Biomineralization pathways in calcifying dinoflagellates: Uptake, storage in MgCaP-rich bodies and formation of the shell

Abstract

Little is known about shell formation of calcareous dinoflagellates, despite the fact that they are one of the major calcifying organisms of the phytoplankton. Here, calcitic cyst formation in two representative members of calcareous dinoflagellates is investigated using cryo-electron microscopy (cryo-SEM and cryo-FIB-SEM) in combination with micro-Raman and infrared spectroscopy. Only calcein-AM and not calcein enters these cells, indicating active uptake of calcium and other divalent cations. Multifunctional vacuoles containing crystalline inclusions are observed, and the crystals are identified as anhydrous guanine in the β-form. The same vacuolar enclosures contain dense magnesium-, calcium-, and phosphorous-rich mineral bodies. These bodies are presumably secreted into the outer matrix where calcite forms. Calcite formation occurs via multiple independent nucleation events, and the different crystals grow with preferred orientation into a dense reticular network that forms the mature calcitic shell. We suggest a biomineralization pathway for calcareous dinoflagellates that includes (1) active uptake of calcium through the membranes, (2) deposition of Mg²⁺- and Ca²⁺-ions inside disordered MgCaP-rich mineral bodies, (3) secretion of these bodies to the inter-membrane space, and (4) Formation and growth of calcite into a dense reticulate network. This study provides new insights into calcium uptake, storage and transport in calcifying dinoflagellates. STATEMENT OF SIGNIFICANCE: Little is known about the shell formation of calcareous dinoflagellates, despite the fact that they are one of the major calcifying organisms of the phytoplankton. We used state-of-the-art cryo-electron microscopy (cryo-SEM and cryo-FIB-SEM) in combination with micro-Raman spectroscopy to provide new insights into mineral formation in calcifying dinoflagellates. To date, intracellular crystalline calcite was assumed to be involved in calcite shell formation. Surprisingly, we identify these crystalline inclusions as anhydrous guanine suggesting that they are not involved in biomineralization. Instead, a key finding is that MgCaP-rich bodies are probably secreted into the outer matrix where the calcite shell is formed. We suggest that these bodies are an essential part of Ca-uptake, -storage and -transport and propose a new biomineralization model.

Keywords: Biomineralization; Calcitic cysts; Cryo-FIB-SEM; Dinoflagellates; Guanine crystals.