Harmful Algae 10 (2011) 480488

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Harmful Algae
journal homepage: www.elsevier.com/locate/hal

The green macroalga, Ulva lactuca, inhibits the growth of seven common harmful algal bloom species via allelopathy
Ying Zhong Tang, Christopher J. Gobler *
School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, NY 11794-5000, USA

A R T I C L E I N F O

A B S T R A C T

Article history: Received 3 December 2010 Received in revised form 15 March 2011 Accepted 16 March 2011 Available online 26 March 2011 Keywords: HABs Ulva Macroalgae Allelopathy

Harmful algal blooms (HABs) are a signicant threat to sheries, public health, and economies around the world, and both HABs and macroalgae are often promoted by nutrient loading. We report on experiments examining the effects of the macroalga, Ulva lactuca, collected from estuaries of NY, USA, on the growth of seven common HAB species: Aureococcus anophagefferens, Chattonella marina, Cochlodinium polykrikoides, Karlodinium venecum, Karenia brevis, Prorocentrum minimum and PseudoNitzschia multiseries. Fresh thalli of U. lactuca added at environmentally realistic levels (mg L1) were capable of lysing or strongly inhibiting the growth of all seven HAB species in a dose-dependent manner within controlled laboratory experiments during which high nutrient levels, low bacterial levels, and common pH levels among treatments and controls were maintained. The dramatic allelopathic effects of extracts of dried and powdered U. lactuca with and without post-extraction heat treatment on the HAB species demonstrated that U. lactuca contains heat-stable allelochemicals that play a major role in the observed allelopathic effects. The addition of live U. lactuca thalli in bottle and mesocosm experiments conducted in the eld during blooms of A. anophagefferens (brown tide; >105 cells mL1) consistently yielded a signicant (p < 0.05) and often large (>50%) reduction in cell densities in $48 h. Our ndings combined with the well-known nutrient removal capacity of seaweeds collectively suggest that the use of macroalgae may be a promising mitigation strategy for HABs in coastal ecosystems. 2011 Elsevier B.V. All rights reserved.

1. Introduction Harmful algal blooms (HABs) are a signicant threat to sheries, public health, and economies around the world. The frequency and impacts of HABs have intensied in recent decades and anthropogenic nutrient loading has been implicated in this expansion (Anderson et al., 2008; Heisler et al., 2008). Another group of autotrophs promoted by eutrophication in coastal ecosystems is macroalgae (Valiela et al., 1997; Valiela and Cole, 2002; Valiela, 2006). Many species of macroalgae are capable of rapid growth in the presence of high nutrient concentration and have a high assimilative capacity for nutrients thus act as nutrient sinks (Valiela et al., 1997; Neori et al., 2004; Zertuche-Gonzalez et al., 2009). Like HABs, the overgrowth of macroalgae can be also considered harmful, as they can outcompete and replace seagrass beds, cover other critical benthic habitats, and promote diel hypoxia in estuaries (Valiela et al., 1997; Valiela and Cole, 2002; Valiela, 2006; Liu et al., 2009).

* Corresponding author. Tel.: +1 631 632 5043; fax: +1 631 632 5070. E-mail addresses: christopher.gobler@stonybrook.edu, cgobler@notes.cc.sunysb.edu (C.J. Gobler). 1568-9883/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2011.03.003

Recent studies, largely in Asia, have demonstrated that macroalgae can have strong growth-inhibiting effects on phytoplankton in general, and multiple HAB species, in particular. Multiple species of the green macroalgal genus, Ulva, including Ulva fasciata, Ulva pertusa, and Ulva linza have displayed the ability to cause rapid lysis of Prorocentrum micans, Prorocentrum donghaiense, Heterosigma akashiwo (Jin and Dong, 2003; Jin et al., 2005; Nan et al., 2004, 2008; R.J. Wang et al., 2007; Y. Wang et al., 2007) and to reduce the growth of Alexandrium tamarense and Chaetoceros gracile (Nan et al., 2004). The red coralline macroalga, Corallina pilulifera, has displayed algicidal activity against several harmful algae including Cochlodinium polykrikoides, Karenia mikimotoi, Akashiwo sanguinea, A. tamarense, and H. akashiwo (Jeong et al., 2000; R.J. Wang et al., 2007). Other macroalgae that have been shown to inhibit the growth of HAB species include Ecklonia kurome, Gracilaria lemaneiformis, and Sargassum thunbergii (Koki et al., 2003; Liu et al., 2006; Lu et al., 2008; R.J. Wang et al., 2007; Y. Wang et al., 2007). The impacts of macroalgae on HABs have been observed from both co-culturing and, in limited cases, from administering macroalgae extracts (Jeong et al., 2000; Jin et al., 2005; R.J. Wang et al., 2007). Two studies have demonstrated that polyunsaturated fatty acids isolated from U. fasciata and U. pertusa have effects on HABs similar to those observed for extracts and whole macroalgae,

