Methodological 'lenses' influence the characterization of phytoplankton dynamics in a coastal upwelling ecosystem.

New technologies enable the opportunity to improve our monitoring and understanding of marine phytoplankton communities. However, careful consideration for how different methodological approaches, or 'lenses', influence our interpretation of phytoplankton ecology is important, particularly when drawing conclusions about change over time or space. Using both high-throughput 18S rRNA gene sequencing and microscopy, we explored how phytoplankton community structure varied over the course of a year within a nearshore semi-enclosed coastal embayment along the Central Coast of California. The seasonal shift in the relative community dominance (i.e., diatom vs. dinoflagellate dominance) was captured in the microscopy results but not effectively captured in the molecular-based findings. However, the molecular approach explained more of the variability in composition across seasons relative to the microscopy approach. Temporal dynamics of specific bloom-forming taxa also differed between the molecular and microscopy results. Overall, the observed differences between the molecular- and microscopy-derived characterization of phytoplankton dynamics suggest that the approaches are best suited to answer different research questions. Moreover, the approaches complement each other for a more comprehensive perspective of a coastal phytoplankton ecosystem. Therefore, identifying the biases of each approach within natural communities is necessary to effectively and accurately characterize phytoplankton communities.

Amino Acid Analog Induces Stress Response in Marine Synechococcus.

Characterizing the cell-level metabolic trade-offs that phytoplankton exhibit in response to changing environmental conditions is important for predicting the impact of these changes on marine food web dynamics and biogeochemical cycling. The time-selective proteome-labeling approach, bioorthogonal noncanonical amino acid tagging (BONCAT), has potential to provide insight into differential allocation of resources at the cellular level, especially when coupled with proteomics. However, the application of this technique in marine phytoplankton remains limited. We demonstrate that the marine cyanobacteria Synechococcus sp. and two groups of eukaryotic algae take up the modified amino acid l-homopropargylglycine (HPG), suggesting that BONCAT can be used to detect translationally active phytoplankton. However, the impact of HPG addition on growth dynamics varied between groups of phytoplankton. In addition, proteomic analysis of Synechococcus cells grown with HPG revealed a physiological shift in nitrogen metabolism, general protein stress, and energy production, indicating a potential limitation for the use of BONCAT in understanding the cell-level response of Synechococcus sp. to environmental change. Variability in HPG sensitivity between algal groups and the impact of HPG on Synechococcus physiology indicates that particular considerations should be taken when applying this technique to other marine taxa or mixed marine microbial communities. IMPORTANCE Phytoplankton form the base of the marine food web and substantially impact global energy and nutrient flow. Marine picocyanobacteria of the genus Synechococcus comprise a large portion of phytoplankton biomass in the ocean and therefore are important model organisms. The technical challenges of environmental proteomics in mixed microbial communities have limited our ability to detect the cell-level adaptations of phytoplankton communities to a changing environment. The proteome labeling technique, bioorthogonal noncanonical amino acid tagging (BONCAT), has potential to address some of these challenges by simplifying proteomic analyses. This study explores the ability of marine phytoplankton to take up the modified amino acid, l-homopropargylglycine (HPG), required for BONCAT, and investigates the proteomic response of Synechococcus to HPG. We not only demonstrate that cyanobacteria can take up HPG but also highlight the physiological impact of HPG on Synechococcus, which has implications for future applications of this technique in the marine environment.

The rhizosphere microbiome plays a role in the resistance to soil-borne pathogens and nutrient uptake of strawberry cultivars under field conditions.

Microbial-root associations are important to help plants cope with abiotic and biotic stressors. Managing these interactions offers an opportunity for improving the efficiency and sustainability of agricultural production. By characterizing the bacterial and archaeal community (via 16S rRNA sequencing) associated with bulk and rhizosphere soil of sixteen strawberry cultivars in two controlled field studies, we explored the relationships between the soil microbiome and plant resistance to two soil-borne fungal pathogens (Verticillium dahliae and Macrophomina phaseolina). Overall, the plants had a distinctive and genotype-dependent rhizosphere microbiome with higher abundances of known beneficial bacteria such as Pseudomonads and Rhizobium. The rhizosphere microbiome played a significant role in the resistance to the two soil-borne pathogens as shown by the differences in microbiome between high and low resistance cultivars. Resistant cultivars were characterized by higher abundances of known biocontrol microorganisms including actinobacteria (Arthrobacter, Nocardioides and Gaiella) and unclassified acidobacteria (Gp6, Gp16 and Gp4), in both pathogen trials. Additionally, cultivars that were resistant to V. dahliae had higher rhizosphere abundances of Burkholderia and cultivars resistant to M. phaseolina had higher abundances of Pseudomonas. The mechanisms involved in these beneficial plant-microbial interactions and their plasticity in different environments should be studied further for the design of low-input disease management strategies.

