Reevaluation of Production of Paralytic Shellfish Toxin by Bacteria Associated with Dinoflagellates of the Portuguese Coast

Paralytic shellfish toxins (PSTs) are potent neurotoxins produced by certain dinoflagellate and cyanobacterial species. The autonomous production of PSTs by bacteria remains controversial. In this study, PST production by two bacterial strains, isolated previously from toxic dinoflagellates, was evaluated using biological and analytical methods. Analyses were performed under conditions determined previously to be optimal for toxin production and detection. Our data are inconsistent with autonomous bacterial PST production under these conditions, thereby challenging previous findings for the same strains.     

PRODUCTION OF PARALYTIC SHELLFISH TOXINS BY APHANIZOMENON SP. LMECYA 31 (CYANOBACTERIA)1

We examined intracellular and extracellular paralytic shellfish toxins (PST) in a strain of Aphanizomenon sp. (LMECYA31) isolated from a Portuguese freshwater reservoir throughout the growth cycle and under different conditions affected by temperature and nitrate and phosphate availability. PST concentrations and compositions were greatly influenced by cell density, growth stage, and temperature and nutrients conditions. On a per‐cell basis results showed (1) the enhancement of PST cell quota after the end of exponential growth phase in nutrient replete batch cultures, (2) the absence of a PST increment at late growth stages under phosphate limitation, (3) a rise in PST maximum cell quota under nitrate depletion, and (4) the enhancement of toxin production at higher temperatures. The relative proportion of the four toxins detected, neoSTX, dcSTX, STX and GTX5, also changed within and between culture settings. While growing under phosphate rich media cells produced mainly GTX5 and neoSTX, whereas under phosphate limitation the proportion of STX and dcSTX increased substantially with culture age. Large amounts of extracellular toxins were found in the culture medium, increasing during culture time. Extracellular toxin composition in each culture was fairly constant and always similar to the intracellular composition found at late stages of growth. This further supported other research that indicates that PSTs are released to the water through cell lysis, and a significant concentration of PST may be expected to remain in the water upon the collapse of a toxic bloom or after cells removal by water treatment.     

Regulation of phenol sulfotransferase expression in cultured bovine bronchial epithelial cells by hydrocortisone

One conjugative pathway for the inactivation of endogenous and exogenous hydroxylated aromatic compounds is catalyzed by phenol (aryl) sulfotransferases (PSTs), which esterify phenolic acceptors with sulfate. The tracheobronchial epithelium is commonly exposed to phenolic drugs and pollutants, and metabolic sulfation and PST activity in this tissue have been previously demonstrated. To determine what factors may control PST expression, extracts of serum-free, growth factor-supplemented cultures of bovine bronchial epithelial cells were assayed for PST activity and PST antigen. The most significant finding was dose-dependent, apparent stimulated expression by hydrocortisone (EC₅₀ = 4 nM, maximal stimulation at 20 nM). Time-course experiments, however, revealed progressive loss of PST in the absence of corticosteroid. After decay of extant PST in steroid-free medium, hydrocortisone reinduced the expression of PST three to fivefold. Western blots using mouse anti-bovine PST revealed corresponding increases in 32 kDa PST protein levels in response to hydrocortisone. Steady state kinetic analyses indicated apparent K_m values of 1—3 μM for 2-naphthol regardless of culture conditions. These results suggest that detoxification of phenolic compounds by sulfation may be regulated by corticosteroids.     

Paralytic shellfish toxins or spirolides? The role of environmental and genetic factors in toxin production of the Alexandrium ostenfeldii complex

