Frances M. Van Dolah

Mortality of sea lions along the central California coast linked to a toxic diatom bloom.

Over 400 California sea lions (Zalophus californianus) died and many others displayed signs of neurological dysfunction along the central California coast during May and June 1998. A bloom of Pseudo-nitzschia australis (diatom) was observed in the Monterey Bay region during the same period. This bloom was associated with production of domoic acid (DA), a neurotoxin that was also detected in planktivorous fish, including the northern anchovy (Engraulis mordax), and in sea lion body fluids. These and other concurrent observations demonstrate the trophic transfer of DA resulting in marine mammal mortality. In contrast to fish, blue mussels (Mytilus edulus) collected during the DA outbreak contained no DA or only trace amounts. Such findings reveal that monitoring of mussel toxicity alone does not necessarily provide adequate warning of DA entering the food web at levels sufficient to harm marine wildlife and perhaps humans.

Microarray validation: factors influencing correlation between oligonucleotide microarrays and real-time PCR

Quantitative real-time PCR (qPCR) is a commonly used validation tool for confirming gene expression results obtained from microarray analysis; however, microarray and qPCR data often result in disagreement. The current study assesses factors contributing to the correlation between these methods in five separate experiments employing two-color 60-mer oligonucleotide microarrays and qPCR using SYBR green. Overall, significant correlation was observed between microarray and qPCR results (ρ=0.708, p<0.0001, n=277) using these platforms. The contribution of factors including up — vs. down-regulation, spot intensity, ρ-value, fold-change, cycle threshold (Ct), array averaging, tissue type, and tissue preparation was assessed. Filtering of microarray data for measures of quality (fold-change and ρ-value) proves to be the most critical factor, with significant correlations of ρτ;0.80 consistently observed when quality scores are applied.

Brevetoxicosis: Red tides and marine mammal mortalities

Potent marine neurotoxins known as brevetoxins are produced by the ‘red tide’ dinoflagellate Karenia brevis. They kill large numbers of fish and cause illness in humans who ingest toxic filter-feeding shellfish or inhale toxic aerosols. The toxins are also suspected of having been involved in events in which many manatees and dolphins died, but this has usually not been verified owing to limited confirmation of toxin exposure, unexplained intoxication mechanisms and complicating pathologies. Here we show that fish and seagrass can accumulate high concentrations of brevetoxins and that these have acted as toxin vectors during recent deaths of dolphins and manatees, respectively. Our results challenge claims that the deleterious effects of a brevetoxin on fish (ichthyotoxicity) preclude its accumulation in live fish, and they reveal a new vector mechanism for brevetoxin spread through food webs that poses a threat to upper trophic levels.

Saxitoxin Puffer Fish Poisoning in the United States, with the First Report of Pyrodinium bahamense as the Putative Toxin Source

Background From January 2002 to May 2004, 28 puffer fish poisoning (PFP) cases in Florida, New Jersey, Virginia, and New York were linked to the Indian River Lagoon (IRL) in Florida. Saxitoxins (STXs) of unknown source were first identified in fillet remnants from a New Jersey PFP case in 2002. Methods We used the standard mouse bioassay (MBA), receptor binding assay (RBA), mouse neuroblastoma cytotoxicity assay (MNCA), Ridascreen ELISA, MIST Alert assay, HPLC, and liquid chromatography-mass spectrometry (LC-MS) to determine the presence of STX, decarbamoyl STX (dc-STX), and N-sulfocarbamoyl (B1) toxin in puffer fish tissues, clonal cultures, and natural bloom samples of Pyrodinium bahamense from the IRL. Results We found STXs in 516 IRL southern (Sphoeroides nephelus), checkered (Sphoeroides testudineus), and bandtail (Sphoeroides spengleri) puffer fish. During 36 months of monitoring, we detected STXs in skin, muscle, and viscera, with concentrations up to 22,104 μg STX equivalents (eq)/100 g tissue (action level, 80 μg STX eq/100 g tissue) in ovaries. Puffer fish tissues, clonal cultures, and natural bloom samples of P. bahamense from the IRL tested toxic in the MBA, RBA, MNCA, Ridascreen ELISA, and MIST Alert assay and positive for STX, dc-STX, and B1 toxin by HPLC and LC-MS. Skin mucus of IRL southern puffer fish captive for 1-year was highly toxic compared to Florida Gulf coast puffer fish. Therefore, we confirm puffer fish to be a hazardous reservoir of STXs in Florida’s marine waters and implicate the dinoflagellate P. bahamense as the putative toxin source. Conclusions Associated with fatal paralytic shellfish poisoning (PSP) in the Pacific but not known to be toxic in the western Atlantic, P. bahamense is an emerging public health threat. We propose characterizing this food poisoning syndrome as saxitoxin puffer fish poisoning (SPFP) to distinguish it from PFP, which is traditionally associated with tetrodotoxin, and from PSP caused by STXs in shellfish.

