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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.

Membrane proteins, found at the junctions between the outside world and the inner workings of the cell, play important roles in human disease and are used as biosensors. More than half of all therapeutics directly affect membrane protein function while nanopores enable DNA sequencing. The structural and functional characterisation of membrane proteins is therefore crucial. However, low levels of naturally abundant protein and the hydrophobic nature of membrane proteins makes production difficult. To maximise success, high-throughput strategies were developed that rely upon simple screens to identify successful constructs and rapidly exclude those unlikely to work. Parameters that affect production such as expression host, membrane protein origin, expression vector, fusion-tags, encapsulation reagent and solvent composition are screened in parallel. In this way, constructs with divergent requirements can be produced for a variety of structural applications. As structural techniques advance, sample requirements will change. Single-particle cryo-electron microscopy requires less protein than crystallography and as cryo-electron tomography and time-resolved serial crystallography are developed new sample production requirements will evolve. Here we discuss different methods used for the high-throughput production of membrane proteins for structural biology.

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 $\sim$ 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.

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.

Many transmembrane receptors have a desensitized state, in which they are unable to respond to external stimuli. The family of microbial rhodopsin proteins includes one such group of receptors, whose inactive or dark-adapted (DA) state is established in the prolonged absence of light. Here, we present high-resolution crystal structures of the ground (light-adapted) and DA states of Archaerhodopsin-3 (AR3), solved to 1.1 Å and 1.3 Å resolution respectively. We observe significant differences between the two states in the dynamics of water molecules that are coupled via H-bonds to the retinal Schiff Base. Supporting QM/MM calculations reveal how the DA state permits a thermodynamic equilibrium between retinal isomers to be established, and how this same change is prevented in the ground state in the absence of light. We suggest that the different arrangement of internal water networks in AR3 is responsible for the faster photocycle kinetics compared to homologs., Archaerhodopsin-3 (AR3) mutants are commonly used in optogenetics for neuron silencing and membrane voltage sensing. High-resolution crystal structures show that desensitization of the AR3 photoreceptor occurs when internal hydrogen-bonded water networks are modified in response to changes in chromophore isomerization.

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.

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, robotic environmental (e)DNA samplers now make it possible for biological observations to match the scale and quality of abiotic measurements collected by automated sensor networks. Merging these automated data streams may allow for improved insight into biotic responses to environmental change and stressors. Here, we merged eDNA data collected by robotic samplers installed at three U.S. Geological Survey (USGS) streamgages with gridded daily weather data, and daily water quality and quantity data into a cloud-hosted database. The eDNA targets were a rare fish parasite and a more common salmonid fish. We then used computationally expedient Bayesian hierarchical occupancy models to evaluate associations between abiotic conditions and eDNA detections and to simulate how uncertainty in result interpretation changes with the frequency of autonomous robotic eDNA sample collection. We developed scripts to automate data merging, cleaning and analysis steps into a chained-step, workflow. We found that inclusion of abiotic covariates only provided improved insight for the more common salmonid fish since its DNA was more frequently detected. Rare fish parasite DNA was infrequently detected, which caused occupancy parameter estimates and covariate associations to have high uncertainty. Our simulations found that collecting samples at least once per day resulted in more detections and less parameter uncertainty than less frequent sampling. Our occupancy and simulation results together demonstrate the advantages of robotic eDNA samplers and how these samples can be combined with easy to acquire, publicly available data to foster real-time biosurveillance and forecasting.

Cancers, neurodegenerative and infectious diseases remain some of the leading causes of deaths worldwide. The structure-guided drug design is essential to advance drug development for these important diseases. One of the key challenges in the structure determination workflow is the production of eukaryotic membrane proteins (drug targets) of high quality. A number of expression systems have been developed for the production of eukaryotic membrane proteins. In this chapter, an optimized detailed protocol for transient transfection and expression of eukaryotic membrane proteins in Expi293F cells is presented. Testing expression and purification on a small scale allow optimizing conditions for sample preparation for downstream structural (cryo-EM) elucidation.

