Interpretation of southern hemisphere humpback whale diet via stable isotopes; implications of tissue-specific analysis

**Background:** Southern hemisphere humpback whales (SHHWs) are capital breeders that rely on intensive summer feeding on Antarctic krill (Euphausia superba) to sustain annual migrations. They serve as sentinel species for monitoring Southern Ocean ecosystem change. Bulk stable isotope analysis (BSIA) of δ13C and δ15N is commonly used to investigate cetacean diet, with blubber and skin being the most frequently sampled tissues via remote biopsy. However, critical comparison of tissue-specific isotopic signals has been lacking, creating uncertainty about whether these tissues can be used interchangeably for accurate dietary determination.

**Methods:** Between 2008 and 2018, 171 paired blubber and skin biopsy samples were collected from free-swimming SHHWs of the E1 stock off North Stradbroke Island, southeast Queensland (27°26 S, 153°34 E) during annual migrations. Blubber tissue underwent solvent lipid extraction using a modified Bligh and Dyer methanol-dichloromethane-water method prior to BSIA. Skin tissue was mathematically lipid-corrected using the Fry mass balance approach with a discrimination factor of 8.92‰, previously determined for this population. Samples were analyzed using a Europa EA-GSL interfaced to a SERCON Hydra 20-20 isotope ratio mass-spectrometer, with standard deviations of 0.1‰ for δ13C and 0.15‰ for δ15N. Statistical analyses included Wilcoxon matched-pair signed rank tests and Kruskal-Wallis tests with α=0.05. Tissue-specific krill ranges and trophic positions were calculated using published krill isotope values and a trophic fractionation factor of 2.82‰ derived from fin whale skin.

**Key Results:** Significant differences were observed between lipid-adjusted blubber and skin for both δ13C (p=0.0001) and δ15N (p=0.0001). Lipid-extracted blubber showed greater variability (δ13C mean=-26.40‰, SD=1.83, range -31.56 to -20.77; δ15N mean=7.65‰, SD=1.14, range 3.65-13.87) compared to lipid-corrected skin (δ13C mean=-25.35‰, SD=0.60, range -27.35 to -23.52; δ15N mean=6.81‰, SD=0.46, range 5.21-8.22). Inter-annual analysis (2013-2018) revealed significant differences in δ13C between tissues in 2016 (p=0.0216), 2017 (p=0.0335), and 2018 (p=0.0001), and in δ15N in 2013 (p=0.0003), 2014 (p=0.0001), and 2017 (p=0.0001). Trophic position estimates differed significantly between tissues (Welch t-test p=2.2e-16), with blubber TP=3.65±0.47 and skin TP=3.29±0.16. Krill space analysis showed that in 2013, only 29.2% of blubber data points fell within the expected krill range compared to 100% of skin data points; in 2015, 53.3% of blubber versus 96.7% of skin data points fell within the krill range.

**Clinical Implications:** These findings demonstrate that blubber and skin tissues from the same individual cannot be used interchangeably for stable isotope-based dietary analysis in SHHWs. The greater variability in blubber δ15N values may reflect methodological artefacts from solvent extraction (potential removal of amino acids) rather than true dietary variation, while the lower variability in skin may underestimate actual foraging plasticity. This has significant implications for long-term monitoring programs like the Humpback Whale Sentinel Program, as tissue choice directly influences interpretation of diet composition, trophic position, and foraging behavior. The study recommends developing standardized protocols including multiple lipid standardization approaches and solvent extraction trials to improve the reliability of cetacean dietary monitoring in rapidly changing polar ecosystems.