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Effects of different nitrate and phosphate concentrations on the growth and toxin production of an Alexandrium tamarense strain collected from Drake Passage
Marine Environmental Research 81 (2012) 62e69
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Effects of different nitrate and phosphate concentrations on the growth and toxin production of an Alexandrium tamarense strain collected from Drake Passage Thomas Chun-Hung Lee 1, Oi-Ting Kwok 1, Kin-Chung Ho, Fred Wang-Fat Lee* School of Science and Technology, The Open University of Hong Kong, Hong Kong, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 April 2012 Received in revised form 24 August 2012 Accepted 27 August 2012
Nitrate (N) and phosphate (P) are believed to be two of the most important nutrients for the growth and toxin production for Alexandrium species. The study of the growth and toxicity characteristics of the Alexandrium spp. under the change of N and P can help us to understand the dynamics of algal bloom and toxification events in natural environments. A strain of Alexandrium tamarense (designated as Kci) was successfully isolated from the Drake Passage in 2001 and the clonal culture has been kept in our laboratory (Ho et al., 2003, 2012). In order to extend our understanding on the growth physiology and toxicity of this A. tamarense strain, growth and cellular toxin content were examined in unialgal batch cultures under different concentrations of N and P. The effects of variable N, P concentrations on growth, cellular toxicity (fg STXeq. cell1), and toxin composition (% molar) were determined in both exponential and stationary growth phases. The toxin profile, determined by high-performance liquid chromatography with fluorescence detection (HPLC-FD), was found to be remained relatively stable and was consistently dominated by the N-sulfocarbamoyl C-toxins (>90%) under different conditions and growth phases. There were also trace amounts of other carbamate gonyautoxins consistently expressed. The cellular toxicity varied under different N and P concentrations, as well as different growth stages. A positive correlation was observed between cellular toxicity and N concentrations, but the toxicity was enhanced when P was depleted. Both cell densities and growth rate of the cells were severely suppressed under Nor P-depletion. However, the biovolume of the cells tended to be larger at N- or P-depleted cultures. Results from the present study provide valuable insight for the ecophysiology of Alexandrium species in the coastal ecosystem of Drake Passage. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Alexandrium Drake Passage Growth Nitrogen Phosphate Toxicity
1. Introduction Dinoflagellate genus Alexandrium is a well-known harmful algal bloom (HAB) causative agent and paralytic shellfish poisoning (PSP) toxins producer in many coastal regions around the world (Anderson et al., 2012; Hallegraeff, 1993, 2003). There are more than 30 morphologically defined species in this genus and more than half of them are known to be toxic (Anderson et al., 2012). Contamination of shellfishes and fishes with such PSP neurotoxins would negatively affect the shellfish and aquaculture industries (Anderson et al., 2012; Hallegraeff, 1993). In addition, it would cause human illness or even death after consumption of shellfishes or fishes that have accumulated the toxins through their diet. One of the first recorded fatal cases of human poisoning after the
* Corresponding author. Tel.: þ852 2768 6868; fax: þ852 27891170. E-mail address: wfl[email protected] (F.W.-F. Lee). 1 Authors with equal contributions. 0141-1136/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.marenvres.2012.08.009
consumption of shellfishes contaminated with the neurotoxins was in 1793 (Hallegraeff, 2003). Such causative alkaloid PSP toxins are so potent that even a very small quantity (about 500 mg) can be fatal to human. On a global scale, nearly 2000 cases of human poisoning with nearly 15% mortality through fishes or shellfishes consumption are reported each year (Hallegraeff, 2003). The increased distributions of Alexandrium spp. have become a global problem with the increase of algal blooms in frequency, intensity, and geographic distribution (Anderson et al., 2012; Hallegraeff, 1993). Although a strong relationship between frequencies of algal blooming and the nutrient load of coastal waters have been suggested (Paerl, 1997; Smayda, 1990), the algal blooming mechanism for Alexandrium spp. is complex and far from understood (Anderson et al., 2012). In addition, it is difficult to generalize the relationship between Alexandrium blooms and nutrition, because Alexandrium spp. has been reported to be able to grow in a wide spectrum of nutrient availability conditions (Anderson et al., 2012). However, nutrient loadings could lead to dramatic changes in the N, P concentrations as well as the N:P supply ratios. Many studies have
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been carried out to investigate the effects of the N to P supply ratios on the growth rate, cell density, cell size, and/or toxin composition/ production in Alexandrium spp., e.g. (Frangopulos et al., 2004; Hwang and Lu, 2000; Leong and Taguchi, 2004; Lim et al., 2010; Touzet et al., 2007; Wang and Hsieh, 2002; Xu et al., 2012). In general, the cellular toxicity of Alexandrium spp. increased with increasing N:P supply ratios (Murata et al., 2006), indicating that cellular toxicity might be stimulated by relative P-deficiency. In addition, N-limitation has been reported to decrease both growth and toxin production of Alexandrium spp. (Anderson et al., 1990b; Flynn et al., 1994; Wang and Hsieh, 2002, 2005). On the other hand, P-limitation has generally been reported to decrease growth of Alexandrium spp. but increase in cellular toxicity (Anderson et al., 1990b; Boyer et al., 1987; Flynn et al., 1994; John and Flynn, 2002; Maestrini et al., 2000; Wang and Hsieh, 2002). However, some investigations have shown different results, for example John and Flynn (2000) showed that cellular toxicity of Alexandrium fundyense would only be affected if N is colimiting with phosphorus limitation (John and Flynn, 2000). Therefore, the toxicity of Alexandrium tamarense could be affected by both N and P concentrations and expected to vary with the N:P supply ratios. It is important to understand how PSP toxins are produced by different geographical prevalent Alexandrium spp. under various environmental factors. A toxic A. tamarense strain (designated as Kci) was isolated from Drake Passage in 2001 (Ho et al., 2003). This was the first A. tamarense found in such unusual regions and might be a potential key species leading to the massive death of penguins in Falkland Islands in December 2002 to January 2003 (Ho et al., 2003). To increase our understandings on the ecophysiology of Alexandrium species in the coastal ecosystem of Drake Passage, the present study was undertaken to investigate the growth and toxinproducing physiology of this strain under the effects of different nitrate and phosphate concentrations. 2. Materials and methods 2.1. Culture conditions Non-axenic A. tamarense was isolated from Drake Passage (sampling site 20, in waters near the southern end of South America) of Southern Ocean in 2001 (Ho et al., 2003). Individual cells from the field samples collected were picked under the microscope before being cultured in L1 seawater based medium (Keller et al., 1987). A monoculture (designated as Kci) was successfully established and kept in the Environmental Laboratory of The Open University of Hong Kong. Culture was kept at exponential growth phase by transferring to new medium every week in a ratio of 1:10 (v/v). Vegetative cells from cultures in mid- or lateexponential phases of growth were inoculated into freshly prepared culture medium. Possible bacterial contamination of the culture was checked by regular microscopic examination. To allow comparison of the results obtained from the present study to others, the cultures were maintained at optimal growing conditions: 20 C under a 12-h light:12-h dark cycle at a light intensity of 120 mE m2 s1 provided by cool white fluorescent tubes in a Conviron growth chamber. The conditions were optimal for algal growth of this strain. 2.2. Cell counts and growth curve For the growth curve determination, roughly 105 cells were harvested from mid-log phase culture by centrifugation (1500 g for 10 min at room temperature), washed twice with sterile seawater and inoculated into L1-medium to yield an initial cell density of 1000 cells ml1. Cell density was measured at the same
