Cytotoxic assessment of the regulated, co-existing mycotoxins aflatoxin B1, fumonisin B1 and ochratoxin, in single, binary and tertiary mixtures

Aflatoxin B1 (AFB1), ochratoxin A (OTA) and fumonisin B1 (FB1) are contaminants which have been shown to regularly co-occur in a range of foods. However, only a small number of studies have evaluated the interactive effect of binary and tertiary mycotoxins. The present study evaluated the effects of low levels of each mycotoxin in combination at their EU regulatory limits. Toxic effect with respect to cell viability was measured by MTT and neutral red assays, assessing mitochondria and lysosome integrities respectively. Individual toxicity showed that OTA (10 μg/ml) was the most cytotoxic mycotoxin in all three cell lines studied (caco-2, MDBK and raw 264.7). Binary combinations were cytotoxic to the MDBK cell line in the order [OTA/FB1] > [AFB1/FB1] > [AFB1/OTA], whilst all effects observed were classified as being additive. Tertiary combinations of AFB1, FB1 and OTA at the EU regulatory limits were tested and not found to exhibit measurable cytotoxicity in MDBK, caco-2 or raw 264.7 cells. However by increasing these concentrations above the legal limits to OTA (3 μg/ml), FB1 (8 μg/ml) and AFB1 (1.28 μg/ml), cytotoxicity was observed with up to 26% reduction in cell viability and synergistic effects were evident with regard to mitochondrial integrity. © 2014 Elsevier Ltd. All rights reserved.

Phylogeographic analysis of the red seaweed Palmaria palmata reveals a Pleistocene marine glacial refugium in the English Channel

Phylogeography has provided a new approach to the analysis of the postglacial history of a wide range of taxa but, to date, little is known about the effect of glacial periods on the marine biota of Europe. We have utilized a combination of nuclear, plastid and mitochondrial genetic markers to study the biogeographic history of the red seaweed Palmaria palmata in the North Atlantic. Analysis of the nuclear rDNA operon (ITS1-5.8S-ITS2), the plastid 16S-trnI-trnA-23S-5S, rbcL-rbcS and rpl12-rps31-rpl9 regions and the mitochondrial cox2–3 spacer has revealed the existence of a previously unidentified marine refugium in the English Channel, along with possible secondary refugia off the southwest coast of Ireland and in northeast North America and/or Iceland. Coalescent and mismatch analyses date the expansion of European populations from approximately 128 000 bp and suggest a continued period of exponential growth since then. Consequently, we postulate that the penultimate (Saale) glacial maximum was the main event in shaping the biogeographic history of European P. palmata populations which persisted throughout the last (Weichselian) glacial maximum (c. 20 000 bp) in the Hurd Deep, an enigmatic trench in the English Channel.

Contribution of the UV component of natural sunlight to photoinhibition of photosynthesis in six species of subtidal brown and red seaweeds.

Field-collected specimens of three species of Laminaria and three species of subtidal red algae (Delesseria sanguinea, Plocamium cartilagineum and Phyllophora pseudoceranoides) were exposed to natural summer sunlight on Helgoland (southern North Sea) for up to 4 h at 15 °C. Dark-adapted variable fluorescence (Fv : Fm) was measured immediately after these treatments, and following 6, 24 and 48 h of recovery in moderate irradiances of white light. The response of plants to the full spectrum of natural sunlight was compared with that to PAR alone, UV-A + visible, UV-A + UV-B, or UV-A alone. The Fv : Fm values of all species were reduced to minimal values after 4 h in all of these treatments, but those of the more resistant species (Laminaria spp. and P. pseudoceranoides) were higher after shorter exposures to UV radiation alone than to PAR with or without UV. The recovery of Fv : Fm in all species was also more rapid in the two treatments that contained UV radiation alone than in those that included PAR. These results suggest that it is the high irradiances of PAR in natural sunlight which are responsible for the photoinhibition of photosynthesis of subtidal seaweeds and that the current ambient irradiances of UV radiation (either UV-B or UV-A) in northern temperate latitudes would not contribute significantly to this photoinhibition.

Do vesicle cells of the red alga Asparagopsis (Falkenbergia stage) play a role in bromocarbon production?

