Environmental Sample Processor Research Articles

Using a long‐range autonomous underwater vehicle to find and sample harmful algal blooms in Lake Erie

AbstractCyanobacterial harmful algal blooms (CyanoHABs) in the Great Lakes pose risks to residential drinking water use, fisheries, and recreation. Active mitigation of these risks requires rapid detection of CyanoHABs and quantification of the toxins they produce. Here, we present a method of using a long‐range autonomous underwater vehicle (LRAUV) equipped with a 3rd‐generation Environmental Sample Processor (3G‐ESP) to search for and adaptively sample areas of high chlorophyll potentially representative of CyanoHAB biomass. In August 2021, this method was used in western Lake Erie. The experiment highlighted the effectiveness of the LRAUV autonomous search‐and‐sample methodology, and demonstrated how an interdisciplinary team located in different states virtually coordinated LRAUV operations and directed sampling activities via Internet connectivity using shared, web‐based situational awareness tools. The advancements made provide a foundation for future work to increase LRAUV autonomy and adaptiveness for CyanoHAB studies and monitoring in both freshwater and marine settings.

Autonomous eDNA collection using an uncrewed surface vessel over a 4200‐km transect of the eastern Pacific Ocean

AbstractThe collection of environmental DNA (eDNA) samples is often laborious, costly, and logistically difficult to accomplish at high frequency in remote locations and over large geographic areas. Here, we addressed those challenges by combining two robotic technologies: an uncrewed surface vessel (USV) fitted with an automated eDNA sample collection device to survey surface waters in the eastern North Pacific Ocean from Alameda, CA to Honolulu, HI. USV Surveyor SD 1200 (Saildrone) carrying the Environmental Sample Processor (ESP) collected 2‐L water samples by filtration followed by RNAlater preservation at regular intervals over a 4200‐km, 29‐day transit. Sixty samples (52 field and 8 controls) were acquired and used to estimate the concentration of specific genes and assess eukaryotic diversity via targeted qPCR and metabarcoding of the cytochrome oxidase subunit I (COI) gene, respectively. Comparisons of control samples revealed important considerations for interpreting results. Samples stored at ambient temperatures onboard Surveyor over the length of the voyage had less total recoverable DNA and specific target gene concentrations compared to the same material immediately flash‐frozen after collection and stored in a laboratory. In contrast, the biodiversity of the COI genes in those samples was similar regardless of sample age and storage condition. COI genes affiliated with 40 eukaryotic phyla were found in native samples collected during the voyage. The distribution and dominance of those phyla varied across different regions, with some taxa spanning large continuous stretches >2000 km, while others were only detected in a single sample. This work highlights the utility and potential of using USVs fitted with autonomous eDNA sample collection devices to improve ocean exploration and support large, basin‐scale, systematic biodiversity surveys. Results of this study also inform future technical considerations for using automated eDNA samplers to acquire material and store it over prolonged periods under prevailing environmental conditions.

Lake Erie field trials to advance autonomous monitoring of cyanobacterial harmful algal blooms

Biomolecular analyses are used to investigate the dynamics of cyanobacterial harmful algal blooms (cyanoHABs), with samples collected during monitoring often analyzed by qPCR and sometimes amplicon and metagenomic sequencing. However, cyanoHAB research and monitoring programs face operational constraints due to the reliance on human resources for sample collections. To address this impediment, a third-generation Environmental Sample Processor (3G ESP) integrated with a long-range autonomous underwater vehicle (LRAUV) was tested during seasonal blooms of Microcystis in western Lake Erie (WLE) in 2018 and 2019. The LRAUV-3G ESP successfully performed flexible, autonomous sampling across a wide range of cyanoHAB conditions, and results indicated equivalency between autonomous and manual methods. No significant differences were found between LRAUV-3G ESP and manual sample collection and handling methods in the 12 parameters tested. Analyzed parameters included concentrations of total cyanobacteria and microcystin toxin gene via qPCR; relative abundances of bacterial amplicon sequence variants (ASVs) from 16S rRNA gene amplicon sequencing; and community diversity measures from both 16S amplicon and metagenomic sequencing. The LRAUV-3G ESP provided additional sampling capacity and revealed differences between field seasons for bacterial taxa and concentrations of total cyanobacteria and microcystin toxin gene. Metagenomic analysis of multiple microcystin toxin genes corroborated the use of the mcyE gene as a proxy for the genomic potential of WLE cyanoHABs to produce microcystin. Overall, this study provides support for the use of autonomous ‘omics capability in WLE to help expand the spatial and temporal coverage of cyanoHAB monitoring operations.

