Biologia, Bratislava, 61/Suppl. 20: S491—S498, 2006 Section Zoology DOI: 10.2478/s11756-007-0070-z Chlorophyll content of Plešné Lake phytoplankton cells studied with image analysis Jiří Nedoma 1 & Linda Nedbalová 2,3 1 Biology Centre of the Academy of Sciences of the Czech Republic, Institute of Hydrobiology, Na Sádkách 7, CZ-37005 České Budějovice, Czech Republic; e-mail: nedoma@hbu.cas.cz 2 Charles University in Prague, Faculty of Science, Department of Ecology, Viničná 7, CZ-12844 Praha 2, Czech Republic; e-mail: lindane@natur.cuni.cz 3 Institute of Botany, Academy of Sciences of the Czech Republic, Dukelská 135, CZ-37982 Třeboň, Czech Republic Abstract: Using image analysis, chlorophyll autofluorescence was measured in single cells of green alga Monoraphidium dybowskii and in filaments of cyanobacteria (Pseudanabaena sp. and Limnothrix sp.) in the vertical profile of small acidified mountain lake Plešné jezero (Plešné Lake) from May to November of 2003. Cell chlorophyll autofluorescence was converted to cell chlorophyll content using a conversion factor determined by comparing the total autofluorescence of phytoplankton in a microscope field with spectrophotometrically determined total chlorophyll concentration; the conversion factor did not differ between epilimnion (0.5 m depth) and hypolimnion (9 m depth). Vertical patterns of chlorophyll concentration and of cellular chlorophyll content depended on water column mixing: during the period of stable thermal stratification, a metalimnetic peak in total chlorophyll concentration was present and cellular chlorophyll contents in the metalimnion and hypolimnion were notably elevated compared to the surface. Monotonous vertical profiles of both total chlorophyll concentration and cell chlorophyll content were typical for the period of water column overturn. During the stratification period, hypolimnetic Monoraphidium cell chlorophyll content was on average twice as high (maximum difference 2.7-fold) compared to surface values (of 3.2–12.9 fg μm -3 ), while in filamentous cyanobacteria (surface cell chlorophyll content of 2.2–13.3 fg μm -3 ), the difference was much higher – six-fold on average, with an 11.6–fold maximum value. The values measured with image analysis in 2003 were compared to unpublished values of total phytoplankton biomass-specific chlorophyll concentrations obtained using manual phytoplankton biomass determination and spectrophotometric chlorophyll measurement in 1998 at the same locality. Good agreement was found in seasonal patterns and vertical profiles of chlorophyll between both seasons. Key words: Cellular chlorophyll content, image analysis, acidified lake, vertical profile, Monoraphidium, filamentous cyanobacteria. Introduction The chlorophyll content of phytoplankton cells (as ex- pressed per unit of carbon or per unit of biovolume) is known to be highly variable depending on a number of factors including taxonomic affiliation, cell size, light history, temperature, nutrient content, and growth rate – to mention only the most important factors (e.g., Gei- der, 1987; Geider et al., 1998). It is no surprise then that in both freshwater and marine environments, phy- toplankton chlorophyll content changes in the course of the season as well as along horizontal profiles, and par- ticularly throughout vertical profiles of water columns that involve strong gradients in light, temperature, and nutrient availability. Interspecies differences in cellular chlorophyll con- tent and in the extent and kinetics of its regulation (especially light acclimation) have been studied exten- sively in cultures (e.g., Fisher et al., 1996; Six et al., 2004). However, the contribution of species-specific changes to bulk changes in phytoplankton chlorophyll content in situ have so far been inferred from indirect evidence only (Felip & Catalan, 2000; Veldhuis & Kraay, 2004). Rapid and high-throughput chlorophyll assays have lately been based on the measurement of chloro- phyll autofluorescence, applied in (spectro)fluorometry and flow cytometry (Neale et al., 1989), however, the applicability of these measurements at the level of sin- gle species is limited. Fluorescence microscopy coupled to image analysis provides a suitable alternative in this respect, as long as species are recognizable on autoflu- orescence images. Acidified lakes are generally characterised by simplified foodwebs and reduced biodiversity result- ing from acidification stress (Vrba et al., 1996, 2003). In the lake Plešné jezero (Plešné Lake), a small acidified lake in the Bohemian Forest (Šu- mava, B¨ohmerwald), more than 95% of phytoplank- ton biomass is consistently formed by three dominant c 2006 Institute of Zoology, Slovak Academy of Sciences