration of all toxins (toxin content) in one isolate can vary with growth conditions, but that the relative abundance of each toxin (toxin composition) does not change. We demonstrate here that dramatic changes in toxin composition do occur in one isolate of Alexandrium fundyense. In nitrogen- and phosphorus-limited semi-continuous cultures, toxin composition varied systematically with growth rate. When cells grew slowly under severe nutrient limitation, toxin composition was dominated by one or at most two toxin epimer pairs; as nutrient stresses eased at higher growth rates, the toxin profiles became more heterogeneous. Steady-state, sustained nitrogen limitation favored the production of toxins C 1,2 and GTX I,IV, whereas phosphorus limitation produced cells with high relative abundance of GTX II,III. STX reached its highest relative abundance when growth was most rapid. The lack of observed compositional changes in most past studies is probably not due to inherent differences in toxin biosynthetic pathways between the strains of Alexandrium examined, but rather to differences in the physiology of cells grown under different culturing modes (batch vs semi-continuous), methods of toxin analysis, and dominant toxins in the particular isolates examined.

Transcriptome Profiling of a Toxic Dinoflagellate Reveals a Gene-Rich Protist and a Potential Impact on Gene Expression Due to Bacterial Presence

Background Dinoflagellates are unicellular, often photosynthetic protists that play a major role in the dynamics of the Earths oceans and climate. Sequencing of dinoflagellate nuclear DNA is thwarted by their massive genome sizes that are often several times that in humans. However, modern transcriptomic methods offer promising approaches to tackle this challenging system. Here, we used massively parallel signature sequencing (MPSS) to understand global transcriptional regulation patterns in Alexandrium tamarense cultures that were grown under four different conditions. Methodology/Principal Findings We generated more than 40,000 unique short expression signatures gathered from the four conditions. Of these, about 11,000 signatures did not display detectable differential expression patterns. At a p-value < 1E-10, 1,124 signatures were differentially expressed in the three treatments, xenic, nitrogen-limited, and phosphorus-limited, compared to the nutrient-replete control, with the presence of bacteria explaining the largest set of these differentially expressed signatures. Conclusions/Significance Among microbial eukaryotes, dinoflagellates contain the largest number of genes in their nuclear genomes. These genes occur in complex families, many of which have evolved via recent gene duplication events. Our expression data suggest that about 73% of the Alexandrium transcriptome shows no significant change in gene expression under the experimental conditions used here and may comprise a “core” component for this species. We report a fundamental shift in expression patterns in response to the presence of bacteria, highlighting the impact of biotic interaction on gene expression in dinoflagellates.

Toxin production of Alexandrium minutum (Dinophyceae) from the Bay of Plenty, New Zealand.

Paralytic shellfish toxins of two clonal cultures of Alexandrium minutum isolated during the 1993 toxic shellfish events in the Bay of Plenty, New Zealand, were analyzed using high-performance liquid chromatography. Toxin composition profiles of both cultures showed neosaxitoxin (> 65 mole%) as the principal toxin, with saxitoxin and gonyautoxins (GTX1-4) as minor components. Neither C-toxins (C1-4) nor GTX5-6 were detectable in the two isolates. Bay of Plenty isolates of A. minutum have a unique toxin profile not found in any other isolates of this species that have been characterized. This weakens the hypothesis that A. minutum was recently introduced to New Zealand waters by ballast water or other long-distance transport mechanisms, and argues instead that the species was endemic to the area, but not noticed in the past. The average toxicity of the cultures was 8.8 and 11.0 pg saxitoxin equiv. cell-1 with acetic acid or HCl extraction, respectively. These are at the high end of the range of toxicity reported for A. minutum strains from around the world, and on a cell volume basis are comparable to the most toxic strains of the Alexandrium tamarense group. The toxin profile of A. minutum most closely matches that of mussels and to a lesser degree tuatua harvested from the Bay during the 1993 outbreak, but is quite different from the profile measured in scallops and pipi. Plausible mechanisms for bioconversion of the ingested algal toxins within the latter two shellfish species can be proposed, but it seems more likely that either other strains of A. minutum or other saxitoxin-producing dinoflagellates were ingested by those shellfish. This study established that A. minutum from the Bay of Plenty contains saxitoxins, has a unique toxin composition compared to all other isolates of this species, and was responsible for at least part of the PSP toxicity measured in shellfish during the 1993 outbreak.

