Paul Dufour
email: paul.dufour80@gmail.com

Christophe De Franceschi

Paul Doniol-Valcroze

Frédéric Jiguet

Maya Guéguen

Julien Renaud

Sébastien Lavergne

Pierre-André Crochet

Uncovering a new westward migration route in an Asian passerine bird

Keywords: Climate change, seasonal migration, geolocators, vagrancy, molt, Richard's Pipit

The evolution of migration routes in birds remains poorly understood as

Results and Discussion

Evidence for a new migration route

Like many other Siberian birds that normally winter in southern Asia, Richard's Pipits have long been known to reach Western Europe in small numbers in autumn. [START_REF] Rabøl | Reversed migration as the cause of westward vagrancy by four Phylloscopus warblers[END_REF][START_REF] Cramp | The Birds of the Western Palearctic Volume III[END_REF][START_REF] Alstrom | Pipits and Wagtails of Europe, Asia and North-America Helm[END_REF] The occurrence of such "accidental" or "lost" birds outside their normal distribution range, which are mostly juvenile birds that will never return to their breeding grounds, is often referred to as vagrancy. [START_REF] Alerstam | Ecological causes and consequences of bird orientation[END_REF] However, the pattern of Richard's Pipit occurrence has recently changed in southwestern Europe (Figure 1). In France for example, the number of records increased from three to six birds annually between 1981 and 1993 to a maximum of 144 birds in 2018 (Figure 2A) and this increase is apparent in several localities in southern France that have been consistently monitored since the 1950s or 1980s; the maximum flock size also increased during the same 1981-2019 period (Figure S1). The number of sightings of other Siberian vagrant species in France has increased to a much lesser extent (average regression coefficient over time, 3.47 in Richard's Pipit vs. 0.05 ± 0.057 for other vagrants; Figure 2B, Table S1), excluding that this increase simply reflect a simultaneous increase in identification skills and observation pressure. [START_REF] Gilroy | Vagrancy theories: Are autumn vagrants really reverse migrants?[END_REF][START_REF] Jiguet | Climate change and rates of vagrancy of Siberian bird species to Europe[END_REF] The first overwintering in Europe has been documented in 1991 in southern France, [START_REF] Dulau | Nouveau cas d'hivernage du Pipit de Richard Anthus richardi dans le sud de la France[END_REF] after which overwintering and staging was reported from an increasing number of sites (Figure S1). The species has now become a regular winter visitor to Southern and Western France, as well as to Spain, Portugal and Italy. [START_REF] Biondi | Record numbers of wintering Richard's Pipit in the Western Palearctic[END_REF] Two non-exclusive hypotheses can explain this recent increase: the number of vagrants reaching Europe has increased or Richard's Pipit has established a new migration route, in accordance with the "pseudo-vagrancy" hypothesis of Gilroy and Lees (2003). [START_REF] Gilroy | Vagrancy theories: Are autumn vagrants really reverse migrants?[END_REF][START_REF] Bearhop | Assortative Mating as a Mechanism for Rapid Evolution of a Migratory Divide[END_REF][START_REF] Rolshausen | Contemporary Evolution of Reproductive Isolation and Phenotypic Divergence in Sympatry along a Migratory Divide[END_REF] To disentangle these explanations, we studied the species during three wintering periods along the Mediterranean coast of France and Spain (Figure 1). Because the vast majority of vagrant passerines are birds in their first post-natal migration, we specifically looked for the presence of adults (birds more than one year old). Moreover, since most migratory passerines are faithful to their migratory routes and wintering areas, [START_REF] Stanley | Repeat Tracking of Individual Songbirds Reveals Consistent Migration Timing but Flexibility in Route[END_REF][START_REF] Van Wijk | Repeatability of individual migration routes, wintering sites, and timing in a long-distance migrant bird[END_REF] we marked individuals to test if they were returning the next winter. Both the presence of adults and of returning individuals would support the migration hypothesis over the vagrancy hypothesis. Among 81 birds examined in the hand in southern France and northeastern Spain, 28 (34.6 %) were found to be adults: a high proportion similar to that observed in regular wintering species in Southern Europe (e.g. European Robin Erithacus rubecula [START_REF] Catry | Winter segregation of migrant European robins Erithacus rubecula in relation to sex, age and size[END_REF] ) and supporting the migration hypothesis. In addition, from the 68 individuals that were color-ringed during the first two seasons, 11 were re-sighted or re-trapped the following winters (four of them over two consecutive seasons; Table S2), providing further empirical support to the migration hypothesis.

To determine whether birds that winter in France originate from the known breeding area or from a previously undetected breeding population, we equipped seven individuals with Global-Location-Sensors (GLS) during winter 2019/2020 (Table S2). The following winter, we re-captured three of these individuals (recapture is needed for data retrieval with GLS). All three birds spent the summer at the western edge of the known breeding range in a small area of the Novosibirsk Oblast in Russia (Figure 3) where light data indicates that at least one individual engaged in breeding behavior (Figure 3F), supporting the hypothesis that these birds were regular migrants rather than vagrants. During the spring migration, the three individuals used three different stopover locations for more than 24 hours; the second-year individual first headed north and stopped in Sweden before redirecting eastward to reach a likely breeding site where it lingered until the GLS stopped (Figure 3E). These three individuals thus traveled 5750, 7045, and 7320 km during their spring migration between Southern France and Siberia along a migratory route that crosses countries where the species is rarely sighted.

To determine whether regular migrants are also present in other parts of the western non-breeding range (Figure 1), we tested for the presence of adult birds in these locations using pictures from citizen-science databases to estimate the age-ratio. On good quality pictures, firstgeneration wing coverts (grown in the nest) can easily be distinguished from second-generation feathers (see Figure S2), allowing safe ageing of birds with first-generation coverts as young bird (i.e. less than one-year-old). However, some young birds molt all wing coverts during the autumn, resulting in an adult-type plumage in winter that cannot be separated from adults without in-hand examination. To estimate the proportions of the different molt stages among young birds, we examined in-hand and aged 78 birds caught in France and Northeastern Spain, to classify them into three molt phenotypes (full-juvenile, extended-molt, and adult-type, see Figure S2), all of which can be determined based on pictures. Of 50 young birds examined, six were of the full-juvenile type (12%), 39 belonged to the extended-molt type (78%), and five to the adult-type (10%; see Figure S2 for details). Within 33 adult-type birds (which of course include all adults), five (15 %) were found to be young birds upon in-hand examination. Molt phenotype thus provides an imprecise but meaningful information on age. We then scored pictures of 331 individuals across Europe and North Africa to estimate age-ratio variation. In Northern Europe, adult-type birds are rare (10% in autumn, 15.2% in winter) and their number is not significantly different from the number of adult-type birds expected if all birds were young birds (7 adult-type birds out of 46 scored individuals versus 5 adult-type birds among 50 in-hand aged juveniles; Fisher's exact test p-value = 0.55). In southwestern Europe and North Africa, however, adult-type birds are more common (48.5% in autumn, 59% in winter, Figure S3), significantly more common than expected if all birds were young (winter period: 57 adulttype out of 97; Chi-square test = 54.1, p-value < 0.001). The proportions of adult-type molt phenotypes in citizen-science pictures thus demonstrate the regular occurrence of adult birds, and hence likely regular migrants and wintering individuals, in the Iberian Peninsula, North Africa (Morocco), western France, and Italy.

Can vagrancy promote the emergence of new migratory routes?

The change in status of Richard's Pipit from vagrant to regular migrant in southwestern Europe opens up the question of the role of vagrancy in the establishment of new migration routes.

