Di-nitrogen (N₂) fixers, also called diazotrophs, are able to reduce atmospheric N₂ into bioavailable nitrogen, giving them an advantage in open ocean regions with low dissolved inorganic nitrogen concentrations. The focus of this thesis are three lineages of symbiotic heterocystous filamentous types (het-1, het-2 and het-3), that associate with several genera of microalgae called diatoms (collectively referred to as Diatom Diazotroph Associations, DDAs). Other major cyanobacterial diazotrophs in the ocean are the filamentous Trichodesmium spp., and the unicellular UCYN-A, UCYN-B and UCYN-C. Although widespread in the tropics and subtropics, and first described in the early 20th century, the DDAs are an understudied group of diazotrophs. Hence, our knowledge of their distribution, abundance, activity, and how these are constrained by the environment is limited.

Initially we investigated the abundances and distributions of eight cyanobacterial diazotrophs, and two proposed micro-algal hosts of UCYN-A1 and A2, in the western tropical south Pacific (WTSP), using quantitative polymerase chain reaction (qPCR). Trichodesmium spp. was the most abundant diazotroph and het-1 was the most abundant DDA symbiont. Using correlation analysis a distinct vertical separation was observed between UCYN-A and the other diazotrophs (Trichodesmium spp., UCYN-B and DDA symbionts). The most influential environmental parameter on the diazotroph abundances in the WTSP was temperature, and in order to investigate this further we compiled qPCR data from 11 publicly available datasets from four ocean basins. Using a weighted meta-analysis we found that temperature was a robust factor governing the diazotroph abundances (except for UCYN-A) across ocean basins.

Attempting to identify differences in environmental impacts on two of the DDA symbiont strains (het-1 and het-2), we applied a new statistical tool called piecewise structural equation model, on qPCR abundance data from the western tropical North Atlantic. We saw that the two strains had a direct positive interaction between each other, but two parameters (salinity and dissolved inorganic phosphorous) differed. Based on a direct positive effect of salinity on het-1, and an indirect negative effect of salinity on het-2, we concluded that het-2 prefers intermediate salinities (30-35 PSU), which is consistent with where observations of het-2 blooms have been made.

Although DDA and UCYN-A symbionts both are major contributors of new N, and are symbiotic, they have several unique differences. The host partners differ in phylogeny (diatom vs prymnesiophyte), size (80-250 vs 7-10 µm) and the symbiotic life history (colonial vs solitary). The larger, colonial nature of DDAs make them difficult to collect, and hence they are often under-sampled and undetected. In fact, after reviewing 46 qPCR studies we found that < 30% of the studies (13 out of 46) quantified all three DDA symbionts, compared to UCYN-A (96%, 44 out of 46).

In order to study the DDA symbiont gene expressions we developed a highly specific DDA symbiont microarray (748 probes), which was applied on samples collected in the South China Sea. Although the gene expression levels were highly variable, we observed an upregulation of the nifH gene (for N₂ fixation) in the night. Investigating environmental impact on overall gene expression levels, we found that fluorescence, temperature and salinity was most important. Temperature and salinity also constrained abundances, but fluorescence could be seen as a proxy for either other phytoplankton or light availability, suggesting that daylight and host influence DDA symbiont gene expression levels.

The results of this thesis broaden our understanding of the DDAs and how their ambient environment influences them. It has also opened up new possibilities for in depth analysis of these complex environmental impacts. Lastly, it has provided new analysis tools for further development on how the symbionts and hosts potentially impact each other’s activities.

Some N₂-fixing cyanobacteria form symbiosis with diverse protists. In the plankton two groups of diazotrophic symbioses are described: (1) a collective group of diatoms which associate with heterocystous cyanobacteria (Diatom Diazotroph Associations, DDA), and (2) the microalgal prymnesiophyte Braarudosphaera bigelowii and its relatives which associate with the unicellular cyanobacterium Candidatus Atelocyanobacterium thalassa (hereafter as UCYN-A). Both symbiotic systems co-occur, and in both partnerships the symbionts function as a nitrogen (N) source. In this perspective, we provide a brief comparison between the DDAs and the prymnesiophyte-UCYN-A symbioses highlighting similarities and differences in both systems, and present a bias in the attention and current methodology that has led to an under-detection and under-estimation of the DDAs.

