Different Elemental Compositions and Potential Functions of Vacuoles in Bolivina spissa (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.