Short-term effects of different nitrogen substrates on growth and toxin production of dinoflagellate Alexandrium catenella Balech (strain ACDH)

Harmful Algae 12 (2011) 46–54

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Short-term effects of different nitrogen substrates on growth and toxin production of dinoflagellate Alexandrium catenella Balech (strain ACDH) Tian-Shen Li a,b, Ren-Cheng Yu a,*, Ming-Jiang Zhou a a b

Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, PR China Monitoring Center of Marine Environment, Beihai, Guangxi, 536000, PR China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 2 February 2009 Received in revised form 26 August 2011 Accepted 26 August 2011 Available online 2 September 2011

Blooms of Alexandrium catenella were frequently found in sea areas adjacent to the estuary of the Changjiang River in spring of the last several years, often in association with large-scale blooms of Prorocentrum donghaiense. It was considered that the increasing intensity of these dinoflagellate blooms was related to the incremental nutrient influx from the Changjiang River over the last 40 years, especially the ‘‘excess’’ nitrogen (N) input. A nutrient-addition experiment was then designed to study the responses of a strain (ACDH) of A. catenella, isolated from the East China Sea, to the addition of different N substrates, including nitrate, ammonium and urea. It was found that N-starved cells of strain ACDH could use all the three N substrates as the sole N source to grow, with similar specific growth rate and maximum cell density. All the three N substrates could elevate cellular toxin content shortly after the addition of nutrients, but cellular toxin content of the urea group was much lower than the nitrate group and the ammonium group. In the following days, the cellular toxin content of the urea group was even lower than the N-limited control group, but cellular toxin contents of the nitrate group and ammonium group were still comparable to the control. It was presumed, based on these findings, that a relatively slower N uptake and assimilation rate occurred in the urea group, and urea–N adsorbed was preferentially utilized for growth rather than toxin synthesis. Therefore, urea in natural seawater could promote the formation of algal blooms, but decrease the toxicity, of strain ACDH. ß 2011 Elsevier B.V. All rights reserved.

Keywords: Alexandrium catenella Ammonium Harmful algal bloom Nitrate Paralytic shellfish poisoning Urea

1. Introduction Some species in genus Alexandrium can produce paralytic shellfish poisoning (PSP) toxins, which pose potent threats on the health of human-beings, mariculture industry and natural ecosystems. Toxic Alexandrium species are widely distributed around the world, and their population dynamics and potential impacts have been extensively studied since the 1980s (for example, Anderson et al., 2005). Blooms of Alexandrium catenella were found in sea areas adjacent to the estuary of the Changjiang River in 2002, 2004, 2005 and 2006, during cruises supported by the National Basic Research Priority Program (the Ecology and Oceanography of Harmful Algal Bloom in China, CEOHAB). The blooms, with maximum cell density up to 106 cells/L and areas over 1000 km2, were often in association with large-scale blooms of Prorocentrum donghaiense in spring (Lu and Goebel, 2001). Studies during the last several years indicated that occurrence of the large-scale dinoflagellate blooms was closely related to the serious eutrophication caused by nutrients

* Corresponding author. Tel.: +86 532 82898590; fax: +86 532 82898590. E-mail address: [email protected] (R.-C. Yu). 1568-9883/$ – see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2011.08.011

discharged from the Changjiang River (Yangtze River), as well as the unique physical oceanographic conditions in this region (Zhou and Zhu, 2006). Large amounts of nutrients, especially N, were brought into the sea by the Changjiang River, which altered nutrient concentrations and compositions in the Changjiang River estuary and its adjacent coastal waters. The nitrate concentration in this area increased more than 2 times, and the N:P ratio increased from about 16 to over 30, during the last 40 years (Wang, 2006; Zhou et al., 2008). Coastal eutrophication, and its close relationship with global expanding of harmful algal blooms (HABs), have been extensively studied and reviewed by many authors in the last several decades (Ryther and Dunstan, 1971; Smith, 1984; Smayda, 1990; Anderson et al., 2002). In many coastal areas, N is considered as the first limiting macronutrient for the primary production (Anderson et al., 2002). Therefore, input of N is critical for the formation of high-biomass algal blooms, while in freshwater, phosphorus (P) is considered more important for the formation of algal blooms, especially those caused by cyanobacteria. N in seawater exists in different forms, such as dissolved inorganic nitrogen (DIN) compounds like nitrate, nitrite and ammonium, dissolved organic nitrogen (DON) compounds like urea, free amino acids and protein, and particulate N (Antia et al., 1991). DIN has been considered as

