Abstract
Despite the oceanographic and geological significance of coccolithophores, the cellular mechanisms that underlie the intracellular production and subsequent secretion of their CaCO3 coccoliths remain poorly understood. Tools for labelling coccoliths and coccospheres in order to track their production would be of great value. We therefore evaluated the use of calcein, a derivative of fluorescein, as a method to fluorescently label coccoliths. The calcein method readily labelled pre-existing coccospheres in a range of coccolithophore species, including diploid and haploid life history phases, without compromising the coccolith structure. Calcite staining was verified though epifluorescence and confocal microscopy, and both stained and unstained cells and coccoliths were readily distinguished using flow cytometry. The fluorescence of stained coccoliths was retained for > 3 d allowing us to confirm the polar secretion of coccoliths by distinguishing pre-existing coccoliths from the accumulation and distribution of non-fluorescent coccoliths produced after calcein exposure. The calcein treatment had no significant effect on photosynthetic physiology, external calcite morphology, or growth rates of the cells over an 8 d period. The calcein staining method therefore represents a simple non-invasive, non-toxic optical technique to ‘tag’ calcium carbonate coccoliths and track their production in response to environmental manipulations or pharmacological treatments. Moreover, calcein staining of the coccosphere allowed for heterogenous patterns of calcification, growth, and cell division to be detected in a population of cells. This is the first description of the use of calcein to stain the biomineral structures of calcifying phytoplankton and this approach has the potential to be applied to detailed cytological investigations as well as high-throughput analysis of cultured cells or field populations.
Highlights
Biomineralization in Marine SystemsCoccolithophores are a group of calcifying unicellular marine phytoplankton recognized for production of extracellular calcium carbonate (CaCO3) plates known as coccoliths
Lower and mid optical sections were acquired though each group of cells that were subsequently scored for calcein staining
We examined the utility of calcein as a non-toxic marker for coccolithophore calcification and established that this fluorescent probe is suitable for labeling of both hetero- and holococcoliths across a range of species
Summary
Biomineralization in Marine SystemsCoccolithophores are a group of calcifying unicellular marine phytoplankton recognized for production of extracellular calcium carbonate (CaCO3) plates known as coccoliths. Coccolithophore calcification plays a critical role in carbon export dynamics (Balch, 2018) as the calcite structures facilitate formation of organic aggregates in marine snow, and coccolith sedimentation results in calcareous deposits on the ocean floor which gave rise to major chalk formations over geological time-scales (De Vargas et al, 2007). In spite of their global importance, the cellular mechanism by which coccolithophores produce their CaCO3 coccoliths, the regulation of this process, and the coordination of coccolith production with cellular metabolism remain poorly understood. Coccolithophores exhibit a haplo-diplontic life cycle with motile haploid cells producing calcified scales comprising numerous simple calcite rhombohedra suggesting a distinct calcification mechanism (see Taylor A.R. et al, 2017 and references therein)
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