Epiphytic abundance and toxicity of Prorocentrum lima populations in the Fleet Lagoon, UK

Planktonic Dinophysis spp. and epiphytic Prorocentrum lima (Ehrenberg) Dodge are known dinoflagellate producers of okadaic acid (OA) and dinophysistoxins (DTX), causative phycotoxins of diarrhetic shellfish poisoning (DSP). Underestimation of toxic dinoflagellates associated with a toxic event may be due to the lack of sampling of species with epiphytic and epibenthic strategies, such as P. lima. As Dinophysis spp. is not found in the Fleet Lagoon, Dorset, but previous DSP events have closed the Crassostrea gigas oyster farm, P. lima is the most likely causative organism. A field assay for separating microalgal epiphytes and concentrating wild cells on to filters was successfully applied to sub-samples of a variety of macroalgae and macrophytes (seagrass) collected from the Fleet during summer 2002. P. lima was present in increasing cell densities on most substratum species, over the sampling period, from 10² to 10³ cells g⁻¹ fresh weight (FW) plant biomass. LC–MS analysis detected OA and DTX-1 in extracts of wild P. lima cells, in ratios characteristic of P. lima strains previously isolated from the Fleet. No toxins, however, were detected in oyster flesh.     

DSP toxin production de novo in cultures of Dinophysis acuminata (Dinophyceae) from North America

For decades, many aspects of Dinophysis biology have remained intractable due to our inability to maintain these organisms in laboratory cultures. Recent breakthroughs in culture methods have opened the door for detailed investigations of these important algae. Here, for the first time, we demonstrate toxin production in cultures of North American Dinophysis acuminata, isolated from Woods Hole, MA. These findings show that, despite the rarity of Dinophysis-related DSP events in North America, D. acuminata from this area has the ability to produce DSP toxins just as it does in other parts of the world where this species is a major cause of DSP toxicity. In our cultures, D. acuminata cells were observed feeding on Myrionecta rubra using a peduncle. Culture extracts were analyzed using LC–MS/MS, providing unequivocal evidence for the toxin DTX1 in the Dinophysis cultures. In addition, a significant amount of an okadaic acid diol ester, OA-D8, was detected. These results suggest that this Dinophysis isolate stores much of its OA as a diol ester. Also, toxin PTX-2 and a hydroxylated PTX-2 with identical fragmentation mass spectrum to that of PTX-11, but with a different retention time, were detected in this D. acuminata culture. This demonstration of toxin production in cultured North American Dinophysis sets the stage for more detailed studies investigating the causes of geographic differences in toxicity. It is now clear that North American Dinophysis have the ability to produce DSP toxins even though they only rarely cause toxic DSP events in nature. This may reflect environmental conditions that might induce or repress toxin production, genetic differences that cause modifications in toxin gene expression, or physiological and biochemical differences in prey species.     

DSP toxin production de novo in cultures of Dinophysis acuminata (Dinophyceae) from North America

For decades, many aspects of Dinophysis biology have remained intractable due to our inability to maintain these organisms in laboratory cultures. Recent breakthroughs in culture methods have opened the door for detailed investigations of these important algae. Here, for the first time, we demonstrate toxin production in cultures of North American Dinophysis acuminata, isolated from Woods Hole, MA. These findings show that, despite the rarity of Dinophysis-related DSP events in North America, D. acuminata from this area has the ability to produce DSP toxins just as it does in other parts of the world where this species is a major cause of DSP toxicity. In our cultures, D. acuminata cells were observed feeding on Myrionecta rubra using a peduncle. Culture extracts were analyzed using LC–MS/MS, providing unequivocal evidence for the toxin DTX1 in the Dinophysis cultures. In addition, a significant amount of an okadaic acid diol ester, OA-D8, was detected. These results suggest that this Dinophysis isolate stores much of its OA as a diol ester. Also, toxin PTX-2 and a hydroxylated PTX-2 with identical fragmentation mass spectrum to that of PTX-11, but with a different retention time, were detected in this D. acuminata culture. This demonstration of toxin production in cultured North American Dinophysis sets the stage for more detailed studies investigating the causes of geographic differences in toxicity. It is now clear that North American Dinophysis have the ability to produce DSP toxins even though they only rarely cause toxic DSP events in nature. This may reflect environmental conditions that might induce or repress toxin production, genetic differences that cause modifications in toxin gene expression, or physiological and biochemical differences in prey species.     

