Tissue-specific Isotopic Incorporation Turnover Rates and Trophic Discrimination Factors in the Freshwater Shrimp Macrobrachium borellii (Crustacea: Decapoda: Palaemonidae).

The interpretation of isotopic data in ecology requires knowledge about two factors: turnover rate and the trophic discrimination factor, which have not been well described in freshwater shrimps. We performed a 142-day diet shift experiment on 174 individuals of the omnivorous shrimp Macrobrachium borellii, measured their growth, and temporally serially sampled muscle and hepatopancreas tissue to quantify carbon and nitrogen incorporation rates and isotope discrimination factors. Shrimps were fed with artificial diets (δ13C = -26.1‰, δ15N= 2.1‰) for 45 days in attempt to standardize the shrimps' initial δ13C and δ15N values for subsequent experiments. Shrimps were then fed with another artificial diet (δ13C = -16.1‰, δ15N = 15.8‰) and the change in δ13C and δ15N was observed for a period of 97 days. The trophic discrimination factor (∆) for δ13C was significantly higher in hepatopancreas (0.7 ± 0.36‰) than in muscle (-0.1 ± 0.83‰); however, the opposite was the case for δ15N (1.7 ± 0.43‰ and 3.6 ± 0.42‰, respectively). In the hepatopancreas the mean residence time (τ) of 13C was 26.3 ± 4.3 days compared to a residence time of 16.6 ± 5.51 days for δ15N, whereas the τ in muscle was 75.8 ± 25 days for δ13C and 40 ± 25 days for δ15N. The rate of incorporation of carbon into muscle was higher than that predicted by allometric equations relating isotopic incorporation rate to body mass that was developed previously for invertebrates. Our results support ranges of traditional trophic discrimination factor values observed in muscles samples of different taxa (∆15N around 3‒3.5‰ and ∆13C around 0‒1‰), but our work provides evidence that these traditionally used values may vary in other tissues, as we found that in the hepatopancreas ∆15N is around 1.7‰.

Isotopic incorporation rates and discrimination factors in mantis shrimp crustaceans.

Stable isotope analysis has provided insights into the trophic ecology of a wide diversity of animals. Knowledge about isotopic incorporation rates and isotopic discrimination between the consumer and its diet for different tissue types is essential for interpreting stable isotope data, but these parameters remain understudied in many animal taxa and particularly in aquatic invertebrates. We performed a 292-day diet shift experiment on 92 individuals of the predatory mantis shrimp, Neogonodactylus bredini, to quantify carbon and nitrogen incorporation rates and isotope discrimination factors in muscle and hemolymph tissues. Average isotopic discrimination factors between mantis shrimp muscle and the new diet were 3.0 ± 0.6 ‰ and 0.9 ± 0.3 ‰ for carbon and nitrogen, respectively, which is contrary to what is seen in many other animals (e.g. C and N discrimination is generally 0-1 ‰ and 3-4 ‰, respectively). Surprisingly, the average residence time of nitrogen in hemolymph (28.9 ± 8.3 days) was over 8 times longer than that of carbon (3.4 ± 1.4 days). In muscle, the average residence times of carbon and nitrogen were of the same magnitude (89.3 ± 44.4 and 72.8 ± 18.8 days, respectively). We compared the mantis shrimps' incorporation rates, along with rates from four other invertebrate taxa from the literature, to those predicted by an allometric equation relating carbon incorporation rate to body mass that was developed for teleost fishes and sharks. The rate of carbon incorporation into muscle was consistent with rates predicted by this equation. Our findings provide new insight into isotopic discrimination factors and incorporation rates in invertebrates with the former showing a different trend than what is commonly observed in other animals.

Spatiotemporal mutualistic model of mistletoes and birds.