Visualization of probiotics via epifluorescence microscopy and fluorescence in situ hybridization (FISH).

Aerobic plate counts, the standard for bacterial enumeration in the probiotic industry, can be biased towards fast-growing organisms that replicate on synthetic media and can significantly underestimate total bacterial abundance. Culture-independent approaches such as fluorescence in situ hybridization (FISH) hold promise as a means to rapidly and accurately enumerate bacteria in probiotic products. In addition, FISH has the potential to more accurately represent bacterial growth dynamics in the environment in which products are applied without imposing additional growth constraints that are required for enumeration via plate counts. In this study, we designed and optimized three new FISH probes to visualize and quantify Bacillus amyloliquefaciens, Bacillus pumilus, and Bacillus licheniformis within probiotic products. Microscopy-based estimates were consistent or higher than label claims for Pediococcus acidilactici, Pediococcus pentosaceus, Lactobacillus plantarum, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus pumilus in both a direct fed microbial (DFM) product as well as a crop microbial biostimulant (CMB) product. Quantification with FISH after a germination experiment revealed the potential for this approach to be used after application of the product.

A low-cost, accessible, and high-performing Arduino-based seawater pH control system for biological applications.

In the last two decades, the need for seawater pH control methodologies paralleled the rise in attention to the biological impacts of ocean acidification. Many effective and high-performing systems have been created, but they are often expensive, complex, and difficult to establish. We developed a system that is similarly high performing, but at a low cost and with a simple and accessible design. This system is controlled by an Arduino Nano, an open-source electronics platform, which regulates the flow of CO2 gas through electric solenoid valves. The Arduino and other inexpensive materials total ∼$150 (plus CO2 gas and regulator), and a new treatment can be added for less than $35. Easy-to-learn code and simple wire-to-connect hardware make the design extremely accessible, requiring little time and expertise to establish. The system functions with a variety of pH probes and can be adapted to fit a variety of experimental designs and organisms. Using this set up, we were able to constrain seawater pH within a range of 0.07 pH units. Our system thus maintains the performance and adaptability of existing systems but expands their accessibility by reducing cost and complexity.

SSU-rRNA Gene Sequencing Survey of Benthic Microbial Eukaryotes from Guaymas Basin Hydrothermal Vent.

Microbial eukaryotes have important roles in marine food webs, but their diversity and activities in hydrothermal vent ecosystems are poorly characterized. In this study, we analyzed microbial eukaryotic communities associated with bacterial (Beggiatoa) mats in the 2,000 m deep-sea Guaymas Basin hydrothermal vent system using 18S rRNA gene high-throughput sequencing of the V4 region. We detected 6,954 distinct Operational Taxonomic Units (OTUs) across various mat systems. Of the sequences that aligned with known protistan phylotypes, most were affiliated with alveolates (especially dinoflagellates and ciliates) and cercozoans. OTU richness and community structure differed among sediment habitats (e.g. different mat types and cold sediments away from mats). Additionally, full-length 18S rRNA genes amplified and cloned from single cells revealed the identities of some of the most commonly encountered, active ciliates in this hydrothermal vent ecosystem. Observations and experiments were also conducted to demonstrate that ciliates were trophically active and ingesting fluorescent bacteria or Beggiatoa trichomes. Our work suggests that the active and diverse protistan community at the Guaymas Basin hydrothermal vent ecosystem likely consumes substantial amounts of bacterial biomass, and that the different habitats, often defined by distances of just a few 10s of cm, select for particular assemblages and levels of diversity.

Interrogating marine virus-host interactions and elemental transfer with BONCAT and nanoSIMS-based methods.

While the collective impact of marine viruses has become more apparent over the last decade, a deeper understanding of virus-host dynamics and the role of viruses in nutrient cycling would benefit from direct observations at the single-virus level. We describe two new complementary approaches - stable isotope probing coupled with nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorescence-based biorthogonal non-canonical amino acid tagging (BONCAT) - for studying the activity and biogeochemical influence of marine viruses. These tools were developed and tested using several ecologically relevant model systems (Emiliania huxleyi/EhV207, Synechococcus sp. WH8101/Syn1 and Escherichia coli/T7). By resolving carbon and nitrogen enrichment in viral particles, we demonstrate the power of nanoSIMS tracer experiments in obtaining quantitative estimates for the total number of viruses produced directly from a particular production pathway (by isotopically labelling host substrates). Additionally, we show through laboratory experiments and a pilot field study that BONCAT can be used to directly quantify viral production (via epifluorescence microscopy) with minor sample manipulation and no dependency on conversion factors. This technique can also be used to detect newly synthesized viral proteins. Together these tools will help fill critical gaps in our understanding of the biogeochemical impact of viruses in the ocean.

