A Multi-Tissue, Multi-Species Assessment of Lipid and Urea Stable Isotope Biases in Mesopredator Elasmobranchs

Abstract

The application of stable isotope analysis (SIA) is increasing in elasmobranch trophic ecology, but inconsistency remains in terms of the tissue pre-treatment methods chosen to remove biases introduced by lipids and urea. SIA of a range of non-lethally extracted tissues from a diverse group of elasmobranchs, including mesopredators, is increasing, yet most studies assume that isotope biases from lipid and urea are the same across tissues and species. To determine tissue- and species-specific isotope biases across treatment methods, three tissues and their components [muscle, fin, and blood separated into plasma and red blood cells (RBC)] were non-lethally extracted from three species of mesopredatory elasmobranchs and subjected to one of three treatment methods: (1) deionized water rinse [DW], (2) chloroform/methanol lipid extraction [LE], or (3) deionized water followed by chloroform/methanol [DW+LE]. In muscle δ13C, all treatments displayed minimal variation (∼ 0‰) but large increases in δ15N (∼ 1‰) indicated urea removal. Fin δ13C values decreased with DW but increased with LE and DW+LE, whilst all treatments increased fin δ15N (∼ 0.5‰), suggesting removal of both lipid and urea. Plasma δ13C and δ15N displayed high individual variation; large decreases in δ13C (∼−0.8‰) across all treatments, but particularly DW, suggested the removal of 13C-enriched compounds while a small increase in δ15N (∼ 0.2‰) suggested minimal urea removal. In RBC, all treatments showed small δ13C declines (∼−0.5‰), with no difference in δ15N, suggesting minimal removal of 13C-enriched compounds and urea. For muscle and fin, DW+LE is the most appropriate treatment to standardize δ13C and δ15N consistently across individuals and tissues. The large individual variation in treatment effects on plasma suggests it is unsuitable for current treatment methods. Consistent treatment effects for RBC allow for DW+LE standardization, however, broader species-specific effects are unknown. The importance of treatment choice for accurately estimating prey contributions to elasmobranch diet was highlighted using Bayesian stable isotope mixing model comparisons, with prey contributions varying significantly among treatments. This variability suggests that ecological inferences from elasmobranch tissue SIA are not robust to different treatment methods. It is recommended that studies employ standardized corrections using a combined DW+LE treatment where applicable.

Parasitic Copepods as Biochemical Tracers of Foraging Patterns and Dietary Shifts in Whale Sharks (Rhincodon typus Smith, 1828)

“…Lipids and urea in whale shark dermal tissues can lower δ 13 C and δ 15 N values [48], leading to bias in analyses. To remove urea, dermal tissue samples were submerged in 1.4 mL of deionized water in 1.5 mL Eppendorf tubes, vortexed for 5 min, and placed in a 30 • C water bath for 24 h. Eppendorf tubes were then removed from the water bath, centrifuged at 2000 rpm for 10 min, and the excess water decanted.…”

Trophic ecology shapes spatial ecology of two sympatric predators, the great hammerhead shark (Sphyrna mokarran) and bull shark (Carcharhinus leucas)

“…Fifty samples of seven reference materials were used for d 13 C, and 54 samples of eight reference materials for d 15 N. Results are expressed in the delta (d) notation as per mil (‰), so that: dX = (R sample /R standard -1) × 10 3 , where dX is the stable isotope composition of the sample, R sample is our sample's molar ratio of the heavy to light element, and R standard that ratio in the international standard material. Lipids and urea were not extracted from shark samples prior to analysis, because lipid/urea treatments can influence d 13 C and d 15 N, and magnitude and direction of this effect is variable among studies, taxa, and tissues (e.g., Carlisle et al, 2017;Crook et al, 2019;Bennett-Williams et al, 2022). Also, it has recently been shown that lipid/urea extraction does not significantly affect elasmobranch muscle d 13 C (Bennett- Williams et al, 2022).…”

“…Lipids and urea were not extracted from shark samples prior to analysis, because lipid/urea treatments can influence d 13 C and d 15 N, and magnitude and direction of this effect is variable among studies, taxa, and tissues (e.g., Carlisle et al, 2017;Crook et al, 2019;Bennett-Williams et al, 2022). Also, it has recently been shown that lipid/urea extraction does not significantly affect elasmobranch muscle d 13 C (Bennett- Williams et al, 2022).…”

Effects of ethanol storage and lipid extraction on stable isotope compositions of twelve pelagic predators

“…Understanding the effects of treatments ET and ET+LE on predator muscle tissue stable isotope values is valuable for accurately interpreting data in food-web studies. Removing these biases is essential for acquiring standardized stable isotope values among different species, and a reasonable adjustment could be helpful for the use of the valuable biopsy samples obtained from remote areas and archived collections (Kim and Koch, 2012;Li et al, 2016;Bennett-Williams et al, 2022). In this study, the results for twelve pelagic predators suggested that both treatments ET and ET+LE caused inconsistent effects in d 13 C, d 15 N values, and C/N ratios during the 28 days of preservation and then could remain stable throughout the experiment, yielding new insights into the effects of these treatments.…”

“…Since the effects of treatment ET on muscle d 13 C and d 15 N values of marine organisms were reported to be variable and inconsistent among species, conducting SIA directly using treatment ET samples was not recommended (Barrow et al, 2008;Boecklen et al, 2011;Kim and Koch, 2012). In addition, lipid extraction (LE) was recommended as a standardization step prior to SIA since lipids were 13 C-depleted relative to proteins and carbohydrates and could potentially cause the d 13 C values of organisms to be negatively biased relative to their diet with increasing lipid content (Wessels and Hahn, 2010;Elliott and Elliott, 2016;Li et al, 2016;Bennett-Williams et al, 2022). However, there was less evidence to indicate whether treatment LE affected the stable isotope values of treatment ET samples.…”