Oceanic microorganisms can rapidly respond to environmental variability. Determining how physical and biological processes control microbial distributions, abundances, and metabolic dynamics is challenging. Here, we used autonomous underwater vehicles capable of Lagrangian feature tracking and in situ sampling, in combination with ship-based measurements, to examine diel to weekly scale changes in microbial transcription and biogeochemistry in the deep chlorophyll maximum (DCM) of a mesoscale cyclonic eddy. Nearly 20% of total transcript expression showed diel periodicity, highlighting the importance of the diurnal cycle on phytoplankton metabolism in the dim waters of the DCM. Eddy-induced isopycnal uplift increased nutrient concentrations and caused upward displacement of the DCM, driving increased picoeukaryotic cell abundances and transcriptional activity of nitrate-incorporating photoautotrophs. As the eddy weakened, the DCM deepened and transcriptional activity shifted towards chemolithoautotrophic ammonia-oxidizing archaea. The temporal dynamics observed demonstrate how plankton communities rapidly respond to both diel variation and stochastic mesoscale disturbances.

The field of environmental DNA (eDNA) analysis has revolutionized our ability to detect and monitor biodiversity in aquatic and terrestrial ecosystems. However, traditional eDNA sampling methods often present limitations in terms of temporal and spatial coverage, resulting in a loss of resolution associated with infrequent events or those prohibitive to onsite fieldwork. In recent years, the emergence of autonomous eDNA sampling technology has provided researchers with a powerful tool for collecting high-resolution genetic data, overcoming many of the challenges associated with manual sample acquisition. This review focuses exclusively on eDNA technologies designed for the collection and preservation of water samples, to provide a comprehensive overview of the current landscape of aquatic autonomous eDNA sampling technology and instrumentation. A new era of instrument development and capabilities is emerging; the result of knowledge gained through experience with long-tested marine biological observation instrumentation. Lastly, we highlight current research to develop an in situ eDNA analytical capability, as well as explore the challenges and future prospects associated with this rapidly evolving field.

Multiple autonomous underwater vehicles (AUVs) working in collaboration can achieve scientific goals more effectively than independently operated vehicles. In this article, we present a case of using two long-range AUVs (LRAUVs) for persistent environmental DNA (eDNA) sampling of a targeted feature. Each LRAUV was equipped with a third-generation environmental sample processor (3G-ESP), a robotic instrument for acquiring and processing water samples for molecular analysis. Each 3G-ESP can collect and process 60 samples. For continuous and persistent eDNA sampling of vertically migrating organisms at a targeted depth layer, we deployed two LRAUVs which alternately triggered the ESP, extending the total time of collecting samples. We developed a method of coordinated sampling by time shift and a collaborative sampling method that uses acoustic handshakes. In the time-shift method, each vehicle switched between two behaviors: sample collection at the targeted depth and spiraling over a large depth range to make contextual measurement. The second vehicle's mission started later than the first vehicle's by a time shift equal to the duration of one sampling event, such that at a given time one vehicle sampled at the targeted depth while the other vehicle spiraled up and down. In the acoustic-handshake method, the two LRAUVs exchanged sample-start and sample-end messages. On receiving vehicle #1’s sample-end message, vehicle #2 triggered a sampling event and transmitted a sample-start message to vehicle #1. Then, vehicle #1 waited for vehicle #2’s sample-end message before triggering the next sampling event, and so forth. The time-shift method is simple, whereas the acoustic-handshake method is accurate and adaptive. Both methods were demonstrated in experiments in Monterey Bay.

