Combining ¹³C, ¹⁵N, and ²H tracers to measure feeding and metabolic activity in marine, shallow-water sponges – A pilot study

Shallow-water sponges can reach high densities and have several functions in the ecosystem. They also have a fast cell turnover and therefore can serve as test animals for the development of methods to measure the metabolic activity of individual organisms. Here, we measured the feeding and metabolic activity (i.e., uptake of ²H) of Halichondria panicea using a triple stable isotope labeling experiment with ¹³C and ¹⁵N-enriched bacteria as substrate and ²H from deuterated water (²H₂O). Sponges were collected in the Eastern Scheldt (North Sea) and incubated in 1 % (volume/volume) ²H-enriched seawater in the presence of ¹³C- and ¹⁵N-enriched inactivated bacteria substrate for 12 h. Water samples for the analysis of dissolved inorganic carbon (DIC), ¹³C-DIC, and inorganic nutrients were taken at the beginning and end of the incubations, while oxygen concentration was recorded continuously. Sponges were sampled for bulk analyses of ¹³C, ¹⁵N, and ²H in sponge tissue, and for ¹³C and ²H incorporation into phospholipid-derived fatty acids (PLFAs). Sponges incorporated ¹³C, ¹⁵N, and ²H into bulk sponge tissue. The PLFAs extracted from the sponges contained on average 5.61 μg ¹³C g⁻¹ dry mass (DM) sponge and 5.43 ng ²H g⁻¹ DM sponge. Most ¹³C derived from the substrate bacteria was incorporated into bacteria-specific PLFAs of the sponge microbiome; ²H, in comparison, was mostly built in sponge-specific PLFAs. H. panicea were in a similar condition as starving specimens from the Baltic Sea which suggests that the large bivalve stocks in the Eastern Scheldt outcompete this sponge for food.