Combined effects of warming and acidification on accumulation and elimination dynamics of paralytic shellfish toxins in mussels Mytilus galloprovincialis.

Harmful algal blooms (HAB) have been increasing in frequency and intensity most likely due to changes on global conditions, which constitute a significant threat to wild shellfish and its commercial farming. This study evaluated the impact of increasing seawater temperature and acidification on the accumulation/elimination dynamics of HAB-toxins in shellfish. Mytilus galloprovincialis were acclimated to four environmental conditions simulating different climate change scenarios: i) current conditions, ii) warming, iii) acidification and iv) interaction of warming with acidification. Once acclimated, mussels were exposed to the paralytic shellfish toxins (PSTs) producing dinoflagellate Gymnodinium catenatum for 5 days and to non-toxic diet during the subsequent 10 days. High toxicity levels (1493 µg STX eq. kg-1) exceeding the safety limits were determined under current conditions at the end of the uptake period. Significantly lower PSP toxicity levels were registered for warming- and acidification-acclimated mussels (661 and 761 µg STX eq. kg-1). The combined effect of both warming and acidification resulted in PSP toxicity values slightly higher (856 µg STX eq. kg-1). A rapid decrease of toxicity was observed in mussels at the current conditions after shifting to a non-toxic diet, which was not noticed under the predicted climate change scenarios. Variability of each PST analogue, measured throughout the experiment, highlighted different mechanisms are associated with changes of each environmental factor, although both resulting in lower toxicity. Warming-acclimated mussels showed lower accumulation/elimination rates, while acidification-acclimated mussels showed higher capability to accumulate toxins, but also a higher elimination rate preventing high toxicity levels. As different mechanisms are triggered by warming and acidification, their combined effect not leads to a synergism of their individual effects. The present work is the first assessing the combined effect of climate change drivers on accumulation/elimination of PSTs, in mussels, indicating that warming and acidification may lead to lower toxicity values but longer toxic episodes. PSTs are responsible for the food poisoning syndrome, paralytic shellfish poisoning (PSP) in humans. This study can be considered as the first step to build models for predicting shellfish toxicity under climate change scenarios.

Toxin profile of Gymnodinium catenatum (Dinophyceae) from the Portuguese coast, as determined by liquid chromatography tandem mass spectrometry.

The marine dinoflagellate Gymnodinium catenatum has been associated with paralytic shellfish poisoning (PSP) outbreaks in Portuguese waters for many years. PSP syndrome is caused by consumption of seafood contaminated with paralytic shellfish toxins (PSTs), a suite of potent neurotoxins. Gymnodinium catenatum was frequently reported along the Portuguese coast throughout the late 1980s and early 1990s, but was absent between 1995 and 2005. Since this time, G. catenatum blooms have been recurrent, causing contamination of fishery resources along the Atlantic coast of Portugal. The aim of this study was to evaluate the toxin profile of G. catenatum isolated from the Portuguese coast before and after the 10-year hiatus to determine changes and potential impacts for the region. Hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) was utilized to determine the presence of any known and emerging PSTs in sample extracts. Several PST derivatives were identified, including the N-sulfocarbamoyl analogues (C1-4), gonyautoxin 5 (GTX5), gonyautoxin 6 (GTX6), and decarbamoyl derivatives, decarbamoyl saxitoxin (dcSTX), decarbamoyl neosaxitoxin (dcNeo) and decarbamoyl gonyautoxin 3 (dcGTX3). In addition, three known hydroxy benzoate derivatives, G. catenatum toxin 1 (GC1), GC2 and GC3, were confirmed in cultured and wild strains of G. catenatum. Moreover, two presumed N-hydroxylated analogues of GC2 and GC3, designated GC5 and GC6, are reported. This work contributes to our understanding of the toxigenicity of G. catenatum in the coastal waters of Portugal and provides valuable information on emerging PST classes that may be relevant for routine monitoring programs tasked with the prevention and control of marine toxins in fish and shellfish.

Invasive clams (Ruditapes philippinarum) are better equipped to deal with harmful algal blooms toxins than native species (R. decussatus): evidence of species-specific toxicokinetics and DNA vulnerability.