Health and Ecological Impacts of Harmful Algal Blooms: Risk Assessment Needs

The symposium session, Indicators for Effects and Predictions of Harmful Algal Blooms, explored the current state of indicators used to assess the human health and ecological risks caused by harmful algal blooms, and highlighted future needs and impediments that must be overcome in order to provide a complete risk assessment of their impacts. Six recognized human poisoning syndromes resulting from algal toxins (paralytic, neurotoxic, amnesic, diarrhetic shellfish poisonings, ciguatera fish poisoning, and putative estuary associated syndrome) impact human health through consumption of contaminated seafood, direct contact with bloom water, or inhalation of aerosolized toxin. Thorough health risk assessment for the variety of algal toxins is hampered to varying degrees because either the toxin has not been identified or indicators for exposure and effects remain poorly defined. Predicting the occurrence and determining the impacts of harmful algal blooms in coastal ecosystems are the two major ecological risk assessment needs. In the former case, the hazard is the suite of conditions that trigger bloom initiation, magnify bloom intensity or support bloom longevity, whereas in the latter case, the hazard is the algal toxin. In both cases, indicators (of triggering mechanisms, exposure, and effects) are better defined for some HAB species and toxins than others, but are by no means complete.

How are climate and marine biological outbreaks functionally linked

Since the mid-1970s, large-scale episodic events such as disease epidemics, mass mortalities, harmful algal blooms and other population explosions have been occurring in marine environments at an historically unprecedented rate. The variety of organisms involved (host, pathogens and other opportunists) and the absolute number of episodes have also increased during this period. Are these changes coincidental? Between 1972 and 1976, a global climate regime shift took place, and it is manifest most clearly by a change in strength of the North Pacific and North Atlantic pressure systems. Consequences of this regime shift are: (1) prolonged drought conditions in the Sahel region of Africa; (2) increased dust supply to the global atmosphere, by a factor of approximately four; (3) increased easterly trade winds across the Atlantic; (4) increased eolian transport of dust to the Atlantic and Caribbean basins; and (5) increased deposition of iron-rich eolian dust to typically iron-poor marine regions. On the basis of well-documented climate and dust observations and the widely accepted increase in marine outbreak rates, this paper proposes that the increased iron supply has altered the micronutrient factors limiting growth of opportunistic organisms and virulence of pathogenic microbes, particularly in macronutrient-rich coastal systems.

MICROARRAY ANALYSIS OF DIURNAL- AND CIRCADIAN-REGULATED GENES IN THE FLORIDA RED-TIDE DINOFLAGELLATE KARENIA BREVIS (DINOPHYCEAE)1

The photoperiod plays a central role in regulating the physiology and behavior of photosynthetic phytoplankton, and many of these processes are controlled by an underlying circadian rhythm. In dinoflagellates, circadian rhythms have been shown to depend largely on posttranscriptional regulation. However, the extent to which dinoflagellates modulate transcript levels to regulate gene expression in response to light and dark has not been addressed. Here we utilized an oligonucleotide microarray containing probes to 4629 unique genes from the Florida red‐tide dinoflagellate Karenia brevis (C. C. Davis) G. Hansen et Moestrup to characterize global gene expression patterns over a 24 h day in cells exposed to a 16:8 light:dark cycle (LD treatment) and cells under constant light (LL treatment). We determined that 9.8% of the queried genes were differentially expressed during LD, while 3% of genes were differentially expressed over the 24 h day when exposed to LL. Most genes exhibited either peak or minimum expression in early dark phase. Of the 453 differentially expressed genes in LD, 104 were assigned predicted annotations based on BLASTX searches. Few of the identifiable genes that were differentially expressed in LD appear to be under circadian control, as their cyclical expression did not persist in LL. Analysis of coordinately expressed genes revealed several novel insights into dinoflagellate gene expression, including an unusually high representation of genes involved in post‐transcriptional processing of RNA and protein turnover, the regulation of PSII genes in response to light and dark, and the absence of transcriptional regulation of cell‐cycle genes.

