Labelling halophilic Archaea using C and N stable isotopes: a potential tool to investigate haloarchaea consumption by metazoans.

The use of stable isotope (SI) labelling and tracing of live diets is currently considered one of the most comprehensive tools to detect their uptake and assimilation by aquatic organisms. These techniques are indeed widely used in nutritional studies to follow the fate of specific microbial dietary components, unraveling trophic interactions. Nevertheless, to the current date our understanding of aquatic trophic relationships has yet to include a whole domain of life, the Archaea. The aim of the present research was, therefore, to describe a halophilic Archaea (haloarchaea) labelling procedure, using the SI C and N, to enable the application of SI tracing in future studies of haloarchaea consumption by aquatic metazoans. To this end, three C enriched carbon sources and two N enriched nitrogen sources were tested as potential labels to enrich cells of three haloarchaea strains when supplemented to the culture medium. Our overall results indicate C-glycerol as the most effective carbon source to achieve an efficient C enrichment in haloarchaea cells, with ΔδC values above 5000‰ in all tested haloarchaea strains. As for N enriched nitrogen sources, both (NH)SO and NHCl seem to be readily assimilated, also resulting in efficient N enrichment in haloarchaea cells, with ΔδN values higher than 20,000‰. We believe that the proposed methodology will allow for the use of SI labelled haloarchaea biomass in feeding tests, potentially providing unambiguous confirmation of the assimilation of haloarchaea biomass by aquatic metazoans.