1. Measuring the metabolic rate of marine animals in their natural environment is challenging, impeding our understanding of their physiological ecology. Recently, a novel metabolic proxy, namely the δ¹³C values of biogenic carbonates (otoliths), was experimentally validated in teleost fishes. Cephalopods possess several types of biogenic carbonates, such as statolith, cuttlebone, and other internal and external shells, which are all potential metabolic recorders, but few have been evaluated. 2. To test the feasibility of the δ¹³C metabolic proxy in cephalopods, we conducted a temperature-controlled experiment with the pharaoh cuttlefish Sepia pharaonis to assess whether the proportion of metabolically derived carbon (C_resp) incorporated into statoliths and cuttlebones increases with metabolism in a warm environment. Moreover, we conducted multiple-species analysis by using the published δ¹³C values of various biogenic carbonates to evaluate the consistency of the proxy among cephalopod species and between cephalopods and teleost fishes. 3. In the temperature-controlled laboratory experiment, C_resp values calculated from statoliths and cuttlebones increased with an increase of environmental temperature. C_resp values did not differ between statoliths and cuttlebones. Moreover, the C_resp values of cephalopod biogenic carbonates had a similar range to those of fish otoliths and were correlated with metabolism-related factors, such as ambient temperature, body mass, ontogeny, and functional behaviours, strengthening the feasibility of their use. 4. The δ¹³C metabolic proxy reflects the total energy used in the natural environment, and it can be transformed into the oxygen consumption rate for a broader comparison with other species and for further evaluation with current theories related to metabolic ecology. However, experimental validation is highly recommended because the relationship between the oxygen consumption rate and C_resp values derived from biogenic carbonates of cephalopods might be structure- and species-specific. Our study revealed that the newly developed metabolic proxy provides a valuable alternative approach to studying ecophysiology in cephalopods, and further development might broaden its applicability. --

Ten cephalopod species with δ¹³C and δ¹⁸O values from the literature were used in analyses of this study. δ¹³C and δ¹⁸O values were derived from external (Nautilus and Argonauta) and internal shells (ram’s horn squids and cuttlefishes) as well as statoliths (squids). The experienced temperature was extracted from the same study. If recorded temperatures were absent in previous studies, we estimated temperatures from the reported δ¹⁸O values by using the temperature-dependant equations.

The C_resp value was estimated based on the δ¹³C values of biogenic carbonates by using the two-component mixing model. In the model, the δ¹³C_DIC values were based on published data in the same sampling area. The δ¹³C_diet values were derived from the δ¹³C values in other tissues/structures (muscle, beaks, or organic matter in biogenic carbonate) minus assumed trophic enrichment factor 1, but for planktivores, the δ¹³C_diet values were obtained from the δ¹³C values of zooplankton in the sample areas according to published spatial ocean modelling.

The parameters in the dataset are described in the README file and the source of isotope values is shown in the dataset.