Dinoflagellates of the Alexandrium ostenfeldii complex (A. ostenfeldii, A. peruvianum) are capable of producing different types of neurotoxins: paralytic shellfish toxins (PSTs), spirolides and gymnodimines, depending on the strain and its geographic origin. While Atlantic and Mediterranean strains have been reported to produce spirolides, strains originating from the brackish Baltic Sea produce PSTs. Some North Sea, USA and New Zealand strains contain both toxins. Causes for such intraspecific variability in toxin production are unknown. We investigated whether salinity affects toxin production and growth rate of 5 A. ostenfeldii/peruvianum strains with brackish water (Baltic Sea) or oceanic (NE Atlantic) origin. The strains were grown until stationary phase at 7 salinities (6–35), and their growth and toxin production was monitored. Presence of saxitoxin (STX) genes (sxtA1 and sxtA4 motifs) in each strain was also analyzed. Salinity significantly affected both growth rate and toxicity of the individual strains but did not change their major toxin profile. The two Baltic Sea strains exhibited growth at salinities 6–25 and consistently produced gonyautoxin (GTX) 2, GTX3 and STX. The two North Sea strains grew at salinities 20–35 and produced mainly 20-methyl spirolide G (20mG), whereas the strain originating from the northern coast of Ireland was able to grow at salinities 15–35, only producing 13-desmethyl spirolide C (13dmC). The effects of salinity on total cellular toxin concentration and distribution of toxin analogs were strain-specific. Both saxitoxin gene motifs were present in the Baltic Sea strains, whereas the 2 North Sea strains lacked sxtA4, and the Irish strain lacked both motifs. Thus sxtA4 only seems to be specific for PST producing strains. The results show that toxin profiles of A. ostenfeldii/peruvianum strains are predetermined and the production of either spirolides or PSTs cannot be induced by salinity changes. However, changes in salinity may lead to changed growth rates, total cellular toxin concentrations as well as relative distribution of the different PST and spirolide analogs, thus affecting the actual toxicity of A. ostenfeldii/peruvianum populations.     

A review of the global distribution of Alexandrium minutum (Dinophyceae) and comments on ecology and associated paralytic shellfish toxin profiles,with a focus on Northern Europe

Alexandrium minutum is a globally distributed harmful algal bloom species with many strains that are known to produce paralytic shellfish toxins (PSTs) and consequently represent a concern to human and ecosystem health. This review highlights that A. minutum typically occurs in sheltered locations, with cell growth occurring during periods of stable water conditions. Sediment characteristics are important in the persistence of this species within a location, with fine sediments providing cyst deposits for ongoing inoculation to the water column. Toxic strains of A. minutum do not produce a consistent toxin profile, different populations produce a range of PSTs in differing quantities. Novel cluster analysis of published A. minutum toxin profiles indicates five PST profile clusters globally. Some clusters are grouped geographically (Northern Europe) while others are widely spread. Isolates from Taiwan have a range of toxin profile clusters and this area appears to have the most diverse set of PST producing A. minutum populations. These toxin profiles indicate that within the United Kingdom there are two populations of A. minutum grouping with strains from Northern France and Southern Ireland. There is a degree of interconnectivity in this region due to oceanic circulation and a high level of shipping and recreational boating. Further research into the interrelationships between the A. minutum populations in this global region would be of value.     

Characterization of paralytic shellfish toxins in seawater and sardines (<Emphasis Type="Italic">Sardina pilchardus</Emphasis>) during blooms of <Emphasis Type="Italic">Gymnodinium catenatum</Emphasis>

The re-emergence of Gymnodinum catenatum blooms after a 10 year hiatus of absence initiated the present investigation. This study aims to evaluate the exposure of small pelagic fishes to paralytic shellfish toxins (PST) during blooms of G. catenatum. Sardines (Sardina pilchardus) were selected as a representative fish species. In order to assess toxin availability to fish, both intracellular PSTs (toxin retained within the algal cells) and extracellular PSTs (toxin found in seawater outside algal cells) were quantified, as well as toxin levels within three fish tissue matrices (viscera, muscle and brain). During the study period, the highest cell densities of G. catenatum reached 2.5 × 10⁴ cells l⁻¹ and intracellular PST levels ranged from 3.4 to 398 ng STXeq l⁻¹ as detected via an enzyme linked immunosorbent assay (ELISA). Measurable extracellular PSTs were also detected in seawater (0.2–1.1 μg STXeq l⁻¹) for the first time in Atlantic waters. The PST profile in G. catenatum was determined via high performance liquid chromatography with fluorescence detection (HPLC-FLD) and consisted mostly of sulfocarbamoyl (C1+2, B1) and decarbamoyl (dcSTX, dcGTX2+3, dcNEO) toxins. The observed profile was similar to that reported previously in G. catenatum blooms in this region before the 10-year hiatus. Sardines, planktivorous fish that ingest a large number of phytoplankton cells, were found to contain PSTs in the viscera, reaching a maximum of 531 μg STXeq kg⁻¹. PSTs were not detected in corresponding muscle or brain tissues. The PST profile characterized in sardine samples consisted of the same sulfocarbamoyl and decarbamoyl toxins found in the algal prey with minor differences in relative abundance of each toxin. Overall, the data suggest that significant biotransformation of PSTs does not occur in sardines. Therefore, planktivorous fish may be a good tracer for the occurrence of offshore G. catenatum blooms and the associated PSTs produced by these algae.     

