Impact of the neurotoxin ß-N-methylamino-L-alanine on the diatom Thalassiosira pseudonana using metabolomics.

The neurotoxin ß-N-methylamino-L-alanine (BMAA) has emerged as an environmental factor related to neurodegenerative diseases. BMAA is produced by various microorganisms including cyanobacteria and diatoms, in diverse ecosystems. In the diatom Phaeodactylum tricornutum, BMAA is known to inhibit growth. The present study investigated the impact of BMAA on the diatom Thalassiosira pseudonana by exposing it to different concentrations of exogenous BMAA. Metabolomics was predominantly employed to investigate the effect of BMAA on T. pseudonana, and MetaboAnalyst (https://www.metabo-analyst.ca/) was used to identify BMAA-associated metabolisms/pathways in T. pseudonana. Furthermore, to explore the unique response, specific metabolites were compared between treatments. When the growth was obstructed by BMAA, 17 metabolisms/pathways including nitrogen and glutathione (i.e. oxidative stress) metabolisms, were influenced in T. pseudonana. This study has further determined that 11 out of 17 metabolisms/pathways could be essentially affected by BMAA, leading to the inhibition of diatom growth.

Evidence of 2,4-diaminobutyric acid (DAB) production as a defense mechanism in diatom Thalassiosira pseudonana.

The neurotoxic secondary metabolite ß-N-methylamino-L-alanine (BMAA) and its structural isomer 2,4-diaminobutyric acid (DAB) are known to be produced by various phytoplankton groups. Despite the worldwide spread of these toxin producers, no obvious role and function of BMAA and DAB in diatoms have been identified. Here, we investigated the effects of biotic factors, i.e., predators and competitors, as possible causes of BMAA and/or DAB regulation in the two diatom species Phaeodactylum tricornutum and Thalassiosira pseudonana. DAB was specifically regulated in T. pseudonana by the presence of predators and competitors. The effects of DAB on both diatoms as competitors and on the copepod Tigriopus sp. as predator at individual and at population levels were examined. The toxic effects of DAB on the growth of T. pseudonana and the population of Tigriopus sp. were significant. The effect of DAB as a defensive secondary metabolite is assumed to be environmentally relevant depending on the number of the copepods. The results show a potential function of DAB that can play an important role in defense mechanisms of T. pseudonana.

Effect and function of ß-N-methylamino-L-alanine in the diatom Phaeodactylum tricornutum.

The neurotoxin ß-N-methylamino-L-alanine (BMAA) is an environmental factor connected to neurodegenerative diseases. BMAA can be produced by various microorganisms (e.g. bacteria, cyanobacteria, dinoflagellates and diatoms) present in diverse ecosystems. No previous study has revealed the function of BMAA in diatoms. In the present study, we combined physiological data with metabolomic and transcriptional data in order to investigate the effect and function of BMAA in the diatom Phaeodactylum tricornutum. P. tricornutum, exposed to different concentrations of exogenous BMAA, showed concentration dependent responses. When the concentration of supplemented BMAA was sufficient to arrest the growth of P. tricornutum, oxidative stress and obstructed carbon fixation were obtained from the specific metabolite and transcriptional data. Results also indicated increased concentration of intracellular chlorophyll a and alterations in the GS-GOGAT cycle, whereas the urea cycle was suppressed. We therefore conclude that BMAA represents a toxic metabolite able to control the growth of P. tricornutum by triggering oxidative stress, and further influencing photosynthesis and nitrogen metabolisms.

Transfer of the Neurotoxin ß-N-methylamino-l-alanine (BMAA) in the Agro-Aqua Cycle.

The neurotoxic non-protein amino acid ß-N-methylamino-l-alanine (BMAA) is connected to the development of neurodegenerative diseases. BMAA has been shown to accumulate in aquatic ecosystems, and filter-feeding molluscs seem particularly susceptible to BMAA accumulation. The blue mussels farmed along the Swedish coastline in the Baltic Sea are, due to their small size, exclusively used to produce feed for chicken and fish in the agro-aqua cycle. We have investigated the possible biotransfer of BMAA from mussels, via mussel-based feed, into chickens. Chickens were divided into two groups, the control and the treatment. BMAA was extracted from the muscle, liver, brain, and eye tissues in both chicken groups; a UPLC-MS/MS method was subsequently used to quantify BMAA. The results indicate detectable concentrations of BMAA in both chicken groups. However, the BMAA concentration in chicken was 5.65 times higher in the treatment group than the control group, with the highest concentration found in muscle tissue extracted from the treatment group chickens. These data suggest that there is a BMAA transfer route within the agro-aqua cycle, so further investigation is recommended before using mussel-based feed in the chicken industry.

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.

The effect of exogenous ß-N-methylamino-l-alanine (BMAA) on the diatoms Phaeodactylum tricornutum and Thalassiosira weissflogii.

