Interactive effects of urea and lipid content confound stable isotope analysis in elasmobranch fishes

TLDR: The results highlight the importance of urea and lipid extraction and demonstrate the confounding effects of these compounds, making it impossible to use C:N of non-urea-extracted samples as a basis for adjustment of δ13C.

Abstract: Stable isotope analysis (SIA) is becoming a commonly used tool to study the ecology of elasmobranchs. However, the retention of urea by elasmobranchs for osmoregulatory purposes may bias the analysis and interpretation of SIA data. We examined the effects of removing urea and lipid on the stable isotope composition of 14 species of sharks, skates, and rays from the eastern North Pacific Ocean. While effects were variable across taxa, removal of urea generally increased δ15N and C:N. Urea removal had less influence on δ13C, whereas extracting urea and lipid generally increased δ15N, C:N, and δ13C. Because C:N values of nonextracted tissues are often used to infer lipid content and adjust δ13C, shifts in C:N following urea extraction will change the inferred lipid content and bias any mathematical adjustment of δ13C. These results highlight the importance of urea and lipid extraction and demonstrate the confounding effects of these compounds, making it impossible to use C:N of non-urea-extracted samples as ...

Figures

Figure 3: Relationships between the C:N of urea-extracted tissue (treatment U, C:Nu) and changes in δ 13C between the U and UL treatments (∆δ13C = δ13CU - δ 13CUL). Lines show selected best fit modeled relationship between C:Nu and ∆δ 13C using best fit models based on AICc, which for groups with relatively low lipid content and C:Nu (batoids and all sharks except for dogfish) was a linear model, but for groups with a higher lipid content and C:Nu was based on the two parameter model from Fry (2002).

Figure 4: Conceptual diagram showing relative effects of urea and lipid extraction on elasmobranch muscle tissue. Axes show relative change in δ13C and δ15N following different treatments, and color bar shows C:N. The secondary axes show the relative effect of urea and lipid removal on δ13C and δ15N, with the size of arrows indicating relative magnitude of effect. Urea extraction will generally increase δ15N and C:N, and potentially also affect δ13C as 13C enriched urea is removed. Lipid extraction does not influence δ15N, but increases δ13C and reduces C:N. The degree to which the different treatments affect δ13C, δ15N (depicted by the magnitude and direction of the “Urea extraction” and “Urea & lipid extraction” arrows) and C:N (depicted by the shading gradient within each treatment arrow) will vary based on the urea and lipid (dashed arrow) content of the tissue.

Table 1: Effects of urea and lipid extraction on stable isotope composition of various sharks (top panel) 563 and batoids (bottom panel). Treatments are C (control), U (urea-extracted), UL (urea & lipid-extracted). 564 C test shows results of statistical comparisons between the control (C) and U and UL treatments, whereas 565 U test shows comparison between U and UL (ns = not significant, * = significant, p <0.05). Note that for 566 taxa with low sample sizes (<=3) we did not test for statistical differences. 567 568 569 570 571 572

Figure 1: Differences in δ13C, δ15N, and C:N between urea-extracted and control samples (A) and urea- and lipid-extracted and control samples (B) in sharks. Statistically significant differences between control and U and UL treatments are indicated (* p <= 0.05). Species are Prionace glauca (PG), Squalus suckleyi (SS), Triakis semifasciata (TS), Isurus oxyrinchus (IO), Lamna ditropis (LD), and Carcharodon carcharias (CC).We did not test for differences in TS due to low sample size.

Figure 2: Differences in δ13C, δ15N, and C:N between urea-extracted and control samples (A) and urea- and lipid- extracted and control samples (B) in batoids. Statistically significant differences between control and U and UL treatments are indicated (* p <= 0.05). Species are Bathyraja aleutica (BA), Bathyraja interrupta (BI), Beringraja binoculata (BB),Raja rhina (RR), Myliobatis californica (IO), Gymnura marmorata (TS), Pteroplatytrygon violacea (PV), and Urobatis halleri (UH). We did not test for differences in BI, MC and GM due to low sample sizes.

