Nitrogen fixation and transfer in open ocean diatom-cyanobacterial symbioses

Abstract

Many diatoms that inhabit low-nutrient waters of the open ocean live in close association with cyanobacteria. Some of these associations are believed to be mutualistic, where N(2)-fixing cyanobacterial symbionts provide N for the diatoms. Rates of N(2) fixation by symbiotic cyanobacteria and the N transfer to their diatom partners were measured using a high-resolution nanometer scale secondary ion mass spectrometry approach in natural populations. Cell-specific rates of N(2) fixation (1.15-71.5 fmol N per cell h(-1)) were similar amongst the symbioses and rapid transfer (within 30 min) of fixed N was also measured. Similar growth rates for the diatoms and their symbionts were determined and the symbiotic growth rates were higher than those estimated for free-living cells. The N(2) fixation rates estimated for Richelia and Calothrix symbionts were 171-420 times higher when the cells were symbiotic compared with the rates estimated for the cells living freely. When combined, the latter two results suggest that the diatom partners influence the growth and metabolism of their cyanobacterial symbionts. We estimated that Richelia fix 81-744% more N than needed for their own growth and up to 97.3% of the fixed N is transferred to the diatom partners. This study provides new information on the mechanisms controlling N input into the open ocean by symbiotic microorganisms, which are widespread and important for oceanic primary production. Further, this is the first demonstration of N transfer from an N(2) fixer to a unicellular partner. These symbioses are important models for molecular regulation and nutrient exchange in symbiotic systems.

Figure 1

Blue light excitation (450–490 nm) images of field collected diatom–cyanobacteria symbioses. The diatom frustules are not easily seen under epi-fluorescence microscopy (except in d), however, the excitation patterns of the cyanobacterial symbionts are clearly different and yellow/orange from their diatom partners (red). (a) Two Hemiaulus membranaceus diatoms with two Richelia intracellularis associated to each diatom. The chlorophyll a within the chloroplast (c) of the diatom fluoresces red, whereas the pigments in the vegetative (v) cells and the terminal heterocyst (h) of Richelia fluoresce yellow–orange. (b) The apical end of a Rhizosolenia clevei diatom with two associated trichomes of R. intracellularis (c) A chain of Chaetoceros spp. diatoms with Calothrix rhizosoleniae attached to the spines (not visible). (d) A chain of Climacodium frauenfeldianum diatoms associated with yellow-fluorescing unicellular cyanobacteria (cyanobionts).

Figure 2

The images of ¹⁵N/¹⁴N ratios are shown for symbiont-containing Hemiaulus. The ¹⁵N/¹⁴N ratio is shown for Hemiaulus–Richelia symbioses at time 0 (a), 30 min (b) and 48 h (c). Inset panels a–c are the epi-fluorescent images taken before the nanoSIMS analyses. The numbers and markings in the figure define regions of interest, which were used for calculating ¹⁵N/¹⁴N ratios. Scale bars are 5 μm.

Figure 3

The images of ¹⁵N/¹⁴N ratios are shown for Chaetoceros-Calothrix and the Climacodium-cyanobiont symbioses. The ¹⁵N/¹⁴N ratio is shown in A and C for Chaetoceros–Calothrix and the Climacodium–Crocosphaera symbioses, respectively. Note the ‘hotspots' (white arrows) of enrichment within the individual cyanobiont (Crocosphaera) cells of Climacodium (c). The corresponding total ion content images images for the same symbioses in a and c are shown in b and d, respectively. The numbers and markings in the Figure define regions of interest, which were used for calculating ¹⁵N/¹⁴N ratios. Scale bars are 5 μm.

Figure 4

Summary of results from nanoSIMS analysis. (a) The atom % of the ¹⁵N/¹⁴N ratios for ROIs of individual Hemiaulus–Richelia are shown as a function of incubation time in both long and short-term experiments. (b) The atom % of the ¹⁵N/¹⁴N ratios for ROIs of individual Hemiaulus–Richelia symbioses from the short-term experiments and represents the values within the red box shown in a. Note that the Richelia enrichment values are given as ratios estimated in the heterocyst and the vegetative cells. The dashed red line indicates the value for a co-occurring non-symbiotic diatom.