Cell cycle patterns and estimates of in situ division rates of dinoflagellates of the genus Dinophysis by a postmitotic index

Abstract

A cell cycle-analysis method based on morphological recognition of cytokinesis and sulcal list regeneration was chosen to estimate in situ division rates (µ) of 4 dinoflagellate species of the genus Dinophysis, associated with diarrhetic shellfish poisoning (DSP), following 2 different models. Sampling over 24 h was conducted on 4 mini-cruises in the Galician rías during spring and autumn proliferations of these species. Frequencies of paired and recently divided cells in integrated water samples (0 to 20 m) were measured at 30, 60, or 120 min intervals. Cellular division was phased in D. acuminata, D. acuta, D. caudata and D. tripos, but the shape of the phase fraction curves and the values of estimated division rates varied considerably between seasons and cruises for the same species. Frequencies of paired plus recently divided cells were maximal at dawn in D. acuminata, and 2 to 3 h later in the other species. The results presented here confirm that the cytokinetic (paired) phase can be very fast in Dinophysis spp. (0.3 to 2.7 h), but sulcal list regeneration was shown to be a more stable process and an unambiguous marker of cellular division. This 'postmitotic index' allowed estimates of µ at low field concentrations (10 2 to 10 3 cell l -1 ) of the target species and required a short time for sample processing (1 to 2 h per sample). Moderate (0.24) to high (0.57) values of µ were found under oceanographic conditions considered unfavourable for growth of Dinophysis spp., and the phase in the population growth season seemed to be a key factor affecting this value. A critical revision of previous results of asynchronous division obtained in cell cycle studies of Dinophysis spp. is presented. It is suggested that monitoring the content of DNA per cell through the cell cycle in Dinophysis spp. is not a reliable method until a reasonable knowledge on the nuclear behaviour during sexual processes and other nonmitotic processes is available for these species, and that even accepting that mitosis is a non-return process, cell division may be arrested in one of its phases, adding further inconsistencies to µ measurements based on quantification of DNA per cell.

Vertical distribution of division rates in coastal dinoflagellate Dinophysis spp. populations: implications for modelling

“…The sample should be collected during the appropriate time-window within which peak division is observed. In the case of Dinophysis acuta and D. caudata, the appropriate time has been confirmed, in several cycle studies, to be 2 to 3 h following sunrise (Reguera et al 2003. …”

“…Vertical hauls and bottle samples from the cell maxima were collected every 2 h from 13:00 to 17:00 h, hourly from 17:00 to 04:00 h and every half hour from 04:00 to 12:00 h (the time window when cellular division processes take place in Dinophysis spp. ; Reguera et al 2003). Depths at which to sample the cell maxima were chosen after reading the CTD profiles and performing quick on-board microscopic observation of concentrated bottle samples from depths where density discontinuities and/or in vivo fluorescence were observed.…”

“…Bulk measurements using chemical indicators such as chlorophyll a concentration and primary production are unsuitable for growth studies of these rare TPA species since they reflect the physiology not of the target organisms but of the whole plankton community. Estimates of μ from in situ cell cycle studies are time-consuming, but provide realistic information on the division capabilities of the species in question (Reguera et al 2003).…”

“…Bulk measurements using chemical indicators such as chlorophyll a concentration and primary production are unsuitable for growth studies of these rare TPA species since they reflect the physiology not of the target organisms but of the whole plankton community. Estimates of μ from in situ cell cycle studies are time-consuming, but provide realistic information on the division capabilities of the species in question (Reguera et al 2003).Estimates of μ in field populations of Dinophysis spp. have been obtained either from incubations, on deck or in diffusion chambers (Garcés et al 1997) or from direct in situ high frequency sampling with a mitotic index approach (Table 1).…”

“…from all Lugol's-fixed samples cells were counted by the Utermöhl (1931) method under a Zeiss (AXIOVERT 135) inverted microscope, after sedimentation from 25 ml columns. Vertical distributions of physical parameters and cell densities were plotted with the Microcal ORI-GIN and Golden Software SURFER contour software.Estimates of division rates: In situ division rates were estimated from the frequency of dividing (paired) and recently divided (incomplete development of the left sulcal list) cells, which were recognized by their distinct morphology as described in Reguera et al (2003), following the model of Carpenter & Chang (1988): (1) where μ is the daily average specific division rate, f c (t i ) is the frequency of cells in the cytokinetic (or paired cells) phase (c) and f r (t i ) is the half frequency of cells in the recently divided (r) phase in the i th sample. T c and T r are the duration of the c and r phases, considered as terminal events (sensu Carpenter & Chang 1988) in the present study; n is the number of samples taken in a 24 h cycle; and t s is the sampling interval in hours.…”