Search

International audience; The comprehension of microbial interactions is one of the key challenges in marine microbial ecology. This study focused on exploring chemical interactions between the toxic dinoflagellate Prorocentrum lima and a filamentous fungal species, Aspergillus pseudoglaucus, which has been isolated from the microalgal culture. Such interspecies interactions are expected to occur even though they were rarely studied. Here, a co-culture system was designed in a dedicated microscale marine-like condition. This system allowed to explore microalgal-fungal physical and metabolic interactions in presence and absence of the bacterial consortium. Microscopic observation showed an unusual physical contact between the fungal mycelium and dinoflagellate cells. To delineate specialized metabolome alterations during microalgal-fungal co-culture metabolomes were monitored by highperformance liquid chromatography coupled to high-resolution mass spectrometry. In-depth multivariate statistical analysis using dedicated approaches highlighted (1) the metabolic alterations associated with microalgal-fungal co-culture, and (2) the impact of associated bacteria in microalgal metabolome response to fungal interaction. Unfortunately, only a very low number of highlighted features were fully characterized. However, an up-regulation of the dinoflagellate toxins okadaic acid and dinophysistoxin 1 was observed during co-culture in supernatants. Such results highlight the importance to consider microalgal-fungal interactions in the study of parameters regulating toxin production.

International audience; The coprophilous ascomycete Podospora anserina is known to have a high potential to synthesize a wide array of secondary metabolites (SMs). However, to date, the characterization of SMs in this species, as in other filamentous fungal species, is far less than expected by the functional prediction through genome mining, likely due to the inactivity of most SMs biosynthesis gene clusters (BGCs) under standard conditions. In this work, our main objective was to compare the global strategies usually used to deregulate SM gene clusters in P. anserina, including the variation of culture conditions and the modification of the chromatin state either by genetic manipulation or by chemical treatment, and to show the complementarity of the approaches between them. In this way, we showed that the metabolomics-driven comparative analysis unveils the unexpected diversity of metabolic changes in P. anserina and that the integrated strategies have a mutual complementary effect on the expression of the fungal metabolome. Then, our results demonstrate that metabolite production is significantly influenced by varied cultivation states and epigenetic modifications. We believe that the strategy described in this study will facilitate the discovery of fungal metabolites of interest and will improve the ability to prioritize the production of specific fungal SMs with an optimized treatment.

Using marine-derived fungi as a source of lipid production holds promise as an alternative to industrial lipid production for health and nutrition in the future. In the present study, this strain showed a high production of lipid, about plus 80% of the total lipids in the host-derived medium. Gas chromatography analysis of fungal lipids revealed the presence of saturated (mainly palmitic acid C16: 0 and stearic acid C18: 0) and unsaturated fatty acid (mainly linoleic acid C18: 2, oleic acid C18: 1). These findings suggest this marine-derived fungus is a promising source for lipid production in various industrial applications. In particular, a fraction containing glycolipids of the crude extract exhibited potential cytotoxic activity on human oral epidermal carcinoma cell lines. This result is very interesting to further isolate and determine the molecular structure of bioactive glycolipids.

