Low Domoic Acid Research Articles

Reduced representation sequencing accurately quantifies relative abundance and reveals population-level variation in Pseudo-nitzschia spp.

Certain species within the genus Pseudo-nitzschia are able to produce the neurotoxin domoic acid (DA), which can cause illness in humans, mass-mortality of marine animals, and closure of commercial and recreational shellfisheries during toxic events. Understanding and forecasting blooms of these harmful species is a primary management goal. However, accurately predicting the onset and severity of bloom events remains difficult, in part because the underlying drivers of bloom formation have not been fully resolved. Furthermore, Pseudo-nitzschia species often co-occur, and recent work suggests that the genetic composition of a Pseudo-nitzschia bloom may be a better predictor of toxicity than prevailing environmental conditions. We developed a novel next-generation sequencing assay using restriction site-associated DNA (2b-RAD) genotyping and applied it to mock Pseudo-nitzschia communities generated by mixing cultures of different species in known abundances. On average, 94% of the variance in observed species abundance was explained by the expected abundance. In addition, the false positive rate was low (0.45% on average) and unrelated to read depth, and false negatives were never observed. Application of this method to environmental DNA samples collected during natural Pseudo-nitzschia spp. bloom events in Southern California revealed that increases in DA were associated with increases in the relative abundance of P. australis. Although the absolute correlation across time-points was weak, an independent species fingerprinting assay (Automated Ribosomal Intergenic Spacer Analysis) supported this and identified other potentially toxic species. Finally, we assessed population-level genomic variation by mining SNPs from the environmental 2bRAD dataset. Consistent shifts in allele frequencies in P. pungens and P. subpacifica were detected between high and low DA years, suggesting that different intraspecific variants may be associated with prevailing environmental conditions or the presence of DA. Taken together, this method presents a potentially cost-effective and high-throughput approach for studies aiming to evaluate both population and species dynamics in mixed samples.

Analysis of Toxin Amnesiac by High Performance Chromatography Liquid in Bivalves of the Dakhla Region

In this study we performed an ASP analysis in bivalve molluscs of the Dakhla region, especially mussels and oysters. This work was performed between March and June 2015. The sample was conducted in four sites in the Dakhla region, namely Boutalha, Duna Blanca, Puertitto and Oum Labouir. After analysis by HPLC / UV it was shown that the greatest percentage of toxicity of domoic acid (DA) was recorded in June, followed by the months of April and May, while the month of Mars was marked by a low DA toxicity. Key word: ASP, Domoic Acid, toxicity, HPLC/UV, Pseudo-Nitzshia, efflorescence, phytoplankton.

Low Dose Domoic Acid Influences Spontaneous Behavior in Adult Rats

Domoic acid (DA) is a potent marine neurotoxine present in seafood. Intoxication by DA causes gastrointestinal symptoms like vomiting and diarrhoea and also the so-called amnesic shellfish poisoning (inflicting memory impairment and seizures). Since exposure to non-convulsive doses is relevant to the human health, we investigated the effect of low dose DA administration in adult Wistar rats. Rats were administered with DA at the dose 1.0 mg/kg and their behavior was monitored for one hour in three sessions. The first session started immediately after DA administration. The second and third session started one and two weeks later. After the third session, the histochemical analysis of the hippocampi of the animals was conducted (Fluoro-Jade B, bis-benzimide). DA increased time spent by locomotion and distance travelled in the second half of the first session and this effect was pronounced during the second and third session. Exploratory rearing was decreased by DA administration in the first half of the first session. DA influenced the grooming in biphasic manner (decrease followed by an increase of time spent by grooming). This biphasic trend was observed even two weeks after the DA administration. Histochemistry of DA treated rats did not confirm the presence of apoptotic bodies, Fluoro-Jade B positive cells were not found neither in CA1 nor CA3 area of the hippocampi. Our study revealed that a low dose of DA affect short and long-term the spontaneous behavior of rats without inducing neuronal damage.

