Juan Rofes
email: juan.rofes@mnhn.fr

Sophie Cersoy

Antoine Zazzo

Aurélien Royer

Pierre-Yves Nicod

Véronique Laroulandie

Mathieu Langlais

Yvan Pailler

Céline Leandri

Franck Leandri

Matthieu Lebon

Anne Tresset

Detecting stratigraphical issues using direct radiocarbon dating from small-mammal remains Running title: Radiocarbon dating small mammal remains

Keywords: archaeology, absolute dates, small mammals, stratigraphic sequences, intrusive episodes

Frequently preserved in archaeological and paleontological sites, the tiny size of smallmammal remains favors percolations into underlying layers along stratigraphic sequences. This is one of the multiple post-depositional processes that may affect the integrity of the original deposits and therefore the subsequent scientific interpretations. Recent developments in sample preparation offer the possibility of detecting intrusive episodes through the absolute dating of minute amounts of bone (down to 10 mg), meaning that isolated elements (such as mandibles in this case) are sufficient to obtain reliable radiocarbon dates if collagen is moderately to well preserved. The radiocarbon dates obtained here for small-mammal bones (recovered from pre-Bølling to recent deposits) and their comparison with previous dates obtained from other sources (large-mammal bones, charcoal, botanical samples, etc.), with different protocols and instruments, illustrate the potential of small-mammal dating to reveal (and eventually contribute a solution to) stratigraphical issues in different archaeological contexts.

Introduction

Small-vertebrate remains are frequently preserved in archaeological and palaeontological deposits such as caves and rock shelters, sometimes in association with human remains and artifacts. Contrary to their larger counterparts, which are usually a product of human selection, the small-vertebrate accumulations mainly result from digestion or storing by their predators (i.e., birds of prey, small carnivores). Despite unavoidable filters due to specific predators (e.g., [START_REF] Mellet | Scatological origin of microvertebrate fossil accumulations[END_REF][START_REF] Andrews | Owls, Caves and Fossils: Predation, Preservation and Accumulation of Small Mammal Bones in Caves, with an Analysis of the Pleistocene Cave Faunas from Westbury-Sub-Mendip[END_REF], they reasonably well reflect local biocenoses.

Presence/absence and relative abundances of small-mammal species can thus be used as proxies for biochronology (e.g., [START_REF] Cuenca-Bescós | Biochronology of Spanish Quaternary small vertebrate faunas[END_REF], 2015) and for the reconstruction of both past environments (e.g., [START_REF] López-García | Palaeoenvironmental and palaeoclimatic reconstruction of the Latest Pleistocene-Holocene sequence from Grotta del Romito (Calabria, southern Italy) using the small-mammal assemblages[END_REF][START_REF] Rofes | The long paleoenvironmental sequence of Santimamiñe (Bizkaia, Spain): 20,000 years of small mammal record from the latest Late Pleistocene to the middle Holocene[END_REF][START_REF] Rofes | Combining Small-Vertebrate, Marine and Stable-Isotope Data to Reconstruct Past Environments[END_REF][START_REF] Royer | How complex is the evolution of small mammal communities during the Late Glacial in southwest France?[END_REF] and biogeographical histories (e.g., [START_REF] Cucchi | The Changing Pace of Insular Life: 5000 Years of Microevolution in the Orkney Vole (Microtus arvalis orcadensis)[END_REF][START_REF] Rofes | Postglacial recolonization and Holocene diversification of Crocidura suaveolens (Mammalia, Soricidae) on the north-western fringe of the European continent[END_REF].

Such interpretations are based on the working hypothesis of a strict association between small vertebrates and archaeological remains, implying a good archaeological coherence of the analyzed assemblages. However, this is not always the case. Multiple kinds of postdepositional processes can affect the stratigraphy of a given site by altering the integrity of the deposits (e.g., [START_REF] Wood | 9-A Survey of Disturbance Processes in Archaeological Site Formation[END_REF][START_REF] Texier | A propos des processus de formation des sites préhistoriques / About prehistoric site formation processes[END_REF] and therefore the subsequent scientific interpretations. For instance, caves and rock shelters are propitious places for small and medium size animals to nest or build burrows, which may significantly modify and pollute faunal assemblages [START_REF] Dalland | The evaluation of a prehistoric mound damaged by rabbit burrowing at Maryton Law, Angus[END_REF][START_REF] Mallye | Badger (Meles meles) remains within caves as an analytical tool to test the integrity of stratified sites: the contribution of Unikoté Cave (Pyrénées-Atlantiques, France)[END_REF][START_REF] Pelletier | Identifying fossil rabbit warrens: Insights from a taphonomical analysis of a modern warren[END_REF].

Absolute dates are crucial for the reconstruction of past environments: they constitute diachronic anchors for the signals tracing the evolution of climate and habitats in a given location. They are also essential for biochronology and biogeography, to explore the accuracy of chronological biomarkers, in the first case, and to test the reliability of previous hypothesis (e.g., from phylogeography), in the second. Finally, they allow the evaluation of faunal assemblages regarding the contemporaneity of archaeological remains and species (e.g., [START_REF] Costamagno | Reexamining the timing of reindeer disappearance in southwestern France in the larger context of late glacial faunal turnover[END_REF][START_REF] Pelletier | Rabbits in the grave! Consequences of bioturbation on the Neandertal "burial" at Regourdou (Montignac-sur-Vézère, Dordogne)[END_REF][START_REF] Royer | A Brief Note on the Presence of the Common Hamster during the Late Glacial Period in Southwestern France[END_REF].

Accelerator mass spectrometers (AMS) have revolutionized the field of archaeology since their inception, allowing absolute dates to be obtained for the last 50 ky [START_REF] Reimer | IntCal13 and Marine13 Radiocarbon Age Calibration Curves 0-50,000 Years cal BP[END_REF].

However, routine AMS dating still requires 60-200 mg of bone, depending on preservation state, which far exceeds the mass of isolated elements of the skeleton of many small mammals, such as rodents, shrews and bats. Here, we present 15 radiocarbon dates obtained from minute amounts of bone (less than 25 mg) of small mammals, using a Mini Carbon Dating System (MICADAS) and following a recently developed optimized protocol (Cersoy et al., 2017a(Cersoy et al., , 2017b)). The samples come from four archaeological sites with different chronologies, chrono-culturally spanning from the upper Magdalenian to recent times. We compare these new dates with previous ones obtained from different materials (e.g., largeand small mammal bones, charcoal, plants) in different laboratories, using other protocols, to check for agreements. Departing from this comparison, our purpose is to evaluate how and to what extent this new procedure can detect stratigraphical issues in archaeological sequences and contribute to their solution.

Materials and Methods

Archaeological sites

The following sites are all located in rock shelters at different altitudes, with the exception of Beg ar Loued, which is an open-air household settlement next to the coast. The location of the sites is shown in Figure 1.

The site of Peyrazet (Creysse, Lot) has five stratigraphic levels, chronologically spanning from the pre-Bølling period (end of Heinrich Stadial 1) to the Preboreal. The site contains archaeological material from the recent Laborian (=Final Paleolithic; a lithic techno-complex occurring during the transition between the Younger-Dryas and the Preboreal in southwest France [START_REF] Langlais | Le Laborien récent de la grotte-abri de Peyrazet (Creysse, Lot, France)[END_REF]), the Azilian (during the Allerød chronozone), and the Upper Magdalenian (during the Bølling) [START_REF] Langlais | La grotte-abri de Peyrazet (Creysse, Lot, France): une séquence tardiglaciaire pour le Quercy[END_REF]. Eight radiocarbon dates from reindeer, roe deer and water vole bones confirmed this chronology [START_REF] Langlais | Le Laborien récent de la grotte-abri de Peyrazet (Creysse, Lot, France)[END_REF][START_REF] Costamagno | Reexamining the timing of reindeer disappearance in southwestern France in the larger context of late glacial faunal turnover[END_REF][START_REF] Royer | How complex is the evolution of small mammal communities during the Late Glacial in southwest France?[END_REF].

The settlement of Beg ar Loued (Molène Island, Brittany) was built during the Early Bronze Age. The radiocarbon dated shrew mandible here comes from the infill of a dry-stone oval building, which, according to several radiocarbon dates obtained from other sources (charcoal and large-mammal bones for the most part), was likely occupied from 2200 to 1800 years cal BC [START_REF] Pailler | Landscape Evolution and Human Settlement in the Iroise Sea (Brittany, France) during the Neolithic and Bronze Age[END_REF][START_REF] Pailler | Une maison sous les dunes: Beg ar Loued, Ile Molène, Finistère. Identité et adaptations des groups humaines en mer d'Iroise à la transition des IIIe-IIe millénaires avant notre ère[END_REF].

The rock shelter of Lano (Castagnicia, Haute-Corse, Corsica) was used for funerary purposes, as revealed by the presence of three wooden coffins found inside. The coffins contained human remains dating to the Bronze Age, based on radiocarbon dating of the human bones and wood from a coffin [START_REF] Leandri | La cavité sépulcrale de Laninca (Lano, Haute-Corse)[END_REF]. In 2016, a test pit was conducted at roughly 2 m from the entrance of the cave. The stratigraphic section was divided into stratigraphic units (US), which, from bottom to top of the sequence, were named US 12 to US 5. The layers D1-D5, from a previous test pit made in 2015, were correlated with US 5 and 6 of this new stratigraphy, being D1 the uppermost and D5 the lowest.

The Grande Rivoire site (Sassenage, Isère, Auvergne-Rhône-Alpes) has been divided in several excavation sectors. The small mammal remains dated in this study come from the NR16-21 and SU16-22 sectors. They comprise five different chrono-cultural units, listed here from earliest to latest: Second Mesolithic, transition from Second Mesolithic to Early Neolithic, Early Neolithic, Middle Neolithic 1 and Middle Neolithic 2. These units were subdivided in turn into subunits ("décapages") and several radiocarbon dates from charcoal, large-mammal bones, seeds and hazelnut were obtained for them, all supporting the chronology [START_REF] Rofes | Datations radiocarbones sur des restes de musaraignes[END_REF].

Samples

Fifteen mandibles of shrews (Soricidae, Mammalia) from the above-mentioned locations have been radiocarbon dated (Table 1). The mandibles belong to the species Crocidura suaveolens, C. russula, C. leucodon, Neomys anomalus and Sorex gr. araneus-coronatus, weighing between 10.5 and 25.0 mg as displayed in Table 2. They were part of natural accumulations of small vertebrates, mainly contributed by birds of prey (i.e., rejection pellets). The taxonomical identifications were performed by one of us (JR), based on diagnostic morphological characters.

