Paleoproteomics characterization of fossil arthropod parasitiformes amber inclusions

Paleoproteomics characterization of fossil arthropod parasitiformes amber inclusions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Paleoproteomics characterization of fossil arthropod parasitiformes amber inclusions Jose de la Fuente, Margarita Villar, Agustín Estrada-Peña, Laura Tormo, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6123337/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Paleoproteomics is the proteomics study of ancient proteins, which may be better preserved than DNA in fossil inclusions and particularly in amber fossilized tree resins. However, only yeast proteins were identified in the only paleoproteomics analysis conducted in amber fossil inclusions. In this study, we developed and applied a paleoproteomics approach to study fossil arthropod parasitiformes inclusions in two Burmese (also known as Kachin) amber (Cretaceous, ca. 99 mya) pieces. The results supported the identification of Cornupalpatum sp. and the first report of fossil Holothyrida, Neothyridae at both morphological and molecular levels. Identified proteins such as Actin (Neothyridae and Cornupalpatum sp.), Ubiquitin ( Tetranychus urticae and Ixodes ricinus ), Triosephosphate isomerase ( Aceria tosichella ), NADH-ubiquinone oxidoreductase and Elongation factor 1-alpha (Neothyridae) were analyzed to evaluate evolutionary trees with possible functional implications. These results provide a paleoproteomics approach to complement morphological studies of the molecular evolution of parasitiformes. Biological sciences/Evolution/Evolutionary theory Biological sciences/Biological techniques/Proteomic analysis amber arthropod fossil paleontology paleoproteomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The study of fossil bones suggested that DNA could not possibly survive much more than 100,000 years unless samples have been preserved frozen or crystalized ( 1 ), conditions that are difficult to find for fossil arthropods. Additionally, some results have been questioned due to possible contamination or replacement of the original tissues by modern organisms thus questioning whether DNA can survive for millions of years even if isolated from amber specimens ( 2 – 5 ). Recently, the isolation of samples from 250 million years ago (mya) salt crystal inclusions ( 6 ), ca. 105 mya Cretaceous Albian Spanish fossil feathers in amber ( 7 ) and fossil biomineral proteins from the Pleistocene fossil invertebrate, stony coral Orbicella annularis ( 8 ) allowed the analysis of amino acid contents in these samples. Furthermore, the identification of collagen peptide sequences in the skeleton of Tyrannosaurus rex ( 9 ) and noncollagenous proteins in compact bones from Iguanodon bernis-sartensis ( 10 ) suggested that at least some proteins may be better preserved than DNA over geological time. Furthermore, a proteomics analysis of a Pleistocene mammoth femur revealed hundreds of putative ancient bone proteins ( 11 ) and Wadsworth and Buckley ( 12 ) investigated the longevity of protein survival in ancient bone samples. Other studies have shown the analysis of fossils in amber based on amino acid racemisation levels ( 13 , 14 ). These findings suggested that amber fossilized tree resins may provide the environment to better preserve proteins during millions of years. Amber diterpenes rapidly dehydrate included specimens, which together with its antimicrobial properties contribute to preservation of these inclusions. The analysis of ancient proteins contributes to evolutionary paleontological studies ( 15 ). In this context, paleoproteomics is the proteomics study of ancient proteins. Some of these proteins may be highly conserved across evolution with key functional implications ( 16 ) while others may be scarcely represented in modern organisms and can be used for molecular de-extinction biomedical applications ( 17 – 21 ). Fossil arthropods have been described in amber inclusions with morphological characterization of multiple Ixodidae (e.g., 22–28). However, paleoproteomics was previously applied only once to amber fossil inclusions with the identification of yeast proteins ( 14 ). To advance in paleoproteomics, in this study we developed a proteomics approach to study amber inclusions of fossil arthropods. The results showed how the characterization of proteins from fossil arthropods complement morphological studies and provide relevant information on the molecular evolution of Parasitiformes. Results Morphological identification of fossil amber inclusions Two Burmese (also known as Kachin) amber (Cretaceous, ca. 99 mya) pieces with arthropod parasitiformes inclusions were used in this study (Figs. 1 A and 1 B). Morphological analysis identified on each piece an Holothyrida, Neothyridae (Fig. 1 A, Supplementary Morphological Data 1, Supplementary Declaration of legal origin 1) and an Ixodida Cornupalpatum sp. (Fig. 1 B, Supplementary Morphological Data 2, Supplementary Declaration of legal origin 2). The Neothyridae are arthropod scavengers ( 29 ) and was found together with insect inclusions in the amber from which it was separated for analysis (Fig. 1 A). These insects were used as controls and with morphological preliminary classification as lacewing (order Neuroptera, family Chrysopidae) larva, probably a first instar (Supplementary Fig. 1, Supplementary Declaration of legal origin 1) and a thrip (order Thysanoptera, family Thripidae, putative Frankliniella sp.), but difficult to confirm due to state of the inclusion (Supplementary Fig. 2, Supplementary Declaration of legal origin 1). Ticks (Ixodida) are hematophagous Acari uncommon on trees and seldom found in amber. Paleoproteomics analysis of Holothyrida, Neothyridae identified in amber inclusion The paleoproteomics analysis identified nine proteins associated with Neothyridae gen. sp. (Elongation factor 1-alpha), Tetranychus urticae (Ubiquitin-like domain-containing protein), Allothyrus sp. (NADH-ubiquinone oxidoreductase chain 3), Ixodes ricinus (Putative ubiquitin/40s ribosomal protein s27a fusion), Tetranychus evansi (Actin isoform X2), Tetranychus urticae (Histone H4), Aceria tosichella (Actin and Triosephosphate isomerase) and Demodex folliculorum (Actin) (Supplementary Table 1). The sequence with highest coverage was Neothyridae gen. sp. Elongation factor 1-alpha (71.3%; Table S1 ), thus supporting morphological identification of Holothyrida, Neothyridae. Additionally, taxonomy analysis together with feeding behavior and geographic origin of the acariformes associated with identified proteins supported the presence of Holothyrida arthropod scavengers in Myanmar, Burma from where amber piece originated ( 29 , 30 ) (Fig. 2 ). Focusing on the Neothyridae gen. sp. Elongation factor 1-alpha sequence phylogenetic analysis, although related to some tick and mite species, Neothyridae form a separate clade within Holothyrida mites (Fig. 3 , Supplementary Fig. 3). Three Actin sequences were identified in different mite species (Supplementary Table 1) and taxonomy tree analysis showed that these proteins are highly conserved in arthropods and not reported in Holothyrida, thus suggesting the possibility that these Actin sequences my belong to Holothyrida (Supplementary Fig. 4). Additionally, and using paleoproteomics analysis of Holothyrida, Neothyridae as a model, a chimeric protein was constructed with random inclusion of all identified peptide sequences with 1–10% FDR. The chimeric protein was used for Blastp analysis, and the results identified conserved domains with Translation elongation factor (TEF1) superfamily highly represented (covering 50.5% of the chimeric protein sequence). Accordingly, the TEF with which Holothyrida, Neothyridae was identified have the highest E-values of protein domain hits (2.09e-144 to 1.56e-25) (Supplementary Fig. 5). Paleoproteomics analysis of Ixodida Cornupalpatum sp. identified in amber inclusion The paleoproteomics analysis identified only one protein matching to Haemaphysalis longicornis Actin with 10.6% coverage (Fig. 4 , Supplementary Table 1). Protein phylogenetic analysis identified H. longicornis as the most represented species within the same clade with other tick and mite species (Fig. 4 ). The analysis of the H. longicornis Actin secondary structure evidenced