Sea lice (Lepeophtherius salmonis) detection and quantification around aquaculture installations using environmental DNA.

The naturally occurring ectoparasite salmon lice (Lepeophtherirus salmonis) poses a great challenge for the salmon farming industry, as well as for wild salmonids in the Northern hemisphere. To better control the infestation pressure and protect the production, there is a need to provide fish farmers with sensitive and efficient tools for rapid early detection and monitoring of the parasitic load. This can be achieved by targeting L. salmonis DNA in environmental samples. Here, we developed and tested a new L. salmonis specific DNA-based assay (qPCR assay) for detection and quantification from seawater samples using an analytical pipeline compatible with the Environmental Sample Processor (ESP) for autonomous water sample analysis of gene targets. Specificity of the L. salmonis qPCR assay was demonstrated through in-silico DNA analyses covering sequences of different L. salmonis isolates. Seawater was spiked with known numbers of nauplii and copepodite free-swimming (planktonic) stages of L. salmonis to investigate the relationship with the number of marker gene copies (MGC). Finally, field samples collected at different times of the year in the vicinity of a salmon production farm in Western Norway were analyzed for L. salmonis detection and quantification. The assay specificity was high and a high correlation between MGC and planktonic stages of L. salmonis was established in the laboratory conditions. In the field, L. salmonis DNA was consequently detected, but with MGC number below that expected for one copepodite or nauplii. We concluded that only L. salmonis tissue or eDNA residues were detected. This novel study opens for a fully automatized L. salmonis DNA quantification using ESP robotic to monitor the parasitic load, but challenges remain to exactly transfer information about eDNA quantities to decisions by the farmers and possible interventions.

An Autonomous Platform for Near Real-Time Surveillance of Harmful Algae and Their Toxins in Dynamic Coastal Shelf Environments

Efforts to identify in situ the mechanisms underpinning the response of harmful algae to climate change demand frequent observations in dynamic and often difficult to access marine and freshwater environments. Increasingly, resource managers and researchers are looking to fill this data gap using unmanned systems. In this study we integrated the Environmental Sample Processor (ESP) into an autonomous platform to provide near real-time surveillance of harmful algae and the toxin domoic acid on the Washington State continental shelf over a three-year period (2016–2018). The ESP mooring design accommodated the necessary subsystems to sustain ESP operations, supporting deployment durations of up to 7.5 weeks. The combination of ESP observations and a suite of contextual measurements from the ESP mooring and a nearby surface buoy permitted an investigation into toxic Pseudo-nitzschia spp. bloom dynamics. Preliminary findings suggest a connection between bloom formation and nutrient availability that is modulated by wind-forced coastal-trapped waves. In addition, high concentrations of Pseudo-nitzschia spp. and elevated levels of domoic acid observed at the ESP mooring location were not necessarily associated with the advection of water from known bloom initiation sites. Such insights, made possible by this autonomous technology, enable the formulation of testable hypotheses on climate-driven changes in HAB dynamics that can be investigated during future deployments.

