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Control of domoic acid toxin expression in Pseudo-nitzschia multiseries by copper and silica: Relevance to mussel aquaculture in New England (USA)
Marine Environmental Research 83 (2013) 23e28
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Control of domoic acid toxin expression in Pseudo-nitzschia multiseries by copper and silica: Relevance to mussel aquaculture in New England (USA) M. Soledad Fuentes a, b, Gary H. Wikfors a, * a b
National Oceanographic and Atmospheric Administration, National Marine Fisheries Service, Northeast Fisheries Science Center, 212 Rogers Avenue, Milford, CT 06460, USA Algenol Biofuels, 16121 Lee Rd, Fort Myers, FL 33912, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 June 2012 Received in revised form 11 October 2012 Accepted 17 October 2012
The production of the toxin Domoic Acid (DA) by the diatoms Pseudo-nitzschia spp. is affected by several environmental factors, among them copper and silica. The effects of these nutrients upon DA production have been studied individually, but not in combination. There is evidence, however, that in diatoms copper can enter the cell via the silicic-acid transport site. The goal of this study was to analyze the effect of the interaction between copper and silicic-acid supply upon DA production in Pseudo-nitzschia multiseries. The study was motivated by concerns about the risk of toxigenic Pseudo-nitzschia spp. impacting mussel aquaculture in New England (USA). The results of the present study do not indicate that copper uses the silicic acid transport site to enter the cell; nevertheless, there is an interaction between these two nutrients that produces a synergistic affect upon toxin production. A small increase in copper, without a simultaneous increase in silicate, as well as an increase in both copper and silicate, leads to DA up-regulation. Furthermore, the field component of this study reports the presence of species of Pseudonitzschia on the New England coast that are capable of producing DA. Together these findings indicate that risk of DA impacting mussel aquaculture along the coast of New England would be increased by an unusual enrichment of copper in the vicinity of mussel farms. Published by Elsevier Ltd.
Keywords: Pseudo-nitzschia Domoic acid Copper Silicate
1. Introduction Mussel aquaculture is a growing industry, with world production dominated by Europe, China, Canada and the United States (FAO, 2012). Three species of mussel are cultured in North America, Mytilus trossulus and Mytilus galloprovincialis on the West Coast, and Mytilus edulis on the East Coast (Hilbish et al., 2000). East-Coast production of this species has increased enormously during the last two decades. The main production center of eastern North America is Prince Edward Island (PEI), Canada, which produces nearly 20 thousand tonnes annually, according to the Fisheries and Oceans Canada (2010). As most of the eastern US mussel market is supplied by imports from Canada, the US government, through H.R. 2010, the National Offshore Aquaculture Act of 2007, provided support for the commercial fishing industry to stimulate a sustainable, offshore aquaculture industry in New England and nationwide. Consequently, mussel aquaculture is a developing industry in New England. Shellfish harvests, including those from mussel farming, are affected every year by Harmful Algal Blooms (HABs), which can
* Corresponding author. Tel.: þ1 203 882 6525; fax: þ1 203 882 6570. E-mail address: [email protected] (G.H. Wikfors). 0141-1136/$ e see front matter Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.marenvres.2012.10.005
cause long-term harvest closures resulting in great economic losses. In the northeastern United States, HAB episodes are a serious and recurrent problem, mainly associated with Paralytic Shellfish Poisoning (PSP) (Hoagland et al., 2002). Several monitoring networks have been implemented to manage the impact of PSP blooms. One of the most notorious human poisoning events attributable to HABs occurred in 1987 when more than 100 people became ill and three persons died after consuming mussels harvested off PEI (Bates et al., 1998). These mussels were found to be contaminated by domoic acid (DA) produced by the diatom Pseudonitzschia multiseries. Recent evidence suggests that Pseudo-nitzschia, a genus that blooms regularly on the US west coast, is expanding in range and has also been detected more frequently off the East Coast of United States. Species of Pseudo-nitzschia have been reported in Massachusetts Bay (Villareal et al., 1994), Narragansett Bay (Hargraves et al., 1993), and recently in Florida, Chesapeake Bay, and Maine (Phlips et al., 2004; Marshall et al., 2005; Van Dolah and Leighfield, unpublished data). In the February, 2007 issue of Plankton News (http://www.chbr.noaa.gov/pmn/_docs/ PlanktonNews/PlanktonNews_February2007.pdf.), a bloom of Pseudo-nitzschia also was reported in North Carolina. Furthermore, Shuler et al. (2012) reported the presence of Pseudo-nitzschia spp. from North Carolina through northern Florida, with six bloom
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events (three of them with detectable levels of DA) throughout nine years of sampling. Given that this genus apparently is expanding throughout the North American East Coast, it is crucial to assess risks to protect the public from Amnesic Shellfish Poisoning (ASP) that can result from the ingestion of DA-contaminated shellfish. Domoic acid is a tricarboxylated amino acid e an analog of the neurotransmitter L-glutamic acid e which binds to the glutamate receptor, causing neuronal depolarization in the brain resulting in permanent memory loss in mammals in severe cases. Blooms of DA-producing Pseudo-nitzschia spp. have been associated with mortalities of both wildlife and humans (reviewed by Lelong et al., 2012a; Trainer et al., 2012). Presence of Pseudo-nitzschia spp. in the water, though, does not necessarily indicate that DA is being produced that could contaminate shellfish and make them unfit for human consumption. Production of DA by Pseudo-nitzschia spp. is extremely variable, depending upon species (Lelong et al., 2012a; Trainer et al., 2012), strain, and physiological status of the population. Furthermore, the interaction between algae and bacteria has been shown to enhance DA production (Bates et al., 1995). This toxin variability makes monitoring and management of shellfishharvesting activities very challenging. Research has started to address the question of how specific environmental conditions and resulting physiological changes may influence DA production in these diatoms. Suggested environmental stimuli leading to DA production in Pseudo-nitzschia spp. include macronutrients such as silica. Low concentrations of silica have been reported to increase DA production (Pan et al., 1996; Amato et al., 2010), which may involve diversion of energy from cell division to production of toxin. In addition, micronutrients also have been reported to influence DA production (Rue and Bruland, 2001; Wells et al., 2005). Experiments have shown that iron levels are important in the promotion of Pseudo-nitzschia spp. blooms (Hutchins et al., 1998; Tsuda et al., 2003; Silver et al., 2010; Trick et al., 2010), and it has been hypothesized that DA could be involved indirectly in the acquisition of iron by facilitating copper uptake (Wells et al., 2005). Usually, in eukaryotic organisms, copper is taken up by membraneassociated copper-importers (Balamurugan and Schaffner, 2006); however, Rueter et al. (1981) suggested that copper might also enter diatom cells using the silicic-acid transport site, as copper inhibits silicic acid uptake. If copper uses the silicic-acid transport system, then high copper could induce silica limitation, thereby increasing DA production. It is, however, not clear yet how the interaction of these two nutrients affects production of DA in Pseudo-nitzschia spp. In aquaculture settings, copper is used as an algicide in gear paint (Schiff et al., 2003), as a feed supplement (Clearwater et al., 2002), and to treat and prevent fungal and bacterial diseases (Fernandes et al., 2009). Various human practices, from shipping to aquaculture itself, may change water chemistry locally in ways that could potentially induce DA production by endemic Pseudo-nitzschia spp. populations. It is necessary, therefore, to understand environmental triggers for up-regulation of DA production to assess the risk of ASP affecting this industry as it develops. Thus far, the role of DA in copper uptake, or otherwise how the copperuptake system works, is not known. Furthermore, there have been no studies evaluating the effects of copper and silica interaction upon DA toxicity in Pseudo-nitzschia, which motivated the present research. In this study we investigated how the interaction of silica and copper affects toxin production by a culture of Pseudo-nitzschia multiseries so that the risk of DA toxicity impacting the developing mussel-aquaculture industry can be managed effectively. We chose to use P. multiseries in this study not only because this is one of the most studied species of Pseudo-nitzschia, but also because the entire genome sequence for this species will soon be available
