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View the pdf version of this report ECOHAB PNW 1 CRUISE REPORT B. Hickey, N. Kachel, A. MacFadyen, N. Adams, W. Cochlan, L. Connell, E. Lessard, V. Trainer, C. Trick, M. Wells Area of Operations
1. Regional Surveys (ECOHAB PNW team) 2. Drift Surveys (MacFadyen, Hickey, drifters; whole team for water samples) 3. Drifter Deployments (MacFadyen, Geier, Hickey, Fredericks) 4. Satellite Imagery (Woodruff, Stumpf, Geier) 5. Laboratory Analyses
b) Sandwich Hybridization Assay (Connell) c) RTC/SFSU Research Activities (Cochlan, Herndon, Ladizinsky) d) Trick Research Group (Trick, McClintock) e) Trainer Group (Trainer, Adams, Bill) f) Wells Group (Wells, Hughes) Acknowledgements List of Tables and Figures ECOHAB PNW 1
Arrive Seattle, WA, June 23, 2003
San Francisco State University University of Maine University of Washington University of Western Ontario
Principle Investigators
Dr. Laurie Connell, University of Maine Dr. Evelyn Lessard, University of Washington Dr. Vera. Trainer, NOAA/Northwest Fisheries Science Center Dr. Charles Trick, University of Western, London, Ontario, Canada Dr. Mark Wells, University of Maine
Brian Bill, NOAA/Northwest Fisheries Science Center Michael Foy, University of Washington Julian Herndon, San Francisco State University Margaret Hughes, University of Maine Nicolas Ladizinsky, San Francisco State University Amy MacFadyen, University of Washington The purpose of this cruise was to measure the physical, chemical and physiological conditions under which the algae Pseudo-nitzschia produce the toxin domoic acid, and when the toxin is released into the environment. We attempted to observe the conditions under which the released domoic acid moves toward the coast of Washington, where it can be taken up by shellfish. Such occurrences lead to closure of beaches to razor clam collection to avoid outbreaks of amnesic shellfish poisoning. Measurements made included continuous surface water properties, temperature, salinity, fluorescence, as well as discrete surface samples for particulate and dissolved domoic acid, chlorophyll concentration, and identification of phytoplankton species. In these surveys profile data taken with the CTD (conductivity, temperature, depth) included extra sensors that measured fluorescence, photosynthetically active radiation (PAR), beam attenuation (light transmission), and oxygen concentration. During CTD casts discrete samples were taken for chlorophyll and nutrient analyses. Several times during the cruise, an iron pump was used to measure vertical profiles of iron concentration. On deck incubations of phytoplankton for growth experiments, as well as shipboard laboratory analyses of the plankton were conducted. Satellite tracked drifters were released both near the Juan de Fuca eddy, and near the coast of Washington. The ship followed these drifters for several days each, so that the same parcels of water could be resampled as they aged, and thus measure in situ changes in the physical, chemical and biologic constituents. Additional drifters were deployed to estimate the ultimate fate of eddy water. The ship track and sampling stations are shown in Figure 1.
