Jessica Makin and Professor Kent Hatch, Integrative Biology

Introduction

Stable isotope research is becoming more widely used in physiological ecology. Atoms of the same atomic number but different atomic weights are called isotopes. Nitrogen (N) has two isotopes, 14N and 15N, with the latter being the heavier isotope. The assumption is that 14N is excreted at a higher rate than 15N. It is also thought that this should cause an increase in 15N of the protein tissues and preferential excretion of 14N of an animal when the animal is in the process of breaking down proteins.

Many of these assumptions are based on a study of eight cows (Steele 1977). This is problematic because “assimilation and metabolic pathways that lead to fractionation differ among animals” (Gannes et al. 1997). Cows and lizards use different metabolic pathways to excrete nitrogenous wastes. Cows excrete urea and lizards excrete uric acid, a white semi-solid suspension via the kidneys. It is therefore possible that the 15N/14N ratios in the excrements of these two groups differ relative to their diets and the 15N/14N ratio in the excrements may not differ significantly from the d15N in the diet. We tested this hypothesis using side-blotched lizards (Uta stansburiana) (experiment #1).

Secondarily, a study suggests animals in dry climates maintain a higher 15N/14N ratio in their tissues than similar animals in humid climates (Ambrose 1993). Uric acid formation requires only a small amount of water to create. Animals that excrete uric acid are usually adapted to conditions of limited water. We decided to test a hypothesis that water deprived animals show an increase in 15N/14N within and across species based on the two findings above (experiment #2). We used two species of lizard: side-blotched lizards, an arid climate lizard, and American anoles (Anolis carolinensis), a lizard typically found in humid climates.

Materials and Methods

In October 2002, we obtained 36 side-blotched lizards from St. George, Utah. They were divided equally among three large aquaria, and fed a diet of crickets ad libitum. In May 2003, we separated the lizards and broke off a piece of tail from each. We collected feces and uric acid over a period of two weeks. Feces, uric acid, tail tissue, and crickets were analyzed for nitrogen and carbon content using a Costech elemental analyzer and a Finnigan Delta II Plus mass spectrometer. Each sample weighed less than 2 mg and was placed in a small Costech tin capsule in preparation for analysis. To test whether 14N is preferentially excreted compared to 15N, we compared the d15N of the diet (crickets) to the uric acid, feces, and tissue (tail). In July 2003, we tested the hypothesis that water deprivation causes a fractionation in N isotopes using 6 side-blotched lizards and 7 American anoles. Both groups were fed ad libitum on a diet of crickets. For two weeks, the control group was given water ad libitum and the treatment group was not given water unless necessary for the animal’s survival. Again, we measured the d15N of the crickets, tail, uric acid, and feces using the same analytical procedure as the first experiment.

Results

Experiment 1. The tail was 15N enriched compared to diet, but less than other studies. Both uric acid and feces were 15N depleted relative to diet. Carbon isotope values showed no significant difference from the diet.

Experiment 2. Water deprived animals show an increase in 15N/14N ratio in the feces when compared to the controls. The 15N/14N ratios of the uric acid of the lizards did not differ between the treatment and control groups. Carbon isotopes did not differ significantly in any area. The two different species of lizard differed in their 15N/14N ratios. There was no difference in the reaction of the treatment between species

Conclusions

Nitrogenous wastes appear to be depleted of 15N relative to diet across taxa. Our study supports the conclusion that body tissue is enriched in 15N relative to diet across taxa. Uric acid d15N levels in animals deprived of water do not differ significantly from the d15N in the uric acid of animals with an abundance of water. Our study suggests that the metabolic pathways of urea formation and uric acid formation both preferentially excrete 14N. More studies could be conducted using different species.

References

• Ambrose, S. H., and L. Norr. 1993. Experimental evidence for the relationship of the carbon isotope ratios of the whole diet and dietary protein to those of one collagen and carbonate, p. 1- 37. J.B. Lambert and G. Grupe [eds.]. In Prehistoric human bone: archeology at the molecular level. Springer-Verlag, Berlin.
• DeNiro, M.J., and S. Epstein. 1977. Mechanism of carbon isotope fractionation associated with lipid synthesis. Science. 197:261-263.
• Gannes, L.Z., D. O’Brien, and C. Martinez del Rio. 1997. Stable isotopes in animal ecology: assumptions, caveats, and a call for more laboratory experiments. Ecology. 78:1271-1276.
• Hatch, K.A., B. Pinshow, and J.R. Speakman. 2002. Carbon isotope ratios in exhaled CO2 can be used to determine not just present, but also past diets. J. Comp. Physiol. B. 172:263-268.
• Magnusson, W.E., A.P. Lima, A.S. Faria, R.L. Victoria, and L. A. Luiz. 2001. Size and carbon acquisition in lizards from Amazonian savanna: Evidence from isotope analysis. Ecology. 6:1772-1780.
• Steele, K.W., and R.M. Daniel. 1977. Fractionation of nitrogen isotopes by animals: a further complication to the use of variations in the natural abundance of 15N for tracer studies.