The abundance and distribution of cyanobacterial diazotrophs were quantified in two regions (Melanesian archipelago, MA; and subtropical gyre, SG) of the western tropical South Pacific using nifH quantitative polymerase chain reaction (qPCR) assays. UCYN-A1 and A2 host populations were quantified using 18S rRNA qPCR assays including one newly developed assay. All phylotypes were detected in the upper photic zone (0-50 m), with higher abundances in the MA region. Trichodesmium and UCYN-B dominated and ranged from 2.18 x 10(2) to 9.41 x 10(6) and 1.10 x 10(2) to 2.78 x 10(6) nifH copies L-1, respectively. Het-1 (symbiont of Rhizosolenia diatoms) was the next most abundant (1.40 x 10(1)-1.74 x 10(5) nifH copies L-1) and co-occurred with het-2 and het-3. UCYN-A1 and A2 were the least abundant diazotrophs and were below detection (bd) in 63 and 79, respectively, of 120 samples. In addition, in up to 39% of samples in which UCYN-A1 and A2 were detected, their respective hosts were bd. Pairwise comparisons of the nifH abundances and various environmental parameters supported two groups: a deep-dwelling group (45 m) comprised of UCYN-A1 and A2 and a surface group (0-15 m) comprised of Trichodesmium, het-1 and het-2. Temperature and photosynthetically active radiation were positively correlated with the surface group, while UCYN-A1 and A2 were positively correlated with depth, salinity, and oxygen. Similarly, in a meta-analysis of 11 external datasets, all diazotrophs, except UCYN-A were correlated with temperature. Combined, our results indicate that conditions favoring the UCYN-A symbiosis differ from those of diatom diazotroph associations and free-living cyanobacterial diazotrophs.

Here we report N-2 fixation rates from a similar to 4000 km transect in the western and central tropical South Pacific, a particularly undersampled region in the world ocean. Water samples were collected in the euphotic layer along a west to east transect from 160 degrees E to 160 degrees W that covered contrasting trophic regimes, from oligotrophy in the Melanesian archipelago (MA) waters to ultraoligotrophy in the South Pacific Gyre (GY) waters. N-2 fixation was detected at all 17 sampled stations with an average depth-integrated rate of 631 +/- 286 mu mol Nm(-2) d(-1) (range 196-1153 mu mol Nm(-2) d(-1)) in MA waters and of 85 +/- 79 mu mol Nm(-2) d(-1) (range 18-172 mu mol Nm(-2) d(-1)) in GY waters. Two cyanobacteria, the larger colonial filamentous Trichodesmium and the smaller UCYN-B, dominated the enumerated diazotroph community (>80 %) and gene expression of the nifH gene (cDNA > 10(5) nifH copies L-1) in MA waters. Single-cell isotopic analyses performed by nanoscale secondary ion mass spectrometry (nanoSIMS) at selected stations revealed that Trichodesmium was always the major contributor to N-2 fixation in MA waters, accounting for 47.1-83.8% of bulk N-2 fixation. The most plausible environmental factors explaining such exceptionally high rates of N-2 fixation in MA waters are discussed in detail, emphasizing the role of macro- and micro-nutrient (e.g., iron) availability, seawater temperature and currents.

