Detection of Pfiesteria spp. by PCR in surface sediments collected from Chesapeake Bay tributaries (Maryland)

In 1997 blooms of Pfiesteria piscicida occurred in association with fish kills and human health problems in tributaries of the Chesapeake Bay (Maryland) and the scientific and media response resulted in large economic losses in seafood sales and tourism. These events prompted the Maryland Department of Natural Resources (MDNR) to begin monitoring for Pfiesteria spp. in water column samples. Real-time PCR assays targeted to the 18S rRNA gene were developed by our laboratories and utilized in conjunction with traditional microscopy and fish kill bioassays for detection of these organisms in estuarine water samples. This monitoring strategy aided in determining temporal and spatial distribution of motile forms of Pfiesteria spp. (i.e. zoospores), but did not assess resting stages of the dinoflagellates’ life cycle. To address this area, a 3-year study was designed using real-time PCR assays for analysis of surface sediment samples collected from several Chesapeake Bay tributaries. These samples were tested with the real-time PCR assays previously developed by our laboratories. The data reported herein suggest a strong positive association between presence of Pfiesteria spp. in the sediment and water column, based on long-term water column monitoring data. P. piscicida is detected more commonly in Maryland's estuarine waters than Pfiesteria shumwayae and sediment ‘cyst beds’ may exist for these organisms.     

Direct uptake of nitrogen by Pfiesteria piscicida and Pfiesteria shumwayae, and nitrogen nutritional preferences

The rates of uptake of a range of forms of nitrogenous nutrients were measured in cultures of Pfiesteria piscicida and Pfiesteria shumwayae maintained at varying physiological states. The measured rates of dissolved N uptake under some conditions approached the rates of N uptake that are achieved through phagotrophy. Rates of dissolved N uptake by P. piscicida contributed <10% of the cellular N of flagellated cells feeding on algae, but were equal to or greater than phagotrophic N acquisition in cells recently removed from fish cultures. Specific N uptake rates (V, h⁻¹) were higher for cells that were maintained on algal prey for long periods (months) than those that were grown with live fish. However, rates of N uptake on a cellular basis for cells grown on or recently removed from fish were comparable to those maintained on algal prey, likely reflecting differences in the sizes of cells of different physiological condition. Preferences for form of N generally followed a decreasing trend of amino acids > urea > NH₄⁺ > NO₃⁻. Nitrate consistently was not a preferred form of N. Although Pfiesteria spp. are often found in eutrophic environments, the relationship between Pfiesteria spp. and nutrient availability is likely to be primarily indirect, mediated through the production of various prey on which Pfiesteria spp. feed. These findings also confirm, however, that when dissolved N concentrations are elevated, they can contribute to the supplemental nutrition of these cells, and thus may provide a significant source of N to Pfiesteria spp. in nature.     

Pfiesteria piscicida and Pfiesteria shumwayae in coastal waters of Long Island, New York, USA

Water and sediment samples were collected during summer and early fall 1999–2004 from coastal waters of New York State, USA, to test for the presence of Pfiesteria piscicida and Pfiesteria shumwayae. Physical and chemical conditions were characterized, and real-time polymerase chain reaction assays were conducted. Both species were relatively common and found at most sites at least once, and the frequency of positive assays was higher in sediments than in the water column. In a subset of the data from Suffolk County, Long Island, the presence of Pfiesteria was related to high chlorophyll a and relatively high nutrient concentrations. Partial SSU rDNA sequences of four PCR amplicons generated using P. shumwayae primers indicated two sequences: three were identical to GenBank P. shumwayae entries, but one showed enough sequence difference (15 positions in a 454 bp amplicon) to suggest a possible new species. Three isolates were tested for toxicity, and one was found to kill fish in bioassays. Despite the widespread presence of both Pfiesteria species and demonstration of potential to harm fish, no blooms of these dinoflagellates have been observed, nor has there been evidence of Pfiesteria-related fish or human health problems in these waters, likely related to colder temperatures than optimal for Pfiesteria species.     