Large angle misorientation, also known as reverse migration [START_REF] Rabøl | Reversed migration as the cause of westward vagrancy by four Phylloscopus warblers[END_REF] or mirror-image migration, [START_REF] Desante | Annual Variability in the Abundance of Migrant Landbirds on Southeast Farallon Island, California[END_REF] has been proposed to explain the occurrence of Siberian vagrants in Europe, particularly for populations breeding in the western part of their distribution [START_REF] Jiguet | Climate change and rates of vagrancy of Siberian bird species to Europe[END_REF][START_REF] Thorup | Reverse migration as a cause of vagrancy[END_REF][START_REF] Thorup | Vagrancy of Yellow-browed Warbler Phylloscopus inornatus and Pallas's Warbler Ph. proregulus in north-west Europe: Misorientation on great circles?[END_REF] (but see [START_REF] Lees | First record of Blyth's Pipit Anthus godlewskii for Micronesia[END_REF] ). Such errors in migratory orientation have usually dramatic consequences on fitness, [START_REF] Delmore | Hybrid songbirds employ intermediate routes in a migratory divide[END_REF] especially in passerines where the energy cost of long migratory flights is very high, [START_REF] Alerstam | Long-distance migration: evolution and determinants[END_REF] and vagrants are therefore usually thought to perish. [START_REF] Alerstam | Ecological causes and consequences of bird orientation[END_REF][START_REF] Ralph | Disorientation and Possible Fate of Young Passerine Coastal Migrants[END_REF] The ability of vagrants to establish new migration routes may thus depend crucially on the wintering conditions they encounter in the areas where they end up (i.e., overwinter survival) and on their ability to perform a successful return journey and transmit their migration route to their offspring (via genetic inheritance as migration direction is genetically determined in songbirds, e.g. [START_REF] Helbig | Inheritance of migratory direction in a bird species: a cross-breeding experiment with SE-and SW-migrating blackcaps (Sylvia atricapilla)[END_REF][START_REF] Lundberg | Genetic differences between willow warbler migratory phenotypes are few and cluster in large haplotype blocks[END_REF][START_REF] Delmore | The Genetics of Seasonal Migration and Plumage Color[END_REF] ).

In Richard's Pipit, the main wintering grounds are in Southern and Southeast Asia (Figure 1). [START_REF] Alstrom | Pipits and Wagtails of Europe, Asia and North-America Helm[END_REF] Thus, the most parsimonious hypothesis is that the species' ancestral wintering grounds are in Asia and that the European wintering range represents a derived state that was recently established, or at least recently increased. To investigate the factors that may have favored the observed change in migration strategy, we examined the wintering niche conditions in the ancestral and derived ranges. When citizen-science occurrences data were plotted in a two-dimensional space defined by PCA axes on climatic and habitat variables (Figure 4A), [START_REF] Alstrom | Pipits and Wagtails of Europe, Asia and North-America Helm[END_REF][START_REF] Thorup | Resource tracking within and across continents in long-distance bird migrants[END_REF] we found that the niche of the European wintering range is broadly similar but not identical to the niche used in Asia: niches overlapped with a Schoener's D value of 0.31 (D = 0: no overlap, D = 1: complete overlap) and 68.8% of the European niche was included within the Asian niche.

We also performed two niche distribution models, one calibrated in Europe and one calibrated in Asia, then projected them under current conditions (Figure 4B,S4). The projection of the Asian calibrated model under current conditions over Europe mainly predict wintering in western Iberian Peninsula and North Africa (Figure 4B), confirming that the species encounter slightly different (but still overlapping) climatic conditions in southern France and northeastern Spain compared to the ancestral wintering areas in Asia. To test for recent changes in European wintering conditions, we projected a European calibrated model over two periods in 1961-1990 and 1990-2018, and found an extension in suitable wintering areas (mostly due to increasing temperature; see Figure S5) between these periods, especially in northern Spain, southern and western France and Italy (Figure 4C). Vagrant Richard's Pipits migrating west from Asia may thus have found suitable environmental conditions to survive the winter in large areas of North Africa and Iberian Peninsula (see [START_REF] Cramp | The Birds of the Western Palearctic Volume III[END_REF] ), after which environmental changes (here climate change) might have increase wintering opportunities for Richard's Pipit in southern Europe.

If these results suggest that the emergence of new migratory routes can ensue from the survival and successful return migration of vagrants to Europe, to what extent natural selection may explain the emergence and spread of this new wintering range remains to be investigated.

In particular, although proximate mechanisms of vagrancy remain largely unknown to date, it should be verified that vagrancy is to some degree hardwired (otherwise this new migratory orientation could not be transmitted to young) and that some forms of reproductive isolation exist between individuals migrating west and east (otherwise panmixia would prevent the stability of migration to Europe). [START_REF] Bearhop | Assortative Mating as a Mechanism for Rapid Evolution of a Migratory Divide[END_REF][START_REF] Rolshausen | Contemporary Evolution of Reproductive Isolation and Phenotypic Divergence in Sympatry along a Migratory Divide[END_REF] We suppose that a migratory divide could exist in this species with individuals at the western margin of the species' range, which should constitute a distinct population, all having a westerly migratory route, [START_REF] Irwin | Birds of Two Worlds: The Ecology and Evolution of Migration[END_REF][START_REF] Møller | Migratory divides and their consequences for dispersal, population size and parasitehost interactions[END_REF] but this would need to be tested by deploying data loggers in breeding populations.

Ancient literature suggests that this species may have been observed with some regularity in few European localities during the 19 th century. [START_REF] Campayo | Histoire naturelle du de ṕartement des Pyre ńe és-Orientales[END_REF][START_REF] Gätke | Heligoland as an Ornithological Observatory. The result of fiftty years' experience[END_REF] Although we showed that Richard's Pipit numbers have undoubtedly increased in recent decades, particularly in southwestern Europe and probably due to changing temperatures, we cannot rule out the hypothesis that a westward migratory route had existed for a long time and that we observed an increased use of this route in recent decades.

Concluding remarks

In this study, we show that the occurrence of Richard's Pipit in southwestern Europe is, at least in part, due to a population performing a regular seasonal migration, and cannot solely be explained by disoriented vagrant individuals. While the role of vagrancy in colonization of new breeding range has been documented in several species, [START_REF] Holt | Definite Breeding of the Glossy Ibis in Louisiana[END_REF][START_REF] Veit | Dispersal, Population Growth, and the Allee Effect: Dynamics of the House Finch Invasion of Eastern North America[END_REF][START_REF] Massa | On how Cattle Egret (Bubulcus ibis) spread to the Americas: meteorological tools to assess probable colonization trajectories[END_REF][START_REF] Dufour | Multilocus barcoding confirms the occurrence of Elegant Terns in Western Europe[END_REF] our results support the idea that vagrancy may also promote the establishment of new migratory routes. [START_REF] Gilroy | Vagrancy theories: Are autumn vagrants really reverse migrants?[END_REF] This idea was also proposed to explain one of the few known contemporary changes [START_REF] Winkler | Long-Distance Range Expansion and Rapid Adjustment of Migration in a Newly Established Population of Barn Swallows Breeding in Argentina[END_REF][START_REF] Areta | Rapid adjustments of migration and life history in hemisphere-switching cliff swallows[END_REF] in migration route in passerines: the change from southwestern to northwestern orientation in central European Blackcap Sylvia atricapilla, [START_REF] Berthold | Rapid microevolution of migratory behaviour in a wild bird species[END_REF][START_REF] Bearhop | Assortative Mating as a Mechanism for Rapid Evolution of a Migratory Divide[END_REF][START_REF] Rolshausen | Contemporary Evolution of Reproductive Isolation and Phenotypic Divergence in Sympatry along a Migratory Divide[END_REF] selected as a response to changing environment that increased overwinter survival. [START_REF] Plummer | Is supplementary feeding in gardens a driver of evolutionary change in a migratory bird species?[END_REF][START_REF] Van Doren | Human activity shapes the wintering ecology of a migratory bird[END_REF] Increased overwinter survival, helped by climate change, may also explain recent increases in autumn and winter numbers of other Siberian species in Europe (e.g. Yellow-browed Warbler Phylloscopus inornatus, Siberian Chiffchaff Phylloscopus collybita tristis, Olive-backed Pipit Anthus hodgsoni) that may also be currently establishing new secondary migration routes to Europe. The lack of records of these species during the spring migration was perceived as undermining the "pseudo-vagrancy" theory [START_REF] Gilroy | Vagrancy theories: Are autumn vagrants really reverse migrants?[END_REF] but we have shown here that such spring return migration can be particularly under-detected. The Richard's Pipit might thus be the most prominent example of a phenomenon that could change current paradigms on the flexibility and constraints of avian migration. Indeed, there is no passerine species confirmed to undertake such longitudinal migration to European wintering grounds and most similar routes concern species with East African wintering grounds that probably spread eastwards from an ancestral breeding range in Europe to breed in the Eastern Palearctic but return to winter in same locations. [START_REF] Bairlein | Cross-hemisphere migration of a 25 g songbird[END_REF][START_REF] Sokolovskis | Ten grams and 13,000km on the wing -route choice in willow warblers Phylloscopus trochilus yakutensis migrating from Far East Russia to East Africa[END_REF] Such examples of apparent failure to discover new, less energetically demanding, wintering areas explain why migration orientation is usually perceived as highly constrained in passerines. [START_REF] Delmore | Hybrid songbirds employ intermediate routes in a migratory divide[END_REF][START_REF] Helbig | Inheritance of migratory direction in a bird species: a cross-breeding experiment with SE-and SW-migrating blackcaps (Sylvia atricapilla)[END_REF] Our results suggest that we need to temper this view and that vagrancy due to misorientation during migration is more than a mere natural curiosity without much scientific interest.

