Dietary and homeostatic controls of Zn isotopes in rats: A controlled-feeding experiment and modelling approach.

The stable isotope composition of zinc (δ66Zn), which is an essential trace metal for many biological processes in vertebrates, is increasingly used in ecological, archeological, and paleontological studies to assess diet and trophic level discrimination among vertebrates. However, the limited understanding of dietary controls and isotopic fractionation processes on Zn isotope variability in animal tissues and biofluids limits precise dietary reconstructions. The current study systematically investigates the dietary effects on Zn isotope composition in consumers using a combined controlled-feeding experiment and box-modelling approach. For this purpose, 21 rats were fed one of seven distinct animal- and plant-based diets and a total of 148 samples including soft and hard tissue, biofluid, and excreta samples of these individuals were measured for δ66Zn. Relatively constant Zn isotope fractionation is observed across the different dietary groups for each tissue type, implying that diet is the main factor controlling consumer tissue δ66Zn values, independent of diet composition. Furthermore, a systematic δ66Zn diet-enamel fractionation is reported for the first time, enabling diet reconstruction based on δ66Zn values from tooth enamel. In addition, we investigated the dynamics of Zn isotope variability in the body using a box-modelling approach, providing a model of Zn isotope homeostasis and inferring residence times, while also further supporting the hypothesis that δ66Zn values of vertebrate tissues are primarily determined by that of the diet. Altogether this provides a solid foundation for refined (paleo)dietary reconstruction using Zn isotopes of vertebrate tissues.

Reconstructing Pleistocene Australian herbivore megafauna diet using calcium and strontium isotopes.

Isotopes in fossil tooth enamel provide robust tools for reconstructing food webs, which have been understudied in Australian megafauna. To delineate the isotopic composition of primary consumers and understand dietary behaviour at the base of the food web, we investigate calcium (Ca) and strontium (Sr) isotope compositions of Pleistocene marsupial herbivores from Wellington Caves and Bingara (New South Wales, Australia). Sr isotopes suggest small home ranges across giant and smaller marsupial herbivores. Ca isotopes in Pleistocene marsupial herbivores cover the same range as those in modern wombats and placental herbivores. Early forming teeth are depleted in heavy Ca isotopes compared to late-forming teeth of a given individual, suggesting a weaning signal. Distinct Ca compositions between taxa can be interpreted as dietary niches. Some niches conform to previous dietary reconstructions of taxa, while others provide new insights into niche differentiation across Australian herbivores. Combined with the small roaming ranges suggested by Sr isotopes, the Ca isotope niche diversity suggests rich ecosystems, supporting a diversity of taxa with various diets in a small area.

Determination of water balance maintenance in Orcinus orca and Tursiops truncatus using oxygen isotopes.

The secondary adaptation of Cetacea to a fully marine lifestyle raises the question of their ability to maintain their water balance in a hyperosmotic environment. Cetacea have access to four potential sources of water: surrounding salt oceanic water, dietary free water, metabolic water and inhaled water vapour to a lesser degree. Here, we measured the 18O/16O oxygen isotope ratio of blood plasma from 13 specimens belonging to two species of Cetacea raised under human care (four killer whales Orcinus orca, nine common bottlenose dolphins Tursiops truncatus) to investigate and quantify the contribution of preformed water (dietary free water, surrounding salt oceanic water) and metabolic water to Cetacea body water using a box-modelling approach. The oxygen isotope composition of Cetacea blood plasma indicates that dietary free water and metabolic water contribute to more than 90% of the total water input in weight for cetaceans, with the remaining 10% consisting of inhaled water vapour and surrounding water accidentally ingested or absorbed through the skin. Moreover, the contribution of metabolic water appears to be more important in organisms with a more lipid-rich diet. Beyond these physiological and conservation biology implications, this study opens up questions that need to be addressed, such as the applicability of the oxygen isotope composition of cetacean body fluids and skeletal elements as an environmental proxy of the oxygen isotope composition of present and past marine waters.

Inferring odontocete life history traits in dentine using a multiproxy approach (δ15 N, δ44/42 Ca and trace elements).