ADCP lines: ~4200 km Flow-Through system track with T,S,FL sensors: ~4200 km CTD casts: 249 Satellite-tracked buoy deployments: 7 (one was picked up and re-deployed)
Nutrient samples: 121 stations Microzooplankton samples: 23 profiles, plus 8 dilution experiments Phytoplankton/Domoic acid samples: 250 stations Fe samples (pumped): 6 profiles, 24 from 10 m depth only Zooplankton net tows: 5 The ECOHAB 1 cruise was remarkably successful, especially considering the number of new technologies utilized by the investigators. Evelyn Lessard was testing a FlowCAM, an imaging cytometer, to rapidly identify and count plankton > 5 µm. Charlie Trick was using a cell sorting flow cytometer for field studies. Vera Trainer used a 96 well plate format for rapid high-throughput particulate toxin analysis. Laurie Connell used specific molecular probes for Pseudo-nitzschia (PN) to rapidly determine plankton species. Mark Wells used a custom-made iron pumping system to acquire uncontaminated water samples. He was also using a newly designed chemiluminescent assay instrument for iron and copper determination. Bill Cochlan was using a flow injection analysis system to analyze dissolved nutrient samples at sea and in near real time. Some hardware problems occurred in the first 5 days. However these were all overcome with the help of shoreside support staff and the onboard marine technician, Daryl Swenson. One major problem was the loss of the towed iron fish on the first day of sampling. We believe the fish hit a submerged log. Iron samples were collected the next day using a small boat. Luckily, two of the ship's engineers, Hank Hazen and Chip Milard, constructed a new fish from materials onboard the ship. This fish performed well throughout the remainder of the cruise. The study included obtaining multi disciplinary data from a large scale grid (Section 1), sampling water properties while following a drifter (Section 2), deployment of surface drifters (Section 3), satellite imagery (Section 4), and laboratory studies using water collected at selected sites (Section 5). Moored arrays were deployed to provide time series of currents and water properties, including total domoic acid, plankton assemblages, and numbers of PN, from May to October, bracketing the first two survey cruises (Section 6). The setting of cruise sampling events within the wind setting (upwelling or downwelling favorable) is shown in Figure 2. The sequence of weather conditions was almost ideal, allowing a variety of water and plankton conditions to be sampled. Surveys and sampling were performed under strong, persistent upwelling conditions (the first half of the cruise), downwelling conditions (3.5 days only) and then weak upwelling conditions (the last week). Over 250 data profiles were obtained. Satellite imagery (SST and chlorophyll) was obtained on a number of days due to the generally good weather. Cruise activities were recorded in a sequential "Event" log (Table 1) from which summary tables discussed below were derived. 1. Regional Surveys (ECOHAB PNW team) The large scale survey grid was designed to include areas influenced by the Strait of Juan de Fuca, the Juan de Fuca eddy region and the coastal upwelling region off the Washington coast (Fig. 3). Data collected on surveys included conductivity (C), temperature (T), light transmission, PAR, oxygen and fluorescence (Fl) profiles, and bottle samples for chlorophyll, sandwich hybridization assays, whole cell fluorescence assays, particulate domoic acid, dissolved domoic acid, samples for scanning electron microscopy of PN species, plankton and macronutrients, all at selected depths. Surface net tows for qualitative community assessment were taken at all survey stations. Water samples containing PN were placed in medium for isolation and culturing in the laboratory. Underway data included T, S and Fl pumped from a depth of about 4 m near the ship's bow as well as ADCP current profiles from both a 75 khz Ocean Survey broadband RDI ADCP and a 150 khz narrowband RDI ADCP. Preliminary water property maps and sections are given in Appendix A (T, S, Fl maps at selected depths, including both underway and CTD data) and Appendix B (T, S, density, Fl transects versus depth for all transects, 0-100 m and 0-500 m scales). A list of CTD stations organized by sample line and including bottle sample types taken is given in Table 2. Lines were sampled in whichever direction made best use of ship time. Also note that occasional short (1-4 hours) time gaps occurred due to rough weather and also due to the necessity of providing a more stable platform for bio-chemical sampling. CTD profiles were taken to 500 m where possible. Deeper data were taken on LP and LC lines on Survey 1, and on the LP and LAB lines on Survey 2. Chlorophyll, particulate and dissolved domoic acid and plankton samples were taken near surface, 5 m, 10 m and at the chlorophyll maximum. On line LAB of Survey 3, however, domoic acid and plankton samples were taken at the surface, 5 m, 10 m, 15 m, 30 m, 50 m to obtain deeper vertical profiles. Macro nutrients were taken generally at the surface, 5 m, 10 m, 15 m, 30 m, 50 m, 100 m, 200 m, 250 m (occasionally), 300 m, 500 m and ~5 meters above bottom