Effect of vegetables on human phenolsulfotransferases in relation to their antioxidant activity and total phenolics

Epidemiology studies have shown that consumption of fruits and vegetables is associated with the prevention of chronic diseases such as cancer and cardiovascular disease. Induction of cellular phase II detoxifying enzymes is associated with cancer preventive potential. Phenolsulfotransferases (PSTs) are traditionally known as phase II drug-metabolizing or detoxifying enzymes that facilitate the removal of drugs and other xenobiotic compounds. Phenolic acids are known to increase the activities of PSTs. In the present study, human HepG2 cells were used as model to investigate the influence of twenty vegetables on human PST activity and to evaluate the relationships to their antioxidant activity and total phenolics content. The result showed that PST-P activity was significantly (p < 0.01) induced by asparagus, broccoli, cauliflower, celery and eggplant, whereas PST-M activity was induced by asparagus, broccoli, carrot, eggplant and potato at a concentration of 100 microg/ml. The vegetable extracts that induced both forms of PSTs activities were found to have higher antioxidant capacities and total phenolic content in the oxygen radical absorbance capacity (ORAC) and Folin-Ciocalteu assay. The major polyphenols in broccoli, the most potential inducer in both forms of PSTs activities, was antioxidant phenolic acids. HPLC retention times and standard spiked indicated the presence of gallic acid, p-hydroxybenzoic acid, p-coumaric acid, gentisic acid and ferulic acid in broccoli. The overall effect of vegetables tested on the activity of PST-P was well correlated to their ORAC value and total phenolics content (r= 0.82, p < 0.05 and r = 0.78, p < 0.05). These results imply that vegetables have a capability of inducing PST activity, and the PST induction may be possibly ascribed to antioxidant phenolic acids in vegetable extracts.     

PARALYTIC SHELLFISH TOXINS ARE RESTRICTED TO FEW SPECIES AMONG AUSTRALIA'S TAXONOMIC DIVERSITY OF CULTURED MICROALGAE1

There are at least 40,000 species of microalgae in the aquatic environment. Fifteen species of marine dinoflagellates and freshwater cyanobacteria are known to produce paralytic shellfish toxins (PSTs) and represent a threat to human and/or livestock health. Although known toxic species are regularly monitored, the wider cross‐section of microalgae has not been systematically tested for PSTs. Advances in rapid screening techniques have resulted in the development of highly sensitive and specific methods to detect PSTs, including the sodium channel and saxiphilin binding assays. These assays were used in this study in 96‐well formats to screen 234 highly diverse isolates of Australian freshwater and marine microalgae for PSTs. The screening assays detected five toxic species, representing one freshwater cyanobacterium (Anabaena circinalis Rabenhorst) and four species of marine dinoflagellates (Alexandrium minutum Halim, A. catenella Balech, A. tamarense Balech, and Gymnodinium catenatum Graham). Liquid chromatography‐fluorescence detection was used to identify 14 saxitoxin analogues across the five species, and each species exhibited distinct toxin profiles. These results indicate that PST production is restricted to a narrow range of microalgal species found in Australian waters.     

Relative importance of nitrogen sources,algal alarm cues and grazer exposure to toxin production of the marine dinoflagellate Alexandrium catenella

Dinoflagellate paralytic shellfish toxin (PST) production is mediated by several abiotic and biotic factors. This study compared the relative importance of nitrogen source and concentration, prey alarm cues and grazer presence on toxin production of the marine dinoflagellate Alexandrium catenella (Group I, strain BF-5). In separate assays run under either nutrient-replete (F/2 medium) or nutrient-depleted (filtered seawater) conditions, PST production of A. catenella was measured as a function of varying concentrations of added nitrogen sources (ammonium and urea), alarm cues from lysed conspecific (A. catenella Group I strains) and interspecific (the diatom, Thalassiosira weissflogii, and the green flagellate, Tetraselmis sp.) algae, and the presence of a grazer (the copepod Acartia hudsonica). Results showed that addition of ammonium or urea did not increase PST production. Unexpectedly, interspecific alarm cues increased toxin production but conspecific ones did not. Grazer presence dramatically induced PST production in A. catenella, irrespective of nutrient conditions, and this effect was an order of magnitude greater than any of the other variables tested. These results corroborate previous studies on grazer-induced PST production, and support the hypothesis that grazer-induced toxin production is not an experimental artifact, but rather a prey defense mechanism.     

