We assess the application of the second-generation Environmental Sample Processor (ESP) for the detection of harmful algal bloom (HAB) species in field and laboratory settings using two molecular probe techniques: a sandwich hybridization assay (SHA) and fluorescent in situ hybridization (FISH). During spring 2006, the first time this new instrument was deployed, the ESP successfully automated application of DNA probe arrays for various HAB species and other planktonic taxa, but non-specific background binding on the SHA probe array support made results interpretation problematic. Following 2006, the DNA array support membrane that we were using was replaced with a different membrane, and the SHA chemistry was adjusted. The sensitivity and dynamic range of these modifications were assessed using 96-well plate and ESP array SHA formats for several HAB species found commonly in Monterey Bay over a range of concentrations; responses were significantly correlated (p < 0.01). Modified arrays were deployed in 2007. Compared to 2006, probe arrays showed improved signal:noise, and remote detection of various HAB species was demonstrated. We confirmed that the ESP and affiliated assays can detect HAB populations at levels below those posing human health concerns, and results can be related to prevailing environmental conditions in near real-time. *Corresponding author: E-mail: dgreenfield@belle.baruch.sc.edu We thank the engineering technicians and machinists at MBARI for their invaluable help and dedication toward instrument development. We also greatly appreciate the hard work and dedication from the crew of the R/V Zephyr and Moss Landing Marine Laboratory, Small Boat Operations, and help from Drs. W. Jones and C. Preston. The authors gratefully acknowledge Drs. Z. Wang and T. Roth for the extraction and analysis of domoic acid in discrete water samples. This project was funded in part by the Monterey Bay Aquarium Research Institute from funds allocated by the David and Lucille Packard Foundation, the National Science Foundation, and the National Aeronautics and Space Administration (OCE-0314222, CCF424599, and NNG06GB34G to CAS, and OCE-0314089 to GJD). Disclaimers: Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. This publication does not constitute an endorsement of any commercial product or intend to be an opinion beyond scientific or other results obtained by the National Oceanic and Atmospheric Administration (NOAA). No reference shall be made to NOAA, or this publication furnished by NOAA, to any advertising or sales promotion which would indicate or imply that NOAA recommends or endorses any proprietary product mentioned herein, or which has as its purpose an interest to cause the advertised product to be used or purchased because of this publication. Limnol. Oceanogr.: Methods 6, 2008, 667-679 © 2008, by the American Society of Limnology and Oceanography, Inc. LIMNOLOGY and 1998; Olson and Sosik 2007), the optical plankton discriminator (Kirkpatrick et al. 2000; Robbins et al. 2006) , the Autonomous Microbial Genosensor , and the Laser In Situ Scattering and Transmissometer (Rienecker et al. 2008) . These and related instruments can be used independently or integrated with larger scale observing systems (e.g., Ryan et al. 2005; Paul et al. 2007; Babin et al. 2008) . In that light, we consider here how the Environmental Sample Processor (ESP) can be applied toward detection of harmful algae in situ (Scholin et al. , 2008 . The ESP is a device that allows users to collect discrete water samples from the ocean subsurface, concentrate particulates, and apply molecular probe-based assays remotely (Roman et al. 2007) . Sample manipulations are carried out in reaction chambers called pucks, which are loaded into and removed from various stations using robotic mechanisms. The ESP currently uses DNA probes in a sandwich hybridization assay (SHA) format and antibody probes in a competitive ELISA format. The ESP also has the capability to archive samples for various laboratory analyses, including fluorescent in situ hybridization (FISH; e.g., Goffredi et al. 2006; Greenfield et al. 2006; Jones et al. 2008) . The "first-generation" (1G) ESP underwent field trials over a several year period from [2001][2002][2003][2004][2005] in Monterey Bay and the Gulf of Maine. Those experiments focused on validating the basic instrument design and molecular assays (Goffredi et al. 2006; Scholin et al. 2008) . Results from those trials guided design iterations for the "second-generation" (2G) ESP, which underwent field deployments during spring 2006. In concert with engineering refinement, one of our goals with the 2G ESP was to achieve a more quantitative framework. To that end, we conducted