Characterization of Ichthyocidal Activity of Pfiesteria piscicida: Dependence on the Dinospore Cell Density

The ichthyocidal activity of Pfiesteria piscicida dinospores was examined in an aquarium bioassay format by exposing fish to either Pfiesteria-containing environmental sediments or clonal P. piscicida. The presence of Pfiesteria spp. and the complexity of the microbial assemblage in the bioassay were assessed by molecular approaches. Cell-free water from bioassays that yielded significant fish mortality failed to show ichthyocidal activity. Histopathological examination of moribund and dead fish failed to reveal the skin lesions reported elsewhere. Fish larvae within “cages” of variable mesh sizes were killed in those where the pore size exceeded that of Pfiesteria dinospores. In vitro exposure of fish larvae to clonal P. piscicida indicated that fish mortality was directly proportional to the dinospore cell density. Dinospores clustered around the mouth, eyes, and operculi, suggesting that fish health may be affected by their direct interaction with skin, gill epithelia, or mucous surfaces. Molecular fingerprinting revealed the presence of a very diverse microbial community of bacteria, protists, and fungi within bioassay aquaria containing environmental sediments. Some components of the microbial community were identified as potential fish pathogens, preventing the rigorous identification of Pfiesteria spp. as the only cause of fish death. In summary, our results strongly suggest (i) that this aquarium bioassay format, which has been extensively reported in the literature, is unsuitable to accurately assess the ichthyocidal activity of Pfiesteria spp. and (ii) that the ichthyocidal activity of Pfiesteria spp. is mostly due to direct interactions of the zoospores with fish skin and gill epithelia rather than to soluble factors.     

Toxigenic Pfiesteria species—Updates on biology,ecology, toxins,and impacts

The genus Pfiesteria includes two toxigenic species, Pfiesteria piscicida and Pfiesteria shumwayae, that are thinly thecate dinoflagellates with apparently cosmopolitan distribution, especially in shallow, poorly flushed, eutrophic estuaries. They are heterotrophic prey generalists that typically feed via phagotrophy and prefer live fish or their fresh tissues as food. They can also engage in limited mixotrophy through temporary retention of kleptochloroplasts from algal prey. Toxicity is highly variable among strains, ranging from apparently nontoxic to highly toxic. Some strains produce a group of hydrophilic toxins with metal-mediated free radical production. Various metals can be involved in the toxin congeners, and the purified toxins are highly labile. These toxins can adversely affect mammalian cells as well as fish. Toxic strains are capable of killing fish by both toxins and physical attack from feeding upon epidermis and other tissues. Non-inducible strains do not produce sufficient toxin to kill fish, but some are capable of causing larval fish death by physical attack. From 1991 to 1998, Pfiesteria spp. were linked to major kills of juvenile Atlantic menhaden (Brevoortia tyrannus), mostly at densities of ≥4(3) × 10² to 10³ (rarely, 10⁴) flagellate cells mL⁻¹. These kills mainly occurred in the second largest and largest estuaries on the U.S. mainland, especially two main tributaries of the Albemarle-Pamlico Estuarine System, following decades of hurricane-free conditions. Between kills, Pfiesteria abundance was low in surface waters (<10 cells mL⁻¹), and the available evidence suggests that the populations were mostly in the lower water column and within surficial sediments. Apparently highly sensitive to scouring effects from major storms, Pfiesteria populations have been sparse in the affected estuaries since several hurricanes struck the Albemarle-Pamlico in the late 1990s. Recent research highlights include characterization of a novel group of Pfiesteria toxins, culture of a toxigenic strain on a sterile fish cell line, axenic culture on a semi-defined medium, the discovery of a new mode of heterotrophic feeding in dinoflagellates as manifested by Pfiesteria, and other advances in understanding the nutritional ecology and prey acquisition of these harmful dinoflagellates.     