Freshwater cyanobacterial harmful algal blooms (CHABs) are a well-known global public health threat. Monitoring and early detection of CHAB toxins are currently accomplished using labor-intensive sampling techniques and subsequent shore-based analyses, with results typically reported 24-48 h after sample collection. We have developed and implemented an uncrewed, autonomous mobile sampler-analytical system capable of conducting targeted in situ toxin measurements in < 2 h. A surface plasmon resonance (SPR) instrument was combined with the environmental sample processor (ESP) to fully automate detection and quantification of particle-associated cyanobacterial microcystins (pMC). This sensor-sampler system was integrated with a long-range autonomous underwater vehicle (LRAUV) and deployed in western Lake Erie for field trials in the summer of 2021. The LRAUV was remotely piloted to acquire samples at selected locations within and adjacent to a CHAB. Sixteen pMC measurements ranging from 0.09 to 0.55 mu g/L lake water were obtained over a 14-day period without recovery of the LRAUV. The SPR/ESP/LRAUV system complements existing satellite, aerial, and manual sampling CHAB survey techniques, and could be used to enhance predictive models that underpin bloom and toxicity forecasts. This system is also extensible to detection of other algal toxins in freshwater and marine environments, with its near real-time assessment of bloom toxin levels potentially offering additional socioeconomic benefits and public health protection in a variety of settings.

Abstract The 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.

Environmental DNA (eDNA) data make it possible to measure and monitor biodiversity at unprecedented resolution and scale. As use-cases multiply and scientific consensus grows regarding the value of eDNA analysis, public agencies have an opportunity to decide how and where eDNA data fit into their mandates. Within the United States, many federal and state agencies are individually using eDNA data in various applications and developing relevant scientific expertise. A national strategy for eDNA implementation would capitalize on recent scientific developments, providing a common set of next-generation tools for natural resource management and public health protection. Such a strategy would avoid patchwork and possibly inconsistent guidelines in different agencies, smoothing the way for efficient uptake of eDNA data in management. Because eDNA analysis is already in widespread use in both ocean and freshwater settings, we focus here on applications in these environments. However, we foresee the broad adoption of eDNA analysis to meet many resource management issues across the nation because the same tools have immediate terrestrial and aerial applications.

Cyanobacterial 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.

Ocean microbes are the foundation of marine food webs, regulating carbon cycling and ecosystem dynamics. How they proliferate, die, move, and interact is regulated by physical, chemical, and biological factors that are dynamic and challenging to quantify in the natural environment. A significant limitation in many marine field studies is the inability to continuously sample the ever-changing ocean environment over space and time. In this study, we integrated spatiotemporal and multi-omic sample collection in an intensive sampling effort of phytoplankton ecology in Monterey Bay, California during the spring of 2021. Sampling methods coupled: (1) manual shipboard CTD sampling, (2) autonomous sampling using a Long-Range Autonomous Underwater Vehicle (LRAUV) equipped with an Environmental Sampling Processor (ESP), and (3) high-resolution physical measurements by an autonomous vertical profiler (Wirewalker). Sampling occurred as upwelling waned alongside declining domoic acid (DA) and low abundances of toxigenic Pseudo-nitzschia . Conditions needed to spark a widespread and toxic Pseudo-nitzschia bloom were absent, yet low-level DA was driven by similar mechanisms to those causing elevated DA. Three DA biosynthetic intermediate molecules were reported in the environment for the first time. Both shipboard and ESP sampling approaches identified DA biosynthetic gene expression at frontal zones. DA and expression of dabA , the gene encoding the first committed step of DA biosynthesis, were higher in association with recently upwelled water that supplied nutrients for growth and DA biosynthesis. Detection of subtle variations in dab gene expression in response to environmental variation provide a window into the ecological dynamics underpinning major toxic events. Graphical Abstract

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.