Figure titles and legends

Figure 1. Distribution ranges and migratory movements of Richard's Pipit

Breeding, migrating and ancestral non-breeding ranges are taken from [START_REF]IUCN Red List for birds[END_REF] . Suspected new nonbreeding areas are defined from occurrences collected from the Global Biodiversity Information Facility (GBIF) for countries that regularly host the species in winter. [START_REF] Alstrom | Pipits and Wagtails of Europe, Asia and North-America Helm[END_REF][START_REF] Biondi | Record numbers of wintering Richard's Pipit in the Western Palearctic[END_REF][START_REF] Biondi | Nuovi dati sullo svernamento e la fenologia del Calandro maggiore Anthus richardi in Italia (1995-2004)[END_REF] Locations of the monitored sites are indicated by blue dots in the zoomed panel, with the size representing the mean annual numbers of birds caught per season. The yellow arrow illustrates the suspected new migratory route that birds should undertake to reach Southern Europe. Note that there is no clear evidence that central Asian populations migrate through or west of the Himalayas to reach ancestral non-breeding areas and could instead head a south/south-west direction thereby avoiding the Gobi Desert, the Tibetan Plateau, and the Himalayas. [START_REF] Alstrom | Pipits and Wagtails of Europe, Asia and North-America Helm[END_REF][START_REF] Delany | Passerine Migration across the Himalayas[END_REF] Illustration © Per Alström. Related to Table S2. S1. To test whether the species increasing trend is entirely driven by an increase in observer effort, we compared linear regression coefficients of number of individuals over time for Richard's Pipit and other vagrants of eastern origin. As these data have always been collected using the same methodology by the French Birds Rarities Committee, we assumed no reporting bias over time and we considered these data as an approximation of observer effort. We excluded the rarest species from the dataset (less than 5 records over the 1981-2019 period; the list of species and numbers of individuals can be found in Table S1). We repeated these analyses for the Mediterranean region of southern France (defined as departments bordering the Mediterranean Sea) as this region hosted the first and most of the wintering sites and includes several localities where observation pressure had remained strong over this period.

In addition, to test whether the trend can be explained by the establishment of a new migration route, we collected flock size data in France over the same period since an increase in group size can only result from actual increase in number rather than increase in probability of detection of the species. To avoid double-counting, we considered, per season and on a given year, the maximal flock size recorded in sites known to host the species during winter and each flock size record in other sites.

Finally, we reported the number of sites frequented by the species in winter between 1981 and 2019. By gridding the whole France territory in cells of 10 x 10 kilometers, we evaluated the number of new grid cells frequented each year between 1981 and 2019. We studied this trend for the winter period (December-February) and the migration period (September-November and March-May).

Ringing campaigns

We caught Richard's Pipits over the course of three wintering periods between 2018 and 2021 on several locations along the Mediterranean coast of France and Spain (see Figure 1 and details in Table S2). Since capture success of the capture sessions was strongly dependent on weather, the effort of capture was heterogeneous between sites and between wintering seasons. Birds were attracted with conspecific call playback and caught using vertical monofilament mist nets or (in Spain only) spring traps, mainly at dawn or dusk. Due to a very variable attraction success of call playback, capture rates were also very variable and null on several sessions. All captured birds were measured, ringed with a metal (left leg) and a Darvic band (engraved with a unique alphanumeric code; right leg), photographed, and a feather sample (one greater covert) was taken before birds were released after ca. 5 min of handling. There is no known reliable way to determine the sex in this species from in-hand examinations and we thus have no information on the sex of the birds we caught.

Birds ageing and molt scoring

First-generation feathers can be easily recognized from field observations or photographs as they show a characteristic thin whitish edge (Figure S2). In contrast, second-generation coverts are indistinguishable from adult (i.e. at least two-year old birds) feathers and the only way to separate young birds (i.e. less than one-year-old) with advanced molt (i.e. having molted most of their wing coverts) from older birds is to examine the shape and color of their primary coverts (Figure S2). [START_REF] Alstrom | Pipits and Wagtails of Europe, Asia and North-America Helm[END_REF] As a consequence, it is necessary to handle young birds with advanced molt to age them accurately. Birds that were caught were thus separated into adult and young birds and we measured the molt extent in the latter age class. Since birds were caught mostly between November and February, they had already achieved their autumn molt. We assessed the molt extent within feathers that are apparent when a bird is perched (i.e. with the wing closed). We thus counted the number of molted feathers in median secondary coverts (from 0 to 8), in greater secondary coverts (from 0 to 10) and in tertials (from 0 to 3) on the right wing. The molt score varied from 0 (no molted feathers) to 21 (completely molted; adult birds logically had a score of 21; Figure S2).

Based on the molt patterns observed during captures, we consider that a young bird with a molt score above 18 cannot be safely distinguished from an adult bird from field observations or photographs. We thus defined an adult-type phenotype for this type of individuals and identified, in total, three phenotypes that we called full-juvenile, extensive and adult-type molt phenotypes. The first molt phenotype called full-juvenile corresponds to young birds that have not molted after fledging (score of 0-1). The extended post-juvenile molt phenotype concerns birds that have replaced part of their first-generation medium coverts (from 0 to 8), tertiaries (from 0 to 3), and up to eight greater coverts (score of 6-18). The adult-type molt phenotype grouped adults and young birds having replaced most of their first-generation covert feathers and which may retain a maximum of one first-generation tertial and some first-generation greater coverts (score of 19-21; Figure S2).

Assessing the geographical distribution of molt phenotypes from pictures

We collected georeferenced pictures on citizen-science databases and Internet picture repositories across different European and North African countries that regularly host the species in autumn and/or during the wintering period. After February, young birds that have not finished molting in the autumn can resume their molt and become similar to adult birds, hence we only collected data associated with pictures obtained between 08 (2013) and 02 (2019). We selected good quality pictures of perched birds showing the secondary coverts and tertials correctly. When several photos were available for a single location where several birds were reported, we allocated photos to different individuals by comparing photos (individual birds usually differ in details of molt or plumage); when in doubt, only one picture was selected from the available sample. We thus obtained 349 pictures from 331 individuals (presumably all different from the birds we caught ourselves) from 17 European and North African countries.

Details on picture collections and websites consulted are available in Table S3. We assigned each bird to one of the three molt phenotypes identified during captures based on its molt score.

For the winter period, we assessed whether the proportions of molt phenotypes in northwestern Europe (Norway, Sweden, Denmark, Germany, the Netherlands, the United-Kingdom, Ireland, Switzerland and Western France) and southwestern Europe (Southern France, Italy, Catalonia, Spain, Portugal, Morocco) are different from the expected proportions under the full vagrancy hypothesis (i.e. under the hypothesis that only young birds are seen in Europe) by performing a Fisher' exact or Chi-square test on contingency tables summarizing observations (the choice of the test depending on the number of observations per category). We thus compared the proportions of molt phenotypes from the 50 young birds examined in the hand with the proportions of molt phenotypes estimated from pictures in Northwestern and Southwestern Europe. A significant test would indicate that the proportions of molt phenotypes differ from those observed in a sample of juvenile birds. If only vagrant birds were involved, we would expect that all or most are juveniles.

