Combining13C,15N, and2H to measure feeding and metabolic activity in marine, shal-low-water sponges – A pilot study
Authors: Stratmann Tanja , Sahonero-Canavesi Diana X. , van der Meer Marcel T. J. 
Published: December 2024

AbstractBackgroundShallow-water sponges can reach high densities and have several functions in the ecosystem, such as, providing microhabitats for other species or being involved in benthic-pelagic coupling. They also have a fast cell turnover so they can serve as test animals for the development of new methods to measure the metabolic activity of individual organisms. Here, we measured the feeding and metabolic activity of the common intertidal spongeHalichondria paniceausing a triple stable isotope labeling experiment with a13C and15N-enriched bacteria as substrate and2H from deuterated water. We also constrained pathways of phospholipid-derived fatty acid (PLFA) biosynthesis in the sponge and its microbiome.MethodsSponges were collected in the Eastern Scheldt (North Sea) and incubated in 1%2H-enriched seawater in the presence of13C- and15N-enriched substrate of inactivated bacteria for 12 h. Water samples for the analysis of dissolved inorganic carbon (DIC),13C-DIC, and inorganic nutrients were taken at the begin and the end of the incubations, while oxygen concentration in the water was recorded continuously. Seawater incubations with the addition of substrate bacteria served as blanks and dead sponges incubated in 1%2H-enriched seawater served as controls for the incorporation of2H into inactive tissue. At the end of the experiment, sponges were sampled for bulk analyses of13C,15N, and2H in sponge tissue, and for13C and2H incorporation into phospholipid-derived fatty acids (PLFAs).ResultsSponges consumed oxygen, nitrite, nitrate, and silicon, while they excreted ammonia and released13C-DIC. They incorporated 4.82 μmol13C mmol C−1d−1, 2.46 μmol15N mmol C−1d−1, and 0.49 μmol2H mmol C−1d−1into bulk sponge tissue. The PLFAs extracted from the sponges contained on average 5.61 μg13C g−1dry mass (DM) sponge and 5.43 ng2H g−1DM sponge. Most13C was incorporated into bacteria-specific PLFAs derived from the substrate bacteria;2H, in comparison, was mostly built in sponge-specific PLFAs.ConclusionH. paniceafrom the Eastern Scheldt 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. During the incubation experiment, however, the sponges were well fed as indicated by the silicon and oxygen uptake rates. The significantly higher uptake rates of2H by living sponges compared to dead sponges proved that deuterated water can be used to measure the metabolic activity of individual filter-feeding specimens, but more test with species of diverse biological traits and growth stages are recommended. Additionally, we suggest to focus on compound-specific2H uptake, as these data were less affected by1H exchange of non-covalently bound1H. Assessing13C- and2H-enriched PLFAs of the incubatedH. paniceaalso revealed that the sponge microbiome produced the bacteria-specific PLFAsi-C14:0,ai-C15:0,i-C17:0, andai-C17:0 using C from the substrate bacteria. Theai-C15:0 was subsequently elongated and desaturated to build the sponge-specific PLFAai-C25:2. Other sponge-specific PLFAs were formed using C14:0 and C16:0 from the sub-strate bacteria as precursors.