Water-soluble extracts of the diatom Thalassiosira rotula induce aberrations in embryonic tubulin organisation of the sea urchin Paracentrotus lividus

Eggs and embryos of the sea urchin Paracentrotus lividus were used as a mod el to study the effect at the cellular level of potential anti-mitotic comp ounds extracted from the diatom Thalassiosira rotula. Eggs and embryos incu bated in a water-soluble diatom extract, corresponding to 5 x 10(6) and 10( 7) cells ml(-1), were totally blocked (i.e. cell division was blocked) at t he one-cell stage. At lower concentrations (2.5 and 1.25 x 10(6) cells ml(- 1)), the first mitotic division was inhibited in 32 +/- 26% and 25 +/- 3.5% of the zygotes, respectively, demonstrating the dose-dependent effect of d iatom extracts on sea urchin development. Immunofluorescence dyes, specific for DNA and alpha-tubulin subunits, were used to stain nuclei and microtub ules in sea urchin embryos during various phases of development. Images wit h the confocal laser scanning microscope showed that tubulin was not organi sed in filaments at the sperm aster and cortex levels, and that the pronucl ei were not fused in embryos incubated soon after fertilisation with water- soluble diatom extracts corresponding to 10(7) cells ml(-1). At lower diato m-extract concentrations (4 x 10(6) cells ml (-1)), fusion of the pronuclei occurred but the mitotic spindle was not formed. Microtubules were clearly de-polymerised and the chromatin appeared globular and compacted at the ce ntre of the cell. A similar structure was observed for sea urchin embryos i ncubated with 0.1 mM colchicine, a potent anti-mitotic compound. When sea u rchin embryos were incubated in water-soluble diatom extracts at different times prior to the first mitotic division, microtubules appeared de-polymer ised at each step, from pronuclear fusion to telophase, and cell division w as blocked. At the histological level, embryos incubated with 4 x 10(6) cel ls ml(-1) diatom extract showed nuclear fragmentation without cytokinesis. The possible use of sea urchin embryos as a bioassay to test for other unkn own compounds with cytotoxic activity in phytoplankton species is discussed .