Simon Chollet
email: simon.chollet@univ-rennes1.fr

Morgane Maillard

Juliane Schörghuber

Sue J Grayston

Jean-Louis Martin

Running Head: Deer slow down litter decomposition Deer slow down litter decomposition by reducing litter quality in a temperate forest

Keywords: decomposition, deer, forests, functional traits, Home-field Advantage, litter diversity effects, macro and meso fauna, top-down control

Litter decomposition is a key process that allows the recycling of nutrients within ecosystems. In temperate forests the role of large herbivores in litter decomposition remains a subject of debate. To address this question, we used two litterbag experiments in a quasi-experimental situation resulting from the introduction of Sitka black-tailed deer Odocoileus hemionus sitkensis on forested islands of Haida Gwaii (Canada). We investigated the two main pathways by which deer could modify litter decomposition: change in litter quality and modification of decomposer communities. We found that deer presence significantly reduced litter mass loss after one year, mainly through a reduction in litter quality. This mass loss reflected a 30 and 28 % lower loss of carbon (C) and nitrogen (N), respectively. The presence of deer also reduced the ability of decomposers to breakdown carbon, but not nitrogen. Indeed, litter placed on an island with deer lost 5% less carbon after one year of decomposition than did litter decomposing on an island without deer. This loss in ability to decompose litter in presence of deer was outweighed by the differences in mass loss associated with the effect of deer on litter quality. Additional effects of feces deposition by deer on the decomposition process were also significant but minor. These results suggest that the effects continental-scale dramatic increases in deer populations may have on broad-scale patterns of C and N cycling deserve closer attention.

Introduction

Deer and the functioning of temperate forests

By the mid-20 th century wild ungulates, essentially deer, were largely missing from temperate forests of Europe and eastern North America, a result of hunting and/or loss of favorable land cover [START_REF] Mcshea | The Science of Overabundance: Deer Ecology and Population Management[END_REF][START_REF] Apollonio | European Ungulates and Their Management in the 21st Century[END_REF]. Since then, changes in hunting regulations, in land-uses, and the absence of their natural predators [START_REF] Côté | Ecological Impacts of Deer Overabundance[END_REF][START_REF] Milner | Temporal and spatial development of red deer harvesting in Europe: biological and cultural factors[END_REF], allowed a dramatic rebound in deer populations. In less than a century the deer population in Europe is estimated to more than 15 million, and 30 million white-tailed deer (Odocoileus virginianus) alone inhabit North America [START_REF] Mcshea | The Science of Overabundance: Deer Ecology and Population Management[END_REF][START_REF] Apollonio | European Ungulates and Their Management in the 21st Century[END_REF]. This rebound brought deer to the forefront of ecological thinking [START_REF] Terborgh | Trophic Cascades: Predators, Prey, and the Changing Dynamics of Nature[END_REF].

Initially research focused on the consequences of deer recovery on forest vegetation, beginning with impacts on tree regeneration and growth [START_REF] Gill | A Review of Damage by Mammals in North Temperate Forests: 3. Impact on Trees and Forests[END_REF]. More recent studies focused on aboveground understory community functioning [START_REF] Horsley | White-tailed deer impact on the vegetation dynamics of a northern hardwood forest[END_REF][START_REF] Royo | Pervasive interactions between ungulate browsers and disturbance regimes promote temperate forest herbaceous diversity[END_REF]) with a focus on effects cascading on invertebrates, birds and small mammals (e.g. Chollet andMartin 2013, Foster et al. 2014)]. In the latter part of the 20 th century, the effect of large herbivores on processes of nutrient cycling in temperate forests also drew attention [START_REF] Pastor | Moose Browsing and Soil Fertility in the Boreal Forests of Isle Royale National Park[END_REF][START_REF] Frank | Ungulate Vs. Landscape Control of Soil C and N Processes in Grasslands of Yellowstone National Park[END_REF][START_REF] Ritchie | Herbivore Effects on Plant and Nitrogen Dynamics in Oak Savanna[END_REF][START_REF] Olofsson | Role of litter decomposition for the increased primary production in areas heavily grazed by reindeer: a litterbag experiment[END_REF][START_REF] Tanentzap | Carbon storage in terrestrial ecosystems: do browsing and grazing herbivores matter?[END_REF]. However, the repercussions of high deer densities on belowground patterns and processes are still insufficiently understood [START_REF] Bardgett | Herbivore-Mediated Linkages Between Aboveground and Belowground Communities[END_REF][START_REF] Hobbie | Interactive effects of plants, decomposers, herbivores, and predators on nutrient cycling. Page Trophic ecology: Bottom-up and top-down interactions across aquatic and terrestrial systems[END_REF]. These belowground effects will partly result from the effects deer have on litter decomposition, a pivotal link between aboveground primary production and belowground processes [START_REF] Chapin | Principles of Terrestrial Ecosystem Ecology[END_REF]. Contrary to grasslands or boreal forests, there are still only a few studies on how large herbivores affect litter decomposition in temperate forest ecosystems.

Deer and litter decomposition

Deer may modify litter decomposition by affecting litter quality. Through plant removal, combined with selective foraging, deer modify plant community composition, plant stoichiometry, as well as the relative contribution of canopy and understory vegetation to litter composition [START_REF] Côté | Ecological Impacts of Deer Overabundance[END_REF]).

Specifically, deer induce the production of anti-herbivore compounds by plants, which decrease litter

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This article is protected by copyright. All rights reserved quality for decomposers [START_REF] Harrison | How browsing by red deer impacts on litter decomposition in a native regenerating woodland in the Highlands of Scotland[END_REF]. In addition, deer promote unpalatable plant species through preferential browsing of the most palatable species [START_REF] Pastor | Moose Browsing and Soil Fertility in the Boreal Forests of Isle Royale National Park[END_REF]. Because some of the traits controlling litter palatability and decomposability are similar, such a long-term plant community shift is expected to change litter decomposability [START_REF] Bardgett | Herbivore-Mediated Linkages Between Aboveground and Belowground Communities[END_REF]. Overall, the expected reductions in litter quantity and quality should slow down decomposition processes and nutrient cycling [START_REF] Bardgett | Herbivore-Mediated Linkages Between Aboveground and Belowground Communities[END_REF]. Deer also release dung and urine, a source of organic matter more easily decomposable than recalcitrant plant litter [START_REF] Ruess | Grazing and the Dynamics of Nutrient and Energy Regulated Microbial Processes in the Serengeti grasslands[END_REF].

Conversely to the reduction in litter quality due to the modification of the plant community, waste deposition by deer may speed up litter decomposition [START_REF] Bardgett | Herbivore-Mediated Linkages Between Aboveground and Belowground Communities[END_REF].

Deer may also modify litter decomposition by affecting decomposers ability to degrade litter (i.e. differences in decomposition rates of a given litter due to decomposers, [START_REF] Keiser | Disentangling the mechanisms underlying functional differences among decomposer communities[END_REF]). The long-term changes deer generate in the vegetation could indeed trigger changes in decomposer communities that specialize them in decomposing the type of litter that falls locally, potentially compensating the aforementioned slowing down of decomposition rate. Such specialization of the decomposers for local litter is known as "home-field advantage" (HFA), and could play an important role in the decomposition process [START_REF] Gholz | Long-term dynamics of pine and hardwood litter in contrasting environments: toward a global model of decomposition[END_REF][START_REF] Austin | There's no place like home? An exploration of the mechanisms behind plant litter-decomposer affinity in terrestrial ecosystems[END_REF]). Yet, studies explicitly evaluating at the same time the role of the shift in vegetation induced by herbivores and the role of HFA are scarce and provided contrasting results (see Olofsson andOksanen 2002, Penner and[START_REF] Penner | Litter Decomposition in Yellowstone Grasslands: The Roles of Large Herbivores, Litter Quality, and Climate[END_REF].

Besides the abovementioned effects of deer on decomposers ability via vegetation replacement, decomposers may also be affected directly by deer. Dung and urine represent a source of inorganic nitrogen for soil decomposers that enhances their development [START_REF] Sitters | The Stoichiometry of Nutrient Release by Terrestrial Herbivores and Its Ecosystem Consequences[END_REF]. This may affect the structure and functioning of the decomposer communities, soil fauna [START_REF] Andriuzzi | Responses of belowground communities to large aboveground herbivores: Meta-analysis reveals biome-dependent patterns and critical research gaps[END_REF] and soil microorganisms [START_REF] Cline | Soil microbial communities and elk foraging intensity: implications for soil biogeochemical cycling in the sagebrush steppe[END_REF][START_REF] Eldridge | Competition drives the response of soil microbial diversity to increased grazing by vertebrate herbivores[END_REF], in ways that increase the rate of litter decomposition [START_REF] Handa | Consequences of biodiversity loss for litter decomposition across biomes[END_REF]. Deer are also suspected to affect edaphic properties by increasing soil temperature and salinity through exposure of bare soil after vegetation removal by browsing, or by increasing soil compaction through trampling, with effects on soil water and oxygen content [START_REF] Schrama | Herbivore trampling as an alternative pathway for explaining differences in nitrogen mineralization in moist grasslands[END_REF]. Such modifications of the soil may also alter the decomposer ability to degrade litter.

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A quasi-experimental context to test predictions on how deer affect litter decomposition

Although the multiplicity of pathways through which deer can affect litter decomposition is recognized (see [START_REF] Bardgett | Herbivore-Mediated Linkages Between Aboveground and Belowground Communities[END_REF] for a conceptual model), our knowledge is mainly based on the independent study of single pathways. This has led to apparent contradictions in results. To better identify the mechanisms behind the effect of deer on litter decomposition we designed a study that combined approaches to better assess of the relative effects of different pathways. Our study design benefitted from the long-term effort to use the late 19 th century introduction of Sitka blacktailed deer Odocoileus hemionus sitkensis to the Haida Gwaii archipelago (British Columbia, Canada, [START_REF] Golumbia | Lessons from the islands: introduced species and what they tell us about how ecosystems work[END_REF]) as an unplanned experiment on trophic interactions. Deer colonized most, but not all, islands of the archipelago. This resulted in a situation with, side by side, islands colonized by deer, and islands never colonized. This unique situation allowed to demonstrate that, in presence of deer, and independently of island size, deer herbivory was the main factor structuring plant, invertebrate and songbird communities, overwhelming other biotic or abiotic factors [i.e. island area, soil and micro-habitat diversity [START_REF] Martin | Interaction among deer browsing, hunting, and tree regeneration[END_REF][START_REF] Gaston | Species-area relationships and the impact of deer-browse in the complex phytogeography of the Haida Gwaii archipelago (Queen Charlotte Islands)[END_REF][START_REF] Martin | Top-down and bottom-up consequences of unchecked ungulate browsing on plant and animal diversity in temperate forests: lessons from a deer introduction[END_REF], Chollet et al. 2013b)]. In addition recurrent experimental culls on some islands allowed to document a partial recovery of the aboveground vegetation and songbirds [START_REF] Chollet | Positive plant and bird diversity response to experimental deer population reduction after decades of uncontrolled browsing[END_REF].