Determination of diarrhetic shellfish toxins in various dinoflagellate species

Sixteen species of unialgal samples of dinoflagellate, either wild or cultured, were tested for production of diarrhetic shellfish toxins such as okadaic acid (OA), dinophysistoxin-1 (DTX1), and pectenotoxins (PTXs). Determination of micro-quantities of the toxins was facilitated by fluorometry and UV HPLC. Seven Dinophysis species were confirmed to produce either OA or DTX1, or both. Toxin content and composition varied regionally and seasonally. Intraspecies variation was also observed among cultured strains of Prorocentrum lima. PTX2 was the only toxin detected among PTX family, and D. fortii was the only species to contain this toxin. author for correspondence     

Seasonality of Dinophysis spp. and Prorocentrum lima in Black Sea phytoplankton and associated shellfish toxicity

Plankton surveys, between 2001 and 2005 along the Russian Caucasian Black Sea Coast, revealed Dinophysis rotundata, D. caudata and Prorocentrum lima as the most ubiquitous of the known dinoflagellates associated with diarrhetic shellfish poisoning (DSP). Dinophysis spp. were first observed during the spring phytoplankton succession and persist throughout the late summer phytoplankton peak. The highest total concentration, 3000 cells/L, of D. rotundata and D. caudata was observed in April 2001. Unlike Dinophysis, P. lima was rarely observed in plankton samples but closely followed storm events with maximum cell counts of P. lima occurred in July 2002.The presence of Dinophysis in mussel (Mytilus galloprovincialis) hepatopancreas correlated with concentration with Dinophysis observed in the plankton samples. Conversely, P. lima could be found in most hepatopancreas samples collected during the May to October period. Therefore, planktonic concentration of P. lima does not reflect its availability for and consumption by shellfish.Samples of mussel hepatopancreas, from August 2002, with a corresponding Dinophysis concentration of 250 cells/L and no observable P. lima, were found to contain 0.03 ng OAE/g. This sample analyses by LC-MS/MS displayed okadaic acid (OA) and related congeners (DTX1) along with the pectinotoxins (PTX2 and PTX2sa). Highest observed levels of P. lima-induced DSP-toxicity in hepatopancreas was 0.41 g OA-equivalents/g corresponded to the highest observed planktonic cell counts of P. lima, 300 cell/L in August 2001. Cultures isolated from this sample were found to produce OA, DTX1 and their related diol esters.These data reveal a threat, represented by DSP-toxic species, at Black Sea coasts, and provide grounds for the introduction of phycotoxin control measures in the region.     

LC–MS/MS analysis of okadaic acid analogues and other lipophilic toxins in single-cell isolates of several Dinophysis species collected in Hokkaido,Japan

Quantification of diarrhetic shellfish poisoning (DSP) toxins (okadaic acid analogues), and other lipophilic toxins in single-cell isolates of the dinoflagellates Dinophysis fortii, D. acuminata, D. mitra, D. norvegica, D. tripos, D. infundibulus and D. rotundata, collected in coastal waters Hokkaido, Japan in 2005, was carried out by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Okadaic acid (OA), dinophysistoxin-1 (DTX1), 7-O-palmitoyldinophysistoxin-1 (DTX3), pectenotoxin-1 (PTX1), pectenotoxin-11 (PTX11), pectenotoxin-2 (PTX2), pectenotoxin-6 (PTX6), pectenotoxin-2 seco-acid (PTX2sa), yessotoxin (YTX) and 45-hydroxyyessotoxin (45-OHYTX) were quantified by LC–MS/MS. PTX2 was the dominant toxin in D. acuminata, D. norvegica and D. infundibulus whereas both DTX1 and PTX2 were the principal toxins in D. fortii. None of the toxins were detected in D. mitra, D. rotundata and D. tripos. These results suggest that D. fortii is the most important species responsible for DSP contamination of bivalves in Hokkaido. This is the first finding of PTX2 in D. infundibulus, and confirms the presence of PTX2 in Japanese D. acuminata and D. norvegica collected from natural seawater.     

