James M. Birch

Near real-time, autonomous detection of marine bacterioplankton on a coastal mooring in Monterey Bay, California, using rRNA-targeted DNA probes

A sandwich hybridization assay (SHA) was developed to detect 16S rRNAs indicative of phylogenetically distinct groups of marine bacterioplankton in a 96-well plate format as well as low-density arrays printed on a membrane support. The arrays were used in a field-deployable instrument, the Environmental Sample Processor (ESP). The SHA employs a chaotropic buffer for both cell homogenization and hybridization, thus target sequences are captured directly from crude homogenates. Capture probes for seven of nine different bacterioplankton clades examined reacted specifically when challenged with target and non-target 16S rRNAs derived from in vitro transcribed 16S rRNA genes cloned from natural samples. Detection limits were between 0.10-1.98 and 4.43- 12.54 fmole ml(-1) homogenate for the 96-well plate and array SHA respectively. Arrays printed with five of the bacterioplankton-specific capture probes were deployed on the ESP in Monterey Bay, CA, twice in 2006 for a total of 25 days and also utilized in a laboratory time series study. Groups detected included marine alphaproteobacteria, SAR11, marine cyanobacteria, marine group I crenarchaea, and marine group II euryarchaea. To our knowledge this represents the first report of remote in situ DNA probe-based detection of marine bacterioplankton.

Underwater application of quantitative PCR on an ocean mooring.

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.

Autonomous Application of Quantitative PCR in the Deep Sea: In Situ Surveys of Aerobic Methanotrophs Using the Deep-Sea Environmental Sample Processor

Recent advances in ocean observing systems and genomic technologies have led to the development of the deep-sea environmental sample processor (D-ESP). The D-ESP filters particulates from seawater at depths up to 4000 m and applies a variety of molecular assays to the particulates, including quantitative PCR (qPCR), to identify particular organisms and genes in situ. Preserved samples enable laboratory-based validation of in situ results and expanded studies of genomic diversity and gene expression. Tests of the D-ESP at a methane-rich mound in the Santa Monica Basin centered on detection of 16S rRNA and particulate methane monooxygenase (pmoA) genes for two putative aerobic methanotrophs. Comparison of in situ qPCR results with laboratory-based assays of preserved samples demonstrates the D-ESP generated high-quality qPCR data while operating autonomously on the seafloor. Levels of 16S rRNA and pmoA cDNA detected in preserved samples are consistent with an active community of aerobic methanotrophs near the methane-rich mound. These findings are substantiated at low methane sites off Point Conception and in Monterey Bay where target genes are at or below detection limits. Successful deployment of the D-ESP is a major step toward developing autonomous systems to facilitate a wide range of marine microbiological investigations.

Simultaneous monitoring of faecal indicators and harmful algae using an in-situ autonomous sensor.

Faecal indicator bacteria (FIB) and harmful algal blooms (HABs) threaten the health and the economy of coastal communities worldwide. Emerging automated sampling technologies combined with molecular analytical techniques could enable rapid detection of micro‐organisms in‐situ, thereby improving resource management and public health decision‐making. We evaluated this concept using a robotic device, the Environmental Sample Processor (ESP). The ESP automates in‐situ sample collection, nucleic acid extraction and molecular analyses. Here, the ESP measured and reported concentrations of FIB (Enterococcus spp.), a microbial source‐tracking marker (human‐specific Bacteriodales) and a HAB species (Psuedo‐nitzschia spp.) over a 45‐day deployment on the Santa Cruz Municipal Wharf (Santa Cruz, CA, USA). Both FIB and HABs were enumerated from single in‐situ collected water samples. The in‐situ qPCR efficiencies ranged from 86% to 105%, while the limit of quantifications during the deployment was 10 copies reaction−1. No differences were observed in the concentrations of enterococci, the human‐specific marker in Bacteroidales spp., and P. australis between in‐situ collected sample and traditional hand sampling methods (P > 0·05). Analytical results were Internet‐accessible within hours of sample collection, demonstrating the feasibility of same‐day public notification of current water quality conditions.

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.