A mathematical model which incorporates the spatial dispersal and interaction dynamics of mistletoes and birds is derived and studied to gain insights of the spatial heterogeneity in abundance of mistletoes. Fickian diffusion and chemotaxis are used to model the random movement of birds and the aggregation of birds due to the attraction of mistletoes, respectively. The spread of mistletoes by birds is expressed by a dispersal operator, which is typically a convolution integral with a dispersal kernel. Two different types of kernel functions are used to study the model, one is a Dirac delta function which reflects the special case that the spread behavior is local, and the other one is a general non-negative symmetric function which describes the nonlocal spread of mistletoes. When the kernel function is taken as the Dirac delta function, the threshold condition for the existence of mistletoes is given and explored in terms of parameters. For the general non-negative symmetric kernel case, we prove the existence and stability of spatially nonhomogeneous equilibria. Numerical simulations are conducted by taking specific forms of kernel functions. Our study shows that the spatial heterogeneous patterns of mistletoes are related to the specific dispersal pattern of birds which carry mistletoe seeds.

The isotopic composition and insect content of diet predict tissue isotopic values in a South American passerine assemblage.

We analyzed the carbon and nitrogen isotopic values of the muscle, liver, and crop contents ("diet") of 132 individuals of 16 species of Chilean birds. The nitrogen content of diet was tightly correlated with the fraction of gut contents represented by insects relative to plant material. The carbon and nitrogen isotopic values of diet, liver, and muscle were all linearly correlated, implying high temporal consistency in the isotopic value of the diet of these birds. However, δ(15)N was not significantly related with the percentage of insects in diet. These results cast doubt on the applicability of the use of (15)N enrichment to diagnose trophic level in, at least some, terrestrial ecosystems. However, the residuals of the relationship relating the isotopic value of bird tissues with those of their diet were weakly negatively correlated with insect intake. We hypothesize that this negative correlation stems from the higher quality of protein found in insects relative to that of plant materials. Finally, our data corroborated a perplexing and controversial negative relationship between tissue to diet isotopic discrimination and the isotopic value of diet. We suggest that this relationship is an example of the commonly observed regression to the mean effect that plagues many scientific studies.

Isotopic incorporation rates for shark tissues from a long-term captive feeding study.

Stable isotope analysis has provided insight into the dietary and habitat patterns of many birds, mammals and teleost fish. A crucial biological parameter to interpret field stable isotope data is tissue incorporation rate, which has not been well studied in large ectotherms. We report the incorporation of carbon and nitrogen into the tissues of leopard sharks (Triakis semifasciata). Because sharks have relatively slow metabolic rates and are difficult to maintain in captivity, no long-term feeding study has been conducted until the point of isotopic steady state with a diet. We kept six leopard sharks in captivity for 1250 days, measured their growth, and serially sampled plasma, red blood cells and muscle for stable carbon and nitrogen isotope analysis. A single-compartment model with first-order kinetics adequately described the incorporation patterns of carbon and nitrogen isotopes for these three tissues. Both carbon and nitrogen were incorporated faster in plasma than in muscle and red blood cells. The rate of incorporation of carbon into muscle was similar to that predicted by an allometric equation relating isotopic incorporation rate to body mass that was developed previously for teleosts. In spite of their large size and unusual physiology, the rates of isotopic incorporation in sharks seem to follow the same patterns found in other aquatic ectotherms.

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

The analysis of hydrogen stable isotopes (δD) 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 δD 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 ((2)H) and a well-studied isotope, (13)C, for blood plasma solids and red blood cells. Tissues differed in both the relative contribution of diet to tissue δD 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.

Spatiotemporal variation of mistletoes: a dynamic modeling approach.

Mistletoes are common aerial stem-parasites and their seeds are dispersed by fruit-eating birds. In the mutually beneficial relationships between mistletoes and bird species that disperse mistletoes' seeds, the preference of birds for infected trees influences the spread of mistletoes and the spatiotemporal pattern formation of mistletoes. We formulate a deterministic model to describe the dynamics of mistletoes in an isolated patch containing an arbitrary number of trees. We establish concrete criterions, expressed in terms of the model parameters, for mistletoes establishing in this area. We conduct numerical simulations based on a field study to reinforce and expand our results.

The influence of drinking water on the deltaD and delta18O values of house sparrow plasma, blood and feathers.

