Microdiversity and temporal dynamics of marine bacterial dimethylsulfoniopropionate genes.

Dimethylsulfoniopropionate (DMSP) is an abundant organic sulfur metabolite produced by many phytoplankton species and degraded by bacteria via two distinct pathways with climate-relevant implications. We assessed the diversity and abundance of bacteria possessing these pathways in the context of phytoplankton community composition over a 3-week time period spanning September-October, 2014 in Monterey Bay, CA. The dmdA gene from the DMSP demethylation pathway dominated the DMSP gene pool and was harboured mostly by members of the alphaproteobacterial SAR11 clade and secondarily by the Roseobacter group, particularly during the second half of the study. Novel members of the DMSP-degrading community emerged from dmdA sequences recovered from metagenome assemblies and single-cell sequencing, including largely uncharacterized gammaproteobacteria and alphaproteobacteria taxa. In the DMSP cleavage pathway, the SAR11 gene dddK was the most abundant early in the study, but was supplanted by dddP over time. SAR11 members, especially those harbouring genes for both DMSP degradation pathways, had a strong positive relationship with the abundance of dinoflagellates, and DMSP-degrading gammaproteobacteria co-occurred with haptophytes. This in situ study of the drivers of DMSP fate in a coastal ecosystem demonstrates for the first time correlations between specific groups of bacterial DMSP degraders and phytoplankton taxa.

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.

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.

Ecological divergence of syntopic marine bacterial species is shaped by gene content and expression.

Identifying mechanisms by which bacterial species evolve and maintain genomic diversity is particularly challenging for the uncultured lineages that dominate the surface ocean. A longitudinal analysis of bacterial genes, genomes, and transcripts during a coastal phytoplankton bloom revealed two co-occurring, highly related Rhodobacteraceae species from the deeply branching and uncultured NAC11-7 lineage. These have identical 16S rRNA gene amplicon sequences, yet their genome contents assembled from metagenomes and single cells indicate species-level divergence. Moreover, shifts in relative dominance of the species during dynamic bloom conditions over 7 weeks confirmed the syntopic species' divergent responses to the same microenvironment at the same time. Genes unique to each species and genes shared but divergent in per-cell inventories of mRNAs accounted for 5% of the species' pangenome content. These analyses uncover physiological and ecological features that differentiate the species, including capacities for organic carbon utilization, attributes of the cell surface, metal requirements, and vitamin biosynthesis. Such insights into the coexistence of highly related and ecologically similar bacterial species in their shared natural habitat are rare.

A Raman spectral reference library of potential anthropogenic and biological ocean polymers.

Microplastics have been extensively documented in marine ecosystems and food webs with devastating impacts. To solve this global crisis, identifying the polymer composition is key for resolving the material origin, geographic source, and ecosystem life cycle of ocean plastics. Visually based techniques, importantly, are not diagnostic. Raman spectroscopy is an increasingly preferred identification method for its accuracy and reduced likelihood of misinterpretation, though it can be inaccessible due to cost of paywalled spectral libraries and availability of relevant polymer spectra for comparison. Here, we provide an open-access reference library of high-quality, broad-spectrum Raman spectra of major polymer categories germane to marine environments. The library includes high-quality spectra from: (a) pristine anthropogenic polymers newly sourced from manufacturers (n = 40), (b) weathered anthropogenic polymers collected from used consumer, beachcast, agricultural, and fishery sources (n = 22), and (c) biological polymers representing diverse marine taxa, trophic levels, and tissues (n = 17). We hope this reference library can help this rapidly expanding scientific community and facilitate progress in the global plastic pollution crisis.

A system of coordinated autonomous robots for Lagrangian studies of microbes in the oceanic deep chlorophyll maximum.

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.

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.

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.

Microbial metagenomes and metatranscriptomes during a coastal phytoplankton bloom.

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.

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.

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.

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.

Multiplexed reverse transcriptase PCR assay for identification of viral respiratory pathogens at the point of care.

We have developed a nucleic acid-based assay that is rapid, sensitive, and specific and can be used for the simultaneous detection of five common human respiratory pathogens, including influenza virus A, influenza virus B, parainfluenza virus types 1 and 3, respiratory syncytial virus (RSV), and adenovirus groups B, C, and E. Typically, diagnosis on an unextracted clinical sample can be provided in less than 3 h, including sample collection, preparation, and processing, as well as data analysis. Such a multiplexed panel would enable rapid broad-spectrum pathogen testing on nasal swabs and therefore allow implementation of infection control measures and the timely administration of antiviral therapies. We present here a summary of the assay performance in terms of sensitivity and specificity. The limits of detection are provided for each targeted respiratory pathogen, and result comparisons were performed on clinical samples, our goal being to compare the sensitivity and specificity of the multiplexed assay to the combination of immunofluorescence and shell vial culture currently implemented at the University of California-Davis Medical Center hospital. Overall, the use of the multiplexed reverse transcription-PCR assay reduced the rate of false-negative results by 4% and reduced the rate of false-positive results by up to 10%. The assay correctly identified 99.3% of the clinical negatives and 97% of the adenovirus, 95% of the RSV, 92% of the influenza virus B, and 77% of the influenza virus A samples without any extraction performed on the clinical samples. The data also showed that extraction will be needed for parainfluenza virus, which was only identified correctly 24% of the time on unextracted samples.

The influence of wing-wake interactions on the production of aerodynamic forces in flapping flight.