The genus Pseudo-nitzschia (Bacillariophyceae) in New Zealand: analysis of the last decade's monitoring data

The diatom genus Pseudo-nitzschia blooms throughout New Zealand's coastal waters. More than a decade of monitoring data were analysed in this study and seasonality was a significant factor in the appearance of Pseudo-nitzschia blooms. Austral spring and summer were the main bloom periods, although there were regional differences. Between January 2000 and August 2011, 8.4% of 29,000 seawater samples analysed for Pseudo-nitzschia exceeded the voluntary trigger level for biotoxin testing (10×104 cells l−1), and these were analysed by fluorescent in situ hybridisation (FISH) assays. Pseudo-nitzschia fraudulenta and P. pseudodelicatissima (low domoic acid [DA] producers) were the dominant bloom formers throughout New Zealand (each contributing 25%) followed by the highly toxic P. australis (10%). Shellfish flesh testing for DA was triggered on 8477 occasions between 2000 and 2011, but no samples exceeded the regulatory limit (20 mg/kg); the maximum concentration was 13 mg/kg (Marlborough Sounds, mid-winter 2010).

Coastal upwelling linked to toxic Pseudo-nitzschia australis blooms in Los Angeles coastal waters, 2005–2007

Harmful algal blooms dominated by the diatom Pseudo-nitzschia spp. have become a perennial but variable event within surface waters near the greater Los Angeles area. Toxic blooms during spring seasons from 2005 to 2007 varied strongly in their overall toxicity and duration. Differences in bloom dynamics were linked to differences in storm-induced river discharge following episodic rain events and coastal upwelling, both major coastal processes that led to the injection of nutrients into coastal surface waters. Heavy river runoff during early 2005, a record-rainfall year, favored a phytoplankton community mainly comprised of algal taxa other than Pseudo-nitzschia. The spring bloom during 2005 was associated with low domoic acid surface concentrations and minor contributions of (mainly) P. delicatissima to the diatom assemblage. In contrast, highly toxic P. australis-dominated blooms during spring seasons of 2006 and 2007 were linked to strong upwelling events. River discharge quotas in 2006 and 2007, in contrast to 2005, fell well below annual averages for the region. Surface toxin levels were linked to colder, more saline (i.e. upwelled) water over the 3-year study, but no such consistent relationship between domoic acid levels and other physiochemical parameters, such as macronutrient concentrations or nutrient ratios, was observed.

Gene expression studies for the analysis of domoic acid production in the marine diatom Pseudo-nitzschia multiseries.

BackgroundPseudo-nitzschia multiseries Hasle (Hasle) (Ps-n) is distinctive among the ecologically important marine diatoms because it produces the neurotoxin domoic acid. Although the biology of Ps-n has been investigated intensely, the characterization of the genes and biochemical pathways leading to domoic acid biosynthesis has been limited. To identify transcripts whose levels correlate with domoic acid production, we analyzed Ps-n under conditions of high and low domoic acid production by cDNA microarray technology and reverse-transcription quantitative PCR (RT-qPCR) methods. Our goals included identifying and validating robust reference genes for Ps-n RNA expression analysis under these conditions.ResultsThrough microarray analysis of exponential- and stationary-phase cultures with low and high domoic acid production, respectively, we identified candidate reference genes whose transcripts did not vary across conditions. We tested eleven potential reference genes for stability using RT-qPCR and GeNorm analyses. Our results indicated that transcripts encoding JmjC, dynein, and histone H3 proteins were the most suitable for normalization of expression data under conditions of silicon-limitation, in late-exponential through stationary phase. The microarray studies identified a number of genes that were up- and down-regulated under toxin-producing conditions. RT-qPCR analysis, using the validated controls, confirmed the up-regulation of transcripts predicted to encode a cycloisomerase, an SLC6 transporter, phosphoenolpyruvate carboxykinase, glutamate dehydrogenase, a small heat shock protein, and an aldo-keto reductase, as well as the down-regulation of a transcript encoding a fucoxanthin-chlorophyll a-c binding protein, under these conditions.ConclusionOur results provide a strong basis for further studies of RNA expression levels in Ps-n, which will contribute to our understanding of genes involved in the production and release of domoic acid, an important neurotoxin that affects human health as well as ecosystem function.