Bioaccumulation of PSTs produced by Alexandrium ostenfeldii in the northern Baltic Sea

Bioaccumulation of paralytic shellfish toxins (PSTs) produced by the dinoflagellate Alexandrium ostenfeldii was investigated in the northern Baltic Sea. The study was based on the assumption that the toxins released during high magnitude blooms of A. ostenfeldii will accumulate in the biota at the bloom site, especially in bivalves. To test this, experiments with blue mussels (Mytilus trossulus) exposed to toxic A. ostenfeldii in field conditions were carried out together with a field survey aimed to quantify natural distribution of PSTs in the biota. As hypothesized, PSTs accumulated in the tissues of the blue mussels during the incubations. Toxins were also detected in natural bivalve communities at the bloom site, the highest toxin concentrations found in the small Cerastoderma glaucum individuals, exceeding the EC safety limit for shellfish consumption. Relatively high total toxin concentrations were also detected from fish (Perca fluviatilis). These are the first records of PST transfer in the food web of the northern Baltic Sea.     

Differential roles of human monoamine (M)-form and simple phenol (P)-form phenol sulfotransferases in drug metabolism

Cytosolic sulfotransferases (STs) are traditionally known as Phase II drug-metabolizing or detoxifying enzymes that facilitate the removal of drugs and other xenobiotic compounds. In this study, we carried out a systematic investigation on the sulfation of drug compounds by two major human phenol STs (PSTs), the monoamine (M)-form and simple phenol (P)-form PSTs. Activity data obtained showed the differential substrate specificity of the two enzymes for the thirteen drug compounds tested. Kinetic studies revealed that the M-form PST displayed stereoselectivity for the chiral drug, isoproterenol. The effects of divalent metal cations on the activity of the M-form and P-form PSTs toward representative drug compounds were quantitatively evaluated. Results obtained indicated that the drug-sulfating activities of the two human PSTs were partially or completely inhibited or stimulated by the ten divalent metal cations tested at a 5 mM concentration. The two enzymes appeared to be less sensitive to the effects of physiologically more abundant metal cations such as Mg(2+) and Ca(2+), but more sensitive to the detrimental effects of other metal cations that may enter the body as environmental contaminants.     

Biochemical Characterization of a Polysialyltransferase from Mannheimia haemolytica A2 and Comparison to Other Bacterial Polysialyltransferases

Polysialic acids are bioactive carbohydrates found in eukaryotes and some bacterial pathogens. The bacterial polysialyltransferases (PSTs), which catalyze the synthesis of polysialic acid capsules, have previously been identified in select strains of Escherichia coli and Neisseria meningitidis and are classified in the Carbohydrate-Active enZYmes Database as glycosyltransferase family GT-38. In this study using DNA sequence analysis and functional characterization we have identified a novel polysialyltransferase from the bovine/ovine pathogen Mannheimia haemolytica A2 (PST_Mh). The enzyme was expressed in recombinant form as a soluble maltose-binding-protein fusion in parallel with the related PSTs from E. coli K1 and N. meningitidis group B in order to perform a side-by-side comparison. Biochemical properties including solubility, acceptor preference, reaction pH optima, thermostability, kinetics, and product chain length for the enzymes were compared using a synthetic fluorescent acceptor molecule. PST_Mh exhibited biochemical properties that make it an attractive candidate for chemi-enzymatic synthesis applications of polysialic acid. The activity of PST_Mh was examined on a model glycoprotein and the surface of a neuroprogenitor cell line where the results supported its development for use in applications to therapeutic protein modification and cell surface glycan remodelling to enable cell migration at implantation sites to promote wound healing. The three PSTs examined here demonstrated different properties that would each be useful to therapeutic applications.     

Inhibition and binding studies of coenzyme A and bovine phenol sulfotransferase.

