Effect of UV-B radiation and humic substances on growth and motility of Gyrodinium aureolum

Size: px

Start display at page:

Download "Effect of UV-B radiation and humic substances on growth and motility of Gyrodinium aureolum"

David Willis
5 years ago
Views:

1 1570 Notes dexter and E. R. Leadbetter [eds.], Bacteria in nature. V. 2. Plenum. PORTER, K. G., AND Y. S. FEIG The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr. 25: PROCTOR, L.M., AND J.A. FUHRMAN Viralmortality of marine bacteria and cyano-bacteria. Nature 343: SIERACKI, M.E.,P.W. JOHNSON,AND J.McN. SIEBURTH Detection, enumeration, and sizing of plank- tonic bacteria by image-analyzed epifluorescence microscopy. Appl. Environ. Microbial. 49: ZIMMERMAN, R., AND L.-A. MEYER-REIL A new method for fluorescence staining of bacterial populations on membrane filters. Kiel. Meeresforsch. 30: Submitted: 14 July 1992 Accepted: 2 January 1993 Revised: 27 January 1993 Limnol. Oceanogr., 38(7), 1993, ,bytheAmetican Society of Limnology and Oceanography, Inc Effect of UV-B radiation and humic substances on growth and motility of Gyrodinium aureolum Abstract- We investigated the effect of ultraviolet-b radiation ( nm) on the growth and motility of the marine dinoflagellate Gyrodinium aureolum in the presence of humic substances. The specific growth rate after 7 d of the W-B,, (biologically effective radiation) exposure (2.15 kj m-2dp ) decreased by 92% compared to the control. The growth rate for W-B treated cells increased by 18, 26, and 10% when cells were cultured in medium enriched with humic substances (1.7, 4.2, and 8.3 mg liter I). However, the growth rate was lower at the highest humic substance than at the two lower concentrations. When humic substances were used as a filter to shade the organisms from W-B radiation without substantial changes in the amount of white light, instead of being added to the medium, the growth rate increased with the concentration of humic substances. W-B radiation had little or no effect on the motility of G. aureolum, whether humic substances were added or not. Phototactic orientation of the UV- B-treated cells was significantly weaker than the control to actinic white light ( nm, 47 m - ). The presence of humic substances during treatment with UV-B radiation reduced the inhibition of the positive phototaxis in G. aureolum. The stratospheric ozone layer protects organisms on earth from ultraviolet-c ( nm) and high levels of ultraviolet-b radiation ( nm). The increased UV-B radiation reaching the earth s surface as a result of chlorofluorocarbons (CFCs)-dependent ozone depletion could have negative effects on living Acknowledgments We thank Lena Carlsson for technical assistance, Silja Petersen-Mahrt for computer assistance, and Ian Max Moller for reading the manuscript. organisms. Smith et al. (1992) measured a minimum of 6-l 2% reduction in primary production associated with O3 depletion in October 1990 over the Southern Ocean. Since marine phytoplankton are responsible for half of the CO, fixation on earth and since these organisms form the first step in the marine food web, increased radiation could have negative ecological and economical effects. Increased acidification of soils and lakes can also affect living organisms. Decreasing ph of soil and water often leads to precipitation of metals. Agricultural activity could cause humic substances to leach from the soil and end up in coastal waters (R. C. Petersen pers. comm.). Humic substances in natural waters differ widely with time and place. Rivers on the Swedish west coast have concentrations of mg liter-l and the Laholm Bay into which many of these rivers discharge has 1.4 mg liter- of humic substances (Graneli et al. 1985). Values from the Gdansk Bay (Baltic Sea) are between 1 and 3 mg liter-l. Laboratory and field experiments have demonstrated that humic substances affect growth productivity and nutrition in marine phytoplankton. Although the nature of humic substances is not so well understood, some important characteristics are that they can act as chelators and ion exchange resins and affect the nutrient dynamics of surface waters (Petersen et al. 1987). Humic substances can form complexes with metals and increase or decrease their toxicity. The toxicity of humic substances is due to their