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suggesting these compounds could be active agents against the HAB species (Alamsjah et al., 2005, 2008). Studies unambiguously demonstrating the allelopathic effects of macroalgae on HABs, however, have been rare (Korner and Nicklisch, 2002; Gross et al., 2003, 2007). In addition, all of the research to date examining the effects of macroalgae on HABs has been conducted in Asia, although many macroalgae including species of Ulva are globally distributed along coastlines. This study reports on the effects of live thalli and extracts of the macroalga, Ulva lactuca, isolated from NY, USA, on the growth of seven HAB species (Aureococcus anophagefferens, Chattonella marina, Pseudo-Nitzschia multiseries, Cochlodinium polykrikoides, Karenia brevis, Karlodinium venecum, and Prorocentrum minimum) within controlled laboratory, eld, and mesocosms experiments. These seven species commonly form HABs in coastal waters in the US and around the world. Results demonstrate that allelochemicals produced by U. lactuca are capable of lysing or strongly inhibiting these HABs and thus that this macroalga has the potential to impact the occurrence of HABs. 2. Materials and methods 2.1. Microalgal cultures (target HAB species) The targeted HAB species included in the study were 4 species of dinoagellates (P. minimum, C. polykrikoides, K. venecum, and K. brevis), one species of the brown tide pelagophyte (Anophagefferens anophagefferens), a species of the toxigenic diatom P. multiseries, and one species of raphidophyte, C. marina. The cultures of P. minimum (strain CCMP696), K. brevis CCMP2228, and A. anophagefferens (strains CCMP1984 and CCMP1707) were obtained from the ProvasoliGuillard National Center for Culture of Marine Phytoplankton (Maine, USA). The culture of C. marina (strain Chatt1) from Singapore coastal water was kindly provided by M.J. Holmes (National University of Singapore), the diatom P. multiseries CLNN21 was isolated from the Bay of Fundy, Canada and kindly provided by Steve Bates (Fisheries and Oceans Canada), and the cultures of C. polykrikoides (strain CP1) and K. venecum (strain FR-6) were isolated by YZ Tang from Flanders Bay and the Forge River, respectively, two estuaries on Long Island, NY, USA; their identity has been conrmed via genetic sequencing (Tang et al., 2010). The three cultures of P. minimum CCMP696 and A. anophagefferens strains CCMP1984 and CCMP1707 were also initially isolated from estuarine waters of Long Island, NY, USA. All the microalgal cultures except P. multiseries were maintained in GSe medium (G medium supplemented with 1 108 M selenium) (Doblin et al., 1999), made with autoclaved and sterile ltered (0.22 mm) seawater with a salinity of $30 PSU. The diatom P. multiseries was maintained in GSe medium supplemented with Si at the same concentration in f/2 medium. Cultures were maintained at 21 8C in an incubator with a 12:12 h light:dark cycle, illuminated by a bank of uorescent lights that provided a light intensity of $100 mmol quanta m2 s1 to cultures. An antibiotic solution (a mixture of 10,000 I.U. penicillin and 10,000 mg mL1 streptomycin; Mediatech. Inc., Hemdon, VA) was added into the medium of all cultures except for K. venecum FR-6 immediately before inoculation, with a nal concentration of 1% to minimize contamination by bacteria. This antibiotic mixture had no negative effects on the growth and survivorship of these microalgae although K. venecum cannot survive this antibiotic solution (Tang et al., 2010). Periodic DAPI-staining of cultures was performed and conrmed an absence of bacteria in cultures. An experiment using A. anophagefferens CCMP1984 as target species was performed with and without addition of antibiotics solution (nal 1%) into culture medium to assess the effect of the macroalga on HAB