We investigated the relationships between the δdeuterium (δD) and the δ(18)oxygen (δ(18)O) of drinking water and the δD and δ(18)O 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 ((18)O in blood plasma), found linear relationships between the δD and δ(18)O 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 δ(18)O values of feathers, red blood cells and plasma solids. In feathers and red blood cells, δD and δ(18)O 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 δ(18)O values of different tissues must account for inter-tissue discrimination factors; and (3) δD/δ(18)O 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.

The fate of carbon in growing fish: an experimental study of isotopic routing.

The application of stable isotope analysis to ecology requires estimating the contribution of different isotopic sources to the isotopic signatures of an animal's tissues using mixing models. These models make the physiologically unrealistic assumption that assimilated nutrients are disassembled into their elemental components and that these atoms are then reassembled into biomolecules. We quantified the extent to which mixing models yield erroneous results with an experiment using Nile tilapia (Oreochromis niloticus). The tilapia were fed synthetic diets that varied in protein content and in which the carbon isotopic composition of protein differed widely from that of carbohydrates and lipids. We predicted that dietary protein would contribute disproportionately to the carbon in muscle, whereas the nonprotein components of diet would contribute disproportionately to lipids. Our experiment also allowed us to test the conjecture of a positive correlation between the (15)N enrichment in tissues and protein intake. As predicted, the contribution of protein carbon to muscle was higher than that expected by the assumptions of isotopic mixing in all treatments except that with the lowest dietary protein content. We hypothesized that the unexpectedly high contribution of nonprotein carbon to muscle was the result of assimilating both dispensable and indispensable amino acids synthesized by the fishes' gut microbiota. Although we expected the contribution of carbon in nonprotein dietary ingredients to be higher than expected from a mixing model, we found that protein contributed more than expected, probably as a result of differences in amino acid composition between diet and tissues, which led to excess carbon used for lipid synthesis. Finally, our results verified the positive relationship between dietary protein content and the enrichment in tissue (15)N. Assuming perfect mixing in field isotopic studies can lead to erroneous inferences about the relative contributions of different sources to an animal's diet.

Dietary and isotopic specialization: the isotopic niche of three Cinclodes ovenbirds.

By comparing the isotopic composition of tissues deposited at different times, we can identify individuals that shift diets over time and individuals with constant diets. We define an individual as an isotopic specialist if tissues deposited at different times have similar isotopic composition. If tissues deposited at different times differ in isotopic composition we define an individual as an isotopic generalist. Individuals can be dietary generalists but isotopic specialists if they feed on the same resource mixture at all times. We assessed the degree of isotopic and dietary specialization in three related Chilean bird species that occupy coastal and/or freshwater environments: Cinclodes oustaleti, Cinclodes patagonicus, and Cinclodes nigrofumosus. C. oustaleti individuals were both isotopic and dietary generalists. Tissues deposited in winter (liver and muscle) had distinct stable C (delta(13)C) and stable N isotope ratio (delta(15)N) values from tissues deposited in the summer (wing feathers) suggesting that birds changed the resources that they used seasonally from freshwater habitats in the summer to coastal habitats in the winter. Although the magnitude of seasonal isotopic change was high, the direction of isotopic change varied little among individuals. C. patagonicus included both isotopic specialists and generalists, as well as dietary specialists and generalists. The isotopic composition of the feathers and liver of some C. patagonicus individuals was similar, whereas that of others differed. In C. patagonicus, there were large inter-individual differences in the magnitude and the direction of seasonal isotopic change. All individuals of C. nigrofumosus were both isotopic and dietary specialists. The distribution of delta(13)C and delta(15)N values overlapped broadly among tissues and clustered in a small, and distinctly intertidal, region of delta space. Assessing individual specialization and unraveling the factors that influence it, have been key questions in animal ecology for decades. Stable isotope analyses of several tissues in appropriate study systems provide an unparalleled opportunity to answer them.

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.

Should we use one-, or multi-compartment models to describe (13)C incorporation into animal tissues?