laboratory studies using the diatom Pseudo-nitzschia australis, applying both the SHA and FISH techniques to assess the performance of the instrument (Greenfield et al. 2006) . The work presented here builds on those laboratory tests, focusing on the first field deployments of the new instrument. Our specific objectives were 3-fold: first, to document a transition from the previous DNA probe array membrane support (Predator, Pall Corp.) to reinforced nitrocellulose (Optitran; Schleicher & Schuell); second, to evaluate this transition by comparing the sensitivity and dynamic range of 96-well plate and ESP array SHA formats; and, third, to evaluate the performance of the ESP and affiliated assays for detection of HABs in a field setting during spring of 2006 and 2007. We demonstrated that the ESP can detect targeted HAB species in situ, and we show that trends in ESP results are corroborated qualitatively by trends in phytoplankton abundances elucidated by periodic water sampling. (clone CAWR04; Tyrrell et al. 2001 ) was grown at 18°C, all other conditions were as above. Pseudo-nitzschia spp. enumeration is described in Greenfield et al. (2006) . A. catenella was counted by sampling five replicate 1.8 mL aliquots of log-phase culture, staining aliquots with 1 drop of acid Lugol's iodine solution, and enumerating cells by light microscopy using a Sedgewick Rafter chamber. H. akashiwo was enumerated according to M. Adachi (unpubl. data) as follows. One hundred microliters of log-phase culture were added to triplicate 3.9 mL 0.2 μm filtered sea water (FSW) aliquots for a 1:40 dilution. To kill cells without rupturing their cell membranes, diluted culture was heated to 45°C in a water bath for ~3 min to stop cell motility then placed immediately on ice for ~2 min. Aliquots (n = 3, 1 mL each) from each of the three tubes were subsequently counted under a light microscope using a Sedgewick Rafter chamber for a total of 9 discrete counts. To collect samples for SHA analysis, logphase culture was concentrated on to 25 mm, 0.65 μm pore size hydrophilic Durapore (Millipore) filters using gentle vacuum (5 mm Hg). Membranes were rolled and placed in cryovials (Nalge Company), sample-side facing in, and capped vials were stored in liquid nitrogen until needed. Preparation and processing of DNA probe arrays for remote detection of HABs-A detailed description of SHA in the DNA probe array format, as well as methods for array printing and processing are described elsewhere (Greenfield et al. 2006 ). For the current study, capture probes used for 2006 deployments included Pseudo-nitzschia australis, (auD1; Table 1), Heterosigma akashiwo (Het1), P. multiseries/pseudodelicatissima (muD2), and Alexandrium tamarense/catenella (NA1). For work conducted during 2006, the control probe (AlexComp) spotted on the arrays was diluted 1:1000, 1:2000, 1:4000, and 1:8000 (Greenfield et al. 2006) . During 2007 laboratory studies and field deployments, an additional capture probe for P. multiseries (muD1) was included, and only a 1:1000 dilution of the AlexComp control was used. Normalization of conjugate activity-We discovered that activity of the antibody-HRP conjugate employed in the SHA (hereafter referred to as "conjugate") was not always consistent between batches, even when prepared and diluted using a consistent protocol (data not shown; after Greenfield et al. 2006) . The conjugate catalyzes colorimetric and chemiluminescent reactions that equate to abundance of target molecules. Because one of our goals is to achieve a more quantitative framework, it is essential to ensure that the reactivity of the working solution of this reagent is consistent among synthesis lots. For that reason, we developed a quality control procedure to adjust conjugate reactivity based on a known concentration of a synthetic target sequence referred to as a "linker." A linker is an oligonucleotide complimentary to both capture and signal probes (see also Goffredi et al. 2006; Greenfield et al. Field applications of the ESP: 2006 -2007 668 Greenfield et al. 2006) . To prepare the linker, a stock (1 ng μL -1 in 1xTE) of NA1S/ALXS alt L, henceforth NA1-L (Table 1) , was diluted to 25 pg mL -1 in 0.2 μm-filtered 3 M GuSCN lysis buffer within 1 h of use to give a 96-well plate SHA target A 450 of ~0.7 (Greenfield et al. 2006) . For the ESP array SHA format, a stock (1 ng μL -1 in 1xTE) of auD1/PSDS L, henceforth auD1-L (the complement of capture and signal probes for P. australis; Table 1 ), was prepared as above to give a target A 450 of ~0.3. When all other reaction conditions are as reported previously, overall reactivity of the SHA toward a linker is governed by the activity of the conjugate. To prepare the conjugate, lyophilized