species in the presence of bacteria. All culture transfers were conducted within a sterile, class-100 laminar ow hood. 2.2. Collection, treatment, and maintenance of macroalgae Fresh thalli of the macroalga U. lactuca were collected from the Old Fort Pond, a shallow estuary on eastern Long Island, NY. Thalli were liberally washed with 0.2 mm ltered seawater and then placed in GSe medium containing 4% antibiotics mixture overnight in an incubator. The 4% antibiotics-containing GSe medium was then changed several times over several days. Vegetative culture of macroalga was maintained in vessels containing GSe medium and 1% antibiotics under the conditions described above for cultures of HAB species. Periodic DAPI-staining of cultures was performed and conrmed an absence of bacteria in cultures. 2.3. Laboratory experiments 2.3.1. Experiments using fresh thalli of U. lactuca The thalli of U. latuca were cut into measured, rectangular pieces of 1 cm 1 cm, 2 cm 2 cm, 3 cm 3 cm, and 4 cm 4 cm and administered into 250-mL asks containing 50 mL cultures of the targeted species, A. anophagefferens, C. marina, P. multiseries, P. minimum, C. polykrikoides, K. venecum, and K. brevis. Care was taken to excise the middle sections of U. latuca, ensuring the vegetative portion of thalli were used in experiments. Consequently, thalli never formed or released gametes during any experiment reported here. The placement of 1 cm 1 cm U. lactuca in 50 mL culture is equivalent to single layer of macroalgae covering the bottom of a 0.5 m deep water, a density that is often exceeded in many shallow estuaries and tributaries across the northeast US coast (Valiela et al., 1997; Valiela and Cole, 2002; Valiela, 2006; authors pers. obs.). Triplicate thalli not used in experiments were immediately scanned using SigmaScan1 software to conrm macroalgal area, and then dried in 65 8C for 24 h and weighed for dry weight. One cm2 of wet U. lactuca was, on average, 2.27 0.3 mg dry weight. Densities of the HAB species used in experiments generally represented bloom and sometimes pre-bloom cell abundances, that were species specic (Gobler et al., 2005, 2007, 2008; Anderson et al., 2008). Control cultures without macroalgae were established at each phytoplankton cell density and all treatments were administered in triplicates. All asks were incubated as described for culture maintenance. Growth of phytoplankton within asks was monitored daily over 5 days via cell counts and chlorophyll uorescence measurements using a TD-700 uoremeter (Turner Design). Flasks were also monitored for pH levels using high resolution pH paper (0.1 units) during experiments to assure cultures were not cross contaminated via a pH probe and with an electrode (Thermo Scientic Orion Star SeriesTM Benchtop pH meter; 0.001 unit; calibrated prior each use with NIST traceable standards) at the end of experiments. The two methods were in good agreement (5%). Upon termination of the experiments, culture aliquots (5 mL) were removed, ltered through a combusted (450 8C for two h) glass ber lter, and analyzed for nitrate using standard methods (Parsons et al., 1984); Measurements of light in larger experimental asks with and without U. lactuca thalli at the experimental levels used scaled to larger volumes measured with a LiCor PAR sensor, indicated that light intensity did not vary signicantly among treatments at the levels administered as, in a manner consistent with an ecosystem setting, thalli were on the bottom of the asks, while the light source was from above asks. All cultures used for experiments were in early or midexponential phase growth with high levels of ambient nutrients still present. Additions of GSe medium were made to all

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experiments, and measured nitrate concentrations exceeded 50 mM during all time points of all experiments and were often more than 100 mM. Given that these concentrations are more than an order of magnitude above the half saturation constant of nitrate for growth of most HAB species (Smayda, 1997; Gobler et al., unpublished data), all experiments were performed under nutrient replete conditions. In addition, pH levels were ranged between 7.8 and 9.5 in treatment with macroalgae a range equivalent to the changes in pH in monocultures for each HAB species used in this study in the absence of macroalgae, suggesting treatment effects were not associated with changes in pH. 2.3.2. Experiments using thalli-free medium and extracts of the dried powder of macroalgae To assess whether macroalgae release compounds that inhibit HABs, the thalli of U. lactuca were removed from the cultures that were subsequently ltered through sterile, polycarbonate 0.22 mm lter, resupplemented with nutrients to full strength GSe medium, and used for experiments. To determine whether U. lactuca contained stable, allelopathic compounds, thalli were dried at 65 8C for 24 h and ground into a ne powder with an acid-washed (10% HCl), liberally rinsed (deionized water), and dried mortar and pestle. The dry powder (3.6 g) was suspended in 100 mL GSe medium in a ask, sonicated on ice for 10 min using a high power sonicator (Ultrasonic Power Corporation, IL, USA), and placed in a refrigerator (4 8C) two weeks for extraction. The slurry of Ulva was then centrifuged and the supernatant was collected for experiments. Since the pH of the slurry was 7.5, a portion of the collected extract was adjusted to pH, 8.5, a typical level for microalgal cultures, before used for experiments. In addition, a part of the extract was sealed in a 50 mL centrifuge tubes and placed in a boiling water bath for 15 m. The target HAB cultures were then inoculated into macroalgae ltrate (50% as nal content) or the extracts at varying dilutions made with GSe medium (0.1 10% = 363600 mg L1) and a full GSe medium control. 2.3.3. Experiments with partitioned macroalgal thalli and target HAB species Another approach used to examine the mechanism by which U. lactuca impacts HAB growth and survival was the use of a partitioned chamber (Tang and Gobler, 2009). The modied plastic (high density polyethylene, HDPE) chamber contains 30 compartments (4 cm 4 cm 4 cm) in which pairs of two adjacent compartments are separated with 5 mm-mesh nylon membrane. A snap-shut lid for the chamber allowed for UV-sterilization prior to experiments, complete sealing of the chamber during experiments, and easy access to all individual compartments at the start and end of all experiments. For each pair of partitioned compartments connected through the 5 mm membrane, 30 mL of three target HAB species (C. polykrikoides, P. minimum, and K. venecum) was added at one side and a piece of U. lactuca (3 cm 3 cm) and 30 mL fresh GSe medium were added to the other side. The chamber was then incubated as described above for 72 h (triplicates for both treatments and GSe control). At the beginning and end of experiments, samples were pipetted from both sides simultaneously by two people and cell densities of HAB species wee enumerated microscopically. 2.4. Field experiments 2.4.1. Bottle incubation experiments Surface seawater was collected in 20 L carboys from the Quantuck Bay at 28 May, 11 June, and 22 June 2008, and from the Great South Bay at 30 May, 6 June, and 20 June 2008 during blooms of the brown tide alga, A. anophagefferens. Within 1 h of collection, six, 1.1 L, acid-washed, polycarbonate bottles were lled with