(http://genome.jgi-psf.org/Psemu1/Psemu1.home.html). It is, however, important to consider species variability in toxin production, as well as species-specific sensitivity to nutrient stress (Lelong et al., 2012a), before generalizing the results from this study. We used a cultured isolate of P. multiseries to: 1) determine possible effects of copper concentrations on DA production; and 2) characterize the copper-uptake system by analyzing copper and silicate uptake under different concentrations of external copper and silicate. In addition, we sampled proposed mussel farming areas on the New England coast for the presence of potentially toxigenic Pseudo-nitzschia species. 2. Materials and methods 2.1. Cultures and culture experiments Pseudo-nitzschia multiseries strain (CCL69) was used as a model organism. This axenic strain was obtained from the Culture Collection of Algae and Protozoa (CCAP, Scotland, United Kingdom). Batch cultures were maintained in artificial seawater enriched with nutrients at f/2 (Guillard, 1975) concentrations at 16 1 C on a 12 h:12 h light:dark cycle with light intensity of 110 mmol photons m2 s1. A factorial experiment was designed to analyze possible interactions between copper and silica upon the production of DA in this species. Several nutrient concentrations (20e300 mM silica and 0e 3.92 107 M copper) were tested to determine an appropriate range based upon the growth rate of P. multiseries. The factorial grid consisted of three concentrations of copper: Low (no copper enrichment); Sufficient (pCu 14.7 ¼ 1.96 108 Cu2þ M); and High (pCu 13.4 ¼ 3.92 107 Cu2þ M) and three concentrations of sodium silicate: low (75 mM), sufficient (150 mM), and high (300 mM), with N ¼ 4 in each treatment block. Cupric ion activities were determined using the chemical equilibrium program MINEQL (Environmental Research Software). Pseudo-nitzschia multiseries cells were collected from an exponentially-growing culture and, to remove any copper or silicate left in the media, the cells were centrifuged at 1000 g for 10 min at 16 C. The overlying water was decanted, and the cells were washed twice with artificial seawater purified in a Chelex 100 ion-exchange resin (Bio-Rad). Five-hundred-ml polystyrene culture flasks containing 250 ml of Aquil medium (Price et al., 1988/89) were inoculated with a starting cell count of approximately 5,000 cells ml1. The cultures were maintained at the temperature, light intensity, and light-cycle conditions indicated above. The growth of each culture was determined three times a week for two weeks from flow-cytometer counts (FACScan, BD BioSciences). Samples of 300 ml were taken from each culture and vortexed before being measured in the flow cytometer so that individual cells rather than groups of cells were counted. Separation of cells from chains was verified in a light microscope (Zeiss Axioscop 2 MOT Plus). Using a flow rate of 60 ml min1, each sample was counted for 30 s. Cells were distinguished based upon particle size (Forward Scatter) and chlorophyll fluorescence (FL3 detector, >650 nm). Nutrient concentrations were quantified with a TRAACS 800 autoanalyzer (Bran and Luebbe), following the protocol described by Hansen and Koroleff (1999). The determination of silica is based upon formation of a blue silicomolybdic complex (detection limit 0.10 mM). Silica uptake was calculated by subtracting final dissolved silicate concentrations from the initial amount. Copper concentrations were quantified by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) (detection limit, Cu < 8.5e09 M), which was performed by an external contract laboratory (Applied Speciation and Consulting LLC). DA concentrations were quantified 2 days after the beginning of the stationary phase using a cELISA kit (detection limit 300 pg DA ml1) (Biosense
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Laboratories) following the protocol provided by the manufacturer. Samples taken from each treatment were divided into duplicates of equal volume. Cells in one of the duplicates were disrupted by sonication (2 min) and later filtered through a 0.22-mm celluloseacetate filter (Sartorius). The filtrate was used to obtain total DA (TDA) concentrations. Dissolved DA (DDA, extracellular) concentrations were obtained by filtering the other sample duplicate without disrupting the cells. Intracellular DA (IDA) concentrations were obtained by subtracting DDA from TDA content. The absorbance for the ELISA assay was read in a microplate reader (Perkin Elmer LS 50 B) at 450 nm.
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Additionally, seawater samples were enriched with nutrients at f/ 10 concentrations and incubated under phytoplankton growth conditions described above. Enriched samples were screened periodically for Pseudo-nitzschia spp. under the light microscope. Micropipette isolation was used to obtained mono-specific cultures of Pseudo-nitzschia sp. Upon isolation, cells were grown in f/2 medium. Genomic DNA isolated from each mono-specific culture was extracted and then sequenced using a 3130xl Genetic Analyzer (Applied Biosystems). A Blast-search was used for species identification. 2.4. Statistical analysis
2.2. Media preparation Aquil basal medium was prepared according to Price et al. (1988/ 89). The synthetic seawater with major nutrients, except silicate, vitamins, and EDTA-trace metals, was passed through a chromatographic column packed with Chelex 100 resin. A separate column was used for silicate purification. Vitamins were not chelex treated. The trace-metal solution was prepared copper-free. Copper stock solutions were prepared individually to modify concentrations. All reagents used in media preparation were trace-metal grade. All glassware and pipette tips used in media preparation and during culture handling were acid washed and then rinsed with Milli-Q water. 2.3. Assessment of the micronutrient status of Pseudo-nitzschia populations in mussel-farming areas Field samples were collected from April to October 2009, and from February to August 2010, at two sites, one off the coast of Hampton, New Hampshire (Isles of Shoals farm) the other between Cape Cod and Martha’s Vineyard, Massachusetts (proposed farming sites) in New England. Copper and silicate concentrations were measured as described above. Samples were screened for Pseudonitzschia under the light microscope and by PCR amplification of the ITS1 internal-transcribed spacer. One liter of each sample was filtered through a 0.45-mm pore-size, 47-mm-diameter, mixedcellulose filter (Whatman). Genomic DNA was extracted from the filters using the DNeasy Plant Mini Kit (Qiagen) following manufacturer instructions. Extracted DNA then was amplified with genus-specific primers (PnAll F/R; Hubbard et al., 2008). Standard PCR 25-ml reactions (12.5 ml Master Mix for PCR from BioRad, 1 ml of each of the two primers at10 mM, and 9.5 ml of doubledistilled water) were performed on a Primus 25 (MWGAG BIOTECH) Thermal Cycler. The PCR products were visualized in a 2% agarose gel. Genomic DNA of P. multiseries was used as a positive control, and sterile, nanopure water served as a negative control.
Main effects and the interaction between silicate and copper were analyzed for each response variable using two-way ANOVA. Tukey’s post hoc test was used to determine individual treatment differences (StatGraphics 5. Manugistics). A level of confidence of 95% was used to determine significance. 3. Results 3.1. Effects of copper and silica concentrations on growth and DA production of Pseudo-nitzschia multiseries in culture Experimental silicate concentrations affected maximum cell counts in cultures significantly. Higher cell densities were observed in treatments with high concentrations of silicate (Fig. 1). Copper concentration, on the other hand, did not affect the maximal culture density of P. multiseries significantly. Importantly, the interaction between copper and silicate did affect final cell count significantly. When concentrations of both copper and silicate were constrained, P. multiseries cultures reached the lowest final cell densities. On the contrary, when the concentrations of both nutrients were high, cultures reached the highest cell densities. Silica-uptake results suggested that copper does not use the silicate uptake system in Pseudo-nitzschia multiseries. If copper used the silica-uptake system, uptake of silica would have decreased with increasing copper concentration. Instead, a synergistic effect was observed when the two nutrients were limiting population growth (significant MANOVA interaction term) (Fig. 2). Uptake of silicate in these nutrient-limited cultures was an order of magnitude higher than in the rest of the experimental treatments. The highest Total DA (TDA) per cell, as well as Dissolved DA (DDA) per cell, concentrations occurred in cultures grown under high silicate/high copper and low silicate/sufficient copper. Cultures grown under sufficient silica/sufficient copper showed the highest proportion of TDA to DDA per cell (Fig. 3). The highest
Fig. 1. 3D plot of the effect of silicate (mM) and copper concentration (M) on maximum cell concentration.