Sample selection: optimized sample yield and ATR-FTIR estimation of collagen content

Prior to radiocarbon dating, we estimated the collagen content of the samples using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), following the procedure described in [START_REF] Lebon | Rapid Quantification of Bone Collagen Content by ATR-FTIR Spectroscopy[END_REF]. Due to their small size, only three samples could be examined (JR1, JR 16 and JR 24, Table 1). Briefly, less than one mg of crushed bone was placed on the diamond ATR accessory (Golden Gate Single Reflection Diamond ATR accessory, Specac, France) and analysed with a Vertex 70 FT-IR spectrometer (Bruker Optics, France). ATR spectra (Figure 2) were obtained by the accumulation of 128 scans in the wavenumber range 4000-400 cm -1 with a spectral resolution of 2 cm -1 . During acquisition, ν 3 PO 4 peaks were normalized to an absorbance of 0.5. Data treatment (linear baseline correction and amide I /phosphate ν 3 (PO 4 ) bands measurement) was performed using OPUS software (Bruker Optics, France). Collagen contents were estimated from the amide I/PO 4 ratio using equations reported in [START_REF] Lebon | Rapid Quantification of Bone Collagen Content by ATR-FTIR Spectroscopy[END_REF], taking standard deviations into account. Note that this method is not reliable for quantification below the 4% yield threshold, and collagen detection is impossible below 3%.

The amount of bone sample needed to perform the radiocarbon date can be predicted using a chart (Figure 3). In the X-axis, the collagen yield in % is reported. Using the 0.2 mgC curve, which is the threshold for the commercially available AGE3 graphitization unit, the minimum amount of bone required for a radiocarbon date can be read on the Y-axis. Two more curves are reported, corresponding to 0.5 mgC and 1 mgC: they show the limit for radiocarbon dating on (more widespread) conventional AMS. This limit gives the amount of bone needed at worst to obtain a radiocarbon date: 18 mg for MICADAS dating (after graphitization) and 40 mg for conventional AMS dating. On the other hand, maximum collagen content in modern fresh bones is indicated (20-25 %) [START_REF] Schoeninger | Detection of bone preservation in archaeological and fossil samples[END_REF].

Collagen content for the three samples varies between 5.2 and 8.0 % indicating that a minimum amount of 6.3 to 9.6 mg of bone is required, if all the bone collagen content is extractible (meaning that the collagen content is equal to the collagen extraction yield), to obtain the minimum 0.2 mg of carbon for radiocarbon dating analysis on ECHoMICADAS (dotted curve). As mandibles weighed between 10.5 and 25 mg (as stated in Table 2), we applied the extraction protocol to all the samples.

Collagen extraction and radiocarbon dating

Collagen extraction and radiocarbon dating were performed following Cersoy et al. (2017aCersoy et al. ( , 2017b)). Briefly, mandibles were immersed in 0.2 M hydrochloric acid at 4 °C for 2 days to perform demineralization. The remaining decalcified collagen was rinsed with Milli-Q water and purified in 0.1 M NaOH at 4 °C for 2 days to eliminate soil contaminants. Finally, collagen was rinsed with 0.2 M HCl and then Milli-Q water before gelatinization in 0.06 M hydrochloric acid at 90 °C for one hour, and purification by glass filtering. For this last step, specific glass vacuum filtration devices, with 1.6 microns maximum pore size, where designed and manufactured by Ellipse (France). Freeze-dried collagen was transferred to tin capsules after resuspension in ultrapure water and combustion was performed in an Elemental Analyzer (EA). All samples, except JR 15, were graphitized on the commercial compact graphitization system AGE 3 (Ion plus, Switzerland) (Wacker et al., 2010a). Due to the low amount of collagen extracted from JR 15 bone sample, a gas interface system (GIS) was used [START_REF] Wacker | A versatile gas interface for routine radiocarbon analysis with a gas ion source[END_REF]. Following combustion in the EA, sample CO 2 was adsorbed on a zeolite trap before being released and expanded to the syringe of the GIS [START_REF] Ruff | On-line Radiocarbon Measurements of Small Samples Using Elemental Analyzer and MICADAS Gas Ion Source[END_REF][START_REF] Wacker | A versatile gas interface for routine radiocarbon analysis with a gas ion source[END_REF]. For all samples, dating was performed on the compact AMS ECHoMICADAS at Gif-sur-Yvette (France) (Wacker et al., 2010b).

Results

The radiocarbon dates of the samples are reported in Table 2. Collagen yields range between 3.1 and 15.4 %, above the 1% threshold for datable samples, confirming the moderately to good preservation of the collagen in the bone samples. We were thus able to extract enough collagen for radiocarbon dating (corresponding to 0.188 to 0.862 mgC). C/N ratios range between 3.2 and 3.6, within the 2.9-3.6 limits suitable for radiocarbon dating [START_REF] Deniro | Postmortem preservation and alteration of in vivo bone isotope ratios in relation to palaeodietary reconstruction[END_REF][START_REF] Van Klinken | Bone collagen quality indicators for palaeodietary and radiocarbon measurements[END_REF].

Peyrazet

Comparing the dates in this study with those previously obtained for Level 4 and Level 2 (Table 3), we observe that, with regard to Level 4, there is an agreement between our dates (12940±70 14C a BP, 12960±70 14C a BP) and the one previously performed on a selection of water vole (Arvicola sp.) bones dated altogether (12960±70 14C a BP; Royer, 2016) in another laboratory (Lyon) with conventional extraction and classical AMS dating. The rest of the dates available for this unit (Ly-6437 to Ly-13447 in Table 3) are also consistent with the new ones and with the chrono-cultural attribution (Upper Magdalenian).

However, there is a significant difference in Level 2 between our dates (1585±30 14C a BP, 1475±30 14C a BP) and the one previously performed on a metapodial of red deer (Cervus elaphus) considered to be in situ (9780±45 14C a BP; [START_REF] Langlais | Le Laborien récent de la grotte-abri de Peyrazet (Creysse, Lot, France)[END_REF]. Another date newly obtained for the site (11790±230 14 C a BP) is also in agreement with a previous one obtained for a roe deer (Capreolus capreolus) metacarpal (11810±50 14C a BP), both remains from the same stratigraphical interface between levels 3 and 4.

Beg ar Loued

A mandible of Neomys anomalus gave a date of 3650±30 14C a BP (ECHo-1258). This date is somewhat older than a previous one, performed on a Microtus agrestis mandible (3177±35 14C a BP, UB-6925) found in the same scree layer inside the house (Table 3) [START_REF] Pailler | Une maison sous les dunes: Beg ar Loued, Ile Molène, Finistère. Identité et adaptations des groups humaines en mer d'Iroise à la transition des IIIe-IIe millénaires avant notre ère[END_REF].

Lano

The comparison of our dates (Table 3) for layers D4 (post-bomb) and D1 (515±20 14C a BP) with those previously obtained from the Poznan Radiocarbon Laboratory for layers D5 (postbomb) and D3 (1155±30 14C a BP), the latter two from selections of small-vertebrate bones with a different technique, allows us to state that: 1) layers D1 and D3 are in good stratigraphical order, D1 dating to the 15th century and D3 to the 8th-10th centuries; 2) D4 and D5 are modern; 3) these data in turn allow to infer that the sequence D5-D1 presents a "stratigraphic inversion", where layers D4 and D5 are more recent than D3-D1; and 4) considering that the US 6 and 5 nearly coincide with layers D5-D1 (see Lano section on Materials and Methods), these units may also present a stratigraphic inversion.

Grande Rivoire

Comparing our seven dates to the corpus previously obtained from other materials (i.e., largemammal bones, charcoal, plants), by different laboratories (Lyon-Saclay and Beta Analytics), we observe that they all agree with the general chronostratigraphic framework. Some of our dates (5070±40 14C a BP [d88], 5760±40 14C a BP [d111], 6510±40 14C a BP[d142]) are almost identical to those already obtained on the same layers (see Table 4), whereas others (7130±40 14C a BP [d143], 7600±50 14C a BP [d147], 6750±50 14C a BP [d28]) are a few centuries older without being in contradiction with the general framework. Finally, it should be noticed that the date obtained for the layer d22 of the sector SU16-22 (6410±45 14C a BP), attributed to the early Neolithic by the excavators, is also coherent with that of the layer d26 (6510±40 14C a BP), which is directly underlying.

It should be noted that three of the oldest dates appear to have a higher C/N ratio, above 3.5. Nonetheless, hardly this can explain the discrepancy observed for two reasons: (1) usually, contamination makes the sample appear younger (and not older) because contaminants come from the soil organic matter whose 14C activity is younger than the bone remains (as recently demonstrated in [START_REF] Zazzo | High-precision dating of ceremonial activity around a large ritual complex in Late Bronze Age Mongolia[END_REF] and ( 2) other samples have also high C/N ratio but they are in good agreement with the paired date, like, for instance, JR 16 (from Peyrazet, described above), which is older and therefore more prone to contamination. Hence, we think the discrepancy observed is rather due to a stratigraphic issue at Grande Rivoire rather than to contamination.

Discussion

In Peyrazet, the perfect agreement between the new dates and those previously obtained in another laboratory for Level 4 (Upper Magdalenian) confirms the reliability of the new protocol. At the same time, these direct dates attest the oldest postglacial presence of N. anomalus in western Europe (see Table 3). The two dates from the interface between Levels 4 and 3 (Upper Magdalenian/Azilian), confirm one another despite the large uncertainty associated with our measurement (±230 14C a) for the mandible of C. cf. suaveolens (due to the low amount of extractible collagen). To our knowledge, this is the oldest direct postglacial evidence (i.e., 14175-13129 cal a BP) for this taxon in western Europe up to now, giving an age well within the Bølling-Allerød warm pulse (c. 14700 to c. 12700 a BP).

Notably, it confirms a recently proposed scenario for the postglacial recolonization of this species from the Iberian Peninsula [START_REF] Rofes | Postglacial recolonization and Holocene diversification of Crocidura suaveolens (Mammalia, Soricidae) on the north-western fringe of the European continent[END_REF]. There is a notorious lack of absolute dates for small mammal taxa to support postglacial recolonization models inferred by other means (e.g., molecular phylogeography), thus those recently obtained for the N. anomalus and C. cf. suaveolens of Peyrazet will certainly be of great biogeographical and biochronological value.

The difference between our dates and those previously obtained regarding Level 2, can be explained by the presence of bioturbation in upper deposits. The archaeological materials seem relatively homogeneous from a cultural standpoint, although protohistoric ceramics have been found in these upper levels [START_REF] Langlais | Le Laborien récent de la grotte-abri de Peyrazet (Creysse, Lot, France)[END_REF]. The integrity of the small vertebrate assemblages was questioned due to the association of black rat (Rattus rattus) and European hamster (Cricetus cricetus) remains [START_REF] Langlais | Le Laborien récent de la grotte-abri de Peyrazet (Creysse, Lot, France)[END_REF]. If the first species is clearly not attested in France before the Antiquity [START_REF] Pascal | Invasions biologiques et extinctions : 11000 ans d'histoire des vertébrés en France[END_REF], the second one suggests that these materials could be Younger-Dryas in origin, as it has been recently demonstrated for the hamster remains in Combe Cullier [START_REF] Royer | A Brief Note on the Presence of the Common Hamster during the Late Glacial Period in Southwestern France[END_REF]. The recent radiocarbon dates obtained from the two shrew remains (c. 5 th -7 th centuries AD) thus strongly suggest the presence of more recent intrusive remains in this level, presumably brought from upper layers by burrowing activities. Further inferences made from this level should then be taken with caution.

