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View the pdf version of this report. ECOHAB PNW 3 CRUISE REPORT B. Hickey, V. Trainer, W. Cochlan, E. Lessard,
C. Trick, M. Wells, A. MacFadyen, N. Adams, Area of Operations
1. Regional Surveys (ECOHAB PNW team) 2. Drift Surveys (MacFadyen, Hickey, drifters; whole team for water samples) 3. Drifter Deployments (MacFadyen, Hickey, Postel, Leithauser) 4. Satellite Imagery (Woodruff, Stumpf) 5. Laboratory Analyses
b) Sandwich Hybridization Assay (Trainer) c) RTC/SFSU Research Activities (Cochlan, Herndon, Ladizinsky) d) Trick Research Group (Trick, McClintock, Beall) e) Trainer Group (Trainer, Adams, Baugh, Bush, Day, Bill) f) Wells Group (Wells, Pickell, Hardy, Hughes) Acknowledgements List of Tables and Figures
ECOHAB PNW 3
Arrive Seattle, WA, September 28, 2003
San Francisco State University University of Maine University of Washington University of Western Ontario
Principle Investigators Dr. Vera Trainer, NOAA/Northwest Fisheries Science Center James Postel, University of Washington Amy MacFadyen, University of Washington (Ph. D.) Herb Bergamini, Northwest School 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 toxic cells advect towards the coast of Washington, where they are consumed 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, analysis of community assemblage 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. A trace metal clean underway sampling system was used to measure iron concentrations on board, and to collect samples for later multi-element determination . On deck incubations of phytoplankton for growth and grazing experiments, as well as shipboard laboratory analyses of the plankton were conducted. Satellite tracked drifters were released in the strait, near the Juan de Fuca eddy and off the coast of Washington to delineate patterns and speeds of surface flows in the eddy area, as well as to determine the ultimate fate of eddy water. Drift studies were also performed, during which the ship followed strategically placed drifters allowing the same parcels of water to be resampled as they aged, and thus measure in situ changes in the physical, chemical and biologic constituents. The ship track and sampling stations are shown in Figure 1. ADCP lines: Chlorophyll
samples: >200 stations and deck-board experiments
(~1500 samples) The ECOHAB 3 cruise was highly successful: Pseudo-nitzschia (PN) blooms of unprecedented high densities and toxicities in the region persisted throughout the cruise. This situation provides an important contrast to the 2003 cruises, in which PN were present, but never a dominant species of the phytoplankton assemblage. The longevity and toxicity of the blooms as well as their large spatial extent provided a rare opportunity for studying toxic blooms in situ. The study included obtaining multi disciplinary data from a large scale grid (Section 1), sampling water properties and plankton while following a drifter (Section 2), deployment of surface drifters (Section 3), satellite imagery (Section 4), and laboratory studies using water/plankton collected at selected sites (Section 5). The setting of cruise sampling events with respect to wind direction (upwelling or downwelling favorable) is shown in Figure 2. For simplicity we characterize the wind patterns into three periods: predominant downwelling (1), intermittent weak upwelling/downwelling (2) and weak, persistent upwelling (3). In general, the weather conditions were much more downwelling favorable in contrast with both cruises in the prior year. This allowed us to reduce our effort on sampling a large scale grid—no dramatic weather changes occurred to warrant extensive repeat sampling until late in the cruise. Moreover the slower current speeds allowed us to successfully follow and sample at drifters for extensive periods. Thus more emphasis was placed on time changes following patches of water, in particular, following patches with high PN density and high particulate domoic acid (DA) concentrations. Upwelling favorable winds occurred briefly near September14 and September 18-20. However winds during these periods were either too weak or too brief to cause significant changes in the density field (although surface drifters did respond to these events). On the other hand, the change to more persistent upwelling after September 23 resulted in higher air temperatures and warming of surface layers. After September 23 weak upwelling winds persisted through the remainder of the cruise—to investigate chnages, one coastal and one eddy transect were re-sampled. Over 200 data profiles were obtained. Satellite imagery [Sea surface temperature (SST) and chlorophyll] was not generally available due to the generally poor weather. However a collage of three SST images were made to