Understanding rates of isotopic incorporation and discrimination factors between tissues and diet is an important focus of ecologists seeking to use stable isotopes to track temporal changes in diet. We used a diet-shift experiment to measure differences among tissues in (13)C incorporation rates in house sparrows (Passer domesticus). We predicted faster incorporation rates in splanchnic than in structural tissues. We also assessed whether isotopic incorporation data were better supported by the one-compartment models most commonly used by ecologists or by multi-compartment models. We found large differences in the residence time of (13)C among tissues and, as predicted, splanchnic tissues had faster rates of isotopic incorporation and thus shorter retention times than structural tissues. We found that one-compartment models supported isotopic incorporation data better in breath, excreta, red blood cells, bone collagen, and claw tissues. However, data in plasma, intestine, liver, pectoralis muscle, gizzard, and intestine tissues supported two-compartment models. More importantly, the inferences that we derived from the two types of models differed. Two-compartment models estimated longer (13)C residence times, and smaller tissue to diet differences in isotopic composition, than one-compartment models. Our study highlights the importance of considering both one- and multi-compartment models when interpreting laboratory and field isotopic incorporation studies. It also emphasizes the opportunities that measuring several tissues with contrasting isotopic residence times offer to elucidate animal diets at different time scales.

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

The rate at which an animal's 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 (15)N 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 (15)N in plasma proteins and blood cells. The diet with the highest protein content led to a (15)N retention time of 21 and 5 days for cells and plasma, respectively. In contrast, average (15)N 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 factor Delta(15)N=delta(15)N(tissues)-delta(15)N(diet) 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 (15)N relative to diet. In contrast, ureteral urine was either significantly depleted of (15)N in birds fed the diet with the lowest protein content or did not differ in delta(15)N 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.

The intake responses of three species of leaf-nosed Neotropical bats.

Flower-visiting bats encounter nectars that vary in both sugar composition and concentration. Because in the new world, the nectars of bat-pollinated flowers tend to be dominated by hexoses, we predicted that at equicaloric concentrations, bats would ingest higher volumes of hexoses than sucrose-containing nectars. We investigated the intake response of three species of Neotropical bats, Leptonycteris curasoae, Glossophaga soricina and Artibeus jamaicensis, to sugar solutions of varying concentrations (292, 438, 584, 730, 876, and 1,022 mmol L(-1)) consisting of either sucrose or 1:1 mixtures of glucose and fructose solutions. Bats did not show differences in their intake response to sucrose and 1:1 glucose-fructose solutions, indicating that digestion and absorption in bat intestines are designed under the principle of symmorphosis, in which no step is more limiting than the other. Our results also suggest that, on the basis of energy intake, bats should not prefer hexoses over sucrose. We used a mathematical model that uses the rate of sucrose hydrolysis measured in vitro and the small intestinal volume of bats to predict the rate of nectar intake as a function of sugar concentration. The model was a good predictor of the intake responses of L. curasoae and G. soricina, but not of A. jamaicensis.

Inference in ecology and evolution.

Most ecologists and evolutionary biologists continue to rely heavily on null hypothesis significance testing, rather than on recently advocated alternatives, for inference. Here, we briefly review null hypothesis significance testing and its major alternatives. We identify major objectives of statistical analysis and suggest which analytical approaches are appropriate for each. Any well designed study can improve our understanding of biological systems, regardless of the inferential approach used. Nevertheless, an awareness of available techniques and their pitfalls could guide better approaches to data collection and broaden the range of questions that can be addressed. Although we should reduce our reliance on significance testing, it retains an important role in statistical education and is likely to remain fundamental to the falsification of scientific hypotheses.

Hummingbirds rely on both paracellular and carrier-mediated intestinal glucose absorption to fuel high metabolism.

Twenty years ago, the highest active glucose transport rate and lowest passive glucose permeability in vertebrates were reported in Rufous and Anna's hummingbirds (Selasphorus rufus, Calypte anna). These first measurements of intestinal nutrient absorption in nectarivores provided an unprecedented physiological foundation for understanding their foraging ecology. They showed that physiological processes are determinants of feeding behaviour. The conclusion that active, mediated transport accounts for essentially all glucose absorption in hummingbirds influenced two decades of subsequent research on the digestive physiology and nutritional ecology of nectarivores. Here, we report new findings demonstrating that the passive permeability of hummingbird intestines to glucose is much higher than previously reported, suggesting that not all sugar uptake is mediated. Even while possessing the highest active glucose transport rates measured in vertebrates, hummingbirds must rely partially on passive non-mediated intestinal nutrient absorption to meet their high mass-specific metabolic demands.