Figure 4 displays the stratigraphy of Peyrazet (4A) and synthetizes all our findings (4B), including agreements and disagreements among dates.

The discrepancy of dates in Beg ar Loued may be due to the different periods in which the remains were naturally deposited inside the structure. The Neomys mandible could date from the foundation of the building during the Early Bronze Age, whereas the Microtus mandible could date from the time of abandonment of the household during the Middle Bronze Age.

There are several potential reasons for their coexistence in the same scree level, including the activity of burrowing animals. Another straightforward explanation is that the shrew mandible was deposited over an elevated stone-wall which posteriorly crumbled.

In Lano, the presence of R. rattus in the lowermost levels of the sequence (US 12), indicates that the accumulation postdates the 4 th -2 nd centuries BC, a period during which the black rat was introduced into Corsica [START_REF] Pascal | Invasions biologiques et extinctions : 11000 ans d'histoire des vertébrés en France[END_REF]. The absence of endemic taxa, such as Prolagus, Asoriculus, Rhagamys or Tyrrenicola, also indicates that this deposit is contemporary or postdates the extinction of the native fauna of the island between the 2 nd and the 13 th centuries AD. The stratigraphic sequence studied is thus clearly posterior to the deposit of the wooden coffins, which date from the first millennium BC [START_REF] Leandri | La cavité sépulcrale de Laninca (Lano, Haute-Corse)[END_REF]. It is too early to propose an explanation regarding the "stratigraphical inversion" detected. Works on the site continues and excavators will be reaching the end of the cavity soon. That will allow identification of the initial area of the deposit and perhaps the reason for the inversion, a phenomenon which is not surprising given the complexity of sedimentary processes in caves.

In Grande Rivoire, the seven ECHo dates are consistent with the general chrono-stratigraphy of the deposit (see Table 4 where they are displayed in stratigraphic order), even if there are three (ECHo-1247, ECHo-1696and ECHO-1702) which are older (~3 centuries) than the measurements made on hazelnuts (Lyon-13971 and Lyon-13972) or bones (Beta 28246 and Beta-255118) from the same levels. For the two results dating back to the Mesolithic/ Neolithic transition (ECHo-1247 and ECHo-1702), the lag of a few centuries could be due to the slow formation of the levels in question, well evidenced by the sedimentological analyzes [START_REF] Nicod | Continuités et ruptures culturelles entre chasseurs mésolithiques et chasseurs néolithiques en Vercors: analyse préliminaire des niveaux du Mésolithique récent et du Néolithique ancien sans céramique de l'abri-sous-roche de la Grande Rivoire (Sassenage, Isère). In Dynamismes et rythmes évolutifs des sociétés de la Préhistoire récente[END_REF]. However, the good general coherence between the new dates and those previously obtained with other techniques, accredits also in this case the reliability of the new protocols. Moreover, these new dates show that the natural accumulations of small vertebrates in the different units do not result from modern contamination or postsedimentary rearrangements. They instead come from in situ accumulations contemporary to the formation of the deposits. This validates any further stratigraphic, biogeographical and/or palaeoenvironmental interpretation made from the small-mammal associations of this site. It should be mentioned that the dates obtained from charred hazelnuts (layers d143 and d147) are clearly more recent than the new dates from the same layers. This might be explained either by contamination or intrusions from upper units.

Conclusions

The radiocarbon dates obtained here for small-mammal bones and their comparison with previous dates obtained from other sources (e.g., large-mammal and other small-vertebrate bones, charcoal, botanical samples, etc.), with different protocols and instruments, show the potential of small-vertebrate dating to reveal (and eventually contribute a solution to) stratigraphical issues in different archaeological contexts, from pre-Bølling to recent times.

Our results show that chronological, environmental and/or biogeographical inferences based on the small-mammal assemblages from Levels 3 and 4 of Peyrazet, and the entire stratigraphical sequence of Grande Rivoire, are trustworthy. We cannot state the same for Level 2 of Peyrazet, the infill of the structure of Beg ar Loued, or the stratigraphy of Lano.

Caution is advisable in the latter cases.

The tiny size of small-mammal remains favors percolations into underlying layers along stratigraphic sequences: this is a well-known phenomenon in archaeological deposits. The new technique offers a straightforward possibility of detecting intrusive episodes through the absolute dating of minute amounts of bone (down to 10 mg in this study), meaning that isolated elements (such as shrew mandibles in this case) are enough to obtain reliable radiocarbon dates if collagen is moderately to well preserved. Besides, this technique is far less destructive than previous ones, which require a minimum of 60 mg of bone to obtain results, meaning collections of bones of small vertebrates with the possible admixture of noncontemporaneous materials. Moreover, for bones of sufficient size, an ATR-FTIR prescreening can estimate quickly, with just 1 mg of bone, whether a sample contains sufficient collagen to be successfully radiocarbon dated or not, well before any further extraction or intense laboratory pretreatment. Table 2. Radiocarbon dating of the shrew samples. Yield is estimated as the ratio (in %) of the total amount of collagen recovered from the amount of initial bone used for extraction. The carbon mass corresponds to the amount of carbon detected following combustion in the Elemental Analyzer and used to produce the graphite target. ECHo n° corresponds to the target numbers. *Four dates (three from Grande Rivoire and one from Peyrazet) appear to have a C/N ratio equal to or higher than 3.5. **Due to the low amount of collagen for this sample (JR 15), 14C content was measured via a gas interface system (GIS) which explains the higher value of the error on the 14C age.

Figure 1 .

 1 Figure 1. Location of the archaeological sites.

Figure 2 .

 2 Figure 2. Prescreening ATR-FTIR spectra of selected shrew bone samples: Neomys anomalus from Peyrazet (JR 16), Sorex gr. araneus-coronatus from Grande Rivoire (JR 24) and Neomys anomalus from Beg ar Loued (JR 1). The two bands of interest for collagen preservation study [ν 3 (PO 4 ) mineral and amide I organic bands] are indicated by arrows.

Figure 3 .

 3 Figure 3. Radiocarbon dating feasibility chart based on the carbon concentration in bone collagen (40%). Y-axis corresponds to the minimum bone weight needed for a reliable radiocarbon date. Xaxis corresponds to the collagen content within the bone as estimated by FTIR. Dotted line: to obtain 0.2 mgC. Solid line: 0.5 mgC. Dashed line: conventional 1 mgC. Samples JR 16, JR 1 and JR 24 are reported on the chart, considering the standard error for FTIR estimation (±1.2 %).

Figure 4 .

 4 Figure 4. (A) Archaeological profile of Peyrazet showing the chrono-cultural stratigraphic units. (B) Synthesis of the radiocarbon dates discussed in the text, including those newly obtained from shrews (blue) and those from other large and small mammals (black).

Acknowledgments

This paper is dedicated to the memory of our courageous colleague Anne Tresset, who contributed the seminal idea for the research and who sadly passed away in January 2019, during the writing of the manuscript. The research was accomplished through the financial support of a Marie Curie Intra-European Fellowship (PIEF-GA-2013-629604 of the European Commission) from which JR was the Fellow and AT the scientist in charge. JR is also a member of the Atapuerca Project PGC2018-093925-B-C33 (MINECO, Spain). Peyrazet field work obtains financial support from DRAC-SRA Occitanie and Conseil Général du Lot. The work was also supported by a State aid, managed by the French National Research Agency (ANR), namely LabEx ANR-10-LABX-0003-BCDiv, within the framework of "Investissements d'avenir" program number ANR-11-IDEX-0004-02. We are also very grateful to C. Gauthier, E. Kaltnecker, F. Thil and N. Tisnerat-Laborde, from the ECHoMICADAS team, for the radiocarbon dates (graphitization, AMS measurements and data reduction). Fiona Brock and another anonymous reviewer substantially contributed to improve the paper.


Khaled Fadhlaoui

Marie-Edith Arnal

Matthieu Martineau

Paméla Camponova

Bernard Ollivier

Paul W O'toole

Jean-François Brugère

MINI

Keywords: Trimethylamine oxide TMAO, Cardiovascular disease CVD, Archaebiotics, Trimethylamine TMA, Trimethylaminuria TMAU, Live biotherapeutic products LBP

Trimethylamine (TMA) and its oxide TMAO are important biomolecules involved in disease-associated processes in humans (e.g., trimethylaminuria and cardiovascular diseases). TMAO in plasma (pTMAO) stems from intestinal TMA, which is formed from various components of the diet in a complex interplay between diet, gut microbiota, and the human host. Most approaches to prevent the occurrence of such deleterious molecules focus on actions to interfere with gut microbiota metabolism to limit the synthesis of TMA. Some human gut archaea however use TMA as terminal electron acceptor for producing methane, thus indicating that intestinal TMA does not accumulate in some human subjects. Therefore, a rational alternative approach is to eliminate neo-synthesized intestinal TMA. This can be achieved through bioremediation of TMA by these peculiar methanogenic archaea, either by stimulating or providing them, leading to a novel kind of next-generation probiotics referred to as archaebiotics. Finally, specific components which are involved in this archaeal metabolism could also be used as intestinal TMA sequesters, facilitating TMA excretion along with stool. Referring to a standard pharmacological approach, these TMA traps could be synthesized ex vivo and then delivered into the human gut. Another approach is the engineering of known probiotic strain in order to metabolize TMA, i.e., live engineered biotherapeutic products. These alternatives would require, however, to take into account the necessity of synthesizing the 22nd amino acid pyrrolysine, i.e., some specificities of the genetics of TMA-consuming archaea. Here, we present an overview of these different strategies and recent advances in the field that will sustain such biotechnological developments.

Key points

• Some autochthonous human archaea can use TMA for their essential metabolism, a methyl-dependent hydrogenotrophic methanogenesis.

• They could therefore be used as next-generation probiotics for preventing some human diseases, especially cardiovascular diseases and trimethylaminuria. • Their genetic capacities can also be used to design live recombinant biotherapeutic products.

• Encoding of the 22nd amino acid pyrrolysine is necessary for such alternative developments.