Recent advances in technology have enabled an unprecedented development of underwater research, extending from near shore to the deepest regions of the globe. However, monitoring of biodiversity is not fully implemented in political agendas and biological observations in the deep ocean have been even more limited in space and time. The Foresight Workshop on Advances in Ocean Biological Observations: a sustained system for deep-ocean meroplankton was convened to to foster advances in the knowledge on deep-ocean invertebrate larval distributions and improve our understanding of fundamental deep-ocean ecological processes such as connectivity and resilience of benthic communities to natural and human-induced disturbance. This Meroplankton Observations Workshop had two specific goals: 1) review the state-of-the-art instrumentation available for meroplankton observations; 2) develop a strategy to implement technological innovations for in-situ meroplankton observation. Presentations and discussions are summarised in this report covering: i) key challenges and priorities for advancing the knowledge of deep-sea larval diversity and distribution: ii) recent developments in technology and future needs for plankton observation, iii) data integration and oceanographic modelling; iv) synergies and added value of a sustained observation system for meroplankton; v) steps for developing a sustained observation system for deep-ocean meroplankton and plans to maximise collaborative opportunities.

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.

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.

An ecologically and economically disruptive harmful algal bloom (HAB) affected much of the northeast Pacific margin in 2015, during a prolonged oceanic warm anomaly. Caused by diatoms of the genus Pseudo-nitzschia, this HAB produced the highest particulate concentrations of the biotoxin domoic acid (DA) ever recorded in Monterey Bay, California. Bloom inception followed strong upwelling during the spring transition, which introduced nutrients and eliminated the warm anomaly locally. Subsequently, moderate and intermittent upwelling created favorable conditions for growth and accumulation of HAB biomass, which was dominated by a highly toxigenic species, P. australis. High cellular DA concentrations were associated with available nitrogen for DA synthesis coincident with silicate exhaustion. This nutrient influence resulted from two factors: (1) disproportionate depletion of silicate in upwelling source waters during the warm anomaly, the most severe depletion observed in 24 years, and (2) silicate uptake by the dense diatom bloom.

A 3-week diversion of the Orange County Sanitation District effluent discharge into nearshore waters off Newport Beach, CA constituted a considerable injection of secondarily-treated effluent into the coastal ecosystem. The location ≈1.6 km from shore, shallow water depth (≈16 m), volume and nutrient content of the discharge (≈5.3 × 10 8 L day −1 of effluent with inorganic nitrogen concentration >2 mM) during the diversion raised concerns regarding the potential for stimulating phytoplankton blooms and, in particular, blooms of toxic species. Remarkably, phytoplankton standing stocks during the event and shortly thereafter did not reach values associated even with minor blooms historically observed in the region (generally −1 ), although shifts in community composition were observed. Diatom abundances increased early during the diversion, dinoflagellates, phototrophic picoplanktonic eukaryotes and other algae increased mid-diversion, and cyanobacteria ( Synechococcus , Prochlorococcus ) increased near the end of the diversion. Concentrations of domoic acid (a phycotoxin commonly present in the area) remained near or below detection throughout the diversion, and abundances of potentially-harmful algal species were unresponsive. Bacterial biomass increased during the diversion, and equaled or exceeded total phytoplankton biomass in most samples. Abundances of microbial grazers were also elevated during the diversion. We speculate that nutrient uptake by the bacterial biomass, acting in concert with or a response to a negative effect of disinfection byproducts associated with chlorination on phytoplankton physiology, played a significant role in muting the response of the phytoplankton to nutrients released in the effluent.

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.

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., Design Type(s)transcription profiling design • sequence assembly objective • biodiversity assessment objectiveMeasurement Type(s)transcription profiling assay • marine metagenome • microbial communityTechnology Type(s)RNA sequencing • DNA sequencing • amplicon sequencingFactor Type(s)assay protocol • temporal_instantSample Characteristic(s)marine metagenome • Monterey Bay • ocean biome Machine-accessible metadata file describing the reported data (ISA-Tab format)

One of the greatest challenges to understanding ecosystem dynamics in the open ocean is predicting where and when hot spots of biological activity exist and then observing and sampling those features for days to weeks, as they move in a complicated trajectory. It is difficult to apply traditional research vessel-based techniques at the relevant temporal and spatial scales when the biological features could be spinning in an eddy, rising and falling on internal waves, or even actively migrating. To address this challenge the Monterey Bay Aquarium Research Institute (MBARI) is developing an observing system based on a collaborating group of autonomous underwater vehicles (AUVs) and an Unmanned Surface Vehicle (USV). Building on the capabilities of the Long Range AUV (LRAUV) and a Liquid Robotics SV-3 Wave Glider, MBARI has begun deploying arrays of LRAUVs configured with complementary sensors that work together as an Open Ocean, Autonomous Vehicle-based Lagrangian Observatory.