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Fig. 2. 3D plot of the effect of silicate and copper concentration (mM) on silicate uptake (moles of Si per cell).
absolute DDA (DA ml1) occurred in the high Si/high Cu treatment (Fig. 4). 3.2. DA production by Pseudo-nitzschia populations in musselfarming areas In the field, silicate and copper concentrations were always undetectable by the methods of analysis used in this study (detection limit, Si 0.10 107 M, Cu 8.5 109 M). Pseudo-nitzschia spp. cells were identified twice (April 14 and July 7, 2009) in samples from Hampton, New Hampshire. On both occasions, the cells were detected in field samples first microscopically and then by PCR amplification of the ITS1 fragment. On one of these occasions (April 14), mucous aggregations were plentiful. These mucous aggregations contained many empty phytoplankton cell walls and frustules, and dinoflagellates cysts, and vegetative cells of Pseudonitzschia-like diatoms were also observed within these aggregates. The best match of the blast-search for the two species isolated from the samples collected July 7, 2009 at the New Hampshire site corresponded to Pseudo-nitzschia granii (Fig. 5A) (length ¼ 39.98 1.81 mm, width ¼ 2.1 0.38 mm) for one strain and Pseudo-nitzschia delicatissima for the other strain (Fig. 5B) (length ¼ 49.66 8.3 mm, width ¼ 2.2 0.38 mm). Although DA was below detection in raw field samples, laboratory cultures of both species tested positive for DA production, with concentrations of over 1 pg cell1 (P. cf. granii ¼ 1.10 0.04 TDA; P. cf. delicatissima ¼ 2.61 0.82 TDA). In Cape Cod, Massachusetts Pseudo-nitzschia sp. was detected once (May 11, 2009) by PCR amplification. No Pseudo-nitzschia strains were isolated.
Fig. 3. Plot of the effect of silicate and copper concentration on total domoic acid (DA) per cell, dissolved domoic acid (DDA, extracellular), and intracellular domoic acid (IDA).
4. Discussion The factorial design of our culture experiment revealed a significant interaction between silica and copper in regulating domoic-acid production by Pseudo-nitzschia multiseries. Silica is a major structural component of cell walls in diatoms (Round et al., 1990), and thus, silica availability is expected to control growth. Copper at high concentrations is toxic (Lelong et al., 2012b), but growth was not diminished by the concentrations used in the present study, implying that the concentrations used were not high enough to inhibit growth. The highest copper concentration used by Lelong et al. (2012b), though, was 1.53 times higher than the highest concentration used in the present study. Lelong et al. (2012b), however, reported that cultures exposed to 2 107 Cu2þ M did not grow; whereas, the present study shows that growth was not inhibited in cultures grown in 3.92 107 Cu2þ M. A possible explanation for the difference in copper toxicity might be that the media used for the experimental treatments in this study was Aquil media; whereas, Lelong et al. (2012b) used f/2 media. Chemical speciation, as well as trace metal contamination and precipitate formation, is controlled in Aquil medium but not in f/2-enriched natural seawater. Bioavailability of copper, therefore, might had been higher in the study by Lelong et al. (2012b) than in the present study. Copper is an essential component of many cellular proteins; therefore, limited availability of this metal results in growth limitation (Peers et al., 2005). In our experiment, responses of P. multiseries to copper and silica when both were either high or low were amplified
Fig. 4. Plot the effect of silicate and copper concentration on absolute domoic acid (DDA ml1).
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Fig. 5. A) P. cf. granii, and B) P. cf. delicatissima isolated from Hampton, New Hampshire.
compared to the responses to individual nutrients. The highest cell densities were obtained when the concentrations of both nutrients were high. The lowest final cell count, on the other hand, occurred when both nutrients were scarce. A synergistic effect of copper and silica upon silica uptake was also observed. Silica-uptake increased enormously (an order of magnitude higher than in the other treatments) in cultures wherein both nutrients were synergistically co-limiting growth. Hutchins and Bruland (1998) found that silicic acid:nitrate (Si:N) uptake ratio was two-to-three times higher in diatoms stressed by iron starvation than in diatoms under ironreplete conditions. These authors suggested that iron starvation led to more-silicified, faster-sinking diatoms. Various studies have documented high cell densities of Pseudo-nitzschia sp. in deep waters (Rines et al., 2002; Sekula-Wood et al., 2009), and Pseudonitzschia blooms are thought to be initiated from seed populations present beneath the euphotic zone (Trainer et al., 2000). Furthermore, we tested the survival of P. multiseries under cold and dark conditions and found that this species is capable of surviving such conditions by using energy stored in the form of lipid (Fuentes et al., unpublished data). Hence, increased silica uptake under nutrientstarvation may be a strategy to access nutrients in deep waters. Additionally, Silica-uptake results suggested that copper does not use the silicate uptake system in Pseudo-nitzschia multiseries. If copper used the silica-uptake system, uptake of silica would have decreased with increasing copper concentration. Also, limitation of silica by copper would have increased DA concentrations in treatments with higher copper concentrations and low silica. The highest TDA and DDA per cell concentrations, instead, occurred in high silicate/high copper and low silicate/sufficient copper treatments. Previous studies have shown increased production of DA with decreasing silica concentrations (Pan et al., 1996; Amato et al., 2010) and with copper toxicity (Maldonado et al., 2002; Wells et al., 2005). But, as mentioned above, the levels of copper used in our study were not toxic to P. multiseries. Rue and Bruland (2001) reported that DA is capable of chelating copper because its chemical structure is similar
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to the phytosiderophore mugenic acid. Later, Maldonado et al. (2002) demonstrated that, with high copper concentrations, higher DA amounts were released into the medium compared to DA release with lower concentrations of the metal. Based upon those results, Maldonado et al. (2002) suggested that DA chelates copper, reducing its availability and thus toxicity. As the higher absolute DDA occurred in the high Si/high Cu treatment, it is possible, therefore, that DA prevented toxicity, and consequently growth inhibition, in treatments with high copper. A more recent study (Lelong et al., 2012b), though, reported that elevated copper concentrations did not induce DA production in P. multiseries. Also, DA did not protect the cell against copper toxicity in this study. A probable cause for the contrasting results of Lelong et al. (2012b) might be inter-strain variability. Furthermore, cultures mixed with bacteria in natural seawater enriched media were used in that study, therefore, a direct comparison between the present study that used a bacteria-free strain cultured in AQUIL and Lelong et al. (2012b) cannot be made. Production of DA has been speculated to be a mechanism for prompt elimination of excess photosynthetic energy that is no longer used for growth and when metabolism decreases (Bates, 1998). In the present study, when both copper and silicate were scarce, growth was depressed and silica uptake increased considerably compared to the other treatments. Our interpretation is that excess photosynthetic energy was used for silica uptake rather than toxin production. Even though low silicate/high copper concentrations increased TDA, the highest TDA occurred in high silicate/ high copper and low silicate/sufficient copper cultures. Low silicate/low copper cultures did not have the highest TDA, perhaps because these cultures were using energy for silicate uptake rather than toxin production. Turning to the field component of the present study, two species of Pseudo-nitzschia capable of producing DA were detected in sites off the coast of New Hampshire where commercial blue-mussel aquaculture is being developed. The identification of these species, however, is only based on the best match of a blast search. Currently, not all species of Pseudo-nitzschia have the internal transcribed spacer (ITS) sequence in public