Autotrophic and heterotrophic acquisition of carbon and nitrogen by a mixotrophic chrysophyte established through stable isotope analysis.

Collectively, phagotrophic algae (mixotrophs) form a functional continuum of nutritional modes between autotrophy and heterotrophy, but the specific physiological benefits of mixotrophic nutrition differ among taxa. Ochromonas spp. are ubiquitous chrysophytes that exhibit high nutritional flexibility, although most species generally fall towards the heterotrophic end of the mixotrophy spectrum. We assessed the sources of carbon and nitrogen in Ochromonas sp. strain BG-1 growing mixotrophically via short-term stable isotope probing. An axenic culture was grown in the presence of either heat-killed bacteria enriched with 15N and 13C, or unlabeled heat-killed bacteria and labeled inorganic substrates (13C-bicarbonate and 15N-ammonium). The alga exhibited high growth rates (up to 2 divisions per day) only until heat-killed bacteria were depleted. NanoSIMS and bulk IRMS isotope analyses revealed that Ochromonas obtained 84-99% of its carbon and 88-95% of its nitrogen from consumed bacteria. The chrysophyte assimilated inorganic 13C-carbon and 15N-nitrogen when bacterial abundances were very low, but autotrophic (photosynthetic) activity was insufficient to support net population growth of the alga. Our use of nanoSIMS represents its first application towards the study of a mixotrophic alga, enabling a better understanding and quantitative assessment of carbon and nutrient acquisition by this species.

Phytoplankton production and taxon-specific growth rates in the Costa Rica Dome.

During summer 2010, we investigated phytoplankton production and growth rates at 19 stations in the eastern tropical Pacific, where winds and strong opposing currents generate the Costa Rica Dome (CRD), an open-ocean upwelling feature. Primary production (14C-incorporation) and group-specific growth and net growth rates (two-treatment seawater dilution method) were estimated from samples incubated in situ at eight depths. Our cruise coincided with a mild El Niño event, and only weak upwelling was observed in the CRD. Nevertheless, the highest phytoplankton abundances were found near the dome center. However, mixed-layer growth rates were lowest in the dome center (∼0.5-0.9 day-1), but higher on the edge of the dome (∼0.9-1.0 day-1) and in adjacent coastal waters (0.9-1.3 day-1). We found good agreement between independent methods to estimate growth rates. Mixed-layer growth rates of Prochlorococcus and Synechococcus were largely balanced by mortality, whereas eukaryotic phytoplankton showed positive net growth (∼0.5-0.6 day-1), that is, growth available to support larger (mesozooplankton) consumer biomass. These are the first group-specific phytoplankton rate estimates in this region, and they demonstrate that integrated primary production is high, exceeding 1 g C m-2 day-1 on average, even during a period of reduced upwelling.

Phytoplankton production and grazing balances in the Costa Rica Dome.

We investigated phytoplankton production rates and grazing fates in the Costa Rica Dome (CRD) during summer 2010 based on dilution depth profiles analyzed by flow cytometry and pigments and mesozooplankton grazing assessed by gut fluorescence. Three community production estimates, from 14C uptake (1025 ± 113 mg C m-2 day-1) and from dilution experiments analyzed for total Chla (990 ± 106 mg C m-2 day-1) and flow cytometry populations (862 ± 71 mg C m-2 day-1), exceeded regional ship-based values by 2-3-fold. Picophytoplankton accounted for 56% of community biomass and 39% of production. Production profiles extended deeper for Prochlorococcus (PRO) and picoeukaryotes than for Synechococcus (SYN) and larger eukaryotes, but 93% of total production occurred above 40 m. Microzooplankton consumed all PRO and SYN growth and two-third of total production. Positive net growth of larger eukaryotes in the upper 40 m was balanced by independently measured consumption by mesozooplankton. Among larger eukaryotes, diatoms contributed ∼3% to production. On the basis of this analysis, the CRD region is characterized by high production and grazing turnover, comparable with or higher than estimates for the eastern equatorial Pacific. The region nonetheless displays characteristics atypical of high productivity, such as picophytoplankton dominance and suppressed diatom roles.

Microbial eukaryotic distributions and diversity patterns in a deep-sea methane seep ecosystem.