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time each day in a 1 mL aliquot removed and fixed with 10 ml Lugol’s solution. Cells were counted under a light microscope using a Sedgwick-Rafter cell counter. The whole experiment was repeated three times, with triplicates in each run. The specific growth rate (m) for the exponential growth phase was calculated using the following equation:
m ¼
ln N1 ln N0 t1 t0
where N0 and N1 are the cell density reading at time t0 and t1. The effects of nitrate and phosphate concentrations on growth and toxin production of Kci were investigated. The cells were cultured in the same conditions as described above, except the L1medium was altered by adding different amount of nitrate (0, 264 and 880 mM) and phosphate (0, 18, 36 mM) in the nitrate- and phosphate-experiments respectively. No effort was made to remove residual amount of N and P from the natural seawater used. Nitrate was added aseptically to yield the following concentration gradient: 0, 264 and 880 mM, in the nitrate experiment. In the P experiment, concentration gradient was set to 0, 18, 36 mM. 2.3. Determination of cell diameter and cell volume Each cell culture sample was fixed in Lugol’s fixation solution. Measurement was performed using a Nikon eclipse TS100 inverted microscope with Nikon digital sight under 400 magnifications. Samples for measurements were taken from day 10 cultures. Measurement was calibrated with micrometer. The mean cell diameter and volume were calculated from a total of at least 80 measurements randomly from the sample. Mean cell volume (mm3) was calculated with the assumption of spherical shape of cells using the following equation (Hillebrand et al., 1999):
v ¼
p 6
d3
where d is the diameter of cells. 2.4. Extraction and analysis of toxins Five hundred milliliters of cells were collected during mid-log and stationary phase. The cells were centrifuged at 3600 rpm for 5 min. The supernatant was discarded and the cell pellet was stored in 80 C until toxin extraction. 0.3 mL of 0.05 M acetic acid was added to each sample and the sample was then homogenized on ice for 10 min. The breakage of cells was confirmed by microscopic examination. The toxin extract was made up to 1 mL and centrifuged at 13,000 rpm for 3 min. Toxin analysis was performed mainly according to Oshima’s HPLC Post-column derivatization method by Waters H-class UPLC system coupled with Waters Post Column Reaction Module (Oshima, 1995). Toxin derivatives were separated by Waters Nova Pak C18 HPLC column (75 mm 3.9 mm i.d.). The mobile phase used to identify the GTX toxins group was constituted by 1.5 mM sodium octanesulfonate (OSA) in 10 mM ammonium phosphate (pH 7.0 with NH4OH). The mobile phase used to identify the STX toxins group and NeoSTX toxins group was made by 0.5 mM OSA in 10 mM ammonium phosphate (pH 7.2 with NH4OH). The mobile phase to identify the dcGTX2, 3 toxins group was constituted by 2.0 mM sodium heptanesulfonate (HSA) in 10 mM ammonium phosphate (pH 7.1 with NH4OH). The mobile phase to identify the C toxins group was constituted by 2.0 mM tetrabutylammonium dihydrogenphosphate in 10 mM ammonium phosphate (pH 6.5 with NH4OH). The flow rate was set at 0.5 mL/ min while the column temperature was adjusted to 40 C. A
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solution of 7 mM periodic acid and 50 mM sodium phosphate (pH 9.0 with 1 N KOH) was introduced to the column and eluted in a biocompatible mixing Tee at a flow rate of 0.15 mL/min controlled by a Waters Reagent Manager. The resulting mixture was heated to 75 C by Waters Temperature Control Module II while passing through a teflon reaction coil. The reaction mixture was then acidified in another mixing Tee with 0.5 M acetic acid (flow rate 0.15 mL/min and in ambient environment). The fluorescent eluted derivatives were monitored at 330 and 390 nm excitation-emission wavelengths, respectively.
3. Results and discussion 3.1. Growth, toxicity, and the toxin profile of the strain The Alexandrium genus has been known to produce potent neurotoxins which cause paralytic shellfish poisoning (PSP). It is a very common HAB causative agent and is cosmopolitan species that widely spread throughout many regions of the world (Hallegraeff, 2003). However, report of Alexandrium cells found in waters of regions in Drake Passage is greatly limited. Until 2001, a toxic A. tamarense was found in various regions in the Drake Passage (Site 1 to 23, from Punta Arenas to the South Shetland Islands bypassing the southern waters of the Falkland Islands) (Ho et al., 2003). A strain of A. tamarense was identified and isolated from live samples collected in waters of the Drake Passage (site 20) of the Southern Ocean in a research cruise by Ho et al. (2003). The research team found that the cells would survive at seawater temperatures ranging from 4.85 C to 8.13 C (measured in situ) during the research cruise, which shows the persistence of this Alexandrium spp. in tolerating temperature variations (Ho et al., 2003). Monoculture of the strain, designated as Kci, has been successfully established and kept in the Environmental Laboratory of The Open University of Hong Kong (Fig. 1). The strain was subjected to the growth and toxicity analysis in the present study. Results of the growth curve (in cells ml1), cellular toxicity (in fg STXeq. cell1) and toxin composition (in mol %) in an optimal growing condition are shown in Fig. 2. The growth pattern of cells grown in batch cultures gave a typical growth curve with three different distinctive growth phases. As indicated in the growth curve, inoculated cells require 1 day for adaptation (lag phase) prior entering the log phase. The cells grew exponentially between day 2 and day 12, with specific growth rate (m) of 0.38 (Fig. 2a). Cells reached a maximum cell capacity of around 22,000 cells ml1 on day 14. Cell size measurement showed that the
average cell diameter and volume of the A. tamarense strain Kci were 28.86 1.12 mm and 12,635 1430 mm3 respectively (Table 1). In general, previous studies reported that the toxicity of the cells in the exponential growth phase was shown to be higher than that of the stationary phase (Anderson et al., 1994, 1990b; Boyer et al., 1987; Cembella et al., 1987; Flynn et al., 1994; Hamasaki et al., 2001; Lim and Ogata, 2005; Parkhill and Cembella, 1999; Wang and Hsieh, 2002, 2005). Our results presented a similar pattern whereas the toxicity was found to be higher at the exponential growth phase than that at the stationary growth phase. Nevertheless, the toxin compositions of this strain grow in exponentialand stationary-growth phase were highly similar to each other. Only less than 1% of dc-GTX2 toxin was decreased when cells entered the stationary phase. This toxic strain produced unusually high proportions of the low potency N-sulfocarbamoyl C-toxins (>94%, with ratio of C2 to C1 toxin is around 2:1). Only trace amounts of GTX and dc-GTX toxins were detected (<6%, including dcGTX-2, dcGTX-3, GTX 2, GTX 3 and GTX 5) (Fig. 2b), while other PSP toxins were not detected. Among these trace amounts of carbamate toxins, dcGTX-2 was dominant. Although toxicity studies of Alexandrium spp. from regions of Drake Passage/ Southern Ocean is greatly limited, a number of studies have been carried out on the Alexandrium spp. from the regions of Argentine Sea, which is closely affiliated to the site of cells collection for the present study (Carreto et al., 2001, 1996; Esteves et al., 1992; Gayoso, 2001; Montoya et al., 2010). Their results showed that, under nutrient replete conditions, the C1 and C2 toxins were predominant in almost all strains analyzed. For instance, Montoya et al. (2010) have analyzed 10 cultured A. tamarense strains from the Argentine Sea (Montoya et al., 2010). The cultures were harvested for toxin analysis during the mid-log growth phase. Among the 10 strains isolated from Argentine, cell toxicity ranged from 1.81 to 10.3 pg STX equiv. cell1 (Montoya et al., 2010). Comparatively, the Drake Passage strain Kci was several times less toxic (w0.6 pg STX equiv. cell1) to those observed in their studied strains. This was due in part, to the strain Kci principally exhibited a relatively high content of the low potency N-sulfocarbamoyl C-toxins (w94%). On the other hand, the observed differences in toxicity among regions may be attributed to differential geographic distribution of the subpopulations of A. tamarense cells. In addition, other factors such as environmental and growing parameters (Etheridge and Roesler, 2005), culture-associated bacteria (Uribe and Espejo, 2003) and long term routine cell culturing maintenance (Cho et al., 2008; Wang et al., 2005, 2006), may also potentially leading to the discrepancies observed. Although most of the Argentine strains were also predominated with C-toxins (Carreto
Fig. 1. (a) Light microscopic and (b) scanning electronic microscopic (SEM) photo of Drake Passage A. tamarense strain Kci.