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the major form of N available for the growth of phytoplankton in the sea. More recently, however, people tend to include organic compounds of N, among which urea is the most important component, in studies concerning the relationship between eutrophication and HABs (Glibert et al., 2006). Different species of algae have different preferences in selection of their N sources (Syrett, 1981; Thompson et al., 1989). The availability of various forms of N can therefore affect the growth of the specific algal species and the succession of phytoplankton community (Eppley and Peterson, 1979; Kokkinakis and Wheeler, 1988; Berg et al., 1997). Generally, diatoms favor nitrate as the nitrogen source, while dinoflagellates prefer ammonium or DON (Collos et al., 2004). Many dinoflagellate blooms, including those caused by toxic species, are believed to be related to the availability of reduced forms of N, such as ammonium and urea (Lomas et al., 2001; Glibert et al., 2001, 2006). In the review of Glibert et al. (2006), it was indicated that the global increase of documented incidences of PSP from 1970s to 2000s was similar to the global increase of urea use. The potential impacts of toxic Alexandrium blooms can also be affected by nutrients, such as N, through their effects on toxin production. PSP toxins are a suite of N-rich compounds, and sufficient N supply is essential for the biosynthesis of PSP toxins. Many studies found that N deficiency could decrease the PSP toxin yield and cellular toxin content in toxic species of Alexandrium (Anderson et al., 1990; MacIntyre et al., 1997; Yu et al., 2001; Leong et al., 2004), while excess N availability under P-limitation condition could increase the cellular toxin content (Boyer et al., 1987; Anderson et al., 1990; Flynn et al., 1994; Bechemin et al., 1999; John and Flynn, 2000; Yu et al., 2001; Wang and Hsieh, 2001, 2002). Therefore, to evaluate and predict potential impacts of the toxic algal blooms of Alexandrium spp, it is critical to fully understand the effects of nutrients on toxin production characteristics of the toxic algae. In China, the dynamics and potential impacts of A. catenella blooms in the Changjinag River estuary and adjacent coastal waters are still not very well understood. It was suggested that some new outbreaks of algal blooms, especially those caused by Alexandrium species, have little relationship to the nutrient enrichment, since some blooms appeared in pristine waters without significant nutrient input from anthropogenic origins (Anderson et al., 2002). However, the blooms of A. catenella in the Changjiang River estuary and adjacent coastal waters will be inevitably affected by the serious eutrophication status, especially the ‘‘excess’’ nitrogen input from the Changjiang River (Wong et al., 1998). And there might be rapid switches in nature among different N sources available, so the effects of different N sources on the growth and toxin production of A. catenella may represent a ‘‘real world’’ situation. How the dynamics, as well as potential impacts of the blooms, would be affected by the increasing N input need to be studied in detail. Therefore, a nutrient-addition experiment was designed to study the responses of strain ACDH of A. catenella, which was isolated from this region, to different nitrogen substrates in the present study. 2. Materials and methods

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autoclaved natural seawater from the seawater supplying system in the Institute of Oceanology, which was pumped from a pristine sea area and pretreated by sand filtration and ozone sterilization. The experiment was carried out with 500 ml flasks, each containing 300 ml culture medium. Four groups, i.e. nitrate group, ammonium group, urea group, and control group, were set up in the experiment. Each group had 40 flasks, where 3 flasks were used for cell-counting, and the rest were used for toxin analysis, cellular C and N analysis, and chlorophyll a (Chl a) analysis. At the beginning of experiment, algae collected at the exponential stage were inoculated into the medium in each flask enriched with Nreduced f/2-Si nutrient solution (the final concentration of nitrate in this medium was 5 mM). The initial cell densities in all the flasks were around 400 cells ml1. The flasks were then put in an environmental chamber and cultured at 20  1 8C and 80 umol m2 s1 in a light:dark cycle of 14:10. After the nitrate in the medium was depleted, as indicated by the decrease of algal densities in the flasks, nitrate, ammonium and urea were added, respectively, to the corresponding group as N substrate. The final concentrations of the three N substrates were all set to be 50 mM N. For the control group, no N substrate was added. During the experiment, the cell densities were monitored and algae samples were collected for the analyses of cellular content of carbon (C), N, Chl a and PSP toxins. 2.2. Determination of cell densities For determination of cell densities, 2 ml algae culture was collected from each of the three flasks in the four groups, after mixing the culture medium gently in the flask. The algal cells were fixed with a drop of Lugol’s solution, and then counted with a Sedgewick-Rafter counting chamber under an inverted microscope. Specific growth rate (m, day1) was calculated from the cell counting results over the exponential phase using the following formula:

m¼

ðln Nt  ln N0 Þ ðt  t 0 Þ

where Nt and N0 are average cell densities at the time of starting point t0 and end point t of the exponential phase, respectively. 2.3. Cellular C and N analyses For analyses of cellular C and N contents, 100 ml of triplicate samples were collected one time prior to nutrient addition (day 13) and three times in each group (days 14, 16, 20). Cell densities were determined using the procedure described in Section 2.2. The algae were filtered on 2.5 cm Whatman GF/C glass–fiber filters precombusted at 450 8C for 3 h. Another filter dipped in the filtrate of each sample was used as blank to reduce the effect of dissolved organic C and N on the analytical results. The filters were dried in an oven at 60 8C for 4 h, and stored at room temperature in a desiccator until analysis. Particulate organic C and N were analyzed with the element analyzer PE240C, using a modified method of Yang and Liu (1995).