Simple Summary Membrane proteins are essential to all forms of life. Millions of membrane proteins are found in the lipid membrane layer that surrounds cells, and in the lipid membrane layers that surround smaller cellular compartments. Many medicines interact with membrane proteins; these include drugs that treat cancer, heart disease and pain. Research into membrane proteins is therefore important to the design and development of new medicines. Membrane proteins are difficult to work with, partly because they are so small. However, using techniques such as X-ray crystallography and electron microscopy, structural biologists, including those at the Membrane Protein Laboratory, are able to see the atomic detail of membrane proteins. There has been great progress in the field of membrane protein structural biology over the past fifteen years. Here, we review the recent advances in membrane protein structural biology, highlight key methods and give an overview of techniques. We also discuss the challenges that remain in this field, and suggest areas for future research. Abstract Membrane proteins are essential components of many biochemical processes and are important pharmaceutical targets. Membrane protein structural biology provides the molecular rationale for these biochemical process as well as being a highly useful tool for drug discovery. Unfortunately, membrane protein structural biology is a difficult area of study due to low protein yields and high levels of instability especially when membrane proteins are removed from their native environments. Despite this instability, membrane protein structural biology has made great leaps over the last fifteen years. Today, the landscape is almost unrecognisable. The numbers of available atomic resolution structures have increased 10-fold though advances in crystallography and more recently by cryo-electron microscopy. These advances in structural biology were achieved through the efforts of many researchers around the world as well as initiatives such as the Membrane Protein Laboratory (MPL) at Diamond Light Source. The MPL has helped, provided access to and contributed to advances in protein production, sample preparation and data collection. Together, these advances have enabled higher resolution structures, from less material, at a greater rate, from a more diverse range of membrane protein targets. Despite this success, significant challenges remain. Here, we review the progress made and highlight current and future challenges that will be overcome.

The overarching logistical challenge in microbial oceanography is acquiring enough samples to provide meaningful scientific interpretation. The number of samples collected during ship expeditions is limited by weather, time on station, and budget. Here we describe a robotic, autonomous vehicle platform equipped with a unique sampling instrument that mitigates some of these constraints. In a joint cruise on the R/V Falkor, the Monterey Bay Aquarium Research Institute and the University of Hawaii deployed two of these vehicles in a mesoscale eddy north of the island of Maui. One vehicle collected contextual measurements while circling a freely drifting sampling vehicle. On the sampling vehicle we implemented several behaviors, including sampling every three hours for a 4-day underwater drift while maintaining position within the deep chlorophyll maximum layer (~100m). Results demonstrate the ability to remain with features of interest and point to an exciting future of long-term, directed, persistent sampling.

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.

The active transport of glycolytic pyruvate across the inner mitochondrial membrane is thought to involve two mitochondrial pyruvate carrier subunits, MPC1 and MPC2, assembled as a 150 kDa heterotypic oligomer. Here, the recombinant production of human MPC through a co-expression strategy is first described; however, substantial complex formation was not observed, and predominantly individual subunits were purified. In contrast to MPC1, which co-purifies with a host chaperone, we demonstrated that MPC2 homo-oligomers promote efficient pyruvate transport into proteoliposomes. The derived functional requirements and kinetic features of MPC2 resemble those previously demonstrated for MPC in the literature. Distinctly, chemical inhibition of transport is observed only for a thiazolidinedione derivative. The autonomous transport role for MPC2 is validated in cells when the ectopic expression of human MPC2 in yeast lacking endogenous MPC stimulated growth and increased oxygen consumption. Multiple oligomeric species of MPC2 across mitochondrial isolates, purified protein and artificial lipid bilayers suggest functional high-order complexes. Significant changes in the secondary structure content of MPC2, as probed by synchrotron radiation circular dichroism, further supports the interaction between the protein and ligands. Our results provide the initial framework for the independent role of MPC2 in homeostasis and diseases related to dysregulated pyruvate metabolism.

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.