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Fig. 2. The (a) growth and toxicity, and (b) toxin composition of the cells grown in normal growing condition.
et al., 2001, 1996; Esteves et al., 1992; Gayoso, 2001; Montoya et al., 2010), substantial amount of other PSP toxin derivatives, including STX, neoSTX and GTX 1e4, were also detected. In contrast, no STX/ neoSTX and only trace amounts of GTX toxins were detected in the strain Kci. Interestingly, the toxin profile of strain Kci is highly similar to A. tamarense cells isolated from Hong Kong waters reported previously (Xu et al., 2012). The Hong Kong strains also predominately produce C-toxins with only trace amount of dc-GTX2,3, and
Table 1 Variations of cell size of the A. tamarense Kci at different concentrations of nitrate and phosphate. Concentrations Nitrate (mm)
Phosphate (mm)
880 880 264 880 0
36 18 36 0 36
Cell diameter (mm) S.D. 28.86 28.78 28.61 29.86 36.25
1.12 1.46 1.60 1.84 1.30
Cell volume (mm) S.D.
12,635.02 12,578.21 12,370.94 14,092.83 25,037.53
1430.69 1893.66 2131.83 2611.20 2686.62
Remarks: Samples for measurements were taken from day 10 cultures.
the toxin composition remained constant for all of the growing conditions that were tested (Xu et al., 2012). Several studies have suggested that the toxin profiles are conservative characteristic of various strains and could be used as specific marker for species identification (Anderson et al., 1994; Boyer et al., 1987; Flynn et al., 1994). However, this issue is still in debate because some other studies found that the toxin composition of Alexandrium spp. often varies with different growing conditions (Anderson et al., 1990a; Hamasaki et al., 2001). The toxin profile which dominated with C-toxins (>90%) of the present strain is very different from the toxin profiles of A. tamarense from many other geographical locations. This is not surprising, because toxin composition has been shown to vary widely among different A. tamarense strains (Anderson et al., 1994; Cembella et al., 1987). However, further studies on more Alexandrium spp. from the same regions of Drake Passage are required to confirm the uniqueness of its toxicological characteristics. 3.2. Effects of nitrate The availability of N source is one of the most important factors governing the growth and biosynthesis of PSP toxins in
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Alexandrium spp. It is believed to be a critical factor for toxin production because the PSP toxins are N-rich compounds (around 5e10% of the total cellular N in the cell) (MacIntyre et al., 1997). The effects of nitrate at the initial phosphate concentration of 36 mM on the cell growth and toxin production were investigated (Fig. 3). Only very little growth was observed in the cultures without the addition of N source (cell density <3000 cells ml1) (Fig. 3a). The growth was increased by the increase of nitrate concentration. Maximum cell density of 16,000 cells ml1 was attained when culture started with 264 mM nitrate. Further increase in nitrate to 880 mM would result in an additional increase in growth, whereas a maximum cell density of about 23,000 cells ml1 was achieved. Cell sizes in both nitrate supplied conditions (i.e. at 264 mM and 880 mM) were very similar but bigger cells were observed in nitrate depleted cultures (Table 1). The mechanism of the increase of the cell size under nutrient limitations was not well understood. However, cell size of A. tamarense was showed to be dependent on the type and concentration of N source supply previously (Leong and Taguchi, 2004) and cell size of several Alexandrium spp. was shown to be increased by the increase of N:P ratios as well (Lim et al., 2010). Some authors also suggested that the increase in cell biovolume might be attributed to the arrest of cells in G1 phase of the cell cycle (John and Flynn, 2000; TaroncherOldenburg et al., 1997; Vaulot et al., 1996). Although no effect of nitrate on cellular toxicity was observed in some studies (Hall, 1982; Parkhill and Cembella, 1999), in general, it is well documented that N deficiency decreased toxin production in Alexandrium cells (Anderson
et al., 1990b; Flynn et al., 1994; John and Flynn, 2000; Wang and Hsieh, 2002). Our results were consistent with these previous findings that the toxicity of the cells increased with the increase in nitrate concentrations (Fig. 3b). The maximum toxicity at the nitrate concentration of 880 mM was around 1.5-fold and 3-fold higher than the toxicity at the nitrate concentrations of 264 mM and 0 mM respectively. However, only slightly increase of the cellular toxicity was observed in stationary phase with the increase in nitrate concentrations from 264 mM to 880 mM. Toxin composition in cells remained relatively constant with the change of nitrate concentrations, with C-toxins as the dominant toxins in all cases (Fig. 3c). However, the ratio of C2:C1 toxins was slightly increased in the N-depleted culture. This is not surprised that sufficient supply of N appeared to be particularly important for growth and high toxin production for Alexandrium strain. This is because both proteins/enzymes and PSP toxins compose high molecular contents of N. In addition, photosynthetic efficiency would be greatly limited during N deprivation, which in turn affects the cell growth. Therefore, it is reasonable to believe that intracellular pools of N would be allocated to the maintenance of basic and essential cellular functions, and the activation of N demanding metabolic pathways (Touzet et al., 2007). For example, two abundant plastid proteins, rubisco II and NAP50, were found to be degraded in the Alexandrium spp. during N-depletion (Lee et al., 2009). The redirection of the metabolic pathway for cell survival requires synthesis of new proteins and enzymes, thereby making insufficient levels of N for the toxin synthesis.
Fig. 3. The (a) growth, (b) toxicity, and (c) toxin composition of the cells grown in different nitrate concentrations.
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3.3. Effects of phosphate Similar to the results of the effects of nitrate on the growth of the strain, the growth of the cells increased with the increase in starting phosphate concentration (Fig. 4a). Only very little growth was observed when there was no phosphate added to the culture (<4000 cells ml1). The maximum cell densities were similar (w22,000 cells ml1) at the phosphate concentration of 18 mM and 36 mM. The cell biovolume at the phosphate concentration of 18 mM and 36 mM was very similar, but the cells became slightly larger in cultures without the addition of phosphate (Table 1). Similar results were also observed in some other studies, e.g. (Lim et al., 2010; Touzet et al., 2007). Some researchers suggested that the increase of cell biovolume under P limitations could possibility due to the synthesis of other non-P compounds when the cells stopped to undergo cell division (John and Flynn, 2000; Vaulot et al., 1996). The toxicity appeared to be stimulated by low phosphate concentration. The highest toxicity was found in the cells grown under phosphate depletion (Fig. 4b). The relatively lower toxicity observed at the phosphate concentrations of 18 mM and 36 mM was very close to each other, indicating that phosphate was not really inhibitory to the toxin production. Enhancement of the PSP toxin production by cells grown in phosphate deficiency conditions has been well documented in the toxin-producing physiology studies for Alexandrium spp. conducted by others (Anderson et al., 1990b; Bechemin et al., 1999; Boyer et al., 1987; Flynn et al., 1994; John and
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Flynn, 2000; Wang and Hsieh, 2001, 2002, 2005; Xu et al., 2012). The PSP toxin molecules do not contain phosphorus; mechanism by which phosphate regulates toxin production is not well understood at the moment. However, some researchers suggested that phosphate deficiency might suppress the cell division and possibility leading to an increase in the availability of intracellular arginine, therefore allowing the cells to utilize more available arginine which is a presumed precursor in PSP toxin production (Anderson et al., 1990b; Shimizu et al., 1984). On the other hand, several researches reported that the toxin composition in Alexandrium spp. varied with growth phases and culturing conditions (Anderson et al., 1990b; Boyer et al., 1987; Flynn et al., 1994; Hamasaki et al., 2001). For example, Anderson et al. (1990a,b) showed that A. fundyense predominately produced C1, 2 and GTX 1, 4 toxins when the cells grown in nitrogen limitation. However, the toxin composition was predominately replaced by the production of GTX 2, 3 toxins when the cells grown in phosphorus limitation. Our data on the A. tamarense strain Kci indicate that toxin composition does not vary significantly with growth phases (Fig. 2c), influence of nitrate (Fig. 3c) as well as phosphate (Fig. 4c). In all cases, C-toxins remained as the dominate toxin (>90%). Similar to the results observed in N-depletion, only slightly increase in the ratio of C2:C1 toxins (w1e2%) was observed at low phosphate conditions (Fig. 4c). The results were in agreement with numerous studies found in the literature. Other studies indicated that toxin composition is constant and independent of various culture conditions
Fig. 4. The (a) growth, (b) toxicity, and (c) toxin composition of the cells grown in different phosphate concentrations.
