The 2008 Texas Dinophysis ovum bloom: Distribution and toxicity

Harmful Algae 9 (2010) 190–199

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The 2008 Texas Dinophysis ovum bloom: Distribution and toxicity Kathleen M. Swanson a, Leanne J. Flewelling b, Meridith Byrd c, Alex Nunez d, Tracy A. Villareal a,* a

University of Texas Marine Science Institute, 750 Channel View Drive, Port Aransas, TX 78373, United States Florida Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute, 100 8th Avenue SE, St. Petersburg, FL 33701, United States c Texas Parks and Wildlife, P.O. Box 688, Port O’Connor, TX 77982, United States d Natural Resource Center, 6300 Ocean Drive, NRC Suite 2501, Corpus Christi, TX 78412, United States b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 19 June 2009 Received in revised form 2 October 2009 Accepted 7 October 2009

In early 2008, oyster beds along the Texas coast were closed due to the confirmed presence of okadaic acid (OA), the toxin responsible for diarrhetic shellfish poisoning (DSP). This marked the first shellfish bed closure in the United States due to both elevated concentrations of Dinophysis and DSP intoxicated shellfish. The population was dominated by Dinophysis ovum. In offshore samples, high concentrations (10,960 and 26,040 cells L 1) were observed in the southern coastal areas in mid to late February prior to the closure, and decreased through late February and early March. Dinophysis abundance increased at the northern areas 1–2 weeks later, although the sampling was insufficient to resolve if this was truly temporally distinct. Dinophysis was patchy at stations inshore of barrier islands, and many stations showed few or no Dinophysis. The high offshore abundance, and inshore bay distributions with maxima near barrier island passes, as well as the timing and magnitude of different peak concentrations, supports an offshore, southern origin for the Dinophysis bloom with advection into the bays. There was no correlation between abundance and environmental parameters, although cell density peaked offshore around 17–18 8C. Inshore occurrences showing a wider temperature range distribution for Dinophysis. Toxin content per Dinophysis cell (OA equiv. cell 1) showed no general pattern with time or conditions. Offshore and inshore maximum OA concentrations were high, but the bulk of the values fell within limits reported previously. Prorocentrum lima was not present, and the data suggests little or no contribution to the OA toxicity by Prorocentrum. With the addition of DSP, the northwestern Gulf of Mexico has among the highest diversity of harmful algal bloom (HAB) events in N. America, including ciguatera, Karenia brevis-neurotoxic shellfish poisoning (NSP), and brown tides. Domoic acid has been reported as well. NSP closures typically occur in late summer or fall. With the potential threat of DSP in the late winter or early spring now, the oyster industry in the NW Gulf of Mexico faces the potential for closures at virtually any time of the year, and possibly over most of the year. ß 2009 Elsevier B.V. All rights reserved.

Keywords: Diarrhetic shellfish poisoning Dinophysis Harmful algal blooms Global epidemic Okadaic acid Toxicity

1. Introduction Diarrhetic shellfish poisoning (DSP) is caused by the ingestion of shellfish contaminated with toxins from the dinoflagellates Dinophysis and Prorocentrum. First described in the 1960s, symptoms include gastrointestinal distress, such as diarrhea, nausea, and vomiting, with onset from 3 to 12 h following ingestion of the contaminated organism (Aune and Yndestad, 1993). No DSP-related human deaths have been recorded, and recovery time for intoxication is around 3 days (Blanco et al., 2005; Hallegraeff, 2003). DSP has been reported worldwide, with Southern Europe (Mediterranean) (Poletti et al., 1998; PavelaVrancic et al., 2002), Scandinavia (Kumagai et al., 1986; Godhe

* Corresponding author. Tel.: +1 361 749 6732. E-mail address: [email protected] (T.A. Villareal). 1568-9883/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2009.10.001

et al., 2002), Japan (Yasumoto et al., 1978), Chile, Thailand, Australia, New Zealand, and Canada (Nova Scotia) being the major areas affected (Hallegraeff, 2003). Okadaic acid (OA), the main toxin responsible for DSP, was originally isolated from the sponge Halichondria okadai by Tachibana et al. (1981), and later identified by Murata et al. (1982). Two dinoflagellate genera, Dinophysis and Prorocentrum, are known to produce toxins of the OA group (Yasumoto et al., 1984; Lee et al., 1989). Dinophysis spp. is a genus found in open and coastal waters of the world, including bay waters of Japan (Nishitani et al., 2002), the Aegean Sea (NE Mediterranean Sea) (Poletti et al., 1998; Koukaras and Nikolaidis, 2004), North Sea (Peperzak et al., 1996), off the Portuguese coast (Palma et al., 1998), and the fjords of Sweden (Lindahl et al., 2007). Dinophysis spp. are usually in low to moderate concentrations (100–10,000 cells L 1) (Reguera and Pizarro, 2008) when they cause DSP events, but exceptional red water blooms are recorded to occur (Dahl et al.,

