320  2000 American Society for Photobiology 0031-8655/00 $5.00+0.00 Photochemistry and Photobiology, 2000, 72(3): 320–326 A Novel Express Bioassay for Detecting Toxic Substances in Water by Recording Rhodopsin-Mediated Photoelectric Responses in Chlamydomonas Cell Suspensions ¶ Elena G. Govorunova, Ilya M. Altschuler, Donat-P. Ha ¨ der* and Oleg A. Sineshchekov² Chair of Physico-Chemical Biology, Biology Faculty, Moscow State University, Moscow, Russia Received 8 February 2000; accepted 9 June 2000 ABSTRACT The influence of Cu 2 , Zn 2 , Cd 2 , Pb 2 and formalde- hyde on rhodopsin-mediated photoelectric responses in the green flagellate Chlamydomonas reinhardtii was in- vestigated using three modifications of a recently devel- oped population method for electrical recording (in non- oriented, phototactically preoriented (PO) and gravitact- ically preoriented cell suspensions). The addition of the heavy metal ions at concentrations several times lower than those known to affect swimming velocity and other physiological parameters in photosynthetic flagellates led to a rapid (one to several minutes) inhibition of the re- sponses. Formaldehyde induced a significant temporary increase in the gravi-orientation of the cells simulta- neously with an inhibition of their photoelectric cascade, photo-orientation and motility. The signals recorded in PO suspensions were more sensitive to all tested toxic substances than those recorded from nonoriented cells and indicated a switch from negative to positive photo- taxis in the presence of the toxic substances. Of the two major components of the photoelectric cascade, the re- generative response was more sensitive to the tested heavy metal ions, but not to formaldehyde, than the pho- toreceptor current. The results obtained show that mea- surement of the photoinduced electrical responses in Chlamydomonas cell suspensions is a powerful novel bio- assay for testing environmental pollutants in water sam- ples. INTRODUCTION The swimming behavior of photosynthetic flagellates is con- trolled by a variety of external factors including light and gravity (1). Photo and gravitaxis are fast, highly specific be- havioral responses that can be easily recorded and quantified. Chlamydomonas reinhardtii and Euglena gracilis are the best investigated model microorganisms in the field of pho- ¶Posted on the website on 21 July 2000. *Permanent address: Institut fu ¨r Botanik und Pharmazeutische Biol- ogie, Friedrich-Alexander-Universita ¨t, Erlangen, Germany. ²To whom correspondence should be addressed at: Chair of Physi- co-Chemical Biology, Biology Faculty, Moscow State University, 119899 Moscow, Russia. E-mail: olegsinesh@yahoo.com totaxis research. Recently extensive studies have been un- dertaken on the gravitaxis in these species (2,3; Sineshche- kov, O. A., M. Lebert and D.-P. Ha ¨der, submitted). Both photo and gravitaxis are strongly affected by low concentrations of toxic substances such as heavy metal ions and organic molecules. The phototactic orientation, swim- ming velocity and percentage of motile cells in Euglena (4) and Haematococcus (5) are inhibited by low concentrations of heavy metal ions. Young Euglena cells show positive gravitaxis, which changes to negative within minutes after the addition of certain heavy metal ions (6). On the basis of these studies, a standard bioassay for detecting toxic sub- stances in water has been developed using a fully automated real time image analysis system (7,8). The bioassay allows simultaneous assessment of several physiological parame- ters, including the percentage of motile cells, swimming ve- locity, cell shape and precision of photo and gravitaxis in the motile flagellate E. gracilis. In Chlamydomonas and related species of green flagellat- ed algae, the generation of a cascade of photoinduced trans- membrane currents is a key component of the signal trans- duction chain for phototaxis (9–11). Two major components of this cascade were initially observed in individual cells by means of the suction pipette technique (12). The inward pho- toreceptor current (PC)‡ is the earliest so far detected event linked to photoexcitation of the receptor rhodopsin. It is gen- erated in the eyespot region of the cell and is responsible for phototaxis. When depolarization of the membrane in- duced by PC achieves a critical threshold, a voltage-driven regenerative response (RR) is triggered, which involves a massive influx of Ca 2+ ions across the flagellar membrane and leads to the photophobic response of the cell. A recently developed method allows the recording of photoinduced electrical responses in a suspension of freely swimming fla- gellates (13). This versatile method is technically simple, highly sensitive and can be applied to a wide range of spe- cies regardless of their size and structure. Moreover, partic- ular modifications of this method enable instant probing of the precision of orientation of the cells caused by external stimuli, including light and gravity. The purpose of this study was to investigate the influence ‡Abbreviations: GO, gravitactically preoriented; PC, photoreceptor current; PO, phototactically preoriented; RR, regenerative re- sponse; UL, unilateral.