DIEL PHASING OF THE CELL‐CYCLE IN THE FLORIDA RED TIDE DINOFLAGELLATE, GYMNODINIUM BREVE

The diel cycle is a key regulator of the cell‐cycle in many dinoflagellates, but the mechanisms by which the diel cycle entrains the cell‐cycle remain poorly understood. In this study, we describe diel phasing of the cell‐cycle in the Florida red tide dinoflagellate Gymnodinium breve Davis, determine the diel cue which serves to entrain the cell‐cycle, and provide evidence for the presence of cyclin‐dependent kinase (CDK), a cell‐cycle regulator which may be responsive to this cue. Four laboratory isolates from the West Coast of Florida were compared. When grown on a 16:8 h LD cycle, all isolates displayed phased cell division, with the S‐phase beginning 6–8 h into the light phase, and mitosis following 12–14 h later, as determined by flow cytometry. A naturally occurring bloom of G. breve, studied over one diel cycle, displayed diel cell‐cycle phasing similar to that in the laboratory cultures, with the S‐phase beginning during daylight and the peak of mitosis occurring approximately 4 h after sunset. In the laboratory cultures, the dark/light “dawn” transition was found to provide the diel cue which serves to entrain the G. breve cell‐cycle, whereas the light/ dark “dusk” transition did not appear to be involved. Evidence for the presence of CDK in G. breve was obtained using two approaches: (1) identification of a 34‐kDa protein, immunoreactive to an antibody against a conserved amino acid sequence (α‐PSTAIR) unique to the CDK protein family and (2) inhibition of the cell‐cycle by olomoucine, a selective CDK inhibitor. Together, these results provide the basis from which one can begin addressing mechanisms by which the diel cycle regulates the cell‐cycle in G. breve.

The role of domoic acid in abortion and premature parturition of California sea lions (Zalophus californianus) on San Miguel Island, California.

Domoic acid is a glutaminergic neurotoxin produced by marine algae such as Pseudo-nitzschia australis. California sea lions (Zalophus californianus) ingest the toxin when foraging on planktivorous fish. Adult females comprise 60% of stranded animals admitted for rehabilitation due to acute domoic acid toxicosis and commonly suffer from reproductive failure, including abortions and premature live births. Domoic acid has been shown to cross the placenta exposing the fetus to the toxin. To determine whether domoic acid was playing a role in reproductive failure in sea lion rookeries, 67 aborted and live-born premature pups were sampled on San Miguel Island in 2005 and 2006 to investigate the causes for reproductive failure. Analyses included domoic acid, contaminant and infectious disease testing, and histologic examination. Pseudo-nitzschia spp. were present both in the environment and in sea lion feces, and domoic acid was detected in the sea lion feces and in 17% of pup samples tested. Histopathologic findings included systemic and localized inflammation and bacterial infections of amniotic origin, placental abruption, and brain edema. The primary lesion in five animals with measurable domoic acid concentrations was brain edema, a common finding and, in some cases, the only lesion observed in aborted premature pups born to domoic acid–intoxicated females in rehabilitation. Blubber organochlorine concentrations were lower than those measured previously in premature sea lion pups collected in the 1970s. While the etiology of abortion and premature parturition was varied in this study, these results suggest that domoic acid contributes to reproductive failure on California sea lion rookeries.

Transcriptional profiling and inhibition of cholesterol biosynthesis in human T lymphocyte cells by the marine toxin azaspiracid.

Azaspiracid-1 (AZA-1) is a marine biotoxin reported to accumulate in shellfish from several countries, including eastern Canada, Morocco, and much of western Europe, and is frequently associated with severe gastrointestinal human intoxication. As the mechanism of action of AZA-1 is currently unknown, human DNA microarrays and qPCR were used to profile gene expression patterns in human T lymphocyte cells following AZA-1 exposure. Some of the early (1 h) responding genes consisted of transcription factors, membrane proteins, receptors, and inflammatory genes. Four- and 24-h responding genes were dominated by genes involved in de novo lipid biosynthesis of which 17 of 18 involved in cholesterol biosynthesis were significantly up regulated. The up regulation of synthesis genes was likely in response to the ca. 50% reduction in cellular cholesterol, which correlated with up regulated protein expression levels of the low-density lipoprotein receptor. These data collectively detail the inhibition of de novo cholesterol synthesis, which is the likely cause of cytotoxicity and potentially a target pathway of the toxin.