ImmunoPure Peroxidase Conjugated IgG Fraction Mouse Anti-Digoxin (Pierce Biotechnology), was resuspended to a theoretical 0.8 mg mL -1 with distilled water (Sigma) then diluted 1:500 with Guardian™ Peroxidase Conjugate Stabilizer/Diluent/Blocker (S/D/B). Aliquots of that 1:500 reagent batch were compared against a reference batch of the same reagent known to have the desired reactivity using the 96-well plate SHA. The new 1:500 batch of conjugate was then diluted accordingly with S/D/B to match the referenced standard (this generally equated to ~1:700-1:800 for the 96-well plate and ~1:1300-1:2000 for the ESP array SHA. The ESP array conjugate activity is used at a weaker concentration than the 96-well plate format because preliminary experiments have shown that array background (region where no probe spotting occurred) increases when conjugate levels are higher (data not shown). For the spring 2007 ESP deployment and laboratory tests, specific conjugate activities for SHA in the 96-well plate and ESP array formats were 1:750 and 1:1776, respectively. Transition to new ESP array membrane support and standard curves-Generation of standard curves using both the 96-well plate and ESP array SHA formats followed methods described previously (Greenfield et al. 2006) . However, the previous ESP array membrane support, Predator (Pall Corporation), was discontinued as a commercial product at the end of 2006 so a new array support was required. A transition to Optitran BA-S 83 Reinforced Nitrocellulose Membrane (Whatman, Schleicher & Schuell), hereafter referred to as 'Optitran', was implemented. The method for printing DNA probes on Optitran was identical to Predator. Application of Optitran-based arrays in the ESP, however, differed slightly from Predator in that the membrane was clamped tight in the puck, no backing filter was used, and the array was initially treated with Western Blocking Solution (Sigma) before addition of lysate (1 mL, 8 min) and then substituted for wash during array processing (Goffredi et al. 2006; Greenfield et al. 2006) . Conjugate activity was determined as above. To generate standard curves for the 96-well plate SHA format, frozen filters (n = 5) containing known cell numbers of target species were thawed to room temperature then immediately lysed and analyzed (Greenfield et al. 2006) . P. australis lysates were serially diluted in lysis buffer to yield ~8 × 10 To generate standard curves using the ESP, sample filters (n = 3) were homogenized, lysates were diluted as appropriate, then the lysate was split such that one aliquot was analyzed using the 96-well plate SHA and the other aliquot of that same lysate was analyzed using the ESP array. The muD2 and muD1 capture probes both react with P. multiseries (Miller and Scholin 1998; Scholin et al. 1999 ) so standard curves for both were performed simultaneously using the same cultured samples. For all standard curves, results of the 96-well plate SHA were used to estimate the concentration of target species supplied to the ESP array. Array spot intensity was determined by subtracting the average of four equal-sized background regions surrounding individual capture and control probe spots, then mean (± SE) spot intensity per probe was reported for images at a 60 s exposure at 1 × 1 CCD camera binning. Field sampling scheme-During spring 2006 and 2007, the ESP was deployed for ~2-3 weeks periods in Monterey Bay, California, next to the CIMT mooring M0 (36.83N, 121.90W, depth ~70 m) such that the ESP sampling inlet remained between 5 and 7 m below the surface. In 2006, Deployment 1 was 16-27 March and Deployment 2 was 10-23 April while our 2007 deployment occurred 17 May-11 June. For all deployments, the ESP remotely automated sample collection and processing of DNA arrays, one DNA array per day, for either HABs, invertebrate larvae, or bacteria. Competitive ELISA (cELISA) arrays for domoic acid (DA) were processed on the same days as HAB arrays. The development and application of the cELISA arrays will be described separately (Doucette et al. in prep. Concurrent environmental data were obtained from two sources: one conductivity, temperature, depth profiler (CTD; Seabird Electronics Inc.) affixed to the ESP and data from the M0 mooring. ESP CTD data were used during Deployment 1 whereas M0 data are presented for 27 March-23 April. Parameters used for this study included surface water temperature (°C) and salinity (‰). In situ ESP sampling and sample archival-The process by which the ESP collects and processes samples in situ is detailed elsewhere (Goffredi et al. 2006; Roman et al. 2007; Jones et al. 2008 ). For our study, whole water samples (400 to 1000 mL for HAB arrays) were processed and