Very little is known about chemical interactions between fungi and their mollusc host within marine environments. Here, we investigated the metabolome of a Penicillium restrictum MMS417 strain isolated from the blue mussel Mytilus edulis collected on the Loire estuary, France. Following the OSMAC approach with the use of 14 culture media, the effect of salinity and of a mussel-derived medium on the metabolic expression were analysed using HPLC-UV/DAD-HRMS/MS. An untargeted metabolomics study was performed using principal component analysis (PCA), orthogonal projection to latent structure discriminant analysis (O-PLSDA) and molecular networking (MN). It highlighted some compounds belonging to sterols, macrolides and pyran-2-ones, which were specifically induced in marine conditions. In particular, a high chemical diversity of pyran-2-ones was found to be related to the presence of mussel extract in the culture medium. Mass spectrometry (MS)- and UV-guided purification resulted in the isolation of five new natural fungal pyran-2-one derivatives—5,6-dihydro-6S-hydroxymethyl-4-methoxy-2H-pyran-2-one (1), (6S, 1’R, 2’S)-LL-P880β (3), 5,6-dihydro-4-methoxy-6S-(1’S, 2’S-dihydroxy pent-3’(E)-enyl)-2H-pyran-2-one (4), 4-methoxy-6-(1’R, 2’S-dihydroxy pent-3’(E)-enyl)-2H-pyran-2-one (6) and 4-methoxy-2H-pyran-2-one (7)—together with the known (6S, 1’S, 2’S)-LL-P880β (2), (1’R, 2’S)-LL-P880γ (5), 5,6-dihydro-4-methoxy-2H-pyran-2-one (8), (6S, 1’S, 2’R)-LL-P880β (9), (6S, 1’S)-pestalotin (10), 1’R-dehydropestalotin (11) and 6-pentyl-4-methoxy-2H-pyran-2-one (12) from the mussel-derived culture medium extract. The structures of 1-12 were determined by 1D- and 2D-MMR experiments as well as high-resolution tandem MS, ECD and DP4 calculations. Some of these compounds were evaluated for their cytotoxic, antibacterial, antileishmanial and in-silico PTP1B inhibitory activities. These results illustrate the utility in using host-derived media for the discovery of new natural products.

The comprehension of microbial interactions is one of the key challenges in microbial ecology. The present study focuses on studying the chemical interaction between the toxic dinoflagellate Prorocentrum lima PL4V strain and associated fungal strains (two Penicillium sp. strains and three Aspergillus sp) among which the Aspergillus pseudoglaucus strain MMS1589 was selected for further co-culture experiment. Such rarely studied interaction (fungal-microalgal) was explored in axenic and non-axenic conditions, in a dedicated microscale marine environment (hybrid solid/liquid conditions), to delineate specialized metabolome alteration in relation to the P. lima and A. pseudoglaucus co-culture in regard to the presence of their associated bacteria. Such alteration was monitored by high-performance liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). In-depth analysis of the resulting data highlighted (1) the chemical modification associated to fungal-microalgal co-culture, and (2) the impact of associated bacteria in microalgal resilience to fungal interaction. Even if only a very low number of highlighted metabolites were fully characterised due to the poor chemical investigation of the studied species, a clear co-culture induction of the dinoflagellate toxins okadaic acid and dinophysistoxin 1 was observed. Such results highlight the importance to consider microalgal microbiome to study parameters regulating toxin production. Finally, a microscopic observation showed an unusual physical interaction between the fungal mycelium and the dinoflagellates.

Serratia sp. cause food losses and waste due to spoilage; it is noteworthy that they represent a dominant population in seafood. The main spoilage associated species comprise S. liquefaciens, S. grimesii, S. proteamaculans and S. quinivorans, also known as S. liquefaciens-like strains. These species are difficult to discriminate since classical 16S rRNA gene-based sequences do not possess sufficient resolution. In this study, a phylogeny based on the short-length luxS gene was able to speciate 47 Serratia isolates from seafood, with S. proteamaculans being the main species from fresh salmon and tuna, cold-smoked salmon, and cooked shrimp while S. liquefaciens was only found in cold-smoked salmon. The genome of the first S. proteamaculans strain isolated from the seafood matrix (CD3406 strain) was sequenced. Pangenome analyses of S. proteamaculans and S. liquefaciens indicated high adaptation potential. Biosynthetic pathways involved in antimicrobial compounds production and in the main seafood spoilage compounds were also identified. The genetic equipment highlighted in this study contributed to gain further insights into the predominance of Serratia in seafood products and their capacity to spoil.

