Keywords:

Earthworms are known to have a major impact on organic matter dynamics in soils. The precise dynamics of carbon incorporation and/or decomposition in soil under the influence of earthworms still need to be investigated. In a mesocosm experiment, the fate of Ryegrass root and shoot litter was monitored in the soil, in the presence and absence of anecic earthworms Lumbricus terrestris L. Residues were 13 C labelled and deposited onto the soil surface. Incorporation of 13 C in surface casts and in the 0-20 and 40-60 cm soil layers was monitored 1, 2, 4, 8, 24 and 54 weeks after adding labelled litter. Organic carbon content and δ 13 C values were obtained for all samples, allowing the determination of the percentage of carbon derived from labelled litter (Clab). Roots and shoots were incorporated in the 0-20 cm soil layer during the year of experiment, Clab reaching 11.4 % of the soil organic carbon after 54 weeks. On the contrary, no significant contribution from labelled residues was observed in the 40-60 cm layer. Roots decomposed at a slower rate compared to shoots. Litter incorporation was observed in casts from the very first weeks of experiment (Clab from 34.8 to 51.4 % after 2 weeks). In the soil, a significant effect of earthworms on the Clab was detected after 24 weeks. Earthworms accelerated root and shoot decomposition in the soil. They also enhanced, in the presence of shoot residues, the decomposition of the organic matter originally present in the soil. However, after one year, earthworms smoothed the difference between residue types in casts and to a lesser extent in soil, revealing their capacity to enhance the decomposition of both roots and shoots.

Soil organic matter (SOM) is (1) a source of nutrients for soil organisms, (2) a key determinant of soil fertility, structuration and evolution and (3) a potential sink of carbon (C) in a context of CO2 concentration increase in the atmosphere [START_REF] Jobbágy | The vertical distribution of soil carbon and its relation to climate and vegetation[END_REF]. SOM has been widely studied but the process of its incorporation and dynamics in soils remains unclear due to its complexity [START_REF] Stockmann | The knowns, known unknowns and unknowns of sequestration of soil organic carbon[END_REF]. The main source of SOM is plant residues, which encompass plant above-ground parts (dead leaves and shoots) deposited on the soil surface and below-ground parts (dead roots and rhizodeposits) within the soil [START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF]. Residues can either be mineralized, releasing CO2 to the atmosphere, or incorporated into soil in the form of organic compounds. After incorporation, these compounds can be mineralized, transferred in deeper layers and/or stored in soil.

Residue decomposition and incorporation into soil depend on numerous abiotic and biotic factors. Abiotic factors mainly correspond to soil climate (temperature and moisture) and physico-chemical characteristics (texture and clay mineralogy). These factors have a critical influence on organo-mineral interactions, impacting organic matter decomposition and accumulation in soil. Biotic factors encompass residue quality, activity of soil fauna and microorganisms [START_REF] Cortez | Field decomposition of leaf litters: relationships between decomposition rates and soil moisture, soil temperature and earthworm activity[END_REF][START_REF] Oades | The retention of organic matter in soils[END_REF]. Soil microorganisms, which comprise, among others, bacteria and fungi, mediate the processes of organic compound mineralization and transformation in soil. Thus, they have a direct implication on residue decomposition [START_REF] Kuzyakov | Microbial hotspots and hot moments in soil: Concept & review[END_REF]. Under relatively constant climatic and edaphic conditions, abiotic factors often influence litter decomposition at large time and space scales, while biotic factors act at shorter time and space scales [START_REF] Lavelle | A Hierarchical model for decomposition in terrestrial ecosystems: application to soils of the humid tropics[END_REF][START_REF] Swift | Decomposition in terrestrial ecosystems[END_REF], Residue quality (i.e. physico-chemical features including chemical composition, anatomical characteristics, etc.) influences their decomposition by macro and microorganisms (Lavelle et al., 1993). While above-ground biomass has long been considered as the main source for SOM, roots are currently recognized as essential contributors to stable organic C. The relative contribution from root and shoot litter to SOM has been the subject of many studies during the last decades [START_REF] Balesdent | Major contribution of roots to soil carbon storage inferred from maize cultivated soils[END_REF][START_REF] Comeau | Carbon input from 13C-labeled crops in four soil organic matter fractions[END_REF][START_REF] Gale | Carbon Dynamics of Surface Residue-and Root-derived Organic Matter under Simulated No-till1Reference to trade names and companies is made for information purposes only and does not imply endorsement by the USDA[END_REF][START_REF] Lu | Carbon dynamics of rhizodeposits, root-and shoot-residues in a rice soil[END_REF][START_REF] Mambelli | Relative contribution of foliar and fine root pine litter to the molecular composition of soil organic matter after in situ degradation[END_REF][START_REF] Puget | Short-Term Dynamics of Root-and Shoot-Derived Carbon from a Leguminous Green Manure[END_REF][START_REF] Rasse | Is soil carbon mostly root carbon? Mechanisms for a specific stabilisation[END_REF] and it is now recognized that roots decompose at a slower rate compared to shoots. However, the reasons explaining the slower decomposition of roots and its higher contribution to soil carbon pool remain debated. Are they driven by the chemical composition, physical protection and/or physico-chemical protection of roots in soils [START_REF] Rasse | Is soil carbon mostly root carbon? Mechanisms for a specific stabilisation[END_REF]?

The contribution from these 3 factors on C dynamics is particularly dependent on residue location in soil (Coppens et al., 2006a, b;[START_REF] Tahir | In situ roots decompose faster than shoots left on the soil surface under subtropical no-till conditions[END_REF]. For example, Coppens et al. (2006a) found that, after 9 weeks of experiment, new organic C storage in soil was increased when rapeseed residues were deposited onto the soil surface compared to residues incorporated in the 0-10 cm layer. In order to investigate the impact of the chemical compositions of residues, the latter must be placed in the same conditions [START_REF] Rasse | Is soil carbon mostly root carbon? Mechanisms for a specific stabilisation[END_REF]. Thus, in the present study, both roots and shoots were deposited onto the soil surface.

Among biotic factors, earthworms play a key role in soil structuration, residue decomposition and C cycling [START_REF] Blouin | A review of earthworm impact on soil function and ecosystem services[END_REF][START_REF] Bossuyt | Protection of soil carbon by microaggregates within earthworm casts[END_REF]. Earthworms feed on both mineral (soil) and organic matter (litter, humus and microorganism), in different proportion depending on their ecological category. For example, anecic earthworms, which represent the dominant earthworm biomass in temperate regions, feed on litter deposited on the soil surface and transfer it along vertical burrows which can reach 1-2 meters [START_REF] Lee | Earthworms: their ecology and relationships with soils and land use[END_REF]. During ingestion, residues are fragmented and the preexisting soil microstructures destroyed.

Mineral and organic elements are mixed, complexed with mucus and released in the form of organo-mineral aggregates called casts [START_REF] Lee | Earthworms: their ecology and relationships with soils and land use[END_REF][START_REF] Six | A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics[END_REF]. This process creates nuclei for the formation of organo-mineral aggregates in soil. Thus, earthworms promote the formation of biogenic macroaggregates [START_REF] Bossuyt | Protection of soil carbon by microaggregates within earthworm casts[END_REF], which are generally more stable than non-biogenic aggregates [START_REF] Six | A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics[END_REF][START_REF] Zangerlé | Do earthworms and roots cooperate to build soil macroaggregates? A microcosm experiment[END_REF]. Earthworms are highly mobile in soil and induce heterogeneous resource distribution [START_REF] Decaëns | Macroecological patterns in soil communities[END_REF].