2.1. Algal culture conditions 2.4. Chl a analysis Strain ACDH of A. catenella, offered by the CEOHAB project, was used in this experiment. It was germinated from the cyst collected during the cruise in 2002 in the East China Sea. The strain was maintained in the Key Laboratory of Marine Ecology and Environmental Sciences, Qingdao, and cultured in the f/2-Si medium (Guillard and Ryther, 1962) at 20  1 8C, under a light:dark cycle of 14:10. The medium used for algae culture was prepared with

For analysis of Chl a, 100 ml of triplicate samples were collected one time prior to nutrient addition (day 13) and six times after nutrient addition in the nitrate group, the ammonium group and the urea group (days 14, 16, 18, 20, 23, 26). While for the control group, only four times (days 14, 16, 18, 20) were collected, due to the extremely low cell densities at the late stage of experiment in

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this group. Cell densities were determined using the procedure described in Section 2.2. Algae filtered on 2.5 cm Whatwan GF/C filters were stored at 20 8C prior to analysis. The method of Yentsch and Menzel (1963) was followed for extraction of Chl a. Each filter was extracted with 10 ml of 90% acetone, and stored in the darkness for 12–24 h at 20 8C. The samples were then centrifuged at 4000 rpm for 10 min and the Chl a values were measured using a Turner Designs 127TD-700 spectrofluorometer, previously calibrated with standard Chl a solution. The excitation wave length and emission wave length were 440 nm and 685 nm, respectively. 2.5. PSP toxins analyses For analyses of PSP toxins, 200 ml of duplicate samples were collected one time prior to nutrient addition (day 13) and seven times after nutrient addition in the nitrate group, the ammonium group and the urea group (days 14, 16, 18, 20, 23, 26, 29), respectively. While for the control group, only four times (days 14, 16, 18, 20) were collected. Cell densities were determined using the same procedure as described previously. The algae were filtered onto 2.5 cm Whatman GF/C filters under low pressure vacuum. The filter was put into a 7 ml centrifuge tube and stored at 20 8C prior to analysis. For extraction of PSP toxins, 4 ml 0.05 N acetic acid solution was added into each centrifuge tube, and the mixture was sonicated with a probe sonicator (200 W) in an icecold bath for 5 min. The samples were then centrifuged at 10,000 rpm for 10 min. 1 ml supernatant was collected and filtered through a 0.22 mm membrane filter. The filtrate was used for PSP toxin analysis with HPLC. Due to the lack of C toxin standards, the samples were hydrolyzed to convert C toxins into the corresponding GTX toxins, using the following procedure: 68 ml of 1 N HCl was added to 300 ml sample solution, the mixture was kept at 90 8C for 15 min, then, 150 ml 1 M CH3COONa solution was added (Yu et al., 1998). Both hydrolyzed and original samples were analyzed with HPLC using the method of Oshima et al. (1989), with slight modifications. Two mobile phases (flow rate 0.7 ml min1) were used in separation of GTX toxins and STX toxins, respectively. For analyses of GTX toxins, the mobile phase was 1 mM sodium heptanesulfonic acid in 10 mM ammonium phosphate buffer (pH 7.1). For analyses of STX toxins, the mobile phase was 1.5 mM sodium heptanesulfonic acid in 30 mM ammonium phosphate buffer (pH 7.1), containing 5% acetonitrile. Through comparison of GTXs’ concentrations in the hydrolyzed and the original samples, the concentrations of C toxins were calculated. PSP toxin standards, including gonyautoxin 1, 2, 3, 4, 5 (GTX1, 2, 3, 4, 5), decarbomyl gonyautoxin 2, 3 (dcGTX2, 3), saxitoxin (STX), neosaxitoxin (neo), were from the Institute for Marine Bioscience, National Research Council, Canada.

Fig. 1. Growth curves of A. catenella (strain ACDH) in different groups (the arrowhead indicating the time for addition of new nitrogen substrates).

reached the stationary phase in 5 days after inoculation, with the cell densities around 3000  300 cell ml1. To further deplete the N stored in the cells, the cultures were maintained until day 12, when the cell densities began to decrease. The depletion of N was also confirmed by the analysis of nitrate in the medium. Different N substrates, including nitrate, ammonium and urea, were then added to the corresponding group, respectively, to make the final concentration of N in the medium increased to 50 mM N on day 13. The addition of all the three N substrates could promote the growth of N-starved cells of strain ACDH. In the three N-enriched groups, cell densities started to increase 2 days after the addition of nutrients, and reached the maximum in another 3 days. After that, the cell densities kept constant for about 7 days, then decreased till the end of the experiment. The specific growth rates and the maximum cell densities of the three N-enriched groups were listed in Table 1. The nitrate group had the highest values of specific growth rate and maximum cell density, followed by the ammonium group and the urea group. However, the differences among the three groups, either of specific growth rate (ANOVA, p > 0.05) or of maximum cell density (ANOVA, p > 0.05), were not significant. In the control group, the cell density kept decreasing till the end of experiment. It was suggested from these results that A. catenella could utilize all the three N substrates as a sole N source to grow. 3.2. Cellular N and C contents