Abstract A greater understanding of eDNA behavior in the environment is needed before it can be employed for ecosystem monitoring applications. The objectives of this study were to use autonomous sampling to conduct long‐term, high‐frequency monitoring of the eDNA of native salmonid species in a Californian coastal stream, describe temporal variation of eDNA on multiple scales and identify environmental factors that drive this variation, and evaluate the ability of the eDNA datasets to detect rare species and represent organismal abundance. Using high‐throughput autonomous environmental sample processors (ESPs) and qPCR, we enumerated eDNA concentrations from 674 water samples collected at subdaily intervals over 360 days at a single site. We detected eDNA from two imperiled salmonids (coho salmon Oncorhynchus kisutch and steelhead/rainbow trout O. mykiss) in most samples; O. kisutch eDNA was generally in lower concentration and more variable than O. mykiss eDNA. High‐frequency (i.e., subdaily and daily) variability in salmonid eDNA concentrations showed occasional patchiness (i.e., large differences between consecutive samples), while seasonal differences were observed consistent with the ecology of the species at this site. Salmonid eDNA concentrations were significantly associated with creek discharge, photoperiod, and whether the creek mouth was open or closed by a seasonal sandbar. The release of hatchery‐origin O. kisutch parr into the stream was associated with a significant increase in eDNA concentration for the remainder of the study. We compared eDNA signals with fish abundance data collected from traps located at the site. Fish were detected more often by eDNA than from trapping. Significant positive associations between fish abundance and eDNA concentrations were observed for O. mykiss; however, no such associations were observed for O. kisutch. This study adds to our knowledge on the occurrence and behavior of fish eDNA in lotic systems and informs future biomonitoring efforts using automated sampling technology.

The use of environmental DNA (eDNA) for studying the ecology and variability of life in the sea is reviewed here in the context of US interagency Marine Biodiversity Observation Network (MBON) projects. Much of the information in this paper comes from samples collected within US National Marine Sanctuaries. The field of eDNA is relatively new but growing rapidly, and it has the potential to disrupt current paradigms developed on the basis of existing measurement methods. After a general review of the field, we provide specific examples of the type of information that eDNA provides regarding the changing distribution of life in the sea over space (horizontally and vertically) and time. We conclude that eDNA analyses yield results that are similar to those collected using traditional observation methods, are complementary to them, and because of the breadth of information provided, have the potential to improve conservation and management practices. Moreover, through technology development and standardization of methods, eDNA offers a means to scale biological observations globally to a level similar to those currently made for ocean physics and biogeochemistry. This scaling can ultimately result in a far better understanding of global marine biodiversity and contribute to better management and sustainable use of the world ocean. Improved information management systems that track methods and associated metadata, together with international coordination, will be needed to realize a global eDNA observation network.

The deep chlorophyll maximum (DCM) layer is an ecologically important feature of the open ocean. The DCM cannot be observed using aerial or satellite remote sensing; thus, in situ observations are essential. Further, understanding the responses of microbes to the environmental processes driving their metabolism and interactions requires observing in a reference frame that moves with a plankton population drifting in ocean currents, i.e., Lagrangian. Here, we report the development and application of a system of coordinated robots for studying planktonic biological communities drifting within the ocean. The presented Lagrangian system uses three coordinated autonomous robotic platforms. The focal platform consists of an autonomous underwater vehicle (AUV) fitted with a robotic water sampler. This platform localizes and drifts within a DCM community, periodically acquiring samples while continuously monitoring the local environment. The second platform is an AUV equipped with environmental sensing and acoustic tracking capabilities. This platform characterizes environmental conditions by tracking the focal platform and vertically profiling in its vicinity. The third platform is an autonomous surface vehicle equipped with satellite communications and subsea acoustic tracking capabilities. While also acoustically tracking the focal platform, this vehicle serves as a communication relay that connects the subsea robot to human operators, thereby providing situational awareness and enabling intervention if needed. Deployed in the North Pacific Ocean within the core of a cyclonic eddy, this coordinated system autonomously captured fundamental characteristics of the in situ DCM microbial community in a manner not possible previously.

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 realtime 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 similar 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.