We made use of these contrasts in deer browsing history in two decomposition experiments designed to test five predictions: 1) decomposition of litter collected from an island without deer would be faster than decomposition of the litter collected from an island with deer because of a decreased in litter quality in presence of deer (litter quality effect); 2) decomposition of a given category of litter (deer present or not) will be favored on the island from which that litter was collected (HFA effect); 3) decomposition ability would be higher in presence of deer because of changes in microbial communities in response to a more recalcitrant litter (decomposer ability effect); 4) presence of deer excrement would locally speed up the decomposition of plant litter; 5) decomposition pattern found on an island with partial understory recovery (cull island) would fall in between what we observe on the islands with and without deer.

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Materials and Methods

The archipelago and deer effect on the vegetation

Haida Gwaii is characterized by a humid temperate-oceanic climate, with mean annual temperature of 8.5°C and precipitation that varies greatly from 1,350 mm on the east coast, where this study took place, to 7,000 mm on the west coast [START_REF] Banner | A Field Guide to Ecosystem Classification and Identification for Haida Gwaii[END_REF]). The archipelago is covered by temperate rainforests dominated, at low elevation, by western hemlock (Tsuga heterophylla), western redcedar (Thuja plicata), and Sitka spruce (Picea sitchensis). The selected study sites belonged all to the Coastal Western Hemlock Wet Hypermaritime biogeoclimatic variant [Biogeoclimatic Ecosystem Classification, code CWHwh1, British Columbia Ministry of Forests, Lands, and Natural Resource Operations 2010)] which covers 49% of the archipelago, and ranges from sea-level to 350 m in elevation [START_REF] Banner | A Field Guide to Ecosystem Classification and Identification for Haida Gwaii[END_REF].

Because of the extensive effect deer had on the understory vegetation, islands can be segregated today into three categories of browsing histories and their associated vegetation patterns. Islands without deer, which are characterized by a diverse and lush understory vegetation dominated by broad leaved shrubs and ferns, producing a diverse and abundant litter. Islands where deer have been present for over 70 years, which are characterized by an open understory dominated by bryophytes where litter is dominated by conifer leaves [START_REF] Stockton | A natural experiment on the effects of high deer densities on the native flora of coastal temperate rain forests[END_REF][START_REF] Martin | Top-down and bottom-up consequences of unchecked ungulate browsing on plant and animal diversity in temperate forests: lessons from a deer introduction[END_REF]. Finally, islands where deer have also been present for over 70 years but have been subjected to recurrent deer culls. Their understory is characterized by an intermediate cover of vegetation [START_REF] Chollet | Positive plant and bird diversity response to experimental deer population reduction after decades of uncontrolled browsing[END_REF].

Study sites and plot selection

We selected three islands in Laskeek Bay (52°53'12"N, 131°35'20"W, Appendix S1 in Supporting Information), Low, Reef and Louise Island. We chose sampling and experimental sites with similar bedrock geology, which was part of the Yakoun formation, consisting of porphyritic andesite agglomerate, calcareous scoraceous lapilli tuff and volcanic sandstone and agglomerate (Sutherland Brown 1968). The soil type was organic and classified into the Folisol order (Soil Classification Working Group / Groupe de travail sur la classification des sols 1998). These islands were representative of the patterns of deer impacts we documented at the scale of the archipelago [START_REF] Martin | Top-down and bottom-up consequences of unchecked ungulate browsing on plant and animal diversity in temperate forests: lessons from a deer introduction[END_REF][START_REF] Chollet | Long-term consequences of invasive deer on songbird communities: Going from bad to worse?[END_REF], Appendix S2 for a synthesis of previous studies). Low Island, 9.6 ha, that

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This article is protected by copyright. All rights reserved had never been colonized by deer, Louise Island, 25,000 ha, that has had deer for over 70 years [START_REF] Vila | Can we reconstruct browsing history and how far back? Lessons from Vaccinium parvifolium Smith in Rees[END_REF]) and had a current deer density estimated at 30 deer / km², and Reef Island, 249 ha, that had also been colonized by deer more than 70 years ago, but its deer population had been regularly culled between 1997 and 2010. At the time of study Reef Island had a deer population density estimated at about 15 deer / km² and a partially recovered understory vegetation [START_REF] Chollet | Positive plant and bird diversity response to experimental deer population reduction after decades of uncontrolled browsing[END_REF].

All islands were covered by tree species characteristic of the Coastal Western Hemlock Wet Hypermaritime biogeoclimatic zone, typical of the archipelago lowlands. Our emphasis on selecting sites similar in parent material prevented us from also controlling for island size. It also allowed us to minimize pseudo-replication by duplicating the experiment on additional islands, a duplication, however, that would have been a logistical challenge exceeding our means. To avoid potential biases due to elevation we selected all sampling plots at low elevation on the three islands with most plot elevations less than 50 m asl and never exceeding 100 m asl. Adjacent plots had to be separated by at least 100 m. On each island, and within the constraints just stated, we randomly established fifteen 10 m x 10 m forest interior plots, leading to a total of 45 plots.

Above and belowground characteristics in relation to deer presence

In each plot we sampled the vegetation by estimating the percent cover of vascular plants and bryophytes using the Londo scale [START_REF] Londo | The decimal scale for releves of permanent quadrats[END_REF]). We measured soil bulk density at the surface of the soil with five replicate measures per plot. For this, we collected soil with a 5.4 cm depth x 4.1 cm diameter (71.29 cm 3 ) copper core hammered into the soil using a mallet. We took care to not change the structure of the soil while sampling. We removed any coarse woody-debris from core samples and subtracted their volume from the volume of the core. We then dried soil at 105°C for 24h to obtain a value for bulk density expressed as g of dried soil per cm 3 of fresh soil.

We used data on soil pH, C:N and organic horizon depth collected in the course of a sister study in plots located in the same area on these same islands (Maillard et al. unpublished data). This data was collected from five plots on Low Island, five plots on Louise Island and six plots on Reef Island.

We sampled soil within these plots with a 2.5 cm diameter x 30 cm depth core collecting approximately 100 cores per plot. They were mixed and sieved with a 5 mm sieve to ensure homogenization, as recommended for soil with high content of organic matter [START_REF] Haynes | Stability of soil aggregates in relation to organic constituents and soil water content[END_REF]. We measured soil pH in a 0.01 M CaCl2 solution using a 1:10 ratio (air dried soil: solution).

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This article is protected by copyright. All rights reserved We determined soil C:N ratio from 3 mg of freeze-dried and ground soil using an Elementar Vario El Cube Analyzer. In each plot we measured the depth of the soil's organic horizon from a soil pit dug within the plot.

Experimental design and protocol

To analyze how the prolonged presence of abundant deer affected litter decomposition, we designed two litterbag experiments centered on the three browsing treatments available: deer absent (no browsing, our reference), deer culled (intermediate browsing), and deer present (severe browsing).

We measured litter decomposition rates using the litterbag method. We made 15 cm x 15 cm bags using polypropylene mesh with two different mesh sizes. We used the litterbags with a 0.2 mm x 0.2 mm mesh size to target the decomposition solely due to the soil microfauna and microorganisms.

We used litterbags with a 3.7 x 4.45 mm mesh size to assess the additional effect of the mesofauna and macrofauna on litter decomposition. For these large mesh litterbags, we used a 0.2 mm x 0.2 mm mesh size on the bottom of the bag to avoid litter spillage.

To obtain our litter samples, we collected summer senescent leaves from plant species with a percent cover on the plots greater than 5 %. In total, we sampled 18, 20 and 17 plants species, respectively, on the island with no browsing, on the island with an intermediate level of browsing, and on the island with a severe level of browsing (Appendix S3). We dried these litter samples at 30°C for a week before using them.

Experiment 1 -We assessed the effect of deer presence on litter decomposition at the scale of an island through a reciprocal litterbag translocation experiment involving litter from the three islands representative of the three browsing treatments: "no", "intermediate" and "severe" browsing. We produced, for each plot, three identical litterbags for each of the two mesh-sizes. Each of these six litterbags contained, in the same proportion as in the plot, plant material from all plant species covering more than 5 % of the plot area. As a result, our litterbags account for the variability in vegetation cover within each island. We fixed the total mass of litter per litterbag at 4 g. Hence, we calculated the mass of litter from each plant species in a given litterbag according to its relative abundance in the plot. For each mesh size, we placed one of the three litterbags on the plot the litter came from ("home"), and placed the two remaining bags on one plot on each of the two remaining islands ("away"). This translocation allowed us to independently test for the relative effects of home-

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field advantage, of decomposer ability (soil properties and decomposer community composition), and of litter quality on litter decomposition (Appendix S1).

Experiment 2 -To investigate the influence of deer feces on litter decomposition rate within a litterbag, we used a standardized litter. We chose litter from one of the dominant tree species on all islands, P. sitchensis. To avoid any biases from potential inter-treatment differences in spruce litter quality, we used, for this set of litterbags, a mix of P. sitchensis litter collected from all three islands.

In order to standardize feces quality, we collected fresh deer feces from another island with deer, also situated in Laskeek Bay (East Limestone, 48 ha). We used this material to place on each of the 45 plots one fine-mesh litterbag containing 2 g of deer feces and 2 g of the standardized litter and two fine-mesh litterbags containing controls, one filled only with 5 g of deer feces and one filled only with 2 g of P. sitchensis litter. We repeated this with coarse-mesh litterbags.

