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Metatranscriptomics and metagenomics data sets benchmarked with internal standards were used to characterize the expression patterns for biogeochemically relevant bacterial and archaeal genes mediating carbon, nitrogen, phosphorus and sulfur uptake and metabolism through the salinity gradient of the Amazon River Plume. The genes were identified in 48 metatranscriptomic and metagenomic data sets summing to >500 million quality-controlled reads from six locations in the plume ecosystem. The ratio of transcripts per gene copy (a direct measure of expression made possible by internal standard additions) showed that the free-living bacteria and archaea exhibited only small changes in the expression levels of biogeochemically relevant genes through the salinity and nutrient zones of the plume. In contrast, the expression levels of genes in particle-associated cells varied over orders of magnitude among the stations, with the largest differences measured for genes mediating aspects of nitrogen cycling (nifH, amtB and amoA) and phosphorus acquisition (pstC, phoX and phoU). Taxa varied in their baseline gene expression levels and extent of regulation, and most of the spatial variation in the expression level could be attributed to changes in gene regulation after removing the effect of shifting taxonomic composition. We hypothesize that changes in microbial element cycling along the Amazon River Plume are largely driven by shifting activities of particle-associated cells, with most activities peaking in the mesohaline regions where N2 fixation rates are elevated.

As part of the ANACONDAS program, we investigated the role of the Amazon plume in stimulating offshore nitrogen fixation and export production during the river's high-discharge period (May–June 2010). Using the shipboard underway system, we performed high-resolution sampling of over 450,000 km2 of surface waters, characterizing the distribution of nutrients, phytoplankton, particulate organic matter (POM), and stable carbon and nitrogen isotopes of POM in the offshore plume. We found distinct regional variations in diazotroph communities, with the Diatom-Diazotroph Associations (DDA) Hemiaulus hauckii – Richelia intracellularis dominating the low N : P mesohaline waters to the northwest of the plume axis and Trichodesmium spp. primarily occupying oceanic waters to the east. Nutrient availability broadly shaped diazotroph distributions along the salinity gradient, but habitat longevity may also play a role in the finer-scale distributions of communities, particularly of DDAs. H. hauckii and Trichodesmium spp. affected the nitrogen and carbon budgets in fundamentally different ways within the plume-influenced regions, with H. hauckii making much greater contributions to the particulate nitrogen pool and to CO2 drawdown than Trichodesmium spp., leading to much higher export fluxes. Our findings provide an important constraint on the role of the Amazon plume in creating distinct niches and roles for diazotrophs in the nutrient and carbon budgets of the western tropical North Atlantic.

The plasticity of nitrogen specific net growth efficiency (NGE) in marine mesozooplankton is currently unresolved, with discordant lines of evidence suggesting that NGE is constant, or that it varies with nitrogen source, food availability, and food quality in marine ecosystems. Specifically, the fate of nitrogen from nitrogen fixation is poorly known. We use 15N : 14N ratios in plankton in combination with hydrological data, nutrient profiles, and nitrogen fixation rate measurements to investigate the relationship between new nitrogen sources and the nitrogen specific NGE in three plankton communities along the outer Amazon River plume. The NGE of small (200–500 μm) mesozooplankton was estimated from the δ15N differences between particulate nitrogen and zooplankton using an open system Rayleigh fractionation model. The transfer efficiency of nitrogen among larger (> 500 μm) mesozooplankton was estimated from the change in δ15N as a function of zooplankton size. The Amazon River was not a significant source of bioavailable nitrogen anywhere in our study region, and subsurface nitrate was the primary new nitrogen source for the outer shelf community, which was dominated by diatoms. N2 fixation was the principal new nitrogen source at sites of high diatom diazotroph association abundance and at oceanic sites dominated by Trichodesmium spp. and Synechococcus spp. Although we found clear spatial differences in food quantity, food quality, and diazotroph inputs into mesozooplankton, our data show no significant differences in mesozooplankton nitrogen transfer efficiency and NGE (for latter, mean ± SD: 59 ± 10%) among sites.