bloom water, while an aliquot of water for each sample was preserved with 1% glutaraldehyde solution and stored at 4 8C for later quantication. All six bottles had 20 mM nitrate and 1.25 mM phosphate added to ensure nutrient replete conditions. For three bottles, one piece of fresh thalli of U. lactuca (3 cm 3 cm) was added, while the other three were used as control. The bottles were then placed in the Old Fort Pond, a semi-enclosed estuary contiguous with Quantuck and Great South Bay at the Stony Brook-Southampton Marine Science Center ensuring bottles were incubated under near ambient light and temperature conditions. After 48 h, water samples were taken from each bottle and preserved with 1% glutaraldehyde solution and stored at 4 8C for later quantication of A. anophagefferens. 2.4.2. Mesocosm experiments A mesocosm experiment was also conducted with six, 300-L oating tanks to examine effect of U. lactuca on A. anophagefferens under ambient conditions. The experimental tanks are the same height (1.2 m) as the mean depth of NYs 100-km, south shore lagoon system and were held in the Old Fort Pond, allowing them to maintain the same temperature and light level as the estuarine system that hosts HABs (Gobler et al., 2007). Seawater from an A. anophagefferens bloom (cell density 2.58 105 cells mL1) was collected from Quantuck Bay on 24 June 2008, using the RV Peconic from the Stony Brook-Southampton eet of vessels. The collected bloom water was equally distributed into the six tanks, with three of them receiving 50 g wet weight of fresh U. lactuca thalli and the other 3 set as control (no addition of Ulva). The Ulva was placed in mesh cages that were adhered to the sides of mesocosms to prevent the macroalgae from being mixed into water circulation pumps, sinking, oating or altering the light regime during the experiment. Water circulator pumps (Rio1 180 Mini Aqua Pump, pumping rate: 456 L h1) were then placed in each tank to ensure mixing of the water column. During the96 h experiment, 10 mM ammonium and 0.5 mM orthophosphate was added daily. Water samples were removed at noon local time each day from each tank for the enumeration of A. anophagefferens cell densities. Samples were preserved with 1% glutaraldehyde solution and stored at 4 8C before quantication. Onset HOBO data loggers were placed at the bottom of each mesocosm and recorded light and temperature every 10 min during experiments and conrmed there were no signicant differences in light or temperatures among tanks. 2.5. Quantication of A. anophagefferens To quantify A. anophagefferens cell densities, two methods were employed depending on the sample source. Monocultures preserved in Lugols iodine were quantied using a hemacytometer and light microscope. Field samples were quantied using a monoclonal antibody (MAb) technique that has been adapted to a colorimetric, enzyme-linked immunosorbent assay (ELISA) format performed in a 96-well plate (Caron et al., 2003). This technique provides more accurate and rapid detection of A. anophagefferens cells in mixed algal samples over both the immunouorescent staining with a polyclonal antibody (PAb) method and traditional microscopy techniques since A. anophagefferens is small and nondistinct making it impossible to distinguish from other picoplankton in eld samples (Sieburth et al., 1988; Anderson et al., 1989; Caron et al., 2003; Gobler et al., 2005). This assay yielded a 96 15% recovery of samples spiked with levels of A. anophagefferens similar to those used in experiments (3 105 cells mL1), a methodological relative standard deviation of 8.1 6.6%, and a detection limit of 3.6 2.1 103 cells mL1. Dense bloom samples were diluted to fall between the detection limit and the highest standard with a solution of 0.2 mm ltered seawater in 1% glutaraldehyde.

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2.6. Statistics The signicance of variance between treatments and control or among treatments was tested with one-way ANOVA, or t-test using the software SigmaStat 3.11 (Systat Software Inc.). The signicance level was set at 0.05 for all tests unless otherwise stated. 3. Results 3.1. Effects of fresh macroalgal isolates on HAB species The addition of fresh thalli of U. lactuca resulted in signicant loss of microalgal biomass compared to the respective control treatCell density (x 10 cells mL )
6 5 4 3 2 1 0 0h 24 h 48 h 72 h 120 h
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ments during a ve day incubation for all seven HAB species examined: A. anophagefferens (two strains CCMP1984 and CCMP1707), C. marina, P. multiseries, P. minimum, C. polykrikoides, K. venecum, and K. brevis (Fig. 1AH; p < 0.0001; ANOVA or t-test). No difference in growth inhibiting effect was observed between treatments with antibioticsantimycotic addition and that without addition for the target species A. anophagefferens (p > 0.05, ANOVA; Fig. 1A). The two strains of A. anophagefferens CCMP1984 and CCMP19707 were equally sensitive to U. lactuca with both displaying more than 80% inhibition in growth compared with the respective controls in 5 d by 180 mg dry wt. L1 of fresh U. lactuca (Fig. 1A and B). Higher levels of U. lactuca resulted in signicantly larger growth inhibition or biomass loss than lower levels for A. anophagefferens
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40 Control Ulva 45 mg/L 30 Ulva 180 mg/L Ulva 400 mg/L 20 Ulva 725 mg/L