Tracking the breeding range of Richard's Pipits with archival light loggers

During the 2019-2020 winter period, we deployed seven INTIGEO P65C2-7 Global-Location-Sensors (GLS) in Southern France (Migrate Technology, 0.74 g; Covid-19 lockdown stopped the fieldwork season in early 2020). GLS are small electronic devices that record time and light intensity, enabling the calculation of the approximate position of the logger given the duration of the day and the time of solar noon, both depending on latitude and longitude for a given calendar day. [START_REF] Lisovski | Geolocation by light: accuracy and precision affected by environmental factors[END_REF] Due to the small body size of the Richard's Pipit, there were no other technology available at the time to track the movements of the species during the annual cycle. Since GLS do not remotely transmit positions, they have to be retrieved to download the data and birds must thus be captured again. Due to the equivalent day length everywhere on Earth at the autumnal and spring equinoxes, latitudinal positions within 15 days on each side of the two equinox dates cannot be estimated accurately. [START_REF] Phillips | Seasonal sexual segregation in two Thalassarche albatross species: competitive exclusion, reproductive role specialization or foraging niche divergence?[END_REF] The error of GLS locations during migration is known to vary up to 200 km [START_REF] Phillips | Seasonal sexual segregation in two Thalassarche albatross species: competitive exclusion, reproductive role specialization or foraging niche divergence?[END_REF] but the precision increases (in the order of a few dozen kilometers) when the bird stays several days at the same location [START_REF] Rakhimberdiev | Comparing inferences of solar geolocation data against highprecision GPS data: annual movements of a double-tagged black-tailed godwit[END_REF] . Using the same technique as explained above, we equipped four adults and three young birds with leg-loop harnesses made from rubber bands to attach the loggers. The device and fitting material represented less than 3% of a bird's weight (average weight: 31.9 ± 2.2 g, n=78; unpublished data). We retrieved three devices the next winter, containing data from at least one complete migration per bird (Table S2). Return rates of GLS-equipped birds did not differ obviously from those that have been colored-ringed during winter 2018-2019 and re-sighted during winter 2018-2019 (42% of returning GLS-equipped bird and 33% of returning color-ringed individuals, but note small sample sizes).

We followed the method described in Lisovski et al. (2020) [START_REF] Lisovski | Lightlevel geolocator analyses: A user's guide[END_REF] to analyze the light data recorded by GLS devices. We first looked for changes in light intensity pattern that could provide evidence for an incubating behavior (i.e., for a bird hidden in the vegetation). Then, we used the TwGeos R package [START_REF] Lisovski | TwGeos: Basic data processing for light based geolocation archival tags[END_REF] to define twilights with the preprocessLight function, using a threshold of 2.5 lux. We manually removed obviously erroneous twilights and applied an additional automated screening using the twilightEdit function (settings were window = 4, outlier.mins = 45 and stationary.mins = 25). We then used the FLightR package [START_REF] Rakhimberdiev | FLightR: an R package for reconstructing animal paths from solar geolocation loggers[END_REF] to determine migration timing. Since, the package requires calibration periods during which the bird is stationary in a known location, we ran the model a first time using the month of March as calibration period and with a particle filter of 10 4 particles (color-ringed birds were observed throughout March on their wintering site, pers. obs). We refined the calibration period using the plot_lon_lat function (plot of latitude and longitude in horizontal layout). We defined the FLightR search grid between 35° S and 65° N latitude and 0° W and 100° E longitude and ran the particle filter with the recommended 10 6 particles. We estimated the breeding location, migration timing, potential stationary periods and their locations using the stationary.migration.summary function by defining a minimum duration of stationary period of 24 hours and testing different values of minimum probability of movement (prob.cutoff). We estimated the migratory route and the distance travelled via the shortest route (i.e. a great circle route; distHaversine function in the geosphere R package) [START_REF] Hijmans | Geosphere: spherical trigonometry[END_REF] between these different stationary locations.

Wintering niche comparison

To compare environmental niches of birds wintering in their ancestral Asian areas and birds wintering in Europe, we collected occurrences of Richard's Pipit from the Global Biodiversity Information Facility (https://www.gbif.org/). We selected occurrence data for the wintering period alone (from December to February) and from countries known to host the species regularly during this period (Europe and North Africa: France, Spain, Portugal, Italy, Morocco; Asia: Taiwan, Thailand, Hong-Kong, China, India, Myanmar, Vietnam, Laos, Cambodia, Sri Lanka; see [START_REF] Alstrom | Pipits and Wagtails of Europe, Asia and North-America Helm[END_REF] ). We retained verified and georeferenced occurrences, resulting in 1,268 occurrences for Europe and North Africa and 6, 717 occurrences for Asia, derived from citizen science databases widely used by the ornithological community (mostly eBird.org, iNaturalist.org, observation.org and the various Biolovision national reporting systems). For Europe, these databased are usually well curated by validation committees that operate at local (regional) or national scales. Data were then thinned prior to analyses to reduce the effect of biased species occurrence collections, using the spThin R package [START_REF] Aiello-Lammens | spThin: an R package for spatial thinning of species occurrence records for use in ecological niche models[END_REF] with a thinning parameter of 0.5 kilometers, leaving a total of 1,638 occurrences.

Since environmental conditions seem to play a key role in the selection of species' wintering grounds, particularly for finding the resources necessary for winter survival, [START_REF] Thorup | Resource tracking within and across continents in long-distance bird migrants[END_REF] we collected several climatic and habitat variables related to the species preference. [START_REF] Alstrom | Pipits and Wagtails of Europe, Asia and North-America Helm[END_REF] We downloaded the temperature and precipitations of the winter months (between December and February), an aridity index (the ratio of the mean annual precipitation on the mean annual potential evapo-transpiration) and a discrete variable characterizing open and closed habitats.

The climatic data were collected from the CHELSA database v1.2 (http:// chelsa-climate.org) at a resolution of 30 arc seconds, with monthly mean temperature and precipitation averaged over the period 1979-2013. [START_REF] Karger | Climatologies at high resolution for the earth's land surface areas[END_REF] The aridity index was collected from the CGIAR-CSI Database (http://www.cgiar-csi.org) at a resolution of 30 arc seconds for the period 1970-2000 [START_REF] Zomer | Climate change mitigation: A spatial analysis of global land suitability for clean development mechanism afforestation and reforestation[END_REF] and habitat variable from the Global Land Cover Map (GlobCover) [START_REF] Arino | Global Land Cover Map for 2009[END_REF] . The habitat variable was coded as "open" and "closed" habitats based on GlobCover categories; crop and mosaic habitats with more than 50% of cropland or grassland were considered as open habitats whereas mosaic habitats with less than 50% of cropland or grassland and more than 50% of forest or scrubland were considered as closed habitats. We checked that variables were not collinear (correlation coefficients < 0.50) using occurrences in both European and Asian regions, resulting in five variables to represent environmental variation among regions: PRW (precipitation during winter months), TW (temperature of winter months), AR (aridity), OH (open-habitats) and CH (close-habitats).

Environmental niche variation was evaluated using principal component analysis (PCA) implemented in the ade4 R package. [START_REF] Chessel | The ade4 package.R package[END_REF] We assessed the variation between the European-North African and Asian wintering ranges by comparing the coordinates of occurrences in a twodimensional environmental space (PC1 x PC2). We evaluated the niche overlap between the Asian and the European niches using Schoener's D. [START_REF] Schoener | Nonsynchronous Spatial Overlap of Lizards in Patchy Habitats[END_REF] We also evaluated the percentage of inclusion of the European niche within the Asian niche by measuring the intersection between each convex hull defined by 100% of occurrences.

To investigate if vagrant birds may have found suitable wintering conditions in Europe, we performed niche distribution models with the BIOMOD R package [START_REF] Thuiller | BIOMOD -a platform for ensemble forecasting of species distributions[END_REF][START_REF] Thuiller | BIOMOD -optimizing predictions of species distributions and projecting potential future shifts under global change[END_REF] implemented in the biomod2 R package. For each region, an ensemble of projections from four statistical models was obtained, including random forest (RF), generalized linear models (GLM), generalized additive models (GAM) and general boosting method (GBM). [START_REF] Araujo | Ensemble forecasting of species distributions[END_REF] Models were calibrated for the baseline period using 70% of observations randomly sampled from the initial data and evaluated against the remaining 30% data using the true skill statistic (TSS) [START_REF] Allouche | Assessing the accuracy of species distribution models: prevalence, kappa and the true skill statistic (TSS)[END_REF] and the area under the curve (ROC). [START_REF] Swets | Measuring the accuracy of diagnostic systems[END_REF] For each model, the relative importance of each environmental variable was assessed by calculating the Pearson's correlation between the standard predictions (i.e. fitted values) and the predictions after randomly permuting the values of the variable. The ensemble forecasting was then built by assigning weight to each individual model depending on its quality (TSS) and taking the weighted sum of occurrence probabilities. Both calibrated models were projected under current conditions over Europe -North Africa and Asia.

To evaluate changes in suitability across Europe over the period 1960-2018, we also performed niche distribution models using the same methodology with environmental variables available for this time period. We thus downloaded the temperature and precipitation of the winter months (between December and February) from the CHELSA database v1.2 (http:// chelsa-climate.org) at a resolution of 30 arc seconds, with monthly mean temperature and precipitation averaged for the 1961-2018 period. [START_REF] Karger | Climatologies at high resolution for the earth's land surface areas[END_REF] The aridity index (CGIAR-CSI Database) and habitat variable from the Global Land Cover Map (GlobCover) [START_REF] Arino | Global Land Cover Map for 2009[END_REF] were not available for the entire study period. Therefore, we calculated the De Martonne index [START_REF] De Martonne | Aréisme et indice d'aridité[END_REF]

Figure 2 .