An Advanced Laser Fluorometer (ALF) capable of discriminating several phytoplankton pigment types was utilized in conjunction with microscopic data to map the distribution of phytoplankton communities in the Amazon River plume in May–June-2010, when discharge from the river was at its peak. Cluster analysis and Non-metric Multi-Dimensional Scaling (NMDS) helped distinguish three distinct biological communities that separated largely on the basis of salinity gradients across the plume. These three communities included an ‘‘estuarine type’’ comprised of a high biomass mixed population of diatoms, cryptophytes and green-water Synechococcus spp. located upstream of the plume, a ‘‘mesohaline type’’ made up largely of communities of Diatom-Diazotroph Associations (DDAs) and located in the northwestern region of the plume and an ‘‘oceanic type’’ in the oligotrophic waters outside of the plume made up of Trichodesmium and Synechococcus spp. Although salinity appeared to have a substantial influence on the distribution of different phytoplankton groups, ALF and microscopic measurements examined in the context of the hydro-chemical environment of the river plume, helped establish that the phytoplankton community structure and distribution were strongly controlled by inorganic nitrate plus nitrite (NO3 +N O 2) availability whose concentrations were low throughout the plume. Towards the southern, low-salinity region of the plume, NO3 +N O 2 supplied by the onshore flow of subsurface (80 m depth) water, ensured the continuous sustenance of the mixed phytoplankton bloom. The large drawdown of SiO3 and PO4 associated with this ‘‘estuarine type’’ mixed bloom at a magnitude comparable to that observed for DDAs in the mesohaline waters, leads us to contend that, diatoms, cryptophytes and Synechococcus spp., fueled by the offshore influx of nutrients also play an important role in the cycling of nutrients in the Amazon River plume.

Interannual variability in the abundances of blue crab (Callinectes sapidus) in populations along the U.S. east coast is well documented, but the mechanisms driving these fluctuations remain poorly understood. Using principal component analysis and dynamic factor analysis we quantified the patterns in variability and the degree of synchrony among blue crab populations along the U.S. east coast to gain insight into the mechanisms regulating the dynamics of these populations. We determined that a latitudinal pattern in the variability in abundance among the states existed and that a combination of the Gulf Stream Index, southern winter temperature, and larval mixing in the coastal ocean may be important drivers for the observed fluctuations of blue crab. The blue crab population in the Chesapeake Bay appeared to be an anomaly in that its abundance did not match the latitudinal trend seen in the other states. Understanding the dynamics of blue crab throughout its range may help managers determine which population responses reflect local dynamics and which may reflect shared, regional responses.

The Amazon River has the largest discharge of all rivers on Earth, and its complex plume system fuels a wide array of biogeochemical processes, across a large area of the western tropical North Atlantic. The plume thus stimulates microbial processes affecting carbon sequestration and nutrient cycles at a global scale. Chromosomal gene expression patterns of the 2.0 to 156 μm size-fraction eukaryotic microbial community were investigated in the Amazon River Plume, generating a robust dataset (more than 100 million mRNA sequences) that depicts the metabolic capabilities and interactions among the eukaryotic microbes. Combining classical oceanographic field measurements with metatranscriptomics yielded characterization of the hydrographic conditions simultaneous with a quantification of transcriptional activity and identity of the community. We highlight the patterns of eukaryotic gene expression for 31 biogeochemically significant gene targets hypothesized to be valuable within forecasting models. An advantage to this targeted approach is that the database of reference sequences used to identify the target genes was selectively constructed and highly curated optimizing taxonomic coverage, throughput, and the accuracy of annotations. A coastal diatom bloom highly expressed nitrate transporters and carbonic anhydrase presumably to support high growth rates and enhance uptake of low levels of dissolved nitrate and CO2. Diatom-diazotroph association (DDA: diatoms with nitrogen fixing symbionts) blooms were common when surface salinity was mesohaline and dissolved nitrate concentrations were below detection, and hence did not show evidence of nitrate utilization, suggesting they relied on ammonium transporters to aquire recently fixed nitrogen. These DDA blooms in the outer plume had rapid turnover of the photosystem D1 protein presumably caused by photodegradation under increased light penetration in clearer waters, and increased expression of silicon transporters as silicon became limiting. Expression of these genes, including carbonic anhydrase and transporters for nitrate and phosphate, were found to reflect the physiological status and biogeochemistry of river plume environments. These relatively stable patterns of eukaryotic transcript abundance occurred over modest spatiotemporal scales, with similarity observed in sample duplicates collected up to 2.45 km in space and 120 minutes in time. These results confirm the use of metatranscriptomics as a valuable tool to understand and predict microbial community function., Gordon and Betty Moore Foundation (River Ocean Continuum of the Amazon Project. Grants GBMF 2293 and 2928), National Science Foundation (U.S.) (Grant NSF-OCE 0934095)

