In a previous work (Lage et al. submitted), the effect of copper in the viability (esterase activity) of Amphidinium carterae was evaluated with the fluorescein diacetate (FDA) assay (1) by flow cytometry. Cells of A. carterae exposed to 2.8 şM estimated liable copper (15.8 şM total copper) showed an unexpected increase in the levels of green fluorescence (GFI) after loading with FDA when compared to cells not exposed to copper or exposed to lower concentrations of this metal. Other changes also underwent by these cells were inhibition of growth and loss of motility as well as morphological alterations similar to those described by Sampayo (2) during the encystment of A. carterae. Trying to confirm the existence of a relation between the high levels of GFI and encystment, which may occur in stationary cultures, where culture conditions become stressful, the FDA assay was performed in A. carterae in exponential and stationary growth phases, both in the absence and after exposure to 2.8 şM estimated liable copper.

A. carterae in exponential growth showed, after loading with FDA, a homogeneous GFI population (P1). In late stationary growth (37 days), two populations could be defined, one (about 74%) with lower levels of GFI (P1) and the second (about 14%) exhibiting higher levels of GFI (P2) (7-fold higher in this population compared to P1). These two populations were also clearly evident (37% of P1 and 55% of P2) in stationary A. carterae cells that have been exposed to a sublethal copper concentration for 25 days (6.5-fold difference in the GFI between the two populations.) When cells from these three cultures were exposed for 30 min. to 2.8 şM estimated labile copper and subsequently loaded with FDA , a GFI homogeneous population more or less coincident with population P2 appeared, with, respectively, an increase of 8-, 10- and 6-fold comparative to the fluorescence levels of the correspondent P1 populations.

The results observed in cells stressed by copper and in temporary "quiescent" stage, could be interpreted as an increase in their metabolic activity. However our results go against the assumption that these kind of cells have a reduced metabolism (1). We believe that these high GFI levels are, most probably, due to an increase in the cellular pH which may occur during the process of encystment.

Work supported by JNICT. (1) Dorsey J., Yentsch, C.M., Mayo, S. & McKenna, C. 1989. Cytometry 10: 622-628; (2) Sampayo, M.A.M. 1985. In "Toxic Dinofl." Anderson, White & Baden (eds). Elsevier Scientific Publ. pp. 125-130.