Autonomous in situ sampling and analysis of eDNA using an Environmental Sample Processor (ESP)

Monitoring of marine offshore biodiversity is expensive and has traditionally relied on invasive techniques like net fishing or direct observations that can only be conducted in calm seas by experts. Analysis of environmental DNA (eDNA) is a non-invasive method and can easily be collected from sea water by water filtration followed by DNA extraction. Due to the fast degradation time in sea water it is considered a good proxy for present living biodiversity. It allows direct identification of species based on their unique DNA sequence and is cheaper compared to traditional methods, which often are carried out from dedicated fishing or research vessels. However, while eDNA collection may reduce operational cost of offshore sampling, it still relies on boat time. Thus, traditional eDNA sampling still presents substantial costs for offshore biodiversity monitoring. This may reduce the number of samples that can be collected and analysed, limiting the sampling to single ‘time shots’, which may not give an adequate picture of the present biodiversity. The 2nd generation Environmental Sample Processor (2G-ESP) is an autonomous sampler/analyser of eDNA. It can collect, extract and analyse eDNA samples in situ using quantitative PCR (qPCR) or store filters for subsequent laboratory analysis after deployment. The instrument can be deployed directly on the seabed or in pelagic configuration where it can operate for several months depending on power supply and power consumption, while it is controlled by, and sends back results to scientists on land. These unique features make the 2G-ESP an interesting candidate for offshore monitoring of marine biodiversity, as well as a potential early warning/detection system e.g. for invasive species. Moreover, the possibility to preserve filters aboard makes it possible to investigate temporal changes of full biological communities by applying metabarcoding techniques on the collected samples. Here we present the major results of three years of work testing the potential use of a second generation Environmental Sample Processor (2G-ESP) for marine monitoring. These include both practical and analytical issues that we have encountered along the way, as well as results on target species detection and temporal analysis using qPCR and metabarcoding methods.

Robotic environmental DNA bio-surveillance of freshwater health

Autonomous water sampling technologies may help to overcome the human resource challenges of monitoring biological threats to rivers over long time periods and across large geographic areas. The Monterey Bay Aquarium Research Institute has pioneered a robotic Environmental Sample Processor (ESP) that overcomes some of the constraints associated with traditional sampling since it can automate water sample filtration and preservation of the captured material. The ESP was originally developed for marine environment applications. Here we evaluated whether the ESP can provide reliable, timely information on environmental (e)DNA detections of human and fish pathogens and introduced fishes at U.S. Geological Survey streamgage sites in freshwater rivers. We compared eDNA collected via ESP at high frequency (e.g., every 3 h) with manual eDNA collections collected at lower frequency (e.g., weekly). We found that water samples filtered and preserved by ESPs successfully detected the DNA of human pathogens, fish pathogens and introduced fishes. Both ESP and manually collected samples provided similar information about target DNA presence. We suggest that the greatest current benefit of the ESP is the cost savings of high frequency, bio-surveillance at remote or hard to access sites. The full potential of robotic technologies like the ESP will be realized when they can more easily execute in situ analyses of water samples and rapidly transmit results to decision-makers.

Remote, autonomous real-time monitoring of environmental DNA from commercial fish

Environmental DNA (eDNA) is increasingly used for monitoring marine organisms; however, offshore sampling and time lag from sampling to results remain problematic. In order to overcome these challenges a robotic sampler, a 2nd generation Environmental Sample Processor (ESP), was tested for autonomous analysis of eDNA from four commercial fish species in a 4.5 million liter mesocosm. The ESP enabled in situ analysis, consisting of water collection, filtration, DNA extraction and qPCR analysis, which allowed for real-time remote reporting and archival sample collection, consisting of water collection, filtration and chemical preservation followed by post-deployment laboratory analysis. The results demonstrate that the 2G ESP was able to consistently detect and quantify target molecules from the most abundant species (Atlantic mackerel) both in real-time and from the archived samples. In contrast, detection of low abundant species was challenged by both biological and technical aspects coupled to the ecology of eDNA and the 2G ESP instrumentation. Comparison of the in situ analysis and archival samples demonstrated variance, which potentially was linked to diel migration patterns of the Atlantic mackerel. The study demonstrates strong potential for remote autonomous in situ monitoring which open new possibilities for the field of eDNA and marine monitoring.