imaged according to Greenfield et al. (2006) with the following modifications: the conjugate was diluted 1:5000 using S/D/B during 2006 but during 2007, conjugate was normalized to a reference activity as above and wash buffer was replaced by Western Blocking Solution. Shortly after collecting a water sample for the HAB array, the ESP collected and preserved an additional 50 mL aliquot. Upon retrieval of the ESP, archived samples were quartered and processed for FISH following Greenfield et al. (2006) . Archived samples may be stored for up to ~1 mo without loss of signal in a FISH assay (Miller and Scholin 2000) . Fluorescein-labeled oligonucleotides used for this study are listed in Table 1 . Ground truthing ESP generated data-Results of HAB array and FISH analyses from ESP-archived samples were compared against laboratory versions of the same types of analyses using water samples collected manually near the ESP and, when possible, coincident with instrument sampling. To collect samples manually, we affixed a davit with a hand-operated winch on a Boston whaler; this enabled deployment of a profiling CTD and two 5 L Niskin bottles at approximately 5 m and 7 m depth. Water samples were returned to the laboratory within 1 h then combined into one depth-integrated 10 L total volume. Since our deployments involved three types of DNA probe arrays (bacteria, invertebrate larvae, HABs), one DNA array per day, ground truth sampling dates did not always coincide with dates when the ESP processed HAB arrays. Manual sample processing proceeded as follows. Upon return to the laboratory, water (up to 1000 mL, sample volume matched ESP sample volume when possible) was concentrated on a 0.65 μm pore size hydrophilic Durapore filter in replicates of 4 or more. Triplicate filters were immediately processed using the 96-well plate SHA (Greenfield et al. 2006 ; Table 1 ). Replicate filtered samples were stored in liquid nitrogen until needed. Samples were collected for FISH in 10 mL aliquots (n = 9-12), then filtered and processed using a 13 mm filtration manifold as per Scholin (1998, 2000) . For DA, two samples of 400 mL each were collected on a 0.65 μm pore size hydrophilic Durapore membranes and stored in liquid nitrogen until extracted and analyzed. DA was ) were mixed 9:1 (v/v) with a rabbit polyclonal anti-DA antibody diluted in HBS-EP buffer (1:300, v/v; GE HealthcareBiacore). Each sample/antibody mixture was injected (150 μL at 25 μL min -1 ) onto a CM5 sensor chip (GE Healthcare-Biacore) containing immobilized DA. Antibody not bound by DA in solution was bound to the chip surface causing a change in refractive index. Quantification was based on a calibration curve prepared using DA certified reference material (Inst. for Marine Biosciences) and ranging from 1 × 10 -3 to 1 × 10 4 ng DA mL -1 . DA concentrations in each sample were calculated using Biacore Q and GraphPad Prism (v. 4) software. Remote detection of HAB species using the ESP: 2006-Based on laboratory trials that used the Predator array support prior to the 2006 field season, P. australis was known to exhibit a predictable relationship between cell concentration and probe array response between 2 × 10 3 to 6.4 × 10 4 cells mL -1 lysate (Greenfield et al. 2006) . It was also known that the HAB arrays could be used to detect P. multiseries/pseudodelicatissima, A. catenella, and H. akashiwo (e.g., Scholin et al. 2008), but the relationship between probe array reactivity and organism abundance was not well characterized prior to the start of field operations. The raw score for interpreting species presence and relative abundance based on the HAB arrays was taken as signal for a given capture probe minus background, as measured by CCD counts. Signals from capture probes that were not significantly higher than background (ANOVA, single factor, α = 0.05) or that were less than background were recorded as nondetect. Of all the DNA probes on the HAB array, only the P. australis assay was considered potentially quantitative given the established standard curve (Greenfield et al. 2006) . During 2006 field trials, surface water temperature exhibited a general warming trend 16 March to 23 April, but ranged between 11°C and 14.6°C (Fig. 1a) . Salinity ranged between 22.90‰ and 33.15‰, but was typically ~33‰ with the exception of a rain event during the first week of April (Fig.  1b) . Results of HAB arrays using the Predator array support membrane during Deployment 1 showed that signal intensi- ties for all four capture probes were below background levels for both HAB array dates (Fig. 2a) . During Deployment 2, HAB arrays exhibited a weak positive signal for Het1 (H. akashiwo) on 10 April, but negative signals for all other capture