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(Anderson et al., 1994; Boyer et al., 1987; Cembella et al., 1987; Flynn et al., 1994; Franco et al., 1994; Hall, 1982; Ogata et al., 1987). However, toxin analysis on more A. tamarense isolates in the same regions should be done before such toxin profile can be conclusively used as the “biochemical marker” to represent the A. tamarense in the Drake Passage. 4. Conclusion Although effects of N and P concentrations on growth and toxin production of various Alexandrium spp. have been extensively studied, relevant study of A. tamarense in the coastal waters of the regions of Drake Passage is greatly limited. The present study reported the growth and toxicity of A. tamarense isolated from Drake Passage under the effects of various N and P concentrations. Our results were consistent with the observations reported by many other authors that deficiency of nitrate decreases both cell growth and toxin production, while deficiency of phosphate decreases cell growth but enhances the production of toxin. However, toxin composition of the cells remains relatively stable in different growth phases and culturing conditions, with C-toxins (>90%) as the dominated toxins. This reaffirms that the stability of toxin composition is greatly species/strain dependant. In order to enhance our understandings in the physiological mechanism of the growth and toxin production of Alexandrium spp., it is important to obtain data on toxin variability in response to environmental factors in each geographical region. The present study not only provided valuable insight on the growth physiology and toxicity analysis of the Drake Passage Alexandrium spp., but also helps to evaluate the potential deleterious effect of the Alexandrium spp. in the regions of Southern Ocean. Acknowledgments This project (project no. 09/2.3) was funded by a research grant from The Open University of Hong Kong. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.marenvres.2012.08.009. References Anderson, D.M., Alpermann, T.J., Cembella, A.D., Collos, Y., Masseret, E., Montresor, M., 2012. The globally distributed genus Alexandrium: multifaceted roles in marine ecosystems and impacts on human health. Harmful Algae 14, 10e35. Anderson, D.M., Kulis, D.M., Doucette, G.J., Gallagher, J.C., Balech, E., 1994. Biogeography of toxic dinoflagellates in the genus Alexandrium from the northeastern United-States and Canada. Mar. Biol. 120, 467e478. Anderson, D.M., Kulis, D.M., Sullivan, J.J., Hall, S., 1990a. Toxin composition variations in one isolate of the dinoflagellate Alexandrium fundyense. Toxicon 28, 885e893. Anderson, D.M., Kulis, D.M., Sullivan, J.J., Hall, S., Lee, C., 1990b. Dynamics and physiology of saxitoxin production by the dinoflagellates Alexandrium spp. Mar. Biol. 104, 511e524. Bechemin, C., Grzebyk, D., Hachame, F., Hummert, C., Maestrini, S.Y., 1999. Effect of different nitrogen/phosphorus nutrient ratios on the toxin content in Alexandrium minutum. Aquat. Microb. Ecol. 20, 157e165. Boyer, G.L., Sullivan, J.J., Andersen, 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, 123e128. Carreto, J.I., Carignan, M.O., Montoya, N.G., 2001. Comparative studies on mycosporine-like amino acids, paralytic shellfish toxins and pigment profiles of the toxic dinoflagellates Alexandrium tamarense, Alexandrium catenella and Alexandrium minutum. Mar. Ecol. Prog. Ser. 223, 49e60. Carreto, J.I., Elbusto, C., Sancho, H., Carignan, M.O., Yasumoto, T., Oshima, Y., 1996. Comparative studies on paralytic shellfish toxin profiles of marine snails, mussels and an Alexandrium tamarense isolate from the Mar del coast (Argentina). Rev. Invest. Des. Pesq. 10, 101e107.
Cembella, A.D., Sullivan, J.J., Boyer, G.L., Taylor, F.J.R., Andersen, R.J., 1987. Variation in paralytic shellfish toxin composition within the Protogonyaulax tamarensise catenella species complex e red tide dinoflagellates. Biochem. Syst. Ecol. 15, 171e186. Cho, Y., Hiramatsu, K., Ogawa, M., Omura, T., Ishimaru, T., Oshima, Y., 2008. Nontoxic and toxic subclones obtained from a toxic clonal culture of Alexandrium tamarense (Dinophyceae): toxicity and molecular biological feature. Harmful Algae 7, 740e751. Esteves, J.L., Santinelli, N., Sastre, V., Diaz, R., Rivas, O., 1992. A toxic dinoflagellate bloom and PSP production associated with upwelling in Golfo-nuevo, Patagonia, Argentina. Hydrobiologia 242, 115e122. Etheridge, S.M., Roesler, C.S., 2005. Effects of temperature, irradiance, and salinity on photosynthesis, growth rates, total toxicity, and toxin composition for Alexandrium fundyense isolates from the Gulf of Maine and Bay of Fundy. DeepSea Res. II Top. Stud. Oceanogr. 52, 2491e2500. 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 or phosphorus stress. Mar. Ecol. Prog. Ser. 111, 99e109. Franco, J.M., Fernandez, P., Reguera, B., 1994. Toxin profiles of natural-populations and cultures of Alexandrium minutum Halim from Galician (Spain) coastal waters. J. Appl. Phycol. 6, 275e279. Frangopulos, M., Guisande, C., deBlas, E., Maneiro, I., 2004. Toxin production and competitive abilities under phosphorus limitation of Alexandrium species. Harmful Algae 3, 131e139. Gayoso, A.M., 2001. Observations on Alexandrium tamarense (Lebour) Balech and other dinoflagellate populations in Golfo Nuevo, Patagonia (Argentina). J. Plankton Res. 23, 463e468. Hall, S., 1982. Toxins and Toxicity of Protogonyaulax from the Northeast Pacific. University of Alaska, Fairbanks, p. 196. Hallegraeff, G.M., 1993. A review of harmful algal blooms and their apparent global increase. Phycologia 32, 79e99. Hallegraeff, G.M., 2003. Harmful Algal Blooms: a Global Overview, Manual on Harmful Marine Microalgae. UNESCO Publishing, pp. 25e50. Hamasaki, K., Horie, M., Tokimitsu, S., Toda, T., Taguchi, S., 2001. Variability in toxicity of the dinoflagellate Alexandrium tamarense isolated from Hiroshima Bay, western Japan, as a reflection of changing environmental conditions. J. Plankton Res. 23, 271e278. Hillebrand, H., Durselen, C.D., Kirschtel, D., Pollingher, U., Zohary, T., 1999. Biovolume calculation for pelagic and benthic microalgae. J. Phycol. 35, 403e424. Ho, K.C., Kang, S.H., Lam, I.H.Y., Hodgkiss, I.J., 2003. Distribution of Alexandrium tamarense in Drake Passage and the threat of harmful algal blooms in the Antarctic Ocean. Ocean Polar Res. 24, 625e631. Ho, K.C., Lee, T.C.H., Kwok, O.T., Lee, F.W.F., 2012. Phylogenetic analysis on a strain of Alexandrium tamarense collected from Antarctic Ocean. Harmful Algae 15, 100e108. Hwang, D.F., Lu, Y.H., 2000. Influence of environmental and nutritional factors on growth, toxicity, and toxin profile of dinoflagellate Alexandrium minutum. Toxicon 38, 1491e1503. John, E.H., Flynn, K.J., 2000. Growth dynamics and toxicity of Alexandrium fundyense (Dinophyceae): the effect of changing N: P supply ratios on internal toxin and nutrient levels. Eur. J. Phycol. 35, 11e23. John, E.H., Flynn, K.J., 2002. Modelling changes in paralytic shellfish toxin content of dinoflagellates in response to nitrogen and phosphorus supply. Mar. Ecol. Prog. Ser. 225, 147e160. Keller, M.D., Selvin, R.C., Claus, W., Guillard, R.R.L., 1987. Media for the culture of oceanic ultraphytoplankton. J. Phycol. 23, 633e638. Lee, F.W.F., Morse, D., Lo, S.C.L., 2009. Identification of two plastid proteins in the dinoflagellate Alexandrium affine that are substantially down-regulated by nitrogen-depletion. J. Proteome Res. 8, 5080e5092. Leong, S.C.Y., Taguchi, S., 2004. Response of the dinoflagellate Alexandrium tamarense to a range of nitrogen sources and concentrations: growth rate, chemical carbon and nitrogen, and pigments. Hydrobiologia 515, 215e224. Lim, P.T., Leaw, C.P., Kobiyama, A., Ogata, T., 2010. Growth and toxin production of tropical Alexandrium minutum Halim (Dinophyceae) under various nitrogen to phosphorus ratios. J. Appl. Phycol. 22, 203e210. Lim, P.T., Ogata, T., 2005. Salinity effect on growth and toxin production of four tropical Alexandrium species (Dinophyceae). Toxicon 45, 699e710. MacIntyre, J.G., Cullen, J.J., Cembella, A.D., 1997. Vertical migration, nutrition and toxicity in the dinoflagellate Alexandrium tamarense. Mar. Ecol. Prog. Ser. 148, 201e216. Maestrini, S.Y., Bechemin, C., Grzebyk, D., Hummert, C., 2000. Phosphorus limitation might promote more toxin content in the marine invader dinoflagellate Alexandrium minutum. Plankton Biol. Ecol. 47, 7e11. Montoya, N.G., Fulco, V.K., Carignan, M.O., Carreto, J.I., 2010. Toxin variability in cultured and natural populations of Alexandrium tamarense from southern South America e evidences of diversity and environmental regulation. Toxicon 56, 1408e1418. Murata, A., Leong, S.C., Nagashima, Y., Taguchi, S., 2006. Nitrogen:phosphorus supply ratio may control the protein and total toxin of dinoflagellate Alexandrium tamarense. Toxicon 48, 683e689. Ogata, T., Ishimaru, T., Kodama, M., 1987. Effect of water temperature and lightintensity on growth-rate and toxicity change in Protogonyaulax tamarensis. Mar. Biol. 95, 217e220.