2.6. Statistical analyses Statistical analyses were performed with SPSS using one-way analysis of variance (ANOVA). The homogeneity of variance was first tested for observations in different treatments, and F test for one-way ANOVA was performed to test the significance of difference among treatments. LSD test (equal variances) was then used to test the significance of difference between two specific treatments, if significant difference was found among treatments. 3. Results 3.1. Cell densities The growth curves of A. catenella were shown in Fig. 1. Due to the reduced concentration of nitrate in the medium, the cultures

The responses of cellular N and C contents to the addition of three different N substrates were shown in Fig. 2. Samples were collected on days 14, 16 and 20, corresponding to early exponential stage, late exponential stage and stationary stage, respectively, after addition of the nutrients.

Table 1 The specific growth rates and maximum cell densities of A. catenella grown on different nitrogen substrates (values are mean  standard error). Nitrogen substrates

Specific growth rate (day1)

Maximum cell density (cell ml1)

Nitrate Urea Ammonium

0.43  0.06 0.30  0.02 0.36  0.06

7300  700 6500  670 7100  850

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the four groups was not significant (ANOVA, p > 0.05). When the cells entered the late exponential stage on day 16, the cellular C contents further decreased and the difference between each Nenriched group and the control became significant (LSD test, p < 0.05). At the stationary stage on day 20, the cellular C contents of the nutrient-enriched groups became comparable to that of the control (ANOVA, p > 0.05). The molar ratio of C and N in the control group was about 18, which was much higher than the Redfield ratio (6.6). After the addition of different N substrates, the molar ratio of C and N decreased significantly to 6.8, 8.8 and 7.7 in the nitrate group, the urea group and the ammonium group, respectively, on day 14. At the stationary stage on day 20, the molar ratio of C and N increased to the value about 14 in the three N-enriched groups, suggesting that the added N substrates were almost depleted. 3.3. Chl a contents The responses of Chl a content to the addition of different N substrates in strain ACDH were shown in Fig. 3. Similar patterns were observed among the three N-enriched groups. There was a remarkable increase of Chl a content on day 14, one day after the addition of nutrients. The Chl a contents continued to increase and peaked on day 16, when the cultures reached the late exponential stage. The maximum Chl a contents of the three N-enriched groups were 7.6, 7.5 and 6.8 pg cell1 in the nitrate group, the ammonium group and the urea group, respectively. From day 16, the Chl a contents kept decreasing till the end of experiment, to a level comparable to the control group. During the whole experiment, difference of Chl a contents among the three N-enriched groups was not significant (ANOVA, p > 0.05). The cellular Chl a content of the control group kept relatively constant, ranging from 3.1 to 3.8 pg cell1. 3.4. Toxin contents and composition Responses of cellular toxin content of strain ACDH to the pulse of different N substrates were shown in Fig. 4. The three N-enriched groups showed a similar pattern in variation of cellular toxin contents. The cellular toxin contents reached the maximum on day 14, one day after the addition of nutrients, and then decreased quickly. However, among the three N-enriched groups, the nitrate

Fig. 2. Effects of different nitrogen substrates on cellular N content and cellular C content of strain ACDH of A. catenella (A: cellular N content; B: cellular C content).

The cellular N contents of the three N-enriched groups increased significantly one day after the addition of nutrients, from 9.0 pmol cell1 to 23.0 pmol cell1, 16.0 pmol cell1 and 19.2 pmol cell1 in the nitrate group, the urea group and the ammonium group, respectively. Similar to the responses in growth characteristics, the nitrate group had the highest value, followed by the ammonium group and the urea group. However, when the algae entered the late exponential stage on day 16, the cellular N contents decreased to a level only slightly higher than the control. The urea group had the highest value, followed by the nitrate group and the ammonium group. At the stationary stage on day 20, cellular N contents of the three N-enriched groups were almost the same as that of the control group. The cellular C contents of A. catenella in the control group on days 14, 16 and 20 were higher than that observed on day 13, due to the enlarged cells of A. catenella formed under nutrient limitation conditions. For the three N-enriched groups, the cellular C contents were a little bit lower than the control group on day 14, one day after the addition of nutrients, but the difference among

Fig. 3. Effects of different nitrogen substrates on cellular Chl a content of strain ACDH of A. catenella.