The fate of diazotroph (N-2 fixers) derived carbon (C) and nitrogen (N) and their contribution to vertical export of C and N in the western tropical South Pacific Ocean was studied during OUTPACE (Oligotrophy to UlTra-oligotrophy PACific Experiment). Our specific objective during OUTPACE was to determine whether autocatalytic programmed cell death (PCD), occurring in some diazotrophs, is an important mechanism affecting diazotroph mortality and a factor regulating the vertical flux of organic matter and, thus, the fate of the blooms. We sampled at three long duration (LD) stations of 5 days each (LDA, LDB and LDC) where drifting sediment traps were deployed at 150, 325 and 500m depth. LDA and LDB were characterized by high chlorophyll a (Chl a) concentrations (0.2-0.6 mu g L-1) and dominated by dense biomass of the filamentous cyanobacterium Trichodesmium as well as UCYN-B and diatom-diazotroph associations (Rhizosolenia with Richelia-detected by microscopy and het-1 nifH copies). Station LDC was located at an ultra-oligotrophic area of the South Pacific gyre with extremely low Chl a concentration (similar to 0.02 mu g L-1) with limited biomass of diazotrophs predominantly the unicellular UCYN-B. Our measurements of biomass from LDA and LDB yielded high activities of caspase-like and metacaspase proteases that are indicative of PCD in Trichodesmium and other phytoplankton. Metacaspase activity, reported here for the first time from oceanic populations, was highest at the surface of both LDA and LDB, where we also obtained high concentrations of transparent exopolymeric particles (TEP). TEP were negatively correlated with dissolved inorganic phosphorus and positively coupled to both the dissolved and particulate organic carbon pools. Our results reflect the increase in TEP production under nutrient stress and its role as a source of sticky carbon facilitating aggregation and rapid vertical sinking. Evidence for bloom decline was observed at both LDA and LDB. However, the physiological status and rates of decline of the blooms differed between the stations, influencing the amount of accumulated diazotrophic organic matter and mass flux observed in the traps during our experimental time frame. At LDA sediment traps contained the greatest export of particulate matter and significant numbers of both intact and decaying Trichodesmium, UCYN-B and het-1 compared to LDB where the bloom decline began only 2 days prior to leaving the station and to LDC where no evidence for bloom or bloom decline was seen. Substantiating previous findings from laboratory cultures linking PCD to carbon export in Trichodesmium, our results from OUTPACE indicate that nutrient limitation may induce PCD in high biomass blooms such as displayed by Trichodesmium or diatom-diazotroph associations. Furthermore, PCD combined with high TEP production will tend to facilitate cellular aggregation and bloom termination and will expedite vertical flux to depth.

Di-nitrogen (N₂) fixation plays a crucial role in oceanic carbon and nitrogen cycles and is important for marine biogeochemistry on regional and global scales. N₂-fixing (diazotrophs) cyanobacteria are considered to contribute the most to marine nitrogen fixation, and thereby fuel the surrounding phytoplankton communities with bioavailable ammonia. Distribution and activity of phytoplankton, including diazotrophs, are largely driven by environmental conditions and temperature is often considered the main influence. A greater understanding of the environmental conditions that govern the diazotrophs, is vital to accurate predictions and estimations for the marine nitrogen budget, as well as understanding their impact on the nitrogen and carbon cycles.

Therefore, the primary aim of this thesis was to determine abundances and distribution patterns for various cyanobacterial diazotrophs in two different regions that in spite of their hydrological differences are prime regions for diazotrophy. Moreover, we attempted to identify the environmental conditions that govern cyanobacterial diazotrophs abundance.

In the first study we observed a clear separation of the unicellular diazotroph UCYN-A from the other diazotrophs, including the other unicellular types (UCYN-B, UCYN-C) in the Western Tropical South Pacific. The main driver of the vertical distribution of diazotrophs was based on a temperature-depth gradient, which was similar to the findings of our meta-analysis which included 11 additional datasets. Using newly and previously designed primers and probe sets for the UCYN-A1 and A2 hosts, we also observed discrepancies in detection and abundance for the two UCYN-A symbiotic strains (A1 and A2) and their respective hosts, which is in contrast to the current understanding of a highly specific and obligate partnership. Lastly, cross-hybridization tests revealed that the qPCR assay targeting the UCYN-A2 strain also enumerated UCYN-A1, demonstrating the difficulty in quantifying closely related strains.