As the ingested residues contain higher concentration of organic C than soil, they modify the incorporation and the stock of C in aggregates [START_REF] Arai | Changes in soil aggregate carbon dynamics under no-tillage with respect to earthworm biomass revealed by radiocarbon analysis[END_REF][START_REF] Fonte | Interactive effects of plants and earthworms on the physical stabilization of soil organic matter in aggregates[END_REF][START_REF] Fonte | Influence of earthworm activity on aggregate-associated carbon and nitrogen dynamics differs with agroecosystem management[END_REF][START_REF] Hong | How do earthworms influence organic matter quantity and quality in tropical soils?[END_REF] and along the soil profile [START_REF] Jégou | Burrowing activity of the earthworms Lumbricus terrestris and Aporrectodea giardi and consequences on C transfers in soil[END_REF]. The net impact of earthworms on soil C strongly varies depending on the studied spatial and time scale. During gut transit and in fresh casts, mineralization of organic C is accelerated. Indeed, in earthworm gut, the physical protections of the organic matter originally present in the soil are broken and plant residues are fragmented [START_REF] Lavelle | Small-scale and large-scale effects of endogeic earthworms on soil organic matter dynamics in soils of the humid tropics[END_REF]. Moreover, the presence of water and intestinal mucus creates conditions favorable to mineralization by the microorganisms present in the earthworm gut and fresh casts [START_REF] Brown | Regulation of soil organic matter dynamics and microbial activityin the drilosphere and the role of interactionswith other edaphic functional domains[END_REF][START_REF] Drake | As the worm turns: The earthworm gut as a transient habitat for soil microbial biomes[END_REF][START_REF] Martin | Les mucus intestinaux de ver de terre moteur de leurs interactions avec la microflore[END_REF]Vidal et al., 2016a). At month or year scale, drying and ageing casts, lead to C stabilization [START_REF] Brown | Regulation of soil organic matter dynamics and microbial activityin the drilosphere and the role of interactionswith other edaphic functional domains[END_REF][START_REF] Lavelle | Small-scale and large-scale effects of endogeic earthworms on soil organic matter dynamics in soils of the humid tropics[END_REF][START_REF] Martin | Short-and long-term effects of the endogeic earthworm Millsonia anomala (Omodeo) (Megascolecidae, Oligochaeta) of tropical savannas, on soil organic matter[END_REF]. Residue decomposition rate by earthworms also depends on residue palatability [START_REF] Cortez | C and N transfer in soil with or without earthworms fed with 14C-and 15N-labelled wheat straw[END_REF]. For example, it has generally been observed that earthworms fed mainly on shoots rather than roots [START_REF] Bouché | Fonction des lombriciens; II: Recherches méthodologiques pour l'analyse du sol ingéré (étude du peuplement de la station RCP-165/PBI)[END_REF][START_REF] Curry | The feeding ecology of earthworms -A review[END_REF]. However, little is known about the incorporation and decomposition of root residues by earthworms in soil [START_REF] Curry | The feeding ecology of earthworms -A review[END_REF][START_REF] Zangerlé | Do earthworms and roots cooperate to build soil macroaggregates? A microcosm experiment[END_REF].

Artificial isotopic labelling of residues allows precise monitoring of the residues introduced (at a given date and place) and distinguishing the added organic matter from that initially present in the soil [START_REF] Brüggemann | Carbon allocation and carbon isotope fluxes in the plant-soil-atmosphere continuum: a review[END_REF][START_REF] Comeau | Carbon input from 13C-labeled crops in four soil organic matter fractions[END_REF][START_REF] Fahey | Earthworm effects on the incorporation of litter C and N into soil organic matter in a sugar maple forest[END_REF][START_REF] Klumpp | Long-term steady state 13 C labelling to investigate soil carbon turnover in grasslands[END_REF][START_REF] Thompson | Pulse-labelling a cover crop with 13 C to follow its decomposition in soil under field conditions[END_REF]. Studies using artificial labelling have focused on (1) the impact of residue quality (roots vs. shoots or plant species) on organic C incorporation and decomposition into soil [START_REF] Blair | Decomposition of 13C and 15N labelled plant residue materials in two different soil types and its impact on soil carbon, nitrogen, aggregate stability, and aggregate formation[END_REF][START_REF] Comeau | Carbon input from 13C-labeled crops in four soil organic matter fractions[END_REF][START_REF] Mambelli | Relative contribution of foliar and fine root pine litter to the molecular composition of soil organic matter after in situ degradation[END_REF][START_REF] Rubino | Carbon input belowground is the major C flux contributing to leaf litter mass loss: Evidences from a 13 C labelled-leaf litter experiment[END_REF][START_REF] Williams | Distribution and fate of 13C-labeled root and straw residues from ryegrass and crimson clover in soil under western Oregon field conditions[END_REF] or (2) the influence of earthworms on shoot decomposition and incorporation into soil and the consequences on soil C storage [START_REF] Fahey | Earthworm effects on the incorporation of litter C and N into soil organic matter in a sugar maple forest[END_REF][START_REF] Fonte | Influence of earthworm activity on aggregate-associated carbon and nitrogen dynamics differs with agroecosystem management[END_REF][START_REF] Stromberger | Distinct microbial and faunal communities and translocated carbon in Lumbricus terrestris drilospheres[END_REF]. The above mentioned studies have proven that, taken separately, these two biotic factors (earthworm and residue quality) have a crucial impact on soil C cycling. However, is the diverging fate of root and shoot residues in soil modified in the presence of earthworms? In this study, a one year mesocosm experiment was set up to monitor the fate of root and shoot residues in the presence and absence of earthworms. Residues were artificially 13 C labelled and deposited onto the soil surface.

Materials and Methods

Soil characteristics and mesocosm experiment

The surface layer of a soil was collected on a permanent grassland, largely dominated by Ryegrass, in the North of France (Aux-Marais, Oise). The mean annual precipitation and temperature in the region were 650 mm and 11°C, repectively. The soil collected is a loamysand soil with characteristics presented in Table 1. The experiment was performed in six PVC containers (length -80 cm, diameter -40 cm) filled with ca. 75 L of soil. It took place in a greenhouse, under controlled conditions (air conditioning), with natural light and the soil was kept at approximately 13°C and 23 % of humidity. The soil was homogenized and sieved at 4 mm in order to obtain homogenous columns of soil. Before starting the experiment, the soil containers were pre-incubated during 6 months in the same conditions as during the experiment. This allowed passing the bacterial flush induced by drying-rewetting of soil [START_REF] Van Gestel | Microbial biomass responses to soil drying and rewetting: The fate of fast-and slow-growing microorganisms in soils from different climates[END_REF][START_REF] Franzluebbers | Flush of carbon dioxide following rewetting of dried soil relates to active organic pools[END_REF] and/or soil physical disturbance during sieving [START_REF] Datta | Effect of soil sieving on respiration induced by low-molecular-weight substrates[END_REF]. The soil macrofauna was first removed manually during sieving and remaining earthworms were extracted from the soil using an electrical device during preincubation, as described in [START_REF] Weyers | Construction of an electrical device for sampling earthworm populations in the field[END_REF]. Each container was weekly vaporized with water (1L/week) and seedlings or mosses in mesocosms were eliminated.

The six treatments were identified as (1) Root-E for the mesocosm with root residues and earthworms (2) Root-NE for the mesocosm with root residues and without earthworms (3) Shoot-E, for the mesocosm with shoot and earthworms (4) Shoot-NE for the mesocosm with shoot and without earthworms (5) Control-E, for the mesocosm without plant residues and with earthworms and (6) Control-NE for the mesocosm without plant residues and without earthworms (Fig. 1).

Earthworms and root-shoot

The common anecic earthworm Lumbricus terrestris L. was used. Earthworms were provided by the SARL Lombri'carraz (France) and were received at adult stage, with an average weight of 4.5 g. They were pre-conditioned during one week in the soil used for the experiment. At the beginning of the experiment, 6 earthworms per container were deposited on the top of three of the mesocosms. Mean living mass was 26.8 ± 0.5 g per container (around 48 ind./m 2 and 224 g fresh wt/m 2 ). This low density was chosen in accordance to [START_REF] Fründ | Using earthworms as model organisms in the laboratory: Recommendations for experimental implementations[END_REF] review, showing that the typical earthworm density (all species concerned) in temperate pastures was 300-1000 ind./m 2 (50-100 g fresh wt/m 2 ). Thus, as L.terrestris is a territorial earthworm, these choices favored their survival in conditions closest to reality. By the end of the experiment, the soil volume extracted during the year of sampling represents approximatively 6 % of the initial soil volume. Thus, it is assumed that earthworms were not limited in their movement along the experiment.

Plants of Italian Ryegrass (Lolium multiflorum Lam.) were artificially labelled with 13 C at the Atomic Energy and Alternative Energies Commission (CEA) in Cadarache (France). In order to obtain homogeneous material, plants were grown under a controlled and constant 13 CO2 enriched atmosphere. Immediately after 13 C labelling, fresh roots and shoots were separated, dried and chopped. They were chopped during 40 seconds with a laboratory blender (Waring Commercial) in order to obtain residues of few millimeters. The mean δ 13 C values were 1324 ‰ (± 43) and 1632 ‰ (± 16) for the roots and shoots, respectively (Table 2). Few hours after earthworms had penetrated the soil, roots and shoots were similarly applied on the soil surface. Thus, the impact of the chemical composition of roots vs shoots was tested, excluding any influence of their location in the soil. Two mesocosms each contained 250 g of shoots, two additional contained 250 g of roots and the latest two were used as control. [START_REF] Curry | The feeding ecology of earthworms -A review[END_REF] reviewed that L.terrestris could ingest 17 mg of dry matter/g fresh wt/day. However, the quantity of litter ingested can increase when the quality of litter decreases. Thus, 250 g of litter (i.e. around 25 mg of dry matter/g fresh wt/day) was chosen as a good compromise to favor earthworm survival.