 2 Figure 2. Trends in Richard's Pipit occurrences in France during the last decades (A) Barplot representing the temporal evolution of the number of Richard's Pipits observed in France between 1981 and 2019. (B) Graphic comparing the trends in the number of Richard's Pipits (red line) versus other vagrant species of eastern origin (black line) regularly observed in France over the same period (1981-2019). Other vagrant species were used as a proxy for the evolution of the observer effort. Regression lines and coefficients are shown. For vagrant species, the coefficient represents the mean value for all vagrant species. Related to Figure S1, TableS1.

Figure 3 .

 3 Figure 3. Migration routes, stopovers and breeding sites of three GLS-tracked Richard's Pipits Individuals are identified by their GLS numbers: BW367 (A-B), BW376 in (C-D) and BW372 (E). Panel (F) indicates the light intensity (log-transformed) recorded by GLS BW372 in May-June. Blue dots indicate the wintering location in Southern France (Labruguière for BW376 and BW372, Lespignan for BW367) where the GLS were deployed. Pink dots indicate the estimated breeding location from the retrieved GLS (55.35 N ± 0.8 SD, 77.53 E ± 1.3 SD, 54.45 N ± 0.4, 79.82 E ± 0.6, and 56.74 N ± 0.3, 79.54 E ± 0.9 for the three birds, respectively), at the limit of the known breeding distribution represented by the pink polygon.[START_REF]IUCN Red List for birds[END_REF] The change of pattern in light intensity data (F) suggests that this individual was likely incubating and thus engaged in breeding behavior. Other dots (with error bars corresponding to standard errors of longitude and latitude estimates) indicate stopover sites, where the bird stayed more than 24 h.

Figure 4 .

 4 Figure 4. Wintering niches comparisons and niche-based distribution modelling (A) Niche comparison between wintering areas in Europe and North Africa (dark blue) and Southeast Asia (light blue). The convex hulls indicate the prevalence (25%, 50%, 75%, and 100% of locations included) of the environmental conditions. Small dots indicate GBIF occurrences and black-circled dots represent the centroids of these occurrences. The correlation circle (inset) indicates the loading of environmental variables onto the first two PCA axes: PRW (precipitation during winter months), TW (temperature of winter months), AR (aridity), OH (open-habitats) and CH (closed-habitats). The first axis (PC1, 39.6% of total variance) differentiated the environmental spaces between wintering ranges with warmer and less-humid winters in Asia than in Europe. The second axis (PC2, 26.4%) is associated with the opening of habitats, with the Asian niche of the species being characterized by more closed habitats than in Europe. (B) Map representing the projection over Europe and North Africa under current conditions of the niche-model calibrated with Asian occurrences. Colors from white to green represent an increasing scale of suitability. The coastal plain in northwest Morocco and to a lesser degree the Portugal coast and the coastal plain area between Alger and Tunis (in Algeria and Tunisia) corresponds to the environmental conditions experienced by Richard's Pipit on their ancestral wintering areas. (C) Map representing the change of suitability based on the projection over two periods (1961-1990 and 1990-2018) of a niche-model calibrated with European occurrences. Dark green represents gain of suitable areas between these periods. Related to Figure S4, S5.

  as an aridity index, from monthly precipitation and annual temperature data for the 1961-2018 period. But we finally found high collinearity (correlation coefficients > 0.50) with precipitations data. The calibrated model based on two environmental variables (PRW and TW) was projected over Europe under conditions of the 1961-1990 and the 1990-2018 periods.

  Fig Th Eu on con 75% env occ cen im firs win AR hab

Acknowledgments

We are grateful to: Loïs Rancilhac for his help in starting up this project; Marc Illa, Albert Burgas, Oriol Clarabuch, Thomas Dagonet, Stephan Tillo and other ringers for their invaluable assistance in the capture of birds; Benjamin Long, Philippe Bailleul and all the people who took part in the bird's monitoring; Janne Aalto, Luca Bergamaschi, Christian Leth, Julien Mazenauer and Louis Sallé for their help in the data collection; Per Alström, Yosef Kiat, Lukas Jenni for their constructive comments on the ecology of the species and molt timing; Anne Charmantier for her help in logistical support; James Fox for help with geolocators; Christoph Haag for his feedback and recommendations on the draft. For providing access to the study sites, we are grateful to: Réserve naturelle de Crau, CEN PACA, ville de Fréjus, Groupe Ornithologique du Roussillon (GOR), Communauté de communes La Domitienne, Departament de Territori i Sostenibilitat, Aiguamolls de l'Empordà Natural Park, Montgrí, Illes Medes and Baix Ter Natural Park. For collecting and sharing their data, we thank: Ligue de Protection des Oiseaux (LPO), Comité d'Homologation National (CHN) and Comité des Migrateurs Rares (CMR). This work has been supported by a PhD grant to P.D. awarded by the LabEx OSUG@2020 (Investissements d'avenir -ANR10LABX56) and a field grant for young researchers to P.D awarded by the Société Française d'Ecologie et d'Evolution (SFE 2 ).

Data and code availability statement

Raw geolocator data have been archived on Movebank.org (study ID: 1720094461).

Occurrences data are from published or downloadable online sources (e.g. Global Biodiversity

Author contributions

P-A.C., S.L. and P.D. designed the study. C.d.F., P.D-V., F.J. and P.D. performed the fieldwork. J.R. M.G., P.D-V. and P.D. collected occurrences and environmental data. M.G. and P.D. ran the analyses. P.D. wrote the first version of the paper and all authors contributed substantially to the revisions.

Declaration of interests

The authors declare no competing interests.

STAR Methods

Resource availability

Lead contact

Further information and requests for resources should be directed to and will be fulfilled by the Lead Contact, Paul Dufour (paul.dufour80@gmail.com).

Material availability

This study did not generate new unique reagents.

Information Facility). Environmental data used in this paper are freely available and downloadable from the web, the R code for running niche modeling analyses is available on Zenodo: https//doi.org/10.5281/zenodo.5500364.

Experimental model and subject details

We captured, measured, and tagged Richard's Pipits Anthus richardi during the non-breeding period in different localities of France and Spain (see Table S2). Birds were handled with permission from MNHN-CRBPO (reference program #989) for France and SEO-BirdLife (reference program #SF/0106/2019 and #SF/0103/2020) for Spain.

Methods details

Estimating trends in Richard's Pipit occurrence

To check whether the number of Richard's Pipits occurring in Europe actually increased over the last few decades, we studied the trend in occurrence numbers in France between 1981 and 2019. We focused on France because the country hosted the first documented wintering record for Europe [START_REF] Dulau | Nouveau cas d'hivernage du Pipit de Richard Anthus richardi dans le sud de la France[END_REF] and because data have been meticulously collected over a period of about 30 years.

The French Birds Rarities Committee collected data between 1981 and 2005 when the species was still rare to scarce. From the 1990s onwards, numbers of occurrences started to increase and data on the species were collected by another committee between 2005 and 2010, after which the citizen-science databases took over. To avoid double-counting, we used the


Lélia Abad

Pierre Chauvelot

Estelle Audoux

Camille Andre

Jérôme Josse

Céline Dupieux

Sebastien Lustig

Tristan Ferry

Paul O Verhoeven

Alan Diot

Sébastien Lustig

Frédéric Laurent

Florent Valour
email: florent.valour@chu-lyon.fr.

Lysosomal alkalization to potentiate eradication of intra-osteoblastic Staphylococcus aureus in the bone and joint infection setting

Keywords: Antimicrobial, Bone and joint infection, Intracellular activity, lysosomal alkalization, Staphylococcus aureus

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Staphylococcus aureus represents the leading cause of bone and joint infection (BJI), and is responsible for difficult-to-treat infections related to several persistence mechanisms [START_REF] Lew | Osteomyelitis[END_REF][START_REF] Kapadia | Periprosthetic joint infection[END_REF][START_REF] Ferry | Virulence determinants in Staphylococcus aureus and their involvement in clinical syndromes[END_REF]. In particular, its ability to persist within non-professional phagocytic bone cells subverts the host immune system and the action of most antimicrobials [START_REF] Valour | Delta-toxin production deficiency in Staphylococcus aureus: a diagnostic marker of bone and joint infection chronicity linked with osteoblast invasion and biofilm formation[END_REF][START_REF] Josse | Staphylococcus aureus vs. osteoblast: relationship and consequences in osteomyelitis[END_REF]. Consequently, the ability of antimicrobials to eradicate the intracellular S. aureus reservoir may have a significant impact on the risk of BJI chronicity and relapse.