a stable protein that may be associated with conservation in fossil inclusion with most of the identified peptides located in highly stable protein regions (Supplementary Fig. 6). As observed in Holothyrida, Actin sequence has not been reported in Cornupalpatum sp. and thus identified peptides by paleoproteomics may belong to this species. Paleoproteomics analysis of amber control samples. To provide additional support to obtained results, two amber samples were processed as controls. The insect inclusions identified together with Holothyrida, Neothyridae (Fig. 1 A, Supplementary Fig. 1, Supplementary Fig. 2) were included, and amber obtained from the same piece but without inclusions was processed as a negative control. The results in insect inclusions identified Frankliniella intonsa Actin in accordance with morphological evaluation but with a highly conserved protein in insects (> 97% identity and E-value = 0.0) (Supplementary Fig. 7). A peptide (NH 2 -TILDDINR-COOH) exclusively found in Fly agaric fungi, Amanita muscaria , was also identified (Supplementary Fig. 8A) with intense and well assigned mass spectrometry (MS) spectrum (Supplementary Fig. 8B). Additionally, an intermediate filament protein ON3 was identified matching Culex pipiens (Supplementary Fig. 9A), but this protein showed a 95% percent identity to zebrafish Danio rerio keratin, type II cytoskeletal 5 protein (Supplementary Fig. 9B). Three additional proteins were identified with one peptide each matching insect species red flour beetle ( Tribolium castaneum ), scarce chaser dragonfly ( Ladona fulva ) and sandfly ( Lutzomyia longipalpis ) (Supplementary Fig. 9C). UniProt BLAST analysis with ensembled protein with three insect-derived peptides resulted in significant homology only with wasp Ceratosolen solmsi marchali (Order: Hymenoptera, Family: Agaonidae) uncharacterized protein (Supplementary Fig. 9D). Neuroptera-derived proteins were not identified. The analysis of negative control amber without inclusions against different databases were similar and showed only one protein that appeared as “REVERSED”, meaning “FALSE” (Supplementary Fig. 10). Discussion Taken together, the results of morphological and paleoproteomics studies supported the identification of Holothyrida, Neothyridae and Ixodida Cornupalpatum sp. in the amber fossil inclusions as part of the molecular evolutionary tree of Parasitiformes (Fig. 5 ) reconstructed based on published studies ( 25 , 27 , 31 – 33 ). The Holothyrida, Neothyridae was identified at both morphological and molecular levels. Cornupalpatum sp. was morphologically identified and although paleoproteomics identified an H. longicornis Actin, these two genera are evolutionary closely related (Fig. 5 ) and Actin protein sequences identified in both amber inclusions may belong to Holothyrida, Neothyridae and Cornupalpatum sp., respectively. Of these fossil arthropod parasitiformes, only Cornupalpatum spp. have been reported before in amber ( 22 ), thus providing the first evidence for fossil Holothyrida, Neothyridae. The analysis in control amber samples with insect inclusions identified F. intonsa Actin in support to morphological results. Frankliniella spp. such as F. occidentalis interact with fungi ( 34 ) with digestion of fungal spores ( 35 ), which may be associated with the Fly agaric fungi peptide identified. Other Frankliniella spp. such as F. zizaniophila are known as aquatic grass-inhabiting thrip ( 36 ) and thus “contaminated” fish residues may be present as identified zebrafish keratin. The presence of other insect-derived peptides agrees with morphological uncertainties in the putative morphologically identified Frankliniella sp. and the absence of Neothyridae-derived peptides. Negative control amber without inclusions provided negative results after MS analysis as expected. Overall, these results support the methodology developed and applied here for paleoproteomics studies of arthropod amber fossil inclusions. The study of multiple amber inclusions by paleoproteomics can advance the characterization of molecular evolution in parasitiformes. This cross disciplinary research between morphological and molecular studies was applied for the analysis of amber inclusions before (morphology) and after full destruction (paleoproteomics) of the specimens. This fact renders the presented paleoproteomics approach only truly complementary of morphological analyses when multiple specimens of a given taxon/species are available and so destructive sampling is affordable. Identified proteins such as Ubiquitin-like domain-containing protein (Fig. 2 , Supplementary Table 1) are highly conserved (Supplementary Table 2) with multiple functions in different cellular processes including immune response, autophagy, transcription and cellular differentiation ( 37 ) and with possible biomedical applications as therapeutic targets ( 38 ). In contrast, homologues to Aceria tosichella Triosephosphate isomerase protein sequence involved in glycolysis (Fig. 2 , Supplementary Table 1) were not identified in other species including humans (Supplementary Table 2). Although Triosephosphate isomerase 1 (TPI1; U3KPZ0) has been reported in humans as potential target in cancer ( 39 ), amino acid sequence differences suggest possible molecular de-extinction biomedical applications ( 17 – 21 ). Another consideration is the Alpha-gal Syndrome (AGS), an allergy to non-primate mammalian meat and derived products associated with tick bites and IgE antibodies to the oligosaccharide galactose-α-1,3-galactose (alpha-gal) (reviewed by 40). The hypothesis is that hominids evolved through catastrophic selection events such as the inactivation of alpha-gal synthesis associated with antibody-mediated improved protection to infection by pathogens with this modification ( 41 , 42 ). Tick proteins recognized by IgE antibodies in patients with anaphylactic reactions to tick bites but not by healthy individuals included proteins identified by paleoproteomics in Neothyridae with (NADH-ubiquinone oxidoreductase; Fig. 2 , Supplementary Table 1) and without (Elongation factor 1-alpha; Fig. 2 , Supplementary Table 1) alpha-gal modifications and with a role of these proteins in anaphylaxis to tick bites ( 43 ). These results suggested the possibility of applying paleoproteomics for the identification and characterization of proteins in fossil arthropod parasitiformes that may be involved in host-ectoparasite coevolutionary processes and diseases with potential positive and negative effects ( 44 ). In conclusion, the application of paleoproteomics analysis described here to arthropod amber inclusions advances in the characterization of fossil proteins in the molecular evolution of parasitiformes to complement morphological studies while providing potentially relevant functional information. Limitations of the study Despite these advances in ancient phylogenies, current limitations of paleoproteomics are based on the limited size and genetic information of ancient amino acid sequences recovered from fossil inclusions ( 45 ). Consequently, the phylogenetic analyses may not be sufficiently confident if only based on paleoproteomics data. Future directions may include development of more efficient protein extraction and mass spectrometry resolution together with the combination with other methods such as morphological analyses as shown here. As recently discussed ( 45 , 46 ), sustainable sampling and international collaborations to preserve fossil inclusions and address ethical concerns is important to consider for future studies. Methods Amber inclusions Arthropod inclusions (Holothyrida, Neothyridae; https://keys.lucidcentral.org/keys/mites/qmites/html/Holothyrida.htm ) with insects (putative lacewing larva, Neuroptera, Chrysopidae and a thrip, Thysanoptera, Thripidae, Frankliniella sp.) and ( Cornupalpatum sp.) in Burmese or Kachin (Burma, Myanmar) amber (Cretaceous, ca. 99 mya) were used for the study. Amber without inclusions and derived from the piece with Holothyrida, Neothyridae and insects was used as negative control (Fig. 1 A, Fig. 1 B). Amber pieces originated from KGJ Collection (Ciudad Real, Spain) in which coauthor JF is included, and were dated to Cretaceous by radiometric analysis ( 47 ) (Holothyrida, Neothyridae, 119.13 ± 0.92 mya; Cornupalpatum sp., 100.33 ± 0.51 