Phenol sulfotransferases (PSTs, EC 2.8.2.1) catalyze sulfonyl group transfer from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to the hydroxyl oxygen of aromatic acceptor substrates. The structural overlap between PAPS and coenzyme A (CoA) suggested a possible role of this common acyl carrier in modulating PST activity. To test this hypothesis, purified recombinant bovine PST was examined by kinetic and affinity chromatographic approaches. After demonstrating PST enzyme inhibition by CoA, systematic variation of CoA and PAPS concentrations indicated simple competitive inhibition with K(i) = 1. 3 microM. PST bound to CoA-agarose, attached via the pantetheinyl thiol group, was eluted with PAP but not by 2-naphthol. This observation was consistent with the pattern of inhibition. Additional members of the sulfotransferase superfamily, as well as acylated CoAs, should be further investigated.     

A plate-based high-throughput activity assay for polysialyltransferase from Neisseria meningitidis

Polysialyltransferases (PSTs) assemble polysialic acid (PSA) and have been implicated in many biological processes. For example, certain bacteria such as neuroinvasive Neisseria meningitidis decorate themselves in a PSA capsule to evade the innate immune system. Identifying inhibitors of PSTs therefore represents an attractive therapeutic goal and herein we describe a high-throughput, robust, and sensitive microtiter-plate-based activity assay for PST from N. meningitidis. A trisialyl lactoside (GT3) serving as the acceptor substrate was immobilized on a 384-well plate by click chemistry. Incubation with PST and CMP-sialic acid for 30 min resulted in polysialylation. The immobilized PSA was then directly detected using a green fluorescent protein (GFP)-fused PSA-binding protein consisting of the catalytically inactive double mutant of an endosialidase (GFP-EndoNF DM). We report very good agreement between kinetic and inhibition parameters obtained with our on-plate assay versus our in-solution validation assay. In addition we prove our assay is robust and reliable with a Z′ score of 0.79. All aspects of our assay are easily scalable owing to optimization trials that allowed immobilization of acceptor substrates prepared from crude reaction mixtures and the use of cell lysates. This assay methodology enables large-scale PST inhibitor screens and can be harnessed for directed evolution screens.     

Recent trends in paralytic shellfish toxins in Puget Sound,relationships to climate,and capacity for prediction of toxic events

Temporal and spatial trends in paralytic shellfish toxins (PSTs) in Puget Sound shellfish and their relationships with climate are investigated using long-term monitoring data since 1957. Data are selected for trend analyses based on the sensitivity of shellfish species to PSTs and their depuration rates, and the frequency of sample collection at individual sites. These criteria limit the analyses to the shellfish species Mytilus edulis at 20 sites from 1993 to 2007. Blue mussel toxicity is highly variable, but typically exceeds the regulatory limit for human consumption from July to November annually, with most closures occurring early in fall. Using blue mussel data only, we find no robust evidence to suggest that the frequency, magnitude, duration, or geographic scope of PST events in Puget Sound increased between 1993 and 2007. However, there is a significant basin-wide trend for closures to occur earlier in the year. There are no significant correlations between annual indices of mussel toxicity and aspects of the local and large-scale climate. Case studies of daily variations in local environmental factors leading up to exceptionally toxic events identify a combination of conditions that generally precedes most closures from 1993 to 2007. These results suggest that periods of warm air and water temperatures and low streamflow on sub-seasonal timescales may facilitate toxin accumulation in mussels. No relationships were found between water residence times in the surface layer and either streamflow or mussel toxicity. Recommendations are made for future monitoring to improve forecasting of PST risks in Puget Sound, an important region for recreational, commercial, and tribal subsistence shellfish harvesting.     