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Fig. 4. Effects of different nitrogen substrates on cellular toxin content of strain ACDH of A. catenella.

group and the ammonium group showed a remarkable increase in cellular toxin contents on day 14, with the maximum values of 14 fmol cell1 and 11 fmol cell1, respectively. For the urea group, only a slight increase of cellular toxin content was observed. On days 16, 18 and 20, the cellular toxin content of the urea group became much lower than any of the other groups, including the control, but the differences were not statistically significant (ANOVA, p > 0.05), probably due to the discrepant data of the duplicate samples in the control group. At the late stationary stage, the data on cellular toxin content in the control group was not available, due to the extremely low cell densities. For the three N-enriched groups, the data on toxin content became discrepant at the late stationary stage, but it could be seen that the difference on cellular toxin content among the three groups was less evident. C1 and C2 were the most predominant toxins in strain ACDH of A. catenella, although trace amounts of GTX4, GTX5, GTX6, neo, GTX3, GTX1 and STX could also be detected in some samples. No significant difference was found on toxin composition among the different groups during the experiment (Fig. 5). However, in the urea group, a decreased predominance of C1 and C2 could be observed at the late stage of experiment, compared to the other groups. The percentage of C1 and C2 to the total cellular toxin content became less than 80% in the urea group, while the percentages of the other groups were all above 80% during the experiment.

Fig. 5. Effects of different nitrogen substrates on toxin compositions of strain ACDH of A. catenella (A: the control group; B: the nitrate group; C: the urea group; D: the ammonium group).

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4. Discussion This study tried to understand the responses of strain ACDH of A. catenella, a bloom causative species in sea areas adjacent to the estuary of the Changjiang River, to the addition of different N substrates, from the points of growth, cellular C, N and Chl a contents, as well as the toxin production characteristics. It was found that all the three N substrates could be used to promote the growth of strain ACDH. However, the effect of urea was different from nitrate and ammonium in affecting toxin production characteristics. It seems that urea was more readily to be used for the growth of strain ACDH, rather than toxin production.

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or not for strain ACDH to use urea simply based on this experiment, since the natural seawater used was not analyzed for nickel in this experiment. But the results indicated that, even if nickel was necessary for the assimilation of urea, the concentration of nickel in natural seawater was high enough for strain ACDH to grow on urea. The same conditions have been used for the study of A. minutum, and it was found that A. minutum could not grow on urea (Zhang et al., 2005). Therefore, there might be factors other than nickel affecting the capability in utilizing urea among the different species in genus Alexandrium. 4.2. Responses of cellular N, C and Chl a to different nitrogen substrates

4.1. Growth responses of strain ACDH to different nitrogen substrates The highest specific growth rate of ACDH found in this experiment is 0.43 day1 in the nitrate group. This is comparable to the maximum specific growth rate (0.47–0.55 day1) reported previously about a strain of A. catenella isolated from Japan under nutrient sufficient conditions (Matsuda et al., 1999). Ammonium and urea can also promote the growth of strain ACDH, but the specific growth rates are slightly lower than that of the nitrate group. This is probably due to the long-term maintenance of strain ACDH in the laboratory using nitrate as a sole nitrogen source, which makes the algae adaptable for growing with nitrate. Despite the exhaustion of nitrate in the medium prior to the addition of new N substrates, the biochemical mechanism for utilizing nitrate in the cells will not change in a short period of time. Therefore, strain ACDH responds more efficiently in using nitrate to grow compared to urea and ammonium. Ammonium has been considered as a more favorable N source for the growth of dinoflagellates, however, high concentration of ammonium will be toxic to the algae and could inhibit the growth (Zhang et al., 2005). In the current study, ammonium was used at a concentration of 50 mM and showed no negative effect on the growth of strain ACDH. For natural phytoplankton community, urea can represent a significant proportion of total nitrogen source (Solomon et al., 2010). It was found that urea represent 52% of the total nitrogen assimilated for the phytoplankton collected in Great South Bay in a previous study (Kaufman et al., 1983). Urea can be used by many algal species as a sole nitrogen source (Dyhrman and Anderson, 2003). However, capacity of algae in utilizing urea varied among different genera and species, even clones within a species (McCarthy, 1972). Antia et al. (1975) studied 26 species of algae and found that 23 species could use urea as nitrogen source. McCarthy and Eppley (1972) examined 40 algal clones of marine phytoplankton and found over half of the clones could grow on urea. Actually, two scientists tested 5 microalgal species in their experiment and found that all the 5 species could use urea as the sole nitrogen source Berman and Chava (1999). The assimilation of urea in algae depends on the function of either nickel-dependent urease or adenosine triphosphate (ATP) urea amidolyase. It was indicated that most marine phytoplankton, with the exception of chlorophytes, utilized urea through the nickel-dependent urease (Dyhrman and Anderson, 2003). For algal species in genus Alexandrium, many contradictory results were reported on their capability in utilizing urea as nitrogen source. Zhang et al. (2005) showed that Alexandrium minutum isolated from Taiwan could not use urea. Collos et al. (2004) found that A. catenella could grow on urea. But Matsuda et al. (1999) found one isolate of A. catenella from Japan could not use urea as the sole nitrogen source. Dyhrman and Anderson (2003) showed that Alexandrium fundyense was able to grow on urea in the presence of nickel, a critical cofactor for the function of urease. The current study also supported that strain ACDH of A. catenella could use urea as a sole nitrogen source. However, it was not possible to assess whether nickel was critical

