Elimination of 15N-thymidine after oral administration in human infants.

BACKGROUND: We have developed a new clinical research approach for the quantification of cellular proliferation in human infants to address unanswered questions about tissue renewal and regeneration. The approach consists of oral 15N-thymidine administration to label cells in S-phase, followed by Multi-isotope Imaging Mass Spectrometry for detection of the incorporated label in cell nuclei. To establish the approach, we performed an observational study to examine uptake and elimination of 15N-thymidine. We compared at-home label administration with in-hospital administration in infants with tetralogy of Fallot, a form of congenital heart disease, and infants with heart failure. METHODS: We examined urine samples from 18 infants who received 15N-thymidine (50 mg/kg body weight) by mouth for five consecutive days. We used Isotope Ratio Mass Spectrometry to determine enrichment of 15N relative to 14N (%) in urine. RESULTS/FINDINGS: 15N-thymidine dose administration produced periodic rises of 15N enrichment in urine. Infants with tetralogy of Fallot had a 3.2-fold increase and infants with heart failure had a 4.3-fold increase in mean peak 15N enrichment over baseline. The mean 15N enrichment was not statistically different between the two patient populations (p = 0.103). The time to peak 15N enrichment in tetralogy of Fallot infants was 6.3 ± 1 hr and in infants with heart failure 7.5 ± 2 hr (mean ± SEM). The duration of significant 15N enrichment after a dose was 18.5 ± 1.7 hr in tetralogy of Fallot and in heart failure 18.2 ± 1.8 hr (mean ± SEM). The time to peak enrichment and duration of enrichment were also not statistically different (p = 0.617 and p = 0.887). CONCLUSIONS: The presented results support two conclusions of significance for future applications: (1) Demonstration that 15N-thymidine label administration at home is equivalent to in-hospital administration. (2) Two different types of heart disease show no differences in 15N-thymidine absorption and elimination. This enables the comparative analysis of cellular proliferation between different types of heart disease.

Foraging consistency of coral reef fishes across environmental gradients in the central Pacific.

We take advantage of a natural gradient of human exploitation and oceanic primary production across five central Pacific coral reefs to examine foraging patterns in common coral reef fishes. Using stomach content and stable isotope (δ15N and δ13C) analyses, we examined consistency across islands in estimated foraging patterns. Surprisingly, species within the piscivore-invertivore group exhibited the clearest pattern of foraging consistency across all five islands despite there being a considerable difference in mean body mass (14 g-1.4 kg) and prey size (0.03-3.8 g). In contrast, the diets and isotopic values of the grazer-detritivores varied considerably and exhibited no consistent patterns across islands. When examining foraging patterns across environmental contexts, we found that δ15N values of species of piscivore-invertivore and planktivore closely tracked gradients in oceanic primary production; again, no comparable patterns existed for the grazer-detritivores. The inter-island consistency in foraging patterns within the species of piscivore-invertivore and planktivore and the lack of consistency among species of grazer-detritivores suggests a linkage to different sources of primary production among reef fish functional groups. Our findings suggest that piscivore-invertivores and planktivores are likely linked to well-mixed and isotopically constrained allochthonous oceanic primary production, while grazer-detritivores are likely linked to sources of benthic primary production and autochthonous recycling. Further, our findings suggest that species of piscivore-invertivore, independent of body size, converge toward consuming low trophic level prey, with a hypothesized result of reducing the number of steps between trophic levels and increasing the trophic efficiency at a community level.

Predicting species' vulnerability in a massively perturbed system: the fishes of Lake Turkana, Kenya.