databases (e.g. GenBank). Therefore, TEM micrographs, with accompanying morphometrics, must also be done on that species to confirm species identity. Unfortunately, further identification of these species was not possible in the present study; EM was not performed before isolates were lost. Nevertheless, the results of this study indicate the existence of a potential risk of shellfish contamination with DA in New England because two Pseudo-nitzschia species with different morphologies and sequence results were found and confirmed to be capable of producing DA. At the time of sampling in both New Hampshire and Massachusetts, the Pseudo-nitzschia populations were not producing measurable quantities of DA, indicating that environmental conditions were not conducive to up-regulation of domoic-acid synthesis pathways in the diatoms. Variation in DA content within Pseudo-nitzschia spp. cells from both natural populations and cultures has been described previously, and nutrient status of cells has been implicated in toxin variation (Bates and Trainer, 2008). Our results showed clearly that the low silicate and copper concentrations present in the New Hampshire mussel-farming site were not conducive to high DA production in the model species. At low silicate levels, however, a small increase in copper led to a doubling in DA content in P. multiseries cultures. A concurrent increase in silicate mitigated the toxin increase, suggesting a mechanism by which DA production can remain low to moderate when all nutrients, including both silicate and copper, are increased simultaneously, e.g. during upwelling of deep water or following runoff from land. At very high copper and silicate levels, however, DA content also increased significantly, possibly in this case
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because copper was approaching inhibitory levels and DA was being up-regulated to chelate copper. 5. Conclusions This study demonstrated the presence of at least two species of Pseudo-nitzschia capable of producing DA in sites off the coast of New Hampshire where commercial blue-mussel aquaculture is being developed. This finding underscores the importance of anticipating the potential for mussel farming in this area to be impacted by Amnesic Shellfish Poisoning (ASP). Overall, the results of this experiment suggest that a small increase in copper, without a simultaneous increase in silicate, could precipitate conditions conducive to DA up-regulation in endemic populations in New England mussel-farming areas. The main finding of this study, though, is that care must be taken to minimize activities that may lead to local copper increases in New England mussel-farming sites where potentially-toxigenic Pseudonitzschia species are endemic. Acknowledgments We appreciate the cooperation of Scott Lindell and Richard Langan, who provided water samples from Massachusetts and New Hampshire, respectively. We thank Jennifer Alix, Shannon Meseck, and Steven Pitchford for assisting with algal culturing and chemical analyses. This project was supported by the NOAA National Aquaculture Program, through a National Research Council PostDoctoral Fellowship to M.S.F., and by the Northeast Fisheries Science Center of the National Marine Fisheries Service. References Amato, A., Ludeking, A., Kooistra, W.H.C.F., 2010. Intracellular domoic acid production in Pseudo-nitzschia multistriata isolated from the Gulf of Naples (Tyrrhenian Sea, Italy). Toxicon 55, 157e161. Balamurugan, K., Schaffner, W., 2006. Copper homeostasis in eukaryotes: teetering on a tightrope. Biochimica et Biophysica Acta 1763, 737e746. Bates, S.S., Trainer, V.L., 2008. The ecology of harmful diatoms. In: Granéli, E. (Ed.), Ecology of Harmful Algae. In: Turner, J.T. (Ed.),, Series: Ecological Studies, vol 189. SpringereVerlag, Berlin, Heidelberg, New York, pp. 81e93. Bates, S.S., Douglas, D.J., Doucette, G.J., Léger, C., 1995. Enhancement of domoic acid production by reintroducing bacteria to axenic cultures of the diatom Pseudonitzschia multiseries. Natural Toxins 3, 428e435. Bates, S.S., Garrison, D.L., Horner, R.A., 1998. Bloom dynamics and physiology of domoic-acid-producing Pseudo-nitzschia species. In: Anderson, D.M., Cembella, A.D., Hallegraeff, G.M. (Eds.), Physiological Ecology of Harmful Algal Blooms. SpringereVerlag, Berlin, Germany, pp. 267e292. Bates, S.S., 1998. Ecophysiology and metabolism of ASP toxin production. In: Anderson, D.M., Cembella, A.D., Hallegraeff, G.M. (Eds.), Physiological Ecology of Harmful Algal Blooms. SpringereVerlag, Berlin, Germany, pp. 405e426. Clearwater, S.J.A., Farag, M., Meyer, J.S., 2002. Bioavailability and toxicity of dietborne copper and zinc to fish. Comparative Biochemistry and Physiology Part C 132, 269e313. FAO Fisheries and Aquaculture, 2012, http://www.fao.org/fishery/culturedspecies/ Mytilus_edulis/en (August 6, 2012). Fernandes, D., Bebianno, M.J., Porte, C., 2009. Assessing pollutant exposure in cultured and wild sea bass (Dicentrarchus labrax) from the Iberian Peninsula. Ecotoxicology 18, 1043e1050. Fisheries and Oceans Canada, 2010. Cultured Aquatic Species Information Programme Mytilus edulis (Linnaeus, 1758). Canadian Aquaculture Production Statistics. Retrieve from: http://www.dfo-mpo.gc.ca/stats/aqua/aqua10-eng. htm (July 30, 2012). Guillard, R.R.L., 1975. Culture of phytoplankton for feeding marine invertebrates. In: Smith, W.L., Chanley, M.H. (Eds.), Culture of Marine Invertebrate Animals. Plenum Press, New York, pp. 26e60. Hansen, H.P., Koroleff, F., 1999. Determination of nutrients. In: Grasshoff, K., Kremling, K., Ehrhardt, M. (Eds.), Methods of Seawater Analysis, third ed. WileyVCH, Weinheim, New York, USA, pp. 159e228. Hargraves, P.E., Zhang, J., Wang, R., Shimizu, Y., 1993. Growth characteristics of the diatoms Pseudo-nitzschia pungens and P. fraudulenta exposed to ultraviolet radiation. Hydrobiology 269/270, 207e212. Hilbish, T.J., Mullinax, A., Dolven, S.I., Meyer, A., Koehn, R.K., Rawson, P.D., 2000. Origin of the anti-tropical distribution pattern in marine mussels (Mytilus spp.): routes and timing of transequatorial migration. Marine Biology 136, 69e77.
Hoagland, P., Anderson, D.M., Kaoru, K., White, A.W., 2002. The economic effects of harmful algal blooms in the United States: estimates, assessment issues, and information needs. Estuaries 25, 819e837. Hubbard, K.A., Rocap, G., Armbrust, E.V., 2008. Inter- and intraspecific community structure within the diatom genus Pseudo-nitzschia (Bacillariophyceae). Journal of Phycology 44, 637e649. Hutchins, D.A., Bruland, K.W., 1998. Iron-limited diatom growth and Si:N uptake ratios in a coastal upwelling regime. Nature 393, 561e564. Hutchins, D.A., DiTullio, G., Zhang, Y., Bruland, K.W., 1998. An iron limitation mosaic in the California upwelling regime. Limnology and Oceanography 43, 1037e1054. Lelong, A., Hégaret, H., Soudant, P., Bates, S.S., 2012a. Pseudo-nitzschia (Bacillariophyceae) species, domoic acid and amnesic shellfish poisoning: revisiting previous paradigms. Phycologia 51, 168e216. Lelong, A., Jolley, D.F., Soudant, P., Hégaret, H., 2012b. Impact of copper exposure on Pseudo-nitzschia spp. physiology and domoic acid production. Aquatic Toxicology 118-119, 37e47. Maldonado, M.T., Hughes, M., Rue, E., Wells, M.L., 2002. The effect of Fe and Cu on the growth and domoic acid production of Pseudo-nitzschia multiseries and Pseudo-nitzschia australis. Limnology and Oceanography 47, 515e526. Marshall, H.G., Burchardt, L., Lacouture, R., 2005. A review of phytoplankton composition within Chesapeake Bay and its tidal estuaries. Journal of Plankton Research 27, 1083e1102. Pan, Y., Subba Rao, D.V., Mann, K.H., Brown, R.G., Pocklington, R., 1996. Effects of silicate limitation on the production of domoic acid, a neurotoxin, by the diatom Pseudo-nitzschia multiseries. I. Batch culture studies. Marine EcologyProgress Series 131, 225e233. Peers, G., Quesnel, S.