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

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Abstract
Background: Shallow-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 sponge Halichondria panicea using a triple stable isotope labeling experiment with a 13 C and 15 N-enriched bacteria as substrate and 2 H from deuterated water. We also constrained pathways of phospholipid-derived fatty acid (PLFA) biosynthesis in the sponge and its microbiome. Methods: Sponges were collected in the Eastern Scheldt (North Sea) and incubated in 1% 2 H-enriched seawater in the presence of 13 C- and 15 N-enriched substrate of inactivated bacteria for 12 h. Water samples for the analysis of dissolved inorganic carbon (DIC), 13 C-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% 2 H-enriched seawater served as controls for the incorporation of 2 H into inactive tissue. At the end of the experiment, sponges were sampled for bulk analyses of 13 C, 15 N, and 2 H in sponge tissue, and for 13 C and 2 H incorporation into phospholipid-derived fatty acids (PLFAs). Results: Sponges consumed oxygen, nitrite, nitrate, and silicon, while they excreted ammonia and released 13 C-DIC. They incorporated 4.82 μmol 13 C mmol C −1 d −1 , 2.46 μmol 15 N mmol C −1 d −1 , and 0.49 μmol 2 H mmol C −1 d −1 into bulk sponge tissue. The PLFAs extracted from the sponges contained on average 5.61 μg 13 C g −1 dry mass (DM) sponge and 5.43 ng 2 H g −1 DM sponge. Most 13 C was incorporated into bacteria-specific PLFAs derived from the substrate bacteria; 2 H, in comparison, was mostly built in sponge-specific PLFAs. Conclusion: H. panicea from 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 of 2 H 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-specific 2 H uptake, as these data were less affected by 1 H exchange of non-covalently bound 1 H. Assessing 13 C- and 2 H-enriched PLFAs of the incubated H. panicea also revealed that the sponge microbiome produced the bacteria-specific PLFAs i -C14:0, ai -C15:0, i -C17:0, and ai -C17:0 using C from the substrate bacteria. The ai -C15:0 was subsequently elongated and desaturated to build the sponge-specific PLFA ai -C25:2. Other sponge-specific PLFAs were formed using C14:0 and C16:0 from the sub-strate bacteria as precursors.
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