DETECTION OF THE TOXIC DINOFLAGELLATE ALEXANDRIUM FUNDYENSE (DINOPHYCEAE) WITH OLIGONUCLEOTIDE AND ANTIBODY PROBES: VARIABILITY IN LABELING INTENSITY WITH PHYSIOLOGICAL CONDITION

 The toxic dinoflagellate Alexandrium fundyense Balech was grown under temperature‐ and nutrient‐limited conditions, and changes in labeling intensity on intact cells were determined for two probe types: an oligonucleotide probe targeting rRNA and a monoclonal antibody (MAb) targeting a cell surface protein. In nutrient‐replete batch culture, labeling with the rRNA probe was up to 400% brighter during exponential phase than during stationary phase, whereas MAb labeling did not change significantly with growth stage at the optimal growth temperature. In cultures grown at suboptimal, low temperatures, there was a significant difference between labeling intensity in stationary versus exponential phase for both probe types, with exponential cells labeling brighter with the rRNA probe and slightly weaker with the MAb. The decrease in rRNA probe labeling with increasing culture age was likely due to lower abundance of the target nucleic acid, as extracted RNA varied in a similar manner. With the MAb and the rRNA probes, slower growing cultures at low, nonoptimal temperature labeled 35% and 50% brighter than cells growing faster at warmer temperatures. Some differences in labeling intensity per cell disappeared when the data were normalized to surface area or volume, which indicated that the number of target antigens or rRNA molecules was relatively constant per unit area or volume, respectively. Slow growth accompanying phosphorus and nitrogen limitation resulted in up to a 400% decrease in labeling intensity with the rRNA probe compared to nutrient‐replete levels, whereas the MAb labeling intensity increased by a maximum of 60%. With both probes, labeling was more intense under phosphorus limitation than under nitrogen limitation, and for all conditions tested, labeling intensity was from 600% to 3600% brighter with the MAb than with the rRNA probe. Thus, it is clear that significant levels of variability in labeling intensity can be expected with both probe types because of the influence of environmental conditions and growth stage on cellular biochemistry, cell size,rRNA levels, and the number or accessibility of cell surface proteins. Of the two probes tested, the rRNA probe was the most variable, suggesting that in automated, whole‐cell assays, it can be used only in a semiquantitative manner. For manual counts, the human eye will likely accommodate the labeling differences. The MAb probe was less variable, and thus should be amenable to both manual and automated counts.

Paralytic shellfish poisoning in southern China

The rapidly expanding mariculture and commercial region along the southern coast of China has experienced sporadic outbreaks of paralytic shellfish poisoning for nearly 30 years, yet virtually nothing is known of the nature of that toxicity or of the causative organisms. This study presents the first direct comparisons of the high performance liquid chromatography toxin composition profiles of shellfish implicated in paralytic shellfish poisoning outbreaks in Daya Bay with Alexandrium tamarense cultures established from those waters. The three cultures that were analyzed produced an unusually high proportion of the low potency N-sulfocarbamoyl toxins C1 and C2 (nearly 90% of the total), and only trace quantities of the other saxitoxin derivatives. Total toxicity was thus very low with mild acid extraction, ranging between 7.2 and 12.7 fmole cell-1, or 0.7-0.9 pg saxitoxin equiv. cell-1. Following acid hydrolysis using the standard AOAC extraction method, the dominant toxins in the cultures were gonyautoxins 2 and 3 and decarbamoyl gonyautoxins 2 and 3. Total potency increased fourfold to 2.6-3.4 pg saxitoxin equiv. cell-1 following acid hydrolysis. These cultures are thus at the low end of the range of toxicities recorded for members of the A. tamarense species complex. Two scallop samples and one mussel sample collected from Daya Bay during paralytic shellfish poisoning episodes in 1990 and 1991 were also analyzed following the AOAC extraction procedure. The toxin profiles were similar for the three shellfish samples, in that the same suite of toxins were present in each, but the relative proportion of those toxins varied. The dominant toxins were gonyautoxins 2 and 3 and toxins C1-C4. Total toxicity was 336 and 654 micrograms saxitoxin per 100 g meat for the scallop samples, and 723 for the mussels. Toxins C3,4 were present in the shellfish at up to 22 mole%, but were not detected in cultures, even when mild acid was used for extraction. Despite the otherwise similar nature of the culture versus the shellfish toxin signatures, the presence of C3,4 indicates that another strain or species of Alexandrium, or possibly a paralytic shellfish poisoning-producing species of another genus was responsible for the 1990 and 1991 paralytic shellfish poisoning outbreaks in Daya Bay. Since the cultures analyzed were of low intrinsic toxicity, A. tamarense may be more widespread along the south coast of China than is suggested by the sporadic pattern of past paralytic shellfish poisoning outbreaks. Blooms with high cell density are required to generate sufficient toxin to be dangerous. The alarming increase in algal blooms in Chinese waters due to persistent and growing pollution may make these low toxicity populations more problematic in the future.