-.A., Price, N.M., 2005. Copper requirements for iron acquisition and growth of coastal and oceanic diatoms. Limnology Oceanography 50, 1149e1158. Phlips, E.J., Badylak, S., Youn, S., Keller, K., 2004. The occurrence of potentially toxic dinoflagellates and diatoms in a subtropical lagoon, the Indian River Lagoon, Florida, USA. Harmful Algae 3, 39e49. Price, N.M., Harrison, G.I., Hering, J.G., Hudson, R.J., Nirel, P.M.V., Palenik, B., Morel, F.M.M., 1988/89. Preparation and chemistry of the artificial algal culture medium Aquil. Biological Oceanography 6, 443e461. Rines, J.E.B., Donaghay, P.L., Dekshenieks, M.M., Sullivan, J.M., Twardowski, M.S., 2002. Thin layers and camouflage: hidden Pseudo-nitzschia populations in a fjord in the San Juan Islands, Washington, USA. Marine Ecology-Progress Series 225, 123e137. Round, F.E., Crawford, R.M., Mann, D.G., 1990. The Diatoms, Biology and Morphology of the Genera. Cambridge University Press, Cambridge. Rue, E., Bruland, K.W., 2001. Domoic acid binds iron and copper: a possible role for the toxin produced by the marine diatom Pseudo-nitzschia. Marine Chemistry 76, 127e134. Rueter, J.G., Chisholm, S.W., Morel, F.M.M., 1981. Effects of copper toxicity on silicic acid uptake and growth in Thalassiosira pseudonana. Journal of Phycology 17, 270e278. Schiff, K.C., Diehl, D., Valkirs, A., 2003. Copper Emissions from Antifouling Paint on Recreational Vessels. Technical Report 405, Southern California Coastal Water Research Project. Sekula-Wood, E., Schnetzer, A., Benitez-Nelson, C.R., Anderson, C., Berelson, W.M., Brzezinski, M.A., Burns, J.M., Caron, D.A., Cetinic, I., Ferry, J.L., Fitzpatrick, E., Jones, B.H., Miller, P.E., Morton, S.L., Schaffner, R.A., Siegel, D.A., Thunell, R., 2009. Rapid downward transport of the neurotoxin domoic acid in coastal waters. Nature Geoscience 2, 272e275. Shuler, A.J., Paternoster, J., Brim, M., Nowocin, K., Tisdale, T., Neller, K., Cahill, J.A., Leighfield, T.A., Fire, S., Wang, Z., Morton, S., 2012. Spatial and temporal trends of the toxic diatom Pseudo-nitzschia in the Southeastern Atlantic United States. Harmful Algae 17, 6e13. Silver, M.W., Bargu, S., Coale, S.L., Benitez-Nelson, C.R., Garcia, A.C., Roberts, K.J., Sekula-Wood, E., Bruland, K.W., Coale, K.H., 2010. Toxic diatoms and domoic acid in natural and iron-enriched waters of the oceanic Pacific. Proceedings of the National Academy of Sciences of the United States of America 107, 20762e 20767. Trainer, V.L., Adams, N.G., Bill, B.D., Stehr, C.M., Wekell, J.C., Moeller, P., Busman, M., Woodruff, D.L., 2000. Domoic acid production near California coastal upwelling zones, June 1998. Limnology and Oceanography 45, 1818e1833. Trainer, V.L., Bates, S.S., Lundholm, N., Thessen, A.E., Cochlan, W.P., Adams, N.G., Trick, C.G., 2012. Pseudo-nitzschia physiological ecology, phylogeny, toxicity, monitoring and impacts on ecosystem health. Harmful Algae 14, 271e300. Trick, C.G., Bill, B., Cochlan, W.P., Wells, M.L., Trainer, V.L., Pickell, L.D., 2010. Iron enrichment stimulates toxic diatom production in High Nitrate, Low Chlorophyll areas. Proceedings of the National Academy of Science of the United States of America 107, 5887e5892. Tsuda, A., Takeda, S., Saito, H., Nishioka, J., Nojiri, Y., Kudo, I., Kiyosawa, H., Shiomoto, A., Imai, K., Ono, T., Shimamoto, A., Tsumune, D., Yoshimure, T., Aono, T., Hinuma, A., Kinugasa, M., Suzuki, K., Sohrin, Y., Noiri, Y., Tani, H., Deguchi, Y., Tsurushima, N., Ogawa, H., Fukami, K., Kuma, K., Saino, T., 2003. A mesoscale iron enrichment in the western subarctic Pacific induces a large centric diatom bloom. Science 300, 958e961. Villareal, T.A., Roelke, D.L., Fryxell, G.A., 1994. Occurrence of the toxic diatom Nitzschia pungens f. multiseries in Massachusetts Bay. Marine Environmental Research 37, 417e423. Wells, M.L., Trick, C.G., Hughes, W.P., Trainer, V.L., 2005. Domoic acid: the synergy of iron, copper, and the toxicity of diatoms. Limnology and Oceanography 50, 1908e1917.
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Marine Environmental Research journal homepage: www.elsevier.com/locate/marenvrev
Control of domoic acid toxin expression in Pseudo-nitzschia multiseries by copper and silica: Relevance to mussel aquaculture in New England (USA) M. Soledad Fuentes a, b, Gary H. Wikfors a, * a b
National Oceanographic and Atmospheric Administration, National Marine Fisheries Service, Northeast Fisheries Science Center, 212 Rogers Avenue, Milford, CT 06460, USA Algenol Biofuels, 16121 Lee Rd, Fort Myers, FL 33912, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 June 2012 Received in revised form 11 October 2012 Accepted 17 October 2012
The production of the toxin Domoic Acid (DA) by the diatoms Pseudo-nitzschia spp. is affected by several environmental factors, among them copper and silica. The effects of these nutrients upon DA production have been studied individually, but not in combination. There is evidence, however, that in diatoms copper can enter the cell via the silicic-acid transport site. The goal of this study was to analyze the effect of the interaction between copper and silicic-acid supply upon DA production in Pseudo-nitzschia multiseries. The study was motivated by concerns about the risk of toxigenic Pseudo-nitzschia spp. impacting mussel aquaculture in New England (USA). The results of the present study do not indicate that copper uses the silicic acid transport site to enter the cell; nevertheless, there is an interaction between these two nutrients that produces a synergistic affect upon toxin production. A small increase in copper, without a simultaneous increase in silicate, as well as an increase in both copper and silicate, leads to DA up-regulation. Furthermore, the field component of this study reports the presence of species of Pseudonitzschia on the New England coast that are capable of producing DA. Together these findings indicate that risk of DA impacting mussel aquaculture along the coast of New England would be increased by an unusual enrichment of copper in the vicinity of mussel farms. Published by Elsevier Ltd.
Keywords: Pseudo-nitzschia Domoic acid Copper Silicate
1. Introduction Mussel aquaculture is a growing industry, with world production dominated by Europe, China, Canada and the United States (FAO, 2012). Three species of mussel are cultured in North America, Mytilus trossulus and Mytilus galloprovincialis on the West Coast, and Mytilus edulis on the East Coast (Hilbish et al., 2000). East-Coast production of this species has increased enormously during the last two decades. The main production center of eastern North America is Prince Edward Island (PEI), Canada, which produces nearly 20 thousand tonnes annually, according to the Fisheries and Oceans Canada (2010). As most of the eastern US mussel market is supplied by imports from Canada, the US government, through H.R. 2010, the National Offshore Aquaculture Act of 2007, provided support for the commercial fishing industry to stimulate a sustainable, offshore aquaculture industry in New England and nationwide. Consequently, mussel aquaculture is a developing industry in New England. Shellfish harvests, including those from mussel farming, are affected every year by Harmful Algal Blooms (HABs), which can
* Corresponding author. Tel.: þ1 203 882 6525; fax: þ1 203 882 6570. E-mail address: [email protected] (G.H. Wikfors). 0141-1136/$ e see front matter Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.marenvres.2012.10.005
cause long-term harvest closures resulting in great economic losses. In the northeastern United States, HAB episodes are a serious and recurrent problem, mainly associated with Paralytic Shellfish Poisoning (PSP) (Hoagland et al., 2002). Several monitoring networks have been implemented to manage the impact of PSP blooms. One of the most notorious human poisoning events attributable to HABs occurred in 1987 when more than 100 people became ill and three persons died after consuming mussels harvested off PEI (Bates et al., 1998). These mussels were found to be contaminated by domoic acid (DA) produced by the diatom Pseudonitzschia multiseries. Recent evidence suggests that Pseudo-nitzschia, a genus that blooms regularly on the US west coast, is expanding in range and has also been detected more frequently off the East Coast of United States. Species of Pseudo-nitzschia have been reported in Massachusetts Bay (Villareal et al., 1994), Narragansett Bay (Hargraves et al., 1993), and recently in Florida, Chesapeake Bay, and Maine (Phlips et al., 2004; Marshall et al., 2005; Van Dolah and Leighfield, unpublished data). In the February, 2007 issue of Plankton News (http://www.chbr.noaa.gov/pmn/_docs/ PlanktonNews/PlanktonNews_February2007.pdf.), a bloom of Pseudo-nitzschia also was reported in North Carolina. Furthermore, Shuler et al. (2012) reported the presence of Pseudo-nitzschia spp. from North Carolina through northern Florida, with six bloom
