1 Effects of nutrients, salinity, pH and light:dark cycle on the production of reactive oxygen species in the alga Chattonella marina Wenhua Liua, Doris W.T. Aua, Donald M. Andersonb, Paul K.S. Lama and Rudolf S.S.Wua1 aCentre for Coastal Pollution and Conservation City University of Hong Kong, Kowloon Hong Kong SAR, China b Biology Department Woods Hole Oceanographic Institution Woods Hole, MA 02543, United States of America 1Corresponding author: Rudolf S.S. Wu Centre for Coastal Pollution and Conservation City University of Hong Kong, Kowloon Hong Kong SAR, China P.R, Tel: 852 2788 7401 Fax: 852 2788 7406 Email: [email protected] 2 Abstract Experiments were carried out to investigate the effects of nutrients, salinity, pH and light:dark cycle on growth rate and production of reactive oxygen species (ROS) by Chattonella marina, a harmful algal bloom (HAB) species that often causes fish kills. Different nitrogen forms (organic-N and inorganic-N), N:P ratios, light:dark cycles and salinity significantly influenced algal growth, but not ROS production. However, iron concentration and pH significantly affected both growth and ROS production in C. marina. KCN (an inhibitor of mitochondrial respiration) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (an inhibitor of photosynthesis) had no significant effects on ROS production. Vitamin K (a plasma membrane electron 3 shuttle) enhanced ROS production while its antagonist, dicumarol, decreased ROS production. Taken together, our results suggest that ROS production by C. marina is related to a plasma membrane enzyme system regulated by iron availability but is independent of growth, photosynthesis, availability of macronutrients, salinity and irradiance. Keywords: Chattonella marina; reactive oxygen species; iron; nutrient; pH; physical parameters; plasma membrane; redox Abbreviations: DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; DIN, dissolved inorganic nitrogen; DON, dissolved organic nitrogen; Glu, glutamic acid; HAB, 3 harmful algal bloom; PHPA, p-hydroxyphenyl acetic acid; PSP, paralytic shellfish poisoning; ROS, reactive oxygen species; RUBISCO, ribulose-1-5-biphosphate carboxylase; SOD, superoxide dismutase 4 Introduction Chattonella marina, a harmful algal bloom (HAB) species, is able to produce reactive oxygen species (ROS, including superoxide anion radicals, O ⎯•, hydrogen 2 peroxide, H O , and hydroxyl radicals, OH·) (Oda et al., 1998) at levels 100 times 2 2 higher than those produced by most algal species (Marshall et al. 2002). In photosynthetic organisms, ROS are continuously produced as byproducts through various metabolic pathways localized in mitochondria, chloroplasts, and peroxisomes (Apel and Hirt, 2004). Photosynthetic organisms can also generate ROS by activating various oxidases and peroxidases in response to environmental stresses such as pathogens (Peng and Kuc, 1992), drought (Moran et al., 1994), light intensity (Karpinski et al., 1997) and contaminants such as paraquat (Iturbe-Ormaetxe et al., 1998). In C. marina, production of ROS has been related to growth phase, and maximum production was found during the exponential growth phase (Kawano et al., 1996). Production of superoxide anion (O ⎯•) was found to be suppressed by iron 2 deficiency (Kawano et al., 1996), and irradiance also appears to play a role in O ⎯• 2 production in C. marina (Marshall et al., 2001; 2002). In Heterosigma akashiwo, another raphidophycean flagellate, iron depletion and an increase in temperature (from 7°C to 30°C) enhanced ROS production (Twiner and Trick 2000). Nevertheless, factors affecting ROS production in raphidophycean flagellates remain unclear. 5 C. marina has caused mass mortalities of fish and great economic losses in many countries (Marshall et al., 2002), and ROS production has been implicated as one of the major factors leading to fish kills (Kawano et al., 1996; Oda et al., 1995). Although the toxic mechanism of this alga remains unclear, it is generally accepted that nutrient availability is one of the key factors in determining the toxicity of HAB species (e.g., Boyer et al., 1987). Furthermore, N:P ratio has also been shown to play an important role in algal toxicity (Hall, 1982; Boyer et al., 1987; Anderson et al., 1990). For example, phosphorus stress increases saxitoxin production and hence toxicity in Alexandrium spp. (Béchemin et al., 1999; John and Flynn, 2000), while toxicity in laboratory cultures of other microalgae (e.g. Prymnesium parvum and Chrysochromulina polylepis) has been shown to increase when nitrogen or phosphorus become limiting (Johansson and Granéli, 1999). Field studies also provide evidence that certain HAB species only occur within a certain range of N:P ratios. For example, Phaeocystis blooms were only found when N:P ratio decreased below the Redfield ratio of 1:16 (Riegman et al., 1992). While the vast majority of previous studies on algal toxicity have focused on dissolved inorganic nitrogen (DIN), recent work has revealed that dissolved organic nitrogen (DON, including urea, dissolved free amino acids, and nucleic acids) may also serve as important nitrogen sources for many phytoplankton species, including 6 HAB-causative species (Anderson et al., 2002; Berman and Bronk, 2003). If and in what way DON affects growth and ROS production in C. marina remains unknown. Availability of micronutrients, especially iron, has also been implicated as an important factor in the bloom of some HAB species (Bruland et al., 2001; Maldonado et al., 2002), and an increase in toxicity in Microcystis aeruginosa was found when iron became limiting (Lukac and Aegerter, 1993). Toxicity of HAB species (e.g. Alexandrium catenella and Heterosigma akashiwo) is also affected