Pseudoalteromonas Bacteria Are Capable of Degrading Paralytic Shellfish Toxins

Marine bacterial isolates cultured from the digestive tracts of blue mussels (Mytilus edulis) contaminated with paralytic shellfish toxins (PSTs) were screened for the ability to reduce the toxicity of a PST mixture. Seven isolates reduced the overall toxicity of the algal extract by ≥90% within 3 days. These isolates shared at least 99% 16S rRNA gene sequence similarity with five Pseudoalteromonas spp. Phenotypic tests suggested that all are novel strains of Pseudoalteromonas haloplanktis.Among the marine algal biotoxins identified to date; paralytic shellfish toxins (PSTs) constitute the most serious threat to the safety of the food supply, mainly due to their high acute toxicities and the absence of antidotes or effective medical treatments (8). Paralytic shellfish poisoning is caused by ingestion of one or more of the chemically related PSTs (see Fig. S1 in the supplemental material). PSTs are mainly produced by marine dinoflagellates, including Alexandrium spp., Gymnodinium catenatum, and Pyrodinium bahamense var. compresssum (16). Since bivalve molluscs filter-feed on marine algae, they tend to concentrate PSTs largely, but not exclusively, in their digestive organs (7, 9, 10, 29). Not affected by commercial sterilization (14, 18) or cooking, PSTs present significant risks to the food supply, particularly during periods of toxic algal blooms. Practical methods for PST detoxification of living shellfish do not exist (5).Transformations of PSTs by bacteria have been reported in the literature (23-25, 31, 35, 36, 38); early studies focused on the conversion of hydroxysulfate carbamate derivatives (gonyautoxins 1 and 4) to the more highly toxic saxitoxin (STX) (23-25). In addition, several reports have noted the high capacity of the digestive gland for PST transformation (12, 28, 32, 39), suggesting the presence of toxin-transforming enzymes and/or microorganisms in bivalve molluscs. The partial degradation of gonyautoxins 1 and 4 and C1/C2 by marine bacteria has also been reported (38). In addition, Stewart et al. (37) discovered the bacterial degradation of domoic acid (another marine toxin that causes amnesic shellfish poisoning), collectively suggesting that bacteria might play a role in the elimination of marine toxins from toxic bivalve molluscs. The capacity to catabolize domoic acid is greater in cultures isolated from blue mussels that rapidly eliminate domoic acid than in bacterial isolates from bivalves known to retain the toxin for longer time periods (e.g., scallops), suggesting these bacteria play a role in the elimination of marine toxins.Recently, we reported the kinetics of PST destruction for a group of marine bacteria isolated from toxic blue mussels (11). Here we report the phenotypic and taxonomic characterization of these unique marine bacteria.     

Environmental profile of Spanish porcelain stoneware tiles

Purpose

Porcelain stoneware tile (PST) is currently the ceramic tile of greatest commercial and innovation interest. An environmental life cycle assessment of different varieties of PST was undertaken to enable hotspots to be identified, strategies to be defined, differences between PST varieties to be evaluated and guidance for PST manufacturers to be provided in choosing the Environmental Product Declaration (EPD) programme that best suited their needs according to grouping criteria.

Methods

Analysis of previous information allowed three main parameters (thickness, glaze content and mechanical treatment) to be identified in order to encompass all PST variations. Fifteen varieties of PST were thus studied. The coverage of 1 m² of household floor surface with the different PST varieties for 50 years was defined as functional unit. The study sets out environmental data whose traceability was verified by independent third parties for obtaining 14 EPDs of PST under Spanish EPD programmes.

Results and discussion

The study presents PST inventory analysis and environmental impact over the entire life cycle of the studied PST varieties. The natural gas consumed in the manufacturing stage accounted for more than 70% abiotic depletion–fossil fuels and global warming; electricity consumption accounted for more than 60% ozone layer depletion, while the electricity generated by the cogeneration systems avoided significant environmental impacts in the Spanish power grid mix. The variations in PST thickness, amount of glaze and mechanical treatments were evaluated. The PST variety with the lowest environmental impact was the one with the lowest thickness, was unglazed and had no mechanical treatments. Similarly, the PST variety with the highest environmental impact was the one with the greatest thickness, was glazed and had been mechanically treated.

Conclusions

The PST life cycle stage with the highest environmental impact was the manufacturing stage. The main hotspots found were production and consumption of energy and raw materials extraction. Variation in thickness was a key factor that proportionally influenced almost all studied impact categories; the quantity of glaze strongly modified abiotic depletion–elements and eutrophication, while the mechanical treatments contributed mainly to ozone depletion. The study of all PST varieties led to the important conclusion, against the current trend, that differences among them were found to be so significant that declaring a number of PSTs within the same EPD is not directly possible, and it needs preliminary verification to ensure compliance with the product category rule.