Effect of Biotic and Abiotic Factors on In Vitro Proliferation, Encystment, and Excystment of Pfiesteria piscicida

Pfiesteria spp. are mixotrophic armored dinoflagellates populating the Atlantic coastal waters of the United States. They have been a focus of intense research due to their reported association with several fish mortality events. We have now used a clonal culture of Pfiesteria piscicida and several new environmental isolates to describe growth characteristics, feeding, and factors contributing to the encystment and germination of the organism in both laboratory and environmental samples. We also discuss applied methods of detection of the different morphological forms of Pfiesteria in environmental samples. In summary, Pfiesteria, when grown with its algal prey, Rhodomonas sp., presents a typical growth curve with lag, exponential, and stationary phases, followed by encystment. The doubling time in exponential phase is about 12 h. The profiles of proliferation under a standard light cycle and in the dark were similar, although the peak cell densities were markedly lower when cells were grown in the dark. The addition of urea, chicken manure, and soil extracts did not enhance Pfiesteria proliferation, but crude unfiltered spent aquarium water did. Under conditions of food deprivation or cold (4°C), Pfiesteria readily formed harvestable cysts that were further analyzed by PCR and scanning electron microscopy. The germination of Pfiesteria cysts in environmental sediment was enhanced by the presence of live fish: dinospores could be detected 13 to 15 days earlier and reached 5- to 10-times-higher peak cell densities with live fish than with artificial seawater or f/2 medium alone. The addition of ammonia, urea, nitrate, phosphate, or surprisingly, spent fish aquarium water had no effect.     

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.     

Growth responses of the mixotrophic dinoflagellates, Cryptoperidiniopsis sp. and Pfiesteria piscicida, to light under prey-saturated conditions

Recent research emphasis on the ecology of Pfiesteria spp. (Dinophyceae) has led to recognition of several morphologically similar heterotrophic dinoflagellates that often co-occur with Pfiesteria spp. in estuaries along the United States Atlantic coast. These include cryptoperidiniopsoid dinoflagellates, which resemble Pfiesteria spp. in having complex life cycles that include zoospores capable of kleptoplastidy. To examine and compare the role of kleptoplastidy in Cryptoperidiniopsis sp. and Pfiesteria piscicida, we tested the effects of irradiance on growth under prey-saturated (Storeatula major, Cryptophyceae) conditions. Growth of Cryptoperidiniopsis was strongly influenced by light intensity while no major effects were observed in P. piscicida. In Cryptoperidiniopsis, highest cell numbers and specific growth rates, but lowest specific cryptophyte consumption rates, were found at the highest light intensity tested (100 μmol photons m⁻² s⁻¹). A growth model was developed and used to estimate that the average half-life of chloroplasts ingested by Cryptoperidiniopsis decreased 3.4-fold from 12.6 h at high light to 3.7 h in the dark. These results show that light strongly enhances specific growth rate and growth efficiency of Cryptoperidiniopsis feeding on cryptophytes, and suggest that retained kleptochloroplasts may play a quantitatively significant role in carbon and energy metabolism of this organism. Differences in the effects of light between Cryptoperidiniopsis and P. piscicida may reflect different nutritional strategies, and allow these closely related dinoflagellates to occupy different niches and co-exist.     

Relative contribution of exotoxin and micropredation to icthyotoxicity of two strains of Pfiesteria shumwayae (Dinophyceae)

The mechanism by which Pfiesteria shumwayae (Glasgow and Burkholder) kills fish is controversial. Several studies have implicated a Pfiesteria-associated exotoxin in fish mortality while other studies indicate that physical attack of dinoflagellates on fish (micropredation) and not exotoxin is responsible. We examined the ichthyotoxicity of two strains of P. shumwayae (CAAE 101272 and CCMP 2089) in a bioassay system that exposed test fish to the dinoflagellates both with and without direct contact in the same aquarium at the same time. Dinoflagellate-free supernatants from both strains were also tested for toxicity. The results showed that direct contact between P. shumwayae and fish significantly enhanced fish mortality with both strains (P < 0.001). About 87.5% and 100% of fish died when exposed directly to CAAE 101272 and CCMP 2089, respectively. When protected from direct contact with Pfiesteria cells, 19% of the fish exposed to CAAE 101272 and 6% of those exposed to CCMP 2089 died. No deaths were observed in controls. Supernatant killed fish when obtained from cultures of CAAE 101272 but not when obtained from CCMP 2089.Analysis of variance showed that, for both strains, fish mortality in Pfiesteria-inoculated bioassays was significantly higher than control bioassays both with and without direct contact (P < 0.001). Differences between strains were not significant (P = 0.3). These results indicate that both strains are associated with exotoxin production. However, the dominant and most consistent mechanism of fish mortality observed in this study required physical contact between fish and Pfiesteria cells.     