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1996). Within a species, regional differences in toxicity exist (Hoshiai et al., 1997). For example, in European waters, Dinophysis acuminata cell concentrations reach 100–40,000 cells L 1, often contaminating mussels leading to human health concerns (Lee et al., 1989; Hawkey, 2003). In contrast, the same species were reported in U.S. waters (Chesapeake Bay) at high numbers (236,000 cells L 1), with undetectable or trace toxicities (Marshall et al., 2002). The identification of a D. acuminata complex in the late 1980s (Lassus and Bardouil, 1991), with both warm and cold water members suggests that taxonomic heterogeneity as well as environmental effects on toxin production (Hawkey, 2003) contribute to toxic bloom variability. The management of Dinophysis spp. and the ability to predict blooms is complicated by its mixotrophic status (Jacobson and Andersen, 1994; Park et al., 2006) and species-specific response to hydrography (Escalera et al., 2006). Unlike many other HABs, nutrient concentrations are not directly linked to biomass increase (Delmas et al., 1992; Godhe et al., 2002; Nishitani et al., 2002). Dinophysis blooms in North America have previously occurred off the coasts of Canada (Gaspe´ Coast) (Cembella, 1989), the northeastern United States (Maranda and Shimizu, 1987; Tango et al., 2002), and Mexico (Gulf of California) (Ochoa et al., 1997). D. acuminata, a potentially toxic species, occurs regularly in the lower St. Lawrence estuary and along the east coast of Canada (Cembella, 1989). Further south, in Narragansett Bay (USA) D. acuminata occurs annually, but at concentrations insufficient to cause oyster toxicity (Maranda and Shimizu, 1987). Prior to 2008, there had been one precautionary DSP-related oyster bed closure in the Potomac River and Chesapeake Bay. Between February and March 2002, D. acuminata cell concentrations reached 236,000 cells L 1, and an immediate shellfish closure was implemented (Marshall et al., 2002). However, only trace concentrations of OA were found in oyster meat, and the shellfish beds were re-opened (Tango et al., 2002). The apparent low toxicity of the bloom was noteworthy. Until the 2008 Texas bloom, Dinophysis had not been considered a problem in the U.S. In March, 2008, a number of Texas bays, including Aransas, Corpus Christi, and Copano, were closed to shellfish harvesting (Texas Department of State Health Services, 2008) due to the presence of DSP toxins. This marked the first time in the United States that shellfish beds had been closed due to both a high Dinophysis as well as shellfish contaminated with OA. Early warning provided by an Imaging FlowCytobot (Campbell et al., in press) detected Dinophysis cells (1–5 cells mL 1) in early February at Port Aransas, Texas. By late February, Dinophysis spp. numbers increased to more than 100 cells mL 1 at Port Aransas, Texas (February 25, 2008). Dinophysis was confirmed by microscopic examination (Fig. 1) and water samples were found to contain 5.9 ng OA mL 1 (Campbell et al., in press). Discolored water at Rockport, Texas contained >3000 cells mL 1 on March 6th, 2008. Shellfish assays (Texas Department of State Health Services) confirmed toxification of oysters at up to 68.1 mg OA per 100 g, and a closure followed quickly. This prevented a potentially large DSP outbreak since the annual Fulton Oysterfest (focusing on local oysters) was scheduled for the following weekend. All bays were re-opened for shellfish harvest by April 8, 2008. In order to understand more about the origins, dynamics, and toxicity of this novel bloom, a sampling program was implemented in the local bays after the initial detection. We used samples from an existing offshore monitoring program as well as samples collected during the bloom. This provided information on abundance toxicity, and environmental conditions during the bloom decline, and permitted some conclusions as to the origin of the bloom population. This is a complementary study to Campbell et al. (in press) where they describe the initial detection by automated imaging of flora at Port Aransas, Texas.

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Fig. 1. Photograph of D. ovum from reported bloom.

