The brevetoxin and brevenal composition of three Karenia brevis clones at different salinities and nutrient conditions

Harmful Algae 9 (2010) 39–47

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The brevetoxin and brevenal composition of three Karenia brevis clones at different salinities and nutrient conditions Danelle K. Lekan 1, Carmelo R. Tomas * University of North Carolina Wilmington, Center for Marine Science, 5600 Marvin K. Moss Lane, Wilmington, NC 28409, United States

A R T I C L E I N F O

A B S T R A C T

Article history: Received 30 June 2009 Received in revised form 29 July 2009 Accepted 29 July 2009

Karenia brevis forms extensive, annual blooms in the Gulf of Mexico releasing potent neurotoxins (brevetoxins) having significant impacts on human health, mortalities of marine mammals, birds and fish. This study examines toxin composition and growth of three clones of K. brevis grown at different nutrient conditions, salinities and temperatures. The three clones studied were K. brevis Wilson, SP3 Ntox and SP3 S-tox clones. Brevetoxins (PbTx-1, -2, -3, -6 and -9) and brevenal were examined using LC– MS/MS. Dialysis studies clearly indicated that PbTx-1, -2 and brevenal were found within intact cells while the other toxins were released in the surrounding medium. Nutrient limitation did not show consistent effects on toxin profiles (brevenal and PbTx-1, -2, -3, -6 and -9) but toxin profiles were significantly related to clone type. The Wilson clone produced a significantly greater amount of brevenal than either SP3 clone. The SP3 clones each responded differently to nutrient limitation in toxin fractions produced. Growth at differing salinities and temperatures confirmed previous reports of K. brevis preference of higher salinities and elevated temperatures but was variable for toxins. These results support the notion that clonal differences in toxin production and profile appears to be less dependent on environmental variables but more related to inherent genetic variations resulting in the differences in clonal responses observed. ß 2009 Elsevier B.V. All rights reserved.

Keywords: Brevenal Brevetoxin profiles Gulf of Mexico Karenia brevis LC–MS/MS Nutrients Red tides SP3 N-tox SP3 S-tox Wilson clone

1. Introduction The Gulf of Mexico has been plagued by recurrent blooms of toxic dinoflagellate Karenia brevis for well over a century. Persistent annual blooms, particularly for the west coast of Florida, were extensively described by Steidinger (2009). In this historical review, blooms are documented from Florida, Texas and Mexico with references to explorer’s journals from 1648 through the most recent studies of this decade. Initiation of K. brevis blooms were described as occurring in the oligotrophic offshore waters of the West Florida Shelf (Tester and Steidinger, 1997) and further concentrated by a complex of physical fronts, winds, currents and tides (Weisberg et al., 2009). The phototactic behavior of K. brevis also aids in this transport resulting in displacement of surface populations (Steidinger, 1975; Heil, 1986; Tester et al., 1991). The bloom initiation zone reported by Steidinger (2009) was 18–74 km offshore with depths of 12–37 m in the mid shelf region. This area was postulated to be a ‘‘seed bed’’ from which blooms develop. However, despite the many years that K. brevis has been in

* Corresponding author. Tel.: +1 910 962 2385; fax: +1 910 962 2410. E-mail address: [email protected] (C.R. Tomas). 1 Present address: Victoria University, Coastal Ecology Laboratory, 396 The Esplanade, Island Bay, Wellington 6023, New Zealand. 1568-9883/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2009.07.004

culture (over 50 years for the Wilson clone), no cysts have ever been documented, leaving a more likely scenario that blooms begin from a low level, motile population found year round (Geesey and Tester, 1993) in the Gulf of Mexico. Frontal zone accumulations (Steidinger, 2009; Weisberg et al., 2009) are further mechanisms for accumulating and transporting the critical mass of cells from which blooms can form. The tolerance to salinity of K. brevis was first described as a narrow range from 27 to 37, with less than optimum growth at salinities below 24 or above 44 (Aldrich and Wilson, 1960; Vargo, 2009). This narrow salinity range suggested a ‘‘salinity barrier’’ of 24 or less for this species. In the past decade, several blooms of K. brevis occurred near the mouth of the Mississippi River and the Florida Panhandle, where freshwater outflow resulted in significantly lower salinities (Dortch et al., 1998; Maier Brown et al., 2006). The evidence of blooms occurring in low salinity waters raises the questions whether salinity is truly a barrier in the bloom development of Karenia spp., and how do lower salinities affect growth and toxin production. Temperature appears to be a much more conservative variable, with optimal growth between 20 and 28 8C (for most laboratory studies) with a lower limit for growth at 7 8C (Vargo, 2009). For field populations, a minimum temperature of 5 8C was reported for blooms of K. brevis in the South Atlantic Bight region (Tester et al., 1991). In the laboratory, temperatures giving good growth were