Persistent Sampling of Vertically Migrating Biological Layers by an Autonomous Underwater Vehicle Within the Beam of a Seabed-Mounted Echosounder

Many pelagic animals, such as krill, lanternfish, and cephalopods, migrate to deep water at dawn to avoid visual predators during daylight hours and move up toward the sea surface at dusk to search for food. This behavior is termed “diel vertical migration.” Migrating animals graze on phytoplankton or zooplankton and in turn serve as food for higher trophic levels, hence providing a key mechanism for carbon export via this migration. These animals are often observed as sound-scattering layers by echosounders, but the animals causing the acoustic scattering are difficult to identify using acoustics alone. In a spring 2019 experiment in Monterey Bay, we deployed autonomous underwater and surface vehicles over a seabed-mounted upward-looking echosounder to collect environmental DNA (eDNA) with the goal of identifying the vertically migrating animals. The echosounder was installed at 890-m depth on the Monterey Accelerated Research System (MARS) seabed cabled ocean observatory, providing real-time data of acoustic backscatter from the full water column. One long-range autonomous underwater vehicle (LRAUV) carrying a Third-Generation Environmental Sample Processor (3G-ESP) acquired water samples from a sequence of layers from near surface down to ~ 290 m as directed by the distribution of animals observed by the echosounder. During the sampling of each layer, the LRAUV ran on a tight circular yo-yo trajectory directly above the echosounder, remaining in its beam by acoustically tracking a station-keeping Wave Glider on the sea surface marking the echosounder's latitude and longitude. The persistent and simultaneous acoustic observation and eDNA acquisition enables identification of animals at precise locations to better understand their vertical migration behaviors. We present the methods and the system performance in the experiment.

Metatranscriptomic Analysis of Oil-Exposed Seawater Bacterial Communities Archived by an Environmental Sample Processor (ESP).

The use of natural marine bacteria as “oil sensors” for the detection of pollution events can be suggested as a novel way of monitoring oil occurrence at sea. Nucleic acid-based devices generically called genosensors are emerging as potentially promising tools for in situ detection of specific microbial marker genes suited for that purpose. Functional marker genes are particularly interesting as targets for oil-related genosensing but their identification remains a challenge. Here, seawater samples, collected in tanks with oil addition mimicking a realistic oil spill scenario, were filtered and archived by the Environmental Sample Processor (ESP), a fully robotized genosensor, and the samples were then used for post-retrieval metatranscriptomic analysis. After extraction, RNA from ESP-archived samples at start, Day 4 and Day 7 of the experiment was used for sequencing. Metatranscriptomics revealed that several KEGG pathways were significantly enriched in samples exposed to oil. However, these pathways were highly expressed also in the non-oil-exposed water samples, most likely as a result of the release of natural organic matter from decaying phytoplankton. Temporary peaks of aliphatic alcohol and aldehyde dehydrogenases and monoaromatic ring-degrading enzymes (e.g., ben, box, and dmp clusters) were observed on Day 4 in both control and oil-exposed and non-exposed tanks. Few alkane 1-monooxygenase genes were upregulated on oil, mostly transcribed by families Porticoccaceae and Rhodobacteraceae, together with aromatic ring-hydroxylating dioxygenases, mostly transcribed by Rhodobacteraceae. Few transcripts from obligate hydrocarbonoclastic genera of Alcanivorax, Oleispira and Cycloclasticus were significantly enriched in the oil-treated exposed tank in comparison to control the non-exposed tank, and these were mostly transporters and genes involved in nitrogen and phosphorous acquisition. This study highlights the importance of seasonality, i.e., phytoplankton occurrence and senescence leading to organic compound release which can be used preferentially by bacteria over oil compounds, delaying the latter process. As a result, such seasonal effect can reduce the sensitivity of genosensing tools employing bacterial functional genes to sense oil. A better understanding of the use of natural organic matter by bacteria involved in oil-biodegradation is needed to develop an array of functional markers enabling the rapid and specific in situ detection of anthropogenic pollution.