probes. On 18 April and 21 April, positive signals were detected for Het1, auD1 (P. australis), and muD2 (P. multiseries/pseudodelicatissima). The NA1 probe (A. catenella) did not return a positive signal on any sampling date. Trends in detection of HABs using the 96-well plate SHA typically followed trends recorded by the ESP (Figs. 2a, b) . These results were based upon a conservative lower limit of detection (LLD) of 0.094 at A 450 for lysis buffer only (study range of 0.052-0.094). For example, the 96-well plate SHA produced negative results for the beginning of Deployment 1, coincident with negative HAB arrays. During Deployment 2, a slight positive reaction for Het1 (0.155 OD ± 0.011) was observed on 10 April, and on 18 April, positive signals were observed for auD1, muD2, and Het1, similar to the array format. Estimates of P. australis levels can be drawn from the 96-well plate SHA values using previously reported standard curves (Greenfield et al. 2006) . Abundances ranged from below the detection limit (10, 13 April) to a maximum of ~1.3 × 10 4 cells L -1 on 21 April corresponding to a 96-well plate SHA value of 0.167 OD ± 0.032 SE. Trends in cell abundances using FISH for samples archived using both the ESP, and field samples collected manually and processed using the filtration manifold approaches were similar during both deployments (Fig. 2c, d ). While overall estimates varied between the two FISH approaches by up to a factor of 2, this discrepancy could be explained by estimates of cell density being below the established, reliable lower limit of detection on all sampling dates (Miller and Scholin 1998; Greenfield et al. 2006) . Some discrepancies, however, between ESP-generated results and data obtained from routine water sampling did occur. For example, a positive signal was detected for the probe NA1 on 18 and 21 April using the 96-well plate, but HAB array results were negative. Such observations were not entirely unexpected because each entails a fundamentally different mode of sample acquisition. The ESP draws in water through a single intake at a fixed depth over a period of up tõ 1 h, and sample intake depth may vary due to tides and current. Moreover, samples archived for FISH using the ESP are collected ~1 h after preparing the previous sample for a corresponding array. In contrast, manual sample collections were generally done while the ESP was collecting a sample for SHA. The 5 L Niskin bottles were tripped roughly 5 m and 7 m from the surface, and those water samples were combined upon return to the laboratory. The 10 L sample thus represents an integrated water volume from a discrete time frame at two depths. As a consequence, samples collected by the ESP and those collected manually are never directly "matched;" differences in the phytoplankton community structure between the two sampling modes are therefore possible. This problem was understood, but the only way to obtain truly matched samples was for divers to collect an equivalent volume of water from near the ESP intake over the same time period the instrument was sampling. This was not possible during this study so we had to rely on shipboard collections; thus, potential discrepancies between ESP and manually collected samples were unavoidable. Nevertheless, general trends observed with the instrument can be corroborated qualitatively by returning discrete samples to the laboratory. Domoic acid (DA) was detected from manual collections on all HAB sampling dates of Deployment 2, but was lowest (4.7 ng mL -1 ) on 13 April, coincident with negative results on the 96-well plate SHA for Pseudo-nitzchia spp. (Fig. 2b,e) . DA was highest toward the end of the deployment, and this coincided with HAB array detection of Pseudo-nitzschia spp. (Fig. 2a,e) . Assay performance following transition to new array membrane support and assay chemistry-Pall Corporation discontinued production of the Predator membrane in 2006, so further field and laboratory studies using that support were terminated. To assess the applicability of the replacement membrane, Optitran BA-S 83, standard curves were generated for both the 96-well plate and ESP array SHA formats using cultured representatives of target HAB species (Fig. 3) . Cell concentrations employed reflected ranges that are either at or below levels typically considered threatening given a water sample volume of 400 mL and lysate volume of 2 mL. For example, DA production by P. australis and P. multiseries generally does not pose healthrelated or ecological threats at abundances < 2.5 × 10 4 cells L -1 : 2006-2007 673 Fig. 3 . SHA standard curves using both the 96-well benchtop assay (n = 5 replicate wells ± SE) and HAB array spot intensity (n = 8 array spots ± SE) for A. catenella probe NA-1 (A), P. australis probe auD1 (B), P. multiseries/pseudodelicatissima probe muD2 (C), P. multiseries probe muD1 (D), and H. akashiwo probe Het1 (E). Note difference in dynamic range (y-axes) among targets. (Tyrrell et al. 2002) . The relationships between cell concentrations using both SHA formats were statistically significant and highly correlated (ANOVA, single factor, α = 0.05; Fig. 3 ). Both assay formats yielded a linear response to an increasing number of target cells as enumerated using microscopy, so direct comparison between the 96-well plate and ESP SHA array formats can be made for matched samples. While the sensitivity and dynamic range differed considerably among DNA probes, the majority of probes enabled detection of cell densities that, within a laboratory setting, are well within ranges of those relevant for human health and wildlife management concerns. Surprisingly, P. multiseries probe muD1 exhibited a lower detection level in the HAB array than the 96-well plate SHA (Fig. 3d) . Historically, muD1 is considered a weakly reacting probe in both SHA and FISH formats relative to muD2, which targets both P. multiseries and P. pseudodelicatissima (Scholin et al. 1999; Miller and Scholin 2000) . In our study, the difference between muD1 and muD2 was particularly evident because all experiments used the same P. multiseries lysate and both capture probes. For example, P. multiseries (5 × 10 6 cells well -1 ) muD2 was above the linear portion of the curve, here operationally defined as OD ~0.1 to 3.0, but that same lysate was within the linear range of muD1 (Figs. 3c,d ). At ≤ 6.25 × 10 3 cells well -1 , P. multiseries was not detected by muD1 using the 96-well plate, but was detectable in the HAB array. In contrast, such low concentrations were detected in both SHA formats by muD2. These observations suggest that the best use for muD1 may be to determine if muD2 is targeting P. multiseries or P. pseudodelicatissima in the HAB array when the target species occur in low numbers. MuD1 is likely only effective quantitatively in the 96-well plate SHA when P. multiseries rRNA content in cellular homogenates exceeds the linear portion of the curve for muD2, such as during blooms. Array-to-array variability and reproducibility of the SHA was assessed using A. catenella as a representative species. Target lysate concentrations represented high, medium, and low levels as 500, 125, and 31 cells well -1 , respectively (Fig. 4) . Some array-to-array variability was found, and the 96-well plate format appeared slightly more sensitive to small changes in A. catenella densities than did the HAB array. Nonetheless, a strong correlation was found between HAB array spot intensity and cellular lysate concentration (R 2 = 0.9686; Fig. 4 ), and this correlation was highly significant (ANOVA, single-factor, α = 0.05, P < 0.001). Some probe cross-reactivity was observed in the DNA array SHA format when samples had high concentrations of Pseudonitzschia spp. (Table 2 ). By comparison, no HAB array crossreactivity was observed when arrays were presented with samples containing high concentrations of A. catenella or H. akashiwo. In the 96-well plate SHA format, no cross-reactivity was observed for any probe when non-target species were present at the highest concentration used on the corresponding standard curve. These results indicate that false-positive signals for some Pseudo-nitzschia spp. could be returned by the ESP during mixed Pseudo-nitzschia spp. blooms and suggests that further assay refinement may be necessary. This also reinforces the need characterize performance of the ESP empirically and establish how the detection method functions in a . Circles represent replicate (n = 3) arrays, and error bars represent SE of CCD counts per array. Table 2 . Matrix of cross-reactivity observed while generating standard curves using ESP arrays Capture probe on ESP array refers to probe that exhibited cross-reactivity and lysate target refers to capture probe corresponding to species homogenate. +++ = cross reactivity observed at top three cellular lysate concentrations, no cross reactivity observed at lower concentrations; ++ = cross reactivity only observed at highest two cellular lysate concentrations; + = cross reactivity observed at highest cellular lysate concentrations only; -= no cross reactivity for any cellular lysate concen tration; N/A = not applicable.
Greenfield et al. have developed a robotic platform to perform laboratory-type measurements of algae in the field REF .
Field applications of the second-generation Environmental Sample Processor (ESP) for remote detection of harmful algae: 2006-2007
Limnology and Oceanography-methods
Chemistry