Integral membrane proteins are among the most fascinating and important biomolecules as they play a vital role in many biological functions. Knowledge of their atomic structures is fundamental to the understanding of their biochemical function and key in many drug discovery programs. However, over the years, structure determination of integral membrane proteins has proven to be far from trivial, hence they are underrepresented in the protein data bank. Low expression levels, insolubility and instability are just a few of the many hurdles one faces when studying these proteins. X-ray crystallography has been the most used method to determine atomic structures of membrane proteins. However, the production of high quality membrane protein crystals is always very challenging, often seen more as art than a rational experiment. Here we review valuable approaches, methods and techniques to successful membrane protein crystallisation.

An observational study was performed in the central Southern California Bight in Spring 2010 to understand the relationship between seasonal spring phytoplankton blooms and coastal processes that included nutrient input from upwelling, wastewater effluent plumes, and other processes. Multi-month Webb Slocum glider deployments combined with MBARI environmental sample processors (ESPs), weekly pier sampling, and ocean color data provided a multidimensional characterization of the development and evolution of harmful algal blooms (HABs). Results from the glider and ESP observations demonstrated that blooms of toxic Pseudo-nitzschia sp. can develop offshore and subsurface prior to their manifestation in the surface layer and/or near the coast. A significant outbreak and surface manifestation of the blooms coincided with periods of upwelling, or other processes that caused shallowing of the pycnocline and subsurface chlorophyll maximum. Our results indicate that subsurface populations can be an important source for “seeding” surface Pseudo-nitzschia HAB events in southern California.

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.

Using autonomous molecular analytical devices embedded within an ocean observatory, we studied harmful algal bloom (HAB) ecology in the dynamic coastal waters of Monterey Bay, California. During studies in 2007 and 2008, HAB species abundance and toxin concentrations were quantified periodically at two locations by Environmental Sample Processor (ESP) robotic biochemistry systems. Concurrently, environmental variability and processes were characterized by sensors co-located with ESP network nodes, regional ocean moorings, autonomous underwater vehicle surveys, and satellite remote sensing. The two locations differed in their long-term average physical and biological conditions and in their degree of exposure to episodic wind-forced variability. While anomalously weak upwelling and strong stratification during the 2007 study favored toxigenic dinoflagellates (Alexandrium catenella), anomalously strong upwelling during the 2008 study favored toxigenic diatoms (Pseudo-nitzschia spp.). During both studies, raphidophytes (Heterosigma akashiwo) were detected within a similar range of concentrations, and they reached higher abundances at the relatively sheltered, stratified site. During 2008, cellular domoic acid reached higher concentrations and was far more variable at the shallower ESP node, where phytoplankton populations were influenced by resuspended sediments. Episodic variability caused by wind forcing, lateral mixing, internal waves, and subsurface phytoplankton layers influenced ESP detection patterns. The results illustrate the importance of mobilizing HAB detection on autonomous platforms that can intelligently target sample acquisition as a function of environmental conditions and biological patch encounter.

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.

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.

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.

UNLABELLED 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. SIGNIFICANCE AND IMPACT OF THE STUDY This study presents the first report of in-situ qPCR enumeration of both faecal indicators and harmful algal species in coastal marine waters. We utilize a robotic device for in-situ quantification of enterococci, the human-specific marker in Bacteriodales and Pseudo-nitzschia spp. from the same water samples collected and processed in-situ. The results demonstrate that rapid, in-situ monitoring can be utilized to identify and quantify multiple health-relevant micro-organisms important in water quality monitoring and that this monitoring can be used to inform same-day notifications.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

SUMMARYWe 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.

SYNOPSIS. During feeding in anurans, the mouth opens while the tongue, which is attached to the mandible at the front of the mouth, rotates forward. Due to the relative simplicity of its anatomy and the complexity of its motion, tongue protraction in frogs presents an ideal system for exploring the neural control of multijoint movements. In this study, we used a forward dynamic, rigid body model with four segments and two muscles to investigate open loop control of tongue protraction in the Australian white-lipped tree frog, Litoria caerulea. Model parameters include the mass distribution, initial position and initial angular velocity of each segment and the anatomy and physiology of each muscle. Model variables include the level of muscle activation at each time step and impulsive torques to open and close the mouth. The model gives X,Y coordinates of each segment and joint angles at each time step as output. The model was tested using scaled, normalized EMG signals and impulsive joint torques to predict the paths of the lower jaw tip and tongue tip. Predicted paths were compared to experimentally observed paths using Pearson product-moment correlation coefficients. Simulations demonstrate that the genioglossus muscles likely play a minor role, if any, in determining the trajectory of the tongue in most anurans. Most of the force for tongue protraction comes from angular momentum transferred to the tongue by the opening jaws. In anurans, tongue protraction is dynamically stable and will occur as long as the musculoskeletal elements are in the correct initial position.