T.C.-H. Lee et al. / Marine Environmental Research 81 (2012) 62e69 Oshima, Y., 1995. Postcolumn derivatization liquid-chromatographic method for paralytic shellfish toxins. J. AOAC Int. 78, 528e532. Paerl, H.W., 1997. Coastal eutrophication and harmful algal blooms: importance of atmospheric deposition and groundwater as ‘new’ nitrogen and other nutrient sources. Limnol. Oceanogr. 42, 1154e1165. Parkhill, J.P., Cembella, A.D., 1999. Effects of salinity, light and inorganic nitrogen on growth and toxigenicity of the marine dinoflagellate Alexandrium tamarense from northeastern Canada. J. Plankton Res. 21, 939e955. Shimizu, Y., Norte, M., Hori, A., Genenah, A., Kobayashi, M., 1984. Biosynthesis of saxitoxin analogues: the unexpected pathway. J. Am. Chem. Soc. 106, 6433e6434. Smayda, T.J., 1990. Novel and Nuisance Phytoplankton Blooms in the Sea: Evidence for a Global Epidemic, Toxic Marine Phytoplankton. Elsevier Science, New York, pp. 29e40. TaroncherOldenburg, G., Kulis, D.M., Anderson, D.M., 1997. Toxin variability during the cell cycle of the dinoflagellate Alexandrium fundyense. Limnol. Oceanogr. 42, 1178e1188. Touzet, N., Franco, J.M., Raine, R., 2007. Influence of inorganic nutrition on growth and PSP toxin production of Alexandrium minutum (Dinophyceae) from Cork Harbour, Ireland. Toxicon 50, 106e119. Uribe, P., Espejo, R.T., 2003. Effect of associated bacteria on the growth and toxicity of Alexandrium catenella. Appl. Environ. Microbiol. 69, 659e662.
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Vaulot, D., LeBot, N., Marie, D., Fukai, E., 1996. Effect of phosphorus on the Synechococcus cell cycle in surface Mediterranean waters during summer. Appl. Environ. Microbiol. 62, 2527e2533. Wang, D.Z., Hsieh, D.P.H., 2001. Dynamics of C2 toxin and chlorophyll-a formation in the dinoflagellate Alexandrium tamarense during large scale cultivation. Toxicon 39, 1533e1536. Wang, D.Z., Hsieh, D.P.H., 2002. Effects of nitrate and phosphate on growth and C2 toxin productivity of Alexandrium tamarense CI01 in culture. Mar. Pollut. Bull. 45, 286e289. Wang, D.Z., Hsieh, D.P.H., 2005. Growth and toxin production in batch cultures of a marine dinoflagellate Alexandrium tamarense HK9301 isolated from the South China Sea. Harmful Algae 4, 401e410. Wang, D.Z., Jiang, T.J., Hsieh, D.P.H., 2005. Toxin composition variations in cultures of Alexandrium species isolated from the coastal waters of southern China. Harmful Algae 4, 109e121. Wang, D.Z., Zhang, S.G., Gu, H.F., Chan, L.L., Hong, H.S., 2006. Paralytic shellfish toxin profiles, and toxin variability of the genus Alexandrium (Dinophyceae) isolated from the Southeast China Sea. Toxicon 48, 138e151. Xu, J., Ho, A.Y.T., He, L., Yin, K., Hung, C., Choi, N., Lam, P.K.S., Wu, R.S.S., Anderson, D.M., Harrison, P.J., 2012. Effects of inorganic and organic nitrogen and phosphorus on the growth and toxicity of two Alexandrium species from Hong Kong. Harmful Algae 16, 89e97.
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Effects of different nitrate and phosphate concentrations on the growth and toxin production of an Alexandrium tamarense strain collected from Drake Passage Thomas Chun-Hung Lee 1, Oi-Ting Kwok 1, Kin-Chung Ho, Fred Wang-Fat Lee* School of Science and Technology, The Open University of Hong Kong, Hong Kong, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 April 2012 Received in revised form 24 August 2012 Accepted 27 August 2012
Nitrate (N) and phosphate (P) are believed to be two of the most important nutrients for the growth and toxin production for Alexandrium species. The study of the growth and toxicity characteristics of the Alexandrium spp. under the change of N and P can help us to understand the dynamics of algal bloom and toxification events in natural environments. A strain of Alexandrium tamarense (designated as Kci) was successfully isolated from the Drake Passage in 2001 and the clonal culture has been kept in our laboratory (Ho et al., 2003, 2012). In order to extend our understanding on the growth physiology and toxicity of this A. tamarense strain, growth and cellular toxin content were examined in unialgal batch cultures under different concentrations of N and P. The effects of variable N, P concentrations on growth, cellular toxicity (fg STXeq. cell1), and toxin composition (% molar) were determined in both exponential and stationary growth phases. The toxin profile, determined by high-performance liquid chromatography with fluorescence detection (HPLC-FD), was found to be remained relatively stable and was consistently dominated by the N-sulfocarbamoyl C-toxins (>90%) under different conditions and growth phases. There were also trace amounts of other carbamate gonyautoxins consistently expressed. The cellular toxicity varied under different N and P concentrations, as well as different growth stages. A positive correlation was observed between cellular toxicity and N concentrations, but the toxicity was enhanced when P was depleted. Both cell densities and growth rate of the cells were severely suppressed under Nor P-depletion. However, the biovolume of the cells tended to be larger at N- or P-depleted cultures. Results from the present study provide valuable insight for the ecophysiology of Alexandrium species in the coastal ecosystem of Drake Passage. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Alexandrium Drake Passage Growth Nitrogen Phosphate Toxicity
1. Introduction Dinoflagellate genus Alexandrium is a well-known harmful algal bloom (HAB) causative agent and paralytic shellfish poisoning (PSP) toxins producer in many coastal regions around the world (Anderson et al., 2012; Hallegraeff, 1993, 2003). There are more than 30 morphologically defined species in this genus and more than half of them are known to be toxic (Anderson et al., 2012). Contamination of shellfishes and fishes with such PSP neurotoxins would negatively affect the shellfish and aquaculture industries (Anderson et al., 2012; Hallegraeff, 1993). In addition, it would cause human illness or even death after consumption of shellfishes or fishes that have accumulated the toxins through their diet. One of the first recorded fatal cases of human poisoning after the
* Corresponding author. Tel.: þ852 2768 6868; fax: þ852 27891170. E-mail address: wfl[email protected] (F.W.-F. Lee). 1 Authors with equal contributions. 0141-1136/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.marenvres.2012.08.009
consumption of shellfishes contaminated with the neurotoxins was in 1793 (Hallegraeff, 2003). Such causative alkaloid PSP toxins are so potent that even a very small quantity (about 500 mg) can be fatal to human. On a global scale, nearly 2000 cases of human poisoning with nearly 15% mortality through fishes or shellfishes consumption are reported each year (Hallegraeff, 2003). The increased distributions of Alexandrium spp. have become a global problem with the increase of algal blooms in frequency, intensity, and geographic distribution (Anderson et al., 2012; Hallegraeff, 1993). Although a strong relationship between frequencies of algal blooming and the nutrient load of coastal waters have been suggested (Paerl, 1997; Smayda, 1990), the algal blooming mechanism for Alexandrium spp. is complex and far from understood (Anderson et al., 2012). In addition, it is difficult to generalize the relationship between Alexandrium blooms and nutrition, because Alexandrium spp. has been reported to be able to grow in a wide spectrum of nutrient availability conditions (Anderson et al., 2012). However, nutrient loadings could lead to dramatic changes in the N, P concentrations as well as the N:P supply ratios. Many studies have