coincide with our grid sampling period. Cruise activities were recorded in a sequential “Event” log (Table 1) from which summary tables discussed below were derived. The cruise was diverted to Neah Bay on two occasions (September 13 and 20) in order to exchange personnel and re-supply clean water. A malfunctioning clean water system (Millipore) required WHOI to send out additional filters. Carboys of water from the Lessard laboratory were brought to the ship on these exchanges. The shortage of Millipore water during peak usage times delayed nutrient analyses and other studies. This was the largest initial problem on this cruise. The shipboard water shortage was partially resolved by assistance from the ship’s engineering staff and the additional resin cartridges. 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, FlowCAM samples, 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 as well as ADCP current profiles from a 150 khz narrowband RDI ADCP. An ISUS nitrate sensor was tested during this cruise. Underway data should be treated with caution up to about September 16, 2030 GMT. During the first part of the cruise, sensors were mounted in their usual position on Atlantis, forward and near the bow thruster at a depth of about 5 m. Comparison with salinity and temperature data from the CTD indicated two problems: first, salinities were too low by about 1 psu and temperature, although closer to the CTD value, was low by about 0.5oC; second, salinity data had very large (several psu) fluctuations when the ship was on station. The latter were attributed to the use of the bow thruster. Subsequently (September 16, 2030 GMT) all sensors were moved to a position mid ship and a water depth of about 4 m. Although this eliminated the fluctuations it did not eliminate the mean offset problem, nor did use of a different sensor. Sensors are Falmouth Scientific CTDs. Finally, a thorough cleaning of the area in which the sensors were housed was done and salinity and temperature means closely approached those on the CTD. Sensors were left in this position for the remainder of the cruise. We did not perform a systematic evaluation of other underway sensors such as the fluorometer. The ISUS nitrate sensor, mounted with the other underway sensors, did not function well during this cruise. Its behaviour was similar to that on ECOHAB2: large offsets occurred. On ECOHAB2 calibration data were sufficient to compute a regression and recover the data. On this cruise, however, offsets appeared to change frequently so that there is little hope of obtaining useful data from this instrument. Note that calibration prior to installation on the ship was extremely tight. NMFS personnel will continue to work with the manufacturer to address these issues. 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. Note that occasional short (1-4 hours) time gaps occurred due to the necessity of obtaining water/plankton samples for bio-chemical incubations. CTD profiles were taken to 500 m where possible. Deeper data were taken on the KB and LB lines on repeat line sampling following the main survey (only). Chlorophyll a, particulate and dissolved DA and plankton samples (for both microscope and molecular probe analysis) were taken near the surface, 5 m, and 10 m (and at 15 m at stations after the initial grid survey). DA samples were also taken at 15, 30 and 50 m at many stations, in particular, at drift stations. Flow cytometry and HPLC pigment samples were taken at 5 m depth. Macro nutrients were taken generally at the surface, 5 m, 10 m, 15 m, 30 m, 50 m, 100 m, 200 m, 500 m and ~5-10 meters above bottom if the bottom was less than 500 m deep. At canyon stations 5 m and 15 m samples were omitted. In the strait survey, samples were taken also at 150 m. On transects, macro 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 most 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). 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 depths). The CTD data were partially edited onboard ship. Shipboard editing included replacing downcast data with upcast data at several early stations (casts 7-9): at these stations the CTD pump was inadvertently not turned on in the upper water column. The edited 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 (Hickey group). 