Experimental procedure—pulse-chase labelling experiment

In order to assess inorganic N and C assimilation, we performed a pulse-chase isotope labelling experiment with three treatments. Treatments included symbiotic individuals incubated under light conditions (SymL), symbiotic individuals incubated in darkness (SymD), and aposymbiotic individuals incubated in light (ApoL) (Fig. ​(Fig.1).1). Prior to the experiment, five symbiotic and five aposymbiotic specimens were sampled to establish natural stable isotope ratios (T0; Fig. ​Fig.1).1). The remaining specimens (30 symbiotic and 15 aposymbiotic) were randomly assigned to one of the three treatments. The experiment consisted of an incubation with tracers (pulse), followed by a tracer-free incubation (chase). All incubations were performed in glass jars (‘incubation chamber’; IKEA Korken, ~1L) in incubators (MLR-352, Panasonic) at 28°C. Light was adjusted to 150 μmol photons m⁻² s⁻¹.

Experimental set-up. Boxes depict treatments (colour) and number of specimens (n). Treatments include symbiotic-light (SymL, orange), symbiotic-dark (SymD, grey), and aposymbiotic-light (ApoL, pink). Each vertical line represents a sampling event (T0, T1, T2, and T3) for 5 specimens from each treatment

At the start of the experiment, each medusae was transferred to an incubation chamber filled with ¹³C and ¹⁵N enriched seawater (Sigma-Aldrich; 117 μM NaH¹³CO₃, 98 atom % ¹³C; 1.18 mM Na¹⁵NO₃, 98 atom % ¹⁵N). Dissolved oxygen (DO) was measured for each chamber (YSI® ProODO™ optical DO sensor, Yellow Springs, USA), then chambers were closed airtight without any remaining bubbles and randomly arranged in the incubators. After ~5 h, chambers were opened one by one and DO measured (Fig. ​(Fig.1).1). Each medusa was rinsed with filtered ASW and five specimens from each treatment (SymL, SymD, and ApoL; n = 15) were sampled (T1). Sampled individuals were measured (bell diameter using standard callipers), rinsed with abundant MilliQ water, wrapped in sterile aluminium foil, frozen at −80°C for about 30 min, and stored at −20°C until further processing. Meanwhile, the incubation chambers were carefully cleaned (bleach and MilliQ) and filled with filtered (tracer-free) ASW. The remaining medusae (n = 30) were transferred back into the chambers for the chase part of the experiment. Chambers were closed and handled as described above. After ~3 h, five individuals from each treatment (n = 15) were sampled (T2, ‘chase3h’), and the remaining individuals (n = 15) were sampled after another 3 h (T3, ‘chase6h’).