Sampling and analyses

Samples were collected at seven time steps: before and 1, 2, 4, 8, 24 and 54 weeks after the addition of roots or shoots and earthworms. At each time step and for each mesocosm, three replicates of soil samples were collected at 0-20 and 40-60 cm with an aluminum auger (height: 100 cm, diameter: 2.5 cm), after putting aside plant residues. Few grams of 3-4 earthworm cast fragments were collected on the soil surface in the mesocosms containing earthworms in order to obtain one composite sample for each time step. During the first 6 months of experiment, most casts were fresh when collected. Casts collected after 54 weeks were starting ageing and drying. The casts collected in the mesocosms Root-E, Shoot-E and

Control-E will be identified as Cast-Root, Cast-Shoot and Cast-Control, respectively. All samples were freeze-dried and ground. After 54 weeks, roots and shoots remaining at the soil surface (i.e. for Root-NE and Shoot-NE) were collected and weighted.

Organic carbon and δ 13 C analyses

Organic C (Corg) and δ 13 C were measured on all soil replicates with Elemental Analysis -Isotope Ratio Mass Spectrometry (EA-IRMS) Vario pyro cube -Micromass Isoprime. The standard gas was calibrated in relation to the international standard (PeeDee Belemnite-PDB). For the casts, the analysis was repeated three times. All samples were decarbonated before the analysis, using the method developed by [START_REF] Harris | Acid fumigation of soils to remove carbonates prior to total organic carbon or Carbon-13 isotopic analysis[END_REF]. Briefly, subsamples of soil (~40 mg) and casts (~15 mg) were placed in open silver capsules (8*5 mm). Samples were placed in a microtiter plate and 15 µL of ultra-pure water were added. They were placed in a vacuum desiccator containing a 100 mL beaker of HCl (12 M). Samples were exposed to HCl vapor during 6 hours and then placed under vacuum, without HCl, to remove HCl potentially trapped in the sample. Samples were then transferred to an oven at 40°C for 15 hours.

Isotopic composition of samples is expressed in δ 13 C relative to the PDB standard (Equation 1):

δ 13 C (‰) = [(Rsample -Rstandard) / Rstandard] * 1000
(1)

Results

Observation of mesocosms during the year of experiment gave first indications on the incorporation of the labelled residues. After 24 weeks of experiment, no more shoot residues were observed in the Shoot-E mesocosm whereas, in the Root-E mesocosm, root residues were still observed on the soil surface, occupying less than 10 % of the surface. After one year of experiment, no more residues were observed at the soil surface in the mesocosm containing earthworms. On the contrary, in the absence of earthworms, after one year, some remaining residues were still visible at the soil surface, for either root or shoot residues. They were roughly separated from the soil and weighted. They accounted for ca. 30 % of the initial weight. After one week, casts were observed in Shoot-E. However, no casts were identified until two weeks in the mesocosm with root residues. In spite of regular removal of seedling and mosses during the experiment, some mosses developed in the Control-NE mesocosm, from 24 weeks until the end of the experiment.

Carbon derived from root and shoot residues

Soil samples

The C derived from labelled litter (Clab) increased continuously in the 0-20 cm soil layer along the year of experiment, reaching between 0.96 and 11.4 % after 54 weeks, for Shoot-E and Root-NE, respectively (Fig. 2A). From the beginning of the experiment to the 8 th week, there were no significant differences between mesocosms, with a mean Clab of 0.40 %. After 24 weeks, Clab was significantly lower for Shoot-E (0.85 %) than Root-E (3.00 %). After 54 weeks, Clab was significantly higher in Root-NE (11.4 %) than in the other mesocosms. Thus, two general trends could be observed after 24 and 54 weeks: (1) Clab in Root-NE and Shoot-NE tended to be higher than Root-E and Shoot-E, respectively and (2) Clab in Root-E and NE tended to be higher than Shoot-E and NE, respectively. Contrary to other mesocosms, Clab in Shoot-E barely varied from 8 th week until the end of experiment, with values of 1.13, 0.85 and 0.96 % after 8, 24 and 54 weeks, respectively. There was no statistically significant differ-ence of Clab between mesocosms in the 40-60 cm soil layer during the year of experiment (Fig. 2B). However, at 54 weeks Clab reached 0.2 % in Root-E and Shoot-E whereas it remained lower than 0.05 % without earthworms.

Earthworm casts

For all the time steps, Clab measured in casts was higher than Clab in soil, with a mean Clab of 37 % with respect to 1.3 % for soils, all time steps and mesocosms included (Fig. 3). Clab in Cast-Shoot reached 34.9 % in average during the first month of experiment. Clab reached a maximum after 8 weeks for shoots (58.1 %) and after 8 and 24 weeks for roots (51.8 %). Clab then decreased during the last six months of experiment, reaching around 12 % for both

Cast-Root and Cast-Shoot after 54 weeks.

Organic carbon content in soil and cast samples

Soil samples

Due to the very low litter C incorporation for the 40-60 cm soil layer, the following of this section is focused on the 0-20 cm layer. In mesocosms containing shoots, with or without earthworms, organic C content tended to be lower than the organic C content before the experiment (RCorg x ≤ 1), for all time steps (Fig. 4 and Table S1). On the contrary, in mesocosms containing roots, with or without earthworms, organic C content tended to be higher than at the beginning of the experiment (RCorg x ≥ 1). RCorg reached 1.2 after 54 weeks in Root-NE.

RCorg was significantly different for Root-E and Shoot-E after 24 weeks (1.1 vs 0.85).This observation was also made between Root-NE and Shoot-NE but to a lesser extent. For example, after 24 weeks, RCorg was 1.1 and 0.89 for Root-NE and Shoot-NE, respectively (Fig. 4).

Earthworm casts

The organic C measured in casts was always higher than the soil organic C measured before the experiment (RCorg >>1) (Fig. 5). For Cast-Control, the RCorg was stable for all the time steps, with a mean value of 1.3. Both Cast-Root and Cast-Shoot, RCorg remained stable at ca. 1.8, during the first month of experiment before increasing to their maximum at 8 weeks and subsequently decreasing until the end of the experiment. When Cast-Root and Cast-Shoot are compared, their RCorg are similar during the first month and it becomes higher for Cast-Root after 8 (3.6 vs 2.7) and 24 (2.8 vs 2.3) weeks of experiments before reaching again similar values after 54 weeks (1.5 and 1.6).

Discussion

The proportion of C derived from the labelled litter into the 0-20 cm soil layer after one year of experiment (Clab between 0.96 and 11.4 % depending on the mesocosm, (Fig. 2A)) is consistent with previous studies [START_REF] Kammer | Mineralisation, leaching and stabilisation of 13$ C-labelled leaf and twig litter in a beech forest soil[END_REF][START_REF] Rubino | Carbon input belowground is the major C flux contributing to leaf litter mass loss: Evidences from a 13 C labelled-leaf litter experiment[END_REF].

Residues placed on the soil surface have either been (1) totally mineralized, (2) left intact at the soil surface and/or (3) transferred to the soil either in form of soluble compounds or as undecomposed fragmented pieces [START_REF] Cortez | Field decomposition of leaf litters: earthworm-microorganism interactions -the ploughing-in effect[END_REF][START_REF] Rubino | Carbon input belowground is the major C flux contributing to leaf litter mass loss: Evidences from a 13 C labelled-leaf litter experiment[END_REF]. Thus, the 13 C signal measured in soil could either derived from the residues themselves (soluble or small fragments) or from microorganisms that have degraded the residues. No significant differences are observed between the mesocosms until the 24 th week of experiment. The absence of significant earthworm impact during the first 8 weeks can be explained by the experimental conditions. Indeed, the low density of earthworms and the localized sampling to preserve soil structure in mesocosms, accounted for the great variability of data and the late significant impact of earthworms. Differences induced by root or shoot residues were not detected until 6 months. This could be explained by the experimental conditions. Indeed, as high volumes of soil were used in the present study, an important quantity of labelled C was needed to reveal a significant difference between samples.

Root vs. shoot effect

For the last two time periods, the Clab of mesocosms containing roots was higher than those with shoots, although the difference was not systematically statistically significant (Fig 2 .A).

The higher contribution to SOM of root compared to shoot residues was also supported by the soil RCorg results after 54 weeks in Root-NE and Shoot-NE (1.20 and 1.05, respectively).

These results are coherent with William et al. (2006) who found, after having incubated roots and shoots of ryegrass in soil for 10 months, a C contribution of 5.5 % and 1.5 %, respectively. Two explanations can be put forward to explain the higher root-derived C input in soil. First, roots could be more transferred from residues into soil (as dissolved OM or small fragments), compared to shoots. However, this explanation is very unlikely, considering the characteristics of roots compared to shoots, as detailed in the second explanation.