This study aims to assess and compare the kinetics (accumulation/elimination) of the marine biotoxins okadaic acid (OA) and dinophysistoxin-1 (DTX1), between native (Ruditapes decussatus) and invasive (Ruditapes philippinarum) clam species, and their genotoxic effects and DNA recover capacity after, exposure to toxic dinoflagellates Prorocentrum lima. Clams were fed with P. lima for 5 days and then to non-toxic algae (post-exposure) during other 5 days. Toxin concentrations determined in clams by LC-MS/MS were related with DNA damage and repair assessment through the comet and base excision repair (BER) assays, respectively. Differential accumulation patterns were observed between the invasive and native species. The invasive species consistently and progressively accumulated the toxins during the first 24 h of exposure, while the native clams showed drastic variations in the toxin accumulation. Nevertheless, at the end of a 5 days of exposure period, the native clams presented higher toxin concentrations, nearly reaching the legal regulatory limit for human consumption. In addition, native clams were vastly affected by OA and DTX1, presenting an increment in the DNA damage since the first day, with a correspondent increase in the repair activity. On the other hand, invasive clams were not affected by the dinoflagellate toxins, exhibiting only some signs of the challenge, namely an increase in the DNA repair mechanisms in the post-exposure period. Invasive clams R. philippinarum are better adapted to cope with harmful algal blooms and OA-group toxins than native species. These results may increase farming interest and may lead to new introductions of the invasive clams. In sympatry sites, exposure to OA-group toxins may unbalance clams species biomass and distribution as exposure to toxic dinoflagellates affects the native clams from cellular to a population level, representing a significant threat to development and maintenance of R. decussatus populations.

A machine learning approach for genome-wide prediction of morbid and druggable human genes based on systems-level data.

BACKGROUND: The genome-wide identification of both morbid genes, i.e., those genes whose mutations cause hereditary human diseases, and druggable genes, i.e., genes coding for proteins whose modulation by small molecules elicits phenotypic effects, requires experimental approaches that are time-consuming and laborious. Thus, a computational approach which could accurately predict such genes on a genome-wide scale would be invaluable for accelerating the pace of discovery of causal relationships between genes and diseases as well as the determination of druggability of gene products. RESULTS: In this paper we propose a machine learning-based computational approach to predict morbid and druggable genes on a genome-wide scale. For this purpose, we constructed a decision tree-based meta-classifier and trained it on datasets containing, for each morbid and druggable gene, network topological features, tissue expression profile and subcellular localization data as learning attributes. This meta-classifier correctly recovered 65% of known morbid genes with a precision of 66% and correctly recovered 78% of known druggable genes with a precision of 75%. It was than used to assign morbidity and druggability scores to genes not known to be morbid and druggable and we showed a good match between these scores and literature data. Finally, we generated decision trees by training the J48 algorithm on the morbidity and druggability datasets to discover cellular rules for morbidity and druggability and, among the rules, we found that the number of regulating transcription factors and plasma membrane localization are the most important factors to morbidity and druggability, respectively. CONCLUSIONS: We were able to demonstrate that network topological features along with tissue expression profile and subcellular localization can reliably predict human morbid and druggable genes on a genome-wide scale. Moreover, by constructing decision trees based on these data, we could discover cellular rules governing morbidity and druggability.

DNA damage and oxidative stress responses of mussels Mytilus galloprovincialis to paralytic shellfish toxins under warming and acidification conditions - Elucidation on the organ-specificity.