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 data require more extensive processing and will be provided later this year (Foreman group). Preliminary water property maps and sections are included in the PI data secion (T, S, O2, Chl, Fl surface maps at selected depths and T, S, density, Fl, O2 transects versus depth for all transects, 0-100 m and 0-500 m scales). Note that contours of fluorescence voltage differ from those in prior years. Maps of relative abundance of PN at the surface are also included. The CTD data are organized into three groups: Survey 1 (September 9-September 16), drifter stations (September 17-26) and re-sampled transects plus canyon and strait transects. Note that the LP line was sampled once prior to the entire grid survey, with the hope of sampling before and after downwelling conditions. Grid stations sampled in each period are shown in Figure 4a,b. Survey 1, which took place during persistent and sometimes strong downwelling favorable winds was the only complete survey (Fig. 4a). The lack of strong or persistent upwelling meant that conditions during the survey as well as following it were unlikely to change significantly. Note however that surface velocities as measured by our drifters always responded to changes from downwelling to upwelling with directional changes onshore or offshore (in the surface Ekman layer). This complete downwelling survey (our first for ECOHAB PNW) allows us to contrast upwelling and downwelling nutrient and flow patterns (although between years). In general, the shelf break coastal jet was weaker than in prior years’ surveys, likely because of the unusually persistent downwelling conditions that would allow the seasonal upwelling pattern to weaken prematurely. Consequently, surface flow patterns were generally weaker and the eddy was more retentive than in our prior surveys. Drift DA started at the start of weather period 2 (intermittent and weak upwelling and downwelling; Fig. 2) and continued through the remainder of the cruise into the third weather period (weak but persistent upwelling). The water for drift DA was taken from the northeast edge of the eddy. This drift lasted for 10 days, with the drifter remaining in the eddy the entire time. Samples were taken as frequently as 3 hr intervals. Drift DB was initiated as an “add on” to a drifter that had been deployed the first day of the cruise with no intention of following it with samples. It proved so interesting that we initiated sampling on September 17, returning to it roughly every 24 hr. Drift DC was also an “afterthought”. It was deployed at a location that in subsequent analyses we found to have high domoic acid. This drifter effectively followed a “hot” filament from the eddy down the coast. We sampled it on three occasions distributed throughout the cruise. The Kalaloch line was sampled twice after the complete grid survey. One the first occasion, winds were downwelling favorable; on the second, winds had been weakly upwelling favorable for about 2 days (Fig.2). CTD/nutrient transects were made also along the axis and along Juan de Fuca strait during the weak but persistent upwelling period (Fig. 4b). The same canyon section was also sampled in June and September 2003. Deeper nutrients were collected on these sections. The beginning of the strait data collection occurred at the start of flood, continuing through flood and ebb. The canyon data were collected through flood and ebb, with the end of ebb coinciding with the stations near the mouth of the strait. Note that this is the first continuous transect made through the canyon and strait. In 2003 both regions were sampled, but the two sections were not connected in time. Some Preliminary Results: The complete survey clearly captured the coastal downwelling that was occurring during most of the sampling period (PI data, surface maps). Fresher water was observed along most of the Washington coast. From Kalaloch south the freshest water was observed slightly offshore of the coast: this is the signature of a plume from the Columbia that has been slightly displaced offshore in the intermittent weak upwelling and downwelling that occurred just prior to these observations. Fresher water was also observed off the Vancouver Island coast: this is the signature of water emanating from the strait, forming the Vancouver Island Coastal Current that typically hugs the Vancouver Island coast. The Juan de Fuca eddy is evident as a saltier, colder region centered offshore and slightly south of the strait. Both temperature and salinity indicate a tongue of this water extending down the Washington coast. The surface fluorescence during the first survey period showed high fluorescence in the region corresponding to the eddy and the tongue directed down the Washington coast. This result is best seen by comparing salinity and fluorescence maps—higher fluorescence occurs roughly in the region of slightly higher salinity.
Psuedo- nitzschia cells and particulate DA reached the highest
levels observed over the broadest area thus far in any of our cruises.
This made sampling for water for experiments much easier. In general
highest
DA concentration and PN counts were observed in the colder water
of the eddy and in the filament extending south to the Washington
shelf.