Environmental DNA: An Emerging Tool in Ecological Assessment.

Environmental DNA (eDNA), as a recent research hotspot in environmental science, the use of eDNA in biological monitoring has the advantages of sensitivity and time/labor saving. The eDNA technology combined with scientific advancement has been applied in investigations of target species (such as invasive species, endangered species and other rare species), biomass, and biodiversity. In addition, ecotoxicology studies and environmental pollution impact assessments based on the development of eDNA technology have gradually emerged in recent years. In this article, we summarizes the application of eDNA in ecological assessment, include species diversity assessment and chemical contamination impacts assessment, provide guiding questions for study design. We additionally discuss current challenges associated with eDNA. Finally, looking to the future, we discuss the opportunities of eDNA technology in environmental protein, environmental sample processor and ecogenomic sensors.

Autonomous Tracking and Sampling of the Deep Chlorophyll Maximum Layer in an Open-Ocean Eddy by a Long-Range Autonomous Underwater Vehicle

Phytoplankton communities residing in the open ocean, the largest habitat on Earth, play a key role in global primary production. Through their influence on nutrient supply to the euphotic zone, open-ocean eddies impact the magnitude of primary production and its spatial and temporal distributions. It is important to gain a deeper understanding of the microbial ecology of marine ecosystems under the influence of eddy physics with the aid of advanced technologies. In March and April 2018, we deployed autonomous underwater and surface vehicles in a cyclonic eddy in the North Pacific Subtropical Gyre to investigate the variability of the microbial community in the deep chlorophyll maximum (DCM) layer. One long-range autonomous underwater vehicle (LRAUV) carrying a third-generation Environmental Sample Processor (3G-ESP) autonomously tracked and sampled the DCM layer for four days without surfacing. The sampling LRAUV's vertical position in the DCM layer was maintained by locking onto the isotherm corresponding to the chlorophyll peak. The vehicle ran on tight circles while drifting with the eddy current. This mode of operation enabled a quasi-Lagrangian time series focused on sampling the temporal variation of the DCM population. A companion LRAUV surveyed a cylindrical volume around the sampling LRAUV to monitor spatial and temporal variation in contextual water column properties. The simultaneous sampling and mapping enabled observation of DCM microbial community in its natural frame of reference.

In situ Autonomous Acquisition and Preservation of Marine Environmental DNA Using an Autonomous Underwater Vehicle

Environmental DNA (eDNA) can be used to identify macroorganisms and describe biodiversity, and thus has promise to supplement biological monitoring in marine ecosystems. Despite this promise, scaling sample acquisition to the size and temporal scales needed for effective monitoring would require prohibitively large investments in time and human resources. To improve upon these problems, here we test the efficacy of an autonomous eDNA sampling system and compare results obtained to traditional eDNA sampling methods. The autonomous sampling instrument consisted of the Environmental Sample Processor, (ESP) coupled to an autonomous underwater vehicle (AUV). We tested equivalency between the ESP and traditional eDNA sampling methods by comparing the quantification of eDNA across a broad range of taxa, from microbes (SAR11), phytoplankton (Pseudo-nitzschia spp.), invertebrates (krill: Euphausia pacifica) to vertebrates (anchovy: Engraulis mordax). No significant differences in eDNA densities were observed between the sample collection and filtration methods. eDNA filters collected by the ESP were preserved and stable for 21 days, the typical deployment length of the instrumentation. Finally, we demonstrated the unique capabilities of an autonomous, mobile ESP during a deployment near Monterey Bay, CA, by remotely and repeatedly sampling a water mass over 12 hours. The development of a mobile ESP reveals the promise of utilizing eDNA measurements to observe complex biological processes.