Commonly affected by changes in climate and environmental conditions, coastal areas are very dynamic environments where shellfish play an important ecological role. In this study, the oxidative stress and genotoxic responses of mussels (Mytilus galloprovincialis) exposed to paralytic shellfish toxin (PST) - producing dinoflagellates Gymnodinium catenatum were evaluated under i) current conditions (CC: 19 °C; pH 8.0), ii) warming (W: 24 °C; pH 8.0), iii) acidification (A:19 °C; pH 7.6) and iv) combined effect of warming and acidification (WA: 24 °C; pH 7.6). Mussels were fed with G. catenatum for 5 days, and to a non-toxic diet during the following 10 days. A battery of oxidative stress biomarkers and comet assay was performed at the peak of toxin accumulation and at the end of the post-exposure phase. Under CC, gills and hepatopancreas displayed different responses/vulnerabilities and mechanisms to cope with PST. While gills presented a tendency for lipid peroxidation (LPO) and genetic damage (expressed by the Genetic Damage Indicator - GDI), hepatopancreas seems to better cope with the toxins, as no LPO was observed. However, the mechanisms involved in hepatopancreas protection were not enough to maintain DNA integrity. The absence of LPO, and the antioxidant system low responsiveness, suggests DNA damage was not oxidative. When exposed to toxic algae under W, toxin-modulated antioxidant responses were observed in both gills and hepatopancreas. Simultaneous exposure to the stressors highlighted gills susceptibility with a synergistic interaction increasing DNA damage. Exposure to toxic algae under A led to genotoxicity potentiation in both organs. The combined effect of WA did not cause relevant interactions in gills antioxidant responses, but stressors interactions impacted LPO and GDI. Antioxidant responses and LPO pointed out to be modulated by the environmental conditions in hepatopancreas, while GDI results support the dominance of toxin-triggered process. Overall, these results reveal that simultaneous exposure to warming, acidification and PSTs impairs mussel DNA integrity, compromising the genetic information due to the synergetic effects. Finally, this study highlights the increasing ecological risk of harmful algal blooms to Mytilus galloprovinciallis populations.

Characterization of intracellular and extracellular saxitoxin levels in both field and cultured Alexandrium spp. samples from Sequim Bay, Washington.

Traditionally, harmful algal bloom studies have primarily focused on quantifying toxin levels contained within the phytoplankton cells of interest. In the case of paralytic shellfish poisoning toxins (PSTs), intracellular toxin levels and the effects of dietary consumption of toxic cells by planktivores have been well documented. However, little information is available regarding the levels of extracellular PSTs that may leak or be released into seawater from toxic cells during blooms. In order to fully evaluate the risks of harmful algal bloom toxins in the marine food web, it is necessary to understand all potential routes of exposure. In the present study, extracellular and intracellular PST levels were measured in field seawater samples (collected weekly from June to October 2004-2007) and in Alexandrium spp. culture samples isolated from Sequim Bay, Washington. Measurable levels of intra- and extra-cellular toxins were detected in both field and culture samples via receptor binding assay (RBA) and an enzyme-linked immunosorbent assay (ELISA). Characterization of the PST toxin profile in the Sequim Bay isolates by pre-column oxidation and HPLC-fluorescence detection revealed that gonyautoxin 1 and 4 made up 65 +/- 9.7% of the total PSTs present. Collectively, these data confirm that extracellular PSTs are present during blooms of Alexandrium spp. in the Sequim Bay region.

Presence and persistence of the amnesic shellfish poisoning toxin, domoic acid, in octopus and cuttlefish brains.

Domoic acid (DA) is a neurotoxin that causes degenerative damage to brain cells and induces permanent short-term memory loss in mammals. In cephalopod mollusks, although DA is known to accumulate primarily in the digestive gland, there is no knowledge whether DA reaches their central nervous system. Here we report, for the first time, the presence of DA in brain tissue of the common octopus (Octopus vulgaris) and the European cuttlefish (Sepia officinalis), and its absence in the brains of several squid species (Loligo vulgaris, L. forbesi and Todarodes sagittatus). We argue that such species-specific differences are related to their different life strategies (benthic/nektobenthic vs pelagic) and feeding ecologies, as squids mainly feed on pelagic fish, which are less prone to accumulate phycotoxins. Additionally, the temporal persistence of DA in octopus' brain reinforces the notion that these invertebrates can selectively retain this phycotoxin. This study shows that two highly-developed invertebrate species, with a complex central nervous system, where glutamatergic transmission is involved in vertebrate-like long-term potentiation (LTP), have the ability of retaining and possibly tolerating chronic exposure to DA, a potent neurotoxin usually acting at AMPA/kainate-like receptors. Here, we filled a gap of information on whether cephalopods accumulated this neurotoxin in brain tissue, however, further studies are needed to determine if these organisms are neurally or behaviourally impaired by DA.

Native (Ruditapes decussatus) and non-indigenous (R. philippinarum) shellfish species living in sympatry: Comparison of regulated and non-regulated biotoxins accumulation.