Field and mesocosm trials on passive sampling for the study of adsorption and desorption behaviour of lipophilic toxins with a focus on OA and DTX1

It has been demonstrated that polymeric resins can be used as receiving phase in passive samplers designed for the detection of lipophilic marine toxins at sea and was referred to as solid phase adsorption toxin tracking (SPATT). The present study describes the uptake and desorption behaviour of the lipophilic marine toxins okadaic acid (OA) and dinophysistoxin-1 (DTX1) from Prorocentrum lima cultures by five styrene—divinylbenzene based polymeric resins Sepabeads^® SP850, Sepabeads^® SP825L, Amberlite^® XAD4, Dowex^® Optipore^® L-493 and Diaion^® HP-20. All resins accumulated OA and DTX1 from the P. lima culture with differences in adsorption rate and equilibrium rate. Following statistical evaluation, HP-20, SP850 and SP825L demonstrated similar adsorption rates. However, possibly due to its larger pore size, the HP-20 did not seem to reach equilibrium within 72 h exposure as opposed to the SP850 and SP825L. This was confirmed when the resins were immersed at sea for 1 week on the West Coast of Ireland. Furthermore, this work also presents a simple and efficient extraction method suitable to SPATT samplers exposed to artificial or natural culture media.     

Palatability and fatality of the dinoflagellate <Emphasis Type="Italic">Prorocentrum lima</Emphasis> to <Emphasis Type="Italic">Artemia salina</Emphasis>

Prorocentrum lima is a toxic alga that produces both intra-cellular and extra-cellular toxins, including okadaic acid (OA) and dinophysistoxins (DTXs). Nauplii of the brine shrimp Artemia salina were exposed to both the cell and cell-free culture medium of P. lima in order to test the hypotheses that the extra-cellular medium is toxic to brine shrimp and that the P. lima cell is palatable but fatal to it. Artemia cysts incubated in the cell-free medium hatched, but mortalities were recorded for nauplii that hatched in, and metanuaplii exposed to, test solutions (autoclaved filtered seawater + cell-free medium) that contained at least 50% of the cell-free medium. Animals exposed to cells of P. lima readily fed on the cells. Some, especially among the Day 1 nauplii, ingested only one cell before dying, while others ingested more than one cell, up to six cells in the case of Day 3 nauplii, before dying. Day 3 nauplii were readily and heavily impacted by the P. lima cells. Survival analysis was used to evaluate survivorship of Day 1 to Day 3 nauplii exposed to cells of P. lima. Estimates were made of tD50s for the different age groups. Comparisons of the tD50s showed that the tD50s for Day 1 and Day 2 nauplii did not vary significantly, but they each varied significantly from the tD50 for the Day 3 nauplii. The possible ecological implications of the findings are discussed.     