Second, root-derived C decomposed at a slower rate than shoot-derived C, as evidenced in numerous studies [START_REF] Beuch | Effect of the Organic Residues of Miscanthus × giganteus on the Soil Organic Matter Level of Arable Soils[END_REF][START_REF] Gale | Carbon Dynamics of Surface Residue-and Root-derived Organic Matter under Simulated No-till1Reference to trade names and companies is made for information purposes only and does not imply endorsement by the USDA[END_REF][START_REF] Scheu | Decomposition of roots and twigs: Effects of wood type (beech and ash), diameter, site of exposure and macrofauna exclusion[END_REF][START_REF] Shi | Decomposition of roots and shoots of perennial grasses and annual barley-separately or in two residue mixes[END_REF][START_REF] Steffens | Translocation of 13Clabeled leaf or root litter carbon of beech (Fagus sylvatica L.) and ash (Fraxinus excelsior L.) during decomposition -A laboratory incubation experiment[END_REF]. Indeed, the higher mineralization of shoots compared to roots can be explained by (1) the higher water soluble C concentration in shoots [START_REF] Beuch | Effect of the Organic Residues of Miscanthus × giganteus on the Soil Organic Matter Level of Arable Soils[END_REF][START_REF] Bird | Fine Roots vs. Needles: A Comparison of 13C and 15N Dynamics in a Ponderosa Pine Forest Soil[END_REF][START_REF] Shi | Decomposition of roots and shoots of perennial grasses and annual barley-separately or in two residue mixes[END_REF], which is rapidly transferred to soil and decomposed by microorganisms [START_REF] Shi | Decomposition of roots and shoots of perennial grasses and annual barley-separately or in two residue mixes[END_REF]; (2) the higher chemical recalcitrance of roots due to higher concentration of resistant molecules such as lignin, tannins and suberins [START_REF] Gleixner | 1.15 -Plant Compounds and Their Turnover and Stabilization as Soil Organic Matter[END_REF][START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF][START_REF] Rasse | Is soil carbon mostly root carbon? Mechanisms for a specific stabilisation[END_REF]. The higher relative abundance of lignin-derived compounds in the roots used in the present study compared to shoots, was confirmed by Vidal et al. (2016b) using molecular characterization and (3) The lower C/N ratio of shoots vs. roots (15 vs. 31 for the litter used in this study -Table 2), which is generally related to an increased rate of litter decomposition (e.g. Nguyen [START_REF] Nguyen Tu | Early degradation of plant alkanes in soils: A litterbag experiment using 13 C-labelled leaves[END_REF][START_REF] Taylor | Nitrogen and lignin content as predictors of litter decay rates: a microcosm test[END_REF][START_REF] Witkamp | Decomposition of leaf litter in relation to environment, microflora, and microbial respiration[END_REF].Thus, the present results highlight the importance of the chemical composition on residue decomposition. It can be suggested that the difference between root and shoot degradation is mainly driven by soluble carbon content at short term (first weeks of experiments), whereas, the chemical recalcitrance of roots probably becomes the dominant factor at longer terms, as suggested in the literature (e.g. [START_REF] Trinsoutrot | Biochemical Quality of Crop Residues and Carbon and Nitrogen Mineralization Kinetics under Nonlimiting Nitrogen[END_REF][START_REF] Machinet | Impact of plant cell wall network on biodegradation in soil: role of lignin composition and phenolic acids in roots from 16 maize genotypes[END_REF].

Effect of earthworms on residue fate 4.2.1. Earthworms tended to accelerate residue incorporation and decomposition

No more residues were observed at the soil surface after the year of experiment in the mesocosm containing earthworms compared to the one without earthworms. Thus, residues had probably been incorporated in soil or casts and/or mineralized. Although absolute quantification was not achieved, it can reasonably be suggested that earthworms accelerated decomposition of residues in soil. Thus, part of the 13 C was probably removed from the system in the form of 13 CO2 (microbial and/or earthworm respiration). Indeed, from 6 months of experimentation, the Clab in soil tended to be lower in the presence of earthworms, for both root and shoot residues (Fig. 2A). This can be explained by the direct impact of earthworms which assimilate and mineralize part of the labelled organic C during the ingestion of residues, as suggested by [START_REF] Daniel | Leaf-litter consumption and assimilation by juveniles of Lumbricus terrestris L. (Oligochaeta, Lumbricidae) under different environmental conditions[END_REF]. Moreover, decomposition and mineralization are governed by microorganisms acting directly on particulate residues, or on soluble compounds in the soil matrix [START_REF] Gaillard | Carbon, nitrogen and microbial gradients induced by plant residues decomposing in soil[END_REF]. In the presence of earthworms, this phenomenon can be amplified as the ingestion of residues by earthworms generally favors microbial activity [START_REF] Amador | Microbiological characterization of the structures built by earthworms and ants in an agricultural field[END_REF][START_REF] Brown | How do earthworms affect microfloral and faunal community diversity?[END_REF][START_REF] Frouz | Microbial properties of soil aggregates created by earthworms and other factors: spherical and prismatic soil aggregates from unreclaimed post-mining sites[END_REF][START_REF] Parle | A Microbiological Study of Earthworm Casts[END_REF]Vidal et al., 2016a).

Castsa key role in residue decomposition

Cast and soil samples presented different trends of organic C incorporation and decomposition (fig. 2.A vs. 3 and fig 4 vs. 5). As expected, the Clab and Corg are higher in casts compared to soil. The organic C content of casts was 1.3 to 3.6 higher compared to the soil. Similarly, [START_REF] Mariani | What happens to earthworm casts in the soil? A field study of carbon and nitrogen dynamics in Neotropical savannahs[END_REF] observed that the C content in anecic casts collected in burrows of a Colombian pasture, was twice higher compared to bulk soil. This can be explained by the feeding behavior of earthworms, especially anecic ones, which incorporate fresh residues and/or organic-rich compounds in casts [START_REF] Decaëns | Carbon and nitrogen dynamics in ageing earthworm casts in grasslands of the eastern plains of Colombia[END_REF][START_REF] Frouz | Microbial properties of soil aggregates created by earthworms and other factors: spherical and prismatic soil aggregates from unreclaimed post-mining sites[END_REF][START_REF] Hong | How do earthworms influence organic matter quantity and quality in tropical soils?[END_REF][START_REF] Zangerlé | Do earthworms and roots cooperate to build soil macroaggregates? A microcosm experiment[END_REF][START_REF] Zhang | Comparison of organic compounds in the particle-size fractions of earthworm casts and surrounding soil in humid Laos[END_REF]. The maximum value for Clab (58.1% in Cast-Shoot, after 8 weeks of incubation) is in agreement with a previous study which reported that half of the C in L.terrestris casts was derived from labelled litter after 246 days of experiment, adding labelled litter every day [START_REF] Jégou | Effects of four ecological categories of earthworms on carbon transfer in soil[END_REF].

A chronology can be proposed, explaining residue incorporation by earthworms in casts (Figure 6), based on Clab and RCorg x results (Fig. 3 and5). After a strong increase within the first week of experiment, both Clab and RCorg in casts remained stable during the four following weeks. In this phase, earthworms have probably combined direct and indirect incorporation of residues. During direct incorporation, residues are ingested and pass through the earthworm gut [START_REF] Shipitalo | Chemistry and micromorphology of aggregation in earthworm casts[END_REF]. Indirect incorporation, also called 'pre-oral litter decomposition', consists in a 'ploughing-in' of residues, i. e. casts are deposited on residues which are not ingested, initiating microbial decomposition [START_REF] Cortez | Field decomposition of leaf litters: earthworm-microorganism interactions -the ploughing-in effect[END_REF]). An increase in Clab and RCorg was observed between 4 and 8 weeks. After few months, earthworms are able to re-ingest their casts in which residues have been partially decomposed by microorganisms [START_REF] Swift | Decomposition in terrestrial ecosystems[END_REF][START_REF] Jiménez | Earthworm communities in native savannas and man-made pastures of the Eastern Plains of Colombia[END_REF][START_REF] Brown | Regulation of soil organic matter dynamics and microbial activityin the drilosphere and the role of interactionswith other edaphic functional domains[END_REF]. Thus, between 4 and 8 weeks of experiment, the 'ploughing-in' and ingestion of undecomposed labelled residues still present at the soil surface were probably associated to the re-ingestion of previously labelled casts (from 1 to 4 weeks), increasing the Clab. During the last 6 months, Clab and RCorg highly decreased, no more litter was present at the surface of the mesocosms containing earthworms and litter, and surface casts were starting ageing and drying. Thus, all the residues have been either incorporated in the soil and casts, or mineralized. The decrease in Clab and RCorg indicated that labelled residues incorporated in casts sampled at 54 weeks have been partially mineralized. This decomposition was enhanced by bacteria and fungi as observed on Cast-Shoot NanoSIMS images after 24 weeks of experiment (Vidal et al., 2016a).