Thus, to implement these two experiments, we placed a total of 12 litterbags on each plot including 6 fine-mesh litterbags [3 for experiment 1 (1 home and 2 away) and 3 for experiment 2 (1 feces only + 1 P. sitchensis only + 1 with feces and P. sitchensis)] and a similar set of 6 litterbags for the coarse-mesh litterbags. As a result, we had a total of 540 litterbags (12 bags * 45 plots) for the full design. All litter bags were identified and numbered with aluminum tags and placed randomly on the surface of the forest floor on each plot. We used U pins at each corner of the bag to hold them in place. We placed litterbags on the three islands in July 2017, and collected them one year later in July 2018. We considered a one-year duration optimal to incorporate the full range of weather conditions.

The remoteness of the islands and associated logistical challenges to access the sites, especially from fall to spring, prevented designing an experiment with a partial collection of litterbags during the year to better assess the kinetics of decomposition. After collection (all but 35 bags were retrieved) we dried the litterbag contents at 70°C for 48h prior to weighing the contents and then performing chemical analyses.

Mass, Carbon and Nitrogen loss in litterbags

To assess litter mass loss over a year in each litterbag we subtracted the final mass of the bag's content from its initial mass. We weighed the quantity of foreign material accumulated after a year in a control coarse-mesh litterbag displaced in each plot. We then subtracted this mass of foreign material from the final mass in coarse mesh litterbags, in order to correct for contamination. To assess

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This article is protected by copyright. All rights reserved C and N loss over a year, we calculated C and N contents of samples with an Elementar Vario El Cube Analyzer (Elementar, Langenselbold, Germany) using 3.5 mg of ground material. We first calculated the initial C and N contents of dried litter using eight individuals of each plant species (vascular and bryophytes) from each island/browsing treatment. Based on these values, and on the relative proportion of each litter in the bags, we calculated the initial C and N content for each litterbag. We also measured initial C and N contents of eight deer pellet groups that were previously dried at 70°C for 48h.

At the end of the experiment we finely ground the dried litter from the fine-mesh litterbags only, to remain within our budget limitations, and measured C and N content. We calculated carbon and nitrogen loss by subtracting the amount of carbon and nitrogen remaining in litter after one year of decomposition from the initial estimates based on our calculations of initial C and N content of the litter material.

Statistical analysis

In order to evaluate the effect of deer herbivory on plant community composition we used a Correspondence Analysis on our data of plant species cover per plot, and performed a between class analysis [START_REF] Dray | The ade4 Package: Implementing the Duality Diagram for Ecologists[END_REF]. We evaluated the significance of the class effect with a permutation test. To assess the initial litter C:N ratios at each plot we calculated the Community Weighted Mean (CWM) of this initial litter C:N ratio using the formula

𝐶𝑊𝑀 𝑖 = 𝑗 ∑ 1 (𝐶 𝑗 × 𝑝 𝑗 )/ 𝑗 ∑ 1 (𝑁 𝑗 × 𝑝 𝑗 )
where i represents the plot, j the plant species on this plot, C j and N j the C and N content of the corresponding litter, and p j the relative abundance of the corresponding plant species on the plot.

We used one-way ANOVA with permutation tests to compare litter CWM C:N ratio, soil bulk density, soil pH, soil C:N ratio and organic horizon depth among the three islands. We used the multiple comparison post-hoc test with the function kruskalmc from the package pgirmess.

To assess differences in the rate of litter mass loss and of C and N loss in both experiments, we calculated the percent differences among treatments in litter mass loss and in C and N loss relative to the no-browsing treatment using the formula:

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This article is protected by copyright. All rights reserved Analyses of decomposition experiment 1 -We used a two-way ANOVA to compare litter mass loss, carbon loss and nitrogen loss from litter after one year among the three origins of litter (15 replicates from each island with no, intermediate or severe deer browsing) and among treatment categories (island with no, intermediate or severe deer browsing).

To disentangle the relative importance of the two main ways deer may modify C and N decomposition, namely litter quality and decomposer ability, we used the Decomposer Ability Regression Test proposed by [START_REF] Keiser | Disentangling the mechanisms underlying functional differences among decomposer communities[END_REF] using SAS 9.4 (SAS Institute, Cary, NC). This method statistically discriminates among effects of litter quality (here defined as how rapidly a litter is decomposed regardless of decomposition site), ability [i.e. how rapidly a litter is decomposed at one site regardless of litter quality (includes the effect of soil abiotic conditions and the ability of the decomposer communities)] and home-field advantage [i.e. the acceleration of litter decomposition when litter is placed in the site it comes from (home) and where it can potentially benefit from a local specialization of the decomposer community]. The regression model defines the rate of decomposition of observation i (Y i ) by three parameters: litter quality (Litter l ), soil ability (Soil s ), and HFA (Home h ) which are dummy variables that equal 1 or 0, respectively, depending on the presence or absence of the litter mixture, soil community and home combination (in observation i). The parameters to be estimated are β l , γ s and η h [START_REF] Keiser | Disentangling the mechanisms underlying functional differences among decomposer communities[END_REF]. The average decomposition across all data (i) in a dataset, after controlling for litter, soil and home combinations, is represented by the intercept (α), and the error term is defined by ε. The β l and γ s are restricted to prevent collinearity.

𝑌 𝑖 = ∝ + ∑ 𝑁 𝑙 = 1 𝛽 𝑙 𝐿𝑖𝑡𝑡𝑒𝑟 𝑙 𝑖 + ∑ 𝑀 𝑠 = 1 𝛾 𝑠 𝑆𝑜𝑖𝑙 𝑠 𝑖 + ∑ 𝐾 𝐻 = 1 ηℎ𝐻𝑜𝑚𝑒 ℎ 𝑖 + 𝜀 𝑖
Compared to the classically used Structural Equation Modeling (SEM), the Decomposer Ability Regression Test offers the additional possibility to explicitly test home-field advantage and maximizes the information extracted from the litter transplant experiment [START_REF] Keiser | Disentangling the mechanisms underlying functional differences among decomposer communities[END_REF].

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This article is protected by copyright. All rights reserved To explore the reasons for differences in litter quality caused by deer, we performed linear models between C or N loss and litter CWM C:N ratio.

Analyses of decomposition experiment 2 (feces manipulation) -We used a two-way ANOVA to compare mass loss, C loss, and N loss of Picea sitchensis, feces and the combination of both.

For all analyses in which homoscedasticity and normality of the distribution of the residues were not respected, we used ANOVA with permutation tests instead of classical ANOVA (lmPerm package, Wheeler 2010). We used the R 3.4.1 environment (R Core Team 2020) for all statistical analyses (except Decomposer Ability Regression Test).

Results

Deer modify aboveground and belowground characteristics

The first axis of the Correspondence Analysis on vegetation cover in the plots significantly discriminated the plant species composition and abundance in the plots according to the intensity of deer browsing (Fig. 1A, Monte-Carlo permutation test: p-value < 0.001). In the absence of deer, vegetation cover was higher and there was greater shrub diversity and heterogeneity among plots (Appendix S3). The vegetation from plots with severe deer browsing was characterized by a high cover and diversity of bryophytes (Fig. 1A) and limited variation in plant community among plots.

Plots under intermediate deer browsing showed intermediate plant species diversity and cover, and intermediate heterogeneity among plot. We found no significant difference in the initial C:N ratio of the plant litter among deer browsing treatments (Fig. 1B, p-value = 0.2). Soil bulk density was significantly higher on plots from islands with deer (Fig. 1C, p-value < 0.001). Soil pH was significantly different among deer browsing treatments (Fig. 1D, p-value = 0.04 however statistical power was not sufficient to discriminate which treatment is different in the post-hoc test). Soil C:N was not significantly different among treatments (Fig. 1E, p-value = 0.32). Depth of the organic horizon measured in the plots decreased by 44 % with increasing browsing intensity (Fig. 1F, p-value = 0.005).

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Litter mass loss in experiment 1

Litter mass loss was highest in litterbags with litter from the island with no deer and lowest in litterbags with litter from the island with severe browsing pressure (Fig. 2A). In fine-mesh litterbags average litter mass loss was 55% in litter from the islands without deer, 43% for litter from islands with intermediate browsing pressure and 34% in litter from islands with severe browsing pressure. This pattern held, whatever the island category litterbags were placed on (Fig. 2A). Variation, due to the context in which a given category of litter was placed, had only a little influence, and there was no significant effect of home-field advantage (Fig. 2B). Mass loss was significantly affected by the place of decomposition (Fig. 2A, F = 113.36, p-value = 0.05). This pattern was due to a significantly enhanced ability of the micro-fauna and microorganisms from plots on the island without deer to decompose litter (Fig. 2C, p-value = 0.004). Litter quality, was the main driver of carbon loss (Fig. 2D). Litter mixes originating from the island with no deer had the best quality index (highest loss after one year), followed by the litter mixes originating from the island with intermediate deer browsing and then by litter mixes originating from the island with severe deer browsing (Fig. 2D).

In coarse-mesh litterbags the overall patterns of decomposition were similar to those from finemesh litterbags (compare Fig. 2 and Appendix S4), although variability among plots within sites was greater (Appendix S4). As a result, mass loss in litter mixes originating from islands with intermediate and severe deer browsing were, respectively, 25% and 39% lower than in litter mixes originating from the plots on the island without deer (p-value < 0.001) (Appendix S4). There was no evidence for home-field advantage in large mesh litter bags (Appendix S4). Place of decomposition significantly affected litter mass loss (Appendix S4, F = 4.54, p-value = 0.01). The ability of the micro-fauna and microorganisms to decompose litter was significantly lower for litter placed in plots exposed to severe browsing (Appendix S4, p-value = 0.009). As observed for fine-mesh litterbags, litter quality had a significant effect on litter decomposition in coarse-mesh bags (Appendix S4, p-value < 0.001).