2. Materials and methods Following the detection of a Dinophysis bloom occurring along the coast of Texas, a combination of offshore (Gulf of Mexico) and inshore (landward of the barrier islands) sampling effort began. Offshore samples were needed to gain an understanding of the geographic distribution. Two sets of initial inshore sampling provided a broad range of inshore distribution and abundance. Once these general patterns were established, a weekly, more intensive sampling regime began. Since there was no historical data to base the sampling on, this initial effort was purely descriptive.

Fig. 2. Five major areas sampled along the Texas coast: 17, Sabine Pass; 18, Bolivar Road Pass; 19, Cavallo Pass; 20, Port Aransas Pass; 21, Brazos Santiago Pass. The circled area represents area of local bay sampling around Port Aransas, Texas shown in Fig. 3.

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2.1. Offshore samples Offshore surface-water samples (Fig. 2) were collected by the Texas Parks and Wildlife Department (TPWD) twice monthly as part of an ongoing Monitoring and Event Response for Harmful Algal Blooms (MERHAB) sampling for Karenia brevis studies (Stumpf et al., 2003). One sample in each region was collected within the first half of the month, with a second during the last half at five major areas along the Texas coast that corresponded with passes into prominent bay systems (Fig. 2). Within each of these major areas, there was a grid system that extended nine nautical miles offshore (the Texas territorial sea; 16.7 s km), and identified minor sampling sites. These minor sites were determined randomly. At each site, environmental parameters (salinity, temperature, and dissolved oxygen; Yellow Springs Instruments, Inc., Yellow Springs, OH, USA, 600XL-B-O) and location (latitude and longitude) were recorded, and surface samples for dissolved nutrients, toxin content per cell, and Lugol’s preserved cell counts were taken (Throndsen, 1978). Dinophysis was enumerated on 25– 50 mL settled volume (Utermo¨hl, 1958). These cell concentrations from offshore samples are reported as cells L 1 to differentiate them from the Sedgewick-Rafter cell count (given in units of cells mL 1). The timing and protocols were fixed and could not be altered in response to the bloom. 2.2. Regional sampling Samples from the major bay systems along the entire Texas coast (Fig. 2) were collected by TPWD as a separate sampling effort for regional, inshore waters (n = 130) that used scheduled finfish survey trips. As a result, the sampling frequency and location was driven by the finfish survey requirements. Surface-water samples were preserved in Lugol’s (Throndsen, 1978), and counted as above for inshore samples. These samples provided a regional inshore characterization of the where Dinophysis occurred during this period.

Fig. 3. Inshore sampling sites and sampling dates. 13 March 2009, Corpus Christi Bay; 18 March 2009, Northern sites; Weekly, began on 27 March 2009; MissionAransas National Estuarine Research Reserve, bi-monthly.

of most immediate practical use to them. To differentiate these counts from the higher volume settled offshore samples, the Sedgwick-Rafter counts are reported as cells mL 1. 2.4. Chemical analysis

2.3. Local bay sampling Following detection of the Dinophysis spp. bloom by the Imaging FlowCytoBot (IFCB) in the Port Aransas ship channel, water samples were collected in Corpus Christi, Redfish, Aransas, Copano, San Antonio, and Matagorda Bays from (13–18 March; Fig. 3). For logistical reasons, surface water (2 L) was collected from docks, jetties or the shore, and was subsampled for nutrient, chlorophyll a, cell counts, and toxin analyses and termed the Corpus Christi Bay (Corpus Christi Bay) and Northern sampling (Redfish, Aransas, Copano, San Antonio, and Matagorda Bays). Temperature, salinity, dissolved oxygen (D.O.), and pH, were measured at each station using a YSI-600 XLM, multi-parameter water quality monitor (Yellow Springs Instruments, Inc., Yellow Springs, OH, USA). By the end of March, four permanent sampling sites were determined and the weekly sampling began until the end of the bloom (Fig. 3). Sites were sampled for 2 weeks following the last Dinophysis spp. cell seen in the sample. Sampling was also completed twice monthly at the Mission-Aransas National Estuarine Research Reserve (NERR) (Fig. 3) on scheduled trips for permanent station servicing, and at a location within the upper Laguna Madre (27.41708N, 97.36478W) sampled as part of a seagrass monitoring program. High sediment loads precluded use of settled samples and only 1 mL of the Lugol’s preserved sample was examined using a Sedgewick-Rafter Cell counting slide. While the Sedgwick-Rafter counts lack statistical power, this methodology is used routinely by the state health units responsible for shellfish bed closures and was