A slight effect in the 40-60 cm soil layer

A slight increase in Clab was observed in the 40-60 cm soil layer during the 54 weeks in the presence of earthworms, although not statistically significant (Fig. 2B). This was observed in other studies which found that below 2 or 5 cm, labelled litter incorporation was not significant, at least for short time scales [START_REF] Jégou | Effects of four ecological categories of earthworms on carbon transfer in soil[END_REF][START_REF] Kammer | Mineralisation, leaching and stabilisation of 13$ C-labelled leaf and twig litter in a beech forest soil[END_REF][START_REF] Rubino | Carbon input belowground is the major C flux contributing to leaf litter mass loss: Evidences from a 13 C labelled-leaf litter experiment[END_REF], mainly due to the limited migration of soluble C in deeper soil layers [START_REF] Thompson | Pulse-labelling a cover crop with 13 C to follow its decomposition in soil under field conditions[END_REF]. However, the burrows of L. terrestris are considered as permanent structures that can reach deep soil layers (>1 m) [START_REF] Jégou | Burrowing activity of the earthworms Lumbricus terrestris and Aporrectodea giardi and consequences on C transfers in soil[END_REF][START_REF] Don | Organic carbon sequestration in earthworm burrows[END_REF]. Thus, earthworms may have contributed to the incorporation of labelled organic compounds into the 40-60 cm soil layer directly, by covering their burrow walls with mucus and belowground casts, both enriched in 13 C [START_REF] Jégou | Burrowing activity of the earthworms Lumbricus terrestris and Aporrectodea giardi and consequences on C transfers in soil[END_REF] or to a lesser extent, indirectly, by creating preferential paths for labelled water soluble C circulation [START_REF] Don | Organic carbon sequestration in earthworm burrows[END_REF]. The Clab increase was probably not significant because earthworm influence is localized on burrow walls and their periphery, and casts [START_REF] Don | Organic carbon sequestration in earthworm burrows[END_REF]. Jegou et al. (2000) showed in an experiment using 13 C labelled litter that below the 2 cm soil layer, the C litter enrichment in the soil surrounding burrows was barely detectable. Thus, the 13 C signal was probably diluted in the soil as earthworm burrows were not sampled separately.

Effect of earthworms on the fate of root vs. shoot

Earthworms minimized the diverging fate of root and shoot residues

In soil samples, the difference in Clab and RCorg between root and shoot mesocosms with earthworms, after 54 weeks, was weaker than in the absence of earthworms (Fig. 2A and3),

underlying their ability to enhance root decomposition, as mentioned by [START_REF] Cortez | Do earthworms eat living roots?[END_REF]. [START_REF] Hopkins | Lumbricus terrestris counteract the effects of modified lignin biosynthesis on the decomposition of tobacco plant residues[END_REF] have compared decomposition of natural tobacco roots and modified tobacco roots with altered lignin structure and composition (i.e. less resistant to decomposition). They found that earthworms reduced the difference between both residue types for C mineralization. Earthworms can enhance the decomposition of recalcitrant residues and smooth the difference between residues of various qualities, probably by facilitating the access of plant residues to microorganisms. Indeed, a mutualistic relation is maintained between bacteria and earthworms in their gut, which enabled the decomposition of highly complex molecules [START_REF] Barois | Changes in respiration rate and some physicochemical properties of a tropical soil during transit through Pontoscolex corethrurus (glossoscolecidae, oligochaeta)[END_REF][START_REF] Trigo | Changes in respiration rate and some physicochemical properties of soil during gut transit through Allolobophora molleri (Lumbricidae, Oligochaeta)[END_REF].

The difference between root and shoot residues was also weak in casts. Cast-Root and Cast-Shoot both exhibited similar evolutionary trends for Clab and RCorg x (Figs. 3 and5).

However, the observation of the mesocosms at the different time steps revealed some differences. After one week of experiment, no cast could be observed on the soil surface in the mesocosm containing roots and the litter appeared unaffected. Moreover, after 24 weeks, while shoots had totally disappeared from the soil surface in Shoot-E, roots were still visible in Root-E. Thus, in our experimental conditions, dead roots in early stage of decomposition seemed less palatable for earthworms than shoots, confirming observations of [START_REF] Zangerlé | Do earthworms and roots cooperate to build soil macroaggregates? A microcosm experiment[END_REF]. [START_REF] Coq | Earthworm activity affects soil aggregation and organic matter dynamics according to the quality and localization of crop residues-An experimental study (Madagascar)[END_REF] also observed that less casts were produced with woody twigs of soybean compared with small straws of rice, underlining the importance of residue palatability for earthworm ingestion. This can be partly explained by the high C/N ratio of roots in comparison to shoots as it is known that earthworms consume preferentially organic compounds with low C/N [START_REF] Curry | The feeding ecology of earthworms -A review[END_REF]. However, after 2 weeks, the Clab increases rapidly in Cast-Root, underlying the capacity of earthworms to process roots [START_REF] Cortez | Do earthworms eat living roots?[END_REF]. After 54 weeks, no more litter was observable in both mesocosms and the Clab and RCorg were not significantly different for casts containing roots or shoots (Figs 3 and5). Thus, even though earthworms prefer feeding on shoots, they are able to incorporate roots in casts, where the decomposition is activated at a comparable rate to that observed with shoots.

Fate of organic matter initially present in soil

In the presence of earthworms, during the first 6 months, the RCorg x is higher in Root-E than in Shoot-E (Table S1), with a significant difference after 6 months of experiment (Fig. 4).

Moreover, the Corg content in Shoot-E is generally lower than that measured before the experiment in the same mesocosm (RCorg x ≤ 1). This difference likely reflects a priming effect in this mesocosm, leading to a high mineralization of the organic matter originally present in the soil. This is also observed to a lesser extent with shoots without earthworms. The enhanced SOM mineralization in the presence of fresh residues has been reported in many studies [START_REF] Kuzyakov | Priming effects: Interactions between living and dead organic matter[END_REF]. Indeed, microorganisms depend on the energy present in fresh residues to decompose the recalcitrant organic matter present in the soil [START_REF] Fontaine | Fungi mediate long term sequestration of carbon and nitrogen in soil through their priming effect[END_REF][START_REF] Fontaine | Stability of organic carbon in deep soil layers controlled by fresh carbon supply[END_REF]. For example, [START_REF] Lu | Carbon dynamics of rhizodeposits, root-and shoot-residues in a rice soil[END_REF] observed that the simple presence of fresh residues, either root or shoot, favored the degradation of native soil organic matter after 8 weeks of experiment, probably due to the stimulation of the microbial activity. The soil fauna, including earthworms, was also mentioned as inducing a priming effect in soil by [START_REF] Lavelle | Mutualism and biodiversity in soils[END_REF]. More recently, [START_REF] Coq | Earthworm activity affects soil aggregation and organic matter dynamics according to the quality and localization of crop residues-An experimental study (Madagascar)[END_REF] showed that total soil C had decreased after 5 months of incubation of rice or soybean shoot residues, in the presence of endogeic earthworms. [START_REF] Fontaine | Fungi mediate long term sequestration of carbon and nitrogen in soil through their priming effect[END_REF] measured the effect of a 13 C labelled cellulose input in a soil during 161 days. They showed that, after one month, respiration of unlabelled CO2 reached a maximum, revealing a priming effect. A continuous and slow decrease in unlabelled CO2 respiration was then observed until the end of the experiment. This decrease in priming effect is in agreement with the present results showing that, after one year, RCorg was around 1 for Shoot-E and Shoot-NE. The suggested priming effect observed for the mesocosm containing shoots was not observed for the one containing roots. Indeed, during the first 6 months of experiment, the Corg tended to be higher in mesocosm containing roots than at the beginning of the experiment (RCorg ≥ 1) Thus, in the present study, the priming effect induced by root residues seems to be lower compared to that of shoot residues during the first six months of experiment. This priming might also be compensated by the higher preservation of root-derived carbon coupled to reduced activation of microbial activity compared to shoot residues [START_REF] Shi | Decomposition of roots and shoots of perennial grasses and annual barley-separately or in two residue mixes[END_REF]. This process is smoothed after one year;

supporting comment from [START_REF] Rasse | Is soil carbon mostly root carbon? Mechanisms for a specific stabilisation[END_REF] that the preferential shoot priming effect on SOM mineralization cannot explain the higher root-derived C in soil, as observed after one year on Fig. 2A. It has to be noted that the increase in RCorg observed for Control-NE after 24 and 54 weeks can be explained by the development of mosses in this mesocosm from 24 weeks until the end of the experiment, potentially increasing the Corg concentration in the 0-20 cm soil layer.