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˜ a and Villareal, commonly observed from 15 to 30 8C range (Magan 2006). The question of nutrients for supporting blooms is a particularly important one. Vargo (2009) reviews laboratory and field data to determine the role of nutrients in supporting blooms on the West Florida Shelf region of the Gulf of Mexico. The oligotrophic nature of the shelf region, where blooms originate, has standing stocks of dissolved inorganic nitrogen and dissolved inorganic phosphorus at very low levels that remained unchanged from bloom to nonbloom periods (Heil et al., 2001). The utilization of dissolved organic nitrogen and phosphorus are both potential sources of nutrients for sustaining populations on the Shelf. However, the combination of nutrients does not appear able to sustain the biomass formed in these blooms. In addition, the role of trace metals, particularly iron, may play an indirect role via diazotroph nitrogen fixation (Vargo et al., 2008; Vargo, 2009 and references therein). The link between blooms of Trichodesmium followed by Karenia blooms was offered as evidence for this phenomenon (Walsh and Steidinger, 2001). Further evidence is offered by Walsh et al. (2009) who argue that the mixotrophic nature of Karenia and a contribution from dead fish can assist in maintaining the blooms Florida in and their transport to the South Atlantic Bight. The major concern regarding transport of K. brevis blooms in the near coastal waters is its potent toxins that can cause mass mortalities of fishes, impact marine mammals, birds, and humans through neurotoxic shellfish poisoning (NSP) and respiratory irritation (Rounsefell and Nelson, 1966; Kirkpatrick et al., 2004). A feature of brevetoxins of particular concern is that they are effective in inducing toxicity at very low concentrations (Naar et al., 2002). Because of this feature, brevetoxins can have complex interactions through the food chain resulting in a transfer through the various components of the marine ecosystem (Landsberg et al., 2009). Human NSP, caused by the ingestion of brevetoxin contami˜ a et al., nated shellfish is debilitating but usually non-fatal (Magan 2003). K. brevis blooms are a concern in coastal areas particularly since this fragile, unarmored dinoflagellate easily lyses because of wave action, releasing cell fragments with brevetoxins (Kirkpatrick et al., 2004). Bubble-mediated transport of aerosols through wind carries the toxin inland (Pierce et al., 1989, 1990) where human inhalation results in respiratory irritation, stinging eyes, stinging ˜ a et al., 2003). Brevetoxins nose, and a dry, choking cough (Magan are potent respiratory toxins, posing particular threats to people with underlying respiratory problems. Despite the abundance of information existing from field and laboratory studies on K. brevis, the factors that influence toxin composition and its abundance remain unresolved. For some harmful species like Prorocentrum lima (Tomas and Baden, 1993), Prymnesium parvum (Johansson and Grane´li, 1999a) and Chrysochromulina polylepis (Dahl et al., 2005; Johansson and Grane´li, 1999b; Edvardsen et al., 1990) phosphorus limitation increases toxicity through stimulation of toxin production. Other environmental factors, such as salinity also impact toxin production. Maier Brown et al. (2006) noted salinity affected cellular and excreted toxin concentrations in K. brevis, with the highest concentrations at salinities of 20 followed by 40 and 30, respectively. While these findings suggest that salinity can influence toxin production, the relationship remains unclear. Baden and Tomas (1988) found differences in brevetoxin toxic fractions from the Wilson clone and clones of K. brevis isolated from a bloom in Corpus Christi Bay, Texas. The toxin profiles varied in spite of using the same growth conditions and were associated most closely with the clone analyzed. The toxicity of K. brevis cells depends on the toxic fractions present, further influenced by the strain, growth phase, and environmental factors (Baden and Tomas, 1988; Bourdelais et al.,