The flow structure that is largely responsible for the good performance of insect wings has recently been identified as a leading-edge vortex. But because such vortices become detached from a wing in two-dimensional flow, an unknown mechanism must keep them attached to (three-dimensional) flapping wings. The current explanation, analogous to a mechanism operating on delta-wing aircraft, is that spanwise flow through a spiral vortex drains energy from the vortex core. We have tested this hypothesis by systematically mapping the flow generated by a dynamically scaled model insect while simultaneously measuring the resulting aerodynamic forces. Here we report that, at the Reynolds numbers matching the flows relevant for most insects, flapping wings do not generate a spiral vortex akin to that produced by delta-wing aircraft. We also find that limiting spanwise flow with fences and edge baffles does not cause detachment of the leading-edge vortex. The data support an alternative hypothesis-that downward flow induced by tip vortices limits the growth of the leading-edge vortex.

Scaling predictions pioneered by A.V. Hill state that isometric changes in kinematics result from isometric changes in size. These predictions have been difficult to support because few animals display truly isometric growth. An exception to this rule is said to be the toads in the genus Bufo, which can grow over three orders of magnitude. To determine whether skull shape increases isometrically, I used linear measurements and geometric morphometrics to quantify shape variation in a size series of 69 skulls from the marine toad, B. marinus. Toads ranged in body mass from 1.8 gm to a calculated 1,558.9 gm. Of all linear measurements (S/V length, skull width, skull length, levator mass, depressor mass, adductor foramen area), only the area of the adductor foramen increased faster than body mass; the remaining variables increased more slowly. In addition, modeling the lower jaw as a lever-arm system showed that the lengths of the closing in- and out-levers scaled isometrically with body mass despite the fact that the skull itself is changing allometrically. Geometric morphometrics discerned areas of greatest variability with increasing body mass at the rear of the skull in the area of the squamosal bone and the adductor foramen. This increase in area of the adductor foramen may allow more muscle to move the relatively greater mass of the lower jaw in larger toads, although adductor mass scales with body mass. If B. marinus feeds in a similar manner to other Bufo, these results imply that morphological allometry may still result in kinematic isometry. J. Morphol. 241:115–126, 1999. © 1999 Wiley-Liss, Inc.

Size and shape are fundamental features of organisms. Ideally, the methods used to describe size and shape must be sensitive enough to detect small differences, and at the same time provide interpretation that is visually satisfying. I compared shape of wings among Eptesicus fuscus, Myotis lucifugus, Pipistrellus hesperas , and Tadarida brasiliensis using three methods of morphometric analysis; principal-components analysis on traditional measures of lengths and areas, principal-components analysis of interlandmark distances, and relative-warp analysis. These species were chosen to test the efficacy of each morphometric analysis at discovering and describing differences in both size and shape. Although principal-components analyses provided similar aggregations of species based on variable loadings, only relative-warps analysis provided clear pictures as to how species differ. The molossid bat, T. brasiliensis , had narrower wings than the vespertilionid bats; this shape was not due to compression of the entire wing, but to a complicated rotation around the fifth digit. The vespertilionid bats were difficult to discern based on wing shape alone, but the warp analysis showed that P. Hesperus and M. lucifugus are more similar in shape of wing than either is to E. fuscus . Each method of analysis of shape has strengths, but relative-warp analysis provided both the power to discriminate among minor differences in shape of species within families, and the graphical capability to display those differences in a visually pleasing way.

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.

We tested two models of adaptive offspring sex-ratio that predict opposite optimal reproductive strategies for female white-tail deer (Odocoileus virginianus). Trivers and Willard's model predicts that does (females) in particularly good condition should produce sons, and Williams refined their model to make specific predictions about optimal offspring number/sex choices. Verme's model results in very different predictions because of very different assumptions about which sex of offspring can best benefit from high levels of maternal resources. We found clear support for the Trivers and Willard/Williams model when we analyzed data from road-killed does, and we furthermore question several of the assumptions of the Verme model.