The Rhodophyceae (red algae) are an established source of volatile halocarbons in the marine environment. Some species in the Bonnemaisoniaceae have been reported to contain large amounts of halogens in structures referred to as vesicle cells, suggesting involvement of these specialised cells in the production of halocarbons. We have investigated the role of vesicle cells in the accumulation and metabolism of bromide in an isolate of the red macroalga Asparagopsis (Falkenbergia stage), a species known to release bromocarbons. Studies of laboratory-cultivated alga, using light microscopy, revealed a requirement of bromide for both the maintenance and formation of vesicle cells. Incubation of the alga in culture media with bromide concentrations below 64 mg l-1 (the concentration of Br- in seawater) resulted in a decrease in the proportion of vesicle cells to pericentral cells. The abundance of vesicle cells was correlated with bromide concentration below this level. Induction of vesicle cell formation in cultures of Falkenbergia occurred at concentrations as low as 8 mg l-1, with the abundance of vesicle cells increasing with bromide concentration up to around 100 mg l-1. Further studies revealed a positive correlation between the abundance of vesicle cells and dibromomethane and bromoform production. Interestingly, however, whilst dibromomethane production was stimulated by the presence of bromide in the culture media, bromoform release remained unaffected suggesting that the two compounds are formed by different mechanisms.

rbcL sequences indicate a single evolutionary origin of multinucleate cells in the red algal tribe Callithamnieae.

In the Ceramiaceae, one of the largest families of the red algae, there are from 1 to 4000 nuclei in each vegetative cell, but each tribe is homogeneous with respect to the uninucleate/multinucleate character state, except for the Callithamnieae. The goals of this study were to analyze rbcL gene sequences to clarify the evolution of taxa within the tribe Callithamnieae and to evaluate the potential evolutionary significance of the development of multinucleate cells in certain taxa. The genus Aglaothamnion, segregated from Callithamnion because it is uninucleate, was paraphyletic in all analyses. Callithamnion (including Aristothamnion) was monophyletic although not robustly so, apparently due to variations between taxa in rate of sequence evolution. Morphological synapomorphies were identified at different depths in the tree, supporting the molecular phylogenetic analysis. The uninucleate character state is ancestral in this tribe. The evolution of multinucleate cells has occurred once in the Callithamnieae. Multiple nuclei in each cell may combine the benefits of small C values (rapid cell cycle) with large cells (permitting morphological elaboration) while maintaining a constant ratio of nuclear volume: cytoplasmic volume.

rbcL sequences reveal multiple cryptic introductions of the Japanese red alga Polysiphonia harveyi.

In Europe, the last 20 years have seen a spectacular increase in accidental introductions of marine species, but it has recently been suggested that both the actual number of invaders and their impacts have been seriously underestimated because of the prevalence of sibling species in marine habitats. The red alga Polysiphoniaharveyi is regarded as an alien in the British Isles and Atlantic Europe, having appeared in various locations there during the past 170 years. Similar or conspecific populations are known from Atlantic North America and Japan. To choose between three competing hypotheses concerning the origin of P. harveyi in Europe, we employed rbcL sequence analysis in conjunction with karyological and interbreeding data for samples and isolates of P. harveyi and various congeners from the Pacific and North Atlantic Oceans. All cultured isolates of P. harveyi were completely interfertile, and there was no evidence of polyploidy or aneuploidy. Thus, this biological species is both morphologically and genetically variable: intraspecific rbcL divergences of up to 2.1% are high even for red algae. Seven rbcL haplotypes were identified. The four most divergent haplotypes were observed in Japanese samples from Hokkaido and south-central Honshu, which are linked by hypothetical 'missing' haplotypes that may be located in northern Honshu. These data are consistent with Japan being the centre of diversity and origin for P. harveyi. Two non-Japanese lineages were linked to Hokkaido and Honshu, respectively. A single haplotype was found in all North Atlantic and Mediterranean accessions, except for North Carolina, where the haplotype found was the same as that invading in New Zealand and California. The introduction of P. harveyi into New Zealand has gone unnoticed because P. strictissima is a morphologically indistinguishable native sibling species. The sequence divergence between them is 4–5%, greater than between some morphologically distinct red algal species. Two different types of cryptic invasions of P. harveyi have therefore occurred. In addition to its introduction as a cryptic sibling species in New Zealand, P. harveyi has been introduced at least twice into the North Atlantic from presumed different source populations. These two introductions are genetically and probably also physiologically divergent but completely interfertile.