2.4.1. Nutrients and chlorophyll a Replicate samples were filtered, frozen and for nutrients using a Lachat QuikChem 8000 (Lachat Instruments, Milwaukee, WI) at University of Texas Marine Science Institute (UTMSI). Duplicate samples for chlorophyll were also filtered and frozen, and later measured on methanol-extracted samples using a Turner Designs TD-700 Fluorometer (Turner Designs, Sunnyvale, CA, USA) equipped with a non-acidification kit (Welschmeyer and Naughton, 1994). 2.4.2. Phosphatase inhibition assay The colorimetric protein phosphatase inhibition assay (PPIA) used was developed by Tubaro et al. (1990). Two modifications were completed to the assay’s protocol: (1) 50 mM Trizma HCl was used instead of 40 mM Trizma HCl and (2) Tween 20 (0.05%) was added to the buffer solution. Toxin was extracted twice with 10 mL of an 80:20 MeOH:MilliQ water mixture was added to the test tubes containing the filters. For inshore samples, 250 mL of water was run through a 1.0 mm Millipore glass fiber filter. For the offshore samples, two filters (=80 mL filtered), were placed into a single test tube, and a combined extraction was performed. Following extraction, the methanol was evaporated, toxins were re-suspended in assay buffer, and the assay was completed. Toxin content per cell was estimated by multiplying the ng OA equiv. mL 1 (provided by assay results) by the amount of MeOH:MilliQ water mixture used divided by the amount of water filtered for the sample. This number was then divided by the density of cells in the water at the time of sampling. All statistical

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analysis was completed on JMP1 7.0.1 2007 (Cary, NC). Similar to OA and its derivatives, microcystin and nodularin (both produced by cyanobacteria) are hepatotoxic peptides that have PP2A inhibitory activity (Boland et al., 1993; Chen et al., 1993; Luu et al., 1993). Neither of these compounds should be located at high concentrations in the Gulf of Mexico. However, because other compounds that can inhibit the enzyme, such as chemical trapped in the sediment that is on the filters, cannot be ruled out, the specificity of the results cannot positively be identified as OA, and OA equivalents (OA equiv.) is used instead.

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average recordings of 34.1  1.2. The northern sites sampled on March 22nd had the lowest average salinity of 17.2  4.8 and the lowest individual reading of 10.2. Nutrient concentrations were highly variable among the inshore stations (data not shown), and reflected the complex local patterns of marshes, mudflats and dredged ship channels with tidal flow dominated by wind-driven circulation. Chlorophyll a from the Northern, Lower Laguna Madre, Weekly, and MissionAransas NERR surveys was within the ranges expected for these areas, with monthly ranges of 9.79  2.47 mg L 1 in March to 6.08  0.92 mg L 1 in May.

3. Results 3.3. Dinophysis concentration 3.1. Offshore hydrography A total of 48 samples were collected from the five major offshore areas from January to May 2008. Environmental conditions were consistent with general seasonal trends. Water temperatures increased from January through May (14.4  0.5 to 24.7  0.5 8C; Fig. 4a). The salinity decreased slightly from 30.1  0.8 in January to 26.4  1.8 in May (Fig. 4b). Salinity and temperature increased southward along the coast. Orthophosphate, silicate, and nitrite/nitrate concentration all decreased with latitude. No chlorophyll measurements were completed for offshore samples since these filters were used for toxin analysis. 3.2. Inshore hydrography Texas bays are shallow, usually averaging <4–5 m depth, and are dominated by wind-driven circulation. The shore/pier collected samples are considered representative of the nearby bay waters, although there is always the possibility of a stronger benthic influence on the waters immediately adjacent to the shore. Inshore samples were only collected during March, April, and May with sampling starting after the oyster bed closure. The average water temperatures during those months were 19.6  0.4, 24.2  0.2, and 23.4  0.2 8C, respectively (Fig. 4a). Monthly average salinity landward of the barrier islands remained between 24 and 26 (Fig. 4b). The Lower Laguna Madre sites were the most saline inshore areas, with