S. aureus has been shown to persist within phagocytes through phagolysosomal replication and escape to gain the cytoplasm [START_REF] Flannagan | Intracellular replication of Staphylococcus aureus in mature phagolysosomes in macrophages precedes host cell death, and bacterial escape and dissemination[END_REF][START_REF] Krauss | Staphylococcus aureus infects osteoclasts and replicates intracellularly[END_REF][START_REF] Strobel | Post-invasion events after infection with Staphylococcus aureus are strongly dependent on both the host cell type and the infecting S. aureus strain[END_REF]. It also notably affects autophagy, a part of the innate immune response against bacterial infections corresponding to a catabolic process leading to autophagosome formation, a compartment that encloses intracytoplasmic bacteria. It involves SQSTM1/p62 (sequestosome 1), a ubiquitin receptor essential to this selective degradation, and LC3, a membrane protein participating to phagosomal elongation and maturation. To eradicate intracellular bacteria, the autophagosome and lysosome merge, generating the autolysosome, a digestive hybrid organelle with an acidic environment [START_REF] Sokolovska | Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function[END_REF][START_REF] Dikic | Mechanism and medical implications of mammalian autophagy[END_REF]. Interestingly, the impaired intracellular antimicrobial activity relies on the poor cellular penetration of some molecules, their low distribution in the subcellular location of S. aureus and the intracellular emergence of small colony variants (SCVs), but also on drug inactivation by the acidic pH of intracellular organelles [START_REF] Dupieux | Intraosteoblastic activity of daptomycin in combination with oxacillin and ceftaroline against MSSA and MRSA[END_REF][START_REF] Baudoux | Combined effect of pH and concentration on the activities of gentamicin and oxacillin against Staphylococcus aureus in pharmacodynamic models of extracellular and intracellular infections[END_REF]. Consequently, some studies have considered lysosomal alkalization to restore intracellular antibiotic activity [START_REF] Maurin | Phagolysosomal alkalinization and the bactericidal effect of antibiotics: the Coxiella burnetii paradigm[END_REF][START_REF] Maurin | Phagolysosomal alkalinization and intracellular killing of Staphylococcus aureus by amikacin[END_REF][START_REF] Leimer | Nonstable Staphylococcus aureus small-colony variants are induced by low pH and sensitized to antimicrobial therapy by phagolysosomal alkalinization[END_REF]. Alkalization also appeared to prevent SCV emergence [START_REF] Leimer | Nonstable Staphylococcus aureus small-colony variants are induced by low pH and sensitized to antimicrobial therapy by phagolysosomal alkalinization[END_REF][START_REF] Vulin | Prolonged bacterial lag time results in small colony variants that represent a sub-population of persisters[END_REF]. Thus, lysosomotropic alkalinizing agents might help targeting S. aureus intraosteoblastic reservoir in the BJI setting. The aim of this study was to compare the intra-osteoblastic activity of anti-staphylococcal agents alone or in combination with lysosomotropic alkalinizing agents in an in vitro model of osteoblast infection.

MATERIAL AND METHODS

Study drugs.

All antimicrobials and the three lysosomotropic agents (i.e., amantadine, ammonium chloride [NH4Cl] and hydroxychloroquine) were used after dilution in NaCl 0.9% (Table 1).

Bacterial strains.

All experiments were performed using three MSSA strains: i) 6850 (ATCC 53657), highly virulent strain isolated from patient with osteomyelitis described by Balwit et al. [START_REF] Balwit | Gentamicin-resistant menadione and hemin auxotrophic Staphylococcus aureus persist within cultured endothelial cells[END_REF]; and ii) clinical isolates n°1 and n°2 collected from patients with recurrent osteomyelitis in our institution.

MICs at pH5 and pH7 determination. MICs of drugs included in the study were determined by broth microdilution following the recommendation of the French Committee for Antimicrobial Susceptibility Testing (CA-SFM). Of note, pH recommended for routine MIC determination is 7.2-7.4 (CA-SFM). Here, Mueller-Hinton broths (MHB, Sigma-Aldrich, Saint-Louis, USA) were adjusted to pH 5 and 7 to mimic cytosolic and lysosomal compartments.

Lysosomal pH measurement from MG63 cells. Measurement of lysosomal pH from human

osteoblastic cell line MG63 (CRL-1427) [START_REF] Pautke | Characterization of osteosarcoma cell lines MG-63, Saos-2 and U-2 OS in comparison to human osteoblasts[END_REF] was performed using LysoSensor Yellow/Blue DND-160 (Invitrogen, Carlsbad, USA), a lysosomotropic pH indicator dye, exhibiting a pH-dependent excitation at 329 nm and 384 nm and allowing the ratiometric assessment of pH changes in acidic organelles [START_REF] Liu | Restoration of Lysosomal pH in RPE Cells from Cultured Human and ABCA4-/-Mice: Pharmacologic Approaches and Functional Recovery[END_REF]. Uninfected MG63 cells were seeded at 10,000 cells per well into 96-well tissue culture plates in 100 µL of growth medium (DMEM + 10 % fetal calf serum) and cultured for 24 h. Cells were incubated with increasing concentrations (2.5, 5, 10 and 20 µg/mL) of lysosomotropic agents for various incubation time (0.5h, 2h, 4h, 24h, 48h and 72h). When incubation exceeded 24 h, medium was changed daily. Then, medium was removed and 50 µL of Lysosensor (dilution 2:1000 in NaCl 0.9%) was added in each well for a 3-min incubation. The fluorescence was quantified using a microplate reader (TECAN Infinite® 200 PRO) to determine lysosomal pH. A calibration curve was performed in duplicate with uninfected cells incubated with a dedicated buffer (KCl 100mM, 20mM NaCl, 20mM Mes, 20µM monensin, 10µM nigericin) at pH 4.5, 5.0, 5.5, 6.0 and 6.5.

Cytotoxicity measurement.

A colorimetric assay based on the enzymatic reduction of 3-[4,5dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to MTT-formazan was used to determine the cells viability as previously described [START_REF] Abad | Antibiofilm and intraosteoblastic activities of rifamycins against Staphylococcus aureus: promising in vitro profile of rifabutin[END_REF]. For each condition, the absorbance was normalized on "untreated cells (0 mg/L)" corresponding to 100%.

Intracellular activity of antibiotics and impact on infection-induced cytotoxicity. Intracellular

activities of levofloxacin, clindamycin, cotrimoxazole and daptomycin with or without hydroxychloroquine were assessed in an in vitro model of osteoblast infection, as previously described [START_REF] Abad | Antibiofilm and intraosteoblastic activities of rifamycins against Staphylococcus aureus: promising in vitro profile of rifabutin[END_REF].

Bacterial strains were cultured in tryptic soy broth at 37°C with shaking for 6 h before storage at -80°C in individual cryovials containing 1×10 10 CFU/mL in PBS (Gibco) containing 10 % glycerol [START_REF] Diep | Polymorphonuclear leukocytes mediate Staphylococcus aureus Panton-Valentine leukocidininduced lung inflammation and injury[END_REF].

For each experiment, one cryovial was used to prepare standardized bacterial suspension in DMEM + 10 % fetal calf serum added to osteoblasts for 2 h at a multiplicity of infection (MOI) of 100 bacteria/cell. Cells were then incubated for 1 h with growth medium supplemented with 10 mg/L of lysostaphin (Sigma-Aldrich) to kill the remaining extracellular bacteria. Infected cells were then incubated for 24h with growth medium containing the tested antimicrobials with or without hydroxychloroquine at 20 mg/L. Antimicrobials were used at the bone concentrations reached during standard human therapy [START_REF] Thabit | Antibiotic penetration into bone and joints: An updated review[END_REF] : daptomycin (20 mg/L), clindamycin (7 mg/L), cotrimoxazole (40/8 mg/L) and levofloxacin (10 mg/L). For each condition, lysostaphin at 10 mg/L was added to rapidly kill the bacteria released upon host cell lysis. Cells were lysed by a 10-min incubation with 1 mL of sterile water, then serial dilutions of lysates were spiral-plated (Spiral®, Interscience, St Nom la Bretèche, France) on TSA (Trypcase Soy Agar, Biomérieux, Marcy-l'Étoile, France) to quantify intracellular bacteria. The impact on infection-induced cytotoxicity was determined with the MTT assay, as described above.