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been carried out to investigate the effects of the N to P supply ratios on the growth rate, cell density, cell size, and/or toxin composition/ production in Alexandrium spp., e.g. (Frangopulos et al., 2004; Hwang and Lu, 2000; Leong and Taguchi, 2004; Lim et al., 2010; Touzet et al., 2007; Wang and Hsieh, 2002; Xu et al., 2012). In general, the cellular toxicity of Alexandrium spp. increased with increasing N:P supply ratios (Murata et al., 2006), indicating that cellular toxicity might be stimulated by relative P-deficiency. In addition, N-limitation has been reported to decrease both growth and toxin production of Alexandrium spp. (Anderson et al., 1990b; Flynn et al., 1994; Wang and Hsieh, 2002, 2005). On the other hand, P-limitation has generally been reported to decrease growth of Alexandrium spp. but increase in cellular toxicity (Anderson et al., 1990b; Boyer et al., 1987; Flynn et al., 1994; John and Flynn, 2002; Maestrini et al., 2000; Wang and Hsieh, 2002). However, some investigations have shown different results, for example John and Flynn (2000) showed that cellular toxicity of Alexandrium fundyense would only be affected if N is colimiting with phosphorus limitation (John and Flynn, 2000). Therefore, the toxicity of Alexandrium tamarense could be affected by both N and P concentrations and expected to vary with the N:P supply ratios. It is important to understand how PSP toxins are produced by different geographical prevalent Alexandrium spp. under various environmental factors. A toxic A. tamarense strain (designated as Kci) was isolated from Drake Passage in 2001 (Ho et al., 2003). This was the first A. tamarense found in such unusual regions and might be a potential key species leading to the massive death of penguins in Falkland Islands in December 2002 to January 2003 (Ho et al., 2003). To increase our understandings on the ecophysiology of Alexandrium species in the coastal ecosystem of Drake Passage, the present study was undertaken to investigate the growth and toxinproducing physiology of this strain under the effects of different nitrate and phosphate concentrations. 2. Materials and methods 2.1. Culture conditions Non-axenic A. tamarense was isolated from Drake Passage (sampling site 20, in waters near the southern end of South America) of Southern Ocean in 2001 (Ho et al., 2003). Individual cells from the field samples collected were picked under the microscope before being cultured in L1 seawater based medium (Keller et al., 1987). A monoculture (designated as Kci) was successfully established and kept in the Environmental Laboratory of The Open University of Hong Kong. Culture was kept at exponential growth phase by transferring to new medium every week in a ratio of 1:10 (v/v). Vegetative cells from cultures in mid- or lateexponential phases of growth were inoculated into freshly prepared culture medium. Possible bacterial contamination of the culture was checked by regular microscopic examination. To allow comparison of the results obtained from the present study to others, the cultures were maintained at optimal growing conditions: 20 C under a 12-h light:12-h dark cycle at a light intensity of 120 mE m2 s1 provided by cool white fluorescent tubes in a Conviron growth chamber. The conditions were optimal for algal growth of this strain. 2.2. Cell counts and growth curve For the growth curve determination, roughly 105 cells were harvested from mid-log phase culture by centrifugation (1500 g for 10 min at room temperature), washed twice with sterile seawater and inoculated into L1-medium to yield an initial cell density of 1000 cells ml1. Cell density was measured at the same
63
time each day in a 1 mL aliquot removed and fixed with 10 ml Lugol’s solution. Cells were counted under a light microscope using a Sedgwick-Rafter cell counter. The whole experiment was repeated three times, with triplicates in each run. The specific growth rate (m) for the exponential growth phase was calculated using the following equation:
m ¼
ln N1 ln N0 t1 t0
where N0 and N1 are the cell density reading at time t0 and t1. The effects of nitrate and phosphate concentrations on growth and toxin production of Kci were investigated. The cells were cultured in the same conditions as described above, except the L1medium was altered by adding different amount of nitrate (0, 264 and 880 mM) and phosphate (0, 18, 36 mM) in the nitrate- and phosphate-experiments respectively. No effort was made to remove residual amount of N and P from the natural seawater used. Nitrate was added aseptically to yield the following concentration gradient: 0, 264 and 880 mM, in the nitrate experiment. In the P experiment, concentration gradient was set to 0, 18, 36 mM. 2.3. Determination of cell diameter and cell volume Each cell culture sample was fixed in Lugol’s fixation solution. Measurement was performed using a Nikon eclipse TS100 inverted microscope with Nikon digital sight under 400 magnifications. Samples for measurements were taken from day 10 cultures. Measurement was calibrated with micrometer. The mean cell diameter and volume were calculated from a total of at least 80 measurements randomly from the sample. Mean cell volume (mm3) was calculated with the assumption of spherical shape of cells using the following equation (Hillebrand et al., 1999):
v ¼
p 6
d3
where d is the diameter of cells. 2.4. Extraction and analysis of toxins Five hundred milliliters of cells were collected during mid-log and stationary phase. The cells were centrifuged at 3600 rpm for 5 min. The supernatant was discarded and the cell pellet was stored in 80 C until toxin extraction. 0.3 mL of 0.05 M acetic acid was added to each sample and the sample was then homogenized on ice for 10 min. The breakage of cells was confirmed by microscopic examination. The toxin extract was made up to 1 mL and centrifuged at 13,000 rpm for 3 min. Toxin analysis was performed mainly according to Oshima’s HPLC Post-column derivatization method by Waters H-class UPLC system coupled with Waters Post Column Reaction Module (Oshima, 1995). Toxin derivatives were separated by Waters Nova Pak C18 HPLC column (75 mm 3.9 mm i.d.). The mobile phase used to identify the GTX toxins group was constituted by 1.5 mM sodium octanesulfonate (OSA) in 10 mM ammonium phosphate (pH 7.0 with NH4OH). The mobile phase used to identify the STX toxins group and NeoSTX toxins group was made by 0.5 mM OSA in 10 mM ammonium phosphate (pH 7.2 with NH4OH). The mobile phase to identify the dcGTX2, 3 toxins group was constituted by 2.0 mM sodium heptanesulfonate (HSA) in 10 mM ammonium phosphate (pH 7.1 with NH4OH). The mobile phase to identify the C toxins group was constituted by 2.0 mM tetrabutylammonium dihydrogenphosphate in 10 mM ammonium phosphate (pH 6.5 with NH4OH). The flow rate was set at 0.5 mL/ min while the column temperature was adjusted to 40 C. A
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solution of 7 mM periodic acid and 50 mM sodium phosphate (pH 9.0 with 1 N KOH) was introduced to the column and eluted in a biocompatible mixing Tee at a flow rate of 0.15 mL/min controlled by a Waters Reagent Manager. The resulting mixture was heated to 75 C by Waters Temperature Control Module II while passing through a teflon reaction coil. The reaction mixture was then acidified in another mixing Tee with 0.5 M acetic acid (flow rate 0.15 mL/min and in ambient environment). The fluorescent eluted derivatives were monitored at 330 and 390 nm excitation-emission wavelengths, respectively.
