Different Elemental Compositions and Potential Functions of Vacuoles in <i>Bolivina spissa</i> (Foraminifera, Rhizaria) Based on Cryo‐SEM‐EDS Analyses

Benthic Foraminifera exhibit diverse adaptations to low oxygen (O₂) environments, including denitrification, a rare trait among eukaryotes. Denitrifying species store intracellular nitrate (NO₃ ^-), possibly within vacuoles, and contribute significantly to the global marine nitrogen (N) cycle. Additionally, widespread phosphate (PO₄ ^3-) accumulation suggests a role in supporting metabolism under O₂-depleted conditions. However, the organelles storing NO₃ ^- and PO₄ ^3- remain unknown, limiting the mechanistic understanding of these alternative metabolic pathways. To investigate the intracellular NO₃ ^- and PO₄ ^3- localization in the benthic foraminifera Bolivina spissa, experimental incubations under varying O₂ and NO₃ ^- conditions followed by cryogenic fixation and scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) analyses were carried out. Most vacuoles were enriched in N relative to the surrounding cytoplasm, likely representing the intracellular NO₃ ^- reservoir. The elemental mapping also confirmed phosphorus (P) enrichment in organelles resembling acidocalcisomes, likely as PO₄ ^3-, which may serve as a readily available energy source used over NO₃ ^- storage during the transition between aerobic and anaerobic respiration. Additionally, barium-rich vacuoles of unknown function(s) display a unique spatial distribution. This study emphasizes the effectiveness of cryogenic techniques in elucidating metabolic processes in foraminifers and other large and/or testate unicellular organisms, particularly for studying soluble compounds that have rarely been investigated.