Assessing the effects of lipid extraction and lipid correction on stable isotope values (δ¹³C and δ¹⁵N) of blubber and skin from southern hemisphere humpback whales

Abstract

The coupled analysis of δ 13 C and δ 15 N stable isotope values of blubber and skin biopsy samples is widely used to study the diet of free-ranging cetaceans.Differences in the lipid content of these tissues can affect isotopic variability because lipids are depleted in 13 C, reducing the bulk tissue 13 C/ 12 C. This variability in carbon isotope values can be accounted for either by chemically extracting lipids from the tissue or by using mathematical lipid normalisation models.Methods: This study examines (a) the effects of chemical lipid extraction on δ 13 C and δ 15 N values in blubber and skin of southern hemisphere humpback whales, (b) whether chemical lipid extraction is more favourable than mathematical lipid correction and (c) which of the two tissues is more appropriate for dietary studies.Strategic comparisons were made between chemical lipid extraction and mathematical lipid correction and between blubber and skin tissue δ 13 C and δ 15 N values, as well as C:N ratios. Six existing mathematical normalisation models were tested for their efficacy in estimating lipid-free δ 13 C for skin.Results: Both δ 13 C and δ 15 N values of lipid-extracted skin (δ 13 C: À25.57‰, δ 15 N: 6.83‰) were significantly higher than those of bulk skin (δ 13 C: À26.97‰, δ 15 N: 6.15‰). Five of the six tested lipid normalisation models had small error terms for predicting lipid-free δ 13 C values. The average C:N ratio of lipid-extracted skin was within the lipid-free range reported in other studies, whereas the average C:N ratio of blubber was higher than previously reported.Conclusions: These results highlight the need to account for lipids when analysing δ 13 C and δ 15 N values from the same sample. For optimised dietary assessments using parallel isotope analysis from a single sample, we recommend the use of unextracted skin tissue. δ 15 N values should be obtained from unextracted skin, whereas δ 13 C values may be adequately lipid corrected by a mathematical correction.

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

“…The mass-balance mathematical lipid correction model proposed by Fry (2002) relies on precision, accuracy and reliability in predicting the lipid-free δ 13 C values. The model is based on C:N ratios and thus lipid content, which was estimated to have a mean standard error of ~0.05 in predicting lipid-free δ 13 C values for skin tissue of E1 humpback whales (3). A study by Groß et al (2021) specifically calculated the discrimination value ‘D’ for skin tissue of individual E1 humpback whales to be applied in the mass balance correction model, which gave a ‘D’ value of 8.92 ‰ and a C:N LM of 3.1.…”

“…As the tissues were obtained from the same individual whale, the extent of the variability in both isotope signatures was not expected. There is limited research on the comparison of δ 13 C and δ 15 N values between HW blubber and skin tissue, however, a significant difference between the two tissues either for one or both isotopes has been documented (e.g Groß et al, 2021; Todd et al, 1997). However, the reasons for this variation are not clear, and thus we attempt to evaluate several factors that may have contributed to the significant differences found in this study.…”

“…The solvent lipid extraction of blubber tissue was completed using a modified methanol-dichloromethane-water (2:1:0.8 v/v/v MeOH/CH 2 Cl 2 /H 2 O) method pioneered by Bligh and Dyer (1959), as described in detail elsewhere (e.g. Groß et al, 2021).…”

“…Southern hemisphere humpback whales ( Megaptera novaeangliae ; SHHWs) have been implemented as a sentinel species for the circumpolar surveillance of pollution and climate change in the Southern Ocean (1,2). As capital breeders, these populations rely on intensive summer feeding on Antarctic krill ( Euphausia superba; hereafter ‘krill’; Groß et al, 2021; Waugh et al, 2012), to sustain their annual winter migrations to lower latitudes for breeding and calving. The narrow foraging niche of SHHWs, results in a distilled connection between ecosystem productivity and energetic provisioning (both prey type and foraging success; Castrillon and Bengtson Nash, 2020).…”

“…The first methods is the physical removal of lipid fractions through solvent extraction prior to BSIA. Alternatively, where the relationship between lipid-containing and lipid-depleted tissues of a species is known, mathematical corrections have been developed and applied (Post et al, 2007; Ryan et al, 2012, Groß et al 2021).…”