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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 spatial and temporal scales needed for effective monitoring would require prohibitively large investments in time and human resources. To address this challenge, we evaluated 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 techniques by comparing the quantification of eDNA across a broad range of taxa, from microbes (SAR11), phytoplankton (Pseudo-nitzschia spp.), and invertebrates (krill: Euphausia pacifica) to vertebrates (anchovy: Engraulis mordax). No significant differences in eDNA densities were observed between the two 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 h. The development of a mobile ESP demonstrates the promise of utilizing eDNA measurements to observe complex biological processes in the ocean absent a human presence.

The advent of ocean observatories is creating unique opportunities for deploying novel sensor systems. We are exploring that potential through the development and application of the Environmental Sample Processor (ESP). ESP is an electromechanical/fluidic system designed to collect discrete water samples, concentrate microorganisms, and automate application of molecular probe technologies. Development and application of ESP grew from extensive partnerships galvanized by the National Oceanographic Partnership Program. Near-real-time observations are currently achieved using low-density DNA probe and protein arrays. Filter-based sandwich hybridization methodology enables direct detection of ribosomal RNA sequences diagnostic for groups of bacteria and archaea, as well as a variety of invertebrates and harmful algal species. An antibody-based technique is used for detecting domoic acid, an algal biotoxin. To date, ESP has been deployed in ocean waters from the near surface to 1000 m. Shallow-water deployments demonstrated application of all four types of assays in single deployments lasting up to 30 days and provided the first remote detection of such phylogenetically diverse organisms and metabolites on one platform. Deep-water applications focused on detection of invertebrates associated with whale falls, using remotely operated vehicle-based operations lasting several days. Current work emphasizes incorporating a four-channel, real-time polymerase chain reaction module, extending operations to 4000-m water depth, and increasing deployment duration.

The Environmental Sample Processor (ESP) is a device that allows for the underwater, autonomous application of DNA and protein probe array technologies as a means to remotely identify and quantify, in situ, marine microorganisms and substances they produce. Here, we added functionality to the ESP through the development and incorporation of a module capable of solid-phase nucleic acid extraction and quantitative PCR (qPCR). Samples collected by the instrument were homogenized in a chaotropic buffer compatible with direct detection of ribosomal RNA (rRNA) and nucleic acid purification. From a single sample, both an rRNA community profile and select gene abundances were ascertained. To illustrate this functionality, we focused on bacterioplankton commonly found along the central coast of California and that are known to vary in accordance with different oceanic conditions. DNA probe arrays targeting rRNA revealed the presence of 16S rRNA indicative of marine crenarchaea, SAR11 and marine cyanobacteria; in parallel, qPCR was used to detect 16S rRNA genes from the former two groups and the large subunit RuBisCo gene (rbcL) from Synecchococcus. The PCR-enabled ESP was deployed on a coastal mooring in Monterey Bay for 28 days during the spring-summer upwelling season. The distributions of the targeted bacterioplankon groups were as expected, with the exception of an increase in abundance of marine crenarchaea in anomalous nitrate-rich, low-salinity waters. The unexpected co-occurrence demonstrated the utility of the ESP in detecting novel events relative to previously described distributions of particular bacterioplankton groups. The ESP can easily be configured to detect and enumerate genes and gene products from a wide range of organisms. This study demonstrated for the first time that gene abundances could be assessed autonomously, underwater in near real-time and referenced against prevailing chemical, physical and bulk biological conditions.