RATIONALE: Understanding the interactions between marine mammals and their environment is critical for ecological and conservation purposes. Odontocetes offer a continuous record of their life history from birth as recorded in annual increments of their tooth dentine. Because dentine is not remodeled and contains collagen, nitrogen stable isotope compositions (δ15 N) reflect nursing and weaning events, life history traits that would otherwise be impossible to retrieve in such elusive marine animals. Yet, capturing the magnitude and temporal changes in these events is constrained by tooth size and sampling resolution. Moreover, historical and fossil specimens undergo collagen decay, hence the need to develop the measurements of other proxies. METHODS: Here, we present a multiproxy approach to investigate the use of Ca isotope compositions (δ44/42 Ca) in relation to δ15 N and laser ablation profiles for different trace metal (Ba, Mg, Sr, Zn) concentrations across the dentine of a single individual of the common bottlenose dolphin Tursiops truncatus. RESULTS: To help interpret the dentine data, we provide milk elemental compositions and δ44/42 Ca values for two odontocete individuals. We discuss the observed changes in δ44/42 Ca across the dentine as potential markers of birth, weaning interval, incidental ingestion of seawater, trophic level and physiology. Incidental ingestion of seawater during nursing induces a positive offset in δ44/42 Ca values recorded in the early formed dentine. CONCLUSIONS: Life history parameters of individual marine mammals are extremely difficult to retrieve due to limitations in observing specimens in the wild and the methodology presented here offers new ecological and paleoecological perspectives.

Calcium isotopes as a biomarker for vascular calcification in chronic kidney disease.

Calcium balance is abnormal in adults with chronic kidney disease (CKD) and is associated with the development of vascular calcification. It is currently not routine to screen for vascular calcification in CKD patients. In this cross-sectional study, we investigate whether the ratio of naturally occurring calcium (Ca) isotopes, 44Ca and 42Ca, in serum could be used as a noninvasive marker of vascular calcification in CKD. We recruited 78 participants from a tertiary hospital renal center: 28 controls, 9 subjects with mild-moderate CKD, 22 undertaking dialysis and 19 who received a kidney transplant. For each participant, systolic blood pressure, ankle brachial index, pulse wave velocity, and estimated glomerular filtration rate were measured, along with serum markers. Calcium concentrations and isotope ratios were measured in urine and serum. While we found no significant association between urine Ca isotope composition (noted δ44/42Ca) between the different groups, δ44/42Ca values in serum were significantly different between healthy controls, subjects with mild-moderate CKD and those undertaking dialysis (P < 0.01). Receiver operative characteristic curve analysis shows that the diagnostic utility of serum δ44/42Ca for detecting medial artery calcification is very good (AUC = 0.818, sensitivity 81.8% and specificity 77.3%, P < 0.01), and performs better than existing biomarkers. Although our results will need to be verified in prospective studies across different institutions, serum δ44/42Ca has the potential to be used as an early screening test for vascular calcification.

Exploring the K isotope composition of Göttingen minipig brain regions, and implications for Alzheimer's disease.

Natural stable metal isotopes have shown utility in differentiation between healthy and diseased brain states (e.g. Alzheimer's disease, AD). While the AD brain accumulates some metals, it purges others, namely K (accompanied by increased serum K, suggesting brain-blood transferal). Here, K isotope compositions of Göttingen minipig brain regions for two AD models at midlife are reported. Results indicate heavy K isotope enrichment where amyloid beta (Aß) accumulation is observed, and this enrichment correlates with relative K depletion. These results suggest preferential efflux of isotopically light K+ from the brain, a linkage between brain K concentrations and isotope compositions, and linkage to Aß (previously shown to purge cellular brain K+). Brain K isotope compositions differ from that for serum and brain K is much more abundant than in serum, suggesting that changes in brain K may transfer a measurable K isotope excursion to serum, thereby generating an early AD biomarker.

Lactation and gestation controls on calcium isotopic compositions in a mammalian model.

Lactation and gestation are among the physiological events that trigger the most intense changes in body calcium (Ca) fluxes. Along with the composition of the animal 2021 diet, these events are suspected to impact the Ca isotopic composition of Ca body reservoirs but their dynamics are poorly understood. In this study, we monitored a group of domestic sows across a full reproduction cycle. We collected tissues and fluids (blood, urine, milk, colostrum, umbilical blood, adult and piglet bones) at different steps of gestation and lactation, and analyzed their Ca isotopic compositions (i.e. δ44/42Ca) by means of multi-collector inductively coupled plasma mass spectrometry. Among other results, we report the first observations of Ca isotopic fractionation between maternal and umbilical blood (Δ44/42Caumbilical blood-sow blood = -0.18 ± 0.11‰, n = 3). Our data also highlight that gestation and lactation periods are characterized by small diet-bone Ca isotopic offsets (Δ44/42Cabone-diet = -0.28 ± 0.11‰, n = 3), with 44Ca-enriched blood compositions during nursing (Δ44/42Canursing blood-gestation blood = $+ 0.42{\rm{\,\,}}_{ - 0.12}^{ + 0.11}$‰, n = 3). Under the light of an up-to-date mammalian box model, we explored different scenarios of gestation and lactation Ca fluxes experienced by a sow-like animal. These simulations suggest that gestation changes on body δ44/42Ca values may result from the intensification of Ca absorption by the animal, whereas the production of 44Ca-depleted milk is the main driver for the 44Ca enrichment in blood during lactation. In addition, our results also support that bone mineralization could be associated with a more restricted Ca isotopic fractionation than previously envisioned. Together, these results refine the framework of Ca isotope applications, notably regarding the monitoring of human bone balance and the study of species and ecosystems from the present and the past.

