ECOHAB-PNW: Cruise 2 Report

ECOHAB PNW 2 CRUISE REPORT
R/V Wecoma W0308C
Aug 30 - Sept 19, 2003

B. Hickey, V. Trainer, N. Adams, A. MacFadyen, S. Geier, W. Cochlan,
E. Lessard, M. Wells

Area of Operations
Itinerary
Participating Organizations
Chief Scientist
Personnel
Cruise Objectives
Operations
Samples Collected
Cruise Summary

Introduction

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, Fredericks)

5. Laboratory Analyses

a) Lessard Group (Lessard, Olson, Bernhardt)

b) Sandwich Hybridization Assay (Trainer, Connell)

c) RTC/SFSU Research Activities (Cochlan, Herndon, Ladizinsky)

d) Trick Research Group (Trick, McClintock)

e) Trainer Group (Trainer, Adams, Baugh, Bush, Ray)

f) Wells Group (Wells, Pickell)

6) Moored Sensor Arrays: (Hickey, Thomson)

Acknowledgements

List of Tables and Figures

ECOHAB PNW 2
CRUISE REPORT
R/V Wecoma W0308C
Aug 30 - Sep19, 2003
B. Hickey, V. Trainer, N. Adams, A. MacFadyen, S. Geier, W. Cochlan, E. Lessard, M. Wells

Area of Operations

Coastal Waters off Washington State and Vancouver Island

Itinerary

Participating Organizations

Chief Scientist

Dr. Barbara M. Hickey, School of Oceanography, University of Washington

Personnel

Nicolaus Adams, NOAA/Northwest Fisheries Science Center, Cruise Chief
Principle Investigators

Staff

Nicolaus Adams, NOAA/Northwest Fisheries Science Center
Susan Geier, University of Washington
Julian Herndon, San Francisco State University
Nicolas Ladizinsky, San Francisco State University
Keri Baugh, NOAA/Northwest Fisheries Science Center
Jason Ray, Saigene Co.

Students

Nicolaus Adams, University of Washington (Ph. D.)
Amy MacFadyen, University of Washington (Ph. D.)
Brady Olson, University of Washington (Ph. D.)
Liza McClintock, University of Western Ontario (Ms.)
Lisa Pickell, University of Maine (Ph. D.)
Sheri Floge, University of Maine (Ms.)
Jeannie Bush, University of Washington (B.S.)
Megan Bernhardt, University of Washington (B.S.)

Cruise Objectives

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 amnesiatic 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. An iron pump was used to measure 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 in the strait, near the Juan de Fuca eddy and off 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. The ship track and sampling stations are shown in Figure 1.

Operations

ADCP lines: ~4100 km
Flow-Through system track with T,S,FL sensors: ~4100 km
CTD casts: 228
Mooring light repair
Satellite-tracked buoy deployments: 7 (one recovered and redeployed)

Samples Collected

Chlorophyll samples: 167 stations
Nutrient samples: 121 stations
Microzooplankton samples: 15 profiles, 8 dilution experiments
Phytoplankton/Domoic acid samples: 236 stations
Fe samples (pumped): 4 profiles, >60 from 10 m depth
Zooplankton net tows: 21

Cruise Summary

Introduction

The ECOHAB 2 cruise was highly successful. Several of the new technologies that had problems on the first cruise were solved prior to or during the second cruise. For example, the FlowCAM, an imaging cytometer used to rapidly identify and count plankton > 5 µm, was operational after a factory retrofit (Lessard). The custom-made iron pumping system worked flawlessly so that uncontaminated water samples could be obtained throughout the cruise (Wells). Iron measurements were obtained at sea for the first time using was also a newly designed chemiluminescent assay instrument (Wells). The flow injection analysis system was used to analyze dissolved nutrient samples at sea and in near real time (Cochlan).
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).

The setting of cruise sampling events with respect to wind direction (upwelling or downwelling favorable) is shown in Figure 2. The sequence of weather conditions allowed 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 or downwellling conditions (the last week); overall similar to the sequence on the June ECOHAB PNW cruise. 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.

The cruise was broken September 10 when we came to Neah Bay to exchange staff in Trainer’s group via a small boat. A failed light was repaired on the EH1 buoy at the same time.