2002). The intensity of the toxic effects can vary from bloom-tobloom, allowing small blooms with low cell densities to be highly toxic while large blooms with higher cell densities may be only slightly toxic (Bourdelais et al., 2002). A mechanism of mediating cellular toxicity of K. brevis was suggested to involve brevenal, a naturally occurring brevetoxin antagonist (Bourdelais et al., 2004). Brevenal is thought to act on site 1 of the sodium channel while brevetoxins act on site 5 of the same channel. Even though brevenal and brevetoxins act on different sodium channel sites, the effects of brevenal appear to take precedence over the brevetoxin (Purkerson-Parker et al., 2000). Abraham et al. (2005) using toxininduced bronchioconstriction in allergic sheep confirmed the action of brevenal in alleviating the symptoms of sheep exposed to PbTx-2 and -3. To better define the interaction of clonal differences, environmental factors and the toxicity of K. brevis, this study examined the brevetoxin and brevenal fractions produced from three clones of K. brevis grown at differing nutrient limitation, temperature and salinity. 2. Materials and methods 2.1. Strain information The cultures used for these studies were the Wilson clone, originally isolated by W. Wilson 1953 from John’s pass Florida and two Texas clones labeled SP3 S-tox and SP3 N-tox obtained from Dr. Ed Buskey, UTMSI Port Aransas, Texas. The SP3 clones were originally isolated by Suzanne Pargee in Dr. T. Villareal’s laboratory from a bloom off South Padre Island, Texas in 1999 (Loret et al., 2002). The N-tox (non-toxic) and S-tox (super-toxic) designations were assigned from unpublished studies done elsewhere (T. Villareal, personal communication) and will be used here to represent each of the SP3 clones. All clones were grown in the same media and under the same conditions to allow accurate comparisons. Extractions were specifically made during stationary phase when the effects of nutrient limitation were strongest and cellular toxin was known to at its highest levels and not at mid log phase cells where lower cellular brevetoxin levels were expected (Baden and Tomas, 1988). 2.2. Growth study Growth and toxin experiments of the three clones of K. brevis were conducted using L1 media (Guillard and Hargraves, 1993) modified by the elimination of Si and the addition of V8 vitamins (Provasoli et al., 1957). The cultures were initially grown at 20 8C, salinity of 33 and a 14:10 h light:dark cycle having a fluence rate of 66 mmol quanta m 2 s 1 cool white fluorescent light. Media for the growth studies was made from low nutrient Gulf Stream water diluted with high purity deionized water (DIW) prior to sterilization for salinity treatments of 20, 25, 30, 35 and 39. The ambient nutrient levels of the Gulf Stream water were always below 1 mM L 1. Three experimental nutrient ratios at each salinity were used with N:P 16:1, 58 mM NO3 + 3.63 mM PO4 , P-limited (N:P 80:1, 80 mM NO3 + 1 mM PO4 ), and N-limited (N:P 1:1, 16 mM NO3 + 16 mM PO4 ) treatment. The growth experiments were run 20, 25 and 30 8C to encompass the range of environmentally realistic temperatures. Three replicates from each of the temperature/salinity matrix were inoculated in 60 mL culture tubes to give an initial fluorometric reading on day 0 and in vivo fluorescence was measured at the same time daily until experimental cultures reached stationary phase or were terminated at 30 days. A Turner Designs 10 AU fluorometer equipped with standard filters was used for these measurements. Periodic samples for cell counts were taken and preserved in Lugol’s for later counting. To verify the

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relationship between in vivo fluorescence and cell number from which growth was determined, fluorescence values over a span of cell densities for each culture was analyzed using linear regression analysis. The regression coefficients (r2  0.98) were obtained for each clone (Fig. 1). Samples for cell counts were taken at specific time intervals and preserved in Lugol’s iodine solution. Counts were made using the Utermo¨hl settling method (Utermo¨hl, 1931; Sournia, 1978) on a minimum of 300 cells per count using a Nikon Diaphot inverted microscope. Three counts were performed for each sample treatment and used to determine the growth patterns and for correcting toxin values to obtain per cell values. Daily fluorescence data as the log2 (fluorescence + 1) were taken for three replicates of each experimental treatment and plotted against time to yield growth curves. The growth curves for each clone at the nutrient and salinity treatments at 20 8C were used to compare patterns of each clone. Growth rates (k) were calculated using 3 data points from steepest slope of the log growth phase giving regression coefficients of r2 values = 0.99 or better. This was accomplished from a least squares regression equation using the LM Program in the statistical software R (R Core Development Team, 2008). To test the significance (p < 0.05) of any treatment on toxins, an Analysis of Variance (ANOVA) was performed. All one-, two- and three-way interactions were included and assessed for significance. Normality of residuals

Fig. 1. Calibration of cell density (cells 106 L 1) versus relative fluorescence for three clones of Karenia brevis: (A) SP3 N-tox, (B) SP3 S-tox and (C) Wilson clones.