2 Notes 1571 phenolic and ionizable organic composition and humic substances become more toxic at lower ph. Humic substances can absorb UV and blue light and be a possible source of nitrogen when N deficiency occurs (Graneli et al. 1985). The formation of superoxid (O,-)- a negatively charged radical-and other reactive intermediates is also a possibility when UV light is absorbed by humic substances. There have been several reports of blooms of the dinoflagellate Gyrodinium aureolum Hulburt in northern European waters. G. aureolum is the most common red tide dinoflagellate species in these waters. Following in the wake of these red tides, there has been mass mortality of various fish and invertebrate species. We therefore need to know more about the behavior of these organism under different environmental conditions. Our aim was to investigate the effect of UV-B radiation and humic substances on growth and motility of G. aureolum. In all experiments, we used G. aureolum (Ba 6). The organisms were isolated from the Kattegat and obtained from E. Graneli. The cells were grown in ASP, medium (Provasoli et al. 1957; Provasoli 1963) as modified by Ekelund and Hader (1988). The humic substance was a commercial soil extract (Aldrich humic acid, sodium salt, EGA- Chemie, catalog no. H1,675-2). The humic substance extract was dissolved in modified ASP, medium to a concentration of 0. 1 g liter-l and filtered through a sterilized membrane (ME28 pore-size, 1.2 pm). The algae were cultured in 1 OO-ml Erlenmeyer flasks or in Petri dishes. For the control treatment ordinary glass flasks and Petri dishes (glass) were used; quartz glass was used for treatments with UV-B radiation. The cells were grown in culture chambers at 20 C with 16 : 8 L/D cycle. The Erlenmeyer flasks were placed at 45 and the Petri dishes at a 90 angle to the light. The algae suspensions were cultured in two ways. First, growth in 50 ml of modified ASP2 medium as control and in medium supplemented with three different concentrations of humic substances (1.7,4.2, and 8.3 mg liter- ). Second, the growth experiment was also done with cells cultured in 50 ml of modified ASP2 medium in Petri dishes with another Petri dish placed above, containing 10 ml of medium without humic substances (control) and with different concentrations of humic substances (1.7, 4.2, and 8.3 mg liter- ). The organisms for specific growth rate were cultured for 7 d. Specific growth rate was calculated with a hematocytometer at the start and after 7 d. A sample was taken from each Erlenmeyer flask and Petri dish and counted. If we know the initial number of cells (No) and the number of cells at the end of the period (N), we can calculate the specific growth rate with the formula p = log (N/N,)k/t, where t is the time in days and k is a constant (=3.3222). For significance calculations, the t-test was used. The organisms were given two different light treatments. In the first, control white light from four 400-W metal halogen lamps (Power Star HQIL Osram) with an irradiance of 19.5 W m-2 ( nm) was used. In the second, control + 1 h of UV-B radiation per day from one 40-W sunlamp (FS40, Westinghouse Elec. Corp.) was used. The biological effective radiation, UV-B,,, was 2.15 kj m-2d-1. In comparison, the biologically effective radiation of natural sunlight conditions from Lund (5 5.7 N, 13.4 E) on a sunny day is 0.55 kj m-2d- l. The UV-B,, was obtained with the calculations of Bjiirn and Murphy (198 5), based on the gen- eralized plant action spectrum to UV-B radiation (Caldwell 197 1). To predict biological damage, it is necessary to use action spectra for various biological effects. The plant action spectrum may not be the best action spectrum for our purpose, but there is none for the motility of dinoflagellates. Radiation was measured with a spectroradiometer (model 742, Optronics Lab.) interfaced with a Hewlett- Packard 85 computer. Photo-orientation and motility determina- tions were carried out with organisms grown in different concentrations of humic substance and light treatments (control and UV-B radiation) for 3 d. Phototaxis is defined as movement in a direction dependent on the light direction. Movement of organisms toward the light source or away from it is called positive and negative phototaxis (Hader 1988). The samples were dark-adapted for 30 min before measurements. Phototactic orientation and motility were determined with a fully automatic computer-controlled video analysis sys-