Toxigenic phytoplankton and concomitant toxicity in the mussel Choromytilus meridionalis off the west coast of South Africa

The variability of toxigenic phytoplankton and the consequent uptake and loss of toxins by the mussel Choromytilus meridionalis was investigated in the southern Benguela at the event scale (3–10 days) in response to the upwelling–downwelling cycle. Phytoplankton and mussel samples were collected daily (20 March–11 April 2007) from a mooring station (32.04°S; 18.26°E) located 3.5 km offshore of Lambert's Bay, within the St Helena Bay region. Rapid changes in phytoplankton assemblages incorporated three groups of toxigenic phytoplankton: (1) the dinoflagellate Alexandrium catenella; (2) several species of Dinophysis, including Dinophysis acuminata, Dinophysis fortii, Dinophysis hastata and Dinophysis rotundata; and (3) members of the diatom genus Pseudo-nitzschia. Analysis of phytoplankton concentrates by LC–MS/MS or LC-FD provided information on the toxin composition and calculated toxicity of each group. Several additional in vitro assays were used for the analysis of toxins in mussels (ELISA, RBA, MBA for PSP toxins; and ELISA for DSP toxins). Good correspondence was observed between methods except for the MBA, which provided significantly lower (approximately 2-fold) estimates of PSP toxins. PSP and DSP toxins both exceeded the regulatory limits in Choromytilis meridionalis, but ASP toxins were undetected. Differences were observed in the composition of both PSP and DSP toxins in C. meridionalis from that of the ingested dinoflagellates (PSP toxins showed an increase in STX, C1,2, and traces of dcSTX and GTX1,4 and a decrease in NEO; DSP toxins showed an increased in DTX1, and traces of PTX2sa, and a decrease in OA). The rate of loss of PSP toxins following dispersal of the A. catenella boom was 0.12 d⁻¹. Variation in the loss rates of different PSP toxins contributed to the change in toxin profile in C. meridionalis. Prediction of net toxicity in shellfish of the nearshore environment in the southern Benguela is limited due to rapid phytoplankton community changes, high variability in cellular toxicity, and the selective uptake and loss of toxins, and/or transformation of toxins.     

Pectenotoxin and okadaic acid-based toxin profiles in Dinophysis acuta and Dinophysis acuminata from New Zealand

The major pectenotoxin and okadaic acid group toxins in Dinophysis acuta and Dinophysis acuminata cell concentrates, collected from various locations around the coast of the South Island of New Zealand (NZ), were determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS). PTX2 and PTX11 were the major polyether toxins in all Dinophysis spp. cell concentrates. D. acuta contained PTX11 and PTX2 at concentrations of 4.7–64.6 and 32.5–107.5 pg per cell, respectively. The amounts of PTX11 and PTX2 in D. acuminata were much lower at 0.4–2.1 and 2.4–25.8 pg per cell, respectively. PTX seco acids comprised only 4% of the total PTX content of both D. acuta and D. acuminata. D. acuta contained low levels of OA (0.8–2.7 pg per cell) but specimens from the South Island west coast also contained up to 10 times higher levels of OA esters (7.0–10.2 pg per cell). Esterified forms of OA were not observed in D. acuta specimens from the Marlborough Sounds. D. acuta did not contain any DTX1 though all D. acuminata specimens contained DTX1 at levels of 0.1–2.4 pg per cell. DTX2 was not present in any New Zealand Dinophysis spp. specimens. Although the total toxin content varied spatially and temporally, the relative proportions of the various toxins in different specimens from the same location appeared to be relatively stable. The total PTX/total OA ratios in different isolates of D. acuta were very similar (mean±S.E.: 14.9±1.9), although the Marlborough Sounds D. acuminata isolates had a higher total PTX/total OA ratio (mean±S.E.: 22.7±2.4) than the Akaroa Harbour isolates (8.0). No evidence of azaspiracids were detected in these specimens. These results show that the LC–MS/MS monitoring of plankton for PTX group toxins (e.g. PTX2) and their derivatives (e.g. PTX2 seco acid) may provide a sensitive, semi-quantitative, indicator of the presence of more cryptic OA group toxins (e.g. OA esters).     