Conclusion

This mesocosm experiment allowed monitoring the fate of root and shoot residues in soil and earthworm casts. Residues deposited onto the soil surface were continuously incorporated in the 0-20 cm soil layer during the year of experiment. In agreement with previous studies, root residues incorporated in the 0-20 cm soil layer tended to decompose at a slower rate than shoot residues, underlining the essential role of litter chemical composition in the organic C fate. Earthworms tended to accelerate both root and shoot residue decomposition in soil. The activity of earthworms also induced a slight, but not statistically significant, transfer of C derived from residues in the deeper soil layer (40-60 cm), without any distinction between roots and shoots. In earthworm casts, root and shoot residues presented similar incorporation and decomposition of Corg trends. A chronology for cast formation and evolution was proposed, based on 13 C and organic C results. Three phases were identified: (1) the first month: mainly incorporation of fresh residues in casts, (2) until 6 months: re-ingestion of labelled casts and incorporation of fresh residues and finally (3) after 6 months: mainly microbial decomposition of residues in casts. This study provides new elements on the role of earthworms on incorporation and decomposition of organic C from roots and shoots into soil. Earthworms minimized the diverging fate of root and shoot residues, after the year of experiment, in both soil and cast samples. Thus, the presence of earthworms tended to enhance the decomposition of structures recognized as more chemically resistant. Moreover, during the first six months of experiment, the presence of earthworms and shoot residues enhanced the decomposition of soil organic C initially present in the soil. After one year, earthworms had accelerated the incorporation and decomposition of plant residues in the soil. However, after this period, the 13 C signal had not decreased in the soil and had only started to decrease in casts. Thus, longer term experiments are needed to investigate the stabilization of C-derived from root and shoot residues in the presence of earthworms.

Moreover, while these results bring new light on the fate of root and shoot litter in the presence of earthworms, they further require field-based experiment, including other earthworm ecological categories, to be generalized. Table S1. Organic carbon content relative to the soil organic carbon content at the beginning of the experiment (RCorg) (n=3). Numbers in parentheses indicate the standard deviation for the 3 samples. Bold letters represent statistical results of Kruskal-Wallis test, different letters indicating significant difference between the six mesocosms, for a given time step. Statistically significant differences were observed after 2 and 24 weeks of experiment.

Earthworms

Fate of 13 C

 13 labelled root and shoot residues in soil and anecic earthworm casts: a mesocosm experiment A.Vidal a , K. Quenea a,* , M. Alexis a , T. T. Nguyen Tu a , J. Mathieu b , V. Vaury b , S. Derenne a a UMR Milieux environnementaux, transferts et interactions dans les hydrosystèmes et les sols (METIS), UMR 7619, UPMC, CNRS, EPHE, 4 place Jussieu, F-75252 Paris, France b UMR IEES-Paris, UMR 7618, UPMC, UPEC, CNRS, INRA, IRD, AgroParisTech,

Fig. 1 .Fig. 2 .Fig. 3 .

 123 Fig. 1. Experimental design. E: with earthworms, NE: without earthworms

Fig. 4 .

 4 Fig. 4. Organic carbon content relative to the soil organic carbon content at the beginning of

Fig. 5 .

 5 Fig. 5. Organic carbon content relative to the soil organic carbon at the beginning of the ex-

Fig. 6 .

 6 Fig. 6. Simplified representation of the proposed chronology explaining residue incorporation

  Fig.1

Table 1 -

 1 Bulk features of the soil used for the experiment

	Soil characteristics	Values
	Clay (< 2 μm)	189 g.kg -1
	Loam (2-50 μm)	248 g.kg -1
	Sand (50-2000 μm)	563 g.kg -1
	Total carbonates (CaCO3) 19.0 g.kg -1
	CEC Metson	9.90 cmol.kg -1
	Organic Carbon	12.1 g.kg -1
	Nitrogen	1.30 g.kg -1
	δ 13 C	-28.1 ‰

Table 2 -

 2 Initial litter characterization (n=13). Numbers in parentheses indicate the standard deviation for the 13 samples.

		δ 13 C (‰) C (g.kg -1 ) N (g.kg -1 ) C:N
	Root	1324 (42) 402 (40)	13.1 (1.2) 30.7 (3.1)
	Shoot	1632 (16) 408 (6.3) 27.7 (2.3) 14.8 (1.1)

Acknowledgements

Patrick Dumont is acknowledged for the management of the UPMC greenhouse. This study benefits from the financial support from the EC2CO program from the French National Institute of Sciences of the Universe (CNRS / INSU). We thank anonymous reviewers for their constructive comments.

Where R is the 13 C/ 12 C ratio of the sample or the standard.

The proportion of C derived from the labelled litter in the soil or casts (Clab), was expressed in percentage by applying equation 2:

Where δs is the δ 13 C value of soil samples or of casts with labelled litter, δc is the δ 13 C value of the control soil sample or control casts without litter, δl is the δ 13 C value of the labelled litter.

In order to overcome the initial variability of the soil organic C measured in the different mesocosms, all the organic C contents were normalized using equation 3:

Where RCorg x represents the trend in organic C content ratio of C after x weeks, Cx is the organic C content after x weeks of experiment and C0 is the soil organic C content before adding earthworms and residues, for the corresponding mesocosm. Thus, values lower or higher than one express a decrease or increase in organic C content in comparison to the beginning of the experiment, respectively.

A similar formula was applied for the casts, where Cx is the organic C content in cast and C0

is the organic C content in the soil before adding earthworms and residues, for the corresponding mesocosm. Thus, values higher than one indicate that the organic C content is higher in the casts compared to the initial soil.

Statistical analysis

Statistical analyses were realized using the R statistical software. As data were not normally distributed (Shapiro-Wilk test), samples from different mesocosms were compared using the non-parametric Kruskal-Wallis test followed by a pairwise comparison of groups from the package 'RcmdrPlugin.coin'. For all analyses, statistical significance was set as α = 0.05.


Véronique Leberre
email: leberre@insa-toulouse.fr

Eric Baranowski

Martine Deplanche

Lidwine Trouilh

Jean Marie Franc ¸ois

Detection of minority variants within bovine respiratory syncytial virus populations using oligonucleotide-based microarrays

Keywords: Oligo-microarrays, BRSV, Discrimination

Microarray technology, originally developed for highly parallel examination of gene expression is regarded as a potential tool in prognosis and diagnosis. With respect to a discrimination analysis, difference as small as one nucleotide base can be distinguished using oligonucleotide-based microarrays. However, this degree of specificity is dependent on several parameters, including the size of the oligoprobes and the sequence context of the probes (e.g. local melting temperature), hybridization conditions and to some extent the chemistry of the glass slides onto which the probes are deposited. Using bovine respiratory syncytial virus (BRSV) as a model study, an oligonucleotide-based microarray approach was developed to measure the relative abundance of a particular single nucleotide variant within mixed BRSV populations. Using this technology, we show that it is possible to discriminate at a rate of 1%, minority variants in a BRSV population.

Introduction

Human and bovine respiratory syncytial viruses (HRSV and BRSV) are closely related paramyxoviruses responsible for severe lower respiratory tract disease in infants and calves, respectively. The bronchiolitis and pneumonia caused by BRSV in calves are similar clinically, pathologically, and epidemiologically to the disease caused by HRSV in children.

Because of the high mutation rate operating during RNA genome replication, BRSV populations are genetically heterogeneous [START_REF] Deplanche | In vivo evidence for quasispecies distributions in the bovine respiratory syncytial virus genome[END_REF] and exhibit an important potential for variation which is reflected in its antigenic diversity. Despite evidence of the highly heterogeneous nature of RNA viral populations, the consensus sequence is often the unique criteria used to characterize these genome populations. Work with different viral systems has highlighted the significance of the mutant spectrum -the distribution of variants continuously generated during the error-prone replication of RNA genomesfor the study of RNA viral quasispecies and their adaptability to changing environments [START_REF] Domingo | Viruses as quasispecies: biological implications[END_REF]. Analysis of the mutant spectrum can also provide valuable information on virus response to antiviral treatments [START_REF] Domingo | Quasispecies and its impact on viral hepatitis[END_REF], and may find applications for the quality control of attenuated vaccines, including the risk associated with virulence reversion [START_REF] Amexis | Quantitative mutant analysis of viral quasispecies by chip-based matrix-assisted laser desorption/ionization timeof-flight mass spectrometry[END_REF]. This calls for methods capable of determining distributions of mutations within RNA virus genome populations that should be rapid and cost-effective, but preferentially high throughput. Sequencing of a viral genomic region from a collection of biological clones (virus from individual plaques formed on cell monolayers) and/or molecular clones (RT-PCR products cloned in E. coli) is a common method to generate coarse-grained pictures of the mutant spectrum. Alternatively, Q-PCR assays have been developed to determine the frequency of particular mutant genomes within viral populations. Recently, a number of Q-PCR-derived methods have been developed for the assessment of genetic stability and quantitative monitoring of genetic changes in RNA viruses of clinical interest [START_REF] Wood | Laboratory tests for live attenuated poliovirus vaccines[END_REF][START_REF] Amexis | Quantitative mutant analysis of viral quasispecies by chip-based matrix-assisted laser desorption/ionization timeof-flight mass spectrometry[END_REF]. The MAPREC method, which combines Q-PCR to enzyme restriction cleavage, has proven to be a sensitive method [START_REF] Chumakov | RNA sequence variants in live poliovirus vaccine and their relation to neurovirulence[END_REF], yet is restricted to a limited number of known mutations [START_REF] Wood | Laboratory tests for live attenuated poliovirus vaccines[END_REF]. [START_REF] Amexis | Quantitative mutant analysis of viral quasispecies by chip-based matrix-assisted laser desorption/ionization timeof-flight mass spectrometry[END_REF] proposed to combine MAPREC with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (DNA Mas-sArray). However, major disadvantages of these methods are that they are comparatively labour intensive, low throughput and expensive.