3 1572 Notes 6 I I 1 I I Fig. 1. Specific growth rate of Gyrodinium aweolum after 7 d. Algal cultures enriched with different concentrations of humic substances. Control (without UV-B radiation) and UV-B radiation. Error bars indicate SE. Fig. 2. Specific growth rate of Gyrodinium aureolum after 7 d. Algal cultures with humic substances in Petri dishes above the cell cultures. Control (without UV-B radiation) and UV-B radiation. Error bars indicate SE. tern. The program was compiled by D.-P. Hader and coworkers with algorithms developed for cell detection and tracking (Hader and Lebert 1985). A far-red-sensitive video camera (FA-74- IN Newvicon) was mounted over a Nikon Optiphot microscope (4 x objective) and the builtin lamp used in combination with an infraredtransmitting cut-off filter (RG780 nm, 2 mm, Schott and Gen.) to produce the monitoring irradiation. To induce the phototactic movements, we used a slide projector equipped with an Osram lamp (24 V, 150 W). The received actinic white light had an irradiance of 47 W m-2 ( nm). The organisms were introduced into a flat-glass cuvette (7 x 20 x 1 mm), which was placed under the microscope. Histograms were calculated from the raw data and the degree of phototactic orientation was measured with Rayleighs (r) test. The Rayleighs test is based on the equation r = [(E sin a)2 + (Z cos a)2 n] /2. Here a is the angle in which the organism is moving, and n is the number of organisms. The r value can vary between 0 and 1; a value close to 0 indicates totally random movement, while a value near 1 describes the movement of all cells in one direction. The test is independent of the specific direction of movement and can be used for both positive and negative phototaxis. All phototactic measurements were made in actinic white light or in darkness. In each test, 1,000 organisms were studied and all experiments were replicated at least 5 times. Cell motility was calculated from tests of 1,000 organisms. The same equipment as in the phototactic measurements was used, except for the slide projector. All tests were replicated at least 5 times. To investigate whether there was a change in the absorbance of the humic substance due to UV-B radiation, the different concentrations of humic substances were exposed to the light treatment as described earlier. Absorption spectra were measured ( nm) at day 1 and 7 with a spectrophotometer (Shimadzu 2 10). Quartz cuvettes were used with ASP, medium as reference. UV-B radiation decreased the growth rate of G. aureolum, which partly recovered when humic substances were added to the medium (Fig. 1). The recovery of growth could be due to the absorbing ability of the humic substances in the UV-B region. Humic substances had a positive effect up to a concentration of 4.2 mg liter-l, but the growth rate decreased slightly at higher concentrations. A positive effect of G. aureolum grown in humic substances has been reported by Prakash and Rashid (1969). At the highest concentration of humic substances, absorbance was greater, but

4 Notes 1573 a. b. Fig. 3. Circular histograms of the phototactic orientation in Gyrodinium aureofum exposed to unilateral white light (47 WmP2) or kept in the dark. [a.] Control + light, Y = [b.] UV-B + light, Y = [c.] UV-B mg liter humic substances + light, Y = [d.] Control + dark, Y = UV-B radiation supplied for 1 h daily (3 d), corresponding to UV-B,, of 2.15 kj m-2d- I. Arrows indicate direction of incident light. there was also a toxic effect. A possible explanation is that the humic substances contain toxic phenolic groups (Petersen et al. 1987; Gjessing 1976). These products are formed when large molecules of humic substances are broken into smaller parts. This breakdown may be initiated by UV radiation (Gjessing 1976). The interaction between humic substances and UV radiation is still unclear; it is possible that UV radiation, by splitting molecules of humic substances, also provides marine microorganisms with nutrients that cause red tides. We used Aldrich humic acid, which could have different effects than a humic acid from a lake or marine environment. The positive results on growth rate with the

5 1574 Notes d b g s = ii a m l l- 20 I. I, I * I, I, I, % Motile cells Fig. 4. Linear histograms of the velocity distribution of Gyrodinium aureolum. The cells were grown without and with different concentrations of humic substances (HS). A-Control; B-UV-B; C-control mg liter HS; D- UV-B mg liter- HS; E-control mg liter- HS; F-UV-B mg liter HS; G-control mg liter-l HS; H-UV-B mg liter-l HS. highest concentration of humic substance used as a filter (Fig. 2) are most likely due to the effect of absorption or reflection of the UV-B radiation. The gradual increase in growth with increasing concentration of humic substances is an indication of this. At different concentrations, t-tests showed significant differences (P < 0.0 1) between control and UV-B-exposed organisms. The highest growth rates for the controls (no UV-B radiation) were achieved when cells were cultured in ASP, medium without humic substances; growth with humic substances added to the medium (Fig. 1) or used as a filter (Fig. 2) showed lower values. The explanation is probably reflection of white light by particles. The absorbance spectra of the three concentrations of humic substances did not show any differences to UV-B radiation after 7 d (data not shown). G. aureolum revealed positive phototaxis to unilateral white light (47 W m-2) (Fig. 3a), but UV-B-treated cells showed significantly lower r-values than the controls (Table 1, Fig. 3b). The positive phototaxis of G. aureolum was significantly higher for organisms cultured in UV-B radiation and humic substances (Fig. 3c) compared to UV-B-treated cells without humic substances (Fig. 3b). The higher r-values for the UV-B-treated cells grown with humic substances shows that humic substances had a protecting effect due to absorbance in the UV-B region. Measurements in the dark showed a random distribution (Fig. 3d). The UV-B-treated cells with and without humic Table 1. Phototactic orientation (r-values+_se, n=5) of Gyrodinium aureolum treated with UV-B radiation (UV- B,, 2.15 kj m-* d-l) and different concentrations of humic substances. r-values indicate phototactic response to unilateral white light of 47 W m-* (light) and to darkness. Humic substance (mg liter- ) Treatment Control Control light 0.26kO kO kO kO.03 UV-B light O.l2t a Control dark 0.05* _ t_o.o UV-B dark a

