TY - JOUR
AU1 - Bearhop,, Stuart
AU2 - Teece, Mark, A.
AU3 - Waldron,, Susan
AU4 - Furness, Robert, W.
AB - The use of nitrogen (15N/14N) and carbon (13C/12C) stable isotopes to infer trophic relationships in food webs has become a common and widely accepted technique (see Michener and Schell 1994). Stable-isotope ratios in the tissues of consumers reflect those in their diets in a predictable fashion, being isotopically enriched in 15N (up to 4‰) and 13C (usually ca. 1‰; DeNiro and Epstein 1981, Peterson and Fry 1987). Thus, unlike conventional dietary studies, stable-isotope analyses reflect assimilated (vs. merely ingested) foodstuffs. In addition, depending on the tissue chosen, dietary information spanning different temporal scales can be obtained (Hobson and Clark 1992a). Typical proteins used in such analyses are bone collagen and muscle (e.g. Hobson 1987, 1990; Hobson et al. 1994), which usually require the sacrifice of live animals or the opportunistic sampling of carcasses. Isotopic analyses can also be performed on tissues that can be sampled nondestructively, such as blood, feathers, and hair (Hobson and Clark 1993, Thompson and Furness 1995, Bearhop et al. 1999). Nondestructive sampling is desirable when dealing with rare species, when individuals are part of ongoing studies, and when investigating variation within individuals over time. However, there are consistent differences in isotopic signatures among tissue types (e.g. Tieszen et al. 1983). Some of this variation can be linked to differences in tissue metabolic rates, but some reflects differences among tissues in biochemical composition (e.g. Hobson and Clark 1992a). Lipid presents a particular problem. The lipid component of a given tissue type can be quite variable among individuals and generally is depleted in 13C compared with whole tissues (e.g. Tieszen et al. 1983). For these reasons, it is common practice to remove lipids from tissue samples where they may be present (Hobson 1987, 1990; Hobson and Clark 1992b; Thompson and Furness 1995). One of the most obvious ways to take nondestructive samples from animals is to obtain blood. The lipid component of blood generally is very low and is carried mostly in the plasma (Deuel 1955). Researchers have either analyzed whole blood (Hobson and Clark 1993, Hobson et al. 1997) or removed the serum fraction and analyzed the cells (Ben-David et al. 1997). However, the concentration of uric acid and urea (the end products of protein catabolism) reach substantial levels in blood plasma, particularly in times of high protein turnover such as during growth (Skadhauge 1983, Wolf et al. 1985, Alonso et al. 1991). Indeed, the concentration of uric acid in blood plasma is close to its solubility limit, and in avian urine, it grossly exceeds this limit and is present in colloid suspension (Skadhauge 1983). The mechanism by which 15N enrichment occurs is largely due to the excretion of isotopically light nitrogen (14N) in nitrogenous waste (Peterson and Fry 1987). Thus, if blood levels of urea or uric acid are elevated, then whole blood measurements will appear to be depleted in 15N. To date, no workers have assessed the influence that the lipid component and the uric acid/urea content may have on the isotopic signature of whole blood. In this paper, we compare the isotope signatures measured in whole blood of Great Skua (Catharacta skua) adults and chicks (more than 28 days old) with those of the same samples after they have been subjected to an aggressive lipid and uric acid/urea extraction technique. As top predators in many marine ecosystems, seabirds frequently have been used in isotopic studies (Hobson 1987, 1990; Hobson et al. 1994; Thompson and Furness 1995). Seabirds tend to share a number of life-history characteristics, so the Great Skua should be a good model for seabirds in general. Moreover, because elevated levels of uric acid have been reported in the blood of chicks from a wide range of taxa (including pelecaniforms [Wolf et al. 1985], ciconiiforms [Alfonso et al. 1991], and galliforms [Featherston 1969]), it might be expected that this phenomenon would apply to a great many species. Methods Great Skuas were