Physical, chemical and microbiological quality of ice used to cool drinks and foods in Greece and its public health implications

Ice used for direct human consumption or to preserve foods and cool down drinks can be contaminated with pathogenic microorganisms and may potentially become a vehicle for consumer’s infection. To evaluate physical, chemical and microbiological quality of commercial ice and ice used for fish and seafood, 100 ice samples collected at 10 different retail points in the region of Epirus were studied. The following microbiological parameters were determined: Total coliforms, fecal coliforms, Salmonella spp., Shigella spp., Yersinia spp., Escherichia coli, Campylobacter sp., Vibrio cholerae, Aeromonas spp., Pseudomonas aeruginosa and Clostridium perfringens.E. coli was detected in 22% and coliforms were detected in 31% of samples. Samples in which coliforms were detected fail to meet the microbiological criteria specified by the drinking water legislation.Aeromonas spp., Shigella spp., Campylobacter sp. and V. cholerae were not detected. Spore forms of C. perfringens were prevalent at 35% and the psychotropic bacterium’s P. aeruginosa and Yersinia spp. were found only at three samples each.The presence of large numbers of coliforms as well as of other pathogenic strains suggested that commercial ice and ice used to make cool drinks or in preservation of fish and seafood may represent a potential hazard to the consumer. In view of the results reported herein, it is highly recommended that national regulatory guidelines should be established for the production of ice as long as regular inspections.     

Comparative toxicity of Pfiesteria spp., prolonging toxicity of P. piscicida in culture and evaluation of toxin(s) stability

Toxicity of Pfiesteria piscicida (strain CAAE #2200) in the presence of fish (juvenile hybrid tilapia, Oreochromis sp., total length 3–6 cm) has been maintained in the laboratory for 19 months by serial transfer of toxic cells using a modified maintenance protocol. Toxicity was re-induced when toxin-producing P. piscicida cells were separated from fish and cultured on algal prey for 50 days and then re-introduced to new tanks containing fish. We confirmed toxicity in a strain of P. shumwayae (strain CAAE #101272). Toxicity to fish was demonstrated in culture filtrates (0.2 μm) derived from cultures of both Pfiesteria spp., however, it was markedly reduced in comparison to unfiltered water. Filtrates retained toxic activity when stored at −20 °C for up to 6 months. Toxicity to fish was retained when filtrates were held at room temperature for 48 h, at 70 °C for 30 min or at 88–92 °C for 2 h. P. piscicida killed all finfish species tested. Grass shrimp (Paleomonetes pugio; adult 2–3 cm), blue crab (Callinectes sapidus; juvenile 4–7 cm) and brine shrimp (Artemia sp.; 18–24 h post-hatch) were unaffected by concentrations of toxin(s) that killed juvenile tilapia in 4–24 h. Ichthyotoxic activity of filtrates from fish-killing cultures and stability of the toxic activity were similar among P. piscicida and P. shumwayae. These results confirm previously reported observations on toxicity of P. piscicidaand P. shumwayae to finfish. We have maintained toxicity in the laboratory for longer periods than have previously been routinely achieved, and we have demonstrated that the toxic activity is heat stable. In contrast to previous studies with other toxic P. piscicida strains, we did not observe toxic activity to blue crabs or other crustaceans.     