Cell-based measurements to assess physiological status of Pseudo-nitzschia multiseries, a toxic diatom

Diatoms of the genus Pseudo-nitzschia are potentially toxic microalgae, whose blooms can trigger amnesic shellfish poisoning. The purpose of this study was to test and adapt different probes and procedures in order to assess the physiological status of Pseudo-nitzschia multiseries at the cell level using flow cytometry. To perform these analyses, probes and procedures were first optimized for concentration and incubation time. The percentage of dead Pseudo-nitzschia cells, the metabolic activity of live cells and their intracellular lipid content were then measured following a complete growth cycle. In addition, chlorophyll autofluorescence and efficiency of photosynthesis (quantum yield) were monitored. The concentration and viability of bacteria present in the medium were also assessed. Domoic acid (DA) was quantified as well. Just before the exponential phase, cells exhibited high metabolic activity, but low DA content. DA content per cell became most abundant at the beginning of the exponential phase when lipid storage was high, which provided a metabolic energy source, and when they were surrounded by a high number of bacteria (high bacteria/ P. multiseries ratio). These physiological measurements tended to decrease during exponential phase and until stationary phase, at which time P. multiseries cells did not contain any DA nor store any lipids, and started to die.

Angler exposure to domoic acid via consumption of contaminated fishes

Domoic acid (DA) is a neurotoxin that causes amnesic shellfish poisoning, and fish are recog- nized vectors of DA to marine fauna. However, the exposure of anglers through consumption of DA- contaminated fish is unknown. We measured DA in 11 fish species targeted by Santa Cruz Wharf (SCW) anglers in Monterey Bay, California, USA, and sur- veyed anglers regarding their fish consumption pat- terns. In addition, we used California mussel Mytilus californianus DA data provided by the state of Califor- nia and our measurements of DA in seawater to exam- ine the associations between DA in fish viscera versus in mussels and seawater. DA was detected in the vis- cera of 7 fish species commonly consumed by anglers, and toxin uptake in fishes varied according to their diet. DA was almost entirely in the viscera, with low DA concentrations detected in muscle tissue. The majority of anglers (58% of 565) reported consuming their catch, with a small fraction ingesting the viscera. Total DA concentrations in fish decreased significantly after 11 mo storage at -20°C. DA concentration in sea- water and California mussels was correlated with DA in the viscera of some but not all fish groups. We con- clude that SCW anglers who consume their catch are exposed to asymptomatic DA doses, and that exposure is a function of the species and parts consumed, as well as storage methods and DA levels in the seawater when the fish are caught.

Feeding mechanics as the basis for differential uptake of the neurotoxin domoic acid by oysters, Crassostrea virginica, and mussels, Mytilus edulis

The neurotoxin domoic acid (DA), produced by diatoms Pseudo-nitzschia spp., is transferred to humans via consumption of contaminated bivalves. This study examines feeding mechanisms, namely reduced filtration, pre-ingestive rejection and poor absorption, that might explain the comparatively low DA levels commonly found in oysters during toxic Pseudo-nitzschia blooms. Clearance rate (CR), absorption efficiency (AE) of organic matter and selective rejection in pseudofeces of oysters ( Crassostrea virginica) and mussels ( Mytilus edulis) were investigated in relation to the DA levels accumulated during 2-wk, simultaneous exposure to toxic Pseudo-nitzschia multiseries. Effects of temperature and P. multiseries cell size were also tested to identify conditions, if any, under which oysters can accumulate unsafe DA levels. Oysters accumulated 3.0–7.5× less DA than mussels from a short-celled P. multiseries clone (length = 24 μm) at 12 °C. This was related to the 7.4–8.5× lower CRs determined for oysters relative to mussels at this temperature. Exposure to a longer-celled P. multiseries clone (81 μm) resulted in up to 70× lower toxin levels in oysters compared to mussels, which was attributed to differential feeding selectivity. Mussels were unable to discriminate between long- and short-celled P. multiseries clones from a mixed suspension, whereas oysters were previously shown to preferentially reject long cells (>70 μm) in pseudofeces. Both bivalves selectively rejected P. multiseries cells from mixed suspensions containing a flagellate but not another diatom. AE of organics from P. multiseries cells by oysters and mussels was comparably low (42 and 39%, respectively) and thus unlikely to explain their differential DA accumulation. CR and DA uptake by oysters were negligible at ≤4 °C but increased with increasing temperature up to 18 °C, although mean DA levels barely attained the regulatory limit (20 μg g −1) when oysters were exposed to long P. multiseries cells. The maximum DA levels accumulated by mussels (320 μg g −1) and oysters (44 μg g −1) exposed to short P. multiseries cells in our study support the inter-specific differences in toxicity during Pseudo-nitzschia blooms, which are expected to be exacerbated at lower temperatures and when long cells or chains are dominant. Additionally, when alternate, non-diatom phytoplankton species are present, both bivalves can feed selectively and thus accumulate much lower DA levels than those predicted from their overall CRs. Our results provide support for the evaluation of species-specific management of DA-contaminated shellfish and need to be considered in modeling DA toxin kinetics of the two target species.