Introduction

The human gut microbiota contains about as many cells as human ones themselves [START_REF] Sender | Revised estimates for the number of human and bacteria cells in the body[END_REF]. It is a complex mix of microbes from the three domains of life (Bacteria, Archaea, and Eukarya) which differs between human subjects [START_REF] Arumugam | Enterotypes of the human gut microbiome[END_REF][START_REF] Donaldson | Gut biogeography of the bacterial microbiota[END_REF]Human Microbiome Project 2012). It is considered similar to a human organ (the "forgotten organ") that participates in several important physiological processes in humans, encompassing notably nutrition (digestion of fibers, vitamins, and short-chain fatty acids synthesis) and protection against pathogens, as well as having endocrine functions [START_REF] Clarke | Minireview: gut microbiota: the neglected endocrine organ[END_REF][START_REF] O'hara | The gut flora as a forgotten organ[END_REF]. It has co-evolved with its host and provides genes and functions not encoded by the human host, with microbial genes accounting about 100 times more than the human genome itself [START_REF] Arumugam | Enterotypes of the human gut microbiome[END_REF][START_REF] Qin | A human gut microbial gene catalogue established by metagenomic sequencing[END_REF]. All of this leads one to consider humans and their microbes as a biological entity, a holobiont [START_REF] Simon | Hostmicrobiota interactions: from holobiont theory to analysis[END_REF]. Bacteria are the most represented microbial part of the gut microbiota, and also the most studied: they account for trillions in each individual and represent about 98% of all microbes [START_REF] Zhernakova | Population-based metagenomics analysis reveals markers for gut microbiome composition and diversity[END_REF]. Eukaryotic cells are far less abundant with yeasts being predominant among them. In-between, archaea account in mean for 1-2% of all microbes with high variations among subjects [START_REF] Zhernakova | Population-based metagenomics analysis reveals markers for gut microbiome composition and diversity[END_REF]. While the diversity of these human-hosted archaea is likely still underestimated, they are mainly anaerobes performing methanogenesis [START_REF] Bang | Archaea: forgotten players in the microbiome[END_REF][START_REF] Gaci | Archaea and the human gut: new beginning of an old story[END_REF][START_REF] Moissl-Eichinger | Archaea are interactive components of complex microbiomes[END_REF]. The main representative of such organisms in the gut is the Methanobacteriales representative Methanobrevibacter smithii [START_REF] Miller | Isolation of Methanobrevibacter smithii from human feces[END_REF].

Less than a decade ago, a small organic plasma molecule was identified as an important risk factor for cardiovascular disease (CVD), plasma trimethylamine oxide (pTMAO) [START_REF] Wang | Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease[END_REF]. It originates exclusively from the metabolic action of the gut microbiota on some various nutrients leading to one deleterious intestinal intermediary compound further oxidized by the liver. Although the role of pTMAO is a matter of debate as for example high fat-fed mice exposed to TMAO have improved glucose tolerance [START_REF] Cho | Trimethylamine-N-oxide: friend, foe, or simply caught in the cross-fire?[END_REF][START_REF] Dumas | Microbial-host co-metabolites are prodromal markers predicting phenotypic heterogeneity in behavior, obesity, and impaired glucose tolerance[END_REF][START_REF] Jaworska | TMA (trimethylamine), but not its oxide TMAO (trimethylamine-oxide), exerts haemodynamic effects: implications for interpretation of cardiovascular actions of gut microbiome[END_REF], pTMAO has been shown to be directly or indirectly involved in various mechanisms that drives cardiovascular events, like contribution to the formation of atherosclerotic plaques, foam cells, and platelet activation [START_REF] Warrier | The TMAO-generating enzyme flavin monooxygenase 3 is a central regulator of cholesterol balance[END_REF][START_REF] Zhu | Gut microbial metabolite TMAO enhances platelet hyperreactivity and thrombosis risk[END_REF]) (see (Tang and Hazen 2017;[START_REF] Zeisel | Trimethylamine N-oxide, the microbiome, and heart and kidney disease[END_REF] for a review). Since, pTMAO has also been linked to various diseases through this "diet-microbe morbid union" [START_REF] Rak | The diet-microbe morbid union[END_REF].Plasma TMAO is, for example, also adversely associated to chronic renal diseases [START_REF] Tang | Gut microbiota-dependent trimethylamine N-oxide (TMAO) pathway contributes to both development of renal insufficiency and mortality risk in chronic kidney disease[END_REF], nonalcoholic fatty liver disease NAFLD [START_REF] Chen | Associations of gutflora-dependent metabolite trimethylamine-N-oxide, betaine and choline with non-alcoholic fatty liver disease in adults[END_REF], type-2 diabetes [START_REF] Shan | Association between microbiota-dependent metabolite trimethylamine-N-oxide and type 2 diabetes[END_REF]Tang et al. 2017), or colorectal and prostate cancer [START_REF] Mondul | Metabolomic analysis of prostate cancer risk in a prospective cohort: the alpha-tocopherol, beta-carotene cancer prevention (ATBC) study[END_REF][START_REF] Xu | A genome-wide systems analysis reveals strong link between colorectal cancer and trimethylamine N-oxide (TMAO), a gut microbial metabolite of dietary meat and fat[END_REF]. Also, it has been recently shown that the gut microbial intermediary compound itself, trimethylamine (TMA) has haemodynamic effects that TMAO does not have, with a significant increased arterial blood pressure [START_REF] Jaworska | TMA (trimethylamine), but not its oxide TMAO (trimethylamine-oxide), exerts haemodynamic effects: implications for interpretation of cardiovascular actions of gut microbiome[END_REF]. At last, TMA is also involved directly in a rare inherited metabolic disorder named trimethylaminuria (TMAU) or fish odor syndrome in addition to also acquired/transient cases [START_REF] Christodoulou | Trimethylaminuria: an under-recognised and socially debilitating metabolic disorder[END_REF][START_REF] Humbert | Trimethylaminuria: the fish-odour syndrome[END_REF][START_REF] Messenger | A review of trimethylaminuria:(fish odor syndrome)[END_REF]. This is a psychosocially distressing disease (see below for reasons and mechanisms).

This peculiar case by which the gut microbiota contributes to disease provides an example on which specific, original, and rational strategies could be applied, which in turn can provide new biotechnological approaches for other (gut microbiota function-derived) diseases.

The gut microbiota and intestinal TMA standing at the crossroads At the crossroads between diet and plasma, TMAO/TMA stands the gut microbiota (Fig. 1). The latter generates intestinal TMA, next absorbed by the human host, conveyed into the liver by the portal vein where it is normally oxidized into pTMAO. This oxidation is driven by hepatic flavine monoxygenases and more specifically FMO3 [START_REF] Bennett | Trimethylamine-Noxide, a metabolite associated with atherosclerosis, exhibits complex genetic and dietary regulation[END_REF][START_REF] Warrier | The TMAO-generating enzyme flavin monooxygenase 3 is a central regulator of cholesterol balance[END_REF][START_REF] Zhu | Flavin monooxygenase 3, the host hepatic enzyme in the metaorganismal trimethylamine N-oxidegenerating pathway, modulates platelet responsiveness and thrombosis risk[END_REF]. The elimination of pTMAO will next be the result of renal excretion [START_REF] Gessner | Contribution of multidrug and toxin extrusion protein 1 (MATE1) to renal secretion of trimethylamine-N-oxide (TMAO)[END_REF]. In some cases, however, impairment of this liver oxidation avoids total or partial conversion of TMA into TMAO, leading to significant levels of TMA in blood which further diffuses in body fluids and is excreted through sweat and breath in addition to urine. The main cause of FMO3 deficiency (i.e., of liver TMA oxidation) relies on mutations in the fmo3 gene, leading to a recessive hereditary metabolic disease, trimethylaminuria (TMAU) [START_REF] Dolphin | Missense mutation in flavin-containing mono-oxygenase 3 gene, FMO3, underlies fish-odour syndrome[END_REF]. TMA is a volatile molecule which characterizes fish spoilage, resulting from the microbial reduction of TMAO present in high level in seafood for osmoregulation [START_REF] Ghaly | Fish spoilage mechanisms and preservation techniques[END_REF]. Humans are very sensitive to this rotten fish (mal)odor, which is interpreted as a protective reflex acquired through evolution, preventing us to eat spoiled food. Consequently, diet does not provide TMA directly because food would be perceived as improper to consume by humans. However, a bad consequence of the distasteful perception of TMA is for patients suffering from TMAU, as they have to handle socially their unpleasant body odor: this often leads to impaired social life, isolation, and depression of patients [START_REF] Christodoulou | Trimethylaminuria: an under-recognised and socially debilitating metabolic disorder[END_REF][START_REF] Mackay | Trimethylaminuria: causes and diagnosis of a socially distressing condition[END_REF].

While no TMA is directly consumed, at least four different nutrients are involved in the generation of TMA in the human gut (Fig. 1). First, TMAO itself is such a diet component mainly found in seafood. It is an intestinal TMA precursor through reduction by gut bacteria (and next oxidized by the liver back into TMAO), providing an example of metabolic retro conversion in humans [START_REF] Hoyles | Metabolic retroconversion of trimethylamine N-oxide and the gut microbiota. Microbiome 6(1):73 Human Microbiome Project C (2012) Structure, function and diversity of the healthy human microbiome[END_REF]. Involved gut bacteria encompass various species with members of Enterobacteriaceae as main contributors (e.g., Escherichia coli and Klebsiella pneumoniae). Other dietary quaternary ammonium compounds that contribute to intestinal TMA are mainly choline-lecithin and L-carnitine [START_REF] Al-Waiz | The exogenous origin of trimethylamine in the mouse[END_REF][START_REF] Koeth | Intestinal microbiota metabolism of Lcarnitine, a nutrient in red meat, promotes atherosclerosis[END_REF][START_REF] Wang | Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease[END_REF]) and also some others [START_REF] Jameson | Metagenomic data-mining reveals contrasting microbial populations responsible for trimethylamine formation in human gut and marine ecosystems[END_REF]. They all require specific enzymatic bacterial activities to be converted into intestinal TMA. Some of these enzymes have only very recently been identified and described [START_REF] Craciun | Microbial conversion of choline to trimethylamine requires a glycyl radical enzyme[END_REF][START_REF] Martinez-Del Campo | Characterization and detection of a widely distributed gene cluster that predicts anaerobic choline utilization by human gut bacteria[END_REF][START_REF] Zhu | Carnitine metabolism to trimethylamine by an unusual Rieske-type oxygenase from human microbiota[END_REF]). Also, the potential of each individual to convert these compounds into TMA is highly dependent on their gut microbiota composition, which shows a high inter-individual variation. For example, subjects with vegan diet were shown to not convert (or less) L-carnitine into TMA [START_REF] Koeth | Intestinal microbiota metabolism of Lcarnitine, a nutrient in red meat, promotes atherosclerosis[END_REF]: it is likely that the diet habits of such subjects lead to a counter-selection of L-carnitine metabolizing gut bacteria over time. Moreover, gut bacteria that support TMA synthesis from these various compounds are from various phylogenetic lineages supposedly due to lateral gene transfers having occurred during (gut bacteria) evolution. In consequence, it remains highly speculative to deduce from the gut microbiota composition (i.e., obtained by 16S rRNA gene sequencing) its ability to generate intestinal TMA. This is likely less speculative from metagenomics data from which the presence and proportion of microbial genes involved in TMA synthesis are determined [START_REF] Borrel | Genomics and metagenomics of trimethylamineutilizing Archaea in the human gut microbiome[END_REF][START_REF] Jameson | Metagenomic data-mining reveals contrasting microbial populations responsible for trimethylamine formation in human gut and marine ecosystems[END_REF][START_REF] Rath | Potential TMA-producing bacteria are ubiquitously found in mammalia[END_REF].