if the bottom was less than 500 m deep. At canyon stations, 5 m and 10 m samples were omitted and deeper samples were taken instead to investigate upwelling of nutrients from deeper depths (400 and 450 m). On survey grids, nutrients were taken in most cases at the two stations closest to shore on a line and then every other station on each line. Chlorophyll samples were taken at every station except the LA line in Survey 3, where they were taken only at nutrient stations. Upper water column iron samples were taken at selected stations (Tables 1 and 2 ). These samples were obtained by weighting the iron "fish" below the surface (~10 m) while towing at a slow speed. Samples typically were taken as the ship left station. Water was pumped for roughly 15 minutes to flush the lines thoroughly before samples were taken. Vertical iron profiles were obtained at several stations by lowering the fish to the target depths (typically 10, 15, 30 and 100 m depth) while maintaining a slow forward speed. The data are organized into three periods: Survey 1 (June 3-11), Survey 2 (June 12-16) and Survey 3 (June 17-22) (Fig. 2). Survey grid stations sampled in each period are shown in Figure 4a,b,c,d. The first survey (Fig. 4a), which took place during persistent and strong upwelling favorable winds and unseasonably warm, sunny weather, was the most complete survey and included two drift studies (DA and DB). Drift DB started at the end of Survey 1 and continued into Survey 2, where most of the drift occurred. The downwelling period (Survey 2, Fig. 4b) was short. Consequently only some of the northern lines could be sampled. While performing a drift survey (DB) CTD transects were made also along the axis and across Juan de Fuca canyon (Fig, 4d). The weak upwelling period (Survey 3, Fig. 4c) was sufficiently long to sample two southern lines and three northern lines, with a drift study in the southern region (DC). The CTD data were partially edited onboard ship. These data were used to construct the preliminary maps and sections appended to the report. Following the cruise, salinity calibration will be performed and more detailed editing completed. Although water property spatial patterns are likely robust, actual values may change slightly following the final editing which we hope to complete this fall. ADCP and water property data require more extensive processing and will be provided later this year. Some Preliminary Results: The first survey clearly captured the strong coastal upwelling that was occurring during that period (Appendix A, surface maps). The coldest, saltiest water near the coast was observed at the northern end of the grid. This result should be interpreted with caution since the strength and duration of upwelling was likely increasing in the direction of our sampling (south to north). The underway salinity and temperature data maps are very similar to the near surface maps constructed from the CTD data. In the first survey cold water at the surface appeared to emanate from the strait. However, the freshest water was observed in a band running north-northwest offshore of the upwelling zone and was not connected with the strait at the surface. We note that the first survey was performed during a period of neap tides, when surface outflow and hence salinity from the strait would be reduced. The surface fluorescence during the first survey showed two regions of high values-one offshore of the strait and southeast of Barkley Sound, the other, off the northern Washington coast. Between these two maxima was a region of lower fluorescence that appeared to emanate from the strait. This low fluorescence region was observed also in several of the chlorophyll satellite images. Low chlorophyll appears to emanate from the strait and "wrap around" the higher chlorophyll water. Although it is tempting to think that the two regions of high surface chlorophyll had been bisected by outflowing strait water, a quick look at salinity patterns suggest that this is not the case-the salinity corresponding to most of the high fluorescence region off the Washington coast is much higher than that of the high fluorescence region off Barkley Sound, suggesting that they reside in different water masses. Moreover the fluorescence sections given in Appendix B show subsurface maxima in lines off the Washington coast, but not generally off Barkley Sound. A subsurface chlorophyll maximum is typical of coastal upwelling regions. The eddy center-defined as the region of maximum property "doming", varied between depths. In general, the eddy center was closer to the strait at shallower depths. Because density is controlled by salinity in this region, salinity is a better indicator of density differences and hence current patterns. At 100 m on Survey 1 the eddy center was at about 48o 20' N 125o 15' W, near the center as defined by multiple eddy tracks deployed in other years and in fall rather than in June. Thus, as we hoped, the mooring E3 appears well placed to monitor currents and water properties slightly away from the center where we expect currents to be less variable. Significant differences in water property patterns were observed between the first survey period, an upwelling period, and the second survey, a downwelling period. Three lines off northern Washington and Vancouver Island were repeated in Survey 2 (Fig. 4b). Surface temperatures dropped by several degrees, possibly indicative of mixing during the storm. 