mya) (Declarations of legal origin S1 and S2). The tick Cornupalpatum sp. showed damage of the body preventing an accurate determination to the species level (Fig. 1 A). Image capture and analysis High-resolution images were captured with Confocal microscopy and Ct scan tomography. For confocal microscopy, the amber inclusions were imaged using a Leica TCS SPE DM 5500 CSQ V-Vis (Mannheim, D-68165, Germany) at the Natural History Museum of Madrid (MNCN-CSIC). The images were acquired with a solid-state laser operating at 488 nm, a 10X eye piece, HCX PL FLUOTAR 5X/0.15, ACS APO 10X/0.3 dry objectives and the Leica Application Suite Advanced Fluorescence software (Leica MM AF 1.4). Fluorescence emissions were collected from approximately 10 nm above the excitation wavelength up to 800 nm. Laser power for acquisition was set by viewing the fluorescence emission and increasing the power until the rate of increase in fluorescence appeared to have slowed. The photomultiplier gain for acquisition was then set by viewing the image and increasing the gain until signal overload was detected, at which point the gain was reduced slightly. The 532 nm laser was used for general visions and the 635 nm for used for the jaws. Pixels matrices of 1024 × 1024, two-dimensional mode, with speed of 400 Hz, and frame average of 4 were acquired for each Z-step of 0.68 micras at a zoom setting of 1.5. An Airy unit setting of 1 was routinely used for the observation pinhole. Tomography was applied to amber piece with Cornupalpatum sp. using a Nikon XTH 160 micro-CT X-ray scanner (Tokyo, Japan) to obtain high-quality 3D images of the inclusion. The images were generated with a molybdenum target and an X-ray voltage of 117 kV and 55 µA, obtaining a voxel size of 3.6 µm. During tomography, projections were collected every 0.12 degrees in duplicate, thus improving the signal/noise ratio and with an integration time of 708 msec. Low-resolution images were captured with a Leica (L`Hospitalet de Llobregat, Barcelona, Spain) M80 routine stereo microscope using a 1X PLAN objective and a 2X-6X zoom ( https://www.leica-microsystems.com/products/light-microscopes/stereo-microscopes/p/leica-m80/ ) and a Carl Zeiss stereomicroscope (SteREO Discovery V12, Munich, Germany) using the ZEN 2 pro software. Microscope images were analyzed using Image J program ( https://imagej.net/ij/ ) and pencil sketch of images using IOimageonline.co ( https://pencilsketch.imageonline.co/index.php ). Processing amber inclusions and proteomics Amber pieces were scrubbed in 5% SDS with a brush and abundantly rinsed first in water and then in 100% methanol. The amber was covered with liquid nitrogen and fractured with a sterile ceramic pestle. Amber fragments without inclusions were discarded and arthropod inclusions were triturated to appearance of powder. Triturates were extracted in 50 µl Laemmli sample buffer by applying 10 cycles of sonication followed by vortex. Subsequently, samples were heated to 90 ºC for 5 min, centrifuged at 12,000 x g for 5 min and supernatants were collected, concentrated on-gel and trypsin digested as previously described ( 48 ). The resulting tryptic peptides were desalted onto OMIX Pipette tips C18 (Agilent Technologies, Santa Clara, CA, USA), dried down and stored at − 20°C until mass spectrometry analysis. Samples were resuspended in 10 µl of 2% acetonitrile − 5% acetic acid in water and analyzed by reverse-phase liquid chromatography coupled online to mass spectrometry (RP-LC-MS/MS) using an Ekspert™ nLC 415 system coupled with a 6600 TripleTOF mass spectrometer (AB Sciex, Framingham, MA, USA) through Information-Dependent Acquisition (IDA). The peptides were concentrated in a 0.1 × 20 mm C18 RP precolumn (Thermo Scientific, Waltham, MA, USA) with a flow rate of 5 µl/min during 10 min in solvent A. Then, peptides were separated in a 0.075 × 150 mm C18 RP column (Eksigent, part of AB Sciex) with a flow rate of 300 nl/min. Peptides elution was done in a 60-min gradient from 5–30% solvent B followed by a 10-min gradient from 30–60% solvent B (Solvent A: 0.1% formic acid in water, solvent B: 0.1% formic acid in acetonitrile) and directly injected into the mass spectrometer for analysis. 2 µl of each independent sample were analyzed by duplicate. The mass spectrometer was set to scanning full spectra from 350 m/z to 1400 m/z (250 ms accumulation time) followed by up to 50 MS/MS scans (100–1500 m/z). Candidate ions with a charge state between + 2 and + 5 and counts per second above a minimum threshold of 100 were isolated for fragmentation. One MS/MS spectrum was collected for 100 ms, before adding those precursor ions to the exclusion list during 15 s (mass spectrometer operated by Analyst R TF 1.7, AB Sciex). Dynamic background subtraction was turned off. Data were acquired in high sensitivity mode with rolling collision energy on and a collision energy spread of 5. Data analysis The IDA MS raw files for each sample were combined (2 runs) and subjected to database search in unison using ProteinPilot software v. 5.0.1 (AB Sciex) with the Paragon algorithm. Spectra identification was performed by searching against the following Uniprot databases. For Holothyrida, Neothyridae, Database Mite (accessed on May 28, 2024, UniProtKB 159,503 entries; https://www.uniprot.org/uniprotkb?query=mite ) and then Database Holothyrida (accessed on May 28, 2024, UniProtKB 18 entries; https://www.uniprot.org/uniprotkb?query=holothyrida ). For Cornupalpatum sp., Database Tick (Ixodidae) (accessed on May 23, 2024, UniProtKB 289,360 entries; https://www.uniprot.org/uniprotkb?query=ixodidae ). Controls with insect inclusions and negative control amber without inclusions derived from the piece with Holothyrida, Neothyridae and insects were processed in the same manner. For insects, spectra identification was performed by searching against the Insecta Uniprot database (accessed on May 28, 2024, and revised on January 20, 2025 UniProtKB 6,898,282 entries; https://www.uniprot.org/uniprotkb?query=taxonomy_name:insecta ). Negative control amber pieces were analyzed by searching against all databases used for Holothyrida, Neothyridae and insects. The search parameters were: iodoacetamide cysteine alkylation, trypsin digestion and gel-based ID as special factor, identification focus on biological modification and thorough ID as search effort. The detected protein threshold was set at 0.05. An independent False Discovery Rate (FDR) analysis with the target-decoy approach provided by ProteinPilot™, was used to assess the quality of identifications. Positive identifications were considered when identified proteins reached a 1% global FDR. Protein Blast (Blastp), conserved domains, phylogenetic and taxonomic analyses were conducted at National Center for Biotechnological Information (NCBI) ( https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome ; https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=2762511 ) and UniProt ( https://www.uniprot.org/blast ). Protein structure was modelled using Swiss-Model ( https://swissmodel.expasy.org ). The mass spectrometry proteomics database was deposited to the ProteomeXchange Consortium via the PRIDE ( 49 ) partner repository with the dataset identifier PXD055542. Declarations Data Availability The data supporting the findings in this paper and its Supporting Information are available. The mass spectrometry proteomics database was deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD055542. Acknowledgments This research was funded by Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación MCIN/AEI/10.13039/501100011033, Spain and EU-FEDER (Grant BIOGAL PID2020-116761GB-I00), and and Junta de Comunidades de Castilla-La Mancha and FEDER (grant PROBIOHEALTH SBPLY/23/180225/000086). 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Tick-host conflict: immunoglobulin E antibodies to tick proteins in patients with anaphylaxis to tick bite. Oncotarget 8 , 20630–20644. https://doi.org/10.18632/oncotarget.15243. de la Fuente J. (2024). Catastrophic selection: the other side of the coin. Ann. Med. 56 , 2391014. https://doi.org/10.1080/07853890.2024.2391014. Paterson, R. S., Madupe, P. P., & Cappellini, E. (2025). Paleoproteomics sheds light on million-year-old fossils. Nat. Rev. Mol. Cell Biol. 26 , 1–2. https://doi.org/10.1038/s41580-024-00803-2. Ortega R. P. (2022). Violent conflict in Myanmar linked to boom in amber studies. Science 378 , 10–11. https://doi.org/10.1126/science.adf1843. Shi G., Grimaldi D.A., Harlow G.E., Wang J., Wang J., Yang M., Lei W., Li Q., Li X. (2012). Age constraint on Burmese amber based on U-Pb dating of zircons. Cretaceous Res. 37 , 155-163. https://doi.org/10.1016/j.cretres.2012.03.014. Villar, M., Ayllón, N., Alberdi, P., Moreno, A., Moreno, M., Tobes, R., Mateos-Hernández, L., Weisheit, S., Bell-Sakyi, L., & de la Fuente, J. (2015). Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells. Mol. Cell. Proteomics 14 , 3154–3172. https://doi.org/10.1074/mcp.M115.051938. Perez-Riverol, Y., Bai, J., Bandla, C., García-Seisdedos, D., Hewapathirana, S., Kamatchinathan, S., Kundu, D. J., Prakash, A., Frericks-Zipper, A., Eisenacher, M., Walzer, M., Wang, S., Brazma, A., & Vizcaíno, J. A. (2022). The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res. 50 , D543–D552. https://doi.org/10.1093/nar/gkab1038. Additional Declarations There is NO Competing Interest. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6123337","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":422245293,"identity":"d387d7dc-dc91-4ce7-8b2d-b9dad4b9fe12","order_by":0,"name":"Jose de la Fuente","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIiWNgGAWjYBACxgYQycYgw8B8ACwgx8DAQ5wWHga2BCAjgcGYoBYIQNKS2EBIC3N7+8MHDGU2PPxtPIaPC3/YpG84fvbggw8MdnK6DTgc1nPG2IDhXBqPxDEeY+MZCWm5G87kJRvOYEg2NjuAQ8uMHDYJxrbDPAz3e8ykeRIO5244kANkMBxI3IZTS/rzH4xt/3nkj/GY/+ZJ+J9ucP4NIS0JZgyMbQd4DI7xmDHzJBxIMLhByBagXyQYziXzGB5jK5bmSUs2nHnjjbHhDAPcfjEEhtgHhjI7ObljzBs/89jYyfOdzzF88KHCTg6nlgZgQP9BFlEAqzTArhwE5DFFGnCrHgWjYBSMgpEJAG06WJiOQjoTAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-7383-9649","institution":"Institute for Game and Wildlife Research","correspondingAuthor":true,"prefix":"","firstName":"Jose","middleName":"de la","lastName":"Fuente","suffix":""},{"id":422245294,"identity":"d9fa6756-a5ed-4ca7-a6ac-9b11e219b8d9","order_by":1,"name":"Margarita Villar","email":"","orcid":"https://orcid.org/0000-0003-4172-9079","institution":"Institute of Research in Game Resources","correspondingAuthor":false,"prefix":"","firstName":"Margarita","middleName":"","lastName":"Villar","suffix":""},{"id":422245295,"identity":"71fc9954-c36c-4a3c-9b15-1f9328f6f2b5","order_by":2,"name":"Agustín Estrada-Peña","email":"","orcid":"https://orcid.org/0000-0001-7483-046X","institution":"University of Zaragoza","correspondingAuthor":false,"prefix":"","firstName":"Agustín","middleName":"","lastName":"Estrada-Peña","suffix":""},{"id":422245296,"identity":"625868ce-78c7-4862-89de-edf5b0977430","order_by":3,"name":"Laura Tormo","email":"","orcid":"","institution":"Museo Nacional Ciencias Naturales, CSIC","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Tormo","suffix":""},{"id":422245297,"identity":"cddd3be0-993d-4034-b794-a318e16ccd54","order_by":4,"name":"Cristina Paradela","email":"","orcid":"","institution":"Museo Nacional Ciencias Naturales, CSIC","correspondingAuthor":false,"prefix":"","firstName":"Cristina","middleName":"","lastName":"Paradela","suffix":""},{"id":422245298,"identity":"cfce1d83-3459-47e9-9d46-43b1374869cf","order_by":5,"name":"Almudena González-García","email":"","orcid":"","institution":"Institute for Game and Wildlife Research","correspondingAuthor":false,"prefix":"","firstName":"Almudena","middleName":"","lastName":"González-García","suffix":""},{"id":422245299,"identity":"2b0489b0-6346-4764-bf4e-91bf296bea03","order_by":6,"name":"David Fernández-Castellanos","email":"","orcid":"","institution":"Institute for Game and Wildlife Research","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Fernández-Castellanos","suffix":""}],"badges":[],"createdAt":"2025-02-27 18:25:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6123337/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6123337/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77568927,"identity":"d2540dfc-069f-46c6-8334-d75b9a224494","added_by":"auto","created_at":"2025-03-03 08:02:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":586004,"visible":true,"origin":"","legend":"\u003cp\u003eArthropod Parasitiformes amber inclusions. (A) Holothyrida, Neothyridae. A scavenger mite found together with insect inclusions in the amber from which it was separated for analysis. The figures provide details of the general look of the piece, measurements, and critical details for identification. Insect inclusions and amber without inclusions derived from this amber piece were processed as controls. (B) \u003cem\u003eCornupalpatum\u003c/em\u003e tick species for which stereomicroscope and Scan Ct images show the damage of the body preventing an accurate determination to the species level.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6123337/v1/cce073b7b11c5e1047e5011c.png"},{"id":77570730,"identity":"55d61ae2-dd33-4573-af2e-3480782670d8","added_by":"auto","created_at":"2025-03-03 08:18:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":339190,"visible":true,"origin":"","legend":"\u003cp\u003eTaxonomy, feeding behavior and geographic origin of the identified acariformes associated with Holothyrida, Neothyridae identified in amber inclusion. Proteins were identified on Database Mite (accessed on Feb 27, 2024, UniProtKB 159,483 results; https://www.uniprot.org/uniprotkb?query=mite). Percent coverage by identified peptides is shown for each protein.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6123337/v1/a2e28307d3f7d7a1c6f1793b.png"},{"id":77568933,"identity":"aa6989bd-5d2e-467b-85c1-40fb3e014842","added_by":"auto","created_at":"2025-03-03 08:02:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":478411,"visible":true,"origin":"","legend":"\u003cp\u003eNeothyridae gen. sp. Elongation factor 1-alpha (A0A2H4RIF7). Sequence and phylogenetic analysis of the protein identified with highest coverage (244/342 amino acids, 71.3%). Neothyridae form a separate clade within Holothyrida mites. High-resolution phylogenetic tree is on Supplementary Fig. 1.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6123337/v1/af9d5d28d5dac5e7f97f0622.png"},{"id":77568932,"identity":"fa6ed902-2554-42fe-a23e-f47f94db4256","added_by":"auto","created_at":"2025-03-03 08:02:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":441446,"visible":true,"origin":"","legend":"\u003cp\u003eTaxonomy of the acariformes based on phylogenetic analysis with identified\u003cem\u003e Haemaphysalis longicornis\u003c/em\u003eActin Q6X4W3 sequence associated with \u003cem\u003eCornupalpatum\u003c/em\u003e sp. Tick and mite Actin protein sequences are highly related, but \u003cem\u003eH. longicornis\u003c/em\u003e is the most represented species with 3 entries.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6123337/v1/80d2495dbfdfedb93308587f.png"},{"id":77568929,"identity":"0b7a1e39-8236-4b2d-a74a-be562566049d","added_by":"auto","created_at":"2025-03-03 08:02:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":61043,"visible":true,"origin":"","legend":"\u003cp\u003eReconstruction of the tree of tick (Ixodida) and mite (Holothyrida) evolution. Arthropods identified in fossil amber inclusions by morphological (Neothyridae and Cornupalpatum) and paleoproteomics (Neothyridae and \u003cem\u003eHaemaphysalis\u003c/em\u003e) analyses are highlighted. Fossil inclusions have been previously identified in amber for Ixodida including Cornupalpatum but not for Neothyridae.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6123337/v1/86c8a31f1adbaa48d69dac25.png"},{"id":78293059,"identity":"e4103ac6-4175-450c-93b2-6d131c0499b0","added_by":"auto","created_at":"2025-03-11 17:43:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2612662,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6123337/v1/cfea61be-0736-49eb-974d-a439e2dd993c.pdf"},{"id":77570434,"identity":"bdafc792-66c4-4efd-88db-581eac4e27f3","added_by":"auto","created_at":"2025-03-03 08:10:46","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":28175,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalinformationlegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-6123337/v1/9de171aa4962b1a606360ed7.docx"},{"id":77568948,"identity":"e34c240e-305a-46bf-b2e8-d71060b87428","added_by":"auto","created_at":"2025-03-03 08:02:47","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11423881,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6123337/v1/fc10603c93de4963b4f62b00.