A strategy designed rationally from the mechanisms: the role of human archaea

Acting on the gut microbiota metabolism to limit pTMAO From above, many potential ways to limit circulating pTMAO and/or intestinal TMA can be imagined. However, most of them have serious disadvantages and side effects. A first idea can be to reduce the amounts of TMA-generating nutrients by selecting foods that provide lower quantities of these different molecules. However, these compounds are present in so many different foods that it makes difficult to address this question, However, intestinal TMA is not an end-product of the gut microbiota in some humans who host particular methanogenic archaea (down-right), which use TMA (+H 2 ) for their methanogenesis, an essential metabolism for their survival. Methyltransferases used for this metabolism are indicated, with an emphasis of the first necessary one, MttB, a pyrrolysinecontaining protein. See text for further details especially at the population level. Some foods are however more prone to allow such kind of intervention: for example, eggs are known to contribute a lot for choline-lecithin, red meat for L-carnitine and choline-lecithin, seafood for TMAO and glycine-betaine, and also vegetables and seeds (e.g., quinoa, wheat, or spinach for glycine-betaine). Even more important, these nutrients by themselves, if they are not metabolized into intestinal TMA, are beneficial to humans or even essential. For example, choline is an essential nutrient whose deficiency leads to severe liver dysfunctions with steatosis and neurological disorders [START_REF] Ueland | Choline and betaine in health and disease[END_REF][START_REF] Zeisel | Choline deficiency[END_REF]. At the opposite side, we could imagine the development and use of drugs inhibiting pTMAO synthesis in the liver: this could be achieved by FMO 3 inhibitors to avoid oxidation of gut-generated TMA. TMAU patients would not benefit from such a drug, and this would also generate a transient fish odor syndrome as a side effect for others. Therefore, acting on the gut microbiota and on its metabolism itself seems the lonely reliable mean, and for which various complementary ways are possible. This encompasses notably prebiotics and probiotics, but also drugs or food components that would have a (selective) inhibitory action on bacterial enzymes involved in gut TMA synthesis. Among possible drugs, 3,3-dimethyl-1-butanol or DMB shows interesting inhibitory properties of bacterial TMA lyases [START_REF] Wang | Non-lethal inhibition of gut microbial trimethylamine production for the treatment of atherosclerosis[END_REF]. This approach allows now a rational screening of possible inhibitors as recently illustrated by S. Hazen and collaborators [START_REF] Roberts | Development of a gut microbetargeted nonlethal therapeutic to inhibit thrombosis potential[END_REF]. Together with the development of drugs targeting specifically some gut microbiota functions, a recent review in this journal described microbes and probiotics associated to decreased pTMAO [START_REF] Din | Amelioration of TMAO through probiotics and its potential role in atherosclerosis[END_REF]). However, this review overlooked another, microbiome-based way to limit pTMAO, namely the usage of a unique metabolic property of some human-associated archaea.

Naturally occurring human methanoarchaea use TMA

Human archaea colonize any human inner or outer surface, as bacteria do even if it is most often in a lesser extent (Pausan et al. 2019). They are of various genera depending on the human biogeographic area, remain likely underestimated, as well considering their amount, diversity, metabolisms, functions, and contributions in each ecosystem [START_REF] Bang | Archaea: forgotten players in the microbiome[END_REF][START_REF] Gaci | Archaea and the human gut: new beginning of an old story[END_REF][START_REF] Koskinen | First insights into the diverse human Archaeome: specific detection of archaea in the gastrointestinal tract, lung, and nose and on skin[END_REF]). In the gut, these archaea are mainly strict anaerobes producing methane with the property to use H 2 as electron donor, i.e., being all hydrogenotrophs. They thus act on one major end-product of the bacterial gut fermentations, H 2 , and therefore allow to keep low pressure of H 2 and in consequence, improve fermentations yields performed by fermentative bacteria that would otherwise been inhibited. According to some large-scale metagenomics studies, they represent in mean 1-2% of the gut microbes [START_REF] Zhernakova | Population-based metagenomics analysis reveals markers for gut microbiome composition and diversity[END_REF]) but being highly variable in amount among subjects. In the human gut, the main representative is Methanobrevibacter smithii, a Methanobacteriales-affiliated species with a high prevalence (considered to be hosted by more than 50% of people with variations according to the considered population): it generates methane using H 2 as electron donor and CO 2 as electron acceptor.

Considering the aim of this article, there are two interesting features of human gut methanogens. First, none has been shown to be pathogenic, which is also the feature of any archaea towards plants or any animal [START_REF] Cavicchioli | Pathogenic archaea: do they exist?[END_REF][START_REF] Chaudhary | Methanogens in humans: potentially beneficial or harmful for health[END_REF][START_REF] Gaci | Archaea and the human gut: new beginning of an old story[END_REF][START_REF] Moissl-Eichinger | Archaea are interactive components of complex microbiomes[END_REF]). In humans, some have been associated to pathologies (e.g., periodontis) but with no evidence of a direct role on them [START_REF] Horz | Relationship between methanogenic archaea and subgingival microbial complexes in human periodontitis[END_REF][START_REF] Lepp | Methanogenic Archaea and human periodontal disease[END_REF][START_REF] Sogodogo | Methanogens as emerging pathogens in anaerobic abscesses[END_REF]. However, another methanoarchaeon of the order Methanobacteriales, namely Methanosphaera stadtmanae, has strong inflammatory properties, able to activate human monocyte-derived dendritic cells [START_REF] Bang | The intestinal ar chaea Methan ospha era s tadtmana e and Methanobrevibacter smithii activate human dendritic cells[END_REF][START_REF] Vierbuchen | The human-associated archaeon Methanosphaera stadtmanae is recognized through its RNA and induces TLR8-dependent NLRP3 inflammasome activation[END_REF]. This is not the case for other human gut methanogens tested so far [START_REF] Bang | Immunogenic properties of the human gut-associated archaeon Methanomassiliicoccus luminyensis and its susceptibility to antimicrobial peptides[END_REF]. Second, some specific archaea are able to produce methane using H 2 as electron donor and TMA as terminal electron acceptor. This unique metabolism was initially deduced from genomic insights of a human archaeon, Candidatus (Ca.) Methanomethylophilus alvus Mx1201 [START_REF] Borrel | Genome sequence of "Candidatus Methanomethylophilus alvus" Mx1201, a methanogenic archaeon from the human gut belonging to a seventh order of methanogens[END_REF]), known to be a representative of a new, 7th methanogenic archaeal taxonomic order (Borrel et al. 2013b;[START_REF] Mihajlovski | A putative new order of methanogenic Archaea inhabiting the human gut, as revealed by molecular analyses of the mcrA gene[END_REF]. Interestingly, a first isolate of this new order named Methanomassiliicoccales was obtained from a human stool sample, Methanomassiliicoccus luminyensis [START_REF] Dridi | Methanomassiliicoccus luminyensis gen. nov., sp. nov., a methanogenic archaeon isolated from human faeces[END_REF][START_REF] Iino | Candidatus Methanogranum caenicola: a novel methanogen from the anaerobic digested sludge, and proposal of Methanomassiliicoccaceae fam. nov. and Methanomassiliicoccales ord. nov., for a methanogenic lineage of the class Thermoplasmata[END_REF]. This microorganism was demonstrated to convert in vitro TMA into methane using H 2 according to the metabolic pathway schematically shown in Fig. 1. This was also confirmed using culture enrichments of Ca. M. alvus [START_REF] Borrel | Genomics and metagenomics of trimethylamineutilizing Archaea in the human gut microbiome[END_REF][START_REF] Brugere | Archaebiotics: proposed therapeutic use of archaea to prevent trimethylaminuria and cardiovascular disease[END_REF] as well with a first isolate of this species, Ca. Methanomethylophilus alvus strain Mx-05 (W. Ben Hania, K. Fadhlaoui et al., in prep.).

Archaea, potential but neglected next-generation probiotics

Distinctive features of Methanomassiliicoccales

Methanogenesis is the essential and the only energyyielding metabolic pathway for any methanoarchaeon, functionally similar to the cellular respiration in other anaerobes (i.e., an electron transport coupled to translocations of protons, with next an ATP synthase using this proton (or Na + ) motive force to generate ATP). Methanomassiliicoccales have several interesting features that are not found in other methanogens (Borrel et al. 2013b;[START_REF] Borrel | Comparative genomics highlights the unique biology of Methanomassiliicoccales, a Thermoplasmatales-related seventh order of methanogenic archaea that encodes pyrrolysine[END_REF]Sollinger et al. 2016), encompassing an unusual methanogenesis and energy conservation process coupled to it [START_REF] Borrel | Comparative genomics highlights the unique biology of Methanomassiliicoccales, a Thermoplasmatales-related seventh order of methanogenic archaea that encodes pyrrolysine[END_REF][START_REF] Kroninger | Evidence for the involvement of two heterodisulfide reductases in the energyconserving system of Methanomassiliicoccus luminyensis[END_REF]Kröninger et al. 2019). Most of hydrogenotrophic methanogens use CO 2 as electron acceptor leading to the terminal product CH 4 by the pathway named hydrogenotrophic methanogenesis [START_REF] Ferry | The chemical biology of methanogenesis[END_REF][START_REF] Thauer | Methanogenic archaea: ecologically relevant differences in energy conservation[END_REF]. There are a few exceptions regarding hydrogenotrophs unable to reduce CO 2 into methane, the main being Methanosphaera spp. (belonging to the order Methanobacteriales as does M. smithii), which can oxidize hydrogen using methanol as terminal electron acceptor. All members of Methanomassiliicoccales however are exceptions, not having the hydrogenotrophic methanogenesis pathway, therefore being unable to perform methanogenesis from CO 2 : they all use C1 compounds as electron acceptors, with some distinctions among them. So far, all of them can use methanol, while some of them can also use monomethylamine (MMA) [START_REF] Lang | New mode of energy metabolism in the seventh order of methanogens as revealed by comparative genome analysis of "Candidatus Methanoplasma termitum[END_REF], and among them, some can also use TMA (and dimethylamine DMA). In fact, such a metabolism on methylamines relies on a rare and original genetic trait (see chapter 5.1). Some other methanogens perform other kind of methanogenesis, either using C1 compounds as electron donors instead of H 2 (methylotrophs, performing a methylotrophic methanogenesis), or acetate (aceticlastic methanogens) [START_REF] Costa | Metabolic versatility in methanogens[END_REF][START_REF] Garcia | Taxonomic, phylogenetic, and ecological diversity of methanogenic Archaea[END_REF][START_REF] Lyu | Methanogenesis[END_REF].