We have recently shown that single nucleotide changes can be distinguished using oligonucleotide-based microarrays [START_REF] Le Berre | Dendrimeric coating of glass slides for sensitive DNA microarrays analysis[END_REF], suggesting that monitoring of genetic heterogeneity within viral quasispecies could be achieved using DNA microarrays. This technique permits simultaneous interrogation with a large number of probe sequences with high robustness and at a lower price per assay. The degree of specificity can be influenced by different parameters, including the size of the oligoprobes or their sequence context (e.g. local melting temperature), hybridization conditions and to some extent the chemistry of the glass slides onto which the probes are deposited. The literature is relatively vague with respect to these technical questions, as for instance probe sizes from 13 up to 50 mers have been used to diagnose various viral species [START_REF] Warsen | Simultaneous discrimination between 15 fish pathogens by using 16S ribosomal DNA PCR and DNA microarrays[END_REF][START_REF] Ryabinin | Microarray assay for detection and discrimination of Orthopoxvirus species[END_REF][START_REF] Martin | Microarray-based identification of antigenic variants of foot-and-mouth disease virus: a bioinformatics quality assessment[END_REF][START_REF] Palacios | Panmicrobial oligonucleotide array for diagnosis of infectious diseases[END_REF].

Using BRSV as a model study, we investigated optimal conditions to discriminate between parental and mutants of BSRV at a specific genome position using DNA oligo-microarrays. Using this technology, we show that it is possible to discriminate at a rate of 1%, single nucleotide minority variants in a BRSV population.

Materials and methods

Virus populations

BRSV populations W2BT10 and W2HEp58 derived from strain W2-00131 [START_REF] Deplanche | In vivo evidence for quasispecies distributions in the bovine respiratory syncytial virus genome[END_REF]. Virus population W2BT10 was obtained upon propagation of BRSV W2-00131 in bovine turbinate (BT; ATCC reference CRL1390) cells for 10 passages. BRSV W2HEp58 was derived from population W2BT10 following virus amplification in baby hamster kidney 21 (BHK-21; ATCC reference CCL-10) cells and long-term propagation (58 passages) in human epithelial 2 (HEp-2; ATCC reference CCL-23) cells.

Oligonucleotides design

Two sets of oligonucleotides specific to BRSV genomic region encoding phosphoprotein P (GeneBank accession no. AF092942) were designed (Table 1). The probe lengths of the first set was 14-15 mers and a second set from 19-23. Unless otherwise stated, these probes were flanked with a 15 mer poly dT at their 5 -extremity. The single nucleotide that discriminates between BRSV parental (W2BT10) and the BRSV mutant (W2HEp58) genomes (position 2686 on the RNA genome) was always positioned at the centre of the 14-22 mers oligonu- cleotides (not taking into account the flanking spacer). Two additional BRSV-specific oligonucleotides (F70-23 and F218-19) mapping outside of phosphoprotein P coding region were used as a control. The oligonucleotides were purchased from Operon.

Preparation of target DNA

Target DNA was obtained by PCR amplification from plasmid DNA or cDNA templates using labelled oligonucleotides 5 -Cy5-GGAAACTATAGAAACATTTGACAATAA-3 (BRSV genome position 2659-2685) and 5 -Cy5-ATATGAGTAGCT-TGATTCTTCTTC-3 (BRSV genome position 2710-2687). cDNA templates were produced by retrotranscription (RT) of BRSV genomic RNA using oligonucleotide N2.1 as described by [START_REF] Valarcher | Evaluation of a nested reverse transcription-PCR assay based on the nucleoprotein gene for diagnosis of spontaneous and experimental bovine respiratory syncytial virus infections[END_REF]. To construct plasmid DNA templates encoding BRSV sequences derived from W2BT10 (parental) and W2HEp58 (mutant) populations, BRSV genomic region spanning nucleotides 1959-3333 was amplified by RT-PCR using oligonucleotides 5 -TGGTCATGCCAGCGTACAAGC-3 and 5 -CATAGGAACCCATATTGTRAGAGATGC-3 and cloned into pCRII-TOPO vector (Invitrogen). Plasmid DNA construction was verified by nucleotide sequencing of the PCRamplified BRSV fragments. Heterogeneous target DNA samples were produced by PCR amplification using different ratios of parental and mutant BRSV cDNAs as DNA templates (Table 2). A SYBR green-based Q-PCR assay was used to standardize BRSV cDNA templates derived from populations W2BT10 (parental) and W2HEp58 (mutant). Oligonucleotides 5 -ATTCGTACATTTTGGCATTGCTC-3 and 5 -TTCCTTC-WACCCTACTACCTCCTCT-3 were designed to amplify a 59 bp product from BRSV cDNA (BRSV genome position 1809-1867) using Primer Express v2.0 software (Applied Biosystems). The reaction was carried out using the qPCR ROX-&GO Green reaction mix (QBIOgene). Thermal cycling was performed with the GeneAmp 5700 (Applied Biosystems) under the following conditions: 95 • C for 15 min followed by 40 cycles of 94 • C for 15 s and 59 • C for 30 s. Standard curves were generated from 10-fold dilutions of plasmid DNA template encoding parental BRSV sequence.

Microarray printing

Lyophylised oligonucleotides were dissolved in the Nexterion TM Spot solution from Schott-0.005% SDS (20 M). They were printed on epoxy-silane-derivatized glass slides (Nexterion ® HiSens E from Schott) by mechanical spotting with a robot (Q-Array mini from Genetix). Further technical information relating to this robot can be found at P.O. Brown's website (http://cmgm.stanford.edu/pbrown/mguide/index.html). The print head on the arrayer uses 'quill' pins SMP3 from Arraylt, whose contact on the surface delivers about 1 nl of DNA solution per spot, with a size diameter of about 100 m under this condition. Oligoprobes were spotted in quadruplicate, with a centre to centre distance of 250 m. An anchoring Cy3-labelled oligonucleotide derived from yeast HSP12 gene (5 -Cy3-GACACGACCGGAAACATATT-3 ) was spotted in quadruplicate, on the right side of the probes according to Fig. 1. Twelve replicates of this grid were spotted on the slide. The oligo-microarrays were kept in a plastic container and stored in a desiccator at room temperature until use.

Hybridization and scanning

Pre-hybridization of the printing area was carried out by immersing array in Pronto TM Universal Pre-hybridization Solution (Corning) for 30 min at 37 • C. The slides were then transferred to a fresh container and wash for 2 min with a 0.2× SSC solution, then transferred to another container and wash for 2 min with 0.1× SSC, then dried by centrifugation. Hybridization was carried out at 37 • C or 55 • C for 2 h or 4 h in the Eppendorf ® ThermoStat Plus equipped with exchangeable thermoblock for four slides. A silicon joint was stick on the slide to delimit the 12 zones of hybridization (Fig. 1). A 3 l solution of PCR target was mixed with 40 l of Pronto TM short oligohybridization solution (Corning) and with 3 l of sonicated DNA salmon sperm. After denaturation at 95 • C for 5 min, the mix was transferred onto the delimited zone of the printed slide. Post-hybridization washes were carried out by incubating the slides under agitation, 5 min in a 2× SSC/0.1% SDS solution, followed by 2 min in a 0.2× SSC solution, then 2 min into 0.1× SSC solution. The slide was then dried by centrifugation. Fluorescence signals were detected by a laser scanner (GenePix 4000A from Axon Instrument) and analysed with the GenePix 6.01 software.

Results

Probe length is a key factor for microarray detection of single nucleotide variations in BRSV genome

To reveal the presence of single nucleotide variants in BRSV genome populations by oligonucleotide-based microarrays, target DNAs were amplified from BRSV phosphoprotein P genomic region cloned into E. coli plasmids. Phosphoprotein P sequences were derived from two BRSV populations (W2BT10 and W2HEp58), which are characterized by a T/G polymorphism at genome position 2686.