We used two-dimensional digital particle image velocimetry (DPIV) to visualize flow patterns around the flapping wing of a dynamically scaled robot for a series of reciprocating strokes starting from rest. The base of the wing was equipped with strain gauges so that the pattern of fluid motion could be directly compared with the time history of force production. The results show that the development and shedding of vortices throughout each stroke are highly stereotyped and influence force generation in subsequent strokes. When a wing starts from rest, it generates a transient force as the leading edge vortex (LEV) grows. This early peak, previously attributed to added-mass acceleration, is not amenable to quasi-steady models but corresponds well to calculations based on the time derivative of the first moment of vorticity within a sectional slice of fluid. Forces decay to a stable level as the LEV reaches a constant size and remains attached throughout most of the stroke. The LEV grows as the wing supinates prior to stroke reversal, accompanied by an increase in total force. At stroke reversal, both the LEV and a rotational starting vortex (RSV) are shed into the wake, forming a counter-rotating pair that directs a jet of fluid towards the underside of the wing at the start of the next stroke. We isolated the aerodynamic influence of the wake by subtracting forces and flow fields generated in the first stroke, when the wake is just developing, from those produced during the fourth stroke, when the pattern of both the forces and wake dynamics has reached a limit cycle. This technique identified two effects of the wake on force production by the wing: an early augmentation followed by a small attenuation. The later decrease in force is consistent with the influence of a decreased aerodynamic angle of attack on translational forces caused by downwash within the wake and is well explained by a quasi-steady model. The early effect of the wake is not well approximated by a quasi-steady model, even when the magnitude and orientation of the instantaneous velocity field are taken into account. Thus, the wake capture force represents a truly unsteady phenomenon dependent on temporal changes in the distribution and magnitude of vorticity during stroke reversal.

Unsteady forces and flows in low Reynolds number hovering flight: two-dimensional computations vs robotic wing experiments.

We compare computational, experimental and quasi-steady forces in a generic hovering wing undergoing sinusoidal motion along a horizontal stroke plane. In particular, we investigate unsteady effects and compare two-dimensional (2D) computations and three-dimensional (3D) experiments in several qualitatively different kinematic patterns. In all cases, the computed drag compares well with the experiments. The computed lift agrees in the cases in which the sinusoidal changes in angle of attack are symmetrical or advanced with respect to stroke positions, but lags behind the measured 3D lift in the delayed case. In the range of amplitudes studied here, 3-5 chords, the force coefficients have a weak dependence on stroke amplitude. As expected, the forces are sensitive to the phase between stroke angle and angle of attack, a result that can be explained by the orientation of the wing at reversal. This dependence on amplitude and phase suggests a simple maneuver strategy that could be used by a flapping wing device. In all cases the unsteady forces quickly reach an almost periodic state with continuous flapping. The fluid forces are dominated by the pressure contribution. The force component directly proportional to the linear acceleration is smaller by a factor proportional to the ratio of wing thickness and stroke amplitude; its net contribution is zero in hovering. The ratio of wing inertia and fluid force is proportional to the product of the ratio of wing and fluid density and the ratio of wing thickness and stroke amplitude; it is negligible in the robotic wing experiment, but need not be in insect flight. To identify unsteady effects associated with wing acceleration, and coupling between rotation and translation, as well as wake capture, we examine the difference between the unsteady forces and the estimates based on translational velocities, and compare them against the estimate of the coupling between rotation and translation, which have simple analytic forms for sinusoidal motions. The agreement and disagreement between the computed forces and experiments offer further insight into when the 3D effects are important. A main difference between a 3D revolving wing and a 2D translating wing is the absence of vortex shedding by a revolving wing over a distance much longer than the typical stroke length of insects. No doubt such a difference in shedding dynamics is responsible in part for the differences in steady state force coefficients measured in 2D and 3D. On the other hand, it is unclear whether such differences would have a significant effect on transient force coefficients before the onset of shedding. While the 2D steady state force coefficients underpredict 3D forces, the transient 2D forces measured prior to shedding are much closer to the 3D forces. In the cases studied here, the chord is moving between 3 to 5 chords, typical of hovering insect stroke length, and the flow does not appear to separate during each stroke in the cases of advanced and symmetrical rotation. In these cases, the wing reverses before the leading edge vortex would have time to separate even in 2D. This suggests that the time scale for flow separation in these strokes is dictated by the flapping frequency, which is dimensionally independent. In such cases, the 2D unsteady forces turn out to be good approximations of 3D experiments.

Force production and flow structure of the leading edge vortex on flapping wings at high and low Reynolds numbers.

The elevated aerodynamic performance of insects has been attributed in part to the generation and maintenance of a stable region of vorticity known as the leading edge vortex (LEV). One explanation for the stability of the LEV is that spiraling axial flow within the vortex core drains energy into the tip vortex, forming a leading-edge spiral vortex analogous to the flow structure generated by delta wing aircraft. However, whereas spiral flow is a conspicuous feature of flapping wings at Reynolds numbers (Re) of 5000, similar experiments at Re=100 failed to identify a comparable structure. We used a dynamically scaled robot to investigate both the forces and the flows created by a wing undergoing identical motion at Re of approximately 120 and approximately 1400. In both cases, motion at constant angular velocity and fixed angle of attack generated a stable LEV with no evidence of shedding. At Re=1400, flow visualization indicated an intense narrow region of spanwise flow within the core of the LEV, a feature conspicuously absent at Re=120. The results suggest that the transport of vorticity from the leading edge to the wake that permits prolonged vortex attachment takes different forms at different Re.