BACKGROUND AND TROPHIC DIVERSITY STUDY: Lake Turkana is an understudied desert lake shared by Kenya and Ethiopia. This system is at the precipice of large-scale changes in ecological function due to climate change and economic development along its major inflowing river, the Omo River. To anticipate response by the fish community to these changes, we quantified trophic diversity for seven ecological disparate species (Alestes baremose, Hydrocynus forskalli, Labeo horie, Lates niloticus, Oreochromis niloticus, Synodontis schall, and Tilapia zillii) using stable isotopes. Based on their marked morphological differentiation, we postulated that dietary niches of these species would be similar in size but show little overlap. The degree of trophic diversity varied greatly among the species studied, refuting our hypothesis regarding dietary niche size. Oreochromis niloticus and L. niloticus had the highest trophic diversity and significantly larger dietary niches than T. zillii, A. baremose and H. forskalli. Low overlap among the dietary niches of the seven species, with the exception of the synodontid catfish S. schall, is consistent with our second hypothesis. PREDICTING SPECIES' VULNERABILITY: Breeding vulnerability was highest among those species with the lowest trophic diversity. We predict that in suffering two strikes against them, A. baremose, H. forskalli, T. zillii, and L. horie will be most affected by the highly altered Lake Turkana ecosystem and that O. niloticus, L. niloticus and S. schall will be least affected. Low vulnerability among O. niloticus and L. niloticus is promising for the future of the lake's fishery, but the third most important fishery species (L. horie) will be highly vulnerable to impending ecosystem change. T. zillii should be treated as separate from O. niloticus in the fishery given higher sensitivity and a different ecological role. We see potential for expansion of the fishery for S. schall but don't recommend the development of a fishery for A. baremose and H. forskalli.

Nutrient routing in omnivorous animals tracked by stable carbon isotopes in tissue and exhaled breath.

Omnivorous animals feed on several food items that often differ in macronutrient and isotopic composition. Macronutrients can be used for either metabolism or body tissue synthesis and, therefore, stable C isotope ratios of exhaled breath (delta(13)C(breath)) and tissue may differ. To study nutrient routing in omnivorous animals, we measured delta(13)C(breath) in 20-g Carollia perspicillata that either ate an isotopically homogeneous carbohydrate diet or an isotopically heterogeneous protein-carbohydrate mixture. The delta(13)C(breath) converged to the delta(13)C of the ingested carbohydrates irrespective of whether proteins had been added or not. On average, delta(13)C(breath) was depleted in (13)C by only ca. -2 per thousand in relation to the delta(13)C of the dietary carbohydrates and was enriched by +8.2 per thousand in relation to the dietary proteins, suggesting that C. perspicillata may have routed most ingested proteins to body synthesis and not to metabolism. We next compared the delta(13)C(breath) with that of wing tissue (delta(13)C(tissue)) in 12 free-ranging, mostly omnivorous phyllostomid bat species. We predicted that species with a more insect biased diet--as indicated by the N isotope ratio in wing membrane tissue (delta(15)N(tissue))--should have higher delta(13)C(tissue) than delta(13)C(breath) values, since we expected body tissue to stem mostly from insect proteins and exhaled CO(2) to stem from the combustion of fruit carbohydrates. Accordingly, delta(13)C(tissue) and delta(13)C(breath) should be more similar in species that feed predominantly on plant products. The species-specific differences between delta(13)C(tissue) and delta(13)C(breath) increased with increasing delta(15)N(tissue), i.e. species with a plant-dominated diet had similar delta(13)C(tissue) and delta(13)C(breath) values, whereas species feeding at a higher trophic level had higher delta(13)C(tissue) than delta(13)C(breath) values. Our study shows that delta(13)C(breath) reflect the isotope ratio of ingested carbohydrates, whereas delta(13)C of body tissue reflect the isotope ratio of ingested proteins, namely insects, supporting the idea of isotopic routing in omnivorous animals.

Effects of sample preparation on stable isotope ratios of carbon and nitrogen in marine invertebrates: implications for food web studies using stable isotopes.

Trophic ecology has benefitted from the use of stable isotopes for the last three decades. However, during the last 10 years, there has been a growing awareness of the isotopic biases associated with some pre-analytical procedures that can seriously hamper the interpretation of food webs. We have assessed the extent of such biases by: (1) reviewing the literature on the topic, and (2) compiling C and N isotopic values of marine invertebrates reported in the literature with the associated sample preparation protocols. The factors considered were: acid-washing, distilled water rinsing (DWR), sample type (whole individuals or pieces of soft tissues), lipid content, and gut contents. Two-level ANOVA revealed overall large and highly significant effects of acidification for both delta(13)C values (up to 0.9 per thousand decrease) and delta(15) N values (up to 2.1 per thousand decrease in whole individual samples, and up to 1.1 per thousand increase in tissue samples). DWR showed a weak overall effect with delta(13)C increments of 0.6 per thousand (for the entire data set) or decrements of 0.7 per thousand in delta(15) N values (for tissue samples). Gut contents showed no overall significant effect, whereas lipid extraction resulted in the greatest biases in both isotopic signatures (delta(13)C, up to -2.0 per thousand in whole individuals; delta(15)N, up to +4.3 per thousand in tissue samples). The study analyzed separately the effects of the various factors in different taxonomic groups and revealed a very high diversity in the extent and direction of the effects. Maxillopoda, Gastropoda, and Polychaeta were the classes that showed the largest isotopic shifts associated with sample preparation. Guidelines for the standardization of sample preparation protocols for isotopic analysis are proposed both for large and small marine invertebrates. Broadly, these guidelines recommend: (1) avoiding both acid washing and DWR, and (2) performing lipid extraction and gut evacuation in most cases.