3.3.1. Offshore Dinophysis species were found at all five offshore sampling sites (reported in cells L 1). While initially identified as D. acuminata, subsequent molecular work indicated Dinophysis ovum was the dominant species (Campbell et al., in press). D. caudata was also present, but never in samples with more than five Dinophysis cells mL 1, and became significant only at the end of May. We will refer to this event as a D. ovum bloom, recognizing that other taxa were present in low numbers. D. ovum was first observed at the two most southerly areas, and these areas had the highest average and individual cell counts (Area 20; 26,000 cells L 1 on February 20th; Fig. 5). Bolivar Road Pass, Area 18, contained the lowest average concentration of cells at 300  140 cells L 1 (Fig. 5). In general, the highest D. ovum abundance was observed in February, with a gradual decline through April (Figs. 5 and 6). By April, cell concentration had declined significantly, and by May D. ovum was no longer present in our counts (Fig. 5). 3.3.2. Inshore No Dinophysis spp. were recorded in the samples collected from the Corpus Christi Bay sampling on March 13th (Fig. 6). Three of the four Northern bay sites (Fig. 6) on March 20th had non-detectable concentrations, while Port O’Connor inshore contained 6 cells mL 1 (Fig. 6). Elevated cell numbers were observed at all weekly sampling sites on 27 March. Rockport Harbor contained the highest cell counts (97 and 107 cells mL 1 on 18 Mar. 18th and 4 April, respectively), and the longest presence of Dinophysis (Fig. 6). Cell concentrations decreased steadily at all weekly sites with no cells present in the final 2 weeks of sampling (22 April and 2 May; Figs. 6 and 7). The Texas Parks and Wildlife Department bay samples (n = 130; 20 March to April 15) showed that Dinophysis was largely restricted to San Antonio to Corpus Christi bay, with a majority of the coast-wide water samples containing no Dinophysis cells (72 samples no observable cells; Fig. 8). No cells were observed in Sedgwick-Rafter counts of Sabine Lake, Matagorda, and Galveston Bay or Laguna Madre samples. 3.4. DSP toxins

Fig. 4. Average ( standard error) of temperature (a) and salinity (b) for offshore and inshore sampling (Jan.–May 2008) (offshore n = 48, inshore n = 52).

OA was below detection limits in 26 of 47 offshore samples and 43 of 61 inshore samples. Concentrations of OA equiv. per cell ranged from non-detectable to 73 and 45 pg OA equiv. cell 1 for offshore (Brazos Santiago Pass, 21 March) and inshore (Rockport, 22 March), respectively. The need to use both replicate filters for toxin content per cell analysis and loss of several samples during the initial screen coupled with the inaccuracy of Sedgwick-Rafter counts suggest that values reported are best considered a general sense rather than a precise measure toxin content per cell. Both cell concentrations and okadaic acid, for inshore and offshore samples, were elevated throughout March (Figs. 5 and 7). Although there was considerable temporal variation in toxin content per cell (Figs. 5 and 7), maximum mean Dinophysis toxin content per cell

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Fig. 5. OA eq. rg cell 1 and D. ovum cells mL 1 for offshore samples collected from January to May 2008 (n = 48). a, Sabine Pass; b, Bolivar Road Pass; c, Cavallo Pass; d, Port Aransas Pass; e, Brazos Santiago Pass. Note different scale for D. ovum cells for Port Aransas Pass and Brazos Santiago Pass.

did not vary significantly with location (Fig. 9) and clustered in the 25–30 pg OA equiv. cell 1 range for non-zero observations. There were no correlations between environmental parameters (temperature, pH, depth, D.O., or salinity) and combined inshore/ offshore Dinophysis cell concentrations or toxin content per cell. In the offshore samples, D. ovum cell concentrations and toxin content per cell peaked at a water temperature of 17–18 8C (Fig. 10). For the inshore samples, most samples that contained cell counts were collected in waters with temperatures of 18–25 8C. Nutrients were poorly correlated with D. ovum concentrations.

4. Discussion The reactive nature of the sampling and rapidly changing conditions restricted the types of data that could be collected, but the broad features of this bloom can be described: (1) The event was relatively short-lived, and largely in decline when this sampling began. (2) Dinophysis was abundant offshore prior to the first observations at Port Aransas by Campbell et al. (in press), and suggest an offshore origin for the bloom. (3) The ranges of environmental conditions during the bloom were similar to other

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Fig. 6. Inshore D. ovum concentrations during the sampling period of March, April, and May 2008 (n = 52). Sampling began after the bloom had been detected, and finished after two consecutive weeks of zero cell detections. Corpus Christi Bay and the northern sites were only sampled once.