Carbon and Nitrogen loss in fine-mesh litterbags in experiment 1

The loss of carbon in the litterbags after one year was highest for litter representative of the vegetation on islands without deer and lowest for litter representative of the vegetation on islands with the most severe browsing pressure (Fig. 2E, F = 108.78, p-value < 0.001), a pattern consistent with the pattern of litter mass loss. When compared to the carbon loss observed in litter originating from the island

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This article is protected by copyright. All rights reserved with no deer, carbon loss after one year was 12% lower in litter collected from the island with intermediate browsing, and 30% lower in litter collected from the island with severe browsing, this was independent of the incubating (i.e. island) context (Fig. 2E). Home-field advantage was not significant (Fig. 2F), indicating that decomposers were not more efficient in decomposing litter carbon when it originated from their own environment rather than from other sites. The ability of the micro-fauna and microorganisms to decompose carbon was significantly higher for litter placed in plots without deer than in plots on islands with deer (Fig. 2G, p-value = 0.008). Indeed, carbon loss after one year was 5% lower in litterbags incubated in plots on the island with severe deer browsing than carbon loss observed in plots from the island without deer. Litter quality was the main driver of carbon loss. Litter mixes originating from the island with no deer had the best quality index (highest loss after one year), followed by the litter mixes originating from the island with intermediate deer browsing and then by litter mixes originating from the island with severe deer browsing (Fig. 2H).

Litter mixes from the island without deer had significantly higher nitrogen loss than litter mixes from islands with deer (Fig. 2I). However, unlike C loss, nitrogen loss was lower for litter mixes originating from the island with intermediate deer browsing than that originating from the island with severe deer browsing (45% and 28% respectively, Fig. 2E, F = 17.53, p-value < 0.001). We detected no home-field advantage for N loss (Fig. 2J). In addition, none of the decomposer communities were better at decomposing and releasing nitrogen (Fig. 2K). Similar to what we observed for carbon, litter quality was the main driver of nitrogen loss in litter bags. However, conversely to carbon, the lowest rate of N loss after one year was observed in litter from the island with intermediate browsing pressure (Figs 2H and 2L).

Carbon and nitrogen loss in litterbags after one year were significantly and negatively related to the initial C:N ratio Community Weighted Mean (CWM) of the litter (Fig. 3). For litter carbon loss, the initial litter C:N CWM explained only 10% of its variation (Appendix S5). Conversely, litter nitrogen loss was strongly linked to the initial litter C:N CWM, which explained 50% of its variability (Appendix S5).

Feces decomposition in experiment 2

The addition of feces enhanced the mass loss in P. sitchensis litter by 29% in fine-mesh (p-value <0.001, Fig. 4A) and marginally in coarse-mesh litterbags (p-value = 0.05, Appendix S6). In fine-

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mesh litterbags place of decomposition had marginal effect on feces mass loss (p-value = 0.06, Fig. 4A). In coarse-mesh litterbags mass loss from feces was 15% higher on islands with deer than on islands without deer (Appendix S6, p-value < 0.001). Feces addition significantly increased the C and N loss in P. sitchensis litter after one year, by 31% for carbon and 47% for nitrogen (p-value < 0.001 for both). There were no differences among place of decomposition in carbon and nitrogen loss from feces (p-value = 0.26 and p-value = 0.66 respectively, Fig. 4B andC). The ability of the decomposer community (i.e. decomposition place) had no effect on carbon loss (p-value = 0.587, Fig. 4B).

However, for nitrogen loss in P. sitchensis litter, the presence of deer feces significantly improved the decomposition ability of the decomposer community in the plots from the islands with deer (p-value < 0.001, Fig. 4C).

Discussion

Deer slow down decomposition mainly by changing understory plant communities

In our study, we demonstrated that deer, through their impact on the understory vegetation, control the overall quality of litter reaching the forest floor, and consequently the decomposition process. We indeed demonstrated that this decrease in litter quality is the overriding factor governing litter decomposition in our study sites, rather than soil properties, composition of the soil decomposer community, or home-field advantage (Fig. 2).

The dramatic decrease in litter quality we observed is the result of an alteration in the understory plant community (Figs. 1A,2H and 2L). Intense and prolonged deer browsing dramatically changed the understory plant composition and cover, resulting in an up to 90% reduction in understory shrub cover (Appendix S3, Fig. 1A). We found that this reduction in litter quality, and the associated modifications in the decomposition pattern, were only partly driven by the variation in the litter CWM C:N ratio (Fig. 3). This suggests that other parameters of litter quality not measured in our study, such as lignin or anti-herbivore compounds, were also involved in overall litter quality decline in presence of deer. Consequently we thereafter refer to the term 'litter quality' as a composite variable calculated as proposed by [START_REF] Keiser | Disentangling the mechanisms underlying functional differences among decomposer communities[END_REF], and representing the rate of decomposition independent of decomposer ability and home-field advantage. The modifications of the understory vegetation we found not only confirm the severe impact of deer on Haida Gwaii [START_REF] Martin | Top-down and bottom-up consequences of unchecked ungulate browsing on plant and animal diversity in temperate forests: lessons from a deer introduction[END_REF],

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This article is protected by copyright. All rights reserved Chollet et al. 2013a), but are consistent with results in numerous other temperate forests [START_REF] Côté | Ecological Impacts of Deer Overabundance[END_REF][START_REF] Boulanger | Ungulates increase forest plant species richness to the benefit of non-forest specialists[END_REF], suggesting that effect of deer on the decomposition process through vegetation community shift may be widespread.

The major change in litter quality caused by deer browsing resulted, after one year, in an overall reduction in litter mass loss in presence of deer. This translated into a 30% lower carbon and a 28% lower nitrogen loss in litter from the island with severe browsing when compared to C and N losses in litter from the island with no browsing (Figs. 2E and2I). This strong control of litter quality on C, and especially on N loss, contrasts with the previous assumption that vegetation changes may affect nitrogen and phosphorus dynamics less than the dynamics of carbon (Bryant et al. 1983, Wardle et al. 2002).

The prevailing role of decreased litter quality in litter decomposition we found in our system is in agreement with the microcosm study of [START_REF] Harrison | How browsing by red deer impacts on litter decomposition in a native regenerating woodland in the Highlands of Scotland[END_REF]. They showed that decomposition of birch (Betula pubescens) litter, originating from inside deer exclosures (unbrowsed) in the Scottish Highlands decomposed faster than litter from outside of the exclosures (browsed), irrespective of the soil origin (inside or outside of exclosures). Conversely, [START_REF] Olofsson | Role of litter decomposition for the increased primary production in areas heavily grazed by reindeer: a litterbag experiment[END_REF], in a field translocation experiment assessing the decomposition of four plant species dominating the vegetation of lightly and heavily grazed tundra, demonstrated a positive effect of reindeer herbivory on decomposition rate.

Decline in litter quality affects C and N cycles differently

The decline in litter quality affected carbon and nitrogen cycles differently. For carbon, litter quality decreased as deer browsing intensity increased (Fig. 2H). For nitrogen, litter quality was poorer in the intermediate deer browsing than on the island with severe browsing (Fig. 2L). The decline in litter C loss could be explained by the shift from an understory dominated by more decomposable species (shrubs) towards an understory of less decomposable species (conifers and bryophytes) as the level of deer browsing intensity increased. Conifers and bryophytes are known to have slow decomposition rates due to low N content and high concentrations of structural carbohydrates and aromatic compounds [START_REF] Cornwell | Plant species traits are the predominant control on litter decomposition rates within biomes worldwide[END_REF][START_REF] Turetsky | Trade-offs in resource allocation among moss species control decomposition in boreal peatlands[END_REF]. The presence of these secondary compounds may largely explain the lower decomposition of the litter from islands with deer and thus the slight effect of CWM of litter C:N (≈10% of variation explained, Fig. 3). The contrasting result we obtained

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for N loss from litter after one year suggests that the vegetation shift caused by deer had different consequences for nitrogen mineralization. Although there was no overall significant difference in CWM litter C:N ratio among deer herbivory treatments, the intermediate treatment had the highest values of C:N (Fig. 3), which explains the lowest N loss values observed in this treatment (≈50% of the variation is explained by CWM litter C:N).

Deer also modify decomposer ability

Although the decrease in litter quality caused by deer herbivory was identified as the main driver of the rate of litter mass and nutrient loss, several other changes in the soil decomposer communities affected nutrient cycling. Decomposers from the island without deer had a greater ability to decompose the carbon present in litter, but not nitrogen (Figs. 2G and 2K). The contrast between C and N decomposition among islands when using fine-mesh litterbags, and the similarity in C and N decomposition when using coarse-mesh litterbags (Appendix S4), suggest that the observed decomposition patterns are more likely explained by biotic differences in soils (i.e. differences in decomposer community) than by the effect of abiotic modifications such as higher soil compaction (Fig. 1C). A possible explanation for the observed contrasts in litter decomposition may be a switch in the bacterial:fungal ratio in presence of deer. In fact the disappearance of base-rich shrubs and their replacement by species with high concentrations of phenolic compounds (e.g. bryophytes) as a result of deer browsing may have increased the dominance of fungi which require less calcium and magnesium for growth [START_REF] Prescott | Litter decomposition: what controls it and how can we alter it to sequester more carbon in forest soils?[END_REF]. This change in decomposer community structure would favor the formation of a mor humus, in which up to 30% of the litter mass is converted to humus rather than decomposing [START_REF] Prescott | Litter decomposition: what controls it and how can we alter it to sequester more carbon in forest soils?[END_REF]. In addition the dramatic reduction in shrub cover may have reduced root exudation which stimulates bacterial activity [START_REF] Ekberg | Rhizospheric influence on soil respiration and decomposition in a temperate Norway spruce stand[END_REF]). Contrary to carbon, the ability of decomposers to decompose nitrogen in litter did not vary among islands with different patterns of deer herbivory (Fig. 2K). This may be explained by the selection of microorganisms better able to exploit N in environments where this element is the most limiting ("nitrogen mining hypothesis", [START_REF] Craine | Microbial Nitrogen Limitation Increases Decomposition[END_REF] compensating for the switch in bacterial:fungal ratio. This hypothesis is supported by our control experiment, where we used a standardized quality of litter (Picea sitchensis), and found a greater ability of decomposers to decompose N in litter samples incubated on sites with deer (Fig. 4C).