Mechanisms contributing to low domoic acid uptake by oysters feeding on Pseudo-nitzschia cells. II. Selective rejection

Oysters accumulate relatively low levels of domoic acid (DA) compared to other bivalves. Mafra et al. (2009, in this Theme Section) identified feeding mechanisms of oysters that may lead to low DA accumulation during monospecific blooms of Pseudo-nitzschia multiseries. However, several differ- ent species of Pseudo-nitzschia, as well as other diatoms and flagellates, may co-occur during a bloom. Therefore, the present study investigates pre-ingestive feeding processes that operate when oysters Crassostrea virginica are exposed to mixed phytoplankton assemblages containing P. multiseries of varying cell length. Guided by video-endoscopy, material transported along the ventral and dorsal gill tracts was sampled and analyzed to determine the site for sorting of microalgae on the pallial organs. There was no preferential rejection of P. multiseries in pseudofeces when oysters were exposed to the alga in a mixed suspension with other diatom species (Thalassiosira weissflogii or Chaetoceros muelleri). In contrast, P. multiseries was preferentially rejected when mixed with the flagellates Iso- chrysis galbana or Rhodomonas lens, suggesting a qualitative mechanism for particle sorting. This occurred on the gills, followed by further selection on the palps. Oysters also preferentially rejected larger P. multiseries cells (82 to 90 µm) relative to smaller ones (24 to 28 µm) on the gills, while no fur- ther selection based on size occurred on the palps. This effect is attributed to the fact that P. multiseries cells with a length that exceeds the width of the principal filament aperture (ca. 68 µm) are more likely directed to the ventral tract and rejected in pseudofeces. These findings offer an additional explanation for the relatively low DA levels found in oysters during natural Pseudo-nitzschia spp. blooms.

Mechanisms contributing to low domoic acid uptake by oysters feeding on Pseudo-nitzschia cells. I. Filtration and pseudofeces production

AB Aquatic Biology Contact the journal Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections AB 6:201-212 (2009) - DOI: https://doi.org/10.3354/ab00121 Theme Section: Directions in bivalve feeding Mechanisms contributing to low domoic acid uptake by oysters feeding on Pseudo-nitzschia cells. I. Filtration and pseudofeces production Luiz L. Mafra Jr.1,2,*, V. Monica Bricelj3, Christine Ouellette1, Claude Léger4, Stephen S. Bates4 1Institute for Marine Biosciences, National Research Council, 1411 Oxford St., Halifax, Nova Scotia B3H 3Z1, Canada 2Department of Biology, Dalhousie University, Halifax, Nova Scotia B3H 4J1, Canada 3Institute of Marine and Coastal Sciences, Rutgers University, 93 Lipman Dr., New Brunswick, New Jersey 08901, USA 4Gulf Fisheries Centre, Fisheries and Oceans Canada, PO Box 5030, Moncton, New Brunswick E1C 9B6, Canada *Email: luiz.mafra@nrc-cnrc.gc.ca ABSTRACT: Bivalve molluscs feeding on toxigenic Pseudo-nitzschia spp. are the main vector of domoic acid (DA) to humans. Although different oyster species rarely exceed the internationally adopted regulatory level for shellfish harvesting closures (20 µg g–1), these are often applied to all bivalve species in an affected area. This study examines the influence of diet composition and Pseudo-nitzschia multiseries cell size, density and toxicity on oyster feeding rates to determine potential pre-ingestive mechanisms that may lead to low DA accumulation in the oysters Crassostrea virginica. Clearance rate (CR) and pseudofeces production of juvenile oysters were quantified under varying laboratory conditions representative of natural Pseudo-nitzschia blooms. Oysters filtered increasing amounts of P. multiseries cells as cell density increased, but ingestion was limited by pseudofeces production at concentrations >10400 cells ml–1 (ca. 6 mg dry weight l–1). Oysters significantly reduced their CR when fed both toxic and non-toxic P. multiseries clones in unialgal suspensions compared to Isochrysis galbana, and this rapid grazing inhibition was not related to growth stage, cell size, or exposure time. When offered mixed suspensions containing equivalent cellular volumes of the 2 species, however, relatively high CR was restored. Therefore, we suggest that DA intake by oysters from mono-specific Pseudo-nitzschia blooms would be limited by a persistently reduced CR and rejection of cells in pseudofeces. When an alternative, good source of food is present in a mixed phytoplankton assemblage with P. multiseries, no CR inhibition is expected and DA intake will be regulated by pre-ingestive particle selection on the feeding organs, as demonstrated in Mafra et al. (2009, in this Theme Section) KEY WORDS: Pseudo-nitzschia multiseries · Domoic acid · Oyster · Crassostrea virginica · Feeding · Filtration · Pseudofeces Full text in pdf format PreviousNextCite this article as: Mafra Jr LL, Bricelj VM, Ouellette C, Léger C, Bates SS (2009) Mechanisms contributing to low domoic acid uptake by oysters feeding on Pseudo-nitzschia cells. I. Filtration and pseudofeces production. Aquat Biol 6:201-212. https://doi.org/10.3354/ab00121 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AB Vol. 6. Online publication date: March 12, 2009 Print ISSN: 1864-7782; Online ISSN: 1864-7790 Copyright © 2009 Inter-Research.