A first set of oligonucleotide probes ranging from 14 to 17 mers were designed with the discriminating nucleotide positioned at the centre of the probe (Table 1) and printed on epoxy-activated glass slides. This short oligonucleotide strategy was chosen in accordance with a recent report showing the detection of antigenic variants of foot-and-mouth disease virus (FMDV) using 15-mers oligoprobes with oligo (dT)15 spacer [START_REF] Martin | Microarray-based identification of antigenic variants of foot-and-mouth disease virus: a bioinformatics quality assessment[END_REF]. Hybridization with Cy5-labelled target DNA with these short oligoprobes was not specific, and neither addition of a poly dT spacer at oligonucleotide 5 extremity, nor elevation of the hybridization temperature (65 • C) could improve the signal intensity and the specificity of the hybridization on the parental DNA target. Therefore, these experiments were repeated using longer oligonucleotides from 19 to 23 mer spotted on Nexterion ® HiSens E (Schott) slide. In these conditions, 2 h hybridization at 37 • C was sufficient to obtain an intense fluorescence signal at each spot, although the absolute intensity was dependent upon the length of the oligonucleotide probes, with a maximal hybridization signal on the 23 mer probes (Fig. 2). However, discrimination between T2686 and G2686 target DNAs, as determined by the T/G signal ratio, was optimal with the 19 mer oligoprobes (Fig. 3). The T/G signal ratio was not enhanced by using purified target DNA (PCR products), whereas target signal intensities were strongly reduced after the purification procedure, probably as a result of the loss of labelled material. As expected, no hybridization was observed between Cy5-labelled target DNA and oligoprobes designed on the F70 and F218 BRSV genomic regions, as they mapped outside of the phosphoprotein P coding region. 2, and were hybridized on a 21 mer oligoprobes at 55 • C for 2 h. (B) Corresponds to a zoom of the histogram (A) for heterogeneous samples 10, 1 and 0. The fluorescent signals were captured using laser scanner Axon GenePix 4000A with a PMT of 600 at 635 nm and 450 at 532 nm. The discrimination was expressed as the ratio of hybridization signal between BRSV parental and mutant genome.

Sensitivity limit for discrimination of mutant variants within BRSV populations

Data given above showed that the parental and the mutant BRSV genome at position 2686 could be discriminated using 19 or 21 mer oligonucleotides. The discriminative sensitivity was quantified by mixing different proportions of parental and mutant cDNA before performing a labelled PCR on this prepared mix (Table 2). These PCR mixtures were subsequently hybridized to a microchip made of 21-mer oligonucleotides. As shown in Fig. 4, a proportion of as low as 1% of the parental amplicon in the total BRSV amplicon population gave a statistically significant, elevated fluorescent signal in comparison to residual fluorescent signal obtained with a population made of 100% of the mutant amplicon. This discrimination rate was around 10 times higher than the one reported by [START_REF] Proudnikov | Analysis of mutations in oral poliovirus vaccine by hybridization with generic oligonucleotide microchips[END_REF] for the population of oral poliovirus vaccine, probably because these authors used a generic microchips consisting in all possible hexamers flanked at both ends of an octanucleotide for detection of the neurovirulent mutants in oral poliovirus vaccine. The discrimination rate between two different targets DNA hybridizing the same probes was also comparable to that of a same target hybridizing two oligoprobes that differed from each other by a single point mutation [START_REF] Le Berre | Dendrimeric coating of glass slides for sensitive DNA microarrays analysis[END_REF].

Discussion

Recent progress in pathogen genomics and microarray technology has allowed the development of high throughput tools for the rapid and accurate detection of human, animal and plant pathogens. [START_REF] Volokhov | Identification of Listeria species by microarray-based assay[END_REF] used oligonucleotide microarrays for the detection and discrimination of six species among the Listeria genus based on sequence differences in the iap gene. Microarrays bearing 40-70 mer oligonucleotides were also designed for the genotyping of human and plant viral pathogens [START_REF] Wang | Microarray-based detection and genotyping of viral pathogens[END_REF][START_REF] Bystricka | Oligonucleotide-based microarray: a new improvement in microarray detection of plant viruses[END_REF]. More recently, [START_REF] Palacios | Panmicrobial oligonucleotide array for diagnosis of infectious diseases[END_REF] reported the development of a panmicrobial microarray (GreeneChipPm) consisting of 60-mer oligonucleotides probes for diagnosis of a large collection of pathogens.

In all cases, these microarrays were designed for the detection of various microbes or species and the specificity was based on the oligoprobes design. Nevertheless, the current literature reveals few quantitative DNA microarray-based analyses of mutant genome distributions within viral populations. This question has been addressed in this present case study aiming at quantifying the heterogeneity of RNA genomes in viral populations. The strategy that used short 21 mer oligonucleotides was found to be effective, because the discrimination rate between the two genomes was around 1%. This resolution was in the same order of magnitude as a classical RT-PCR and the recently developed MAPREC technique.

Taken together, these results should encourage future development for more complex oligonucleotide arrays to investigate the genetic variability within viral population in a high throughput manner, and to use them for the screening of viral isolates from various sources, including healthy carriers, animals and plants.

Fig. 1 .

 1 Fig. 1. Scheme of probes spotting on epoxy-activated glass slides. Oligonucleotides were spotted in quadruplicate, with a centre to centre distance of 250 m. A Cy3-labelled yeast HSP12 oligonucleotide (5 -Cy3-GACACGAC-CGGAAACATATT-3 ) was spotted in quadruplicate at the right side of square to allow anchoring of the feature for image capture by the GenePix6.0 software. This pattern was repeated 12 times to fit with the 16 well-superstructure seal strips from Schott Nexterion used in the hybridization step.

Fig. 2 .

 2 Fig. 2. Hybridization patterns of BRSV parental (A) and mutant (B) target DNA on the oligonucleotides arrays. The Cy5-labelled targets were produced by RT-PCR on a BRSV genomic region encompassing position 2686. These amplicons were hybridized at 37 • C for 4 h. Images were captured by the laser scanner Genepix 4000A at a PMT of 600 at 635 nm and 450 at 532 nm.

Fig. 3 .

 3 Fig.3. Influence of the probe sizes on the hybridization specificity of BRSV parental and mutant genome. The target BRSV wild type (A) and BRSV mutant (B) were produced as in Fig.2. They were hybridized at 37 • C for 4 h and the specificity was expressed as the ratio of the BRSV parental hybridization signal vs. that of the mutant on each oligoprobe.

Fig. 4 .

 4 Fig. 4. Discrimination rate of BRSV parental cDNA vs. BRSV mutant cDNA. The Cy5-labelled targets from BRSV parental and mutant genome were produced by RT-PCR as in Fig. 2. The parental and mutant amplicons were mixed at various ratios as indicated in Table2, and were hybridized on a 21 mer oligoprobes at 55 • C for 2 h. (B) Corresponds to a zoom of the histogram (A) for heterogeneous samples 10, 1 and 0. The fluorescent signals were captured using laser scanner Axon GenePix 4000A with a PMT of 600 at 635 nm and 450 at 532 nm. The discrimination was expressed as the ratio of hybridization signal between BRSV parental and mutant genome.

Table 1

 1 Oligonucleotides specific to BRSV genome

	Oligonucleotide	Sequence (5 →3 ) a	Genomic
			position b
	P112-1	TTCTTCATTATTGT	2692-2679
	P112-2	TTCTTCCTTATTGT	2692-2679
	P112-3	CAATAATGAAGAAG	2680-2693
	P112-4	CAATAAGGAAGAAG	2680-2693
	P112-5	ACAATAATGAAGAAG	2679-2693
	P112-6	ACAATAAGGAAGAAG	2679-2693
	P112-7	TTCTTCATTATTGTC	2692-2678
	P112-8	TTCTTCCTTATTGTC	2692-2678
	P112-9	CAATAATGAAGAAGA	2680-2694
	P112-10	CAATAAGGAAGAAGA	2680-2694
	P112-19 wt	TGACAATAATGAAGAAGAA	2677-2695
	P112-19 mut	TGACAATAAGGAAGAAGAA	2677-2695
	P112-21 wt	TTGACAATAATGAAGAAGAAT	2676-2696
	P112-21 mut	TTGACAATAAGGAAGAAGAAT	2676-2696
	P112-23 wt	TTTGACAATAATGAAGAAGAATC	2675-2697
	P112-23 mut	TTTGACAATAAGGAAGAAGAATC	2675-2697
	F70-23	AATGTGTGTAAAAGTACTGATTC	5768-5790
	F218-19	CCAACATAGTAACTGTGAT	6213-6231

a The mutated position is underlined. b The numbering corresponds to the reference sequence of BRSV genome (GeneBank accession no. AF092942).

Table 2

 2 Parental and mutant BRSV cDNAs ratios in heterogeneous target DNA samples

	Heterogeneous sample	cDNAs ratio a
	100	100:0
	99	99:1
	90	93:7
	50	60:40
	10	15:85
	1	1:99
	0	0:100

a [BRSV WT cDNA]/[BRSV mutant cDNA].

Acknowledgments

Dr. Andrew Mc Donagh from the Imperial College of London is acknowledged for his English proofreading of the manuscript, and Mr. Moulignie for his excellent technical assistance. This work was supported in part by the ACI 'Microbiologie 2003' of the French Ministry of Education and Research to Eric Baranowski and by the 'Reseau National des Genopoles' to Veronique Le Berre and Jean-Marie Franc ¸ois.