Natural products (NPs) are considered as a cornerstone for the generation of bioactive leads in drug discovery programs. However, one of the major limitations of NP drug discovery program is "rediscovery" of known compounds, thereby hindering the rate of drug discovery efficiency. Therefore, in recent years, to overcome these limitations, a great deal of attention has been drawn towards understanding the role of microorganisms' co-culture in inducing novel chemical entities. Such induction could be related to activation of genes which might be silent or expressed at very low levels (below detection limit) in pure-strain cultures under normal laboratory conditions. In this review, chemical diversity of compounds isolated from microbial co-cultures, is discussed. For this purpose, chemodiversity has been represented as a chemical-structure network based on the "Tanimoto Structural Similarity Index". This highlights the huge structural diversity induced by microbial co-culture. In addition, the current trends in microbial co-culture research are highlighted. Finally, the current challenges (1 - induction monitoring, 2 - reproducibility, 3 - growth time effect and 4 - up-scaling for isolation purposes) are discussed. The information in this review will support researchers to design microbial co-culture strategies for future research efforts. In addition, guidelines for co-culture induction reporting are also provided to strengthen future reporting in this NP field.

Two new unusual C25 steroid epimers, 24-O-methyl-24-epi-cyclocitrinol (1) and 24-O-methylcyclocitrinol (2), together with the known oxylipin α-dimorphecolic acid (3), were isolated from the extract of cultures of a Penicillium ubiquetum MMS330 strain sampled from the blue mussel Mytilus edulis. The structures of the new compounds were established based on 1D and 2D NMR spectral analyses and CP3 conformation analysis. LC–MS dereplication of other C25 steroids in culture extracts was also performed on the basis of their HRMS/MS fragmentation patterns, allowing to detect other analogues. Biological activities of compounds 1 and 2 were evaluated as a mixture on the two human cancer cell lines KB and MCF-7.

International audience

Penicillium ubiquetum MMS330 isolated from the blue mussel Mytilus edulis collected on the Loire estuary in France was here investigated. As very few secondary metabolites have been documented for this species, its metabolome was studied following the OSMAC approach to enhance as many biosynthetic pathways as possible. Interestingly, HPLC-HRMS based hierarchical clustering analysis together with MS/MS molecular networking highlighted the selective overproduction of some structurally related compounds when the culture was performed on seawater CYA (Czapek Yeast extract Agar) medium. Mass-guided purification from large scale cultivation on this medium led to the isolation of nine meroterpenoids including two new analogues, 22-deoxyminiolutelide A (1) and 4-hydroxy-22-deoxyminiolutelide B (2), together with seven known compounds (3–9). The structures of 1 and 2 were elucidated on the basis of HR-ESIMS and NMR spectroscopic data analysis. Furthermore, NMR signals of 22-deoxyminiolutelide B (3) were reassigned.

In a context of growing resistance to classical antifungal therapy, the design of new drugs targeting alternative pathways is highly expected. Benzofuro[3,2-d]pyrimidine derivatives, derived from (-)-cercosporamide, were synthesized and evaluated as potential Candida albicans PKC inhibitors in the aim of restoring susceptibility to azole treatment. Co-administration assay of benzofuropyrimidinedione 23 and fluconazole highlighted a synergistic effect on inhibition of cell growth of a Candida albicans resistant strain.

In order to assess the putative toxigenic risk associated with the presence of fungal strains in shellfish-farming areas, Penicillium strains were isolated from bivalve molluscs and from the surrounding environment, and the influence of the sample origin on the cytotoxicity of the extracts was evaluated. Extracts obtained from shellfish-derived Penicillia exhibited higher cytotoxicity than the others. Ten of these strains were grown on various media including a medium based on mussel extract (Mytilus edulis), mussel flesh-based medium (MES), to study the influence of the mussel flesh on the production of cytotoxic compounds. The MES host-derived medium was created substituting the yeast extract of YES medium by an aqueous extract of mussel tissues, with other constituent identical to YES medium. When shellfish-derived strains of fungi were grown on MES medium, extracts were found to be more cytotoxic than on the YES medium for some of the strains. HPLC-UV/DAD-MS/MS dereplication of extracts from Penicillium marinum and P. restrictum strains grown on MES medium showed the enhancement of the production of some cytotoxic compounds. The mycotoxin patulin was detected in some P. antarcticum extracts, and its presence seemed to be related to their cytotoxicity. Thus, the enhancement of the toxicity of extracts obtained from shellfish-derived Penicillium strains grown on a host-derived medium, and the production of metabolites such as patulin suggests that a survey of mycotoxins in edible shellfish should be considered. Significance and Impact of the Study Penicillium strains isolated from bivalve molluscs produce extracts exhibiting a higher cytotoxicity than extracts from Penicillium strains isolated from the surrounding marine environment. The use of a mussel-based medium for cultures of some shellfish-derived strains enhances the cytotoxicity of extracts when compared with classical media. The production of cytotoxic compounds and of the mycotoxin patulin on such a host-derived medium highlights a potential health risk for shellfish consumers.