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Paleoproteomics characterization of fossil arthropod parasitiformes amber inclusions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe study of fossil bones suggested that DNA could not possibly survive much more than 100,000 years unless samples have been preserved frozen or crystalized (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), conditions that are difficult to find for fossil arthropods. Additionally, some results have been questioned due to possible contamination or replacement of the original tissues by modern organisms thus questioning whether DNA can survive for millions of years even if isolated from amber specimens (\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecently, the isolation of samples from 250\u0026nbsp;million years ago (mya) salt crystal inclusions (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), ca. 105 mya Cretaceous Albian Spanish fossil feathers in amber (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) and fossil biomineral proteins from the Pleistocene fossil invertebrate, stony coral \u003cem\u003eOrbicella annularis\u003c/em\u003e (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) allowed the analysis of amino acid contents in these samples. Furthermore, the identification of collagen peptide sequences in the skeleton of \u003cem\u003eTyrannosaurus rex\u003c/em\u003e (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) and noncollagenous proteins in compact bones from \u003cem\u003eIguanodon bernis-sartensis\u003c/em\u003e (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) suggested that at least some proteins may be better preserved than DNA over geological time. Furthermore, a proteomics analysis of a Pleistocene mammoth femur revealed hundreds of putative ancient bone proteins (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) and Wadsworth and Buckley (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) investigated the longevity of protein survival in ancient bone samples. Other studies have shown the analysis of fossils in amber based on amino acid racemisation levels (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). These findings suggested that amber fossilized tree resins may provide the environment to better preserve proteins during millions of years. Amber diterpenes rapidly dehydrate included specimens, which together with its antimicrobial properties contribute to preservation of these inclusions.\u003c/p\u003e \u003cp\u003eThe analysis of ancient proteins contributes to evolutionary paleontological studies (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). In this context, paleoproteomics is the proteomics study of ancient proteins. Some of these proteins may be highly conserved across evolution with key functional implications (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) while others may be scarcely represented in modern organisms and can be used for molecular de-extinction biomedical applications (\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFossil arthropods have been described in amber inclusions with morphological characterization of multiple Ixodidae (e.g., 22\u0026ndash;28). However, paleoproteomics was previously applied only once to amber fossil inclusions with the identification of yeast proteins (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo advance in paleoproteomics, in this study we developed a proteomics approach to study amber inclusions of fossil arthropods. The results showed how the characterization of proteins from fossil arthropods complement morphological studies and provide relevant information on the molecular evolution of Parasitiformes.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMorphological identification of fossil amber inclusions\u003c/h2\u003e \u003cp\u003eTwo Burmese (also known as Kachin) amber (Cretaceous, ca. 99 mya) pieces with arthropod parasitiformes inclusions were used in this study (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Morphological analysis identified on each piece an Holothyrida, Neothyridae (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Supplementary Morphological Data 1, Supplementary Declaration of legal origin 1) and an Ixodida \u003cem\u003eCornupalpatum\u003c/em\u003e sp. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Supplementary Morphological Data 2, Supplementary Declaration of legal origin 2). The Neothyridae are arthropod scavengers (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) and was found together with insect inclusions in the amber from which it was separated for analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). These insects were used as controls and with morphological preliminary classification as lacewing (order Neuroptera, family Chrysopidae) larva, probably a first instar (Supplementary Fig.\u0026nbsp;1, Supplementary Declaration of legal origin 1) and a thrip (order Thysanoptera, family Thripidae, putative \u003cem\u003eFrankliniella\u003c/em\u003e sp.), but difficult to confirm due to state of the inclusion (Supplementary Fig.\u0026nbsp;2, Supplementary Declaration of legal origin 1). Ticks (Ixodida) are hematophagous Acari uncommon on trees and seldom found in amber.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePaleoproteomics analysis of Holothyrida, Neothyridae identified in amber inclusion\u003c/h3\u003e\n\u003cp\u003eThe paleoproteomics analysis identified nine proteins associated with Neothyridae gen. sp. (Elongation factor 1-alpha), \u003cem\u003eTetranychus urticae\u003c/em\u003e (Ubiquitin-like domain-containing protein), \u003cem\u003eAllothyrus\u003c/em\u003e sp. (NADH-ubiquinone oxidoreductase chain 3), \u003cem\u003eIxodes ricinus\u003c/em\u003e (Putative ubiquitin/40s ribosomal protein s27a fusion), \u003cem\u003eTetranychus evansi\u003c/em\u003e (Actin isoform X2), \u003cem\u003eTetranychus urticae\u003c/em\u003e (Histone H4), \u003cem\u003eAceria tosichella\u003c/em\u003e (Actin and Triosephosphate isomerase) and \u003cem\u003eDemodex folliculorum\u003c/em\u003e (Actin) (Supplementary Table\u0026nbsp;1). The sequence with highest coverage was Neothyridae gen. sp. Elongation factor 1-alpha (71.3%; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), thus supporting morphological identification of Holothyrida, Neothyridae. Additionally, taxonomy analysis together with feeding behavior and geographic origin of the acariformes associated with identified proteins supported the presence of Holothyrida arthropod scavengers in Myanmar, Burma from where amber piece originated (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFocusing on the Neothyridae gen. sp. Elongation factor 1-alpha sequence phylogenetic analysis, although related to some tick and mite species, Neothyridae form a separate clade within Holothyrida mites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Supplementary Fig.\u0026nbsp;3). Three Actin sequences were identified in different mite species (Supplementary Table\u0026nbsp;1) and taxonomy tree analysis showed that these proteins are highly conserved in arthropods and not reported in Holothyrida, thus suggesting the possibility that these Actin sequences my belong to Holothyrida (Supplementary Fig.\u0026nbsp;4).\u003c/p\u003e \u003cp\u003eAdditionally, and using paleoproteomics analysis of Holothyrida, Neothyridae as a model, a chimeric protein was constructed with random inclusion of all identified peptide sequences with 1\u0026ndash;10% FDR. The chimeric protein was used for Blastp analysis, and the results identified conserved domains with Translation elongation factor (TEF1) superfamily highly represented (covering 50.5% of the chimeric protein sequence). Accordingly, the TEF with which Holothyrida, Neothyridae was identified have the highest E-values of protein domain hits (2.09e-144 to 1.56e-25) (Supplementary Fig.\u0026nbsp;5).