Analytical results of cellular N content of strain ACDH in our experiment are consistent with those reported previously on A. catenella by Collos et al. (2004) and Matsuda et al. (1996). Rapid and significant increases of cellular N contents were observed in all the three N-enriched groups one day after the addition of nutrients. The accumulation process can be very fast for nitrate and ammonium, and it has been found that the algae adsorbed enough N in less than 2 h to meet its growth requirements over 24 h (Collos, 1986; Collos et al., 2004). It seems that A. catenella can store high amounts of N before cell division, exhibiting a storage strategy rather than a growth strategy in response to the nutrient pulse. It was found that cellular N content in the urea group was the lowest on day 14, but became the highest on day 16, suggesting a much lower accumulation rate of cellular N compared to nitrate and ammonium. Actually, high cellular N content observed in the urea group on day 14 can also be different from the nitrate group and the ammonium group in nature. In the nitrate group and ammonium group, high cellular N contents reflect increasing assimilation and storage of cellular N. However, for the urea group, high cellular N content might only be an indicator of high amount of urea taken up, rather than assimilated, in the cells. It was indicated previously that the uptake of urea in algae could be distinct from the assimilation process (Antia et al., 1991). Even the algal species unable to grow on urea could uptake it in a similar pattern to those capable of utilizing urea (McCarthy, 1972). Carbon is an essential element for algal growth and reproduction. It was found that the cellular C content correlated well with cell volume in many previous studies on Alexandrium spp. (Leong and Taguchi, 2004). Active cell division of Alexandrium under optimal conditions will produce smaller cells, leading to the decrease of cellular C content. However, when the cell division is limited, the cells become larger, and the cellular C content also increases. In the current study, it was found that the addition of all the three N substrates significantly decreased the cellular C contents in comparison to the N-limited control group, indicating an active growth after the nutrient addition to the N-starved cells. Chl a represents one of the most important N-rich compounds in algal cells. Cellular Chl a content of strain ACDH analyzed in the current study was lower than those analyzed in previous studies (Carlsson et al., 1998; Montecino et al., 2001; Carignan et al., 2002; Navarro et al., 2006), but comparable to the values of Alexandrium tamarense treated with similar concentration of nitrogen (Leong and Taguchi, 2004). The addition of three N substrates to the Nstarved cells led to a rapid increase of cellular Chl a content in strain ACDH, suggesting that all the three N substrates could be utilized efficiently for the algal growth. 4.3. Responses of toxin production to different nitrogen substrates Total cellular toxin content analyzed in the current study in strain ACDH was between 4.3 and 14.0 fmol cell1. The values were lower than those reported previously for the same species

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isolated from other regions (Siu et al., 1997; Sekiguchi et al., 2001; Yoshida et al., 2001; Se´chet et al., 2003; MacKenzie et al., 2004; Kim et al., 2005; Navarro et al., 2006), but a little higher than that analyzed of the same isolate by Wu et al. (2005). The variation of cellular toxin content among different geographical isolates of the same species was often reported. High values of cellular toxin content observed in the current study were only found in the samples collected shortly after the addition of N substrates, particularly in the nitrate and ammonium groups. The addition of all the three N substrates to N-starved cells of strain ACDH could affect cellular toxin content, but the responses of cellular toxin content to the three N substrates were different. The addition of both nitrate and ammonium led to quick increases of cellular toxin contents 1 day after the addition of nutrients. This was consistent with many previous results, which indicated that elevated toxin contents could be caused by the enrichment of nitrate or ammonium to the nitrogen-starved cells of toxic Alexandrium (Anderson et al., 1990; MacIntyre et al., 1997; Leong et al., 2004). After 3 days, the cellular toxin contents rapidly decreased to a level comparable to the N-limited control group. The decrease in cellular toxin content correlated well with cellular N content, suggesting that the toxin content has a direct relationship with cellular N content. Both Anderson et al. (1990) and Flynn et al. (1994) have suggested that PSP toxin production of Alexandrium spp. related to cellular N content when nitrate or ammonium was used as nitrogen source. In contrast to nitrate and ammonium, the addition of urea led to a slight increase in cellular toxin content 1 day after the addition of nutrient. After 3 days, the cellular toxin content decreased dramatically, and the toxin level was much lower than the Nlimited control group. Similar phenomenon has been found in previous studies, which showed that the cellular toxin contents of A. fundyense and A. catenella grown on urea was much lower than the toxin content of nitrate-starved cells (Dyhrman and Anderson, 2003; Leong et al., 2004). This different response of strain ACDH to the addition of three N substrates was probably due to the different metabolism processes of nitrate, ammonium and urea. Both nitrate and ammonium can be actively absorbed by algae. Nitrate adsorbed can be further reduced to ammonium in the cell through the function of nitrate reductase. Therefore, the active adsorption of either nitrate or ammonium by N-starved cells will lead to the accumulation of ammonium in the cells, supplying sufficient ammonium not only for the synthesis of protein and chlorophyll prerequisite for algal growth, but also for PSP toxin production. This will lead to the elevated cellular toxin content before cell division, as observed in the current experiment 1 day after the addition of either nitrate or ammonium. It will compensate the ‘‘dilution’’ effect caused by cell division, and make the cellular toxin contents in nitrate and ammonium groups comparable to those in the control group in the following days. The metabolism of urea, however, was different from nitrate and ammonium. The metabolism of urea in algae was reviewed in detail by Antia et al. (1991), although still little information is available on urea metabolism in dinoflagellates. It was found that most of the urea taken up into the algal cells, either through active uptake or passive diffusion, was in the form of free urea (Rees and Syrett, 1979). Based on the model for urea uptake and assimilation in Thalassiosira pseudonana proposed by Price and Harrison (1988), there was a rapid influx of urea at first, followed by a much reduced influx at the late stage. Intracellular metabolism of urea produced ammonia/ammonium, which was excreted out of the cells. The excreted ammonia/ammonium could be re-adsorbed in the form of ammonium together with urea. However, the ammonium was preferentially absorbed, while the uptake of urea was inhibited by the existence of ammonium. Urea uptake increased only when the