Full text

Draft
Interactive effects of urea and lipid content confound stable
isotope analysis in elasmobranch fishes
Journal:
Canadian Journal of Fisheries and Aquatic Sciences
Manuscript ID
cjfas-2015-0584.R2
Manuscript Type:
Article
Date Submitted by the Author:
19-Jul-2016
Complete List of Authors:
Carlisle, Aaron; Hopkins Marine Station of Stanford University,
Litvin, Steven; Stanford University
Madigan, Daniel; Harvard University, Center for the Environment
Lyons, Kady; University of Calgary
Bigman, Jennifer; Simon Fraser University
Ibarra, Melissa; University of California Davis
Bizzarro, Joseph; Moss Landing Marine Laboratories
Keyword:
stable isotopes, urea, lipid, mathematical lipid correction, C:N
https://mc06.manuscriptcentral.com/cjfas-pubs
Canadian Journal of Fisheries and Aquatic Sciences

Draft
1
Interactive effects of urea and lipid content confound stable isotope analysis in
1
elasmobranch fishes
2
3
Aaron B. Carlisle
1*
, Steven Y. Litvin
1
, Daniel J. Madigan
2
, Kady Lyons
3
, Jennifer S. Bigman
4
, Melissa 4
Ibarra
5
& Joseph J. Bizzarro
6
5
6
1
Hopkins Marine Station of Stanford University, 120 Oceanview Blvd, Pacific Grove, CA, USA 93950 7
2
Harvard University Center for the Environment, 24 Oxford Street, Cambridge, MA, USA 02138 8
3
University of Calgary, 2500 University Dr NW, Calgary, Alberta, Canada T2N 4N1 9
4
Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, Canada V5A 1S6 10
5
University of California Davis, One Shields Avenue, Davis, CA, USA 95616 11
6
Moss Landing Marine Laboratories, 8272 Moss Landing Road, Moss Landing, CA 95039 12
*
Corresponding author 13
14
Aaron B. Carlisle – aaroncar@stanford.edu
15
Steven Y. Litvin – litvin@stanford.edu 16
Daniel J. Madigan – danieljmadigan@fas.harvard.edu
17
Kady Lyons – kady.lyons@sbcglobal.net
18
Jennifer S. Bigman – jbigman@sfu.ca
19
Melissa Ibarra – mibarra08@yahoo.com
20
Joseph J. Bizzarro – jbizzarro@mlml.calstate.edu 21
22
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24
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29
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Abstract 30
Stable isotope analysis (SIA) is becoming a commonly used tool to study the ecology of elasmobranchs. 31
However, the retention of urea by elasmobranchs for osmoregulatory purposes may bias the analysis and 32
interpretation of SIA data. We examined the effects of removing urea and lipid on the stable isotope 33
composition of fourteen species of sharks, skates, and rays from the eastern North Pacific Ocean. While 34
effects were variable across taxa, removal of urea generally increased δ
15
N and C:N. Urea removal had 35
less influence on δ
13
C, whereas extracting urea and lipid generally increased δ
15
N and C:N while also 36
increasing δ
13
C. Because C:N values of non-extracted tissues are often used to infer lipid content and 37
adjust δ
13
C, shifts in C:N following urea extraction will change the inferred lipid content and bias any 38
mathematical adjustment of δ
13
C. These results highlight the importance of urea and lipid extraction and 39
demonstrate the confounding effects of these compounds, making it impossible to use C:N of non-urea-40
extracted samples as a diagnostic tool to estimate and correct for lipid content in elasmobranch tissues. 41
42
Keywords: Stable isotopes, urea, lipid, carbon, nitrogen, C:N, elasmobranch, mathematical lipid 43
correction, elasmobranch 44
45
46
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48
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Introduction 56
Stable isotope analysis (SIA) uses the stable isotope composition of organismal tissue to 57
understand a diverse suite of biological and ecological processes. SIA is increasingly being used to 58
investigate the ecology of marine taxa (Peterson and Fry 1987, Michener and Kaufman 2007), including 59
sharks, skates, and rays (elasmobranchs) (Hussey et al. 2012b). Since SIA makes inferences based on the 60
chemical composition of tissues, certain compounds found in specific taxa can interfere with analysis and, 61
therefore, conclusions. Here, we investigate the effects of urea and lipid extraction on tissues from 62
fourteen elasmobranch species and report results that demonstrate the necessity to account for these 63
compounds when using SIA in elasmobranch studies. 64
The physiology and anatomy of elasmobranchs present unique challenges when applying SIA to 65
study their ecology. In particular, elasmobranchs retain urea ((NH
2
)
2
CO) and trimethylamine oxide 66
(TMAO (C
3
H
9
NO)) in their tissues for osmoregulatory processes (Ballantyne 1997, Olson 1999, Hazon et 67
al. 2003). This retention of urea can differentially bias stable isotope results depending upon the tissue 68