Accumulation of domoic acid and its effect on juvenile king scallop Pecten maximus (Linnaeus, 1758)

Juvenile king scallops Pecten maximus of 1.23 cm shell height were exposed to domoic acid (DA) and the toxin accumulation and effects of bearing this toxin were investigated. After feeding on a formulated feed containing about 0.19 pg DA per particle for 7 days, the juvenile scallops incorporated a relatively low DA burden of 15.9 ng g − 1 , which was similar to those exposed to 10 and 50 ng ml − 1 dissolved DA. The maximum DA accumulation, 302.5 ng g − 1 whole tissue, was achieved by scallops maintained in 100 ng ml − 1 dissolved DA for 18 days. No significant effect of exposure to DA was observed on feeding rate (on microalgae), shell valve activity (shell clatter), righting response, or the secretion of byssus threads. However, negative impacts on growth rate and survival were found for scallops exposed to DA-spiked feed. This is the first study on DA accumulation in juvenile P. maximus and on the negative effect of DA on this life history stage.

Tissue distribution and neurotoxic effects of domoic acid in a prominent vector species, the northern anchovy Engraulis mordax

The planktivorous northern anchovy is a prominent vector of the phycotoxin domoic acid (DA) to organisms at higher trophic levels, including fish-eating seabirds and mammals. Although there are abundant data reporting DA-induced excitotoxic symptoms in higher vertebrates, to date there has been no reported evidence of neurotoxic effects in lower vertebrate vectors such as fish. To explain this apparent lack of toxicity, it has been suggested that DA may not reach the brain in anchovies and/or that fish are not as sensitive neurologically to DA. In the present study, intracoelomic (IC) injection of DA, at doses ranging from 1 to 14 μg DA g−1 total fish weight, resulted in severe neurotoxic symptoms such as spinning, disorientation, inability to school, and mortality, indicating that anchovies are neurologically susceptible and that DA crosses the blood–brain barrier in fish. An ED50 of 3.2 μg DA g−1 total body weight was determined via IC injection of DA in 83 anchovies. Comparable intraperitoneal injection studies with mice, rats, and monkeys report similar DA-induced neurotoxic symptoms at doses near 3.2 μg DA g−1, suggesting a similar neurologic sensitivity and mechanism of toxicity between anchovies and mammals. DA tissue distribution measurements from freshly collected field-exposed anchovies and orally gavaged anchovies indicate that DA uptake from the gastrointestinal tract does occur. Levels as high as 1,175 μg DA g−1 were measured in anchovy viscera, while muscle and brain tissue DA levels were 3 orders of magnitude lower, indicating low but measurable DA uptake. Further evidence is needed to confirm that uptake is sufficient during field events to induce symptoms in anchovies. Our work provides the first reported evidence of neurotoxic symptoms in fish and suggests that anchovies may be affected by DA during toxic diatom blooms. If sufficient uptake occurs, DA-induced neurotoxic symptoms and mortality may make fish easier prey targets, thereby selecting for the highest toxin levels transferred, as well as providing a possible pathway for the transfer of DA to benthic communities.