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and homogeneous variances were verified through residual and normal probability plots. If an interaction was not found to be significant, the data was collapsed to remove that variable to increase the power of detecting other significant differences. 2.3. Toxin studies Since Karenia was not amenable for separation by centrifugation or routine filtration, a dialysis method was determined to be the most gentle to avoid cell lysis. To determine toxin profile within the cells versus that in external media, six Spectra/Por1 7 dialysis membranes (MWCO 3500 Da) were rinsed with DIW and filled with 50 mL of NH-15 media (Gates and Wilson, 1960), and securely sealed prior to being suspended in a 4-week-old 8 L culture of K. brevis (Wilson clone) grown in NH-15 media. At hours 0, 6, 12, 24 and 48, one dialysis tube was harvested and extracted for brevetoxins according to the protocol described below. At the last sampling (48 h) when the last dialysis tube was harvested, a whole culture sample (cells and media) was taken and extracted. The extracts were process by the LC–MS/MS methods described below. Cultures of 0.5 L of each clone were grown at 20 8C (0.5 8C) in a constant temperature water bath and at salinities of 25, 35 and 39 with nutrient N:P balanced, P-limited and N-limited treatments. Toxin analyses were performed on each culture treatment when daily cell counts indicated the culture had reached stationary phase, where our preliminary experiments and those of others (Baden and Tomas, 1988; Maier Brown et al., 2006) indicated the greatest per cell toxin concentration, and the full impact of the nutrient limitation was expected. Aliquots of 50–100 mL of whole culture with known cell densities were sequentially extracted three times with HPLC grade ethyl acetate (EtOAc). Cells were disrupted using an Autotune sonicator for 90 s at an amplitude of 50 and output setting of 12 on a mixture of whole culture with 30 mL EtOAc. The mixture was transferred to a 250 mL separatory funnel with an additional 30 mL EtOAc and the layers separated after mixing. The combined organic layers were washed with deionized water and the organic layer concentrated to dryness using a Heidolph Laboratory 4000 rotary evaporator with a water bath temperature of 37 8C. The dried sample was resuspended in 5 mL of ACS grade acetone and incubated at 4 8C for 30 min to precipitate waxes, fats, and proteins. The cooled extract was filtered through a 0.2 mm Magna nylon filter and the filtrate evaporated to dryness. The sample was redissolved in 98% acetonitrile (ACN), 2% water, 0.1% formic acid in a volume proportional to the cell density of the original culture. A fixed-size aliquot was transferred to a 300 mL polyspring insert inside a 1.8 mL amber glass vial with a PTFE-lined rubber septum. LC–MS/MS analyses were performed using an Agilent (Santa Clara, CA) 1100 HPLC system coupled with a Q TRAP (ABI MDS/ SCIEX, Ontario, Canada) quadrupole/linear ion trap mass spectrometer with a Turbo Spray source controlled by Analyst 4.0 software (MDS, Concord, Ontario, Canada). All solvents were HPLC grade. Separation was achieved on a Varian Microsorb 100-3 C18 column (3 mm; 100 mm  2 mm) and a binary mobile phase system consisting of 98% water, 2% ACN, 0.1% formic acid (A) and 98% ACN, 2% water, 0.1% formic acid (B). For each sample, 4 mL were injected and subjected to the following stepped gradient program: 60% B, 110 mL min 1 (0–5 min); 99% B, 140 mL min 1 (5–12 min); 60% B, 110 mL min 1 (12–20 min). The mass spectrometer was operated at a capillary voltage of 5 kV and a source temperature of 300 8C. Compound peak areas measured for the extracted culture samples were quantitated for each species using calibration curves constructed from injection on the day of each analysis of a series of known concentrations of purified standards for PbTx-1, -2, -3, -6,

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Fig. 2. Chemical structures of PbTx-1 (A, backbone brevetoxin) and PbTx-2, -3, -6, -9 (B, backbone brevetoxins), CBA, carboxylic acid and brevenal detected in this study.

and -9 and for brevenal and PbTx-2 carboxylic acid (CBA) (Fig. 2). Because PbTx-6 and CBA are isobaric and hence not distinguishable by molecular mass alone, CBA was included as a set of standards to make it possible to distinguish the two by their differing retention times. The m/z values used to quantitate the various compounds are shown in Table 1. The peak areas for the molecular ions and the fragments due to loss of H2O were summed for quantitation of each compound. A linear fit through zero with 1/x weighting (as determined appropriate from residual plots) of peak area versus mass injected was calculated for each set of calibration standards. In all cases R2 values 0.99 were achieved. For each analyte, the concentration (pg mL 1) calculated from the calibration curve was converted to pg cell 1 based on the cell density in the original cultures.

2.4. Statistical analyses An ANOVA was run to determine significant (p < 0.05) effects on toxin production of individual PbTx-1, -2, -3, -6 and -9, total PbTx2 + 3 + 9 and brevenal in the three clones at 20 8C, salinities of 25, 35 and 39 and nutrient conditions of balanced, P-limited and N-limited. A Tukey’s pair wise comparison between variables was run to more closely examine where toxin production was significantly affected by the clone and/or environmental factors. A MANOVA of PbTx-2, -3, -9 from combined effects of salinity–clone, clone–nutrient and nutrient–salinity, was run to determine if the environmental variables studied significantly affected toxin production. 3. Results 3.1. Dialysis toxin studies

Table 1 Monitored m/z values for parent and derivative brevetoxins (PbTx) and brevenal from Karenia brevis used as reference standards in analysis with LC–MS/MS. Compound

PbTx-1 PbTx-2 PbTx-3 PbTx-6 PbTx-9 Brevenal CBA

H2O (amu)

Mass + H (amu)