Metabolic substrate use and the turnover of endogenous energy reserves in broad-tailed hummingbirds (Selasphorus platycercus).

We fed broad-tailed hummingbirds (Selasphorus platycercus) diets of contrasting carbon isotope composition and measured changes in the delta(13)C of expired breath through time. By measuring the delta(13)C in the breath of fed and fasted birds we were able to quantify the fraction of metabolism fueled by assimilated sugars and endogenous energy reserves. These measurements also allowed us to estimate the fractional turnover of carbon in the hummingbirds' energy reserves. When hummingbirds were feeding, they fueled their metabolism largely ( approximately 90%) with assimilated sugars. The rate of carbon isotope incorporation into the energy reserves of hummingbirds was higher when birds were gaining as opposed to losing body mass. The average residence time of a carbon atom in the hummingbirds' energy reserves ranged from 1 to 2 days.

Osmoregulatory capacity and the ability to use marine food sources in two coastal songbirds (Cinclodes: Furnariidae) along a latitudinal gradient.

Cinclodes nigrofumosus and C. oustaleti are two closely related songbirds that inhabit the northern Chilean coast during the austral fall and winter. This stretch spans a dramatic north to south latitudinal gradient in rainfall and temperature. Whereas C. nigrofumosus lives exclusively on coastal environments, C. oustaleti shifts seasonally from coastal environments to inland freshwater ones. We used the delta13C of these two species' tissues to investigate whether the reliance on marine versus terrestrial sources varied from the hyper-arid north to the wet south. We also investigated latitudinal variation in the renal traits that mediate how these birds cope with dehydration and a salty marine diet. Both species increased the incorporation of terrestrial carbon, as measured by delta13C, as terrestrial productivity increased southwards. However, C. nigrofumosus had consistently more positive (i.e. more marine) and less variable delta13C values than C. oustaleti. The osmoregulatory traits of both species varied with latitude as well. Urine osmolality decreased from extremely high values in the north to moderate values in the south, while C. nigrofumosus produced more concentrated urine than C. oustaleti. In both species, the proportion of kidney devoted to medullary tissue decreased from north to south, and kidney size increased significantly with latitude. Cinclodes nigrofumosus had larger kidneys with larger proportions of medullary tissue than C. oustaleti. C. nigrofumosus and C. oustaleti are terrestrial organisms subsidized by a rich marine environment where it is adjacent to an unproductive terrestrial. Variation in the reliance on marine food sources seems to be accompanied by adjustments in the osmoregulatory mechanisms used by these birds to cope with salt and dehydration.

Are the low protein requirements of nectarivorous birds the consequence of their sugary and watery diet? A test with an omnivore.

Nectar-feeding birds have remarkably low nitrogen requirements. These may be due either to adaptation to a low-protein diet or simply to feeding on a fluid diet that minimizes metabolic fecal nitrogen losses. We measured minimal nitrogen requirements (MNR) and total endogenous nitrogen loss (TENL) in the omnivorous European starling Sturnus vulgaris, fed on an artificial nectar-like fluid diet of varying concentrations of sugar and protein. The MNR and TENL of the birds were similar and even slightly higher than allometrically expected values for birds of the starlings' mass (140% and 103%, respectively). This suggests that the low measured nitrogen requirements of nectar-feeding birds are not simply the result of their sugary and watery diets but a physiological adaptation to the low nitrogen input. We also measured the effect of water and protein intake on the nitrogenous waste form in the excreta and ureteral urine in European starlings. Neither high water intake nor low protein intake increased the fraction of nitrogen excreted as ammonia. Ammonia was excreted at consistently low levels by the starlings, and its concentration was significantly higher in ureteral urine than in excreta. We hypothesize that ureteral ammonia was reabsorbed in the lower intestine, indicating a postrenal modification of the urine.