Nutrition or detoxification: why bats visit mineral licks of the Amazonian rainforest.

Many animals in the tropics of Africa, Asia and South America regularly visit so-called salt or mineral licks to consume clay or drink clay-saturated water. Whether this behavior is used to supplement diets with locally limited nutrients or to buffer the effects of toxic secondary plant compounds remains unclear. In the Amazonian rainforest, pregnant and lactating bats are frequently observed and captured at mineral licks. We measured the nitrogen isotope ratio in wing tissue of omnivorous short-tailed fruit bats, Carollia perspicillata, and in an obligate fruit-eating bat, Artibeus obscurus, captured at mineral licks and at control sites in the rainforest. Carollia perspicillata with a plant-dominated diet were more often captured at mineral licks than individuals with an insect-dominated diet, although insects were more mineral depleted than fruits. In contrast, nitrogen isotope ratios of A. obscurus did not differ between individuals captured at mineral lick versus control sites. We conclude that pregnant and lactating fruit-eating bats do not visit mineral licks principally for minerals, but instead to buffer the effects of secondary plant compounds that they ingest in large quantities during periods of high energy demand. These findings have potential implications for the role of mineral licks for mammals in general, including humans.

Blood-sucking bugs as a gentle method for blood-collection in water budget studies using doubly labelled water.

During doubly-labelled water (DLW) experiments, blood collection by venous puncture may traumatize animals and consequently affect the animals' behaviour and energy budget. Recent studies have shown that blood-sucking bugs (Triatominae; Heteroptera) can be used instead of conventional needles to obtain blood from animals. In this paper, we validate the bug method in captive nectar-feeding bats, Glossophaga soricina, for water budget analysis by comparing the daily water flux estimated with the DLW method with values measured by an energy balance method. As the mean daily water flux of the DLW method was not significantly deviating from the expected value, blood-sucking bugs may substitute more invasive methods of blood collection in DLW experiments. Based on the DLW estimates, daily energy and water intake rates were calculated and compared to values measured with the energy balance method. The DLW method and the energy balance method yielded on average similar results regarding the daily energy intake (DLW method: 48.8+/-14.2 kJ d(-1) versus energy balance method: 48.1+/-9.9 kJ d(-1)) and daily water intake (DLW method: 13.7+/-2.4 mL d(-1) versus energy balance method: 14.7+/-3.0 mL d(-1)). Based on the calculated water and sugar intake per day, we estimated the sugar concentration of ingested nectar to equal on average 16.2+/-2.4% (mass/mass), which fell close to the measured sugar concentration of 17% (mass/mass) bats fed on during the experiment. We conclude that it is possible to extrapolate mean daily energy and water intake for animal groups, populations and species based on DLW estimates, but due to the large variance of results (low accuracy), it seems inadequate to calculate values for single individuals.

The energetics of trading nuptial gifts for copulations in katydids.

During copulation, male Isophya kraussi transfer a large nuptial gift to females. In this study, we hypothesized that the energy content of spermatophores should meet the energy requirements of both body maintenance and egg production of females. We measured the field metabolic rate of male and female I. kraussi using the doubly labeled water method and the energy content of spermatophores and male bodies with microbomb calorimetry. The energy content of male nuptial gifts averaged 0.66+/-0.09 kJ, approximately 20% of the total body energy content of male I. kraussi (3.24+/-0.26 kJ). Field metabolic rates averaged 0.41+/-0.17 kJ d(-1) (n = 8) for males and 0.30+/-0.15 kJ d(-1) (n = 5) for female I. kraussi. Thus, the energy content of spermatophores exceeded the daily energy requirements of existence in male I. kraussi. A single nuptial gift provides for all energy requirements of females for 1 or 2 d, depending on their activity, egg production, and ambient temperature. Because the shortest known remating interval of female katydids varies between 1 and 3 d, female I. kraussi could theoretically exist exclusively on spermatophores to meet their nutritional requirements.