Mass

[M+H]+

[M+H H2O]+

867.5 895.5 897.5 911.5 899.5 657.5 911.5

849.5 877.3 879.5 893.5 881.5 639.5 893.5

The study using dialysis tubing employed an 8-week-old 10 L culture of K. brevis. The data from the extraction of 6 dialysis tubes originally containing the standard media and suspended in the K. brevis culture is presented in Fig. 3. One of the most potent toxins, PbTx-1, and brevenal were found exclusively in the whole sample (cells + media) and were not present in any of the dialysis tubes (media) incubated over the 48 h study period. PbTx-2 concentrations were very high relative to the other brevetoxins where 99.5% of PbTx-2 was detected inside cells and only 0.5% of PbTx-2 in the media. Likewise, PbTx-1 and brevenal were found in lesser quantities, but only inside the cells, confirming whole culture

D.K. Lekan, C.R. Tomas / Harmful Algae 9 (2010) 39–47

Fig. 3. Brevetoxin and brevenal (pg mL 1) from organic extractions of whole culture (media plus cells) and from media in dialysis tubing suspended in a Karenia brevis culture over an equilibration period of 0, 6, 12, 24 and 48 h.

extractions accurately estimated cellular PbTx-1, -2 and brevenal. Only 19% of total PbTx-3 and 0.4% of PbTx-6 was found in the final external media sample (Fig. 3), while PbTx-9 and CBA were not detected in any of the dialysis samples. These results confirmed that whole culture extractions accurately reflect cellular toxin PbTx-1, -2 and brevenal, whereas PbTx-3 and -6 represent a much lower concentration of combined toxin from inside and outside cells. 3.2. Toxins in stressed populations The effects of salinity and nutrient limitation on production of PbTx-1, -2, and -3 for each clone are shown in Fig. 3. Among the

Fig. 4. Concentration of combined brevetoxins (PbTx-1, -2 and -3) pg cell 39, and at N:P balanced, N-limited and P-limited nutrient ratios.

1

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measured toxins (PbTx-1, -2, -3, -6, and -9), production of PbTx-2 consistently exceeded 20 pg cell 1 in all three clones. PbTx-3, the next most abundant toxin, was an order of magnitude less at 0.6– 6.9 pg cell 1. The less abundant PbTx-1 exhibited more clonal variation with elevation in N-limited and P-limited N-tox and S-tox cultures (2.3–3.9 pg cell 1), but values rarely exceeding 1.0 pg cell 1 in the Wilson clone at nutrient limited conditions. Concentrations of PbTx-6 and PbTx-9 were minimal in all samples (0–0.9 and 0–0.2 pg cell 1, respectively). Combining all treatments, 18 of the 27 (67%) samples had PbTx-2 values exceeding 12 pg cell 1, a value commonly used as an average cellular concentration for the Wilson clone (Baden and Tomas, 1988). The greatest combined toxin production on a per cell basis for the parent toxins, PbTx-1, -2 and -3, was observed in the N-limited N-tox clone at a salinity of 39 and for the N-limited Wilson clone at salinity of 39, with values of 32.8 and 31.9 pg cell 1, respectively (Fig. 4). PbTx-1 and -2 concentrations were elevated in P-limited cultures at salinities of 35 and 39 for both N-tox and S-tox clones (26.2 and 29.8, 26.4 and 27.8 pg cell 1, respectively) and at a salinity of 25 and 39 for the N-limited Wilson clone (31.8 and 26.4 pg cell 1, respectively). The lowest combined cellular toxin value of 3.4 pg cell 1 was found for the P-limited S-tox clone at a salinity of 25. Except for two P-limited treatments at salinities of 35 and 39, the combined cellular toxin content of the S-tox clone was less than that found in the same treatments for N-tox and Wilson clones (Fig. 4). Regarding brevenal production, the Wilson clone produced the greatest concentration of brevenal followed by the S-tox and N-tox clones (Fig. 5A). The greatest brevenal concentrations of 5.3 and 4.1 pg cell 1 occurred in the N-limited Wilson clone at salinities of 25 and 39, respectively. The highest brevenal values in the S-tox clone exceeded 1.0 pg cell 1 in P-limiting treatments at salinities of 35 and 39. Brevenal concentrations for the N-tox clone were highest at a salinity of 39 with values between 0.8 and 0.9 pg cell 1. The lowest brevenal concentrations for S-tox and N-tox clone occurred in the salinity of 25. When examined as a

from Karenia brevis SP3 N-tox, SP3 S-tox and Wilson clones grown at 20 8C in salinities of 25, 35 and

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Fig. 5. The brevenal concentration (A) and the ratio of brevenal to PbTx-1 + 2 (B) produced in three clones of Karenia brevis (SP3 N-tox, SP3 S-tox and Wilson) grown at 20 8C in salinities of 25, 35 and 39, and N:P balanced, N-limited and P-limited nutrient treatments.

ratio of brevenal to PbTx-2 and -3 (Fig. 5B), both SP3 clones had ratios that were below 0.06 with the majority found at <0.05. In contrast, the Wilson clone had ratio’s exceeding 0.13 with a maximum exceeding 0.17. ANOVA analysis of the brevetoxin fractions and brevenal concentrations of N-tox, S-tox and Wilson clones at different nutrient treatments and salinities (Table 2) gave significant p-