Effects of nutrient enrichment on surface microbial community gene expression in the oligotrophic North Pacific Subtropical Gyre.

Marine microbial communities are critical for biogeochemical cycles and the productivity of ocean ecosystems. Primary productivity in the surface ocean is constrained by nutrients which are supplied, in part, by mixing with deeper water. Little is known about the time scales, frequency, or impact of mixing on microbial communities. We combined in situ sampling using the Environmental Sample Processor and a small-scale mixing experiment with lower euphotic zone water to determine how individual populations respond to mixing. Transcriptional responses were measured using the MicroTOOLs (Microbiological Targets for Ocean Observing Laboratories) microarray, which targets all three domains of life and viruses. The experiment showed that mixing substantially affects photosynthetic taxa as expected, but surprisingly also showed that populations respond differently to unfiltered deep water which contains particles (organisms and detritus) compared to filtered deep water that only contains nutrients and viruses, pointing to the impact of biological interactions associated with these events. Comparison between experimental and in situ population transcription patterns indicated that manipulated populations can serve as analogs for natural populations, and that natural populations may be frequently or continuously responding to nutrients from deeper waters. Finally, this study also shows that the microarray approach, which is complementary to metatranscriptomic sequencing, is useful for determining the physiological status of in situ microbial communities.

Collecting and processing samples in remote and dangerous places: the Environmental Sample Processor as a case study

Abstract Collecting water samples in remote or dangerous places can help identify chemical spills, discover clandestine weapons production, or determine if there has been natural or human-caused biological contamination of waterways. These collections can be expensive or put humans at risk due to the nature of the locale or the detection target. Such sample collection challenges are similar to those faced in oceanography, where accessibility and the physical realities of remoteness, corrosiveness, and pressure, place severe requirements on instrumentation, especially for unattended operations over long periods of time. The Monterey Bay Aquarium Research Institute (MBARI) has been at the forefront of developments that push forward sample collection and processing capabilities in the ocean. Specifically, the development of the environmental sample processor (ESP), a microbiology laboratory-in-a-can, has allowed extended presence with high frequency sampling. When deployed, the ESP filters water to collect particles, then either preserves those particles, or creates a homogenate for molecular analysis. Originally designed for detecting harmful algae blooms and the toxins they produce, the ESP now has expanded analytical capabilities. A newer version of the ESP is now being tested on an autonomous underwater vehicle, providing never-before-seen mobility and unprecedented access to the top 300 m of the ocean.

The Quest to Develop Ecogenomic Sensors: A 25-Year History of the Environmental Sample Processor (ESP) as a Case Study

The Quest to Develop Ecogenomic Sensors: A 25-Year History of the Environmental Sample Processor (ESP) as a Case Study

Sandwich hybridization probes for the detection of Pseudo-nitzschia (Bacillariophyceae) species: An update to existing probes and a description of new probes

New sandwich hybridization assay (SHA) probes for detecting Pseudo-nitzschia species (P. arenysensis, P. fraudulenta, P. hasleana, P. pungens) are presented, along with updated cross-reactivity information on historical probes (SHA and FISH; fluorescence in situ hybridization) targeting P. australis and P. multiseries. Pseudo-nitzschia species are a cosmopolitan group of diatoms that produce varying levels of domoic acid (DA), a neurotoxin that can accumulate in finfish and shellfish and transfer throughout the food web. Consumption of infected food sources can lead to illness in humans (amnesic shellfish poisoning; ASP) and marine wildlife (domoic acid poisoning; DAP). The threat of human illness, along with economic loss from fishery closures has resulted in the implementation of monitoring protocols and intensive ecological studies. SHA probes have been instrumental in some of these efforts, as the technique performs well in complex heterogeneous sample matrices and has been adapted to benchtop and deployable (Environmental Sample Processor) platforms. The expanded probe set will enhance future efforts towards understanding spatial, temporal and successional patterns in species during bloom and non-bloom periods.