International audience; A new chlorinated sesquiterpenoid analogue of fumagillin, ligerin (1), was isolated from a marine-derived strain of Penicillium, belonging to the subgenus Penicillium, along with the known compounds penicillic acid (2), orcinol, and orsellinic acid. Chemical structures were established by an interpretation of spectroscopic data including IR, UV, and HRESIMS, together with analyses of 1D and 2D NMR spectra and X-ray analysis for the determination of the absolute configuration. Ligerin (1) displayed strong inhibitory activity against an osteosarcoma cell line. This is the first report of the isolation of a fumagillin analogue from a marine-derived Penicillium strain.

International audience

This work aimed at studying metabolome variations of marine fungal strains along their growth to highlight the importance of the parameter “time” for new natural products discovery. An untargeted time-scale metabolomic study has been performed on two different marine-derived Penicillium strains. They were cultivated for 18 days and their crude extracts were analyzed by HPLC-DAD-HRMS (High Performance Liquid Chromatography-Diode Array Detector-High Resolution Mass Spectrometry) each day. With the example of griseofulvin biosynthesis, a pathway shared by both strains, this work provides a new approach to study biosynthetic pathway regulations, which could be applied to other metabolites and more particularly new ones. Moreover, the results of this study emphasize the interest of such an approach for the discovery of new chemical entities. In particular, at every harvesting time, previously undetected features were observed in the LC-MS (Liquid Chromatography-Mass Spectrometry) data. Therefore, harvesting times for metabolite extraction should be performed at different time points to access the hidden metabolome.

During our search for marine-derived fungi from shellfish farming areas along the Algerian western coast, more than 250 strains were isolated from mussels (Mytilus galloprovincialis) and their immediate environment (marine sediments and seawater). A majority of the strains isolated belonged to four genera: Penicillium (55.1%), Aspergillus (8.3%), Trichoderma (5.8%), and Cladosporium (3.7%). Extracts of 91 strains were assayed to evaluate their accute toxicity on Artemia salina larvae. Twenty-four percent of the extracts tested appeared active with different levels of toxicity. There were few toxigenic strains in those isolated from sediments whereas a majority of strains isolated from mussels exhibited toxicity. Highest activities were exhibited by strains belonging to the genus Penicillium. Their presence in shellfish farming areas confirmed the necessity to pay a peculiar attention to shellfish contamination by such fungi.

Penicillium expansum is a ubiquitous species for which there are only few reports for chemical investigation in marine environments. Among thenumerous secondary metabolites produced by this species, communesins represent a new class of cytotoxic and insecticidal indole alkaloids. In this study, we investigated a marine P. expansum strain exhibiting neuroactivity on a Diptera larvae bioassay. Bio-guided purification led to the isolation and the identification of communesin B as the main active compound by HRMS and 1 H and 13 C NMR. Liquid chromatography analyses with detection by electrospray ionization coupled with tandem mass spectrometry (LC/ESI-MS/MS) and high-resolution tandem mass spectrometry (LC/HRMS/MS) allowed the identification and characterization of four other known communesins (A, D, E and F) in the crude extract. A fragmentation modelfor dimethyl epoxide communesins was proposed after detailed interpretation oftheir MS/MS spectra. Further analyses of the extract using the modelled fragmentations led to the detection of seven new communesins found as minor compounds. Chemical structural elucidation of these new derivatives is discussed based on their fragmentation characteristics. Copyright #2009 John Wiley & Sons, Ltd.