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Fig. 1. Growth-inhibition effects of fresh thalli of U. lactuca on target HAB species (A) A. anophagefferens strain CCMP1984, (B) A. anophagefferens CCMP1707, (C) C. marina, (D) P. multiseries, (E) P. minimum, (F) C. polykrikoides, (G) K. venecum, and (H) K. brevis. The sign + in (A) indicates treatments with addition of antibioticsantimycotic solution (1% nal concentration). In (E), two initial cell densities of P. minimum were used: 0.85 104 cells mL1 (Low) and 6.46 104 cells mL1 (High). The amounts of fresh thalli of U. lactuca are expressed as mg dry wt. L1. Data points are means SD (n = 3).

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(Fig. 1A), C. marina (Fig. 1C), P. multiseries (Fig. 1D), P. minimum (Fig. 1E), C. polykrikoides (Fig. 1F), K. venecum (Fig. 1G), and K. brevis (Fig. 1H) (ANOVA, p < 0.01). Comparing the results of treatments using the same amount of U. lactuca, A. anophagefferens and K. brevis exhibited higher sensitivity than K. venecum, C. marina, P. multiseries, and P. minimum to the macroalga. For example, 72 h exposure to 725 mg L1 of U. lactuca caused 75, 50, 38, 32, 29, and 24% of biomass reductions in A. anophagefferens CCMP1984, K. brevis, K. venecum, C. marina, P. multiseries, and P. minimum, respectively, in comparison with their respective controls (Fig. 1A, CE, G and H). In 120 h of co-culturing with 180 and 400 mg L1 of U. lactuca, C. polykrikoides was experienced only a 12 and 29% growth-inhibition, respectively, in comparison with the control (p < 0.001, ANOVA; Fig. 1F), indicating that C. polykrikoides was inhibited by U. lactuca but to a lesser extent than other HAB species. 3.2. Effects of thalli-free medium and extracts of the dried powder of macroalgae on HAB species Extracts of U. lactuca were equally or more potent in inhibiting the growth of HABs compared to live thalli (Fig. 2). The relative sensitivities of the HAB species to extracts differed from those displayed for the macroalgal thalli and the lowest concentrations of extract needed to inhibit the growth of the HAB species differed

among the species examined: 36 mg dry wt. L1 for C. marina (Fig. 2B) and P. multiseries (Fig. 2C), 180 mg dry wt. L1 for A. anophagefferens (Fig. 2A), P. minimum (Fig. 2D), 360 mg dry wt. L1 for K. venecum (Fig. 2E), and >360 mg dry wt. L1 for K. brevis (Fig. 2F). Except for P. minimum, almost all cells of other HAB species inhibited by the extracts were lysed after 5 d. Three days of growth followed by a dramatic decrease in cell density was observed in A. anophagefferens (Fig. 2A) and C. marina (Fig. 2B) at concentrations above 180 mg dry wt. L1 (A. anophagefferens) or >36 mg dry wt. L1 (C. marina), while a monotonic growth (at low Ulva concentrations) or decrease (at higher Ulva concentrations) was observed in P. multiseries, P. minimum, K. venecum, and K. brevis (Fig. 2C, D, E, and F). In the experiments using GSe medium with additions of the ltered (0.22 mm) U. lactuca culture medium (nal concentration 25, 50, and 75%), no signicant growth-inhibiting effect was observed in the three species examined: A. anophagefferens, C. marina, and P. minimum (data not shown). The results of experiments using the U. lactuca extract after adjustment of pH to 8.5 showed similar cell lysis effects in the four target species, A. anophagefferens, P. minimum, K. venecum, and C. marina during 48 h incubations and no live cells were observed under an inverted microscope for all test species at 48 h, although many cells of P. minimum were still intact (data not shown). For experiments using the extract of U. lactuca that was boiled, cell lysis effects similar to that described above were observed in two HAB species, A. anophagefferens and C. marina during 120 h

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Fig. 2. Growth-inhibition effects of the aqueous extract of U. lactuca (without pH adjustment) on HAB species. (A) A. anophagefferens, (B) C. marina, (C) P. multiseries, (D) P. minimum, (E) K. venecum, and (F) K. brevis. A concentration of 180 mg L1 of dry biomass was equivalent to a dilution of 0.5% extract or 80 cm2 L1 of fresh thalli. Data points are means SD (n = 3).