The numerically abundant diatom is a strain of P. pseudodelicatissima
that was not recognized by the specific muD2 molecular probe. Intermittent and relatively weak winds persisted over much of the cruise (Fig. 2). Hence, in contrast to prior ECOHAB cruises no attempt was made to resample major portions of the grid. Persistence increased after September 18, with a weak upwelling event of about 2 days followed by weak downwelling for about 2 days (weather period 2). The KB line was re-sampled at this point, with the intention of capturing a well developed Columbia plume after the considerable period of downwelling over the prior 2 weeks. The hope was to determine whether the plume had significant DA and PN and whether PN might be subducted beneath the plume. A longer period of weak upwelling (weather period 3) extended through the end of the cruise (September 23-28). The KB and LB lines were re-sampled and the canyon and strait transects were sampled during this period. 2. Drift Surveys (Amy MacFadyen, Barbara Hickey, drifters; whole team for water samples) Three drift studies were performed, following water patches with satellite-tracked Brightwaters drifters. To avoid regions of high current shear, no drogues were used—the drifters average the top meter of the flow field. Note that Mark Wells’ iron fish samples were taken slightly deeper (~ 8-10 m) due to requirements of the iron pumping fish; Lessard sampled at 2-4 m to obtain ideal light levels. Deployment and recovery times and deployment location are listed in Table 3. Note that drifts are being numbered sequentially beginning each ECOHAB year. CTD profiles and bottle casts were taken at the start of each drift and water was collected for incubation experiments. CTD profiles and nutrients were taken at 3-24 hour intervals. The deckboard grow-out incubations (Wells/Cochlan/Trick) during drifts 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 chelators/ligands (desferal, domoic acid) manipulations. Incubation bottle and in-situ samples were taken for Chl a, nutrients, cell counts, species composition, rates of carbon uptake (P vs. E), DA concentrations. Additionally, samples for Fe uptake were taken for the deckboard experiments. Deckboard dilution experiments (Lessard) were run for 24 hours with water collected once or twice each day; eleven experiments were performed at the Drift A time series. Samples for size-fractionated chlorophyll, picoplankton, nanoplankton and microplankton, macronutrients, dissolved and particulate DA were taken in each experiment. Experimental manipulations included the addition of DA, Fe, and macronutrients. The first drift (Drifter A, 3818 and 3900) was begun on September 17 on the eastern edge of the eddy in a region with both high PN densities and significant DA (Fig. 5a). The drifter was followed for 9 days and the drift was only aborted because the cruise was ending. The drifter was replaced with an expendable drifter (#3900) on September 26 after the last CTD cast at Drifter A. This drift was our most successful to date, allowing us to follow a large dense patch of toxic PN through growth and senescence. Cell numbers and DA levels were potentially the highest ever measured in this region. As the bloom progressed, another species, believed to be of the genus Chattonella, dominated the assemblage. Between the first and last sampling the drifter circumnavigated most of the eddy. A time series of all parameters was taken with sufficient resolution (generally3 hr) to resolve changes in properties due to internal tidal fluctuations. Tidal fluctuations were extremely irregular in period and amplitude and affected all water properties, even at the sea surface. Two longer periods of 3 hourly sampling were accomplished—one for 36 hours, the second for 24 hr (the latter included iron measurement and characterization of phytoplankton assemblage with depth). As the drift continued, sampling intervals were reduced to 12 and even 24 hr periods. However, even then, 3-4 three hour samples were taken when we returned to the drifter in order to separate long term trends from tidal changes. Sampling successfully resolved both long term trends and tidal scale relationships between physical forcing and biological responses. The second drift (DB, 9123, 3918 and 3901) was begun September 17 at the location of a drifter (#9123) that had been deployed near LAB 6 on September 10 as part of our initial cruise deployment of three expendable drifters (Fig. 5b). The drifter had been deployed on what was expected to be the southwest portion of the eddy. Based on the previous year we expected that drifter to move southwest and then southward as part of the coastal upwelling shelf break jet. In contrast to September 2003, the eddy was much more isolated from the coastal front in 2004 and this drifter never left the eddy. As with the first drift, the water followed by DB after sampling began was in a patch of high PN and high particulate DA. However, nutrient levels in surface layers were initially higher, and were higher at depth, consistent with the fact that this drift location was over the eddy where isopycnals dome. The drifter was sampled on July 17, beginning drift DB. On September 18 this drifter was replaced with drifter #3918 and CTDs were performed at this drifter at approximately 24 hr intervals until the end of the cruise. On September 26 this drifter was replaced with the expendable drifter #3901. Overall, the patch of water flowed by these drifters remained in the eddy for at least 20 days, performing an entire circuit of the eddy. The third drift (DC, 3884) was actually begun earlier than the other two drifts. However we had not decided to make it a “drift” until high DA levels were found in the CTD taken at the deployment site (Fig. 5c). Hence it is labeled as the third drift, rather than the first. The drifter was deployed at station CF05, in the presence of high densities of PN as well as high particulate DA. This drifter was followed intermittently throughout the cruise. Satellite imagery indicate that this drifter was located in a filament that extended southward from the eddy along the mid Washington coast. This is the type of filament (high in DA) hypothesized to move onshore during fall storms to impact the coastal clams. 