Co-registered Geochemistry and Metatranscriptomics Reveal Unexpected Distributions of Microbial Activity within a Hydrothermal Vent Field

Despite years of research into microbial activity at diffuse flow hydrothermal vents, the extent of microbial niche diversity in these settings is not known. To better understand the relationship between microbial activity and the associated physical and geochemical conditions, we obtained co-registered metatranscriptomic and geochemical data from a variety of different fluid regimes within the ASHES vent field on the Juan de Fuca Ridge. Microbial activity in the majority of the cool and warm fluids sampled was dominated by a population of Gammaproteobacteria (likely sulfur oxidizers) that appear to thrive in a variety of chemically distinct fluids. Only the warmest, most hydrothermally-influenced flows were dominated by active populations of canonically vent-endemic Epsilonproteobacteria. These data suggest that the Gammaproteobacteria collected during this study may be generalists, capable of thriving over a broader range of geochemical conditions than the Epsilonproteobacteria. Notably, the apparent metabolic activity of the Gammaproteobacteria—particularly carbon fixation—in the seawater found between discrete fluid flows (the intra-field water) suggests that this area within the Axial caldera is a highly productive, and previously overlooked, habitat. By extension, our findings suggest that analogous, diffuse flow fields may be similarly productive and thus constitute a very important and underappreciated aspect of deep-sea biogeochemical cycling that is occurring at the global scale.

Development of a mobile ecogenomic sensor

Modern ocean microbial research utilizes advanced molecular analytical techniques, such as polymerase chain reaction (PCR), DNA and protein probe arrays, and nucleic acid sequencing (etc.). Applying or at least initiating these techniques at the point and time of sample collection can enhance their effectiveness. To that end, in-situ sample processing and real-time molecular detection schemes have been implemented using deployable autonomous systems that can be operated in diverse ocean environments from shallow coastal waters to the deep sea. Such devices have been termed “ecogenomic sensors.” The size of these instruments currently requires that they be moored in a fixed location or passively mobile, drifting at fixed depth and observing microbial communities in a moving frame of reference with ocean currents. With the highly dynamic motion of open water and microbial life, the next frontier of ocean microbial research requires the improved capability of an actively mobile asset. A mobile ecogenomic sensor encompasses a fully maneuverable vehicle with weeks of persistence, environmental data analysis, detection of physical and biological features, autonomous sampling and in situ analysis, and near-real-time data reporting. This system is now being developed by integrating three components: a compact molecular analytical instrument (the 3rd generation Environmental Sample Processor), a long-range autonomous underwater vehicle, and software algorithms for AUV-based feature detection and sampling. A summary of the system and its initial application is presented.

Tracking and sampling of a phytoplankton patch by an autonomous underwater vehicle in drifting mode

Phytoplankton patches in the coastal ocean have important impacts on the patterns of primary productivity, the survival and growth of zooplankton and fish larvae, and the development of harmful algal blooms (HABs). We desire to observe microscopic life in a phytoplankton patch in its natural frame of reference (which is moving with the ocean current), thereby permitting resolution of time-dependent evolution of the population. To achieve this goal, we have developed a method for a Tethys-class long range autonomous underwater vehicle (AUV) (which has a propeller and a buoyancy engine) to detect, track, and sample a phytoplankton patch in buoyancy-controlled drifting mode. In this mode, the vehicle shuts off its propeller and actively controls its buoyancy to autonomously find the peakchlorophyll layer, stay in it, and trigger water sampling in the layer. In an experiment in Monterey Bay, CA in July 2015, the Makai AUV, which was equipped with a prototype 3rd-generation Environmental Sample Processor (3G-ESP), ran the algorithm to autonomously detect the peak-chlorophyll layer, and drifted and triggered ESP samplings in the layer.

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.

Diversity and toxicity of Pseudo-nitzschia species in Monterey Bay: Perspectives from targeted and adaptive sampling

Monterey Bay, California experiences near-annual blooms of Pseudo-nitzschia that can affect marine animal health and the economy, including impacts to tourism and commercial/recreational fisheries. One species in particular, P. australis, has been implicated in the most toxic of events, however other species within the genus can contribute to widespread variability in community structure and associated toxicity across years. Current monitoring methods are limited in their spatial coverage as well as their ability to capture the full suite of species present, thereby hindering understanding of HAB events and limiting predictive accuracy. An integrated deployment of multiple in situ platforms, some with autonomous adaptive sampling capabilities, occurred during two divergent bloom years in the bay, and uncovered detailed aspects of population and toxicity dynamics. A bloom in 2013 was characterized by spatial differences in Pseudo-nitzschia populations, with the low-toxin producer P. fraudulenta dominating the inshore community and toxic P. australis dominating the offshore community. An exceptionally toxic bloom in 2015 developed as a diverse Pseudo-nitzschia community abruptly transitioned into a bloom of highly toxic P. australis within the time frame of a week. Increases in cell density and proliferation coincided with strong upwelling of nutrients. High toxicity was driven by silicate limitation of the dense bloom. This temporal shift in species composition mirrored the shift observed further north in the California Current System off Oregon and Washington. The broad scope of sampling and unique platform capabilities employed during these studies revealed important patterns in bloom formation and persistence for Pseudo-nitzschia. Results underscore the benefit of expanded biological observing capabilities and targeted sampling methods to capture more comprehensive spatial and temporal scales for studying and predicting future events.