On this cruise samples were also taken at three sites in Puget Sound on the return to port. Toxic Pseudo-nitzschia (PN) blooms had been reported in these areas a few days prior to our sampling. Beaches had been closed to shellfish harvesting due to DA levels above the regulatory limit. These were the first DA related closures in Puget Sound The areas in which the PN bloom was found were near Admiralty Inlet.

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. An ISUS nitrate sensor was tested for the first time during this cruise and provided some underway data after problems with equipment calibration were resolved. Preliminary water property maps and sections are given in Appendix A (T, S, O2, Chl, Fl maps at selected depths) 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 the LP line on Survey 1 (only). Chlorophyll, particulate and dissolved domoic acid and plankton samples were taken near surface, 5 m, 10 m and at the chlorophyll maximum. 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 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 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 depth).

The data are organized into four periods: Survey 1 (September 1-6), Survey 2 (September 6-12), Survey 3 (September 12-17) and Survey 4 (September 17-20; strait and Puget Sound samples only) (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 warm, sunny weather, was the most complete survey. Drift DD started at the start of Survey period 2 and continued through the downwelling period. The water for drift DD was taken from the southwest edge of the eddy or in the shelf break coastal jet region (ie; we believe, “aged “water). The downwelling period (Survey 2, Fig. 4b) was shorter and also some time was spent following drift DD. Consequently none of the southern lines could be sampled. The biologists took water for on deck experiments at LA2 during this period. This station was in strait/eddy water. The weaker, less intermittent upwelling period (Survey 3, Fig. 4c) was sufficiently long to sample three southern lines; a new line farther south offshore of Grays Harbor (GH) was added to ensure that we were sampling ahead of a toxic “hot spot” that had been discovered (see below). The LP line was sampled twice; once, during the weak upwelling, and a second time after stronger upwelling to see whether the cap of freshwater from the Columbia plume preventing upwelling under weaker wind conditions, had finally moved offshore (it had; see below).

The usefulness of (a) near real time domoic acid analyses and (b) simultaneous physical data were clearly demonstrated on this cruise. A region of high DA was discovered after the Chief Scientist requested immediate analysis of DA samples for a station showing high PN. After this discovery the patch was relocated by estimating drift path from our satellite-tracked drifters . A drift station was begun (DE) and indeed the domoic acid levels were extremely high in this patch. The drift was terminated when it became clear that this patch would not likely return to the coast as the plume from the Columbia River was on its shoreward side.
CTD/nutrient transects were made also along the axis and across Juan de Fuca canyon during weak upwelling conditions (Fig, 4d). The same sections were made in June under weak downwelling conditions. Note that on both June and September ECOHAB cruises deeper nutrients were collected on these sections to provide input for modeling studies.
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 (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 and water property data require more extensive processing and will be provided later this year (Foreman group).

Some Preliminary Results (Hickey):

The first survey clearly captured the strong coastal upwelling that was occurring during that period (Appendix A, surface maps). The saltiest water was observed near the coast. Cold water at the surface appeared to emanate from the strait and also from the coastal upwelling region.

The surface chlorophyll during the first survey showed two regions of high values—one offshore of the strait and southeast of Barkley Sound, the other, off the sourthern Washington coast. As in the June ECOHAB cruise, between these two maxima was a region of lower chlorophyll that appeared to emanate from the strait. This low chlorophyll region was observed also in several of the chlorophyll satellite images. Low chlorophyll appears to emanate from the strait. The eddy center—defined as the region of maximum property “doming”, varied between depths during the first survey period.

As in the June cruise, during intermittent and weak upwelling (Survey 3) the Columbia River plume occupied the stations nearest the coast at least from La Push south, preventing upwelling on the inner shelf. This was confirmed by a series of inner shelf stations from Grays Harbor to La Push. The La Push line was repeated in Survey 3 to determine whether winds of 20 m s-1 were sufficient to move the plume offshore; they were.

2. Drift Surveys (Amy MacFadyen, Barbara Hickey, drifters; whole team for water samples)

Two drift surveys were performed, following water patches with a GPS-tracked Brightwaters drifter. Drogues were set at 5 m to best accommodate disparate sampling strategies. For example, Wells samples were taken a bit deeper (~ 8-10 m) due to requirements of the iron pumping fish; Lessard sampled at 3-5 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 with drifts on the first ECOHAB cruise. 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 generally at 12 hour intervals.