3. Drifter Deployments (Amy MacFadyen, Barbara Hickey, James Postel, Brent Leithauser) Several Davis-type Brightwater drifter 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. Data were stored at the University of Washington and also transmitted to the ship by Sue Geier, Ryan McCabe, Nancy Kachel and Neil Banas. Drifter location and water temperatures are available at 30 minute intervals during deployment. Six drifters will continue to collect data until about the end of October. Two drifters were deployed at the beginning of the cruise: one (#9124) just inside the strait; a second (#22248) near the expected center of the eddy; a third (#9123) near the southwest edge of the eddy (Fig. 6). All three drifters were deployed prior to any real time information about the eddy location. Only the drifter deployed inside the strait (#9124) escaped the eddy during the cruise period: this drifter apparently entered the Vancouver Island Coastal Current. Although it moved southward around Barkley Sound, it continued northwestward along the Vancouver Island coast during the cruise. The drifter deployed near the center of the eddy (#22248) remained in the eddy for the entire cruise, making a complete circuit of the eddy over that time. The drifter deployed near the southwest edge of the eddy (#9123) as well as its replacements (# 3918 and # 3901) also remained in the eddy for the 21 days of the cruise. This drifter remained within the eddy for some time after the cruise as well. Near the end of the cruise when upwelling became more persistent some of the drifters did escape the eddy, heading south along the Washington coast (Fig. 6). 4. Satellite Imagery (Dana Woodruff, Rick Stumpf) Satellite imagery during the cruise was provided by two groups who sent data to the WHOI FTP site—Dana Woodruff from Battelle Northwest Laboratory provided SST imagery and surface chlorophyll imagery was provided by Rick Stumpf at NOAA. Susan Geier and Nancy Kachel (Hickey group) assessed data quality for the shipboard group prior to putting on the ship’s FTP site. The available imagery and an assessment of its quality are listed in Table 4. In general, because of the predominantly downwelling wind conditions, few good images were available during the sampling of the large scale grid. We were able to have three SST images combined for that period, with reasonable results. Some good images were obtained midway through the drift DA (September 19-20). Those chlorophyll and temperature images illustrated the filament moving down the coast in which the DC drifter was imbedded. a) Lessard Group (Evelyn Lessard, Brady Olson, Mike Foy, Megan Bernhardt) The main goal of this component of ECOHAB PNW is to determine the role of grazers in PN population dynamics and DA production. We used the dilution technique to experimentally alter grazing rate and nutrient recycling to determine the effects of grazers on the net growth rate of the whole and size fractionated phytoplankton community, specific species and groups of phytoplankton, and the production of dissolved and particulate DA. These experiments also provide estimates of the in situ growth rates of PN and other phytoplankton. 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 (Vera Trainer) The goal of this aspect of ECOHAB PNW was to validate the specificity and to continue field testing of PN sandwich hybridization assays (SHA) that will eventually be used to identify and enumerate HAB species in near real-time from environmental samples. In the SHA, 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. During this cruise, at selected survey stations where numerous PN were seen and at all drifter stations (in general, at 0, 5, 10 m depth), 400 ml of seawater were filtered onto a 0.65 µm, 25 mm Durapore membrane filters (Millipore). These filters were placed into plastic test tubes and frozen at –80 oC until analyzed. SHA was carried out using pre-dispensed reagents in 96-well microtiter plates. Cell lysate was prepared by adding filtered cells to Sample Solution Premix and incubating the cells within a 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. Two capture probes will be tested on samples collected during this cruise with two primary PN species as targets. The auD1 probe targets P. australis and muD2 probe targets P. pseudodelicatissima and P. multiseries. Because P. multiseries has rarely been observed in Washington coastal samples, we hope that the muD2 probe will primarily target P. pseudodelicatissima. Cell numbers of either P. australis or P. pseudodelicatissima will be determined by comparing sample absorbance values with known PN cell numbers generated from cultured cells. Standard curves have been generated from serial dilutions of cultured cells from cruise samples collected in the same region in 2003.