ammonium in the medium decreased to a certain level. This process would slow down the uptake rate of urea, making it much slower compared with nitrate and ammonium. It could be reasonably presumed that this gradual supply of ammonium converted from the hydrolysis of urea would be preferentially used for growth of algae rather than synthesis of toxins, since PSP toxins were secondary metabolites not necessary for algal growth. Therefore, only a slight increase in cellular toxin content was observed in the current experiment, shortly after the addition of urea. After cell division, the increased toxin content could not compensate the ‘‘dilute’’ effect caused by active cell division, leading to lower cellular toxin content compared to the N-limited cells in the control group. Dyhrman and Anderson (2003) have indicated, based on their findings, that field population of A. fundyense or A. catenella using urea as a nitrogen source in low nitrate environment could be less toxic. Our results also support this finding. Only slight variation of toxin composition among the three nitrogen substrates was found during the experiment. Many previous studies found that toxin composition of toxic Alexandrium spp. was relatively stable and independent of culture conditions (Boyer et al., 1987; Hall, 1982; Flynn et al., 1994; Parkhill and Cembella, 1999). Therefore, toxin composition could serve as a population-specific marker. However, some studies showed that the toxin composition could also be affected by culture conditions, especially nutrient status (Flynn et al., 1994; Hamasaki et al., 2001), although not as significant as cellular toxin content. In the current study, only a slight decrease of predominance of C1,2 in the urea group was found. A significant decrease of the percentage of C1,2 has been observed in urea-grown A. catenella by Dyhrman and Anderson (2003), and it was indicated that the decreased predominance of C1,2 was related to the effect of urea, or the nickel added to their culture medium. Nickel could inhibit the activity of N-sulfotransferase enzyme, which catalyze the sulfation reaction of GTX2.3 to C1,2. In the current study, no nickel was added to the culture medium. The decreasing predominance was probably caused by the addition of urea. 4.4. Implication for dynamics and potential impacts of A. catenella blooms in the East China Sea The estuary of the Changjiang River and adjacent coastal waters is among the most serious eutrophicated sea areas along the Chinese coast, and the Changjiang River contributes significantly to the eutrophication process in this region. Based on an investigation in 1998 (Shen, 2004), the total N influx from the Changjiang River into the sea is estimated to be 2.84 million metric tons, and nitrate is the major form of N from the Changjiang River, accounting half of the total N flux into the sea. Therefore, nitrate should be the major factor affecting the dynamics and potential impacts of A. catenella blooms, compared to other forms of nitrogen in seawater. The increasing nitrate flux from the Changjiang River over the last 40 years significantly contributes to the increasing scale and intensity of dinoflagellate blooms in the sea. Although studies indicated that large amount of organic nitrogen was also brought into the sea by the Changjiang River, there was little information available on the composition of organic nitrogen in seawater. Data on urea in this sea area are also very limited. Due to the high concentration of nitrate in seawater, effects of urea on dynamics and potential impacts of A. catenella blooms should be less visible. Research works are still needed to study the potential effects of urea on dinoflagellate blooms in this area. 5. Conclusion In the present study, we showed that the strain ACDH of A. catenella, which was isolated from the East China Sea, could use