Effect of small-scale shear on grazing and growth of the dinoflagellate Pfiesteria piscicida

Low turbulence environments have been hypothesized to be necessary for toxic outbreaks of the heterotrophic/mixotrophic dinoflagellate Pfiesteria piscicida. A toxic Pfiesteria outbreak occurred in the shallow waters (the flats) of the lower Pocomoke River, MD, USA, in 1997. During August 1999 and May and August 2000, data were collected with a Shallow Water ADV Turbulence Tripod (SWATT) to estimate turbulent shear at a location monitored for Pfiesteria on the flats. Approximately 78% of the observed shears were ≤1 s⁻¹ and 98% were ≤2 s⁻¹. Densities of Pfiesteria-like dinoflagellates were low in the Pocomoke River during 1999 and 2000 and no toxic outbreaks occurred. In the laboratory, Couette cylinders were used to determine the effect of small-scale shear on feeding and growth of cultured P. piscicida growing on cryptophyte prey. Shear of 1 s⁻¹ had little or no effect on feeding or growth but 3 s⁻¹ reduced feeding and survival/growth of Pfiesteria zoospores. Small, rapidly dividing Pfiesteria cells were most susceptible to negative effects of shear. Pfiesteria appears to be more sensitive to small-scale shear than are most autotrophic dinoflagellates. However, in the lower Pocomoke River, turbulent shear is rarely high enough to inhibit the growth of non-toxic Pfiesteria zoospores. Toxic functional types (TOX-A and TOX-B) may be more sensitive to small-scale shear; it will be important to determine the responses of these types of Pfiesteria in order to predict the affects of wind and tidal mixing on toxic outbreaks.     

On the control of HAB species using low biosurfactant concentrations

Biosurfactants have been suggested as a method to control harmful algal blooms (HABs), but warrant further and more in-depth investigation. Here we have investigated the algicidal effect of a biosurfactant produced by the bacterium Pseudomonas aeruginosa on five diverse marine and freshwater HAB species that have not been tested previously. These include Alexandrium minutum (Dinophycaee), Karenia brevis (Dinophyceae), Pseudonitzschia sp. (Bacillariophyceae), in marine ecosystems, and Gonyostomum semen (Raphidophyceae) and Microcystis aeruginosa (Cyanophyecae) in freshwater. We examined not only lethal but also sub-lethal effects of the biosurfactant. In addition, the effect of the biosurfactant on Daphnia was tested. Our conclusions were that very low biosurfactant concentrations (5 μg mL⁻¹) decreased both the photosynthesis efficiency and the cell viability and that higher concentrations (50 μg mL⁻¹) had lethal effects in four of the five HAB species tested. The low concentrations employed in this study and the diversity of HAB genera tested suggest that biosurfactants may be used to either control initial algal blooms without causing negative side effect to the ecosystem, or to provoke lethal effects when necessary.     

Grazing activity of Pfiesteria piscicida (Dinophyceae) and susceptibility to ciliate predation vary with toxicity status

Variability has been reported in the toxicity potential of Pfiesteria piscicida that is partly a function of the history of exposure to live fish. Grazing properties of P. piscicida and its susceptibility to ciliate predation were compared in three functional types or toxicity states of this species: actively toxic cultures, cultures with temporary loss of demonstrable toxicity, and cultures with no demonstrable toxicity. Pronounced differences in predator–prey interactions were found between actively toxic cultures and cultures with reduced toxicity. When grown with Rhodomonas sp. (Cryptophyceae) prey, specific growth rates were relatively low in actively toxic cultures under both relatively high and low irradiances. In the cultures with reduced toxicity, prey chloroplast material was apparent in nearly 100% of dinoflagellate cells 3 h after feeding, while chloroplast inclusions were found in <40% of actively toxic cells for ≤16 h (high light) and ≤23 h (low light). These results suggest a relatively high reliance on phagotrophic carbon assimilation and more rapid response to algal prey availability in Pfiesteria cells with lower toxicity. Grazing by two euplotid benthic ciliates (Euplotes vannus and E. woodruffi) on P. piscicida also varied among functional types. Grazing on actively toxic P. piscicida cells did not occur, whereas net positive ingestion rates were calculated for the other prey cultures. These results support concurrent experimental findings that a natural assemblage of microzooplankton displayed lower grazing potential on actively toxic P. piscicida than on cultures with reduced toxicity. In summary, pronounced differences in trophic interactions were found between actively toxic cultures and those with reduced or undetectable toxicity, providing additional evidence of the importance of cellular toxicity in the trophic ecology of Pfiesteria.     