     

Parallel Analyses of Alexandrium catenella Cell Concentrations and Shellfish Toxicity in the Puget Sound

Alexandrium catenella is widespread in western North America and produces a suite of potent neurotoxins that cause paralytic shellfish poisoning (PSP) in humans and have deleterious impacts on public health and economic resources. There are seasonal PSP-related closures of recreational and commercial shellfisheries in the Puget Sound, but the factors that influence cell distribution, abundance, and relationship to paralytic shellfish toxins (PSTs) in this system are poorly described. Here, a quantitative PCR assay was used to detect A. catenella cells in parallel with state shellfish toxicity testing during the 2006 bloom season at 41 sites from April through October. Over 500,000 A. catenella cells liter⁻¹ were detected at several stations, with two main pulses of cells driving cell distribution, one in June and the other in August. PSTs over the closure limit of 80 μg of PST 100 per g of shellfish tissue were detected at 26 of the 41 sites. Comparison of cell numbers and PST data shows that shellfish toxicity is preceded by an increase in A. catenella cells in 71% of cases. However, cells were also observed in the absence of PSTs in shellfish, highlighting the complex relationship between A. catenella and the resulting shellfish toxicity. These data provide important information on the dynamics of A. catenella cells in the Puget Sound and are a first step toward assessing the utility of plankton monitoring to augment shellfish toxicity testing in this system.Various species of the dinoflagellate genus Alexandrium, including members of the species complex comprising Alexandrium catenella, Alexandrium fundyense, and Alexandrium tamarense, produce saxitoxins and a number of related derivatives (1). Shellfish that ingest toxic Alexandrium cells accumulate these potent neurotoxins, which can then lead to paralytic shellfish poisoning (PSP) in human consumers of shellfish. As such, paralytic shellfish toxins (PSTs) pose a serious threat to both public health and economically important fisheries (16). Within the Alexandrium genus, A. catenella is widespread in the northwestern part of North America, including the Puget Sound, and is responsible for seasonal harmful algal blooms (HABs) in this region (17). In the Puget Sound, recreational shellfish harvesters collect nearly 2 million pounds of clams and oysters annually, and Washington is also a leading producer of farmed bivalve shellfish in the United States, generating an estimated $77 million in sales a year and supporting thousands of jobs (13).PSTs are not a new problem in the Pacific Northwest; events have been documented as far back as the late 18th century (17). Currently, the Sentinel Monitoring Program of the Washington State Department of Health (WADOH) is in place to provide systematic early warning of harmful levels of PSTs, with caged mussels sampled at as many as 70 sites throughout all basins of Puget Sound at roughly 2-week intervals. Analysis of this long-term shellfish monitoring data indicates that maximum PST levels and PST-related closures have increased over the past 20 years, reaching >10,000 μg of PST per 100 g of shellfish tissue in multiple years and resulting in significant negative impacts on shellfisheries in the region (17).To date, monitoring efforts in the Puget Sound have focused on measuring the level of PSTs present in shellfish tissue. Existing programs do not typically monitor for phytoplankton species composition or abundance. Information on A. catenella distribution and seasonal dynamics is limited for this region, despite its potential value for monitoring and understanding toxic A. catenella blooms and their impacts. Toward this end, we used a previously developed high-throughput quantitative PCR (qPCR) method (5, 6) to detect and enumerate A. catenella cells. We couple this specific and sensitive detection method for A. catenella with PST monitoring efforts to examine changes in A. catenella populations and accompanying shellfish toxicity in the Puget Sound. The data, collected from April through October, span nearly all of the 2006 A. catenella bloom season in the region. These results provide important information on the abundance and dynamics (e.g., possible source populations) of A. catenella cells during a bloom season and on their relationship to PSTs in shellfish. This effort represents a first step toward assessing the utility of plankton monitoring to augment shellfish toxicity testing in this region.     

Technological properties of bakers’ yeasts in durum wheat semolina dough

Properties of 13 Saccharomyces cerevisiae strains isolated from different sources (traditional sourdoughs, industrial baking yeasts etc.) were studied in dough produced with durum wheat (Sicilian semolina, variety Mongibello). Durum wheat semolina and durum wheat flour are products prepared from grain of durum wheat (Triticum durum Desf.) by grinding or milling processes in which the bran and germ are essentially removed and the remainder is comminuted to a suitable degree of fineness. Acidification and leavening properties of the dough were evaluated. Strains isolated from traditional sourdoughs (DSM PST18864, DSM PST18865 and DSM PST18866) showed higher leavening power, valuable after the first and second hours of fermentation, than commercial baking yeasts. In particular the strain DSM PST 18865 has also been successfully tested in bakery companies for the improvement of production processes. Baking and staling tests were carried out on five yeast strains to evaluate their fermentation ability directly and their resistance to the staling process. Amplified fragment length polymorphism (fAFLP) was used to investigate genetic variations in the yeast strains. This study showed an appreciable biodiversity in the microbial populations of both wild and commercial yeast strains.