Effects of lipid and urea extraction on stable isotope values (δ¹³C and δ¹⁵N) of two batoids: A call for more species‐specific investigations

Abstract

The presence of lipids and urea in elasmobranch tissues can affect carbon (δ13C) and nitrogen (δ15N) stable isotope values, introducing bias in food web interpretations. Information on how lipids and urea affect δ13C/δ15N is only available for < 5% of ~ 1150 described elasmobranch species and results are highly variable among existing studies. Here, we investigate the effects of lipid and urea extraction on δ13C, δ15N, and C:N ratios in muscle and blood plasma of two batoids (Pastinachus ater and Himantura australis) and examine the influence of lipid/urea extraction on isotopic niche size and overlap. Lipid extraction (LE) did not affect δ13C, δ15N, or C:N ratios, suggesting low lipid content. Urea extraction (UE), however, increased δ15N and C:N in muscle. The ~ 1‰ δ15N increase represents a shift of ~ 0.5 trophic levels relative to bulk samples, highlighting the importance of UE to accurately assess trophic positions. Although there was no effect of any treatment on niche size, the probability of P. ater occurring within the niche of H. australis increased following UE. Overall, results suggest that urea should be removed from muscle prior to analysis, but LE is not required. Given the interspecific variability in the effects of lipid/urea on elasmobranch δ13C/δ15N, more studies are needed to assess the effects of lipid and urea on a broader range of species to produce a generalized understanding. Where no species‐specific data are available, we recommend pilot samples are analyzed to determine if LE is needed prior to preparation of the overall sample set.

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

“…The shift suggests DW is removing compounds that are 13 C-enriched exacerbating their influence on the δ 13 C value. The large decreases in δ 13 C correlate with previous studies on elasmobranch plasma (Kim and Koch, 2012;Crook et al, 2019). As LE and DW+LE have an almost identical effect but a smaller shift in δ 13 C, this suggests that the LE component is potentially also removing lipids or other 13 C-depleted compounds.…”

“…Plasma δ 15 N values are extremely variable across the treatments, but they do display a general positive shift. Increases in δ 15 N have also been displayed in leopard sharks (Triakis semifasciata) (Kim and Koch, 2012), whereas two batoid species displayed decreases in δ 15 N (Crook et al, 2019). This might indicate that 15 N-depleted urea is being removed from the plasma, but the removal of 15 Nenriched amino acids is masking the effect of urea removal on the δ 15 N value.…”

“…Urea removal from elasmobranch muscle tissue has been extensively researched-a deionized water rinse (DW) is recommended (Kim and Koch, 2012;Li et al, 2016;Carlisle et al, 2017). For other tissues, DW has been shown to have no significant effect on dermis tissue δ 15 N of two stingray species (Shipley et al, 2017) and urea extraction on blood and its components is not recommended as it decreases δ 15 N values (Kim and Koch, 2012;Crook et al, 2019), potentially due to the removal of free-form amino acids (Kim and Koch, 2012) found in plasma (Bedford, 1983). The combination of urea and lipid extraction is well investigated with recommendations provided for elasmobranch muscle for multiple species (Li et al, 2016) and whole blood from one species (Connan et al, 2019), but effects on fin, plasma, and RBC remain understudied.…”

Trophic niche of Australian cownose rays (Rhinoptera neglecta) and whitespotted eagle rays (Aetobatus ocellatus) along the east coast of Australia

“…Elasmobranch tissues, especially those of batoids, are often assumed to have low lipid content and typically do not require lipid correction if C:N ratios are <3.5 (Carlisle et al ., 2017; Crook et al ., 2019; Li et al ., 2016; Post et al ., 2007). Lipid content in the present study ranged from 3.29 to 4.67 ( R. neglecta 3.81 ± 0.01, A. ocellatus 4.01 ± 0.01).…”

“…Samples were considered adequately homogenized when no large particles (>2 mm) remained. Lipid extractions were not conducted under the premise of low lipid content in batoids (Crook et al ., 2019; Hussey et al ., 2011; Olin et al ., 2014). Additionally, although these fin clips are comprised of skin, ceratotrichia and other tissues (Hussey et al ., 2011), acid washes were not conducted because they would have resulted in excessive loss of tissue for some smaller samples.…”