3. Results and discussion 3.1. Growth, toxicity, and the toxin profile of the strain The Alexandrium genus has been known to produce potent neurotoxins which cause paralytic shellfish poisoning (PSP). It is a very common HAB causative agent and is cosmopolitan species that widely spread throughout many regions of the world (Hallegraeff, 2003). However, report of Alexandrium cells found in waters of regions in Drake Passage is greatly limited. Until 2001, a toxic A. tamarense was found in various regions in the Drake Passage (Site 1 to 23, from Punta Arenas to the South Shetland Islands bypassing the southern waters of the Falkland Islands) (Ho et al., 2003). A strain of A. tamarense was identified and isolated from live samples collected in waters of the Drake Passage (site 20) of the Southern Ocean in a research cruise by Ho et al. (2003). The research team found that the cells would survive at seawater temperatures ranging from 4.85 C to 8.13 C (measured in situ) during the research cruise, which shows the persistence of this Alexandrium spp. in tolerating temperature variations (Ho et al., 2003). Monoculture of the strain, designated as Kci, has been successfully established and kept in the Environmental Laboratory of The Open University of Hong Kong (Fig. 1). The strain was subjected to the growth and toxicity analysis in the present study. Results of the growth curve (in cells ml1), cellular toxicity (in fg STXeq. cell1) and toxin composition (in mol %) in an optimal growing condition are shown in Fig. 2. The growth pattern of cells grown in batch cultures gave a typical growth curve with three different distinctive growth phases. As indicated in the growth curve, inoculated cells require 1 day for adaptation (lag phase) prior entering the log phase. The cells grew exponentially between day 2 and day 12, with specific growth rate (m) of 0.38 (Fig. 2a). Cells reached a maximum cell capacity of around 22,000 cells ml1 on day 14. Cell size measurement showed that the
average cell diameter and volume of the A. tamarense strain Kci were 28.86 1.12 mm and 12,635 1430 mm3 respectively (Table 1). In general, previous studies reported that the toxicity of the cells in the exponential growth phase was shown to be higher than that of the stationary phase (Anderson et al., 1994, 1990b; Boyer et al., 1987; Cembella et al., 1987; Flynn et al., 1994; Hamasaki et al., 2001; Lim and Ogata, 2005; Parkhill and Cembella, 1999; Wang and Hsieh, 2002, 2005). Our results presented a similar pattern whereas the toxicity was found to be higher at the exponential growth phase than that at the stationary growth phase. Nevertheless, the toxin compositions of this strain grow in exponentialand stationary-growth phase were highly similar to each other. Only less than 1% of dc-GTX2 toxin was decreased when cells entered the stationary phase. This toxic strain produced unusually high proportions of the low potency N-sulfocarbamoyl C-toxins (>94%, with ratio of C2 to C1 toxin is around 2:1). Only trace amounts of GTX and dc-GTX toxins were detected (<6%, including dcGTX-2, dcGTX-3, GTX 2, GTX 3 and GTX 5) (Fig. 2b), while other PSP toxins were not detected. Among these trace amounts of carbamate toxins, dcGTX-2 was dominant. Although toxicity studies of Alexandrium spp. from regions of Drake Passage/ Southern Ocean is greatly limited, a number of studies have been carried out on the Alexandrium spp. from the regions of Argentine Sea, which is closely affiliated to the site of cells collection for the present study (Carreto et al., 2001, 1996; Esteves et al., 1992; Gayoso, 2001; Montoya et al., 2010). Their results showed that, under nutrient replete conditions, the C1 and C2 toxins were predominant in almost all strains analyzed. For instance, Montoya et al. (2010) have analyzed 10 cultured A. tamarense strains from the Argentine Sea (Montoya et al., 2010). The cultures were harvested for toxin analysis during the mid-log growth phase. Among the 10 strains isolated from Argentine, cell toxicity ranged from 1.81 to 10.3 pg STX equiv. cell1 (Montoya et al., 2010). Comparatively, the Drake Passage strain Kci was several times less toxic (w0.6 pg STX equiv. cell1) to those observed in their studied strains. This was due in part, to the strain Kci principally exhibited a relatively high content of the low potency N-sulfocarbamoyl C-toxins (w94%). On the other hand, the observed differences in toxicity among regions may be attributed to differential geographic distribution of the subpopulations of A. tamarense cells. In addition, other factors such as environmental and growing parameters (Etheridge and Roesler, 2005), culture-associated bacteria (Uribe and Espejo, 2003) and long term routine cell culturing maintenance (Cho et al., 2008; Wang et al., 2005, 2006), may also potentially leading to the discrepancies observed. Although most of the Argentine strains were also predominated with C-toxins (Carreto
Fig. 1. (a) Light microscopic and (b) scanning electronic microscopic (SEM) photo of Drake Passage A. tamarense strain Kci.
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Fig. 2. The (a) growth and toxicity, and (b) toxin composition of the cells grown in normal growing condition.
et al., 2001, 1996; Esteves et al., 1992; Gayoso, 2001; Montoya et al., 2010), substantial amount of other PSP toxin derivatives, including STX, neoSTX and GTX 1e4, were also detected. In contrast, no STX/ neoSTX and only trace amounts of GTX toxins were detected in the strain Kci. Interestingly, the toxin profile of strain Kci is highly similar to A. tamarense cells isolated from Hong Kong waters reported previously (Xu et al., 2012). The Hong Kong strains also predominately produce C-toxins with only trace amount of dc-GTX2,3, and
Table 1 Variations of cell size of the A. tamarense Kci at different concentrations of nitrate and phosphate. Concentrations Nitrate (mm)
Phosphate (mm)
880 880 264 880 0
36 18 36 0 36
Cell diameter (mm) S.D. 28.86 28.78 28.61 29.86 36.25
1.12 1.46 1.60 1.84 1.30
Cell volume (mm) S.D.
12,635.02 12,578.21 12,370.94 14,092.83 25,037.53
1430.69 1893.66 2131.83 2611.20 2686.62
Remarks: Samples for measurements were taken from day 10 cultures.
the toxin composition remained constant for all of the growing conditions that were tested (Xu et al., 2012). Several studies have suggested that the toxin profiles are conservative characteristic of various strains and could be used as specific marker for species identification (Anderson et al., 1994; Boyer et al., 1987; Flynn et al., 1994). However, this issue is still in debate because some other studies found that the toxin composition of Alexandrium spp. often varies with different growing conditions (Anderson et al., 1990a; Hamasaki et al., 2001). The toxin profile which dominated with C-toxins (>90%) of the present strain is very different from the toxin profiles of A. tamarense from many other geographical locations. This is not surprising, because toxin composition has been shown to vary widely among different A. tamarense strains (Anderson et al., 1994; Cembella et al., 1987). However, further studies on more Alexandrium spp. from the same regions of Drake Passage are required to confirm the uniqueness of its toxicological characteristics. 3.2. Effects of nitrate The availability of N source is one of the most important factors governing the growth and biosynthesis of PSP toxins in
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Alexandrium spp. It is believed to be a critical factor for toxin production because the PSP toxins are N-rich compounds (around 5e10% of the total cellular N in the cell) (MacIntyre et al., 1997). The effects of nitrate at the initial phosphate concentration of 36 mM on the cell growth and toxin production were investigated (Fig. 3). Only very little growth was observed in the cultures without the addition of N source (cell density <3000 cells ml1) (Fig. 3a). The growth was increased by the increase of nitrate concentration. Maximum cell density of 16,000 cells ml1 was attained when culture started with 264 mM nitrate. Further increase in nitrate to 880 mM would result in an additional increase in growth, whereas a maximum cell density of about 23,000 cells ml1 was achieved. Cell sizes in both nitrate supplied conditions (i.e. at 264 mM and 880 mM) were very similar but bigger cells were observed in nitrate depleted cultures (Table 1). The mechanism of the increase of the cell size under nutrient limitations was not well understood. However, cell size of A. tamarense was showed to be dependent on the type and concentration of N source supply previously (Leong and Taguchi, 2004) and cell size of several Alexandrium spp. was shown to be increased by the increase of N:P ratios as well (Lim et al., 2010). Some authors also suggested that the increase in cell biovolume might be attributed to the arrest of cells in G1 phase of the cell cycle (John and Flynn, 2000; TaroncherOldenburg et al., 1997; Vaulot et al., 1996). Although no effect of nitrate on cellular toxicity was observed in some studies (Hall, 1982; Parkhill and Cembella, 1999), in general, it is well documented that N deficiency decreased toxin production in Alexandrium cells (Anderson
et al., 1990b; Flynn et al., 1994; John and Flynn, 2000; Wang and Hsieh, 2002). Our results were consistent with these previous findings that the toxicity of the cells increased with the increase in nitrate concentrations (Fig. 3b). The maximum toxicity at the nitrate concentration of 880 mM was around 1.5-fold and 3-fold higher than the toxicity at the nitrate concentrations of 264 mM and 0 mM respectively. However, only slightly increase of the cellular toxicity was observed in stationary phase with the increase in nitrate concentrations from 264 mM to 880 mM. Toxin composition in cells remained relatively constant with the change of nitrate concentrations, with C-toxins as the dominant toxins in all cases (Fig. 3c). However, the ratio of C2:C1 toxins was slightly increased in the N-depleted culture. This is not surprised that sufficient supply of N appeared to be particularly important for growth and high toxin production for Alexandrium strain. This is because both proteins/enzymes and PSP toxins compose high molecular contents of N. In addition, photosynthetic efficiency would be greatly limited during N deprivation, which in turn affects the cell growth. Therefore, it is reasonable to believe that intracellular pools of N would be allocated to the maintenance of basic and essential cellular functions, and the activation of N demanding metabolic pathways (Touzet et al., 2007). For example, two abundant plastid proteins, rubisco II and NAP50, were found to be degraded in the Alexandrium spp. during N-depletion (Lee et al., 2009). The redirection of the metabolic pathway for cell survival requires synthesis of new proteins and enzymes, thereby making insufficient levels of N for the toxin synthesis.