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events (three of them with detectable levels of DA) throughout nine years of sampling. Given that this genus apparently is expanding throughout the North American East Coast, it is crucial to assess risks to protect the public from Amnesic Shellfish Poisoning (ASP) that can result from the ingestion of DA-contaminated shellfish. Domoic acid is a tricarboxylated amino acid e an analog of the neurotransmitter L-glutamic acid e which binds to the glutamate receptor, causing neuronal depolarization in the brain resulting in permanent memory loss in mammals in severe cases. Blooms of DA-producing Pseudo-nitzschia spp. have been associated with mortalities of both wildlife and humans (reviewed by Lelong et al., 2012a; Trainer et al., 2012). Presence of Pseudo-nitzschia spp. in the water, though, does not necessarily indicate that DA is being produced that could contaminate shellfish and make them unfit for human consumption. Production of DA by Pseudo-nitzschia spp. is extremely variable, depending upon species (Lelong et al., 2012a; Trainer et al., 2012), strain, and physiological status of the population. Furthermore, the interaction between algae and bacteria has been shown to enhance DA production (Bates et al., 1995). This toxin variability makes monitoring and management of shellfishharvesting activities very challenging. Research has started to address the question of how specific environmental conditions and resulting physiological changes may influence DA production in these diatoms. Suggested environmental stimuli leading to DA production in Pseudo-nitzschia spp. include macronutrients such as silica. Low concentrations of silica have been reported to increase DA production (Pan et al., 1996; Amato et al., 2010), which may involve diversion of energy from cell division to production of toxin. In addition, micronutrients also have been reported to influence DA production (Rue and Bruland, 2001; Wells et al., 2005). Experiments have shown that iron levels are important in the promotion of Pseudo-nitzschia spp. blooms (Hutchins et al., 1998; Tsuda et al., 2003; Silver et al., 2010; Trick et al., 2010), and it has been hypothesized that DA could be involved indirectly in the acquisition of iron by facilitating copper uptake (Wells et al., 2005). Usually, in eukaryotic organisms, copper is taken up by membraneassociated copper-importers (Balamurugan and Schaffner, 2006); however, Rueter et al. (1981) suggested that copper might also enter diatom cells using the silicic-acid transport site, as copper inhibits silicic acid uptake. If copper uses the silicic-acid transport system, then high copper could induce silica limitation, thereby increasing DA production. It is, however, not clear yet how the interaction of these two nutrients affects production of DA in Pseudo-nitzschia spp. In aquaculture settings, copper is used as an algicide in gear paint (Schiff et al., 2003), as a feed supplement (Clearwater et al., 2002), and to treat and prevent fungal and bacterial diseases (Fernandes et al., 2009). Various human practices, from shipping to aquaculture itself, may change water chemistry locally in ways that could potentially induce DA production by endemic Pseudo-nitzschia spp. populations. It is necessary, therefore, to understand environmental triggers for up-regulation of DA production to assess the risk of ASP affecting this industry as it develops. Thus far, the role of DA in copper uptake, or otherwise how the copperuptake system works, is not known. Furthermore, there have been no studies evaluating the effects of copper and silica interaction upon DA toxicity in Pseudo-nitzschia, which motivated the present research. In this study we investigated how the interaction of silica and copper affects toxin production by a culture of Pseudo-nitzschia multiseries so that the risk of DA toxicity impacting the developing mussel-aquaculture industry can be managed effectively. We chose to use P. multiseries in this study not only because this is one of the most studied species of Pseudo-nitzschia, but also because the entire genome sequence for this species will soon be available
(http://genome.jgi-psf.org/Psemu1/Psemu1.home.html). It is, however, important to consider species variability in toxin production, as well as species-specific sensitivity to nutrient stress (Lelong et al., 2012a), before generalizing the results from this study. We used a cultured isolate of P. multiseries to: 1) determine possible effects of copper concentrations on DA production; and 2) characterize the copper-uptake system by analyzing copper and silicate uptake under different concentrations of external copper and silicate. In addition, we sampled proposed mussel farming areas on the New England coast for the presence of potentially toxigenic Pseudo-nitzschia species. 2. Materials and methods 2.1. Cultures and culture experiments Pseudo-nitzschia multiseries strain (CCL69) was used as a model organism. This axenic strain was obtained from the Culture Collection of Algae and Protozoa (CCAP, Scotland, United Kingdom). Batch cultures were maintained in artificial seawater enriched with nutrients at f/2 (Guillard, 1975) concentrations at 16 1 C on a 12 h:12 h light:dark cycle with light intensity of 110 mmol photons m2 s1. A factorial experiment was designed to analyze possible interactions between copper and silica upon the production of DA in this species. Several nutrient concentrations (20e300 mM silica and 0e 3.92 107 M copper) were tested to determine an appropriate range based upon the growth rate of P. multiseries. The factorial grid consisted of three concentrations of copper: Low (no copper enrichment); Sufficient (pCu 14.7 ¼ 1.96 108 Cu2þ M); and High (pCu 13.4 ¼ 3.92 107 Cu2þ M) and three concentrations of sodium silicate: low (75 mM), sufficient (150 mM), and high (300 mM), with N ¼ 4 in each treatment block. Cupric ion activities were determined using the chemical equilibrium program MINEQL (Environmental Research Software). Pseudo-nitzschia multiseries cells were collected from an exponentially-growing culture and, to remove any copper or silicate left in the media, the cells were centrifuged at 1000 g for 10 min at 16 C. The overlying water was decanted, and the cells were washed twice with artificial seawater purified in a Chelex 100 ion-exchange resin (Bio-Rad). Five-hundred-ml polystyrene culture flasks containing 250 ml of Aquil medium (Price et al., 1988/89) were inoculated with a starting cell count of approximately 5,000 cells ml1. The cultures were maintained at the temperature, light intensity, and light-cycle conditions indicated above. The growth of each culture was determined three times a week for two weeks from flow-cytometer counts (FACScan, BD BioSciences). Samples of 300 ml were taken from each culture and vortexed before being measured in the flow cytometer so that individual cells rather than groups of cells were counted. Separation of cells from chains was verified in a light microscope (Zeiss Axioscop 2 MOT Plus). Using a flow rate of 60 ml min1, each sample was counted for 30 s. Cells were distinguished based upon particle size (Forward Scatter) and chlorophyll fluorescence (FL3 detector, >650 nm). Nutrient concentrations were quantified with a TRAACS 800 autoanalyzer (Bran and Luebbe), following the protocol described by Hansen and Koroleff (1999). The determination of silica is based upon formation of a blue silicomolybdic complex (detection limit 0.10 mM). Silica uptake was calculated by subtracting final dissolved silicate concentrations from the initial amount. Copper concentrations were quantified by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) (detection limit, Cu < 8.5e09 M), which was performed by an external contract laboratory (Applied Speciation and Consulting LLC). DA concentrations were quantified 2 days after the beginning of the stationary phase using a cELISA kit (detection limit 300 pg DA ml1) (Biosense
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Laboratories) following the protocol provided by the manufacturer. Samples taken from each treatment were divided into duplicates of equal volume. Cells in one of the duplicates were disrupted by sonication (2 min) and later filtered through a 0.22-mm celluloseacetate filter (Sartorius). The filtrate was used to obtain total DA (TDA) concentrations. Dissolved DA (DDA, extracellular) concentrations were obtained by filtering the other sample duplicate without disrupting the cells. Intracellular DA (IDA) concentrations were obtained by subtracting DDA from TDA content. The absorbance for the ELISA assay was read in a microplate reader (Perkin Elmer LS 50 B) at 450 nm.