Remote, subsurface detection of the algal toxin domoic acid onboard the Environmental Sample Processor: Assay development and field trials

The ability to detect harmful algal bloom (HAB) species and their toxins in real- or near real-time is a critical need for researchers studying HAB/toxin dynamics, as well as for coastal resource managers charged with monitoring bloom populations in order to mitigate their wide ranging impacts. The Environmental Sample Processor (ESP), a robotic electromechanical/fluidic system, was developed for the autonomous, subsurface application of molecular diagnostic tests and has successfully detected several HAB species using DNA probe arrays during field deployments. Since toxin production and thus the potential for public health and ecosystem effects varies considerably in natural phytoplankton populations, the concurrent detection of HAB species and their toxins onboard the ESP is essential. We describe herein the development of methods for extracting the algal toxin domoic acid (DA) from Pseudo-nitzschia cells (extraction efficiency >90%) and testing of samples using a competitive ELISA onboard the ESP. The assay detection limit is in the low ng/mL range (in extract), which corresponds to low ng/L levels of DA in seawater for a 0.5 L sample volume acquired by the ESP. We also report the first in situ detection of both a HAB organism (i.e., Pseudo-nitzschia) and its toxin, domoic acid, via the sequential (within 2–3 h) conduct of species- and toxin-specific assays during ESP deployments in Monterey Bay, CA, USA. Efforts are now underway to further refine the assay and conduct additional calibration exercises with the aim of obtaining more reliable, accurate estimates of bloom toxicity and thus their potential impacts.     

A fish kill of massive proportion in Kuwait Bay, Arabian Gulf, 2001: the roles of bacterial disease, harmful algae, and eutrophication

In August and September 2001, Kuwait Bay, a semi-enclosed embayment of the Arabian Gulf, experienced a massive fish kill involving over >2500 metric tons of wild mullet (Liza klunzingeri), due to the bacterium Streptococcus agalactiae. In the Bay, this event was preceded by a small fish kill (100–1000 dead fish per day) of gilthead sea bream (Sparus auratus) in aquaculture net pens associated with a bloom of the dinoflagellate Ceratium furca. Sea bream were found to be culture positive for S. agalactiae, but did not show any visible signs of disease. Unusually warm temperatures (up to 35 °C) and calm conditions prevailed during this period. As the wild fish kill progressed, various harmful algae were observed, including Gymnodinium catenatum, Gyrodinium impudicum, and Pyrodinium bahamense var. compressum. Cell numbers of G. catenatum and G. impudicum exceeded 10⁶ l⁻¹ in some locations. All fish tested below the limits of detection for paralytic shellfish poisoning (PSP) and brevetoxins. Clams (Circe callipyga) were positive for PSP but at levels below regulatory limits. Nutrient concentrations, both inorganic and organic, were highly variable with time and from site to site, reflecting inputs from sewage outfalls, the aquaculture operations, a high biomass of decomposing fish, and other sources. It is hypothesized that many factors contributed to the initial outbreak of the bacterial disease, including unusual warm and calm conditions. The same factors, as well as enriched nutrient conditions, also apparently were conducive to the subsequent HAB outbreaks. The detection of PSP, while below regulatory limits, warrants further monitoring to protect human health.     

