The actinorhizal plant Datisca glomerata (Datiscaceae, Cucurbitales) establishes a root nodule symbiosis with actinobacteria from the earliest branching symbiotic Frankia clade. A subfamily of a gene family encoding nodule-specific defensin-like cysteine-rich peptides is highly expressed in D. glomerata nodules. Phylogenetic analysis of the defensin domain showed that these defensin-like peptides share a common evolutionary origin with nodulespecific defensins from actinorhizal Fagales and with nodule-specific cysteine-rich peptides (NCRs) from legumes. In this study, the family member with the highest expression levels, DgDef1, was characterized. Promoter-GUS studies on transgenic hairy roots showed expression in the early stage of differentiation of infected cells, and transient expression in the nodule apex. DgDef1 contains an N-terminal signal peptide and a C-terminal acidic domain which are likely involved in subcellular targeting and do not affect peptide activity. In vitro studies with E. coli and Sinorhizobium meliloti 1021 showed that the defensin domain of DgDef1 has a cytotoxic effect, leading to membrane disruption with 50% lethality for S. meliloti 1021 at 20.8 μM. Analysis of the S. meliloti 1021 transcriptome showed that, at sublethal concentrations, DgDef1 induced the expression of terminal quinol oxidases, which are associated with the oxidative stress response and are also expressed during symbiosis. Overall, the changes induced by DgDef1 are reminiscent of those of some legume NCRs, suggesting that nodule-specific defensin-like peptides were part of the original root nodule toolkit and were subsequently lost in most symbiotic legumes, while being maintained in the actinorhizal lineages. 

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.

Diatom diazotroph associations (DDAs) are a widespread marine planktonic symbiosis between several diatom genera and di-nitrogen (N₂)-fixing bacteria. Combining single cell confocal microscopy observations and molecular genetic approaches on individual field collected cells, we determined the phylogenetic diversity, distribution and evolution of the DDAs. Confocal analyses coupled with 3-D imaging re-evaluated the cellular location of DDA symbionts. DDA diversity was resolved by paired gene sequencing (18S rRNA and rbcL genes, 16S rRNA and nifH genes). A survey using the newly acquired sequences against public databases found sequences with high similarity (99–100%) to either host (18S rRNA) or symbiont (16S rRNA) in atypical regions for DDAs (high latitudes, anoxic basin and copepod gut). Concatenated phylogenies were congruent for the host and cyanobacteria sequences and implied co-evolution. Time-calibrated trees dated the appearance of N₂ fixing planktonic symbiosis from 100–50Mya and were consistent with the symbiont cellular location: symbioses with internal partners are more ancient. An ancestral state reconstruction traced the evolution of traits in DDAs and highlight that the adaptive radiation to the marine environment was likely facilitated by the symbiosis. Our results present the evolutionary nature of DDAs and provide new genetic and phenotypic information for these biogeochemically relevant populations.

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.

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.

Epiphytic diatoms on seaweeds and seagrasses were collected from tropical regions in the western Pacific Ocean (i.e., Siladen Island, Celebes Sea, Indonesia; Guam; Palau). Fifteen species of Diploneis are described in detail with light and scanning electron microscopy including six species new to science, D. cerebrum, D. claustra, D. craticula, D. crispa, D. rimosa and D. weissflogiopsis. Valve structure in Diploneis is particularly complicated, and two new distinctive structures are described for the first time: (1) central external raphe endings with flaps or rolled flaps and (2) internal globular papillae near the marginal ends of the virgae. In addition, we confirm that openings on the inner surface of the longitudinal canals are of taxonomic value. The structural complexities encountered in Diploneis require the study of a great many more taxa before a meaningful revision of the genus can be undertaken. Its diversity in warm waters is a rich resource for new Diploneis taxa to further our understanding of the genus.