Another unique feature among Methanomassiliicoccales is their phylogenetic situation split into two main clades reflecting roughly an ecological distribution. One clade, to which Ca. M. alvus belongs, is exclusively formed of several candidate species and genera that are to be found in digestive tracts of animals, encompassing insects, poultry, pigs, ruminants, and humans (Borrel et al. 2013b;[START_REF] Mihajlovski | A putative new order of methanogenic Archaea inhabiting the human gut, as revealed by molecular analyses of the mcrA gene[END_REF][START_REF] Mihajlovski | Molecular evaluation of the human gut methanogenic archaeal microbiota reveals an age-associated increase of the diversity[END_REF][START_REF] Paul | Methanoplasmatales," Thermoplasmatales-related archaea in termite guts and other environments, are the seventh order of methanogens[END_REF][START_REF] Soellinger | Phylogenetic and genomic analysis of Methanomassiliicoccales in wetlands and animal intestinal tracts reveals clade-specific habitat preferences[END_REF]. It forms the host-associated clade or HAc [START_REF] Borrel | Genomics and metagenomics of trimethylamineutilizing Archaea in the human gut microbiome[END_REF][START_REF] Soellinger | Phylogenetic and genomic analysis of Methanomassiliicoccales in wetlands and animal intestinal tracts reveals clade-specific habitat preferences[END_REF]. The other clade is formed of members found in environments and not in or on an host, with however at least one interesting exception (see below, Ca. Methanomassiliicoccus intestinalis Mx01): all other are freely found in various anaerobic environments like seafloors, freshwater sediments, wetlands, and peatlands for example [START_REF] Borrel | Genomics and metagenomics of trimethylamineutilizing Archaea in the human gut microbiome[END_REF]Borrel et al. 2013b;[START_REF] Soellinger | Phylogenetic and genomic analysis of Methanomassiliicoccales in wetlands and animal intestinal tracts reveals clade-specific habitat preferences[END_REF]. It forms the free-living clade FLc to which Methanomassiliicoccus luminyensis B10 belongs. The latter was, however, isolated from human stools [START_REF] Dridi | Methanomassiliicoccus luminyensis gen. nov., sp. nov., a methanogenic archaeon isolated from human faeces[END_REF], so that it remains unclear if the natural habitat of this species is really (human or other animal) digestive environments, or is another one, and so if this species was only transiently present (or exceptionally living) in this human subject. This species has never been clearly demonstrated again to live within the human gut: it has sometimes been found in other studies using PCR/qPCR tools in humans, which is likely the result of a cross-signal with another Methanomassiliicoccales from this clade, Candidatus Methanomassiliicoccus intestinalis, also named Mx-01 (Borrel et al. 2013a): different strains of Mx-01, phylogenetically closely related to M. luminyensis have been identified in various studies and several subjects (e.g., [START_REF] Borrel | Genomics and metagenomics of trimethylamineutilizing Archaea in the human gut microbiome[END_REF][START_REF] Mihajlovski | Molecular evaluation of the human gut methanogenic archaeal microbiota reveals an age-associated increase of the diversity[END_REF])), forming one notable exception among FLc members. Therefore, it is assumed that M. luminyensis is not a typical representative of the gut-associated Methanomassiliicoccales, which could affect its efficacy to thrive in the human gut.

Using these natural properties to design probiotics from the archaeal domain: archaebiotics

If we sum up what is mentioned above, TMA is a deleterious molecule synthesized by gut microbes originally believed to be an end-product of the diet-derived metabolism of the gut microbiota. It seems not to be the case in subjects who house some peculiar archaea belonging to the order Methanomassiliicoccales, which have the property to convert TMA into methane (see below for arguments). Methane can be considered as an inert gas for the host, especially at the amount that would be derived from TMA. These archaea are well adapted to this environment, at least for members from the host-associated clade. Moreover, they are very likely safe: this can be first presumed from our knowledge about the archaeal world (no pathogens described so far in humans, animals or plants). Also, M. luminyensis, which very likely does not usually thrive in humans, does not lead in vitro to activation of human monocyte-derived dendritic cells [START_REF] Bang | Immunogenic properties of the human gut-associated archaeon Methanomassiliicoccus luminyensis and its susceptibility to antimicrobial peptides[END_REF]), a difference with another human-hosted archaeon, Methanosphaera stadtmanae [START_REF] Bang | The intestinal ar chaea Methan ospha era s tadtmana e and Methanobrevibacter smithii activate human dendritic cells[END_REF][START_REF] Vierbuchen | The human-associated archaeon Methanosphaera stadtmanae is recognized through its RNA and induces TLR8-dependent NLRP3 inflammasome activation[END_REF]). Based on this, it is logical to propose the use of such archaea as live biotherapeutic agents [START_REF] Brugere | Archaebiotics: proposed therapeutic use of archaea to prevent trimethylaminuria and cardiovascular disease[END_REF], referred as "Archaebiotics," or probiotics from the domain Archaea. Several data described below support the functionality of such an approach.

In vivo in the human gut, TMA is very likely used by Methanomassiliicoccales which have the genetic potential to use it. In fact, it may be suggested that TMA use is restricted to these microorganisms, conferring them an ecological niche in the human digestive tract: from the Eldermet cohort, an Irish cohort of elderly people [START_REF] Claesson | Composition, variability, and temporal stability of the intestinal microbiota of the elderly[END_REF][START_REF] Claesson | Gut microbiota composition correlates with diet and health in the elderly[END_REF], it has been shown that the TMA level in stools was significantly higher in people in whom no Methanomassiliicoccales were detected, or who had Methanomassiliicoccales (genetically) not able to use trimethylamines, as determined by qPCR and metagenomics data of more than 100 subjects [START_REF] Borrel | Genomics and metagenomics of trimethylamineutilizing Archaea in the human gut microbiome[END_REF]) (Fig. 2a). Moreover, the mean level of intestinal TMA was lower when subjects were hosting more than 10 8 TMA-using Methanomassiliicoccales (based on a molecular metabolic marker) than less than 10 8 , corresponding also with a higher significance of this difference in intestinal TMA level with subjects not hosting Methanomassiliicoccales or not TMAusing Methanomassiliicoccales. Unfortunately, no data are available from this study to know if this lower level of TMA in stools is also associated with lower TMAO level in plasma.

The archaebiotics concept was recently tested on mice [START_REF] Ramezani | Gut colonization with methanogenic archaea lowers plasma trimethylamine N-oxide concentrations in apolipoprotein E -/mice[END_REF]. In preliminary experiments, laboratory mice were treated with antibiotics to favor persistence/ implantation of archaea, and fed with a choline-enriched diet. Tested archaea were various mesophilic methanogens provided by a unique gavage of about 10 8 cells after antibiotics treatments. With the exception of Methanobrevibacter smithii (a human-hosted archaeon), they were all known to use TMA for their methanogenesis. Also, they were all known to not thrive in the human (and mouse) gut, with possibly the exception of Methanomassiliicoccus luminyensis which belongs to the free-living clade (see above). This species was the unique axenic cultivated representative from Methanomassiliicoccales available for experimentation by authors. Surprisingly, gavage with the human archaeon M. smithii resulted in significant lower pTMAO levels all along the experiment (days 2, 10, and 30 after the unique gavage) compared to controls. It also improved the health status of ApoE -/-mice compared to controls, i.e., less aortic/atherosclerotic lesions. As it is an archaeon that does not Methanomassiliicoccales was observed, more pronounced for subjects hosting higher amounts of these archaea (derived from Fig. 3d in [START_REF] Borrel | Genomics and metagenomics of trimethylamineutilizing Archaea in the human gut microbiome[END_REF]. Only mean values and statistical significance are indicated. b Archaebiotics in mice: Raj et collaborators reported the beneficial effect of some archaea on plasma TMAO levels in mice [START_REF] Ramezani | Gut colonization with methanogenic archaea lowers plasma trimethylamine N-oxide concentrations in apolipoprotein E -/mice[END_REF]): after antibiotics treatment followed by a unique gavage with some different methanogens, pTMAO was assayed at different time points (days 2, 10, and 30 after inoculation, respectively indicated as D2, D10, and D30). Some archaea and time points (reported at left) gave significant differences compared to pTMAO in control mice (which are reported here).

From the results given in the original work (text + figures), an efficacy index for each species of methanogens is calculated to take into account the amount of archaea which were implemented: this index corresponds to the fold of reduction of pTMAO (pTMAO ratios between control and tests) divided by the amount of archaea per μg of stools. The Methanomassiliicoccales M. luminyensis, which has a significant effect at the 3 time points, is highlighted (gray box)

use TMA, the beneficial effects of M. smithii can be attributed to a modification/modulation of the gut microbiota metabolic activity, with an observed change in its bacterial composition compared to controls [START_REF] Ramezani | Gut colonization with methanogenic archaea lowers plasma trimethylamine N-oxide concentrations in apolipoprotein E -/mice[END_REF]. M. smithii was also shown to colonize the mouse gut, at a higher level compared to any other tested archaea. Other (TMA-using) archaea gave significantly lower pTMAO levels, but this effect was detectable only for the first days of experiments after the unique gavage: in fact, these archaea were not retrieved (or at very low levels) at day 30, indicating no colonization for most of them and a continuous disappearance all along the experiment. One exception was however Methanomassiliicoccus luminyensis, which was retrieved in stools at low level from the beginning to the end of the experiment. If we analyze more deeply data provided by Ramezani et coll. [START_REF] Ramezani | Gut colonization with methanogenic archaea lowers plasma trimethylamine N-oxide concentrations in apolipoprotein E -/mice[END_REF]) and using numerical approximations from the figures and text, we can compare the effectiveness of each archaeal species which lowered significantly pTMAO taking into account the count of archaea retrieved in mice stools at each time points (Fig. 2b). M. smithii has in fact a significant effect of pTMAO level associated to a high count of M. smithii in stools. This corresponds to a lower reduction of pTMAO per cell of M. smithii varying across time points: this argues for an indirect action of M. smithii on intestinal TMA/pTMAO, likely by acting on the gut microbial composition. Interestingly, the use of the Methanomassiliicoccales representative M. luminyensis gave a significant reduction of pTMAO all over the 30 days of experiments. It had however a low count in stools, but one of the highest and quite similar efficacy per cell at the three time points (see Box in Fig. 2b). This indicates that M. luminyensis has a strong effect that can be detected at even low colonization level, likely by acting directly on intestinal TMA through a conversion into methane. These results are in fact in line with observations made in humans of the Eldermet cohort [START_REF] Borrel | Genomics and metagenomics of trimethylamineutilizing Archaea in the human gut microbiome[END_REF]. Moreover, the low level of M. luminyensis observed in mice stools may be the result of a methanogen not well adapted to thrive in the gut, especially in mice: the isolation of Ca. Methanomethylophilus alvus (W. Ben Hania, K. Fadhlaoui et al., in prep.) gives now the opportunity to carry out similar experiments with a human-hosted Methanomassiliicoccales from the host-associated clade, which should indicate more precisely the interest (or not) to follow up a development and use of archaebiotics.