The first drift (DD, 3818) was begun on the western edge of the eddy in “aged” water at LA10. The drifter was followed for 2 days and aborted since the water mass had changed. Following the drift, the drifter was left in the water until the cruise end and then recovered. The second drift (DE, 3918) was begun offshore of the coastal upwelling region just south of KB5, halfway to the CB line, after relocating a patch with high PN. Although the first drift water had reasonably high levels of particulate DA, the second drift had extremely high levels of particulate DA. Interestingly, the final paths of the two drifts merged (Fig. 6) so that one might speculate that water from the first drift drifted to the site of the second drift.

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.

3. Drifter Deployments (Amy MacFadyen, Sue Geier, Barbara Hickey, Bill Fredericks)

Two Davis-type Clearwater GPS drifters and one 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 UW and also transmitted to the ship by Bill Fredericks. Deployment times and locations are listed in Table 3. Drifter location and water temperatures are available at 30 minute intervals during deployment. The three remaining drifters will continue to collect data until about the end of October.

Two drifters were deployed at the beginning of the cruise inside the strait. One (3901) was drogued at 20 m, the other had no drogue (3885). The pathways of the two drifters differed radically. The deeper drifter was caught in the nearshore Vancouver Island Coastal Current, staying next to the coast past Barkely Sound, where it was recovered. The surface drifter exited the strait farther from the coast, made one small circuit of the eddy, a second larger circuit and a partial third circuit. It then entered the strait where it became caught in kelp and was subsequently recovered.

Drifters deployed in the north portion of the eddy failed to join the eddy (Fig. 6). Particularly during downwelling (survey 2), the drifters showed little organized motion. However, three drifters did eventually join the coastal front (DD 3818, 3900 and 3943, which was deployed deliberately in the front); these have continued down the coast. 3943 moved somewhat onshore during downwelling later in the cruise, whereas its companion (3900) did not; likely it was impeded by the Columbia plume. The drifters remaining in the water after the cruise period moved onshore and northward in the first large fall storm as expected. These drifters did not turn south again; rather, they both beached on the Vancouver Is. coast where they were recovered through the efforts of the Canadian Coast Guard and Lighthouse keepers. This contrasts with the June period, when drifters remaining after the cruise headed down to Oregon and even California.

4. Satellite Imagery (Dana Woodruff, Rick Stumpf, Bill Fredericks)

Satellite imagery during the cruise was provided by two groups who sent data to the OSU ftp site—Dana Woodruff from Battelle Northwest Laboratory provided SST imagery and surface chlorophyll imagery was provided by Rick Stumpf at NOAA. Bill Fredericks (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. Images also confirmed that in the weaker, more intermittent upwelling period of Survey 3 upwelled water was not reaching the surface anywhere near the coast. Comparison of SST images at the beginning and end of the cruise showed that the eddy had expanded greatly following a brief downwelling event. The chlorophyll images, which had better spatial coverage, were also very useful, although less frequent on this cruise. 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 as relative fluorescence. We used some patterns to select in situ sampling sites.

5. Laboratory Analyses

a) Lessard Group (Evelyn Lessard, Brady Olson, Megan Bernhardt)

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 three main tools: the dilution experiment, copepod grazing experiments 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 copepod 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:

