Symbioses between eukaryotes and nitrogen (N₂)-fixing cyanobacteria (or diazotrophs) are quite common in the plankton community. A few genera of diatoms (Bacillariophyceae) such as Rhizosolenia, Hemiaulus and Chaetoceros are well known to form symbioses with the heterocystous diazotrophic cyanobacteria Richelia intracellularis and Calothrix rhizosoleniae. The latter are also called diatom-diazotroph associations, or DDAs. Up to now, the prokaryotic partners have been morphologically and genetically characterized, and the phylogenetic reconstruction of the well conserved nifH gene (encodes for the nitrogenase enzyme) placed the symbionts in 3 clusters based on their host-specificity, i.e. het-1 (Rhizosolenia-R. intracellularis), het-2 (Hemiaulus-R. intracellularis), and het-3 (Chaetoceros-C- rhizosoleniae). Conversely, the diatom-hosts, major representative of the phytoplankton community and crucial contributors to the carbon (C) biogeochemical cycle, have been understudied.

The first aim of this thesis was to genetically and morphologically characterize the diatom-hosts, and to reconstruct the evolutionary background of the partnerships and the symbiont integration in the host. The molecular-clock analysis reconstruction showed the ancient appearance of the DDAs, and the traits characterizing the ancestors. In addition, diatom-hosts bearing internal symbionts (with more eroded draft genomes) appeared earlier than diatom-hosts with external symbionts. Finally a blast survey highlighted a broader distribution of the DDAs than expected.

The second aim of this thesis was to compare genetic and physiological characteristics of the DDAs symbionts with the other eukaryote-diazotroph symbiosis, i.e. prymnesiophyte-UCYN-A (or Candidatus Atelocyanobacterium thalassa). The genome comparison highlighted more genes for transporters in het-3 (external symbiont) and in the UCYN-A based symbiosis, suggesting that symbiont location might be relevant also for metabolic exchanges and interactions with the host and/or environment. Moreover, a summary of methodological biases that brought to an underestimation of the DDAs is reported.

The third aim of this thesis was to determine the distribution of the DDAs in the South Pacific Ocean using a quantitative polymerase chain reaction (qPCR) approach and to outline the environmental drivers of such distribution. Among the het-groups, het-1 was the most abundant/detected and co-occurred with the other 2 symbiotic strains, all responding similarly to the influence of abiotic factors, such as temperature and salinity (positive and negative correlation, respectively). Globally, Trichodesmium dominated the qPCR detections, followed by UCYN-B. UCYN-A phylotypes (A-1, A-2) were detected without their proposed hosts, for which new oligonucleotides were designed. The latter suggested a facultative symbiosis. Finally, microscopy observations of the het-groups highlighted a discrepancy with the qPCR counts (i.e. the former were several order of magnitudes lower), leading to the idea of developing a new approach to quantify the DDAs.  

The fourth aim of this thesis was to develop highly specific in situ hybridization assays (CARD-FISH) to determine the presence of alternative life-stages and/or free-living partners. The new assays were applied to samples collected in the South China Sea and compared with abundance estimates from qPCR assays for the 3 symbiotic strains. Free-living cells were indeed detected along the transect, mainly at deeper depths. Free-living symbionts had two morphotypes: trichomes and single-cells. The latter were interpreted as temporary life-stages. Consistent co-occurrence of the 3 het-groups was also found in the SCS and application of a SEM model predicted positive interactions between the het groups. We interpreted the positive interaction as absence of intra-specific competition, and consistent with the previous study, temperature and salinity were predicted as major drivers of the DDAs distribution.

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.