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.