Table 2 Significant p-values for three-, two- and one-way ANOVA for brevetoxin and brevenal concentrations for the Wilson, SP3 N-tox and SP3 S-tox clones of Karenia brevis at a temperature of 20 8C and salinities of 25, 35 and 39. Variable

PbTx 1

Clone Salinity

0.0004 0.0268

Brevenal 2 0.0097 –

3 – 0.0154

6 0.0187 –

9 0.0498 –

Total* 0.0196 –

<0.0001 –

* PbTx-2 + 3 + 9; nutrient, clone–salinity, clone–nutrient and salinity–nutrient significant if <0.05.

values (<0.05) based on the clone type for PbTx-1, -2, -6, -9 and brevenal. PbTx-1 was also significant for both clone and salinity while PbTx-3 was significant for salinity only. MANOVA analyses of PbTx-2, -3 and -9 showed significant differences across the entire group based on the clone studied or the salinity used (Table 3).

Table 3 Wilks’ Lambda values for MANOVA analysis on combined PbTx-2, -3, -9 toxin production for SP3 N-tox, SP3 S-tox and Wilson clones a temperature of 20 8C, salinities of 25, 35 and 39 and balanced, P-limited and N-limited nutrient conditions. Variable

Wilks’ Lambda

Significance

Clone Salinity Nutrient Clone–salinity Clone–nutrient Salinity–nutrient

0.0012 0.0148 0.2841 0.1526 0.3035 0.4744

*

*

Significance at 0.05 level.

*

– – – –

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3.3. Growth patterns Salinity and temperature treatment of each clone gave three general patterns for growth (Fig. 5). For less than optimal conditions (salinity of 20 and temperature 20 8C), cultures initially grew slightly and by the third day began to decline regardless of the nutrient treatment as shown for the SP3 N-tox clone (Fig. 6A). Under more favorable conditions a salinity of 30 and temperature of 25 8C, both SP3 clones increased rapidly during the first 15 days (Fig. 6B) where the N-limited treatment generally resulted in a rapid decline in cells during the remainder of the 30day growth period and both the P-limited and balanced treatments continued growth, reaching stationary phase at day 30, as shown for SP3 S-tox (Fig. 6B). The Wilson clone differed slightly in its growth pattern at optimal temperatures and salinity in the

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Table 4 Growth rates (k) for Karenia brevis clones (SP3 N-tox, SP3 S-tox and Wilson) at temperatures of 20, 25 and 30 8C, salinities of 25, 30, 35 and 39, in balanced, Plimited and N-limited nutrient conditions. Clone

N-tox N-tox N-tox N-tox N-tox N-tox N-tox N-tox N-tox N-tox S-tox S-tox S-tox S-tox S-tox S-tox S-tox S-tox S-tox S-tox Wilson Wilson Wilson Wilson Wilson Wilson Wilson Wilson a

Temperature

20 20 20 20 25 25 25 25 30 30 20 20 20 20 25 25 25 25 30 30 20 20 20 20 25 25 25 25

Salinity

25 30 35 39 25 30 35 39 35 39 25 30 35 39 25 30 35 39 35 39 25 30 35 39 25 30 35 39

Growth rates (k) Balanceda

P-limiteda

N-limiteda

0.15 0.17 0.23 0.20 0.13 0.25 0.28 0.23 0.16 0.19 0.14 0.16 0.20 0.18 0.07 0.17 0.21 0.25 0.18 0.19 0.07 0.15 0.10 0.13 0.12 0.14 0.18 0.14

0.16 0.18 0.23 0.18 0.11 0.26 0.22 0.24 0.11 0.14 0.15 0.15 0.19 0.17 0.06 0.20 0.13 0.28 0.16 0.20 0.05 0.12 0.12 0.09 0.13 0.14 0.14 0.10

0.15 0.17 0.20 0.16 0.13 0.29 0.24 0.22 0.13 0.14 0.17 0.19 0.21 0.20 0.05 0.18 0.24 0.27 0.20 0.19 0.05 0.14 0.13 0.10 0.10 0.15 0.16 0.13

Balanced = 16:1 N:P, P-limited = 80:1, N-limited = 1:1.