Future developments: genetics of archaea as a tool for next-generation probiotics

It is shown from above that only archaea, and among them, only some methanoarchaea are currently known to have properties to remediate TMA in the gut, with a few of them naturally occurring in human gut. It is however imaginable that some of these metabolic properties could be used to provide alternative solutions in human health. Among these solutions, the specificity of enzymes used in this methylotrophic metabolism could be used to sequester TMA in the human gut, in a similar but more specific way than charcoal does, this last being sometimes used for TMAU patients to lower TMA absorption. The first archaeal enzyme of this pathway, MttB, can be considered for such an application. This is the one that recognizes TMA to further transfer a methyl-group to several other methyltransferases (a corrinoid protein MttC, next another methyltransferase MttA which methylates coenzyme M; see Fig. 1).

Pyrrolysine, a necessary unusual amino acid in anaerobic TMA metabolism

MttB is a (dimeric) enzyme which has however a striking feature, from which depends its biological activity. For substrate specificity and functional activity, this protein has to possess pyrrolysine (Pyl) in its active site [START_REF] Hao | A new UAG-encoded residue in the structure of a methanogen methyltransferase[END_REF], hence losing its specificity [START_REF] Craciun | Microbial conversion of choline to trimethylamine requires a glycyl radical enzyme[END_REF]. Pyrrolysine is the 22nd proteinogenic amino acid discovered at the beginning of the twenty-first century [START_REF] Atkins | Biochemistry. The 22nd amino acid[END_REF][START_REF] Srinivasan | Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNA[END_REF]. It is found quite exclusively in some archaea in which it is intimately linked to methylamines metabolism, especially the methanogenesis from MMA, DMA, and TMA [START_REF] Brugère | Pyrrolysine in archaea: a 22nd amino acid encoded through a genetic code expansion[END_REF]. Its synthesis, encoding, phylogenetic origin, and biological properties raise many questions considering archaea and more specifically Methanomassiliicoccales [START_REF] Brugère | Pyrrolysine in archaea: a 22nd amino acid encoded through a genetic code expansion[END_REF]. This peculiar amino acid and the system which allows its encoding have led to new biotechnological applications, especially in synthetic biology and for quite any kind of cells (for a recent review, see [START_REF] Tharp | tRNA(Pyl): structure, function, and applications[END_REF]). In short, this unusual proteinogenic 22nd amino acid is synthesized from one usual amino acid, lysine, which is used twice through 3 reactions catalyzed by 3 different enzymes (PylB, PylC, and PylD) (Fig. 3) [START_REF] Krzycki | Function of genetically encoded pyrrolysine in corrinoid-dependent methylamine methyltransferases[END_REF][START_REF] Krzycki | The direct genetic encoding of pyrrolysine[END_REF]. This amino acid is next encoded by the usual way at ribosomes, using a specific tRNA whose anticodon is complementary to the UAG amber codon (usually a stop codon). This tRNA acts as an UAG stop codon suppressor: The mechanisms underlying the insertion in nascent proteins (rather than a stall in-between the mRNA and the ribosome) is still unclear but is likely the result of a competition between archaeal releasing factor aRF and Pyl-tRNA at the ribosome in archaea [START_REF] Zhang | Pyrrolysine and selenocysteine use dissimilar decoding strategies[END_REF]). As mentioned above, Pyl is a necessary component of an active MttB protein, so that a biotechnological production of MttB has to rely to organisms or artificial/bioengineered systems/ cells that can encode (and synthesize) Pyl.

Producing and using components from archaea for a pharmaceutical approach

We therefore propose to engineer microbial cells factories that would be exploited for the controlled production of MttB, next used to be delivered in the human gut to sequester TMA before its absorption like charcoal does unspecifically (Fig. 3). It has been shown for a long time that, for example, E. coli could be transformed by the Pyl genetic components [START_REF] Longstaff | A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine[END_REF][START_REF] Namy | Adding pyrrolysine to the Escherichia coli genetic code[END_REF]. For this purpose, MtmB, a Pylprotein which recognizes MMA was synthesized by E. coli as a reporter of the functionality of the Pyl-system in this nonnaturally Pyl-encoding bacterium. Recently also, Pyl-tRNA/ Pyl-tRNA synthetase pair from Ca. Methanomethylophilus alvus was shown to have even better yields for site-directed insertion of non-natural amino acids in proteins (Beránek et al. 2019;[START_REF] Willis | Mutually orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs[END_REF]. We successfully produced MtmB with the Pylsystem of Ca. M. alvus (unpublished data) and are now engineering various E. coli recombinant bacterial cells to produce and/or secrete MttB. This cell or medium/purified protein will be next used in vitro to determine its affinity towards TMA with the aim to sequester TMA in the human gut: properly delivered in the gut, it would link neo-synthesized TMA before its absorption by the gut epithelium and its diffusion to the liver, so either lowering pTMAO (which would prevent cardiovascular disease) or lowering TMA (which would lowered symptoms for TMAU subjects). Therefore, the genetics of these specific TMAmetabolizing archaea could be used to engineer MttB factoring cells, as producers of a protein drug. It is naturally able to synthesize and encode Pyl, the 22nd amino acid through an UAG (amber) suppressing tRNA, which allows the synthesis of a functional methyltransferase specific to TMA (MttB, see also Fig. 1). Therefore, this organism (or any other MttB-encoding archaeon) can be used as a live biotherapeutic archaeon (1) which will convert TMA into methane through its methanogenesis (Archaebiotics). Alternatively, this kind of archaeon can also be used to extract a functional MttB protein that can be further used as a pharmaceutical delivered in the human gut in order to catch TMA (2). Such a protein production and purification can be achieved through the engineering of bacterial cells (or yeasts) factories (2′) with the need of the correct synthesizing and encoding of Pyl. The production of Pyl-containing methyltransferases has already been achieved in E. coli. At last, and alternatively, such an engineered bacterial cell can originate from a known probiotics. This next-generation probiotics can next be used as a live recombinant biotherapeutic product able to synthesize and deliver MttB in situ in the gut to sequester TMA (3), accordingly to what has been already developped for E. coli Nissle 1917 [START_REF] Isabella | Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria[END_REF] Upgrading bacterial probiotics with archaeal components: live bioengineered biotherapeutic agents

While the in vitro synthesis of MttB as a drug is a feasible option, leading in fact to quite a standard pharmaceutical approach, the functionality of such a system in human remains to be tested: especially, the delivery of MttB, whose integrity has to be kept long enough to fix TMA, is a determining step. Another option can be the in situ synthesis of TMA in the human gut, by providing factory cells rather than the protein itself. Live recombinant biotherapeutic agents have been recently shown to be a functional option using a standard probiotic cell that was bioengineered to synthesize and deliver active enzymes in the gut [START_REF] Kurtz | Translational development of microbiome-based therapeutics: kinetics of E. coli Nissle and engineered strains in humans and nonhuman primates[END_REF]. As an example, the probiotic strain E. coli Nissle 1917 was recently engineered to express specific proteins in gut of mice and monkeys in response to anoxic environments: it allows the expression of enzymes able to convert phenylalanine (Phe) into trans-cinnamate (further converted into hypurate by the host), therefore depleting (diet-derived) Phe [START_REF] Isabella | Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria[END_REF]. This is of particular interest in phenylketonuria, a genetic disease in which unmetabolized Phe can lead to neurotoxicity. This case has many similarity to the one described for intestinal TMA and cardiovascular disease or TMAU. It is now known that E. coli survives to the expression of an UAG non-sense codon suppressing tRNA and that the archaeal Pyl-system is functional in this organism. Therefore, it can be proposed first to engineer the probiotic strain E. coli Nissle 1917 to synthesize and encode Pyl, as well as to synthesize (a functionnal) MttB. Other probiotic strains can also to be considered (Fig. 3): the live bioengineered strain, e.g., a recombinant E. coli Nissle 1917 would be given as live biotherapeutic product, which would synthesize an active TMA sequestering protein when reaching the gut.

Concluding remarks

So far, microorganisms belonging to the domain Archaea remain neglected candidates as potential probiotics: no archaeon has been used as a live biotherapeutic product in humans or in any other animal so far, while archaea have specific features that are of great interest for the prevention of important human diseases including cardiovascular diseases and trimethylaminuria. We already know that these properties apparently contribute to limit deleterious effects consecutive to TMA synthesis by bacteria in the gut [START_REF] Borrel | Genomics and metagenomics of trimethylamineutilizing Archaea in the human gut microbiome[END_REF]. If not used directly for their properties as live biotherapeutic product (i.e., Archaebiotics), archaea provide unique features that could be engineered in other microbes to provide either pharmaceuticals or live biotherapeutic products. Moreover, it has to be highlighted that so far, no archaeon has been reported

as being pathogenic for any plant or animal, and interestingly for humans. This gives value to the use of them for therapeutic purposes, especially as some are natural inhabitants of most host-associated microbiomes. Archaea-based research for alternative solutions in human, animal, or plant health is just reaching its infancy, providing promising new strategies for prophylactics and therapeutics of some important diseases.

Fig. 1

 1 Fig. 1 Origin and fate of intestinal TMA. Intestinal TMA originates from various nutrients from diet (1) which are further metabolized in the gut by bacteria from the microbiota (2). Nutrients are indicated in bold, bacterial genes encoding necessary enzymatic activities in red and italics. TMA is next absorbed and either oxidized into pTMAO in the liver or not, contributing respectively to cardiovascular disease or trimethylaminuria (3).

Fig. 2

 2 Fig.2Influence of Methanomassiliicoccales on fecal TMA and plasma TMAO. a In the subjects from the ELDERMET cohort, a significantly lower fecal TMA level for subjects hosting MttB-encoding Methanomassiliicoccales was observed, more pronounced for subjects hosting higher amounts of these archaea (derived from Fig.3din[START_REF] Borrel | Genomics and metagenomics of trimethylamineutilizing Archaea in the human gut microbiome[END_REF]. Only mean values and statistical significance are indicated. b Archaebiotics in mice: Raj et collaborators reported the beneficial effect of some archaea on plasma TMAO levels in mice[START_REF] Ramezani | Gut colonization with methanogenic archaea lowers plasma trimethylamine N-oxide concentrations in apolipoprotein E -/mice[END_REF]): after antibiotics treatment followed by a unique gavage with some different methanogens, pTMAO was assayed at different time points (days 2,

Fig. 3

 3 Fig. 3 Biotechnological developments using archaea and genetics of archaea in order to lower intestinal TMA and prevent TMAU and CVD. The Methanomassiliicoccales archaeon Candidatus Methanomethylophilus alvus is represented in the up center of the figure.It is naturally able to synthesize and encode Pyl, the 22nd amino acid through an UAG (amber) suppressing tRNA, which allows the synthesis of a functional methyltransferase specific to TMA (MttB, see also Fig.1). Therefore, this organism (or any other MttB-encoding archaeon) can be used as a live biotherapeutic archaeon (1) which will convert TMA into methane through its methanogenesis (Archaebiotics). Alternatively, this kind of archaeon can also be used to extract a

Acknowledgements

JFB acknowledges Dominic S. Raj from The George Washington University School of Medicine, Washington, USA for communications about mice experiments with archaea (Fig. 2b).