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nitrate, ammonium and urea for growth. However, the effects of three nitrogen substrates on toxin production were different. The nutrient pulse of both nitrate and ammonium led to dramatic increases of cellular PSP toxin contents, however, only a slight increase was observed shortly after the addition of urea. The longterm utilization of urea would significantly decrease the cellular toxin content. It seems that urea is more readily to be used by strain ACDH for the growth, rather than toxin production. It is believed that the urea in natural seawater can promote the formation of ACDH blooms, but decrease the cellular toxin content of this strain. Acknowledgements The study was supported by the National High Technology Research and Development Program of China (2006AA09Z178), the National Basic Research Program (973 program) of China (2010CB428705), and the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (no. 40821004). The strain ACDH of Alexandrium catenella used in this study was offered by the CEOHAB project. We thank Mr. Liu Qun and Yu Lidong for their assistance in analysis of cellular contents of N, C and PSP toxins.[SS] References Anderson, D.M., Glibert, P.M., Burkholder, J.M., 2002. Harmful algal blooms and eutrophication: nutrient sources, composition, and consequences. Estuaries 25, 704–726. Anderson, D.M., Kulis, D.M., Sullivan, J.J., Hall, S., Lee, C., 1990. Dynamics and physiology of saxitoxin production by the dinoflagellates Alexandrium spp. Mar. Biol. 104, 511–524. Anderson, D.M., McGillicuddy Jr., D.J., Townsend, D.W., Turner, J.T., 2005. Preface. Deep-Sea Res. II 52, 2365–2368. Antia, N.J., Berland, B.R., Bonin, D.J., Maestrini, S.Y., 1975. Comparative evaluation of certain organic and inorganic sources of nitrogen for phototrophic growth of marine microalgae. J. Mar. Biol. Assoc. U.K. 55, 519–539. Antia, N.J., Harrison, P.J., Oliveira, L., 1991. The role of dissolved organic nitrogen in phytoplankton nutrition, cell biology and ecology. Phycologia 30, 1–89. Bechemin, C., Grzebyk, D., Hachame, F., Hummert, C., Maestrini, S.Y., 1999. Effect of different nitrogen/phosphorous nutrient ratios on the toxin content in Alexandrium minutum. Aquat. Microb. Ecol. 20, 157–165. Berg, G.M., Glibert, P.M., Lomas, M.L., Burford, M., 1997. Organic nitrogen uptake and growth by the Chrysophyte Aureococcus anophagefferens during a brown tide event. Mar. Biol. 129, 377–387. Berman, T., Chava, S., 1999. Algal growth on organic compounds as nitrogen sources. J. Plank. Res. 21, 1423–1437. Boyer, G.L., Sullivan, J.J., Anderson, R.J., Harrison, P.J., Taylor, F.J.R., 1987. Effects of nutrient limitation on toxin production and composition in the marine dinoflagellate Protogonyaulax tamarensis. Mar. Biol. 96, 123–128. Carignan, M.O., Montoya, N.G., Carreto, J.I., 2002. Long-term effects of ultraviolet radiation on the composition of pigment and mycosporine-like amino acids (MAAs) composition in Alexandrium catenella. In: Arzul, G. (Ed.), Aquaculture, Environment and Marine Phytoplankton. Proc. Ifremer Symposium, Brest, 21– 23 May 2001, no. 34, pp. 191–207. Carlsson, P., Edling, H., Be´chemin, C., 1998. Interactions between a marine dinoflagellate (Alexandrium catenella) and a bacterial community utilizing riverine humic substances. Aquat. Microb. Ecol. 16, 65–80. Collos, Y., 1986. Time-lag algal growth dynamics: biological constraints on primary production in aquatic environments. Mar. Ecol. Prog. Ser. 33, 193–206. Collos, Y., Gagne, C., Laabir, M., Vaquer, A., 2004. Nitrogenous nutrition of Alexandrium catenella (dinophyceae) in cultures and in Thau Lagoon, Southern France. J. Phycol. 40, 96–103. Dyhrman, S.T., Anderson, D.M., 2003. Urease activity in cultures and field populations of the toxic dinoflagellate Alexandrium. Limnol. Oceanogr. 48, 647–655. Eppley, R.W., Peterson, B.J., 1979. Particulate organic matter flux and planktonic new production in the deep ocean. Nature 282, 677–680. Flynn, K., Franco, J.M., Fernandez, P., Reguera, B., Zapata, M., Wood, G., Flynn, K.J., 1994. Changes in toxin content, biomass and pigments of the dinoflagellate Alexandrium minutum during nitrogen refeeding and growth into nitrogen and phosphorus stress. Mar. Ecol. Prog. Ser. 111, 99–109. Glibert, P.M., Harrison, J., Heil, C., Seitzinger, S., 2006. Escalating worldwide use of urea – a global change contributing to coastal eutrophication. Biogeochemistry 77, 441–446. Glibert, P.M., Magnien, R., Lomas, M.W., Alexander, J., Fan, C., Haramoto, E., Trace, M., Kana, T.M., 2001. Harmful algal blooms in the Chesapeake and coastal bays of Maryland, USA: comparison of 1997, 1998 and events. Estuaries 24, 875–883.

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