In order to enhance the knowledge of the putative toxinic risk linked to mycotoxin excretion in shellfish farming areas, the influence of seawater salinity was studied on 2 marine-derived Aspergillus fumigatus strains. This fungal species produces gliotoxin, an epipolythiodioxopiperazine immunosuppressive mycotoxin that can be accumulated in the meat of cultured blue mussel (Mytilus edulis), and could be responsible for disease when ingested. Two marine strains were grown in vitro both on a non-saline and a saline culture media and were compared with 13 terrestrial strains to observe the effects of seawater on fungal growth and gliotoxin excretion in the exudate produced. Daily measurement of the colony areas showed that the seawater salinity significantly reduced the rate of growth of all the strains. Marine and terrestrial strains appeared to be almost similar as regards the appearance, growth and gliotoxin excretion, but the marine strains exudation seemed to be less influenced by seawater salinity than the terrestrial strains. Seawater salinity, however, enhanced exudation and gliotoxin excretion by A. fumigatus, and thus seems to be an aggravating factor for the toxicity of this species in the marine environment.

Mammea coumarins are isoprenylated 4-alkyl or 4-phenylcoumarins. Their distribution is limited to 3 Clusiaceae / Calophyllaceae genera. We recently reported on their presence in Mammea neurophylla bark extracts, where they exhibited anti-AGE properties associated with a prevention of the endothelial dysfunction. About 120 mammea coumarins were already described so, in order to focus further phytochemical analysis on original or bio-active compounds, we developed a methodology to facilitate the detection and identification of compounds of interest. Our aim was to develop a LC-DAD–ESI-MS n method for rapid, sensitive and simple analysis of the mammea coumarins in calophyllaceous/clusiaceous species. For that, full LC-DAD–MS n data were acquired from 11 4-phenylcoumarins previously isolated in our laboratory. Bark, leaves and fruits of M. neurophylla were then extracted with DCM using an ASE apparatus. Extracts were finally analyzed through LC-DAD–HRMS n and UV and MS profiles were compared to our database as well as literature data. Detected new compounds were isolated and their structures elucidated through 1 H, 13 C and 2D NMR analysis. Finally, 24 known mammea coumarins were dereplicated from bark, leaf and fruit DCM extracts of M. neurophylla and the structure of 4 unreported compounds could be predicted. In particular, the structures of mammea A/AA 9-hydroxyCycloF and mammea A/AB 9-hydroxyCycloF were confirmed after purification and extensive NMR analyses. By comparison of UV and mass fragmentation data from a small library of reference compounds, LC-DAD–HRMS n analysis of mammea coumarins in crude extracts allows the structure prediction of novel or bio-active compounds. This useful guiding-tool could be easily applied to other Clusiaceae / Calophyllaceae phytochemical analysis.

When cultured in a marine solid medium, a strain of Aspergillus fumigatus (Fresenius) isolated from a shellfish-farming area in the Loire estuary (France) produced a highly cytotoxic exudate. To identify the origin of this activity, a cytotoxicity test on KB cells was used to monitor the purification of the exudate, together with electrospray/ion trap/mass spectrometry (ESI/IT/ MS n ) to detect and identify the toxic compound. After three purification stages, a comparison of fullscan analyses of the last six fractions showed that a monocharged compound at m/z 349 was present only in the active fraction, corresponding to the sodium adduct of gliotoxin [C13H14N2O4S2+Na] + . Isotopic distribution determination showed that the m/z 349 product possessed two sulphur atoms and multi-stage fragmentation confirmed the hypothesis. MS/MS analysis exhibited the characteristic gliotoxin loss of the disulphide intracyclic bridge. MS 3 analysis revealed four main ions and confirmed the identity of the m/z 349 ion. This study points out that the combined use of a KB cells bioassay and ESI/IT/MS n allows a fast and very specific detection and elucidation of unidentified cytotoxic products in natural samples. This method does not require total purification, and it allowed us to report the first detection of gliotoxin production in marine conditions. D 2002 Elsevier Science B.V. All rights reserved.