sampled on Foula, Shetland (60°08′N, 2°05′W), during summer 1996 and on St. Kilda, Western Isles (57°49′N, 8°35′W), during summer 1997. Blood was collected from the tarsal vein using a 23-gauge needle and transferred to a glass vial, where it was stored frozen until analysis. Prior to analysis, samples of whole blood were freeze dried and powdered. Part of each dried sample was subjected to a standard technique for the extraction of lipids using a soxhlet apparatus with refluxing chloroform for 4 to 6 h. Unfortunately, the samples were not weighed. All δ13C and δ15N analyses were carried out by continuous-flow isotope ratio mass spectrometry (CF-IRMS) using a Europa Anca 20–20 C/N/S apparatus. Samples were loaded into miniature tin cups (4 × 6 mm) for combustion. A typical run consisted of a reference sample, duplicate analyses of two standards (internal laboratory carbon standard and IAEA-N-2), and then 8 to 10 samples. This sequence was repeated throughout the run, allowing a correction to be made for drift, if necessary. Isotope ratios are expressed in parts per thousand (‰) according to the following equation: where X is 15N or 13C and r is the corresponding ratio 15N/14N or 13C /12C. The rstandard for 13C is Pee Dee belemnite and for 15N is atmospheric nitrogen. Measurements of δ13C and δ15N are precise and accurate to ±0.2‰ and ±0.4‰, respectively. To adjust for test-wise error, we applied a sequential Bonferroni adjustment for α (α = 0.006 for 8 statistical tests). Results The δ13C signatures of blood from adult Great Skuas did not change significantly after washing in hot chloroform (before, x̄ = −17.1 ± SD of 0.6‰, n = 23; after, x̄ = −17.2 ± 0.8‰, n = 23; paired t = 1.61, P = 0.122). The same was true of δ15N values in adult blood (before, x̄ = 12.6 ± 0.9‰, n = 21; after, x̄ = 12.6 ± 0.9‰, n = 21; paired t = 0.76, P = 0.94). The δ13C values of chick blood also remained unaffected by the treatment (before, x̄ = −17.6 ± 0.6‰, n = 30; after, x̄ = −17.7 ± 0.9‰, n = 30; paired t = 0.43, P = 0.43). However, the mean δ15N value of chick blood increased significantly after treatment (before, x̄ = 11.6 ± 0.5‰, n = 30; after, x̄ = 12.8 ± 0.7‰, n = 30; paired t = 11.5, P < 0.001). Before extraction with hot chloroform, the mean δ13C value of adult whole blood was lower than that of chicks (t = 2.9, df = 51, P = 0.005), as was the mean δ15N value (t = 4.3, df = 29, P < 0.001). However, after treatment with hot chloroform, we detected no significant differences between blood from adults and chicks (δ13C, t = 1.9, df = 51, P = 0.052; δ15N, t = 1.1, df = 49, P = 0.27). Discussion Given that other researchers have measured the 13C content of lipid as being depleted relative to whole tissues (e.g. Tieszen et al. 1983), carbon-isotope ratios suggest that the lipid component of adult blood is so small that it does not significantly affect the whole-tissue isotope signatures. These findings are supported by studies that determined that the lipid component of avian blood is less than 1% of the total wet mass (e.g. Wolf et al. 1985, Rosa et al. 1993, Sartori et al. 1995); hence, it is unlikely to be of sufficient magnitude to influence the overall δ13C value. The pattern for skua chicks appears to be similar, which is somewhat surprising because young birds tend to have larger stores of lipid than adults (see Blem 1990), and one might have expected to detect a similar pattern in blood. However, even if lipid levels in chick blood were much higher than those in adult blood, they would still comprise only a small proportion of the whole tissue and as such would probably not be substantial enough to affect the δ13C ratio significantly. The results for 15N were different than those for 13C. After washing in hot chloroform, δ15N values in chick blood were significantly higher than those of whole tissue, whereas no such difference was detected in adult blood. Moreover, after the aggressive extraction procedure, the mean δ15N signature of chick blood did not differ significantly from that of adult blood. These observations suggest that washing whole blood with hot chloroform results in the removal of isotopically light nitrogenous compounds, which