Eight dilution growth and grazing experiments and eight macrozooplankton grazing experiments. In these experiments, we followed changes in >5 mm and total chlorophyll, particulate DA, dissolved DA, PN species abundance using whole cell and sandwich hybridization assays, and macronutrients. We analyzed the chlorophylls onboard; Cochlan’s group analyzed the nutrients. A set of whole cell and sandwich hybridization assays were performed onboard (see below). Experimental manipulations included dissolved DA additions, combined macro/micro nutrient additions, and macronutrient only additions.
High frequency abundance estimates of PN and other plankton with the FlowCAM. After exhibiting serious problems last cruise, the FlowCAM was repaired by the manufacturer, and performed much better on this cruise. Discrete samples from the Fe pump at every other station along three transect lines were analyzed with the FlowCAM, as well as selected stations in the study grid throughout the surveys. The data files were stored and will be edited and calibrated in the lab to obtain quantitative counts. Replicate fixed samples were taken for microscopic enumerations and calibration of the FlowCAM. During surveys, the FlowCAM proved particular useful for a quick assessment of PN abundance and community composition at the surface and at depth.
Grazing on Pseudo-nitzschia australis measured with rRNA probes. Brady Olson ran both whole cell and sandwich hybridization (SHA) analyses shipboard using P. australis probes on a set of samples from a macrozooplankton grazing experiment. A robust signal was detected with the SHA, and the trend in the signal (expected to correlate with abundance) in treatments with and without copepods and with and without added nutrients indicated that copepods were actively grazing on P. australis and that P.australis was nutrient limited. The whole cell hybridization on replicate samples worked extremely well; P. australis lit up brightly with the probe, and a minimum of background and non-specific staining. Microscopically enumerated probed P.australis showed the same trends as the SHA, indicating that the SHA is quantitatively detecting the P. australis target. These are very exciting results.
Vertical profiles of micro- and nanoplankton and macrozooplankton. We took preserved plankton samples at a number of stations on the large scale survey, and at the beginning and end of drift stations for microscopic determination of autotrophic and heterotrophic nanoplankton, and heterotrophic/mixotrophic dinoflagellates and ciliates. Twenty one vertical net tows with a 200 µm mesh net were also done for enumerating macrozooplankton.

b) Sandwich Hybridization Assay (Vera Trainer, Laurie Connell)

The goal of this aspect of ECOHAB PNW was to initiate field testing of Pseudo-nitzschia 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 stations where numerous Pseudo-nitzschia were seen, 2 liters of seawater were filtered onto a 5 µm, 25 mm Durapore membrane filters (Millipore). These filters were placed into plastic test tubes and frozen at –80 oC until analyzed in the lab. SHA will be carried out using pre-dispensed reagents in 96-well microtiter plates. Cell lysate is prepared by adding filtered cells to Sample Solution Premix and incubating the cells within Lysis Tube (thin wall tube) at 80ºC for 5 minutes. Cell lysates are then loaded into the Universal Processor (Affirm Corp.) for processing. The optical density (OD) of the colorimetric product is 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. AU targets P. australis and WA001 targets P. pseudodelicatissima.
Cell numbers of either P. australis or P. pseudodelicatissima will be determined by comparing sample absorbance values with known Pseudo-nitzschia cell numbers generated from cultured cells. Standard curves will be generated from serial dilutions of cultured cells that will be isolated from cruise samples.

c) RTC/SFSU Research Activities (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 four (4) depths (0, 5, 10, 15 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 dissolved inorganic nutrients were also determined at the drifter stations (at 12-h intervals), during deep canyon profiles, and at a series of vertical stations (5) 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 access the role of trace metal (Cu and Fe) availability on the growth of PN and domoic acid production. During these experiments (conducted in collaboration with Wells and Trick’s research group), bacterial heterotrophic productivity (³H-leucine uptake method) was measured daily to evaluate the relationship between micro-nutrient availability and bacterial degradation (or possible stimulation) of domoic acid production by PN. Bacterial abundance estimates, to be determined at the UWO 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 saturated nitrate tracer concentrations (20 µM) to estimate maximal nitrate uptake potential as an indicator of phytoplankton community physiological “health”. During selected incubation experiments, the potential uptake rates of both new (nitrate) and regenerated nitrogen substrates (urea and ammonium) were determined to access nitrogenous uptake preference by the size-fractionated plankton communities (total and > 5 µm in size). 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:

Dissolved Nutrients: Over 90% of collected samples were analyzed onboard and draft (uncorrected) concentrations made available daily. This enabled working maps of nutrients to be developed that helped guide further sampling strategies. The remainder will be available by Nov 1, 2003 using automated and manual colorimetric methods.
Phytoplankton Biomass: All initial survey grid samples, drifter profiles and onboard deck experiments were analyzed onboard, and are currently available in draft form.
Bacterial Productivity: Radio-isotope samples (³H) will be analyzed using liquid scintillation counting at the RTC, rates should be estimated by Dec. 1.
Photosynthetic Efficiency: Radio-isotope samples (¹⁴C) were prepared on board for liquid scintillation counting ashore at RTC; P-E curves should be generated by Dec. 1.
New and Regenerated Production: Samples must be returned to RTC for mass spectrometric analysis, and may be available prior to Dec 1, depending on the scheduled availability of the RTC mass spectrometer.