International audience; Ligerin is a natural chlorinated merosesquiterpenoid related to fumagillin exhibiting a selective antiproliferative activity against osteosarcoma cell lines and an in vivo antitumor activity in a murine model. Semisynthesis of ligerin analogs was performed in order to study the effects of the C3-spiroepoxide substitution by a halogenated moiety together with the modulation of the C6 chain. Results showed that all derivatives exhibited an in vitro antiproliferative activity against osteosarcoma cell lines and that chlorohydrin compounds were equally or more active than their spiroepoxy analogs. Among semisynthetic analogs, the parent compound 1 was the best candidate for further studies since it exhibited higher or equivalent activity compared to TNP470 against SaOS2 and MG63 human osteosarcoma cells with a four times weaker toxicity against HFF2 human fibroblasts. Quantitative videomicroscopy analysis was conducted and allowed a better understanding of the mechanism of its antiproliferative activity.

International audience

International audience

Pinnatoxin G (PnTX-G) is a marine toxin belonging to the class of cyclic imines and produced by the dinoflagellate Vulcanodinium rugosum. In spite of its strong toxicity to mice, leading to the classification of pinnatoxins into the class of “fast-acting toxins”, its hazard for human health has never been demonstrated. In this study, crude extracts of V. rugosum exhibited significant cytotoxicity against Neuro2A and KB cells. IC50 values of 0.38 µg mL−1 and 0.19 µg mL−1 were estimated on Neuro2A cells after only 24 h of incubation and on KB cells after 72 h of incubation, respectively. In the case of Caco-2 cells 48 h after exposure, the crude extract of V. rugosum induced cell cycle arrest accompanied by a dramatic increase in double strand DNA breaks, although only 40% cytotoxicity was observed at the highest concentration tested (5 µg mL−1). However, PnTX-G was not a potent cytotoxic compound as no reduction of the cell viability was observed on the different cell lines. Moreover, no effects on the cell cycle or DNA damage were observed following treatment of undifferentiated Caco-2 cells with PnTX-G. The crude extract of V. rugosum was thus partially purified using liquid-liquid partitioning and SPE clean-up. In vitro assays revealed strong activity of some fractions containing no PnTX-G. The crude extract and the most potent fraction were evaluated using full scan and tandem high resolution mass spectrometry. The dereplication revealed the presence of a major compound that could be putatively annotated as nakijiquinone A, N-carboxy-methyl-smenospongine or stachybotrin A, using the MarinLit™ database. Further investigations will be necessary to confirm the identity of the compounds responsible for the cytotoxicity and genotoxicity of the extracts of V. rugosum.

International audience; Peptaibols are small linear fungal peptides which are produced in the marine environment. They exhibit neurotoxicity by forming pores in neuronal membranes. This work describes their combine effect with domoic acid, a neurotoxic phycotoxin, on Diptera larvae. The Acute toxicity bioassay on this biological model was tested with a panel of different toxins (microbial, algal or fungal). It allowed the discrimination of neurotoxins and non-neurotoxic toxins, and an evaluation of the toxicity level (MED and ED50) which were correlated with published LD50 in mice for neurotoxins tested. The highest activities on this test were found for Na+ channel blockers tetrodotoxin (ED50 = 0.026 mg/kg) and saxitoxin (ED50 = 0.18 mg/kg). Domoic acid was less active with an ED50 = 7.6 mg/kg. For synergism study, longibrachin-A-I, a 20-mer peptaibol isolated from cultures of a marine-derived strain of Trichoderma longibrachiatum Rifai was chosen. Bioassay results confirmed its neuroactivity. Its level of toxicity (ED50 = 270 mg/kg) was lower than those of phycotoxins tested but higher than mycotoxin ones. Injected together, longibrachin-A-I and domoic acid exhibited an increase of their activities. With doses of longibrachin-A-I below its Minimal Effective Dose (MED), the synergism factor which expresses the enhancement of domoic acid toxicity could reach 34.5. Both domoic acid and longibrachin-A-I are acting on ion channels and pores in neuronal membranes which contribute to the intake of Ca2+ into cells.