Accepted Article

This article is protected by copyright. All rights reserved Interestingly, we also found that the inclusion of the soil macro and mesofauna (hereafter fauna) in litter decomposition, via the use of coarse-mesh litterbags, affected litter mass loss (Appendix S4). Specifically, we found that litter decomposition was reduced on the island with the highest browsing intensity, suggesting a negative effect of deer on the faunal decomposer communities. Previous studies documented negative effects of large herbivores on the abundance and diversity of the soil fauna (see review by [START_REF] Andriuzzi | Responses of belowground communities to large aboveground herbivores: Meta-analysis reveals biome-dependent patterns and critical research gaps[END_REF]), but the consequences on litter were not studied. The negative effect we observed could be due to a reduction in both the abundance and the activity of the soil fauna through several mechanisms. Directly, through soil trampling by deer which might reduce soil fauna habitat through soil physical compaction and its reduction of soil pore size [START_REF] Beylich | Evaluation of soil compaction effects on soil biota and soil biological processes in soils[END_REF]. Indirectly, the reduction of litter quality by deer might be responsible for a slowing down of soil faunal abundance and activity for which litter quality is known to be a controlling factor [START_REF] Hendriksen | Leaf litter selection by detritivore and geophagous earthworms[END_REF][START_REF] García-Palacios | Climate and litter quality differently modulate the effects of soil fauna on litter decomposition across biomes[END_REF]. As most previous studies on the effects of large herbivores on decomposition focused mainly on the role of microbes, we feel more attention needs to be paid to the role of the soil fauna in order to better understand ecosystem nutrient cycling.

Dung and urine deposition effects on litter decomposition

We found that soil fauna contributed significantly to the decomposition of deer feces, and observed evidence of home-field advantage (HFA). Indeed, deer feces decomposition in our study was more rapid on islands with deer (home) than on the island without (away), but only when including the effects of fauna (coarse-mesh bags, Appendix S6). We infer that deer have a positive effect on macrofauna decomposing dung. Such a positive effect of a deer species on this specialized fauna has been recently demonstrated in Japan where [START_REF] Iida | Large herbivores affect forest ecosystem functions by altering the structure of dung beetle communities[END_REF] found a positive relationship between the populations of dung beetles and deer density. In our study, we demonstrated that litter fauna, but not microorganisms, were selected for the decomposition of a particular litter type. This result highlights the importance of soil fauna in HFA, in agreement with the review of [START_REF] Austin | There's no place like home? An exploration of the mechanisms behind plant litter-decomposer affinity in terrestrial ecosystems[END_REF] that suggests a significant role of soil meso and macro detritivores for this mechanism.

A large proportion of what we know on the effect of high-quality litter deposition (dung and urine) by large herbivores on nutrient cycling comes from the study of domestic animals and/or grassland ecosystems [START_REF] Mcnaughton | Promotion of the Cycling of Diet-Enhancing Nutrients by African Grazers[END_REF][START_REF] Frank | Ungulate Vs. Landscape Control of Soil C and N Processes in Grasslands of Yellowstone National Park[END_REF][START_REF] Christenson | Winter climate change implications for decomposition in northeastern forests: comparisons of sugar maple litter with herbivore fecal inputs[END_REF].

Accepted Article

This article is protected by copyright. All rights reserved We demonstrate that, in the temperate forests we studied, dung decomposed faster, and released a larger proportion of nitrogen, than observed for plant litter. Also, the addition of feces, whatever the mesh size, increased the rate of Picea sitchensis decomposition, increasing C loss by 31% and N loss by 47% (Fig. 4). This may be explained by the presence of labile nutrients in dung, which enhance the development of microbial communities, increasing rates of nutrient cycling [START_REF] Bardgett | Dynamic interactions between soil animals and microorganisms in upland grassland soils amended with sheep dung: a microcosm experiment[END_REF]. However, despite these results, we found that high quality litter deposition did not affect overall decomposer ability (no higher decomposer ability on islands with deer dung/urine, Fig. 2G and2K). This contradicts recent studies which demonstrated that feces deposition enhanced plant productivity and soil nutrient availability [START_REF] Barthelemy | Strong Responses of Subarctic Plant Communities to Long-Term Reindeer Feces Manipulation[END_REF][START_REF] Wang | Feces nitrogen release induced by different large herbivores in a dry grassland[END_REF]). The explanation for the lack of effect in the forests we studied likely rests with the patchy distribution of solitary deer, in contrast to herding species like reindeer or livestock, and thus reflects the patchy, and limited, amounts of dung deposited locally at any given time, amounts that appear not to be sufficient to influence the nutrient cycling at the ecosystem level [START_REF] Pastor | Moose, Microbes, and the Boreal Forest[END_REF].

Conclusion

Our results show that in temperate forests abundant deer may play an important role in ecosystem functioning, modifying not only aboveground characteristics, but also those belowground, and reducing nutrient cycling. In the last few decades, the awareness and knowledge of the effects of overabundant deer on aboveground communities has been growing worldwide [START_REF] Côté | Ecological Impacts of Deer Overabundance[END_REF][START_REF] Takatsuki | Effects of sika deer on vegetation in Japan: A review[END_REF]). Our study suggests that these aboveground changes can potentially be at the root of modifications in nutrient cycling in temperate forest ecosystems. In addition, it has to be emphasized that our results are likely an underestimation of effects as we did not account the dramatic effect deer have on the quantity of litter reaching the forest floor. For example, in Europe the current 10 million roe deer Capreolus capreolus, in addition to the increasing populations of other ungulates, represent a standing biomass estimated at 0.75 billion kg that consumes ≈20 million tons of green vegetation each year [START_REF] Apollonio | European Ungulates and Their Management in the 21st Century[END_REF]. Consequently, there is a critical need to expand our results to other temperate forests to assess the overall consequences increasing deer populations have on broad scale nitrogen cycling in soils [START_REF] Hobbie | Interactive effects of plants, decomposers, herbivores, and predators on nutrient cycling. Page Trophic ecology: Bottom-up and top-down interactions across aquatic and terrestrial systems[END_REF] and their potential influence on global carbon storage [START_REF] Tanentzap | Carbon storage in terrestrial ecosystems: do browsing and grazing herbivores matter?[END_REF]. [START_REF] Keiser | Disentangling the mechanisms underlying functional differences among decomposer communities[END_REF]. HFA = Home-Field-Advantage. Ability = decomposer ability, or the difference in decomposition rates of the same type of litter exposed to different decomposer communities [START_REF] Keiser | Disentangling the mechanisms underlying functional differences among decomposer communities[END_REF].

Accepted Article

Litter quality = difference in decomposition rates among different litter types exposed to the same decomposer community [START_REF] Keiser | Disentangling the mechanisms underlying functional differences among decomposer communities[END_REF].

Accepted Article

This article is protected by copyright. All rights reserved

  100 -(𝑥 𝑏𝑟𝑜𝑤𝑠𝑖𝑛𝑔 × 100)/𝑥 𝑛𝑜 𝑏𝑟𝑜𝑤𝑠𝑖𝑛𝑔 Where is the litter, C, or N mass loss in litterbags from either the intermediate or the severe 𝑥 𝑏𝑟𝑜𝑤𝑠𝑖𝑛𝑔 browsing treatment, and the litter, C, or N mass loss in litterbags from the no browsing 𝑥 𝑛𝑜 𝑏𝑟𝑜𝑤𝑠𝑖𝑛𝑔 treatment.

Figure 1 .

 1 Figure 1. Effect of deer herbivory on aboveground (A, B) and belowground (C to F) parameters. Shades of dots and barplots represent the deer browsing treatment with: light grey = no browsing (deer absent), grey = intermediate (deer present for over 70 years but exposed to significant culls) and dark grey = severe browsing (deer present for over 70 years and not exposed to hunting). Small letters on each barplot indicate differences tested by non-parametric post-hoc test. Panel A -Correspondence Analysis on the vegetation data collected at each plot. Dots, squares and lozenges represent the coordinates of the plots from the islands with no browsing, intermediate browsing and severe browsing, respectively. Arrows indicate the species contributions to axes (one arrow per species). Plant species are classified according to their functional group; Panel B -Community Weighted Mean (CWM) of C:N ratio of the plant community; Panel C -Soil bulk density; Panel D -Soil pH; Panel E -Soil C:N ratio; Panel F -Organic horizon depth.

Figure 2 .

 2 Figure 2. Decomposition rate of the plant community litter among deer browsing categories for mass (top), carbon (middle) and nitrogen (bottom) in fine-mesh litterbags in the translocation experiment. Shades of barplots represent the deer browsing intensity with: light grey = no browsing (deer absent), grey = intermediate (deer present for over 70 years but exposed to significant culls) and dark grey = severe browsing (deer present for over 70 years but not exposed to hunting, highest deer density). Asterisks indicate estimates significantly different from zero with *<0.05, ** <0.01, ***<0.001. Fine letters in each bar plots indicate differences tested by post-hoc test. Panel A, Panel E and Panel I represent mass, carbon and nitrogen loss after one year among treatments respectively with bars grouped according to litter origin and shades corresponding to the category of deer browsing of the location where the litter bags were placed. Panel B-D, F-H and J-L represent the parameter estimates (± SE) calculated using the Decomposer Ability Regression Test proposed by Keiser et al. (2014).

Figure 3 .

 3 Figure 3. Linear regression of carbon (left) and nitrogen (right) loss variation with plant C:N Community Weighted Mean. Shades of dots represent the deer browsing intensity on the island where the litter came from, with: light grey = no browsing (deer absent), grey = intermediate (deer present for over 70 years but exposed to significant culls) and dark grey = severe browsing (deer present for over 70 years but not exposed to hunting, highest deer density). The shapes of the symbols refer to the browsing category of the island where we placed the litterbags. Details on regressions models are given in Appendix S5.

Figure 4 .

 4 Figure 4. Mass, carbon and nitrogen loss in Picea sitchensis litter with and without the addition of feces and in feces alone, in relation to browsing treatment in fine-mesh litterbags. Shades of barplots refer to the deer browsing category of the place of decomposition with: light grey = no browsing (deer absent), grey = intermediate (deer present for over 70 years but exposed to significant culls) and dark grey = severe deer browsing (deer present for over 70 years but not exposed to hunting, highest deer density). Panel A -Mass loss; Panel B -Carbon loss; Panel C -Nitrogen loss. Small letters above each barplot indicate differences tested by post-hoc test.

