A non‐lethal approach to estimate whole‐body 13C and 15N stable isotope ratios of freshwater amphipods using walking legs

To analyze the stable isotope ratios of small‐bodied invertebrates, the entire animal is typically sacrificed and processed, which is problematic for threatened or endangered species. Appendages which are regenerated could be used to infer whole‐body isotope ratios, but differences in turnover rates and isotopic signatures among tissues may confound such an approach. We tested the hypothesis that the δ¹³C and δ¹⁵N of whole‐body tissue for freshwater amphipods could be predicted from the δ¹³C and δ¹⁵N of walking legs, with the goal of estimating body δ¹³C and δ¹⁵N of Gammarus acherondytes, a United States federally endangered species. To test this, we analyzed the δ¹³C and δ¹⁵N of walking legs and bodies of five species of amphipods from geographically distant areas (Idaho, Illinois, and Washington) in the United States. The general relationships of whole‐body isotope ratios of C and N as a function of leg isotope ratios were linear and had slopes of one. In the range of the data, leg δ¹³C was slightly lower than body δ¹³C, indicating some tissue‐specific fractionation, while δ¹⁵N was similar for legs and bodies. Our data suggest that legs can be used to predict body isotope ratios in freshwater amphipods. This approach provides an additional tool to help researchers understand the biology of small, endangered invertebrates without sacrificing individuals. This is especially useful in cave ecosystems where populations are naturally sparse.