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Fig. 3. Growth-inhibition and cell lysis effects of the extract of U. lactuca dry powder after water-bath boiling for 15 m on the HAB species of (A) A. anophagefferens, (B) C. marina. Data points are means SD (n = 3).

incubations (Fig. 3A and B). Collectively, these results demonstrated that the observed growth inhibition and cell lysis in test HAB species were not caused by pH shock and that the putatively involved allelochemicals are heat stable. 3.3. Chamber experiments Physical contact between U. lactuca and HAB species was not required for the macroalgae to inhibit the growth of the microalgae. After 96 h in the partitioned chamber, the growth of C. polykrikoides, P. minimum, and K. venecum separated from U. lactuca by a 5-mm mesh was signicantly reduced by 40%, 41%, and 29%, respectively, compared to each control treatment (p < 0.001, t-tests; Fig. 4). 3.4. Experiment with bloom populations of A. anophagefferens From 28 May through 22 June 2008, six bottle incubation experiments were conducted with bloom water collected from the
5 140 120 4 100 3

Fig. 5. Effects of U. lactuca live thalli on A. anophagefferens in 6 bottle co-incubation experiments in the eld. The addition of U. lactuca thalli was 9 cm2 L1, or 20 mg dry wt. L1. Experiment duration was 72 h. Data points are means SD (n = 3).

Cell density (103 cells mL-1)

Quantuck Bay and Great South Bay containing between 2.0 and 10 105 cells mL1 of A. anophagefferens (Fig. 5). Compared with both the initial and control cell concentrations of A. anophagefferens, the addition of fresh thalli of U. lactuca (3 cm L1 3 cm L1) yielded signicant reductions in the cell densities in all six experiments performed (p < 0.05, One way ANOVA; Fig. 5). The percent reduction in cell density relative to controls ranged from 22% to 57% (Fig. 5). U. lactuca also restricted the growth of A. anophagefferens during the mesocosm experiment conducted in the Old Fort Pond from 24 to 28 June 2008 using bloom water collected from the Quantuck Bay on 24 June 2008 (Fig. 6). During the 4-d experiment, the cell density of A. anophagefferens in the mesocosms with U. lactuca was reduced from 2.6 105 cells mL1 to 0.15 105 cells mL1 (94%; Fig. 6; p < 0.0001). In stark contrast, there was a slight increase (42%) in the no-U. lactuca control during the rst 24 h after which cell density of A. anophagefferens did not change appreciably from the initial concentration, remaining > 2.4 105 cells mL1 for the duration of the experiment (Fig. 6).

Cell density (x10 cells mL )

80 60 40

-1

5 Control 4 3 2 1 0 0 1 2 3 4 Ulva

1 20 0 Treat Control 0 Treat Control Treat Control

C. polykrikoides

P. minimum

K. veneficum

Fig. 4. Growth-inhibition effects of fresh thalli of U. lactuca on HAB species C. polykrikoides, P. minimum, and K. venecum in the indirect-contact experiments conducted with the chambers partitioned through a 5 mm-mesh membrane. The symbol * indicates signicant difference between the treatment (indirect exposure to U. lactuca) and the control (t-test, p < 0.01). Data points are means SD (n = 3).

Time (day)
Fig. 6. Effect of U. lactuca live thalli on A. anophagefferens in mesocosm experiments in the eld. Data points are means SD (n = 3).

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4. Discussion This study demonstrated that the green macroalga, U. lactuca, is capable of restricting the growth of seven species of phytoplankton that form HABs across the globe. The occurrence of these impacts in the presence of high nutrient concentrations, undetectable bacterial concentrations, and normal pH levels implicates allelochemicals as a causative mechanism in the growth reduction of HABs. The ability of aqueous extracts of macroalgae or live thalli of macroalgae connected to HABs through a 5-mm mesh to restrict HAB growth demonstrated that the dry powder of U. lactuca contains water-soluble and heat-stable allelochemical(s) responsible for inhibiting the growth or lysis of the cells of microalgae and that direct contact is not required for the effects of U. lactuca on these HAB species to occur. Collectively, these results provide new insight into the ecological role of macroalgae such as U. lactuca in the occurrence of HABs. 4.1. Mechanisms for the observed effects and potential of application There are multiple mechanisms that may account for the growth-inhibiting effects of U. lactuca on the HAB species. Macroalgae are known to display an enormous capacity for the assimilation of nutrients (Valiela et al., 1997; Neori et al., 2004; Zertuche-Gonzalez et al., 2009) and are purposefully used in many parts of the world to reduce nutrients levels in coastal waters (Neori et al., 2004; Carmona et al., 2006; Chopin et al., 2001, 2008). Given that many HABs are directly or indirectly promoted by nutrients (Heisler et al., 2008; Anderson et al., 2008), macroalgae may reduce the occurrence of HABs by reducing the levels of nutrients available in the water column. High-level nutrients assimilation certainly occurred in our experiments conducted with the fresh thalli of U. lactuca (Table 1), where the NO3 concentrations during experiments were signicantly reduced from $1000 mM to $100 mM at the end of experiments. Importantly, however, differences between control and U. lactuca treatments were small and not signicant (Table 1). Moreover, concentrations always remained above the nitrate half-saturation constants for these and other HABs (Smayda, 1997). Collectively, these ndings suggest that the observed negative effects of U. lactuca on the HAB species were not due to nutrient limitation of the microalgae or nutrient competition between micro- and macro-algae. High rates of photosynthesis may drawdown CO2 and increase pH levels in treatments with U. lactuca compared to control treatments and high pH has been previously invoked to account for the allelopathic affects of some autotrophs (Schmidt and Hansen, 2001; Lundholm et al., 2005). The pH levels in the co-cultures of microalgae and live U. lactuca, however, were equivalent to those in the macroalga-free controls (range 7.89.1). Studies have also documented that there are high levels of bacteria growing epiphytically on macroalgae (Imai et al., 2006) and that some bacteria have algicidal properties (Mayali and Azam, 2004; Frazier et al., 2007). However, our use of antibiotics kept concentrations of planktonic bacteria to undetectable levels. In addition, for A. anophagefferens, the growth-inhibiting effect of live U. lactuca was nearly identical with and without the antibioticantimycotic addition (Fig. 1A), suggesting the observed effect on A. anophagefferens was not caused by bacteria or fungi. Finally, but more

