Mitotic indices of zooxanthellae: a comparison of techniques based on nuclear and cell division frequencies

Hitherto, studies of mitotic indices in zooxanthellae have involved identification of doublet cells in the mitohc phase of cytokinesis. This note presents comparative results for zooxanthellae in the temperate sea anemone Anemonia viridis Forskal based upon measurements in both cytokinesis and karyokinesis, provides some evidence for the relative duration of these phases of mitosis in A. viridis zooxanthellae and highlights the potential benefits of deriving mitotic indices during karyokinesis. Measurement of mitotic index in cell populations involves a n assessment of the proportion of cells which are observed in mitosis (Mitchison 1971). Such measurements have been widely used by those monitoring growth in both animal and plant tissues, though the techniques employed have varied widely depending on the study organism, most methods calculating mitotic index on the basis of the fraction of cells showing paired nuclei or 2 mitotic figures per cell. Studies of mitosis in symbiotic algae (zooxanthellae) resident in coelenterates have, in contrast, used an index based upon the fraction of cells appearing as doublets with a distinct cell plate (Wilkerson et al. 1983, 1987, Cook & D'Elia 1987, Hoegh Guldberg et al. 1987, Steen & Muscatine 1987). A major aim in these studies has been the calculation of algal population growth rates and doubling times using equations derived from phased cell division in marine phytoplankton (Weiler & Chisholm 1976, McDuff & Chisholm 1982, Wilkerson e t al. 1983). In such a context cell plate measurements have provided reasonable estimates of in sj tu algal growth rates that have significantly advanced our understanding of the effects of nutrients on the dynamics of symbiotic algae (Muscatine et al. 1989); temporal patterns of algal division (Wilkerson et al. 1988); and mechanisms of algal regulation (Trench 1987). More recently the balance between the coelenterate host and its symbiotic algae has been shown to be very sensitive to environmental factors such as temperature increase (Glynn & D'Croz 1990), temperature decrease (Muscatine et al. 1991), salinity and irradiance (HoeghGuldberg & Smith 1989). As a result it has been suggested (Brown 1988) that a better understanding of the mitotic cell cycle in symbiotic algae could not only lead to an improved quantitative estimate of 'stress' in coelenterates but also clarification of cause/effect relationships between mitotic responses and specific pollutants. In the present note the advantages of monitoring mitotic indices for these purposes, using an index which is based on the number of cells showing mitotic figures, are presented using the temperate sea anemone Anemonia vjridis Forskal. Materials and methods. Anemones were collected from Millport, Isle of Cumbrae, Scotland, and were maintained under laboratory conditions as described in Suharsono & Brown (1992) in recirculated, artificial seawater at 15'C. The anemones were fed weekly on fresh Mytilus edulis (1 g wet weight mussel flesh per anemone). Algal division in Anernonia viridis has previously been shown to be asynchronous (Suharsono 1990), resembling that in the temperate anemone Anthopleura elegantissima which showed considerable variability over the course of a day (Wilkerson et al. 1983). In the present experiment algal division was therefore measured over a n 11 h period, between 09:OO and 20:oo h. In the present experiment 2 methods of measuring mitotic index in Anemonia viridis zooxanthellae were compared one involved identification of 2 mitotic figures per cell (using the Feulgen staining of nuclear DNA) and the other, the counting of doublet cells a s used in the majority of coelenterate studies (Wilkerson O Inter-Research 1992 100 Mar Ecol. Prog. Ser. 89: 99-102, 1992 et al. 1983). Feulgen staining of nuclear material is a standard cytochemical technique (Boon & Drijver 1986) and one that has been used to measure nuclear division in both plants (Clowes & Juniper 1968) and animals, including coelenterate material (Muscatine & Neckelmann 1981). Three tentacles were sacrificed from each of 3 anemones at hourly intervals. The tentacles were fixed in 3:l alcohol/glacial acetic acid for 30 rnin and were then transferred to 70 "A alcohol and stored at 4 "C. Each tentacle was then hydrolysed in 1M HC1 at 60°C for 30 min followed by 10 rnin at 20°C. Serial washing of tentacles in distilled water was carried out to remove any acid; tentacles were then placed in Feulgen stain for 3 h. They were subsequently macerated on a slide in 45 Oh (v/v) acetic acid before being examined as a squash preparation under an Olympus phase contrast microscope at 1 0 0 0 ~ magnification. A total of 500zooxanthellae were counted per slide and the number of algae showing 2 mitotic figures within the cell recorded. This value was then expressed as a percentage of the totalnumber of cells counted to provide a mitotlc index based on karyolunesis (nuclear division). Using the same tentacle samples the numbers of cells showing a cell plate were also scored and a mitotic index value calculated based on cytokinesis (cell divi-