by physical and chemical factors such as irradiance (Ono et al., 2000), pH and salinity (Siu et al., 1997). Conceivably, toxicity and ROS production by C. marina may similarly be affected by these factors. Both laboratory and field evidence showed that toxicity of C. marina varies considerably under different culture/field conditions. Nevertheless, no systematic studies have been carried out thus far to determine how physical and chemical factors may affect ROS production in C. marina. Notably, environmental factors may also directly affect algal toxicity (Boyer et al., 1987; John and Flynn, 2000) or indirectly affect algal toxicity by affecting growth rate (Parkhill and Cembella, 1999). It has been proposed that C. marina may generate ROS through an NADPH-dependent pathway, and an enzymatic system analogous to neutrophil NADPH oxidase was identified in the plasma membrane of C. marina (Kim et al., 7 2000). Many redox enzymes located in the plant plasma membrane play a significant role in nitrate and ferric reduction (Berczi and Moller, 2000). In plants, ROS are mainly byproducts from the electron transport chains in chloroplasts (Asada, 1999), mitochondria and the plasma membrane (cytochrome b-mediated electron transfer) (Elstner, 1987). It is likely that these organelles are also sites for ROS production in C. marina, but this has yet to be demonstrated. . The objective of this study is to carry out a systematic and comprehensive study to investigate the effects of macro- and micro-nutrient availability, nutrient ratio, nutrient forms, irradiance, pH and salinity on ROS production and related growth rate in C. marina. Attempts were also made to identify the site of ROS production in this HAB species. 8 Materials and methods Algae and culture conditions The Chattonella marina (Subrahmanyan) Hara et Chihara (NIES-3) stock culture was kindly provided by Prof. M. Watanabe of the National Institute of Environmental Studies (NIES), Japan. Seawater used in this study was collected from a clean site in Hong Kong and filtered through a 0.22 µm filter before being used for culture medium preparation. Unless otherwise stated, all cultures of C. marina were grown in seawater-K-medium (Keller and Guillard 1985; salinity: 30‰; pH: 7.8–8.2) at 22–24°C under a 12:12-h light:dark cycle with light intensity of ~42 µmol photons.m-2.s-1. Algal growth rate Cell number was estimated using a hemacytometer every two days over a period of 8 days, and growth rate was calculated by the difference in cell number between two consecutive sampling intervals, using the equation given by Guillard (1973): Growth rate K' = ln (N / N ) / (t - t ) (1) 2 1 2 1 Where: N and N = biomass at t and t respectively. Divisions per day were 1 2 1 2 calculated as K' / ln2. The maximum growth rate during the entire study period was presented and compared between treatments in this study. 9 Effects of nutrient availability (N, P and Fe) on growth rate and ROS production Three experiments were carried out to determine ROS production under different DON:DIN (atomic) ratio, N:P (atomic) ratio and iron concentrations. In the first experiment, glutamic acid (Glu) and urea were individually used as the source of DON, and nitrate plus ammonium (atomic ratio 18:1, identical to that in K-medium) was used as the source of DIN. The total nitrogen concentration (0.95 mM) was also kept identical to that in K-medium. Algal growth and ROS production were determined for 6 different combinations of medium DON:DIN (0:100, 20:80, 40:60, 60:40, 80:20 and 100:0). In the second experiment, algal growth and ROS production under 6 different N:P ratios (4:1, 8:1, 16:1, 32:1, 64:1 and 128:1) (Yin et al., 2000) were determined, with the total concentration of N+P kept constant at 0.95 mM N+0.03 mM P as in the original K-medium. In the third experiment, algal growth and ROS production were measured under five iron concentrations (0, 0.02x10-8, 0.2x10-8, 1x10-8 and 5x10-8 M, each with Na -EDTA 10 times that in K-medium to ensure 2 bioavailabilty). In each treatment of the above three experiments, triplicate cultures of C. marina were grown in 5 mL medium in 12-well microplates with an initial cell density of ~500 cells.mL-1. No stirring was provided since C. marina grows best under static conditions. Cell numbers were counted every 2 days (viz. day 2, 4, 6, 8, 10 10 and 12) using a hemacytometer. Samples were collected on days 4, 6 and 8 for ROS measurements. All samplings were non-repetitive and algal samples in each well were used only once to avoid possible disturbance to the algae. Effects of physical parameters on growth rate and ROS production Three sets of experiments were carried out to investigate effects of light:dark cycles, pH and salinity on growth rate and ROS production of C. marina. In each treatment, triplicates of C. marina were grown in 5 mL of K-medium in a 12-well microplate with an initial cell density of ~500 cells.mL-1. In the first experiment, the C. marina culture was placed inside controlled environmental chambers with five different light:dark cycles (light hour:dark hour: 16:8, 14:10, 12:12, 10:14 and 8:16). In the second experiment, the initial pH of the culture was adjusted to 7.5, 8.0, 8.5, 9.0 and 9.5 with 1 M HCl or NaOH prior to the experiment. In the third experiment, C. marina was grown under 5 different salinities (30, 25, 20, 15 and 10‰) by diluting seawater with milli-Q water before nutritents were added. Cell density was measured at 2 d intervals from day 0, and ROS were measured at days 4, 6 and 8, as decribed below. Similar to the nutrient availability experiment, all samplings were non-repetitive and algal samples in each well were used only once to avoid possible disturbance to the algae.