F. Baldi1, A. Malej2, A. Minacci1, C. Milanesi1, R. Vignani1
1) Department of Environmental Biology, University of Siena, via P. A. Mattioli, 4 ; 53100 Siena. 2) National Institute of Biology, Marine Biological Station Piran, Fornace 41, 6330 Piran, Slovenia.
The extensive mucilage appeared in the North Adriatic Sea in 1988, 1989, 1991 after a long period. These catastrophic events in the row enhanced studies on the origin of this phenomenon, which was described for the first time in the 1729. Different hypotheses have been proposed so far but not one can reproduces mucilage under laboratory conditions. The mucilage is chemically constituted by heterogeneous polysaccharides. This finding was related to a special algal exudation. It was recently demonstrated that the polysaccharide matrix was probably formed also from lysed cells of diatoms (Baldi et al., 1997). Intracellular polysaccharides such as carbon storage materials and polysaccharidic envelopes could contribute strongly to mucilage formation. Investigations were undertaken to demonstrate the presence of virus in the frozen and formaline stored mucilage in 1991 and in fresh sample of 1997.
The stored mucilage under buffered formaline was fixed and observed with a Zeiss EM 9A transmission electron microscope. Frozen mucilage was thawed and immediately stained with Yo-Pro-1 {4-[3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylmethyledene]-1-(3’-trimethylammoniumpropyl)-quinoliniumdiiodide} (Molecular Probes Inc.) as regards as Hennes and Suttle protocol (1995). The distribution fluorescence viral particles were than observed with scanning confocal laser microscopy (SCLM). Isolation of viral DNA and restriction enzymes analysis was also performed according to the method by Cottrell and Suttle (1991).
The TEM analyses of formaline stored mucilage showed the presence of virus-particles with appropriated geometric shape and size (˜70 nm) in eukaryotic organisms producing intracellular holes. TEM images of virus particles were not conclusive since their shapes were not well preserved. The use of YO-PRO-1 molecular probe, to determine viral DNA particles was performed with SCLM microscopy. This technique showed the presence of many nucleic acid particles similar to virus in washed sample of frozen mucilage. Control analysis of sample of Phoeocystis pouchetii infected by PPV (kindly donated by M. Heldal) has been used as reference sample confirm that fluorescence particles could be virus. So a further technique was used on the basis of molecular biology protocol. The digestion of viral DNA with enzymes BamHI and EcoRI produced a restriction pattern if it was compared with standard lambda/Hind III. Spectrometric analysis of viral DNA at 260 nm and 280 nm showed a high impurity of our DNA preparation. The presence of polysaccharides make the DNA extraction more difficult. A high degradation of DNA molecules was observed more in the frozen samples than in the formaline sample These results are encouraging for supporting the viral infection of diatoms.
References
Baldi F., Minacci A., Saliot A., Mejanelle L., Mozetic P., Turk V. and Malej A. 1997. Mar. Ecol. Prog. Ser. 153: 45-57
Cottrell M.T. and C.A. Suttle (1991). Mar. Ecol. Prog. Ser. 78:1-9.
Hennes K. P. Suttle C.A. (1995). Limnol. Oceanogr. 40:1050-1055.