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First published online August 25, 2003

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A marine diatom-derived aldehyde induces apoptosis in copepod and sea urchin embryos

Giovanna Romano^1,*, Gian Luigi Russo², Isabella Buttino¹, Adrianna Ianora¹ and Antonio Miralto¹

¹ Stazione Zoologica `Anton Dohrn' Villa Comunale 1 - 80121 Napoli, Italy
² Istituto Scienze dell'Alimentazione, Consiglio Nazionale delle Ricerche - 83100 Avellino, Italy

View larger version (84K): [in a new window]   Fig. 1. Calanus helgolandicus embryos after TUNEL staining and observed by confocal laser scanning microscopy in fluorescence (A,C,E,G) and transmitted (B,D,F,H) light. (A) Fluorescent, three-dimensional image of an embryo produced by females fed for 10 days with the diatom Thalassiosira rotula. Nuclei are positively stained (green) by the TUNEL. Bar, 39.7 µm. (B) The same embryo as in A observed in transmitted light. Bar, 42.1 µm. (C) Fluorescent three-dimensional image of a C. helgolandicus embryo produced by female fed non-diatom Prorocentrum minimun (PRO) algae for 24 h then incubated for 1 h in 5 µg ml-1 DD. Nuclei (green) are positively stained by TUNEL. Bar, 40 µm. (D) Embryo in C observed in transmitted light. (E) Three-dimensional image of the embryo produced by female fed 24 PRO. Nuclei are not stained in green and appear as black shadows. Bar, 42.2 µm. (F) The same embryo as in E observed in transmitted light. Bar, 40.3 µm. (G) Three-dimensional fluorescent image of a TUNEL-positive control embryo obtained by female fed for 24 h with PRO and incubated in DNase to simulate apoptosis. Nuclei are stained in green as in A and C. Bar, 34.6 µm. (H) The same embryo as in G observed in transmitted light. Bar, 34.6 µm.

View larger version (68K): [in a new window]   Fig. 2. (A) PCR amplification of DNA isolated from copepod embryos using the 16SAR and 16SBR primers, as reported in Materials and methods. The arrow indicates the presence of a single PCR product of approximately 430 bp. The presence (+) or absence (-) of template DNA is shown above. (B) DNA laddering of two DNA samples (6, 18) isolated from copepod embryos treated with 5 µg ml-1 of DD for 3 and 1 h, respectively. Sample 31 represents an untreated control. Semiquantitative analysis of the fragmentation extent is reported in Table 1. M, markers (DNA Ladder 100; from top to bottom: 1000, 900, 800, 700, 600, 500, 400, 300, 200 bp).

View larger version (18K): [in a new window]   Fig. 3. Induction of membrane blebbing following DD treatment. Sea urchin embryos were treated with the indicated concentrations of DD and the percentage of cell blebbing was determined every 30 min. Values (means ± S.D.; N=600) are the results of three different experiments.

View larger version (98K): [in a new window]   Fig. 4. Sea urchin embryo observed by confocal laser scanning microscopy in fluorescent (A,C,E) and in transmitted (B,D,F) light. (A) Fluorescent three-dimensional image of the untreated embryo (16-blastomere stage) stained with TUNEL. Bar, 30.6 µm. (B) The same embryo as in A observed in transmitted light. Bar, 28.2 µm. (C) Fluorescent three-dimensional image of the sea urchin embryo incubated at the 8-blastomere stage in 5 µg ml-1 DD for 60 min and stained with TUNEL. Bar, 29.8 µm. (D) The same embryo as in C observed in transmitted light. Bar, 26.8 µm. (E) Fluorescent three-dimensional image of the sea urchin embryo incubated at the 8-blastomere stage in 5 µg ml-1 DD for 120 min and stained with TUNEL. Bar, 30.6 µm. (F) The same embryo as in E observed in transmitted light. Bar, 28.9 µm.

View larger version (18K): [in a new window]   Fig. 5. Percentage of TUNEL-fluorescent apoptotic sea urchin embryos observed by confocal laser scanning microscopy. 8-blastomere embryos were incubated in 5 µg ml-1 DD or 5 µg ml-1 decanal for 30, 60, 90 and 120 min, and then stained with TUNEL. See Materials and methods for details. Values (means ± S.D.; N=200) are the results of three different experiments.

View larger version (16K): [in a new window]   Fig. 6. Caspase-3-like activity in sea urchin embryos. Caspase-3-like activity was measured as described in Materials and methods. The amount of the fluorescent AFC moiety released during the reaction was related to the incubation time in DD. Values (means ± S.D.) are the results of three different experiments.

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