Evolution of Saxitoxin Synthesis in Cyanobacteria and Dinoflagellates

Dinoflagellates produce a variety of toxic secondary metabolites that have a significant impact on marine ecosystems and fisheries. Saxitoxin (STX), the cause of paralytic shellfish poisoning, is produced by three marine dinoflagellate genera and is also made by some freshwater cyanobacteria. Genes involved in STX synthesis have been identified in cyanobacteria but are yet to be reported in the massive genomes of dinoflagellates. We have assembled comprehensive transcriptome data sets for several STX-producing dinoflagellates and a related non-toxic species and have identified 265 putative homologs of 13 cyanobacterial STX synthesis genes, including all of the genes directly involved in toxin synthesis. Putative homologs of four proteins group closely in phylogenies with cyanobacteria and are likely the functional homologs of sxtA, sxtG, and sxtB in dinoflagellates. However, the phylogenies do not support the transfer of these genes directly between toxic cyanobacteria and dinoflagellates. SxtA is split into two proteins in the dinoflagellates corresponding to the N-terminal portion containing the methyltransferase and acyl carrier protein domains and a C-terminal portion with the aminotransferase domain. Homologs of sxtB and N-terminal sxtA are present in non-toxic strains, suggesting their functions may not be limited to saxitoxin production. Only homologs of the C-terminus of sxtA and sxtG were found exclusively in toxic strains. A more thorough survey of STX+ dinoflagellates will be needed to determine if these two genes may be specific to SXT production in dinoflagellates. The A. tamarense transcriptome does not contain homologs for the remaining STX genes. Nevertheless, we identified candidate genes with similar predicted biochemical activities that account for the missing functions. These results suggest that the STX synthesis pathway was likely assembled independently in the distantly related cyanobacteria and dinoflagellates, although using some evolutionarily related proteins. The biological role of STX is not well understood in either cyanobacteria or dinoflagellates. However, STX production in these two ecologically distinct groups of organisms suggests that this toxin confers a benefit to producers that we do not yet fully understand.

Fiber-Optic Microarray for Simultaneous Detection of Multiple Harmful Algal Bloom Species

ABSTRACT Harmful algal blooms (HABs) are a serious threat to coastal resources, causing a variety of impacts on public health, regional economies, and ecosystems. Plankton analysis is a valuable component of many HAB monitoring and research programs, but the diversity of plankton poses a problem in discriminating toxic from nontoxic species using conventional detection methods. Here we describe a sensitive and specific sandwich hybridization assay that combines fiber-optic microarrays with oligonucleotide probes to detect and enumerate the HAB species Alexandrium fundyense, Alexandrium ostenfeldii, and Pseudo-nitzschia australis. Microarrays were prepared by loading oligonucleotide probe-coupled microspheres (diameter, 3 μm) onto the distal ends of chemically etched imaging fiber bundles. Hybridization of target rRNA from HAB cells to immobilized probes on the microspheres was visualized using Cy3-labeled secondary probes in a sandwich-type assay format. We applied these microarrays to the detection and enumeration of HAB cells in both cultured and field samples. Our study demonstrated a detection limit of approximately 5 cells for all three target organisms within 45 min, without a separate amplification step, in both sample types. We also developed a multiplexed microarray to detect the three HAB species simultaneously, which successfully detected the target organisms, alone and in combination, without cross-reactivity. Our study suggests that fiber-optic microarrays can be used for rapid and sensitive detection and potential enumeration of HAB species in the environment.