In the second study we distinguished the environmental conditions favoring two closely related heterocystous cyanobacterial strains of Richelia intracellularis (het-1 and het-2) which associate with two different diatom hosts (Rhizosolenia and Hemiaulus, respectively) in the Western Tropical North Atlantic (WTNA). In general, the Amazon River (AR) plume heavily influenced het-1 and het-2 abundances; higher densities were quantified at stations with mesohaline sea surface salinities and maximum abundances were detected in the sub-surface, below the freshwater discharge. However, maximum abundances at oceanic sea surface salinity stations were observed nearer to the surface. A piecewise Structural Equation Model (SEM) was developed and identified turbidity as an important factor governing het-1 and het-2 abundance. In addition, het-1 and het-2 distribution pattern was influenced by dissolved inorganic phosphorus (DIP) concentration and salinity. Het-1 tended to penetrate deeper waters and was favored by increased salinity, while the opposite was true for het-2.

The results of this thesis contribute to a better understanding of marine cyanobacterial diazotrophs’ ecological niches and will prove useful in future predictions and research on nitrogen fixation, and the role of cyanobacterial diazotrophs in marine biogeochemistry.

Diatom diazotroph associations (DDAs) are important components in the world's oceans, especially in the western tropical north Atlantic (WTNA), where blooms have a significant impact on carbon and nitrogen cycling. However, drivers of their abundances and distribution patterns remain unknown. Here, we examined abundance and distribution patterns for two DDA populations in relation to the Amazon River (AR) plume in the WTNA. Quantitative PCR assays, targeting two DDAs (het-1 and het-2) by their symbiont's nifH gene, served as input in a piecewise structural equation model (SEM). Collections were made during high (spring 2010) and low (fall 2011) flow discharges of the AR. The distributions of dissolved nutrients, chlorophyll-a, and DDAs showed coherent patterns indicative of areas influenced by the AR. A symbiotic Hemiaulus hauckii-Richelia (het-2) bloom (> 10(6) cells L-1) occurred during higher discharge of the AR and was coincident with mesohaline to oceanic (30-35) sea surface salinities (SSS), and regions devoid of dissolved inorganic nitrogen (DIN), low concentrations of both DIP (> 0.1 mu mol L-1) and Si (> 1.0 mu mol L-1). The Richelia (het-1) associated with Rhizosolenia was only present in 2010 and at lower densities (10-1.76 x 10(5) nifH copies L-1) than het-2 and limited to regions of oceanic SSS (> 36). The het-2 symbiont detected in 2011 was associated with H. membranaceus (> 10(3) nifH copies L-1) and were restricted to regions with mesohaline SSS (31.8-34.3), immeasurable DIN, moderate DIP (0.1-0.60 mu mol L-1) and higher Si (4.19-22.1 mu mol L-1). The piecewise SEM identified a profound direct negative effect of turbidity on the het-2 abundance in spring 2010, while DIP and water turbidity had a more positive influence in fall 2011, corroborating our observations of DDAs at subsurface maximas. We also found a striking difference in the influence of salinity on DDA symbionts suggesting a niche differentiation and preferences in oceanic and mesohaline salinities by het-1 and het-2, respectively. The use of the piecewise SEM to disentangle the complex and concomitant hydrography of the WTNA acting on two biogeochemically relevant populations was novel and underscores its use to predict conditions favoring abundance and distributions of microbial populations.

Three heterocystous cyanobacterial strains (two Richelia intracellularis: RintHH01, RintRC01, Calothrix rhizosoleniae: CalSC01) are known to form highly specific, intimate associations with several lineages of diatoms. Collectively these symbioses are unique since the cellular location varies from internal, partially integrated, and fully external; hence the immediate environment of the symbiont differs. We investigated the environmental gene expression levels of the three strains using a targeted mRNA microarray comprised of 748 genes on environmental samples collected in the South China Sea. Approximately half of the total genes (47%, 354 of the 748 genes) were expressed above background. The most highly transcribed genes were involved in photosynthesis, N₂ fixation and potassium homeostasis, and strain differences were identifiable. The most important environmental parameters on the symbiont gene expression levels were fluorescence, temperature and beam transmission. However, salinity and oxygen impacted gene expression levels of one symbiont strain, RintHH01 differently (positively), possibly due to its different cellular location. Our results suggest that differences in gene expression patterns and environmental conditions influence the three closely related symbiont strains, and are likely related to their cellular location in their respective host diatoms.