Nathan Wolf

Isotopic ecology ten years after a call for more laboratory experiments

About 10 years ago, reviews of the use of stable isotopes in animal ecology predicted explosive growth in this field and called for laboratory experiments to provide a mechanistic foundation to this growth. They identified four major areas of inquiry: (1) the dynamics of isotopic incorporation, (2) mixing models, (3) the problem of routing, and (4) trophic discrimination factors. Because these areas remain central to isotopic ecology, we use them as organising foci to review the experimental results that isotopic ecologists have collected in the intervening 10 years since the call for laboratory experiments. We also review the models that have been built to explain and organise experimental results in these areas.

The influence of drinking water on the δD and δ18O values of house sparrow plasma, blood and feathers

SUMMARY We investigated the relationships between the δdeuterium (δD) and the δ18oxygen (δ18O) of drinking water and the δD and δ18O of blood plasma, red blood cells and feathers in house sparrows (Passer domesticus) fed on diets with identical hydrogen and oxygen isotopic compositions and five isotopically distinct drinking water treatments. We expected and, with only one exception (18O in blood plasma), found linear relationships between the δD and δ18O values of drinking water and those of bird tissues. The slopes of these relationships, which estimate the percentage contributions of drinking water to the tissue isotopic signatures, were lower than those of previous studies. We found significant differences in the δD and δ18O values of feathers, red blood cells and plasma solids. In feathers and red blood cells, δD and δ18O values were linearly correlated. Our results have three implications for isotopic field studies: (1) if the isotopic composition of drinking water differs from that of food, its effect on tissue isotope values can confound the assignment of animals to a site of origin; (2) comparisons of the δD and δ18O values of different tissues must account for inter-tissue discrimination factors; and (3) δD/δ18O linear relationships are probably as prevalent in animal systems as they are in geohydrological systems. These relationships may prove to be useful tools in animal isotopic ecology.

Dietary protein influences the rate of 15N incorporation in blood cells and plasma of Yellow-vented bulbuls (Pycnonotus xanthopygos).

SUMMARY The rate at which an animals tissues incorporate the isotopic composition of food determines the time window during which ecologists can discern diet changes. We investigated the effect of protein content in the diet on the incorporation rate of 15N into the plasma proteins and blood cells of Yellow-vented bulbuls (Pycnonotus xanthopygos). Using model comparison analyses, we found that one-compartment models described incorporation data better than two-compartment models. Dietary protein content had a significant effect on the residence time of 15N in plasma proteins and blood cells. The diet with the highest protein content led to a 15N retention time of 21 and 5 days for cells and plasma, respectively. In contrast, average 15N retention time in the cells and plasma of birds fed on the diet with the lowest protein was 31 and 7 days, respectively. The isotopic discrimination factorΔ 15N=δ15Ntissues–δ15Ndiet was also dependent on dietary protein content, and was lowest in birds fed the diet with the highest protein content. Blood, plasma and excreta were enriched in 15N relative to diet. In contrast, ureteral urine was either significantly depleted of 15N in birds fed the diet with the lowest protein content or did not differ in δ15N from the diets with the intermediate and high protein content. Thus, isotopic incorporation rates and tissue-to-diet discrimination factors cannot be considered fixed, as they depend on diet composition.

An experimental exploration of the incorporation of hydrogen isotopes from dietary sources into avian tissues

SUMMARY The analysis of hydrogen stable isotopes (∂D) is a potentially powerful tool for studying animal ecology. Unlike other stable isotopes used in ecological research, however, we are less familiar with the physiological processes that influence the incorporation of hydrogen isotopes from dietary resources to animal tissues. Here we present the results of a controlled feeding experiment utilizing Japanese quail (Cortunix japonica) that was designed to: (1) estimate the relative contributions of diet to the ∂D signatures of blood plasma, red blood cells, intestine, liver, muscle and feathers; (2) investigate possible differences among these same tissues in diet to tissue discrimination; and (3) explore the differences in incorporation dynamics between deuterium (2H) and a well-studied isotope, 13C, for blood plasma solids and red blood cells. Tissues differed in both the relative contribution of diet to tissue ∂D and diet to tissue discrimination. The average residence time of both hydrogen and carbon was significantly lower in plasma solids than in red blood cells. The average residence time of hydrogen was significantly lower than that of carbon in plasma solids, but not in red blood cells. Although the average residence times of hydrogen and carbon were positively correlated, the correlation was weak. Hence the incorporation of carbon seems to be a poor predictor of that of hydrogen.

Effects of Trophic Level and Metamorphosis on Discrimination of Hydrogen Isotopes in a Plant-Herbivore System

The use of stable isotopes in ecological studies requires that we know the magnitude of discrimination factors between consumer and element sources. The causes of variation in discrimination factors for carbon and nitrogen have been relatively well studied. In contrast, the discrimination factors for hydrogen have rarely been measured. We grew cabbage looper caterpillars (Trichoplusia ni) on cabbage (Brassica oleracea) plants irrigated with four treatments of deuterium-enriched water (δD = −131, −88, −48, and −2‰, respectively), allowing some of them to reach adulthood as moths. Tissue δD values of plants, caterpillars, and moths were linearly correlated with the isotopic composition of irrigation water. However, the slope of these relationships was less than 1, and hence, discrimination factors depended on the δD value of irrigation water. We hypothesize that this dependence is an artifact of growing plants in an environment with a common atmospheric δD value. Both caterpillars and moths were significantly enriched in deuterium relative to plants by ∼45‰ and 23‰ respectively, but the moths had lower tissue to plant discrimination factors than did the caterpillars. If the trophic enrichment documented here is universal, δD values must be accounted for in geographic assignment studies. The isotopic value of carbon was transferred more or less faithfully across trophic levels, but δ15N values increased from plants to insects and we observed significant non-trophic 15N enrichment in the metamorphosis from larvae to adult.