Fig. 3. The (a) growth, (b) toxicity, and (c) toxin composition of the cells grown in different nitrate concentrations.
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3.3. Effects of phosphate Similar to the results of the effects of nitrate on the growth of the strain, the growth of the cells increased with the increase in starting phosphate concentration (Fig. 4a). Only very little growth was observed when there was no phosphate added to the culture (<4000 cells ml1). The maximum cell densities were similar (w22,000 cells ml1) at the phosphate concentration of 18 mM and 36 mM. The cell biovolume at the phosphate concentration of 18 mM and 36 mM was very similar, but the cells became slightly larger in cultures without the addition of phosphate (Table 1). Similar results were also observed in some other studies, e.g. (Lim et al., 2010; Touzet et al., 2007). Some researchers suggested that the increase of cell biovolume under P limitations could possibility due to the synthesis of other non-P compounds when the cells stopped to undergo cell division (John and Flynn, 2000; Vaulot et al., 1996). The toxicity appeared to be stimulated by low phosphate concentration. The highest toxicity was found in the cells grown under phosphate depletion (Fig. 4b). The relatively lower toxicity observed at the phosphate concentrations of 18 mM and 36 mM was very close to each other, indicating that phosphate was not really inhibitory to the toxin production. Enhancement of the PSP toxin production by cells grown in phosphate deficiency conditions has been well documented in the toxin-producing physiology studies for Alexandrium spp. conducted by others (Anderson et al., 1990b; Bechemin et al., 1999; Boyer et al., 1987; Flynn et al., 1994; John and
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Flynn, 2000; Wang and Hsieh, 2001, 2002, 2005; Xu et al., 2012). The PSP toxin molecules do not contain phosphorus; mechanism by which phosphate regulates toxin production is not well understood at the moment. However, some researchers suggested that phosphate deficiency might suppress the cell division and possibility leading to an increase in the availability of intracellular arginine, therefore allowing the cells to utilize more available arginine which is a presumed precursor in PSP toxin production (Anderson et al., 1990b; Shimizu et al., 1984). On the other hand, several researches reported that the toxin composition in Alexandrium spp. varied with growth phases and culturing conditions (Anderson et al., 1990b; Boyer et al., 1987; Flynn et al., 1994; Hamasaki et al., 2001). For example, Anderson et al. (1990a,b) showed that A. fundyense predominately produced C1, 2 and GTX 1, 4 toxins when the cells grown in nitrogen limitation. However, the toxin composition was predominately replaced by the production of GTX 2, 3 toxins when the cells grown in phosphorus limitation. Our data on the A. tamarense strain Kci indicate that toxin composition does not vary significantly with growth phases (Fig. 2c), influence of nitrate (Fig. 3c) as well as phosphate (Fig. 4c). In all cases, C-toxins remained as the dominate toxin (>90%). Similar to the results observed in N-depletion, only slightly increase in the ratio of C2:C1 toxins (w1e2%) was observed at low phosphate conditions (Fig. 4c). The results were in agreement with numerous studies found in the literature. Other studies indicated that toxin composition is constant and independent of various culture conditions
Fig. 4. The (a) growth, (b) toxicity, and (c) toxin composition of the cells grown in different phosphate concentrations.
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(Anderson et al., 1994; Boyer et al., 1987; Cembella et al., 1987; Flynn et al., 1994; Franco et al., 1994; Hall, 1982; Ogata et al., 1987). However, toxin analysis on more A. tamarense isolates in the same regions should be done before such toxin profile can be conclusively used as the “biochemical marker” to represent the A. tamarense in the Drake Passage. 4. Conclusion Although effects of N and P concentrations on growth and toxin production of various Alexandrium spp. have been extensively studied, relevant study of A. tamarense in the coastal waters of the regions of Drake Passage is greatly limited. The present study reported the growth and toxicity of A. tamarense isolated from Drake Passage under the effects of various N and P concentrations. Our results were consistent with the observations reported by many other authors that deficiency of nitrate decreases both cell growth and toxin production, while deficiency of phosphate decreases cell growth but enhances the production of toxin. However, toxin composition of the cells remains relatively stable in different growth phases and culturing conditions, with C-toxins (>90%) as the dominated toxins. This reaffirms that the stability of toxin composition is greatly species/strain dependant. In order to enhance our understandings in the physiological mechanism of the growth and toxin production of Alexandrium spp., it is important to obtain data on toxin variability in response to environmental factors in each geographical region. The present study not only provided valuable insight on the growth physiology and toxicity analysis of the Drake Passage Alexandrium spp., but also helps to evaluate the potential deleterious effect of the Alexandrium spp. in the regions of Southern Ocean. Acknowledgments This project (project no. 09/2.3) was funded by a research grant from The Open University of Hong Kong. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.marenvres.2012.08.009. References Anderson, D.M., Alpermann, T.J., Cembella, A.D., Collos, Y., Masseret, E., Montresor, M., 2012. The globally distributed genus Alexandrium: multifaceted roles in marine ecosystems and impacts on human health. Harmful Algae 14, 10e35. Anderson, D.M., Kulis, D.M., Doucette, G.J., Gallagher, J.C., Balech, E., 1994. Biogeography of toxic dinoflagellates in the genus Alexandrium from the northeastern United-States and Canada. Mar. Biol. 120, 467e478. Anderson, D.M., Kulis, D.M., Sullivan, J.J., Hall, S., 1990a. Toxin composition variations in one isolate of the dinoflagellate Alexandrium fundyense. Toxicon 28, 885e893. Anderson, D.M., Kulis, D.M., Sullivan, J.J., Hall, S., Lee, C., 1990b. Dynamics and physiology of saxitoxin production by the dinoflagellates Alexandrium spp. Mar. Biol. 104, 511e524. Bechemin, C., Grzebyk, D., Hachame, F., Hummert, C., Maestrini, S.Y., 1999. Effect of different nitrogen/phosphorus nutrient ratios on the toxin content in Alexandrium minutum. Aquat. Microb. Ecol. 20, 157e165. Boyer, G.L., Sullivan, J.J., Andersen, 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, 123e128. Carreto, J.I., Carignan, M.O., Montoya, N.G., 2001. Comparative studies on mycosporine-like amino acids, paralytic shellfish toxins and pigment profiles of the toxic dinoflagellates Alexandrium tamarense, Alexandrium catenella and Alexandrium minutum. Mar. Ecol. Prog. Ser. 223, 49e60. Carreto, J.I., Elbusto, C., Sancho, H., Carignan, M.O., Yasumoto, T., Oshima, Y., 1996. Comparative studies on paralytic shellfish toxin profiles of marine snails, mussels and an Alexandrium tamarense isolate from the Mar del coast (Argentina). Rev. Invest. Des. Pesq. 10, 101e107.
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