2. Drift Surveys (MacFadyen, Hickey, drifters; whole team for water samples) Three drift surveys were performed. Deployment and recovery times and deployment location are listed in Table 3. The goal was to follow patches of water from (a) the eddy and (b) the coastal upwelling region, examining water properties as the patches aged. The first two drifts were attempts to follow water from the eddy-however, in the one case, the drifter was lost before escaping the eddy (DA); in the second case, the drifter stayed in the eddy and went round it (DB). In the third drift (DC) the drifter never left the coastal upwelling zone. Drifter tracks and CTD stations taken during the drifts are shown in Figure 5 a,b,c. The first drift study (DA) took place during strong upwelling (Survey 1). The drift was begun 1-2 miles east of station LAB6. The second drift (DB) took place at the end of the strong persistent upwelling period and continued through most of the storm (Survey 2). The drift began near LAB4. The third drift (DC) took place during weak upwelling in Survey 3. CTD profiles and bottle casts were taken at the start of each drift and water was collected for incubation experiments. A Brightwaters GPS-type drifter was deployed, drogued at 5 m for DA and DB, but at 10 m for DC. CTD profiles were taken at 6 hour intervals for roughly a day to accumulate data on tidal changes, then at 12 hour intervals until the end of a drift. In drift DC, CTD profiles were taken at 3 hour intervals for the complete drift. That drift was aborted after 18 hours when the drifter entered water shallower than its drogue. The deckboard grow-out incubations (Wells/Cochlan/Trick) were typically run for 4-5 days. Water was collected at the time the drifter was deployed. Treatments for the deckboard experiments included both metal (Fe, Cu) and chelator (desferal, domoic acid) manipulations. Incubation bottle and in-situ samples were taken for Chl a, nutrients, cell composition and domoic acid concentrations. Samples for bacterial productivity and Fe uptake measurements were additionally taken for the deckboard experiments. Deckboard dilution experiments (Lessard) were run for 24 hours with water collected at the beginning, middle and end of each drifter survey. Samples for size-fractionated chlorophyll, picoplankton, nanoplankton and microplankton, macronutrients, dissolved and particulate DA and sandwich hybridization assays were taken in each experiment. Experimental manipulations included the addition of DA, Fe and macronutrients. The first drift, which occurred near the edge of the eddy (Fig. 5a) had much lower near surface nutrients than the second drift, which was more towards the eddy center (Fig. 5b). The first drift was aborted when the drifter transmission failed and the drifter was lost. The DB drift started south of the eddy center but moved northwest and then around the eddy towards the northeast after crossing the mouth of the strait (Fig. 5b). The final drift was begun nearshore near LP1 under weak upwelling conditions (Fig. 5c). In spite of the upwelling, a "lid" of Columbia River plume water remained over the nearshore water throughout the drift (see sections in Appendix B). The origin of this water in the plume was confirmed with underway surveys south towards the Columbia mouth, with CTD transects and with satellite turbidity imagery, graciously provided by R. Stumpf"s group. We speculate that the plume was particularly strong due to the occurrence of spring tides. Also June is the month of maximum seasonal outflow from the Columbia. Isopleths below the shallow plume layer showed clear evidence of upwelling, and nutrients were available at deeper depths but not at the surface. Surprisingly, the drifter remained in shallow water, traveling rapidly down the coast and generally crossing into shallower water (Fig. 5c). This lack of offshore movement was likely a result of the lid of plume water and/or the 10 m drogue depth. 3. Drifter Deployments (MacFadyen, Geier, Hickey, Fredericks) Three surface Davis-type Clearwater GPS drifters were deployed to delineate patterns and speeds of surface flows in the eddy area, as well as to determine the ultimate fate of eddy water. Drifter deployment and recovery times and deployment locations are given in Table 3. Two drifters were deployed near the eddy center. The third drifter was deployed in the mouth of the strait with the hope of tracking the pathway of water exiting the strait. Data were stored at UW and also transmitted to the ship by Susan Geier. Deployment times and locations are listed in Table 3. Drifter location and water temperatures are available at 30 minute intervals during deployment. These three drifters will continue to collect data until about the end of August. The two drifters deployed during the strong upwelling event (3819, blue track; 3861, red track) traveled southsoutheast at speeds of 15-20 miles per day (Fig. 6). Although the second drifter was deployed several miles east of the