decisively, our experiments conducted with aqueous extracts of U. lactuca were similar and in some cases more dramatic than the effects observed with live thalli, excluding the possible effects of nutrients competition and limitation, pH change, and microbial contamination. Therefore, the negative effects (growth inhibition and cell lysis) of U. lactuca on A. anophagefferens, K. venecum, C. polykrikoides, C. marina, P. minimum, K. brevis, and P. multiseries during our experiments must have been primarily contributed by allelopathic effects of U. lactuca. Based on prior research conducted with species of Ulva from Asia, some of the allelochemicals produced by this alga may include polyunsaturated fatty acids (Alamsjah et al., 2005, 2008). Dithiolane and trithiane compounds isolated and identied from Characean species were also observed to cause allelopathic effects on epiphytic diatoms and other phytoplankton (Wium-Andersen et al., 1982). Our results demonstrated that the dried or heated extracts of U. lactuca were equally or more potent than the equivalent thalli, an observation consistent with the hypothesis that polyunsaturated fatty acids or organosulfur compounds are active allelopathic agents. The enhanced potency of the extracts also suggests that allelochemicals in cells were more concentrated than the portion released into the environment. The ability of live thalli within partitioned chambers to restrict HAB growth but the inability of ltered Ulva culture medium to do so suggests allelochemicals released by live thalli may degrade rapidly. Thus, the HAB-inhibiting effects observed in live thalli were likely a consequence of a low but sustained level of release of the allelochemicals. Additionally, the Ulva thalli used for all experiments besides the ltrate experiment were mechanically cut or disrupted, a processes that may enhance the production and/or release of allelochemicals (Reigosa et al., 1999). As such, the inability of the ltrate to impact HABs may have been due to an absence of mechanical disturbance of the macroalga. Importantly, within an ecosystem setting, the mechanisms by which U. lactuca inhibit HABs may include allelopathy, nutrient scavenging, algicidal bacteria, and pH changes (Schmidt and Hansen, 2001; Carmona et al., 2006; Imai et al., 2006). Nevertheless, further research is required to identify the precise allelopathic compounds produced by U. lactuca. 4.2. Ecological relevance of allelopathic effects of U. lactuca Our eld-based experiments demonstrated that U. lactuca can reduce the densities of eld populations of HABs, at least in the case of A. anophagefferens, an observation consistent with the spatial distribution of brown tides in NY. Specically, while A. anophagefferens achieves high densities within the open waters of estuaries, their cell densities decline by orders of magnitudes in shallow tributaries where salinities are not signicantly different from neighboring, open estuarine sites, but high densities of macroalgae are present. For example, during the A. anophagefferens bloom of 2008, cell densities in Old Fort Pond, NY, where dense stands of U. lactuca were present and used for the experiments present in this study, were <104 cells mL1 while the open waters of neighboring Shinnecock Bay exceeded 105 cells mL1 (SCDHS, 2008; C. Gobler, unpubl.). While such differences in A. anophagefferens cell abundance were previously ascribed to higher levels of inorganic nutrients in tidal tributaries (Gobler et al., 2005), the current study suggests that lower A. anophagefferens abundances in

Table 1 The residual concentrations of NO3 in the cultures of three HAB species with and without live U. lactuca thalli (725 mg dry wt. L1) after 120 h cultivation. The concentrations of NO3 in the full strength GSe medium is 1980 mM. P. minimum control NO3 (mM) 80 23 P. minimum + Ulva 89 10 A. anophagefferens control 52 0.4 A. anophagefferens + Ulva 151 45 K. venecum control 146 53 K. venecum + Ulva 140 61