Although chemosynthetic ecosystems are known to support diverse assemblages of microorganisms, the ecological and environmental factors that structure microbial eukaryotes (heterotrophic protists and fungi) are poorly characterized. In this study, we examined the geographic, geochemical and ecological factors that influence microbial eukaryotic composition and distribution patterns within Hydrate Ridge, a methane seep ecosystem off the coast of Oregon using a combination of high-throughput 18S rRNA tag sequencing, terminal restriction fragment length polymorphism fingerprinting, and cloning and sequencing of full-length 18S rRNA genes. Microbial eukaryotic composition and diversity varied as a function of substrate (carbonate versus sediment), activity (low activity versus active seep sites), sulfide concentration, and region (North versus South Hydrate Ridge). Sulfide concentration was correlated with changes in microbial eukaryotic composition and richness. This work also revealed the influence of oxygen content in the overlying water column and water depth on microbial eukaryotic composition and diversity, and identified distinct patterns from those previously observed for bacteria, archaea and macrofauna in methane seep ecosystems. Characterizing the structure of microbial eukaryotic communities in response to environmental variability is a key step towards understanding if and how microbial eukaryotes influence seep ecosystem structure and function.

Biophysical basis for convergent evolution of two veil-forming microbes.

Microbes living in stagnant water typically rely on chemical diffusion to draw nutrients from their environment. The sulfur-oxidizing bacterium Thiovulum majus and the ciliate Uronemella have independently evolved the ability to form a 'veil', a centimetre-scale mucous sheet on which cells organize to produce a macroscopic flow. This flow pulls nutrients through the community an order of magnitude faster than diffusion. To understand how natural selection led these microbes to evolve this collective behaviour, we connect the physical limitations acting on individual cells to the cell traits. We show how diffusion limitation and viscous dissipation have led individual T. majus and Uronemella cells to display two similar characteristics. Both of these cells exert a force of approximately 40 pN on the water and attach to boundaries by means of a mucous stalk. We show how the diffusion coefficient of oxygen in water and the viscosity of water define the force the cells must exert. We then show how the hydrodynamics of filter-feeding orient a microbe normal to the surface to which it attaches. Finally, we combine these results with new observations of veil formation and a review of veil dynamics to compare the collective dynamics of these microbes. We conclude that this convergent evolution is a reflection of similar physical limitations imposed by diffusion and viscosity acting on individual cells.

Methane Seep Carbonates Host Distinct, Diverse, and Dynamic Microbial Assemblages.

UNLABELLED: Marine methane seeps are globally distributed geologic features in which reduced fluids, including methane, are advected upward from the subsurface. As a result of alkalinity generation during sulfate-coupled methane oxidation, authigenic carbonates form slabs, nodules, and extensive pavements. These carbonates shape the landscape within methane seeps, persist long after methane flux is diminished, and in some cases are incorporated into the geologic record. In this study, microbial assemblages from 134 native and experimental samples across 5,500 km, representing a range of habitat substrates (carbonate nodules and slabs, sediment, bottom water, and wood) and seepage conditions (active and low activity), were analyzed to address two fundamental questions of seep microbial ecology: (i) whether carbonates host distinct microbial assemblages and (ii) how sensitive microbial assemblages are to habitat substrate type and temporal shifts in methane seepage flux. Through massively parallel 16S rRNA gene sequencing and statistical analysis, native carbonates are shown to be reservoirs of distinct and highly diverse seep microbial assemblages. Unique coupled transplantation and colonization experiments on the seafloor demonstrated that carbonate-associated microbial assemblages are resilient to seep quiescence and reactive to seep activation over 13 months. Various rates of response to simulated seep quiescence and activation are observed among similar phylogenies (e.g., Chloroflexi operational taxonomic units) and similar metabolisms (e.g., putative S oxidizers), demonstrating the wide range of microbial sensitivity to changes in seepage flux. These results imply that carbonates do not passively record a time-integrated history of seep microorganisms but rather host distinct, diverse, and dynamic microbial assemblages. IMPORTANCE: Since their discovery in 1984, the global distribution and importance of marine methane seeps have become increasingly clear. Much of our understanding of methane seep microorganisms-from metabolisms to community ecology-has stemmed from detailed studies of seep sediments. However, it has become apparent that carbonates represent a volumetrically significant habitat substrate at methane seeps. Through combined in situ characterization and incubation experiments, this study demonstrates that carbonates host microbial assemblages distinct from and more diverse than those of other seep habitats. This emphasizes the importance of seep carbonates as biodiversity locales. Furthermore, we demonstrate that carbonate-associated microbial assemblages are well adapted to withstand fluctuations in methane seepage, and we gain novel insight into particular taxa that are responsive (or recalcitrant) to changes in seep conditions.