first drifter and a few days later, the second drifter moved rapidly to the same pathway as the first, indicating the existence of a strong front in this region. Both of these tracks were very similar to drifter tracks in two previous years in September, indicating the robust nature of the eddy and the coastal front. The front was confirmed by our CTD surveys and by satellite imagery. During the downwelling event that occurred mid cruise both drifters moved toward shore. The drifter near Kalaloch moved about 10 miles across the shelf to within 7 miles of the beach where it turned northward (see red circular path). It traveled another 20 miles north along the coast until the winds again turned to upwelling favorable. It again moved offshore to almost the identical pathway the second drifter had traveled. The drifter that was close to the Columbia River mouth when the storm occurred moved only slightly onshore, likely being impeded by the strong fronts bounding the Columbia plume. The drifter slowed during the storm but subsequently continued south along the shelf following the isobath direction. All three drifters moed offshore near Haceta/Stonewall banks in Oregon and proceeded south. Two drifters passed in to California before going offline (Fig. 6). The drifter deployed in the strait (3817, green track) was deployed at maximum ebb during spring tides. It was deployed in the center of the strait in the hopes of avoiding transit in the near coast Vancouver Island Coastal Current. To our surprise, the drifter initially went upstrait, then crossed to the south side of the strait before exiting the strait westward. It then milled about in tidal motions in the region near the mouth of the strait for several days before finally turning south, like its predecessors, following the coastal front (Fig. 6). The drifter tracks illustrate that the location of the coastal front off Washington and northern Oregon was relatively fixed throughout the cruise-this dramatically illustrates that eddy water, and, surprisingly, water exiting the Strait of Juan de Fuca, could impact much of the US west coast. 4. Satellite Imagery (Woodruff, Stumpf, Geier) Satellite imagery during the cruise was provided by two groups who sent data to the OSU ftp site-Dana Woodruff from Battelle Northwest provided SST imagery and surface chlorophyll was provided by Rick Stumpf at NOAA. Susan Geier (Hickey group) assessed data quality for the shipboard group, emailing Dr. Hickey with daily recommendations. The available imagery and an assessment of its quality are listed in Table 4. Both data sets proved to be valuable tools during the cruise. In particular, SST images were useful in locating upwelled water and, more important, in showing changes in surface eddy expression. For example, the eddy as captured by our survey lines appeared to move nearshore during the first part of our cruise. We subsequently confirmed this with the imagery. The images also confirmed that in the weak upwelling period of Survey 3 upwelled water was not reaching the surface anywhere near the coast. This information helped us change strategy and move back to the eddy before our final shipboard samples. The chlorophyll images, which had better spatial coverage, were the most useful. These images showed low chlorophyll water exiting the strait and swirling around the eddy. The patterns appeared to have a good relationship to the patterns we were observing shipboard in relative fluorescence. We used some patterns to select in situ sampling sites. a) Lessard Group (Evelyn Lessard, Brady Olson, Michael Foy) The main goal of this component of ECOHAB PNW is to determine the role of grazers in PN population dynamics and domoic acid (DA) production. We are using two main tools: the dilution experiment and species-specific rRNA probes. The dilution experiment allows us to experimentally alter grazing pressure and determine grazing effects on net growth rate of the whole and size fractionated phytoplankton community, as well as specific species/groups of phytoplankton, dDA and pDA production. The rRNA probes allow us to identify specific grazers on PN (protist and zooplankton) and, with further development, specific grazing rates. We also took FlowCAM and fixed samples to follow the in situ spatial and temporal changes in the protist grazing community in relation to PN and hydrography. On this cruise, we performed the following:
b) Sandwich Hybridization Assay (Laurie Connell) The goal of this aspect of ECOHAB PNW was to initiate field testing of PN sandwich hybridization assays used to identify and enumerate HAB species in near real-time from environmental samples. Extracted nucleic acids from cell lysates are assayed with two oligonucleotide probes, a capture probe and signal probe. The capture probe immobilizes target sequence from the crude cell extract onto a dextran-coated solid support. A "sandwich" hybrid complex is formed when the immobilized target sequence is transferred to a second solution containing a dig-labeled signal probe. SHA products are detected using an anti-dig antibody conjugated to horseradish peroxidase. The horseradish peroxidase reacts with a substrate to generate a blue colorimetric product, the