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First published online July 26, 2004
Journal of Experimental Biology 207, 2935-2946 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01105

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Cytotoxicity of diatom-derived oxylipins in organisms belonging to different phyla

Sven Adolph¹, Stéphane Bach², Marc Blondel², Anne Cueff², Marjolaine Moreau², Georg Pohnert¹, Serge André Poulet^2,*, Thomas Wichard¹ and Alga Zuccaro³

¹ Max-Planck Institute, Hans-Knöll-Str. 8, D-07745 Jena, Germany, ² Station Biologique, CNRS, Mer et Santé (FRE 2775), INSU, UPMC, PO Box 74, 29682 Roscoff, France and ³ Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany

View larger version (61K): [in a new window]   Fig. 8. Calanus helgolandicus (crustacean). (A) Post-ingestion effect of the noxious diatom Thalassiosira rotula, provided to spawning females. Dose-dependent values (mean ± S.D.) of the hatching success are the results of triplicate observations for each diet treatment in batch samples of 30-100 eggs each. The control diet (test number 1, with the dinoflagellate Prorocentrum minimum) does not contain any noxious unsaturated aldehydes. Values of the potential production of two dominant unsaturated aldehydes (2,4-octadienal and 2,4,7-decatrienal combined), measured as the mean aldehyde production (2 fmol cell-1) of these major unsaturated aldehydes in a T. rotula initial culture (T. Wichard, S. Poulet and G. Pohnert, unpublished). Potential yields of these aldehydes are calculated in relation to the equivalent diatom cell concentrations in female diets (test numbers 2-5). (B) Micrographs of a normal embryo at the two-blastomere stage produced by females fed the dinoflagellate diet (1), blockage of cell division in abnormal embryos produced by females fed the diatom diet at >103 cells ml-1 (2), fluorescent micrograph of a similar abnormal embryo stained with Hoechst 33342, specific to DNA, showing two nuclei blocked in the egg matrix (3). (C) Fate of N1 stage naplius produced by females fed two different diets [1 and 2, dinoflagellate (104 cells ml-1); 3, toxic diatom (104 cells ml-1)], sampled on day 5 during an 8-day incubation period. Light micrograph of a normal larva (1). Fluorescent confocal micrographs of normal (2) and apoptotic (arrow) (3) larvae double-stained with FITC-Annexin V + propidium iodide. Size of eggs=172 ±4µm; size of larvae=208±10 µm. Scale bars in B and C apply to 1, 2 and 3.

View larger version (35K): [in a new window]   Fig. 1. Vibrio splendidus (bacterium). Effects of the aldehydes decanal (1=66 µg disk-1, 2=6.6 µg disk-1) and 2E,4E-decadienal (1=33.3 µg disk-1, 2=6.6 µg disk-1, 3=0.66 µg disk-1) on cell proliferation, shown by the growth inhibition zone around the disk at different dilutions. Comparisons with DMSO and two antibiotics (15 µg disk-1 chloramphenicol and 30 µg disk-1 gentamycin) are shown.

View larger version (97K): [in a new window]   Fig. 2. Saccharomyces cerevisiae (fungus). erg6 cells are insensitive to decanal and 4Z-decenal but are highly sensitive to 2E,4E-decadienal and 2E-decenal, as indicated by the growth inhibition halo around the filter where these molecules were spotted. Concentration of each aldehyde tested was 9.1 µg disk-1. ERG6wt cells (wild-type strain) are not sensitive to any of these molecules.

View larger version (68K): [in a new window]   Fig. 3. Sphaerechinus granularis (echinoderm). (A) Dose-dependent effects of decanal (circles) and 2E,4E-decadienal (triangles) on cell division during the early embryogenic phase (four blastomeres) in 2.5 h-old embryos. Values are means of three replicate measurements. Standard error (<3% of the mean) is not shown. (B) Light microscope photographs of (1) normal divided embryos observed either in seawater controls or in 2E,4E-decadienal (<5 µmol l-1) and decanal (<80 µmol l-1) test solutions, (2) abnormal embryos presenting totally blocked or abnormal cell divisions in 2E,4E-decadienal (>10 µmol l-1) and (3) blocked embryos presenting intoxication features with decanal (>80 µmol l-1). Egg size: 95 ±6 µm. Scale bar applies to 1, 2 and 3.

View larger version (30K): [in a new window]   Fig. 4. Crassostrea gigas (mollusc). (A) Dose-dependent response of oyster haemocytes incubated in seawater (SW, controls), DMSO (solvent control), 2E,4E-decadienal and decanal. Values (mean ± S.D.) are the estimates of the proportions of abnormal, round cells reflecting the impact of these treatments on the cytoskeleton. (B) Fluorescent micrographs of normal (1) and abnormal (2) cytoskeleton revealed in rhodamine-phalloidin-stained cells.

View larger version (32K): [in a new window]   Fig. 5. Crassostrea gigas (mollusc). (A) Dose-dependent response of oyster haemocytes incubated in seawater (SW, controls), DMSO (solvent control), 2E,4E-decadienal and decanal. Values (mean ± S.D.) are the proportions of abnormal, apoptotic cell degradations reflecting the noxious impact of the treatments on the haemocytes. (B) Fluorescent micrographs of normal (1) and apoptotic (2) FITC-Annexin V-stained haemocytes.

View larger version (25K): [in a new window]   Fig. 6. Crassostrea gigas (mollusc). (A) Response of oyster haemocytes incubated in 2E,4E-decadienal or decanal (at concentrations of 2 and 50 µmol l-1) and DMSO (solvent control). Values (mean ± S.E.M.) are given for three replicate tests, showing the proportions of blood cells presenting phagocytosis inhibition with each treatment. (B) Fluorescent confocal micrographs of three consecutive optical sections of normal (1) and inhibited (2) haemocytes, related to presence (P) or absence of fluorescent phagocytosed beads observed inside the cytoplasm. N, cell nucleus.

View larger version (24K): [in a new window]   Fig. 7. Crassostrea gigas (mollusc). Concentration-dependent inhibition of the luminol-dependent chemiluminescence response of oyster haemocytes from a common pool of cells (106 cells ml-1) by aldehydes (at concentrations of 2 and 50 µmol l-1). For each treatment, values are the results of triplicate measurements of the chemiluminescence responses, within 15 min before and 45 min after addition of the stimulatory Zymosan particles (arrow).