Predator–prey interactions between a planktonic ciliate Strombidium sp. (Ciliophora, Oligotrichida) and the dinoflagellate Pfiesteria piscicida (Dinamoebiales, Pyrrophyta)

The functional response of a planktonic ciliate, Strombidium sp. feeding on the dinoflagellate Pfiesteria piscicida non-toxic zoospores (NTZ) was experimentally studied with four different prey concentrations (43–3153 cells ml⁻¹). Data from direct observations (NTZ inside individual Strombidium sp.) was used to calculate predator–prey specific ingestion and clearance rates. The ingestion rates varied between 0.68 and 14.26 NTZ ind⁻¹ h⁻¹, and with the predator–prey specific handling time of 2.83 min the U_max was 21.18 NTZ ind⁻¹ h⁻¹. The increase in the prey concentration between approximately 700 and 3000 NTZ ml⁻¹ did not increase the uptake of prey, and at the lowest Pfiesteria NTZ concentrations the feeding efficiency of Strombidium sp. was lowered, possibly indicating a situation of threshold feeding. When data from direct observations of ingested Pfiesteria NTZ were compared with values of total NTZ loss from the experimental water during the experiment, ingestion was found to represent only a fraction of the total NTZ loss in the presence of ciliates. This discrepancy was concluded to be due to other grazer related factors than actual ciliate grazing. The control of the initial growth of Pfiesteria community, in a pre-bloom situation, would require only a small ciliate abundance (less than 5 ml⁻¹), but when the Pfiesteria NTZ are scarce, relatively more ciliates are needed to limit the population growth of the dinoflagellate community because of the apparent feeding threshold. It is concluded that the formation of non-toxic P. piscicida blooms require periods of low grazing pressure or a means to escape grazing.     

Phytoplankton composition of the Kandalaksha Gulf, Russian White Sea: Dinophysis and lipophilic toxins in the blue mussel (Mytilus edulis)

Dinophysis acuminata and D. norvegica were observed in plankton net samples during the summer of 2002 from the Kandalaksha Gulf in the White Sea (North European Russia). Prorocentrum lima was found as an epiphyte on subtidal macroalgae in August, but not observed in plankton net samples. Protein phosphatase 2A (PP2A) inhibition measured 127.8 ng OA-equivalent/g of mussel (Mytilus edulis) hepatopancreas from samples collected a few days after when Dinophysis was recorded at a density of 1550 cells L⁻¹. Liquid chromatography–mass spectrometry confirmed presence of several classes of lipophilic shellfish toxins associated with Dinophysis spp. in the mussels including okadaic acid, dinophysistoxin-1, pectenotoxins and yessotoxins. No azaspiracid was detected. This represents the first identification of phycotoxicity in the White Sea.     

Boron deprivation decreases liver S-adenosylmethionine and spermidine and increases plasma homocysteine and cysteine in rats

Two experiments were conducted with weanling Sprague–Dawley rats to determine whether changes in S-adenosylmethionine utilization or metabolism contribute to the diverse responses to boron deprivation. In both experiments, four treatment groups of 15 male rats were fed ground corn-casein based diets that contained an average of 0.05 mg (experiment 1) or 0.15 mg (experiment 2) boron/kg. In experiment 2, some ground corn was replaced by sucrose and fructose to increase oxidative stress. The dietary variables were supplemental 0 (boron-deprived) or 3 (boron-adequate) mg boron/kg and different fat sources (can affect the response to boron) of 75 g corn oil/kg or 65 g fish (menhaden) oil/kg plus 10 linoleic acid/kg. When euthanized at age 20 (experiment 1) and 18 (experiment 2) weeks, rats fed the low-boron diet were considered boron-deprived because they had decreased boron concentrations in femur and kidney. Boron deprivation regardless of dietary oil increased plasma cysteine and homocysteine and decreased liver S-adenosylmethionine, S-adenosylhomocysteine, and spermidine. Plasma concentration of 8-iso-prostaglandin F_2α (indicator of oxidative stress) was not affected by boron, but was decreased by feeding fish oil instead of corn oil. Fish oil instead of corn oil decreased S-adenosylmethionine, increased spermidine, and did not affect S-adenosylhomocysteine concentrations in liver. Additionally, fish oil versus corn oil did not affect plasma homocysteine in experiment 1, and slightly increased it in experiment 2. The findings suggest that boron is bioactive through affecting the formation or utilization of S-adenosylmethionine. Dietary fatty acid composition also affects S-adenosylmethionine formation or utilization, but apparently through a mechanism different from that of boron.