The effect of nutrients on carbon and nitrogen fixation by the UCYN-A-haptophyte symbiosis

Abstract

Symbiotic relationships between phytoplankton and N2-fixing microorganisms play a crucial role in marine ecosystems. The abundant and widespread unicellular cyanobacteria group A (UCYN-A) has recently been found to live symbiotically with a haptophyte. Here, we investigated the effect of nitrogen (N), phosphorus (P), iron (Fe) and Saharan dust additions on nitrogen (N2) fixation and primary production by the UCYN-A-haptophyte association in the subtropical eastern North Atlantic Ocean using nifH expression analysis and stable isotope incubations combined with single-cell measurements. N2 fixation by UCYN-A was stimulated by the addition of Fe and Saharan dust, although this was not reflected in the nifH expression. CO2 fixation by the haptophyte was stimulated by the addition of ammonium nitrate as well as Fe and Saharan dust. Intriguingly, the single-cell analysis using nanometer scale secondary ion mass spectrometry indicates that the increased CO2 fixation by the haptophyte in treatments without added fixed N is likely an indirect result of the positive effect of Fe and/or P on UCYN-A N2 fixation and the transfer of N2-derived N to the haptophyte. Our results reveal a direct linkage between the marine carbon and nitrogen cycles that is fuelled by the atmospheric deposition of dust. The comparison of single-cell rates suggests a tight coupling of nitrogen and carbon transfer that stays balanced even under changing nutrient regimes. However, it appears that the transfer of carbon from the haptophyte to UCYN-A requires a transfer of nitrogen from UCYN-A. This tight coupling indicates an obligate symbiosis of this globally important diazotrophic association.

Figure 1

Mean bulk N₂ fixation rates (a), as well as mean UCYN-A nifH gene and nifH transcript abundance (b) with s.e. from incubation experiments across nutrient treatments as described in the Materials and Methods.

Figure 2

Visualization of the UCYN-A–haptophyte association according to the probe-conferred ¹⁹F signal (left panels a, d, g) and single-cell activities based on isotope ratios of C (¹³C/¹²C=middle panels b, e, h) and N (¹⁵N/¹⁴N=right panels c, f, i) within different nutrient amendment incubation experiments. Inset panels on the left side show the corresponding epifluorescence images of the UCYN-A cells (green signal) and its associated haptophyte cell (red signal), as well as DAPI staining (blue signal) based on double CARD–FISH approach taken before nanoSIMS analysis. NanoSIMS images refer to different nutrient amendment incubation experiments: (a–c) Control=no nutrient added, (d–f) N=NH₄NO₃ addition, (g–i) DII=4 mg l⁻¹ Saharan dust. Warmer colors represent higher abundance of the heavier isotopes.

Figure 3

Mean biovolumes with s.e. for (a) UCYN-A and (b) the associated haptophyte cells in the different treatments as described in the Materials and Methods. The asterisks indicate a statistically significant difference compared with the control. No statistics were performed on results from the PFe treatment because only one UCYN-A–haptophyte association was found. Dashed lines indicate mean values of control measurements.

Figure 4

NanoSIMS measurements for the association between UCYN-A and their associated haptophytes from the nutrient amendment experiments. The panels on the left side represent the isotope enrichment in AT% for individual cells for ¹³C (a, UCYN-A; c, haptophyte) and ¹⁵N (e, UCYN-A; g, haptophyte). The panels on the right side (b, d, f, h) show the corresponding single-cell activity in fmol cell^–1 h^–1 for CO₂ and N₂ fixation, as well as C and N transfer rates for individual UCYN-A and partner haptophyte cells, calculated based on obtained nanoSIMS values (AT%) and cell dimension analysis. Treatments are as described in Materials and Methods. The asterisk symbol indicates means that are significantly different from the control at a P<0.05 significance level. No statistics were performed on the PFe treatment. Dashed lines indicate mean values of control measurements.

Figure 5

Single-cell enrichments and rates for (a, b) carbon in AT% ¹³C and fmol C cell^–1 h^–1, respectively, and for (c, d) nitrogen in AT% ¹⁵N and fmol N cell^–1 h^–1, respectively, within individual associations between UCYN-A and its corresponding haptophyte partner cell across all treatments. Dashed lines represent regression lines and their corresponding r-values are depicted within each panel. Treatments are as described in Materials and Methods.

Figure 6

NanoSIMS measurements visualizing the ‘unknown structure' found attached to a UCYN-A–haptophyte association within the NP treatment. (a) The probe-conferred ¹⁹F signal and the corresponding epifluorescence images of the UCYN-A cells (green signal) and its associated haptophyte cell (red signal), as well as DAPI staining (blue signal) based on double CARD–FISH approach taken before nanoSIMS analysis (small inset panel). (b) Carbon enrichment as ¹³C/¹²C, (c) nitrogen enrichment as ¹⁵N/¹⁴N, (d) black and white image of the DAPI signals, (e) carbon and nitrogen distribution as ¹²C/¹⁴N * 1000 and (f) sulfur as ³²S. An unknown structure attached to the UCYN-A association was observed that is highly enriched in C, but lower in N and had a weak DAPI signal (b, c, d). In addition to carbon and nitrogen (b, c, e) the structure contained sulfur (f). Such a structure was only found when inorganic nitrogen was added. Warmer colors represent higher abundance of the heavier isotopes. Brighter white DAPI signals indicate stronger staining because of more DNA.