Corresponding whole cell assays were performed using complementary
auD1 and muD2 probes (see Section e below). c) RTC/SFSU Research Group (Bill Cochlan, Julian Herndon, Nick Ladizinsky) The primary objective of this component 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 three depths (0, 5, 10 m) and, after the survey grid, at 15 m as well. 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 dissolved inorganic nutrients were also determined at the drifter stations, during deep canyon profiles, and at a series of vertical stations in Juan de Fuca Strait on the return transit. Samples from the Juan de Fuca transit were also analyzed for ammonium and urea, in addition to the standard inorganic nutrients. 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. 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) were determined for all incubator experiments (described below) and drifter stations. A series of eleven shipboard incubation experiments (termed ‘grow-outs’) were designed to assess the role of trace metal (Cu and Fe) availability on the growth of PN and domoic acid production. Bacterial abundance estimates, to be determined at the University of Western Ontario using flow cytometry [Becton Dickinson, FACSCalibur] on preserved samples, will be used to calculate specific bacterial productivity. Photosynthetic-irradiance (P-E) curves were generated from short-term (1h) 14C uptake experiments using a photosynthetron during the grow-out experiments; these results will be used to describe the efficiency and capacity of phytoplankton photosynthesis with respect to light intensity. P-E curves were generated for most shipboard incubation experiments at the middle and end of the 3 or 4-day grow-out incubations. Potential new production rates were determined using the 15N-tracer technique using saturating and tracer concentrations of nitrate, ammonium and urea (10 and 0.1 µm respectively) to estimate maximal nitrate, ammonium and urea uptake potential as an indicator of phytoplankton community physiological “health”. Size-fractionated phytoplankton biomass estimates (as previously described) were determined for all metal and chelator treatments on all days of the incubation experiments. Expected Results:
d) Trick Research Group (Charlie Trick, Liza McClintock, Benjamin Beall) Our contribution to the ECOHAB project is two-fold: 1) to provide flow cytometric analysis (FCM) and HPLC pigment analysis to characterize the community assemblage; and 2) to provide experimental evidence of factors that either increase the competitive ability of PN or increase the level of domoic acid per cell. Samples for FCM and HPLC were collected at the 5 m depth at all stations on the grid survey as well as the second LB line survey. This will allow for quantitative analysis of bacteria, cyanobacteria, and nanoplankton communities, complemented by pigment analysis to characterize the phytoplankton assemblage which 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. 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 ~ 1-2 months since they preserve poorly. Maps of reconstructed photosynthetic communities will be available soon thereafter. In our second major contribution to the cruise mandate, the personnel from the Cochlan, Wells and Trick labs carried out deckboard incubation growth experiments. For Liza McClintock’s Masters thesis, six of these experiments were designed and conducted to observe the response of the aging PN bloom and associated community (following Drifter A) to increased iron and copper stresses. All labs offered their expertise to the common goal of all growth experiments (biomass formation, nutrient drawdown measurements, DA analysis (particulate and dissolved), community structure changes, bacterial and phytoplankton productivity, photosynthetic efficiency and iron [59Fe] uptake 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 fully utilize the macro-nutrients and grow effectively. An additional component to our research on the ECOHAB 3 cruise was to gain understanding of the sedimentation characteristics of the phytoplankton community within the Juan de Fuca eddy region. Samples were collected at 5 m depth during the survey and during the drifter DA experiment. For Ben Beall’s Masters research, the sinking rate and the potential for aggregate formation were both examined in order to understand the magnitude and causes of phytoplankton sedimentation. e) Trainer Group (Vera Trainer, Nicolaus Adams, Keri Baugh, Jeannie Bush, Sheryl Day, Brian Bill) At each survey and drift station, samples were routinely taken at 0, 5, 10 m for measurement of particulate and dissolved levels of DA, 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, eddy, and coast stations, depth profiles of cells and toxins were done at some of the following depths: 0, 5, 10, 20, 30, 50 m. Particulate domoic acid was analyzed by filtering 1 L seawater through a Nucleopore HA filter (0.45 micron pore size). Filters were minced in 5 ml distilled water with a thin metal spatula 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 DA in a sample from a cloned glutamate receptor. Each plate of samples is compared to known DA standards analyzed on the same plate. Endogenous glutamate was digested prior to sample analysis using glutamate dehydrogenase. Whole cell hybridization assay Up to 60 ml sample was filtered and fixed with saline-ethanol for 2 h. Then a specific P. australis probe (auD1, Texas Red labeled) and muD2 probe (fluorescein labeled) 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. Slides were kept in the dark for cell counting in our land-based laboratory. Dissolved domoic acid These samples were filtered through a 0.45 mm syringe filter and refrigerated until analysis. Selected samples from survey “hot spots” were analyzed at 0, 5, 10 m and complete sets of dissolved DA were analyzed at drifter stations. A commercially available enzyme-linked immunosorbent assay with picomolar sensitivity was used for these analyses (Biosense Corporation). 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.