N-limited cells reaching stationary phase on day 20 while Plimited and balanced cultures continued growth reaching stationary phase between days 25 and 30 (Fig. 6C). 3.4. Growth rates Growth of the three clones at different salinities and temperatures exhibited three general patterns. k Ranged from 0.05 to 0.29 (Table 4). The highest growth rates (k  0.25 day 1), were found exclusively in the SP3 clones. The Wilson clone had particularly low growth rates, <0.18 day 1, for all treatments. The highest growth rate for all treatments and clones was 0.29 day 1, observed for the SP3 N-tox clone in N-limited conditions at 25 8C in a salinity of 30 (Table 4). The lowest growth rates for each clone were generally found in the lower salinity treatments. Growth rates were low at salinity 25 at 25 8C in the SP3 S-tox clone with 0.07, 0.06 and 0.05 day 1 found in balanced, P-limited and Nlimited nutrients treatments respectively, and in the Wilson clone in a salinity of 25 at 20 8C with 0.07, 0.05 and 0.05 day 1 in balanced, P-limited and N-limited treatments respectively (Table 4). 4. Discussion

Fig. 6. Growth patterns of Karenia brevis SP3 N-tox, SP3 S-tox and Wilson clones at salinities of 20 and 30 at temperatures of 20 and 25 8C under N:P balanced, Nlimiting and P-limiting growth conditions; (A) steadily decline of SP3 N-tox clones at sub optimal conditions, (B) normal growth for Sp3 S-tox clone at N:P balanced, Plimited and N-limited cultures and (C) normal growth curve for Wilson clone grown at N:P balanced, N-limited and P-limiting conditions.

The retention of the brevetoxin within cells versus secretion into the external medium is not a trivial matter. The two most potent brevetoxins, PbTx-1 and PbTx-2, were found almost exclusively within intact cells (Fig. 3). PbTx-1 and less observed PbTx-7 and -10 (not analyzed), consist of an A type backbone structure and is frequently more potent than other brevetoxins (Baden, 1989; Ramsdell, 2008). The production of PbTx-1 varied with clone (N-tox and S-tox), and although not significantly different from the balanced nutrient treatments, levels in both P- and N-limited cells tended to be elevated. The Wilson clone

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differed markedly in that it had barely measurable levels of PbTx-1. Along with this toxin, the predominant amounts of PbTx-2 were found only within cells. While more potent than the other brevetoxins, the relative amount of PbTx-1 was much lower than PbTx-2 suggesting toxicity in natural samples is a function of a complex mixture of toxins and not the presence of one toxin alone. PbTx-2, and -3 were the most abundant toxins having the B backbone. PbTx-6 and -9 were found in lesser amounts or not detected. Confirmation that both PbTx-1 and -2 were located within cells with only minor amounts in the external medium is consistent with the notion that both are parent toxins from which the others are formed. Their potency is highest and any transformation to another form results in a lessening of potency (Baden, 1989). PbTx-3, the most water soluble form and a direct derivative from PbTx-2, was the most abundant brevetoxin outside cells liberated into the external media followed by PbTx-6 at lower concentrations. As modified forms of PbTx-2, their concentrations and potency were less than the parent toxin. Toxin production was significantly related to clonal type. The Wilson clone produced the most PbTx-2, followed by the SP3 N-tox clone, while the SP3 S-tox clone produced the least. This pattern was not modified by nutrient treatment. While changes in toxin levels were found in the nitrogen and phosphorus stressed cultures of a given clone, no statistically significant differences were found among clones. Comparison of compound levels for all clones and growth conditions showed clonal type to be the most significant factor with salinity apparently affecting primarily PbTx-1 and PbTx-3. A similar result was reported by Baden and Tomas (1988) comparing the toxin profiles from the Wilson clone and several isolates of K. brevis from Florida and a bloom in Corpus Christi Bay, Texas. This also clearly showed that the labels for the SP3 clones as N-tox and S-tox are misleading and non-toxic (N-tox) and supertoxic (S-tox) are not consistent with the toxin contents of the SP3 clones. The Wilson clone produced comparable levels of PbTx-2 and -3, as did the N-tox and S-tox clones at salinities 35. The cellular amounts of these toxins for all clones tested here far exceeded those reported by McNabb et al. (2006) and were more consistent with the results reported by Errera et al. (2009). These results also emphasize that remarkably the Wilson clone has retained its toxin production in spite of being maintained in cultivation for over 56 years, contrasting some observations related to loss of toxicity with time reported for other toxic species maintained in cultivation (Furey et al., 2007; Baden, personal communication). Brevenal, the brevetoxin antagonist (Bourdelais et al., 2004) occurred in all three clones tested; however, the brevenal production in the Wilson clone far exceeded that of the other clones (Fig. 4A). Brevenal production among clones varied significantly, with the Wilson clone producing greater amounts of brevenal than either of the SP3 clones in any treatment studied. Salinity appeared to affect brevenal production in the Wilson clone in which greater amounts were produced at lower salinities, while the SP3 clones produced a relatively greater amount at higher salinities (Fig. 5A). The highest brevenal concentrations in the Wilson clone (4.1 and 5.3 pg cell 1) far exceeded that in the other clones and were significantly greater than those reported by Errera et al. (2009). For purified toxins and brevenal, Abraham et al. (2005), showed brevenal mitigated toxin-induced bronchoconstriction in allergic sheep exposed to PbTx-2 and -3. The IC50 reported for brevenal on a combination of the most abundant brevetoxins, PbTx-2 and -3, was 10 and 44 pg mL 1, respectively, with >80% inhibition occurring at 100-fold molar ratios of brevenal to brevetoxin (Abraham et al., 2005). Our studies reporting higher levels of brevenal produced by K. brevis clones relative to those of Errera et al. (2009) indicated that brevenal was never produced in sufficient quantities to neutralize cellular PbTx-2 and -3 concen-