Validation of a non-invasive blood-sampling technique for doubly-labelled water experiments.

Two techniques for bleeding small mammals have been used in doubly-labeled water (DLW) studies, including vena puncture and the use of starved nymphal stages of hematophagous reduviid bugs (Reduviidae, Hemiptera). In this study, we tested the validity of using reduviid bugs in doubly-labeled water experiments. We found that the isotope enrichment in initial blood samples collected with bugs was significantly lower compared to isotope enrichment in blood samples obtained using vena puncture. We therefore used the desiccation method for estimating total body water (TBW) in DLW experiments because TBW calculated using the isotope dilution method was overestimated when blood samples were collected using reduviid bugs. In our validation experiment with nectar-feeding bats (Glossophaga soricina), we compared estimates of daily energy expenditure (DEE) using DLW with those derived from the energy balance method. We considered Speakman's equation (controlling for 25% fractionated water loss) as the most appropriate for our study animal and calculated DEE accordingly. On average, DEE estimated with DLW was not significantly different from the mean value obtained with the energy balance method (mean deviation 1.2%). We conclude that although bug hemolymph or intestinal liquids most likely contaminate the samples, estimates of DEE are still valid because the DLW method does not depend on absolute isotope enrichments but on the rate of isotope decrease over time. However, dilution of blood with intestinal liquids or hemolymph from a bug may lead to larger variation in DEE estimates. We also tested how the relative error of DLW estimates changed with varying assumptions about fractionation. We used three additional equations for calculating DEE in DLW experiments. The basic equation for DLW experiments published by Lifson and McClintock (LM-6) assumes no fractionation, resulted in an overestimate of DEE by 10%. Nagy's equation (N-2) controls for changes in body mass but not for fractionation. Using Nagy's equation, DEE was overestimated by 8%. Under the assumption that 50% of total water flux fractionates, the alternative equation by Lifson and McClintock (LM-35) DEE was underestimated by 5%. The best fit between estimates of DEE based on DLW and energy balance measurements was derived by assuming that 32% of total water flux (TWF) is fractionated. We conclude that the outcome of DLW experiments is sensitive to assumptions regarding evaporative water loss, and thus recommend Speakman's equation 7.17 for use with bats.

Low turnover rates of carbon isotopes in tissues of two nectar-feeding bat species.

Stable isotopes of carbon are commonly used to characterize dietary preferences in animals. Because turnover rates of carbon isotopes are related to metabolic rate, we wanted to determine the rates at which carbon isotopes are exchanged in tissues of two species of nectar-feeding bats (Leptonycteris curasoae and Glossophaga soricina), both of which have relatively high mass-specific metabolic rates. To test the hypothesis that isotope turnover is higher in nectar-feeding bats, because of their high mass-specific metabolic rates, than in other eutherian mammals, we conducted diet-switching experiments and chose three target tissues (hair, wing membrane and blood) to evaluate the isotopic turnover rates. We made the following predictions: (1) isotopic composition should change towards higher delta(13)C-values due to the turnover of carbon isotopes of C(3) origin with those of C(4)/CAM origin; (2) the turnover rates of carbon isotopes would differ between the three types of tissues in the following order of decreasing turnover rates: blood>wing membrane>hair; and (3) turnover rates of nectar-feeding bats should exceed those reported for other small mammals because of the high mass-specific metabolic rate of nectar-feeding bats. Compared to the initial diet, target tissues were enriched in heavy carbon isotopes by 2.8 per thousand in L. curasoae and by 2.6 per thousand in G. soricina. After changing the diet from C(3) to C(4)/CAM origin we found an increase in abundance of (13)C in blood and wing membrane in all experimental subjects. The estimated half life of carbon isotope turnover ranged from 100 to 134 days and did not differ significantly between blood and wing membrane, nor did it differ between the two species. The low turnover rate in wing membrane may reflect its specific composition and the relatively low temperature of this tissue, and long-lived erythrocytes in bat blood may be responsible for the low turnover rate of carbon isotopes in blood. The turnover rate of stable carbon isotopes in hair was low in L. curasoae and undetectable in G. soricina, which may be explained by the seasonal growth of the hair in these two species. Because both species are small (10 and 25 g, respectively) and nectar-feeding bats have higher mass-specific metabolic rates than bats in temperate regions or similar sized terrestrial mammals, our findings of low turnover rates were unexpected.