intensity of which is representative of the target cells present in the original sample. When acidified this product turns yellow. SHA was carried out using pre-dispensed reagents in 96-well microtiter plates. Environmental samples were filtered onto a 5 µm, 25 mm Durapore membrane filters (Millipore). Cell lysate were prepared by adding filtered cells to Sample Solution Premix then incubating the cells within Lysis Tube (thin wall tube) at 80ºC for 5 minutes. Cell lysates were then loaded into the Universal Processor (Affirm Corp.) for processing. The optical density (OD) of the colorimetric product was then read using a 96-well plate spectrophotometer. Four capture probes were field tested during this cruise with four primary PN species as targets. AU targets P. australis, MuD1 targets P. multiseries, 006 targets P. pseudodelicatisima, and WA001 targets P. delicatisima. After initial tests for general background and basic sensitivity, 1L samples were concentrated from each sample for use with each probe. Results were encouraging. However cell numbers in samples cannot be determined until isolates of cells collected from this cruise are cultured, tested and standard curves are produced. Some Preliminary Results:
Bottom line--these probes show promise after cell numbers are ground-truthed using standard microscopy and cell counts. c) RTC/SFSU Research Activities (William Cochlan, Julian Herndon, Nicholas Ladizinsky) The primary objective of this componenet of ECOHAB PNW is to examine the relationship between elevated concentrations of the pennate diatom PN and its toxin domoic acid, and ambient concentrations of macro-nutrients and phytoplankton biomass. At each station, phytoplankton biomass levels were estimated from chlorophyll a (Chl a) concentrations determined using in vitro fluorometry (aboard ship) after extraction for 24 h with 90% acetone. Chl a samples generally were collected at four (4) depths (0, 5, 10 m, and the depth of the chlorophyll maximum). At every second station, dissolved inorganic nutrients were collected at 0, 5, 10, 15, 30, 50, 100, 200 m and near bottom) and analyzed using appropriate colorimetric methods for determination of nitrate, phosphate, and silicate with a Lachat Instruments QuickChem 8000 Series Flow Injection Automated Ion Analyzer. Both Chl a and nutrients were determined at the two most-shoreward stations of each sampling line. Vertical profiles of nutrients were also analyzed at a series of four (4) drifter stations at either 6- or 12-h intervals in addition to determination of size-fractionated biomass: total planktonic community, as collected on Whatman GF/F filters (nominal pore-size of 0.7 µm), and cells > 5 µm in size (Poretics silver membranes). Dissolved nutrients were determined at the beginning (time-zero) and end (time-final) of all of the dilution experiments performed by Lessard's research group. During a series of four shipboard incubation experiments (outlined by Trick and Wells), bacterial heterotrophic productivity (3H-leucine uptake method) was measured daily to evaluate the relationship between micro-nutrient (Fe, Cu) availability and bacterial degradation (or possible stimulation) of domoic acid production by PN. Bacterial abundance estimates, determined by Trick using flow cytometry [Becton Dickinson, FACSCalibur], will be used to calculate specific bacterial productivity. Potential new production was determined using the 15N-tracer technique using saturated nitrate tracer concentrations (10 or 20 µM) to estimate maximal nitrate uptake potential as an indicator of phytoplankton community physiological "health". Size-fractionated phytoplankton biomass (as described above) was determined for all metal treatments on all days of the incubation experiments. Expected Results:
d) Trick Research Group (Charlie Trick, Liza McClintock) Our contribution to the ECOHAB project is two-fold: 1) to provide flow cytometric analysis of the communities; and 2) to provide experimental evidence of factors that either increase the competitive ability of PN or increase the level domoic acid per cell. For flow cytometric (FCM) analysis we brought on-board the flow cytometer with cell sorting capabilities. Initial experiments using our flow cytometer indicated that we were able to assay samples for the presence of bacteria, cyanobacteria (phycoerythrin-containing and phycocyanin-containing prokaryotes), picoeukaryotes and nanoeukaryotes. However we were unable with the setup presently installed in the flow cytometer to quantify these groups of organisms since the large chain-forming diatoms physically impeded the flow of seawater sample into the assay chamber. Without our ability to quantify samples we resorted to the backup procedure of collecting and freezing samples for analysis back in the laboratory. We will be able to solve the problem back in the lab using one of two procedures: modifying the sampling orifice to allow for the sampling and assay of larger cells or using small volume centrifugal filters, remove the largest cells that cause the blockage, followed by normal FCM analysis. For our analysis we have collected FCM samples from all of the survey points (for depths of 0, 5, 10 m and the depth of maximum