Penicillium expansum is a ubiquitous species for which there are only few reports for chemical investigation in marine environments. Among the numerous secondary metabolites produced by this species, communesins represent a new class of cytotoxic and insecticidal indole alkaloids. In this study, we investigated a marine P. expansum strain exhibiting neuroactivity on a Diptera larvae bioassay. Bio-guided purification led to the isolation and the identification of communesin B as the main active compound by HRMS and 1H and 13C NMR. Liquid chromatography analyses with detection by electrospray ionization coupled with tandem mass spectrometry (LC/ESI-MS/MS) and high-resolution tandem mass spectrometry (LC/HRMS/MS) allowed the identification and characterization of four other known communesins (A, D, E and F) in the crude extract. A fragmentation model for dimethyl epoxide communesins was proposed after detailed interpretation of their MS/MS spectra. Further analyses of the extract using the modelled fragmentations led to the detection of seven new communesins found as minor compounds. Chemical structural elucidation of these new derivatives is discussed based on their fragmentation characteristics.

Different peptaibols were observed in both fresh and frozen marine sediments collected from a marine area devoted to bivalve culture (Fier d'Ars, Atlantic coast, France). The identification of the peptaibols was based on a three-step mass-spectrometric analysis: observation of doubly charged ions with a characteristic isotopic profile, cleavage and observation of C- and N-terminal fragments, and partial sequencing of the N-terminal segments. The MS characteristics indicated numerous similarities between the peptaibols detected and those produced by different strains of Trichoderma species isolated from fresh sediments. Peptaibols were also detected in mussel samples collected at the same site. This constitutes the first observation of contamination of the marine human-food chain by fungal metabolites. Since peptaibols were readily observed both in fresh sediments and in samples kept frozen for several years, these compounds can be considered as stable markers of the development of Trichoderma in the marine environment.

A marine strain of Trichoderma longibrachiatum isolated from blue mussels (Mytilus edulis) was investigated for short peptaibol production. Various 11-residue peptaibols, obtained as microheterogenous mixtures after a chromatographic fractionation, were identified by positive mass spectrometry fragmentation (ESI-IT-MS(n), CID-MS(n) and GC/EI-MS). Thirty sequences were identified, which is the largest number of analogous sequences so far observed at once. Twenty-one sequences were new, and nine others corresponded to peptaibols already described. These peptaibols belonged to the same peptidic family based on the model Ac-Aib-xxx-xxx-xxx-Aib-Pro-xxx-xxx-Aib-Pro-xxol. They were named trichobrachin A when the residue in position 2 was an Asn, and trichobrachin C when it was a Gln. Major chromatographic sub-fractions, corresponding to purified peptaibols, were assayed for their cytotoxic activity. Trichobrachin A-IX and trichobrachin C exhibited the highest activities. There was an exponential relation between their relative hydrophobicity and their cytotoxicity on KB cells.

A strain of Aspergillus fumigatus has been isolated from sediments of a mussel bed. When cultured in hyper saline conditions (with sea-water), it produces a cytotoxic and immunosuppressive toxin, gliotoxin, which is excreted in an exudate. In order to know if this toxin could represent a risk for shellfish consumers, an experiment of bioaccumulation of gliotoxin in mussel has been carried out. After 6 days of contamination, toxin was accumulated in the meat of the mussels, at a level up to 2.9 microg/mg of extract weight, with a mode of contamination different to the classical digestive process described for a majority of marine toxins, but similar to the contamination mode of domoic acid.