Authors' contributions JFB and PWOT are inventors on patent WO2014082773A1 co-owned by their respective universities. PWOT is a founder of 4D Pharma Cork Ltd., a role that has not constrained or influenced this review. All the authors contributed to the written of the manuscript and the conception and design of figures. All the authors read and approved the manuscript.

Funding information JFB is supported by a grant from the Agence Nationale de la Recherche of the French government through the program "Investissements d'Avenir" (16-IDEX-0001 CAP 20-25), through an "Innovation-Research" grant from Hub Innovergne. Work in PWOT's lab is supported by an award from Science Foundation Ireland to APC Microbiome Ireland.

Compliance with ethical standards

Conflict of interests The authors declare that they have no conflict of interest.

Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


F Renaud

O Gauthier-Lafaye

A Monmayrant

E Popov

A.-L Fehrembach

S Calvez

S Gregory Jones

Titania A R Schmidt

Kenneth M Suzuki

Frequency doubling in Cavity-Resonator Integrated Grating Filter

Keywords: second harmonic generation, guided-mode resonance filters, lithium niobate, cavity-resonant integrated grating filter, nonlinear frequency conversion

Guided Mode Resonant Filters (GMRFs) have long been studied as a support surface for nonlinear optical interactions due to their intrinsically high Q-factor. However, their operation relies on a non-localized and large-area guided mode that limits the achievable power density and requires complex phase-matching approaches. Conversely, photonic crystal nano-cavities have shown promising results due to both their high-Q factors to enhance the pump field and their localized nature that allows phase-matching-free implementation and high power density excitation. However, their intrinsic small size restricts the supported input power and hinders the coupling efficiency of the pump into the mode.

In this paper, we report the first experimental demonstration of continuous-wave second harmonic generation in a Cavity-Resonator Integrated Grating Filter (CRIGF). This intermediary device, which can be described as a cavityenhanced finite-size GMRF or, equivalently, as a low-index-contrast photonic crystal micro-cavity, will be shown to offer a practical route to nonlinear interactions with viable power (<20 mW) and excitation conditions (surface excitation with a ~10-µm-waist spot size). In practice, the devices under study make use of a lithium-niobate on insulator (LNOI) waveguide with a nanostructured silicon nitride upper cladding as a pragmatic way to implement a high second-order nonlinearity platform with established processing technology. The already-demonstrated versatility of the CRIGF design (demonstrated at wavelengths of 850 nm using S 3 iN 4 /SiO 2 platform, 4.5 µm with the GaAs/AlGaAs technology and, here, at 1.55 µm with the LiNbO 3 platform) coupled to the electro-optical tuning afforded by lithium niobate system makes this approach extremely promising for pixelated non-linear systems.

INTRODUCTION

Guided Mode Resonant Filters (GMRFs) have been studied as devices for nonlinear optical interactions due to their intrinsically high Q-factor [START_REF] Blau | Grating-assisted phase-matched second-harmonic generation from a polymer waveguide[END_REF][START_REF] Siltanen | Strong enhancement of second-harmonic generation in all-dielectric resonant waveguide grating[END_REF] . Given that their principle is based on non-localized and large-area guided mode(s), to ensure an efficient nonlinear conversion, the excitation beam needs to have a low-divergence and be wide and thereby the operation is inherently limited by the achievable power density. Furthermore, to achieve the constructive harmonic generation over such large surfaces, the phase-matching condition has to be strictly met. Conversely, photonic crystal nano-cavities have shown promising results due to both their high-Q factors to enhance the pump field and their localized nature that allows phase-matching-free implementation and high power density excitation [START_REF] Mccutcheon | Experimental demonstration of second-order processes in photonic crystal microcavities at submilliwatt excitation powers[END_REF][START_REF] Rivoire | Second harmonic generation in gallium phosphide photonic crystal nanocavities with ultralow continuous wave pump power[END_REF] . However, their intrinsic small size restricts the supported input power and hinders the coupling efficiency of the pump into the mode.

In this paper, we report the first experimental demonstration of continuous-wave second harmonic generation (SHG) in a low-index-contrast photonic crystal micro-cavity or equivalently in cavity-enhanced finite-size GMRF that is also known as a Cavity-Resonant Integrated Grating Filter (CRIGF) [START_REF] Kintaka | Cavity-resonatorintegrated guided-mode resonance filter for aperture miniaturization[END_REF][START_REF] Buet | High angular tolerance and reflectivity with narrow bandwidth cavity-resonator-integrated guided-mode resonance filter[END_REF] . Practically, the device was made using a hybrid lithium niobate and silicon-oxynitride technology.

DESIGN

As shown on Figure 1, the considered CRIGF structure relies on a lithium-niobate-on-insulator (LNOI) substrate since this technological platform combines a high non-linear susceptibility (of 33pm/V) with an ability to make highconfinement but low-loss waveguides [START_REF] Poberaj | Lithium niobate on insulator (LNOI) for micro-photonic devices[END_REF][START_REF] Boes | Status and Potential of Lithium Niobate on Insulator (LNOI) for Photonic Integrated Circuits[END_REF][START_REF] Wu | Long Low-Loss-Litium Niobate on Insulator Waveguides with Sub-Nanometer Surface Roughness[END_REF] . Here, the sample consists of a 297-nm-thick X-cut lithium-niobate core on a 2µm-thick SiO2-coated lithium-niobate substrate of similar orientation.

We chose to use two additional layers (made of Si 3 N 4 and SiO 2 ) to form the upper-cladding of the waveguide such that we could create an anti-reflective multilayer stack at the fundamental wavelength of design (1555 nm) under surfacenormal excitation and choose the bandwidth of the Distributed Bragg Reflectors that sets the micro-cavity. From these thicknesses, we defined the coupling grating and DBR constitutive parameters based on standard effective index calculation of the TE-polarized mode and set the phase section length to obtain a resonance at the design wavelength. The resulting parameters are listed in Table 1 and Figure 1. Further details on the design procedure can be found in [START_REF] Calvez | Thermally-tunable cavity resonatorintegrated guided-mode resonance filters[END_REF] .

Parameter Value

Grating coupler period (nm) 865

Grating coupler number of periods 10.5

Grating coupler fill factor 0.45 DBR period (nm) 432

DBR number of periods 400 DBR fill factor 0.5

Phase section length (nm)

Using RCWA calculations and assuming a mode-matched excitation and a loss-less waveguide, the CRIGF is shown to have a ~1 nm bandwidth with a maximum reflectivity of 85.3% and minimum transmission of 14.7% (see Figure 2). The generated second harmonic signal was also calculated under the undepleted pump approximation and is shown on Figure 2 to be exalted at the device resonance. Further information on the calculations are available in reference [START_REF] Renaud | Second-harmonic-generation enhancement in cavity resonator integrated grating filters[END_REF] together with a comparison to the expected results from substrate-only and GMRF-based generation. To complement this information, we also computed the substrate-radiating SHG pattern presented in Figure 3 by selecting the modal content whose wavevectors are above the light cone. The SHG emission diagram exhibits three peaks that can be assigned to the extraction of the SHG signal by the grating-coupler section (through the 0 th and ±1 st orders).

FABRICATION

The device fabrication started from the LNOI planar waveguide described above obtained from NanoLN. The first layer of the waveguide upper-cladding layer, made of Si 3 N 4 , was deposited using an Inductive-Coupled-Plasma Plasma-Enhanced Chemical Vapor Deposition system. The designed gratings were subsequently patterned into this layer using nano-imprint lithography [START_REF] Pelloquin | Soft mold NanoImprint Lithography: a versatile tool for subwavelength grating applications[END_REF] and dry etching with the LiNbO 3 top surface acting as an etch-stop interface. The device fabrication was completed by the deposition of a 323-nm-thick SiO 2 upper cladding layer on top of the grating structure and of a 266-nm-thick SiO 2 single-layer anti-reflective coating on the rear-side of the substrate to avoid back-reflection.

CHARACTERISATION

The sample was characterized using the setup shown in Figure 4. The tunable source delivered up to 17 mW of fundamental signal at a wavelength tunable from 1500 to 1630 nm with a linewidth of ~100 kHz and was optically relayed using a telescope system to ensure mode-matched excitation of the CRIGF. The generated second harmonic signal was collected using a NA=0.15 lens, separated from the pump using a dichroic filter and analyzed using a visible spectrum analyzer. In the absence of an InGaAs camera, the 980nm beam served as a guide to align the 1550 nm beam and was turned off during the measurements. As shown in Figure 5, the second harmonic signal is observed to increase significantly at a wavelength of 1555 nm corresponding to the device resonance. We were also able to establish that a fraction of the SHG signal is also diffracted at ~±60°, in agreement with the above-displayed calculated radiation pattern. Polarization analysis reveals that the second harmonic emission is TE-polarized as expected and accounted for in the numerical simulations. Finally, the measurement of the performance on three devices with scaled designs (corresponding grating coupler periods of 845, 865 and 885 nm) enabled to demonstrate the linear dependence of the SHG wavelength to the device design parameter (or equivalently resonant wavelength), consistent with cavity-enhanced SHG operation.

Figure 7. SHG tunability with CRIGF design parameter (here taken to be the grating coupler period).

CONCLUSION

We demonstrated successful continuous-wave SHG enhancement in CRIGF structures made using a hybrid lithiumniobate/silicon oxynitride technology. Future prospects will be to look for improved nonlinear conversion performance using a combination of optimised SHG-CRIGF designs and improved fabrication processes.

Figure 1 .

 1 Figure 1. Schematic of the CRIGF structure under study

Figure 2 .

 2 Figure 2. Calculated spectral reflectivity (black) and transmission (grey) of the designed CRIGF.

Figure 3 .

 3 Figure 3. Radiation pattern in air of the second harmonic signal for the considered CRIGF.

Figure 4 .

 4 Figure 4. Setup used for that characterisation of the considered CRIGF.

Figure 5 .

 5 Figure 5. CRIGF SHG spectral characteristic reccorded at normal incidence.

Figure 6 .

 6 Figure 6. Measurement of the SHG polarisation state (a=0° for analysis polarizer along the grating lines).

Table 1 .

 1 Parameters of the CRIGF nano-structured elements.

ACKNOWLEDGEMENTS

The authors would like to thank NanoLN for providing the LNOI sample onto which the device was fabricated and acknowledge that part of the work was funded by the ANR ASTRID "RESON" and also the technical support from the LAAS-CNRS micro and nanotechnologies platform, member of the French RENATECH of cleanroom facilities.