6 Notes 1575 substance also showed a random distribution in the dark (Table 1). The effect of UV-B radiation on the motility of G. aureolum in different concentrations of humic substances showed no significant differences (Fig. 4). The control cells moved slightly faster than the UV-B-treated cells, and this was most pronounced for cells grown with the highest concentration of humic substances. A negative effect of UV-B radiation on growth and motility of microorganisms has been shown previously (Hider 1985; Ekelund 199 1). The observation that photo-orientation is affected, but not motility, indicates that the photoreceptor for phototactic orientation may be more sensitive to UV-B radiation than the target molecule for motility. Another explanation is that UV-B radiation only affects the longitudinal flagellum, which seems to be involved in the phototactic reorientation of the cell; the lateral flagellum, which is responsible for forward locomotion (Hand and Schmidt 1975), remains unaffected. The difficulties in simulating natural light regimes in the laboratory are due to the great variation of natural radiation in the water column, the amount of dissolved substances, and other parameters such as vertical mixing. The present results indicate that phytoplankton are protected from the effects of UV-B radiation by humic substances. This protection may be connected with observations that photoinhibition of phytoplankton in productive waters with high levels of humic substances is less pronounced (Kirk 1983). The penetration of UV-B radiation is maximal in ocean waters having minimum concentrations of Chl and dissolved organic material (Smith and Baker 1979). Smith et al. (1992) also showed that the ozone hole increases the effective penetration of UV-B radiation by -7 m and that it is possible to detect UV-B radiation at depths of m. To predict the effects of ozone depletion on phytoplankton, we need more data from different marine and fresh-water environments. Corresponding author. Tom Nielsen Nils G.A. Ekelund Department of Plant Physiology University of Lund Box 7007 S Lund, Sweden References BJ~RN, L. O., AND T. M. MURPHY Computer calculation of solar ultraviolet radiation at ground level. Physiol. Veg. 23: CALDWELL, M. M Solar UV irradiation and growth and development of higher plants, p. 13 l-l 77. Zn A. C. Giese [ed.], Photophysiology. Academic. EKELUND, N. G. A The effects of UV-B radiation on dinoflagellates. J. Plant Physiol. 138: , AND D.-P. HADER Photomovement and photobleaching in two Gyrodinium species. Plant Cell Physiol. 29: 1109-l 114. GJESSING, E. T Physical and chemical characteristics of aquatic humus. Ann Arbor Sci. GRAN~LI,E.,L. EDLER, D. GEDZIOROWSKA,AND U.NYMAN Influence of humic and fulvic acids on Prorocentrum minimum, p Zn D. M. Andersson et al. [eds.], Toxic dinoflagellates. Elsevier. HADER, D.-P Effects of UV-B radiation on motility and photobehavior in the green flagellate glena gracilis. Arch. Microbial. 141: Eu Ecological consequences of photomovements in microorganisms. J. Photochem. Photobiol. B. 1: AND M. LEBERT Real time computer conirolled tracking of motile microorganisms. Photothem. Photobiol. 42: HAND, W. G., AND J. A. SCHMIDT Phototactic orientation by the marine dinoflagellate Gyrodinium dorsum Kofoid. 2. Flagellar activity and overall response mechanism. Protozoology. 22: KIRK, J. T Light and photosynthesis in aquatic ecosystems. Cambridge. PETERSEN, R. C., JR., A. HARGEBY, AND A. KULLBERG The biological importance of humic material in acidified waters. Natl. Swed. Environ. Protect. Bd. PRAKASH, A., AND M. A. RASHID The influence of humic substances on coastal phytoplankton productivity, p Zn A. Ayala-Castanares and F. B. Phleger [eds.], Coastal lagoons. UNAM-UNESCO. PROVASOLI, L Growing marine seaweeds, p Zn Proc. 3rd Int. Seaweed Symp. Pergamon. -, J.J.A. MCLAUGHLIN,AND M.R. DROOP The development of artificial media for marine algae. Arch. Microbial. 25: SMITH, R. C., AND S. B. BAKER Penetration of UV-B and biologically effective dose-rates in natural waters. Photochem. Photobiol. 29: 3 1 l AND OTHERS Ozone depletion: Ultraviolet radiation and phytoplankton biology in antarctic waters. Science 225: Submitted: 26 February 1992 Accepted: 26 January 1993 Revised: 24 February 1993

Impact of UV-B Radiation on Haematococcus pluvialis Flotow Isolated from Himachal Pradesh under Laboratory Conditions

Volume 3, Issue 11 April 2015 581 RESEARCH ARTICLE ISSN: 2278-5213 Impact of UV-B Radiation on Haematococcus pluvialis Flotow Isolated from Himachal Pradesh under Laboratory Conditions G. Kavitha, C. Kurinjimalar,