Impact of hydroxychloroquine on the autophagic flux. MG63 cells were infected with the 6850 strain and exposed to hydroxychloroquine at 2.5, 5, 10 and 20 mg/L for 24h. Cell lysates were centrifuged at 10,000 rpm for 10 min at 4°C. Proteins concentrations of the supernatants were determined by BCA assay (Kit Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific). Proteins lysates were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membrane by electroblotting (BioRad, Hercules, USA). Membranes were blocked with 5 % non-fat milk in PBS-Tris buffer for 1 h, followed by incubation with rabbit primary antibody targeting LC3 (Cell Signaling, Danvers, USA), SQSTM1 (Cell Signaling) and actin (used as reference protein, Cell Signaling) at 4°C overnight. After three steps of washing, membranes were incubated with secondary anti-rabbit goat antibody (HRP, Thermo Fisher Scientific) at room temperature for 1 h. Clarity Western Blot ECL substrate (BioRad) was used for protein detection.

Statistical methods. Data were summarized as means and their 95% confidence intervals (95%CI).

Conditions were compared using a nonparametric two-sided Mann-Whitney U test. A p-value < 0.05 was considered significant. All analyses were performed using Prism software (GraphPad, San Diego, USA).

RESULTS

Susceptibility studies and choice of the tested antimicrobials.

MICs of the three S. aureus strains (6850, clinical isolates n°1 and 2) are presented in Table 1. As shown by significant increased MICs (i.e., at least 2 dilutions) at pH 5 compared to pH 7, acidic pH impaired the activity of clindamycin, cotrimoxazole, levofloxacin, ofloxacin, rifabutin, daptomycin, fosfomycin and tigecyclin.

Alkalization kinetics and cytotoxicity of the lysosomotropic agents. Lysosomal pH of uninfecteduntreated cells was 4.8 (95%CI, 4.5-5.0). Alkalization kinetics (0-72 h) for increasing concentrations of NH4Cl, hydroxychloroquine and amantadine are reported in Figure 1.B, 1.C and 1.D respectively.

Regardless of the tested concentration and exposition time, amantadine induced a slight lysosomal pH rise, reaching a maximum of 6.0 (95%CI, 5.3-6.6) at 10 mg/L after 72 h of incubation. NH4Cl at 20 mg/L allowed to reach a maximal pH of 8.1 (95%CI, 7.4-8.8) after 2 h, but with high variations observed over time. For hydroxychloroquine, a reproducible dose-effect was observed at all timepoints, with a maximal effect reached after 2 h and remaining stable over time. At the maximal concentration of 20 mg/L, the lysosomal pH rose to 7.0 (95%CI, 6.8-7.3) after 2 h.

In parallel, cytotoxicity kinetics of the three lysosomotropic agents were explored (Figure 2 and supplementary data table 2). Amantadine and NH4Cl decreased cell viability from and over 10 mg/L for all incubation times. Hydroxychloroquine was significantly cytotoxic at 20 mg/L only after 72 h.

The alkalinizing and cytotoxicity profiles of hydroxychloroquine led us to use this drug at 20 mg/L for 24 h when tested in combination with antimicrobials in our in vitro osteoblast infection model.

Intraosteoblastic activity of antimicrobials with or without hydroxychloroquine and impact on infection-induced cytotoxicity.

At the tested concentrations, daptomycin, cotrimoxazole, clindamycin and levofloxacin with or without hydroxycholoroquine (20 mg/L) had no significant cytotoxicity on uninfected cells (supplementary Figure 1). mg/L, increasing the bactericidal effect of levofloxacin to -1.8 log10CFU/100,000 cells (95%CI; -1.6 to -2.8; p=0.015) (Figure 4B).

Impact of hydroxychloroquine on autophagy.

Compared to untreated infected cells (mean LC3-II/actin ratio, 0.15), the relative level of LC3-II trended to increase until 10 mg/L of hydroxychloroquine, with a mean ratio 0.53 for 2.5 mg/L, 0.62 for 5 mg/L, 1.14 for 10 mg/L and 0.44 for 20 mg/L (Figure 4A). While less pronounced, SQSTM1/actin ratios followed the same trend, measured at 0.47, 0.57, 0.66, 0.65 and 0.43 when hydroxychloroquine concentration increased from 0 to 20 mg/L (Figure 4A).

DISCUSSION

The present study represents the first evaluation of various lysosomotropic alkalinizing agents used as adjuvants to conventional antistaphylococcal drugs in an in vitro model of osteoblast infection.

The preliminary experiments performed allowed us to set the best conditions to establish the proof of concept of alkalization in our model. First, MIC determinations at pH 5 and 7 confirmed that low lysosomal pH might impair the activity of many antimicrobials. Impacted by acid pH and being the most antimicrobial prescribed in BJIs, daptomycin, cotrimoxazole, clindamycin and levofloxacin were chosen for further evaluation in the osteoblast infection model. Second, based on its high alkalinizing potential with low cytotoxicity at the concentration and time of exposition tested, hydroxychloroquine was chosen as the better adjuvant to be tested in combination with antibiotics. Indeed, after 24 hours of incubation at 20 mg/L, hydroxychloroquine allowed the lysosomal pH to be increased from 4.8 to 7.0. Similarly, Maurin et al. reported an increase of lysosomal pH from 4.8 to 5.3, 5.7 and 6.8 in a murine macrophage model using amantadine (1 mg/L), chloroquine (1 mg/L) and ammonium chloride (1 mg/mL) [START_REF] Maurin | Phagolysosomal alkalinization and the bactericidal effect of antibiotics: the Coxiella burnetii paradigm[END_REF].

Regarding the impact of lysosomal alkalization, the addition of hydroxychloroquine to antimicrobials allowed a significant increase of their bactericidal effects, without inter-strain variability. Data are scarce in the literature regarding antibiotic potentiation by alkalinizing agents toward intracellular S. aureus killing. Our results complete the preliminary results, showing that chloroquine (1 mg/L) associated with amikacin increased intramacrophagic S. aureus inoculum reduction [START_REF] Maurin | Phagolysosomal alkalinization and intracellular killing of Staphylococcus aureus by amikacin[END_REF]. Additionally, using human lung epithelial cells, Leimer et al. showed that flucloxacillin associated with chloroquine, decreased significantly the proportion of intracellular SCVs [START_REF] Leimer | Nonstable Staphylococcus aureus small-colony variants are induced by low pH and sensitized to antimicrobial therapy by phagolysosomal alkalinization[END_REF]. Together, these data support the use of this adjuvant therapy against intracellular S. aureus.

On the other hand, some antimicrobials with increased activity at acidic pH could be impeded by alkalinizing therapy. In particular, a 8 to 42-fold increase in rifampin MICs at pH 7 compared to pH 5 was observed, which can constitute an significant limitation of the use of alkalinizing strategies according to the crucial role of rifampin in orthopedic device-associated infection outcome [START_REF] Senneville | Outcome and Predictors of Treatment Failure in Total Hip/Knee Prosthetic Joint Infections Due to Staphylococcus aureus[END_REF][START_REF] Murillo | A large multicenter study of methicillin-susceptible and methicillin-resistant Staphylococcus aureus prosthetic joint infections managed with implant retention[END_REF]. However, rifampin MICs evaluated at neutral pH remains far below the intraosseous concentrations reached with standard therapeutic dosages. Additionally, other rifamycins could be proposed as an alternative, and especially rifabutin, that could theoretically benefit from alkalization and present higher antibiofilm and intracellular activities [START_REF] Abad | Antibiofilm and intraosteoblastic activities of rifamycins against Staphylococcus aureus: promising in vitro profile of rifabutin[END_REF].

