Determining the appropriate pretreatment procedures and the utility of liver tissue for bulk stable isotope (δ13C and δ15N) studies in sharks

Stable-isotope analysis (SIA) provides a valuable tool to address complex questions pertaining to elasmobranch ecology. Liver, a metabolically active, high turnover tissue (similar to 166 days for 95% turnover), has the potential to reveal novel insights into recent feeding/movement behaviours of this diverse group. To date, limited work has used this tissue, but ecological application of SIA in liver requires consideration of tissue preparation techniques given the potential for high concentrations of urea and lipid that could bias delta C-13 and delta N-15 values (i.e., result in artificially lower delta C-13 and delta N-15 values). Here we investigated the effectiveness of (a) deionized water washing (WW) for urea removal from liver tissue and (b) chloroform-methanol for extraction of lipids from this lipid rich tissue. We then (a) established C:N thresholds for deriving ecologically relevant liver isotopic values given complications of removing all lipid and (b) undertook a preliminary comparison of delta C-13 values between tissue pairs (muscle and liver) to test if observed isotopic differences correlated with known movement behaviour. Tests were conducted on four large shark species: the dusky (DUS, Carcharhinus obscurus), sand tiger (RAG, Carcharias taurus), scalloped hammerhead (SCA, Sphyrna lewini) and white shark (GRE, Carcharodon carcharias). There was no significant difference in delta N-15 values between lipid-extracted (LE) liver and lipid-extracted/water washed (WW) treatments, however, WW resulted in significant increases in %N, delta C-13 and %C. Following lipid extraction (repeated three times), some samples were still biased by lipids. Our species-specific C:N thresholds provide a method to derive ecologically viable isotope data given the complexities of this lipid rich tissue (C:N thresholds of 4.0, 3.6, 4.7 and 3.9 for DUS, RAG, SCA and GRE liver(LEWW) tissue, respectively). The preliminary comparison of C:N threshold corrected liver and muscle delta C-13 values corresponded with movement/habitat behaviours for each shark; minor differences in delta C-13 values were observed for known regional movements of DUS and RAG (delta C-13(Diffs) = 0.24 +/- 0.99 parts per thousand and 0.57 +/- 0.38 parts per thousand, respectively), while SCA and GRE showed greater differences (1.24 +/- 0.63 parts per thousand and 1.08 +/- 0.71 parts per thousand, respectively) correlated to large-scale movements between temperate/tropical and pelagic/coastal environments. These data provide an approach for the successful application of liver delta C-13 and delta N-15 values to examine elasmobranch ecology.

Automated summary

Stable-isotope analysis in liver tissue of sharks provides valuable insights into their recent feeding and movement behaviors, but careful consideration of tissue preparation techniques is necessary to avoid bias from urea and lipid concentrations. Specific C:N thresholds in liver tissue are crucial for deriving ecologically relevant isotope data, and preliminary comparisons of delta C-13 values between muscle and liver tissues can offer insights into movement and habitat behaviors of different shark species.