Use of Highly Specific Molecular Markers Reveals Positive Correlation between Abundances of Mesodinium cf. major and Its Preferred Prey, Teleaulax amphioxeia, During Red Water Blooms in the Columbia River Estuary.

In a previous study, Teleaulax amphioxeia-the preferred prey of Mesodinium in the Columbia River estuary-were undetectable within intense annual blooms, suggesting blooms are prey-limited or prey are acquired outside of bloom patches. We used a novel molecular approach specifically targeting the prey (i.e., Unique Sequence Element [USE] within the ribosomal RNA 28S D2 regions of T.amphioxeia nucleus and nucleomorph) in estuarine water samples acquired autonomously with an Environmental Sample Processor integrated within a monitoring network (ESP-SATURN). This new approach allowed for both more specific detection of the prey and better constraint of sample variability. A positive correlation was observed between abundances of M. cf. major and T.amphioxeia during bloom periods. The correlation was stronger at depth (>8.2m) and weak or nonexistent in the surface, suggesting that predator-prey dynamics become uncoupled when stratification is strong. We confirmed exclusive selectivity for T.amphioxeia by M. cf. major and observed the incorporation of the prey nucleus into a 4-nuclei complex, where it remained functionally active. The specific biomarker for T.amphioxeia was also recovered in M. cf. major samples from a Namibian coastal bloom, suggesting that a specific predator-prey relationship might be widespread between M. cf. major and T.amphioxeia.

Small Things Considered

Transcriptomic methods make it possi-ble to take a snapshot of the processes occurring in a mixed microbial popula-tion; sampling over a time course can transform that image into a motion pic-ture. That's the goal: a molecular video of what is being done, by whom, and when, out in the environment. But there is a catch: for continuity of the frames in your movie, you need to sample the same population at each time point. When your target population is drifting effortlessly in the marine environment, and your bucket is tethered to a sluggish ship, the ocean current presents you with a new population each time you capture some water. Ed DeLong, with postdocs Liz Ottesen and Frank Ayl-ward, and colleagues from the Monterey Bay Aquarium Research Institute, solved this problem by using a free-drifting, ro-botic Environmental Sample Processor (ESP). The ESP, suspended 23 m below a drifting float on the surface in the North Pacific Subtropical Gyre (NPSG), could “go with the flow,” and collect and pre-serve samples of microbes ranging from 0.22–5 μm in size at specified time points. Physical measurements from the environment were recorded in real time, and RNA from the recovered samples was analyzed by reverse transcription and sequencing, and mapped back to the genomes of ocean denizens.

Recovery and identification of Pseudo-nitzschia (Bacillariophyceae) frustules from natural samples acquired using the environmental sample processor.

Many species within the diatom genus Pseudo-nitzschia are difficult to distinguish without applying molecular analytical or microscopy-based methods. DNA, antibody and lectin probes have previously been used to provide rapid and specific detection of species and strains in complex field assemblages. Recently, however, well-documented cryptic genetic diversity within the group has confounded results of DNA probe tests in particular. Moreover, the number of species descriptions within the genus continues to increase, as do insights into toxin production by both new and previously described species. Therefore, a combination of classical morphological techniques and modern molecular methodologies is needed to resolve ecophysiological traits of Pseudo-nitzschia species. Here, we present an approach to recover and identify frustules from sample collection filters used for toxin analysis onboard the Environmental Sample Processor (ESP), an in situ sample collection and analytical platform. This approach provides a new and powerful tool for correlating species presence with toxin detected remotely and in situ by the ESP, and has the potential to be applied broadly to other sampling configurations. This new technique will contribute to a better understanding of naturally occurring Pseudo-nitzschia community structure with respect to observed domoic acid outbreaks.