d) Trick Research Group (Charlie Trick, Liza McClintock)

Our contribution to the ECOHAB project is two-fold: 1) to provide flow cytometric analysis (FCM) of the communities; 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 flow cytometric analysis were preserved by freezing, and will be returned to UWO for quantitative analysis of bacteria, cyanobacteria, picoplankton and nanoplankton communities. FCM samples were collected from all of the survey stations (for depths of 0, 5, 10 m and the depth of maximum chlorophyll layer), at several deep samples, and at the drifter stations (at 12-h intervals).

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. 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. As part of her Master’s research Liza McClintock collected samples for protein analysis from various areas of the transect survey, upwelling zones, downwelling zones, edges of the eddy and at selected drifter sites. These samples will be analyzed ashore for induced cell wall proteins unique to a hypothesized Fe/Cu reductase protein; this protein is thought to assist in the transport of domoic acid-mediated iron assimilation.

In our second major contribution to the cruise mandate, the personnel from the Cochlan, Wells and Trick labs carried out eleven incubator studies – termed “grow-out” experiments, two of these experiments were designed and conducted primarily by graduate students Liza McClintock (UWO) and Lisa Pickell (UM). All labs offered their expertise to the common goal (biomass formation, nutrient drawdown measurements, DA analysis (particulate and dissolved), community structure changes, bacterial and phytoplankton productivity, nitrogen 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.

e) Trainer Group (Vera Trainer, Nicolaus Adams, Keri Baugh, Jeannie Bush, Jason Ray)

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. 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 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. Slides were kept in the dark for cell counting in our land-based laboratory.

Dissolved domoic acid

These samples were refrigerated and will be analyzed in the land-based laboratory using an enzyme-linked immunosorbent assay in samples where high numbers of Pseudo-nitzschia are seen.

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, isolates from the eddy and nearshore regions will be used to assess the genetic diversity among certain PN species. This will be used to make a preliminary determination of the relatedness between PN populations in the eddy and nearshore regions.

f) Wells Group (Mark Wells, Lisa Pickell)

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 sixty 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 four 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. Problems encountered in determining the analytical blank during the first cruise were solved during this cruise and preliminary results were obtained for more than 40 surface and deepwater samples. While these determinations await verification against more standard analyses of retained samples our shore-based laboratory, the results show oceanographically consistent patterns in Fe distributions.

6. Moored Sensor Arrays: (Barbara Hickey, Rick Thomson)

Three arrays of moored sensors were deployed May 9-11 from the CCGS J. P. Tully and were successfully recovered September 30-October 4 from the CCGS J. P. Tully. Deployment and recovery 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).

Initial inspection of data suggest close to 100% data collection for in-water sensors. One ADCP only ran for a month when the battery leaked. This battery apparently was part of a bad batch of batteries that have also leaked in the shop. A light went out on the buoy in the strait, a result of water in the battery box due to a poor factory-supplied seal. Batteries were replaced and the light reactivated by Bill Fredericks (Hickey group) and Daryl Swenson (the ship marine technician) on the ECOHAB 2 cruise.

Acknowledgements

We would like to thank the captain and crew of the R/V Wecoma for their support and extra effort that made the September 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 in advance for their help in mooring recovery in October. 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 were made possible by Canadian support to Rick Thomson at IOS.

List of Tables and Figures

Table 1 Event log
Table 2 CTD stations organized by sample line and date, showing types of bottle samples taken as well as associated surface iron samples.
Table 3 Drifter deployment locations and times
Table 4 Dates and file name of available satellite imagery
Table 5 Mooring locations, bottom depths, deployment times and satellite PTT ID

Fig. 1 Cruise track with sampling stations.
Fig. 2 Time series of shipboard vector winds
Fig. 3 Theoretical survey grid and locations of moored arrays
Fig. 4 (a,b,c,d) CTD cast numbers for the Surveys 1-3 and the final survey lines
Fig. 5 (a,b) Drifter tracks during Drifts D-E with CTD cast numbers
Fig. 6 Trajectories of expendable drifters deployed on the cruise
Fig. 7 Mooring schematic