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regions with dense stands of U. lactuca could be due to allelopathic inhibition of this HAB. Our results regarding U. lactuca and HABs t within the ecosystem-level phase shifts that have occurred in estuaries across the globe during the twentieth century. Specically, there have been more frequent and intense algal blooms, including HABs (Kemp et al., 2005; Gobler et al., 2005; Anderson et al., 2008), associated with the overharvest of shellsh (Jackson et al., 2001; Cerrato et al., 2004; Kemp et al., 2005) and increased anthropogenic nutrient loading (Heisler et al., 2008; Anderson et al., 2008) in recent decades. These blooms have resulted in a concomitant decline in light penetrating to the benthos (Dennison, 1988; Newell and Koch, 2004; Wall et al., 2008) with negative consequences for benthic autotrophs (Newell and Koch, 2004; MacIntyre et al., 2004; Wall et al., 2008), including macroalgae (Valiela et al., 1997). If HABs promoted by higher nutrient loading and lower bivalve ltration shade out macroalgae populations that had previously been present and had restricted HAB growth via allelopathy, nutrient competition, large pH variation, and algicidal bacteria (Valiela et al., 1997; Schmidt and Hansen, 2001; Imai et al., 2006), a positive feedback loop may now be in effect (Sunda et al., 2006). More intense blooms have developed, creating more turbid water, and thus fewer macroalgae, leading to reduced allelopathic growth restriction of HABs by macroalgae permitting more intense HABs that will further light limit of benthic autotrophs. Therefore, if macroalgal populations and/or bivalve populations are purposefully restored in a HAB-affected estuary, this pattern might occur in reverse in the future. Beyond the experimental evidence that has already been accumulated demonstrating that some macroalgae can inhibit the growth of HABs (Jeong et al., 2000; Jin and Dong, 2003; Koki et al., 2003; Nan et al., 2004, 2008; Jin et al., 2005; Liu et al., 2006; R.J. Wang et al., 2007; Y. Wang et al., 2007; Lu et al., 2008; this study), there are many aspects of macroalgae that may make them an attractive option for the prevention, control, and mitigation of HABs. Firstly, and perhaps most importantly, macroalgae are a natural component of coastal waters and thus their use to control HABs would not involve the introduction of a biotic or abiotic entity or process that was non-native to estuarine ecosystems. Next, there is precedent for the purposeful use and deployment of macroalgae in coastal waters as a nutrient mitigation strategy (Neori et al., 2004; Yang et al., 2006). Large scale deployments of 10s of square kilometers of aquacultured macroalgae are common in Asia (Pereira and Yarish, 2009). In some cases, macroalgae are deployed simply to restrict nutrient concentrations and uxes to coastal waters (Neori et al., 2004; Yang et al., 2006; Zhou et al., 2006). In other scenarios, macroalgae have become a principal component of integrated multi-trophic aquaculture (IMTA) systems whereby macroalgae are cultivated with aquacultured sh and/or shellsh with the macroalgae often being harvested for commercial use and subsequent prot (Kraemer et al., 2004; Neori et al., 2004; Abreu et al., 2009). In some cases, U. lactuca has been successfully deployed within large scale IMTA systems to both reduce nutrient levels and to feed abalone (Robertson-Andersson et al., 2008; Bolton et al., 2009). For several species of herbivorous sh, Ulva is a preferred food source (Tolentino-Pablico et al., 2008). As such, IMTA systems with macroalgae could have the combined benets of reducing nutrients, mitigating HABs, providing feed for some aquaculture animals, and/or being a harvested product. Finally, the purposeful deployment of macroalgae should be approached with care. U. lactuca is used as feed for some aquacultured animals (Robertson-Andersson et al., 2008; Bolton et al., 2009) and Ulva is a preferred food source for some herbivorous sh (Tolentino-Pablico et al., 2008). Consistent with these ndings, we have co-cultured live thalli of U. lactuca with larval shellsh (bay scallops Argopecten irradians) and juvenile

nsh (Cyprinodon variegates) at high abundances (>2 g dry wt. L1) and have found no change in the survival of these animals compared to the controls without macroalgae (one week exposure; data not shown). Importantly, however, the overgrowth of wild macroalgae can cover critical benthic habitats and promote diel hypoxia in estuaries (Valiela et al., 1997; Valiela and Cole, 2002; Nelson et al., 2003; Valiela, 2006; Liu et al., 2009). Hence, deployment of this species may be best suited for enclosed, aquaculture scenarios or within settings where the U. lactuca can be contained off of the benthos and harvested and removed as it grows. Since desiccated Ulva can inhibit the growth of some larval bivalves (Nelson et al., 2003; this study), the use of U. lactuca extracts in an ecosystem setting should be approached with extreme caution and would require further research. Lastly, it is likely that other macroalgal species, including those with commercial value but not deleterious ecosystem impacts, may also be adapted to for the purpose of prevention and control of HABs and certainly warrants further investigation.

Acknowledgements We acknowledge the nancial support from NOAA, the Suffolk County Department of Health Services, Ofce of Ecology and the New Tamarind Foundation. We thank Drs. MJ Holmes (National University of Singapore, Singapore), Claude Leger, and Steve Bates (Fisheries and Oceans Canada) for supplying cultures. We also thank Dr. Maria Alejandra Marcoval, Matthew Harke, Ryan Wallace, Ayodeji Ajayi, Heidi Mittelsdorf, Florian Koch, and Elyse Walker for technical assistance.[SS] References
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