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Additionally, seawater samples were enriched with nutrients at f/ 10 concentrations and incubated under phytoplankton growth conditions described above. Enriched samples were screened periodically for Pseudo-nitzschia spp. under the light microscope. Micropipette isolation was used to obtained mono-specific cultures of Pseudo-nitzschia sp. Upon isolation, cells were grown in f/2 medium. Genomic DNA isolated from each mono-specific culture was extracted and then sequenced using a 3130xl Genetic Analyzer (Applied Biosystems). A Blast-search was used for species identification. 2.4. Statistical analysis
2.2. Media preparation Aquil basal medium was prepared according to Price et al. (1988/ 89). The synthetic seawater with major nutrients, except silicate, vitamins, and EDTA-trace metals, was passed through a chromatographic column packed with Chelex 100 resin. A separate column was used for silicate purification. Vitamins were not chelex treated. The trace-metal solution was prepared copper-free. Copper stock solutions were prepared individually to modify concentrations. All reagents used in media preparation were trace-metal grade. All glassware and pipette tips used in media preparation and during culture handling were acid washed and then rinsed with Milli-Q water. 2.3. Assessment of the micronutrient status of Pseudo-nitzschia populations in mussel-farming areas Field samples were collected from April to October 2009, and from February to August 2010, at two sites, one off the coast of Hampton, New Hampshire (Isles of Shoals farm) the other between Cape Cod and Martha’s Vineyard, Massachusetts (proposed farming sites) in New England. Copper and silicate concentrations were measured as described above. Samples were screened for Pseudonitzschia under the light microscope and by PCR amplification of the ITS1 internal-transcribed spacer. One liter of each sample was filtered through a 0.45-mm pore-size, 47-mm-diameter, mixedcellulose filter (Whatman). Genomic DNA was extracted from the filters using the DNeasy Plant Mini Kit (Qiagen) following manufacturer instructions. Extracted DNA then was amplified with genus-specific primers (PnAll F/R; Hubbard et al., 2008). Standard PCR 25-ml reactions (12.5 ml Master Mix for PCR from BioRad, 1 ml of each of the two primers at10 mM, and 9.5 ml of doubledistilled water) were performed on a Primus 25 (MWGAG BIOTECH) Thermal Cycler. The PCR products were visualized in a 2% agarose gel. Genomic DNA of P. multiseries was used as a positive control, and sterile, nanopure water served as a negative control.
Main effects and the interaction between silicate and copper were analyzed for each response variable using two-way ANOVA. Tukey’s post hoc test was used to determine individual treatment differences (StatGraphics 5. Manugistics). A level of confidence of 95% was used to determine significance. 3. Results 3.1. Effects of copper and silica concentrations on growth and DA production of Pseudo-nitzschia multiseries in culture Experimental silicate concentrations affected maximum cell counts in cultures significantly. Higher cell densities were observed in treatments with high concentrations of silicate (Fig. 1). Copper concentration, on the other hand, did not affect the maximal culture density of P. multiseries significantly. Importantly, the interaction between copper and silicate did affect final cell count significantly. When concentrations of both copper and silicate were constrained, P. multiseries cultures reached the lowest final cell densities. On the contrary, when the concentrations of both nutrients were high, cultures reached the highest cell densities. Silica-uptake results suggested that copper does not use the silicate uptake system in Pseudo-nitzschia multiseries. If copper used the silica-uptake system, uptake of silica would have decreased with increasing copper concentration. Instead, a synergistic effect was observed when the two nutrients were limiting population growth (significant MANOVA interaction term) (Fig. 2). Uptake of silicate in these nutrient-limited cultures was an order of magnitude higher than in the rest of the experimental treatments. The highest Total DA (TDA) per cell, as well as Dissolved DA (DDA) per cell, concentrations occurred in cultures grown under high silicate/high copper and low silicate/sufficient copper. Cultures grown under sufficient silica/sufficient copper showed the highest proportion of TDA to DDA per cell (Fig. 3). The highest
Fig. 1. 3D plot of the effect of silicate (mM) and copper concentration (M) on maximum cell concentration.
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Fig. 2. 3D plot of the effect of silicate and copper concentration (mM) on silicate uptake (moles of Si per cell).
absolute DDA (DA ml1) occurred in the high Si/high Cu treatment (Fig. 4). 3.2. DA production by Pseudo-nitzschia populations in musselfarming areas In the field, silicate and copper concentrations were always undetectable by the methods of analysis used in this study (detection limit, Si 0.10 107 M, Cu 8.5 109 M). Pseudo-nitzschia spp. cells were identified twice (April 14 and July 7, 2009) in samples from Hampton, New Hampshire. On both occasions, the cells were detected in field samples first microscopically and then by PCR amplification of the ITS1 fragment. On one of these occasions (April 14), mucous aggregations were plentiful. These mucous aggregations contained many empty phytoplankton cell walls and frustules, and dinoflagellates cysts, and vegetative cells of Pseudonitzschia-like diatoms were also observed within these aggregates. The best match of the blast-search for the two species isolated from the samples collected July 7, 2009 at the New Hampshire site corresponded to Pseudo-nitzschia granii (Fig. 5A) (length ¼ 39.98 1.81 mm, width ¼ 2.1 0.38 mm) for one strain and Pseudo-nitzschia delicatissima for the other strain (Fig. 5B) (length ¼ 49.66 8.3 mm, width ¼ 2.2 0.38 mm). Although DA was below detection in raw field samples, laboratory cultures of both species tested positive for DA production, with concentrations of over 1 pg cell1 (P. cf. granii ¼ 1.10 0.04 TDA; P. cf. delicatissima ¼ 2.61 0.82 TDA). In Cape Cod, Massachusetts Pseudo-nitzschia sp. was detected once (May 11, 2009) by PCR amplification. No Pseudo-nitzschia strains were isolated.
Fig. 3. Plot of the effect of silicate and copper concentration on total domoic acid (DA) per cell, dissolved domoic acid (DDA, extracellular), and intracellular domoic acid (IDA).
4. Discussion The factorial design of our culture experiment revealed a significant interaction between silica and copper in regulating domoic-acid production by Pseudo-nitzschia multiseries. Silica is a major structural component of cell walls in diatoms (Round et al., 1990), and thus, silica availability is expected to control growth. Copper at high concentrations is toxic (Lelong et al., 2012b), but growth was not diminished by the concentrations used in the present study, implying that the concentrations used were not high enough to inhibit growth. The highest copper concentration used by Lelong et al. (2012b), though, was 1.53 times higher than the highest concentration used in the present study. Lelong et al. (2012b), however, reported that cultures exposed to 2 107 Cu2þ M did not grow; whereas, the present study shows that growth was not inhibited in cultures grown in 3.92 107 Cu2þ M. A possible explanation for the difference in copper toxicity might be that the media used for the experimental treatments in this study was Aquil media; whereas, Lelong et al. (2012b) used f/2 media. Chemical speciation, as well as trace metal contamination and precipitate formation, is controlled in Aquil medium but not in f/2-enriched natural seawater. Bioavailability of copper, therefore, might had been higher in the study by Lelong et al. (2012b) than in the present study. Copper is an essential component of many cellular proteins; therefore, limited availability of this metal results in growth limitation (Peers et al., 2005). In our experiment, responses of P. multiseries to copper and silica when both were either high or low were amplified
Fig. 4. Plot the effect of silicate and copper concentration on absolute domoic acid (DDA ml1).
M.S. Fuentes, G.H. Wikfors / Marine Environmental Research 83 (2013) 23e28
Fig. 5. A) P. cf. granii, and B) P. cf. delicatissima isolated from Hampton, New Hampshire.