are present in high concentrations in chick blood but in low amounts in adult blood. Excreted nitrogenous compounds, such as urea and uric acid, are isotopically depleted in 15N (Peterson and Fry 1987). Assessment of plasma concentrations of these components in adults and chicks of the White Stork (Ciconia ciconia) yielded higher overall concentrations in chicks (Alfonso et al. 1991). Furthermore, in Brown Pelicans (Pelecanus occidentalis) and domestic fowl (Gallus gallus), large chicks tend to have elevated levels of uric acid in blood due to their large daily food intake (Featherston 1969, Wolf et al. 1985). Uric acid is the end product of protein catabolism and is produced mostly in the kidney and liver, and the serum concentration of uric acid is employed as an indicator of rates of protein catabolism (Stevens 1996). Rates of protein turnover are substantially higher during growth periods, with muscle protein turnover of 30% per day in chicks versus 5% per day in adults (Swick 1982). We suggest that the aggressive extraction technique using refluxing hot chloroform removes uric acid from whole blood. The solubility of uric acid in chloroform increases substantially with temperature from ca. 0.11 mM at 25°C to more than 0.25 at 50°C (M. Teece unpubl. data). Furthermore, in a study of mono- and dimethyl uric acids in incubation media, a mixture of chloroform and 2-propanol (85:15 v/v) was used to extract the compounds of interest (Benchekroun et al. 1990). Because uric acid is assumed to be isotopically light (Petersen and Fry 1987) and is present in substantially higher concentrations in chicks than in adults (Alfonso et al. 1991), removal of this compound during the extraction procedure would account for the observed results. Our results indicate that there is no need to extract lipids prior to isotopic analysis of avian blood. However, if protein turnover in the individuals being studied is thought to be high, then the possibility of elevated uric acid levels should be considered, particularly when comparing the diets of adults and young. It is worth noting that adult birds also can experience periods of high protein turnover, for example during rapid molt or egg formation (Blem 1990), at which time they also may have high levels of uric acid in their blood. Therefore, the influence of elevated concentrations of nitrogenous waste on the δ15N of blood must be considered when assessing trophic status based solely on the measurement of bulk tissue. Acknowledgments We thank Tony Fallick and Julie Dougans from the Life Sciences Community Stable Isotope Facility, which is funded by a consortium of Scottish Universities and the Natural Environmental Research Council. Fieldwork was made possible by the Holborn family in Foula and by Scottish Natural Heritage in St. Kilda. Dan Greenwood, Rob Malsom, Catherine Gray, Ben Ross, and Paulo Catry gave invaluable assistance in the field. We also thank David Thompson. SB was funded by a University of Glasgow Scholarship and the Louise Hiom Trust. Literature Cited Alonso , J. C. , V. Huecas , J. A. Alonso , M. Abelenda , R. Munoz-Pulido , and M. L. Puerta . 1991 . Hematology and blood chemistry of adult White Storks (Ciconia ciconia). Comparative Biochemistry and Physiology 98A : 395 – 397 . Google Scholar Crossref Search ADS WorldCat Bearhop , S. , D. R. Thompson , S. Waldron , I. C. Russell , G. Alexander , and R. W. Furness . 1999 . Stable isotopes indicate the extent of freshwater feeding by cormorants Phalacrocorax carbo shot at inland fisheries in England. Journal of Applied Ecology 36 : 75 – 84 . Google Scholar Crossref Search ADS WorldCat Ben-David M. R. W. Flynn , and D. M. Schell . 1997 . 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Google Scholar Crossref Search ADS WorldCat © The American Ornithologists' Union, 2000
TI - Influence of Lipid and Uric Acid on δ13C and δ15N Values of Avian Blood: Implications for Trophic Studies
JF - Auk: Ornithological Advances
DO - 10.1093/auk/117.2.504
DA - 2000-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/influence-of-lipid-and-uric-acid-on-13c-and-15n-values-of-avian-blood-yDS7uA3Stg
SP - 504
VL - 117
IS - 2
DP - DeepDyve
ER -