We investigated expression of genes mediating elemental cycling at the microspatial scale in the ocean's largest river plume using, to our knowledge, the first fully quantitative inventory of genes and transcripts. The bacterial and archaeal communities associated with a phytoplankton bloom in Amazon River Plume waters at the outer continental shelf in June 2010 harbored ∼ 1.0 × 10(13) genes and 4.7 × 10(11) transcripts per liter that mapped to several thousand microbial genomes. Genomes from free-living cells were more abundant than those from particle-associated cells, and they generated more transcripts per liter for carbon fixation, heterotrophy, nitrogen and phosphorus uptake, and iron acquisition, although they had lower expression ratios (transcripts ⋅ gene(-1)) overall. Genomes from particle-associated cells contributed more transcripts for sulfur cycling, aromatic compound degradation, and the synthesis of biologically essential vitamins, with an overall twofold up-regulation of expression compared with free-living cells. Quantitatively, gene regulation differences were more important than genome abundance differences in explaining why microenvironment transcriptomes differed. Taxa contributing genomes to both free-living and particle-associated communities had up to 65% of their expressed genes regulated differently between the two, quantifying the extent of transcriptional plasticity in marine microbes in situ. In response to patchiness in carbon, nutrients, and light at the micrometer scale, Amazon Plume microbes regulated the expression of genes relevant to biogeochemical processes at the ecosystem scale.

The nutrient-rich waters of the Amazon River plume (ARP) support dense blooms of diatom-diazotroph assemblages (DDAs) that introduce large quantities of new nitrogen to the planktonic ecosystem and, unlike other nitrogen-fixers, are likely to directly fuel vertical carbon flux. To investigate the factors controlling DDA blooms, we develop a five phytoplankton (cyanobacteria, diatoms, unicellular microbial diazotrophs, DDAs, and Trichodesmium), two zooplankton model and embed it within a 1/6° resolution physical model of the tropical and subtropical Atlantic. The model generates realistic DDA blooms in the ARP and also exhibits basin-wide primary production, nitrogen fixation, and grazing rates consistent with observed values. By following ARP water parcels with synthetic Lagrangian drifters released at the river mouth we are able to assess the relative impacts of grazing, nutrient supply, and physical forcing on DDA bloom formation. DDA bloom formation is stimulated in the nitrogen-poor and silica-rich water of the ARP by decreases in grazing pressure when mesozooplankton (which co-occur in high densities with coastal diatom blooms) concentrations decrease. Bloom termination is driven primarily by silica limitation of the DDAs. In agreement with in situ data, this net growth niche for DDAs exists in a salinity range from ∼20–34 PSU, although this co-occurrence is coincidental rather than causative. Because net growth rates are relatively modest, bloom formation in ARP water parcels depends critically on the time spent in this ideal habitat, with high DDA biomass only occurring when water parcels spent >23 days in the optimal habitat niche.

[1] Offshore tropical river plumes are associated with areas of high N2 fixation (diazotrophy) and biological carbon drawdown. Episodic blooms of the diatom Hemiaulus hauckii and its diazotrophic cyanobacterial symbiont Richelia intracellularisare believed to dominate that carbon drawdown, but the mechanism is not well understood. We report primary productivity associated with blooms of these diatom-diazotroph assemblages (DDAs) in the offshore plume of the Amazon River using simultaneous measurements of O2/Ar ratios and the triple-isotope composition of dissolved O2. In these blooms, we observe peaks in net community productivity, but relatively small changes in gross primary productivity, suggesting that DDA blooms increase the ecosystem carbon export ratio more than twofold. These events of enhanced export efficiency lead to biological uptake of dissolved inorganic carbon and silicate, whose longer mixed-layer residence times otherwise obscure the differential impact of DDAs. The shorter-term rate estimates presented here are consistent with the results derived from longer-term geochemical tracers, confirming that DDAs drive a significant biological CO2 pump in tropical oceans.

