Periodic Reporting for period 1 - AROUSE (Assessing the impact of climate fluctuations on hibernation phenology using novel dental biomarkers)
Periodo di rendicontazione: 2023-08-01 al 2025-07-31
Recent increases in global temperatures have been severely affecting the hibernation patterns of small mammals. Many species now tend to emerge too early in spring, potentially altering individual fitness and posing risks to population viability. Hibernation is a key physiological mechanism of metabolic reduction that allows small endothermic mammals to survive harsh seasonal climates, and it is associated with increased survival rates and the evolution of slow life histories.
Previous research has investigated modern small-mammal hibernation from neurological, ethological, and physiological perspectives. However, despite its importance for understanding the link between climate change and phenology, the direct influence of long-term paleoclimatic fluctuations on small-mammal torpor and physiology has remained unexplored. Over the past ~50,000 years, several abrupt climate change events marked the transition from glacial to interglacial periods, profoundly impacting the life histories of mammals, including humans.
The AROUSE project addresses this gap by examining the hibernation of small mammals from a novel, interdisciplinary perspective. It focuses on the micro-chemistry and histomorphometry of well-dated fossil and modern Alpine marmot (Marmota marmota) incisors, spanning from the Late Pleistocene to the Holocene, thus encompassing the Last Glacial Maximum (LGM). Marmot odontoskeletal remains are abundant in archaeological contexts across this timeframe, providing an exceptional archive for the study of physiological adaptation.
Through this approach, the project aims to generate unprecedented insights into the physiology, adaptation, and ecology of marmot hibernation during critical climatic transitions. The results are expected to inform multiple disciplines, including ethology, conservation biology, animal physiology, paleontology, paleoanthropology, archaeology, and modern medicine, supporting broader societal objectives related to climate change adaptation and biodiversity resilience. The expected impacts are significant in scale and scope. The identification of robust chemical biomarkers for hibernation and torpor will open new avenues in chronobiology, conservation planning, and the study of metabolic regulation, potentially informing medical research on suspended animation and metabolic disorders. Beyond scientific advances, the project also enhances awareness of how past biotic responses to climate variability can inform present-day conservation and climate strategies.
Previous research has investigated modern small-mammal hibernation from neurological, ethological, and physiological perspectives. However, despite its importance for understanding the link between climate change and phenology, the direct influence of long-term paleoclimatic fluctuations on small-mammal torpor and physiology has remained unexplored. Over the past ~50,000 years, several abrupt climate change events marked the transition from glacial to interglacial periods, profoundly impacting the life histories of mammals, including humans.
The AROUSE project addresses this gap by examining the hibernation of small mammals from a novel, interdisciplinary perspective. It focuses on the micro-chemistry and histomorphometry of well-dated fossil and modern Alpine marmot (Marmota marmota) incisors, spanning from the Late Pleistocene to the Holocene, thus encompassing the Last Glacial Maximum (LGM). Marmot odontoskeletal remains are abundant in archaeological contexts across this timeframe, providing an exceptional archive for the study of physiological adaptation.
Through this approach, the project aims to generate unprecedented insights into the physiology, adaptation, and ecology of marmot hibernation during critical climatic transitions. The results are expected to inform multiple disciplines, including ethology, conservation biology, animal physiology, paleontology, paleoanthropology, archaeology, and modern medicine, supporting broader societal objectives related to climate change adaptation and biodiversity resilience. The expected impacts are significant in scale and scope. The identification of robust chemical biomarkers for hibernation and torpor will open new avenues in chronobiology, conservation planning, and the study of metabolic regulation, potentially informing medical research on suspended animation and metabolic disorders. Beyond scientific advances, the project also enhances awareness of how past biotic responses to climate variability can inform present-day conservation and climate strategies.
The first work package of the project focused on collecting modern and fossil marmot incisors and studying their dental enamel through histomorphometry. Initially, we developed a method to embed and thin-section the long, curved marmot incisors to improve the readability of growth marks. During the secondment at Sapienza University, within the framework of the ERC StG MOTHERS, histomorphometry analyses allowed us to establish a chronological framework for marmot incisors by studying the daily secretion rates of ameloblasts, based on accentuated lines within the dental enamel. In addition, in some specimens, a higher frequency of stress lines likely corresponds to periods of hibernation—though this remains a topic requiring further investigation. In collaboration with the ERC StG MOTHERS and the MSCA-IF TULAR, we also developed a protocol to optimize ancient tooth sampling for histology and subsequent biogeochemical analyses, focusing on minimizing destructive techniques and enabling specimen restoration.
The second work package focused on liquid-chromatography mass spectrometry analyses of marmot enamel to develop a proteomics-based method for sex determination. During the reporting period, we were only able to initiate work on this topic. In collaboration with UNIMORE, we established a laboratory protocol for amelogenin-based sex estimation using nanoLC-MS. Starting with well-established human proteome extraction and analysis methods, we adapted the approach to other animals, including elephants, equids, bovines, and rodents. The methodology included enamel sampling, digestion, protein purification, nanoLC-MS/MS analyses and bioinformatic. We successfully developed a robust sex estimation protocol for these species, although it has so far been tested on a limited number of individuals.