Additionally, up to 100 monoclonal isolates from the
eddy and nearshore regions will be used to assess the genetic
diversity
among certain
PN species using microsatellite DNA markers. This information
will be used to make a preliminary determination of the
relationship between PN populations in the eddy and nearshore regions
(Nicolaus Adams,
Master’s
thesis). f) Wells Group (Mark Wells, Lisa Pickell, Kathy Hardy, 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. Over eighty 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 three deep (= 100 m) profiles. 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. Iron concentrations were highest in the nearshore shallow shelf regions and decreased in the offshore direction. Values in Juan de Fuca strait and along the Vancouver Island Coastal Current were very high (> 5 nm). Values obtained by flow injection analysis after short term (12 h) acidification will be compared with independent determinations on acidified samples by high resolution Inductively Coupled Plasma Mass Spectrometry. 6. Moored Sensor Arrays: (Barbara Hickey, Rick Thomson, Susan Geier, Tom Juhasz, Jim Johnson) One of the many components of this project was to design and maintain three surface moorings to collect time series data of water properties and currents and in situ meteorological events in the Juan de Fuca eddy region. A summary of the deployment and recovery times and positions for each mooring is included in Table 5. Deployment Cruise The deployment cruise, 2004-07, on the Canadian Coast Guard Offshore Research and Survey Vessel John P. Tully was scheduled for April 26-May 9, 2004. The ECOHAB moorings were scheduled to be deployed on the second leg of the cruise. Bill Fredericks and Jim Johnson from the University of Washington and Nick Adams from the Northwest Fisheries Science Center drove the equipment to the Institute of Ocean Sciences, prepared it for deployment and loaded it on the CCGS John P. Tully. The Tully left the dock at Patricia Bay, Vancouver Island, B.C. on May 1, 2004 with Jim Johnson serving as Dr. Hickey’s representative on the cruise. Tom Juhasz from the Institute of Ocean Sciences was the Chief Scientist under the direction of Dr. Rick Thompson. The first two moorings to be deployed were EH3-2004 in the Eddy and EH2-2004 off the Washington Coast. They were successfully deployed on May 3, and 4. The last mooring to be deployed was EH1-2004 in the Straits of Juan de Fuca. The currents were so strong the surface buoy was partially pulled under. The buoy was brought back on deck and a 36 inch Scottsman float was secured inside the bridle to give it increased flotation. The mooring was successfully deployed May 8, 2004. The Tully returned to Patricia Bay May 9, 2004 and Mr. Johnson returned to Seattle. Moorings were equipped with an ARGOS Satellite transmitter so that their positions could be checked from shore. The moorings were also equipped with Coast Guard approved lights. In order to insure the EH1-2004 mooring in the Straits did not lose power to its light Anthony Odell checked its visibility several times over the course of the summer. The lights on the other two moorings are solar powered. Mooring Recovery Cruise Sue Geier and Anthony Odell served as representatives from Dr. Hickey's group on the mooring recovery cruise, 2004-29. Tom Juhasz from the Institute of Ocean Sciences was the Chief Scientist under the direction of Dr. Rick Thompson. Ms. Geier and Mr. Odell joined the second leg of the cruise on Sept. 16, 2004. The CCGS John P. Tully left the dock at the Institute of Ocean Sciences, Patricia Bay early the next morning. All 3 moorings were successfully recovered on Sept. 18 and 19. The CCGS John P. Tully returned to the dock at the Institute of Ocean Sciences on September 23, 2004 and Ms. Geier and Mr. Odell returned to Seattle. We would like to thank the captain and crew of the R/V Atlantis for their support and extra effort that made the September 2004 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 and Susan Geier in advance for their help in mooring recovery in September. 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 recovery on the Tully was made possible by Canadian support to Richard Thomson at the Institute of Ocean Sciences. Table
1 Event log Fig.
1 Cruise track with sampling stations.
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