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePaleoproteomics analysis of Ixodida\u003c/b\u003e \u003cb\u003eCornupalpatum\u003c/b\u003e \u003cb\u003esp. identified in amber inclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe paleoproteomics analysis identified only one protein matching to \u003cem\u003eHaemaphysalis longicornis\u003c/em\u003e Actin with 10.6% coverage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplementary Table\u0026nbsp;1). Protein phylogenetic analysis identified \u003cem\u003eH. longicornis\u003c/em\u003e as the most represented species within the same clade with other tick and mite species (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The analysis of the \u003cem\u003eH. longicornis\u003c/em\u003e Actin secondary structure evidenced a stable protein that may be associated with conservation in fossil inclusion with most of the identified peptides located in highly stable protein regions (Supplementary Fig.\u0026nbsp;6).\u003c/p\u003e \u003cp\u003eAs observed in Holothyrida, Actin sequence has not been reported in \u003cem\u003eCornupalpatum\u003c/em\u003e sp. and thus identified peptides by paleoproteomics may belong to this species.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePaleoproteomics analysis of amber control samples.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo provide additional support to obtained results, two amber samples were processed as controls. The insect inclusions identified together with Holothyrida, Neothyridae (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;1, Supplementary Fig.\u0026nbsp;2) were included, and amber obtained from the same piece but without inclusions was processed as a negative control. The results in insect inclusions identified \u003cem\u003eFrankliniella intonsa\u003c/em\u003e Actin in accordance with morphological evaluation but with a highly conserved protein in insects (\u0026gt;\u0026thinsp;97% identity and E-value\u0026thinsp;=\u0026thinsp;0.0) (Supplementary Fig.\u0026nbsp;7). A peptide (NH\u003csub\u003e2\u003c/sub\u003e-TILDDINR-COOH) exclusively found in Fly agaric fungi, \u003cem\u003eAmanita muscaria\u003c/em\u003e, was also identified (Supplementary Fig.\u0026nbsp;8A) with intense and well assigned mass spectrometry (MS) spectrum (Supplementary Fig.\u0026nbsp;8B). Additionally, an intermediate filament protein ON3 was identified matching \u003cem\u003eCulex pipiens\u003c/em\u003e (Supplementary Fig.\u0026nbsp;9A), but this protein showed a 95% percent identity to zebrafish \u003cem\u003eDanio rerio\u003c/em\u003e keratin, type II cytoskeletal 5 protein (Supplementary Fig.\u0026nbsp;9B). Three additional proteins were identified with one peptide each matching insect species red flour beetle (\u003cem\u003eTribolium castaneum\u003c/em\u003e), scarce chaser dragonfly (\u003cem\u003eLadona fulva\u003c/em\u003e) and sandfly (\u003cem\u003eLutzomyia longipalpis\u003c/em\u003e) (Supplementary Fig.\u0026nbsp;9C). UniProt BLAST analysis with ensembled protein with three insect-derived peptides resulted in significant homology only with wasp \u003cem\u003eCeratosolen solmsi marchali\u003c/em\u003e (Order: Hymenoptera, Family: Agaonidae) uncharacterized protein (Supplementary Fig.\u0026nbsp;9D). Neuroptera-derived proteins were not identified. The analysis of negative control amber without inclusions against different databases were similar and showed only one protein that appeared as \u0026ldquo;REVERSED\u0026rdquo;, meaning \u0026ldquo;FALSE\u0026rdquo; (Supplementary Fig.\u0026nbsp;10).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTaken together, the results of morphological and paleoproteomics studies supported the identification of Holothyrida, Neothyridae and Ixodida \u003cem\u003eCornupalpatum\u003c/em\u003e sp. in the amber fossil inclusions as part of the molecular evolutionary tree of Parasitiformes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) reconstructed based on published studies (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). The Holothyrida, Neothyridae was identified at both morphological and molecular levels. \u003cem\u003eCornupalpatum\u003c/em\u003e sp. was morphologically identified and although paleoproteomics identified an \u003cem\u003eH. longicornis\u003c/em\u003e Actin, these two genera are evolutionary closely related (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) and Actin protein sequences identified in both amber inclusions may belong to Holothyrida, Neothyridae and \u003cem\u003eCornupalpatum\u003c/em\u003e sp., respectively.\u003c/p\u003e \u003cp\u003eOf these fossil arthropod parasitiformes, only \u003cem\u003eCornupalpatum\u003c/em\u003e spp. have been reported before in amber (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), thus providing the first evidence for fossil Holothyrida, Neothyridae.\u003c/p\u003e \u003cp\u003eThe analysis in control amber samples with insect inclusions identified \u003cem\u003eF. intonsa\u003c/em\u003e Actin in support to morphological results. \u003cem\u003eFrankliniella\u003c/em\u003e spp. such as \u003cem\u003eF. occidentalis\u003c/em\u003e interact with fungi (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) with digestion of fungal spores (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), which may be associated with the Fly agaric fungi peptide identified. Other \u003cem\u003eFrankliniella\u003c/em\u003e spp. such as \u003cem\u003eF. zizaniophila\u003c/em\u003e are known as aquatic grass-inhabiting thrip (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) and thus \u0026ldquo;contaminated\u0026rdquo; fish residues may be present as identified zebrafish keratin. The presence of other insect-derived peptides agrees with morphological uncertainties in the putative morphologically identified \u003cem\u003eFrankliniella\u003c/em\u003e sp. and the absence of Neothyridae-derived peptides. Negative control amber without inclusions provided negative results after MS analysis as expected.\u003c/p\u003e \u003cp\u003eOverall, these results support the methodology developed and applied here for paleoproteomics studies of arthropod amber fossil inclusions. The study of multiple amber inclusions by paleoproteomics can advance the characterization of molecular evolution in parasitiformes. This cross disciplinary research between morphological and molecular studies was applied for the analysis of amber inclusions before (morphology) and after full destruction (paleoproteomics) of the specimens. This fact renders the presented paleoproteomics approach only truly complementary of morphological analyses when multiple specimens of a given taxon/species are available and so destructive sampling is affordable.\u003c/p\u003e \u003cp\u003eIdentified proteins such as Ubiquitin-like domain-containing protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Supplementary Table\u0026nbsp;1) are highly conserved (Supplementary Table\u0026nbsp;2) with multiple functions in different cellular processes including immune response, autophagy, transcription and cellular differentiation (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) and with possible biomedical applications as therapeutic targets (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). In contrast, homologues to \u003cem\u003eAceria tosichella\u003c/em\u003e Triosephosphate isomerase protein sequence involved in glycolysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Supplementary Table\u0026nbsp;1) were not identified in other species including humans (Supplementary Table\u0026nbsp;2). Although Triosephosphate isomerase 1 (TPI1; U3KPZ0) has been reported in humans as potential target in cancer (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e), amino acid sequence differences suggest possible molecular de-extinction biomedical applications (\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Another consideration is the Alpha-gal Syndrome (AGS), an allergy to non-primate mammalian meat and derived products associated with tick bites and IgE antibodies to the oligosaccharide galactose-α-1,3-galactose (alpha-gal) (reviewed by 40). The hypothesis is that hominids evolved through catastrophic selection events such as the inactivation of alpha-gal synthesis associated with antibody-mediated improved protection to infection by pathogens with this modification (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Tick proteins recognized by IgE antibodies in patients with anaphylactic reactions to tick bites but not by healthy individuals included proteins identified by paleoproteomics in Neothyridae with (NADH-ubiquinone oxidoreductase; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Supplementary Table\u0026nbsp;1) and without (Elongation factor 1-alpha; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Supplementary Table\u0026nbsp;1) alpha-gal modifications and with a role of these proteins in anaphylaxis to tick bites (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). These results suggested the possibility of applying paleoproteomics for the identification and characterization of proteins in fossil arthropod parasitiformes that may be involved in host-ectoparasite coevolutionary processes and diseases with potential positive and negative effects (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn conclusion, the application of paleoproteomics analysis described here to arthropod amber inclusions advances in the characterization of fossil proteins in the molecular evolution of parasitiformes to complement morphological studies while providing potentially relevant functional information.