[1] Climatological satellite observations in the tropical North Atlantic generally show a wintertime surface chlorophyll-a (Chl-a) maximum except over a broad region in the western North Atlantic that has a summer Chl-a maximum. This region also shows decoupling between Chl-a and vertical nutrient flux, based on the positive relationship between sea surface height anomaly (SSH), sea surface temperature, and Chl-a. An analogous summer Chl-a maximum is simulated in a model including a dynamic representation of Trichodesmium and N2-fixation, but not in runs without. These results suggest that the growth is fuelled by N2-fixation. Using the observed summertime increase in Chl-a and the model efficiency for N2-fixation transfer to phytoplankton biomass, we calculate a nitrogen fixation rate of 220 μmol N m−2day−1 in this region. This constitutes the first satellite observation of the effect of nitrogen fixation on Chl-a, and may ultimately provide a means of deriving new global N2-fixation estimates.

[1] In this paper we use a biological-physical model with an explicit representation of Trichodesmium to examine the influence of N2 fixation in the Atlantic. Three solutions are examined, one where the N2 fixation rate has been set to observed levels, one where the rate has been increased to levels comparable to geochemical estimates, and one with no N2 fixation. All solutions are tuned to reproduce satellite surface chlorophyll concentrations, so that differences in the runs are manifested in productivity and export. Model runs with N2 fixation have different phytoplankton production and export distributions than runs without. Over the Atlantic basin the ecosystem “fixes” nitrogen at the rate of 1.47 × 1012 mol N yr−1, when tuned to observed phytoplankton and Trichodesmium biomass. This rate is comparable to the lower range of direct estimates of 1.3–2.2 × 1012mol N yr−1 [Capone et al., 1997; J. N. Galloway et al., manuscript in preparation, 2003; D. Capone et al., New nitrogen input in the tropical North Atlantic Ocean by nitrogen fixation, submitted to Nature, 2004, hereinafter referred to as Capone et al., submitted manuscript, 2004] but less than geochemical indirect estimates over a reduced domain (2.0 × 1012 mol N yr−1 [Gruber and Sarmiento, 1997] versus 0.55 × 1012 mol N yr−1 for the model). The nitrogen from N2 fixation increases new production by 30% and total production by 5%. However, it does not supplement upwelled nitrate sufficiently to bring production and export into line with remote sensing and geochemically derived estimates. Simulations with N2 fixation rates comparable to geochemical estimates show that reasonable phytoplankton concentrations can be maintained if export is increased. Moreover, phytoplankton productivity increases to values approaching remote-sensing-based estimates in the oligotrophic ocean. However, Trichodesmium biomass may be higher than observed.

[1] In this paper we use a coupled, 3-dimensional, biological-physical model, which includes an explicit, dynamic representation of Trichodesmium, to predict the distribution of Trichodesmium and rates of N2-fixation in the tropical and subtropical Atlantic Ocean. It is shown that the model reproduces the approximate observed meridional distribution of Trichodesmium in the Atlantic and elevated concentrations in specific coastal and open ocean regions where this organism is known to occur. The model also appears to reproduce the observed seasonality of Trichodesmium populations at higher latitudes (highest concentrations in summer and fall), but this seasonal cycle may be too pronounced at low latitudes. High and persistent Trichodesmium concentrations and rates of N2-fixation are generated by the model in the Gulf of Guinea off of Africa. This unexpected finding appears to be confirmed by historical measurements. In general, increased Trichodesmium concentrations develop in regions where the mixed layer is relatively thin (resulting in high mean light levels) and dissolved inorganic nitrogen (DIN) concentrations and phytoplankton biomass are low for extended periods of time. The model-predicted Trichodesmium distributions are therefore very sensitive to the fidelity of the physical model's representation of mixed layer depth variability, and upwelling intensity, and the biological model's estimated DIN and phytoplankton concentrations. The model generates a three-step successional sequence where (1) high DIN concentrations due to upwelling and/or mixing stimulate phytoplankton growth, followed by (2) Trichodesmium growth after DIN depletion and phytoplankton decline, followed by (3) enhanced phytoplankton growth due to new nitrogen inputs from N2-fixation. This sequence develops in response to seasonal variations in mixing in the southwestern North Atlantic and in response to upwelling along the coast of Africa and the equator. We interpret this sequence as representing a diatom- Trichodesmium-flagellate succession, which is consistent with observed species successions off of northwest Africa and in the Gulf of Mexico. The results presented in this paper lead us to conclude that our model includes the primary factors that dictate when and where Trichodesmium and N2-fixation occurs in the Atlantic. Moreover, it appears that our model reproduces some of the major effects that diazotrophically-derived inputs of new nitrogen have on the pelagic ecosystem.