Occurrence of Prorocentrum lima,a DSP toxin-producing species from the Atlantic coast of Canada

A species of Prorocentrum (Dinophyta, Prorocentrales), isolated from a phytoplankton net sample from the Atlantic coast of Nova Scotia, has been brought into unialgal culture. The sample was collected at an aquaculture site immediately following an incident of diarrhetic shellfish poisoning (DSP) due to the consumption of contaminated mussels. This clonal isolate has been identified as P. lima, based on its morphological characteristics. Analysis of the culture extract, using high performance liquid chromatography (HPLC) with fluorescence detection, indicated the presence of the DSP toxins, okadaic acid (OA) and dinophysistoxin-1 (DTX-1).     

Toxins and other chemical constituents from Prorocentrum lima

The marine phytoplankton Prorocentrum lima is one of the toxic and harmful microalgae which can cause red tides. In chemical investigation on cultured strain P. lima PL11, seven compounds were isolated and identified by spectroscopic data, including three typical shellfish toxins okadaic acid (OA, 1), OA methyl ester (2), and prorocentrolide (3), three terpenoids (4–6), and one polyketide (7). Compounds 5 and 6 should be derived from carotenoid fucoxanthin. Compounds 4–7 were isolated from this genus of microalgae for the first time.     

Whole‐cell biotransformation of oleanolic acid by free and immobilized cells of Nocardia iowensis: Characterization of new metabolites

In this study, Nocardia iowensis was used to transform oleanolic acid (OA) into oleanane derivatives. The first derivative, which was found after 24 h of cultivation, was the known and already described OA methyl ester. After 1 week, two other derivatives (oleanonic acid methyl ester and an unknown metabolite) were identified as new products of a biotransformation by N. iowensis. These oleanane metabolites were characterized by HPLC, HPLC‐ESI‐MS, and HPLC‐¹H NMR spectroscopy. The biotransformation was performed by suspended and immobilized cells (ICs) of N. iowensis. Cells immobilized in alginate beads were used in order to prepare a continuous process. The substrate uptake of free and ICs was similar, whereas the peak area of OA methyl ester of the ICs was only about 10% of the native cells. However, the final product (oleanonic acid methyl ester) concentrations were similar in both approaches, whereas the unknown metabolite 3 was only detected transiently in the medium of ICs. Based on these results, a new biosynthetic pathway for the biotechnological production of oleanonic acid methyl ester is proposed.     

INTER‐ AND INTRASPECIFIC RELATIONSHIPS BETWEEN NUCLEAR DNA CONTENT AND CELL SIZE IN SELECTED MEMBERS OF THE CENTRIC DIATOM GENUS THALASSIOSIRA (BACILLARIOPHYCEAE)1

The enormous species diversity of diatoms correlates with the remarkable range of cell sizes in this group. Nuclear DNA content relates fundamentally to cell volume in other eukaryotic cells. The relationship of cell volume to G1 DNA content was determined among selected members of the genus Thalassiosira, one of the most species‐rich and well‐studied centric diatom genera. Both minimum and maximum species‐specific cell volume correlated positively with G1 DNA content. Phylogeny based on 5.8 S and ITS rDNA sequences indicated that multiple changes in G1 DNA content and cell volume occurred in Thalassiosira evolution, leading to a 1,000‐fold range in both parameters in the group. Within the Thalassiosira weissflogii (Grunow) G. A. Fryxell et Grunow species complex, G1 DNA content varied 3‐fold: differences related to geographic origin and time since isolation; doubling and tripling of G1 DNA content occurred since isolation in certain T. weissflogii isolates; and subcultures of T. weissflogii CCMP 1336 diverged in DNA content by 50% within 7 years of separation. Actin, β‐tubulin, and Spo11/TopVIA genes were selected for quantitative PCR estimation of haploid genome size in subclones of selected T. weissflogii isolates because they occur only once in the T. pseudonana Hasle et Heimdal genome. Comparison of haploid genome size estimates with G1 DNA content suggested that the most recent T. weissflogii isolate was diploid, whereas other T. weissflogii isolates appeared to be polyploid and/or aneuploid. Aberrant meiotic and mitotic cell divisions were observed, which might relate to polyploidization. The structural flexibility of diatom genomes has important implications for their evolutionary diversification and stability during laboratory maintenance.     