The third work package focused on high-spatial resolution elemental and isotopic analyses of marmot incisor enamel using laser ablation ICP mass spectrometry. We tested and refined protocols for highly accurate elemental and isotope analyses on well-characterized human and animal dental specimens, which could then be applied to marmot teeth. Previously sectioned dental specimens were analyzed along the enamel-dentine junction, following tooth growth, to reconstruct the chemo-history of both modern and fossil marmots. In modern specimens, we developed a new method for retrospectively identifying the tooth hibernation zone based on strontium-over-calcium and barium-over-calcium elemental ratios. However, many—but not all—of the fossil teeth showed elevated diagenetic markers, such as REEs and U, which may limit their reliability. In addition, the accumulation of heavy metals along enamel growth, may serve as a new way to track metal exposure in the environment over time, at a weekly resolution.
Preliminary results of the AROUSE project were presented in two main (international) conferences. Two open-access publications resulting from side projects were also published in peer-reviewed journals. Two core manuscripts are in preparation and anticipated to be published in the next few years.
The second work package focused on liquid-chromatography mass spectrometry analyses of marmot enamel to develop a proteomics-based method for sex determination. During the reporting period, we were only able to initiate work on this topic. In collaboration with UNIMORE, we established a laboratory protocol for amelogenin-based sex estimation using nanoLC-MS. Starting with well-established human proteome extraction and analysis methods, we adapted the approach to other animals, including elephants, equids, bovines, and rodents. The methodology included enamel sampling, digestion, protein purification, nanoLC-MS/MS analyses and bioinformatic. We successfully developed a robust sex estimation protocol for these species, although it has so far been tested on a limited number of individuals.
The third work package focused on high-spatial resolution elemental and isotopic analyses of marmot incisor enamel using laser ablation ICP mass spectrometry. We tested and refined protocols for highly accurate elemental and isotope analyses on well-characterized human and animal dental specimens, which could then be applied to marmot teeth. Previously sectioned dental specimens were analyzed along the enamel-dentine junction, following tooth growth, to reconstruct the chemo-history of both modern and fossil marmots. In modern specimens, we developed a new method for retrospectively identifying the tooth hibernation zone based on strontium-over-calcium and barium-over-calcium elemental ratios. However, many—but not all—of the fossil teeth showed elevated diagenetic markers, such as REEs and U, which may limit their reliability. In addition, the accumulation of heavy metals along enamel growth, may serve as a new way to track metal exposure in the environment over time, at a weekly resolution.
Preliminary results of the AROUSE project were presented in two main (international) conferences. Two open-access publications resulting from side projects were also published in peer-reviewed journals. Two core manuscripts are in preparation and anticipated to be published in the next few years.
The AROUSE project allowed the development of novel methodologies combining histological and elemental analyses of animal teeth. Specifically, it established a new histo-chronological framework for marmot incisor enamel, both to study enamel growth and to guide subsequent high-temporal-resolution LA-ICPMS analyses. Additionally, a new laboratory for (paleo)proteomic analysis of animal dental enamel and amelogenin-based sex determination was launched.
AROUSE also developed an innovative method for the retrospective study of hibernation in small-mammal dental enamel, based on metabolic and dietary fluctuations over time reflected in specific metal concentrations along the tooth. In this context, enamel Sr and Ba contents proved to be particularly valuable for reconstructing the life histories of small-mammal hibernators and may eventually allow researchers to determine both the timing and metabolic impact of hibernation periods. Unfortunately, the early conclusion of the project limited the application of this new methodology to a broader set of archaeological and fossil specimens, constraining our inferences on past hibernation phenology. Further research involving a larger sample of fossil marmot incisors will be essential to assess whether climate fluctuations affected marmot hibernation strategies. Moreover, combining SHRIMP and LA-IRMS oxygen and carbon isotope analyses will help refine these inferences.
Finally, AROUSE methodological advancements in elemental analysis of synanthropic animal enamel have opened up new possibilities for environmental monitoring. In particular, the exploitation of ever-growing incisors, such as those found in rodents, offers a strong tool for studying temporal variations in heavy metal exposure in the environment. These teeth, which grow continuously throughout the animal's life, can serve as natural archives, recording fluctuations in environmental contaminants over time.
AROUSE also developed an innovative method for the retrospective study of hibernation in small-mammal dental enamel, based on metabolic and dietary fluctuations over time reflected in specific metal concentrations along the tooth. In this context, enamel Sr and Ba contents proved to be particularly valuable for reconstructing the life histories of small-mammal hibernators and may eventually allow researchers to determine both the timing and metabolic impact of hibernation periods. Unfortunately, the early conclusion of the project limited the application of this new methodology to a broader set of archaeological and fossil specimens, constraining our inferences on past hibernation phenology. Further research involving a larger sample of fossil marmot incisors will be essential to assess whether climate fluctuations affected marmot hibernation strategies. Moreover, combining SHRIMP and LA-IRMS oxygen and carbon isotope analyses will help refine these inferences.
Finally, AROUSE methodological advancements in elemental analysis of synanthropic animal enamel have opened up new possibilities for environmental monitoring. In particular, the exploitation of ever-growing incisors, such as those found in rodents, offers a strong tool for studying temporal variations in heavy metal exposure in the environment. These teeth, which grow continuously throughout the animal's life, can serve as natural archives, recording fluctuations in environmental contaminants over time.