Variability in the routing of dietary proteins and lipids to consumer tissues influences tissue-specific isotopic discrimination.

RATIONALE The eco-physiological mechanisms that govern the incorporation and routing of macronutrients from dietary sources into consumer tissues determine the efficacy of stable isotope analysis (SIA) for studying animal foraging ecology. We document how changes in the relative amounts of dietary proteins and lipids affect the metabolic routing of these macronutrients and the consequent effects on tissue-specific discrimination factors in domestic mice using SIA. We also examine the effects of dietary macromolecular content on a commonly used methodological approach: lipid extraction of potential food sources. METHODS We used carbon ((13) C) and nitrogen ((15) N) isotopes to examine the routing of carbon from dietary proteins and lipids that were used by mice to biosynthesize hair, blood, muscle, and liver. Growing mice were fed one of four diet treatments in which the total dietary content of C4 -based lipids (δ(13) C = -14.5‰) and C(3) -based proteins (δ(13) C = -27‰) varied inversely between 5% and 40%. RESULTS The δ(13) C values of mouse tissues increased by approximately 2-6‰ with increasing dietary lipid content. The difference in δ(13) C values between mouse tissues and bulk diet ranged from 0.1 ± 1.5‰ to 2.3 ± 0.6‰ for all diet treatments. The mean (±SD) difference between the δ(13) C values of mouse tissues and dietary protein varied systematically among tissues and ranged from 3.1 ± 0.1‰ to 4.5 ± 0.6‰ for low fat diets and from 5.4 ± 0.4‰ to 10.5 ± 7.3‰ for high fat diets. CONCLUSIONS Mice used some fraction of their dietary lipid carbon to synthesize tissue proteins, suggesting flexibility in the routing of dietary macromolecules to consumer tissues based on dietary macromolecular availability. Consequently, all constituent dietary macromolecules, not just protein, should be considered when determining the relationship between diets and consumer tissues using SIA. In addition, in cases where animals consume diets with high lipid contents, non lipid-extracted prey samples should be analyzed to estimate diets using SIA.

The Relationship between Drinking Water and the Hydrogen and Oxygen Stable Isotope Values of Tissues in Japanese Quail (Cortunix japonica)

ABSTRACT. Developing an understanding of how differences in the dietary ecology and physiology of different species can influence the incorporation of hydrogen and oxygen from resources into consumer tissues is an important factor to consider when designing, or interpreting data from, ecological studies using stable isotope analyses of these elements. Here, we present the results of an experiment designed to examine the relationship between the &dgr;2H and &dgr;18O of drinking water and the &dgr;2H and &dgr;18O values of body water, blood plasma, red blood cells, intestine, liver, muscle, and feathers of Japanese Quail (Cortunix japonica). Because Japanese Quail have high drinking-water requirements compared with many other bird species, we hypothesized that the relative contribution of drinking water to tissue &dgr;2H and &dgr;18O values would be higher than that of bird species with lower drinking-water requirements. Our results demonstrate that the contribution of drinking water to the &dgr;2H and &dgr;18O values of tissues is generally higher in Japanese Quail than in other birds with lower drinking-water rates. However, we failed to find significant relationships between drinking-water isotope values and tissue isotope values in many of the tissues that we examined. We suspect that this lack of significant relationships is the result of variation in tissue isotope values caused by differences in drinking-water consumption rates among individual birds. Given these results, we recommend that researchers use caution when interpreting data from ecological investigations using &dgr;2H and &dgr;18O analyses.

Environmental and Physiological Influences to Isotopic Ratios of N and Protein Status in a Montane Ungulate in Winter

Winter severity can influence large herbivore populations through a reduction in maternal proteins available for reproduction. Nitrogen (N) isotopes in blood fractions can be used to track the use of body proteins in northern and montane ungulates. We studied 113 adult female caribou for 13 years throughout a series of severe winters that reduced population size and offspring mass. After these severe winters, offspring mass increased but the size of the population remained low. We devised a conceptual model for routing of isotopic N in blood in the context of the severe environmental conditions experienced by this population. We measured δ15N in three blood fractions and predicted the relative mobilization of dietary and body proteins. The δ 15N of the body protein pool varied by 4‰ and 46% of the variance was associated with year. Annual variation in δ15N of body protein likely reflected the fall/early winter diet and winter locations, yet 15% of the isotopic variation in amino acid N was due to body proteins. Consistent isotopic differences among blood N pools indicated that animals tolerated fluxes in diet and body stores. Conservation of body protein in caribou is the result of active exchange among diet and body N pools. Adult females were robust to historically severe winter conditions and prioritized body condition and survival over early investment in offspring. For a vagile ungulate residing at low densities in a predator-rich environment, protein restrictions in winter may not be the primary limiting factor for reproduction.