Fig. 7. OA eq. rg cell 1 and abundances for samples collected inshore after mid-March (n = 27). All sampling ended on May 2nd. a. sRockport; b. Crabman; c. Port Aransas; d. Conn Brown Harbor; e. Mission Aransas NERR.

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Fig. 8. Dinophysis ovum distribution for regional sampling sites (n = 130). Samples were collected from 21 March -15 April 2009. First number indicates number of samples containing D. ovum, second represents the number of samples collected.

D. ovum blooms. (4) Cell toxicities were generally comparable with those reported for similar species in other waters. The unique occurrence of this event adds another toxic species to the list of HAB events that are now known from the western Gulf of Mexico, and occurs in a season that could exacerbate problems facing the oyster industry. We will elaborate on these conclusions below. Elevated offshore concentrations occurred during early to midFebruary off Brazos Santiago Pass and Port Aransas Pass prior to the detection by the Imaging FlowCytobot (Campbell et al., in press). Based on our limited sampling, it seems likely that the bloom

Fig. 9. Average OA eq. rg cell

1

spread up the coast although the timing of the offshore samples was not ideal to resolve this and several key missing samples in February are problematic. The geographic locations and timing of inshore cell occurrences also support the idea that the bloom occurred off the coast first and moved inshore. D. ovum was concentrated around the Port Aransas Ship Channel (e.g. Aransas Bay, Crabman, Conn Brown Harbor, and Port Aransas), while sampling sites that were farther from the Gulf of Mexico source water contained few or no Dinophysis (e.g. Corpus Christi Bay, Goose Island, Copano Bay, and the Lower Laguna Madre). This is consistent with the general circulation patterns of these bays where limited exchange occurs primarily at the passes, and residence time of the inner bays increases with distance from the passes (Ward, 1997). The only northern, inshore sampling site that contained cells was Port O’Connor with 6 cells mL 1 and a salinity of 31.45. The similarity to salinity and abundance at nearby Cavallo Pass (4880 and 500 cells L 1 on 4 and 20 March) suggests that these cells had been advected in from offshore as well. Samples collected for oyster toxicity sampling indicated a discontinuous inshore Dinophysis distribution concentrated near passes in the barrier islands (K. Wiles, Texas Department of State Health Services, pers. comm.). Both the regional and weekly surveys noted concentrations decreased to both the north and south of Port Aransas Pass suggesting that the cells were transported into the bays from offshore populations. In general, concentrations >50 cells mL 1 from inshore samples were associated with small harbors and embayments. The highest cell densities observed (>3000 cells mL 1) were in a red water streak in Rockport Harbor, where water circulation, harbor orientation and the constant winds may have concentrated the cells in a manner consistent with the vertical migration mechanisms (Maestrini, 1998). These small harbors are highly restricted and dredged to greater depths than much of the surrounding area. While not directly measured, they are more likely to temperature stratify than the surrounding open water. A similar situation may have occurred in the Antifer area in France, where D. acuminata concentrations were found to increase in densities due to a prevalent southwesterly wind (Lassus et al., 1993). In Rockport Harbor, the prevailing southeastern winds coming off Aransas Bay push the water into the harbor, trapping and concentrating cells. This is in contrast to Conn Brown Harbor, which is much less open and bounded by spoil islands upwind. Campbell et al.’s (in press) continuous sampling noted dividing cells in the incoming populations only during a short time at the inception of the

for all offshore (black symbols) and inshore (grey symbols). Error bars represent standard error.

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Fig. 10. D. ovum abundances and okadaic acid eq. per D. ovum cell for all offshore samples (n = 48) plotted against temperature.