trations suggested by the sheep studies. Indeed, the brevenal:PbTx-1 + 2 ratios (Fig. 5B) in the Wilson clone, the most prolific brevenal producer never exceeded 0.18 in contrast to the ratio of 100 needed for protection in the study with sheep. Within the temperature, salinity and nutrient matrix of our experiments, as well as those reported by Errera et al. (2009) for brevenal production, ratios approaching those required to neutralize brevetoxins were never observed. Unless there is some as yet to be discovered environmental condition that enhances brevenal production, it appears unlikely that, at environmentally realistic concentrations, brevenal could be produced in sufficient quantities to be an effective modulator of toxicity. In its purified form, however, there is no question that brevenal offers an exciting potential in alleviating the bronchioconstriction induced by exposure to brevetoxins. Relative to the influence of environmental factors on toxin production, the ANOVA statistical analysis consistently found significant differences between clones. Salinity was the only environmental factor to differ significantly across the toxin group from the B backbone of PbTx-2, -3 and -9 in the MANOVA. We did, however, in this case again find the clone studied was shown to be significantly different. In most cases, growth below a salinity of 25 was generally minimal and rarely exceeded k = 0.16 day 1. The patterns observed with the SP3 and Wilson clones were more ˜ a and Villareal (2006), who found higher similar to those of Magan growth rates of 0.30 day 1 for the Wilson clone at 20 8C and a salinity of 25; a rate of 0.36 day 1 at a salinity of 30, and 0.32 day 1 at a salinity of 35 and 0.26 day 1 at a salinity of 39. 5. Conclusions The Karenia species in our experiments produced variable toxin profiles suggesting a physiology which, to some extent, reflected the variations in bloom toxicity observed in field and experimental laboratory studies. The clonal variability in toxin in our study, agreed with that observed by Baden and Tomas (1988) also as reported by Errera et al. (2009). The dilemma posed by these observations is to what extent is the environment a modifier of toxicity? Environmental conditioning in K. brevis appears to play a secondary role in influencing toxin from what presumably are genetically distinct populations as those reported by Loret et al. (2002). Nutrient stress or salinity/temperature treatments did not yield significant differences in toxin composition among clones. This implies that the nutrient environment can influence biomass, but not necessarily cellular toxin levels. The environmental role of brevenal needs to be further explored and unless there is an enhanced production of that compound it may not effectively modulate toxicity in a natural bloom. Future work requires the examination of the intraspecific genetic variability of a bloom population to help define the responses observed in overall bloom toxicity. Similar variability was presented in the literature review by Burkholder and Glibert (2006) of a number of HAB species including K. brevis. Acknowledgements We thank Dr. Daniel Baden, Dr. Edward Buskey and Dr. Tracy Villareal for providing the Wilson and SP3 clones used in this study. Dr. D. Baden provided all the toxin standards used in calibration of the LC–MS/MS and Dr. Andrea Bourdelais assisted in developing methods of organic extractions and tutelage in use of the mass spectrometer. Dr. Susan J. Simmons gave invaluable assistance with statistical analyses. This work was supported by the Center for Disease Control and Prevention grant # 0150407 awarded to Dr. Carmelo R. Tomas, through the North Carolina Department of Health and Human Services. Support for this work also came in

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part from the P01 ES 10594 (NIEHS, NIH, DHHS) grant awarded to Dr. D. Baden and UNCW’s MARBIONC program. We thank anonymous reviewers, for their constructive comments and suggestions. This work was submitted in partial fulfillment of a Masters in Marine Science for D.K. Lekan, successfully defended in July, 2008.[TS] References Abraham, W., Bourdelais, A.J., Sabater, J.R., Ahmed, A., Lee, T.A., Serebriakov, I., Baden, D.G., 2005. Airway responses to aerosolized brevetoxins in an animal model of asthma. American Journal of Respiratory and Critical Care Medicine 171, 26–34. Aldrich, D.V., Wilson, W.B., 1960. The effect of salinity on growth of Gymnodinium breve Davis. Biological Bulletin 119, 57–64. Baden, D.G., 1989. Brevetoxins: unique polyether dinoflagellate toxins. Journal of the Federation of American societies for Experimental Biology 3, 1807–1817. Baden, D.G., Tomas, C.R., 1988. Variations in major toxin composition for six clones of Ptychodiscus brevis. Toxicon 26, 961–963. 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