Finally, we highlighted that the antimicrobial activity potentiation related to lysosomal alkalization was not the only mechanism of hydroxychloroquine on staphylococcal intracellular reservoir. Indeed, and despite the absence of intrinsic antibacterial activity, hydroxychloroquine alone was able to significantly reduce S. aureus intracellular inoculum. Interestingly, hydroxychloroquine has been reported to interact with autophagic pathways, mostly in the oncology setting [START_REF] Plantone | Current and future use of chloroquine and hydroxychloroquine in infectious, immune, neoplastic, and neurological diseases: a minireview[END_REF][START_REF] Mauthe | Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion[END_REF][START_REF] Jacquin | Pharmacological modulators of autophagy activate a parallel noncanonical pathway driving unconventional LC3 lipidation[END_REF]. The observed increase in LC3-II starting at the lowest concentrations of hydroxychloroquine are in favor of an induction of autophagy. In addition a weak SQSTM1 accumulation advocate for a simultaneous inhibition of phagolysosome formation, as described for other cell types [START_REF] Jacquin | Pharmacological modulators of autophagy activate a parallel noncanonical pathway driving unconventional LC3 lipidation[END_REF][START_REF] Lin | Chloroquine and hydroxychloroquine inhibit bladder cancer cell growth by targeting basal autophagy and enhancing apoptosis[END_REF]. As hydroxychloroquine improved intracellular bacteria eradication starting at 2.5 mg/L but fostered the antimicrobials activity from 10 mg/L only, we therefore assumed a combined effect of the induction of the autophagic flux at low concentrations, followed by antimicrobial potentiation by lysosomal alkalization at higher concentrations. Of note, if hydroxychloroquine alone induced a significant reduction of intracellular inoculum at low concentrations, it could not prevent infection-induced cytotoxicity. This paradoxical result might rely on the absence of intrinsic effect of hydroxychloroquine on bacterial growth and toxin secretion. Finally, the usual posology of hydroxychloroquine as lifelong treatment for rheumatoid arthritis and lupus erythematosus is 400-800 mg/day, providing blood concentrations between 1 and 2.5 mg/L [START_REF] Munster | Hydroxychloroquine concentration-response relationships in patients with rheumatoid arthritis[END_REF][START_REF] Costedoat-Chalumeau | Hydroxychloroquine in systemic lupus erythematosus: results of a French multicentre controlled trial (PLUS Study)[END_REF]. The use of higher daily doses -even for shorter duration -during BJI treatment to improve eradication of bacteria intracellular is likely not be possible due to tolerance issues. Nevertheless, the present data represents a proof of concept in cellular model and other lysosomotropic agents such as Lys05, a novel 4-aminoquinoline with an improved ability to accumulate in the lysosomes [START_REF] Amaravadi | Lys05: a new lysosomal autophagy inhibitor[END_REF], may have more potent alkalinizing effect and could be an alternative to hydroxychloroquine.

To conclude, hydroxychloroquine as adjuvant to antimicrobials may improve the eradication of the intraosteoblastic S. aureus reservoir by both restoring intracellular activity of some antimicrobial and interfering with the bacterial subversion of autophagy. This new clinical perspective requires now in vivo confirmation of its effectiveness and toxicity.

TABLES

Figure 1 .

 1 Figure 1. Modulation of lysosomal pH by various lysosomotropic agents using increasing

Figure 2 .

 2 Figure 2. Cytotoxicity-induced kinetics for ammonium chloride (A), hydroxychloroquine (B) and

Figure 3 .

 3 Figure 3. Impact of daptomycin (20 mg/L), clindamycin (7 mg/L), cotrimoxazole (40/8 mg/L) and

Figure 4 .

 4 Figure 4. Impact of increasing concentration of hydroxychloroquine on autophagy markers, and

Table 1 .

 1 In vitro MICs of the study drugs at pH 5 and 7.

	MICs (mg/L)	6850		Clinical isolate n°1 Clinical isolate n°2
		pH 7	pH 5	pH 7	pH 5	pH 7	pH 5
	Antimicrobials
	Cefazolin 1	0.125	0.125	0.0625	0.03	0.5	0.03
	Clindamycin 2	8	>32	4	>32	4	32
	Cotrimoxazole 3	0.5	2	1	4	1	4
	Daptomycin 4	1	4	2	4	2	4
	Doxycycline 5	0.125	0.0625 0.5	0.125	0.5	0.125
	Fosfomycin 6	0.25	2	0.5	1	8	16
	Levofloxacin 7	0.25	2	0.125	0.5	0.125	1
	Linezolid 2	4	4	2	2	2	2
	Ofloxacin 8	0.5	2	0.25	2	0.5	2
	Oxacillin 9	0.0625 0.0625 0.0625	0.03	<0.015 <0.015
	Rifabutin 8	<0.0035 0.125	0.007	0.5	0.007	0.5
	Rifampin 6	0.0075 0.0009 0.03	0.007	0.015	0.0015
	Rifapentine 8	0.007	<0.0035 0.015	0.007	0.015	0.0035
	Teicoplanin 8	0.5	0.0625 0.25	2	0.5	1
	Tigecycline 8	0.25	1	0.0625	1	0.0625 1
	Vancomycin 10	2	1	1	0,5	0.25	0.125
	Lysosomotropic agents
	Amantadine 8	>100	>100	>100	>100	>100	>100
	Hydroxychloroquine 8 >100	>100	>100	>100	>100	>100
	NH4Cl 8	>100	>100	>100	>100	>100	>100
	Grey boxes: increased MICs at pH 5 by at least 2 dilutions.
	Providers: 1 Mylan, Canonsburg, USA; 2 Pfizer, New York, USA; 3 Roche, Bâle, Switzerland; 4 Novartis,
	Bâle, Switzerland; 5 Sandoz, Bâle, Switzerland; 6 Sanofi, Paris, France; 7 Fresenius Kabir, Bad Homburg
	vor der Höhe, Germany; 8 Sigma-Aldrich, Saint-Quantin Fallavier, France; 9 Bristol-Myrs-Squibb, New
	York, USA; 10 Sandoz, Holzkirchen, Germany.

ACKNOWLEDGEMENTS

We thank Claire Mitchell and Nestor Mas Gomez for their help on the protocol of Lysosensor.

FUNDING

This study received the support of an institutional grant for "Young Investigators" from the Hospices Civils de Lyon.

After the 3h infection step, the intracellular inoculum was 2.5 x 10 5 CFU/ 100,000 cells (95%CI, 1.6 x 10 5 -3.5 x 10 5 ). Without treatment, the number of intraosteoblastic bacteria was 1.2 x 10 5 CFU/ 100,000 cells (95%CI, 7.6 x 10 4 -1.6 x 10 5 ) after 24h (Figure 3A), and the infected cell viability was reduced by 55.7% (95%CI, 48.7-62.7) after 24 h in comparison with uninfected cells (p<10 -4 ) (Figure 3B).

Hydroxychloroquine alone (20 mg/L) did not reduce infection-induced cytotoxicity but significantly decreased the intracellular inoculum by 0.36 (95%CI, 0.18-0.66; p<10 -4 ) log10CFU/100,000 cells.

Daptomycin, cotrimoxazole, clindamycin and levofloxacin alone reduced S. aureus intraosteoblastic inoculum by 0.14 (95%CI, 0.01-0.34; p<10 -3 ), 0.25 (95%CI, 0.12-0.43; p<10 -4 ), 0.16 (95%CI, 0.004-0.39; p<10 -4 ) and 1.18 (95%CI, 1.04-1.38; p<10 -4 ) log10CFU/100,000 cells, respectively. Adding hydroxychloroquine (20 mg/L) led to multiply bactericidal effect of antimicrobial alone by 2.7 for daptomycin, cotrimoxazole and clindamycin, and 6.5 for levofloxacin, corresponding to an inoculum reduction of 0.50 (95%CI, 0.30-0.84), 0.73 (95%CI, 0.59-0.96), 0.59 (95%CI, 0.46-0.78) and 1.8 (95%CI, 1.66-2.1) log10CFU/100,000 cells, (p<10-4), respectively (Figure 3A).

Compared to untreated cells, daptomycin, cotrimoxazole, clindamycin and levofloxacin alone increased significantly cell viability by 28.0% (95%CI, 9.0-46.9; p=0.021), 42.0% (95%CI, 26.0-58.0; p=0.002); 83.1% (95%CI, 66.4-99.5; p<10 -4 ) and 50.6% (95%CI, 29.6-71.6; p<10 -3 ), respectively.

Compared to antibiotics alone, the adjuvant uses of hydroxychloroquine did not significantly increase the cell viability (Figure 3B).

Additional experiments were designed to evaluate the combined dose-effect of hydroxychloroquine and antimicrobials using increasing concentrations of hydroxychloroquine and one antimicrobial.

Levofloxacin was chosen because it was the most potent antimicrobials against intracellular S. aureus based on previous results. From 2.5 µg/mL, hydroxychloroquine alone reduced the intracellular inoculum -0.35 log10CFU/100,000 cells (95%CI, -0.22 to -0.55; p=0.002) with no significant difference until 10 mg/L. Levofloxacin alone decreased -1.3 log10CFU/100,000 cells (95%CI; -1.1 to -1.8; p=0.002) the intracellular inoculum. Potentiation by hydroxychloroquine was significant from 10 TRANSPARENCY DECLARATIONS