The native Ruditapes decussatus and the non-indigenous Ruditapes philippinarum are an important target of shellfish industries. The aim of this study was to compare an invader with a native species living in sympatry in the view of marine biotoxins accumulation. Samples were analysed for regulated and non-regulated biotoxins. The consistently occurrence of okadaic acid-group toxins and BMAA, may cause human health problems and economical losses. A strong positive relationship was observed between species, with significantly higher DSP toxicity in R. decussatus. Similar toxin profiles dominated by DTX3 in both species suggests similar metabolic pathways. Lower DSP toxicity in R. philippinarum may favour their cultivation, but a tendency for higher levels of the non-regulated BMAA was observed, indicating risks for consumers that are not monitored. This study highlights the need to better understand the physiological responses and adaptations allowing similar species exposed to the same conditions to present different toxicity levels.

In vitro bioaccessibility of the marine biotoxin okadaic acid in shellfish.

Okadaic acid (OA) and their derivatives are marine toxins responsible for the human diarrhetic shellfish poisoning (DSP). To date the amount of toxins ingested in food has been considered equal to the amount of toxins available for uptake by the human body. In this study, the OA fraction released from the food matrix into the digestive fluids (bioaccessibility) was assessed using a static in vitro digestion model. Naturally contaminated mussels (Mytilus galloprovincialis) and donax clams (Donax sp.), collected from the Portuguese coast, containing OA and dinophysistoxin-3 (DTX3) were used in this study. Bioaccessibility of OA total content was 88% and 75% in mussels and donax clams, respectively. Conversion of DTX3 into its parent compound was verified during the simulated digestive process and no degradation of these toxins was found during the process. This is the first study assessing the bioaccessibility of OA-group toxins in naturally contaminated seafood. This study provides relevant new data that can improve and lead to more accurate food safety risk assessment studies concerning these toxins.

Accumulation, transformation and tissue distribution of domoic acid, the amnesic shellfish poisoning toxin, in the common cuttlefish, Sepia officinalis.

Domoic acid (DA) is a phycotoxin produced by some diatoms, mainly from the Pseudo-nitzschia genus, and has been detected throughout the marine food web. Although DA has been frequently found in cephalopod prey such as crustaceans and fish, little is known about DA accumulation in these molluscs. This study presents the first data showing relevant concentrations of DA detected in the common cuttlefish, Sepia officinalis, which is one of the most studied cephalopod species in the world. Domoic acid was consistently found throughout 2003 and 2004 in the digestive gland of cuttlefish reaching concentrations of 241.7 microg DA g(-1). The highest DA values were detected during spring and summer months, periods when Pseudo-nitzschia occur in the plankton. In fact, Pseudo-nitzschia blooms preceded the highest DA concentrations in cuttlefish. Evaluation of DA tissue distribution showed elevated DA concentrations in the digestive gland and branchial hearts. Further, DA isomers comprised a relevant percentage of the toxin profile, indicating degradation and biotransformation of the toxin in the branchial hearts. The common cuttlefish, like other cephalopod species, plays a central position in the food web and might be a new DA vector to top predators like marine mammals. Human intoxications are not expected since DA was only seldom detected in the mantle and even then in very low levels (max 0.7 microg DA g(-1)). However, in some countries whole juvenile animals are consumed (i.e. without evisceration) and in this case they might represent a risk to human health. This study contributes to understanding the occurrence of phycotoxins in cephalopods and reveals a new member of the marine food web able to accumulate DA.

A potential vector of domoic acid: the swimming crab Polybius henslowii Leach (Decapoda-brachyura).

The swimming crab Polybius henslowii may play an important role in the movement of the amnesic shellfish toxin, domoic acid (DA), through the marine food chain. High DA concentrations have been determined in crab samples harvested along the Portuguese coast during the summer of 2002, reaching a level of 323.1 microg DA/g crab tissue. Toxin distribution in the different crab organs showed levels as high as 571.6 microg DA/g in the visceral tissues. Levels of toxin 4-12 times lower were detected in the remaining tissues. This crab might be a prominent vector of the toxin to higher trophic levels, including fishes, sea birds and even humans. In Portugal P. henslowii is commercialised during the summer in some local markets. DA concentrations were found close to the legal limit of 20 microg/g in samples purchased at Figueira da Foz market. The crabs are boiled prior to reaching the consumers. The cooking process was evaluated. Determination of toxin losses during the cooking process showed a toxin reduction higher than 50%. DA was determined by HPLC-UV and confirmed by spectra acquired with diode-array detector.