\u003c/p\u003e\n\u003ch3\u003eLimitations of the study\u003c/h3\u003e\n\u003cp\u003eDespite these advances in ancient phylogenies, current limitations of paleoproteomics are based on the limited size and genetic information of ancient amino acid sequences recovered from fossil inclusions (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Consequently, the phylogenetic analyses may not be sufficiently confident if only based on paleoproteomics data. Future directions may include development of more efficient protein extraction and mass spectrometry resolution together with the combination with other methods such as morphological analyses as shown here. As recently discussed (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e), sustainable sampling and international collaborations to preserve fossil inclusions and address ethical concerns is important to consider for future studies.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAmber inclusions\u003c/h2\u003e \u003cp\u003eArthropod inclusions (Holothyrida, Neothyridae; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://keys.lucidcentral.org/keys/mites/qmites/html/Holothyrida.htm\u003c/span\u003e\u003cspan address=\"https://keys.lucidcentral.org/keys/mites/qmites/html/Holothyrida.htm\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with insects (putative lacewing larva, Neuroptera, Chrysopidae and a thrip, Thysanoptera, Thripidae, \u003cem\u003eFrankliniella\u003c/em\u003e sp.) and (\u003cem\u003eCornupalpatum\u003c/em\u003e sp.) in Burmese or Kachin (Burma, Myanmar) amber (Cretaceous, ca. 99 mya) were used for the study. Amber without inclusions and derived from the piece with Holothyrida, Neothyridae and insects was used as negative control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Amber pieces originated from KGJ Collection (Ciudad Real, Spain) in which coauthor JF is included, and were dated to Cretaceous by radiometric analysis (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e) (Holothyrida, Neothyridae, 119.13 \u0026plusmn; 0.92 mya; \u003cem\u003eCornupalpatum\u003c/em\u003e sp., 100.33 \u0026plusmn; 0.51 mya) (Declarations of legal origin S1 and S2). The tick \u003cem\u003eCornupalpatum\u003c/em\u003e sp. showed damage of the body preventing an accurate determination to the species level (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImage capture and analysis\u003c/h3\u003e\n\u003cp\u003eHigh-resolution images were captured with Confocal microscopy and Ct scan tomography. For confocal microscopy, the amber inclusions were imaged using a Leica TCS SPE DM 5500 CSQ V-Vis (Mannheim, D-68165, Germany) at the Natural History Museum of Madrid (MNCN-CSIC). The images were acquired with a solid-state laser operating at 488 nm, a 10X eye piece, HCX PL FLUOTAR 5X/0.15, ACS APO 10X/0.3 dry objectives and the Leica Application Suite Advanced Fluorescence software (Leica MM AF 1.4). Fluorescence emissions were collected from approximately 10 nm above the excitation wavelength up to 800 nm. Laser power for acquisition was set by viewing the fluorescence emission and increasing the power until the rate of increase in fluorescence appeared to have slowed. The photomultiplier gain for acquisition was then set by viewing the image and increasing the gain until signal overload was detected, at which point the gain was reduced slightly. The 532 nm laser was used for general visions and the 635 nm for used for the jaws. Pixels matrices of 1024 \u0026times; 1024, two-dimensional mode, with speed of 400 Hz, and frame average of 4 were acquired for each Z-step of 0.68 micras at a zoom setting of 1.5. An Airy unit setting of 1 was routinely used for the observation pinhole. Tomography was applied to amber piece with \u003cem\u003eCornupalpatum\u003c/em\u003e sp. using a Nikon XTH 160 micro-CT X-ray scanner (Tokyo, Japan) to obtain high-quality 3D images of the inclusion. The images were generated with a molybdenum target and an X-ray voltage of 117 kV and 55 \u0026micro;A, obtaining a voxel size of 3.6 \u0026micro;m. During tomography, projections were collected every 0.12 degrees in duplicate, thus improving the signal/noise ratio and with an integration time of 708 msec. Low-resolution images were captured with a Leica (L`Hospitalet de Llobregat, Barcelona, Spain) M80 routine stereo microscope using a 1X PLAN objective and a 2X-6X zoom (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.leica-microsystems.com/products/light-microscopes/stereo-microscopes/p/leica-m80/\u003c/span\u003e\u003cspan address=\"https://www.leica-microsystems.com/products/light-microscopes/stereo-microscopes/p/leica-m80/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and a Carl Zeiss stereomicroscope (SteREO Discovery V12, Munich, Germany) using the ZEN 2 pro software. Microscope images were analyzed using Image J program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.net/ij/\u003c/span\u003e\u003cspan address=\"https://imagej.net/ij/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and pencil sketch of images using IOimageonline.co (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pencilsketch.imageonline.co/index.php\u003c/span\u003e\u003cspan address=\"https://pencilsketch.imageonline.co/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eProcessing amber inclusions and proteomics\u003c/h3\u003e\n\u003cp\u003eAmber pieces were scrubbed in 5% SDS with a brush and abundantly rinsed first in water and then in 100% methanol. The amber was covered with liquid nitrogen and fractured with a sterile ceramic pestle. Amber fragments without inclusions were discarded and arthropod inclusions were triturated to appearance of powder. Triturates were extracted in 50 \u0026micro;l Laemmli sample buffer by applying 10 cycles of sonication followed by vortex. Subsequently, samples were heated to 90 \u0026ordm;C for 5 min, centrifuged at 12,000 x g for 5 min and supernatants were collected, concentrated on-gel and trypsin digested as previously described (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). The resulting tryptic peptides were desalted onto OMIX Pipette tips C18 (Agilent Technologies, Santa Clara, CA, USA), dried down and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until mass spectrometry analysis. Samples were resuspended in 10 \u0026micro;l of 2% acetonitrile \u0026minus;\u0026thinsp;5% acetic acid in water and analyzed by reverse-phase liquid chromatography coupled online to mass spectrometry (RP-LC-MS/MS) using an Ekspert\u0026trade; nLC 415 system coupled with a 6600 TripleTOF mass spectrometer (AB Sciex, Framingham, MA, USA) through Information-Dependent Acquisition (IDA). The peptides were concentrated in a 0.1 \u0026times; 20 mm C18 RP precolumn (Thermo Scientific, Waltham, MA, USA) with a flow rate of 5 \u0026micro;l/min during 10 min in solvent A. Then, peptides were separated in a 0.075 \u0026times; 150 mm C18 RP column (Eksigent, part of AB Sciex) with a flow rate of 300 nl/min. Peptides elution was done in a 60-min gradient from 5\u0026ndash;30% solvent B followed by a 10-min gradient from 30\u0026ndash;60% solvent B (Solvent A: 0.1% formic acid in water, solvent B: 0.1% formic acid in acetonitrile) and directly injected into the mass spectrometer for analysis. 