Subsamples (95 ml) taken from the incubation bottles were fixed with sterile-filtered formaldehyde (MilliporeSigma) at a final concentration of 1.85% (v/v) for >1 h at 4 °C, then concentrated with 0.6 µm pore-size polycarbonate filters (MilliporeSigma) under gentle vacuum, air-dried and stored at −80 °C. UCYN-A1 and UCYN-A2 symbioses were targeted using 5′-horseradish peroxidase-labeled oligonucleotide probes (Biomers, Inc., Ulm/Donau, Germany), using helper and competitor probes for both symbionts and hosts (Biomers). Protocols for CARD-FISH hybridizations followed procedures described in detail by Cabello et al. 2016.
Samples were visualized, transferred, and mapped to facilitate nanoSIMS analyses according to protocols detailed in Mills et al. 2020. Individual symbioses were analyzed on a Cameca nanoSIMS 50 L at the Stanford Nano Shared Facilities (Stanford, CA). Once targets were located using the charged-coupled device camera and the secondary electron image, image fields were rastered with a 16 keV Cesium primary ion beam (~5 pA) focused into ca. 120 nm spot diameter (256 × 256 pixels, dwell time 1 ms per pixel). Images of 12C−, 13C−, 12C14N−and 12C15N− were measured over 30–100 planes with a mass resolving power of ca. 8000. Regions of interest were defined around UCYN-A and host cells using Look@nanoSIMS. Isotope ratios of UCYN-A and haptophyte cells were calculated as described in Mills et al. 2020.
Methodology described in depth in Turk-Kubo et al. (2021)
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