Acknowledgments

This study is part of the long-term research produced on Haida Gwaii by the Research Group on Introduced Species (RGIS). We want to thank Catch Catomeris, Maria Continentino, Yonadav Anbar, Barb Roswell and Max Bullock for their support in the field. This research was financially supported by the France Canada Research Fund (FCRF), University of Rennes 1 ("Défis scientifiques émergents"), the French Embassy in Canada, the French consulate in Vancouver and the Mitacs Globalink Research Award. The Research Group on Introduced Species provided financial and logistic support. The Laskeek Bay Conservation Society provided logistic support as did many members of the Haida Gwaii communities. We also acknowledge two anonymous reviewers for their useful comments on an earlier version of the manuscript. Statement of authorship: SC and MM contributed equally to this work; SC conceived the ideas and designed the methodology in close interaction with MM; SC, MM, JS and J-LM collected the data; MM and SC analyzed the data; SC and MM led the writing of the manuscript. All authors contributed critically to the drafts and gave final approval for publication.

Accepted Article

This article is protected by copyright. All rights reserved Wardle, D. A., K. I. Bonner, and G. M. Barker. 2002. Linkages between plant litter decomposition, litter quality, and vegetation responses to herbivores. Functional Ecology 16:585-595. Wheeler, B. 2010. Package "lmPerm." Accepted Article

Supporting Information

Additional supporting information may be found online at: [link to be added in production].


Nathan Jeger-Madiot
email: nathan.jeger-madiot@espci.fr

Lousineh Arakelian
email: lousineh.arakelian@aphp.fr

Niclas Setterblad

Patrick Bruneval

Mauricio Hoyos

Jérôme Larghero

Jean-Luc Aider

Self-organization and culture of Mesenchymal Stromal Cell spheroids in acoustic levitation

Keywords: Multi-trap acoustofluidic device, Cell culture in acoustic levitation, Scaffold-free 3D culture, Mesenchymal Stromal Cells, Spheroids

In recent years, 3D cell culture models such as spheroid or organoid technologies have known important developments. Many studies have shown that 3D cultures exhibit better biomimetic properties compared to 2D cultures. These properties are important for in-vitro modeling systems, as well as for in-vivo cell therapies and tissue engineering approaches. A reliable use of 3D cellular models still requires standardized protocols with well-controlled and reproducible parameters. To address this challenge, a robust and scaffold-free approach is proposed, which relies on multi-trap acoustic levitation. This technology is successfully applied to Mesenchymal Stromal Cells (MSCs) maintained in acoustic levitation over a 24-hour period. During the culture, MSCs spontaneously self-organized from cell sheets to cell spheroids with a characteristic time of about ten hours. Each acoustofluidic chip could contain up to 30 spheroids in acoustic levitation and four chips could be ran in parallel, leading to the production of 120 spheroids per experiment. Various biological characterizations showed that the cells inside the spheroids were viable, maintained the expression of their cell surface markers and had a higher differentiation capacity compared to standard 2D culture conditions. These results open the path to long-time cell culture in acoustic levitation of cell sheets or spheroids for any type of cells.

Introduction

3D cell cultures have shown closer biological properties to cells in a physiological context than 2D cell cultures [START_REF] Abbott | Biology's new dimension[END_REF][START_REF] Pampaloni | The third dimension bridges the gap between cell culture and live tissue[END_REF]. Therefore they have become one of the main research and development topics in cell biology of recent years [START_REF] Laurent | Convergence of microengineering and cellular self-organization towards functional tissue manufacturing[END_REF]. Nevertheless, the clinical translation of these models has been very limited due to a lack of reproducibility of the results [START_REF] Edmondson | Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors[END_REF]. Most protocols rely on the use of poorly defined hydrogels which affect cells with their chemical and biomechanical properties [START_REF] Kratochvil | Engineered materials for organoid systems[END_REF].

Alternatively, several scaffold-free methods, such as hanging drop [START_REF] Sart | Multiscale cytometry and regulation of 3D cell cultures on a chip[END_REF], non-adhesive surface [START_REF] Fennema | Spheroid culture as a tool for creating 3D complex tissues[END_REF] or rotating bioreactor [START_REF] Ferreira | Design of spherically structured 3D in vitro tumor models-Advances and prospects[END_REF], have shown spheroid fabrication as 3D cell culture models. However, some disadvantages such as variation in spheroid size and shape, or lack of cell-matrix interactions still remain unsolved [START_REF] Cui | Advances in multicellular spheroids formation[END_REF].

Thanks to its excellent biocompatibility and versatility, acoustic approaches are promising tools for contactless manipulation of cells [START_REF] Olofsson | Ultrasonic Based Tissue Modelling and Engineering[END_REF]. Besides the various applications of cell focusing or separation [START_REF] Laurell | Chip integrated strategies for acoustic separation and manipulation of cells and particles[END_REF], acoustic methods have been used for tissue engineering and in vitro techniques [START_REF] Evander | Noninvasive Acoustic Cell Trapping in a Microfluidic Perfusion System for Online Bioassays[END_REF]. Many works [START_REF] Liu | Functional three-dimensional HepG2 aggregate cultures generated from an ultrasound trap: Comparison with HepG2 spheroids[END_REF][START_REF] Hultström | Proliferation and viability of adherent cells manipulated by standing-wave ultrasound in a microfluidic chip[END_REF] studied cell behavior with a short-time ultrasound exposure, classically around one hour and used it to instantaneously shape complex geometries like layer [START_REF] Tait | Engineering multi-layered tissue constructs using acoustic levitation[END_REF], multi-layer [START_REF] Bouyer | A Bio-Acoustic Levitational (BAL) Assembly Method for Engineering of Multilayered, 3D Brain-Like Constructs, Using Human Embryonic Stem Cell Derived Neuro-Progenitors[END_REF] or spheroid [START_REF] Kuznetsova | Multiple three-dimensional mammalian cell aggregates formed away from solid substrata in ultrasound standing waves[END_REF] in order to culture it in usual way.

Here, we report the fabrication and the culture of several spheroids over a 24-hour period in an acoustofluidic chip with a well-controlled cell culture environment. The acoustic forces act on the suspended cells [START_REF] Settnes | Forces acting on a small particle in an acoustical field in a viscous fluid[END_REF] by clustering and trapping them in multiple disk-like layers in acoustic levitation, located at the different pressure nodes of a resonant cylindrical cavity [START_REF] Bruus | Theoretical microfluidics[END_REF]. After a self-organization step, the spheroids could be maintained and cultured in levitation, with a system of perfused culture medium.

We designed an acoustofluidic chip made of a PDMS (Polydimethylsiloxane) body bonded to a microscope cover-glass. We chose PDMS material because it is highly biocompatible and permeable to the air, making it ideal for cell culture [START_REF] Torino | PDMS-Based Microfluidic Devices for Cell Culture[END_REF]. It is also transparent, allowing optical accessibility and is easy to process.

A 2 MHz ultrasonic transducer (Figure 1a) was inserted into the PDMS body, in direct contact with the fluid, closing the cylindrical cavity. The microscope cover-glass, facing the transducer, acts as an efficient acoustic reflector and allows an optical access to visualize the cell clusters. The geometry of our chip has been optimized in order to increase the number of acoustic traps per chip, up to 30, and to be easily parallelizable.

In this study we have used four chips in parallel, leading to the culture of 120 cell spheroids per experiment. To maintain sterility, the chips are single use and disposable.

We first demonstrated the functionality of our system using a suspension of 10µm polystyrene beads close to the targeted cells dimensions. As soon as the acoustic generator was turned on, the particles were trapped in the acoustic nodes as monolayers and remained stable as long as needed. This result is illustrated for a 90 hours experiment on the video movieBeads.mp4.

We then evaluated our system for culturing human cells. MSCs are primary cells that can been isolated from various tissue sources including the umbilical cords, adipose tissue and the bone marrow. They are characterized by their ability to adhere to plastic, by the expression of certain surface markers, as well as their capacity to differentiate into adipocytes, osteoblasts and chondrocytes. These cells are used in many clinical and tissue engineering applications. We decided to use this cell model as a proof of concept for our system. We isolated these cells from human adipose tissue.

Results

hour acoustic levitation of MSCs and selforganization into spheroids

For the acoustic levitation experiments, the cells were injected in the cavity at a concentration of 1.5 million cells/ml and cultured for 24 hours. The setup has been optimized to allow a constant flow of nutrients. The cell culture medium was injected at a rate of 20µL/h, which allowed constant medium renewal without disturbing the cells aggregates in acoustic levitation. The observation of the cells' collective behavior in levitation showed that they first formed monolayers in the same way as for the beads. Then, gradually, during the first few hours, they spontaneously reorganized and formed spheroids of a typical diameter close to 450µm (Figure 1b, movieCellLevi-tationEx1.mp4 and movieCellLevitationEx2.mp4). This behavior was simultaneously observed in all the cell layers in acoustic levitation. This illustrated the self-organizational behavior of living cells, which was not observed with polystyrene beads.

Time evolution of the spheroid sizes

The dynamics of the self-organization into spheroids was studied through image processing of the video time series. The axial R z and radial R r dimensions of the spheroids were measured for every spheroid and averaged at each time step. We show on Figure 1c the time evolution of the mean values < R z > (t) and < R r > (t). One can see that after 12 hours, all the cell sheets have turned into stable spheroids elongated along the radial dimension. The average diameter of the spheroids, obtained on 120 samples, is 400µm.

Viability and proliferation of the MSCs spheroids after the 24 hour acoustic levitation

We evaluated the viability of the cells after a 24 hours culture in acoustic levitation using calcein and propidium iodide, which showed that the majority of the cells were alive at the surface of the spheroid (Figure 1d). However, due to the optical limitation of the confocal microscopy or substrate diffusion, it was not possible to visualize the core of the spheroids with this technique. We therefore evaluated the integrity of the structures by histology. Hematoxylin and eosin (H&E) staining showed that the spheroids were filled structures, with no necrosis in their cores (Figure 1e). Furthermore, the intact form of the cell nuclei indicated that they were not apoptotic. The evaluation of cell proliferation by Ki67 staining showed that most cells were not proliferative but a few cells still stained positive, despite being in levitation (Figure 1f). These results indicate a behavior of the cells inside the spheroids close to that of cells within tissues, which are mainly quiescent while only a few cells proliferate [START_REF] Langhans | Three-Dimensional in Vitro Cell Culture Models in Drug Discovery and Drug Repositioning[END_REF]. The whole time-lapse is available on the movieCellLevitationEx1.mp4. At the beginning, the cells were trapped in monolayers. Then, the cells sheets contracted and the cells self-organized to reach a stable spheroid shape. (c) Time-evolution of the axial Rz and the radial Rr dimensions study of the cells aggregates, averaged over 76 individuals, computed from the time-lapse observations. At t = 0h, widths Rr ranged from 0,75 to 1,5 mm depending on the aggregate location. However, all the heights were similar because of the monolayer shape. After 15h of levitation, every MSC aggregates have reached a stable spheroid shape (Rz ≈ Rr). (d) Confocal and fluorescence imagery of a spheroid just after the acoustic levitation. A live/dead kit was used to label the cells. Because of the limited optical penetration or the substrate diffusion, only the cells at the surface were visible and a majority of them showed a living signal. (e) Histology of MSC spheroids. After a 24h levitation, spheroids were collected from the chip and were immediately fixed in parafolmaldehyde 4% overnight. The spheroids were then embedded in fibrin network (described by the symbol * on the images) for an easy handling of paraffin inclusion and histological preparation. Spheroids were stained with H&E for general structure evaluation and (f) Ki67 for proliferation.