2 \u0026micro;l of each independent sample were analyzed by duplicate. The mass spectrometer was set to scanning full spectra from 350 m/z to 1400 m/z (250 ms accumulation time) followed by up to 50 MS/MS scans (100\u0026ndash;1500 m/z). Candidate ions with a charge state between +\u0026thinsp;2 and +\u0026thinsp;5 and counts per second above a minimum threshold of 100 were isolated for fragmentation. One MS/MS spectrum was collected for 100 ms, before adding those precursor ions to the exclusion list during 15 s (mass spectrometer operated by Analyst R TF 1.7, AB Sciex). Dynamic background subtraction was turned off. Data were acquired in high sensitivity mode with rolling collision energy on and a collision energy spread of 5.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eThe IDA MS raw files for each sample were combined (2 runs) and subjected to database search in unison using ProteinPilot software v. 5.0.1 (AB Sciex) with the Paragon algorithm. Spectra identification was performed by searching against the following Uniprot databases. For Holothyrida, Neothyridae, Database Mite (accessed on May 28, 2024, UniProtKB 159,503 entries; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/uniprotkb?query=mite\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/uniprotkb?query=mite\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and then Database Holothyrida (accessed on May 28, 2024, UniProtKB 18 entries; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/uniprotkb?query=holothyrida\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/uniprotkb?query=holothyrida\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). For \u003cem\u003eCornupalpatum\u003c/em\u003e sp., Database Tick (Ixodidae) (accessed on May 23, 2024, UniProtKB 289,360 entries; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/uniprotkb?query=ixodidae\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/uniprotkb?query=ixodidae\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Controls with insect inclusions and negative control amber without inclusions derived from the piece with Holothyrida, Neothyridae and insects were processed in the same manner. For insects, spectra identification was performed by searching against the Insecta Uniprot database (accessed on May 28, 2024, and revised on January 20, 2025 UniProtKB 6,898,282 entries; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/uniprotkb?query=taxonomy_name:insecta\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/uniprotkb?query=taxonomy_name:insecta\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Negative control amber pieces were analyzed by searching against all databases used for Holothyrida, Neothyridae and insects. The search parameters were: iodoacetamide cysteine alkylation, trypsin digestion and gel-based ID as special factor, identification focus on biological modification and thorough ID as search effort. The detected protein threshold was set at 0.05. An independent False Discovery Rate (FDR) analysis with the target-decoy approach provided by ProteinPilot\u0026trade;, was used to assess the quality of identifications. Positive identifications were considered when identified proteins reached a 1% global FDR. Protein Blast (Blastp), conserved domains, phylogenetic and taxonomic analyses were conducted at National Center for Biotechnological Information (NCBI) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp\u0026amp;PAGE_TYPE=BlastSearch\u0026amp;LINK_LOC=blasthome\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp\u0026amp;PAGE_TYPE=BlastSearch\u0026amp;LINK_LOC=blasthome\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=2762511\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=2762511\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and UniProt (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/blast\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/blast\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Protein structure was modelled using Swiss-Model (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swissmodel.expasy.org\u003c/span\u003e\u003cspan address=\"https://swissmodel.expasy.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The mass spectrometry proteomics database was deposited to the ProteomeXchange Consortium via the PRIDE (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e) partner repository with the dataset identifier PXD055542.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings in this paper and its Supporting Information are available. The mass spectrometry proteomics database was deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD055542.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by Ministerio de Ciencia e Innovaci\u0026oacute;n/Agencia Estatal de Investigaci\u0026oacute;n MCIN/AEI/10.13039/501100011033, Spain and EU-FEDER (Grant BIOGAL PID2020-116761GB-I00), and and Junta de Comunidades de Castilla-La Mancha and FEDER (grant PROBIOHEALTH SBPLY/23/180225/000086).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.F. and M.V. conception of the project and designed research; M.V., A.E.-P., L.T., C.P., A.G.-G., D.F.-C., and J.F. conceived methodology and performed research; J.F., M.V. and A.E.-P. performed the investigation, analyzed data and visualization; J.F., A.E.-P., and M.V. wrote the original draft of the manuscript; L.T. and C.P. contributed to reviewing and editing. All authors approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSmejkal, G. B., Poinar, G. O., \u0026amp; Righetti, P. G. Will amber inclusions provide the first glimpse of a Mesozoic proteome?. \u003cem\u003eExpert Rev. Proteomics\u003c/em\u003e\u003cstrong\u003e6\u003c/strong\u003e, 1\u0026ndash;4 (2009). https://doi.org/10.1586/14789450.6.1.1.\u003c/li\u003e\n \u003cli\u003eAustin, J. J., Ross, A. J., Smith, A. B., Fortey, R. A., \u0026amp; Thomas, R. H. Problems of reproducibility--does geologically ancient DNA survive in amber-preserved insects?. \u003cem\u003eProc. Biol. 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Nucleic Acids Res. \u003cem\u003e50\u003c/em\u003e, D543\u0026ndash;D552. https://doi.org/10.1093/nar/gkab1038.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"amber, arthropod, fossil, paleontology, paleoproteomics","lastPublishedDoi":"10.21203/rs.3.rs-6123337/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6123337/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePaleoproteomics is the proteomics study of ancient proteins, which may be better preserved than DNA in fossil inclusions and particularly in amber fossilized tree resins. However, only yeast proteins were identified in the only paleoproteomics analysis conducted in amber fossil inclusions. In this study, we developed and applied a paleoproteomics approach to study fossil arthropod parasitiformes inclusions in two Burmese (also known as Kachin) amber (Cretaceous, ca. 99 mya) pieces. The results supported the identification of \u003cem\u003eCornupalpatum\u003c/em\u003e sp. and the first report of fossil Holothyrida, Neothyridae at both morphological and molecular levels. Identified proteins such as Actin (Neothyridae and \u003cem\u003eCornupalpatum\u003c/em\u003e sp.), Ubiquitin (\u003cem\u003eTetranychus urticae\u003c/em\u003e and \u003cem\u003eIxodes ricinus\u003c/em\u003e), Triosephosphate isomerase (\u003cem\u003eAceria tosichella\u003c/em\u003e), NADH-ubiquinone oxidoreductase and Elongation factor 1-alpha (Neothyridae) were analyzed to evaluate evolutionary trees with possible functional implications. These results provide a paleoproteomics approach to complement morphological studies of the molecular evolution of parasitiformes.\u003c/p\u003e","manuscriptTitle":"Paleoproteomics characterization of fossil arthropod parasitiformes amber inclusions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-03 08:02:42","doi":"10.21203/rs.3.rs-6123337/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c2346e02-c2bf-474f-b42e-719f0b41b6e8","owner":[],"postedDate":"March 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":45009280,"name":"Biological sciences/Evolution/Evolutionary theory"},{"id":45009281,"name":"Biological sciences/Biological techniques/Proteomic analysis"}],"tags":[],"updatedAt":"2025-03-11T17:35:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-03 08:02:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6123337","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6123337","identity":"rs-6123337","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}