bloom, supporting the notion that the inshore populations did not actively divide. By the end of April, cell densities had decreased at all sites and shellfish beds had been re-opened. Inorganic nutrients and Dinophysis abundance was not correlated, results similar to previous work (Delmas et al., 1992; Godhe et al., 2002; Nishitani et al., 2002). Temperature and salinity during the bloom were generally within the ranges for Dinophysis reported for Greek coastal waters (Koukaras and Nikolaidis, 2004) where the peak concentration occurred between 11 and 17 8C. These values correspond closely with peak concentrations observed in the open Gulf of Mexico during this study (Fig. 10). Although a strong negative correlation has been observed between Dinophysis spp. concentrations and salinity in the range of 20–32.5 (Godhe et al., 2002), no such correlations were observed during this study. Since there was apparently little active growth occurring during the 2008 Texas bloom, the distributions were dominated by mixing processes that did not favor exchange with lower salinity water deep in the bays (Ward, 1997). We can only speculate as to what terminated the bloom. Copepods are reported to graze Dinophysis (Maneiro et al., 2000; Wexels-Riser et al., 2003), Escalera et al. (2007) documented Dinophysis spp. in food vacuoles of Noctiluca scintillans. Noctiluca was noted in both inshore and offshore samples towards the end of the Texas Coast bloom, and discolored water due to decaying Noctiluca was seen at the Crabman sampling site in late April (Villareal, unpubl. obs.). However, we have no data on whether Dinophysis was present in Noctiluca in our samples. Our average values for inshore samples containing detectable OA have comparable values to those reported by Raho et al. (2008), although the offshore values exceed what they reported. D. ovum cell toxicities in this study ranged from non-detectable to 72.63 pg OA equiv. cell 1; however, the lower number of replicates coupled with the use of Sedgwick-Rafter counts suggests that the extreme values are likely artifacts. Prorocentrum was present at high concentrations in many samples, but the evidence suggests that it played no major role in toxicity. Seventeen samples with greater than 1 cell mL 1 of Prorocentrum had no measurable OA equiv. However, one sample with detectable OA equiv. concentrations (Cavallo Pass on 4 April 2008) had no Dinophysis but contained over 9000 Prorocentrumkl spp. cells L 1. Most studies have identified Dinophysis spp. as the causative agent of DSP events, but in rare cases P. lima has been known to cause intoxication (Blanco et al., 2005; Morton et al., 2009). We saw no members of the P. lima complex (Aligizaki et al., 2009) in our study. Prorocentrum is a common member of the Texas coastal flora, and

since the OA occurrence appeared and disappeared with the Dinophysis bloom, it seems likely that Dinophysis was the OA source. We found no consistent pattern of toxin content per cell during the bloom (Figs. 5 and 7). Toxin content per cell increased as cells declined at four of the five offshore passes (Fig. 5). Inshore samples showed both increase and decrease of toxin content per cell as the bloom declined (Fig. 7). It seems likely that toxin content per cell was more than a simple function of bloom stage. Recent work has indicated that toxin can persist in plankton concentrates well after a bloom (Pizarro et al., in press), a report consistent with our observations of OA with few or no Dinophysis present. Although the early detection of the bloom resulted in no known cases of DSP, the occurrence of okadaic acid toxicity is a serious concern for the Gulf of Mexico. The 2008 event is the first documented closure of shellfish beds in the U.S. due to OA. Texas Department of State Health Services does not routinely monitor for OA, and unlike K. brevis blooms, there is no clear environmental signal (dead fish and respiratory irritation) that triggers shellfish monitoring. Five Dinophysis spp. cells mL 1 is now used as a threshold for oyster testing for OA, but abundance is not routinely monitored. Texas experiences significant economic losses due to Karenia red tides (Evans and Jones, 2001), with blooms occurring in ˜ a et al., 2003). Since Dinophysis the summer to late fall (Magan bloomed in the late winter, there is now a potential for spring closures due to DSP, and summer/fall closures due to neurotoxic shellfish poisoning. D. ovum (D. acuminata complex) has been recorded in archived offshore samples (Swanson and Villareal in prep.) but there is no evidence of shellfish toxicity. The Gulf of Mexico is experiencing a flowering of HAB events, and with the addition of DSP can claim among the highest diversity of HABs in N. America (see www.whoi.edu/redtide/page.do?pid=14898). Domoic acid has been repeatedly detected in the plankton (Dickey et al., 1992). Ciguatoxicity is present in fish from both the Flower Gardens Banks National Marine Sanctuary (T.A. Villareal and R.L. Dickey, unpubl. data) and along the Texas coast (Villareal et al., 2006, 2007). A seafood safety advisory is now in place for the Flower Garden Banks (Food and Drug Administration, 2008). The Texas coast also claims the longest running HAB in the scientific literature, the Texas Brown Tide (Buskey et al., 2001). Added to the long-standing K. brevis problem, and occasional Alexandrium monilatum blooms (Buskey et al., 1996), this increase leaves little doubt that HAB diversity and intensity is more apparent in the Gulf of Mexico than in the past decades, parallel with global trends in HABs (Smayda, 1990).

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