Differentiation of the MSCs spheroids

The spheroids were then reseeded in culture plates (Figure 2a), and their attachment and spreading were observed with a video microscope (Nikon Biostation, moviePh.mp4 and movieGFP.mp4). The spheroids maintained their potential to re-adhere to plastic and MSCs were shown to proliferate and spread out. In these conditions, MSCs expressed the standard surface markers of mesenchymal cells, thus showing that levitation did not alter these properties (Figure 2b). Furthermore, differentiation into adipocytes after attachment to plastic showed that the cells closest to the center of the spheroid had the highest potential of differentiation and lipid accumulation (Figure 2c). The number of adipocytes observed with the spheroid was much higher than the control condition in 2D culture. When differentiated along the osteoblastic lineage, MSCs cultured in levitation showed higher number and a larger size of osteoblast nodules than in 2D control conditions (Figure 2c), indicating that the physical effects induced by levitation increased the potential of osteoblastic differentiation.

Discussion

We hereby demonstrate the development of a robust and reproducible cell culture system based on acoustic levitation using a PDMS chip and an ultrasound transducer, with the possibility of a constant flow, which allows culture medium renewal. Even though previous studies have shown the possibility of using acoustic levitation for cell sheet and aggregate forma-tion, these studies were limited to a very short treatment period usually less than one hour [START_REF] Olofsson | Ultrasonic Based Tissue Modelling and Engineering[END_REF]. Only a few studies pushed the culture time beyond 24 hours. However, either the device was limited to a single aggregate [START_REF] Li | Application of an acoustofluidic perfusion bioreactor for cartilage tissue engineering[END_REF] or the method produced small aggregates with no levitation (< 150µm) [START_REF] Christakou | Ultrasonic three-dimensional on-chip cell culture for dynamic studies of tumor immune surveillance by natural killer cells[END_REF]. Thanks to its versatility and ease of implementation, our cell culture in multi-trap acoustic levitation (CCMAL) device allows the production and the culture of hundreds of large spheroids with a reproducible size. The CCMAL setup can be easily scaled up to culture thousands of spheroids in acoustic levitation in parallel. It is well adapted for MSC cultures, preserving their viability, proliferation and characteristic surface markers. It also increases their potential of differentiation. It has been previously shown that high density of MSCs in 2D culture, as well as spheroid formation in low attachment dishes [START_REF] Yamaguchi | Mesenchymal stem cell spheroids exhibit enhanced invitro and in-vivo osteoregenerative potential[END_REF], or the application of ultrasound [START_REF] Ebisawa | Ultrasound enhances transforming growth factormediated chondrocyte differentiation of human mesenchymal stem cells[END_REF] enhances their osteoblastic potential. In our system, the rapid formation of spheroids mimics both high confluency of 2D cultures and spheroid formation owing to the acoustic confinement (aggregation induced by the axial and transverse components of the ARF). This approach opens the path to a better understanding of these phenomena and application to many other different types of cells. It also demonstrates the use of acoustic levitation as a new method of long-term, reproducible cell culture process for scaffold-free tissue engineering purposes such as organoid formation and multicellular co-cultures.

Materials and methods

Acoustofluidic setup

For experiment, we ran four cell culture in multi-trap acoustic levitation (CCMAL) devices in parallel in order to increase the number of spheroids and maximize the cell preparation. One has to control the acoustic levitation process, the optical observation, the fluid perfusion as well as the biological part from the cell preparation to the post-levitation analysis and the collect of all samples.

The ultrasonic waves were generated by a transducer driven by an arbitrary waveform generator (Handyscope HS5 from TiePie Engineering™) monitored by a computer. Each output of the signal generators supplied two ultrasonic transducers (2 MHz SignalProcessing™) with a sinusoidal waveform of amplitude 7V pp and frequency 2.15MHz. The wave parameters were chosen to optimize the levitation process while avoiding undesired phenomena like acoustic streaming.

The chip is composed by a PDMS (Polydimethylsiloxane) body bonded on microscope cover-glass and a mono-element ultrasonic transducer. The geometry of the PDMS chip is designed to allow the insertion of the ultrasonic transducer inside the PDMS body. The transducer closed the acoustofluidic cavity. One side of the resonant cavity is ultrasound emitter, in contact with the fluid, avoiding acoustic losses through various interfaces, while on the other side the microscope cover-glass acts as a powerful acoustic reflector and allows an optical access. The overall acoustic resonant cavity is simple and efficient.

To shape the PDMS body, we use a negative mold made with a 3D printer (3D Form 2 from Formlabs company). Then the PDMS (RTV 615) is poured in and put in the oven at 70°C for 24h. After unmolding the solid PDMS body is bounded on a cover-glass with a plasma cleaner.

Because of this fabrication process, the geometry of the chip is highly versatile. We settled on two geometries. The first chip (see Figure 3a) includes the levitation cavity with 11 nodes, a channel and a simple bubble trap. Our manufacture process makes the sidewall perfectly transparent to visible optical waves and allows the observation from the side of the acoustic levitation process over the entire cavity. To increase the spheroid number from 11 up to 27 we designed a second chip.

The geometry (see Figure 3b) consists in a cylindrical well of diameter D = 5mm and height 10mm. The optical access is still possible but with a lower quality. To monitor the formation of the cells sheets and the spheroids self-organization from the side view, we used four usb video microscopes (Edge and AM 2111 from Dinolite company).

The culture medium was continuously perfused in the chip with a syringe pump (Pump 11 Elite from Harvard Apparatus™) in a withdrawal mode with a flowrate of 20µL/h small enough to avoid any perturbations inside the acoustofluidic cavity.

Image processing for the study of the spheroid size

In order to characterize the time evolution of the sizes of the spheroids, we developed a Matlab image analysis code. It works automatically with a few user-defined parameters. In particu-lar, we manually defined the regions of interest (ROIs) for each spheroid. Then an ellipsoid is fitted on the binarized image. The section of the original image passing through the ellipse center is selected in the axial and radial directions. We stored the width of the 70% thresholded curves of the X and Y sections (See movieImageProcessing.mp4). For the whole image sequence, ROIs were defined automatically from the ellipsoid characteristics of the previous image. We obtained the time evolution of the axial and radial dimensions of the spheroid with a one-minute time step. We finally applied a moving average of 30 points to smooth the signal fluctuations. Afterwards, the average and the standard-deviation were computed.

MSC isolation and characterization

MSCs were isolated from adipose tissue of the thigh of a healthy donor, after a signed consent, according to the French regulations. MSCs were obtained after the digestion of adipose tissue with collagenase NB6 (Serva electrophoresis). The adipose tissue was placed in 40ml of collagenase NB6 diluted in α-MEM medium at a final concentration of 5µg/ml in a 50ml conical tube (Falcon, Dutscher). After a 2 hour incubation at 37°C, the digested tissue was filtered through a cell strain with pores of 100µm to remove the remaining tissue. The strained cells were then centrifuged and plated in a cell factory (Thermofisher™, Nunc™) for expanding in MSC culture medium composed of α-MEM (Gibco™) supplemented with 10% fetal bovine serum (FBS, Biowest) and 1% antibiotic / antimycotic mix (Anti-Anti 100x, Gibco™).

At the second passage, cells were characterized by flow cytometry using a panel of MSC positive markers including CD29-PE (MACS Miltenyi), CD73-PE (BD), CD90-FITC (BD), CD105-PE (BD), and negative markers including CD31-FITC (BD) and CD34-APC (BD). Unstained cells and cells stained with control isotypes have been used as negative control.

MSC characterization after acoustic levitation

After 24h of acoustic levitation, the spheroids were collected and immediately fixed overnight in paraformaldehyde 4% diluted in PBS. They were then embedded in a fibrin gel to facilitate manipulation for preparing samples. The fibrin gel containing the spheroids was included in paraffin and 5µm thick sections were cut. The slices were stained with H&E for general structure. Ki67 immunohistochemistry was performed with anti-ki67 antibody (Agilent-Dako) 3-step immunoperoxidase technique.

MSC viability and adherence after acoustic levitation

To evaluate the capacity of MSC spheroids, which also shows their viability, they were placed in 24 well dishes and were observed during 24h in an Incuyte. Cell viability was further evaluated with a LIVE/DEAD™Viability/Cytotoxicity Kit (Invitrogen). The components (calcein and propidium iodide) were diluted in PBS according to the provided instructions of the kit and the spheroids were incubated in the solution for 30 mins in an Ibidi™dish prior to observation with a confocal microscope (Zeiss LSM 780 AiryScan).

Evaluation of MSC surface markers and potential of differentiation

After 24h of acoustic levitation, MSC spheroids were collected and reseeded in 75cm 2 for expanding in MSC culture medium. About a week later, cells were detached and evaluated by flow cytometry using a panel of MSC positive and negative markers including CD31, CD34, CD29, CD73, CD90 and CD105. Data were acquired and analyzed with an Attune flow cytometer (Ther-mofisher™).

The potential of differentiation of the aggregates into adipocytes and osteoblasts was also evaluated. For this purpose, the spheroids were collected and reseeded in a 24 wells plate at a density of one spheroid / well in MSC expansion medium. A 2D culture of cells at the same passage that had not undergone levitation was also used as control.