DSP toxin contents inDinophysis fortii and scallops collected at Mutsu Bay,Japan

Cell densities of toxic phytoplankton species responsible for diarrhetic shellfish poisoning (DSP) were monitored at a sampling site in Mutsu Bay, Japan, in 1995.Dinophysis fortii almost completely dominated the toxic phytoplankton community. Okadaic acid (OA) and dinophysistoxin-1 (DTX1) contents in bothD. fortii cells and midgut glands of scallops collected at the same sampling site were determined by HPLC — fluorometry. DTX1 was detected fromD. fortii and scallops. The contents of DTX1 inD. fortii changed markedly during the experimental periods (5–252 pg cell^–1). The highest concentration of DTX1 in the midgut glands of scallops coincided with the period of relatively high cell densities ofD. fortii with the highest content of DTX1 (252 pg cell^–1). The results demonstrate that toxin content in the cells is an important factor affecting the toxicity of shellfish.     

The binding of okadaic acid analogs to recombinant OABP2.1 originally isolated from the marine sponge Halichondria okadai

The binding between [24-³H]okadaic acid (OA) and a recombinant OA binding protein OABP2.1 was examined using various OA analog, including methyl okadaate, norokadanone, 7-deoxy OA, and 14,15-dihydro OA, 7-O-palmitoyl DTX1, to investigate the structure activity relationship. Among them, 7-O-palmitoyl DTX1, which is one of the diarrhetic shellfish poisoning (DSP) toxins identified in shellfish, displayed an IC₅₀ for [24-³H]OA binding at 51 ± 6.3 nM (Mean ± SD). In addition, a synthetic compound, N-pyrenylmethyl okadamide, exhibited its IC₅₀ at 10 ± 2.9 nM (Mean ± SD). These results suggested that the recombinant OABP2.1 and the N-pyrenylmethyl okadamide might be core substances in a novel assay for the DSP toxins.     

GROWTH INHIBITION AND TOXICITY OF THE DIATOM ALDEHYDE 2‐TRANS, 4‐TRANS‐DECADIENAL ON THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)1

A common aldehyde present in marine and freshwater diatoms, 2‐trans, 4‐trans‐decadienal (A3), is involved in the wound‐activated response of diatoms to copepod grazing. Upon breakage of the diatom cell membrane, aldehydes are enzymatically produced by the rapid conversion of precursors and strongly impact copepod reproduction by impairing egg production and hatching success, inducing teratogenic embryos modifications. In this study, A3 was assayed with the marine diatom Thalassiosira weissflogii (Grunow) Fryxell et Hasle. The aldehyde concentration necessary to reduce 50% growth rate (EC₅₀) was 0.29 mg·L^?1. Decadienal was found to inhibit T. weissflogii cell growth in a dose‐ and time‐dependent manner, with irreversible effects after 24 h of exposure. Decadienal induced a degenerative process, through modifications of cell membrane characteristics, interference with cell cycle progression, and with cell metabolic activity, leading to cell death. A preferential action of A3 on dividing cells was observed. Photosynthetic efficiency significantly decreased upon exposure to the aldehyde, paralleled by an increase in diatoxanthin, suggesting a protective role of this xanthophyll, usually involved in photoprotection. Dying cells exhibited the morphological and biochemical features that bear close resemblance to apoptosis of mammalian cells, including cell shrinkage, chromatin condensation, and degradation of nuclear DNA to nucleosomal size fragments. These data are the first direct evidence to show aldehydes are toxic to diatoms. We suggest a possible nontoxic role of such compounds as chemical signals of unfavorable conditions within the phytoplankton communities, which may be relevant for the population dynamics of diatoms during blooms.     