chlorophyll). We have supplemented this collection with several deep samples and samples collected at the previously described depths from the drifter sites (samples once-a-day). Overall, we have collected more than 2000 FCM samples for analysis of the indicated cell groups. In addition to the FCM samples, at each of the survey sites (and at each of the indicated depths) we collected cells for pigment analysis. Pigment analysis will be performed using our HPLC isolation-and-characterization methods. This method uses the presence or absence of the taxon-specific pigments (often referred to as the "minor or accessory" pigments) in relation to the ubiquitous photosynthetic pigments (chlorophyll) to describe the phytoplankton community structure. We recognize that diatom-rich communities (where the presence of PN and other diatoms brings great joy to this research group) are the focus of this study (and are easily described using light microscopy.) Our analysis by HPLC will establish the composition of the communities before and after the presence of the diatom communities, thus serving as an important oceanographic descriptor. These samples will be analyzed within the next month since they preserve poorly. Maps of reconstructed photosynthetic communities will be available prior to the September cruise. In our second contribution to the cruise mandate, the personnel from the Cochlan, Wells and Trick lab carried our several incubator studies - termed "grow-out" experiments. All labs offered their expertise to the common goal (biomass formation, nutrient drawdown measurements, DA analysis (particulate and dissolved), community structure changes, bacterial productivity, nitrogen and iron uptakes rates). The overall foundation of these grow-out experiments was aimed at elucidating the factors that influence the initiation, formation and/or maintenance of PN blooms or DA levels (either cellular or extracellular). For every cruise we may have different hypotheses to test but the working hypothesis for this set of experiments was that PN benefits from producing DA because DA serves as an iron and/or copper chelator. Thus in the presence of macronutrients (either in upwelling sites or in the areas of high nutrients associated with the Juan de Fuca eddy) DA would act as an iron chelator, ensuring that the cells would have a supply of iron as iron concentrations diminish, either through colloid formation or utilization. Alternatively DA could serve as a copper chelator, reducing the levels of cupric ion to less inhibitory levels, allowing PN to utilize the nutrients and grow. Four grow-out experiments were performed (two from upwelling areas, two from the Juan de Fuca feature). Samples were collected at a place and time where some PN were present in the water column but the levels were still lower than anticipated (allowing "room" for these cells to grow to higher densities - we refer to this as having "bloom potential"). Using a combinatorial experimental design we followed communities in bottles amending with the appropriate nutrients, chelator and/or copper. While analysis will take time, we should be able to evaluate the role of these inducers on DA production and community structure before the September cruise. e) Trainer Group (Vera Trainer, Nicolaus Adams, Brian Bill) At each survey and drift station, samples were routinely taken at 0, 5, 10 m and chlorophyll maximum for measurement of particulate and dissolved levels of domoic acid. Samples were taken at the surface and chlorophyll maximum for whole cell counts of PN, enumeration of PN size classes, and scanning electron microscopy for species determination in selected samples. A net tow was taken at every station to rapidly determine the presence or absence of PN and their relative abundance. At selected drifter and eddy stations, depth profiles of cells and toxins were done at some of the following depths: 0, 5, 10, 20, 30, 50 m. When large PN were numerous, samples were analyzed for whole cell hybridization to P. australis species-specific molecular probe. These samples are designated as VT in Table 1. Particulate domoic acid was analyzed by filtering 1 L seawater through 2-3 Nucleopore HA filters (0.45 micron pore size). Filters were minced in 5 ml distilled water with a glass pipet and sonicated for 2 h in a bath sonicator to lyse cells. An aliquot of each sample was analyzed using a receptor binding assay in 96-well plate format using a multiwell harvester and Top Count scintillation counter. The receptor binding assay uses the displacement of [3H]kainate by domoic acid in a sample from a cloned glutamate receptor. Each plate of samples is compared to known domoic acid standards analyzed on the same plate. Endogenous glutamate was digested prior to sample analysis using glutamate dehydrogenase. Whole cell hybridization assay Up to 25 ml sample was filtered and fixed with saline-ethanol for 2 h. Then a specific P. australis probe (auD1) was incubated with samples from several depths and compared to uniC (positive universal species control) and uniR (negative control) probes. Positively labeled cells on each filter were counted using fluorescence microscopy. Dissolved domoic acid Several control experiments