After allowing the cells to adhere and expand for three days, the cell culture medium was replaced by adipocyte differentiation medium or osteoblast differentiation medium (StemX-Vivo™, R&D systems). MSC expansion medium was also used as the control condition. After a two-weeks culture, cells were fixed with 4% paraformaldehyde. Adipocyte differentiation was permeabilized with isopropanol for 5 mins and the lipid vesicles were stained with oil red-O (Sigma-Aldrich). Osteoblasts were stained with alizarin red (Sigma-Aldrich).

Supplementary informations

Acoustofluidic principles

The acoustic levitation describes the fact that an object is suspended in a medium by the action of the Acoustic Radiation Force (ARF) against the gravity. The ARF results from the time-averaged acoustic quantities from scattering of the acoustic waves on the objects. The ARF is explained by the second order perturbation theory of the Navier-stokes equation. The first analytical models of the ARF applied to particles date back in 1934 and make strong simplifying hypothesis like considering incompressible spheres [START_REF] Karlsen | Forces acting on a small particle in an acoustical field in a thermoviscous fluid[END_REF]. Then, the compressibility was taken into account by the Yosioka model [START_REF] Hasegawa | Acoustic radiation force on fused silica spheres, and intensity determination[END_REF] but with sound field assumptions (considering a plane progressive or a standing-plane wave). Gor'kov [START_REF] Gorkov | On the forces acting on a small particle in an acoustic field in an ideal fluid[END_REF] summarized and generalized the previous models with the following assumptions: the surrounding fluid is inviscid and the particles are much smaller than the acoustic wavelength λ.

In our experiments we used the axial and the transverse components of the ARF: the axial component moves the cells toward the pressure nodes, while the transverse components forces the cells to gather at the location of maximum acoustic energy, roughly at the center of the cylindrical cavity. The location and strength of the acoustic traps can be adjusted by changing the acoustic frequency f ac , as shown by [START_REF] Dron | Varying the agglomeration position of particles in a micro-channel using Acoustic Radiation Force beyond the resonance condition[END_REF]. The acoustic levitation process is mainly due to the primary ARF given by the equation [START_REF] Abbott | Biology's new dimension[END_REF].

F ac = π 4 < E ac > kd 3 p F Y sin(2kz) e z (1)
with < E ac > for time-average of the acoustic energy density, k = 2π λ the wave number of the acoustic plane wave with frequency f, c m the acoustic celerity in the medium, d p the particle or cell diameter, and F Y the contrast factor. The z variable represents the axial position. The cells are affected by the primary ARF because of their acoustic contrast with the suspending medium quantified by the acoustic contrast factor F Y :

F Y = 1 + 2 3 (1 -ρm ρc ) 2 + ρm ρc - ρ m c 2 f 3ρ c c 2 c (2)
with c p the acoustic celerity in the cell material, c m the acoustic celerity in the medium, ρ c the cell density and ρ m the medium density.

The amplitude of the primary ARF typically ranges between 10 -14 and 10 -12 N (here for a 5V pp input signal, the force is about 5 × 10 -11 N).

In general, for cells and micro-organisms like bacteria, the contrast factor is positive thus they move toward the pressure nodes. The closer the cells go near the theoretical node, and the weaker the axial primary ARF will be until cancelling itself when reaching the pressure node. From this observation, the transverse ARF becomes significant and is responsible of the cell aggregation in the nodal plane. For a radially symmetric acoustic wave, the transverse ARF can be described in the nodal plane with the equation equation ( 3)

F T = d 3 p 3(ρ c -ρ m ) ρ m + 2ρ c ∇ < E ac > (3)
In this work we use a cylindrical transducer. The radial acoustic energy gradient is directed toward the axis center, where the acoustic energy is the highest. This region can be considered as an acoustic trap where any cells with a positive contrast factor, as well as most living micro-organisms, even motile like bacteria [START_REF] Gutiérrez-Ramos | Induced clustering of Escherichia coli by acoustic fields[END_REF], can be trapped and contained within the acoustic force field [START_REF] Friend | Microscale acoustofluidics: Microfluidics driven via acoustics and ultrasonics[END_REF].

A supplementary short-range force, called Bjerkness or Secondary Radiation Force (SRF), attracts cells close to each other until clustering. This is an inter-cells force due to the scattered acoustic field by the other cells. When cells are lined up perpendicular to the wave propagation (located in the nodal plane for instance) the force is attractive and can be expressed by equation ( 4) with v ac the acoustic velocity at the plane position of the cells and d inter the distance inter-cells. The combination of these forces allows the creation of layers of cells at every pressure node. In this work we used a robust approach to obtain efficient levitation and aggregation, which consists in generating pseudo standing waves in a resonant cavity. Indeed, to maximize the emitted energy, the acoustic wave is confined between the transducer and the reflector. To obtain constructive interferences between the emitted and the reflected wave, the height H of the cavity must be equal to a multiple of the half-wavelength H = n λac 2 .

Note

The article layout was adapted from the latex template for GigaScience Journal Manuscript Soumissions under the Creative Commons CC BY 4.0. The authors of this template are OUP and Overleaf. https://www.overleaf.com/latex/templates/ template-for-gigascience-journal-manuscript-submissions/ shgtrssvbjhs

Figure 1 .

 1 Figure 1. Self-organization of MSCs cells sheets into spheroid shapes and biological viability. (a) Presentation of the experimental setup. A computer drives the wave generation and the acquisition of images. The transducer converts the electrical energy into acoustic energy inside the chip cavity and produced the acoustic levitation of cells over multiple acoustic pressure nodes. A USB digital microscope took side-view pictures and time-lapses of the cell sheets in levitation and selforganization into spheroids. A syringe pump allows a continuous perfusion of the cell medium during the 24h acoustic manipulation. The whole setup, apart from the computer and the generator, was put in an incubator for a well-controlled cell culture environment. (b) Time lapse snapshots of the cell self-organization.The whole time-lapse is available on the movieCellLevitationEx1.mp4. At the beginning, the cells were trapped in monolayers. Then, the cells sheets contracted and the cells self-organized to reach a stable spheroid shape. (c) Time-evolution of the axial Rz and the radial Rr dimensions study of the cells aggregates, averaged over 76 individuals, computed from the time-lapse observations. At t = 0h, widths Rr ranged from 0,75 to 1,5 mm depending on the aggregate location. However, all the heights were similar because of the monolayer shape. After 15h of levitation, every MSC aggregates have reached a stable spheroid shape (Rz ≈ Rr). (d) Confocal and fluorescence imagery of a spheroid just after the acoustic levitation. A live/dead kit was used to label the cells. Because of the limited optical penetration or the substrate diffusion, only the cells at the surface were visible and a majority of them showed a living signal. (e) Histology of MSC spheroids. After a 24h levitation, spheroids were collected from the chip and were immediately fixed in parafolmaldehyde 4% overnight. The spheroids were then embedded in fibrin network (described by the symbol * on the images) for an easy handling of paraffin inclusion and histological preparation. Spheroids were stained with H&E for general structure evaluation and (f) Ki67 for proliferation.

Figure 2 .

 2 Figure 2. Biological evaluation of MSCs in levitation. (a) Pictures of a spheroid recovered after the 24h levitation and reseeded in a classical culture plate. 2h after the levitation, the spheroid began to adhere to the plastic surface. Then, during the 8 following days, the cells spread out onto the plate. (b) Evaluation of MSC cell surface markers by flow cytometry. After a 24h levitation, the spheroids were expanded in polystyrene dishes. A week later, cells were detached, stained and analyzed for the expression of classic MSC markers by flow cytometry. Here the results are expressed as the percentage of positive cells. (c) Differentiation of MSCs into adipocytes and osteoblasts. After a 24h acoustic levitation, spheroids were reseeded in 24 well plates. Once they adhered, they were treated with MSC expansion medium (control), adipocyte or osteoblast differentiation media. A 2D confluent culture of cells, which did not undergo acoustic levitation was used as a control condition. Adipocyte differentiation was revealed by the staining of lipid vesicles by oil-red O and osteoblasts were revealed by alizarin-red staining.

Figure 3 .

 3 Figure 3. (a) Chip geometry 1 (b) Chip geometry 2 (c) Schema of the multilayer acoustic cavity (d) The suspension of cells particles undergoes the Acoustic Radiation Force (ARF). The cells are first focused in the nodal plane and then, trapped by the transverse component of the ARF. The cavity dimensions are adjusted to have multiple clusters. Example of the levitation and aggregation process: (e) Suspended particles are injected in the cavity. Here is 15µm diameter polystyrene beads. (f) Axial acoustic radiation forces are dominating and bring the particles in the pressure nodal plane. (g) Therefore, the transverse forces trap particles in a circular monolayer.

F r = π(ρ c -ρ m ) 2 v 2 ac d 6 c 96ρ m d 4 inter

 64

Acknowledgements

The authors would like to thank Briac Thierry and Françoise Remangeon for the collect and the donation of MSCs, and especially Briac for his support on the ethical rules.

Funding

This work was supported by grants from Région Ile-de-France (DIM ELICIT) and Recherche Hospitalo-Universitaire iLite (Agence Nationale de la Recherche).

The authors benefited from access to the microfluidic plateform of "Institut Pierre-Gilles de Gennes" supported by the program "Investissements d'Avenir" ANR-10-EQPX-34.

Ethical statement

We declare that all methods in the manuscript were carried out in accordance with relevant guidelines and regulations given by Advanced Healthcare Biomaterials. All procedures involving patients were conduct according to the Helsinki Declaration.

Mesenchymal Stromal Cells were isolated from the adipose tissue of a male donor, obtained at Necker-Enfants Malades hospital in Paris, France. The adipose tissue was the surgical leftover and was used for research purposes after a signed consent from the donor and his parents as the legal tutors, according to the French bioethical and medical research regulations. In France, according to the law "loi Jardé" (article L. 1121-1 of the public health code) governing scientific research on human subjects and tissue samples, the surgical leftover can be used for scientific research without the prior approval of an ethical committee.

Author's Contributions

N.J. and L.A designed and performed experiments and wrote the manuscript, N.S. designed and performed experiments, P.B. performed the immunohistochemistry analysis, M.H. supervised the project, J.L. and J-L.A. conceived and supervised the project, and reviewed the manuscript.

Competing Interests

The authors declare that they have no competing interests