Liquid chromatography high‐resolution mass spectrometry for fatty acid profiling

Quantification of fatty acids has been crucial to elucidate lipid biosynthesis pathways in plants. To date, fatty acid identification and quantification has relied mainly on gas chromatography (GC) coupled to flame ionization detection (FID) or mass spectrometry (MS), which requires the derivatization of samples and the use of chemical standards for annotation. Here we present an alternative method based on a simple procedure for the hydrolysis of lipids, so that fatty acids can be quantified by liquid chromatography mass spectrometry (LC‐MS) analysis. Proper peak annotation of the fatty acids in the LC‐MS‐based methods has been achieved by LC‐MS measurements of authentic standard compounds and elemental formula annotation supported by ¹³C isotope‐labeled Arabidopsis. As a proof of concept, we have compared the analysis by LC‐MS and GC‐FID of two previously characterized Arabidopsis thaliana knock‐out mutants for FAD6 and FAD7 desaturase genes. These results are discussed in light of lipidomic profiles obtained from the same samples. In addition, we performed untargeted LC‐MS analysis to determine the fatty acid content of two diatom species. Our results indicate that both LC‐MS and GC‐FID analyses are comparable, but that because of higher sensitivity and selectivity the LC‐MS‐based method allows for a broader coverage and determination of novel fatty acids.     

A proteomics assay to detect eight CBRN‐relevant toxins in food

A proteomics assay was set up to analyze food substrates for eight toxins of the CBRN (chemical, biological, radiological and nuclear) threat, namely ricin, Clostridium perfringens epsilon toxin (ETX), Staphylococcus aureus enterotoxins (SEA, SEB and SED), shigatoxins from Shigella dysenteriae and entero‐hemorragic Escherichia coli strains (STX1 and STX2) and Campylobacter jejuni cytolethal distending toxin (CDT). The assay developed was based on an antibody‐free sample preparation followed by bottom‐up LC‐MS/MS analysis operated in targeted mode. Highly specific detection and absolute quantification were obtained using isotopically labeled proteins (PSAQ standards) spiked into the food matrix. The sensitivity of the assay for the eight toxins was lower than the oral LD50 which would likely be used in a criminal contamination of food supply. This assay should be useful in monitoring biological threats. In the public‐health domain, it opens the way for multiplex investigation of food‐borne toxins using targeted LC‐MS/MS.     

Pattern of Proteins after Heat Shock and UV-B Radiation of some Temperate Marine Diatoms and the Antarctic Odontella weissflogii

Synthesis of stress proteins after heat shock and different periods of UV-B radiation were investigated with marine diatom species from the North Sea Ditylum brightwellii, Lithodesmium variabile, Odontella sinensis, Thalassiosira rotula and the Antarctic diatom Odontella weissfloggii from the Weddell Sea. Algae were grown in an artifical sea-water medium under controlled laboratory conditions: light/dark regime of 12:12 h (7.2 W m^?2), normal air (0.035 vol.% CO₂) and 18° or 4 °C. All the tested diatom species can produce heat shock proteins (HSPS) of the 70 kDa family by in vivo labelling with [³⁵S]-methionine. The same results were obtained for Odontella sinensis, Ditylum brightwellii and Odontella weissflogii by estimation of the in vitro translation products with poly-A-mRNA isolated from these organisms. However, Odontella weissflogii, a species relatively insensitive to UV-B irradiance, did not synthesize UV-induced HSPS, whereas the UV-sensitive diatom Odontella sinensis, as well as Lithodesmium variabile, produced all the observed HSPS after UV-B exposure. In addition, a protein of 43 kDa was found after UV-B irradiance of the temperate Odontella sinensis. The temperate marine diatom Thalassiosira rotula synthesized 70 kDa and 5 7 kDa proteins after a heat shock and a UV-B exposure of 2 h, but a 40 kDa protein could not be detected, whereas a 60 kDa protein was found after 2 h UV-B exposure. The results are discussed in view of a possible adaptation of O. weissflogii to an enhanced UV dose.