Isotopic calcium biogeochemistry of MIS 5 fossil vertebrate bones: application to the study of the dietary reconstruction of Regourdou 1 Neandertal fossil.

The calcium isotopic composition (δ44/42Ca) of bone and tooth enamel can be used for dietary reconstructions of extant and extinct mammals. In natural conditions, the δ44/42Ca value of bone and teeth varies according to dietary intake with a constant isotopic offset of about -0.6‰. Owing to the poor conservation of collagen, carbon (C), and nitrogen (N) isotopic compositions of the Regourdou Mousterian site (MIS 5, Dordogne, France) previously failed to provide any paleodietary information. Therefore, to reconstruct the trophic chain, we have measured calcium (Ca) isotopes from fossil bone samples of the fauna from the Regourdou site, as well as from three bone samples of the Regourdou 1 Neandertal specimen. The results show a taxon-dependent patterning of the Ca isotopic compositions: herbivores generally have higher δ44/42Ca values than carnivores. All the δ44/42Ca values of Regourdou 1 are low (<-1.6‰), placing this specimen amid carnivores. Using a bone-muscle Ca isotopic offset determined on extant animals, we further show that the δ44/42Ca value of the Regourdou 1 diet, and that of most carnivores, cannot be accounted for by the consumption of meat only, as plants and meat have indistinguishable δ44/42Ca values. Mass balance calculations indicate that the low δ44/42Ca values of the Neandertal's carnivorous diet are explained by the ingestion of bone marrow containing as little as 1% trabecular bone. Our results show that the Regourdou 1 Neanderthal consumed a mixture of various herbivorous prey, as well as trabecular bone, which probably occurred when marrow was ingested, by accident or intentionally.

Calcium isotopic ecology of Turkana Basin hominins.

Diet is a major driver of hominin evolution, but most of the geochemical evidence relies on carbon isotopes (δ13C). Here, we report enamel stable calcium isotope (δ44/42Ca) values against δ13C values for several hominins and co-existing primates in the Turkana Basin area, circa 4 to 2 Ma. Australopithecus anamensis clusters with mammal browsers, Kenyanthropus platyops is distinct from A. anamensis in foraging into more open environments and the coexisting Theropithecus brumpti encompasses both the grazer and omnivore/carnivore domains. Early Homo is remarkable for its wide distribution in δ44/42Ca values, possibly reflecting omnivorous and opportunistic preferences. Paranthropus boisei is uniquely distributed in the δ13C versus δ44/42Ca iso-space being distinct from all other hominins from the Turkana Basin area as well as from the co-existing Theropithecus oswaldi. Several hypotheses are explored to discuss the unique δ44/42Ca values of Paranthropus boisei including significant differences observed with δ44/42Ca values recently reported for P. robustus from South Africa, questioning the monophyly of this genus.

Calcium isotopic patterns in enamel reflect different nursing behaviors among South African early hominins.

Nursing is pivotal in the social and biological evolution of hominins, but to date, early-life behavior among hominin lineages is a matter of debate. The calcium isotopic compositions (δ44/42Ca) of tooth enamel can provide dietary information on this period. Here, we measure the δ44/42Ca values in spatially located microsized regions in tooth enamel of 37 South African hominins to reconstruct early-life dietary-specific variability in Australopithecus africanus, Paranthropus robustus, and early Homo. Very low δ44/42Ca values (<-1.4‰), indicative of milk consumption, are measured in early Homo but not in A. africanus and P. robustus. In these latter taxa, transitional or adult nonmilk foods must have been provided in substantial quantities relative to breast milk rapidly after birth. The results suggest that early Homo have continued a predominantly breast milk-based nursing period for longer than A. africanus and P. robustus and have consequently more prolonged interbirth interval.

Spatial distribution of trace element Ca-normalized ratios in primary and permanent human tooth enamel.