ß-N-methylamino-l-alanine (BMAA), a non-protein amino acid with neurodegenerative features, is known to be produced by cyanobacteria, diatoms and a dinoflagellate. BMAA research has intensified over the last decade, and knowledge has been gained about its bioaccumulation in aquatic and terrestrial ecosystems, toxic effects in model organisms and neurotoxicity in vivo and in vitro. Nevertheless, knowledge of the actual physiological role of BMAA in the producing species or of the ecological factors that regulate BMAA production is still lacking. A few studies propose that BMAA functions to signal nitrogen depletion in cyanobacteria. To investigate whether BMAA might have a similar role in diatoms, two diatom species - Phaeodactylum tricornutum and Thalassiosira weissflogii - were exposed to exogenous BMAA at environmental relevant concentrations, i.e. 0.005, 0.05 and 0.5µM. BMAA was taken up in a concentration dependent manner in both species in the BMAA free fraction and in the protein fraction of T. weissflogii. As a result of the treatments, the diatom cells at some of the time points and at some of the BMAA concentrations exhibited lower concentrations of chlorophyll a and protein, in comparison to controls. At the highest (0.5µM) concentration of BMAA, extracellular ammonia was found in the media of both species at all time points. These results suggest that BMAA interferes with nitrogen metabolism in diatoms, possibly by inhibiting ammonium assimilation via the GS/GOGAT pathway.

BMAA extraction of cyanobacteria samples: which method to choose?

ß-N-Methylamino-L-alanine (BMAA), a neurotoxin reportedly produced by cyanobacteria, diatoms and dinoflagellates, is proposed to be linked to the development of neurological diseases. BMAA has been found in aquatic and terrestrial ecosystems worldwide, both in its phytoplankton producers and in several invertebrate and vertebrate organisms that bioaccumulate it. LC-MS/MS is the most frequently used analytical technique in BMAA research due to its high selectivity, though consensus is lacking as to the best extraction method to apply. This study accordingly surveys the efficiency of three extraction methods regularly used in BMAA research to extract BMAA from cyanobacteria samples. The results obtained provide insights into possible reasons for the BMAA concentration discrepancies in previous publications. In addition and according to the method validation guidelines for analysing cyanotoxins, the TCA protein precipitation method, followed by AQC derivatization and LC-MS/MS analysis, is now validated for extracting protein-bound (after protein hydrolysis) and free BMAA from cyanobacteria matrix. BMAA biological variability was also tested through the extraction of diatom and cyanobacteria species, revealing a high variance in BMAA levels (0.0080-2.5797 µg g(-1) DW).

Biotransfer of ß-N-methylamino-L-alanine (BMAA) in a eutrophicated freshwater lake.

ß-N-Methylamino-L-alanine (BMAA), a neurotoxic non-protein amino acid, plays a significant role as an environmental risk factor in neurodegenerative diseases, such as amyotrophic lateral sclerosis. BMAA producers occur globally, colonizing almost all habitats and represent species from distinct phytoplanktonic groups, i.e., cyanobacteria, diatoms, and dinoflagellates. Bioaccumulation of BMAA in invertebrate and vertebrate organisms has also been registered around the globe. In the Baltic Sea, BMAA has been detected in several commercial fish species, raising the question of the bioaccumulation of BMAA in Swedish limnic systems. Here we find the presence of BMAA in water samples from Lake Finjasjön and identify its bioaccumulation patterns in both plankti-benthivorous and piscivorous fish, according to fish species, total weight, gender, and season of collection. For the first time, a large number of fish individuals were used in order to draw conclusions on BMAA bioaccumulation in a closed ecological community based on a statistical approach. We may, therefore, conclude that feeding patterns (plankti-benthivorous) and increased age of fish may lead to a higher tissue concentration of BMAA.

BMAA in shellfish from two Portuguese transitional water bodies suggests the marine dinoflagellate Gymnodinium catenatum as a potential BMAA source.

The neurotoxin ß-N-methylamino-l-alanine (BMAA) and its putative role in multiple neurodegenerative diseases have been intensely studied since 2005 when the toxin was discovered to be produced by worldwide-distributed cyanobacterial species inhabiting terrestrial, marine, brackish, and freshwater ecosystems. Recently, BMAA production was also associated with one eukaryotic group, namely, diatoms, raising questions about its production by other phytoplanktonic groups. To test for BMAA bioavailability in ecosystems where abundant phytoplanktonic blooms regularly occur, samples of filter-feeding shellfish were collected in two Portuguese transitional water bodies. BMAA content in cockles (Cerastoderma edule) collected weekly between September and November 2009 from Ria de Aveiro and at least once a month from May to November from Ria Formosa, fluctuated from 0.079±0.055 to 0.354±0.066µg/g DW and from below the limit of detection to 0.434±0.110µg/g DW, respectively. Simultaneously to BMAA occurrence in cockles, paralytic shellfish toxins were detected in shellfish as a result of Gymnodinium catenatum blooms indicating a possible link between this marine dinoflagellate and BMAA production. Moreover, considerable high BMAA levels, 0.457±0.186µg/g DW, were then determined in a laboratory grown culture of G. catenatum. This work reveals for the first time the presence of BMAA in shellfish from Atlantic transitional water bodies and consubstantiate evidences of G. catenatum as one of the main sources of BMAA in these ecosystems.