type examined (Hazon et al. 2008, Kim and Koch 2011, Hussey et al. 2012b, Churchill et al. 2015). As a 69
waste product, urea is expected to have low δ
15
N values (Minagawa and Wada 1984, Balter et al. 2006) 70
because
14
N is preferentially concentrated in urea by deaminases and transaminases (Gannes et al. 1998). 71
We were unable to find any comparable data on TMAO, but as a waste product it also would be expected 72
to be depleted in
15
N. As a result, the relative concentrations of urea and TMAO in a tissue may influence 73
the δ
15
N value of that tissue. As urea and TMAO (hereafter referred to together as urea) both contain 74
carbon, they could potentially affect δ
13
C. Kim and Koch (2011) reported that the carbon in urea is 75
enriched in δ
13
C in some terrestrial taxa; however information on the isotopic composition of these waste 76
products, especially in aquatic taxa, remains lacking. Further complicating the effect of urea on SIA is its 77
varying concentration within organisms, which is influenced by a variety of factors including tissue type 78
(Ballantyne 1997), ambient salinity (Hazon et al. 2003, Pillans et al. 2005) and diet (Wood et al. 2010). 79
Information on how to address the effects of urea on SIA results is needed, both in terms of appropriate 80
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sample treatment methodology and data interpretation (Martinez del Rio et al. 2009, Logan and 81
Lutcavage 2010, Kim and Koch 2011, Hussey et al. 2012b, Li et al. 2016). 82
In addition to the potential effect of urea on the stable isotope composition of elasmobranchs, the 83
presence of lipids is known to influence the δ
13
C values of tissues (Post et al. 2007, Martinez del Rio et al. 84
2009, Hussey et al. 2012a). Because lipids are depleted in
13
C relative to protein, the presence of lipid in 85
tissues can bias δ
13
C values and increase the tissue carbon-to-nitrogen ratio (C:N) (Pinnegar and Polunin 86
1999, Post et al. 2007). Tissue samples with high lipid concentrations have lower δ
13
C values than 87
samples of the same tissue with lipids removed (Post et al. 2007). To account for variation in lipids across 88
tissue types, researchers either chemically extract or mathematically correct for lipids based on the tissue 89
C:N, which has been used as a proxy for relative lipid content in tissues (Post et al. 2007). 90
The influence of lipid content on SIA data of elasmobranch tissues has been relatively well 91
studied (Kim and Koch 2011, Hussey et al. 2012a) compared to that of urea (Hussey et al. 2012b). Logan 92
and Lutcavage (2010) and Kim and Koch (2011) directly assess the effects of urea extraction on SIA data 93
of elasmobranchs. Logan and Lutcavage (2010) reported no effect of urea extraction on elasmobranch 94
tissues, whereas Kim and Koch (2011) reported a significant increase in δ
15
N in urea-extracted tissues. 95
However, treatment methods differed between studies, with Kim and Koch (2011) using a more extensive 96
deionized water (DIW) extraction, which potentially resulted in more complete urea removal. Given that 97
lipid has a high C:N and urea has low C:N (0.5), removal of these compounds will influence tissue C:N. 98
Several studies examining the effect of lipid extraction on elasmobranch tissue noted increases in δ
15
N 99
and C:N following lipid extraction in a manner consistent with the removal of urea, suggesting that lipid 100
extraction may effectively remove urea as well as lipid (Hussey et al. 2010, Kim and Koch 2011, Hussey 101
et al. 2012a, Churchill et al. 2015, Li et al. 2016). However Kim and Koch (2011) reported that 102
elasmobranch tissues should have both urea and lipid-extracted to obtain the most reliable results. Li et al. 103
(2016) recently conducted the most thorough study of the interactive effects of urea and lipid to-date, 104
examining the effects of urea and lipid extraction on six species of pelagic sharks. They reported 105
significant increases in δ
15
N and C:N following lipid extraction, urea extraction, and lipid and urea 106
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Journal: Canadian Journal of Fisheries and Aquatic Sciences Manuscript ID cjfas-2015-0584. R2 Manuscript Type: Article Date Submitted by the Author: 19-Jul-2016 Complete List of Authors: Carlisle, Aaron ; Hopkins Marine Station of Stanford University, Litvin, Steven ; Stanford University Madigan, Daniel ; Harvard University, Center for the Environment Lyons, Kady ; University of Calgary Bigman, Jennifer ; Simon Fraser University Ibarra, Melissa ; University of California Davis Bizzarro, Joseph ; Moss Landing Marine Laboratories Keyword: stable isotopes, urea, lipid, mathematical lipid correction, C: N