The trace elements distribution embedded in tooth enamel offers a means to study exposure of toxic metals and allows the reconstruction of dietary behaviors. The quantification of most of the elements with a spatial high resolution (~50µm) is routinely achieved using laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS). However, the lack of a comprehensive framework of trace elements distribution in enamel jeopardizes any endorsed sampling strategy using LA-ICPMS. The present work is an effort to improve our knowledge on this issue. We studied a suite of 22 sectioned teeth with known dietary history, including 12 3rd molars from 12 living individuals and 10 primary teeth from 3 living individuals. Using LA-ICPMS, we measured Ca, Cu, Zn, Ni, Sr, Ba and Pb variations along 2 or 3 rasters from cervical to occlusal enamel. Calcium concentrations are lower in primary than in permanent teeth and do not vary spatially within a tooth suggesting that enamel matures homogeneously before eruption. The Pb/Ca ratio does not vary within tooth enamel and between primary and permanent tooth enamel. The Cu/Ca and Ni/Ca ratios do not vary within tooth enamel but discriminate primary from permanent tooth enamel. The Zn/Ca ratios are higher in permanent than in primary tooth enamel, and increase up to an order of magnitude in the last hundred of microns at the enamel surface. The Sr/Ca and Ba/Ca ratios are higher in permanent than in primary tooth enamel, and decrease from the enamel-dentine junction towards outer enamel in permanent but not in primary tooth enamel. Considering the Ca-normalized intra-tooth variations of Zn, Sr and Ba, we recommend to perform laser ablation rasters along the enamel-dentine junction because this area is likely to retain most of the original and complete chemical information related to individual's life.

Assessing human weaning practices with calcium isotopes in tooth enamel.

Weaning practices differ among great apes and likely diverged during the course of human evolution, but behavioral inference from the fossil record is hampered by a lack of unambiguous biomarkers. Here, we show that early-life dietary transitions are recorded in human deciduous tooth enamel as marked variations in Ca isotope ratios (δ44/42Ca). Using a sequential microsampling method along the enamel growth axis, we collected more than 150 enamel microsamples from 51 deciduous teeth of 12 different modern human individuals of known dietary histories, as well as nine enamel samples from permanent third molars. We measured and reconstructed the evolution of 44Ca/42Ca ratios in enamel from in utero development to first months of postnatal development. We show that the observed variations of δ44/42Ca record a transition from placental nutrition to an adult-like diet and that Ca isotopes reflect the duration of the breastfeeding period experienced by each infant. Typically, the δ44/42Ca values of individuals briefly or not breastfed show a systematic increase during the first 5-10 mo, whereas individuals with long breastfeeding histories display no measurable variation in δ44/42Ca of enamel formed during this time. The use of Ca isotope analysis in tooth enamel allows microsampling and offers an independent approach to tackle challenging questions related to past population dynamics and evolution of weaning practices in hominins.

Calcium Isotopic Evidence for Vulnerable Marine Ecosystem Structure Prior to the K/Pg Extinction.

The collapse of marine ecosystems during the end-Cretaceous mass extinction involved the base of the food chain [1] up to ubiquitous vertebrate apex predators [2-5]. Large marine reptiles became suddenly extinct at the Cretaceous-Paleogene (K/Pg) boundary, whereas other contemporaneous groups such as bothremydid turtles or dyrosaurid crocodylomorphs, although affected at the familial, genus, or species level, survived into post-crisis environments of the Paleocene [5-9] and could have found refuge in freshwater habitats [10-12]. A recent hypothesis proposes that the extinction of plesiosaurians and mosasaurids could have been caused by an important drop in sea level [13]. Mosasaurids are unusually diverse and locally abundant in the Maastrichtian phosphatic deposits of Morocco, and with large sharks and one species of elasmosaurid plesiosaurian recognized so far, contribute to an overabundance of apex predators [3, 7, 14, 15]. For this reason, high local diversity of marine reptiles exhibiting different body masses and a wealth of tooth morphologies hints at complex trophic interactions within this latest Cretaceous marine ecosystem. Using calcium isotopes, we investigated the trophic structure of this extinct assemblage. Our results are consistent with a calcium isotope pattern observed in modern marine ecosystems and show that plesiosaurians and mosasaurids indiscriminately fall in the tertiary piscivore group. This suggests that marine reptile apex predators relied onto a single dietary calcium source, compatible with the vulnerable wasp-waist food webs of the modern world [16]. This inferred peculiar ecosystem structure may help explain plesiosaurian and mosasaurid extinction following the end-Cretaceous biological crisis.