were performed using an enzyme-linked immunosorbent assay for domoic acid using a specific antibody developed in rabbit. Replicate sample variability was high, therefore these samples will be frozen and analyzed upon return to the lab. Pseudo-nitzschia culturing At stations throughout the cruise where PN cells were present, a drop of sample was placed in f/2 medium for isolation and culturing upon return to the lab. PN cells will be allowed to grow in artificial seawater medium and growth and toxin production will be determined for several isolates. This will allow us to understand the relative levels of dissolved and particulate toxin each species is contributing to our cruise samples. f) Wells Group (Mark Wells, Peggy Hughes) The primary goals of this ECOHAB PNW component on this cruise were to collect seawater samples for determining the distribution of dissolved Fe concentrations in and around the Juan de Fuca eddy, and to field test a new flow injection analysis instrument for online determinations of dissolved Fe and Cu concentrations in surface and deep waters. Fifty-five water samples were collected using a trace metal clean tow-fish deployed from the ships' main boom. These collections included both surface (underway) samples and six deep (= 100 m) profiles. The original tow-fish and 10 m of Kevlar-reinforced tubing was lost to an underwater obstacle early in the cruise, but a replacement fish was fabricated by the Assistant Engineers and was successfully deployed for the remainder of the cruise. Flow injection analysis proved to be highly sensitive (detection limits for Fe of < 50 pM). Cross interferences of the dual chemiluminescent methods for Fe and Cu were tested and shown to be insignificant. Alternate column techniques were tested but were found to be much less effective than the direct (non-column) method. The analyses show oceanographically consistent patterns in Fe distributions. However, difficulties in accurate determination of the analytical blanks limited the on-board use of the instrument. The root of this problem was determined, and several approaches were identified for testing on return to the laboratory. Water samples will be returned to U. Maine for dual determination of Fe and Cu by both FIA and Inductively-Coupled Plasma Mass Spectrometry methods. Work also was done in testing Vera's new antibodies for the detection of dissolved domoic acid with cELISA. These tests found the method to lack precision and accuracy at sea, in contrast to runs on-shore. More than 80 dissolved and particulate fractions have been collected from the incubation experiments for analysis of domoic acid on our return. In addition, a 2 day experiment was conducted to determine the photodegradation kinetics of domoic acid in the deckboard incubation bottles. 6. Moored Sensor Arrays: (Barbara Hickey, Richard Thomson) Three arrays of moored sensors were deployed May 9-11 from the R/V Tully. Deployment times and locations are listed in Table 5. The moored arrays were designed to collect time series of winds, above surface and subsurface PAR, currents and water properties throughout the water column, plankton, and domoic acid between June and October, spanning the period of both ECOHAB PNW cruises. Deployments from the CCGS J. P. Tully were made under the supervision of Richard Thomson; the primary marine technicians were Tom Juhasz from the Institute of Ocean Sciences and Jim Johnson from the University of Washington. Sensor set up was primarily done by Susan Geier at the University of Washington. Wind sensors were provided by and set up by the Institute of Ocean Sciences. Nicolaus Adams set up the Aqua Monitors. Bill Fredericks prepared the toroidal buoys, lights, satellite transmitters and towers. Locations of the moorings (EH1, EH2, and EH3) are shown in Figure 3. The moorings (Fig. 7) consist of toroidal surface buoys supporting wind and PAR sensors (above water), a Sea-Bird MicroCAT 37 (C,T) at 1 m, 15 m (C,T) and 7 meters above bottom, a Sea-Bird 16 (C,T) with fluorometry and PAR at 4 m, Sea-Bird 39s (T) at 5, 20 and 40 m, a downward looking 300 khz ADCP at 5 m, an InterOcean S4 current meter at 4 m and an EnviroTech Aqua Monitor at 4 m. The Aqua Monitor was set to acquire samples every 3 days; 1% formalin was added to sample bags prior to deployment. These samples will be analyzed to produce time series of phytoplankton abundance and total domoic acid using enzyme-linked immunosorbent assay (ELISA). We would like to thank the captain and crew of the R/V Wecoma for their support and extra effort that made the June 2003 cruise successful. We thank the crew and officers of CCGS J.P Tully and the IOS/OSAP/UW mooring team of Tom Juhasz, Dave Spears ad Jim Johnson for their help in mooring deployments. This research was supported through the Ecology and Oceanography of Harmful Algal Blooms program by National Oceanographic and Atmospheric Administration/Coastal Ocean Program Award No. NA17OP2789 and National Science Foundation Award No. 0234587. Mooring deployments on the Tully were made possible by Canadian support to Rick Thomson at IOS. Table
1 Event log Fig.
1 Cruise track with sampling stations.
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