compared to the responses to individual nutrients. The highest cell densities were obtained when the concentrations of both nutrients were high. The lowest final cell count, on the other hand, occurred when both nutrients were scarce. A synergistic effect of copper and silica upon silica uptake was also observed. Silica-uptake increased enormously (an order of magnitude higher than in the other treatments) in cultures wherein both nutrients were synergistically co-limiting growth. Hutchins and Bruland (1998) found that silicic acid:nitrate (Si:N) uptake ratio was two-to-three times higher in diatoms stressed by iron starvation than in diatoms under ironreplete conditions. These authors suggested that iron starvation led to more-silicified, faster-sinking diatoms. Various studies have documented high cell densities of Pseudo-nitzschia sp. in deep waters (Rines et al., 2002; Sekula-Wood et al., 2009), and Pseudonitzschia blooms are thought to be initiated from seed populations present beneath the euphotic zone (Trainer et al., 2000). Furthermore, we tested the survival of P. multiseries under cold and dark conditions and found that this species is capable of surviving such conditions by using energy stored in the form of lipid (Fuentes et al., unpublished data). Hence, increased silica uptake under nutrientstarvation may be a strategy to access nutrients in deep waters. Additionally, Silica-uptake results suggested that copper does not use the silicate uptake system in Pseudo-nitzschia multiseries. If copper used the silica-uptake system, uptake of silica would have decreased with increasing copper concentration. Also, limitation of silica by copper would have increased DA concentrations in treatments with higher copper concentrations and low silica. The highest TDA and DDA per cell concentrations, instead, occurred in high silicate/high copper and low silicate/sufficient copper treatments. Previous studies have shown increased production of DA with decreasing silica concentrations (Pan et al., 1996; Amato et al., 2010) and with copper toxicity (Maldonado et al., 2002; Wells et al., 2005). But, as mentioned above, the levels of copper used in our study were not toxic to P. multiseries. Rue and Bruland (2001) reported that DA is capable of chelating copper because its chemical structure is similar
27
to the phytosiderophore mugenic acid. Later, Maldonado et al. (2002) demonstrated that, with high copper concentrations, higher DA amounts were released into the medium compared to DA release with lower concentrations of the metal. Based upon those results, Maldonado et al. (2002) suggested that DA chelates copper, reducing its availability and thus toxicity. As the higher absolute DDA occurred in the high Si/high Cu treatment, it is possible, therefore, that DA prevented toxicity, and consequently growth inhibition, in treatments with high copper. A more recent study (Lelong et al., 2012b), though, reported that elevated copper concentrations did not induce DA production in P. multiseries. Also, DA did not protect the cell against copper toxicity in this study. A probable cause for the contrasting results of Lelong et al. (2012b) might be inter-strain variability. Furthermore, cultures mixed with bacteria in natural seawater enriched media were used in that study, therefore, a direct comparison between the present study that used a bacteria-free strain cultured in AQUIL and Lelong et al. (2012b) cannot be made. Production of DA has been speculated to be a mechanism for prompt elimination of excess photosynthetic energy that is no longer used for growth and when metabolism decreases (Bates, 1998). In the present study, when both copper and silicate were scarce, growth was depressed and silica uptake increased considerably compared to the other treatments. Our interpretation is that excess photosynthetic energy was used for silica uptake rather than toxin production. Even though low silicate/high copper concentrations increased TDA, the highest TDA occurred in high silicate/ high copper and low silicate/sufficient copper cultures. Low silicate/low copper cultures did not have the highest TDA, perhaps because these cultures were using energy for silicate uptake rather than toxin production. Turning to the field component of the present study, two species of Pseudo-nitzschia capable of producing DA were detected in sites off the coast of New Hampshire where commercial blue-mussel aquaculture is being developed. The identification of these species, however, is only based on the best match of a blast search. Currently, not all species of Pseudo-nitzschia have the internal transcribed spacer (ITS) sequence in public databases (e.g. GenBank). Therefore, TEM micrographs, with accompanying morphometrics, must also be done on that species to confirm species identity. Unfortunately, further identification of these species was not possible in the present study; EM was not performed before isolates were lost. Nevertheless, the results of this study indicate the existence of a potential risk of shellfish contamination with DA in New England because two Pseudo-nitzschia species with different morphologies and sequence results were found and confirmed to be capable of producing DA. At the time of sampling in both New Hampshire and Massachusetts, the Pseudo-nitzschia populations were not producing measurable quantities of DA, indicating that environmental conditions were not conducive to up-regulation of domoic-acid synthesis pathways in the diatoms. Variation in DA content within Pseudo-nitzschia spp. cells from both natural populations and cultures has been described previously, and nutrient status of cells has been implicated in toxin variation (Bates and Trainer, 2008). Our results showed clearly that the low silicate and copper concentrations present in the New Hampshire mussel-farming site were not conducive to high DA production in the model species. At low silicate levels, however, a small increase in copper led to a doubling in DA content in P. multiseries cultures. A concurrent increase in silicate mitigated the toxin increase, suggesting a mechanism by which DA production can remain low to moderate when all nutrients, including both silicate and copper, are increased simultaneously, e.g. during upwelling of deep water or following runoff from land. At very high copper and silicate levels, however, DA content also increased significantly, possibly in this case
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because copper was approaching inhibitory levels and DA was being up-regulated to chelate copper. 5. Conclusions This study demonstrated the presence of at least two species of Pseudo-nitzschia capable of producing DA in sites off the coast of New Hampshire where commercial blue-mussel aquaculture is being developed. This finding underscores the importance of anticipating the potential for mussel farming in this area to be impacted by Amnesic Shellfish Poisoning (ASP). Overall, the results of this experiment suggest that a small increase in copper, without a simultaneous increase in silicate, could precipitate conditions conducive to DA up-regulation in endemic populations in New England mussel-farming areas. The main finding of this study, though, is that care must be taken to minimize activities that may lead to local copper increases in New England mussel-farming sites where potentially-toxigenic Pseudonitzschia species are endemic. Acknowledgments We appreciate the cooperation of Scott Lindell and Richard Langan, who provided water samples from Massachusetts and New Hampshire, respectively. We thank Jennifer Alix, Shannon Meseck, and Steven Pitchford for assisting with algal culturing and chemical analyses. This project was supported by the NOAA National Aquaculture Program, through a National Research Council PostDoctoral Fellowship to M.S.F., and by the Northeast Fisheries Science Center of the National Marine Fisheries Service. References Amato, A., Ludeking, A., Kooistra, W.H.C.F., 2010. Intracellular domoic acid production in Pseudo-nitzschia multistriata isolated from the Gulf of Naples (Tyrrhenian Sea, Italy). Toxicon 55, 157e161. Balamurugan, K., Schaffner, W., 2006. Copper homeostasis in eukaryotes: teetering on a tightrope. Biochimica et Biophysica Acta 1763, 737e746. Bates, S.S., Trainer, V.L., 2008. The ecology of harmful diatoms. In: Granéli, E. (Ed.), Ecology of Harmful Algae. In: Turner, J.T. (Ed.),, Series: Ecological Studies, vol 189. SpringereVerlag, Berlin, Heidelberg, New York, pp. 81e93. Bates, S.S., Douglas, D.J., Doucette, G.J., Léger, C., 1995. Enhancement of domoic acid production by reintroducing bacteria to axenic cultures of the diatom Pseudonitzschia multiseries. Natural Toxins 3, 428e435. Bates, S.S., Garrison, D.L., Horner, R.A., 1998. Bloom dynamics and physiology of domoic-acid-producing Pseudo-nitzschia species. In: Anderson, D.M., Cembella, A.D., Hallegraeff, G.M. (Eds.), Physiological Ecology of Harmful Algal Blooms. SpringereVerlag, Berlin, Germany, pp. 267e292. Bates, S.S., 1998. Ecophysiology and metabolism of ASP toxin production. In: Anderson, D.M., Cembella, A.D., Hallegraeff, G.M. (Eds.), Physiological Ecology of Harmful Algal Blooms. SpringereVerlag, Berlin, Germany, pp. 405e426. Clearwater, S.J.A., Farag, M., Meyer, J.S., 2002. Bioavailability and toxicity of dietborne copper and zinc to fish. Comparative Biochemistry and Physiology Part C 132, 269e313. FAO Fisheries and Aquaculture, 2012, http://www.fao.org/fishery/culturedspecies/ Mytilus_edulis/en (August 6, 2012). Fernandes, D., Bebianno, M.J., Porte, C., 2009. Assessing pollutant exposure in cultured and wild sea bass (Dicentrarchus labrax) from the Iberian Peninsula. Ecotoxicology 18, 1043e1050. Fisheries and Oceans Canada, 2010. Cultured Aquatic Species Information Programme Mytilus edulis (Linnaeus, 1758). Canadian Aquaculture Production Statistics. Retrieve from: http://www.dfo-mpo.gc.ca/stats/aqua/aqua10-eng. htm (July 30, 2012). Guillard, R.R.L., 1975. Culture of phytoplankton for feeding marine invertebrates. In: Smith, W.L., Chanley, M.H. (Eds.), Culture of Marine Invertebrate Animals. Plenum Press, New York, pp. 26e60. Hansen, H.P., Koroleff, F., 1999. Determination of nutrients. In: Grasshoff, K., Kremling, K., Ehrhardt, M. (Eds.), Methods of Seawater Analysis, third ed. WileyVCH, Weinheim, New York, USA, pp. 159e228. Hargraves, P.E., Zhang, J., Wang, R., Shimizu, Y., 1993. Growth characteristics of the diatoms Pseudo-nitzschia pungens and P. fraudulenta exposed to ultraviolet radiation. Hydrobiology 269/270, 207e212. Hilbish, T.J., Mullinax, A., Dolven, S.I., Meyer, A., Koehn, R.K., Rawson, P.D., 2000. Origin of the anti-tropical distribution pattern in marine mussels (Mytilus spp.): routes and timing of transequatorial migration. Marine Biology 136, 69e77.
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