In this paper, a molecular analytical approach for detecting hydrocarbonoclastic bacteria in water is suggested as a proxy measurement for tracking petroleum discharges in industrialized or pristine aquatic environments. This approach is tested for general application in cold marine regions (freezing to 5 °C). We used amplicon sequencing and qPCR to quantify 16S rRNA and GyrB genes from oleophilic bacteria in seawater samples from two different crude oil enrichments. The first experiment was conducted in a controlled environment using laboratory conditions and natural North Sea fjord seawater (NSC) at a constant temperature of 5 °C. The second was performed in the field with natural Arctic seawater (ARC) and outdoor temperature conditions from -7 °C to around 4 °C. Although the experimental conditions for NSC and ARC differed, the temporal changes in bacterial communities were comparable and reflected oil biotransformation processes. The common bacterial OTUs for NSC and ARC had the highest identity to Colwellia rossensis and Oleispira antarctica rRNA sequences and were enriched within a few days in both conditions. Other typical oil degrading bacteria such as Alcanivorax (n-alkane degrader) and Cycloclasticus (polycyclic aromatic hydrocarbons degrader) were rapidly enriched only in NSC conditions. Both the strong correlation between Oleispira SSU gene copies and oil concentration, and the specificity of the Oleispira assay suggest that this organism is a robust bioindicator for seawater contaminated by petroleum in cold water environments. Further optimization for automation of the Oleispira assay for in situ analysis with a genosensing device is underway. The assay for Colwellia quantification requires more specificity to fewer Colwellia OTUs and a well-established dose-response relationship before those taxa are used for oil tracking purposes.

Metagenomic and metatranscriptomic time-series data covering a 52-day period in the fall of 2016 provide an inventory of bacterial and archaeal community genes, transcripts, and taxonomy during an intense dinoflagellate bloom in Monterey Bay, CA, USA. The dataset comprises 84 metagenomes (0.8 terabases), 82 metatranscriptomes (1.1 terabases), and 88 16S rRNA amplicon libraries from samples collected on 41 dates. The dataset also includes 88 18S rRNA amplicon libraries, characterizing the taxonomy of the eukaryotic community during the bloom. Accompanying the sequence data are chemical and biological measurements associated with each sample. These datasets will facilitate studies of the structure and function of marine bacterial communities during episodic phytoplankton blooms.

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.

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.

Phytoplankton and associated microbial communities provide organic carbon to oceanic food webs and drive ecosystem dynamics. However, capturing those dynamics is challenging. Here, an in situ, semi-Lagrangian, robotic sampler profiled pelagic microbes at 4 h intervals over ~2.6 days in North Pacific high-nutrient, low-chlorophyll waters. We report on the community structure and transcriptional dynamics of microbes in an operationally large size class (>5 μm) predominantly populated by dinoflagellates, ciliates, haptophytes, pelagophytes, diatoms, cyanobacteria (chiefly Synechococcus), prasinophytes (chiefly Ostreococcus), fungi, archaea, and proteobacteria. Apart from fungi and archaea, all groups exhibited 24-h periodicity in some transcripts, but larger portions of the transcriptome oscillated in phototrophs. Periodic photosynthesis-related transcripts exhibited a temporal cascade across the morning hours, conserved across diverse phototrophic lineages. Pronounced silica:nitrate drawdown, a high flavodoxin to ferredoxin transcript ratio, and elevated expression of other Fe-stress markers indicated Fe-limitation. Fe-stress markers peaked during a photoperiodically adaptive time window that could modulate phytoplankton response to seasonal Fe-limitation. Remarkably, we observed viruses that infect the majority of abundant taxa, often with total transcriptional activity synchronized with putative hosts. Taken together, these data reveal a microbial plankton community that is shaped by recycled production and tightly controlled by Fe-limitation and viral activity.

The organic sulfur compound dimethylsulfoniopropionate (DMSP) is synthesized by numerous species of marine phytoplankton, and its volatile degradation products are a major source of biogenic sulfur to the atmosphere. A massive bloom of the dinoflagellate Akashiwo sanguinea occurred in Monterey Bay, CA, USA, in the fall of 2016 and led to exceptionally high seawater DMSP concentrations that peaked at 4,240 nM. Bacterial consumption rates showed that only a small fraction of the DMSP standing stock flowed through the dissolved DMSP pool per day, contributing to the high DMSP concentrations and creating conditions conducive to production of dimethylsulfide (DMS). Conservative calculations of DMS yield from this persistent A. sanguinea bloom suggest substantial regional‐scale inputs of DMS‐sulfur to the atmosphere. Other recently reported major coastal blooms of A. sanguinea, along with indications that this species may benefit from climate change conditions, reveal a mechanism that could alter oceanic contributions to atmospheric sulfur pools.